Prolonged fasting on the lipolytic activity of isolated adipocytes of the rat epididymis

Male Wistar rats, 6-8 week old, were fasted for 72 hours. The in vitro lipolytic activity of epididymal adipocytes was measured in the presence of adrenalin (a alpha and beta adrenergic agonist), isoprenaline (a pure beta agonist), theophylline (a phosphodiesterase inhibitor) or UK 14304 (a alpha 2 adrenoceptor agonist) associated with adenosine deaminase. The basal lipolytic activity, expressed per 100 mg lipids, was higher in fasted adipocytes than in fed ones. Its stimulation by adrenalin or isoproterenol was decreased by fast. The effects of these drugs were more potentiated by theophylline in fasted adipocytes than in fed ones. The UK 14304 inhibition of adenosine deaminase-stimulated lipolysis was about 20% in fasted adipocytes and 50% in fed adipocytes. The in vitro resistance of fasted adipocytes to the lipolytic effect of adrenalin or isoproterenol may be related to the hypothyroid status of fasted rats.     

Age-related norepinephrine induced lipolytic response of isolated rat adipocytes

1. Groups of male Long Evans rats were sacrificed at the ages of 10, 16, 20, 24, 28, 32, 38 and 45 weeks. 2. The two epididymal fat pads from each rat in each group (4-5 rats) were excised for the preparation of adipocytes. 3. Cell suspensions were incubated in triplicate with each of seven norepinephrine concentrations ranging from 0.5694 to 569,400 nM. 4. Lipolytic responses are expressed as nmol glycerol released/microgram DNA/90 min. 5. The animals reached a peak response between the ages of 20 and 32 weeks. 6. Aging resulted in a gradual increase in the apparent affinity (Km) of the response yielding system for norepinephrine. 7. Initially an increase in the lipolytic capacity of the cells in response to norepinephrine, is observed, as reflected by the Vmax values up to an age of 20 weeks. 8. Vmax then stays relatively constant at elevated levels up to an age of 32 weeks, followed by an abrupt decrease with further aging.     

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.     

Stress-induced alteration in the lipolytic response to beta-adrenoceptor agonists in rat white adipocytes.

We analysed the sensitivity to beta-adrenoceptor agonists in epididymal adipose cells from rats submitted to a stress protocol previously reported to induce alterations in sensitivity to catecholamines in cardiac tissue from rats. Food intake and body weight were lower, whereas adipocytes basal lipolysis was higher (control: 0.59 +/- 0.04; stress: 1.00 +/- 0.11, micromol glycerol/100 mg total lipids/100 min) in stressed compared to control rats. The responses to isoprenaline (pD(2) control: 7.46 +/- 0.11; stress: 8.11 +/- 0.17), adrenaline (pD(2) control: 5.78 +/- 0. 20; stress: 6.13 +/- 0.18), and salbutamol (pD(2) control: 5.64 +/- 0.28; stress: 5.92 +/- 0.34) were sensitized, and the lipolytic responses to norepinephrine (pD(2) control: 6.98 +/- 0.13; stress: 6. 41 +/- 0.12) and to BRL37344 (pD(2) control: 8.43 +/- 0.19; stress: 7.54 +/- 0.21) were desensitized. Responses to the higher concentration (100 microm) of isoprenaline (control: 1.80 +/- 0.18; stress: 2.24 +/- 0.10 micromol glycerol/100 mg total lipids/100 min), epinephrine (control: 1.64 +/- 0.17; stress: 2.24 +/- 0.14 micromol glycerol/100 mg total lipids/100 min), salbutamol (control: 0.65 +/- 0.11; stress: 1.21 +/- 0.41 micromol glycerol/100 mg total lipids/100 min), and d-butyryl-cAMP (control: 1.59 +/- 0.17; stress: 2.72 +/- 0.25) were significantly enhanced in adipocytes from stressed rats. pD(2) or maximum response to CGP12177 were not altered. Supersensitivity to isoprenaline was abolished by 50 nm ICI118,551 but was not modified by 100 nm metoprolol. However, subsensitivity to norepinephrine and to BRL37344 was abolished by 100 nM metoprolol. Our results suggest that in epididymal adipocytes from stressed rats there is a desensitization of the response to adrenoceptor agonists mediated by beta(1)-adrenoceptors together with a sensitization of the response mediated by beta(2)-adrenoceptors. beta(3)-adrenoceptors seem to be resistant to the stress effect.     

Short-term regulation of adiponectin secretion in rat adipocytes

Adiponectin belongs to the group of biologically active substances secreted by adipocytes and referred to as adipokines. Disturbances in its secretion and/or action are thought to be involved in the pathogenesis of some metabolic diseases. However, regulation of adiponectin secretion is poorly elucidated. In the present study, short-term regulation of adiponectin secretion in primary rat adipocytes was investigated. Isolated rat adipocytes were incubated in Krebs-Ringer buffer containing 5 mM glucose and insulin alone or in the combination with epinephrine, dibutyryl-cAMP, adenosine A(1) receptor antagonist (DPCPX), palmitate, 2-bromopalmitate or inhibitor of mitochondrial electron transport (rotenone). Adipocyte exposure for 2 h to insulin (1-100 nM) significantly increased secretion of adiponectin compared with secretion observed without insulin. Furthermore, secretion of adiponectin from adipocytes incubated with glucose and insulin was reduced by 1 and 2 microM epinephrine, but not by 0.25 and 0.5 microM epinephrine. Under similar conditions, 1 and 2 mM dibutyryl-cAMP substantially diminished secretion of adiponectin, whereas 0.5 mM dibutyryl-cAMP was ineffective. Secretion of adiponectin was found to be effectively decreased by DPCPX. Moreover, adipocyte exposure to rotenone also resulted in a substantial diminution of secretory response of adipocytes incubated for 2 h with glucose and insulin. It was also demonstrated that palmitate and 2-bromopalmitate (0.06-0.5 mM) failed to affect secretion of leptin. The obtained results indicated that in short-term regulation of adiponectin secretion, insulin and epinephrine exert the opposite effects. These effects appeared as early as after 2 h of exposure. Moreover, deprivation of energy or blockade of adenosine action substantially decreased secretion of adiponectin.     

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.     

Discrepancies in lipolytic activities induced by beta-adrenoceptor agonists in human and rat adipocytes

A number of catecholamine and non-catecholamine beta-adrenoceptor agonists, including the lipolytically selective compound BRL 37344, were compared for lipolytic activity on human and rat adipocytes. On rat adipocytes, all compounds were full agonists, BRL 37344 being the most potent. On human adipocytes, only the catecholamines were full beta-adrenoceptor agonists. The other compounds were partial agonists, with intrinsic activities declining in the order fenoterol greater than salbutamol greater than clenbuterol greater than BRL 37344. This was the case with FFA- as well as with glycerol-production. Addition of 20 microM phentolamine did not enhance BRL 37344 activity. The isoprenaline- and BRL 37344-induced lipolysis on rat white adipocytes was stereoselectively antagonized by enantiomers of alprenolol, with atypical low potencies and stereoselectivity. It was concluded that (1) human and rat adipocyte beta-adrenoceptors mediating lipolysis are not essentially different, (2) partial agonism in human adipocytes is not explained by enhanced re-esterification and (3) BRL 37344 selectively stimulates rat adipocyte lipolysis.     

Translocation of hormone-sensitive lipase and perilipin upon lipolytic stimulation of rat adipocytes

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the beta-adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.     

Glucose as a lipolytic agent: studies on isolated rat adipocytes

In order to elucidate the direct effect of glucose on lipolysis in isolated rat adipocytes, cells were incubated in a buffer with different concentrations of this sugar: 2, 8 or 16 mmol/l. The increase in glucose concentration from 2 mmol/l to 8 or 16 mmol/l enhanced basal lipolysis by 30% and 47%, respectively. Epinephrine-induced lipolysis (1 micromol/l) was also increased by 31% and 32%, when glucose concentration was increased from 2 mmol/l to 8 or 16 mmol/l, respectively. The rise in lipolysis caused by glucose was restricted by H-89 (an inhibitor of protein kinase A, 30 micromol/l), but insulin (1 nmol/l) had no inhibitory action. The augmentation of lipolysis by glucose did not require its metabolism (as demonstrated using 2-deoxyglucose) and was due to the action of this sugar on the final steps of the lipolytic cascade, particularly on protein kinase A. However, short-term exposure of adipocytes to higher glucose concentrations did not restrict the inhibitory action of insulin on lipolysis induced by epinephrine.     

Adrenergic receptors are not mediators in the lipolytic effect of somatostatin in rat adipocytes

Beta-adrenergic receptors have been purported to act as possible mediators in the lipolytic effect of somatostatin in vivo. Investigations with isolated rat adipocytes studying the lipolytic activity of somatostatin (1.7 x 10(-7) M), glucagon (8.1 x 10(-8 M) and norepinephrine (10(-6) M), have shown that the lipolytic effect stimulated by somatostatin is not altered by 10(-5) M propranolol (beta-antagonist); is significantly enhanced by 10(-5) M isoproterenol (beta-agonist) and is not altered by the addition of 10(-6) M phenoxybenzamine (alpha-antagonist) or 10(-6) M phenylephrine (alpha-agonist). Similar results were found when lipolysis was stimulated by glucagon, whereas the lipolytic effect stimulated by norepinephrine was blocked by propranolol. These results indicate that the direct lipolytic effect of somatostatin on isolated rat adipocytes does not seem to be mediated through mechanisms involved with adrenergic receptors.     

Natriuretic peptides: a new lipolytic pathway in human adipocytes.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.     

Analysis of lipolytic protein trafficking and interactions in adipocytes

This work examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis during brief cAMP-dependent protein kinase A (PKA) activation. Double label immunofluorescence analysis of 3T3-L1 adipocytes indicated that PKA activation increases the translocation of hormonesensitive lipase (HSL) to perilipin A (Plin)-containing droplets and increases the colocalization of adipose tissue triglyceride lipase (Atgl) with its coactivator, Abhd5. Imaging of live 3T3-L1 preadipocytes transfected with Aquorea victoria-based fluorescent reporters demonstrated that HSL rapidly and specifically translocates to lipid droplets (LDs) containing Plin, and that this translocation is partially dependent on Plin phosphorylation. HSL closely, if not directly, interacts with Plin, as indicated by fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) experiments. In contrast, tagged Atgl did not support FRET or BiFC with Plin, although it did modestly translocate to LDs upon stimulation. Abhd5 strongly interacted with Plin in the basal state, as indicated by FRET and BiFC. PKA activation rapidly (within minutes) decreased FRET between Abhd5 and Plin, and this decrease depended upon Plin phosphorylation. Together, these results indicate that Plin mediates hormone-stimulated lipolysis via direct and indirect mechanisms. Plin indirectly controls Atgl activity by regulating accessibility to its coactivator, Abhd5. In contrast, Plin directly regulates the access of HSL to substrate via close, if not direct, interactions. The differential interactions of HSL and Atgl with Plin and Abhd5 also explain the findings that following stimulation, HSL and Atgl are differentially enriched at specific LDs.     

Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.

Phosphatidylinositol (PtdIns) 3-kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzyme in insulin signaling, we studied the activation of PtdIns-3-kinase in adipocytes, a major insulin target tissue for glucose transport and utilisation. To analyze possible interactions of the enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti-phosphotyrosine antibodies, (b) two antibodies to the 85-kDa subunit of PtdIns-3-kinase (p85) and (c) an antibody to the 185-kDa major insulin receptor substrate (p185). We show that in cell extracts from adipocytes exposed to insulin, and after immunoprecipitation with an anti-phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns-3-kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns-3-kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns-3-kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrate, such as p185, which could function as a link between the insulin receptor and PtdIns-3-kinase. The PtdIns-3-kinase was activated within 1 min of exposure to insulin, and the half-maximal effect was reached at the same concentration, i.e. 3 nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns-3-kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns-3-kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin-induced recruitment of the PtdIns-3-kinase from the cytosol to the membrane. Finally, we used isoproterenol, which exerts antagonistic effects on insulin action. This drug was found to inhibit both the PtdIns-3-kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns-3-kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin-stimulated PtdIns-3-kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.     

Studies of glucagon resistance in large rat adipocytes: 125I-labeled glucagon binding and lipolytic capacity

This study is concerned with potential modifications of large fat cells from adult rats (400-450 g) that make them resistant to stimulation by glucagon. The lipolytic capacity and (125)I-labeled glucagon-binding capability of these cells were compared with these properties of small glucagon-sensitive cells from young rats (130-160 g). As determined by maximal stimulation with theophylline, dibutyryl cAMP, or epinephrine, the lipolytic capacity of large cells was not markedly different from small cells, which suggests that an alteration contributing to glucagon insensitivity is not present in the enzymes involved with hormone-mediated lipolysis. Glucagon-binding studies did indicate a difference between the two cell types. Both large cells and particulate fractions from large cells bound less (125)I-labeled glucagon than small cells or small-cell particles. That diminished binding is not a consequence of glucagon degradation is indicated by the similar amounts of (125)I-labeled glucagon degraded by both cell types. The decrease in (125)I-labeled glucagon binding was not as marked as the decrease in lipolytic response to glucagon stimulation. This lack of correlation and the relationship between elevated phosphodiesterase levels and glucagon insensitivity described in the accompanying report suggest that diminished binding explains only in part the marked resistance to glucagon found in large cells.     

Correction by dexamethasone treatment of the altered lipolytic cascade induced by adrenalectomy in rat adipocytes

The ability of glucocorticoid-treatment to reverse the metabolic alterations caused by adrenalectomy in rat adipocytes was studied. Correction of the enhanced adenosine antilipolytic effect and of the defect in lipolysis, cyclic AMP and adenylate cyclase responsiveness to guanine nucleotides were all achieved after a 24 h dexamethasone treatment, whereas correction of the defect in beta-adrenoceptor-density required a 48 h treatment. The latter treatment, however, failed to reverse the defect in both the adenylate cyclase catalytic activity and protein content per fat cell. These different kinetics of restoration indicate that correction by dexamethasone of the defective cyclic AMP and lipolytic responses on one hand and of the guanine nucleotide control of adenylate cyclase on the other one are two related phenomenoms.     

Characterization of lipolytic responses of isolated white adipocytes from hamsters.

1. Lipolysis by isolated white adipocytes from hamsters, as measured by glycerol production, was stimulated by corticotropin, isopropylnorepinephrine (INE), norepinephrine, or epinephrine (EPI), in a dose-dependent fashion. 2. Lipolysis was stimulated by five inhibitors of cyclic 3',5'-adenosine monophosphate phosphodiesterase: caffeine, theophylline, 1-methyl-3-isobutyl xanthine, 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazolo-(3,4,-b)-pyridine-5-carboxylic acid ethyl ester (SQ 20009), and 4-(3,4-dimethoxybenzyl)-2-imidazolidinone (Ro 7-2956). Caffeine-stimulated lipolysis consistently attained higher rates than did hormone-stimulated lipolysis. However, when cells were stimulated by both caffeine and a hormone, lipolytic rates were consistently lower than those attained under the influence of caffeine alone. 3. Isolated white adipocytes from hamsters were sensitive to both alpha- and beta-adrenergic antagonists. The beta-adrenergic antagonist propranolol could completely inhibit norepinephrine-stimulated glycerol production. The alpha-adrenergic antagonist phentolamine, on the other hand, had a biphasic effect on the cells. At 5-10(-7) M or 5-10(-6) M, phentolamine enhanced norepinephrine-stimulated lipolysis, while concentrations higher than 5-10(-5) M caused inhibition. 4. The effects of two different concentrations of six antilipolytic agents, prostaglandin E1, nicotinic acid, phenylisopropyladenosine, 5-methylpyrazole-3-carboxylic acid, adenosine and insulin, were measured. With the exception of insulin, all of these agents showed much more potent inhibition of caffeine-stimulated lipolysis than of hormone-stimulated lipolysis. Insulin, in contrast, showed only modest inhibition of hormone-stimulated lipolysis and virtually no inhibition of caffeine-stimulated lipolysis.