Biomedical subjects

J L Treadway

Publications and source records attributed to J L Treadway.

30 records · Page 2Linked to original sources

PubMed1991

Transdominant inhibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors.

Classical insulin and insulin-like growth factor I (IGF-I) receptors exist as well defined alpha 2 beta 2 heterotetrameric complexes that are assembled from two identical alpha beta heterodimeric half-receptor precursors. Recent evidence suggests that insulin and IGF-I half-receptors can heterologously assemble to form alpha 2 beta 2 insulin/IGF-I hybrid receptor complexes in vivo and in vitro. We have utilized hybrid receptor complexes to examine ligand-stimulated transmembrane signaling of wild-type insulin (alpha beta INS.WT) or IGF-I (alpha beta IGF.WT) half-receptors assembled with a kinase-defective insulin half-receptor mutant (alpha beta INS.A/K). In vitro assembly of either (alpha beta)IGF.WT/(alpha beta)INS.A/K or (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptors resulted in decreased substrate protein kinase activity. The degree of protein kinase inactivation directly correlated with the amount of immunologically cross-reactive hybrid receptors formed. In contrast to substrate kinase activity, insulin-stimulated autophosphorylation of the (alpha beta)INS.WT/(alpha beta)INS.A/K hybrid receptor complex was completely unaffected in comparison to the wild-type (alpha beta)INS.WT/(alpha beta)INS.WT receptor. To assess a molecular basis for this difference, autophosphorylation of a hybrid receptor composed of a truncated beta-subunit insulin half-receptor with the kinase-defective half-receptor, (alpha beta)INS. delta CT/(alpha beta)INS.A/K, demonstrated the exclusive autophosphorylation of the (alpha beta)INS.A/K half-receptor beta subunit. These results demonstrate that ligand-dependent substrate phosphorylation by insulin and IGF-I holoreceptors requires interactions between two functional beta subunits within the alpha 2 beta 2 heterotetrameric complex and occurs through an intramolecular trans-phosphorylation reaction.

Cell Membrane↗

PubMed1990

The endogenous functional turkey erythrocyte and rat liver insulin receptor is an alpha 2 beta 2 heterotetrameric complex.

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.

Animals↗

PubMed1990

Failure of prior low-intensity exercise to potentiate the thermic effect of glucose.

We have previously shown that following recovery from 45 min exercise at 67% maximum oxygen consumption (VO2max) the thermic effect of a glucose load is increased by 65% over that observed on a non-exercise day (Young et al. 1986). The purpose of this study was to determine if potentiation of the thermic effect of glucose by prior exercise is dependent on exercise intensity. The thermic response to a 1674 kJ glucose load was measured in five subjects in the absence of exercise (control) and following recovery from 45 min cycling exercise at each of three intensities: low (34% VO2max), moderate (54% VO2max), and high (75% VO2max). The average percentage increase in oxygen consumption over baseline due to glucose ingestion was similar for the control (9.9%, SE 2.0%), and the low- (10.2%, SE 0.9%) and moderate- (12.6%, SE 1.2%) intensity exercise conditions, while a significant increase in average VO2 was observed after the high-intensity condition (18.0%, SE 2.3%, P less than 0.05). The total energy expenditure (kJ) over baseline for 3 h was also similar for the control (84.5, SE 11.7), and the low-(100.0, SE 9.2) and moderate- (118.8, SE 5.0) intensity exercise conditions. The thermic response following high-intensity exercise (146.4 kJ, SE 13.4) was significantly greater than that observed in the control (P less than 0.01) or low-intensity (P less than 0.05) exercise conditions. These findings demonstrate that unlike prior high-intensity exercise (75% VO2max), low- or moderate-intensity exercise (i.e., 34% or 54% VO2max) fails to potentiate the thermic effect of a glucose load.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1990

Effect of insulin on protein synthesis and degradation in skeletal muscle after exercise.

This study examined whether insulin stimulation of protein synthesis and inhibition of protein degradation is enhanced after exercise. The isolated perfused rat hindquarter preparation was used to evaluate net protein breakdown, myofibrillar protein degradation, and protein synthesis. Thirty minutes after treadmill exercise of high and moderate intensity, rates of tyrosine release were increased by 58 and 25%, respectively. Insulin at 75 microU/ml had no effect on these increases after intense exercise; however, 20,000 microU/ml of insulin totally inhibited this increase. Cycloheximide increased the tyrosine release in both control and exercised rat muscle. It also abolished the difference between them, suggesting that the increase in tyrosine release after exercise is caused by an inhibition of protein synthesis. Phenylalanine incorporation into protein was marginally depressed (22%, P = NS) in the white gastrocnemius muscle after intense exercise. Insulin at 200 microU/ml stimulated protein synthesis in these rats, but no more than it did in a nonexercised control group. Failure to observe a greater effect of insulin on protein metabolism was also noted when rat muscle was studied 150 min after intense exercise and after contractions induced by electrical stimulation of the sciatic nerve. These findings suggest that after exercise or electrically induced contractions the enhanced ability of insulin to stimulate hexose and amino acid transport is not paralleled by an increase in its ability to stimulate protein synthesis or inhibit protein degradation.

Animals↗

PubMed1989

Assembly of insulin/insulin-like growth factor-1 hybrid receptors in vitro.

Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.

Adenosine Triphosphate↗

PubMed1989

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: interactions between alpha beta heterodimeric receptor halves.

Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗

PubMed1989

Regulation of the insulin receptor kinase by hyperinsulinism.

A murine fibroblast cell line transfected with human insulin receptor cDNA, NIH 3T3 HIR3.5, was observed to display insulin-induced down-regulation of insulin-binding activity in a time- and concentration-dependent manner. Maximal inhibition of insulin-binding activity (54%) occurred within 16 h of exposure to 100 nM insulin in vivo, where in vivo refers to intact cells in tissue culture. The decrease in cellular insulin-binding activity was the consequence of a decrease in the number of cell-associated insulin receptors as determined by Scatchard analysis of insulin binding, 125I-insulin affinity cross-linking, and Western blotting of the insulin receptor beta subunit. Acute insulin treatment in vivo (1-60 min) resulted in the activation of the insulin receptor protein tyrosine kinase as determined by in vitro phosphorylation of glutamic acid:tyrosine (4:1), where in vitro refers to broken cell preparations. This acute in vivo insulin activation of the insulin receptor tyrosine kinase resulted in a greater stimulation (1.4-1.9-fold) of tyrosine kinase activity in the glutamic acid:tyrosine (4:1) assay than the maximal stimulation produced by insulin treatment in vitro. In contrast, long term (24 h) insulin treatment in vivo resulted in a 50-70% decrease in intrinsic protein tyrosine kinase activity of the insulin receptors compared with that of acutely activated (1 min) insulin receptors. Under these conditions, the insulin receptor protein kinase activity remained insulin independent in the in vitro substrate kinase assay. Surprisingly, the insulin-independent activated (1 min in vivo insulin-treated) and uncoupled (24 h in vivo insulin-treated) insulin receptors displayed similar stoichiometries of 32P incorporation into the beta subunit by in vitro autophosphorylation when compared with the control insulin receptors, ranging from 1.5 to 1.8 mol of phosphate incorporated/mol of insulin receptor. Phosphoamino acid analysis demonstrated that the phosphoserine/phosphothreonine content of in vivo 32P-labeled insulin receptors increased markedly within a 1-h exposure to insulin in vivo, whereas insulin-induced receptor desensitization was not apparent until 10-24 h after exposure to insulin. These data suggest that insulin treatment in vivo results initially in the activation of the insulin receptor kinase followed by a subsequent uncoupling of protein kinase activity. This insulin-induced desensitization of the insulin receptor kinase does not correlate with the extent of beta subunit serine/threonine phosphorylation.

Animals↗

PubMed1989

Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle.

Insulin action in skeletal muscle is markedly enhanced for several hours after an acute bout of exercise. The purpose of this study was to examine the possible involvement of the intrinsic tyrosine kinase activity of the insulin receptor in mediating these effects. Red and white muscles were removed from rats either at rest or following a treadmill run (45 min at 18 m/min), and insulin receptors were isolated in partially purified form. Basal and insulin-stimulated receptor kinase activity was higher in red than in white muscle, in agreement with previous studies (J. Biol. Chem. 261: 14939-14944, 1986). There was no effect of exercise on insulin binding, basal and insulin-stimulated receptor autophosphorylation, or basal and insulin-stimulated exogenous kinase activity, in either red or white muscle. Similar data were obtained when phosphatase inhibitors were used during receptor isolation. The structure of insulin receptors isolated from the muscle of exercised and control rats was similar as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity cross-linked insulin receptors. We conclude that enhanced insulin action in muscle during the postexercise state is not related to increased kinase activity of the insulin receptor.

Animals↗

PubMed1989

Decreased glucose uptake in the fetus after maternal exercise.

Glucose uptake by muscle is stimulated by exercise. However, during pregnancy, insulin resistance develops in maternal skeletal muscle as a mechanism to spare glucose for use by the developing fetus. The purpose of this study was to determine whether a bout of exercise adversely affects glucose availability to the fetus by increasing glucose uptake in maternal muscle tissue. Pregnant (P) and nonpregnant (NP) rats were divided into rest and exercise groups. Immediately following a 50-min treadmill run, a bolus of glucose (1 g.kg-1) and tracer [1-3H]2-deoxyglucose was injected i.v., and rats underwent a 60-min i.v. glucose tolerance test (IVGTT), after which they were sacrificed. Although glucose levels were lower for P vs NP rats at all time points, prior exercise did not affect glucose levels in either P or NP rats. Plasma insulin levels were augmented in P compared with NP rats and were not significantly altered by prior exercise. Glucose/tracer accumulation was enhanced in red and white gastrocnemius in both P and NP rats, and in soleus of NP rats, by exercise. However, glucose/tracer accumulation in the fetus was decreased by 40% (P less than 0.01) in the exercised dams. These results indicate that uptake of glucose by the fetus is compromised following exercise, as a result of increased glucose uptake by maternal skeletal muscles.

Animals↗

PubMed1986

Prior exercise potentiates the thermic effect of a carbohydrate load.

It is unclear whether dietary-induced thermogenesis (DIT) is increased after exercise. To test this possibility, six healthy volunteers, male and female, exercised for 45 minutes at 70% of maximal aerobic capacity (VO2 max) in the morning after an overnight fast. Two hours after the end of the exercise, by which time VO2 had returned to near baseline levels, subjects ingested a 100-g glucose load. Blood samples and respiratory gas exchange data were collected over the next three hours. On a separate day on which the subjects did not exercise, the test procedure was repeated. Glucose tolerance and the insulin response to the glucose load were not significantly different between the two trials; however, VO2 increased by 15.5% over baseline on the exercise day, compared with only 8.9% when exercise was not performed. The net increase in energy expenditure for the three-hour period following glucose ingestion was 15 kcal/180 min greater on the exercise than on the control day, with increases upwards of 20 kcal/180 min in several individuals. No correlation was found between the magnitude of exercise-enhanced DIT and VO2 max, suggesting that this effect is independent of the state of training. The results indicate that the thermic effect of exogenous carbohydrate can be potentiated by prior exercise.

Adult↗

PubMed1986

The effects of exercise on milk yield, milk composition, and offspring growth in rats.

This study examined effects of exercise training throughout pregnancy and lactation upon selected indicators of lactational performance in Wistar rats. During the 7 wk prior to mating, experimental female rats were gradually trained to swim for 2 h/day, 5 days/wk with a 3% tail weight; they continued swimming until the 19th day of pregnancy. Control animals remained sedentary. Swimming resumed during days 2-14 of lactation. Food intake was greater in exercised rats during the lactation period. Body weights of the two groups were comparable. Mild yield (day 10), energy content (day 15), and protein and fat concentrations did not differ, but milk of exercised rats had a lower lactose concentration. The exercise regimen had no statistically significant effect on litter size or on offspring weight to day 15. This study indicates that moderate-intensity, aerobic exercise does not markedly affect the lactational performance of ad libitum-fed rats.

Animals↗

PubMed1986

Effects of oral hypoglycemic agent methylpalmoxirate on exercise capacity of streptozocin diabetic rats.

The effect of the oral hypoglycemic agent methylpalmoxirate (methyl-2-tetradecylglycidate), a selective inhibitor of long-chain fatty acid oxidation, on the exercise capacity of diabetic rats was evaluated. Rats were made diabetic by injection of streptozocin (75 mg/kg i.p.), which was confirmed by 4+ glucosuria. Daily oral administration of 2.5 mg/kg for 5 days, or a single dose of 10 mg/kg, of methylpalmoxirate produced a slight, nonsignificant decrease in the ability of diabetic rats to perform strenuous exercise of an intensity that caused exhaustion in less than 30 min. The ability of diabetic rats to perform prolonged, moderately strenuous exercise of an intensity that could be maintained for more than 60 min was not affected by methylpalmoxirate treatment. Methylpalmoxirate normalized plasma glucose concentrations, with resting glucose levels reduced 71% compared with nontreated controls, and did not cause hypoglycemia during prolonged exercise to exhaustion. Hepatic glycogen content was significantly reduced in methylpalmoxirate-treated rats in the fed, resting state, and during exercise, suggesting that liver was forced to oxidize carbohydrate at the expense of carbohydrate storage. Blood ketone levels of methylpalmoxirate-treated rats were reduced by 82% at rest, and exercise-induced ketosis was prevented by drug treatment. Muscle glycogen concentration and the rate of muscle glycogen depletion during exercise were not altered by methylpalmoxirate. It appears that the liver is the major site of action of methylpalmoxirate in diabetic rats when given in low doses.

3-Hydroxybutyric Acid↗