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T M Chan

Publications and source records attributed to T M Chan.

At least 163 records · Page 9Linked to original sources
PubMed1986

Effects of adrenalectomy on binding to and actions of adrenergic receptors.

Adrenalectomy results in significant changes in the mechanism of adrenergic activation of hepatic glycogenolysis. In adrenalectomized rats a greater role for the beta-adrenergic receptor is observed, whereas the alpha 1-adrenergic-mediated phosphorylase activation declines. Our present findings document that adrenalectomy causes a significant decrease in the high-affinity population of the alpha 1-adrenergic receptor labelled with [3H]adrenaline. Our data indicate a large increase in the number of beta-adrenergic binding sites after adrenalectomy. This increase was not consistent with the observed modest increase in the beta-adrenergic-mediated activation of cyclic AMP accumulation and glycogen phosphorylase. When alpha-adrenergic antagonists are present along with the catecholamine, a 100% increase in the adrenaline-mediated accumulation of cyclic AMP in hepatocytes from adrenalectomized rats was observed. Adrenalectomy was also shown to cause a significant increase in the hepatic alpha 2-adrenergic binding sites. These data are consistent with an inhibitory role on the beta-adrenergic-mediated activation of glycogenolysis by the hepatic alpha 2-adrenergic receptor in adrenalectomy.

Adrenalectomy↗
PubMed1986

Impaired insulin receptor phosphorylation in skeletal muscle membranes of db/db mice: the use of a novel skeletal muscle plasma membrane preparation to compare insulin binding and stimulation of receptor phosphorylation.

A method has been developed to isolate skeletal muscle plasma membranes from mice in good yield without harsh extraction procedures. The method involves perfusion of mouse hindquarters with a calcium-deficient buffer containing collagenase and hyaluronidase. This is followed by gentle disruption, filtration, and differential centrifugations. The entire procedure takes about six hours and the yield is approximately 4 mg. protein from 10 g. equivalent of hindquarter muscle. The preparation contained predominantly plasma membranes based on specific activities of marker enzymes, electron microscopic data, and specific binding sites for insulin and a -adrenergic ligand. Studies using such preparations from lean, 4-5 week old and 12-20 week old db/db mice showed marked reduction in the phosphorylation of the 95 kDa subunit of the insulin receptor of the obese mice with no change in insulin binding. In addition, there was a progressive reduction in insulin sensitivity in stimulating receptor phosphorylation in the db/db mice.

Animals↗
PubMed1986

Possible involvement of a hypothalamic dopaminergic receptor in development of genetic obesity in mice.

We have studied the binding of the dopaminergic agonist 2-[5,8-3H]amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene [( 3H]ADTN) to hypothalamic membranes from the genetically diabetic-obese (db/db) mice and their lean littermates. The specific binding of [3H]ADTN was defined as the difference between the radioligand binding to the membranes in the absence or presence of 1 microM (+)-butaclamol. In order to control nonspecific binding, all binding assays were performed in the presence of 0.1 mM catechol and 0.3 mM ascorbic acid. Binding of [3H]ADTN was rapid, dissociable, saturable and stereoselective. (+)-Butaclamol was very potent whereas (-)-butaclamol was ineffective in inhibiting the binding of this radioligand. Concentration-dependent binding experiments and Scatchard analysis of the data yielded dissociation constant (Kd) of 3.5-4.2 nM and number of binding sites (n) equivalent to 170-200 fmol/mg protein for lean mice. For db/db mice, the data yielded a Kd of 4.0-4.7 nM and an n of 400-500 fmol/mg protein. It was also shown that the anorexic drugs, amphetamine and fenfluramine, inhibited [3H]ADTN binding in a dose-dependent manner. Binding parameters, obtained using membranes from mice made obese by parenteral administration of gold thioglucose, were not significantly different from those obtained for the lean mice. It is concluded that the regulation of the hypothalamic dopaminergic receptors may be related to the lesion in the genetically obese mice.

Adenylyl Cyclases↗
PubMed1986

Effect of ciglitazone on glucose uptake and insulin sensitivity in skeletal muscle of the obese (ob/ob) mouse: distinct insulin and glucocorticoid effects.

The oral hypoglycemic agent, ciglitazone, (5-[4-(1-methylcyclohexylmethoxy)benzyl]-thiazolidine-2,4-dione), was fed for nine days to genetically obese (ob/ob) mice aged 5 to 6 weeks. This treatment resulted in a lowering of plasma glucose and circulating insulin levels, but did not cause a fall in plasma corticosterone levels. Basal 2-deoxy-D-glucose uptake by the perfused hindquarters of ob/ob mice was unchanged by ciglitazone feeding. In the presence of 0.1 mU/mL insulin in the perfusion medium, there was a significant increase in the uptake rate of 2-deoxy-D-glucose by the skeletal muscle of ciglitazone-treated ob/ob mice, while there was no insulin effect in untreated ob/ob mice. Insulin at a concentration of 1 mU/mL caused a further stimulation of 2-deoxy-D-glucose transport. However, this response was significantly lower than the maximal stimulation in lean mice. Ciglitazone feeding did not have any effect on [5-3H]-glucose metabolism by the perfused muscle which remained subnormal, suggesting that the posttransport metabolism of glucose was limited by substrate availability. In the perfused mouse liver, net [14C]-glucose production from [14C]-lactate was unchanged by ciglitazone treatment while gluconeogenesis from [14C]-alanine was reduced. These findings show that ciglitazone produces its hypoglycemic effect by improving the insulin sensitivity in skeletal muscle, as others have reported in the adipose tissue. The presence of elevated plasma levels of corticosterone and lower levels of insulin in ciglitazone-treated ob/ob mice suggests that the adrenal glucocorticoids are responsible for the basal defects in glucose transport and the hyperinsulinemia is responsible for the insulin insensitivity.

Adrenal Cortex↗
PubMed1984

Evidence for heterogeneous distribution of alpha 1, alpha 2- and beta-adrenergic binding sites on rat-liver cell surface.

Fractionation of preparations of rat-liver membranes on linear sucrose gradients revealed different profiles for the binding of alpha 1-, alpha 2- and beta-adrenergic radioligands. The peaks of binding activities of [3H]prazosin and [3H]epinephrine were clearly separated from those of [3H]yohimbine and [125I]iodocyanopindolol which appeared at lower sucrose densities. Enzyme marker activities in the sucrose subfractions indicated the presence of plasma membranes in all of the subfractions. Furthermore, the binding peaks of the various adrenergic radioligands cannot be correlated with the presence of membranes derived from microsomes, lysosomes or Golgi apparatus. Pretreatment of rat livers with concanavalin A, in order to prevent the fragmentation of the plasma membranes during isolation, resulted in the shift of the binding of [3H]yohimbine and [125I]iodocyanopindolol to sucrose-gradient subfractions of higher densities, clearly separate from fractions containing microsomes and Golgi apparatus. There was no distinct separation of the binding peaks of prazosin, yohimbine, and cyanopindolol in sucrose-gradient subfractions from concanavalin A-pretreated livers. These results are consistent with the hypothesis that alpha 1-, alpha 2-, and beta-adrenergic binding sites are associated with plasma membranes, and are heterogeneously distributed on the rat-liver cell surface.

Animals↗
PubMed1984

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats.

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.

Adrenalectomy↗
PubMed1984

Glucose transport and metabolism in the perfused hindquarters of lean and obese-hyperglycemic (db/db) mice. Effects of insulin and electrical stimulation.

Changes in glucose transport and metabolism in skeletal muscles of the obese-diabetic mice (db/db) was characterized using the perfused mouse hindquarter preparation. Metabolism of [5-3H]glucose, uptake of 3-O-[methyl-3H]glucose (methylglucose) and [2-14C]deoxyglucose (deoxyglucose) was studied under resting, electrically stimulated contracting, and insulin-stimulated conditions. Basal rate of methylglucose uptake was 255 +/- 18 and 180 +/- 9 microliter/15 min per ml intracellular fluid space for lean and db/db mice, respectively. The V- of methylglucose transport was decreased with no change in Km in the db/db mice. Both electrical stimulation and insulin (1/mU/ml) increased methylglucose uptake rate 2-fold in both lean and obese mice. We observed no significant change in insulin sensitivity in the db/db mice in stimulating methylglucose uptake which was subnormal under all conditions. Similar results were obtained using deoxyglucose. Likewise, uptake of glucose and 3H2O production from [5-3H]glucose were significantly reduced, both at rest and during electrically stimulated contraction in the db/db mouse. However, lactate production in the electrically stimulated db/db mouse preparations was not significantly different from that in the lean mice. These data suggest a major contribution from an impaired glucose transport activity to the reduction in glucose metabolism in the db/db mouse skeletal muscle.

3-O-Methylglucose↗
PubMed1984

Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice.

Glucose transport and insulin resistance in the skeletal muscle of the genetically obese (ob/ob) mouse has been studied in animals at 6-7 wk of age using an in situ perfused hindquarter preparation. Rates of glucose uptake and the uptake of radioactively labeled 2-deoxy-D-glucose were lower in muscles from intact ob/ob mice than in those from their lean littermates. Following adrenalectomy the basal rate of glucose or 2-deoxy-D-glucose uptake in the ob/ob mice was restored to normal. The uptake of 2-deoxyglucose was tested at two insulin concentrations. In lean mice, adrenalectomy had no effect on 2-deoxyglucose uptake. Adrenalectomy, however, restored the responsiveness to insulin in the ob/ob mouse to normal. The activity ratio of muscle glycogen synthase was elevated by insulin but the effect was greater in the lean animals with or without adrenal glands than in the obese mouse. Adrenalectomy in the obese mouse did not correct this defect.

Adrenal Glands↗
PubMed1984

Muscle protein turnover in the perfused hindquarters of lean and genetically obese-diabetic (db/db) mice.

The stability of the perfused mouse hindquarter was assessed for a period of 5 h, and the preparation shown to remain stable for metabolic studies over this time period. Muscle protein synthesis and degradation rates in lean and diabetic-obese (db/db) mice were measured using the in situ perfused hindquarter preparation. The rates of protein synthesis were 48% lower in the muscles of intact db/db mice than in the lean controls when expressed per gram TCA precipitable protein and 46% lower when expressed per gram dry weight. Adrenalectomy, which has been shown to restore the lean body mass of the db/db mice to normal, had the effect of returning protein synthesis rate in muscle of db/db mice to lean control values. Insulin at a dose of 1 mU/ml stimulated protein synthesis in lean mice only, showing that the process of protein synthesis in the db/db mice is also insensitive to insulin. Measurements of the rates of degradation of muscle protein showed no differences between lean and db/db mice. These findings suggest that the decreased lean body mass of db/db mice is the result of a defect in protein synthesis rather than due to altered degradation.

Adrenalectomy↗
PubMed1984

Two-dimensional NMR approaches to the study of protein structure and function.

Two-dimensional Fourier transform methods for homonuclear proton NMR spectroscopy have been introduced by Wüthrich and Ernst as a means of extending assignments in spectra of proteins. Multinuclear two-dimensional approaches also appear promising. We are applying current one- and two-dimensional NMR methods to protein family members that differ from one another by one or more amino acid substitutions. The overall goal is to elucidate relationships among the sequences, structures, and functions of these proteins: for example, to delineate primary structural requirements for changes in observable properties such as conformation, amino acid side chain dynamics, hydrogen exchange dynamics, pK'a values, and oxidation-reduction potentials. The ovomucoids from a variety of species of birds, which include a single set of 12 pairs of third-domain proteins (Mr = 6062 for turkey third domain, similar for others) that differ by single amino acid substitutions, provide a favorable system for the study of the structural and dynamic effects of single amino acid replacements. X-ray crystallographic structures of two ovomucoid third domains are available. Other series of proteins being studied by these methods include the photosynthetic electron transport proteins ferredoxin and plastocyanin.

Amino Acid Sequence↗
PubMed1983

Activation of lipolysis by epinephrine and electrical stimulation in the perfused hindquarters of lean and obese-diabetic (db/db) mice.

The effects of epinephrine and electrical stimulation on lipolysis in the skeletal muscles of lean, and obese-diabetic (db/db) mice were studied using the perfused mouse hindquarter preparation. The rate of glycerol release from the obese mouse preparation was two times that of their lean littermates. Epinephrine sensitivity in the db/db mice was reduced only with respect to lipolytic activation, but was unaltered with respect to tissue cyclic AMP elevation and glycogen phosphorylase activation. Electrical stimulation caused a prompt increase in lactate production in both lean and obese mice and elevated glycerol release only in the obese mice. Glycerol release was also increased in the lean mice by stimulating at voltages exceeding 10 volts. Cyclic AMP levels were unaffected by electrical stimulation. Omission of calcium from the perfusion medium severely blunted lipolytic activation by electrical stimulation, which could be restored by calcium readdition. These results suggest that an impairment in lipolytic activation by epinephrine distal to activation of adenylate cyclase exists in the skeletal muscle of young db/db mice. Despite of this impairment, however, lipolysis in the obese mouse muscle was more sensitive to activation by electrical stimulation through a mechanism unrelated to changes in cyclic AMP, but is dependent on extracellular calcium.

Animals↗
PubMed1982

The effect of Davallina Orientalis on bone healing--a preliminary report.

A crude methanol extract of the herb Davallina Orientalis was given intraperitoneally to test its effect on bone healing in mice. The results indicated that it could increase the breaking strength of a fracture site. In our experimental model, the lower dosage has a better result probably because of less toxicity.

Animals↗
PubMed1981

Mechanisms of hormonal regulation of liver metabolism.

The mechanism of actions of glucagon, alpha- and beta-adrenergic agonists, vasopressin and angiotensin II in the liver proposed in this article are summarized in Fig. 8. The actions of glucagon and beta-adrenergic agonists in liver can be entirely ascribed to their interaction with specific plasma membrane receptors which activate adenylate cyclase leading to the intracellular accumulation of cAMP and activation of cAMP-dependent protein kinase. This enzyme phosphorylates phosphorylase b kinase, glycogen synthase, L-type pyruvate kinase, and other liver proteins resulting in alterations in their activities which can account for several of the known hepatic responses to glucagon. There is no clear evidence that Ca2+ ions are involved in the hepatic actions of this hormone. Glucocorticoids, but not thyroid hormones, are required for normal responsiveness of the liver to glucagon. The steroids do not modify cAMP accumulation or cAMP-dependent protein kinase activation, but may act by modulating the action of the kinase on its substrates. Glucocorticoids and thyroid hormones decrease beta-adrenergic responses in the liver apparently by decreasing the number of beta-receptors. Insulin inhibits the actions of physiological concentrations of glucagon by decreasing cAMP accumulation: its mechanism of action is unknown. The actions of alpha-adrenergic agonists, vasopressin and angiotensin II on the liver resemble those of glucagon, but do not involve accumulation of cAMP or activation of cAMP-dependent protein kinase. These agents appear to act by increasing cytosolic Ca2+ thus altering the activities of Ca2+-sensitive enzymes such as phosphorylase b kinase and calmodulin-dependent glycogen synthase kinase. Their receptors appear to be located exclusively on the plasma membrane and a major mechanism by which they raise cytosolic Ca2+ is by inducing the release of this cation from mitochondria. These considerations imply the existence of an intracellular messenger(s) for these agents which is generated at the plasma membrane in response to receptor activation and exerts effects on mitochondria or perhaps other intracellular structures. Glucocorticoids and thyroid hormones increase alpha-adrenergic responses in the liver apparently by increasing the number of alpha-receptors. Insulin inhibits the responses of the liver to alpha-agonists, but not to vasopressin or angiotensin II.

Adrenergic alpha-Agonists↗
PubMed1980

The role of phosphorylation in the alpha-adrenergic-mediated inhibition of rat hepatic pyruvate kinase.

Phenylephrine in the presence of 1-methyl-3-isobutylxanthine and propanolol caused a 40-50% inhibition of pyruvate kinase (type L) activity in isolated hepatocytes, which was accompanied by a 2-3-fold increase in the phosphate content of the enzyme. These changes were blocked by the alpha-adrenergic antagonist dihydroergocryptine and could not be accounted for by the slight increase in cyclic AMP-dependent protein kinase activity generated by the alpha-adrenergic agonist. It is concluded that a significant component of the inhibition of hepatic pyruvate kinase mediated by alpha-adrenergic agonists can be attributed to a cyclic AMP-independent alteration in the phosphorylation state of the enzyme.

Animals↗
PubMed1979

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.

Adrenalectomy↗
PubMed1979

Glucagon stimulation of liver mitochondrial CO2 fixation utilizing pyruvate generated inside the mitochondria.

Glucagon administration to the intact rat has been shown to stimulate pyruvate metabolism in liver mitochondria, presumably by increasing pyruvate transport into the organelle. In this report, we used alanine in place of pyruvate to examine the possibility that glucagon might stimulate pyruvate carboxylation per se independent of its postulated action on pyruvate transport. In agreement with previous reports, injection of a low dose of glucagon (50 micrograms/kg of rat) increased respiration, ATP synthesis, pyruvate decarboxylation, and CO2 fixation in liver mitochondria subsequently isolated. When alanine was used as a substrate, CO2 fixation, but not decarboxylation, was increased in liver mitochondria isolated from glucagon-treated rats. Pyruvate accumulation under these conditions was significantly lower in the glucagon-treated rat preparation. When mitochondria were incubated in a HCO3- -deficient buffer, pyruvate accumulation was identical in both preparations. The addition of a pyruvate transport inhibitor, alpha-cyanohydroxycinnamate (0.5 mM), inhibited CO2 fixation with pyruvate by 70%, but had no effect when alanine was used. Our data therefore suggest that glucagon stimluates mitochondrial pyruvate carboxylation independent of its possible action on pyruvate transport.

Alanine↗