Jorma J. Ohisalo

Defective Stimulation of Adipocyte Adenylate Cyclase, Blunted Lipolysis, and Obesity in Pseudohypoparathyroidism 1a

ABSTRACT: Adipocyte plasma membranes were isolated from four patients with type la pseudohypoparathyroidism, a disease in which deficiency of the stimulatory guanine nucleotide binding protein Gs has been reported, and from controls. Stimulation of adenylate cyclase by isoproterenol was defective, whereas inhibition of forskolin-stimulated cyclase activity by N6-(phenylisopropyl)adenosine was normal. The patients had low serum FFA concentrations and developed obesity in childhood. These results suggest that pseudohypoparathyroidism 1a is connected with a blunted stimulatory response of adenylate cyclase, possibly because of low Gs activity, and that this blunted response may lead to decreased lipolysis and to obesity.

Hepatotoxicity and absorption of extrahepatic acetaldehyde in rats

Acetaldehyde, the first metabolite of ethanol oxidation, has been proposed as a major initiating factor in ethanol‐induced liver injury. The aims of this study were to examine whether acetaldehyde is absorbable from the digestive tract and whether, when delivered chronically in drinking water, it is capable of inducing liver injury in rats. Acetaldehyde concentrations in the rat portal and peripheral blood were measured by head space gas chromatography after intragastric (5 ml) and intracolonic (3 ml) administration of 20 mM acetaldehyde solution. In the hepatotoxicity study, rats were exposed to acetaldehyde (20 and 120 mM) delivered in drinking water for 11 weeks and histopathological changes in the liver were morphometrically assessed. Peak blood acetaldehyde levels were found at 5 min after acetaldehyde infusion and were 235±11 μμ (mean±SE) after intragastric and 344±83 μμ after intracolonic infusion of 20 mM acetaldehyde solution. The exposure of rats to 120 mM acetaldehyde solution for 11 weeks resulted in the development of fatty liver and inflammatory changes. Morphometric analysis showed significantly more fat accumulation in rats receiving 120 mM acetaldehyde solution (85±2 per cent of hepatocytes occupied by fat) than in rats receiving 20 mM acetaldehyde solution (38±11 per cent) or in controls (36±10 per cent). The dose of extrahepatic acetaldehyde (500 mg/kg per day) producing liver injury corresponds to only around 3 per cent of that derived from hepatic ethanol oxidation in animals receiving an ethanol‐containing totally liquid diet (15 g/kg per day). These results indicate that acetaldehyde delivered via the digestive tract can reach the liver by the portal circulation and that acetaldehyde of extrahepatic origin appears to be more hepatotoxic than acetaldehyde formed during ethanol oxidation within the liver.

Quantitation of inhibitory G-proteins in fat cells of obese and normal-weight human subjects.

Antisera were raised in rabbits against synthetic peptides corresponding to sequences of the guanine nucleotide binding proteins Gi1, Gi2, Gi3 and Go. These and previously described antisera were used to identify different G-proteins in Western blots of human adipocyte plasma membranes and to quantify them using purified recombinant alpha subunits as standards. Go was shown to be absent or << 15 pmol/mg of protein. A band stained by a previously characterized Go antiserum is suggested to be due to nonspecific staining of Gi1. Gi1 and Gi2 were the major G-proteins. Gi1 was present at concentrations of 52 and 18 pmol/mg of protein in lean and obese subjects, respectively, and the concentration was negatively correlated with the body mass index. Gi2 concentrations averaged 64 pmol/mg of protein and there was no correlation to the body mass index. Gi3 levels were much lower (<< 13 pmol/mg of protein) and the presence of this protein could not be demonstrated with certainty. The concentrations of Gi1 and Gi2 are thus over two orders of magnitude higher than those of the receptors whose effects they mediate. The low concentration of Gi1 in adipocyte plasma membranes of obese subjects could in part explain the attenuated inhibitory responses of adenylate cyclase in isolated fat cells in obesity.

Regulation of adenylate cyclase in plasma membranes of human intraabdominal and abdominal subcutaneous adipocytes

Fat cells were isolated from human subcutaneous, omental, and mesenteric adipose tissue. Omental fat cells were the smallest (438 pL), subcutaneous cells were intermediate (494 pL), and mesenteric cells were the largest (600 pL). There was no difference in the stimulation of adenylate cyclase by isoproterenol in plasma membranes of adipocytes prepared from the three sites. N6-(phenylisopropyl)adenosine inhibited 7-deacetyl-6-(N-acetylglycyl)forskolin-stimulated adenylate cyclase activity more potently in subcutaneous than in intraabdominal (especially omental) fat cell membranes. Kd values of the adenosine A1 receptors for 1,3-[3H]dipropyl-8-cyclopentylxanthine were similar in the three fat depots, but the receptor number as calculated per milligram protein was lower in omental than in abdominal subcutaneous adipocytes. Differences in adipocyte size cannot explain regional differences in the regulation of adenylate cyclase.

STIMULATION OF LIPOPROTEIN LIPASE ACTIVITY OF RAT ADIPOSE TISSUE AND POST-HEPARIN PLASMA BY N6-(PHENYLISOPROPYL)ADENOSINE

Adenosine has profound effects on adipose tissue metabolism. It is an inhibitor of 13-adrenergic stimulation of cyclic AMP accumulation [1,2] and lipolysis [2-101 in tire isolated rat fat cell. It also opposes the effects of adrenaline on phosphate uptake [ 11 ]. In vivo, tile nucleoside has been reported to lower plasma free fatty acid and glycerol concentrations [8]. The effects of adenosine are thought to be mediated by two membrane receptors, one requiring an intact ribose (R-site) and tile other one an intact purine moiety [ 12-14] (P-site). Adenosine is rapidly metabolized but its effects on the R-site [12-14] are shared by purine-substituted analogs such as N6-(phenylisopro pyl)adenosine that are not deaminated by the enzyme adenosine deaminase (EC 3.5.4.4) [15]. Lipoprotein lipase (EC 3.1.1.3.4) is the enzyme responsible for the hydrolysis and uptake of triglycerides from circulating chylomicrons and very low density lipoproteins [16-19] . The enzyme is thought to be located on the endothelial surface of the capillary bed of extrahepatic tissues and it can be released into the circulation by intravenous injection of heparin [ 16-20] . Another heparin-releasable ectoenzyme is probably located on the outer surface of the endothelial cells of the liver and it is therefore called hepatic lipase [20]. This enzyme has been suggested to hydrolyze high density lipoprotein phospholipids and triglycerides [21 ]. This work was undertaken to find out ifN6-(phenyl isopropyl)adenosine has effects on lipoprotein lipase activity and, consequently, on the uptake of circulating triglycerides into tissues. Male Lewis albino rats ( 150 -250 g body wt) were used. They were fed ad libitum with a standard chow until injected intraperitoneally with 50-300/al isotonic NaC1 with/without N6-(phenylisopropyl)adenosine, at which time food was withheld. At the times indicated, the animals were anaesthetized with ether and blood was drawn by cardiac puncture into heparinized syringes. For post-heparin plasma lipase assays, blood was drawn 2 min after intravenous injection ofheparin (2000 units/kg body wt). The blood was centrifuged and the resulting plasma was stored at 2 0 ° C until assayed. Free glycerol was estimated enzymatically by using the commercial kit no. 125 032 of BoehringerMannheim GmbH. Tile assays for porst-heparin plasma lipases and the antibody to rat hepatic lipase have been described in [22]. The method used to measure heparin-releasable lipoprotein lipase activity of adipose tissue will be presented in detail in the text. N6-(phenylisopropyl)Adenosine was a gift from Dr Harald Stork of Boehringer-Mannheim GmbH. Glyceroltri-[14C] oleate (49 mCi/mmol) was from the Radiochemical Centre (Amersham). Heparin was from Medica (ttelsinki). All other reagents were from Sigma (St Louis MO).

Modulation of lipolysis by adenosine and Ca2+ in fat cells from hypothyroid rats

Thyroid hormones are required to render peripheral tissues sensitive to fl-adrenergic stimulation. Accordingly, catecholamines fail to stimulate lipolysis in adipocytes isolated from hypothyroid rats [l-6] whereas in normal fat cells the stimulatory effect is thoroughly documented. A decreased number of /3-adrenergic receptors [2], disturbance in transduction of information between receptor and adenylyl cyclase [3], defective adenylyl cyclase [4] and increased phosphodiesterase activity [ 1,5] have all been suggested to be involved in this altered responsiveness. Adenosine accumulates in preparations of isolated rat fat cells and it is an inhibitor of adenylyl cyclase activity [7]. Removal of this endogenous adenosine by added adenosine deaminase results in restoration of the lipolytic response to catecholamine stimulation [6]. The inhibitory effect of adenosine on adenylyl cyclase in broken fat cell preparations was reported dependent on the presence of Ca’+ and abolished by EGTA [8]. In [9] removal of Ca” from the incubation medium resulted in restoration of adrenalinestimulated lipolysis to normal levels in fat cells prepared from hypothyroid rats. This work was undertaken to investigate the possible involvement of external Ca2+ in the hypothyroid insensitivity towards catecholamine stimulation and the dependence on Ca2’ of the inhibitory actions of adenosine in fat cells.

Mechanisms of thyroid hormone control over sensitivity and maximal contractile responsiveness to β-adrenergic agonists in atria

This paper discusses the mechanisms of two basic effects of thyroid hormones on atrial responses to β-adrenergic agonists, i.e. increased inotropic sensitivity and decreased maximal contractile responsiveness. The increased sensitivity of atria to β; adrenergic agonists under thyroid hormones appears to be related to increases in β-adrenoceptor density and Gs/Gi. protein ratio, leading to activation of Gs-mediated pathway, but suppression of Gi.-mediated pathway of adenylate cyclase regulation. Therefore, the i/c concentrations of cAMP and corresponding inotropic responses achieve their maximums at lower doses of β-adrenergic agonist. Thyroid hormones also decrease the expression of phospholamban, but increase the expression of sarcoplasmic reticulum Ca2+-pump. As a result, the basal activity of sarcoplasmic reticulum Ca2+-pump increases, but its β-adrenergic activation through phosphorylation of phospholamban decreases. It is suggested that these changes are causal for decreased maximal inotropic and lusitropic responses of atria to β-adrenergic agonists.(Mol Cell Biochem 184: 419–426, 1998)

Membrane-bound phosphodiesterases in rat myocardium

Isoenzyme‐specific phosphodiesterase (PDE) inhibitors are potential positive inotropic drugs. For evaluating such drugs in experimental models and to understand the physiological roles of the different isoenzymes, it is necessary to know what isoenzymes are present in the tissues studied. Rat myocardium has been reported to be devoid of the particulate cGMP‐inhibited cAMP‐PDE (type III isoenzyme). Here we re‐evaluate the isoenzyme profile of rat myocardium.

On the induction of tyrosine aminotransferase in rat liver by α-methyl-p-tyrosine

Abstract Hepatic tyrosine aminotransferase ( l -tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) is known to be induced by α-methyl-p-tyrosine, a well-known catecholamine depletor, in both intact and adrenalectomized rats. The authors have studied this subject further and their results show that α-methyl-p-tyrosine does not influence the activity of tyrosine aminotransferase in the isolated, perfused liver and that hypophysectomy totally abolishes the induction of the enzyme by this agent. The involvement of hypophyseal hormones is discussed.

Decreased expression of phospholamban is not associated with lower β-adrenergic activation in rat atria

The aim of the study was to find out whether low phospholamban level in atria as compared with ventricles is associated with differences in sarcoplasmic reticular Ca2+-uptake and contractile performance. Relationship between phospholamban and β-adrenergic stimulation in rat left atria and papillary muscles were examined by means of contractile measurements, sarcoplasmic reticular oxalate-supported Ca2+-uptake, and Western blotting of phosphorylated phospholamban. Phosphoprotein determination after β-adrenergic stimulation demonstrated that the levels of Ser16 and Thr17 phosphorylated phospholamban in atria remained at about one-third of that in ventricles. However, comparison of sarcoplasmic reticular Ca2+-uptake in control and isoproterenol perfused preparations demonstrated that the effect of β-adrenergic stimulation on sarcoplasmic reticular Ca2+-uptake was stronger in atrial preparations. Moreover, atria responded to isoproterenol with much larger increases in developed tension, contractility and relaxation rates than papillary muscles. Thus, despite lower level of phospholamban, the β-adrenergic activation of sarcoplasmic reticular Ca2+-uptake and contractile indices are higher in atria.