In vivo suppression of mafA mRNA with siRNA and analysis of the resulting alteration of the gene expression profile in mouse pancreas by the microarray method.

Maf is a family of transcription factor proteins that is characterized by a typical bZip structure, and one of the large mafs, mafA is a strong transactivator of insulin. To explore the role of mafA in the pancreas, we modified the mafA mRNA level in vivo in mice by the RNA interference (siRNA) technique and analyzed the resulting alteration of the expressed gene profile with a microarray system. The mafA expression level in siRNA-treated mice was reduced approximately 60% compared with control-siRNA-treated animals. Microarray analysis revealed changes in the expression level of several genes in the siRNA-treated mice, with prominent down-regulated expression of the genes encoding insulin, glucagon, and adipocytokines, suggesting possible role of mafA in the pathophysiological states of impaired metabolic responses or inflammatory reactions.

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

INTRODUCTION: Although 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. METHODS: The 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). RESULTS: At 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. CONCLUSIONS: These 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.

Decreased bone mineral density at the distal radius, but not at the lumbar spine or the femoral neck, in Japanese type 2 diabetic patients.

The purpose of this study is to assess the association between type 2 diabetes and bone mineral density. This study included 145 Japanese patients (64 men and 81 women) with type 2 diabetes and 95 non-diabetic control subjects (41 men and 54 women) of similar age. We measured bone mineral density (BMD) at the sites with different cortical/cancellous bone ratio (lumbar spine, femoral neck, and distal radius) using dual-energy X-ray absorptiometry. BMD and Z score at the distal radius were significantly lower in type 2 diabetic patients than those in control subjects, and in type 2 diabetic patients, the Z score at the distal radius was lower than that at their own lumbar spine and femoral neck. In type 2 diabetic patients, negative correlation between BMD and the mean HbA1c during the previous 2 years was found significantly at the distal radius in both genders and at the femoral neck in women. These results indicate the selective cortical bone loss in type 2 diabetes and suggest the importance of also determining BMD at the radius and keeping good metabolic control to prevent bone loss in type 2 diabetic patients.

Impaired blood rheology by remnant-like lipoprotein particles: studies in patients with fatty liver disease.

Fatty liver disease (FLD) characterised by a high plasma levels of lipoproteins and remnant-like lipoproteins (RLP) is a risk factor for impaired microvascular blood flow, endothelial cell dysfunction and atherosclerosis. Using an immunoseparation technique with a gel mixture containing human monoclonal antibodies to apo A-I and apo B-100, we separated and measured RLP cholesterol (RLP-C) levels which reflect RLP in patients with FLD (n=20). Whole blood transit time (TT) was determined by a microchannel method (MC-FAN) which allows blood flow to be viewed via a microscope connected to an image display unit. RLP-C levels were higher (P<0.01) in FLD, 15.6 +/- 1.0 mg/dl compared with 4.8 +/- 0.5 mg/dl for controls (n=20). Similarly, TT was longer (P<0.01) in FLD, 284.5 +/- 26.1 sec/100 microl compared with 82.8 +/- 1.0 sec/100 microl for controls. Since the liver is a major site for RLP formation and degradation, it is affected to a greater extent in patients with FLD. It is likely that high levels of RLP can impair microvascular perfusion in the liver tissue and contribute to the development and progression of FLD.

Changes in vasoactive substances during gastric ulcer healing.

In order to study the roles of vasoactive peptides during tissue repair of gastric ulcers, we compared concentrations in tissue surrounding gastric ulcers of endothelin-1(ET-1), adrenomedullin (AM), and transforming growth factor-beta (TGF-beta) among different stages of ulcer development. A total of 82 cases were studied. Ulcers were located in the gastric angulus in 51 cases. All cases were positive for Helicobacter pylori (Hp). Ten cases were in the active stage (GA), 18 were in the healing stage (GH), and 28 were in the scarring stage (GS). As control, 17 cases of Hp-positive gastritis (gast+) and 14 of Hp-negative gastritis (gast-) were studied. The concentrations of endothelin (ET) and TGF-beta were in the order of GH> GA> GS, and those of AM were in the order of GS > GH > GA. On immunostaining, ET stained positively in endothelial cells and vascular smooth muscle cells (VSMCs) during the GH and GS stages, and AM stained positively in histiocytes during GA, GH and GS, and also stained positively in glandular epithelia and smooth muscle fibers during GH and GS. When our results were reviewed with respect to the regulation of vascular tonus and the proliferation of VSMCs, ET and AM were considered to have roles in the regulation of proliferation.

Changes of nitric oxide and growth factors during gastric ulcer healing.

We measured the concentrations of vascular endothelial growth factor (VEGF), nitric oxide and endothelin-1 (ET-1) in the gastric mucosa and examined the relationships between these factors. VEGF, nitric oxide and ET participate in angiogenesis and vascular remodeling, important elements of gastric ulcer healing. We studied cases of gastric ulcer as confirmed by endoscopic examination. All 61 cases in the angulus were positive for Helicobacter pylori (Hp). Fifteen cases were active stage (GA), 23 were healing stage (GH), and 23 were scarring stage (GS). As control, 17 cases of Hp-positive gastritis (gast+) and 14 cases of Hp-negative gastritis (gast-) were studied. Biopsy samples taken from the angulus during endoscopic examination were frozen and sliced into thin sections. ET was measured by enzyme immunoassay, VEGF was measured by enzyme-linked immunosorbent assay (ELISA) and nitric oxide was measured in terms of metabolite oxides of nitrogen (NOx) as described by Griess. ET, VEGF and inducible nitric oxide synthase (iNOS) were immunostained. The GA group had the highest concentration of NOx, suggesting that nitric oxide participates in the early stage of mucosal repair. In the GH group, all three factors showed high concentrations, suggesting that all may be involved in increased production. In the GS group, all three factors were significantly lower than in the GA and GH groups. Immunohistochemical studies showed that the distribution of ET- and iNOS-positive cells differed according to the ulcer stage. In particular, ET- and iNOS-positive cells in the vascular wall were primarily endothelial cells during GA and GH and vascular smooth muscle cells (VSMCs) during GS. These findings suggest that endothelial cells produce increased amounts of ET, nitric oxide and VEGF early in ulcer healing, a period of active endothelial cell repair and angiogenesis. During the scarring stage, vascular remodeling may result from the effects of ET and nitric oxide in regulating the proliferation of VSMCs. Our results suggest that VEGF. nitric oxide and ET participate in angiogenesis, vascular remodeling and mucosal regeneration during ulcer healing.

Clinical manifestations due to a point mutation of the mitochondrial tRNAleu(UUR) gene in five families with diabetes mellitus.

It has been shown that an adenine (A) to guanine (G) transition at position 3243 of the mitochondrial transfer RNA(tRNA)leu(UUR) gene is associated with a subgroup of diabetes mellitus. Therefore, we screened for this transition in 86 patients with non-insulin-dependent diabetes mellitus (NIDDM) in which two or three generations were affected with diabetes, in 14 patients with insulin-dependent diabetes mellitus, and in 9 families with diabetes mellitus and/or associated disorders suggesting mitochondrial gene abnormalities. We failed to identify the mutation in 100 diabetic patients, 86 NIDDM and 14 insulin-dependent diabetes mellitus (IDDM). Out of the latter 9 families, we identified an A to G transition in 14 individuals in 5 families. Diabetes mellitus was shown to be maternally inherited in one family. In 9 of 14 patients with the mutation, insulin was required to treat diabetes mellitus, indicating impaired insulin secretion. A hyperglycemic clamp test performed in one subject revealed significant impairment of insulin secretion, whereas euglycemic clamp test showed normal insulin sensitivity in this patient. The heteroplasmy of the mutant mitochondrial DNA (mtDNA) in leukocytes does not appear to correlate with the severity of diabetes in terms of the insulin therapy required. Body mass index of the affected individuals was less than 23.3. In one family, in addition to diabetes mellitus and hearing loss, hypoparathyroidism was associated with the mutation, suggesting that hypoparathyroidism is caused by the impaired processing and/or secretion of proparathyroid hormone due to the mutation. In addition, the affected subjects presented with proteinuria at the time of diagnosis of diabetes mellitus which appeared not to be related with diabetic nephropathy.

Relationship between recurrence of gastric ulcer and the microcirculation.

We investigated the relationship between microcirculatory disturbance and the host response to Helicobacter pylori infections in gastric ulcer scars to determine the role of endothelin-1 (ET) in ulcer recurrence. The subjects were divided into three groups. The GuS group consisted of patients who had red scarring (S1 stage) at the gastric angle with H. pylori, the gast+ group who had gastritis with H. pylori, and the gast- group who had gastritis without H. pylori. During endoscopic examination, biopsies were taken from the gastric angle. Mucosal ET, nitric oxide (NO), interleukin-8 (IL-8), and RANTES were measured. ET, inducible NO synthase (iNOS), and endothelial constitutive NOS (ecNOS) were immunostained. Mucosal ET and oxides of nitrogen (NOx) were significantly higher in the GuS group than in the other groups. IL-8 was elevated in the GuS and gast+ groups, and RANTES was elevated in the gast+ group (p < 0.01). There was prominent inflammatory cell infiltration in the GuS group. ET-positive cells were found in vascular smooth muscle, gastric epithelium, and gastric smooth muscle. iNOS-positive cells were found in vascular smooth muscle, gastric epithelium, gastric smooth muscle, and inflammatory cells. In conclusion, local inflammation and microcirculatory disturbance persist at the center of the ulcer scar (S1). Decreased cytokine levels and increased ET and NO (mainly synthesized by iNOS) levels suggested that microcirculatory disturbance is a more important factor than immune response in ulcer recurrence.

The significance of the Trp 64 Arg mutation of the beta3-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.

Molecular screening of both the promoter and the protein coding regions in the human ob gene in Japanese obese subjects with non-insulin-dependent diabetes mellitus.

OBJECTIVE: Although the molecular mechanism of obesity has been poorly understood, recent studies indicate that leptin plays a critical role in regulating both food intake and body weight. Because obesity decreases the sensitivity to insulin, the human ob gene is presumed to be one of the candidate genes for non-insulin-dependent diabetes mellitus (NIDDM) associated with obesity. Although the protein coding region in the ob gene has been screened for mutations, the promoter region and the non-coding first exon have not yet been studied. We investigated the involvement of the human ob gene, especially mutations at the promoter region and the non-coding first exon, in the development of NIDDM associated with obesity. SUBJECTS: The study group comprised 60 Japanese obese subjects with NIDDM (body mass index (BMI) 43.6 > or = BMI > or = 26.4, 29.0+/-0.41 (mean+/-S.E.M.)) and 24 obese individuals with impaired glucose tolerance (IGT) (30 > or = BMI > or = 26.4, 27.1+/-0.22). METHODS: Mutations at both the promoter region and all three exons in the human ob gene were screened by the single-stranded conformational polymorphism analysis. When aberrantly migrated bands were recognized, the PCR-amplified DNA fragment was directly sequenced. RESULTS: In the protein coding region a silent mutation in the second exon was detected. The non-coding first exon and the about 100 bp 5'-flanking region of the gene which contains a proximal CCAAT/enhancer-binding protein site were screened, but no mutations were found. CONCLUSION: These results suggest that no mutations in either the promoter region at the about 100 bp 5'-flanking region of the gene, or in any of the three exons, are involved in the development of NIDDM or IGT associated with obesity.

[Blood rheological study in rats with fatty liver--with special reference to effects of ethyl icosapentate].

A blood rheological study was conducted using Kikuchi's micro-channel method in rats with fatty liver. Effects of eicosapentaenoic acid (EPA) on blood rheology were also evaluated. Male SD rats given normal feed served as the control. One group was given choline-deficient feed for 4 weeks (EPA (-) group), while another group was daily given EPA (1000 mg/kg) for 4 weeks together with choline-deficient feed (EPA (+) group). The micro-channel passage time was determined using 100 microliters of whole blood. The passage time significantly increased in the EPA (-) group compared to the control (p < 0.01). It significantly decreased in the EPA (+) group compared to the EPA (-) group (p < 0.01). Findings obtained in the present study suggested that blood rheological factors are related to the development of fatty liver and that EPA inhibits fatty changes of the liver by improving these rheological factors.

Differential activation of mitogen-activated protein kinase by insulin and epidermal growth factor in 3T3-L1 adipocytes: a possible involvement of PI3-kinase in the activation of the MAP kinase by insulin.

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred micromol/l LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5'-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wortmannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3-kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.

Increase in hepatic tissue blood flow by teprenone.

The major objective of the present study was to evaluate mechanisms by which teprenone, a gastric mucosal protecting agent, increases hepatic mucosal blood flow using male Sprague-Dawley rats. Hepatic and gastric blood flow was measured using a laser blood flow meter after administration of teprenone, dissolved in Tween 80, into the inferior vena cava. Teprenone itself increased hepatic and gastric blood flow. It also increased hepatic and gastric blood flow in rats with acute hepatic disorders due to carbon tetrachloride (CCL4) and improved histological changes, such as inflammatory cell infiltration and fatty changes in the liver. The fact that blood endothelin (ET) concentrations increased after administration of teprenone suggest that teprenone has great affinity for ET beta receptors and shows ET beta-receptor antagonist-like effects. Hepatic blood flow decreased after administration of N-nitro-L-arginine methyl ester, a nitric oxide (NO) synthetase inhibitor, suggesting that teprenone increase NO activity. Teprenone was thought to increase hepatic and gastric blood flow by different mechanisms, because it increased gastric mucosal prostaglandin E2 concentrations.

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

Phosphoinositide 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 (p85 alpha). 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.

Augmented expression of obese (ob) gene during the process of obesity in genetically obese-hyperglycemic Wistar fatty (fa/fa) 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 (fa/fa) 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.

Structural organization and chromosomal assignment of the human obese gene.

The obese (ob) gene has been identified through a positional cloning approach; the mutation of this gene causes marked hereditary obesity and diabetes mellitus in mice. We report here the isolation and characterization of the human ob gene. Southern blot analysis demonstrated a single copy of the ob gene in the human genome. The human ob gene spanned approximately 20 kilobases (kb) and contained three exons separated by two introns. The first intron, approximately 10.6 kb in size, occurred in the 5'-untranslated region, 29 base pair (bp) upstream of the ATG start codon. The second intron of 2.3 kb in size was located at glutamine +49. By rapid amplification of 5'-cDNA ends, the transcription initiation sites were mapped 54-57 bp upstream of the ATG start codon. The 172-bp 5'-flanking region of the human ob gene contained a TATA box-like sequence and several cis-acting regulatory elements (three copies of GC boxes, an AP-2-binding site, and a CCAAT/enhancer-binding protein-binding site). By the fluorescence in situ hybridization technique, the ob gene was assigned to human chromosome 7q31.3. This study should establish the genetic basis for ob gene research in humans, thereby leading to the better understanding of the molecular mechanisms underlying 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.

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.