Hirofumi Shigeta

Interleukin-6 polymorphism (-634C/G) in the promotor region and the progression of diabetic nephropathy in type 2 diabetes.

Aims Interleukin‐6 (IL‐6) is a multifunctional cytokine produced by many different cell types, including glomerular mesangial cells. Recently, a novel C/G polymorphism at position −634 in the promotor region of the IL‐6 gene has been reported. The aim of this study was to investigate whether the −634C/G polymorphism is associated with an increased risk for progression to diabetic nephropathy as well as elevated levels of IL‐6 secretion by peripheral blood mononuclear cells.

Troglitazone (CS-045) Ameliorates Albuminuria in Streptozotocin-Induced Diabetic Rats

We studied the effect of troglitazone, a new oral antidiabetic agent that potentiates insulin action and reduces insulin resistance, on albuminuria in streptozotocin (STZ)-treated diabetic rats. Without affecting blood glucose level, blood pressure, and creatinine clearance, troglitazone treatment of diabetic rats significantly decreased the diabetes-associated albuminuria at all time points studied 14 to 12 weeks of treatment: diabetic 510 +/- 161 micrograms/24 h v diabetic treated 112 +/- 34 micrograms/24 h at 12 weeks, P < .05). These data suggest that troglitazone has potential in the treatment of diabetic nephropathy.

Induction of aldose reductase in cultured human microvascular endothelial cells by advanced glycation end products.

Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.

TNF, TNF receptor type 1, and allograft inflammatory factor-1 gene polymorphisms in Japanese patients with type 1 diabetes.

The human leukocyte antigen (HLA) class III region, located on chromosome 6p21, has been regarded as one of the susceptible loci for type 1 diabetes. Because it contains many genes related to inflammatory and immune responses, including tumor necrosis factor (TNF), lymphotoxin-alpha (LT-alpha), and allograft inflammatory factor 1 (AIF-1) genes, it is unclear which gene within the class III region is responsible for the susceptibility to the disease. We sequenced the AIF-1 gene region and detected three novel polymorphisms, all of which were diallelic and localized at introns. Then, we investigated AIF-1, TNF, and LT-alpha gene polymorphisms in 165 patients with type 1 diabetes, consisting of 90 patients with young-onset type 1 diabetes, 75 patients with adult-onset type 1 diabetes, and 200 control patients. We also analyzed TNF receptors type 1 (TNFR1) and type 2 (TNFR2) gene polymorphisms, located on chromosome 12p13 and 1p36, respectively. Although there were significant differences between type 1 diabetes patients and controls in the distributions of TNF promoter polymorphisms at position -1031 and -857, and LT-alpha gene NcoI polymorphism, none of them was independently associated with the disease after two-locus analysis with HLA class II alleles. We detected the significantly increased frequency of the -383C allele, located in the TNFR-1 promoter region, in both young-onset and adult-onset diabetes patients compared with controls. In addition, the -383C allele was found to be associated with higher expression of the TNFR1 gene than that of -383A allele in in vitro expression. These results suggest that the TNFR1 gene region might be a susceptible locus to type 1 diabetes in Japanese.

Immunochemical quantification of crossline as a fluorescent advanced glycation endproduct in erythrocyte membrane proteins from diabetic patients with or without retinopathy

Crossline is a novel advanced glycation endproduct (AGE) which has both a crosslink and fluorescence similar to AGE‐protein in vivo. To assess the association of AGEs to the development of diabetic retinopathy we developed a sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for crossline in blood samples and investigated the association of the development of retinopathy and erythrocyte membrane protein (EMP)‐crossline concentrations in patients with Type 2 diabetes mellitus (Type 2 DM). Crossline formation in EMP exceeded that in haemoglobin and was detectable in normal EMP samples without pretreatment by this ELISA system. Mean (±SE) EMP crossline levels were elevated 1.6‐fold in diabetic patients without retinopathy (7.6 ± 0.5 pmol mg−1, p < 0.005), 2.2‐fold in diabetic patients with non‐proliferative retinopathy (10.5 ± 0.6 pmol mg−1, p < 0.001) and 2.6‐fold in diabetic patients with proliferative retinopathy (12.0 ± 0.6 pmol mg−1, p < 0.001) compared with healthy control subjects (4.7 ± 0.5 pmol mg−1). Type 2 DM patients with retinopathy had significantly higher EMP‐crossline levels than those without retinopathy (p < 0.005). Our data suggest that elevated EMP‐crossline concentrations are associated with the presence of retinopathy in patients with Type 2 DM and EMP‐crossline measured by our ELISA may provide a useful marker for assessing the role of glycation in the development of diabetic retinopathy.

INFLUENCE OF TNF MICROSATELLITE POLYMORPHISMS (TNFA) ON AGE-AT-ONSET OF INSULIN-DEPENDENT DIABETES MELLITUS

The TNF-alpha gene is located in the HLA region and has been implicated in the pathogenesis of Type I (insulin-dependent) diabetes mellitus (IDDM). We investigated the frequency of TNFa microsatellite alleles in 76 young-onset IDDM patients, 65 adult-onset IDDM patients, and 90 control subjects. We also examined the association of these TNFa alleles with HLA-DRB1 alleles, HLA-class I alleles, and TNF-alpha production. The frequency of the TNFa2 and TNFa9 alleles was increased in the young-onset IDDM patients compared to control subjects, but the increased frequency of TNFa2 was not significant after the correction for the number of comparisons was made. We did not find any association of TNFa2 or TNFa9 with any of the HLA-DRB1 alleles. In contrast, the frequency of the TNFa13 allele was decreased in both the young-onset and the adult-onset IDDM patients compared to the control subjects, but the difference lost significance after the correction was made in the adult-onset IDDM. The TNFa13 allele was strongly associated with DRB1*1502. Patients with TNFa2 or TNFa9 had greater TNF-alpha production, while those positive for TNFa13 had lower TNF-alpha production than patients with non-TNFa2, a9, and a13 alleles. These results suggest that TNFa polymorphisms are associated with age-at-onset of IDDM and influence the inflammatory process of pancreatic beta cell destruction in the development of IDDM.

Antibodies to Glutamic Acid Decarboxylase in Japanese Diabetic Patients with Secondary Failure of Oral Hypoglycaemic Therapy

Some patients with non‐insulin‐dependent (Type 2) diabetes mellitus (NIDDM) are positive for antibodies to glutamic acid decarboxylase (anti‐GAD), which have been shown to be a useful marker for the diagnosis and prediction of insulin‐dependent (Type 1) diabetes mellitus (IDDM). Anti‐GAD positive NIDDM patients tend to develop insulin deficiency. We investigated the prevalence of anti‐GAD in 200 NIDDM with secondary failure of oral hypoglycaemic therapy (SF) and 200 NIDDM well controlled by diet and/or sulphonylurea agents (NSF). Twenty‐two of 200 (11 %, p < 0.05) SF patients and 6 of 200 (3 %) NSF patients were anti‐GAD positive. The positive rate for anti‐GAD was as high as 23.8 % in the non‐obese and insulin deficient SF patients. The SF patients with anti‐GAD tended to be non‐obese and to have an impaired release of endogenous insulin. The interval before development of secondary failure was not associated with the presence of anti‐GAD in this study. In conclusion we found that anti‐GAD was positive in as many as 11 % of the SF patients, suggesting that autoimmune mechanisms may play an important role in the pathogenesis of secondary failure of sulphonylurea therapy.

Serum Autoantibodies Against Sulfatide and Phospholipid in NIDDM Patients With Diabetic Neuropathy

OBJECTIVE We investigated the presence of antisulfatide and antiphospholipid antibodies and the relationship between these antibodies and the results of quantitative tests of nerve function in NIDDM patients with diabetic neuropathy. RESEARCH DESIGN AND METHODS Antisulfatide and antiphospholipid antibodies were measured in serum samples obtained from 68 NIDDM patients with diabetic neuropathy by an enzyme-linked immunosorbent assay (ELISA). Each patient was classified into one of three groups based on the combined neuropathy score (determined by the symptom score, the results of autonomic nerve function tests, and the vibration perception test), as follows: mild (n = 26), moderate (n = 22), and severe (n = 20). Nerve conduction studies were performed in a subgroup of 37 patients. RESULTS The antisulfatide antibody was detected in 1 (4%) of 26 patients in the mild group, 4 (18%) of 22 patients in the moderate group, and 8 (40%) of 20 patients in the severe group (P < 0.01 vs. mild group). The antiphospholipid antibody was detected in none of the patients in the mild group, 8 (36%) of 22 patients in the moderate group (P < 0.001 vs. mild group), and 6 (30%) of 20 patients in the severe group (P < 0.01 vs. mild group). The threshold amplitude, determined by the vibration perception test, was significantly higher in antibody-positive patients than in antibody-negative patients: antisulfatide antibody, 55.9 ± 46.8 μm (n = 13) vs. 22.9 ± 13.7 μm (n = 55), P < 0.001; antiphospholipid antibody, 47.2 ± 32.5 μm (n = 14) vs. 24.5 ± 23.2 μm (n = 54), P < 0.01. The conduction velocity of the sural nerve was slower in the antisulfatide antibody-positive group (37.9 ± 11.1 m/s, n = 12) than in the antisulfatide antibody-negative group (45.2 ± 6.0 m/s, n = 19) (P < 0.05). CONCLUSIONS These results suggest that autoimmune nerve destruction may be involved in diabetic neuropathy in NIDDM patients.

Crossline levels in serum and erythrocyte membrane proteins from patients with diabetic nephropathy

Crossline is one of the structurally defined adducts of advanced glycation endproducts (AGEs) which has both a crosslink and fluorescence similar to AGE-protein in vivo. Crossline was measured in serum and erythrocyte membrane proteins (EMP) from 52 type 2 diabetic patients using a specific enzyme-linked immunosorbent assay system. Serum and EMP crossline levels in the diabetic patients were significantly higher than those in normal control. The patients with advanced diabetic nephropathy (serum creatinine levels of more than 1.2 mg/dl) had markedly elevated serum crossline levels compared to those with moderate diabetic nephropathy (clinical proteinuria) (180. 7+/-51.7 vs. 71.8+/-18.4 pmol/ml; P<0.01). On the other hand, there were no significant differences in EMP crossline levels between the two. EMP crossline levels in the patients with moderate diabetic nephropathy (8.8+/-2.9 pmol/mg protein) and those with advanced diabetic nephropathy (9.7+/-3.0 pmol/mg protein) were significantly higher than those without clinical proteinuria (6.4+/-1.9 pmol/mg protein; P<0.01). The present study demonstrated that EMP crossline levels were associated with the presence of nephropathy in patients with type 2 diabetes mellitus. Serum crossline levels were significantly influenced by remaining renal function. The measurement of crossline from a blood sample could provide us with important information for the study of clinical evaluation and pathogenesis of diabetic complications.

High Prevalence of Mitochondrial Diabetes Mellitus in Japanese Patients With Major Risk Factors

To identify diabetes mellitus caused by the mitochondrial gene substitution at genomic nucleotide pair 3243 (M3243A-->G) we selected 87 diabetic patients with high risk factors such as maternal inheritance and hearing loss. Total DNA was extracted from peripheral leukocytes, and mitochondrial DNA fragments containing M3243A-->G were amplified by polymerase chain reaction (PCR). The amplified fragments were digested with a restriction endonuclease Apa1 and analyzed by agarose gel electrophoresis. The incidence of the M3243A-->G mutation was 4.6% (four of 87) in diabetic patients with maternal inheritance and/or hearing loss. In a subgroup with both maternal inheritance and hearing loss, the incidence of the mutation was as high as 21.4% (three of 14). Cardiac disorders were also present in all four diabetic patients with the mutation. This study suggests that maternal inheritance and hearing loss are useful clinical findings to identify diabetic patients with the mutation, and that cardiac involvement is a high risk factor for the M3243A-->G mutation.