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J M May

Publications and source records attributed to J M May.

At least 91 records · Page 5Linked to original sources
PubMed1986

Testing models of insulin binding in rat adipocytes using network thermodynamic computer simulations.

Many different models have been proposed to explain the complex binding behavior of insulin to its receptor, but a systematic comparison of models with experimental results is lacking. We have used network thermodynamic computer simulations to compare models of insulin binding against the results of several experimental tests designed to differentiate between the models. Six models of insulin binding were tested (simple, diffusion-reaction, conversion, dissociation, heterogeneous site, and two-step intramembrane) against results reported in the literature for isolated rat adipocytes. Although still a matter of experimental controversy, the criteria selected for modeling were curvilinear Scatchard plots, bi-or multi-exponential dissociation, insulin-accelerated dissociation, lack of dependence of the overall dissociation constant on receptor number, and receptor reserve. Using a given set of parameter values most appropriate for each model, none was able to account for all of the observed experimental results. This indicates both the complexity of the binding reaction and the need for further model development. The approach of using computer simulations to systematically test models against experimental results affords not only insight into the critical features of a model enabling it to pass a test, but also indicates potential experiments which might differentiate between models.

Adipose Tissue↗
PubMed1985

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes.

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.

3-O-Methylglucose↗
PubMed1985

Binding of agonists and antagonists to beta-adrenoceptors in rat vas deferens: relationship to functional response.

The properties of beta-adrenoceptors in rat vas deferens were examined using radioligand binding assays of 125I-pindolol(125IPIN) and inhibition of electrically-evoked contractions of vas deferens in vitro. 125IPIN labelled a single class of high affinity binding sites with apparently mass action kinetics in membrane preparations of vas deferens with properties consistent with an essentially homogeneous population of beta 2-adrenoceptors. Isoprenaline inhibited electrically evoked (60 V, 1.0 ms, 0.1 Hz) contractions of vas deferens with an EC50 of 18.0 +/- 2.1 nM. KB values for antagonists in competitively antagonizing this response correlated well (r2 = 0.99) with the KD values for inhibition of 125IPIN binding. Inhibition of 125IPIN binding by isoprenaline, adrenaline, noradrenaline and salbutamol was determined under conditions designed to produce high and low affinity agonist binding. In the presence of 10 mM MgCl2, agonists inhibited specific 125IPIN binding with a relatively high potency and low Hill slope, while in the presence of 154 mM NaCl and 300 microM guanosine-5'-triphosphate, agonists inhibited specific 125IPIN binding with a lower potency and an apparent Hill slope closer to 1. To determine which affinity state was relevant to functional receptor stimulation, receptor density was decreased with bromoacetylalprenololmenthane (BAAM). Treatment of membrane preparations with 0.3 microM BAAM produced a 45% decrease in the Bmax for 125IPIN with no change in the apparent KD. Treatment of intact vasa deferentia with increasing concentrations of BAAM resulted in a progressive rightward shift in the dose-response curve to isoprenaline or salbutamol followed by a decreased maximum response. KA values for isoprenaline and salbutamol in activating the functional beta-adrenoceptors were compared with KI values for agonist inhibition of specific 125IPIN binding. The KA values for both agonists were not significantly different from the low affinity KI values, but were significantly different from the high affinity KI values. These data suggest that 1) a homogeneous population of beta 2-adrenoceptors inhibiting contraction of rat vas deferens can be labelled with 125IPIN, 2) there is a substantial beta-adrenoceptor reserve in rat vas deferens; and 3) the initial event in signal transduction by beta-adrenoceptors in rat vas deferens is the binding of agonists to the low affinity form of the receptor which is not complexed with the guanine nucleotide binding protein.

Adrenergic beta-Agonists↗
PubMed1985

Thyroid status and adrenergic receptor subtypes in the rat: comparison of receptor density and responsiveness.

The density and functional responsiveness of adrenergic receptor subtypes were determined in tissues from control, hyperthyroid and hypothyroid rats. There was a decrease in sensitivity to isoproterenol in spontaneously beating right atria, electrically driven left atria and field-stimulated vas deferens associated with hypothyroidism, with no change in maximum response. Hyperthyroidism increased the potency of isoproterenol in right atria, but not in left atria or vas deferens. The maximal response to isoproterenol was greatly reduced in hyperthyroid left atria. The potency of procaterol, a partial agonist at beta adrenergic receptors in right atria, was unaltered in hyper- or hypothyroidism, although the maximum stimulation by procaterol was increased in hyperthyroidism. Scatchard analysis of specific [125I]pindolol binding showed that beta adrenergic receptor density was greater in hyperthyroidism than in hypothyroidism in left atria, right atria, ventricles, vas deferens and cerebral cortex, although the proportions of beta-1 and beta-2 adrenergic receptor subtypes did not change. There was no change in the responsiveness of alpha-1 adrenergic receptors mediating contraction of caudal artery and vas deferens or mediating [3H]inositol phosphate accumulation in cerebral cortex in hyperthyroid or hypothyroid rats, although the maximal contraction of caudal artery was significantly reduced in hyperthyroidism. Scatchard analysis of specific [125I]BE 2254 binding showed that alpha-1 adrenergic receptor density was significantly decreased in the ventricles from hyperthyroid rats and increased in the ventricles of hypothyroid rats, but was unchanged in vas deferens, caudal artery and cerebral cortex. Alpha-2 adrenergic receptor density in cerebral cortex, determined by Scatchard analysis of specific [3H] rauwolscine binding, was not altered in hyperthyroid or hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

In vitro phosphorylation of a synthetic collagen peptide by cyclic AMP-dependent protein kinase.

A synthetic tridecapeptide that corresponds closely to amino acid residues 98 to 110 in chick collagen alpha 1(I) contains several determinants of specificity required for recognition and phosphorylation by the cyclic AMP-dependent protein kinase. The peptide Gly-Leu-Hyp-Gly-Nle-Lys-Gly-His-Arg-Gly-Phe-Ser-Gly was predicted to be a substrate for the cyclic AMP-dependent protein kinase because it contained multiple basic amino acids NH2-terminal to a potentially phosphorylatable seryl residue. When tested as a substrate for the enzyme, the peptide was stoichiometrically phosphorylated. Phosphoserine was identified as the only phosphoamino acid in a partial hydrolysate of the phosphorylated peptide. The peptide and several of its analogs were phosphorylated by the cyclic AMP-dependent protein kinase with Km values from 1 to 6 mM and Vmax values from 1 to 3 mumol of phosphate/min/mg of enzyme. Although the Km of the kinase for the collagen peptide was high, these results confirmed the prediction made from knowledge of the substrate specificity of cyclic AMP-dependent protein kinase. The potential for such a phosphorylation reaction to occur in vivo is discussed.

Amino Acid Sequence↗
PubMed1984

Abnormal islet and adipocyte function in young B-cell-deficient rats with near-normoglycemia.

Sprague-Dawley male rats were injected at 2 days of age with streptozotocin (SZ). At 4 wk of age the fed plasma glucose concentration of the SZ group was 151 +/- 6 mg/dl as compared with 133 +/- 4 for the control group. The fed plasma insulin values were indistinguishable, however. In response to an intraperitoneal glucose challenge the SZ group had marked glucose intolerance and virtually no rise in plasma insulin. After a meal challenge the SZ group also had glucose intolerance, but plasma insulin responses were similar to those of the control. The pancreata of the 4-wk-old rats were perfused in vitro and the SZ group had essentially no response to glucose, but did respond to arginine. Adipocytes of the 4-wk-old SZ rats had impaired glucose conversion to CO2 similar to that seen in the more hyperglycemic 6-wk-old SZ rats. Castration carried out at about 3 wk of age did not influence the hyperglycemia seen at 6 wk of age and later. These data indicate that 4-wk-old SZ rats, while having near-normal plasma glucose levels and normal plasma insulin values, have clearly abnormal B-cell and adipocyte function. With increasing age and weight gain these SZ rats develop frank hyperglycemia.

Adipose Tissue↗
PubMed1983

Glucose utilization in rat adipocytes. The interaction of transport and metabolism as affected by insulin.

The relative contributions of transport and intracellular metabolism of glucose to the control of overall glucose utilization were evaluated in rat adipocytes. Transport of 3-O-methylglucose and hexokinase activity in crude homogenates were measured and the derived kinetic parameters incorporated into network thermodynamic computer simulations. Hexokinase was found to be inhibited in a fully noncompetitive pattern by glucose 6-phosphate (Ki = 0.46 mM). When this feature was incorporated into the computer simulations, they reflected measured rates of overall glucose utilization as well as intracellular glucose 6-phosphate concentrations, both in the presence and absence of insulin. The effect of the hormone was represented in the simulations solely by an increase in the number of hexose carriers. A predominant stimulation of transport rather than metabolism was also suggested by the finding that intracellular glucose concentrations assessed by glucose-induced 3-O-methylglucose counter-transport were higher in the presence than in the absence of insulin over a wide range of extracellular glucose concentrations. Nevertheless, it was also found that insulin induced a significant countertransport gradient while the oxidant H2O2 did not, which suggests that insulin-stimulated metabolism does increase overall glucose utilization independently of effects on transport. These studies show that the kinetic patterns of basal and insulin-stimulated glucose utilization in adipocytes may be generated simply by coupling transport and phosphorylation steps and providing for inhibition of the latter by accumulated glucose 6-phosphate.

3-O-Methylglucose↗
PubMed1982

The simple model of adipocyte hexose transport. Kinetic features, effect of insulin, and network thermodynamic computer simulations.

Kinetic studies of the rat adipocyte hexose transport system were performed using the integrated rate approach and these compared to the simple carrier model of transport. Equilibrium exchange 3-O-methylglucose entry and exit studies showed directional symmetry with Km = overall dissociation constants = 8-10 mM. Comparison of zero-trans and equilibrium exchange entry also revealed similar Km and Vmax values. Insulin pretreatment increased the maximal rate of transport at 20 mM 3-O-methylglucose about 5- to 6-fold with each procedure. Studies of glucose-induced steady state 3-O-methylglucose countertransport provided evidence that carrier permeability and not carrier-substrate dissociation was rate limiting for overall transport. These data, therefore, indicate equal mobility of the loaded and unloaded carriers. Network thermodynamic computer simulations of the simple carrier model using kinetic parameters derived from zero-trans experiments provided good fits of actual data. The effect of insulin was best represented by an increase in total number of carrier units. It is concluded that the adipocyte hexose carrier displays bidirectional symmetry, limitation of transport by carrier movement rather than substrate-carrier interaction, equal rates of movement of loaded and unloaded carriers, and adherence to a simple carrier model in which insulin increases the total number of carrier units.

3-O-Methylglucose↗
PubMed1982

The mechanism of the insulin-like effects of ionic zinc.

The insulin-like effects of ionic zinc (Zn2+) were studied in isolated rat adipocytes. Concentrations of Zn2+ between 250 and 1000 microM stimulated 3-O-methylglucose transport and glucose metabolism to CO2, glyceride-fatty acid, and glyceride-glycerol. Selective stimulation of the pentose phosphate cycle was observed since a Zn2+-induced increase in glucose carbon 1 oxidation persisted even when glucose transport was blocked with 50 microM cytochalasin B or when transport was no longer rate-limiting for metabolism at high concentrations of glucose. Enhanced pentose phosphate cycle activity may have been due to a selective inhibition of glutathione reductase by the ion, which was also accompanied by a fall in cellular glutathione content. Zn2+ also inhibited lipolysis stimulated by the beta-adrenergic agent ritodrine in the absence of glucose. The effects of Zn2+ on glucose oxidation and stimulated rates of lipolysis were inhibited by extracellular catalase, indicating that they were largely a result of H2O2 generation. The H2O2 production appeared for the most part to be caused by zinc-catalyzed autoxidation of sulfhydryl groups present on external cell membranes, although involvement of sulfhydryl groups on bovine serum albumin in the buffer could also have contributed. The insulin-like effects of Zn2+ in adipocytes are therefore caused not only by direct effects of the ion on intracellular metabolism but also by indirect effects related to H2O2 generation.

3-O-Methylglucose↗
PubMed1982

Rat adipocyte utilization of different substrates: effects of cell size and the control of lipogenesis.

The metabolism of labeled glucose, pyruvate and acetate was compared in adipocytes isolated from old, obese rats (greater than 500 g) and young, lean rats (130-150 g). The larger cells from old, obese rats had markedly reduced rates of glucose, pyruvate and acetate conversion to glyceride-fatty acids, indicating that large cell fatty acid formation is reduced at some point beyond the entry of pyruvate and acetate into glucose metabolism. No evidence of a primary block in the pentose phosphate cycle of cells from old, obese rats was found. In spite of diminished glucose metabolism to several products in the large cells, both basal and insulin-stimulated rates of glyceride-glycerol synthesis from glucose and pyruvate were similar in each cell type. This indicates a relative diversion of carbon flow to alpha-glycerophosphate and reesterification in the large cells. Addition of low concentrations of glucose increase glyceride-fatty acid synthesis from acetate (both cell types) or pyruvate carbon (small cells), but decreased glyceride-glycerol synthesis from pyruvate carbon (both cell types). The acceleration of small cell fatty acid synthesis from pyruvate carbon by glucose and insulin was shown to be related to provision of NADPH from glucose metabolism in the pentose cycle. These studies indicate that, although the block in lipogenesis in adipocytes from old, obese rats appears to reside in the pathway of fatty acid synthesis itself, provision of additional alpha-glycerophosphate or NADPH from glucose metabolism may, under certain conditions, increase lipogenesis in cells from old, obese and young, lean rats.

6-Aminonicotinamide↗
PubMed1982

The effect of insulin-stimulated pentose phosphate cycle activity on cellular glutathione content in rat adipocytes.

Insulin is known to stimulate intracellular H2O2 production in rat adipocytes. This H2O2 could in turn stimulate the pentose phosphate cycle by oxidizing GSH and shifting the redox state of the cells. However, insulin had no effects on cell GSH content or GSSG in buffer other than as related to changes in medium glucose. On the contrary, in the presence of an active pentose phosphate cycle, insulin tended to reverse the fall in glutathione content induced with the catalase inhibitor 3-amino-1,2,4-triazole, the oxidant t-butyl hydroperoxide and the sulfhydryl blocker N-ethylmaleimide. It was also found that insulin-stimulated H2O2 production could be blocked under conditions in which the effect of the hormone on the pentose phosphate cycle persisted. These results suggest that stimulation of the pentose phosphate cycle by insulin is not related to increased H2O2 generation, rather that activation of the pentose phosphate cycle by the hormone may provide NADPH for regeneration of depleted GSH.

Adipose Tissue↗
PubMed1982

Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells.

A line of toad kidney cells (A6) in continuous culture was evaluated for ion transport and metabolic responses to insulin. The cells were grown on permeable supports to allow access of the medium to both basolateral and apical sides of the epithelium. Insulin, on the basolateral side only, produced an increase in short-circuit current (Isc) that was maximal at 40-60 min. A concentration-dependent increase in Isc and potential difference (PD) was found in the range of 10-3.2 X 10(3) microunits/ml insulin. The maximal stimulation of Isc and PD was approximately six- and twofold, respectively. After insulin exposure Isc was equivalent to net Na+ transport, indicating active Na+ transport stimulation. Insulin was also found to increase the incorporation of radiolabeled glucose into glycogen. Thus A6 cells exhibit both transepithelial transport and metabolic responses to insulin.

Animals↗
PubMed1982

Triacylglycerol turnover in large and small rat adipocytes: effects of lipolytic stimulation, glucose, and insulin.

Rates of lipolysis and reesterification were determined under various conditions in adipocytes from epididymal fat pads of old, spontaneously obese rats and compared to cells from younger, leaner animals. No differences were observed in lipolytic responsiveness to several concentrations (2--50 microM) of the beta-adrenergic agent ritodrine compared to cells from younger, leaner rats. The large cells showed diminished rates of lipolysis, reesterification, and glyceride-glycerol synthesis from glucose of a submaximal but not maximal insulin concentrations, probably reflecting decreased large cell receptor numbers. In both cell types reesterification measured in the presence of ritodrine progressively rose with increasing concentrations of glucose in the medium. At each glucose concentration (0.5--25 mM) rates of reesterification were similar in each cell type. When maximal concentrations of insulin (5 nM) were also added, at low glucose concentrations (less than 5 mM) there was a similar increase in reesterification in large and small cells. No insulin effect in either cell type was observed at high concentrations of glucose. Although fatty acid synthesis from glucose in the large cells was markedly diminished, glyceride-glycerol synthesis was well maintained, correlating well with calculated reesterification rates. In fact, reesterification was found to be quantitatively very important in determining total triacylglycerol turnover in both cell types. High rates of reesterification might not only allow maintenance of triacylglycerol stores, but could also increase metabolic sensitivity to changes in hormonal or substrate concentrations.

Adipose Tissue↗
PubMed1981

Determinants of gastric inhibitory polypeptide and insulin secretion.

The plasma concentrations of gastric inhibitory polypeptide (IR-GIP), insulin, and glucose were measured in 5 dogs after administration of various constituents of food. IR-GIP responses produced by galactose and fructose were minimal compared to those of glucose. Corn starch caused a significant early increase in insulin and glucose concentrations, although the rise in IR-GIP was delayed. The responses after starch were delayed, possibly due to the necessity for hydrolytic cleavage. Neither individual fatty acids (palmitic, oleic, and linoleic), monoolein, nor glycerol produced increases in IR-GIP comparable to those observed with lesser amounts (on a molar basis) of corn oil alone. None of four amino acids tested orally (arginine, leucine, alanine, and phenylalanine) caused increases in IR-GIP concentrations, but they increased insulin concentrations. Although glucose-induced IR-GIP responses were not inhibited by the amino acids (1 g/kg) tested, increasing concentrations (up to 1 g/kg) of either alanine or arginine caused progressive inhibition of corn-oil-stimulated IR-GIP responses, in spite of the expected amino-acid-induced rises in insulin concentrations. These results confirm the specificity of the IR-GIP secretory response for glucose and triglyceride, and suggest that certain food constituents (such as amino acids) may modify the GIP secretory response to a meal.

Administration, Oral↗
PubMed1980

The insulin-like effects of low molecular weight thiols: role of trace metal contamination of commercial thiols.

During the investigation of insulin-like effects of low molecular weight thiols in rat adipocytes it was found that thiol autoxidation to H2O2 occurred even in the absence of albumin and cells. Since chromatography of the thiols on a chelating ion exchange resin prevented H2O2 formation, it is likely that trace metal contaminations of the thiol preparations catalyzed thiol autoxidation in oxygenated solutions. Incubation of rat adipocytes with 1 mM dithioerythritol and low concentrations of dialyzed albumin stimulated 2-deoxyglucose transport, glucose metabolism to CO2 and lipids, and formate resin or in most instances incubation with EDTA or catalase prevented these effects. The antilipolytic effect of dithioerythritol was also abolished by catalase. The insulin-like effects of thiols therefore appear to be solely mediated by H2O2 generated during thiol autoxidation catalyzed by trace metal contaminants either of albumin or of the commercial thiol preparation itself.

Adipose Tissue↗