Control of endogenous triglyceride breakdown in the mouse diaphragm

The control of endogenous triglyceride breakdown was studied in vitro, in the incubated intact mouse diaphragm. Isoproterenol (2 microgram/ml) produced parallel increases in glycerol and free fatty acid release, and in tissue cyclic AMP levels, suggesting that cyclic AMP mediates the action of the catecholamine on triglyceride mobilization. In addition to cyclic AMP, calcium seems to be involved in the action of isoproterenol because preincubation of hemidiaphragms in the presence of the calcium ionophore A23187 decreased the lipolytic effect of the drug. Insulin (12.5 mU/ml) antagonized the action of isoproterenol on triglyceride breakdown (it decreased glycerol and free fatty acid release) without altering its stimulatory effect on cyclic AMP levels. On the other hand, no detectable effect on lipolysis was observed with carbachol in control and denervated hemidiaphragms, although the latter possess acetylcholine receptors over the entire surface area of the muscle. It was concluded that catecholamines control triglyceride breakdown in muscle while the cholinergic system does not seem to be involved. Cyclic AMP, calcium, and insulin all affect lipolysis in muscle and the interrelationships remain to be elucidated.     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

Roles of cAMP and free fatty acids on the activity of the hexose monophosphate shunt

Summary Basal glucose utilization by isolated rat adipocytes have been found to be increased ten times in the presence of certain preparations of albumin. In these conditions the effects of several adrenergic agonists and related compounds on glucose oxidation, lipolysis and triacylglycerol synthesis in isolated fat cells have been studied. Oxidation of D(1-¹⁴C) glucose in rat adipocytes was almost completely inhibited by norepinephrine and isoproterenol when added to incubated fat cells. Agents able to modify intracellular AMP cyclic levels by different mechanisms display a similar ability to imitate the effect of lipolytic agents. The inhibition of glucose oxidation due to norepinephrine and isoproterenol is partially reverted by propanolol. Under the same conditions in which norepinephrine and isoproterenol markedly reduced glucose conversion to ¹⁴CO₂, they stimulated lipolysis and triacylglycerol synthesis and in this case propanolol also reverted those actions. However, in these experimental conditions, norepinephrine and isoproterenol did not raise CAMP levels 10 min after hormone addition.It is concluded from these data that glucose oxidation through hexose monophosphate shunt, activation of lipolysis and triacylglycerol synthesis in isolated rat fat cells by lipolytic agents occurs by a mechanism(s) that depend(s) on intracellular free fatty acids levels.     

Myocardial triglyceride turnover and contribution to energy substrate utilization in isolated working rat hearts

The objective of this study was to determine the contribution of myocardial triglycerides to overall ATP production in isolated working rat hearts. Endogenous lipid pools were initially prelabeled (pulsed) by perfusing hearts for 60 min with Krebs-Henseleit buffer containing 1.2 mM [1-14C]palmitate. During a subsequent 60-min period (chase), hearts were perfused with either no fat, low fat (0.4 mM [9,10-3H] palmitate), or high fat (1.2 mM [9,10-3H]palmitate). All buffers contained 11 mM glucose. During the "chase," 14CO2 production (a measure of endogenous fatty acid oxidation) and 3H2O production (a measure of exogenous fatty acid oxidation) were determined. Oxidative rates of endogenous fatty acids during the chase were 279 +/- 50, 88 +/- 14, and 88 +/- 8 nmol of [14C]palmitate oxidized per g dry weight.min in the no fat, low fat, and high fat groups, respectively, compared to exogenous palmitate oxidation rates of 0, 361 +/- 68, and 633 +/- 60 nmol of [3H]palmitate/g dry weight.min, in the no fat, low fat, and high fat groups, respectively. Endogenous [14C]palmitate oxidation rates were matched by loss of [14C]palmitate from endogenous myocardial triglycerides. Overall triglyceride content decreased during the no fat and low fat chase perfusion but did not change during the high fat chase. Loss of triglyceride [14C]palmitate during the high fat chase was matched by incorporation of exogenous [3H]palmitate in triglycerides. In a second series of perfusions, three groups of hearts were perfused under similar conditions, except that unlabeled palmitate was used during the "pulse" and that 11 mM [2-3H/U-14C]glucose and unlabeled palmitate was present during the chase. During the chase, both glycolysis (3H2O production) and glucose oxidation (14CO2 production) rates were measured. Rates of glucose oxidation were inversely related to the fatty acid concentration in the perfusate (1257 +/- 158, 366 +/- 40, and 124 +/- 26 nmol of glucose oxidized per min.g dry weight in the no fat, low fat, and high fat groups, respectively), while rates of glycolysis were not significantly different between these groups. Calculation of overall ATP production from both oxidative and glycolytic sources determined that even in the presence of high concentrations of fatty acids, myocardial triglyceride turnover can provide over 11% of steady state ATP production in the aerobically perfused heart. In the absence of fatty acids, myocardial triglyceride fatty acids can become the major energy substrate of the heart.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of adenosine 3':k'-monophosphate accumulation white fat acids, lactate, and beta-hydroxybutyrate.

The large increase in cyclic AMP accumulation by rat white fat cells seen in the presence of lipolytic agents plus methylxanthines and adenosine deaminase was markedly inhibited by lactate. However, lipolysis was unaffected by lactate. Octanoate, hexanoate, heptanoate, and beta-hydroxybutyrate inhibited both cyclic AMP accumulation and lipolysis by rat fat cells. The mechanism by which these acids inhibit lipolysis differs from that for long chain fatty acids such as oleate. Oleate directly inhibited triglyceride lipase activity of homogenized rat adipose tissue. In contrast, octanoate, beta-hydroxybutyrate, and lacatate had no effect on triglyceride lipase activity. Hormone-stimulated adenylate cyclase activity of rat fat cell ghosts was inhibited by oleate and 4mM octanoate but not by 1.6 mM octanoate, heptanoate, hexanoate, beta-hydroxybutyrate or lactate. None of the acids affected the soluble protein kinase activity of rat adipose tissue. There was no stimulation by lactate, butyrate, beta-hydroxybutyrate, or octanoate of the soluble or particulate cyclic AMP antilipolytic action of a short chain acid such as octanoate or hexanoate was not accompanied by any drop in total fat cell ATP. The mechanism by which lactate lowers cyclic AMP but not lipolysis remains to be established.     

G0/G1 Switch Gene 2 Regulates Cardiac Lipolysis

The anabolism and catabolism of myocardial triacylglycerol (TAG) stores are important processes for normal cardiac function. TAG synthesis detoxifies and stockpiles fatty acids to prevent lipotoxicity, whereas TAG hydrolysis (lipolysis) remobilizes fatty acids from endogenous storage pools as energy substrates, signaling molecules, or precursors for complex lipids. This study focused on the role of G₀/G₁ switch 2 (G0S2) protein, which was previously shown to inhibit the principal TAG hydrolase adipose triglyceride lipase (ATGL), in the regulation of cardiac lipolysis. Using wild-type and mutant mice, we show the following: (i) G0S2 is expressed in the heart and regulated by the nutritional status with highest expression levels after re-feeding. (ii) Cardiac-specific overexpression of G0S2 inhibits cardiac lipolysis by direct protein-protein interaction with ATGL. This leads to severe cardiac steatosis. The steatotic hearts caused by G0S2 overexpression are less prone to fibrotic remodeling or cardiac dysfunction than hearts with a lipolytic defect due to ATGL deficiency. (iii) Conversely to the phenotype of transgenic mice, G0S2 deficiency results in a de-repression of cardiac lipolysis and decreased cardiac TAG content. We conclude that G0S2 acts as a potent ATGL inhibitor in the heart modulating cardiac substrate utilization by regulating cardiac lipolysis.     

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.     

Calcium antagonists and lipolysis in isolated rat epididymal adipocytes: effects of tetracaine, manganese, cobaltous and lanthanum ions and D600.

The present study reports the effects on lipolysis occurring in isolated rat epididymal adipocytes of several agents which have each been found to interfere with membrane calcium transport in a variety of tissues. As reported by other workers, the local tetracaine was a strong inhibitor of hormone accelerated but not of basal lipolysis. The bivalent cations Mn2+ and Co2+ were similarly found to inhibit lipolysis stimulated with either epinephrine, ACTH, theophylline or dibutyryl cyclic AMP, whereas basal lipolysis was not markedly altered. This effect of Mn2+ and Co2+ was not mimicked by either Sr2+, Ba2+, Mg2+ or Ca2+. Cyclic AMP levels in adipocytes stimulated with epinephrine or ACTH tended to be higher in the presence of Mn2+ and Co2+. It is concluded, therefore, that Mn2+ and Co2+ inhibit lipolysis by uncoupling cyclic AMP accumulation from activation of triglyceride lipase. In contrast to Mn2+ and Co2+, the calcium antagonists La3+ and D600 were without effect on lipolysis. The antilipolytic effect of tetracaine, Mn2+ and Co2+ was found to persist in the absence of extracellular calcium, suggesting therefore that the antilipolytic effect of these drugs is unrelated to inhibition of calcium influx into adipocytes. The possibility is discussed that lipolytic agents cause an intracellular redistribution of calcium ion and that local anesthetics, Mn2+ and Co2+ interfere with lipolysis by preventing this intracellular redistribution of calcium.     

Free fatty acids as feedback regulators of adenylate cyclase and cyclic 3':5'-AMP accumulation in rat fat cells.

Rat fat cells incubated with lipolytic agents released substances to the medium which acted as feedback regulators of cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. The feedback regulators were not removed by adenosine deaminase. Dialyzed medium that had previously been incubated with fat cells in the presence of norepinephrine markedly inhibited cyclic AMP accumulation by fresh cells, whereas dialyzed medium from control cells did not inhibit cyclic AMP accumulation. The effects of lipolytic agents could be mimicked by adding dialyzed medium previously incubated with fat cells in the presence of oleic acid. This suggested that free fatty acids were the nondialyzable and adenosine deaminase-insensitive inhibitors of cyclic AMP accumulation released to the medium by fat cells incubated with lipolytic agents. The regulatory function of free fatty acids was related to the molar ratio of fatty acid to albumin. Profound inhibition of both lipolysis and cyclic AMP accumulation was seen as the free fatty acid/albumin ratio exceeded 3. The inhibition of cyclic AMP accumulation by oleate was seen as soon as there was a detectable increase in cyclic AMP due to lipolytic agents. Protein kinase activity (in the presence of cyclic AMP) of the infranatant obtained after centrifugation of fat cell homogenates at 48,000 x g was inhibited by medium from cells incubated with lipolytic agents or added oleate. Adenylate cyclase activity of rat fat cell ghosts was also inhibited by dialyzed or nondialyzed medium that previously had been incubated with lipolytic agents or added fatty acids. The direct addition of oleate markedly inhibited adenylate cyclase activity as the free fatty acid/albumin ratio exceeded 2. These data suggest that the prolonged drop in cyclic AMP accumulation seen during the incubation of rat fat cells with lipolytic agents is due to the inhibition of adenylate cyclase. This occurs when the free fatty acid/albumin ratio exceeds 3.     

Relationship between cyclic AMP production and lipolysis induced by forskolin in rat fat cells.

Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) induced both cyclic AMP production and lipolysis in intact fat cells, but stimulated lipolysis without increasing cyclic AMP at a concentration of 10(-5) M. Homogenization of fat cells elicited lipolysis without elevation of cyclic AMP. Forskolin did not stimulate lipolysis in the homogenate. Forskolin stimulated both cyclic AMP production and lipolysis in a cell-free system consisting of endogenous lipid droplets and a lipoprotein lipase-free lipase fraction prepared from fat cells. However, at a concentration of 10(-6) M, it induced lipolysis without increase in the cyclic AMP content in this cell-free system. In the cell-free system, homogenization of the lipid droplets resulted in marked increase in lipolysis to almost the same level as that with 10(-4) M forskolin without concomitant increase in cyclic AMP. Addition of forskolin to a cell-free system consisting of homogenized lipid droplets and lipase did not stimulate lipolysis further. Phosphodiesterase activities were found to be almost the same both in the presence and absence of forskolin in these reaction mixtures. Although 10(-3) M forskolin produced maximal concentrations of cyclic AMP: 6.7 x 10(-7) M in fat cells and 2.7 x 10(-7) M in the cell-free system, 10(-4) M cyclic AMP did not stimulate lipolysis in the cell-free system. In a cell-free system consisting of lipid droplets and the lipase, pyrophosphate inhibited forskolin-induced cyclic AMP production, but decreased forskolin-mediated lipolysis only slightly. Based on these results, mechanism of lipolytic action of forskolin was discussed.     

Effect of NK-104, a new synthetic HMG-CoA reductase inhibitor, on triglyceride secretion and fatty acid oxidation in rat liver.

For the investigation of the mechanism responsible for the hypotriglyceridemic effect of NK-104, a new synthetic inhibitor of HMG-CoA reductase, the rate-limiting enzyme for cholesterol synthesis, isolated rat liver was perfused with or without NK-104 in the presence of exogenous [1-(14)C]oleic acid substrate. Addition of NK-104 tended to increase the ketone body production while it caused a significant decrease in the secretion rate of triglyceride by the perfused liver without affecting uptake of exogenous [1-(14)C]oleic acid. The inhibitor also significantly decreased hepatic triglyceride concentration. The altered triglyceride secretion was accompanied by a concomitant decreased incorporation of exogenous [1-(14)C]oleate into triglyceride. The conversion of exogenous [1-(14)C]oleic acid substrate indicated an inverse relationship between the pathways of oxidation and esterification. No effect of NK-104 on hepatic secretion of cholesterol was observed. These results suggest that NK-104 exerts its hypotriglyceridemic action, primarily by diverting the exogenous free fatty acid to the pathways of oxidation at the expense of esterification.     

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.     

Relationship between the tissue level of cyclic AMP and the fat cell size of human adipose tissue.

The relationship between mean fat cell size, maximal tissue cyclic AMP concentration, and glycerol release was investigated in human subcutaneous adipose tissue incubated in vitro with or without isoprenaline or noradrenaline added at maximal effective concentrations. Basal and stimulated glycerol release and cyclic AMP concentration were each related to the fat cell size. Whether or not the phosphodiesterase inhibitor theophylline was present in the incubation system, basal and noradrenaline-induced cyclic AMP levels were significantly correlated with the fat cell size. The noradrenaline-induced cyclic AMP levels resulted in twice as rapid glycerol release as could be expected from the basal ratio between glycerol release and cyclic AMP. Furthermore, both basal and noradrenaline-induced glycerol release in relation to the cyclic AMP levels were more rapid in enlarge fat cells. It is concluded that basal and catecholamine-induced production of cyclic AMP is related to the fat cell size and that a quantitative relationship exists between rate of lipolysis and maximal tissue levels of cyclic AMP in human adipose tissue. Basal and noradrenaline-induced lipolysis are probably regulated by different mechanisms and the lipolytic sensitivity to cyclic AMP seems increased in large fat cells.     

Intracellular lipase activities in heart and skeletal muscle homogenates. The absence of trierucin cleavage by the heart: a possible biochemical basis for erucic acid lipidosis.

Rat heart and skeletal muscle homogenates were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C12:0) to trierucin (C22:1). It is shown that for all triglycerides esterified with fatty acids from C12 to C18, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity. In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride. Lipolysis of tri-delta-11-eicosenoin (C20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides. Although cleavage of trierucin (C22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased. A mixture of triglycerides did not show preferential hydrolysis of any simple triglyceride. Trierucin was the only triglyceride that did not complete for lipolytic activity and only with heart homogenates, which shows that that lipase(s) do not cleave trierucin. The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Studies on adrenaline-induced lipolysis in artificial lipid micelles.

Lipid micelles were prepared by incubating a mixture of glycerides (triolein, diolein, and monoolein), and lecithin in Krebs-Ringer phosphate buffer at 37 degrees C for 30 min. It was found that adrenaline stimulated the release of free fatty acids in a lipolytic system consisting of the lipid micelles and adipose tissue lipase. Adrenaline did not increase the cyclic AMP content of the reaction mixture. Dibutyryl cyclic AMP, theophylline, and phospholipase C increased the rate of lipolysis in the system but cyclic AMP and phospholipase D did not.     

Surface structure changes of rat adipocytes during lypolysis stimulated by various lypolysis stimulated by various lypolytic agents

A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 μM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.     

Bimodal effect of insulin on hormone-stimulated lipolysis: relation to intracellular 3',5'-cyclic adenylic acid and free fatty acid levels

The present study was undertaken to determine the relationship between the antilipolytic and lipolytic effects of insulin on hormone-stimulated lipolysis and the mechanisms of these reactions. The dose-response curve of norepinephrine-stimulated lipolysis in rat adipocytes was not sigmoidal but biphasic in nature. Intracellular free fatty acid levels were linearly related to lipolytic rate and also described a biphasic profile in response to increments in norepinephrine concentration. Intracellular 3',5'-cyclic AMP levels measured 10 min after addition of increasing concentrations of norepinephrine showed a rise and a plateau followed by a secondary rise. Insulin was antilipolytic at low concentrations of norepinephrine and distinctly lipolytic at high concentrations. The combined antilipolytic and lipolytic effect of insulin is termed the "bimodal" effect of insulin on hormone-stimulated lipolysis. The bimodal effect of insulin correlated positively with changes in peak intracellular 3',5'-cyclic AMP levels. In the presence of glucose, insulin invariably enhanced lipolysis. It is suggested that the antilipolytic effect of insulin is achieved by both inhibition of adenyl cyclase activity and activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase, the net effect being a low accumulation of 3',5'-cyclic AMP. On the other hand, the lipolytic effect of insulin probably reflects enhancement of adenyl cyclase activity to an extent that overrides any activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase activity, resulting in an increase in peak adipocyte 3',5'-cyclic AMP levels.     

Decreased expression and function of adipocyte hormone-sensitive lipase in subcutaneous fat cells of obese subjects.

Decreased lipolytic effect of catecholamines in adipose tissue has repeatedly been demonstrated in obesity and may be a cause of excess accumulation of body fat. However, the mechanisms behind this lipolysis defect are unclear. The role of hormone-sensitive lipase was examined using abdominal subcutaneous adipocytes from 34 obese drug-free and otherwise healthy males or females and 14 non-obese control subjects. The enzyme catalyzes the rate-limiting step of the lipolysis pathway. The maximum lipolytic capacity of fat cells was significantly decreased in obesity when measured using either a non-selective beta-adrenergic receptor agonist (isoprenaline) or a phosphodiesterase resistant cyclic AMP analogue (dibutyryl cyclic AMP). Likewise, enzyme activity, protein expression, and mRNA of hormone-sensitive lipase were significantly decreased in adipocytes of obese subjects. The findings were not influenced by age or gender. The data suggest that a decreased expression of hormone-sensitive lipase in subcutaneous fat cells, which in turn causes decreased enzyme function and impaired lipolytic capacity of adipocytes, is present in obesity. Impaired expression of the hormone-sensitive lipase gene might at least in part explain the enzyme defect.     

Lipolysis and reesterification: effects of some inhibitors of adenosine 3',5'-cyclic monophosphate phosphodiesterase

Theophylline and three lipolytic agents, 2,5-bis(2-chloroethylsulfonyl)-pyrrole-3,4-dicarbonitrile (substituted pyrrole), 2,4-diamino-6-butoxy-s-triazine (substituted triazine), and 2,3-dihydro-5,6-dimethyl-3-oxo-4-pyridazinecarbonitrile (substituted pyridazine), stimulate basal lipolysis in adipose tissue in vitro. They also cause an increased release of free fatty acids, but not glycerol, from adipose tissue in which lipolysis is already maximally stimulated by epinephrine. The four compounds also inhibit cyclic AMP phosphodiesterase and the conversion of [1-(14)C]glucose to (14)CO(2). Evidence is presented that free fatty acids accumulate as the result of inhibited reesterification. The substituted pyridazine and triazine, but not the pyrrole, elevate plasma free fatty acids after oral or intraperitoneal administration in rats.