Gut microbiome-derived metabolite(s) promote skeletal muscle adaptation to exercise

Abstract

Regular exercise yields a multitude of systemic benefits, many of which may be mediated through the gut microbiome. The present disclosure identifies that cecal microbial transplants (CMTs) from exercise-trained vs. sedentary animals provides benefits in reducing skeletal muscle atrophy using a mouse model of unilaterally hindlimb-immobilization. Direct administration of top microbial-derived exerkines from an exercise-trained gut microbiome preserves muscle function and prevented skeletal muscle atrophy.

Description

GUT MICROBIOME-DERIVED METABOLITE(S) PROMOTE SKELETAL MUSCLE ADAPTATION TO EXERCISE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application 63 / 652,985, filed May 29, 2024, the contents of which are hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under 5R21AG071888 and 1R01AR084282 awarded by National Institutes of Health (Department of Health and Human Services). The Government may have certain rights to the invention. BACKGROUND

[0003] The human gut microbiome is a collection of trillions of microorganisms inhabiting the gastrointestinal tract. These microorganisms act as a dynamic extension of the human host, serving many functions essential to the host’s well-being, and dysregulation of the microbiome can severely influence human health. Recent evidence has tied the gut microbiome and microbial-derived metabolites to phenotypic adaptations in skeletal and cardiac muscle. It has been reported that dysbiosis of the gut microbiome results in maladaptation in skeletal muscle in response to exercise-training, demonstrating the importance of the gut microbiome in skeletal muscle adaptation. Given the relationship between the gut microbiome and exercise adaptation, efforts have been dedicated to determine if the benefits of exercise are able to be conferred via the gut microbiome. SUMMARY

[0004] A 1staspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns a method for treating atrophied tissue in a subject comprising administering a cecal microbial transplant from an exercise-trained subject to the subject.

[0005] A 2ndaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1staspect, wherein the cecal microbial transplant comprises a microbiome from cecal tissue or extra-cellular matrix of cecal tissue of the exercise-trained subject.

[0006] A 3rdaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1stor 2ndaspect, wherein the cecal microbial transplant comprises one or more exerkines.

[0007] A 4thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1st, 2nd, or 3rdaspect, wherein the cecal microbial transplant comprises succinate and / or pipecolic acid.

[0008] A 5thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1staspect, wherein the cecal microbial transplant comprises succinate or pipecolic acid or a combination thereof.

[0009] A 6thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 5thaspect, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

[0010] A 7thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 5thaspect, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

[0011] An 8thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1staspect, wherein at least one other agent is administered to the subject.

[0012] A 9thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 8thaspect, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

[0013] A 10thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 1staspect, further comprising physical activity or exercise by the subject.

[0014] An 11thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns a method for treating muscle tissue in a subject comprising administering one or more exerkines to the subject.

[0015] A 12thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 11thaspect, wherein the one or more exerkines are derived from an exercised-trained subject.

[0016] A 13thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 11thor 12thaspect, wherein the one or more exerkines comprises succinate and / or pipecolic acid.

[0017] A 14thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 13thaspect, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

[0018] A 15thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 13thaspect, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

[0019] A 16thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 11thaspect, wherein at least one other agent is administered to the subject.

[0020] A 17thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 16thaspect, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

[0021] An 18thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 11thaspect, further comprising physical activity or exercise by the subject.

[0022] A 19thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns a method for treating muscle tissue in a subject comprising administering succinate and / or pipecolic acid to a subject in need thereof.

[0023] A 20thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 19thaspect, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

[0024] A 21staspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 19thaspect, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

[0025] A 22ndaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 19thaspect, wherein at least one other agent is administered to the subject.

[0026] A 23rdaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 22ndaspect, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

[0027] A 24thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the method of the 19thaspect, further comprising physical activity or exercise by the subject.

[0028] A 25thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns a composition for treating muscle tissue comprising succinate and pipecolic acid.

[0029] A 26thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the composition of the 25thaspect, wherein succinate is present to provide 50 to 200 mg / kg to a subject.

[0030] A 27thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the composition of the 25thaspect, wherein pipecolic acid is present to provide 100 to 300 mg / kg to a subject.

[0031] A 28thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the composition of the 25thaspect, further comprising at least one other agent.

[0032] A 29thaspect of the present disclosure, either alone or in combination with any other aspect as set forth herein, concerns the composition of the 28thaspect, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Fig. 1 shows cecal microbial transplants (CMT) from exercise-trained donors increase microbial abundance and diversity in exercise-naïve recipient mice.

[0034] Fig. 1A shows experimental design showing acclimatization period, timeline of cecal microbial transplants (CMT) from sedentary donors (DON-SED) and exercised donors (DON- EXR) into recipient mice (REC-SED, REC-EXR, respectively), and 10-day unilateral hindlimb immobilization.

[0035] Fig.1B shows effectiveness of CMT measured by percentage of recipient species present in donor.

[0036] Fig.1C shows microbial abundance in REC-SED and REC-EXR. *** = p < 0.001.

[0037] Fig.1D shows Alpha diversity (Chao1) in DON and REC groups. * = p < 0.05; ** = p < 0.01.

[0038] Fig.1E shows Beta diversity shown by principal coordinates analysis (PCoA).

[0039] Fig.1F shows a heat map of relative bacterial abundance in REC-SED and REC-EXR mice.

[0040] Fig. 1G shows a volcano plot showing differentially abundant species between REC-EXR vs REC-SED.

[0041] Fig. 1H shows linear discriminant analysis (LDA) score of predictive bacterial features.

[0042] Figs.1I and 1J shows abundance of Akkermansia muciniphila and Muribaculaceae bacterium DSM 103720 in recipient groups. **** = p < 0.0001.

[0043] Fig.2 shows cecal microbial transplants from exercise-trained donors ameliorate loss in muscle size and function during disuse atrophy induced by hindlimb immobilization.

[0044] Fig.2A shows average running volumes of exercise donor (DON-EXR) groups used for cecal microbial transplants (CMT).

[0045] Figs.2B and 2C show muscle mass of soleus and plantaris in control and casted legs normalized to body weight of mice receiving cecal microbial transplants (CMTs) from sedentary (REC-SED) or exercised (REC-EXR) donors after 10 days of hindlimb immobilization. **** = p < 0.0001.

[0046] Figs.2D and 2E show mean muscle fiber cross-sectional area (CSA) of soleus and plantaris muscles. ** = p < 0.01; **** = p < 0.0001.

[0047] Figs.2F and 2G show percent change in muscle fiber-type specific cross-sectional area (CSA) in soleus and plantaris muscles of REC-SED and REC-EXR groups. * = p < 0.05; ** = p < 0.01.

[0048] Figs.2H, 2I, 2J, and 2K show muscle fiber type proportions in soleus and plantaris muscles of REC-SED and REC-EXR groups. * = p < 0.05.

[0049] Fig.2L comparison of ex vivo soleus force-frequency relationship of REC-SED and REC-EXR groups.

[0050] Fig. 2M comparison of soleus rate of force development across stimulation frequencies of REC-SED and REC-EXR groups.

[0051] Fig.2N comparison of soleus fatigue resistance during repeated contractions of REC-SED and REC-EXR groups.

[0052] Fig.2O shows partial least squares discriminant analysis (PLS-DA) of metabolite profiles from cecal content, serum, and muscle of REC-SED and REC-EXR groups.

[0053] Fig.2P shows mean decrease accuracy of top exercise-associated features of REC- EXR group.

[0054] Fig.2Q shows correlation of pipecolic acid-associated features of REC-EXR group.

[0055] Figs.2R and 2S shows normalized abundance of pipecolic acid and succinate in cecal content, serum, and muscle of REC-SED and REC-EXR groups. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

[0056] Fig.3 shows microbial-derived exerkines pipecolic acid and succinate potentiate the positive effects of cecal microbial transplants on muscle size and function during disuse atrophy and enhance exercise adaptation by elevating exercise performance.

[0057] Fig. 3A comparison of soleus mass of hindlimb immobilized (HLI) leg and contralateral leg (CON) of vehicle (VEH), pipecolic acid (PIP), succinate (SUC), and pipecolic acid and succinate (PAS) treated groups. ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

[0058] Fig.3B comparison of soleus mean muscle fiber cross-sectional area (CSA) of HLI and CON legs of vehicle (VEH), pipecolic acid (PIP), succinate (SUC), and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05; ** = p < 0.01.

[0059] Figs.3C and 3D comparison of soleus type I and IIa muscle fiber CSA of HLI and CON legs of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0060] Figs.3E and 3F comparison of soleus type I and IIa muscle fiber proportions of HLI and CON legs of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups.

[0061] Fig.3G comparison of soleus muscle fiber CSA distribution of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups.

[0062] Fig.3H comparison of ex vivo soleus force-frequency relationship of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0063] Fig. 3I comparison of soleus rate of force development across stimulation frequencies of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0064] Fig. 3J comparison of soleus fatigue resistance during repeated contractions of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups.

[0065] Fig.3K comparison of in vivo plantarflexor force-frequency relationship of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0066] Fig.3L comparison of in vivo plantarflexor maximal torque production of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0067] Fig.3M comparison of soleus mean muscle fiber CSA following exercise with VEH or PAS of vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups. * = p < 0.05.

[0068] Fig. 3N comparison of weekly running distance during progressive weighted wheel running (PoWeR) while administered vehicle (VEH) and pipecolic acid and succinate (PAS) via drinking water.

[0069] Figs. 3O and 3P comparison of ex vivo soleus force-frequency relationship and rate of force development following PoWeR training with vehicle (VEH) and pipecolic acid and succinate (PAS).

[0070] Fig.4 shows microbial-derived exerkines pipecolic acid and succinate elicit their effects through the stabilization of cell energetics, reduction of nucleotide degradation, and preservation of ribosomal integrity.

[0071] Fig. 4A shows a volcano plot of differentially expressed proteins (DEPs) in the gastrocnemius muscle between vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups.

[0072] Figs.4B, 4C, and 4D show enrichment analysis of upregulated and downregulated pathways of DEPs.

[0073] Figs.4E and 4F show protein abundance of eukaryotic translation initiation factor 3 subunit E (EIF3E) and DDB1 and CUL4 associated factor 8 (DCAF8) of VEH and PAS groups. * = p < 0.05.

[0074] Fig. 4G shows pathway analysis of differentially expressed metabolites using untargeted metabolomics.

[0075] Figs. 4H, 4I, and 4J show metabolite abundance of pipecolic acid, adenosine triphosphate (ATP), and hypoxanthine using untargeted metabolomics of VEH and PAS groups. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001.

[0076] Figs.4K and 4L show mitochondrial complex II / III and IV activity of VEH and PAS groups.

[0077] Fig.4M shows C2C12 myotubes with ATP reporter (iATP) following treatment with VEH or PAS.

[0078] Fig. 4N shows ATP abundance in C2C12 myotubes treated with vehicle (VEH), succinate (SUC), pipecolic acid (PIP), or pipecolic acid and succinate (PAS). *** = p < 0.001; **** = p < 0.0001.

[0079] Fig.4O shows citrate synthase (CS) activity in C2C12 myotubes treated with VEH, SUC, PIP, or PAS. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

[0080] Fig. 4P shows representative rRNA traces from vehicle (VEH) and pipecolic acid and succinate (PAS) treated muscles.

[0081] Fig.4Q shows rRNA integrity measured by 28S:18S ratio from vehicle (VEH) and pipecolic acid and succinate (PAS) treated muscles.

[0082] Fig.4R shows proportion of rRNA to total RNA from vehicle (VEH) and pipecolic acid and succinate (PAS) treated muscles. ** = p < 0.01.

[0083] Fig. 4S shows proposed mechanism of action for pipecolic acid and succinate (PAS) treatment in preventing muscle atrophy. ASSA = L-2-alpha-aminoadipic acid 6- semialdehyde; P2C = Δ1-piperideine 2-carboxylate; α-KG = α-ketoglutarate.

[0084] Fig. 5 shows a comparison of sedentary donor (DON-SED) and exercised donor (DON-EXR) gut microbiomes.

[0085] Fig. 5A shows the relative abundances of cecal microbial species between sedentary donors (DON-SED) and exercised donors (DON-EXR).

[0086] Fig.5B shows microbial abundance of DON-SED and DON-EXR groups.

[0087] Fig.5C shows Alpha diversity of DON-SED and DON-EXR groups.

[0088] Fig.5D shows Beta diversity of DON-SED and DON-EXR groups.

[0089] Fig. 5E shows a volcano plot showing differentially abundant species between DON-EXR vs DON-SED.

[0090] Fig. 5F shows linear discriminant analysis (LDA) score of predictive bacterial features in DON-SED and DON-EXR.

[0091] Fig.5G and 5H show abundance of Akkermansia muciniphila and Muribaculaceae bacterium DSM 103720 in DON-SED and DON-EXR groups. **** = p < 0.0001.

[0092] Fig.6 shows comparison of DON-SED and REC-SED gut microbiomes.

[0093] Fig. 6A shows the relative abundances in cecal microbial species between sedentary donors (DON-SED) and recipients of sedentary donor cecal microbial transplants (REC- SED).

[0094] Fig.6B shows microbial abundance of DON-SED and REC-SED groups.

[0095] Fig.6C shows Alpha diversity of DON-SED and REC-SED groups. ** = p < 0.01.

[0096] Fig.6D shows Beta diversity of DON-SED and REC-SED groups.

[0097] Fig. 6E shows a volcano plot showing differentially abundant species between REC-SED vs DON-SED.

[0098] Fig. 6F shows linear discriminant analysis (LDA) score of predictive bacterial features in DON-SED and REC-SED.

[0099] Figs. 6G and 6H show abundance of Akkermansia muciniphila and Dubosiella newyorkensis in SED groups. ** = p < 0.01; *** = p < 0.001.

[0100] Fig.7 shows a comparison of DON-EXR and REC-EXR gut microbiomes.

[0101] Fig. 7A shows the relative abundances in cecal microbial species between the exercised donors (DON-EXR) and recipients of exercised donor cecal microbial transplants (REC- EXR).

[0102] Fig.7B shows microbial abundance of DON-EXR and REC-EXR groups.

[0103] Fig.7C shows Alpha diversity of DON-EXR and REC-EXR groups.

[0104] Fig.7D shows Beta diversity of DON-EXR and REC-EXR groups.

[0105] Fig. 7E shows a volcano plot showing differentially abundant species between REC-EXR vs DON-EXR.

[0106] Fig. 7F shows linear discriminant analysis (LDA) score of predictive bacterial features in DON-EXR and REC-EXR.

[0107] Figs.7G and 7H show abundance of Akkermansia muciniphila and Muribaculaceae bacterium DSM 103720 in EXR groups. * = p < 0.05.

[0108] Fig.8 shows validation of the hindlimb immobilization model.

[0109] Fig.8A shows a 3D-printed cast design and application.

[0110] Figs.8B, 8C, and 8D show muscle mass of soleus, plantaris, and gastrocnemius at 5, 7, and 10 days of immobilization normalized to body weight. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

[0111] Figs. 8E and 8F show percent change in soleus and plantaris mean muscle fiber cross-sectional area (CSA) with immobilization duration. * = p < 0.05; *** = p < 0.001.

[0112] Figs.8G, 8H, and 8I show tibialis anterior (TA) muscle mass and cross-sectional area (CSA) following 10 days and 4 weeks of immobilization.

[0113] Fig.9 shows plantaris muscle mass, fiber size, and fiber type proportion in VEH and PAS treated groups.

[0114] Fig. 9A shows the comparison in plantaris mass following 10-day hindlimb immobilization in vehicle (VEH) and pipecolic acid and succinate (PAS) groups. ** = p < 0.01; *** = p < 0.001.

[0115] Fig.9B shows the comparison in plantaris mean muscle fiber cross-sectional area (CSA) in vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups during 10 days of unilateral immobilization. ** = p < 0.01.

[0116] Figs.9C and 9D show the comparison in plantaris type IIa and IIb / x muscle fiber CSA in vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups during 10 days of unilateral immobilization. * = p < 0.05; ** = p < 0.01.

[0117] Figs.9E and 9F shows the comparison in plantaris type IIa and IIb / x muscle fiber proportions in vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups during 10 days of unilateral immobilization. * = p < 0.05.

[0118] Fig. 10 shows the effects of pipecolic acid and succinate (PAS) treatment on mTORC1 signaling.

[0119] Figs.10A, 10B, and 10C show protein abundance of phosphorylated AKT (pAKT) and total AKT (tAKT) with vehicle (VEH) and pipecolic acid and succinate (PAS) treatment.

[0120] Figs 10D, 10E, and 10F show protein abundance of phosphorylated p70S6K (p- p70S6K) and total p70S6K (t-p70S6K) with vehicle (VEH) and pipecolic acid and succinate (PAS) treatment.

[0121] Fig. 11 shows metabolomic profiling of gastrocnemius muscles of VEH and PAS treated groups.

[0122] Fig. 11A shows a principal component analysis of metabolite profile between vehicle (VEH) and pipecolic acid and succinate (PAS) treated muscles.

[0123] Fig. 11B shows a volcano plot of differentially expressed metabolites in vehicle (VEH) and pipecolic acid and succinate (PAS) treated muscles.

[0124] Fig.12 shows mitochondrial complex protein expression following pipecolic acid and succinate (PAS) treatment.

[0125] Figs.12A, 12B, 12C, 12D, and 12E shows the comparison in protein abundance of mitochondrial complexes I-V in vehicle (VEH) and pipecolic acid and succinate (PAS) treated groups.

[0126] Fig.13 shows ATP Reporter validation.

[0127] Fig.13A shows iATP green fluorescence in C2C12 myotubes under different fed and starved conditions.

[0128] Fig.13B shows experimental design for C2C12 myotube adenosine triphosphate reporter (iATP) assay. MOI = multiplicity of infection. DESCRIPTION

[0129] The present disclosure concerns, in part, administration of exerkines to a subject. As shown herein, administration of exerkines can prevent disuse-atrophy and preserve muscle function. In some aspects, the present disclosure concerns administering the microbiome or the extract thereof of an active subject to a second subject to prevent disuse atrophy and / or preserve muscle function. In some aspects, the microbiome includes exerkines as described herein. It is an aspect of the present disclosure that exerkines produced in the microbiome of the gut of active subjects is of benefit to the muscle, such that the exerkines or material including the exerkines can be administered to subjects in need to assist preserve and / or benefit their muscle tissue.

[0130] In some aspects, the present disclosure includes administering one or more microbial-derived exerkines (MDEs) to a subject. In some aspects, the methods include administering an MDE of at least one of pipecolic acid and / or succinate or a combination thereof to a subject. As set forth herein, administration of these MDEs prevents muscle atrophy and / or preserves muscle function. As set forth in the working examples, oral administration of two key MDEs, pipecolic acid and succinate, prevented disuse-induced atrophy and preserved muscle function in exercise-naïve mice, possibly by preserving cellular energy status and enhancing translational capacity. The findings of the studies herein further define the gut microbiome- skeletal muscle axis and provide the first evidence of MDEs which represent a novel class of exercise mimetics for the treatment of exercise-responsive conditions.

[0131] In some aspects, the present disclosure concerns administration of exerkines to a subject to address muscle tissue issues therein as is described herein. In some aspects, MDEs or exerkines can be administered through transplantation, such as through cecal transplant or cecal microbiome transplant from a subject that is active or exercise-trained. In some aspects, the transplanted material can be prepared and / or purified to isolate the microbiome or concentrate the microbes from the extracted tissue. In some aspects, the extracted tissue can be processed to extract the exerkines. In some aspects, the methods can include administration of exerkines, either derived from the active subject or synthetic or mimetic thereof, such as with the administration of succinate and / or pipecolic acid.

[0132] In some aspects, the present disclosure concerns administration of MDEs or exerkines such as succinate and / or pipecolic acid to a subject. In some aspects, the succinate and / or pipecolic acid is derived from a cecal microbiome. In some aspects, the succinate and / or pipecolic acid is derived synthetically or commercially. In some aspects, succinate is administered to the subject at about 50 to 150 mg / kg / day, including about 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, and 145 mg / kg / day (± 1 / 2 / 3 / 4 mg). In some aspects, pipecolic acid is administered to the subject at about 100 to about 300 mg / kg / day including about 110, 120, 1330, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 mg / kg / day (± 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 mg). For example, as set forth in the examples, the subjects were administered 112.3 ± 26.3 mg / kg / d and 222.8 ± 51.4 mg / kg / d of succinate and pipecolic acid, respectively, and responded positively.

[0133] In some aspects, the present disclosure concerns administering or placing transplanted tissue or extract thereof into a subject. In some aspects, the present disclosure concerns administering to a muscle tissue, either directly or indirectly, cecal microbiome transplant tissue or extract thereof or exerkines derived thereof or succinate and / or pipecolic acids or combinations thereof. The administration can be to a muscle cell or a collection thereof, such as a culture or a tissue. The administration can be either directly or indirectly, such as through the blood stream, to the extracellular matrix of a muscle cell or cells, such that the muscle cell(s) can thereby come into contact and interact. Administration can be either direct or through other known routes such as intravenous, intramuscular, sublingual, subcutaneous, and so forth.

[0134] In some aspects, the administration may include one or more additional agents. Such may include a carrier, such as a pharmaceutically acceptable carrier, an excipient, a vitamin, a surfactant, and / or a pharmaceutical compound or agent. The administration may by in combination, either directly, simultaneously, indirectly, or in sequence with another agent. Such may include agents to target muscle ailments or issues thereof. Such may include causative ailments for muscle issues or ailments and / or symptoms downstream from muscle issues. Such may include steroids, glucocorticosteroids, amino acids, protein, peptides, and / or growth hormone. Such may include administration of stem cells.

[0135] In some aspects, the administration to the subject may be accompanied by a therapy, such as physical therapy and / or exercise. As disclosed herein, the administration can have beneficial effects on muscle tissue. However, coupling the administration with physical exertion will allow endogenous exerkine production as well and provide the opportunity to enhance the effects.

[0136] In some aspects, the present disclosure provides for methods of manipulating the gut biome to affect skeletal muscle. As set forth in the working examples, experiments were designed to interrogate whether gut microbiome manipulations are able to ameliorate skeletal muscle atrophy during disuse. cecal microbial transplants (CMTs) to determine if the gut microbiome of exercise-trained mice could ameliorate muscle atrophy of exercise-naive mice subjected to unilateral hind limb immobilization (HLI) (Fig.1A). The gut microbiome is comprised of the trillions of microorganisms found within the gastrointestinal tract and functions through a dynamic symbiotic relationship to directly impact the health and well-being of the host.Dysregulation of this complex ecosystem can significantly impair host health. Recent evidence has established an interaction between the gut microbiome and skeletal muscle through microbial-derived metabolites. It was reported that dysbiosis of the gut microbiome impaired skeletal muscle adaptation to exercise training. Further, it has been shown that the direct administration of microbial-derived metabolites has beneficial effects on skeletal muscle, underscoring the gut microbiome's importance in muscle adaptation.

[0137] The purpose of this study was to test the hypothesis that, in response to exercise training, the gut microbiome produces exerkines which can be used to treat skeletal muscle atrophy. The study was motivated by the finding that a healthy gut microbiome is necessary for skeletal muscle adaptation to exercise training, presumably through the production of exerkines. To test the hypothesis, cecal microbial transplants (CMTs) were first performed using sedentary donors (DON-SED) or exercise-trained donors (DON-EXR) into exercise-naïve recipient mice (REC- SED and REC-EXR, respectively). Upon finding skeletal muscle disuse atrophy was significantly less in REC-EXR group compared to RED-SED group, untargeted metabolomics was used to identify candidate microbial-derived exerkines (MDEs). The administration of two MDEs (pipecolic acid and succinate) during hind limb immobilization prevented muscle atrophy while preserving muscle function. The effectiveness of MDEs to maintain muscle mass and function during a period of disuse demonstrates their therapeutic potential and establishes a novel class of exercise mimetics. Given the deep richness of the gut microbiome, this work is presented with the expectation that many more MDEs capable of treating exercise-responsive conditions remain to be discovered.

[0138] Cecal Microbial Transplants from Exercise-Trained Donors Increase Microbial Abundance and Diversity in Exercise-Naïve Recipient Mice

[0139] We performed cecal microbial transplants (CMTs) to determine if the gut microbiome of exercise-trained mice could ameliorate muscle atrophy of exercise-naive mice subjected to unilateral hind limb immobilization (HLI) (Fig.1A). Metagenomic sequencing of the cecal contents from donor and recipient groups showed that CMTs resulted in 81.4 ± 6.7% and 79.6 ± 3.8% of donor microbial species were transferred to REC-SED and REC-EXR, respectively, thus demonstrating the effective engraftment of the donor microbiome (Fig.1B) comparable to levels achieved in previous studies. Total microbial abundance was significantly higher in REC- EXR versus REC-SED (p < 0.001) (Fig.1C). Alpha diversity was also higher in REC-EXR comparedto REC-SED (p < 0.01) (Fig.1D). Principal component analysis revealed notable differences in beta diversity between REC-SED and REC-EXR groups (p < 0.01) (Fig. 1E). The taxa abundance heat map further highlights these divergent microbial profiles between the two recipient groups (Fig. 1F). Volcano plot analysis of differentially abundant species showed several significantly altered bacterial populations when comparing REC-EXR versus REC-SED (Fig. 1G). Linear discriminant analysis (LDA) identified key predictive bacterial features distinguishing the recipient groups, with Akkermansia muciniphila showing the strongest association with the REC-EXR group (Fig. 1H). Quantification confirmed significantly higher abundance of both Akkermansia muciniphila (p < 0.0001) (Fig.1I) and Muribaculaceae bacterium DSM 103720 (p < 0.0001) (Fig.1J) in REC- EXR compared to REC-SED mice. For detailed metagenomic comparison between DON, SED, and EXR groups, see Fig.5-7.

[0140] Together, these findings suggest that the transplantation of cecal content from sedentary and exercised hosts into exercise-naïve recipients is sufficient to recapitulate the donor gut microbiome in recipient mice. Moreover, given the accentuated differences in beta diversity between REC groups relative to differences between DON groups (Fig.1E, 5D, 6D, 7D), as well as maintenance of alpha diversity and microbial abundance during transplantation between EXR groups (Fig.1C-D, Fig.5B-C, 6B-C, 7B-C), these data suggest that the differences in microbial profile between donor SED and EXR mice are accentuated following transplantation.

[0141] Cecal Microbial Transplants from Exercise-Trained Donors Ameliorate Loss in Muscle Size and Function During Disuse

[0142] After five weeks of CMT treatment, muscle atrophy was induced through 10 days of unilateral HLI via casting (Fig.1A) using a 3-D printed cast (Fig.8A). The 10-day time point was chosen due to its ability to induce consistent decreases in muscle mass and cross-sectional area (CSA) in the muscles of the posterior compartment of the lower leg (i.e., soleus, plantaris, and gastrocnemius) (Fig.8B-F). The anterior compartment of the lower leg was not impacted by HLI, as there was no change in normalized tibialis anterior muscle mass or fiber CSA (Fig.8G-I), thus assessments focused to the muscles of the posterior compartment.

[0143] Prior to CMT, cecal contents were pooled from groups of three donors matched by total distance ran during the exercise protocol to eliminate exercise volume as a confounding factor (Fig. 2A). Soleus and plantaris (a slow- and fast-twitch muscle, respectively) mass were decreased in both REC-SED and REC-EXR groups following HLI (p < 0.0001) (Fig. 2B-C). Meansoleus muscle fiber CSA was lower with HLI in both REC-SED (-42.7 ± 4.2%) and REC- EXR (-27.7 ± 13.9%) (p < 0.0001) (Fig. 2D), and average plantaris muscle fiber CSA was lower by -29.6 ± 14.7% (p < 0.01) and -20.3 ± 11.0% (p < 0.01), respectively (Fig. 2E). However, there was a significant interaction effect (p < 0.05) (Fig. 2D) between HLI and CMT in the soleus such that skeletal muscle atrophy was 35.1% lower on average in REC-EXR compared to REC-SED (p < 0.01), an effect which is seen in the CSA of both the type I (42.6%; p < 0.05) and IIa (20.6%; p = 0.06) muscle fibers (Fig.2F). There was a trend for a greater reduction in plantaris muscle mean fiber CSA (31.6%; p = 0.09) in REC-EXR, although this was not as apparent in the CSA of type IIa (14.9%) or type IIb muscle fibers (25.3%) (Fig.2G). Other than a small decrease in the proportion of type IIa muscle fibers in the REC-SED group following HLI, there were no changes in muscle fiber type composition in either the soleus or plantaris muscles (Fig.2H-K). These results demonstrate that CMT from exercise-trained mice is able to blunt muscle atrophy in the soleus muscle, with more mild reductions in muscle atrophy of the plantaris muscle.

[0144] To assess whether the reduction in skeletal muscle atrophy resulted in functional outcomes, ex vivo muscle function analyses of the soleus was performed following HLI. The soleus was excised and placed in a myograph where it was stimulated at frequencies ranging from 10-120Hz to assess strength, then stimulated at 60Hz for 150 repeated contractions separated by 2 seconds to assess fatigue resistance. There were no significant differences in specific force generation at any stimulation frequency (Fig. 2L). However, the rate of force development (RFD) was higher across the stimulation battery in the REC-EXR (area under curve p < 0.05) (Fig. 2M), suggesting that the REC-EXR group had accelerated force generation. Moreover, REC-EXR maintained a higher force production relative to the first contraction during the fatigue protocol (area under curve p < 0.0001) (Fig.2N), suggesting that CMTs from the DON- EXR resulted in greater fatigue resistance in the soleus. Together, these data demonstrate that the structural differences between REC-SED and REC-EXR were accompanied by improved muscle function in REC-EXR relative to REC-SED.

[0145] To identify candidate microbial-derived muscle-preserving metabolites transferred through CMT, untargeted metabolomics on cecal content, serum, and gastrocnemius muscle was performed from REC-SED and REC-EXR. Partial Least-Squares Discriminant Analysis (PLS-DA) revealed distinct metabolite profiles between REC-SED and REC-EXR across all sample types (i.e., cecal, serum, muscle) (Fig.2O), demonstrating that transplantation of the exercise-trained gut microbiome alters peripheral tissues in recipient mice, even without exercise. Integrating metabolomic data from all sample types, mean decrease accuracy (MDA) analyses was performed via random forest machine learning to determine predictive accuracy and metabolite importance. This analysis identified pipecolic acid, a lysine metabolite, as the top microbial-derived exerkine (MDE) predictive of REC-EXR (Fig. 2P). Succinate, a TCA cycle intermediate, was among the top MDEs and correlated strongly with pipecolic acid (Fig.2P-Q), along with 2-oxindole-3-acetate. Given the higher level of pipecolic acid and succinate in all sample types (Fig.2R-S), it was sought to establish the efficacy of using MDEs pipecolic acid and succinate to treat muscle atrophy.

[0146] Microbial-Derived Exerkines Pipecolic Acid and Succinate Potentiate the Positive Effects of Cecal Microbial Transplants on Muscle Size and Function During Disuse and Promote Exercise Adaptation

[0147] In order to determine the efficacy of purified MDEs in replicating the benefits of CMTs from exercised donors, pipecolic acid and succinate were administered via edible hydrogels, either separately (PIP and SUC) or in combination (PAS), for 10 days during HLI. Soleus mass decreased in every group (p < 0.01 to 0.0001) (Fig.3A); however, soleus mean muscle fiber CSA was maintained by PAS treatment (Fig.3B) unlike vehicle (VEH) (p < 0.01), PIP (p < 0.01), and SUC groups (p < 0.05). Similar results were observed in the plantaris muscle (Fig. 9A-F). PAS treatment maintained both type I and type IIa CSA of the soleus muscle (Fig. 3C-D) with no changes in muscle fiber-type composition (Fig. 3E-F). Frequency histograms of soleus muscle fiber size revealed a shift towards larger fiber sizes in the PAS group (Fig. 3G). Ex vivo muscle function analyses showed that PAS treatment resulted in significantly higher soleus strength (area under curve p < 0.0001), RFD (area under curve p < 0.0001), and fatigue resistance (area under curve p < 0.0001) relative to VEH (Fig.3H-J). The effects of PAS on muscle function were more robust than those of EXR CMTs, demonstrating the direct administration of purified MDEs potentiated the positive effects of CMT. Additionally, in vivo muscle function analyses were performed by stimulating the plantarflexors (i.e., soleus, plantaris, and gastrocnemius) at frequencies ranging from 10-200Hz. PAS treatment preserved in vivo torque production at 80- 200Hz (p < 0.05; area under curve p < 0.0001) (Fig.3K), as well as maximal torque production (p < 0.05) (Fig.3L), indicating the preservation of the function of entire muscle groups (i.e., plantar flexors) in their native environments. These results demonstrate that MDEs from an exercise-trained gut microbiome can prevent skeletal muscle atrophy and preserve muscle function when directly administered to mice undergoing HLI. Moreover, these data provide convincing evidence that the targeted administration of key microbiome-derived metabolites not only replicate the positive effects of CMTs from exercised donors, but further enhance the beneficial effects, demonstrating exciting therapeutic potential.

[0148] Given the ability of PAS administration to preserve muscle mass and function with disuse atrophy, it was considered if PAS treatment might improve exercise adaptation and performance. Mice were administered either VEH or PAS or via drinking water during six weeks of exercise-training that was modified from a progressive weighted wheel running (PoWeR)Am J Physiol Cell Physiol 316, C649-C654 (2019). doi.org / 10.1152 / ajpcell.00050.2019). Weights (2g / week) were progressively added to a running wheel until the mice were running with an additional 12g during the final week of the protocol. Soleus muscle mean fiber CSA was significantly larger in the PAS group (p < 0.05) (Fig. 3M), indicating a greater hypertrophic response to exercise training compared to VEH group. PAS administration resulted in the maintenance of weekly running volume relative to the first week, while VEH running volume declined (area under curve p < 0.05) (Fig.3N). Thus, the enhanced hypertrophic growth of the PAS group likely reflected this higher training stimulus of mice in the PAS group. The ex vivo muscle function analyses described above were repeated, finding that specific force was unchanged (area under curve p = 0.35) while RFD (area under curve p < 0.05) was higher in mice receiving PAS relative to those receiving VEH (Fig.3O-P). Since the force data is normalized to CSA, it is unsurprising that specific force did not change given the robust increase in CSA with PAS administration. These results demonstrate that PAS administration not only prevented atrophy during disuse but promoted muscle hypertrophy by enhancing exercise capacity.

[0149] Microbial-Derived Exerkines Pipecolic Acid and Succinate Elicit Their Effects Through the Stabilization of Cell Energetics, Reduction of Nucleotide Degradation, and Preservation of Ribosomal Integrity

[0150] To elucidate the mechanism through which PAS treatment exerts its effects on skeletal muscle, we began by performing proteomics on the HLI gastrocnemius muscles from VEH and PAS treated groups. Proteomics analysis revealed 308 differentially expressed proteins (DEPs, p < 0.10): 169 upregulated and 139 downregulated (Fig.4A). To interpret the biologicaland functional implications of these DEPs, Enrichr for gene ontology and pathways enrichment analysis was utilized (Fig.4B-D). Upregulated DEPs showed significant enrichment in cytoplasmic ribosomal proteins, cap-dependent translation initiation, and translation (Fig. 4B). These included numerous structural ribosomal proteins (e.g., RPL3L, RPL4, RPL11, RPL17, RPL22, RPL23A, RPL29, RPL32, RPS16, RPS25), ribosome associated proteins (e.g., RPS6KA), and translation initiation factors (e.g., EIF3C, EIF3D, EIF3E). Notably, EIF3E, a subunit of eukaryotic initiation factor 3 (eIF3) crucial for maintaining sarcomeric structure in skeletal muscle, was higher with PAS treatment (p < 0.05) (Fig.4E). Moreover, upregulated DEPs were enriched for processes such as muscle / striated muscle contraction and muscle / actin-myosin filament sliding, including proteins such as MYH3, MYH4, MYH7, MYH8, TPM1, TPM2, TPM4, ACTA1, and ACTA2 (Fig.4B-C). Downregulated DEPs were enriched for pathways involved in protein metabolism and post-translational protein modification (Fig.4D).

[0151] Notably, DCAF8, a known MuRF1 binding partner that promotes muscle atrophy through proteasomal degradation of sarcomeric proteins, was downregulated (p < 0.05) (Fig.4F). Taken together, these proteomic data suggest that PAS treatment mitigates atrophy by maintaining sarcomeric protein expression, potentially through enhanced protein synthesis and reduced protein degradation. Next, it was assessed whether PAS influenced mTOR signaling as a means of preserving muscle size during atrophy. There were no differences in the expression or phosphorylation of AKT or p70S6K (Fig. 10A-F), suggesting that mTORC1 signaling is not implicated in the mechanism of PAS.

[0152] To further elucidate the effects of PAS treatment on skeletal muscle during atrophy, untargeted metabolomics was performed on the HLI gastrocnemius muscles from VEH and PAS treated groups. There were clear distinctions between VEH and PAS treated groups (Fig. 11A). Pipecolic acid was the top elevated metabolite in the PAS group (Fig. 11B), confirming successful delivery to skeletal muscle. Pathway analysis of differentially expressed metabolites indicated enrichment of the lysine breakdown pathway, of which pipecolic acid is involved, as well as CoA and purine metabolism pathways in the PAS group (Fig. 4G). Along with higher pipecolic acid levels (p < 0.0001) (Fig.4H), significantly higher levels of ATP was observed with PAS treatment (p < 0.05) (Fig. 4I), suggesting increased energy availability. Moreover, hypoxanthine, an adenine nucleotide degradation product, was lower in PAS-treated muscle (p < 0.01) (Fig.4J). This is noteworthy as increased adenine nucleotide degradation is associatedwith skeletal muscle atrophy. Mitochondrial respiration was assessed, finding that the activity of mitochondrial complexes II (p = 0.07) and IV (p = 0.09) (Fig.4K-L) were non-significantly higher in the PAS group, although no accompanying increases in the expression of any of the 5 OXPHOS complexes were observed (Fig.12A-E).

[0153] To confirm the findings demonstrating higher cellular ATP levels with PAS treatment, C2C12 myotubes were cultured and delivered a fluorescent ATP reporter (iATP) via a muscle specific adeno-associated virus (MyoAAV). It was validated that this reporter allowed for the detection of ATP levels in vitro in live cells using 2-deoxy-glucose which cannot be utilized for ATP production (Fig.13A). iATP+ myotubes were then treated with VEH, SUC, PIP, or PAS for three days (Fig.13B) and quantified fluorescence to determine ATP levels. ATP increased with PIP treatment relative to VEH (p < 0.001), and PAS treatment increased ATP abundance more than all other treatments (p < 0.0001) (Fig.4M-N). This increase in ATP was accompanied by an increase in citrate synthase activity in PIP (p < 0.01) and PAS (p < 0.001) relative to VEH (Fig.4O).

[0154] Given that up to 90% of cellular RNA is rRNA, lower hypoxanthine with PAS treatment suggests that PAS decreased degradation of rRNA. To assess RNA integrity, the ratio of 28S to 18S rRNA was measured (Fig.4P). The 28S to 18S rRNA ratio showed a statistical trend favoring PAS-treated muscle (p = 0.06) (Fig.4Q), suggesting lower levels of rRNA degradation. Additionally, the proportion of rRNA relative to total RNA was significantly higher in PAS relative to VEH (p < 0.01) (Fig. 4R). Ribosome biogenesis is an energy-intensive process, and rRNA transcription is tightly coupled to cellular energy status. The observed higher levels of ribosomal proteins, combined with the metabolomic and rRNA data, suggest that PAS treatment may preserve skeletal muscle size and function through the preservation of cellular energy status, the reduction of nucleotide degradation, and the maintenance of ribosomal integrity and enhancement of translational capacity.

[0155] This disclosure presents several findings that advance understanding of the impact that an exercise-trained gut microbiome has on host organ adaptation. First, it is demonstrated that microbiome transfers from exercise-trained donors ameliorate skeletal muscle disuse atrophy and help preserve muscle function during periods of disuse. Next, using an integrative multi-omics approach, key MDEs – pipecolic acid and succinate – are identified with therapeutic potential due to their association with an exercise-trained gut microbiome. Upon direct administration, these MDEs were able to preserve skeletal muscle mass and functionduring disuse, possibly by maintaining sarcomeric protein expression, preserving cellular energy status, and enhancing translational capacity. Studies have demonstrated the ability of an exercise-trained microbiome to confer the benefits of exercise to exercise-naïve hosts. The findings herein support these data and demonstrate that an exercise-trained gut microbiome can elicit phenotypic effects on adult skeletal muscle consistent with exercise preconditioning.

[0156] Likewise, microbial products have been shown to elicit therapeutically relevant effects on host tissues, including skeletal muscle. The work herein specifically identifies microbial products from an exercise-trained microbiome which confer the benefits of exercise phenotypically (i.e., prevent atrophy during disuse) and functionally (i.e., preserve muscle strength, power, and fatigue resistance), thus indicating widespread therapeutic potential and setting the stage for novel drug discovery.

[0157] A concept supported by the findings herein is that the benefits of regular exercise are mediated by exercise-induced changes to the gut microbiome. Studies have assessed the effectiveness of microbial transplants from exercise donors in altering glucose metabolism, obesity, colitis, and exercise capacity. By transplanting microbiota from exercise-responsive and non-responsive humans into mice, Liu et al. demonstrated that the metabolic signature of the donors was replicated in the recipient mice (Cell Metab 31, 77-91 e75 (2020)). This study showed the potential of conferring the metabolic benefits of exercise through the gut microbiome. Herein it is established that microbial transplants from exercise-trained donors affect recipient skeletal muscle in a manner consistent with exercise adaptation, resulting in clinically relevant improvements in muscle function. The work herein further demonstrates the potential of leveraging an exercise-trained gut microbiome to confer the benefits of exercise, a therapeutic avenue which could significantly impact populations with exercise limitations.

[0158] Many factors influencing the success of fecal / cecal microbial transplantation have been postulated, including sample preparation, dosing, and delivery and recipient immune function and gut microbiome composition. Transplantation success is also dependent on the microbial diversity and composition of donor samples, leading to the proposition of so-called “super-donors.” Herein it is demonstrated that donor exercise improves CMT efficiency as demonstrated by the maintenance of microbial abundance and diversity after transplantation relative to sedentary donors. Therefore, it is possible that donor exercise provides a beneficial stimulus for improving the therapeutic efficacy of microbial transplants.

[0159] While transplanting microbiomes from exercised donors has therapeutic potential, a primary goal of this study was to further identify and test the effects of direct administration of the bioactive microbial metabolites. Recently, there have been several microbial-derived metabolites identified with positive effects on skeletal muscle. Urolithin A, a compound derived from the microbial metabolism of ellagitannins, has been shown to improve strength and exercise performance through the regulation of mitophagy in skeletal muscle. Oral supplementation of urolithin A showed promise in improving strength and performance in humans through the improvement of mitochondrial function, although the results vary. More recently, it was reported that nicotinic acid derived from Bifidobacterium adolescentis improved mitochondrial function in skeletal muscle, leading to positive effects on sarcopenia. By leveraging exercise to modify the gut microbiome, then identifying related metabolites, herein it is demonstrated the ability to confer the benefits of exercise through the direct administration of microbial-derived, exercise-responsive metabolites. These metabolites are herein termed “microbial-derived exerkines” (MDEs) for their ability to mediate the systemic adaptations consistent with the benefits of exercise. In the present work, pipecolic acid and succinate were identified as key MDEs. When administered together, these metabolites prevented the loss of skeletal muscle size and function during atrophic conditions. Both pipecolic acid and succinate have been suggested to impact mTORC1 signaling in vitro, although it is shown that administration of PAS had no effect on the activation of key proteins in the mTORC1 signaling pathway (i.e., AKT, p70S6K) in skeletal muscle in vivo. The present work demonstrates promising effects of PAS treatment on ATP levels and mitochondrial complex activity. Succinate has been shown to rescue mitochondrial dysfunction in various tissues, including skeletal muscle. While the effect of pipecolic acid on mitochondrial function has not been elucidated, pipecolic acid, as a product of lysine catabolism, can be metabolized into acetyl-CoA and enter the TCA cycle. It may be that the microbial-driven increase in pipecolic acid and succinate in skeletal muscle in response to exercise (or direct administration of these MDEs) increases substrate flux through the TCA cycle, thereby promoting ATP generation. Ribosome biogenesis is an energy-intensive process, so this maintenance of energy status may be linked to our findings that PAS preserves rRNA content and the expression of ribosomal proteins. Given the negative influence of atrophic conditions on ATP production and ribosomal / translational capacity, this preservation of ribosomal integrity through the promotion of ATP production may explain how PAS is able toprevent skeletal muscle atrophy and preserve muscle function. The ribosome has been postulated as a small-molecule sensor in microorganisms and plants, so it may be that PAS affects the ribosome through direct interaction. Therefore, the present work provides several promising avenues for establishing the precise mechanism of MDE treatment in preventing disuse-induced atrophy in skeletal muscle (Fig.4S). In some aspects, PAS can impact ribosomes directly, for a microbial-derived metabolite to preserve mammalian host ribosomal abundance.

[0160] Several additional considerations should be noted. Pipecolic acid has been shown to increase with exercise in both mice and humans. Additionally, the exercise-trained microbiome likely produces a complex mixture of beneficial metabolites that may act synergistically. Future studies will explore the broader metabolite profile and potential interactions, as well as focus on establishing the effects of MDEs on other tissues affected by exercise (e.g., heart, liver, brain). Nevertheless, the implications of these findings are far- reaching. Given the widespread benefits of exercise for conditions such as cardiovascular disease, Alzheimer's disease, aging, and diabetes, the present results provide a new avenue for the development of therapeutic interventions to treat exercise-responsive conditions. By leveraging an exercise-trained microbiome to identify key metabolites, it is possible confer some exercise benefits to individuals unable to perform physical activity, such as bed-ridden patients or those with mobility limitations. Additionally, MDEs allow to circumvent the gut microbiome entirely via direct administration, which may be particularly relevant in patients receiving broad- spectrum antibiotics. Future research will focus on elucidating the precise mechanisms by which MDE treatment ameliorates disuse atrophy, exploring potential synergies between different MDEs, and establishing the efficacy of an exercise-trained gut microbiome in treating various disease models. Moreover, translational studies will determine the applicability of these findings in human populations. In summary, this present disclosure opens new possibilities for microbiome-based therapies inspired by exercise, potentially revolutionizing our approach to treating muscle wasting and other exercise- responsive conditions.

[0161] The present disclosure therefore demonstrates that microbiome transplants from exercised-trained donors are able to ameliorate skeletal muscle disuse atrophy. Moreover, the administration of MDEs from an exercise-trained microbiome is able to reproduce this preservation of skeletal muscle mass along with the maintenance of muscle function. The present disclosure has demonstrated the ability of the gut microbiome to elicit therapeuticbenefits on target tissues directly. These results demonstrate for the first time that an exercise- trained microbiome and associated metabolites elicit phenotypic effects on adult skeletal muscle corresponding with typical exercise adaptation (i.e., the maintenance of muscle mass during disuse as a result of exercise preconditioning). Further, these findings serve as a proof of concept that some of the benefits of regular exercise are, in part, mediated by the gut microbiome. Given the widespread prescription of exercise as a therapeutic intervention for conditions such as cardiovascular disease, Alzheimer’s disease, aging, diabetes, etc., the findings of the present study provide compelling evidence to support leveraging an exercise-trained microbiome to treat various diseases positively impacted by exercise. EXAMPLES

[0162] Microbial-Derived Exerkines Prevent Skeletal Muscle Atrophy

[0163] Mice

[0164] 4- to 12-month-old female mice on a C57BL / 6J background were used for all experiments. Mice were allowed to acclimate to the university vivarium for >4 weeks to allow the gut microbiome to stabilize. For experiments involving the gut microbiome transfers, mice were randomly assigned to one of the following groups: sedentary donor (DON-SED), exercised donor (DON-EXR), recipient from sedentary donor (REC-SED), or recipient from exercised donor (RED-EXR) (n = 9). During metabolite administration experiments, mice were randomly assigned to receive vehicle (VEH), succinate (SUC), pipecolic acid (PIP), or a combination of both succinate and pipecolic acid (PAS) (n = 5-6). All experiments were conducted using the same groups of mice with the exception of ex vivo muscle function and in vivo muscle function which required additional cohorts. All animal procedures were performed according to national and local guidelines and approved by the Institutional Animal Care and Use Committee (2018-3005) at the University of Kentucky.

[0165] Exercise Training

[0166] Donor mice were subjected to 8 weeks of progressive weighted wheel running (PoWeR). Briefly, mice were singly housed in running wheel cages with free access to the running wheel for one week of acclimation. After acclimation, weights consisting of 2g in week 1, 3g in week 2, 4g in week 3, 5g in weeks 4 and 5, and 6g in weeks 6-8 were added to one side of the wheel. Sedentary mice were singly housed under the same conditions with a locked runningwheel. For experiments involving VEH and PAS treatment during exercise training (n = 3), mice were singly housed in running wheel cages as described above. After acclimation, weights consisting of 2g in week 1, 4g in week 2, 6g in week 3, 7g in week 4, 10g in week 5, and 12g in week 6 were added to one side of the wheel. All running data (distance in km) were acquired via ClockLab software (Actimetrics, Wilmette, IL).

[0167] Cecal Microbial Transplant

[0168] The transplantation of cecal contents to a new host has recently emerged as a successful method of transplanting the gut microbiome in murine models. Cecal contents were collected from exercised and sedentary donor mice, resuspended into sterile PBS at a concentration of 150mg / ml, and stored at -80°C. Prior to the first transfer, recipient mice were treated with polyethylene glycol (PEG) (SLBZ3934; Sigma-Aldrich, St. Louis, MO) to clear the existing microbiome. Mice received four boluses of 200µL of 425g / L PEG via oral gavage over the course of one hour. Cecal contents collected from three exercise-trained or sedentary mice were pooled and transferred via oral gavage (200µL per transfer) to three anesthetized recipient mice six hours after the final PEG treatment. Recipient mice received transfers on three consecutive days, followed by a single transfer per week for the next four weeks, resulting in seven total transfers over the course of five weeks (Fig. 1A). Following the final transfer, mice were unilaterally cast for 10 days to induce skeletal muscle atrophy (Fig.5, see below).

[0169] Microbial-Derived Exerkine Administration

[0170] For experiments involving the direct administration of microbial-derived metabolites, herein known as microbial-derived exerkines (MDEs), mice received either VEH (gel only), SUC (S7501; Sigma-Aldrich), PIP (P2519; Sigma-Aldrich), or PAS via MediGel Sucralose hydrogels (74-02-5022; ClearH2O, Westbrook, ME). Mice received free access to the hydrogels for 10 days during hindlimb immobilization. During this period, mice consumed an average of 112.3 ± 26.3 mg / kg / d and 222.8 ± 51.4 mg / kg / d of succinate and pipecolic acid, respectively. For experiments involving the administration of VEH and PAS during six weeks of exercise training, mice received free access to VEH (2% sucrose) or PAS (146.0. ± 10.9 mg / kg / d succinate, 292.0 ± 21.7 mg / kg / d pipecolic acid [50-194-7204; Fisher Scientific, Waltham, MA], 2% sucrose) through their drinking water.

[0171] Unilateral Hindlimb Immobilization

[0172] A model of hindlimb immobilization utilizing casting was developed and subsequently modified using 3-D printing technology. This model allows the contralateral leg to remain mobile and serve as a control, thus limiting inter-animal variation in muscle size. Briefly, the leg is encapsulated in a plastic, 3-D printed external cast which maintains the leg in gentle flexion at the knee and dorsiflexion at the ankle. The casts were secured via twisty ties to prevent escape. Mice were casted for 10 days in all immobilization experiments.

[0173] Ex Vivo Muscle Function

[0174] Mice were anesthetized via isoflurane and euthanized via cervical dislocation. The soleus of the casted leg was excised and mounted to a force transducer in an organ bath (DMT stuff) in Krebs Ringer buffer (in mM: 119 NaCl, 5.0 KCl, 5.0 NaHCO3, 1.25 CaCl2, 1.0 KH2PO4, 10 HEPES, 1.0 MgSO4; pH 7.2) via tendon clamps and maintained at room temperature. The proximal tendon was secured to the force transducer, while the distal tendon was secured to a length dial (0.1mm increments). Following a 10-minute equilibration period, optimal length (LO) was determined by adjusting the muscle length by increments of 0.1-0.5mm to determine maximal force output in response to 20V, 20ms pulse width stimulations delivered using parallel platinum electrodes and separated by 30 seconds of rest (DMT stuff). The muscle was then stimulated every 3 minutes at LO at 10, 20, 40, 60, 80, 100, and 120 Hz (biphasic, 300ms trains, 0.2ms pulse width, 20V) to generate force-frequency curves. Following another 3-minute rest period, a fatigue resistance protocol was performed where the muscle was subjected to 150 repeated 60Hz stimulations separated by 2 seconds (same parameters above). Afterwards, LO was recorded, the muscle was blot dried, weighed, covered in Tissue-Tek O.C.T. Compound (4583; Sakura Finetek, Torrance, CA), and frozen in liquid nitrogen-cooled isopentane in preparation for immunohistochemistry. Force output was recorded using Labview software () and analyzed using MATLAB (ver. R2024b Update 5). Absolute force output (mN) was normalized to cross-sectional area (CSA) determined via immunohistochemistry (see below) and reported as specific force (N / µm2). Rate of force development (RFD) was calculated as follows: RFD = Δ Force / Δ Time from the beginning of the contraction to peak force.

[0175] In Vivo Muscle Function

[0176] Plantarflexion in vivo isometric peak torque was assessed. Briefly, anesthetized mice were placed in the supine position on a 37°C temperature regulated platform (809c in situ mouse apparatus, Aurora Scientific, Aurora, ON, Canada) and the hindlimb was secured at theknee with the foot taped to a dual-mode lever and motor (300D-300C-LRFP, Aurora Scientific) so that the tibia was parallel to the platform and the knee was held at a 90° angle. Percutaneous needle electrodes placed lateral to the knee were used to stimulate the tibial nerve with an electrical stimulator (High Power Bi-Phase Stimulator, Aurora Scientific). To maximize torque production and eliminate activation of dorsiflexors, needles were adjusted in response to repeated twitches to identify the optimal placement. Peak tetanic torque was recorded at 10, 40, 80, 120, 150, 180, and 200Hz stimulations (0.25s duration, 50mA) using DMC v6.000 and analyzed with MATLAB. Data are reported as normalized to body mass (mN-m / gram of BW).

[0177] Tissue Collection

[0178] Following immobilization, hindlimb musculature (soleus, plantaris, and gastrocnemius) from both lower hindlimbs was carefully excised, weighed, and either covered in Tissue-Tek O.C.T. Compound and snap-frozen in liquid nitrogen-cooled isopentane in preparation for immunohistochemistry (soleus, plantaris), or directly snap-frozen in liquid nitrogen for downstream molecular analyses (gastrocnemius). For SED and EXR experiments specifically, cecal contents and serum were also collected at sacrifice. Cecal contents were immediately snap frozen in liquid nitrogen. To obtain serum, blood was collected, allowed to clot, and centrifuged at 150 x g for 15 minutes. The supernatant was removed and centrifuged again at 300 x g for 15 minutes. Quadriceps muscles were also collected from the ex vivo muscle function cohorts of VEH and PAS mice for complex activity analyses.

[0179] Immunohistochemistry

[0180] Muscle fiber-type staining procedures were carried out. Briefly, unfixed soleus sections were incubated overnight with antibodies against myosin heavy chain types I (BA.D5), IIA (SC.71), and IIB (BF.F3) (1:100; Developmental Studies Hybridoma Bank, Iowa City, IA), as well as rabbit anti-laminin IgG (1:100; L9393; Sigma-Aldrich). The sections were then incubated with fluorescence-conjugated secondary antibodies against the various mouse immunoglobulin subtypes. Muscle sections were imaged using either a Zeiss upright fluorescent microscope (Zeiss AxioImager M1 Oberkochen, Germany) or an Olympus BX61VS Upright Fluorescent Microscope (Evident Scientific, Bethlehem, PA) at 20x magnification.

[0181] MyoVision, an unbiased automated image analysis program developed by our lab, was used to determine myofiber CSA and myonuclear abundance in a blinded manner. Western Blots Gastrocnemius samples were homogenized using a THb Handheld Tissue Homogenizer andHard Tissue Omni Tip Plastic Homogenizer Probes (Omni International, Kennesaw, GA) in RIPA Lysis and Extraction Buffer (786-490; G-Biosciences, St. Louis, MO) with Halt Protease & Phosphatase Inhibitor Cocktail (1861281; ThermoFisher Scientific, Waltham, MA). For quantification of protein abundance, samples were resolved on a 4–12% Bis-Tris Plus Protein Gels (NW04120BOX; ThermoFisher Scientific) and transferred to a PVDF membrane (LC2002; ThermoFisher Scientific). The membrane was blocked with 5% BSA in 0.1% Tween Tris-buffered saline (TBS-T) and incubated at room temperature with primary antibodies against AKT (9272S; Cell Signaling), p-AKT (4051S, Ser473; Cell Signaling), p70S6K (9202S; Cell Signaling), p-p70S6K (9206S, Thr389; Cell Signaling), and mitochondrial complex proteins (45-8099; ThermoFisher Scientific). Blots were developed using enhanced chemiluminescence (Clarity Western ECL Substrate; Bio-Rad, Hercules, CA) and imaged on a Chemidoc imaging system (Bio-Rad).

[0182] Metagenomics

[0183] Microbial DNA was extracted from the cecal contents. Briefly, upon euthanasia, the cecum was excised from DON-SED, DON-EXR, REC-SED, and REC-EXR mice and the contents were carefully removed and immediately frozen in liquid nitrogen. DNA was isolated from cecal content using the PureLink Microbiome DNA purification kit (A29790; ThermoFisher Scientific) according to the manufacturer’s instructions. Samples were sent to Alkek Center for Metagenomic and Microbiome Research (Baylor College of Medicine, Houston, TX) for metagenomic sequencing. Samples were analyzed on a HiSeq X Ten Sequencing System (Illumina, San Diego, CA). Taxonomic classification was performed with MetaPhlan 3.065. One REC-SED sample was excluded due to low read depth. Microbial diversity, profiling, and differential statistics analyses were performed and visualized using the online software MicrobiomeAnalyst 2.066: Chao1 indices were used to determine alpha diversity; principal component analyses (PCoA) based on Jensen-Shannon divergence were used to determine beta diversity; linear discriminant analyses (LED) were used to identify predictive features; DESeq2 package was used to identify differential microbes.

[0184] Untargeted Metabolomics

[0185] Cecal content, serum, and gastrocnemius samples from SED and EXR mice were sent to Metabolon, Inc. (Morrisville, NC) for untargeted metabolite profiling. Samples were analyzed on a ThermoFisher Scientific Q-Exactive spectrometer interfaced with a heated electrospray ionization source and Orbitrap mass analyzer. Metabolites were separated on anACQUITY UPLC Peptide BEH C18 column (100mm x 2.1mm, 1.7µm particle size; 186003555; Waters, Milford, MA). Compounds were identified using Metabolon’s internal library of authenticated standards. Partial least squares discriminant analysis (PLS-DA) and random forest classification were performed using the online software MetaboAnalyst 6.067.

[0186] For VEH and PAS experiments, gastrocnemius samples were pulverized (CryoPREP CP02 Cryogenic Dry Pulverization; Covaris, Woburn, MA), suspended in 1mL of extraction solution (50% HPLC-grade methanol and 1% formic acid), and homogenized using zirconium silicate beads (ZSB10; Next Advance, Inc., Troy, NY) in a Bullet Blender Gold (Next Advance, Inc.). Supernatant was filtered using Captiva EMR-Lipid cartridges (5190-1002; Agilent Technologies, Santa Clara, CA) placed on top of autosampler vials (5190-2280; Agilent Technologies). Samples were then dried and concentrated using a Savant SpeedVac Integrated Vacuum Concentrator System and Kit (SPD2030; ThermoFisher Scientific) at 25°C until a complete pellet was formed (~48 hrs). The pellet was resuspended in 5% acetonitrile and 0.1% formic acid, filtered (0.22µm; 8160; Costar, Glendale, AZ), and stored in polymer feet autosampler inserts (5183-2088; Agilent Technologies) in autosampler vials. Metabolites were separated using a SeQuant ZIC-pHILIC column (150mm x 2.1mm, 5µm particle size; 150454; Millipore Sigma, Burlington, MA). Mobile phase A consisted of 10 mM ammonium acetate in water, pH 9.8 and mobile phase B consisted of 100% methanol. All chemicals were LC-MS grade. The column flow rate was set to 0.15 mL / min, column temperature set at 25°C. Metabolites were separated over a 19-minute gradient from 90% B to 30% B. The column was washed for 5 minutes at 30% B, and then re- equilibrated at 90% B for 8 minutes. Samples were analyzed on a ThermoFisher Scientific Orbitrap Exploris 240 mass spectrometer coupled to a Vanquish Neo UHPLC system (ThermoFisher Scientific). Polarity-switching MS1 only acquisition was acquired for each polarity set at 120,000 FWHM resolution and a scan range was set to 80-1200 Daltons; the automatic gain control (AGC) target was set to ‘Custom’ at 1e6 absolute AGC value and a maximum inject time of 50ms. Spectra were analyzed using Compound Discoverer Software 3.3SP (ThermoFisher Scientific) and searched against ChemSpider databases (BioCyc, Human Metabolome, and KEGG) to identify compound names. PLS-DA, differential statistics, and pathways enrichment analyses were performed and visualized using MetaboAnalystR R package68.

[0187] Proteomics

[0188] Gastrocnemius muscles were suspended in RIPA buffer (786-490; G-Biosciences, St. Louis, MO) and homogenized using an Omni Tissue Homogenizer (Omni International, Kennesaw, GA). After a 10-minute incubation on ice, samples were centrifuged at 10,000 x g for 10 minutes twice. Pierce BCA Protein Assay kits (23225; ThermoFisher Scientific) were used to quantify protein levels. Equivalent protein amounts (20µg) were enzymatically digested using the EasyPep MS Sample Prep Kits (A40006; ThermoFisher Scientific) according to the manufacturer’s protocol. Peptides were resuspended in 5% acetonitrile and 0.1% formic acid and analyzed on a ThermoFisher Scientific Orbitrap Exploris 240 mass spectrometer with an EASY-Spray source housing and a Vanquish Neo UHPLC system. Peptides were separated on an EASY-Spray HPLC column (150mm x 75µm, 2µm particle size; ES904; ThermoFisher Scientific) paired with a PepMap Neo 5μm C18 300μm x 5mm Trap Cartridge (174500; ThermoFisher Scientific). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in acetonitrile. The flow rate was set to 0.3µL / min.

[0189] Peptides were separated over a 90-minute linear gradient from 2-55% mobile phase B. The column was equilibrated for 1.5 mins at 2% mobile phase B. Mobile phase B was increased to 10% over 10 minutes and increased again to 25% over the next 35 minutes. Mobile phase B was increased to 35% over 25 mins followed by 55% mobile phase B for 15 minutes. Following peptide separation, the column was washed at 0.75µL / min at 98% mobile phase B for 8 minutes, followed by column re-equilibration at 2% mobile phase B for 5 minutes. Data was collected using a data-dependent acquisition (DDA) strategy. The instrument was set to 60,000 resolution, with a top N precursor ions in a 3 second cycle time. Data was collected using positive ionization including charge states 2-4. Full scan (MS1) settings were set as follows: (a) scan range: 375-1200 m / z, (b) RF lens (%): 45, (c) AGC target: custom, (d) normalized AGC target (%) 250, (e) maximum injection time mode: custom, and (f) maximum injection time (ms): 20. Peptide fragmentation spectra (MS2) were collected using a normalized HCD collision energy set to 26%, 2 m / z isolation window, 15,000 resolution, normalized AGC target set to 50%, and automatic maximum injection time. Data were analyzed against the Mus musculus UniProt database (downloaded March 2024) using the ThermoFisher Scientific Proteome Discoverer (version 3.1) software and the SEQUEST search algorithm. Proteomic output was analyzed using the R (version 4.3.2)69 package DEP70. One outlier sample was removed from the VEH group. Data were visualized using the EnhancedVolcano package71.

[0190] Electron Transport Chain Complex Activity

[0191] The activity of mitochondrial electron transport chain Complexes II and IV was assessed using frozen quadriceps samples. Samples were homogenized in Mitochondrial Isolation Buffer (MIB) (in mM: 70 sucrose, 220 mannitol, 5 KH2PO4, 5 MgCl2, 1 EGTA, 2 HEPES, 3mM BSA; pH 7.4) using a THb Handheld Tissue Homogenizer and Hard Tissue Omni Tip Plastic Homogenizer Probes (Omni International, Kennesaw, GA). Mitochondria were isolated by differential centrifugation, and protein concentration was determined using a BCA Protein Assay Kit (71285-3; Millipore Sigma). 20µg of protein was loaded into Seahorse XF24 Cell Culture Microplate (100777-004; Agilent Technologies) with 500µL Seahorse Assay Buffer (103575-100, Agilent Technologies) with 2mg / 100mL cytochrome C and spun at 2,000g for 20 minutes with no break at 4°C to adhere mitochondria to the plate.

[0192] Oxygen consumption rate (OCR) was measured in a Seahorse XF24 Analyzer (Agilent) at baseline and after each of the following injections: 1mM succinate and 2μM rotenone; 4 μM antimycin A; 0.5mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and 1mM ascorbic acid (freshly prepared), pH 7.4; 50mM sodium azide. Complex II (C-II) activity was reported as the OCR recording after succinate and rotenone addition (prior to antimycin A injection). Complex IV (C-IV) activity was determined by subtracting the OCR after sodium azide addition from that after ascorbic acid + TMPD.

[0193] rRNA Integrity

[0194] RNA was isolated from gastrocnemius samples from VEH and PAS groups. Samples were homogenized in TRI Reagent (T9424; Millipore Sigma) with zirconium silicate beads (ZSB10; Next Advance, Inc.) using a Bullet Blender Gold (Next Advance, Inc.). After a 10- minute incubation on ice, samples were centrifuged at 10,000 x g for 10 minutes. RNA was isolated using Direct-zol RNA Miniprep Plus Kits (R2070; Zymo Research, Irvine, CA) according to manufacturer’s instructions. Samples were diluted 1:200 and run on an Agilent 2100 Bioanalyzer (Agilent Technologies) using RNA Pico Chips (5067; Agilent Technologies) to assess rRNA quality.

[0195] In vitro iATP Reporter

[0196] An ATP-GFP reporter (iATP) (Plasmid #209651; Addgene) was co-transfected with the capsid (MyoAAV 2A) and helper (pAdDeltaF6, Plasmid #112867, Addgene) plasmids into HEK293T-17 cells using PEI Prime™ (AQ100, Serochem, Bloominton, MN). Viral particles were purified using iodixanol gradient ultracentrifugation and titered by ddPCR using primer / probeset targeting the ITR (TaqMan custom assay, ThermoFisher Scientific). Mouse C2C12 myoblasts (American Type Culture Collection, ATCC, Manassas, VA) were cultured in high glucose containing Dulbecco’s Modified Eagle growth medium (DMEM; 10-013-CM; Corning, Corning, NY) supplemented with 10% (v / v) fetal bovine serum (FB-73; Alkali Scientific) and 100 U / mL penicillin and 100 µg / mL streptomycin (15-140-148, ThermoFisher Scientific). Cells were infected with iATP-MyoAAV at an estimated 103, 104, and 105 multiplicity of infection (MOI) in differentiation medium containing high glucose DMEM and 2% (v / v) horse serum (HS, H1138, Sigma-Aldrich, MO, USA) for 7 days. Cells were imaged using Olympus APEXVIEW APX100 (Evident Scientific) at 20x magnification. To confirm iATP reporters dynamically report ATP levels in cells, we subjected cells to filter sterilized Tyrode’s solution (pH 7.4) containing 1mM MgCl2, 1.8 mM CaCl2, and 5.5mM 2-deoxy-d-glucose, which essentially removes glucose from becoming a source of energy inside the cells thereby depleting cellular ATP abundance (Fig. 13A). Reintroduction of differentiation media containing glucose increased cellular ATP levels overnight but does not return cells to pre-deoxy-glucose levels (Fig.13A).

[0197] For metabolite treatment experiments, mouse C2C12 myoblasts were differentiated and infected with iATP-MyoAAV at 103 MOI (Fig.13B). After 7 days of infection, cells were treated with VEH, PIP (10mM), SUC (2mM), or PAS (10mM and 2mM, respectively) in differentiation media for 72hrs and imaged as described above. Cells were treated with 25nM of MitoTracker Red (M7512; ThermoFisher Scientific) for 30 minutes and washed to control for mitochondrial abundance. Data represents iATP fluorescence intensity normalized to MitoTracker Red fluorescence intensity.

[0198] Citrate Synthase Activity

[0199] Citrate synthase activity in cell homogenates was determined using an enzymatic assay. Briefly, samples were mixed in a buffer containing Tris (100mM, pH 8.0), 5,5’-Dithiobis-(2- nitrobenzoic acid) (DTNB) (10mM) (D8130; Sigma Aldrich), acetyl CoA (30 mM) (A2056; Sigma Aldrich), and oxaloacetic acid (OAA) (10mM) (O4126; Sigma Aldrich). Equal amounts of protein (8µg) were loaded, and enzymatic activity was assessed by measuring the change in absorbance at 412nM over 10 minutes using a Cytation 5 Multimode reader (Biotek, Winooski, VT). The following calculation was used to determine enzymatic activity: U / mg protein = (Δ Absorbance / Δ Time in minutes) / (13.6 × 0.625 × mg protein).

[0200] Statistics

[0201] All statistical analyses were performed using GraphPad Prism version 10.2.3 for Windows (GraphPad Software, La Jolla, CA). Unpaired, one-tailed t-tests were used to test for differences in total microbial abundance, individual microbe abundance, alpha diversity, muscle mass, muscle CSA, torque production, protein and metabolite expression, mitochondrial complex activity, in vivo citrate synthase activity, and rRNA integrity and proportion between respective groups. One-sample t-tests were used to test differences between control and casted muscle CSA in 5, 7, and 10-day immobilized mice. Two-way ANOVAs or mixed-effect analyses (exercise, transplant; treatment, casting) with Fisher’s post hoc analyses were used to test for differences in alpha diversity, muscle mass, muscle CSA, and muscle fiber proportion within and between respective groups. Area under the curve was calculated for all force / torque-frequency, RFD, and fatigue curves and statistically significant differences were tested using multiple, unpaired t-tests. Multiple, unpaired t-tests were used to test for differences in pipecolic acid and succinate abundances between SED and EXR groups in cecal content, serum, and muscle.

[0202] Unpaired one-way ANOVAs with Tukey’s post hoc analyses maximizing power were used to test for differences in ATP abundance and citrate synthase activity in vitro between metabolite treated groups.

[0203] It is further hypothesized that CMT from exercise-trained mice will reduce muscle atrophy of recipient mice by stimulating myofiber protein synthesis via mTORC1 activation. To test this hypothesis, adult female and male mice will receive CMT from either sedentary or exercise-trained mice prior to 10 days of unilateral hind limb immobilization; the contralateral leg will serve as control. To assess muscle atrophy, immunohistochemistry will be used to measure fiber-type-specific cross-sectional area. The rate of protein synthesis will be determined using the SuNSET method with mTORC1 activation assessed by western blot. In vivo muscle function of the plantar flexors will be determined following the immobilization period. If our hypothesis is not supported by the results, single-cell / myonuclear RNA-seq will be performed to identify the cell(s) within muscle affected by exercise-trained CMT.Metagenomic analysis identified bacterial species with higher abundance following CMT from exercise-trained mice. It is hypothesized that a probiotic supplement containing one or more of these bacterial species will ameliorate muscle atrophy induced by hind limb immobilization. To test this hypothesis, adult female and male C57BL / 6J mice will be administered by gavage the proposed probiotic supplement 14 days prior to and throughout unilateral hind limb immobilization. Muscle atrophyand function will be assessed as describe above. Metabolism of confirmed bacterial specie(s) will be characterized by continuous culture and RNA-seq.

[0204] Based on the metabolomic analysis, it is hypothesized that the administration of a post-biotic containing pipecolic acid and / or succinate will significantly reduce muscle atrophy induced by hind limb immobilization. To test this hypothesis, adult female and male C57BL / 6J mice will be administered via food gel pipecolic acid (200 mg / kg / d) and / or succinate (100 mg / kg / d) during 10 days of unilateral hind limb immobilization. Muscle atrophy and function will be assessed as described above.

[0205] Using the gut microbiome to ameliorate mechanical ventilator-induced diaphragm atrophy following SCI (spinal cord injury)

[0206] Mouse model and demographics: All proposed studies will use adult (16 weeks of age) female and male HSA-GFP mouse. The HSA-GFP mouse will be generated by crossing our myofiber-specific Tet-ON mouse (HSA-rtTA) to the TRE-H2b-GFP mouse, thus producing the HSA- rtTA;TRE-H2b-GFP mouse (designated HSA-GFP). The HSA-GFP mouse will allow us to specifically label >95% of all myonuclei with GFP following doxycycline administration (0.5mg / mL for 7 days). GFP+ myonuclei will be isolated by fluorescent-activated cell sorting (FACS) for single-nuclear RNA-sequencing (snRNA-seq). Various modifications of the present disclosure, in addition to those shown and described herein, will be apparent to those skilled in the art of the above description. Such modifications are also intended to fall within the scope of the appended claims.

[0207] Spinal cord injury and diaphragm atrophy: To induce spinal cord injury (SCI) and diaphragm atrophy HSA-GFP mice will undergo C2-hemisection surgery. This is a well- characterized model of SCI and respiratory motor impairment. Following C2 hemisection, these animals will be then placed on a mechanical ventilator (MV) for 12 or 24 hours to induce atrophy.

[0208] Following the completion of the C2-hemisection injury, but prior to MV, mice will be treated with polyethylene glycol to cleanse the bowels in preparation for the transfer of cecal contents from mice that had undergone exercise-training (8 weeks of weighted wheel running) or were sedentary (control treatment). At designated time points post-injury and intervention, the diaphragm muscle will be collected and prepared for: 1) immunohistochemistry to measure the extent of atrophy (or potentially hypertrophy) through assessment of fiber-type specific cross-section myofiber area using dystrophin staining;2) Surface Sensing of Translation (SUNSET) analysis to quantify protein synthesis and Western blot measurement of mTORC1; and 3) FACS isolation of GFP+ myonuclei and subsequent snRNA-seq to measure the impact of cecal transfer on diaphragm myonuclear heterogeneity.

[0209] Upon completion of the injury, mice will be administered pipecolic acid (200 mg / kg) and / or succinate (300 mg / kg) by intraperitoneal injection. At designated time points, diaphragm muscle will be processed and analyzed as described above. major challenge to individuals who suffer a high-level SCI is the rapid atrophy of the diaphragm as a result of mechanical ventilation (MV). The magnitude of diaphragm atrophy is so significant that it creates a barrier to weaning SCI individuals off of MV and thereby reducing the quality of life, as well as incurring significant healthcare costs. Despite the obvious need to prevent diaphragm atrophy there is a paucity of studies investigating this issue. The proposed study seeks to fill this critical gap in knowledge and investigate a novel approach to ameliorating the diaphragm atrophy induced by MV following SCI by leveraging recent findings showing the benefit of the gut microbiome to rescue muscle atrophy. Metabolite analysis identified the microbially-derived metabolites, pipecolic acid and succinate, that may be responsible for mediating the aforementioned beneficial effects on muscle size. These metabolites will also be tested to determine the extent to which they can reduce the magnitude of diaphragm atrophy induced by MV.

[0210] Alzheimer’s Studies

[0211] The 3xTg-AD mouse model will be used and will receive cecal content from exercise-trained mice. It is hypothesized that transferring the gut microbiome from exercise- trained mice to 3xTg-AD mice will delay the onset of cognitive impairment. To test this hypothesis, two-month-old female 3xTg-AD mice will receive, by oral gavage, a slurry derived from the cecal contents of sedentary or exercise-trained mice once per week for eight weeks. We chose to start the treatment at two months of age because the 3xTg-AD mice do not show cognitive impairment until four-months of age. Only female will be used because male 3xTg-AD mice do not present a strong phenotype. Upon completion of the intervention, cognitive function will be assessed in the UK Rodent Behavioral Core facility using protocols established in the Johnson lab for Morris water maze (MWM), novel object recognition (NOR), and the elevated plus maze (EPM). Following testing, mice will be humanely euthanized and the brains carefullyexcised with one hemisphere prepared for immunohistochemistry to assess plaque load and microglia activation i.e., neuroinflammation and the other hemisphere used for biochemical analyses to measure Aβ content and inflammatory markers. Compared to 3x-Tg-AD mice that received cecal transfer from sedentary mice, we anticipate 3x-Tg-AD mice receiving cecal transfer from exercise-trained mice will perform better on the aforementioned cognitive tests with lower Aβ deposition, microglial activation, and expression of pro-inflammatory markers.

[0212] It is further hypothesized that 6-month-old female 3xTg-AD mice receiving cecal contents from exercise-trained mice will have reduced plaque load, neuroinflammation, and cognitive impairments compared to 3xTg-AD mice receiving cecal contents from sedentary mice. Six-month old mice are chosen because 3xTg-AD mice at this age show clear cognitive impairment and Aβ plaque deposition. To test this hypothesis, six-month-old 3xTg-AD mice will receive by oral gavage, a cecal slurry derived from the gut microbiome of either sedentary or exercise-trained mice once per week for 12 weeks. Upon completion of the intervention, cognitive function, IHC and biochemical analyses will be performed as described in Aim 1. While it may not be expected that the intervention will completely reverse the cognitive impairment or brain pathology, it can be anticipated that cognitive function of 3xTg-AD mice receiving cecal contents from exercise-trained mice will be higher and associated with lower Aβ deposition compared to the sedentary-control group.

[0213] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0214] Two-month-old female 3xTg-AD mice on a C57BL / 6J background will be ordered from The Jackson Laboratory. The 3xTg-AD strain is chosen because the strain has a well-defined pathological timeline which allows us to select specific ages to target both the onset and progression of the pathology. Sex will be limited to female due to previous data showing that 3xTg-AD males do not develop robust AD pathology. Additionally, 12-week old wild-type female C57BL / 6J mice (WT) will be ordered to generate cecal content for CMT procedures. All mice will acclimate to UK mouse vivarium for >4 weeks to allow the gut microbiome to stabilize. Based on previous literature regarding the effects of exercise on AD, the sample size will be set at 20 per group to provide sufficient power to detect significant differences in the cognitive tests, IHC and biochemical assays.

[0215] Exercise Training. a novel progressive weighted-wheel-running (PoWeR) model of murine exercise training was developed. PoWeR has been shown to induce robust hypertrophy in the hindlimb musculature; additional strengths of PoWeR training is it is voluntary nature and similarity to human exercise (i.e., episodic, reversible). Mice will be singly housed in running wheel cages with free access to the running wheel. During the first week of training, mice will acclimate to an unloaded running wheel. After acclimation, weights consisting of 2g in week 1, 3g in week 2, 4g in week 3, 5g in weeks 4 and 5, and 6g in weeks 6-8 will be added to one side of the wheel. Running data (km / d) and total running volume (km) will be recorded. Sedentary controls will be singly housed in a locked wheel cage for the duration of the training period.

[0216] Cecal Microbial Transfer (CMT). The transfer of cecal contents to a new host has recently emerged as a successful method of transplanting the gut microbiome in murine models. As previously described, cecal contents will be collected from PoWeR-trained or “sedentary” (i.e., untrained controls) WT donor mice in an anerobic chamber and released into pre-reduced anaerobic dental transport media to be frozen at -80oC. Pooled cecal contents collected from either PoWeR-trained or sedentary mice will be transferred to 3xTg-AD recipient mice via oral gavage once a week for 8 (Aim 1) or 12 (Aim 2) weeks. Prior to transfer, recipient mice will be treated with polyethylene glycol to clear the existing microbiome.

[0217] Cognitive Assessments. Cognitive function will be assessed in the UK Rodent Behavioral Core facilities using protocols established in the Johnson lab for the Morris water maze (MWM), novel object recognition

[0218] (NOR), and the elevated plus maze (EPM).

[0219] Aβ Plaque Quantification. In order to determine regional plaque load, brain sections will be stained for Aβ deposits via incubation with 2 µM X34 for 20 min. Following staining and washing, high resolution (3.45 µM pixel size) IHC images of the entire brain section will be acquired using a Zeiss Axioscan slide scanner. Plaque number, size, and intensity will be quantified using the Johnson lab’s established protocol via HALO software.

[0220] Amyloid β Measures. Brain samples will be homogenized in PBS, followed by 5 M guanidine buffer with protease and phosphatase inhibitors. Homogenates will be centrifuged and the supernatant saved as the PBS soluble fraction.5 M guanidine buffer will be added to the pellet and homogenized. Following centrifugation, the supernatant will be saved as the 5 Mguanidine insoluble fraction. Commercially available ELISAs will be used to determine the levels of Aβ40 and Aβ42 in the PBS and 5 M guanidine fractions.

[0221] Inflammatory Markers. In order to evaluate the inflammatory phenotype of the brain, homogenates will be assessed for levels of interleukin (IL)-1β, IL-2, IL-6, IL-10, and tumor necrosis factor (TNFα), common markers of inflammation, via commercially available multiplex immunoassays.

[0222] Microglial Activation. As another assessment of neuroinflammation, brain sections will be stained for established markers of microglial activation Clec7a, CD74, and CD11c using commercially available antibodies and imaged as described above.

[0223] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components, regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof” means a combination including at least one of the foregoing elements.

[0224] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0225] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The drawings are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and / or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0226] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individual patent, publication, or application was specifically and individually incorporated herein by reference.

[0227] The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure. REFERENCES 1 Fan, Y. & Pedersen, O. Gut microbiota in human metabolic health and disease. Nat Rev 670 Microbiol 19, 55-71 (2021). doi.org / 10.1038 / s41579-020-0433-9 2 Zhao, W. et al. Mannan Oligosaccharides Promoted Skeletal Muscle Hypertrophy through the Gut Microbiome and Microbial Metabolites in Mice. Foods 12 (2023). doi.org / 10.3390 / foods12020357 3 Yan, H. et al. Gut microbiota can transfer fiber characteristics and lipid metabolic profiles of skeletal muscle from pigs to germ-free mice. Sci Rep 6, 31786 (2016). doi.org / 10.1038 / srep31786 4 Giron, M., Thomas, M., Dardevet, D., Chassard, C. & Savary-Auzeloux, I. Gut microbes and muscle function: can probiotics make our muscles stronger? 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Claims

CLAIMS 1. A method for treating atrophied tissue in a subject comprising administering a cecal microbial transplant from an exercise-trained subject to the subject.

2. The method of claim 1, wherein the cecal microbial transplant comprises a microbiome from cecal tissue or extra-cellular matrix of cecal tissue of the exercise-trained subject.

3. The method of claim 1 or 2, wherein the cecal microbial transplant comprises one or more exerkines.

4. The method of claim 1, 2, or 3, wherein the cecal microbial transplant comprises succinate and / or pipecolic acid.

5. The method of claim 1, wherein the cecal microbial transplant comprises succinate or pipecolic acid or a combination thereof.

6. The method of claim 5, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

7. The method of claim 5, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

8. The method of claim 1, wherein at least one other agent is administered to the subject.

9. The method of claim 8, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

10. The method of claim 1, further comprising physical activity or exercise by the subject.

11. A method for treating muscle tissue in a subject comprising administering one or moreexerkines to the subject.

12. The method of claim 11, wherein the one or more exerkines are derived from an exercised-trained subject.

13. The method of claim 11 or 12, wherein the one or more exerkines comprises succinate and / or pipecolic acid.

14. The method of claim 13, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

15. The method of claim 13, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

16. The method of claim 11, wherein at least one other agent is administered to the subject.

17. The method of claim 16, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

18. The method of claim 11, further comprising physical activity or exercise by the subject.

19. A method for treating muscle tissue in a subject comprising administering succinate and / or pipecolic acid to a subject in need thereof.

20. The method of claim 19, wherein succinate is administered to the subject at 50 to 200 mg / kg / day.

21. The method of claim 19, wherein pipecolic acid is administered to the subject at 100 to 300 mg / kg / day.

22. The method of claim 19, wherein at least one other agent is administered to the subject.

23. The method of claim 22, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

24. The method of claim 19, further comprising physical activity or exercise by the subject.

25. A composition for treating muscle tissue comprising succinate and pipecolic acid.

26. The composition of claim 25, wherein succinate is present to provide 50 to 200 mg / kg to a subject.

27. The composition of claim 25, wherein pipecolic acid is present to provide 100 to 300 mg / kg to a subject.

28. The composition of claim 25, further comprising at least one other agent.

29. The composition of claim 28, wherein the at least one other agent comprises a stem cell, a steroid, a glucocoreticosteroid, a growth hormone, a vitamin, an amino acid, a protein, or a combination thereof.

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