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.     

Excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle: evidence against a role for reduced fatty acid oxidation in lipid-induced insulin resistance in rodents

A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including (14)C-palmitate oxidation, palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, beta-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet-fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1alpha, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.     

Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death

Loss of podocytes by apoptosis characterizes the early stages of diabetic nephropathy. To examine its mechanism we studied glomeruli and podocytes isolated from db/db mice with early diabetic nephropathy and albuminuria. Phosphorylation of AKT (protein kinase B, a key survival protein) was found to be lower in the glomeruli of 12 week old db/db compared to db/+ mice. In vitro, insulin phosphorylated AKT solely in podocytes from db/+ mice. Serum deprivation and exposure to tumor necrosis factor-alpha significantly compromised cell viability in podocytes from db/db but not from db/+ mice, and this was associated with a significant decrease in AKT phosphorylation. Inhibition of AKT was necessary to achieve the same degree of cell death in db/+ podocytes. Our study shows that podocyte inability to respond to insulin and susceptibility to cell death may partially account for the decreased podocyte number seen in early diabetic nephropathy.     

Defective oxidative metabolism of heart mitochondria from genetically diabetic mice

Long chain saturated beta-hydroxy fatty acid content and oxidative metabolism were studied in hearts of diabetic mice (C57BL/KsJ db/db) with a progressive cardiomyopathy at intervals of 7, 10, 16, and 26 wk of age. Total beta-hydroxy fatty acid (BHFA) content increases progressively with age in diabetic hearts with a mean value of 143.5 nmol/g dry wt as compared with a mean of 59.6 nmol/g dry wt in control hearts. There was also a redistribution of BHFA in myocardium of diabetic mice when compared with controls, with a relative decrease in beta-hydroxymyristate and an increase of beta-hydroxypalmitate. Oxidative phosphorylation studies using isolated mitochondria from diabetic mice demonstrated depressed state 3 oxidation rates with both palmityl carnitine and pyruvate as substrates. While mitochondrial NADH-oxidase activity was not statistically different from that of controls, there was a significant decrease in mitochondrial total NAD + NADH content in diabetic hearts. In addition, treatment of myocardial tissue with lanthanum demonstrated an abnormal permeability of sarcolemmal, intercalated disc as well as mitochondrial membranes in myocytes of diabetic mice. The data indicate that deficiencies in total NAD + NADH content can account for the depressed state 3 oxidation of palmitylcarnitine and pyruvate in diabetic mice that in turn may explain the abnormal accumulation of BFHA. The latter could play a role in altering the permeability of cardiac cell membranes.     

Leptin activation of corticosterone production in hepatocytes may contribute to the reversal of obesity and hyperglycemia in leptin-deficient ob/ob mice

Glucocorticoids have been implicated as pathophysiological mediators of obesity and insulin resistance and are regulated by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). This enzyme regenerates active corticosterone from inactive 11-keto forms. To assess the role of 11beta-HSD1-mediated synthesis of active corticosterone in leptin-related obesity and diabetes, we examined the peripheral effect of leptin on 11beta-HSD1 activity and gene expression in vivo and in vitro in hepatocytes from ob/ob mice and in liver of streptozotocin (STZ)-treated ob/ob mice. We observed an inverse relationship between hepatic 11beta-HSD1 expression and body weight in ob/ob mice and lean littermates. Leptin treatment of ob/ob mice markedly increased hepatic 11beta-HSD1 activity and mRNA expression. This induction of 11beta-HSD1 expression corresponded to reduced levels of circulating corticosterone and weight loss in ob/ob mice treated with leptin, indicating that impaired hepatic 11beta-HSD1 expression may contribute to the pathogenesis of obesity in ob/ob mice. In addition, leptin treatment of STZ-treated ob/ob mice caused marked increases in hepatic 11beta-HSD1 levels associated with decreased body weight and a significant reduction in hyperglycemia due to pancreatic beta-cell damage. Addition of leptin to ob/ob mouse primary hepatocytes led to a dose-dependent increase in 11beta-HSD1 mRNA expression. In contrast, leptin did not influence 11beta-HSD1 expression in primary hepatocytes from db/db mice, indicating that leptin regulation of 11beta-HSD1 expression is probably mediated by the functional leptin receptor. Thus, leptin appears to be an important metabolic signal that directly activates intrahepatic corticosterone production. These findings suggest that the liver-specific interaction of leptin with 11beta-HSD1 is involved in the development of obesity and insulin resistance in ob/ob mice.     

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.     

Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/+) mice.

During gestation, heterozygous C57BLKS/J-Lepr(db/+) mice develop spontaneous gestational diabetes mellitus (GDM), and the newborn fetuses are macrosomic compared with offspring from wild-type (+/+) mothers. To investigate the effects of the leptin receptor mutation on maternal metabolism and fetal growth during pregnancy, we studied +/+, db/+, and db/+ transgenic mice that overexpress the human GLUT4 gene two- to three-fold (db/+TG6). During pregnancy, fasting plasma glucose and hepatic glucose production were twofold greater in db/+ than +/+ mice, despite similar insulin levels. In skeletal muscle, insulin-stimulated tyrosine phosphorylation was decreased in pregnant +/+ mice, and even more so in db/+ mice: insulin receptor beta (IR-beta), +/+ 34%, db/+ 57% decrease, P<0.05; insulin receptor substrate 1 (IRS-1), +/+ 44%, db/+ 61% decrease, P<0.05; and phosphoinositol (PI) 3-kinase (p85alpha), +/+ 33%, db/+ 65% decrease, P<0.05. Overexpression of GLUT4 in db/+TG6 mice markedly improved glucose-stimulated insulin secretion, by 250%, and increased IRbeta, IRS-1, and p85alpha phosphorylation twofold, despite no change in concentration of these proteins. Plasma leptin concentration increased 40-fold during pregnancy, from 2.2+/-0.5 to 92+/-11 ng/ml and 3.6+/-0.1 to 178+/-34 ng/ml in +/+ and db/+ mice, respectively (P<0.01), but was increased to only 23+/-3 ng/ml in pregnant db/+TG6 mice (P<0.001). Maternal fat mass and energy intake were greater in db/+ mice, and fat mass was reduced by GLUT4 overexpression, independent of food intake. Fetal body weight was increased by 8.1 and 7.9% in db/+ and db/+TG6 mothers, respectively (P<0.05), regardless of fetal genotype, whereas fetuses from db/+TG8 mothers (four- to fivefold overexpression) weighed significantly less compared with pups from +/+ or db/+ mothers (P<0.05). These results suggest that the single mutant db allele effects susceptibility to GDM through abnormalities in insulin receptor signaling, defective insulin secretion, and greater nutrient availability. GLUT4 overexpression markedly improves insulin-signaling in GDM, resulting in increased insulin secretion and improved glycemic control. However, maternal hyperglycemia appears not to be the sole cause of fetal macrosomia. These data suggest that GDM is associated with defects in insulin receptor signaling in maternal skeletal muscle, and this may be an important factor provoking maternal and fetal perinatal complications.     

Control of glycogen synthase and phosphorylase in hepatocytes from diabetic rats. Effects of glucagon, vasopressin, and vanadate

Although glycogen synthase is present in a highly inactivated state in hepatocytes from streptozocin-induced diabetic rats, glucagon, vasopressin, and vanadate are still able to further decrease the basal activity of the enzyme. This inactivation was observed with the low-to-high glucose 6-phosphate activity ratio assay. The inactivation of glycogen synthase occurred concomitantly with the activation of glycogen phosphorylase. When hepatocytes from diabetic rats were incubated with [32P]phosphate and then with the agents and when the 32P-labeled glycogen synthase was immunoprecipitated, we observed that the 32P bound to the 88,000-Mr subunit increased in all cases. All the [32P]phosphate was located in two cyanogen bromide fragments of the enzyme, indicating that the enzyme was phosphorylated at multiple sites. The fragments were precisely those phosphorylated by glycogenolytic hormones in hepatocytes from normal rats. These results demonstrated that hepatic glycogen synthase, although highly inactive, is under potential hormonal control in diabetes and that the enzyme has not reached its maximal level of phosphorylation. Furthermore, they indicated that vanadate behaves as a glycogenolytic agent regarding its effects on glycogen-metabolizing enzymes in hepatocytes from diabetic rats.     

Bone Marrow Transplantation Induces Normoglycemia in a Type 2 Diabetes Mellitus Murine Model

Objective

To study the cellular mechanisms involved in the regression of diabetic nephropathy, bone marrow-derived cells must be identified. The aim of this study was to obtain a diabetic chimeric model with bone marrow cells expressing enhanced green fluorecence protein (EGFP), without modifying the course of diabetic nephropathy.

Materials and Methods

Bone marrow transplantation (BMT) was performed in an obese type 2 diabetic murine model (db/db) owing to a mutation in the leptin receptor gene. Whole bone marrow from female donor C57BL/6 EGFP+ mice was transplanted into 8-week-old C57BL/6 mice and into 8- and 24-week-old female C57BLKS (db/db) EGFP− mice. Recipient mice received total body irradiation (TBI) followed by bone marrow (BM) cell infusion. We tested various irradiation doses (Gy) and numbers of BM cells.

Results

When a low TBI dose and a small number of BM cells were administered, only syngeneic C57BL/6 mice became chimeric, whereas allogeneic db/db mice showed rejection. When Gy dose and BM cells were increased, db/db mice became chimeric. However, 8-week-old db/db mice lost the obese phenotype and became normoglycemic, probably due to peripheral BM cell infiltration. Conversely, 24-week-old db/db mice remained obese showing similar blood glucose values, body weights, albuminuria, and glomerular lesions at nontransplanted db/db mice.

Conclusions

Recipient age greatly influenced the peripheral repopulation after BMT in db/db mice. Only the adult chimeric db/db mice seemed to be a good model to study the cellular mechanisms involved in the regression of diabetic nephropathy.     

Long-term treatment of glucagon-like peptide-1 analog exendin-4 ameliorates diabetic nephropathy through improving metabolic anomalies in db/db mice

Glucagon-like peptide-1 (GLP-1) is a gut incretin hormone and is a new clinically available class of agents for improving of insulin resistance in both animals and humans with type 2 diabetes. These studies aimed to determine whether long-term treatment with a long-acting GLP-1 analog, exendin-4, delayed the progression of diabetes. Male db/db mice and db/m mice at 8 wk of age were treated with exendin-4 for 8 wk, whereas the control db/db mice received only vehicle. Urinary albumin excretion was significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in db/db mice that were treated with 0.5 nmol/kg exendin-4 and control db/db mice (P < 0.005). Intraperitoneal glucose tolerance test was improved in db/db mice that were treated with 1 nmol/kg exendin-4 compared with other groups (P < 0.05). Despite this, fasting blood glucose, glycated hemoglobin, and creatinine concentrations were not significantly different among db/db mice. Renal histology studies further demonstrated that glomerular hypertrophy, mesangial matrix expansion, TGF-beta1 expression, and type IV collagen accumulation and associated glomerular lipid accumulation were significantly decreased in db/db mice that were treated with 1 nmol/kg exendin-4. Furthermore, there were fewer infiltrating inflammatory cells and apoptotic cells in the glomeruli of db/db mice that were treated with 1 nmol/kg exendin-4 compared with those in the other groups accompanied by an increase in the renal immunoreactivity of peroxisome proliferator-activated receptor alpha and GLP-1 receptor-positive cells and a decrease in 24-h urinary 8-hydroxy-deoxyguanosine levels (P < 0.01, respectively) along with decreases in lipid content. Taken together, exendin-4 treatment seems to ameliorate diabetic nephropathy together with improvement of the metabolic anomalies. These results suggest that exendin-4 could provide a therapeutic role in diabetic nephropathy that results from type 2 diabetes.     

Increased glucocorticoid receptor and 11{beta}-hydroxysteroid dehydrogenase type 1 expression in hepatocytes may contribute to the phenotype of type 2 diabetes in db/db mice

Excess tissue glucocorticoid action may contribute to the hyperglycemia and insulin resistance associated with type 2 diabetes, but the associated mechanisms are poorly understood. 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts inactive 11-dehydrocorticosterone into active corticosterone, thus amplifying glucocorticoid receptor-mediated tissue glucocorticoid action, particularly in the liver. To examine the role of tissue glucocorticoid action in type 2 diabetes, we analyzed expression of glucocorticoid receptor and 11beta-HSD1 and their regulation by endogenous hormones in vivo and in vitro in hepatocytes from db/db mice (a model of type 2 diabetes). We observed positive relations between expression of both glucocorticoid receptor and 11beta-HSD1 in liver and insulin sensitivity and expression of PEPCK mRNA in db/db mice and db/+ controls. Increased expression of glucocorticoid receptor and 11beta-HSD1 in the liver of db/db mice was correlated with elevated circulating levels of corticosterone, insulin, and blood glu-cose. Treatment of db/db mice with glucocorticoid antagonist RU486 reversed the increases in the expression of glucocorticoid receptor and 11beta-HSD1 within the liver and attenuated the phenotype of type 2 diabetes. Addition of corticosterone to db/db mouse primary hepatocytes activated expression of glucocorticoid receptor, 11beta-HSD1, and PEPCK, and these effects were abolished by RU486. Incubation of primary hepatocytes with increasing concentrations of glucose caused dose-dependent increases in glucocorticoid receptor and 11beta-HSD1 expression, whereas insulin did not affect the expression of 11beta-HSD1 and glucocorticoid receptor in primary hepatocytes. These findings suggest that activation of glucocorticoid receptor and 11beta-HSD1 expression within the liver may contribute to the development of type 2 diabetes in db/db mice.     

Influence of Hemodialysis on Monoamine Oxidase Kinetics in Platelets from Chronic Schizophrenic Patients

Abstract: Platelet monoamine oxidase (MAO) activity in seven chronic schizophrenic patients undergoing hemodialysis treatment was studied. The treatment was performed in a double-blind crossover design. A significant increase in MAO activity was observed after 5 h of active dialysis, whereas inactive dialysis did not significantly alter the enzyme activity. The increase in MAO activity could be explained by the observed significant decrease in apparent Km values for the amine (phenethylamine) studied. During a treatment period of 8 weeks, the MAO activity was not significantly altered with either active or sham dialysis. No relation between clinical ratings and platelet MAO activity was observed. Plasma from the seven patients was collected before and after 5 h of active or inactive dialysis and incubated with rat brain mitochondrial MAO (RBM-MAO). No difference was noted in the RBM-MAO-activity-regulating properties of the plasma sampled during either sham or active dialysis.     

Inflammatory stress exacerbates the progression of diabetic nephropathy

Objective To investigate the effects of inflammatory stress on the progression of diabetic nephropathy （DN） through making an inflamed animal model of DN. Methods Male db/db mice and db/m mice were randomly divided into four groups: db/m group (control, n=8), casein injected db/m (db/m＋casein, n=8), db/db mice (db/db, n=8), and casein injected db/db mice (db/db＋casein, n=8). Chronic inflammation was induced by subcutaneously injection of 0.5 ml 10% casein to db/m＋casein and db/db＋casein group every another day while db/m and db/db mice as the control were injected with 0.5 ml distilled water. Body weight and 24-hour urinary protein were measured every week. The plasma levels of serum amyloid A (SAA) and tumor necrotic factor-α (TNF-α) were detected by enzyme-linked immuno sorbent assay. Renal pathological changes were evaluated by renal pathological staining and electron microscope. Immunohistochemical staining and Western blotting were used to detect the expression of podocyte related specific proteins and inflammatory cytokines. Results The plasma levels of SAA[(13.83±0.29) mg/L vs (1.52±0.19) mg/L, P＜0.05; (13.84±0.28) mg/L vs (1.67±0.58) mg/L, P＜0.05] and TNF-α[(14.23±1.42) ng/L vs (10.70±1.38) ng/L, P＜0.05; (14.54±1.91) ng/L vs (10.88±1.22) ng/L, P＜0.05] were significantly increased in db/m＋casein and db/db＋casein group compared to that in db/m and db/db group respectively. Furthermore, the 24-hour urinary protein in casein injected db/db mice was markedly increased compared with db/db group. There were more significant renal pathological injuries and podocyte damage in casein injected db/db mice compared with db/db mice whereas these were no difference in casein injected db/m mice compared with db/m mice. Conclusion Inflammatory stress plays important roles in accelerating the progression of DN.     

Association of SH2-containing inositol phosphatase 2 with the insulin resistance of diabetic db/db mice

SH-2-containing inositol 5'-phosphatase 2 (SHIP-2) is a physiologically important lipid phosphatase that functions to hydrolyze phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P3 to PI(3,4)P2 in the negative regulation of insulin signaling. We investigated whether SHIP-2 is associated with the insulin resistance of diabetic db/db mice. The amount of SHIP-2 protein was elevated in quadriceps muscle and epididymal fat tissue, but not in the liver, of db/db mice relative to that in control db/+m mice. In accordance with the enhanced expression of SHIP-2, its localization at the membrane preparation was increased in the skeletal muscle and fat tissue of db/db mice. Insulin stimulation of PI 3-kinase activity was modestly decreased in skeletal muscle, fat tissue, and liver of db/db mice compared with that of db/+m mice. In addition to the modest decrease at the level of PI 3-kinase, the activity of Akt and protein kinase C (PKC)-zeta/lambda, which are downstream molecules of PI 3-kinase, was more severely reduced in the skeletal muscle and fat tissue, but not in liver of db/db mice. Treatment with the insulin-sensitizing agent rosiglitazone decreased the elevated expression of SHIP-2 in the skeletal muscle and fat tissue of db/db mice. Insulin-induced Akt activation and PKC-zeta/lambda phosphorylation were restored to the control level, although insulin-stimulated PI 3-kinase activation was minimally affected in the skeletal muscle and fat tissue of db/db mice. These results indicate that SHIP-2 is a novel molecule associated with insulin resistance in the skeletal muscle and fat tissue, and that insulin-induced activity of the downstream molecules of PI 3-kinase is decreased, at least in part, by the elevated expression of SHIP-2 in diabetic db/db mice.     

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)     

Neutral endopeptidase inhibition in diabetic wound repair

In response to cutaneous injury, sensory nerves release substance P, a proinflammatory neuropeptide. Substance P stimulates mitogenesis and migration of keratinocytes, fibroblasts, and endothelial cells. Neutral endopeptidase (NEP), a cell surface metallopeptidase, degrades substance P. Chronic nonhealing wounds and skin from patients with diabetes mellitus show increased NEP localization and activity. We hypothesized that increased NEP may retard wound healing and that NEP inhibition would improve closure kinetics in an excisional murine wound model. NEP enzyme activity was measured in skin samples from mutant diabetic mice (db/db) and nondiabetic (db/-) littermates by degradation of glutaryl-ala-ala-phe-4-methoxy-2-naphthylamine. Full-thickness 6-mm dorsal excisional wounds treated with normal saline or the NEP inhibitor thiorphan (10 microM or 25 microM) for 7 days were followed until closure. Histological examination and NEP activity were evaluated in a subset of wounds. NEP activity in unwounded db/db skin (20.6 pmol MNA/hr/ microg) significantly exceeded activity in db/-skin (7.9 pmol MNA/hr/ microg; p = 0.02). In db/db mice, 25 microM thiorphan shortened time to closure (18.0 days; p < 0.05) compared to normal saline (23.5 days). NEP inhibition did not alter closure kinetics in db/-mice. While the inflammatory response appeared enhanced in early wounds treated with thiorphan, blinded histological scoring of healed wounds using a semiquantitative scale showed no difference in inflammation. Unwounded skin from diabetic mice shows increased NEP activity and NEP inhibition improved wound closure kinetics without affecting contraction, suggesting that its principal effect was to augment epithelialization.     

Age-related changes in hepatic glycogen metabolism in the genetically diabetic (db/db) mouse

Hepatic glycogen metabolism was investigated in genetically diabetic C57BL/KsJ-db/db mice during their development. Initially, the development of obesity, hyperglycemia, hyperinsulinemia, and hyperglucagonemia in these mice was examined, which illustrated that the diabetes progressed normally. Little difference in hepatic glycogen concentrations was observed, averaging approximately 50 and 60 mg/g liver in diabetic (db/db) and control heterozygote (db/+) mice, respectively. Glycogen synthase activity (total and a-form) was significantly elevated by 5 wk in the diabetic mice relative to controls and reached maximum levels (two-fold higher than controls) around 8-9 wk. This activity then slowly declined during the rest of the 15-wk period examined. Both phosphorylase a and total phosphorylase activities were also elevated by 5 wk, reaching levels twofold higher than controls. These activities did not decline at the end of this 15-wk period, but instead continued to slowly increase. Glycogen synthase a activity showed a positive correlation (r = 0.54, N = 144) with circulating levels of insulin, and a similar correlation was seen for phosphorylase a activity and plasma glucagon levels (r = 0.64, N = 72). Protein kinase and phosphoprotein phosphatase activities were also measured, but no differences were detected between diabetic and control mice. This longitudinal study clarifies some of the changes in hepatic glycogen metabolism that occur during the progression of diabetes in the db/db mouse and indicates a role for circulating insulin and glucagon concentrations on the steady-state activities of glycogen synthase and phosphorylase, respectively.     

Kinetic properties of glycogen synthase and phosphorylase and structural aspects of glycogen in the db/db mouse liver

Kinetic studies were carried out on liver glycogen synthase and phosphorylase isolated from genetically diabetic db/db mice. Glycogen synthase a and b enzymes from diabetic mice had Vmax values 30% and 20% lower, respectively, than the enzymes from normal mice. Glycogen synthase b from diabetic mice also had a 30% lower I0.5 for Pi and ATP at physiologic concentrations of UDP-glucose (0.25 mM) compared with the normal enzyme. Kinetic studies of phosphorylase a showed that, at low glycogen concentrations (0.25 mg/ml), the Vmax of the diabetic enzyme was twofold greater than that of the normal enzyme. This was probably related to the diabetic phosphorylase a having a lower apparent Km for glycogen. This enzyme also had a slightly higher I0.5 for ATP compared with the enzyme from normal mice. Structural studies of liver glycogen isolated from these diabetic mice showed differences from normal mouse glycogen. Both the alpha- and beta-amylase limits were lower in the diabetic glycogen, and the average chain lengths, exterior chain lengths, and interior chain lengths calculated from these limits were all shorter in the glycogen from diabetic mice. Although both normal and diabetic glycogen absorbed light maximally at 430 nm when complexed with iodine, the absolute absorbance value was significantly lower for the diabetic glycogen. These data suggest an altered branching pattern of liver glycogen from the diabetic mice and it is suggested that this altered structure may ultimately influence the activities of glycogen-metabolizing enzymes. These results provide further characterization of the db/db mouse and show heretofore undescribed changes in phosphorylase a kinetics and glycogen structure that occur in diabetes.     

Glomerular localization and expression of Angiotensin-converting enzyme 2 and Angiotensin-converting enzyme: implications for albuminuria in diabetes

Angiotensin-converting enzyme 2 (ACE2) expression has been shown to be altered in renal tubules from diabetic mice. This study examined the localization of ACE and ACE2 within the glomerulus of kidneys from control (db/m) and diabetic (db/db) mice and the effect of chronic pharmacologic ACE2 inhibition. ACE2 co-localized with glomerular epithelial cell (podocyte) markers, and its localization within the podocyte was confirmed by immunogold labeling. ACE, by contrast, was seen only in glomerular endothelial cells. By immunohistochemistry, in glomeruli from db/db mice, strong ACE staining was found more frequently than in control mice (db/db 64.6 +/- 6.3 versus db/m 17.8 +/- 3.4%; P < 0.005). By contrast, strong ACE2 staining in glomeruli from diabetic mice was less frequently seen than in controls (db/db 4.3 +/- 2.4 versus db/m 30.6 +/- 13.6%; P < 0.05). For investigation of the significance of reduced glomerular ACE2 expression, db/db mice were treated for 16 wk with a specific ACE2 inhibitor (MLN-4760) alone or combined with telmisartan, a specific angiotensin II type 1 receptor blocker. At the end of the study, glomerular staining for fibronectin, an extracellular matrix protein, was increased in both db/db and db/m mice that were treated with MLN-4760. Urinary albumin excretion (UAE) increased significantly in MLN-4760-treated as compared with vehicle-treated db/db mice (743 +/- 200 versus 247 +/- 53.9 microg albumin/mg creatinine, respectively; P < 0.05), and the concomitant administration of telmisartan completely prevented the increase in UAE associated with the ACE2 inhibitor (161 +/- 56; P < 0.05). It is concluded that ACE2 is localized in the podocyte and that in db/db mice glomerular expression of ACE2 is reduced whereas glomerular ACE expression is increased. The finding that chronic ACE2 inhibition increases UAE suggests that ACE2, likely by modulating the levels of glomerular angiotensin II via its degradation, may be a target for therapeutic interventions that aim to reduce albuminuria and glomerular injury.