Masato Kasuga

Role of Krüppel-like Factor 15 (KLF15) in Transcriptional Regulation of Adipogenesis

Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor γ (PPARγ) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein β (C/EBPβ) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARγ in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPβ, C/EBPδ, or C/EBPα in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPα acted synergistically to increase the activity of the PPARγ2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR γ expression.

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.

A pilot clinical trial of a new oral hypoglycemic agent, CS-045, in patients with non-insulin dependent diabetes mellitus

CS-045, (+/-)-5-[4-(6-hydroxy-2,5,7,8-tetramkethylchroman-2- ylmethoxy)benzyl]-2,4-thiazolidinedione, lowers plasma glucose in several animal models of non-insulin dependent diabetes mellitus (NIDDM) presumably by increasing insulin sensitivity. Little adverse effect was found in a phase 1 study on healthy male subjects. In order to test its efficacy in lowering plasma glucose in NIDDM in man, a pilot multi-center clinical trial of CS-045 was carried out in 146 patients with NIDDM whose glycemic control was inadequate (FPG greater than 140 mg/dl) on diet and/or other oral hypoglycemic agents. CS-045 was given orally in a daily dose of 200 mg or 400 mg for 12 weeks in addition to the previous treatment. The mean fasting plasma glucose (FPG) and fructosamine began to decrease within 2 weeks and the mean HbA1c within 8 weeks. After 12 weeks, the FPG fell from 192 +/- 41 to 155 +/- 45 mg/dl (P less than 0.01), fructosamine from 3.7 +/- 0.6 to 3.3 +/- 0.6 (P less than 0.01), and HbA1c from 8.9 +/- 1.5 to 8.1 +/- 1.5% (P less than 0.01). The drug was effective in 39% of patients in that FPG fell by more than 20% of the initial value. This rate of efficacy was the same when CS-045 was given alone or together with other oral hypoglycemic agents. The drug was more effective in a dosage of 400 mg than with 200 mg (the rate of efficacy 46% vs 25%) and more effective in obese patients than in lean patients (46% vs 25%).(ABSTRACT TRUNCATED AT 250 WORDS)

Alteration in growth, cell morphology, and cytoskeletal structures of KB cells induced by epidermal growth factor and transforming growth factor-β☆

Long-term biological effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and transforming growth factor-beta (TGF-beta) were examined with human epidermoid carcinoma KB cells. EGF inhibited the growth of KB cells in both serum-containing and serum-free synthetic media by reducing the growth rate and by lowering the saturation density. The cells cultured with EGF showed relatively high motility and grew dispersely as single cells, whereas the cells cultured in the absence of EGF grew in clusters. Although TGF-beta itself did not inhibit the growth of KB cells, it augmented the growth inhibition by EGF. TGF-beta also affected the cell morphology. In the presence of TGF-beta, the cells became flattened and actin stress fibers were well developed compared to those cultured in its absence. The effects of EGF on growth, cell motility, and cell morphology were reversible. Tyrosine phosphorylation of EGF receptors was continuously observed for at least 50 h in the presence of EGF. TGF-beta did not increase the phosphorylation induced by EGF. These results suggested that signals continuously transmitted through EGF receptors caused the changes in cell growth and morphology and that TGF-beta did not act on the cells by modulating binding of EGF to its receptors or activation of the receptor kinase. In contrast to EGF and TGF-beta, neither insulin nor IGF-I affected cell morphology or growth, although KB cells express their receptors and the receptor kinases were also continuously activated during exposure of the cells to insulin or IGF-I.

Insulin-induced tyrosine-phosphorylation in intact rat adipocytes

Insulin-induced tyrosine-phosphorylation in intact isolated rat adipocytes was studied using immunoblotting method with antiphosphotyrosine antibodies. Insulin-stimulated adipocytes were solubilized with Triton X-100. The lysate was incubated with wheat germ agglutinin, then with hydroxylapatite. Insulin stimulated tyrosine-phosphorylation of a 95 KDa protein which adsorbs to wheat germ agglutinin and appears to be the beta-subunit of the insulin receptor. Among the proteins adsorbed to hydroxylapatite, tyrosine-phosphorylation of 170 KDa and 60 KDa proteins was stimulated. 170 KDa was also stimulated by polyclonal anti-insulin receptor antibodies B-10 Ig G, IGF-I and H2O2. The detection of these proteins in rat adipocytes may lead to the elucidation of a common signal transduction pathway in insulin-responsive cells.

Vanadate can replace interleukin 3 for transient growth of factor-dependent cells

A mouse interleukin 3 (IL-3)-dependent cell line, IC2, could survive and proliferate over a 48-h period in the absence of IL-3 when incubated with micromolar concentrations of sodium orthovanadate. The greatest response was obtained at 12.5 microM, as judged by stimulation of cell growth. Vanadate also stimulated synthesis of nucleotides and protein in IC2 cells. After 6 h of culture in the absence of IL-3, the intracellular ATP levels of IC2 cells fell dramatically; this fall was prevented by addition of vanadate to the culture. Studies with recombinant IL-3 revealed that vanadate potentiated the effect of submaximal doses of IL-3 on the growth of IC2 cells, but did not appear to act synergistically with IL-3 to give a maximal proliferative response of the cells. After 48 h of IL-3 replacement with vanadate, IC2 cells could respond to IL-3 with no loss of proliferative integrity. These results suggest that IL-3 dependence of IC2 cells is transiently substituted for by vanadate, and it may be a useful tool to understand the mechanism of action of IL-3.

Identification of erythropoietin receptors on fetal liver erythroid cells

Erythropoietin (EPO) has a central role in the growth and development of erythroid cells. Using a biologically active radioiodinated derivative, EPO receptors were identified on fetal mouse liver cells mostly consisting of erythroid cells. 125I-EPO was cross-linked to two receptors forms with apparent molecular masses of 110 and 95 kilodaltons, respectively and both having similar affinity toward EPO.

Identification of a phosphorylation site of the rat insulin receptor catalyzed by protein kinase C in an intact cell

In two‐dimensional tryptic phosphopeptide mapping, the β‐subunit of the insulin receptor phosphorylated by 12‐O‐tetra‐decanoylphorbol‐13‐acetate in rat hepatoma cells (H‐35) was separated into one phosphothreonine‐containing peptide and several phosphoserine‐containing peptides. The synthetic peptide coding residues 1327–1343 in the C‐terminal region of the rat insulin receptor was phosphorylated at the threonine residue by protein kinase C in a phosphatidylserine and oleoylacetylglycerol dependent manner. Tryptic digest of this phosphopeptide migrated to the same position as the phosphothreonine containing peptide obtained from the β‐subunit in two‐dimensional phosphopeptide mapping. These data suggested that Thr 1336 of the insulin receptor is the site of phosphorylation by protein kinase C in intact cells.

Long-term comparison of oral hypoglycemic agents in diabetic retinopathy. Gliclazide vs. other sulfonylureas.

Gliclazide has been reported to possess the properties of preventing the progression of diabetic retinopathy and of controlling blood glucose levels. This report describes a long-term comparative clinical trial of this agent to assess its efficacy against diabetic retinopathy. One hundred and fifty-nine NIDDM patients with no retinopathy or with simple retinopathy entered this trial. One hundred and nineteen patients receiving other sulfonylurea agents were randomly allocated to two groups (G: gliclazide, SU: other sulfonylureas). Forty patients continued to be treated with diet alone (D group). Finally a total of 60 patients, that is, 21 patients in the G group, 19 patients in the SU group, and 20 patients in the D group, were followed with funduscopic examinations for more than 4 years. The results are summarized as follows. (1) Distribution of background factors between the two drug therapy groups was balanced, but in the D group more male patients and relatively milder cases were involved than in the drug therapy groups. (2) Fasting blood glucose control in the three groups was not significantly different. (3) Funduscopic deterioration was observed less frequently, though not significantly, in the G group than in the other groups. (4) Progression to preproliferative retinopathy was significantly less frequent in the G group than in the SU group. Thus, gliclazide seems to have additional properties compared with other sulfonylurea drugs in preventing deterioration of diabetic retinopathy, and particularly in preventing progression to proliferative retinopathy.

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.