Yukio Horikawa

Variants in KCNQ1 are associated with susceptibility to type 2 diabetes mellitus

We carried out a multistage genome-wide association study of type 2 diabetes mellitus in Japanese individuals, with a total of 1,612 cases and 1,424 controls and 100,000 SNPs. The most significant association was obtained with SNPs in KCNQ1, and dense mapping within the gene revealed that rs2237892 in intron 15 showed the lowest P value (6.7 × 10−13, odds ratio (OR) = 1.49). The association of KCNQ1 with type 2 diabetes was replicated in populations of Korean, Chinese and European ancestry as well as in two independent Japanese populations, and meta-analysis with a total of 19,930 individuals (9,569 cases and 10,361 controls) yielded a P value of 1.7 × 10−42 (OR = 1.40; 95% CI = 1.34–1.47) for rs2237892. Among control subjects, the risk allele of this polymorphism was associated with impairment of insulin secretion according to the homeostasis model assessment of β-cell function or the corrected insulin response. Our data thus implicate KCNQ1 as a diabetes susceptibility gene in groups of different ancestries.

The ratio of leptin to adiponectin can be used as an index of insulin resistance

The level of leptin increases with obesity, whereas that of adiponectin decreases with obesity. It is reported that the ratio of leptin to adiponectin (L/A) is associated with insulin resistance. It is difficult to evaluate insulin resistance in diabetic patients who have a dysfunction of insulin secretion. The aim of this study was to examine whether the L/A ratio is a useful marker for insulin resistance in diabetic patients. We examined L/A in the serum of a total of 139 Japanese patients with type 2 diabetes mellitus (66 women and 73 men) and 7 healthy individuals recruited in our hospital. Changes in the levels of leptin and adiponectin were observed using the oral glucose tolerance test and a hyper- and euglycemic clamp test. Twenty-one patients with type 2 diabetes mellitus were observed for more than 6 months after treatment with pioglitazone, and 31 patients with type 2 diabetes mellitus were observed for more than 6 months after the treatment with metformin. The mean value of L/A in 139 Japanese patients with type 2 diabetes mellitus was 1.22 +/- 1.41 (1.68 +/- 1.76 in women, 0.81 +/- 0.80 in men; P = .0002). In the clamp tests, L/A correlated with glucose infusion rate (GIR) (r(2) = 0.26, P = .0034). The correlation of L/A and GIR indicated a stronger correlation than either leptin (r(2) = 0.144, P = .03) or adiponectin alone (r(2) = 0.023, P = .41), or the homeostasis model assessment of insulin resistance (r(2) = 0.103, P = .08). The average hemoglobin A(1c) (HbA(1c)) improved from 10.2% +/- 1.2% to 9.2% +/- 1.6% (P = .0037) in 6 months after treatment with pioglitazone. Our results indicate pioglitazone to be effective for HbA(1c) improvement in subjects with high L/A and low L/A. The average HbA(1c) improved from 9.2% +/- 0.9% to 8.0% +/- 1.2% (P = .0002) in 6 months after treatment with metformin. Our results indicate metformin to be effective for HbA(1c) improvement in subjects with a low L/A. In conclusion, we demonstrate that L/A is different between male and female subjects. The correlation of L/A and GIR by the euglycemic hyperinsulinemic clamp test suggests that L/A is a useful indicator for the choice of drug to treat diabetes mellitus.

Construction of a prediction model for type 2 diabetes mellitus in the Japanese population based on 11 genes with strong evidence of the association.

Prediction of the disease status is one of the most important objectives of genetic studies. To select the genes with strong evidence of the association with type 2 diabetes mellitus, we validated the associations of the seven candidate loci extracted in our earlier study by genotyping the samples in two independent sample panels. However, except for KCNQ1, the association of none of the remaining seven loci was replicated. We then selected 11 genes, KCNQ1, TCF7L2, CDKAL1, CDKN2A/B, IGF2BP2, SLC30A8, HHEX, GCKR, HNF1B, KCNJ11 and PPARG, whose associations with diabetes have already been reported and replicated either in the literature or in this study in the Japanese population. As no evidence of the gene–gene interaction for any pair of the 11 loci was shown, we constructed a prediction model for the disease using the logistic regression analysis by incorporating the number of the risk alleles for the 11 genes, as well as age, sex and body mass index as independent variables. Cumulative risk assessment showed that the addition of one risk allele resulted in an average increase in the odds for the disease of 1.29 (95% CI=1.25–1.33, P=5.4 × 10−53). The area under the receiver operating characteristic curve, an estimate of the power of the prediction model, was 0.72, thereby indicating that our prediction model for type 2 diabetes may not be so useful but has some value. Incorporation of data from additional risk loci is most likely to increase the predictive power.

Associations of coronary artery calcification and carotid intima-media thickness with plasma concentrations of vascular calcification inhibitors in type 2 diabetic patients

Vascular calcification is frequently accompanied by intima-media thickening, but the associations among these atherosclerotic features and bone-related peptides in diabetic patients are unclear. We enrolled 168 type 2 diabetic patients and 40 non-diabetic subjects consecutively admitted to our hospital. Mean intima-media thickness (mean-IMT) in common carotid arteries was measured by B-mode ultrasonography. Agatston coronary artery calcium score (CACS) was obtained using multidetector-row computed tomography (MDCT). Plasma bone-related peptides osteopontin and osteoprotegerin levels were measured. Diabetic patients had higher mean-IMT (p=0.0002) and log(CACS+1) (p<0.0001) and similar bone-related peptides compared to non-diabetic subjects. In diabetic patients classified into tertiles according to their CACS levels, those with the highest scores showed the highest mean-IMT (p=0.0004) and bone-related peptides (p<0.05) among the groups. log(CACS+1) and mean-IMT were associated (p<0.0001) and were positively correlated with osteopontin (p<0.01) and osteoprotegerin (p<0.01) in diabetic patients. Multivariate analyses revealed that the significant independent determinants of log(CACS+1) were age, duration of diabetes and osteopontin (p<0.0001) and those of mean-IMT were age, hypertension, osteopontin and osteoprotegerin (p<0.0001), respectively. We have demonstrated that vascular calcification in type 2 diabetic patients is frequently accompanied by intima-media thickening, and osteopontin may act as a vascular calcification inhibitor by increasing intima-media thickness.

Large‐scale analysis of glucocorticoid target genes in rat hypothalamus

Insufficient glucocorticoid (GC) signaling is frequently observed in major depressive disorder (MDD). Since emotional and behavioral symptoms are often accompanied by disturbances in hypothalamic systems, GC insufficiency in this region is regarded as important in the pathogenesis of MDD. In this study, 22 early GC‐responsive genes comprising 15 up‐regulated and 7 down‐regulated genes in rat hypothalamus were identified as being regulated at least two‐fold by dexamethasone using microarray with 22u2003599 unique transcripts. Among these 22 genes, five of which are novel GC‐responsive genes, the expression patterns of sgk, bcl6, pdk4, and plekhf1 were examined in vitro in detail, and GC‐responsive regions were identified only within the promoter of sgk. This suggests that glucocorticoid response element‐independent pathways also play a critical role in early GC‐response in hypothalamus. Considering that a number of these GC‐responsive genes are candidate neuronal regulators, this gene list should be useful in clarifying the relationship between GC insufficiency and the pathogenesis of MDD.

Krüppel-like factor-10 is directly regulated by carbohydrate response element-binding protein in rat primary hepatocytes.

Krüppel-like factor (KLF)-10, is a circadian transcriptional regulator, which links the molecular clock to energy metabolism in the liver. Recently, it was reported that Klf-10 expression is induced by glucose stimulation in mouse hepatocytes. We previously reported that carbohydrate response element-binding protein (ChREBP) plays an important role in the regulation of hepatic lipogenic gene expression. Here, we investigate whether ChREBP, a glucose-activated transcription factor, directly regulates Klf-10 mRNA expression in rat primary hepatocytes. We found that both glucose stimulation and adenoviral overexpression of ChREBP induce Klf-10 mRNA expression in rat primary hepatocytes in a dose-dependent manner. Conversely, overexpression of dominant-negative Max-like protein inhibits glucose-induction expression of Klf-10 mRNA. Deletion analysis using rat Klf-10 promoter in the pGL3 vector combined with a chromatin immunoprecipitation assay against the anti-ChREBP antibody demonstrated that the carbohydrate response element is located between -125 bp and -109 bp in the rat Klf-10 promoter. Conversely, adenoviral overexpression of KLF-10 partly inhibits glucose induction of ChREBP target genes in primary hepatocytes. In conclusion, these data suggest that crosstalk between ChREBP and KLF-10 is involved in the regulation of the lipogenic pathway.

Isolation and structure elucidation of tumescenamides A and B, two peptides produced by Streptomyces tumescens YM23-260

Two peptides, tumescenamides A and B, were isolated from the fermentation broth of a marine bacterium, Streptomyces tumescens YM23-260. The structure of tumescenamide A was determined to be a cyclic depsipeptide consisting of α-amino-2-butenoic acid, tyrosine, valine, leucine and threonine, substituted with a 2,4-dimethylheptanoyl residue at the α-NH2 position. Tumescenamide B possesses a 2,4,6-trimethylnonanoyl residue in place of the 2,4-dimethylheptanoyl substituent in tumescenamide A. Tumescenamide A induced reporter gene expression under the control of the insulin-degrading enzyme promoter.

Fat and carbohydrate in western diet contribute differently to hepatic lipid accumulation.

We investigated the contributions of dietary fat and dietary carbohydrate to the development of fatty liver induced by western diet (WD). Compared with WD-fed wild type (WT) mice, livers of WD-fed ChREBP(-/-) mice showed lipid droplets of varying sizes around the hepatic lobules, while hepatic triglyceride and cholesterol contents were only modestly decreased. Inflammation and fibrosis were suppressed in ChREBP(-/-) mice. In addition, compared with WD-fed WT mice, ChREBP(-/-) mice showed decreased β-oxidation, ketogenesis and FGF21 production, increased intestinal lipid absorption, and decreased VLDL secretion. These findings suggest that dietary fat and carbohydrate contribute differently to the development of fatty liver.

The effects of pitavastatin on glucose metabolism in patients with type 2 diabetes with hypercholesterolemia

BACKGROUNDnAlthough there have been several reports that statins cause insulin resistance that leads to the occurrence of type 2 diabetes in Caucasians, there has been no Japanese prospective studies investigating the effects of statins on the glucose metabolism system.nnnMATERIALS AND METHODSnOur subjects were 86 Japanese patients with type 2 diabetes with hypercholesterolemia. Pitavastatin 2mg/day was administered for 12 months and the lipid-related values, glucose metabolism values, and the presence/absence of side effects were investigated.nnnRESULTSnNone of these factors was found to differ between before and after administration of pitavastatin in overall analysis of all subjects. In subgroup analysis, fasting blood glucose showed a decrease in the BMI ≥ 25 group and there was a significant difference between the BMI<25 and BMI ≥2 5 groups (P-values: 0.021 and 0.0036). Although HbA1c showed an increase both in the group switched to pitavastatin and the BMI<25 group (P-values: 0.035 and 0.033) and HOMA-β showed a decrease in the BMI<25 group (P-values: 0.044), there were no significant differences in changes between each divided group and their counterparts.nnnCONCLUSIONnIn the Japanese obese group with BMI ≥ 25, pitavastatin elicited a significant decrease in fasting blood glucose. It is not clear whether or not this is due to improved insulin resistance as a direct effect of pitavastatin, but in contrast to findings in Caucasians pitavastatin does not worsen insulin resistance in Japanese patients with type 2 diabetes complicated by hypercholesterolemia.

Rat glucagon receptor mRNA is directly regulated by glucose through transactivation of the carbohydrate response element binding protein

The glucagon receptor (Gcgr) is essential for maintaining glucose homeostasis in the liver and for stimulating insulin secretion in pancreatic β-cells. Glucose induces rat Gcgr mRNA expression; however, the precise mechanism remains unknown. We previously have studied the role of the carbohydrate response element binding protein (ChREBP), a glucose-activated transcription factor, in the regulation of glucose-stimulated gene expression. The G-box has previously been reported to be responsible for glucose regulation of Gcgr mRNA expression. The G-box comprises two E-boxes separated by 3bp, which distinguishes it from the carbohydrate response element (ChoRE), which has 5-bp spacing between the two E-boxes. In the rat Gcgr promoter, a putative ChoRE (-554bp/-538bp) is localized near the G-box (-543bp/-529bp). In rat INS-1E insulinoma cells, deletion studies of the rat Gcgr promoter show that ChoRE is a minimal glucose response element. Moreover, reporter assays using a pGL3 promoter vector, which harbors ChoRE and chromatin immunoprecipitation assays reveal that ChoRE is a functional glucose response element in the rat Gcgr promoter. Furthermore, In contrast, glucagon partly suppresses glucose-induced expression of Gcgr mRNA. Thus, ChREBP directly regulates rat Gcgr expression in INS-1E cells. In addition, negative feedback looping between ChREBP and GCGR may further contribute to the regulation of glucose-induced gene expression.