A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b.

Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation.

Rab10, a target of the AS160 Rab GAP, is required for insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane.

GLUT4 trafficking to the plasma membrane of muscle and fat cells is regulated by insulin. An important component of insulin-regulated GLUT4 distribution is the Akt substrate AS160 rab GTPase-activating protein. Here we show that Rab10 functions as a downstream target of AS160 in the insulin-signaling pathway that regulates GLUT4 translocation in adipocytes. Overexpression of a mutant of Rab10 defective for GTP hydrolysis increased GLUT4 on the surface of basal adipocytes. Rab10 knockdown resulted in an attenuation of insulin-induced GLUT4 redistribution to the plasma membrane and a concomitant 2-fold decrease in GLUT4 exocytosis rate. Re-expression of a wild-type Rab10 restored normal GLUT4 translocation. The basal increase in plasma-membrane GLUT4 due to AS160 knockdown was partially blocked by knocking down Rab10 in the same cells, further indicating that Rab10 is a target of AS160 and a positive regulator of GLUT4 trafficking to the cell surface upon insulin stimulation.

Insulin-stimulated GLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotide exchange factor Dennd4C.

Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.

A truncation mutation in TBC1D4 in a family with acanthosis nigricans and postprandial hyperinsulinemia.

Tre-2, BUB2, CDC16, 1 domain family member 4 (TBC1D4) (AS160) is a Rab-GTPase activating protein implicated in insulin-stimulated glucose transporter 4 (GLUT4) translocation in adipocytes and myotubes. To determine whether loss-of-function mutations in TBC1D4 might impair GLUT4 translocation and cause insulin resistance in humans, we screened the coding regions of this gene in 156 severely insulin-resistant patients. A female presenting at age 11 years with acanthosis nigricans and extreme postprandial hyperinsulinemia was heterozygous for a premature stop mutation (R363X) in TBC1D4. After demonstrating reduced expression of wild-type TBC1D4 protein and expression of the truncated protein in lymphocytes from the proband, we further characterized the biological effects of the truncated protein in 3T3L1 adipocytes. Prematurely truncated TBC1D4 protein tended to increase basal cell membrane GLUT4 levels (P = 0.053) and significantly reduced insulin-stimulated GLUT4 cell membrane translocation (P < 0.05). When coexpressed with wild-type TBC1D4, the truncated protein dimerized with full-length TBC1D4, suggesting that the heterozygous truncated variant might interfere with its wild-type counterpart in a dominant negative fashion. Two overweight family members with the mutation also manifested normal fasting glucose and insulin levels but disproportionately elevated insulin levels following an oral glucose challenge. This family provides unique genetic evidence of TBC1D4 involvement in human insulin action.

Full intracellular retention of GLUT4 requires AS160 Rab GTPase activating protein.

Insulin controls glucose flux into muscle and fat by regulating the trafficking of GLUT4 between the interior and surface of cells. Here, we show that the AS160 Rab GTPase activating protein (GAP) is a negative regulator of basal GLUT4 exocytosis. AS160 knockdown resulted in a partial redistribution of GLUT4 from intracellular compartments to the plasma membrane, a concomitant increase in basal glucose uptake, and a 3-fold increase in basal GLUT4 exocytosis. Reexpression of wild-type AS160 restored normal GLUT4 behavior to the knockdown adipocytes, whereas reexpression of a GAP domain mutant did not revert the phenotype, providing the first direct evidence that AS160 GAP activity is required for basal GLUT4 retention. AS160 is the first protein identified that is specially required for basal GLUT4 retention. Our findings that AS160 knockdown only partially releases basal GLUT4 retention provides evidence that insulin signals to GLUT4 exocytosis by both AS160-dependent and -independent mechanisms.

Insulin-stimulated phosphorylation of the Rab GTPase-activating protein TBC1D1 regulates GLUT4 translocation.

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.

Rab10 in insulin-stimulated GLUT4 translocation.

In fat and muscle cells, insulin stimulates the movement to and fusion of intracellular vesicles containing GLUT4 with the plasma membrane, a process referred to as GLUT4 translocation. Previous studies have indicated that Akt [also known as PKB (protein kinase B)] phosphorylation of AS160, a GAP (GTPase-activating protein) for Rabs, is required for GLUT4 translocation. The results suggest that this phosphorylation suppresses the GAP activity and leads to the elevation of the GTP form of one or more Rabs required for GLUT4 translocation. Based on their presence in GLUT4 vesicles and activity as AS160 GAP substrates, Rabs 8A, 8B, 10 and 14 are candidate Rabs. Here, we provide further evidence that Rab10 participates in GLUT4 translocation in 3T3-L1 adipocytes. Among Rabs 8A, 8B, 10 and 14, only the knockdown of Rab10 inhibited GLUT4 translocation. In addition, we describe the subcellular distribution of Rab10 and estimate the fraction of Rab10 in the active GTP form in vivo. Approx. 5% of the total Rab10 was present in GLUT4 vesicles isolated from the low-density microsomes. In both the basal and the insulin state, 90% of the total Rab10 was in the inactive GDP state. Thus, if insulin increases the GTP form of Rab10, the increase is limited to a small portion of the total Rab10. Finally, we report that the Rab10 mutant considered to be constitutively active (Rab10 Q68L) is a substrate for the AS160 GAP domain and, hence, cannot be used to deduce rigorously the function of Rab10 in its GTP form.

Substrate specificity and effect on GLUT4 translocation of the Rab GTPase-activating protein Tbc1d1.

Insulin stimulation of the trafficking of the glucose transporter GLUT4 to the plasma membrane is controlled in part by the phosphorylation of the Rab GAP (GTPase-activating protein) AS160 (also known as Tbc1d4). Considerable evidence indicates that the phosphorylation of this protein by Akt (protein kinase B) leads to suppression of its GAP activity and results in the elevation of the GTP form of a critical Rab. The present study examines a similar Rab GAP, Tbc1d1, about which very little is known. We found that the Rab specificity of the Tbc1d1 GAP domain is identical with that of AS160. Ectopic expression of Tbc1d1 in 3T3-L1 adipocytes blocked insulin-stimulated GLUT4 translocation to the plasma membrane, whereas a point mutant with an inactive GAP domain had no effect. Insulin treatment led to the phosphorylation of Tbc1d1 on an Akt site that is conserved between Tbc1d1 and AS160. These results show that Tbc1d1 regulates GLUT4 translocation through its GAP activity, and is a likely Akt substrate. An allele of Tbc1d1 in which Arg(125) is replaced by tryptophan has very recently been implicated in susceptibility to obesity by genetic analysis. We found that this form of Tbc1d1 also inhibited GLUT4 translocation and that this effect also required a functional GAP domain.

Temporal dynamics of tyrosine phosphorylation in insulin signaling.

The insulin-signaling network regulates blood glucose levels, controls metabolism, and when dysregulated, may lead to the development of type 2 diabetes. Although the role of tyrosine phosphorylation in this network is clear, only a limited number of insulin-induced tyrosine phosphorylation sites have been identified. To address this issue and establish temporal response, we have, for the first time, carried out an extensive, quantitative, mass spectrometry-based analysis of tyrosine phosphorylation in response to insulin. The study was performed with 3T3-L1 adipocytes stimulated with insulin for 0, 5, 15, and 45 min. It has resulted in the identification and relative temporal quantification of 122 tyrosine phosphorylation sites on 89 proteins. Insulin treatment caused a change of at least 1.3-fold in tyrosine phosphorylation on 89 of these sites. Among the responsive sites, 20 were previously known to be tyrosine phosphorylated with insulin treatment, including sites on the insulin receptor and insulin receptor substrate-1. The remaining 69 responsive sites have not previously been shown to be altered by insulin treatment. They were on proteins with a wide variety of functions, including components of the trafficking machinery for the insulin-responsive glucose transporter GLUT4. These results show that insulin-elicited tyrosine phosphorylation is extensive and implicate a number of hitherto unrecognized proteins in insulin action.

Adipocytes contain a novel complex similar to the tuberous sclerosis complex.

Recently we identified a novel 250 kDa protein in adipocytes that is a substrate for the insulin-activated protein kinase Akt. We refer to this protein as AS250 for Akt substrate of 250 kDa. AS250 has a predicted GTPase activating protein (GAP) domain at its carboxy terminus. This domain shows some homology to the GAP domains for Rheb at the carboxy terminus of the protein tuberin and for Rap1 in the protein Rap1 GAP. The present study further characterizes AS250. The cDNA sequence for human AS250 is reported, and the sites that undergo phosphorylation upon insulin treatment of adipocytes have been identified by tandem mass spectrometry. We have found that in adipocytes AS250 exists as a complex with a novel protein of 1484 amino acids known as KIAA1219. The complex of AS250 with KIAA1219 is notably similar to the important regulatory complex of the protein tuberin with hamartin (the tuberous sclerosis complex), in the size of its subunits, the location of the GAP domain, and its phosphorylation by Akt. In an effort to detect the cellular role of the AS250/KIAA1219 complex, we generated 3T3-L1 adipocytes that largely lack AS250 by shRNA knockdown and examined several insulin-dependent effects. The knockdown of AS250 had no effect on insulin activation of the kinases, Akt, 70 kDa S6 kinase, or ERK1/2, or on insulin-stimulated actin bundling, and it had only a slight effect on insulin-stimulated GLUT4 translocation.

Increased phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle in response to insulin or contractile activity.

In 3T3-L1 adipocytes, insulin-stimulated GLUT4 translocation requires phosphorylation of the protein designated Akt substrate of 160 kDa (AS160). Both insulin and contractions activate Akt in skeletal muscle. Therefore, we assessed the effects in skeletal muscle of each stimulus on phosphorylation of proteins, including AS160, on the Akt phosphomotif. Isolated rat epitrochlearis muscles were incubated with insulin (for time course and dose response), stimulated to contract, or incubated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) and used to assess the following: serine-phosphorylation of Akt (P-Akt), immunoreactivity with an antibody recognizing the Akt phosphomotif (alpha-phospho-[Ser/Thr] Akt substrate [PAS]), and PAS immunoreactivity of samples immunoprecipitated with anti-AS160. P-Akt peaked at 5 min of insulin, and PAS immunoreactivity subsequently peaked for proteins of 250 kDa (10 min) and 160 kDa (15 min). P-Akt, PAS-160, and PAS-250 increased significantly with 0.6 nmol/l insulin. Contractile activity led to increased P-Akt and PAS immunoreactivity of proteins of 160 and 250 kDa. The 160-kDa protein was confirmed to be AS160 based on elevated PAS immunoreactivity in AS160 immunoprecipitates. Wortmannin inhibited insulin (120 nmol/l) and contraction effects on AS160 phosphorylation. Incubation with AICAR caused increased phosphorylation of AMP-activated protein kinase and AS160 but not Akt. Our working hypothesis is that phosphorylation of these putative Akt substrates is important for some of the insulin and contraction bioeffects.

Insulin-stimulated phosphorylation of the Akt substrate AS160 is impaired in skeletal muscle of type 2 diabetic subjects.

AS160 is a newly described substrate for the protein kinase Akt that links insulin signaling and GLUT4 trafficking. In this study, we determined the expression of and in vivo insulin action on AS160 in human skeletal muscle. In addition, we compared the effect of physiological hyperinsulinemia on AS160 phosphorylation in 10 lean-to-moderately obese type 2 diabetic and 9 healthy subjects. Insulin infusion increased the phosphorylation of several proteins reacting with a phospho-Akt substrate antibody. We focused on AS160, as this Akt substrate has been linked to glucose transport. A 160-kDa phosphorylated protein was identified as AS160 by immunoblot analysis with an AS160-specific antibody. Physiological hyperinsulinemia increased AS160 phosphorylation 2.9-fold in skeletal muscle of control subjects (P < 0.001). Insulin-stimulated AS160 phosphorylation was reduced 39% (P < 0.05) in type 2 diabetic patients. AS160 protein expression was similar in type 2 diabetic and control subjects. Impaired AS160 phosphorylation was related to aberrant Akt signaling; insulin action on Akt Ser(473) phosphorylation was not significantly reduced in type 2 diabetic compared with control subjects, whereas Thr(308) phosphorylation was impaired 51% (P < 0.05). In conclusion, physiological hyperinsulinemia increases AS160 phosphorylation in human skeletal muscle. Moreover, defects in insulin action on AS160 may impair GLUT4 trafficking in type 2 diabetes.

Novel insulin-elicited phosphoproteins in adipocytes.

Akt is a key insulin-activated protein kinase. We searched for Akt substrates in 3T3-L1 adipocytes by means of immunoprecipitation with an Akt phosphomotif-specific antibody (PAS antibody). Four insulin-elicited phosphoproteins were isolated and identified by mass spectrometry. The identity of each protein was established by isolating the protein from lysates of untreated and insulin-treated adipocytes with an antibody specific for the protein and showing that the PAS antibody reacted only with the protein in the immunoprecipitate from insulin-treated cells. These proteins have sizes of 47, 75, 105, and 250 kDa on SDS PAGE, and have been designated pp47, 75, 105, and 250. The effect of inhibitors on the phosphorylation of the proteins, the identified sites of phosphorylation, and in vitro phosphorylation by recombinant Akt further indicated that pp47, 105, and 250 are likely to be Akt substrates, whereas pp75 may not be. pp47 and 105 are novel proteins with no known or predicted function. pp75 was previously found as a protein that associated with the colony-stimulating factor receptor, designated as Fms-interacting protein. pp250 is a novel protein with a predicted GTPase activating protein (GAP) domain for Rheb and/or Rap at its carboxy terminus. The subcellular and tissue distributions of the four proteins were determined.

AS160, the Akt substrate regulating GLUT4 translocation, has a functional Rab GTPase-activating protein domain.

Recently, we described a 160 kDa protein (designated AS160, for Akt substrate of 160 kDa) with a predicted Rab GAP (GTPase-activating protein) domain that is phosphorylated on multiple sites by the protein kinase Akt. Phosphorylation of AS160 in adipocytes is required for insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The aim of the present study was to determine whether AS160 is in fact a GAP for Rabs, and, if so, what its specificity is. We first identified a group of 16 Rabs in a preparation of intracellular vesicles containing GLUT4 by MS. We then prepared the recombinant GAP domain of AS160 and examined its activity against many of these Rabs, as well as several others. The GAP domain was active against Rabs 2A, 8A, 10 and 14. There was no significant activity against 14 other Rabs. GAP activity was further validated by the finding that the recombinant GAP domain with the predicted catalytic arginine residue replaced by lysine was inactive. Finally, it was found by immunoblotting that Rabs 2A, 8A and 14 are present in GLUT4 vesicles. These results indicate that AS160 is a Rab GAP, and suggest novel Rabs that may participate in GLUT4 translocation.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. BIOLOGICAL SIGNIFICANCE: Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results.

κB-Ras proteins regulate both NF-κB-dependent inflammation and Ral-dependent proliferation.

The transformation of cells generally involves multiple genetic lesions that undermine control of both cell death and proliferation. We now report that κB-Ras proteins act as regulators of NF-κB and Ral pathways, which control inflammation/cell death and proliferation, respectively. Cells lacking κB-Ras therefore not only show increased NF-κB activity, which results in increased expression of inflammatory mediators, but also exhibit elevated Ral activity, which leads to enhanced anchorage-independent proliferation (AIP). κB-Ras deficiency consequently leads to significantly increased tumor growth that can be dampened by inhibiting either Ral or NF-κB pathways, revealing the unique tumor-suppressive potential of κB-Ras proteins. Remarkably, numerous human tumors show reduced levels of κB-Ras, and increasing the level of κB-Ras in these tumor cells impairs their ability to undergo AIP, thereby implicating κB-Ras proteins in human disease.

Insulin stimulates the phosphorylation of the exocyst protein Sec8 in adipocytes.

The signal transduction pathway leading from the insulin receptor to stimulate the fusion of vesicles containing the glucose transporter GLUT4 with the plasma membrane in adipocytes and muscle cells is not completely understood. Current evidence suggests that in addition to the Rab GTPase-activating protein AS160, at least one other substrate of Akt (also called protein kinase B), which is as yet unidentified, is required. Sec8 is a component of the exocyst complex that has been previously implicated in GLUT4 trafficking. In the present study, we report that insulin stimulates the phosphorylation of Sec8 on Ser-32 in 3T3-L1 adipocytes. On the basis of the sequence around Ser-32 and the finding that phosphorylation is inhibited by the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, it is likely that Akt is the kinase for Ser-32. We examined the possible role of Ser-32 phosphorylation in the insulin-stimulated trafficking of GLUT4, as well as the TfR (transferrin receptor), to the plasma membrane by determining the effects of overexpression of the non-phosphorylatable S32A mutant of Sec8 and the phosphomimetic S32E mutant of Sec8. Substantial overexpression of both mutants had no effect on the amount of GLUT4 or TfR at the cell surface in either the untreated or insulin-treated states. These results indicate that insulin-stimulated phosphorylation of Sec8 is not part of the mechanism by which insulin enhances the fusion of vesicles with the plasma membrane.

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.