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Aims: Lipid parameters, lipid risk indexes and lipid-related oxidative stress markers (paraoxonase 1 [PON1] and lipid peroxides [LPO]) reflect the actual status of lipid metabolism in type 2 diabetes (T2DM). We hypothesized that relationships of high-density lipoprotein cholesterol (HDL-c) with PON1 and apolipoprotein A1 (ApoA1) and/or PON1 with ApoA1 under conditions of hyperglycaemia and oxidative stress might reveal HDL functionality. We investigated relationships between PON1, LPO, and lipid risk markers in T2DM subjects and compared them with those in healthy subjects., Methods: A total of 107 Caucasian subjects, 67 T2DM outpatients (mean age 52.2 ± 6.9 years) and 40 healthy subjects (mean age 48.1 ± 7.5 years) were included in the study. Serum levels of total cholesterol (CHOL-T), HDL-c, low-density lipoprotein cholesterol (LDL-c), triglycerides (TG), apolipoprotein B (ApoB), ApoA1, LPO, and PON1 activity were measured. Non-HDL-c, ApoB/ApoA1 and non-HDL/HDL (lipid risk indexes) were calculated., Results: Higher levels of TG, LPO (P < 0.0001), nonHDL/HDL and ApoB/ApoA1 (P < 0.001, 0.05, respectively), and lower levels of HDL-c, ApoA1, and PON1 (P < 0.0001) were observed in T2DM subjects than in controls. There is a lack of relationship among PON1, HDL-c, and ApoA1 in T2DM patients. PON1 activity positively correlated with these parameters (P < 0.0001) in controls. Strong correlations between non-HDL-c and ApoB (r = 0.956 vs. 0.756; P < 0.0001), LPO and TG (r = 0.831 vs. 0.739; P < 0.0001) were recorded in both study groups (P < 0.0001)., Conclusions: Impaired anti-oxidant and anti-atherogenic HDL properties associated with weakened PON1 function and lipid peroxidation may contribute to the development of atherosclerosis-related diseases in T2DM., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Aldose reductase (AR) gene C-106T polymorphism may be associated with diabetic nephropathy (DN) susceptibility, but the results of individual studies remain controversial., Objective and Methods: To explore the relationship between AR gene C-106T gene polymorphism and DN in the Eastern Asians with type 2 diabetes mellitus (T2DM) population, we conducted a meta-analysis of 2120 participants from 5 studies. Pooled odds ratio (ORs) and the corresponding 95% confidence interval (95% CI) were evaluated by either a fixed or random-effects models., Results: AR C-106T gene polymorphism was significantly associated with DN in the Eastern Asians population with T2DM under allelic (OR: 1.81, 95% CI: 1.30-2.52, P=0.0005), recessive (OR: 1.88, 95% CI: 1.20-2.97, P=0.006), dominant (OR: 9.22, 95% CI: 2.73-31.12, P=0.0003), homozygous (OR:2.27, 95% CI: 1.43-3.61, P=0.0005), heterozygous (OR: 5.75, 95% CI: 1.96-16.81, P=0.001), and additive genetic models (OR: 2.27, 95% CI: 1.48-3.48, P=0.0002)., Conclusions: In the Eastern Asians with T2DM, the AR gene C-106T gene polymorphism is correlated with an increased risk of DN. The Eastern Asians with the T allele of AR gene C-106T gene polymorphism might be susceptible to DN., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: We aimed to determine serum dipeptidyl peptidase-4 (DPP-4) activity in a group of persons with latent autoimmune diabetes in adults (LADA) and to compare it with persons with type 1, type 2 diabetes and healthy controls., Methods: DPP-4 activity measurement was performed in 67 persons (21 with type 1, 26 type 2 and 19 with LADA) and 13 healthy age and gender matched controls., Results: Persons with LADA showed highest DPP-4 activity among the study groups (32.71±3.55 vs 25.37±2.84 vs 18.57±2.54 vs 18.57±2.61U/L p<0.001). Mean glutamic acid autoantibody in persons with LADA was 164.32±86.28IU/mL. It correlated with DPP-4 activity (r=0.484, p=0.013). Furthermore, DPP-4 activity correlated with waist circumference (r=0.279, p=0.034) and glycated haemoglobin A1c (r=0.483, p<0.001), as well as with LDL cholesterol (r=0.854, p<0.001) and total daily insulin dose (r=0.397, p=0.001). In the multinomial regression analysis DPP-4 activity remained associated with both LADA (prevalence ratio 1.058 (1.012-1.287), p=0.001) and type 1 diabetes (prevalence ratio 1.506 (1.335-1.765), p<0.001) while it did not show an association with type 2 diabetes (prevalence ratio 0.942 (0.713-1.988), p=0.564)., Conclusions: Persons with LADA express higher DPP-4 activity compared to persons with both type 1 and type 2 diabetes. The possible pathophysiological role of DPP-4 in the LADA pathogenesis needs to be further evaluated., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: Maturity-onset diabetes of the young 2 (MODY2) is a form of diabetes that is clinically characterized by early age at onset and mild hyperglycemia, and has a low risk of late complications. It is often underdiagnosed due to its mild symptoms. To date, over 600 different GCK/MODY2 mutations have been reported. Despite only a few de novo mutations having been described, recent studies have reported the detection of a higher frequency of this kind of mutation. Therefore, de novo mutations could be more frequent than previously described. Even though common recommendations regarding the diagnosis of monogenic diabetes include the existence of a strong family history of diabetes, here we describe the study of mutations in two families with a symptomatic individual with clear clinical features of MODY2 but without any family history of diabetes., Methods: Genetic diagnosis in a group of participants with MODY2 characteristics was carried out by direct sequencing of coding regions of the GCK gene and analysis of mutations found using bioinformatics tools., Results: We found two de novo mutations, one of them novel, constituting 14.29% of all the participants who were phenotyped as MODY2., Conclusions: The number of mutations in GCK/MODY2 or even other MODY-related genes is undoubtedly underestimated, as accepted criteria for performing genetic tests include family history of the pathology. These cases illustrate the value of analyzing the GCK gene in patients with clinical features of MODY2, even in the absence of family history of the condition as it is essential for establishing the correct treatment., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

We report a peculiar case of a GCK/MODY2 family in whom monogenic and autoimmune diabetes coexist and exert different effects on clinical pictures, passing from mild hyperglycaemia not requiring any treatment through occasional insulin administration to complete insulin dependent type 1 diabetes., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

A potential adverse effect of dipeptidyl peptidase-4 inhibitors (DPP-4i) on the pancreas remains controversial. We evaluated the DPP-4i effects on pancreatic amylase and lipase activity in patients with type 2 diabetes. These enzymes were slightly but significantly increased, suggesting DPP-4i cause a low-grade inflammatory change in the exocrine pancreas., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Six Brazilian families with mild familial hyperglycaemia have been screened for glucokinase (GCK) mutations. All had mutations that co-segregated with the phenotype. One of the mutations, the deletion 96&#95;98delAAG (p.Lys32del), had not been previously described, reinforcing the worldwide prevalence of GCK MODY and widespread existence of undetected new mutations., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Aim: To investigate the association of functional single nucleotide polymorphisms (SNPs) of the endothelial nitric oxide synthase gene (eNOS) gene (T-786C, G894T) and one variable number tandem repeat polymorphism (aa 27VNTR bb) with reno-protective response to angiotensin converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) therapy in North Indian type 2 diabetic mellitus (T2DM) subjects with cases having diabetic nephropathy (DN) and controls without DN., Method: We genotyped three polymorphisms of eNOS (two SNPs: T-786C, G894T and one 27 VNTR) in T2DM patients with overt nephropathy (cases: n=320) and T2DM patients without overt nephropathy (controls: n=490), using validated PCR-RFLP assays. These 810 North Indian T2DM patients treated with ACEI or ARB after diagnosis were followed up for 3 years. Percent changes in eGFR, urinary albumin excretion (UAE), serum creatinine at the end of 3 years of treatment were taken as end points of renoprotective response., Result: We observed that in normoalbuminuric patients, eNOS -786 CC genotype and haplotypes C-b-G and C-b-T were associated with lesser renoprotective response to ACEI. While, in macroalbuminurics, eNOS -786 CC genotype, haplotypes C-b-G and C-b-T and 27VNTR aa were associated with better renoprotective response to ACEI/ARB., Conclusion: Our results showed that eNOS T-786C CC genotype and 27VNTR individually and in interaction with other eNOS SNPs modulate renoprotective efficacy of ACEI and ARB in T2DM patients, depending on the status of proteinuria., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

We investigated two single nucleotide polymorphisms of the NOS3 gene in type 2 diabetic patients (n=93) and healthy non-diabetic controls (n=76) and their relationship with smoking habits, body mass index, hypertension and dyslipidemia. Results showed that eNOS polymorphism rs891512 (G24943A) is associated with hypertension in Chilean individuals (p<0.05)., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aims: Media calcification is a predictor of cardiovascular mortality in type 2 diabetes mellitus (T2DM). Undercarboxylation of some vitamin K-dependent proteins, due to genetic polymorphisms of VKORC1, can lead to calcification. We examined a potential association between VKORC1 -1639 G>A polymorphism and T2DM and, also, the association of this polymorphism with carotid intima-media thickness (cIMT)., Methods: VKORC1 -1639 G>A polymorphism was determined in 299 T2DM patients and 328 controls of Caucasian origin using PCR-RFLP. cIMT was measured in a subgroup of 118 T2DM patients., Results: The frequency of VKORC1 genotypes between diabetic and nondiabetic subjects differed significantly (p=0.01). VKORC1 genotype was associated with T2DM in an adjusted model (OR 1.36, p=0.009). A statistically significant difference was observed in the maximum value of cIMT among different genotypes. VKORC1 -1639 G>A polymorphism was an independent predictor of cIMT (p=0.029) after adjusting for established risk factors., Conclusions: The association between VKORC1 -1639 G>A polymorphism and risk of T2DM could be due to the higher prevalence of calcification in T2DM patients. This is supported by the independent association between VKORC1 -1639 G>A polymorphism and maximum cIMT in T2DM patients which is likely due to atherosclerosis characterized by increased calcification., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The aim of the present study was to investigate the effects of Syzygium cumini leaf extract (ASc), on Adenosine deaminase (ADA) and Acetylcholinesterase (AChE) activities, and also on oxidative stress parameters in erythrocytes hemolysates (RBCs) and erythrocytes membranes (ghosts) from type 2 diabetics patients (Type 2 DM) under in vitro conditions. Non protein thiol groups (NP-SH), AChE, Catalase (CAT) and Superoxide Dismutase (SOD) activities were measure in RBCs. Further, ADA activity, Thiobarbituric Acid-Reactive Substances (TBARS) levels and protein thiol groups (P-SH) were estimated in ghosts. Also, P-SH and Vitamin C (VIT C) were measure in plasma sample. The results demonstrated that ADA and AChE activities, besides TBARS levels were higher in erythrocytes of Type 2 DM, while SOD activity and NP-SH levels were decreased when compared to control group. ASc, in vitro, reduced ADA and AChE activities and some parameters of oxidative stress. Furthermore, we observed correlations between VIT C and P-SH levels, ADA activity and P-SH levels, as well as NP-SH and TBARS levels in diabetics. The results suggest that ASc in vitro is able to promote the reduction of inflammation and oxidative stress parameters, and act against biochemical changes occurring in Diabetes mellitus (DM)., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Background: Abnormally elevated alanine aminotransferase (ALT) of nonspecific causes is a common outpatient problem. Without considering ethnicity, several studies had suggested that it was associated with insulin resistance (IR)., Objective: To investigate whether nonspecific elevated ALT in Taiwanese population could reflect a likely underlying IR and was associated with impaired fasting glucose or type 2 diabetes mellitus (IFG/T2DM)., Methods: The health examination profiles of 1313 Taiwanese were investigated cross-sectionally. The prevalence and odds ratios (ORs) for IFG/T2DM and metabolic abnormalities in relation to elevated ALT were analyzed., Results: Subjects with metabolic syndrome (MS) all had IFG/T2DM. The elevated ALT significantly correlated with MS and IFG/T2DM (i.e., 19.9-29.2% vs. 7.8% for MS, and 27.0-31.5% vs. 16.1% for IFG/T2DM). However, after excluding MS and adjustment for age and sex, the elevated ALT alone was not consistently associated with IFG/T2DM (36 < ALT ≤ 80 IU/L with OR 0.97, 95% CI 0.58-1.61; 80 < ALT ≤ 120 IU/L with OR 0.55, 95% CI 0.13-2.37; none with ALT > 120 had IFG)., Conclusions: In a cross-sectional analysis of Taiwanese industrial employees, elevated ALT associated with MS, but in subjects who did not meet MS criteria, elevated ALT by itself did not associate with IFG/T2DM., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Aims: We studied the relations of lysophosphatidylcholine (lyso-PC) in LDL with serum lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), paraoxonase and homocysteine thiolactonase (HTLase) activities in patients with type 2 diabetes mellitus., Methods: Lyso-PC was measured by electrospray ionization-liquid chromatography/mass spectrometry. Paraoxonase and HTLase activities were measured with paraoxon and gamma-thiobutyrolactone as substrates, respectively., Results: Serum HTLase and paraoxonase activities were significantly suppressed in diabetic patients (n=96) compared with control (n=25), whereas serum Lp-PLA(2) did not differ in control and diabetic patients. Lyso-PC contents in LDL correlated with serum Lp-PLA(2) activity positively and with serum HTLase activity negatively. Stepwise regression analysis revealed that serum Lp-PLA(2) and HTLase activities independently contributed to lyso-PC contents in LDL. In patients with diabetic nephropathy, lyso-PC contents in LDL were increased with reduced serum HTLase and paraoxonase activities compared with control, while serum Lp-PLA(2) activity did not differ. On the other hand, 3-month treatment with simvastatin reduced both lyso-PC contents in LDL and serum Lp-PLA(2) activity in hypercholesterolemic diabetic patients, while serum HTLase or paraoxonase activities did not change., Conclusions: Increased lyso-PC contents in LDL were associated with the suppressed HTLase activity, and serum Lp-PLA(2) and HTLase activities may be related to lyso-PC in type 2 diabetic patients.

As cyclin-dependant kinase 4 (CDK4) regulates beta cell proliferation, it may confer T2D risk. We tested the CDK4 IVS4-nt40A-->G SNP for association with T2D in Italians (230 cases, 204 control subjects) and excluded a role of the variant in our dataset.

The Met55Val polymorphism in the small ubiquitin-like modifier 4 (SUMO4) gene has been associated with susceptibility not only to type 1 diabetes, but also to type 2 diabetes and diabetic nephropathy. We tried to confirm the association with susceptibility to type 2 diabetes and to investigate its role in diabetic vascular complications. The polymorphism was genotyped in two independent Japanese samples (Wakayama and Tokyo) by the TaqMan method. Susceptibility to type 2 diabetes and prevalence of diabetic vascular complications (coronary heart disease, cerebral infarction, retinopathy, and nephropathy) were evaluated by case-control study and multivariate logistic regression analysis, respectively. There were no significant differences in the frequency of alleles or genotypes between patients and controls. The Val allele, however, was associated with higher prevalence of coronary heart disease in patients in both groups (Wakayama, n=423, odds ratio, 1.64; 95% confidence interval, 1.02-2.64; P=0.041; Tokyo, n=451, odds ratio, 1.58; 95% CI, 1.07-2.34; P=0.021, in an additive model, respectively). No significant associations were observed with other diabetic vascular complications. Although association of the polymorphism with susceptibility to type 2 diabetes or nephropathy was not replicated, an association of the polymorphism with risk of coronary heart disease in type 2 diabetes is suggested.

Maturity onset diabetes of the young (MODY) is a genetically heterogeneous disorder characterized by autosomal dominant inheritance, altered function of pancreatic beta cells and early onset diabetes mellitus, usually before 25 years old. The prevalence of specific mutations of MODY genes differs considerably among different countries. In this study we analyzed 53 index cases from unrelated MODY families who are potential carriers of mutations in GCK gene. In addition, 122 relatives were also studied. We have identified eight new mutations in the GCK gene. One of them is a non-frameshift deletion involving Lysine 143. This amino acid is part of the conserved stretch of basic residues (KHKKL) which spans from residue 140 to 144. The non-frameshift deletion might implicate the affinity of GCK for GCKRP, and potentially the abnormal nuclear localization of GCK. Additional studies should be performed to confirm this possibility.

Aims: Recent evidence indicates that oxidative stress may play an important role in the pathogenesis of insulin resistance and that gene polymorphism (Ala16Val) of manganese superoxide dismutase (MnSOD) may protect against reactive oxygen species (ROS) function. We aimed to test the hypothesis that the Ala16Val variant could be associated with the development of type 2 diabetes., Methods: We examined 523 nondiabetic Japanese-Americans who underwent a 75g oral glucose tolerance test (OGTT) and were followed for an average of 9.9 years. Cox proportional hazard analysis, stratified by category of OGTT, was used to determine whether the Ala16Val polymorphism was a risk factor in the development of type 2 diabetes., Results: During the follow-up period, 65 subjects developed type 2 diabetes. Compared with Ala allele carriers, subjects with a Val homozygote showed significantly higher risk for developing diabetes (stratified hazard ratio=2.05 [95% confidence interval 1.03-4.08]; P=0.041) after adjustment for age, gender, systolic blood pressure, total cholesterol, body mass index, and homeostasis model assessment., Conclusions: We demonstrated that the MnSOD Ala16Val polymorphism might be associated with development of type 2 diabetes among Japanese-Americans. These results suggest that insufficient ROS scavenging might be associated with a susceptibility to glucose intolerance.

We investigated the association between the angiotensin converting enzyme (ACE) insertion/deletion polymorphism and glycemic state. Diabetes mellitus, impaired fasting glucose and mean fasting glucose were not associated with genotype among Hindustani Surinamese, African Surinamese and Dutch participants. Our results cast (further) doubts on the association between ACE and glycemic state.

Aims: Abnormal high-density lipoproteins (HDL) metabolism is a major cardiovascular risk factor in type 2 diabetes mellitus (DM2). Lecithin:cholesterol acyltransferase (LCAT) increases HDL size by transferring 2-acyl groups from lecithin or phosphatidylethanolamine to unesterified cholesterol. The purpose of this study was to determine the independent correlates of LCAT activity in DM2 patients., Methods: A total of 45 (male: 20) consecutive adult DM2 patients aging 50.0+/-7.0 years (range: 40-64 years) with a median diabetes duration of 4 years (range: 2-18) were studied. Exclusion criteria were: smoking, positive history of cardiovascular, thyroid, renal or liver disease, pregnancy, treatment with metformin, insulin, lipid lowering drugs, angiotensin-converting enzyme inhibitors, aspirin or antioxidant supplements. Univariate and multivariate analyses were performed., Results: From a comprehensive list of variables studied, only HbA1c (rho=-0.951) and oxidized LDL (rho=-0.779) had statistically significant correlation with LCAT activity (p<0.001). These two variables were themselves strongly correlated to each other (rho=0.809, p<0.001). To eliminate potential confounding effects, we performed multivariate analysis, where HbA1c emerged as a strong independent predictor of LCAT activity (adjusted OR=-0.928, p<0.001)., Conclusions: Glycemia-induced glycation of HDL decreases LCAT activity. The fact that HbA1c is an accurate measure of glycation and can therefore reflect glycated HDL levels explains the association found in the present study. In conclusion, HbA1c provides an easy-to-assess, accurate measure of LCAT activity in DM2.

The purpose of this study was to determine if immune mechanisms in GAD positive patients' contribute to the pathogenesis of a specific sub-type of Type 2 diabetes. GAD positive (n=8) and GAD negative (n=8) subjects diagnosed with Type 2 diabetes were matched for age, gender, body mass index, duration of diabetes and glycaemic control. All subjects underwent an insulin-modified frequently sampled intravenous glucose tolerance test to measure insulin sensitivity and insulin secretory function with minimal model analysis. In addition, BRIN-BD11 clonal beta-cells were supplemented with patients' sera to determine basal and alanine-stimulated insulin secretion and terminal complement complex (TCC) formation. Both groups were severely insulin resistant (0.56+/-0.17 vs. 0.99+/-0.3310(-4)min(-1)/(microUml(-1)) for GADneg and GADpos, respectively) but the GAD negative subjects had a higher basal (87+/-11 vs. 58+/-14pmoll(-1), p<0.05) and glucose-stimulated insulin secretion (DeltaAUCins 0.96+/-0.12 vs. 0.60+/-0.12pmol/(l(-1)min), p<0.05). In vivo measures of insulin secretion were negatively correlated with TCC formation, independent of antibody status. In conclusion, GAD positive subjects initially diagnosed with Type 2 diabetes are unable to compensate for insulin resistance due to more pronounced beta-cell impairment. TCC formation may be partly responsible for the insulin secretory dysfunction associated with this specific sub-type of Type 2 diabetes.

Background/aims: Loss of circadian blood pressure (BP) variation (i.e., lack of nocturnal BP dip by at least 10mmHg, 'non-dipping') is associated with increased mortality rate in subjects with diabetes. We studied whether angiotensin converting enzyme (ACE) insertion/deletion (I/D) gene polymorphism may play a role in 24-h BP rhythm control., Methods: The study group was 38 normotensive normoalbuminuric type 2 diabetes patients with impaired BP variation, the controls were 51 well-matched type 2 diabetes subjects with normal 24-h BP rhythm. ACE I/D polymorphism, endothelial function and subclinical inflammation parameters (serum endothelin-1, sE-selectin, intercellular and vascular cell adhesion molecules, tumor necrosis factor-alpha) were assessed., Results: ACE DD genotype was found in 20 (53%), ID genotype in 16 (42%), and II genotype in 2 (5%) study group subjects, while 5 (10%) control subjects had DD genotype, 30 (59%) - ID genotype, and 16 (31%) - II genotype (p<0.0001). Study group subjects presented with marked endothelial dysfunction., Conclusion: Impaired circadian blood pressure variation in normotensive normoalbuminuric type 2 diabetes patients is associated with ACE DD genotype and marked endothelial dysfunction when compared to diabetic subjects with normal blood pressure rhythm.

Insulin-degrading enzyme (IDE) is a metalloproteinase which degrades insulin and terminates its action. Homologous deletion of IDE gene resulted in hyperinsulinemia and glucose intolerance in a rat model of type 2 diabetes mellitus. Several genetic association studies examined IDE as a susceptibility gene for type 2 diabetes in European descents. Here we investigated the genetic association of IDE polymorphisms with the risk of type 2 diabetes and its related phenotypes in the Korean population. Among six single nucleotide polymorphisms analyzed, g.-179T>C (OR=1.73, P=0.04), and g.IVS18+99G>A (OR=1.23, P=0.02) revealed borderline association with increased risk of type 2 diabetes. Combining our results with previous data obtained from the European population, g.-179T>C (OR=1.11, P=0.03), and g.IVS24-64A>T (OR=1.18, P=0.005) showed significant association with type 2 diabetes. Haplotype consisting of common alleles of the six polymorphisms was associated with decreased risk of type 2 diabetes (OR=0.82, P=0.02). However, none of the polymorphisms was significantly associated with metabolic phenotypes. We can conclude that variations in IDE might contribute to diabetes susceptibility in the Korean population.

We previously reported that lipoprotein lipase mass levels in preheparin serum (preheparin LPL mass) was significantly lower in type 2 diabetes mellitus compared to healthy subjects and that low preheparin LPL mass may be a high-risk factor of coronary atherosclerosis. The aim of this study was to clarify the effects of metformin on serum lipoprotein lipase mass levels (preheparin LPL mass), adiponectin and lipid metabolism in patients with type 2 diabetes mellitus. Twenty-eight patients with type 2 diabetes mellitus (HbAlc>7.0%), who were already receiving sulfonylurea agents, took metformin 500 mg orally twice daily for 3 months. Fasting blood glucose (FBG), immunoreactive insulin (basal IRI) and HbAlc decreased significantly after metformin treatment. LDL-Rm ratio decreased significantly (from 0.3521+/-0.046 to 0.3339+/-0.030, P<0.05) and preheparin LPL mass increased significantly (from 42.5+/-3.2 to 50.6+/-3.5 ng/ml, P<0.0005), but adiponectin was unchanged. The correlation of a change of LDL-Rm ratio and a change of preheparin LPL mass showed a negative correlation tendency. The changes in LDL-Rm ratio and preheparin LPL mass were independent of the hypoglycemic effect of metformin. These results suggest that metformin may increase LPL production, thereby increasing LDL particle size. These effects might be independent of the hypoglycemic effect of metformin.

Recent studies suggest that blockade of angiotensin type 1 (AT1) receptor may have some effect on glucose and lipoprotein metabolism. Serum level of preheparin lipoprotein lipase (LPL) reflects LPL production mainly in adipocytes and is believed to be related to insulin sensitivity. We studied the effect of a selective AT1 antagonist, valsartan, on glucose, lipid metabolism and the preheparin LPL mass in 55 patients with type 2 diabetes and hypertension. Patients were randomized into a group administered valsartan 80 mg/day for 12 weeks or a group not administered valsartan (control). Blood pressure decreased significantly. HbA1c and TG levels decreased and HDL-C level increased, but these changes tended to be significantly different. TC and LDL-C levels were not significant changes. Preheparin LPL mass increased after valsartan administration compared with control (P = 0.0307), and migration ratio of LDL (LDL-Rm), which correlated negatively with LDL particle size, decreased compared with control (P < 0.0001). DeltaLDL-Rm correlated inversely with Delta preheparin LPL mass (r = -0.459). Among subjects treated with valsartan, greater improvement in preheparin LPL mass and blood pressure was observed in the subgroup with preheparin LPL mass <40 ng/ml. The results of this study suggest that valsartan may enhance LPL production in adipocytes, resulting in enlarged LDL particle size.

Mitochondrial respiratory function in a patient with maternally inherited type 2 diabetes mellitus and hypertrophic cardiomyopathy associated with heteroplasmic mitochondrial DNA (mtDNA) C3310T mutation, which replaces the second amino acid of NADH dehydrogenase 1 (ND1) from a hydrophobic Proline to a hydrophilic Serine, was investigated. Mitochondrial respiratory function solely due to mtDNA C3310T mutation was investigated in cybrid system by the fusion of mtDNA-deleted (rho(0)) HeLa cells and exogenous mtDNA either from the proband or from controls. Total oxygen consumption of the proband cybrid cells was significantly decreased compared with those of controls (2.468+/-0.475 versus 2.871+/-0.484 micromol/h/10(7) cells, p=0.0392). Mitochondrial respiratory chain complex I activity of the proband cybrid cells was also significantly decreased compared with those of controls (0.191+/-0.080 versus 0.288+/-0.113 micromol/h/mg protein, p=0.0223). Furthermore, ATP content in the proband cybrid cells was also significantly decreased compared with those in controls (1.119+/-0.344 versus 1.419+/-0.378 pmol/10(5) cells, p=0.044). The present study indicates that mtDNA C3310T mutation may be a pathogenic mutation of maternally inherited type 2 diabetes mellitus and hypertrophic cardiomyopathy in the proband and the family.

Familial and epidemiological studies have shown that genetic factors play a role in the development and progression of type 2 diabetes mellitus (T2DM). Asian Indians have shown an increasing prevalence of T2DM. Apolipoprotein E (APOE) and Angiotensin-1 converting enzyme (ACE) I/D polymorphisms have been associated with T2DM. This study examined the association of APOE and ACE genes with T2DM patients of Punjab, India. APOE (HhaI) and ACE (I/D) genotypes analysed by polymerase chain reaction were available from 90 patients and 97 random healthy controls. All loci and populations are in Hardy-Weinberg equilibrium. There is no significant association of APOE vis-à-vis T2DM, however APOE*4 allele frequency is low in diabetics (3.9% and 8.8%). DD genotype and *D allele of ACE are associated with T2DM (OR=1.90, p<0.05, and OR=1.58, p<0.05, respectively). Recessive and multiplicative mode of inheritance for *D allele provided the strongest support for the association. Height, weight and BMI did not reveal any significant association with APO or ACE. DD-33 and ID-23 combinations (ACE-APOE) showed higher odds of 2.01 and 2.14, respectively. ACE but not APOE polymorphism is positively associated with T2DM in Indian population, however, the synergistic effects of DD-33 and ID-23 are also evident.

We previously reported that lipoprotein lipase mass level in preheparin serum (preheparin LPL mass) was significantly lower in type 2 diabetes mellitus compared to healthy subjects and increased by conventional insulin therapy using NPH (intermediate-acting) insulin. The aim of this study was to investigate the effects of intensive insulin therapy on preheparin LPL mass. Thirty-two subjects (total group) with type 2 diabetes receiving treatment by NPH insulin injection twice a day in the morning and evening were switched to basal bolus insulin (BBI) therapy (fast-acting insulin after each meal and NPH insulin before bedtime). In 14 subjects, the total daily insulin dose was not change after switching to BBI therapy (iso-dose group). After 3 months of BBI therapy, preheparin LPL mass increased significantly from 47 to 56 ng/ml in total group. Glycosylated hemoglobin and serum triglyceride levels decreased significantly, and high-density lipoprotein-cholesterol increased significantly. Low-density lipoprotein levels did not changed but increase in size was suggested by PAG disc electrophoresis. Similar changes were observed in the iso-dose group. These results suggest that BBI therapy enhances preheparin LPL mass, accompanied by antiatherogenic changes in glucose and lipid metabolism.

Oxidative stress and the gene expression at the transcriptional level of antioxidant enzymes were investigated in two models of diabetes in mice. We used KKAy mice as a model of obese insulin-resistant diabetes, and streptozotocin-induced diabetic mice (STZ mice) as a model of insulin-deficient diabetes. C57BL mice and saline-injected ICR mice were used as the respective non-diabetic controls. To assess oxidative damage, plasma malonedialdehyde (MDA), urine 8-isoprostane and 8-hydroxy deoxyguanosine (8-OHdG) were measured. The mRNA expression of antioxidant enzymes, superoxide dismutase 1 (SOD-1) and glutathione peroxidase 1 (GPx-1) in the kidney and heart were quantified using a real-time polymerase chain reaction. The KKAy mice demonstrated moderate hyperglycemia and hyperlipidemia, and the STZ mice showed severe hyperglycemia and hypolipidemia. The KKAy mice, but not the STZ mice, showed elevated plasma MDA relative to the non-diabetic controls. Urine 8-isoprostane and 8-OHdG in both diabetic mouse groups increased significantly. The urine oxidative stress markers in the severely hyperglycemic STZ mice were higher than those in the moderately hyperglycemic KKAy mice. Although GPx-1 and SOD-1 showed elevated mRNA expression in the KKAy mice in the kidney and heart, in the STZ mice they did not increase compared to the controls. The compensatory up-regulation of the mRNA expression of antioxidant enzymes may be impaired in the insulin-deficient severely hyperglycemic state.

Background and Aim: In vivo and in vitro experimental findings indicate that the hyperglycemic diabetic milieu can induce altered expression of the matrix metalloproteinase (MMP) genes and contribute to imbalances in vascular matrix homeostasis. We examined the plasma levels of enzymes and inhibitors involved in extracellular matrix turnover., Methods: We measured the plasma concentrations of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase 1 (TIMP-1) in 80 type-2 diabetic subjects without uremia and in 80 age-matched controls. In addition, we determined the plasma levels of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and high sensitive(hs) C-reactive protein (CRP) in both groups., Results: Plasma MMP-2, TIMP-1, and hs-CRP concentrations were significantly elevated in diabetic patients as compared to the control subjects (p<0.05). Plasma levels of MMP-2, MMP-9, TIMP-1, VCAM-1, ICAM-1, and hs-CRP were found not to be significantly associated with age, duration of diabetes, blood pressure, or serum lipid concentrations., Conclusions: Plasma MMP-2, TIMP-1 and hs-CRP concentrations were significantly increased in type-2 diabetic patients.

Background: Nitric oxide (NO) is related to the pathogenesis of renal hemodynamics in diabetes mellitus. Endothelial nitric oxide synthase (eNOS) gene polymorphism is considered the deterioration factor for progressive renal disease. It has been reported that an interaction of angiotensin II (Ang II) and NO is involved in the control of glomerular hemodynamics. This study aimed to elucidate the roles of eNOS and the angiotensin-converting enzyme (ACE) gene polymorphism for the progression of type 2 diabetic nephropathy (DN)., Methods: Korean type 2 diabetic patients (n = 177) were studied. eNOS and ACE genotypes were determined by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. They were divided into three groups: group 1 consisted of patients with normoalbuminruia (n = 59), group 2 had microalbuminuria (n = 35) and group 3 had overt nephropathy (n = 83)., Results: Group 3 had a higher frequency of eNOS(GT) than groups 1 and 2. Patients with eNOS(GT) genotype showed more rapid deterioration in renal function, higher incidence of overt nephropathy and lower renal survival than those with eNOS(GG) genotype. However, there was no significant association between the ACE genotypes and DN, and no interaction between eNOS and ACE gene polymorphism., Conclusion: These results imply that eNOS(GT) genotype is associated with the progression of type 2 DN in Korean patients.

We investigated the relationship between diabetic retinopathy (DR) and three polymorphisms, C(-106)T in the aldose reductase (ALR2) gene, 4G/5G in the plasminogen activator inhibitor-1 (PAI-1) gene and C677T in the methylenetetrahydrofolate reductase (MTHFR) gene, in 210 Euro-Brazilian type 2 diabetic patients. Retinopathy was evaluated by funduscopic examination and genotype analysis was performed using the polymerase chain reaction and allele-specific restriction. Retinopathy was detected in 47% of the patients. There were no significant differences in allele or genotype distributions between patients with or without retinopathy for all polymorphisms. Thus, the three polymorphisms are not related to the presence of DR in Euro-Brazilian type 2 diabetic patients.

In the present study, we evaluated O(6)-methylguanine-DNA methyltransferase (MGMT) activity in diabetic patients. The study was performed on 27 patients with Type 1 diabetes, and 42 with Type 2 diabetes. Patients with complications were excluded from the study. 36 non-diabetic volunteers, non-smokers who do not consume alcoholic beverage, were chosen from the medical staff as control subjects. MGMT activity was measured by the transfer of radiolabeled methyl groups from a prepared methylguanine-DNA substrate to the enzyme fraction of leukocyte extract. Leukocyte MGMT activity was significantly reduced in both Type 1 and Type 2 diabetes patients as compared with control subjects (P<0.001). The present study demonstrates decreased MGMT activity in leukocytes from patients with Type 1 and Type 2 diabetes.

Serum paraoxonase (PON) is a high-density lipoprotein-bound enzyme that can prevent oxidation of low-density lipoprotein by hydrolyzing lipid peroxides and thus exert an anti-atherogenic effect. Recent studies have suggested that glutamine(Q isoform)/arginine(R isoform) polymorphism at position 192 of PON(1) gene is associated with macrovascular disease of type 2 diabetes mellitus (T2DM). We re-investigated this relationship using carotid intima-media thickness (IMT) as a surrogate continuous variable for macroangiopathy. The genotype and allele frequency of PON(1) 192 Q/R polymorphism was assayed by polymerase chain reaction-restriction fragment length polymorphism in 152 type 2 diabetic patients and 128 healthy subjects from a population of Chinese Han nationality in ChengDu area. The carotid IMT was measured by B-mode ultrasonography in type 2 diabetic patients. No differences were found in PON(1) gene Q/R polymorphism in the type 2 diabetic patients when compared with the control group. The mean carotid IMT in type 2 diabetic subjects with the QQ, QR and RR genotype was 0.65+/-0.27, 0.83+/-0.27 and 1.05+/-0.32 mm, respectively. One-way ANOVA showed that IMT was significantly greater in the RR subgroup than in both QR and QQ subgroups (P<0.01). Multivariate logistic regression analysis showed R allele to be the main determinant of IMT variability (OR 4.0 95% CI 2.10-7.40 P=0.005). Our data support the view that 192 R allele of PON(1) gene is a risk factor for macrovascular disease of T2DM in Chinese.

The aim of this study was to investigate association of a missense mutation in plasma PAF acetylhydrolase (G994T) with intima media thickness (IMT) of the carotid arteries. One hundred and forty Japanese type 2 diabetic patients aged from 40 to 79 years without severe nephropathy were enrolled in this study. The genotype of the patients was determined by allele specific PCR. IMT of the carotid arteries of the subjects was recorded by B-mode ultrasound imaging. The patients were divided into two groups by genotyping, one carrying two wild alleles (wild group), and another carrying one or two mutant alleles (mutant group). Each group was further divided into two subgroups according to age; one subgroup consisted of 40s or 50s, and another consisted of 60s or 70s. The prevalence of the G994T mutation in the subjects was 28.6% (24.3% heterozygote, and 4.3% homozygote). IMT of the elderly patients of the mutant group was significantly greater (0.98 +/- 0.22 mm, n = 26) than of the elderly patients of the wild group (0.87 +/- 0.20 mm, n = 50, P = 0.0292). There was no significant difference in clinical characteristics between the two subgroups. The results of this study indicate that the missense mutation in plasma PAF acetylhydrolase is associated with development of atherosclerosis in the elderly., (Copyright 2002 Elsevier Science Ireland Ltd.)

The effect of long-term (6 months) administration of voglibose in a dietary mixture (10 ppm) on intestinal disaccharidase activity was examined in non obese type 2 diabetes model Goto-Kakizaki (GK) rats. The postprandial blood glucose level in voglibose-treated GK rats was significantly lower than in untreated GK rats (190+/-19 vs. 250+/-25 mg/dl, P<0.01; 1 h, 212+/-23 vs. 256+/-20, P<0.05; 2 h), and the activities of maltase, sucrase, and isomaltase remained significantly lower throughout the 6 months of voglibose treatment. The expressions of protein and mRNA of sucrase-isomaltase (SI) complex were significantly higher in voglibose-treated GK rats. Voglibose administration then was stopped after 6 months of treatment. The mRNA level and protein level of the SI complex became normalized during the interruption of drug administration, and disaccharidase activities increased almost to the level of the untreated group 1 month after treatment was stopped. After 1 day of re-administration of the drug, however, disaccharidase activities again became significantly inhibited. These results indicate that voglibose may improve glucose tolerance since it inhibits activities of disaccharidases in spite of increasing the expression of them on intestine, furthermore voglibose may be reversible and reproducible through interruption and re-administration.

There exists lipoprotein lipase mass in preheparin serum, even though the activity is scarcely found. We studied the preheparin serum lipoprotein lipase mass levels (prehaparin LPL mass) in type 2 diabetes mellitus patients and the effect of insulin therapy on the levels of preheparin LPL mass. In 40 type 2 diabetes mellitus patients, preheparin LPL mass were measured by the sandwich enzyme-linked immunosorbent assay (ELISA), and were compared with those of non-diabetic healthy control. The correlation between preheparin LPL mass and Hemoglobin A(1c) (HbA(1c)), serum lipids were studied. Preheparin LPL mass were measured before and after insulin therapy. Preheparin LPL mass of type 2 diabetes mellitus patients was significantly lower than that of non-diabetic healthy control. In diabetic patients, preheparin LPL mass were negatively correlated with HbA(1c). Fifteen patients started to take insulin therapy. Preheparin LPL mass increased significantly at 4th week, when fasting blood glucose decreased. These results suggested that preheparin LPL mass was greatly regulated by insulin action.

We screened for mutations in the gene of insulin-sensitive phosphodiesterase 3B (PDE3B), which regulates antilipolytic actions of insulin via reduction of intracellular cyclic AMP levels, in Japanese patients with type 2 diabetes mellitus and lipoatrophic diabetes mellitus using single-stranded conformation polymorphism analysis and Southern analysis and investigated frequencies of variable number of tandem repeats. A silent polymorphism at the Arg463 codon (AGG-->AGA) in exon 4 was identified after examining all 16 exons and exon-intron splicing junctions of the gene. This polymorphism was found in 53 of 100 subjects with type 2 diabetes mellitus, 2 of 5 lipoatrophic diabetic patients and 24 of 50 control subjects, without any significant difference in allele frequency between groups. An EcoRI restriction fragment length polymorphism was identified in patients with type 2 diabetes mellitus and control subjects, again with no differences in occurrence. The allelic distribution of two polymorphic tandem repeats sequences in introns 5 and 12 of the gene did not differ significantly between patients with type 2 diabetes mellitus and control subjects. In conclusion, alterations in the PDE3B gene are unlikely to contribute importantly to the pathogenesis of type 2 diabetes mellitus or lipoatrophic diabetes mellitus in Japan.

Lipid peroxides are thought to be formed by free radicals and may play an important role in the development of atheromatous vascular diseases. The relationship between serum lipids, lipoproteins, lipid peroxides [thiobarbituric acid reactive substances (TBARS)] and erythrocyte antioxidant enzymes [catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD)] was investigated in non-insulin-dependent diabetic patients with and without coronary heart disease (CHD), and a comparison was made for all the above parameters with non-diabetic patients with CHD. Lipid peroxide concentrations were significantly increased in both groups of diabetic patients and also in non-diabetic patients with CHD, compared to those in control subjects. Diabetic patients with CHD had higher levels of TBARS compared to those diabetics without CHD. Hyperlipidaemia and abnormal lipoprotein levels were observed in all three groups of patients. Increased total cholesterol and LDL-cholesterol were observed in diabetics with CHD compared to those without CHD. Among the erythrocyte antioxidant enzymes, CAT activity was increased, GPx activity was decreased and no change was observed in SOD activity in both groups of diabetic patients and non-diabetic patients with CHD compared to those in controls. A clear correlation was observed between the CAT activity and lipid peroxide concentrations in all the diabetic patients. These observations suggest that there are similar abnormalities in lipid metabolism and erythrocyte antioxidant enzymes in diabetic patients and non-diabetic patients with CHD.

We investigated the relationship between advanced diabetic retinopathy (ADR) and an angiotensin-converting enzyme (ACE) gene polymorphism in subjects with type 2 diabetes and ADR, pre-proliferative (PrePDR) or proliferative diabetic retinopathy (PDR) without overt nephropathy. Polymerase chain reactions were used to detect insertion/deletion (I/D) polymorphisms of the ACE gene. There was no difference in the frequency of II, ID, or DD genotypes, or of I and D alleles among subjects with type 2 diabetes without diabetic retinopathy (NDR) or with simple diabetic retinopathy (SDR) and non-diabetic controls. There was also no difference in the frequency of ACE genotypes among subjects with type 2 diabetes with NDR, or SDR and ADR. However, the frequency of the ACE DD genotype in ADR was significantly higher than that in controls (chi(2)=6.64, P=0.036). On the other hand, the frequency of the D allele in ADR was significantly higher than that in controls (chi(2)=6.33, P=0.012), NDR (chi(2)=4.18, P=0.041) and SDR (chi(2)=4. 89, P=0.027), respectively. These results indicate a significant relationship between the presence of the D allele polymorphism in the ACE gene and ADR in Japanese subjects with type 2 diabetes and no overt nephropathy.

Ca(2+)-Mg(2+)-ATPase is an important regulator of intracellular calcium concentration and therefore, of erythrocyte deformability. We have investigated the possible relationship between Ca(2+)-Mg(2+)-ATPase activity (ATPase) and ionized calcium (Ca(2+)), in diabetic patients with peripheral neuropathy. A total of 104 Type 2 diabetic patients (57 neuropathic and 47 non-neuropathic) and 25 non-diabetic subjects were studied. After an overnight fast, blood was taken for Ca(2+)-Mg(2+)-ATPase activity, Ca(2+), Mg(2+), PTH and HbA(1c). The neuropathy study group had significantly lower levels of ATPase, 6.6 (95% CI, 5.6-7.7) nmol/mg/min compared to controls 7. 1 (6.2-8.3) nmol/mg/min, P<0.001 and to diabetic patients without neuropathy 7.0 (6.0-8.1) nmol/mg/min, P<0.001. The study group had also lower levels of Ca(2+) (0.89+/-0.18 mmol/l vs. control 1.08+/-0. 24 mmol/l, P<0.01 and non-neuropathic 0.98+/-0.27 mmol/l, P<0.05) and Mg(2+) (0.73+/-0.13 mmol/l vs. control 0.81+/-0.14 mmol/l, P<0. 05), despite similar PTH levels. In diabetic subjects, no correlation was found between ATPase or Ca(2+) with glucose, HbA(1c), age or duration of diabetes. We conclude that in patients with diabetic neuropathy there are abnormalities of Ca(2+)-Mg(2+)-ATPase activity and Ca(2+). This provides further support for the role of microangiopathy in the pathogenesis of neuropathy.

The ability of insulin to influence activities of various protein kinases and protein phosphatases, that are thought to mediate insulin action, are limited in patients with insulin resistance. Because numerous responses to insulin are affected, we undertook studies to determine whether protein tyrosine phosphatases (PTPs) activities are altered in patients with diabetes syndrome. In order to evaluate abnormal PTP activities, we done a comparative study using erythrocytes from normal and diabetic patients. We determined the activity of the cytosolic acid PTP in basal and insulin-dependent states. Mean basal PTP activities, were found to be significantly higher in diabetics than in normal subjects (type 1 diabetics: 0.36 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g hemoglobin (Hb), P < 0.001; type 2 diabetics: 0.35 +/- 0.01 vs 0.28 +/- 0.01 mmol p-nitrophenolate/h per g Hb, P < 0.001). Insulin, at concentrations above physiological levels (1 mIU/ml), inhibited the PTP activities in erythrocytes from normal subjects (-15 +/- 4.1%, P < 0.01). Insulin could also modulate glycolysis, probably as a consequence of receptor tyrosine kinase activation, inducing phosphorylation of protein band 3 and hence the release of glycolytic enzymes. We have previously reported that a reductase enzyme in human erythrocytes is dependent on glycolysis being significantly activated (+28 +/- 3.1%, P < 0.001) by high insulin levels (1 mIU/ml). Mean basal reductase activities were found to be significantly lower in diabetics than in normal subjects (type 1 diabetics: 0.77 +/- 0.03 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001; type 2 diabetics: 0.77 +/- 0.04 vs 0.97 +/- 0.02 mmol ferrocyanide/20 min per l cells, P < 0.001), indicating altered erythrocyte metabolism in the diabetic patients. High glucose levels were used to mimic hyperglycemia condition, using erythrocytes from normal subjects. At 30 mM glucose, erythrocytic phosphatase activity was stimulated (+32 +/- 4.2%, P < 0.0001), although no effect was observed on the reductase enzyme at the same glucose levels. Results indicated that diabetic disorders appear to be associated with quantitative alterations of erythrocyte acid phosphatase activity and other enzymes that depend on the glycolytic rate (reductase). The overall data suggest that erythrocyte acid phosphatase may have a role in the modulation of glycolytic rates through the control of insulin receptor phosphorylation.

Aldose reductase (AR) protein was measured in peripheral mononuclear cells (PMCs) from 55 patients with type 2 diabetes by a two-site ELISA using anti-human AR monoclonal antibody. AR levels did not correlate with age, duration of diabetes, and HbAlc. Furthermore, no significant differences were found in AR levels between the patients and healthy subjects. Thirty seven patients had at least one of diabetic microangiopathy; retinopathy, neuropathy, or nephropathy. AR levels were significantly higher in the patients with microangiopathy than in those without it (52.3 +/- 15.7 vs. 43.0 +/- 15.2 ng/10(6) cells, P < 0.05). The patients with neuropathy had significantly higher AR levels than those without neuropathy (53.7 +/- 15.8 vs. 42.7 +/- 14.3 ng/l0(6) cells, P < 0.05). The same result applied to the patients with retinopathy (54.5 + 15.4 vs. 44.6 +/- 15.3 ng/10(6) cells, P < 0.05). The AR levels in the patients with nephropathy tended to give a higher value than those without it. However, there were no significant differences between the two (53.9 +/- 3.6 vs. 46.4 +/- 2.6 ng/10(6) cells, NS). These results indicate that AR levels in PMCs from type 2 diabetic patients are associated with the presence of microangiopathy. The measurement of AR proteins in PMCs with this ELISA system is a useful tool for the clinical study of diabetic complications, and would increase our understanding of the pathogenesis of the disease.

The aim of this study was to investigate the effect of sodium orthovanadate on the alterations of human erythrocytes insulin receptor autophosphorylation. Human erythrocytes were incubated with insulin in a cell system and then lysed. The autophosphorylated insulin receptors were measured with the aid of a two-site immunofluorometric assay and using a monoclonal anti-insulin receptor antibody to label the insulin receptors and a monoclonal anti-phosphotyrosine antibody to assess tyrosine phosphorylation. When the erythrocytes were treated with insulin and then reincubated in insulin-free medium, vanadate completely inhibited insulin receptor dephosphorylation, although it had no effect on in vitro receptor autophosphorylation. Thus insulin receptor tyrosine phosphatase activity is postulated to be [% (autophosphorylated insulin receptors with vanadate - autophosphorylated insulin receptors without vanadate)/total insulin receptors] under overall steady conditions in a cell system. Using this assay, the insulin receptor tyrosine phosphatase activities of 25 control and 32 diabetic subjects were studied. There was no significant difference in insulin receptor tyrosine phosphatase activity between control subjects and diabetic subjects (0.173 +/- 0.062 vs 0.209 - +/- 0.057 autophosphorylated insulin receptors units/insulin receptors units). The assay used in this study requires only 0.6 ml of whole blood, and so should be a useful tool for detecting patients who are insulin-resistant due to abnormal insulin receptor tyrosine phosphatase activity.

To compare the clinical usefulness of commercial radioimmunoassay (RIA) kits based on recombinant and pig brain GAD, we measured glutamic acid decarboxylase autoantibody (GADAb) titers in 125 non-obese (body mass index < 24) Japanese diabetics without insulin therapy using two commercial RIA kits based on recombinant human (rh) GAD65 (GADAb Cosmic) and purified pig brain native GAD (RIP Anti-GAD Hoechst). The frequencies of GADAb positivity using these two RIA kits (normal ranges; < 1.3 and < 4.0 U/ml, respectively) were about 4.8 (6/125) and 3.2% (4/125), respectively. The six patients found to be positive with RIA using GADAb Cosmic demonstrated significantly higher prevalence of NIDDM in their parents (P = 0.04), lower beta-cell function estimated by intravenous glucagon loading tests (P = 0.03) and higher prevalence of progression to insulin therapy (P = 0.0001). Five of these six patients slowly progressed to insulin-requiring status within 34 +/- 11 months of follow-up evaluation, and one of these five patients progressed to a completely insulin-dependent status within 30 months from the onset of diabetes. Of these six patients, two demonstrated chronic pancreatitis, three had chronic thyroiditis, and five showed HLA DR4. Interestingly, two of the six patients demonstrated very low GADAb titers (2.3 and 2.9 U/ml), while RIP Anti-GAD Hoechst showed no positivity with the same sera. Based on the binding study after pre-incubation of unlabeled GADs, these low titrated GADAb were elucidated to be true specific reactions to rh GAD65 alone. Moreover, one of the two patients with chronic thyroiditis and HLA DR4 slowly progressed to insulin-requiring status over a period of 45 months. These findings suggest that the measurement of GADAb using a commercial assay kit with rh GAD65 may be more useful to detect non-insulin-dependent type I diabetics among non-obese patients than using a commercial kit with purified pig brain native GAD, especially among those with low GADAb titers.

The level of erythrocyte aldose reductase protein (AR-p) was determined in diabetic patients as well as in 76 healthy controls by a two-site enzyme-linked immunosorbent assay. No significant difference in the mean AR-p level was demonstrated between the healthy and diabetic individuals. Based on the results of seven nerve function tests, 95 non-insulin-dependent diabetes mellitus (NIDDM) patients were classified into two groups: Group I, without demonstrable neuropathy ( < or = 1 abnormal test results); Group II, overt neuropathy ( > or = 2 abnormal results). The AR-p level was significantly higher in Group II than that in Group I. Multivariate logistic regression analysis identified two independent risk factors for overt neuropathy: longer duration of diabetes after clinical diagnosis (odds ratio, 1.15 per year; 95% confidence interval, 1.05-1.25) and a higher level of AR-p (odds ratio, 1.92 per 1 ng mgHb(-1); 95% confidence interval, 1.39-2.65). On 31 patients the AR-p level was re-assessed after a 12-month follow-up period. Irrespective of improved or stable HbA(1c) levels during the follow-up period, no apparent alteration in the level of AR-p was demonstrated. These results suggest that erythrocyte AR-p level may affect the susceptibility or development of diabetic neuropathy in NIDDM patients.

This paper examines the question of whether diabetes in humans is associated with changes in aldose reductase and sorbitol dehydrogenase gene expression. The polyol pathway, which comprises the enzymes aldose reductase and sorbitol dehydrogenase, is recognised to play a central role in the pathogenesis of the diabetic complications. Whilst it is known that experimental diabetes in the rat is associated with increased aldose reductase gene expression, possibly as an osmoregulatory response to hyperglycaemia, little is known about aldose reductase and sorbitol dehydrogenase gene expression in diabetes in humans. White cell aldose reductase mRNA levels were increased in patients with insulin-dependent (by 135%, P < 0.05) and non-insulin-dependent (by 132%, P < 0.05) diabetes compared to levels in healthy volunteers. Levels of glycosylated haemoglobin were also increased (P < 0.001) in diabetes but there was no correlation between white cell aldose reductase mRNA and glycosylated haemoglobin levels. In contrast to aldose reductase, levels of white cell sorbitol dehydrogenase mRNA were not affected by diabetes. These results establish for the first time that diabetic patients show increases in white cell aldose reductase mRNA levels, possibly consistent with increased aldose reductase gene expression. This finding may have implications for the use of aldose reductase inhibitors in the treatment of the diabetic complications.

The activities of FAD-linked glycerophosphate dehydrogenase (m-GDH), glutamate dehydrogenase (GlDH), glutamate-pyruvate transaminase (GPT) and glutamate-oxalacetate transaminase (GOT) were measured in purified populations of CD3+ lymphocytes from 55 control subjects, 62 type-2 diabetics and 50 non-diabetic relatives of the latter patients. The activity of m-GDH was measured by both a radioisotopic procedure and colourimetric technique. As judged from these measurements and relative to the paired value for GlDH, the incidence of abnormally low m-GDH activity was significantly higher in type-2 diabetics than in control subjects. Moreover, the paired ratio in reaction velocity between the colourimetric and radioisotopic assay of m-GDH was abnormally high in patients with low m-GDH activity. Low m-GDH activity often coincided with increased GPT activity in plasma or high GPT/GOT ratio in lymphocytes. No obvious clustering of these anomalies was found in relatives of diabetic patients. These findings suggest that an inherited or acquired genomic defect of m-GDH in lymphocytes, and possibly in pancreatic B-cells, may participate to the pathogenesis of non-insulin-dependent diabetes mellitus.

Mutation of the glucokinase gene has recently been identified as a cause of maturity-onset diabetes of the young (MODY), a subset of non-insulin-dependent diabetes mellitus (NIDDM). However, its role in the wide variety of NIDDM remains controversial due to conflicting reports of association studies, negative results of linkage studies and low prevalence of glucokinase mutations in the common variety of NIDDM. In this study, two (CA)n-microsatellite polymorphisms flanking both ends of the glucokinase gene, termed GCK1 and GCK2, were used to evaluate the role of glucokinase on NIDDM susceptibility of Taiwanese. For GCK1, three alleles (Z,Z+2 and Z+4 with a polymorphic information content index (PIC) of 0.53) and six genotypes were evident in 119 Taiwanese. When compared with control subjects, the NIDDM group had a much less frequency of the Z+2 allele (14.0% vs. 23.9%). In addition, the Z+2 allele was noted to have a marginal protective effect for NIDDM in Taiwanese with the odds ratio of 0.52 (95% confidence interval (C.I.) 0.26-1.03, P = 0.058). For GCK2, four alleles (0, 2, 4 and 6 with a PIC of 0.48) and seven genotypes were identified. There was no significant difference in allele frequency between NIDDM and control groups in the locus of GCK2. Our data were in agreement with reports from American Blacks, Mauritian Creoles, Asian Indians, Japanese and Finnish--that there is a positive association of GCK1 and a negative association of GCK2 with NIDDM. Furthermore, the Z+2 allele was a protective factor for NIDDM in Taiwanese.

Adenosine deaminase (ADA) is suggested to be an important enzyme for modulating the bioactivity of insulin, but its clinical significance in diabetes mellitus (DM) is not yet characterized. We measured the serum levels of ADA isoenzymes (ADA1 and ADA2) in healthy donors (HD, n = 52), insulin-dependent diabetes mellitus (IDDM, n = 53) patients and non-insulin-dependent diabetes mellitus (NIDDM, n = 65) patients. The mean serum level of ADA1 in HD, IDDM or NIDDM patients was, respectively 6.5, 8.1 or 9.5 units/l (P < 0.001 vs. HD) and that of ADA2 in HD, IDDM or NIDDM patients was 7.0, 14.9 (P < 0.001 vs. HD) or 11.2 units/l (P < 0.001 vs. HD), respectively. Normalization of the blood glucose level by the hospitalization was associated with the decrease in ADA2 (but not ADA1) activity in 6 of 8 IDDM or 11 of 12 NIDDM poorly controlled patients. ADA2 (but not ADA1) activity in the poorly controlled NIDDM patients directly correlated with the hemoglobin A1c level (P < 0.002). Measurement of serum ADA2 activity may be important to better understand the clinical aspects of both IDDM and NIDDM. The pathogenic role of elevated ADA activity in the sera of DM patients was addressed.

Antibodies to glutamic acid decarboxylase (anti-GAD) are common in typical insulin-dependent diabetes mellitus, and also identify a sub-group of older persons who are originally misdiagnosed as having non-insulin-dependent disease (NIDDM). The Wanigela people of Papua New Guinea are highly susceptible to diabetes mellitus, with a prevalence of 20.4% in urbanised young adults aged 25-34 years. On the basis of clinical features including the presence of obesity and relatively high insulin concentrations the Wanigelas have NIDDM. To determine whether anti-GAD is present in this high prevalence form of diabetes, and to investigate whether there might be an autoimmune component to the disease, we measured anti-GAD in 93 newly-diagnosed diabetic subjects aged 25-44 years, and in 40 controls with normal glucose tolerance. There was no difference in mean levels of anti-GAD in diabetic subjects and normal controls. Two subjects had borderline elevated anti-GAD levels: one was a normal control, and the other a diabetic. This study shows that anti-GAD is not present in this (and probably other) high prevalence variant of NIDDM. Moreover, the results suggest strongly that diabetes in the Wanigela people is unlikely to have an autoimmune component to its pathogenesis.