Horst Kather

Human fat cell lipolysis is primarily regulated by inhibitory modulators acting through distinct mechanisms.

The effects of adenosine deaminase and of pertussis toxin on hormonal regulation of lipolysis were investigated in isolated human fat cells. Adenosine deaminase (1.6 micrograms/ml) caused a two-to threefold increase in cyclic AMP, which was associated with an increase in glycerol release averaging 150-200% above basal levels. Clonidine, N6-phenylisopropyladenosine, prostaglandin E2, and insulin caused a dose-dependent inhibition of glycerol release in the presence of adenosine deaminase. Pretreatment of adipocytes with pertussis toxin (5 micrograms/ml) for 180 min resulted in a five- to sevenfold increase in cyclic AMP. Glycerol release was almost maximal and isoproterenol caused either no further increase or only a marginal additional increase of lipolysis after pretreatment with pertussis toxin, whereas cyclic AMP levels were 500 times higher than in controls. The effects of antilipolytic agents known to affect lipolysis by inhibition of adenylate cyclase activity, i.e., clonidine, N6-phenylisopropyladenosine, and prostaglandin E2, were impaired. In contrast, the antilipolytic action of insulin was preserved in adipocytes pretreated with pertussis toxin. As in controls, the peptide hormone had no detectable effect on cyclic AMP after pertussis toxin treatment. The findings support the view that the antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action. In addition, the results demonstrate that, upon relief of endogenous inhibition, human fat cell lipolysis proceeds at considerable (adenosine deaminase) or almost maximal (pertussis toxin) rates. A certain degree of inhibition, therefore, appears to be necessary for human fat cell lipolysis to be susceptible for hormonal activation.

Microdetermination of glycerol using bacterial NADH-linked luciferase

A bioluminescent assay for determination of glycerol using bacterial NADH-linked luciferase was developed and successfully applied to measurement of glycerol release from human fat cells. The procedure is based on enzymic conversion of glycerol to 3-phosphoglycerate, which is irreversible in the presence of arsenate, and subsequent determination of NADH formed in the glycerol-phosphate- and glyceraldehyde-3-phosphate dehydrogenase reactions respectively. Bioluminescent determination of glycerol is about 100 times more sensitive than conventional spectrophotometry, thus permitting more than 100 determinations to be carried out on needle biopsy specimens.

Biphasic Effects of Prostaglandin E2 on the Human Fat Cell Adenylate Cyclase

Adenylate cyclase of human fat cell ghosts shows a biphasic response towards prostaglandin E2 with inhibition occurring at nanomolar concentrations of the hormone and stimulation at concentrations beyond 10(-6) mol/liter. The expression of the inhibitory effect is critically dependent on GTP. Under the conditions employed (1 mmol/liter ATP, 5 mmol/liter Mg2+, 30 degrees C) the inhibitory component of prostaglandin E2 became apparent at GTP concentrations exceeding 10(-6) mol/liter. The prostaglandin E2-induced inhibition displayed characteristic features of prostaglandin action in intact fat cells with respect to the effective concentrations and degree of inhibition. It is concluded that prostaglandin E2 is capable of inducing antagonistic effects upon lipolysis via interaction with the membrane-bound adenylate cyclase.

Adrenoceptor of the alpha2‐subtype mediating inhibition of the human fat cell adenylate cyclase

Abstract. In an attempt to characterize the adenylate cyclase‐coupled alpha‐adrenoceptors of human fat cells the effects of various alpha‐adrenergic agonists and antagonists were examined in the presence of 0.05 mmol/1 of propranolol.

Interaction of laxatives with enzymes of cyclic AM P metabolism from human colonic mucosa

Abstract. The mechanism by which laxatives such as dioctyl sodium sulfosuccinate and ricinoleic acid evoke colonic fluid secretion has been suggested to involve mucosal cyclic AMP. Ricinoleic acid and dioctyl sodium sulfosuccinate were tested for their capacity to modulate the key enzymes of cAMP‐metabolism‐ adenylate cyclase and cAMP‐phosphodiesterase‐in human colonic mucosa.

Human colonic adenylate cyclase: effects of bile acids.

Abstract. Three different bile acids—deoxycholic acid. chenodeoxycholic acid and ursodeoxycholic acid‐were tested for their capacity to stimulate the adenylate cyclase in human colonic mucosa.

Chemiluminescent determination of adenosine, inosine, and hypoxanthine/xanthine

A fully automatic method for analysis of adenosine, inosine, and hypoxanthine/xanthine which combines the specificity of enzymatic catalysis and sensitivity of chemiluminescence is presented. The hydrogen peroxide formed by sequential catabolism of purines to uric acid is detected by the oxidation of luminol in the presence of peroxidase. The method takes advantage of the fact that light output in the H2O2/luminol system is transient. By adopting a two-step procedure this feature enables selective determination of adenosine, inosine, and hypoxanthine/xanthine. In step 1 any purines lower in the catabolic sequence than the analyte under study are converted to uric acid. Light emission is allowed to decay to baseline levels. During step 2 the analyte is selectively degraded. The H2O2 formed leads to a new light emission which is proportional to the square of analyte concentration. The method can be performed with commercially available reagents and enzymes and requires minimal processing of biological samples. Excellent agreement has been obtained with HPLC analysis. Sensitivity is in the range of 5-10 nmol/liter in as little as 0.1 ml. More than 200 samples per day can be analyzed by a single operator.

Human Colonic Adenylate Cyclase

The effects of meclofenamic acid on basal and hormone-stimulated adenylate cyclase in human colonic mucosa were tested. It is shown that meclofenamic acid is a relatively specific inhibitor of the stimulatory action of prostaglandins on the level of mucosal adenylate cyclase. This compound did not inhibit significantly the effects of vasoactive intestinal peptide (VIP) on the human enzyme system. VIP18-28 did not act as a partial agonist and did not inhibit the response to the intact VIP. Our studies suggest the existence of distinct receptor sites for both secretagogues (VIP and prostaglandins) in human colonic mucosa.

Islet‐activating protein discriminates the antilipolytic mechanism of insulin from that of other antilipolytic compounds

In vivo administration of islet‐activating protein to rats resulted in an increase in fat cell lipolysis in vitro, which was associated with almost complete resistance of adipocytes towards the antilipolytic effects of N 6‐phenylisopropyladenosine, prostaglandin E2 and nicotinic acid. Concomitantly, the inhibitory effects of these compounds on adenylate cyclase activity in membranes were impaired. In contrast, the antilipolytic action of insulin was not only preserved, but even augmented in cells from rats treated with islet‐activating protein. The data suggest that insulin exerts its antilipolytic effects via mechanisms which are different from those involved in the effects of prostaglandin E2, N 6‐phenylisopropyladenosine and nicotinic acid.

Inhibition of human fat cell adenylate cyclase mediated via alpha‐adrenoceptors

Abstract. Human adipose tissue contains alpha‐ as well as β‐adrenoceptor sites mediating antagonistic catecholamine effects on lipolysis. To characterize the mechanisms of catecholamine action in biochemical terms we have studied the effects of the almost pure β‐adrenoceptor agonist isoproterenol and of the mixed adrenergic agonist adrenaline on human fat cell adenylate cyclase in the presence of alpha‐ and β‐adrenoceptor blocking drugs.