Adrenergic lipolysis in human fat cells: properties and physiological role of alpha-adrenergic receptors (author's transl)

Lipolytic activity of human isolated fat cells from different fat deposits was studied. The purpose of the present investigations was to determine the epinephrine responsiveness, with regard to alpha- and beta-adrenergic receptor site activity, of omental and subcutaneous adipocytes (abdominal or from the lateral part of the thigh). Adipocytes were obtained from normal subjects or from obese subjects on iso- or hypocaloric diets. The lipolytic effect of epinephrine varied according to the fat deposits, while the beta-lipolytic effect of isoproterenol was more stable (Fig. 1). We explored the possible involvement of adrenergic alpha-receptors, in order to explain these results. The potentiating action of phentolamine on epinephrine-induced lipolysis, and the antilipolytic effect of alpha-agonists on basal or theophylline--induced lipolysis, were found to be a good indication of alpha-adrenergic activity. The alpha-adrenergic antilipolytic effect was most prominent in adipose tissue from the lateral part of the thigh, and less noticeable in omental adipocytes. In conclusion, the inability of epinephrine to induce lipolysis, and the epinephrine-induced inhibition of lipolysis observed when the basal rate of FFA release was spontaneously increased in subcutaneous fat-cells of the thigh, could be explained by an increased alpha adrenergic responsiveness (Fig. 2). Moreover, various alpha-adrenergic agonists (phenylephrine, noradrenaline and adrenaline) showed a clear inhibiting effect on theophylline-stimulated adipocytes from the thigh. The pharmacological study of the antilipolytic effect of epinephrine on theophylline-induced lipolysis showed that the inhibition was linked to a specific stimulation of the alpha-receptors of the subcutaneous adipocytes (Fig. 4). From the different sets of experiments, it is shown that the modifications in the lipolytic effect of epinephrine on adipocytes of different areas could be explained by the occurrence of a variable alpha-adrenergic effect initiated by catecholamine. Furthermore, theophylline stimulation of lipolysis provides an accurate system to investigate the alpha-inhibiting effect of catecholamines. Our study was completed by the investigation of the lipolytic activity of subcutaneous fat cells from obese subjects submitted to a hypocaloric diet (800-1 000 Cal/day). An increased alpha-inhibitory effect of epinephrine was shown on the increased basal lipolytic activity observed in the fat cells of obese subjects on a hypocaloric diet (Fig. 5); a similar effect was observed when these adipocytes were stimulated by theophylline. To conclude, these investigations allow the alpha-adrenergic effect to be considered as a regulator mechanism of the in vitro lipolytic activity in human adipose tissue, since the antilipolytic effect is operative whenever the basal rate of lipolysis is increased (spontaneously, after caloric restriction, or with a lipolytic agent such as theophylline).     

Duration of feeding on a sucrose-rich diet determines metabolic and morphological changes in rat adipocytes.

In this work, we studied the effect of a short-term (3 wk) and a long-term (15 wk) administration of a sucrose-rich diet (SRD) to Wistar rats on the morphological aspects and metabolic function of the epididymal adipose tissue that may contribute to the mechanism underlying the impaired glucose homeostasis and insulin resistance. The present work showed the following. 1) There was both a moderate increase of basal lipolysis and a decrease of the antilipolytic action of insulin in the adipocytes of rats fed a SRD for 3 wk. Neither size alterations nor increases in adipose tissue mass were recorded in this period. 2) There was a significant (P < 0.05) increase of epididymal weight after 15 wk on a SRD as well as a hypertrophy of adipocytes with a clear alteration in the cell size distribution. This was accompanied by a significant increase (P < 0.05) of basal and stimulated lipolysis and a marked decrease (P < 0.05) of the antilipolytic action of insulin. Moreover, these changes appear together with a worsening of both impaired glucose homeostasis and insulin resistance. Our results also indicate that the length of time on the SRD plays an important role in the evolution of the adiposity and metabolic changes observed in the fat pad. Furthermore, the latter precedes the detection of adiposity.     

Eicosapentaenoic acid inhibits tumour necrosis factor-α-induced lipolysis in murine cultured adipocytes

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid with beneficial effects in obesity and insulin resistance. High levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α) in obesity promote lipolysis in adipocytes, leading to the development of insulin resistance. Thus, the aims of the present study were to analyze the potential antilipolytic properties of EPA on cytokine-induced lipolysis and to investigate the possible mechanisms involved. The EPA effects on basal and TNF-α-induced lipolysis were determined in both primary rat and 3T3-L1 adipocytes. Treatment of primary rat adipocytes with EPA (100 and 200 μM) significantly decreased basal glycerol release (P<.01) and prevented cytokine-induced lipolysis in a dose-dependent manner (P<.001). Moreover, EPA decreased TNF-α-induced activation of nuclear factor-κB and extracellular-related kinase 1/2 phosphorylation. In addition, the antilipolytic action of EPA was stimulated by the AMP-kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside and blocked by the AMPK-inhibitor compound C. Moreover, we found that EPA stimulated hormone-sensitive lipase (HSL) phosphorylation on serine-565, which further supports the involvement of AMPK in EPA's antilipolytic actions. Eicosapentaenoic acid treatment (24 h), alone and in the presence of TNF-α,? also decreased adipose triglyceride lipase (ATGL) protein content in cultured adipocytes. However, oral supplementation with EPA for 35 days was able to partially reverse the down-regulation of HSL and ATGL messenger RNA observed in retroperitoneal adipose tissue of high-fat-diet-fed rats. These findings suggest that EPA inhibits proinflammatory cytokine-induced lipolysis in adipocytes. This effect might contribute to explain the insulin-sensitizing properties of EPA.     

Inhibition of epinephrine-induced lipolysis in isolated white adipocytes of aging rabbits by increased alpha-adrenergic responsiveness.

The aim of this study was to explain the unresponsiveness of rabbit perirenal adipose tissue to epinephrine. The in vitro lipolytic response to isoproterenol and to epinephrine alone or associated with alpha- or beta-adrenergic blocking agents, was studied in the adipocytes of rabbits of various ages. Epinephrine induces a large glycerol release in young rabbit adipocytes whereas an increase in the rate of lipolysis cannot be shown with adult rabbit fat cells. Moreover, an antilipolytic effect can be shown for low concentrations of epinephrine when the basal rate of lipolysis is high in older rabbit adipocytes. Isoproterenol (beta-adrenomimetic) always exerts a strong adipokinetic effect, thus revealing functional beta-receptor sites. The blockade of alpha-adreneoceptor sites by phentolamine, which has no effect on young rabbits, abolishes the antilipolytic effect and unmasks strong lipolytic effect of epinephrine on aged and normal rabbit adipocytes. The loss of beta-adrenergic responsiveness towards epinephrine in the aging rabbit is linked to the involvement of an increased alpha-adrenergic responsiveness. The stimulation of alpha receptor sites by epinephrine leads to a depressive effect on lipolysis (lack of adipokinetic effect or antilipolytic action).     

Genistein, a plant-derived isoflavone, counteracts the antilipolytic action of insulin in isolated rat adipocytes

Genistein is a phytoestrogen exerting numerous biological effects. Its direct influence on adipocyte metabolism and leptin secretion was previously demonstrated. This study aimed to determine whether genistein antagonizes the antilipolytic action of insulin in rat adipocytes. Freshly isolated adipose cells were incubated for 90 min with epinephrine, epinephrine with insulin and epinephrine with a specific inhibitor of protein kinase A (H-89) at different concentrations of genistein (0, 6.25, 12.5, 25, 50 and 100 μM). Genistein failed to affect epinephrine-induced glycerol release, however, the inhibitory action of insulin on epinephrine-induced lipolysis was significantly abrogated in cells exposed to the phytoestrogen (12.5–100 μM). The increase in insulin concentration did not suppress the genistein effect. Its inhibitory influence on the antilipolytic action of insulin was accompanied by a substantial rise in cAMP in adipocytes. This rise appeared despite the presence of 10 nM insulin in the incubation medium. Further experiments, in which insulin was replaced by H-89, revealed that the antilipolytic action of protein kinase A inhibitor on epinephrine-induced lipolysis was not affected by genistein. This means that genistein counteracted the antilipolytic action of insulin due to the increase in cAMP levels and activation of protein kinase A in adipocytes. The observed attenuation of the inhibitory effect of insulin on triglyceride breakdown evoked by genistein was not related to its estrogenic activities, as evidenced in experiments employing the intracellular estrogen receptor blocker, ICI 182,780. Moreover, it was found that genistein-induced impairment of the antilipolytic action of insulin was not accompanied by changes in the proportion between fatty acids and glycerol released from adipocytes. The ability of genistein to counteract the antilipolytic action of insulin may contribute to the decreased triglyceride accumulation in adipose tissue.     

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents.

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.     

Evolution of the sensitivity of isolated adipocytes of ewes to the antilipolytic action of adenosine during pregnancy and lactation

Four successive biopsies of omental adipose tissue were performed on the 43rd, 100th, 140th days of pregnancy and during the 3rd week of lactation in 6 "Pré-Alpes" ewes. Using incubated isolated adipocytes, we studied the evolution of the antilipolytic effect of adenosine. Adenosine presented a significant inhibitory effect on basal lipolysis only at the end of pregnancy. The antilipolytic effect of adenosine on the stimulated lipolysis by a beta-agonist (isoproterenol 400 nM) increased during pregnancy. The maximal effect was observed 1 week before parturition. The level of inhibition remained high during lactation. The lipolytic effect of adenosine-deaminase was enhanced during pregnancy and lactation and evolved similar to the observed action of adenosine. Results suggest that the antilipolytic action of adenosine may have a physiologic importance.     

Effects of dietary fat and adipose tissue location on insulin action in young boar adipocytes

1. Regulation of lipogenesis and lipolysis by insulin was studied on adipocytes isolated from 100 kg Large white male pigs. Two adipose tissues were studied: subcutaneous and perirenal. Animals were fed either a control low fat diet or a diet containing 14.7% sunflower seed oil. 2. The cell diameter was higher in the group fed the sunflower diet. 3. De novo lipogenesis was decreased for each adipose tissue in the group fed the sunflower diet. The perirenal site had a higher lipogenic activity than subcutaneous site whatever the diet. 4. Insulin did not significantly stimulate lipogenesis but had an important antilipolytic effect on stimulated lipolysis by isoproterenol. 5. The antilipolytic action of insulin was higher in perirenal adipocytes with the control diet. With the sunflower diet, the decrease was about 54.4% for subcutaneous adipocytes, whereas the inhibition was decreased in perirenal adipocytes. Addition of theophylline reversed the antilipolytic action of insulin. 6. Insulin binding was not affected neither by the dietary fat nor by the adipose tissue location. 7. Absence of de novo lipogenesis stimulation by insulin was not due to an impairment in insulin binding. 8. The different effects of dietary fat and adipose tissue location on the antilipolytic action of insulin could not be explained by a modification of insulin binding but rather by a latter event, probably at a post-insulin binding stage.     

Homocysteine suppresses lipolysis in adipocytes by activating the AMPK pathway

Hyperhomocysteinemia (HHcy) is an independent risk factor for coronary artery disease. Emerging evidence suggests that HHcy is also associated with adipocyte tissue dysfunction. One of the principal functions of adipose tissue is to provide energy substrate via lipolysis. In the present study, we investigated the effects of homocysteine (Hcy) on lipolysis in adipocytes. We found that Hcy inhibited release of glycerol and fatty acids, two typical indicators of the lipolytic response, in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner under both basal and isoproterenol-stimulated conditions. In differentiated 3T3-L1 adipocytes, decreased glycerol and free fatty acid (FFA) release was associated with elevation of intracellular TG content. Further studies showed that Hcy-mediated antilipolytic responses were independent of the cyclic AMP-PKA and MEK-ERK1/2 pathways. However, Hcy increased phosphorylation levels of AMP-activated protein kinase (AMPK) and its downstream enzyme acetyl-CoA carboxylase. Compound C, an AMPK inhibitor, abolished Hcy-induced reduction of glycerol and FFA release under both basal and isoproterenol-stimulated conditions. Furthermore, AMPKα1 siRNA reversed Hcy-inhibited glycerol release. Supplementation of exogenous Hcy in the diet for 2 wk lowered circulating glycerol and FFA levels. Moreover, Hcy supplementation was associated with elevated leptin levels and reduced adiponectin levels in plasma. These results show that Hcy inhibits lipolysis through a pathway that involves AMPK activation.     

A change of hormonal regulation of adenylyl cyclase in epididymal adipose tissue of rats with experimental models of diabetes mellitus

One of the key causes of diabetes mellitus (DM) and its complications are hormonal disturbances in functioning of hormonal signaling systems, including the adenylyl cyclase signaling system (ACSS). The goal of this work was to study the functional state and hormonal sensitivity of ACSS in the epididymal adipose tissue of male rats in the 7-month model of mild type 1 DM (DM1), in the 18-month neonatal model of type 2 DM (DM2), and in the taken for comparison model of the 30 day-longs acute DM1. It is shown for the first time that in adipocytes from the epididymal fat of rats with the studied DM models the basal AC activity and its stimulation by forskolin were decreased, which indicates a weakening of the catalytic functions of the enzyme adenylyl cyclase (AC). Stimulation of AC by guanine nucleotides in DM changed to the lesser extent, which argues in favor of preservation of functions of heterotrimeric Gs-proteins in the epididymal fat. In rats with DM1 the sensitivity of AC of adipocytes to agonists of β-adrenergic receptors (β-AR), activators of lipolysis, remained practically unchanged, while in animals with DM2 the AC stimulating effects of β-AR-agonists were reduced or completely blocked, like in the case of β₃-AR-agonists BRL-37344 and CL-316243. In adipocytes of rats with DM1 the AC inhibitory effect of N⁶-cyclopentyladenosine, agonist of type 1 adenosine receptors (Aden₁R), an inhibitor of lipolysis, was attenuated, whereas in DM2 this effect was completely preserved. Thus, in the epididymal adipose tissue of rats with DM1 the antilipolytic AC cascades including Aden₁R were decreased and the stimulation of AC by β-AR-agonists was preserved, whereas in rats with DM2 the β-AR-mediated AC cascades activating lipolysis were reduced, but Aden₁R-mediated AC cascades inhibiting lipolysis did not change. The changes of hormonal regulation of ACSS in adipocytes from the epididymal fat lead to disturbances of the metabolic status of animals with DM1 and DM2 and should be considered in the diagnostics and treatment of DM and its complications.     

Soy hydrolysate enhances the isoproterenol-stimulated lipolytic pathway through an increase in β-adrenergic receptor expression in adipocytes

ABSTRACT

Activation of the adipose lipolytic pathway during lipid metabolism is mediated by protein kinase A (PKA), which responds to β-adrenergic stimulation, leading to increased lipolysis. Soy is well known as a functional food and it is able to affect lipolysis in adipocytes. However, the mechanism by which soy components contribute to the lipolytic pathway remains to be fully elucidated. Here, we show that hydrolyzed soy enhances isoproterenol-stimulated lipolysis and activation of PKA in 3T3-L1 adipocytes. We also found that the expression of β-adrenergic receptors, which coordinate the activation of PKA, is elevated in adipocytes differentiated in the presence of soy hydrolysate. The activity of the soy hydrolysate towards β-adrenergic receptor expression was detected in its hydrophilic fraction. Our results suggest that the soy hydrolysate enhances the PKA pathway through the upregulation of β-adrenergic receptor expression and thereby, increase lipolysis in adipocytes.     

Increased 4-Hydroxynonenal Formation Contributes to Obesity-Related Lipolytic Activation in Adipocytes

Oxidative stress in adipose tissue plays an etiological role in a variety of obesity-related metabolic disorders. We previously reported that increased adipose tissue 4-hydroxynonenal (4-HNE) contents contributed to obesity-related plasma adiponectin decline in mice. In the present study, we investigated the effects of intracellular 4-HNE accumulation on lipolytic response in adipocytes/adipose tissues and underlying mechanisms. In both fully-differentiated 3T3-L1 and primary adipocytes, a 5-hour 4-HNE exposure elevated lipolytic reaction in a dose-dependent manner at both basal and isoproterenol-stimulated conditions, evidenced by significantly increased glycerol and fatty acids releases. This conclusion was corroborated by the comparable observations when the minced human visceral adipose tissues were used. Mechanistic investigations revealed that 4-HNE-stimulated lipolytic activation is multifactorial. 4-HNE exposure quickly increased intracellular cyclic AMP (cAMP) level, which was concomitant with increased phosphorylations of protein kinase A (PKA) and its direct downstream target, hormone sensitive lipase (HSL). Pre-incubation with H89, a potent PKA inhibitor, prevented 4-HNE stimulated glycerol release, suggesting that enhanced lipolytic action in response to 4-HNE increase is mediated mainly by cAMP/PKA signal pathway in adipocytes. In addition to activating cAMP/PKA/HSL pathway, 4-HNE exposure also suppresses AMP-activated protein kinase (AMPK), a suppressive pathway for lipolysis, measured by both Western blotting for phosphorylated form of AMPK and ELISA for enzyme activity. Furthermore, 5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), a pharmacological AMPK activator, alleviated 4-HNE-induced lipolysis, suggesting that AMPK suppression also contributes to 4-HNE elicited lipolytic response. In conclusion, our findings indicate that increased intracellular 4-HNE accumulation in adipocytes/adipose tissues contributes to obesity-related lipolytic activation.     

Differences in lipogenesis and lipolysis in obese and non-obese adult human adipocytes

It has been proposed that differences in adipocyte function and/or metabolism between obese and lean individuals may manifest themselves in functional adipose tissue abnormalities that lead to metabolic disorders in obesity. We studied lipogenesis and lipolysis of omental adipocytes from obese (OB) and non-obese (NOB) humans. The specific activity of the lipogenic marker enzyme G3PDH was 50% lower in total adipocytes of OB compared to that of NOB subjects. Omental adipocytes from OB subjects also had lower basal lipolytic levels, and a lower lipolytic response to beta-adrenergic stimulus. Cholesterol depletion of adipocyte plasma membrane using methyl b-cyclodextrin caused a lipolytic effect on adipocytes of both groups together, but when obese and lean subjects were analyzed separately, the response was significant only in the obese. We present evidence of a different lipogenic and lipolytic profile in obese individuals' omental adipocytes, and propose a relevant role of plasma membrane cholesterol, where the impact of its removal in OB and NOB adipocyte lipolysis differs.     

Acute exposure to long-chain fatty acids impairs {alpha}2-adrenergic receptor-mediated antilipolysis in human adipose tissue

The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.     

Impaired insulin action in subcutaneous adipocytes from women with visceral obesity

Visceral obesity is associated with resistance to the antilipolytic effect of insulin in vivo. We investigated whether subcutaneous abdominal and gluteal adipocytes from viscerally obese women exhibit insulin resistance in vitro. Subjects were obese black and white premenopausal nondiabetic women matched for visceral adipose tissue (VAT), total adiposity, and age. Independently of race and adipocyte size, increased VAT was associated with decreased sensitivity to insulin's antilipolytic effect in subcutaneous abdominal and gluteal adipocytes. Absolute lipolytic rates at physiologically relevant concentrations of insulin or the adenosine receptor agonist N(6)-(phenylisopropyl)adenosine were higher in subjects with the highest vs. lowest VAT area. Independently of cell size, abdominal adipocytes were less sensitive to the antilipolytic effect of insulin than gluteal adipocytes, which may partly explain increased nonesterified fatty acid fluxes in upper vs. lower body obese women. Moreover, increased VAT was associated with decreased responsiveness, but not decreased sensitivity, to insulin's stimulatory effect on glucose transport in abdominal adipocytes. These data suggest that insulin resistance of subcutaneous abdominal and, to a lesser extent, gluteal adipocytes may contribute to increased systemic lipolysis in both black and white viscerally obese women.