Insulin-stimulated Insulin Receptor Research Articles

AMP activated kinase negatively regulates hepatic Fetuin-A via p38 MAPK-C/EBPβ/E3 Ubiquitin Ligase Signaling pathway.

Fetuin-A (Fet-A) is a liver-secreted phosphorylated protein, known to impair insulin signaling, which has been shown to be associated with obesity, insulin resistance, and incident diabetes. Fet-A interacts with the insulin-stimulated insulin receptor (IR) and inhibits IR tyrosine kinase activity and glucose uptake. It has been shown that high glucose increases Fet-A expression through the ERK1/2 signaling pathway. However, factors that downregulate Fet-A expression and their potential mechanisms are unclear. We examined the effect of AMP-activated protein kinase (AMPK) on high-glucose induced Fet-A expression in HepG2 cells, Hep3B cells and primary rat hepatocytes. High glucose increased Fet-A and phosphorylated (Ser312) fetuin-A (pFet-A) expression, which are known to impair insulin signaling. AICAR-induced AMPK activation significantly down-regulated high glucose-induced Fet-A expression and secretion of pFet-A while treatment with Compound C (AMPK inhibitor), SB202190 (p38 MAPK inhibitor) or p38 MAPK siRNA transfection prevented AICAR-induced downregulation of Fet-A expression. In addition, activation of p38 MAPK, by anisomycin, decreased the hepatic expression of Fet-A. Further, we our studies have shown that short-term effect of AICAR-treatment on Fet-A expression was mediated by proteosomal degradation, and long-term treatment of AICAR was associated with decrease in hepatic expression of C/EBP beta, an important transcription factor involved in the regulation of Fet-A. Taken together, our studies implicate a critical role for AMPK-p38 MAPK-C/EBPb-ubiquitin-proteosomal axis in the regulation of the expression of hepatic Fet-A.

Molecular effects of intranasal insulin in the rodent brain: Examination of insulin receptor signaling and the glutamatergic system.

As brain insulin resistance has been identified as a pathological feature of age-related cognitive decline (ARCD), intranasal insulin (INI) is being explored as a potential treatment for patients with ARCD. Previous studies have demonstrated that INI enhances memory, but the underlying mechanisms remain unclear. To investigate the mechanistic basis for the pro-cognitive effects of INI, insulin receptor (IR) signaling and the expression of glutamate receptors and transporters, due to the role of the glutamatergic system in hippocampal synaptic transmission, was examined. Adult male Sprague-Dawley rats received bilateral hippocampal injections of a control lentiviral vector (LV-Con) or a lentivirus containing a selective insulin receptor antisense sequence (LV-IRAS) to induce hippocampal-specific insulin resistance. Seven months later, these animals were administered INI 30 minutes before euthanasia. In a separate cohort of Fischer 344 x Brown Norway F1 hybrid male rats (no virus) we investigated the effects of acute and chronic (10 days) INI dosing paradigms in young (3 months old) and aged (26 months old) rats, with a final INI administration 30 minutes before euthanasia. In both experiments, the hippocampus was processed for immunoblot analysis to assess changes in central IR signaling and phosphorylation/expression of glutamate receptor subunits and transporters. We previously observed that LV-IRAS injection selectively downregulated hippocampal IR expression and insulin-stimulated IR phosphorylation without affecting peripheral insulin sensitivity. Additionally, LV-IRAS animals showed reduced hippocampal basal glutamate levels and decreased phosphorylation/expression of glutamate receptor subunits. In this study, vesicular glutamate transporter 2 (vGluT2) expression, but not vGluT1, was significantly decreased in the hippocampus of LV-IRAS animals after INI administration. In the other cohort, age- and dosing paradigm-dependent effects were observed vis-à-vis IR signaling and glutamate receptor phosphorylation/expression. More studies are needed to fully understand the mechanistic changes following INI and to determine the most effective treatment strategies, but these data indicate that INI may improve cognition through the enhancement of IR signaling in the brain and/or through exerting synaptic effects on glutamate neurotransmission. It is important to understand the mechanism of action, as INI could eventually be used in the broader clinical setting to treat ARCD.

Mechanisms by which adiponectin reverses high fat diet-induced insulin resistance in mice

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2-associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane sn-1,2-DAG-induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.

Prep1 deficiency improves metabolic response in white adipose tissue

Prep1 is a gene encoding for a homeodomain transcription factor which induces hepatic and muscular insulin resistance. In this study, we show that Prep1 hypomorphic heterozygous (Prep1i/+) mice, expressing low levels of protein, featured a 23% and a 25% reduction of total body lipid content and epididymal fat, respectively. The percentage of the small adipocytes (25–75 μm) was 30% higher in Prep1i/+ animals than in the WT, with a reciprocal difference in the large adipose cells (100–150 and >150 μm). Insulin-stimulated insulin receptor tyrosine and Akt serine phosphorylation markedly increased in Prep1i/+ mice, paralleled by 3-fold higher glucose uptake and a significant increase of proadipogenic genes such as C/EBPα, GLUT4, and FABP4. Moreover, T cells infiltration and TNF-α, IFNγ and leptin expression were reduced in adipose tissue from Prep1i/+ mice, while adiponectin levels were 2-fold higher. Furthermore, Prep1i/+ mature adipocytes released lower amounts of pro-inflammatory cytokines and higher amount of adiponectin compared to WT cells. Incubation of murine liver cell line (NMuLi) with conditioned media (CM) from mature adipocytes of Prep1i/+ mice improved glucose metabolism, while those from WT mice had no effect. Consistent with these data, Prep1 overexpression in 3T3-L1 adipocytes impaired adipogenesis and insulin signaling, and increased proinflammatory cytokine secretion. All these findings suggest that Prep1 silencing reduces inflammatory response and increases insulin sensitivity in adipose tissue. In addition, CM from mature adipocytes of Prep1i/+ mice improve metabolism in hepatic cells.

Effect of Dehydroepiandrosterone (DHEA) on Diabetes Mellitus and Obesity.

Type 2 diabetes is a metabolic disorder that is characterized by an impaired capacity to secrete insulin, insulin resistance, or both. Dehydroepiandrosterone (DHEA), a steroid hormone produced by the adrenal cortex, has been reported to have beneficial effects on diabetes mellitus and obesity in animal models. DHEA and DHEA-sulfate (DHEA-S) have been reported to increase not only insulin secretion of the pancreas but also insulin sensitivity of the liver, adipose tissue, and muscle. We investigated the effects of DHEA on glucose metabolism in animal models and reported decrease of liver gluconeogenesis. Recently, we reported the effect of DHEA on the liver and muscle by using insulin-stimulated insulin receptor substrate 1 and 2 (IRS1 and IRS2)-deficient mice. DHEA increased Akt phosphorylation in the liver of C57BL6 IRS1- and IRS2-deficient mice fed with a high-fat diet (HFD), which suggests that the increase in DHEA-induced Akt signaling is sufficient in the presence of IRS1 or IRS2. In addition, other studies have also reported the effect of DHEA on diabetes mellitus in the liver, muscle, adipose tissue, and pancreatic β-cell and its effect on obesity in animal models. A meta-analysis in elderly men and women has found that DHEA supplementation has no effects on blood glucose levels. However, DHEA supplementation to patients with type 2 diabetes has not been fully elucidated. Therefore, further studies are needed to provide greater insight into the effect of DHEA on diabetes and obesity in animal and human models.

Circulating betatrophin is associated with insulin resistance in humans: cross-sectional and interventional studies in vivo and in vitro.

Betatrophin has a closely relationship with metabolism. However, its effect on metabolism disorder remains unclear. This study was comprised of a series of cross-sectional and interventional studies in vivo and vitro. PCOS women with IR and healthy women were recruited from the general population and outpatients. Plasma betatrophin levels were measured with ELISA. Insulin sensitivity was assessed with EHC. Gene expressions at mRNA and protein levels were determined with RT-PCR and Western blotting. Influences of insulin, metformin, rosiglitazone and over- or knockdown-expression of betatrophin were analyzed ex vivo. Our results indicated that IR women had higher betatrophin levels compared with the controls. Circulating betatrophin was positively correlated with BMI, WHR, Fat%, triglyceride, total cholesterol, LDL-C, AUCglucose and AUCinsulin, luteinizing Hormone, FAI and HOMA-IR but negatively with M-value. Metformin treatment in PCOS women with IR led to a reduction of betatrophin levels. Insulin stimulation in hepatocytes increased betatrophin expression. Metformin or rosiglitazone led to a reduction of betatrophin expression in insulin-stimulated hepatocytes. In hepatocytes/macrophages co-culture systems, betatrophin expression was significantly increased, whereas this increase was eliminated by rosiglitazone. In hepatocytes, overexpression and knockdown of betatrophin decreased or increased insulin-stimulated insulin receptor, protein kinase B and insulin receptor substrate-1 phosphorylation respectively. Serum from metformin-treated women with IR decreased betatrophin expression and reinforced insulin signals. Thus, the present study provides the in vivo and in vitro evidence, suggesting that there is a cell cross-talking between hepatocytes with macrophages for the regulating betatrophin and it may be a useful marker for IR and metabolic disorders.

Short-Term Hypoxia-Induced Inhibition of Adipogenesis and Enhancement of Insulin Sensitivity in 3T3-L1 Adipocytes

Obesity is a risk factor of insulin resistance and metabolic disorders. Studies demonstrated that adipose tissue dysfunction may cause insulin resistance in obese individuals. Several pieces of evidence suggested that adipose tissues of obese individuals were in a low-oxygen status compared with the lean control. Other studies demonstrated that exercise re-arranged tissue blood flow, disposing cells to low oxygen concentration and increasing insulin sensitivity. According to the above findings, regulation of hypoxia on insulin sensitivity is still controversial. The purpose of the present study was to investigate the regulatory mechanism of hypoxia on adipogenesis and insulin sensitivity in 3T3-L1 dipocytes. To explore the effect of hypoxia on adipogenesis, 3T3-L1 preadipocytes were incubated in hypoxia status for 6 h in initial adipocyte differentiation. Differentiation efficiency was determined by intracellular triglyceride content measurement, oil red O staining of oil droplets, and expression of adipocyte lipidbinding protein (ALBP/aP2). Expressions of key transcriptional regulators, including those of preadipocyte factor-1 (pref-1), CCAAT/enhancer binding protein α (C/EBPα), and peroxisome proliferator-activated receptor-γ (PPARγ), were measured by immunoblotting. To investigate the effect of hypoxia on insulin sensitivity, differentiated 3T3-L1 adipocytes were preincubated in hypoxia status for 6 h, and then insulin-stimulated phosphorylation of insulin signaling pathways and glucose uptake were measured. To further elucidate the underlying mechanism, we clarify the involvement of hypoxia inducible factor-1α (HIF-1α) on hypoxia-regulated adipogenesis and insulin sensitivity by using HIF-1α inhibitor. Our results showed that hypoxia significantly decreased lipid accumulation and aP2 expression compared with normoxia control. Inhibitory effects of hypoxia on adipocyte differentiation were associated with increased pref-1 expression and decreased PPARγ expression. Hypoxia treatment increased insulinstimulated insulin receptor autophosphorylation and Akt phosphorylation and glucose uptake in 3T3-L1 adipocytes compared with normoxia control. Both hypoxia-inhibited adipogenesis and hypoxia-enhanced insulin sensitivity could be prevented by pretreatment with HIF-1α inhibitor, YC-1. In conclusion, short-term hypoxia suppressed adipogenesis and enhanced insulin sensitivity through HIF- 1α-dependent pathway in 3T3-L1 adipocytes.

Reciprocal regulation of insulin and plasma 5'-AMP in glucose homeostasis in mice.

A previous investigation has demonstrated that plasma 5'-AMP (pAMP) exacerbates and causes hyperglycemia in diabetic mice. However, the crosstalk between pAMP and insulin signaling to regulate glucose homeostasis has not been investigated in depth. In this study, we showed that the blood glucose level was more dependent on the ratio of insulin to pAMP than on the absolute level of these two factors. Administration of 5'-AMP significantly attenuated the insulin-stimulated insulin receptor (IR) autophosphorylation in the liver and muscle tissues, resulting in the inhibition of downstream AKT phosphorylation. A docking analysis indicated that adenosine was a potential inhibitor of IR tyrosine kinase. Moreover, the 5'-AMP treatment elevated the ATP level in the pancreas and in the isolated islets, stimulating insulin secretion and increasing the plasma level of insulin. The insulin administration decreased the 5'-AMP-induced hyper-adenosine level by the up-regulation of adenosine kinase activities. Our results indicate that blood glucose homeostasis is reciprocally regulated by pAMP and insulin.

Plasma fetuin‐A and phosphofetuin‐A (Ser312) responses to a single or short‐term repeated bout of exercise in obese and normal‐weight individuals (1028.2)

Fetuin‐A, an inhibitor of insulin‐stimulated insulin receptor tyrosine kinase (IR‐TK) and glucose uptake, has been shown to be associated with obesity, insulin resistance, and incident diabetes. In circulation, fetuin‐A exists in both phosphorylated and dephosphorylated forms. Phosphorylation status of fetuin‐A has been shown to be critical for its IR‐TK inhibitory activity. The objective of this study was to characterize the effects of single and repeated bouts of exercise on serum total fetuin‐A and phosphofetuin‐A (Ser312) in obese individuals. 31 obese and 11 normal‐weight men underwent a single bout of treadmill walking expending 500 kcals. Insulin, glucose, HOMA, QUICKI, and glucose/insulin ratio were not altered immediately after or 24 hours following a single bout of exercise. Total fetuin‐A concentrations were significantly elevated immediately after exercise, which decreased to pre‐exercise levels in the recovery period (24 hours after the single bout of exercise). Area under the curve for serum total fetuin‐A, phosphofetuin‐A, glucose, and insulin during an oral glucose tolerance test was significantly decreased 24 hours after a single bout of exercise. Next, we investigated the influence of repeated bouts of exercise, expending 350 kcal each session, on four consecutive days. Compared to baseline, insulin and HOMA‐IR were decreased significantly on days 2, 3, and 24 hours following the last bout of exercise. Unlike total fetuin‐A, serum phosphofetuin‐A levels were decreased significantly 24 hours after the last bout of exercise. Taken together, these results suggest that the observed lowering of serum phosphofetuin‐A (Ser312) levels, 24 hours after a single or short‐term repeated bout of exercise, in obese individuals, may contribute to the improvement of insulin sensitivity.Grant Funding Source: Supported by Alabama Agricultural Experiment Station Grants Program

Effect of Metformin on Tyrosine Kinase Autophosphorylation of Erythrocyte Insulin Receptor from Women with Polycystic Ovary Syndrome

Objective To document the possible effect of tyrosine kinase autophosphorylation on erythrocyte insulin-receptor after 12 weeks of metformin administration from the women with polycystic ovary syndrome (PCOS). Methods Thirty non-obese (BMI 2 ) healthy women with normal reproductive cycles were evaluated by conventional criteria as the control, 30 non-obese women with PCOS were categorized as nob-PCOS group, 40 obese women with PCOS (BMI ⩾ 25 kg/m 2 ) were categorized as ob-PCOS group. Subjects with PCOS were given metformin 850 mg/d for 12 weeks. The autophosphorylation of insulin receptor pY1158, pY1162/1163 and insulin receptor-β subunit (IR-β) from the solubilised erythrocyte were detected by ELISA. Results 1) Only the autophosphorylation level of pY1158 in nob-PCOS and ob-PCOS groups was lower (P 0.05), even insulin-stimulated insulin receptor (P>0.05) from both of PCOS groups. 2) After metformin administration, the pY1158, pY1162/pY1163 autophosphorylation levels were increased (P 0.05). In summary, oral administration of metformin led to an significant increase in tyrosine kinase activity in both groups of PCOS. Conclusion The impairment of tyrosine autophosphorylation at pY1158 may contribute to insulin resistance, the tyrosine kinase activity per receptor of solubilised erythrocytes were significantly increased by metformin administration and the effect of metformin on insulin-receptor tyrosine kinase activity appeared to be independent of either of these variables.

Overfeeding a moderate energy diet prepartum does not impair bovine subcutaneous adipose tissue insulin signal transduction and induces marked changes in peripartal gene network expression

Mechanisms regulating subcutaneous adipose tissue (SAT) insulin sensitivity and gene network expression during the peripartal period were evaluated in cows fed to meet or exceed prepartal energy requirements. Holstein cows were dried off at −50 d relative to expected parturition and fed a controlled-energy diet [CON; net energy for lactation=1.24Mcal/kg of dry matter (DM); 36% of DM as wheat straw] until −21 d. Cows were then randomly assigned (n=7/diet) to either the same CON diet or a moderate-energy close-up diet (OVE; net energy for lactation=1.47Mcal/kg of DM) until parturition. Biopsies of SAT were harvested at −10, 7, and 21 d for mRNA expression of 48 genes associated with insulin signaling, adipogenesis, and lipolysis. In vitro basal and insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation (IRS1-PY) was assessed at −10 and 7 d. The OVE led to more positive energy balance and greater serum insulin concentration prepartum. Compared with CON, OVE led to a more drastic increase in serum NEFA and also greater overall serum BHBA postcalving, both of which were associated with greater hepatic total lipid and triacylglycerol concentration. Close-up OVE did not improve any aspect of performance. In prepartal SAT, insulin-stimulated IRS1-PY was greater in OVE than in CON. However, IRS1-PY, serum insulin, and GLUT4 expression decreased postpartum regardless of prepartal treatment, suggesting a more severe state of insulin resistance. The expression of all genes encoding adipogenic regulators (PPARG and ZFP423), most lipogenic enzymes/inducers (FASN, SCD, DGAT2, and INSIG1), and basal-lipolysis regulators (ATGL and ABDH5) was greater at −10 d in OVE than in CON. Whereas adipogenic and basal lipolysis regulator expression remained greater in cows fed OVE by 7 d postpartum, expression of all lipogenic enzymes decreased regardless of diet. Despite those responses, the approximately 3-fold increase in expression of IRS1 and ZFP423 between 7 and 21 d suggested that insulin responsiveness and adipogenic capacity of SAT were partially restored. Expression of the preadipocyte marker DLK1, adiponutrin (PNPLA3), and fibroblast growth factor 21 (FGF21) was undetectable. Results suggested that close-up energy overfeeding did not exacerbate insulin resistance in SAT. Signs of restored insulin responsiveness (upregulation of IRS1, INSIG2, SREBF1, and ZFP423) were apparent as early as 3 wk postpartum. Thus, identifying specific nutrients capable of activating PPARγ after calving in AT might help accelerate its replenishment. A regulatory network encompassing the genes and physiological measurements obtained is proposed.

Using gene expression information obtained by quantitative real-time PCR to evaluate Angus bulls divergently selected for feed efficiency

Residual feed intake (RFI) is a measure of feed efficiency in beef cattle. Young Angus bulls from lines of cattle divergently selected for RFI were used in a gene expression profiling study of the liver. Quantitative real-time PCR (qPCR) assay was used to quantify the differentially expressed genes and the information was used to examine the relationships between the genes and RFI and to classify the bulls into their respective RFI group. Gene expression of 21 genes in liver biopsies from 22 low RFI and 22 high RFI bulls were measured by qPCR. Gene expressions of 14 of the 21 genes were significantly correlated with RFI. The expression of the genes was used in a principal component analysis from which five components were extracted. The five principal components explained 70% of the variation in the dependency structure. The first component was highly correlated (correlation coefficient of 0.69) with RFI. The genes of the glutathione S-transferase Mu family (GSTM1, GSTM2, GSTM4), protocadherin 19 (PCDH19), ATP-binding cassette transporter C4 (ABCC4) and superoxide dismutase 3 (SOD3) are in the xenobiotic pathway and were the key factors in the first principal component. This highlights the important relationship between this pathway and variation in RFI. The second and third principal components were also correlated with RFI, with correlation coefficients of –0.28 and –0.20, respectively. Two of the four important genes of the second principal component work coordinately in the signalling pathways that inhibit the insulin-stimulated insulin receptor and regulate energy metabolism. This is consistent with the observation that a positive genetic correlation exists between RFI and fatness. The important genes in the third principal component are related to the extracellular matrix activity, with low RFI bulls showing high extracellular matrix activity.

Palmitate-Activated Macrophages Confer Insulin Resistance to Muscle Cells by a Mechanism Involving Protein Kinase C θ and ε

BackgroundMacrophage-derived factors contribute to whole-body insulin resistance, partly by impinging on metabolically active tissues. As proof of principle for this interaction, conditioned medium from macrophages treated with palmitate (CM-PA) reduces insulin action and glucose uptake in muscle cells. However, the mechanism whereby CM-PA confers this negative response onto muscle cells remains unknown.Methodology/Principal FindingsL6-GLUT4myc myoblasts were exposed for 24 h to palmitate-free conditioned medium from RAW 264.7 macrophages pre-treated with 0.5 mM palmitate for 6 h. This palmitate-free CM-PA, containing selective cytokines and chemokines, inhibited myoblast insulin-stimulated insulin receptor substrate 1 (IRS1) tyrosine phosphorylation, AS160 phosphorylation, GLUT4 translocation and glucose uptake. These effects were accompanied by a rise in c-Jun N-terminal kinase (JNK) activation, degradation of Inhibitor of κBα (IκBα), and elevated expression of proinflammatory cytokines in myoblasts. Notably, CM-PA caused IRS1 phosphorylation on Ser1101, and phosphorylation of novel PKCθ and ε. Co-incubation of myoblasts with CM-PA and the novel and conventional PKC inhibitor Gö6983 (but not with the conventional PKC inhibitor Gö6976) prevented PKCθ and ε activation, JNK phosphorylation, restored IκBα mass and reduced proinflammatory cytokine production. Gö6983 also restored insulin signalling and glucose uptake in myoblasts. Moreover, co-silencing both novel PKC θ and ε isoforms in myoblasts by RNA interference, but not their individual silencing, prevented the inflammatory response and restored insulin sensitivity to CM-PA-treated myoblasts.Conclusions/Clinical SignificanceThe results suggest that the block in muscle insulin action caused by CM-PA is mediated by novel PKCθ and PKCε. This study re-establishes the participation of macrophages as a relay in the action of fatty acids on muscle cells, and further identifies PKCθ and PKCε as key elements in the inflammatory and insulin resistance responses of muscle cells to macrophage products. Furthermore, it portrays these PKC isoforms as potential targets for the treatment of fatty acid-induced, inflammation-linked insulin resistance.

Resveratrol improves insulin signaling in a tissue-specific manner under insulin-resistant conditions only: in vitro and in vivo experiments in rodents

Resveratrol (RSV) has various metabolic effects, especially with relatively high-dose therapy. However, the ability of RSV to modulate insulin signaling has not been completely evaluated. Here, we determined whether RSV alters insulin signaling in insulin-responsive cells and tissues. The effects of RSV on insulin signaling in 3T3-L1 adipocytes under both insulin-sensitive and insulin-resistant states and in insulin-sensitive tissues of high fat–fed diet-induced obese (DIO) mice were investigated. Insulin-stimulated insulin receptor substrate–1 tyrosine phosphorylation (Y612) was suppressed in RSV-treated adipocytes compared with untreated adipocytes, as was the insulin-stimulated Akt phosphorylation (Ser473). However, under an insulin-resistant condition that was made by incubating 3T3-L1 adipocytes in the conditioned medium from lipopolysaccharide-stimulated LAW264.7 cells, RSV reduced inducible nitric oxide synthase expression and IκBα protein degradation and improved insulin-stimulated Akt phosphorylation (Ser473). In DIO mice, relatively low-dose RSV (30 mg/kg daily for 2 weeks) therapy lowered fasting blood glucose level and serum insulin, increased hepatic glycogen content, and ameliorated fatty liver without change in body weight. The insulin-stimulated Akt phosphorylation was decreased in the liver and white adipose tissue of DIO mice, but it was completely normalized by RSV treatment. However, in the skeletal muscle of DIO mice, insulin signaling was not improved by RSV treatment, whereas the phosphorylation of adenosine monophosphate–activated protein kinase α (Thr172) was improved by it. Our results show that RSV enhances insulin action only under insulin-resistant conditions and suggest that the effect of RSV may depend on the type of tissue being targeted and its metabolic status.

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.

Cyclin-dependent Kinase-5 Is a Key Molecule in Tumor Necrosis Factor-α-induced Insulin Resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.

Activating transcription factor 6 protects insulin receptor from ER stress-stimulated desensitization via p42/44 ERK pathway

Activating transcription factor 6 (ATF6) is a key signal transducer of endoplasmic reticulum stress (ER stress). This study was conducted to clarify the potential role of ATF6 in the insulin signaling pathway under chronic ER stress. ER stress of HEK293 cells was induced with tunicamycin (2 μg/mL). The cells were transfected with ATF6α or ATF6β. The phosphorylation level of insulin receptor (IR) was analyzed using Western blot. The changes in ER stress and ERK signaling pathways were explored using Western blot and quantitative real-time PCR. Tunicamycin-induced chronic ER stress attenuated IR tyrosine phosphorylation in a time-dependent manner, whereas over-expression of ATF6 protected IR from desensitization. ATF6 modulation of IR suppression was associated with insulin-stimulated extracellular signal-regulated kinase (ERK) phosphorylation. The treatment of the cells with a specific ERK inhibitor U0126 (10 μmol/L) mimicked the effect of ATF6 over-expression and restored the insulin-stimulated IR phosphorylation. The treatment of the cells with the ERK activator epidermal growth factor (EGF, 200 ng/mL) decreased the protection effect of ATF6 on IR. Our results demonstrate that ATF6 may serve as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Cannabinoids Inhibit Insulin Receptor Signaling in Pancreatic β-Cells

OBJECTIVEOptimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CB1R) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CB1Rs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action.RESEARCH DESIGN AND METHODSWe measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KCl. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R−/−) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice.RESULTSECs are generated within β-cells, which also express CB1Rs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CB1R results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner.CONCLUSIONSThese findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and proliferation in diabetes.

Hepatic insulin resistance in mice with hepatic overexpression of diacylglycerol acyltransferase 2

Mice overexpressing acylCoA:diacylglycerol (DAG) acyltransferase 2 in the liver (Liv-DGAT2) have been shown to have normal hepatic insulin responsiveness despite severe hepatic steatosis and increased hepatic triglyceride, diacylglycerol, and ceramide content, demonstrating a dissociation between hepatic steatosis and hepatic insulin resistance. This led us to reevaluate the role of DAG in causing hepatic insulin resistance in this mouse model of severe hepatic steatosis. Using hyperinsulinemic-euglycemic clamps, we studied insulin action in Liv-DGAT2 mice and their wild-type (WT) littermate controls. Here, we show that Liv-DGAT2 mice manifest severe hepatic insulin resistance as reflected by decreased suppression of endogenous glucose production (0.8 ± 41.8 vs. 87.7 ± 34.3% in WT mice, P < 0.01) during the clamps. Hepatic insulin resistance could be attributed to an almost 12-fold increase in hepatic DAG content (P < 0.01) resulting in a 3.6-fold increase in protein kinase Cε (PKCε) activation (P < 0.01) and a subsequent 52% decrease in insulin-stimulated insulin receptor substrate 2 (IRS-2) tyrosine phosphorylation (P < 0.05), as well as a 64% decrease in fold increase pAkt/Akt ratio from basal conditions (P < 0.01). In contrast, hepatic insulin resistance in these mice was not associated with increased endoplasmic reticulum (ER) stress or inflammation. Importantly, hepatic insulin resistance in Liv-DGAT2 mice was independent of differences in body composition, energy expenditure, or food intake. In conclusion, these findings strengthen the link between hepatic steatosis and hepatic insulin resistance and support the hypothesis that DAG-induced PKCε activation plays a major role in nonalcoholic fatty liver disease (NAFLD)-associated hepatic insulin resistance.

Chronically Increased S6K1 Is Associated with Impaired IRS1 Signaling in Skeletal Muscle of GDM Women with Impaired Glucose Tolerance Postpartum

The rapidly increasing prevalence of gestational diabetes mellitus (GDM) globally places a growing population at risk for developing type 2 diabetes mellitus (T2DM), particularly those with persistent impaired glucose tolerance (IGT) postpartum. We sought to 1) identify dynamic insulin signaling abnormalities in vivo in a prospective, longitudinal study of GDM women compared to weight-matched pregnant controls both antepartum and postpartum; and 2) determine abnormalities that might distinguish GDM women who normalize their glucose tolerance postpartum from those with persistent IGT. Skeletal muscle biopsies were obtained before and after a 75-g glucose load in nine overweight to obese GDM women and 10 weight-matched pregnant controls antepartum and postpartum. Postpartum biopsies were collected in five weight-matched GDM women with IGT (GDM/IGT). GDM women had decreased skeletal muscle insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine activation and reduced IRS1, concomitant with increased basal IRS1 serine phosphorylation and basal p70 S6-kinase (S6K1) activation, which resolved postpartum. However, GDM/IGT subjects had a persistent impairment in IRS1 activation and increased S6K1 phosphorylation compared to GDM subjects with normal glucose tolerance. This study reveals that women with GDM demonstrate impaired IRS1 signaling associated with increased S6K1 activation in skeletal muscle in vivo. This defect is maintained postpartum in GDM/IGT subjects, despite similar body weights and cytokine levels. Given that GDM women with persistent IGT are at a high risk of developing T2DM, understanding how the nutrient-sensitive mammalian target of rapamycin/S6K1 pathway is chronically activated in GDM may lead to important therapies that could prevent the progression to T2DM.