The Effects of Palmitate on Hepatic Insulin Resistance Are Mediated by NADPH Oxidase 3-derived Reactive Oxygen Species through JNK and p38MAPK Pathways

Elevated plasma free fatty acid (FFA) levels in obesity may play a pathogenic role in the development of insulin resistance. However, molecular mechanisms linking FFA to insulin resistance remain poorly understood. Oxidative stress acts as a link between FFA and hepatic insulin resistance. NADPH oxidase 3 (NOX3)-derived reactive oxygen species (ROS) may mediate the effect of TNF-α on hepatocytes, in particular the drop in cellular glycogen content. In the present study, we define the critical role of NOX3-derived ROS in insulin resistance in db/db mice and HepG2 cells treated with palmitate. The db/db mice displayed increased serum FFA levels, excess generation of ROS, and up-regulation of NOX3 expression, accompanied by increased lipid accumulation and impaired glycogen content in the liver. Similar results were obtained from palmitate-treated HepG2 cells. The exposure of palmitate elevated ROS production and NOX3 expression and, in turn, increased gluconeogenesis and reduced glycogen content in HepG2 cells. We found that palmitate induced hepatic insulin resistance through JNK and p38^MAPK pathways, which are rescued by siRNA-mediated NOX3 reduction. In conclusion, our data demonstrate a critical role of NOX3-derived ROS in palmitate-induced insulin resistance in hepatocytes, indicating that NOX3 is the predominant source of palmitate-induced ROS generation and that NOX3-derived ROS may drive palmitate-induced hepatic insulin resistance through JNK and p38^MAPK pathways.     

Role of FoxO1 in FFA-induced oxidative stress in adipocytes

Reactive oxygen species (ROS) production has recently been established as an essential contributor in the pathogenesis of obesity-associated insulin resistance. The FoxO1 pathway plays a role not only in nutrient sensing but also in regulating ROS production. We exposed adipocytes to free fatty acids (FFA) and demonstrated that FoxO1 protein levels decrease in a dose-dependent manner. The FoxO1 downregulation correlated with an increase in the production of ROS and a proinflammatory adipokine pattern characterized by a decrease in adiponectin and an increase in IL-6, plasminogen activator inhibitor-1, and monocyte chemotactic protein-1 mRNA expression levels. Similarly, a decrease in FoxO1 protein levels was seen in adipocytes of db/db mice compared with controls. Treatment with the sirtuin agonist resveratrol, which translocates FoxO1 to the nucleus, increased FoxO1 protein levels in adipocytes exposed to FFA. This correlated with a decrease in the generation of ROS and a partial reversal of the proinflammatory adipokine pattern. Together these results indicate that the insulin-resistant adipocyte produced by the exposure to a high concentration of fatty acids is characterized by decreased levels of FoxO1. These data also suggest that modulation of the Sirt1/FoxO1 pathway is a potentially useful therapeutic target for the obesity-induced dysfunctional adipocyte.     

Green tea catechins ameliorate adipose insulin resistance by improving oxidative stress

Epidemiological data have suggested that drinking green tea is negatively associated with diabetes, and adipose oxidative stress may have a central role in causing insulin resistance, according to recent findings. The aim of this work is to elucidate a new mechanism for green tea's anti-insulin resistance effect. We used obese KK-ay mice, high-fat diet-induced obese rats, and induced insulin resistant 3T3-L1 adipocytes as models. Insulin sensitivity and adipose reactive oxidative species (ROS) levels were detected in animals and adipocytes. The oxidative stress assay and glucose uptake ability assay were performed, and the effects of EGCG on insulin signals were detected. Green tea catechins (GTCs) significantly decreased glucose levels and increased glucose tolerance in animals. GTCs reduced ROS content in both models of animal and adipocytes. EGCG attenuated dexamethasone and TNF-α promoted ROS generation and increased glucose uptake ability. EGCG also decreased JNK phosphorylation and promoted GLUT-4 translocation. EGCG and GTCs could improve adipose insulin resistance, and exact this effect on their ROS scavenging functions.     

Caloric restriction ameliorates cardiomyopathy in animal model of diabetes

Background

The db/db mouse is an animal model of diabetes in which leptin receptor activity is deficient resulting accelerated cardiomyopathy when exposed to angiotensin (AT). Toll-like receptors 4 and 2 (TLR4, TLR2) are pattern recognition receptors, that recognize pathogen-associated molecular patterns and exacerbate and release inflammatory cytokines. Fetuin A (Fet A) is a fatty acid carrier which affects inflammation and insulin resistance in obese humans and animals through TLRs.The aim of this study was to investigate the effect of caloric restriction (CR) on free fatty acids (FFA) level and the inflammatory response in diabetic cardiomyopathy.

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.     

High lipolytic activity and dyslipidemia in a Spontaneous Hypertensive/NIH Corpulent (SHR/N-cp) rat: a genetic model of obesity and type 2 diabetes mellitus

In order to better understand the link between obesity and type 2 diabetes, lipolysis and its adrenergic regulation was investigated in various adipose depots of obese adult females SHR/N-cp rats. Serum insulin, glucose, free fatty acids (FFA), triglycerides (TG) and glycerol were measured. Adipocytes were isolated from subcutaneous (SC), parametrial (PM) and retroperitoneal (RP) fat pads. Total cell number and size, basal lipolysis or stimulated by norepinephrine (NE) and BRL 37344 were measured in each depot. Obese rats were hyperinsulinemic and hyperglycemic, suggesting high insulin resistance. They presented a marked dyslipidemia, attested by increased serum FFA and TG levels. High serum glycerol levels also suggest a strong lipolytic rate. Obese rats showed an excessive development of all fat pads although a more pronounced effect was observed in the SC one. The cellularity of this depot was increased 8 fold when compared to lean rats, but these fat cells were only 1.5 to 2-fold larger. SC adipocytes showed a marked increase in their basal lipolytic activity but a lack of change in responsiveness to NE or BRL 37344. The association between high basal lipolysis and increased cellularity yields to a marked adipose cell lipolytic rate, especially from the SC region. SHR/N-cp rats were characterized by a hyperplasic type of obesity with an excessive development of the SC depot. The dyslipidemia, attested by an altered serum lipid profile could be attributed to excessive lipolysis that contributes to increased FFA levels, and to early development of insulin resistance through a lipotoxicity effect.     

Anti-diabetic effects of electrolyzed reduced water in streptozotocin-induced and genetic diabetic mice

Oxidative stress is produced under diabetic conditions and is likely involved in progression of pancreatic beta-cell dysfunction found in diabetes. Both an increase in reactive oxygen free radical species (ROS) and a decrease in the antioxidant defense mechanism lead to the increase in oxidative stress in diabetes. Electrolyzed reduced water (ERW) with ROS scavenging ability may have a potential effect on diabetic animals, a model for high oxidative stress. Therefore, the present study examined the possible anti-diabetic effect of ERW in two different diabetic animal models. The genetically diabetic mouse strain C57BL/6J-db/db (db/db) and streptozotocin (STZ)-induced diabetic mouse were used as insulin deficient type 1 and insulin resistant type 2 animal model, respectively. ERW, provided as a drinking water, significantly reduced the blood glucose concentration and improved glucose tolerance in both animal models. However, ERW fail to affect blood insulin levels in STZ-diabetic mice whereas blood insulin level was markedly increased in genetically diabetic db/db mice. This improved blood glucose control could result from enhanced insulin sensitivity, as well as increased insulin release. The present data suggest that ERW may function as an orally effective anti-diabetic agent and merit further studies on its precise mechanism.     

Berberine inhibits PTP1B activity and mimics insulin action

Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.     

Chromium (VI) induces insulin resistance in 3T3-L1 adipocytes through elevated reactive oxygen species generation

Reactive oxygen species (ROS) have been proposed to be involved in the development of insulin resistance, although the exact molecular link between ROS and insulin resistance remains to be determined. Chromium (Cr(VI)) is known as an inducer of ROS. Therefore, this study examined whether Cr(VI) could induce insulin resistance. It demonstrated that Cr(VI) treatment significantly inhibited insulin-stimulated glucose uptake and attenuated insulin signalling. Moreover, Cr(VI) treatment markedly increased the intracellular levels of superoxide anion, hydrogen peroxide and hydroxyl radical. N-acetylcysteine, superoxide dismutase and catalase can block the ROS generation and alleviate the insulin resistance induced by Cr(VI) treatment. In addition, Cr(VI) treatment induced endoplasmic reticulum (ER) stress and JNK activation and these effects were diminished by N-acetylcysteine. These results suggested that ROS generation through Cr(VI) treatment cause ER stress, JNK activation and insulin resistance in adipocytes. Therefore, the oxidative stress could be a potential interventional target for insulin-resistance related diseases.     

Chromium (VI) induces insulin resistance in 3T3-L1 adipocytes through elevated reactive oxygen species generation

Reactive oxygen species (ROS) have been proposed to be involved in the development of insulin resistance, although the exact molecular link between ROS and insulin resistance remains to be determined. Chromium (Cr(VI)) is known as an inducer of ROS. Therefore, this study examined whether Cr(VI) could induce insulin resistance. It demonstrated that Cr(VI) treatment significantly inhibited insulin-stimulated glucose uptake and attenuated insulin signalling. Moreover, Cr(VI) treatment markedly increased the intracellular levels of superoxide anion, hydrogen peroxide and hydroxyl radical. N-acetylcysteine, superoxide dismutase and catalase can block the ROS generation and alleviate the insulin resistance induced by Cr(VI) treatment. In addition, Cr(VI) treatment induced endoplasmic reticulum (ER) stress and JNK activation and these effects were diminished by N-acetylcysteine. These results suggested that ROS generation through Cr(VI) treatment cause ER stress, JNK activation and insulin resistance in adipocytes. Therefore, the oxidative stress could be a potential interventional target for insulin-resistance related diseases.     

Tertiary butylhydroquinone alleviates gestational diabetes mellitus in C57BL/KsJ-Lep db/+ mice by suppression of oxidative stress

Gestational diabetes mellitus (GDM) is a common disorder characterized by abnormal glucose metabolism during pregnancy, affecting 2% to 5% of pregnant women. Currently, clinical treatment for GDM is very limited. The present study was designed to investigate the effect and underlying molecular mechanism of tertiary butylhydroquinone (TBHQ) in a pregnant C57BL/KsJ-Lep db/+ (referred to as db+) GDM mouse model. The results showed that nonpregnant db/+ mice did not show a diabetic phenotype, and TBHQ had no effect on glucose and insulin tolerance in these mice. Moreover, in db/+ pregnant mice exhibiting typical diabetes symptoms, such as hyperglycemia and hypoinsulinemia, TBHQ could remarkably decrease the blood glucose level, increase insulin level, and improve glucose and insulin intolerance. The results also revealed that TBHQ could inhibit oxidative stress in pregnant db/+ mice. Furthermore, TBHQ greatly improved offspring survival rate, glucose metabolism, and insulin tolerance. In addition, TBHQ inhibited oxidative stress by reducing malondialdehyde (MDA) and reactive oxygen species (ROS) levels and increased superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Moreover, we found that TBHQ activated the nuclear factor erythroid 2-related factor 2 (Nrf2), thereby increasing the levels of Nrf2, and ultimately upregulating the expression of heme oxygenase 1 (NO-1) and superoxide dismutase 2 (SOD2). In conclusion, our findings demonstrated that TBHQ alleviated GDM via Nrf2 activation.     

Chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes through oxidative stress

Insulin resistance and hyperinsulinemia are commonly present in obesity and pre-diabetes, and hyperinsulinemia is both a marker and a cause for insulin resistance. However, the molecular link between hyperinsulinemia and insulin resistance remains elusive. The present study examined the effect of chronic insulin treatment on the reactive oxygen species (ROS) production, insulin signalling and insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The results showed that chronic insulin treatment significantly increased the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radical. ROS induced by chronic insulin treatment inhibited insulin signalling and glucose uptake, induced endoplasmic reticulum (ER) stress and JNK activation. Furthermore, these effects were reversed by antioxidants N-acetylcysteine, superoxide dismutase or catalase. These results suggested that ROS, ER stress and JNK pathway are involved in insulin resistance induced by chronic insulin treatment. Therefore, oxidative stress could be a potential interventional target for hyperinsulinemia-induced insulin resistance and related diseases.     

Mild Electrical Stimulation with Heat Shock Ameliorates Insulin Resistance via Enhanced Insulin Signaling

Low-intensity electrical current (or mild electrical stimulation; MES) influences signal transduction and activates phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Because insulin resistance is characterized by a marked reduction in insulin-stimulated PI3K-mediated activation of Akt, we asked whether MES could increase Akt phosphorylation and ameliorate insulin resistance. In addition, it was also previously reported that heat shock protein 72 (Hsp72) alleviates hyperglycemia. Thus, we applied MES in combination with heat shock (HS) to in vitro and in vivo models of insulin resistance. Here we show that 10-min treatment with MES at 5 V (0.1 ms pulse duration) together with HS at 42°C increased the phosphorylation of insulin signaling molecules such as insulin receptor substrate (IRS) and Akt in HepG2 cells maintained in high-glucose medium. MES (12 V)+mild HS treatment of high fat-fed mice also increased the phosphorylation of insulin receptor β subunit (IRβ) and Akt in mice liver. In high fat-fed mice and db/db mice, MES+HS treatment for 10 min applied twice a week for 12–15 weeks significantly decreased fasting blood glucose and insulin levels and improved insulin sensitivity. The treated mice showed significantly lower weight of visceral and subcutaneous fat, a markedly improved fatty liver and decreased size of adipocytes. Our findings indicated that the combination of MES and HS alleviated insulin resistance and improved fat metabolism in diabetes mouse models, in part, by enhancing the insulin signaling pathway.     

Natural COA water inhibits mitochondrial ROS-mediated apoptosis through Plk3 downregulation under STZ diabetic stress in pancreatic β-cell lines

Diabetes from pancreatic β cell death and insulin resistance is a serious metabolic disease in the world. Although the overproduction of mitochondrial reactive oxygen species (ROS) plays an important role in the pathogenesis of diabetes, its specific molecular mechanism remains unclear. Here, we show that the natural Charisma of Aqua (COA) water plays a role in Streptozotocin (STZ) diabetic stress-induced cell death inhibition. STZ induces mitochondrial ROS by increasing Polo-like kinase 3 (Plk3), a major mitotic regulator, in both Beta TC-6 and Beta TC-tet mouse islet cells and leads to apoptosis. Overexpression of Plk3 regulates an increase in mitochondrial ROS as well as cell death, also these events were inhibited by Plk3 gene knockdown in STZ diabetic stimulated-Beta TC-6 cells. Interestingly, we found that natural COA water blocks mitochondrial ROS generation through the reduction of Plk3 and prevents apoptosis in STZ-treated beta cells. Furthermore, using the 3D organoid (ex vivo) system, we confirmed that the insulin secretion of the supernatant medium under STZ treated pancreatic β-cells is protected by the natural COA water. These findings demonstrate that the natural water COA has a beneficial role in maintaining β cell function through the inhibition of mitochondrial ROS-mediated cell death, and it might be introduced as a potential insulin stabilizer.     

Chronic insulin treatment causes insulin resistance in 3T3-L1 adipocytes through oxidative stress

Insulin resistance and hyperinsulinemia are commonly present in obesity and pre-diabetes, and hyperinsulinemia is both a marker and a cause for insulin resistance. However, the molecular link between hyperinsulinemia and insulin resistance remains elusive. The present study examined the effect of chronic insulin treatment on the reactive oxygen species (ROS) production, insulin signalling and insulin-stimulated glucose uptake in 3T3-L1 adipocytes. The results showed that chronic insulin treatment significantly increased the intracellular generation of superoxide anion, hydrogen peroxide and hydroxyl radical. ROS induced by chronic insulin treatment inhibited insulin signalling and glucose uptake, induced endoplasmic reticulum (ER) stress and JNK activation. Furthermore, these effects were reversed by antioxidants N-acetylcysteine, superoxide dismutase or catalase. These results suggested that ROS, ER stress and JNK pathway are involved in insulin resistance induced by chronic insulin treatment. Therefore, oxidative stress could be a potential interventional target for hyperinsulinemia-induced insulin resistance and related diseases.     

Overexpression of glucose-6-phosphate dehydrogenase is associated with lipid dysregulation and insulin resistance in obesity

Glucose-6-phosphate dehydrogenase (G6PD) produces cellular NADPH, which is required for the biosynthesis of fatty acids and cholesterol. Although G6PD is required for lipogenesis, it is poorly understood whether G6PD in adipocytes is involved in energy homeostasis, such as lipid and glucose metabolism. We report here that G6PD plays a role in adipogenesis and that its increase is tightly associated with the dysregulation of lipid metabolism and insulin resistance in obesity. We observed that the enzymatic activity and expression levels of G6PD were significantly elevated in white adipose tissues of obese models, including db/db, ob/ob, and diet-induced obesity mice. In 3T3-L1 cells, G6PD overexpression stimulated the expression of most adipocyte marker genes and elevated the levels of cellular free fatty acids, triglyceride, and FFA release. Consistently, G6PD knockdown via small interfering RNA attenuated adipocyte differentiation with less lipid droplet accumulation. Surprisingly, the expression of certain adipocytokines such as tumor necrosis factor alpha and resistin was increased, whereas that of adiponectin was decreased in G6PD overexpressed adipocytes. In accordance with these results, overexpression of G6PD impaired insulin signaling and suppressed insulin-dependent glucose uptake in adipocytes. Taken together, these data strongly suggest that aberrant increase of G6PD in obese and/or diabetic subjects would alter lipid metabolism and adipocytokine expression, thereby resulting in failure of lipid homeostasis and insulin resistance in adipocytes.     

Mechanisms underlying different responses of plasma triglyceride to high-fat diets in hamsters and mice: Roles of hepatic MTP and triglyceride secretion

Hypertriglyceridemia, closely associated with insulin resistance, is induced on high-fat diets (HFD) in humans but not in mouse models. Mechanisms underlying this species difference are still unclear. Hamsters resemble humans in lipoprotein metabolism. Here by comparing the responses to HFD in hamsters and mice, we found that hepatic TG secretion, MTP expression and plasma free fatty acid (FFA) level were increased in hamsters on HFD feeding but decreased in mice. Although hepatic steatosis and de novo lipogenesis were induced by HFD feeding in both models, cholesterol biosynthesis was inhibited in mice but not in hamsters. Moreover, in insulin deficient state, HFD increased plasma TG level, hepatic TG secretion, MTP expression and plasma FFA level in both models. In summary, distinct changes of MTP expression, in correlation with hepatic TG secretion, underlie the opposite responses of plasma TG levels to high-fat diets in hamsters and mice. Furthermore, hepatic TG secretion and MTP expression seems to be associated with plasma FFA level and cholesterol biosynthesis but not hepatic steatosis or de novo lipogenesis.     

Reversal of Inflammation‐Induced Impairment of Glucose Uptake in Adipocytes by Direct Effect of CB1 Antagonism on Adipose Tissue Macrophages

Macrophage infiltration into adipose tissue (AT‐MP) is thought to induce insulin resistance and diabetes in obesity. Here, we investigated the effect of the antiobesity drug SR141716 (a CB1 antagonist) on macrophage‐mediated inhibition of insulin signaling in adipocytes. THP1 macrophages (THP1) were stimulated in vitro with lipopolysaccharide (LPS) and SR141716 or vehicle. The resulting conditioned medium (CM) was analyzed and incubated on human adipocytes. CM from LPS‐stimulated THP1 inhibited insulin‐induced AKT phosphorylation in adipocytes, in contrast to CM from nonactivated THP1. Moreover, it contained higher concentrations of tumor necrosis factor‐α (TNFα) and lower levels of the anti‐inflammatory cytokine IL‐10. SR141716 reduced TNFα production and increased IL‐10 secretion, resulting in a rescue of insulin signaling in adipocytes. To confirm these findings in vivo, AT‐MP CM from cafeteria diet‐fed or Zucker diabetic fatty (ZDF) rats that had received SR141716 for 3 weeks were isolated, analyzed, and incubated with adipocytes. Cafeteria diet induced macrophage‐mediated inhibition of insulin signaling in adipocytes. Interestingly, SR141716 rescued insulin‐induced glucose uptake in adipocytes. Finally, AT‐MP CM from obese ZDF rats inhibited insulin‐stimulated glucose uptake in adipocytes in contrast to AT‐MP CM from lean ZDF rats. After treatment with SR141716, AT‐MP CM rescued insulin‐induced glucose uptake in adipocytes. In summary, our data indicate that CB1 receptor antagonism in macrophages modified their cytokine production and improved the insulin responsiveness of adipocytes that had been incubated with macrophage CM. Thus, SR141716 ameliorated adipose tissue insulin resistance by direct action on AT‐MP demonstrating a novel peripheral mode of action of CB1 antagonism.