Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during postexercise recovery

Akt substrate of 160 kDa (AS160/TBC1D4) is associated with insulin and contraction-mediated glucose uptake. Human skeletal muscle AS160 phosphorylation is increased during aerobic exercise but not immediately following resistance exercise. It is not known whether AS160 phosphorylation is altered during recovery from resistance exercise. Therefore, we hypothesized that muscle AS160/TBC1D4 phosphorylation and glucose uptake across the leg would be increased during recovery following resistance exercise. We studied 9 male subjects before, during, and for 2 h of postexercise recovery. We utilized femoral catheterizations and muscle biopsies in combination with indirect calorimetry and immunoblotting to determine whole body glucose and fat oxidation, leg glucose uptake, muscle AMPKalpha2 activity, and the phosphorylation of muscle Akt and AS160/TBC1D4. Glucose oxidation was reduced while fat oxidation increased ( approximately 35%) during postexercise recovery (P 相似文献   

The effect of exercise and insulin on AS160 phosphorylation and 14-3-3 binding capacity in human skeletal muscle

AS160 is an Akt substrate of 160 kDa implicated in the regulation of both insulin- and contraction-mediated GLUT4 translocation and glucose uptake. The effects of aerobic exercise and subsequent insulin stimulation on AS160 phosphorylation and the binding capacity of 14-3-3, a novel protein involved in the dissociation of AS160 from GLUT4 vesicles, in human skeletal muscle are unknown. Hyperinsulinemic-euglycemic clamps were performed on seven men at rest and immediately and 3 h after a single bout of cycling exercise. Skeletal muscle biopsies were taken before and after the clamps. The insulin sensitivity index calculated during the final 30 min of the clamp was 8.0 +/- 0.8, 9.1 +/- 0.5, and 9.2 +/- 0.8 for the rest, postexercise, and 3-h postexercise trials, respectively. AS160 phosphorylation increased immediately after exercise and remained elevated 3 h after exercise. In contrast, the 14-3-3 binding capacity of AS160 and phosphorylation of Akt and AMP-activated protein kinase were only increased immediately after exercise. Insulin increased AS160 phosphorylation and 14-3-3 binding capacity and insulin receptor substrate-1 and Akt phosphorylation, but the response to insulin was not enhanced by prior exercise. In conclusion, the 14-3-3 binding capacity of AS160 is increased immediately after acute exercise in human skeletal muscle, but this is not maintained 3 h after exercise completion despite sustained AS160 phosphorylation. Insulin increases AS160 phosphorylation and 14-3-3 binding capacity, but prior exercise does not appear to enhance the response to insulin.     

Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise.

The primary purpose of this study was to determine the effect of prior exercise on insulin-stimulated glucose uptake with physiological insulin in isolated muscles of mice. Male C57BL/6 mice completed a 60-min treadmill exercise protocol or were sedentary. Paired epitrochlearis, soleus, and extensor digitorum longus (EDL) muscles were incubated with [3H]-2-deoxyglucose without or with insulin (60 microU/ml) to measure glucose uptake. Insulin-stimulated glucose uptake for paired muscles was calculated by subtracting glucose uptake without insulin from glucose uptake with insulin. Muscles from other mice were assessed for glycogen and AMPK Thr172 phosphorylation. Exercised vs. sedentary mice had decreased glycogen in epitrochlearis (48%, P < 0.001), soleus (51%, P < 0.001), and EDL (41%, P < 0.01) and increased AMPK Thr172 phosphorylation (P < 0.05) in epitrochlearis (1.7-fold), soleus (2.0-fold), and EDL (1.4-fold). Insulin-independent glucose uptake was increased 30 min postexercise vs. sedentary in the epitrochlearis (1.2-fold, P < 0.001), soleus (1.4-fold, P < 0.05), and EDL (1.3-fold, P < 0.01). Insulin-stimulated glucose uptake was increased (P < 0.05) approximately 85 min after exercise in the epitrochlearis (sedentary: 0.266 +/- 0.045 micromol x g(-1) x 15 min(-1); exercised: 0.414 +/- 0.051) and soleus (sedentary: 0.102 +/- 0.049; exercised: 0.347 +/- 0.098) but not in the EDL. Akt Ser473 and Akt Thr308 phosphorylation for insulin-stimulated muscles did not differ in exercised vs. sedentary. These results demonstrate enhanced submaximal insulin-stimulated glucose uptake in the epitrochlearis and soleus of mice 85 min postexercise and suggest that it will be feasible to probe the mechanism of enhanced postexercise insulin sensitivity by using genetically modified mice.     

AS160 phosphotyrosine-binding domain constructs inhibit insulin-stimulated GLUT4 vesicle fusion with the plasma membrane

AS160 (TBC1D4) is a known Akt substrate that is phosphorylated downstream of insulin action and that leads to regulated traffic of GLUT4. As GLUT4 vesicle fusion with the plasma membrane is a highly regulated step in GLUT4 traffic, we investigated whether AS160 and 14-3-3 interactions are involved in this process. Fusion was inhibited by a human truncated AS160 variant that encompasses the first N-terminal phosphotyrosine-binding (PTB) domain, by either of the two N-terminal PTB domains, and by a tandem construct of both PTB domains of rat AS160. We also found that in vitro GLUT4 vesicle fusion was strongly inhibited by the 14-3-3-quenching inhibitors R18 and fusicoccin. To investigate the mode of interaction of AS160 and 14-3-3, we examined insulin-dependent increases in the levels of these proteins on GLUT4 vesicles. 14-3-3γ was enriched on insulin-stimulated vesicles, and its binding to AS160 on GLUT4 vesicles was inhibited by the AS160 tandem PTB domain construct. These data suggest a model for PTB domain action on GLUT4 vesicle fusion in which these constructs inhibit insulin-stimulated 14-3-3γ interaction with AS160 rather than AS160 phosphorylation.     

Insulin-stimulated phosphorylation of a Rab GTPase-activating protein regulates GLUT4 translocation

Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.     

In vitro simulation of calorie restriction-induced decline in glucose and insulin leads to increased insulin-stimulated glucose transport in rat skeletal muscle

In vivo calorie restriction [CR; consuming 60% of ad libitum (AL) intake] induces elevated insulin-stimulated glucose transport (GT) in skeletal muscle. The mechanisms triggering this adaptation are unknown. The aim of this study was to determine whether physiological reductions in extracellular glucose and/or insulin, similar to those found with in vivo CR, were sufficient to elevate GT in isolated muscles. Epitrochlearis muscles dissected from rats were incubated for 24 h in media with glucose (8 mM) and insulin (80 microU/ml) at levels similar to plasma values of AL-fed rats and compared with muscles incubated with glucose (5.5 mM) and/or insulin (20 microU/ml) at levels similar to plasma values of CR rats. Muscles incubated with CR levels of glucose and insulin for 24 h had a subsequently greater (P < 0.005) GT with 80 microU/ml insulin and 8 mM [(3)H]-3-O-methylglucose but unchanged GT without insulin. Reducing only glucose or insulin for 24 h or both glucose and insulin for 6 h did not induce altered GT. Increased GT after 24-h incubation with CR levels of glucose and insulin was not attributable to increased insulin receptor tyrosine phosphorylation, Akt serine phosphorylation, or Akt substrate of 160 kDa phosphorylation. Nor did 24-h incubation with CR levels of glucose and insulin alter the abundance of insulin receptor, insulin receptor substrate-1, GLUT1, or GLUT4 proteins. These results provide the proof of principle that reductions in extracellular glucose and insulin, similar to in vivo CR, are sufficient to induce an increase in insulin-stimulated glucose transport comparable to the increase found with in vivo CR.     

Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats

Calorie restriction [CR; ~65% of ad libitum (AL) intake] improves insulin-stimulated glucose uptake (GU) and Akt phosphorylation in skeletal muscle. We aimed to elucidate the effects of CR on 1) processes that regulate Akt phosphorylation [insulin receptor (IR) tyrosine phosphorylation, IR substrate 1-phosphatidylinositol 3-kinase (IRS-PI3K) activity, and Akt binding to regulatory proteins (heat shock protein 90, Appl1, protein phosphatase 2A)]; 2) Akt substrate of 160-kDa (AS160) phosphorylation on key phosphorylation sites; and 3) atypical PKC (aPKC) activity. Isolated epitrochlearis (fast-twitch) and soleus (slow-twitch) muscles from AL or CR (6 mo duration) 9-mo-old male F344BN rats were incubated with 0, 1.2, or 30 nM insulin and 2-deoxy-[(3)H]glucose. Some CR effects were independent of insulin dose or muscle type: CR caused activation of Akt (Thr(308) and Ser(473)) and GU in both muscles at both insulin doses without CR effects on IRS1-PI3K, Akt-PP2A, or Akt-Appl1. Several muscle- and insulin dose-specific CR effects were revealed. Akt-HSP90 binding was increased in the epitrochlearis; AS160 phosphorylation (Ser(588) and Thr(642)) was greater for CR epitrochlearis at 1.2 nM insulin; and IR phosphorylation and aPKC activity were greater for CR in both muscles with 30 nM insulin. On the basis of these data, our working hypothesis for improved insulin-stimulated GU with CR is as follows: 1) elevated Akt phosphorylation is fundamental, regardless of muscle or insulin dose; 2) altered Akt binding to regulatory proteins (HSP90 and unidentified Akt partners) is involved in the effects of CR on Akt phosphorylation; 3) Akt effects on GU depend on muscle- and insulin dose-specific elevation in phosphorylation of Akt substrates, including, but not limited to, AS160; and 4) greater IR phosphorylation and aPKC activity may contribute at higher insulin doses.     

Prior serum- and AICAR-induced AMPK activation in primary human myocytes does not lead to subsequent increase in insulin-stimulated glucose uptake

Exposing isolated rat skeletal muscle to 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, a pharmacological activator of AMP-activated protein kinase (AMPK)] plus serum leads to a subsequent increase in insulin-stimulated glucose transport (Fisher JS, Gao J, Han DH, Holloszy JO, and Nolte LA. Am J Physiol Endocrinol Metab 282: E18-E23, 2002). Our goal was to determine whether preincubation of primary human skeletal muscle cells with human serum and AICAR (Serum+AICAR) would also induce a subsequent elevation in insulin-stimulated glucose uptake. Cells were preincubated for 1 h under 4 conditions: 1) without AICAR or serum (Control), 2) with serum, 3) with AICAR, or 4) with Serum+AICAR. Some cells were then collected for immunoblot analysis to assess phosphorylation of AMPK (pAMPK) and its substrate acetyl-CoA carboxylase (ACC). Other cells were incubated for an additional 4 h without AICAR or serum and then used to measure basal or insulin-stimulated 2-deoxyglucose (2-DG) uptake. Level of pAMPK was increased (P < 0.01) for myotubes exposed to Serum+AICAR vs. all other groups. Phosphorylated ACC (pACC) levels were higher for both Serum+AICAR (P < 0.05) and AICAR (P < 0.05) vs. Control and Serum groups. Basal (P < 0.05) and 1.2 nM insulin-stimulated (P < 0.005) 2-DG uptake was higher for Serum vs. all other preincubation conditions at equal insulin concentration. Regardless of insulin concentration (0, 1.2, or 18 nM), 2-DG was unaltered in cells preincubated with Serum+AICAR vs. Control cells. In contrast to results with isolated rat skeletal muscle, increasing the pAMPK and pACC in human myocytes via preincubation with Serum+AICAR was insufficient to lead to a subsequent enhancement in insulin-stimulated glucose uptake.     

The Association of ClipR-59 Protein with AS160 Modulates AS160 Protein Phosphorylation and Adipocyte Glut4 Protein Membrane Translocation

ClipR-59 is a membrane-associated protein and has been implicated in membrane signaling and vesicle trafficking. Recently, we have identified ClipR-59 as an Akt-interacting protein, and we have found that, by interacting with Akt, ClipR-59 modulates Akt subcellular compartmentalization and Akt substrate AS160 phosphorylation, thereby promoting Glut4 membrane translocation. Here, we have further investigated the regulatory effects of ClipR-59 on AS160 phosphorylation and subsequent adipocyte glucose transport. Our data showed that ClipR-59 interacted with AS160, which was mediated by the ankyrin repeats of ClipR-59 and regulated by insulin signaling. Moreover, the data also demonstrated that the interaction of ClipR-59 with AS160 was required for ClipR-59 to modulate Glut4 membrane translocation as ΔANK-ClipR-59, an AS160 interaction-defective mutant, failed to promote AS160 phosphorylation, Glut4 membrane translocation, and glucose transport induced by insulin in 3T3-L1 adipocytes. Because ClipR-59 also interacts with Akt and enhances the interaction between Akt and AS160, we suggest that ClipR-59 functions as a scaffold protein to facilitate Akt-mediated AS160 phosphorylation, thereby regulating glucose transport.     

Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.     

Characterization of the role of the Rab GTPase-activating protein AS160 in insulin-regulated GLUT4 trafficking

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane. In the present study we have conducted a comprehensive proteomic analysis of affinity-purified GLUT4 vesicles from 3T3-L1 adipocytes to discover potential regulators of GLUT4 trafficking. In addition to previously identified components of GLUT4 storage vesicles including the insulin-regulated aminopeptidase insulin-regulated aminopeptidase and the vesicle soluble N-ethylmaleimide factor attachment protein (v-SNARE) VAMP2, we have identified three new Rab proteins, Rab10, Rab11, and Rab14, on GLUT4 vesicles. We have also found that the putative Rab GTPase-activating protein AS160 (Akt substrate of 160 kDa) is associated with GLUT4 vesicles in the basal state and dissociates in response to insulin. This association is likely to be mediated by the cytosolic tail of insulin-regulated aminopeptidase, which interacted both in vitro and in vivo with AS160. Consistent with an inhibitory role of AS160 in the basal state, reduced expression of AS160 in adipocytes using short hairpin RNA increased plasma membrane levels of GLUT4 in an insulin-independent manner. These findings support an important role for AS160 in the insulin regulated trafficking of GLUT4.     

A role for 14-3-3 in insulin-stimulated GLUT4 translocation through its interaction with the RabGAP AS160

Translocation of the insulin-regulated glucose transporter GLUT4 to the cell surface is dependent on the phosphatidylinositol 3-kinase/Akt pathway. The RabGAP (Rab GTPase-activating protein) AS160 (Akt substrate of 160 kDa) is a direct substrate of Akt and plays an essential role in the regulation of GLUT4 trafficking. We have used liquid chromatography tandem mass spectrometry to identify several 14-3-3 isoforms as AS160-interacting proteins. 14-3-3 proteins interact with AS160 in an insulin- and Akt-dependent manner via an Akt phosphorylation site, Thr-642. This correlates with the dominant negative effect of both the AS160(T642A) and the AS160(4P) mutants on insulin-stimulated GLUT4 translocation. Introduction of a constitutive 14-3-3 binding site into AS160(4P) restored 14-3-3 binding without disrupting AS160-IRAP (insulin-responsive amino peptidase) interaction and reversed the inhibitory effect of AS160(4P) on GLUT4 translocation. These data show that the insulin-dependent association of 14-3-3 with AS160 plays an important role in GLUT4 trafficking in adipocytes.     

AS160 Modulates Aldosterone-stimulated Epithelial Sodium Channel Forward Trafficking

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I_sc). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I_sc 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I_sc under basal conditions to ∼80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, β and ε. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3β and ε and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.     

Heterogeneous Effects of Calorie Restriction on In Vivo Glucose Uptake and Insulin Signaling of Individual Rat Skeletal Muscles

Calorie restriction (CR) (consuming ∼60% of ad libitum, AL, intake) improves whole body insulin sensitivity and enhances insulin-stimulated glucose uptake by isolated skeletal muscles. However, little is known about CR-effects on in vivo glucose uptake and insulin signaling in muscle. Accordingly, 9-month-old male AL and CR (initiated when 3-months-old) Fischer 344xBrown Norway rats were studied using a euglycemic-hyperinsulinemic clamp with plasma insulin elevated to a similar level (∼140 µU/ml) in each diet group. Glucose uptake (assessed by infusion of [¹⁴C]-2-deoxyglucose, 2-DG), phosphorylation of key insulin signaling proteins (insulin receptor, Akt and Akt substrate of 160kDa, AS160), abundance of GLUT4 and hexokinase proteins, and muscle fiber type composition (myosin heavy chain, MHC, isoform percentages) were determined in four predominantly fast-twitch (epitrochlearis, gastrocnemius, tibialis anterior, plantaris) and two predominantly slow-twitch (soleus, adductor longus) muscles. CR did not result in greater GLUT4 or hexokinase abundance in any of the muscles, and there were no significant diet-related effects on percentages of MHC isoforms. Glucose infusion was greater for CR versus AL rats (P<0.05) concomitant with significantly (P<0.05) elevated 2-DG uptake in 3 of the 4 fast-twitch muscles (epitrochlearis, gastrocnemius, tibialis anterior), without a significant diet-effect on 2-DG uptake by the plantaris or either slow-twitch muscle. Each of the muscles with a CR-related increase in 2-DG uptake was also characterized by significant (P<0.05) increases in phosphorylation of both Akt and AS160. Among the 3 muscles without a CR-related increase in glucose uptake, only the soleus had significant (P<0.05) CR-related increases in Akt and AS160 phosphorylation. The current data revealed that CR leads to greater whole body glucose disposal in part attributable to elevated in vivo insulin-stimulated glucose uptake by fast-twitch muscles. The results also demonstrated that CR does not uniformly enhance either insulin signaling or insulin-stimulated glucose uptake in all muscles in vivo.     

An amino acid mixture is essential to optimize insulin-stimulated glucose uptake and GLUT4 translocation in perfused rodent hindlimb muscle

The purpose of this study was to investigate whether an amino acid mixture increases glucose uptake across perfused rodent hindlimb muscle in the presence and absence of a submaximal insulin concentration, and if the increase in glucose uptake is related to an increase in GLUT4 plasma membrane density. Sprague-Dawley rats were separated into one of four treatment groups: basal, amino acid mixture, submaximal insulin, or amino acid mixture with submaximal insulin. Glucose uptake was greater for both insulin-stimulated treatments compared with the non-insulin-stimulated treatment groups but amino acids only increased glucose uptake in the presence of insulin. Phosphatidylinositol 3-kinase (PI 3-kinase) activity was greater for both insulin-stimulated treatments with amino acids having no additional impact. Akt substrate of 160 kDa (AS160) phosphorylation, however, was increased by the amino acids in the presence of insulin, but not in the absence of insulin. AMPK was unaffected by insulin or amino acids. Plasma membrane GLUT4 protein concentration was greater in the rats treated with insulin compared with no insulin in the perfusate. In the presence of insulin, amino acids increased GLUT4 density in the plasma membrane but had no effect in the absence of insulin. AS160 phosphorylation and plasma membrane GLUT4 density accounted for 76% of the variability in muscle glucose uptake. Collectively, these findings suggest that the beneficial effects of an amino acid mixture on skeletal muscle glucose uptake, in the presence of a submaximal insulin concentration, are due to an increase in AS160 phosphorylation and plasma membrane-associated GLUT4, but independent of PI 3-kinase and AMPK activation.     

Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.     

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.     

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60-65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160(Thr642) and pFilamin C(Ser2213) without altering pIR(Tyr1162/1163). These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.     

Exercise-induced enhancement of insulin sensitivity is associated with accumulation of M2-polarized macrophages in mouse skeletal muscle

Exercise enhances insulin sensitivity in skeletal muscle, but the underlying mechanism remains obscure. Recent data suggest that alternatively activated M2 macrophages enhance insulin sensitivity in insulin target organs such as adipose tissue and liver. Therefore, the aim of this study was to determine the role of anti-inflammatory M2 macrophages in exercise-induced enhancement of insulin sensitivity in skeletal muscle. C57BL6J mice underwent a single bout of treadmill running (20 m/min, 90 min). Twenty-four hours later, ex vivo insulin-stimulated 2-deoxy glucose uptake was found to be increased in plantaris muscle. This change was associated with increased number of CD163-expressing macrophages (i.e. M2-polarized macrophages) in skeletal muscle. Systemic depletion of macrophages by pretreatment of mice with clodronate-containing liposome abrogated both CD163-positive macrophage accumulation in skeletal muscle as well as the enhancement of insulin sensitivity after exercise, without affecting insulin-induced phosphorylation of Akt and AS160 or exercise-induced GLUT4 expression. These results suggest that accumulation of M2-polarized macrophages is involved in exercise-induced enhancement of insulin sensitivity in mouse skeletal muscle, independently of the phosphorylation of Akt and AS160 and expression of GLUT4.