Jonathan R.S. Arch

Orexins and Orexin Receptors: A Family of Hypothalamic Neuropeptides and G Protein-Coupled Receptors that Regulate Feeding Behavior

The hypothalamus plays a central role in the integrated control of feeding and energy homeostasis. We have identified two novel neuropeptides, both derived from the same precursor by proteolytic processing, that bind and activate two closely related (previously) orphan G protein-coupled receptors. These peptides, termed orexin-A and -B, have no significant structural similarities to known families of regulatory peptides. prepro-orexin mRNA and immunoreactive orexin-A are localized in neurons within and around the lateral and posterior hypothalamus in the adult rat brain. When administered centrally to rats, these peptides stimulate food consumption. prepro-orexin mRNA level is up-regulated upon fasting, suggesting a physiological role for the peptides as mediators in the central feedback mechanism that regulates feeding behavior.

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.

A guide to analysis of mouse energy metabolism

We present a consolidated view of the complexity and challenges of designing studies for measurement of energy metabolism in mouse models, including a practical guide to the assessment of energy expenditure, energy intake and body composition and statistical analysis thereof. We hope this guide will facilitate comparisons across studies and minimize spurious interpretations of data. We recommend that division of energy expenditure data by either body weight or lean body weight and that presentation of group effects as histograms should be replaced by plotting individual data and analyzing both group and body-composition effects using analysis of covariance (ANCOVA).

Effects of single and chronic intracerebroventricular administration of the orexins on feeding in the rat

Two novel hypothalamic neuropeptides, orexin-A and -B, are suggested to regulate feeding. A single intracerebroventricular injection of orexin-A (23.4 nmol), administered 3 h into the light phase, increased feeding in satiated rats and prolonged feeding in fasted rats; it also increased feeding when given 6 h into, but not at the start of, the dark phase. An 8-day intracerebroventricular infusion with orexin-A (18 nmol/day) increased daytime feeding on days 2 and 8, but nocturnal feeding was reduced and there was no change in 24 h intake. Orexin-B had no effects. These results demonstrate a circadian variation in feeding responses to orexin-A.

A selective orexin-1 receptor antagonist reduces food consumption in male and female rats.

A variety of evidence implicates the orexins, especially orexin-A, in the regulation of food intake, but it has not been established whether this effect is mediated by the orexin-1 or orexin-2 receptor. In the present study, a selective orexin-1 receptor antagonist, 1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl urea hydrochloride (SB-334867-A), was administered intraperitoneally to rats under various conditions, and food consumption was subsequently measured over 24 h. In male rats, a single dose of SB-334867-A (30 mg/kg, i.p.) given during the light phase reduced both orexin-A-induced food intake (7 nmol, i.c.v.) and feeding stimulated by an overnight fast for 4 h. When given at the start of the dark phase, food consumption was reduced in both male and female rats over 24 h. Daily injections at the start of the dark phase for 3 days reduced natural feeding in male rats over 24 h on days one and three. These findings demonstrate direct inhibition of orexin-A induced food intake with a selective orexin-1 receptor antagonist. Furthermore, the suppression of nocturnal feeding and food intake stimulated by an overnight fast supports other evidence that orexin-A is involved in the regulation of natural feeding and suggests that orexin-1 receptor antagonists could be useful in the treatment of obesity.

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.

Differential distribution of orexin-A and orexin-B immunoreactivity in the rat brain and spinal cord<

The orexins are recently identified appetite-stimulating hypothalamic peptides. We used immunohistochemistry to map orexin-A and orexin-B immunoreactivity in rat brain, spinal cord, and some peripheral tissues. Orexin-A- and orexin-B-immunoreactive cell bodies were confined to the lateral hypothalamic area and perifornical nuclei. Orexin-A-immunoreactive fibers were densely distributed in the hypothalamus, septum, thalamus, locus coeruleus, spinal cord, and near the ventricles, but absent from peripheral sites investigated. In contrast, orexin-B-immunoreactive fibers were distributed sparsely in the hypothalamus. Orexin cells are strategically sited to contribute to feeding regulation, but their widespread projections suggest that orexins have other physiological roles.

Some mathematical and technical issues in the measurement and interpretation of open-circuit indirect calorimetry in small animals

Indirect calorimetry is increasingly used to investigate why compounds or genetic manipulations affect body weight or composition in small animals. This review introduces the principles of indirect (primarily open-circuit) calorimetry and explains some common misunderstandings. It is not widely understood that in open-circuit systems in which carbon dioxide (CO2) is not removed from the air leaving the respiratory chamber, measurement of airflow out of the chamber and its oxygen (O2) content paradoxically allows a more reliable estimate of energy expenditure (EE) than of O2 consumption. If the CO2 content of the exiting air is also measured, both O2 consumption and CO2 production, and hence respiratory quotient (RQ), can be calculated. Respiratory quotient coupled with nitrogen excretion allows the calculation of the relative combustion of the macronutrients only if measurements are over a period where interconversions of macronutrients that alter their pool sizes can be ignored. Changes in rates of O2 consumption and CO2 production are not instantly reflected in changes in the concentrations of O2 and CO2 in the air leaving the respiratory chamber. Consequently, unless air-flow is high and chamber size is small, or rates of change of O2 and CO2 concentrations are included in the calculations, maxima and minima are underestimated and will appear later than their real times. It is widely appreciated that bigger animals with more body tissue will expend more energy than smaller animals. A major issue is how to compare animals correcting for such differences in body size. Comparison of the EE or O2 consumption per gram body weight of lean and obese animals is misleading because tissues vary in their energy requirements or in how they influence EE in other ways. Moreover, the contribution of fat to EE is lower than that of lean tissue. Use of metabolic mass for normalisation, based on interspecific scaling exponents (0.75 or 0.66), is similarly flawed. It is best to use analysis of covariance to determine the relationship of EE to body mass or fat-free mass within each group, and then test whether this relationship differs between groups.

Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)‐stimulated leptin secretion by activating Gαi. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gαi signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild‐type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild‐type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gαi signalling mediated by GPR43 in SCFA‐stimulated leptin secretion.

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.