Hepatic metabolism of genetically diabetic (db/db) mice. I. Carbohydrate metabolism.

Hepatic carbohydrate metabolism in genetically diabetic mice (db/db) and their normal littermates has been studied. In db/db mice, body water was below normal and declined with age. The liver of db/db mice was abnormally large in relation to the metabolic mass of the body at all ages studied. In db/db mice, hepatic glycogenolysis, glycogen synthesis, glycogen synthetase, and phosphorylase were markedly increased. Gluconeogenesis from alanine or lactate in perfused livers of db/db mice was greater than normal per 100 g body water. Activities of fructose-1, 6-biophosphatase, glucose-6-phosphatase, glucokinase + hexokinase, and pyruvate kinase were elevated in livers of db/db mice. Diabetic mouse livers perfused with lactate showed a markedly reduced concentration of P-enolpyruvate and clear "forward crossover" between fructose-1, 6-P2 and fructose-6-P. In vivo glucose clearance, measured with [3-3H]glucose, in db/db mice was 170% that of normal mice. Data presented indicate that in livers of db/db mice: 1) glucose production is elevated prior to hyperglycemia, 2) glycogen turns over more rapidly, and 3) glycolytic and gluconeogenic enzymes are elevated paradoxically. These abnormalities are discussed from the viewpoint of their etiology.     

Differential effect of statins on diabetic nephropathy in db/db mice

Recent studies suggest a potential benefit of the lipid-lowering medication in the treatment of chronic kidney disease (CKD) such as diabetic nephropathy. Although statins have been widely used to lower serum cholesterol levels, the effect of these drugs on diabetic nephropathy has not been fully elucidated. In the present study, therefore, we addressed the role of different kinds of statins on diabetic nephropathy in db/db mice. Mice were fed with a standard diet with 0.005% (w/w) of pitavastatin, rosuvastatin, and pravastatin for 8 weeks starting from 8 weeks of age. The treatment with statins did not affect the food intake, body weight gain, adiposity, or blood pressure in db/db mice. Treatment with statins also had no effect on plasma lipid levels. In terms of the effect on albuminuria, pitavastatin and rosuvastatin reduced the urinary excretion of albumin by 60 and 40%, respectively, but not pravastatin, suggesting the effect of these two drugs on diabetic nephropathy. Furthermore, pitavastatin and rosuvastatin improved glomerular hypertrophy. All statins treatment improved insulin resistance. In addition, rosuvastatin and pravastatin treatment reduced oxidative stress measured by urinary 8-OHdG level, whereas the statins had no effect on the inflammatory response in the kidney of db/db mice. These results are not consistent with the renoprotective effect of statins. In conclusion, our data suggest that pitavastatin and rosuvastatin can improve diabetic nephropathy through the suppression of glomerular hypertrophy, independent of lipid-lowering or anti-oxidative effects.     

In vitro and in vivo evidence that autoimmune reactivity to collagen develops spontaneously in Schistosoma mansoni-infected mice

Egg-induced granuloma formation in murine schistosomiasis mansoni results from vigorous anti-parasite reaction by activated T cells, macrophages, eosinophils, and fibroblasts. The present study suggests that strain-specific, autoimmune T-cell reactivity directed against host matrix proteins might also contribute to granulomatous hypersensitivity. T cells from infected C57B1/6, but not from CBA or BALB/c mice, proliferative in vitro in response to denatured collagen. T cells from uninfected mice, previously immunized with soluble egg antigen (SEA), did not respond in vitro to collagen. Spleen cells from acutely infected mice, but not chronically infected or uninfected animals, formed granulomas around collagen-coupled polyacrylamide beads in vitro. This response was blocked by anti-collagen antibodies that had no inhibitory effect on in vitro granuloma formation around SEA-coupled beads. In related in vivo studies, granuloma formation was quantitated after iv injection of SEA-, collagen-, or uncoated beads into normal or infected recipients. The mean diameter of lung granulomas induced by collagen-coupled beads in infected mice was significantly greater than the diameter of granulomas around either collagen beads in uninfected mice or uncoated beads in infected mice. these observations indicate that anti-collagen responses develop spontaneously in Schistosoma-infected mice and suggest that such reactivity might play a secondary role in granuloma formation and the pathogenesis of hepatic fibrosis.     

凋亡细胞在db/db自发性糖尿病小鼠颌下腺的分布

目的:观察凋亡细胞在db/db糖尿病小鼠颌下腺中的分布。方法:选取3、4、6、8、10月龄db/db糖尿病小鼠及相应月龄的dh/m~(?)小鼠颌下腺,应用TUNEL标记方法染色后进行图像分析.统计凋亡细胞在颌下腺组织中分布的细胞阳性率。结果:随着糖尿病的发展,颌下腺组织出现腺体萎缩及颗粒曲管数目减少,实质细胞排列不整齐,呈簇状堆集,纤维及血管增多。凋亡细胞在对照组及糖尿病组颌下腺中均有分布,糖尿病组凋亡细胞阳性率高于对照组。糖尿病组与对照组凋亡细胞阳性率随月龄增大均呈增加趋势。结论:db/db糖尿病可导致颌下腺组织萎缩及实质细胞形态学改变;凋亡细胞阳性率在糖尿病组随疾病发展而增加显著。这与糖尿病腺体萎缩和功能受损相一致。     

Ⅱ型糖尿病模型db／db小鼠胰岛α细胞超微结构变化

目的:观察Ⅱ型糖尿病模型 db/db 小鼠胰岛α细胞的超微结构变化,探讨α细胞的病理改变与Ⅱ型糖尿病病因的关系.方法:选取 3、5、8 月龄尾静脉空腹血糖高于10.1 mmol/L,且肥胖的 db/db 自发性精尿病小鼠作为糖尿病组;选取相应年龄段尾静脉空腹血糖低于 6.0 mmol/L,体质量正常的 db/ m 表型正常小鼠作为对照组.于相应年龄段取胰尾,透射电镜观察胰岛α细胞超微结构.结果:精尿病组胰岛α细胞胞质内分泌颗粒数量增多,可见致密芯与界膜间的间隙变窄,有的消失;线粒体和粗面内质网增多,细胞器结构未见明显改变.结论:胰岛α细胞超微结构改变和功能异常可能与Ⅱ型糖尿病的发病有关.     

Gender-dependent attenuation of cardiac potassium currents in type 2 diabetic db/db mice

Single ventricular myocytes were prepared from control db /+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 μ m , 5–9 h). In cells from female db/db mice (12 weeks of age), K⁺ currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocte-specific insulin receptor knockout (CIRKO) mice. Both K⁺ currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K⁺ currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K⁺ currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K⁺ currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin–angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K⁺ currents in cells from both males and females.     

The role of interstitial macrophages in nephropathy of type 2 diabetic db/db mice

Diabetic nephropathy is associated with interstitial macrophage infiltrates, but their contribution to disease progression is unclear. We addressed this question by blockade of chemokine receptor (CCR)1 because CCR1 mediates the macrophage recruitment to the renal interstitium. In fact, when CCR1 was blocked with BL5923, a novel orally available CCR1 antagonist, the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with advanced diabetic nephropathy. Likewise, BL5923 (60 mg/kg, twice a day) orally administered from months 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, transforming growth factor-beta1, and collagen I-alpha1 when compared with untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for interstitial macrophages for tubulointerstitial injury, loss of peritubular microvasculature, interstitial inflammation, and fibrosis in type 2 diabetic db/db mice. These data identify oral treatment with the CCR1 antagonist BL5923 as a potential therapy for late-stage diabetic nephropathy.     

Thermoregulation in the diabetic-obese (db/db) mouse

1. Thermoregulation and non-shivering thermogenesis have been studied in the genetically diabeticobese (db/db) mouse.
2. At all environmental temperatures between 33 and 10°C the body temperature of the diabetic mice was lower than that of the normal littermates, the difference varying from 1.1°C at 33°C to 4.5°C at 10°C.
3. At 4°C the diabetic mice rapidly died (3.2 h) of hypothermia while the normal mice maintained their body temperature within the normal range.
4. At 23°C the diabetic animals exhibited a diurnal rhythm in body temperature which was similar in both phase and amplitude to the controls, but at every point throughout the 24h cycle the temperature of the mutants was lower by 1–2°C.
5. The resting metabolic rate at thermoneutrality (33°C) was higherper whole animal for the diabetics than for the normals. However, at temperatures below thermoneutrality the converse was observed; between 30 and 4°C the RMR of the mutants was lower than the controls by approximately 25%.
6. The capacity for non-shivering thermogenesis in diabetic mice was only one-half that found in normal animals.
7. The diabetic mouse has abnormalities in thermoregulation and non-shivering thermogenesis which are similar to those found in the genetically obese (ob/ob) mouse.

It is concluded that the high metabolic efficiency of the diabetic mouse, like that of the ob/ob mouse, can be explained by a reduced energy expenditure on thermoregulatory thermogenesis; this may represent a primary mechanism for the operation of the “thrifty genotype” associated with obesity and diabetes.     

Mibefradil reduces blood glucose concentration in db/db mice

OBJECTIVE:

Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca²⁺]_i) is a common defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanism in the defective pancreatic β-cell function in type 2 diabetes. T-type Ca²⁺ channel antagonists lower blood glucose in type 2 diabetes, but the mechanism remains unknown.

METHODS:

We examined the effect of the Ca²⁺ channel antagonist mibefradil on blood glucose in male db/db mice and phenotypically normal heterozygous mice by intraperitoneal injection.

RESULTS:

Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from 430.92±20.46 mg/dl to 285.20±5.74 mg/dl in three days. The hypoglycemic effect of mibefradil was reproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca²⁺ channels, but not by the specific L-type Ca²⁺ channel blocker nicardipine. Mibefradil did not show such hypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake were significantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis, immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-type Ca²⁺ channel α-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those from heterozygous animals.

CONCLUSIONS:

Collectively, these results suggest that T-type Ca²⁺ channels are potential therapeutic targets for antidiabetic drugs.     

SPARC在db/db小鼠肾脏组织中高表达

目的 检测db/db鼠肾脏组织中的富含半胱氨酸的酸性分泌蛋白(SPARC)的表达情况.方法 RT-PCR、Western blot及免疫荧光方法检测db/db鼠肾脏组织中的SPARC mRNA及蛋白的表达.结果 SPARC在db/db鼠肾脏组织中呈现高表达.结论 db/db鼠肾脏组织中的SPARC呈现高表达(P＜0.05),可能与糖尿病肾病的发生与发展有关.     

Feeding, activity, and body temperature following 6-hydroxydopamine lesions in diabetes (db/db) mice

Reductions in central catecholamines produced by intraventricular injections of 6-hydroxydopamine (6-OHDA) cause weight loss and decreased plasma glucose in diabetes (db/db) mice. The effects of this treatment were examined in short-term (64-day) and long-term (120-day) survival groups. Diabetes mice treated with 6-OHDA decreased food intake, lost weight, and maintained a lower weight than vehicle-treated controls until vehicle-treated animals began to enter the terminal stages of the syndrome, indicated by a loss of body weight. Diabetes mice given 6-OHDA lost weight despite reduced body temperatures and activity levels. Blood glucose levels were always lower in 6-OHDA than in ad lib fed vehicle-treated db/db mice. The 6-OHDA treatment also improved pancreatic islet granulation. Pair feeding vehicle-treated with 6-OHDA-treated db/db mice did not halt weight gain in the vehicle-treated group. However, measurement of carcass fat indicated similar losses in db/db-6-OHDA mice and vehicle-treated mice when the vehicle group was pair-fed with lean controls. Treatment with 6-OHDA produced long-term improvement in the diabetes syndrome, but the decreased body weight of the 6-OHDA-treated diabetes mice could not be completely accounted for by changes in food intake or measured indices of energy expenditure.     

Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet

Non‐alcoholic fatty liver disease (NAFLD) is one of the commonest liver diseases in Western countries. Although leptin deficient ob/ob and db/db mice are frequently used as murine models of NAFLD, an exhaustive characterization of their hepatic lesions has not been reported to date, particularly under calorie overconsumption. Thus, liver lesions were characterized in 78 ob/ob and db/db mice fed either a standard or high‐calorie (HC) diet, for one or three months. Steatosis, necroinflammation, apoptosis and fibrosis were assessed and the NAFLD activity score (NAS) was calculated. Steatosis was milder in db/db mice compared to ob/ob mice and was more frequently microvesicular. Although necroinflammation was usually mild in both genotypes, it was aggravated in db/db mice after one month of calorie overconsumption. Apoptosis was observed in db/db mice whereas it was only detected in ob/ob mice after HC feeding. Increased apoptosis was frequently associated with microvesicular steatosis. In db/db mice fed the HC diet for three months, fibrosis was aggravated while steatosis, necroinflammation and apoptosis tended to alleviate. This was associated with increased plasma β‐hydroxybutyrate suggesting an adaptive stimulation of hepatic mitochondrial fatty acid oxidation (FAO). Nevertheless, one‐third of these db/db mice had steatohepatitis (NAS ≥ 5), whereas none of the ob/ob mice developed non‐alcoholic steatohepatitis under the same conditions. Steatosis, necroinflammation, apoptosis and fibrosis are modulated by calorie overconsumption in the context of leptin deficiency. Association between apoptosis and microvesicular steatosis in obese mice suggests common mitochondrial abnormalities. Enhanced hepatic FAO in db/db mice is associated with fibrosis aggravation.     

Impaired liver regeneration after partial hepatectomy in db/db mice

Fatty liver is the most common hepatic disorder in humans and supposed to be a cause of poor prognosis after liver transplantation and hepatic resection which could be resulted from impaired liver regeneration. This study was carried out to analyze the process of liver regeneration in db/db mice which show severe steatosis because of abnormal leptin receptor. We performed 70% partial hepatectomy (PH) on db/db mice and normal +m/+m mice, and then sacrificed the animals 1, 2, 3, 5, 7 and 10 days later. The liver samples were weighed and examined histologically or immunohistochemically. As a result, the liver mass restitution was significantly inhibited in db/db mice compared with +m/+m mice. The BrdU labelling index peaked at 2 days after PH in both strains, although the value was lower in db/db mice. After that, interestingly, it decreased to the control level at 5 days in +m/+m mice while the recovery was delayed in db/db mice. Similar sequence was also observed in the PCNA labelling index. In addition, the peak time of the mitosis index was 2 days and 5 days after PH in +m/+m mice and in db/db mice, respectively. Thus, although not significant, the proliferative response of hepatocytes to PH occurred somewhat more transient and sharply in +m/+m mice while it lasted somewhat longer in db/db mice. This suggests that db/db mice may be valuable as one of the animal models for the investigation of the effects of steatosis on the liver regeneration.