Vitamin D Up-regulates Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization Mediated by Cystathionine-γ-lyase (CSE) Activation and H2S Formation in 3T3L1 Adipocytes

A scientific explanation for the beneficial role of vitamin D supplementation in the lowering of glycemia in diabetes remains to be determined. This study examined the biochemical mechanism by which vitamin D supplementation regulates glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mm) in the presence or absence of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) (25, 50 nm), the active form of vitamin D. 1,25(OH)₂D₃ treatment caused significant up-regulation of GLUT4 total protein expression and its translocation to cell surface, and an increase in glucose uptake as well as glucose utilization in HG-treated cells. 1,25(OH)₂D₃ also caused cystathionine-γ-lyase (CSE) activation and H₂S formation in HG-treated adipocytes. The effect of 1,25(OH)₂D₃ on GLUT4 translocation, glucose utilization, and H₂S formation was prevented by propargylglycine, an inhibitor of CSE that catalyzes H₂S formation. Studies using antisense CSE also demonstrated the inhibition of GLUT4 translocation as well as glucose uptake and utilization in 1,25(OH)₂D₃-supplemented CSE-siRNA-transfected adipocytes compared with controls. 1,25(OH)₂D₃ treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared with either insulin or 1,25(OH)₂D₃ alone in HG-treated adipocytes. 1,25(OH)₂D₃ supplementation also inhibited monocyte chemoattractant protein-1 and stimulated adiponectin secretion in HG-treated adipocytes, and this positive effect was prevented in propargylglycine-treated or CSE-knockdown adipocytes. This is the first report to demonstrate that 1,25(OH)₂D₃ up-regulates GLUT4 translocation and glucose utilization and decreases inflammatory markers, which is mediated by CSE activation and H₂S formation in adipocytes. This study provides evidence for a novel molecular mechanism by which 1,25(OH)₂D₃ can up-regulate the GLUT4 translocation essential for maintenance of glucose metabolism.     

Nexilin,a Cardiomyopathy-Associated F-Actin Binding Protein,Binds and Regulates IRS1 Signaling in Skeletal Muscle Cells

Insulin stimulates glucose uptake through a highly organized and complex process that involves movement of the glucose transporter 4 (GLUT4) from intracellular storage sites to the plasma membrane. Previous studies in L6 skeletal muscle cells have shown that insulin-induced activation and assembly of insulin receptor substrate 1 (IRS1) and p85α the regulatory subunit of the Type 1A phosphatidylinositol-3-kinase (PI3K), within remodeled actin-rich membrane structures is critical for downstream signalling mediating the translocation of GLUT4. The mechanism for localization within actin cytoskeletal scaffolds is not known, as direct interaction of IRS1 or p85α with F-actin has not been demonstrated. Here we show that nexilin, a F-actin binding protein implicated in the pathogenesis of familial dilated cardiomyopathies, preferentially binds to IRS1 over IRS2 to influence glucose transport in skeletal muscle cells. Nexilin stably associates with IRS1 under basal conditions in L6 myotubes and this complex is disassembled by insulin. Exposure of L6 myotubes to Latrunculin B disrupts the spatial patterning of nexilin and its transient association with IRS1. Functional silencing of nexilin has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, however it enhances recruitment of p85α to IRS1 resulting in increased PI-3, 4, 5-P₃ formation, coincident with enhanced AKT activation and glucose uptake. By contrast, overexpression of nexilin inhibits transmission of IRS1 signals to AKT. Based on these findings we propose that nexilin may tether IRS1 to actin-rich structures under basal conditions, confining IRS1 signaling to specific subcellular locations in the cell. Insulin-elicited release of this constraint may enhance the efficiency of IRS1/PI3K interaction and PI-3, 4, 5-P₃ production at localized sites. Moreover, the selective binding of nexilin to IRS1 and not IRS2 may contribute to the differential specificity of IRS isoforms in the modulation of GLUT4 trafficking in skeletal muscle cells.     

Effects of 1,25(OH)2D3 and 25(OH)D3 on C2C12 Myoblast Proliferation,Differentiation, and Myotube Hypertrophy

An adequate vitamin D status is essential to optimize muscle strength. However, whether vitamin D directly reduces muscle fiber atrophy or stimulates muscle fiber hypertrophy remains subject of debate. A mechanism that may affect the role of vitamin D in the regulation of muscle fiber size is the local conversion of 25(OH)D to 1,25(OH)₂D by 1α‐hydroxylase. Therefore, we investigated in a murine C2C12 myoblast culture whether both 1,25(OH)₂D₃ and 25(OH)D₃ affect myoblast proliferation, differentiation, and myotube size and whether these cells are able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. We show that myoblasts not only responded to 1,25(OH)₂D₃, but also to the precursor 25(OH)D₃ by increasing their VDR mRNA expression and reducing their proliferation. In differentiating myoblasts and myotubes 1,25(OH)₂D₃ as well as 25(OH)D₃ stimulated VDR mRNA expression and in myotubes 1,25(OH)₂D₃ also stimulated MHC mRNA expression. However, this occurred without notable effects on myotube size. Moreover, no effects on the Akt/mTOR signaling pathway as well as MyoD and myogenin mRNA levels were observed. Interestingly, both myoblasts and myotubes expressed CYP27B1 and CYP24 mRNA which are required for vitamin D₃ metabolism. Although 1α‐hydroxylase activity could not be shown in myotubes, after treatment with 1,25(OH)₂D₃ or 25(OH)D₃ myotubes showed strongly elevated CYP24 mRNA levels compared to untreated cells. Moreover, myotubes were able to convert 25(OH)D₃ to 24R,25(OH)₂D₃ which may play a role in myoblast proliferation and differentiation. These data suggest that skeletal muscle is not only a direct target for vitamin D₃ metabolites, but is also able to metabolize 25(OH)D₃ and 1,25(OH)₂D₃. J. Cell. Physiol. 231: 2517–2528, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.     

Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes

Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-a-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70-75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells.     

Inhibition of mitochondrial respiratory chain in the brain of rats after hepatic failure induced by acetaminophen

To explore the effect of LYRM1 over-expression on basal and insulin-stimulated glucose uptake in rat skeletal muscle cells, and to understand the underlying mechanisms, Rat myoblasts (L6) transfected with either an empty expression vector (pcDNA3.1Myc/His B) or a LYRM1 expression vector were differentiated into myotubes. Glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake into L6 myotubes. Western blotting was performed to assess the translocation of insulin-sensitive glucose transporter 4 (GLUT4). It was also used to measure the phosphorylation and total protein contents of insulin-signaling proteins, such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, phosphatidylinositol-3-kinase (PI3K) p85, Akt, ERK1/2, P38, and JNK. LYRM1 over-expression in L6 myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1, PI3K (p85), and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, P38, and JNK. LYRM1 regulates the function of IRS-1, PI3K, and Akt, and decreases GLUT4 translocation and glucose uptake in response to insulin. These observations highlight the potential role of LYRM1 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity.     

Stimulatory Effect of Balanced Deep-Sea Water Containing Chitosan Oligosaccharides on Glucose Uptake in C<Subscript>2</Subscript>C<Subscript>12</Subscript> Myotubes

Deep-sea water (DSW) and chitosan oligosaccharides (COS) have recently drawn much attention because of their potential medical and pharmaceutical applications. Balanced DSW (BDSW) was prepared by mixing DSW mineral extracts and desalinated water. This study investigated the effects of BDSW, COS, and BDSW containing COS on glucose uptake and their mode of action in mature C₂C₁₂ myotubes. BDSW and COS increased glucose uptake in a dose-dependent manner. BDSW containing COS synergistically increased glucose uptake; this was dependent on the activation of insulin receptor substrate 1 and protein kinase C in insulin-dependent signaling pathways as well as liver kinase B1, AMP-activated protein kinase, and mammalian target of rapamycin in insulin-independent signaling pathways. Quantitative real-time polymerase chain reaction revealed that the expressions of the following genes related to glucose uptake were elevated: glucose transporter 4 (GLUT4), insulin-responsive aminopeptidase, and vesicle-associated membrane protein 2 for abundant proteins of GLUT4 storage vesicles (GSVs); syntaxin 4 and soluble N-ethylmaleimide-sensitive factor attachment protein 23 for trafficking between the plasma membrane and GSVs; and syntaxin 6 and syntaxin 16 for trafficking between GSVs and the trans-Golgi network. Taken together, these results suggest BDSW containing COS has a greater stimulatory effect on glucose uptake than BDSW or COS alone. Moreover, this effect is mediated by the stimulation of diverse signaling pathways via the activation of main signaling molecules related to GSV trafficking.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing GLUT4-myc and AS160_v2

Muscle and fat cells translocate GLUT4 (glucose transporter 4) to the plasma membrane when stimulated by insulin. Usually, this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4 by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (substrate of 160 kDa), namely AS160_v2, and showed that its coexpression with GLUT4 in L6 myoblasts increased the insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which coexpress myc-tagged GLUT4 and AS160_v2, can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC₅₀ values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation, and an image-based assay increases our confidence in the mode of action of the compounds identified.     

Epigallocatechin gallate promotes GLUT4 translocation in skeletal muscle

In this study, we investigated whether epigallocatechin gallate (EGCg) affects glucose uptake activity and the translocation of insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. A single oral administration of EGCg at 75 mg/kg body weight promoted GLUT4 translocation in skeletal muscle of rats. EGCg significantly increased glucose uptake accompanying GLUT4 translocation in L6 myotubes at 1 nM. The translocation of GLUT4 was also observed both in skeletal muscle of mice and rats ex vivo and in insulin-resistant L6 myotubes. Wortmannin, an inhibitor of phosphatidylinositol 3′-kinase, inhibited both EGCg- and insulin-increased glucose uptakes, while genistein, an inhibitor of tyrosine kinase, failed to inhibit the EGCg-increased uptake. Therefore, EGCg may improve hyperglycemia by promoting GLUT4 translocation in skeletal muscle with partially different mechanism from insulin.     

D-Pinitol and myo-Inositol Stimulate Translocation of Glucose Transporter 4 in Skeletal Muscle of C57BL/6 Mice

Diabetes mellitus is a complex disease that is characterized by the defection of insulin sensitivity in such peripheral tissues as skeletal muscle, adipose tissue and liver. We have previously demonstrated that certain inositol derivatives stimulated glucose uptake accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane in L6 myotubes. We investigated in this present study whether an oral intake of D-pinitol (PI) and myo-inositol (MI) would affect GLUT4 translocation in the skeletal muscle of mice. PI or MI at 1 g/kg BW administered orally to mice 30 min before a post-oral injection of glucose at 2 g/kg BW resulted in both PI and MI increasing GLUT4 translocation in the skeletal muscle and lowering the plasma glucose and insulin levels. PI and MI, therefore, have the potential to prevent diabetes mellitus by reducing the postprandial blood glucose level and stimulating GLUT4 translocation in the skeletal muscle.     

Tumor necrosis factor alpha produces insulin resistance in skeletal muscle by activation of inhibitor kappaB kinase in a p38 MAPK-dependent manner

Insulin stimulation produced a reliable 3-fold increase in glucose uptake in primary neonatal rat myotubes, which was accompanied by a similar effect on GLUT4 translocation to plasma membrane. Tumor necrosis factor (TNF)-alpha caused insulin resistance on glucose uptake and GLUT4 translocation by impairing insulin stimulation of insulin receptor (IR) and IR substrate (IRS)-1 and IRS-2 tyrosine phosphorylation, IRS-associated phosphatidylinositol 3-kinase activation, and Akt phosphorylation. Because this cytokine produced sustained activation of stress and proinflammatory kinases, we have explored the hypothesis that insulin resistance by TNF-alpha could be mediated by these pathways. In this study we demonstrate that pretreatment with PD169316 or SB203580, inhibitors of p38 MAPK, restored insulin signaling and normalized insulin-induced glucose uptake in the presence of TNF-alpha. However, in the presence of PD98059 or SP600125, inhibitors of p42/p44 MAPK or JNK, respectively, insulin resistance by TNF-alpha was still produced. Moreover, TNF-alpha produced inhibitor kappaB kinase (IKK)-beta activation and inhibitor kappaB-beta and -alpha degradation in a p38 MAPK-dependent manner, and treatment with salicylate (an inhibitor of IKK) completely restored insulin signaling. Furthermore, TNF-alpha produced serine phosphorylation of IR and IRS-1 (total and on Ser(307) residue), and these effects were completely precluded by pretreatment with either PD169316 or salicylate. Consequently, TNF-alpha, through activation of p38 MAPK and IKK, produces serine phosphorylation of IR and IRS-1, impairing its tyrosine phosphorylation by insulin and the corresponding activation of phosphatidylinositol 3-kinase and Akt, leading to insulin resistance on glucose uptake and GLUT4 translocation.     

Maturation of the regulation of GLUT4 activity by p38 MAPK during L6 cell myogenesis

Insulin stimulates glucose uptake in skeletal muscle cells and fat cells by promoting the rapid translocation of GLUT4 glucose transporters to the plasma membrane. Recent work from our laboratory supports the concept that insulin also stimulates the intrinsic activity of GLUT4 through a signaling pathway that includes p38 MAPK. Here we show that regulation of GLUT4 activity by insulin develops during maturation of skeletal muscle cells into myotubes in concert with the ability of insulin to stimulate p38 MAPK. In L6 myotubes expressing GLUT4 that carries an exofacial myc-epitope (L6-GLUT4myc), insulin-stimulated GLUT4myc translocation equals in magnitude the glucose uptake response. Inhibition of p38 MAPK with SB203580 reduces insulin-stimulated glucose uptake without affecting GLUT4myc translocation. In contrast, in myoblasts, the magnitude of insulin-stimulated glucose uptake is significantly lower than that of GLUT4myc translocation and is insensitive to SB203580. Activation of p38 MAPK by insulin is considerably higher in myotubes than in myoblasts, as is the activation of upstream kinases MKK3/MKK6. In contrast, the activation of all three Akt isoforms and GLUT4 translocation are similar in myoblasts and myotubes. Furthermore, GLUT4myc translocation and phosphorylation of regulatory sites on Akt in L6-GLUT4myc myotubes are equally sensitive to insulin, whereas glucose uptake and phosphorylation of regulatory sites on p38 MAPK show lower sensitivity to the hormone. These observations draw additional parallels between Akt and GLUT4 translocation and between p38 MAPK and GLUT4 activation. Regulation of GLUT4 activity by insulin develops upon muscle cell differentiation and correlates with p38 MAPK activation by insulin.     

Zinc stimulates glucose consumption by modulating the insulin signaling pathway in L6 myotubes: essential roles of Akt–GLUT4, GSK3β and mTOR–S6K1

The present study was performed to evaluate the insulin-like effects of zinc in normal L6 myotubes as well as its ability to alleviate insulin resistance. Glucose consumption was measured in both normal and insulin-resistant L6 myotubes. Western blotting and immunofluorescence revealed that zinc exhibited insulin-like glucose transporting effects by activating key markers that are involved in the insulin signaling cascade (including Akt, GLUT4 and GSK3β), and downregulating members of the insulin signaling feedback cascade such as mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K1). In normal L6 myotubes, zinc enhanced glucose consumption via a mechanism that might involve the activation of Akt phosphorylation, glucose transporter 4 (GLUT4) translocation and GSK3β phosphorylation. In contrast, zinc exerted insulin-mimetic effects in insulin-resistant L6 myotubes by upregulating Akt phosphorylation, GLUT4 translocation and GSK3β phosphorylation, and downregulating the expression of mTOR and S6K1. In conclusion, zinc might enhance glucose consumption by modulating insulin signaling pathways including Akt–GLUT4, GSK3β, mTOR and S6K1.     

Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.     

Dimethyl sulfoxide enhances GLUT4 translocation through a reduction in GLUT4 endocytosis in insulin-stimulated 3T3-L1 adipocytes

Insulin increases muscle and fat cell glucose uptake by inducing the translocation of glucose transporter GLUT4 from intracellular compartments to the plasma membrane. Here, we have demonstrated that in 3T3-L1 adipocytes, DMSO at concentrations higher than 7.5% augmented cell surface GLUT4 levels in the absence and presence of insulin, but that at lower concentrations, DMSO only enhanced GLUT4 levels in insulin-stimulated cells. At a 5% concentration, DMSO also increased cell surface levels of the transferrin receptor and GLUT1. Glucose uptake experiments indicated that while DMSO enhanced cell surface glucose transporter levels, it also inhibited glucose transporter activity. Our studies further demonstrated that DMSO did not sensitize the adipocytes for insulin and that its effect on GLUT4 was readily reversible (t1/2∼12 min) and maintained in insulin-resistant adipocytes. An enhancement of insulin-induced GLUT4 translocation was not observed in 3T3-L1 preadipocytes and L6 myotubes, indicating cell specificity. DMSO did not enhance insulin signaling nor exocytosis of GLUT4 vesicles, but inhibited GLUT4 internalization. While other chemical chaperones (glycerol and 4-phenyl butyric acid) also acutely enhanced insulin-induced GLUT4 translocation, these effects were not mediated via changes in GLUT4 endocytosis. We conclude that DMSO is the first molecule to be described that instantaneously enhances insulin-induced increases in cell surface GLUT4 levels in adipocytes, at least in part through a reduction in GLUT4 endocytosis.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

Characterization of 1,25-dihydroxyvitamin D3-dependent calcium uptake in isolated chick duodenal cells

Summary Thein vivo andin vitro effects of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) on calcium uptake by isolated chick duodenal cells were studied.In vivo, 1,25-(OH)₂D₃ given orally to vitamin D-deficient chicks increased the initial rate of calcium uptake by cells prepared 1 hr after administration of the hormone. The rate was stimulated approximately 100%, 17 to 24 hr after repletion.In vitro, pre-incubation of 1,25-(OH)₂D₃ with cells from D-deficient chicks increased the cellular rate of calcium uptake in a concentration-dependent relationship. Enhancement was found with 10^–15 m, was maximal at 10^–13 m, and was diminished at higher (10^–11 m) concentrations. Stimulation was observed after a pre-incubation period as brief as 1 hr. The potency order for vitamin D₃ analogs was 1,25-(OH)₂D₃=1-(OH)D₃>25-(OH)D₃>1,24,25-(OH)₃D₃>24,25-(OH)₂D₃>D₃. The maximal enhancement in calcium uptake induced by the analogs was the same, only the concentration at which the cell responded was different. The effectiveness of 1,25-(OH)₂D₃ was five orders of magnitude greater than D₃. Kinetically, 1,25-(OH)₂D₃ increased theV _max of calcium uptake; the affinity for calcium (K _m=0.54mm) was unchanged. The enhanced uptake found after the cells were pre-incubated for 2 hr with the hormone was completely blocked by inhibitors of protein synthesis. 1,25-(OH)₂D₃,in vitro, also increased calcium uptake in cells isolated from D-replete chicks. The maximal rates of uptake were the same in cells from D-deficient and D-replete animals. The hormone had no effect of calcium efflux from cells. Calcium uptake in microvillar brush-border membrane vesicles was increased by 1,25-(OH)₂D₃. These findings suggest that thein vitro cell system described in this paper represents an appropriate model to examine the temporal relationships between 1,25-(OH)₂D₃ induction of calcium transport and specific biochemical correlates.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.