Incubation Of Adipocytes Research Articles

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes.

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.

A Comparison of Basal and Insulin stimulated Glucose Transport in Rat Adipocyte Plasma Membranes

Specific D-glucose transport in plasma membranes prepared from control and insulin-treated rat adipocytes was measured using a recently developed dual isotope ([3H]-D-glucose and [14C]-L-glucose), rapid filtration assay which allowed measurements of initial rates at 1 s and 37 degrees. Plasma membranes from insulin-treated adipocytes showed an increase in glucose transport compared with control cells. Saturation kinetic data revealed that the plasma membranes from insulin-treated and control cells had the same Km (26 mM) for glucose transport, whereas insulin treatment increased the Vmax from 4433 pmol/mg protein/s to 9465 pmol/mg proteins/s. Arrhenius plots showed no difference in the energy of activation between control and insulin-stimulated glucose transport states. The optimum pH of both control and insulin-stimulated glucose transport was 7.4. Lower or higher pHs progressively decreased both control and insulin-stimulated glucose transport proportionately. Calcium in the transport assay media did not affect basal or insulin-stimulated glucose transport. However, omission of calcium from the adipocyte incubation media significantly lowered the insulin stimulation by 24% while basal levels were not significantly affected. Insulin specifically bound to the plasma membrane was carried through the fractionation procedure, but removal of this insulin did not alter the stimulated glucose transport. Glucose transport by plasma membranes from control or insulin-treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, dithiothreitol, reduced glutathione, or cytochalasin B. No inhibition of control or insulin-stimulated transport was seen with cytochalasin D or oxidized glutathione. The data presented are consistent with insulin causing the formation of new transport sites similar to the existing basal sites.

Nonesterified fatty acids oppose insulin activation of acetyl CoA carboxylase in rat adipocytes incubated with glucose

1. 1. Incubation of rat adipocytes for 30min with insulin in the presence of glucose led to a significant activation of acetyl CoA carboxylase. 2. 2. This effect of the hormone was not apparent when palmitate was added to the incubation in physiological concentrations.

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine. The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.

Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [ 3H]AMP ([ 3H]cAMP) and washed, and the bound [ 3H]cAMP was estimated. The band of [ 3H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [ 3H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [ 3H]-cAMP was bound in Peak II than in Peak I. The [ 3H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity. Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [ 3H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [ 3H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described. Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.

Identification and subcellular distribution of adipocyte peptides and phosphopeptides.

Subcellular fractions of high purity (including plasma membrane, endoplasmic reticulum, mitochondria, nuclei, and cytoplasm) were prepared from isolated adipocytes, and the peptide components were examined by detergent gel electrophoresis. Each fraction except the endoplasmic reticulum exhibited a unique and reproducible complement of major peptides. Although the endoplasmic reticulum was distinctive in its enzymic markers, its peptide components showed striking homologies with certain species in the plasma membrane and cytoplasm. The two major adipocyte glycopeptides appear to be contained in the plasma membrane, inasmuch as they followed the distribution of 5'-nucleotidase. Incubation of adipocytes with extracellular 32Pi led to a uniform rate of incorporation of 32P into cellular peptides, with steady-state incorporation reached by 2 hours. Plasma membrane, mitochondria, nuclei, and cytoplasm all contained a distinctive complement of from two to five major phosphopeptides of different molecular weights. The majority of endoplasmic reticulum phosphopeptides exhibited molecular weights closely similar to those of certain species in the plasma membrane and cytoplasm. The phosphopeptides of the plasma membrane exhibited the highest absolute 32P incorporation of all phosphopeptides, next was the single major mitochondiral phosphopeptide. All fractions except the mitochondria contained, in addition to the few major phosphopeptides, numerous minor 32P-labeled phosphopeptides.

The Role of Calcium Ion in Epinephrine Activation of Lipolysis

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.

The Stimulation of Adipocyte Plasma Membrane Magnesium Ion-stimulated Adenosine Triphosphatase by Insulin and Concanavalin A

SUMMARY These studies were designed to determine whether insulin affected the ATPase system of the adipocyte plasma mem- brane. The hormone was found to stimulate the Mg2+- ATPase significantly at concentrations as low as 5 microunits per ml and reaching maximum stimulation between 50 to 100 microunits per ml. The effect occurred at the earliest time point measured (2.0 min) and was linear over a 20-min incubation. The maximal mean dBerence in specific activity caused by 100 microunits of insulin per ml was 1.10 f 0.11 pmoles of Pi per mg of protein per 10 min in 25 experiments and was a 12.1% increase over the basal mean specific activity of 9.82 f 0.38. Insulin did not stimulate the sodium and potassiti ion-activated Mg2+ATPase of the plasma membrane nor the Mg2+ATPase of either the adipo- cyte mitochondrial or microsomal fractions. Epinephrine had no effect on the plasma membrane Mg2+ATPase. Pre- incubation of adipocytes with insulin prior to preparation of the plasma membranes or preincubation of the membranes prior to assay did not prevent the insulin stimulation of the Mg2+ATPase. The basal enzyme activity of insulin-pre- treated membranes or of membranes from insulin-pre- treated cells was signi&antly lower than the corresponding controls. Oligomycin and sodium azide significantly lowered the basal Mg2+ATPase specific activity and prevented the insulin stimulation of the enzyme but neither agent blocked the binding of insulin to the plasma membranes. Con- canavalin A mimicked the effect of insulin on the plasma membrane Mg2+ATPase but produced a greater magnitude of change and could overcome the inhibition caused by oligo- mycin and sodium azide. The data suggest that insulin stimulates the Mg2+ATPase after interacting with the mem- brane receptor but the role of this phenomenon in the mech- anism of insulin action remains to be determined. Insulin is known to cause a set

Hormonal Regulation of Adipocyte Enzymes: THE EFFECTS OF EPINEPHRINE AND INSULIN ON THE CONTROL OF LIPASE, PHOSPHORYLASE KINASE, PHOSPHORYLASE, AND GLYCOGEN SYNTHASE

Abstract The effects of epinephrine and insulin on three enzyme systems in adipocytes were investigated with regard to the role of adenosine 3',5'-monophosphate (cyclic AMP) in their control and the possibility that differential regulatory mechanisms might operate beyond cyclic AMP. When rat adipocytes were incubated with increasing concentrations of epinephrine (0.01 to 100 µm) elevation of cyclic AMP and glycerol production, activation of phosphorylase and deactivation of glycogen synthase appeared to be closely correlated. Activatability of hormone-sensitive in cell extracts by cyclic AMP-dependent protein decreased with increasing concentrations of epinephrine indicating conversion of the nonactivated to the activated form during incubation of the cells. Activation of in cell-free extracts by cyclic AMP and MgATP was promptly arrested by addition of protein inhibitor. This finding, together with results of previous studies, rules against the involvement of a lipase kinase analogous to that of phosphorylase in phosphorylase activation. Phosphorylase activities measured between pH 6.0 and 9.2 were unaffected at any concentration of epinephrine. Moreover, activation of phosphorylase in cell-free extracts was not inhibited by addition of protein inhibitor. When adipocytes were incubated in Ca2+-free Krebs-Ringer bicarbonate medium, activation of phosphorylase in response to epinephrine was not impaired. Phosphorylase assayed in cell-free extracts did not require Ca2+ for the activation of endogenous phosphorylase. However, some Ca2+ dependence was shown when exogenous skeletal muscle phosphorylase b was used as substrate. Thus, Ca2+ does not appear to regulate the catalytic activity of adipocyte phosphorylase kinase, nor could any change in the activation state of this enzyme in response to epinephrine be observed. These results distinguish adipocyte phosphorylase from that in muscle and in heart which requires Ca2+ and is clearly convertible to an activated form by epinephrine. Incubation of adipocytes with glucose (10 mm) significantly reduced the phosphorylase activity ratio (activity in the absence of 5'-AMP divided by that in its presence). Epinephrine stimulated increases in lipolysis and phosphorylase activity in the presence or absence of glucose, but its effect on glycogen synthase was apparent only in the presence of glucose. The insulin-induced increase in glycogen synthase activity ratio (activity in the absence of glucose 6-phosphate divided by that in its presence) was also dependent on glucose. Insulin (0.4 nm, 50 microunits per ml), in the presence of glucose, almost completely antagonized the epinephrine-induced increases in lipolysis, activation of phosphorylase and deactivation of glycogen synthase. The antilipolytic action of insulin in cells previously exposed to epinephrine was associated with reversion of the activated to its nonactivated form. However, insulin antagonized the elevation of cyclic AMP induced by epinephrine only marginally. This dissociation between the antagonism of insulin and epinephrine on the activities of the three enzymes on the one hand and the concentration of cyclic AMP on the other hand makes it seem unlikely that the action of insulin is mediated by a reduction in cyclic AMP concentration.