Mechanism of the age - related decrease of epinephrine - stimulated lipolysis in isolated rat ad i pocytes : p - ad renerg ic receptor bind i ng , adenylate cyclase activity , and cyclic AMP accumulation

3-adrenergic binding ( [3H]dihydroalprenolo1), adenylate cyclase activity, and cAMP accumulation were measured in adipocytes to investigate whether the mechanism of decreased hormone-sensitive lipolytic response with age was mediated through membrane-associated events. The dose of epinephrine required for half maximal stimulation of glycerol release (ED,,) was significantly lower in 2-month-old rats (0.8 ? 0.2 pM) than in mature (6and 12-month-old) rats (5.2 ? 1.5 and 6.2 +. 1.5 p M , respectively). In 24-month-old rats the ED,, (0.7 +0.2 pM) was less than in mature rats. Maximum rates of hormone-stimulated glycerol release (per lo6 cells) was highest in the two mature groups and decreased by 50% in the old rats (P < 0.01). Lipolytic changes were independent of cell size. &adrenergic receptor number (50-90 thousand siteslcell) and affinity (K, 4-5 nM) were the same in each age group. ED,, and maximum level of hormone-stimulated adenylate cyclase activity did not change with age. The ED,, of cAMP accumulation of young rats was 3 ? 5 pM compared with 24 ? 4 and 25 ? 5 p,M in 6and 12-month-old rats, respectively. In old rats, the ED,, of cAMP accumulation was 2 -t 1 pM (P < 0.001 compared with mature rats). Maximally stimulated cAMP levels were the same in old and mature animals. Phosphodiesterase activity in the presence and absence of lo-, M isoproterenol did not change with a g e . l The results suggest that age-related decrease of epinephrine-sensitive lipolysis in old rats may be due to altepations of the lipolytic pathway distal to the receptoradenylate cyclase complex and the generation of cyclic AMP. -Dax, E. M., J. S. Partilla, and R. I. Gregerman. Mechanism of the age-related decrease of epinephrine-stimulated lipolysis in isolated rat adipocytes: /3-adrenergic receptor binding, adenylate cyclase activity, and cyclic AMP accumulation. J. Lipid Res. 1981. 22: 934-943. Supplementary key words aging . catecholamine effects [3H]dihydroalprenolo1 . adipocyte membrane * receptor-cyclase system Age-related changes in the /3-adrenergic lipolytic response have been previously reported, but little evidence concerning mechanisms has been presented (1 -4). Yu, Bertrand, and Masoro (1) have shown that catecholamine-induced lipolysis in adipocytes decreased with age, while the age-effect was delayed by dietary restriction. Guidicelli and Pecquery (2) reported a loss of lipolytic response to epinephrine with aging and proposed that this finding was related to a decrease in the number of @adrenergic receptor sites per cell. Forn et al. ( 5 ) reported a decrease of norepinephrine-stimulated lipolysis during maturation in the rat. Decreased adenylate cyclase activity and increased phosphodiesterase were observed and were postulated to explain the age-related alteration of lipolysis. On consideration of the published reports in this area, the fragmentary nature of the information becomes even more apparent. Changes during maturation have not been differentiated from those of aging in the latter part of the life span (4-8). Lipolysis has sometimes been related to cell triglyceride content rather than to cell number (9). Increasing cell size which occurs during maturation has been equated with changes due to aging (4, 5, 7, 10). Some studies have used only a single submaximal dose of hormone and thus fail to examine the issue of sensitivity to hormone (dose-response, 2). Other work has used doses of hormone that may be supramaximal and result in non-specific stimulation of the lipolytic response (8). The present investigation of the nature and mechanism of age-related alteration of lipolysis was an attempt to take into account a number of these ' To whom correspondence should be addressed at the Cerontology Research Center, Baltimore City Hospitals, Baltimore, MD 21224. 934 Journal of Lipid Research Volume 22, 1981 at P E N N S T A T E U N IV E R S IT Y , on F ebuary 3, 2013 w w w .j.org D ow nladed fom issues and variables. Dose-response relationships along with cell size and number have been determined. Groups of animals have been chosen at ages that would allow distinction to be made between changes due to maturation and those related to senescence. Finally, several components of the padrenergic receptor-adenylate cyclase system, viz., p-receptors, adenylate cyclase, cyclic AMP accumulation, and phosphodiesterase activity, have been examined in order to investigate whether these membrane-associated parameters mediate changes in p-adrenergic lipolytic function with age. MATERIALS AND METHODS Male Wistar rats bred and maintained at the Gerontology Research Center were used in this study. The rats have a median life span of 24 months (50% mortality at 24 months) (1 1). The mean weights of a representative group of rats over the period of these experiments at 2, 6, 12, and 24 months were 301 5 15,571 & 13,614 ? 8, and 595 5 15 g, ( m e a t S.E.M.), respectively. (N = 10 in each group). The rats were fed standard laboratory chow with a 5% (by weight) crude fat content. The rats were killed by decapitation or CO, narcosis between 9:30 and 1O:OO AM. There was no difference in results from rats killed by either method. Chemicals used were reagent grade or better. Isotopes were obtained from New England Nuclear. Collagenase (Clostridium histolyticum, Type I) was obtained from Worthington. Preparation and sizing of isolated fat cells Epididymal fat pads from 2-6 animals in each age group (2, 6, 12, and 24 months) were immediately dissected and rinsed in normal saline at 37°C. After weighing the fat pads from each age group, isolated fat cells were prepared according to the method of Rodbell (12) but with the following minor modifications. The fat pads were minced with scissors and placed in plastic conical flasks in Krebs-bicarbonate buffer (3 ml per g wet weight of fat) with 4% bovine serum albumin and 5 mg collagenase per g wet weight of fat. A single batch of collagenase was used throughout these experiments. Collagenase digestion was carried out at 37°C in a metabolic shaker at 100 cycles per minute. Cells were washed twice in fresh Krebs-albumin buffer and allowed to separate from the infranatant by flotation. A 500-pl aliquot of appropriately diluted cells was fixed in osmium tetroxide for counting and sizing in a Coulter counter (13) as previously reported from this laboratory (1 1). The packed cell volume for each preparation was noted for use in calculations to establish the number of adipocytes in each assay. Incubation of isolated fat cells for determination of lipolysis rate and cyclic AMP Packed fat cells were diluted 1 to 4 in Krebs-albumin buffer and 350 p1 was added to polystyrene tubes containing Krebs-albumin buffer and epinephrine to give the desired final concentration of epinephrine in a total volume of 1 ml. After incubation at 37°C with gentle rocking (-60 cycles per min) in a metabolic bath for 1 hr, the tubes were placed on ice. Two hundred pl of cells and infranatant was transferred to tubes containing 20 pl of 2 M HCl for cAMP determinations and 200 p1 of infranatant was reserved for determination of glycerol release. All incubations were carried out in duplicate. The mean numbers of cells in the incubates were 1.4 2 0.4 x lo4, 6.7 2 0.6 x lo3, 6.4 2 0.7 x lo3, and 1.4 2 0.3 x lo4 adipocytes per 200 pl for the 2, 6, 12, and 24-month adipocytes, respectively. The number of experiments is indicated in Table 1. Measurement of lipolysis The rate of lipolysis was measured as glycerol release. Glycerol was measured by the enzymatic method described by Weiland (14).