Biomedical subjects
Publications and source records attributed to J Larner.
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Brief treatment of rat adipocytes with low concentration of trypsin activated both cell membrane and intracellular insulin-sensitive functions in marked contrast H2O2 (1), increase in pH, and oxidized glutathione (papers I and II). Glucose oxidation was activated maximally by trypsin in 30 s and preceded maximal activation of glycogen synthase, which occurred in 60s. Trypsin action to activate glycogen synthase was further enhanced by insulin. Mitochondrial pyruvate dehydrogenase was also rapidly activated by trypsin. With both insulin and trypsin action, mediator generation was directly demonstrated by glycogen synthase phosphoprotein phosphatase activation. Trypsin is thus the most insulin-like of these four agents studied since it acts by the formation of chemical mediator peptide(s) which are similar but not identical to those produced by insulin.
The influence of pH and 3-hydroxybutyrate on insulin binding and action has been studied in cultured human fibroblasts. When the pH of the incubation medium was decreased from 7.6 to 6.8, insulin stimulation of glycogen synthase and total glucose uptake was decreased. The decreased pH induced both an increase in the insulin concentration for half-maximal response, and a decrease in maximal responsiveness. When insulin binding was measured at lower pH, receptor affinity was decreased. The effect of 3-hydroxybutyrate on insulin binding and action was assessed at pH 7.4 and 6.9. In the presence of 3-hydroxybutyrate, insulin binding increased, but insulin action on glycogen synthase and total glucose uptake was unaffected. The data show that insulin action is impaired at lower pH. The decreased sensitivity is probably related to the decrease in insulin binding affinity at lower pH, but decreased maximal responsiveness implies that postreceptor components are also affected by lower pH. The results also suggest that 3-hydroxybutyrate induced a dissociation between binding and response, since an increase of insulin binding was not accompanied by changes in the regulation of glycogen synthase and total glucose uptake.
Commercially available concanavalin A binds Ca2+ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca2+ uptake (defined as an increased association of 45Ca2+ with cells) in rat splenocytes and Ca2+ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in 45Ca2+ uptake over control. The concanavalin A stimulated uptake of 45Ca2+ occurred within minutes, and required concentrations of concanavalin A which promoted [3H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca2+ uptake than concanavalin A. Sodium periodate inhibited Ca2+ uptake at concentrations which promoted 3H-thymidine incorporation into splenocytes. It is concluded that concanavalin A promotes Ca2+ uptake which is not due to binding of 45Ca2+ to concanavalin A. Although the concanavalin A-promoted Ca2+ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by 3H-thymidine incorporation, the role of Ca2+ in this event remains unclear.
Incubation of intact mouse diaphragms with insulin in the absence of glucose resulted in a rapid inhibition of the subsequent cell-free phosphorylation of endogenous protein substrates in tissue extracts. The phosphorylation of added histone was inhibited to a lesser extent. The inhibition was observed both in the absence and in the presence of added cyclic 3'5' adenosine monophosphate. Acrylamide gel electrophoresis of phosphorylation products revealed a number of major phosphorylated polypeptides. The phosphorylation of several polypeptides was inhibited following short treatment with insulin. These results represent a novel experimental approach to the elucidation of the mechanism of the action of insulin and are consistent with our hypothesis that the inhibition of protein kinase activities in the tissue may be the first step in this mechanism.
The incubation of intact mouse diaphragms with insulin caused a dose and time dependent increase in the independent activity of glycogen synthase in tissue extracts. 2-deoxyglucose (2-10 mM) alone markedly stimulated the conversion of glycogen synthase to the independent activity under conditions in which tissue ATP concentrations were not affected. The incubation of diaphragms with both insulin and 2-deoxyglucose resulted in a greater than additive effect. Insulin stimulated the uptake of 2-deoxyglucose into mouse diaphragms, accumulating as 2-deoxyglucose-6-phosphate. The accumulation of 2-deoxyglucose-6-phosphate correlated well with the increase in the independent activity of glycogen synthase and with the activation of glycogen synthase phosphatase in tissue extracts. The uptake of 3-0 methyl glucose was also markedly stimulated by insulin, without affecting the activity of glycogen synthase. Both glucose-6-phosphate and 2-deoxyglucose-6-phosphate stimulated the activation of endogenous glycogen synthase phosphatase activity in muscle homogenates. We conclude that insulin, in addition to its effects in the absence of exogenous sugars, increases the independent activity of glycogen synthase through increased sugar transport resulting in increased concentrations of sugar-phosphates which promote the activity of glycogen synthase phosphatase.
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Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.
Rat hemidiaphragms incubated with epinephrine exhibited increases in cyclic AMP content and protein kinase activity which were proportional to the logarithm of the hormone concentration from 0.1--2 microM. The fraction of glycogen synthase made independent of glucose-6-P for activity (%I) decreased concomitantly, but correlated only with epinephrine concentrations up to 0.2 microM. Insulin (0--100 mU/ml) increased glycogen synthase %I in a dose-dependent manner with no change in cyclic AMP concentration. Protein kinase activity increased slightly at the lowest insulin concentration, then decreased slightly as glycogen synthase %I increased. Insulin was without effect when administered with a supramaximal dose of epinephrine. In the presence of submaximal epinephrine, insulin produced a dose-dependent increase in glycogen synthase %I which correlated with a decrease in protein kinase activity, without changing cyclic AMP. Insulin had no effect on the increases in cyclic AMP produced by varying levels of epinephrine. However, the activation of protein kinase activity by endogenous cyclic AMP was inhibited in the presence of insulin. The glycogen synthase %I response to epinephrine also was less sensitive in the presence of insulin. Insulin antagonizes the activation of cyclic AMP-dependent protein kinase by epinephrine without altering cyclic AMP levels.
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Postprandial and postabsorptive glucose metabolism was studied in a 3-yr-old girl with leprechaunism by substrate and hormonal measurements and by quantifying hepatic glucose output during continuous infusion of D-[6-6-2H2]-glucose. Hepatic glucogen content and the activity of glycogen synthase and phosphorylase were also measured in the post-prandial state on a separate occasion. During the 4-h postprandial state, plasma glucose, alanine, lactate, beta-hydroxybutyrate, and glycerol were normal, as were hepatic glycogen, glycogen synthase, phosphorylase, and hepatic glucose output of 7.5 mg kg-1 min-1. Intravenous injection of glucagon (30 micrograms kg-1) caused an immediate almost 3-fold rise in glucose production consistent with brisk glycogenolysis. During the 8- to 12-h postabsorptive state, however, the patient had elevated levels of glycerol (330-508 microM) and beta-hydroxybutyrate (3291-3801 microM) and decreased levels of glucose 24-29 mg/dl) and alanine (121-135 microM) consistent with a much longer period of fasting in the normal child. Furthermore, hepatic glucose output was reduced to 3.9 mg kg-1 min-1, and iv glucagon injection failed to increase this rate; both of these observations are consistent with a hepatic state generally found only later in fasting in the normal child. From these observations we conclude that the hypoglycemia reported in the leprechaunism syndrome is due to an accelerated fasting state secondary to insulin resistance. As with long-fasted, glycogen-depleted normal children, gluconeogenesis alone is often not capable of adequately meeting the child's large noninsulin-dependent cerebral glucose requirements.
Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromatographed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 leads to 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 leads to 6 and 3 leads to 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 leads to 6 and 3 leads to 6.
The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.
(-) Isoproterenol-stimulated adenylate cyclase activity of washed at parotid membrane preparations from gland slices previously treated with the alpha-adrenergic agonist, methoxamine, was inhibited approximately 30%. The action of methoxamine required exogenous Ca2+ and was blocked by the alpha-adrenergic blocking agent, phentolamine. Incubation of gland slices with carbamylcholine resulted in a dose-dependent inhibition (50%) of (-) isoproterenol-stimulated adenylate cyclase activity in washed membranes. The action of carbamylcholine was independent of exogenous Ca2+ and was blocked by preincubation with atropine. Cholinergic inhibition of parotid adenylate cyclase was compared to the cholinergic inhibition of adenylate cyclase in dog heart homogenates. While carbamylcholine caused a limited (20-30%) inhibition of basal and (-) isoproterenol-stimulated activities when added to dog heart homogenates, it failed to produce any effect in parotid homogenates prepared in an identical manner. Cholinergic inhibition of parotid adenylate cyclase activity was stable and persisted in washed particulate fractions while the inhibition in dog heart homogenates was reversible by washing. Cholinergic inhibition of adenylate cyclase was markedly dependent on the presence of GTP and was abolished when Mn2+ was subsituted for Mg2+ in both systems. Guanyl-5'-yl imidodiphosphate had little effect on the inhibition in parotid preparations but abolished the inhibition in heart homogenates. It is concluded that, in contrast to the cholinergic action in dog heart homogenates, the exposure of parotid slices to carbamylcholine results in a stable lesion in the adenylate cyclase activity.
Deproteinized skeletal muscle extracts free of major nucleotides from control and insulin-treated rats were fractionated and assayed for inhibition of protein phosphorylation by cyclic adenosine monophosphate (AMP)-dependent and -independent protein kinases. A differential effect of insulin on a particular fraction was observed on cyclic AMP-dependent protein kinase but not on cyclic AMP-independent protein kinases. This fraction that inhibited cyclic AMP-dependent protein kinase also stimulated glycogen synthase phosphoprotein phosphatase. It is proposed that this fraction may contain a mediator substance generateed in the presence of insulin.