Cooper B, Gregerman R I
J Clin Invest. 1976 Jan;57(1):161-8. doi: 10.1172/JCI108256.
The possibility has been explored that decreases of adenylate cyclase may explain diminished hormone sensitivity of adipose tissue with aging. Isolated cells were prepared from epididymal fat pads of rats 1-, 2-, 6-, 12-, and 24-mo old, fixed in OSO4, and sized and counted with a Coulter apparatus. Adenylate cyclase was assayed in cell membranes (ghosts) using [alpha-32P] ATP as substrate and expressed as cyclic [32P] AMP/10 min per mg protein or per 10(6) cells. Enzyme activity was determined for the basal state and in the presence of varying concentrations of glucagon, ACTH, epinephrine, and fluoride. Basal activity per cell increased in threefold between 1 and 2 mo with a comparable increase in cell surface area, suggesting synthesis of enzyme along with new cell membrane. Although epinephrine stimulated adenylate cyclase 8-fold and fluoride 12-fold throughout the life-span of the rat, stimulated activity paralleled basal levels, decreasing 60% between 2 and 24 mo per mg protein and 40% between 6 and 24 mo per cell. Glucagon stimulated adenylate cyclase 4.5-fold relative to basal in the 1-mo-old rat, but its effect then rapidly decreased and was absent by 12 mo. The fourfold stimulation by ACTH noted in the 1-mo-old animals decreased gradually with age but was still twice basal at 24 mo. Since no significant change of cell size occurred after 6 mo, diminished hormone sensitivity with senescence cannot be related to cell size. Similar age-related patterns of hormonal activation were evoked by 5'-guanylyl-imidodiphosphate [GMP-P(NH)P], a nucleotide analogue which increased both basal- and hormone-activated enzyme at all ages studied. Dose-response curves to hormones, fluoride, and GMP-P (NH)P were not affected by age. High Mg++ (50 mM) in the presence of GMP-P-(NH)P stimulated adenylate cyclase to levels greater than with fluoride, but a similar loss of activity with aging was still observed. Loss of hormone receptors may partially explain the age-related decreases of glucagon and ACTH-sensitive adenylate cyclase, but decreased basal-, epinephrine-, fluoride-, and GMP-P-(NH) P-stimulated responses suggest loss of the catalytic component of the adenylate cyclase enzyme complex in the aging fat cell membranes.
已经探讨了腺苷酸环化酶减少可能是脂肪组织随衰老激素敏感性降低的原因这一可能性。从1个月、2个月、6个月、12个月和24个月大的大鼠附睾脂肪垫制备分离细胞，用四氧化锇固定，并用库尔特仪器进行大小测量和计数。以[α-32P]ATP为底物在细胞膜（鬼细胞）中测定腺苷酸环化酶活性，并表示为每毫克蛋白质或每10(6)个细胞每分钟的环[32P]AMP量。测定基础状态以及存在不同浓度胰高血糖素、促肾上腺皮质激素、肾上腺素和氟化物时的酶活性。每细胞的基础活性在1至2个月之间增加了三倍，细胞表面积也有类似增加，表明酶与新细胞膜一起合成。尽管在大鼠的整个寿命期内肾上腺素刺激腺苷酸环化酶8倍，氟化物刺激12倍，但刺激活性与基础水平平行，每毫克蛋白质在2至24个月之间下降60%，每细胞在6至24个月之间下降40%。在1个月大的大鼠中，胰高血糖素刺激腺苷酸环化酶相对于基础水平增加4.5倍，但其作用随后迅速下降，到12个月时消失。在1个月大的动物中观察到的促肾上腺皮质激素四倍刺激作用随年龄逐渐降低，但在24个月时仍为基础水平的两倍。由于6个月后细胞大小没有显著变化，衰老过程中激素敏感性降低与细胞大小无关。在所有研究的年龄中，核苷酸类似物5'-鸟苷酰亚胺二磷酸[GMP-P(NH)P]引起了类似的与年龄相关的激素激活模式，它增加了基础和激素激活的酶。对激素、氟化物和GMP-P(NH)P的剂量反应曲线不受年龄影响。在GMP-P(NH)P存在下高镁离子（50 mM）刺激腺苷酸环化酶达到比氟化物更高的水平，但仍观察到随着衰老活性有类似的丧失。激素受体的丧失可能部分解释了与年龄相关的胰高血糖素和促肾上腺皮质激素敏感的腺苷酸环化酶的降低，但基础、肾上腺素、氟化物和GMP-P(NH)P刺激反应的降低表明衰老脂肪细胞膜中腺苷酸环化酶酶复合物的催化成分丧失。
