Yasunao Yoshimasa

Endothelial Nitric Oxide Synthase Gene Is Positively Associated With Essential Hypertension

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

Pathophysiological role of leptin in obesity-related hypertension

To explore the pathophysiological role of leptin in obesity-related hypertension, we examined cardiovascular phenotypes of transgenic skinny mice whose elevated plasma leptin concentrations are comparable to those seen in obese subjects. We also studied genetically obese KKA(y) mice with hyperleptinemia, in which hypothalamic melanocortin system is antagonized by ectopic expression of the agouti protein. Systolic blood pressure (BP) and urinary catecholamine excretion are elevated in transgenic skinny mice relative to nontransgenic littermates. The BP elevation in transgenic skinny mice is abolished by alpha(1)-adrenergic, beta-adrenergic, or ganglionic blockers at doses that do not affect BP in nontransgenic littermates. Central administration of an alpha-melanocyte-stimulating hormone antagonist causes a marked increase in cumulative food intake but no significant changes in BP. The obese KKA(y) mice develop BP elevation with increased urinary catecholamine excretion relative to control KK mice. After a 2-week caloric restriction, BP elevation is reversed in nontransgenic littermates with the A(y) allele, in parallel with a reduction in plasma leptin concentrations, but is sustained in transgenic mice overexpressing leptin with the A(y) allele, which remain hyperleptinemic. This study demonstrates BP elevation in transgenic skinny mice and obese KKA(y) mice that are both hyperleptinemic, thereby suggesting the pathophysiological role of leptin in some forms of obesity-related hypertension.

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.

Cloning of rat uncoupling protein-3 and uncoupling protein-2 cDNAs: their gene expression in rats fed high-fat diet

In order to elucidate energy balance in the skeletal muscle, we cloned cDNA of a homologue of uncoupling protein (UCP) from rat skeletal muscle. We also cloned rat UCP‐2 cDNA from rat brown adipose tissue (BAT). The UCP cloned from rat skeletal muscle showed 57% and 72% identity with rat UCP‐1 and UCP‐2. The mRNA was expressed abundantly in the skeletal muscle, moderately in the BAT, and slightly in the white adipose tissue (WAT) with a major band at 2.5 kb and a minor band at 2.8 kb, while the UCP‐2 gene expression was widely detected in the whole body with substantial levels in the WAT and with slight levels in the skeletal muscle and BAT. The rat UCP cloned in the present study showed 86% identity with the recently cloned human UCP‐3, which was also expressed abundantly in the skeletal muscle with a signal of 2.4 kb. Therefore, the rat UCP was considered to be rat UCP‐3. In rats fed high‐fat diet the UCP‐3 gene expression was augmented 2‐fold in the skeletal muscle while UCP‐2 mRNA levels were increased significantly (1.6‐fold) in the epididymal WAT. Augmented expression of UCPs may provide defense against high‐fat induced obesity and impairment of glucose metabolism.

Satiety effect and sympathetic activation of leptin are mediated by hypothalamic melanocortin system

Leptin is an adipocyte-derived blood-borne satiety factor that decreases food intake and increases energy expenditure, thereby leading to a substantial decrease in body weight. To explore the possible roles of the hypothalamic melanocortin system in leptin action, we examined the effects of intracerebroventricular (i.c.v.) injection of leptin with or without SHU9119, a potent antagonist of alpha-melanocyte stimulating hormone, on food intake, body weight, and mitochondrial uncoupling protein-1 (UCP-1) mRNA expression in the brown adipose tissue (BAT) in rats. A single i.c.v. injection of leptin decreased cumulative food intake and body weight gain, and increased UCP-1 mRNA expression during 3 h at the onset of the dark phase. Inhibition of food intake and body weight change with leptin was reversed by co-injection of SHU9119 in a dose-dependent manner. Co-injection of SHU9119 also inhibited completely the leptin-induced increase in UCP-1 mRNA expression in the BAT. Treatment with SHU9119 alone did not affect food intake, body weight, and UCP-1 mRNA expression in rats. The present study provides evidence that the hypothalamic melanocortin system plays a central role in both satiety effect and sympathetic activation of leptin.

The arcuate nucleus as a primary site of satiety effect of leptin in rats

The obese (ob) gene encodes a fat cell-derived circulating satiety factor (leptin) that is involved in the regulation of energy homeostasis. In the present study, we examined effects of i.c.v. injection of recombinant human leptin on food intake and body weight gain in rats. We also studied effects of direct microinjections of leptin into the arcuate nucleus (Arc), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH). A single i.c.v. injection of recombinant human leptin (0.25-2.0 micrograms/rat) reduced significantly and dose-dependently food intake and body weight gain in rats. Microinjections (0.125-0.5 microgram/site) into the bilateral Arc, VMH, and LH caused dose-related decreases in food intake and body weight gain as compared with vehicle-treated groups with a rank order of potency; Arc > VMH = LH. The present study provides the first direct evidence that the Arc is a primary site of satiety effect of leptin.

NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function

Reactivation of the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression of fetal cardiac genes remains unresolved. Here we show that neuron‐restrictive silencer factor (NRSF), a transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atrial natriuretic peptide, brain natriuretic peptide and α‐skeletal actin, and plays a role in molecular pathways leading to the re‐expression of those genes in ventricular myocytes. Moreover, transgenic mice expressing a dominant‐negative mutant of NRSF in their hearts exhibit dilated cardiomyopathy, high susceptibility to arrhythmias and sudden death. We demonstrate that genes encoding two ion channels that carry the fetal cardiac currents If and ICa,T, which are induced in these mice and are potentially responsible for both the cardiac dysfunction and the arrhythmogenesis, are regulated by NRSF. Our results indicate NRSF to be a key transcriptional regulator of the fetal cardiac gene program and suggest an important role for NRSF in maintaining normal cardiac structure and function.

Trial of insulinlike growth factor I therapy for patients with extreme insulin resistance syndromes

Extreme insulin resistance occurs in patients with primary defects in insulin action at the receptor or postreceptor levels. The condition commonly is associated with acanthosis nigricans and ovarian masculinization. Despite a marked increase in insulin secretion, some patients develop frank diabetes mellitus that does not respond adequately to insulin therapy. Insulinlike growth factor I exerts metabolic effects similar to those of insulin. This study assessed the potential effectiveness of IGF-I as a blood glucose lowering agent in patients with extreme insulin resistance syndromes, including type A insulin resistance, congenital generalized lipodystrophy, and leprechaunism. Among the 11 patients studied, some exhibited mutated insulin receptors, whereas others were suspected to have defects in postreceptor sites. In each patient, plasma glucose levels decreased in response to subcutaneous injections of recombinant human IGF-I (0.1−0.3 mg/kg body wt). The degree of the decrease was roughly comparable with that observed in normal individuals. IGF-I also reduced plasma insulin concentrations. A long-term trial of IGF-I (up to 16 mo) showed that IGF-I (0.1−0.4 mg/kg body wt twice daily) is effective in lowering both fasting and postprandial plasma glucose concentrations with decreases in both fructosamine and HbA1c values. Improvement of acanthosis nigricans was observed in some of the patients. These results suggest that recombinant human IGF-I could be used clinically as a hypoglycemic agent in diabetic patients with extreme insulin resistance in whom insulin treatment is ineffective.

Human leptin receptor gene in obese Japanese subjects: evidence against either obesity-causing mutations or association of sequence variants with obesity.

Summary Leptin is an adipocyte-derived blood-borne satiety factor that acts on its cognate leptin receptor (Ob-R) in the hypothalamus, thereby regulating food intake and energy expenditure. To explore whether mutations in the Ob-R gene cause obesity in humans, we have searched for mutations in the gene for Ob-Rb, a biologically active receptor isoform, in obese Japanese subjects. We have also examined associations between such mutants and obesity in the Japanese. Genomic DNAs were used as templates in polymerase chain reaction (PCR) with primers selected to amplify exons 2 to 20 of the human Ob-Rb gene. Direct sequence analysis of the PCR products revealed 7 nucleotide sequence variants (Lys109Arg, Gln223Arg, Ser343Ser, Ser492Thr, Lys656Asn, Ala976Asp, and Pro1019Pro) in the Ob-Rb coding region from 17 obese Japanese subjects with a family history of obesity (BMI 39.3 ± 8.4 kg/m2). No missense and nonsense mutations were found such as those in Zucker fatty (fa/fa) rats and Koletsky (fak/fak) rats. Nucleotide substitutions occurred at relatively high frequencies at codons 109, 223, 976, and 1019 (79, 91, 100, and 85 %, respectively). Allele frequency of each variant determined by PCR-RFLP and PCR-single strand conformation polymorphism analyses showed no significant differences between 47 obese (BMI 35.1 ± 6.5 kg/m2) and 68 non-obese (BMI 21.6 ± 2.2 kg/m2) subjects. The present study represents the first report of sequence variants of the Ob-Rb gene in the Japanese and provides evidence against either obesity-causing mutations or association of sequence variants with obesity in obese Japanese subjects. [Diabetologia (1997) 40: 1204–1210]