Changes in the blood lactate dehydrogenase measurements in canines and felines following the international standardization of the assay method.

Lactate dehydrogenase (LDH) in blood is measured using the Japanese Society of Clinical Chemistry (JSCC) method in Japan and the International Federation of Clinical Chemistry (IFCC) method in other countries. In human clinical practice, the IFCC method replaced the JSCC method due to international standardization. Moreover, veterinary LDH measurement will also eventually shift to the IFCC method. However, the relationship between the IFCC and JSCC methods for LDH in various animals and whether they can be equated or not have not yet been investigated. This study aimed to present the changes in measurements in canines and felines after switching to the IFCC method. The plasma LDH activity of canines (N=177) and felines (N=115), who visited a secondary care veterinary clinic, was measured using the JSCC and IFCC methods. The IFCC/JSCC ratio was <1.0 in 85% of canines and 88% of felines, indicating that the IFCC method tended to show lower values than the JSCC method, presumably because LDH5 is dominant among the LDH isozymes in canines and felines. The increase in the systematic errors of both assays was in the high value range, with some samples exceeding the error tolerance from near the upper end of the reference range. When switching to the IFCC method for LDH measurement in canines and felines, each institution should consider whether the reference range and clinical diagnostic values established by the JSCC method are appropriate for continued use.

PPARgamma Pro12Ala Pro/Pro and resistin SNP-420 G/G genotypes are synergistically associated with plasma resistin in the Japanese general population.

OBJECTIVE: The Ala allele of the Pro12Ala polymorphism (rs1801282) of peroxisome proliferator-activated receptor gamma (PPARgamma) is protective against type 2 diabetes (T2DM). Resistin, secreted from adipocytes, causes insulin resistance in rodents. Resistin gene expression is reduced by the PPARgamma ligand. We previously reported that subjects with the G/G genotype of a resistin gene single nucleotide polymorphism (SNP) at -420 (rs1862513) had the highest circulating resistin levels, followed by C/G and C/C. The aim of this study was to determine the relationship among PPARgamma Pro12Ala polymorphism, resistin SNP-420, and plasma resistin. DESIGN, PATIENTS AND MEASUREMENTS: We cross-sectionally analysed 2077 community-dwelling subjects attending an annual medical check-up. Genotypes were determined by TaqMan analysis. Fasting plasma resistin was measured using ELISA. RESULTS: Plasma resistin appeared to be higher in subjects with the Pro/Pro genotype of PPARgamma than those with Pro/Ala and Ala/Ala genotypes (mean +/- SE, 11.6 +/- 0.2 vs. 10.4 +/- 0.5 microg/l). Multiple regression analysis, adjusted for age, gender, BMI, and resistin SNP-420, revealed that the Pro/Pro genotype was a positive predictor of plasma resistin (PPARgamma , Pro/Pro vs. Pro/Ala + Ala/Ala, unstandardized regression coefficient (beta) = 1.03, P = 0.0384). The effects of the Pro/Pro genotype of PPARgamma (Pro/Pro vs. Pro/Ala + Ala/Ala) and the G/G genotype of resistin SNP-420 (G/G vs. C/C) on plasma resistin were synergistic (beta = 4.76, P = 0.011). CONCLUSIONS: The PPARgamma Pro12Ala Pro/Pro and resistin SNP-420 G/G genotypes were synergistically associated with plasma resistin, when adjusted for age, gender, and BMI, in the Japanese general population.

Serum resistin is positively correlated with the accumulation of metabolic syndrome factors in type 2 diabetes.

OBJECTIVE: Resistin, secreted from adipocytes, causes insulin resistance in rodents. We reported that the G/G genotype of a resistin gene promoter single nucleotide polymorphism (SNP) at -420 increases type 2 diabetes (T2DM) susceptibility by enhancing promoter activity. We also showed that serum resistin was positively correlated with G at SNP-420, the duration of T2DM, and HbA1c in T2DM. The aim of this study was to determine the relation between serum resistin and factors related to the metabolic syndrome (MetS) in T2DM. DESIGN, PATIENTS AND MEASUREMENTS: We analysed 238 Japanese T2DM subjects (124 males and 114 females, age 60.2 +/- 11.3 years, body mass index (BMI) 24.1 +/- 3.9) whose overnight fasting sera were available. Serum resistin was measured using ELISA. RESULTS: Serum resistin was higher in subjects with either obesity (P = 0.041), low HDL (P = 0.004), high triglycerides (TG) (P = 0.019), hypertension (HT) (P = 0.001) or atherosclerosis (P = 0.012). Simple regression analysis revealed that serum resistin was correlated with lower HDL, TG and high-sensitivity C-reactive protein (hsCRP). Multiple regression analysis (or logistic regression analysis for HT), adjusted for age, gender, BMI and the duration of T2DM, revealed that serum resistin was correlated with lower HDL (P = 0.008), TG (P = 0.041), HT (P = 0.031) and hsCRP (P = 0.004). Serum resistin was positively correlated with the number of MetS factors, independent of age, gender and the duration of T2DM (P < 0.001). Adjustment by either thiazolidinedione (TZD) treatment or hsCRP had no effects on these findings. CONCLUSIONS: Serum resistin was positively correlated with the accumulation of MetS factors in T2DM.

Serum total and high molecular weight adiponectin levels are correlated with the severity of diabetic retinopathy and nephropathy.

OBJECTIVE: Adiponectin is secreted specifically from adipocytes, and improves insulin sensitivity. Of its isoforms, the high molecular weight (HMW) complex is thought to be the most active. The aim of this study was to determine the relationship between serum total or HMW adiponectin and diabetic microangiopathy. DESIGN, PATIENTS AND MEASUREMENTS: We analysed 198 Japanese patients with type 2 diabetes mellitus (T2DM) whose fasting serum samples were available. Serum total adiponectin and HMW adiponectin were measured using an enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum total adiponectin was found to have increased in the advanced stages of diabetic retinopathy (mean +/- SE, none, 6.9 +/- 0.3; simple, 8.3 +/- 1.0; preproliferative, 8.4 +/- 0.8; proliferative, 12 +/- 1.1 mg/l; anovaP = 0.0004) and nephropathy (stage I, 7.0 +/- 0.3; II, 7.7 +/- 0.5; III, 9.5 +/- 0.9; IV, 16 +/- 4.5 mg/l, P < 0.0001). Similarly, serum HMW adiponectin had increased in the advanced stages of retinopathy (3.7 +/- 0.2, 4.6 +/- 0.5, 4.6 +/- 0.6 and 6.9 +/- 0.8 mg/l, respectively, P = 0.0005) and nephropathy (3.7 +/- 0.2, 4.3 +/- 0.4, 5.3 +/- 0.7 and 7.9 +/- 2.2 mg/l, respectively, P = 0.0007). Neither serum total nor HMW adiponectin was correlated with neuropathy. The HMW/total adiponectin ratio was not correlated with microangiopathy. Multiple regression analysis revealed that serum total and HMW adiponectin were independent factors for retinopathy stage (P = 0.0055 and P = 0.0027, respectively) and nephropathy stage (P = 0.0003 and P = 0.0018, respectively), when adjusted for age, gender, body mass index (BMI) and the duration of T2DM. This correlation remained significant when serum creatinine (or estimated glomerular filtration rate) and hypertension were added as independent variables. Treatment with thiazolidinediones (TZDs) did not affect these findings. CONCLUSIONS: Serum total adiponectin and HMW adiponectin were found to be positively correlated with the severity of retinopathy and nephropathy but not with neuropathy in T2DM.

The G/G genotype of a single nucleotide polymorphism at -1066 of c-Jun N-terminal kinase 1 gene (MAPK8) does not affect type 2 diabetes susceptibility despite the specific binding of AP2alpha.

OBJECTIVE: The c-Jun N-terminal kinase 1 (JNK1, mitogen-activated kinase 8; MAPK8) phosphorylates insulin receptor substrate-1 (IRS-1) at serine 307, which induces insulin resistance. MAPK8 activity is increased in obese insulin-resistant mice, whereas mapk8 (-/-) mice show decreased adiposity and improved insulin sensitivity. The aim of this study was to determine the relationship between single nucleotide polymorphisms (SNPs) of MAPK8 and type 2 diabetes (T2DM). DESIGN, PATIENTS AND MEASUREMENTS: Approximately 2 kb of 5' flanking and the coding regions were initially sequenced in 24 Japanese T2DM subjects. Identified SNPs were genotyped in 204 T2DM cases and 201 nondiabetic controls. The function of promoter SNP-1066 (g.-1066G > A, rs10857561) was analysed by electrophoretic mobility shift assay (EMSA) and luciferase assay. SNP-1066 was further genotyped in a total of 498 cases and 407 controls, and in 2075 subjects in the general population. RESULTS: In 204 cases and 201 controls, 11 identified SNPs were not associated with T2DM. These SNPs were in the same linkage disequilibrium (LD) block. The tag SNP-1066 was not associated with T2DM in a total of 498 cases and 407 controls with the power > 80% when the relative risk is > 1.31. Functionally, transcription factor AP2alpha specifically recognized G but not A at -1066. MAPK8 promoter activity was unchanged between G and A. In 2075 subjects, neither body mass index (BMI), fasting plasma glucose (FPG), homeostasis model assessment insulin resistance index (HOMA-IR), nor beta cell function index (HOMA-beta) was associated with SNP-1066. CONCLUSIONS: The G/G genotype of MAPK8 SNP-1066 did not affect T2DM susceptibility despite specific binding of AP2alpha.

Serum resistin is reduced by glucose and meal loading in healthy human subjects.

Resistin is an adipokine that induces insulin resistance in mice; serum concentrations are decreased by fasting and increased by feeding. Adiponectin, another adipokine, improves insulin sensitivity. The aims of this study were to determine the effects of glucose and meal loading on serum resistin and total and high-molecular weight (HMW) adiponectin in humans and to explore potential determinants of fasting serum resistin and of changes in resistin. Serum resistin and total and HMW adiponectin were measured by enzyme-linked immunosorbent assay in young, lean, nondiabetic subjects during 75-g oral glucose tolerance test (OGTT) and meal tolerance test (MTT). Resistin single nucleotide polymorphism (SNP) -420 was typed. Serum resistin was decreased at 60 and 120 minutes during OGTT compared with baseline (n = 36, 1-way repeated-measures analysis of variance, P < .0001; Scheffe, P = .0457 and P < .0001, respectively). Serum resistin was also reduced at 240 minutes during MTT (n = 33, 1-way repeated measures analysis of variance, P < .0001; Scheffe, P = .0002). Multiple regression analysis adjusted for age, sex, and body mass index revealed that the reductions in serum resistin were dependent on baseline resistin levels. Subjects with greater baseline concentrations of resistin experienced more pronounced declines in resistin (OGTT, unstandardized regression coefficient (beta) = -0.19, P = .0005; MTT, beta = -0.63, P < .0001). Serum total and HMW adiponectin was unchanged. Fasting serum resistin was positively correlated with the G allele number of SNP -420 (beta = 7.70, P = .01) and white blood cell count (beta = 0.007, P = .0001) adjusted for age, sex, and body mass index. Therefore, in young, lean, nondiabetic humans, serum resistin was reduced by glucose and meal loading; the reduction in resistin was greater in subjects with higher fasting resistin. Fasting resistin was correlated with SNP -420 and white blood cell count.

[A simple separative method for measuring serum amyloid A in high-density-lipoprotein and low-density-lipoprotein fractions using the phosphotungstic acid-Mg2+ precipitation procedure].

We developed a simple separative method for measuring serum amyloid A (SAA) in both high-density-lipoprotein (HDL) and low-density-lipoprotein (LDL) fractions. It was devised using the SAA agglutination method and phosphotungstic acid-Mg2+ precipitation procedure for evaluating HDL-cholesterol (HDL-C). The new method is also able to detect amyloid A (AA) in each fraction with precision. The results of both the present method and the method using SAA agglutination and the dextran sulfate-Mg2+ precipitation procedure showed a strong correlation when used to measure the level of SAA in the LDL fraction of patients (r = 0.997; p < 0.0001). Reference intervals in normal healthy subjects (n=75) ranged from 0.5 to 4.7 microg/ml in the HDL fraction and from 0.1 to 1.9 microg/ml in the LDL fraction. SAA in the LDL fraction of subjects with hyperlipidemia was significantly higher than in normal subjects and subjects with normal lipidemia. SAA in the HDL fraction and total sera of subjects with hyperlipidemia was significantly higher than in normal subjects; however, it was not higher than in patients with normal lipidemia. The present methods for detecting SAA, especially in the LDL fraction, might benefit from analyzing patho-physiological events in various lipid disorders.

[Clinical significance of small, dense LDL cholesterol in patients with hyperlipidemia and type 2 diabetes].

We investigated the serum levels of small, dense LDL-cholesterol (sd LDL-C) in patients with hyperlipidemia and type 2 diabetes. An analytical assay was used to determine the levels of sd LDL-C, employing a filter method using a separating agent of polyanion and divalent cation natures. Reference intervals of sd LDL-C in normal healthy subjects (n=113) ranged from 8.0 to 42.0 mg/dl. We found a strong correlation between the levels of sd LDL-C and both the ratio of LDL-C/apolipoprotein B and the LDL migration index. The LDL migration index was analyzed using polyacrylamide gel disk electrophoresis. The levels of sd LDL-C in patients with types IIa, IIb and IV hyperlipidemia were significantly higher than those in normal subjects and in patients with normal lipidemia. The levels of sd LDL-C in patients with type IIb were higher than those with types IIa and VI. Examination of patients with polydisperse LDL showed that the levels of nodular and disrupted type sd LDL-C were higher than the levels of symmetry type sd LDL-C. Moreover, the levels of sd LDL-C in patients with type 2 diabetes were higher than those in normal subjects. A high level of sd LDL-C in patients with type 2 diabetes was found to be an indicator of possible complications of hyperlipidemia and lessly related to glycemic control. Therefore, the determination of sd LDL-C levels can be useful in the diagnosis of patients with hyperlipidemia and polydisperse LDL and in patients with type 2 diabetes with complications of hyperlipidemia and atherosclerosis.

Dual Effects of a RETN Single Nucleotide Polymorphism (SNP) at -420 on Plasma Resistin: Genotype and DNA Methylation.

CONTEXT: We previously reported that single nucleotide polymorphism (SNP)-420 C>G (rs1862513) in the promoter region of RETN was associated with type 2 diabetes. Plasma resistin was tightly correlated with SNP-420 genotypes. SNP-420 is a CpG-SNP affecting the sequence of cytosine-phosphate-guanine dinucleotides. OBJECTIVE: To examine whether methylation at SNP-420 affects plasma resistin, we analyzed plasma resistin and methylation at RETN SNP-420. DESIGN AND METHODS: Genomic DNA was extracted from peripheral white blood cells in 2078 Japanese subjects. Quantification of the methylation was performed by pyrosequencing after DNA bisulfite conversion. RESULTS: Methylation at SNP-420 was highest in the C/C genotype (36.9 ± 5.7%), followed by C/G (21.4 ± 3.5%) and G/G (2.9 ± 1.4%; P < 0.001). When assessed in each genotype, methylation at SNP-420 was inversely associated with plasma resistin in the C/C (ß = -0.134, P < 0.001) or C/G (ß = -0.227, P < 0.001) genotype. In THP-1 human monocytes intrinsically having the C/C genotype, a demethylating reagent, 5-aza-dC, decreased the methylation at SNP-420 and increased RETN messenger RNA. SNP+1263 (rs3745369), located in the 3' untranslated region of RETN, was also associated with methylation at SNP-420. In addition, highly sensitive C-reactive protein was inversely associated with methylation at SNP-420 in the C/C genotype, whereas body mass index was positively associated. CONCLUSIONS: Plasma resistin was inversely associated with the extent of methylation at SNP-420 mainly dependent on the SNP-420 genotype. The association can also be explained partially independent of SNP-420 genotypes. SNP-420 could have dual, genetic and epigenetic effects on plasma resistin.

[Clinical significance of serum-glycated apolipoprotein B measured by the digestion LDL sediment fraction with protease].

We previously reported an assay method for serum glycated aplipoprotein B (G-apo B) using protease. The present study demonstrated correlations between serum G-apo B levels and some other serum parameters, from which a clinical significance of the G-apo B in diabetics was deduced. Serum G-apo B determined by the present method was significantly correlated with glyco-albumin and glycohemoglobin. However, no significant difference was observed between G-apo B levels and total cholesterol and the other lipid items. The mean levels of serum G-apo B in type 2 diabetics with or without hyperlipidemia were significantly higher than in normal subjects (p<0.001). In a comparison of type 2 diabetics with and without hyperlipidemia, the G-apo B levels were not significant between the former and the latter. However, those levels were significantly higher in the nodular and disrupted type of LDL than in the symmetry type of LDL. Even more, the G-apo B levels in the nodular type of LDL were significantly higher than in the disrupted type of LDL. Therefore, the G-apo B levels might be considered an independent risk marker of diabetes hyperlipidemia and atherogenesis.

Systematic search for single nucleotide polymorphisms in the FOXC2 gene: the absence of evidence for the association of three frequent single nucleotide polymorphisms and four common haplotypes with Japanese type 2 diabetes.

FOXC2, a forkhead/winged helix transcription factor, represents a promising candidate gene for type 2 diabetes since transgenic mice that specifically overexpress this gene in adipocytes are lean and insulin sensitive. To determine whether there are single nucleotide polymorphisms (SNPs) in this gene that are associated with type 2 diabetes, sequences of the coding and approximately 1 kb of 5' flanking regions in 24 Japanese type 2 diabetic subjects were initially analyzed using PCR direct sequencing, and the regions containing the identified polymorphisms were then examined. In 200 control subjects, three frequent SNPs were found (g. -512C>T [32.3%] and -350G>T [13.0%] in the 5' flanking region and +1548C>T [10.0%] in the 3' flanking region). Linkage disequilibria were found between all three pairs of these SNPs. Of the eight possible haplotypes defined by these SNPs, only four were found. When the frequencies of these SNPs and the four common haplotypes between 195 type 2 diabetic and 200 control subjects were compared, no association was evident. The +898C>T (Pro300Ser), +907C>A (Leu303Met), 1167_1169delCCA (389delHis), and +1251C>A (Ala417Ala) identified in the coding region were rare, although +907C>A could be higher in type 2 diabetic subjects (1.5%) than in control subjects (0.3%). Thus, the SNPs identified in the FOXC2 gene are unlikely to have major effects on susceptibility to Japanese type 2 diabetes.

Systematic search for single nucleotide polymorphisms in the resistin gene: the absence of evidence for the association of three identified single nucleotide polymorphisms with Japanese type 2 diabetes.

Resistin is a novel polypeptide specifically secreted from adipocytes, and its serum levels are increased in obese diabetic mice. Resistin antagonizes insulin and could account for insulin resistance. To determine whether there are single nucleotide polymorphisms (SNPs) in the resistin gene associated with type 2 diabetes, sequences for 24 Japanese type 2 diabetic patients were initially analyzed using PCR direct sequencing. Three SNPs were found in the introns, but none were present in the coding regions. The allele frequencies of genomic -167C>T, +157C>T, and +299G>A in 99 Japanese control subjects were determined to be 3.5, 6.6, and 39.4%, respectively. In each pair of these SNPs, linkage disequilibria were found between either -167C>T and +299G>A or +157C>T and +299G>A. A linkage disequilibrium was also detected among -167C>T, +157C>T, and +299G>A, and only four of the eight possible haplotypes defined by these SNPs were found. A comparison of the frequencies of these SNPs and haplotypes between 99 type 2 diabetes and 99 control subjects revealed no evidence for any association. These identified SNPs, which were in linkage disequilibrium, represent potentially useful tools for searching for their association with specific phenotypes of diabetes.

A simple precipitation method for measuring sialic acid in apolipoprotein B-containing lipoproteins in plasma.

BACKGROUND: A convenient method for the measurement of sialic acid in plasma apoB-containing lipoproteins is described. METHODS: Dextran sulphate-Mg(2+) precipitation and enzymatic sialic acid assay were combined and applied to analysis of plasma from 96 healthy controls and 136 hyperlipidaemic subjects of types IIa (n=46), IIb (n=43), and IV (n=47). RESULTS: The sialic acid concentrations (mean+/-SD) in plasma apoB-containing lipoproteins were 19.4+/-5.9, 24.3+/-4.7 (P<0.0001 versus normal), 23.0+/-4.7 (P<0.0001), 27.9+/-5.2 (P<0.0001), and 22.3+/-3.4 mg/L (P<0.002), for normal, all types of hyperlipidaemia, types IIa, IIb, and IV, respectively. The contents of sialic acid in apoB were 2.03+/-0.41%, 2.09+/-0.35% (no significance versus normal), 1.86+/-0.27% (P<0.0001), 1.97+/-0.26% (P<0.02), and 2.28+/-0.41% (P<0.002), for normal, all types of hyperlipidaemia, types IIa, IIb, and IV, respectively. CONCLUSION: The content of sialic acid in apoB decreased significantly in type IIa but increased in type IV hyperlipidaemia, which may reflect the presence of sialic acid in very low-density lipoprotein apolipoproteins other than apoB. This simple precipitation method will be useful to evaluate the sialic acid content in low-density lipoprotein in hyperlipidaemic subjects, especially of type IIa.

[Convenient assay for glycated apolipoprotein B using protease].

The aim of this study is to develop a convenient method for monitoring glycated apolipoprotein B levels. Serum sample was treated with dextran-magnesium and the resulting precipitates were subjected to glycated albumin assay. Dissolving the precipitates by Triton X-100 and digesting by proteinase K enable the establishment of stable and sensitive assay. Intra- and inter assay coefficients of variation were 1.5-3.5% and 1.6-3.3%, respectively. The serum glycated apolipoprotein B values by present method correlated well with those by enzyme-linked immunosorbent assay (r=0.979). The serum glycated apolipoprotein B values in healthy subjects was 4.14+/-0.51% (mean+/-SD) with no significant difference between men and women and with no age-dependent variation. Patients with type 2 diabetes mellitus had higher serum glycated apolipoprotein B levels than the healthy subjects. This assay should further be investigated to establish the validity of glycated apolipoprotein B measurement in clinical field.

Reduction of phosphodiesterase 3B gene expression in peroxisome proliferator-activated receptor gamma (+/-) mice independent of adipocyte size.

Phosphodiesterase 3B (PDE3B) gene expression is generally reduced in large adipocytes of obese, insulin-resistant mice. This reduced gene expression is restored by peroxisome proliferator-activated receptor (PPAR) gamma ligands accompanied by a reduced fat cell size. To determine whether PDE3B gene expression is regulated by PPAR gamma itself, we analyzed lean PPAR gamma (+/-) mice with adipocyte size comparable to control PPAR gamma (+/+) mice. In adipocytes of PPAR gamma (+/-) mice, PDE3B mRNA and protein were both reduced to 63% of wild-type levels. Basal PDE activity tended to be decreased to 70% of wild-type levels, and, similarly, insulin-induced PDE activity was significantly decreased to 70%. Thus, PPAR gamma is required for PDE3B gene expression independent of adipocyte size.

Differential regulation of gene expression and insulin-induced activation of phosphodiesterase 3B in adipocytes of lean insulin-resistant IRS-1 (-/-) mice.

Phosphodiesterase (PDE) 3B, a major isoform of PDE in adipocytes, mediates the antilipolytic action of insulin. PDE3B gene expression is generally reduced in adipocytes of either monogenic or polygenic rodent models of obese, insulin-resistance. An increased fat cell size, a common feature of obesity, could account for this reduction. Insulin receptor substrate-1 (IRS-1) (-/-) mice are lean with a reduced fat cell size and have insulin resistance due to a primary defect of insulin signaling. To determine whether the regulation of PDE3B gene expression is correlated with fat cell size, we examined this gene expression in adipose tissues of IRS-1 (-/-) mice. In IRS-1 (-/-) mice, PDE3B mRNA and protein levels were increased 1.24- and 1.35-fold those in C57BL/6J control mice, respectively. Independently, the fold induction of PDE activity by insulin (insulin-induced/basal) was 1.7-fold in control mice, but was reduced to 1.35-fold in IRS-1 (-/-) mice. Thus, PDE3B gene expression may be inversely correlated with a fat cell size, whereas insulin-induced PDE3B activation is mediated through IRS-1.

The absence of evidence for major effects of the frequent SNP +299G>A in the resistin gene on susceptibility to insulin resistance syndrome associated with Japanese type 2 diabetes.

Resistin, specifically secreted from adipocytes, antagonizes insulin and represents a promising candidate gene for type 2 diabetes. We reported that a frequent single nucleotide polymorphism (SNP) +299G>A in this gene is not associated with type 2 diabetes. To determine whether this SNP affects insulin resistance syndrome associated with type 2 diabetes, we examined its effects on susceptibility to obesity, hyperlipidemia and hypertension in type 2 diabetic subjects and on susceptibility to type 2 diabetes by interaction with other frequent genes involved in lipid metabolism, namely, beta3-adrenergic receptor (b3AR) Trp64Arg, phosphodiesterase 3B (PDE3B) c.1389G>A or lysosomal acid lipase (LAL) Thr-6Pro. The 99 type 2 diabetic and 99 control subjects were typed by PCR direct sequencing or PCR-RFLP. No differences in frequencies of obesity, hyperlipidemia and hypertension were found between the type 2 diabetic subjects with G/G and those with G/A or A/A genotypes of the resistin SNP. When the combination of the resistin SNP with each of b3AR, PDE3B and LAL SNPs was assessed, no association with type 2 diabetes was evident. Therefore, the frequent SNP +299G>A in the resistin gene is unlikely to have major effects on susceptibility to insulin resistance syndrome associated with type 2 diabetes in Japanese subjects.

Plasma resistin is associated with single nucleotide polymorphisms of a possible resistin receptor, the decorin gene, in the general Japanese population.

Resistin is an adipokine secreted from adipocytes in mice. We previously reported that a single nucleotide polymorphism (SNP) -420 (rs1862513) in the human resistin gene (RETN), is correlated with plasma resistin. Decorin is a multifunctional proteoglycan, and its isoform, lacking 14 amino acids from the N terminal region of mature core decorin, recently was identified as a resistin receptor in mice. To examine whether SNPs in the vicinity of the human decorin gene (DCN) are associated with plasma resistin, we cross-sectionally analyzed six tag SNPs selected around DCN in the same linkage disequilibrium block in 2,078 community-dwelling Japanese subjects. Plasma resistin was associated with the rs7139228, rs7956537, rs516115, and rs3138167 genotypes in DCN. A multiple regression analysis revealed that the genotype of rs7308752 (G/G) or rs516115 (C/C) was associated with decreased plasma resistin after adjusted for age, sex, BMI, and the RETN SNP rs1862513. The effect of rs7139228 and rs1862513 seemed to be additive without synergistic interaction. Therefore, plasma resistin was associated with some tag SNPs around DCN in the general Japanese population. The possibility that human decorin is a human resistin receptor should be pursued.

A at single nucleotide polymorphism-358 is required for G at -420 to confer the highest plasma resistin in the general Japanese population.

Insulin resistance is a feature of type 2 diabetes. Resistin, secreted from adipocytes, causes insulin resistance in mice. We previously reported that the G/G genotype of single nucleotide polymorphism (SNP) at -420 (rs1862513) in the human resistin gene (RETN) increased susceptibility to type 2 diabetes by enhancing its promoter activity. Plasma resistin was highest in Japanese subjects with G/G genotype, followed by C/G, and C/C. In this study, we cross-sectionally analyzed plasma resistin and SNPs in the RETN region in 2,019 community-dwelling Japanese subjects. Plasma resistin was associated with SNP-638 (rs34861192), SNP-537 (rs34124816), SNP-420, SNP-358 (rs3219175), SNP+299 (rs3745367), and SNP+1263 (rs3745369) (P<10(-13) in all cases). SNP-638, SNP -420, SNP-358, and SNP+157 were in the same linkage disequilibrium (LD) block. SNP-358 and SNP-638 were nearly in complete LD (r(2) = 0.98), and were tightly correlated with SNP-420 (r(2) = 0.50, and 0.51, respectively). The correlation between either SNP-358 (or SNP-638) or SNP-420 and plasma resistin appeared to be strong (risk alleles for high plasma resistin; A at SNP-358, r(2) = 0.5224, P = 4.94x10(-324); G at SNP-420, r(2) = 0.2616, P = 1.71x10(-133)). In haplotypes determined by SNP-420 and SNP-358, the estimated frequencies for C-G, G-A, and G-G were 0.6700, 0.2005, and 0.1284, respectively, and C-A was rare (0.0011), suggesting that subjects with A at -358, generally had G at -420. This G-A haplotype conferred the highest plasma resistin (8.24 ng/ml difference/allele compared to C-G, P<0.0001). In THP-1 cells, the RETN promoter with the G-A haplotype showed the highest activity. Nuclear proteins specifically recognized one base difference at SNP-358, but not at SNP-638. Therefore, A at -358 is required for G at -420 to confer the highest plasma resistin in the general Japanese population. In Caucasians, the association between SNP-420 and plasma resistin is not strong, and A at -358 may not exist, suggesting that SNP-358 could explain this ethnic difference.

Hyperresistinemia is associated with coexistence of hypertension and type 2 diabetes.

Numerous studies have demonstrated that high blood pressure substantially increases the risk of microvascular and macrovascular complications in patients with type 2 diabetes mellitus (T2DM). Currently, we found that serum resistin, an adipocyte- and monocyte-derived cytokine, was positively correlated with several components of the metabolic syndrome, including hypertension in T2DM. To investigate the association of resistin with an etiologic difference among subjects with hypertension with T2DM, hypertension without T2DM, and normotensive T2DM, we analyzed 210 subjects, including 91 with hypertension with T2DM, 55 with hypertension without T2DM, and 64 with normotensive T2DM. Serum resistin level was higher in subjects with hypertension with T2DM, followed by subjects with normotensive T2DM and hypertension without T2DM, irrespective of antihypertensive treatment status (20.9+/-17.6 and 14.0+/-8.9 versus 11.2+/-7.6 ng/mL, respectively; P<0.01). Simple regression analysis revealed that resistin positively correlated with blood pressure (systolic blood pressure: r=0.29, P<0.01; diastolic blood pressure: r=0.21, P<0.05) and intima-media thickness (r=0.27; P<0.05) in patients with T2DM but not in subjects with hypertension without T2DM. Multiple regression analysis, adjusted for age, gender, body mass index, fasting glucose, high-density lipoprotein cholesterol, white blood cell counts, and glomerular filtration rate, further revealed that resistin was an independent factor for high blood pressure in patients with T2DM (P<0.05). In vitro gene expression analysis in human coronary endothelial cells revealed that resistin induced fatty acid binding protein, a key molecule of insulin resistance, diabetes, and atherosclerosis. These results suggest that hyperresistinemia would contribute to the pathogenesis of hypertension in patients with T2DM, significantly linked to vascular complications and cardiovascular events.