Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes

During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.     

Immunohistochemical study of monocyte chemoattractant protein-1 in the pancreas of NOD mice following cyclophosphamide administration and during spontaneous diabetes

In type 1 diabetes mellitus (T1DM), the processes which control the recruitment of immune cells into pancreatic islets are poorly defined. Complex interactions involving adhesion molecules, chemokines and chemokine receptors may facilitate this process. The chemokine, monocyte chemoattractant protein-1 (MCP-1), previously shown to be important in leukocyte trafficking in other disease systems, may be a key participant in the early influx of blood-borne immune cells into islets during T1DM. In the non-obese diabetic (NOD) mouse, the expression of MCP-1 protein has not been demonstrated. We employed dual-label immunohistochemistry to examine the intra-islet expression, distribution and cellular source of MCP-1 in the NOD mouse following cyclophosphamide administration. NOD mice were treated with cyclophosphamide at day 72–73 and MCP-1 expression studied at days 0, 4, 7, 11 and 14 after treatment and comparisons were made between age-matched NOD mice treated with diluent and non-diabetes-prone CD-1 mice. Pancreatic expression of MCP-1 was also examined in NOD mice at various stages of spontaneous diabetes. In the cyclophosphamide group at day 0, MCP-1 immunolabelling was present in selective peri-islet macrophages but declined at day 4. It increased slightly at day 7 but was more marked from day 11, irrespective of diabetes development. The pattern of MCP-1 expression in macrophages was different over time in both the cyclophosphamide and control groups. In the cyclophosphamide group, there was a change over time with an increase at day 11. In the control group, there was little evidence of change over time. There was no significant difference in the mean percentage of MCP-1 positive macrophages between the cyclophosphamide-treated diabetic and non-diabetic mice. During spontaneous diabetes in the NOD mouse, only a few peri-islet MCP-1 cells appeared at day 45. These became more numerous from day 65 but were absent at diabetes onset. We speculate that a proportion of early islet-infiltrating macrophages which express MCP-1 may attract additional lymphocytes and macrophages into the early inflamed islets and intensify the process of insulitis.     

Pancreatic Beta cells in very old mice retain capacity for compensatory proliferation

Recent studies suggested that in old mice, beta cells lose their regenerative potential and cannot respond to mitogenic triggers. These studies examined beta cell replication in aged mice under basal conditions and in response to specific stimuli including treatment with the glucagon-like peptide-1 analog exenatide, streptozotocin injection, partial pancreatectomy, and high fat diet. However, it remains possible that the ability to mount a compensatory response of beta cells is retained in old age, but depends on the specific stimulus. Here, we asked whether partial ablation of beta cells in transgenic mice, using doxycycline-inducible expression of diphtheria toxin, triggers a significant compensatory proliferative response in 1-2-year-old animals. Consistent with previous reports, the basal rate of beta cell replication declines dramatically with age, averaging 0.1% in 2-year-old mice. Transient expression of diphtheria toxin in beta cells of old mice resulted in impaired glucose homeostasis and disruption of islet architecture (ratio of beta to alpha cells). Strikingly, the replication rate of surviving beta cells increased 3-fold over basal rate, similarly to the -fold increase in replication rate of beta cells in young transgenic mice. Islet architecture and glucose tolerance slowly normalized, indicating functional significance of compensatory beta cell replication in this setting. Finally, administration of a small molecule glucokinase activator to old mice doubled the frequency of beta cell replication, further showing that old beta cells can respond to the mitogenic trigger of enhanced glycolysis. We conclude that the potential for functionally significant compensatory proliferation of beta cells is retained in old mice, despite a decline in basal replication rate.     

缺氧诱导因子-1α和葡萄糖转运蛋白-1在不同月龄大鼠椎间盘中的表达

目的检测缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)和葡萄糖转运蛋白-1(glucose transporter-1,GLUT-1)在不同月龄大鼠椎间盘纤维环组织中的表达及其相关性,探讨HIF-1α及GLUT-1在椎间盘退变过程中的作用。方法取Wistar大鼠50只,分别以1,3,6,12,18个月龄分为5组。采用免疫组化法及Western blot法检测各组椎间盘中HIF-1α和GLUT-1表达情况。结果随着大鼠月龄的增长,其椎间盘纤维环组织中HIF-1α和GLUT-1的表达也发生变化,由低月龄组(1-3月龄)至成年组(6-12月龄)HIF-1α和GLUT-1表达逐渐减少,而老年组(18月龄)二者表达显著增加。且这种变化有显著统计学意义(P0.01)。纤维环中HIF-1α和GLUT-1的蛋白表达呈正相关。结论HIF-1α、GLUT-1表达水平的变化与椎间盘退变的发生关系密切相关,HIF-1α可以通过上调GLUT-1等相关因子并延缓椎间盘退变,可能作为椎间盘退变治疗研究的切入点。     

Subcongenic analysis of genetic basis for impaired development of invariant NKT cells in NOD mice

Reduced numbers and function of invariant NKT (iNKT) cells partially contribute to type 1 diabetes (T1D) development in NOD mice. Previous linkage analysis identified a genetic locus on chromosome 2 controlling numbers of thymic iNKT cells. Interestingly, this locus resides within the Idd13 region that distinguishes NOD mice from the closely genetically related, but strongly T1D-resistant NOR strain. Thus, we tested if a genetic variant that confers T1D resistance in NOR mice may do so by enhancing iNKT cell numbers. iNKT cells were enumerated by an α-GalCer analog loaded CD1d tetramer in NOD and NOR mice as well as in NOD stocks carrying NOR-derived congenic regions on chromosome 1, 2, or 4. Significantly, more thymic and splenic iNKT cells were present in NOR than NOD mice. The NOR-derived Idd13 region on chromosome 2 contributed the most significant effect on increasing iNKT cell numbers. Subcongenic analyses indicated that at least two genes within the Idd13 region regulate iNKT cell numbers. These results further define the genetic basis for numerical iNKT cell defects contributing to T1D development in NOD mice.     

Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice

Insulin production afforded by hepatic gene therapy (HGT) retains promise as a potential treatment for type 1 diabetes, but successful approaches have been limited. We employed a novel and previously untested promoter for this purpose, glucose transporter-2 (GLUT2) to drive insulin production via delivery by recombinant adeno-associated virus (rAAV). In vitro, the GLUT2 promoter was capable of robust glucose-responsive expression in transduced HepG2 human hepatoma cells. Therefore, rAAV constructs were designed to express the furin-cleavable human preproinsulin B10 gene, under the control of the murine GLUT2 promoter and packaged for delivery with rAAV expressing the type 5 capsid. Streptozotocin-induced diabetic mice were subjected to hepatic portal vein injection immediately followed by implantation of a sustained-release insulin pellet to allow time for transgenic expression. All mice injected with the rAAV5-GLUT2-fHPIB10 virus remained euglycemic for up to 35 days post-injection, with 50% euglycemic after 77 days post-injection. In contrast, mock-injected mice became hyperglycemic within 15 days post-injection following dissolution of the insulin pellet. Serum levels of both human insulin and C-peptide further confirmed successful transgenic delivery by the rAAV5-GLUT2-fHPIB10 virus. These findings indicate that the GLUT2 promoter may be a potential candidate for regulating transgenic insulin production for hepatic insulin gene therapy in the treatment of type I diabetes.     

Glucose-modulated tyrosine nitration in beta cells: Targets and consequences

Hyperglycemia, key factor of the pre-diabetic and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in islets of Langerhans and insulinoma beta cells. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified function in protein folding, energy metabolism, antioxidant capacity, and membrane permeability. Nitration of heat shock protein 60 in vitro was found to decrease its ATP hydrolysis and interaction with proinsulin, suggesting a mechanism by which protein nitration could diminish insulin secretion. This was supported by our finding of a decrease in stimulated insulin secretion following glycolytic stress in cultured cells. Our results reveal that protein tyrosine nitration may be a previously unrecognized factor in beta-cell dysfunction and the pathogenesis of diabetes.     

2-NBDG as a fluorescent indicator for direct glucose uptake measurement

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research.     

Tfh 细胞在NOD小鼠糖尿病发病过程中的作用机制的研究

目的:研究滤泡辅助性T细胞(Follicular Helper T cell,Tfh)在非肥胖性糖尿病小鼠(Non-obese Diabetic mice,NOD)发病过程中的作用机制。方法:实验动物NOD小鼠按血糖值分为胰岛炎组(血糖浓度≤9 mmol/L)及糖尿病组(血糖浓度≥20 mmol/L)。ELISA法检测各组中糖尿病自身抗体谷氨酸脱羧酶抗体(65-kda glutamate decarboxylase antibody,GAD65Ab)、抗胰岛素自身抗体(Insulin autoantibody,IAA)表达水平,Western blot检测B细胞型淋巴瘤6蛋白(B-cell lymphoma 6 protein,Bcl-6)及可诱导共刺激分子(Inducible costimulatory molecule,ICOS)表达,流式细胞仪检测各组外周血及脾脏Tfh细胞水平。结果:糖尿病组NOD鼠自身抗体GAD65Ab(1.21±0.23 nmol/L)、IAA(0.96±0.12 nmol/L)浓度较胰岛炎组(0.32±0.09 nmol/L,0.25±0.06 nmol/L)均有明显升高;糖尿病组NOD鼠Bcl-6及ICOS表达较胰岛炎组NOD鼠有明显升高,外周血和脾脏Tfh细胞水平糖尿病组NOD鼠(24.55%)较胰岛炎组NOD鼠(4.27%)升高明显。结论:NOD小鼠自发糖尿病与自身抗体浓度升高相关,Tfh细胞可能参与NOD鼠糖尿病发生及发展过程。     

不同品系小鼠感染甲型流感病毒肺小血管内血栓形成

目的本实验旨在观察不同品系小鼠感染甲型流感病毒后肺组织内血栓形成的情况。方法使用H1N1病毒A/California/7/2009（CA7）株和H3N2病毒A/Brisbane/10/07株,对BALB/C小鼠、Scid小鼠、NOD/LTJ小鼠、BALB/C-nu小鼠、NOD-Scid小鼠和icosl-KO小鼠经乙醚麻醉后进行滴鼻攻毒。检测小鼠感染后肺组织病毒拷贝数并观察肺组织病理学改变。结果 H1N1和H3N2滴鼻攻毒的各组小鼠均染毒,病理表现为程度略有差异的间质性肺炎。13只H1N1病毒感染小鼠和6只H3N2感染小鼠在肺组织中观察到多个小血管内有血栓形成,血栓成分主要为纤维素和血小板。结论各品系小鼠感染H1N1和H3N2流感病毒后均可能出现肺组织内血栓形成。     

Sphingosine 1-phosphate (S1P) regulates glucose-stimulated insulin secretion in pancreatic beta cells

Recent studies suggest that sphingolipid metabolism is altered during type 2 diabetes. Increased levels of the sphingolipid ceramide are associated with insulin resistance. However, a role for sphingolipids in pancreatic beta cell function, or insulin production, and release remains to be established. Our studies in MIN6 cells and mouse pancreatic islets demonstrate that glucose stimulates an intracellular rise in the sphingolipid, sphingosine 1-phosphate (S1P), whereas the levels of ceramide and sphingomyelin remain unchanged. The increase in S1P levels by glucose is due to activation of sphingosine kinase 2 (SphK2). Interestingly, rises in S1P correlate with increased glucose-stimulated insulin secretion (GSIS). Decreasing S1P levels by treatment of MIN6 cells or primary islets with the sphingosine kinase inhibitor reduces GSIS. Moreover, knockdown of SphK2 alone results in decreased GSIS, whereas knockdown of the S1P phosphatase, Sgpp1, leads to a rise in GSIS. Treatment of mice with the sphingosine kinase inhibitor impairs glucose disposal due to decreased plasma insulin levels. Altogether, our data suggest that glucose activates SphK2 in pancreatic beta cells leading to a rise in S1P levels, which is important for GSIS.     

Discovery of non-glucoside SGLT2 inhibitors

A series of benzothiazinone and benzooxazinone derivatives were discovered as SGLT2 inhibitors. The optimization led to the discovery of compounds 31 and 32, which exhibited similar potency and better SGLT1 selectivity compared to dapagliflozin. These compounds may provide novel promising scaffolds, which are different from phlorizin-based SGLT2 inhibitors.     

Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells

Glucagon-like peptide-1 (GLP-1), a meal-stimulated gastrointestinal insulinotropic hormone inactivated by dipeptidyl peptidase-IV (DPP-IV), is reduced in type 2 diabetic patients. The present study shows that 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPP-IV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity. On the other hand, incubation of purified DPP-IV in a buffer solution added with high glucose does not affect enzyme activity. These results suggest that high glucose increases expression and activity of DPP-IV, possibly contributing to GLP-1 reduction in type 2 diabetic patients.     

葡萄糖转运蛋白-1在胰腺癌组织中的表达及其与细胞增殖和凋亡的相关性研究

目的探讨胰腺癌组织中葡萄糖转运蛋白-1(Glut-1)的表达及其与细胞增殖、凋亡的关系。方法采用免疫组化链霉抗生物素蛋白-过氧化物酶连接法(SP法)检测15例正常胰腺组织和49例胰腺癌组织中Glut-1、增殖细胞核抗原(PCNA)蛋白的表达,并计算增殖指数(PI)。采用原位末端标记法(TUNEL)检测胰腺癌凋亡细胞,计算凋亡指数(AI)。结果Glut-1在正常胰腺组织中不表达,而在胰腺癌组织中阳性表达率为73.47%(36/49),胰腺癌组织中Glut-1的阳性表达率明显高于正常胰腺组织(P<0.01)。正常胰腺组织及癌组织中的AI分别为0.41%±0.13%和5.93%±4.18%,两者比较有显著性差异(P<0.05)。而两组中的增殖指数分别为6.25%±2.59%和32.54%±14.69%,胰腺癌组织的细胞增殖程度明显高于正常胰腺组织(P<0.05)。随胰腺癌细胞Glut-1蛋白表达水平升高,肿瘤细胞增殖活性相应增加,而凋亡则相应减少。Glut-1表达与胰腺癌细胞增殖呈正相关性(r=0.726,P<0.01),与凋亡程度无相关性(r=-0.102,P>0.05)。结论Glut-1在胰腺癌组织中表达增高,Glut-1与细胞的增殖密切相关,可能在胰腺癌的发生发展中起重要作用。     

Molecular target structures in alloxan-induced diabetes in mice

Type 1 diabetes results from irreversible damage of insulin-producing beta-cells. In laboratory animals, diabetes can be induced with alloxan (ALX), a 2,4,5,6-tetraoxopyrimidine. ALX is a potent generator of reactive oxygen species (ROS), which can mediate beta-cell toxicity. However, the initial lesions on essential beta-cell structures are not known. In this study, we report that the glucose transporter 2 (GLUT2) and glucokinase (GK) are target molecules for ALX. Ex vivo, a gradual decrement of both GLUT2 and GK mRNA expression was found in islets isolated from ALX-treated C57BL/6 mice. This reduction was more pronounced for GLUT2 than for GK. The mRNA expression of beta-actin was also slightly affected with time after ALX exposure, the proinsulin mRNA, however, remained unaffected as well as the pancreatic total insulin content. Pretreatment with D-glucose (D-G) protected the mRNA expression of GLUT2 and GK against ALX toxicity and prevented diabetes. Yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. Pretreatment with 5-thio-D-glucose (5-T-G) failed to prevent ALX diabetes, administration of zinc sulfate (Zn(2+))-enriched drinking water, however, reduced ALX-induced hyperglycemia. In conclusion, ALX exerted differential toxicity on beta-cell structures similar to in vitro results reported from this laboratory. Furthermore, the present results differ from those reported for the diabetogen streptozotocin (STZ). Injections of multiple low doses (MLD) of STZ reduced GLUT2 expression only, but failed to affect expression of GK and proinsulin as well as beta-actin as internal control. MLD-STZ diabetes was prevented by pretreatment with both D-G and 5-T-G and administration of Zn(2+)-enriched drinking water. Apparently, ALX and MLD-STZ exert diabetogenicity by different pathways requiring different interventional schedules for prevention.