ERK1/2 activation by angiotensin II inhibits insulin-induced glucose uptake in vascular smooth muscle cells

Clinical evidence suggests a relationship between hypertension and insulin resistance, and cross-talk between angiotensin II (Ang II) and insulin signaling pathways may take place. We now report the effect of Ang II on insulin-induced glucose uptake and its intracellular mechanisms in vascular smooth muscle cells (VSMC). We examined the translocation of glucose transporter-4 (GLUT-4) and glucose uptake in rat aortic smooth muscle cells (RASMC). Mitogen-activated protein (MAP) kinases and Akt activities, and phosphorylation of insulin receptor substrate-1 (IRS-1) at the serine and tyrosine residues were measured by immunoprecipitation and immunoblotting. As a result, Ang II inhibited insulin-induced GLUT-4 translocation from cytoplasm to the plasma membrane in RASMC. Ang II induced extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) activation and IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹⁶. Ang II-induced Ser³⁰⁷ and Ser⁶¹⁶ phophorylation of IRS-1 was inhibited by a MEK inhibitor, PD98059, and a JNK inhibitor, SP600125. Ang II inhibition of insulin-stimulated IRS-1 tyrosyl phophorylation and Akt activation were reversed by PD98059 but not by SP600125. Ang II inhibited insulin-induced glucose uptake, which was also reversed by PD98059 but not by SP600125. It is shown that Ang II-induced ERK1/2 activation inhibits insulin-dependent glucose uptake through serine phophorylation of IRS-1 in RASMC.     

Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells

The renin-angiotensin system (RAS) and reactive oxygen species (ROS) have been implicated in the development of insulin resistance and its related complications. There is also evidence that angiotensin II (Ang II)-induced generation of ROS contributes to the development of insulin resistance in skeletal muscle, although the precise mechanisms remain unknown. In the present study, we found that Ang II markedly enhanced NADPH oxidase activity and consequent ROS generation in L6 myotubes. These effects were blocked by the angiotensin II type 1 receptor blocker losartan, and by the NADPH oxidase inhibitor apocynin. Ang II also promoted the translocation of NADPH oxidase cytosolic subunits p47phox and p67phox to the plasma membrane within 15 min. Furthermore, Ang II abolished insulin-induced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1), activation of protein kinase B (Akt), and glucose transporter-4 (GLUT4) translocation to the plasma membrane, which was reversed by pretreating myotubes with losartan or apocynin. Finally, small interfering RNA (siRNA)-specific gene silencing targeted specifically against p47phox (p47siRNA), in both L6 and primary myotubes, reduced the cognate protein expression, decreased NADPH oxidase activity, restored Ang II-impaired IRS1 and Akt activation as well as GLUT4 translocation by insulin. These results suggest a pivotal role for NADPH oxidase activation and ROS generation in Ang II-induced inhibition of insulin signaling in skeletal muscle cells.     

Angiotensin II induces tyrosine nitration and activation of ERK1/2 in vascular smooth muscle cells

Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.     

Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells

Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno-fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently.     

Vanillin induces adipocyte differentiation in 3T3-L1 cells by activating extracellular signal regulated kinase 42/44

AimsTo investigate the effect of vanillin, a dietary component, on adipocyte differentiation and the mechanism involved in the process using 3T3-L1 murine preadipocytes.Main methodsThe effect of vanillin on adipocyte differentiation was detected by Oil Red O analysis. The activation of extracellular signal regulated kinase 42/44 (ERK 42/44), Akt, expression of the key regulator of adipocyte differentiation peroxisome proliferators-activated receptor (PPARγ) and its target gene glucose transporter 4 (GLUT4) were detected by western blotting. Glucose uptake assay was used to determine the insulin sensitivity of adipocytes differentiated by vanillin treatment. To confirm the role of ERK 42/44 and Akt, Oil Red O analysis was performed with cells differentiated in the presence or absence of ERK inhibitor U0126 or Akt kinase 1/2 inhibitor.Key findingsVanillin induced adipocyte differentiation in 3T3-L1 cells in a dose dependent manner and also increased the expression levels of PPARγ and its target gene GLUT4. The adipocytes differentiated by vanillin exhibited insulin sensitivity as demonstrated by a significant increase in glucose uptake. Vanillin treatment activated the phosphorylation of ERK 42/44 during the initial phase of adipocyte differentiation but there was no significant change in the Akt phosphorylation status.SignificanceThe data show that vanillin induces adipocyte differentiation in 3T3-L1 cells by activating ERK42/44 and these adipocytes are insulin sensitive in nature.     

Activation of ERK5 in angiotensin II-induced hypertrophy of human aortic smooth muscle cells

Extracellular signal-regulated kinase 5 (ERK5), a recently discovered mitogen-activated protein kinase (MAPK), plays a key role in the development and pathogenesis of cardiovascular disease. In order to clarify the pathophysiological significance of ERK5 in vascular remodeling, we investigated ERK5 phosphorylation in hypertrophy of human aortic smooth muscle cells (HASMCs) induced by angiotensin II (Ang II). The AT1 receptor was involved in Ang II-induced ERK5 activity. Hypertrophy was detected by the measurement of protein synthesis with [³H]-Leu incorporation in cultured HASMCs. Ang II rapidly induced phosphorylation of ERK5 at Thr218/Tyr220 residues in a time- and dose-dependent manner. Activation of myocyte enhancer factor-2C (MEF2C) by ERK5 was inhibited by PD98059. Transfecting HASMCs with small interfering RNA (siRNA) to silence ERK5 inhibited Ang II-induced cell hypertrophy. Thus, ERK5 phosphorylation contributes to MEF2C activation and subsequent HASMC hypertrophy induced by Ang II, for a novel molecular mechanism in cardiovascular diseases induced by Ang II.     

Intracellular signaling of angiotensin II-induced p70 S6 kinase phosphorylation at Ser(411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal-regulated kinase, and Akt

Activation of p70 S6 kinase (p70(S6K)) by growth factors requires multiple signal inputs involving phosphoinositide 3-kinase (PI3K), its effector Akt, and an unidentified kinase that phosphorylates Ser/Thr residues (Ser(411), Ser(418), Ser(424), and Thr(421)) clustered at its autoinhibitory domain. However, the mechanism by which G protein-coupled receptors activate p70(S6K) remains largely uncertain. By using vascular smooth muscle cells in which we have demonstrated Ras/extracellular signal-regulated kinase (ERK) activation through Ca(2+)-dependent, epidermal growth factor (EGF) receptor transactivation by G(q)-coupled angiotensin II (Ang II) receptor, we present a unique cross-talk required for Ser(411) phosphorylation of p70(S6K) by Ang II. Both p70(S6K) Ser(411) and Akt Ser(473) phosphorylation by Ang II appear to involve EGF receptor transactivation and were inhibited by dominant-negative Ras, whereas the phosphorylation of p70(S6K) and ERK but not Akt was sensitive to the MEK inhibitor. By contrast, the phosphorylation of p70(S6K) and Akt but not ERK was sensitive to PI3K inhibitors. Similar inhibitory pattern on these phosphorylation sites by EGF but not insulin was observed. Taken together with the inhibition of Ang II-induced p70(S6K) activation by dominant-negative Ras and the MEK inhibitor, we conclude that Ang II-initiated activation of p70(S6K) requires both ERK cascade and PI3K/Akt cascade that bifurcate at the point of EGF receptor-dependent Ras activation.     

Rapid activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes

The serine/threonine kinase Akt2 has been implicated in insulin-regulated glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. However, it remains unclear whether activation of Akt2 is sufficient since a role for alternate signaling pathways has been proposed. Here we have engineered 3T3-L1 adipocytes to express a rapidly inducible Akt2 system based on drug-inducible heterodimerization. Addition of the dimerizer rapalog resulted in activation of Akt2 within 5 min, concomitant with phosphorylation of the Akt substrates AS160 and GSK3. Comparison with insulin stimulation revealed that the level of Akt2 activity observed with rapalog was within the physiological range, reducing the likelihood of off-target effects. Transient activation of Akt2 also increased glucose transport and GLUT4 translocation to the plasma membrane. These results show that activation of Akt2 is sufficient to stimulate GLUT4 translocation in 3T3-L1 adipocytes to an extent similar to insulin.     

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/RalGAPα1^Thr735Ala 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.     

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.     

A Novel Pleckstrin Homology Domain-containing Protein Enhances Insulin-stimulated Akt Phosphorylation and GLUT4 Translocation in Adipocytes

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P₂, PI(3,5)P₂, and PI(3,4,5)P₃, as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.     

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, 2-Arachidonoylglycerol (2-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. 2-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by 2-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of 2-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither 2-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.     

Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars

Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and production of extracellular matrix, which are very important process in skin wound healing and scarring; however, the signaling pathways involved in this process, especially in humans, are less explored. In the present study, we used skin fibroblasts of human hypertrophic scar, which expressed both AT1 and AT2 receptors, and observed that Ang II increased Akt phosphorylation and phosphoinositide 3 kinase (PI 3-K) activity. In addition, the Ang II-induced Akt phosphorylation was blocked by wortmannin, a PI 3-K inhibitor. This Ang II-activated PI 3-K/Akt cascade was markedly inhibited by valsartan, an AT(1) receptor-specific blocker, whereas it was enhanced by PD123319, an AT(2) receptor antagonist. On the other hand, the Ang II- or EGF-induced activation of PI 3-K/Akt was strongly attenuated by AG1478, an inhibitor of epidermal growth factor (EGF) receptor kinase. Moreover, Ang II stimulated tyrosine phosphorylation of EGF receptor and p85alpha subunit of PI 3-K accompanied by an increase in their association, which was inhibited by valsartan, and enhanced by PD123319. The Ang II-induced transactivation of EGF receptor resulted in activation of extracellular signal-regulated kinase (ERK) that was also inhibited by valsartan, and enhanced by PD123319. Taken together, our results showed that AT(1) receptor-mediated activation of PI 3-K/Akt cascades occurs at least partially via the transactivation of EGF receptor, which is under a negative control by AT(2) receptor in hypertrophic scar fibroblasts. These findings contribute to understanding the molecular mechanism of human hypertrophic scar formation.     

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.     

Hypertrophic response to angiotensin II is mediated by protein kinase D-extracellular signal-regulated kinase 5 pathway in human aortic smooth muscle cells

Angiotensin II plays a critical role in hypertrophy of vascular smooth muscle cells, however, the molecular underpinnings remain unclear. The present study indicated that AT₁/PKC/PKD pathway was able to regulate downstream ERK5, affecting pro-hypertrophic responses to Ang II. Ang II-stimulated phosphorylation of ERK5 in a time- and dose-dependent manner in human aortic smooth muscle cells (HASMCs). The pharmacological inhibitors for AT₁ and PKCs significantly inhibited Ang II-induced ERK5 activation, suggesting the involvement of the AT₁/PKC pathway. In particular, PKD was critical for Ang II-induced ERK5 activation since silencing PKD by siRNA markedly inhibited Ang II-induced ERK5 activation. Consequently, we found that Losartan, Gö 6983 and PKD siRNA significantly attenuated ERK5 activated translocation and hypertrophy of HASMCs by Ang II. Taken together, we demonstrated for the first time that Ang II activates ERK5 via the AT₁/PKC/PKD pathway and revealed a critical role of ERK5 in Ang II-induced HASMCs hypertrophy.     

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.     

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.Insulin stimulates glucose transport into adipose and muscle cells by increasing the amount of the GLUT4 glucose transporter at the cell surface by a process termed GLUT4 translocation (1, 2). Unstimulated adipocytes and myotubes sequester GLUT4 in intracellular compartments. Insulin activates signaling cascades that lead to the trafficking of specialized GLUT4 vesicles to the cell membrane and fusion of the vesicles therewith. A key signaling pathway for GLUT4 translocation proceeds from the insulin receptor through the activation of the protein kinase Akt. One Akt substrate that connects signaling to GLUT4 trafficking is the Rab GTPase-activating protein (GAP)³ known as AS160. There is now considerable evidence for the following scheme (2, 3): under basal conditions, AS160 acts as a brake on GLUT4 translocation by maintaining one or more Rab proteins required for translocation in their inactive GDP state; in response to insulin, Akt phosphorylates AS160 and thereby suppresses its GAP activity; as a consequence, the elevation of the GTP form of the Rab proteins occurs, leading to the increased docking and subsequent fusion of the GLUT4 vesicles at the plasma membrane.More recently, we and others have characterized a paralog of AS160 known as TBC1D1 (4–7). Overall, TBC1D1 is 47% identical to AS160, with the GAP domain being 79% identical (4). Its GAP domain has the same Rab specificity as the GAP domain of AS160 (4). TBC1D1 is predominantly expressed in skeletal muscle; its expression in adipocytes is very low (5, 6). Nevertheless, 3T3-L1 adipocytes are a convenient cell type in which to examine the role of proteins in GLUT4 translocation, because insulin causes an ∼10-fold increase in GLUT4 at the cell surface. Previously, we examined the role of TBC1D1 in GLUT4 translocation by overexpressing it in 3T3-L1 adipocytes. Surprisingly, even though insulin led to phosphorylation of TBC1D1 on Akt site(s), ectopic TBC1D1 potently inhibited GLUT4 translocation (4, 5). By contrast, overexpression of AS160 did not inhibit GLUT4 translocation (8). This difference suggested that the regulation of TBC1D1 might be fundamentally different from that of AS160. In the present study, we show that this is not the case. By reducing the level of ectopic TBC1D1, we have obtained evidence that phosphorylation of TBC1D1 on several likely Akt sites relieves the inhibitory effect on GLUT4 translocation. In addition, we have examined the effect of a variant of TBC1D1 genetically associated with obesity on GLUT4 translocation and determined the relative levels of TBC1D1 in muscle biopsies from healthy and type 2 diabetic individuals.     

Oxidative signaling in renal epithelium: Critical role of cytosolic phospholipase A2 and p38(SAPK)

Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38(SAPK), ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38(SAPK) and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38(SAPK) is upstream of ERK1/2 and Akt1, since a p38(SAPK) inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38(SAPK) isoform alpha inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38(SAPK) provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.     

Enhancement of insulin-induced PI3K/Akt/GSK-3beta and ERK signaling by neuronal nicotinic receptor/PKC-alpha/ERK pathway: up-regulation of IRS-1/-2 mRNA and protein in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells treated with nicotine (10 µm for 24 h), phosphorylation of Akt, glycogen synthase kinase‐3β (GSK‐3β) and extracellular signal‐regulated kinase (ERK)1/2 induced by insulin (100 nm for 10 min) was enhanced by ～ 62%, without altering levels of these protein kinases. Nicotine produced time (> 12 h)‐ and concentration (EC₅₀ 3.6 and 13 µm )‐dependent increases in insulin receptor substrate (IRS)‐1 and IRS‐2 levels by ～ 125 and 105%, without altering cell surface density of insulin receptors. In these cells, insulin‐induced tyrosine phosphorylation of IRS‐1/IRS‐2 and recruitment of phosphoinositide 3‐kinase (PI3K) to IRS‐1/IRS‐2 were augmented by ～ 63%. The increase in IRS‐1/IRS‐2 levels induced by nicotine was prevented by nicotinic acetylcholine receptor (nAChR) antagonists, the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)‐ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis‐acetoxymethyl ester, cycloheximide or actinomycin D. Nicotine increased IRS‐1 and IRS‐2 mRNA levels by ～ 57 and ～ 50%, and this was prevented by conventional protein kinase C (cPKC) inhibitor Gö6976, or ERK kinase inhibitors PD98059 and U0126. Nicotine phosphorylated cPKC‐α, thereby increasing phosphorylation of ERK1/ERK2, as demonstrated by using Gö6976, PD98059 or U0126. Selective activation of cPKC‐α by thymeleatoxin mimicked these effects of nicotine. Thus, stimulation of nAChRs up‐regulated expression of IRS‐1/IRS‐2 via Ca²⁺‐dependent sequential activation of cPKC‐α and ERK, and enhanced insulin‐induced PI3K/Akt/GSK‐3β and ERK signaling pathways.