Enhanced activity of hormone-sensitive adenylate cyclase during dietary restriction in the rat: dependence on age and relation to cell size.

Age-related decreases of hormone-sensitive adenylate cyclase activities of rat fat cell plasma membranes (ghosts) have been recently described. Glucagon-sensitive activity was completely lost between 1 and 6 mo, an interval in which fat cell size increases rapidly, while decreased activation by ACTH was gradual over the entire life span of the animal (24 mo), and epinephrine-sensitive enzyme diminished modestly and only during senescence. In the present studies an attempt was made by restricting food intake to assess the importance of changing cell size in the age-related alterations of hormone-sensitive enzyme activities. Enzyme activities were determined before restriction and at monthly intervals for 3 mo for the unstimulated enzyme (basal) and in the presence of maximally stimulating concentrations of glucagon, ACTH, epinephrine, and fluoride. Activities were calculated per milligram ghost protein or per cell. Restriction of food intake for 3 mo starting at 1 or 12 mo produced fat cells equal in size to those of 5-wk-old animals fed ad lib. In young animals restricted for 1 mo, hormone-stimulated activity expressed as fold increase (stimulated/basal) was not merely maintained as the cells were prevented from enlarging, but was enhanced two to three times over the initial values with all three hormones. With continued restriction epinephrine-sensitive activity remained two times increased. Glucagon and ACTH responses subsequently decreased, but even by 3 mo of restriction, responses to the latter hormones, although declining, were still 1.5-3 times greater than the unrestricted controls, regardless of whether activity was expressed as total activity per milligram ghost protein or per cell, or as fold-increase. In the young animals, basal and fluoride-sensitive activities after a 3-mo restriction were unchanged or had decreased only slightly, depending on the base line used. Dietary restriction of adult animals for 3 mo, in contrast to the results in the young, did not increase total hormone-stimulated activity but rather produced either 0% (per milligram protein) or 25% decrease (per cell) for epinephrine-sensitive enzyme, 25 or 50% decrease of ACTH response, and 40 or 60% decreases of basal- and fluoride-stimulated activities. Expression of activities of restricted adults as fold-increase (stimulate/basal) showed an "increase of responsiveness" for all three hormones, but this was a reflection of the marked decrease of basal activity. Nonetheless, the restricted adults showed significant restoration of a small amount of glucagon-sensitive activity (1.8-fold over basal). These results indicate that cell size, per se, is not a dominant factor affecting hormone-responsive adenylate cyclase under conditions of dietary restriction...

Hormone-sensitive fat cell adenylate cyclase in the rat. Influences of growth, cell size, and aging.

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.

Thyroid hormone binding by human serum prealbumin (TBPA). Electrophoretic studies of triiodothyronine-TBPA interaction.

Thyroxine-binding prealbumin (TBPA) in normal human serum has been shown in a polyacrylamide gel electrophoresis system to bind 7-9% of tracer level purified [(125)I]triiodothyronine (T3), and more than 30% of T3 in serum deficient in thyroxinebinding globulin (TBG). The T3-TBPA interaction has been confirmed at pH 9.0 and pH 7.4 in this electrophoretic demonstration of TBPA binding of T3 in serum. Purified human TBPA has also been shown to bind T3. Progressive additions of unlabeled thyroxine (T4) to serum containing tracer [(125)I]T3 displace T3 from TBG, its principal carrier, to TBPA and albumin; however, T4 loading does not lead to significant T3 displacement from TBPA even at T4 levels known to saturate TBPA. Loading of serum with unlabeled T3 results in displacement of more than 50% of [(125)I]T3 from TBPA, as well as from TBG, to albumin. Studies carried out with serum containing diphenylhydantoin (DPH) or MK-185, known inhibitors of T4 binding by TBG, also showed T3 displacement from TBG to TBPA and albumin. Although salicylate and tetraiodothyroacetic acid (TETRAC) displace T4 from sites on TBPA, they have only minimal effects on T3-TBPA interaction.

Adenylate cyclase of human fat cells. Expression of epinephrrine-sensitive activation revealed by 5'guanylyl-imidodiphosphate.

Although catecholamines stimulate lipolysis in human fat cells, activation by epinephrine of adenylate cyclase in human fat cell membranes is not readily observed. The possible role of guanine nucleotides in this reaction has now been examined with human material. Fat cell ghosts were prepared from subcutaneous fat obtained from patients undergoing elective surgery. Adenylate cyclase was assayed with [alpha-32P]ATP as substrate. Fluoride ion stimulated the enzyme 8.3-fold relative to basal levels, but epinephrine activation of cyclase was not statistically significant. GTP did not allow expression of an epinephrine effect. However, the addition of the GTP analogue, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P], along with epinephrine produced 5.7-fold activation of the enzyme (P less than 0.001). GMP-P(NH)P alone was without stimulatory effect. Comparable augmentation by GMP-P (NH) P of adenylate cyclase activity was seen with isoproterenol, norepinephrine, and epinephrine. Propranolol blocked catecholamine-GMP-P (NH) P stimulation of the enzyme, suggesting that the nucleotide-dependent activation of catecholamine-sensitive adenylate cyclase is mediated by beta-receptors. GMP-P(NH)P may prove useful in allowing in vitro demonstration of additional hormone-sensitive adenylate cyclase systems.

Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.

Anions and cations as stimulators of liver adenylate cyclase.

The effects of inorganic salts on adenylate cyclase activities were studied in rat liver homogenates, particulates, and purified membranes. Na+ salts of N-3, NO-2, S2O32-, SO42-, and non-F- halides stimulated homogenates by a maximum of 2---5-fold; half-maximal effects were seen at concentrations ranging from 90 mM (Na2S2O3) to 410 mM (NaNo2). NaIO3 stimulated about 2-fold at relatively low salt concentrations (5---20 mM). Na+ salts were stimulatory using both ATP and the ATP analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-P(NH)P) as enzyme substrate. The time courses of stimulation by NaCl and NaN3 were linear to at least 10 min with any of the three tissue preparations. Although salt effects clearly varied with the different anions, the magnitude and dose-response of stimulation of homogenates by Cl- salts were also dependent on the accompanying alkali cation (Li+, Na+, K+, Rb+, Cs+). MgCl2 at relatively high concentrations (50 mM) enhanced NaCl-stimulated activity in homogenates only slightly at relatively low concentrations of NaCl and not at high concentrations, suggesting a common mechanism of activation by Mg2+ and Na+ salts. NaCl- and NaN3-stimulated activities were unstable in homogenates kept at 0 degree C for 4 h, falling to 35% and 50% of their respective baseline activities. The effects on homogenates and particulates of maximally stimulatory concentrations of NaCl and NaN3 were further enhanced by GTP and the GTP analog 5'-guanylyl-beta, gama-imidodiphosphate (GMP-P(NH)P).

Separation of human renal renin and pseudorenin by affinity chromatography on hemoglobin-Sepharose-2B.

Human renal renin (EC 3.4.99.19) and pseudorenin were easily separated in a single step by affinity chromatography on hemoglobin-Sepharose-2B. Renin and pseudorenin were monitored by their actions on crude and partially purified hog protein renin substrates at neutral and acidic pH and on synthetic labelled polymeric renin substrate. Under the conditions employed (0.1 M sodium acetate (pH 3.5)/1 M sodium chloride at 4 degrees C) renin does not bind to the affinity adsorbent while pseudorenin is effectively bound and can be eluted only after raising the pH to 6.5. Pseudorenin-free renin prepared by this method is devoid of proteolytic activity toward hemoglobin. The chromatographic behaviour of renal pseudorenin on hemoglobin-Sepharose-2B is similar to that of cathepsin D.

Quantitation of epinephrine- and glucagon-sensitive adenylate cyclases of rat liver. Implications of alterations of enzymatic activities during preparation of particulate fractions and membranes.

Stimulated and basal adenylate cyclase activities from livers of young and old rats were lower in particulates than in homogenates. Particulates were compared to homogenates by reconstituting the suspensions to the volume of the homogenates from which they were derived; enzyme activities in paired homogenates and particulates therefore reflected the same amounts of membrane-bound enzyme. The magnitude of the losses of hormone-sensitive activities in particulates was dependent on the age and sex of the animals and the concentrations of hormone. Particulates from 3-month-old animals showed glucagon-(1 . 10(-5) M) and epinephrine-sensitive (1 . 10(-4) M) activities which were 67 and 78% of homogenate activities, respectively; particulates from 24-month-old animals had activities relative to homogenates of 55% for glucagon and as low as 32% for epinephrine. The glucagon dose vs. response curve in particulates and membranes showed maximal activity at 1 . 10(-7) M glucagon while in homogenates activity increased linearly with increasing glucagon concentrations up to 1 . 10(-5) M. Losses of basal and anion-stimulated activities were similar at both ages. Fluoride and azide stimulations relative to basal activities were greater in particulates than in homogenates, while relative epinephrine activity was lower in particulates, suggesting qualitative alteration of adenylate cyclase during preparation of particulates. These studies show that adenylate cyclase activity in rat liver is presently best quantitated in homogenates and suggest caution in comparisons of enzyme activities based on particulates or membranes prepared from animals of differing physiologic states.

Altered glucagon- and catecholamine hormone-sensitive adenylyl cyclase responsiveness in rat liver membranes induced by manipulation of dietary fatty acid intake.

Glucagon-stimulated adenylyl cyclase activity has been shown to change in liver membranes manipulated to alter either their fatty acid composition or fluidity. We examined whether membrane alterations induced by dietary manipulation affected receptor function. Glucagon- and beta-adrenergic-stimulated receptor-adenylyl cyclase systems were examined in liver membranes of rats fed diets containing 10% corn oil, 10% coconut oil (essential FFA deficient), or 8.5% coconut oil with 1.5% corn oil (essential FFA repleat). Basal and maximal nonreceptor-mediated adenylyl cyclase activity (stimulated by NaF, guanylylimidodiphosphate, and forskolin) was the same in membranes of each of the dietary groups, suggesting that Gs-protein and the catalytic unit activity per se were unaltered by the manipulations. Glucagon-stimulated adenylyl cyclase activity increased with increasing unsaturation of dietary fatty acids; activity in coconut oil-fed rats was 527 +/- 30 (mean +/- SEM) pmol/mg.10 min, that in coconut/corn oil-fed rats was 752 +/- 74 pmol/mg.10 min, and that in corn oil-fed rats was 981 +/- 94 pmol cAMP/mg.10 min. [125I]Monoiodoglucagon binding did not increase in parallel to the adenylyl cyclase alterations; coconut oil-fed animals (614 fmol/mg) differed from the other groups (450 and 430 fmol/mg). Isoproterenol (beta-adrenergic)-stimulated adenylyl cyclase activity was also highest in the corn oil-fed animals, but was similar in the other dietary groups, with no difference in other characteristics of [125I]iodopindolol binding between the groups. The results demonstrate that alterations in the glucagon-stimulated adenylyl cyclase response are different from those in the beta-adrenergic adenylyl cyclase response. Further, they suggest that although direct activations of the catalytic unit or its interaction with the guanine nucleotide-sensitive protein are apparently not affected, hormone receptor-mediated adenylyl cyclase activity may be altered by these dietary manipulations.

Beta-adrenergic receptors, glucagon receptors, and their relationship to adenylate cyclase in rat liver during aging.

The beta-adrenergic and glucagon receptor-binding capacities in rat livers from 6-27 months of age were measured to investigate the mechanism of a previously observed rise in beta-adrenergic stimulated adenylate cyclase with increasing age. There was no concomitant increase in glucagon-stimulated adenylate cyclase. In the present study neither glucagon-binding capacity nor glucagon-stimulated adenylate cyclase changed with age. In contrast, the beta-adrenergic receptor capacity, measured in the same membranes by [125I]iodopindolol binding, increased nearly 3-fold from 6.6 +/- 0.6 fmol/mg at 6 months to 19.1 +/- 3.3 fmol/mg at 18-19 months. The increase was directly proportional to the maximum isoproterenol-stimulated adenylate cyclase activity in livers of rats up to 19 months of age. By 24-27 months the binding capacity had increased to 24.9 +/- 3.3 fmol/mg, but there was no further increase in adenylate cyclase activity. Thus, there appeared to be a beta-receptor-adenylate cyclase uncoupling in livers from the senescent animals (25-27 months). The defect could not be demonstrated by studies examining isoproterenol competition of [125I]iodopindolol from agonist-induced high affinity sites on the membranes, a procedure that examines receptor-Ns protein coupling. Activation of adenylate cyclase by the nonhormonal stimulators F- and forskolin did not change with age, indicating that the catalytic unit was not a limiting factor. Since the relationship between the glucagon receptor and adenylate cyclase also remained unaltered, the uncoupling apparently lies in an alteration of the interaction between the beta-adrenergic receptor and the guanine nucleotide-sensitive Ns protein.

Ectopic beta-adrenergic receptors coupled to adenylate cyclase in human adrenocortical carcinomas.

The adenylate cyclase of an adrenocortical carcinoma of the rat is activated not only by ACTH but also by beta-adrenergic agonists, which bind to ectopic beta-adrenergic receptors not present in normal rat adrenal cortex. Previous reports examining possible beta-adrenergic control of adenylate cyclase in human adrenocortical carcinomas failed to demonstrate beta-adrenergic receptor-linked enzyme activity. We studied six human adrenal carcinomas and normal adrenal cortex from three subjects for beta-adrenergic agonist-sensitive adenylate cyclase and beta-adrenergic binding sites. Three of the six carcinomas had adenylate cyclase responses to both ACTH and beta-agonists. Two tumors were ACTH responsive but not beta-agonist responsive; one tumor responded to beta-agonists but not to ACTH. Adenylate cyclase activity of normal adrenal cortex from three subjects was stimulated by ACTH but not by beta-agonists. In membrane preparations from three tumors with beta-agonist-sensitive adenylate cyclase, the radiolabeled beta-adrenergic antagonist [125I]pindolol bound specifically and with high affinity (Kd = 38-83 pM) to a single class of binding sites which showed saturation with ligand concentration, reversibility of binding, pharmacological specificity, and stereospecificity. Normal cortex and one tumor without beta-adrenergic agonist-sensitive adenylate cyclase had no specific binding of [125I]pindolol. These results indicate that malignant transformation of adrenal cortex in man is frequently but not invariably associated with the appearance of ectopic beta-adrenergic receptors functionally linked to adenylate cyclase. Loss of ACTH-responsive adenylate cyclase may also occur simultaneously with the development of beta-adrenergic receptor-linked adenylate cyclase.

Beta adrenergic regulation of rat liver glycogenolysis during aging.

Studies from a number of laboratories demonstrate a biphasic change in beta adrenergic regulation of hepatic glycogenolysis over the life span of the male rat. The beta adrenergic response is prominent in immature animals, declines rapidly during subsequent development to a minimum by the time of young adulthood, and then reemerges during postmaturational development. Age changes in beta adrenergic-responsive adenylate cyclase activity follow a "U"-shaped curve similar to that described by changes in liver glycogenolytic responsiveness during aging. Developmental and postmaturational changes in beta adrenergic-sensitive adenylate cyclase activation are related to parallel alterations in the density of beta adrenergic receptors and also to functional changes in nonreceptor components of the enzyme. The prevailing view that catecholamines stimulate hepatic glycogenolysis by an alpha adrenergic receptor-mediated, cyclic AMP-independent mechanism is based almost entirely on evidence from young adult male rats. We propose that current concepts of alpha adrenergic-responsive liver glycogenolysis underestimate a physiological role for beta adrenergic responsiveness over the majority of the life span.

Mechanism of action of forskolin on adenylate cyclase: effect on bovine sperm complemented with erythrocyte membranes.

The mechanism of action of forskolin stimulation of adenylate cyclase was investigated by examining its effects on the enzyme's Mg2+ activated catalytic unit (C) from bovine sperm, both preceding and following complementation with human erythrocyte membranes as a source of guanine nucleotide regulatory protein (N). Prior to complementation, sperm C was not activated by either NaF (10 mM) or 5'-guanylyl-beta-gamma-imidodiphosphate (Gpp(NH)p, 10 microM), suggesting that functional N was not present in this preparation. Forskolin (100 microM) was also without effect on C. Following complementation of the sperm membranes with those of erythrocytes, Mg2+-dependent sensitivity to forskolin, NaF, and Gpp(NH)p was imparted to C. Our findings are incompatible with the current hypothesis that forskolin stimulates adenylate cyclase by direct activation of C. Rather, the data suggest that the activation process occurs through an effect on N or by augmentation of the interaction between the components of the adenylate cyclase complex.