PubMed Health⌕ Search

Biomedical subjects

D E Peavy

Publications and source records attributed to D E Peavy.

33 records · Page 2Linked to original sources
PubMed1984

Selective effects of inhibitors of hormone processing on insulin action in isolated hepatocytes.

Several compounds known to alter receptor-mediated endocytosis were tested for their effect on insulin action in isolated rat hepatocytes. Dansylcadaverine blocked the inhibitory effect of insulin on hepatic protein degradation, but was without effect on the inhibition produced by high concentrations of amino acids. Dansylcadaverine also was without effect on the stimulatory action of insulin on glucose incorporation into glycogen, but totally blocked the insulin stimulation of alanine uptake. No inhibition of basal or (Bu)2cAMP-stimulated alanine uptake was observed with dansylcadaverine. These results indicate that dansylcadaverine has selective effects on some but not all of insulin's actions. Methylamine also blocked insulin stimulation of alanine transport, although at higher doses, methylamine decreased basal and (Bu)2cAMP-stimulated uptake due to cellular toxicity. Chloroquine, an inhibitor of intracellular insulin processing, had no effect on insulin-stimulated alanine transport. To examine the site of action of dansylcadaverine on insulin processing, the binding (cell-associated radioactivity) and degradation of [125I]iodoinsulin by hepatocytes were examined in the presence and absence of dansylcadaverine. Dansylcadaverine delayed the time required to achieve maximal insulin binding and produced a dose-dependent decrease in cell-mediated insulin degradation. Acid dissociation and proteolytic digestion methods were used to differentiate between [125I]iodoinsulin bound to the cell surface vs. that which had been internalized. Dansylcadaverine did not block internalization as measured by either of these criteria, but did block the increase in cell-associated radioactivity produced by chloroquine, suggesting that dansylcadaverine blocks insulin processing after internalization but before the chloroquine-sensitive step. These data show that some of the hepatic actions of insulin, namely inhibition of protein degradation and stimulation of alanine transport, are blocked by certain inhibitors of cellular hormone processing, suggesting that these actions of insulin may require postreceptor events involving insulin metabolism.

Animals↗
PubMed1984

Receptor binding and biological activity of specifically labeled [125I]- and [127I]monoiodoinsulin isomers in isolated rat adipocytes.

The receptor binding characteristics and biological activity of single site monoiodinated insulin was investigated using isolated rat adipocytes. Pork insulin was iodinated by the lactoperoxidase method, and each of the four monoiodo derivatives (iodotyr-A14, iodotyr-A19, iodotyr-B16, and iodotyr-B26) was isolated by high performance liquid chromatography. Both radioactive 125I-labeled and nonradioactive 127I-labeled iodoinsulins were prepared. In competitive binding experiments in which each 125I-labeled iodoisomer competed for receptor binding at 15 C with its homologous 127I-labeled iodoisomer, the concentrations of homolog required for 50% inhibition of tracer binding were 1.67 +/- 0.06, 2.14 +/- 0.23, and 2.35 +/- 0.27 X 10(-9) M for the B26, A14, and B16 iodoisomers, respectively (mean +/- SEM; n = 3). Scatchard analysis of the homologous competition curves using a two-site model indicated that there was no difference in the total number of specific sites to which each isomer could bind or in their affinity for binding to the low affinity site. However, a significantly (P less than 0.05) higher affinity for (B26)iodoinsulin binding to the high affinity site was observed compared to either the (A14)- or (B16)iodoisomers (Kd values of 1.09 +/- 0.18, 2.09 +/- 0.40, and 2.24 +/- 0.15 X 10(-9) M for B26, A14, and B16, respectively). Degradation of the isomers by adipocytes incubated at 37 C occurred at a rate proportional to their receptor binding affinity. The biological activity of iodoinsulins was also evaluated, based on the ability either to stimulate glucose oxidation or to exert an antilipolytic effect. The 127I-labeled (B26)iodoisomer exhibited the greatest potency in both assays compared to either (A14)- or (B16)iodoinsulins, consistent with its higher apparent affinity for receptor binding. The receptor-binding activity and biological potency of unmodified native insulin and 127I-labeled (A14)-iodoinsulin were directly compared. Identical results were obtained for each in both types of assay, suggesting that (A14)iodoinsulin is a valid tracer for insulin with isolated adipocytes. We conclude that in isolated rat adipocytes, (B26)iodoinsulin has greater activity than either unlabeled insulin or the other iodinated derivatives. (A14)Iodoinsulin is indistinguishable from native insulin; (B16)iodoinsulin has slightly reduced activity, while that of (A19)iodoinsulin is considerably reduced.

Adipose Tissue↗
PubMed1984

The degradation of monoiodotyrosyl insulin isomers by insulin protease.

The four single-site monoiodotyrosyl insulin isomers were synthesized by lactoperoxidase-catalyzed iodination of porcine insulin and were separated from one another by high performance liquid chromatography. The susceptibility of the four isomers (A14-, A19-, B16-, and B26-monoiodotyrosyl insulin) to degradation by purified insulin protease was examined using several different assay methods, including trichloroacetic acid precipitation, immunoprecipitation, and Sephadex G-50 chromatograpy. Using trichloroacetic acid precipitation, isomer susceptibility, determined from the initial rate of hydrolysis, was highest with the A14 isomer, lowest with the A19 isomer, and intermediate and roughly equal with the two B-chain-labeled isomers. Based upon the initial rate of isomer hydrolysis, the Michaelis Menten constant (Km) of insulin protease was higher for the B16 isomer (55 nM) than for the other three isomers, whose Km values were not different from one another (A14 = 24 nM; A19 = 35 nM; B26 = 29 nM). In addition, the values for maximum velocity (Vmax) were higher for the A14 and B26 isomers than for the A19 and B16 isomers. However, during incubation, the order of isomer susceptibility to insulin protease changed to B26 greater than A14 greater than A19 greater than B16. This change in apparent isomer susceptibility was prevented by including in the incubation mixture a rat renal peptidase, which did not degrade the intact isomers, suggesting that insulin protease converted the isomers to trichloroacetic acid-soluble products via trichloroacetic acid-precipitable intermediates. Using the immunoprecipitation assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was degraded more rapidly. Each isomer was converted more rapidly to nonimmunoprecipitable products than to trichloroacetic acid-soluble products, implying that insulin protease converted the isomers to trichloroacetic acid-precipitable, nonimmunoprecipitable intermediates, which it then converted to trichloroacetic acid-soluble form. Using Sephadex G-50 chromatography (SGC) assay, the susceptibility of isomers to hydrolysis did not change during incubation, but remained highest with the A14 isomer, lowest with the A19 isomer, and intermediate with the two B-chain-labeled isomers, of which the B16 isomer was hydrolyzed more rapidly. With the exception of the A19 isomer, isomer hydrolysis appeared faster with SGC assay than with either of the other two assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemical Precipitation↗
PubMed1984

In vitro activity of biosynthetic human proinsulin. Receptor binding and biologic potency of proinsulin and insulin in isolated rat adipocytes.

The receptor binding characteristics and biologic protency of biosynthetic human proinsulin (rDNA) were determined in isolated rat adipocytes and compared with those of insulin. In competition with 125I(A14)-iodoinsulin for binding to adipocyte receptors at 15 degrees C, proinsulin showed a 100-fold lower affinity for binding than did insulin. A proinsulin concentration of 3.2 +/- 0.8 X 10(-7) M was required for 50% inhibition of tracer binding as compared with a concentration of 1.7 +/- 0.3 X 10(-9) M for insulin. These results were confirmed in direct competition studies using proinsulin and 125I-iodoproinsulin. A similar 100-fold difference was also observed in competitive binding experiments conducted at 37 degrees C. The biologic potency of human proinsulin was evaluated by its ability to stimulate glucose incorporation into total fat cell lipid and also by its antilipolytic activity. Glucose incorporation into lipid was half-maximal at a proinsulin concentration of 1.5 +/- 0.4 X 10(-8) M, whereas the same response was observed at an insulin concentration of 5.2 +/- 1 X 10(-11) M. Proinsulin also demonstrated an antilipolytic potency that was less than 1% that of insulin. The time course over which insulin and proinsulin stimulated glucose incorporation into lipid was the same, as was the time course over which the stimulation dissipated after removal of the hormones. No synergism of insulin and proinsulin stimulation of lipogenesis was observed when fat cells were incubated with submaximal concentrations of the two hormones.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1983

Diabetes-induced alterations in liver protein synthesis. Changes in the relative abundance of mRNAs for albumin and other plasma proteins.

Mechanisms responsible for diabetes-induced alterations in liver protein synthesis were investigated in vivo and in perfused liver using Bio-Breeding Worcester (BB/W) control rats, spontaneously diabetic BB/W rats maintained on insulin therapy, and diabetic BB/W rats withdrawn from insulin therapy for 48 h. Withdrawal of insulin therapy in the diabetic rats resulted in marked alterations in a number of parameters related to liver protein synthesis compared to BB/W control or insulin-maintained diabetic rats. Alterations seen in vivo following withdrawal of insulin included changes in the relative concentrations of several plasma proteins, a 40% reduction in total liver RNA relative to DNA, a 5-fold reduction in albumin synthesis relative to the synthesis of total liver proteins, a 5-fold reduction in albumin mRNA relative to total RNA, reductions in the relative abundance of mRNAs for at least four plasma proteins other than albumin, and a relative increase in mRNA for at least one plasma protein. Alterations observed in perfused liver included reductions in total liver protein synthesis (60% of control), albumin production (24% of control), and total secretory protein production (44% of control). All parameters studied were essentially unchanged from BB/W control values when the diabetic rats were maintained on insulin therapy. The results indicate that insulin deficiency leads to marked reductions in liver protein synthesis, particularly the synthesis of albumin and other plasma proteins. The mechanisms responsible for these alterations include changes in the relative abundance of specific mRNAs and a decrease in total cellular RNA.

Animals↗
PubMed1982

The role of amino acids in the regulation of protein synthesis in perfused rat liver. I. Reduction in rates of synthesis resulting from amino acid deprivation and recovery during flow-through perfusion.

The role of perfusate amino acid concentrations in regulating rates of protein synthesis was investigated using the perfused rat liver. Livers from fed rats were perfused with a nonrecirculating medium and the incorporation of [3H]leucine into albumin and total protein was determined under conditions where the leucyl-tRNALeu and perfusate leucine specific activities were equal and constant. During perfusions of less than 1 h, rates of total protein synthesis were sensitive to the concentrations of amino acids in the perfusate. When no exogenous amino acids were provided, rates of synthesis of albumin and total protein were 40% of the maximal rates which were achieved when the medium was supplemented with 5 times the normal plasma concentrations of amino acids. However, rates of synthesis in livers perfused with amino acid-deficient medium rose with extension of the duration of perfusion to 95 min. The defect induced by amino acid deficiency did not appear to result from reductions in the charging of tRNA since no change in the quantities of amino acids bound to tRNA occurred in the amino acid-deficient perfusion. The recovery of protein synthesis with time was prevented by inhibitors of proteolysis suggesting a role for protein degradation in this phenomenon.

Amino Acids↗
PubMed1982

The role of amino acids in the regulation of protein synthesis in perfused rat liver. II. Effects of amino acid deficiency on peptide chain initiation, polysomal aggregation, and distribution of albumin mRNA.

Decreased rates of protein synthesis which occurred in rat livers perfused with amino acid-deficient medium were accompanied by a loss of polysomes and a doubling of concentrations of ribosomal subunits and monomers as compared to unperfused liver or livers perfused with amino acid-supplemented medium. The loss of polysomes was not the result of mRNA degradation because this effect could be reversed by addition of amino acids to the perfusion medium. Instead, loss of polysomes indicated an impairment in peptide chain initiation. To determine whether the block of initiation could be the result of sequestration of mRNA in untranslatable pools, the content of albumin mRNA in membrane-bound and free polysomes and in the non-polysomal fraction isolated on sucrose density gradients was determined by hybridization to an albumin cDNA. In livers perfused with an amino acid-supplemented medium, 90% of the albumin mRNA was found in bound polysomes, and with amino acid-deficient medium, this value was decreased to 80%. Thus, there was no indication of a significant pool of mRNA which was not being translated. The block of initiation was, however, accompanied by a marked change in density and methionine-binding characteristics of the 40 S ribosomal subunit in livers perfused with deficient medium as compared to livers perfused with supplemented medium or to unperfused livers. There was a relative loss of 40 S subunits in a low density form, 1.41 g/cm3, an increase in the proportion in a high density form, 1.48 g/cm3, and a decrease in binding of [35S]methionine to 40 S ribosomal subunits. These changes provide evidence of reduced rates of formation of the 40 S initiation complex.

Amino Acid Sequence↗
PubMed1981

Alterations in albumin secretion and total protein synthesis in livers of thyroidectomized rats.

Perfused rat livers and isolated rat hepatocytes exhibited a 50% decrease in the secretion of both albumin and total secretory proteins after thyroidectomy. In contrast, synthesis of non-secretory proteins was decreased by only 20% from the rates observed in liver preparations from euthyroid rats. These observations suggested a disproportionate effect of thyroidectomy on the synthesis of secretory proteins compared with non-secretory proteins. Disproportionate decreases in the synthesis of albumin in other endocrine-deficient states such as hypophysectomy and diabetes had previously been shown to be associated with decreases of similar magnitude in the relative abundance of albumin-mRNA sequences. In contrast, thyroidectomy did not affect the activity or amount of albumin mRNA in total liver poly(A)-containing RNA when assayed by cell-free translation and by hybridization with complementary DNA, respectively. Furthermore, labelling experiments in vivo demonstrated that albumin synthesis represented 12.9 +/- 0.5% and 12.4 +/- 0.4% of total protein synthesis in livers of thyroidectomized and euthyroid rats respectively. Therefore the fall in secretion of albumin and total secretory protein after thyroidectomy did not appear to be a reflection of disproportionate decreases in the synthesis of these proteins. Instead, defects in steps involved in the post-synthetic processing and secretion of albumin are suggested. A number of comparisons, including ribosome half-transit times, the size distributions of total and albumin-synthesizing polyribosomes, and the fraction of RNA present as inactive ribosomes, provided evidence that the overall decrease in protein synthesis after thyroidectomy was not due to generalized alterations in translational processes. Instead, the decrease in total protein synthesis appeared to reflect the RNA content of the liver, which fell in proportion to th decrease in protein synthesis.

Albumins↗
PubMed1981

Protein synthesis in perfused rat liver following thyroidectomy and hormone treatment.

Rat livers perfused in situ were used to investigate the effects of thyroid hormone deficiency and subsequent hormone replacement on hepatic protein synthesis and secretion. Following surgical thyroidectomy, production of albumin and total secretory proteins was reduced by 50%, whereas the synthesis of retained intracellular proteins was reduced by only 20% compared to the respective control rates. The 28% reduction in the synthesis of total liver proteins (secreted plus retained) was associated with a fall of similar magnitude in the tissue RNA level. Treatment of thyroidectomized rats with various doses of thyroid hormones indicated a regimen that restored the rate of synthesis of total liver proteins and the concentration of RNA to normal levels. This treatment regimen increased the synthesis of retained proteins to 115% of the control rate, but produced only modest increases in the production of albumin and total secretory proteins. These findings indicated an impairment in protein secretion in livers of thyroidectomized rats that persisted after the defect in protein synthesis was corrected by thyroid hormone treatment.

Animals↗
PubMed1978

Correlation of albumin production rates and albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic rats.

We have studied the effects of alloxan-induced diabetes and subsequent insulin replacement on albumin and total hepatic protein synthesis. Diabetes resulted in a reduction to approximately 20% of normal in albumin synthesis relative to the rate of total protein synthesis in vivo and a reduction to 10% in the absolute rate of albumin secretion by perfused livers. In contrast, the synthesis of total secretory protein and retained hepatic protein was affected to a lesser extent by diabetes. Treatment of diabetic rats with insulin restored rates of albumin and total hepatic protein synthesis to normal levels. The molecular basis of these alterations in albumin synthesis was investigated by examining albumin mRNA levels in livers of normal, diabetic, and insulin-treated diabetic animals. The level of albumin mRNA, whether assayed by cell-free translation or by hybridization to a specific complementary DNA probe, was markedly decreased in livers of diabetic animals and was restored to normal by insulin treatment. These changes occurred in parallel with changes in the rates of albumin secretion observed in perfused liver, suggesting that albumin mRNA content is the primary factor responsible for altering rates of albumin synthesis under these conditions.

Albumins↗
PubMed1976

Lack of effect of amino acid concentration on protein synthesis in the perfused rat liver.

The effect of increasing the perfusate concentration of amino acids on the incorporation of labelled valine into protein was followed in perfusions of rat livers lasting for 2h. A fixed amount of labelled and unlabelled valine was added to the perfusate as the other amino acids were increased in multiples of the concentrations normally found in rat plasma. Under these conditions no increase in valine incorporation was observed, which appeared to be in conflict with results published by other workers, However, a different method of labelling from that used here was used in the earlier studies. An increasing amount of a labelled amino acid was added as the concentrations of the unlabelled amino acids were increased in the perfusate. An experiment directly comparing to the two labelling methods produced results that indicated that the apparent increase in liver protein synthesis observed by the other workers could have been due to the method of radioisotope addition. It is therefore concluded that increasing the perfusate concentration of amino acids does not increase amino acid incorporation into liver protein.

Amino Acids↗