Differential regulation of adipocytokine mRNAs by rosiglitazone in db/db mice.

The precise mechanism by which PPARgamma activation by thiazolidinediones (TZDs) improves insulin sensitivity is still unclear. Recent studies have focused on the role of adipocytokines in metabolic control and their regulation by TZDs. In this study, we compared the chronic effects of antihyperglycemic doses of the TZD rosiglitazone, the beta3-adrenoceptor agonist BRL-35135, and the PPARalpha agonist Wy-14,643 on the mRNA expression of adipocytokines in WAT of db/db mice. Rosiglitazone treatment decreased adiponectin and resistin mRNA levels by 57 and 72%, respectively (P < 0.001), with no effect on the level of TNFalpha or RELMalpha transcripts. In comparison, Wy-14,643 reduced adiponectin transcript levels by 31% (P = 0.015) while BRL-35135 increased RELMalpha mRNA expression by 245% (P < 0.001) without effect on the other transcripts. Our results indicate that although a reduction in adiponectin and resistin mRNA levels in WAT by rosiglitazone treatment of diabetic mice may contribute to the antidiabetic effects, an alteration in TNFalpha, adiponectin, resistin, or RELMalpha mRNA expression is not absolutely required for the regulation of blood glucose concentration in the db/db mouse.

Differential vasoconstrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and cynomolgus monkey.

1. Urotensin-II (U-II) and its G-protein-coupled receptor, GPR14, are expressed within mammalian cardiac and peripheral vascular tissue and, as such, may regulate mammalian cardiovascular function. The present study details the vasoconstrictor profile of this cyclic undecapeptide in different vascular tissues isolated from a diverse range of mammalian species (rats, mice, dogs, pigs, marmosets and cynomolgus monkeys). 2. The vasoconstrictor activity of human U-II was dependent upon the anatomical origin of the vessel studied and the species from which it was isolated. In the rat, constrictor responses were most pronounced in thoracic aortae and carotid arteries: -log[EC(50)]s 9.09+/-0.19 and 8.84+/-0.21, R(max)s 143+/-21 and 67+/-26% 60 mM KCl, respectively (compared, for example, to -log[EC(50)] 7.90+/-0.11 and R(max) 142+/-12% 60 mM KCl for endothelin-1 [ET-1] in thoracic aortae). Responses were, however, absent in mice aortae (-log[EC(50)] <6.50). These findings were further contrasted by the observation that U-II was a 'coronary-selective' spasmogen in the dog (-log[EC(50)] 9.46+/-0.11, R(max) 109+/-23% 60 mM KCl in LCX coronary artery), yet exhibited a broad spectrum of vasoconstrictor activity in arterial tissue from Old World monkeys (-log[EC(50)]s range from 8.96+/-0.15 to 9.92+/-0.13, R(max)s from 43+/-16 to 527+/-135% 60 mM KCl). Interestingly, significant differences in reproducibility and vasoconstrictor efficacy were seen in tissue from pigs and New World primates (vessels which responded to noradrenaline, phenylephrine, KCl or ET-1 consistently). 3. Thus, human U-II is a potent, efficacious vasoconstrictor of a variety of mammalian vascular tissues. Although significant species/anatomical variations exist, the data support the hypothesis that U-II influences the physiological regulation of mammalian cardiovascular function.

Mice overexpressing human uncoupling protein-3 in skeletal muscle are hyperphagic and lean.

Uncoupling protein-3 (UCP-3) is a recently identified member of the mitochondrial transporter superfamily that is expressed predominantly in skeletal muscle. However, its close relative UCP-1 is expressed exclusively in brown adipose tissue, a tissue whose main function is fat combustion and thermogenesis. Studies on the expression of UCP-3 in animals and humans in different physiological situations support a role for UCP-3 in energy balance and lipid metabolism. However, direct evidence for these roles is lacking. Here we describe the creation of transgenic mice that overexpress human UCP-3 in skeletal muscle. These mice are hyperphagic but weigh less than their wild-type littermates. Magnetic resonance imaging shows a striking reduction in adipose tissue mass. The mice also exhibit lower fasting plasma glucose and insulin levels and an increased glucose clearance rate. This provides evidence that skeletal muscle UCP-3 has the potential to influence metabolic rate and glucose homeostasis in the whole animal.

Identification of differentially expressed genes in dorsal root ganglia following partial sciatic nerve injury.

Partial sciatic nerve injury, a model of neuropathic pain, elicits a variety of neurochemical, electrophysiological and neuroanatomical changes in primary sensory neurons. We have used the technique of messenger RNA differential display to identify genes with altered expression in these neurons which may contribute to the development of aberrant sensation following such peripheral nerve damage. This approach identified 14 distinct complementary DNA clones, representing transcripts with increased ipsilateral expression in L4/5 dorsal root ganglia, two weeks after unilateral partial ligation of the rat sciatic nerve. Both Zucker diabetic fatty rats and their lean counterparts were used in this study but none of the transcripts identified showed an induction that was confined to one of the two groups. The majority of the clones did not show significant sequence similarity to previously reported genes and therefore may represent novel messenger RNA sequences or, alternatively, unknown regions of partially characterised messenger RNAs. Two of the clones represented transcripts for the known proteins muscle LIM protein and acidic epididymal glycoprotein, neither of which had previously been associated with expression in the nervous system. Reverse transcriptase-polymerase chain reaction analysis and in situ hybridization confirmed that the messenger RNA expression of both muscle LIM protein and acidic epididymal glycoprotein was induced in an ipsilateral-specific manner. Their localisations, examined with in situ hybridization in L5 dorsal root ganglia, were limited in each case to a sub-population of neuronal profiles. Those neuronal profiles that demonstrated muscle LIM protein hybridization were distributed across the profile size range, whereas the distribution of acidic epididymal glycoprotein-positive profiles appeared to be skewed towards smaller profiles. The induction of muscle LIM protein and acidic epididymal glycoprotein in dorsal root ganglia may play an important functional role in the adaptive response of primary sensory neurons following partial sciatic nerve injury.

Acceleration of the development of diabetes in obese diabetic (db/db) mice by nicotinamide: a comparison with its antidiabetic effects in non-obese diabetic mice.

Destruction of pancreatic beta cells has been implicted in the progression to hyperglycemia in type 1 diabetes. While there is evidence of beta-cell loss in type 2 diabetes, its contribution to the development of the diabetic state is undecided. Nicotinamide has defensive effects against toxic insults to the pancreatic islets and confers protection in both human and animal models of type 1 diabetes, but its effects on type 2 diabetes are less well documented. This report describes a comparison of the outcome of chronic oral administration of nicotinamide on the development of diabetes in obese diabetic (db/db) and non-obese diabetic (NOD) mice models of type 2 and type 1 diabetes, respectively. Nicotinamide was administered in the diet (5 g/kg diet) for 12 (db/db) or 24 (NOD) weeks. Over the 12 weeks of the study, control diabetic (db/db) mice became progressively more hyperglycemic and glycosuric, while serum and pancreatic insulin levels decreased compared with those on day 0. In mice treated with nicotinamide, there was a pronounced acceleration in the development of hyperglycemia and glycosuria, as well as a decrease in pancreatic insulin levels, compared with time-matched controls. In addition, the morphology of the pancreatic islets of nicotinamide-treated diabetic (db/db) mice showed an enhanced islet disorganization. By comparison, in NOD mice, nicotinamide prevented the decline in serum and pancreatic insulin levels and maintained normal islet architecture and insulin content. Our data shows that in contrast to its preventative effects on the development of autoimmune diabetes in NOD mice, chronic nicotinamide administration to obese diabetic (db/db) mice markedly accelerated the progression of diabetes. The results of our study caution against the use of nicotinamide in insulin-resistant states, such as type 2 diabetes.

Thermal, but not mechanical, nociceptive behavior is altered in the Zucker Diabetic Fatty rat and is independent of glycemic status.

This study investigated the possible link between developing hyperglycemia and mechanical and/or thermal hyperalgesia in the Zucker Diabetic Fatty (ZDF) rat. When normoglycemic (nonfasting blood glucose levels of 6 mM), 6-week-old ZDF rats were glucose intolerant compared to the nondiabetic Zucker lean control (ZL) rats, but there was no difference in their response to a noxious mechanical (paw pressure test) or thermal (hot plate) stimulus (mechanical nociceptive thresholds: ZDF 176.7+/-14.4 g, ZL 161.7+/-13.3 g; latencies to response to the thermal stimulus: ZDF 13.1+/-1.6 sec, ZL 16.7+/-1.5 sec). Blood glucose levels in untreated ZDF rats increased to 28.4+/-2.9 mM by 20 weeks of age, while ZDF rats treated with the insulin sensitizer, rosiglitazone, and ZL rats remained normoglycemic (< or =8 mM) throughout the study. Hyperglycaemia in ZDF rats was not associated with mechanical hyperalgesia, as the nociceptive threshold remained constant in both the rosiglitazone-treated and untreated ZDF rats and in the ZL rats throughout the study. In contrast, the latency to response to the thermal stimulus increased with time in ZL rats, but remained constant in hyperglycaemic ZDF rats such that the difference reached significance by 9 weeks of age (ZDF 11.6+/-1.7 sec, ZL 21.8+/-2.7 sec, p<0.01) and is consistent with hyperalgesia in the ZDF phenotype. However, this difference was not moderated by maintaining normoglycaemia in rosiglitazone-treated ZDF rats (12.8+/-1.3 sec). Together, the data suggest that hyperglycemia does not play a central role in the development of hyperalgesia in the ZDF rat.

Potential benefit of inhibitors of advanced glycation end products in the progression of type II diabetes: a study with aminoguanidine in C57/BLKsJ diabetic mice.

Prolonged hyperglycemia in type II diabetic patients is linked both with diabetic complications and with further impairment of glucose homeostasis, possibly due to glucose toxicity of the beta cell. While the connection between the accumulation of extracellular advanced glycation end products (AGEs) and the development of complications is well established, it has only recently been suggested that intracellular glycation may be equally adverse and could be involved in the pathogenesis of glucose toxicity in vitro. Aminoguanidine is a recognized inhibitor of the formation of both extracellular and intracellular AGEs. In this study, we show that the development of diabetes, measured by increased water intake and concomitant midday blood glucose levels in type II genetically diabetic mice, is reduced by treatment with aminoguanidine at a dosage of 500 mg/kg/d for 12 weeks in the diet. In addition, at the end of the study, aminoguanidine reduced the decline in serum and pancreatic insulin levels and the degree of pancreatic islet morphological degeneration, all of which are associated with pancreatic insufficiency following prolonged hyperglycemia in this animal model. These results suggest that AGEs may be involved in the aggravation of type II diabetes in vivo and aminoguanidine may be beneficial in its treatment.

A comparison of spasmogenic and relaxant responses in aortae from C57/BL/KsJ diabetic mice with those from their non-diabetic litter mates.

We have investigated the responsiveness of thoracic aorta from the C57/BL/KsJ-db/db mouse (a model of type II diabetes) using a small-vessel myograph. The maximum tension developed in response to phenylephrine was greater in diabetic mice compared with non-diabetic (+/?) mice (2.7 +/- 0.1 and 1.8 +/- 0.1 mN/mm, respectively). Responses to phenylephrine were enhanced in tissues from both phenotypes when preincubated with L-NAME (100 mumol/l) and after the addition of oxyhaemoglobin (3 mumol/l), suggesting that endogenous NO release occurs in both. The maximum relaxation to carbachol was less in db/db mice (32 +/- 4%) than in +/? mice (49 +/- 5%) whilst that to sodium nitroprusside was similar (> 90%). However, the concentration-effect curve to both vasorelaxants in db/db mice lay to the right of that in the +/? mice. These results suggest that the responsiveness of the vasculature is altered in the db/db mouse. Since this mouse is a model of type II diabetes this may be a consequence of hyperglycaemia and/or insulin resistance.

Interactions between imidazoline compounds and sulphonylureas in the regulation of insulin secretion.

1. Imidazoline alpha 2-antagonist drugs such as efaroxan have been shown to increase the insulin secretory response to sulphonylureas from rat pancreatic B-cells. We have investigated whether this reflects binding to an islet imidazoline receptor or whether alpha 2-adrenoceptor antagonism is involved. 2. Administration of (+/-)-efaroxan or glibenclamide to Wistar rats was associated with a transient increase in plasma insulin. When both drugs were administered together, the resultant increase in insulin levels was much greater than that obtained with either drug alone. 3. Use of the resolved enantiomers of efaroxan revealed that the ability of the compound to enhance the insulin secretory response to glibenclamide resided only in the alpha 2-selective-(+)-enantiomer; the imidazoline receptor-selective-(-)-enantiomer was ineffective. 4. In vitro, (+)-efaroxan increased the insulin secretory response to glibenclamide in rat freshly isolated and cultured islets of Langerhans, whereas (-)-efaroxan was inactive. By contrast, (+)-efaroxan did not potentiate glucose-induced insulin secretion but (-)-efaroxan induced a marked increase in insulin secretion from islets incubated in the presence of 6 mM glucose. 5. Incubation of rat islets under conditions designed to minimize the extent of alpha 2-adrenoceptor signalling (by receptor blockade with phenoxybenzamine; receptor down-regulation or treatment with pertussis toxin) abolished the capacity of (+)- and (+/-)-efaroxan to enhance the insulin secretory response to glibenclamide. However, these manoeuvres did not alter the ability of (+/-)-efaroxan to potentiate glucose-induced insulin secretion. 6. The results indicate that the enantiomers of efaroxan exert differential effects on insulin secretion which may result from binding to effector sites having opposite stereoselectivity. Binding of (-)-efaroxan (presumably to imidazoline receptors) results in potentiation of glucose-induced insulin secretion, whereas interaction of (+)-efaroxan with a second site leads to selective enhancement of sulphonylurea-induced insulin release.

Effects of dexamethasone in a model of lung hyperresponsiveness in the rat.

In rats, Sephadex treatment on days 0, 2 and either 4 or 5 resulted in a blood and lung eosinophilia, an increase in lung cell fragility, an increase in the functional activity of peritoneal eosinophils in vitro and a sustained increased responsiveness of lung parenchymal strips to KCl, 5-hydroxytryptamine (5-HT) and carbachol that was not associated with oedema or gross fibrosis. The corticosteroid dexamethasone, when given before each injection of Sephadex, reduced all these effects of Sephadex. When given 30 min after the last injection of Sephadex, dexamethasone had no effect on the number of blood and lung eosinophils but it did reduce the functional activity of peritoneal eosinophils, the increased lung cell fragility and the hyperresponsiveness to 5-HT. Repeated administration of dexamethasone to rats with an established hyperresponsiveness that was no longer associated with cellular inflammation had minimal effects on this hyperresponsiveness.

Effects of isoprenaline, adrenaline and selective alpha 1- and alpha 2- adrenoceptor stimulation on hypoxic pulmonary vasoconstriction in rat isolated perfused lungs.

In the rat pulmonary vasculature perfused with blood in situ vasoconstriction induced by hypoxia was reversed by isoprenaline (doses greater than 1 ng) and adrenaline (doses greater than 30 ng) and exacerbated by phenylephrine but not UK 14304. Doses of adrenaline less than 30 ng had no effect, except in the presence of propranolol (1 microM) or phentolamine (3 microM) when they caused vasoconstriction and vasodilation respectively, showing that, at dose levels less than 30 ng, adrenaline's beta- adrenoceptor vasodilator properties were balanced by its alpha- adrenoceptor vasoconstrictor properties. The pressor effects of adrenaline, in the presence of propranolol, were antagonised by prazosin (0.1 microM) but not by equi-molar concentrations of rauwolscine. These results suggest that the alpha- adrenoceptor agonist property of adrenaline is of benefit to its use as an inhaled bronchodilator because unopposed beta- adrenoceptor stimulation can reverse hypoxic pulmonary vasoconstriction in poorly ventilated regions of the lung, promote further ventilation/perfusion mismatching and lower PaO2. They further suggest that adrenaline affects pulmonary vascular tone in the rat via alpha 1- adrenoceptors, stimulation of alpha 2- adrenoceptors having no effect.

Method for assessing the activity of drugs at beta 1- and beta 2-adrenoceptors in the same animal.

A method is described for assessing the selectivity of compounds for beta 1- and beta 2-adrenoceptors in vivo. The potency of selective beta-adrenoceptor agonists to increase heart rate and decrease uterine contractions in pithed rats and in isolated tissues was determined. The order of potency both in vivo (i.v. route) and in vitro was: isoprenaline greater than noradrenaline greater than salbutamol on heart rate, and isoprenaline greater than salbutamol greater than noradrenaline on uterine relaxation. Fenoterol, salbutamol, and BRL 26830A/28410, but not denopamine, (i.p.) route were more potent stimulants of uterine relaxation than of heart rate in pithed rats and in vitro. The abilities of atenolol (beta 1-selective), ICI 118551 (beta 2-selective) and propranolol (non selective between beta 1- and beta 2-adrenoceptors) to inhibit responses to isoprenaline on heart rate and uterine contractions in vivo were also assessed. The effects of isoprenaline on heart rate were selectively antagonized by atenolol while those on the uterus were selectively antagonized by ICI 118551. These results show that beta 1-adrenoceptors mediate increases in heart rate and that beta 2-adrenoceptors mediate uterine relaxation in the pithed rat. They further show that the activity of compounds at these tissues can be used to assess their selectivity for beta 1- or beta 2-adrenoceptors in vivo.

The beta-adrenoreceptors mediating uterine relaxation throughout the oestrous cycle of the rat are predominantly of the beta 2-subtype.

1 The beta-adrenoreceptor subtype(s) mediating uterine relaxation throughout the 4-day oestrous cycle of the rat were determined by using pharmacological techniques. 2 The order of potency of the test beta-adrenoreceptor agonists in dioestrus, proestrus, oestrus and metoestrus was the same, being isoprenaline = fenoterol greater than salbutamol greater than noradrenaline greater than prenalterol greater than BRL 28410. 3 The pA2 values for the antagonists atenolol and ICI 11851 against both noradrenaline and fenoterol were not altered by the stage of oestrus and were consistent with the receptor being solely of the beta 2-subtype. 4 It is concluded that the beta-adrenoreceptors mediating uterine relaxation are of the beta 2-subtype throughout the natural oestrous cycle.

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.

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 .

Treatment of obesity with thermogenic beta-adrenoceptor agonists: studies on BRL 26830A in rodents.

Studies on BRL 26830A in rodents have shown that thermogenic beta-adrenoceptor agonists have potential for the therapy of obesity. BRL 26830A reduced body weight gain in ob/ob mice and fa/fa rats by reducing lipid accumulation. It had no effect on lean body mass. BRL 26830A did not reduce food intake, its anti-obesity effect being due to stimulation of energy expenditure. This thermic effect was enhanced in the obese animals by repeat dosing. BRL 26830A did not affect body weight gain in the lean counterparts of the obese animals because its thermic effect in lean animals was reduced by repeat dosing. Brown adipose tissue is an important site of BRL 26830A-induced thermogenesis. A single dose of BRL 26830A raised brown adipose tissue temperature, depleted brown adipose tissue lipid and unmasked GDP-binding sites in brown adipose tissue mitochondria. Repeat dosing caused hypertrophy of brown adipose tissue and improved cold tolerance in mice. In-vitro studies showed that the rat brown adipocyte beta-adrenoceptor does not fall into the beta 1/beta 2 classification and BRL 28410, which mediates the biological effects of BRL 26830A in vivo, selectively stimulated the brown adipocyte receptor. It is concluded that BRL 26830A achieves its anti-obesity effect by mimicking natural mechanisms involved in thermogenesis and the control of body weight.