Dynamin-related protein 1 mediates high glucose induced pancreatic beta cell apoptosis

The pancreatic beta cell dysfunction is critical cycle in the pathogenesis of diabetes. Hyperglycemia is one of factors that induce pancreatic beta cell dysfunction, but the underlying mechanisms have not been well elucidated. In this study, we reported that a mitochondrial fission modulator, Dynamin-related protein 1 (Drp-1), plays an important role in high glucose induced beta cell apoptosis. Drp-1 expressed in islet beta cells was increased drastically under hyperglycemia conditions. Induction of Drp-1 expression significantly promoted high glucose induced apoptosis in Drp-1WT (Drp-1 wild type) inducible beta cell line, but not in Drp-1K38A (a dominant negative mutant of Drp1) inducible beta cell line. We further demonstrated that mitochondrial fission, cytochrome C release, mitochondrial membrane potential decreased, caspase-3 activation and generation of reactive oxygen species were enhanced by induction of Drp-1WT, but prevented by Drp-1K38A in pancreatic beta cells under high glucose condition. These results indicated that Drp-1 mediates high glucose induced pancreatic beta cell apoptosis.     

TRB3 Is Involved in Free Fatty Acid-Induced INS-1-Derived Cell Apoptosis via the Protein Kinase C δ Pathway

Chronic exposure to free fatty acids (FFAs) may induce β cell apoptosis in type 2 diabetes. However, the precise mechanism by which FFAs trigger β cell apoptosis is still unclear. Tribbles homolog 3 (TRB3) is a pseudokinase inhibiting Akt, a key mediator of insulin signaling, and contributes to insulin resistance in insulin target tissues. This paper outlined the role of TRB3 in FFAs-induced INS-1 β cell apoptosis. TRB3 was promptly induced in INS-1 cells after stimulation by FFAs, and this was accompanied by enhanced INS-1 cell apoptosis. The overexpression of TRB3 led to exacerbated apoptosis triggered by FFAs in INS-1-derived cell line and the subrenal capsular transplantation animal model. In contrast, cell apoptosis induced by FFAs was attenuated when TRB3 was knocked down. Moreover, we observed that activation and nuclear accumulation of protein kinase C (PKC) δ was enhanced by upregulation of TRB3. Preventing PKCδ nuclear translocation and PKCδ selective antagonist both significantly lessened the pro-apoptotic effect. These findings suggest that TRB3 was involved in lipoapoptosis of INS-1 β cell, and thus could be an attractive pharmacological target in the prevention and treatment of T2DM.     

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.     

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (ΔΨ(m)). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.     

A chemical chaperone 4-PBA ameliorates palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS)

Free fatty acids (FFAs) are believed to be a stimulus to elicit beta cell dysfunction. The present study was undertaken to determine whether endoplasmic reticulum (ER) stress was involved in palmitate-induced inhibition of glucose-stimulated insulin secretion (GSIS) and whether reduction of ER stress using a chemical chaperone restored the GSIS-inhibition. Treatment of INS-1 cells with 300 μM palmitate for 24 h elicited ER stress, showing increased levels of phospho-eIF2α, Bip and spliced XBP, and also induced GSIS-inhibition without reduction of cell viability. Replenishment with 4-phenyl butyric acid (4-PBA) as a chemical chaperone reduced the palmitate-induced-ER stress and significantly reversed the palmitate-induced GSIS-inhibition. Furthermore, 4-PBA ameliorated palmitate-induced GSIS-inhibition in primary rat islet cells. These data suggested that ER stress was involved in FFA-induced GSIS-inhibition and that the FFA-induced beta cell dysfunction could be ameliorated by treatment with a chemical chaperone.     

Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.     

H2S, endoplasmic reticulum stress, and apoptosis of insulin-secreting beta cells

Cystathionine gamma-lyase (CSE) is a key enzyme in the trans-sulfuration pathway, which uses L-cysteine to produce hydrogen sulfide (H2S). Functional changes of pancreatic beta cells induced by endogenous H2S have been reported, but the effect of the CSE/H2S system on pancreatic beta cell survival has not been known. In this study, we demonstrate that H2Sat physiologically relevant concentrations induced apoptosis of INS-1E cells, an insulin-secreting beta cell line. Transfection of INS-1E cells with a recombinant defective adenovirus containing the CSE gene (Ad-CSE) resulted in a significant increase in CSE expression and H2S production. Ad-CSE transfection also stimulated apoptosis. The other two end products of CSE-catalyzed enzymatic reaction, ammonium and pyruvate, had no effects on INS-1E cell apoptosis, indicating that overexpression of CSE may stimulate INS-1E cell apoptosis via increased endogenous production of H2S. Both exogenous H2S (100 microM) and Ad-CSE transfection inhibited ERK1/2 but activated p38 MAPK. Interestingly, BiP and CHOP, two indicators of endoplasmic reticulum (ER) stress, were up-regulated in H2S-and CSE-mediated apoptosis in INS-1E cells. After suppressing CHOP mRNA expression, H2S-induced apoptosis of INS-1E cells was significantly decreased. Inhibition of p38 MAPK, but not of ERK1/2, inhibited the expression of BiP and CHOP and decreased H2S-stimulated apoptosis, suggesting that p38 MAPK activation functions upstream of ER stress to initiate H2S-induced apoptosis. It is concluded that H2S induces apoptosis of insulin-secreting beta cells by enhancing ER stress via p38 MAPK activation. Our findings may help unmask a novel role of CSE/H2S system in regulating pancreatic functions under physiological condition and in diabetes.     

Free Fatty Acid Induces Endoplasmic Reticulum Stress and Apoptosis of β-cells by Ca2+/Calpain-2 Pathways

Dysfunction of β-cells is a major characteristic in the pathogenesis of type 2 diabetes mellitus (T2DM). The combination of obesity and T2DM is associated with elevated plasma free fatty acids (FFAs). However, molecular mechanisms linking FFAs to β-cell dysfunction remain poorly understood. In the present study, we identified that the major endoplasmic reticulum stress (ERS) marker, Grp78 and ERS-induced apoptotic factor, CHOP, were time-dependently increased by exposure of β-TC3 cells to FFA. The expression of ATF6 and the phosphorylation levels of PERK and IRE1, which trigger ERS signaling, markedly increased after FFA treatments. FFA treatments increased cell apoptosis by inducing ERS in β-TC3 cells. We also found that FFA-induced ERS was mediated by the store-operated Ca²⁺ entry through promoting the association of STIM1 and Orai1. Moreover, calpain-2 was required for FFA-induced expression of CHOP and activation of caspase-12 and caspase-3, thus promoting cell apoptosis in β-TC3 cells. Together, these results reveal pivotal roles for Ca²⁺/calpain-2 pathways in modulating FFA-induced β-TC3 cell ERS and apoptosis.     

Effects of human serum albumin complexed with free fatty acids on cell viability and insulin secretion in the hamster pancreatic β-cell line HIT-T15

AimsThe effects of human serum albumin (HSA) complexed with various free fatty acids (FFAs) on ß-cells have not been studied in detail. In this study, we examined the effects of HSA and its mutants on FFA-induced cell viability changes and insulin secretion from the hamster pancreatic insulinoma cell line, HIT-TI5.Main methodsCells were exposed to different FFAs in the presence of HSA or its mutants and/or bovine serum albumin (BSA) for 24 h. Cell viability, apoptosis, insulin secretion, and unbound FFA (FFA_u) levels were determined.Key findingsIn the presence of 0.1 mM HSA, palmitate and stearate induced significant cell death at 0.1 mM or higher, whereas myristate, palmitoleate, oleate, elaidate, linoleate, linoelaidate, and conjugated linoleate showed minimal changes on cell viability. Furthermore, oleate and linoleate were clearly cytoprotective against palmitate-induced cell death. The apoptosis inhibitors, cyclosporin A (csA) and the caspase inhibitor ZVAD-FMK, did not completely prevent FFA-induced cell death, although ZVAD-FMK blocked apoptosis with no differences in the presence of either HSA or BSA. In addition, insulin secretion from the cells was significantly reduced in the presence of HSA/oleate complexes. We also found differential effects of HSA mutants complexed with FFAs on cell viability.SignificanceIn summary, our results showed that saturated FFAs induced more cell death than unsaturated FFAs. Furthermore, modified HSA/FFA interactions caused by mutations of key amino acids involved in the binding of FFA to HSA resulted in changes in cell viability, suggesting a possible role of HSA polymorphism on FFA-induced changes in cellular functions.     

Calcium-independent phospholipase A2 (iPLA2 beta)-mediated ceramide generation plays a key role in the cross-talk between the endoplasmic reticulum (ER) and mitochondria during ER stress-induced insulin-secreting cell apoptosis

Endoplasmic reticulum (ER) stress induces INS-1 cell apoptosis by a pathway involving Ca(2+)-independent phospholipase A(2) (iPLA(2)beta)-mediated ceramide generation, but the mechanism by which iPLA(2)beta and ceramides contribute to apoptosis is not well understood. We report here that both caspase-12 and caspase-3 are activated in INS-1 cells following induction of ER stress with thapsigargin, but only caspase-3 cleavage is amplified in iPLA(2)beta overexpressing INS-1 cells (OE), relative to empty vector-transfected cells, and is suppressed by iPLA(2)beta inhibition. ER stress also led to the release of cytochrome c and Smac and, unexpectedly, their accumulation in the cytosol is amplified in OE cells. These findings raise the likelihood that iPLA(2)beta participates in ER stress-induced apoptosis by activating the intrinsic apoptotic pathway. Consistent with this possibility, we find that ER stress promotes iPLA(2)beta accumulation in the mitochondria, opening of mitochondrial permeability transition pore, and loss in mitochondrial membrane potential (Delta Psi) in INS-1 cells and that these changes are amplified in OE cells. ER stress also led to greater ceramide generation in ER and mitochondria fractions of OE cells. Exposure to ceramide alone induces loss in Delta Psi and apoptosis and these are suppressed by forskolin. ER stress-induced mitochondrial dysfunction and apoptosis are also inhibited by forskolin, as well as by inactivation of iPLA(2)beta or NSMase, suggesting that iPLA(2)beta-mediated generation of ceramides via sphingomyelin hydrolysis during ER stress affect the mitochondria. In support, inhibition of iPLA(2)beta or NSMase prevents cytochrome c release. Collectively, our findings indicate that the iPLA(2)beta-ceramide axis plays a critical role in activating the mitochondrial apoptotic pathway in insulin-secreting cells during ER stress.     

Apoptosis of insulin-secreting cells induced by endoplasmic reticulum stress is amplified by overexpression of group VIA calcium-independent phospholipase A2 (iPLA2 beta) and suppressed by inhibition of iPLA2 beta

The death of insulin-secreting beta-cells that causes type I diabetes mellitus (DM) occurs in part by apoptosis, and apoptosis also contributes to progressive beta-cell dysfunction in type II DM. Recent reports indicate that ER stress-induced apoptosis contributes to beta-cell loss in diabetes. Agents that deplete ER calcium levels induce beta-cell apoptosis by a process that is independent of increases in [Ca(2+)](i). Here we report that the SERCA inhibitor thapsigargin induces apoptosis in INS-1 insulinoma cells and that this is inhibited by a bromoenol lactone (BEL) inhibitor of group VIA calcium-independent phospholipase A(2) (iPLA(2)beta). Overexpression of iPLA(2)beta amplifies thapsigargin-induced apoptosis of INS-1 cells, and this is also suppressed by BEL. The magnitude of thapsigargin-induced INS-1 cell apoptosis correlates with the level of iPLA(2)beta expression in various cell lines, and apoptosis is associated with stimulation of iPLA(2)beta activity, perinuclear accumulation of iPLA(2)beta protein and activity, and caspase-3-catalyzed cleavage of full-length 84 kDa iPLA(2)beta to a 62 kDa product that associates with nuclei. Thapsigargin also induces ceramide accumulation in INS-1 cells, and this response is amplified in cells that overexpress iPLA(2)beta. These findings indicate that iPLA(2)beta participates in ER stress-induced apoptosis, a pathway that promotes beta-cell death in diabetes.     

Evidence against calcium as a mediator of mitochondrial dysfunction during apoptosis induced by arachidonic acid and other free fatty acids

Apoptosis is often accompanied by activation of phospholipase A(2), causing release of free fatty acids (FFAs), which in turn are thought to contribute to the loss of mitochondrial transmembrane potential (Deltapsi(m)). In these experiments, we asked whether calcium plays a role as an intermediate in this process. A total of 14 FFAs were compared for their ability to cause loss of Deltapsi(m) and for their ability to affect levels of intracellular calcium. Among the FFAs, unsaturated FFAs tended to induce apoptosis while saturated FFAs did not. Arachidonic acid (AA) was most damaging, causing loss of Deltapsi(m) and cell death in 8-10 h while linoleic acid, gamma-linolenic acid, and docosapentaenoic also strongly induced apoptosis. Effects of the FFAs on levels of intracellular calcium were very different. Many caused strong calcium responses; however, the ability to induce a strong calcium response was not predictive of ability to induce apoptosis, and overall, we did not find a correlation between apoptosis and calcium induction. Also, verapamil and TMB-8 were able to block the calcium response, but these inhibitors did not prevent loss of Deltapsi(m), indicating that the calcium response is not necessary for FFA-induced loss of Deltapsi(m). In contrast, we found that cyclosporine A could inhibit the AA-induced loss of Deltapsi(m) with both whole cells and isolated mitochondria, confirming that the antimitochondrial effects of FFA can stem from direct effects on the mitochondrial permeability transition pore. Finally, we show that the strong apoptosis-inducing activity of AA may stem from its ability to selectively induce its own release.     

The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase

Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.     

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways

In the present study, we examined the effects of free fatty acids (FFAs) on insulin sensitivity and signaling cascades in the C2C12 skeletal muscle cell culture system. Our data clearly manifested that the inhibitory effects of PKC on insulin signaling may at least in part be explained by the serine/threonine phosphorylation of IRS-1. Both oleate and palmitate treatment were able to increase the Serine³⁰⁷ phosphorylation of IRS-1. IRS-1 Serine³⁰⁷ phosphorylation is inducible which causes the inhibition of IRS-1 tyrosine phosphorylation by either IκB-kinase (IKK) or c-jun N-terminal kinase (JNK) as seen in our proteomic kinases screen. Furthermore, our proteomic data have also manifested that the two FFAs activate the IKKα/β, the stress kinases S6 kinase p70 (p70SK), stress-activated protein kinase (SAPK), JNK, as well as p38 MAP kinase (p38MAPK). On the other hand, the antioxidant, Taurine at 10 mM concentrations was capable of reversing the oleate-induced insulin resistance in myocytes as manifested from the glucose uptake data. Our current data point out the importance of FFA-induced insulin resistance via multiple signaling mechanisms.     

A mitophagic response to iron overload-induced oxidative damage associated with the PINK1/Parkin pathway in pancreatic beta cells

BackgroundIron overload can result in a disorder in glucose metabolism. However, the underlining mechanism through which iron overload induces beta cell death remains unknown.MethodsAccording to the concentration of ferric ammonium citrate (FAC) and N-acetylcysteine, INS-1 cells were randomly divided into four groups: normal control (FAC 0 μM) group, FAC 80 μM group, FAC 160 μM group, FAC 160μM + NAC group. Cell proliferation was assessed by Cell Counting Kit-8. Reactive oxygen species (ROS) level was further evaluated using flow cytometer with a fluorescent probe. The mitochondrial membrane potential was detected by JC-1 kit, and transmission electron microscopy was used to observe the mitochondrial changes. The related protein expressions were detected by western bolt to evaluate mitophagy status.ResultsIt was shown that FAC treatment decreased INS-1 cell viability in vitro, resulted in a decline in mitochondrial membrane potential, increased oxidative stress level and suppressed mitophagy. Furthermore, these effects could be alleviated by the ROS scavenger.ConclusionsWe proved that increased iron overload primarily increased oxidative stress and further suppressed mitophagy via PTEN-induced putative kinase 1/Parkin pathway, resulting in cytotoxicity in INS-1 cells.     

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.