Cyclic AMP metabolism and lipolysis in cultured human adipocyte precursors

Triacylglycerol breakdown (lipolysis) results from a series of reactions culminated by activation of "hormone-stimulated" triacylglycerol lipase, an enzyme unique to adipose tissue. We have studied various components of the lipolytic process in human omental adipocyte precursors differentiating in culture. The levels of cyclic AMP, the "second messenger" of lipolytic hormones, were about sixfold higher in fat cell precursors than those in abdominal skin fibroblasts. L-Isoproterenol resulted in significant elevation of cyclic AMP levels in both cell types. Preincubation of intact adipocyte precursors with insulin resulted in significant enhancement of "low Km" cyclic AMP phosphodiesterase activity; in contrast, this hormone had no effect on fibroblast phosphodiesterase activity, a distinctive biochemical difference despite the morphological similarities between the two cell types during the early stages of adipocyte precursor maturation. Incubation of adipocyte precursors with isoproterenol resulted in the release of fatty acids into the medium, findings indicative of "hormone-stimulated" lipase activity and, hence, the operation of the entire "lipolytic cascade"; isoproterenol-stimulated lipolysis was inhibited by insulin. Release of fatty acids from fibroblasts was not observed. Thus, "hormone-stimulated" lipolysis and insulin stimulation of cyclic AMP phosphodiesterase activity are expressed during early stages of human adipocyte precursor differentiation.     

Cyclic AMP inhibition of protein kinase activity in HeLa 65 plasma membranes.

Demethylisothiocolchicine forms monoclinic crystals, space group P2₁, a = 11.906, b = 8.713, c = 10.004 Å; β = 89.44°; Z = 2. The crystal and molecular structure was determined from 1911 independent x-ray reflections and refined to R = 0.04. The molecule has the same overall shape as active colchicine derivatives but one methoxy group has a different conformation from that found in other compounds.     

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.     

Cyclic AMP and the regulation of cholesterol metabolism.

Cyclic AMP has been implicated to a greater or lesser extent in the regulation of four key enzymes which interact to regulate intracellular cholesterol metabolism; HMG CoA reductase; ACAT; cholesteryl ester hydrolase; and cholesterol 7 alpha hydroxylase. The relationship between these enzymes and the sites where current evidence suggests that cyclic AMP may be involved are summarized in Fig. 3. Cholesterol 7 alpha hydroxylase controls the catabolism of cholesterol to bile acids in the liver, and thus its removal from the body via the bile, but does not have a major role in cholesterol metabolism in extrahepatic tissues. It is clear that cyclic AMP is able to influence the activity of this enzyme in liver sub-cellular fractions and isolated hepatocytes in vitro, and studies in our laboratory have shown that changes in Ca2+ fluxes within the cell may be important in its mechanism of action. Whether or not the cyclic nucleotide has a role regulating cholesterol 7 alpha hydroxylase activity in vivo, however, is not known. HMG CoA reductase is inactivated by phosphorylation both in vitro and in vivo, but although cyclic AMP and glucagon have been shown to inhibit the enzyme, cyclic AMP-dependent protein kinase is not directly involved. The exact mechanism by which the cyclic nucleotide influences the system remains unclear, but it may be related to activation of microsomal phosphatases. The activity of ACAT has been shown to be modulated by phosphorylation in a number of tissues in vitro, but the involvement of cyclic AMP has not been unequivocally demonstrated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca and calmodulin

The effects of Ca²⁺ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [³²P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca²⁺. Maximal incorporation of [³²P] into the protein was achieved with free Ca²⁺ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [³²P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca²⁺ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca²⁺ concentrations between 10⁻⁶ – 10⁻⁵ M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca²⁺ and calmodulin.     

Characterization of calcium binding to adipocyte plasma membranes.

Calcium binding to adipocyte plasma membranes has been assessed by equilibrium dialysis and by membrane filtration techniques. Calcium binding was specific and saturable, displaying two distinct classes of binding sites. The affinity constants and maximum binding capacities in the presence of 0.1 M KCl were 4.5 X 10(4) M-1 and 1.8 nmol/mg of protein and 2.0 X 10(3) M-1 and 13.7 nmol/mg for the high and low affinity sites, respectively. Bound calcium was totally dissociated in the presence of excess calcium within 11.0 min in two distinct phases corresponding to the two classes of sites. Association and dissociation rate constants for the high affinity sites were 7.7 X 10(2) M-1S-1 and 9.2 X 10(-3S-1 respectively. Free energy changes at 24 degrees were +6.4 kcal mol-1 for the high affinity sites and +4.5 kcal mol-1 for the low affinity sites. The high affinity sites demonstrated a pH optimum of 7.0 whereas the binding to the low affinity sites progressively increased between pH 6.0 and 9.0. Low concentrations of MgCl2 (less than 300 muM) enhanced calcium binding slightly, whereas high concentrations of KCl and MgCl2 were noncompetitive inhibitors of calcium binding. Procaine and ruthenium red had no effect on calcium binding and lanthanum was a poor inhibitor of calcium binding. This represents the first report of calcium binding to adipocyte plasma membranes and the first kinetic analysis of calcium binding to biological membranes. The specificity of this calcium-binding system in adipocyte plasma membranes suggests its importance in cellular bioregulation.     

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 K_m 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 > lysophosphatidylcholine > lysophosphatidylserine > phosphatidylserine > 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 K_m 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 K_m cAMP phosphodiesterase activity.     

Calmodulin-sensitive ATP-dependent Ca2+ transport across adipocyte plasma membranes

An ATP-dependent transport system which is active at concentrations of free Ca2+ in the submicromolar range has been identified in adipocyte plasma membranes. The system appears to represent the functional component of the high affinity insulin-sensitive calcium-stimulated, magnesium-dependent adenosine triphosphatase preveiously described in the same preparation (Pershadsingh, H. A., and McDonald, J. M. (1979) Nature 281, 495-497). This ATP-dependent Ca2+ transport pump was stimulated approximately 3-fold by the Ca2+-dependent regulatory protein, calmodulin. This effect was confined to the plasma membrane since a similar effect was undetectable in the fraction enriched in endoplasmic reticulum. Calmodulin stimulation was dose-dependent but saturable with half-maximal activation occurring at 0.72 microgram/ml (43 nM). Calmodulin appeared to stimulate the system primarily by decreasing the apparent half-maximal saturation constant for free Ca2+ from 0.20 +/- 0.04 microM to 0.07 +/- 0.01 microM (n = 3). The Hill coefficient increased from 1.6 +/- 0.2 to 3.2 +/- 0.6 (n = 3), thus showing an increased positive cooperativity which allows the pump to be activated by an exceedingly narrow Ca2+ threshold in the presence of calmodulin. The calmodulin stimulation of the plasma membrane Ca2+ extrusion pump in adipocytes, working in opposition to metabolic signals which increase cytoplasmic Ca2+, could constitute a self-regulating negative feedback device for maintaining a low steady state level of intracellular Ca2+. This feedback system may be of critical importance in regulation of cellular metabolism by insulin.     

Cyclic AMP metabolism in adipose tissue of exercise-trained rats.

Cyclic AMP metabolism in epididymal adipose tissue of exercise-trained rats was examined to determine if training induced changes in cyclic AMP production or inactivation. Beginning at 7 weeks of age, male rats were physically trained by 12 weeks of treadmill running. Pair-fed control rats remained sedentary in their cages for the duration of the experiment. Tissue levels of cyclic AMP were measured in epididymal adipose tissue slices incubated with norepinephrine. Adenyl cyclase was assayed in adipocyte ghost cell prepartions and low-Km phosphodiesterase was assayed in homogenates of adipose tissue. In response to norepinephrine stimulation, tissue cyclic AMP levels were reduced in trained compared to untrained rats. Training increased the ratio of activity of phosphodiesterase relative to adenyl cyclase. The results of this study indicate that cyclic AMP production in response to norepinephrine stimulation is not increased by training and may even be reduced, implying that adipose tissue cyclic AMP levels may be under a greater degree of control in trained rats. Modulation of adipose tissue cyclic AMP levels may function to regulate more closely the duration of lipolysis in exercise-trained rats.     

Antibodies to ovine adipocyte plasma membranes recognize tissue and species specific plasma membrane components

1. Ovine adipocyte plasma membrane (PM) contains three unique proteins that have relative molecular mass of 70, 106, and 110 kD which are lacking in PM from liver, kidney, heart, and red blood cells. 2. Two major proteins on ovine adipocyte PM having molecular mass of 44 and 46 kD which were also present on porcine adipocyte PM. 3. These ovine proteins could not be detected on either rat or chicken adipocyte PM.     

Characterization of polyclonal antibodies against ovine adipocyte plasma membranes.

1. Antisera against ovine adipocyte plasma membranes were developed in a mare. 2. These antisera showed a high degree of specificity to adipocyte plasma membranes and cross-reacted with other tissues. 3. Antisera cross-reactivity can be removed by adsorption of the antiserum with various tissue plasma membranes without significant reduction in their reactivity to adipocyte plasma membranes. 4. Antisera reacted with different affinity to adipocyte plasma membranes from different sites and from different species of animals.     

Cyclic AMP regulation of fructose metabolism in germinatingPilobolus longipes spores

DormantPilobolus longipes spores metabolized fructose primarily to ethanol, CO₂, and trehalose. Cyclic AMP-induced spore activation was accompanied by a large stimulation of glycolytic activity. Mobilization of reserves, which was cyclic AMP dependent, accounted for a portion of the glycolytic product. The remaining product was derived from exogenous fructose. Increases in both fructose transport activity and hexose 6-phosphate levels were associated with 6-deoxyglucose-induced spore activation. Phosphofructokinase-1 activity in spore extracts was almost totally dependent upon fructose, 2,6-bisphosphate. High fructose 2,6-bisphosphate levels were correlated with rapid fructose metabolism. However, fructose alone caused a rise in fructose 2,6-bisphosphate content (sufficient to fully stimulate phosphofructokinase-1 activity) but there was no concurrent stimulation of glycolysis. These results suggest that glycolytic rates are determined mainly by hexose 6-phosphate levels and that cyclic AMP regulation of transport is an important determinant of hexose 6-phosphate concentration.     

Cyclic AMP and the plasma membrane potential in Neurospora crassa.

Diverse treatments, which have been shown by Slayman, C. L. (1977) in Water Relations in Membrane Transport in Plants and Animals (Jungreis, A., Hodges, T. K., Kleinzeller, A., and Schultz, S. G., eds) pp. 69-86, Academic Press, New York, to depolarize the plasma membrane of Neurospora, increase levels of adenosine 3':5'-monophosphate (cyclic AMP) in the organism. The treatments include those producing large transport fluxes of metabolizable or nonmetabolizable compounds, rapid temperature drops, and addition of agents which uncouple oxidative phosphorylation. Severe mechanical stress, which may also act to depolarize the plasma membrane, leads to increases in cyclic AMP. The maximal depolarization appears to precede the maximal cyclic AMP levels. It is proposed that the membrane depolarization produces the increased cyclic AMP levels by stimulating the plasma membrane-bound adenylate cyclase and that cyclic AMP may be important to the maintenance of membrane integrity.     

Cyclic AMP, glucose and cortisol in plasma during surgery.

Cyclic AMP, glucose and cortisol in plasma were measured before, during and after major surgery (hysterectomy, six patients) and minor surgery (tympanoplasty, 10 patients). During major surgery cyclic AMP as well as glucose and cortisol showed a pronounced increase. During minor surgery cyclic AMP, glucose and cortisol levels were significantly lower than in the group undergoing major surgery. It is concluded that the increase of plasma cyclic AMP during operative procedures is related to the severity of the trauma.     

Alterations in microsomal and plasma membranes during liver regeneration.

Investigations have been carried out on the alterations of membrane lipids and some enzyme activities during liver regeneration. The results indicated that 32 h after partial hepatectomy the membrane phospholipids per mg protein were augmented. The cholesterol esters were also increased in both microsomal and plasma membranes. The specific radioactivity of the separate phospholipid fractions, estimated by incorporation of 14C-palmitate into the phospholipid molecules, was higher in membranes from partially hepatectomized rats, compared to sham-operated ones, indicating an enhanced phospholipid synthesis. The content and specific radioactivity of diacylglycerols and triacylglycerols was enhanced in both types of membranes from regenerating liver. Moreover, we observed a fluidization of these membranes, which is illustrated by the decrease of the structural order parameter (SDPH) of the lipid bilayer as well as by the elevation of the excimer to monomer fluorescent ratio (IE/IM). 1,6-Diphenyl-1,3,5-hexatriene and pyrene were used as fluorescent probes for determination of the membranes physical state. Palmitoyl-CoA and oleoyl-CoA synthetase, acyl-CoA: lysophosphocholine and acyl-CoA:lysophosphoethanolamine acyltransferase as well as phospholipase C activities were augmented in membranes from partially hepatectomized rats. The biological significance of these alterations in the process of liver regeneration is discussed.