[Insulin and insulin receptor disorders].
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Diabetes Mellitus↗
Biomedical subjects
M Kasuga
Publications and source records attributed to M Kasuga.
Phorbol ester stimulates the activity of a protein tyrosine phosphatase containing SH2 domains (PTP1C) in HL-60 leukemia cells by increasing gene expression.
The affinity-purified antibody to a protein tyrosine phosphatase (PTP) containing two src homology 2 domains (PTP1C) was generated. The antibody recognized two types of PTP1C (PTP1C-alpha and -beta) of which the molecular sizes were 66 (alpha) and 62 kDa (beta), respectively, and these two types were expressed differentially in various cell types. The immune complex phosphatase assay using the antibody demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) and a vitamin D metabolite increased the PTP activity of immunoprecipitated PTP1C to 230 and 150% of control, respectively. By contrast, neither dimethyl sulfoxide nor retinoic acid significantly affected the PTP activity of PTP1C in HL-60 cells. The time course increment by TPA of PTP1C activity was closely correlated with that of the acquisition by HL-60 cells of a macrophage-like phenotype. In addition, TPA increased the amount of PTP1C detected by immunoblotting and immunoprecipitation and raised the level of expression of PTP1C mRNA in HL-60 cells. The increase of PTP1C mRNA induced by TPA treatment was inhibited by cycloheximide, suggesting that new protein synthesis is required for the increase by TPA of PTP1C mRNA expression. Furthermore, TPA increased the rate of transcription of the PTP1C gene without affecting the stability of PTP1C mRNA. These results suggest that (i) two subtypes of PTP1C may exist and function in various cell types, and (ii) TPA stimulates the PTP activity of PTP1C by increasing the transcription rate of PTP1C gene expression. The possible role of PTP1C in the macrophage differentiation will be also discussed.
Base Sequence↗
A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.
Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.
Amino Acid Sequence↗
Phosphorylation sites of myelin basic protein by a catalytic fragment of non-receptor type protein-tyrosine kinase p72syk and comparison with those by insulin receptor kinase.
Myelin basic protein has been used as a model substrate for determination of substrate recognition motif of various protein kinases. In this report phosphorylated sites of bovine brain myelin basic protein were studied with a catalytic fragment of protein-tyrosine kinase p72syk. Three of four tyrosine residues in myelin basic protein were phosphorylated by this kinase. Major phosphorylated site was 134Y and minor phosphorylated sites were 68Y and 127Y. As the phosphorylation site by p56lck was only 68Y, the recognition motif of p72syk was quite different from that of p56lck. Furthermore, the fact that elution pattern on HPLC of the phosphopeptides obtained by insulin receptor kinase was different from that by p72syk suggested that the recognition motif of p72syk was also different from that of insulin receptor kinase. These results may suggest that each protein-tyrosine kinase has a specific substrate recognition motif.
Amino Acid Sequence↗
Effect of T-cell receptor V beta-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice.
The expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor V beta-specific monoclonal antibodies. No significant age- or sex-related differences were observed in V beta usage by peripheral and splenic T lymphocytes. CD8+ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their V beta gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor V beta segments in the development of diabetes mellitus, T-cell receptor V beta-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-V beta 3 (11 of 22 mice became diabetic, 50%), anti-V beta 5 (9 of 14, 64%), anti-V beta 8 (9 of 21, 43%), anti-V beta 11 (12 of 23, 52%), anti-V beta 14 (7 of 12, 58%), and anti-V beta 5 + anti-V beta 11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor V beta usage between diabetic and non-diabetic cyclophosphamide-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Effect of islet hormones on secretin-stimulated exocrine secretion in isolated perfused rat pancreas.
To clarify the effect of islet hormones on pancreatic ductular cell function, we measured the exocrine secretion elicited by 10 pM secretin in the presence or absence of islet hormones using an isolated perfused rat pancreas model. Insulin significantly increased secretin-stimulated pancreatic juice secretion, but not protein secretion. The potentiating effect of insulin on pancreatic juice secretion was concentration-dependent, and the maximal effect was observed with 1 microM insulin. Ouabain, a specific Na+,K(+)-ATPase inhibitor, caused concentration-dependent inhibition of the potentiating effect of insulin without affecting secretin action. Glucagon (100 nM) significantly inhibited secretin-stimulated pancreatic juice secretion and also tended to inhibit protein secretion. A somatostatin analog, SMS 201-995 (10 nM) significantly inhibited both the pancreatic juice and protein secretion stimulated by secretin. The inhibitory effect of SMS 201-995 was concentration-dependent and was maximal at 1-10 nM. These results demonstrate that insulin potentiates the secretory response to secretin, at least partly by increasing Na+,K(+)-ATPase activity, whereas glucagon and somatostatin inhibit this response. Thus, pancreatic islet hormones regulate the secretory function of pancreatic ductular and centroacinar cells.
Animals↗
In vitro inhibitory effect of somatostatin on secretin action in exocrine pancreas of rats.
BACKGROUND: Exocrine pancreatic function is influenced by pancreatic islet hormones. Although the existence of somatostatin receptors has been shown on pancreatic acinar cells, the in vitro effect of somatostatin on exocrine secretory function has not been established. METHODS: Using isolated rat pancreatic acini, the effect of somatostatin analog SMS 201-995 (SMS) and somatostatin 14 (S-14) on amylase release, cyclic adenosine monophosphate (cAMP) production, and hormone binding were determined. RESULTS: SMS inhibited the potentiating effect of secretin on amylase response to cholecystokinin octapeptide (CCK-8) in a concentration-dependent manner. The inhibitory effects of SMS and S-14 were similar on a molar basis and were observed when vasoactive intestinal polypeptide (VIP) but not 8bromoadenosine 3':5' cyclic monophosphate was used instead of secretin and when carbachol, bombesin, A23187, and 12-O-tetradecanoylphorbol 13-acetate were used instead of CCK-8. SMS inhibited secretin-induced cAMP production, and the dose-inhibition curve for cAMP was similar to that for amylase release. SMS had no influence on 125I-secretin and 125I-VIP binding. CONCLUSIONS: Somatostatin acts directly on acinar cells and inhibits secretin potentiation of secretory response in part by inhibiting secretin-induced cAMP production.
8-Bromo Cyclic Adenosine Monophosphate↗
Cholecystokinin octapeptide analogues suppress food intake via central CCK-A receptors in mice.
To examine the mechanism of the satiety-producing effect of cholecystokinin (CCK) in the central nervous system, we compared the potency of intraperitoneally (ip) or intracerebroventricularly (icv) administered CCK-8 and its analogues on food intake in fasted mice. The icv administration of a small dose of CCK-8 (0.03 nmol/brain) or of Suc-(Thr28, Leu29, MePhe33)-CCK-7 (0.001 nmol/brain) suppressed food intake for 20 min, whereas CCK-8 (1 nmol/kg, which is equivalent to 0.03 nmol/brain) or Suc-(Thr28, Leu29, MePhe33)-CCK-7 (1 nmol/kg) had satiety effect after ip administration. Dose-response studies indicated the following rank order of potency: Suc-CCK-7 > or = Suc-(Thr28, Leu29, MePhe33)-CCK-7 > or = CCK-8 > or = (Nle28,31)-CCK-8 >> desulfated CCK-8 = CCK-4 = 0 in the case of ip administration and Suc-(Thr28, Leu29, MePhe33)-CCK-7 >> Suc-CCK-7 > or = CCK-8 > or = (Nle28,31)-CCK-8 >> desulfated CCK-8 = CCK-4 = 0 in the case of icv administration. The selective CCK-A receptor antagonist MK-329 reversed the inhibitory effect of the centrally as well as peripherally administered CCK-8, or of Suc-(Thr28, Leu29, MePhe33)-CCK-7, whereas the selective CCK-B receptor antagonist L-365260 did not. The icv administered CCK-8 did not appear in the peripheral circulation. These findings suggest the participation of CCK-A receptors in the brain in mediating the satiety effect of CCK and the difference in CCK-A receptors in the brain and peripheral tissues.
Amino Acid Sequence↗
Plasma and cerebroventricular fluid levels of pancreatic polypeptide in the dog: effects of feeding, insulin-induced hypoglycemia, and physical exercise.
We investigated the penetration of plasma pancreatic polypeptide (PP) into the third cerebral ventricular fluid (CSF) of dogs. Plasma and CSF levels of PP were measured by RIA during the iv infusion of PP and during such stimuli as eating, insulin-induced hypoglycemia, and physical exercise. Plasma and CSF levels of insulin and glucose were also measured and compared during eating and insulin-induced hypoglycemia. Plasma glucose increased after feeding and decreased after insulin injection, followed by a corresponding change in CSF glucose without an apparent time lag. CSF insulin insignificantly increased after feeding and the injection of insulin, while CSF PP did not increase despite the marked elevation of plasma PP in response to these stimuli. CSF PP did not increase after the infusion of exogenous PP. Strenuous exercise, however, evoked an increase in both plasma and CSF PP levels; the CSF response was prompt, but more prolonged than that of plasma, suggesting the slow removal of PP from CSF. We conclude that 1) PP and insulin in CSF do not appear to play a major role in the short term regulation of food intake and acute changes in energy metabolism; and 2) PP, probably after entering the brain, may modulate brain function in such physiological situations as strenuous exercise.
Animals↗
[A case of prolonged liver injury caused by low dose of acetaminophen--the involvement of other multiple drugs such as Proheparum and Mazulene-S was considered in this drug-induced hepatitis].
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Acetaminophen↗
[Inhibitory effect of somatostatin analogue, SMS 201-995, on secretin-stimulated exocrine secretion in isolated perfused rat pancreas].
In order to clarify the effect of somatostatin of the ductal secretion of the exocrine pancreas, we measured pancreatic juice and protein secretion stimulated with 10 pM secretin and/or 10 pM cholecystokinin (CCK) in the presence or absence of somatostatin analogue, SMS 201-995 (SMS) utilizing the isolated perfused pancreas of rats. SMS significantly inhibited both pancreatic juice flow and protein output elicited by 10 pM secretin without affecting basal secretion. The inhibitory effect of SMS was dose-dependent and maximal inhibition was observed with 1-10 nM. Half-inhibitory dose of SMS for juice secretion was 140 pM. Because CCK is thought to potentiate secretin action on the ductal system, we examined the effect of SMS on pancreatic secretory response to 10 pM secretin in combination with 10 pM CCK. In the experimental system we used, the amounts of pancreatic juice and protein secreted during a 30-min stimulation with secretin and CCK were additive. SMS inhibited both pancreatic juice and protein secretion to the level comparable with that obtained with either stimulus and SMS. SMS had no effect on CCK-stimulated pancreatic juice secretion but significantly inhibited protein output. The present study demonstrated, therefore, that SMS inhibits ductal secretion in response to physiological concentration of secretin.
Animals↗
Hypertriglyceridemia in nephrotic rats is due to a clearance defect of plasma triglyceride: overproduction of triglyceride-rich lipoprotein is not an obligatory factor.
This study was conducted to determine whether overproduction of triglyceride-rich lipoprotein is an obligatory factor for experimental hypertriglyceridemia in nephrotic rats. Nephrosis was induced in male Wistar rats by administration of 150 mg/kg puromycin aminonucleoside. Nephrotic rats had slightly increased triglyceride secretion rate (TGSR) estimated using Triton WR1339 (0.53 +/- 0.05 vs. 0.45 +/- 0.04 mg/min, P < 0.05 vs. control rats) and marked hypertriglyceridemia (330.4 +/- 78.6 mg/dl). Rats made diabetic by 40 mg/kg streptozotocin were normotriglyceridemic (66.3 +/- 12.1 mg/dl) but had suppressed TGSR (0.33 +/- 0.09 mg/min). Experimental nephrosis was induced in diabetic rats. Their TGSR remained suppressed (0.35 +/- 0.06 mg/min) but they had marked hypertriglyceridemia (296.6 +/- 72.4 mg/dl) suggesting further impairment of triglyceride removal from the circulation in diabetic rats caused by nephrosis. Endogenously radiolabeled very low density lipoprotein (VLDL)-triglyceride from donor rats was reinjected into normal recipient rats. [3H]VLDL from the experimental groups (the rats with nephrosis, diabetes with nephrosis, and diabetes alone) were more slowly cleared by normal rats than VLDL from normal rats. These results suggest that circulating insulin is essential for increased triglyceride secretion in experimental nephrosis and that nephrotic hypertriglyceridemia can be induced only by a triglyceride removal defect. Therefore, hypersecretion of triglyceride-rich lipoprotein is not an obligatory factor for nephrotic hypertriglyceridemia.
Animals↗
[Diabetes mellitus as a risk factor for arteriosclerosis].
Diabetes mellitus is an important risk factor for coronary heart disease and cerebral vascular disease. Moreover, diabetic patients are frequently hyperlipidemic (our recent study has revealed that approximately 70% of diabetic patients are dyslipidemic) and are at a higher risk when complicated with hyperlipidemia. In addition, abnormalities in lipoprotein metabolism, which may promote arteriosclerosis, are seen even in normolipidemic diabetics. One such alteration is an increased cholesterol level in intermediated-density lipoproteins from normolipidemic diabetic patients. Recently, abnormal particles in low-density lipoprotein (LDL) fraction, glycated-LDL and small, dense LDL, are regarded as atherogenic lipoproteins. Several factors promoting arteriosclerosis in diabetics, such as abnormal lipoprotein metabolism, increase of lipoprotein (a), hyperinsulinemia, enhancement of polyol pathway, dysfunction of platelet, are described here.
Arteriosclerosis↗
[Effect of cholecystokinin and secretin on insulin binding to rat pancreatic acini and pancreatic cancer cell line AR42J cells].
In order to clarify the interaction of hormones which exert various effects on the exocrine pancreas, we investigated the effect of cholecystokinin (CCK) and secretin on subsequent insulin binding to pancreatic acini and cultured AR42J cells derived from azaserine-induced acinar cell carcinoma of the pancreas. CCK at concentrations of 100pM-10nM inhibited subsequent 125I-insulin binding to pancreatic acini. 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited 125I-insulin binding whereas A23187 had little effect, suggesting that the inhibitory effect of CCK is mediated by protein kinase C. On the other hand, 100pM-10nM secretin had no effect on subsequent 125I-insulin binding to pancreatic acini, although higher concentrations of forskolin and 8 bromoadenosine 3', 5'-cyclic monophosphate inhibited 125I-insulin binding. In addition, secretin exerted no potentiating effect on the inhibitory effect of CCK on 125I-insulin binding to pancreatic acini. Based on these results, we further investigated the effect of CCK and TPA on subsequent 125I-insulin binding to AR42J cells. In this carcinoma cell line, inhibitory effect of CCK and TPA on insulin binding was completely abolished. The present results suggest, therefore, that hormonal interaction may play an important role in the regulation of exocrine pancreatic function including acinar cell growth.
Animals↗
Biphasic activation of two mitogen-activated protein kinases during the cell cycle in mammalian cells.
We studied mitogen-activated protein kinase (MAPK) activities during the cell cycle of Chinese hamster ovary (CHO) cells using site-specific antibodies against extracellular signal-regulated kinase-1, a 44-kDa MAPK (Boulton, T.G., Yancopoulos, G.D., Gregory, J.S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M.H. (1990) Science 249, 64-67). These antibodies detected two distinct MAPKs (44- and 42-kDa MAPKs) in CHO cells. CHO cells were arrested at metaphase in the M phase by treatment with nocodazole, and activities of MAPKs were analyzed at specific time points after release from arrest. Immune complex kinase assay and renaturation and phosphorylation assay in substrate-containing gel revealed that both 44- and 42-kDa MAPKs had activities in the G1 through S and G2/M phases and were activated biphasically, in the G1 phase and around the M phase. MAPKs were inactivated in metaphase-arrested cells. The amount of MAPKs did not change significantly in the cell cycle. In the G1, S, and G2/M phases, MAPKs were phosphorylated on both tyrosine and threonine residues and dephosphorylated in metaphase-arrested cells. Our data suggest that MAPKs may play some role in the cell cycle other than G0/G1 transition.
Amino Acid Sequence↗
[Metabolic disease and molecular biology].
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Diabetes Mellitus↗
Effects of antiulcer drugs on phosphatidylcholine synthesis in isolated guinea pig gastric glands.
To better understand phosphatidylcholine synthesis in the stomach, we isolated guinea pig gastric glands and examined their [3H]choline incorporation into phosphatidylcholine in response to either antiulcer drugs such as geranylgeranylacetone (GGA) and H2-receptor antagonists or agents that cause phosphatidylcholine synthesis in other tissues. [3H]Choline incorporation was stimulated by GGA, palmitate, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Dibutyryl cyclic-AMP had no effect. By contrast with GGA, famotidine, ranitidine, and cimetidine equipotently inhibited [3H]choline incorporation into phosphatidylcholine. GGA, palmitate, and TPA increased phosphatidyl-[3H]choline and decreased phosphoryl-[3H]choline as compared with control in tissues that had been pulsed with [3H]choline. On the other hand, no more decrease in [3H]choline incorporation at chase periods was observed in pulse-labeled glands in response to each H2-receptor antagonist. The particulate fraction of glands that had been incubated with GGA or palmitate had more CTP-phosphocholine cytidylyltransferase activity than that of glands incubated without agents. A decrease in choline kinase activity was not observed in the cytosolic fraction of glands that had been incubated with cimetidine. These results suggest that GGA and palmitate stimulate phosphatidylcholine synthesis by activating cytidylyltransferase, and H2-receptor antagonists may affect phosphatidylcholine synthesis by inhibiting choline uptake in the gastric glands.
Analysis of Variance↗
M3 muscarinic receptors mediate pepsinogen secretion via polyphosphoinositide hydrolysis in guinea pig gastric chief cells.
The muscarinic receptor system involved in pepsinogen secretion from isolated guinea pig gastric chief cells was investigated by evaluating the effect of muscarinic receptor antagonists on carbamylcholine (CCh)-stimulated chief cell responses. CCh stimulated the hydrolysis of polyphosphoinositide in chief cells at the same concentrations as it stimulated pepsinogen secretion. Each of five different muscarinic receptor antagonists, atropine, pirenzepine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), AF-DX116 and scopolamine, inhibited both pepsinogen secretion and inositol phosphate accumulation stimulated by graded concentrations of CCh. The pA2 values of the antagonists calculated from data on inositol phosphate accumulation and pepsinogen secretion (atropine = scopolamine = 4-DAMP greater than pirenzepine greater than AF-DX116) suggest that the muscarinic acetylcholine receptor in gastric chief cells is the M3 subtype. On the other hand, CCh did not affect the adenylate cyclase/cAMP signaling pathway in gastric chief cells. All pA2 values of the antagonists were also in agreement with the Ki values determined by [3H]NMS binding to chief cells. Furthermore, GTP gamma S reduced [3H]acetylcholine binding to chief cell membranes in a concentration-dependent manner. The present study, therefore, suggests that muscarinic M3 receptors, which may be coupled to a G protein, mediate pepsinogen secretion, probably by activation of the polyphosphoinositide second messenger system in guinea pig gastric chief cells.
Animals↗