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Biomedical subjects

J Larner

Publications and source records attributed to J Larner.

At least 91 records · Page 5Linked to original sources
PubMed1984

Selective inhibition of the insulin-stimulated phosphorylation of the 95,000 dalton subunit of the insulin receptor by TAME or BAEE.

Added N alpha-p-tosyl-l-arginine methyl ester or N alpha-benzoyl-l-arginine ethyl ester inhibited the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of the insulin receptor both in a partially purified insulin receptor fraction from rat adipocytes and in a highly purified insulin receptor preparation from human placenta. N-alpha-p-tosyl-l-lysine chloromethyl ketone, N alpha-p-tosyl-l-lysine methyl ester, or N-acetyl-l-phenylalanine ethyl ester were much less potent, while N-benzoyl-1-alanine methyl ester was without effect. Inhibition of the phosphorylation by the arginine analogues did not require preincubation of the insulin receptor with inhibitors in the presence of insulin prior to phosphorylation. Inhibition by N alpha-p-tosyl-l-arginine methyl ester was decreased by preincubation of the receptor fraction with cold ATP and MnCl2. These results suggest that N alpha-p-tosyl-l-arginine methyl ester inhibits an initial ATP and Mn2+ dependent reaction in insulin-stimulated phosphorylation process.

Adipose Tissue↗
PubMed1984

Purification and partial characterization of a putative mediator of insulin action on cyclic AMP-dependent protein kinase.

An insulin mediator which inhibits cAMP-dependent protein kinase has been purified approximately 1000-2000-fold from skeletal muscle. Following heat treatment, charcoal adsorption and Sephadex G-25 sieving, Sephadex G-15 sieving and HPLC over an anion exchange column were performed. The mediator has characteristics of a relatively low molecular weight peptide or derivatized peptide which acts on cAMP-dependent protein kinase but not on mitochondrial pyruvate dehydrogenase.

Animals↗
PubMed1984

Mechanisms of insulin resistance in cultured fibroblasts from a patient with leprechaunism: impaired post-binding actions of insulin and multiplication-stimulating activity.

The binding and action of insulin and of the insulin-like growth factor, multiplication-stimulating activity (MSA), were studied in cultured skin fibroblasts from an infant with leprechaunism and associated insulin resistance. Three actions of insulin were reduced in the leprechaun cells: activation of glycogen synthase was 30% as great as in control fibroblasts, the increase in 2-deoxyglucose transport was 33% of the control value, and the uptake of alpha-aminoisobutyric acid was sevenfold less sensitive to enhancement. On a molar basis, MSA was at least as effective as insulin in activating glycogen synthase in control fibroblasts; in the patient's cells there was a reduction in activation that paralleled the changes observed with insulin. To localize the site of insulin resistance, the binding of both [125I]-insulin and [125I]-MSA to fibroblasts was measured and found to be reduced in the leprechaun cells. However, the impairment of the actions of insulin and MSA in the patient's cells was not explained solely by the diminished binding of the two polypeptides. Since the hexose transport system and the terminal enzymes studied thus far are intact, the defect is postulated to involve the post-binding coupling mechanism and mediator formation.

Adult↗
PubMed1984

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes.

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).

Adipose Tissue↗
PubMed1983

Insulin-like effect of trypsin on the phosphorylation of rat adipocyte insulin receptor.

Trypsin treatment of a partially purified insulin receptor preparation from rat adipocytes stimulated the phosphorylation of 90,000- and 72,000-Da polypeptides immunoprecipitated by anti-insulin receptor antibody. The phosphorylation of tyrosine residues alone was observed in both polypeptides. Trypsin concentrations which stimulated insulin receptor phosphorylation were the same as those previously shown to activate rat adipocyte glycogen synthase. Trypsin treatment of the insulin receptor fraction also stimulated the phosphorylation of an exogenous substrate of tyrosine kinase similarly to insulin treatment. Trypsin treatment of a highly purified insulin receptor from human placenta also activated the phosphorylation of the receptor-derived peptides. These results suggest that the insulin-stimulated protein kinase, a component of the insulin receptor, was activated by tryptic digestion to phosphorylate polypeptides derived from the insulin receptor itself. Thus, it is suggested that stimulation by trypsin of phosphorylation of the insulin receptor may be related to the insulin-like metabolic actions of trypsin observed in rat adipocytes.

Adipose Tissue↗
PubMed1983

Effect of temperature on insulin binding and action in cultured human fibroblasts.

Insulin stimulation of glycogen synthase activity and insulin binding were measured in fibroblast monolayers at 24, 32, and 37 degrees C. Insulin stimulation of %I glycogen activity increased with increasing temperature. Maximum response was greater at 37 degrees C than at 32 degrees C, and half maximal stimulation required at 2.0 nM insulin at 37 degrees C vs. 10 nM at 32 degrees C. Insulin stimulation of glycogen synthase was greater and somewhat faster at 37 degrees C than at 32 degrees C. No insulin effect was observed at 24 degrees C. 125I-insulin binding to monolayers became maximal in 15 min at 37 degrees C, 60 min at 32 degrees C, and 120 min at 24 degrees C. However, insulin binding decreased with increasing temperature, and this decline was due to decreased numbers of receptors. Insulin binding and stimulation of glycogen synthase were comparable at 32 degrees C, with half maxima at 10 nM, indicating no evidence of "spare" receptors. The data indicate that temperature effects on insulin binding and action in fibroblasts are not directly related. The results also suggest that a rate limiting step(s) of insulin action is temperature sensitive, and that this step is not insulin binding.

Cells, Cultured↗
PubMed1983

Insulin-like effect of vanadate on adipocyte glycogen synthase and on phosphorylation of 95,000 dalton subunit of insulin receptor.

Vanadate enhanced the state of activation of rat adipocyte glycogen synthase in a manner similar to that of insulin. No additional effect was observed when insulin and vanadate were added together. The effect of vanadate, like insulin, was reversed by incubation with epinephrine. Vanadate also enhanced the degree of phosphorylation of the 95,000 dalton subunit of insulin receptor, selectively on tyrosine residues, in the solubilized rat adipocyte insulin receptor system. This demonstrates that insulin and vanadate have similar initial actions on receptor phosphorylation and also act similarly on an intracellular event, namely the activation of glycogen synthase.

Adipose Tissue↗
PubMed1983

Stimulation of maximal intracellular insulin action on glycogen synthase by preincubation of adipocytes with adenosine 5'-triphosphate.

Preincubation of rat adipocytes with ATP further stimulated maximal insulin action on glycogen synthase. Half-maximum concentration of ATP was 5 X 10(-5) M. ATP, ADP, adenosine, inosine, and GTP were effective, while beta-gamma-methylene ATP was without effect. ADP and GTP were less potent than ATP, adenosine, or inosine. Inosine was active without insulin but was without effect in the presence of insulin. The mechanism of action of adenosine was clearly different from ATP. While ATP required both Mg2+ and Ca2+ for effectiveness, adenosine required only Ca2+. The effect of ATP, but not of adenosine, was preserved after cells were washed. The adenosine effect was completely blocked by theophylline, but the ATP effect was inhibited only 40%. The ATP effect was thus not due to adenosine generated by ATP breakdown.

Adenosine↗
PubMed1983

Mediators of postreceptor action of insulin.

Intracellular insulin action has been investigated in terms of the control of glycogen synthesis. The action was systematically traced back to the initial action of insulin at the cell membrane. Insulin activates the rate-controlling enzyme, glycogen synthase. The mechanism of this activation was found to be the control of glycogen synthase by covalent phosphorylation and dephosphorylation. Insulin brought about dephosphorylation and enzyme activation, which led to increased glycogen synthesis. A study of the interconversion reactions led to the discovery of the cyclic adenosine monophosphate dependent (cAMP-dependent) protein kinase which phosphorylates glycogen synthase and inactivates the enzyme, and a phosphoprotein phosphatase which dephosphorylates glycogen synthase and activates it. Studies revealed that insulin does not act on glycogen synthase by decreasing basal tissue concentrations of cAMP (or by altering cyclic guanosine monophosphate (cGMP) tissue concentrations); rather, insulin acts more directly on the cAMP-dependent protein kinase to inactivate the kinase and to desensitize it to the activating action of cAMP. An intracellular mediator of insulin action was hypothesized to carry out this effect on the kinase; therefore, the kinase was used as an assay to search for the putative mediator. Such an insulin-generated mediator was found to be present initially in skeletal muscle and more recently in several insulin-sensitive tissues. Present evidence, although indirect, strongly suggests (1) that the mediator is a small peptide or peptide-like molecule, (2) that probably several mediators (or a family of mediators) are formed rapidly with insulin action; and (3) that they are formed from cell-membrane proteins by a process of limited proteolysis, which is initiated by the binding of insulin to its receptor. The present status of the rapidly developing area of hormone-induced transmembrane signaling via mediators is reviewed.

Animals↗
PubMed1983

'Insulin-like' effects of lithium ion on isolated rat adipocytes. I. Stimulation of glycogenesis beyond glucose transport.

Both insulin and lithium ion stimulated cytochalasin B-sensitive glucose transport in isolated rat adipocytes. As a result of enhanced glucose transport, the incorporation of [14C]glucose into CO2, glycogen and lipid were increased by both agents. However, the action of these two agents was distinguished. Cytochalasin B decreased insulin-stimulated glucose oxidation, glycogenesis and lipogenesis. In contrast, lithium-stimulated glycogenesis was decreased but lithium-stimulated glucose oxidation and lipogenesis were completely blocked. These results indicate that lithium ion in rat adipocytes has a specific insulin-like effect on glycogenesis without affecting glucose oxidation and lipogenesis.

Adipose Tissue↗
PubMed1983

'Insulin-like' effects of lithium ion on isolated rat adipocytes. II. Specific activation of glycogen synthase.

Lithium ion, like insulin, activated adipocyte glycogen synthase with or without glucose in the medium. However, the effect of lithium ion was much greater than that of insulin under both conditions. The lithium-activated glycogen synthase was stable to both Sephadex chromatography and ethanol precipitation of the enzyme, indicating that the effect of lithium ion on glycogen synthase was through covalent modification of the enzyme. Glycogen synthase was significantly activated by lithium ion under conditions where concentrations of cellular ATP were unaffected. The effect of lithium ion on glycogen synthase was rapid and observed at concentrations as low as 1 to 3 mM, reaching a maximum at the concentration of 40 mM. It was thus the most sensitive of all the effects studied (see previous paper). Insulin further stimulated glycogen synthase at low concentrations but not at maximal concentration of lithium ion. Lithium-activated glycogen synthase was inhibited by both epinephrine and dibutyryl cyclic AMP, but was not affected by the removal of extracellular Ca++. Interestingly, lithium ion had no detectable effect on basal pyruvate dehydrogenase as well as on epinephrine-stimulated phosphorylase. The failure of lithium ion to thus mimic insulin actions on pyruvate dehydrogenase and on phosphorylase suggests that the action of lithium ion on glycogen synthase is quite specific and may be mediated by stimulating a phosphatase or by inhibiting a protein kinase acting specifically on glycogen synthase.

Adipose Tissue↗
PubMed1982

A proteolytic mechanism for the action of insulin via oligopeptide mediator formation.

Evidence is presented that the chemical mediator of insulin action is a peptide(s) and most likely glycopeptide(s). The mediator is formed proteolytically because 1) protease inhibitors inhibit insulin action and 2) trypsin mimicks insulin action via mediator formation. Trypsin mediator does not faithfully reproduce the action of insulin mediator, which indicates that the sites of proteolytic cleavage by insulin and trypsin differ. A coordinated multivalent proteolytic mechanism by which insulin acts to trigger an external membrane-bound protease to cleave mediator from a membrane glycoprotein precursor is presented.

Animals↗
PubMed1981

Effect of insulin fragments on biological activity of insulin and desoctapeptide insulin. I. Potentiation of biological activities.

Four derivatives of the insuln B-chain COOH-terminal pentapeptide Arg-Gly-Phe-Phe-Tyr (B22-26) were synthesized and shown to be inactive alone. In the presence of submaximal concentrations of insulin or desoctapeptide insulin, peptides at concentrations of 10(-4) M and higher, markedly stimulated the actions of insulin on rat adipocytes including labeled glucose oxidation, activation of glycogen synthase, and stimulation of 2-deoxyglucose transport. The B-chain COOH-terminal heptapeptide, Gly-Phe-Phe-Tyr-Thr-Pro-Lys (B23-29) was inactive alone or in the presence of submaximal concentrations of insulin or desoctapeptide insulin, suggesting that argnine is required. Dose response curves of insulin, and desoctapeptide insulin for labeled glucose oxidation by rat adipocytes were shifted 1 log concentration unit to the left in the presence of peptide. Peptide shortened the lag time of labeled glucose oxidation and markedly enhanced the rate of 14CO2 production following the lag time. Peptides also enhanced insulin-like activities of concanavalin A and nonsuppressible insulin-like activity P.

Adipose Tissue↗