Insulin-stimulated GLUT4 Translocation Research Articles

GARNL1, a major RalGAP α subunit in skeletal muscle, regulates insulin-stimulated RalA activation and GLUT4 trafficking via interaction with 14-3-3 proteins

Insulin and muscle contraction each stimulate translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle, an important process regulating whole-body glucose homeostasis. RalA mediates insulin-stimulated GLUT4 translocation; however, it is unclear how this small GTPase is regulated in skeletal muscle in response to insulin. Here, we identified GARNL1/RalGAPα1, a major α subunit of the Ral-GTPase activating protein in skeletal muscle, as a protein whose phosphorylation and binding to the regulatory 14-3-3 proteins is stimulated by insulin and also by muscle contraction. The insulin-stimulated interaction with 14-3-3 involved PKB/Akt-mediated phosphorylation of Thr735 on GARNL1/RalGAPα1. Knockdown of GARNL1/RalGAPα1 increased, while overexpression of GARNL1/RalGAPα1Thr735Ala mutant protein decreased, the RalA activation and the RalA-dependent GLUT4 translocation in response to insulin in muscle cells. These findings show that GARNL1/RalGAPα1 is the missing link that connects the insulin-PKB/Akt signaling pathway with the activation of the RalA small GTPase in muscle cells. GARNL1/RalGAPα1 and its phosphorylation and/or binding to 14-3-3s are critical for GLUT4 trafficking through RalA in muscle cells.

Chromium enhances insulin responsiveness via AMPK

Trivalent chromium (Cr3+) is known to improve glucose homeostasis. Cr3+ has been shown to improve plasma membrane-based aspects of glucose transporter GLUT4 regulation and increase activity of the cellular energy sensor 5’ AMP-activated protein kinase (AMPK). However, the mechanism(s) by which Cr3+ improves insulin responsiveness and whether AMPK mediates this action is not known. In this study we tested if Cr3+ protected against physiological hyperinsulinemia-induced plasma membrane cholesterol accumulation, cortical filamentous actin (F-actin) loss and insulin resistance in L6 skeletal muscle myotubes. In addition, we performed mechanistic studies to test our hypothesis that AMPK mediates the effects of Cr3+ on GLUT4 and glucose transport regulation. Hyperinsulinemia-induced insulin-resistant L6 myotubes displayed excess membrane cholesterol and diminished cortical F-actin essential for effective glucose transport regulation. These membrane and cytoskeletal abnormalities were associated with defects in insulin-stimulated GLUT4 translocation and glucose transport. Supplementing the culture medium with pharmacologically relevant doses of Cr3+ in the picolinate form (CrPic) protected against membrane cholesterol accumulation, F-actin loss, GLUT4 dysregulation and glucose transport dysfunction. Insulin signaling was neither impaired by hyperinsulinemic conditions nor enhanced by CrPic, whereas CrPic increased AMPK signaling. Mechanistically, siRNA-mediated depletion of AMPK abolished the protective effects of CrPic against GLUT4 and glucose transport dysregulation. Together these findings suggest that the micronutrient Cr3+, via increasing AMPK activity, positively impacts skeletal muscle cell insulin sensitivity and glucose transport regulation.

CYP2E1 Impairs GLUT4 Gene Expression and Function: NRF2 as a Possible Mediator

Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

5′-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [(3)H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPKα threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPKα threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes.

Synip phosphorylation is required for insulin-stimulated Glut4 translocation and glucose uptake in podocyte [Rapid Communication

Previously we reported that the phosphorylation of Synip on serine 99 is required for Synip dissociation from Syntaxin4 and insulin-stimulated Glut4 translocation in cultured 3T3-L1 adipocytes. We also reported that the dissociated Synip remains anchored to the plasma membrane by binding to Phosphatidylinositol (3,4,5)-triphosphate. Recently Synip was reported to arrest SNARE-dependent membrane fusion as a selective t-SNARE binding inhibitor. In this study, we have found that Synip is expressed in podocytes although at a somewhat lower level than in adipocytes. To determine whether phosphorylation of Synip on serine 99 is required for insulin-stimulated Glut4 translocation and glucose uptake in podocytes we expressed a phosphorylation deficient Synip mutant (S99A-Synip) that inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake in adipocytes. We conclude that serine 99 phosphorylation of Synip is required for Glut4 translocation and glucose uptake in both adipocytes and podocytes, suggesting that defects in Synip phosphorylation may underlie insulin resistance and associated diabetic nephropathy.

Nucleobindin-2 is a positive regulator for insulin-stimulated glucose transporter 4 translocation in fenofibrate treated E11 podocytes.

The physiology of insulin signaling under normal and disease conditions is well studied in classical insulin target tissues, but not in podocytes. To examine insulin stimulation of podocyte GLUT4 translocation, we established a protocol involving treatment with the PPARα agonist fenofibrate to induce E11 podocyte differentiation within 48 hours rather than 7-10 days, which is required for differentiation under the reported protocol. This allowed us to transiently introduce GLUT4 reporter cDNA and RNAi and thereby to examine the regulatory pathway involved. Here we demonstrate that treatment with 200 μM fenofibrate for 36 hours following transfection had a dramatic effect on podocyte morphology, induced several podocyte specific protein expression markers (G protein-coupled receptor 137B, chloride intracellular channel 5, and nephrin) and resulted in insulin-stimulated GLUT4 translocation. In addition, Nucleobindin-2 was found to constitutively associate with Septin 7 (the repressor of GLUT4 translocation), and knockdown of Nucleobindin-2 was found to completely abrogate insulin-stimulated GLUT4 translocation. Together, these data suggest that Nucleobindin-2 may repress Septin7-induced inhibition of insulin-stimulated GLUT4 translocation in podocytes.

Dipalmitoleoylphosphoethanolamine as a PP2A Enhancer Obstructs Insulin Signaling by Promoting Ser/Thr Dephosphorylation of Akt

Background/Aims: The phospholipid phosphatidylethanolamine is implicated in the regulation of a variety of cellular processes. The present study investigated the effect of phosphatidylethanolamines such as 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) on protein phosphatases, Akt1/2 activity, GLUT4 mobilizations, and glucose uptake into cells. Methods: Activity of protein phosphatase 2A (PP2A) was assayed under the cell-free conditions, and Western blotting, intracellular GLUT4 trafficking, and glucose uptake into cells were monitored using differentiated 3T3-L1-GLUT4myc adipocytes. Results: Of the investigated phosphatidylethanolamines, DLPE and DPPE significantly enhanced PP2A activity. DPPE inhibited insulin-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in differentiated 3T3-L1-GLUT4myc adipocytes. DPPE also inhibited insulin-stimulated GLUT4 translocation to the cell surface and reduced insulin-stimulated glucose uptake into adipocytes. Conclusion: The results of the present study indicate that the PP2A enhancer DPPE obstructs insulin signaling by promoting serine/threonine dephosphorylation of Akt1/2, resulting in the suppression of GLUT4 translocation to the cell surface and glucose uptake into adipocytes.

Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle

Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.

Counteracting neuronal nitric oxide synthase proteasomal degradation improves glucose transport in insulin-resistant skeletal muscle from Zucker fa/fa rats

Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect. Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays. nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes. Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.

Lifelong Physical Activity Prevents Aging-Associated Insulin Resistance in Human Skeletal Muscle Myotubes via Increased Glucose Transporter Expression

Both aging and physical inactivity are associated with increased development of insulin resistance whereas physical activity has been shown to promote increased insulin sensitivity. Here we investigated the effects of physical activity level on aging-associated insulin resistance in myotubes derived from human skeletal muscle satellite cells. Satellite cells were obtained from young (22 yrs) normally active or middle-aged (56.6 yrs) individuals who were either lifelong sedentary or lifelong active. Both middle-aged sedentary and middle-aged active myotubes had increased p21 and myosin heavy chain protein expression. Interestingly MHCIIa was increased only in myotubes from middle-aged active individuals. Middle-aged sedentary cells had intact insulin-stimulated Akt phosphorylation however, the same cell showed ablated insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane. On the other hand, middle-aged active cells retained both insulin-stimulated increases in glucose uptake and GLUT4 translocation to the plasma membrane. Middle-aged active cells also had significantly higher mRNA expression of GLUT1 and GLUT4 compared to middle-aged sedentary cells, and significantly higher GLUT4 protein. It is likely that physical activity induces a number of stable adaptations, including increased GLUT4 expression that are retained in cells ex vivo and protect, or delay the onset of middle-aged-associated insulin resistance. Additionally, a sedentary lifestyle has an impact on the metabolism of human myotubes during aging and may contribute to aging-associated insulin resistance through impaired GLUT4 localization.

Fatty acid transport proteins chronically relocate to the transverse-tubules in muscle from obese Zucker rats but are resistant to further insulin-induced translocation

ObjectivesRecently, we have demonstrated that FA transport proteins are located within the t-tubule fraction of rodent muscle, and that insulin stimulation causes their translocation to this membrane fraction. Chronic relocation of the FA transport protein FAT/CD36 to the sarcolemma is observed in obese rodents and humans, and correlates with intramuscular lipid accumulation and insulin resistance. It is not known whether in an obese, insulin resistant state FA transporters also chronically relocate to the t-tubules. Furthermore, it is not known whether the insulin-stimulated translocation of the various FA transport proteins to the t-tubules is impaired in insulin resistance. MethodsSarcolemmal and t-tubule membrane fractions were isolated via differential centrifugation from muscles of lean and obese female Zucker rats during basal or insulin stimulated conditions. FA transport proteins were measured via western blot on both membrane fractions. ResultsOur results demonstrate that in muscle from insulin resistant Zucker rats, FAT/CD36, FABPpm and FATP1 are all increased on the t-tubules in the basal state (+72%, +120%, and +69%, respectively), potentially contributing to the accumulation of intramuscular lipids. Insulin failed to increase the content of the FA transport proteins on either the t-tubule or sarcolemma above the elevated basal levels, analogous to the well characterized impairment of insulin-stimulated GLUT4 translocation to both membrane domains in obesity. ConclusionFA transport proteins chronically relocate to the t-tubule domain in insulin resistant muscle, potentially contributing to lipid accumulation. Further translocation of the FA transport proteins to this domain during insulin stimulation, however, is impaired.

Rac1 Signaling Is Required for Insulin-Stimulated Glucose Uptake and Is Dysregulated in Insulin-Resistant Murine and Human Skeletal Muscle

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 PAKThr423 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.

Ceramides Contained in LDL Are Elevated in Type 2 Diabetes and Promote Inflammation and Skeletal Muscle Insulin Resistance

Dysregulated lipid metabolism and inflammation are linked to the development of insulin resistance in obesity, and the intracellular accumulation of the sphingolipid ceramide has been implicated in these processes. Here, we explored the role of circulating ceramide on the pathogenesis of insulin resistance. Ceramide transported in LDL is elevated in the plasma of obese patients with type 2 diabetes and correlated with insulin resistance but not with the degree of obesity. Treating cultured myotubes with LDL containing ceramide promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation compared with LDL deficient in ceramide. LDL-ceramide induced a proinflammatory response in cultured macrophages via toll-like receptor–dependent and –independent mechanisms. Finally, infusing LDL-ceramide into lean mice reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle. These newly identified roles of LDL-ceramide suggest that strategies aimed at reducing hepatic ceramide production or reducing ceramide packaging into lipoproteins may improve skeletal muscle insulin action.

PAQR3 Modulates Insulin Signaling by Shunting Phosphoinositide 3-Kinase p110α to the Golgi Apparatus

Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3β, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.

Ahsg-fetuin blocks the metabolic arm of insulin action through its interaction with the 95-kD β-subunit of the insulin receptor

We previously have shown that Ahsg, a liver glycoprotein, inhibits insulin receptor (InsR) tyrosine kinase (TK) activity and the ERK1/2 mitogenic signaling arm of insulin signaling. Here we show that Ahsg blocks insulin-stimulated GLUT4 translocation and Akt activation in intact cells (mouse myoblasts). Furthermore, Ahsg inhibits InsR autophosphorylation of highly-purified insulin holoreceptors in a cell-free, ATP-dependent system, with an IC50 within the range of single-chain Ahsg concentrations in human serum. Binding of 125I-insulin to living cells overexpressing the InsR shows a dissociation constant (KD) of 250pM, unaltered in the presence of 300 nM Ahsg. A mutant InsR cDNA encoding the signal peptide, the β-subunit and the furin processing site, but deleting the α-subunit, was stably expressed in HEK293 cells. Treatment with peroxovanadate, but not insulin, dramatically increased the 95 kD β-subunit tyrosine phosphoryation. The level of tyrosine phosphorylation of the 95-kD β-subunit can be driven down sharply by treatment of living HEK293 transfectant cells with physiological doses of Ahsg. Treatment of myogenic cells with Ahsg blunts insulin-stimulated InsR autophosphorylation and AKT phosphorylation. Taken together, we show that Ahsg antagonizes the metabolic functions initiated by InsR activation without interference in insulin binding. The experiments suggest a direct interaction of Ahsg with the InsR ectodomain β-subunit in a mode that does not significantly alter the high-affinity binding of insulin to the holoreceptor's two complementing α-subunits.

Fucosylated chondroitin sulfate from Acaudina molpadioides improves hyperglycemia via activation of PKB/GLUT4 signaling in skeletal muscle of insulin resistant mice

In this study, we investigated the improvement of fucosylated chondroitin sulfate (CHS) from the cucumber Acaudina molpadioides on hyperglycemia in skeletal muscle of insulin resistant mice. CHS, rosiglitazone (RSG), and their combinations were supplemented to high-fat high-sucrose diet (HFSD)-fed C57BL/6J mice for 19 weeks. The results showed that CHS treatment remarkably decreased blood glucose level and insulin resistance. The glucose metabolism-related genes expressions at the transcriptional level were apparently increased in skeletal muscle. Although the total protein expressions of IR-β, IRS-1, PI3K, PKB and GLUT4 in skeletal muscle were not affected, insulin-stimulated GLUT4 translocation and phosphorylation of Tyr-IR-β, Tyr612-IRS-1, p85-PI3K, Ser473-PKB, and Thr308-PKB were significantly increased by CHS supplement. Additionally, combination of CHS and RSG produced synergistic effects on anti-hyperglycemia. These results indicate that CHS can alleviate hyperglycemia via activation of the PKB/GLUT4 signaling pathway in skeletal muscle of insulin resistant mice.

NAADP Mediates Insulin-Stimulated Glucose Uptake and Insulin Sensitization by PPARγ in Adipocytes

Insulin stimulates glucose uptake through the membrane translocation of GLUT4 and GLUT1. Peroxisome proliferator-activated receptor γ (PPARγ) enhances insulin sensitivity. Here, we demonstrate that insulin stimulates GLUT4 and GLUT1 translocation, and glucose uptake, by activating the signaling pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), a calcium mobilizer, in adipocytes. We also demonstrate that PPARγ mediates insulin sensitization by enhancing NAADP production through upregulation of CD38, the only enzyme identified for NAADP synthesis. Insulin produced NAADP by both CD38-dependent and -independent pathways, whereas PPARγ produced NAADP by CD38-dependent pathway. Blocking the NAADP signaling pathway abrogated both insulin-stimulated and PPARγ-induced GLUT4 and GLUT1 translocation, thereby inhibiting glucose uptake. CD38 knockout partially inhibited insulin-stimulated glucose uptake. However, CD38 knockout completely blocked PPARγ-induced glucose uptake in adipocytes and PPARγ-mediated amelioration of glucose tolerance in diabetic mice. These results demonstrated that the NAADP signaling pathway is a critical molecular target for PPARγ-mediated insulin sensitization.

PS9 - 43. Overexpression of vesicle-associated membrane protein-3 (VAMP3) protects against lipid-induced inhibition of insulin-stimulated GLUT4 translocation in cardiomyocytes

Cardiac glucose utilization is regulated by translocation of the glucose transporter GLUT4 from intracellular stores to the sarcolemma. During lipid-induced insulin resistance, the sarcolemmal presence of the fatty acid transporter CD36 increases, resulting in increased fatty acid uptake and elevation of intracellular lipid metabolites, which interfere with insulin-stimulated GLUT4 translocation, and consequently lead to impaired glucose utilization.

Knockdown of NYGGF4 (PID1) rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes

NYGGF4 is a recently identified gene that is involved in obesity-associated insulin resistance. Previous data from this laboratory have demonstrated that NYGGF4 overexpression might contribute to the development of insulin resistance (IR) and to mitochondrial dysfunction. Additionally, NYGGF4 knockdown enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We designed this study to determine whether silencing of NYGGF4 in 3T3-L1 adipocytes could rescue the effect of insulin sensitivity and mitochondrial function induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to ascertain further the mechanism of NYGGF4 involvement in obesity-associated insulin resistance. We found that 3T3-L1 adipocytes, incubated with 5μM FCCP for 12h, had decreased levels of insulin-stimulated glucose uptake and had impaired insulin-stimulated GLUT4 translocation. Silencing also diminished insulin-stimulated tyrosinephosphorylation of IRS-1 and serine phosphorylation of Akt. This phenomenon contrasts with the effect of NYGGF4 knockdown on insulin sensitivity and describes the regulatory function of NYGGF4 in adipocytes insulin sensitivity. We next analyzed the mitochondrial function in NYGGF4-silenced adipocytes incubated with FCCP. NYGGF4 knockdown partly rescued the dissipation of mitochondrial mass, mitochondrial DNA, intracellular ATP synthesis, and intracellular reactive oxygen species (ROS) production occurred following the addition of FCCP, as well as inhibition of mitochondrial transmembrane potential (ΔΨm) in 3T3-L1 adipocytes incubated with FCCP. Collectively, our results suggested that addition of silencing NYGGF4 partly rescued the effect of insulin resistance and mitochondrial dysfunction in NYGGF4 silenced 3T3-L1 adipocytes incubated with FCCP, which might explain the involvement of NYGGF4-induced IR and the development of NYGGF4 in mitochondrial function.

Overexpression of Vesicle-associated Membrane Protein (VAMP) 3, but Not VAMP2, Protects Glucose Transporter (GLUT) 4 Protein Translocation in an in Vitro Model of Cardiac Insulin Resistance

Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.