Matthew V. Sennitt

The mouse SWISS‐2D PAGE database: a tool for proteomics study of diabetes and obesity

A number of two‐dimensional electrophoresis (2‐DE) reference maps from mouse samples have been established and could be accessed through the internet. An up‐to‐ date list can be found in WORLD‐2D PAGE (http://www.expasy.ch/ch2d/2d‐index.html), an index of 2‐DE databases and services. None of them were established from mouse white and brown adipose tissues, pancreatic islets, liver nuclei and skeletal muscle. This publication describes the mouse SWISS‐2D PAGE database. Proteins present in samples of mouse (C57Bl/6J) liver, liver nuclei, muscle, white and brown adipose tissue and pancreatic islets are assembled and described in an accessible uniform format. SWISS‐2D PAGE can be accessed through the World Wide Web (WWW) network on the ExPASy molecular biology server (http://www.expasy.ch/ch2d/).

The rat lipolytic β-adrenoceptor: Studies using novel β-adrenoceptor agonists

EC50 and relative intrinsic activity values were obtained for isoprenaline, fenoterol, salbutamol, prenalterol and three new β-adrenoceptor agonist, BRL 2841, 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 propratolol, 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 β1/β2-adrenoceptor classification.

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 11β-hydroxysteroid dehydrogenase (11β-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 11β-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.

Effect of Rosiglitazone on the Differential Expression of Diabetes-associated Proteins in Pancreatic Islets of C57Bl/6 lep/lep Mice

The insulin sensitizer drug, rosiglitazone, has been shown to have a protective effect on pancreatic islet cell structure and function in animal models of type 2 diabetes. The identification of new molecular targets associated both with islet cell dysfunction and protection is a crucial research goal. In the present study, a proteomics approach has been used to identify such targets. Obese C57Bl/6J lep/lep mice and lean littermates were given the insulin sensitizer drug BRL49653, rosiglitazone. It normalized the impaired glucose tolerance in lep/lep mice but had no significant effect on glucose tolerance in the lean mice. Pancreatic islet polypeptides were arrayed by a two-dimensional gel electrophoresis system that separated more than 2500 individual spots. Three overexpressed and six underexpressed proteins were significant (p < 0.05) between lep/lep and lean mice, and four were modulated significantly (p < 0.05) by the rosiglitazone treatment of the obese mice. The identity of these differentially expressed proteins was made using mass spectrometric analysis and provided evidence that differential expression of actin-binding proteins may be an important aspect of defective islet function. Rosiglitazone increased carboxypeptidase B expression in both lep/lep and normal mice suggesting that this might be an independent effect of rosiglitazone that contributes to improved insulin processing.

Prolonged treatment of genetically obese mice with conjugated linoleic acid improves glucose tolerance and lowers plasma insulin concentration: possible involvement of PPAR activation

BackgroundStudies in rodents and some studies in humans have shown that conjugated linoleic acid (CLA), especially its trans-10, cis-12 isomer, reduces body fat content. However, some but not all studies in mice and humans (though none in rats) have found that CLA promotes insulin resistance. The molecular mechanisms responsible for these effects are unclear, and there are conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-γ (PPARγ) activation and expression. We have conducted three experiments with CLA in obese mice over three weeks, and one over eleven weeks. We have also investigated the effects of CLA isomers in PPARγ and PPARα reporter gene assays.ResultsInclusion of CLA or CLA enriched with its trans-10, cis-12 isomer in the diet of female genetically obese (lepob/lepob) mice for up to eleven weeks reduced body weight gain and white fat pad weight. After two weeks, in contrast to beneficial effects obtained with the PPARγ agonist rosiglitazone, CLA or CLA enriched with its trans-10, cis-12 isomer raised fasting blood glucose and plasma insulin concentrations, and exacerbated glucose tolerance. After 10 weeks, however, CLA had beneficial effects on glucose and insulin concentrations. At this time, CLA had no effect on the plasma TNFα concentration, but it markedly reduced the plasma adiponectin concentration. CLA and CLA enriched with either isomer raised the plasma triglyceride concentration during the first three weeks, but not subsequently. CLA enriched with its trans-10, cis-12 isomer, but not with its cis-9, trans-11 isomer, stimulated PPARγ-mediated reporter gene activity; both isomers stimulated PPARα-mediated reporter gene activity.ConclusionsCLA initially decreased but subsequently increased insulin sensitivity in lepob/lepob mice. Activation of both PPARγ and PPARα may contribute to the improvement in insulin sensitivity. In the short term, however, another mechanism, activated primarily by trans-10, cis-12-CLA, which probably leads to reduced adipocyte number and consequently reduced plasma adiponectin concentration, may decrease insulin sensitivity.

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.

Effects of BRL 26830, a novel β-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.

Effect of a novel thermogenic β-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.

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.

SK&F 104078, a post-functionally selective α2-adrenoceptor antagonist in the human saphenous vein in vitro

SummaryThe present study investigated the effects of SK&F 104078 (6-chloro-9-[(3-methyl-2-butenyl)oxy]-3methyl-1H,2,3,4,-tetrahydro-3-benzazapine) at pre- and post functional α2-adrenoceptors in the human isolated saphenous vein. Noradrenaline (0.001–100 μmol/l) produced concentration-dependent contractions of the human saphenous vein which were competitively antagonised by the α1-adrenoceptor antagonist prazosin (0.01–1.0 μmol/l) and the α2-adrenoceptor antagonist, rauwolscine (0.01–1.0 μmol/l), indicating the presence of both post functional α1- and α2-adrenoceptors in this preparation. The selective α2-adrenoceptor agonist, UK-14,304 (0.01–100 μmol/l) also produced concentration-dependent contractions of the human saphenous vein which were antagonised by both rauwolscine (0.1 μmol/l) and prazosin (0.1 μmol/l). In the presence of angiotensin II (0.05 μmol/l), which itself produced a transient contraction, rauwolscine (0.1 μmol/l) produced a rightward shift of the UK-14,304 concentration-response curve while prazosin (0.1 μmol/l) had no effect. SK&F 104078 (10.0 μmol/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 functional α2-adrenoceptors, exogenous noradrenaline (0.01 and 0.1 gmol/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 μmol/l) and tolazoline (10.0 μmol/l) but not by SK&F 104078 (10.0 gmol/l).Rauwolscine (0.1 μmol/l) produced a small increase in stimulation-evoked [7-3H]-noradrenaline release while both tolazoline and SK&F 104078 failed to produce any enhancement in release in the absence of exogenous agonist atconcentrationsupto10 gmol/l.Insummary, noradrenaline and UK-14,304 contracted the human isolated saphenous vein by an action at both postfunctional α1- and α2-adrenoceptors. These data demonstrate that SK&F 104078 discriminates between post- and pre-junctional α2-adrenoceptors in the human isolated saphenous vein.