The Effects Of Ghrelin On Lipotoxic Apoptosis Of Pancreatic β-cells And Its Molecular Mechanism

Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. In principle, physiologic levels of glucose and lipids are not toxic but essential to normalβ-cell function. However, the prolonged exposure of pancreatic (3-cells to elevated levels of glucose or/and fat signal is associated with impairment of insulin gene expression,inhibition of insulin synthesis and secretion and induction ofβ-cell apoptosis.. A number of studies have shown that fatty acids can induceβ-cell apoptosis in the presence of high glucose. The serine/threonine kinase Akt, also known as protein kinase B (PKB), plays a vital role in regulating mass and function of pancreaticβ-cell. There are growing evidences that activation of PKB phosphorylation is able to prevent pancreatic P-cell from lipotoxicity. To pancreatic P-cells, PKB is not an unique central node in cell signaling downstream of growth factors and cytokines, but a key target of insulin signal system as well.Ghrelin is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, and is mainly secreted from X/A like cells of gastric fungus, as a natural endogenous ligand of the orphan growth hormone secretagogue receptor type la (GHS-Rla), through which it acts as a growth hormone releasing peptide and food intake modulator. The effects of ghrelin are considered to be broadly including energy and glucose homeostasis, regulation of proliferation, apoptosis and differentiation of various normal and neoplastic cells lines, et.al.. Recently, the relationship between ghrelin and pancreaticβ-cells has attracted much attention. Some studies have showed ghrelin-secreting cells namedεcells could be detected in the verges of pancreatic islets, suggesting that ghrelin affects pancreaticβ-cells via both endocrine and paracrine pathway. It becomes increasingly clear that the effect of ghrelin on the regulation of pancreatic P-cells proliferation, apoptosis and function, are mediated by mechanisms dependent and independent of GHS-Rla. Ghrelin could promote cell proliferation and inhibit pancreaticβ-cells apoptosis induced by interferon-r/tumor necrosis factor-a (IFN-r /TNF-a) synergism, as well as doxorubicin-inducedβ-cells apoptosis. PI3K/PKB is supposed as the main signal pathway that mediated protective effect of ghrelin in type 1 diabetes. However, it remains to be elucidated whether ghrelin protects P cells against lipotoxicity-induced apoptosis in type 2 diabetes.Therefore, the aim of the present study is to investigate whether ghrelin preventsβ-cells from lipotoxicity and explore the mechanism behind.Materials and methodsMaterialsMIN6 cells, a widely used pancreaticβ-cell line(passages 11-30); Ghrelin, Fatty acid-free bovine serum albumin(BSA, fraction V), Palmitate, Hoechst33258, MTT, LY294002, SP600125; Antibodies used were anti-phospho-ser473-PKB, anti-total PKB, anti-phospho-JNK1/2, anti-total JNK; TUNEL (in situ cell death detection kit, POD) kit; Caspase-3 activity assay kit; TG GPO-POD assay kit; AnnexinV-PI apoptosis detection kit; The primer of PCR and the agent of demi-quantitate PT-PCR detection.Methods1,Cell cultureMIN6 cells, a widely used pancreaticβ-cell line(passages 11-30), were cultured in Dulbecco's modified eagle's medium (DMEM) containing 25 mM glucose, with 15% fetal bovine serum (FBS),100 U/mL penicillin,100μg/mL streptomycin,100μg/mL L-glutamine,5μL/Lβ-mercaptoethanol in humidified 5% CO2,95%air at 37℃. As grew to 80-85%, the cells were utilized for experiment or passage.2,Cell viability assay Cells were seeded on 96-well plates at a density of 5000 cells per well. For detection, cells were incubated with 5mg/mL MTT for approximately 4h under 5%CO2,37℃. The medium was removed and the formazan product was solubilized with 150μL dimethylsulfoxide. The plates were vibrated on swing bed for 10 min. Viability was assessed by spectrophotometry at 570nm absorbance using a 96-well plate reader.3,Caspase-3 activity assayBriefly, harvested cells were centrifuged at 10,000 rpm/min for 1 min after washed twice by PBS, followed by the addition of 1μL DTT and 100μL lysis buffer. Cell lysates in a 96 well microplate were incubated at 37℃with 5μL of Caspase-3 colorimetric substrate (DEVD-pNA) for 2 h. Absorbances were read by a microplate reader at 405 nm wavelength.4,TUNEL assayTUNEL staining was performed according to the manufacturer's protocol with few modifications, Cell slides were fixed in 4% paraformaldehyde and stained with TUNEL reaction mixture and 50μL converter-horse-radish peroxidase (POD), followed by adding DAB substrate and hematoxylin. Apoptosis indexes were assessed by counting TUNEL positive cells (apoptotic nucleus was brown-stained or black-stained) through light microscope. Apoptosis ratio was TUNEL positive cells over the whole cells5,Hoechst33258 stainingHoechst staining was performed by fixing the cells in 4% paraformaldehyde for 10 min under 4℃and exposing the cell slides to lOug/mL Hoechst 33258 for 10 min at room temperature. Apoptosis indexes were assessed by counting Hoechst positive cells (chromatin condensation or fragmented nuclear membrane) through fluorescent microscope. Apoptosis ratio was Hoechst positive cells over the whole cells.6,AnnexinV-PI staining and Flow cytometry assayHarvested cells were mixed with PBS and counted number.5×104 cells were collected and centrifuged at 1000 r/min for 5 min, followed by adding 195μl Annexin V-FITC binding fluid and 5μl Annexin V-FITC. After recentrifuged at 1000 r/min for 5 min, the cells sample were mixed with 190μl Annexin V-FITC binding fluid and 10μl PI. Apoptosis index were detected and assessed by flow cytometry. Annexin V-FITC and PI respectively present green and red fluorescence.7,Electron microscope analysisAs grown to 80% in 250ml culture flask, the MIN6 cells were fixed with 2.5% Glutaral. Cells sample were assessed by electron microscope.8,Cytoplasmic triglyceride assayCytoplasmic TG was detected with a TG GPO-POD assay kit according to the manufacturer's protocol. In brief, after two washes in PBS, cells were harvested,6×106 cells/mL were lysed by ultrasound, and 10-uL aliquots of cell lysates or standard samples were added to 96-well plates, followed by the addition of 200μL glycerokinase and 70μL glycerophosphate oxidase and developer. Absorbance at 500 nm was measured with a microplate reader. The TG concentration was calculated according to a standard curve.9,RNA isolation and Reverse-transcription polymerase chain reactionReverse-transcription polymerase chain reaction was performed to semi-quantify mRNA expression of SREBPlc, CHOP, BAX, BCL-2 andβ-actin gene.Total RNA was isolated and first strand cDNA synthesized using MMLV reverse transcriptase and hexamers from MIN6 cells. The primer sequences were devised by primer5 software.cDNA(9μL) was amplified by PCR in a 50μL volume with amplitaq gold polymerase and the PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidiumbromide staining. The resulting images were analyzed by Scion Image software.10,Western blotBriefly, protein was extracted with a cell lysis buffer. Protein samples (100μg for p-PKB (ser473) and p-JNK1/2 or 50ug for total PKB, JNK) were separated by SDS-electrophoresis through either 10% gradient polyacrylamide gels and transferred to nitrocellulose membranes, followed by immunoblotting using all primary antibodies according to the manufacturer's instructions. Immuno-detection was developed with ECL advance, and the resulting images were analyzed by Scion Image software. 11,Statistical analysisData are presented as means±SE. Statistical analyses were performed with SPSS using ANOVA. Differences between groups were assessed for significance by Dunnett's test. A p value of less than 0.05 was considered significant.Results1.The effects of lipotoxicity on cells viabiltiy, microstructure and cells apoptosis in pancreaticβ-cells(1) Palmitate-induced lipotoxicity could concentration-dependently decrease cells viability of pancreaticβ-cells.(2)Lipotoxicity of palmitate damaged serevely cell morphous of pancreatic P-cells: nuclear crenation, chromatin condensation, distension of endoplasmic reticulum, engorgement of chondrosome, decreased or disappeared secretory granule in cytoplasm.(3) Palmitate concentration-dependently induced apoptosis of pancreatic P-cells.2.The effects of ghrelin on lipotoxicity-induced apoptosis and deposition of triglyceride in pancreaticβ-cells(1) Cell viability assessed by MTT was decreased by 21% (p<0.05) after 0.4mM palmitate (PA) treatment. Treatment with AG dose-dependently prevented PA-induced toxicity, most effectively by 34% at 100nM (p＜0.01). Cell apoptosis evaluated by caspase3 activity assay was significantly increased in PA group by 61%, while PA /AG (at 100nM) synergism decreased caspase 3 activity by 27% (p<0.05). Apoptosis evaluated by hoechst33258 staining showed, the percentage of apoptosis reached to 47% after PA treatment for 24 hours, while treatment with AG at 100nM reversed apoptosis rate by 16%(p＜0.01).(2)As opposed to the effects of palmitate (p＜0.01), ghrelin significantly decreased the cytoplasmic TG level in BSA-treated or palmitate-treated MIN6 cells in a concentration-dependent manner. A maximal effect was observed at 100nM(p＜0.05). 3.Molecular mechanism of ghrelin protective actions on pancreaticβ-cells against lipotoxicity(1)Exposure of the cells to 0.4mM palmitate for 24 hours caused significant inhibition of PKB phosphorylation (p<0.01), while ghrelin at 100nM induced rapid activation of PKB and reached the maximal effect at 30 min (p<0.01). Ghrelin dose-dependently stimulated activation of PKB phosphorylation and treatment at 100nM produced most effective potentiation(p<0.01). Ghrelin-induced activation of PKB was markedly blocked by PI3K inhibitor, LY294002(p<0.05).Moreover, LY294002 abolished ghrelin cytoprotective activity against palmitate-induced apoptosis,.which evaluated by TUNEL assay and hoechst33258 staining.(2)MIN6 cells were incubated with PA for 24 hours and produced activation of JNK phosphorylation (p<0.01), while ghrelin at 100nM resulted in a transient decrease at 30 min (p<0.05). ghrelin-induced inhibition of JNK phosphorylation was blocked by PI3K inhibitor, LY294002 (p<0.05). SP600125 alone can significantly prevent palmitate induced apoptosis. Combination of SP600125 with AG at 100nM could enhance ghrelin's antiapoptotic effect in MIN6 cells.(3)Consistent with caspase 3 activity assay, palmitate significantly up-regulated BAX, SREBP1C, and CHOP-10 and down-regulatesd BCL-2. However, ghrelin at 100nM downregulated BAX, SREBP1C, and CHOP-10 mRNA expression,but did not affect BCL-2 mRNA expression.Conclusions1.Palmitate-produced lipotoxicity decreases cells viability, changes cells microstructure and induces concentration-dependently in MIN6 pancreaticβ-cells.2.Ghrelin promote pancreatic P-cells growth, increases cells viability and inhibits lipotoxicity-induced cell apoptosis.3.Ghrelin prevents MIN6 cells from apoptosis induced by lipotoxicity via PI3K/PKB pathway.4. Ghrelin attenuates activation of palmitate-induced JNK phosphorylation and JNK inhibitor, SP600125, enhances antiapoptotic effects of ghrelin in MIN6 cells.5. Ghrelin antiapoptosis effect involve mitochondrial pathways under lipotoxic state.6.Ghrelin decreases the cytoplasmic TG level and its antilipotoxicity is associated with endoplasmic reticulum stress pathways.