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Crucial role of the small GTPase Rac1 in insulin-stimulated translocation of glucose transporter 4 to the mouse skeletal muscle sarcolemma.

ABSTRACT: The Rho family GTPase Rac1 has been implicated in the regulation of glucose uptake in myoblast cell lines. However, no evidence for the role of Rac1 has been provided by a mouse model. The purpose of this study is to test the involvement of Rac1 in insulin action in mouse skeletal muscle. Intravenous administration of insulin indeed elicited Rac1 activation in gastrocnemius muscle, suggesting the involvement of Rac1 in this signaling pathway. We then examined whether insulin-stimulated translocation of the facilitative glucose transporter GLUT4 from its storage sites to the skeletal muscle sarcolemma depends on Rac1. We show that ectopic expression of constitutively activated Rac1, as well as intravenous administration of insulin, caused translocation of GLUT4 to the gastrocnemius muscle sarcolemma, as revealed by immunofluorescent staining of a transiently expressed exofacial epitope-tagged GLUT4 reporter. Of particular note, insulin-dependent, but not constitutively activated Rac1-induced, GLUT4 translocation was markedly suppressed in skeletal muscle-specific rac1-knockout mice compared to control mice. Immunogold electron microscopic analysis of endogenous GLUT4 gave similar results. Collectively, we propose a critical role of Rac1 in insulin-dependent GLUT4 translocation to the skeletal muscle sarcolemma, which has heretofore been predicted solely by cell culture studies.

SUBMITTER: Ueda S

PROVIDER: S-EPMC4183928 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Insulin-stimulated translocation of glucose transporter (GLUT) 12 parallels that of GLUT4 in normal muscle.

Project description:ContextGLUT4 is the predominant glucose transporter isoform expressed in fat and muscle. In GLUT4 null mice, insulin-stimulated glucose uptake into muscle was diminished but not eliminated, suggesting that another insulin-sensitive system was present.ObjectiveThis study was intended to determine whether insulin caused GLUT12 translocation in muscle.DesignSix normal volunteers had muscle biopsies before and after euglycemic insulin infusions.SettingInfusions and biopsies were performed in an outpatient clinic.ParticipantsSubjects were nonobese, young adults with no family history of diabetes.Main outcome measuresGLUT12, GLUT4, and GLUT1 proteins were quantified in muscle biopsy fractions. Cultured myoblasts were used to determine whether GLUT12 translocation was phosphatidyl inositol-3 kinase (PI3-K)-dependent.InterventionInsulin was infused at 40 mU/m(2) x min for 3 h.ResultsIn human muscle, insulin caused a shift of a portion of GLUT12 from intracellular low-density microsomes to the plasma membrane (PM) fraction (17% in PM at baseline, 38% in PM after insulin). Insulin increased GLUT4 in PM from 13 to 42%. GLUT1 was predominantly in the PM fractions at baseline and did not change significantly after insulin. L6 myoblasts in culture also expressed and translocated GLUT12 in response to insulin, but inhibiting PI3-K prevented the translocation of GLUT12 and GLUT4.ConclusionsInsulin causes GLUT12 to translocate from an intracellular location to the plasma membrane in normal human skeletal muscle. Translocation of GLUT12 in cultured myoblasts was dependent on activation of PI3-K. GLUT12 may have evolutionarily preceded GLUT4 and now provides redundancy to the dominant GLUT4 system in muscle.

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

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| S-EPMC2781555 | biostudies-literature

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| S-EPMC3528717 | biostudies-literature

TCGAP, a multidomain Rho GTPase-activating protein involved in insulin-stimulated glucose transport.

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| S-EPMC156759 | biostudies-literature

The effects of muscle contraction and insulin on glucose-transporter translocation in rat skeletal muscle.

Project description:The effect of electrically induced muscle contraction, insulin (10 m-units/ml) and electrically-induced muscle contraction in the presence of insulin on insulin-regulatable glucose-transporter (GLUT-4) protein distribution was studied in female Sprague-Dawley rats during hindlimb perfusion. Plasma-membrane cytochalasin B binding increased approximately 2-fold, whereas GLUT-4 protein concentration increased approximately 1.5-fold above control with contractions, insulin, or insulin + contraction. Microsomal-membrane cytochalasin B binding and GLUT-4 protein concentration decreased by approx. 30% with insulin or insulin + contraction, but did not significantly decrease with contraction alone. The rate of muscle glucose uptake was assessed by determining the rate of 2-deoxy[3H]glucose accumulation in the soleus, plantaris, and red and white portions of the gastrocnemius. Both contraction and insulin increased glucose uptake significantly and to the same degree in the muscles examined. Insulin + contraction increased glucose uptake above that of insulin or contraction alone, but this effect was only statistically significant in the soleus, plantaris and white gastrocnemius. The combined effects of insulin + contraction of glucose uptake were not fully additive in any of the muscles investigated. These results suggest that (1) insulin and muscle contraction are mobilizing two separate pools of GLUT-4 protein, and (2) the increase in skeletal-muscle glucose uptake due to insulin + contraction is not due to an increase in plasma-membrane GLUT-4 protein concentration above that observed for insulin or contraction alone.

| S-EPMC1137867 | biostudies-other

GLUT8 is a glucose transporter responsible for insulin-stimulated glucose uptake in the blastocyst.

Project description:Mammalian preimplantation blastocysts exhibit insulin-stimulated glucose uptake despite the absence of the only known insulin-regulated transporter, GLUT4. We describe a previously unidentified member of the mammalian facilitative GLUT superfamily that exhibits approximately 20-25% identity with other murine facilitative GLUTs. Insulin induces a change in the intracellular localization of this protein, which translates into increased glucose uptake into the blastocyst, a process that is inhibited by antisense oligoprobes. Presence of this transporter may be necessary for successful blastocyst development, fuel metabolism, and subsequent implantation. Moreover, the existence of an alternative transporter may explain examples in other tissues of insulin-regulated glucose transport in the absence of GLUT4.

| S-EPMC16542 | biostudies-literature

Rac1 signaling is required for insulin-stimulated glucose uptake and is dysregulated in insulin-resistant murine and human skeletal muscle.

Project description:The actin cytoskeleton-regulating GTPase Rac1 is required for insulin-stimulated GLUT4 translocation in cultured muscle cells. However, involvement of Rac1 and its downstream signaling in glucose transport in insulin-sensitive and insulin-resistant mature skeletal muscle has not previously been investigated. We hypothesized that Rac1 and its downstream target, p21-activated kinase (PAK), are regulators of insulin-stimulated glucose uptake in mouse and human skeletal muscle and are dysregulated in insulin-resistant states. Muscle-specific inducible Rac1 knockout (KO) mice and pharmacological inhibition of Rac1 were used to determine whether Rac1 regulates insulin-stimulated glucose transport in mature skeletal muscle. Furthermore, Rac1 and PAK1 expression and signaling were investigated in muscle of insulin-resistant mice and humans. Inhibition and KO of Rac1 decreased insulin-stimulated glucose transport in mouse soleus and extensor digitorum longus muscles ex vivo. Rac1 KO mice showed decreased insulin and glucose tolerance and trended toward higher plasma insulin concentrations after intraperitoneal glucose injection. Rac1 protein expression and insulin-stimulated PAK(Thr423) phosphorylation were decreased in muscles of high fat-fed mice. In humans, insulin-stimulated PAK activation was decreased in both acute insulin-resistant (intralipid infusion) and chronic insulin-resistant states (obesity and diabetes). These findings show that Rac1 is a regulator of insulin-stimulated glucose uptake and a novel candidate involved in skeletal muscle insulin resistance.

| S-EPMC3661612 | biostudies-other

The exocyst complex regulates insulin-stimulated glucose uptake of skeletal muscle cells.

Project description:Skeletal muscle handles ~80-90% of the insulin-induced glucose uptake. In skeletal muscle, insulin binding to its cell surface receptor triggers redistribution of intracellular glucose transporter GLUT4 protein to the cell surface, enabling facilitated glucose uptake. In adipocytes, the eight-protein exocyst complex is an indispensable constituent in insulin-induced glucose uptake, as it is responsible for the targeted trafficking and plasma membrane-delivery of GLUT4. However, the role of the exocyst in skeletal muscle glucose uptake has never been investigated. Here we demonstrate that the exocyst is a necessary factor in insulin-induced glucose uptake in skeletal muscle cells as well. The exocyst complex colocalizes with GLUT4 storage vesicles in L6-GLUT4myc myoblasts at a basal state and associates with these vesicles during their translocation to the plasma membrane after insulin signaling. Moreover, we show that the exocyst inhibitor endosidin-2 and a heterozygous knockout of Exoc5 in skeletal myoblast cells both lead to impaired GLUT4 trafficking to the plasma membrane and hinder glucose uptake in response to an insulin stimulus. Our research is the first to establish that the exocyst complex regulates insulin-induced GLUT4 exocytosis and glucose metabolism in muscle cells. A deeper knowledge of the role of the exocyst complex in skeletal muscle tissue may help our understanding of insulin resistance in type 2 diabetes.

| S-EPMC6962504 | biostudies-literature

Cassia Abbreviata Enhances Glucose Uptake and Glucose Transporter 4 Translocation in C2C12 Mouse Skeletal Muscle Cells.

Project description:BackgroundThis study aim at assessing C. abbreviata aqueous extracts for its potential to exhibit anti-diabetic activity in skeletal muscle cells. In addition to the toxicological and glucose absorption studies, the action of C. abbreviata extracts on some major genes involved in the insulin signaling pathway was established.MethodsThe in vitro cytotoxic effects C. abbreviata was evaluated on muscle cells using the MTT assay and the in vitro glucose uptake assay conducted using a modified glucose oxidase method described by Van de Venter et al. (2008). The amount of GLUT-4 on cell surfaces was estimated quantitatively using the flow cytometry technique. Real time quantitative PCR (RT-qPCR) was used to determine the expression of GLUT-4, IRS-1, PI3 K, Akt1, Akt2, PPAR-γ.ResultsCytotoxicity tests revealed that all extracts tested at various concentrations were non-toxic (LC50 > 5000). Aqueous extracts of leaves, bark and seeds resulted in a dose-dependent increase in glucose absorption by cells, after 1 h, 3 h and 6 h incubation period. Extracts of all three plant parts had the best effect after 3 h incubation, with the leaf extract showing the best activity across time (Glucose uptake of 29%, 56% and 42% higher than untreated control cells after treatment with 1 mg/ml extract at 1 h, 3 h and 6 h, respectively). All extracts, with the exception 500 µg/ml seed extract, induced a two-fold increase in GLUT-4 translocation while marginally inducing GLUT-10 translocation in the muscle cells. The indirect immunofluorescence confirmed that GLUT-4 translocation indeed occurred. There was an increased expression of GLUT-4, IRS1 and PI3 K in cells treated with insulin and bark extract as determined by the RT-qPCR.ConclusionThe study reveals that glucose uptake involves GLUT-4 translocation through a mechanism that is likely to involve the upstream effectors of the PI3-K/Akt pathway.

| S-EPMC8020231 | biostudies-literature

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