Improved glycemic control in C57Bl/KsJ (db/db) mice after treatment with the thermogenic beta-adrenoceptor agonist, BRL 26830.

BRL 26830, (R*,R*)-(+/-)-methyl-4-(2-[(2-hydroxy-2-phenylethyl)amino]propyl)-benzoa te, is a new type of beta-adrenoceptor receptor agonist that combines antihyperglycemic and thermogenic properties. In C57Bl/KsJ db/db mice, treatment with BRL 26830 (50 mg of the hemifumarate salt/kg diet) decreased blood glucose concentration and normalized water intake. As judged by the normalization of polydipsia, BRL 26830 was effective within 2 days and the effect was maintained throughout a treatment period of up to 11 wk. Treatment of db/db mice with BRL 26830 resulted in an increase in both plasma and pancreatic insulin concentrations and a partial restoration of first-phase insulin secretion by the isolated, perfused pancreas in response to a high (16.7 mM) glucose pulse. Given acutely, BRL 26830 increased energy expenditure in both fed and fasted db/db mice. When given chronically, BRL 26830 increased significantly the dietary and thermoregulatory component of metabolic rate. It is suggested that the antidiabetic and thermogenic properties of BRL 26830 are linked and that blood glucose acts either directly or indirectly as a substrate for thermogenesis.

BRL 35135, a potent and selective atypical beta-adrenoceptor agonist.

BRL 35135, via its active deesterified metabolite BRL 37344, is a potent example of a new group of beta-adrenoceptor agonists that stimulate selectively a novel beta adrenoceptor that was originally shown to be present in brown adipose tissue in rodents. BRL 35135 produces a dose-related increase in energy expenditure in rodents and, in genetically obese (ob/ob) mice, a dose of 0.5 mg.kg-1.d-1 has significant antiobesity activity. This weight loss is entirely due to loss of fat; muscle protein is preserved. In studies in nonobese men, BRL 35135 (0.1 mg/kg) increased both resting metabolic rate and the thermic response to a glucose load. BRL 35135 is effective in improving glucose tolerance in genetically obese (ob/ob) mice and obese Zucker (fa/fa) rats at doses that have no significant antiobesity activity. The improved glucose tolerance is the result of significant improvement in insulin sensitivity. In 10-d studies in obese and diabetic patients, BRL 35135 produced improvements in glucose tolerance and insulin sensitivity.

The effect of 5-methylpyrazole-3-carboxylate and nicotinic acid on abnormalities of carbohydrate metabolism in alloxan-diabetic rat muscle.

The administration of the antilipolytic agents sodium nicotinate (1 mmole/kg i.p.) or sodium 5-methylpyrazole-3-carboxylate (0.5 or 1.0 mmole/kg i.p.) to alloxan-diabetic rats produced a significant reduction in the plasma concentration of free fatty acids and a slight reduction in blood glucose concentration. The concentrations in the freeze-clamped heart of citrate , acetyl CoA, Glucose-6-phosphate and fructose-6-phosphate were increased in untreated alloxan-diabetic rats relative to normoglycaemic controls. Treatment of alloxan-diabetic rats with the antilipolytic agents or insulin (60 U/kg i.p.) lowered these increased concentrations of metabolites in the heart. Treatment of the diabetic rats with the antilipolytic agents also produced an increase in the activity of pyruvate dehydrogenase in heart, but only treatment with 5-methylpyrazole-3-carboxylate had a significant effect on the activity of the enzyme in freeze-clamped soleus muscle.

Effects of BRL 26830, a novel beta-adrenoceptor agonist, on glucose tolerance, insulin sensitivity and glucose turnover in Zucker (fa/fa) rats.

Zucker fa/fa rats exhibit glucose intolerance in comparison with lean Fa/? littermates. A single acute dose of BRL 26830 (2.9 mg/kg p.o.) improved glucose tolerance in Fa/? littermates but exacerbated glucose intolerance in the fa/fa rats. This latter effect occurred in spite of an increase in the plasma insulin concentration. Chronic treatment of Zucker fa/fa rats with BRL 26830 (2.9 mg/kg) for 24 days or more produced a significant reduction in the area under the glucose tolerance curve. In addition, the glucose decay rate (k%) following the administration of insulin intravenously was significantly increased in the BRL 26830-treated rats suggesting that tissue insulin sensitivity was increased. Glucose turnover measurements show that chronic treatment of Zucker fa/fa rats with BRL 26830 produced a significant increase in the rate of glucose utilization integrated over a 3 hr period, but this increase was, in part, off-set by an increase in the endogenous rate of glucose production. The ultimate fate of the extra glucose that is metabolized is not known but it is suggested that it might be used to support the thermogenic response that is also activated by BRL 26830.

Anti-hyperglycaemic action of BRL 26830, a novel beta-adrenoceptor agonist, in mice and rats.

BRL 26830, (R*,R*)-(+/-)-methyl 4-[2-[(2-hydroxy-2-phenylethyl)amino] propyl] benzoate, is a new orally active anti-hyperglycaemic agent. In 24 hr-fasted rats and mice, BRL 26830 decreased the blood glucose concentration following the administration of a subcutaneous glucose load. It also improved oral and intravenous glucose tolerance in 24 hr-fasted rats and decreased the post-prandial blood glucose concentration following the consumption of the complete, milk-based, meal "Nutrament". BRL 26830 produced a dose-related increase in the plasma insulin concentration and since it was inactive in lowering blood glucose in streptozotocin-diabetic rats, it is likely that its acute action on glucose tolerance was through the stimulation of insulin secretion. In contrast to the sulphonylurea, glibenclamide, BRL 26830 had no effect on the blood glucose concentration in 5 hr-fasted rats and only produced a transient reduction in 24 hr-fasted rats. BRL 26830 did not improve glucose tolerance when given acutely to hyperinsulinaemic C57BL/6 ob/ob mice. However, chronic treatment of these mice with BRL 26830 for 14-43 days resulted in a significant improvement in glucose tolerance.

The rat lipolytic beta-adrenoceptor: studies using novel beta-adrenoceptor agonists.

EC50 and relative intrinsic activity values were obtained for isoprenaline, fenoterol, salbutamol, prenalterol and three new beta-adrenoceptor agonists, BRL 28410, BRL 35113 and BRL 35135 on rat white adipocyte lipolysis, rat atrial rate and tension, rat uterus tension and guinea-pig tracheal tension. Fenoterol and salbutamol were selective for tracheal and uterine responses, prenalterol was selective for atrial responses, but BRL 28410, BRL 35113 and BRL 35135 were selective for the adipocyte lipolytic response. pA2 values for propranolol, practolol, ICI 118,551 and sotalol were obtained on adipocytes, atria and trachea. pA2 values for propranolol and sotalol were much lower on adipocytes than on atria or trachea. The pA2 value for practolol was lower on adipocytes than on atria and the pA2 value for ICI 118,551 was lower on adipocytes than on trachea. Both agonist and antagonist studies therefore suggest that the rat adipocyte lipolytic receptor does not fit into the current beta 1/beta 2-adrenoceptor classification.

SK&F 104078, a post-junctionally selective alpha 2-adrenoceptor antagonist in the human saphenous vein in vitro.

The present study investigated the effects of SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3-methyl-1H,2,3,4,- tetrahydro-3-benzazapine) at pre- and post-junctional alpha 2-adrenoceptors in the human isolated saphenous vein. Noradrenaline (0.001-100 mumol/l) produced concentration-dependent contractions of the human saphenous vein which were competitively antagonised by the alpha 1-adrenoceptor antagonist prazosin (0.01-1.0 mumol/l) and the alpha 2-adrenoceptor antagonist, rauwolscine (0.01-1.0 mumol/l), indicating the presence of both post-junctional alpha 1- and alpha 2-adrenoceptors in this preparation. The selective alpha 2-adrenoceptor agonist, UK-14,304 (0.01-100 mumol/l) also produced concentration-dependent contractions of the human saphenous vein which were antagonised by both rauwolscine (0.1 mumol/l) and prazosin (0.1 mumol/l). In the presence of angiotensin II (0.05 mumol/l), which itself produced a transient contraction, rauwolscine (0.1 mumol/l) produced a rightward shift of the UK-14,304 concentration-response curve while prazosin (0.1 mumol/l) had no effect. SK&F 104078 (10.0 mumol/l) under these conditions also produced a rightward shift of the concentration-response curve to UK-14,304, but was at least 100-fold less potent than rauwolscine. At pre-junctional alpha 2-adrenoceptors, exogenous noradrenaline (0.01 and 0.1 mumol/l) induced a concentration-dependent inhibition of stimulation-evoked [7-3H]-noradrenaline release from the human saphenous vein in vitro, which was antagonised by rauwolscine (0.1 mumol/l) and tolazoline (10.0 mumol/l) but not by SK&F 104078 (10.0 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Improvement of glucose tolerance in Zucker diabetic fatty rats by long-term treatment with the dipeptidyl peptidase inhibitor P32/98: comparison with and combination with rosiglitazone.

AIM: The aim of this study was to investigate the effect of long-term treatment with the dipeptidyl peptidase inhibitor P32/98 and its combination with rosiglitazone on blood glucose control and islet of Langerhans histology in male Zucker diabetic fatty (ZDF) rats, when treatment begins before or after the development of overt diabetes. METHODS: ZDF rats were treated with P32/98 from the age of 9, 12 or 15 weeks. Rosiglitazone maleate was given to a separate group from the age of 13 weeks. P32/98 was given to all of these rosiglitazone-treated rats from 16 weeks of age. Rosiglitazone maleate was also given from 16 weeks of age to half the rats that were given P32/98 from 9 weeks of age. The compounds were given by oral gavage until the rats were 14 weeks old and then in the diet. The experiment was terminated at the age of 20-21 weeks. Blood glucose, plasma insulin and oral glucose tolerance were measured at intervals; islet histology was assessed terminally. RESULTS: P32/98 improved glucose tolerance after both single and multiple doses when treatment started at 9 weeks of age, also after the third week of treatment when treatment began at 12 or 15 weeks of age. P32/98 reduced daytime blood glucose when treatment began at 12 weeks. Treatment with rosiglitazone increased food intake and body weight, and after 2 weeks, reduced daytime blood glucose, water intake and the area under the glucose tolerance curve. A single dose of P32/98 markedly improved glucose tolerance in rosiglitazone-treated rats. When treatment had begun at 9 weeks of age, P32/98 stimulated insulin secretion in some glucose tolerance tests. Neither P32/98 nor rosiglitazone affected pancreatic insulin content, nor did they have clear effects on islet histology. CONCLUSION: P32/98 elicited a sustained improvement in glucose tolerance in both prediabetic and diabetic ZDF rats. The effects of P32/98 on glucose and insulin were similar to those of rosiglitazone, and in contrast to rosiglitazone, P32/98 did not increase food intake or body weight. However, neither compound was especially effective at improving diabetes in ZDF rats when treatment began at 9, 12 or 15 (P32/98) or 13 (rosiglitazone) weeks of age.

Effect of a novel thermogenic beta-adrenoceptor agonist (BRL 26830) on insulin resistance in soleus muscle from obese Zucker rats.

Young lean (Fa/?) and obese (fa/fa) rats were treated with the thermogenic beta-adrenoceptor agonist, BRL 26830, for 3 weeks. In lean rats this treatment had no effect on body weight but there was a marked increase in the insulin sensitivity of soleus muscle strips with respect to glycolytic rate. Treatment of obese rats with BRL 26830 produced a small but not significant decrease in body weight but the sensitivity of both glycolysis and glycogen synthesis to insulin was increased so that muscles of treated obese rats showed similar insulin sensitivity to untreated lean rats. It is suggested that such changes are unlikely to be merely a secondary consequence of an anti-obesity action.

Retinoid X receptor agonists have anti-obesity effects and improve insulin sensitivity in Zucker fa/fa rats.

OBJECTIVE: To investigate whether retinoid X receptor agonists act as insulin sensitizers and compare their effects with that of thiazolidinedione BRL 49653 in obese Zucker rats. DESIGN: In two independent studies, obese Zucker rats were dosed orally once daily for 14 days with one of the following treatments: LG 100268 (20 mg/kg), LG 100324 (20 mg/kg), BRL 49653 (3 mg/kg) or vehicle. MEASUREMENTS: Daily food intake and body weight gain, blood glucose, plasma and pancreatic insulin, whole body glucose disposal (by euglycaemic-hyperinsulinaemic clamp) and tissue glucose utilization. RESULTS: The retinoid X receptor agonists (rexinoids) LG 100268 and LG 100324 caused a reduction in the food intake of obese Zucker rats relative to controls and to rats receiving BRL 49653. The two rexinoids also produced a marked decrease in the body weight gain, whereas the growth rate of rats treated with BRL 49653 tended to increase. Both rexinoids and BRL 49653 reduced the plasma insulin concentration of fed rats. LG 100268 and LG 100324 also significantly lowered blood glucose concentrations after 1 week of treatment. The 5 h fasted plasma insulin concentration was significantly lower in the rexinoid-treated groups and the terminal insulin level (at the end of the clamp) tended to be lower in all treated groups compared with animals given the dosing vehicle. However, pancreatic insulin content was not affected by any of the treatments. Under euglycaemic-hyperinsulinaemic clamp conditions, there were no significant differences in the rate of hepatic glucose output and whole body glucose disposal, except that, in experiment 1, BRL 49653 caused significant increase in the glucose infusion rate and muscle glucose utilization. In experiment 2, a similar glucose infusion rate to the controls was achieved in all treatment groups but the steady-state insulin concentration in the treated animals was only about 50% of that in the control animals, despite the fact that all rats received a similar insulin infusion concentration. This suggests that both the rexinoids and BRL 49653 increased insulin clearance. CONCLUSIONS: Chronic administration of retinoid X receptor agonists LG 100268 and LG 100324 to Zucker fa/fa rats reduces food intake and body weight gain, lowers plasma insulin concentrations while maintaining normoglycaemia, indicating an improvement of insulin sensitivity.

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).

The beta-adrenoceptor selectivity profile of BRL 37344 in the pithed rat.

1. Rats were pithed in order to disrupt baroreflex pathways. Heart rate was used as a measure of beta 1-adrenoceptor activity, blood pressure as a measure of beta 2-adrenoceptor activity and oxygen consumption and brown adipose tissue temperature as measures of beta 3-adrenoceptor activity. 2. The effects of the selective beta 3-adrenoceptor agonist BRL 37344 were compared with those of isoprenaline, a non-selective beta-adrenoceptor agonist, and denopamine and salbutamol, which are respectively beta 1 and beta 2-adrenoceptor agonists. 3. Denopamine was 10-fold more potent on heart rate than blood pressure, whilst salbutamol was 18-fold more potent on blood pressure than heart rate. These findings confirm that in this preparation increases in heart rate are predominantly beta 1 adrenoceptor-mediated, whilst blood pressure is beta 2-adrenoceptor-mediated. Further confirmation is provided by the blockade with atenolol, of the chronotropic effect, but not the blood pressure effect, of isoprenaline. 4. BRL 37344 was the most potent beta-adrenoceptor agonist on both oxygen consumption and brown adipose tissue temperature, revealing the beta 3-nature of these responses. Dose-response curves for oxygen consumption and brown adipose tissue temperature were identical, whichever of the beta-adrenoceptor agonists was used. Both systems may be considered equally effective as indicators of beta 3-adrenoceptor agonist activity.

Atypical beta-adrenoceptor on brown adipocytes as target for anti-obesity drugs.

Recent studies suggest that thermogenesis in brown adipose tissue has an important role in the regulation of energy balance. Thermogenesis is effected by noradrenaline released from sympathetic nerve endings; the noradrenaline stimulates beta-adrenoceptors, causing lipolysis, and the released fatty acids then promote the uncoupling of oxidative phosphorylation from electron transport. It has been widely accepted that mammalian beta-adrenoceptors exist as two subtypes, beta 1 and beta 2, and rat brown adipocyte beta-adrenoceptors have been classed as beta 1 or as a mixed beta 1/beta 2 population. The beta 1 subtype predominates in atria, whereas the beta 2 subtype predominates in trachea. However, we have now found a novel group of beta-adrenoceptor agonists that selectively stimulate lipolysis in brown adipocytes. In contrast, isoprenaline, fenoterol and salbutamol are less potent as stimulants of lipolysis than as stimulants of atrial rate or tracheal relaxation. Therefore, beta-adrenoceptors in rat brown adipocytes are of neither the beta 1 nor beta 2 subtypes. Compounds that selectively stimulate brown adipocyte beta-adrenoceptors should have potential as thermogenic anti-obesity agents and this has been demonstrated with BRL 26830A , BRL 33725A and BRL 35135A .

Effects of novel beta-adrenoceptor agonists on carbohydrate metabolism: relevance for the treatment of non-insulin-dependent diabetes.

BRL 26830A, (R*, R*)-(+/-)-methyl 4-[2-[(2-hydroxy-2-phenylethyl) amino] propyl]-benzoate, (E)-2-butenedioate (2:1) salt, is a new antihyperglycemic agent orally active in obese-hyperglycemic animal models. In C57Bl/6 ob/ob mice, BRL 26830A (1 mg/kg) given daily for periods of 14 d-6 weeks produced a marked improvement in glucose tolerance and a reduction in the fasting plasma insulin concentration. Adipocytes prepared from treated mice showed improved insulin responsiveness. In Zucker fa/fa rats, treatment with BRL 26830A (2.9 mg/kg/d for 23 d) produced improvements in both glucose tolerance and whole animal insulin sensitivity, as assessed by rate of fall of blood glucose in response to an intravenous dose of insulin. In C57Bl/KSJ db/db mice, BRL 26830A (admixed with food at 50 mg/kg diet) decreased blood glucose to values similar to those in lean mice. At the end of a 10-week treatment, BRL 26830A-treated mice had a higher plasma and pancreatic insulin content than the untreated db/db mice. In normoglycaemic rats and mice, BRL 26830A increases the plasma insulin concentration and increases glucose disposal. However, in most circumstances, there is a counteracting increase in endogenous glucose synthesis and, therefore, no change in blood glucose occurs. Improvements in glucose tolerance occur in 24-h fasted rats and mice. BRL 26830A has thermogenic activity and it is suggested that this might be linked to increased glucose utilization. Brown adipose tissue (BAT) of C57Bl/KSJ db/db mice has a reduced maximum glycolytic capacity relative to lean littermates. Treatment with BRL 26830A increased the glycolytic capacity 10-fold.

Modulation of susceptibility to weight gain and insulin resistance in low birthweight rats by treatment of their mothers with leptin during pregnancy and lactation.

OBJECTIVES: To investigate whether administration of leptin to rats during pregnancy and lactation affects placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD2) activity and the susceptibility of their offspring to weight gain and insulin resistance. DESIGN: Pregnant rats fed on a low-protein diet were administered leptin or saline by subcutaneous minipump from day 14 of gestation and throughout lactation. A further group was fed a normal diet and given saline. After weaning, the offspring of each group were fed on a normal diet until 6 weeks of age and then half of each group was transferred to a high-fat diet until 12 months of age. RESULTS: Plasma leptin levels were raised two-fold on days 16-18 of pregnancy in the leptin-treated dams, but, despite a constant rate of infusion, at parturition they dipped to control levels before rising again. The activity of placental 11beta-HSD2 was reduced by the low-protein diet; this reduction was prevented by treating the dams with leptin. The male offspring of the saline-treated dams gained more weight and had higher plasma leptin levels on the high fat than the chow diet, but the offspring of the leptin-treated dams did not. Fasting blood glucose and intraperitoneal glucose tolerance at 6 and 12 months of age was unaffected by the high-fat diet, but only the offspring of the leptin-treated dams achieved this control without raised insulin levels. CONCLUSIONS: The rate of leptin clearance appears to increase at parturition. The administration of leptin to rats during late pregnancy and lactation makes their male offspring less susceptible to high-fat-diet-induced weight gain and insulin resistance.

The tissue distribution of the human beta3-adrenoceptor studied using a monoclonal antibody: direct evidence of the beta3-adrenoceptor in human adipose tissue, atrium and skeletal muscle.

OBJECTIVE: To develop a monoclonal antibody that recognises an epitope of the native beta3-adrenoceptor expressed on the extracellular surface of human cells and tissues. DESIGN: A high affinity monoclonal antibody, Mab72c, was raised against the human beta3-adrenoceptor expressed on a transfected mammalian cell line. RESULTS: In CHO (Chinese hamster ovary) cells transfected with beta3-adrenoceptor cDNA, antibody labelling was found to be proportional to receptor density measured by the binding of the radiolabelled beta-adrenoceptor antagonist, [125I]-iodocyanopindolol. The use of Mab 72c has demonstrated the expression of the beta3-adrenoceptor in a variety of human tissues, including gall bladder, prostate and colon, where a mRNA signal had been detected previously. This study also provides the first direct demonstration of the expression of beta3-adrenoceptors in human skeletal muscle, atrium and adipose tissue. CONCLUSION: The development of this antibody represents an important addition to the armentarium of reagents that are available to study the localisation of beta3-adrenoceptors in human tissues.

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities.

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.