Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B(1), did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.     

Protein kinase A increases the binding affinity and the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

Background information. In endocrine cells, IP₃R (inositol 1,4,5‐trisphosphate receptor), a ligand‐gated Ca²⁺ channel, plays an important role in the control of intracellular Ca²⁺ concentration. There are three subtypes of IP₃R that are distributed differentially among cell types. RINm5F cells express almost exclusively the IP₃R‐3 subtype. The purpose of the present study was to investigate the effect of PKA (protein kinase A) on the activity of IP₃R‐3 in RINm5F cells. Results. We show that immunoprecipitated IP₃R‐3 is a good substrate for PKA. Using a back‐phosphorylation approach, we show that endogenous PKA phosphorylates IP₃R‐3 in intact RINm5F cells. [³H]IP₃ (inositol 1,4,5‐trisphosphate) binding affinity and IP₃‐induced Ca²⁺ release activity were enhanced in permeabilized cells that were pre‐treated with forskolin or PKA. The PKA‐induced enhancement of IP₃R‐3 activity was also observed in intact RINm5F cells stimulated with carbachol and epidermal growth factor, two agonists that use different receptor types to activate phospholipase C. Conclusion. The results of the present study reveal a converging step where the cAMP and the Ca²⁺ signalling systems act co‐operatively in endocrine cell responses to external stimuli.     

Resistin induces rat insulinoma cell RINm5F apoptosis

Beta-cell apoptosis induced by adipokines may result in beta-cell dysfunction in type 2 diabetes. Resistin, an adipokine-linked obesity with type 2 diabetes, impairs glucose-stimulated insulin secretion (GSIS) in beta-cells. Presently, the effects of resistin on rat insulinoma cells RINm5F were examined. Treatment of RINm5F with resistin induced cell damage. Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) protected resistin-mediated cytotoxicity in RINm5F. Incubation with resistin up-regulated caspase-3 activity and induced the formation of a DNA ladder. TIMP-1 attenuated these effects. The molecular mechanism of TIMP-1 inhibition of resistin-mediated cytotoxicity appeared to involve Akt phosphorylation and activation of IkB-α phosphorylation. Resistin treatment suppressed Akt phosphorylation and activated IkB-α phosphorylation, which could be attenuated by TIMP-1. We conclude that resistin can induce beta-cell apoptosis and that resistin-related beta-cell apoptosis can be prevented by TIMP-1. C.-l. Gao and D.-y. Zhao contributed equally to this work.     

Protective effect of cyclo(his-pro) on streptozotocin-induced cytotoxicity and apoptosis in vitro

Cyclo(His-Pro) (CHP) is a naturally occurring, cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). CHP was efficiently obtained from soybean meal by hydrolysis with flavourzyme and alcalase. In this study, the effects of CHP on streptozotocin (STZ)-induced beta-cell dysfunction and apoptosis were investigated in rat insulinoma cells (RINm5F) secreting insulin. When the RINm5F cells were treated with 2mM STZ, insulin secretion decreased to approximately 54% that of control cells. However, CHP treatment restored the insulin-secreting activity of RINm5F cells to approximately 71% that of the untreated control cells. Moreover, CHP significantly protected the cells from STZ-mediated cytotoxicity via reduction of nitric oxide (NO) production (2.3-fold) and lipid peroxidation (1.9-fold), which were induced by STZ. Moreover, CHP treatment also attenuated STZ-induced apoptotic events, such as activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in RINm5F cells, indicating that CHP could protect the cells from apoptotic cell death induced by oxidative stress of STZ by increasing the expression of an anti-apoptotic protein, Bcl-2. These results suggest that CHP could be a candidate material for a protective and therapeutic agent against STZ-mediated cytotoxicity and apoptosis.     

Tat-CIAPIN1 protein prevents against cytokine-induced cytotoxicity in pancreatic RINm5F β-cells

Cytokines activate inflammatory signals and are major mediators in progressive β-cell damage, which leads to type 1 diabetes mellitus. We recently showed that the cell-permeable Tat-CIAPIN1 fusion protein inhibits neuronal cell death induced by oxidative stress. However, how the Tat-CIAPIN1 protein affects cytokine-induced β-cell damage has not been investigated yet. Thus, we assessed whether the Tat-CIAPIN1 protein can protect RINm5F β-cells against cytokine-induced cytotoxicity. In cytokine-exposed RINm5F β-cells, the transduced Tat-CIAPIN1 protein elevated cell survivals and reduced reactive oxygen species (ROS) and DNA fragmentation levels. The Tat-CIAPIN1 protein reduced mitogen-activated protein kinases (MAPKs) and NF-κB activation levels and elevated Bcl-2 protein, whereas Bax and cleaved Caspase-3 proteins were decreased by this fusion protein. Thus, the protection of RINm5F β-cells by the Tat-CIAPIN1 protein against cytokine-induced cytotoxicity can suggest that the Tat-CIAPIN1 protein might be used as a therapeutic inhibitor against RINm5F β-cell damage.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子（ｒｈＨＧＦ）对ＣＣｌ４染毒肝细胞的保护作用。结果表明：（１）ｒｈＨＧＦ（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／Ｌ）染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶（ＡＬＴ）、Ｋ＾＋的漏出;（２）表皮生长因子（ＥＧＦ,５０ｎｇ／ｍｌ）和ｒｈＨＧＦ（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.     

dsRNA formed as an intermediate during Coxsackievirus infection does not induce NO production in a beta-cell line with or without addition of IFN-gamma

Virus infection is one environmental factor that has been implicated as a precipitating event initiating beta-cell damage during the development of type 1 diabetes. One aim of this study was to investigate how permissive an insulin-producing beta-cell line, RINm5F, is to enterovirus (EV) infections. A second aim was to study if the viral replicative intermediate, double-stranded RNA (dsRNA), together with IFN-gamma results in nitric oxide (NO) production. Monolayer cultures of RINm5F cells were not permissive to infection with seven different strains of EV. However, when the growth pattern of the beta-cell line changed and the cells started to grow as free-floating RIN cell clusters (RCC), all EV strains replicated. Immunostaining for the Coxsackie-adenovirus-receptor (CAR) detected the protein on the free-floating RIN cell clusters, but not on the RINm5F cells cultured as a monolayer of beta-cells. This shows that the CAR expression can change and/or the CAR protein can be redistributed on the cell surface as a consequence of altered growth pattern thus allowing viral replication in a previously non-permissive beta-cell line. As expected, NO production was significantly increased (p<0.05) by addition of synthetic dsRNA and IFN-gamma to the RCC. In contrast, the dsRNA formed during virus infection with a Coxsackievirus B4 strain (E2) with or without addition of IFN-gamma did not induce NO production in these cells. This indicates that synthetic dsRNA does not mimic a real viral infection in that respect, and suggests an NO-independent mechanism for virus-induced beta-cell damage.     

Conditioned mesenchymal stem cells attenuate progression of chronic kidney disease through inhibition of epithelial‐to‐mesenchymal transition and immune modulation

Mesenchymal stem cells (MSCs) have been shown to improve the outcome of acute renal injury models; but whether MSCs can delay renal failure in chronic kidney disease (CKD) remains unclear. In the present study, the were cultured in media containing various concentrations of basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate to investigate whether hepatocyte growth factor (HGF) secretion could be increased by the stimulation of these growth factors. Then, TGF‐β1‐treated renal interstitial fibroblast (NRK‐49F), renal proximal tubular cells (NRK‐52E) and podocytes were co‐cultured with conditioned MSCs in the absence or presence of ascorbic acid 2‐phosphate to quantify the protective effects of conditioned MSCs on renal cells. Moreover, male Sprague‐Dawley rats were treated with 1 × 10⁶ conditioned MSCs immediately after 5/6 nephrectomy and every other week through the tail vein for 14 weeks. It was found that basic fibroblast growth factor, epidermal growth factor and ascorbic acid 2‐phosphate promoted HGF secretion in MSCs. Besides, conditioned MSCs were found to be protective against TGF‐β1 induced epithelial‐to‐mesenchymal transition of NRK‐52E and activation of NRK‐49F cells. Furthermore, conditioned MSCs protected podocytes from TGF‐β1‐induced loss of synaptopodin, fibronectin induction, cell death and apoptosis. Rats transplanted with conditioned human MSCs had a significantly increase in creatinine clearance rate, decrease in glomerulosclerosis, interstitial fibrosis and increase in CD4⁺CD25⁺Foxp3⁺ regulatory T cells counts in splenocytes. Together, our studies indicated that conditioned MSCs preserve renal function by their anti‐fibrotic and anti‐inflammatory effects. Transplantation of conditioned MSCs may be useful in treating CKD.     

Scoparia dulcis, a traditional antidiabetic plant, protects against streptozotocin induced oxidative stress and apoptosis in vitro and in vivo

Oxidative stress is implicated in the pathogenesis of diabetic complications. The experiments were performed on normal and experimental male Wistar rats treated with Scoparia dulcis plant extract (SPEt). The effect of SPEt was tested on streptozotocin (STZ) treated Rat insulinoma cell lines (RINm5F cells) and isolated islets in vitro. Administration of an aqueous extract of Scoparia dulcis by intragastric intubation (po) at a dose of 200 mg/kg body weight significantly decreased the blood glucose and lipid peroxidative marker thiobarbituric acid reactive substances (TBARS) with significant increase in the activities of plasma insulin, pancreatic superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) in streptozotocin diabetic rats at the end of 15 days treatment. Streptozotocin at a dose of 10 mug/mL evoked 6-fold stimulation of insulin secretion from isolated islets indicating its insulin secretagogue activity. The extract markedly reduced the STZ-induced lipidperoxidation in RINm5F cells. Further, SPEt protected STZ-mediated cytotoxicity and nitric oxide (NO) production in RINm5F cells. Treatment of RINm5F cells with 5 mM STZ and 10 mug of SPEt completely abrogated apoptosis induced by STZ, suggesting the involvement of oxidative stress. Flow cytometric assessment on the level of intracellular peroxides using fluorescent probe 2'7'-dichlorofluorescein diacetate (DCF-DA) confirmed that STZ (46%) induced an intracellular oxidative stress in RINm5F cells, which was suppressed by SPEt (21%). In addition, SPEt also reduced (33%) the STZ-induced apoptosis (72%) in RINm5F cells indicating the mode of protection of SPEt on RIN m5Fcells, islets, and pancreatic beta-cell mass (histopathological observations). Present study thus confirms antihyperglycemic effect of SPEt and also demonstrated the consistently strong antioxidant properties of Scoparia dulcis used in the traditional medicine.     

Dihydroavenanthramide D protects pancreatic β-cells from cytokine and streptozotocin toxicity

Dihydroavenanthramide D (DHAvD) is a synthetic analog to naturally occurring avenanthramide, which is the active component of oat. Although its anti-inflammatory, antiatherosclerotic, and antioxidant effects have been reported, the effect of DHAvD on type 1 diabetes is unknown. Therefore, in this study, the effect of DHAvD on cytokine- or streptozotocin-induced β-cell damage was investigated. Treatment of RINm5F insulinoma cells or isolated islets with IL-1β and IFN-γ induced β-cell damage through a NF-κB-dependent signaling pathway. DHAvD-pretreated RINm5F cells or islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced the inducible form of NO synthase expression, and decreased β-cell destruction and the normal insulin secretion capacity. Furthermore, pretreatment with DHAvD blocked the development of type 1 diabetes in streptozotocin-treated mice. Prior injection with DHAvD maintained a normal range of plasma glucose and insulin, and retained immunoreactivity for insulin in the pancreas. These results suggest that DHAvD may be used to preserve functional β-cell mass.     

KIOM-4 protects RINm5F pancreatic β-Cells against streptozotocin induced oxidative stress <Emphasis Type="Italic">in vitro</Emphasis>

The effect of KIOM-4, a combination of four plant extracts was assessed on streptozotocin (STZ) treated rat insulinoma (RIN5mF) cells in vitro. KIOM-4 scavenged the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and intracellular reactive oxygen species (ROS) induced by STZ. KIOM-4 prevented the STZ-induced DNA damage, which is detected using comet assay, Western blot and lipid peroxidation assays. KIOM-4 inhibited the STZ induced apoptosis, therefore protecting from cell death. Additionally, KIOM-4 induced the activation of catalase and extracellular regulated kinase (ERK). These results suggest that KIOM-4 protects RINm5F cells via radical scavenging activity, the activation of catalase and ERK against STZ induced oxidative stress.     

Ethanol and acetaldehyde potentiate the clastogenicity of ultraviolet light, methyl methanesulfonate, mitomycin C and bleomycin in Chinese hamster ovary cells

Ethanol itself did not induce any apparent chromosome aberrations in Chinese hamster ovary cells. However, posttreatment with ethanol potentiated the chromosome aberrations induced by ultraviolet light (UV), methyl methanesulfonate (MMS), mitomycin C (MMC) or bleomycin (BLM). Chromatid exchanges were predominantly increased in cultures treated with UV, MMS or MMC and then with ethanol, whereas chromosome breaks and chromatid exchange were the major types of aberrations increased in the cultures treated with BLM and ethanol. Posttreatment with acetaldehyde, the major metabolite of ethanol, also potentiated the chromosome aberrations induced by UV, MMS, MMC or BLM. The main types of aberrations potentiated by posttreatment with acetaldehyde were similar to those by posttreatment with ethanol.     

Magnitude-dependent effects of cyclic stretch on HGF- and VEGF-induced pulmonary endothelial remodeling and barrier regulation

Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability.