Drak2 overexpression results in increased beta-cell apoptosis after free fatty acid stimulation

Drak2 is a serine threonine kinase in the death-associated protein family. In this study, we investigated its role in free fatty acid (FFA)-induced islet apoptosis. Drak2 mRNA and protein were rapidly induced in islet beta-cells after FFA stimulation. Such Drak2 upregulation was accompanied by increased beta-cell apoptosis, which was inhibited by Drak2 knockdown using siRNA. Conversely, transgenic (Tg) Drak2 overexpression led to aggravated beta-cell apoptosis triggered by FFA. Drak2 overexpression in islets compromised the increase of anti-apoptotic factors, such as Bcl-2, Bcl-xL and Flip, upon FFA assault. Further in vivo experiments demonstrated that Drak2 Tg mice presented compromised glucose tolerance in a diet-induced obesity model. Our data show that Drak2 is detrimental to islet survival in the presence of excessive lipid.     

Experimental study of rat beta islet cells cultured under simulated microgravity conditions

To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy. Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient trans     

Humic acid induces apoptosis in human premyelocytic leukemia HL-60 cells

It has been shown that humic acid (HA), a phenolic polymer, exhibits pro-oxidant and cytotoxic effects. In this study, HA induction of apoptosis was studied using cultured human premyelocytic leukemia HL-60 cells. Treatment at a range of HA concentrations (50-400 microg/ml) resulted in dose-and time-dependent sequences of events marked by apoptosis, as demonstrated through by apoptotic features such as loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. This HA-induced apoptosis in the HL-60 cells was mainly associated with the release of cytochrome c from the mitochondria. Furthermore, apoptosis in the HL-60 cells was accompanied by the activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a major component in the apoptotic cell death mechanism. Although the HA-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data reported herein reveals that HA exerts antiproliferative action and growth inhibition on HL-60 cells through induction of apoptosis, which may have anticancer properties potentially useful for the development of new drug products.     

Beta-amyloid peptides 1-40betaA and 25-35betaA suppress human amylin-mediated death of RINm5F islet beta-cells with distinct actions on fibril formation

Amyloid deposition is a common feature of Alzheimer's disease and type 2 diabetes related to beta-amyloid peptides (betaA) and human amylin (hA), respectively. Both betaA and hA form aggregates and fibrils and kill cultured cells. To investigate whether betaA and hA display peptide-specific toxicity on cultured islet beta-cells, we examined the effects of (1-40)betaA and (25-35)betaA peptides on hA-mediated cell death and [(125)I-Tyr(37)]hA precipitation. Synthetic hA aggregated in solution and evoked both conformation- and sequence-dependent cell death. While neither (1-40)betaA nor (25-35)betaA was toxic to islet beta-cells, they suppressed hA-evoked cell death in a concentration-dependent and saturable manner. Only (1-40)betaA, but not (25-35)betaA, showed trophic effects on cultured islet beta-cells and inhibited the precipitation of [(125)I]hA caused by hA. These results suggest that (25-35)betaA does not interfere with hA-mediated fibril formation. Suppression of hA-evoked death of cultured pancreatic islet beta-cells by the betaA peptides is likely to occur through a competing interaction at these cells.     

Humic acid induces oxidative DNA damage,growth retardation,and apoptosis in human primary fibroblasts

Humic acid (HA) has been implicated as an etiological factor of Blackfoot disease endemic in the southwest coast of Taiwan. Dysfunction of endothelial cells and vasculopathy have been proposed to explain the onset of ulcerous changes at extremities. However, little is known about the effect of HA on activities of cells in these nonhealing wounds. In the present study, we demonstrate that HA adversely affects the growth properties of fibroblasts, one of the key players in wound repair. HA treatment caused growth arrest and apoptosis in human foreskin fibroblasts (HFF). This was accompanied by a significant increase in the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in cellular DNA. The increased fluorescence in dichlorofluorescin (H2DCF)-stained and HA-treated cells suggests the involvement of reactive oxygen species (ROS) in HA-induced biological effects. Conversely, vitamin E pretreatment, which significantly reduced the 8-OHdG formation in HA-treated cells, alleviated the growth-inhibitory and apoptosis-inducing effects of HA. These results indicate that HA initiates oxidative damages to fibroblasts, and leads to their dwindling growth potential and survival. The present study suggests that HA-induced growth retardation and apoptosis of fibroblasts may play a role in the pathogenesis of Blackfoot disease.     

Comparison of cellular and medium insulin and GABA content as markers for living beta-cells

Experimental and therapeutic use of islet cell preparations could benefit from assays that measure variations in the mass of living beta-cells. Because processes of cell death can be followed by depletion and/or discharge of cell-specific substances, we examined whether in vitro conditions of beta-cell death resulted in changes in tissue and medium content of insulin and of gamma-aminobutyric acid (GABA), two beta-cell-specific compounds with different cellular localization and turnover. Exposure of rat purified beta-cells to streptozotocin (5 mM, 120 min) or to the nitric oxide donor GEA-3162 (GEA; 50 microM, 120 min) caused 80% necrosis within 24 h; at the end of this period, cellular insulin content was not significantly decreased, but cellular GABA content was reduced by 70%; when cultured at basal glucose (6 mM), the toxin-exposed cells did not discharge less insulin but released 80% less GABA in the period 8-24 h. As in rat beta-cell purification, GABA comigrated with insulin during human islet cell isolation. Twenty-four hours after GEA (500 microM, 120 min), human islet cell preparations exhibited 90% dead cells and a 45 and 90% reduction, respectively, in tissue insulin and GABA content; in the period 9-24 h, insulin discharge in the medium was not reduced, but GABA release was decreased by 90%. When rat beta-cells were cultured for 24 h with nontoxic interleukin (IL)-1beta concentrations that suppressed glucose-induced insulin release, cellular GABA content was not decreased and GABA release increased by 90% in the period 8-24 h. These data indicate that a reduction in cellular and medium GABA levels is more sensitive than insulin as a marker for the presence of dead beta-cells in isolated preparations. Pancreatic GABA content also rapidly decreased after streptozotocin injection and remained unaffected by 12 h of hyperglycemia. At further variance with insulin, GABA release from living beta-cells depends little on its cellular content but increases with IL-1beta-induced alterations in beta-cell phenotype.     

Glucose promotes pancreatic islet beta-cell survival through a PI 3-kinase/Akt-signaling pathway

The concentration of glucose in plasma is an important determinant of pancreatic beta-cell mass, whereas the relative contributions of hypertrophy, proliferation, and cell survival to this process are unclear. Glucose results in depolarization and subsequent calcium influx into islet beta-cells. Because depolarization and calcium (Ca(2+)) influx promote survival of neuronal cells, we hypothesized that glucose might alter survival of islet beta-cells through a similar mechanism. In the present studies, cultured mouse islet beta-cells showed a threefold decrease in apoptosis under conditions of 15 mM glucose compared with 2 mM glucose (P < 0.05). MIN6 insulinoma cells incubated in 25 mM glucose for 24 h showed a threefold decrease in apoptosis compared with cells in 5 mM glucose (1.7 +/- 0.2 vs. 6.3 +/- 1%, respectively, P < 0.001). High glucose (25 mM) enhanced survival-required depolarization and Ca(2+) influx and was blocked by phosphatidylinositol (PI) 3-kinase inhibitors. Glucose activation of the protein kinase Akt was demonstrated in both insulinoma cells and cultured mouse islets by means of an antibody specific for Ser(473) phospho-Akt and by an in vitro Akt kinase assay. Akt phosphorylation was dependent on PI 3-kinase but not on MAPK. Transfection of insulinoma cells with an Akt kinase-dead plasmid (Akt-K179M) resulted in loss of glucose-mediated protection, whereas transfection with a constitutively active Akt enhanced survival in glucose-deprived insulinoma cells. The results of these studies defined a novel pathway for glucose-mediated activation of a PI 3-kinase/Akt survival-signaling pathway in islet beta-cells. This pathway may provide important targets for therapeutic intervention.     

高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响

目的:探讨高浓度葡萄糖对体外培养大鼠胰岛细胞凋亡的影响及作用机制。方法:SD大鼠胰岛细胞原代培养,不同浓度的葡萄糖处理后,采用MTT比色法检测细胞存活率,Hoechst-PI染色观察细胞凋亡,电镜观察细胞超微结构改变,RT-PCR方法检测Bax及Bcl-2相关基因mRNA表达水平。结果:5.5 mmol·L~(-1),11.1 mmol·L~(-1)和22.2 mmol·L~(-1)葡萄糖处理后,胰岛细胞活性分别下降到78.08%±2.29%、58.39%±3.13%和36.05%±2.63%,与阴性对照组比较差异具有统计学意义(P0.05);Hoechst-PI染色结果显示随着葡萄糖作用浓度的增加,凋亡细胞数量也增加;RT-PCR显示胰岛细胞经不同浓度葡萄糖处理后,Bax mRNA的表达明显上调,Bcl-2 mRNA表达下调(P0.05);电镜观察结果显示随着葡萄糖作用浓度的增加,胰岛细胞超微结构损害程度依次加重。结论:高浓度葡萄糖能明显引起胰岛细胞活性的下降,诱导凋亡反应的发生,凋亡机制与Bcl-2家族蛋白相关。     

Prolonged culture in low glucose induces apoptosis of rat pancreatic beta-cells through induction of c-myc

Prolonged culture in low-glucose concentrations (相似文献   

Comparative effects of a vasopeptidase inhibitor vs. an angiotensin convertin enzyme inhibitor on cardiomyocyte apoptosis in rats with heart failure

Apoptosis is involved in ventricular remodeling after myocardial infarction (MI). We investigated the effects of the vasopeptidase inhibitor (VPI) omapatrilat on cardiomyocyte apoptosis and compared it to the angiotensin converting enzyme inhibitor (ACEI) captopril in the rat post-MI model and in cultured neonatal rat cardiomyocytes. Wistar males rats surviving 4 h post-MI were assigned to omapatrilat (40 or 80 mg/kg/day), captopril (160 mg/kg/day) or no treatment. After 56 days, hemodynamic measurements were performed (n = 96) and rats were sacrificed. One group had assessment of cardiac remodeling and detection of DNA fragments by in situ end labelling method (ISEL), while the other had morphologic measurements and DNA laddering assessed. In addition, cultured neonatal rat cardiomyocytes (n = 6) were treated for 72 h with vehicle, captopril or omapatrilat in the presence or absence of the apoptosis inducing agent H₂O₂. Omapatrilat and captopril resulted in similar improvements of hemodynamic measurements, ventricular weight and dilatation, cardiac fibrosis and myocardial cell cross-section in large MI rats. Omapatrilat increased scar thickness more than did captopril. All sham-operated groups had little evidence of apoptosis. In the large MI group, there was a significant increase in ISEL-positive cells in the control (0.095 ± 0.016%) and captopril (0.124 ± 0.024%) groups in comparison with control sham-operated (0.006 ± 0.006%), but this increase was limited to the peri-MI area. Omapatrilat (0.012 ± 0.012% for both doses) prevented the increase in apoptosis in the peri-MI area. Also, omapatrilat but not captopril reduced DNA laddering in large MI. Moreover, in cultured neonatal rat cardiomyocytes, omapatrilat but not captopril reduced apoptosis as assessed by DNA laddering. The VPI omapatrilat, with its combination of NEP and ACE inhibition, suppresses cardiomyocyte apoptosis post-MI and in neonatal cultured rat cardiomyocytes more than the ACEI captopril, but this does not result in significant hemodynamic or morphologic differences between omapatrilat and captopril.