Exercise increases TBC1D1 phosphorylation in human skeletal muscle

Exercise and weight loss are cornerstones in the treatment and prevention of type 2 diabetes, and both interventions function to increase insulin sensitivity and glucose uptake into skeletal muscle. Studies in rodents demonstrate that the underlying mechanism for glucose uptake in muscle involves site-specific phosphorylation of the Rab-GTPase-activating proteins AS160 (TBC1D4) and TBC1D1. Multiple kinases, including Akt and AMPK, phosphorylate TBC1D1 and AS160 on distinct residues, regulating their activity and allowing for GLUT4 translocation. In contrast to extensive rodent-based studies, the regulation of AS160 and TBC1D1 in human skeletal muscle is not well understood. In this study, we determined the effects of dietary intervention and a single bout of exercise on TBC1D1 and AS160 site-specific phosphorylation in human skeletal muscle. Ten obese (BMI 33.4 ± 2.4, M-value 4.3 ± 0.5) subjects were studied at baseline and after a 2-wk dietary intervention. Muscle biopsies were obtained from the subjects in the resting (basal) state and immediately following a 30-min exercise bout (70% Vo(2 max)). Muscle lysates were analyzed for AMPK activity and Akt phosphorylation and for TBC1D1 and AS160 phosphorylation on known or putative AMPK and Akt sites as follows: AS160 Ser(711) (AMPK), TBC1D1 Ser(231) (AMPK), TBC1D1 Ser(660) (AMPK), TBC1D1 Ser(700) (AMPK), and TBC1D1 Thr(590) (Akt). The diet intervention that consisted of a major shift in the macronutrient composition resulted in a 4.2 ± 0.4 kg weight loss (P < 0.001) and a significant increase in insulin sensitivity (M value 5.6 ± 0.6), but surprisingly, there was no effect on expression or phosphorylation of any of the muscle-signaling proteins. Exercise increased muscle AMPKα2 activity but did not increase Akt phosphorylation. Exercise increased phosphorylation on AS160 Ser(711), TBC1D1 Ser(231), and TBC1D1 Ser(660) but had no effect on TBC1D1 Ser(700). Exercise did not increase TBC1D1 Thr(590) phosphorylation or TBC1D1/AS160 PAS phosphorylation, consistent with the lack of Akt activation. These data demonstrate that a single bout of exercise regulates TBC1D1 and AS160 phosphorylation on multiple sites in human skeletal muscle.     

Insulin and AMPK regulate FA translocase/CD36 plasma membrane recruitment in cardiomyocytes via Rab GAP AS160 and Rab8a Rab GTPase

The FA translocase cluster of differentiation 36 (CD36) facilitates FA uptake by the myocardium, and its surface recruitment in cardiomyocytes is induced by insulin, AMP-dependent protein kinase (AMPK), or contraction. Dysfunction of CD36 trafficking contributes to disordered cardiac FA utilization and promotes progression to disease. The Akt substrate 160 (AS160) Rab GTPase-activating protein (GAP) is a key regulator of vesicular trafficking, and its activity is modulated via phosphorylation. Our study documents that AS160 mediates insulin or AMPK-stimulated surface translocation of CD36 in cardiomyocytes. Knock-down of AS160 redistributes CD36 to the surface and abrogates its translocation by insulin or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR). Conversely, overexpression of a phosphorylation-deficient AS160 mutant (AS160 4P) suppresses the stimulated membrane recruitment of CD36. The AS160 substrate Rab8a GTPase is shown via overexpression and knock-down studies to be specifically involved in insulin/AICAR-induced CD36 membrane recruitment. Our findings directly demonstrate AS160 regulation of CD36 trafficking. In myocytes, the AS160 pathway also mediates the effect of insulin, AMPK, or contraction on surface recruitment of the glucose transporter GLUT4. Thus, AS160 constitutes a point of convergence for coordinating physiological regulation of CD36 and GLUT4 membrane recruitment.     

Understanding the Mechanism Underlie the Antidiabetic Activity of Oleuropein Using Ex-Vivo Approach

Background:Oleuropein, the main constituent of olive fruit and leaves, has been reported to protect against insulin resistance and diabetes. While many experimental investigations have examined the mechanisms by which oleuropein improves insulin resistance and diabetes, much of these investigations have been carried out in either muscle cell lines or in vivo models two scenarios with many drawbacks. Accordingly, to simplify identification of mechanisms by which oleuropein regulates specific cellular processes, we resort, in the present study, to isolated muscle preparation which enables better metabolic milieu control and permit more detailed analyses.Methods:For this purpose, soleus muscles were incubated for 12 h without or with palmitate (1.5 mM) in the presence or absence of oleuropein (1.5 mM), and compound C. Insulin-stimulated glucose transport, glucose transporter type 4 (GLUT4) translocation, Akt substrate of 160 kDa (AS160) phosphorylation and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation were examined.Results:Palmitate treatment reduced insulin-stimulated glucose transport, GLUT4 translocation and AS160 phosphorylation, but AMPK phosphorylation was not changed. Oleuropein administration (12 h) fully rescued insulin-stimulated glucose transport, but partially restored GLUT4 translocation. However, it fully restored AS160 phosphorylation, raising the possibility that oleuropein may also have contributed to the restoration of glucose transport by increased GLUT4 intrinsic activity. Inhibition of AMPK phosphorylation with compound C (50 µM) prevented oleuropein -induced improvements in insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation.Conclusion:Our results clearly indicate that oleuropein alleviates palmitate-induced insulin resistance appears to occur via an AMPK-dependent mechanism involving improvements in the functionality of the AS160-GLUT4 signaling system.Key Words: AMPK, GLUT4, Muscle, Insulin resistance, Oleuropein     

Regulation of multisite phosphorylation and 14-3-3 binding of AS160 in response to IGF-1, EGF, PMA and AICAR

AS160 (Akt substrate of 160 kDa) mediates insulin-stimulated GLUT4 (glucose transporter 4) translocation, but is widely expressed in insulin-insensitive tissues lacking GLUT4. Having isolated AS160 by 14-3-3-affinity chromatography, we found that binding of AS160 to 14-3-3 isoforms in HEK (human embryonic kidney)-293 cells was induced by IGF-1 (insulin-like growth factor-1), EGF (epidermal growth factor), PMA and, to a lesser extent, AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside). AS160-14-3-3 interactions were stabilized by chemical cross-linking and abolished by dephosphorylation. Eight residues on AS160 (Ser318, Ser341, Thr568, Ser570, Ser588, Thr642, Ser666 and Ser751) were differentially phosphorylated in response to IGF-1, EGF, PMA and AICAR. The binding of 14-3-3 proteins to HA-AS160 (where HA is haemagglutinin) was markedly decreased by mutation of Thr642 and abolished in a Thr642Ala/Ser341Ala double mutant. The AGC (protein kinase A/protein kinase G/protein kinase C-family) kinases RSK1 (p90 ribosomal S6 kinase 1), SGK1 (serum- and glucocorticoid-induced protein kinase 1) and PKB (protein kinase B) displayed distinct signatures of AS160 phosphorylation in vitro: all three kinases phosphorylated Ser318, Ser588 and Thr642; RSK1 also phosphorylated Ser341, Ser751 and to a lesser extent Thr568; and SGK1 phosphorylated Thr568 and Ser751. AMPK (AMP-activated protein kinase) preferentially phosphorylated Ser588, with less phosphorylation of other sites. In cells, the IGF-1-stimulated phosphorylations, and certain EGF-stimulated phosphorylations, were inhibited by PI3K (phosphoinositide 3-kinase) inhibitors, whereas the RSK inhibitor BI-D1870 inhibited the PMA-induced phosphorylations. The expression of LKB1 in HeLa cells and the use of AICAR in HEK-293 cells promoted phosphorylation of Ser588, but only weak Ser341 and Thr642 phosphorylations and binding to 14-3-3s. Paradoxically however, phenformin activated AMPK without promoting AS160 phosphorylation. The IGF-1-induced phosphorylation of the novel phosphorylated Ser666-Pro site was suppressed by AICAR, and by combined mutation of a TOS (mTOR signalling)-like sequence (FEMDI) and rapamycin. Thus, although AS160 is a common target of insulin, IGF-1, EGF, PMA and AICAR, these stimuli induce distinctive patterns of phosphorylation and 14-3-3 binding, mediated by at least four protein kinases.     

Muscle-specific deletion of rictor impairs insulin-stimulated glucose transport and enhances Basal glycogen synthase activity

Rictor is an essential component of mTOR (mammalian target of rapamycin) complex 2 (mTORC2), a kinase complex that phosphorylates Akt at Ser473 upon activation of phosphatidylinositol 3-kinase (PI-3 kinase). Since little is known about the role of either rictor or mTORC2 in PI-3 kinase-mediated physiological processes in adult animals, we generated muscle-specific rictor knockout mice. Muscle from male rictor knockout mice exhibited decreased insulin-stimulated glucose uptake, and the mice showed glucose intolerance. In muscle lacking rictor, the phosphorylation of Akt at Ser473 was reduced dramatically in response to insulin. Furthermore, insulin-stimulated phosphorylation of the Akt substrate AS160 at Thr642 was reduced in rictor knockout muscle, indicating a defect in insulin signaling to stimulate glucose transport. However, the phosphorylation of Akt at Thr308 was normal and sufficient to mediate the phosphorylation of glycogen synthase kinase 3 (GSK-3). Basal glycogen synthase activity in muscle lacking rictor was increased to that of insulin-stimulated controls. Consistent with this, we observed a decrease in basal levels of phosphorylated glycogen synthase at a GSK-3/protein phosphatase 1 (PP1)-regulated site in rictor knockout muscle. This change in glycogen synthase phosphorylation was associated with an increase in the catalytic activity of glycogen-associated PP1 but not increased GSK-3 inactivation. Thus, rictor in muscle tissue contributes to glucose homeostasis by positively regulating insulin-stimulated glucose uptake and negatively regulating basal glycogen synthase activity.     

Artemisia princeps extract promoted glucose uptake in cultured L6 muscle cells via glucose transporter 4 translocation

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser(473) residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr(308)), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5'-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.     

Complementary regulation of TBC1D1 and AS160 by growth factors, insulin and AMPK activators

AS160 (Akt substrate of 160 kDa) and TBC1D1 are related RabGAPs (Rab GTPase-activating proteins) implicated in regulating the trafficking of GLUT4 (glucose transporter 4) storage vesicles to the cell surface. All animal species examined contain TBC1D1, whereas AS160 evolved with the vertebrates. TBC1D1 has two clusters of phosphorylated residues, either side of the second PTB (phosphotyrosine-binding domain). Each cluster contains a 14-3-3-binding site. When AMPK (AMP-activated protein kinase) is activated in HEK (human embryonic kidney)-293 cells, 14-3-3s bind primarily to pSer237 (where pSer is phosphorylated serine) in TBC1D1, whereas 14-3-3 binding depends primarily on pThr596 (where pThr is phosphorylated threonine) in cells stimulated with IGF-1 (insulin-like growth factor 1), EGF (epidermal growth factor) and PMA; and both pSer237 and pThr596 contribute to 14-3-3 binding in cells stimulated with forskolin. In HEK-293 cells, LY294002 inhibits phosphorylation of Thr596 of TBC1D1, and promotes phosphorylation of AMPK and Ser237 of TBC1D1. In vitro phosphorylation experiments indicated regulatory interactions among phosphorylated sites, for example phosphorylation of Ser235 prevents subsequent phosphorylation of Ser237. In rat L6 myotubes, endogenous TBC1D1 is strongly phosphorylated on Ser237 and binds to 14-3-3s in response to the AMPK activators AICAR (5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside), phenformin and A-769662, whereas insulin promotes phosphorylation of Thr596 but not 14-3-3 binding. In contrast, AS160 is phosphorylated on its 14-3-3-binding sites (Ser341 and Thr642) and binds to 14-3-3s in response to insulin, but not A-769662, in L6 cells. These findings suggest that TBC1D1 and AS160 may have complementary roles in regulating vesicle trafficking in response to insulin and AMPK-activating stimuli in skeletal muscle.     

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590). RNAi (RNA interference)-mediated silencing of TBC1D1 elevated basal deoxyglucose uptake by approx. 61% in 3T3-L1 mouse embryo adipocytes, while the suppression of TBC1D4 and RapGAP220 under the same conditions had little effect on basal and insulin-stimulated deoxyglucose uptake. Silencing of TBC1D1 strongly increased expression of the GLUT1 glucose transporter but not GLUT4 in cultured adipocytes, whereas the decrease in TBC1D4 had no effect. Remarkably, loss of TBC1D1 in 3T3-L1 adipocytes activated the mTOR (mammalian target of rapamycin)-p70 S6 protein kinase pathway, and the increase in GLUT1 expression in the cells treated with TBC1D1 siRNA (small interfering RNA) was blocked by the mTOR inhibitor rapamycin. Furthermore, overexpression of the mutant TBC1D1-T590A, lacking the putative Akt/PKB phosphorylation site, inhibited insulin stimulation of p70 S6 kinase phosphorylation at Thr(389), a phosphorylation induced by mTOR. Taken together, our data suggest that TBC1D1 may be involved in controlling GLUT1 glucose transporter expression through the mTOR-p70 S6 kinase pathway.     

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.     

Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle

The main purpose of this study was to determine whether the increased glucose transport (GT) found immediately postexercise (IPEX) or 4 h postexercise (4hPEX) is accompanied by increased phosphorylation of Akt substrate of 160 kDa (AS160, a protein regulator of GLUT4 translocation). Paired epitrochlearis muscles were dissected from rats (sedentary or IPEX, 2-h swim) and used to measure protein phosphorylation and insulin-independent GT. IPEX values exceeded sedentary values for GT and phosphorylations of AS160, AMP-activated protein kinase (pAMPK) and acetyl-CoA carboxylase (pACC) but not for AS160 abundance or phosphorylation of Akt serine (pSerAkt), Akt threonine (pThrAkt), or glycogen synthase kinase-3 (pGSK3). AS160 phosphorylation was significantly correlated with GT (R=0.801, P<0.01) and pAMPK (R=0.655, P<0.05). Muscles from other rats were studied 4hPEX along with sedentary controls. One muscle per rat was incubated without insulin, and the contralateral muscle was incubated with insulin. 4hPEX values exceeded sedentary values for insulin-stimulated GT. The elevated pAMPK and pACC found IPEX had reversed by 4hPEX. Insulin caused a significant increase in pSerAkt, pThrAkt, pGSK3, and AS160 phosphorylation with or without exercise. Exercise significantly increased AS160 phosphorylation, regardless of insulin, with unchanged AS160 abundance. Among the signaling proteins studied, insulin-stimulated GT was significantly correlated only with insulin-stimulated pThrAkt (R=0.720, P<0.0005). The results are consistent with a role for increased AS160 phosphorylation in the increased insulin-independent GT IPEX, and the exercise effects on AS160 phosphorylation and/or pThrAkt at 4hPEX are potentially relevant to the increased insulin-stimulated glucose transport at this time.     

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.     

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.     

Rabs 8A and 14 are targets of the insulin-regulated Rab-GAP AS160 regulating GLUT4 traffic in muscle cells

Insulin causes translocation of glucose transporter GLUT4 to the membrane of muscle and fat cells, a process requiring Akt activation. The Rab GTPase-activating protein (Rab-GAP) AS160 is inhibited upon phosphorylation by insulin-activated Akt, thereby allowing GLUT4 translocation. Although several Rab proteins are detected on GLUT4 vesicles, the target Rabs of AS160 involved in the GLUT4 translocation have not been identified. We test whether Rabs 8A, 10, and 14 (in vitro targets of AS160) rescue the inhibition of GLUT4 translocation caused by 'constitutively active' 4P-AS160 in L6 muscle cells. Coexpression of GFP-tagged Rabs 8A or Rab14 with 4P-AS160 prevented the inhibition of GLUT4 translocation imposed by 4P-AS160. GFP-tagged, constitutively active Rab8A also elicited this rescue. In contrast, neither wild-type nor constitutively active GFP-tagged Rab10 restored GLUT4 translocation. These results suggest that Rab8A and possibly Rab14 may be targets of AS160 leading to GLUT4 translocation in L6 muscle cells.     

IGF-1 deficiency resists cardiac hypertrophy and myocardial contractile dysfunction: role of microRNA-1 and microRNA-133a

This study was designed to examine the impact of insulin-like growth factor-1 (IGF-1) deficiency on abdominal aortic constriction (AAC)-induced cardiac geometric and functional changes with a focus on microRNA-1, 133a and 208, which are specially expressed in hearts and govern cardiac hypertrophy and stress-dependent cardiac growth. Liver-specific IGF-1-deficient (LID) and C57/BL6 mice were subject to AAC. Echocardiographic and cardiomyocyte function were assessed 4 wks later. Haematoxylin and eosin staining was used to monitor myocardial morphology. Western blot and real-time PCR were used to detect protein and miR expression, respectively. Neonatal rat cardiomyocytes (NRCMs) were transfected with miRs prior to IGF-1 exposure to initiate cell proliferation. Immunohistochemistry and [(3)H] Leucine incorporation were used to detect cell surface area and protein abundance. C57 mice subject to AAC displayed increased ventricular wall thickness, decreased left ventricular end diastolic and end systolic dimensions and elevated cardiomyocyte shortening capacity, all of which were attenuated in LID mice. In addition, IGF-1 deficiency mitigated AAC-induced increase in atrial natriuretic factor, GATA binding protein 4, glucose transporter 4 (GLUT4) and Akt phosphorylation. In contrast, neither AAC treatment nor IGF-1 deficiency affected glycogen synthase kinase 3b, mammalian target of rapamycin, the Glut-4 translocation mediator Akt substrate of 160 kD (AS160) and protein phosphatase. Levels of miR-1 and -133a (but not miR-208) were significantly attenuated by AAC in C57 but not LID mice. Transfection of miR-1 and -133a obliterated IGF-1-induced hypertrophic responses in NRCMs. Our data suggest that IGF-1 deficiency retards AAC-induced cardiac hypertrophic and contractile changes via alleviating down-regulation of miR-1 and miR-133a in response to left ventricular pressure overload.     

Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.     

Muscle cells engage Rab8A and myosin Vb in insulin-dependent GLUT4 translocation

Insulin causes translocation of glucose transporter 4 (GLUT4) to the membrane of muscle and fat cells, a process requiring Akt activation. Two Rab-GTPase-activating proteins (Rab-GAP), AS160 and TBC1D1, were identified as Akt substrates. AS160 phosphorylation is required for insulin-stimulated GLUT4 translocation, but the participation of TBC1D1 on muscle cell GLUT4 is unknown. Moreover, there is controversy as to the AS160/TBC1D1 target Rabs in fat and muscle cells, and Rab effectors are unknown. Here we examined the effect of knockdown of AS160, TBC1D1, and Rabs 8A, 8B, 10, and 14 (in vitro substrates of AS160 and TBC1D1 Rab-GAP activities) on insulin-induced GLUT4 translocation in L6 muscle cells. Silencing AS160 or TBC1D1 increased surface GLUT4 in unstimulated cells but did not prevent insulin-induced GLUT4 translocation. Knockdown of Rab8A and Rab14, but not of Rab8B or Rab10, inhibited insulin-induced GLUT4 translocation. Furthermore, silencing Rab8A or Rab14 but not Rab8B or Rab10 restored the basal-state intracellular retention of GLUT4 impaired by AS160 or TBC1D1 knockdown. Lastly, overexpression of a fragment of myosin Vb, a recently identified Rab8A-interacting protein, inhibited insulin-induced GLUT4 translocation and altered the subcellular distribution of GTP-loaded Rab8A. These results support a model whereby AS160, Rab8A, and myosin Vb are required for insulin-induced GLUT4 translocation in muscle cells, potentially as part of a linear signaling cascade. glucose transporter 4; insulin signaling; Rab guanosine 5'-triphosphatases; Rab-guanosine 5'-triphosphatase-activating protein; myosin Vb     

Perturbations of the stress-induced GLUT4 localization pathway in slow-twitch muscles of obese Zucker rats

Past studies have suggested that the stress-induced GLUT4 localization pathway is damaged in fast-twitch muscles (white muscles) of obese subjects. In this study, we used obese rodents in an attempt to determine whether the stress-induced GLUT4 localization pathway is abnormal in slow-twitch muscles (red muscles), which are responsible for most daily activities. Protein expression levels of the intracellular stress sensor AMP-activated protein kinase (AMPK), its upstream kinase LKB1, its downstream protein AS160 and the glucose transporter protein 4 (GLUT4) in the red gastrocnemius muscle were measured under either resting or stress conditions (1 h of swimming or 14% hypoxia) in both lean and obese Zucker rats (n = 7 for each group). At rest, obese rats displayed higher fasting plasma insulin levels and increased muscle AMPK and AS160 phosphorylation levels compared with lean controls. No significant difference was found in the protein levels of LKB1, total GLUT4, or membrane GLUT4 between the obese and lean control groups. After one hour of swimming, AMPK and AS160 phosphorylation levels and the amount of GLUT4 translocated to the plasma membrane were significantly elevated in lean rats but remained unchanged in obese rats relative to their resting conditions. One hour 14% hypoxia did not cause significant changes in the LKB1-AMPK-AS160-GLUT4 pathway in either lean or obese rats. This study demonstrated that the AMPK-AS160-GLUT4 pathway was altered at basal levels and after exercise stimulation in the slow-twitch muscle of obese Zucker rats.     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

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.