Kazuyuki Hamaguchi

Variations in the FTO gene are associated with severe obesity in the Japanese

AbstractVariations in the fat-mass and obesity-associated gene (FTO) are associated with the obesity phenotype in many Caucasian populations. This association with the obesity phenotype is not clear in the Japanese. To investigate the relationship between the FTO gene and obesity in the Japanese, we genotyped single nucleotide polymorphisms (SNPs) in the FTO genes from severely obese subjects [n = 927, body mass index (BMI) ≥ 30 kg/m2] and normal-weight control subjects (n = 1,527, BMI < 25 kg/m2). A case-control association analysis revealed that 15 SNPs, including rs9939609 and rs1121980, in a linkage disequilibrium (LD) block of approximately 50 kb demonstrated significant associations with obesity; rs1558902 was most significantly associated with obesity. P value in additive mode was 0.0000041, and odds ratio (OR) adjusted for age and gender was 1.41 [95% confidential interval (CI) = 1.22–1.62]. Obesity-associated phenotypes, which include the level of plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure were not associated with the rs1558902 genotype. Thus, the SNPs in the FTO gene were found to be associated with obesity, i.e., severe obesity, in the Japanese.

PDX-1 Induces Insulin and Glucokinase Gene Expressions in αTC1 Clone 6 Cells in the Presence of Betacellulin

The pancreatic β- and α-cells are developmentally related to each other but reveal diverse gene expression patterns. Among the two important transcription factors for insulin gene expression, IEF1 is present both in α- and β-cells, but PDX-1/IPF1/STF-1/IDX-1, a homeodomain-containing transcription factor, is present in β-cells but not in α-cells. To elucidate the function of PDX-1 in the expression of β-cell-specific genes, we established stable αTC1 clone 6 (αTC1.6)-derived transfectants expressing PDX-1 and examined the changes in the gene expression patterns in them. The exogenous expression of PDX-1 in αTC1.6 cells alone could induce islet amyloid polypeptide (IAPP) mRNA expression in the cells but not the expression of insulin, glucokinase, or GLUT2 gene. However, when βcellulin was added to the medium, the PDX-1-expressing αTC1.6 cells, but not the control αTC1.6 cells, came to express insulin and glucokinase mRNAs. This did not occur with other growth factors such as epidermal growth factor, transforming growth factor α, and insulin-like growth factor I. GLUT2 mRNA remained undetectable in the PDX-1–expressing αTC1.6 cells. These observations demonstrate the potency of PDX-1 for the expression of the insulin, glucokinase, and IAPP genes and suggest that certain regulatory factors, which can partially be modified by βcellulin, also contribute to the β-cell specificity of gene expression.

Comparison of cytokine effects on mouse pancreatic alpha-cell and beta-cell lines. Viability, secretory function, and MHC antigen expression.

Cytokine effects on permanent cell lines of transformed mouse pancreatic α- and β-cells were compared. The β–tumor cell 1 (βTC1) line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat insulin II promoter) produced insulin predominantly, although small quantities of intracellular glucagon (100:1 insulin to glucagon) were detectable by radioimmunoassay. The αTC1 line (from an adenoma created in transgenic mice expressing the SV40 large T-antigen oncogene under control of the rat preproglucagon promoter) produced not only glucagon but also considerable quantities of insulin (4:1 glucagon to insulin) and preproinsulin mRNA. We therefore cloned αTC1 cells and obtained 12 glucagon-producing clonai cell lines that did not produce levels of insulin detectable by radioimmunoassay. Analysis by Northern blotting of total RNA from two lines, αTC1 clones 6 and 9, confirmed the absence of preproinsulin mRNA. No somatostatin or pancreatic polypeptide was detected by immunohistochemical staining in αTC1 clones 6 or 9 or βTC1 cells. Rat recombinant γ-interferon (IFN-γ; 5–250 U/ml) or mouse recombinant interleukin 1 (IL-1; 1–25 U/ml) individually inhibited DNA synthesis in βTC1 cells after 3 days of treatment. The two cytokines in combination acted synergistically to further depress DNA synthesis and increase cytotoxicity. In contrast, αTC1 clone 9 cells were not sensitive to inhibition of DNA synthesis by each cytokine individually, although glucagon synthesis was inhibited. The combination of these cytokines caused marked inhibition of DNA and glucagon syntheses in αTC1 clone 9 cells. αTC1 clone 9 cells were somewhat more resistant to the cytotoxic action of the combined cytokines than were βTC1 cells. Incubation with 50 U/ml IFN-γ induced class II MHC molecules (I-Ab, I-Ad, and I-Ed) and enhanced the constitutive expression of class I molecules (H-2Kb and H-2Kd) on the cell surfaces of βTC1, uncloned αTC1, and αTC1 clones 6 and 9. Thus, these cell lines are heterozygous for MHC alleles derived from both parental strains used in the construction of the transgenic mice [C57BL/6J (H-2b) and DBA/2J (H-2d) ]. Class II gene transcription induced by IFN-γ was confirmed in βTC1 and αTC1 clone 9 cells by Northern blot analysis with Aα-, Aβ-, Eα-, and Eβ-DNA probes. The differences in sensitivity to the cytotoxic action of cytokines may relate to the preferential destruction of β-cells in the early stage of insulin-dependent diabetes. Because class I and II MHC expression was enhanced or induced by IFN-γ in both the α- and β-cell lines, the ability to induce MHC molecules in itself is not sufficient to explain β-cell– specific autoimmune recognition associated with insulin-dependent diabetes.

Association between obesity and polymorphisms in SEC16B, TMEM18, GNPDA2, BDNF, FAIM2 and MC4R in a Japanese population

There is evidence that the obesity phenotype in the Caucasian populations is associated with variations in several genes, including neuronal growth regulator 1 (NEGR1), SEC16 homolog B (SCE16B), transmembrane protein 18 (TMEM18), ets variant 5 (ETV5), glucosamine-6-phosphate deaminase 2 (GNPDA2), prolactin (PRL), brain-derived neurotrophic factor (BDNF), mitochondrial carrier homolog 2 (MTCH2), Fas apoptotic inhibitory molecule 2 (FAIM2), SH2B adaptor protein 1 (SH2B1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MAF), Niemann-Pick disease, type C1 (NPC1), melanocortin 4 receptor (MC4R) and potassium channel tetramerisation domain containing 15 (KCTD15). To investigate the relationship between obesity and these genes in the Japanese population, we genotyped 27 single-nucleotide polymorphisms (SNPs) in 14 genes from obese subjects (n=1129, body mass index (BMI) ⩾30 kg m−2) and normal-weight control subjects (n=1736, BMI <25 kg m−2). The SNP rs10913469 in SEC16B (P=0.000012) and four SNPs (rs2867125, rs6548238, rs4854344 and rs7561317) in the TMEM18 gene (P=0.00015), all of which were in almost absolute linkage disequilibrium, were significantly associated with obesity in the Japanese population. SNPs in GNPDA2, BDNF, FAIM2 and MC4R genes were marginally associated with obesity (P<0.05). Our data suggest that some SNPs identified by genome-wide association studies in the Caucasians also confer susceptibility to obesity in Japanese subjects.

Role of fatty acid composition in the development of metabolic disorders in sucrose-induced obese rats.

Fatty acids have been shown to be involved in the development of insulin resistance associated with obesity. We used sucrose loading in rats to analyze changes in fatty acid composition in the progression of obesity and the related metabolic disorder. Although rats fed a sucrose diet for 4 weeks had body weights similar to those of control animals, their visceral fat pads were significantly larger, and serum triglyceride levels were higher; however, neither plasma glucose nor insulin levels were significantly higher. After 20 weeks of sucrose loading, body weight and visceral and subcutaneous fat pads had increased significantly compared with those in control rats. Moreover, plasma glucose, Insulin, and triglyceride levels were significantly higher. An analysis of individual fatty acid components in the blood and peripheral tissues demonstrated phase- and tissue-dependent changes. After 20 weeks of sucrose loading, palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9), the major components of monounsaturated fatty acid, showed a ubiquitous increase in plasma and all tissues analyzed. In contrast, linoleic acid (18:2 n-6) and arachidonic acid (20:4 n-6), the major components of polyunsaturated fatty acid in the n-6 family, decreased in plasma and all tissues analyzed. After 4 weeks of sucrose loading, these changes in fatty acid composition were observed only in the liver and plasma and not in fat and muscle. This led us to conclude that elevation of plasma glucose and insulin develop at the late phase of sucrose-induced obesity, when changes in fatty acid composition appear in fat and muscle. Furthermore, changes in fatty acid composition in liver seen after 4 weeks of sucrose loading, when increases in neither plasma glucose nor insulin were detected, suggest that liver may be the initial site of fatty acid imbalance and that aberrations in hepatic fatty acid composition may lead to fatty acid imbalances in other tissues.

Association of variations in the FTO , SCG3 and MTMR9 genes with metabolic syndrome in a Japanese population

Metabolic syndrome is defined as a cluster of multiple risk factors, including central obesity, dyslipidemia, hypertension and impaired glucose tolerance, that increase cardiovascular disease morbidity and mortality. Genetic factors are important in the development of metabolic syndrome, as are environmental factors. However, the genetic background of metabolic syndrome is not yet fully clarified. There is evidence that obesity and obesity-related phenotypes are associated with variations in several genes, including NEGR1, SEC16B, TMEM18, ETV5, GNPDA2, BDNF, MTCH2, SH2B1, FTO, MAF, MC4R, KCTD15, SCG3, MTMR9, TFAP2B, MSRA, LYPLAL1, GCKR and FADS1. To investigate the relationship between metabolic syndrome and variations in these genes in the Japanese population, we genotyped 33 single-nucleotide polymorphisms (SNPs) in 19 genes from 1096 patients with metabolic syndrome and 581 control individuals who had no risk factors for metabolic syndrome. Four SNPs in the FTO gene were significantly related to metabolic syndrome: rs9939609 (P=0.00013), rs8050136 (P=0.00011), rs1558902 (P=6.6 × 10−5) and rs1421085 (P=7.4 × 10−5). rs3764220 in the SCG3 gene (P=0.0010) and rs2293855 in the MTMR9 gene (P=0.0015) were also significantly associated with metabolic syndrome. SNPs in the FTO, SCG3 and MTMR9 genes had no SNP × SNP epistatic effects on metabolic syndrome. Our data suggest that genetic variations in the FTO, SCG3 and MTMR9 genes independently influence the risk of metabolic syndrome.

INSIG2 gene rs7566605 polymorphism is associated with severe obesity in Japanese.

AbstractThe single nucleotide polymorphism (SNP) rs7566605 in the upstream region of the insulin-induced gene 2 (INSIG2) is associated with the obesity phenotype in many Caucasian populations. In Japanese, this association with the obesity phenotype is not clear. To investigate the relationship between rs7566605 and obesity in Japanese, we genotyped rs7566605 from severely obese subjects [n = 908, body mass index (BMI) ≥ 30 kg/m2] and normal-weight control subjects (n = 1495, BMI < 25 kg/m2). A case-control association analysis revealed that rs7566605 was significantly associated with obesity in Japanese. The P value in the minor allele recessive mode was 0.00020, and the odds ratio (OR) adjusted for gender and age was 1.61 [95% confidential interval (CI) = 1.24-2.09]. Obesity-associated phenotypes, which included the level of BMI, plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure, were not associated with the rs7566605 genotype. Thus, rs7566605 in the upstream region of the INSIG2 gene was found to be associated with obesity, i.e., severe obesity, in Japanese.

Cell Adhesion Molecule 1 Is a Novel Pancreatic–Islet Cell Adhesion Molecule That Mediates Nerve–Islet Cell Interactions

BACKGROUND & AIMS Cell adhesion molecule 1 (CADM1), mediates nerve-mast cell attachment and communication through homophilic binding. An immunohistochemical screen showed that CADM1 is expressed in pancreatic islets. Here, we determined the cell types expressing CADM1 and examined its role in nerve-islet cell interactions. METHODS Immunohistochemistry and double-staining immunofluorescence were performed on murine and human pancreases and on islet cell tumors (ICTs). alphaTC6 cells, a murine alpha cell line, were cultured on neurite networks of superior cervical ganglia. Neurite-alphaTC6 cell attachment and communication were examined after nerves were activated specifically by scorpion venom. RESULTS CADM1 was expressed on the plasma membrane in all 4 major types of islet cells, alpha, beta, D, and pancreatic polypeptide in human beings, but primarily in alpha cells in mice. In cocultures, alphaTC6 cell to neurite attachment was inhibited dose-dependently by an anti-CADM1 function-blocking antibody. In response to scorpion venom-evoked nerve activation, 36% of the alphaTC6 cells mobilized Ca(2+), and introduction of a CADM1-targeting small interfering RNA reduced the fraction of responding cells to 7%. In 21 human ICTs, CADM1 was present in the plasma membrane of 7, and the others were negative for CADM1. Six of the CADM1-expressing tumors were functional hormonally, whereas all but 2 of the CADM1-negative tumors were nonfunctional (P = .0032). CONCLUSIONS CADM1 is a novel islet cell adhesion molecule mediating nerve-islet cell interactions. The strong correlation between CADM1 expression and hormonally functional phenotypes suggests that CADM1 is involved in hormone secretion from ICTs.

Polymorphisms in NRXN3, TFAP2B, MSRA, LYPLAL1, FTO and MC4R and their effect on visceral fat area in the Japanese population.

The predominant risk factor of metabolic syndrome is intra-abdominal fat accumulation, which is determined by waist circumference and waist–hip ratio measurements and visceral fat area (VFA) that is measured by computed tomography (CT). There is evidence that waist circumference and waist–hip ratio in the Caucasian population are associated with variations in several genes, including neurexin 3 (NRXN3), transcription factor AP-2β (TFAP2B), methionine sulfoxide reductase A (MSRA), lysophospholipase-like-1 (LYPLAL1), fat mass and obesity associated (FTO) and melanocortin 4 receptor (MC4R) genes. To investigate the relationship between VFA and subcutaneous fat area (SFA) and these genes in the recruited Japanese population, we genotyped 8 single-nucleotide polymorphisms (SNPs) in these 6 genes from 1228 subjects. Multiple regression analysis revealed that gender, age, and rs1558902 and rs1421085 genotypes (additive model) in FTO were significantly associated with body mass index (BMI; P=0.0039 and 0.0039, respectively), SFA (P=0.0027 and 0.0023, respectively) and VFA (P=0.045 and 0.040, respectively). However, SNPs in other genes, namely, NRXN3, TFAP2B, MSRA, LYPLAL1 and MC4R were not significantly associated with BMI, SFA or VFA. Our data suggest that some SNPs, which were identified in genome-wide studies in the Caucasians, also confer susceptibility to fat distribution in the Japanese subjects.

Novel mutations of the ATP7B gene in Japanese patients with Wilson disease

AbstractWilson disease (WD) is an autosomal recessive disorder characterized by copper accumulation in the liver, brain, kidneys, and corneas, and culminating in copper toxication in these organs. In this study, we analyzed mutations of the responsible gene, ATP7B, in four Japanese patients with WD. By direct sequencing, we identified five mutations, of which two were novel, and 16 polymorphisms, of which 6 were novel. The mutations 2871delC and 2513delA shift the reading frame so that truncated abnormal protein is expected. In contrast to these mutations found in patients with hepatic-type of early onset, the mutations A874V, R778L, and 3892delGTC were either missense mutations or inframe 1-amino acid deletion, and occurred in the patients with hepato-neurologic type of late onset. The mutations 2871delC and R778L have been previously reported in a relatively large number of Japanese patients. In particular, R778L is known to be more prevalent in Asian countries than in other countries of the world. Our data are compatible with the hypothesis that the mutations tend to occur in a population-specific manner. Therefore, the accumulation of the types of mutations in Japanese patients with WD will facilitate the fast and effective genetic diagnosis of WD in Japanese patients.