Search Results

You are looking at 1 - 4 of 4 items for

  • Author: T. Kato x
  • Refine by access: Content accessible to me x
Clear All Modify Search
Y Kato
Search for other papers by Y Kato in
Google Scholar
PubMed
Close
,
I Sato
Search for other papers by I Sato in
Google Scholar
PubMed
Close
,
T Ihara
Search for other papers by T Ihara in
Google Scholar
PubMed
Close
,
K Tomizawa
Search for other papers by K Tomizawa in
Google Scholar
PubMed
Close
,
J Mori
Search for other papers by J Mori in
Google Scholar
PubMed
Close
,
M Geshi
Search for other papers by M Geshi in
Google Scholar
PubMed
Close
,
T Nagai
Search for other papers by T Nagai in
Google Scholar
PubMed
Close
,
K Okuda
Search for other papers by K Okuda in
Google Scholar
PubMed
Close
,
T Kato
Search for other papers by T Kato in
Google Scholar
PubMed
Close
, and
S Ueda
Search for other papers by S Ueda in
Google Scholar
PubMed
Close

Biologically active recombinant porcine FSH (rec-pFSH) free from the cognate pituitary glycoprotein hormone LH was produced. It was synthesized by a baculovirus vector-insect cell system using two cDNAs encoding the glycoprotein alpha and FSH beta subunits. Its antigenicity was the same as that of pFSH prepared from the pituitary. Glycosylation of rec-pFSH was shown by tunicamycin treatment but the molecular mass of each subunit was lower than that of pituitary-derived FSH, because of the absence of trimming of terminal sugars in insect cells. Rec-pFSH was secreted into the culture medium at about 1 mg/l and purified in six fractions, because of the heterogeneity of the sugar group, by S-Sepharose and concanavalin A-Sepharose column chromatography. The biological activity of rec-pFSH was examined by measuring its effect on progesterone secretion from porcine granulosa cells and germinal vesicle breakdown (GVBD) of porcine oocytes. It showed adequate activity with respect to progesterone secretion, although some fractions rich in the sugar group showed lower activity than that of pituitary-derived FSH. It exhibited higher GVBD activity than that of pituitary-derived FSH at concentrations as low as 1 ng/ml. These results demonstrate that the baculovirus vector-insect cell system can provide biologically active rec-pFSH.

Free access
M Imae
Search for other papers by M Imae in
Google Scholar
PubMed
Close
,
Z Fu
Search for other papers by Z Fu in
Google Scholar
PubMed
Close
,
A Yoshida
Search for other papers by A Yoshida in
Google Scholar
PubMed
Close
,
T Noguchi
Search for other papers by T Noguchi in
Google Scholar
PubMed
Close
, and
H Kato
Search for other papers by H Kato in
Google Scholar
PubMed
Close

Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in response to fasting also suggest that the regulation of nucleus/cytoplasm translocation actually functions in vivo.

Free access
Y Toyoshima
Search for other papers by Y Toyoshima in
Google Scholar
PubMed
Close
,
Y Ohne
Search for other papers by Y Ohne in
Google Scholar
PubMed
Close
,
SI Takahashi
Search for other papers by SI Takahashi in
Google Scholar
PubMed
Close
,
T Noguchi
Search for other papers by T Noguchi in
Google Scholar
PubMed
Close
, and
H Kato
Search for other papers by H Kato in
Google Scholar
PubMed
Close

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.

Free access
H Tokuda Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan
Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by H Tokuda in
Google Scholar
PubMed
Close
,
K Kato Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan
Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by K Kato in
Google Scholar
PubMed
Close
,
H Natsume Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan
Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by H Natsume in
Google Scholar
PubMed
Close
,
A Kondo Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan
Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by A Kondo in
Google Scholar
PubMed
Close
,
G Kuroyanagi Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan
Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by G Kuroyanagi in
Google Scholar
PubMed
Close
,
R Matsushima-Nishiwaki Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by R Matsushima-Nishiwaki in
Google Scholar
PubMed
Close
,
Y Ito Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by Y Ito in
Google Scholar
PubMed
Close
,
T Otsuka Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by T Otsuka in
Google Scholar
PubMed
Close
, and
O Kozawa Department of Clinical Laboratory, Department of Pharmacology, Department of Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu 474-8511, Japan

Search for other papers by O Kozawa in
Google Scholar
PubMed
Close

We previously demonstrated that thrombin stimulates synthesis of interleukin 6 (IL6), a potent bone resorptive agent, in part via p44/p42 MAP kinase and p38 MAP kinase but not through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) among the MAP kinase superfamily in osteoblast-like MC3T3-E1 cells. In this study, we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in thrombin-stimulated IL6 synthesis in MC3T3-E1 cells. The phosphorylation of p44/p42 MAP kinase, p38 MAP kinase, SAPK/JNK, or AMPK was determined by western blot analysis. The release of IL6 was determined by the measurement of IL6 concentration in the conditioned medium using an ELISA kit. The expression of IL6 mRNA was determined by RT-PCR. Thrombin time dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an inhibitor of AMPK, dose-dependently suppressed the thrombin-stimulated IL6 release in the range between 0.3 and 10 μM. Compound C reduced thrombin-induced acetyl-CoA carboxylase phosphorylation. The IL6 mRNA expression induced by thrombin was markedly reduced by compound C. Downregulation of AMPK by siRNA suppressed the thrombin-stimulated IL6 release. The thrombin-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase was inhibited by compound C, which failed to affect SAPK/JNK phosphorylation. These results strongly suggest that AMPK regulates thrombin-stimulated IL6 synthesis via p44/p42 MAP kinase and p38 MAP kinase in osteoblasts.

Free access