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Biomedical subjects

L Jarett

Publications and source records attributed to L Jarett.

At least 91 records · Page 5Linked to original sources
PubMed1985

Insulin mediator causes dephosphorylation of the alpha subunit of pyruvate dehydrogenase by stimulating phosphatase activity.

Insulin treatment of rats results in an increased amount or activity of insulin mediators in liver and skeletal muscle. These mediators stimulated pyruvate dehydrogenase and inhibited adenylate cyclase. The insulin-generated mediators caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in mitochondria prelabeled with [gamma-32P]ATP. An assay was developed which quantitatively measured mediator activity by determining the rate of alpha-subunit dephosphorylation. The dephosphorylation was directly proportional to the amount of mediator added and was directly related to activation of pyruvate dehydrogenase. The decrease of alpha-subunit phosphorylation resulted from stimulation of pyruvate dehydrogenase phosphatase, since it occurred in the absence of ATP and was inhibited by NaF. These data further delineate the mechanism of insulin mediator activation of pyruvate dehydrogenase.

Adenylyl Cyclases↗
PubMed1985

Insulin mediators from rat skeletal muscle have differential effects on insulin-sensitive pathways of intact adipocytes.

The effects of partially purified insulin-generated mediators from rat skeletal muscle were compared to those of insulin on intact adipocytes. Insulin and insulin mediator stimulated both pyruvate dehydrogenase and glycogen synthase activity of intact adipocytes. In contrast, insulin stimulated glucose oxidation and 3-O-methylglucose transport, whereas insulin-generated mediators had no effect. Insulin-generated mediators cannot account for all the pleiotropic effects of insulin, especially membrane-controlled processes.

3-O-Methylglucose↗
PubMed1985

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors.

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.

Adipose Tissue↗
PubMed1985

Insulin stimulates generation of intracellular mediators in rat heart.

Insulin treatment of rats results in an increased amount or activity of insulin mediators in heart muscle. The mediators stimulated mitochondrial pyruvate dehydrogenase and inhibited glucagon-stimulated adenylate cyclase. The mediators were copurified by ultrafiltration, ethanol extraction, Dowex cation-exchange, and QAE-Sephadex anion-exchange chromatography. The activities of the two mediators were separated by Sephadex G-10 chromatography. Fasting rats for 72 h diminished the mediator response to insulin treatment. These results, taken together with previous reports, indicate that insulin generates a number of mediators which have a ubiquitous tissue distribution. The activity of these mediators, like insulin responsiveness, is altered by the metabolic state of the animal.

Adenylyl Cyclase Inhibitors↗
PubMed1985

Stimulation of pyruvate dehydrogenase activity in intact rat adipocytes by insulin mediator from rat skeletal muscle.

This study compared the effects of insulin and insulin mediator from skeletal muscle of control and insulin-treated rats on intact adipocyte pyruvate dehydrogenase. Increasing insulin concentrations stimulated pyruvate dehydrogenase activity in a biphasic manner with a maximal stimulation at 100 microU/ml which was 2-fold and sustained for up to 1 h. The mediators from control or insulin-treated rats also stimulated pyruvate dehydrogenase of intact adipocytes with the effect increasing in a linear manner up to a 1:10 final dilution. The latter mediator had twice the stimulatory activity as the former. Peak stimulation of pyruvate dehydrogenase by the mediators was attained within 10 min of incubation. The enzyme activity rapidly declined thereafter, with the stimulation by mediator from control rats decreasing at a faster rate than that due to mediator from insulin-treated rats. The stimulatory effect of the mediators on adipocyte pyruvate dehydrogenase was found to be additive to that of insulin. This study demonstrates: 1) that insulin mediator can act on mitochondrial pyruvate dehydrogenase of intact, functional adipocytes as it does on isolated intact or broken mitochondria; 2) that the mediator is degraded by the adipocyte; and 3) that the amount of mediator generated by insulin probably limits the stimulation of pyruvate dehydrogenase by insulin. These findings further substantiate the physiological relevance of this putative insulin second messenger.

Adipose Tissue↗
PubMed1985

Insulin mediator stimulates pyruvate dehydrogenase of intact liver mitochondria.

The effects of putative insulin mediators on the pyruvate dehydrogenase (PDH) activity of intact mitochondria isolated from rat liver were investigated. The mitochondria were judged intact on the basis of electron microscopic examination and demonstrated respiratory control. Only mitochondria having respiratory control ratios of greater than 4, using succinate as a substrate, were used in these studies. Addition of physiologic concentrations of insulin to these mitochondria caused stimulation of PDH activity, attributed to generation of an insulin mediator from plasma membranes contaminating the mitochondrial preparation. Exogenous plasma membranes from rat adipocytes or liver caused further stimulation of PDH activity, which was proportional to the amount of plasma membranes added. Addition of insulin to the mixture of mitochondria and plasma membranes stimulated PDH still further. The stimulation was proportional to the insulin concentration, with maximal effects observed at 50 microU/ml insulin. Partially purified mediators from liver, muscle, H4-II-E hepatoma cells, and IM9 lymphocytes also stimulated PDH activity in intact mitochondria. Mediators prepared from insulin-treated liver, muscle, and cultured hepatoma cells stimulated PDH more than did mediators from the corresponding untreated source. Mediator from insulin-treated IM9 lymphocytes stimulated PDH less than did mediator from untreated IM9 lymphocytes. These findings are consistent with the known effects of insulin on these tissues and with the reported effects of the various mediators on PDH activity in non-intact mitochondria. These observations support the proposal that these mediators are physiologically significant modulators of insulin's effects on PDH activity.

Animals↗
PubMed1984

Effect of adenosine on insulin activation of rat adipocyte pyruvate dehydrogenase.

Adenosine and its analogue N6-phenylisopropyladenosine stimulated pyruvate dehydrogenase activity of isolated rat adipocytes. Maximal stimulation was obtained with concentrations between 50 and 100 mu M, with the effect decreasing at higher concentrations. The effects of insulin on this enzyme was modified by adenosine. The concentration of insulin (10 mu units/ml) that produced almost half-maximal stimulation, had little or no effect, when adenosine deaminase was present. Adenosine also enhanced the effect of suboptimal but not optimal concentrations of insulin. Thus, the mechanism of adenosine action on adipocyte pyruvate dehydrogenase could in some way be similar or related to that of insulin.

Adenosine↗
PubMed1984

Insulin stimulation of phospholipid methylation in isolated rat adipocyte plasma membranes.

Partially purified plasma membranes prepared from rat adipocytes contain N-methyltransferase(s) that utilize(s) S-adenosyl-L-methionine to synthesize phosphatidylcholine from phosphatidylethanolamine. The incorporation of [3H]methyl from S-adenosyl-L-[methyl-3H]methionine into plasma membrane phospholipids was linear with incubation time and plasma membrane protein concentration and was inhibited in a dose-dependent manner by both S-adenosyl-L-homocysteine and 3-deazadenosine. The addition of insulin to plasma membranes stimulated the methylation of endogenous phosphatidylethanolamine, as evidenced by an increase in the levels of phosphatidyl-N-monomethylethanolamine, phosphatidyl-N, N-dimethylethanolamine, and phosphatidylcholine. The effect of insulin was rapid and concentration-dependent, with 100 microunits/ml providing near maximal stimulation. The incorporation of [3H]methyl into phospholipids of control and insulin-stimulated plasma membranes was enhanced by the addition of exogenous methyltransferase substrates phosphatidylethanolamine, phosphatidyl-N-monomethylethanolamine, and phosphatidyl-N,N-dimethylethanolamine. The stimulatory effect of insulin on adipocyte plasma membrane phospholipid methylation may have a physiological role in insulin action.

Adipose Tissue↗
PubMed1983

Comparison of phospholipid effects on insulin-sensitive low Km cyclic AMP phosphodiesterase in adipocyte plasma membranes and microsomes.

Both adipocyte plasma membranes and microsomes possess insulin-sensitive low Km cyclic AMP phosphodiesterase activity. The activity of the enzyme from both sources was susceptible to activation by several anionic phospholipids. Activators of the plasma membrane enzyme were lysophosphatidylglycerol greater than lysophosphatidylcholine greater than lysophosphatidylserine greater than phosphatidylserine greater than phosphatidylglycerol. These same phospholipids activated the microsomal enzyme but the extent of activation by each phospholipid was reversed. Neutral phospholipids and other anionic phospholipids were without effect. The phospholipids had no effect on high Km cAMP phosphodiesterase in either membrane. The results suggest that the phospholipid headgroup was an important determinant for enzyme activation by phospholipid. The increased susceptibility of the plasma membrane enzyme to lysophospholipid may be attributed to a difference in the plasma membrane enzyme compared to the microsomal membrane enzyme or to differences in plasma membrane and microsomal membrane phospholipid composition and their ability to regulate low Km cAMP phosphodiesterase activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

The chemical mediators of insulin action: possible targets for postreceptor defects.

An insulin-sensitive subcellular system was developed from rat adipocytes consisting of plasma membranes and mitochondria. Direct addition of insulin, concanavalin A or anti-insulin receptor antibody to this system resulted in the production of a mediator substance from the plasma membrane that caused dephosphorylation of the alpha subunit of pyruvate dehydrogenase in the mitochondria with concomitant activation of the enzyme. The mediator activated pyruvate dehydrogenase by activating the pyruvate dehydrogenase phosphatase and not by inhibiting the pyruvate dehydrogenase kinase. This was similar to the mechanism by which insulin causes activation of the enzyme in the intact cell. The insulin-sensitive mediator material from the adipocyte plasma membrane was acid-stable with a molecular weight of 1,000 to 1,500. Our laboratory has shown that the mediator that activates pyruvate dehydrogenase was present in intact adipocytes, hepatoma cells, and IM-9 lymphocytes. Insulin altered the amount or activity of the mediator consistent with the effect of the hormone on the cell. Other laboratories have shown similar effects on skeletal muscle and liver. We have shown the mediator to mimic insulin action on the low Km cyclic adenosine monophosphate (AMP) phosphodiesterase and the (calcium++-magnesium++)-adenosine triphosphatase (Ca++-Mg++)-ATPase of adipocyte plasma membranes in addition to pyruvate dehydrogenase. Other laboratories have shown the mediator to activate glycogen synthase. A body of direct and indirect evidence exists that demonstrates that more than one mediator exists. The chemical nature of the mediator is unknown but probably represents a new family of intracellular mediators of hormone action. These mediators may have clinical relevance in postreceptor defects of obesity and type II diabetes (noninsulin-dependent diabetes mellitus).

Adipose Tissue↗
PubMed1983

Quantitative ultrastructural analysis of receptor-mediated insulin uptake into adipocytes.

Monomeric ferritin-insulin was used as an ultrastructural marker to determine by quantitative electron microscopy the time course and route of insulin uptake in rat adipocytes. To approximate steady state membrane binding conditions prior to any internalization, adipocytes were prefixed with glutaraldehyde and incubated for 30 min with 70 nM monomeric ferritin-insulin. Electron micrographs of these cells showed that the ferritin-insulin particles were predominantly in small groups of receptor sites on the plasma membrane and in pinocytotic-like invaginations of the plasma membrane. Significant amounts of ferritin-insulin were observed in cytoplasmic vesicles of unfixed cells as early as 2 min and in multivesicular bodies and lysosome-like structures within 5 to 10 min after the addition of the ligand. Ferritin-insulin accumulation reached steady state levels in the cytoplasmic vesicles in 5 to 10 min and in the lysosome-like structures in 15 min. Little ferritin-insulin was bound to coated pits, and the relative paucity of coated pits found in adipocytes suggested that these specialized endocytotic structures have a relatively insignificant role in insulin uptake in fat cells. Quantitative analysis of the uptake process suggested that a proportion of the insulin internalized by the cell may not be transported to lysosomes, but may be recycled along with the insulin receptor to the plasma membrane.

Adipose Tissue↗
PubMed1983

Phospholipids and the regulation of pyruvate dehydrogenase from rat adipocyte mitochondria.

Aqueous dispersions of 4 out of 9 phospholipids added individually to the mitochondrial fraction from rat adipocytes altered the activity of pyruvate dehydrogenase in a dose-dependent manner from 1 to 300 microM. Phosphatidylserine increased and phosphatidylcholine, phosphatidylinositol and phosphatidylinositol-4-phosphate decreased enzyme activity. The stimulation of pyruvate dehydrogenase induced by phosphatidylserine may be reversed to below basal activity by phosphatidylinositol-4-phosphate and to basal activity by NaF, a pyruvate dehydrogenase phosphatase inhibitor. The inhibition of pyruvate dehydrogenase induced by phosphatidylinositol-4-phosphate may be restored to basal levels by the addition of calcium. These results suggest that phosphatidylserine activates pyruvate dehydrogenase activity through activation of the phosphatase, perhaps forming a phosphatidylserine-calcium complex. The inhibition by phosphatidylinositol-4-phosphate may be mediated by disruption of the enzyme complex. The phospholipids may play a physiological role in the regulation of pyruvate dehydrogenase activity.

Adipose Tissue↗
PubMed1983

Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes.

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Partial disruption of naturally occurring groups of insulin receptors on adipocyte plasma membranes by dithiothreitol and N-ethylmaleimide: the role of disulfide bonds.

In this ultrastructural study, monomeric ferritin-insulin was used to further elucidate the role of disulfide bonds in maintaining the natural groups of insulin receptors on adipocyte plasma membranes. Dithiothreitol (1 mM) caused partial disruption of the occupied receptor groups with an increase in single receptors to greater than 50% of total occupied receptors. N-Ethylmaleimide (1 mM) disrupted the groups to the same extent as dithiothreitol and the effect was partly additive with the dithiothreitol effect. The magnitude of the disruption caused by dithiothreitol or N-ethylmaleimide was similar to that caused by cytochalasin B. Dithiothreitol, a reducing agent, caused a marked increase in binding of insulin to the plasma membranes while N-ethylmaleimide and cytochalasin B, both thiol reagents, had little if any effect on insulin binding. These data suggest that two different sets of disulfide bonds are involved. One set was susceptible to both reducing and thiol reagents and responsible for holding the receptor groups together, and the other set was susceptible to reducing agents only and related to the increased insulin binding caused by dithiothreitol. A proposed model is discussed.

Adipose Tissue↗
PubMed1982

Inhibition by bacitracin of rat adipocyte plasma membrane degradation of 125I-insulin is associated with an increase in plasma membrane bound insulin and a potentiation of glucose oxidation by adipocytes.

The present study demonstrated that at physiological concentrations of insulin bacitracin inhibited the degradation of specifically bound insulin by enzymes located in the rat adipocyte plasma membrane. Bacitracin increased the amount of intact insulin specifically bound to the plasma membrane and potentiated the stimulation of adipocyte glucose oxidation by submaximal concentrations of the hormone. In contrast to agents such as chloroquine, which inhibit lysosomal degradation of internalized insulin, bacitracin was shown by two approaches to inhibit a degradative process localized to the adipocyte plasma membrane. Cyanide and 2,4-dinitrophenol, agents which inhibit energy requiring endocytosis, had no effect on the bacitracin inhibition of cellular degradation of 125I-insulin. Bacitracin directly inhibited 125I-insulin degradation by isolated plasma membranes at similar concentrations and to a similar extent as found with cells. The degradative process inhibited by bacitracin accounted for the majority of cellular degradation of the hormone. The increased 125I-insulin bound to adipocytes was shown to be intact by gel chromatographic analysis and was localized to the plasma membrane by direct and indirect approaches. Bacitracin increased 125I-insulin specifically bound to isolated plasma membranes as early as 2 min. The 125I-insulin bound to adipocytes in the presence of bacitracin was completely dissociable by the addition of 8 microM unlabeled insulin whereas a significant portion of 125I-insulin bound to chloroquine-treated cells could not be dissociated. Bacitracin slowed dissociation of 125I-insulin from the cells. Bacitracin increased the 125I-insulin binding to cells in the presence and absence of cyanide and 2,4-dinitrophenol. Bacitracin potentiated the stimulation of adipocyte glucose oxidation at submaximal concentrations of insulin.

Adipose Tissue↗