Peptides derived from Ruminococcus torques

Abstract

The present invention relates to polypeptides derived from Ruminococcus torques, as well as polypeptide fragments and variants thereof, useful for the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, and bone disorders, and to host cells comprising said polypeptides, polypeptide fragments or variants thereof for use as probiotics or live biopharmaceutical products (LBPs).

Description

[Technical field]

[0001] The present invention relates to polypeptides derived from Ruminococcus torques, as well as polypeptide fragments and variants thereof, useful for the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, and bone disorders, and to host cells comprising said polypeptides, polypeptide fragments or variants thereof for use as probiotics or live biopharmaceutical products. [Background technology]

[0002] Over the past decade, advances in epidemiological, physiological, ecological, and omics-based human research, complemented by cellular and mechanistic studies in animals, have led to the idea that microbial communities mediate a significant portion of the environmental effects on human health. 1,2 These non-pathogenic (i.e., commensal and mutualistic) microorganisms, collectively referred to as the microbiota, include vast numbers of interacting bacteria, archaea, bacteriophages, eukaryotic viruses, and fungi that coexist on human surfaces and in all body cavities. The collection of all microbial genes in and on an individual (i.e., the microbiome) represents a gene repertoire that is more than an order of magnitude higher in genes than the human nuclear genome. 3 .

[0003] The majority of microbes that inhabit humans reside in the distal gut where they play important roles in training host immunity, digesting food, regulating enteroendocrine function and neural signaling, modifying the action and metabolism of drugs, removing toxins, and releasing multiple microbial compounds that affect the host. 1,2 There are many examples of human gut bacterial strains. One of them is Ruminococcus torques. In human metagenomes, the contribution of specific strains of R. torques can reach 10% of the total relative abundance. 8 The bacteria are associated with mucous membranes and are able to break down mucus.

[0004] One prime example of several bacterial compounds known to modulate host metabolism is Amuc_1100, a protein present in the outer membrane of the symbiotic gut bacterium Akkermansia muciniphila. 4 The bacteria has been associated with improved metabolism in preclinical studies when administered in either raw or pasteurized form. 4 In a recent human pilot intervention, the formulation of A. muciniphila was shown to be tolerable without side effects and to alleviate metabolic abnormalities in overweight and obese patients with some clinical relevance. 5 .

[0005] According to the World Health Organization (WHO), obesity has tripled since 1975, with more than 1.9 billion adults being overweight in 2016, of which more than 650 million were obese. Obesity is closely linked to other conditions such as hypertension, fatty liver disease (FLD), and metabolic syndrome, with indications such as type 2 diabetes (T2D). The WHO estimates that the number of people with diabetes has increased four-fold since 1980. Today, 422 million people worldwide have diabetes, the majority of whom suffer from T2D, which is caused by overweight, obesity, and lack of physical activity.

[0006] Other diseases that affect many people include muscular and skeletal diseases, disorders and injuries. Bone loss or weakening of bones is a common disorder, especially in countries with an aging population. In some countries, up to 70% of people over 80 years old suffer from osteoporosis. In the following, some muscle disorders, namely neuromuscular disorders such as muscular dystrophies, cause skeletal muscle weakening and destruction over time. Prognosis for muscular dystrophies and other neuromuscular disorders ranges from mild to severe, depending on the specific cause.

[0007] Thus, there is a clear need in the art for improved treatment and prevention of the above-mentioned diseases and disorders. Such approaches to treatment can identify organisms and compounds from the gut microbiome that promote health through positive effects on host metabolism. Summary of the Invention

[0008] The present invention relates to polypeptides and their uses for the treatment and / or prevention of metabolic, muscle and bone disorders, as well as host cells comprising the polypeptides, polypeptide fragments and variants thereof for use as probiotics or as live biopharmaceutical products (LBPs).

[0009] The inventors have demonstrated that bacterial peptides derived from Ruminococcus torques, as well as fragments and variants of the peptides, may be effective in the treatment and prevention of metabolic disorders, muscle disorders and injuries, and bone disorders.

[0010] Thus, provided herein is an isolated polypeptide having a length of less than 200 amino acids comprising or consisting of an amino acid sequence selected from the group consisting of: a) the amino acid sequence according to SEQ ID NO: 4 and / or SEQ ID NO: 19; b) variants of SEQ ID NO:4 and / or SEQ ID NO:19, which have at least 60%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95% sequence identity to SEQ ID NO:4 and / or SEQ ID NO:19, but less than 99% sequence identity to SEQ ID NO:4 and / or SEQ ID NO:19; c) variants of SEQ ID NO: 4 and / or SEQ ID NO: 19 having 1 to 40 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, such as 5, 10, 15, 20, 25, 30 or 35 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; d) a fragment of SEQ ID NO: 4 and / or SEQ ID NO: 19 having a length of at least 10 amino acids or a variant of said fragment having 1 to 5 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, respectively, such as 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, wherein the polypeptide has a length of less than 50 amino acids; e) an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by truncation at the N-terminus of at least one amino acid, such as 1 to 67 amino acids, for example 1 to 60 amino acids, for example 1 to 50 amino acids, for example 1 to 40 amino acids, for example 1 to 30 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, or a variant thereof having 1 to 10 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; f) an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by truncation at the C-terminus of at least one amino acid, such as 1 to 21 amino acids, such as 1 to 20 amino acids, such as 1 to 15 amino acids, such as 1 to 10 amino acids, such as 1 to 5 amino acids, or a variant thereof having 1 to 30 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, such as 1, 5, 10, 15, 20 or 25 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; g) an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by a truncation at the N-terminus of at least one amino acid, such as 1 to 67 amino acids, for example 1 to 60 amino acids, such as 1 to 50 amino acids, for example 1 to 40 amino acids, for example 1 to 30 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, and a truncation at the C-terminus of at least one amino acid, such as 1 to 21 amino acids, for example 1 to 20 amino acids, for example 1 to 15 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, wherein said polypeptide is an amino acid sequence having a length of at least 10 amino acids, or a variant thereof having 1 to 5 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, such as 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; h) the amino acid sequence according to SEQ ID NO: 5 and / or SEQ ID NO: 20; i) variants of SEQ ID NO:5 and / or SEQ ID NO:20, which have at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95% sequence identity to SEQ ID NO:5 and / or SEQ ID NO:20, but less than 99% sequence identity to SEQ ID NO:5 and / or SEQ ID NO:20; j) variants of SEQ ID NO:5 and / or SEQ ID NO:20, having 1 to 10 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, wherein the polypeptide has a length of less than 50 amino acids; k) a fragment of SEQ ID NO: 5 and / or SEQ ID NO: 20 comprising at least 10 consecutive amino acids of SEQ ID NO: 5 and / or SEQ ID NO: 20, or a variant thereof having 1 to 5 amino acid substitutions relative to SEQ ID NO: 5 and / or SEQ ID NO: 20, such as 1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO: 5 and / or SEQ ID NO: 20, wherein the polypeptide has a length of less than 50 amino acids; l) fragments of SEQ ID NO: 19, selected from the group consisting of SEQ ID NOs: 27, 33, 34, 35, 36, 37 and 95, and their respective variants having 1 to 3 amino acid substitutions relative to SEQ ID NO: 19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 19, wherein the polypeptide has a length of less than 50 amino acids; m) fragments of SEQ ID NO: 4, selected from the group consisting of SEQ ID NO: 107, 108, 109, 110, 111, 165 and 168, and respective variants thereof having 1 to 3 amino acid substitutions relative to SEQ ID NO: 4, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4, wherein the polypeptide has a length of less than 50 amino acids; n) fragments of variants of SEQ ID NO: 19, selected from the group consisting of SEQ ID NO: 173, 176, 181 and 188, and respective variants thereof having 1 to 3 amino acid substitutions relative to SEQ ID NO: 19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 19, wherein said polypeptide has a length of less than 50 amino acids; o) fragments of SEQ ID NO: 4, said fragments being selected from the group consisting of SEQ ID NO: 193, 196, 201 and 208, and respective variants thereof having 1 to 3 amino acid substitutions relative to SEQ ID NO: 4, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4, wherein said polypeptide has a length of less than 50 amino acids; p) fragments of SEQ ID NO: 19, selected from the group consisting of SEQ ID NO: 210, 211, 212, 213, 229, 232, 233, 234 and 235, and their respective variants having 1 to 3 amino acid substitutions relative to SEQ ID NO: 19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 19, wherein the polypeptide has a length of less than 50 amino acids; q) Fragments of SEQ ID NO: 4, selected from the group consisting of SEQ ID NOs: 243, 244, 245, 246, 262, 265, 266, 267 and 268, and respective variants thereof having 1 to 3 amino acid substitutions relative to SEQ ID NO: 4, such as 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4, wherein said polypeptide has a length of less than 50 amino acids.

[0011] Further provided herein are isolated polynucleotides encoding the polypeptides of the present invention.

[0012] The present specification also provides a vector comprising a polynucleotide according to the present invention.

[0013] Further provided herein is a host cell comprising the polynucleotide and / or vector according to the invention.

[0014] Also provided herein are pharmaceutical compositions comprising the polypeptides, polynucleotides, vectors and / or host cells according to the invention.

[0015] Also provided herein is a feed composition comprising the polypeptide, conjugate, polynucleotide, vector and / or host cell, and which also comprises one or more of prebiotics, probiotics, live biopharmaceutical products (LBPs), synbiotics, proteins, lipids, carbohydrates, vitamins, fiber and / or nutrients, such as feed minerals.

[0016] Further provided herein is a polypeptide, conjugate, polynucleotide, vector, host cell and / or pharmaceutical composition according to the invention for use as a medicament.

[0017] Further provided herein is a host cell according to the invention for use as a probiotic or live biopharmaceutical product (LBP).

[0018] Also provided herein is the use of a polypeptide, conjugate, vector and / or host cell according to the invention as a food ingredient or as a food or beverage additive.

[0019] Further provided herein is the use of a host cell according to the invention as a probiotic or live biopharmaceutical product (LBP). Also provided herein are polypeptides, conjugates, polynucleotides, vectors, host cells and / or pharmaceutical compositions according to the invention for use in the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, and / or bone disorders.

[0020] Further provided herein is the use of a polypeptide, conjugate, vector, host cell and / or pharmaceutical composition according to the invention in the manufacture of a medicament for treating metabolic disorders, muscle disorders and injuries, and / or bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and / or osteopetrosis.

[0021] Also provided herein is a method for the treatment of metabolic disorders, muscle disorders and injuries, and / or bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and / or osteopetrosis, comprising administering to an individual in need thereof a polypeptide, conjugate, polynucleotide, vector, host cell, and / or pharmaceutical composition according to the invention. [Brief description of the drawings]

[0022] [Figure 1]1 shows the amino acid sequence alignment of human FNDC5, bacterial FNDC5-like protein, human irisin, and RUCILP2, respectively. Identical amino acid residues between RUCILP2 and human irisin are represented by asterisks, with low and high similarities represented in the period and colon, respectively. Multiple sequence alignment of amino acids in human FNDC5, bacterial FNDC5-like protein, human irisin, and RUCILP2, respectively, was performed using the open access tool Clustal Ω (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to determine the number of identical and conserved residues. Thus, FIG. 1 shows the level of amino acid sequence similarity of human FNDC5, bacterial FNDC5-like protein, human irisin, and RUCILP2, respectively. [Diagram 2] Detection of dimerized RUCILP1 and RUCILP2 in the culture medium. In the left panel, Coomassie brilliant blue staining of proteins on polyvinylidene fluoride blot membranes showed equal loading of proteins in each well. In the right panel, Western blot for FNDC5 in the culture medium of Ruminoccocus torgues (RT)-ATCC27756 (n=3) and the control strain RT-ATCC35915 (n=3), each with a bacterial cell density of about 1010 per ml of culture medium after 3 days of anaerobic culture. Thus, the results in this figure suggest that the Ruminoccocus torgues (RT)-ATCC27756 strain releases a dimeric form of RUCILP2 into the growth culture medium. [Diagram 3] The predicted structure of RUCILP2 and its alignment with irisin are shown. The open source software I-TASSER was used for protein structure modeling by iterative threading assembly simulation. The open access PyMOL (v2.1.1) tool was applied as a visualization tool for the predicted 3D structures. This figure suggests that RUCILP2 is a structural analogue of irisin. [Figure 4]Docking models of the interaction between integrin αV / β5 receptor and RUCILP2 (left panel) and irisin (right panel), respectively, are shown. The putative integrin-binding regions of amino acids 60-76 and 101-118, respectively, are shown in dark color. The binding residues of RUCILP2 are closer to integrin αV / β5 receptor than irisin. The binding ability of RUCILP2 and irisin to integrin αV / β5 receptor was evaluated by computational analysis in Autodock (v4.2.6). The final complex structure of the docking model was shown by PyMOL (v2.1.1). This figure suggests the binding of RUCILP2 to integrin αV / β5 receptor. [Diagram 5] Figure 1 shows the visualization of predicted binding sites in RUCILP2 for integrin αV / β5 receptor. The ZDOCK web server ZDOCK (https: / / zdock.umassmed.edu / ) was applied to predict the top-ranked model of RUCILP2 and integrin αV / β5 receptor complex. This model was visualized in the PyMOL (v2.1.1) program and showed that RUCILP2 binds to integrin αV / β5 receptor at amino acid residues V7, E9, and E58, respectively. [Figure 6] Co-immunoprecipitation on nickel ion column to verify the binding of recombinant RUCILP2 to αV / β5 integrin receptor complex. 100 nM of Fc-fused RUCILP2 was incubated with 5 nM of His-tagged αV / β5 integrin receptor, followed by immunoprecipitation using nickel-nitrilotriacetic acid (Ni-NTA) agarose. Precipitated integrin and co-precipitated irisin were analyzed by immunoblot analysis. Elution shows a mixture of detached integrin and RUCILP2 from the integrin-RUCILP2 complex. Before loading, the sample was a mixture of co-incubation of RUCILP2 with integrin receptor. Thus, this experiment shows a direct interaction between RUCILP2 and integrin αV / β5. [Figure 7]We show the application of double-stranded RNAscope-based mRNA in situ hybridization arrays to identify signal dots of integrin αV / β5 receptors (ITGAV and ITGB5 mRNAs) in the submucosa of normal human colon. Two target mRNAs were stained as signal dots in red (ITGAV) and green (ITGB5), respectively. The experimental setup included Polr2a (RNA polymerase II subunit A, red) as a positive control and PPIB (peptidyl prolyl isomerase B, green) as a positive control, as well as DapB (dihydrodipicolinate reductase) as a negative control probe set. Images were acquired using a Zeiss AxioScan with a 20x objective. The visualized signals indicate the presence of integrin αV / β5 receptors in human colon tissue. [Figure 8] Figure 1 shows a double-stranded RNAscope-based mRNA in situ hybridization array to identify signal dots (submucosal) of integrin αV / β5 receptor (ITGAV and ITGB5 mRNA) and cocaine- and amphetamine-regulated transcript protein (CART) in normal human colon. The two target mRNAs are stained as signal dots in red (ITGAV and ITGB5) and green (CART). The experimental setup included DapB (dihydrodipicolinate reductase) as a negative control probe set. Images were acquired using a Zeiss AxioScan with a 20x objective. [Figure 9]Exposure of recombinant RUCILP2 to human visceral white preadipocytes or mouse inguinal white adipocytes upregulates the expression of genes involved in thermogenesis / browning. Top panel: mRNA expression levels of adipocyte differentiation marker genes such as Ucp1, Pparγ1, Dio2, and Cox2 and brown adipocyte-selective genes such as Cpt1b and Ebf2 on human white preadipocytes (HWPs). Human white preadipocytes were cultured until 80% confluent and switched to differentiation medium (containing 0.3ug / ml fetal bovine serum, 8ug / ml d-biotin, 0.5ug / ml insulin, and 400ng / ml dexamethasone). The differentiation process into mature adipocytes was completed after 12–14 days. Treatment of RUCILP2 began on day 3 of differentiation. After 14 days of differentiation, cells were harvested and gene expression was quantified by q-PCR. All gene expression levels were normalized to the gene expression level of TATA-binding protein (TBP). Lower panel: Stromal vascular fraction cells derived from mouse inguinal white adipose tissue were differentiated into adipocytes and treated with recombinant RUCILP2 at the indicated doses for 4 days, respectively. Graph shows qPCR of the indicated gene expression. For integrin inhibitor treatment, cells were treated with 10 μM cRGDyK (Selleckchem, #S7844) for 10 min, washed with PBS, and then treated with RUCILP2. Data are shown as mean + / - SEM of one representative experiment performed in technical triplicate. Statistical significance was determined by unpaired two-tailed Student's t-test. *, p<0.05 vs. 0 nM RUCILP2. [Figure 10] Recombinant RUCILP2 reduces lipid content in adipocytes as shown by Oil Red O staining. Staining was performed on 10% formalin-fixed adipocytes according to the manufacturer's protocol. [Figure 11]Recombinant RUCILP2 stimulates sclerostin expression in the MLO-Y4 (mouse long bone cell-Y4) cell line. Cells were incubated for 4 h in FreeStyle293 medium and treated for 16 h with the indicated concentrations of RUCILP2 or irisin (upper panel), followed by addition of vehicle (phosphate-buffered saline) or 10 μM of the integrin inhibitor cRGDyK for 10 min pretreatment (lower panel). Data are presented as mean ± SEM, n = 3 wells / group. Significant differences between the two groups were assessed using an unpaired two-tailed Student's t-test. Upper panel, *, p<0.05; **, p<0.01; #, p<0.05; ##, p<0.01, compared to the blank group. Lower panel, *, p<0.05, compared to the vehicle group. [Figure 12] Recombinant RUCILP2 induces myotube formation in mouse C2C12 myoblasts. C2C12 myotubes were treated with the indicated doses of RUCILP2 overnight and myotube images were collected at 10x magnification. [Figure 13] Recombinant RUCILP2 treatment reduces expression of genes involved in gluconeogenesis in HepG2 hepatocytes, increases expression of genes involved in intestinal integration in Caco-2 cells, and stimulates gene expression in H9C2 cardiomyoblasts, respectively. Cells were treated with various doses of RUCILP2 and cells were harvested for q-PCR quantification for the indicated genes. For integrin inhibitor treatment, cells were treated with 10 μM cRGDyK (Selleckchem, #S7844) for 10 min, washed with PBS, and then treated with RUCILP2. Data are shown as mean + / - SEM of one representative experiment performed in technical triplicate. Statistical significance was determined by unpaired two-tailed Student's t-test. *, p<0.05 vs. 0 nM RUCILP2. [Figure 14]Recombinant RUCILP2 stimulates glucagon-like peptide-1 (GLP-1) secretion in perfused rat colon when RUCILP2 is perfused through the luminal route. In the left panel, GLP-1 secretion from isolated perfused rat colon in the presence of RUCILP2 is mean ± SEM, n = 6 in each group. In the right panel, baseline is subtracted from total GLP-1 output during 10 min (12-21 min) of luminal infusion. **, p < 0.01 using Student's t-test. [Figure 15] Recombinant RUCILP2 stimulates peptide YY (PYY) secretion in perfused rat colon when RUCILP2 is perfused through the luminal route. In the left panel, PYY secretion from isolated perfused rat colon in the presence of RUCILP2 is mean ± SEM, n = 6 in each group. In the right panel, baseline is subtracted from total PYY output during 10 min (12–21 min) of luminal infusion. **, p < 0.01, using Student's t-test. [Figure 16] Recombinant RUCILP2 stimulates somatostatin secretion in perfused rat colon when RUCILP2 is perfused through the luminal route. In the left panel, somatostatin secretion from isolated perfused rat colon in the presence of RUCILP2 is mean ± SEM, n = 6 in each group. In the right panel, baseline is subtracted from total somatostatin output during 10 min (12–21 min) of luminal infusion. **, p < 0.05, using Student's t-test. [Figure 17] Daily intraperitoneal injection of recombinant RUCILP2 for 7 days in mice fed normal chow induces the expression of genes involved in thermogenesis and decreases the expression of genes involved in adipogenesis without affecting the expression of marker genes for lipolysis. Recombinant RUCILP2 was injected intraperitoneally at a concentration of 1 mg / kg daily for 1 week into 9-week-old wild-type C57BL / 6N mice. mRNA levels of the indicated genes were analyzed by qRT-PCR. Data are expressed as mean ± SEM, *, p<0.05; **, p<0.01; ***, p<0.001 when compared to the phosphate-buffered saline (PBS) group, n=9 animals / group. [Figure 18] Figure 1 shows the weight change of mice treated with oral gavage of different strains of the species Ruminococcus torques. R3-LD, R. torques ATCC35915, 5x107 colony forming units per 100 μl dose; R3-HD, R. torques ATCC35915, 5x108 colony forming units per 100 μl dose; R2-LD, R. torques ATCC27756, 5x107 colony forming units per 100 μl dose; R2-HD, R. torques ATCC27756, 5x108 colony forming units per 100 μl dose; HK-R2-HD, heat-killed R. torques ATCC27756, 5x108 colony forming units per 100 μl dose. Data are expressed as mean + / - SEM. Thus, this figure shows that oral (gavage) administration of live or pasteurized Rumminoccocus torques strains has no significant effect on weight development in mice fed a chow diet (Altromin1328 diet) over a period of 8 weeks. [Figure 19] Oral gavage of mice with Ruminococcus torques ATCC27756 strain, which synthesizes RUMTOR_00181, reduces fat mass and increases lean body mass in mice. Mice were fed normal chow and the intervention lasted for 8 weeks. Magnetic resonance imaging scans of body composition in the indicated groups of mice were performed according to the manufacturer's tutorial. R3-LD, R.torquesATCC35915, 5x107 colony forming units per 100μl dose; R3-HD, R.torquesATCC35915, 5x108 colony forming units per 100μl dose; R2-LD, R.torquesATCC27756, 5x107 colony forming units per 100μl dose; R2-HD, R.torquesATCC27756, 5x108 colony forming units per 100μl dose; HK-R2-HD, heat-killed R.torquesATCC27756, 5x108 colony forming units per 100μl dose. Data are presented as mean + / - SEM. *, p<0.05, *, p<0.01, when compared to PBS group as determined by unpaired two-tailed Student's t-test. [Figure 20]Figure 1 shows the weight progression of mice fed a high-fat diet treated by oral gavage with different strains of the species Ruminococcus torques. RT3, R. torques ATCC 35915, the gene encoding RUMTOR_00181 is absent, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC 27756, the gene encoding RUMTOR_00181 is present, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC 27756, the gene encoding RUMTOR_00181 is present, 5x109 colony forming units per 100 μl dose. Data are presented as mean + / - SEM (10 mice / group). *, p<0.05, RT2 vs. RT3; #, p<0.05, RT2 vs. sterile phosphate-buffered saline; &, p<0.05, RT2 vs. heat-killed RT2. Statistical significance was determined using unpaired two-tailed t-tests. Thus, this figure shows that oral (gavage) supplementation with live Rumminoccocus torques strain significantly reduces weight development in mice fed a high-fat diet over an 8-week period (Research Diet, D12451i). [Figure 21]RUMTOR_00181-producing Ruminococcus torques (RT ATCC27756) strain improves glucose tolerance. Mice were fed normal chow. Glucose tolerance experiments were performed at week 6. The left figure shows the intraperitoneal glucose tolerance test curves 6 weeks after oral gavage of RUMTOR_00181-producing R. torques strain. The right figure shows the area under the curve of the glucose tolerance test (GTT). R3-LD, R.torques ATCC35915, 5x107 colony forming units per 100 μl dose; R3-HD, R.torques ATCC35915, 5x108 colony forming units per 100 μl dose; R2-LD, R.torques ATCC27756, 5x107 colony forming units per 100 μl dose; R2-HD, R.torques ATCC27756, 5x108 colony forming units per 100 μl dose; HK-R2-HD, heat-killed R.torques ATCC27756, 5x108 colony forming units per 100 μl dose. Data are expressed as mean + / - SEM. * indicates p<0.05 using Student's t-test. ip means intraperitoneal injection. GTT means glucose tolerance test. AUC means area under the curve (here the curve of blood glucose excursion). [Figure 22]RUMTOR_00181-producing Ruminococcus torques (RT ATCC27756) strain improves glucose tolerance. Mice were fed a high-fat diet. Intraperitoneal glucose tolerance tests were performed 6 weeks after oral gavage with R. torques strains: RT3, R. torques ATCC35915, RUMTOR_00181-encoding gene absent, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC27756, RUMTOR_00181-encoding gene present, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC27756, RUMTOR_00181-encoding gene present, 5x109 colony forming units per 100 μl dose. Data are presented as mean + / - SEM (10 mice / group). *, p<0.05, ***, p<0.001, RT2 vs. RT3; #, p<0.05, RT2 vs. sterile phosphate-buffered saline; &, p<0.05, RT2 vs. heat-killed RT2. Statistical significance was determined by unpaired two-tailed t-test. ip means intraperitoneal injection. GTT means glucose tolerance test. [Diagram 23]Oral gavage of the RUMTOR_00181-producing R. torques strain (RTATCC27756) for 8 weeks in mice fed regular chow activated the expression of genes involved in thermogenesis and decreased the expression of adipogenic genes in inguinal adipocytes. q-PCR quantification was performed on RNA extracted from mouse inguinal adipose tissue. R3-LD, R.torques ATCC35915, 5x107 colony forming units per 100μl dose; R3-HD, R.torques ATCC35915, 5x108 colony forming units per 100μl dose; R2-LD, R.torques ATCC27756, 5x107 colony forming units per 100μl dose; R2-HD, R.torques ATCC27756, 5x108 colony forming units per 100μl dose; HK-R2-HD, heat-killed R.torques ATCC27756, 5x108 colony forming units per 100μl dose. Data are expressed as mean + / - SEM. n=3-4 mice / group. *, p<0.05 vs. R.torques ATCC35915, 5x108 colony forming units per 100μl group. [Figure 24] Oral gavage of RUMTOR_00181-producing R.torques strain (RTATCC27756) for 8 weeks in mice fed regular chow increases tibial cortical thickness. Left: 3D images of midshaft tibia cross sections from each group. Right: cortical thickness collected from 3D images from each group. *, false discovery rate corrected p<0.05. R3-LD, R.torques ATCC35915, 5x107 colony forming units per 100μl dose; R3-HD, R.torques ATCC35915, 5x108 colony forming units per 100μl dose; R2-LD, R.torques ATCC27756, 5x107 colony forming units per 100μl dose; R2-HD, R.torques ATCC27756, 5x108 colony forming units per 100μl dose; HK-R2-HD, heat-killed R.torques ATCC27756, 5x108 colony forming units per 100μl dose. Data are expressed as mean + / - SEM. n=3-4 mice / group. *, p<0.05 determined by Student's t-test. [Diagram 25] Representative chromatograms showing the presence of RUCILP2 in fasting human plasma are shown. Parallel reaction monitoring (PRM) elution profile of the y-ions of the unique tryptic RUCILP2 peptide (EAAGYNVYVDGVK) found in human plasma samples. The top panel is a PRM trace of the fragment ions of the light peptide found in the human plasma sample (box a), and the bottom panel is a PRM trace of the fragment ions of 10.0 femtomoles of a heavy isotope-labeled (Lys13C6,15N4) synthetic peptide (internal standard, IS) spiked into the fractionated human plasma sample (box b). The retention time of each peptide is labeled on the x-axis, and the y-axis represents the relative intensity of each fragment ion peak. One milliliter human plasma samples were depleted of albumin and immunoglobulin G after deglycosylation. Deglycosylated plasma was resolved by SDS-PAGE and the molecular weight region corresponding to fully deglycosylated RUCILP2 (10-15 kDa) was excised and then digested in-gel overnight before LC-MS / MS detection. Based on a comparative analysis of the relative intensities of fragment ions found in treated human plasma and those found in plasma spiked with a heavy isotope-labeled internal standard, we estimate that the interindividual concentration of RUCILP2 in human plasma varies between 10-100 pg / ml. [Figure 26] Amino acid sequence alignment of RUCILP2 and 21-AABP2. Multiple sequence alignment was performed by Clustal Omega. The sequence of 21-AABP2 is highlighted in grey. [Figure 27]The molecular docking model of 21-AABP2 and integrin αV / β5 receptor is shown. The dotted lines indicate the hydrogen bonds formed by the two ligands, and the binding site of 21-AABP2 is indicated by the amino acid residue code. The best ZDOCK web server (ZDOCK (https: / / zdock.umassmed.edu / ) predicted model of RUCILP2 and integrin αV / β5 receptor complex was visualized with the PyMOL (v2.1.1) program, showing the binding amino acid residues of 21-AABP2 to integrin αV / β5 receptor at Y5, F6, E8, and N17, respectively. This docking model predicted not only the binding site of 21-AABP2 and integrin αV / β5 receptor, but also the potential binding. [Figure 28]21-AABP2 promotes the expression of genes involved in thermogenesis / browning in human visceral white preadipocytes (HWP), induces the expression of key genes controlling thermogenesis in mouse inguinal preadipocytes, and stimulates the expression of genes involved in myogenesis in mouse C2C12 myoblasts. Human visceral fat-derived white preadipocytes (C-12732, PromoCell) were cultured to 80% confluence and switched to differentiation medium (0.3 ug / ml fetal calf serum (FCS), 8 ug / ml d-biotin, 0.5 ug / ml insulin, 400 ng / ml dexamethasone) in the presence of 15 nM 21-AABP2. The differentiation process into mature adipocytes was completed after 14 days. After 14 days of differentiation, cells were harvested and the indicated genes were quantified by q-PCR. Inguinal adipose tissue from 6-week-old wild-type C57BL / 6J female mice was dissected, washed with PBS, minced, and digested in PBS containing 10 mM CaCl2, 2.4 U / ml Dispase II (Roche), and 10 mg / ml Collagenase D (Roche) for 1 h at 37°C. After adding warm DMEM / F12 (1:1) containing 10% FCS, the digested tissue was filtered through a 70 mm cell strainer and centrifuged at 600 x g for 10 min. The pellet was resuspended in 40 ml DMEM / F12 (1:1) containing 10% FCS, filtered through a 40 mm cell strainer, and then centrifuged at 600 x g for 10 min. Pelleted inguinal stromal vascular cells were grown to confluence and split into 12-well plates. Cells were induced to differentiate by treatment with 1 mM rosiglitazone, 5 mM dexamethasone, and 0.5 mM isobutylmethylxanthine for 2 days. Cells were then maintained in 1 mM rosiglitazone for 4 days, with medium changed every other day. During 6 days of differentiation, cells were treated with 15 nM 21-AABP2 every other day. Cells were harvested after 6 days of differentiation, and thermogenic genes were quantified by q-PCR. C2C12 myoblasts (CRL-1772, ATCC) were cultured until 80% confluent and switched to differentiation medium (containing 2% horse serum). Treatment with 21-AABP2 began on day 2 of differentiation. Cells were harvested after 4 days of differentiation, and expression of myogenic genes was quantified by q-PCR. Data are shown as mean + / - SEM of one representative experiment performed in biological triplicates.Statistical significance was determined by unpaired two-tailed Student's t test, *, p<0.05. [Figure 29] 21-AABP2 increases myotube development in C2C12 mouse myoblasts. Representative images of myoblasts (CRL-1772, ATCC) at 24 h of differentiation in the presence of phosphate-buffered saline (PBS, blank) or 21-AABP2 (15 nM) are shown. Images presented are from one representative experiment performed in biological triplicates. [Diagram 30] Insulin release from immortalized rat insulinoma INS-1 cells is increased after 21-AABP2 stimulation. INS-1 cells (832 / 13, ThermoFisher) were grown in RPM1640 medium until they reached 70% confluence, then switched to RPM1640 medium supplemented with 15 nM 21-AABP2 and incubated for 12 h. Insulin concentrations in the cell culture medium supernatants were measured by MSD rat / mouse insulin ELISA kit. Data are shown as mean + / - SEM of one representative experiment performed in biological triplicates. Statistical significance was determined by unpaired two-tailed Student's t-test, *, p<0.05. [Diagram 31] High-quality 3D structure of RUMTOR_00181 protein. SP, signal peptide; TD, transmembrane domain; FNIII, fibronectin type III domain. Blue ribbons indicate unannotated regions. Protein structure was modeled using artificial intelligence algorithms AlphaFold222 via ColabFold23 and MMseqs224 to predict protein structure using multiple sequence alignments23. [Diagram 32]Alignment of irisin to RUCILP1 and RUCILP2 is shown. (A) A total of 27 amino acids (88 aa) from RUCILP1 are identical to those of irisin. (B) Thirty amino acid residues from RUCILP2 (87 aa) are shown to be identical to those of irisin. Identical residues between the two sequences are indicated by asterisks, and areas of low and high similarity are represented by period and colon, respectively. [Diagram 33] Alignment of the RUCILP1 and RUCILP2 sequences is shown. A total of 65 amino acids of RUCILP1 (88aa) are identical to those of RUCILP2 (87aa). Identical residues between the two sequences are indicated by asterisks, and areas of low and high similarity are represented by period and colon, respectively. [Diagram 34] Figure 2 shows the proposed topology of the RUMTOR_00181 protein and trypsin / LysC-dependent cleavage for release of RUCILP1 and RUCILP2 into the bacterial extracellular space. aa, amino acid residue; K, lysine; LysC, an endoproteinase that cleaves proteins at the C-terminal side of lysine residues. [Diagram 35]Oral gavage of mice with Ruminococcus torques ATCC27756 strain, which synthesizes RUMTOR_00181, reduced fat mass and increased lean body mass in mice fed a chow diet for 8 weeks. Mice were fed regular chow and the intervention continued for 8 weeks. Magnetic resonance imaging scans (MRI) of body composition in the indicated groups of mice were performed according to the manufacturer's tutorial. PBS, phosphate buffered saline; R3-LD, R.torquesATCC35915, 5x107 colony forming units per 100 μl dose; R3-HD, R.torquesATCC35915, 5x108 colony forming units per 100 μl dose; R2-LD, R.torquesATCC27756, 5x107 colony forming units per 100 μl dose; R2-HD, R.torquesATCC27756, 5x108 colony forming units per 100 μl dose; HK-R2-HD, heat-killed R.torquesATCC27756, 5x108 colony forming units per 100 μl dose. Data are expressed as mean + / - SEM. *, p<0.05, and **, p<0.01 determined by unpaired two-tailed Student's t-test. [Diagram 36] Oral gavage of ATCC27756 Ruminoccocus torques strain synthesizing RUMTOR_00181 reduces inguinal and epididymal fat tissue weights in mice fed a high-fat diet. PBS, phosphate-buffered saline; RT3, R. torques ATCC35915, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC27756, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC27756, 5x109 colony forming units per 100 μl dose. Data are presented as mean + / - SEM for 10 mice per group; iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue. Data are presented as mean + / - SEM. *, p<0.05, determined using one-way ANOVA followed by Turkey post-hoc correction. [Figure 37]Oral gavage of RUMTOR_00181-producing ATCC27756 Ruminoccocus torques strain activates thermogenesis, reduces adipogenesis, enhances lipolysis, and downregulates inflammation in adipose tissue of mice fed a high-fat diet. PBS, phosphate-buffered saline; RT3, R. torques ATCC35915, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC27756, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC27756, 5x109 colony forming units per 100 μl dose. Data are shown as mean + / - SEM using 10 mice / group. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001, determined using one-way ANOVA followed by Tukey post-hoc correction. [Figure 38] Oral gavage with RUMTOR_00181-producing ATCC27756 Ruminoccocus torques strain reduced adipocyte size in the inguinal fat of mice fed a high-fat diet, as visualized by hematoxylin and eosin staining.PBS, phosphate-buffered saline;RT3, R. torques ATCC35915, 5x109 colony forming units per 100 μl dose;heat-killedRT2, heat-killed R. torques ATCC27756, 5x109 colony forming units per 100 μl dose;RT2, R. torques ATCC27756, 5x109 colony forming units per 100 μl dose. [Figure 39]Oral gavage of RUMTOR_00181-producing ATCC27756 Ruminoccocus torques strain enhances the expression of the browning marker UCP1 at the protein level in inguinal white adipose tissue of mice fed a high-fat diet. PBS, phosphate-buffered saline; RT3, R. torques ATCC35915, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC27756, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC27756, 5x109 colony forming units per 100 μl dose. Data are shown as mean + / - SEM using 6 mice / group. **, p<0.01; ***, p<0.001, determined using one-way ANOVA followed by Tukey post-hoc correction. [Diagram 40] Oral gavage of RUMTOR_00181-producing ATCC 27756 Ruminoccocus torques strain activates bone formation in the distal femur of mice fed a high-fat diet. The top panel shows representative 3D cross-sectional images of femurs, and the bottom panel shows a summary comparison of cortical thickness collected from 3D (left) and 2D (right) images. PBS, phosphate-buffered saline; RT3, R. torques ATCC 35915, 5x109 colony forming units per 100 μl dose; heat-killed RT2, heat-killed R. torques ATCC 27756, 5x109 colony forming units per 100 μl dose; RT2, R. torques ATCC 27756, 5x109 colony forming units per 100 μl dose. Data are shown as mean + / - SEM with 6 mice per group. *, p<0.05; **, p<0.01; ***, p<0.001, determined using one-way ANOVA followed by Tukey post-hoc correction. [Diagram 41]Images of SPOT peptide microarray (μSPOT) assays of RUCILP1 and RUCILP2 binding to the integrin αV / β5 receptor are shown. (A) Image of a cellulose membrane without synthetic 15mer peptides after direct incubation with 6x-His antibody. (B) Representative images of cellulose membranes with attached synthetic libraries of 15mer peptides of RUCILP1 (left) and RUCILP2 (right), respectively, after interacting with the integrin αV / β5 receptor and subsequent incubation with 6x-His antibody. [Figure 42A] 1 shows the results of a systematic screen to identify the binding epitope between RUCILP and the integrin αV / β5 receptor. Relative integrin αV / β5 (2.5 nM) binding of 15mer peptides of RUCILP1 (SEQ ID NOs: 22-95). [Figure 42B] 1 shows the results of a systematic screening to identify the binding epitope of RUCILP to the integrin αV / β5 receptor. Binding of a 15mer peptide of RUCILP2 (SEQ ID NO: 96-168) to integrin αV / β5 (2.5 nM). Data are presented as the mean ± SD of triplicate quantifications. [Diagram 43] AlphaFold 3D structure of RUCILP. (A) Structure of RUCILP1 predicted by AlphaFold. (B) Structure of RUCILP2 predicted by AlphaFold. (C) Crystal structure of irisin. (D) Electrostatic surface representation of RUCILP1 (top) and RUCILP2 (bottom). In A-C, the loops responsible for binding to the integrin αV / β5 receptor are marked in red. In D, the red region indicates the flexible C-terminus of both proteins. [Diagram 44]Shown are the in vitro effects of RUCILP1 (Panel A) and RUCILP2 (Panel B) in gene expression assays of various cell types. mRNA expression levels of brown adipocyte-selective and white adipocyte marker genes in mouse 3T3-L1 fibroblasts, bone remodeling maker genes in mouse osteoblasts, and myotube formation genes in mouse myoblasts upon treatment with RUCILP1 (A) and RUCILP2 (B). * indicates p<0.05 using one-way ANOVA followed by Dunnett's post-hoc correction. Data are presented as mean ± SEM, n=3 wells / group. [Diagram 45] 1 shows the effect of 21-AABP1 on mouse 3T3-L1 fibroblasts. Data are presented as mean ± SEM, n=3 wells / group. [Figure 46] Figure 1 shows the effect of RUCILP1 and RUCILP2 on gene expression in vivo. Recombinant RUCILP was injected intraperitoneally into 8-week-old wild-type C57BL / 6N mice at a concentration of 1 mg / kg daily for 1 week. mRNA levels of the indicated genes in subcutaneous white adipose tissue (SWAT) and liver were analyzed by qRT-PCR. n=6 animals / group. * indicates p<0.05 using one-way ANOVA followed by Dunnett's post-hoc correction. Data are presented as mean ± SEM. [Figure 47A] Alanine scanning of the RUCILP1 19mer epitope against the integrin αV / β5 receptor. Relative binding of integrin αV / β5 (2.5 nM) to the alanine scanning library of RUCILP1 19mer epitopes (SEQ ID NOs: 169-188). [Figure 47B] Figure 1 shows alanine scanning of RUCILP2 19mer epitopes against the integrin αV / β5 receptor. Relative binding of integrin αV / β5 (2.5 nM) to the alanine scanning library of RUCILP2 19mer epitopes (SEQ ID NOs: 189-208). Data are presented as the mean ± SEM of triplicate quantifications. [Figure 48A]1 shows a truncation scan of the RUCILP1 19mer epitope against the integrin αV / β5 receptor. Relative binding of integrin αV / β5 (2.5 nM) to a truncation scanning library of RUCILP1 19mer epitopes (SEQ ID NOs: 209-241). [Figure 48B] Truncation scanning of RUCILP2 19mer epitopes against the integrin αV / β5 receptor. Relative binding of integrin αV / β5 (2.5 nM) to the truncation scanning library of RUCILP2 19mer epitopes (SEQ ID NOs: 242-274). Dark bars highlight enhanced binding hits after subtraction of background signal. Data are presented as the mean ± SD of triplicate quantifications. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] definition Amino acid substitution - As used herein, the term "amino acid substitution" refers to a change from one amino acid to a different amino acid in a peptide, polypeptide or protein. The substitution may be a conservative substitution, where an amino acid is replaced with another amino acid having similar properties. The substitution may be a non-conservative substitution, where an amino acid is replaced with another amino acid having different properties. The properties of the amino acid include, for example, the charge, polarity, acidity, size and hydrophobicity of the amino acid.

[0024] Bone Disorders - As used herein, the term "bone disorders" refers to a subgroup of musculoskeletal disorders, diseases, injuries and conditions that affect human bone. In particular, it refers to osteoporosis, osteogenesis imperfecta and osteopetrosis. Osteoporosis can be subdivided into primary and secondary osteoporosis. Primary osteoporosis is the most common form of the disease and includes postmenopausal osteoporosis (type I) and senile osteoporosis (type II). There are many causes of secondary bone loss (osteoporosis), including side effects from various drug therapies, endocrine disorders, eating disorders, immobilization, bone marrow-related disorders, gastrointestinal or biliary disorders, renal diseases, and cancer.

[0025] Identity - The term identity with respect to a polynucleotide or polypeptide is defined herein as the percentage of nucleic acids or amino acids that are identical to the corresponding naturally occurring nucleic acid or amino acid residues in a candidate sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Neither 5' or 3' extensions or insertions (in the case of nucleic acids) nor N' or C' extensions or insertions (in the case of polypeptides) result in a loss of identity. Alignment methods and computer programs are well known in the art.

[0026] Live Biopharmaceutical Product (LBP) - As used herein, the term "live biopharmaceutical product" or "LBP" refers to a biological product that is: i) living microorganisms, including, for example, bacteria or yeast; ii) applicable to the prevention, treatment or cure of a human disease or condition; and iii) It is not a vaccine, fecal microbiota transfer or gene therapy agent.

[0027] Metabolic Disorders - As used herein, the term "metabolic disorders" refers to disorders that negatively alter the body's processing and distribution of macronutrients such as proteins, fats, and carbohydrates. In particular, as used herein, the term "metabolic disorders" refers to diseases, disorders, and conditions associated with metabolic syndrome.

[0028] Metabolic syndrome--The term "metabolic syndrome" as used herein refers to a series of pathological complications including obesity, hypertension, hyperglycemia, high serum triglycerides, and low serum high density lipoprotein, as well as cardiovascular disease, FLD, prediabetes, and T2D. As used herein, "metabolic syndrome" also includes related concepts such as syndrome X, insulin resistance syndrome, visceral fat syndrome, and multiple risk factor syndrome. In the present invention, the prevention or treatment of metabolic syndrome means preventing or treating the occurrence of symptoms in at least one pathological condition selected from the group of pathological conditions described above.

[0029] Muscle disorders - As used herein, the term "muscle disorders" refers to a subgroup of musculoskeletal disorders, diseases, injuries and conditions, as well as neuromuscular disorders that affect human joints and muscles. Specifically, it refers to muscular dystrophies such as Duchenne muscular dystrophy, amyotrophic lateral sclerosis (ALS), Lambert-Eaton syndrome (Lambert-Eaton myasthenic syndrome), myasthenia gravis, polymyositis, and peripheral neuropathy.

[0030] Prediabetes - As used herein, the term "prediabetes" refers to a condition characterized by elevated blood glucose levels. Many, but not all, patients with prediabetes will develop T2D. Prediabetes may be diagnosed by hemoglobin A1C, fasting glucose measurement, or glucose tolerance test, which indicate that prediabetes is an A1C of 5.7-6.4%, a fasting glucose level of 100-125 mg / dl, and a 2-hour glucose level on an oral glucose tolerance test (OGTT) of 140-199 mg / dl.

[0031] Treatment - The term "treatment" as used herein may refer to any type of treatment. Treatment may be a curative treatment, treatment may be a treatment that alleviates and / or reduces the effects of the disease, injury and / or disorder being treated. Treatment may also be a treatment that delays the progression and / or occurrence of the disease, injury and / or disorder being treated. Treatment may also be a prophylactic / preventative, i.e., a treatment that eliminates or reduces the risk of developing the disease, injury and / or disorder disclosed herein.

[0032] Polypeptides The present invention relates to polypeptides derived from fibronectin type III domain-containing protein 5 (FNDC5) of the enterobacterial strain Ruminococcus torques or RUMTOR_00181 (UniProtID: A5KIY5). In particular, the present invention relates to polypeptides comprising a fragment of the FNDC5 polypeptide or RUMTOR_00181 (RUCILP1; RUCILP2; Ruminococcus torques irisin-like peptide 1 or 2), as well as variants and fragments thereof, such as the 21 amino acid fragment of RUCILP1 (21-AABP1; 21 amino acid bacterial peptide 1) or RUCILP2 (21-AABP2; 21 amino acid bacterial peptide 2), fragments and variants of 21-AABP2 or 21AABP1.

[0033] Thus, provided herein is an isolated polypeptide having a length of less than 200 amino acids comprising or consisting of an amino acid sequence selected from the group consisting of: a. the amino acid sequence according to SEQ ID NO:4 and / or SEQ ID NO:19; b. a variant of SEQ ID NO:4 and / or SEQ ID NO:19, which has at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:4 and / or SEQ ID NO:19, but less than 99% sequence identity to SEQ ID NO:4 and / or SEQ ID NO:19; c. A variant of SEQ ID NO:4 and / or SEQ ID NO:19 having 1 to 40 amino acid substitutions relative to SEQ ID NO:4 and / or SEQ ID NO:19, such as 5, 10, 15, 20, 25, 30 or 35 amino acid substitutions relative to SEQ ID NO:4 and / or SEQ ID NO:19; d. A fragment of SEQ ID NO: 4 and / or SEQ ID NO: 19 having a length of at least 10 amino acids, or a variant of said fragment having 1-5 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, respectively, such as 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, wherein the polypeptide has a length of less than 50 amino acids, or a variant thereof; e. an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by truncation at the N-terminus of at least one amino acid, such as 1 to 67 amino acids, for example 1 to 60 amino acids, for example 1 to 50 amino acids, for example 1 to 40 amino acids, for example 1 to 30 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, or a variant thereof having 1 to 10 amino acid substitutions with respect to SEQ ID NO: 4 and / or SEQ ID NO: 19, for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions with respect to SEQ ID NO: 4 and / or SEQ ID NO: 19; f. an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by truncation at the C-terminus of at least one amino acid, such as 1 to 21 amino acids, such as 1 to 20 amino acids, such as 1 to 15 amino acids, such as 1 to 10 amino acids, such as 1 to 5 amino acids, or a variant thereof having 1 to 30 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, such as 1, 5, 10, 15, 20 or 25 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; g. an amino acid sequence which differs from SEQ ID NO: 4 and / or SEQ ID NO: 19 by a truncation at the N-terminus of at least one amino acid, such as 1 to 67 amino acids, for example 1 to 60 amino acids, for example 1 to 50 amino acids, for example 1 to 40 amino acids, for example 1 to 30 amino acids, for example 1 to 20 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, and a truncation at the C-terminus of at least one amino acid, such as 1 to 21 amino acids, for example 1 to 20 amino acids, for example 1 to 15 amino acids, for example 1 to 10 amino acids, for example 1 to 5 amino acids, wherein said polypeptide is an amino acid sequence having a length of at least 10 amino acids, or a variant thereof having 1 to 5 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 4 and / or SEQ ID NO: 19; h. an amino acid sequence according to SEQ ID NO:5 and / or SEQ ID NO:20; i. variants of SEQ ID NO:5 and / or SEQ ID NO:20, which have at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% sequence identity to SEQ ID NO:5 and / or SEQ ID NO:20, but less than 99% sequence identity to SEQ ID NO:5 and / or SEQ ID NO:20; j. A variant of SEQ ID NO:5 and / or SEQ ID NO:20 having 1 to 10 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, wherein the polypeptide has a length of less than 50 amino acids; k. A fragment of SEQ ID NO:5 and / or SEQ ID NO:20 comprising at least 10 consecutive amino acids of SEQ ID NO:5 and / or SEQ ID NO:20, or a variant thereof having 1 to 5 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, such as 1, 2, 3 or 4 amino acid substitutions relative to SEQ ID NO:5 and / or SEQ ID NO:20, wherein the polypeptide has a length of less than 50 amino acids; l. Fragments of SEQ ID NO:19, selected from the group consisting of SEQ ID NOs:27, 33, 34, 35, 36, 37 and 95, and their respective variants having 1 to 3 amino acid substitutions relative to SEQ ID NO:19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO:19, wherein the polypeptide has a length of less than 50 amino acids; m. fragments of SEQ ID NO:4 selected from the group consisting of SEQ ID NO:107, 108, 109, 110, 111, 165 and 168, and respective variants thereof having 1-3 amino acid substitutions relative to SEQ ID NO:4, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO:4, wherein the polypeptide has a length of less than 50 amino acids; n. Fragments of variants of SEQ ID NO: 19, selected from the group consisting of SEQ ID NOs: 173, 176, 181 and 188, and respective variants thereof having 1-3 amino acid substitutions relative to SEQ ID NO: 19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO: 19, wherein said polypeptide has a length of less than 50 amino acids; o. fragments of SEQ ID NO:4 selected from the group consisting of SEQ ID NOs:193, 196, 201 and 208, and respective variants thereof having 1-3 amino acid substitutions relative to SEQ ID NO:4, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO:4, wherein the polypeptide has a length of less than 50 amino acids; p. fragments of SEQ ID NO:19 selected from the group consisting of SEQ ID NOs:210, 211, 212, 213, 229, 232, 233, 234 and 235, and respective variants thereof having 1-3 amino acid substitutions relative to SEQ ID NO:19, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO:19, wherein said polypeptide has a length of less than 50 amino acids; q. Fragments of SEQ ID NO:4 selected from the group consisting of SEQ ID NOs:243, 244, 245, 246, 262, 265, 266, 267 and 268, and respective variants thereof having 1 to 3 amino acid substitutions relative to SEQ ID NO:4, for example 1, 2 or 3 amino acid substitutions relative to SEQ ID NO:4, wherein the polypeptide has a length of less than 50 amino acids.

[0034] In one embodiment, the polypeptide has a length of at least 10 amino acids, e.g., at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least 100 amino acids.

[0035] Thus, in one embodiment, the polypeptide has a length of at least 15 amino acids. In one embodiment, the polypeptide has a length of at least 20 amino acids. In one embodiment, the polypeptide has a length of at least 25 amino acids. In one embodiment, the polypeptide has a length of at least 30 amino acids. In one embodiment, the polypeptide has a length of at least 35 amino acids. In one embodiment, the polypeptide has a length of at least 40 amino acids. In one embodiment, the polypeptide has a length of at least 45 amino acids. In one embodiment, the polypeptide has a length of at least 50 amino acids. In one embodiment, the polypeptide has a length of at least 55 amino acids. In one embodiment, the polypeptide has a length of at least 60 amino acids. In one embodiment, the polypeptide has a length of at least 65 amino acids. In one embodiment, the polypeptide has a length of at least 70 amino acids. In one embodiment, the polypeptide has a length of at least 75 amino acids. In one embodiment, the polypeptide has a length of at least 80 amino acids. In one embodiment, the polypeptide has a length of at least 85 amino acids. In one embodiment, the polypeptide has a length of at least 90 amino acids. In one embodiment, the polypeptide has a length of at least 95 amino acids. In one embodiment, the polypeptide has a length of at least 100 amino acids.

[0036] In one embodiment, the polypeptide has a length of less than 150 amino acids, such as less than 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, or 25, 20, 15 or 10. Thus, in one embodiment, the polypeptide has a length of less than 150 amino acids.

[0037] In one embodiment, the polypeptide has a length of less than 140 amino acids. In one embodiment, the polypeptide has a length of less than 130 amino acids. In one embodiment, the polypeptide has a length of less than 120 amino acids. In one embodiment, the polypeptide has a length of less than 110 amino acids. In one embodiment, the polypeptide has a length of less than 100 amino acids. In one embodiment, the polypeptide has a length of less than 95 amino acids. In one embodiment, the polypeptide has a length of less than 90 amino acids. In one embodiment, the polypeptide has a length of less than 85 amino acids. In one embodiment, the polypeptide has a length of less than 80 amino acids. In one embodiment, the polypeptide has a length of less than 75 amino acids. In one embodiment, the polypeptide has a length of less than 70 amino acids. In one embodiment, the polypeptide has a length of less than 65 amino acids. In one embodiment, the polypeptide has a length of less than 60 amino acids. In one embodiment, the polypeptide has a length of less than 55 amino acids. In one embodiment, the polypeptide has a length of less than 50 amino acids. In one embodiment, the polypeptide has a length of less than 45 amino acids. In one embodiment, the polypeptide has a length of less than 40 amino acids. In one embodiment, the polypeptide has a length of less than 35 amino acids. In one embodiment, the polypeptide has a length of less than 30 amino acids. In one embodiment, the polypeptide has a length of less than 25 amino acids. In one embodiment, the polypeptide has a length of less than 20 amino acids. In one embodiment, the polypeptide has a length of less than 15 amino acids. In one embodiment, the polypeptide has a length of less than 10 amino acids.

[0038] In one embodiment, the polypeptide has an amino acid length of 10-200, such as 10-150, such as 10-100, such as 10-80, such as 10-50, such as 10-30, such as 10-15, such as 25-75, such as 25-60, such as 30-80, such as 40-70, such as 15-30, such as 15-25, such as 18-23, such as 20-22, such as 50-150, such as 50-100, such as 70-100, such as 80-90, such as 85-90, such as 86-88.

[0039] Thus, in one embodiment, the polypeptide has a length of between 10 and 200 amino acids. In one embodiment, the polypeptide has a length of 10 to 150 amino acids. In one embodiment, the polypeptide has a length of between 10 and 100 amino acids. In one embodiment, the polypeptide has a length of 10 to 80 amino acids. In one embodiment, the polypeptide has a length of 10 to 50 amino acids. In one embodiment, the polypeptide has a length of 10 to 30 amino acids. In one embodiment, the polypeptide has a length of 10-15 amino acids. In one embodiment, the polypeptide has a length of 25 to 75 amino acids. In one embodiment, the polypeptide has a length of 25 to 60 amino acids. In one embodiment, the polypeptide has a length of 30 to 80 amino acids. In one embodiment, the polypeptide has a length of 40-70 amino acids. In one embodiment, the polypeptide has a length of 15 to 30 amino acids. In one embodiment, the polypeptide has a length of 15 to 25 amino acids. In one embodiment, the polypeptide has a length of 18 to 23 amino acids. In one embodiment, the polypeptide has a length of 20-22 amino acids. In one embodiment, the polypeptide has a length of 50 to 150 amino acids. In one embodiment, the polypeptide has a length of 50-100 amino acids. In one embodiment, the polypeptide has a length of between 70 and 100 amino acids. In one embodiment, the polypeptide has a length of 80-90 amino acids. In one embodiment, the polypeptide has a length of 85 to 95 amino acids. In one embodiment, the polypeptide has a length of 86-88 amino acids.

[0040] In one embodiment, a variant of the polypeptide has at least 60% sequence identity to SEQ ID NO:4 or SEQ ID NO:19, such as at least 61% identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, to SEQ ID NO:4 or SEQ ID NO:19. such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity, such as 100% sequence identity.

[0041] Thus, in one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 61% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 62% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 63% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 64% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 65% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 66% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 67% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the variant of the polypeptide has at least 68% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 69% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 70% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 71% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 72% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 73% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 74% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 75% sequence identity to SEQ ID NO:4 or SEQ ID NO:19.In one embodiment, the polypeptide variant has at least 76% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 77% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 78% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 79% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 80% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 81% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 82% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 83% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 84% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 85% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 86% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 87% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 88% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 89% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 90% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, a variant of the polypeptide has at least 91% sequence identity to SEQ ID NO:4 or SEQ ID NO:19.In one embodiment, the polypeptide variant has at least 92% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 93% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 94% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 95% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 96% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 97% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 98% sequence identity to SEQ ID NO:4 or SEQ ID NO:19. In one embodiment, the polypeptide variant has at least 99% sequence identity to SEQ ID NO:4 or SEQ ID NO:19.

[0042] In one embodiment, a variant of the polypeptide has less than 99% sequence identity to SEQ ID NO:4 or SEQ ID NO:19.

[0043] In one embodiment, the polypeptide variant has 1 to 25 amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO: 19, for example, 1 to 20, such as 1 to 15, for example 1 to 10, for example 1 to 5, for example 1 to 3, for example 10 to 20, for example 5 to 15, for example 5 to 10 amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO: 19.

[0044] Thus, in one embodiment, a variant of the polypeptide has from 1 to 25 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0045] In one embodiment, a variant of the polypeptide has from 1 to 20 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0046] In one embodiment, a variant of the polypeptide has between 1 and 15 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0047] In one embodiment, a variant of the polypeptide has from 1 to 10 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0048] In one embodiment, a variant of the polypeptide has from 1 to 5 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0049] In one embodiment, a variant of the polypeptide has one to three amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0050] In one embodiment, a variant of the polypeptide has between 10 and 20 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0051] In one embodiment, a variant of the polypeptide has between 5 and 15 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0052] In one embodiment, a variant of the polypeptide has between 5 and 10 amino acid substitutions compared to SEQ ID NO:4 or SEQ ID NO:19.

[0053] In some embodiments, the isolated polypeptide comprises both the sequences of SEQ ID NO:4 and SEQ ID NO:19, or each variant or fragment thereof as described herein.

[0054] In one embodiment, a variant of the polypeptide has at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:20, such as at least 61% identity, such as at least 62% identity, such as at least 63% identity, such as at least 64% identity, such as at least 65% identity, such as at least 66% identity, such as at least 67% identity, such as at least 68% identity, such as at least 69% identity, such as at least 70% identity, such as at least 71% identity, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, to SEQ ID NO:5 or SEQ ID NO:20. such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity, such as 100% sequence identity.

[0055] Thus, in one embodiment, the variant of the polypeptide has at least 60% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 61% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 62% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 63% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 65% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 65% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 66% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 67% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the variant of the polypeptide has at least 68% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 69% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 70% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 71% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 72% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 73% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 75% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 75% sequence identity to SEQ ID NO:5 or SEQ ID NO:20.In one embodiment, the polypeptide variant has at least 76% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 77% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 78% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 79% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 80% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 81% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 82% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 83% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 85% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 85% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 86% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 87% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 88% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 89% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, a variant of the polypeptide has at least 91% sequence identity to SEQ ID NO:5 or SEQ ID NO:20.In one embodiment, the polypeptide variant has at least 92% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 93% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 95% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 95% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 96% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 97% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 98% sequence identity to SEQ ID NO:5 or SEQ ID NO:20. In one embodiment, the polypeptide variant has at least 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:20.

[0056] In one embodiment, a variant of the polypeptide has less than 99% sequence identity to SEQ ID NO:5 or SEQ ID NO:20.

[0057] In one embodiment, the polypeptide variant has 1 to 5 amino acid substitutions compared to SEQ ID NO: 5 or SEQ ID NO: 20, or, for example, 1 to 4, such as 1 to 3, for example 2 to 4, such as 2 to 5, for example 3 to 5 amino acid substitutions compared to SEQ ID NO: 5 or SEQ ID NO: 20.

[0058] Thus, in one embodiment, a variant of the polypeptide has from 1 to 5 amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0059] In one embodiment, a variant of the polypeptide has from 1 to 4 amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0060] In one embodiment, a variant of the polypeptide has one to three amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0061] In one embodiment, a variant of the polypeptide has 2-4 amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0062] In one embodiment, a variant of the polypeptide has 2-5 amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0063] In one embodiment, a variant of the polypeptide has 3 to 5 amino acid substitutions compared to SEQ ID NO:5 or SEQ ID NO:20.

[0064] In some embodiments, the isolated polypeptide comprises both the sequences of SEQ ID NO:5 and SEQ ID NO:20, or each variant or fragment thereof as described herein.

[0065] In one embodiment, the polypeptide fragment comprises or consists of an amino acid sequence according to positions 7 to 16 of SEQ ID NO: 4, which corresponds to SEQ ID NO: 6, or a variant thereof having 1 to 5 amino acid substitutions compared to SEQ ID NO: 4, for example 1, 2, 3, 4 or 5 amino acid substitutions compared to SEQ ID NO: 4.

[0066] In one embodiment, the polypeptide fragment comprises or consists of an amino acid sequence according to positions 27 to 39 of SEQ ID NO: 4, which corresponds to SEQ ID NO: 7, or a variant thereof having 1 to 6 amino acid substitutions compared to SEQ ID NO: 4, for example 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to SEQ ID NO: 4.

[0067] In one embodiment, the polypeptide fragment comprises or consists of an amino acid sequence according to positions 43 to 56 of SEQ ID NO: 4, which corresponds to SEQ ID NO: 8, or a variant thereof having 1 to 6 amino acid substitutions compared to SEQ ID NO: 4, for example 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to SEQ ID NO: 4.

[0068] In one embodiment, the amino acid substitution is a conservative substitution. A conservative amino acid substitution is a substitution of an amino acid in a polypeptide with a given amino acid having similar biochemical properties, such as similar size, charge, hydrophobicity and / or polarity. Such substitutions often have a smaller effect on polypeptide function compared to non-conservative substitutions. Examples of conservative amino acid substitutions can be found in the table below. [Table 1]

[0069] In other embodiments, amino acid substitution is non-conservative substitution.The data herein show that it may be beneficial to substitute acidic amino acid residues with basic amino acid residues.Thus, in certain embodiments, substitution comprises one or more substitutions of acidic amino acid residues with basic amino acid residues.

[0070] The polypeptide fragment may be a 15 amino acid fragment of RUCILP1. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:27. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:33. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:34. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:35. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:36. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:37. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:95. In some embodiments, the disclosure provides a variant of a polypeptide according to any of SEQ ID NOs:27, 33, 34, 35, 36, 37 or 95, wherein the variant has 1, 2 or 3 amino acid substitutions compared to the sequence from which it is derived, i.e., SEQ ID NOs:27, 33, 34, 35, 36, 37 or 95.

[0071] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of SEQ ID NO:19 (RUCILP1), wherein the fragment is selected from the group consisting of SEQ ID NOs:27, 33, 34, 35, 36, 37 and 95, and their respective variants having one to three amino acid substitutions relative to SEQ ID NO:19, such as one, two or three amino acid substitutions relative to SEQ ID NO:19.

[0072] The polypeptide fragment may be a 15 amino acid fragment of RUCILP2. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 107. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 108. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 109. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 110. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 111. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 165. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 168. In some embodiments, the disclosure provides a variant of a polypeptide according to any one of SEQ ID NOs: 107, 108, 109, 110, 111, 165 or 168, wherein the variant has 1, 2 or 3 amino acid substitutions compared to the sequence from which it is derived, i.e., SEQ ID NOs: 107, 108, 109, 110, 111, 165 or 168.

[0073] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of SEQ ID NO:4 (RUCILP2), wherein the fragment is selected from the group consisting of SEQ ID NOs:107, 108, 109, 110, 111, 165 and 168, and respective variants having one to three amino acid substitutions relative to SEQ ID NO:4, such as one, two or three amino acid substitutions relative to SEQ ID NO:4.

[0074] In some embodiments, the polypeptide fragment is a 19 amino acid fragment of RUCILP1 in which one amino acid is substituted with an alanine. This fragment may have increased binding affinity to the integrin αV / 5β receptor compared to the same fragment in which none of the amino acids are modified. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 173. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 176. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 181. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 188. In some embodiments, the disclosure provides a variant of the polypeptide according to any one of SEQ ID NOs: 173, 176, 181 or 188, wherein the variant has one, two or three amino acid substitutions compared to the sequence from which it is derived.

[0075] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of a variant of SEQ ID NO: 19 (RUCILP1), the fragment being selected from the group consisting of SEQ ID NOs: 173, 176, 181 and 188, and respective variants having one to three amino acid substitutions relative to SEQ ID NO: 19, such as one, two or three amino acid substitutions relative to SEQ ID NO: 19.

[0076] In some embodiments, the polypeptide fragment is a 19 amino acid fragment of RUCILP2 in which one amino acid is substituted with an alanine. This fragment may have increased binding affinity to the integrin αV / 5β receptor compared to the same fragment in which none of the amino acids are modified. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 193. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 196. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 201. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO: 208. In some embodiments, the disclosure provides a variant of the peptide according to any one of SEQ ID NOs: 193, 196, 201 or 208, wherein the variant has one, two or three amino acid substitutions compared to the sequence from which it is derived.

[0077] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of a variant of SEQ ID NO:4 (RUCILP2), the fragment being selected from the group consisting of SEQ ID NOs:193, 196, 201 and 208, and respective variants having one to three amino acid substitutions relative to SEQ ID NO:4, such as one, two or three amino acid substitutions relative to SEQ ID NO:4.

[0078] In some embodiments, the polypeptide fragment is a fragment of RUCILP1. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:210. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:211. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:212. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:213. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:229. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:232. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:233. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:234. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:235. In some embodiments, the disclosure provides a variant of a peptide according to any one of SEQ ID NOs:210, 211, 212, 213, 229, 232, 233, 234 or 235, wherein the variant has one, two or three amino acid substitutions compared to the sequence from which it is derived.

[0079] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of SEQ ID NO:19 (RUCILP1), wherein the fragment is selected from the group consisting of SEQ ID NOs:210, 211, 212, 213, 229, 232, 233, 234 and 235, and their respective variants having one to three amino acid substitutions relative to SEQ ID NO:19, such as one, two or three amino acid substitutions relative to SEQ ID NO:19.

[0080] In some embodiments, the polypeptide fragment is a fragment of RUCILP2. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:243. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:244. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:245. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:246. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:262. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:265. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:266. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:267. In some embodiments, the polypeptide fragment consists of an amino acid sequence according to SEQ ID NO:268. In some embodiments, the disclosure provides a variant of a peptide according to any one of SEQ ID NOs:243, 244, 245, 246, 262, 265, 266, 267 or 268, wherein the variant has one, two or three amino acid substitutions compared to the sequence from which it is derived.

[0081] In some embodiments, the disclosure provides an isolated polypeptide having a length of less than 50 amino acids comprising or consisting of a fragment of SEQ ID NO:4 (RUCILP2), wherein the fragment is selected from the group consisting of SEQ ID NOs:243, 244, 245, 246, 262, 265, 266, 267 and 268, and respective variants having one to three amino acid substitutions relative to SEQ ID NO:4, such as one, two or three amino acid substitutions relative to SEQ ID NO:4.

[0082] The present inventors have identified specific amino acids that may be involved in the interaction of RUCILP2 (SEQ ID NO: 4) / 21-AABP2 (SEQ ID NO: 5) with the integrin αV / β5 receptor. In particular, residues 7, 9, and 58 of SEQ ID NO: 4 and residues 5, 6, and 8 of SEQ ID NO: 5 are believed to have any role in the RUCILP2-integrin αV / β5 receptor and 21-AABP2-integrin αV / β5 receptor interactions, respectively.

[0083] Thus, in one embodiment, the polypeptide comprises a V at amino acid position 7 of SEQ ID NO:4, or a conservative substitution thereof, such as M, I, Y, F or L; and / or an E at amino acid position 9 of SEQ ID NO:4, or a conservative substitution thereof, such as Q, D, K, N, H or R; and / or an E at amino acid position 58 of SEQ ID NO:4, or a conservative substitution thereof, such as Q, D, K, N, H or R. In one embodiment, the polypeptide comprises a V at amino acid position 7 of SEQ ID NO:4, or a conservative substitution thereof, such as M, I, Y, F or L; an E at amino acid position 9 of SEQ ID NO:4, or a conservative substitution thereof, such as Q, D, K, N, H or R; and an E at amino acid position 58 of SEQ ID NO:4, or a conservative substitution thereof, such as Q, D, K, N, H or R.

[0084] In another embodiment, the polypeptide comprises a Y at amino acid position 5 of SEQ ID NO:5, or a conservative substitution thereof, such as F, W, M, I, V or L; and / or an F at amino acid position 6 of SEQ ID NO:5, or a conservative substitution thereof, such as M, Y, I, L, W or V; and / or an E at amino acid position 8 of SEQ ID NO:5, or a conservative substitution thereof, such as Q, D, K, N, H or R; and / or an N at amino acid position 17 of SEQ ID NO:5, or a conservative substitution thereof, such as D, S or Q. In one embodiment, the polypeptide comprises a Y at amino acid position 5 of SEQ ID NO:5, or a conservative substitution thereof, such as F, W, M, I, V or L; and / or an F at amino acid position 6 of SEQ ID NO:5, or a conservative substitution thereof, such as M, Y, I, L, W or V; and / or an E at amino acid position 8 of SEQ ID NO:5, or a conservative substitution thereof, such as Q, D, K, N, H or R; and / or an N at amino acid position 17 of SEQ ID NO:5, or a conservative substitution thereof, such as D, S or Q.

[0085] The polypeptides disclosed herein may be further modified, for example by attachment of one or more moieties, thereby providing the conjugates of the invention. Such modifications may improve the properties of the polypeptide, and thus the stability, membrane permeability and / or half-life of the polypeptide in vivo. Thus, in one embodiment, the polypeptide comprises one or more moieties conjugated to the polypeptide, and optionally, the polypeptide and the one or more moieties are conjugated to each other by a linker.

[0086] The one or more moieties can be any type of moiety. In one embodiment, the one or more moieties are selected from the group consisting of alkenes, alkyls, aryls, heteroaryls, fatty acids, polyethylene glycols (PEGs), saccharides, and polysaccharides. In one embodiment, the alkyls contain 1-12 carbon atoms, for example 1-6 carbon atoms. In one embodiment, the alkenes contain 1-12 carbon atoms, for example 1-6 carbon atoms. In one embodiment, the fatty acids contain 1-12 carbon atoms, for example 1-6 carbon atoms.

[0087] A polypeptide can form any kind of complex, such as a dimer and / or a multimer. A polypeptide dimer is formed by two polypeptide monomers that are non-covalently connected. A polypeptide multimer is formed by more than two polypeptide monomers. Thus, in one embodiment, the polypeptide is a dimer. In another embodiment, the polypeptide is a multimer.

[0088] The inventors have shown that the polypeptides presented herein and their fragments and variants have significant effects on various processes at both the organismal and cellular levels, such as cell signaling, peptide secretion and gene expression. For example, the inventors have shown that the polypeptides disclosed herein bind to at least one integrin receptor, namely, the αV / β5 integrin receptor. Thus, in one embodiment, the polypeptides can bind to the αV / β5 integrin receptor.

[0089] Integrin receptors, or integrins, are transmembrane receptors that facilitate cell-cell and extracellular matrix adhesion. Upon ligand binding, integrins activate signaling pathways that mediate cell signals such as regulation of the cell cycle, organization of the intracellular cytoskeleton, and recruitment of new receptors to the cell membrane. Integrins are obligate heterodimers composed of α and β subunits.

[0090] The aV class of integrins is a receptor that can be present in bone cells and adipose tissue. Using double-stranded RNAscope-based mRNA in situ hybridization arrays targeting the signal dots of integrin αV / β5 receptor (ITGAV and ITGB5mRNA), the present inventors further demonstrated that integrin αV / β5 receptor is present in the submucosa of human colonic tissue.

[0091] Adipose tissue, or body fat, is composed primarily of adipocytes. The two main types of adipose tissue are white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is responsible for energy storage, such as the storage of triglycerides, while BAT is a specialized form of adipose tissue that is important for adaptive thermogenesis in humans and other mammals. Browning of WAT, also called "browning," occurs when adipocytes within WAT depots develop characteristics of BAT. Brown-like adipocytes take on a multilocular appearance (containing several lipid droplets) and increase the expression of several proteins, such as uncoupling protein 1 (UCP1). These normal energy-storing adipocytes then become energy-releasing adipocytes.

[0092] The present inventors have shown that the polypeptides disclosed herein, as well as fragments and variants of the polypeptides, induce thermogenesis in white adipocytes. In other words, the polypeptides induce browning of WAT. In particular, the polypeptides induce the expression of genes involved in thermogenesis in adipocytes, and reduce adipogenesis, such as by reducing the expression of genes involved in adipogenesis. Thus, in one embodiment, the polypeptides induce thermogenesis in white adipocytes, for example by inducing the expression of genes involved in thermogenesis. In one embodiment, the polypeptides induce the mRNA expression of one or more genes selected from the group consisting of Ucp1, Pparγ1, Dio2, Cox2, Cpt1b, and Ebf2 in human white preadipocytes, where the mRNA expression level is quantified by q-PCR. In one embodiment, the polypeptide induces in vivo mRNA expression of one or more genes involved in thermogenesis, wherein the genes are selected from the group consisting of UCP1, Dio1, Elovl3, Cidea, Cox2 and Prdm16, and the mRNA expression levels are quantified by q-PCR.

[0093] In one embodiment, the polypeptide reduces the lipid content of adipocytes, for example by reducing the expression of genes involved in adipogenesis. In one embodiment, the polypeptide reduces lipid content in adipocytes, where the reduction is measured using Oil Red O staining. In one embodiment, the polypeptide reduces the in vivo mRNA expression of one or more genes involved in adipogenesis, such as Acaca, Scd1, and / or Fasn, where the mRNA expression levels are quantified by q-PCR.

[0094] Large WAT deposits are closely related to obesity and metabolic syndrome. In addition to obesity, patients with metabolic syndrome often have high blood pressure, hyperglycemia, high serum triglycerides, and low serum high-density lipoprotein (HDL). Metabolic syndrome is also closely related to insulin resistance, T2D, FLD, impaired intestinal barrier junctions, and cardiovascular disease.

[0095] The inventors have shown that the polypeptides and fragments and variants thereof disclosed herein act on several factors, such as genes and hormones, associated with metabolic syndrome. For example, the inventors have shown that the polypeptides stimulate the secretion of glucagon-like peptide-1 (GLP-1), insulin, peptide-YY (PYY) and somatostatin, which induces weight loss and improves glucose tolerance in vivo.

[0096] Thus, in one embodiment, the polypeptide stimulates the secretion of GLP-1 and glucagon-like peptide-2 (GLP-2). In one embodiment, the polypeptide stimulates the intestinal luminal release of GLP-1 and GLP-2. GLP-1 is a peptide hormone that can promote insulin secretion in a glucose-dependent manner. GLP-1 also promotes insulin gene transcription, mRNA stability and biosynthesis, thereby ensuring replenishment of insulin stores in beta cells to prevent secretory exhaustion. In the stomach, GLP-1 inhibits gastric emptying, acid secretion and motility, which overall reduce appetite. GLP-1 is secreted in equimolar amounts with glucagon-like peptide-2 (GLP-2).

[0097] In one embodiment, the polypeptide stimulates insulin secretion. In one embodiment, the polypeptide stimulates insulin release from INS-1 cells. Insulin is a peptide hormone produced by the beta cells of pancreatic islets and released into the blood in response to food intake. It is considered the main anabolic hormone of the human body. Insulin regulates carbohydrate, fat, and protein metabolism by promoting glucose absorption from the blood into liver, fat, and skeletal muscle cells. High blood insulin concentrations strongly inhibit or lack glucose production and secretion by the liver. Reduced or absent insulin activity results in diabetes, hereafter referred to as T2D.

[0098] In one embodiment, the polypeptide stimulates secretion of PYY. In one embodiment, the polypeptide stimulates the luminal release of PYY into the intestinal tract. PYY is a short peptide released from cells in the ileum and colon in response to feeding. In the blood, intestine, and other elements of the periphery, PYY acts to reduce appetite.

[0099] In one embodiment, the polypeptide stimulates the secretion of somatostatin. In one embodiment, the polypeptide stimulates the luminal release of somatostatin into the intestinal tract. Somatostatin is a peptide hormone secreted by delta cells in the digestive system. It reduces the rate of gastric emptying and inhibits the release of pancreatic hormones such as insulin and glucagon secretion.

[0100] In one embodiment, the polypeptide improves glucose tolerance.Glucose tolerance is defined as the ability to process glucose load.Glucose intolerance, which may be seen in the majority of patients with metabolic syndrome, is defined as the impaired ability to process glucose.Methods for testing glucose tolerance are well known in the art, and include, for example, subjecting a subject to an oral glucose load and measuring circulating glucose before and after.

[0101] In one embodiment, the polypeptide reduces the expression of genes involved in gluconeogenesis. In one embodiment, the polypeptide reduces the mRNA expression of G6pase and / or Pepck in HepG2 cells, wherein the mRNA expression level is quantified using q-PCR.

[0102] In one embodiment, the polypeptide enhances intestinal barrier junction. In one embodiment, the polypeptide increases the mRNA expression of genes involved in intestinal integration, such as Ocln and / or ZO-1, in Caco-2 cells, where the mRNA expression level is quantified using q-PCR. The intestinal barrier ensures sufficient containment of luminal contents in the intestine and retains the ability to absorb nutrients. Intestinal barrier junction dysfunction is related to many health conditions, including diabetes, such as metabolic syndrome, FLD and T2D.

[0103] In one embodiment, the polypeptide induces weight loss. In one embodiment, the polypeptide reduces fat mass and increases lean mass in a subject, such as a mouse, rat, or human. In one embodiment, the polypeptide induces weight loss in a subject with obesity. Obesity is a medical condition in which excess body fat accumulates to a degree that can have negative health effects. Humans are generally considered to be obese or obese with a BMI (body mass index) of 30 kg / m 2 If it is higher, it is considered obese. 25-30kg / m 2 A person with a BMI of 0.05 to 0.25 is defined as overweight. As mentioned above, obesity, and to some extent overweight, is correlated with various diseases and conditions, here cardiovascular diseases, musculoskeletal disorders, T2D and FLD.

[0104] Osteocytes are the most common cells in mature bone tissue. Osteocytes synthesize sclerostin, which can antagonize bone formation and increase bone resorption by reducing osteoblast formation and osteoblast activity. Lack of sclerostin expression in bone has been found to be a factor in high bone mass in sclerosteosis. Furthermore, irisin has been shown to be able to regulate bone formation by improving the secretion of sclerostin. In various skeletal disorders, including osteoporosis, the ability to form mature bone tissue is impaired, which leads to bone fragility and increased risk of fracture. The inventors have shown that the polypeptides disclosed herein stimulate bone formation, which increases the cortical thickness of the tibia. Thus, in one embodiment, the polypeptide stimulates bone formation, for example, by stimulating the expression of sclerostin in bone cells. In one embodiment, the polypeptide induces mRNA expression of the gene encoding sclerostin in MLO-Y4 (mouse long bone-Y4) cells, where the mRNA expression level is quantified using q-PCR. In another embodiment, the polypeptide increases tibia cortical thickness.

[0105] In one embodiment, the polypeptide induces cardiomyogenesis. Cardiomyogenesis includes the proliferation of bone marrow stem cells, which then differentiate into cardiomyocytes. In one embodiment, the polypeptide increases and increases the mRNA expression of FST (follistatin) in H9C2 cardiomyocytes, where the mRNA expression level is quantified using q-PCR.

[0106] In one embodiment, the polypeptide induces myotube formation and muscle formation. In one embodiment, the polypeptide increases the number of myotubes formed. In one embodiment, the polypeptide increases myotube formation in C2C12 myoblasts. In one embodiment, the polypeptide increases the mRNA expression of genes involved in myogenesis, such as Mymk and / or caveolin-3, where the mRNA expression level is quantified by q-PCR. Myogenesis is the formation of skeletal muscle tissue, i.e., myogenesis. Muscles are generally formed by fusing myoblasts into myotubes. Myogenesis is often impaired in patients with musculoskeletal disorders and muscular dystrophies, such as Duchenne muscular dystrophy patients. Impaired muscle formation or muscle weakness has also been reported in ALS, Lambert-Eaton syndrome, myasthenia gravis, and polymyositis.

[0107] Nucleic Acids / Vector / Host Cells Also provided herein are isolated polynucleotides that encode the polypeptides provided in the "Polypeptides" section.

[0108] In one embodiment, the isolated polynucleotide is selected from the group consisting of SEQ ID NOs: 9-18, e.g., SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18. In a preferred embodiment, the polypeptide is selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

[0109] Also provided is a vector comprising the polynucleotides presented herein. The vector can be any type of vector. In one embodiment, the vector is an expression vector, such as an expression vector selected from the group consisting of a bacterial expression vector, a mammalian expression vector, and an insect expression vector. In one embodiment, the expression vector is an E. coli expression vector, such as a pGEX-4T-1 expression vector, or an insect expression vector, such as a SF9-insect expression vector.

[0110] Further provided is a host cell comprising the polynucleotides presented herein. The host cell may be any type of host cell capable of expressing and secreting the polypeptides encoded by the polynucleotides disclosed herein. In some embodiments, the host cell is a cell naturally occurring in the human gut microbiota. In one embodiment, the host cell is selected from the group consisting of Lactobacillus, Lactococcus, Escherichia coli, Bacillus subtilis, Pseudomonas putida, Saccharomyces cerevisiae, and Ruminococcus torques. In a preferred embodiment, the host cell is selected from the group consisting of Escherichia coli and Ruminococcus torques.

[0111] The polynucleotides and / or vectors described herein may not be naturally contained in the host cell. Thus, in some embodiments, the polynucleotide contained in the host cell is heterologous to the host cell. In some embodiments, the vector contained in the host cell is heterologous to the host cell. In some embodiments, the polynucleotide and / or vector contained in the host cell is heterologous to the host cell.

[0112] In some embodiments, the host cell is Ruminococcus torques ATCC27756. In some embodiments, the host cell is Ruminococcus torques AM22-16. In some embodiments, the host cell is Ruminococcus torques aa_0143. In some embodiments, the host cell is Ruminococcus torques2789STDY5834841.

[0113] Pharmaceutical Compositions In one embodiment, the invention provides a pharmaceutical composition comprising a polypeptide, conjugate, polynucleotide, vector and / or host cell as described herein. The pharmaceutical composition may also comprise a naturally occurring protein, including a polypeptide, such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot: A5KIY5) set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding the protein, or a host cell comprising the vector or polynucleotide.

[0114] The pharmaceutical composition may further comprise one or more pharma- ceutically acceptable excipients and / or other additives.

[0115] The pharmaceutical compositions may further comprise one or more additional active ingredients suitable for the treatment of the indications disclosed herein.

[0116] Medical Use The data presented herein indicates that the polypeptides described herein or naturally occurring proteins comprising the polypeptides, such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) represented by SEQ ID NO: 21, and fragments and variants thereof, are effective in the treatment and prevention of metabolic disorders, muscle disorders and injuries, and bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta and osteopetrosis.

[0117] Thus, provided herein are polypeptides, conjugates, polynucleotides, vectors, host cells, and / or pharmaceutical compositions according to the invention for use as a medicament. In some embodiments, there is provided a naturally occurring protein comprising a polypeptide, such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) as set forth in SEQ ID NO:21, or a vector or polynucleotide encoding the protein, or a host cell comprising the vector or polynucleotide, for use as a medicament.

[0118] In one embodiment, the polypeptide, conjugate, polynucleotide, vector, host cell and / or pharmaceutical composition according to the invention is for use in the treatment of a metabolic disorder. In one embodiment, the metabolic disorder is selected from the group consisting of metabolic syndrome, obesity, pre-diabetes, T2D and FLD. In some embodiments, there is provided a naturally occurring protein comprising a polypeptide such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) as set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding the protein, or a host cell comprising the vector or polynucleotide, for use in the treatment of a metabolic disorder, such as a metabolic disorder selected from the group consisting of metabolic syndrome, obesity, pre-diabetes, T2D and FLD.

[0119] In one embodiment, the polypeptide, conjugate, polynucleotide, vector, host cell and / or pharmaceutical composition according to the invention is for use in treating muscle disorders and / or muscle damage. In some embodiments, a naturally occurring protein, including a polypeptide such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding a polypeptide, or a host cell comprising the vector or polynucleotide, is provided for use in treating a metabolic disorder. In one embodiment, the muscle disorder is selected from the group consisting of muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, and peripheral neuropathy.

[0120] In one embodiment, the polypeptide, conjugate, polynucleotide, vector, host cell and / or pharmaceutical composition according to the invention is for use in treating a bone disorder. In some embodiments, a naturally occurring protein, including a polypeptide such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot: A5KIY5) set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding the polypeptide, or a host cell comprising the vector or polynucleotide, is provided for use in treating a bone disorder. In a preferred embodiment, the bone disorder is selected from the group consisting of osteoporosis, osteogenesis imperfecta, and osteopetrosis.

[0121] Thus, provided herein are polypeptides, conjugates, polynucleotides, vectors, host cells, and / or pharmaceutical compositions for use in the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, and / or bone disorders. In some embodiments, provided are naturally occurring proteins, including polypeptides such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot: A5KIY5) set forth in SEQ ID NO: 21, or vectors or polynucleotides encoding polypeptides, or host cells comprising the vectors or polynucleotides, for use in the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, and / or bone disorders.

[0122] Further provided herein are polypeptides, conjugates, polynucleotides, vectors, host cells and / or pharmaceutical compositions according to the invention for use in the treatment and / or prevention of diseases, disorders and conditions selected from the group consisting of metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and osteopetrosis. In some embodiments, there is provided a naturally occurring protein comprising a polypeptide, such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) set forth in SEQ ID NO:21, or a vector or polynucleotide encoding the polypeptide, or a host cell comprising the vector or polynucleotide, for use in the treatment and / or prevention of a disease, disorder, and condition selected from the group consisting of metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and osteopetrosis.

[0123] Also provided herein is the use of the polypeptides, conjugates, polynucleotides, vectors, host cells and / or pharmaceutical compositions in the manufacture of a medicament for treating metabolic disorders, muscle disorders and injuries, and / or bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and osteopetrosis. In some embodiments, there is provided the use of a naturally occurring protein, including a polypeptide, such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding the polypeptide, or a host cell comprising the vector or polynucleotide, in the manufacture of a medicament for the treatment of a metabolic disorder, muscle disorder and injury, and / or bone disorder, such as, for example, metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta and osteopetrosis.

[0124] Also provided herein is a method for the treatment of metabolic disorders, muscle disorders and injuries, and / or bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and / or osteopetrosis, comprising administering to an individual in need thereof a polypeptide, conjugate, polynucleotide, vector, host, and / or pharmaceutical composition according to the invention. In some embodiments, there is provided a method for the treatment of metabolic disorders, muscle disorders and injuries, and / or bone disorders, e.g., metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and osteopetrosis, the method comprising administering to an individual in need thereof a naturally occurring protein comprising a polypeptide, e.g., the Ruminococcus torques protein RUMTOR_00181 (Uniprot:A5KIY5) set forth in SEQ ID NO: 21, or a vector or polynucleotide encoding the polypeptide, or a host cell comprising the vector or polynucleotide.

[0125] The polypeptides, conjugates, polynucleotides, vectors, hosts, and / or pharmaceutical compositions are administered in a therapeutically effective amount.Similarly, naturally occurring proteins, including polypeptides such as the Ruminococcus torques protein RUMTOR_00181 (Uniprot: A5KIY5) set forth in SEQ ID NO: 21, or vectors or polynucleotides encoding the polypeptides, or host cells containing the vectors or polynucleotides, are administered in a therapeutically effective amount.

[0126] In one embodiment, the individual or subject is a mammal, preferably a human.

[0127] In one embodiment, the present disclosure provides Ruminococcus torques for use in the treatment of metabolic disorders, muscle disorders and injuries, and / or bone disorders, such as metabolic syndrome, obesity, pre-diabetes, T2D, FLD, cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, ALS, Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta, and osteopetrosis.

[0128] Use of probiotics or live biopharmaceutical products The data presented herein indicates that naturally occurring proteins, including polypeptides such as the polypeptides described herein, Ruminococcus torques RUMTOR_00181 (Uniprot:A5KIY5), and fragments and variants thereof, are useful when included within probiotics or live biopharmaceutical products (LBPs) or administered as feed compositions.

[0129] Thus, in some embodiments, a) a polypeptide or conjugate as described elsewhere herein; b) a RUMTOR_00181 polypeptide comprising or consisting of: i. a polypeptide according to SEQ ID NO: 21; or ii. A variant of SEQ ID NO: 21 having at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity to said polypeptide; c) a polynucleotide described elsewhere herein; d) a polynucleotide encoding a RUMTOR_00181 polypeptide; e) a vector as described elsewhere herein, f) a vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide, and / or g) a host cell according to any one of items 24 to 26, and / or h) a host cell comprising: i. a polynucleotide encoding a RUMTOR_00181 polypeptide; and / or ii. a vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide, Here, the feed composition optionally further comprises one or more of prebiotics, probiotics, synbiotics, proteins, lipids, carbohydrates, vitamins, fiber, and / or nutrients, such as feed minerals.

[0130] In some embodiments, there is also provided a host cell for use as a probiotic or as a live biopharmaceutical product (LBP), comprising: a) a polypeptide or conjugate as described elsewhere herein, and / or a RUMTOR_00181 polypeptide comprising or consisting of: i. a polypeptide according to SEQ ID NO: 21; or ii. A variant of SEQ ID NO: 21 having at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity to said polypeptide; b) a polynucleotide described elsewhere herein and / or a polynucleotide encoding a RUMTOR_00181 polypeptide; and / or c) A vector described elsewhere herein and / or a vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide.

[0131] In another aspect, there is provided the use of a host cell as a probiotic or as a live biopharmaceutical product (LBP), comprising: a) a polypeptide or conjugate as described elsewhere herein, and / or a RUMTOR_00181 polypeptide comprising or consisting of: i. a polypeptide according to SEQ ID NO: 21; or ii. A variant of SEQ ID NO: 21 having at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity to said polypeptide; b) a polynucleotide described elsewhere herein and / or a polynucleotide encoding a RUMTOR_00181 polypeptide; and / or c) A vector described elsewhere herein and / or a vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide.

[0132] Also embodiments provide for the use as a food ingredient or as a food or beverage additive of a polypeptide or conjugate as described elsewhere herein, or a RUMTOR_00181 polypeptide comprising or consisting of: i. a polypeptide according to SEQ ID NO: 21; or ii. A variant of SEQ ID NO: 21 having at least 85%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity to said polypeptide; a polynucleotide described elsewhere herein or a polynucleotide encoding a RUMTOR_00181 polypeptide; a vector described elsewhere herein or a vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide; A host cell as described elsewhere herein or comprising: i. a polynucleotide encoding a RUMTOR_00181 polypeptide; or ii. A vector comprising a polynucleotide encoding a RUMTOR_00181 polypeptide.

[0133] In some embodiments a variant of SEQ ID NO:21 has at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity to SEQ ID NO:21.

[0134] Administering a probiotic, live biopharmaceutical product (LBP) or feed composition to a subject has various health benefits. In some embodiments, administration of a probiotic, LBP or feed composition provides the benefits described elsewhere herein for administration of an isolated polypeptide.

[0135] In some embodiments, administration of the probiotics, LBP or feed composition reduces body fat mass, for example by reducing adipocyte size in white adipose tissue. In some embodiments, administration of the probiotics, LBP or feed composition increases lean body mass.

[0136] In some embodiments, administration of the probiotic, LBP or feed composition increases thermogenesis in adipose tissue, e.g., as measured by increased expression of mRNAs encoding thermogenic markers (e.g., Ucp1, Cidea, or Dio2 or corresponding mRNAs). In some embodiments, administration of the probiotic, LBP or feed composition results in decreased adipogenesis, including fat, e.g., decreased fat lipid production, as measured by decreased expression of mRNAs encoding genes involved in adipogenesis, including fat (e.g., Fasn, Scd1, and Acaca or corresponding genes). In some embodiments, administration of the probiotic, LBP or feed composition increases the protein level of UCP1 in adipose tissue.

[0137] In some embodiments, administration of the probiotic, LBP or feed composition improves glucose tolerance. In some embodiments, administration of the probiotic, LBP or feed composition increases bone mass. EXAMPLES

[0138] Example 1 - Discovery and characterization of novel polypeptide hormones released from common commensal bacterial strains in the human gut microbiome Main results Applying an in silico (alignment) approach, we searched for partial sequence homology with 118 mammalian polypeptide hormones, metabolism-related cytokines, and neuropeptides in approximately 5,600 complete prokaryotic genomes. We identified 118 peptides and cytokines by searching the scientific literature published in the fourth week of 2019. One alignment hit identified a bacterial pre-polypeptide, fibronectin type III protein containing domain 5 (FNDC5), which has relatively high homology to a known human precursor protein. 6Human FNDC5, which consists of 212 amino acids including a signal peptide, is mainly expressed in skeletal muscles, where it is cleaved after acute exercise to become a myokine (irisin, which consists of 112 amino acids). 7 .

[0139] The bacterial FNDC5-like protein contains 142 amino acids, of which 66% show similarity to human FNDC5 (calculated by dividing the number of amino acids of bacterial FNDC5-like proteins with identical or similar chemical structures to human FNDC5 by the length of the bacterial FNDC5-like protein and multiplying by 100%). The bacterial FNDC5-like protein is expressed by four strains of the commensal Ruminococcus torques (RT) species, which, together with 20 known strains, are prevalent and highly abundant (up to 10%) in the human gut microbiota. 8 The four RT strains harboring genes encoding FNDC5-like proteins were predicted to synthesize an 87 amino acid polypeptide with an overall amino acid sequence similarity of 64% and amino acid identity of 32% to human irisin (calculated by dividing the number of amino acids in the bacterial FNDC5-like protein that have the same or similar chemical structure as human irisin by the length of the bacterial FNDC5-like protein and multiplying by 100%, Figure 1). The enzyme(s) that cleave the human FNDC5 and bacterial FNDC5-like proteins are unknown. 7 We created an enzymatically cleaved bacterial fragment FNDC5-like protein for Ruminococcus torques irisin-like peptide 2 (RUCILP2).

[0140] In bacterial culture experiments with one of the four RT strains carrying the desired FNDC5-like sequence, RT-ATCC27756, and one of the control strains without the specific sequence, RT-ATCC35915, we documented that the RT strain carrying the sequence for the synthesis of RUCILP2 released the polypeptide into the culture medium (Fig. 2 ).

[0141] Irisin and the αV / β5 (ITGAV / ITGB5) integrin receptor 9A docking model of the interaction with RUCILP2 was applied to evaluate the putative 3D structure of RUCILP2 (Figure 3). The estimated free energy of RUCILP2 receptor binding was -1.43 kcal / mol, suggesting the existence of high binding affinity between RUCILP2 and the αV / β5 integrin receptor, which is ubiquitously expressed (Figure 4).

[0142] By applying the ZDOCK prediction tool and the PyMOL program, we predicted amino acids V7, E9, and E58 of RUCILP2 as binding sites to the αV / β5 integrin receptor (Figure 5). Using recombinant RUCILP2, we experimentally demonstrated the binding of the ligand to the αV / β5 integrin receptor (Figure 6). In addition to the in silico analysis, the presence of the αV / β5 integrin receptor in human colon was visualized by RNAscope-based mRNA in situ hybridization and immunostaining (Figures 7 and 8).

[0143] Materials and Methods Bioinformatics analysis: The reference prokaryotic genome database was downloaded from NCBI (ftp: / / ftp.ncbi.nlm.nih.gov / blast / db) and tBLASTn was used to search 118 peptide and cytokine amino acid sequences from Uniprot (https: / / www.uniprot.org / ) reported in the Supplementary table with a threshold e-value ≤ 0.1. Alignment analysis showed that RUCILP2 is predicted to be synthesized from an FNDC5-like precursor as an 87 amino acid protein with an overall 64% amino acid sequence homology and 32% amino acid sequence identity to irisin. Multiple sequence alignment of amino acids in human FNDC5, FNDC5-like bacterial proteins, human irisin and RUCILP2 was performed using the open access tool Clustal Ω (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to determine the number of identical and conserved residues. The 3D structure of RUCILP2 was predicted using the open access website tool I-TASSER (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ). The binding ability of RUCILP22 and irisin to integrin αV / β5 receptor was evaluated by computational analysis in ZDOCK (https: / / zdock.umassmed.edu / ). The final complex structure of the docking model was shown by PyMOL (v2.1.1). The direct binding interactions in the complex were visualized by PyMOL (v2.1.1).

[0144] Experimental verification: (1) Release of RUCILP2 in cultured RT cells In a culture experiment of one of the RT strains carrying the desired FNDC5-like protein sequence, RT-ATCC27756, and one of the control strains without the specific sequence, RT-ATCC35915, it was recorded that the RT strain carrying the sequence for the synthesis of RUCILP2 released the polypeptide into the culture medium (Figure 2). The detailed experimental protocol was as follows:

[0145] (1) Add 100 μl of bacterial protein extraction reagent (Thermo Scientific, 90080, lysozyme and DNase I (Fisher Scientific, 181610), 1 mM dithiothreitol (Sigma, 10197777001), 0.5 mM phenylmethylsulfonyl fluoride (Sigma, 10837091001), and phosphatase inhibitor cocktail (Fisher Scientific, 78440) per ml of resuspended cell culture and mix by pipetting up and down. (2) Incubate the cells at room temperature for 15 minutes. (3) Centrifuge at 13,000 revolutions per minute (rpm) for 10 minutes to obtain the supernatant. (4) Pierce Rapid Gold BCA Protein Assay was performed for each standard or bacterial protein lysate sample, and 20 μL was distributed in duplicate into a 96-well microplate. Pierce Rapid Gold BCA Protein Assay (Fisher Scientific, A53225) working reagent was prepared by mixing 50 parts of Reagent A and 1 part of Reagent B, and 200 μl of working reagent was added to each well with a multichannel pipette and mixed thoroughly for 30 seconds on a plate shaker. The plate was incubated at room temperature for 5 minutes, after which the absorbance was detected at 480 nm on a Thermo Scientific™ Multiskan™ GO microplate spectrophotometer. Unknown protein concentrations were measured using the standard curve. (5) Protein extracts (30 μg) were incubated at 98°C for 10 minutes, resolved on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) gel, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk, and incubated overnight with rabbit anti-FNDC5 antibody (Abcam, ab131390). (6) The membranes were incubated with anti-rabbit immunoglobulin G (IgG) secondary antibody (Fisher Scientific, G21234) and visualized by enhanced chemiluminescence. Equal loading was confirmed using Coomassie Brilliant Blue staining of PVDF membranes (PVDF, Millipore, IPFL85R).

[0146] (2) Co-immunoprecipitation with nickel ion column to verify the binding of RUCILP2 to the αV / β5 integrin receptor complex Step 1: Binding of integrins to RUCILP2. 100 nM of Fc-tagged RUCILP2 was incubated with 5 nM of the indicated his-tagged integrin avb5 in a final volume of 500 ul in 1.5 ml Protein LoBind tubes (Eppendorf, 022431081) for 5 / 30 / 90 min at room temperature under rotation. Step 2: After spinning, a Ni spin column (ThermoFisher Scientific, R901-01) was applied to obtain immunoprecipitated integrins. In detail, 500 μl of FC-RUCILP2-integrin-His protein complex was added to the column. Mix end-over-end for 15 min at 4°C. After washing the column twice, it was eluted twice and the eluted samples were stored. Step 3: Further elution: Add 200 μl of SDS-PAGE loading buffer to the column and pipette up and down to remove an aliquot of resin from the spin column. Incubate at 70°C for 5 minutes to release any proteins remaining on the resin after elution. Load and analyze proteins by SDS-PAGE. Most proteins are recovered by this procedure, whether they bind to nickel or to the agarose beads themselves. Step 4: Samples from each step were incubated at 70° C. for 10 min to allow dissociation of RUCILP2 and integrins. Step 5: Analyze by SDS-PAGE. Load and analyze the protein mix (FC-RUCILP2-integrin-His protein complex (before loading onto the column), flow through the column, wash and elute) by SDS-PAGE. Precipitated integrin was detected by immunoblot analysis against the his-tag. Co-precipitated RUCILP2 was detected by immunoblot analysis against the FC-tag. Each sample was loaded onto both gels, but probed with anti-his-tag integrin and anti-FC integrin αV / β5 primary antibodies, respectively.

[0147] (3) Visualization of integrin αV / β5 (ITGAV / ITGB5) receptors in human colon tissue samples using RNAscope-based mRNA in situ hybridization (ISH) and immunostaining. Samples: ITGAV / ITGB5 mRNA ISH analysis was performed on three paraffin samples of normal human colon tissue obtained from BioIVT. Sections were stained with hematoxylin and eosin to confirm that the tissue contained both colonic mucosa and colonic wall with the presence of ganglia / neuronal cells. Performance of the dual assay: The experimental setup included both positive and negative control probe sets. The two target mRNAs were stained as red and green signal dots, which became more diffuse precipitates when overrepresented. The ISH signal dots were visualized as red staining (ITGAV) and green staining (ITGB5), respectively. The RNAscope probes are shown with indications of the detection channels and associated chromogens. [Table 2] Images were acquired using a Zeiss AxioScan with a 20x objective. Representative areas were selected and presented.

[0148] Example 2 - Effects of RUCILP2: Cellular (in vitro) and rodent (in vivo) studies Main results We provided evidence of the metabolic effects of recombinant RUCILP2 in both cellular and mouse experiments. Thus, we found that equimolar (cellular studies) doses (in vivo studies) of RUCILP2 and irisin had similar effects in a dose-dependent manner on both the expression of genes important for thermogenesis and browning in human and mouse preadipocytes (Figure 9). RUCILP2 inhibited the expression of genes controlling adipogenesis in adipocytes (Figures 10 and 17). RUCILP2 produced a significant stimulatory effect on osteogenesis (Figure 11) and myogenesis (Figure 12). In hepatocytes, the hormone inhibited the expression of genes controlling gluconeogenesis (Figure 13). Furthermore, RUCILP2 increased the expression of genes involved in the intestinal barrier function and markers of cardiomyogenesis in intestinal epithelial cells (Figure 13). The cellular effects of RUCILP2 were blocked by pretreatment with CycloRGDyK, a nonspecific inhibitor of integrin receptors. In live rat colonic perfusion experiments, RUCILP2 exerted a stimulatory effect on the intestinal luminal release of glucagon-like peptide-1 (GLP-1, FIG. 14), glucagon-like peptide-2 (GLP-2), peptide YY (PYY, FIG. 15), and somatostatin (FIG. 16) following intraluminal injection.

[0149] Materials and Methods (1) Recombinant synthesis of 6-His-tagged RUCILP2 in Escherichia coli The target DNA sequence of the 87 amino acid RUCILP2 polypeptide was codon-optimized and synthesized for Escherichia coli. The synthesized sequence was cloned into the vector pET-30a(+) with a 6-His-tag for protein expression in E. coli strain BL21 star(DE3) transformed with the recombinant plasmid. One colony was inoculated into Terrific Broth (TB) medium containing the relevant antibiotic, the culture was incubated at 37°C and 200 rpm, and then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Expression was monitored using SDS-PAGE. Recombinant BL21 star(DE3) stored in glycerol was inoculated into TB medium containing the relevant antibiotic and cultivated at 37°C. When the OD600 reached approximately 1.2, the cell culture was induced with IPTG at 37°C / 4 hours. The cells were harvested by centrifugation. The cell pellet was resuspended in lysis buffer and then sonicated. The supernatant after centrifugation was then retained for purification. The target protein was obtained by one-step purification using Ni column. The target protein was maintained in 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0, then sterilized with a 0.22 μm filter and stored in aliquots. The concentration was determined by Bicinchoninic Acid (BCA)™ protein assay using bovine serum albumin (BSA) as a standard. The purity and molecular weight of the protein were determined by standard SDS-PAGE as well as confirmation by Western blotting. The protein was diluted in sterile phosphate buffered saline (PBS) and used for cell culture experiments and in vivo injection.

[0150] (2) Effect of recombinant RUCILP2 on human visceral white preadipocytes Human white preadipocytes were cultured until 80% confluent and then switched to differentiation medium (containing 0.3 ml / ml fetal bovine serum, 8 μg / ml d-biotin, 0.5 μg / ml insulin, and 400 ng / ml dexamethasone). The differentiation process into mature adipocytes was completed after 12–14 days. Treatment with RUCILP2 and irisin was started on day 3 of differentiation. For integrin complex inhibition, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 min, washed with PBS, and then treated with RUCILP2 and irisin, respectively. After 14 days of differentiation, cells were harvested and thermogenic genes were quantified by quantitative polymerase chain reaction (q-PCR).

[0151] (3) Effect of recombinant RUCILP2 on mouse inguinal preadipocytes Inguinal adipose tissue from 10-week-old wild-type C57BL / 6J male mice was dissected, washed with Dulbecco's modified Eagle's medium (D-MEM) containing 1% penicillin-streptomycin (P / S) solution, minced, and digested in D-MEM containing 2% BSA, 0.2% type 1 collagenase (1% P / S) for 1 h at 37° C. The digested tissue was then centrifuged at 400 g for 5 min at room temperature. The pellet was resuspended in 10 ml of D-MEM containing 10% FBS and 1% P / S and filtered through a 200 μm cell strainer. Inguinal stromal vascular cells were split onto type I collagen-coated 12-well plates, grown to confluence, and then induced to differentiate by treatment with 1 μM rosiglitazone, 86 nM insulin, 0.1 μM dexamethasone, 1 nM triiodo-L-thyronine (T3), and 250 μM methylisobutylxanthine.

[0152] Two days after induction, cells were switched to induction medium in the presence of 15nM recombinant RUCILP2, or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or saline for 2 days. Then, medium was changed every other day and cells were maintained in 86nM insulin and 1nMT3 in the presence of recombinant RUCILP2 or commercial recombinant irisin or saline at the indicated concentrations for 4 days. To inhibit integrin complexes, cells were treated with 10μM cRGDyK for 10 minutes before being treated with recombinant RUCILP2 or commercial recombinant irisin or saline every 2 days during 6 days of differentiation. Cells were harvested for qRT-PCR analysis as described in the protocol for gene expression analysis.

[0153] Oil Red O staining of lipids in adipocytes was performed according to the following protocol: (1) Fixation - Remove the medium and gently wash the cells twice with PBS. Add formalin (10%) to the cells and incubate for 30 minutes. (2) Discard the formalin and wash the cells twice with sterile water. Add 60% isopropanol to the cells and incubate for 5 minutes. (3) Discard the 60% isopropanol and evenly cover the cells with Oil Red O working solution. Rotate the plate or dish and incubate for 15 minutes. (4) Discard the Oil Red O solution and wash the cells four times with water until no excess staining is observed. (5) Add hematoxylin to the cells and incubate for 1 minute. Discard the hematoxylin and wash the cells 4 times with water. (6) The cells are covered with sterile water and observed under a microscope. Lipid droplets appear red and nuclei appear blue.

[0154] (4) Effect of recombinant RUCILP2 on mouse long bone cell-Y4 (MLO-Y4) cell line MLO-Y4 cells were kindly provided by Prof Moustapha Kassem, University of Southern Denmark. Cells (mouse osteocyte-like cell line) were seeded on type I collagen-coated 6-well plates in Minimum Essential Medium (α-MEM, Fisher Scientific, #15430584) supplemented with 2.5% fetal bovine serum (FBS, Fisher Scientific, #11550356), 2.5% bovine serum (Hyclone, SH30072.03) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37°C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days.

[0155] At 60% confluence, the medium was switched to FreeStyle293 expression medium after washing with warm PBS. After 4 hours of incubation, cells were treated with recombinant RUCILP2 or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or saline for 24 hours. For integrin inhibitor treatment, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 minutes, washed with PBS, and treated with recombinant RUCILP2 or commercial recombinant irisin or saline. After treatment, MLO-Y4 cells were harvested for qRT-PCR analysis of sclerostin mRNA levels as described in gene expression analysis.

[0156] (5) Effects of recombinant RUCILP2 on immortalized mouse myoblasts and C2C12 cell lines C2C12 cells were seeded in 12-well plates under DMEM / F-12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12, Fisher Scientific, #11524436) supplemented with 10% FBS (fetal bovine serum, Fisher Scientific, #11550356) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37°C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days.

[0157] At approximately 80% confluence, 10% fetal bovine serum was replaced with 2% horse serum to induce C2C12 myoblasts to differentiate into myotubes. After 24 hours (differentiation day 1), cells were treated with recombinant RUCILP2 or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or saline. For integrin inhibitor treatment, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 minutes, washed with PBS, and treated with recombinant RUCILP2 or commercial recombinant irisin or saline. On day 3, cells were refreshed with the same medium as day 1. After 6 hours of treatment on day 3, C2C12 cells were harvested for qRT-PCR analysis.

[0158] (6) Effect of recombinant RUCILP2 on immortalized hepatoma cells, HepG2 cell line HepG2 cells were seeded in 12-well plates under DMEM / F-12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12, Fisher Scientific, #11524436) supplemented with 10% FBS (fetal bovine serum, Fisher Scientific, #11550356) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37 °C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days.

[0159] At 70% confluence, HepG2 cells were incubated with 18 mM glucosamine (GlcN) in serum-free medium for 18 hours to induce insulin resistance, and then treated with recombinant RUCILP2 or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or saline for 24 hours. For integrin inhibitor treatment, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 minutes, washed with PBS, and treated with recombinant RUCILP2 or commercial recombinant irisin or saline. After treatment, HepG2 cells were harvested for qRT-PCR analysis.

[0160] (7) Effect of recombinant RUCILP2 on immortalized human colon adenocarcinoma cells, Caco-2 cell line Commercially available Caco-2 cells (ATCC, #HTB-37) were seeded in 12-well plates in EMEM medium (Eagle's Minimum Essential Medium, ATCC, #30-2003) supplemented with 20% FBS (fetal bovine serum, Fisher Scientific, #11550356) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37°C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days.

[0161] To induce hypoxia / reoxygenation (H / R) cell culture model at 70% confluence, Caco-2 cells were cultured in EMEM medium (without glucose and FBS) and exposed to hypoxic conditions (94% N2, 5% CO2 and 1% O2) at 37°C for 120 min. The cells were then treated with the indicated concentrations of recombinant RUCILP2 or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or PBS for 24 h immediately after the start of reoxygenation. For integrin inhibitor treatment, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 min, washed with PBS, and treated with recombinant RUCILP2 or commercial recombinant irisin or PBS. After treatment, Caco-2 cells were harvested for qRT-PCR analysis of the mRNA levels of intestinal epithelial barrier-related genes.

[0162] (8) Effect of recombinant RUCILP2 on H9C2 cell line Commercially available H9C2 cells (ATCC, #CRL-1446) were seeded in 12-well plates under DMEM / F-12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12, Fisher Scientific, #11524436) supplemented with 10% FBS (fetal bovine serum, Fisher Scientific, #11550356) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37°C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days.

[0163] At 70% confluence, cells were treated with the indicated concentrations of recombinant RUCILP2, commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) and PBS for 24 hours. For integrin inhibitor treatment, cells were treated with 10 μM CycloRGDyK (Selleckchem, #S7844) for 10 minutes, washed with PBS, and treated with recombinant RUCILP2, commercial recombinant irisin, and PBS. After treatment, H9C2 cells were harvested for qRT-PCR analysis of cardiomyocyte mRNA levels and differentiation-related genes.

[0164] (9) Effect of recombinant RUCILP2 on hormone release in the perfused live rat colon. Animals: Male Wistar rats (approximately 250 g) were obtained from Janvier (Le Genest-Saint-Isle, France) and housed with 2-4 rats / cage. Rats had free access to water and standard chow and were acclimated to a 12:12 h light / dark cycle for 1 week.

[0165] Ethical considerations: The study was conducted in accordance with the EU Directive 2010 / 63 / EU, as well as Danish legislation governing animal experimentation (1987) and the guidelines of the National Institute for Health, and with the approval of the Danish Animal Experiments Inspectorate (2018-15-0201-01397) and the local ethical committee (EMED, P20-058).

[0166] Isolation and perfusion of the proximal rat colon: On the day of the experiment, non-fasted rats were anesthetized by subcutaneous injection of Hypnorm / midazolam (0.0158 mg fentanyl citrate + 0.5 mg fluanisone + 0.25 mg midazolam / 100 g). When reflexes were eliminated, the mouse was placed on a heating plate (37°C) and the abdominal cavity was opened. The colon was isolated by ligating the vascular supply to the cecum, small intestine, spleen, stomach, kidneys, and celiac artery, which allowed the isolation of the most proximal part of the colon up to the part immediately proximal to the entrance of the inferior mesenteric artery (approximately 10 cm). A plastic tube was placed into the colonic lumen and the colon was gently flushed with isotonic saline (room temperature) to remove the luminal contents. A constant luminal flow of saline was applied by a syringe pump (0.15 ml / min) throughout the experimental protocol. A catheter was inserted into the abdominal aorta, which was ligated proximally to the superior mesenteric artery, and the intestine was vascularly perfused with heated (37°C) and oxygenated (95% O2 and 5% CO2) perfusion buffer at a constant flow rate of 3 ml / min using a single-pass perfusion system (UP100, Hugo Sachs Harvard Apparatus, Germany). A metal catheter was inserted into the venous port to collect the venous effluent. As soon as adequate flow was evident, the rats were euthanized by puncture of the diaphragm. The intestine was perfused for 25 min before the start of the experimental protocol to allow equilibration of the system. Each protocol started with a baseline period followed by the addition of test substances applied intra-arterially through a catheter inserted into the luminal tube or the aorta. The venous effluent was collected for 1 min via the drainage catheter using a fraction collector. Effluent samples were immediately placed on ice and stored at -20°C until analysis. Perfusion pressure was monitored throughout the experiment as an indicator of colonic health.

[0167] Perfusion buffer: The perfusion buffer consisted of modified Krebs-Ringer bicarbonate buffer supplemented with 3.5 mmol / L glucose, 0.1% (w / v) bovine serum albumin (catalog number 1.12018.0500, Merck, Denmark), 5% (w / v) dextran T-70 (to balance oncotic pressure; Pharmacosmos, Denmark), 5 mmol / L each of fumarate, pyruvate, and glutamate (Sigma Aldrich, Brondby, Denmark) and 10 μmol / L 3-isobutyl-1-methylxanthine (IBMX, catalog number 5879, Sigma Aldrich).

[0168] Hormonal measurements: Peptide hormones were measured using in-house radioimmunoassays: total GLP-1 (sum of 7-36NH2, 9-36NH2 and potential mid-terminal truncated fragments) was measured using a C-terminal specific antibody (code no. 89390) targeting the amidated form of GLP-1. Total PYY (PYY1-36+PYY3-36) was measured with a porcine antiserum (catalog no. T-4093; Bachem). Somatostatin was measured using a side-viewing antibody (code no. 1758-5) that detects all bioactive forms of somatostatin.

[0169] (10) Effect of recombinant RUCILP2 after intraperitoneal injection in chow-fed mice Mice (20 wild-type male C57BL / 6N, 8 weeks old, Janvier) were housed singly at 23±1°C with a 12-h light / dark cycle and free access to food and water. After 7 days of acclimation on a standard chow diet (Altromin1328 diet), a fixed grain-based (soybean, wheat, corn) formula lacking nitrosamines and free of alfalfa and seafood / animal food to avoid stress, mice were intraperitoneally injected daily for 7 days with 1 mg / kg recombinant RUCILP2 and saline, respectively. All animals were then sacrificed and subcutaneous fat pads and liver tissues were harvested. The mRNA levels of thermogenic genes such as Ucp1, Elovl3, Cidea, and Prdm16, and adipogenic genes such as Acaca and Fasn in inguinal fat were analyzed by qRT-PCR as described below.

[0170] RNA extraction and real-time PCR analysis: Total RNA was extracted from tissues using Trizol reagent (Invitrogen) according to the manufacturer's instructions, followed by concentration measurements. 1 μg of RNA was transcribed into cDNA using a reverse transcription system (Promega). Real-time PCR was performed using the LC480 detection system (Roche Diagnostics) and SYBR Green I Supermix (Takara). Samples were run in duplicate on one 384-well reaction plate. Data were normalized to the housekeeping Rpl36, TBP or GAPDH genes and analyzed according to the ΔΔCT method.

[0171] Example 3 - Systemic effects of RUMTOR_00181-producing and non-RUMTOR_00181-producing RT strains on mice fed a chow diet Main results The effect of oral gavage, twice weekly for 8 weeks, with either the RUMTOR_00181-producing RT strain (VPI B2-51, ATCC) or the non-RUMTOR_00181-producing RT strain (VPI 13831, ATCC) was compared in mice fed a chow diet. Oral gavage of the RUMTOR_00181-producing RT strain was found to reduce fat mass and increase lean mass (Figure 19), without any effect on mouse body weight gain (Figure 18). Meanwhile, the thermogenic program in inguinal adipose tissue was activated, followed by a decrease in adipogenesis (Figure 23). Glucose tolerance was improved (Figure 21), and furthermore, cortical bone density was increased (Figure 24). In mice fed a high-fat diet, the live RUMTOR_00181-producing Ruminococcus torques strain -ATCC27756- (RT2) was found to reduce body weight gain during the 8-week intervention (Figure 20). Furthermore, glucose tolerance tests by intraperitoneal injection showed that the RUMTOR_00181 producer -ATCC27756-(RT2)- improved glucose tolerance in vivo in mice fed a high-fat diet (Figure 22).

[0172] Materials and Methods Intervention study in mice carrying RUMTOR_00181-producing and non-RUMTOR_00181-producing RT strains For the intervention study in mice fed a chow diet, 8-week-old male C57BL / 6N mice (SPF; specific pathogen-free grade, Janvier) were housed singly and given free access to a chow diet (Altromin 1328 diet) containing 11% fat, 24% protein, and 65% carbohydrate under a strict 12-h light cycle and water. They were then divided into six groups and fed either sterile PBS, live RT-ATCC35915 (5 × 10 in sterile PBS), or water. 7 colony forming units (CFU) / 100 μl), live RT-ATCC35915 (5 × 10 in sterile PBS 8 CFU / 100 μl), live RT-ATCC27756 (5 × 10 in sterile PBS) 7 CFU / 100 μl), live RT-ATCC27756 (5 × 10 in sterile PBS) 8CFU / 100 μl), and heat-killed RT-ATCC27756 (5 × 10 8 CFU / 100μl, 70℃, 30min) twice a week for 8 weeks. Body weight was measured before each gavage. For the intervention study in mice fed a high-fat diet, 8-week-old male C57BL / 6N mice (specific pathogen-free grade, Janvier) were group-housed under a strict 12-hour light cycle with free access to a high-fat diet containing: 45kcal% fat, 20kcal% protein, and 35kcal% carbohydrate (Research Diet, D12451i), and water. They were then divided into four groups and fed sterile PBS, live RT-ATCC35915 (5x10 in sterile PBS), and water. 9 colony forming units (CFU) / 100 μl), live RT-ATCC27756 (5x10 in sterile PBS 9 CFU / 100 μl) and heat-killed RT-ATCC27756 (5x10 in sterile PBS 9 Mice were gavaged with 100 μl of 10 ... A portion of the adipose tissue and one of the tibiae were fixed in 4% paraformaldehyde in PBS for histological analysis or micro-CT scanning.

[0173] For glucose tolerance testing after 6 weeks of bacterial treatment, all mice were returned to standard drinking water, fasted for 4 hours, weighed, and then administered one bolus of glucose via intraperitoneal injection (2 g glucose / kg body weight).Blood samples were taken from the tail vein at 0, 15, 30, 60, and 120 min for blood glucose measurement using a glucose meter (LifeScan).

[0174] Example 4 - Proteomic assay for the measurement of RUCILP2 in human plasma Main results The developed targeted proteomic assay showed that RUCILP2 circulates in human plasma with an inter-individual concentration interval of 10-100 pg / ml measured from six individuals (Figure 25).

[0175] Materials and Methods Protocol for preparing proteomics samples (1) Human plasma samples (1 ml) were depleted of albumin and IgG using a ProteoExtract kit (Millipore, 122642). 1.1 Dilute the desired amount of sample with 10x binding buffer and water. 1.2 Install a column or column assembly for use with a syringe tip filter. 1.3 For column equilibration, fill a syringe with 6-10 ml of 1X binding buffer. Remove any trapped air from the syringe before connecting to the column. 1.4 By applying gentle pressure, a volume of 2 ml per column of 1X Binding Buffer can be passed through the resin bed. Discard the flow-through. 1.5 Fill a new syringe with the diluted sample. By applying gentle pressure, allow the diluted sample to pass through the column. Collect the column(s) flow-through. NOTE: Count up to 5 between two consecutive drops to allow sufficient contact time between the sample and the resin. 1.6 Connect the syringe from step 3 filled with 1X Binding Buffer to the column(s). By applying gentle pressure, a volume of 2 ml per column of buffer can be passed through the resin bed. Collect the column(s) flow-through as the wash fraction. Combine the wash fraction with the previously collected flow-through as the depleted sample. (2) The depleted sample was then concentrated using a 3 kilodalton (kDa) molecular weight cutoff spin filter column (Millipore, UFC900324). (3) Deglycosylation of plasma was performed using the Protein Deglycosylation Mix II kit (New England Biolabs) under denaturing reaction conditions. 3.1 Dissolve 100 μg of glycoprotein in 40 μl of water. 3.2 Add 5 μl of Deglycosylation Mix Buffer 2. 3.3 Incubate at 75°C for 10 minutes and then cool. 3.4 Add 5 μl of Protein Deglycosylation Mix II and mix gently. 3.5 Incubate the reaction at 25℃ (room temperature) for 30 minutes. 3.6 Shift the reaction to 37°C and incubate for 1 hour.

[0176] In-gel digestion step (1) Deglycosylated plasma samples (300 μg) were reduced with 10 mM dithiothreitol (DTT, Sigma), alkylated with 50 mM iodoacetamide, and then resolved by SDS-PAGE using 4%-12% NuPAGE Bis-Tris precast gels (Biorad 4-12% Criterion™ XT Bis-Tris Protein Gels, 18 wells, 30 μl, #3450124). (2) The gel was Coomassie stained and the fragment was excised from the 10-15 kDa region. (3) The gel pieces were destained, dehydrated with 100% acetonitrile, and dried in vacuum. (4) Resuspend the dried residue in 200 μl of Tris-urea buffer (8 M urea (sigma) / 0.1 M Tris-HCl, pH 8.5), add to a centrifugal spin filtration unit (0.5 ml, molecular weight cut-off 10 kDa) and centrifuge at 10,000 g until less than 10 μl of sample remains in the filter. This typically requires a centrifugation time of 10-15 min. This is true for all further centrifugation steps. (5) Add 200 μl of UA to the filtration unit and repeat centrifugation. (6) Discard the flow-through from the collection tube. (7) Add 100 μl of DB (0.05 M Tris-HCl, pH 8.5) to the filtration unit and centrifuge at 10,000 g for 10 minutes. Repeat this step twice. (8) Add 100 μl of 50 mM ammonium bicarbonate (Sigma-Aldrich) and 500 ng of sequencing grade trypsin (Promega) directly onto the filtration membrane and incubate at 37° C. overnight. (9) Add 20 μl of 1x internal standard mix (=2 pmoles) directly to the filtration membrane. (10) After 12 hours, centrifuge the filtration unit at 10,000 g until the solution has completely passed through the filtration membrane (approximately 5 minutes). (11) Add 100 μl of digestion buffer (0.05 M Tris-HCl, pH 8.5) and centrifuge the filtration unit at 10,000 g until all the liquid has passed through, where the sample volume should be approximately 200 μl. (12) Peptides are desalted using Millipore C18 ZipTips (Sigma). (13) Peptides were eluted with 5 μl of 70% acetonitrile and 1% formic acid and then dried using a speedvac.

[0177] Liquid chromatography-mass spectrometry (LC-MS) detection steps Mass spectrometry data were collected using a Q Exactive or LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled with a Famos Autosampler (LC Packings) and an Accela 600 liquid chromatography (LC pump (Thermo Fisher Scientific). Peptides were separated on a 100 μm i.d. microcapillary column packed with approximately 0.5 cm of Magic C4 resin (5 μm, Michrom Bioresources) followed by approximately 20 cm of Accucore C18 resin (1.6 μm, Thermo Fisher Scientific). For each analysis, approximately 4 μl was loaded onto the column. Peptides were separated using a 50 min gradient of 8% to 30% acetonitrile / 0.125% formic acid at a flow rate of approximately 250 nl / min.

[0178] The parallel reaction monitoring (PRM) step of targeted mass spectrometry PRM analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) with the following parameters: full MS scan from 400 to 700 Thomson (Th) at Orbitrap resolution 70,000 (m / z 200), automatic gain control (AGC) target 5x10 6 , Maximum injection time 500 ms. After a full MS scan, a resolution of 35,000 (m / z 200) (AGC target 5x10) was performed as triggered by the scheduled target list. 6 25–50 PRM scans were performed at 100 s (maximum injection time 500 ms). The PRM method employed isolation of targeted ions with a 2Th (Thomson) isolation window fragmented at a normalized collision energy (NCE) of 25. MS / MS scans were acquired with a starting mass range of 100 m / z and obtained as profile spectrum data type. Precursor and fragment ions were quantified using Skyline version 3.1.

[0179] Data Dependency Acquisition Step For data-dependent acquisition using Q Exactive, the scan sequence started with an Orbitrap MS1 spectrum with the following parameters: resolution 70,000, scan range 400–1,400 Th, automatic gain control (AGC) target 5x10. 6 , maximum injection time 250 ms, and centroid spectrum data type. MS 2 The top 20 precursors were selected for analysis, which was performed with the following parameters: resolution 17,500, AGC 1 × 10 5 The data was acquired using a centroid spectrum data type consisting of high-energy collision dissociation (HCD) with a maximum injection time of 60 ms, an isolation window of 2 Th, and an NCE of 25. The underfill ratio was set to 9%, which is 1.5x10 5 In addition, unassigned singly charged species are identified by the MS 2 were excluded from the analysis and dynamic exclusion was set to automatic.

[0180] Data-dependent acquisition using the Linear Trap Quadrupole (LTQ) Orbitrap Elite, the first analyzer MS1, survey scans were performed in the Orbitrap in the range 300–1,500 Th, with 3x10 4 This was followed by collision-induced dissociation (CID)-MS using a precursor isolation window of 2Th. 2 -The 10 most intense ions (TOP10) were selected for fragmentation. AGC settings were adjusted for the survey scan and MS 2 Scans are 3x10 each 6 Ion and 2.5x10 5 The ions whose intensity reached the threshold of 500 counts were analyzed by MS. 2 The maximum ion accumulation time was 1,000 ms for the survey MS scan and 1,000 ms for the MS scan. 2 The scan time was set to 250 ms. Singly charged species and ions with undetermined charge state were analyzed using MS 2 MS 2Ions within a 10 parts per million (ppm) m / z window surrounding the ion selected for were excluded from further selection for fragmentation for 120 s.

[0181] Peptide and protein identification steps After mass spectrometry data acquisition, Thermo Fisher RAW files were converted to eXtensible Markup Language (mzXML) format and processed using a set of software tools developed in-house for the analysis of proteomics data sets. All precursors selected for MS / MS fragmentation were confirmed using algorithms that detect and correct errors in monoisotopic peak assignments and refine the mass measurement of precursor ions. All MS / MS spectra were then exported as individual .DTA files and searched using the Sequest algorithm. These spectra were searched against a database containing the sequences of all human proteins reported by Uniprot in both forward and reverse directions. Common contaminating protein sequences (e.g., human keratin, porcine trypsin) were included as well. The following parameters were selected to identify peptides from non-enriched peptide samples: precursor mass tolerance of 25 ppm, product ion mass tolerance of 0.02 Da, no enzymatic digestion, and a maximum of two tryptic deletion cleavages. Variable modifications: oxidation of methionine (+15.994915) and deamidation of asparagine (0.984016), fixed modification: carbamidomethylation of cysteine ​​(+57.021464). The AScore algorithm was implemented to quantify the confidence with which each deamidation modification could be assigned to a specific residue in each peptide. Peptides with an AScore >13 were considered to be localized to a specific residue (p<0.05).

[0182] Example 5-21 - Identification and in vitro functional characterization of AABP2 Main results One of the trypsin cleavage fragments of RUCILP2, 21-amino acid bacterial peptide 2 (21-AABP2, FIG. 26), was predicted as the only fragment peptide with a higher hydrophobicity score than RUCILP2, which means that 21-AABP2 may exhibit higher protein structural or functional stability than its precursor, RUCILP2. Furthermore, 21-AABP2 was predicted to bind to the RUCILP2 receptor (αV / β5 integrin receptor, FIG. 27). Peptide 21-AABP2 was shown to be an inducer of key genes regulating thermogenesis in human visceral white preadipocytes and mouse inguinal preadipocytes (FIG. 28). In mouse myoblasts, 21-AABP2 promoted myogenesis and myotube formation (FIG. 29). Furthermore, 21-AABP2 stimulated insulin release from a rat insulinoma cell line (FIG. 30).

[0183] Materials and Methods Prediction of hydrophobicity scores of RUCILP2 and its tryptic cleavage fragment peptides Theoretical proteolytic cleavage of RUCILP2 after trypsin digestion was processed by PeptideMass (https: / / web.expasy.org / peptide_mass / ). Peptide hydrophobicity was predicted by PEPTIDE 2.0 using default settings (https: / / www.peptide2.com / N_peptide_hydrophobicity_hydrophilicity.php).

[0184] Predicted interaction of 21-AABP2 with αV / β5 integrin receptors The 3D protein structure of 21-AABP2 was predicted by PEP-FOLD. RUCILP2 or 21-AABP2 was docked to αV / β5 receptor using ZDOCK server according to the provider's guidelines. The complex 3D structure was visualized by PyMOL. Docking model of 21-AABP2 and integrin receptor was performed by ZDOCK server, and the complex with the highest docking score was selected as the best binding model.

[0185] Synthesis of recombinant 21-AABP2 in Escherichia coli The target DNA sequence of 21 amino acids 21-AABP2 was optimized and synthesized. The synthesized sequence was cloned into the vector pET-30a(+) with a 6-His-tag for protein expression in E. coli strain BL21 star(DE3) transformed with the recombinant plasmid. One colony was inoculated into Terrific Broth (TB) medium containing the relevant antibiotic, the culture was incubated at 37°C, 200 rpm, and then induced with isopropyl β-D-1-thiogalactopyranoside (IPTG). Expression was monitored using SDS-PAGE. Recombinant BL21 star(DE3) stored in glycerol was inoculated into TB medium containing the relevant antibiotic and cultivated at 37°C. When the OD600 reached about 1.2, the cell culture was induced with IPTG at 37°C / 4 hours. The cells were harvested by centrifugation. The cell pellet was resuspended in lysis buffer and then sonicated. The supernatant after centrifugation was then retained for purification. The target protein was obtained by one-step purification using a Ni column. The target protein was maintained in 50 mM Tris-HCl, 150 mM NaCl, 10% glycerol, pH 8.0, then sterilized with a 0.22 μm filter and stored in aliquots. The concentration was determined by Bicinchoninic Acid (BCA)™ protein assay using bovine serum albumin (BSA) as a standard. The purity and molecular weight of the protein were determined by standard SDS-PAGE as well as confirmation by Western blotting. The protein was diluted in sterile phosphate-buffered saline (PBS) and used for cell culture experiments.

[0186] Cellular metabolic effects of 21-AABP2 (1) Recombinant 21-AABP2 promotes the expression of key genes for thermogenesis and browning in human visceral white preadipocytes. Human visceral fat-derived white preadipocytes (C-12732, PromoCell) were cultured until 80% confluent and switched to differentiation medium (0.3μg / ml fetal calf serum (FCS), 8ug / ml d-biotin, 0.5ug / ml insulin, 400ng / ml dexamethasone) in the presence of 15nM 21-AABP2. The differentiation process into mature adipocytes was completed after 14 days. After 14 days of differentiation, cells were harvested and thermogenesis-related genes such as Ucp1 and Lhx8 were quantified by q-PCR.

[0187] (2) Recombinant 21-AABP2 induces the expression of key genes regulating thermogenesis in mouse inguinal preadipocytes. Inguinal adipose tissue from 6-week-old wild-type C57BL / 6J female mice was dissected, washed with PBS, minced, and digested in PBS containing 10 mM CaCl2, 2.4 U / ml Dispase II (Roche), and 10 mg / ml Collagenase D (Roche) for 1 h at 37°C. After adding warm DMEM / F12 (1:1) containing 10% FCS, the digested tissue was filtered through a 70 mm cell strainer and centrifuged at 600 x g for 10 min. The pellet was resuspended in 40 ml DMEM / F12 (1:1) containing 10% FCS, filtered through a 40 mm cell strainer, and then centrifuged at 600 x g for 10 min. Pelleted inguinal stromal vascular cells were grown to confluence and split into 12-well plates. Cells were induced to differentiate by treatment with 1 mM rosiglitazone, 5 mM dexamethasone, and 0.5 mM isobutylmethylxanthine for 2 days. Cells were then maintained in 1 mM rosiglitazone for 4 days, with medium changed every other day. During 6 days of differentiation, cells were treated with 15 nM 21-AABP2 every other day. After 6 days of differentiation, cells were harvested and thermogenic genes such as Ucp1, Cidea, Elovl3, Dio2, and Pgc1α were quantified by q-PCR.

[0188] (3) Recombinant 21-AABP2 stimulates myogenesis and myotube formation in mouse C2C12 myoblasts. C2C12 myoblasts were cultured until 80% confluent and switched to differentiation medium (containing 2% horse serum). Treatment with 21-AABP2 was initiated on day 2 of differentiation. Representative images of myotubes formed at 24 h of differentiation in the presence of PBS (blank) or 21-AABP2 (15 nM) were captured. Cells were harvested 4 days after differentiation and the expression of myogenic genes, such as Mymk and caveolin-3, was quantified by q-PCR.

[0189] (4) Recombinant 21-AABP2 stimulates insulin release from immortalized rat insulinoma cells, INS-1 cells. INS-1 cells were grown in RPMI1640 medium (11875093, ThermoFisher Scientific) until they reached 70% confluency, then switched to RPMI1640 medium supplemented with 15 nM 21-AABP2 and incubated for 12 h. Insulin concentrations in the cell culture medium supernatants were measured by MSD Rat / Mouse Insulin ELISA Kit (Merck Millipore).

[0190] Example 6 - Physiological effects of oral gavage with live RUMTOR_00181 producing RT2 strain on mice fed a chow or high fat diet Overview Through a comprehensive bioinformatics search for the presence of human hormone-like sequences in publicly available prokaryotic genomes, we identified two significant homologies, both from the CoDing Sequence (CDS) annotated as RUMTOR_00181 (Uniprot:A5KIY5), within the genome of Ruminococcus torques ATCC27756. The genome information of R. torques ATCC27756 has been deposited at NCBI as a reference genome and is used as the type strain for the bacterial species R. torques.

[0191] AlphaFold2 22The 3D structure of the RUMTOR_00181 protein predicted by shows a signal peptide at the N-terminus, two fibronectin type III (FNIII) domains, and one hydrophobic domain that is likely inserted into the membrane, followed by a 7 amino acid C-terminal domain (Figure 31).

[0192] The species R. torques has 26 reported strains. We found the presence of predicted proteins with high homology to the RUMTOR_00181 protein in the genomes of three additional strains of R. torques. Among the genes encoding RUMTOR_00181 and RUMTOR_00181-like proteins in four R. torques strains, we conserve 1,260 of 1,271 amino acid residues. Pairwise comparisons show RUMTOR_00181-like sequences in R. torques AM22-16, R. torques aa_0143, and R. torques 2789 STDY5834841. These are 99.5% (1,265 of 1,271), 99.5% (1,265 of 1,271), and 99.4% (1,263 of 1,271), respectively, identical to RUMTOR_00181 of R. torques ATCC27756.

[0193] Interestingly, two FNIII domains in RUMTOR_00181 show 30.7% and 34.5% homology identity to the recently discovered myokine irisin, respectively (Figure 32). Therefore, the two FNIII-containing domains were named Ruminococcus torques irisin-like peptide 1 (abbreviated RUCILP1) and RUCILP2, which have 88 and 87 amino acid residues, respectively.

[0194] By applying the EMBOSS Needle pairwise sequence alignment program (https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ), pairwise alignment of RUCILP1 to RUCILP2 showed that RUCILP1 has 73.9% (65 / 88) identity to RUCILP2 (Figure 33).

[0195] RUCILP1 and RUCILP2 are believed to be released by the RUMTOR_00181 producer via extracellular trypsin / LysC-dependent proteolytic cleavage at K961, K1050, K1122, and K1220, respectively (Figure 34).

[0196] Considering that RUCILP2 has a relatively high identity to irisin, we recombinantly synthesized RUCILP2 from Escherichia coli and investigated its physiological effects. In cell and animal experiments, we showed that recombinant RUCILP2 and irisin at equimolar concentrations (cell studies) or doses (in vivo studies) have similar effects on both the expression of key genes for thermogenesis and browning in mouse and human visceral adipocytes. RUCILP2 enhances the expression of leptin in adipocytes and inhibits the expression of genes controlling lipogenesis in adipocytes and hepatocytes. Furthermore, in hepatocytes, the hormone inhibits the expression of genes controlling gluconeogenesis. RUCILP2 stimulates insulin biosynthesis, and in live rat colon perfusion experiments, RUCILP2 shows a strong stimulatory effect on the luminal release of GLP-1, GLP-2, PYY, and somatostatin.

[0197] Below we summarize the main findings of two interventions in which mice on a C57BL / 6N background (specific pathogen-free grade) aged 8 weeks at the start of the study were orally supplemented with a live RUMTOR_00181-producing strain. One intervention was chow-fed mice. The other intervention was high-fat-fed mice. Each intervention was carried out for 8 weeks.

[0198] Main results The effects of oral gavage of either live RUMTOR_00181-producing R.torques ATCC27756 (RT2) or live non-RUMTOR_00181-producing R.torques ATCC35915 (RT3) strains twice weekly for 8 weeks were compared in C57BL / 6N mice fed a chow diet. Oral gavage of the RT2 strain reduced fat mass and increased lean body mass (Figure 19), but did not affect weight gain in mice over the study period (Figure 18). Meanwhile, gene expression analysis showed that the thermogenic program in inguinal adipose tissue was activated concomitantly with reduced adipose tissue lipogenesis (Figure 23). Glucose tolerance was improved (Figure 21), as well as increased cortical thickness in the proximal tibia (Figure 24).

[0199] In mice fed a high-fat diet (HFD), live RT2 strain was found to reduce body weight gain during the 8-week intervention (Figure 19). Meanwhile, magnetic resonance imaging (MRI) scans of body composition showed that RT2 supplementation significantly reduced mouse fat mass and increased lean mass (Figure 35). It is concluded that RT2 colonization reduced fat accumulation over time in HFD-fed mice, as shown by the reduced weight of inguinal and epididymal white adipose tissue mass in RT2-supplemented mice (Figure 36). Furthermore, intraperitoneal glucose tolerance tests showed that RT2 strain improved glucose tolerance in HFD-fed mice (Figure 22). Genetic analysis of inguinal fat showed that the expression of mRNAs encoding thermogenic markers such as Ucp1, Cidea, and Dio2 was enhanced in RT2-forced mice, while genes involved in adipose adipogenesis such as Fasn, Scd1, and Acaca were attenuated. Consistently, we found activation of lipolysis in subcutaneous white adipose tissue cells of RT2-supplemented mice. Moreover, causes of white adipose tissue inflammation such as Tnf-α, Mcp-1, and F4 / 80 in HFD-fed mice were suppressed in response to RT2 intervention (Figure 37). Histological analysis of inguinal fat revealed that adipocyte size was substantially smaller in HFD mice gavaged with live RT2 than in HFD mice gavaged with phosphate-buffered saline (PBS) or heat-killed RT2 or live RT3 strains (Figure 38). Apart from the reduction in white adipose tissue adipocyte size, the expression of UCP1 at the protein level was enhanced in inguinal adipose tissue (Figure 39). Notably, we detected a significantly higher distal femoral bone mass in HFD-fed mice after intervention with RT2 (Figure 40).

[0200] Materials and Methods Cultivation of Ruminococcus torques strains RUMTOR_00181-positive R. torques ATCC27756 (RT2) and RUMTOR_00181-negative R. torques ATCC35915 (RT3) strains were purchased from the ATCC Bacteriology Collection and cultured overnight under anaerobic conditions (95% N2, 5% H2) in ATCC medium #1589 containing modified shredded meat (Anaerobe Systems #AS-813) with 1% glucose.

[0201] For oral gavage in mice, cultures of both strains were centrifuged at 6,000 g for 10 min, washed twice with phosphate-buffered saline (PBS), and anaerobically incubated at 5 × 10 in anaerobic PBS containing 20% ​​(vol / vol) glycerol. 10 The cells were concentrated to colony forming units / ml (CFU / ml).

[0202] Bacterial counts were determined using tryptic soy medium (ATCC medium #260) containing 5% defibrinated sheep blood and 1.5% agar. 10 The RT2 strain concentrated by CFU / ml was autoclaved for 15 min at 121° C. Viability was confirmed by culture, which showed that the heat-killed RT2 did not grow at all, whereas the live RT2 strain grew well.

[0203] Before oral administration to mice, thaw the bacterial stock solution and divide it into 5x10 7 CFU / ml, 5x10 8 CFU / ml and 5x10 9 Diluted to CFU / ml. Protocol for intervention in mice All mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) on a C57BL / 6N background (specific pathogen-free grade). Animal experiments were performed using approved protocols of the Danish Animal Experiments Inspectorate (license number: 2018-15-0201-01491) and the University of Copenhagen (project number: P20-392). The mice were housed under the following conditions: unless otherwise stated, all mice were housed in an enriched environment at 23 ± 1 °C with free access to food and tap water and a 12-h light / dark cycle. Male mice were used for all in vivo studies. Mice were acclimated to the above environment for 1 week on a standard chow diet before any experiments were performed. Acclimation was performed in open cages in a temperature-controlled room (Memmert, HPP750). Mice were group-housed unless the relevant phenotyping strategy (indirect calorimetry) required singly housing.

[0204] For the chow-fed mouse intervention study, 8-week-old male C57BL / 6N mice (specific pathogen-free grade, Janvier) were housed singly under a strict 12-h light cycle with free access to chow chow (see description below) and water. They were then divided into six groups and gavaged twice weekly for 8 weeks with the following: sterile PBS, low-dose live RT3 (RT3-LD, 5x10 in sterile PBS), 10x10 in PBS, ... 7 colony forming units (CFU) / 100 μl), high dose live RT3 (RT3-HD, 5x10 in sterile PBS 8 CFU / 100μl), low dose live RT2 (RT2-LD, 5x10 in sterile PBS) 7 CFU / 100 μl), high dose live RT2 (RT2-HD, 5x10 in sterile PBS 8 CFU / 100 μl), and heat-killed RT2 (HK-RT2, 5x10 8 CFU / 100 μl sterile PBS solution, 121° C. for 15 min. Body weight was measured before each oral gavage.

[0205] For the intervention study in mice fed a high-fat diet, 8-week-old male C57BL / 6N mice (specific pathogen-free grade, Janvier) were group-housed under a strict 12-h light cycle with free access to high-fat diet (see below) and water. They were then divided into four groups and given either sterile PBS, live RT3 (5x10 in sterile PBS), or 100 mg / kg of water per day. 9 colony forming units (CFU) / 100 μl), live RT2 (5x10 in sterile PBS 9 CFU / 100 μl) and heat-killed RT2 (5x10 in sterile PBS 9 Mice were gavaged with 100 μl of 10 ...

[0206] Diet The standard chow diet (Altromin 1328 diet) contains 11% fat, 24% protein and 65% carbohydrate. This diet is a fixed mix of alfalfa and fish / animal metal free, nitrosamine free grain based (soybean, wheat, corn). The high-fat diet (Research Diet, D12451i) was formulated with 45 kcal% fat (lard oil and soybean oil), 20 kcal% protein (casein), and 35 kcal% carbohydrate (sucrose, Rodex 10, and starch).

[0207] Obtaining tissue samples Tissue samples (liver, interscapular brown adipose tissue, inguinal white adipose tissue, testicular white adipose tissue, jejunum, ileum and proximal colon) were dissected, weighed and stored at −80° C. for subsequent analysis. A portion of the adipose tissue, as well as one of the tibia and femur, were fixed in PBS containing 4% paraformaldehyde for histological analysis or micro-CT scanning.

[0208] Glucose tolerance test For glucose tolerance testing after 6 weeks of bacterial treatment, all mice were returned to standard drinking water, fasted for 4 hours, weighed, and then administered one bolus of glucose via intraperitoneal injection (2 g glucose / kg body weight).Blood samples were taken from the tail vein at 0, 15, 30, 60, and 120 min for blood glucose measurement using a glucose meter (LifeScan).

[0209] Histological analysis Inguinal white adipose tissue (iWAT) drops of mice were fixed in 4% paraformaldehyde / 1xPBS overnight at 4°C, then immersed in 100% ethanol for 24 hours and embedded in paraffin. To measure adipocyte size, adipose tissue paraffin sections were stained with hematoxylin and eosin (H&E staining). Images were obtained under a bright-field microscope. Representative images of each group are shown in this study, and the cell diameter of adipocytes was measured from H&E stained slides using open source ImageJ software.

[0210] RNA extraction and quantification For total RNA extraction of tissue samples isolated from mice, each piece of frozen tissue sample (weighing approximately 50 mg) was homogenized using one round of sterile and stainless steel beads (QIAGEN, #69989) after adding 500 μl of QIAzol lysis reagent. After homogenization, the samples were centrifuged at 12,000 g for 15 min at 4° C., and the supernatant was collected. RNA extraction was then performed according to the instructions provided by the manufacturer of the RNeasy mini kit (QIAGEN, #74106), followed by measurement of RNA purity and concentration by a NanoDrop™ 2000 / 2000c spectrophotometer (Thermo Fisher, #ND2000CLAPTOP). A total of 1 μg of RNA was used for reverse transcription into cDNA using the High-Capacity RNA-to-cDNA™ Kit (Fisher Scientific, #10704217) following the protocol and heating program. cDNA samples were premixed with Precision® PLUS Master Mix (Primer Design, #PPLUS-machine type) and then subjected to real-time PCR using the LightCycler® 480 System (Roche Diagnostics). For each gene indicated, samples were run in white 384-well plates and the ΔΔCt method was used to quantify RNA expression levels.

[0211] Western blot analysis Total proteins from iWAT were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich) premixed with a cocktail containing protease and phosphatase inhibitors (Sigma-Aldrich). Extracted proteins were measured with the Pierce BCA Protein Assay kit (Thermo Fisher Scientific) prior to SDS-PAGE on 4–20% polyacrylamide gels, diluted with loading dye, and heated at 96°C for 10 min. Proteins were then subjected to an immunoblot assay with UCP1 (ab10983, Abcam) and β-actin antibody (ab115777, Abcam) as an internal control. A LAS 4000 (Life Science) system was used to visualize the membranes, following the provider's guide.

[0212] Micro-CT analysis of mouse proximal tibia and distal femur High-resolution desktop micro-computed tomography imaging (Skyscan1172, Bruker) was used to scan the proximal tibias of mice fed a chow diet and the distal femur of mice fed a HFD. Morphometric analysis of the cortical microstructure of the proximal tibia and distal femur was performed as follows: X-ray voltage 50 kV, X-ray current 200 μA, filter 0.5 mm aluminum, image pixel size 4–5 μm, camera resolution field width 1,280 pixels, tomography rotation 180° / 360°, rotation step 0.3–0.5°, frame averaging 1–2, and scan duration 30–50 min.

[0213] The metaphysis-osteoblast cortex was selected for the growth plate. A slice of the cross section was selected as the growth plate reference slice as follows: moving slice by slice from the metaphysis / diaphysis towards the growth plate, from one corner of the cross section to the other, a point is reached where a clear "bridge" of low density cartilage (chondrocyte seam) is established. This bridge is established by the disappearance of the last band of thin primary cancellous bone that interrupts the chondrocyte seam. This landmark allows the definition of a reference level for the growth plate. The cortical volume of interest was then defined relative to this reference level.

[0214] The cortical area started approximately 2.15 mm (500 image slices) from the growth plate level in the metaphyseal direction and extended another 0.43 mm (100 image slices) from this position. 3D and 2D morphometric parameters were calculated for selected regions of interest (ROI) of the cortex. 3D parameters were based on the analysis of a marching-cubes model with rendered surfaces. 2D area and perimeter calculations were based on the Pratt algorithm. Structural thickness in 3D was calculated using the local thickness or "sphere-fitting" method, and a structural model index (a measure of the relative prevalence of plates and rods) was derived according to the method of Hildebrand and Ruegsegger. The degree of anisotropy was calculated by the mean intercept method. Rendered 3D models were constructed for the 3D representation of the cortical analysis area. Model construction was by the "Double time cubes" method, a modification of the marching cubes method. Cortical morphological parameters measured by micro-CT included 3D cortical thickness (Ct.Th, mm), 2D cortical cross-sectional thickness (Ct.Cs.Th mm), cortical periosteal periphery (Ct.Pe.Pm, mm), cortical endosteal periphery (Ct.En.Pm, mm), and cortical cross-sectional area (Ct.Ar, mm). 2 ), polar moment of inertia (MMI(p), mm 4 ), eccentricity (Ecc), and cortical porosity (Ct.Po, %).

[0215] All measurements were performed blinded to the examiner.

[0216] Example 7 - Identification of binding epitopes in RUCILP1 and RUCILP2 to the integrin αV / β5 receptor using a SPOT peptide microarray assay As shown, RUCILP1 and RUCILP2 can exert multiple metabolic enhancing effects by binding to the integrin αV / β5 receptor. This example aimed to identify the binding epitopes of both proteins to the receptor by performing an unbiased semi-quantitative SPOT peptide microarray (μSPOT) assay.

[0217] Materials and Methods Synthesis of 15mer peptides of both proteins for μSPOT assay μSPOT peptide array 25(CelluSpots, Intavis AG, Cologne, Germany) were synthesized on acid-labile amino-functionalized cellulose membrane disks (Intavis AG) (minimum loading 1.0 μmol / cm) containing 9-fluorenylmethyloxycarbonyl-β-alanine (Fmoc-β-Ala) linkers using a RePepSL synthesizer (Intavis AG). Synthesis was initiated by Fmoc deprotection using 20% ​​piperidine in N-methylpyrrolidone (NMP) (1x2 and 1x4 μL, 3 and 5 min, respectively), followed by washing with dimethylformamide (DMF, 7x100 μL / disk) and ethanol (EtOH, 3x300 μL / disk). The disk loading was reduced to 50% by using a mixture of Fmoc-Gly-OH and Boc-Gly-OH (0.25M:0.25M / NMP). All couplings were accomplished using 1.2 μL of a coupling solution containing pre-activated amino acids (AA, 0.5 M) with ethyl 2-cyano-2-(hydroxyimino)acetate oxyma (1.5 M) and N,N'-diisopropylcarbodiimide (DIC, 1.1 M) in NMP (2:1:1, AA:oxyma:DIC). Couplings were performed six times (5 min, 10 min, 20 min, 30 min, 30 min, and 30 min, respectively), after which the membranes were capped twice with capping mixture (5% acid anhydride in NMP) and subsequently washed with DMF (7x100 μL / disc). After chain elongation, final Fmoc deprotection was performed with 20% piperidine in NMP (3x4 μL, 5 min each), followed by six washes with DMF, followed by N-terminal acetylation with capping mixture (3x4 μL, 5 min each) and final washes with DMF (7x100 μL / disc) and EtOH (7x200 μL / disc). The dried cellulose membrane discs were transferred to a 96 deep-well block and treated with side-chain deprotection solution consisting of 80% TFA, 12% DCM, 5% HO, and 3% TIPS (150 μL / well) for 1.5 h at room temperature (rt). The deprotection solution was then removed and the discs were solubilized overnight at room temperature using a solvation mixture consisting of 88.5% TFA, 4% trifluoromethanesulfonic acid (TFMSA), 5% HO, and 2.5% TIPS (250 μL / well).The resulting peptide-cellulose conjugates were precipitated with ice-cold ether (700 μL / well), spun down at 1000 rpm for 90 min, and the pellets formed were then further washed with ice-cold ether. The resulting pellets were redissolved in DMSO (250 μL / well) to obtain final stocks, which were transferred to 384-well plates and printed (in duplicate) onto white coated Celluspots blank slides (76x26mm, IntavisAG) using a SlideSpotter robot (IntavisAG).

[0218] Visualization and analysis of μSPOT assays After rinsing the peptide array slides with 100 mM phosphate-buffered saline (PBS) (pH 7.4), the arrays were blocked with 3% bovine serum albumin (BSA) in PBS for >2 h at room temperature. These arrays were then incubated with His-tagged integrin receptors (2.5 nM) for 1 h at room temperature under blocking conditions. After washing 5x1 min with blocking buffer, the slides were probed with HRP-conjugated 6X-His tag antibody (1:10000, ab184607, abcam) for 0.5 h at room temperature. Finally, the slides were washed 3x1 min with PBS, 2x1 min with PBST, and 2x1 min with PBS at room temperature. The washed arrays were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) with a Fusion FX SPectra multimodal imaging platform (Vilber). The resulting blots were analyzed using array analysis software (Active Motif). This defines an error range for each data set by comparing the intensities of each peptide overlap on the analyzed array.

[0219] result First, we generated a library of 15-mer peptides covering the entire sequence of both proteins to systematically map their interactions with the integrin αV / β5 receptor, resulting in 74 and 73 peptides for RUCILP1 (SEQ ID NOs: 22-95) and RUCILP2 (SEQ ID NOs: 96-168), respectively, that were chemically synthesized on acid-labile, amino-functionalized, cellulose membrane discs.

[0220] Prior to initial screening, we verified that the secondary antibodies did not result in non-specific binding ("background binding") to coated microscope slides (Figure 41A). We then screened μSPOT peptide arrays with recombinantly expressed His-tagged integrin αV / β5 (2.5 nM) and semi-quantitatively assessed the relative binding affinities of the resulting peptides by subsequent incubation with horseradish peroxidase (HRP)-conjugated 6X-His antibody (Figure 41B).

[0221] After visualization of the peptide array, residues 12 ETSAKVSWKNAADGKEAAG 30 (SEQ ID NO: 169; RUCILP1) and 12 ETSAKASWKNAADGKEAAG 30 (SEQ ID NO: 183; RUCILP2) (Figures 42A and 42B). Furthermore, both proteins 74 ESAKSEKVEFTTVKK 88 (SEQ ID NO: 95; RUCILP1) and 73 NESVKSEKVTFKTLK 87 The very C-terminal sequence of (SEQ ID NO: 168; RUCILP2) showed relatively high affinity for integrin receptors (Figures 42A and 42B).

[0222] In the N-terminus of both proteins, the binding regions identified were consistent with AlphaFold modeling, which predicted that the loops were in the same region (Figures 43A and 43B).

[0223] Flexible loops are segments of proteins that group together secondary structure elements and are typically found on the surface of the protein. They are primarily responsible for interactions with other proteins, such as putative receptors.

[0224] Indeed, the identified binding regions in the two proteins correspond to loops in irisin that may interact with the same integrin receptor. 26 (Figure 43C). Our AlphaFold model also predicted the C-tails of both proteins as flexible elements located on the surface of the proteins (Figure 43D). Therefore, it is reasonable to find additional binding hits to receptors at the C-terminus of both proteins.

[0225] conclusion The SPOT peptide microarray assay allows experimental validation of in silico predictions of protein binding to its receptor. Preliminary in silico predictions show that residues in both proteins 69 DAA 71 However, the results of this experiment do not support the predicted binding in RUCILP1, whereas in RUCILP2, a 15-mer peptide containing two alanine residues in the predicted loop was identified. 70 AAGNESVKSEKVTFK 84 (SEQ ID NO: 165), which showed significant binding to the αV / β5 integrin receptor.

[0226] Example 8 - Direct biological activity comparison of RUCILP1 and RUCILP2 in vitro and in vivo RUCILP2 has been shown to have multiple positive effects on metabolism in both cellular and rodent studies. Here, we directly compared the effects of RUCILP1 and RUCILP2 both in vitro and in vivo.

[0227] Materials and Methods Cell culture experiments 3T3-L1 cells (a mouse fibroblast cell line) were split into 12-well plates, grown to confluence, and then treated with 86 nM insulin, 0.1 μM dexamethasone, and 250 μM methylisobutylxanthine to induce differentiation. Two days after induction, cells were switched to induction medium in the presence of recombinant RUCILP, commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix Pharmaceuticals, #067-29A), or phosphate-buffered saline (PBS) for 2 days. Media was then changed every other day for 6 days, and cells were maintained in 86 nM insulin for 4 days in the presence of the indicated concentrations of recombinant RUCILP, 21-AABP1, commercial recombinant irisin, or PBS. Cells were then harvested for q-PCR analysis as described in standard protocols for gene expression analysis.

[0228] MLO-Y4 cells (mouse osteocyte-like cell line) were seeded on type I collagen-coated 6-well plates under Minimum Essential Medium (α-MEM, Fisher Scientific, #15430584) supplemented with 2.5% fetal bovine serum (FBS, Fisher Scientific, #11550356), 2.5% bovine serum (Hyclone, SH30072.03) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37°C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days. At 60% confluence, the medium was switched to FreeStyle293 expression medium after washing with warmed PBS. After 4 hours of incubation, cells were treated with recombinant RUCILP or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or PBS for 24 hours. After treatment, MLO-Y4 cells were harvested for qRT-PCR analysis of sclerostin mRNA levels as described in standard gene expression analysis protocols.

[0229] C2C12 cells (mouse myoblast cell line) were seeded in 12-well plates under DMEM / F-12 medium (Dulbecco's modified Eagle's medium / nutrient mixture F-12, Fisher Scientific, #11524436) supplemented with 10% FBS (fetal bovine serum, Fisher Scientific, #11550356) and 1% penicillin-streptomycin (Fisher Scientific, #11548876). Cell cultures were maintained at 37 °C in a humidified chamber containing 5% CO2, and the culture medium was changed every 2-3 days. At approximately 80% confluence, the 10% fetal bovine serum was replaced with 2% horse serum to induce C2C12 myoblasts to differentiate into myotubes. After 24 hours (differentiation day 1), cells were treated with recombinant RUCILP or commercial recombinant irisin (Sigma, #SRP8039-10UG and Phoenix pharmaceuticals, #067-29A) or PBS. On day 3, cells were refreshed with the same medium as on day 1. After 6 hours of treatment on day 3, cells were harvested for qRT-PCR analysis of mRNA levels of the indicated genes.

[0230] Experiments in mice All mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France) on a C57BL / 6N background (specific pathogen-free grade). Animal experiments were performed using approved protocols of the Danish Animal Experiments Inspectorate (license number: 2018-15-0201-01491) and the University of Copenhagen (project number: P20-392). The mice were housed under the following conditions: unless otherwise stated, all mice were housed in an enriched environment at 23 ± 1 °C with free access to food and tap water and a 12-h light / dark cycle. Male mice were used for all these in vivo studies. Mice were acclimated to the above environment for 1 week on a standard chow diet before any experiments were performed. Acclimation was performed in open cages in a temperature-controlled room (Memmert, HPP750). Mice were group-housed unless the relevant phenotyping strategy (indirect calorimetry) required singly housing.

[0231] In the RUCILP intervention study, male mice (6 / group, 8 weeks old) fed a standard chow diet were treated with daily intraperitoneal injections of 1 mg / kg recombinant RUCILP1, RUCILP2, or saline for 1 week. All animals were then sacrificed and the subcutaneous fat pads and livers were harvested. The mRNA levels of the indicated genes in the subcutaneous fat and liver were analyzed by qRT-PCR as described in standard protocols for gene expression analysis.

[0232] Gene expression analysis in cells and mouse tissues Total RNA from cells or tissues was extracted using QIAzol lysis reagent (QIAGEN) according to the instructions provided by the manufacturer of the RNeasy mini kit (QIAGEN), and then RNA purity and concentration were measured by a NanoDrop2000 spectrophotometer (Thermo Scientific). A total of 1 μg of RNA was used for reverse transcription to cDNA using the iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories). Samples were premixed with Precision® PLUS Master Mix (Primer Design) and then subjected to real-time PCR using the LC480 system (Roche Diagnostics). For each gene indicated, samples were run in duplicate in 384-well plates and expression was quantified using the ΔΔCt method after normalization to the housekeeping Rpl36 or GAPDH genes.

[0233] result Direct comparison of the biological activities of RUCILP1 and RUCILP2 as measured in vitro First, we examined the dose-response effect of RUCILP on gene expression in mouse fibroblasts (3T3-L1), mouse osteoblasts (MLO-Y4), and mouse myoblasts (C2C12).

[0234] To assess the effect on adipocyte browning, the expression of Ucp1, Prdm16, Pgc1a, Dio2, and Cox2 was measured, and the expression of AdipoQ as a marker for white adipocytes was measured. The expression of sclerostin was measured as a marker for osteogenesis; markers of myotube formation included the expression of Heyl, Sox3, and Stat3.

[0235] As shown in Figure 44, in mouse fibroblasts, both RUCILPs were observed to dose-dependently upregulate adipocyte browning markers while downregulating adiponectin expression. In bone cells, RUCILP1 and RUCILP2 increase sclerostin expression levels at a dose of 150 nM. In myoblasts, Heyl expression shows a more pronounced dose-dependent response to RUCILP2 stimulation than after RUCILP1 exposure, whereas the other two myotube formation markers (Sox8 and Stat3) show less dose-dependent responses.

[0236] In addition to RUCILP1 and RUCILP2, 21-AABP1, a 21 amino acid fragment derived from RUCILP1, was also tested on 3T3-L1 fibroblasts (Figure 45), but despite a clear trend towards upregulation of browning markers consisting of Ucp1, Prdm16, and Dio2, the effect was not considered significant.

[0237] Comparative study of RUCILP1 and RUCILP2 as measured in vivo in C57 / b6 mice RUCILP1 or RUCILP2 were injected into the peritoneum of C57 / b6 mice at 1 mg / kg body weight for 7 days, and isotonic saline was administered in the same manner as the control. After dissociation of subcutaneous white adipose tissue (SWAT) and quantitative PCR-based measurements of thermogenesis and white adipocyte markers, we found that both RUCILPs showed comparable effects on thermogenic markers such as Ucp1, Prdm16, and Dio2 (Figure 46). However, there was no significant decrease in the expression of white adipocyte gene markers, i.e., AdipoQ and Ppara, with RUCILP1 exposure, whereas RUCILP2 exposure reduced the expression of these two genes in white adipocytes. In mouse hepatocytes, exposure to RUCILP2 reduced the expression of genes involved in gluconeogenesis, i.e., G6pase and Pepck1, whereas exposure to RUCILP1 had no effect.

[0238] conclusion In vitro experiments found that exposure of mouse fibroblasts (3T3-L1), mouse osteoblasts (MLO-Y4), and mouse myoblasts (C2C12) to equivalent concentrations of recombinant RUCILP1 or RUCILP2 induced similar overall effects on the expression of selected genes.

[0239] 21-AABP1 has no significant in vitro effect on browning gene expression. In vivo mouse studies of the effects of RUCILP1 and RUCILP2 showed that both peptides have comparable stimulatory effects on thermogenesis in subcutaneous white adipose tissue (SWAT). However, RUCILP2 reduces the expression of white genes in SWAT and gluconeogenic gene markers in the liver, whereas RUCILP1 has no such effect.

[0240] Example 9 - Alanine scan to identify specific residues involved in binding to integrin receptors and truncation scan to determine the minimum peptide length required for binding activity of fragments derived from RUCILP1 and RUCILP2

[0241] After finding that RUCILP1 and RUCILP2 can bind to the integrin αV / β5 receptor via a 19-mer binding epitope, we performed the following: 1) an alanine scan of the peptide library to identify the specific amino acid residues involved in binding; and 2) a truncation scan of the peptide library to predict the shortest peptide that maintains binding activity, and then performed a SPOT peptide microarray (μSPOT) assay to visualize and quantify the binding affinity.

[0242] Materials and Methods Synthesis of alanine-scanning and truncation-scanning libraries of both 19mer epitopes for μSPOT assays μSPOT peptide array 22(CelluSpots, Intavis AG, Cologne, Germany) were synthesized on acid-labile amino-functionalized cellulose membrane disks (Intavis AG) (minimum loading 1.0 μmol / cm) containing 9-fluorenylmethyloxycarbonyl-β-alanine (Fmoc-β-Ala) linkers using a RePepSL synthesizer (Intavis AG). Synthesis was initiated by Fmoc deprotection using 20% ​​piperidine in N-methylpyrrolidone (NMP) (1x2 and 1x4 μL, 3 and 5 min, respectively), followed by washing with dimethylformamide (DMF, 7x100 μL / disk) and ethanol (EtOH, 3x300 μL / disk). The disk loading was reduced to 50% by using a mixture of Fmoc-Gly-OH and Boc-Gly-OH (0.25M:0.25M / NMP). All couplings were accomplished using 1.2 μL of a coupling solution containing pre-activated amino acids (AA, 0.5 M) with ethyl 2-cyano-2-(hydroxyimino)acetate oxyma (1.5 M) and N,N'-diisopropylcarbodiimide (DIC, 1.1 M) in NMP (2:1:1, AA:oxyma:DIC). Couplings were performed six times (5 min, 10 min, 20 min, 30 min, 30 min, and 30 min, respectively), after which the membranes were capped twice with capping mixture (5% acid anhydride in NMP) and subsequently washed with DMF (7x100 μL / disc). After chain elongation, final Fmoc deprotection was performed with 20% piperidine in NMP (3x4 μL, 5 min each), followed by six washes with DMF, followed by N-terminal acetylation with capping mixture (3x4 μL, 5 min each) and final washes with DMF (7x100 μL / disc) and EtOH (7x200 μL / disc). The dried cellulose membrane discs were transferred to a 96 deep-well block and treated with side-chain deprotection solution consisting of 80% TFA, 12% DCM, 5% HO, and 3% TIPS (150 μL / well) for 1.5 h at room temperature (rt). The deprotection solution was then removed and the discs were solubilized overnight at room temperature using a solvation mixture consisting of 88.5% TFA, 4% trifluoromethanesulfonic acid (TFMSA), 5% HO, and 2.5% TIPS (250 μL / well).The resulting peptide-cellulose conjugates were precipitated with ice-cold ether (700 μL / well), spun down at 1000 rpm for 90 min, and the pellets formed were then further washed with ice-cold ether. The resulting pellets were redissolved in DMSO (250 μL / well) to obtain final stocks, which were transferred to 384-well plates and printed (in duplicate) onto white coated Celluspots blank slides (76x26mm, IntavisAG) using a SlideSpotter robot (IntavisAG).

[0243] Visualization and analysis of μSPOT assays After rinsing the peptide array slides with phosphate-buffered saline (PBS) (pH 7.4), the arrays were blocked with PBS containing 3% bovine serum albumin (BSA) for >2 h at room temperature. These arrays were then incubated with His-tagged integrin receptors (2.5 nM) for 1 h at room temperature under blocking conditions. After washing 5x1 min with blocking buffer, the slides were probed with HRP-conjugated 6X-His tag antibody (1:10000, ab184607, abcam) for 0.5 h at room temperature. Finally, the slides were washed 3x1 min with PBS, 2x1 min with PBST, and 2x1 min with PBS at room temperature. The washed arrays were visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) with a Fusion FX SPectra multimodal imaging platform (Vilber). The resulting blots were analyzed using array analysis software (Active Motif). This defines an error range for each data set by comparing the intensities of each peptide overlap on the analyzed array.

[0244] result To identify the amino acid residues critical for binding between the two RUCILPs and integrin receptors, we generated an alanine-scanning library of the identified binding epitopes in both RUCILPs, in which each amino acid residue in the 19-mer epitope was replaced with alanine, and the binding affinity of the peptides to the integrin receptors was compared with that of the wild type.

[0245] As shown in Figure 47, after systematic screening of mutant peptides, we found that lysine substitutions in both RUCILP1 and RUCILP2 resulted in a significant loss of receptor affinity, indicating the importance of basic residues within the binding epitopes of both RUCILPs.

[0246] Furthermore, we found that substitution of acidic residues (glutamic acid and aspartic acid) or tryptophan with alanine increased binding affinity.

[0247] These results are interpreted as follows: (1) Amino acid residues in the 19-mer epitope that may be important for maintaining binding affinity: RUCILP1: 12 ETSAKVSWKNAADGKEAAG 30 (SEQ ID NO:169) RUCILP2: 12 ETSAKASWKNAADGKEAAG 30 (SEQ ID NO:183) (2) Amino acid residues in the 19-mer epitope that may potentially enhance binding affinity after substitution with alanine: RUCILP1: 12 ETSAKVSWKNAADGKEAAG 30 (SEQ ID NO:169) RUCILP2: 12 ETSAKASWKNAADGKEAAG 30 (SEQ ID NO:183)

[0248] We then performed a truncation scan of the identified binding epitopes to determine the minimum length required to maintain core binding activity. This library was created through systematic truncation of peptide sequences from each end. As shown in Figure 48, we found that for both binding epitopes, the N-terminal amino acid residues are more important than the C-terminal residues to maintain core binding activity to integrin receptors.

[0249] When compared to the 19-mer peptide, the 15-mer peptide showed higher binding affinity, indicating that the truncated peptide may exhibit tighter binding to the integrin receptor. Using a truncation scan, it was shown that the shortest peptide exhibiting high binding affinity was: RUCILP1: 12 ETSAKVSWK 20 (SEQ ID NO:235) RUCILP2: 12 ETSAKASWK 20 (SEQ ID NO:268)

[0250] Consideration We report that this results in the identification of three lysines (K 16 , K 20 and K 26 Of the three lysine residues, the C-terminal lysine residues (K 26 ) is a flexible loop involved in binding to integrin receptors ( 17 ADGK 20 ) has previously been predicted to be involved in

[0251] Our alanine scan results not only support the AlphaFold predictions, but also highlight the importance of basic amino acid residues, especially lysines, for maintaining binding affinity to integrin receptors.

[0252] Very importantly, we found that substitution of four acidic residues significantly increased binding affinity, providing potential sites for peptide modification to further improve affinity.

[0253] In a truncation scan, the 19-mer epitope was truncated to a peptide containing 9 amino acid residues with relatively good binding properties, which could serve as a potential drug-derived analogue for both RUCILP1 and RUCILP2 while retaining many of the key features of RUCILP1 and RUCILP2.

[0254] [Table 3] TIFF2024522359000005.tif168159References 1.Lynch, SV & Pedersen, O. The human intestinal microbiome in health and disease. N. Engl. J. Med. 375, 2369-2379 (2016). 2.Fan, Y. & Pedersen, O. Gut microbiota in human metabolic health and disease. Nat. Rev. Microbiol. 19, 55-71 (2021). 3.Qin, J. et al. A human gut microbial gene catalog established by metagenomic sequencing.Nature 464, 59-65 (2010). 4.Plovier, H. et al. A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice.Nat. Med. 23, 107-113 (2017). 5.Depommier, C. et al. Supplementation with Akkermansia muciniphila in overweight and obese human volunteers: a proof-of-concept exploratory study.Nat. Med. 25, 1096-1103 (2019). 6.NCBI Fibronectin type III domain-containing protein 5 isoform 2 preproprotein [Homo sapiens].NCBI (2016). 7.Bostrom, P. et al. A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature 481, 463-468 (2012). 8.Kraal, L., Abubucker, S., Kota, K., Fischbach, M.A. & Mitreva, M. The prevalence of species and strains in the human microbiome: a resource for experimental efforts.PLoS ONE 9, e97279 (2014). 9.Kim, H. et al. Irisin mediates effects on bone and fat via αV integrin receptors.Cell 175, 1756-1768. e1717 (2018). 10.Nie, Y. & Liu, D. N-Glycosylation is required for FDNC5 stabilization and irisin secretion.Biochem. J. 474, 3167-3177 (2017). 11.Schumacher , MA , Chinnam , N. , Ohashi , T. , Shah , RS & Erickson , HP The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation.J. Biol. Chem. Rev. 288, 33738–33744 (2013). 12.Khanal, P., Jia, Z. & Yang, X. Cysteine ​​residues are essential for dimerization of Hippo pathway components YAP2L and TAZ.Sci. Rep. 8, 1-12 (2018). 13.Ronda, L., S. Bruno, S. Bettati, P. Storici & Mozzarelli, A. From protein structure to function via single crystal optical spectroscopy.Front. Mol. Biosci. 2, 12 (2015). 14.Schumacher , MA , Chinnam , N. , Ohashi , T. , Shah , RS & Erickson , HP The Structure of Irisin Reveals a Novel Intersubunit β-Sheet Fibronectin Type III (FNIII) Dimer.J. Biol. Chem. Rev. 288, 33738–33744 (2013). 15.Albrecht, E. et al. Irisin: Still chasing shadows.Mol. Metab. 34, 124–135 (2020). 16.Roth, Z., Yehezkel, G. & Khalaila, I. Identification and quantification of protein glycosylation.Int.J.Carbohydr. Chem. 2012 (2012). 17.Li, D. et al. Distinct functions of PPARγ isoforms in regulating adipocyte plasticity.Biochem. Biophys. Res. Commun. 481, 132-138 (2016). 18.Li, W. et al. High potency of a bivalent human VH domain in SARS-CoV-2 animal models.Cell 183, 429-441. e416 (2020). 19.Zhang, D. et al. Review of Research on the Role of Irisin in Tumors.OncoTargets Ther. 13, 4423 (2020). 20.Christiansen, C.B. et al. The impact of short-chain fatty acids on GLP-1 and PYY secretion from the isolated perfused rat colon.Am. J. Physiol. Gastrointest. Liver Physiol. 315, G53-G65 (2018). 21.Liu, T.-Y. et al. Irisin inhibits hepatic gluconeogenesis and increases glycogen synthesis via the PI3K / Akt pathway in type 2 diabetic mice and hepatocytes.Clin. Sci. 129, 839-850 (2015). 22. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold.Nature 596, 583-589 (2021). 23.Mirdita, M. et al. ColabFold-Making protein folding accessible to all. (2021). 24.Steinegger, M. & Soding, J. MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets.Nat. Biotechnol. 35, 1026-1028 (2017). 25.Dikmans, A., Beutling, U., Schmeisser, E., Thiele, S. & Frank, R. SC2: A novel process for manufacturing multipurpose high-density chemical microarrays.Qsar Comb Sci 25, 1069-1080, doi:10.1002 / qsar.200640130 (2006). 26.Schumacher, M. A., Chinnam, N., Ohashi, T., Shah, R. S. & Erickson, H. P. The structure of Irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: Implications for receptor activation.J. Biol. Chem. 288, 33738-33744, doi:10.1074 / jbc.M113.516641 (2013).

Claims

1. An isolated polypeptide having an amino acid length of less than 200, comprising or consisting of an amino acid sequence selected from the group consisting of: a. The amino acid sequence according to SEQ ID NO: 4 or SEQ ID NO: 19; b. A variant of SEQ ID NO: 4 or SEQ ID NO: 19, having at least 75% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 19, but having less than 99% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 19; c. A variant of SEQ ID NO: 4 or SEQ ID NO: 19, having 1 to 20 amino acid substitutions relative to SEQ ID NO: 4 or SEQ ID NO: 19; d. An amino acid sequence different from SEQ ID NO: 4 or SEQ ID NO: 19 by truncation at the N-terminus of 1 to 10 amino acids, or a variant thereof having 1 to 10 amino acid substitutions relative to SEQ ID NO: 4 or SEQ ID NO: 19; e. An amino acid sequence different from SEQ ID NO: 4 or SEQ ID NO: 19 by truncation at the C-terminus of 1 to 10 amino acids, or a variant thereof having 1 to 10 amino acid substitutions relative to SEQ ID NO: 4 or SEQ ID NO: 19; f. An amino acid sequence different from SEQ ID NO: 4 or SEQ ID NO: 19 by truncation at the N-terminus of 1 to 10 amino acids and truncation at the C-terminus of 1 to 10 amino acids, or a variant thereof having 1 to 5 amino acid substitutions relative to SEQ ID NO: 4 or SEQ ID NO: 19; g. The amino acid sequence according to SEQ ID NO: 5 or SEQ ID NO: 20; h. A variant of SEQ ID NO: 5 or SEQ ID NO: 20, having at least 75% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 20, but having less than 99% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 20; i. A variant of SEQ ID NO: 5 or SEQ ID NO: 20, having 1 to 5 amino acid substitutions relative to SEQ ID NO: 5 or SEQ ID NO: 20, wherein the polypeptide has an amino acid length of less than 50.

2. The polypeptide according to claim 1, wherein the polypeptide has a. an amino acid length of at least 15, b. an amino acid length of less than 100, or c. an amino acid length of 15 to 100 .

3. The polypeptide according to claim 1, wherein the variant has 1 to 10 amino acid substitutions compared to SEQ ID NO: 4 or SEQ ID NO:

19.

4. The polypeptide according to claim 1, wherein the variant has 1 to 3 amino acid substitutions as compared to SEQ ID NO: 5 or SEQ ID NO:

20.

5. The polypeptide according to claim 1, wherein the amino acid substitution is a conservative substitution.

6. The polypeptide according to claim 1, wherein the polypeptide comprises the following: a) V at amino acid position 7 of SEQ ID NO: 4, or a conservative substitution thereof, or E at amino acid position 9 of SEQ ID NO: 4, or a conservative substitution thereof, or E at amino acid position 58 of SEQ ID NO: 4, or a conservative substitution thereof, or b) Y at amino acid position 5 of SEQ ID NO: 5, or a conservative substitution thereof, or F at amino acid position 6 of SEQ ID NO: 5, or a conservative substitution thereof, or E at amino acid position 8 of SEQ ID NO: 5, or a conservative substitution thereof, or N at amino acid position 17 of SEQ ID NO: 5, or a conservative substitution thereof.

7. The conjugate comprising the polypeptide according to claim 1, wherein the polypeptide comprises one or more moieties conjugated to the polypeptide, and the one or more moieties are selected from the group consisting of alkyl, aryl, heteroaryl, olefin, fatty acid, polyethylene glycol (PEG), saccharide, and polysaccharide.

8. The polypeptide according to claim 1 or the conjugate according to claim 7, wherein the following are possible for the polypeptide: a) Binding to the αV / β5 integrin receptor; b) Inducing thermogenesis in white adipocytes; c) Decreasing the lipid content of adipocytes; d) Stimulating bone formation; e) Inducing cardiomyogenesis; f) Inducing myotube formation and myogenesis in myoblasts; g) Enhancing the intestinal barrier junction; h) Stimulating the secretion of glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2); i) Stimulating insulin secretion; j) Stimulating the secretion of peptide-YY (PYY); k) Stimulating the secretion of somatostatin; l) Inducing weight loss; m) Improving glucose tolerance, or n) Increasing the cortical thickness of the tibia.

9. An isolated polynucleotide encoding the polypeptide according to claim 1.

10. A vector comprising the polynucleotide according to claim 9.

11. A host cell comprising the polynucleotide according to claim 9.

12. A pharmaceutical composition comprising the following: A) The polypeptide according to claim 1 or the conjugate according to claim 7; B) A polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; C) The polynucleotide according to claim 9; D) A polynucleotide encoding a polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; E) The vector according to claim 10; F) A vector comprising a polynucleotide encoding a polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; G) The host cell according to claim 11; or H) A host cell comprising: a) A polynucleotide encoding a polypeptide comprising or consisting of: i. The polypeptide according to SEQ ID NO: 21, or ii. A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; or b) A vector comprising a polynucleotide encoding a polypeptide comprising or consisting of: i. The polypeptide according to SEQ ID NO: 21, or ii. A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide.

13. A) The polypeptide according to claim 1 or the conjugate according to claim 7; B) A polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; C) The polynucleotide according to claim 9; D) A polynucleotide encoding a polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; E) The vector according to claim 10; F) A vector comprising the polynucleotide encoding a polypeptide comprising or consisting of: a) The polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; G) A host cell according to claim 11; or H) A host cell comprising: a) A polynucleotide encoding a polypeptide comprising or consisting of: i. A polypeptide according to SEQ ID NO: 21, or ii. A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; or b) A vector comprising a polynucleotide encoding a polypeptide comprising or consisting of: i. A polypeptide according to SEQ ID NO: 21, or ii. A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide, a feed composition comprising one or more of prebiotics, probiotics, live biopharmaceutical products (LBPs), symbiotics, proteins, lipids, carbohydrates, vitamins, fibers, nutrients, or feed minerals.

14. The pharmaceutical composition according to claim 12 for use as a medicament.

15. A host cell comprising the following for use as a probiotic or as a live biopharmaceutical product (LBP): A) The polypeptide according to claim 1 or the conjugate according to claim 7; B) A polypeptide comprising or consisting of: a) A polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; C) The polynucleotide according to claim 9; D) A polynucleotide encoding a polypeptide comprising or consisting of: a) A polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide; E) The vector according to claim 10; or F) A vector comprising a polynucleotide encoding a polypeptide comprising or consisting of: a) A polypeptide according to SEQ ID NO: 21, or b) A variant of SEQ ID NO: 21 having at least 85% sequence identity to said polypeptide.

16. A medicament comprising the polypeptide according to claim 1 or the conjugate according to claim 7 for use in the treatment and / or prevention of metabolic disorders, muscle disorders and injuries, or bone disorders.

17. A medicament comprising the polypeptide according to claim 1 or the conjugate according to claim 7 for use in the treatment or prevention of a disease, disorder or condition selected from the group consisting of metabolic syndrome, obesity, prediabetes, type 2 diabetes (T2D), fatty liver disease (FLD), cardiovascular disease, muscular dystrophy, Duchenne muscular dystrophy, amyotrophic lateral sclerosis (ALS), Lambert-Eaton syndrome, myasthenia gravis, polymyositis, peripheral neuropathy, osteoporosis, osteogenesis imperfecta and marble bone disease.

18. Use of the feed composition according to claim 13 as a food ingredient or as a food or beverage additive.

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