Lucy Pickavance

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.

Increased neuropeptide Y secretion in the hypothalamic paraventricular nucleus of obese (fa/fa) Zucker rats

NPY is synthesized in the hypothalamic arcuate nucleus (ARC), and NPY injected into the paraventricular nucleus (PVN), the main site of NPY release, induces hyperphagia and reduces energy expenditure. Hypothalamic NPY mRNA and NPY levels are increased in fatty Zucker rats, consistent with increased NPY release. This could explain the hyperphagia and reduced energy expenditure, which lead to obesity in the fatty Zucker rat. We have therefore compared NPY secretion in the PVN of conscious fatty and lean Zucker rats using push-pull sampling. The NPY secretory profile was consistently higher in fatty Zucker rats than in lean rats throughout the 3-h study period (P < 0.01), and mean NPY secretion over the whole 3 h was increased 2-fold in the fatty rats (P < 0.001). We conclude that fatty Zucker rats have increased NPY release in the PVN. This observation further supports the hypothesis that increased activity of the NPYergic ARC-PVN pathway may contribute to obesity in the fatty Zucker syndrome.

The serotonergic agent fluoxetine reduces neuropeptide y levels and neuropeptide y secretion in the hypothalamus of lean and obese rats

Evidence suggests that serotonin and neuropeptide Y neurons in the hypothalamus, which respectively inhibit and stimulate food intake, may interact to control energy homoeostasis. We therefore investigated the effects of fluoxetine, which inhibits serotonin reuptake, on food intake and the activity of the neuropeptide Yergic arcuato-paraventricular projection in lean Wistar and Zucker rats. We also studied its effects in obese Zucker rats, in which obesity is postulated to be due to overactivity of the arcuato-paraventricular projection. Fluoxetine significantly reduced food intake in lean and obese rats, both during continuous subcutaneous infusion and (10 mg/kg/day for seven days) and acutely after a single injection (10 mg/kg). Fluoxetine also significantly reduced neuropeptide Y levels in the paraventricular nucleus, a major site of neuropeptide Y release which is highly sensitive to the appetite-stimulating actions of neuropeptide Y. Push-pull sampling in lean and fatty Zucker rats showed that neuropeptide Y secretion in the paraventricular nucleus was significantly reduced after acute fluoxetine treatment. Furthermore, seven days fluoxetine treatment prevented the significant increases in hypothalamic neuropeptide Y messenger RNA which were induced in lean rats by food restriction which precisely matched the hypophagia induced by the drug. We conclude that fluoxetine inhibits various aspects of the activity of the neuropeptide Yergic arcuato-paraventricular neurons, and suggest that reduced neuropeptide Y release in the paraventricular nucleus may mediate, at least in part, the drugs hypophagic action. We further suggest that serotonin may influence food intake and energy balance by inhibiting the arcuato-paraventricular projection, and that the two neurotransmitters may act together to regulate feeding and energy homoeostasis.

Hyperphagia in cold-exposed rats is accompanied by decreased plasma leptin but unchanged hypothalamic NPY

Chronic cold exposure stimulates sympathetically driven thermogenesis in brown adipose tissue (BAT), resulting in fat mobilization, weight loss, and compensatory hyperphagia. Hypothalamic neuropeptide Y (NPY) neurons are implicated in stimulating food intake in starvation, but may also suppress sympathetic outflow to BAT. This study investigated whether the NPY neurons drive hyperphagia in rats that have lost weight through cold exposure. Rats exposed to 4°C for 21 days weighed 14% less than controls maintained at 22°C ( P < 0.001). Food intake increased after 3 days and remained 10% higher thereafter ( P < 0.001). Increased BAT activity was confirmed by 64, 96, and 335% increases in uncoupling protein-1 mRNA at 2, 8, and 21 days. Plasma leptin decreased during prolonged cold exposure. Cold-exposed rats showed no significant changes in NPY concentrations in any hypothalamic regions or in hypothalamic NPY mRNA at any time. We conclude that the NPY neurons are not activated during cold exposure. This is in contrast with starvation-induced hyperphagia, but is biologically appropriate since enhanced NPY release would inhibit thermogenesis causing potentially lethal hypothermia. Other neuronal pathways must therefore mediate hyperphagia in chronic cold exposure.

Effects of olanzapine in male rats: enhanced adiposity in the absence of hyperphagia, weight gain or metabolic abnormalities

Many of olanzapines (OLZ) actions in humans related to weight regulation can be modelled in female rats (Cooper et al., 2005). Such effects include weight gain, hyperphagia, enhanced visceral adiposity and elevated Levels of insulin and adiponectin. As sex differences have been reported in the effects of antipsychotic drugs, including OLZ, in rats, the current study extended our study in female rats by directly comparing the actions of OLZ in maLes using identical methodology. Individually housed male Han Wistar rats were administered OLZ twice daily (i.p.), at 0, 1, 2, and 4 mg/kg over 21 days. Both differences from, and simiLarities to, the data obtained in females were obtained. Males treated with OLZ showed reduced weight gain, enhanced visceral adiposity and reduced Lean muscle mass. There were no accompanying changes in food or water intake. OLZ did not induce changes in plasma Levels of insulin, Leptin or gLucose. Significant elevation of adiponectin was observed. OLZ-treated males dispLayed elevated prolactin and suppressed testosterone. OLZs effects in humans can very clearly be most validly modelled in female rats, although the cause(s) of the sex difference in OLZs actions in rats are not clear. However, the finding that significantly enhanced adiposity is seen in both male and female rats, in other animal species (mice and dogs) and in humans suggests that studies in male rats of OLZs effects may be of value, by highlighting the consistent ability of OLZ to increase visceral adiposity. It is hypothesized that such adiposity is a key, clinically relevant, common component of OLZs actions which may be, at Least partially, independent of both OLZinduced weight gain and hyperphagia, and which is induced reliably in male and female rats and other animal species. Possible mechanisms involved in the effects reported are discussed.

The dual PPARα/γ agonist, ragaglitazar, improves insulin sensitivity and metabolic profile equally with pioglitazone in diabetic and dietary obese ZDF rats

1 In 6‐ and 10‐week‐old obesity‐prone (fa/fa) Zucker diabetic fatty (ZDF) rats, effects of prevention and intervention therapies, respectively, were compared between PPARα/γ agonist, ragaglitazar (RAGA) and separate PPARγ and α agonists, pioglitazone (PIO) and bezafibrate (BF). 2 In a separate study, lean (+/+) ZDF rats fed highly palatable chow to induce dietary obesity and insulin resistance were treated similarly. To test insulin‐secretory capacity, all animals underwent a hyperglycaemic clamp. 3 Insulin sensitivity was improved equally by RAGA and PIO in fa/fa rats subjected to both prevention and intervention treatments (e.g., prevention HOMA‐IR: −71 and −72%, respectively), as was hyperglycaemia (both −68%). BF had no effect on either parameter in any study. Plasma lipids were markedly reduced (by 48–77%) by RAGA in all studies, equivalent to PIO, but to a greater extent than BF. 4 RAGA improved β‐cell function (HOMA‐β) more than three‐fold with prevention and intervention therapies, whereas PIO showed improvement only in intervention therapy. Consistent with improved insulin sensitivity, glucose infusion rate during the clamp was 60% higher in RAGA‐treated animals subjected to prevention therapy, but there was little additional insulin‐secretory response, suggesting that insulin secretion was already maximal. 5 Thus, RAGA and PIO equally improve metabolic profile in ZDF rats, particularly when administered early in the course of diabetes. They also improve β‐cell function, although this is better demonstrated through indices incorporating fasting insulin and glucose concentrations than through the hyperglycaemic clamp technique in this model.

Down-regulation of orexin gene expression by severe obesity in the rats: studies in Zucker fatty and Zucker diabetic fatty rats and effects of rosiglitazone

Orexins (hypocretins) are lateral hypothalamic neuropeptides implicated in regulating feeding and the sleep-wake cycle. To study their possible relevance to obesity and diabetes, we measured hypothalamic prepro-orexin mRNA levels in obese, normoglycemic Zucker fatty (fa/fa) and in hyperglycemic, non-obese Zucker diabetic fatty (ZDF) rats. Hypothalamic prepro-orexin mRNA concentrations in Zucker fatty rats were 31% lower than those in lean controls (0. 69+/-0.06 vs. 1.00+/-0.10 arbitrary units, P<0.05), but did not differ between ZDF diabetic rats and non-diabetic controls. Treatment of ZDF diabetic rats with rosiglitazone (1 or 3 mg/kg body weight daily for 13 weeks) normalized plasma glucose and significantly reduced plasma insulin, while leptin levels were 67% higher than in untreated ZDF rats (20.2+/-0.5 vs. 12.1+/-2.5, P<0. 001). Rosiglitazone treatment markedly enhanced weight gain compared with untreated ZDF rats (final weight 732+/-13 g vs. 409+/-13 g, P<0. 001) even though they were restricted to the same food intake. Rosiglitazone-treated ZDF rats had significantly lower hypothalamic prepro-orexin mRNA levels (0.68+/-0.07 arbitrary units) than both non-diabetic lean controls (1.00+/-0.10, P=0.02) and untreated diabetics (1.03+/-0.14, P=0.03). Our data suggest that prepro-orexin gene expression may be suppressed by substantial weight gain. Obesity-related signals that might mediate this effect have not been identified, but plasma leptin, insulin and glucose are not obviously involved.

Increased feeding in fatty Zucker rats by the thiazolidinedione BRL 49653 (rosiglitazone) and the possible involvement of leptin and hypothalamic neuropeptide Y.

1 The thiazolidinedione BRL 49653 (rosiglitazone) induces hyperphagia and weight gain in obese, insulin‐resistant fatty Zucker rats but not in lean insulin‐sensitive rats. We investigated whether these responses might involve neuropeptide Y (NPY), leptin and insulin. 2 BRL 49653 (1 mg kg−1 day−1, orally) was given for 7 or 20 days to fatty and lean Zucker and Wistar rats. 3 In lean rats of either strain, BRL 49653 had no effect on food intake, body weight, plasma insulin and corticosterone, NPY or NPY mRNA levels. 4 Fatty rats given BRL 49653 showed a 30% increase in food intake and accelerated body weight gain (both P<0.01) after 7 and 20 days, but without significant changes in regional hypothalamic NPY or NPY mRNA levels. 5 Plasma leptin levels were twice as high in untreated fatty Zucker rats as in lean rats (P<0.01), but were unaffected by BRL 49653 given for 20 days. However, BRL 49653 reduced insulin levels by 42% and increased corticosterone levels by 124% in fatty rats (both P<0.01). 6 Hyperphagia induced in fatty Zucker rats by BRL 49653 does not appear to be mediated by either a fall in circulating leptin levels or increased activity of hypothalamic NPYergic neurones. The fall in plasma insulin and/or rise in corticosterone levels during BRL 49653 treatment may be involved, consistent with the postulated role of these hormones in the control of food intake.

The serotonin (5-HT) antagonist methysergide increases neuropeptide Y (NPY) synthesis and secretion in the hypothalamus of the rat

NPY is synthesized in neurons of the hypothalamic arcuate nucleus (ARC) which project to the paraventricular nucleus (PVN), an important site of NPY release. Serotonin (5-HT) has been suggested to induce satiety and 5-HT fibers contact NPY neurons in the ARC and PVN, suggesting that 5-HT could inhibit the ARC-PVN projection. Methysergide is a 5-HT antagonist which stimulates feeding in rats and increases NPY levels in the hypothalamus. To clarify the effects of methysergide on NPY, we examined its effects on NPY synthesis and on NPY secretion in the PVN using push-pull sampling. Hypothalamic NPY mRNA levels were measured in rats (n = 8/group) given either saline or methysergide (10 mg/kg) and killed after 4 h or after 7 days. Food intake was increased by 33% in the acute study and by 9% in the 7-day study (both P < 0.01). NPY mRNA levels were 80% higher in the 7-day study (P < 0.05) and unchanged in the acute study. NPY secretion was measured over a 3-h period after an i.p. injection of methysergide or saline (10 mg/kg, n = 12) with a flow rate of 15 microliters/min. Mean NPY secretion in the methysergide-injected rats was increased by 34% (P < 0.01). We conclude that methysergide induced feeding is associated with increased activity of the NPY neurons in the ARC-PVN projection. This is consistent with our previous findings suggesting that the NPYergic ARC-PVN projection may mediate, at least in part the effects of 5-HT on feeding and energy balance.

A fat-enriched, glucose-enriched diet markedly attenuates adiponectin mRNA levels in rat epididymal adipose tissue.

Adiponectin levels are decreased in subjects with obesity, diabetes and coronary artery disease. In the present study, we have investigated whether the decrease in the levels and mRNA expression of adiponectin is due to obesity or to the diet itself. Wistar rats were either fed standard laboratory chow throughout (controls) or given a fat-enriched, glucose-enriched diet (diet-fed) for 2 days or 16 weeks. After 2 days of diet feeding, total body weight, fat pad masses and the plasma levels of glucose, insulin and leptin were all comparable between the two groups, while plasma NEFA (non-esterified fatty acid) and triacylglycerol levels were increased in the diet-fed animals (P<0.01 for both). There was a marked (P<0.01) decrease in plasma adiponectin levels. After 16 weeks of diet feeding, diet-fed rats had significantly higher body weight, fat pad mass and plasma levels of leptin, adiponectin, NEFA and triacylglycerol (P<0.001 for all) compared with chow-fed controls, whereas plasma levels of glucose and insulin were similar in the two groups. After 2 days of diet feeding, there were no significant changes in Ob mRNA levels in epididymal fat, whereas there was a marked decrease in adiponectin mRNA levels. After 16 weeks of diet feeding, rats had significantly increased levels of Ob mRNA, but decreased adiponectin mRNA levels, in epididymal fat compared with the chow-fed group (P<0.001 for both). These findings suggest that obesity per se is not a factor in the decreased adiponectin levels observed in obese subjects. We propose that the lipid profile of the plasma and/or the constituents of the diet consumed by rats may contribute to adiponectin levels more than obesity per se.