I SURVIVE: inter-rater reliability of three physical functional outcome measures in intensive care unit survivors.

PURPOSE: We prospectively assessed inter-rater reliability of three physical function measures in Canadian intensive care unit (ICU) survivors in the inpatient setting. METHODS: We enrolled patients who had an ICU length of stay of ≥ three days, were mechanically ventilated for ≥ 24 hr, and were ambulating independently before hospital admission. Weekly from ICU discharge to hospital discharge, two trained frontline clinicians, blinded to each other's findings, independently performed the Physical Function ICU Test-scored (PFIT-s; score out of 10), 30-sec sit-to-stand (30STS; # of stands), and two-minute walk test (2MWT; distance in m), all within 24 hr. We calculated the intraclass correlation coefficient (ICC), standard error of measurement (SEM), and minimal detectable change (MDC90). RESULTS: We enrolled 42 patients. PFIT-s: in 36 patients with 66 paired scores, the ICC was 0.78 (95% confidence interval [CI], 0.66 to 0.86), the SEM was 1.04, and the MDC90 was 2.42. 30STS: in 35 patients with 67 paired scores, the ICC was 0.85 (95% CI, 0.76 to 0.90), the SEM was 1.91, and the MDC90 was 4.45. 2MWT: in 35 patients with 58 paired scores, the ICC was 0.78 (95% CI, 0.66 to 0.87), the SEM was 20.87, and the MDC90 was 48.69. CONCLUSION: These three measures show good inter-rater reliability when used by trained frontline clinicians to assess physical function in ICU survivors in the inpatient setting.

RéSUMé: OBJECTIF: Nous avons évalué de manière prospective la fidélité interobservateurs de trois mesures de la capacité physique fonctionnelle auprès de survivants des unités de soins intensifs (USI) canadiennes dans un cadre hospitalier. MéTHODE: Nous avons recruté des patients qui avaient séjourné à l'USI ≥ trois jours, avaient été sous ventilation mécanique ≥ 24 h et se déplaçaient de façon autonome avant leur admission à l'hôpital. Entre le congé de l'USI et le congé de l'hôpital, deux cliniciens de première ligne formés ont réalisé chaque semaine et de manière indépendante trois tests : le PFIT (Test de la capacité physique fonctionnelle à l'USI - Physical Function ICU Test-scored; score sur 10), le test de 30 sec pour se lever d'une position assise (30STS; # de fois debout), et le test de marche pendant deux minutes (2MWT; distance en m). Les cliniciens n'avaient pas accès aux résultats de leur collègue. Nous avons calculé le coefficient de corrélation intraclasse (CCI), l'erreur type sur la mesure (ETM), et le changement minimal détectable (CMD90). RéSULTATS: Nous avons recruté 42 patients, et observé les résultats suivants : pour le PFIT : chez 36 patients avec 66 scores appariés, le CCI était de 0,78 (intervalle de confiance [IC] 95 %, 0,66 à 0,86), l'ETM de 1,04, et le CMD90 de 2,42; pour le test 30STS : chez 35 patients avec 67 scores appariés, le CCI était de 0,85 (IC 95 %, 0,76 à 0,90), l'ETM de 1,91, et le CMD90 de 4,45; pour le 2MWT : chez 35 patients avec 58 scores appariés, le CCI était de 0,78 (IC 95 %, 0,66 à 0,87, l'ETM de 20,87, et le CMD90 de 48,69. CONCLUSION: Ces trois mesures affichent une bonne fidélité interobservateurs lorsqu'elles sont utilisées par des cliniciens de première ligne formés afin d'évaluer la capacité physique fonctionnelle des survivants de l'USI dans un cadre hospitalier.

Elevated C-Peptides, Abdominal Obesity, and Abnormal Adipokine Profile are Associated With Higher Gleason Scores in Prostate Cancer.

BACKGROUND: Prostate cancer development is associated with numerous lifestyle factors (i.e., physical activity, nutrition intake) and metabolic perturbations. These factors have been studied independently; here, we used an integrative approach to characterize these lifestyle and metabolic parameters in men undergoing diagnostic prostate biopsies. METHODS: We prospectively evaluated 51 consecutive men for body composition, metabolic factors including glucose- and lipid-related measures, as well as lifestyle factors prior to prostate biopsy. Evaluations were performed in a blinded manner and were subsequently related to biopsy outcomes for: (i) presence or absence of cancer; and (ii) where cancer was present, Gleason score. RESULTS: Serum C-peptide concentrations were significantly greater in participants with Gleason scores ≥4 + 3 (2.8 ± 1.1 ng/ml) compared to those with Gleason 3 + 3 (1.4 ± 0.6 ng/ml) or Gleason 3 + 4 (1.3 ± 0.8 ng/ml, P = 0.002), suggesting greater insulin secretion despite lack of differences in fasting glucose concentrations. Central adiposity, measured by waist circumference, was significantly greater in participants with Gleason ≥4 + 3 (110.1 ± 7.4 cm) compared to those with Gleason 3 + 4 (102.0 ± 9.5 cm, P = 0.028). Men with Gleason ≥4 + 3 also had significantly greater leptin concentrations than those with lower Gleason scores (Gleason ≥4 + 3: 15.6 ± 3.3 ng/ml vs. Gleason 3 + 4: 8.1 ± 8.1 ng/ml, P < 0.05) and leptin:adiponectin ratio (Gleason ≥4 + 3: 9.7 ± 6.1 AU, Gleason 3 + 4: 2.9 ± 3.2, Gleason 3 + 3: 2.4 ± 2.1 AU, P = 0.013). CONCLUSIONS: We profiled a cluster of obesity-related metabolic perturbations (C-peptide, central adiposity, leptin, and leptin:adiponectin ratios) which may associate with more aggressive prostate cancer histology. Prostate 77:211-221, 2017. © 2016 Wiley Periodicals, Inc.

Sleeve Gastrectomy Does Not Cause Hypertrophy and Reprogramming of Intestinal Glucose Metabolism in Rats.

BACKGROUND: Clinical studies have shown similar rapid improvements in body mass and glycemic control after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG). Evidence suggests that adaptive intestinal tissue growth and reprogramming of intestinal glucose disposal play a key role in the beneficial effects on glucose homeostasis after RYGB, but it is not known whether such adaptive changes also occur after sleeve gastrectomy. METHODS: High-fat diet-induced obese rats were subjected to either VSG or RYGB, and intestinal growth and functional adaptations were assessed by using morphometric, immunohistochemical, and immuno-blot techniques, 3 months after surgery or sham surgery. RESULTS: The cross-sectional areas of the Roux and common limbs are significantly increased after RYGB compared with sham surgery (Roux limb: 17.1 ± 4.0 vs. 5.5 ± 0.1 mm(2); common limb: 11.7 ± 0.6 vs. 5.1 ± 0.5 mm(2); p < 0.01), but the cross-sectional area of the corresponding jejunum is not different from controls after VSG. Similarly, mucosal thickness and the number of GLP-1 cells are not increased after VSG. Protein expression of hexokinase II is increased fourfold (p < 0.01) in the Roux limb after RYGB, but not in the jejunum after VSG. CONCLUSIONS: Adaptive hypertrophy and reprogramming of glucose metabolism in the small intestine are not necessary for VSG to improve body composition and glycemic control. The similar beneficial effects of VSG and RYGB on glucose homeostasis might be mediated by different mechanisms.

Monoclonal antibody targeting of fibroblast growth factor receptor 1c ameliorates obesity and glucose intolerance via central mechanisms.

We have generated a novel monoclonal antibody targeting human FGFR1c (R1c mAb) that caused profound body weight and body fat loss in diet-induced obese mice due to decreased food intake (with energy expenditure unaltered), in turn improving glucose control. R1c mAb also caused weight loss in leptin-deficient ob/ob mice, leptin receptor-mutant db/db mice, and in mice lacking either the melanocortin 4 receptor or the melanin-concentrating hormone receptor 1. In addition, R1c mAb did not change hypothalamic mRNA expression levels of Agrp, Cart, Pomc, Npy, Crh, Mch, or Orexin, suggesting that R1c mAb could cause food intake inhibition and body weight loss via other mechanisms in the brain. Interestingly, peripherally administered R1c mAb accumulated in the median eminence, adjacent arcuate nucleus and in the circumventricular organs where it activated the early response gene c-Fos. As a plausible mechanism and coinciding with the initiation of food intake suppression, R1c mAb induced hypothalamic expression levels of the cytokines Monocyte chemoattractant protein 1 and 3 and ERK1/2 and p70 S6 kinase 1 activation.

Thromboembolic events in patients with urothelial carcinoma undergoing neoadjuvant chemotherapy and radical cystectomy.

OBJECTIVES: Patients receiving cisplatin are at high risk of thromboembolic events (TEEs). The objective of this study was to assess the effect of cisplatin-based neoadjuvant chemotherapy (NCT) on the incidence of perioperative TEEs in patients undergoing radical cystectomy. METHODS AND MATERIALS: We analyzed a consecutive sample of 202 patients with urothelial carcinoma treated with radical cystectomy between 2005 and 2013. Data were collected retrospectively by reviewing medical records. Median follow-up was 16.9 months. Events of interest were defined as venous or arterial TEEs occurring from the date of diagnosis to 30 days after surgery. TEE incidence among patients treated with NCT and cystectomy was compared with that among patients treated with cystectomy alone using Fisher exact test and Cox proportional hazards regression. Proportional hazards regression was also used to assess whether TEE is a predictor of cancer progression and survival. RESULTS: Of 202 patients, 17 (8.4%) developed a TEE, including 8 of 42 (19.1%) treated with NCT and cystectomy and 9 of 160 (5.6%) treated with cystectomy alone (risk ratio = 3.39, 95% CI: 1.39-8.24). After adjustment for observation time, there remained an association between treatment with NCT and risk of TEE (hazard ratio = 2.40; 95% CI: 0.92-6.27; P = 0.07). Overall, 7 events occurred before cystectomy and 10 occurred postoperatively. Among patients treated with NCT, 6 of 8 events occurred before cystectomy. Detection of TEE was clinically significant as preoperative TEE was found to be an independent predictor of progression and cancer-specific mortality (adjusted hazard ratio = 3.91, 95% CI: 1.34-11.45). The main limitations of our study are its retrospective data collection and small absolute number of events. CONCLUSIONS: TEE occurs commonly in patients with urothelial carcinoma undergoing NCT. Preoperative TEE is an independent predictor of progression and cancer-specific mortality.

Vagal innervation of intestine contributes to weight loss After Roux-en-Y gastric bypass surgery in rats.

BACKGROUND: It is conceivable that overstimulation of chemo- and mechano-sensors in the Roux and common limbs by uncontrolled influx of undigested nutrients after Roux-en-Y gastric bypass surgery (RYGB) could lead to exaggerated satiety signaling via vagal afferents and contribute to body weight loss. Because previous clinical and preclinical studies using vagotomy came to different conclusions, the aim was to examine the effects of selective and histologically verified celiac branch vagotomy on reduced food intake and body weight loss induced by RYGB. METHODS: Male Sprague-Dawley rats underwent either RYGB + celiac branch vagotomy (RYGB/VgX, n=15), RYGB + sham celiac branch vagotomy (RYGB/Sham VgX; n=6), Sham RYGB + celiac branch vagotomy (Sham/VgX; n=6), or sham RYGB + sham celiac branch vagotomy (Sham/Sham; n=6), and body weight, body composition, and food choice were monitored for 3 months after intervention. RESULTS: In rats with RYGB, histologically confirmed celiac branch vagotomy significantly moderated weight loss during the first 40 days after surgery, compared to either sham or failed vagotomy (P<0.05). In contrast, celiac branch vagotomy slightly, but non-significantly, reduced body weight gain in sham RYGB rats compared to sham/sham rats. Furthermore, the significant food intake suppression during the first 32 days after RYGB (P<0.05) was also moderated in rats with verified celiac branch vagotomy. CONCLUSIONS: The results suggest that signals carried by vagal afferents from the mid and lower intestines contribute to the early RYGB-induced body weight loss and reduction of food intake.

Reversible hyperphagia and obesity in rats with gastric bypass by central MC3/4R blockade.

OBJECTIVE: To test the commonly held assumption that gastric bypass surgery lowers body weight because it limits the ability to eat large amounts of food. METHODS: Central melanocortin signaling was blocked by ICV infusion of the melanocortin-3/4 receptor antagonist SHU9119 for 14 days in rats whose high-fat diet-induced obesity had been reversed by Roux-en-Y gastric bypass surgery. RESULTS: SHU9119 increased daily food intake (+ 100%), body weight (+30%), and fat mass (+50%) in rats with RYGB, surpassing the presurgical body weight and that of saline-treated sham-operated rats. Doubling of food intake was entirely due to increased meal frequency, but not meal size. After termination of SHU9119, body weight promptly returned to near preinfusion levels. In sham-operated rats, SHU9119 produced even larger increases in food intake and body weight. CONCLUSIONS: RYGB rats do not settle at a lower level of body weight because they cannot eat more food as they can easily double food intake by increasing meal frequency. The reversible obesity suggests that RYGB rats actively defend the lower body weight. However, because both RYGB and sham-operated rats responded to SHU9119, central melanocortin signaling is not the critical mechanism in RYGB rats responsible for this defense.

GLP-1 receptor signaling is not required for reduced body weight after RYGB in rodents.

Exaggerated GLP-1 and PYY secretion is thought to be a major mechanism in the reduced food intake and body weight after Roux-en-Y gastric bypass surgery. Here, we use complementary pharmacological and genetic loss-of-function approaches to test the role of increased signaling by these gut hormones in high-fat diet-induced obese rodents. Chronic brain infusion of a supramaximal dose of the selective GLP-1 receptor antagonist exendin-9-39 into the lateral cerebral ventricle significantly increased food intake and body weight in both RYGB and sham-operated rats, suggesting that, while contributing to the physiological control of food intake and body weight, central GLP-1 receptor signaling tone is not the critical mechanism uniquely responsible for the body weight-lowering effects of RYGB. Central infusion of the selective Y2R-antagonist BIIE0246 had no effect in either group, suggesting that it is not critical for the effects of RYGB on body weight under the conditions tested. In a recently established mouse model of RYGB that closely mimics surgery and weight loss dynamics in humans, obese GLP-1R-deficient mice lost the same amount of body weight and fat mass and maintained similarly lower body weight compared with wild-type mice. Together, the results surprisingly provide no support for important individual roles of either gut hormone in the specific mechanisms by which RYGB rats settle at a lower body weight. It is likely that the beneficial effects of bariatric surgeries are expressed through complex mechanisms that require combination approaches for their identification.

Longitudinal assessment of food intake, fecal energy loss, and energy expenditure after Roux-en-Y gastric bypass surgery in high-fat-fed obese rats.

BACKGROUND: The efficacy of Roux-en-Y gastric bypass (RYGB) surgery to produce weight loss has been well-documented, but few studies have measured the key components of energy balance, food intake, and energy expenditure longitudinally. METHODS: Male Sprague-Dawley rats on a high-fat diet underwent either RYGB, sham operation, or pair feeding and were compared to chow-fed lean controls. Body weight and composition, food intake and preference, energy expenditure, fecal output, and gastric emptying were monitored before and up to 4 months after intervention. RESULTS: Despite the recovery of initially decreased food intake to levels slightly higher than before surgery and comparable to sham-operated rats after about 1 month, RYGB rats maintained a lower level of body weight and fat mass for 4 months that was not different from chow-fed age-matched controls. Energy expenditure corrected for lean body mass at 1 and 4 months after RYGB was not different from presurgical levels and from all other groups. Fecal energy loss was significantly increased at 6 and 16 weeks after RYGB compared to sham operation, and there was a progressive decrease in fat preference after RYGB. CONCLUSIONS: In this rat model of RYGB, sustained weight loss is achieved by a combination of initial hypophagia and sustained increases in fecal energy loss, without change in energy expenditure per lean mass. A shift away from high-fat towards low-fat/high-carbohydrate food preference occurring in parallel suggests long-term adaptive mechanisms related to fat absorption.

"Liking" and "wanting" of sweet and oily food stimuli as affected by high-fat diet-induced obesity, weight loss, leptin, and genetic predisposition.

Cross-sectional studies in both humans and animals have demonstrated associations between obesity and altered reward functions at the behavioral and neural level, but it is unclear whether these alterations are cause or consequence of the obese state. Reward behaviors were quantified in male, outbred Sprague-Dawley (SD) and selected line obesity-prone (OP) and obesity-resistant (OR) rats after induction of obesity by high-fat diet feeding and after subsequent loss of excess body weight by chronic calorie restriction. As measured by the brief access lick and taste-reactivity paradigms, both obese SD and OP rats "liked" low concentrations of sucrose and corn oil less, but "liked" the highest concentrations more, compared with lean rats, and this effect was fully reversed by weight loss in SD rats. Acute food deprivation was unable to change decreased responsiveness to low concentrations but eliminated increased responsiveness to high concentrations in obese SD rats, and leptin administration in weight-reduced SD rats shifted concentration-response curves toward that seen in the obese state in the brief access lick test. "Wanting" and reinforcement learning as assessed in the incentive runway and progressive ratio lever-pressing paradigms was paradoxically decreased in both obese (compared with lean SD rats) and OP (compared with OR rats). Thus, reversible, obesity-associated, reduced "liking" and "wanting" of low-calorie foods in SD rats suggest a role for secondary effects of the obese state on reward functions, while similar differences between select lines of OP and OR rats before induction of obesity indicate a genetic component.

High-fat intake induced by mu-opioid activation of the nucleus accumbens is inhibited by Y1R-blockade and MC3/4R- stimulation.

Nucleus accumbens mu-opioid receptor activation can strongly stimulate intake of high-fat food in satiated rats, and one of the mechanisms involves activation of lateral hypothalamic orexin neurons and orexin receptor-1 signaling in the mesolimbic dopamine system. Here, we tested the potential contribution of NPY/Y1R and alpha-MSH/MC3/4R-signaling to accumbens-induced high-fat feeding. Prior administration of the selective Y1R antagonist 1229U91 or the MC3/4R agonist MTII into the lateral ventricle (LV) dose-dependently decreased high-fat intake induced by nucleus accumbens injection of the mu-opioid receptor agonist DAMGO. Both drugs also decreased high-fat feeding induced by switching rats from regular chow to high-fat diet, but less efficiently than when DAMGO-induced. Administration of 1229U91 directly into the PVH also suppressed DAMGO-induced high-fat intake, but a higher dose was required. The results suggest that NPY/Y1R signaling in the PVH and other forebrain sites is necessary for accumbens DAMGO to elicit high-fat intake, and that forebrain MC3/4R signaling can suppress it.

A potential role for hypothalamomedullary POMC projections in leptin-induced suppression of food intake.

Melanocortin-3/4 receptor ligands administered to the caudal brain stem potently modulate food intake by changing meal size. The origin of the endogenous ligands is unclear, because the arcuate nucleus of the hypothalamus and the nucleus of the solitary tract (NTS) harbor populations of proopiomelanocortin (POMC)-expressing neurons. Here we demonstrate that activation of hypothalamic POMC neurons leads to suppression of food intake and that this suppression is prevented by administration of a melanocortin-3/4 receptor antagonist to the NTS and its vicinity. Bilateral leptin injections into the rat arcuate nucleus produced long-lasting suppression of meal size and total chow intake. These effects were significantly blunted by injection of SHU-9119 into the fourth ventricle, although SHU-9119 increased meal size and food intake during the first, but not the second, 14-h observation period. Leptin effects on meal size and food intake were abolished throughout the 40-h observation period by injection of SHU-9119 into the NTS at a dose that by itself had no effect. Neuron-specific tracing from the arcuate nucleus with a Cre-inducible tract-tracing adenovirus in POMC-Cre mice showed the presence of labeled axons in the NTS. Furthermore, density of alpha-melanocyte-stimulating hormone-immunoreactive axon profiles throughout the NTS was decreased by approximately 70% after complete surgical transection of connections with the forebrain in the chronic decerebrate rat model. The results further support the existence of POMC projections from the hypothalamus to the NTS and suggest that these projections have a functional role in the control of food intake.

Meal patterns, satiety, and food choice in a rat model of Roux-en-Y gastric bypass surgery.

Gastric bypass surgery efficiently and lastingly reduces excess body weight and reverses type 2 diabetes in obese patients. Although increased energy expenditure may also play a role, decreased energy intake is thought to be the main reason for weight loss, but the mechanisms involved are poorly understood. Therefore, the aim of this study was to characterize the changes in ingestive behavior in a rat model of Roux-en-Y gastric bypass surgery (RYGB). Obese (24% body fat compared with 18% in chow-fed controls), male Sprague-Dawley rats maintained for 15 wk before and 4 mo after RYGB or sham-surgery on a two-choice low-fat/high-fat diet, were subjected to a series of tests assessing energy intake, meal patterning, and food choice. Although sham-operated rats gained an additional 100 g body wt during the postoperative period, RYGB rats lost approximately 100 g. Intake of a nutritionally complete and palatable liquid diet (Ensure) was significantly reduced by approximately 50% during the first 2 wk after RYGB compared with sham surgery. Decreased intake was the result of greatly reduced meal size with only partial compensation by meal frequency, and a corresponding increase in the satiety ratio. Similar results were obtained with solid food (regular or high-fat chow) 6 wk after surgery. In 12- to 24-h two-choice liquid or solid diet paradigms with nutritionally complete low- and high-fat diets, RYGB rats preferred the low-fat choice (solid) or showed decreased acceptance for the high-fat choice (liquid), whereas sham-operated rats preferred the high-fat choices. A separate group of rats offered chow only before surgery completely avoided the solid high-fat diet in a choice paradigm. The results confirm anecdotal reports of "nibbling" behavior and fat avoidance in RYGB patients and provide a basis for more mechanistic studies in this rat model.

Phenotype of neurons in the nucleus of the solitary tract that express CCK-induced activation of the ERK signaling pathway.

The satiating potency of CCK has been well characterized, including its mediation by capsaicin-sensitive vagal primary afferents. We have previously shown that peripherally administered CCK activates the MAPK-signaling cascade in a population of nucleus of the solitary tract (NTS) neurons and that preventing ERK1/2 phosphorylation partly attenuates CCK's satiating potency. The aim of this study was to identify the neurochemical phenotypes of the NTS neurons that exhibit CCK-induced activation of ERK1/2. Using confocal microscopy, we demonstrate that intraperitoneal CCK administration increases the number of neurons that express phosphorylated ERK1/2 (pERK1/2) in the medial and commissural subnuclei of the NTS and that CCK-induced expression of ERK1/2 is increased in tyrosine hydroxylase-immunoreactive neurons. Using Western blot analysis, we show that the robust increase in tyrosine hydroxylase phosphorylation obtained with intraperitoneal CCK is significantly attenuated in rats pretreated with the ERK-pathway blocker U0126 injected into the 4th ventricle. In addition, CCK injections increased pERK1/2 expression in POMC neurons in the NTS. In contrast, only the rare GAD67, neuronal nitric oxide synthase, and leptin-responsive neuron exhibited CCK-induced pERK immunoreactivity. We conclude that activation of POMC-immunoreactive neurons and tyrosine hydroxylase activity via the ERK-signaling pathway in the NTS likely contributes to CCK's satiating effects.

Increased adiposity on normal diet, but decreased susceptibility to diet-induced obesity in mu-opioid receptor-deficient mice.

The mu-opioid receptor encoded by the Oprm1 gene plays a crucial role in the mediation of food reward and drug-induced positive reinforcement, but its genetic deletion has been shown to provide food intake-independent, partial protection from diet-induced obesity. We hypothesized that mu-opioid receptor-deficient mice would show an even greater, intake-dependent, resistance to high-fat diet-induced obesity if the diet comprises a sweet component. We generated an F2 population by crossing the heterozygous offspring of homozygous female Oprm1(-/-) mice (on a mixed C57BL/6 and BALB/c genetic background) with male inbred C57BL/6 mice. Groups of genotyped wild-type (WT) and homozygous mutant (KO) males and females were fed either control chow or a high caloric palatable diet consisting of sweet, liquid chocolate-flavored Ensure together with a solid high-fat diet. Food intake, body weight, and body composition was measured over a period of 16 weeks. Unexpectedly, male, and to a lesser extent female, KO mice fed chow for the entire period showed progressively increased body weight and adiposity while eating significantly more chow. In contrast, when exposed to the sweet plus high-fat diet, male, and to a lesser extent female, KO mice gained significantly less body weight and fat mass compared to WT mice when using chow fed counterparts for reference values. Male KO mice consumed 33% less of the sweet liquid diet but increased intake of high-fat pellets, so that total calorie intake was not different from WT animals. These results demonstrate a dissociation of the role of mu-opioid receptors in the control of adiposity for different diets and sex. On a bland diet, normal receptor function appears to confer a slightly catabolic predisposition, but on a highly palatable diet, it confers an anabolic metabolic profile, favoring fat accretion. Because of the complexity of mu-opioid gene regulation and tissue distribution, more selective and targeted approaches will be necessary to fully understand the underlying mechanisms.

Orexin signaling in the ventral tegmental area is required for high-fat appetite induced by opioid stimulation of the nucleus accumbens.

The overriding of satiety and homeostatic control mechanisms by cognitive, rewarding, and emotional aspects of palatable foods may contribute to the evolving obesity crisis, but little is known about neural pathways and mechanisms responsible for crosstalk between the "cognitive" and "metabolic" brain in the control of appetite. Here we show that neural connections between the nucleus accumbens and hypothalamus might be part of this link. Using the well known model of selective stimulation of high-fat intake induced by intra-accumbens injection of the mu-opioid receptor agonist D-Ala2-N-Me-Phe4-gly5-ol-enkephalin (DAMGO), we demonstrate that orexin signaling in the ventral tegmental area is important for this reward-driven appetite to override metabolic repletion signals in presatiated rats. We further show that accumbens DAMGO in the absence of food selectively increases the proportion of orexin neurons expressing c-Fos in parts of the perifornical hypothalamus and that neural projections originating in DAMGO-responsive sites of the nucleus accumbens make close anatomical contacts with hypothalamic orexin neurons. These findings suggest that direct accumbens-hypothalamic projections can stimulate hypothalamic orexin neurons, which in turn through orexin-1 receptor signaling in the ventral tegmental area and possibly other sites interfaces with the motivational and motor systems to increase intake of palatable food.

Brainstem mechanisms integrating gut-derived satiety signals and descending forebrain information in the control of meal size.

Ingestive behavior is controlled by a complex interplay between signals conveying availability of (1) potentially ingestible food in the environment, (2) digestible food in the alimentary canal, (3) circulating fuels and (4) stored fuels. Each of these four classes of signals interact with specific sensors and neural circuits whose integrated output determines when food intake is initiated and when it is stopped. Because the final common path responsible for oromotor control is contained within complex neural pattern generators within the brainstem and is intimately linked to sensory information from the alimentary canal, at least part of the integration between the four classes of signals is thought to take place at the level of the caudal brainstem. Here we show that CCK, representing a class 2, or direct signal, and MC4-melanocortin receptor activity, representing a second order class 3/4, or indirect signal, converge in the nucleus of the solitary tract where they modulate activity of the mitogen-activated, extracellular-signal regulated kinases 1 and 2 (ERK) pathway to determine the level of satiation. Blockade of this signaling pathway attenuates suppression of deprivation-induced food intake by intraperitoneal CCK and fourth ventricular MTII injection. Additional findings suggest that specific ERK-phosphorylation sites on ion channels and enzymes involved in catecholamine synthesis of NTS neurons may be involved in ERK-mediated satiation and meal termination. Longer-term downstream effects of ERK activation might involve CREB-mediated gene transcription known to produce plasticity changes in neurocircuitry that could determine inter-meal intervals and the size of future meals.

Melanocortinergic modulation of cholecystokinin-induced suppression of feeding through extracellular signal-regulated kinase signaling in rat solitary nucleus.

Signals from the gut and hypothalamus converge in the caudal brainstem to control ingestive behavior. We have previously shown that phosphorylation of ERK1/2 in the solitary nucleus (NTS) is necessary for food intake suppression by exogenous cholecystokinin (CCK). Here we test whether this intracellular signaling cascade is also involved in the integration of melanocortin-receptor (MCR) mediated inputs to the caudal brainstem. Using fourth ventricular-cannulated rats and Western blotting of NTS tissue, we show that the MC4R agonist melanotan II (MTII) rapidly and dose-dependently increases phosphorylation of both ERK1/2 and cAMP response element-binding protein (CREB). Sequential administration of fourth ventricular MTII and peripheral CCK at doses that alone produced submaximal stimulation of pERK1/2 produced an additive increase. Prior fourth ventricular administration of the MC4R antagonist SHU9119 completely abolished the CCK-induced increases in pERK and pCREB and, in freely feeding rats, SHU9119 significantly increased meal size and satiety ratio. Prior administration of the MAPK kinase inhibitor U0126 abolished the capacity of MTII to suppress 2-h food intake and significantly decreased MTII-induced ERK phosphorylation in the NTS. Furthermore, pretreatment with the cAMP inhibitor, cAMP receptor protein-Rp isomer, significantly attenuated stimulation of pERK induced by either CCK or MTII. The results demonstrate that activation of the ERK pathway is necessary for peripheral CCK and central MTII to suppress food intake. The cAMP-->ERK-->CREB cascade may thus constitute a molecular integrator for converging satiety signals from the gut and adiposity signals from the hypothalamus in the control of meal size and food intake.

Orexin-A projections to the caudal medulla and orexin-induced c-Fos expression, food intake, and autonomic function.

Orexin-expressing neurons in the hypothalamus project throughout the neuraxis and are involved in regulation of the sleep/wake cycle, food intake, and autonomic functions. Here we specifically analyze the anatomical organization of orexin projections to the dorsal vagal complex (DVC) and raphe pallidus and effects on ingestive behavior and autonomic functions of local orexin-A administration in nonanesthetized rats. Retrograde tracing experiments revealed that as many as 20% of hypothalamic orexin neurons project to the DVC, where they form straight varicose axon profiles, some of which are in close anatomical apposition with tyrosine hydroxylase (TH)-, glucagon-like peptide-1-, gamma-aminobutyric acid-, and nitric oxide synthase-immunoreactive neurons in a nonselective manner. Similar contacts were frequently observed with neurons of the nucleus of the solitary tract whose activation by gastrointestinal food stimuli was demonstrated by the expression of nuclear c-Fos immunoreactivity. Orexin-A administration to the fourth ventricle induced significant Fos-expression throughout the DVC compared with saline control injections, with about 20-25% of TH-ir neurons among the stimulated ones. Fourth ventricular orexin injections also significantly stimulated chow and water intake in nonfood-deprived rats. Direct bilateral injections of orexin into the DVC increased intake of palatable high-fat pellets. Orexin-ir fibers also innervated raphe pallidus. Fourth ventricular orexin-A (1 nmol) activated Fos expression in the raphe pallidus and C1/A1 catecholaminergic neurons in the ventral medulla and increased body temperature, heart rate, and locomotor activity. The results confirm that hypothalamomedullary orexin projections are involved in a variety of physiological functions, including ingestive behavior and sympathetic outflow.

Orexin inputs to caudal raphé neurons involved in thermal, cardiovascular, and gastrointestinal regulation.

Orexin-expressing neurons in the lateral hypothalamus with their wide projections throughout the brain are important for the regulation of sleep and wakefulness, ingestive behavior, and the coordination of these behaviors in the environmental context. To further identify downstream effector targets of the orexin system, we examined in detail orexin-A innervation of the caudal raphe nuclei in the medulla, known to harbor sympathetic preganglionic motor neurons involved in thermal, cardiovascular, and gastrointestinal regulation. All three components of the caudal raphe nuclei, raphe pallidus, raphe obscurus, and parapyramidal nucleus, are innervated by orexin-A-immunoreactive fibers. Using confocal microscopy, we demonstrate close anatomical appositions between varicose orexin-A immunoreactive axon profiles and sympathetic premotor neurons identified with either a transneuronal retrograde pseudorabies virus tracer injected into the interscapular brown fat pads, or with in situ hybridization of pro-TRH mRNA. Furthermore, orexin-A injected into the fourth ventricle induced c-Fos expression in the raphe pallidus and parapyramidal nucleus. These findings suggest that orexin neurons in the hypothalamus can modulate brown fat thermogenesis, cardiovascular, and gastrointestinal functions by acting directly on neurons in the caudal raphe nuclei, and support the idea that orexin's simultaneous stimulation of food intake and sympathetic activity might have evolved as a mechanism to stay alert while foraging.