The selectivity in vitro of the stereoisomers of the beta-3 adrenoceptor agonist BRL 37344.

The stimulation by BRL 37344 of lipolysis in rat adipose tissues, and of relaxation of the rat distal colon, is mediated by the beta-3 adrenoceptor. The stereochemical requirements of the beta-3 adrenoceptor are poorly understood. The activities of the four stereoisomers of BRL 37344 (i.e., two pairs of diastereoisomers) on three beta-3 adrenoceptor-mediated responses (brown and white adipose tissue lipolysis and relaxation of distal colon) have been determined and compared with those responses mediated by beta-1 adrenoceptors (increase in atrial rate) and beta-2 adrenoceptors (uterine relaxation). The potency order for the stereoisomers (RR>RS=SR>>SS) was the same for all tissues, regardless of whether the response was mediated by beta-1, beta-2 or beta-3 adrenoceptors. These results indicate that both chiral centers are determinants of agonist potency at all three subtypes of the beta adrenoceptor. Furthermore, agonist activity at beta-1, beta-2 and beta-3 adrenoceptors resides predominantly with the RR enantiomer. Finally, the RR enantiomer of BRL 37344 was a more potent agonist in brown adipocytes (EC50 = 3.3 +/- 0.8 nM) than in white adipocytes (EC50 = 5.7 +/- 0.9 nM) or colon (EC50 = 27.5 +/- 7.7 nM).

Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cell-surface GLUT4 as measured by photoaffinity labeling.

(+/-)-5-([4-[2-Methyl-2(pyridylamino)ethoxy]phenyl]methyl) 2,4-thiazolidinedione (BRL 49653) is a new potent antidiabetic agent that improves insulin sensitivity in animal models of NIDDM. In C57BL/6 obese (ob/ob) mice, BRL 49653, included in the diet for 8 days, improved glucose tolerance. The half-maximal effective dose was 3 mumol/kg diet, which is equivalent to approximately 0.1 mg/kg body wt. Improvements in glucose tolerance were accompanied by significant reductions in circulating triacylglycerol, nonesterified fatty acids, and insulin. The insulin receptor number of epididymal white adipocytes prepared from obese mice treated with BRL 49653 (30 mumol/kg diet) for 14 days was increased twofold. The affinity of the receptor for insulin was unchanged. In the absence of added insulin, the rates of glucose transport in adipocytes from untreated and BRL 49653-treated obese mice were similar. Insulin (73 nmol/l) produced only a 1.5-fold increase in glucose transport in adipocytes from control obese mice, whereas after BRL 49653 treatment, insulin stimulated glucose transport 2.8-fold. BRL 49653 did not alter the sensitivity of glucose transport to insulin. The increase in insulin responsiveness was accompanied by a 2.5-fold increase in the total tissue content of the glucose transporter GLUT4. Glucose transport in adipocytes from lean littermates was not altered by BRL 49653. To establish the contribution of changes in glucose transporter trafficking to the BRL 49653-mediated increase in insulin action, the cell-impermeant bis-mannose photolabel 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-mannos++ +-4-yloxy) -2-[2-3H]-propylamine was used to measure adipocyte cell-surface-associated glucose transporters.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of adenylyl cyclase in goldfish brain.

The rate of production of cAMP by the adenylyl cyclase enzyme from goldfish brain was linear with time and with protein concentration. In agreement with mammalian adenylyl cyclase systems the enzyme is divalent cation dependent, being activated in the presence of either Mg2+ or Mn2+. Forskolin also stimulated the rate of reaction in a dose-dependent manner with a half-maximal effect of 1 microM. The activated enzyme was inhibited by high concentrations of Ca2+ but was independent of Na+ concentration. The presence of guanine nucleotide binding proteins (G-proteins) was demonstrated by the fact that both NaF and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated the basal rate. In addition, the p[NH]ppG dose-response curve of the forskolin-stimulated enzyme was biphasic, similar to that observed for other systems. At low concentrations of p[NH]ppG a small inhibition was observed while higher concentrations produced a stimulation. These data suggest that the goldfish brain adenylyl cyclase enzyme complex includes both stimulatory and inhibitory G-proteins in addition to the catalytic unit. A series of known and putative goldfish neurotransmitter substances failed to either stimulate or inhibit the adenylyl cyclase activity. The endogenous neurotransmitters which interact with this second messenger system remain to be determined.

Correlation of beta 3-adrenoceptor-induced activation of cyclic AMP-dependent protein kinase with activation of lipolysis in rat white adipocytes.

The lipolytic action of the beta 3-adrenoceptor-selective agonist 4-[2-[(2-hydroxy-2-(3-chlorophenyl)ethyl)-amino]propyl]-phenoxyacetic acid (BRL 37344) was compared to that of isoprenaline in adipocytes derived from rat white adipose tissue. Concentration-response curves for activation of lipolysis by each agonist correlated well with the dose-response curves for activation of cAMP-dependent protein kinase (A-Kinase). Addition of propranolol at a concentration (0.1 microM) sufficient to block beta 1- and beta 2-adrenoceptors did not affect the stimulation of either parameter by BRL 37344 or isoprenaline, indicating that lipolysis was predominantly dependent on beta 3-adrenoceptor stimulation. Blockade of beta 3-adrenoceptors by 3 microM propranolol antagonized both A-Kinase activation and glycerol release. Activation of lipolysis by BRL 37344 was blocked by treatment of the cells with N-[2-p-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide (H89) a potent and selective isoquinolinesulphonamide inhibitor of A-Kinase activity. Taken together, these results indicate that lipolysis in rat white adipocytes is primarily controlled by beta 3-adrenoceptors, and that cyclic AMP generation alone is responsible for activation of lipolysis in this tissue.

Demonstration of inhibitory guanine nucleotide regulatory protein (Gi) function in liver and hepatocyte membranes from streptozotocin-treated rats.

By using a defined plasma-membrane preparation, functional inhibition of adenylate cyclase activity by the inhibitory G-protein (Gi) was observed in liver and hepatocyte membranes from rats made diabetic by streptozotocin. These observations contrast with previous reports which have shown a defect in Gi in this diabetic animal model. These results suggest that Gi function is not impaired in the livers of streptozotocin-treated rats and that plasma-membrane preparation procedures should be clearly defined before ascribing Gi defects to a pathological state such as diabetes.

Evaluation of inhibitory guanine nucleotide regulatory protein Gi function in hepatocyte and liver membranes from obese Zucker (fa/fa) rats and their lean (Fa/?) littermates.

Previous studies have shown that hepatocyte and liver membranes from insulin resistant animals exhibit an impairment of inhibitory guanine nucleotide binding regulatory protein, Gi function, such that a Gi defect may contribute towards the diabetic syndrome. In the current studies, it is shown that the demonstration of Gi activity in liver and hepatocyte membranes is dependent critically on the membrane preparation technique. A technique is defined that allows functional Gi activity to be demonstrated in liver and hepatocyte membranes from both lean (Fa/?) and obese (fa/fa) Zucker rats. Consequently, previous reports on the loss of Gi function in insulin resistant states require revaluation.

Lymphocyte enzyme activities in East African blacks: decrease in 5'nucleotidase and possible relation to immunosuppression.

Microanalysis of subcellular organelle marker enzymes was applied to cryopreserved lymphocytes (obtained and processed in the field) from East African blacks with moderate to severe malnutrition and subject to locally endemic parasitic and infectious diseases. An initial study demonstrated that activities of these enzymes, with the partial exception of catalase, were stable to cryopreservation. Cryopreserved and thawed lymphocyte specimens (1 to 3 X 10(6) viable cells) from 26 Africans and 20 Caucasian controls were studied. There was a highly significant decrease in 5'nucleotidase activity in these African subjects. Activity of another plasma membrane enzyme, gamma-glutamyl transferase, and of marker enzymes for other intracellular organelles, was not significantly different between the two groups, indicating that the nucleotidase alteration is highly specific. 5'Nucleotidase activity in a group of 17 East African blacks of high socio-economic status lay between the values obtained in the other two groups and was not significantly different from either. Further studies on 5'nucleotidase showed no evidence that the enzyme is functionally different in Africans. The differences in activity of this enzyme in Africans may reflect the known immuno-suppressive effects of infectious disease and malnutrition or may have a genetic basis which may in turn be associated with the pathogenesis of secondary immunodeficiency.