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.

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.

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.

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.

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.

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.

Inhibitory effect of ursodeoxycholic acid on the progression of chronic hepatic disorders with special reference to increases in blood flow.

Ursodeoxycholic acid (UDCA), which is commonly used as a cholesterol-gallstone-dissolving agent, is now expected to be effective not only for primary biliary cirrhosis but for chronic active hepatitis as well. In this study, we administered 0.1% ethionine-added, choline-deficient diet for 8 weeks to male Sprague-Dawley rats to prepare an animal model of chronic hepatic disorders. At the same time, UDCA (50 mg/kg/day) was administered orally to these animals, and its effects on the liver, including effects on hepatic blood flow determined with a laser Doppler blood flow meter, were evaluated. Hepatic blood flow increased significantly in the UDCA group compared with the untreated groups. Transaminase levels decreased significantly in the UDCA group compared with the untreated group. Histologic differences were noted between the UDCA and untreated groups in histopathologic examinations of the liver, with liver cirrhosis or early liver cirrhosis being present in the untreated group, compared with only chronic active hepatitis in the UDCA group. These findings suggest that UDCA is able to prevent or inhibit the progression of chronic hepatic disorders, an effect that may be due in part to increases in hepatic blood flow.

Experimental use of pravastatin in patients with primary biliary cirrhosis associated with hypercholesterolemia.

Primary biliary cirrhosis (PBC) is a refractory liver disease for which no medical treatment has been established. The investigators administered 20 mg/day of pravastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, to 2 PBC patients with hypercholesterolemia (1010 and 306 mg/dl) for 3 years and 10 months in order to decrease the blood concentration of bile acids and prevent adverse effects on the hepatocellular membrane. The drug markedly decreased not only cholesterol levels but also total bile acid levels, producing particularly pronounced decreases in cholic acid and chenodeoxycholic acid. Histologically, progression was inhibited in one patient, whereas improvement was seen in the other. Bile duct enzymes and other biochemical parameters showed improvement in both cases. General pruritus and blepharal and palmar xanthoma also improved. These findings suggest that pravastatin may be useful in the treatment of PBC associated with hypercholesterolemia.

Role of eicosapentaenoic acid in lipid metabolism in the liver, with special reference to experimental fatty liver.

Choline-deficient feed was given to three groups (n = 7 in each) of male Sprague-Dawley rats for 4 weeks to induce the development of fatty liver. In addition, two of the groups received eicosapentaenoic acid (EPA), 1000 mg/kg/d, administered orally either for all 4 weeks or for only the last 2 weeks of the study, respectively. The third group received the choline-deficient diet but no EPA. The untreated control group (n = 7) received only normal feed. The efficacy of EPA in preventing fatty liver was assessed based on the evaluation of pathologic and biochemical parameters and hepatic blood flow. EPA markedly improved fatty liver, probably due to both direct effects (inhibition of the synthesis of triglyceride in the liver) and indirect effects (increased hepatic blood flow). Decreased blood flow due to sinusoidal block is responsible for the progression of fatty liver. EPA has been shown to decrease thromboxane A2 production and blood viscosity and to enhance red cell deformability. These effects are thought to have contributed to the increases in hepatic blood flow.

Effect of interferon therapy in a patient with chronic active hepatitis type C associated with interstitial pneumonia and rheumatoid arthritis: a case report.

Interferon (IFN) is widely used to treat patients with chronic hepatitis types B and C but has been found to occasionally aggravate rheumatoid arthritis (RA) or cause interstitial pneumonia. We administered 6 MIU/d IFN-beta by intravenous injection for 6 weeks to a 55-year-old man with chronic active hepatitis type C associated with RA and interstitial pneumonia. Transaminase levels rapidly returned to normal after treatment and hepatitis C virus-RNA (nested RT-PCR method) was negative on completion of treatment. No significant adverse reactions or aggravation of RA or interstitial pneumonia occurred. These findings suggest that use of IFN-beta in the treatment of patients with chronic hepatitis type C associated with RA and/or interstitial pneumonia presents no problem if appropriate precautions are taken.

Salivary immunoreactive human epidermal growth factor (IR-hEGF) in patients with peptic ulcer disease.

EGF is reported to have a potent protective effect on peptic ulcer formation in rats. In this study, we measured the IR-hEGF concentrations in the saliva of normal human subjects and patients with peptic ulcer disease or non-peptic ulcer gastroduodenal disease. In normal subjects, the level of salivary IR-hEGF was highest in the early morning, and the values in individuals on different days showed small variations. There were no sex differences or age-related changes in the salivary IR-hEGF levels. The concentrations of the peptide were lower in patients in the active (0.96 +/- 26 ng/ml, mean +/- SE, n = 4) and healing stages (1.06 +/- 0.24 ng/ml, n = 8) of peptic ulcer disease as compared with those in normal subjects (3.19 +/- 0.46 ng/ml, n = 47). No significant differences in salivary IR-hEGF levels were observed between normal subjects and patients in the scaring stage of peptic ulcer disease (2.40 +/- 0.42 ng/ml, n = 21), or those with gastric cancer (2.44 +/- 0.27 ng/ml, n = 21) and atrophic or superficial gastritis (2.31 +/- 0.34 ng/ml, n = 28). Although the pathophysiological significance of these lower salivary IR-hEGF levels in patients with peptic ulcer disease is unclear, it is possible that the low level of hEGF in saliva may decrease the resistance of the mucosa to physicochemical stress, and thus participate in the development of the diseases.

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.

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.

A case report of epithelioid leiomyosarcoma of transverse mesocolon: diagnosis and treatment.

A case of epithelioid leiomyosarcoma of the transverse mesocolon in a 45-year-old man was reported. The patient had a rapidly growing mass in the left upper quadrant. Ultrasonography, gastrointestinography, and abdominal computed tomography showed that the mass was separated from the pancreas, the gastrointestinal tract, and the retroperitoneal organs. Preoperatively the primary origin of this tumor was related to the transverse mesocolon. On laparotomy the tumor of 5cm by 6cm by 3cm in size was found in the anterior left of the transverse mesocolon and the mass was resected entirely. The patient is well 18 months after surgical treatment with no evidence of recurrence.

An experience of ischemic limb salvage associated with myositis ossificans of the left thigh.

A case of ischemic limb salvage associated with myositis ossificans of the left thigh in a 66-year-old man was reported. The patient had a medical history of cerebral palsy and a cervical spinal cord injury, and had an operative past history of hip arthroplasty for fracture of the left femoral neck 10 years before. He showed ischemic symptoms such as paleness, coldness, and loss of the left dorsal arterial pulsation in the left toe, and had a rapidly growing mass in the left thigh. Roentgenography and computed tomography showed a mass 10 cm by 10 cm by 8 cm in size with severe calcification in the left quariceps muscle. Occlusion of the left common femoral artery was found in the arteriogram. Surgery was carried out in order to establish an accurate diagnosis and to rescue the left lower limb. The arterial pulsation was recovered as the result of completely resecting the left quariceps muscle tumor. The pathohistological diagnosis was of myositis ossificans in the quariceps muscle of the thigh. Etidronate disodium was administered in order to prevent a recurrence postoperatively. The patient has been well for the 13 months since surgery.

A case report of substernal goiter.

A case of substernal goiter is reported. A 78-year-old female was admitted to our hospital with no symptoms. Chest roentgenography on admission showed that a mass of 3 by 5 cm in size with calcification located in the substernal region. Computed tomography of the chest and aortography revealed that the mass was attached to the trachea, but the connection to the great vessels was not clear. Pathological findings of the incisional biopsy specimen showed thyroid tissue with no evidence of malignancy. Our clinical diagnosis was substernal goiter. Surgery was not carried out in this case, based on the literature. Surgery is indicated in case of malignancy or in cases with severe illness such as respiratory disorder and superior vena cava syndrome.

[Clinical studies and sputum levels of ceftriaxone once daily administration in respiratory tract infection].

Ceftriaxone (CTRX), a new cephalosporin, was investigated by once daily administration for its clinical efficacy and safety on respiratory tract infections. The results obtained are summarized as follows: 1. Clinical responses to CTRX of a total of 39 cases with respiratory tract infections were excellent in 12 cases, good in 23, fair in 3, poor in 1 with an efficacy rate of 89.7%. Against acute bronchitis, lung abscess, bronchiectasis, chronic bronchitis and obstructive pneumonia, efficacy rates were 100%. 2. Serum levels and urinary excretion rates of CTRX were investigated in 2 cases after intravenous drip infusion of the drug at doses of 1 g and 2 g, respectively. Although urinary excretion rate tended to decrease with the deterioration of renal functions, prolongation of serum half-life was slight in those patients with normal liver function. In 1 case, it remained at 1.9 micrograms/ml at 12 hours and in another at 0.9 microgram/ml at 22 hours in sputum. According to the results, it appears that once daily administration of CTRX is effective and well tolerated in patients with acute respiratory infections.

[Effect of endothelin on gastric mucosal lesion in rats].

Gastric mucosal blood flow and gastric mucosal prostaglandin E2 (PGE2) and prostacyclin (PGI2) were investigated in male Wistar rats intraarterially injected with endothelin (ET), an endothelium-derived vasoconstrictor peptide. Immediately following ET (4 nmol/kg) administration, gastric mucosal blood flow decreased. Then 30 min later, the blood flow reached the minimum, but PGE2 and PGI2 showed the highest value. PGE2 showed a tendency to decrease 90 min later, while PGI2 continued to show high value. There were redness and hemorrhagic damage in the gastric mucosa. Endogenous PGs were presumed to be relate to the regulation of the development of the mucosal damage owing to decrease in the blood flow after ET administration.

[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.