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Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes

Abstract

Both obesity and sarcopenia are frequently associated in ageing, and together may promote the progression of related conditions such as diabetes and frailty. However, little is known about the pathophysiological mechanisms underpinning this association. Here we show that systemic alanine metabolism is linked to glycaemic control. We find that expression of alanine aminotransferases is increased in the liver in mice with obesity and diabetes, as well as in humans with type 2 diabetes. Hepatocyte-selective silencing of both alanine aminotransferase enzymes in mice with obesity and diabetes retards hyperglycaemia and reverses skeletal muscle atrophy through restoration of skeletal muscle protein synthesis. Mechanistically, liver alanine catabolism driven by chronic glucocorticoid and glucagon signalling promotes hyperglycaemia and skeletal muscle wasting. We further provide evidence for amino acid–induced metabolic cross-talk between the liver and skeletal muscle in ex vivo experiments. Taken together, we reveal a metabolic inter-tissue cross-talk that links skeletal muscle atrophy and hyperglycaemia in type 2 diabetes.

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Fig. 1: Alanine metabolism and liver ALT isoform expression is upregulated in obesity and type 2 diabetes.
Fig. 2: Silencing of both, but not individual, liver ALT isoforms affects systemic alanine and glucose homeostasis after diverse nutritional challenges.
Fig. 3: Silencing of both, but not individual, liver alanine aminotransferase isoforms retards hyperglycaemia in mouse models of obesity-related type 2 diabetes.
Fig. 4: Disrupting the heightened liver alanine catabolism improves skeletal muscle size and function in mouse models of type 2 diabetes.
Fig. 5: A glucocorticoid–liver glucocorticoid receptor axis links heightened liver alanine catabolism with hyperglycaemia and muscle atrophy in type 2 diabetes.
Fig. 6: Chronic liver glucagon action affects hyperglycaemia and skeletal muscle atrophy via hepatic alanine catabolism.
Fig. 7: Disrupting the heightened liver alanine catabolism reverses the reduced skeletal muscle protein synthesis and branched-chain amino acid levels in type 2 diabetes.

Data availability

Correspondence and requests for materials including data that support the findings of this study should be made to the corresponding author. Source data are provided with this paper.

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Acknowledgements

The authors thank J. Fuhrmeister, A. Maida and T. Gantert (A170, DKFZ), L. Figur (A171, DKFZ), as well as J. Hetzer and D. Heide (F180, DKFZ) for experimental support. J.S. was supported by a fellowship from the Helmholtz International Graduate School for Cancer Research. M.H. was supported by an ERC Consolidator grant (HepatoMetaboPath), SFBTR 209 (Liver Cancer) and SFBTR179. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 667273. This work was funded by the Helmholtz Future Topic ‘Aging and metabolic reprogramming’ (to S.H.). This work was was also funded by a project grant from the EFSD/Lilly European Diabetes Research Programme, as well as a Medical Research Grant from the Sir Edward Dunlop Medical Research Foundation (to A.J.R.).

Author information

Authors and Affiliations

Authors

Contributions

Project conceptualization, administration and management: A.J.R. Resources: J.G.O., M.B., S.H., O.J.M., M.A.K., M.H. and A.J.R. Investigation: J.G.O., J.S., K.V.S., K.M.R.-T., R.D.R., A.Z., A.J., L.M., M.B., M.H. and A.J.R. Software and formal analysis: P.R. and A.J.R. Writing, original draft: A.J.R. Writing, editing: J.G.O., P.M.R., J.S., R.D.R., M.B., S.H., O.J.M., M.A.K., M.H. and A.J.R. Visualization: P.R., M.H. and A.J.R. Funding acquisition: S.H., M.H. and A.J.R.

Corresponding author

Correspondence to Adam J. Rose.

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All authors declare no competing interests.

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Peer review information Nature Metabolism thanks Sue Bodine, Kei Sakamoto and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt.

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Extended data

Extended Data Fig. 1 Alanine metabolism and liver ALT isoform expression is upregulated in obesity and type 2 diabetes. Related to Fig. 1.

a, Serum alanine levels in wildtype C57Bl/6 J (wt) and obese/diabetic BKS-db/db mice from ad libitum feeding (fed), 24 h fasted (fasted) and 24 h fasted, 6 h refeeding (refed). Data are mean ± SEM, N = 4/group. Effect of genotype: * P > 0.05. Effect of nutritional state versus fed: # P < 0.05. b, Serum alanine levels in New Zealand Black (NZB) and New Zealand Obese (NZO) mice from 24h fasting (fasted) and 24h fasted, 6h refeeding (refed). Data are mean ± SEM, N = 4/group. Effect of genotype: * P > 0.05. Effect of nutritional state vs. fasted: # P < 0.05. c, Plasma alanine levels during an intraperitoneal alanine tolerance test (ipATT) in mice on low fat diet (LFD) or an obesogenic high-fat diet with (HFD-STZ) or without (HFD) streptozotocin (STZ) pre-treatment to exacerbate the progression of frank diabetes. Data are mean ± SEM, N = 6/group. Different than LFD: * P > 0.05. Different than HFD: # P < 0.05. d, Blood glucose levels in mice as in C. e, Plasma insulin levels during an intraperitoneal alanine tolerance test (ipATT) in C57Bl/6J (WT) and BKS-db/db (db/db) mice. N = 4/group. Genotype difference: *P < 0.05. f: Plasma insulin levels of mice as in C. g, Blood glucose levels during an intraperitoneal pyruvate tolerance test (ipPTT) in C57Bl/6J (WT) and BKS-db/db (db/db) mice. N = 4/group. Genotype difference: *P < 0.05. h, Blood glucose levels during an intraperitoneal alanine tolerance test (ipPTT) in in mice on low fat diet (LFD) or an obesogenic high-fat diet with (HFD-STZ) or without (HFD) streptozotocin (STZ) pre-treatment to exacerbate the progression of frank diabetes. Data are mean ± SEM, N = 6/group. Different than LFD: * P > 0.05. Different than HFD: # P < 0.05. i, Blood glucose levels from type 2 diabetic (T2D; N = 11) and age-matched normal glucose tolerant (NGT; N = 12) individuals undergoing a mixed-meal tolerance test (MMTT). *: cohort difference, *p < 0.05, **p < 0.01, ***P < 0.001. #: effect of time, #p < 0.05, ##p < 0.01, ###P < 0.001. j, Blood plasma insulin levels of individuals as in I. k, Blood plasma glucagon levels of individuals as in I. l, Blood plasma cortisol levels of individuals as in I. Statistical tests: A, B: 2-way ANOVA with Holm-Sidak posthoc hoc tests. C-L: 2-way repeated measures ANOVA with Holm-Sidak posthoc hoc tests.

Extended Data Fig. 2 Silencing of both, but not individual, liver ALT isoforms affects systemic alanine and glucose homeostasis after diverse nutritional challenges. Related to Fig. 2.

a, Liver mRNA expression from mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) and alanine-glyoxylate aminotransferase (Agxt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 6/group. b, Representative western blots of GPT isoforms as well as the housekeeping protein heat shock protein 90 (HSP90) from mice as in A. 3 samples/group were analyzed and are shown. c, Body mass of mice as in A. d, Liver mass of mice as in A. e, Gastrocnemius complex skeletal muscle (GCM) mass of mice as in A. f, Perigonadal white adipose tissue (pgWAT) mass of mice as in A. g, Food intake during a 6h refeeding period following 24h fasting from mice as in A. h, Plasma alanine levels from mice as in G. i, Blood glucose levels from mice as in G. j, Plasma lactate levels from mice as in G. k, Plasma glycerol levels rom mice as in G. l, Plasma non-esterified fatty acid (NEFA) levels from mice as in G. m, Plasma ketone body levels from mice as in G. n, Liver mRNA levels from mice pre-treated with adeno-associated viruses to express a negative control micro-RNA (NC miR) and green fluorescent protein (GFP), Gpt and Gpt2-specfific miRs, human Gpt and Gpt2 mRNAs (HsGpt OE, HsGpt2 OE), and mutants of human Gpt and Gpt2 mRNAs to produce enzymatically inactive proteins (HsGptmut OE, HsGPT2mut OE). . Data are mean ± SEM, N = 6/ group. o, Liver mRNA expression of glutamic-pyruvic transaminase (Gpt) isoforms in mice fed a ketogenic diet with hepatocyte selective AAV-miR mediated silencing of Gpt isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 6/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. p, Body mass from mice as in O. q, Liver mass from mice as in O. r, Serum triglyceride (TG) levels from mice as in O. s, Serum NEFA levels from mice as in O. t, Serum glycerol levels from mice as in O. u, Serum cholesterol levels from mice as in O. v, The change (Δ) in fat mass as determined by ECHO-MRI before and after ketogenic diet feeding from mice as in O. w, Blood glucose levels in mice in the ad libitum fed state following adaptation to a protein-enriched (80%E) diet with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 6/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. x, Body mass from mice as in W. y, Liver mass from mice as in W. z, Gastrocnemius complex skeletal muscle (GCM) mass from mice as in W. Statistical tests: C, D, E, F, P, Q, R, S, T, U, V: 1-way ANOVA with Holm-Sidak posthoc hoc tests. I, J, K, L, M, N: 2-way repeated measures ANOVA with Holm-Sidak posthoc hoc tests. W, X, Y, z: Students t-tests.

Source data

Extended Data Fig. 3 Silencing of both, but not individual, liver alanine aminotransferase isoforms retards hyperglycaemia in mouse models of obesity-related type 2 diabetes. Related to Fig. 3.

a, Liver glutamic-pyruvic transaminase isoform mRNA expression in obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of Gpt isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 6/group. b, Liver GPT activity from mice as in A. Different than NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Different than Gpt or Gpt2 miR: ### P < 0.001. c, Blood glucose levels during an intraperitoneal alanine tolerance test (ipATT) in obese/diabetic BKS-db/db mice as in A. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. d, Body mass of mice as in A. e, Liver mass of mice as in A. f, Blood glucose levels after an overnight fast and following a 6h refeed from mice as in A. g, Food intake during a 6h refeeding period following fasting from mice as in F. h, Ad libitum fed serum triglyceride (TG) levels from mice as in A. i, Serum ketone body (KB) levels from mice as in H. j, Serum non-esterified fatty acid (NEFA) levels from mice as in H. k, Serum cholesterol levels from mice as in H. l, Liver GPT activity in lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 4/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. m, Plasma insulin levels of mice as in Fig. 3b during an alanine tolerance test (ipATT). Data are mean ± SEM, N = 4/ group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. n, Blood glucose levels during an intraperitoneal pyruvate tolerance test (ipPTT) in lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 4/ group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. o, Blood glucose levels during an intraperitoneal glycerol tolerance test (ipGyTT) of mice as in N. p, Representative western blots images of liver glucose-6-phosphatase (G6PC), phosphoenolpyruvate carboxykinase 1 (PCK1), GPT isoforms, as well as the housekeeping protein vinculin (VCL) in lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. 3 samples per group were analyzed and are shown. q, Liver GPT activity of mice on an obesogenic high-fat diet with (HFD-STZ) or without (HFD) streptozocin (STZ) pre-treatment to exacerbate the progression of frank diabetes; with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. Data are mean ± SEM, N = 6/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of STZ: # P < 0.05, ## P < 0.01, ### P < 0.001. r, Micrographs of liver glutamic-pyruvic transaminase (GPT) immunohistochemical staining of sections taken from HFD-STZ mice as in Q. Shown are 2 representative images taken from 2 individual mice per group (2/group were stained and imaged). Scale bar: 200µm. Statistical tests: B, D, E, G-K: 1-way ANOVA with Holm-Sidak posthoc hoc tests. C, F, M-O: 2-way repeated measures ANOVA with Holm-Sidak posthoc hoc tests. L, Q: 2-way ANOVA with Holm-Sidak posthoc hoc tests.

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Extended Data Fig. 4 Disrupting the heightened liver alanine catabolism improves skeletal muscle size and function in mouse models of type 2 diabetes. Related to Fig. 4.

a, Body mass of lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 4/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. b, Lean body mass as determined by ECHO-MRI of mice as in A. c, Fat body mass as determined by ECHO-MRI of mice as in A. d, Body mass of mice on an obesogenic high-fat diet with (HFD-STZ) or without (HFD) streptozocin (STZ) pre-treatment to exacerbate the progression of frank diabetes; with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. Data are mean ± SEM, N = 6/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of STZ: # P < 0.05, ## P < 0.01, ### P < 0.001. e, Lean body mass as determined by ECHO-MRI of mice as in D. f, Fat body mass as determined by ECHO-MRI of mice as in D. Statistical tests: A, B, C, D, E, F: 2-way ANOVA with Holm-Sidak posthoc hoc tests.

Extended Data Fig. 5 A glucocorticoid–liver glucocorticoid receptor axis links heightened liver alanine catabolism with hyperglycaemia and muscle atrophy in type 2 diabetes. Related to Fig. 5.

a, Serum corticosterone levels in lean New Zealand Black (NZB) and obese/diabetic New Zealand Obese (NZO) mice. Data are mean ± SEM, N = 6/group. d, Liver GPT activity of mice chronically treated with the synthetic glucocorticoid dexamethasone (Dex; 1mg/kg per day, 14d) or vehicle control (Veh). Data are mean ± SEM, N = 7/group. Effect of Dex: # P < 0.05, ## P < 0.01, ### P < 0.001. c, Liver glutamic-pyruvic transaminase (Gpt) isoform mRNA expression of C57Bl/6N in mice chronically treated with the synthetic glucocorticoid dexamethasone (Dex; 1mg/kg per day, 14d) or vehicle control (Veh) with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA.. Data are mean ± SEM, N = 8/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of Dex: # P < 0.05, ## P < 0.01, ### P < 0.001. d, Blood glucose levels of mice as in C. e, Gastrocnemius complex (GCM), triceps brachii (TB) and tibialis anterior (TA) skeletal muscle mass’ of mice as in C. f, Forelimb grip strength of mice as in C. g, Body mass of mice as in C. h, Liver mass of mice as in C. i, Perigonadal white adipose tissue (pgWAT) mass of mice as in C. j, Liver and perigonal white adipose tissue mass’ of mice chronically treated with the synthetic glucocorticoid dexamethasone (Dex; 1mg/kg per day, 14d) or vehicle control (Veh) with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 8/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of Dex: # P < 0.05, ## P < 0.01, ### P < 0.001. k, ENSEMBL genome browser images of DNAse hypersensitivity sites as well as CEBPb and GR ChIP-seq peaks of the Gpt and Gpt2 gene, and flanking regions, in mouse liver. Statistical tests: A, B: Students t-tests (two sided). C, D, E, F, G, H, I, J: 2-way ANOVA with Holm-Sidak posthoc hoc tests.

Extended Data Fig. 6 Chronic liver glucagon action affects hyperglycaemia and skeletal muscle atrophy via hepatic alanine catabolism. Related to Fig. 6.

a, Messenger RNA (mRNA) levels of glutamic-pyruvic transaminase (Gpt) and Gpt2 in precision cut liver slices treated ex vivo with the synthetic glucocorticoid dexamethasone (DEX), glucagon (GCG) or a combination thereof (DEX + GCG). N = 6 individual slices per treatment group. One way ANOVA: different than VEH: * p < 0.05, ** p < 0.01, *** p < 0.001. b, Western blot images of liver glutamic-pyruvic transaminase (GPT), GPT2, and loading control heat shock protein 90 (HSP90) of C57Bl6/J (Bl6/J) and obese/diabetic C57BKS mice with homozygous leptin receptor mutation (BKS-db/db) chronically treated with a glucagon receptor antagonist (REMD). 2 samples per group were analyzed and are shown. c, Body mass of mice of C57Bl/6J mice chronically (3wk) treated with the acyl-glucagon (acyl-GCG; 1 nmol/g/d) or vehicle control (Veh) with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 8/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of Dex: # P < 0.05, ## P < 0.01, ### P < 0.001. d, Liver and perigonadal white adipose tissue (PGWAT) mass’ of mice of C57Bl/6J mice chronically treated with the acyl-glucagon (acyl-GCG; 1 nmol/g/d) or vehicle control (Veh) with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 8/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of Dex: # P < 0.05, ## P < 0.01, ### P < 0.001. Statistical tests: A, D: 1-way ANOVA with Holm-Sidak posthoc hoc tests. C: 2-way repeated meaures ANOVA with Holm-Sidak posthoc hoc tests.

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Extended Data Fig. 7 Disrupting the heightened liver alanine catabolism reverses the reduced skeletal muscle protein synthesis and branched-chain amino acid levels in type 2 diabetes. Related to Fig. 7.

a, Gastroconemius complex skeletal muscle mRNA expression of transcripts of atrophy related genes Trim63 (aka Murf1), Fbxo32 (aka MAFbx or Atrogin-1), Ddit4 (aka Redd1), Klf15 and Foxo3a from lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. Data are from mice 6 wk after AAV administration. NC: negative control. miR: micro-RNA. GCM: gastrocnemius complex muscle. TB: Triceps brachii. TA: tibialis anterior. Data are mean ± SEM, N = 4/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. b, Skeletal muscle mass’ of overnight fasted, 4h refed lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. Study was conducted 10d after AAV administrations. NC: negative control. miR: micro-RNA. GCM: gastrocnemius complex muscle. TB: Triceps brachii. TA: tibialis anterior. Data are mean ± SEM, N = 4/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. c, In vivo protein synthesis rate calculated from mixed muscle 3H-phenylalanine incorporation of mice as in B. d, Gastrocnemius complex skeletal muscle (GCM) Gpt isoform mRNA expression in lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 4/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. e, Ex vivo extensor digitorum longus (EDL) skeletal muscle protein synthesis rate during co-culture and cross-co-culture with liver slices. Tissues were taken from lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. In one condition, media was collected after culture with db/db liver for 3h after which it was dialyzed (1 KDa cutoff) against normal media to normalize metabolites such as amino acids but retain large molecules such as peptides. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 3/group with 2 technical replicates per treatment condition. Effect of Liver miR: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of muscle genotype: # P < 0.05, ## P < 0.01, ### P < 0.001. f, Serum insulin like growth factor 1 (IGF1) levels of BKS-db/db mice as in D. g, Serum follistatin (FST) levels of BKS-db/db mice as in D. h, Serum fibroblast growth factor 21 (FGF21) levels of BKS-db/db mice as in D. i, Serum insulin levels of BKS-db/db mice as in D. j, Gastrocnemius complex skeletal muscle (GCM) Valine (Val) and Leucine/Isoleucine (Leu/Ile) concentrations in obese/diabetic BKS-db/db mice with hepatocyte selective AAV-miR mediated silencing of glutamic-pyruvic transaminase (Gpt) isoforms. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 6/group. Effect of miR vs. NC miR: * P < 0.05, ** P < 0.01, *** P < 0.001. k, Ex vivo extensor digitorum longus (EDL) skeletal muscle protein synthesis rate during co-culture and cross-co-culture with liver slices with (+5mM Ala) or without (Con) treatment with 5mM alanine in the media. Tissues were taken from lean C57Bl/6J (Bl6) and age-matched obese/diabetic BKS-db/db mice. NC: negative control. miR: micro-RNA. Data are mean ± SEM, N = 3/group with 2 technical replicates per treatment condition. Effect of muscle genotype: * P < 0.05, ** P < 0.01, *** P < 0.001. Effect of 5mM Ala: # P < 0.05, ## P < 0.01, ### P < 0.001. l, Immunoblot images showing phospho-Thr389-p70S6 kinase 1, phospho-Thr37/46-4E binding protein 1, and the loading control vinculin of gastrocnemius skeletal muscle samples from mice as in B. 3 samples per group were analyzed and are shown. Statistical tests: A, B, C, D, J: 2-way ANOVA with Holm-Sidak posthoc hoc tests. E, F, G, H, K: Student’s t-test. I: 1-way ANOVA with Holm-Sidak posthoc hoc tests.

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Okun, J.G., Rusu, P.M., Chan, A.Y. et al. Liver alanine catabolism promotes skeletal muscle atrophy and hyperglycaemia in type 2 diabetes. Nat Metab 3, 394–409 (2021). https://doi.org/10.1038/s42255-021-00369-9

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