Hitomi Fukuda

Transcriptional regulation of leptin gene promoter in rat

To investigate the DNA regulatory sequences required for stimulation and suppression of leptin gene expression, primary cultured hepatocytes and adipocytes of rats were transfected with plasmids containing the 5′‐flanking sequences of the rat leptin gene fused to the luciferase gene. When two copies of the sequences spanning nucleotides −101 to −83 of the leptin promoter were used for transfection, the reporter activity significantly increased in the presence of glucose/insulin in comparison with glucose alone. The glucose/insulin stimulation of the transcription was inhibited by addition of polyunsaturated fatty acids. These results were similar to those found earlier for the transcription of the fatty acid synthase, FAS(−57/−35) and ATP citrate‐lyase, ACL(−64/−41) genes. Cotransfection studies in the cells with a Sp1 expression vector and leptin (−101/−83) constructs showed the inactivation of the leptin promoter by Sp1. Gel mobility shift assays using an end‐labeled leptin(−101/−83) construct as a probe revealed that nuclear factor(s) from rat liver or adipose tissue specifically formed complexes with the sequence. The DNA‐protein complexes were common to the glucose/insulin‐responsive regions of the leptin, ACL and FAS genes, suggesting that these genes are coordinately regulated. In addition, by antibody supershift assays, the transcription factor Sp1 was found to bind the GC‐rich region located between nucleotides −101 and −83 of the leptin gene. Mutational analysis of this region showed that the sequence of the region was critical for glucose/insulin stimulation of transcription. Thus, we postulated that the region from −101 to −83 of the leptin gene is responsible for glucose/insulin stimulation of transcription, and that Sp1 is somehow involved in this regulation.

Transcriptional regulation of fatty acid synthase gene and ATP citrate-lyase gene by Sp1 and Sp3 in rat hepatocytes1

When two copies of the sequences spanning −57 to −35 of the fatty acid synthase (FAS) or −64 to −41 of the ATP citrate‐lyase (ACL) gene linked to a reporter gene were transfected into primary cultured hepatocytes, the reporter activities significantly increased in response to insulin/glucose treatment. In cotransfection experiments of the FAS(−57/−35) with the Sp1 or Sp3 expression vector, the reporter activities of transcription were suppressed by Sp1 and stimulated by Sp3. In the cotransfection experiments of ACL(−64/−41), the activities were suppressed by Sp1 but were unchanged by Sp3. A similar effect of Sp1 and Sp3 on transcription was seen in mRNA concentrations and enzyme activities of endogenous FAS and ACL. Moreover, the mRNA concentrations and enzyme activities of endogenous acetyl‐CoA carboxylase were suppressed by Sp1 and greatly increased by Sp3. Gel mobility super shift assays using antibodies against Sp1 or Sp3 revealed the binding of the transcription factors Sp1 and Sp3 with the GC rich regions located within FAS(−57/−35) and ACL(−64/−41) genes. The formation of DNA‐protein complexes was decreased in rats fed a high‐carbohydrate diet in comparison with that in fasted rats, but feeding the corn oil diet inhibited this decrease. In Western immunoblotting assay, however, the amount of Sp1 and Sp3 remained unchanged in the dietary conditions. Therefore, the binding of DNA‐protein complexes was not due to changes in the amount of Sp1 and Sp3 but to changes in the binding activity, suggesting that these transcription factors may be an important determinant of lipogenic enzyme expression.

Differences in labelled triolein turnover after oral administration between liver and adipose tissue of rats.

To investigate exogenous triacylglycerol turnover, the time courses for labelled triolein in the liver, plasma and epididymal adipose tissue (adipose tissue) after oral administration to rats fed a fat-free or 10 % corn oil diet for 3 d after fasting overnight were examined for 10 d. After the administration of labelled triolein to rats fed the fat-free diet, the incorporation (dpm/g) into total lipids of the liver and adipose tissue each reached the maximum in 8 h and was seven times higher in the adipose tissue than in the liver. The half-lives of total lipid radioactivities during the decreasing phases were 0.39 and 2.58 d, respectively, in the rapid and slow phases of the decay curve in the liver, and 4.78 d in only one phase of the adipose tissue. Radioactivity after administration of labelled triolein was mostly found in the oleic acid in the tissues. The half-life of oleic acid was 3.92 d in the adipose tissues. These half-lives were similar in both dietary groups. Thus, although dietary corn oil reduced the triolein incorporation to cellular lipids in comparison to the fat-free diet, it did not affect these half-lives. The labelled triacylglycerol-oleic acid stayed abundantly intact for a long time in the adipose tissue and was scarcely changed to other fatty acids, whereas it was slightly incorporated into total lipids and quickly metabolized in the liver. Non-essential fatty acids may be mostly endogenous in the liver but may be exogenous and endogenous in adipose tissue.

Okara ameliorates glucose tolerance in GK rats

Okara, a food by-product from the production of tofu and soy milk, is rich in three beneficial components: insoluble dietary fiber, β-conglycinin, and isoflavones. Although isoflavones and β-conglycinin have recently been shown to improve glucose tolerance, the effects of okara have not yet been elucidated. Therefore, we herein investigated the effects of okara on glucose tolerance in Goto-Kakizaki (GK) rats, a representative animal model of Japanese type 2 diabetes. Male GK rats were fed a 10% lard diet with or without 5% dry okara powder for 2 weeks and an oral glucose tolerance test was performed. Rats were then fed each diet for another week and sacrificed. The expression of genes that are the master regulators of glucose metabolism in adipose tissue was subsequently examined. No significant differences were observed in body weight gain or food intake between the two groups of GK rats. In the oral glucose tolerance test, increases in plasma glucose levels were suppressed by the okara diet. The mRNA expression levels of PPARγ, adiponectin, and GLUT4, which up-regulate the effects of insulin, were increased in epididymal adipose tissue by the okara diet. These results suggest that okara provides a useful means for treating type 2 diabetes.