Increase in uncoupling protein-2 mRNA expression by BRL49653 and bromopalmitate in human adipocytes.

Uncoupling protein-2 (UCP2) is a novel mitochondrial protein that may be involved in the control of energy expenditure. We have previously reported an upregulation of adipose tissue UCP2 mRNA expression during fasting in humans. Analysis of changes in metabolic parameters suggested that fatty acids may be associated with the increased UCP2 mRNA level. Culture of human adipose tissue explants was used to study in vitro regulation of adipocyte UCP2 gene expression. A 48-h treatment with BRL49653 and bromopalmitate, two potent activators of PPARgamma, resulted in a dose-dependent increase in UCP2 mRNA levels. The induction by BRL49653 was rapid (from 6 h) and maintained up to 5 days. TNFalpha provoked a 2-fold decrease in UCP2 mRNA levels. Human recombinant leptin did not affect UCP2 mRNA expression. The data support the hypothesis that fatty acids are involved in the control of adipocyte UCP2 mRNA expression in humans.

Regulation of hormone-sensitive lipase expression by glucose in 3T3-F442A adipocytes.

Hormone-sensitive lipase (HSL) catalyzes the rate-limiting step in adipocyte lipolysis. We have studied the effect of glucose and long chain fatty acids on the HSL mRNA content of 3T3-F442A adipocytes. Exposure of the cells for 32 h to a medium without glucose led to a decrease by a factor of 2.5-3 in the HSL mRNA without a change in C/EBP alpha mRNA or triglyceride content of the adipocytes. The reduction in HSL mRNA level was accompanied by a decrease in HSL total activity. The effect of glucose exposure was observed after 24 h of exposure and was reversible. Treatment of the adipocytes with oleate did not affect HSL expression. These data show that glucose modulates HSL gene expression and hence could participate in the regulation of adipose tissue lipolysis.

Comparison of hormone-sensitive lipase activity in visceral and subcutaneous human adipose tissue.

The possible role of hormone-sensitive lipase (HSL) in determining regional differences in lipolysis activation in humans was studied in vitro. Small adipose tissue biopsies were obtained from the abdominal sc and omental regions during surgery in 21 subjects spanning a wide range of body mass index (22-50 kg/m2). In lipolysis experiments, isolated fat cells were incubated with lipolytic agents acting at different levels in the lipolytic cascade. The activity and messenger ribonucleic acid expression of HSL were determined. The maximum lipolytic capacity was higher in sc than in omental fat cells as were HSL activity and messenger ribonucleic acid expression. The maximum lipolysis rate was significantly correlated to HSL activity. This is in accordance with the role of HSL as the rate-limiting step of lipolysis. However, adipocytes were 24% larger in the sc than in the omental region, and the lipolysis rate was significantly correlated to fat cell size regardless of either the region of origin or gender. This indicates that the regulation of HSL activity in healthy subjects, which appears to occur at a transcriptional level, is to a large extent dependent on fat cell size.

Adipose tissue lipolysis and hormone-sensitive lipase expression during very-low-calorie diet in obese female identical twins.

Eight pairs of obese female monozygotic twins were subjected to a 4-week, very-low-calorie diet (VLCD) that induced a decrease in mean body mass index from 32.9 +/- 1.1 to 29.7 +/- 1.1 kg/m2. Infusion of the beta-adrenergic agonist, isoproterenol, induced an increase in plasma levels of nonesterified fatty acids and glycerol that was more pronounced during than before VLCD. sc fat biopsies were obtained before and during VLCD to study adipocyte lipolysis. beta-adrenergic sensitivity was moderately improved during VLCD. Basal and stimulated lipolyses, and hormone-sensitive lipase activity and protein levels were increased during VLCD. Before VLCD, intrapair resemblance was found for basal and stimulated lipolysis rates. In response to the treatment, intrapair resemblance was observed for basal lipolysis and for lipolysis stimulated with agents acting on plasma membrane receptors. These results suggest that the increase of basal lipolysis during VLCD is caused by an increase of hormone-sensitive lipase expression. They support the notion that the genotype may play a role in regulating the changes of adipose tissue lipolysis rates observed during VLCD.

Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

Adipocyte lipolysis in normal weight subjects with obesity among first-degree relatives.

In this study we investigated whether fat cell lipolysis could be involved in the aetiology of obesity by comparing non-obese subjects with (Hob) or without (Hnorm) a family trait for overweight. A family history of obesity was present when at least one of the first-degree relatives had body mass index of 27 kg/m2 or more. Twenty-seven healthy, drug-free non-obese adult subjects were investigated; 13 were Hob and the remaining 14 were Hnorm. Eleven Hob had at least one obese parent. Isolated fat cells from abdominal subcutaneous adipose tissue were incubated in vitro. Glycerol release (lipolysis index), mRNA levels and enzymatic activity of hormone-sensitive lipase and radioligand binding to beta 1- and beta 2-adrenoceptors were determined. The lipolytic effects of noradrenaline (major endogenous lipolytic agent), isoprenaline (a non-selective beta-adrenoceptor agonist), forskolin (a direct activator of adenylyl cyclase) and dibutyryl cyclic AMP (activating protein kinase and thereby hormone-sensitive lipase) were reduced by about 50% (p from 0.001 to 0.01). The maximum activity of hormone-sensitive lipase was reduced 50% in Hob (p < 0.05) and correlated with the lipolytic responsiveness of fat cells in the whole population (r = 0.71). However, there was no difference between the groups in steady-state mRNA levels for the enzyme. Beta 1-->, beta 2- and alpha 2-adrenoceptor sensitivity as well as beta 1- and beta 2-adrenoceptor numbers were normal in Hob. Fasting plasma insulin was 49.1 and 32.6 pmol/l, respectively in Hob and Hnorm (p = 0.01). There was, however, no significant correlation between lipolysis in vitro and plasma insulin. Thus, lipolytic catecholamine resistance in fat cells, at least partly due to impaired function of hormone-sensitive lipase, is an adipocyte abnormality associated with a family tendency to obesity.

Expression of hormone-sensitive lipase in the human colon adenocarcinoma cell line HT29.

Hormone-sensitive lipase expression was studied in the human colon adenocarcinoma cell line, HT29. Diacylglycerol lipase and cholesterol esterase [corrected] activities in HT29 cells were inhibited by known inhibitors of hormone-sensitive lipase (diethyl-p-nitrophenyl phosphate, NaF and HgCl2) to the same extent as in human adipocytes. A polyclonal antiserum directed against rat hormone-sensitive lipase inhibited 89% of HT29 cell lipase activity. HT29 hormone-sensitive lipase was the same size as the adipocyte enzyme as was its mRNA. Complete homology between mRNA sequences in HT29 and adipocyte was demonstrated using ribonuclease protection assay. These data are consistent with the expression of a protein closely related, if not identical, to the enzyme expressed in human adipose tissue. HT29 is the first human cell line where hormone-sensitive lipase expression has been shown.

In vivo upregulation of adipocyte alpha 2-adrenoceptors by androgens is consequence of direct action on fat cells.

Adipose precursor cells from male hamsters were exposed to testosterone in primary culture. The effect of the sex steroid on alpha 2-adrenoceptor (AR) expression was studied. Testosterone enhanced alpha 2-AR expression during adipose conversion to an identical extent to that determined in vivo without modification of the differentiation state as estimated by glycerol-3-phosphate dehydrogenase activity. The expression of preadipocyte alha 2-ARs from female hamster was also submitted to the testosterone control. The upregulating effect was androgen specific, since only testosterone and dihydrotestosterone were efficient in inducing an increase of alpha 2-AR density, whereas estradiol, dexamethasone, progesterone, and androstenedione were without any action. To conclude, androgens act directly on fat cells by upregulating alpha 2-AR expression. The effects are not mediated by aromatization into estrogens and probably involve androgen receptor interactions.

Desensitization of beta-1 and beta-2, but not beta-3, adrenoceptor-mediated lipolytic responses of adipocytes after long-term norepinephrine infusion.

The beta-3 adrenoceptor protein lacks most of the potential phosphorylation sites for beta adrenoceptor kinase and protein kinase A. In addition, it exhibits a lower affinity toward norepinephrine than beta-1 or beta-2 adrenoceptors. It is thus expected that beta-3 adrenoceptors could be less implicated in desensitization processes than the beta-1 or beta-2 adrenoceptors. An attempt to demonstrate the physiological relevance of this prediction was performed by using fat cells having a beta-3 adrenergic responsiveness or not (hamster and guinea pig). The influence of prolonged in vivo exposure to norepinephrine on the beta adrenoceptor-mediated lipolytic responses was investigated in both species. In control guinea pigs, isoproterenol, norepinephrine and epinephrine were fully lipolytic, whereas BRL 37344 [(R',R')-4-2[2-((2[(3-chlorophenyl)-2-hydroxyethyl]amino] propyl)phenyl]phenoxyacetic acid], CGP 12177](+-)-4-(3-tertiarybutylamino-2-hydroxypropoxy)- benzimidazole-2-on hydrochloride] and other beta-3 agonists were inefficient, whereas hamster adipocytes exhibited maximal response to the beta-3 agonists. Blockade of the lipolytic effect of isoproterenol in the guinea pig gave a rank order of beta antagonists [CGP 20712A (1-[2-(3-carbamoyl-4-hydroxyphenoxy)ethylamino]-3-4-(1-methyl-4-tr i-fluoro-methyl-1H-imidazol-2-yl)phenoxy-2-propanol methanesulfonate] > bupranolol > or = propranolol >> ICI 118551 [erythrodl-1-(7- methylindan-4-yloxy)-3-isopropylaminobutan-2-ol)] in agreement with that of a beta-1 effect. In contrast, the selective beta-1 antagonist CGP 20712A did not counteract the effect of BRL 37344 in hamsters and bupranolol was the best beta antagonist tested; a result arguing for the predominance of a beta-3 component in the adrenergic activation of lipolysis, as in rat fat cells. In treated guinea pigs (6-day treatment with osmotic minipumps delivering norepinephrine at the rate of 5 micrograms/min/kg), the adrenocorticotropic hormone dose-response curve was identical to that of controls, but the curves for isoproterenol, norepinephrine and epinephrine were flattened and shifted to the right. A down-regulation of beta-1 and beta-2 adrenoceptors was evidenced by a reduction in [3H]CGP 12177 high-affinity binding sites. In treated hamsters, compared to the controls, there was no change in the lipolytic response to the beta adrenergic agonists. Other protocols of chronic exposure to norepinephrine (e.g., daily injections) at different doses were also unable to reduce the beta-lipolytic effect in the hamster fat cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Drop in the "atypical" beta-adrenergic response and modification of the beta/alpha 2-adrenoceptor balance in fat cells from aging rabbits.

Previous studies have shown a strong reduction of catecholamine-induced lipolysis in perirenal white fat cells in aging rabbits. The molecular basis of this observation was explored on scapular and perirenal adipocytes from 45 and 300- to 500-day-old rabbits. ACTH and forskolin were used to define the maximal lipolytic potencies of the adipocytes. beta-Adrenergic responsiveness was explored with isoproterenol and specific agonists of the "atypical" beta-adrenoceptor (beta-AR) (BRL37344 and (+/-)CGP12177); beta 1/beta 2-ARs were identified with [125I]cyanopindolol. alpha 2-adrenergic responses were evaluated with the full alpha 2-agonist, UK14304. The alpha 2 AR number was determined with the alpha 2-antagonist radioligand [3H]RX821002. Whatever the fat deposit, the relative order of lipolytic potency of the beta-agonists was: isoproterenol greater than BRL37344 greater than (+/-)CGP12177. As previously reported for catecholamines, the maximal lipolytic response initiated by isoproternol decreased with aging; the stronger reduction was observed in perirenal adipocytes compared to subscapular adipocytes. The most striking observation concerns the parallel and complete disappearance of the lipolytic responses induced by the atypical beta-agonists (BRL37344 and (+/-)CGP12177) and the preservation of a residual action of isoproterenol (30% of that described in young animals) which was attributed to the stimulation of beta 1/beta 2-ARs. The number of beta 1/beta 2-AR binding sites was practically equivalent whatever the fat deposite and the age of the animals. alpha 2-Adrenergic responsiveness and alpha 2-adrenergic receptor number were increased with aging in the various deposits but the stronger changes were observed in the perirenal adipocytes where epineprine initiated a biphasic effect on lipolysis (antilipolytic and then lipolytic). To conclude, the reduction of catecholamine-induced lipolysis observed in the rabbit fat cells with aging can be explained by changes in the atypical beta-AR/alpha 2-AR balance. First, a loss of responsiveness to the atypical beta-adrenergic agents was observed (it is impossible for the moment to distinguish between the loss of atypical beta-AR binding sites and their putative uncoupling from the adenylate cyclase system) whereas beta 1/beta 2-AR-mediated responses were maintained. Second, an increment of alpha 2-adrenergic responsiveness and of the alpha 2-AR binding sites accompanied aging and fattening. In the absence of, or after a strong reduction of the atypical beta-AR component of the lipolytic response in fat cells of aged rabbits, epinephrine exerts a biphasic effect on lipolysis, demonstrating the changes occurring in the atypical beta-AR/alpha 2-AR balance.(ABSTRACT TRUNCATED AT 400 WORDS)

Late expression of alpha 2-adrenergic-mediated antilipolysis during differentiation of hamster preadipocytes.

In order to study the ontogenesis of the beta- and alpha 2-adrenergic control of lipolysis during the adipose conversion process, a model based on preadipocytes isolated from the stromal-vascular fraction of hamster adipose tissue was developed. When cultured in an ITT (insulin, transferrin, triiodothyronine) medium supplemented with 2% fetal calf serum, adipose precursors differentiated into adipose-like cells. On 8-day-post-confluent differentiating preadipocytes, the rank order of potency of activation of lipolysis by various beta-adrenergic agonists (BRL37344 greater than norepinephrine = isoproterenol greater than epinephrine greater than fenoterol) was equivalent to that determined in mature adipocytes isolated from adult hamster adipose tissue. On 8-day-post-confluent differentiating preadipocytes, phenylisopropyladenosine (A1-adenosine agonist) and prostaglandin E1 evoked a strong antilipolytic response whereas that evoked by UK 14304 and clonidine (alpha 2-adrenergic agonists) remained undetectable at this step of differentiation. The activity of UK 14304 and clonidine only appeared on 20- to 25-day-post-confluent differentiating preadipocytes. They induced dose-dependent antilipolysis with a maximal effect reaching 80-85% inhibition of adenosine deaminase-stimulated lipolysis. Their action was blocked by increased concentrations of different alpha 2-adrenergic antagonists with the following order of potency, RX 821002 greater than phentolamine much greater than yohimbine. This order of potency was similar to that determined on mature adipocytes isolated from adult hamsters. Both the density of the alpha 2-adrenoceptors, identified with the selective alpha 2-adrenergic radioligand [3H]RX-821002 (19 +/- 1 vs. 30 +/- 1 fmol/mg protein: P less than 0.01) and the amount of Gi proteins identified by pertussis toxin-catalyzed ADP-ribosylation (31 +/- 4 vs. 43 +/- 4% of the amount defined in mature fat cells from adult hamsters: P less than 0.05) were significantly increased between 8 days and 20-25 days after confluence, explaining the late emergence of the alpha 2-adrenergic control of lipolysis during preadipocyte differentiation. In conclusion, the late emergence of the alpha 2-adrenergic control of lipolysis, which is also supported by previous data obtained in vivo that demonstrated the age and/or the fat cell size dependence of alpha 2-adrenoreceptor expression in mature adipocytes, allows the alpha 2-adrenoceptor to be considered as a marker of adipocyte hypertrophy.

Photoperiodic control of adipocyte alpha 2-adrenoceptors in Syrian hamsters: role of testosterone.

The impact of photoperiodic manipulations and testosterone treatments on the adipocyte alpha 2-adrenergic (alpha 2-AR), beta-adrenergic (beta-AR), and A1-adenosine (A1-R) responsiveness, was explored in male Syrian hamsters (Mesocricetus auratus). Moreover, binding studies were performed with appropriate alpha 2-AR, beta-AR, and A1-R radioligands to study receptor changes. Animals were kept for 12 weeks in long day photoperiod (LD: 16 h light (L)-8 h dark (D)), in short day photoperiod (SD: 6L-18D), or in short photoperiod with testosterone treatments (1 mg/animal/day sc) 10 days before sacrifice (SD+T). The antilipolytic effect of the full alpha 2-AR agonist UK14304 and the specific binding of the alpha 2-AR radioligands [3H] RX821002 (antagonist) and [3H]UK14304 were significantly reduced in SD hamsters compared with LD hamsters. The alpha 2-site number and alpha 2-AR responsiveness were completely restored in SD+T hamsters. Whatever the experimental conditions the adipocyte beta-AR receptivity (lipolytic response of isoproterenol and [125I]cyanopindolol binding), and the A1-R receptivity (antilipolytic response initiated by (-)phenylisopropyladenosine and [3H]dipropyl-8-cyclopentylxanthine and [3H]phenylisopropyladenosine binding) remained unchanged. Moreover, the kidney and brain alpha 2-AR densities identified with [3H]RX821002 were not significantly different in LD, SD or SD+T hamsters. These results were obtained without any modification of animal weight, white adipose tissue weight, or white fat cell size. We conclude that, in the Syrian hamster, the expression of the adipocyte alpha 2-AR is under the control of the photoperiod by a testosterone-dependent mechanism probably mediated through the hypothalamic-pituitary axis, without any alteration of the animal fat stores.

Discrimination between alpha 2-adrenoceptors and [3H]idazoxan-labelled non-adrenergic sites in rabbit white fat cells.

Previous results indicated that the rabbit could represent a suitable model for investigations on the functional role of alpha 2-adrenoceptors in fat cells, but the characterization of these receptors was not resolved yet. In the present report, imidazoline compounds were used to attempt a better definition of rabbit adipocyte alpha 2-receptivity by means of lipolysis and binding studies. Lipolysis data showed that UK14304 is a full alpha 2-adrenoceptor agonist promoting a strong antilipolysis in rabbit fat cells. Moreover, the methoxy derivative of idazoxan, RX821002, is a more potent antagonist of UK14304-induced antilipolysis than idazoxan or yohimbine. Whereas [3H]yohimbine failed to bind at rabbit adipocyte alpha 2-adrenoceptors, [3H]UK14304 and [3H]RX821002 are valuable tools to study this receptor. Analysis of binding data demonstrated that [3H]UK14304 labels the high-affinity-state receptor while [3H]RX821002 binds to the whole alpha 2-adrenergic population. Inhibition studies of [3H]RX821002 and [3H]UK14304 binding by various compounds confirmed the alpha 2-adrenergic nature of the sites labelled by both radioligands. The other alpha 2-adrenoceptor radioligand, [3H]idazoxan, labelled binding sites which are insensitive to catecholamines. Competition studies of [3H]idazoxan binding with imidazoline derivatives revealed structure-activity relationships different from those of alpha 2-adrenoceptors. The most striking observation is that substitutions in the 2-position of idazoxan markedly reduce the affinity for the non-adrenergic sites, whereas the alpha 2-potency is increased or unchanged.

Lipolytic action of a new alpha-2 adrenergic antagonist of the piperazinopyrimidine family: RP 55462.

Recent investigations have demonstrated the in vitro lipid mobilizing effects induced by alpha-2 adrenergic antagonist administration and have focused attention on the putative therapeutic interest of such compounds in the treatment of obesity as adjuvants in caloric restriction programs. We studied the impact of RP 55462 [6-chloro-4-(isopropylamino)-5-(methyl)-2, piperazinopyrimidine], a new alpha-2 adrenergic antagonist compound of the piperazinopyrimidine family, on fat cell function. The alpha-2-blocking properties of this agent, which had been defined initially on the brain were confirmed on adipocytes. RP 55462 competed with [3H]yohimbine binding sites on human fat cell membranes and inhibited the antilipolytic action of alpha-2-agonist compounds (UK 14304, clonidine and epinephrine) in human and hamster fat cells. It was also noticed that RP 55462 alone was able to activate lipolysis in isolated fat cells from various species (man, rat, hamster and dog). Moreover, the lipolytic response induced by isoproterenol or synacthene was largely amplified in the presence of RP 55462 in rat fat cells which are the least alpha-2 adrenergic responsive tested. RP-55462-dependent stimulation of lipolysis was not affected by the presence of other alpha-2 adrenergic antagonists (idazoxan, yohimbine or phentolamine). Intravenous administration of RP 55462 in alert dogs promoted an increment in plasma nonesterified acid concentrations reflecting its lipid mobilizing action. In summary this study focuses attention on a new alpha-2-antagonist compound which exhibits an in vivo lipid mobilizing action which could be attributable to its alpha-2 adrenergic antagonist properties. Inasmuch as the lipolytic activity of RP 55462, revealed in in vitro studies, seems to be independent from its alpha-2 adrenolytic properties; further studies are required to define the mechanism of such a lipolytic effect as well as its possible involvement in in vivo conditions.

Mechanism of lipolytic action of a new alpha-2 adrenergic antagonist of the piperazinopyrimidine family: RP 55462.

The in vivo lipid mobilizing effect of alpha-2 adrenergic antagonist has been demonstrated previously. This has attracted attention to the putative interest of such compounds in a lipid-mobilizing strategy. RP 55462 [6-chloro-4-(isopropylamino)-5-(methyl)-2 piperazinopyrimidine], a piperazinopyrimidine derivative, has already been shown to exert alpha-2 adrenergic antagonist actions on fat cell function in vitro. Moreover, RP 55462 exhibits a direct in vitro lipolytic action which is independent of its alpha-2-blocking potency. When administered i.v. RP 55462 is also able to induce an increment in plasma nonesterified levels in dogs. The mechanism of action of RP 55462 was studied and the nature of its lipomobilizing effect was explored. RP 55642-dependent lipolysis was not affected by beta adrenergic blockers on rat fat cells and RP 55462 had no direct effect on adenylylcyclase activity on fat cell membranes. Moreover, RP 55462 did not compete with [3H]phenyl isopropyl adenosine binding (A1-adenosine receptor agonist) on fat cell membranes. In fact, RP 55462 inhibited, in a dose-dependent manner, the cyclic AMP (cAMP)-dependent phosphodiesterase (PDE) activity in rat adipose tissue. Several derivatives with the piperazinopyrimidine structure also inhibited cAMP-dependent PDE activity and exerted lipolytic effects. A short structure-activity study was performed with various derivatives. In dogs, by contrast with yohimbine, the in vivo lipid mobilizing effect of RP 55462 was not abolished by pretreatment with propranolol, and lasted longer. It is concluded that the in vivo lipolytic activity of RP 55462 is connected with its ability to inhibit cAMP-dependent PDE activity; a property of several piperazinopyrimidine derivatives. The lipid mobilizing effect induced in vivo by RP 55462 results from a combination of its alpha-2 adrenergic antagonist properties and its direct lipolytic action mediated by cAMP-dependent PDE inhibiting effects.