The gut microbiota Microbiome signaling pathways influences skeletal muscle mass and function in mice

Authors: Shawon Lahiri1,2, ‡,*, Hyejin Kim3, Isabel Garcia-. Perez4, Musarrat M.aisha Reza1,5, Katherine A. Martin3, Parag Kundu3,6, Laura M. Cox7, Joel Selkrig3, †, Joram M. Posma4, Hongbo Zhang8,§, Parasuraman Padmanabhan3, Catherine Moret2, Balázs Gulyás9,10, Martin. J. Blaser11,12, Johan Auwerx8, Elaine Holmes4,13, Jeremy Nicholson4,14 , Walter Wahli2,3,153 !, and Sven Pettersson1,3,6, !*

Affiliations:

1Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Sweden.

2Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.

3Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.

4Division of Computational and Systems Medicine, Department of Surgery and Cancer, Sir Alexander Fleming Building, Imperial College London, SW72AZ, UK.

4MRC-NIHR National Phenome Centre, Computational and Systems Medicine, Department of Surgery and Cancer, Imperial College of London, UK.

5 School of Biological Sciences, Nanyang Technological University, Singapore. 

6Singapore Center for Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, Singapore. 

7Ann Romney Center for Neurologic Diseases, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA.

8Laboratory of Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

9Department of Neuroscience and Mental Health, Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore.

10Depertment of Clinical Neuroscience, Karolinska Institutet, Sweden.

11Department of Medicine, New York University School of Medicine, New York, NY 10016, USA.

12Medical Service, VA New York Harbor Healthcare System, New York, NY 10010, USA.

13Division of Computational and Systems Medicine, Department of Surgery and Cancer, Sir Alexander Fleming Building, Imperial College London, SW72AZ UK

14Australian National Phenome Center, Murdoch University, WA 6150

153INRA ToxAlim Integrative Toxicology and Metabolism UMR1331, Chemin de Tournefeuille, Toulouse Cedex, France.

‡ Current affiliation: Swedish Research Council, Sweden.

† Current affiliation: European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany.

§ Current affiliation: Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education; The Department of Histology and Embryology of Zhongshan School of Medicine, Sun-Yat Sen University, Guangzhou, China

! Shared last authorships

*Corresponding authors

Overline: Gut Microbiota

One Sentence Summary: Gut microbes transplanted into germ free mice improve Gut microbes influence skeletal muscle mass and strength function.

Accessible Summary

Skeletal muscle is not only important for locomotion but also for regulating metabolic function. This study has elucidated the functional interactions between gut microbiota and skeletal muscle in mice. We identified genes and signaling pathways involved in the regulation of skeletal muscle mass and function that respond to cues from the gut microbiota. Additional biochemical and functional analysis has also revealed influence of gut microbiota in regulation of the communication between nerves and skeletal muscle. Our findings open for better understanding of the role of gut microbiota in mechanisms underlying loss of muscle mass and diseases affecting skeletal muscle.

Abstract:

The functional interactions between the gut microbiota es and the host are important for host physiology, homeostasis and sustained health. Skeletal muscle of gUsing the germ-free mice, that lack gut microbiota(GF) mouse-model system, we have identified a gut microbiome–muscle axis that regulates skeletal muscle mass and function. GF mice displayed reduced skeletal muscle mass, which correlated with skeletal muscle atrophy, decreased expression of insulin-like-growth factor 1 (Igf1) and reduced transcription of genes associated with skeletal muscle growth and mitochondrial function when compared to pathogen-free mice that had gut microbiota. and decreased Igf1 expression including a set of other key transcription factors related to skeletal muscle growth and mitochondrial function. Interestingly, we observed a rescue of skeletal muscle mass and reduced oxidative metabolic capacity when GF mice were exposed to gut microbes. Complementary NMR spectrometry analysis of skeletal muscle, liver, and serum from germ-free mice revealed multiple changes in the metabolites amounts of amino acids, including of the amino acid metabolic pathway dominated by glycine and and alanine compared to the metabolite spectrum observed in pathogen-free mice . .Germ-freeGF mice also had displayed reduced serum choline, the precursor of acetylcholine, the a key neurotransmitter synapse mediator between muscle and nerve at the neuromuscular junction (NMJ). Reduced expression of Rapsyn and Lrp4, two proteins important for neuromuscular junction assembly and function were also observed in muscle from germ-free mice. Transplanting germ-free mice with gut microbiota from pathogen-free mice, resulted in Reduced expression of Rapsyn and Lrp4, key molecules required for NMJ assembly and thus optimal NMJ function, was also observed in GF mice. an increase in skeletal muscle mass, reduction in atrophy markers, improved oxidative metabolic capacity of muscle and elevated expression of the neuromuscular junction assembly genes Rapsyn and Lrp4. Exposing They were restored when GF mice were exposed to gut microbes. Further exposing germ-freeGF mice to microbial metabolites such as to microbial metabolites (short chain fatty acids, in part ) in part reversed the skeletal muscle impairments phenotype. Our results elucidate asuggest a role for the gut microbiotaes in regulating skeletal muscle mass and function in mice., relevant for future design of gut microbiome based therapeutics to target myopathies.Comment by Orla Smith [2]: Author, correct?

Introduction

SSkeletal muscle function is essential for locomotion and body posture. These functionsis are regulated by the central nervous system through neurovia transmission at the neuromuscular junctions (NMJs). NMJs , which are highly specialized chemical synapses formed between motor neurons and skeletal muscle fibers (1). Skeletal muscle displays marked high plasticity being able to respond by responding to a variety of environmental cues, such as exercise and nutrition. Skeletal muscle is It is also the major site of insulin-stimulated glucose uptake and fatty acid oxidation emphasizing its key role in metabolism. RNot surprisingly, reduced skeletal muscle mass and function are associated with metabolic disorders (2) and sarcopenia (3), underscoring its role in maintenance of health.

It has is now wellbeen established that the gut microbiotaes influences host health in partpartially due to its their co-evolvement with the host to meet mutually beneficial biochemical and biological needs (4). There are many studies While publications investigating how on the gut microbiota gut microbes influences ing liver and intestinal metabolism, immunity, and behavior has been published (5–8). However,, few studies have reported surprisingly little is reported on how the gut microbiota gut microbes regulates ing one of the dominant metabolic organs in the body, skeletal muscle. Here, we present evidence from germ-free and pathogen-free mice of gut microbiome-muscle axis that the gut microbiota influences skeletal muscle mass and , function. and communication between muscles and nerves possible through the NMJs.

Results

The gutGut microbiotaes modulateinfluence on skeletal muscle mass in mice

Germ-free (GF) mice (GF) that lacked gut microbiota displayed reduced weight of skeletal muscle mass compared to pathogen–free (PF) mice(PF) control mice that had gut microbiota (p<0.01; Fig. 1A). . Transplanting Exposing gGerm-freeF mice with the gut microbiota of to gut microbespathogen-free mice (henceforth referred as conventionalized germ-free mice, C-GF) (C-GF group) restored muscle mass in similar to that observed for PF micethe transplanted animals (C-GF) (p<0.05; Fig. 1A). Histological examination of the tibialis anterior a (fast oxidative) muscle (tibialis anterior, TA) revealed a trend towards fewer but, larger muscle fibers (>2000–3000 m2 cross-sectional fiber area) in gGerm-freeF mice compared to pathogen-free mice versus PF muscle (Fig. S1A and B). In addition, reduced expression of the myosin heavy chain genes (MyHC IIa, IIb, and IIx ) (Fig. 1B) were as observed in gGerm-freeF muscle compared to pathogen-free tibialis anterior TA muscle (p<0.05; Fig. 1B). A similar trend was also observed in the soleus ( slow-oxidative) muscle (soleus) and fast-glycolytic (the extensor digitorum longus (fast-glycolytic) , EDL) muscle of gGerm-freeF mice compared to pathogen-free mice s, (p<0.01; Fig. S1C and D). Furthermore, we observed increased expression expression analyses of E3 ubiquitin ligases in GF TA muscle tissue revealed up-regulation of Atrogin-1 and MuRF-1, encoding E3 ubiquitin ligases, known to be involved in muscle induce atrophy, in the tibialis anterior muscle of germ-free mice compared to pathogen-free mice. Both gene and protein expression of Atrogin-1 and MuRF-1 were increased in GF vs PF TA muscle (p<0.01; Fig. 1C, 1E, and 1F). Transplanting germ-free mice with the gut microbiota of pathogen-free mice reduced the eOf note, expression of both MuRF-1 and Atrogin-1 in were reduced anterior tibialis muscle of when GFthe C-GF mice (p<0.01; Fig. 1C). The expression of A were exposed to microbes (C-GF), indicating the influence of gut microbes on skeletal muscle mass. Atrogin-1 and MuRf-1 are known to be are regulated by FoxO transcription factors (9). While elevated expression of. An elevated expression of FoxO3 expression was was also observed in the tibialis anterior GF TA muscle of germ-free mice, the expression was normalized in the conventionalized germ-free mice that was restored upon exposure to gut microbes (C-GF) (p<0.05; Fig. 1C). The energy sensor AMP-activated protein kinase ( AMPK) controls muscle fiber size by through activating the FoxO-mediated protein degradation pathway (10). Further analysis of GF TAthe tibialis anterior muscle from germ-free mice revealed an increase in phosphorylation of the catalytic domain (Thr172) of AMPK the AMP-activated protein kinase (AMPK) (Fig. 1E and F)), suggesting that the AMPK-FoxO3-atrogenes cascade could be one possible signaling pathway to explain, at least in part, the atrophy observed in GF muscle tissues. Similar elevation in AMPK phosphorylation in gastrocnemius muscle of GF mice fed on a Western diet for 5 weeks was previously reported ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1073/pnas.0605374104","ISSN":"0027-8424","PMID":"17210919","abstract":"The trillions of microbes that colonize our adult intestines function collectively as a metabolic organ that communicates with, and complements, our own human metabolic apparatus. Given the worldwide epidemic in obesity, there is interest in how interactions between human and microbial metabolomes may affect our energy balance. Here we report that, in contrast to mice with a gut microbiota, germ-free (GF) animals are protected against the obesity that develops after consuming a Western-style, high-fat, sugar-rich diet. Their persistently lean phenotype is associated with increased skeletal muscle and liver levels of phosphorylated AMP-activated protein kinase (AMPK) and its downstream targets involved in fatty acid oxidation (acetylCoA carboxylase; carnitine-palmitoyltransferase). Moreover, GF knockout mice lacking fasting-induced adipose factor (Fiaf), a circulating lipoprotein lipase inhibitor whose expression is normally selectively suppressed in the gut epithelium by the microbiota, are not protected from diet-induced obesity. Although GF Fiaf-/- animals exhibit similar levels of phosphorylated AMPK as their wild-type littermates in liver and gastrocnemius muscle, they have reduced expression of genes encoding the peroxisomal proliferator-activated receptor coactivator (Pgc-1alpha) and enzymes involved in fatty acid oxidation. Thus, GF animals are protected from diet-induced obesity by two complementary but independent mechanisms that result in increased fatty acid metabolism: (i) elevated levels of Fiaf, which induces Pgc-1alpha; and (ii) increased AMPK activity. Together, these findings support the notion that the gut microbiota can influence both sides of the energy balance equation, and underscore the importance of considering our metabolome in a supraorganismal context.","author":[{"dropping-particle":"","family":"Bäckhed","given":"Fredrik","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Manchester","given":"Jill K","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Semenkovich","given":"Clay F","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gordon","given":"Jeffrey I","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Proceedings of the National Academy of Sciences of the United States of America","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2007","1","16"]]},"page":"979-84","title":"Mechanisms underlying the resistance to diet-induced obesity in germ-free mice.","type":"article-journal","volume":"104"},"uris":["http://www.mendeley.com/documents/?uuid=8289dd09-791b-3748-9f5e-cfd6819384f3"]}],"mendeley":{"formattedCitation":"(11)","plainTextFormattedCitation":"(11)","previouslyFormattedCitation":"(11)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(11), but not from mice in the normal physiological setting as shown in our study. It has beenis reported that Atrogin-1 expression regulates MyoD expression in skeletal muscle under atrophy atrophy conditions (112). We also observed noted reduced gene expression of key muscle genes that biomarkers regulateing skeletal muscle differentiation, such as MyoD and Myogenin, MyoD and Myogenin in gGerm-freeF skeletal muscle tissue compared to pPathogen-freeF skeletal muscle (p<0.01; Fig. 1D). Similar trends inof increased expression of atrophy markers such as Atrogin-1 and MuRF-1 with a concomitant down-regulation of MyoD expression were also observed in slow oxidative (soleus (slow oxidative) muscle) and fast glycolytic (extensor digitorum longus (fast glycolytic) , EDL) muscle of germ-free mices (p<0.001; Fig. S1 E-H). These results suggest that the phenotypic changes observed in GF conditions are not unique to muscle with specific metabolic characteristic but have a consistent impact on skeletal muscle as a consequence of the absence of gut microbes. Comment by Orla Smith: Please spell out germ free throughout the whole text to reduce the number of acronymsComment by Orla Smith [2]: Fig. 1 needs to be split into two figures as it is just too cramped. Please change the orange color (to anything but red) and please don’t use the split scale. Please renumber subsequent figuresComment by Orla Smith: Cite p value hereComment by Orla Smith: Cite p valueComment by Orla Smith: Spell out AMPK the first time it is usedComment by Orla Smith: Rest of this sentence belongs in the discussionComment by Orla Smith: This sentence has been deleted as it belongs in the discussionComment by Orla Smith: Cite p valuesComment by Orla Smith: Cite p valuesComment by Orla Smith: Last sentence has been deleted as it belongs in the discussion

Glucocorticoids are known to also induce skeletal muscle atrophy underin various pathological conditions (123). The transcription factor Kruppel Like Factor 15 (KLF15) ), a transcription factor, is one of the target genes activated by the glucocorticoid receptor and is involved in the regulation of several metabolic processes in skeletal muscle including, including the up-regulation of branched-chain aminotransferase 2 (BCAT2), which in turn induces the degradation of branched-chain amino acids (BCAAs) degradation (134). A surge in of corticosterone concentrations was observed in serum of gGerm-freeF mice (p<0.05; Fig. 2A1G), along with up-regulation of Klf15 expression in GF tibialis anterior TA muscle of germ-free (p<0.01; Fig. 2B1H). The observed GF muscle atrophy in gGerm-freeF mice was also associated with an activation of the enzymes involved in the BCAA catabolic pathway. BCAAs are trans-aminated by BCAT2 to generate branched-chain α-keto acids, which in turn are subjected to oxidative decarboxylation by branched-chain α-keto acid dehydrogenase (BCKDH) to produce Coenzyme A esters. BCKDH activity is negatively regulated by the BCKDH kinase (BCKDK). Increased gene expressions of Bcat and Bckdh were were observed in GF tibialis anterior muscle of germ-free mice, whereas ile the expression of the inhibitor, Bckdk, was reduced suggesting , supporting increased BCAA catabolism inin skeletal muscle tissue of gGerm-free miceF TA muscle (p<0.05; Fig. 2C1I). Comment by Orla Smith [2]: Authr, correct?Comment by Shawon: OK

Skeletal muscle mass is maintained by the balance between protein synthesis and protein degradation. To monitor potential decline in the protein synthesis pathway, we assessed the Igf1-Akt-mTOR growth-promoting pathway. While the Eexpression of the insulin-like-growth factor 1 gene (Igf1) in GF muscle was impaired in tibialis anterior muscle of germ-free mice, the Igf1 expression was normalized when (Fig.1J), but was restored germ-free mice were transplanted with gut microbiota of pathogen-free mice (p<0.01; Fig. 2D). Notably, upon exposure to microbes. Of note, Igf1 protein levels remain quantities remained unaltered in the serum of gGerm-freeF mice (Fig. S1I), suggesting an additional Igf1 regulatory loop(s), possibly involving the Igf binding proteins (IGFBPs) that are known to regulate Igf1 functions. that regulate Igf-mediated functions. Indeed, of the six IGFBPs, we observed exelevation of expression of pression of the Igfbp3 gene in tibialis anterior muscle of was induced in gGerm-freeF mice muscle tissue compared to pPathogen-freeF mice and C-GFC-GF mice (p<0.01; Fig. 2D1J),, in line with a previous observation (145). Increased expression of Elevated Igfbp3 expression is known to exert an inhibitory effect on skeletal muscle growth suggesting on regulatory loop to explain the reduced muscle mass in skeletal muscle of germ-free mice (156). However, Tthe activation profile of Akt, mTOR, and its downstream effector, the S6 ribosomal protein, were unaffected in muscles from germ-free mice GF muscle tissue (Fig. S1J and K). These data imply that multiple factors could activate the atrophy program in GF skeletal muscle. Comment by Orla Smith [2]: Author, correct?Comment by Shawon: OKComment by Orla Smith [2]: Sentence deleted as it belongs in the discussion

Altered metabolism Alterations of metabolic sensors in GF skeletal muscle of Germ-free mice

Given that Since skeletal muscle of tissue in gGerm-freeF mice showed clear signs of atrophy, we next investigated the oxidative metabolic capacity of skeletal muscle, which is important for energy provision for muscle function. Histological staining revealed reduced activity of the mitochondrial enzyme succinateic dehydrogenase (SDH) (Fig. 32A) and expression of the Sdh gene (p<0.05; Fig. 3B) in germ-free muscle compared to that observed in muscle from pathogen-free mice. ), which further validates the reduced expression of Sdh gene expression in GF muscle compared to PF (Fig. 2B). MMitochondrial DNA content was also reduced in gGerm-free F muscle,s but was restored when transplanted with gut microbiota from pathogen-free mice became restored after exposure to gut microbes (p<0.05; Fig. 32C). Additionally, we observed reduced gene expression of key mitochondrial biogenesis markers, such as peroxisome proliferator-activated receptor gamma coactivator1-alpha (Pgc1α) and mitochondrial transcription factor A (Tfam) in in GF sskeletal muscle of germ-free mice such as, Peroxisome proliferator-activated receptor gamma coactivator1-alpha (Pgc1α) and mitochondrial transcription factor A (Tfam) were observed in GF muscle (p<0.05; Fig. 32D). Moreover, we noticed reduced expression of genes encoding different mitochondrial oxidative phosphorylation complexes encoding genes in skeletal muscle of gGerm-freeF mice muscles, including genes encoding for cytochrome oxidase subunits of complex IV (CoxVa, CoxVIIb, and CytC), known to be which are involved in the electron transport chain (p<0.05; Fig. 32E). Similar observations supporting dysfunctional mitochondrial biogenesis program and oxidative capacity were also observed noted in two other skeletal muscle subtypes, soleus (oxidative) and extensor digitorum longus (glycolytic) EDL muscle of germ-free mice subtypes (p<0.01; Fig. S2 A-D). The as well as from protein expression profile of the different oxidative phosphorylation (OXPHOS) complexes of the electron transport chain (as observed in Fig S6 A) were also altered in muscle from germ-free mice. Together, these observations suggest a reduced oxidative capacity in both glycolytic and oxidative skeletal muscle in GF mice. DDespite a possible reduction in oxidative metabolic capacity, gGerm-free F mice performeded as well as pathogen-free mice exposed to gut microbiota (Fig. S2E) when challenged to exercise until exhaustion (Fig. S2E).

These results prompted us to undertake further metabolic analyses of germ-free mice. Analysis of serum metabolic markers revealed reduced quantities of glucose and insulin in gGerm-freeF mice compared to pPathogen-freeF mice (p<0.05; Fig. S2F). Further investigation of Addressing further metabolic pathways for fatty acid and glucose metabolism in gGerm-freeF mice muscle, revealed reduced that the expression of glycolytic genes encoding the enzymes phosphofructokinase (Pfk), pyruvate kinase (Pk), lactate dehydrogenase (Ldh), and pyruvate dehydrogenase (Pdh) in tibialis anterior muscle from germ-free mice. (p<0.05; Fig. 3F)were reduced in GF TA muscle Expressions of these enzymes were restored when germ-free mice were transplanted with the gut microbiota of pathogen-free mice but became normalized in C-GF mice (p<0.01; (Fig. 32F). A similar trend of reduced expression of glycolytic genes was also observed in the soleus and extensor digitorum longus EDL muscles of gGerm-freeF mice (p<0.01; Fig. S2GD and HE). No differences in gene expression of genes encoding medium-chain acyl-CoA dehydrogenase (Mcad) and muscle specific carnitine palmitoyltransferase1b (mCpt1b) involved in fatty acid oxidation (Fig. 32G) as well as phosphorylation of acetyl Co-A carboxylase (Fig. S2I and J) were observed in GF tibialis anterior TA muscle of germ-free mice. along with phosphorylation of acetyl Co-A carboxylase (ACC) (Fig. S2I and J). Cholesterol and free fatty acids in serum also remained unaltered in germ-free mice compared to pathogen-free mice (Fig. S2F). Further analysis of monitoring glucose uptake in GFskeletal muscles, mice using FDG-PET/MRI imaging, scanning revealed no obvious differences in glucose activity uptake for the back muscles and hindleg muscles of of germ-free GF when compared to pathogen-free mice mice vs PF mice (Fig. S2K and L). Similarly, unaltered glucose uptake in skeletal muscle of microbiota-depleted mice was reported in a recent study utilizing hyperinsulinemic-euglycemic clamp ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/nm.3994","ISSN":"1546-170X","PMID":"26569380","abstract":"Brown adipose tissue (BAT) promotes a lean and healthy phenotype and improves insulin sensitivity. In response to cold or exercise, brown fat cells also emerge in the white adipose tissue (WAT; also known as beige cells), a process known as browning. Here we show that the development of functional beige fat in the inguinal subcutaneous adipose tissue (ingSAT) and perigonadal visceral adipose tissue (pgVAT) is promoted by the depletion of microbiota either by means of antibiotic treatment or in germ-free mice. This leads to improved glucose tolerance and insulin sensitivity and decreased white fat and adipocyte size in lean mice, obese leptin-deficient (ob/ob) mice and high-fat diet (HFD)-fed mice. Such metabolic improvements are mediated by eosinophil infiltration, enhanced type 2 cytokine signaling and M2 macrophage polarization in the subcutaneous white fat depots of microbiota-depleted animals. The metabolic phenotype and the browning of the subcutaneous fat are impaired by the suppression of type 2 cytokine signaling, and they are reversed by recolonization of the antibiotic-treated or germ-free mice with microbes. These results provide insight into the microbiota-fat signaling axis and beige-fat development in health and metabolic disease.","author":[{"dropping-particle":"","family":"Suárez-Zamorano","given":"Nicolas","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Fabbiano","given":"Salvatore","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Chevalier","given":"Claire","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stojanović","given":"Ozren","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Colin","given":"Didier J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Stevanović","given":"Ana","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Veyrat-Durebex","given":"Christelle","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tarallo","given":"Valentina","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Rigo","given":"Dorothée","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Germain","given":"Stéphane","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ilievska","given":"Miroslava","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Montet","given":"Xavier","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Seimbille","given":"Yann","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Hapfelmeier","given":"Siegfried","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Trajkovski","given":"Mirko","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature medicine","id":"ITEM-1","issue":"12","issued":{"date-parts":[["2015","12","16"]]},"page":"1497-501","title":"Microbiota depletion promotes browning of white adipose tissue and reduces obesity.","type":"article-journal","volume":"21"},"uris":["http://www.mendeley.com/documents/?uuid=c9f23f20-ec27-3fc2-958a-78c90b680422"]}],"mendeley":{"formattedCitation":"(17)","plainTextFormattedCitation":"(17)","previouslyFormattedCitation":"(17)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(17). However, we observed an increased accumulation of glycogen in GF quadriceps (muscle (quadriceps; fast glycolytic) muscles of germ-free mice (p<0.001; Fig. 32H), implying possible impaired utilization of glucose by the quadriceps muscle by germ-free mice. the GF muscle.Comment by Orla Smith [2]: This sentence has been deleted as it belongs in the discussion

To further corroborate our findings, we assessed whether disruption of the gut microbiota–host environment, using antibiotics had effects on skeletal muscle. impart similar consequences on skeletal muscle. We monitored muscle samples from pPathogen-free F mice exposed to low-dose penicillin in order to examine consequences of (LDP) to disruption of host metabolic functions mediated by alterations in by altering microbial community composition (168, 179) . Whereas low-dose penicillin increased LDP imposed negative effects on muscle health by elevating FoxO3 gene expression and reduced ing MyoD gene expression in skeletal muscle of pathogen-free mice s (p<0.05; Fig. S3A), ; However, the expression of Atrogin-1, and MuRf-1, expressions remained unaffected as were Pgc1α and Tfam genes remained largely unaffected under these experimental conditions (Fig. S3A and B). However, Interestingly, markers of the electron transport chain genes (CoxVa, CoxVIIb, and CytC) were down regulated in pathogen-free mice treated with low-dose penicillin the LDP-treated group (p<0.05; Fig. S3C) indicating reduced oxidative metabolic capacity in skeletal muscle. Thus, exposing PF mice to low-doses of antibiotic appear to in part reduce oxidative metabolic capacity in skeletal muscle. A recent study showing that mice treated with the antibiotic metronidazole display skeletal muscle atrophy and changes in expression of metabolic genes further support our observations ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.3390/ijms19082418","abstract":"

Antibiotics lead to increased susceptibility to colonization by pathogenic organisms, with different effects on the host-microbiota relationship. Here, we show that metronidazole treatment of specific pathogen-free (SPF) mice results in a significant increase of the bacterial phylum Proteobacteria in fecal pellets. Furthermore, metronidazole in SPF mice decreases hind limb muscle weight and results in smaller fibers in the tibialis anterior muscle. In the gastrocnemius muscle, metronidazole causes upregulation of Hdac4, myogenin, MuRF1, and atrogin1, which are implicated in skeletal muscle neurogenic atrophy. Metronidazole in SPF mice also upregulates skeletal muscle FoxO3, described as involved in apoptosis and muscle regeneration. Of note, alteration of the gut microbiota results in increased expression of the muscle core clock and effector genes Cry2, Ror-β, and E4BP4. PPARγ and one of its important target genes, adiponectin, are also upregulated by metronidazole. Metronidazole in germ-free (GF) mice increases the expression of other core clock genes, such as Bmal1 and Per2, as well as the metabolic regulators FoxO1 and Pdk4, suggesting a microbiota-independent pharmacologic effect. In conclusion, metronidazole in SPF mice results in skeletal muscle atrophy and changes the expression of genes involved in the muscle peripheral circadian rhythm machinery and metabolic regulation.

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Altered NMR spectroscopy reveals distinct changes in metabolites in skeletal muscle, liver, and serum of in Germ-freeF mice

To characterize the metabolic phenotype of gGerm-freeF mice in detail, proton NMR spectra were measured at 600 MHz for a series of skeletal muscle, liver, and serum samples from germ-free mice, pathogen-free mice and conventionalized germ-free mice with gut microbiota from pathogen-free mice (Fig. 43). Cross-validated principal component analysis and orthogonal partial least squares analysis (OPLS-DA) models revealed showed strong differences in 1H NMR metabolic profiles for skeletal muscle, liver, and serum from between gGerm-freeF mice compared to vs pPathogen-freeF mice and from gGerm-freeF mice compared to conventionalized germ-free mice transplanted with gut microbiota from pathogen-free mice vs C-GF 1H NMR metabolic profiles of skeletal muscle, liver, and serum respectively (Fig. S4A, B and C). Transplanting germ-free mice with gut microbiota from pathogen-free mice normalized the metabolite profiles to that observed in pathogen-free mice. However, no differences were found PF and C-GF samples were compared. Below we report the results for:

Skeletal muscle. Our metabolic profiling identified large high quantities of amino acids such as alanine and glycine in skeletal muscle of gGerm-freeF mice skeletal muscle (Fig. 43A and Table 1). The gene expression of the gene encoding alanine transaminase (Alt) ) that results in transamination of alanine was also increased in muscle of gGerm-freeF mice muscle in comparison to their PF counterparts ( p<0.01; Fig. S5A). Typically, these amino acids - alanine and glycine, and their carbon skeletons, enter different metabolic pathways to generate energy. However, we did not observe any change in there was no significant change in ATP concentrations in the skeletal muscle of gGerm-free mice F mice when compared to the levels in pPathogen-free mice and C-GF mice transplanted with gut microbiota from pathogen-free mice F and C-GF mice (Fig. S5B). We next tested whether higher alanine levels in the muscle of in germ-freeGF mice muscle served as a source for hepatic gluconeogenesis through the glucose–alanine shuttle. Reduced alanine (Fig. 3B and Table 1) along with low expression of the gene encoding glucose-6-phosphatase gene (G6Pase), a gluconeogenic marker, were observed in the liver of germ-freeGF mice when compared to the pathogen- free mice liver (p<0.05; Fig. S5C).

A large high amount of glycine was observed in skeletal muscle of gGerm-free miceF skeletal muscle (Fig. 3A and Table 1). Apart from a possible increase in protein degradation of muscle tissue, glycine can be derived from intermediates of glycolysis through diversion of the glycolytic intermediate 3-phosphoglycerate into the serine synthesis pathway and by the ultimate conversion of serine to glycine (1821). However, no changes in expression of genes encoding key enzymes of this intermediate pathway, such as , phosphoglycerate dehydrogenase (Phgdh) and serine hydroxymethyltransferases (Shmt1 and Shmt2) , involved in this were intermediate pathway was observed in the muscle of gGerm-freeF mice muscle (Fig. S5D). Glycine can also be generated from choline via betaine, dimethylglycine, and sarcosine. Reduced quantities of choline and dimethylglycine along with higher amounts of glycine were found in the serum of gGerm-free miceF serum (Fig. 43C and Table 1).

In GF-liver, intermediates in the choline metabolic pathway, - glycerophosphocholine and betaine, were also reduced in the liver of germ-free mice (Fig. 43B and Table 1). However, However, no changes in gene expression of the genes encoding the enzymes involved in theose intermediate steps---- choline dehydrogenase (Chdh), betaine homocysteine s-methyltransferase (Bhmt), Bhmt2, and sarcosine dehydrogenase (Sardh) were observed in the skeletal muscle of gGerm-freeF mice when compared to pathogen-free miceskeletal muscle (Fig. S5E). While these findings warrant more detailed analyses, our observations imply that elevated quantities of amino acids such as glycine and alanine might be the result of the catabolic program in GF muscle to serve as an alternate energy source when living microbes don’t colonize the host. Furthermore, a recent study revealed activated metabolic pathways of amino acid biosynthesis specifically, glycine, in response to mitochondrial stress ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1083/jcb.201702058","ISBN":"1540-8140 (Electronic) 0021-9525 (Linking)","PMID":"28566324","abstract":"Mitochondrial stress activates a mitonuclear response to safeguard and repair mitochondrial function and to adapt cellular metabolism to stress. Using a multiomics approach in mammalian cells treated with four types of mitochondrial stressors, we identify activating transcription factor 4 (ATF4) as the main regulator of the stress response. Surprisingly, canonical mitochondrial unfolded protein response genes mediated by ATF5 are not activated. Instead, ATF4 activates the expression of cytoprotective genes, which reprogram cellular metabolism through activation of the integrated stress response (ISR). Mitochondrial stress promotes a local proteostatic response by reducing mitochondrial ribosomal proteins, inhibiting mitochondrial translation, and coupling the activation of the ISR with the attenuation of mitochondrial function. Through a trans-expression quantitative trait locus analysis, we provide genetic evidence supporting a role for Fh1 in the control of Atf4 expression in mammals. Using gene expression data from mice and humans with mitochondrial diseases, we show that the ATF4 pathway is activated in vivo upon mitochondrial stress. Our data illustrate the value of a multiomics approach to characterize complex cellular networks and provide a versatile resource to identify new regulators of mitochondrial-related diseases.","author":[{"dropping-particle":"","family":"Quirós","given":"Pedro M.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Prado","given":"Miguel A.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zamboni","given":"Nicola","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"D'Amico","given":"Davide","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Williams","given":"Robert W.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Finley","given":"Daniel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gygi","given":"Steven P.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Auwerx","given":"Johan","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Cell Biology","id":"ITEM-1","issue":"7","issued":{"date-parts":[["2017"]]},"page":"2027–2045","title":"Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals","type":"article-journal","volume":"216"},"uris":["http://www.mendeley.com/documents/?uuid=faa38788-5e19-4043-82a3-27cb36bd57ff"]}],"mendeley":{"formattedCitation":"(22)","plainTextFormattedCitation":"(22)","previouslyFormattedCitation":"(22)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(22). Higher glycine in GF skeletal muscle could probably be result of mitochondrial stress due to mitochondrial dysfunction as observed in GF muscle.Comment by Orla Smith [2]: Last part of this paragraph has been deleted as it belongs in the discussion

Liver. The metabolite profiles of liver in germ-freeGF mice liver were was characterized by high taurine, hypotaurine, and tauro-conjugated bile acids (taurocholic acid) affecting bile acid metabolism. , reflecting the influence of the gut microbiome in regulating bile acid metabolism ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/msb.2008.56","ISSN":"1744-4292","PMID":"18854818","abstract":"To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.","author":[{"dropping-particle":"","family":"Claus","given":"Sandrine P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tsang","given":"Tsz M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wang","given":"Yulan","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cloarec","given":"Olivier","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Skordi","given":"Eleni","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Martin","given":"François-Pierre","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Rezzi","given":"Serge","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ross","given":"Alastair","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kochhar","given":"Sunil","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Holmes","given":"Elaine","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Nicholson","given":"Jeremy K","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Molecular Systems Biology","id":"ITEM-1","issued":{"date-parts":[["2008","10","14"]]},"page":"219","title":"Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes","type":"article-journal","volume":"4"},"uris":["http://www.mendeley.com/documents/?uuid=fb82174b-aa2b-370b-8dd7-075b66f552a8"]}],"mendeley":{"formattedCitation":"(23)","plainTextFormattedCitation":"(23)","previouslyFormattedCitation":"(23)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(23). We also observed increased dimethylamine, a product of choline degradation in the liver of gGerm-freeF mouse liver ((Fig. 43B, and Table 1). Furthermore, glycerophosphocholine, betaine (tri-methyl-glycine), and oxidized glutathione were also present in lower amounts in liver of gGerm-freeF mice liver, indicating potential perturbation of the cysteine metabolic pathway even though the amount of sarcosine remained was high. In addition, glutamine, alanine, leucine, and valine along with glutamate were reduced in the liver of gGerm-free mice as compared to F liverpathogen-free mice. Pyruvate was lower in the liver of gGerm-free mice as Fcompared to pathogen-free mice liver. E and profiling of gene expression profiling of genes encoding enzymes involved in mitochondrial oxidative metabolism as well as glucose metabolism revealed reduced expression of several genes down-regulation of several marker genes oinvolved inf these pathways s indicating metabolic dysfunction perturbations inin liver of germ-free mice GFcompared to that of pathogen-free mice liver (p<0.01; Fig. S5F, and G).Comment by Orla Smith [2]: Rest of the sentence belongs in the discussionComment by Orla Smith [2]: Author, correct?Comment by sven pettersson: correct

Serum. Glycine amounts were was high in serum and muscle (same as in muscle) of gGerm-freeF mice compared to pathogen-free or conventionalized mice transplanted with gut microbiota of pathogen-free mice in comparison to PF and C-GF mice (Fig. 43C, and Table 1). However, amounts of choline, trimethylamine, and dimethylglycine were lower in serum of gGerm-freeF serumcompared to pathogen-free and transplanted germ-free mice. In . In addition, acetate, and pyruvate were also low in GF serum along with and 3-hydroxybutyrate were lower in serum of germ-free mice. , suggesting inefficient energy metabolism in GF mice. We also observed differences in serum lipid profiles with high amounts of Very-Low-Density-Lipoproteins ( VLDL) and low amounts of Low-Density-Lipoproteins ( LDL) quantities in gGerm-freeF mice compared to pathogen-free mice, in agreement with an earlier report (1924). Overall, the metabolomics data indicates disrupted energy homeostasis in germ-freeGF mice compared to pathogen-free mice, condition with a marked perturbation of the amino acid metabolic pathway, as illustrated in the metabolome-network map (Fig. 5 4).Comment by Orla Smith [2]: Spell outComment by Orla Smith [2]: Spell outComment by Orla Smith [2]: This needs to be made into a supplementary figure. Please renumber subsequent figuresComment by sven pettersson: I disagree as this capture elegantly the scope of the metabonomic analysis We should keep this

Altered expression of Neuro-Muscular Junction (NMJ) genes in Neuro-Muscular Junction (NMJ) genes are affected in GGerm-freeF mice

Given that we observed low quantities of choline in the serum of GF mice (Fig. 4C, Table 1), and that choline is a substrate for the derivation of the neurotransmitter acetylcholine and essential for membrane integrity (20), we screened an array of molecules that affect NMJ development and function including acetylcholine receptors (21). Choline is essential for membrane integrity and as substrate for the derivation of acetylcholine, a key signaling molecule of the NMJs ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISSN":"0022-3042","PMID":"7452263","abstract":"Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with alpha-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.","author":[{"dropping-particle":"","family":"Jope","given":"R S","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Jenden","given":"D J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of neurochemistry","id":"ITEM-1","issue":"2","issued":{"date-parts":[["1980","8"]]},"page":"318-25","title":"The utilization of choline and acetyl coenzyme A for the synthesis of acetylcholine.","type":"article-journal","volume":"35"},"uris":["http://www.mendeley.com/documents/?uuid=fb7bafa1-b2d2-34ea-94bd-ef8b9da0df37"]}],"mendeley":{"formattedCitation":"(25)","plainTextFormattedCitation":"(25)","previouslyFormattedCitation":"(25)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(25). Since we noted low quantities of choline in serum of GF mice (Fig. 3C and Table 1), we screened an array of molecules that affect NMJ development and function. Of these, the different acetylcholine receptors (Chrn) are important for the acetylcholine-mediated transmission of nerve impulses at the NMJs ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"ISSN":"0036-8075","PMID":"10521340","abstract":"Quantitative fluorescence imaging was used to study the regulation of acetylcholine receptor (AChR) number and density at neuromuscular junctions in living adult mice. At fully functional synapses, AChRs have a half-life of about 14 days. However, 2 hours after neurotransmission was blocked, the half-life of the AChRs was now less than a day; the rate was 25 times faster than before. Most of the lost receptors were not quickly replaced. Direct muscle stimulation or restoration of synaptic transmission inhibited this process. AChRs that were removed from nonfunctional synapses resided for hours in the perijunctional membrane before being locally internalized. Dispersed AChRs could also reaggregate at the junction once neurotransmission was restored. The rapid and reversible alterations in AChR density at the neuromuscular junction in vivo parallel changes thought to occur in the central nervous system at synapses undergoing potentiation and depression.","author":[{"dropping-particle":"","family":"Akaaboune","given":"M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Culican","given":"S M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Turney","given":"S G","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Lichtman","given":"J W","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Science (New York, N.Y.)","id":"ITEM-1","issue":"5439","issued":{"date-parts":[["1999","10","15"]]},"page":"503-7","title":"Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo.","type":"article-journal","volume":"286"},"uris":["http://www.mendeley.com/documents/?uuid=9d99faa8-1bbb-307f-a8e2-a0e43c0760a4"]}],"mendeley":{"formattedCitation":"(26)","plainTextFormattedCitation":"(26)","previouslyFormattedCitation":"(26)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(26). We Interestingly, we observed reduced reduced gene expression of genes encoding different acetylcholine receptor Chrn subunits (including Chrna1, Chrnb, Chrne, and Chrnd) in GF tibialis anterior muscle of germ-free mice (p<0.05; Fig. 6A) suggesting a potential impairment of assembly of acetylcholine receptors. TA muscle (Fig. 5A). We, therefore, monitored the expression of Furthermore, we probed for molecules rgenes associated with equired in the formation,development, maturation, and maintenance of NMJs, including. Rapsyn, a 43kDa receptor-associated synaptic protein of the synapse is necessary for acetylcholine receptor cluster formation relevant for NMJ development (227) and . Another protein important in NMJ formation is the low-density lipoprotein receptor related protein 4 (Lrp4), both reported to be thatimportant for the development and affects the assembly of acetylcholine receptors in NMJs (238). Expression of both Rapsyn and Lrp4 genes were reduced in GF mice, and this change was reversed when germ-free mice were transplanted with the gut microbiota of pathogen-free mice (p<0.05; Fig. 6B). Expressions of both Rapsyn and Lrp4 were reduced in GF mice, which reversed after exposure to gut microbes (C-GF) (Fig. 5B). Lrp4 associates with muscle-specific kinase (MuSK) to form a receptor complex necessary for agrin to bind (249). While Although we we noted elevated noted an elevation of gene expression of the MuSK gene, no change in expression of the , its ligand agrin (Agrn gene, encoding its ligand agrin, was observed in muscle of ) remained unaltered in gGerm-free miceF muscle (p<0.05; Fig. 65B). The While further experiments, including physiological experiments are required to reach definitive conclusion, our data suggest that the communication between muscle cells and nerve cells via the NMJs may be impaired in GF condition. Rreduced expressions of the gene encoding troponins in skeletal musle of germ-free mice suggested possiblegenes (Tnn) are consistent with impairment in possible myofiber contractility impairment in GF skeletal muscle (p<0.01; Fig. 65C). We, therefore, evaluated muscle strength in germ-free mice. Germ-free GF mice displayed reduced muscle strength compared in comparison to muscle from the pPathogen-freeF mice when examined in a for weightss test (p<0.05; Fig. 65D). Germ-free mice GF mice also exhibited reduced locomotor and rearing activity compared to pPathogen-freeF mice (p<0.01, Fig. 65E and p<0.001 Fig 6F).

Bacterial metabolites influence skeletal muscle mass in Germ-free mice

Germ-freeF mice have an elevated hypothalamic–pituitary–adrenal axis (HPA) activity as shown by, reflected through increased serum corticosterone (Fig. 2A1G). We therefore used an established in vitro cell culture model (2530) to study the effects of microbial bacterial metabolites on muscle against atrophy, induced by glucocorticoidss. DAs can be seen, dexamethasone treatment induced Atrogin-1 expression in differentiated C2C12 myotubes in vitro (p<0.001; Fig. 76A). A Of interest, a cocktail of short chain fatty acids (SCFAs), such as those generated by microbial fermentation of dietary polysaccharides, reduced the effect of dexamethasone-induced effect on Atrogin-1 expression (p<0.01; Fig. 76A). We also observed that SCFAs enhanced the expression of Pgc1a, Tfam (p<0.01; Fig. 76B), and that of CoxVa and CoxVIIb when administered to differentiated C2C12 myotubes in vitro (p<0.01; Fig. 76C), indicating that microbial metabolites can positively regulate oxidative metabolism in skeletal muscle of mice muscle under these experimental conditions. In addition, we monitored the effect of the SCFAs cocktail on both basal and maximal cellular respiration in C2C12 myotubes. Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP) stimulated respiration in mitochondria by uncoupling ATP synthesis from electron transport in C2C12 myotubes (Fig. S6 B and C). Under In FCCP-stimulationed conditions, treatment withwith the SCFA cocktail resulted in decreased oxygen consumption rate (p<0.01; OCR; Fig. S6 B) and an increased extracellular acidification rate (p<0.001; ECAR; Fig. S6 C) in C2C12 myotubes implying a metabolic switch from oxidative phosphorylation to a glycolytic mode of energy production. Bacterially produced SCFAs (and not glucose) were shown to be the primary energy source for colonocytes, but not for other tissues (e.g., liver, heart, kidney or testis) ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.cmet.2011.02.018","ISSN":"1932-7420","PMID":"21531334","abstract":"The microbiome is being characterized by large-scale sequencing efforts, yet it is not known whether it regulates host metabolism in a general versus tissue-specific manner or which bacterial metabolites are important. Here, we demonstrate that microbiota have a strong effect on energy homeostasis in the colon compared to other tissues. This tissue specificity is due to colonocytes utilizing bacterially produced butyrate as their primary energy source. Colonocytes from germfree mice are in an energy-deprived state and exhibit decreased expression of enzymes that catalyze key steps in intermediary metabolism including the TCA cycle. Consequently, there is a marked decrease in NADH/NAD(+), oxidative phosphorylation, and ATP levels, which results in AMPK activation, p27(kip1) phosphorylation, and autophagy. When butyrate is added to germfree colonocytes, it rescues their deficit in mitochondrial respiration and prevents them from undergoing autophagy. The mechanism is due to butyrate acting as an energy source rather than as an HDAC inhibitor.","author":[{"dropping-particle":"","family":"Donohoe","given":"Dallas R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Garge","given":"Nikhil","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Zhang","given":"Xinxin","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sun","given":"Wei","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"O'Connell","given":"Thomas M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bunger","given":"Maureen K","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bultman","given":"Scott J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Cell metabolism","id":"ITEM-1","issue":"5","issued":{"date-parts":[["2011","5","4"]]},"page":"517-26","title":"The microbiome and butyrate regulate energy metabolism and autophagy in the mammalian colon.","type":"article-journal","volume":"13"},"uris":["http://www.mendeley.com/documents/?uuid=5afc489a-494d-303f-93ac-f8af59e5aa9e"]},{"id":"ITEM-2","itemData":{"DOI":"10.1371/journal.pone.0046589","ISSN":"1932-6203","PMID":"23029553","abstract":"A prodigious number of microbes inhabit the human body, especially in the lumen of the gastrointestinal (GI) tract, yet our knowledge of how they regulate metabolic pathways within our cells is rather limited. To investigate the role of microbiota in host energy metabolism, we analyzed ATP levels and AMPK phosphorylation in tissues isolated from germfree and conventionally-raised C57BL/6 mice. These experiments demonstrated that microbiota are required for energy homeostasis in the proximal colon to a greater extent than other segments of the GI tract that also harbor high densities of bacteria. This tissue-specific effect is consistent with colonocytes utilizing bacterially-produced butyrate as their primary energy source, whereas most other cell types utilize glucose. However, it was surprising that glucose did not compensate for butyrate deficiency. We measured a 3.5-fold increase in glucose uptake in germfree colonocytes. However, (13)C-glucose metabolic-flux experiments and biochemical assays demonstrated that they shifted their glucose metabolism away from mitochondrial oxidation/CO(2) production and toward increased glycolysis/lactate production, which does not yield enough ATPs to compensate. The mechanism responsible for this metabolic shift is diminished pyruvate dehydrogenase (PDH) levels and activity. Consistent with perturbed PDH function, the addition of butyrate, but not glucose, to germfree colonocytes ex vivo stimulated oxidative metabolism. As a result of this energetic defect, germfree colonocytes exhibited a partial block in the G(1)-to-S-phase transition that was rescued by a butyrate-fortified diet. These data reveal a mechanism by which microbiota regulate glucose utilization to influence energy homeostasis and cell-cycle progression of mammalian host cells.","author":[{"dropping-particle":"","family":"Donohoe","given":"Dallas R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wali","given":"Aminah","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Brylawski","given":"Bruna P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bultman","given":"Scott J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"PloS one","editor":[{"dropping-particle":"","family":"Bereswill","given":"Stefan","non-dropping-particle":"","parse-names":false,"suffix":""}],"id":"ITEM-2","issue":"9","issued":{"date-parts":[["2012","9","28"]]},"page":"e46589","title":"Microbial regulation of glucose metabolism and cell-cycle progression in mammalian colonocytes.","type":"article-journal","volume":"7"},"uris":["http://www.mendeley.com/documents/?uuid=834743a1-9215-35ef-ba1a-78bec314fc8d"]},{"id":"ITEM-3","itemData":{"ISSN":"0002-9513","PMID":"1122009","abstract":"The ADP:O ratios and State 3 (ADP-stimulated) and State 4 (controlled) rates of succinate, beta-hydroxybutyrate, isocitrate, glutamate, pyruvate + malate, alpha-ketoglutarate, and ascorbate + N,N,N',N'-tetramethylphenylenediamine (TMPD) oxidation were examined in liver mitochondria from germ-free and conventional rats of both Lobund Wistar (100-day-old) and Fisher (120-day-old) strains. The State 3 respiration rates of isolated mitochondria from germ-free and conventional rats were comparable except for the rate of succinate oxidation in the Wistar rats, which was significantly lower (approx. 10%). The State 4 respiration rates were generally lower in mitochondria isolated from germ-free Fisher rats (approx. 8%) and significantly lower (approx. 18%) in germ-free Wistar rats. The ADP:O ratios were similar in germ-free and conventional rats. Serum thyroxine concentrations indicated delayed maturation of thyroid function in young germ-free rats, but adult animals had concentrations similar to those found in conventional rats. The results indicate that, although absence of a microflora results in a 20-30% reduction in metabolic rate, the germ-free state has little influence on the functional respiration or oxidative phosphorylation of mitochondria isolated from the liver of the adult rat.","author":[{"dropping-particle":"","family":"Sewell","given":"D L","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Wostmann","given":"B S","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gairola","given":"C","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Aleem","given":"M I","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"The American journal of physiology","id":"ITEM-3","issue":"2","issued":{"date-parts":[["1975","2"]]},"page":"526-9","title":"Oxidative energy metabolism in germ-free and conventional rat liver mitochondria.","type":"article-journal","volume":"228"},"uris":["http://www.mendeley.com/documents/?uuid=723c1ec6-d780-3701-99d0-440aedcbe140"]}],"mendeley":{"formattedCitation":"(31–33)","plainTextFormattedCitation":"(31–33)","previouslyFormattedCitation":"(31–33)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(31–33). However, additional studies are warranted in this regard in GF skeletal muscle.

We also treated GF mice in vivo with a cocktail of SCFAs. We observed We also performed in vivo treatment of GF mice with a cocktail of SCFAsa and observed a trend of increased of weight gain in skeletal muscle mass, when treated with SCFAs particularly in the case of the gastrocnemius muscle (p<0.05; Fig. 76D) in germ-free mice treated with SCFAs compared to untreated germ-free mice in comparison to GF mice. Furthermore, In agreement with this observation, in vivo treatment of germ-free mice with SCFAss treatment, resulted in reduced the expression of Atrogin-1 (p<0.0001; Fig. 76E) although expression of in TA muscle, though interestingly, MuRf-1 was higher expression was higher in tibialis anterior TA muscle of SCFAs treated GF mice (p<0.001; Fig. 76E). We also noted However, with SCFAs treatment increased expression of MyoD , a marker associated with myogenesis, in tibialis anterior was elevated in TA muscle of SCFA-treated germ-free mice compared to untreated germ-free mice. indicating enhanced myogenesis (p<0.05; Fig. 76E). However, we did not Nobserve changes in o alterations in gene expressionons of genes encoding the mitochondrial transcription factors the mitochondrial transcription factors (Pgc1α and Tfam (; Fig. 76F) or markers of the electron transport chain proteins (CoxVa, CoxVIIb, and CytC; (p<0.01; Fig. 76G) were observed in the tibialis anterior TA muscle of SCFA-s treated gGerm free F mice compared to untreated micemice in comparison to GF mice. Importantly, SCFAs treatment improved muscle ular strength of gGerm-free F mice in the when examined for weights s test compared to untreated GF mice (Fig. 76H). No alterations in gene expression of the NMJ markers, Rapsyn and MuSK, were observed in tibialis anterior muscle of SCFA- treated mice compared to untreated GF mice TA muscles (Fig. 76I). These observations indicate that replenishment with SCFAs in part revert impaired muscle functions observed in GF mice. However, additional microbial products other than SCFAs might also be involved in skeletal muscle growth and function.Comment by Orla Smith [2]: This belongs in the discussion and has been deleted

Discussion

Here we show that the gut microbiota contributes to modulate We have identified a gut microbiome-muscle axis that regulates skeletal muscle mass and function in mice. Germ-free mice Further, GF muscle tissue displayed reduced muscle mass and signs of muscle muscle atrophy through increased expression of FoxO, Atrogin-1, Murf-1 and MyoD along with with reduced muscle strength.

Activation of AMPK in germ-free mouse muscle further suggests that the AMPK-FoxO3-atrogenes cascade could be one possible signaling pathway to explain, at least in part, the atrophy observed in germ-free muscle tissues. Similar elevation in AMPK phosphorylation in gastrocnemius muscle of germ-free mice fed on a Western diet for 5 weeks was previously reported (26), but not from mice in the normal physiological setting as shown in our study. In addition, , activating the FoxO, Atrogin-1, and MyoD pathway, resulting in reduced muscle weight. Skeletal muscle growth also depends on IGF1-mediated cascades, which operates in an autocrine/paracrine manner. Oour results suggest alteration in observations suggest involvement of locally produced IIGF1 protein expression locally in in the muscle of germ-free mice, GF skeletal muscle rather than peripheral effects of circulating IGF-1. The However, the protein synthesis mechanism through the Akt-mTOR pathway appeared to be unaffected in muscle of germ-free mice. This remained unaffected suggestsing that protein degradation exceeded protein synthesis, which in part could contribute to the reduced , which resulted in the loss of skeletal muscle mass observed in in germ-free miceGF mice. Our observations of increased corticosterone concentrations in serum, induction of followed by induced Klf15 gene expression and activation of the BCAA pathway indicate a possible HPA-glucocorticoid driven atrophy of skeletal muscle mass in germ-free miceprovides one mechanistic insight to the possible pathways resulting in reduced skeletal muscle mass in GF mice. Skeletal muscle atrophy observed in mice maintained under germ-free conditions, thus, might be the consequence of dysregulation in several signaling pathways controlling skeletal muscle mass. Skeletal muscle atrophy as observed in GF conditions thus might be the consequence of not a single but, of multifactorial deregulation in signaling pathways controlling skeletal muscle mass. The reduced levels of choline in serum, along with marked changes of gene expression of Marked changes observed in the expression of NMJ linked proteins markers of NMJin muscle of s of gGerm-free mice, defective locomotion and F muscle correlate with reduced concentrations of choline, locomotion and grip strength capacity, are therefore possible additional factors affecting muscle mass and function in germ-free mice. Furthermore, our observations from muscles of different metabolic subtypes suggest that the phenotypic changes observed in germ-free mice are not unique to muscle with a specific metabolic characteristic but have a consistent impact on skeletal muscle as a consequence of the absence of gut microbiota. consistent with a defective assembly of NMJs resulting in reduced muscle function. Impairment of NMJs has been suggested to be one mechanism underlying loss of muscle mass in the elderly ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1523/JNEUROSCI.3590-11.2011","ISSN":"1529-2401","PMID":"22016524","abstract":"Vertebrate neuromuscular junctions are highly stable synapses, retaining the morphology they achieve in early postnatal development throughout most of life. However, these synapses undergo dramatic change during aging. The acetylcholine receptors (AChRs) change from smooth gutters into fragmented islands, and the nerve terminals change similarly to be varicosities apposed to these islands. These changes have been attributed to a slow deterioration in mechanisms maintaining the synapse. We have used repeated, vital imaging to investigate how these changes occur in the sternomastoid muscle of aging mice. We have found, contrary to expectation, that individual junctions change infrequently, but change, when it occurs, is sudden and dramatic. The change mimics that reported previously for cases in which muscle fibers are deliberately damaged: most of the AChRs present disappear rapidly and are replaced by a new set of receptors that become fragmented. The fiber segment underneath the synapse has centrally located nuclei, showing that this segment has undergone necrosis, quickly regenerated, and been reinnervated with an altered synapse. We show that necrotic events are common in aged muscle and have likely been missed previously as a cause of the alterations in aging because central nuclei are a transient phenomenon and the necrotic events at the junction infrequent. However, the changes are permanent and accumulate over time. Interventions to reduce the neuromuscular changes during aging should likely focus on making muscle fibers resistant to injury.","author":[{"dropping-particle":"","family":"Li","given":"Yue","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"il","family":"Lee","given":"Young","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Thompson","given":"Wesley J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"The Journal of neuroscience : the official journal of the Society for Neuroscience","id":"ITEM-1","issue":"42","issued":{"date-parts":[["2011","10","19"]]},"page":"14910-9","title":"Changes in aging mouse neuromuscular junctions are explained by degeneration and regeneration of muscle fiber segments at the synapse.","type":"article-journal","volume":"31"},"uris":["http://www.mendeley.com/documents/?uuid=c94c61fa-8470-3e6a-ac2c-fe77e9b6deff"]}],"mendeley":{"formattedCitation":"(34)","plainTextFormattedCitation":"(34)","previouslyFormattedCitation":"(34)"},"properties":{"noteIndex":0},"schema":"https://github.com/citation-style-language/schema/raw/master/csl-citation.json"}(34). Our findings may have implications for future gut microbiome–based therapeutic strategies aiming to support muscle growth during aging and attenuate frailty and sarcopenia. Comment by Orla Smith [2]: Where did you show this? I think this is an overstatementComment by Shawon: We have shown this through tissue-specific gene expression levels of Igf-1 and Igfbps (Fig 2D) and Igf1 protein levels estimation in serum (Fig S1.I). Please check. We have provided evidence to this sentence.Comment by Orla Smith [2]: Author this is speculation and has been deleted as your study is all in mice and we don’t know if the same situation holds for humans

Gut microbes biosynthesize amino acids and make them available to the host (3527). AIntegrated analyses of skeletal muscle, liver, and serum metabolites metabolomes revealed altered amino acid metabolism metabolic networks in GF mice. Glycine and alanine were increased in the discriminant metabolites identified in GF compared to PF mouse skeletal muscle. Glycine has important implications in skeletal muscle wasting (2836), acts as an energy source for muscle (2937) and is also induced in response to mitochondrial stress (2230). BCAA catabolism is associated with skeletal muscle protein breakdown, as and was observed in GF muscle tissue and concordant with correlated with low leucine and valine concentrations in the liver tissue and serum of germ-free mice. Decreased quantities of BCAAs, as seen in liver and serum of gGerm-freeF mice, have been (valine and leucine), are associated with severe catabolic conditions, such as cachexia in and terminal stages of cancer (318, 329). BCAAs not only serve as an alternate energy substrate but also are known to improve nitrogen retention and protein synthesis (3340). They represent the major nitrogen source for alanine synthesis in muscle. Prior to their its usage as fuel and being fed into the tricarboxylic acid cycle, BCAAs undergo a series of trans-amination steps eventually resulting in production of alanine. High quantities of alanine along with increased Alt gene expression observed in msucle of gGerm-freeF mice muscle might thus be a consequence of muscle protein breakdown resulting in increased bioavailability of amino acids to cope with the stress generated due to the absence of gut microbiota gut microbes. Comment by Orla Smith [2]: Author, correct?Comment by Orla Smith [2]: Author, correct?

Many interactions between the host and its the gut microbiota me are mediated by SCFAs generated from the bacterial fermentation of dietary polysaccharides (34-36). From our observations, SCFAs support, in part, skeletal muscle function by preventing atrophy and increasing muscular strength. However, the diverse biosynthetic activity of the gut microbiota me beyond other than providing SCFAs to be used by the host as an energy source may explain why supplementingonly SCFAs supplementation s alone toin germ-freeGF mice could not fully rescue the muscle impairment phenotype. However, the current study has not implicated on a specific signaling mechanism involved through which the gut microbes modulate skeletal muscle function. Microbes and their metabolites are likely to engage multiple converging pathways to regulate host muscle growth and function. Additional microbial products other than SCFAs might also be involved in skeletal muscle growth and function. Our and this study has elucidated several more than one underlying molecular mechanisms supporting regulation of skeletal muscle mass and function in gGerm-freeF mice.. Further experiments are therefore warranted. While, germ-free mice,

While use of germ free mice as an animal model used to study microbe-host interactions is an artificial system, these mice devoid of exposure to microbes, , provide opportunities to study analyses using GF mice offers the possibility to identify and understand microbe-host interactions in vivo. Deficits in the physiological and metabolic processes due to maintaining mice under germ-free conditions upbringing along with less developed organ functions in GF mice are well documented and, are likely to include also skeletal muscle. The use of low dose antibiotic treatment helped us to corroborate support our observations in from germ-freeGF mice that implied the lack of gut microbiota in reduced muscle mass and function. A recent study showing that mice treated with the antibiotic metronidazole display skeletal muscle atrophy and changes in expression of metabolic genes further support our observations (37). However, the muscle tissue samples collected from the antibiotic experiment was obtained from pathogen-free mice used in this experimental setup came from animals raised in a different animal facility and hence, finding should be interpreted cautiously. While our observations also suggest that the communication between muscle cells and nerve cells via the NMJs may be impaired in germ-free condition, further experiments, including physiological experiments are required to reach definitive conclusion. Additional studies are also warranted to identify specific bacterially produced metabolites or other microbial products to influence skeletal muscle growth and function. In conclusion, we have demonstrated the existence of a gut microbiota-skeletal muscle axis that opens for further mechanistic and physiological studies for a better understanding of mechanisms regulating this highly important metabolic organ by the gut microbiota. The observations reported here should thus be interpreted cautiously. In conclusion, we have identified a gut microbiome-skeletal muscle axis that has considerable implications to influence disease- and/or age-related loss of skeletal muscle mass and function and support sustained health.Comment by Orla Smith [2]: Author, please expand on discussion of limitations in the final paragraphComment by Orla Smith [2]: I don’t understand this sentence. Please clarify. You mean you had two colonies of GF mice??

Materials and MethodsComment by Orla Smith [2]: Please integrate supplementary materials and methods into main materials and methods

Study design

: The objective of thise study was to elucidate decipher the functional interactions between the gut microbiotaes and the host in the regulation of ng skeletal muscle mass and function. Muscle was It was assessed in germ-free mice and pathogen-free mice maintained on autoclaved R36 Lactamin chow (Lactamin), provided with sterile drinking water ad libitum, and kept under 12-h light/dark cycles in state-of-art germ-free animal housing facilities. by Sexamining skeletal muscle mass was examined and through characterizing differences in gene expression of genes encoding different cell signaling molecules and pathways that regulate muscle mass were characterized. Detailed metabolic characterization was also performed using utilizing NMR spectrometric analyses of skeletal muscle, along with liver and serum samples. The influence of the gut microbiota me on NMJs neuromuscular junction (NMJ) was also examined by analyzing expression of through NMJ proteins and testing mouse muscle strength in the expression along with grip strength test in germ-free mice compared to pathogen-free mice. The gut microbiota’s role in muscle mass and function was further analysed by transplanting The observations were further corroborated exposing gGerm-free F mice with the gut microbiota of pathogen-free animals or treating them with to gut microbes and microbial metabolites (short chain fatty acids). We further examined the effects alterations of gut microbiota. composition on muscle by treating pathogen-free mice with if alterations in gut microbiota composition using antibiotic (low-dose penicillin. Three independent cohorts of pathogen-free, germ-free and conventionalized germ-free groups have been used with a minimum of 5 mice included in each experimental group. Blinding of researchers about the knowledge of group allocation has been ensured along with randomization of the groups to limit the occurrence of conscious and unconscious bias during experimentation. ) also affects skeletal muscle markers.Comment by Orla Smith [2]: Need to state how the animals were housed differently in one sentence and then expand under animals subheading

Animals

. Germ-freeF and pathogen-freePF C57BL/6J male mice (6 to 8 weeks old) were raised in the Core Facility for Germ-Free Research, Karolinska Institutet, Sweden, and LKC School of Medicine GF research facility, Singapore, in accordance with the regulatory standards of each institution. All experiments were approved by respective local ethical committees. Germ-free mice were raised in special sterile isolators. Isolator sterility was analyzed weekly by plating fecal homogenates onto different types of agar plates to detect aerobic and anaerobic bacteria and fungi. To transplant germ-free with gut microbiota of pathogen-free mice, germ-free mice were conventionalized (C-GF) post-weaning by gavage of 100 µl of homogenate generated by dissolving two fecal pellets from pathogen-free mice in 1 ml of PBS. After gavage, C-GF mice were co-housed with pathogen-free mice for 21 days before they were sacrificed. The control group was gavaged with sterile PBS. Mice were sacrificed under isoflurane anesthesia. The behavioral assays were conducted in accordance with national guidelines for the care and use of laboratory animals for scientific purposes, with approved protocols from the Institutional Animal Care and Use Committees of Duke-NUS Graduate Medical School, Singapore. All animals were maintained on autoclaved R36 Lactamin chow (Lactamin), provided with sterile drinking water ad libitum, and kept under 12-h light/dark cycles. Three independent cohorts of pathogen-freePF, germ-freeGF and C-GFgerm-free mice transplanted with gut microbiota of pathogen-free mice groups have been performedwere used with a minimum of n=55 mice in each group. The behavioral assays were conducted in accordance with national guidelines for the care and use of laboratory animals for scientific purposes, with approved protocols from the Institutional Animal Care and Use Committees of Duke-NUS Graduate Medical School, Singapore. Details are provided in the supplementary methods section.Comment by Orla Smith [2]: Please move this information from the supp material into main materials and methods

SCFAs treatment of germ-free mice. Germ-free mice (6–8 weeks of age) were treated with a cocktail of SCFAs as per a previously published protocol (38). Briefly, mice (n=6 per group) were fed a mix of SCFAs (67.5 mM sodium acetate, 40 mM sodium butyrate, 25.9 mM sodium propionate) in their drinking water for a period of 4 weeks. No decrease in water intake was noted, and mice were monitored for signs of dehydration. Sterility of the GF and SCFAs-treated GF cohort was assessed twice a week and maintained throughout this study. After 4 weeks, mice were euthanized by inhalation of isoflurane and sacrificed, and muscles were harvested. TA muscles were used for qPCR analyses.

Antibiotic treatment.C57BL/6J mice were given low-dose penicillin (1 µg/g body weight, LDP) via drinking water or no antibiotics (control) for 4 weeks post-weaning as previously reported (16).

Weights test.

. Weight tests were performed to measure muscular strength as described previously (3941). Details of the test are provided in the supplementary methods section. Briefly, each mouse was held by the middle of the tail and slowly lowered to grasp the first weight (26 g) in a well-lit fume hood. Upon grasping the wire scale, mice were raised until the weight was fully raised above the bench. The criterion was met if the mouse could hold the weight for 3 s. If the weight was dropped in before 3 s, the time was noted, and the mouse rested for 10 s before performing a repeat trial. Each mouse was allowed a maximum of 5 trials before being assigned the maximum time achieved; if it successfully held the weight for 3 s, it was allowed to progress to the next heaviest weight. The apparatus comprised 6 weights, weighing 26g, 33g, 44g, 63g, 82g, and 100g. All cage mates of each group were tested using a given weight before progressing to the next-heaviest weight. The total score for each mouse was then calculated as the product of the number of links in the heaviest chain held for the full 3 s, multiplied by the time (s) it was held.

Activity in the open field task.

. Locomotor and rearing activities were monitored for individual mice using an automated Omnitech Digiscan apparatus (AccuScan Instruments, Columbus, OH, USA), as described previously (402). Mice were monitored in the open field for 60 min. Locomotion was measured as total distance traveled and rearing as vertical activity.

Treadmill running task.

. Eight-week-old mice were subjected to exercise on a treadmill (Columbus, OH, USA) using a previously reported exercise regimen (413). Mice in all groups were acclimated to moderate treadmill running (10 m/min for 15 min; 5 incline) for 2 days. After acclimation, for the exercise test, mice ran on a treadmill set at a gradually increasing speed from 0 to 15 m/min and then maintained at a constant speed until exhaustion. Mice were removed from the treadmill upon exhaustion and had free access to food and water after the running protocol was completed. Mice were sacrificed 3 h after the exercise test.

PET-MRI. Mice were fasted for 4 h prior to the imaging session. They were imaged under anesthesia using a nanoScan PET/MRI scanner (Mediso, Hungary) under anesthesia (42). The whole body MRI images were acquired with the 1 T MRI component of the nanoScan PET/MRI scanner. All images and data analyses for the FDG PET images were performed using PMOD (version 3.5; PMOD Technologies).

PET acquisition: A 40 min long 3D static PET scan was performed after IV injection of FDG; the animals were moving freely during the waiting period. The injected radioactivity was 10.0–15 MBq, and the maximum injected volume was 0.1 ml.

MRI acquisition. A 35-mm mouse whole body coil was used for the MRI scan. 0.8 mm slices were obtained using a 2D T1 SD coronal sequence with 100-mm square FOV, 256 × 256 matrix, 1034-ms repetition time (TR), 12.4-ms echo time (TE), and 90 degree flip angle.

Image analysis: The PET images were first automatically registered to the MRI images using the FUSION tool in PMOD. The MRI images were then used for the manual delineation of volumes of interest (VOI). VOIs for hind leg muscle and back muscle were used for the analysis.

Antibiotic treatment

. C57BL/6J mice were given low-dose penicillin (1 µg/g body weight, LDP) via drinking water or no antibiotics (control) for 4 weeks post-weaning as previously reported ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1016/j.cell.2014.05.052","ISSN":"1097-4172","PMID":"25126780","abstract":"Acquisition of the intestinal microbiota begins at birth, and a stable microbial community develops from a succession of key organisms. Disruption of the microbiota during maturation by low-dose antibiotic exposure can alter host metabolism and adiposity. We now show that low-dose penicillin (LDP), delivered from birth, induces metabolic alterations and affects ileal expression of genes involved in immunity. LDP that is limited to early life transiently perturbs the microbiota, which is sufficient to induce sustained effects on body composition, indicating that microbiota interactions in infancy may be critical determinants of long-term host metabolic effects. In addition, LDP enhances the effect of high-fat diet induced obesity. The growth promotion phenotype is transferrable to germ-free hosts by LDP-selected microbiota, showing that the altered microbiota, not antibiotics per se, play a causal role. These studies characterize important variables in early-life microbe-host metabolic interaction and identify several taxa consistently linked with metabolic alterations. PAPERCLIP:","author":[{"dropping-particle":"","family":"Cox","given":"Laura M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Yamanishi","given":"Shingo","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Sohn","given":"Jiho","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"V","family":"Alekseyenko","given":"Alexander","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Leung","given":"Jacqueline M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Cho","given":"Ilseung","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Kim","given":"Sungheon G","non-dropping-p