The glucose-lowering effects of the PDE4 inhibitors roflumilast and roflumilast-N-oxide in db/db mice

Aims/hypothesis  

The cAMP-degrading phosphodiesterase 4 (PDE4) enzyme has recently been implicated in the regulation of glucagon-like peptide-1 (GLP-1), an incretin hormone with glucose-lowering properties. We investigated whether the PDE4 inhibitor roflumilast elevates GLP-1 levels in diabetic db/db mice and whether this elevation is accompanied by glucose-lowering effects.     

Coenzyme Q10 prevents GDP-sensitive mitochondrial uncoupling, glomerular hyperfiltration and proteinuria in kidneys from db/db mice as a model of type 2 diabetes

Aims/hypothesis  

Increased oxygen consumption results in kidney tissue hypoxia, which is proposed to contribute to the development of diabetic nephropathy. Oxidative stress causes increased oxygen consumption in type 1 diabetic kidneys, partly mediated by uncoupling protein-2 (UCP-2)-induced mitochondrial uncoupling. The present study investigates the role of UCP-2 and oxidative stress in mitochondrial oxygen consumption and kidney function in db/db mice as a model of type 2 diabetes.     

Treatment with brain natriuretic peptide prevents the development of cardiac dysfunction in obese diabetic db/db mice

Aims/hypothesis

Obesity and diabetes increase the risk of developing cardiovascular diseases and heart failure. These metabolic disorders are generally reflected by natriuretic peptide system deficiency. Since brain natriuretic peptide (BNP) is known to influence metabolism and cardioprotection, we investigated the effect of chronic exogenous BNP treatment on adverse myocardial consequences related to obesity and diabetes.

Methods

Ten-week-old C57BL/KsJ-db/db obese diabetic mice (db/db) and their lean control littermates (db/+) were treated with BNP (0.6 μg kg^?1 h^?1) or saline for 12 weeks (n?=?10/group). Serial blood and tomography analysis were performed. Cardiac function was determined by echocardiography, and biochemical and histological heart and fat analyses were also performed.

Results

BNP treatment resulted in an average increase in plasma BNP levels of 70 pg/ml. An improvement in the metabolic profile of db/db mice was observed, including a reduction in fat content, increased insulin sensitivity, improved glucose tolerance and lower blood glucose, despite increased food intake. db/db mice receiving saline displayed both early systolic and diastolic dysfunction, whereas these functional changes were prevented by BNP treatment. The cardioprotective effects of BNP were attributed to the inhibition of cardiomyocyte apoptosis, myocardial fibrosis, cardiac hypertrophy and the AGE–receptor for AGE (RAGE) system as well as normalisation of cardiac AMP-activated protein kinase and endothelial nitric oxide synthase activities.

Conclusions/interpretation

Our results indicate that chronic BNP treatment at low dose improves the metabolic profile and prevents the development of myocardial dysfunction in db/db mice.     

Intracellular sodium increase and susceptibility to ischaemia in hearts from type 2 diabetic db/db mice

Aims/hypothesis An important determinant of sensitivity to ischaemia is altered ion homeostasis, especially disturbances in intracellular Na⁺ handling. As no study has so far investigated this in type 2 diabetes, we examined susceptibility to ischaemia–reperfusion in isolated hearts from diabetic db/db and control db/+ mice and determined whether and to what extent the amount of increase during a transient period of ischaemia could contribute to functional alterations upon reperfusion. Methods Isovolumic hearts were exposed to 30-min global ischaemia and then reperfused. ²³Na nuclear magnetic resonance (NMR) spectroscopy was used to monitor and ³¹P NMR spectroscopy to monitor intracellular pH (pH_i). Results A higher duration of ventricular tachycardia and the degeneration of ventricular tachycardia into ventricular fibrillation were observed upon reperfusion in db/db hearts. The recovery of left ventricular developed pressure was reduced. The increase in induced by ischaemia was higher in db/db hearts than in control hearts, and the rate of pH_i recovery was increased during reperfusion. The inhibition of Na⁺/H⁺ exchange by cariporide significantly reduced gain at the end of ischaemia. This was associated with a lower incidence of ventricular tachycardia in both heart groups, and with an inhibition of the degeneration of ventricular tachycardia into ventricular fibrillation in db/db hearts. Conclusions/interpretation These findings strongly support the hypothesis that increased plays a causative role in the enhanced sensitivity to ischaemia observed in db/db diabetic hearts.     

The secretory deficit in islets from db/db mice is mainly due to a loss of responding beta cells

Aims/hypothesis

We used the db/db mouse to determine the nature of the secretory defect in intact islets.

Methods

Glucose tolerance was compared in db/db and wild-type (WT) mice. Isolated islets were used: to measure insulin secretion and calcium in a two-photon assay of single-insulin-granule fusion; and for immunofluorescence of soluble N-ethylmaleimide-sensitive factor attachment proteins (SNAREs).

Results

The 13–18-week-old db/db mice showed a diabetic phenotype. Isolated db/db islets showed a 77% reduction in insulin secretion induced by 15 mmol/l glucose and reductions in the amplitude and rise-time of the calcium response to glucose. Ionomycin-induced insulin secretion in WT but not db/db islets. Immunofluorescence showed an increase in the levels of the SNAREs synaptosomal-associated protein 25 (SNAP25) and vesicle-associated membrane protein 2 (VAMP2) in db/db islets, but reduced syntaxin-1A. Therefore, db/db islets have both a compromised calcium response to glucose and a compromised secretory response to calcium. Two-photon microscopy of isolated islets determined the number and distribution of insulin granule exocytic events. Compared with WT, db/db islets showed far fewer exocytic events (an 83% decline at 15 mmol/l glucose). This decline was due to a 73% loss of responding cells and, in the remaining responsive cells, a 50% loss of exocytic responses per cell. An assay measuring granule re-acidification showed evidence for more recaptured granules in db/db islets compared with WT.

Conclusions/interpretation

We showed that db/db islets had a reduced calcium response to glucose and a reduction in syntaxin-1A. Within the db/db islets, changes were manifest as both a reduction in responding cells and a reduction in fusing insulin granules per cell.     

Tissue-specific dysregulation of hexose-6-phosphate dehydrogenase and glucose-6-phosphate transporter production in db/db mice as a model of type 2 diabetes

Aims/hypothesis  

Tissue-specific amplification of glucocorticoid action through 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) affects the development of the metabolic syndrome. Hexose-6-phosphate dehydrogenase (H6PDH) mediates intracellular NADPH availability for 11β-HSD1 and depends on the glucose-6-phosphate transporter (G6PT). Little is known about the tissue-specific alterations of H6PDH and G6PT and their contributions to local glucocorticoid action in db/db mice.     

Prevention of decline in renal function in the diabetic db/db mouse

Summary We recently reported that when diabetic db/db mice, which develop glomerular pathology resembling that in human diabetes mellitus, are treated with monoclonal antibodies (A717) that neutralize the effects of excess glycated albumin, there is an amelioration of mesangial expansion, renal overexpression of mRNAs encoding for the extracellular matrix proteins collagen IV and fibronectin, and proteinuria. These findings suggested that A717 might also retard the development of compromised renal function in this animal model. To examine this possibility, serum creatinine and blood urea nitrogen (BUN) were measured in diabetic db/db mice and their non-diabetic db/m littermates before and after an 8-week course of treatment with A717 or irrelevant murine immunoglobulin (MIg). Early in the course of diabetes, BUN and serum creatinine concentrations did not significantly differ from those in the db/m littermates, but were significantly increased after 10 weeks of sustained hyperglycaemia. Treatment of db/db mice with A717 prevented the rise in creatinine and attenuated the elevation in BUN. A717 also prevented the decrease in creatinine clearance observed in diabetic compared with non-diabetic animals (2.2±0.8 vs 4.1±0.3 vs 5.0±1.1 ml/h in db/db vs db/db-A717 vs db/m, respectively). MIg did not alter the change in renal function with time in db/db mice. Taken together with our previous results, the present findings indicate that the diabetic db/db mouse develops changes in renal function and structure that parallel the course of human diabetic nephropathy in nature and chronology and demonstrate, for the first time, that therapy directed against increased glycated albumin can prevent the decline in renal function in this rodent model of genetic diabetes.Abbreviations MIg Murine immunoglobulin - BUN blood urea nitrogen - ACE angiotensin converting enzyme - AGE advanced glycation end product     

The human glucagon-like peptide-1 analogue liraglutide preserves pancreatic beta cells via regulation of cell kinetics and suppression of oxidative and endoplasmic reticulum stress in a mouse model of diabetes

Aims/hypothesis  

We investigated the molecular mechanism by which the human glucagon-like peptide-1 analogue liraglutide preserves pancreatic beta cells in diabetic db/db mice.     

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesis  

The TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.     

Insulin-like growth factor I (IGF-1) supplementation prevents diabetesinduced alterations in coenzymes Q<Subscript>9</Subscript> and Q<Subscript>10</Subscript>

Diabetes, which causes enhanced oxidative stress, is a multifactorial disease that leads to deleterious effects in many organ systems within the body. Ubiquinones (coenzyme Q₉ and Q₁₀) are amphipathic molecular components of the electron transport chain that function also as endogenous antioxidants and attenuate the diabetes-induced decreases in antioxidant defense mechanisms. Insulin-like growth factor 1 (IGF-1) is considered to be an “essential surviving factor”, the level and function of which are compromised in diabetes. This study investigated the impact of IGF-1 supplementation on ubiquinone levels in a rat model of type I diabetes. Adult male Sprague-Dawley rats were divided into four groups: control, control plus IGF-1, diabetic and diabetic plus IGF-1. Diabetic animals received a single intravenous injection of streptozotocin (STZ, 55 mg/kg). IGF-1 supplementation groups received a daily intraperitoneal dose of 3 mg IGF-1 per kilogram body weight for 7 weeks. Coenzyme Q₉ and Q₁₀ levels were assessed by ultraviolet detection on high pressure liquid chromatography. STZ caused a significant reduction in body weight and an elevation in blood glucose level, which were not prevented by IGF-1 supplementation. In addition Q₉ and Q₁₀ levels in diabetic liver were significantly elevated. IGF-1 supplementation prevented liver alterations in Q₁₀ but not Q₉ levels. Q₉ and Q₁₀ levels in diabetic kidney were significantly depressed, and these deleterious effects were abolished by IGF-1 treatment. These data suggest that IGF-1 antagonizes the diabetes-induced alterations in endogenous antioxidants including coenzyme Q₁₀, and hence may have a therapeutic role in diabetes. Received: 28 March 2002 / Accepted in revised form: 5 December 2002 Correspondence to J. Ren     

Beneficial effects of phlorizin on diabetic nephropathy in diabetic db/db mice

AimsThis study observes the effects of phlorizin on diabetic nephrology in db/db diabetic mice and explores possible underlying mechanisms.MethodsSixteen diabetic db/db mice and eight age-matched db/m mice were divided into three groups: vehicle-treated diabetic group (DM group), diabetic group treated with phlorizin (DMT group) and normal control group (CC group). Phlorizin was given in normal saline solution by intragastric administration for 10 weeks. Differentially expressed proteins in three groups were identified using iTRAQ quantitative proteomics and the data were further analyzed with ingenuity pathway analysis.ResultsThe body weight and serum concentrations of fasting blood glucose (FBG), advanced glycation end products (AGEs), total cholesterol, triglycerides, blood urea nitrogen, creatinine and 24-h urine albumin were increased in the DM group compared to those of the CC group (P < 0.05), and they were decreased by treatment with phlorizin (P < 0.05). Morphologic observations showed phlorizin markedly attenuated renal injury. Phlorizin prevented diabetic nephropathy by regulating the expression of a series of proteins involved in renal and urological disease, molecular transport, free radical scavenging, and lipid metabolism.ConclusionsPhlorizin protects mice from diabetic nephrology and thus may be a novel therapeutic approach for the treatment of diabetic nephrology.     

Studies in the diabetic mutant mouse: V. Glucose tolerance in mice homozygous and heterozygous for the diabetes (db) gene

Summary Mice homozygous and heterozygous for the diabetes (db) gene were studied to determine: 1. whether latent carbohydrate intolerance is present in young normoglycemic diabetic mutants (db/db); 2. whether normoglycemic food restricted diabetic mutants are carbohydrate intolerant; and 3. whether mice heterozygous for thedb gene (db/+) manifest abnormalities in glucose tolerance, serum IRI levels or body weights. Blood glucose levels were determined 0, 1/2, 1, 2 and 3 h following intraperitoneal administration of 2 mg glucose/g body weight. Normals (+/+) and diabetics (db/db) showed similar glucose tolerance curves during the first two weeks of life; however, both were markedly glucose intolerant compared to normal adult mice. At 3 weeks a small number of mutants had higher 3 h levels than any achieved in normal mice. By 4 weeks the average value for diabetics prior to glucose loading (0 time) was significantly (P < 0.02) elevated (db/db — 144 mg glucose/100 ml, +/+ = 124 mg glucose/100 ml). Although food restriction reduced blood glucose concentration at 0 time, persistence of carbohydrate intolerance was readily demonstrable following glucose loading. — Abnormalities in heterozygotes (db/+), 3 to 16 months of age, were primarily restricted to male mice, which showed moderate, but statistically significant elevations in blood glucose both at 0 time and following glucose administration. Forty percent of male heterozygotes had higher serum IRI levels than any observed in normal control males. Body weights of male heterozygotes were significantly greater (P<0.01) than those in agematched normals.USPHS Research Career Development Awardee, Grant K4-AM-7394.     

Monocyte chemoattractant protein-1-induced tissue inflammation is critical for the development of renal injury but not type 2 diabetes in obese db/db mice

Aims/hypothesis Tissue macrophage accumulation is thought to induce insulin resistance during obesity and stimulate the progression of diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a potent stimulator of macrophage recruitment. It is increased in adipose tissue during obesity and in diabetic kidneys, suggesting that inflammation of these tissues may be MCP-1-dependent. Based on these findings, the aim of this study was to examine whether a deficiency in MCP-1 would alter the development of type 2 diabetes and its renal complications. Materials and methods The role of MCP-1 in the progression of type 2 diabetes and its associated renal injury was assessed in obese db/db mice that were deficient in the gene encoding MCP-1 (Ccl2). Results The incidence and development of type 2 diabetes were similar in Ccl2 ^+/+ and Ccl2 ^−/− db/db mice between 8 and 32 weeks of age. Body mass, hyperglycaemia, hyperinsulinaemia, glucose and insulin tolerance, plasma triacylglycerol and serum NEFA were not different between these strains. Pathological changes in epididymal adipose tissue, including increases in macrophage accumulation and Tnfa mRNA and reductions in Adipoq mRNA, were unaffected by the absence of MCP-1. In contrast, kidney macrophage accumulation and the progression of diabetic renal injury (albuminuria, histopathology, renal fibrosis) were substantially reduced in Ccl2 ^−/− compared with Ccl2 ^+/+ db/db mice with equivalent diabetes. Conclusions/interpretation Our study demonstrates that MCP-1 promotes type 2 diabetic renal injury but does not influence the development of obesity, insulin resistance or type 2 diabetes in db/db mice. MCP-1 plays a critical role in inflammation of the kidney, but not adipose tissue, during the progression of type 2 diabetes.     

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.     

miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes

Aims/hypothesis

As microRNA-21 (miR-21) plays a pathological role in fibrosis, we hypothesised that it may be a therapeutic target for diabetic nephropathy.

Methods

Abundance of miR-21 was examined in diabetic kidneys from db/db mice. The therapeutic potential of miR-21 in diabetic kidney injury was examined in db/db mice by an ultrasound-microbubble-mediated miR-21 small hairpin RNA transfer. In addition, the role and mechanisms of miR-21 in diabetic renal injury were examined in vitro under diabetic conditions in rat mesangial and tubular epithelial cell lines by overexpressing or downregulating miR-21.

Results

In db/db mice, a mouse model of type 2 diabetes, renal miR-21 at age 20 weeks was increased twofold compared with db/m ⁺ mice at the same age, and this increase was associated with the development of microalbuminuria and renal fibrosis and inflammation. More importantly, gene transfer of miR-21 knockdown plasmids into the diabetic kidneys of db/db mice at age 10 weeks significantly ameliorated microalbuminuria and renal fibrosis and inflammation at age 20 weeks, revealing a therapeutic potential for diabetic nephropathy by targeting miR-21. Overexpression of miR-21 in kidney cells enhanced, but knockdown of miR-21 suppressed, high-glucose-induced production of fibrotic and inflammatory markers. Targeting Smad7 may be a mechanism by which miR-21 regulates renal injury because knockdown of renal miR-21 restored Smad7 levels and suppressed activation of the TGF-β and NF-κB signalling pathways.

Conclusions/interpretation

Inhibition of miR-21 might be an effective therapy for diabetic nephropathy.     

Role of MKK3–p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice

Aims/hypothesis  Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. Methods  Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. Results   Mkk3 ^+/+ db/db and Mkk3 ^−/− db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 ^+/+ db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 ^−/− db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 ^−/− db/db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 ^−/− db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. Conclusions/interpretation  MKK3–p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.     

Coenzyme Q10 improves endothelial dysfunction of the brachial artery in Type II diabetes mellitus

Aim/hypothesis: We assessed whether dietary supplementation with coenzyme Q₁₀ improves endothelial function of the brachial artery in patients with Type II (non-insulin-dependent) diabetes mellitus and dyslipidaemia. Methods: A total of 40 patients with Type II diabetes and dyslipidaemia were randomized to receive 200 mg of coenzyme Q₁₀ or placebo orally for 12 weeks. Endothelium-dependent and independent function of the brachial artery was measured as flow-mediated dilatation and glyceryl-trinitrate-mediated dilatation, respectively. A computerized system was used to quantitate vessel diameter changes before and after intervention. Arterial function was compared with 18 non-diabetic subjects. Oxidative stress was assessed by measuring plasma F₂-isoprostane concentrations, and plasma antioxidant status by oxygen radical absorbance capacity. Results: The diabetic patients had impaired flow-mediated dilation [3.8 % (SEM 0.5) vs 6.4 % (SEM 1.0), p = 0.016], but preserved glyceryl-trinitrate-mediated dilation, of the brachial artery compared with non-diabetic subjects. Flow-mediated dilation of the brachial artery increased by 1.6 % (SEM 0.3) with coenzyme Q₁₀ and decreased by –0.4 % (SEM 0.5) with placebo (p = 0.005); there were no group differences in the changes in pre-stimulatory arterial diameter, post-ischaemic hyperaemia or glyceryl-trinitrate-mediated dilation response. Coenzyme Q₁₀ treatment resulted in a threefold increase in plasma coenzyme Q₁₀ (p < 0.001) but did not alter plasma F₂-isoprostanes, oxygen radical absorbance capacity, lipid concentrations, glycaemic control or blood pressure. Conclusion/interpretation: Coenzyme Q₁₀ supplementation improves endothelial function of conduit arteries of the peripheral circulation in dyslipidaemic patients with Type II diabetes. The mechanism could involve increased endothelial release and/or activity of nitric oxide due to improvement in vascular oxidative stress, an effect that might not be reflected by changes in plasma F₂-isoprostane concentrations. [Diabetologia (2002) 45: 420–426] Received: 14 August 2001 and in revised form: 15 November 2001     

Coenzyme Q10 administration increases brain mitochondrial concentrations and exerts neuroprotective effects

Coenzyme Q₁₀ is an essential cofactor of the electron transport chain as well as a potent free radical scavenger in lipid and mitochondrial membranes. Feeding with coenzyme Q₁₀ increased cerebral cortex concentrations in 12- and 24-month-old rats. In 12-month-old rats administration of coenzyme Q₁₀ resulted in significant increases in cerebral cortex mitochondrial concentrations of coenzyme Q₁₀. Oral administration of coenzyme Q₁₀ markedly attenuated striatal lesions produced by systemic administration of 3-nitropropionic acid and significantly increased life span in a transgenic mouse model of familial amyotrophic lateral sclerosis. These results show that oral administration of coenzyme Q₁₀ increases both brain and brain mitochondrial concentrations. They provide further evidence that coenzyme Q₁₀ can exert neuroprotective effects that might be useful in the treatment of neurodegenerative diseases.