Lipolysis In Rat Adipocytes Research Articles

Mechanism of pervanadate stimulation and potentiation of insulin-activated glucose transport in rat adipocytes: dissociation from vanadate effect.

Previous studies have shown that the combination of vanadate and H2O2 generates peroxide(s) of vanadate (pervanadate) that is able to mimic insulin in stimulating lipogenesis or protein synthesis and inhibiting lipolysis in rat adipocytes. Here we report that pervanadate is a potent trigger of 3-O-methylglucose transport in rat adipocytes, with an effective concentration of 5 microM and a maximum at 20 microM. Moreover, pervanadate produced an additional activation of approximately 60% on glucose influx in cells treated with maximally activating concentrations of insulin. Vanadate was ineffective in potentiating insulin-stimulated glucose uptake. Quercetin, a bioflavonoid that inhibits insulin receptor tyrosine kinase, blunted this effect of pervanadate. Treatment of adipocytes with pervanadate inhibited protein phosphotyrosyl phosphatase activity of cell extracts in a dose-dependent manner, with an ID50 of 5 microM and complete inhibition at 80 microM. In contrast, vanadate (1-800 microM) did not appreciably inhibit cell phosphotyrosyl phosphatases. The inhibitory effect of pervanadate correlated with the increase in protein phosphotyrosine accumulation, as determined by Western blotting with antiphosphotyrosine antibodies. The most prominent phosphotyrosine-containing band detected in pervanadate-treated adipocytes was that of autophosphorylated insulin receptor, identified by immunoblotting or immunoprecipitation with antiinsulin receptor antibodies. The addition of insulin to pervanadate-treated adipocytes (20 microM) caused a further increase (approximately 70%) in receptor autophosphorylation. In a cell-free system using partially purified insulin receptor devoid of tyrosine phosphatase activity, pervanadate did not stimulate the receptor autophosphorylation or interfere with the stimulating effect of insulin. These results suggest that 1) pervanadate triggers glucose uptake by increasing autophosphorylation of insulin receptor, preventing its dephosphorylation; 2) under physiological conditions, cellular protein phosphotyrosyl phosphatase activity is high, thereby significantly opposing insulin-mediated hexose transport; and 3) pervanadate has the unique ability to markedly increase maximal cell responsiveness in stimulating glucose transport achieved at a saturating insulin concentration. These findings suggest a possible clinical application in the management of glucose uptake in pathological conditions of insulin resistance and hyperinsulinemia.

Malarial toxic antigens synergistically enhance insulin signalling

Hypoglycaemia is a major complication of severe malaria [(1990) Trans. Roy. Soc. Trop. Med. 84 (suppl. 2) 1–65], especially cerebral malaria, in which it is associated with increased mortality [(1990) Lancet 336, 1039–1043; (1989) Quart. J. Med. (New series) 71, 441–459]; however, the mechanisms responsible have not been fully explained. Preparations containing toxic malaria antigens (TMA) released by blood stage Plasmodium yoelii malaria parasites have been shown to induce hypoglycaemia in mice lasting at least 8 h [(1992) Clin. Exp. Immunol. (in press)]. Here we report that TMAs can act synergistically with insulin in both stimulating lipogenesis and inhibiting lipolysis in rat adipocytes in vitro, and, furthermore, that they act synergistically with insulin in the induction of hypoglycaemia in vivo.

CAMP-dependent protein kinase activation mediated by β3-adrenergic receptors parallels lipolysis in rat adipocytes

Catecholamine-induced lipolysis is chiefly mediated through the recently characterized β 3-adrenergic receptor (AR) in rat adipocytes. Discrepancies between the ability of β 3-AR agonists to stimulate adenylyl cyclase and the resulting lipolysis were recently reported. cAMP-dependent protein kinase (A-kinase) activation induced by these agonists was compared to lipolysis. Agonist potencies were similar for A-kinase activity ratios and lipolysis. The same A-kinase activity ratio to lipolysis relationship was found for the β 3-AR agonists tested.

Prostanoid-induced inhibition of lipolysis in rat isolated adipocytes: Probable involvement of EP 3 receptors

Sulprostone, enprostil and 16,16 dimethyl PGE 2 have all been found to be potent inhibitors of lipolysis induced by 3-isobutyl-1-methyl-xanthine (IBMX) in rat isolated adipocytes. The potency of sulprostone and enprostil in particular indicates that the response is likely to be mediated through either EP 3 or EP 1-receptors. However, the EP 1-receptor blocking drug, AH6809, had no effect on the antilipolytic response to either PGE 2 or sulprostone. We therefore conclude that the receptors mediating prostanoid-induced inhibition of lipolysis in rat adipocytes must principally be of the EP 3 sub-type.

Endotoxicosis modulates cytosolic free calcium and basal and ACTH-stimulated lipolysis in rat adipocytes

Lipolytic rates and intracellular Ca 2+ concentration ([Ca 2+] i) were determined under basal conditions and upon stimulation with adrenocorticotropic hormone (ACTH), norepinephrine (NE) and insulin (I), in adipocytes isolated from control and acutely endotoxin (ET)-treated rats ( 1 mg 100 g body weight, LD 50 at 6 h). [Ca 2+] i measurements were done using the fluorescent Ca 2+-indicator Fura-2. NE and ACTH, but not I, produced a marked increase of [Ca 2+] i in cells of both control and ET-treated rats. ET treatment elicited a significant increase in [Ca 2+] i of resting cells, and enhanced the ACTH effect on this parameter. The changes in lipolytic activity correlated well with changes of [Ca 2+] i induced by ACTH. The results indicate that ET-induced alterations in intracellular calcium homeostasis of adipocytes may contribute to the mediation of effects on fat mobilization during endotoxemia.

Studies on hormonal regulation of lipolysis and lipogenesis in fat cells of various mammalian species

1. 1. Corticotropin-stimulated lipolysis in adipocytes of rats, mice, hamsters, guinea pigs and rabbits. Melanotropins elicited high lipolytic activity only in guinea pig and rabbit adipocytes. Opiate peptides were active only in rabbit adipocytes. Pituitary and chorionic gonadotropins and somatotropin were lipolytic in guinea pig adipocytes. Other hormones tested including prolactin, somatostatin, substance P, neurotensin, angiotensin II, thyrotropin releasing hormone and pancreatic polypeptide were devoid of lipolytic activity in all of the adipocytes studied. 2. 2. In the rabbit adipocytes γ-melanotropin was lipolytic only at high doses. At these doses the peptide inhibited the lipolytic response to a high dose of corticotropin. 3. 3. Lipolysis stimulated by vasoactive intestinal peptide and epinephrine in rat adipocytes was antagonized by insulin. The lipolytic hormones corticotropin, epinephrine, vasoactive intestinal peptide and secretin suppressed basal and insulin-stimulated lipogenesis.

A1-adenosine receptor-mediated inhibition of adipocyte adenylate cyclase and lipolysis in Zucker rats

Hormone-stimulated lipolysis is reduced in genetically obese rodents and may contribute to the increased adiposity characteristic of the obese state. Endogenously released adenosine, acting via the A1 receptor coupled to the inhibitory guanosine 5'-triphosphate binding protein, Gi, provides a tonic inhibition of lipolysis in rat adipocytes. Removal of this inhibition by the addition of adenosine deaminase frequently results in maximal lipolytic activity. Adipocytes isolated from lean Zucker (Fa/?) rats responded normally to adenosine deaminase, where lipolysis in adipocytes from obese Zucker (fa/fa) rats remained approximately 50% inhibited. Adipocyte adenylate cyclase was equally responsive to activation by forskolin, but lipolytic hormones were significantly less effective in stimulating adenosine 3',5'-cyclic monophosphate (cAMP) production in the obese adipocytes. These cells also exhibited an increased sensitivity to inhibition by the adenosine agonist, N6-(L-2-phenylisopropyl)-adenosine, either in combination with forskolin or beta-adrenergic hormone stimulation. Treatment of isolated adipocytes with pertussis toxin, which uncouples receptor-mediated Gi function, had little effect in cells from lean rats but increased isoproterenol stimulated cAMP production of cells from obese rats to levels observed in the lean cells. In addition, the adenosine A1 antagonist, 8-phenyltheophylline, increased cAMP and lipolytic activity in the obese adipocytes while having little significant effect in the lean adipocytes. These results suggest that hormonal control of lipolysis is altered in the obese Zucker rat because of an alteration in A1-adenosine receptor-mediated inhibition of adenylate cyclase.

The Insulin-Like Effect of Vanadate on Lipolysis in Rat Adipocytes Is Not Accompanied by an Insulin-Like Effect on Tyrosine Phosphorylation*

Tyrosine phosphorylation of the insulin receptor and other intracellular proteins in rat adipocytes was examined using an immunoblot technique with antiphosphotyrosine antibody. Insulin at 10(-7) M increased the tyrosine phosphorylation of the 95K subunit of the insulin receptor (15-fold) and proteins of 180K (7-fold) and 60K (23-fold). Increases in insulin-dependent phosphorylation of the three proteins were detectable at 10(-10) M insulin and attained steady state within 30 sec of insulin (10(-7) M) addition. Small effects of insulin (less than 30% increases) were observed on proteins of 120K and 53K. In contrast to insulin, the effects of vanadate on tyrosine phosphorylation were small and nonspecific. Vanadate increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit and the 120K and 60K proteins similarly, with increases of 1.5- to 3-fold at 1 mM and 2-fold or less at 200 and 50 microM. Vanadate-dependent tyrosine phosphorylation of the 180K protein increased to a maximum of only 30% at 200 microM. Tyrosine phosphorylation of the 53K protein was somewhat larger, approaching 4-fold at 1 mM vanadate. The concentration of insulin and vanadate that inhibited isoproterenol-dependent lipolysis were not comparable to those that increased tyrosine phosphorylation. Vanadate at 1 mM was more potent as an antilipolytic agent than 10(-9) M insulin (93% vs. 81%), yet increased tyrosine phosphorylation of the 95K insulin receptor beta-subunit only as effectively as 10(-10) M insulin (which inhibited lipolysis only 42%). The dissimilar responses were even more pronounced when antilipolysis was compared to tyrosine phosphorylation of the 180K and 60K proteins. For example, insulin at 10(-9) M increased tyrosine phosphorylation of the 180K protein 2.9-fold, while 1 mM vanadate had a negligible effect (10% increase). Thus, vanadate exerts an insulin-like effect on lipolysis, yet its effects on tyrosine phosphorylation differ from those of insulin.

Evidence that phorbol ester-activated pathways are not directly involved in the action of insulin in rat adipocytes

The effects of 12-phorbol 13-myristate acetate (PMA) on glucose transport, glucose metabolism and lipolysis in rat adipocytes were examined. Alone, PMA (100 ng/ml) stimulated 2-deoxyglucose transport, glucose oxidation and lipogenesis by an amount corresponding to about 30–50% of the maximal insulin effect. The effect of PMA on the insulin-stimulated processes was additive at all insulin concentrations. Adenosine deaminase-stimulated lipolysis was enhanced by about 50% by PMA (100 ng/ml). Thus, in contrast to insulin, PMA had a lipolytic effect. The antilipolytic effect of insulin was unaffected by the presence of PMA, both ED 50 and maximal inhibition of insulin were unchanged. In conclusion, it is suggested that phorbol esters may activate pathways other than those activated by insulin in rat adipocytes.

Lipid peroxidation inhibits norepinephrine-stimulated lipolysis in rat adipocytes. Reduction of beta-adreno-ceptor number

The effect of lipid peroxidation on lipolysis depends on the intactness of the adipocyte plasma membrane. In the intact cells, the norepinephrine-stimulated lipolysis was inhibited, while the basal one was elevated. In the lysed cells, lipid peroxidation had no effect upon hormone-stimulated lipolysis, but the basal one was strongly inhibited. The effects of free radical damage (iron plus ascorbate ions) were compared to those of malondialdehyde, a non-radical product of lipid peroxidation. Although qualitatively similar, deterioration of plasma membrane induced by malondialdehyde was much lower than by free radicals. The changes in lipolytic response to norepinephrine were accompanied by a drastic reduction in the number of beta-adrenergic receptors.

Role of growth hormone in the adaptations of lipolysis in rat adipocytes during recovery from lactation.

Removal of the litter from lactating rats results in a fall in the rate of noradrenaline-stimulated lipolysis of adipocytes. This adaptation can be prevented by administration of growth hormone (somatotropin) to such rats and mimicked by injecting lactating rats with an antiserum to growth hormone, whereas lowering serum prolactin by injecting bromocriptine had no effect. The anti-lipolytic effect of adenosine is increased during lactation and is still increased by 2 days after litter removal. Injection of growth hormone into lactating rats decreased slightly the response to adenosine, whereas injection of growth hormone into rats after removal of their litters resulted in a much greater decrease in the response to adenosine, to that found in virgin and pregnant rats.

Studies on a protein kinase inhibitor-insensitive, phospholipase C-sensitive pathway of lipolysis in rat adipocytes

Endogenous lipid droplets were prepared by subjecting fat cells to hypotonic shock and Triton X-100 treatment. The endogenous lipid droplets were found to show lipolysis in response to epinephrine, but not to show lipogenesis from glucose in response to insulin. These results indicated that the preparation of endogenous lipid droplets did not contain any intact fat cells capable of insulin-stimulated lipogenesis. Results with these endogenous lipid droplets showed that protein kinase inhibitor inhibited protein kinase-mediated hormone-sensitive lipase activity but did not reduce epinephrine-induced lipolysis. Cyclic AMP and dibutyryl cyclic AMP induced lipolytic activity in the presence of 80 mM KCl and their activities were not inhibited by protein kinase inhibitor. Phospholipase C inhibited epinephrine, cyclic AMP and dibutyryl cyclic AMP-induced lipolysis, but did not affect the lipolytic activity of either the activated or non-activated form of hormone-sensitive lipase. These results indicate the existence of a protein kinase inhibitor-insensitive and phospholipase C-sensitive lipolytic pathway in rat adipocytes.

Effect of Dietary Saturated Fatty Acids on Hormone-Sensitive Lipolysis in Rat Adipocytes

The objective of this work was to examine the mechanism by which dietary saturated fatty acids, as compared with polyunsaturated fatty acids, lower hormone-sensitive lipolysis in rat adipocytes. Rats were fed a purified diet containing 14% of a fat with a high concentration of either saturated fatty acids (coconut oil or beef fat) or polyunsaturated fatty acids (safflower oil) as a control. In addition, each diet contained 2% corn oil. The animals were fed these diets for 4 wk. Norepinephrine-stimulated lipolysis was 50% lower when diets rich in saturated fatty acids, regardless of their chain length, were fed than when a diet containing a high concentration of polyunsaturated fatty acids was fed. The specific activities of adenylate cyclase, 3′,5′-cyclic nucleotide (cAMP) phosphodiesterase and hormone-sensitive lipase were lower when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. Accumulation of cAMP upon stimulation with 10-5M norepinephrine was lower when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. Moreover, adipocytes were larger when saturated fatty acids were fed than when polyunsaturated fatty acids were fed. The data obtained suggest that dietary saturated fats exert their inhibitory effect on hormone-stimulated lipolysis by influencing several points in the lipolytic cascade.

Lipolysis in rat adipocytes during recovery from lactation. Response to noradrenaline and adenosine.

The replenishment of lipid reserves of adipocytes following the removal of litters from lactating rats is associated with a 3-fold decrease in both the response of lipolysis to noradrenaline and the maximum rate of lipolysis (measured in the presence of noradrenaline plus adenosine deaminase); these adaptations do not appear to result from the changes in serum prolactin and insulin concentrations that occur on litter removal.

CAMP-dependent protein kinase and lipolysis in rat adipocytes. I. Cell preparation, manipulation, and predictability in behavior.

With the use of -cAMP/+cAMP activity ratios of cAMP-dependent protein kinase (A-kinase) in fat cell extracts as an index of cellular cAMP concentrations, it is apparent from both the current literature and from data presented in this paper that classical cell isolation procedures yield cells whose behavior is unpredictable from day to day. Herein, procedures are described for isolating adipocytes, preparing cytosolic extracts, and assaying A-kinase that result in kinase activity ratios in isolated cells equal to those in the fat pad from which cells are derived, approximately 0.05. An important modification in the procedure is the inclusion of 200 nM exogenous Ado in all cell manipulation media, and the data indicate that variable removal of contaminating endogenous Ado accounts for unpredictable results with standard cell isolation techniques. A further benefit of Ado inclusion is greatly reduced cell lysis. Acute removal of Ado with adenosine deaminase results in rapid elevation of A-kinase activity ratios and lipolysis which, in fasted animals, equals that achieved with lipolytic hormones. Cells from fed animals exhibit poor predictability in behavior. Moreover, A-kinase activity ratios exhibit seasonal tendencies in response to Ado removal, with cells isolated in spring being more activated than cells isolated later in the year. The information and procedures in this paper form the basis for succeeding papers on the regulation of adipocyte metabolism by hormones.

CAMP-dependent protein kinase and lipolysis in rat adipocytes. III. Multiple modes of insulin regulation of lipolysis and regulation of insulin responses by adenylate cyclase regulators.

The relationship between cAMP-dependent protein kinase (A-kinase) activity ratios and lipolysis in the presence of insulin was compared to the standard relationship between these two parameters established with a variety of adenylate cyclase modulators (Honnor, R. C., Dhillon, G., and Londos, C. (1985) J. Biol. Chem. 260, 15130-15138). Three phases of insulin action were observed. First, when tested in control cells exhibiting A-kinase activity ratios up to approximately 0.25, insulin inhibition of lipolysis could be accounted for by the decrease in A-kinase activity. Second, in cells exhibiting A-kinase activity ratios greater than 0.3, the decrease in kinase activity by insulin did not account for the decrease in lipolysis. Finally, as the A-kinase activity ratio approached 0.6 the insulin effect on lipolysis was lost. The data suggest that protein phosphatase activation accounts for the cAMP-independent insulin action. Moreover, the insulin effect not accounted for by a decrease in A-kinase activity appears to be elicited only upon elevation of A-kinase activity. The method by which cells were stimulated determined the IC50 for insulin inhibition of: 1) A-kinase activity ratios, 2) lipolysis explained by the decrease in A-kinase activity ratios, and 3) lipolysis not explained by a decrease in A-kinase activity ratios. For all three parameters, cells stimulated by lipolytic hormones were approximately 5 times more sensitive to insulin than cells stimulated by incubation in a ligand-free environment achieved with adenosine deaminase; insulin IC50 values were approximately 120 and 600 pM, respectively. Such data establish a link between insulin actions in modifying cAMP concentrations and in modifying events apparently independent of changes in cAMP. It is proposed that the receptors and regulatory components associated with adipocyte adenylate cyclase are associated also with components of the insulin response system separate from cyclase.

CAMP-dependent protein kinase and lipolysis in rat adipocytes. II. Definition of steady-state relationship with lipolytic and antilipolytic modulators.

The steady-state relationship between the activation state of cAMP-dependent protein kinase (A-kinase) and lipolysis has been defined quantitatively. A-kinase activation was assessed by measuring the ( +/- cAMP) activity ratio in adipocyte extracts, and lipolysis was determined by measuring glycerol release from cells. Both processes were stimulated either by incubating cells in a ligand-free environment achieved with adenosine deaminase or by addition of lipolytic hormones. A response spectrum was obtained with a variety of adenylate cyclase stimulators and inhibitors, both receptor- and nonreceptor-mediated. Regardless of the ligands used to manipulate adipocyte activity, lipolysis varied from nil to maximal as the A-kinase activity ratio varied from approximately 0.05 to 0.3-0.35. These data provide a quantitative description of the steady-state relationship between A-kinase activity and lipolysis and indicate that the various lipolytic and antilipolytic agents tested act on the lipolytic process exclusively by altering adenylate cyclase activity and, thus, cellular cAMP concentrations. The data reveal also that transient "peaking" of cAMP, as measured by A-kinase activity ratios, is not an inherent feature of adipocyte metabolism. Moreover, the concentration requirements for lipolytic hormone action are critically dependent on the ambient concentration of antilipolytic agents, and t concentration requirements for antilipolytic agents are dependent on the extent to which cells are stimulated. The data in this paper provide the basis for assessing the relationship between A-kinase activity ratio and lipolysis in the presence of insulin (Londos, C., Honnor, R. C., and Dhillon, G. S. (1985) J. Biol. Chem. 260, 15139-15145).

Effect of alkaline cations on cyclic 3′ 5′-AMP stimulated lipolysis in rat adipocytes

In Krebs-Ringer phosphate medium, cyclic AMP had little effect on production of free fatty acids by fat cells in vitro, whereas dibutyryl cyclic AMP or epinephrine stimulated the production of free fatty acids. On the other hand, although under non-physiological condition, cyclic AMP was found to stimulate the lipolysis in simple KC1-Tris medium. The stimulation level was similar to that elicited by dibutyryl cyclic AMP. Cyclic AMP also stimulated lipolysis in LiCl-Tris or RbCl-Tris medium, but not in NaCl-Tris medium. In KC1-Tris medium, addition of divalent alkaline cations (Mg 2+, Ca 2+ and Sr 2+, at 40 mM) completely inhibited the stimulation by cyclic AMP, but not those by dibutyryl cyclic AMP and epinephrine. No cyclic AMP-induced lipolysis was observed in homogenized or freeze-thawed cells.

Comparative effects of cholera and Bordetella pertussis toxins on cyclic AMP and GTP levels and on lipolysis in rat adipocytes incubated in vitro

The respective effects of cholera and Bordetella pertussis toxins were studied in time and concentration dependent experiments, following glycerol and fatty acid release, GTP and cAMP levels. Cholera toxin, after a lag time of 30 min, stimulated linearly GTP and cAMP accumulation and lipolysis (maximal effect: 2-fold increase at 5 μg ml ). Pertussis toxin presented a biphasic effect both in time and concentration dependent studies. Up to a maximum reached after 2 h with 1.4 units LPF ml the stimulation affected GTP (3 fold) and cAMP (7 fold) levels, glycerol and fatty acid release (15 fold). Beyond this, an inhibition occurred, yielding a decrease towards basal values of GTP and cAMP content whereas the glycerol and fatty acid release was stopped. These results, which are the first reporting the fluctuation of the GTP content of intact cells challenged with bacterial toxins, show a close relationship between GTP and cyclic AMP levels and lipolytic activity.

Effect of phenylephrine on lipolysis in rat adipocytes: No evidence for an α-adrenergic mechanism

1. 1. Phenylephrine. a strong α 1-adrenergic agonist, exerted a concentration dependent antilipolytic effect against isoproterenol-activated lipolysis in rat adipocytes with the effect decreasing as the isoproterenol concentration increased. 2. 2. The α-adrenergic antagonists phentolamine and phenoxybenzamine did not reverse phenylephrinc's antilipolytic effect. 3. 3. Phenylephrine alone activated lipolysis at concentrations above 10 −5M and at 5 × 10 −4 M the rate of lipolysis was increased 3.4-fold. Propranolol abolished this effect. 4. 4. In the presence of sub-maximum concentrations of dibutyryl cyclic-AMP (< 10 −4M), 10 −4M phenylephrine increased the rate of lipolysis above that activated by dibutyryl cyclic-AMP alone. At maximum dibutyryl cyclic-AMP concentrations, or in the presence of propranolol, phenylephrine had no effect on dibutyryl cyclic-AMP-dependent lipolysis. 5. 5. There is no evidence to support an α 1-adrenergic mechanism for regulation of lipolysis in the rat adipocyte. All effects of the α-adrenergic agonist phenylephrine appear to be due to its weak β -adrenergic activity.