Free Fatty Acid-induced Apoptosis Research Articles

Yap1 plays a protective role in suppressing free fatty acid-induced apoptosis and promoting beta-cell survival

Mammalian pancreatic β-cells play a pivotal role in development and glucose homeostasis through the production and secretion of insulin. Functional failure or decrease in β-cell number leads to type 2 diabetes (T2D). Despite the physiological importance of β-cells, the viability of β-cells is often challenged mainly due to its poor ability to adapt to their changing microenvironment. One of the factors that negatively affect β-cell viability is high concentration of free fatty acids (FFAs) such as palmitate. In this work, we demonstrated that Yes-associated protein (Yap1) is activated when β-cells are treated with palmitate. Our loss- and gain-of-function analyses using rodent insulinoma cell lines revealed that Yap1 suppresses palmitate-induced apoptosis in β-cells without regulating their proliferation. We also found that upon palmitate treatment, re-arrangement of F-actin mediates Yap1 activation. Palmitate treatment increases expression of one of the Yap1 target genes, connective tissue growth factor (CTGF). Our gain-of-function analysis with CTGF suggests CTGF may be the downstream factor of Yap1 in the protective mechanism against FFA-induced apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-016-0258-5) contains supplementary material, which is available to authorized users.

Activation of AMP-activated protein kinase by metformin protects human coronary artery endothelial cells against diabetic lipoapoptosis.

BackgroundThe prevalence of type 2 diabetes (T2D) among adults worldwide is rapidly increasing, and in patients with diabetes the major cause of death is macrovascular disease. Endothelial cells play an important role in maintaining vascular homeostasis. Free fatty acids, which are elevated in T2D, have previously been shown to induce endothelial dysfunction and apoptosis of endothelial cells, which is considered as an important and early factor in the onset of atherosclerosis and cardiovascular disease. Metformin, which is used as first line treatment of T2D patients, is believed to exert its pharmacological effects through activation of AMP-activated protein kinase, which has emerged as a new potential target in reversing endothelial dysfunction.MethodsHere we studied the protective effect of metformin against free fatty acid-induced apoptosis of human coronary artery endothelial cells (HCAECs) by assessing DNA fragmentation and cleaved caspase 3 levels. We also attempted to elucidate the underlying mechanisms by investigating the involvement of AMP-activated protein kinase, p38 MAPK and eNOS. Generation of reactive oxygen species by free fatty acid exposure was also examined.ResultsOur results suggest that metformin protects HCAECs from lipoapoptosis, an effect that involves eNOS and p38 MAPK, downstream of AMPK signaling, but not as previously suggested through suppression of reactive oxygen species.ConclusionThe protective effect of metformin against free fatty acid induced apoptosis is potentially clinically relevant as metformin is first line treatment for patients with T2D, a patient group which is rapidly increasing and carries a high burden of cardiovascular disease.

Activation of AMP-activated protein kinase by metformin protects human coronary artery endothelial cells against diabetic lipoapoptosis

The prevalence of type 2 diabetes (T2D) among adults worldwide is rapidly increasing, and in patients with diabetes the major cause of death is macrovascular disease. Endothelial cells play an important role in maintaining vascular homeostasis. Free fatty acids, which are elevated in T2D, have previously been shown to induce endothelial dysfunction and apoptosis of endothelial cells, which is considered as an important and early factor in the onset of atherosclerosis and cardiovascular disease. Metformin, which is used as first line treatment of T2D patients, is believed to exert its pharmacological effects through activation of AMP-activated protein kinase, which has emerged as a new potential target in reversing endothelial dysfunction. Here we studied the protective effect of metformin against free fatty acid-induced apoptosis of human coronary artery endothelial cells (HCAECs) by assessing DNA fragmentation and cleaved caspase 3 levels. We also attempted to elucidate the underlying mechanisms by investigating the involvement of AMP-activated protein kinase, p38 MAPK and eNOS. Generation of reactive oxygen species by free fatty acid exposure was also examined. Our results suggest that metformin protects HCAECs from lipoapoptosis, an effect that involves eNOS and p38 MAPK, downstream of AMPK signaling, but not as previously suggested through suppression of reactive oxygen species. The protective effect of metformin against free fatty acid induced apoptosis is potentially clinically relevant as metformin is first line treatment for patients with T2D, a patient group which is rapidly increasing and carries a high burden of cardiovascular disease.

JNK inhibitor SP600125 suppresses free fatty acid-induced apoptosis of hepatic stellate cells

JNK inhibitor SP600125 suppresses free fatty acid-induced apoptosis of hepatic stellate cells

Exendin-4 Protects Murine Pancreatic β-Cells from Free Fatty Acid-Induced Apoptosis Through PI-3K Signaling

Introduction. Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by the L-cells of the distal intestine that has proliferative and anti-apoptotic actions on the β-cell. Methods. In this study, exendin-4, a long-acting GLP-1 receptor agonist, was studied as a novel agent to suppress apoptosis in pancreatic β-cells and to protect against free fatty acid (FFA)-induced cytotoxicity. Results. Exendin-4 significantly reduced the percentage of cells that underwent apoptosis when β-cells were exposed to FFA. Exendin-4 increased the levels of P-Akt and Bcl-2 proteins in FFA-induced β-cells, and this effect was blocked by Wortmannin. Conclusions. These results suggest that phosphoinositide-3 kinase signaling is involved in the modulation of β-cell apoptosis which is induced by exendin-4. Taken together, our findings provide a new mechanism for the modulation of exendin-4 in the pathological processes underlying FFA-induced diabetes mellitus.

Vaspin protects vascular endothelial cells against free fatty acid-induced apoptosis through a phosphatidylinositol 3-kinase/Akt pathway

Vaspin, an adipocytokine recently identified in a rat model of type 2 diabetes, has been suggested to have an insulin-sensitizing effect. However, the exact mechanism underlying this action has not been fully elucidated. Furthermore, the specific function of vaspin is largely unknown, especially in vascular cells. We examined whether vaspin affects the insulin-signaling pathway in cultured endothelial cells and is capable of preventing free fatty acid (FFA)-induced apoptosis in endothelial cells through its insulin sensitizing effect, specifically, through its stimulatory effect on PI3-kinase/Akt signaling pathways. Vaspin significantly increased Akt phosphorylation and prevented the impairment of Akt phosphorylation by linoleic acid (LA) in insulin-stimulated endothelial cells, which effects were abolished by pretreatment with the PI3-kinase inhibitor, Wortmannin. Moreover, pretreatment with vaspin prevented LA-induced apoptosis in insulin-stimulated endothelial cells; this anti-apoptotic effect of vaspin was also eliminated by pretreatment with Wortmannin. The present study indicates that vaspin protects vascular endothelial cells from FFA-induced apoptosis through upregulation of the PI3-kinase/Akt signaling pathway. Our study is the first to demonstrate that vascular cells can be targets of vaspin. Our results further suggest that vaspin could have beneficial effects on the atherosclerosis.

The Role of Uncoupling Protein 2 in the Apoptosis Induced by Free Fatty Acid in Rat Cardiomyocytes

The apoptotic loss of cardiomyocytes contributes to numerous cardiovascular disorders. Evidence suggests that free fatty acids induce cellular apoptosis, and recent studies have shown that free fatty acids dramatically elevate mRNA levels of uncoupling protein 2 (UCP2) in some cell lines. In this study, we investigated the possibility that free fatty acids induce the expression of UCP2 through the peroxisome proliferator-activated receptor pathway, thereby increasing cell apoptosis in adult rat cardiomyocytes. Primary cultured adult rat cardiomyocytes exposed to free fatty acids exhibited a dose-dependent increase in apoptosis. Quantitative real-time reverse transcription-polymerase chain reaction and Western blotting showed significant increases in the level of UCP2 expression at 6, 12, and at 24 hours after treatment of adult rat cardiomyocytes with free fatty acids. Expression of UCP2 was suppressed with RNA interference, and knockdown of UCP2 attenuated free fatty acid-induced apoptosis in the cardiomyocytes. In summary, free fatty acids induced UCP2 expression through peroxisome proliferator-activated receptor alpha in adult rat cardiomyocytes.

Roles of Inhibitors of Poly(ADP-ribose) Polymerase in Protecting Rat RINm5F Cell Line against Free Fatty Acid-induced Apoptosis

Chronic exposure to high levels of free fatty acid (FFA) has adverse effects on the function of pancreatic beta-cell, with a consequent increase in the production of reactive oxygen species. Poly (ADP-Ribose) polymerase (PARP-1) overactivation leads to massive NAD(+) consumption and ATP depletion with induction of cellular necrosis under high reactive oxygen species. In the present study, we investigated whether inhibitors of poly(ADP-ribose) polymerase were capable of protecting beta-cells from death induced by extended exposure to FFA. RINm5F cell line was cultured in the presence of FFA (palmitate) in order to induce apoptosis, and then cells were treated with low-potency poly(ADP-ribose) polymerase inhibitor (3-aminobenzamide) or potent poly(ADP-ribose) polymerase inhibitor (PJ34). In order to explore whether poly(ADP-ribose) polymerase inhibitors could inhibit the apoptosis induced by FFA, expression of PARP-1 was measured by RT-qPCR and Western blot, while the apoptosis of RINm5F cells were analyzed by flow cytometry and Tdt-mediated dUTP Nick-End Labeling(TUNEL). Low-potency poly(ADP-ribose) polymerase inhibitor (3-aminobenzamide) significantly suppressed the impaired insulin secretion and FFA-induced apoptosis (P<0.01). However, potent poly(ADP-ribose) polymerase inhibitor (PJ34) had no significant effects on FFA-induced apoptosis (P>0.05). Moreover, low-potency inhibitors of PARP-1 increased PDX-1 expression down-regulated by FFA-treatment. These findings suggested that low-potency inhibitors of poly(ADP-ribose) polymerases could protect rat RINm5F cell line against free fatty acid-induced apoptosis, and it was through regulatory pathway of regulating PDX-1 expression.

Protection of INS-1 cells from free fatty acid-induced apoptosis by inhibiting the glycogen synthase kinase-3

To examine the role of glycogen synthase kinase 3 (GSK-3) in the apoptosis of pancreatic beta-cells to better understand the pathogenesis and to find new approach to the treatment of type 2 diabetes, apoptosis was induced by oleic acid (OA) in INS-1 cells and the activity of GSK-3 was inhibited by LiCl. The PI staining and flow cytometry were employed for the evaluation of apoptosis. The phosphorylation level of GSK-3 was detected by Western blotting. The results showed that OA at 0.4 mmol/L could cause conspicuous apoptosis of INS-1 cells and the activity of GSK-3 was significantly increased. After the treatment with 24 mmol/L of LiCl, a inhibitor of GSK-3, the OA-induced apoptosis of INS-1 cells was lessened and the phosphorylation of GSK-3 was increased remarkably. It is concluded that GSK-3 activation plays an important role in OA-induced apoptosis in pancreatic beta-cells and inhibition of the GSK-3 activity can effectively protect INS-1 cells from the OA-induced apoptosis. Our study provides a new experimental basis and target for the clinical treatment of type-2 diabetes.

Identification of a WD40 Repeat-Containing Isoform of PHIP as a Novel Regulator of β-Cell Growth and Survival

The pleckstrin homology domain-interacting protein (PHIP) was originally identified as a 902-amino-acid (aa) protein that regulates insulin receptor-stimulated GLUT4 translocation in skeletal-muscle cells. Immunoblotting and immunohistological analyses of pancreatic beta-cells reveal prominent expression of a 206-kDa PHIP isoform restricted to the nucleus. Herein, we report the cloning of this larger, 1,821-aa isoform of PHIP (PHIP1), which represents a novel WD40 repeat-containing protein. We demonstrate that PHIP1 overexpression stimulates insulin-like growth factor 1-dependent and -independent proliferation of beta-cells, an event which correlates with transcriptional upregulation of the cyclin D2 promoter and the accumulation of cyclin D2 protein. RNA interference knockdown of PHIP1 in INS-1 cells abrogates insulin receptor substrate 2 (IRS2)-mediated DNA synthesis, providing for a specific role for PHIP1 in the enhancement of IRS2-dependent signaling responses leading to beta-cell growth. Finally, we provide evidence that PHIP1 overexpression blocks free fatty acid-induced apoptosis in INS-1 cells, which is accompanied by marked activation of phosphoprotein kinase B (PKB)/AKT and the concomitant inhibition of caspase-9 and caspase-3 cleavage. Our finding that the restorative effect of PHIP1 on beta-cell lipotoxicity can be attenuated by the overexpression of dominant-negative PKB suggests a key role for PKB in PHIP1-mediated cytoprotection. Taken together, these findings provide strong support for PHIP1 as a novel positive regulator of beta-cell function. We suggest that PHIP1 may be involved in the induction of long-term gene expression programs to promote beta-cell mitogenesis and survival.

Selective Inhibition of Eukaryotic Translation Initiation Factor 2α Dephosphorylation Potentiates Fatty Acid-induced Endoplasmic Reticulum Stress and Causes Pancreatic β-Cell Dysfunction and Apoptosis

Free fatty acids cause pancreatic beta-cell apoptosis and may contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum stress. Reductions in eukaryotic translation initiation factor (eIF) 2alpha phosphorylation trigger beta-cell failure and diabetes. Salubrinal selectively inhibits eIF2alpha dephosphorylation, protects other cells against endoplasmic reticulum stress-mediated apoptosis, and has been proposed as a beta-cell protector. Unexpectedly, salubrinal induced apoptosis in primary beta-cells, and it potentiated the deleterious effects of oleate and palmitate. Salubrinal induced a marked eIF2alpha phosphorylation and potentiated the inhibitory effects of free fatty acids on protein synthesis and insulin release. The synergistic activation of the PERK-eIF2alpha branch of the endoplasmic reticulum stress response, but not of the IRE1 and activating transcription factor-6 pathways, led to a marked induction of activating transcription factor-4 and the pro-apoptotic transcription factor CHOP. Our findings demonstrate that excessive eIF2alpha phosphorylation is poorly tolerated by beta-cells and exacerbates free fatty acid-induced apoptosis. This modifies the present paradigm regarding the beneficial role of eIF2alpha phosphorylation in beta-cells and must be taken into consideration when designing therapies to protect beta-cells in type 2 diabetes.

Hepatocyte growth factor protects rat RINm5F cell line against free fatty acid-induced apoptosis by counteracting oxidative stress

Type 2 diabetes is characterized by peripheral insulin resistance, pancreatic beta-cells dysfunction, and decreased beta-cell mass with increased rate of apoptosis. Chronic exposure to high levels of free fatty acids (FFAs) has detrimental effects on beta-cell function and survival. FFAs have adverse effects on mitochondrial function, with a consequent increase in the production of reactive oxygen species. Hepatocyte growth factor (HGF) plays a critical role in promoting beta-cell survival. In the present study, we investigated whether HGF was capable of protecting beta-cells from death induced by prolonged exposure to FFAs. RINm5F cell line was cultured in the presence of FFAs (oleate:palmitate 2:1) for 72 h in order to induce apoptosis. Simultaneous administration of HGF and FFAs significantly suppressed the impaired insulin secretion and FFA-induced apoptosis. Specifically, HGF exerted its protective effect by counteracting: (i) the overproduction of either hydrogen peroxide and superoxide anion, (ii) the reduction of intracellular gamma-glutamylcysteinylglycine level, and (iii) the depolarization of mitochondrial membrane, induced by prolonged FFAs exposure. These effects appear to be mediated by bcl-2 and phosphatidylinositol 3 kinase (PI3K)/Akt pathways. Indeed, HGF increased mRNA and protein expression of bcl-2 downregulated by FFAs-treatment; moreover, pre-treatment with the specific PI3-kinase inhibitor LY294002, significantly abolished the protective effect of HGF. In conclusion, in rat insulin-producing RINm5F cells, HGF exerts its prosurvival effect by counteracting the increased intracellular oxidative stress and, consequently, by inhibiting apoptosis induced by chronic exposure to FFAs.

The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits β-cell apoptosis in vitro

We here show that GLP-1 and the long-acting GLP-1 analogue, liraglutide, interfere with diabetes-associated apoptotic processes in the β-cell. Studies using primary neonatal rat islets showed that native GLP-1 and liraglutide inhibited both cytokine- and free fatty acid-induced apoptosis in a dose-dependent manner. The anti-apoptotic effect of liraglutide was mediated by the GLP-1 receptor as the specific GLP-1 receptor antagonist, exendin(9–39), blocked the effects. The adenylate cyclase activator, forskolin, had an anti-apoptotic effect similar to those of GLP-1 and liraglutide indicating that the effect was cAMP-mediated. Blocking the PI3 kinase pathway using wortmannin but not the MAP kinase pathways by PD98059 inhibited the effects of liraglutide. In conclusion, GLP-1 receptor activation has anti-apoptotic effect on both cytokine, and free fatty acid-induced apoptosis in primary islet-cells, thus suggesting that the long-acting GLP-1 analogue, liraglutide, may be useful for retaining β-cell mass in both type 1 and type 2 diabetic patients.

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on β-cells. It has been suggested that the β-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in β-cells following exposure to FFA.