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. Author manuscript; available in PMC: 2017 Jan 12.
Published in final edited form as: Appl Physiol Nutr Metab. 2016 Aug 16;41(11):1208–1211. doi: 10.1139/apnm-2016-0326

Inhibition of Akt2 phosphorylation abolishes the calorie restriction-induced improvement in insulin-stimulated glucose uptake by rat soleus muscle

Naveen Sharma 1,4, Edward B Arias 1, Gregory D Cartee 1,2,3
PMCID: PMC5228618  NIHMSID: NIHMS840556  PMID: 27786542

Abstract

Calorie restriction (CR; ~60–65% of ad libitum, AL, consumption) can enhance insulin-stimulated glucose uptake (ISGU) in predominantly slow-twitch skeletal muscles (e.g., soleus) by an incompletely understood mechanism. We used an Akt inhibitor (MK-2206) to eliminate CR’s effect on insulin-stimulated Akt2 phosphorylation in isolated rat soleus muscles. We found long-term CR-enhanced ISGU was abolished by eliminating the CR-effect on Akt2 phosphorylation, suggesting the CR-induced benefit on ISGU in the predominantly slow-twitch soleus relies on enhanced Akt2 phosphorylation.

Keywords: glucose transport, insulin signaling, dietary restriction, slow-twitch muscle, protein kinase B

INTRODUCTION

Skeletal muscle, a heterogeneous tissue composed of multiple fiber types, is the major tissue for insulin-stimulated glucose disposal (DeFronzo 1988). A well-described health-related benefit of calorie restriction (CR; ~60% of ad libitum, AL, consumption) is improved insulin sensitivity (McCurdy et al. 2005; Sharma et al. 2012c; Sharma et al. 2012b). Insulin-stimulated glucose uptake (ISGU), which is enhanced with CR (McCurdy et al. 2005; Sharma et al. 2011; Sequea et al. 2012; Sharma et al. 2012a; Sharma et al. 2015; Wang et al. 2016b), is attributed to enhanced activation of the serine/threonine kinase Akt (McCurdy et al. 2003; McCurdy et al. 2005; Sharma et al. 2011; Sequea et al. 2012; Sharma et al. 2012a; Sharma et al. 2012c; Sharma et al. 2012d; Sharma et al. 2015; Wang et al. 2016a; Wang et al. 2016b). Consistent with the fact that Akt2 is the major Akt isoform for ISGU (Bae et al. 2003; Sakamoto et al. 2006), previous studies demonstrated that CR increases insulin-stimulated Akt2 activation (McCurdy et al. 2003; McCurdy et al. 2005; Sequea et al. 2012; Sharma et al. 2012a; Wang et al. 2016b).

There are muscle-specific differences in CR-effects. In male Akt2-knockout (Akt2-KO) mice, Akt2 is essential for the CR-effect on ISGU in the predominantly fast-twitch extensor digitorum longus (EDL) (McCurdy et al. 2005). In contrast, ISGU of the soleus (largely slow-twitch) from the same Akt2-KO mice was greater for the CR versus AL group. A caveat of this study was that Akt2 expression was eliminated in all tissues throughout the development of Akt2-KO animals. Subsequently, we studied isolated rat epitrochlearis (predominantly fast-twitch) muscles (Sharma et al. 2012a; Wang et al. 2016a) incubated with a selective Akt-inhibitor, MK-2206 (Hirai et al. 2010; Tan et al. 2011), at a dose that reduced Akt2 phosphorylation of insulin-treated muscles from CR rats to levels similar to AL muscles. Preventing the CR-induced increase in Akt2 phosphorylation eliminated the CR-effect on epitrochlearis ISGU. We now extend this research by evaluating the Akt-inhibitor MK-2206’s effect on insulin-stimulated Akt2 phosphorylation and glucose uptake of isolated soleus from CR and AL rats. We hypothesized that preventing CR’s effect on Akt2 phosphorylation in the soleus would reduce ISGU in the CR group.

MATERIALS AND METHODS

Materials for SDS-PAGE and immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Anti-phospho Akt Thr308 (pAktThr308), anti-phospho Akt Ser473 (pAktSer473), and anti-rabbit IgG horseradish peroxidase conjugate were from Cell Signaling Technology (Danvers, MA, USA). Anti-Akt2 was from R&D Biosystems (Minneapolis, MN, USA). Anti-phospho AS160Thr642 (pAS160Thr642) was from B-Bridge International (Cupertino, CA, USA). Anti-AS160 and anti-sheep IgG horseradish peroxidase conjugate were from EMD Millipore (Billerica, MA, USA). Phospho-Akt antibodies against the Thr308 and Ser473 sites recognize Thr309 and Ser474 on Akt2, respectively. MK-2206 was from Selleck Chemicals (Houston, TX, USA). [3H]-2-Deoxy-D-glucose ([3H]-2-DG) and [14C]-mannitol were from PerkinElmer (Boston, MA, USA).

Animal care procedures were approved by the University of Michigan Committee on Use and Care of Animals. Male Fisher-344xBrown Norway rats were obtained from the National Institute on Aging (NIA) Calorie Restricted Rodent Colony. Data from these rats on CR-effects in the predominantly fast-twitch epitrochlearis have been published (Sharma et al. 2012a). Calorie restriction was initiated at 14weeks-old in CR rats (AL rats had free access to NIH31 chow; CR: NIH31/NIA fortified chow at ~60–65% of AL), until euthanization at ~9 months-old (CR duration of ~25 weeks).

Muscle dissection and incubation have been described (Sharma et al. 2011; Sharma et al. 2012a). Soleus strips were incubated (30 min; gassed with 95% O2/5% CO2 at 35 °C) in vials containing 2 ml Krebs Henseleit Buffer (KHB) supplemented with 0.1% bovine serum albumin (BSA), 2 mmol/L sodium pyruvate, 6 mmol/L mannitol, and DMSO or MK-2206 (0.1 μmol/L). Muscles were transferred to vials containing the identical solution as the previous step, ±1.2 nmol/L insulin (30 min). Muscles were transferred to a vial containing 2 ml KHB/BSA, the same concentration of MK-2206 and insulin as the previous step, 1mM 2-DG (2.25 mCi/mmol [3H]-2-DG), and 9 mmol/L mannitol (0.022 mCi/mmol [14C]-mannitol) for 20 min. Muscles were blotted, trimmed, freeze-clamped, and stored (−80°C). Muscles were homogenized and processed for immunoprecipitation and immunoblotting (Sharma et al. 2011; Sharma et al. 2012a), and [3H]-2-Deoxy-D-glucose (2-DG) uptake (Hansen et al. 1994). One-way ANOVA was used for statistical comparison (SigmaPlot version 11.0; San Jose, CA). Data are mean ±SEM. A P value <0.05 was statistically significant.

RESULTS

Immunoblotting

There was no significant diet-related difference for muscles incubated without insulin and MK-2206 for pan-Akt Thr308-phosphorylation (Fig. 1A), Akt2 Thr309-phosphorylation (Fig. 1B), pan-Akt Ser473-phosphorylation (Fig. 1C), or Akt2 Ser474-phosphorylation (Fig. 1D). For insulin-stimulated muscles without MK-2206, phosphorylation of AktThr308, Akt2Thr309, AktSer473 and Akt2Ser474 of CR exceeded (P<0.05) AL. Incubation of insulin-stimulated CR muscles with MK-2206 reduced (P<0.05) pan-AktThr308, Akt2Thr309, pan-AktSer473 and Akt2Ser474 phosphorylation versus insulin-stimulated CR muscles without MK-2206. Pan-AktSer473 phosphorylation was significantly (P<0.05) reduced in insulin-stimulated CR muscles with MK-2206 versus AL insulin-stimulated muscles (Fig. 1C). There was no significant diet-related difference for muscles incubated without insulin and MK-2206 for AS160 Thr642-phosphorylation (Fig. 2A). There were also no differences among insulin-stimulated groups for AS160 Thr642-phosphorylation. There were no group differences for total Akt2 or AS160 abundance (data not shown).

Figure 1.

Figure 1

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Immunoblot analysis of Akt phosphorylation in rat soleus muscle. (A) pan-Akt Thr308 phosphorylation (pAktThr308). (B) Akt2 Thr309 phosphorylation (pAkt2Thr309), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-AktThr308 antibody, (C) pan-Akt Ser473 phosphorylation (pAktSer473), and (D) Akt2 Ser474 phosphorylation (pAkt2Ser474), muscle lysates were immunoprecipitated with Akt2 antibody prior to immunoblotting with phospho-AktSer473 antibody. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P < 0.05) different from each other. Values are means ± SE, n = 6–10 per treatment group. All lanes are from the same gel, however, the representative blots have a vertical line separating the final 2 lanes indicating that the original blot has been re-ordered to match the respective bar graphs.

Figure 2.

Figure 2

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AS160 Thr642-phosphorylation (pAS160Thr642) and 2-deoxy-D-glucose (2-DG) uptake in rat soleus muscle. (A) pAS160Thr642. (B) 2-DG uptake. Filled bars are muscles from AL-fed rats, open bars are muscles from CR-fed rats, and hatched bar are muscles from CR-fed rats incubated with MK-2206. “ns” indicates no statistical difference between AL and CR groups in the absence of insulin. Insulin-stimulated groups with matching letters are not statistically different, and groups with differing letters are statistically (P < 0.05) different from each other. Values are means ± SE, n = 12–18 per treatment group.

2-DG Uptake

There was no significant diet-related difference in 2-DG uptake for muscles without insulin and MK-2206 (Fig. 2B). For insulin-stimulated muscles without MK-2206, 2-DG uptake of CR exceeded (P<0.05) AL. Incubation of insulin-stimulated CR muscles with MK-2206 reduced (P<0.05) 2-DG uptake versus insulin-stimulated CR muscles without MK-2206. 2-DG uptake was not significantly different in insulin-stimulated AL muscles versus insulin-stimulated CR muscles with MK-2206.

DISCUSSION

We were successful in using a selective Akt-inhibitor to eliminate the CR-induced increase in pan-Akt and Akt2 phosphorylation on each of the sites important for enzyme activity and increasing ISGU. Rapid Akt2 inhibition avoided the caveats associated with the life-long, whole body Akt2-KO model used previously (McCurdy et al. 2005). Together with the concomitant MK-2206-associated elimination of the CR-induced increase in ISGU, the current data provides compelling new evidence that insulin-stimulated Akt2 phosphorylation is essential for the CR-induced enhancement in ISGU by the predominantly slow-twitch rat soleus. The current results in the soleus are similar to results for the predominantly fast-twitch rat epitrochlearis using MK-2206 in the same rats (Sharma et al. 2012a). These results support the idea that, at least in rats, long-term CR relies on an Akt2-dependent mechanism for improving ISGU by both slow-twitch and fast-twitch muscles.

McCurdy et al. used Akt2-KO mice to probe Akt2’s role in CR-induced increase in ISGU (McCurdy et al. 2005). The magnitude of ISGU in the soleus of male CR mice was reduced versus wild-type CR mice, but a significant CR-effect on ISGU was found in Akt2-KO animals, suggesting a possible Akt2-independent mechanism. The differing results in the current study versus the earlier study might be related to species differences or compensatory mechanisms with whole-body elimination of Akt2 in Akt2-KO mice.

During a euglycemic-hyperinsulinemic clamp, CR increased glucose uptake by several predominantly fast-twitch muscles, but it did not significantly increase glucose uptake by the soleus (Sharma et al. 2014). The isolated muscle model provides valuable information about mechanisms that are inherent to the muscle itself, whereas the clamp results reflect both muscle and systemic (vascular, endocrine and neural) mechanisms. In the soleus of AL animals during a clamp, there is evidence that glucose transport across the sarcolemma is not a rate-limiting step, whereas it is rate-limiting in fast-twitch muscles (Halseth et al. 2001; Petersen et al. 2003). Other experimental differences may have contributed to the divergent results, including the duration and concentration for insulin exposure in the clamp (145 min with ~0.84 nmol/L) versus the isolated soleus (50 min with 1.2 nmol/L).

AS160 is the Akt2 substrate that has been most convincingly linked to increased ISGU (Sano et al. 2003; Cartee 2015). In the rat epitrochlearis, CR produces increased phosphorylation of AS160 (Sharma et al. 2011; Sharma et al. 2012a). However, insulin-stimulated AS160 phosphorylation was not enhanced above AL controls in the soleus in this study or an earlier study of 9 month-old rats undergoing the same CR protocol (Sharma et al. 2011; Sharma et al. 2012d). These results suggest CR might rely on greater phosphorylation of Akt2 substrates besides AS160 in the soleus. It would also be valuable to determine if CR alters AS160’s subcellular localization and/or binding to regulatory proteins which modify AS160 function (Stockli et al. 2011; Tan et al. 2012). The current data support the idea that the CR-effect on Akt2 is necessary for the CR-improvement in ISGU, but it uncertain if greater Akt2 activation is sufficient for CR-induced elevation in ISGU. It is possible that CR-effects on ISGU require additional changes in Akt2-independent processes.

Acknowledgments

This research was supported by NIA Grants AG-010026 and AG-013283.

Footnotes

Conflict of interest statement

The authors declare that there are not conflicts of interest.

References

  1. Bae SS, Cho H, Mu J, Birnbaum MJ. Isoform-specific regulation of insulin-dependent glucose uptake by Akt/protein kinase B. J Biol Chem. 2003;278:49530–49536. doi: 10.1074/jbc.M306782200. [DOI] [PubMed] [Google Scholar]
  2. Cartee GD. Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated glucose transport of skeletal muscle. Diabetologia. 2015;58:19–30. doi: 10.1007/s00125-014-3395-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. DeFronzo RA. Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes. 1988;37:667–687. doi: 10.2337/diab.37.6.667. [DOI] [PubMed] [Google Scholar]
  4. Halseth AE, Bracy DP, Wasserman DH. Functional limitations to glucose uptake in muscles comprised of different fiber types. Am J Physiol Endocrinol Metab. 2001;280:E994–999. doi: 10.1152/ajpendo.2001.280.6.E994. [DOI] [PubMed] [Google Scholar]
  5. Hansen PA, Gulve EA, Holloszy JO. Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol (1985) 1994;76:979–985. doi: 10.1152/jappl.1994.76.2.979. [DOI] [PubMed] [Google Scholar]
  6. Hirai H, Sootome H, Nakatsuru Y, Miyama K, Taguchi S, Tsujioka K, et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol Cancer Ther. 2010;9:1956–1967. doi: 10.1158/1535-7163.MCT-09-1012. [DOI] [PubMed] [Google Scholar]
  7. McCurdy CE, Cartee GD. Akt2 is essential for the full effect of calorie restriction on insulin-stimulated glucose uptake in skeletal muscle. Diabetes. 2005;54:1349–1356. doi: 10.2337/diabetes.54.5.1349. [DOI] [PubMed] [Google Scholar]
  8. McCurdy CE, Davidson RT, Cartee GD. Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle. Am J Physiol Endocrinol Metab. 2003;285:E693–700. doi: 10.1152/ajpendo.00224.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Petersen HA, Fueger PT, Bracy DP, Wasserman DH, Halseth AE. Fiber type-specific determinants of Vmax for insulin-stimulated muscle glucose uptake in vivo. Am J Physiol Endocrinol Metab. 2003;284:E541–548. doi: 10.1152/ajpendo.00323.2002. [DOI] [PubMed] [Google Scholar]
  10. Sakamoto K, Arnolds DE, Fujii N, Kramer HF, Hirshman MF, Goodyear LJ. Role of Akt2 in contraction-stimulated cell signaling and glucose uptake in skeletal muscle. Am J Physiol Endocrinol Metab. 2006;291:E1031–1037. doi: 10.1152/ajpendo.00204.2006. [DOI] [PubMed] [Google Scholar]
  11. Sano H, Kane S, Sano E, Miinea CP, Asara JM, Lane WS, et al. Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation. J Biol Chem. 2003;278:14599–14602. doi: 10.1074/jbc.C300063200. [DOI] [PubMed] [Google Scholar]
  12. Sequea DA, Sharma N, Arias EB, Cartee GD. Calorie restriction enhances insulin-stimulated glucose uptake and Akt phosphorylation in both fast-twitch and slow-twitch skeletal muscle of 24-month-old rats. J Gerontol A Biol Sci Med Sci. 2012;67:1279–1285. doi: 10.1093/gerona/gls085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, et al. Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats. Am J Physiol Endocrinol Metab. 2011;300:E966–978. doi: 10.1152/ajpendo.00659.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sharma N, Arias EB, Sequea DA, Cartee GD. Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction’s effect on glucose uptake in skeletal muscle. Biochim Biophys Acta. 2012a;1822:1735–1740. doi: 10.1016/j.bbadis.2012.07.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sharma N, Castorena CM, Cartee GD. Greater insulin sensitivity in calorie restricted rats occurs with unaltered circulating levels of several important myokines and cytokines. Nutr Metab (Lond) 2012b;9:90. doi: 10.1186/1743-7075-9-90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sharma N, Castorena CM, Cartee GD. Tissue-specific responses of IGF-1/insulin and mTOR signaling in calorie restricted rats. PLoS One. 2012c;7:e38835. doi: 10.1371/journal.pone.0038835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sharma N, Sequea DA, Arias EB, Cartee GD. Greater insulin-mediated Akt phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in soleus of calorie-restricted rats. Am J Physiol Regul Integr Comp Physiol. 2012d;303:R1261–1267. doi: 10.1152/ajpregu.00457.2012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sharma N, Sequea DA, Castorena CM, Arias EB, Qi NR, Cartee GD. Heterogeneous effects of calorie restriction on in vivo glucose uptake and insulin signaling of individual rat skeletal muscles. PLoS One. 2014;8:e65118. doi: 10.1371/journal.pone.0065118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sharma N, Wang H, Arias EB, Castorena CM, Cartee GD. Mechanisms for independent and combined effects of calorie restriction and acute exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats. Am J Physiol Endocrinol Metab. 2015;308:E603–612. doi: 10.1152/ajpendo.00618.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stockli J, Fazakerley DJ, James DE. GLUT4 exocytosis. J Cell Sci. 2011;124:4147–4159. doi: 10.1242/jcs.097063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tan S, Ng Y, James DE. Next-generation Akt inhibitors provide greater specificity: effects on glucose metabolism in adipocytes. Biochem J. 2011;435:539–544. doi: 10.1042/BJ20110040. [DOI] [PubMed] [Google Scholar]
  22. Tan SX, Ng Y, Burchfield JG, Ramm G, Lambright DG, Stockli J, et al. The Rab GTPase-activating protein TBC1D4/AS160 contains an atypical phosphotyrosine-binding domain that interacts with plasma membrane phospholipids to facilitate GLUT4 trafficking in adipocytes. Mol Cell Biol. 2012;32:4946–4959. doi: 10.1128/MCB.00761-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang H, Arias EB, Cartee GD. Calorie restriction leads to greater Akt2 activity and glucose uptake by insulin-stimulated skeletal muscle from old rats. Am J Physiol Regul Integr Comp Physiol. 2016a;310:R449–458. doi: 10.1152/ajpregu.00449.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wang H, Sharma N, Arias EB, Cartee GD. Insulin Signaling and Glucose Uptake in the Soleus Muscle of 30-Month-Old Rats After Calorie Restriction With or Without Acute Exercise. J Gerontol A Biol Sci Med Sci. 2016b;71:323–332. doi: 10.1093/gerona/glv142. [DOI] [PMC free article] [PubMed] [Google Scholar]

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