Peter S. Widdowson

Inhibition of Food Response to Intracerebroventricular Injection of Leptin Is Attenuated in Rats With Diet-Induced Obesity

The fat-derived hormone, leptin, is thought to regulate adipose tissue mass by acting on the brain to reduce food intake and increase thermogenesis. We have produced obesity in rats more than 8 weeks old by feeding a high-calorie diet and have then examined the inhibitory effect of intracerebroventricularly injected recombi-nant murine leptin on their food intake versus control rats. In control rats, randomized injections of leptin (0.5, 2.0, or 10.0 μg) or sterile saline vehicle into the lateral ventricle produced a dose-dependent reduction in normal laboratory diet consumed 1, 4, and 24 h after the lights were turned off. However, in diet-induced obesity, the dose-dependent inhibition of food intake was observed at 1 h only, and the effect was attenuated. Switching the diet-induced obese rats to a normal laboratory diet 1 week before injections of leptin were commenced resulted in a reduction in the daily food consumption. These data suggest that rats made obese by feeding a high-calorie diet override the normal satiety effects of leptin since when they are returned to a normal laboratory diet, they reduce their calorie intake, possibly as a result of a restoration of the satiety effects of endogenous leptin. However, the fact that the hypophagic response to exogenous leptin is impaired in these rats at this time suggests some residual impairment of the satiety signal, perhaps caused by reduced receptor sensitivity and/or near total occupation of receptors by endogenous leptin molecules, levels of which are raised in plasma.

Therapeutic index for rosiglitazone in dietary obese rats: separation of efficacy and haemodilution

The blood glucose‐lowering efficacy of rosiglitazone (RSG) and the mechanisms of associated weight gain were determined in dietary obese rats (DIOs). DIO and chow‐fed rats received RSG 0.3–30 mg kg−1 daily for 21 days. In DIOs, plasma glucose and insulin concentrations were reduced by RSG at dosages of 3 and 10 mg kg−1, respectively. Homeostasis model assessment (HOMA) indicated the threshold for a reduction of insulin resistance was 1 mg kg−1. Neither glucose nor insulin levels were affected by treatment in chow‐fed rats. RSG 0.3 mg kg−1 lowered free fatty acids (FFAs) in DIOs, whereas for plasma triglycerides (TGs), the threshold was 3 mg kg−1. By contrast, the threshold for reducing packed red cell volume (PCV) and increasing cardiac mass was 10 mg kg−1. Thus, the therapeutic index for RSG in DIOs was >3 and 10. Energy intake and weight gain increased in treated DIOs (by 20% and 50 g, at 30 mg kg−1) and chow‐fed rats (by 25% and 35 g, at 30 mg kg−1). In DIOs, these increases coincided with falls in plasma leptin (40% lower at 30 mg kg−1) and insulin (43% lower at 30 mg kg−1). By contrast, in chow‐fed rats, weight gain and hyperphagia occurred without changes in either leptin or insulin. However, reductions in FFAs below 0.4–0.3 mM were associated with hyperphagia and weight gain in DIO and chow‐fed rats. We conclude that increased energy intake and body weight did not attenuate the improved metabolism evoked by RSG in DIO rats, and that insulin action was enhanced at a dose >3 fold below the threshold for causing haemodilution and cardiac hypertrophy in DIO rats.

Down-regulation of cannabinoid-1 (CB-1) receptors in specific extrahypothalamic regions of rats with dietary obesity: a role for endogenous cannabinoids in driving appetite for palatable food?

Agonists at cannabinoid-1 (CB-1) receptors stimulate feeding and particularly enhance the reward aspects of eating. To investigate whether endogenous cannabinoids might influence appetite for palatable food, we compared CB-1 receptor density in the forebrain and hypothalamus, between rats fed standard chow (n=8) and others given palatable food (n=8) for 10 weeks to induce dietary obesity. CB-1 receptor density was significantly decreased by 30-50% (P<0.05) in the hippocampus, cortex, nucleus accumbens and entopeduncular nucleus of diet-fed rats. Furthermore, CB-1 receptor density in the hippocampus, nucleus accumbens and entopeduncular nucleus was significantly inversely correlated with intake of palatable food (r(2)=0.25-0.35; all P<0.05). By contrast, CB-1 receptor binding in the hypothalamus was low and not altered in diet-fed rats. CB-1 receptor down-regulation is consistent with increased activation of these receptors by endogenous cannabinoids. Acting in areas such as the nucleus accumbens and hippocampus, which are involved in the hedonic aspects of eating, cannabinoids may therefore drive appetite for palatable food and thus determine total energy intake and the severity of diet-induced obesity. However, cannabinoids in the hypothalamus do not appear to influence this aspect of eating behaviour.

Effect of a selective neuropeptide Y Y2 receptor antagonist, BIIE0246 on neuropeptide Y release

We have examined the selective neuropeptide Y Y(2) receptor antagonist, (S)-N(2)-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6h)-oxodibenz[b, e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl]acetyl ]-N-[2-[1 ,2-dihydro-3,5(4H)-dioxo-1,2-diphenyl-3-H-1,2, 4-triazol-4-yl]ethyl]-argininamid (BIIE0246) on neuropeptide release from rat hypothalamic slices in vitro. BIIE0246 prevented neuropeptide Y-(13-36)-induced reduction in basal and K(+)-stimulated neuropeptide Y release. Addition of BIIE0246 alone enhanced K(+)-stimulated neuropeptide release, without affecting basal release. These data are consistent with anatomical and functional studies suggesting a pre-synaptic role for neuropeptide Y Y(2) receptors in regulating rat hypothalamic neuropeptide Y release in the rat.

Regulation of neuropeptide Y release by neuropeptide Y receptor ligands and calcium channel antagonists in hypothalamic slices.

Abstract : Neuropeptide Y (NPY) is an important regulator of energy balance in mammals through its orexigenic, antithermogenic, and insulin secretagogue actions. We investigated the regulation of endogenous NPY release from rat hypothalamic slices by NPY receptor ligands and calcium channel antagonists. High‐potassium stimulation (60 mM) of the slices produced a calcium‐dependent threefold increase in NPY release above basal release. The Y2 receptor agonists NPY(13‐36) and N‐acetyl[Leu28, Leu31]NPY(24‐36), the Y4 agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human pancreatic polypeptide (hPP) significantly reduced both basal and stimulated NPY release. NPY(13‐36)‐induced reduction of NPY release could be partially prevented in the presence of the weak Y2 antagonist T4‐[NPY(33‐36)]4, whereas the hPP‐ and rPP‐induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium‐stimulated release when administered alone. The calcium channel inhibitors ω‐conotoxin GVIA (N‐type), ω‐agatoxin TK (P/Q‐type), and ω‐conotoxin MVIIC (Q‐type) all significantly inhibited potassium‐stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both ω‐conotoxin GVIA and ω‐agatoxin TK together completely inhibited the potassium‐stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium‐dependent, involving N‐, P‐, and Q‐type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY‐containing nerve terminals.

Increased binding at 5-HT1A, 5-HT1B, and 5-HT2A receptors and 5-HT transporters in diet-induced obese rats

5-Hydroxytryptamine (5-HT, serotonin), synthesized in midbrain raphe nuclei and released in various hypothalamic sites, decreases food intake but the specific 5-HT receptor subtypes involved are controversial. Here, we have studied changes in the regional density of binding to 5-HT receptors and transporters and the levels of tryptophan hydroxylase, in rats with obesity induced by feeding a palatable high-energy diet for 7 weeks. We mapped binding at 5-HT receptor subtypes and transporters using quantitative autoradiography and determined tryptophan hydroxylase protein levels by Western blotting. In diet-induced obese (DiO) rats, specific binding to 5-HT(1A) receptors ([3H]8-OH-DPAT) was significantly increased in the dorsal and median raphe by 90% (P<0.01) and 132% (P<0.05), respectively, compared with chow-fed controls. 5-HT(1B) receptor binding sites ([125I]cyanopindolol) were significantly increased in the hypothalamic arcuate nucleus (ARC) of DiO rats (58%; P<0.05), as were 5-HT(2A) receptor binding sites ([3H]ketanserin) in both the ARC (44%; P<0.05) and lateral hypothalamic area (LHA) (121%; P<0.05). However, binding to 5-HT(2C) receptors ([3H]mesulgergine) in DiO rats was not significantly different from that in controls in any hypothalamic region. Binding to 5-HT transporters ([3H]paroxetine) was significantly increased (P<0.05) in both dorsal and median raphe, paraventricular nuclei (PVN), ventromedial nuclei (VMH), anterior hypothalamic area (AHA) and LHA of DiO rats, by 47%-165%. Tryptophan hydroxylase protein levels in the raphe nuclei were not significantly different between controls and DiO rats. In conclusion, we have demonstrated regionally specific changes in binding to certain 5-HT receptor subtypes in obesity induced by voluntary overeating of a palatable diet. Overall, these changes are consistent with reduced 5-HT release and decreased activity of the 5-HT neurons. Reduction in the hypophagic action of 5-HT, possibly acting at 5-HT(1A), 5-HT(1B) and 5-HT(2A) receptors, may contribute to increased appetite in rats presented with highly palatable diet.

[3H]-idazoxan binding to rabbit cerebral cortex recognises multiple imidazoline I2-type receptors: pharmacological characterization and relationship to monoamine oxidase.

1 In rabbit cerebral cortical homogenates, saturation analysis of [3H]‐idazoxan, an α2‐adrenoceptor antagonist, revealed high affinity binding to a single site with high density. Competition experiments demonstrated that the [3H]‐idazoxan recognition site was insensitive to the catecholamines, adrenaline and noradrenaline and possessed a low affinity for the α2‐ and α1‐adrenoceptor antagonists, rauwolscine, yohimbine and prazosin, suggesting that the site was not an adrenoceptor. Mapping [3H]‐idazoxan binding sites in the forebrain of rabbits by autoradiography, showed high densities of I2 sites in the medial preoptic area and in the stria terminalis. Moderate binding was found in caudate nucleus, putamen, cerebral cortex and hippocampus. 2 The imidazolines cirazoline, naphazoline, guanabenz and BRL44408 along with amiloride, which is structurally related to the imidazolines, all had high affinity for the [3H]‐idazoxan site, suggesting that the site was related to the I2 imidazoline‐recognition site described by other groups. However, the imidazolines, clonidine and UK‐14,304 and the structurally related rilmenidine all had a low affinity for the binding site, showing that [3H]‐idazoxan was not binding to the I1 imidazoline‐recognition site found in rat, bovine and human medulla oblongata. 3 Naphazoline, guanabenz, clonidine and amiloride competition studies had Hill slopes which were significantly different from unity (P < 0.01) and computer analysis showed that the [3H]‐idazoxan binding data could be best fitted to a model which considers binding to two sites (P < 0.01). One site has a high affinity for idazoxan, cirazoline, naphazoline, guanabenz and amiloride and a moderate affinity for BRL44408 and clonidine (70% of binding) and the second site (30% of binding) has a high affinity for idazoxan and cirazoline, but a lower affinity for naphazoline, guanabenz, amiloride, BRL44408 and clonidine. 4 Experiments using [3H]‐RX821002, in contrast to [3H]‐idazoxan, clearly demonstrated the presence of a single type of α2‐adrenoceptor in rabbit cortex with a pharmacological profile which is similar to the α2A‐adrenoceptor possessing a high affinity for yohimbine, rauwolscine, BRL44408 and oxymetazoline, but a lower affinity for prazosin. 5 The monoamine oxidase inhibitors, clorgyline, pargyline and deprenyl had at least a ten fold lower affinity at the rabbirt cortex I2 site as compared to their known affinity at monoamine oxidase suggesting that the I2 site is not related to the active site of the enzyme, monoamine oxidase. In addition, the peripheral benzodiazepine ligands, PK‐11195 or Ro 5–4864 both had very low affinities at the I2 site in rabbit cortex suggesting that the [3H]‐idazoxan binding was not to the peripheral benzodiazepine binding site.

Improved metabolic status and insulin sensitivity in obese fatty (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats treated with the thiazolidinedione, MCC-555

We examined the effect of chronic (21 days) oral treatment with the thiazolidinedione, MCC‐555 ((±)‐5‐[{6‐(2‐fluorbenzyl)‐oxy‐2‐naphy}methyl]‐2,4‐thiazolidinedione) on metabolic status and insulin sensitivity in obese (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats which display an impaired glucose tolerance (IGT) or overt diabetic symptoms, respectively. MCC‐555 treatment to obese Zucker rats (10 and 30 mg kg−1) and diabetic ZDF rats (10 mg kg−1) reduced non‐esterified fatty acid concentrations in both rat strains and reduced plasma glucose and triglyceride concentrations in the obese Zucker rats. Liver glycogen concentrations were significantly increased by chronic MCC‐555 treatment in both obese Zucker rats (30 mg kg−1 day−1) and diabetic ZDF rats (10 mg kg−1 day−1), as compared with vehicle‐treated lean and obese rats and there was a significant increase in hepatic glycogen synthase activity in MCC‐555‐treated diabetic ZDF rats as compared to vehicle‐treated controls. During a euglycaemic hyperinsulinaemic clamp, MCC‐555‐treated obese Zucker rats and diabetic ZDF rats required significantly higher glucose infusion rates to maintain stable glucose concentrations (2.01 ± 0.19 mg min−1 and 6.42 ± 1.03 mg min−1, respectively) than vehicle‐treated obese controls (0.71 ± 0.17 mg min−1 and 2.09 ± 0.71 mg min−1; P<0.05), demonstrating improved insulin sensitivity in both Zucker and ZDF rats. MCC‐555 treatment also enhanced insulin‐induced suppression of hepatic glucose production in ZDF rats as measured using infusions of [6‐3H]‐glucose under clamp conditions. In conclusion, we have demonstrated that MCC‐555 improves metabolic status and insulin sensitivity in obese Zucker and diabetic ZDF rats. MCC‐555 may prove a useful compound for alleviating the metabolic disturbances and IGT associated with insulin resistance in man.

S 15535: a highly selective benzodioxopiperazine 5-HT1A receptor ligand which acts as an agonist and an antagonist at presynaptic and postsynaptic sites respectively

The novel benzodioxopiperazine, S 15535 (4-(benzodioxan-5-yl)1-(indan-2- yl)piperazine), displayed high affinity for 5-HT1A binding sites (1.8 nM) whereas its affinity was 100-fold lower at other 5-HT receptor types, at alpha 1, alpha 2- and beta-adrenoceptors and at dopamine D1 and D2 receptors. In vivo, S 15535 (0.16-10 mg/kg s.c.) acted as an antagonist at postsynaptic 5-HT1A receptors in completely blocking the flat-body posture and hypothermia elicited by the 5-HT1A receptor agonist, 8-OH-DPAT. It had no effect when applied alone. At presynaptic 5-HT1A receptors, S 15535 acted as an agonist in inhibiting striatal accumulation of 5-hydroxytryptophan (0.04-0.63 mg/kg s.c.) and in spiperone reversibly reducing electrical activity of the dorsal raphe nucleus (0.004-0.031 mg/kg i.v.). At doses up to 40.0 mg/kg s.c., S 15535 neither inhibited methylphenidate-induced gnawing nor elicited ptosis suggesting a lack of antagonist properties at, respectively, dopamine D2 receptors and alpha 1-adrenoceptors. In conclusion, S 15535 is a potent 5-HT1A ligand which acts, in vivo, as a highly selective agonist and antagonist at presynaptic and postsynaptic 5-HT1A receptors, respectively.

The development of overt diabetes in young Zucker Diabetic Fatty(ZDF) rats and the effects of chronic MCC-555 treatment

1 Young (6‐week‐old) pre‐diabetic Zucker Diabetic Fatty (ZDF) rats displaying impaired glucose tolerance (IGT), moderate hyperglycaemia and hyperinsulinaemia were treated with the novel thiazolidinedione, MCC‐555, for 28 days, during which time β‐cell failure and progression to overt diabetes occurs. 2 Treated ZDF rats exhibited consistently lower blood glucose levels than vehicle‐treated diabetic controls, with a delayed rise and lower plateau levels. MCC‐555 maintained plasma insulin levels throughout the treatment period, whereas these fell by 40% in untreated ZDF rats. 3 The rise in body weight was maintained in MCC‐555‐treated rats, whereas vehicle‐treated rats exhibited blunted body weight gain after 8 weeks of age. Daily food intake was higher in diabetic, as compared to non‐diabetic rats, but treatment did not modify food intake in diabetic rats. Water intake was lower in treated ZDF rats, concomitant with lowering of blood glucose. 4 The hyperinsulinaemic‐euglycaemic clamp technique was applied to all rats after treatment to examine the effects of MCC‐555 on insulin sensitivity. The glucose infusion rate to maintain normoglycaemia was lower in diabetic than in non‐diabetic rats, demonstrating reduced glucose entry into insulin‐sensitive tissues in diabetic rats. Increased glucose infusion rates were required to maintain euglycaemia in treated diabetic rats, demonstrating increased insulin sensitivity in these animals. 5 In conclusion, chronic MCC‐555 treatment of young ZDF rats displaying IGT attenuates the development of overt diabetes through improved insulin sensitivity and maintenance of β‐cell function. MCC‐555 may thus be beneficial in humans with IGT, to prevent or delay the progression of diabetes.