Adrenaline but not noradrenaline is a determinant of exercise-induced lipid mobilization in human subcutaneous adipose tissue.

The relative contribution of noradrenaline (norepinephrine) and adrenaline (epinephrine) in the control of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise was evaluated in men treated with a somatostatin analogue, octreotide. Eight lean and eight obese young men matched for age and physical fitness performed 60 min exercise bouts at 50% of their maximal oxygen consumption on two occasions: (1) during i.v. infusion of octreotide, and (2) during placebo infusion. Lipolysis and local blood flow changes in SCAT were evaluated using in situ microdialysis. Infusion of octreotide suppressed plasma insulin and growth hormone levels at rest and during exercise. It blocked the exercise-induced increase in plasma adrenaline while that of noradrenaline was unchanged. Plasma natriuretic peptides (NPs) level was higher at rest and during exercise under octreotide infusion in lean men. Under placebo, no difference was found in the exercise-induced increase in glycerol between the probe perfused with Ringer solution alone and that with phentolamine (an alpha-adrenergic receptor antagonist) in lean subjects while a greater increase in glycerol was observed in the obese subjects. Under placebo, propranolol infusion in the probe containing phentolamine reduced by about 45% exercise-induced glycerol release; this effect was fully suppressed under octreotide infusion while noradrenaline was still elevated and exercise-induced lipid mobilization maintained in both lean and obese individuals. In conclusion, blockade of beta-adrenergic receptors during exercise performed during infusion of octreotide (blocking the exercise-induced rise in adrenaline but not that of noradrenaline) does not alter the exercise-induced lipolysis. This suggests that adrenaline is the main adrenergic agent contributing to exercise-induced lipolysis in SCAT. Moreover, it is the combined action of insulin suppression and NPs release which explains the lipolytic response which remains under octreotide after full local blockade of fat cell adrenergic receptors. For the moment, it is unknown if results apply specifically to SCAT and exercise only or if conclusions could be extended to all forms of lipolysis in humans.

Real-time RT-PCR for the detection of beta-adrenoceptor messenger RNAs in small human endomyocardial biopsies.

Quantification of mRNAs from extremely small human samples remains a challenge. Requiring minimal amounts of tissue and no post-reaction manipulation, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is an attractive method to quantitatively assess the expression of rare mRNAs. We evaluated the applicability of the technique on RNA extracted from human endomyocardial biopsies and isolated cardiomyocytes, and compared the technique to the RT-competitive PCR approach. Primers and probes were designed to amplify the three subtypes of human beta -adrenoceptors (beta1-, beta2- and beta3 AR), as well as reference genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hypoxanthine-guanine phosphoribosyltransferase (HPRT), and the oncogene ABL by real-time RT-PCR. Specific primers and a deleted competitor were synthetized to compare the quantitation of the beta 3 AR mRNA expression by RT-competitive PCR. We validated the technique on human cardiomyocytes either freshly isolated or selectively excised from fixed sections of human myocardium by Laser Capture Microdissection. The standard curves obtained for the cDNA's analysed showed mean slopes comprised between -3.3 and -3.7. Inter- and intra-assay variability of gene quantitation was reflected by mean values of the variance coefficients of Ct of 4.84+/-1.13% and 2.73+/-0.39% or 3.32+/-1.03% and 2.21+/-0.24% (corresponding to percent variances of copy numbers of 83.07+/-12.72% and 34.45+/-9.03% or 47.40+/-8.59% and 23.83+/-3.16%) for human beta3 AR and GAPDH genes, respectively. The expression of GAPDH, HPRT and ABL mRNA was characterized by a very low dispersion of individual values across cardiac pathologies, suggesting that these genes may be used as reference genes in quantitative PCR studies. Finally, we applied the technique to detect rare mRNAs, such as beta -AR mRNAs, from small human endomyocardial biopsies and even isolated cardiomyocytes. Real-time RT-PCR is appropriate to quantitate rare messenger RNAs, including in extremely small human tissue samples. This method appears very promising for futures studies of gene expression in several pathophysiological conditions, including heart failure.

Triiodothyronine-mediated up-regulation of UCP2 and UCP3 mRNA expression in human skeletal muscle without coordinated induction of mitochondrial respiratory chain genes.

Triiodothyronine (T3) increases mitochondrial respiration and promotes the uncoupling between oxygen consumption and ATP synthesis. T3 effect is mediated partly through transcriptional control of genes encoding mitochondrial proteins. We determined the effect of T3 on mRNA levels of uncoupling proteins (UCP) and proteins involved in the biogenesis of the respiratory chain in human skeletal muscle and on UCP2 mRNA expression in adipose tissue. Ten young, healthy males received 75 to 100 5g of T3 per day for 14 days. The increase in plasma-free T3 levels was associated with an increase of resting metabolic rate and a decrease of respiratory quotient. In skeletal muscle, treatment with T3 induced a twofold increase of both UCP2 and UCP3 mRNA levels (p c oxidase subunits 2 and 4, nuclear respiratory factor 1, mitochondrial transcription factor A, and the co-activator PGC1 did not change during the treatment. In adipose tissue, UCP2 mRNA levels increased threefold. The direct effect of T3 on skeletal muscle an d adipose tissue UCP2 and UCP3 mRNA expression was demonstrated in vitro in human primary cultures. Our data show that T3 induces UCP2 and UCP3 mRNA expression in humans. In skeletal muscle, UCP regulation by T3 is not associated with the transcriptional regulation of respiratory chain proteins.

Lack of skeletal muscle uncoupling protein 2 and 3 mRNA induction during fasting in type-2 diabetic subjects.

Skeletal muscle uncoupling protein 2 and 3 (UCP-2 and UCP-3) mRNA levels are increased during calorie restriction in lean and nondiabetic obese subjects. In this work, we have investigated the effect of a 5-day hypocaloric diet (1,045 kJ/day) on UCP-2 and UCP-3 gene expression in the skeletal muscle of type-2 diabetic obese patients. Before the diet, UCP-2 and UCP-3 mRNA levels were more abundant in diabetic than in nondiabetic subjects. The long (UCP-3(L)) and short (UCP-3(S)) forms of UCP-3 transcripts were expressed at similar levels in nondiabetic subjects, but UCP-3(S) transcripts were twofold more abundant than UCP-3(L) transcripts in the muscle of diabetic patients. Calorie restriction induced a two- to threefold increase in UCP-2 and UCP-3 mRNA levels in nondiabetic patients. No change was observed in type-2 diabetic patients. Variations in plasma nonesterified fatty acid level were positively correlated with changes in skeletal muscle UCP-3(L) (r = 0.6, P < 0.05) and adipose tissue hormone-sensitive lipase (r = 0.9, P < 0.001) mRNA levels. Lack of increase in plasma nonesterified fatty acid level and in hormone-sensitive lipase upregulation in diabetic patients during the diet strengthens the hypothesis that fatty acids are associated with the upregulation of uncoupling proteins during calorie restriction.

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) expression in adipose tissue and skeletal muscle in humans.

Uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) are mitochondrial proteins that may play a role in the control of energy expenditure by uncoupling respiration from ATP synthesis. The present review focuses on data obtained in humans. UCP2 is widely expressed in the body, whereas UCP3 expression is restricted to skeletal muscle. Positive correlations have been reported between UCP2 mRNA concentrations in adipose tissue, UCP3 mRNA concentrations in skeletal muscle, and components of the metabolic rate. Fasting induces an up-regulation of UCP2 and UCP3 mRNA expression. In vivo and in vitro studies suggest that fatty acids could modulate uncoupling protein gene expression. The putative relationship between obesity, energy expenditure and uncoupling protein expression, and the unexpected rise in UCP2 and UCP3 mRNA concentrations during short-term fasting, are discussed in view of the recent data obtained in rodents and cell lines.

Cloning and mRNA tissue distribution of human PPARgamma coactivator-1.

OBJECTIVES: To determine human PPARgamma coactivator-1 (PGC-1) amino acid sequence and to study PGC-1 mRNA tissue distribution. PGC-1 is a novel transcriptional coactivator of nuclear receptors that may play a role in the control of thermogenesis. SUBJECTS: Subcutaneous adipose tissue was obtained from six obese and five lean male subjects. Vastus lateralis skeletal muscle was obtained from seven lean and six obese subjects undergoing a 5-day severe calorie restriction. Other tissue biopsies were from nonobese nondiabetic subjects. METHODS: Human PGC-1 was cloned from a skeletal muscle cDNA library. A reverse transcription-competitive polymerase chain reaction assay was developed to determine PGC-1 mRNA levels in human tissues. RESULTS: The human amino acid sequence showed 95% identity with mouse PGC-1. PGC-1 mRNA was expressed at very low levels in the small and large intestines and white adipose tissue. Heart, kidney, liver and skeletal muscle showed higher mRNA levels. The degree of obesity did not affect PGC-1 mRNA levels in adipose tissue while lean subjects expressed more PGC-1 mRNA than obese subjects in skeletal muscle. A 5-day severe calorie restriction induced PGC-1 mRNA expression in skeletal muscle of obese but not of lean subjects. CONCLUSION: PGC-1 shows a restricted tissue expression that suggests a tissue-specific role in the control of gene transcription and possible interaction with various members of the PPAR family. The lower expression of skeletal muscle PGC-1 in obesity could contribute to an alteration of mitochondrial gene expression.

mRNA expression of the long and short forms of uncoupling protein-3 in obese and lean humans.

Uncoupling protein-3 (UCP3) is a mitochondrial protein expressed in skeletal muscle, an important site of thermogenesis in humans. By uncoupling respiration from ATP synthesis, UCP3 might be involved in the control of energy expenditure. Two transcripts encoding long (UCP3L) and short (UCP3S) form are generated from the human UCP3 gene. UCP3S is predicted to encode a protein which lacks the C-terminus of UCP3L, a region which contains motifs critical for uncoupling activity. We have investigated the regulation of UCP3L and UCP3S mRNAs in lean and obese humans. A specific reverse transcription-competitive polymerase chain reaction assay was developed to separately quantify the two mRNAs. Each transcript represents half of total UCP3 mRNA in 16 vastus lateralis muscle samples. The amounts of UCP3L and UCP3S mRNAs did not differ between obese and lean subjects. The effect of fasting was studied in six lean and seven obese subjects maintained on a hypocaloric diet (1045 kJ/d) for 5 days. Calorie restriction results in an approximately threefold increase of UCP3L and UCP3S mRNA levels. The induction was similar in lean and obese subjects. The data suggest that there is no major alteration of UCP3 gene expression and regulation at the level of transcription and alternative splicing in skeletal muscle of obese subjects.

Uncoupling protein-2 messenger ribonucleic acid expression during very-low-calorie diet in obese premenopausal women.

Uncoupling protein-2 (UCP2) is a mitochondrial protein expressed in a wide range of human tissues. By uncoupling respiration from ATP synthesis, UCP2 might be involved in the control of energy expenditure. We have investigated UCP2 gene expression in human adipose tissue. In eight subjects, we found a positive correlation (r = 0.91, P < 0.002) between subcutaneous and visceral fat depots UCP2 messenger RNA (mRNA) levels, suggesting that UCP2 mRNA level in subcutaneous adipose tissue is a good index of UCP2 gene expression in whole body adipose tissues. The effect of a 25-day very-low-calorie diet un UCP2 mRNA level and resting metabolic rate was investigated in eight obese premenopausal women. There was no difference in UCP2 mRNA levels before and during the diet. After 25 days of hypocaloric diet, a positive correlation was found between adipose tissue UCP2 mRNA level and resting metabolic rate adjusted for lean body mass (r = 0.82, P < 0.01). These results show that very-low-calorie diet, unlike short-term fasting, is not associated with an induction in UCP2 mRNA expression, and that adipose tissue UCP2 mRNA levels may be related to variations in resting energy expenditure in humans.

High expression of leptin by human bone marrow adipocytes in primary culture.

Adipocytes participate in the microenvironment of the bone marrow (BM), but their exact role remains to be determined. It has recently been shown that leptin, a hormone secreted from extramedullary adipocytes, could be involved in hematopoiesis. Therefore we have developed a primary culture system of human BM adipocytes to characterize their differentiation and determine whether leptin is also secreted from these adipocytes. BM cells were cultured with fetal calf and horse sera. In the presence of dexamethasone, cells with vesicles containing lipids appeared within 15 days. They expressed glycerol phosphate dehydrogenase activity and a lipolytic activity in response to isoproterenol, but expressed neither the adrenergic beta3 receptor nor the mitochondrial uncoupling protein UCP1. The addition of insulin alone to the culture media did not promote adipocyte differentiation. Leptin was expressed and secreted at high levels during adipocyte differentiation. Acute exposure of differentiated adipocytes to insulin had little effect on leptin expression whereas forskolin strongly inhibited it. These results show that although human BM adipocytes differ from extramedullary adipose tissues in their sensitivity to different effectors, they are a secondary source of leptin production. They suggest that BM adipocytes could contribute to hematopoiesis via the secretion of leptin in the vicinity of hematopoietic stem cells.

Kupffer cells are a dominant site of uncoupling protein 2 expression in rat liver.

The mechanisms underlying thermogenesis in liver are not well understood. They may involve proteins related to the mitochondrial uncoupling protein (UCP1) of brown adipocytes. In this paper, it is demonstrated that UCP1 is not expressed in any liver cell type of rat while UCP2, a recently cloned homologue of UCP1, is expressed at a very high level in Kupffer cells but not in hepatocytes. This high level of expression of UCP2 in Kupffer cells allowed cross immunoreactivity with antibodies directed against UCP1. This cross reactivity was confirmed by the detection of UCP2 with anti-UCP1 antibody, in western blotting analysis of transfected yeasts expressing rat UCP2. The high level expression of UCP2 in Kupffer cells suggests a particular function of UCP2 in macrophages.

Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans.

Uncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.

Characterization of a melatonin binding site in Siberian hamster brown adipose tissue.

Melatonin has been shown, in various rodent species, to mediate photoperiodic effects on body weight and, consequently, fat mass. Pharmacological investigations indicated that the brown adipose tissue of Siberian hamsters possesses a melatonin binding site with a dissociation constant of 570+/-300 pM and a density of 3.2+/-1.8 fmol/mg protein. This binding site can also be detected on mature brown adipocyte membranes. The rank order of potency of a variety of drugs to displace 2-[125I]iodomelatonin from binding sites on Siberian hamster brown adipose tissue was as follows: 2-iodomelatonin > melatonin = prazosin > GR135531 (5-methoxycarbonylamino-N-acetyltryptamine) > N-acetylserotonin > 6-chloromelatonin > S20304 (N-(2-(1-naphthyl)ethyl)cyclobutanecarboxamide) >> methoxamine, phenylephrine, serotonin. Mel(1a) mRNA was not detected by RT-PCR (reverse transcription-polymerase chain reaction) in brown adipose tissue. Melatonin had no effect on either basal or stimulated lipolysis. Moreover, melatonin did not modify intracellular cAMP accumulation or inositol phosphate content. Together, these results suggest that the melatonin binding site characterized in brown adipose tissue is clearly different from the Mel(1) cloned subtype and has some features different from those of the Mel2 subtype.

Evidence for numerous brown adipocytes lacking functional beta 3-adrenoceptors in fat pads from nonhuman primates.

Brown adipose tissue (BAT) is involved in the control of energy balance and has been demonstrated to be activated through beta 3-adrenoceptor (beta 3-AR) occupation in rodents. The ability to specifically activate energy expenditure via this receptor is of great interest for the treatment of obesity. Nevertheless, the extent of BAT and the presence of a functional beta 3-AR in humans are now debated, and this situation is difficult to clarify for evident practical and ethical reasons. We investigated the occurrence of brown adipocytes in fat deposits of prepubertal baboons using antibodies raised against uncoupling protein (UCP) in Western blotting and immunocytology experiments. UCP was detected in all types of fat pads studied and was revealed in multilocular cells. Pericardiac and axillary adipose tissues displayed large amounts of UCP and can be assimilated to typical BAT. Most of the other pads looked like white adipose tissue, but exhibited areas with clusters of brown adipocytes and, thus, can be assimilated to the convertible adipose tissue as previously described in rodents. The presence of beta 3-ARs was evaluated by both beta 2-agonist-stimulated lipolysis and messenger ribonucleic acid (mRNA) expression studies. There was no significant lipolytic effect of any of the beta 3-AR agonists tested (SR 58611A, BRL 37344, CGP 12177, or CL 316243) in either white or brown tissues. PCR analysis demonstrated that beta 3-AR mRNA expression is not related to the UCP content of fat pads and that beta 3-AR expression is low. This study demonstrates the presence of great proportions of brown adipocytes in adipose tissue and the heterogeneity of the fat pads in baboons. The lack of a metabolic effect of beta 3-agonists combined with the weak expression of beta 3-AR mRNAs raise the question of the role of beta 3-ARs in adipose tissues of primates.

Coupling of inhibitory receptors with Gi-proteins in hamster adipocytes: comparison between adenosine A1 receptor and alpha 2-adrenoceptor.

Adenosine A1 receptors and alpha 2-adrenoceptors are both potent inhibitors of adipocyte lipolysis when activated by their agonists. The aim of this work was to compare the coupling of these receptors to the Gi-proteins in hamster adipocytes. The adenosine A1 receptor was characterized with the antagonist [3H]dipropyl-cyclopentyl-xanthine ([3H]DPCPX) and the agonist [3H](-)-phenylisopropyladenosine ([3H]PIA). It was demonstrated by [32P]ADP-ribosylation with pertussis toxin and immunoblotting that Gi1, Gi2 and Gi3 are expressed in hamster adipocytes. Partial ADP-ribosylation of Gi-proteins by pertussis toxin, acting on the intact cells or on the adipocyte membranes, demonstrated that the adenosine A1 receptor was less sensitive to the disappearance of functional Gi-proteins than the alpha 2-adrenoceptor. These results are in accordance with the weak sensitivity of the binding of the agonist [3H]PIA to guanine nucleotides and seem to confirm that the adenosine A1 receptor is strongly and differently coupled than the alpha 2-adrenoceptor to the Gi-proteins in hamster adipocyte membranes.

Distribution of PYY receptors in human fat cells: an antilipolytic system alongside the alpha 2-adrenergic system.

The antilipolytic effect of peptide YY (PYY) and neuropeptide Y has recently been shown in human adipocytes. PYY receptors were investigated in three human adipose deposits. A greater number of 125I-labeled PYY binding sites was found in femoral adipocyte membranes (maximal binding = 40 +/- 4 fmol/mg protein; dissociation constant = 0.3 +/- 0.1 nM) when compared with mammary and pericolonic adipose tissue. PYY receptors, like alpha 2-adrenergic sites, were largely expressed in femoral fat cells. Such a distribution was not specific either to inhibitory or to stimulating adenylyl cyclase systems since adenosine A1 and beta-adrenergic receptors were more numerous in pericolonic adipocytes. On isolated adipocytes, PYY (10(-7) M) inhibited lipolysis by 58 +/- 2% in femoral and 14 +/- 4% in pericolonic fat cells; epinephrine had the following similar response: 62 +/- 5 and 26 +/- 8%, respectively. A close relationship between the number of alpha 2-sites and PYY sites and the antilipolytic effects initiated by PYY and an alpha 2-agonist was observed. No significant differences were noted in the amount of Gi proteins in femoral and pericolonic adipocyte membranes.

Coupling of the alpha 2-adrenergic receptor to the inhibitory G-protein Gi and adenylate cyclase in HT29 cells.

Previous studies have established that the human colon carcinoma cell line HT29 expresses an alpha 2-adrenergic receptor of the alpha 2A subtype, which is negatively coupled to adenylate cyclase. The purpose of the present study was to examine the mechanisms of alpha 2-adrenergic signal transduction in these cells. [32P]ADP-ribosylation with pertussis toxin and immunoblots using antibodies specific for the Gi alpha-subunits indicated that two distinct Gi-proteins (Gi2 and Gi3) were present in HT29-cell membranes. Treatment of intact cells with pertussis toxin resulted in a time-dependent decrease in the amount of [32P]ADP-ribosylatable Gi2 and Gi3, which coincided with a diminution in the number of alpha 2-adrenergic receptors in high-affinity state for agonists and with a progressive loss of ability of UK14304 to inhibit forskolin-stimulated accumulation of cyclic AMP. When membranes were [32P]ADP-ribosylated with cholera toxin in the absence of exogenous added guanine nucleotides, radioactivity was incorporated into a 45 kDa polypeptide representing Gs, as well as into 40-41 kDa polypeptides corresponding to Gi3 and Gi2. The amount of radioactivity incorporated into the two GiS under basal conditions was decreased by addition of the alpha 2-antagonist RX821002. It was not significantly affected by addition of clonidine (partial alpha 2-agonist), but was doubled by the addition of UK14304 (full alpha 2-agonist). This effect was blocked by RX821002. Study of adenylate cyclase activity indicated that preincubation of HT29 membranes with the antibody AS/7 (anti-alpha i1/alpha i2), but not with the antibody EC/2 (anti-alpha i3), attenuated the inhibitory effect of UK14304 on forskolin-stimulated adenylate cyclase. These data demonstrate that the alpha 2A-adrenergic receptor is coupled to both Gi2 and Gi3, and identify Gi2 as the major mediator of inhibition of adenylate cyclase in HT29 cells.

A1 adenosine receptors in the human fat cell: tissue distribution and regulation of radioligand binding.

Identification of adenosine A1 receptor binding sites was performed using the antagonist, [3H]1,3-dipropyl-8-cyclopentyl-xanthine ([3H]DPCPX), and the agonist [3H](-)-N6-R-phenylisopropyladenosine ([3H](-)-PIA) on human adipocyte membranes from different anatomical localizations (i.e. abdominal, femoral and omental fat deposits). Despite the strong antilipolytic effect initiated by various adenosine analogs, the human adipocytes were poorly equipped in adenosine A1 receptors whatever the anatomical location (Bmax less than or equal to 95 fmol/mg of protein for the antagonist [3H]DPCPX and less than or equal to 72 fmol/mg for the agonist [3H](-)-PIA). There was no marked difference between the three fat deposits in terms of maximal binding for both radioligands. Saturation and competition experiments showed that the proportion of receptors in the high-affinity state for the agonists was very high (70-91%), but only 33-44% of them were guanine nucleotide-sensitive. Moreover the guanine nucleotides were shown to enhance the specific binding of the antagonist [3H]DPCPX by decreasing its KD value. These binding properties are strongly different from those of another Gi-coupled receptor on the human fat cell, the alpha 2A-adrenoceptor, and indicates that the adenosine A1 receptor and the alpha 2-adrenoceptor could be differentially coupled with Gi proteins in the human fat cell.

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.

Non-adrenergic sites for imidazolines are not directly involved in the alpha 2-adrenergic antilipolytic effect of UK 14304 in rat adipocytes.

The binding of the alpha 2-agonist [3H]UK 14304 on Wistar rat adipocyte membranes was separated in two distinct components: one was displaceable by adrenaline or other alpha 2-adrenergic agents and possessed the characteristics of alpha 2-adrenoceptors while the other, non-adrenergic in nature, was only recognized by some imidazoline derivatives [3H]idazoxan binding shared the same characteristics. The non-adrenergic sites labeled by both radioligands are similar to those described for [3H]idazoxan on other tissues such as brain cortex, smooth muscle and kidney. Even though they were about 10-fold more numerous than the true alpha 2-adrenoceptors, the non-adrenergic binding sites were not directly involved in the antilipolytic action of UK 14304 since alpha 2-antagonists devoid of interaction with these sites (yohimbine, phentolamine) totally blocked the UK 14304 effect. However, the existence of such a type of site impairs direct quantification of alpha 2-adrenoceptors in rat adipocytes. The use of [3H]RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)imidazoline) allowed an accurate quantification of rat adipocyte alpha 2-adrenoceptors (Bmax = 35 +/- 2 fmol/mg protein, Kd = 2.6 +/- 0.6 nM) since it did not interact with non-adrenergic binding sites and exhibited the highest alpha 2-blocking properties among the various alpha 2-antagonists tested. [3H]RX 821002 binding analysis revealed that alpha 2-adrenoceptors are, on rat adipocytes; (i) less numerous than in other species well known for their alpha 2-adrenergic inhibitory regulation of lipolysis (human, hamster, rabbit); (ii) slightly different in nature from the receptors of these species since they had weaker affinity for clonidine and yohimbine; and however (iii) not of the typical alpha 2-B subtype since the affinity of prazosin was lower than that of oxymetazoline in displacing [3H]RX 821002 or [3H]yohimbine binding.

New tools for human fat cell alpha-2A adrenoceptor characterization. Identification on membranes and on intact cells using the new antagonist [3H]RX821002.

The pharmacology of the alpha-2 adrenoceptor of the human adipocyte was improved by using some new alpha-2 antagonists from different chemical families (imidazolines, benzazepines and benzofuroquinolizines) in biological and binding assays. Moreover, investigations were also carried out to define the binding properties of a new imidazolinic antagonist, RX821002 [2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline], which could be a potential radioligand. [3H]RX821002 binding was very rapid and reversible. Saturation isotherms indicated that [3H]RX821002 labeled, with high affinity, a homogeneous population of noninteracting binding sites with a mean Kd of 0.98 +/- 0.05 nM (n = 6). The binding of [3H]RX821002 on the human fat cell alpha-2 adrenoceptor displayed a specificity which is strictly similar to that obtained with [3H]rauwolscine and which is classical for an alpha-2 A adrenoceptor. The binding parameters of [3H]RX821002 were compared with those obtained with the classical alpha-2 antagonist [3H]yohimbine. Analysis of the data indicate: 1) that [3H]RX821002 exhibited higher affinity; 2) that the nonspecific binding of [3H]RX821002 was very low; 3) that the total number of sites (maximum binding values) defined with [3H]RX821002 was significantly higher than that defined with [3H]yohimbine. This difference was not due to a specific preferential labeling of one of the two affinity states of the receptor, but suggested that [3H]yohimbine does not label the whole receptor population; 4) that [3H]RX821002 specific binding was less sensitive to magnesium chloride and GTP than [3H]yohimbine binding; and 5) that [3H]RX821002 can be used suitably for identification of alpha-2 adrenoceptors on the intact adipocyte.(ABSTRACT TRUNCATED AT 250 WORDS)