Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

Changes in dietary sodium consumption modulate GLUT4 gene expression and early steps of insulin signaling

Previous studies have shown that chronic salt overload increases insulin sensitivity, while chronic salt restriction decreases it. In the present study we investigated the influence of dietary sodium on 1) GLUT4 gene expression, by No the n and Western blotting analysis; 2) in vivo GLUT4 protein translocation, by measuring the GLUT4 protein in plasma membrane and microsome, before and after insulin injection; and 3) insulin signaling, by analyzing basal and insulin-stimulated tyrosine phosphorylation of insulin receptor (IR)-beta, insulin receptor substrate (IRS)-1, and IRS-2. Wistar rats we e fed no mal-sodium (NS-0.5%), low-sodium (LS-0.06%), o high-sodium diets (HS-3.12%) fo 9 wk and were killed under pentobarbital anesthesia. Compared with NS ats, HS ats inc eased (P < 0.05) the GLUT4 protein in adipose tissue and skeletal muscle, whereas GLUT4 mRNA was increased only in adipose tissue. GLUT4 expression was unchanged in LS ats compared with NS ats. The GLUT4 translocation in HS ats was higher (P < 0.05) both in basal and insulin-stimulated conditions. On the other hand, LS ats did not increase the GLUT4 translocation after insulin stimulus. Compared with NS ats, LS ats showed reduced (P < 0.01) basal and insulin-stimulated tyrosine phosphorylation of IRS-1 in skeletal muscle and IRS-2 in live, whereas HS ats showed enhanced basal tyrosine phosphorylation of IRS-1 in skeletal muscle (P < 0.05) and of IRS-2 in live. In summary, increased insulin sensitivity in HS ats is elated to increased GLUT4 gene expression, enhanced insulin signaling, and GLUT4 translocation, whereas decreased insulin sensitivity of LS ats does not involve changes in GLUT4 gene expression but is elated to impaired insulin signaling.     

Maternal protein restriction during early lactation induces GLUT4 translocation and mTOR/Akt activation in adipocytes of adult rats

Epidemiological and experimental studies have demonstrated that early postnatal nutrition has been associated with long-term effects on glucose homeostasis in adulthood. Recently, our group demonstrated that undernutrition during early lactation affects the expression and activation of key proteins of the insulin signaling cascade in rat skeletal muscle during postnatal development. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity in peripheral tissues, we investigated the insulin signaling in adipose tissue. Adipocytes were isolated from epididymal fat pads of adult male rats that were the offspring of dams fed either a normal or a protein-free diet during the first 10 days of lactation. The cells were incubated with 100 nM insulin before the assays for immunoblotting analysis, 2-deoxyglucose uptake, immunocytochemistry for GLUT4, and/or actin filaments. Following insulin stimulation, adipocytes isolated from undernourished rats presented reduced tyrosine phosphorylation of IR and IRS-1 and increased basal phosphorylation of IRS-2, Akt, and mTOR compared with controls. Basal glucose uptake was increased in adipocytes from the undernourished group, and the treatment with LY294002 induced only a partial inhibition both in basal and in insulin-stimulated glucose uptake, suggesting an involvement of phosphoinositide 3-kinase activity. These alterations were accompanied by higher GLUT4 content in the plasma membrane and alterations in the actin cytoskeleton dynamics. These data suggest that early postnatal undernutrition impairs insulin sensitivity in adulthood by promoting changes in critical steps of insulin signaling in adipose tissue, which may contribute to permanent changes in glucose homeostasis.     

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.     

Palmitate stimulates glucose transport in rat adipocytes by a mechanism involving translocation of the insulin sensitive glucose transporter (GLUT4).

In rat adipocytes, palmitate: a) increases basal 2-deoxyglucose transport 129 +/- 27% (p less than 0.02), b) decreases the insulin sensitive glucose transporter (GLUT4) in low density microsomes and increases GLUT4 in plasma membranes and c) increases the activity of the insulin receptor tyrosine kinase. Palmitate-stimulated glucose transport is not additive with the effect of insulin and is not inhibited by the protein kinase C inhibitors staurosporine and sphingosine. In rat muscle, palmitate: a) does not affect basal glucose transport in either the soleus or epitrochlearis and b) inhibits insulin-stimulated glucose transport by 28% (p less than 0.005) in soleus but not in epitrochlearis muscle. These studies demonstrate a potentially important differential role for fatty acids in the regulation of glucose transport in different insulin target tissues.     

Insulin action in skeletal muscle from patients with NIDDM

Insulin resistance in peripheral tissues is a common feature of non insulin-dependent diabetes mellitus (NIDDM). The decrease in insulin-mediated peripheral glucose uptake in NIDDM patients can be localized to defects in insulin action on glucose transport in skeletal muscle. Following short term in vitro exposure to both submaximal and maximal concentrations of insulin, 3-O-methylglucose transport rates are 40-50% lower in isolated skeletal muscle strips from NIDDM patients when compared to muscle strips from nondiabetic subjects. In addition, we have shown that physiological levels of insulin induce a 1.6-2.0 fold increase in GLUT4 content in skeletal muscle plasma membranes from control subjects, whereas no significant increase was noted in NIDDM skeletal muscle. Impaired insulin-stimulated GLUT4 translocation and glucose transport in NIDDM skeletal muscle is associated with reduced insulin-stimulated IRS-1 tyrosine phosphorylation and PI3-kinase activity. The reduced IRS-1 phosphorylation cannot be attributed to decreased protein expression, since the IRS-1 protein content is similar between NIDDM subjects and controls. Altered glycemia may contribute to decreased insulin-mediated glucose transport in skeletal muscle from NIDDM patients. We have shown that insulin-stimulated glucose transport is normalized in vitro in the presence of euglycemia, but not in the presence of hyperglycemia. Thus, the circulating level of glucose may independently regulate insulin stimulated glucose transport in skeletal muscle from NIDDM patients via a down regulation of the insulin signaling cascade.     

α-Lipoic acid protects 3T3-L1 adipocytes from NYGGF4 (PID1) overexpression-induced insulin resistance through increasing phosphorylation of IRS-1 and Akt

NYGGF4 (also called PID1) was demonstrated that it may be related to the development of obesity-related IR. We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using α-Lipoic acid (LA) treatment, which could facilitate glucose transport and utilization in fully differentiated adipocytes. Our data showed that the LA pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the LA pretreatment. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with LA strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. These results suggest that LA protects 3T3-L1 adipocytes from NYGGF4-induced IR partially through increasing phosphorylation of IRS-1 and Akt and provide evidence that NYGGF4 may be a potential target for the treatment of obesity and obesity-related IR.     

Enhanced insulin action on glucose transport and insulin signaling in 7-day unweighted rat soleus muscle.

Hindlimb suspension (HS), a model of simulated weightlessness, enhances insulin action on glucose transport in unweighted rat soleus muscle. In the present study, we tested the hypothesis that these changes in glucose transport in 3- and 7-day HS soleus of juvenile, female Sprague-Dawley rats were due to increased functionality of insulin signaling factors, including insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase (PI3-kinase), and Akt. Insulin-stimulated (2 mU/ml) glucose transport was significantly (P < 0.05) enhanced in 3- and 7-day HS soleus by 59 and 113%, respectively, compared with weight-bearing controls. Insulin-stimulated tyrosine phosphorylation of IR and Ser(473) phosphorylation of Akt was not altered by unweighting. Despite decreased (34 and 64%) IRS-1 protein in 3- and 7-day HS soleus, absolute insulin-stimulated tyrosine phosphorylation of IRS-1 was not diminished, indicating relative increases in IRS-1 phosphorylation of 62 and 184%, respectively. In the 7-day HS soleus, this was accompanied by increased (47%) insulin-stimulated IRS-1 associated with the p85 subunit of PI3-kinase. Interestingly, the enhanced insulin-stimulated glucose transport in the unweighted soleus was not completely inhibited (89-92%) by wortmannin, a PI3-kinase inhibitor. Finally, protein expression and activation of p38 MAPK, a stress-activated serine/threonine kinase associated with insulin resistance, was decreased by 32 and 18% in 7-day HS soleus. These results indicate that the increased insulin action on glucose transport in the 7-day unweighted soleus is associated with increased insulin signaling through IRS-1 and PI3-kinase and decreased p38 MAPK protein expression. However, PI3-kinase-independent mechanisms must also play a small role in this adaptive response to HS.     

Sennidin stimulates glucose incorporation in rat adipocytes

A novel small molecule compound which exerts insulin mimetic is desirable. Dozens of natural products that have quinone, naphthoquinone, or anthraquinone structure, were tested by a glucose incorporation assay. We found that sennidin A, anthraquinone derivative, stimulated glucose incorporation to near level of maximal insulin-stimulated and sennidin B, a stereoisomer of sennidin A, also stimulated, but the activity of sennidin B was lower than sennidin A. Sennidin A-stimulated glucose incorporation was completely inhibited by wortmannin. Sennidin A did not induce tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), but induced phosphorylation of Akt and glucose transporter 4 (GLUT4) translocation. Our results suggest that in rat adipocytes, sennidin A stimulates glucose incorporation in the phosphatidylinositol 3-kinase (PI3K)- and Akt-dependent, but in the IR/IRS1-independent manner.     

Infusion of beta-endorphin improves insulin resistance in fructose-fed rats.

In an attempt to probe the effect of beta-endorphin on insulin resistance, we used Wistar rats that were fed fructose-rich chow to induce insulin resistance. Insulin action on glucose disposal rate (GDR) was measured using the hyperinsulinemic euglycemic clamp technique, in which glucose (variable), insulin (40 mU/kg/min), and beta-endorphin (6 ng/kg/min) or vehicle were initiated simultaneously and continued for 120 min. A marked reduction in insulin-stimulated GDR was observed in fructose-fed rats compared to normal control rats. Infusion of beta-endorphin reversed the value of GDR, which was inhibited by naloxone and naloxonazine each at doses sufficient to block opioid mu-receptors. Opioid mu-receptors may therefore be activated by beta-endorphin to improve insulin resistance. Next, soleus muscle was isolated to investigate the effect of beta-endorphin on insulin signals. Insulin resistance in rats induced by excess fructose was associated with the impaired insulin receptor (IR), tyrosine autophosphorylation, and insulin receptor substrate (IRS)-1 protein content in addition to the significant decrease in IRS-1 tyrosine phosphorylation in soleus muscle. This impaired glucose transportation was also due to signaling defects that included an attenuated p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) and Akt serine phosphorylation. However, IR protein levels were not markedly changed in rats with insulin resistance. beta-endorphin infusion reversed the fructose-induced decrement in the insulin-signaling cascade with increased GDR. Apart from IR protein levels, infusion of beta-endorphin reversed the decrease in protein expression for the IRS-1, p85 regulatory subunit of PI3-kinase, and Akt serine phosphorylation in soleus muscle in fructose-fed rats. The decrease in insulin-stimulated protein expression of glucose transporter subtype 4 (GLUT 4) in fructose-fed rats returned to near-normal levels after beta-endorphin infusion. Infusion of beta-endorphin may improve insulin resistance by modulating the insulin-signaling pathway to reverse insulin responsiveness.     

Depletion of mitochondrial DNA causes impaired glucose utilization and insulin resistance in L6 GLUT4myc myocytes

Mitochondrial dysfunction contributes to a number of human diseases, such as hyperlipidemia, obesity, and diabetes. The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the pathogenesis of diabetes. To elucidate the association of cellular mtDNA content and insulin resistance, we produced L6 GLUT4myc myocytes depleted of mtDNA by long term treatment with ethidium bromide. L6 GLUT4myc cells cultured with 0.2 mug/ml ethidium bromide (termed depleted cells) revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. Interestingly, the mtDNA-depleted cells showed a drastic decrease in basal and insulin-stimulated glucose uptake, indicating that L6 GLUT4myc cells develop impaired glucose utilization and insulin resistance. The repletion of mtDNA normalized basal and insulin-stimulated glucose uptake. The mRNA level and expression of insulin receptor substrate (IRS)-1 associated with insulin signaling were decreased by 76 and 90% in the depleted cells, respectively. The plasma membrane (PM) GLUT4 in the basal state was decreased, and the insulin-stimulated GLUT4 translocation to the PM was drastically reduced by mtDNA depletion. Moreover, insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B were drastically reduced in the depleted cells. Those changes returned to control levels after mtDNA repletion. Taken together, our data suggest that PM GLUT4 content and insulin signal pathway intermediates are modulated by the alteration of cellular mtDNA content, and the reductions in the expression of IRS-1 and insulin-stimulated phosphorylation of IRS-1 and Akt2/protein kinase B are associated with insulin resistance in the mtDNA-depleted L6 GLUT4myc myocytes.     

Resistin inhibits glucose uptake in L6 cells independently of changes in insulin signaling and GLUT4 translocation

Elevated levels of resistin have been proposed to cause insulin resistance and therefore may serve as a link between obesity and type 2 diabetes. However, its role in skeletal muscle metabolism is unknown. In this study, we examined the effect of resistin on insulin-stimulated glucose uptake and the upstream insulin-signaling components in L6 rat skeletal muscle cells that were either incubated with recombinant resistin or stably transfected with a vector containing the myc-tagged mouse resistin gene. Transfected clones expressed intracellular resistin, which was released in the medium. Incubation with recombinant resistin resulted in a dose-dependent inhibition of insulin-stimulated 2-deoxyglucose (2-DG) uptake. The inhibitory effect of resistin on insulin-stimulated 2-DG uptake was not the result of impaired GLUT4 translocation to the plasma membrane. Furthermore, resistin did not alter the insulin receptor (IR) content and its phosphorylation, nor did it affect insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation, its association with the p85 subunit of phosphatidylinositol (PI) 3-kinase, or IRS-1-associated PI 3-kinase enzymatic activity. Insulin-stimulated phosphorylation of Akt/protein kinase B-alpha, one of the downstream targets of PI 3-kinase and p38 MAPK phosphorylation, was also not affected by resistin. Expression of resistin also inhibited insulin-stimulated 2-DG uptake when compared with cells expressing the empty vector (L6Neo) without affecting GLUT4 translocation, GLUT1 content, and IRS-1/PI 3-kinase signaling. We conclude that resistin does not alter IR signaling but does affect insulin-stimulated glucose uptake, presumably by decreasing the intrinsic activity of cell surface glucose transporters.     

Adenovirus-mediated overexpression of IRS-1 interacting domains abolishes insulin-stimulated mitogenesis without affecting glucose transport in 3T3-L1 adipocytes.

Activated insulin receptor (IR) interacts with its substrates, IRS-1, IRS-2, and Shc via the NPXY motif centered at Y960. This interaction is important for IRS-1 phosphorylation. Studies using the yeast two-hybrid system and sequence identity analysis between IRS-1 and IRS-2 have identified two putative elements, the PTB and SAIN domains, between amino acids 108 and 516 of IRS-1 that are sufficient for receptor interaction. However, their precise function in mediating insulin's bioeffects is not understood. We expressed the PTB and SAIN domains of IRS-1 in HIRcB fibroblasts and 3T3-L1 adipocytes utilizing replication-defective adenoviral infection to investigate their role in insulin signalling. In both cell types, overexpression of either the PTB or the SAIN protein caused a significant decrease in insulin-induced tyrosine phosphorylation of IRS-1 and Shc proteins, IRS-1-associated phosphatidylinositol 3-kinase (PI 3-K) enzymatic activity, p70s6k activation, and p44 and p42 mitogen-activated protein kinase (MAPK) phosphorylation. However, epidermal growth factor-induced Shc and MAPK phosphorylation was unaffected by the overexpressed proteins. These findings were associated with a complete inhibition of insulin-stimulated cell cycle progression. In 3T3-L1 adipocytes, PTB or SAIN expression extinguished IRS-1 phosphorylation with a corresponding 90% decrease in IRS-1-associated PI 3-K activity. p70s6k is a downstream target of PI 3-K, and insulin-stimulated p70s6k was inhibited by PTB or SAIN expression. Interestingly, overexpression of either PTB or SAIN protein did not affect insulin-induced AKT activation or insulin-stimulated 2-deoxyglucose transport, even though both of these bioeffects are inhibited by wortmannin. Thus, interference with the IRS-1-IR interaction inhibits insulin-stimulated IRS-1 and Shc phosphorylation, PI 3-K enzymatic activity, p70s6k activation, MAPK phosphorylation and cell cycle progression. In 3T3-L1 adipocytes, interference with the IR-IRS-1 interaction did not cause inhibition of insulin-stimulated AKT activation or glucose transport. These results indicate a bifurcation or subcompartmentalization of the insulin signalling pathway whereby some targets of PI 3-K (i.e., p70s6k) are dependent on IRS-1-associated PI 3-K and other targets (i.e., AKT and glucose transport) are not. IR-IRS-1 interaction is not essential for insulin's effect on glucose transport, and alternate, or redundant, pathways exist in these cells.     

Role of Rho-kinase in regulation of insulin action and glucose homeostasis

Accumulating evidence indicates an important role for serine phosphorylation of IRS-1 in the regulation of insulin action. Recent studies suggest that Rho-kinase (ROK) is a mediator of insulin signaling, via interaction with IRS-1. Here we show that insulin stimulation of glucose transport is impaired when ROK is chemically or biologically inhibited in cultured adipocytes and myotubes and in isolated soleus muscle ex vivo. Inactivation of ROK also reduces insulin-stimulated IRS-1 tyrosine phosphorylation and PI3K activity. Moreover, inhibition of ROK activity in mice causes insulin resistance by reducing insulin-stimulated glucose uptake in skeletal muscle in vivo. Mass spectrometry analysis identifies IRS-1 Ser632/635 as substrates of ROK in vitro, and mutation of these sites inhibits insulin signaling. These results strongly suggest that ROK regulates insulin-stimulated glucose transport in vitro and in vivo. Thus, ROK is an important regulator of insulin signaling and glucose metabolism.     

Monoclonal antibody to six transmembrane epithelial antigen of prostate-4 influences insulin sensitivity by attenuating phosphorylation of P13K (P85) and Akt: Possible mitochondrial mechanism

We examined the effects of anti-six-transmembrane epithelial antigen of the prostate-4 (STEAP4) antibodies on glucose transport in mature adipocytes and determined the mechanism of insulin resistance in obesity. Western blotting was performed to determine STEAP4 expression, to assess translocation of insulin-sensitive glucose transporter 4 (GLUT4), and to measure phosphorylation and total protein content of insulin-signaling proteins. Confocal laser microscopy and flow cytometry were used to detect intracellular reactive oxygen species (ROS) and fluctuations in mitochondrial membrane potential (ΔΨ). ATP production was measured by using a luciferase-based luminescence assay kit. After the application of anti-STEAP4 antibodies at 0.002?mg/mL, adipocytes exhibited reduced insulin-stimulated glucose transport by attenuating the phosphorylation of IRS-1, PI3K (p85), and Akt. The antibodies also potentially increase the level of ROS and decrease cellular ATP production and ΔΨ. In conclusion, (i) STEAP4 regulates the function of IRS-1, PI3K, and Akt and decreases insulin-induced GLUT4 translocation and glucose uptake; (ii) ROS-related mitochondrial dysfunction may be related to a reduced IRS-1 correlation with the PI3K signaling pathway, leading to insulin resistance. These observations highlight the potential role of STEAP4 in glucose homeostasis and possibly in the pathophysiology of type 2 diabetes related to obesity and may provide new insights into the mechanisms of insulin resistance in obesity.     

Myricetin, a naturally occurring flavonol, ameliorates insulin resistance induced by a high-fructose diet in rats

The present study was conducted to explore the effects of myricetin on insulin resistance in rats fed for 6 weeks with a diet containing 60% fructose. Repeated intravenous (i.v.) injection of myricetin (1 mg/kg per injection, 3 times daily) for 14 days was found to significantly decrease the high glucose and triglyceride levels in plasma of fructose chow-fed rats. Also, the higher degree of insulin resistance in fructose chow-fed rats as measured by homeostasis model assessment of basal insulin resistance was significantly decreased by myricetin treatment. Myricetin increased the whole-body insulin sensitivity in fructose chow-fed rats, as evidenced by the marked elevation of composite whole-body insulin sensitivity index during the oral glucose tolerance test. Myricetin was found to reverse the defect in expression of insulin receptor substrate-1 (IRS-1) and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) in soleus muscle of fructose chow-fed rats under the basal state, despite the protein expression of insulin receptor (IR). Increased basal phosphorylation of IR and IRS-1 as well as Akt was observed in parallel. The reduced level of insulin action on phosphorylation of IR, IRS-1 and Akt in soleus muscle of fructose chow-fed rats was reversed by myricetin treatment. Furthermore, myricetin treatment improved the defective insulin action on the translocation of glucose transporter subtype 4 (GLUT 4) in insulin-resistant soleus muscle. These findings indicate that myricetin improves insulin sensitivity through the enhancement of insulin action on IRS-1-associated PI 3-kinase and GLUT 4 activity in soleus muscles of animals exhibiting insulin resistance.     

An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 microM). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane.     

G protein-coupled receptor kinase 2 mediates endothelin-1-induced insulin resistance via the inhibition of both Galphaq/11 and insulin receptor substrate-1 pathways in 3T3-L1 adipocytes

G protein-coupled receptor kinases (GRKs) regulate seven-transmembrane receptors (7TMRs) by phosphorylating agonist-activated 7TMRs. Recently, we have reported that GRK2 can function as a negative regulator of insulin action by interfering with G protein-q/11 alpha-subunit (Galphaq/11) signaling, causing decreased glucose transporter 4 (GLUT4) translocation. We have also reported that chronic endothelin-1 (ET-1) treatment leads to heterologous desensitization of insulin signaling with decreased tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and Galphaq/11, and decreased insulin-stimulated glucose transport in 3T3-L1 adipocytes. In the current study, we have investigated the role of GRK2 in chronic ET-1-induced insulin resistance. Insulin-induced GLUT4 translocation was inhibited by pretreatment with ET-1 for 24 h, and we found that this inhibitory effect was rescued by microinjection of anti-GRK2 antibody or GRK2 short interfering RNA. We further found that GRK2 mediates the inhibitory effects of ET-1 by two distinct mechanisms. Firstly, adenovirus-mediated overexpression of either wild-type (WT)- or kinase-deficient (KD)-GRK2 inhibited Galphaq/11 signaling, including tyrosine phosphorylation of Galphaq/11 and cdc42-associated phosphatidylinositol 3-kinase activity. Secondly, ET-1 treatment caused Ser/Thr phosphorylation of IRS-1 and IRS-1 protein degradation. Overexpression of KD-GRK2, but not WT-GRK2, inhibited ET-1-induced serine 612 phosphorylation of IRS-1 and restored activation of this pathway. Taken together, these results suggest that GRK2 mediates ET-1-induced insulin resistance by 1) inhibition of Galphaq/11 activation, and this effect is independent of GRK2 kinase activity, and 2) GRK2 kinase activity-mediated IRS-1 serine phosphorylation and degradation.     

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.