PPARalpha agonist fenofibrate improves diabetic nephropathy in db/db mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy.     

Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice

Macrophage dysfunction is a likely mechanism underlying common diabetic complications such as increased susceptibility to infection, accelerated atherosclerosis, and disturbed wound healing. There are no available studies on the function of tissue macrophages in diabetes in humans. We have therefore studied peritoneal macrophages from diabetic type 2-like db/db mice. We found that the release of tumor necrosis factor-alpha and interleukin-1beta from lipopolysaccharide plus interferon-gamma-stimulated macrophages and vascular endothelial growth factor from both stimulated and nonstimulated macrophages was significantly reduced in diabetic animals compared with nondiabetic controls. Nitric oxide production from the stimulated db/db macrophages was significantly higher than that in the db/+ cultures, whereas there was no difference in their ability to generate reactive oxygen species. When studied both at light and electron microscopic levels, macrophages in diabetic animals had an altered morphological appearance compared with those of normal controls. We conclude that the function and morphology of the macrophages are disturbed in db/db mice and that this disturbance is related to the mechanisms underlying common inflammatory and degenerative manifestations in diabetes.     

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.     

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.     

Empagliflozin alleviates podocytopathy and enhances glomerular nephrin expression in db/db diabetic mice

BACKGROUNDModern guidelines recommend sodium-glucose cotransporter-2 (SGLT2) inhibitors as the preferred antihyperglycemic agents for patients with type 2 diabetes and chronic kidney disease. However, the mechanisms underlying the renal protective effect of SGLT2 inhibitors are not fully understood.AIMTo estimate the effect of the SGLT2 inhibitor, empagliflozin (EMPA), on the structure of podocytes and nephrin expression in glomeruli in db/db diabetic mice.METHODSWe treated 8-wk-old male db/db mice with EMPA (10 mg/kg/d) or vehicle for 8 wk. Age-matched male db/+ mice were included as non-diabetic controls. Parameters of body composition, glycemic and lipid control, and plasma concentrations of leptin, insulin and glucagon were assessed. We evaluated renal hypertrophy as kidney weight adjusted to lean mass, renal function as plasma levels of creatinine, and albuminuria as the urinary albumin-to-creatinine ratio (UACR). Renal structures were studied by light and transmission electron microscopy with a focus on mesangial volume and podocyte structure, respectively. Glomerular nephrin and transforming growth factor beta (TGF-β) were assessed by immunohistochemistry.RESULTSSevere obesity and hyperglycemia developed in db/db mice prior to the start of the experiment; increased plasma concentrations of fructosamine, glycated albumin, cholesterol, leptin, and insulin, and elevated UACR were detected. Mesangial expansion, glomerular basement membrane thickening, and increased area of TGF-β staining in glomeruli were revealed in vehicle-treated mice. Podocytopathy was manifested by effacement of foot processes; nephrin-positive areas in glomeruli were reduced. EMPA decreased the levels of glucose, fructosamine and glycated albumin, UACR, kidney hypertrophy, mesangial expansion, glomerular basement membrane thickening, and glomerular TGF-β staining, alleviated podocytopathy and restored glomerular staining of nephrin.CONCLUSIONThese data indicate that EMPA attenuates podocytopathy in experimental diabetic kidney disease. The anti-albuminuric effect of EMPA could be attributed to mitigation of podocyte injury and enhancement of nephrin expression.     

Reduction in glucagon receptor expression by an antisense oligonucleotide ameliorates diabetic syndrome in db/db mice

Excess glucagon levels contribute to the hyperglycemia associated with type 2 diabetes. Reducing glucagon receptor expression may thus ameliorate the consequences of hyperglucagonemia and improve blood glucose control in diabetic patients. This study describes the antidiabetic effects of a specific glucagon receptor antisense oligonucleotide (GR-ASO) in db/db mice. The ability of GR-ASOs to inhibit glucagon receptor mRNA expression was demonstrated in primary mouse hepatocytes by quantitative real-time RT-PCR. Intraperitoneal administration of GR-ASO at a dosage of 25 mg/kg twice a week in db/db mice for 3 weeks resulted in 1) decreased glucagon receptor mRNA expression in liver; 2) decreased glucagon-stimulated cAMP production in hepatocytes isolated from GR-ASO-treated db/db mice; 3) significantly reduced blood levels of glucose, triglyceride, and free fatty acids; 4) improved glucose tolerance; and 5) a diminished hyperglycemic response to glucagon challenge. Neither lean nor db/db mice treated with GR-ASO exhibited hypoglycemia. Suppression of GR expression was also associated with increased ( approximately 10-fold) levels of plasma glucagon. No changes were observed in pancreatic islet cytoarchitecture, islet size, or alpha-cell number. However, alpha-cell glucagon levels were increased significantly. Our studies support the concept that antagonism of glucagon receptors could be an effective approach for controlling blood glucose in diabetes.     

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.     

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor activator, improves diabetes and other metabolic abnormalities and preserves beta-cell function in db/db mice

Muraglitazar, a novel dual (alpha/gamma) peroxisome proliferator-activated receptor (PPAR) activator, was investigated for its antidiabetic properties and its effects on metabolic abnormalities in genetically obese diabetic db/db mice. In db/db mice and normal mice, muraglitazar treatment modulates the expression of PPAR target genes in white adipose tissue and liver. In young hyperglycemic db/db mice, muraglitazar treatment (0.03-50 mg . kg(-1) . day(-1) for 2 weeks) results in dose-dependent reductions of glucose, insulin, triglycerides, free fatty acids, and cholesterol. In older hyperglycemic db/db mice, longer-term muraglitazar treatment (30 mg . kg(-1) . day(-1) for 4 weeks) prevents time-dependent deterioration of glycemic control and development of insulin deficiency. In severely hyperglycemic db/db mice, muraglitazar treatment (10 mg . kg(-1) . day(-1) for 2 weeks) improves oral glucose tolerance and reduces plasma glucose and insulin levels. In addition, treatment increases insulin content in the pancreas. Finally, muraglitazar treatment increases abnormally low plasma adiponectin levels, increases high-molecular weight adiponectin complex levels, reduces elevated plasma corticosterone levels, and lowers elevated liver lipid content in db/db mice. The overall conclusions are that in db/db mice, the novel dual (alpha/gamma) PPAR activator muraglitazar 1) exerts potent and efficacious antidiabetic effects, 2) preserves pancreatic insulin content, and 3) improves metabolic abnormalities such as hyperlipidemia, fatty liver, low adiponectin levels, and elevated corticosterone levels.     

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.     

db/db小鼠胰岛的分离与特征分析

目的 分离db/db小鼠胰岛,并对其特征进行检测和分析。方法 采用胶原酶V对10只db/db小鼠胰岛分离进行逆行胰管灌注和消化,用Ficoll-1077和Ficoll-1119进行胰岛的不连续密度梯度纯化,对分离的胰岛进一步进行手工挑选,并用DTZ进行胰岛和纯度鉴定,用透射电镜观察胰岛内部分泌颗粒等情况。结果 经本方法分离可得到db/db小鼠胰岛数量为（122.4±6.6）个/只,当量为（483.6±82.3）IEQ/只,与ICR小鼠胰岛分离结果差异有统计学意义（P<0.05）;db/db小鼠胰岛DTZ染色后显淡红色;胰岛大小指数为（3.96±0.64）,显著大于ICR小鼠胰岛（P<0.05）;透射电镜下显示β细胞中的胰岛素分泌颗粒减少,分泌颗粒颜色较浅。结论 采用本方法可分离得到db/db小鼠胰岛,为后续开展基于胰岛功能和特征变化的2型糖尿病患者治疗研究提供一种参考。     

The effect of chronic alpha-glycosidase inhibition on diabetic nephropathy in the db/db mouse

Acarbose, a complex oligosaccharide, is a potent competitive inhibitor of sucrase and decreases postprandial hyperglycemia when administered with food. To evaluate its potential for metabolic control and prevention of diabetic nephropathy, groups of gentically diabetic mice (C57 BLKsJ db/db) were treated with Acarbose for 10 wk. Control mice received normal chow and experimental groups were given Acarbose prepared as a drug-food mixture in doses of 10, 20, and 40 mg/100 g of food. Acarbose did not influence fasting blood glucose, food intake, or the normal development of obesity in the mice. Urinary glucose excretion and glycosylated hemoglobin was significantly reduced in animals receiving high-dose Acarbose (40 mg/100 g food). Immunopathologic examination of the kidneys showed a dose-dependent decrease in glomerular mesangial immunoglobulin deposition. By light microscopy, glomerular mesangial thickening was significantly reduced in the group receiving high-dose Acarbose (40 mg/100 g food). To the extent that Acarbose improves metabolic control in the db/db mouse, chronic treatment with this agent produces a dose-dependent amelioration of diabetic nephropathy. Alphaglycosidase inhibition may be a useful adjunctive therapy for blood glucose control in diabetes mellitus.     

Brain-derived neurotrophic factor improves blood glucose control and alleviates fasting hyperglycemia in C57BLKS-Lepr(db)/lepr(db) mice

Systemic administration of brain-derived neurotrophic factor (BDNF) decreases nonfasted blood glucose in obese, non-insulin-dependent diabetic C57BLKS-Lepr(db)/lepr(db) (db/db) mice, with a concomitant decrease in body weight. By measuring percent HbA1c in BDNF-treated and pair-fed animals, we show that the effects of BDNF on nonfasted blood glucose levels are not caused by decreased food intake but reflect a significant improvement in blood glucose control. Furthermore, once established, this effect can persist for weeks after cessation of BDNF treatment. Oral glucose tolerance tests were performed to examine the effects of BDNF on blood glucose control in the fasted state and after an oral glucose challenge. BDNF treatment normalized fasting blood glucose from initially hyperglycemic levels and also showed evidence for beneficial, although less marked, effects on the ability to remove exogenous glucose from blood. One means to lower fasting blood glucose is to reduce the glucose output of peripheral tissues that normally play a part in the maintenance of fasting hyperglycemia. Because the liver is the major endogenous source of glucose in blood during fasting, and because hepatic weight and glucose output are increased in type 2 diabetes, we evaluated the effects of BDNF on liver tissue. BDNF reduced the hepatomegaly present in db/db mice, in association with reduced liver glycogen and reduced liver enzyme activity in serum, supporting the possible involvement of liver tissue in the mechanism of action for BDNF.     

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.     

Molecular mimicry between insulin and retroviral antigen p73. Development of cross-reactive autoantibodies in sera of NOD and C57BL/KsJ db/db mice

Enzyme-linked immunosorbent assay (ELISA) was used to study temporal development of murine autoantibodies against insulin and both type C and intracisternal type A retroviral antigens. The nonobese diabetic (NOD) mouse, a model for autoimmune, insulin-dependent diabetes, was compared with a related, but diabetes-resistant, strain, nonobese normal (NON). Similarly, C57BL/KsJ db/db mice (insulin-resistant model of insulin-dependent diabetes and obesity) were compared with diabetes-resistant C57BL/6 db/db mice. NOD mice developed much higher autoantibody titers than did NON mice. Whereas type C autoantibodies in NOD developed to peak titer shortly after mice were weaned, autoantibodies against insulin and p73 (group-specific antigen of the intracisternal type A particle) did not develop until shortly before, or concomitant with, the development of hyperglycemia. Two NOD mice not developing hyperglycemia during the 40-wk study period were distinguished from the mice developing diabetes by a delayed onset of insulin (but not p73) autoantibodies. Our findings suggest that in NOD mice, the appearance of insulin and p73 autoantibodies signifies that extensive underlying necrosis of beta-cells occurred. C57BL/KsJ db/db mice (with extensive beta-cell necrosis and early hyperglycemia) developed much higher autoantibody titers to insulin and p73 than did the diabetes-resistant C57BL/6 db/db mice. However, the presence of autoantibodies in normoglycemic C57BL/KsJ +/db controls demonstrated that elevated autoantibody titers alone were insufficient to produce diabetes in this model. Absorption studies indicated that autoantibodies against p73 recognized a common epitope on insulin and IgE-binding factor. The potential significance of this molecular mimicry is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.