Biomedical subjects
Publications and source records attributed to L Jarett.
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Plasma membranes from insulin or insulin mimicker (hydrogen peroxide, anti-insulin receptor antibody, and concanavalin A) treated adipocytes showed an increase in glucose transport compared to control cells due to an increase in Vmax and not due to alteration in Km. Arrhenius plots showed no difference in the energy of activation between control and insulin or insulin mimicker stimulated glucose transport states. Glucose transport by plasma membranes from control or treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, reduced glutathione, or cytochalasin B. The data indicate that the increased transport resulted from addition of new transport sites similar to the sites existing in the basal state.
A quantitative morphological analysis of insulin uptake into adipocytes was undertaken to determine the structural basis for chloroquine-induced increases in intracellular insulin. Adipocytes were incubated with ferritin-labeled insulin in the presence or absence of 50 microM chloroquine at 37 degrees C for 2-90 min and the uptake of the hormone conjugate was determined quantitatively. Quantitative morphometry of cellular organelles also was performed. Chloroquine treatment of adipocytes incubated with 70 nM ferritin-labeled insulin resulted in: (i) a 120% increase in the number of lysosomes in the cytoplasm; (ii) a 75% increase in the average concentration of ferritin-labeled insulin in a lysosome; and (iii) a 25% increase in the percentage of lysosomes containing ferritin-labeled insulin. The cumulative result of these effects was a substantial increase in the amount of intact intracellular hormone within the lysosomes. These morphological data are consistent with biochemical data concerning chloroquine-induced accumulation of 125I-labeled insulin in adipocytes.
A new, simplified, and highly reproducible method for preparing biologically active monomeric ferritin-insulin that can be used as a high-resolution ultrastructural marker for occupied insulin receptors is described. The ferritin-insulin conjugate was stable, and contained negligible free insulin and low levels of free ferritin. The hormone conjugate behaved identically to native insulin in both binding assays and biological activity (glucose oxidation) assays. The immunological and biological activities of the insulin in the conjugate were equal. Biogel 1.5 column purification was used to yield a product monomeric in ferritin.
Studies with a subcellular system demonstrated that the interaction of insulin with the adipocyte plasma membrane resulted in the generation from the plasma membrane of a mediator that activated mitochondrial pyruvate dehydrogenase (EC 1.2.4.1). The insulin-sensitive chemical mediator from the plasma membrane has been partially characterized. It has a molecular weight of 1000-1500. The chemical mediator has been extracted from skeletal muscle, adipocytes, hepatoma cells, and IM-9 lymphocytes. Insulin increased the amount or activity of the mediator in the first three cell types, whereas insulin decreased the activity or amount of the mediator in IM-9 lymphocytes. These insulin-induced variations were consistent with the biological responses of these cells to insulin treatment. The activities of insulin-sensitive enzymes, including pyruvate dehydrogenase, adipocyte low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and adipocyte plasma membrane [Ca2+ + Mg2+]-ATPase were shown to be altered by the chemical mediator. The mediator may act by altering various protein kinases and phosphoprotein phosphatases that modulate the state of phosphorylation and activity of these enzyme systems. The existence of two mediators is proposed. The first may mediate dephosphorylation of various substrates, and the second may influence phosphorylation.
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A computerized quantitative technique was used to analyze the distribution of ferritininsulin receptor sites on rat adipocytes and the effects of cytochalasin B on groups of receptor sites. Computer analysis of separation distances between receptor sites established that insulin receptor sites on adipocytes did not have a random distribution but have a distinct tendency to exist in groups with a maximum separation distance between particles of 400 A. A peak in the distribution of separation distances occurred at 100-200 A. Cytochalasin B, but not cytochalasin D, treatment of adipocytes resulted in a decrease in the number of large groups of receptor sites and a corresponding increase in single and paired receptor sites without affecting the separation distance between the remaining grouped receptors. This suggested that when cytochalasin B disrupted the bond holding receptor sites together, it caused complete disruption. These observations provided additional information on the ultrastructural characteristics of the insulin receptor. Further application of these techniques to the analysis of insulin receptors may provide the necessary structural correlates to the biochemically observed differences in insulin action in other tissues and diseased states.
The endoplasmic reticulum of adipocytes can accumulate calcium by an active transport system linked to (Ca2+ + Mg2+)-ATPase activity (Black, B. L., Jarett, L., and McDonald, J. M. (1980) Biochim. Biophys. Acta 596, 359-371). The ability of this transport system to regulate intracellular calcium levels was tested by incubating adipocytes with various concentrations of calcium and measuring the calcium transport activity in subsequently isolated endoplasmic reticulum vesicles (microsomes). The calcium transport system responded to changes in extracellular calcium concentration; 2.5 mM calcium elicited a transport rate 21 to 34% greater than that of microsomes from cells incubated in the absence of calcium. The stimulatory effect of extracellular calcium was maximum at 2.5 mM, was reduced in magnitude at higher calcium concentrations, was demonstrable after a cell incubation period as short as 5 min, and was maintained for an additional 60 min. The transport system also could be modulated by preincubating isolated microsomes in calcium-containing medium prior to assay of calcium transport. Preincubation with low calcium concentrations elicited a 22 to 33% stimulation of subsequent calcium uptake with half-maximal stimulation at 0.19 +/- 0.02 microM free calcium. Preincubation with high calcium (200 microM) resulted in a 28% inhibition of calcium uptake. The stimulatory effect required ATP during the preincubation period and was modulated by ATP in the 0.05 to 0.5 mM range. The stimulation was not due to changes in efflux of calcium or in affinity of the transport system for calcium. The (Ca2+ + Mg2+)-ATPase activity of a deoxycholate-treated microsomal preparation was increased under preincubation conditions identical with those eliciting stimulation of calcium uptake, suggesting that modulation of enzyme activity produced the stimulatory effect. The results indicate that the endoplasmic reticulum plays a homeostatic role in control of intracellular calcium levels and suggest that the homeostatic response is mediated through interaction of the organelle with calcium and ATP.
The purpose of this study was to determine whether exercise training alters the sensitivity and responsiveness to insulin of glucose uptake and oxidation in fat cells. Female rats were exercised by swimming 6 h/day, 5 days/wk for 12 wk. The swimmers' fat cells were smaller than those of sedentary controls of the same age and similar body weight. A larger amount of insulin was specifically bound by fat cells of the trained rats because of an increase in the number of insulin receptors. The rates of 2-deoxyglucose uptake and of glucose oxidation were higher in fat cells of trained compared with sedentary rats at all insulin concentrations. A maximal insulin stimulus resulted in rates of sugar uptake and oxidation that were about sixfold higher in trained than in sedentary rats' fat cells. This greater responsiveness to insulin could not be explained by the increase in insulin binding but appears to be mediated by adaptation/s) at a step(s) beyond the binding of insulin to its receptors. Our findings suggest that fat cells of exercise-trained animals are adapted for rapid replenishment of energy stores.
Comparison was made of the distribution of the insulin receptor sites on adipocyte and liver plasma membranes by using ferritin-insulin. Two-thirds of the occupied insulin receptors on adipocytes occurred in groups of two or more whereas up to two-thirds of the receptors on liver occurred as single receptors. Ferritin-insulin did not cause aggregation of the receptor sites in either tissue. The naturally occurring groups of receptors on adipocyte membranes may play a role in the greater sensitivity of adipocytes to insulin.
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Calcium uptake by adipocyte endoplasmic reticulum was studied in a rapidly obtained microsomal fraction. The kinetics and ionic requirements of Ca2+ transport in this preparation were characterized and compared to those of (Ca2+ + Mg2+)-ATPase activity. The time course of Ca2+ uptake in the presence of 5 mM oxalate was nonlinear, approaching a steady-state level of 10.8--11.5 nmol Ca2+/mg protein after 3--4 min of incubation. The rate of Ca2+ transport was iM oxalate. The calculated initial rate of calcium uptake was 18.5 nmol Ca2+/mg protein per min. The double reciprocal plot of ATP concentration against transport rate was nonlinear, with apparent Km values of 100 muM and 7 muM for ATP concentration ranges above and below 50 muM, respectively. The apparent Km values for Mg2+ and Ca2+ were 132 muM and 0.36--0.67 muM, respectively. The energy of activation was 23.4 kcal/mol. These kinetic properties were strikingly similar to those of the microsomal (Ca2+ + Mg2+)-ATPase. The presence of potassium was required for maximum Ca2+ transport activity. The order of effectiveness of monovalent cations in stimulating both Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity was K+ greater than Na+ = NH4+ greater than Li+. Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity were both inhibited 10--20% by 6 mM procaine and less than 10% by 10 mM sodium azide. Both processes were completely inhibited by 3 mM dibucaine or 50 muM p-chloromercuribenzene sulfonate. The results indicate that Ca2+ transport in adipocyte endoplasmic reticulum is mediated by a (Ca2+ + Mg2+)-ATPase and suggest an important role for endoplasmic reticulum in control of intracellular Ca2+ distribution.
Preliminary characterization of calcium binding was determined in a highly-enriched islet-cell plasma membrane fraction using a membrane filtration technique. Equilbrium calcium binding was specific, concentration dependent and saturable. Scatchard analysis indicated the existence of more than one class of calcium binding sites. The affinity constants and maximum binding capacities were 1.14 X 10(5) M-1 and 1.2 picomol/microgram protein and 1.17 X 10(3) M-1 and 64.8 picomol/microgram for the high and low affinity sites, respectively. Bound 45Ca2+ was dissociate from the plasma membranes in a biphasic manner in the presence of excess unlabelled calcium.
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The addition of insulin to a mixture of plasma membrane and mitochondrial fractions from rat adipocytes results in a decrease in the phosphorylation of a mitochondrial protein identified as the alpha subunit of pyruvate dehydrogenase [pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] (Seals, J. R., McDonald, J. M. & Jarett, L. (1979) J. Biol. Chem. 254, 6991-6996). This study confirms the prediction that a corresponding increase in pyruvate dehydrogenase activity can be effected by insulin treatment of this preparation. Incubation of the plasma membrane/mitochondria mixture with ATP inhibited pyruvate dehydrogenase activity as measured in a subsequent enzyme assay. The presence of insulin during this incubation with ATP resulted in a 24.5% stimulation of enzyme activity compared to incubation without insulin (n = 9, P < 0.001). The effect was specific for biologically active insulin and was insulin dose-dependent in the physiological range of insulin. Supermaximal doses of insulin produced reduced effects. An insulin effect of similar magnitude could also be observed when the plasma membrane/mitochondria mixture was incubated without ATP. Two insulin mimickers, concanavalin A and antibody to insulin receptor, stimulated pyruvate dehydrogenase by 30.4% (n = 6, P <0.001) and 28.1% (n = 8, P<0.001), respectively. Both of these agents also produced reduced effects at supermaximal concentrations. The effects of all three agents required plasma membranes and could not be produced by treatment of mitochondria alone. The results suggest that a mechanism common to all three agents is responsible for transmitting the stimulation from the plasma membrane to the mitochondrial components of the mixture.