Nobuhiko Takahashi

Adiponectin acts in the brain to decrease body weight.

Adiponectin (ADP) is an adipocyte hormone involved in glucose and lipid metabolism. We detected a rise in ADP in cerebrospinal fluid after intravenous (i.v.) injection, consistent with brain transport. In contrast to leptin, intracerebroventricular (i.c.v.) administration of ADP decreased body weight mainly by stimulating energy expenditure. Full-length ADP, mutant ADP with Cys39 replaced with serine, and globular ADP were effective, whereas the collagenous tail fragment was not. Lep ob/ob mice were especially sensitive to i.c.v. and systemic ADP, which resulted in increased thermogenesis, weight loss and reduction in serum glucose and lipid levels. ADP also potentiated the effect of leptin on thermogenesis and lipid levels. While both hormones increased expression of hypothalamic corticotropin-releasing hormone (CRH), ADP had no substantial effect on other neuropeptide targets of leptin. In addition, ADP induced distinct Fos immunoreactivity. Agouti (A y/a) mice did not respond to ADP or leptin, indicating the melanocortin pathway may be a common target. These results show that ADP has unique central effects on energy homeostasis.

Activation of PPARγ inhibits cell growth and induces apoptosis in human gastric cancer cells

We investigated the expression of peroxisome proliferator‐activated receptor γ (PPARγ) and the role of PPARγ in cell growth in human gastric cancer cells. Reverse transcription‐polymerase chain reaction, Northern blot and Western blot analyses showed that a human gastric cancer cell line, MKN45, expressed PPARγ mRNA and protein. Luciferase assay in MKN45 cells showed that troglitazone, a selective ligand for PPARγ, transactivated the transcription of a peroxisome proliferator response element‐driven promoter. Troglitazone or pioglitazone, selective ligands for PPARγ, inhibited the growth of MKN45 cells in a dose‐dependent manner. Co‐incubation of MKN45 cells with troglitazone induced DNA ladder formation. These results suggest that human gastric cancer cells express PPARγ and that activation of PPARγ inhibits cell growth and induces apoptosis in gastric cancer cells.

PPARγ ligand-induced apoptosis through a p53-dependent mechanism in human gastric cancer cells

We have recently demonstrated that the PPARγ ligand troglitazone induced cell growth arrest and evoked apoptosis in a gastric cancer cell line, MKN–45. Since in general, p53 plays an important role in the induction of apoptosis and growth inhibition, we tried to clarify whether or not p53 mediates troglitazone‐induced apoptosis and growth arrest in gastric cancer cells. Troglitazone increased the number of apoptoic cells in MKN‐28, MKN‐45 and MKN‐74, but not in KATO‐III cells. The troglitazone‐induced apo‐ptotic change was significantly reduced by coincubation with bisphenol A digycidyl ether (BADGE), a synthetic PPARy antagonist, in MKN‐74 cells, suggesting that PPARγ mediates the apo‐ptotic effect of troglitazone. Since KATO‐III lacks the p53 gene, we speculated that p53 might be implicated in the PPARγ ligand‐induced apoptosis. Western blot analysis revealed that p53 expression was increased by troglitazone in a time‐dependent manner in MKN‐74 cells, further suggesting that p53 may mediate the ap‐optotic process induced by troglitazone. We next established a dominant‐negative p53 mutant by stable transfection of p53 mutant into MKN‐74 cells. In the dominant‐negative p53 mutant cells, troglitazone failed to induce apoptosis, strongly supporting the hypothesis that p53 indeed mediates the process of the troglitazone‐induced apoptosis. In the dominant‐negative p53 mutant cells, troglitazone significantly induced cell growth arrest and increased expression of p27Kip1 protein, which is thought to be the key molecule to evoke growth arrest, suggesting that p53 is not involved in the growth inhibition by troglitazone. All these results suggest that p53 mediates the PPARy ligand‐induced apoptosis, but not the cell growth inhibition. (Cancer Sci 2003; 94: 338–343)

Growth arrest by troglitazone is mediated by p27Kip1 accumulation, which results from dual inhibition of proteasome activity and Skp2 expression in human hepatocellular carcinoma cells

In our study, we examined whether human hepatocellular carcinoma (HCC) expresses peroxisome proliferator‐activated receptor γ (PPARγ) and the effects of PPAR γ activation by its selective ligands on cell growth and cell invasion in HCC cells. RT‐PCR and Western blot analysis revealed that HCC‐derived cell lines, HepG2 and HLF, express PPARγ mRNA and protein. Luciferase assay in HLF cells showed that troglitazone, a selective ligand for PPAR γ, transactivated the transcription of a peroxisome proliferator response element‐driven promoter in a dose‐dependent manner, suggesting that the expressed PPARγ functions as a transcriptional factor. Not only troglitazone but pioglitazone dose‐dependently inhibited cell growth in HepG2 and HLF cells. Invasion assay using a transwell chamber demonstrated that troglitazone also inhibited cell invasion in HCC cells. To examine the mechanism of the troglitazone‐induced growth inhibition, we determined p27Kip1, a cyclin dependent kinase inhibitor, expression by Western blot analysis in troglitazone‐treated HLF cells. Troglitazone increased p27Kip1 in time‐ and dose‐dependent manners, suggesting that p27Kip1 may be involved in the growth inhibition by troglitazone in HLF cells. To further examine the mechanism of the troglitazone‐induced p27Kip1 protein accumulation, 2 major systems for regulation of p27Kip1 protein, proteasome activity and Skp2, an F‐box protein that targets p27Kip1 for degradation, were evaluated. Troglitazone potently inhibited proteasome activity and decreased Skp2 protein levels. All these results suggest that human HCC cells express functional PPAR γ and PPARγ activation resulted in growth inhibition. The growth inhibition was mediated by p27Kip1 accumulation, which is induced by both inhibition of ubiquitylation of p27Kip1 and reduction of degradation activity of p27Kip1 by proteasome.

Inhibition of cell invasion and morphological change by troglitazone in human pancreatic cancer cells.

BackgroundWe have recently demonstrated that peroxisome proliferator activated receptor (PPAR) γ activation by its selective ligand, troglitazone, potently inhibited cell proliferation in human pancreatic cancer cells. The present study was performed to clarify the role of PPARγ in cell invasion/motility in human pancreatic cancer cells.MethodsCell invasive activity was assessed by an in vitro invasion assay, using a Transwell chamber, and by a wound-healing assay, in the human pancreatic cancer cell lines, PK-1 and PK-9. Cell morphology and actin structure were evaluated by phase-contrast and fluorescence microscopy.ResultsPPARγ activation by troglitazone inhibited cell invasion and cell migration in PK-1 and PK-9 cells. We also examined the effect of troglitazone on cell morphology and actin structure because of its effect on cell motility. The size of PK-1 and PK-9 cells that had been incubated with troglitazone became smaller, and the in shape changed from flat to spindle, followed by round. The troglitazone-induced cell rounding was reversible by replacement with troglitazone-free medium. Rhodamine-phalloidin staining revealed a decreased number of actin filaments in PK-1 cells treated with troglitazone. In cells treated with mycalolide B, an actin depolymerizing agent, troglitazone failed to induce cell rounding.ConclusionsThese results suggest that PPARγ activation by troglitazone inhibited cell motility and changed cell morphology through modulating actin organization.

An inverse correlation between serum leptin levels and hemoglobin A1c in patients with non-insulin dependent diabetes mellitus

We measured serum leptin concentrations in 70 patients with diabetes mellitus to investigate the relationship between serum leptin levels and glycemic control. A positive correlation between serum leptin levels and body mass index or plasma insulin was obtained as reported previously. The present study also demonstrated an inverse association of serum leptin levels with hemoglobin A1c (HbA1c). Multiple regression analysis revealed that HbA1c was an independent determinant of serum leptin levels. These results suggest that HbA1c may be a factor to influence serum leptin levels and that hyperglycemia for a long period or poorly controlled diabetes may reduce leptin levels.

Delayed gastric emptying by Helicobacter pylori lipopolysaccharide in conscious rats

The present study was carried out to investigatethe possibility that lipopolysaccharide deprived fromHelicobacter pylori may alter gastric motility. Toaddress the question, we examined the effect of H. pylori lipopolysaccharide on gastricemptying in conscious rats. Gastric emptying wasevaluated by the phenol red method. Time-course anddose-related effects of intraperitoneal administrationof H. pylori lipopolysaccharide were investigated.Intraperitoneal injection of H. pylorilipopolysaccharide significantly suppressed gastricemptying of a liquid meal in a dose-dependent manner.The inhibitory action of H. pylori lipopolysaccharide wasobserved 2, 4, 8, or 12 hr after the injection. Theseresults suggest for the first time that H. pylorilipopolysaccharide may suppress gastric emptying in along-lasting fashion. It is also suggested that H. pylorimay influence gastric function through its cell wallstructure named lipopolysaccharide.

A selective orexin-1 receptor antagonist, SB334867, blocks 2-DG-induced gastric acid secretion in rats.

We have previously demonstrated that intracisternal orexin-A potently stimulated gastric acid secretion through the vagus nerve. Considering its stimulatory action on feeding, we hypothesized that orexin-A is a candidate mediator of cephalic phase gastric secretion. It has also been suggested that the stimulation of acid by central orexin-A may be mediated by orexin 1 receptor (OX1R) in the brain. In the present study, we tried to clarify whether endogenously released orexin-A in the brain indeed plays a physiological role in gastric secretion. To address the question, the effects of OX1R antagonist on gastric acid secretion was examined in rats. Intraperitoneal administration of SB334867, a specific OX1R antagonist, by itself did not change gastric acid secretion in pylorus-ligated conscious rats. Pretreatment with SB334867 in a dose of 10 mg/kg completely blocked the stimulated acid output by intracisternal orexin-A but not thyrotropin-releasing hormone, suggesting that SB334867 specifically blocked the action of orexin-A in the brain. 2-Deoxy-D-glucose (2-DG)-induced stimulation of gastric acid output was significantly blocked by pretreatment with intraperitoneal administration of SB334867. These results suggest that endogenously released orexin-A in the brain plays a vital role in central regulation of gastric secretion. Since 2-DG induces central glucoprivation as a hunger state, the present study furthermore supports the speculation that orexin-A may be an important molecule that triggers the cephalic phase gastric acid secretion.

Troglitazone Induces G1 Arrest by p27Kip1 Induction That Is Mediated by Inhibition of Proteasome in Human Gastric Cancer Cells

We examined in the present study whether human gastric cancer cells express peroxisome proliferator‐activated receptor γ (PPARγ), the effect of PPARy activation by troglitazone, a selective ligand, on cellular growth, and the mechanism of the growth arrest by troglitazone in gastric cancer cells. RT‐PCR, northern blot and western blot analysis demonstrated that all four tested human gastric cancer cell lines, MKN–28, MKN–45, MKN–74 and KATO‐III, expressed PPARγ mRNA and protein. WST–1 assay and flow cytometric analysis revealed that troglitazone inhibited the growth and induced G1 arrest in all four gastric cancer cell lines. To examine the role of p27Kip1, a cyclin‐dependent kinase inhibitor, in the G1 arrest by troglitazone, we determined p27Kip1 protein expression by western blot analysis in gastric cancer cells that had been treated with troglitazone. Troglitazone increased p27Kip1 in all four gastric cancer cell lines. Since it has been reported that the ubiquitin‐proteasome system plays a vital role in the degradation of p27Kip1 protein, we evaluated the hypothesis that inhibition of proteasome mediates the troglitazone‐induced p27Kip1 accumulation. Lactacystin, a proteasome inhibitor, inhibited cell growth and increased p27Kilp1 expression in MKN–74 cells. It was further demonstrated that troglitazone inhibited proteasome activity in a dose‐dependent manner in MKN–74 cells. All these results suggest that troglitazone inhibited proteasome activity, followed by induction of p27Kipl, which arrests cells at the Gl phase of the cell cycle in gastric cancer cells. The troglitazone‐mediated inhibition of the proteasome suggests a novel mechanism for the anti‐proliferative effect of this agent in cancer cells.

A Jak2 inhibitor, AG490, reverses lipin-1 suppression by TNF-α in 3T3-L1 adipocytes

Lipin-1 is a multifunctional metabolic regulator, involving in triacylglycerol and bioactive glycerolipids synthesis as an enzyme, transcriptional regulation as a coactivator, and adipogenesis. In obesity, adipose lipin-1 expression is decreased. Although lipin-1 is implicated in the pathogenesis of obesity, the mechanism is still not clear. Since TNF-alpha is deeply involved in the pathogenesis of obesity, insulin resistance, and diabetes, here we investigated the role of TNF-alpha on lipin-1 expression in adipocytes. Quantitative PCR studies showed that TNF-alpha suppressed both lipin-1A and -1B isoform expression in time- and dose-dependent manners in mature 3T3-L1 adpocytes. A Jak2 inhibitor, AG490, reversed the suppressive effect of TNF-alpha on both lipin-1A and -1B. In contrast, NF-kappaB, MAPKs, ceramide, and beta-catenin pathway tested were not involved in the mechanism. These results suggest that TNF-alpha could be involved in obesity-induced lipin-1 suppression in adipocytes and Jak2 may play an important role in the mechanism.