Increased 4-Hydroxynonenal Formation Contributes to Obesity-Related Lipolytic Activation in Adipocytes

Oxidative stress in adipose tissue plays an etiological role in a variety of obesity-related metabolic disorders. We previously reported that increased adipose tissue 4-hydroxynonenal (4-HNE) contents contributed to obesity-related plasma adiponectin decline in mice. In the present study, we investigated the effects of intracellular 4-HNE accumulation on lipolytic response in adipocytes/adipose tissues and underlying mechanisms. In both fully-differentiated 3T3-L1 and primary adipocytes, a 5-hour 4-HNE exposure elevated lipolytic reaction in a dose-dependent manner at both basal and isoproterenol-stimulated conditions, evidenced by significantly increased glycerol and fatty acids releases. This conclusion was corroborated by the comparable observations when the minced human visceral adipose tissues were used. Mechanistic investigations revealed that 4-HNE-stimulated lipolytic activation is multifactorial. 4-HNE exposure quickly increased intracellular cyclic AMP (cAMP) level, which was concomitant with increased phosphorylations of protein kinase A (PKA) and its direct downstream target, hormone sensitive lipase (HSL). Pre-incubation with H89, a potent PKA inhibitor, prevented 4-HNE stimulated glycerol release, suggesting that enhanced lipolytic action in response to 4-HNE increase is mediated mainly by cAMP/PKA signal pathway in adipocytes. In addition to activating cAMP/PKA/HSL pathway, 4-HNE exposure also suppresses AMP-activated protein kinase (AMPK), a suppressive pathway for lipolysis, measured by both Western blotting for phosphorylated form of AMPK and ELISA for enzyme activity. Furthermore, 5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), a pharmacological AMPK activator, alleviated 4-HNE-induced lipolysis, suggesting that AMPK suppression also contributes to 4-HNE elicited lipolytic response. In conclusion, our findings indicate that increased intracellular 4-HNE accumulation in adipocytes/adipose tissues contributes to obesity-related lipolytic activation.     

Homocysteine suppresses lipolysis in adipocytes by activating the AMPK pathway

Hyperhomocysteinemia (HHcy) is an independent risk factor for coronary artery disease. Emerging evidence suggests that HHcy is also associated with adipocyte tissue dysfunction. One of the principal functions of adipose tissue is to provide energy substrate via lipolysis. In the present study, we investigated the effects of homocysteine (Hcy) on lipolysis in adipocytes. We found that Hcy inhibited release of glycerol and fatty acids, two typical indicators of the lipolytic response, in primary adipocytes and fully differentiated 3T3-L1 adipocytes in a dose-dependent manner under both basal and isoproterenol-stimulated conditions. In differentiated 3T3-L1 adipocytes, decreased glycerol and free fatty acid (FFA) release was associated with elevation of intracellular TG content. Further studies showed that Hcy-mediated antilipolytic responses were independent of the cyclic AMP-PKA and MEK-ERK1/2 pathways. However, Hcy increased phosphorylation levels of AMP-activated protein kinase (AMPK) and its downstream enzyme acetyl-CoA carboxylase. Compound C, an AMPK inhibitor, abolished Hcy-induced reduction of glycerol and FFA release under both basal and isoproterenol-stimulated conditions. Furthermore, AMPKα1 siRNA reversed Hcy-inhibited glycerol release. Supplementation of exogenous Hcy in the diet for 2 wk lowered circulating glycerol and FFA levels. Moreover, Hcy supplementation was associated with elevated leptin levels and reduced adiponectin levels in plasma. These results show that Hcy inhibits lipolysis through a pathway that involves AMPK activation.     

Hormonal control of lipolysis from the white adipose tissue of hibernating jerboa (Jaculus orientalis)

1. Plasma glucose, glycerol, free fatty acids and total lipid content of the white adipose tissue were measured in euthermic and hibernating jerboa. 2. During hibernation, plasma glucose and glycerol were low compared to the euthermic animals, whereas there was no obvious difference in plasma free fatty acids. The white adipose tissue lipid content was strongly reduced in the hibernating state. 3. The effect of lipolytic hormones (norepinephrine and glucagon) and antilipolytic hormone (insulin) on in vitro glycerol release by adipose tissue isolated from hibernating or euthermic jerboa has been studied. 4. The white adipose tissue from hibernating jerboa presented a higher sensitivity to norepinephrine and glucagon than that of euthermic jerboa; insulin did not modify either basal glycerol release or lipolysis induced by the two lipolytic hormones at low temperatures (7 degrees C) and during the rewarming (from 7 degrees C to 37 degrees C) of the tissue slices. 5. These results suggested that white adipose tissue constitutes an important source of substrates derived from lipolysis during hibernation.     

Lipolysis and reesterification: effects of some inhibitors of adenosine 3',5'-cyclic monophosphate phosphodiesterase

Theophylline and three lipolytic agents, 2,5-bis(2-chloroethylsulfonyl)-pyrrole-3,4-dicarbonitrile (substituted pyrrole), 2,4-diamino-6-butoxy-s-triazine (substituted triazine), and 2,3-dihydro-5,6-dimethyl-3-oxo-4-pyridazinecarbonitrile (substituted pyridazine), stimulate basal lipolysis in adipose tissue in vitro. They also cause an increased release of free fatty acids, but not glycerol, from adipose tissue in which lipolysis is already maximally stimulated by epinephrine. The four compounds also inhibit cyclic AMP phosphodiesterase and the conversion of [1-(14)C]glucose to (14)CO(2). Evidence is presented that free fatty acids accumulate as the result of inhibited reesterification. The substituted pyridazine and triazine, but not the pyrrole, elevate plasma free fatty acids after oral or intraperitoneal administration in rats.     

Hormones and triacylglycerol metabolism under normoxic and ischemic conditions

Summary Fatty acids, the preferred substrate in normoxic myocardium, are derived from either exogenous or endogenous triacylglycerols. The supply of exogenous fatty acids is dependent of the rate of lipolysis in adipose tissue and of the lipoprotein lipase activity at the coronary vascular endothelium. A large part of the liberated fatty acids is reesterified with glycerol-3-phosphate and converted to triacylglycerols. Endogenous lipolysis and lipogenesis are intracellular compartmentalized multienzyme processes of which individual hormone-sensitive steps have been demonstrated in adipose tissue. The triacylglycerol lipase is the rate-limiting enzyme of lipolysis and glycerol-3-phosphate acyltransferase and possibly phosphatidate phosphohydrolase are the rate-limiting enzymes of lipogenesis. The hormonal regulation of both processes in heart is still a matter of dispute. Triacylglycerol lipase activity in myocardial tissue has two intracellular sources: 1, the endoplasmic reticular and soluble neutral lipase, and 2. the lysosomal acid lipase. Studies in our laboratory have indicated that whereas lipolysis is enhanced during global ischemia and anoxia, overall lipolytic enzyme activities in heart homogenates were not altered. In addition we were unable to demonstrate alterations in tissue triacylglycerol content and glycerol-3-phosphate acyltransferase activity under these conditions. Lipolysis, is subject to feedback inhibition by product fatty acids. Therefore all processes leading to an increased removal of fatty acids from the catalytic site of the lipase will stimulate lipolysis. These studies will be reviewed. In addition, studies from our department have demonstrated the capacity of myocardial lysosomes to take up and degrade added triacylglycerol-particles in vitro. Such a process, stimulated by Ca²⁺ and stimulated by acidosis, offers another physiological target for hormone actions.     

A low-protein, high-carbohydrate diet increases the adipose lipid content without increasing the glycerol-3-phosphate or fatty acid content in growing rats

The amount of triacylglycerol (TAG) that accumulates in adipose tissue depends on 2 opposing processes: lipogenesis and lipolysis. We have previously shown that the weight and lipid content of epididymal (EPI) adipose tissue increases in growing rats fed a low-protein, high-carbohydrate (LPHC) diet for 15 days. The aim of this work was to study the pathways involved in lipogenesis and lipolysis, which ultimately regulate lipid accumulation in the tissue. De novo fatty acid synthesis was evaluated in vivo and was similar for rats fed an LPHC diet or a control diet; however, the LPHC-fed rats had decreased lipoprotein lipase activity in the EPI adipose tissue, which suggests that there was a decreased uptake of fatty acids from the circulating lipoproteins. The LPHC diet did not affect synthesis of glycerol-3-phosphate (G3P) via glycolysis or glyceroneogenesis. Glycerokinase activity - i.e., the phosphorylation of glycerol from the hydrolysis of endogenous TAG to form G3P - was also not affected in LPHC-fed rats. In contrast, adipocytes from LPHC animals had a reduced lipolytic response when stimulated by norepinephrine, even though the basal adipocyte lipolytic rate was similar for both of the groups. Thus, the results suggest that the reduction of lipolytic activity stimulated by norepinephrine seems essential for the TAG increase observed in the EPI adipose tissue of LPHC animals, probably by impairment of the process of activation of lipolysis by norepinephrine.     

Lack of feedback regulation of cyclic 3':5'-AMP accumulation by free fatty acids in chicken fat cells.

Fat cells isolated from the mesenteric adipose tissue of chickens (pullets) responded to glucagon with an increase in lipolysis and a sustained rise in cyclic adenosine 3':5'-monophosphate (cyclic AMP) over a 30-min incubation. The prolonged accumulation of cyclic AMP due to glucagon in chicken fat cells was primarily intracellular. In addition, there was little increase in cyclic AMP accumulation due to theophylline alone or potentiation of the increase due to glucagon. These data indicate that chicken fat cells, unlike rat fat cells, are relatively insensitive to theophylline. Neither lipolysis nor cyclic AMP accumulation by chicken fat cells was inhibited by free fatty acid to albumin ratios (3 to 7) which markedly reduced both events in rat fat cells. However, in the absence of albumin from the medium, lipolysis in chicken fat cells was reduced, but not to the same extent as in rat fat cells. Chicken fat cells did accumulate more intracellular free fatty acids in response to lipolytic agents than did rat fat cells. The uptake of oleate by rat and chicken fat cells was identical. Glucagon-induced accumulation of cyclic AMP by chicken fat cell ghosts was unaffected by added oleate. Under identical conditions glucagon-induced adenylate cyclase activity of rat fat cell ghosts was markedly inhibited by added oleate. Triglyceride lipase activity of the pH 5.2 precipitate from a 40,000 x g infranatant of homogenized fat cells from chickens was less sensitive than that from rat fat cells to the ratio of oleate to albumin. These results suggest that the maintenance of cyclic AMP levels in chicken fat cells incubated with lipolytic agents results from the relative insensitivity of chicken fat cells to free fatty acid inhibition of cyclic AMP accumulation.     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

Summary Hormone stimulated lipolysis of mouse and rabbit adipocytes as measured by both free fatty acid and glycerol release, is proportionally elevated with increase in the adipocyte cAMP level up to 1 nmole/g. The correlation coefficients are 0.94 and 0.97 for FFA/cAMP and glycerol/cAMP respectively. Increments in cAMP greater than 1 nmole/g show no correlation with increase in lipolysis. The release of lipolytic products, glycerol and free fatty acids, from white adipocytes in response to ACTH, epinephrine or morepinephrine was measured using radiochemical assays in short term incubation systems, with cAMP levels measured at the same time and from the same cell sample. Under the conditions studied, epinephrine is a more effective lipolytic hormone than ACTH in mouse adipocyte, and ACTH is more effective than epinephrine in rabbit adipocyte. The effect of catecholamines on the rabbit adipocyte is not modified by phentolamine (10 μM), but it is potentiated by 1-methyl-3-isobutyl xanthine (0.1 mM). The results suggest that cAMP mediates the action of these lipolytic hormones in white adipocytes of mouse and rabbit.     

Comparative effects of cholera and Bordetella pertussis toxins on cyclic AMP and GTP levels and on lipolysis in rat adipocytes incubated in vitro

The respective effects of cholera and Bordetella pertussis toxins were studied in time and concentration dependent experiments, following glycerol and fatty acid release, GTP and cAMP levels. Cholera toxin, after a lag time of 30 min, stimulated linearly GTP and cAMP accumulation and lipolysis (maximal effect: 2-fold increase at 5 micrograms/ml). Pertussis toxin presented a biphasic effect both in time and concentration dependent studies. Up to a maximum reached after 2 h with 1.4 units LPF/ml the stimulation affected GTP (3 fold) and cAMP (7 fold) levels, glycerol and fatty acid release (15 fold). Beyond this, an inhibition occurred, yielding a decrease towards basal values of GTP and cAMP content whereas the glycerol and fatty acid release was stopped. These results, which are the first reporting the fluctuation of the GTP content of intact cells challenged with bacterial toxins, show a close relationship between GTP and cyclic AMP levels and lipolytic activity.     

Ledakrin effect on free fatty acid and glycerol mobilization from epididymal adipose tissue of rat in vitro

It was demonstrated that Ledakrin, an antitumour drug, causes mobilization of free fatty acids and glycerol from the epididymal adipose tissue of rat in vitro. The disproportion observed in the release of glycerol and free fatty acids following Ledakrin administration suggested a biphasic effect of this drug, with initial stimulation and later inhibition of the processes of lipolysis and lipogenesis. Blockade of adrenergic membrane receptors (with propranolol or regitine) abolished the lipolytic effect of Ledakrin.     

Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue

Glucose utilization was studied in isolated fat cells prepared from rat adipose tissue which had been cultured for 18 hr in TC 199 medium. When 1% bovine serum albumin (BSA) was in the culture medium, basal rates of (14)CO(2) and [(14)C]triglyceride production from [1-(14)C]glucose were markedly depressed and there was no effect of insulin. With 4% BSA, basal (14)CO(2) production was the same as in cells prepared from fresh tissue and basal triglyceride production was greatly increased. Insulin effect on these cells was minimal. One-minute uptake of [(14)C]2-deoxyglucose was stimulated by 800-1000% in fresh cells and 300-500% in cells cultured with either 1% or 4% BSA. Oxidation of [U-(14)C]glucose showed a much smaller impairment in cultured cells than for [1-(14)C]glucose, suggesting that the pentose phosphate shunt was more severely impaired than glycolysis. Glyceride-glycerol production was increased in cultured cells relative to preculture (fresh) cells. There was no effect of insulin in the culture medium in any of these systems. Rates of free fatty acid and glycerol release were markedly increased in cultured cells, especially when insulin was present in the culture medium. The acute antilipolytic effect of insulin was retained, so that insulin in the test incubation decreased lipolysis by 40-80%. Nevertheless, cell-associated fatty acids were increased in cultured cells and FFA/albumin ratios in the medium often reached potentially toxic levels. The reduction in pentose phosphate shunt activity, lipogenesis, and insulin effect resembles other models of insulin insensitivity. The impaired metabolism is probably due to an intracellular defect. A possible toxic role of either intracellular or extracellular fatty acids cannot be excluded. This system should be a useful model in which to study the cellular mechanisms of insulin insensitivity in adipocytes.-Bernstein, R. S. Insulin insensitivity and altered glucose utilization in cultured rat adipose tissue.     

Role of PAT proteins in lipid metabolism

One of the central reactions in bodily energy metabolism is lipolysis in adipocytes, the reaction that governs the release of stored fatty acids from the adipocyte triacylglycerol pool, which constitutes the major energy reserve in animals. These fatty acids are then transported by serum albumin to various tissues to supply their energy requirements. This reaction was previously thought to result from phosphorylation and activation of hormone-sensitive lipase by protein kinase A (PKA) but is now known to be governed by a translocation of the lipase from the cytosol to the surface of the intracellular lipid droplet that houses the reservoir of TAG. This droplet is coated with perilipin A, which is also phosphorylated by PKA in response to lipolytic stimuli, and phosphorylation of perilipin A is essential for HSL translocation and stimulated lipolysis.     

Human glucagon and vasoactive intestinal polypeptide (VIP) stimulate free fatty acid release from human adipose tissue in vitro

Glucagon, vasoactive intestinal polypeptide and secretin are strong stimulators of lipolysis in adipose tissue from laboratory animals. Yet, in human adipose tissue these data could not be confirmed under comparable experimental conditions. Using pH stat titration, an advanced in vitro test system for evaluating lipolysis, it was possible to demonstrate lipolytic activity for glucagon down to a concentration of 10(-8) mol/l. This is comparable to the minimal effective doses in rat adipose tissue and corresponds to the effect of equimolar concentrations of noradrenaline in man. Secretin with an amino acid sequence very similar to glucagon was not lipolytically active, while VIP stimulated free fatty acid release in a concentration of 10(-6) mol/l. Since the minimal effective dose of glucagon is only 30 times greater than the plasma levels a physiological significance of these finding may be suggested. The lipolytic activity of VIP seems to be only of pharmacological interest.     

Intracellular accumulation of free fatty acids in isolated white adipose cells

A simple, rapid, and accurate method was developed for measuring intracellular FFA levels in isolated white adipose cells using sucrose-(14)C or inulin carboxyl-(14)C as nontransportable, nonutilizable markers of the extracellular space. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose-(14)C or inulin carboxyl-(14)C retained in the floating packed adipose cells. In this way the FFA content of the adipose cells could be corrected for contamination by FFA bound to extracellular albumin. With this technique the initial events in hormone-activated lipolysis were studied under conditions of maximal and constant rates of triglyceride hydrolysis. The FFA content of isolated adipocytes of fed rats was 0.5 micro mole/g cell lipid. On addition of norepinephrine in the presence of medium albumin, the concentration of intracellular FFA rapidly increased and reached a plateau at a concentration of 2-2.5 micro moles/g cell lipid. In the presence of medium albumin an initial lag in glycerol release occurred and this was attributed to partial hydrolysis of triglyceride with retention of lower glycerides. After 5 min of incubation FFA and glycerol output was constant. In the absence of medium albumin norepinephrine-stimulated lipolysis was reduced more than 90% and extracellular FFA release was not detected. Nevertheless, intracellular FFA accumulation was identical to that seen in the presence of albumin. The data suggest that most of this intracellular pool of FFA is bound to cytoplasmic constituents.     

Short-term fasting and lipolytic activity in rat adipocytes.

The aim of this experiment was to study the influence of 18-hour food deprivation on basal and stimulated lipolysis in adipocytes obtained from young male Wistar rats. Fat cells from fed and fasted rats were isolated from the epididymal adipose tissue by collagenase digestion. Adipocytes were incubated in Krebs-Ringer buffer (pH 7.4, 37 degrees C) without agents affecting lipolysis and with different lipolytic stimulators (epinephrine, forskolin, dibutyryl-cAMP, theophylline, DPCPX, amrinone) or inhibitors (PIA, H-89, insulin). After 60 min of incubation, glycerol and, in some cases, also fatty acids released from adipocytes to the incubation medium were determined. Basal lipolysis was substantially potentiated in cells of fasted rats in comparison to adipocytes isolated from fed animals. The inhibition of protein kinase A activity by H-89 partially suppressed lipolysis in both groups of adipocytes, but did not eliminate this difference. The agonist of adenosine A (1) receptor also did not suppress fasting-enhanced basal lipolysis. The epinephrine-induced triglyceride breakdown was also enhanced by fasting. Similarly, the direct activation of adenylyl cyclase by forskolin or protein kinase A by dibutyryl-cAMP resulted in a higher lipolytic response in cells derived from fasted animals. These results indicate that the fasting-induced rise in lipolysis results predominantly from changes in the lipolytic cascade downstream from protein kinase A. The antagonism of the adenosine A (1) receptor and the inhibition of cAMP phosphodiesterase also induced lipolysis, which was potentiated by food deprivation. Moreover, the rise in basal and epinephrine-stimulated lipolysis in adipocytes of fasted rats was shown to be associated with a diminished non-esterified fatty acids/glycerol molar ratio. This effect was presumably due to increased re-esterification of triglyceride-derived fatty acids in cells of fasted rats. Comparing fed and fasted rats for the antilipolytic effect of insulin in adipocytes revealed that short-term food deprivation resulted in a substantial deterioration of the ability of insulin to suppress epinephrine-induced lipolysis.     

Medium‐Chain Fatty Acids Attenuate Agonist‐Stimulated Lipolysis,Mimicking the Effects of Starvation

Objective: To test the hypothesis that incorporation of medium‐chain fatty acids (FAs) into adipocyte triglycerides alters intracellular lipolysis. Research Methods and Procedures: 3T3‐L1 adipocytes were pretreated with octanoate for various incubation periods. After the removal of exogenous FAs, cells were incubated with different lipolytic agonists. To determine the effects on lipolysis, we measured the following: the release of glycerol and FAs, lipase activity, protein levels of hormone‐sensitive lipase (HSL), and perilipin A; translocation of HSL; phosphorylation of perilipin A; and levels of cellular adenosine triphosphate, cyclic adenosine monophosphate, and H₂O₂. To compare the effects of starvation with those caused by octanoate pretreatment, we measured glycerol release and H₂O₂ generation in rat adipocytes of starved donors. Results: Pretreatment of adipocytes with octanoate in vitro increased basal lipolysis but decreased the cellular response for agonists. The same effects were seen in starvation in vivo. Preincubation with octanoate for 48 hours did not affect basal lipase activity, HSL, and perilipin protein levels, but it reduced agonist‐stimulated perilipin phosphorylation and HSL translocation toward fat droplets. This was associated with a reduction in basal cellular adenosine triphosphate levels and agonist‐stimulated cyclic adenosine monophosphate generation. Starvation and octanoate pretreatment both increased intracellular H₂O₂ concentrations, which might also contribute to the inhibition on agonist‐stimulated lipolysis. Discussion: Pretreatment with octanoate seems to induce changes in adipocyte lipolysis in a pattern mimicking the effects of starvation. Such changes could contribute, in part, to weight loss in animals and humans associated with dietary medium‐chain FAs.     

STRUCTURE-FUNCTION RELATIONSHIPS IN THE ADIPOSE CELL : III. Effects of Bovine Serum Albumin on the Metabolism of Glucose and the Release of Nonesterified Fatty Acids and Glycerol by the Isolated Adipose Cell

Serum albumin stimulates the uptake of U-glucose-¹⁴C and the incorporation of ¹⁴C-counts into triglyceride glycerol and inhibits the incorporation of ¹⁴C-counts into triglyceride fatty acids by isolated adipose cells; insulin and epinephrine enhance these effects. In the absence of hormones, these responses to albumin increase with increasing albumin concentration. In the presence of insulin, a qualitatively similar pattern of increasing responses to albumin is observed; the enhancement of each response by insulin is, however, only slightly potentiated by higher albumin concentrations. In contrast, in the presence of epinephrine, these responses to albumin are maximal at the lowest albumin concentration tested, 0.1%; the enhancement of each response by epinephrine is similarly maximal at 0.1% albumin, but decreases rapidly as the albumin concentration is raised. Increasing serum albumin concentrations do, however, stimulate the release of fatty acids and glycerol by epinephrine-treated cells increasingly until a plateau, determined by the epinephrine dose, is reached. These data support the suggestion that intracellular fatty acid levels function in the regulation of adipose cell activity, and further suggest that serum albumin plays a role in determining the metabolic fate of these fatty acids.     

Triglyceride Storage Disease: A Disorder of Lipolysis in Adipose Tissue in Two Patients

An obese patient was studied whose adipose tissue showed a significant reduction in release of glycerol (P < 0·05) when stimulated by isoprenaline despite normal rises in tissue levels of cyclic-AMP. However, lipolysis was stimulated by a fast of 14 days as judged by weight loss and a rise in plasma fatty acids from 0·4 to 1·2 mM. The defect in this patient may be familial since her obese daughter also showed diminished release of glycerol from adipose tissue on three occasions when stimulated by isoprenaline, despite normal rises in levels of cyclic-AMP.     

Parathyroid hormone-induced lipolysis in human adipose tissue

Relative lipolytic activity of human parathyroid hormone-(1-34) (hPTH-(1-34], hPTH-(3-34), desamino-Ser1-hPTH-(1-34), and rat PTH-(1-34) was compared in human subcutaneous adipose tissues in vitro. Human PTH-(1-34), rat PTH-(1-34), and desamino-Ser1-hPTH-(1-34) stimulated in vitro lipolysis significantly above basal level at the concentration of 10(-6) M. Average increments of lipolytic rate were 2.39, 1.82, and 0.87 mumol/g per 2 hr, respectively, being significantly different among the three groups. On the other hand, hPTH-(3-34)-induced lipolytic rate was 0.83 +/- 0.18 mumol/g per 2 hr, not significantly different from the basal level (0.71 +/- 0.20 mumol/g per 2 hr). The effect of hPTH-(3-34) on glycerol release stimulated by hPTH-(1-34), isoproterenol, or forskolin was subsequently investigated. Human PTH-(3-34) produced a dose-dependent inhibition of hPTH-(1-34)-stimulated lipolysis. In contrast, isoproterenol- and forskolin-induced lipolytic rates were not influenced by hPTH-(3-34). The effect of propranolol on hPTH-(1-34)- or isoproterenol-induced lipolysis was also studied. Propranolol dose-dependently inhibited isoproterenol-induced lipolysis but had no effect on lipolysis stimulated by hPTH-(1-34). These results suggest that the amino acids at positions 1 (serine) and 2 (valine) of PTH are critical for the stimulation of lipolysis in human adipose tissue. Human PTH-(1-34) causes lipolysis after binding to receptors distinct from beta-adrenergic receptors of fat cells and possibly hPTH-(3-34) inhibits hPTH-(1-34)-stimulated lipolysis by competing at the level of PTH receptor.