Michika Shigemoto

Human Obese Gene Expression: Adipocyte-Specific Expression and Regional Differences in the Adipose Tissue

The obese (ob) gene, the mutation of which results in severe hereditary obesity and diabetes in mice, has recently been isolated through positional cloning. In this study, we isolated a full-length human ob complementary DNA (cDNA) clone and examined the tissue distribution of ob gene expression in humans. The nucleotide sequences of the human ob cDNA coding region were 83% identical to those of the mouse and rat ob cDNA coding regions. Analysis of the deduced amino acid sequences revealed that the human ob protein is a 166–amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively. Northern blot analysis using the cloned human ob cDNA fragment as a probe identified a single messenger RNA (mRNA) species 4.5 kb in size found abundantly in the adipose tissues obtained from the subcutaneous, omental, retroperitoneal, perilymphatic, and mesenteric fat pads. However, no significant amount of ob mRNA was present in the brain, heart, lung, liver, stomach, pancreas, spleen, small intestine, kidney, prostate, testis, colon, or skeletal muscle. The ob mRNA level in the adipose tissue varied from region to region even in the same individual. Furthermore, in the human adipose tissue, ob gene expression occurred in mature adipocytes rather than in stromal-vascular cells. This study is the first report of the elucidation of ob gene expression in human tissues, thereby leading to better understanding of the physiological and clinical implications of the ob gene.

Molecular cloning of rat obese cDNA and augmented gene expression in genetically obese Zucker fatty (fa/fa) rats.

The obese (ob) gene has recently been isolated through a positional cloning approach, the mutation of which causes a marked hereditary obesity and diabetes mellitus in mice. In the present study, we isolated rat ob cDNA and examined the tissue distribution of the ob gene expression in rats. We also studied the gene expression in genetically obese Zucker fatty (fa/fa) rats. The rat ob gene product, a 167 amino acid protein with a putative signal sequence, was 96 and 83% homologous to the mouse and human ob proteins, respectively. Northern blot analysis using the rat ob cDNA probe identified a single mRNA species of 4.5 kb in size in the adipose tissue, while no significant amount of ob mRNA was present in other tissues in rats. The ob gene was expressed in the adipose tissue with region specificities. The rank order of the ob mRNA level in the adipose tissue was epididymal, retroperitoneal, and pericardial white adipose tissue > mesenteric and subcutaneous white adipose tissue > or = interscapular brown adipose tissue. The ob gene expression occurred in mature adipocytes rather than in stromalvascular cells isolated from the rat adipose tissue. Expression of the ob gene was markedly augmented in all the adipose tissue examined in Zucker fatty (fa/fa) rats at the stage of established obesity. The present study leads to the better understanding of the physiologic and pathophysiologic roles of the ob gene.

Augmented expression of obese (ob) gene during the process of obesity in genetically obese-hyperglycemic Wistar fatty (falfa) rats

Expression of the obese (ob) gene is up‐regulated in the adipose tissue in several obese rodent models. To study the regulation of the ob gene expression during the development of obesity, we examined the ob gene expression in genetically obese‐hyperglycemic Wistar fatty (falfa) rats at several stages of obesity. The ob mRNA levels in the adipose tissue from Wistar fatty rats was unequivocally augmented and continued to rise in the process of obesity. Furthermore, the ob gene expression in this obese model was much more rapidly enhanced in the mesenteric fat than in the subcutaneous fat. Moreover, the ob gene expression was more greatly augmented in the mesenteric fat than the lipoprotein lipase gene expression. These results suggest the presence of obesity‐linked and region‐specific regulation of the ob gene expression.

The significance of the Trp 64 Arg mutation of the β3-adrenergic receptor gene in impaired glucose tolerance, non—insulin-dependent diabetes mellitus, and insulin resistance in Japanese subjects☆

It has been reported that the Trp 64 Arg mutation of the human beta3-adrenergic receptor (beta3-AR) gene is related to an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and features of insulin resistance and weight gain in morbidly obese patients. However, such findings have not been consistent in varying ethnic populations. In the present study, we investigated the frequency of the Trp 64 Arg mutation of the human beta3-AR gene in Japanese control subjects (n = 253) and in NIDDM (n = 314) and impaired glucose tolerance (IGT) patients (n = 100). We compared the frequency of the mutation with the body-mass index (BMI) in these groups and with the metabolic clearance rate (MCR) of glucose in the NIDDM patients. A Trp 64 Arg mutation was observed in 36.7%, 31.6%, and 37.0% of the control, NIDDM, and IGT subjects, respectively. The frequency of the homozygotes for the mutation was 4.3%, 4.8%, and 3.0%, respectively. Neither the genotype frequency (Trp/Arg, Arg/Arg) nor the frequency of the mutated allele was significantly different among the three groups. The BMI of the subjects with the mutation was not significantly higher than that of the subjects without the mutation in each group. Furthermore, the allele frequency (A) was not different among the subjects with different BMIs (BMI < 22.0, 22.0 < or = BMI < or = 26.4, BMI > 26.4) in each group. In a separate group of NIDDM patients, the MCR of the subjects with intermediate BMIs (22.0 < or = BMI < or = 26.4) with the mutation tended to be lower than that of those without the mutation. In addition, the MCR of the subjects with the mutation in this group was significantly lower compared with that of those with a BMI less than 22. These results indicate that the Trp 64 Arg mutation of the beta3-AR gene may not contribute to the development of NIDDM or be a determinant of obesity in the Japanese population. However, the mutation may contribute to insulin resistance in NIDDM patients with an intermediate BMI.

Negative correlation between bone mineral density and TSH receptor antibodies in male patients with untreated Graves' disease

IntroductionAlthough it has been established that hyperthyroidism leads to reduced bone mineral density (BMD), with accelerated bone turnover promoting bone resorption in female patients, there is a dearth of data for male patients with hyperthyroidism. This study evaluated BMD and bone metabolism in male patients with Graves’ disease.MethodsThe study included 56 Japanese male patients with newly diagnosed Graves’ disease and 34 normal Japanese male control subjects of similar age and body mass index. We used dual energy x-ray absorptiometry to measure BMD at sites with different cortical/cancellous bone ratios (lumbar spine, femoral neck, and distal radius).ResultsAt the lumbar spine and the distal radius, BMD and T-scores were significantly lower for patients than for controls. At the femoral neck, on the other hand, the same values were relatively, but not significantly, lower in patients than in controls. However, Z-scores at all three sites were significantly lower for patients than for controls. The Z -score at the distal radius of patients was significantly lower than that at their lumbar spine and femoral neck. In addition, Z-score at the distal radius correlated negatively with age, free thyroxine, thyroid stimulating hormone receptor antibodies, thyroid stimulating antibody, and urinary N-terminal telopeptide of type I collagen normalized by creatinine.ConclusionsThese results indicate a high prevalence of cortical bone loss in male patients with Graves’ disease, especially elderly patients. We conclude that BMD measurement is crucial in all Graves’ disease patients regardless of their gender and that the radial BMD as well as BMD at the lumbar spine and femoral neck should be monitored to effectively prevent bone loss and subsequent fracture.

Insulin-induced activation of phosphoinositide 3-kinase in Fao cells

SummaryPhosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85α). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4–6% of the total PI3-kinase, and their specific activity was 11–14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.