Lipolysis In Rat Fat Cells Research Articles

Anti-lipase and lipolytic activities of ursolic acid isolated from the roots of Actinidia arguta

The aim of this study was to investigate the anti-obestic effects of ursolic acid isolated from the roots of Actinidia arguta, as well as the mechanism of action of this compound. This was conducted by testing whether ursolic acid inhibited the elevation of the rat plasma triacylglycerol levels after oral administration of a lipid emulsion containing corn oil in rats. Ursolic acid prevented the elevation of plasma triacylglycerol levels 2 h after oral administration of the lipid emulsion at a dose of 100 mg/kg. Furthermore, ursolic acid inhibited phosphodiesterase activity in vitro with an IC(50) of 51.21 muM and enhanced lipolysis in rat fat cells. We suggest that the inhibitory effects of ursolic acid, isolated from the roots of A. arguta, on obesity, might be attributable to the inhibition of lipid absorption through the inhibition of pancreatic lipase and by enhancing lipolysis in fat cells.

NoIn VitroEffects of Fatty Acids on Glucose Uptake, Lipolysis or Insulin Signaling in Rat Adipocytes

Elevated plasma levels of free fatty acids (FFA) can produce insulin resistance in skeletal muscle tissue and liver and, together with alterations in beta-cell function, this has been referred to as lipotoxicity. This study explores the effects of FFAs on insulin action in rat adipocytes. Cells were incubated 4 or 24 h with or without an unsaturated FFA, oleate or a saturated FFA, palmitate (0.6 and 1.5 mM, respectively). After the culture period, cells were washed and insulin effects on glucose uptake and lipolysis as well as cellular content of insulin signaling proteins (IRS-1, PI3-kinase, PKB and phosphorylated PKB) and the insulin regulated glucose transporter GLUT4 were measured. No significant differences were found in basal or insulin-stimulated glucose uptake in FFA-treated cells compared to control cells, regardless of fatty acid concentration or incubation period. Moreover, there were no significant alterations in the expression of IRS-1, PI3-kinase, PKB and GLUT4 following FFA exposure. Insulin's ability to stimulate PKB phosphorylation was also left intact. Nor did we find any alterations following FFA exposure in basal or cAMP-stimulated lipolysis or in the ability of insulin to inhibit lipolysis. The results indicate that oleate or palmitate does not directly influence insulin action to stimulate glucose uptake and inhibit lipolysis in rat fat cells. Thus, lipotoxicity does not seem to occur in the fat tissue itself.

Relationships between lipolysis induced by various lipolytic agents and hormone-sensitive lipase in rat fat cells

Norepinephrine induced lipolysis in rat fat cells, in vitro, in a time- and concentration-dependent manner, without concomitantly increasing hormone-sensitive lipase (HSL) activity. It also induced, time and concentration dependently, HSL translocation from the cytosol to the lipid droplets in fat cells. Isoproterenol, forskolin, dibutyryl cyclic AMP, and theophylline also induced lipolysis in fat cells, but did not stimulate HSL activity. These agents also induced HSL translocation from the cytosol to the lipid droplets in fat cells: about 80% to 90% of all HSL was located in lipid droplets after incubation for 1 h. These results suggest that the critical event in lipolytic activation of fat cells induced by lipolytic agents is not an increase in the catalytic activity of HSL but translocation of HSL to its substrate on the surfaces of lipid droplets in fat cells.—Morimoto, C., K. Kameda, T. Tsujita, and H. Okuda. Relationships between lipolysis induced by various lipolytic agents and hormone-sensitive lipase in rat fat cells. J. Lipid Res. 2001. 42: 120–127.

Mechanism of the stimulatory action of okadaic acid on lipolysis in rat fat cells

Okadaic acid was found to induce concentration- and time-dependent lipolysis in rat fat cells in the absence of lipolytic hormones, but it did not significantly increase the total hormone-sensitive lipase (HSL) activity in these fat cells, the activity of HSL extracted from fat layer and that of HSL in the supernatant of homogenized fat cells. Western blotting of fat cell homogenate fractions with an antiserum raised against synthetic peptide derived from rat HSL showed that HSL protein shifted from the supernatant to the fat layer in response to okadaic acid, which increased the HSL protein content on the fat layer and concomitantly reduced that of the supernatant, concentration- and time-dependently. Sonication of the fat cells abolished their responsiveness to okadaic acid. The lipolytic action of okadaic acid was examined and its site was identified using a cell-free system comprising lipid droplets isolated from rat fat cells and HSL. Okadaic acid induced lipolysis in this cell-free system and sonication of the lipid droplets caused disappearance of lipolytic action of okadaic acid. Okadaic acid failed to stimulate lipolysis in a cell-free system comprising HSL and artificial lipid droplets (trioleoylglycerol emulsified with gum arabic) instead of lipid droplets isolated from rat fat cells. These results suggest that okadaic acid does not increase the catalytic activity of HSL but induces translocation of HSL to the lipid droplets isolated from rat fat cells. The site of the lipolytic action of okadaic acid in relation to the interaction between HSL and lipid droplet is discussed. —Morimoto, C., A. Kiyama, K. Kameda, H. Ninomiya, T. Tsujita, and H. Okuda. Mechanism of the stimulatory action of okadic acid on lipolysis in rat fat cells.

Norepinephrine-augmenting lipolytic effectors from Astilbe thunbergii rhizomes.

An EtOAc-soluble fraction from a 80% Me2CO extract of the rhizomes of Astilbe thunbergii enhanced norepinephrine-induced lipolysis in rat fat cells, while an EtOAc-insoluble fraction had no effect. The active substances isolated from the EtOAc-soluble fraction of the rhizomes were identified as eucryphin (1), bergenin (2), and astilbin (3), which enhanced norepinephrine-induced lipolysis at concentrations of 10-1000 microgram/mL, while they themselves did not cause lipolysis. Furthermore, these compounds slightly stimulated adrenocorticotrophic hormone-induced lipolysis and inhibited insulin-induced lipogenesis from glucose.

Norepinephrine-induced lipolysis in rat fat cells from visceral and subcutaneous sites: role of hormone-sensitive lipase and lipid droplets.

Norepinephrine-induced lipolysis was examined in visceral (epididymal and omental) and subcutaneous (abdominal and femoral) fat cells in rats. Hormone-induced lipolysis was considerably higher in the visceral fat cells than in the subcutaneous cells. A higher hormone-sensitive lipase (HSL) activity was present in the visceral than in the subcutaneous fat cells. Endogenous lipid droplets were prepared from rat visceral (epididymal and omental) and subcutaneous (abdominal and femoral) fat cells and norepinephrine-induced lipolysis was examined in a cell-free system consisting of the lipid droplets prepared from fat cells from each site and a fixed amount of HSL from rat epididymal adipose tissue. In this cell-free system, norepinephrine induced a higher rate of lipolysis with lipid droplets from the visceral than from the subcutaneous fat cells. These findings suggest that the difference in norepinephrine-induced lipolysis between visceral and subcutaneous fat cells may be due to the differences in HSL activity and lipid droplet character at each site.

Beta-Adrenergic receptors in rat fat cells and their relationship with lipolysis.

Norepinephrine stimulated lipolysis in rat fat cells while (-)-alprenolol completely inhibited this lipolysis. (-)-Alprenolol competed for (-)-[3H] dihydroalprenolol (DHA) binding sites on rat fat cells. The specific (-)-[3H]DHA binding sites identified by competition with (-)-alprenolol were found to be transferred to the solubilized supernatant during preparation of endogenous lipid droplets from the fat cells. Although the lipid droplets did not exhibit specific (-)-[3H]DHA binding, norepinephrine induced lipolysis in a cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). Norepinephrine-induced lipolysis in the cell-free system was inhibited by propranolol and (-)-alprenolol, but not by phenoxybenzamine. The lipolytic action of norepinephrine and the anti-lipolytic actions of propranolol and (-)-alprenolol disappeared after sonication of the lipid droplets in the cell-free system. These results suggest that the adrenergic receptor concerned with lipolysis in fat cells may not be a specific (-)-[3H]DHA binding site, but may be closely related to the lipid droplets.

Mechanism of norepinephrine-induced lipolysis in isolated adipocytes: evidence for its lipolytic action inside the cells

Norepinephrine elicited remarkable lipolysis in rat fat cells, but did not stimulate lipolysis in a fat cell homogenate. We prepared endogenous lipid droplets from fat cells and established a norepinephrine-sensitive cell-free system consisting of the lipid droplets and hormone-sensitive lipase (HSL). In the cell-free system, hydrolysis of the lipid droplets was markedly increased by addition of norepinephrine but homogenization of the droplets caused loss of norepinephrine responsiveness. These results suggest that the site of the lipolytic action of norepinephrine may be the lipid droplets. When 3T3-L1 fat cell monolayers were exposed to 3H-norepinephrine for 2 or 4 min, many silver grains, representing the localization of labeled norepinephrine, were observed in autoradiograms of cell sections. The grains were found in the cytoplasm and along the margin of the lipid droplets. These results also suggest that norepinephrine may be incorporated into fat cells and exhibit its lipolytic action inside the cells.

Role of substrate in catecholamine-induced lipolysis in rat fat cells.

Role of substrate in catecholamine-induced lipolysis in rat fat cells.

Influence of Ovariectomy and Regional Fat Distribution on the Membranous Transducing System Controlling Lipolysis in Rat Fat Cells*

The mechanism by which sex steroid hormones modulate the lipolytic process in fat cells is still not completely understood, particularly at the level of the membrane. As estrogens seem to have regional influence on adipose tissue, we have compared the effects of long term ovariectomy (3 weeks) on the membranous transducing system controlling lipolysis in two rat fat deposits, the femoral sc and the parametrial adipose tissue. These experiments demonstrate the following. 1) Compared to parametrial adipocytes, sc adipocytes have a reduced cAMP production and a decreased beta-adrenoceptor number associated with a reduced lipolytic activity. 2) In parametrial adipocytes, ovariectomy induces a decrease in cAMP production and in both the beta-agonist-stimulated response and the catalytic activity of adenylate cyclase in association with a decreased lipolysis. In contrast, these parameters are unaltered by ovariectomy in sc adipocytes. This study, which reveals site-related differences in the sensitivity of the fat cell adenylate cyclase system to ovarian status, suggests that these differences may explain some of the sex-related regional specificities of adipose tissue metabolism and distribution.

Effects of active compounds isolated from Angelica shikokiana on lipid metabolism in fat cells

It was found that the EtOAc fraction of the roots of Angelica shikokiana inhibited adrenaline-induced lipolysis in rat fat cells, while having no effect on ACTH-induced lipolysis. On the other hand, the MeOH fraction was found to inhibit ACTH-induced lipolysis but not to inhibit adrenaline-induced lipolysis. The active substances isolated from this root were elucidated to be psoralen (I), bergapten (II), isopteryxin(3'-angeloyloxy-4'-acetoxy-3', 4'-dihydroseselin) (III) and 3'-epoxyangeloyloxy-4'-acetoxy-3',4'-dihydroseselin (IV), respectively. The two furocoumarins, psoralen and bergapten, were found to enhance ACTH- and adrenaline-induced lipolysis in fat cells respectively. In contrast, a coumarin derivative of khellactone type, isopteryxin (III), was found to inhibit the adrenaline-induced lipolysis but with no effect on ACTH-induced lipolysis and insulin-induced lipogenesis from glucose. The other coumarin of khellactone type, 3'(R), 4'(R)-3'-epoxyangeloyloxy-4'-acetoxy-3', 4'-dihydroseseline (IV), was found to inhibit the insulin-induced lipogenesis from glucose but it had no effect on adrenaline- and ACTH-induced lipolysis.

Increased adenylate cyclase catalytic activity explains how estrogens “in vivo” promote lipolytic activity in rat white fat cells

Estradiol administration (5 μg per day × 4 days) to ovariectomized rats resulted in a 60–70 % increase in the maximal lipolytic response of their white adipocytes to isoproterenol, epinephrine, IBMX and forskolin. These altered lipolytic responses were accompanied by parallel changes in the intracellular cyclic AMP levels found in response to 1 mM IBMX alone (+ 106 %) or combined with submaximal concentrations of isoproterenol (+ 205 %), epinephrine (+ 190 %) and forskolin (235 %). Studies of the adenylate cyclase activity revealed an overall increase in the stimulatory responsiveness of the enzyme (+ 150 to + 200 %) after the estradiol-treatment, regardless of the stimulatory agents tested (GTP, GppNHp, fluoride, isoproterenol, ACTH, forskolin). Finally, the finding of a 2-fold enhancement of the Mn 2+ (± GDP β S)-stimulated adenylate cyclase activity after the estradiol-treatment strongly suggests that increased activity of the catalytic subunit of this enzyme is the likely mechanism whereby estrogens promote lipolysis in rat fat cells.

Prostacyclin and lipolysis in rat fat cells

In rat fat cells incubated for 15 min at 37° and pH 8.5, glycerol release was highly stimulated both by norepinephrine and by theophylline. Prostacyclin (PGI 2) (10 −8−10 −7M) did not alter the basal rate of glycerol release but potentiated the lipolytic effect of 2 × 10 −6M norepinephrine. The rate of norepinephrine-induced glycerol release was increased by PGI 2 during 10 min of incubation and then maintained for the next 5 min. Lipolysis induced by concentrations of norepinephrine which produced maximal effects was not altered by PGI 2. PGI 2 (10 −7−10 −6 M) also potentiated the effect of 5 × 10 −4 M theophylline on glycerol release, but antagonized the stimulation induced by a maximally effective concentration of the methylxanthine (2 × 10 −3M). Incubation of the cells with norepinephrine in the presence of 2 × 10 −4 or 5 × −4 M theophylline caused a loss of the potentiating effect of PGI 2 on norepinephrine-induced lipolysis. In the presence of 10 −3M theophylline, the lipolytic action of norepinephrine was inhibited by PGI 2. In fat cells incubated with adenosine deaminase (0.5 U/ml), 2.5 × 10 −7M PGI 2 did not alter the response to 5 × 10 −4M theophylline and inhibited the effect of norepinephrine both in the absence and in the presence of theophylline. The present results show that, under appropriate experimental conditions, PGI 2 may act as a lipolytic agent in isolated fat cells and that some kind of interaction exists between stimulation of methylxanthine-sensitive adenosine receptors and stimulation of PGI 2 receptors.

Cyclic nucleotides and lipolysis in rat fat cells. Interaction between calcium ionophore A23187 and FCCP, uncoupler of oxidative phosphorylation

The interaction between calcium ionophore A23187 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) has been studied at the level of cyclic-AMP (cAMP), cyclic-GMP (cGMP) and lipolysis in isolated rat fat cells. Ionophore A23187 (1–10 μM) stimulated cGMP accumulation and glycerol release without affecting cAMP level. FCCP (1–100 μM) inhibited the effect of A23187 on cGMP level and glycerol release, but did not affect or increase cAMP. Thus a correlation exists between the changes of cGMP levels and lipolysis and a dissociation of lipolysis from cAMP.

Adenosine actions and adenosine receptors after 1 week treatment with caffeine.

After one week treatment with caffeine (20 mg/kg i.p.) the number of adenosine receptors, as determined by specific binding of (3H)-L-PIA, in rat cerebral cortical membranes was increased by about 25%. Cyclic AMP accumulation induced by adenosine analogues in slices of rat hippocampus was unaffected by caffeine treatment. The inhibition of lipolysis in rat fat cells by 2-chloro-adenosine was similarly unaffected. The potency of caffeine as an antagonist of these adenosine-receptor mediated effects was not altered by caffeine treatment. It is concluded that at least some adenosine receptors are up-regulated as a consequence of prolonged caffeine treatment, but that the increase in receptor number is not related to changes in at least two effects of adenosine and caffeine.

The influence of sexual hormones on lipogenesis and lipolysis in rat fat cells.

The insulin-stimulated conversion of glucose to fatty acids (fatty acid synthesis) and the maximally norepinephrine-stimulated lipolysis were studied in isolated fat cells from normal male and female rats, ovariectomized rats and sexual hormone-treated normal and ovariectomized rats. The fatty acid synthesis and the lipolysis oscillated considerably more in fat cells from female rats than in fat cells from male rats. This was found to be due to the oestrous cycle, since the fatty acid synthesis was high in prooestrus and low in both oestrus and dioestrus, while the lipolysis was higher in oestrus and prooestrus than in dioestrus. Oestradiol treatment of both female and male rats and testosterone treatment of male rats for three days lowered the fatty acid synthesis and increased the lipolysis. The metabolic oscillation disappeared in ovariectomized rats, and the fat cells from these animals showed a metabolic pattern comparable with that found in prooestrus. Even when ovariectomized rats were treated with oestradiol or progesterone only three hours before the examination a significant lower fatty acid synthesis was found in oestradiol-treated animals compared with the progesterone-treated animals. Since these rats were fasted three hours before the experiments, the results were not due to differences in food intake. It is concluded that fat cell metabolism is influenced by sexual hormones. The results are compatible with the hypothesis that the variation in food intake due to sexual hormones is secondary to the metabolic changes.

Effect of adenosine, adenosine analogues and drugs inhibiting adenosine inactivation on lipolysis in rat fat cells.

It has been suggested that adenosine may be a physiologically important modulator of lipolysis. In the present study it was found that adenosine inhibited lipolysis stimulated by low (0.03 micrometer) concentrations of noradrenaline (NA). Lipolysis stimulated by higher concentrations (0.3 and 3 micrometer) of NA was inhibited to a minor degree or not at all. Theophylline (1 micromete)-induced lipolysis was inhibited by adenosine (IC50 approximately 10 micrometer). Inhibition of theophylline-induced lipolysis was tested for several analogues of adenosine. Some N6-substituted adenosine analogues and 2-Cl-adenosine were more potent inhibitors. Adenine-nucleotides (ATP, ADP, AMP) were about equipotent with adenosine. Several adenosine analogues, including its breakdown products were considerably less potent or ineffective. None of the analogues tested inhibited the action of adenosine. Dipyridamol, dilazep and papaverine, which inhibit the uptake of adenosine into cells, caused only a slight enhancement of the antilipolytic effect of adenosine. None of the analogues inhibited the effect of adenosine. It is concluded that adenosine can inhibit lipolysis due to low, "physiological" concentrations of noradrenaline and of low concentration of theophylline via an action on a receptor structure on the cell surface which exhibits structural specificity.

Effects of the adrenergic agonists isoprenaline and noradrenaline and the α-blocking agents dihydroergotamine and phentolamine on the lipolysis in isolated fat cells of the rat, guinea pig, dog and man

The actions of noradrenaline and isoprenaline and of the α-adrenoceptor antagonists dihydroergotamine (DHE) and phentolamine, on lipolysis in isolated fat cells from the rat, guinea pig, dog and man have been investigated. In isolated rat fat cells the efficacy of noradrenaline in stimulating lipolysis was equal to that of isoprenaline. In guinea pig, dog and, particularly in human fat cells the maximal effects elicited by noradrenaline were lower than those elicited by isoprenaline. In these tissues the efficacy of noradrenaline was elevated to isoprenaline levels in the presence of low concentrations of DHE or phentolamine. DHE alone produced a weak stimulation of lipolysis in rat fat cells and an intense stimulation in canine fat cells; phentolamine was without effect. In concentrations of 10 −5 M and above both DHE and phentolamine inhibited catecholamine stimulated lipolysis in fat cells from all species tested, probably as a result of unspecific effects.

Insulin-like actions of nickel and other transition-metal ions in rat fat-cells.

NiC12 (1-6mM) decreased adrenaline and glucagon-stimulated lipolysis in rat fat-cells, and also considerably stimulated [U-14C]glucose incorporation into fat-cell lipids. 2. These insulin-like effects were also observed with CuCl, CuCl2, CoCl2 and (to a lesser extent) with MnCl2. 3. NiCl2 was less effective in mimicking insulin effects on [U-14C]fructose metabolism than on glucose utilization. 4. It is tentatively suggested that these transition-metal ions may mimic actions of insulin at the fat-cell plasma membrane which decrease lipolysis and stimulate glucose transport, but do not mimic certain other effects of the hormone on intracellular metabolic processes. 5. These results are discussed with reference to suggestions that redistributions of cellular Ca2+ are associated with insulin action in fat-cells.