NOX3-derived reactive oxygen species promote TNF-α-induced reductions in hepatocyte glycogen levels via a JNK pathway

TNF-α-induced insulin resistance is associated with generation of reactive oxygen species (ROS). This study aims at defining the link between ROS production and hepatic insulin resistance. Treatment with TNF-α increased ROS generation through activating NADPH oxidase 3 (NOX3) in HepG2 hepatocytes. Down-regulation of NOX3 using siRNA prevented TNF-α-induced decrease of cellular glycogen. In the cells treated with TNF-α, there were NOX3-dependent activation of JNK, inhibition of IRS1 and phosphorylation of AKT/PKB and GSK. In conclusion, the effects of TNF-α on hepatic insulin resistance appear to be, at least in part, mediated by NOX3-derived ROS through a JNK pathway.     

Palmitate Induces Insulin Resistance in H4IIEC3 Hepatocytes through Reactive Oxygen Species Produced by Mitochondria

Visceral adiposity in obesity causes excessive free fatty acid (FFA) flux into the liver via the portal vein and may cause fatty liver disease and hepatic insulin resistance. However, because animal models of insulin resistance induced by lipid infusion or a high fat diet are complex and may be accompanied by alterations not restricted to the liver, it is difficult to determine the contribution of FFAs to hepatic insulin resistance. Therefore, we treated H4IIEC3 cells, a rat hepatocyte cell line, with a monounsaturated fatty acid (oleate) and a saturated fatty acid (palmitate) to investigate the direct and initial effects of FFAs on hepatocytes. We show that palmitate, but not oleate, inhibited insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 2 and serine phosphorylation of Akt, through c-Jun NH₂-terminal kinase (JNK) activation. Among the well established stimuli for JNK activation, reactive oxygen species (ROS) played a causal role in palmitate-induced JNK activation. In addition, etomoxir, an inhibitor of carnitine palmitoyltransferase-1, which is the rate-limiting enzyme in mitochondrial fatty acid β-oxidation, as well as inhibitors of the mitochondrial respiratory chain complex (thenoyltrifluoroacetone and carbonyl cyanide m-chlorophenylhydrazone) decreased palmitate-induced ROS production. Together, our findings in hepatocytes indicate that palmitate inhibited insulin signal transduction through JNK activation and that accelerated β-oxidation of palmitate caused excess electron flux in the mitochondrial respiratory chain, resulting in increased ROS generation. Thus, mitochondria-derived ROS induced by palmitate may be major contributors to JNK activation and cellular insulin resistance.Insulin is the major hormone that inhibits gluconeogenesis in the liver. Visceral adiposity in obesity causes hepatic steatosis and insulin resistance. In an insulin-resistant state, impaired insulin action allows enhancement of glucose production in the liver, resulting in systemic hyperglycemia (1) and contributing to the development of type 2 diabetes. In addition, we have demonstrated experimentally that insulin resistance accelerated the pathology of steatohepatitis in genetically obese diabetic OLETF rats (2). In contrast, lipid-induced oxidative stress caused steatohepatitis and hepatic insulin resistance in mice (3). In fact, steatosis of the liver is an independent predictor of insulin resistance in patients with nonalcoholic fatty liver disease (4).It remains unclear whether hepatic steatosis causally contributes to insulin resistance or whether it is merely a resulting pathology. Excessive dietary free fatty acid (FFA)² flux into the liver via the portal vein may cause fatty liver disease and hepatic insulin resistance. Indeed, elevated plasma FFA concentrations correlate with obesity and decreased target tissue insulin sensitivity (5).Experimentally, lipid infusion or a high fat diet that increases circulating FFA levels promotes insulin resistance in the liver. Candidate events linking FFA to insulin resistance in vivo are the up-regulation of SREBP-1c (6), inflammation caused by activation of c-Jun amino-terminal kinase (JNK) (7) or IKKβ (8), endoplasmic reticulum (ER) stress (9), ceramide (10, 11), and TRB3 (12).However, which event is the direct and initial target of FFA in the liver is unclear. Insulin resistance induced by lipid infusion or a high fat diet is complex and may be accompanied by alterations not restricted to the liver, making it difficult to determine the contribution of FFAs to hepatic insulin resistance. For example, hyperinsulinemia and hyperglycemia secondary to the initial event also may contribute to the development of diet-induced insulin resistance in vivo (6).To address the early event(s) triggering the development of high fat diet- or obesity-induced insulin resistance, we investigated the molecular mechanism(s) underlying the direct action of FFA on hepatocytes to cause insulin resistance in vitro, using the rat hepatocyte cell line H4IIEC3. We found that mitochondria-derived reactive oxygen species (ROS) were a cause of palmitate-induced insulin resistance in hepatocytes.     

In vivo and in vitro application of black soybean peptides in the amelioration of endoplasmic reticulum stress and improvement of insulin resistance

AimsHepatic endoplasmic reticulum (ER) stress plays a key role in the development of obesity-induced insulin resistance. This study evaluated the effects of peptides from black soybean (BSP) on ER stress and insulin signaling in vitro and in vivo.Main methodsUsing C2C12 myotubes or HepG2 cells, we evaluated the effects of BSP on the expression of proteins involved in insulin signaling and in the ER stress response in insulin-sensitive or insulin-resistant cells. BSP was given orally to db/db mice for 5 weeks to investigate its antidiabetic effects in vivo and the underlying mechanisms.Key findingsBSP increased GLUT4 translocation and glucose transport in myotubes and stimulated Akt-mediated glycogen synthase kinase-3β (GSK-3β) and Foxo1 phosphorylation in HepG2 cells. BSP significantly restored the suppression of insulin-mediated Akt phosphorylation in insulin-resistant cells. BSP significantly inhibited the activation of ER stress-responsive proteins by thapsigargin. BSP also significantly reduced blood glucose and improved glucose tolerance in db/db mice. The serum lipid profile (triglyceride and high-density lipoprotein concentrations) improved concomitantly with the BSP-induced downregulation of hepatic fatty acid synthase expression in db/db mice. Consistent with the results observed in HepG2 cells, BSP downregulated the elevated hepatic ER stress response in diabetic mice concomitantly with an increased expression of phospho-Foxo1.SignificanceA peptide mixture, BSP, showed beneficial effects through multiple mechanisms involving the suppression of hepatic ER stress and restoration of insulin resistance, suggesting that it has potential as an antidiabetic agent.     

Salsalate and Adiponectin Improve Palmitate-Induced Insulin Resistance via Inhibition of Selenoprotein P through the AMPK-FOXO1α Pathway

Selenoprotein P (SeP) was recently identified as a hepatokine that induces insulin resistance (IR) in rodents and humans. Recent clinical trials have shown that salsalate, a prodrug of salicylate, significantly lowers blood glucose levels and increases adiponectin concentrations. We examined the effects of salsalate and full length-adiponectin (fAd) on the expression of SeP under hyperlipidemic conditions and explored their regulatory mechanism on SeP. In palmitate-treated HepG2 cells as well as high fat diet (HFD)-fed male Spraque Dawley (SD) rats and male db/db mice, SeP expression and its regulatory pathway, including AMPK-FOXO1α, were evaluated after administration of salsalate and salicylate. Palmitate treatment significantly increased SeP expression and aggravated IR, while knock-down of SeP by siRNA restored these changes in HepG2 cells. Palmitate-induced SeP expression was inhibited by both salsalate and salicylate, which was mediated by AMPK activation, and was blocked by AMPK siRNA or an inhibitor of AMPK. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assay showed that salsalate suppressed SeP expression by AMPK-mediated phosphorylation of FOXO1α. Moreover, fAd also reduced palmitate-induced SeP expression through the activation of AMPK, which results in improved IR. Both salsalate and salicylate treatment significantly improved glucose intolerance and insulin sensitivity, accompanied by reduced SeP mRNA and protein expression in HFD-fed rats and db/db mice, respectively. Taken together, we found that salsalate and adiponectin ameliorated palmitate-induced IR in hepatocytes via SeP inhibition through the AMPK-FOXO1α pathway. The regulation of SeP might be a novel mechanism mediating the anti-diabetic effects of salsalate and adiponectin.     

NADPH oxidase 2 plays a critical role in dysfunction and apoptosis of pancreatic β-cells induced by very low-density lipoprotein

In type 2 diabetes, pancreatic β-cells cannot secret enough insulin compensate for insulin resistance, which are often accompanied by abnormality in lipid metabolism such as hypertriglyceridemia. It is reported that oxidative stress is involved in pancreatic β-cell dysfunction. However, molecular mechanisms linking between excessive generations of reactive oxygen species (ROS) and β-cell dysfunction and apoptosis induced by high levels of very low-density lipoprotein (VLDL) are poorly understood. In this study, we test the hypothesis that NADPH oxidase 2 (NOX2)-derived ROS may play a key role in dysfunction and apoptosis of pancreatic β-cell induced by VLDL. Our results show that the ApoCIII transgenic mice displayed increased serum TG levels, enhanced generation of ROS and impaired insulin content in pancreatic β-cells. In vitro, the treatment of pancreatic NIT-1 cells with 1?mg/ml VLDL for 12?h stimulated NOX2-derived ROS generation, decreased expression and secretion of insulin. Furthermore, we found that VLDL induced dysfunction and apoptosis of pancreatic β-cells through JNK and p53 pathways, which were rescued by siRNA-mediated NOX2 reduction. In conclusion, our data demonstrate a critical role of NOX2-derived ROS in dysfunction and apoptosis through JNK and p53 pathways in pancreatic β-cells induced by VLDL.     

NADPH oxidase 2-derived reactive oxygen species mediate FFAs-induced dysfunction and apoptosis of β-cells via JNK, p38 MAPK and p53 pathways

Dysfunction of β-cell is one of major characteristics in the pathogenesis of type 2 diabetes. The combination of obesity and type 2 diabetes, characterized as 'diabesity', is associated with elevated plasma free fatty acids (FFAs). Oxidative stress has been implicated in the pathogenesis of FFA-induced β-cell dysfunction. However, molecular mechanisms linking between reactive oxygen species (ROS) and FFA-induced β-cell dysfunction and apoptosis are less clear. In the present study, we test the hypothesis that NOX2-derived ROS may play a critical role in dysfunction and apoptosis of β-cells induced by FFA. Our results show that palmitate and oleate (0.5 mmol/L, 48 h) induced JNK activation and AKT inhibition which resulted in decreased phosphorylation of FOXO1 following nuclear localization and the nucleocytoplasmic translocation of PDX-1, leading to the reducing of insulin and ultimately dysfunction of pancreatic NIT-1 cells. We also found that palmitate and oleate stimulated apoptosis of NIT-1 cells through p38MAPK, p53 and NF-κB pathway. More interestingly, our data suggest that suppression of NOX2 may restore FFA-induced dysfunction and apoptosis of NIT-1 cells. Our findings provide a new insight of the NOX2 as a potential new therapeutic target for preservation of β-cell mass and function.     

NEFA‐induced ROS impaired insulin signalling through the JNK and p38MAPK pathways in non‐alcoholic steatohepatitis

The aim of this study was to investigate the changes in hepatic oxidative phosphorylation (OXPHOS) complexes (COs) in patients and cows with non‐alcoholic steatohepatitis (NASH) and to investigate the mechanism that links mitochondrial dysfunction and hepatic insulin resistance induced by non‐esterified fatty acids (NEFAs). Patients and cows with NASH displayed high blood NEFAs, TNF‐α and IL‐6 concentrations, mitochondrial dysfunction and insulin resistance. The protein levels of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), mitofusin‐2 (Mfn‐2) and OXPHOS complexes (human: COI and COIII; cow: COI‐IV) were significantly decreased in patients and cows with NASH. NEFA treatment significantly impaired mitochondrial function and, increased reactive oxygen species (ROS) production, and excessive ROS overactivated the JNK and p38MAPK pathways and induced insulin resistance in cow hepatocytes. PGC‐1α and Mfn‐2 overexpression significantly decreased the NEFA‐induced ROS production and TNF‐α and IL‐6 mRNA expressions, reversed the inhibitory effect of NEFAs on mitochondrial function and attenuated the overactivation of the ROS‐JNK/p38MAPK pathway, alleviated insulin resistance induced by NEFAs in cow hepatocytes and HepG2 cells. These findings indicate that NEFAs induce mitochondrial dysfunction and insulin resistance mediated by the ROS‐JNK/p38MAPK pathway. PGC‐1α or Mfn‐2 overexpression reversed the lipotoxicity of NEFAs on mitochondrial dysfunction and insulin resistance. Our study clarified the mechanism that links hepatic mitochondrial dysfunction and insulin resistance in NASH.     

Lysophosphatidylcholine as an effector of fatty acid-induced insulin resistance

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα_i, reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.     

Dehydroepiandrosterone decreases elevated hepatic glucose production in C57BL/KsJ-db/db mice

Dehydroepiandrosterone (DHEA) is known to improve hyperglycemia in diabetic db/db mice that are obese and insulin resistant. In a previous study, we reported that DHEA suppresses the elevated hepatic gluconeogenic glucose-6-phosphatase (G6Pase) activity and gene expression in C57BL/KsJ-db/db mice. In the present study, we evaluated the total amount of gluconeogenesis using NaH[(14)C]CO(3) and hepatic glucose production using fructose as a substrate in primary cultured hepatocytes. Despite hyperinsulinemia, the glucose production of db/db mice in the total body and hepatocytes was elevated as compared to their heterozygote littermate C57BL/KsJ-db/+m mice. Administration of DHEA significantly decreased the blood glucose level and increased the plasma insulin level in db/db mice. Administration of DHEA decreased the elevated total body and hepatic glucose production in db/db mice. In addition, the glucose production in the primary cultured hepatocytes of db/db mice was decreased significantly by the direct addition of DHEA or DHEA-S to the medium. These results suggest that administration of DHEA suppresses the elevated total body and hepatic glucose production in db/db mice, and this effect on the liver is considered to result from increased plasma insulin and DHEA or DHEA-S itself.     

Oxamate Improves Glycemic Control and Insulin Sensitivity via Inhibition of Tissue Lactate Production in db/db Mice

Oxamate (OXA) is a pyruvate analogue that directly inhibits the lactate dehydrogenase (LDH)-catalyzed conversion process of pyruvate into lactate. Earlier and recent studies have shown elevated blood lactate levels among insulin-resistant and type 2 diabetes subjects and that blood lactate levels independently predicted the development of incident diabetes. To explore the potential of OXA in the treatment of diabetes, db/db mice were treated with OXA in vivo. Treatment of OXA (350–750 mg/kg of body weight) for 12 weeks was shown to decrease body weight gain and blood glucose and HbA1c levels and improve insulin secretion, the morphology of pancreatic islets, and insulin sensitivity in db/db mice. Meanwhile, OXA reduced the lactate production of adipose tissue and skeletal muscle and serum lactate levels and decreased serum levels of TG, FFA, CRP, IL-6, and TNF-α in db/db mice. The PCR array showed that OXA downregulated the expression of Tnf, Il6, leptin, Cxcr3, Map2k1, and Ikbkb, and upregulated the expression of Irs2, Nfkbia, and Pde3b in the skeletal muscle of db/db mice. Interestingly, LDH-A expression increased in the islet cells of db/db mice, and both treatment of OXA and pioglitazone decreased LDH-A expression, which might be related to the improvement of insulin secretion. Taken together, increased lactate production of adipose tissue and skeletal muscle may be at least partially responsible for insulin resistance and diabetes in db/db mice. OXA improved glycemic control and insulin sensitivity in db/db mice primarily via inhibition of tissue lactate production. Oxamic acid derivatives may be a potential drug for the treatment of type 2 diabetes.     

Ceramide generation is sufficient to account for the inhibition of the insulin-stimulated PKB pathway in C2C12 skeletal muscle cells pretreated with palmitate.

We have employed C2C12 myotubes to investigate lipid inhibition of insulin-stimulated signal transduction and glucose metabolism. Cells were preincubated for 18 h in the absence or presence of free fatty acids (FFAs) and stimulated with insulin, and the effects on glycogen synthesis and signaling intermediates were determined. While the unsaturated FFAs oleate and linoleate inhibited both basal and insulin-stimulated glycogen synthesis, the saturated FFA palmitate reduced only insulin-stimulated glycogen synthesis, and was found to inhibit insulin-stimulated phosphorylation of glycogen synthase kinase-3 and protein kinase B (PKB). However, no effect of palmitate was observed on tyrosine phosphorylation, p85 association, or phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates. In contrast, palmitate promoted phosphorylation of mitogen-activated protein MAP) kinases. Ceramide, a derivative of palmitate, has recently been associated with similar inhibition of PKB, and here, ceramide levels were found to be elevated 2-fold in palmitate-treated C2C12 cells. Incubation of C2C12 cells with ceramide closely reproduced the effects of palmitate, leading to inhibition of glycogen synthesis and PKB and to stimulation of MAP kinase. We conclude that palmitate-induced insulin resistance occurs by a mechanism distinct from that of unsaturated FFAs, and involves elevation of ceramide by de novo synthesis, leading to PKB inhibition without affecting IRS-1 function.     

Protective effect of metformin against palmitate-induced hepatic cell death

Lipotoxicity causes hepatic cell death and therefore plays an important role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metformin, a first-line anti-diabetic drug, has shown a potential protective effect against NAFLD. However, the underlying mechanism is still not clear. In this study, we aim to understand the molecular mechanism of the protective effect of metformin in NAFLD, focusing on lipotoxicity. Cell death was studied in HepG2 cells and primary rat hepatocytes exposed to palmitate and metformin. Metformin ameliorated palmitate-induced necrosis and apoptosis (decreased caspase-3/7 activity by 52% and 57% respectively) in HepG2 cells. Metformin also reduced palmitate-induced necrosis in primary rat hepatocytes (P < 0.05). The protective effect of metformin is not due to reducing intracellular lipid content or activation of AMPK signaling pathways. Metformin and a low concentration (0.1 μmol/L) of rotenone showed moderate inhibition on mitochondrial respiration indicated by reduced basal and maximal mitochondrial respiration and proton leak in HepG2 cells. Moreover, metformin and rotenone (0.1 μmol/L) preserved mitochondrial membrane potential in both HepG2 cells and primary rat hepatocytes. In addition, metformin and rotenone (0.1 μmol/L) also reduces reactive oxygen species (ROS) production and increase superoxide dismutase 2 (SOD2) expression. Our results establish that metformin AMPK-independently protects against palmitate-induced hepatic cell death by moderate inhibition of the mitochondrial respiratory chain, recovering mitochondrial function, decreasing cellular ROS production, and inducing SOD2 expression, indicating that metformin may have beneficial actions beyond its glucose-lowering effect and also suggests that mitochondrial complex І may be a therapeutic target in NAFLD.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.     

Hepatic deficiency of selenoprotein S exacerbates hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is closely associated with insulin resistance (IR) and type 2 diabetes mellitus (T2DM), which are all complex metabolic disorders. Selenoprotein S (SelS) is an endoplasmic reticulum (ER) resident selenoprotein involved in regulating ER stress and has been found to participate in the occurrence and development of IR and T2DM. However, the potential role and mechanism of SelS in NAFLD remains unclear. Here, we analyzed SelS expression in the liver of high-fat diet (HFD)-fed mice and obese T2DM model (db/db) mice and generated hepatocyte-specific SelS knockout (SelS^H-KO) mice using the Cre-loxP system. We showed that hepatic SelS expression levels were significantly downregulated in HFD-fed mice and db/db mice. Hepatic SelS deficiency markedly increased ER stress markers in the liver and caused hepatic steatosis via increased fatty acid uptake and reduced fatty acid oxidation. Impaired insulin signaling was detected in the liver of SelS^H-KO mice with decreased phosphorylation levels of insulin receptor substrate 1 (IRS1) and protein kinase B (PKB/Akt), which ultimately led to disturbed glucose homeostasis. Meanwhile, our results showed hepatic protein kinase Cɛ (PKCɛ) activation participated in the negative regulation of insulin signaling in SelS^H-KO mice. Moreover, the inhibitory effect of SelS on hepatic steatosis and IR was confirmed by SelS overexpression in primary hepatocytes in vitro. Thus, we conclude that hepatic SelS plays a key role in regulating hepatic lipid accumulation and insulin action, suggesting that SelS may be a potential intervention target for the prevention and treatment of NAFLD and T2DM.Subject terms: Metabolic syndrome, Obesity     

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.     

Jejunal Proteins Secreted by db/db Mice or Insulin-Resistant Humans Impair the Insulin Signaling and Determine Insulin Resistance

Background

Two recent studies demonstrated that bariatric surgery induced remission of type 2 diabetes very soon after surgery and far too early to be attributed to weight loss. In this study, we sought to explore the mechanism/s of this phenomenon by testing the effects of proteins from the duodenum-jejunum conditioned-medium (CM) of db/db or Swiss mice on glucose uptake in vivo in Swiss mice and in vitro in both Swiss mice soleus and L6 cells. We studied the effect of sera and CM proteins from insulin resistant (IR) and insulin-sensitive subjects on insulin signaling in human myoblasts.

Methodology/Principal Findings

db/db proteins induced massive IR either in vivo or in vitro, while Swiss proteins did not. In L6 cells, only db/db proteins produced a noticeable increase in basal ⁴⁷³Ser-Akt phosphorylation, lack of GSK3β inhibition and a reduced basal ³⁸⁹Thr-p70-S6K1 phosphorylation. Human IR serum markedly increased basal ⁴⁷³Ser-Akt phosphorylation in a dose-dependent manner. Human CM IR proteins increased by about twofold both basal and insulin-stimulated ⁴⁷³Ser-Akt. Basal ⁹Ser-GSK3β phosphorylation was increased by IR subjects serum with a smaller potentiating effect of insulin.

Conclusions

These findings show that jejunal proteins either from db/db mice or from insulin resistant subjects impair muscle insulin signaling, thus inducing insulin resistance.