Methods and compositions for inducing differentiation of human brown adipocyte progenitor cells

Abstract

This invention relates to compositions and methods for recruiting brown adipocytes in vitro and in vivo from brown adipocyte progenitor cells found in human skeletal muscle. Methods for treating metabolic diseases are also provided. Furthermore, methods for treating hypothermia are provided. In some embodiments, the brown adipocyte recruiter is a human protein or peptide. In other embodiments, the brown adipocyte recruiter may be a non-human protein or peptide. In other embodiments, the brown adipocyte recruiter is a small molecule or a natural product.

Description

[0001] Cross-references to related applications

[0002] This invention claims priority and benefit to U.S. Provisional Application No. 61 / 966,496, filed February 24, 2014, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to compositions and methods for increasing brown adipocytes and / or brown adipocyte quality in conditions such as type 2 diabetes, obesity, insulin resistance, and dyslipidemia. Specifically, the invention identifies and describes compounds that increase or promote the differentiation of brown adipose tissue (BAT) progenitor cells separated from skeletal muscle into brown adipocytes. Furthermore, the invention identifies and describes compounds that interact with gene products involved in the regulation of brown adipocyte differentiation and / or quality. Additionally, the invention provides methods for identifying compounds for the prevention and treatment of type 2 diabetes, obesity, insulin resistance, and dyslipidemia, and their therapeutic applications. This invention is intended for the study, prevention, and treatment of various metabolic diseases such as obesity, type 2 diabetes, insulin resistance, and dyslipidemia.

[0004] background

[0005] The prevalence of obesity is closely associated with an increased prevalence of diabetes, hypertension, coronary heart disease, cancer, and other diseases. White adipose tissue serves to store lipids and is associated with obesity. Brown adipose tissue ("BAT") plays the opposite role. It is specifically dedicated to lipid burning and energy dissipation as heat. In fact, brown adipocytes contain a large number of mitochondria (where cellular combustion occurs) and uniquely express uncoupling protein-1 ("UCP1"). UCP1 acts as an uncoupling agent for oxidative phosphorylation, leading to energy dissipation as heat. The sympathetic nervous system stimulates mitochondria development and UCP1 expression and activity. In rodents, the thermogenic effects associated with BAT increase after exposure to low temperatures (e.g., to prevent hypothermia) or lead to overheating, burning excess absorbed fat, and preventing weight gain. BAT also improves insulin sensitivity by altering susceptibility to weight gain and by consuming large amounts of glucose. Therefore, it plays an important role in maintaining body temperature, energy balance, and glucose metabolism.

[0006] Transgenic animal studies support the potential anti-obesity properties of BAT. For example, genetic ablation of BAT has been reported to induce obesity, while increased BAT content and / or function (and / or UCP1 expression) has been reported to promote a lean and healthy phenotype. Specifically, mice with higher BAT content gained less weight and had higher insulin sensitivity than control mice. Recently, long-acting ectopic BAT formulations have been demonstrated in mouse muscle to exhibit a genetic mechanism protecting against weight gain and metabolic syndrome.

[0007] Although UCP1 has been reported to play a role in controlling energy balance in rodents, and BAT expressing UCP1 is present in human newborns, it was long believed that physiologically relevant UCP1 expression was absent in adults. Indeed, BAT expressing UCP1 was thought to disappear early in life, and adults were believed to be devoid of BAT. However, recent studies have confirmed that BAT is actually maintained in most adults, albeit at significantly lower levels than in newborns and children.

[0008] Therefore, it is necessary to carefully identify and study methods that provide more BAT and / or stimulate UCP1 expression in adults for the research, prevention and treatment of various metabolic diseases such as obesity, type 2 diabetes, insulin resistance, dyslipidemia and type 1 diabetes.

[0009] In, for example, PCT Publications WO2009151541 and WO2013071063, the applicant previously identified cells capable of differentiating into brown adipocytes in various tissues, both of which are incorporated herein by reference in their entirety. However, there remains a need for agents (e.g., compounds, proteins, biological products, etc.) capable of, for example, inducing UCP gene expression, promoting the in vitro differentiation of BAT progenitor cells into brown adipocytes, promoting the in vivo differentiation of BAT progenitor cells into brown adipocytes, or combinations of these activities.

[0010] Overview

[0011] This invention provides compositions for recruiting or producing brown adipocytes in vitro and in vivo from BAT progenitor cells found in human skeletal muscle. These agents, or combinations thereof, can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro or in vivo, or both in vitro and in vivo. Furthermore, these agents can be used to treat metabolic diseases in patients, including but not limited to obesity, diabetes, insulin resistance, hyperlipidemia, and other conditions.

[0012] This invention is partly based on the discovery that different proteins, peptides, and small molecules (collectively, reagents) play important roles in the differentiation of BAT progenitor cells. Specifically, the different reagents disclosed herein have been found to significantly induce the differentiation of BAT progenitor cells isolated from human skeletal muscle into mature, functional brown adipocytes. Treatment of these BAT progenitor cells with one or more of these different reagents induces brown adipocyte differentiation. In some cases, treatment with the reagents for a period of time (e.g., several hours, 1 day, 2 days, 3 days, or less or longer) followed by introduction into an adipogenic medium leads to brown adipocyte differentiation. In other cases, treatment with the reagents concurrently with and / or subsequently with the introduction of an adipogenic medium results in brown adipocyte differentiation.

[0013] Because brown adipose tissue (BAT) is uniquely used for energy expenditure, the agent described herein has potential applications in treating obesity and related disorders such as diabetes. This agent can also be used to reduce fat storage in subjects, including food animals, thereby improving, for example, their meat quality.

[0014] Therefore, in one aspect, the present invention is characterized by a method for treating a subject, such as reducing the subject's fat storage or weight. The method includes administering a therapeutically effective amount of the present invention's reagent or combination of reagents to the subject.

[0015] In other respects, this article is characterized as a treatment method for subjects requiring reduction of fat storage or weight, comprising administering a reagent-activated population of BAT progenitor cells, wherein the reagent-activated progenitor cell population undergoes brown adipocyte generation. The method may optionally include identifying subjects requiring reduction of fat storage or weight.

[0016] In other respects, this article includes methods for improving the insulin sensitivity of subjects (e.g., subjects resistant to insulin). The methods include administering a reagent and / or a reagent-activated population of BAT progenitor cells, wherein the reagent-activated BAT progenitor cell population undergoes brown adipocyte formation. The methods may optionally include identifying subjects requiring improved insulin sensitivity.

[0017] On the other hand, this article features a method for regulating the function or development of brown adipose tissue (e.g., promoting BAT adipocyte generation) in a subject. The method includes administering a reagent and / or a reagent-activated population of BAT progenitor cells to the subject, wherein the reagent-activated progenitor cell population undergoes brown adipocyte generation.

[0018] In some embodiments, the methods described herein may include implanting a reagent-activated population of BAT progenitor cells into a subject. The reagent-activated cells may be implanted directly or delivered in the form of a scaffold, matrix, or other implantable device to which cells can attach (examples include carriers made from, for example, collagen, fibronectin, elastin, cellulose acetate, nitrocellulose, polysaccharides, fibrin, gelatin, self-assembled small peptides, and combinations thereof). Typically, the methods involve implanting a reagent-activated population of BAT progenitor cells comprising a sufficient number of cells to promote an increase in the mass of brown adipocytes in the subject, for example, an increase of at least 1%, such as 2%, 5%, 7%, 10%, 15%, 20%, 25%, or more.

[0019] In some embodiments, the method includes assessing the level of BAT adipocyte generation in the subject by exposing BAT progenitor cells isolated from the subject to one or more of the reagents described herein. BAT differentiation can be assessed by measuring any of the following: for example, BAT markers, for example, uncoupling protein (UCP) expression, such as UCP1; BAT morphology (e.g., visual observation of cells using a microscope); or BAT thermodynamics, such as the activity of cytochrome oxidase, enzymatic units of Na+-K+-ATPase, or other enzymes involved in BAT thermogenicity.

[0020] Typically, the object can be a mammal. In some embodiments, the object is a human object, such as an obese human object. In some embodiments, the object is a non-human mammal, such as a laboratory animal, companion animal, or livestock, such as a dairy cow, pig, or sheep raised for food. Typically, when a protein or peptide is used to recruit brown adipocytes from BAT progenitor cells, the protein or peptide will be derived from the same or related species as the object, such as a human, cat, dog, dairy cow, pig, or sheep. The protein or peptide may also be heterologous to the object.

[0021] In some embodiments, the method includes assessing one or more of the following: weight, white adipose tissue storage, brown adipose tissue storage, adipose tissue morphology, insulin levels, insulin metabolism, glucose levels, thermogenic capacity, and cold sensitivity. The assessment can be performed before, during, and / or after administration of the reagent and / or reagent-induced BAT progenitor cells. For example, the assessment can be performed at least 1, 2, 4, 7, 14, 21, 30 days or more, or less, before and / or after administration.

[0022] In some embodiments, the method includes one or more additional treatment rounds of BAT progenitor cells induced by a reagent or implantation reagent, thereby, for example, increasing the mass of brown adipose cells, and subsequently, for example, maintaining or further reducing obesity in the subject.

[0023] In some embodiments, when using protein or peptide reagents, BAT progenitor cells may be genetically engineered to stably or transiently express higher levels of the protein or peptide. The cells may be, for example, cultured mammalian cells, such as human cells. The recombinant protein or peptide used for expression is typically derived from the same or related species as the BAT progenitor cells, such as human proteins or peptides expressed in human cells. The recombinant protein or peptide may also be heterologous to the BAT progenitor cells.

[0024] In other respects, this document provides the use of one or more of the reagents described herein for promoting the differentiation of BAT progenitor cells into brown adipocytes and / or inducing the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα and / or COX IV in BAT progenitor cells in vitro or in vivo, or both in vivo and in vitro.

[0025] This document also provides the use of one or more of the agents described herein for the treatment of one or more diseases or conditions selected from the group consisting of: overweight, obesity, insulin resistance, diabetes, hyperinsulinemia, hypertension, hyperlipidemia, hepatic steatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, bulimia, endocrine disorders, triglyceride storage disorders, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease. Methods of treating the aforementioned diseases are also provided, including administering one or more of the agents described herein to a subject in need.

[0026] On the other hand, it relates to pharmaceutical compositions, including one or more reagents described herein and pharmaceutically acceptable excipients, diluents or carriers.

[0027] One specific aspect relates to the use of a reagent in recruiting brown adipocytes from BAT progenitor cells isolated from human skeletal muscle, wherein said reagent is selected from one or more of the following:

[0028] Antihistamines such as famotidine;

[0029] Antidopaminergic agents such as sulpiride hydrochloride, thiophene, or spirocyclopiperidone;

[0030] Microtubule ligands such as colchicine;

[0031] Rauvolfia alkaloids or derivatives such as reserpine or silcoline;

[0032] Potassium channel ligands, such as minoxidil;

[0033] Calcium channel antagonists such as felodipine;

[0034] Probenecid;

[0035] Prostaglandin F2 (PGF2) derivatives such as 9β,11α-PGF2 or 9α,11β-PGF2;

[0036] Peptides derived from the pituitary adenylate cyclase-activated polypeptide (PACAP) gene, such as the PACAP precursor of 55aa (aa 25-79).

[0037] Flavonoids, such as quinolone;

[0038] Fibroblast growth factor (FGF), such as FGF7, FGF10, or FGF13;

[0039] Transient receptor potential melanin-inhibin 8 (TRPM8) ligands such as menthol or ethelin;

[0040] Korean frog dermatitis extract;

[0041] Stromal cell-derived factor 1 (SDF-1), such as isotype SDF-1γ;

[0042] Cyclooxygenase inhibitors, such as difluorophenyl salicylic acid;

[0043] Biguanides, such as metformin;

[0044] Phosphodiesterase inhibitors, such as PDE3 inhibitors, such as cyanidin;

[0045] Stimulants of soluble guanylate cyclase (sGC), such as rosigmata;

[0046] b-type natriuretic peptide (BNP);

[0047] Ciliary neurotrophic factor (CNTF);

[0048] Interleukin-6 (IL-6);

[0049] Orexin B; and

[0050] α2-adrenergic receptor agonists, such as guanifacin hydrochloride.

[0051] In some embodiments, the reagent is selected from one or more of the following:

[0052] Antihistamines such as famotidine;

[0053] Antidopaminergic agents such as sulpiride hydrochloride or thiophene;

[0054] Microtubule ligands such as colchicine;

[0055] Rauvolfia alkaloids or derivatives such as reserpine or silcoline;

[0056] Potassium channel ligands, such as minoxidil;

[0057] Calcium channel antagonists such as felodipine;

[0058] Probenecid;

[0059] Prostaglandin F2 (PGF2) derivatives such as 9β,11α-PGF2 or 9α,11β-PGF2;

[0060] Peptides derived from the pituitary adenylate cyclase-activated polypeptide (PACAP) gene, such as the PACAP precursor of 55aa (aa 25-79).

[0061] Flavonoids, such as quinolone;

[0062] Fibroblast growth factor (FGF), such as FGF7 or FGF10;

[0063] Transient receptor potential melanin-inhibin 8 (TRPM8) ligands such as menthol or ethelin;

[0064] Korean frog dermatitis extract;

[0065] stromal cell-derived factor 1 (SDF-1), such as allotype SDF-1γ; and

[0066] Cyclooxygenase inhibitors, such as difluorophenyl salicylic acid.

[0067] In some embodiments, the reagent can induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro or in vivo, or both in vivo and in vitro.

[0068] In some embodiments, the reagent may have one or more biological activities selected from the group consisting of:

[0069] (a) Causes an increase or decrease in one or more of the following: β3-adrenergic receptor (β3-AR), solute carrier family 2, glucose transporter member 4 (SLC2A4), very long chain fatty acid protein 3 elongation (ELOVL3), CD36 antigen, type II iodinated thyroxine deiodinase (DIO2), BMP5, BMP6, FGF7, FGF10, FGF13, FGF21, fatty acid binding protein 7 (FABP7), CXCL12, atypical chemokine receptor 3 (ACKR3), insulin-like growth factor binding protein 4 (IGFBP4), pituitary adenylate cyclase activating polypeptide (PACAP), adenylate cyclase... The enzymes included: α-carboxylase 4 (ADCY4), cell death activator CIDE-A (CIDEA), secretory coil-associated protein 1 (SRFP1), SRFP2, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor D (VEGF-D), transforming growth factor β-2 (TGFB2), cAMP-specific 3',5'-cyclic phosphodiesterase 4B (PDE4B), cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D), high-affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A (PDE7A), and cAMP-specific 3',5'-cyclic phosphodiesterase 7B (PDE7B).

[0070] (b) Causes increased heat production in brown adipose tissue and / or skeletal muscle tissue;

[0071] (c) Causes increased insulin sensitivity in skeletal muscle, white adipose tissue, or liver;

[0072] (d) Causes increased glucose tolerance;

[0073] (e) Causes an increase in basal respiration, maximum respiratory rate, or uncoupled respiration;

[0074] (f) Causes an increase in metabolic rate; and

[0075] (g) Causes a reduction in hepatic steatosis.

[0076] In some embodiments, the reagent may cause an increase or decrease in one or more of the following: β3-adrenergic receptor (β3-AR), solute carrier family 2, glucose transporter member 4 (SLC2A4), very long chain fatty acid protein 3 elongation (ELOVL3), CD36 antigen, and type II iodinated thyroxine deiodinase (DIO2).

[0077] In some embodiments, the reagent can modulate the metabolic response of a subject or prevent or treat metabolic disorders in a subject. In some embodiments, the metabolic disorder may be one or more of the following: obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatic steatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, bulimia, endocrine disorders, triglyceride storage disorders, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.

[0078] On the other hand, the method relates to a method for promoting brown adipose tissue formation in a desired subject, the method comprising administering a reagent selected from one or more of the reagents described herein to the subject. In some embodiments, the method may further comprise modulating the subject's metabolic response and / or preventing or treating metabolic disorders in the subject. The metabolic disorders may be one or more of the following: obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatic steatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, bulimia, endocrine disorders, triglyceride storage disorders, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease. In some embodiments, the method further comprises contacting the subject's cells with the reagent and optionally transplanting the cells into the subject after the contacting step. In some embodiments, the cells may be BAT progenitor cells isolated from human skeletal muscle. The cells may be CD34 positive and / or CD31 negative.

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials described herein are used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Attached Figure Description

[0080] Figure 1 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on PPARγ2 mRNA expression.

[0081] Figure 2 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on PPARγ2 mRNA expression.

[0082] Figure 3 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on UCP1 mRNA expression.

[0083] Figure 4 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on UCP1 mRNA expression.

[0084] Figure 5This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0) on PPARγ2 mRNA expression.

[0085] Figure 6 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0) on PPARγ2 mRNA expression.

[0086] Figure 7 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0) on PPARγ2 mRNA expression.

[0087] Figure 8 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on PPARγ2 mRNA expression.

[0088] Figure 9 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on UCP1 mRNA expression.

[0089] Figure 10 This demonstrates the effect of FGF7 and FGF10 (both 100 nM, incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on UCP1 mRNA expression.

[0090] Figure 11 This demonstrates the effect of FGF7 and FGF10 (both 100 nM, incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on PPARγ2 mRNA expression.

[0091] Figure 12 This demonstrates the effect of FGF7 (1 nM, incubated with brown adipocyte progenitor cells from day 0 to day 3) on UCP1 mRNA expression. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0092] Figure 13 This demonstrates the effect of FGF7 (1 nM, incubated with brown adipocyte progenitor cells from day 0 to day 3) on PPARγ2 mRNA expression. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0093] Figure 14This demonstrates the effect of FGF10 (10 nM, incubated with brown adipocyte progenitor cells from day -3 to day 0) on UCP1 mRNA expression. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0094] Figure 15 This demonstrates the effect of FGF10 (10 nM, incubated with brown adipocyte progenitor cells from day -3 to day 0) on PPARγ2 mRNA expression. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0095] Figure 16 The effects of FGF7 (1 nM, incubated with brown adipocyte progenitor cells from day 0 to day 3) or FGF10 (10 nM, incubated with said cells from day -3 to day 0) on UCP1 mRNA expression were shown. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0096] Figure 17 The effects of FGF7 (1 nM, incubated with brown adipocyte progenitor cells from day 0 to day 3) or FGF10 (10 nM, incubated with said cells from day -3 to day 0) on PPARγ2 mRNA expression were shown. Rosiglitazone (rosi, 1 μM), bone morphogenetic protein-7 (bmp7, 6 nM), or both rosi and bmp7 were incubated with cells from day -3 to day 0.

[0097] Figure 18 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on PPARγ2 mRNA expression.

[0098] Figure 19 This demonstrates the effects of different reagents (incubated with brown adipocyte progenitor cells from day -3 to day 0 and from day 0 to day 3) on UCP1 mRNA expression.

[0099] Figures 20A-20C The image shows a fluorescence micrograph revealing the results of immunohistochemical (IHC) analysis of UCP1 protein expression (FITC, green) and the number of cell nuclei (DAPI, blue). CD31- cells differentiated for 8 days in minimal differentiation medium (MDM) after exposure to rosiglitazone (1 μM) were deeply differentiated into brown adipocytes expressing high levels of UCP1. Figure 20A Cells not exposed to rosiglitazone showed much lower levels of differentiation and UCP1 expression. Figure 20BCells maintained in proliferation medium (EGM-2) were undifferentiated and did not express UCP1. Figure 20C ).

[0100] Figure 21 The fluorescence signals of BODIPY 500 / 510C1,C12 cells after induction differentiation were shown in the images after brown adipocyte progenitor cells were cultured for 9 days under different conditions (rosi and BMP-7 were used from day -3 to day 0).

[0101] Figure 22 Fluorescence microscopy results revealed the BODIPY assay results (BODIPY500 / 510C1,C12, green) of intracellular lipid droplet formation. CD31- cells were differentiated for 8 days in minimal differentiation medium (MDM) 3 days after exposure to rosiglitazone (1 μM) (day -3 to day 0).

[0102] Figures 23A-23H An optical micrograph of CD31- cells shows brown adipocyte differentiation induced by treatment with several reagents that promote brown adipocyte formation. Figure 23A : Carrier (DMSO). Figure 23B Rosiglitazone Figure 23C :BMP7. Figure 23D Difluorobenzene salicylic acid. Figure 23E :Xi Keping. Figure 23F Kaempferol. Figure 23G Probenecid. Figure 23H Sulfamethoxazole. Invention Details

[0104] As used herein, “reagent-activated” means that BAT progenitor cells have been treated with one or more of the reagents described herein and are at least partially directed to differentiate into brown adipocytes. The cells may be autologous, allogeneic, or xenogeneic. “Brown adipocyte generation” means the generation of brown adipocytes from BAT progenitor cells in vivo, in vitro, or partially in vivo and partially in vitro. It should be understood that brown adipocyte generation can be induced; that is, so-called “BAT progenitor cells” are not necessarily directed to differentiate into brown adipocytes before being induced, for example, by one or more of the reagents described herein, and can be reprogrammed or transdifferentiated from stem cells or somatic cells into brown adipocytes. “Recruiting brown adipocytes” means promoting or increasing the differentiation of BAT progenitor cells into brown adipocytes, and / or increasing the in vivo and / or in vitro content or concentration of brown adipocytes.

[0105] This document provides reagents (e.g., compounds, proteins, biological products, etc.) that promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce in vivo, in vitro, or both in vivo and in vitro expression of the UCP1 gene. These reagents can be identified by screening compounds, proteins, biological products, etc. For example, in some embodiments, BAT progenitor cells (e.g., those isolated from human skeletal muscle) can be used to screen reagents for their ability to induce UCP gene expression and / or differentiate into brown adipocytes. Reagents identified in this manner can be used for various research, diagnostic, and therapeutic purposes, including, for example, the treatment of metabolic diseases such as obesity, type 2 diabetes, insulin resistance, and dyslipidemia. In some embodiments, reagents identified by the tests described herein are optimized for improving their physicochemical and / or pharmacokinetic properties.

[0106] The expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COXIV in BAT progenitor cells, both in vitro and in vivo, can be enhanced according to the methods described herein. In some embodiments, exposure to adipogenic medium can be used to stimulate increased expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COXIV in BAT progenitor cells.

[0107] Therefore, in some embodiments, the following agents or combinations thereof may be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα and / or COX IV in BAT progenitor cells in vitro or in vivo, or both in vivo and in vitro: PDE3 inhibitors (e.g., cyanidazol), PDE4 inhibitors (e.g., cyclophosphamide), prostaglandin F2 (PGF2) derivatives such as 9β,11α-prostaglandin F2 or 9α,11β-prostaglandin F2, and peptides (e.g., a portion) derived from the pituitary adenylate cyclase activating polypeptide (PACAP, ADCYAP1, UniProt P18509) gene, such as 55aa(aa PACAP propeptide (25-79), BDNF (brain-derived neurotrophic factor), TGR5 agonists such as oleanolic acid, BMP-7, flavonoids such as kaempferol (KMP, CAS number 520-18-3), soluble guanylate cyclase (sGC) stimulants such as riociguat (BAY 63-2521, CAS 625115-55-1), fibroblast growth factors (e.g., FGF7 (fibroblast growth factor-7, KGF, keratinocyte growth factor), FGF10 (fibroblast growth factor-10, KGF-2, keratinocyte growth factor-2), or FGF13 (fibroblast growth factor-13)), BNP (β-natriuretic peptide), TRPM8 (CMRI) ligands such as menthol or eserin, bufotenoids such as those from toads (UniProt P84214), CNTF (ciliary neurotrophic factor, UniProt P05231), interleukin-6 (IL-6), orexin B, SDF-1γ (CXCL12), or guanifacine hydrochloride.

[0108] In addition, in other embodiments, other agents or combinations thereof that can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce UCP1 expression include prostaglandin J2 (PGJ2), 24(S)-hydroxycholesterol, various forms of vitamin D, such as 1,25-dihydroxyvitamin D3 or 24,25-dihydroxyvitamin D3, and cyclooxygenase inhibitors such as difluorophenylsalicylic acid.

[0109] In other embodiments, additional agents or combinations thereof that can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce UCP1 expression include bone morphogenetic proteins such as BMP5 (bone morphogenetic protein 5, UniProt P22003) and BMP6 (bone morphogenetic protein 6, UniProt P22004), platelet-derived growth factor receptor-like protein (PDGFRL, UniProt Q15198), vascular endothelial growth factor D (VEGF-D, FIGF, UniProt O43915), CYTL1 (cytokine-like protein 1, UniProt Q9NRR1), SCG2 (secretory granulin-2, UniProt P13521), NPTX2 (neuronal pentamin-2, UniProt P47972), OLFML2B (olfglobulin-like protein 2B, UniProt Q68BL8), and TFPI2 (tissue factor pathway inhibitor 2, UniProt). P48307), IFNE (interferon ε, UniProt Q86WN2), prostaglandin F2-α receptor (PTGFR, prostaglandin FP receptor, UniProt P43088) ligands such as prostaglandin F2, CNTF (ciliary neurotrophic factor), interleukin-6 (IL-6, UniProt P05231), interleukin-15 (IL-15, UniProt P40933), CXCL12 (chemokine (CXC motif) ligand 12), stromal cell-derived factor 1, SDF1, UniProt P48061 isotype SDF-1g / UniProt P48061-3), and / or atypical chemokine receptor 3 (ACKR3, CMKOR1, CXCR7, GPR159, RDC1, UniProt P25106) ligands such as SDF1 (CXCL12).

[0110] In other embodiments, other agents or combinations thereof that can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce UCP1 expression include biguanides such as metformin, antihistamines such as famotidine, antidopaminers such as sulpiride hydrochloride, spiropiperidone, thioethorazine, microtubule regulators such as colchicine, rauwolf alkaloids or derivatives such as reserpine or sicoplanin, potassium channel ligands such as minoxidil, probenecid, or calcium channel antagonists such as felodipine.

[0111] In some embodiments, treatment of a subject (including a human subject) with one or more of the reagents described herein results in increased production of UCP1 mRNA or protein in the subject's skeletal muscle. For example, in some embodiments, treatment of a subject with rosiglitazone induces the appearance or differentiation of brown adipocytes in skeletal muscle, increases the expression of the UCP1 gene in brown adipocytes present in or near skeletal muscle (between muscle fibers, on the surface and / or adjacent to skeletal muscle tissue), or both. In some embodiments, the appearance or differentiation of brown adipocytes in skeletal muscle can be induced in subjects with metabolic diseases. Brown adipocytes provide glucose reservoirs with high mitochondrial and cellular respiration and fatty acid oxidation rates, dissipating energy into heat (uncoupled oxidative phosphorylation). The subject's metabolic rate can be increased and body weight can be reduced through induction. Induction of brown adipocyte appearance or differentiation can also improve insulin sensitivity, glycemic homeostasis, and cardiovascular disease risk factors. Brown adipocytes can also secrete factors for achieving healthy energy balance and low body fat levels, increasing insulin sensitivity, and improving glycemic homeostasis or cardiovascular health.

[0112] Therefore, in some embodiments, the reagents described herein, or combinations thereof, can be used in therapeutic subjects, including human subjects. In some aspects, these reagents can promote the differentiation of BAT progenitor cells into brown adipocytes. In other aspects, these reagents can induce, either in vitro or in vivo, or both in vivo and in vitro, the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COXIV in BAT progenitor cells.

[0113] In some areas, the metabolic diseases that can be treated include obesity, type II diabetes, insulin resistance, hyperinsulinemia, hypertension, hyperlipidemia, hepatic steatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, bulimia, endocrine disorders, triglyceride storage disorders, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.

[0114] In other embodiments, the reagent can be used to activate isolated autologous BAT progenitor cells, which are then used to treat subjects, including human subjects.

[0115] Identifying molecular pathways

[0116] Gene chip studies were conducted to identify molecular pathways that play a role in the differentiation of CD31-progenitor cells into brown adipocytes and / or the induction of UCP1 expression. CD31-cells were isolated from human skeletal muscle biopsies as previously described in WO2013071063, which is incorporated herein by reference, and were used in the following two studies: (1) a cAMP study: CD31-cells differentiated according to WO2013071063 and incorporated herein by reference (control) + addition of a loading agent (control 1 sample) or cAMP (cAMP sample); and (2) a rosiglitazone study: CD31-cells differentiated according to WO2013071063, except that rosiglitazone was omitted from the adipogenic medium (control 2 sample). In this study, rosiglitazone was added only to the second sample (rosiglitazone sample). As stated above, these reagents have been shown to promote the differentiation of CD31-cells into brown adipocytes and UCP1 expression.

[0117] Total RNA was purified from these samples, and transcriptional profiles were assessed using an Illumina human WG-6 bead array (Expression Analysis, Inc., Durham, NAT). Results were analyzed using IngenuityPathway Analysis 7.0 (experimental version). These results were used to determine which molecular pathways are involved in CD31-cell differentiation into brown adipocytes, and more importantly, which molecular pathways can be used to develop agents that promote brown adipocyte emergence and UCP1 expression.

[0118] Based on this work, the following mechanisms and reagents were found to promote the development from BAT progenitor cells to brown adipocytes: PPARγ ligands (e.g., rosiglitazone), PDE3 inhibitors (e.g., cyanidin), PDE4 inhibitors (e.g., cyclophosphamide), BMP7 (bone morphogenetic protein 7, UniProt P18075), BMP5 (bone morphogenetic protein 5, UniProt P22003), BMP6 (bone morphogenetic protein 6, UniProt P22004), FGF7 (fibroblast growth factor-7, KGF, keratinocyte growth factor), FGF10 (fibroblast growth factor-10, KGF-2, keratinocyte growth factor-2), BNP (β-natriuretic peptide), FGF13 (fibroblast growth factor-13), BDNF (brain-derived neurotrophic factor), and stimulants of soluble guanylate cyclase (sGC) (e.g., rosiglitazone (BAY 63-2521, CAS)). 625115-55-1), TRPM8 (CMRI) ligands (e.g., menthol, ethelin), platelet-derived growth factor receptor-like protein (PDGFRL, UniProt Q15198), vascular endothelial growth factor D (VEGF-D, FIGF, UniProt O43915), CYTL1 (cytokine-like protein 1, UniProt Q9NRR1), SCG2 (secretory granulin-2, UniProt P13521), NPTX2 (neuronal pentamericin-2, UniProt P47972), OLFML2B (olfylglobulin-like protein 2B, UniProt Q68BL8), TFPI2 (tissue factor pathway inhibitor 2, UniProt P48307), IFNE (interferon ε, UniProt Q86WN2), and prostaglandin F2-α receptor (PTGFR, prostaglandin FP receptor, UniProtP43088) ligands such as prostaglandin F2.Other mechanisms / reagents identified based on gene chip data that can promote the development from BAT progenitor cells to brown adipocytes include: peptides derived from the pituitary adenylate cyclase activating polypeptide (PACAP, ADCYAP1, UniProtP18509) gene, such as 55 aaa (aa 25-79) PACAP propeptide (200 nM-2 μM), CNTF (ciliary neurotrophic factor), interleukin-6 (IL-6, UniProt P05231), interleukin-15 (IL-15, UniProt P40933), CXCL12 (chemokine (CXC motif) ligand 12), stromal cell-derived factor 1 (SDF1, UniProt P48061) isotype SDF-1g / UniProt P48061-3) and / or atypical chemokine receptor 3 (ACKR3, CMKOR1, CXCR7, GPR159, RDC1, UniProt... Ligands of P25106 include SDF1 (CXCL12).

[0119] Screening for potential regulators of human UCP1 mRNA

[0120] CD31-cells can be used as a tool to identify agents (e.g., compounds, proteins, biopharmaceuticals, etc.) that induce these cells to differentiate into brown adipocytes or regulate UCP1 expression. For example, RT-PCR-based methods can be used to measure UCP1 mRNA levels, which can be affected by certain reagents.

[0121] This allows for the identification of reagents that can enhance the differentiation of CD31- cells into brown adipocytes and / or increase UCP1 expression by enhancing UCP1 gene transcription and / or by stabilizing UCP1 transcripts.

[0122] For example, PPARγ ligands such as rosiglitazone can be used to promote the differentiation of CD31-progenitor cells into brown adipocytes. Figure 1-19 (20A, 21, 22). Other examples use recombinant proteins, such as human BMP-7 (…). Figure 1-19 ,twenty one).

[0123] The powerful method disclosed prior in WO2013071063 and incorporated herein by reference, detects the differentiation of CD31- cells into brown adipocytes by simultaneously quantifying mRNA material corresponding to brown adipocyte markers UCP1, adipocyte marker PPARγ2, and the housekeeping gene cyclophilin A used as a control.

[0124] This method allows for the analysis of large numbers of samples to identify agents that enhance the differentiation of CD31- cells into brown adipocytes. Upon differentiation into brown adipocytes, CD31- cells express very high levels of UCP1 and PPARγ2 mRNA in response to a given level of cyclophilin A. UCP1 and PPARγ2 mRNA levels normalized to cyclophilin A mRNA levels indicate the degree of CD31- cell differentiation into brown adipocytes, regardless of the total cell count in the sample.

[0125] Therefore, the quantification of UCP1, PPARγ2 and cyclophilin A mRNA by multiplex TaqMan real-time PCR was used to quantify the differentiation of CD31- cells into brown adipocytes.

[0126] Using this method, identify or confirm that the following reagents promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro or in vivo, or both in vitro and in vivo: PPARγ ligands such as rosiglitazone, PDE3 inhibitors (e.g., cyanidazol), PDE4 inhibitors (e.g., cyclophosphamide), prostaglandin F2 (PGF2) derivatives such as 9β,11α-prostaglandin F2, and peptides derived from pituitary adenylate cyclase activating polypeptide (PACAP, ADCYAP1, UniProt P18509) genes such as 55aa(aa PACAP propeptide (25-79), BDNF (brain-derived neurotrophic factor), TGR5 agonists such as oleanolic acid, BMP-7, flavonoids (KMP, CAS number 520-18-3), soluble guanylate cyclase (sGC) stimulants such as rosigmatide (BAY 63-2521, CAS 625115-55-1), FGF7 (fibroblast growth factor-7, KGF, keratinocyte growth factor), FGF10 (fibroblast growth factor-10, KGF-2, keratinocyte growth factor-2), BNP (β-type natriuretic peptide), TRPM8 (CMRI) ligands such as menthol or eserin, bufotalin, CNTF (ciliary neurotrophic factor), interleukin-6 (IL-6), orexin B, SDF-1γ (CXCL12), and FGF13 (fibroblast growth factor-13).

[0127] Unless otherwise stated, all analytical and molecular biological grade organic and inorganic chemicals were purchased from Sigma Chemicals, Inc. (St. Louis, NY), Life Technologies, Inc. (Grand Island, NY), GenScript, Prospec, LifeTein, Inc., and AnaSpec. Rosiglitazone was purchased from Cayman Chemical (#71742) and recombinant human BMP7 (rhBMP7) was purchased from R&D Systems, Inc. (100 μg / ml, 6.3 μM, #354-BP-010).

[0128] Cell culture

[0129] Cells at 10,000 / cm 2 Cells were seeded on 0.2% gelatin-coated plates (48-well tissue culture, Chemglass#CLS-3500-048) and cultured at 37°C in endothelial cell growth medium-2 (EGM2) (Bullet Kit growth medium, Lonza#CC-3162) until passage (2-4 days) and until differentiation (an additional 10-16 days). After 2 or 3 days in EGM2, the medium was replaced with adipogenic medium to induce cell differentiation, which was modified according to Rodriguez et al.

[21] and may or may not contain a differentiation inducer (e.g., PPARγ agonist). The above MDM contains: a mixture of DMEM / Ham's F-1250 / 50 (3.151 g / L, 17.5 mM D-glucose, 3.651 g / L L-glutamine) (Cellgro#10-090-CV), 5 μg / ml (0.86 μM) insulin, 1 μM dexamethasone, 100 μM 3-isobutyl-1-methylxanthine, 0.2 nM 3,3',5-triiodo-L-methylformamide, 10 μg / ml (127 nM) transferrin, and 1% penicillin-streptomycin. If rosiglitazone is used as a differentiation inducer, it can be added at 1 μM or any other concentration sufficient to induce BAT progenitor cells to differentiate into brown adipocytes.

[0130] For cell expansion studies, fusion cells grown in EGM2 medium were detached at 37°C by treatment with trypsin-EDTA for 3-5 minutes, then separated at a 1:3 or 1:4 ratio and cultured as described above.

[0131] Quantification of UCP1 and PPARγ2 mRNA was performed using quantitative reverse transcription and real-time PCR.

[0132] Total RNA was prepared from cells using the PureLink RNA Isolation Kit (Invitrogen #12183-016). First-strand cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and random primers.

[0133] Quantitative real-time PCR was performed using the following: StepOnePlus, Applied Biosystems. TM Custom TaqMan gene expression probes and primers for instruments, TaqMan gene expression master mix (Applied Biosystems, Inc. #4369016), and human uncoupling protein-1 "UCP1" (GenBank NM_021833) and human peptidylprolyl isomerase A "cyclophilin A" (GenBank NM_021130). Customized TaqMan gene expression reagents were also developed for the simultaneous measurement of peroxisome proliferator-activated receptor γ, transcript variant 2 (PPARγ2) in multiplexed form (with UCP1 and cyclophilin A) (GenBank NM_015869): UCP1FAM-MGB probe: TCA AGG GGT TGG TAC CTT CC (SEQ ID NO.:1), positive primer: CAC TAA CGAAGG ACC AAC GG (SEQ ID NO.:2), and antisense primer: TTC CAG GAT CCA AGT CGC AA (SEQ ID NO.:3). Cyclophilin A NED-MGB probe: ACT GCC AAG ACT GAG TGG TT (SEQ ID NO.:4), positive primer: CAA ATG CTG GAC CCA ACA CA (SEQ ID NO.:5), and antisense primer: TCA CTT TGC CAA ACACCA CA (SEQ ID NO.:6). PPARγ2VIC-MGB probe: TCA CAA GAA ATG ACC ATG GTT G (SEQ ID NO.:7), positive primer: AGC GAT TCC TTC ACT GAT ACA C (SEQ ID NO.:8), and negative primer: CCAGAA TGG CAT CTC TGT GT (SEQ ID NO.:9).

[0134] Cyclophilin A was used as a control to account for any variations due to reverse transcription efficiency. Arbitrary units (based on Cts) were determined by normalizing target mRNA levels to cyclophilin A mRNA levels.

[0135] Statistical analysis

[0136] Data are presented as mean ± SEM. Significance was assessed using an unpaired Stein t-test. Significance was set at p < 0.05.

[0137] Cell morphology images

[0138] Cell images were taken using a handheld digital camera (Nikon Coolpix 950) and an inverted microscope (Nikon TMS) for cell culture observation; images were optimized using Adobe Photoshop Elements 8 with automatic contrast and automatic level.

[0139] The chapter headings and subheadings used herein are for organizational purposes only and are not intended to limit the subject matter in any way. Furthermore, while the invention has been described in conjunction with various embodiments, it is not intended to limit the invention to these embodiments. Rather, those skilled in the art will understand that the invention encompasses various alternatives, modifications, and equivalents.

[0140] Quantitative analysis of UCP1 protein

[0141] The differentiation of brown adipocyte progenitor cells into brown adipocytes can be detected by immunohistochemistry (IHC) quantification of UCP1 protein.

[0142] CD31- cells were cultured and differentiated into brown adipocytes using adipogenic differentiation medium lacking rosiglitazone (MDM) or containing 1 μM rosiglitazone (RDM). After 15 days of differentiation, cells were fixed in 4% paraformaldehyde PBS at pH 7.4 and incubated with UCP1 antibody (Abcam ab23841) and Alexafluor 488 goat anti-rabbit antibody to quantify relative UCP1 levels (green) according to a standard protocol. Cell nuclei were labeled with 5 μM MAPI (blue) for 10 minutes prior to cell fixation. Each treatment condition was evaluated three times in 96-well plates, corresponding to approximately 360–480 wells for each data point. InCell 1000 Developer Toolbox software was used to develop automated cell detection scripts for measuring UCP1 signal intensity, using both nuclear and cytoplasmic detection algorithms. As readings, the total intracellular UCP1 signal intensity was used, normalized to cell number.

[0143] In some embodiments, the reagents or combinations thereof identified using this technique include: famotidine, sulpiride hydrochloride, guanifacine hydrochloride, reserpine, minoxidil, spiroperidolone, difluorobenzyl salicylic acid, sicoplanin, probenecid, metformin, thiophenepiprozin, colchicine, and felodipine.

[0144] Detection of brown adipocyte differentiation

[0145] Neutral lipids labeled with the BODIPY fluorescent dye are incorporated into cytoplasmic lipid droplets, allowing for the analysis of cellular fatty acid uptake and adipogenesis differentiation via fluorescence cell imaging. Cells are used... Incubate at 500 / 510C12 (Molecular Probes #D-3823) for 3-6 hours, then incubate on a microplate-based high-throughput, high-content, bright-field, and fluorescence cell imaging and analysis system (Cyntellect). Imaging can be performed on a GE Healthcare InCell Analyzer.

[0146] Other implementation methods

[0147] The various aspects of this invention can be used alone, in combination, or in various arrangements not specifically discussed in the foregoing embodiments, and therefore their application is not limited to the details and arrangements of the components shown in the foregoing description or the accompanying drawings. For example, an aspect of one embodiment can be combined with aspects of other embodiments in any way.

[0148] The sequential terms “first,” “second,” “third,” etc., used in the claims to modify the elements do not imply any priority, precedence, or hierarchy of one element relative to another, or the temporal order of the multiple actions of the method, but are merely used as markers to distinguish one element with a certain name from another element with the same name (but only in terms of the use of sequential terms) to differentiate the multiple elements.

[0149] Furthermore, the words and terms used herein are for descriptive purposes and not restrictive. The use of “comprising,” “including,” or “having,” “containing,” “involving,” and variations thereof in this document means to cover the items listed thereafter and their equivalents, as well as additional items.

[0150] This invention provides novel compositions and others capable of recruiting brown adipocytes in vitro and in vivo. Although specific embodiments of the invention have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon reading this specification. The full scope of the invention should be determined by reference to the appended claims together with the full scope of their equivalents, and the description together with such variations.

[0151] Incorporated by reference

[0152] All publications, patents and patent applications used in this document are incorporated herein by reference in their entirety for all purposes, as if each publication, patent or patent application were specifically described and incorporated herein by reference.

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Claims

1. A method for recruiting brown adipocytes from BAT progenitor cells isolated from human skeletal muscle in vitro, wherein the method comprises culturing BAT progenitor cells in vitro in the presence of fibroblast growth factor (FGF) 7.

2. The method of claim 1, wherein FGF7 can induce the expression of UCP1, FABP4(aP2), PPARg2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells.

3. The method of claim 1, wherein FGF7 has one or more biological activities selected from the group consisting of: (a) Causes an increase or decrease in one or more of the following: β3-adrenergic receptor (β3-AR), solute carrier family 2, glucose transporter member 4 (SLC2A4), very long chain fatty acid protein 3 elongation (ELOVL3), CD36 antigen, type II iodinated thyroxine deiodinase (DIO2), BMP5, BMP6, FGF7, FGF10, FGF13, FGF21, fatty acid binding protein 7 (FABP7), CXCL12, atypical chemokine receptor 3 (ACKR3), insulin-like growth factor binding protein 4 (IGFBP4), pituitary adenylate cyclase activating peptide (PACAP), adenylate cyclase 4 (ADCY4), cell death activator CIDE-A (CIDEA), secretory coil-associated protein 1 (SRFP1), SRFP2, brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor D (VEGF-D), transforming growth factor β-2 (TGFB2), cAMP-specific 3',5'-cyclic phosphodiesterase 4B (PDE4B), cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D), high-affinity cAMP-specific 3',5'-cyclic phosphodiesterase 7A (PDE7A), and cAMP-specific 3',5'-cyclic phosphodiesterase 7B (PDE7B); (b) Causes increased heat production in brown adipose tissue and / or skeletal muscle tissue; (c) Causes increased insulin sensitivity in skeletal muscle, white adipose tissue, or liver; (d) Causes increased glucose tolerance; (e) Causes an increase in basal respiration, maximum respiratory rate, or uncoupled respiration; (f) Causes an increase in metabolic rate; and (g) Causes a reduction in hepatic steatosis.

4. The method of claim 3, wherein FGF7 causes an increase or decrease in one or more of the following: β3-adrenergic receptor (β3-AR), solute carrier family 2, glucose transporter member 4 (SLC2A4), very long chain fatty acid protein 3 elongation (ELOVL3), CD36 antigen, and type II iodinated thyroxine deiodinase (DIO2).

5. Use of the composition in the preparation of a medicament for the treatment of obesity, diabetes and insulin resistance in desired subjects, said composition comprising a differentiator composed of FGF7.

Images