诺沙坦改善非胰岛素依赖型糖尿病大鼠胰岛素敏感性的作用机制

本文研究血管紧张素Ⅱ受体拮抗剂诺沙坦对非胰岛素依赖型糖尿病(non-insulin-dependent diabetes mellitus,NIDDM)大鼠胰岛素敏感性的改善作用,并探讨其作用机制。从饮水中给予正常或高脂喂养加小剂量链脲佐菌素(STZ)诱发的NIDDM大鼠诺沙坦(4 mg/kg),连续6周。分离骨骼肌,用免疫印迹法检测诺沙坦对胰岛素受体底物1(insulin receptor substrate 1,IRS-1)、蛋白激酶B(protein kinase B,PKB)和葡萄糖转运因子4(glucose transporter 4,GLUT4)的表达,以及IRS-1的磷酸化、IRS-1与磷脂酰肌醇3激酶(phosphatidylinositol(PI)3-kinase)的结合。口服葡萄糖耐量试验表明,口服诺沙坦可改善糖尿病大鼠胰岛素敏感性。在骨骼肌组织,NIDDM和正常大鼠的IRS-1、PKB和GLUT4蛋白表达无差异,且不受诺沙坦处理的影响。NIDDM大鼠胰岛素刺激后的骨骼肌IRS-1酪氨酸磷酸化水平、PI 3-kinase结合IRS-1的活性和PKB活性较对照组显著降低(P<0.01),且不能被诺沙坦改善。诺沙坦显著增加NIDDM大鼠肌细胞质膜(plasma membrane,PM)和T管(T-tubules,TT)胰岛素诱导的GLUT4的 含量(P<0.05)。与该结果一致的是,诺沙坦处理的NIDDM大鼠血糖水平较未处理NIDDM大鼠下降(P<0.05)。结果表明,诺沙坦可改善胰岛素抵抗状态,主要是通过非PI 3-kinase依赖的     

The effect of diet on the Vervet monkey endocrine pancreas

Vervet monkeys (Cercopithecus aethiops) used for pancreatic endocrine cell distribution studies were found to have been maintained on different diets. Although the effect of dietary changes on the exocrine pancreas has been described in several animals, little, apart from the effect of malnutrition, has been reported for the endocrine pancreas. Reported here are pancreatic endocrine cell distributions in monkeys on a standard diet (n ? 3) compared with monkeys on an atherogenic diet (n = 3). Quantitation of immunolabelled pancreatic endocrine cell types revealed a significant 80% increase in A (glucagon) cell volume in monkeys on an atherogenic diet concomitant with a significant reduction in B (insulin) cell volume to approximately 60% of normal. This reflects a pattern of events that occurs in non-insulin dependent diabetes. An accompanying reduction in PP (pancreatic polypeptide) cell volumes supports our hypothesis that altering A and PP cell volumes could reflect differential gene expression in those cells in the adult in which glucagon and PP are co-localized.     

Islet Endothelial Cells Induce Glycosylation and Increase Cell-surface Expression of Integrin β1 in β Cells

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin β1. A wide array of integrin β subunits was detected in βTC3 and NIT-1 insulinomas as well as in primary islets, with integrin β1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin β1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer β-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin β1 in PIs. Antibody-mediated blocking of integrin β1 led to alterations in β-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin β1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in β cells.     

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2′-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.     

Mitochondrial uncoupling protein-2 is not involved in palmitate-induced impairment of glucose-stimulated insulin secretion in INS-1E insulinoma cells and is not needed for the amplification of insulin release

We have recently shown that overnight exposure of INS-1E insulinoma cells to palmitate in the presence of high glucose causes defects in both mitochondrial energy metabolism and glucose-stimulated insulin secretion (GSIS). Here we report experiments designed to test the involvement of mitochondrial uncoupling protein-2 (UCP2) in these glucolipotoxic effects. Measuring real-time oxygen consumption in siRNA-transfected INS-1E cells, we show that deleterious effects of palmitate on the glucose sensitivity of mitochondrial respiration and on the coupling efficiency of oxidative phosphorylation are independent of UCP2. Consistently, palmitate impairs GSIS to the same extent in cells with and without UCP2. Furthermore, we knocked down UCP2 in spheroid INS-1E cell clusters (pseudoislets) to test whether or not UCP2 regulates insulin secretion during prolonged glucose exposure. We demonstrate that there are no differences in temporal GSIS kinetics between perifused pseudoislets with and without UCP2. We conclude that UCP2 is not involved in palmitate-induced impairment of GSIS in INS-1E insulinoma cells and is not needed for the amplification of insulin release. These conclusions inform ongoing debate on the disputed biochemical and physiological functions of the beta cell UCP2.     

MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

The tandem of free radicals and methylglyoxal

Methylglyoxal is an alpha-oxoaldehyde inevitably produced from triose-phosphate intermediates of phosphorylating glycolysis, and also from amino acids and acetone. Recently, the attention has been focused on the involvement of free radicals in methylglyoxal toxicity. In this review, a summary of the relationship between methylglyoxal metabolism and free radical production is presented, extending discussion from the possible metabolic routes to the toxicological events by reviewing the role of free radicals in both generation and degradation of this 1,2-dicarbonyl as well as in the modification of biological macromolecules, and focusing on the action of methylglyoxal upon cellular glutathione content. Methylglyoxal-provoked free radical generation involving reactive oxygen species (ROS), reactive nitrogen species (RNS) as well as organic radicals like methylglyoxal radial or crosslinked protein radical as potential risk factors to tissue damage propagation, is thoroughly discussed. Special attention is paid to the potential therapeutic interventions. The paper arrives at the conclusion that a tight junction exists between methylglyoxal toxicity and free radical (particularly ROS) generation, though the toxicity of 1,2-dicarbonyl evolves even under anaerobic conditions, too. The events follow a sequence beginning with carbonyl stress essential for the toxicity, leading to free radical formation and finally ending in either apoptosis or necrosis. Both oxidative and nitrosative stress play important but not indispensable role in the development of methylglyoxal toxicity.