Mitsuo Itakura

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.

Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

Hypoplasia of pancreatic islets in transgenic mice expressing activin receptor mutants.

Activin, a member of the TGF-beta superfamily, regulates the growth and differentiation of a variety of cell types. Based on the expression of activin in pancreatic rudiments of rat embryos and stimulation of insulin secretion from adult rat pancreatic islets by activin, activin is implicated in the development and function of islets. To examine the significance of activin signaling in the fetal and postnatal development of islets, transgenic mice expressing a dominant negative form of activin receptor (dn-ActR) or a constitutively active form of activin receptor (ActR-T206D) in islets were generated together with the transgenic mice expressing intact activin receptor (intact ActR) as a negative control. Transgenic mice with both dn-ActR and ActR-T206D showed lower survival rates, smaller islet area, and lower insulin content in the whole pancreas with impaired glucose tolerance when compared with transgenic mice with intact ActR or littermates, but they showed the same alpha cell/beta cell ratios as their littermates. In addition to islet hypoplasia, the insulin response to glucose was severely impaired in dn-ActR transgenic mice. It is suggested that a precisely regulated intensity of activin signaling is necessary for the normal development of islets at the stage before differentiation into alpha and beta cells, and that activin plays a role in the postnatal functional maturation of islet beta cells.

Rare mutations of the Gs alpha subunit gene in human endocrine tumors. Mutation detection by polymerase chain reaction—primer-introduced restriction analysis

Background. The Gs alpha (Gsα) gene can be activated to the putative oncogene gsp by specific point mutations at codons 201 or 227. Such mutations have been reported in growth hormone (GH)‐secreting pituitary adenomas and thyroid tumors. To clarify the role of Gsα gene in human endocrine tumors, 197 tumors were screened for point mutations at codons 201 or 227 of the Gsα gene.

Prevention of adoptively transferred diabetes in nonobese diabetic mice with IL-10-transduced islet-specific Th1 lymphocytes. A gene therapy model for autoimmune diabetes.

Four pancreatic islet-specific CD4+ helper T (Th) 1 (Th1) clones and two Th1 clones transduced with an SRalpha promoter-linked murine IL-10 (mIL-10) cDNA of 2.0-6.0 x 10(6) cells were adoptively transferred to nonobese diabetic (NOD) mice at age 8 d. Cyclophosphamide (CY) was administered at age 37 d (plus CY), and the incidence of diabetes and the histological grade of insulitis were examined at age 47 d. After the adoptive transfer of IL-10-transduced Th1 cells, polymerase chain reaction (PCR) and reverse-transcription (RT)-PCR detected the neo gene and the retrovirus vector-mediated IL-10 mRNA in situ in recipient islets, respectively. RT-PCR detected the decrease of IFN-gamma mRNA relative to IL-10 mRNA in IL-10-transduced Th1 clones in vitro and also in recipient islets. All four wild type Th1 clones plus CY induced the insulitis grade of 2.75 and diabetes in 66% of recipient NOD mice. IL-10-transduced two Th1 clones plus CY induced periinsulitis with the grade of 1.43 and diabetes in 8.0%. The 1:1 mixture of wild type Th1 cells and IL-10-transduced Th1 cells plus CY induced periinsulitis with the grade of 1.85 and diabetes in 20%. The suppression of diabetes through decreasing IFN-gamma mRNA by the tissue-specific delivery of IL-10 to pancreatic islets with IL-10-transduced Th1 cells affords us the starting basis to develop the gene therapy for autoimmune diabetes.

The Novel Gene Encoding a Putative Transmembrane Protein Is Mutated in Gnathodiaphyseal Dysplasia (GDD)

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.

Central role for aldose reductase pathway in myocardial ischemic injury

Aldose reductase (AR), a member of the aldo‐keto reductase family, has been implicated in the development of vascular and neurological complications of diabetes. Recently, we demonstrated that aldose reductase is a component of myocardial ischemic injury and that inhibitors of this enzyme protect rat hearts from ischemia‐reperfusion injury. To rigorously test the effect of aldose reductase on myocardial ischemia‐reperfusion injury, we used transgenic mice broadly overexpressing human aldose reductase (ARTg) driven by the major histocompatibility complex I promoter. Hearts from these ARTg or littermate mice (WT) (n=6 in each group) were isolated, perfused under normoxic conditions, then subjected to 50 min of severe low flow ischemia followed by 60 min of reperfusion. Creatine kinase (CK) release (a marker of ischemic injury) was measured during reperfusion; left ventricular developed pressure (LVDP), end diastolic pressure (EDP), and ATP were measured throughout the protocol. CK release was significantly greater in ARTg mice compared with the WT mice. LVDP recovery was significantly reduced in ARTg mice compared with the WT mice. Furthermore, ATP content was higher in WT mice compared with ARTg mice during ischemia and reperfusion. Infarct size measured by staining techniques and myocardial damage evaluated histologically were also significantly worse in ARTg mice hearts than in controls. Pharmacological inhibition of aldose reductase significantly reduced ischemic injury and improved functional recovery in ARTg mice. These data strongly support key roles for AR in ischemic injury and impairment of functional and metabolic recovery after ischemia. We propose that interventions targeting AR may provide a novel adjunctive approach to protect ischemic myocardium.—Hwang, Y. C., Kaneko, M., Bakr, S., Liao, H., Lu, Y., Lewis, E. R., Yan, S., Ii, S., Itakura, M., Rui, L., Skopicki, H., Homma, S., Schmidt, A. M., Oates, P. J., Szabolcs, M., Ramasamy, R. Central role for aldose reductase pathway in myocardial ischemic injury. FASEB J. 18, 1192–1199 (2004)

Overexpression of HMGA2 relates to reduction of the let-7 and its relationship to clinicopathological features in pituitary adenomas

High-mobility group A2 is highly expressed during embryogenesis and in various benign and malignant tumors. Recent studies report that high-mobility group A2 is negatively regulated by the let-7 microRNAs (miRNAs) family in vitro. The development of pituitary adenomas in high-mobility group A2 transgenic mice showed that high-mobility group A2 may be involved in pituitary tumorigenesis. However, no studies have investigated the clinical significance of high-mobility group A2 and its relationship to the let-7 miRNA family in human pituitary adenomas. Using immunohistochemistry, we analyzed high-mobility group A2 expression with respect to various clinicopathologic factors in 98 pituitary adenomas. Overexpression of high-mobility group A2 was observed in 39% (38/98) of pituitary adenomas compared with normal adenohypophysial tissue and was frequently found in adenomas including prolactin (PRL), adrenocorticotrophic hormone, or follicle-stimulating hormone/luteinizing hormone and in null cell adenomas, but relatively rare in growth hormone (GH) and mixed GH/PRL adenomas. High-mobility group A2 expression was significantly associated with tumor invasion (P<0.05) and was significantly higher in grade IV than in grades I, II, and III adenomas (P<0.05). High levels of high-mobility group A2 expression were more frequently observed in macroadenomas than in microadenomas (P<0.05). High levels of high-mobility group A2 expression also significantly correlated with the proliferation marker Ki-67 (P<0.0001). Real-time quantitative RT-PCR analysis was carried out to evaluate the expression of let-7 in 55 pituitary adenomas. Subsequently, decreased expression of let-7 was confirmed in 23 of 55 (42%) adenomas and was correlated with high-grade tumors (P<0.05). An inverse correlation between let-7 and high-mobility group A2 expression was evident (R=−0.33, P<0.05). These findings support a causal link between let-7 and high-mobility group A2 whereby loss of let-7 expression induces high-mobility group A2 upregulation that represents an important mechanism in pituitary tumorigenesis and progression.

A missense mutant myostatin causes hyperplasia without hypertrophy in the mouse muscle

Myostatin, which is a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle formation. Double-muscled Piedmontese cattle have a C313Y mutation in myostatin and show increased skeletal muscle mass which resulted from an increase of myofiber number (hyperplasia) without that of myofiber size (hypertrophy). To examine whether this mutation in myostatin gene affects muscle development in a dominant negative manner, we generated transgenic mice overexpressing the mutated gene. The transgenic mice exhibited dramatic increases in the skeletal muscle mass resulting from hyperplasia without hypertrophy. In contrast, it has been reported that a myostatin mutated at its cleavage site produces hypertrophy without hyperplasia in the muscle. Thus, these results suggest that (1) the myostatin containing the missense mutation exhibits a dominant negative activity and that (2) there are two types in the dominant negative form of myostatin, causing either hypertrophy or hyperplasia.

Abrogation of autoimmune diabetes in nonobese diabetic mice and protection against effector lymphocytes by transgenic paracrine TGF-beta1.

Paracrine effect of transforming growth factor-beta1 (TGF-beta1) on autoimmune insulitis and diabetes was studied by transgenic production of the active form of porcine TGF-beta1 (pTGF-beta1) in pancreatic islet (islet) alpha cells in nonobese diabetic (NOD) mice under the control of rat glucagon promoter (RGP) (NOD-RGP-TGF-beta1). None of 27 NOD-RGP-TGF- beta1 mice developed diabetes by 45 wk of age, in contrast to 40 and 71% in male and female nontransgenic mice, respectively. None of the NOD-RGP-TGF-beta1 mice developed diabetes after cyclophosphamide (CY) administration. Adoptive transfer of splenocytes of NOD-RGP-TGF-beta1 mice to neonatal NOD mice did not transfer diabetes after CY administration. Adoptive transfer of three types of diabetogenic lymphocytes to NOD-RGP-TGF-beta1 and nontransgenic mice after CY administration led to the lower incidence of diabetes in NOD-RGP-TGF-beta1 mice versus that in nontransgenic mice: 29 vs. 77% for diabetogenic splenocytes, 25 vs. 75% for islet beta cell-specific Th1 clone cells, and 0 vs. 50% for islet beta cell-specific CD8(+) clone cells, respectively. Based on these, it is concluded that autoimmune diabetes in NOD mice is not a systemic disease and it can be completely prevented by the paracrine TGF-beta1 in the islet compartment through protection against CD4(+) and CD8(+) effector lymphocytes.