Contraction-Mediated Glucose Transport in Skeletal Muscle Is Regulated by a Framework of AMPK, TBC1D1/4, and Rac1.

The two closely related RabGTPase-activating proteins (RabGAPs) TBC1D1 and TBC1D4, both substrates for AMPK, play important roles in exercise metabolism and contraction-dependent translocation of GLUT4 in skeletal muscle. However, the specific contribution of each RabGAP in contraction signaling is mostly unknown. In this study, we investigated the cooperative AMPK-RabGAP signaling axis in the metabolic response to exercise/contraction using a novel mouse model deficient in active skeletal muscle AMPK combined with knockout of either Tbc1d1, Tbc1d4, or both RabGAPs. AMPK deficiency in muscle reduced treadmill exercise performance. Additional deletion of Tbc1d1 but not Tbc1d4 resulted in a further decrease in exercise capacity. In oxidative soleus muscle, AMPK deficiency reduced contraction-mediated glucose uptake, and deletion of each or both RabGAPs had no further effect. In contrast, in glycolytic extensor digitorum longus muscle, AMPK deficiency reduced contraction-stimulated glucose uptake, and deletion of Tbc1d1, but not Tbc1d4, led to a further decrease. Importantly, skeletal muscle deficient in AMPK and both RabGAPs still exhibited residual contraction-mediated glucose uptake, which was completely abolished by inhibition of the GTPase Rac1. Our results demonstrate a novel mechanistic link between glucose transport and the GTPase signaling framework in skeletal muscle in response to contraction.

Histone deacetylase 5 regulates interleukin 6 secretion and insulin action in skeletal muscle.

OBJECTIVE: Physical exercise training is associated with increased glucose uptake in skeletal muscle and improved glycemic control. HDAC5, a class IIa histone deacetylase, has been shown to regulate transcription of the insulin-responsive glucose transporter GLUT4 in cultured muscle cells. In this study, we analyzed the contribution of HDAC5 to the transcriptional network in muscle and the beneficial effect of muscle contraction and regular exercise on glucose metabolism. METHODS: HDAC5 knockout mice (KO) and wild-type (WT) littermates were trained for 8 weeks on treadmills, metabolically phenotyped, and compared to sedentary controls. Hdac5-deficient skeletal muscle and cultured Hdac5-knockdown (KD) C2C12 myotubes were utilized for studies of gene expression and glucose metabolism. Chromatin immunoprecipitation (ChIP) studies were conducted to analyze Il6 promoter activity using H3K9ac and HDAC5 antibodies. RESULTS: Global transcriptome analysis of Hdac5 KO gastrocnemius muscle demonstrated activation of the IL-6 signaling pathway. Accordingly, knockdown of Hdac5 in C2C12 myotubes led to higher expression and secretion of IL-6 with enhanced insulin-stimulated activation of AKT that was reversed by Il6 knockdown. Moreover, Hdac5-deficient myotubes exhibited enhanced glucose uptake, glycogen synthesis, and elevated expression levels of the glucose transporter GLUT4. Transcription of Il6 was further enhanced by electrical pulse stimulation in Hdac5-deficient C2C12 myotubes. ChIP identified a ∼1 kb fragment of the Il6 promoter that interacts with HDAC5 and demonstrated increased activation-associated histone marker AcH3K9 in Hdac5-deficient muscle cells. Exercise intervention of HDAC5 KO mice resulted in improved systemic glucose tolerance as compared to WT controls. CONCLUSIONS: We identified HDAC5 as a negative epigenetic regulator of IL-6 synthesis and release in skeletal muscle. HDAC5 may exert beneficial effects through two different mechanisms, transcriptional control of genes required for glucose disposal and utilization, and HDAC5-dependent IL-6 signaling cross-talk to improve glucose uptake in muscle in response to exercise.

TBC1D4 Is Necessary for Enhancing Muscle Insulin Sensitivity in Response to AICAR and Contraction.

Muscle insulin sensitivity for stimulating glucose uptake is enhanced in the period after a single bout of exercise. We recently demonstrated that AMPK is necessary for AICAR, contraction, and exercise to enhance muscle and whole-body insulin sensitivity in mice. Correlative observations from both human and rodent skeletal muscle suggest that regulation of the phosphorylation status of TBC1D4 may relay this insulin sensitization. However, the necessity of TBC1D4 for this phenomenon has not been proven. Thus, the purpose of this study was to determine whether TBC1D4 is necessary for enhancing muscle insulin sensitivity in response to AICAR and contraction. We found that immediately after contraction and AICAR stimulation, phosphorylation of AMPKα-Thr172 and downstream targets were increased similarly in glycolytic skeletal muscle from wild-type and TBC1D4-deficient mice. In contrast, 3 h after contraction or 6 h after AICAR stimulation, enhanced insulin-stimulated glucose uptake was evident in muscle from wild-type mice only. The enhanced insulin sensitivity in muscle from wild-type mice was associated with improved insulin-stimulated phosphorylation of TBC1D4 (Thr649 and Ser711) but not of TBC1D1. These results provide genetic evidence linking signaling through TBC1D4 to enhanced muscle insulin sensitivity after activation of the cellular energy sensor AMPK.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP.

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Deletion of the RabGAP TBC1D1 Leads to Enhanced Insulin Secretion and Fatty Acid Oxidation in Islets From Male Mice.

The Rab guanosine triphosphatase-activating protein (RabGAP) TBC1D1 has been shown to be a key regulator of glucose and lipid metabolism in skeletal muscle. Its function in pancreatic islets, however, is not yet fully understood. Here, we aimed to clarify the specific impact of TBC1D1 on insulin secretion and substrate use in pancreatic islets. We analyzed the dynamics of glucose-stimulated insulin secretion (GSIS) and lipid metabolism in isolated islets from Tbc1d1-deficient (D1KO) mice. To further investigate the underlying cellular mechanisms, we conducted pharmacological studies in these islets. In addition, we determined morphology and number of both pancreatic islets and insulin vesicles in ß-cells using light and transmission electron microscopy. Isolated pancreatic islets from D1KO mice exhibited substantially increased GSIS compared with wild-type (WT) controls. This was attributed to both enhanced first and second phase of insulin secretion, and this enhanced secretion persisted during repetitive glucose stimuli. Studies with sulfonylureas or KCl in isolated islets demonstrated that TBC1D1 exerts its function via a signaling pathway at the level of membrane depolarization. In line, ultrastructural analysis of isolated pancreatic islets revealed both higher insulin-granule density and number of docked granules in ß-cells from D1KO mice compared with WT controls. Like in skeletal muscle, lipid use in isolated islets was enhanced upon D1KO, presumably as a result of a higher mitochondrial fission rate and/or higher mitochondrial activity. Our results clearly demonstrate a dual role of TBC1D1 in controlling substrate metabolism of the pancreatic islet.

"Deletion of both Rab-GTPase­activating proteins TBC1D1 and TBC1D4 in mice eliminates insulin- and AICAR-stimulated glucose transport [corrected].

The Rab-GTPase­activating proteins TBC1D1 and TBC1D4 (AS160) were previously shown to regulate GLUT4 translocation in response to activation of AKT and AMP-dependent kinase [corrected]. However, knockout mice lacking either Tbc1d1 or Tbc1d4 displayed only partially impaired insulin-stimulated glucose uptake in fat and muscle tissue. The aim of this study was to determine the impact of the combined inactivation of Tbc1d1 and Tbc1d4 on glucose metabolism in double-deficient (D1/4KO) mice. D1/4KO mice displayed normal fasting glucose concentrations but had reduced tolerance to intraperitoneally administered glucose, insulin, and AICAR. D1/4KO mice showed reduced respiratory quotient, indicating increased use of lipids as fuel. These mice also consistently showed elevated fatty acid oxidation in isolated skeletal muscle, whereas insulin-stimulated glucose uptake in muscle and adipose cells was almost completely abolished. In skeletal muscle and white adipose tissue, the abundance of GLUT4 protein, but not GLUT4 mRNA, was substantially reduced. Cell surface labeling of GLUTs indicated that RabGAP deficiency impairs retention of GLUT4 in intracellular vesicles in the basal state. Our results show that TBC1D1 and TBC1D4 together play essential roles in insulin-stimulated glucose uptake and substrate preference in skeletal muscle and adipose cells.