Inducible nitric-oxide synthase and NO donor induce insulin receptor substrate-1 degradation in skeletal muscle cells

Chronic inflammation plays an important role in insulin resistance. Inducible nitric-oxide synthase (iNOS), a mediator of inflammation, has been implicated in many human diseases including insulin resistance. However, the molecular mechanisms by which iNOS mediates insulin resistance remain largely unknown. Here we demonstrate that exposure to NO donor or iNOS transfection reduced insulin receptor substrate (IRS)-1 protein expression without altering the mRNA level in cultured skeletal muscle cells. NO donor increased IRS-1 ubiquitination, and proteasome inhibitors blocked NO donor-induced reduction in IRS-1 expression in cultured skeletal muscle cells. The effect of NO donor on IRS-1 expression was cGMP-independent and accentuated by concomitant oxidative stress, suggesting an involvement of nitrosative stress. Inhibitors for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and c-Jun amino-terminal kinase failed to block NO donor-induced IRS-1 reduction, whereas these inhibitors prevented insulin-stimulated IRS-1 decrease. Moreover iNOS expression was increased in skeletal muscle of diabetic (ob/ob) mice compared with lean wild-type mice. iNOS gene disruption or treatment with iNOS inhibitor ameliorated depressed IRS-1 expression in skeletal muscle of diabetic (ob/ob) mice. These findings indicate that iNOS reduces IRS-1 expression in skeletal muscle via proteasome-mediated degradation and thereby may contribute to obesity-related insulin resistance.     

Effects of physiological quercetin metabolites on interleukin-1β-induced inducible NOS expression

Cytokines released by inflammatory cells around the pancreatic islets are implicated in the pathogenesis of diabetes mellitus. Specifically, interleukin-1β (IL-1β) is known to be involved in islet β-cell damage by activation of nuclear factor-κB (NF-κB)-mediated inducible nitric oxide synthase (iNOS) gene expression. Though most flavonoids are shown to have various beneficial effects, little is known about the anti-inflammatory effects of their metabolites. Therefore, we investigated the effects of quercetin and its metabolites quercetin 3'-sulfate, quercetin 3-glucuronide and isorhamnetin 3-glucuronide on IL-1β-stimulated iNOS gene expression in RINm5F β-cells. The nitrite level, iNOS protein and its mRNA expression levels and iNOS promoter activity were measured. In addition, IκBα protein phosphorylation, nuclear translocation of nuclear factor-κB (NF-κB) and NF-κB DNA binding activity were determined. Adenosine 5'-triphosphate disodium salt-induced insulin release was also measured. Quercetin significantly reduced IL-1β-induced nitrite production, iNOS protein and its mRNA expression levels, and it also inhibited IL-1β-induced IκBα phosphorylation, NF-κB activation and iNOS promoter activity. Additionally, quercetin significantly restored the inhibition of insulin secretion by IL-1β. Meanwhile, quercetin metabolites did not show any effect on IL-1β-induced iNOS gene expression and also on insulin secretion. Therefore, in terms of iNOS expression mechanism, dietary ingestion of quercetin is unlikely to show anti-inflammatory effects in rat islet β-cells exposed to IL-1β.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

Characterisation of a human liver cystathionine beta synthase mRNA sequence corresponding to the c.[833T>C;844_845ins68] mutation in CBS gene

Endoplasmic reticulum (ER) homeostasis is crucial for β-cell function and survival. Direct as well as indirect evidence has pointed toward Ca²⁺ as an important determinant of interleukin-1β (IL-1β)-induced β-cell dysfunction and apoptosis. In the present study, we show that IL-1β-induced apoptosis and necrosis in primary rat β-cells and MIN6 cells largely depends on ER stress, ER Ca²⁺ release, and c-Jun N-terminal kinase (JNK) activation. β-cells also showed marked sensitivity to apoptosis induced by sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA) blockers, thapsigargin and cyclopiazonic acid (CPA). IL-1β induced ER Ca²⁺ release, which was paralleled by an IL-1β-dependent induction of JNK activation and the ER stress response, including activation of PRK (RNA-dependent protein kinase)-like ER kinase (PERK). Furthermore, reduced activation of JNK, utilizing JNK inhibitor SP600125, resulted in significant protection from IL-1β- or thapsigargin-induced apoptosis via ER stress. In conclusion, our results suggest that the IL-1β-induced depletion of ER Ca²⁺ and activation of the ER stress via JNK pathway are potential contributory mechanisms to β-cell death.     

Liver-specific inducible nitric-oxide synthase expression is sufficient to cause hepatic insulin resistance and mild hyperglycemia in mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.     

Inactivation of glycogen synthase kinase-3β is required for osteoclast differentiation

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3β in osteoblasts is well characterized as a negative regulator of β-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3β inactivation upon receptor activator of NF-κB ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3β activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3β (GSK3β-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3β (GSK3β-K85R) or small interfering RNA (siRNA)-mediated GSK-3β silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3β further confirmed the negative role of GSK-3β in osteoclast formation. We also show that overexpression of the GSK3β-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3β-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3β in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.     

Suppression of NADPH oxidase 2 substantially restores glucose-induced dysfunction of pancreatic NIT-1 cells

Defects in insulin secretion by pancreatic cells and/or decreased sensitivity of target tissues to insulin action are the key features of type 2 diabetes. It has been shown that excessive generation of reactive oxygen species (ROS) is linked to glucose-induced β-cell dysfunction. However, cellular mechanisms involved in ROS generation in β-cells and the link between ROS and glucose-induced β-cell dysfunction are poorly understood. Here, we demonstrate a key role of NADPH oxidase 2 (NOX2)-derived ROS in the deterioration of β-cell function induced by a high concentration of glucose. Sprague-Dawley rats were fed a high-fat diet for 24 weeks to induce diabetes. Diabetic rats showed increased glucose levels and elevated ROS generation in blood, but decreased insulin content in pancreatic β-cells. In vitro, increased ROS levels in pancreatic NIT-1 cells exposed to high concentrations of glucose (33.3 mmol·L(-1)) were associated with elevated expression of NOX2. Importantly, decreased glucose-induced insulin expression and secretion in NIT-1 cells could be rescued via siRNA-mediated NOX2 reduction. Furthermore, high glucose concentrations led to apoptosis of β-cells by activation of p38MAPK and p53, and dysfunction of β-cells through phosphatase and tensih homolog (PTEN)-dependent Jun N-terminal kinase (JNK) activation and protein kinase B (AKT/PKB) inhibition, which induced the translocation of forkhead box O1 and pancreatic duodenal homeobox-1, followed by reduced insulin expression and secretion. In conclusion, NOX2-derived ROS could play a critical role in high glucose-induced β-cell dysfunction through PTEN-dependent JNK activation and AKT inhibition.     

Flavonoids protect pancreatic beta-cells from cytokines mediated apoptosis through the activation of PI3-kinase pathway

The preventive effects of four phenolic compounds against cytokines-induced β-cell destruction were assessed in this study. Treatment of INS-1 (832/13) cells with pro-inflammatory cytokine mixtures (interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)) resulted in an increased apoptosis. While resveratrol or myricetin failed to prevent cell apoptosis, quercetin or naringenin treatment exhibited an about 40% less in cell death induced by cytokines-mediated damage. This protective effect of quercetin or naringenin might be mediated partially via the activation of the downstream pAkt and pBad pathways, an outcome which was abolished by pretreatment with a specific PI3-kinase inhibitor. Cellular protein levels of p-p38 MAPK and inducible NO synthase (iNOS) were enhanced after cytokines addition; however, the presence of quercetin or naringenin could not suppress their expression. While cytokines induced MnSOD, quercetin or naringnin did not further enhance expression of this protective protein. In addition, the loss of mitochondria membrane potential (MMP) after cytokines treatment might be partially corrected with quercetin or naringenin. However, none of the phenolic compounds tested in this study reversed the blunted glucose-stimulated insulin secretion after cytokines treatment. These results suggest that quercetin or naringenin might possibly be able to protect β-cells from cytokines toxicity by enhancing cell survival through PI3-kinase pathway, independent of p-p38 MAPK or iNOS.     

Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the "priming kinase." It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH(2)-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/ob mice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.     

Hepatitis B virus X protein regulates hepatic glucose homeostasis via activation of inducible nitric oxide synthase

Dysregulation of liver functions leads to insulin resistance causing type 2 diabetes mellitus and is often found in chronic liver diseases. However, the mechanisms of hepatic dysfunction leading to hepatic metabolic disorder are still poorly understood in chronic liver diseases. The current work investigated the role of hepatitis B virus X protein (HBx) in regulating glucose metabolism. We studied HBx-overexpressing (HBxTg) mice and HBxTg mice lacking inducible nitric oxide synthase (iNOS). Here we show that gene expressions of the key gluconeogenic enzymes were significantly increased in HepG2 cells expressing HBx (HepG2-HBx) and in non-tumor liver tissues of hepatitis B virus patients with high levels of HBx expression. In the liver of HBxTg mice, the expressions of gluconeogenic genes were also elevated, leading to hyperglycemia by increasing hepatic glucose production. However, this effect was insufficient to cause systemic insulin resistance. Importantly, the actions of HBx on hepatic glucose metabolism are thought to be mediated via iNOS signaling, as evidenced by the fact that deficiency of iNOS restored HBx-induced hyperglycemia by suppressing the gene expression of gluconeogenic enzymes. Treatment of HepG2-HBx cells with nitric oxide (NO) caused a significant increase in the expression of gluconeogenic genes, but JNK1 inhibition was completely normalized. Furthermore, hyperactivation of JNK1 in the liver of HBxTg mice was also suppressed in the absence of iNOS, indicating the critical role for JNK in the mutual regulation of HBx- and iNOS-mediated glucose metabolism. These findings establish a novel mechanism of HBx-driven hepatic metabolic disorder that is modulated by iNOS-mediated activation of JNK.     

Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.     

Knockdown of apoptosis signal-regulating kinase 1 modulates basal glycogen synthase kinase-3β kinase activity and regulates cell migration

GSK-3β is a basally active kinase. Axin forms a complex with GSK-3β and β-catenin; this complex promotes the GSK-3β-dependent phosphorylation of β-catenin, thereby inducing its degradation. However, the inhibition of GSK-3β provokes cell migration via the dysregulation of β-catenin. In this study, we determined that the level of apoptosis signal-regulating kinase 1 (ASK1) was lower in a metastatic breast cancer cell line, compared to that of non-metastatic cancer cell lines and the knockdown of ASK1 not only induces β-catenin activation via the inhibition of GSK-3β and collapsing the subsequent protein complex by regulating Axin dynamics, but also stimulates cell migration. Together, the blockage of the GSK-3β-β-catenin pathway resulting from the knockdown of ASK1 modulates the migration of breast cancer cells.     

实时监测高通量低能量激光照射诱导GSK-3β的核质穿梭

糖原合成酶激酶-3(glycogen synthase kinase-3, GSK-3)是一种丝/苏氨酸蛋白激酶.哺乳动物细胞中主要存在GSK-3α与GSK-3β两种亚型.以前的研究认为GSK-3是一种单一的磷酸化糖原合成酶的激酶,可以抑制糖原的合成.最近的研究表明GSK-3可以磷酸化50多种底物,进而调节细胞的多种生理过程,包括细胞结构的改变、代谢,基因表达及细胞凋亡.本文主要研究在高通量低能量激光(high fluence low-power laser irradiation,HF-LPLI)照射下,GSK-3β在活细胞中的动态分布变化.应用荧光蛋白融合蛋白GFP-GSK-3β,在人神经胶质母细胞瘤细胞(U-87)中实时监测高通量低能量激光照射下GSK-3β的动态行为.实验结果显示,120 J/cm~2 的氦氖激光照射后,GSK-3β在9 h时进入细胞核,并维持在核内近2 h, 随后GSK-3β又从细胞核转位到细胞质中. 这表明高通量低能量激光照射激活了GSK-3β.同时,实验结果预示了高通量低能量激光照射可能激活GSK-3β并且参与调控了p53、β-catenin、Myc等相关的转录因子.进一步的研究将探讨在高通量低能量激光照射下,GSK-3β具体调控的转录因子以及调控机制.     

Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade

Adiponectin is the most abundant adipokine secreted from adipocytes. Accumulating evidence suggests that the physiological roles of adiponectin go beyond its metabolic effects. In the present study, we demonstrate that adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) are expressed in adult hippocampal neural stem/progenitor cells (hNSCs). Adiponectin treatment increases proliferation of cultured adult hNSCs in a dose- and time-dependent manner, whereas apoptosis and differentiation of adult hNSCs into neuronal or glial lineage were not affected. Adiponectin activates AMP-activated protein kinase and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in adult hNSCs. Pretreatment with the p38MAPK inhibitor SB203580, but not the AMP-activated protein kinase inhibitor Compound C, attenuates adiponectin-induced cell proliferation. Moreover, adiponectin induces phosphorylation of Ser-389, a key inhibitory site of glycogen synthase kinase 3β (GSK-3β), and this effect can be blocked by inhibition of p38MAPK with SB203580. Levels of total and nuclear β-catenin, the primary substrate of GSK-3β, were increased by adiponectin treatment. These results indicate that adiponectin stimulates proliferation of adult hNSCs, via acting on GSK-3β to promote nuclear accumulation of β-catenin. Thus, our studies uncover a novel role for adiponectin signaling in regulating proliferation of adult neural stem cells.