Phospholipid methyltransferase activity in diabetic rat fat cells: effect of isoproterenol and insulin

The effects of isoproterenol and insulin on phospholipid methyltransferase (PLMT) activity were investigated in adipocytes from control and streptozotocin-diabetic rats. PLMT activity was assayed by measuring the rate of incorporation of ³H-methyl groups from S-adenosyl-L-[methyl-³H] methionine into phospholipids. Basal PLMT activity was higher in adipocytes from diabetic animals. Treatment of adipocytes with isoproterenol induced a concentration-dependent stimulation of PLMT activity. In control adipocytes, the maximal effect was obtained at 100 nM isoproterenol with 2.3 fold increase in PLMT activity and a half maximal effect at 25 nM. In adipocytes from diabetic rats, a lower dose of isoproterenol (10 nM), caused 1.2 fold increase with a half maximal effect at 4 nM. Addition of 100 nM insulin inhibited basal PLMT activity and the stimulatory effect of isoproterenol in both types of adipocytes. The -adrenergic blocking agent propranolol inhibited the stimulatory effect of isoproterenol on PLMT activity in control and diabetic adipocytes. Intracellular concentration of cAMP was higher in diabetic adipocytes but decreased to normal values after incubation in the presence of insulin.     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Stimulation and inhibition of lipolysis in isolated rat adipocytes: evidence for age-related changes in responses to forskolin and PGE1

The ability of a number of hormones to activate cellular responses in a variety of cells declines with age. The mechanisms responsible for these alterations are complex and incompletely understood. Rat adipocytes have served as an important model to study blunted responses to stimulatory hormones which function by activating cAMP accumulation. We have previously found that the blunted lipolytic response of adipocytes from older rats to the beta adrenergic receptor agonist isoproterenol appeared to be due to a lessened ability of isoproterenol to activate cAMP accumulation. Further, the blunted response to isoproterenol was apparently caused by an accentuated inhibition of lipolysis, mediated by adenosine receptors activated by endogenously released adenosine. The present studies were designed to test and extend those conclusions. We have utilized forskolin to augment the cAMP accumulation that occurs in the presence of isoproterenol. Isoproterenol-activated lipolysis was greater in adipocytes from 2 month old rats compared with those from 12 month old rats (603 +/- 32 vs 450 +/- 29 nmol/10(5) cells/hr, P less than 0.01). However, in the presence of forskolin (10(-6) M), there was no significant difference in the response to isoproterenol between the two groups (646 +/- 23 vs 615 +/- 29 nmoles/10(5) cells/hr). As we had seen previously, the adenosine receptor agonist phenylisopropyladenosine more effectively inhibited lipolysis in the adipocytes from older rats. We now also find that PGE1 more efficaciously inhibits lipolysis in the cells from older rats. These data confirm that diminished cAMP accumulation in adipocytes from older rats appears to be a rate-limiting alteration in the regulation of lipolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of leptin secretion from white adipocytes by free fatty acids

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.     

Genistein restricts leptin secretion from rat adipocytes

The isoflavones--genistein and daidzein -- compounds found in high concentrations in soy play an important role in prevention of many diseases and affect some metabolic pathways. In the performed experiment it was demonstrated that genistein (5mg/kg b.w.) administered intragastrically for three days to male Wistar rats substantially diminished blood leptin level. Studies with isolated rat adipocytes revealed that this phytoestrogen strongly restricted leptin secretion from these cells. These effects were not accompanied by any changes in leptin gene expression in adipocytes. Daidzein-- an analogue of genistein -- used at similar concentrations did not affect blood leptin concentration, leptin secretion and expression of its gene. To determine the influence of genistein and daidzein on leptin release, adipocytes isolated from the epididymal fat tissue were incubated for 2h in Krebs--Ringer buffer. Leptin secretion stimulated by glucose with insulin was significantly diminished by genistein (0.25--1mM). This effect of genistein may arise from several aspects of its action in adipocytes documented in the literature such as the inhibition of glucose transport and metabolism, the attenuation of insulin signalling, the inhibition of cAMP phosphodiesterase and the stimulation of lipolysis. However, the bypassing of the restrictive action of genistein on glucose transport and glycolysis (by the use of alanine instead of glucose) and on insulin action (by the use of nicotinic acid) was not sufficient to restore leptin secretion from isolated adipocytes. It was also demonstrated that the restriction of the stimulatory influence of genistein on cAMP/protein kinase A (PKA) pathway (by the inhibition of PKA activity) did not improve leptin release. Results obtained in our experiments point at the restriction of glucose metabolism following formation of pyruvate as the pivotal reason of the inhibitory action of genistein on leptin release.     

A dual component analysis explains the distinctive kinetics of cAMP accumulation in brown adipocytes

The mechanism behind the distinctive non-Michaelis-Menten, bell-shaped kinetics of cAMP accumulation in brown adipocytes (which underlies the similar kinetics of UCP1 and beta(1)-adrenoreceptor gene expression) was investigated. A theoretical dual component analysis indicated that the observed dose-response curves could be constructed as the resultant of a stimulatory and an inhibitory component. Experimentally, inhibition of the alpha(1)-component of the norepinephrine response revealed the underlying existence of a much larger stimulatory beta(3)-component which displayed monophasic Michaelis-Menten kinetics. The inhibitory alpha(1)-component (which was also monophasic but had a 2-fold higher EC(50)) was mediated via an increase in [Ca(2+)](i); the protein kinase C pathway was not involved. The [Ca(2+)](i) increase which resulted in massive inhibition of cAMP accumulation was very low: <100 nM. The [Ca(2+)](i) signal stimulated a calmodulin-controlled phosphodiesterase, possibly PDE-1. The acquirement of this specific interaction pattern between beta- and alpha(1)-adrenergic stimulation was thus part of the differentiation program of the brown adipocytes. It was concluded that an array of synergistic or inhibitory alpha(1)/beta interactions occur in the adrenergic regulation of this cell type which is unique in its dependence upon adrenergic stimulation for cellular proliferation, differentiation, and metabolic function.     

Selective regulation by pertussis toxin of insulin-induced activation of particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes

Incubation of 3T3-L1 adipocytes with insulin or isoproterenol for 10 min increased particulate "low Km" cAMP phosphodiesterase activity by 42% and 50%, respectively. Pertussis toxin catalyzed the [32P]-ADP ribosylation of a 41,000 dalton protein in adipocyte particulate fractions; prior incubation of adipocytes with toxin markedly reduced incorporation of radiolabel. Exposure of adipocytes to pertussis toxin (0.3 microgram, 18 hr) increased glycerol production and inhibited activation of cAMP phosphodiesterase by insulin, but not by isoproterenol. These results suggest that pertussis toxin can interfere with receptor-mediated processes that stimulate cAMP hydrolysis as well as those that inhibit cAMP formation.     

Regulation of insulin secretion by cAMP in rat islets of Langerhans permeabilised by high-voltage discharge

Adenosine 3',5-cyclic monophosphate (cAMP) was shown to stimulate insulin secretion from electrically permeabilised islets of Langerhans incubated in Ca2+/EGTA buffers. cAMP-induced insulin secretion occurred in the presence of either sub-stimulatory (50 nM) or stimulatory (greater than 100 nM) concentrations of Ca2+. Similar effects on secretion were obtained in response to 8-bromo-cAMP (8-Br-cAMP) or the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine. Forskolin (0.2-20 microM) increased adenylate cyclase activity and enhanced insulin secretion from the permeabilised islets. These results suggest that, in electrically permeabilised islets, cAMP-induced insulin secretion is not dependent on changes in cytosolic Ca2+.     

Natriuretic peptides: a new lipolytic pathway in human adipocytes.

Atrial natriuretic peptide (ANP) receptors have been described on rodent adipocytes and expression of their mRNA is found in human adipose tissue. However, no biological effects associated with the stimulation of these receptors have been reported in this tissue. A putative lipolytic effect of natriuretic peptides was investigated in human adipose tissue. On isolated fat cells, ANP and brain natriuretic peptide (BNP) stimulated lipolysis as much as isoproterenol, a nonselective beta-adrenergic receptor agonist, whereas C-type natriuretic peptide (CNP) had the lowest lipolytic effect. In situ microdialysis experiments confirmed the potent lipolytic effect of ANP in abdominal s.c. adipose tissue of healthy subjects. A high level of ANP binding sites was identified in human adipocytes. The potency order defined in lipolysis (ANP > BNP > CNP) and the ANP-induced cGMP production sustained the presence of type A natriuretic peptide receptor in human fat cells. Activation or inhibition of cGMP-inhibited phosphodiesterase (PDE-3B) (using insulin and OPC 3911, respectively) did not modify ANP-induced lipolysis whereas the isoproterenol effect was decreased or increased. Moreover, inhibition of adenylyl cyclase activity (using a mixture of alpha(2)-adrenergic and adenosine A1 agonists receptors) did not change ANP- but suppressed isoproterenol-induced lipolysis. The noninvolvement of the PDE-3B was finally confirmed by measuring its activity under ANP stimulation. Thus, we demonstrate that natriuretic peptides are a new pathway controlling human adipose tissue lipolysis operating via a cGMP-dependent pathway that does not involve PDE-3B inhibition and cAMP production.     

Endothelin-1 inhibits resistin secretion in 3T3-L1 adipocytes

Resistin is an adipocyte-derived hormone whose role in the development of insulin resistance is controversial. Endothelin-1 (ET-1) is a 21 amino acid peptide demonstrated to possess vasoconstrictor, positive inotropic, mitogenic, and metabolic properties. In numerous disease states, including congestive heart failure, obesity, and diabetes, elevated levels of ET-1 have been reported and are thought to contribute to the pathology of the disease. A recent study demonstrated that ET-1 induces the expression and stimulates the secretion of the adipose tissue-derived hormone leptin. However, the effect of ET-1 on resistin secretion has not been determined. To characterize the effect of ET-1 on resistin secretion, 3T3-L1 fibroblasts were differentiated into adipocytes and allowed to mature for 14 days. Cells were incubated for 24h with ET-1 (1-100 nM), insulin (1-100 nM), insulin+ET-1 (100 nM I+E) or the appropriate vehicle or antagonist. At the end of the incubation period, resistin secretion was determined in the media by immunoblotting and densitometric analysis. ET-1 (1-100 nM) significantly decreased basal resistin secretion by 49% (1 nM), 43% (10nM), and 59% (100 nM). Insulin (1-100 nM) produced a concentration-dependent increase in resistin secretion from 3T3-L1 adipocytes (1 nM-42%, 10nM-55%, and 100 nM-86% vs. control). Insulin-stimulated resistin secretion (100 nM) was almost completely inhibited (94%) by ET-1 (100 nM). The effects of ET-1 on resistin protein secretion were inhibited by co-incubation with the ET(A) receptor antagonist BQ-610. In conclusion, our studies demonstrate that basal and hormonal stimulation of resistin secretion by insulin are inhibited by ET-1. Such findings demonstrate that resistin secretion is regulated in a similar manner to other adipose tissue factors, including leptin, in 3T3-L1 adipocytes. In addition, our findings suggest that vascular factors such as ET-1 may regulate whole body energy metabolism through adipocyte-derived hormones, including leptin and resistin.     

A preadipocyte clonal line from mouse brown adipose tissue. Short- and long-term responses to insulin and beta-adrenergics

A clonal cell line has been established from the interscapular brown adipose tissue (BAT) of the C57 BL/6J +/+ mouse. The line, designated BFC-1, is aneuploid and exhibits both morphological and biochemical properties characteristics of mature adipocytes. Adipose conversion begins after confluence and is accompanied by an early emergence of lipoprotein lipase; a later emergence of glycerol-3-phosphate dehydrogenase and acid: CoA ligase; an increase in the average triglyceride content. Adipose conversion, estimated by activities of enzyme markers, is enhanced at any given time by the continuous presence in the culture medium of insulin and triiodothyronine, both within their physiological range of concentrations. In addition to both hormones, chronic exposure of confluent cells to beta-adrenergics brings similar long-term effects on adipose conversion. The uptake of labelled 2-deoxyglucose by differentiated BFC-1 cells is stimulated by insulin; the half-maximum effect is observed at 1 nM insulin. Differentiated BFC-1 cells, in which endogenous triglycerides have been prelabelled on the fatty acid moiety, do respond to beta-adrenergics by releasing radioactive fatty acids. The agonist potency order and the EC50 value for each agonist are BRL 37344 (0.5 nM) greater than isoproterenol (1.5 nM) greater than norepinephrine (3 nM) greater than epinephrine (7 nM) greater than salbutamol (15 nM). The half-maximally and maximally effective concentrations of corticotropin to stimulate lipolysis are found to be 4 and 100 nM, respectively. The lipolytic response to isoproterenol is counteracted by prior addition of insulin or simultaneous addition of propranolol. Parallel studies performed on Ob17 cells, a clonal line established from mouse white adipose tissue (Négrel et al., Proc natl acad sci US 75 (1978) 6054), show that the agonist potency order and the EC50 value for each agonist are BRL 37344 (3 nM) greater than isoproterenol (10 nM) greater than norepinephrine (20 nM) greater than epinephrine (40 nM). Thus both BFC-1 cells and Ob17 cells show an atypical beta-adrenoreceptor similar to that described in rat adipocytes (Arch et al., Nature 309 (1984) 163), but the sensitivity of BFC-1 cells toward beta-agonists is found to be 6-fold higher than that of Ob17 cells. Thus the BFC-1 line represents a useful model for the study of short- and long-term responses to beta-adrenergics.     

Insulin antagonism of catecholamine stimulation of fatty acid transport in the adipocyte. Studies on its mechanism of action

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of catecholamines was mediated by a beta-receptor interaction and cAMP (Abumrad, N.A., Park, C.R., and Whitesell, R. R. (1986) J. Biol. Chem. 261, 13082-13086). In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the cAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phosphodiesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 microM) of the phosphodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 microgram/ml) gradually abolished insulin's antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 microM methylisobutylxanthine. Also consistent with involvement of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin's responsiveness. Treatment of cells with adenosine deaminase (1 unit/ml) enhanced the effect of norepinephrine by about 30%. A 10-fold higher range of insulin concentrations was then required to produce inhibition of fatty acid transport. The effect of adenosine removal was reversed by addition of phenylisopropyladenosine (500 nM), which is resistant to hydrolysis by the deaminase. Finally, exposure of insulin-treated cells (1 nM for 5 min) to dinitrophenol (1 mM for 5 min) reversed insulin action, consistent with reports of reversal of insulin's activation of the phosphodiesterase. In conclusion, our studies support the involvement of cAMP lowering in insulin's antagonism of fatty acid transport stimulation in the adipocyte.     

Regulation of adiponectin and leptin secretion and expression by insulin through a PI3K-PDE3B dependent mechanism in rat primary adipocytes

Adiponectin is intimately involved in the regulation of insulin sensitivity, carbohydrate and lipid metabolism, and cardiovascular functions. The circulating concentration of adiponectin is decreased in obesity and Type 2 diabetes. The present study attempts to elucidate the mechanisms underlying the regulation of adiponectin secretion and expression in rat primary adipocytes. The beta-agonist, isoprenaline, decreased adiponectin secretion and expression in a dose-dependent manner in primary adipocytes. Importantly, such an inhibitory effect could be blocked by insulin. The opposing effects of isoprenaline and insulin could be explained by differential regulation of intracellular cAMP levels, since cAMP analogues suppressed adiponectin secretion and expression in a fashion similar to isoprenaline, and insulin blocked the inhibitory effects of the cAMP analogue hydrolysable by PDE (phosphodiesterase). A specific PDE3 inhibitor, milrinone, and PI3K (phosphoinositide 3-kinase) inhibitors abolished the effects of insulin on adiponectin secretion and expression. In the same studies, leptin secretion and expression displayed a similar pattern of regulation to adiponectin. We conclude that insulin and beta-agonists act directly at the adipocytes in opposing fashions to regulate the production of adiponectin and leptin, and that a PI3K-PDE3B-cAMP pathway mediates the effects of insulin to restore beta-agonist/cAMP-suppressed secretion and expression of these two adipokines.     

Prolonged fasting on the lipolytic activity of isolated adipocytes of the rat epididymis

Male Wistar rats, 6-8 week old, were fasted for 72 hours. The in vitro lipolytic activity of epididymal adipocytes was measured in the presence of adrenalin (a alpha and beta adrenergic agonist), isoprenaline (a pure beta agonist), theophylline (a phosphodiesterase inhibitor) or UK 14304 (a alpha 2 adrenoceptor agonist) associated with adenosine deaminase. The basal lipolytic activity, expressed per 100 mg lipids, was higher in fasted adipocytes than in fed ones. Its stimulation by adrenalin or isoproterenol was decreased by fast. The effects of these drugs were more potentiated by theophylline in fasted adipocytes than in fed ones. The UK 14304 inhibition of adenosine deaminase-stimulated lipolysis was about 20% in fasted adipocytes and 50% in fed adipocytes. The in vitro resistance of fasted adipocytes to the lipolytic effect of adrenalin or isoproterenol may be related to the hypothyroid status of fasted rats.     

T3 and Triac inhibit leptin secretion and expression in brown and white rat adipocytes

Leptin regulates appetite, inhibits food intake, and seems to increase energy expenditure. We investigated the effect of triiodothyroacetic acid (Triac), a metabolite of T3, which seems to be more thermogenic than T3, on leptin secretion and mRNA expression. Rat primary cultures of white and brown adipocytes were treated with increasing concentrations of Triac and T3. The effect of different types of serum and insulin concentrations was also tested. Serum inhibited leptin secretion and mRNA expression. Leptin secretion was also clearly inhibited by Triac and T3 in a dose-dependent manner and with similar potency. In the presence of norepinephrine (NE), Triac and T3 had a similar inhibitory effect, but the inhibition was almost complete in white adipocytes. Parallel results were found at the mRNA level, where Triac and T3 had similar inhibitory potency, both alone and with NE. We also show that insulin induced dose- and time-dependent increases in leptin secretion, reaching maximum levels at 0.5 and 3 nM insulin for white and brown adipocytes, respectively. Leptin secretion was higher in white than in brown adipocytes. The increases in leptin secretion were preceded by increases in leptin mRNA. In conclusion, these data demonstrate for the first time that Triac, like T3 and serum, inhibits leptin secretion and expression in white and brown adipocytes, whereas insulin has the opposite effect.     

beta-Adrenergic stimulated lipolysis in pony adipocytes is exclusively via a beta2-subtype and is not affected by lactation

Catecholamines are important lipolytic agents in horses and ponies but the nature of the adrenergic receptor subtype distribution in their adipocytes is uncertain. A first objective was to identify the beta-adrenergic receptor subtype(s) present in adipocytes from horses and ponies. A second objective was to evaluate if the lipolytic responsiveness of isolated adipocytes to beta-adrenergic agonists is altered during lactation, a condition known to affect markedly maternal fat metabolism. Isoproterenol and salbutamol elicited strong lipolytic responses in adipocytes isolated from horse and pony subcutaneous adipose tissue. There were weak lipolytic responses to norepinephrine, dobutamine and BRL37344. The weak lipolytic response to NE compared to isoproterenol or salbutamol suggests an antilipolytic action from alpha2-adrenergic receptors. The relative order of potency for the beta-adrenergic agonists was isoproterenol>/=salbutamol>dobutamine=BRL37344. There was expression of beta2-adrenergic receptor mRNA in pony and horse adipose tissues, as estimated by relative RT-PCR, but no expression of mRNAs for beta1- or beta3-adrenergic receptors. Early lactation did not alter the lipolytic responses to beta-adrenergic agonists, nor the expression of beta2-adrenergic receptor mRNA. Thus, these results indicate a dominant if not exclusive presence of beta2-adrenergic receptors in pony and horse adipocytes that is not affected by lactation.     

Adrenergic control of lipogenesis and lipolysis in the chicken in vitro

The adrenergic inhibition of lipogenesis and stimulation of lipolysis in the avian has been examined using chicken hepatocytes and adipose tissue explants in vitro. Lipogenesis was inhibited by adrenergic agonists: epinephrine (alpha + beta) greater than isoproterenol (beta 1/beta 2) greater than norepinephrine (alpha 1/alpha 2, beta 1) greater than metaproterenol (beta 2), phenylephrine (alpha 1). Dobutamine (beta 1 agonist) and dopamine (dopaminergic agonist) did not significantly affect [14C]acetate incorporation into lipid, while clonidine and para-aminoclonidine (alpha 2 agonists) were slightly stimulatory. Lipolysis in young and adult chicken adipose tissue was stimulated by epinephrine, isoproterenol, phenylephrine, dobutamine and metaproterenol, but was inhibited by clonidine and para-aminoclonidine. Both the antilipogenic and lipolytic effects of epinephrine were partially blocked by phentolamine (alpha 1 = alpha 2 antagonist) or propranolol (beta 1 = beta 2 antagonist), but completely inhibited by phentolamine and propranolol administered together.     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.