A new mitochondrial pH biosensor for quantitative assessment of pancreatic ß-cell function.

Mitochondrial pH is a key determinant of mitochondrial energy metabolism. We have developed a new fluorescence-based ratiometric pH biosensor using a chloride-insensitive and hydrogen-sensitive probe for direct, quantitative and bleaching-free measurement in a living cell. Fusing this biosensor with a mitochondrial localization signal (MTpHGV) allowed us to determine mitochondrial pH. This new system was applied to measure mitochondrial pH in pancreatic ß-cells, in which mitochondrial function plays a pivotal role in insulin secretion. Rat INS1 cells and mouse MIN6 cells are transfected with MTpHGV stably to monitor mitochondrial pH. While carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment rapidly decreased mitochondrial pH in cultured rat MTpHGV-INS-1 cells, MTpHGV-MIN6 cells showed a rapid increase. These data suggest that MTpHGV probe exist in matrix side in INS-1 cells, but on the outer side of mitochondrial inner membrane in MIN6 cells. Moreover, while MTpHGV-INS-1 cells showed a rapid increase of pH by glucose stimulation, mitochondrial pH decreased quickly by glucose stimulation in all MTpHGV-MIN6 cells examined and recovered smoothly. Perfusion study of glucose load in MTpHGV-MIN6 cells under aminooxyacetate (AOA) or 100µM diazoxide showed that this mitochondrial pH acidification was dependent on nicotinamide adenine dinucleotide (NADH) shuttle, but independent from KATP channel. This new system for measuring mitochondrial pH is sensitive across the range of physiologic conditions and may be a useful tool for evaluating mitochondrial function in living cells.

Localization of hepatocyte nuclear factor-4α in the nucleolus and nucleus is regulated by its C-terminus.

UNLABELLED: Aims/Introduction: Mutations in hepatocyte nuclear factor-4α (HNF4α) lead to various diseases, among which C-terminal deletions of HNF4α are exclusively responsible for maturity onset diabetes of the young 1 (MODY1). MODY is an autosomal dominant disease characterized by a primary defect in insulin response to glucose, suggesting that the C-terminus of HNF4α is important for pancreatic ß-cell function. To clarify the role of the C-terminus of HNF4α, changes in cellular localization and the binding ability to its regulator were examined, specifically in the region containing Q268, which deletion causes MODY1. MATERIALS AND METHODS: Cellular localization of mutant HNF4α were examined in monkey kidney 7 (COS7), Chinese hamster ovary, rat insulinoma and mouse insulinoma cells, and their binding activity to other proteins were examined by fluorescence resonance energy transfer (FRET) in COS7 cells. RESULTS: Although wild-type HNF4α was localized in the nucleoplasm in transfected cultured cells, Q268X-HNF4α was located predominantly in the nucleolus. Deletion analysis of the C-terminus of HNF4α showed that the S337X-HNF4α mutant, and other mutants with shorter amino acid sequences (S337-K194), were mostly localized in the nucleolus. HNF4α mutants with amino acid sequences shorter than the W192X-HNF4α mutant gradually spread to the nucleoplasm in accordance with their lengths. The A250X-HNF4α mutant was capable of causing the accumulation of HNF4α or the small heterodimer partner (SHP), one of the HNF4α regulators, in the nucleolus. However, the R154X-HNF4α mutant did not have binding ability to wild-type HNF4α or SHP, and thus was seen in the nucleus. CONCLUSIONS: The C-terminus sites might play a key role in facilitating the nucleolar and subnucleolar localization of HNF4α. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00210.x, 2012).

Identification of KCNJ15 as a susceptibility gene in Asian patients with type 2 diabetes mellitus.

Recent advances in genome research have enabled the identification of new genomic variations that are associated with type 2 diabetes mellitus (T2DM). Via fine mapping of SNPs in a candidate region of chromosome 21q, the current study identifies potassium inwardly-rectifying channel, subfamily J, member 15 (KCNJ15) as a new T2DM susceptibility gene. KCNJ15 is expressed in the beta cell of the pancreas, and a synonymous SNP, rs3746876, in exon 4 (C566T) of this gene, with T allele frequency among control subjects of 3.1%, showed a significant association with T2DM affecting lean individuals in three independent Japanese sample sets (p = 2.5 x 10(-7), odds ratio [OR] = 2.54, 95% confidence interval [CI] = 1.76-3.67) and with unstratified T2DM (p = 6.7 x 10(-6), OR = 1.76, 95% CI = 1.37-2.25). The diabetes risk allele frequency was, however, very low among Europeans in whom no association between this variant and T2DM could be shown. Functional analysis in human embryonic kidney 293 cells demonstrated that the risk allele of the synonymous SNP in exon 4 increased KCNJ15 expression via increased mRNA stability, which resulted in the higher expression of protein as compared to that of the nonrisk allele. We also showed that KCNJ15 is expressed in human pancreatic beta cells. In conclusion, we demonstrated a significant association between a synonymous variant in KCNJ15 and T2DM in lean Japanese patients with T2DM, suggesting that KCNJ15 is a previously unreported susceptibility gene for T2DM among Asians.

Hepatic steatosis rather than visceral adiposity is more closely associated with insulin resistance in the early stage of obesity.

The aim of the present study was to investigate the specific relationship between hepatic steatosis and insulin resistance in the early stage of obesity. Among general health examinees who received an ultrasound scanning, 131 subjects without fatty liver (non-FL group) and 142 subjects with fatty liver (FL group) were selected so that both groups were matched for age, sex, body mass index, and % body fat. The FL group was then subdivided into 2 groups according to the severity of steatosis by ultrasound. Insulin resistance was assessed by homeostasis model assessment for insulin resistance, serum high-molecular-weight (HMW) adiponectin, and insulin-like growth factor binding protein 1 concentrations. Unexpectedly, the non-FL group showed higher waist circumference than the FL group. Nevertheless, homeostasis model assessment for insulin resistance as well as conventional insulin resistance indexes such as serum insulin, free fatty acid, and triglyceride levels demonstrated a stepwise increase, and HMW adiponectin and insulin-like growth factor binding protein 1 demonstrated a stepwise decrease with increasing degree of hepatic steatosis. Overall, insulin resistance markers correlated with obesity indexes, but only HMW adiponectin no longer showed any meaningful correlation in the presence of fatty liver. The prevalence of BP, fasting serum glucose, and high-density lipoprotein cholesterol above or below cutoff points and subjects having 2 or more metabolic syndrome components were higher in the moderate to severe FL group compared to the non-FL group. In conclusion, these results in nondiabetic and relatively normal-body mass index subjects suggest that hepatic steatosis is independently associated with insulin resistance regardless of extrahepatic adiposity and might be the earliest event in pathogenesis of the metabolic syndrome.