Robin E. Buckingham

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.

Abnormal Cardiac and Skeletal Muscle Energy Metabolism in Patients With Type 2 Diabetes

Background It is well known that patients with type 2 diabetes have increased risk of cardiovascular disease, but it is not known whether they have underlying abnormalities in cardiac or skeletal muscle high‐energy phosphate metabolism. Methods and Results We studied 21 patients with type 2 diabetes with no evidence of coronary artery disease or impaired cardiac function, as determined by echocardiography, and 15 age‐, sex‐, and body mass index‐matched control subjects. Cardiac high‐energy phosphate metabolites were measured at rest using 31P nuclear magnetic resonance spectroscopy (MRS). Skeletal muscle high‐energy phosphate metabolites, intracellular pH, and oxygenation were measured using 31P MRS and near infrared spectrophotometry, respectively, before, during, and after exercise. Although their cardiac morphology, mass, and function appeared to be normal, the patients with diabetes had significantly lower phosphocreatine (PCr)/ATP ratios, at 1.50±0.11, than the healthy volunteers, at 2.30±0.12. The cardiac PCr/ATP ratios correlated negatively with the fasting plasma free fatty acid concentrations. Although skeletal muscle energetics and pH were normal at rest, PCr loss and pH decrease were significantly faster during exercise in the patients with diabetes, who had lower exercise tolerance. After exercise, PCr recovery was slower in the patients with diabetes and correlated with tissue reoxygenation times. The exercise times correlated negatively with the deoxygenation rates and the hemoglobin (Hb)A1c levels and the reoxygenation times correlated positively with the HbA1c levels. Conclusions Type 2 diabetic patients with apparently normal cardiac function have impaired myocardial and skeletal muscle energy metabolism related to changes in circulating metabolic substrates. (Circulation. 2003;107:3040‐3046.)

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.

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.

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.

Inhibition by glibenclamide of the vasorelaxant action of cromakalim in the rat.

1 In rat isolated thoracic aortic rings pre‐contracted with noradrenaline (10−6 m), cromakalim (3 × 10−7‐3 × 10−5 m) produced concentration‐related relaxation. This effect was progressively inhibited by increasing concentrations of the anti‐diabetic sulphonylurea drug, glibenclamide (10−6‐10−5 m). 2 In rat isolated portal veins, cromakalim (3 × 10−8‐10−6 m) produced concentration‐related inhibition of the spontaneous contractive activity and glibenclamide (3 × 10−7‐3 × 10−6 m) prevented this inhibitory action in a concentration‐dependent manner. 3 In both rat aortic rings and portal veins, cromakalim (10−5 m) stimulated 86Rb efflux. Prior exposure to glibenclamide (10−7‐10−6 m) produced a concentration‐related inhibition of this response. 4 In conscious rats, cromakalim, 0.075 mg kg−1 i.v., produced a rapid and sustained fall in arterial blood pressure which was not influenced by pretreatment (2 h) with a large oral dose of glibenclamide (100 mg kg−1). 5 In conscious rats, the hypotensive action of cromakalim, 0.075 mg kg−1 i.v., was abolished by pretreatment (30 min) with glibenclamide, 20 mg kg−1, given by the intravenous route. 6 The results suggest that the vasorelaxant and hypotensive actions of cromakalim involve a K+ channel which can be inhibited by glibenclamide, but which may be distinct from the ATP‐sensitive K+ channel of the pancreatic β‐cell.

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.

Characterisation of the neuropeptide Y receptor that mediates feeding in the rat : a role for the Y5 receptor?

Food intake was measured in freely fed rats following intracerebroventricular administration of neuropeptide Y (NPY) and several of its analogues and antagonists to investigate the hypothesis that the NPY Y5 receptor mediates feeding. Rat NPY (rNPY), rNPY(2-36) and rNPY(3-36) produced similar feeding responses over the dose range 0.7-7.0 nmol. Rat peptide YY (rPYY) was more potent and at least as efficacious as rNPY. [Leu31 Pro34]-rNPY (agonist potency: Y1 > Y5 > Y4 = y6) and human pancreatic polypeptide (hPP) produced flatter dose-response curves, suggesting partial agonism at the receptor(s). rNPY(13-36) (agonist potency: Y2 > Y5) had little activity and rPP was inactive. [D-Trp32]-NPY was a weak orexigenic agent given alone and, consistent with partial agonism, it markedly antagonised the response to porcine NPY (pNPY). Similarly, the receptor antagonist (Y1 > Y4) 1229U91 stimulated feeding slightly, and markedly inhibited rNPY-induced feeding. In contrast to a previous report, BIBP 3226 (70 nmol), another Y1 receptor antagonist, failed to inhibit the response to rNPY. Our data in vivo are inconsistent with findings that hPP, [Leu31 Pro34]-rNPY and [D-Trp32]-rNPY are full agonists at the rat cloned Y5 receptor. Thus, whilst the Y5 receptor may be involved, its participation as the sole receptor mediating the orexigenic action of NPY in the rat remains unproven.