Differential changes in islet amyloid polypeptide (amylin) and insulin mRNA expression after high-fat diet-induced insulin resistance in C57BL/6J mice

Islet amyloid, derived from islet amyloid polypeptide (IAPP or amylin), frequently occurs in type 2 diabetes. Availability of this peptide for amyloid formation may be enhanced by increased islet expression of IAPP. In the insulin resistant state, euglycemia is maintained by hypersecretion of insulin. Whether     

链脲佐菌素诱导C57BL/6小鼠发生糖尿病视网膜病变

目的研究糖尿病视网膜病变小鼠的眼底变化。方法给6周龄的C57BL/6小鼠腹腔注射链脲佐菌素(STZ)建立糖尿病模型。模型建立后5 w和10 w,分别行眼内压测量和视网膜电图(ERG)检测以及免疫荧光血管染色检查。结果与对照组小鼠相比较,糖尿病小鼠的眼压升高(P<0.01),ERG的a、b波振幅下降,免疫荧光染色可见糖尿病小鼠视网膜血管异常。结论糖尿病会导致小鼠眼底发生异常改变。这些病理改变可能是糖尿病视网膜病变的重要组成部分。     

Calcium and calmodulin changes with ageing in C57BL/6J mice

Male C57BL/6J mice ranging in age from 50 to 1186 days were used to measure total calcium and calmodulin concentrations. The increase in calcium between 0 and 1,000 days of age was 260% for kidney, followed by brain (189%), heart (173.5%), lung (106.5%) and liver (78.5%). Calcium in femur declined by 28.2%. The calmodulin content of liver increased with ageing. Both liver and kidney calmodulin concentrations declined early in life followed by ageing-related increases. Brain, lung and heart calmodulin concentrations did not change significantly with ageing. We conclude that changes in calcium homeostasis are not reflected in calmodulin changes. The loss of calcium in bone is consistent with the occurrence of osteoporosis in ageing C57 mice.     

Protection of nonobese diabetic mice from autoimmune diabetes by reduction of islet mass before insulitis.

Nonobese diabetic mice spontaneously develop diabetes that is caused by autoimmune cell-mediated destruction of pancreatic beta cells. Here we report that surgical removal of 90% of pancreatic tissue before onset of insulitis induced a long-term diabetes-free condition in nonobese diabetic mice. Pancreatectomy after development of moderate insulitis had no effect on the course of diabetes. The effect of pancreatectomy was abrogated with subsequent development of diabetes by infusion of islet-cell-specific T lymphocytes and by transplantation of pancreatic islets. Lymphocytes from pancreatectomized diabetes-free mice exhibited low response to islet cells but responded normally to alloantigens. These results suggest that the islet cell mass plays a critical role in development of autoimmune diabetes.     

Developmental changes in the hypoxic ventilatory response in C57BL/6 mice.

C57BL/6 mice are the strain into which most null mutations for neurotransmitters or their receptors are backcrossed. A number of these transgenic mice have recently been shown to have an abnormal respiratory phenotype; however, the postnatal development of the ventilatory response to hypoxia has not been characterized in C57BL/6 mice. The effect of 8% oxygen for 5 min was examined in mice at five periods from P1 to P30 using a body plethysmograph. Neonatal and juvenile animals from P7 to P30 showed a biphasic pattern in hypoxia in which the increase in minute ventilation achieved in the first min declined towards baseline by the fifth minute and was decreased below baseline in the first minute of return to air breathing. In contrast P1-P3 C57BL/6 mice had a sustained increase in both respiratory frequency and tidal volume and their minute volume remained above baseline on return to air. The decline in oxygen consumption, measured in the fifth minute of hypoxia, was not different in P1-P3 mice compared to P8-P10. These results suggest that the earliest response to hypoxia of the respiratory system in this strain is not characterized by a time dependent depression as seen in older animals and in species whose motor systems are relatively more developed at birth.     

Diet promotes beta-cell loss by apoptosis in prediabetic nonobese diabetic mice.

Diet as an environmental factor influences age of onset in models of spontaneous insulin-dependent diabetes mellitus. We reported recently that a protein-rich diet accelerated diabetes incidence in nonobese diabetic (NOD) mice. In the present study, we investigated the effect of diet on beta-cells and glucose metabolism in NOD mice before diabetes onset. Three different diets were maintained from 4 weeks on: low fat (LF; 12% fat, 21% protein, and 68% carbohydrates), high fat (HF; 39% fat, 17% protein, and 43% carbohydrate), and high fat-high protein (HFHP; 43% fat, 38% protein, and 19% carbohydrates) diet. The cumulative incidence of diabetes was 92% for HFHP (P < 0.01 vs. LF), 80% for HF (P = NS), and 65% for the LF cohort. At 20 weeks of age insulin secretion in the isolated pancreas was doubled for the HF diet and 4.4 times higher for the HFHP-fed mice compared with the LF group. Feeding HF and HFHP reduced total glucose utilization during continuous insulin infusion (1 mU/kg) by 34% (P < 0.05). HFHP, but not HF, diet elevated endogenous glucose production by 48% (P < 0.05) compared with that in the LF group. Beta-cell mass, estimated by imaging analysis, was initially high in young HFHP-fed mice, aged 10 weeks, but declined rapidly thereafter [HFHP, 1.6 +/- 0.2 (P < 0.05 vs. LF); HF, 2.4 +/- 0.4 (P = NS vs. LF); LF, 2.1 +/- 0.5 mg at 30 weeks]. A reduction of beta-cell mass was associated with HF 14% (P < 0.05 vs. LF) and HFHP 82% (P < 0.01 vs. LF) more apoptotic beta-cells at 30 weeks. Depending on age, 1.2-3.1 of 1000 beta-cells were in a stage of proliferation without significant differences among the dietary groups. In conclusion, HFHP diet was associated with impaired glucose metabolism and high insulin release followed by enhanced diabetes incidence. Diabetes was promoted by increased rate of cell death over beta-cell neogenesis.     

Estrogenic stimulation of hypothalamic-limbic system metabolism in ageing diabetic C57BL/KsJ mice.

The therapeutic influences of estrogen treatment on age- and diabetes-related declines in regional brain glucose utilization (RBGU) rates were evaluated in 8- to 20-week-old female C57BL/KsJ normal (+/?) and diabetic (db/db) mice. Following either oil vehicle (oil: 0.1 ml) or estradiol (E: 1 microgram/3.5 days) treatments starting at 3 weeks of age, RBGU rates were subsequently determined at 8, 12, 16 and 20 weeks of age. A gradual decline in the basal rate of brain glucose utilization was observed in all control (oil- and E-treated) groups between 8 and 20 weeks. Expression of the hyperglycemic-obese diabetes syndrome in db/db mice resulted in a significant reduction in RBGU rates between 8 and 20 weeks relative to control values. In estrogen-sensitive hypothalamic, septal and amygdaloid regions, E therapy modulated RBGU rates in db/db mice relative to oil-treated diabetics, but did not significantly alter utilization rates in +/? mice. In cortical samples, E therapy had no significant influence on glucose utilization rates in either control or diabetic groups. A noticeable pattern of maturation-associated decline in CNS glucose utilization rates in all brain regions resulted in comparable regional metabolic indices being exhibited by all groups at 20 weeks of age, with the exception of the diabetes-associated exacerbation of RBGU rates in the oil-treated db/db group. These data demonstrate that the normal development-related decline in regional brain carbohydrate metabolism is accelerated by the diabetes syndrome, and that E therapy can modulate the syndrome-associated suppression of glucose utilization in steroid-sensitive CNS loci. These data suggest that the depressive influences of the diabetes syndrome on brain carbohydrate utilization rates may be therapeutically modified in recognized CNS regions possessing steroid-sequestering, metabolically responsive neurons.     

Aging is associated with increased T-cell chemokine expression in C57BL/6 mice

To better understand the contribution of the chemokine system in immune senescence, we determined the aging effect on CD4+ and CD8+ T-cell chemokine expression by microarray screening and ribonuclease protection assays. Compared with young C57BL/6 mice, freshly isolated CD4+ cells from aged mice express increased level of interferon-gamma-inducible protein 10 (IP-10), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated upon activation, normal T-cell expressed and secreted (RANTES), and lymphotactin (Ltn). T-cell receptor (TCR)/coreceptor stimulation up-regulates MIP-1alpha, MIP-1beta, and Ltn, and down-regulates IP-10 and RANTES expression in CD4+ T cells. A similar increase in chemokine expression was demonstrated in the CD8+ T cell. Enzyme-linked immunosorbent assays confirmed increased T-cell chemokine protein production in old CD4+ and CD8+ T cells. Finally, supernatant of cultured T cells from old animals caused an enhanced leukocyte chemotaxis response compared with that from young animals, suggesting that the age-related difference in T-cell chemokine expression has an important functional consequence.     

泡球蚴感染C57BL/6小鼠肝脏CD34表达的动态变化

目的 观察泡球蚴(Em)感染C57BL/6小鼠肝脏CD34微血管密度(MVD)的动态变化。方法 将40只C57BL/6小鼠随机分为实验组和对照组,实验组通过肉眼肝叶穿刺感染泡球蚴(Em),对照组通过注射等量PBS。分别于感染后30 d、60 d、90 d及120 d后取小鼠肝脏组织,苏木素-伊红(HE)染色观察肝脏的病理组织学改变,免疫组织化学染色观察泡球蚴感染后CD34-MVD的动态改变。结果 实验组泡球蚴组织各时间点中CD34-MVD分别为(39.10±11.84)/HP、(66.80±11.08)/HP、(111.20±8.00)/HP和(56.14±7.12)/HP,实验组泡球蚴周围肝组织分别为 (1.14±0.82)/HP、(1.56±0.92)/HP、(2.32±1.43)/HP和(1.38±0.82)/HP ;对照组分别为(1.00±0.94)/HP、(1.30±1.06)/HP、(2.00±1.15)/HP和(1.10±0.87)/HP。实验组与对照组比较,差异均有统计学意义(P<0.01);实验组泡球蚴组织内比较,差异有统计学意义(F=94.63,P<0.001);实验组泡球蚴周围肝组织内比较,差异无统计学意义(F=3.24,P>0.05);对照组内比较,差异无统计学意义(F=1.98,P>0.05)。结论 泡球蚴浸润性生长过程中可能伴随着血管新生,血管新生可能为其生长重要的机制之一。     

Inflammatory and antioxidant gene expression in C57BL/6J mice after lethal and sublethal ozone exposures

Ozone (O3) is a highly reactive and toxic oxidant pollutant. The objective of this study is to compare cytokine, chemokine, and metallothionein (Mt) changes elicited by lethal and sublethal exposure to ozone in a genetically sensitive strain of mice. Eight-week-old C57BL/6J mice were exposed to 0.3 ppm ozone for 0, 24, or 96 hours; 1.0 ppm ozone for 0, 1, 2, or 4 hours; or 2.5 ppm ozone for 0, 2, 4, or 24 hours. After 24 hours of exposure to 0.3 ppm ozone, increases in mRNA abundance were detected for messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2. These increases persisted through 96 hours of exposure. At this time point messages encoding lymphotactin (Ltn) and metallothionein were also increased. After 4 hours of 1.0 ppm ozone exposure, increases in mRNA abundance were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, and interleukin (IL)-6. Mt mRNA abundance was increased after 1 hour of exposure and persisted through 4 hours, although the magnitude of the alterations increased. After 2 hours of 2.5 ppm ozone exposure, increases were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, IL-6, and Mt. These increases persisted through 4 hours of exposure. Lung weights of mice exposed to 2.5 ppm ozone for 24 hours were approximately 2 times greater than air-exposed mice. At this dose lethality occurred by 36 hours. Increased mRNAs for eotaxin, MIP-1 alpha, MIP-2, and Mt were to a higher magnitude than were detected after 2 and 4 hours of exposure. Messages encoding IL-12, IL-10, interferon (IFN)-gamma, IL-1 alpha, IL-1 beta, and IL-1Ra were unaltered at all time points and doses examined. Our results demonstrate dose- and time-dependent changes in chemokine, cytokine, and Mt mRNA abundance and that early acute changes may be predictive of subacute and chronic responses to ozone.     

Limits of a deletion spanning Tlr4 in C57BL/10ScCr mice

Proceeding from our observation that LPS-unresponsive mice of the strain C57BL/10ScCr mice fail to express the Tlr4 gene [Poltorak A, He X. Smirnova I et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998; 282: 2085], we have defined the exact limits of a deletion encompassing Tlr4 in the C57BL/10ScCr genome. The deletion removes 74723 bp of DNA, with reference to the control strain 129/J (from which the complete sequence of the Tlr4 locus was obtained). There is no inserted element, and no re-arrangement of the chromosome (e.g. inversion or translocation) in the immediate region of Tlr4; the deletion removes only one recognizable gene. Hence, other immunological anomalies that have been identified in C57BL/10ScCr mice (a non-healing phenotype in Leishmania inoculation and failure to produce interferon-gamma in response to numerous microbial infections) must be ascribed to one of two causes. Mutation(s) at other loci may be responsible for these defects. Alternatively, Tlr4 locus deletion may have phenotypic consequences that exceed the well known blockade of LPS signal transduction.     

Fas ligand-mediated mechanisms are involved in autoimmune destruction of islet beta cells in non-obese diabetic mice

Aims/hypothesis. A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes. Methods. We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated. Results. We found 58 % (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4⁺ T cells, CD8⁺ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies. Conclusion/interpretation. These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes. [Diabetologia (2000) 43: 1149–1156] Received: 31 March 2000 and in revised form: 5 June 2000     

Age-related changes in hypothalamic-pituitary-adrenal axis activity of male C57BL/6J mice

As there is little known about age-related changes in the hypothalamic-pituitary-adrenal (HPA) axis of mice, we determined the daily patterns of corticosterone secretion every 2 h, together with adrenocorticotropic hormone (ACTH) release and central HPA axis markers in the morning and evening of 3-, 9- and 16-month-old male C57BL/6J mice. We observed that: (i) corticosterone secretion showed a distinct age-related circadian pattern. During the light period this was expressed by relative hypercorticism in 9-month-old mice and relative hypocorticism in 16-month-old mice. ACTH was elevated at 16 months of age; (ii) mineralocorticoid (MR) and glucocorticoid receptor (GR) mRNA expression in the hippocampus was significantly decreased in 9-month-old mice, whereas in 16-month-old mice, expression was similar to young animals. Circadian variation was modest in all age groups; (iii) the parvocellular hypothalamic paraventricular nucleus (PVN) expressed very high vasopressin mRNA, which was subject to circadian variation in 3- and 9-month-old mice. Furthermore, significant levels of MR mRNA were expressed in the PVN. In conclusion, basal HPA axis activity and expression of its central regulatory markers are age-dependent in mice. This suggests that the capacity to adjust to environmental demands is either a function of age, or depends on different dynamics of the HPA axis.     

醛糖还原酶遗传缺失可显著减缓C57BL/6小鼠糖尿病肾病进程

目的 研究醛糖还原酶在糖尿病肾病发生、发展中的作用.方法 健康雄性8周龄野生型C57BL/6小鼠(WT,n=6)、醛糖还原酶基因敲除型C57BL/6小鼠(KO,n=6)和醛糖还原酶双转基因型C57BL/6小鼠(BT,n=6)腹腔注射40μg/g链脲佐菌素,诱导糖尿病模型.另18只小鼠(每种品系各6只)注射枸橼酸盐缓冲液,作为对照.17周后测定体重、肾重、血糖、血清甘油三酯、胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、尿素氮、肌酐、肾小球滤过率、尿白蛋白等指标.采用过碘酸雪夫染色观察肾小球组织形态学变化,应用Western blot和免疫组织化学法检测肾皮质中胶原蛋白Ⅳ和转化生长因子-β1蛋白表达量,选用Pep Tag非放射性蛋白激酶C检测方法检测肾皮质细胞质和细胞膜中蛋白激酶C活性.多组间数据比较采用单因素方差分析.结果 醛糖还原酶基因敲除显著改善了糖尿病小鼠的生理生化指标、肾皮质、肾小球组织形态以及肾功能.糖尿病KO小鼠和糖尿病BT小鼠尿白蛋白分别较WT小鼠下降了43%[分别为(1.49±0.26)和(2.62±0.34)mg/g肌酐,F=20.8,P＜0.01]和48%[分别为(1.36±0.12)和(2.62±0.34)mg/g肌酐,F=20.8,P＜0.01].同时,醛糖还原酶基因缺失亦抑制了高糖对肾皮质蛋白激酶C和转化生长因子-β1的激活.结论 醛糖还原酶基因缺失显著改变了糖尿病肾病的进程,抑制醛糖还原酶可能有助于糖尿病肾病的防治.     

Glomerular hyperfiltration during the onset of diabetes mellitus in two strains of diabetic mice (C57BL/6Jdb/db and C57BL/KsJdb/db)

Summary GFR estimated by the total clearence of⁵¹Cr-EDTA increases from 41 to 60 ml/min·m² at the onset and during the development of marked hyperglycaemia and obesity in female diabetic mice (db/db) of the C57BL/6J and the C57BL/KsJ strains (blood sugar rises from 160 to 400 mg/100 ml). GFR decreases slowly in older diabetic mice (>120 days old) approaching the control values (42±7 ml/ min·m²) and then decreases still further. The total clearance of¹⁴C-hippuric acid is unaltered indb/db mice and controls between 45 and 150 days of age (96±20 ml/min·m²). This suggests no alteration of RPF during the development of the diabetic syndrome. The glomerular hyperfiltration of diabetic mice lasts until they are 120 days old and shows no correlation with the different blood glucose levels typical for diabetic mice (db/db) of the two strains.     

Genetic control of endogenous C-type virus production in pancreatic acinar cells of C57BL/He and C57BL/6J mice.

Electron microscopy revealed very active production of C-type virus particles in the pancreatic acinar cells of untreated normal adult mice of the C57BL/He strain. In C57BL/6J mice, a similar picture was observed after a single intraperitoneal injection of dexamethasone. No viruses were observed in the pancreas of untreated or dexamethasone-treated BALB/c and C3Hf mice. F1 hybrids of both C57BL strains with C3Hf mice produced viruses in the same manner and quantity as the C57BL parents, whereas hybrids with BALB/c mice were entirely negative. Approximately 50% of mice of the first backcross generation of (BALB/c times C57BL/He)F1 hybrids with C57BL/He mice were active producers of C-type particles, while the other 50% were negative. It is suggested that a regulator gene that controls C-type virus production does not function in the pancreatic cells of either C57BL strain, and that BALB/c mice can provide hybrids with an active regulator.     

Body temperature of C57BL/6J mice with age

On the basis of a study of rectal temperature in a group of 180, C57BL/6J male mice, ranging in age from 3 months to 30 months, the following conclusions were drawn: 1) The positive correlation between body weight and body temperature typical for rodents was found only for young adults of the C57BL/6J strain; 2) body temperature of male C57BL/6J did not appear to decline until about 23.5 months, after which there was a significant negative correlation (r = -0.53) between age and temperature.     

紫外线辐照致弱日本血吸虫尾蚴诱导C57BL/6 小鼠皮肤早期免疫应答动态变化的研究

目的观察紫外线辐照致弱日本血吸虫尾蚴经耳廓免疫C57BL/6小鼠后,小鼠皮肤组织早期免疫应答的动态变化.方法 98只小鼠测定双侧耳廓厚度后,14只作为0 d组不感染/免疫,其余平均分为2组.一组经双侧耳廓分别感染正常尾蚴150 条/耳, 另一组经双侧耳廓分别免疫紫外线辐照致弱尾蚴150条/耳.感染/免疫后第1、2、4、7、14、21天分别剖杀2组小鼠各7只,测定耳廓厚度.取感染处的耳廓组织,左侧进行培养,收集上清检测相应细胞因子.右侧纵切为二,一半进行HE染色观察炎症反应;另一半应用免疫组化技术观察皮肤组织中IL-12和CD11c分子的表达.结果正常尾蚴感染的小鼠皮肤炎症反应在第7天达到高峰后逐渐下降,第21天基本恢复;而辐照尾蚴免疫小鼠耳廓皮肤在第14天炎症反应才达高峰,第21天反应仍然十分强烈.尾蚴穿皮后第4天,组织中有较高水平的CD11c及IL-12分子表达.皮片培养细胞因子定量检测也显示：与Th1细胞应答相关的IL-12、IFN-γ及Th2型因子IL-10早期在正常、辐照尾蚴差异无显著性.结论和正常尾蚴相比,辐照致弱尾蚴诱导的皮肤免疫应答持续时间长、强度高,但两者诱导Th细胞向不同方向极化并不发生于免疫应答启动阶段.