Modulation of palmitate-induced endoplasmic reticulum stress and apoptosis in pancreatic β-cells by stearoyl-CoA desaturase and Elovl6

Induction of endoplasmic reticulum (ER) stress and apoptosis by elevated exogenous saturated fatty acids (FAs) plays a role in the pathogenesis of β-cell dysfunction and loss of islet mass in type 2 diabetes. Regulation of monounsaturated FA (MUFA) synthesis through FA desaturases and elongases may alter the susceptibility of β-cells to saturated FA-induced ER stress and apoptosis. Herein, stearoyl-CoA desaturase (SCD)1 and SCD2 mRNA expression were shown to be induced in islets from prediabetic hyperinsulinemic Zucker diabetic fatty (ZDF) rats, whereas SCD1, SCD2, and fatty acid elongase 6 (Elovl6) mRNA levels were markedly reduced in diabetic ZDF rat islets. Knockdown of SCD in INS-1 β-cells decreased desaturation of palmitate to MUFA, lowered FA partitioning into complex neutral lipids, and increased palmitate-induced ER stress and apoptosis. Overexpression of SCD2 increased desaturation of palmitate to MUFA and attenuated palmitate-induced ER stress and apoptosis. Knockdown of Elovl6 limited palmitate elongation to stearate, increasing palmitoleate production and attenuating palmitate-induced ER stress and apoptosis, whereas overexpression of Elovl6 increased palmitate elongation to stearate and palmitate-induced ER stress and apoptosis. Overall, these data support the hypothesis that enhanced MUFA synthesis via upregulation of SCD2 activity can protect β-cells from elevated saturated FAs, as occurs in prediabetic states. Overt type 2 diabetes is associated with diminished islet expression of SCD and Elovl6, and this can disrupt desaturation of saturated FAs to MUFAs, rendering β-cells more susceptible to saturated FA-induced ER stress and apoptosis.     

SDF-1α inhibits hypoxia and serum deprivation-induced apoptosis in mesenchymal stem cells through PI3K/Akt and ERK1/2 signaling pathways

Bone marrow-derived mesenchymal stem cells (BMSCs) have been demonstrated to be a promising cell sources for cardiac regeneration. Poor survival rate of transplanted BMSCs in infarcted myocardium attenuated its clinical application. It’s reported that stromal-derived factor-1 (SDF-1) could protect progenitor cells including endothelial progenitor cells and embryonic stem cells from apoptosis. But little is known whether SDF-1α protein has the same protective effects on BMSCs under conditions of hypoxia and serum deprivation (hypoxia/SD). In present study, we verified that SDF-1α (0.50–2.0 μg/ml) inhibited hypoxia/SD induced apoptosis of BMSCs through mitochondrial pathway. After administration of SDF-1α, the loss of mitochondrial membrane potential and cytochrome c released from mitochondria to cytosol were significantly inhibited, and caspase 3 activity also declined. Furthermore, the effect of SDF-1α on mitochondrial pathway was neutralized by using PI3K inhibitor (Wortmannin) and ERK1/2 inhibitor (U0126). Our observations suggested that SDF-1α inhibits hypoxia/SD induced BMSCs apoptosis through PI3K/Akt and ERK1/2 signaling pathways. These data also imply that the anti-apoptotic effect mediated by SDF-1α may enhance cell survival after cell transplantation.     

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.     

Blockade of sphingosine 1-phosphate receptor 2 signaling attenuates streptozotocin-induced apoptosis of pancreatic β-cells

Sphingosine 1-phosphate (S1P) is a potent sphingolipid mediator that acts through five cognate G protein-coupled receptors (S1P₁-S1P₅) and regulates many critical biological processes. Recent studies indicated that S1P at nanomolar concentrations significantly reduces cytokine-induced apoptosis of pancreatic β-cells in which genes for S1P₁-S1P₄ are co-expressed. However, the S1P receptor subtype(s) involved in this effect remains to be clarified. In this study, we investigated the potential role of S1P₂ in streptozotocin (STZ)-induced apoptosis of pancreatic β-cells and progression of diabetes. S1P₂-deficient (S1P₂^-/-) mice displayed a greater survive ability, lower blood glucose levels, and smaller numbers of TUNEL-positive apoptotic β-cells to administration of a high dose of STZ than wild-type (WT) mice. S1P₂^-/- mice showed higher insulin/glucose ratios (an index of relative insulin deficiency) and larger insulin-positive islet areas to administration of a low dose of STZ than WT mice. Moreover, administration of JTE-013, a S1P₂-specific antagonist, to WT mice ameliorated STZ-induced blood glucose elevation and reduced the incidence of diabetes. Our findings indicate that blockade of S1P₂ signaling attenuates STZ-induced apoptosis of pancreatic β-cells and decreases the incidence of diabetes.     

Prevention of TGF-β-induced apoptosis by interlukin-4 through Akt activation and p70S6K survival signaling pathways

In this study, we demonstrate that interleukin-4 (IL-4) protects human hepatocellular carcinoma (HCC) cell line Hep3B from apoptosis induced by transforming growth factor-β (TGF-β). Further investigation of IL-4-transduced signaling pathways revealed that both insulin response substrate 1 and 2 (IRS-1/-2) and extracellular signal-regulated kinase (ERK) pathways were activated after IL-4 stimulation. The IRS-1/-2 activation was accompanied by the activation of phosphotidylinositol-3-kinase (PI3K), leading to Akt and p70 ribosomal protein S6 kinase (p70S6K). Interestingly, a protein kinase C (PKC) inhibitor, Gö6976, inhibited the phosphorylation of Akt, suggesting that the Akt activation was PKC-dependent. Using specific inhibitors for PI3K or ERK, we demonstrated that the PI3K pathway, but not the ERK pathway, was required for protection. The constitutively active form of PI3K almost completely rescued TGF-β-induced apoptosis, further supporting the importance of the PI3K pathway in the protective effect of IL-4. Furthermore, a dominant negative Akt and/or Gö6976 only partially blocked the anti-apoptotic effect of IL-4. Similarly, rapamycin, which interrupted the activation of p70S6K, also only partially blocked the protective effect of IL-4. However, in the presence of both rapamycin and dominant negative Akt with or without Gö6976, IL-4 almost completely lost the anti-apoptotic effect, suggesting that both Akt and p70S6K pathways were required for the protective effect of IL-4 against TGF-β-induced apoptosis.     

Pyrroloquinoline quinine protects HK-2 cells against high glucose-induced oxidative stress and apoptosis through Sirt3 and PI3K/Akt/FoxO3a signaling pathway

High glucose(HG)-induced oxidative stress and apoptosis in renal tubular epithelial cells play an important role in the pathogenesis of diabetic nephropathy. Pyrroloquinoline quinine (PQQ), a new B vitamin, has been demonstrated to be important in antioxidant and anti-apoptotic effects. However, its effect on HK-2?cells and the potential mechanism are rarely investigated. In this study, we investigated that PPQ had protective effects against HG-induced oxidative stress damage and apoptosis in vitro model of diabetic nephropathy. PPQ at 10, 100, 500, 1000 and 10000?nM could protect HK-2?cell from HG-induced inhibition. The protective effects of PQQ were associated with increasing the level of antioxidants(SOD2, CAT), inhibition of reactive oxygen species(ROS) production, and dependent modulation of Bcl-2 family proteins. PPQ significantly upregulated the protein and mRNA expression of Sirtuin3(Sirt3) in HG-induced HK-2?cells. PPQ also reduced apoptosis in HG-induced HK-2?cells by the PI3K/Akt/FoxO3a signal pathway. As down-regulated sirt3 or inhibitory the activity of PI3K/Akt/FoxO3a pathway, the protective effects of PPQ were weakened. In conclusion, our data suggest that PPQ achieves the protective effects through PI3K/Akt/FoxO3a pathway and dependent modulation of Sirt3.     

Activation of PPARδ up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic β-cells

Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor δ (PPARδ) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic β-cells. After HIT-T15 cells (a β-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPARδ), we found that administration of GW increased the expression of PPARδ mRNA. GW-induced activation of PPARδ up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4 (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPARδ plays an important role in protecting pancreatic β-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.     

Nodal induces apoptosis through activation of the ALK7 signaling pathway in pancreatic INS-1 β-cells

We demonstrated previously that the activation of ALK7 (activin receptor-like kinase-7), a member of the type I receptor serine/threonine kinases of the TGF-β superfamily, resulted in increased apoptosis and reduced proliferation through suppression of Akt signaling and the activation of Smad2-dependent signaling pathway in pancreatic β-cells. Here, we show that Nodal activates ALK7 signaling and regulates β-cell apoptosis. We detected Nodal expression in the clonal β-cell lines and rodent islet β-cells. Induction of β-cell apoptosis by treatment with high glucose, palmitate, or cytokines significantly increased Nodal expression in clonal INS-1 β-cells and isolated rat islets. The stimuli induced upregulation of Nodal expression levels were associated with elevation of ALK7 protein and enhanced phosphorylated Smad3 protein. Nodal treatment or overexpression of Nodal dose- or time-dependently increased active caspase-3 levels in INS-1 cells. Nodal-induced apoptosis was associated with decreased Akt phosphorylation and reduced expression level of X-linked inhibitor of apoptosis (XIAP). Remarkably, overexpression of XIAP or constitutively active Akt, or ablation of Smad2/3 activity partially blocked Nodal-induced apoptosis. Furthermore, siRNA-mediated ALK7 knockdown significantly attenuated Nodal-induced apoptosis of INS-1 cells. We suggest that Nodal-induced apoptosis in β-cells is mediated through ALK7 signaling involving the activation of Smad2/3-caspase-3 and the suppression of Akt and XIAP pathways and that Nodal may exert its biological effects on the modulation of β-cell survival and β-cell mass in an autocrine fashion.     

Integrin β1 mediates vaccinia virus entry through activation of PI3K/Akt signaling

Vaccinia virus has a broad range of infectivity in many cell lines and animals. Although it is known that the vaccinia mature virus binds to cell surface glycosaminoglycans and extracellular matrix proteins, whether additional cellular receptors are required for virus entry remains unclear. Our previous studies showed that the vaccinia mature virus enters through lipid rafts, suggesting the involvement of raft-associated cellular proteins. Here we demonstrate that one lipid raft-associated protein, integrin β1, is important for vaccinia mature virus entry into HeLa cells. Vaccinia virus associates with integrin β1 in lipid rafts on the cell surface, and the knockdown of integrin β1 in HeLa cells reduces vaccinia mature virus entry. Additionally, vaccinia mature virus infection is reduced in a mouse cell line, GD25, that is deficient in integrin β1 expression. Vaccinia mature virus infection triggers the activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling, and the treatment of cells with inhibitors to block P13K activation reduces virus entry in an integrin β1-dependent manner, suggesting that integrin β1-mediates PI3K/Akt activation induced by vaccinia virus and that this signaling pathway is essential for virus endocytosis. The inhibition of integrin β1-mediated cell adhesion results in a reduction of vaccinia virus entry and the disruption of focal adhesion and PI3K/Akt activation. In summary, our results show that the binding of vaccinia mature virus to cells mimics the outside-in activation process of integrin functions to facilitate vaccinia virus entry into HeLa cells.     

ATF3 represses PDX-1 expression in pancreatic β-cells

The downregulation of PDX-1 expression plays an important role in development of type 2 diabetes. However, the negative regulator of PDX-1 expression is not well known. In this study, we analyzed the mouse PDX-1 promoter to characterize the effects of ATF3 on PDX-1 expression in pancreatic β-cells. Both thapsigargin treatment, an inducer of ER stress, and ATF3 expression decreased PDX-1 expression in pancreatic β-cells, MIN6N8. Furthermore, they also repressed the activity of −4.5 Kb promoter of mouse PDX-1 gene. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of 0.9 Kb PDX-1 promoter, whereas it did not affect the activity of 0.7 Kb PDX-1 promoter, suggesting that ATF3 responsive element is located between the −903 and −702. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds directly to the promoter region spanning from −759 to −738. Moreover, mutation of the putative ATF/CRE site between −752 and −745 abrogated ATF3-mediated transrepression of the PDX-1 promoter. PDX-1 was decreased in MIN6N8 cells treated with high glucose or high palmitate, whereas ATF3 was increased, indicating that ATF3 plays a role in hyperglycemia or hyperlipidemia-mediated downregulation of PDX-1 expression. Collectively, these results demonstrate that ATF3 represses PDX-1 expression via binding to an ATF3-responsive element in its promoter, which plays an important role in suppression of pancreatic β-cells function.     

β-Catenin mediates cyclic strain-stimulated cardiomyogenesis in mouse embryonic stem cells through ROS-dependent and integrin-mediated PI3K/Akt pathways

Wnt/β-catenin signaling regulates various cellular events involved in the proliferation and differentiation and these events are affected sensitively by applying to mechanical stimuli. However, the mechanisms by which mechanical force stimulates cardiomyogenesis are not extensively explored. In this study we investigated the cellular mechanisms by which β-catenin signaling regulates cardiac differentiation of strain-subjected embryonic stem (ES) cells. The application of cells to cyclic strain increased beating cardiomyocyte foci with the attendant increases of Cx 43 and Nkx 2.5 proteins. Anti-oxidants such as vitamin C or N-acetyl cysteine (NAC) blocked the strain-mediated increases of Cx 43, Nkx 2.5, and α5/β1 integrins. These anti-oxidants also suppressed the activation of phosphoinositide 3-kinase (PI3K) and Akt in cyclic strain-subjected cells. Western blot analysis revealed that PI3K is a critical downstream effector of β1 integrin signaling and mediates Cx 43 and Nkx 2.5 expression in cyclic strain-applied ES cells. Cyclic strain increased the expression of β-catenin and stimulated its nuclear translocation from the cytosol, which was prevented by anti-oxidant treatment. In addition, the application to cyclic strain increased mRNA expression of β-catenin target genes, Axin2 and c-myc, as well as the phosphorylation of glycogen synthase kinase-3β. Furthermore, the blockage of β-catenin by its specific siRNA transfection diminished the cellular levels of Cx 43 and Nkx 2.5 proteins and the number of beating cardiomyocyte foci. Collectively, these results suggest that β-catenin-mediated signaling is required for cyclic strain-stimulated cardiomyogenesis through ROS-dependent and integrin-mediated PI3K-Akt signaling cascades.     

Control by Ca2+ of mitochondrial structure and function in pancreatic β-cells

Mitochondria play a central role in glucose metabolism and the stimulation of insulin secretion from pancreatic β-cells. In this review, we discuss firstly the regulation and roles of mitochondrial Ca²⁺ transport in glucose-regulated insulin secretion, and the molecular machinery involved. Next, we discuss the evidence that mitochondrial dysfunction in β-cells is associated with type 2 diabetes, from a genetic, functional and structural point of view, and then the possibility that these changes may in part be mediated by dysregulation of cytosolic Ca²⁺. Finally, we review the importance of preserved mitochondrial structure and dynamics for mitochondrial gene expression and their possible relevance to the pathogenesis of type 2 diabetes.     

The cardioprotective effect of dihydromyricetin prevents ischemia–reperfusion-induced apoptosis in vivo and in vitro via the PI3K/Akt and HIF-1α signaling pathways

Reperfusion therapy is widely used to treat acute myocardial infarction (AMI). However, further injury to the heart induced by rapidly initiating reperfusion is often encountered in clinical practice. A lack of pharmacological strategies in clinics limits the prognosis of patients with myocardial ischemia-reperfusion injury (MIRI). Dihydromyricetin (DMY) is one of the most abundant components in vine tea, commonly known as the tender stems and leaves of Ampelopsis grossedentata. The aim of this study was to evaluate the cardioprotection of DMY against myocardial ischemia-reperfusion (I/R) injury and to further investigate the underlying mechanism. An I/R injury was induced by left anterior descending coronary artery occlusion in adult male rats in vivo and a hypoxia–reoxygenation (H/R) injury in H9c2 cardiomyocytes in vitro. We found that DMY pretreatment provided significant protection against I/R-induced injury, including enhanced antioxidant capacity and inhibited apoptosis in vivo and in vitro. This effect correlated with the activation of the PI3K/Akt and HIF-1α signaling pathways. Conversely, blocking Akt activation with the PI3K inhibitor LY294002 effectively suppressed the protective effects of DMY against I/R-induced injury. In addition, the PI3K inhibitor partially blocked the effects of DMY on the upregulation of Bcl-2, Bcl-xl, procaspase-3, -8, and -9 protein expression and the downregulation of HIF-1α, Bnip3, Bax, Cyt-c, cleaved caspase-3, -8, and -9 protein expression. Collectively, these results showed that DMY decreased the apoptosis and necrosis by I/R treatment, and PI3K/Akt and HIF-1α plays a crucial role in protection during this process. These observations indicate that DMY has the potential to exert cardioprotective effects against I/R injury and the results might be important for the clinical efficacy of AMI treatment.     

Celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-Jun N-terminal kinase and suppression of PI3 K/Akt signaling pathways

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.     

Pyrrolidine dithiocarbamate protects pancreatic β-cells from oxidative damage through regulation of FoxO1 activity in type 2 diabetes rats

Pyrrolidine dithiocarbamate （PDTC） can lower the bloot glucose level and improve the insulin sensitivity in diabeti, rats. However, the mechanisms underlying this effect o PDTC treatment in diabetic rats remained uncertain, h this study, we evaluated the mechanisms by which PDT（ conferred protection against oxidative damage to pancreat ic islet β-cells in rats with experimental type 2 diabete mellitus （DM）. DM in the rats was elicited by long-tern high-fat diet accompanied with a single intraperitonea （i.p.） injection of a low dose of streptozotocin. After a 7-da1 administration of PDTC （50 mg/kg/day i.p.）, blood glucos levels were measured and pancreatic tissues were collecte / for the determination of various biochemical and enzyma 1 ic activities using immunohistochemistry, immunofluoresI cence, and western blot techniques. The percentage o 1 apoptotic pancreatic islet β-cells was detected by flow cyto metry. The results showed that diabetic rats had elevate blood glucose levels and insulin resistance, accompanieq with an increase in malondialdehyde content, nitrotyrosin production, and inducible nitric oxide synthase expression A decrease in superoxide dismutase and glutathione pero idase activities was also observed in DM rats, culminatin with elevated β-cell apoptosis. PDTC treatment significantl reduced the oxidative damage and the β-cell apoptosi and also increased the insulin production through down-reg lating FoxO1 acetylation and up-regulating nuclear PDX- level. These data suggested that PDTC can protect islet βcells from oxidative damage and improve insulin productio through regulation of PDX-1 and FoxO1 in a DM rat model.