Sciatic nerve ATPase activity is unaffected in diabetic mutant C57Bl/Ks (db/db) mice

We investigated composite (total), Mg2+-(ouabain-resistant), and Na+-K+-(ouabain-inhibited) ATPase in sciatic nerves of diabetic mutant C57Bl/Ks (db/db) and age-related littermate (db/+) control mice at various ages (16, 26, and 40 wk). This is the first report indicating that nerve ATPase activities measured in vitro showed no deficit in mice with spontaneous diabetes. Thus, diabetic neuropathy in the genetically diabetic mouse may occur in the absence of Na+-K+-ATPase abnormalities, which were previously described in association with polyol pathway impairment in experimental diabetic and BB Wistar rats. Control and diabetic groups treated with ganglioside mixture for 30 days before death presented statistically insignificant differences. Therefore, the beneficial effect of gangliosides described in C57Bl/Ks (db/db) mice on electrophysiological and morphometrical parameters must be due to different pharmacological activities rather than to prevention of the decay or better maintenance of ATPase activity.     

Alteration of phosphoinositide metabolism, protein phosphorylation, and carbohydrate levels in sciatic nerve from Wistar fatty diabetic rats

Sciatic nerve from the Wistar fatty diabetic (FD) rat, a prospective model for non-insulin-dependent diabetes mellitus, was investigated to determine the content of carbohydrates and to measure the incorporation of 32P into phosphoinositides and proteins. This strain has been shown to develop structural abnormalities in nerves and to exhibit reduced conduction velocity. Males became diabetic between the ages of 8 and 10 wk and were maintained together with lean sibling controls until the animals were either 22 or 44 wk old. Throughout this period, FD rats displayed moderate hyperglycemia. The carbohydrate profile of FD rat sciatic nerve exhibited modest increases in glucose, fructose, and sorbitol levels and significantly reduced myo-inositol concentrations, which were comparable at both ages. When nerves from 22-wk-old animals were incubated with [32P]orthophosphate and incorporation of radioactivity into phospholipids was measured, an increase in isotope uptake into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate in the distal portions of tissue from the FD rat was observed. This effect was more pronounced in nerves from 44-wk-old rats. Phosphorylation of the major myelin protein P0 was 70% higher in the most distal portion of FD sciatic nerve from 22-wk-old animals. A comparable rise in phosphorylation of P0 as well as the large (P1) and small (Pr) myelin basic proteins occurred in nerves from 44-wk-old rats. In these animals, an approximately 50% decrease in the uptake of 32P into P0 and P1 in the most proximal region of FD nerve was also apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Renoprotective effect of transforming growth factor beta activator kinase 1 inhibitor in diabetic db/db mice and its mechanism

Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 (TAK1) inhibitor 5Z-7-oxozeaenol (OZ) in diabetic db/db mice and the mechanism. Methods Twenty-four male db/db mice were randomly divided into two groups: db/db mice (db/db, n=12) and db/db mice with 5Z-7-oxozeaenol treatment (db/db+OZ, n=12). Another group of wild type mice (n=12) was held as the control group. OZ 2 mg/kg was administrated by intraperitoneal injection every other day. At week 8 and 12 after 5Z-7-oxozeaenol treatment, blood glucose (BG), body weight (BW), kidney weight (KW) and urinary albumin excretion rate (UAER) were evaluated. Kidney pathological lesions were detected by light and electron microscopy. NF-κB p65, monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α) were detected by immunohistochemistry. Western blotting was used to detect p-TAK1, TAB1, p-p38MAPK and IL-1β expression, while ICAM-1 and MCP-1 mRNA levels were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were higher (P＜0.01) in db/db mice group, while BW, KW and UAER levels were significantly decreased in db/db+OZ group compared with that in db/db mice group (P＜0.05). In week 8 and 12 db/db mice, glomerular volume and extracellular matrix were increased, while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor. Immunohistochemistry showed that NF-κB p65, MCP-1 and TNF-α expression levels were apparently increased in db/db mice group compared with that in control group (P＜0.05) and were significantly inhibited by TAK1 inhibitor (P＜0.05). Western blotting showed that p-TAK1, TAB1, p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group (P＜0.05) and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Moreover, real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db+OZ group than that in db/db mice group (P＜0.05). Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-κB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.     

Regulation of melatonin on Toll-like receptor 4 signaling in diabetic db/db mice kidneys

Objective To investigate the regulation of melatonin (MT) on Toll-like receptor 4 (TLR4) signaling in diabetic db/db mice kidneys. Methods The 48 10-week-old male db/db mice were randomly divided into db/db group, db/db+MT 50 μg/kg group, db/db+MT 100 μg/kg group and db/db+MT 200 μg/kg group, each consisting of 12 mice. These mice received i.p. injections of MT These mice received i.p. injections of MT [dissoved in phosphate buffer solution (PBS)/ dimethylsulfoxide (DMSO) solution, given every day]. Alternatively, 12 db/m mice served as the control group. db/m and db/db group were injected i.p. with the same volume of PBS/DMSO solution. The animals were sacrificed after 12 weeks of dosage administration. Blood glucose (BG), body weight (BW), kidney weight (KW) and 24 h urinary albumin excretion rate (UAER) were determined; Kidney pathological lesions were evaluated by renal pathological staining. Immunohistochemistry of renal TLR4, NF-κB p65, and ED-1 was performed to determine the immunoreactivity. Western blotting was used to detect the expression of renal TLR4, myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF-3) and NF-κB p65, while the mRNA expressions of renal tumor necrosis factor -α (TNF-α) and monocyte chemotactic protein-1 (MCP-1) were evaluated by real-time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were much higher in db/db mice group (P＜0.01), while KW in db/db+MT (100, 200 μg/kg) groups and UAER level in db/db+MT (50, 100, 200 μg/kg) groups were distinctly decreased compared with those in db/db group (P＜0.01). In week 12 db/db mice, the glomerular mesangial expansion index and tubulointerstitial injury index were increased compared with those in db/m mice (P＜0.01). The above kidney histopathologic lesions were distinctly ameliorated by 50, 100, 200 μg/kg MT (P＜0.05). Immunohistochemistry intensity of renal TLR4, NF-κB p65 and ED-1 displayed obvious differences between db/m mice and db/db mice (P＜0.01), and that were remarkably decreased in db/db+MT (50, 100, 200 μg/kg) mice compared with db/db mice (P＜0.05). Western blotting showed that the protein expression of renal TLR4, MyD88, TRIF, IRF-3 and NF-κB p65 were stronger in db/db group compared with those in db/m group (P＜0.05) and weaker in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.05). Futhermore, the mRNA expressions of renal MCP-1 and TNF-α were higher in db/db group compared with those in db/m group (P＜0.01) and lower in db/db+MT (50, 100, 200 μg/kg) groups compared with those in db/db group (P＜0.01). Conclusion Melatonin may partly down-regulate TLR4 signaling pathway to inhibit Inflammatory reaction and alleviate kidney injury in diabetic db/db mice.     

Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes

The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with [32P]orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of 32P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with [32P]-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.     

Inhibiting albumin glycation ameliorates diabetic nephropathy in the db/db mouse

Albumin modified by Amadori glucose adducts stimulates the expression of extracellular matrix proteins by glomerular mesangial and endothelial cells, and has been mechanistically linked to the pathogenesis of diabetic nephropathy. To test the hypothesis that inhibiting the formation of glycated albumin might beneficially influence the development of kidney disease in diabetes, we treated diabetic db/db mice for 12 weeks with a low-molecular-weight compound (EXO-226) that impedes the condensation of free glucose with lysine epsilon-amino groups in albumin. Administration of EXO-226 (3 mg/kg) twice daily by gavage normalized the plasma concentration of glycated albumin within days after initiation of treatment and maintained glycated albumin within the normal range throughout the study, despite persistent and severe hyperglycemia. Urine albumin excretion, which was markedly increased at the start of the study (age 12 weeks), was significantly reduced in treated diabetic animals compared with their untreated diabetic littermates. The fall in creatinine clearance that was observed in untreated diabetic animals was prevented in diabetic littermates that received treatment. Compared with the nondiabetic controls, the amount of glomerular mesangial matrix was threefold greater in untreated diabetic mice; in contrast, the mesangial matrix fraction was only 1. 5 times that of nondiabetic controls in the treated diabetic animals, representing a reduction in mesangial matrix accumulation of more than 50%. EXO-226 also reduced the overexpression of mRNA encoding for alpha1 (IV) collagen in renal cortex of db/db mice. We conclude that normalization of plasma glycated albumin concentrations with the glycation inhibitor EXO-226 ameliorates the glomerular structural and functional abnormalities associated with diabetic nephropathy in the db/db mouse.     

Quantitative analysis of pancreatic proinsulin mRNA in genetically diabetic (db/db) mice

C57BL/KsJ db/db mice develop hyperphagic obesity and nonketotic diabetes similar to non-insulin-dependent diabetes mellitus in humans. Initially the mice demonstrate an abundant beta-cell mass and hyperinsulinemia, which is followed by apparent beta-cell loss. As an index of insulin synthesis, this study assesses pancreatic proinsulin mRNA, measured by dot hybridization to cloned cDNA, during the development of diabetes in the mice. Changes in proinsulin mRNA from 5 to 13 wk of age are compared with serum insulin, pancreatic insulin content, and blood glucose. In control (+/db) mice, total proinsulin mRNA and pancreatic insulin content increased with age. Both changes were proportional to an increase in body weight. Obesity, hyperglycemia, and hyperinsulinemia were evident in diabetic (db/db) mice at 5 wk of age. Although pancreatic insulin content was comparable to that in the +/db controls at 5 wk, a fourfold relative elevation of proinsulin mRNA was observed. Despite an increase in body weight, proinsulin mRNA concentration and total proinsulin mRNA fell to levels similar to those of the control mice at 10 and 13 wk, associated with a loss of hyperinsulinemia, a mild decrease in pancreatic insulin content, and a marked increased in fasting blood glucose. A separate group of db/db mice was pair fed with the +/db controls from 4 to 13 wk. These diet-restricted diabetic mice were heavier than control mice and gained weight with age, but they weighed less than the unrestricted mice at all ages. Compared with the unrestricted db/db mice, a more modest fasting hyperglycemia was apparent, and a persistent hyperinsulinemia was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用

目的 观察肾素抑制剂阿利吉仑对2型糖尿病db/db小鼠肾脏损伤的保护作用。 方法 8周龄db/db和db/m小鼠行单侧肾切除,16周进入实验,分为4组：db/m小鼠对照组（db/m组）、db/db糖尿病小鼠对照组（db/db组）、db/db糖尿病小鼠阿利吉仑3 mg&#8226;kg-1&#8226;d-1治疗组（db/db+A3组）和db/db糖尿病小鼠阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗组(db/db+A25组)。阿利吉仑溶于磷酸盐缓冲液（PBS,350 mg/L）皮下泵入（0.25 μl/h）给药,疗程４周。治疗前后检测体质量、血糖、糖化血红蛋白、尿蛋白量、血压水平;PAS染色观察肾脏组织学变化;ELISA法检测肾皮质转化生长因子β1（TGF-β1）和纤溶酶原激活抑制因子1（PAI-1）含量;间接免疫荧光检测肾小球Ⅳ型胶原（ColⅣ）和纤连蛋白（FN）表达;实时定量PCR检测TGF-β1、PAI-1、ColⅣ、FN和肾素mRNA表达;放射免疫法检测肾皮质肾素活性和血管紧张素Ⅱ（AngⅡ）水平。 结果 与db/m组小鼠比较,db/db组小鼠有大量蛋白尿,肾小球细胞外基质沉积增加,TGF-β1、PAI-1、ColⅣ和FN蛋白及mRNA表达增加,同时肾皮质肾素mRNA、肾素活性和AngⅡ水平增高（均P < 0.05）。阿利吉仑25 mg&#8226;kg-1&#8226;d-1治疗在没有影响血压情况下,显著减少db/db小鼠24 h尿蛋白量,减少肾小球细胞外基质沉积,减少TGF-β1、PAI-1、ColⅣ、FN蛋白和mRNA表达,同时降低肾皮质肾素活性和AngⅡ水平（均P < 0.05）。 结论 阿利吉仑对2型糖尿病db/db小鼠肾脏损伤有保护作用。     

T-lymphopenia and T-cell imbalance in diabetic db/db mice

The diabetic db/db mice of the C57 BL/KsJ strain display anti-islet immunity, thymic dysfunction, and lymphopenia. In the present work, lymphocytes, T-cells, and T-cell subsets were enumerated in thymus and spleen from diabetic db/db mice and their db/ + heterozygote littermates from the 10th day to the 10th month of life. A significant lymphopenia was detected in thymus and spleen from the second month on, involving specifically the T-cell compartment, as assessed by use of a monoclonal anti-Thy1 antibody in indirect fluorescence. The study of T-cell subsets by monoclonal anti-Lyt1 and anti-Lyt2 antibodies revealed a significant increase in Lyt1+ cells and a decrease in Lyt2+ cells, with a corresponding increase of the Lyt1+/Lyt2+ ratio. These anomalies appeared early in life, and were apparently linked neither with the degree of hyperglycemia nor with weight loss or infection. The T-cell depletion in thymus was more pronounced in young male (less than 3 mo) than in young female db/db mice. These alterations may correspond to an increase in the helper/suppressor-cytotoxic ratio and could be linked with the thymic anomalies present in these mice, contributing to the development of anti-islet autoimmunity.     

N-acetyl-seryl-aspartyl-lysyl-proline prevents renal insufficiency and mesangial matrix expansion in diabetic db/db mice

We have previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is a tetrapeptide hydrolyzed by ACE, inhibits the transforming growth factor-beta (TGF-beta)-induced expression of extracellular matrix proteins via inhibition of the Smad signaling in human mesangial cells. To test in vivo the antifibrotic efficacy of Ac-SDKP, we examined whether long-term Ac-SDKP treatment can prevent renal insufficiency and glomerulosclerosis in diabetic db/db mice. Diabetic db/db mice or nondiabetic db/m mice were treated with Ac-SDKP for 8 weeks using osmotic minipumps. The treatment with Ac-SDKP increased plasma Ac-SDKP concentrations by approximately threefold in both groups but did not affect the blood glucose levels. Histologically, the increased glomerular surface area, mesangial matrix expansion, and overproduction of extracellular matrix proteins in db/db mice were significantly inhibited by Ac-SDKP. Furthermore, Ac-SDKP treatment normalized the increased plasma creatinine value in db/db mice, whereas the albuminuria in Ac-SDKP-treated db/db mice was somewhat decreased as compared with nontreated db/db mice, although the difference was not statistically significant. In addition, the nuclear translocation of Smad3 was inhibited by Ac-SDKP. These results demonstrate that long-term Ac-SDKP treatment ameliorates renal insufficiency and glomerulosclerosis in db/db mice via inhibition of TGF-beta/Smad pathway, suggesting that Ac-SDKP could be useful in the treatment of diabetic nephropathy.     

Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice

Diabetic nephropathy is a leading cause of end-stage renal failure and is a growing concern given the increasing incidence of type 2 diabetes. Diabetic nephropathy is associated with progressive kidney macrophage accumulation and experimental studies suggest that intercellular adhesion molecule (ICAM)-1 facilitates kidney macrophage recruitment during type 1 diabetes. To ascertain the importance of ICAM-1 in promoting type 2 diabetic nephropathy, the development of renal injury in ICAM-1 intact and deficient db/db mice with equivalent hyperglycemia and obesity between ages 2 and 8 mo was examined and compared with results with normal db/+ mice. Increases in albuminuria (11-fold), glomerular leukocytes (10-fold), and interstitial leukocytes (three-fold) consisting of predominantly CD68+ macrophages were identified at 8 mo in diabetic db/db mice compared with nondiabetic db/+ mice. In comparison to db/db mice, ICAM-1-deficient db/db mice had marked reductions in albuminuria at 6 mo (77% downward arrow) and 8 mo (85% downward arrow). There was also a significant decrease in glomerular (63% downward arrow) and interstitial (83% downward arrow) leukocytes in ICAM-1-deficient db/db mice, which were associated with reduced glomerular hypertrophy and hypercellularity and tubular damage. The development of renal fibrosis (expression of TGF-beta1, collagen IV, and interstitial alpha-smooth muscle actin) was also strikingly attenuated in the ICAM-1-deficient db/db mice. Additional in vitro studies showed that macrophage activation by high glucose or advanced glycation end products could promote ICAM-1 expression on tubular cells and macrophage production of active TGF-beta1. Thus, ICAM-1 appears to be a critical promoter of nephropathy in mouse type 2 diabetes by facilitating kidney macrophage recruitment.     

Mechanisms of [Ca2+]i transient decrease in cardiomyopathy of db/db type 2 diabetic mice

Cardiovascular disease is the leading cause of death in the diabetic population. However, molecular mechanisms underlying diabetic cardiomyopathy remain unclear. We analyzed Ca2+-induced Ca2+ release and excitation-contraction coupling in db/db obese type 2 diabetic mice and their control littermates. Echocardiography showed a systolic dysfunction in db/db mice. Two-photon microscopy identified intracellular calcium concentration ([Ca2+]i) transient decrease in cardiomyocytes within the whole heart, which was also found in isolated myocytes by confocal microscopy. Global [Ca2+]i transients are constituted of individual Ca2+ sparks. Ca2+ sparks in db/db cardiomyocytes were less frequent than in +/+ myocytes, partly because of a depression in sarcoplasmic reticulum Ca2+ load but also because of a reduced expression of ryanodine receptor Ca2+ channels (RyRs), revealed by [3H]ryanodine binding assay. Ca2+ efflux through Na+/Ca2+ exchanger was increased in db/db myocytes. Calcium current, I(Ca), triggers sarcoplasmic reticulum Ca2+ release and is also involved in sarcoplasmic reticulum Ca2+ refilling. Macroscopic I(Ca) was reduced in db/db cells, but single Ca2+ channel activity was similar, suggesting that diabetic myocytes express fewer functional Ca2+ channels, which was confirmed by Western blots. These results demonstrate that db/db mice show depressed cardiac function, at least in part, because of a general reduction in the membrane permeability to Ca2+. As less Ca2+ enters the cell through I(Ca), less Ca2+ is released through RyRs.     

microRNA-215在糖尿病肾病小鼠肾组织中的表达及意义

目的 探讨microRNA-215( miR-215)在糖尿病肾病(DN)小鼠肾组织中的表达变化规律及在DN发病中的作用.方法 选择4周龄的2型糖尿病肾病db/db小鼠(实验组)和db/m小鼠(对照组),采用实时荧光定量PCR法动态检测8、12及16周龄时肾组织miR-215的表达变化;实时荧光定量PCR和Western印迹法、免疫组化法测定连环蛋白β互动蛋白1( CTNNBIP1)的mRNA及蛋白的表达;双荧光素酶报告法确证miR-215对CTNNBIP1表达的直接调控作用.结果 (1)随着周龄的增加,db/db小鼠肾小球逐渐肥大、节段性系膜细胞增生和系膜基质积聚.(2)与同周龄的db/m小鼠比较,8、12及16周龄的db/db小鼠体质量(BW)、血糖( Glu)及24 h尿白蛋白排泄量(UAE)均显著增加(均P＜0.05).(3)随着周龄的增加,db/db小鼠肾脏组织miR-215表达显著高于同周龄的db/m小鼠(P＜0.05).(4)与同周龄的db/m小鼠比较,db/db小鼠肾脏组织CTNNBIP1 mRNA和蛋白均显著降低(均P＜0.05).(5)应用双荧光素酶报告法证实,miR-215可显著抑制CTNNBIP1的表达(P＜0.01).结论 miR-215表达上调可能通过抑制CTNNBIP1的表达参与DN的发生、发展.     

Aldose reductase-deficient mice are protected from delayed motor nerve conduction velocity, increased c-Jun NH2-terminal kinase activation, depletion of reduced glutathione, increased superoxide accumulation, and DNA damage

The exaggerated flux through polyol pathway during diabetes is thought to be a major cause of lesions in the peripheral nerves. Here, we used aldose reductase (AR)-deficient (AR(-/-)) and AR inhibitor (ARI)-treated mice to further understand the in vivo role of polyol pathway in the pathogenesis of diabetic neuropathy. Under normal conditions, there were no obvious differences in the innervation patterns between wild-type AR (AR(+/+)) and AR(-/-) mice. Under short-term diabetic conditions, AR(-/-) mice were protected from the reduction of motor and sensory nerve conduction velocities observed in diabetic AR(+/+) mice. Sorbitol levels in the sciatic nerves of diabetic AR(+/+) mice were increased significantly, whereas sorbitol levels in the diabetic AR(-/-) mice were significantly lower than those in diabetic AR(+/+) mice. In addition, signs of oxidative stress, such as increased activation of c-Jun NH(2)-terminal kinase (JNK), depletion of reduced glutathione, increase of superoxide formation, and DNA damage, observed in the sciatic nerves of diabetic AR(+/+) mice were not observed in the diabetic AR(-/-) mice, indicating that the diabetic AR(-/-) mice were protected from oxidative stress in the sciatic nerve. The diabetic AR(-/-) mice also excreted less 8-hydroxy-2'-deoxyguanosine in urine than diabetic AR(+/+) mice. The structural abnormalities observed in the sural nerve of diabetic AR(+/+) mice were less severe in the diabetic AR(-/-) mice, although it was only mildly protected by AR deficiency under short-term diabetic conditions. Signs of oxidative stress and functional and structural abnormalities were also inhibited by the ARI fidarestat in diabetic AR(+/+) nerves, similar to those in diabetic AR(-/-) mice. Taken together, increased polyol pathway flux through AR is a major contributing factor in the early signs of diabetic neuropathy, possibly through depletion of glutathione, increased superoxide accumulation, increased JNK activation, and DNA damage.     

Decreased sarcoplasmic reticulum activity and contractility in diabetic db/db mouse heart

Although it is known that insulin-dependent (type 1) diabetes results in depressed contractile performance associated with diminished sarcoendoplasmic reticular Ca2+-ATPase (SERCA2a) activity, findings in insulin-resistant (type 2) diabetes suggest a less clear association. The db/db insulin-resistant mouse model exhibits decreased cardiac performance both in situ and in isolated ex vivo working hearts. In this study, contractile performance and calcium transients were measured in Langendorff-perfused hearts and isolated cardiac myocytes. Diabetic (db/db) mouse hearts demonstrated decreased rates of contraction, relaxation, and pressure development. Calcium transients from isolated myocytes revealed significantly lower diastolic and systolic levels of calcium in diabetic hearts. Furthermore, the decay rate of the calcium transient was significantly reduced in diabetic myocytes, suggesting a diminished capacity for cytosolic calcium removal not associated with a change in sodium-calcium exchanger activity. Calcium leakage from the sarcoplasmic reticulum (SR) measured using tetracaine was significantly increased in diabetic myocytes. Western blot analysis indicated only a small decrease in SERCA2a expression in diabetic mice, but a large increase in phospholamban expression. Expression of the ryanodine receptor did not differ between groups. In conclusion, the decreased contractile function observed in the db/db diabetic mouse model appears to be related to decreased calcium handling by the SR.     

Altered beta-endorphin, Met- and Leu-enkephalins, and enkephalin-containing peptides in pancreas and pituitary of genetically obese diabetic (db/db) mice during development of diabetic syndrome

We have recently shown that in addition to beta-endorphin the opioid peptides Met- and Leu-enkephalin and their apparent precursors are localized in islet endocrine cells of the rat pancreas. To begin evaluating a possible role for these pancreatic opiates in the pathophysiology of genetic diabetes in rodents, immunoreactive beta-endorphin and Met- and Leu-enkephalins were measured in acetic acid extracts of pancreas and pituitary of C57BL/KsJ db/db mice and their lean littermates. Groups of animals were studied during three phases of development of the diabetic syndrome in the mutant mice: at 4 (hyperinsulinemic and prediabetic); 6, 9, and 12 (frankly obese and diabetic); and 30 (hypoinsulinemic) wk of age. Elevations or decreases (P less than .05) were found in db/db mice (vs. lean littermates) as follows: pituitary content of Met-enkephalin was twofold higher at all ages studied; pituitary free Leu-enkephalin was lower at 4 wk and reversed to higher at 6-30 wk; pancreatic beta-endorphin was 30% lower at 4 wk and reversed to threefold higher at 6-12 wk; Met- and Leu-enkephalin-containing larger peptides were elevated at one or more points between 6 and 12 wk in both the pancreas and the pituitary. Thus, the onset of overt obesity between 4 and 6 wk of age was accompanied by a marked rise in both pancreatic beta-endorphin and pituitary Leu-enkephalin; similar elevations in these parameters have been reported previously in C57BL/6J ob/ob mice at approximately 12 wk of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevention and reversal of diabetic nephropathy in db/db mice treated with alagebrium (ALT-711)

BACKGROUND: Alagebrium (ALT-711) has been shown to improve renal dysfunction in animal models of diabetes. METHODS: To test its effects in diabetic nephropathy (DN), ALT-711 was administered (1 mg/kg daily i.p.) to 9-week-old female db/db mice (n = 15, group A1) for 3 weeks and to 3-month-old (n = 15, group A2), 7-month-old (n = 7, group A3), and 12-month-old (n = 5, group A4) female db/db mice for 12 weeks, while a similar number of diabetic and nondiabetic mice were used as controls. The epsilonN-carboxymethyllysine (CML) levels in serum, urine, skin, and kidney tissue were measured by enzyme-linked immunosorbent assay. The renal morphometric parameters were assessed by electron and light microscopy. RESULTS: By the 3rd week of treatment, the serum CML level decreased by 41%, and the urinary CML concentration increased by 138% from baseline, while the urinary albumin/creatinine ratio was lower (p < 0.05) in diabetic and nondiabetic group A1 mice. After 3 months of treatment, serum, skin, and kidney CML levels and urinary albumin/creatinine ratio were lower (p < 0.05) and the urinary CML levels higher (p < 0.05) in treated group A2, A3, and A4 animals compared with groups which received phosphate-buffered saline, with a similar pattern observed in nondiabetic mice. The renal morphological parameters characteristic of DN decreased in treated compared with untreated mice. CONCLUSION: Alagebrium may prevent, delay, and/or reverse established DN in db/db mice by reducing the systemic advanced glycation end product pools and facilitating the urinary excretion of advanced glycation end products.     

Age-dependent changes in metabolism,contractile function,and ischemic sensitivity in hearts from db/db mice

Glucose and palmitate metabolism and contractile function were measured with ex vivo perfused working hearts from control (db/+) and diabetic (db/db) female mice at 6, 10-12, and 16-18 weeks of age. Palmitate oxidation was increased by 2.2-fold in 6-week-old db/db hearts and remained elevated in 10- to 12- and 16- to 18-week-old hearts. Carbohydrate oxidation was normal at 6 weeks but was reduced to 27 and 23% of control at 10-12 and 16-18 weeks, respectively. At 6 weeks, db/db hearts exhibited a slight reduction in mechanical function, whereas marked signs of dysfunction were evident at 10-12 and 16-18 weeks. Mechanical function after ischemia-reperfusion was examined in hearts from male mice; at 6 weeks, db/db hearts showed normal recovery, whereas at 12 weeks it was markedly reduced. Fatty acid oxidation was the predominant substrate used after reperfusion. Thus, diabetic db/db hearts exhibit signs of a progressive cardiomyopathy; increased fatty acid oxidation preceded reductions in carbohydrate oxidation. Postischemic recovery of function was reduced in db/db hearts, in parallel with age-dependent changes in normoxic contractile performance. Finally, peroxisome proliferator-activated receptor-alpha treatment (3 weeks) did not affect sensitivity to ischemia-reperfusion, even though carbohydrate oxidation was increased and palmitate oxidation was decreased.