IRS-1 Ser307 Research Articles

DNAJB3/HSP-40 cochaperone improves insulin signaling and enhances glucose uptake in vitro through JNK repression.

Heat shock response (HSR) is an essential host-defense mechanism that is dysregulated in obesity-induced insulin resistance and type 2 diabetes (T2D). Our recent data demonstrated that DNAJB3 was downregulated in obese human subjects and showed negative correlation with inflammatory markers. Nevertheless, DNAJB3 expression pattern in diabetic subjects and its mode of action are not yet known. In this study, we showed reduction in DNAJB3 transcript and protein levels in PBMC and subcutaneous adipose tissue of obese T2D compared to obese non-diabetic subjects. Overexpression of DNAJB3 in HEK293 and 3T3-L1 cells reduced JNK, IRS-1 Ser-307 phosphorylation and enhanced Tyr-612 phosphorylation suggesting an improvement in IRS-1 signaling. Furthermore, DNAJB3 mediated the PI3K/AKT pathway activation through increasing AKT and AS160 phosphorylation. AS160 mediates the mobilization of GLUT4 transporter to the cell membrane and thereby improves glucose uptake. Using pre-adipocytes cells we showed that DNAJB3 overexpression caused a significant increase in the glucose uptake, possibly through its phosphorylation of AS160. In summary, our results shed the light on the possible role of DNAJB3 in improving insulin sensitivity and glucose uptake through JNK repression and suggest that DNAJB3 could be a potential target for therapeutic treatment of obesity-induced insulin resistance.

Lipid-induced Muscle Insulin Resistance Is Mediated by GGPPS via Modulation of the RhoA/Rho Kinase Signaling Pathway

Elevated circulating free fatty acid levels are important contributors to insulin resistance in the muscle and liver, but the underlying mechanisms require further elucidation. Here, we show that geranylgeranyl diphosphate synthase 1 (GGPPS), which is a branch point enzyme in the mevalonic acid pathway, promotes lipid-induced muscle insulin resistance through activation of the RhoA/Rho kinase signaling pathway. We have found that metabolic perturbation would increase GGPPS expression in the skeletal muscles of db/db mice and high fat diet-fed mice. To address the metabolic effects of GGPPS activity in skeletal muscle, we generated mice with specific GGPPS deletions in their skeletal muscle tissue. Heterozygous knock-out of GGPPS in the skeletal muscle improved systemic insulin sensitivity and glucose homeostasis in mice fed both normal chow and high fat diets. These metabolic alterations were accompanied by activated PI3K/Akt signaling and enhanced glucose uptake in the skeletal muscle. Further investigation showed that the free fatty acid-stimulated GGPPS expression in the skeletal muscle was able to enhance the geranylgeranylation of RhoA, which further induced the inhibitory phosphorylation of IRS-1 (Ser-307) by increasing Rho kinase activity. These results implicate a crucial role of the GGPPS/RhoA/Rho kinase/IRS-1 pathway in skeletal muscle, in which it mediates lipid-induced systemic insulin resistance in obese mice. Therefore, skeletal muscle GGPPS may represent a potential pharmacological target for the prevention and treatment of obesity-related type 2 diabetes.

Titanium dioxide nanoparticles increase plasma glucose via reactive oxygen species-induced insulin resistance in mice.

There have been few reports about the possible toxic effects of titanium dioxide (TiO2 ) nanoparticles on the endocrine system. We explored the endocrine effects of oral administration to mice of anatase TiO2 nanoparticles (0, 64 and 320 mg kg(-1) body weight per day to control, low-dose and high-dose groups, respectively, 7 days per week for 14 weeks). TiO2 nanoparticles were characterized by scanning and transmission electron microscopy (TEM) and dynamic light scattering (DLS), and their physiological distribution was investigated by inductively coupled plasma. Biochemical analyzes included plasma glucose, insulin, heart blood triglycerides (TG), free fatty acid (FFA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and reactive oxygen species (ROS)-related markers (total SOD, GSH and MDA). Phosphorylation of IRS1, Akt, JNK1, and p38 MAPK were analyzed by western blotting. Increased titanium levels were found in the liver, spleen, small intestine, kidney and pancreas. Biochemical analyzes showed that plasma glucose significantly increased whereas there was no difference in plasma insulin secretion. Increased ROS levels were found in serum and the liver, as evidenced by reduced total SOD activity and GSH level and increased MDA content. Western blotting showed that oral administration of TiO2 nanoparticles induced insulin resistance (IR) in mouse liver, shown by increased phosphorylation of IRS1 (Ser307) and reduced phosphorylation of Akt (Ser473). The pathway by which TiO2 nanoparticles increase ROS-induced IR were included in the inflammatory response and phosphokinase, as shown by increased serum levels of TNF-α and IL-6 and increased phosphorylation of JNK1 and p38 MAPK in liver. These results show that oral administration of TiO2 nanoparticles increases ROS, resulting in IR and increasing plasma glucose in mice.

Alternate Phosphorylation/O-GlcNAc Modification on Human Insulin IRSs: A Road towards Impaired Insulin Signaling in Alzheimer and Diabetes.

Impaired insulin signaling has been thought of as important step in both Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM). Posttranslational modifications (PTMs) regulate functions and interaction of insulin with insulin receptors substrates (IRSs) and activate insulin signaling downstream pathways via autophosphorylation on several tyrosine (TYR) residues on IRSs. Two important insulin receptor substrates 1 and 2 are widely expressed in human, and alternative phosphorylation on their serine (Ser) and threonine (Thr) residues has been known to block the Tyr phosphorylation of IRSs, thus inhibiting insulin signaling and promoting insulin resistance. Like phosphorylation, O-glycosylation modification is important PTM and inhibits phosphorylation on same or neighboring Ser/Thr residues, often called Yin Yang sites. Both IRS-1 and IRS-2 have been shown to be O-glycosylated; however exact sites are not determined yet. In this study, by using neuronal network based prediction methods, we found more than 50 Ser/Thr residues that have potential to be O-glycosylated and may act as possible sites as well. Moreover, alternative phosphorylation and O-glycosylation on IRS-1 Ser-312, 984, 1037, and 1101 may act as possible therapeutic targets to minimize the risk of AD and T2DM.

IKKε is key to induction of insulin resistance in the hypothalamus, and its inhibition reverses obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Melatonin rescues 3T3-L1 adipocytes from FFA-induced insulin resistance by inhibiting phosphorylation of IRS-1 on Ser307.

Melatonin is biosynthesized in the pineal gland and secreted into the bloodstream. Evidences indicate a role of melatonin in the regulation of glucose metabolism. The objective of this study was to investigate the effect of melatonin on insulin sensitivity in insulin resistant adipocytes. Following a preincubation with melatonin or vehicle for 30 min, insulin resistant cells of 3T3-L1 adipocytes were induced by palmitic acids (300 μM, 6 h). Our results showed that palmitic acids inhibited both the basal and insulin-stimulated uptake of [(3)H]-2-Deoxyglucose, down-regulated the levels of IRS-1 and GLUT-4. However, compared to the vehicle group, melatonin pre-treatment increased significantly the uptake of [(3)H]-2-Deoxyglucose as well as the level of GLUT-4, and decreased phosphorylated IRS-1 (Ser307) although total IRS-1 did not change significantly. These data suggest that palmitic acids impair insulin signal via down-regulating the expressions of IRS-1 and GLUT-4; whereas melatonin can ameliorate insulin sensitivity by inhibiting Ser307 phosphorylation in IRS-1 and increasing GLUT-4 expressions in insulin resistant 3T3-L1 adipocytes. We conclude that melatonin regulates the insulin sensitivity and glucose homeostasis via inhibiting Ser-phosphorylation and improving function of IRS-1.

Tauroursodeoxycholic Acid Mitigates High Fat Diet-Induced Cardiomyocyte Contractile and Intracellular Ca2+ Anomalies

ObjectivesThe endoplasmic reticulum (ER) chaperone tauroursodeoxycholic acid (TUDCA) has exhibited promises in the treatment of obesity, although its impact on obesity-induced cardiac dysfunction is unknown. This study examined the effect of TUDCA on cardiomyocyte function in high-fat diet-induced obesity.MethodsAdult mice were fed low or high fat diet for 5 months prior to treatment of TUDCA (300 mg/kg. i.p., for 15d). Intraperitoneal glucose tolerance test (IPGTT), cardiomyocyte mechanical and intracellular Ca2+ property, insulin signaling molecules including IRS-1, Akt, AMPK, ACC, GSK-3β, c-Jun, ERK and c-Jun N terminal kinase (JNK) as well as ER stress and intracellular Ca2+ regulatory proteins were examined. Myocardial ultrastructure was evaluated using transmission electron microscopy (TEM).ResultsHigh-fat diet depressed peak shortening (PS) and maximal velocity of shortening/relengthenin as well as prolonged relengthening duration. TUDCA reversed or overtly ameliorated high fat diet-induced cardiomyocyte dysfunction including prolongation in relengthening. TUDCA alleviated high-fat diet-induced decrease in SERCA2a and phosphorylation of phospholamban, increase in ER stress (GRP78/BiP, CHOP, phosphorylation of PERK, IRE1α and eIF2α), ultrastructural changes and mitochondrial permeation pore opening. High-fat diet feeding inhibited phosphorylation of AMPK and promoted phosphorylation of GSK-3β. TUDCA prevented high fat-induced dephosphorylation of AMPK but not GSK-3β. High fat diet promoted phosphorylation of IRS-1 (Ser307), JNK, and ERK without affecting c-Jun phosphorylation, the effect of which with the exception of ERK phosphorylation was attenuated by TUDCA.ConclusionsThese data depict that TUDCA may ameliorate high fat diet feeding-induced cardiomyocyte contractile and intracellular Ca2+ defects through mechanisms associated with mitochondrial integrity, AMPK, JNK and IRS-1 serine phosphorylation.

Effect of Gelam Honey on the Oxidative Stress-Induced Signaling Pathways in Pancreatic Hamster Cells

Background. Oxidative stress induced by reactive oxygen and nitrogen species is critically involved in the impairment of β-cell function during the development of diabetes. Methods. HIT-T15 cells were cultured in 5% CO2 and then preincubated with Gelam honey extracts (20, 40, 60, and 80 µg/mL) as well as quercetin (20, 40, 60, and 80 µM), prior to stimulation by 20 and 50 mM of glucose. Cell lysate was collected to determine the effect of honey extracts and quercetin on the stress activated NF-κB, MAPK pathways, and the Akt (ser473) activated insulin signaling pathway. Results. HIT-T15 cells cultured under hyperglycemic conditions demonstrated insulin resistance with a significant increase in the levels of MAPK, NF-κB, and IRS-1 serine phosphorylation (ser307); however, Akt expression and insulin contents are significantly decreased. Pretreatment with quercetin and Gelam honey extract improved insulin resistance and insulin content by reducing the expression of MAPK, NF-κB, and IRS-1 serine phosphorylation (ser307) and increasing the expression of Akt significantly. Conclusion. Gelam honey-induced differential expression of MAPK, NF-κB, IRS-1 (ser307), and Akt in HIT-T15 cells shows that Gelam honey exerts protective effects against diabetes- and hyperglycemia-induced oxidative stress by improving insulin content and insulin resistance.

NOD1 activation induces innate immune responses and insulin resistance in human adipocytes

The innate immune-receptor nucleotide oligomerization domain (NOD) protein recognizes intracellular bacterial peptidoglycan. Activation of the innate immune system contributes to the development and progression of insulin resistance. The present study aimed to determine the presence of NOD1 and NOD2 in human adipose cells as well as to assess their functionality. Subcutaneous abdominal fat from obese subjects was biopsied and characterized for NOD expression using quantitative real-time PCR (qPCR). Human adipocytes were stimulated with iE-DAP (NOD1-specific ligand), and NOD1, proinflammatory cytokine production and nuclear factor (NF)-κB activation were quantified using qPCR, enzyme-linked immunosorbent assay (Elisa) and luciferase assay. Insulin-stimulated glucose uptake was determined by measuring 2-deoxy-D-[(3)H] glucose uptake. Expression and phosphorylation of IRS-1, Akt and JNK were evaluated using Western blotting. NOD1/NOD2 mRNA expression was induced during adipocyte differentiation and enhanced in human adipose depots. Stimulation of isolated human adipocytes with iE-DAP induced NF-κB p65 nuclear translocation and a marked increase in proinflammatory cytokine production, including MCP-1, IL-6 and IL-8. NOD1 activation weakened insulin signal transduction as revealed by increased JNK and IRS-1 Ser307 phosphorylation, inhibited IRS-1 tyrosine phosphorylation, and reduced insulin-induced phosphorylation of Akt on Ser473 and Thr308 in human adipocytes. Moreover, NOD1 activation reduced insulin-induced glucose uptake, leading to insulin resistance. These results suggest that NOD1 signaling could be one of the links between innate immunity and insulin resistance in human adipocytes. This study provides supporting evidence for NOD1 protein as a component of innate immunity involved in insulin resistance.

Molecular pathways for glucose homeostasis, insulin signaling and autophagy in hepatitis C virus induced insulin resistance in a cellular model

Chronic HCV infection induces insulin resistance (IR). We studied this in a persistently infected cell line with defects in glucose homeostasis resulting from the phosphorylation of glycogen synthase (GS Ser641) and GS kinase isoform 3β (GSK 3βSer9). Reversal of these effects in cells cured of HCV with interferon supports viral specificity. Insulin signaling was disrupted by IRS-1 Ser312 phosphorylation and dysregulation of the Akt pathway. In infected cells, active autophagy was revealed by the formation of LC3 puncta or by increased levels (50–200%) of the markers Beclin 1 and conjugated Atg5/Atg12. Inhibition of autophagy by 3-methyl-adenine (3-MA) reduced Beclin1 levels, inhibited IRS-1 Ser312 or GS Ser641 phosphorylation and decreased viral load. Furthermore, IRS-1 Ser312 and Beclin1 were co-immunoprecipitated suggesting that they interact. It thus appears that HCV infection disturbs glucose homeostasis or insulin signaling to induce IR and also elicits autophagy that may contribute to this process.

Modulation of gut microbiota by antibiotics improves insulin signalling in high-fat fed mice

A high-fat dietary intake induces obesity and subclinical inflammation, which play important roles in insulin resistance. Recent studies have suggested that increased concentrations of circulating lipopolysaccharide (LPS), promoted by changes in intestinal permeability, may have a pivotal role in insulin resistance. Thus, we investigated the effect of gut microbiota modulation on insulin resistance and macrophage infiltration. Swiss mice were submitted to a high-fat diet with antibiotics or pair-feeding for 8 weeks. Metagenome analyses were performed on DNA samples from mouse faeces. Blood was collected to determine levels of glucose, insulin, LPS, cytokines and acetate. Liver, muscle and adipose tissue proteins were analysed by western blotting. In addition, liver and adipose tissue were analysed, blinded, using histology and immunohistochemistry. Antibiotic treatment greatly modified the gut microbiota, reducing levels of Bacteroidetes and Firmicutes, overall bacterial count and circulating LPS levels. This modulation reduced levels of fasting glucose, insulin, TNF-α and IL-6; reduced activation of toll-like receptor 4 (TLR4), c-Jun N-terminal kinase (JNK), inhibitor of κ light polypeptide gene enhancer in B cells, kinase β (IKKβ) and phosphorylated IRS-1 Ser307; and consequently improved glucose tolerance and insulin tolerance and action in metabolically active tissues. In addition, there was an increase in portal levels of circulating acetate, which probably contributed to an increase in 5'-AMP-activated protein kinase (AMPK) phosphorylation in mice. We observed a striking reduction in crown-like structures (CLS) and F4/80(+) macrophage cells in the adipose tissue of antibiotic-treated mice. These results suggest that modulation of gut microbiota in obesity can improve insulin signalling and glucose tolerance by reducing circulating LPS levels and inflammatory signalling. Modulation also appears to increase levels of circulating acetate, which activates AMPK and finally leads to reduced macrophage infiltration.

Role of β-Adrenergic Receptor Regulation of TNF-α and Insulin Signaling in Retinal Müller Cells

The goal of this study was to determine the relationship of TNF-α and the downregulation of insulin receptor signaling in retinal Müller cells cultured under hyperglycemic conditions and the role of β-adrenergic receptors in regulating these responses. Retinal Müller cells were cultured in normal (5 mM) or high (25 mM) glucose until 80% confluent and then were reduced to 2% serum for 18 to 24 hours. The cells were then treated with 10 μM salmeterol followed by Western blot analysis or ELISA. For TNF-α inhibitory studies, the cells were treated with 5 ng/mL of TNF-α for 30 minutes or by a 30-minute pretreatment with TNF-α followed by salmeterol for 6 hours. In the TNF-α short hairpin (sh)RNA experiments, the cells were cultured until 90% confluent, followed by transfection with TNF-α shRNA for 18 hours. TNF-α-only treatments of Müller cells resulted in significant decreases of tyrosine phosphorylation of the insulin receptor and Akt in high-glucose conditions. Salmeterol (10 μM), a β-2-adrenergic receptor agonist, significantly increased phosphorylation of both insulin receptor and Akt. TNF-α shRNA significantly decreased phosphorylation of IRS-1(Ser307), which was further decreased after salmeterol+TNF-α shRNA. Both TNF-α shRNA and salmeterol significantly reduced death of the retinal Müller cells. These studies demonstrate that β-adrenergic receptor agonists in vitro can restore the loss of insulin receptor activity noted in diabetes. By decreasing the levels of TNF-α and decreasing the phosphorylation of IRS-1(Ser307) while increasing tyrosine phosphorylation of insulin receptor, these results suggest a possible mechanism by which restoration of β-adrenergic receptor signaling may protect the retina against diabetes-induced damage.

Inhibition of obesity-induced hepatic ER stress by early insulin therapy in obese diabetic rats

To understand the mechanism by which early insulin therapy improves insulin sensitivity in type 2 diabetes, we investigated endoplasmic reticulum (ER) stress in the liver of type 2 diabetic rats. A high fat diet plus a low dose of streptozotocin (STZ) in Sprague-Dawley (SD) rats was implemented to create an animal model mimicking diabetes. After 3 weeks of insulin treatment, the rats were examined for insulin sensitivity and ER stress in the liver. To investigate insulin sensitivity within the liver, serine phosphorylation of IRS-1 (Ser307) and Akt (Ser473) and expression of gluconeogenic genes, PEPCK and G6Pase, were tested. Protein levels of ER stress markers, such as immunoglobulin binding protein (Bip), inositol-requiring protein 1 alpha (IRE1α), and unspliced and spliced x-box binding protein-1 (XBP-1), were determined to assess ER stress. In the diabetic (DM) group, IRS-1 phosphorylation was increased (P < 0.05), Akt phosphorylation was reduced (P < 0.05), expression of PEPCK and G6Pase was elevated (P < 0.05), and ER stress markers were up-regulated (P < 0.05) relative to the non-diabetic rats. In the insulin (INS) therapy group, all of aforementioned changes were attenuated or reversed (P < 0.05). In addition, c-Jun N-terminal kinase (JNK) activity and SREBP-1 expression were decreased (P < 0.05). Adipose tissue mass was increased (P < 0.05). These data suggest that short-term insulin therapy relieved ER stress and enhanced insulin sensitivity in the liver of diabetic rats. The mechanism is likely related to fat redistribution from liver to adipose tissue. These cellular and molecular responses may represent a mechanism for improvement of insulin sensitivity in type 2 diabetic rats by insulin therapy.

Dysregulation of Glycogen Synthase Kinase-3 in Skeletal Muscle and the Etiology of Insulin Resistance and Type 2 Diabetes

Insulin resistance of glucose transport and metabolism in insulin-sensitive tissues is a primary defect leading to the development of type 2 diabetes. While the etiology of insulin resistance is multifactorial, one factor associated with reduced insulin action is enhanced activity of the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) in skeletal muscle, liver, and adipose tissue. GSK-3 is involved in numerous cellular functions, including glycogen synthesis, protein synthesis, gene transcription, and cell differentiation. Evidence from muscle and fat cell lines and in skeletal muscle from a variety of obese rodent models and from type 2 diabetic humans supports a role of GSK-3 overactivity in the development of insulin resistance of glucose transport and glycogenesis. Studies utilizing highly selective GSK-3 inhibitors indicate that GSK-3 overactivity in obesity is associated with enhanced IRS-1 serine phosphorylation and defective IRS-1-dependent signaling, ultimately resulting in reduced GLUT-4 translocation and glucose transport activity in skeletal muscle. A role of GSK-3 overactivity in the exaggerated hepatic glucose production of type 2 diabetes has also been reported. Recent studies have demonstrated that oxidative stress, resulting from enhanced exposure to oxidants, causes impaired insulin signaling and insulin resistance of skeletal muscle glucose transport, in part due to reduced suppression of GSK-3 activity and increased IRS-1 Ser(307) phosphorylation. The evidence to date supports an important role of GSK-3 dysfunction in the multifactorial etiology of insulin resistance in skeletal muscle. GSK-3 remains an important target for interventions designed to improve insulin action in obesity-associated insulin resistance and type 2 diabetes.

Altered hepatic insulin signalling in male offspring of obese mice.

Individuals exposed in utero to maternal obesity have increased risk of developing type 2 diabetes mellitus and obesity in adulthood. The molecular mechanisms underlying this association are unknown. We have therefore used a murine model of maternal obesity, in which the offspring of obese dams develop hyperinsulinaemia by 3 months of age indicative of insulin resistance. Here, we investigate the effects of maternal diet-induced obesity on the expression/phosphorylation of key hepatic insulin signalling proteins and the expression of anti-oxidant enzymes in male offspring. At 3 months of age, offspring of obese dams had decreased levels of insulin receptor substrate (IRS) 1 (P < 0.01), whereas the ratio of phosphorylated IRS1 Ser307 to total IRS1 was significantly increased (P < 0.001), suggesting that it was less active. Protein expression of the PI3K p85α subunit was decreased (P < 0.01) and there was a tendency for phosphorylation of Akt at Ser473 to be reduced (P = 0.08) in the offspring of obese dams. protein kinase Cζ (P < 0.001) and glycogen synthase kinase 3β (P < 0.05) levels were increased in these animals in comparison with controls. Maternal obesity also resulted in increased phosphorylation of p38 mitogen-activated protein kinase at Thr180/Tyr182 (P < 0.01) and raised c-Jun N-terminal kinase 1 expression (P < 0.5) in the offspring. The expression of antioxidant enzymes was also affected by maternal obesity with CuZnSOD (P < 0.001) and glutathione reductase (P < 0.05) being increased, whereas glutathione peroxidase 1 was reduced (P < 0.05) in the offspring. We conclude that maternal obesity leads to alterations in hepatic insulin signalling protein expression and phosphorylation. These molecular changes may contribute to the development of insulin resistance.

Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine

Sepsis and lipopolysaccharide (LPS) may decrease skeletal muscle protein synthesis by impairing mTOR (mammalian target of rapamycin) activity. The role of mTOR in regulating muscle protein synthesis was assessed in wild-type (WT) and mTOR heterozygous (+/-) mice under basal conditions and in response to LPS and/or leucine stimulation. No difference in body weight of mTOR(+/-) mice was observed compared with WT mice; whereas whole body lean body mass was reduced. Gastrocnemius weight was decreased in mTOR(+/-) mice, which was attributable in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR(+/-) mice to the same extent. Reduced muscle protein synthesis in mTOR(+/-) mice under basal and LPS-stimulated conditions was associated with lower 4E-BP1 and S6K1 phosphorylation. LPS also decreased PRAS40 phosphorylation and increased phosphorylation of raptor and IRS-1 (Ser(307)) to the same extent in WT and mTOR(+/-) mice. Muscle atrogin-1 and MuRF1 mRNA content was elevated in mTOR(+/-) mice under basal conditions, implying increased ubiquitin-proteasome-mediated proteolysis, but the LPS-induced increase in these atrogenes was comparable between groups. Plasma insulin and IGF-I as well as tissue expression of TNFalpha, IL-6, or NOS2 did not differ between WT and mTOR(+/-) mice. Finally, whereas LPS impaired the ability of leucine to stimulate muscle protein synthesis and 4E-BP1 phosphorylation in WT mice, this inflammatory state rendered mTOR(+/-) mice leucine unresponsive. These data support the idea that the LPS-induced reduction in mTOR activity is relatively more important in regulating skeletal muscle mass in response to nutrient stimulation than under basal conditions.

Direct inhibition by angiotensin II of insulin-dependent glucose transport activity in mammalian skeletal muscle involves a ROS-dependent mechanism

No previous study has investigated how the vaso-constrictive peptide Ang II impacts insulin action in isolated mammalian skeletal muscle. We investigated the molecular actions of Ang II on insulin signalling and glucose transport in skeletal muscle from lean Zucker rats. Soleus strips were incubated with insulin (5 mU/ml) and/or Ang II (500 nM) for 2 hours. Ang II caused significant (p < 0.05) inhibition of insulin-stimulated glucose transport (39%) and decreased phosphorylation of Akt Ser473 (37%) and glycogen synthase kinase-3β Ser9 (42%) without affecting phosphorylation of IRS-1 Ser307 or p38 MAPK. We used the superoxide dismutase mimetic, tempol (1 mM), to determine if reactive oxygen species (ROS) contribute to Ang II-mediated insulin resistance. Tempol partially reversed (42%) Ang II-induced inhibition of insulin-stimulated glucose transport. These results indicate that Ang II inhibits distal insulin signalling and insulin-stimulated glucose transport in isolated mammalian skeletal muscle, and that this effect is partially mediated by ROS.

Skeletal muscle protein balance in mTOR heterozygous mice in response to endotoxin and leucine

We assessed the role of mTOR in regulating muscle protein synthesis in wild‐type (WT) and mTOR heterozygous (+/−) mice under basal conditions, and in response to endotoxin (LPS) and/or leucine. Lean body mass was decreased in mTOR+/− vs WT mice. Gastrocnemius weight was decreased in mTOR+/− mice due in part to a reduced rate of basal protein synthesis. LPS decreased muscle protein synthesis in WT and mTOR+/− mice to the same extent. Changes in protein synthesis in mTOR+/− mice under basal and LPS‐stimulated conditions were associated with reduced 4E‐BP1 and S6K1 phosphorylation. LPS also decreased PRSA40 phosphorylation and increased phosphorylation of raptor and IRS‐1 (Ser307) to the same extent in WT and mTOR+/− mice. Muscle atrogin‐1 and MuRF1 mRNAs were elevated in mTOR+/− mice under basal conditions implying increased proteolysis, and the LPS‐induced increase in these atrogenes was comparable between groups. Differences in basal muscle protein balance were not due to differences in plasma insulin or IGF‐I, nor to tissue TNFα, IL‐6 or NOS2. LPS impaired leucine's ability to increase muscle 4E‐BP1 and S6K1 phosphorylation in WT mice and this leucine resistance was exacerbated in mTOR+/− mice. Our data support the contention that sepsis‐induced reductions in mTOR activity are central in regulating skeletal muscle mass under basal conditions and especially in response to nutrient stimulation. (GM38032)

Effects of Bushen Tongmai recipe on expression of IRS-1 Ser307in polycystic ovarian syndrome rats accompanying with insulin resistance

To investigate the effects of Bushen Tongmai recipe on the phospharylation expression of insulin receptor substrate-1 Ser307 (IRS-1 Ser307) in insulin target tissues of polycystic ovarian syndrome (PCOS) rats accompanying with insulin resistance (IR). Rats of model of PCOS accompanying with IR were randomly divided into the model group and the traditional Chinese medicine (TCM) group. Meanwhile, a group of 13 rats of the same age was considered as the normal control group. The TCM group was administered with BSTMR. Half of each group was given insulin injection through portal vein in late estrus. Another half didnt receive insulin injection. The phospharylation levels of IRS-1 Ser307 in liver and fatty tissues were measured by Western blot. The phospharylation level of IRS-1 Ser307 in ovary tissue was measured by immunohistochemistry. The phospharylation expressions of IRS-1 Ser307 in liver, fat and ovary in the model group were higher than those in the normal group (P<0.05, P<0.01); those criteria in the TCM group were lower than those in the model group (P<0.05). After insulin stimulation, the phospharylation expressions of IRS-1 Ser307 were higher than those before insulin stimulation (P<0.05). Bushen Tongmai recipe could attenuate the phospharylation expression of IRS-1 Ser307 and then enhance insulin signal transduction, which may be one of the mechanisms of Bushen Tongmai recipe in improving PCOS accompanying with IR.

Functional Analysis of Long-chain Acyl-CoA Synthetase 1 in 3T3-L1 Adipocytes

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a approximately 15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.