GLP-1-related proteins attenuate the effects of mitochondrial membrane damage in pancreatic ß cells.

Glucagon-like peptide (GLP)-1 analog based therapies are used not only for their insulinotropic effects, but also for their pleiotropic effects that improve pancreatic ß cell function. Liraglutide is a long acting derivative of human GLP-1(7-37), which is a cleavage product encompassing amino acids 7-37 of GLP-1. In this study, we examined whether Liraglutide treatment restore the glucose-stimulated mitochondrial response of ß cells with chemically induced mitochondrial damage. We tested three GLP-1-related proteins: human GLP-1(1-37), GLP-1(7-37) and Liraglutide. To measure changes of the mitochondrial pH quantitatively in real-time, we have developed a bioengineered ß cell line. We generated a mitochondrial damaged model by treating ß cells with ethidium bromide (EtBr; 0.5 or 1 µg/mL for 48 h). EtBr treatment reduced the response to 25 mM glucose in mitochondrial pH in a dose- and time-dependent manner. GLP-1(7-37) (100 nM) enhanced the response of mitochondria to glucose stimulation in undamaged ß cells. Preincubation with Liraglutide (1 nM) or GLP-1 (100 nM) for 3h recovered the mitochondrial response to glucose in damaged ß cells, however, GLP-1(7-37) (100 nM) did not. When GLP-1(7-37) was administered in stepwise increments (i.e., starting with 20 nM to reach 100 nM in 3h), similar recovery of the mitochondrial function was observed. The results suggest that Liraglutide is effective to recover glucose-stimulated mitochondrial response in damaged ß cells.

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.

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.

Low resting energy expenditure in postmenopausal Japanese women with type 2 diabetes mellitus.

OBJECTIVE: Resting energy expenditure (REE) is an important tool in nutrition management, especially in type 2 diabetes mellitus (T2DM). The predicted REE (pREE) was reported to be inaccurate, compared with measured REE (mREE) in Japanese T2DM patients. Despite the accuracy of REE, measured via indirect calorimetry (mREE), the technique is demanding. This study evaluated the associated clinical factors of the difference between pREE and mREE in Japanese patients with T2DM. METHODS: Forty-nine Japanese patients with T2DM but no severe complications (32 men and 17 women) were enrolled. mREE was determined via indirect calorimetry. RESULTS: Participants average age was 56.3 ± 11.0 years, body mass index was 25.2 ± 3.6 kg/m2, and HbA1c was 9.6 ± 1.6%. The mean mREE was 1099 ± 212 kcal/day. Age, body mass index, hemoglobin, and uric acid levels were all associated with mREE by simple regression; of these, body weight was the significant factor in the multiple regression analysis. When the patients were divided into tertiles, the average mREE values were lower than the pREE values for each group. The difference between mREE and pREE was largest in the lowest value group, whose subjects were mostly women aged over 50 years. This group of women showed significantly lower mREE (904 ± 121 kcal) in comparison with men in the same age group, with 26% overestimation of pREE, even when the equation that yielded the closest mREE value was used. CONCLUSION: The previously reported pREE overestimates mREE in Japanese patients with T2DM, especially in postmenopausal women.

Alterations of serum Th1 and Th2 chemokines by combination therapy of interferon-gamma and narrowband UVB in patients with mycosis fungoides.

BACKGROUND: Mycosis fungoides (MF) is a T cell neoplasm with elevation of serum Th2 chemokines. Although interferon-gamma (IFN-gamma) administration and narrowband-UVB (NB-UVB) phototherapy are used for the treatment of MF, a combination therapy of these two modalities is not fully established. OBJECTIVES: To define whether the combination of IFN-gamma and NB-UVB affects the balance of serum levels of Th1 and Th2 chemokines in patients with MF. METHODS: Twelve patients with MF received intravenous or intramuscular injections of recombinant IFN-gamma (rIFN-gamma) or natural IFN-gamma (nIFN-gamma) in combination with NB-UVB phototherapy. As control, three MF patients were treated with NB-UVB monotherapy. At the beginning and cessation of therapy, the concentrations of serum Th2 chemokines, TARC/CCL17 and MDC/CCL22, and Th1 chemokines, IP-10/CXCL10 and MIG/CXCL9 were measured by ELISA. RESULTS: Before treatment, not only Th2 chemokines but also Th1 chemokines were elevated in the patients. Whereas no significant changes were observed in the levels of TARC and MDC, IP-10 and MIG were further elevated by the combination of IFN-gamma and NB-UVB. On the other hand, NB-UVB monotherapy did not change the level of either Th1 or Th2 chemokine. CONCLUSIONS: The combination of IFN-gamma and NB-UVB elevated serum Th1 chemokines but unaffected Th2 chemokines. Since NB-UVB monotherapy could not affect the chemokine levels, the effect of the combination therapy is attributable to IFN-gamma. Given the role of Th1 chemokines for tumor-attacking T cell recruitment at the early stage of MF, the therapy may exert a beneficial effect for early MF.

Aberrant aquaporin 5 expression in the sweat gland in aquagenic wrinkling of the palms.

Aquagenic wrinkling of the palms (AWP) is an uncommon disease characterized by the rapid and transient formation of edematous whitish plaques on the palms on exposure to water. Although this disease is occasionally accompanied by hyperhidrosis, the pathophysiology of AWP remains unknown. Herein we describe a patient with AWP. The location of wrinkling was limited to the areas positive for iodine-starch test after water exposure, which suggests that AWP is etiologically related to hyperhidrosis. Histologic examination revealed hyperplastic and papillated eccrine glandular epithelium with the enlarged diameter of eccrine coils. Immunohistochemically, while aquaporin 5 (AQP5), one of the water channel AQP families, was present exclusively in the dark cells of sweat glands of healthy donors, an aberrant AQP5 staining, extending to the clear cells, was found in the patient with AWP. The hyperplastic glandular epithelium and aberrant AQP5 staining in the patient's sweat glands suggest that AWP stems from dysregulation of sweating.

Role of vitamin D in energy and bone metabolism in postmenopausal women with type 2 diabetes mellitus: A 6-month follow-up evaluation.

AIMS/INTRODUCTION: Resting energy expenditure was associated with a serum bone turnover marker in postmenopausal women with type 2 diabetes (T2DMPW) in the present cross-sectional study. To clarify the fundamental pathological factor for the correlation of bone metabolism and basal metabolism in type 2 diabetes, a 6-month prospective follow-up study was carried out with supplementation of vitamin D. MATERIALS AND METHODS: A total of 44 T2DMPW were enrolled. The following factors were evaluated at the beginning and the end of the summer: procollagen type 1 N-terminal propeptide, carboxy-terminal collagen crosslinks-1, intact parathyroid hormone and 25-hydroxyvitamin D (25[OH]D), as well as diabetic complications, body composition, respiratory quotient and resting energy expenditure. A total of 23 patients with low 25(OH)D levels (˂20 ng/mL) were instructed to increase vitamin D levels by lifestyle change. Among them, 15 patients with osteoporosis were also administered alfacalcidol. RESULTS: Serum 25(OH)D increased in 25 patients and decreased in 19 patients. Patients who did not receive the study intervention at the start tended to have a decreased 2525(OH)D level; therefore, the average 25(OH)D level of all patients was not changed. Changes in resting energy expenditure were positively correlated with those of procollagen type 1 N-terminal propeptide/carboxy-terminal collagen crosslinks-1. Changes in the respiratory quotient correlated with the mean glycated hemoglobin levels; procollagen type 1 N-terminal propeptide levels positively correlated with serum 25(OH)D after the intervention. These correlations were prominent in patients with increased 25(OH)D and those with alfacalcidol supplementation. CONCLUSIONS: Restoration of vitamin D level might be a prerequisite for a normal correlation between bone and basal metabolism in T2DMPW. Lifestyle intervention for retention of vitamin D level is important even in summer, in T2DMPW.

Pancreatic developmental defect evaluated by celiac artery angiography in a patient with MODY5.

The hepatocyte nuclear factor 1ß gene (HNF1B) is responsible for maturity-onset diabetes of the young type 5 (MODY5), which is characterized by early-onset diabetes mellitus and urogenital malformations. HNF1B is expressed during visceral endoderm formation. We identified a disruption of the great pancreatic artery in a patient with MODY5 with no pancreatic body or tail. Our finding supports the significance of HNF1B in the development of the pancreas.

Mapping genes influencing type 2 diabetes risk and BMI in Japanese subjects.

We have carried out an autosomal genome scan for genes contributing to the development of type 2 diabetes and affecting BMI in the Japanese population (164 families, 256 affected sib-pairs). We found 12 regions that showed nominally significant multipoint evidence of linkage with type 2 diabetes (i.e. logarithm of odds [LOD] score >0.59, P < 0.05): chromosome 1 29.9 cM; chromosome 2 169.6 and 236.8 cM; chromosome 4 104.9 cM; chromosome 5 114.8 cM; chromosome 6 42.3 cM; chromosome 8 15.3 and 93.3 cM; chromosome 9 140.0 cM; chromosome 11 131.6 cM; chromosome 17 36.1 cM; and chromosome 21 48.0 cM. Twelve regions showed nominal multipoint evidence for linkage with log-transformed BMI (lnBMI): chromosome 2 167.9 and 210.5 cM; chromosome 3 185.7 cM; chromosome 4 118.9 and 145.6 cM; chromosome 5 131.9 cM; chromosome 7 7.4 cM; chromosome 10 70.0 cM; chromosome 15 12.8 cM; chromosome 16 30.0 cM; and chromosome 17 47.8 and 100.2 cM. Although none of the regions achieved genome-wide levels of significance, simulation studies showed that we observed more linkage signals than expected if there were no loci contributing to type 2 diabetes or BMI. Eight of the regions showing nominal evidence for linkage with type 2 diabetes have been reported in other genome scans, and seven of the regions showing linkage with lnBMI have shown linkage with BMI and BMI-related traits in other studies. Thus, our results may replicate findings in other studies. They may also indicate new regions of the genome that are involved in the regulation of blood glucose levels or body weight.

Association between basal metabolic function and bone metabolism in postmenopausal women with type 2 diabetes.

OBJECTIVE: Diabetes is a risk factor for osteoporosis, and glycemic control is critical during osteoporosis treatment in patients with type 2 diabetes (T2D). However, diabetic therapies have potentially adverse effects on bone metabolism. Additionally, biomarkers for bone metabolism are directly affected by drug therapies for osteoporosis. This study examined resting energy expenditure (REE) and respiratory quotient (RQ) as indices of bone metabolism in postmenopausal Japanese women with T2D. METHODS: Forty-six postmenopausal Japanese women with T2D were examined. Procollagen type 1 N-terminal propeptide (P1NP, a fasting serum bone formation marker) and carboxy-terminal collagen cross-links-1 (CTX-1, a resorption marker) were evaluated, along with intact parathyroid hormone, 25-hydroxyvitamin D (25[OH]D), urine microalbumin, motor nerve conduction velocity, sensory nerve conduction velocity, R-R interval, body composition, REE, RQ, and bone mineral density at the nondominant distal radius. RESULTS: The mean T-score was low with high variance (-1.7 ± 1.6), and 18 patients (39%) met the criteria for osteoporosis. REE was positively correlated with body mass index (ß = 0.517; r(2) = 0.250), serum calcium (ß = 0.624; r(2) = 0.200), glycated hemoglobin A1C for the previous 6 mo (ß = 0.395; r(2) = 0.137), and the serum P1NP/CTX-1 ratio (ß = 0.380; r(2) = 0.144). RQ was positively correlated with serum 25(OH)D (ß = 0.387; r(2) = 0.131). CONCLUSION: The basal metabolic rate and diabetic pathophysiology are interrelated with bone turnover.

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).

Genetic variants in the calpain-10 gene and the development of type 2 diabetes in the Japanese population.

Variation in the gene encoding the cysteine protease calpain-10 has been linked and associated with risk of type 2 diabetes. We have examined the effect of three polymorphisms in the calpain-10 gene (SNP-43, Indel-19, and SNP-63) on the development of type 2 diabetes in the Japanese population in a pooled analysis of 927 patients and 929 controls. We observed that SNP-43, Indel-19, and SNP-63 either individually or as a haplotype were not associated with altered risk of type 2 diabetes with the exception of the rare 111/221 haplogenotype (odds ratio (OR) =3.53, P=0.02). However, stratification based on the median age-at-diagnosis in the pooled study population (<50 and > or =50 years) revealed that allele 2 of Indel-19 and the 121 haplotype were associated with reduced risk in patients with later age-at-diagnosis (age-at-diagnosis > or =50 years OR=0.82 and 0.80, respectively; P=0.04 and 0.02). Thus, variation in the calpain-10 gene may affect risk of type 2 diabetes in Japanese, especially in older individuals.

Nuclear translocation of SHP and visualization of interaction with HNF-4alpha in living cells.

Mutations in small heterodimer partner (SHP) and hepatocyte nuclear factor 4alpha (HNF4alpha) are associated with mild obesity and diabetes mellitus, respectively. Both receptors work together to determine the normal pancreatic beta-cell function. We examined their subcellular localization and interaction in living cells by tagging them with yellow and cyan variants of green fluorescent protein (GFP) variants. Expressed SHP resided only in the cytoplasm in COS-7 cells which lacks HNF4alpha, but predominantly in the nucleus in insulinoma cells (MIN6). HNF4alpha was localized exclusively in the nuclei of both cells, coexpressed with HNF4alpha in COS-7 cells, redistributed in the nucleus, depending on the amount of HNF4alpha. We found fluorescence resonance energy transfer between GFP-tagged SHP and HNF4alpha, indicating a specific close association between them in the nucleus. The results strongly suggest that SHP exists primarily in the cytoplasm and is translocated into the nucleus on interacting with its nuclear receptor partner HNF4alpha.