Diabetes Volume 64, June 2015 1901 Gregory D. Cartee AMPK-TBC1D4–Dependent Mechanism for Increasing Insulin Sensitivity of Skeletal Muscle Diabetes 2015;64:1901–1903 | DOI: 10.2337/db15-0010 Insulin resistance in skeletal muscle, the major site of Although much is unknown about the mechanisms for insulin-stimulated blood glucose clearance, is a primary improved insulin sensitivity after exercise, elevated phos- defect in the development of type 2 diabetes. Accordingly, phorylation of the Rab-GTPase protein known as TBC1D4 there is great interest in interventions that improve (also called Akt substrate of 160 kDa, AS160) has emerged muscle insulin sensitivity. A single exercise session can as an attractive candidate to participate in the persistent substantially enhance muscle insulin sensitivity for up to increase in insulin-stimulated glucose uptake. Enhanced 24–48 h postexercise (1–11), but full discernment of the TBC1D4 phosphorylation, which is crucial for insulin- specific processes responsible for this health benefit stimulated GLUT4 translocation (13), tracks closely with remains elusive. Illuminating these mechanisms may be the increased glucose uptake by insulin-stimulated muscle helpful for designing optimally effective exercise proto- during the hours after acute exercise (7–11,14–16). If cols. Furthermore, because of the very limited exercise prior AICAR treatment is found to also produce greater COMMENTARY capacity of many insulin-resistant people, these insights TBC1D4 phosphorylation in insulin-stimulated muscles, it may enable the development of other therapies that im- would suggest that AICAR and exercise may share a com- prove insulin sensitivity by engaging the exercise-induced mon mechanism to improve insulin sensitivity. mechanisms. In the current issue of Diabetes, Kjøbsted et al. (17) aim In vivo exercise influences many physiological sys- to determine if AMPK activation is essential for the tems, making it challenging to isolate the specificevents AICAR-induced increase in muscle insulin sensitivity. Iso- that account for improved insulin sensitivity. A strategy lated extensor digitorum longus (EDL) and soleus muscles to eliminate systemic exercise responses is to electrically from mice were studied 4–6 h after 50 min of AICAR stimulate the contraction of isolated rodent skeletal incubation. Prior AICAR exposure caused greater insulin- muscles. Using this approach, Gao et al. (5) discovered stimulated glucose uptake in the EDL, but not the soleus. that ex vivo contractions could induce subsequently To learn if AMPK activation was required for improved greater insulin-stimulated glucose uptake. Although ex insulin sensitivity, additional experiments used EDL muscles vivo contraction is much less complicated than in vivo from three different genetically modified mouse models in exercise, it still produces complex effects on calcium, ten- which muscle AMPK activity was greatly reduced. Prior sion, high-energy phosphates, glycogen and lipids, meta- AICAR exposure led to greater insulin-stimulated glucose bolic intermediates, redox potential, reactive oxygen and uptakeinmusclesfromwild-typemicewithnormalAMPK nitrogen species, and many enzymes. activity, but not in AMPK-deficient muscles. Furthermore, An even simpler strategy is to incubate an isolated for insulin-stimulated muscles from wild-type mice, but muscle with a chemical that triggers a limited subset of not insulin-stimulated muscles from AMPK-deficient exercise’s numerous consequences without causing muscle mice, prior AICAR stimulation led to greater TBC1D4 contraction. AMPK, which is stimulated by exercise/con- phosphorylation on Thr649 and Ser711. In tibialis anterior traction, is also stimulated by AICAR. Fisher et al. (12) muscles expressing TBC1D4 that was mutated so that it found that incubation of isolated rat muscles with AICAR could not be phosphorylated on Ser711, there was an at- produced a subsequent increase in insulin-stimulated glu- tenuated insulin effect on TBC1D4 Thr649 phosphoryla- cose uptake, suggesting that AMPK activation can lead to tion. Given that AMPK can phosphorylate TBC1D4 on increased insulin sensitivity in muscle. Ser711 (18) and that Thr649 phosphorylation is crucial Muscle Biology Laboratory, School of Kinesiology; Department of Molecular & © 2015 by the American Diabetes Association. Readers may use this article as Integrative Physiology; and Institute of Gerontology, University of Michigan, Ann long as the work is properly cited, the use is educational and not for profit, and Arbor, MI the work is not altered. Corresponding author: Gregory D. Cartee, [email protected]. See accompanying article, p. 2042. 1902 Commentary Diabetes Volume 64, June 2015 Figure 1—AMPK activity was increased in isolated muscles after brief (50 min) AICAR incubation, and AMPK was previously shown to phosphorylate TBC1D4 on Ser711. Six hours after AICAR treatment, insulin produced greater TBC1D4 Ser711 and Thr 649 phosphorylation (pSer711-TBC1D4 and pThr 649-TBC1D4, respectively) along with increased glucose uptake compared with insulin-stimulated muscles without AICAR pretreatment. Muscles expressing TBC1D4 that was mutated to prevent phosphorylation on Ser711 had attenuated insulin-stimulated pThr 649-TBC1D4 (a site known to be crucial for insulin-stimulated glucose uptake). Prior effects of AICAR on TBC1D4 phosphorylation and insulin-stimulated glucose uptake were absent in genetically modified muscles that were AMPK deficient. The working hypothesis is that AICAR activation of AMPK leads to greater pSer711-TBC1D4, which facilitates elevated pThr 649-TBC1D4 in subsequently insulin-stimulated muscles, leading to greater insulin-mediated glucose uptake. for TBC1D4 regulation of insulin-stimulated glucose up- phosphorylation and insulin sensitivity in insulin-resistant take (13,19), the authors speculate that the ability of muscle? AICAR to elevate TBC1D4 Ser711 phosphorylation may Kjøbsted et al. (17) provide compelling evidence that play a role in AICAR’s AMPK-dependent effect on insulin AMPK activation is essential for AICAR-induced elevation sensitivity (Fig. 1). of muscle insulin sensitivity. The effects of prior AICAR The key and novel aspect of the experimental design exposure on TBC1D4 phosphorylation and glucose uptake of the current study was the use of muscles with normal in insulin-stimulated muscle were reminiscent of the AMPK activity and muscles that were AMPK deficient. In effects of in vivo exercise on these outcomes. Together addition, the effect of AICAR on the activity of multiple with earlier research demonstrating that acute exercise AMPK isoforms was examined, and the greatest response activates AMPK and leads to a subsequent increase in in- was found for the a2b2g3 heterotrimer. Furthermore, sulin sensitivity, the current observations provide the basis prior AICAR exposure led to greater insulin-stimulated glu- for the working hypothesis that the exercise-induced in- cose uptake concomitant with elevated TBC1D4 phos- crease in insulin sensitivity is regulated by an AMPK- phorylation on Thr649 and Ser711.TheresultsforTBC1D4 TBC1D4 signaling mechanism. phosphorylation and insulin-stimulated glucose uptake were reminiscent of earlier observations after acute ex- ercise (8–11,15). Funding. G.D.C. was supported by a grant from the National Institutes of This study raises new questions that will require Health (R01-DK-071771). Duality of Interest. No potential conflicts of interest relevant to this article further research. First, AICAR treatment activated were reported. AMPK without enhancing subsequent insulin sensitivity in the soleus. This observation differs from the results of References earlier research in which prior exercise led to enhanced 1. Richter EA, Garetto LP, Goodman MN, Ruderman NB. Muscle glucose insulin-stimulated glucose uptake in the soleus of either metabolism following exercise in the rat: increased sensitivity to insulin. J Clin mice or rats (11,20). AICAR is a useful, but complemen- Invest 1982;69:785–793 tary, tool rather than a substitute for directly studying 2. Mikines KJ, Sonne B, Farrell PA, Tronier B, Galbo H. Effect of physical exercise. It would now be valuable to use AMPK-deficient exercise on sensitivity and responsiveness to insulin in humans. Am J Physiol 1988;254:E248–E259 models to test if normal AMPK activation is essential for 3. Cartee GD, Young DA, Sleeper MD, Zierath J, Wallberg-Henriksson H, the postexercise effect on insulin-stimulated glucose up- Holloszy JO. Prolonged increase in insulin-stimulated glucose transport in muscle take by multiple muscles, including the soleus. Second, after exercise. Am J Physiol 1989;256:E494–E499 649 insulin-stimulated TBC1D4 Thr phosphorylation was 4. Cartee GD, Holloszy JO. Exercise increases susceptibility of muscle attenuated in tibialis anterior muscles expressing glucose transport to activation by various stimuli. Am J Physiol 1990;258: TBC1D4 that was mutated to prevent Ser711 phosphor- E390–E393 ylation, but insulin-stimulated glucose uptake was not 5. Gao J, Gulve EA, Holloszy JO. Contraction-induced increase in muscle assessed, nor was the effect of AICAR pretreatment eval- insulin sensitivity: requirement for a serum factor. Am J Physiol 1994;266: uated. 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