Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status.

Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high-fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative (125)I-labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high-fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. (125)I-labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. (125)I-Labelled leptin was reduced by leptin challenge and increased by high-fat feeding. Ob-Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob-Rb expression in the ARC, but not after high-fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob-Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high-fat diet. Thus, early loss of leptin sensitivity after high-fat feeding is unrelated to down-regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease (125)I-labelled leptin binding and the loss of ability of leptin to up-regulate Ob-Rb expression in the ARC after high-fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high-fat diet.

Dietary supplementation with conjugated linoleic acid plus n-3 polyunsaturated fatty acid increases food intake and brown adipose tissue in rats.

The effect of supplementation with 1% conjugated linoleic acid and 1% n-3 long chain polyunsaturated fatty acids (CLA/n-3) was assessed in rats. Food intake increased with no difference in body weights. White adipose tissue weights were reduced whereas brown adipose tissue and uncoupling protein-1 expression were increased. Plasma adiponectin, triglyceride and cholesterol levels were reduced while leptin, ghrelin and liver weight and lipid content were unchanged. Hypothalamic gene expression measurements revealed increased expression of orexigenic and decreased expression of anorexigenic signals. Thus, CLA/n-3 increases food intake without affecting body weight potentially through increasing BAT size and up-regulating UCP-1 in rats.

Solid and liquid obesogenic diets induce obesity and counter-regulatory changes in hypothalamic gene expression in juvenile Sprague-Dawley rats.

Contemporary foods and beverages that constitute the diets of adults and children almost certainly contribute to the obesity problem. To develop a model of childhood obesity, we examined the effects of feeding juvenile rats 2 solid diets, either alone or in combination [nonpurified control diet (C), high-energy (HE), or C+HE] with or without the liquid supplement Ensure (EN). Rats were fed C until 4 wk of age and then were assigned to 1 of 6 weight-matched groups that were fed C, HE, C+HE, C+EN, HE+EN, or C+HE+EN for 5 wk. EN accelerated weight gain and increased energy intake and adiposity irrespective of the solid diet consumed. Serum leptin concentrations were increased after the consumption of all diets when compared with C rats, but there was dissociation between leptin levels and adiposity. The type of solid diet had no effect on the expression of a panel of hypothalamic genes except for glutamate-decarboxylase-67. EN decreased mRNA for agouti-related peptide and neuropeptide Y in the arcuate nucleus and DYN in the paraventricular nucleus. Dynorphin and CART mRNA were decreased in the supraoptic retrochiasmatic nucleus. The reduction in orexigenic signaling in the hypothalamus suggests that overconsumption of EN is sensed by the hypothalamus but that any initiated physiological responses fail to compensate effectively and may be negated or overwhelmed by other systems. Providing diets in solid and liquid form, with choice, mimics more closely the human environment. Understanding the interactions between these diets and peripheral and central energy balance systems could be crucial in unraveling the events underlying human obesity and its early development.

Circulating hormones and hypothalamic energy balance: regulatory gene expression in the Lou/C and Wistar rats.

To ascertain the mechanisms underlying low caloric intake and low body weight in the Lou/C rat, the circulating hormone levels and gene expression of hypothalamic peptides and receptors important in energy balance and the induction of suppressor of cytokine signalling 3 (SOCS3) gene expression in response to leptin challenge were compared with Wistar rats. Plasma leptin levels were lower in the Lou/C rat, as were levels of rat corticosterone, TSH and T4 but not T3. Ghrelin levels were higher in the Lou/C rat. Total leptin receptor (Ob-R) and the long form of the leptin receptor (Ob-Rb) gene expression were lower in the arcuate (ARC) and ventromedial nuclei (VMN) in Lou/C rat. Ghrelin receptor expression in the ARC and VMN was lower in Lou/C than in Wistar rats. However, agouti gene-related peptide (AgRP) and neuropeptide Y (NPY) gene expression were higher in the Lou/C rat. There was no difference in the level of cocaine- and amphetamine-regulated transcript gene expression in the ARC, but both were higher in the paraventricular nuclei of the Lou/C breed. There was no difference in Ob-R gene expression in, or [(125)I]leptin binding to, the choroid plexus. SOCS3 mRNA induction in response to leptin was lower in the Lou/C rat. This study reveals that the comparatively low plasma leptin, TSH and T4 levels, and high ghrelin levels together with high levels of AgRP and NPY gene expression seen in the Lou/C rat are indicative of a strong drive to eat and decreased energy expenditure, which are in direct opposition to the comparatively low body weight and adiposity of this rat strain.

Negative energy balance and leptin regulate neuromedin-U expression in the rat pars tuberalis.

Central neuromedin U (NMU) functions in energy balance, the hypothalamic-pituitary-adrenal axis, LH release and circadian rhythmicity. In rats, high levels of NMU occur in the hypothalamic suprachiasmatic nuclei and the pars tuberalis of the pituitary. NMU expression in the pars tuberalis appears to be downregulated in the Zucker fatty (fa/fa) rat, lacking functional leptin receptors. In contrast, in the dorsomedial (DMH) nuclei of the mouse, NMU expression is higher in the ob/ob mouse, lacking leptin, and is upregulated by fasting. However, leptin appears not to change NMU gene expression in either the mouse DMH or the rat pars tuberalis. Thus, the present study aims to better identify factors influencing central NMU expression in the rat pars tuberalis. Sprague-Dawley rats were fasted and/or challenged with intracerebroventricular leptin or ghrelin and gene expression was measured using real-time reverse transcriptase-PCR and quantitative in situ hybridisation with riboprobes specific for NMU and NMU receptor (NMU-R2). NMU expression in the rat pars tuberalis was elevated by fasting. Ghrelin administration had no effect on the level of NMU expression, but leptin was found to diminish the expression in a concentration- and time-dependent manner. NMU-R2 expression was unchanged in any of the groups measured. These results suggest that NMU expression in rat pars tuberalis is upregulated in states of negative energy balance, and this may be mediated indirectly by changes in leptin levels. These results demonstrate a link between energy balance and NMU expression in the pars tuberalis of the pituitary.

Effect of flavour of liquid Ensure diet supplement on energy intake in male SD rats.

Outbred male Sprague-Dawley rats were provided with one of the four flavours of the liquid diet, Ensure, in addition to chow pellets, to examine whether differences in flavour lead to differences in energy intake i.e. degree of over-consumption. For half the rats, the Ensure supplement was provided for 14 days and then withdrawn for the final 8 days of the study, whereas the remaining animals were allowed to consume Ensure for 22 days. All four flavours of Ensure, chocolate, vanilla, coffee and asparagus, induced a sustained increase in daily energy intake of approximately 15%. There was an effect of flavour on initial consumption of the Ensure diet, with coffee and asparagus flavours being consumed less avidly than vanilla or chocolate. However, this effect was short-lived. Overall, there was no effect of flavour on body weight gain, energy intake from Ensure, total energy intake, body composition, or measured blood hormones and metabolites. Withdrawal of Ensure resulted in reductions in body weight gain, total energy intake, fat but not lean tissue mass, and concentrations of blood leptin, non-esterified fatty acids and triglycerides, but there was no effect of the flavour of Ensure previously supplied on any of these parameters. The ability of the liquid diet, Ensure, to stimulate long-term caloric over-consumption is not due to its flavouring. Rather, other attributes of Ensure must be more important, such as its intrinsic flavour, liquid formulation, macronutrient composition, and ease of ingestion, digestion and absorption.

Regulation of growth hormone secretagogue receptor gene expression in the arcuate nuclei of the rat by leptin and ghrelin.

The anorexigenic and orexigenic hormones leptin and ghrelin act in opposition to one another. When leptin signaling is reduced, as in the Zucker fatty rat, or when circulating ghrelin is increased during fasting, the effect of ghrelin becomes more dominant, indicating an influence of both hormones on ghrelin action. This effect could be mediated via the level of expression of ghrelin receptor (growth hormone secretagogue receptor [GHS-R]). For testing this, GHS-R expression was measured using in situ hybridization in Zucker fatty versus lean rats; in fed versus fasted (48 h) rats, treated with either ghrelin or leptin; and in GH-deficient, dwarf versus control rats. In the arcuate nuclei of the Zucker fatty rat and in fasted rats, GHS-R expression is significantly increased. A single leptin intracerebroventricular injection attenuated the fasting-induced increase in GHS-R but had no effect in fed rats 2 h after injection, whereas leptin infusion for 24 h or longer significantly decreased GHS-R expression in fed rats. Ghrelin significantly increased GHS-R expression but not in dwarf rats. These results show that the level of GHS-R expression in the ARC is reduced by leptin and increased by ghrelin and that the effect of ghrelin may be GH dependent.

Effect of retinoic acid on leptin, glycerol, and glucose levels in mature rat adipocytes in vitro.

To elucidate the effects of retinoic acids (RAs) on adipogenesis and insulin sensitivity, we treated mature adipocytes with two different kinds of RA, 9-cis-RA and all-trans-RA. Both 9-cis- and all-trans-RA inhibited the secretion of leptin. However, the inhibition was significantly decreased at a higher dose of each RA. The inhibitory effect of 9-cis-RA was synergistically enhanced by the addition of rosiglitazone, a synthetic ligand for peroxisome proliferator-activated receptor (PPAR) gamma. 9-cis-RA also leads to adipogenesis in a dose-dependent manner. On the contrary, all-trans-RA does not increase adipogenesis in a dose-dependent manner. To clarify the antidiabetic effects of RA, glucose uptake was assessed by estimating glucose concentrations in the medium. 9-cis-RA reduced glucose levels in the culture media, but all-trans-RA did not. In conclusion, all-trans-RA does not alter adipogenesis and glucose uptake but does inhibit leptin secretion. 9-cis-RA, however, seems to increase both adipogenesis and glucose uptake through activation of the retinoid X receptor/PPARgamma heterodimer.

Hypothalamic gene expression is altered in underweight but obese juvenile male Sprague-Dawley rats fed a high-energy diet.

The incidence of obesity, with its associated health risks, is on the increase throughout the western world affecting all age groups, including children. The typical western diet is high in fat and sugar and low in complex carbohydrates. This study looks at the effects of feeding an equivalent high-energy (HE) diet to growing rats. Juvenile male Sprague-Dawley rats that were fed an HE (18.9 kJ/g) diet starting approximately 10 d after weaning gained less weight than littermates fed a nonpurified (14 kJ/g) diet. Despite an initial hyperphagia following the change in diet, HE rats also consumed less energy. Although they exhibited reduced weight gain, HE rats were relatively obese; fat pad weights were elevated for all 4 dissected depots. HE-fed rats exhibited symptoms of developing metabolic syndrome with elevated plasma concentrations of glucose, triglycerides, nonesterified fatty acids, insulin, and leptin. In addition, leptin receptor gene expression in the hypothalamic arcuate nucleus (ARC) and ventromedial nucleus of HE rats was reduced. Consistent with the elevated serum leptin and other peripheral signals in HE rats, hypothalamic gene expression for the orexigenic neuropeptides, neuropeptide Y (ARC and dorsomedial nucleus), and agouti-related peptide (AgRP), was reduced. This reduction in orexigenic signaling and decline in energy intake is consistent with an apparent attempt to counter the further development of an obese state in rats consuming an energy-dense diet. The juvenile Sprague-Dawley rat has potential in the development of a model of childhood diet-induced obesity.

Normal distribution of body weight gain in male Sprague-Dawley rats fed a high-energy diet.

OBJECTIVE: To investigate the effect of a high-energy (HE) diet on caloric intake, body weight, and related parameters in outbred male Sprague-Dawley (SD) rats. RESEARCH METHODS AND PROCEDURES: Twenty-eight SD rats were fed either chow (C) for 19 weeks or HE diet for 14 weeks and then C for 5 weeks. Blood hormones and metabolites were assayed, and expression of uncoupling protein-1 and hypothalamic energy-balance-related genes were determined by Northern blotting and in situ hybridization, respectively. RESULTS: HE rats gained body weight more rapidly than C animals with a range of weight gains, but there was no evidence that weight gain was bimodally distributed. Caloric intake was transiently elevated after introduction of the HE diet. Transfer of HE rats back to C resulted in a drop in caloric intake, but a stable body weight. In terminal analysis, two of four dissected adipose tissue depots were heavier in rats that had previously been fed HE diet. Blood leptin, insulin, glucose, and nonesterified fatty acids were not different between the groups. Uncoupling protein-1 mRNA was elevated in interscapular brown adipose tissue from HE rats. There was a trend for agouti-related peptide mRNA in the hypothalamic arcuate nucleus to be higher in HE rats. DISCUSSION: Contrary to other studies of the SD rat on HE diet, body weight and other measured parameters were normally distributed. There was no segregation into two distinct populations on the basis of susceptibility to diet-induced obesity. This characteristic may be dependent on the breeding colony from which animals were sourced.