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