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Cited by 167Metabolism, Diabetes, and CancerMuscle Physiology and DisordersMuscle metabolism and nutrition

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The role of skeletal muscle Akt in the regulation of muscle mass and glucose homeostasis

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Jaiswal, N., Gavin, M.G., Quinn, W.J., Luongo, T.S., Gelfer, R.G., Baur, J.A., Titchenell, P.M.·2019·Molecular Metabolism
DOI 10.1016/j.molmet.2019.08.001PMC PMC6822261
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Abstract

OBJECTIVE: Skeletal muscle insulin signaling is a major determinant of muscle growth and glucose homeostasis. Protein kinase B/Akt plays a prominent role in mediating many of the metabolic effects of insulin. Mice and humans harboring systemic loss-of-function mutations in Akt2, the most abundant Akt isoform in metabolic tissues, are glucose intolerant and insulin resistant. Since the skeletal muscle accounts for a significant amount of postprandial glucose disposal, a popular hypothesis in the diabetes field suggests that a reduction in Akt, specifically in skeletal muscle, leads to systemic glucose intolerance and insulin resistance. Despite this common belief, the specific role of skeletal muscle Akt in muscle growth and insulin sensitivity remains undefined. METHODS: We generated multiple mouse models of skeletal muscle Akt deficiency to evaluate the role of muscle Akt signaling in vivo. The effects of these genetic perturbations on muscle mass, glucose homeostasis and insulin sensitivity were assessed using both in vivo and ex vivo assays. RESULTS: Surprisingly, mice lacking Akt2 alone in skeletal muscle displayed normal skeletal muscle insulin signaling, glucose tolerance, and insulin sensitivity despite a dramatic reduction in phosphorylated Akt. In contrast, deletion of both Akt isoforms (M-AktDKO) prevented downstream signaling and resulted in muscle atrophy. Despite the absence of Akt signaling, in vivo and ex vivo insulin-stimulated glucose uptake were normal in M-AktDKO mice. Similar effects on insulin sensitivity were observed in mice with prolonged deletion (4 weeks) of both skeletal muscle Akt isoforms selectively in adulthood. Conversely, short term deletion (2 weeks) of skeletal muscle specific Akt in adult muscles impaired insulin tolerance paralleling the effect observed by acute pharmacological inhibition of Akt in vitro. Mechanistically, chronic ablation of Akt induced mitochondrial dysfunction and activation of AMPK, which was required for insulin-stimulated glucose uptake in the absence of Akt. CONCLUSIONS: Together, these data indicate that chronic reduction in Akt activity alone in skeletal muscle is not sufficient to induce insulin resistance or prevent glucose uptake in all conditions. Therefore, since insulin-stimulated glucose disposal in skeletal muscle is markedly impaired in insulin-resistant states, we hypothesize that alterations in signaling molecules in addition to skeletal muscle Akt are necessary to perturb glucose tolerance and insulin sensitivity in vivo.

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Figure 1 Skeletal muscle Akt1 functionally compensates in the absence of Akt2 to preserve insulin signaling in skeletal muscles. (A) Western Blot of Akt2 and total Akt in soleus and gastrocnemius muscle from mice with specific deletion of Akt2 and both Akt1 and Akt2. (B) Body weight of control and experimental mice (n = 34 for control, 5 for M-Akt2KO and 12 for M-AktDKO mice). (C) Muscle weight from different muscle depots of control and experimental mice (n = 22 for control, n = 8 for M-Akt2KO and n = 24 for M-AktDKO mice). (D) Representative image of skeletal muscles isolated from control and M-AktDKO mice. (E) Western Blot for phosphorylation of Akt, Akt2, PRAS40, S6, 4EBP1 and HSP90 in Gastrocnemius muscle from control and experimental mice either unstimulated or stimulated with insulin (2 U/kg) for 20 min following an overnight fast. (*p < 0.05, ****p < 0.0001 vs. control).
Figure 1 Skeletal muscle Akt1 functionally compensates in the absence of Akt2 to preserve insulin signaling in skeletal muscles. (A) Western Blot of Akt2 and total Akt in soleus and gastrocnemius muscle from mice with specific deletion of Akt2 and both Akt1 and Akt2. (B) Body weight of control and experimental mice (n = 34 for control, 5 for M-Akt2KO and 12 for M-AktDKO mice). (C) Muscle weight from different muscle depots of control and experimental mice (n = 22 for control, n = 8 for M-Akt2KO and n = 24 for M-AktDKO mice). (D) Representative image of skeletal muscles isolated from control and M-AktDKO mice. (E) Western Blot for phosphorylation of Akt, Akt2, PRAS40, S6, 4EBP1 and HSP90 in Gastrocnemius muscle from control and experimental mice either unstimulated or stimulated with insulin (2 U/kg) for 20 min following an overnight fast. (*p < 0.05, ****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central
Figure 2 M-AktDKO mice display modest abnormalities in glucose homeostasis. (A) Blood glucose and plasma insulin level (n = 11 for control, 5 for M-Akt2KO and 7 for M-AktDKO mice) in ad libitum state. (B) Intraperitoneal glucose tolerance test (2 g/kg) (n = 17 for control, 6 for M-Akt2KO and n = 11 for M-AktDKO mice). (C) Plasma Insulin level in fasted or 15 min after insulin injection (n = 5–13). (D) Insulin tolerance test (0.75 U/kg) (n = 10 for control, 5 for M-Akt2KO and 7 for M-AktDKO mice) (***p < 0.001, ****p < 0.0001 vs. control).
Figure 2 M-AktDKO mice display modest abnormalities in glucose homeostasis. (A) Blood glucose and plasma insulin level (n = 11 for control, 5 for M-Akt2KO and 7 for M-AktDKO mice) in ad libitum state. (B) Intraperitoneal glucose tolerance test (2 g/kg) (n = 17 for control, 6 for M-Akt2KO and n = 11 for M-AktDKO mice). (C) Plasma Insulin level in fasted or 15 min after insulin injection (n = 5–13). (D) Insulin tolerance test (0.75 U/kg) (n = 10 for control, 5 for M-Akt2KO and 7 for M-AktDKO mice) (***p < 0.001, ****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central
Figure 3 M-AktDKO mice have reduced whole-body glucose turnover and normal insulin-stimulated uptake in skeletal muscle. Hyperinsulinemic-euglycemic clamps were performed in control and M-AktDKO mice following 5 h fasting using a 2.5 mU/kg/min infusion of insulin. (A) Steady state glucose infusion rate, (B) rate of glucose disposal, (C) glucose infusion rate normalized to lean mass, (D) glucose disposal rate normalized to lean mass, (E) hepatic glucose production during basal and insulin portion of the clamp, (F) hepatic glucose production normalized to lean mass, and (G) in vivo glucose uptake in skeletal muscle during hyperinsulinemic-euglycemic clamp (n = 4–9). (**p < 0.01, ***p < 0.001 vs. controls, ****p < 0.0001 vs. control).
Figure 3 M-AktDKO mice have reduced whole-body glucose turnover and normal insulin-stimulated uptake in skeletal muscle. Hyperinsulinemic-euglycemic clamps were performed in control and M-AktDKO mice following 5 h fasting using a 2.5 mU/kg/min infusion of insulin. (A) Steady state glucose infusion rate, (B) rate of glucose disposal, (C) glucose infusion rate normalized to lean mass, (D) glucose disposal rate normalized to lean mass, (E) hepatic glucose production during basal and insulin portion of the clamp, (F) hepatic glucose production normalized to lean mass, and (G) in vivo glucose uptake in skeletal muscle during hyperinsulinemic-euglycemic clamp (n = 4–9). (**p < 0.01, ***p < 0.001 vs. controls, ****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central
Figure 4 Skeletal muscle from M-AktDKO mice exhibit normal PI3K-dependent insulin stimulated glucose uptake. (A–B) Ex vivo insulin-stimulated glucose uptake was measured in EDL (A) or soleus (B) from control and M-AktDKO mice with/without pretreatment of wortmannin (1 μM) (n = 7–13). (C) Western blot of Glut1 and Glut4 protein from plasma membrane fraction of hind limb and whole cell Lysate (WCL) of gastrocnemius muscles from control and M-AktDKO mice stimulated with insulin (2 U/kg) for 20 min. (D) Western Blot analysis of phosphorylated AS160, PAS-160 and TBC1D1 in gastrocnemius muscle from control and M-AktDKO mice fasted overnight and treated with BSA or insulin (2 U/kg) for 20 min (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control).
Figure 4 Skeletal muscle from M-AktDKO mice exhibit normal PI3K-dependent insulin stimulated glucose uptake. (A–B) Ex vivo insulin-stimulated glucose uptake was measured in EDL (A) or soleus (B) from control and M-AktDKO mice with/without pretreatment of wortmannin (1 μM) (n = 7–13). (C) Western blot of Glut1 and Glut4 protein from plasma membrane fraction of hind limb and whole cell Lysate (WCL) of gastrocnemius muscles from control and M-AktDKO mice stimulated with insulin (2 U/kg) for 20 min. (D) Western Blot analysis of phosphorylated AS160, PAS-160 and TBC1D1 in gastrocnemius muscle from control and M-AktDKO mice fasted overnight and treated with BSA or insulin (2 U/kg) for 20 min (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central
Figure 5 Inducible deletion of Akt in adult skeletal muscle have time responsive effect on glucose tolerance and insulin sensitivity. (A) Western Blot for Akt2 in Quadriceps of M-indAktKO 2 and 4 week post tamoxifen. (B) Western blot for phosphorylation of Akt, Akt2, pPRAS40, phosphorylation of S6, S6 and HSP90 in Gastrocnemius muscle from M-indControl and M-indAktDKO animals either unstimulated or stimulated with insulin (2 U/kg) for 20 min after overnight fasting. (C) Muscle weight from different muscle depots of M-indControl and M-indAktDKO mice (n = 50–60 for M-indControl and n = 20–24 for M-indAktDKO mice). (D–E) Intraperitoneal glucose tolerance test (2 g/kg) (n = 22 for M-indControl and n = 20 for M-indAktDKO) and Insulin tolerance test (0.75 U/kg) (n = 22 for M-indControl and n = 19 for M-indAktDKO) 2 week post tamoxifen. (F–G) Intraperitoneal glucose tolerance test (2 g/kg) (n = 11 for M-indControl and n = 7 for M-indAktDKO mice) and Insulin tolerance test (0.75 U/kg) (n = 11 for M-indControl n = 8 for M-indAktDKO mice) 4 week post tamoxifen. (**p < 0.01, ****p < 0.0001 vs. control).
Figure 5 Inducible deletion of Akt in adult skeletal muscle have time responsive effect on glucose tolerance and insulin sensitivity. (A) Western Blot for Akt2 in Quadriceps of M-indAktKO 2 and 4 week post tamoxifen. (B) Western blot for phosphorylation of Akt, Akt2, pPRAS40, phosphorylation of S6, S6 and HSP90 in Gastrocnemius muscle from M-indControl and M-indAktDKO animals either unstimulated or stimulated with insulin (2 U/kg) for 20 min after overnight fasting. (C) Muscle weight from different muscle depots of M-indControl and M-indAktDKO mice (n = 50–60 for M-indControl and n = 20–24 for M-indAktDKO mice). (D–E) Intraperitoneal glucose tolerance test (2 g/kg) (n = 22 for M-indControl and n = 20 for M-indAktDKO) and Insulin tolerance test (0.75 U/kg) (n = 22 for M-indControl and n = 19 for M-indAktDKO) 2 week post tamoxifen. (F–G) Intraperitoneal glucose tolerance test (2 g/kg) (n = 11 for M-indControl and n = 7 for M-indAktDKO mice) and Insulin tolerance test (0.75 U/kg) (n = 11 for M-indControl n = 8 for M-indAktDKO mice) 4 week post tamoxifen. (**p < 0.01, ****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central
Figure 6 M-indAktDKO mice display a fiber type specific effect on glucose uptake. (A–B) Ex vivo insulin-stimulated 2-deoxyglucose uptake in EDL (A) or soleus (B) from M-indControl and M-indAktDKO mice with/without pretreatment of wortmannin (1 μM) (n = 20 for control and insulin, n = 4 for wortmannin). (C) Western blot of Glut1 and Glut4 protein and HSP90 in Plasma membrane fraction of hind limb and whole cell lysate of gastrocnemius muscles from M-indControl and M-indAktDKO mice stimulated with insulin (2 U/kg) for 20 min after overnight fast. (D) Western Blot analysis of phosphorylated AS160, TBC1D1 and HSP90 in gastrocnemius muscle from M-indControl and M-indAktDKO mice fasted overnight and treated with BSA or insulin (2 U/kg) for 20 min (*p < 0.05, **p < 0.01, ****p < 0.0001 vs. control; #p < 0.05 vs control insulin injected).
Figure 6 M-indAktDKO mice display a fiber type specific effect on glucose uptake. (A–B) Ex vivo insulin-stimulated 2-deoxyglucose uptake in EDL (A) or soleus (B) from M-indControl and M-indAktDKO mice with/without pretreatment of wortmannin (1 μM) (n = 20 for control and insulin, n = 4 for wortmannin). (C) Western blot of Glut1 and Glut4 protein and HSP90 in Plasma membrane fraction of hind limb and whole cell lysate of gastrocnemius muscles from M-indControl and M-indAktDKO mice stimulated with insulin (2 U/kg) for 20 min after overnight fast. (D) Western Blot analysis of phosphorylated AS160, TBC1D1 and HSP90 in gastrocnemius muscle from M-indControl and M-indAktDKO mice fasted overnight and treated with BSA or insulin (2 U/kg) for 20 min (*p < 0.05, **p < 0.01, ****p < 0.0001 vs. control; #p < 0.05 vs control insulin injected).CC BY-NC-ND · PubMed Central
Figure 7 Akt deletion induces mitochondrial dysfunction and activates AMPK, which is required for insulin-stimulated glucose uptake. (A) Western blot analysis for TOM20 and HSP90 from whole cell lysates of gastrocnemius muscle. (B) mRNA expression from quadricep muscle (n = 7 control and n = 7 M-AktDKO). (C) Oroborus high-capacity respirometer analysis of isolated mitochondria from control and M-AktDKO gastrocnemius muscle (n = 7 control and n = 7 M-AktDKO). (D) relative ATP levels in gastrocnemius muscle of control and M-AktDKO mice (n = 4 control and n = 5 M-AktDKO). (E) Western blot analysis of phosphorylated AMPK and AMPK from control and M-AktDKO gastrocnemius muscle. (F–G) Ex vivo insulin-stimulated 2-deoxyglucose uptake in soleus (F) or EDL (G) from control and M-AktDKO mice with/without pretreatment of compound C (20 μM) (n = 6 for control, 7 for insulin and 7 for compound C). (*p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001 vs. control).
Figure 7 Akt deletion induces mitochondrial dysfunction and activates AMPK, which is required for insulin-stimulated glucose uptake. (A) Western blot analysis for TOM20 and HSP90 from whole cell lysates of gastrocnemius muscle. (B) mRNA expression from quadricep muscle (n = 7 control and n = 7 M-AktDKO). (C) Oroborus high-capacity respirometer analysis of isolated mitochondria from control and M-AktDKO gastrocnemius muscle (n = 7 control and n = 7 M-AktDKO). (D) relative ATP levels in gastrocnemius muscle of control and M-AktDKO mice (n = 4 control and n = 5 M-AktDKO). (E) Western blot analysis of phosphorylated AMPK and AMPK from control and M-AktDKO gastrocnemius muscle. (F–G) Ex vivo insulin-stimulated 2-deoxyglucose uptake in soleus (F) or EDL (G) from control and M-AktDKO mice with/without pretreatment of compound C (20 μM) (n = 6 for control, 7 for insulin and 7 for compound C). (*p < 0.05, **p < 0.01, ***p < 0.001****p < 0.0001 vs. control).CC BY-NC-ND · PubMed Central

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  1. Moller, 2019, The role of p-21 activated kinases (PAKs) in glucose homeostasis and skeletal muscle glucose uptake, BioRxiv

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Metadata

Authors
Jaiswal, N., Gavin, M. G., Quinn, W. J., Luongo, T. S., Gelfer, R. G., Baur, J. A., & Titchenell, P. M.
Affiliations
University of Pennsylvania (US)
Corresponding author
Titchenell, P.M.
ORCID
Venue
Molecular Metabolism
Publisher
Elsevier BV
Published
2019-10
Volume
28
Pages
1-13
ISSN
2212-8778 (ISSN-L)
DOI
10.1016/j.molmet.2019.08.001
PMC
PMC6822261
OpenAlex
W2964701486
Cited by
167
References
81
Citations by year
2026: 10 · 2025: 28 · 2024: 27 · 2023: 20 · 2022: 40 · 2021: 23 · 2020: 18 · 2019: 1
FWCI
6.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Metabolism (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • University of Pennsylvania
  • National Institutes of Health (P30 19525, K01 111715)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolism, Diabetes, and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Muscle Physiology and Disorders
  • Health Sciences › Nursing › Nutrition and Dietetics › Muscle metabolism and nutrition
Keywords
AKT2, Akt isoforms, muscle mass, muscle atrophy, glucose homeostasis, insulin resistance, insulin sensitivity, insulin-stimulated glucose uptake, mitochondrial dysfunction, AMPK, skeletal muscle insulin signaling
MeSH terms
Animals, Glucose, Homeostasis, Humans, Male, Weight Gain, Muscle, Skeletal, Proto-Oncogene Proteins c-akt, Mice

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