Lack of alpha(2)-adrenergic antilipolytic effect during exercise in subcutaneous adipose tissue of trained men.

The aim of this study was to investigate the involvement of the antilipolytic alpha(2)-adrenergic receptor pathway in the regulation of lipolysis during exercise in subcutaneous abdominal adipose tissue (SCAAT). Seven trained men and 15 untrained men were studied. With the use of microdialysis, the extracellular glycerol concentration was measured in SCAAT at rest and during 60 min of exercise at 50% of maximal oxygen consumption. One microdialysis probe was perfused with Ringer solution; the other was supplemented with phentolamine (alpha(2)-adrenergic receptor antagonist). No differences in baseline extracellular or plasma glycerol concentrations were found between the two groups. The exercise-induced extracellular and plasma glycerol increase was higher in trained compared with untrained subjects (P < 0.05). Addition of phentolamine to the perfusate enhanced the exercise-induced response of extracellular glycerol in untrained subjects but not in trained subjects. The exercise-induced increase in plasma norepinephrine and epinephrine concentrations and the decrease in plasma insulin were not different in the two groups. These in vivo findings demonstrate higher exercise-induced lipolysis in trained compared with untrained subjects and show that, in trained subjects, the alpha(2)-mediated antilipolytic action is not involved in the regulation of lipolysis in SCAAT during exercise.     

Activation of alpha(2)-adrenergic receptors impairs exercise-induced lipolysis in SCAT of obese subjects

With the use of the microdialysis method, exercise-induced lipolysis was investigated in subcutaneous adipose tissue (SCAT) in obese subjects and compared with lean ones, and the effect of blockade of alpha(2)-adrenergic receptors (ARs) on lipolysis during exercise was explored. Changes in extracellular glycerol concentrations and blood flow were measured in SCAT in a control microdialysis probe at rest and during 60-min exercise bouts (50% of heart rate reserve) and in a probe supplemented with the alpha(2)-AR antagonist phentolamine. At rest and during exercise, plasma norepinephrine and epinephrine concentrations were not different in obese compared with lean men. In the basal state, plasma and extracellular glycerol concentrations were higher, whereas blood flow was lower in SCAT of obese subjects. During exercise, the increase of plasma glycerol was higher in obese subjects (115 +/- 35 vs. 65 +/- 21 micromol/l). Oppositely, the exercise-induced increase in extracellular glycerol concentrations in SCAT was five- to sixfold lower in obese than in lean subjects (50 +/- 14 vs. 318 +/- 53 micromol/l). The exercise-induced increase in extracellular glycerol concentration was not significantly modified by phentolamine infusion in lean subjects but was strongly enhanced in the obese subjects and reached the concentrations found in lean sujects (297 +/- 46 micromol/l). These findings demonstrate that the physiological stimulation of SCAT adipocyte alpha(2)-ARs during exercice-induced sympathetic nervous system activation contributes to the blunted lipolysis noted in obese men.     

Expression of human alpha 2-adrenergic receptors in adipose tissue of beta 3-adrenergic receptor-deficient mice promotes diet-induced obesity

Catecholamines play an important role in controlling white adipose tissue function and development. beta- and alpha 2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte alpha 2/beta-AR balance in obesity, and it has been proposed that increased alpha 2-ARs in adipose tissue with or without decreased beta-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte alpha 2/beta-AR balance was genetically manipulated in mice. Human alpha 2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (-/-) or heterozygous (+/-) for a disrupted beta 3-AR allele. Mice expressing alpha 2-ARs in fat, in the absence of beta 3-ARs (beta 3-AR -/- background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of alpha2-ARs, the absence of beta 3-ARs, and a high fat diet. Of note, obese alpha 2-transgenic beta 3 -/- mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased alpha 2/beta-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (alpha 2 and beta 3-AR) and diet interact to influence fat mass.     

Effect of carbohydrate ingestion on adipose tissue lipolysis during long-lasting exercise in trained men

To study whethersucrose administration acts on lipid mobilization during prolongedexercise, we used subcutaneous abdominal adipose tissue microdialysisin eight well-trained subjects submitted at random to two 100-minexercises (50% maximal aerobic power) on separate days. After 50 minof exercise, the subjects ingested either a sucrose solution (0.75 g/kgbody wt) or water. By using a microdialysis probe, dialysate wasobtained every 10 min from the subjects at rest, during exercise, andduring a 30-min recovery period. During exercise without sucrose,plasma and dialysate glycerol increased significantly. With sucrose,the response was significantly lower for dialysate glycerol(P < 0.05). Plasma free fatty acidlevel was lower after sucrose than after water ingestion(P < 0.05). With water ingestion,plasma catecholamines increased significantly, whereas insulin fell(P < 0.05). With sucrose ingestion,the epinephrine response was blunted, whereas the insulin level wassignificantly increased. In conclusion, the use of adipose tissuemicrodialysis directly supports a lower lipid mobilization duringexercise when sucrose is supplied, which confirms that the availabilityof carbohydrate influences lipid mobilization.

     

Urinary urea nitrogen excretion during the hyperinsulinemic euglycemic clamp in type 1 diabetic patients and healthy subjects

The hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry (IC) is used for estimation of insulin-stimulated substrate utilization. Calculations are based on urinary urea nitrogen excretion (UE), which is influenced by correct urine collection. The aims of our study were to improve the timing of urine collection during the clamp and to test the effect of insulin on UE in patients with type 1 diabetes (DM1; n=11) and healthy subjects (C; n=11). Urine samples were collected (a) over 24 h divided into 3-h periods and (b) before and during two-step clamp (1 and 10 mIU.kg(-1).min(-1); period 1 and period 2) combined with IC. The UE during the clamp was corrected for changes in urea pool size (UEc). There were no significant differences in 24-h UE between C and DM1 and no circadian variation in UE in either group. During the clamp, serum urea decreased significantly in both groups (p<0.01). Therefore, UEc was significantly lower as compared to UE not adjusted for changes in urea pool size both in C (p<0.001) and DM1 (p<0.001). While UE did not change during the clamp, UEc decreased significantly in both groups (p<0.01). UEc during the clamp was significantly higher in DM1 compared to C both in period 1 (p<0.05) and period 2 (p<0.01). The UE over 24 h and UEc during the clamp were statistically different in both C and DM1. We conclude that urine collection performed during the clamp with UE adjusted for changes in urea pool size is the most suitable technique for measuring substrate utilization during the clamp both in DM1 and C. Urine collections during the clamp cannot be replaced either by 24-h sampling (periods I-VII) or by a single 24-h urine collection. Attenuated insulin-induced decrease in UEc in DM1 implicates the impaired insulin effect on proteolysis.     

Ontogenetic studies on adrenergic lipolysis in rat adipocytes from subcutaneous adipose tissue

The in vitro release of free fatty acids from small adipocytes (mean diameter 24.4 +/- 0.4 micrometer; 11-42 micrometer) isolated from subcutaneous adipose tissue of rats aged 5, 14, 21 and 35 days was induced by l-isoprenaline (ISO), l-noradrenaline (NA) or l-adrenaline (ADR). The results were related to a cell count of 10(6) and compared to results in adult rats [both sexes, fed ad libitum, mean weight 180-210 g). The cell yield decreased with development, i.e. it was highest in the youngest rats. In adult rats, increasing catecholamine (CA) concentrations produced pronounced concentration-dependent lipolysis in the potency order ISO greater than NA greater than ADR which is typical of beta 1-adrenergic functions. Qualitatively the same results in 5- and 35-day-old rats were observed, the latter showing greater sensitivity towards CA. Practically no lipolytic response was recorded in 14 and 21-day-old animals, however. Not one of the CA, nor any of the concentrations used, could evoke lipolysis in these animals. The experiments were carried out simultaneously on different age groups, technical errors can therefore be excluded.     

Cross-regulation between G-protein-coupled receptors. Activation of beta 2-adrenergic receptors increases alpha 1-adrenergic receptor mRNA levels.

Stimulation of DDT1 MF-2 vas deferens cells with epinephrine resulted in a time- and dose-dependent loss of alpha 1-adrenergic receptor-specific ligand binding. Regulation of alpha 1-adrenergic receptor mRNA was characterized. In monolayer culture, cells displayed 0.7 +/- 0.05 amol of alpha 1-adrenergic receptor mRNA/microgram of total cellular RNA. Epinephrine, which acts at both alpha 1- and beta 2-adrenergic receptors of DDT1 MF-2 cells, induced a short term (2-8 h) increase (50-70%) in the abundance of alpha 1-adrenergic receptor mRNA. Propranolol, a beta 2-adrenergic receptor antagonist, attenuated the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA but did not affect the decrease in alpha 1-adrenergic receptor-specific ligand binding. Phentolamine, an alpha 1-adrenergic receptor antagonist, did not attenuate the epinephrine-mediated increase in alpha 1-adrenergic receptor mRNA at 4 h but did block the decrease in alpha 1-adrenergic receptor-specific ligand binding. The half-life of the alpha 1-adrenergic receptor mRNA was approximately 7 h in untreated cells as well as in cells challenged with epinephrine. The epinephrine-promoted increase in alpha 1-adrenergic receptor mRNA was found to result from cross-regulation via beta 2-adrenergic receptors. Cholera toxin, forskolin, as well as the cyclic AMP analog CPT cAMP (8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate) increased the alpha 1-adrenergic receptor mRNA at 4 h, as did epinephrine in the presence of alpha 1-antagonists but not in the presence of a beta-adrenergic antagonist. This is the first report of heterologous up-regulation of mRNA levels of adrenergic receptors. Cross-regulation between alpha 1- and beta 2-adrenergic receptor-mediated pathways at 4 h occurs at the level of mRNA whereas later down-regulation of alpha 1-receptor mRNA and binding proceed via agonist activation of alpha 1-adrenergic receptors.     

Regional variation in adipose tissue lipolysis in lean and obese men.

Biopsies of adipose tissue were obtained from two subcutaneous regions (abdominal and femoral) in a sample of 54 men (32 obese and 22 lean subjects). Clonidine-induced antilipolysis in femoral adipose cells was similar in both groups, whereas subcutaneous abdominal adipocytes of obese individuals showed a higher alpha 2-adrenergic response than did subcutaneous abdominal adipose cells from lean subjects. In addition, epinephrine had a biphasic effect in subcutaneous abdominal adipocytes from obese individuals, as it induced antilipolysis at low concentrations, and a net lipolytic response at higher doses. In contrast, the physiological amine promoted lipolysis in subcutaneous abdominal adipose cells of lean subjects. Epinephrine- and clonidine-induced antilipolysis of subcutaneous abdominal adipocytes was positively associated with the level of subcutaneous abdominal fat measured by computed tomography (CT). Finally, men with a high alpha 2-adrenergic response of subcutaneous abdominal fat cells were fatter than those with a low alpha 2-adrenergic component. These results suggest that, in men with a wide range of body fatness, variations in the lipolytic response of subcutaneous abdominal adipose cells to epinephrine appear to involve changes in the functional balance between alpha 2- and beta-adrenoceptors.     

Omental and subcutaneous adipose tissue steroid levels in obese men

We examined plasma and fat tissue sex steroid levels in a sample of 28 men aged 24.8-62.2 years (average BMI value of 46.3 +/- 12.7 kg/m(2)). Abdominal adipose tissue biopsies were obtained during general or obesity surgery. Omental and subcutaneous adipose tissue steroid levels were measured by gas chromatography and chemical ionization mass spectrometry after appropriate extraction procedures. BMI and waist circumference were negatively correlated with plasma testosterone (r = -0.49 and -0.50, respectively, p < 0.01) and dihydrotestosterone (r = -0.58 and -0.56, respectively, p < 0.01), and positively associated with estrone levels (r = 0.64 and 0.62, respectively, p < 0.001). Regional differences in adipose tissue steroid levels were observed for dihydrotestosterone (p < 0.005), androstenedione (p < 0.0001) and dehydroepiandrosterone levels (p < 0.05), which were all significantly more concentrated in omental versus subcutaneous fat. Positive significant associations were found between circulating level of a steroid and its concentration in omental and subcutaneous adipose tissue, for estrone (r = 0.72 and 0.57, respectively, p < 0.01), testosterone (r = 0.66 and 0.58, respectively, p < 0.01) and dihydrotestosterone (r = 0.58 and 0.45, respectively, p < 0.05). Positive correlations were observed between plasma dehydroepiandrosterone-sulfate and omental (r = 0.56, p < 0.01) as well as subcutaneous adipose tissue dehydroepiandrosterone level (r = 0.38, p = 0.05). Positive significant associations were found between omental adipocyte responsiveness to positive lipolytic stimuli (isoproterenol, dibutyryl cyclic AMP and forskolin) and plasma or omental fat tissue androgen levels. In conclusion, although plasma androgen or estrogen levels are strong correlates of adipose tissue steroid content both in the omental and subcutaneous fat depots, regional differences may be observed. Androgen concentration differences in omental versus subcutaneous adipose tissue suggest a depot-specific impact of these hormones on adipocyte function and metabolism.     

Catecholamine and insulin control of lipolysis in subcutaneous adipose tissue during long-term diet-induced weight loss in obese women

The aim of this study was to investigate the evolution of the adrenergic and insulin-mediated regulation of lipolysis during different phases of a 6-mo dietary intervention. Eight obese women underwent a 6-mo dietary intervention consisting of a 1-mo very low-calorie diet (VLCD) followed by a 2-mo low-calorie diet (LCD) and 3-mo weight maintenance (WM) diet. At each phase of the dietary intervention, microdialysis of subcutaneous adipose tissue (SCAT) was performed at rest and during a 3-h hyperinsulinemic euglycemic clamp. Responses of dialysate glycerol concentration (DGC) were determined at baseline and during local perfusions with adrenaline or adrenaline and phentolamine before and during the last 30 min of the clamp. Dietary intervention induced a body weight reduction and an improved insulin sensitivity. DGC progressively decreased during the clamp, and this decrease was similar during the different phases of the diet. The adrenaline-induced increase in DGC was higher at VLCD and LCD compared with baseline condition and returned to prediet levels at WM. In the probe with adrenaline and phentolamine, the increase in DGC was higher than that in the adrenaline probe at baseline and WM, but it was not different at VLCD and LCD. The results suggest that the responsiveness of SCAT to adrenaline-stimulated lipolysis increases during the calorie-restricted phases due to a reduction of the α(2)-adrenoceptor-mediated antilipolytic action of adrenaline. At WM, adrenaline-stimulated lipolysis returned to the prediet levels. Furthermore, no direct relationship between insulin sensitivity and the diet-induced changes in the regulation of lipolysis was found.     

Comparison between surrogate indexes of insulin sensitivity and resistance and hyperinsulinemic euglycemic clamp estimates in mice

Insulin resistance contributes to the pathophysiology of diabetes, obesity, and their cardiovascular complications. Mouse models of these human diseases are useful for gaining insight into pathophysiological mechanisms. The reference standard for measuring insulin sensitivity in both humans and animals is the euglycemic glucose clamp. Many studies have compared surrogate indexes of insulin sensitivity and resistance with glucose clamp estimates in humans. However, regulation of metabolic physiology in humans and rodents differs and comparisons between surrogate indexes and the glucose clamp have not been directly evaluated in rodents previously. Therefore, in the present study, we compared glucose clamp-derived measures of insulin sensitivity (GIR and SI(Clamp)) with surrogate indexes, including quantitative insulin-sensitivity check index (QUICKI), homeostasis model assessment (HOMA), 1/HOMA, log(HOMA), and 1/fasting insulin, using data from 87 mice with a wide range of insulin sensitivities. We evaluated simple linear correlations and performed calibration model analyses to evaluate the predictive accuracy of each surrogate. All surrogate indexes tested were modestly correlated with both GIR and SI(Clamp). However, a stronger correlation between body weight per se and both GIR and SI(Clamp) was noted. Calibration analyses of surrogate indexes adjusted for body weight demonstrated improved predictive accuracy for GIR [e.g., R = 0.68, for QUICKI and log(HOMA)]. We conclude that linear correlations of surrogate indexes with clamp data and predictive accuracy of surrogate indexes in mice are not as substantial as in humans. This may reflect intrinsic differences between human and rodent physiology as well as increased technical difficulties in performing glucose clamps in mice.     

Differences of subcutaneous adipose tissue topography between type-2 diabetic men and healthy controls

Men with noninsulin-dependent diabetes mellitus (type 2 DM) provide a different subcutaneous body fat distribution and a concentration of fatness on the upper trunk compared with healthy subjects. However, subcutaneous fat distribution is always measured in an inaccurate and/or very simplified way (e.g., by caliper), and to date, there exists no study reporting on the exact and complete subcutaneous adipose tissue distribution of type 2 DM men. A new optical device, the LIPOMETER, enables the nonivasive, quick, and safe determination of the thickness of subcutaneous adipose tissue layers at any given site of the human body. The specification of 15 evenly distributed body sites allows the precise measurement of subcutaneous body fat distribution, so-called subcutaneous adipose tissue topography (SAT-Top). SAT-Tops of 21 men with clinically proven type 2 DM (mean age of 57.5 +/- 6.7 years) and 111 healthy controls of similar age (mean age 59.0 +/- 5.4 years) were measured. In this paper, we describe the precise SAT-Top differences of these two groups and we present the multidimensional SAT-Top information condensed in a two-dimensional factor value plot. In type 2 DM men, especially in the upper trunk, SAT-Top is significantly increased (up to +50.7% at the neck) compared with their healthy controls. One hundred eleven of the 132 individuals (84.1%) are correctly classified (healthy or type 2 DM) by their subcutaneous fat pattern by stepwise discriminant analysis.     

Identification of structurally distinct alpha 2-adrenergic receptors

Recent studies involving a variety of membrane receptors and ion channels indicate that diversity exists among these proteins as evidenced by tissue-specific and developmentally related expression of different isoforms. Alpha 2-Adrenergic receptors, plasma membrane proteins involved in sympathetic neurotransmission, may similarly represent a nonhomogeneous class of binding sites based on the following observations. First, their activation can elicit a wide variety of effector cell responses, which are apparently triggered by at least three different signal transduction mechanisms. Second, alpha 2-adrenergic receptors in various tissues and species exhibit marked differences in their ligand recognition properties. To determine if heterogeneity of the receptor protein itself is involved in generating this diversity, we structurally characterized the alpha 2-adrenergic receptor in two tissues that exhibit the greatest differences in ligand recognition properties, neonatal rat lung and human platelet. We report here that these differences in ligand recognition are maintained after partial receptor purification (50-100-fold) and are associated with distinct differences in the physical and structural properties of the receptor protein. The human platelet and neonatal rat lung receptor differ in the apparent molecular weight of their hormone-binding subunits (human platelet, Mr approximately 64,000 versus neonatal rat lung, Mr approximately 44,000) as well as in the number or type of their associated oligosaccharide moieties. The observed diversity is consistent with expression of isoforms of the alpha 2-adrenergic receptor and suggests the presence of more than one gene encoding similar but distinct receptor proteins.     

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.