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Research Notes: HungerBiochem Biophys Res Commun. 2007 Aug 3. Hypothalamic neurons monitor peripheral energy status and produce signals to adjust food intake and energy expenditure to maintain homeostasis. However, the molecular mechanisms by which these signals are generated remain unclear. Fluctuations in the level of hypothalamic malonyl-CoA are known to serve as an intermediary in regulating energy homeostasis and it has been proposed that the brain-specific carnitine palmitoyltransferase-1c (CPT1c) serves as a target of malonyl-CoA in the central nervous system (CNS). Here, we report that CPT1c is widely expressed in neurons throughout the CNS including the hypothalamus, hippocampus, cortex, and amygdala. CPT1c is enriched in neural feeding centers of the hypothalamus with mitochondrial localization as an outer integral membrane protein. Ectopic over-expression of CPT1c by stereotactic hypothalamic injection of a CPT1c adenoviral vector is sufficient to protect mice from body weight gain when fed a high-fat diet. These findings show that CPT1c is appropriately localized in regions and cell types to regulate energy homeostasis and that its over-expression in the hypothalamus is sufficient to protect mice from adverse weight gain caused by high-fat intake. Clin Endocrinol (Oxf). 2007 Feb. Objective: Ghrelin and polipeptide YY (PYY) are involved in the regulation of food intake. We evaluated these two peptides and their possible relationship in adult patients with Prader-Willi syndrome (PWS). Patients: Seven patients with PWS, 16 age-sex-BMI matched obese and 42 age-sex matched lean subjects. Design and measurements: Fasting plasma PYY and ghrelin levels were measured in all subjects and, postprandially until 6 h, in seven matched subjects of each group. Results: Fasting ghrelin levels were higher in PWS than in the other two groups. Fasting PYY levels were lower in patients with PWS than in lean subjects but similar to those in obese subjects. The postprandial decrease in ghrelin concentrations was lower in PWS as compared to the other two groups and therefore the 6-h-postprandial area under the curve (AUC) for ghrelin was higher in PWS than in obese subjects. PYY response after the meal was blunted in patients with PWS, but not in the other two groups that showed a peak at 60 min The AUC for PYY was lower in PWS as compared to the other two groups. Fasting PYY levels correlated negatively with fasting ghrelin levels and with ghrelin AUC and they were the only predictor for ghrelin AUC (beta = -0.464, P = 0.034). The increase in PYY correlated negatively with the decrease in ghrelin at times 60 min and 120 min in PWS. Conclusions: In PWS, the low decrease in postprandial ghrelin levels could be related to the low fasting PYY concentrations and their blunted postprandial response. Diabetes. 2007 Jan. The ability for the brain to sense peripheral fuel availability is mainly accomplished within the hypothalamus, which detects ongoing systemic nutrients and adjusts food intake and peripheral metabolism as needed. Here, we hypothesized that mitochondrial reactive oxygen species (ROS) could trigger sensing of nutrients within the hypothalamus. For this purpose, we induced acute hypertriglyceridemia in rats and examined the function of mitochondria in the hypothalamus. Hypertriglyceridemia led to a rapid increase in the mitochondrial respiration in the ventral hypothalamus together with a transient production of ROS. Cerebral inhibition of fatty acids-CoA mitochondrial uptake prevented the hypertriglyceridemia-stimulated ROS production, indicating that ROS derived from mitochondrial metabolism. The hypertriglyceridemia-stimulated ROS production was associated with change in the intracellular redox state without any noxious cytotoxic effects, suggesting that ROS function acutely as signaling molecules. Moreover, cerebral inhibition of hypertriglyceridemia-stimulated ROS production fully abolished the satiety related to the hypertriglyceridemia, suggesting that hypothalamic ROS production was required to restrain food intake during hypertriglyceridemia. Finally, we found that fasting disrupted the hypertriglyceridemia-stimulated ROS production, indicating that the redox mechanism of brain nutrient sensing could be modulated under physiological conditions. Altogether, these findings support the role of mitochondrial ROS as molecular actors implied in brain nutrient sensing. Diabetes. 2007 Jan. Ghrelin is a potent orexigenic and adipogenic hormone that strongly influences fat deposition and the generation of hunger in obesity. Indeed, hyperghrelinemia appears to promote an increase in food intake as seen in Prader-Willi Syndrome (PWS). Exendin (Ex)-4 is an agonist of the glucagon-like peptide (GLP)-1 receptor (GLP-1r) that has anorexigenic and fat-reducing properties. Here, we report that Ex-4 reduces the levels of ghrelin by up to 74% in fasted rats. These effects are dose dependent and long lasting (up to 8 h), and they can be detected after both central and peripheral administration of Ex-4. Suppression of ghrelin was neither mimicked by GLP-1(7-36)-NH(2) nor blocked by the GLP-1r antagonist Ex-(9-39). Moreover, it was independent of the levels of leptin and insulin. The decrease in ghrelin levels induced by Ex-4 may explain the reduced food intake in fasted rats, justifying the more potent anorexigenic effects of Ex-4 when compared with GLP-1. As well as the potential benefits of Ex-4 in type 2 diabetes, the potent effects of Ex-4 on ghrelin make it tempting to speculate that Ex-4 could offer a therapeutic option for PWS and other syndromes characterized by substantial amounts of circulating ghrelin. J Clin Endocrinol Metab. 2006 Dec 27. Context: Fasting levels of plasma ghrelins are grossly elevated in children with Prader-Willi syndrome (PWS). The cause of this elevation and the regulation of ghrelins in PWS is largely unknown. The regulatory role of individual nutritional components and of growth hormone (GH) is not well characterised. Objective: We investigated the influence of GH on acylated (aGhr) and total ghrelin (tGhr) concentrations before and after an oral glucose load, and on insulin resistance in PWS children. Design, Patients and Interventions: In a clinical follow-up study, plasma ghrelins were measured during an oral glucose tolerance test, and parameters of insulin resistance were determined in 28 PWS children before and/or 1.18 (0.42 - 9.6) years (median, range) after start of GH therapy (0.035 mg/kg body weight/d). Main Outcome Measures: Fasting and post glucose concentrations of aGhr and tGhr, and HOMA2 insulin resistance were the main outcome measures. Setting: The study was conducted in a single center (University Children's Hospital). Results: High fasting (1060 +/- 292 [SD] pg/ml; n = 12) and post glucose trough (801 +/- 303 pg/ml; n = 10) tGhr concentrations in GH-untreated PWS children were found to be decreased in the GH treated group (fasting 761 +/- 247 pg/ml, n = 24, P = 0.006; post glucose 500 +/- 176 pg/ml, n = 20; P = 0.006). In contrast, aGhr concentrations and insulin resistance were not changed by GH treatment. Both aGhr and tGhr concentrations were decreased by oral carbohydrate administration, independent of the GH treatment status. Conclusions: Our results indicate that in PWS children aGhr and tGhr are differentially regulated by GH. J Neurol Neurosurg Psychiatry. 2006 Dec 8. Background: Individuals with Prader-Willi syndrome (PWS) exhibit severe disturbances of appetite regulation, including delayed meal termination, early return of hunger after a meal, seeking and hoarding food, and eating of non-food substances. Brain pathways involved in control of appetite in humans are thought to include the hypothalamus, frontal cortex (including orbitofrontal, ventromedial prefrontal, dorsolateral prefrontal, and anterior cingulate areas), insula, and limbic and paralimbic areas. We hypothesized that the abnormal appetite in PWS results from aberrant reward processing of food stimuli in these neural pathways. Methods: We compared functional MRI (fMRI) blood-oxygen level dependent (BOLD) responses while viewing pictures of food in eight adults with PWS and eight normal weight adults after ingestion of an oral glucose load. Results: Subjects with PWS demonstrated significantly greater BOLD activation in the ventromedial prefrontal cortex than controls when viewing food pictures. No significant differences were found in serum insulin, glucose, or triglyceride levels between the groups at the time of the scan. Conclusions: Individuals with PWS had an increased BOLD response in the ventromedial prefrontal cortex compared to normal weight controls when viewing pictures of food after an oral glucose load. These findings suggest that an increased reward value for food may underlie the excessive hunger in PWS, and support the significance of the frontal cortex in modulating the response to food in humans. Our findings in the extreme appetite phenotype of PWS support the importance of the neural pathways that guide reward-related behavior in modulating the response to food in humans. Physiol Behav. 2006 Aug 30. Ghrelin, an acylated upper gastrointestinal peptide, is the only known orexigenic hormone. Considerable evidence implicates ghrelin in mealtime hunger and meal initiation. Circulating levels decrease with feeding and increase before meals, achieving concentrations sufficient to stimulate hunger and food intake. Preprandial ghrelin surges occur before every meal on various fixed feeding schedules and also among individuals initiating meals voluntarily without time- or food-related cues. Ghrelin injections stimulate food intake rapidly and transiently, primarily by increasing appetitive feeding behaviors and the number of meals. Preprandial ghrelin surges are probably triggered by sympathetic nervous output. Postprandial suppression is not mediated by nutrients in the stomach or duodenum, where most ghrelin is produced. Rather, it results from post-ingestive increases in lower intestinal osmolarity (information probably relayed to the foregut via enteric nervous signaling), as well as from insulin surges. Consequently, ingested lipids suppress ghrelin poorly compared with other macronutrients. Beyond a probable role in meal initiation, ghrelin also fulfills established criteria for an adiposity-related hormone involved in long-term body-weight regulation. Ghrelin levels circulate in relation to energy stores and manifest compensatory changes in response to body-weight alterations. Ghrelin crosses the blood-brain barrier and stimulates food intake by acting on several classical body-weight regulatory centers, including the hypothalamus, hindbrain, and mesolimbic reward system. Chronic ghrelin administration increases body weight via diverse, concerted actions on food intake, energy expenditure, and fuel utilization. Congenital ablation of the ghrelin or ghrelin-receptor gene causes resistance to diet-induced obesity, and pharmacologic ghrelin blockade reduces food intake and body weight. Ghrelin levels are high in Prader-Willi syndrome and low after gastric bypass surgery, possibly contributing to body-weight alterations in these settings. Extant evidence favors roles for ghrelin in both short-term meal initiation and long-term energy homeostasis, making it an attractive target for drugs to treat obesity and/or wasting disorders. Peptides. 2006 Jul. We have evaluated the effects of fatty acid chain length on ghrelin, peptide YY (PYY), glucagon-like peptide-2 (GLP-2) and pancreatic polypeptide (PP) secretion and hypothesized that intraduodenal administration of a dodecanoic ("C12"), but not decanoic ("C10"), acid would decrease plasma ghrelin and increase PYY, GLP-2 and PP concentrations. Plasma hormone concentrations were measured in seven healthy men during 90-min intraduodenal infusions of: (i) C12, (ii) C10 or (iii) control (rate: 2 ml/min, 0.375 kcal/min for C12/C10) and after a buffet-meal consumed following the infusion. C12 markedly suppressed plasma ghrelin and increased both PYY and GLP-2 (all P < 0.05) compared with control and C10, while C10 had no effect. Both C10 and C12 increased PP concentrations slightly (P < 0.05). We conclude that the effects of intraduodenal fatty acids on ghrelin, PYY and GLP-2 secretion are dependent on their chain length. Obesity (Silver Spring). 2006 Jun. Objective: Prader-Willi syndrome (PWS) is a genetic disorder associated with developmental delay, obesity, and obsessive behavior related to food consumption. The most striking symptom of PWS is hyperphagia; as such, PWS may provide important insights into factors leading to overeating and obesity in the general population. We used functional magnetic resonance imaging to study the neural mechanisms underlying responses to visual food stimuli, before and after eating, in individuals with PWS and a healthy weight control (HWC) group. Research methods and procedures: Participants were scanned once before (pre-meal) and once after (post-meal) eating a standardized meal. Pictures of food, animals, and blurred control images were presented in a block design format during acquisition of functional magnetic resonance imaging data. Results: Statistical contrasts in the HWC group showed greater activation to food pictures in the pre-meal condition compared with the post-meal condition in the amygdala, orbitofrontal cortex, medial prefrontal cortex (medial PFC), and frontal operculum. In comparison, the PWS group exhibited greater activation to food pictures in the post-meal condition compared with the pre-meal condition in the orbitofrontal cortex, medial PFC, insula, hippocampus, and parahippocampal gyrus. Between-group contrasts in the pre- and post-meal conditions confirmed group differences, with the PWS group showing greater activation than the HWC group after the meal in food motivation networks. Discussion: Results point to distinct neural mechanisms associated with hyperphagia in PWS. After eating a meal, the PWS group showed hyperfunction in limbic and paralimbic regions that drive eating behavior (e.g., the amygdala) and in regions that suppress food intake (e.g., the medial PFC). Cell Metab. 2006 May. Ghrelin and leptin are suggested to regulate energy homeostasis as mutual antagonists on hypothalamic neurons that regulate feeding behavior. We employed reverse genetics to investigate the interplay between ghrelin and leptin. Leptin-deficient mice (ob/ob) are hyperphagic, obese, and hyperglycemic. Unexpectedly, ablation of ghrelin in ob/ob mice fails to rescue the obese hyperphagic phenotype, indicating that the ob/ob phenotype is not a consequence of ghrelin unopposed by leptin. Remarkably, deletion of ghrelin augments insulin secretion in response to glucose challenge and increases peripheral insulin sensitivity; indeed, the hyperglycemia exhibited by ob/ob mice is markedly reduced when ob/ob mice are bred onto the ghrelin(-/-) background. We further demonstrate that ablation of ghrelin reduces expression of Ucp2 [uncoupling protein 2] mRNA in the pancreas, which contributes toward enhanced glucose-induced insulin secretion. Hence, chronically, ghrelin controls glucose homeostasis by regulating pancreatic Ucp2 expression and insulin sensitivity. J Clin Invest. 2006 Apr. Short-term overfeeding blunts the central effects of fatty acids on food intake and glucose production. This acquired defect in nutrient sensing could contribute to the rapid onset of hyperphagia and insulin resistance in this model. Here we examined whether central inhibition of lipid oxidation is sufficient to restore the hypothalamic levels of long-chain fatty acyl-CoAs (LCFA-CoAs) and to normalize food intake and glucose homeostasis in overfed rats. To this end, we targeted the liver isoform of carnitine palmitoyltransferase-1 (encoded by the CPT1A gene) by infusing either a sequence-specific ribozyme against CPT1A or an isoform-selective inhibitor of CPT1A activity in the third cerebral ventricle or in the mediobasal hypothalamus (MBH). Inhibition of CPT1A activity normalized the hypothalamic levels of LCFA-CoAs and markedly inhibited feeding behavior and hepatic glucose fluxes in overfed rats. Thus central inhibition of lipid oxidation is sufficient to restore hypothalamic lipid sensing as well as glucose and energy homeostasis in this model and may be an effective approach to the treatment of diet-induced obesity and insulin resistance. Int J Obes (Lond). 2006 Feb. Objective: To investigate the neural basis of the abnormal eating behaviour in Prader-Willi syndrome (PWS), using brain imaging. We predicted that the satiety response in those with PWS would be delayed and insensitive to food intake. Design and participants: The design of this study was based on a previous investigation of the neural activation associated with conditions of fasting and food intake in a nonobese, non-PWS group. The findings were used to generate specific hypotheses regarding brain regions of interest for the current study, in which 13 adults with PWS took part (mean +/- s.d. age = 29 +/- 6; BMI = 31.5 +/- 5.1; IQ = 71 +/- 8, six were female). Measurements: Regional cerebral blood flow was measured using positron emission tomography in three sessions: one following an overnight fast and two following disguised energy controlled meals of similar volume and appearance - one of 1674 kJ (400 kcal) and another of 5021 kJ (1200 kcal). Subjective ratings of hunger, fullness and desire to eat, and blood plasma levels of glucose, insulin, leptin, ghrelin and PYY were measured before and after each imaging session. Results: The neural representation of hunger, after an overnight fast, was similar to that found in nonobese individuals in the control study. In contrast, after food intake, the patterns of neural activation previously associated with satiety were not found, even after the higher-energy load. Lateral and medial orbitofrontal cortical activation was associated with consumption of the 400- and 1200-kcal meals, respectively. The medial orbitofrontal activation, however, was only found in those who had shown a large percentage change in fullness ratings following the higher-energy meal. Conclusion: We conclude that there is a dysfunction in the satiety system in those with PWS. These findings suggest that brain regions associated with satiety are insensitive even to high-energy food intake in those with the syndrome. This may be the neural basis of the hyperphagia seen in PWS. Ann Nutr Metab. 2006. AIM: The purpose of this study was to investigate the response of postprandial acylated ghrelin to changes in macronutrient composition of meals in healthy adult males. METHODS: A randomized crossover study was performed. Ten healthy adult males were recruited. All subjects received, on separate occasions, a high-carbohydrate (HC), a high-fat (HF), and a high-protein (HP) meal. Blood samples were collected before and 15, 30, 60, 120, and 180 min following the ingestion of each meal. Plasma acylated ghrelin as well as serum insulin, glucose, and triglycerides were measured. RESULTS: The levels of acylated ghrelin fell significantly following the three meals. The HC meal induced the most significant decrease in postprandial ghrelin secretion (-15.5 +/- 2.53 pg/ml) as compared with HF (-8.4 +/- 2.17 pg/ml) and HP (-10.0 +/- 1.79 pg/ml) meals (p < 0.05). However, at 180 min, the HP meal maintained significantly lower mean ghrelin levels (29.7 +/- 3.56 pg/ml) than both HC (58.4 +/- 5.75 pg/ml) and HF (45.7 +/- 5.89 pg/ml) meals and lower levels than baseline (43.4 +/- 5.34 pg/ml) (p <0.01). The postprandial insulin levels increased to significantly higher levels following the HC meal (+80.6 +/- 11.14 microU/ml) than following both HF (37.3 +/- 4.82 microU/ml) and HP (51.4 +/- 6.00 microU/ml) meals (p < 0.001). However, at 180 min, the mean insulin levels were found to be significantly higher following the HP meal (56.4 +/- 10.80 microU/ml) as compared with both HC (30.9 +/- 4.31 microU/ml) and HF (33.7 +/- 4.42 microU/ml) meals (p < 0.05). Acylated ghrelin was also found to be negatively correlated with circulating insulin levels, across all meals. CONCLUSIONS: These results indicate that the nutrient composition of meals affects the extent of suppression of postprandial ghrelin levels and that partial substitution of dietary protein for carbohydrate or fat may promote longer-term postprandial ghrelin suppression and satiety. Our results also support the possible role of insulin in meal-induced ghrelin suppression. Am J Physiol Endocrinol Metab. 2005 Dec. Stimulation of cholecystokinin and glucagon-like peptide-1 secretion by fat is mediated by the products of fat digestion. Ghrelin, peptide YY (PYY), and pancreatic polypeptide (PP) appear to play an important role in appetite regulation, and their release is modulated by food ingestion, including fat. It is unknown whether fat digestion is a prerequisite for their suppression (ghrelin) or release (PYY, PP). Moreover, it is not known whether small intestinal exposure to fat is sufficient to suppress ghrelin secretion. Our study aimed to resolve these issues. Sixteen healthy young males received, on two separate occasions, 120-min intraduodenal infusions of a long-chain triglyceride emulsion (2.8 kcal/min) 1) without (condition FAT) or 2) with (FAT-THL) 120 mg of tetrahydrolipstatin (THL, lipase inhibitor), followed by a standard buffet-style meal. Blood samples for ghrelin, PYY, and PP were taken throughout. FAT infusion was associated with a marked, and progressive, suppression of plasma ghrelin from t = 60 min (P < 0.001) and stimulation of PYY from t = 30 min (P < 0.01). FAT infusion also stimulated plasma PP (P < or = 0.01), and the release was immediate. FAT-THL completely abolished the FAT-induced changes in ghrelin, PYY, and PP. In response to the meal, plasma ghrelin was further suppressed, and PYY and PP stimulated, during both FAT and FAT-THL infusions. In conclusion, in healthy humans, 1) the presence of fat in the small intestine suppresses ghrelin secretion, and 2) fat-induced suppression of ghrelin and stimulation of PYY and PP is dependent on fat digestion. Excerpts from the full text article: A number of gastrointestinal peptides play a role in the regulation of energy intake in humans, including cholecystokinin (CCK) (19, 27), glucagon-like peptide-1 (GLP-1) (13, 17), peptide YY (PYY) (4), pancreatic polypeptide (PP) (5), and ghrelin (42). The secretion of CCK, GLP-1, and PYY from intestinal cells, and PP from the pancreas, is stimulated by meal ingestion or infusion of nutrients into the small intestine (10, 18, 21, 29). In contrast, ghrelin is secreted by the stomach, and plasma concentrations increase during fasting and are suppressed by a meal (7, 8). Plasma ghrelin concentrations decrease rapidly following food ingestion, and there is evidence that ghrelin plays a role in meal initiation (7, 8). Intravenously administered ghrelin has been shown to stimulate appetite and increase food intake in humans (42). Both carbohydrate and fat, when ingested orally, suppress ghrelin secretion (9, 15), whereas protein may stimulate ghrelin secretion (9) or have no effect (15). Although until recently it has been unclear whether the suppressive effect of carbohydrate and fat on ghrelin secretion is mediated by the presence of nutrients in the stomach, the small intestine, and/or the circulation, recent animal (41) and human (30) studies indicate that the interaction of nutrients with the small intestine is important in the glucose-induced modulation of ghrelin secretion. In rats, the prevention of gastric emptying with a pyloric cuff abolished the suppression of ghrelin secretion by intragastric glucose (41). Moreover, in healthy older humans, both intragastric and intraduodenal glucose infusions suppress ghrelin secretion with no difference between them (34). We (11) have recently established that the stimulation of both CCK and GLP-1 by a duodenal fat infusion is dependent on the interaction of the products of fat digestion with the gut. Inhibition of fat digestion by concomitant administration of the lipase inhibitor tetrahydrolipstatin (THL) completely abolished the increases in plasma CCK and GLP-1 concentrations induced by duodenal infusion of a long-chain triglyceride emulsion in healthy subjects (11). Intraduodenal infusion of the triglyceride emulsion was also associated with a reduction in perceptions of appetite as well as a decrease in energy intake at a buffet meal consumed immediately following the infusion compared with the condition in which fat digestion was inhibited (11). The stimulation of phasic and tonic pyloric pressures by intraduodenal triglyceride was also attenuated by lipase inhibition (11). PYY and PP are both members of the pancreatic polypeptide family of peptides. PYY is secreted predominantly from endocrine cells in the ileum and colon and PP from endocrine cells in the pancreas, both in response to all three macronutrients, fat, carbohydrate, and protein, with fat being the most potent stimulus and carbohydrate the least potent or, possibly, impotent (2, 16, 29, 33). Enteral administration of free fatty acids, including dodecanoate (3) or oleate (22), are known to be potent stimuli of PYY secretion. Both PYY and PP, when infused intravenously, have been shown to reduce appetite and energy intake in healthy humans (4, 5), suggesting an important role for these peptides in the regulation of appetite. Because, as discussed, the appetite suppressant effect of duodenal lipid is dependent on fat digestion (11), it is possible that fat digestion is also a prerequisite for fat-induced suppression of ghrelin and stimulation of PYY and PP. The suppression of ghrelin by fat is not mediated by an increase in plasma concentrations of free fatty acids (30). Although the effects on CCK and GLP-1 secretion in our previous study were striking (11), it has so far been unclear whether these findings could also be extrapolated to ghrelin, PYY, and PP. This hypothesis has, hitherto, not been evaluated. We have now assayed the plasma samples from our previous study (11), in which we had evaluated the role of fat digestion on appetite and energy intake, antropyloroduodenal motility, and plasma CCK and GLP-1 concentrations. We hypothesized that, in response to duodenal fat infusion, 1) plasma ghrelin concentrations would be suppressed, and 2) the suppression of plasma ghrelin and stimulation of PYY and PP would be attenuated when triglyceride digestion is inhibited. Subjects and methods [...] Results Plasma Ghrelin Concentrations Effect of duodenal infusion. There was a significant treatment x time interaction (P < 0.001) for plasma ghrelin concentrations (Fig. 1A). Infusion of FAT was associated with a marked, and progressive, suppression of plasma ghrelin concentrations, which was significant from t = 60 min (P 0.001); in contrast, FAT-THL had no effect on plasma ghrelin. Plasma ghrelin concentrations were lower during infusion of FAT compared with infusion of FAT-THL from t = 45 min (P < 0.01). Effect of meal. During both conditions, plasma ghrelin concentrations decreased further following meal ingestion (time effects: P < 0.001) but remained significantly lower following infusion of FAT compared with FAT-THL (treatment effect: P = 0.001). Plasma PYY Concentrations Effect of duodenal infusion. There was a significant treatment x time interaction (P < 0.001) for plasma PYY concentrations (Fig. 1B). Infusion of FAT was associated with a marked, and progressive, rise in plasma PYY concentrations, which was significant from t = 30 min (P < 0.01); in contrast, infusion of FAT-THL did not affect plasma PYY concentrations. Plasma PYY concentrations were higher during infusion of FAT compared with FAT-THL from t = 15 min (P < 0.01). Effect of meal. Plasma PYY concentrations further rose during both conditions (time effects: P < 0.01) at t = 165 min, and were significantly higher following infusion of FAT compared with FAT-THL (treatment effect: P < 0.01). Plasma PP Concentrations Effect of duodenal infusion. There was a significant effect of treatment (P < 0.01) on plasma PP concentrations (Fig. 1C). During infusion of FAT, PP concentrations were higher from t = 15 min and then plateaued compared with infusion of FAT-THL; infusion of FAT-THL had no effect on plasma PP concentrations. Effect of meal. There was a significant treatment x time interaction (P = 0.01) for plasma PP concentrations. Plasma PP rose during both conditions (time effects: P < 0.001) but was higher following infusion of FAT-THL compared with infusion of FAT (P < 0.01). Discussion Our study establishes that, in healthy humans, 1) the presence of fat in the proximal small intestine is sufficient to suppress ghrelin secretion, and 2) the fat-induced suppression of ghrelin and stimulation of PYY and PP secretion are dependent on fat digestion. Although it is known that meal ingestion suppresses ghrelin (8) and that this effect is induced by both fat and carbohydrate, but not protein (9, 15), it has been unclear whether the stimulus was the presence of nutrients in the stomach, small intestine, and/or postabsorptive factors. There is evidence that gastric distension per se does not reduce ghrelin (34, 39, 41) in that, although an oral glucose load suppressed ghrelin secretion in healthy humans, a water load of identical volume had no effect (39). The effect of oral glucose on ghrelin secretion is probably accounted for by the action of glucose in the small intestine rather than a "gastric" effect. In rats, a gastric glucose load did not suppress ghrelin secretion when gastric emptying was prevented by a pyloric cuff (41). A recent study from our laboratory (34) indicates that gastric and duodenal glucose infusions suppress ghrelin to a similar degree in healthy older subjects. In considering the potential role of postabsorptive factors, intravenous infusion of glucose suppresses ghrelin in both rats (14) and humans (39), whereas intravenous infusion of a fat emulsion with subsequent elevation of free fatty acids had no effect in humans (30), but suppressed plasma ghrelin in rats (14). These latter observations argue against a role for postabsorptive factors in fat- but perhaps not glucose-induced suppression of ghrelin in humans. The current study is the first to demonstrate that duodenal infusion of a long-chain triglyceride emulsion potently suppresses ghrelin secretion in healthy young men, indicating that in humans ghrelin is sensitive to digested fats, an effect apparently not mediated by an increase in blood free fatty acids (30). The stimulation of PYY and PP by small intestinal fat is well documented (22, 26, 29). Our study also provides additional insights into the role of fat digestion products in the modulation of gastrointestinal peptide secretion. Inhibition of fat digestion abolished the suppression of ghrelin and the stimulation of PYY and PP secretion, indicating that fat digestion products, i.e., free fatty acids, play an important role. Although the current findings in relation to the regulation of ghrelin, PYY, and PP secretion are novel, they are consistent with previous observations that fat digestion is a prerequisite for the slowing of gastric emptying (6, 36, 38), proximal gastric relaxation (12), stimulation of pyloric and suppression of antral pressures (11), stimulation of CCK, GLP-1, and GIP (glucose-dependent insulinotropic polypeptide) secretion in healthy subjects and type 2 diabetes (11, 32, 36, 38), suppression of appetite perceptions (11), induction of upper gastrointestinal symptoms (12), and suppression of energy intake (11, 31). Although our study did not include a formal control condition (i.e., administration of THL alone), there is no evidence that THL per se has any effects on the gastrointestinal tract, including gastrointestinal hormone secretion; furthermore, systemic absorption of THL is known to be very low (personal communication, Dr. Jacques Bailly, Hoffmann-La Roche, Basel, Switzerland). Although lipase inhibition had substantial effects on the antropyloroduodenal motor responses to duodenal lipid (11), it is most unlikely that these would account for the observed hormonal responses. It is of interest that the pattern of suppression of ghrelin and stimulation of PYY and PP by the small intestinal triglyceride infusion differed markedly. The fall in ghrelin and rise in PYY appeared to be progressive between t = 0 and 120 min, whereas PP rose to a maximum within 15 min and did not change after that time. These discrepant responses provide insights into the potential small intestinal mechanisms that may modulate the secretion of these hormones. PYY-containing cells predominate in the distal small intestine and colon, and PYY secretion is initiated either by direct luminal contact of nutrients with the endocrine cells (3, 22, 25) and/or indirectly through neurohumoral signals (23, 25). The rise in PYY was relatively prompt and, hence, most unlikely due exclusively to direct exposure of the distal small intestine. In dogs, CCK, which is released from enteroendocrine cells located in the proximal small intestine, has been shown to modulate PYY release (23), and in our original report (11) we described that plasma CCK concentrations rose significantly within 15 min of commencing the duodenal triglyceride infusion but decreased after 30 min; this is consistent with the concept that the initial rise in PYY is due to a link between the proximal small intestine with PYY-releasing cells in the distal small intestine and that the continued rise of PYY throughout the infusion reflects the direct contact of lipid with the distal small intestine. The profile obtained for PYY plasma concentrations in our study closely resembles that observed after a meal (2). As the emulsion containing THL resulted in only 75%, the observed total abolition of PYY secretion and ghrelin suppression suggests that a critical threshold concentration or load for luminal fatty acids is required for these effects. Conversely, there was a progressive suppression of ghrelin secretion over the infusion period. Hence, for both PYY and ghrelin, our data suggest that a critical nutrient load and/or exposure of a specific length or region of intestine to nutrient is required for their stimulation. This is not surprising, as studies in experimental animals have provided convincing evidence for a role of nutrient load, as well as the length of intestine exposed to nutrient, in the regulation of both gastric emptying and food intake (24, 28). In terms of the regulation of ghrelin secretion from the small intestine, it remains to be determined what factors and pathways may be involved in feeding back luminal signals to the ghrelin-secreting cells in the stomach. It has recently been demonstrated that intravenous PYY suppresses ghrelin secretion (4), suggesting that PYY and ghrelin may operate in a negative feedback relationship. In contrast to PYY and ghrelin secretion, there was a prompt rise in PP in response to the duodenal triglyceride infusion with no further increase over the course of the infusion. This pattern is consistent with that observed in response to meal ingestion (1) and suggests that regulation of PP secretion is confined to the proximal small intestine and that the critical nutrient load required for secretion had been exceeded. Alternatively, it is possible that a further increase in PP throughout the infusion was suppressed by negative feedback mechanisms induced by other peptides released from the distal small intestine. In this study, THL was administered with the duodenal lipid infusion, which was ceased immediately before the meal. Because THL is known to only inhibit fat digestion from the meal it is ingested with (or in the case of this study, the duodenal lipid infusion), it was to be expected that meal ingestion would have an additional effect on hormone secretion. Although both PYY and ghrelin secretions were modified markedly by the duodenal lipid infusion, meal ingestion had only a moderate additional effect. PP secretion was, in contrast, stimulated markedly by the meal. This may suggest that, in contrast to PYY and ghrelin, gastric distension, enhanced by the greater amount of food eaten during the FAT-THL condition, may be a more important stimulus for PP. Indeed, moderate gastric distension with a balloon (volume: 600 ml) has been shown to cause a substantial (71%) increase in PP secretion in healthy volunteers (20). Alternatively, it is possible that the other macronutrients, carbohydrate and protein, are more important stimuli for PP secretion than fat. It remains perplexing that protein, the most satiating of the three macronutrients, does not decrease ghrelin (9). However, it needs to be recognized that interpretation of the meal-induced changes in plasma hormone concentrations in our study is limited by the fact that energy intake was variable between subjects and 15% greater following lipase inhibition [as reported previously (11), energy intake after infusion of FAT-THL (5,999 ± 1,433 kJ) was greater than after infusion of FAT (5,177 ± 1,740 kJ, P < 0.05)] and that the changes in hormones are likely to be dependent on the premeal values, which were markedly different for ghrelin and PYY but not PP. There is persuasive evidence that ghrelin, PYY, and PP play a role in the regulation of food intake, ghrelin as a stimulant (8, 42), and PYY and PP as suppressants (4, 5). Given that, as our data demonstrate, the secretion of all three hormones is modulated by fat and the inhibition of fat digestion abolishes this modulation, ghrelin, PYY, and PP may contribute to the suppression of energy intake by fat (11). Such a relationship has been established for CCK, in that the CCK-A receptor antagonist loxiglumide attenuated the inhibitory effects of an intraduodenally administered long-chain fatty acid, oleic acid, on energy intake (27). In summary, our study demonstrates that, in healthy adults, 1) proximal small intestinal exposure to nutrients, in this case long-chain triglycerides, is sufficient to suppress ghrelin secretion; 2) fat digestion is required for the suppression of ghrelin and stimulation of PYY and PP secretion; and 3) the effects of small intestinal fat on ghrelin and PYY secretion occur progressively, indicative of modulation by mechanisms that depend on the nutrient load and length of intestine exposed to nutrient. Clin Sci (Lond). 2005 Oct. The aim of the present study was to investigate the postprandial effect of diet composition on circulating acylated ghrelin levels in healthy women. A randomized cross-over study of three experimental treatments was performed. A total of 11 healthy young women of normal body weight completed the study. All 11 subjects consumed three iso-energetic meals of different macronutrient composition, a balanced meal (50% carbohydrates, 30% fat and 20% protein), a high-fat meal (45% carbohydrates, 45% fat and 10% protein) and a high-protein meal (45% carbohydrates, 20% fat and 35% protein), for breakfast on separate days. The test meals were administered 1 month apart. Blood samples were withdrawn immediately before and at 15, 30, 60, 120 and 180 min after the test meal for measurement of plasma acylated ghrelin, as well as serum glucose, insulin and triacylglycerol (triglyceride) levels. Acylated ghrelin fell significantly after ingestion of both the balanced and high-protein meals. Ghrelin persisted at significantly lower levels than baseline for a longer duration following the high-protein meal (P<0.05 at 15, 30, 60 and 120 min) compared with the balanced meal (P<0.05 at 30 and 60 min). Moreover, acylated ghrelin levels correlated negatively with the postprandial insulin levels. In conclusion, postprandial changes in acylated plasma ghrelin depend on the macronutrient composition of the meal and are possibly influenced by insulin. Endocrinology. 2005 Oct. Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS. Note: This study is possibly the most important investigation of the underlying biochemical basis for PWS yet. A journal post where I describe the findings in lay terms is here. Excerpts from the full text article: Introduction Prader-Willi Syndrome (PWS) is characterized by a distinct biphasic clinical phenotype. Infants show failure to thrive, including severe muscle hypotonia, respiratory and feeding difficulties, and hypogonadotropic hypogonadism (1), whereas children and adults display short stature, mild to moderate mental retardation with behavioral abnormalities, hyperphagia, and severe obesity (2, 3, 4). Indeed, much of the pathology that results in morbidity and mortality in PWS, including type 2 diabetes and cardiovascular disease, is secondary to obesity (3). A number of endocrine abnormalities occur in children and adults with PWS, including GH (3, 5, 6, 7, 8, 9), IGF-I, and IGF binding protein (IGFBP)-3 deficiencies (3, 8), and insulin levels are relatively decreased compared with individuals with common obesity (10, 11). In contrast, fasting levels of circulating ghrelin, a gut hormone believed to act on hypothalamic arcuate nucleus neurons to modulate feeding (12), are grossly elevated in children and adults with PWS, which may play a major role in the hyperphagia and obesity (13, 14, 15). Although the hormonal and clinical abnormalities in PWS have led to the idea of a primary hypothalamic origin for the disorder (2, 9, 16), hormonal and metabolic studies have not been reported in newborns with PWS and the precise pathophysiology for the disorder remains unknown. PWS is caused by loss of function of a unique set of 10 known imprinted, paternally expressed genes from human chromosome 15q11-q13 (4). Seventy-five percent of cases are associated with a 4- to 5-Mb deletion of this region that encompasses the imprinted genes and a set of five or nine nonimprinted loci (4, 17). The other 25% of PWS cases typically have maternal uniparental disomy or, rarely, an imprinting defect (4). The PWS imprinted candidate genes include three intronless genes (NDN, MAGEL2, and MKRN3), a complex polycistronic locus (SNURF-SNRPN), and five subfamilies of box C/D snoRNAs (4). SNRPN encodes a core spliceosomal protein in postnatal neurons (18). Although some biochemical properties for the other PWS-region gene products are known or have been proposed based on sequence homologies, the precise functions of these genes and the links with clinical aspects of the syndrome are not known. The PWS chromosome region is conserved in mouse chromosome 7C, and three PWS mouse models have been generated with maternal uniparental disomy, an imprinting defect (ID), or a paternally derived chromosome deletion. These models share a very similar phenotype that models the first stage of the human syndrome and includes hypotonia, respiratory difficulties, failure to thrive, and early postnatal lethality (19, 20, 21). Because there is reduced fetal movement and a severe clinical course in the newborn with PWS, we hypothesize a fetal developmental abnormality in PWS leading to the neonatal failure to thrive with the adult metabolic phenotype resulting from a failure in compensatory mechanisms. To begin to test this hypothesis, in the present study, we use a transgenic PWS (TgPWS) deletion mouse model to evaluate endocrinological and metabolic abnormalities in late fetal life and during the failure-to-thrive period. TgPWS mice display fetal and neonatal growth retardation associated with insulin/IGF axis abnormalities. Because the fetal changes predate the postnatal failure to thrive, this may identify a primary mechanism. Subsequently endocrine and metabolic deficiencies peak at postnatal day (P)2, leading to dramatic hypoglycemia and severe failure to thrive in TgPWS neonates, mimicking the clinical course in untreated infants with PWS. [...] Results Metabolic and endocrine evaluations Growth. TgPWS mice have a transgene insertion/deletion transmitted through CD-1 TgAS (transgenic Angelman syndrome mouse model) males, with half of the offspring being TgPWS and half being WT. Despite their relatively normal appearance at birth, TgPWS mice were significantly growth retarded at E18 (P = 0.0007), and after birth they did not grow at the same rate as their WT littermates [F(3, 1) = 444, P < 0.0001]. Consequently, at P5 TgPWS pups reached only half the weight of their WT littermates (Fig. 2A). At E18, placental weights did not differ between genotypes (TgPWS, 0.11 ± 0.007 g, n = 5; WT, 0.13 ± 0.005 g, n = 9; P = 0.14). Similar postnatal growth patterns for a small number of PWS ID mice have been reported (20), except that the majority of mutants in that study died within 48 h (20). In contrast, in our study the majority (80%) of TgPWS mice survived beyond P2 but then usually died between P5 and P7... The difference is likely due to the use of inbred (20) vs. outbred (our study) strains of mice, with the earlier lethality potentially associated with Ndn deficiency and a respiratory deficit that is pronounced in some inbred strains of mice (27). Body composition. From P1 to P4, TgPWS contained a greater proportion of body water than the WT mice [F(3, 1) = 10.53, P = 0.001] (Fig. 2B). At P4, percent water content was the highest in TgPWS, compared with WT littermates (P = 0.04). Body protein percentage was unchanged from P1 to P3, but by P4 protein content was higher in TgPWS vs. WT (P = 0.01) (Fig. 2C). Triglycerides, comprising the majority of total body fat content, did not differ at birth (P1). However, overall body triglyceride percentage was decreased in TgPWS [F(3, 1) = 40.87, P = 0.0001] and was most pronounced at P2 (P = 0.0003) (Fig. 2D). Liver glycogen. Liver glycogen content was similar at E18 and P1 (Fig. 3A) between TgPWS and WT pups. Both groups of mice had a significant reduction in hepatic glycogen content between P1 and P2 (76 and 91% for WT and TgPWS, respectively; P < 0.0003). Overall, postnatal WT pups had more glycogen in liver than did TgPWS pups [F(3, 1) = 13.6, P = 0.004]. This difference was most pronounced at P2 and P3 (P < 0.01). Plasma glucose. There was no difference in plasma glucose levels between TgPWS and WT pups at E18 or P1 (Fig. 3B). However, at P2 glucose concentration dropped in TgPWS pups and thereafter was significantly lower, compared with WT mice [F(3, 1) = 100.7, P = 0.0001] (Fig. 3B). Plasma insulin. Plasma insulin levels were significantly lower in TgPWS vs. WT mice in fetal life at E18 (P = 0.04) and from P1 to P4 [F(3, 1) = 44.9, P = 0.0001] (Fig. 3C). Insulin levels were extremely low (<0.15 ng/ml, the detection limit of the assay) in 75 and 100% of TgPWS pups at P2 and P3, respectively. Plasma glucagon. The concentration of plasma glucagon was also lower in TgPWS, compared with WT mice [F(3, 1) = 7.4, P = 0.008] (Fig. 3D). Similar to insulin levels, glucagon was also very low (<20 pg/mg, the detection limit of the assay) in TgPWS plasma from P2 to P4. Corticosterone. Corticosterone was significantly higher in TgPWS vs. WT mice at all postnatal days [F(3, 1) = 9.31, P = 0.003] but not at E18 (Fig. 3E). The normal fall in corticosterone levels that occurs after birth was blunted in TgPWS mice (P < 0.0001). Ghrelin. There was no difference in plasma ghrelin levels between TgPWS and WT mice at P1 and P2, but for P3, P4 and P5, ghrelin concentration was significantly higher in TgPWS pups (P = 0.0003) (Fig. 3F). Triglycerides. Between P1 and P4, plasma triglyceride concentrations in TgPWS pups were 3559% of those in WT pups [F(3, 1) = 91.2, P = 0.0001] (Fig. 4A). Between P1 and P2, there were substantial increases in both WT (+182%; P = 0.0001) and TgPWS (+387%; P = 0.0001) pups. Overall plasma free fatty acids were also significantly reduced in TgPWS mice [F(3, 1) = 19.8, P = 0.0001] and were most profoundly reduced on P1 and P4 (P < 0.05) (Fig. 4B). Concentrations of free fatty acids increased substantially between P1 and P2 in both WT (+95%; P = 0.0001) and TgPWS (+159%; P = 0.0001) pups. In contrast, plasma levels of ketones were higher in TgPWS than in WT pups from P1 to P4 [F(3, 1) = 6.9, P = 0.01] (Fig. 4C). Liver lipids. Paralleling the increase in plasma triglyceride and free fatty acid levels, liver lipid content also increased from P1 to P2 in both WT and TgPWS pups (Fig. 4D). However, this increase was markedly blunted in TgPWS mice (97%, P = 0.0001, and 47%, P < 0.0001, respectively). Overall, between P1 and P4, liver lipid contents were higher in WT pups than TgPWS pups [F(3, 1) = 10.5, P < 0.002]. Liver ATP. Liver ATP content of WT pups did not change between P1 and P4 (Fig. 4E). There was also no difference between WT and TgPWS pups on P1. In contrast, in TgPWS pups, liver ATP content significantly increased from the first to the second day of life in TgPWS mice (P < 0.02) but then fell back to P1 levels by P3. Overall, liver ATP contents were higher in TgPWS pups than WT pups [F(3, 1) = 7.3, P < 0.01]. Between P1 and P4, liver phosphorylation potential decreased gradually in WT pups (P = 0.0001) (Fig. 4F), most likely due to a gradual increase in liver inorganic phosphate content (P = 0.0001) (data not shown). Overall, TgPWS pups had higher liver phosphorylation potential than did WT pups [F(3, 1) = 6.0, P < 0.02], which was highest at P2 and P3 (P < 0.01). Hepatic IGF system gene expression We examined hepatic expression of the genes encoding IGF-I, IGFBP-3, and IGFBP-1 because plasma levels are often reported abnormal in humans with PWS (3, 8, 28, 29). QRT-PCR analysis showed a 2.6-fold decreased level of Igf1 mRNA at P1 (P = 0.002) and a 2.2-fold decreased level at P5 (P = 0.006) but no difference at E18 in TgPWS vs. WT liver (Fig. 5A). Hepatic mRNA levels of Igfbp3, which encodes the protein that binds nearly 75% of circulating IGF-I (30), did not differ between TgPWS and WT mice at all three time points (Fig. 5B). In contrast, Igfbp1 mRNA levels were consistently up-regulated in postnatal TgPWS mice, by 2.3-fold, compared with WT at P1 (P = 0.02), and 5.3-fold at P5 (P = 0.002). However, there was no difference between TgPWS and WT at E18 (P = 0.8) (Fig. 5C). Discussion We measured metabolic and hormonal parameters in a TgPWS deletion mouse model in fetal life and early postnatal life to assess the abnormalities that underlie the neonatal phenotype and premature lethality in these mice. We found a constellation of abnormalities in TgPWS mice that are consistent with the clinical presentation in newborns or children with PWS, including growth retardation, insulin/IGF-I deficiencies, and hyperghrelinemia. Additional findings of abnormal glucose and energy homeostasis and pancreatic insufficiency, beginning in fetal life, provide new insights into the pathophysiological basis of PWS. The most surprising finding of our study was the deficit in insulin and glucagon observed in fetal and postnatal TgPWS mice, suggesting that this model of PWS in mice induces a primary pancreatic defect. To determine whether TgPWS mice have defects in pancreatic development and function, we are currently examining islet architecture and ί- and -cell mass and performing analyses of global gene expression in the pancreas, compared with WT mice. Our findings have important implications for the pathophysiology of type 2 diabetes that is so often observed in patients with PWS. It has generally been hypothesized that diabetes in this population is secondary to profound obesity; however, a number of studies have demonstrated that insulin levels are actually much lower than expected for the degree of the increase in body mass index (3, 10, 11). Glucose levels were normal in fetal TgPWS mice, suggesting that placental function is normal in these mice. The fetal requirement for glucose is met almost, if not entirely, by transplacental transport from the mother to the fetus (31, 32). At birth, there is an abrupt loss of the maternal supply of substrates and nutrients and the newborn has to mobilize glucose and other substrates to meet its energy needs. This is achieved primarily through breakdown of glycogen. A number of factors initiate liver glycogenolysis at birth; however, the precise mechanisms have not been completely elucidated. Whereas insulin and glucagon play a role in the breakdown of glycogen, increased plasma catecholamine levels have been suggested to be the primary mediators of the sudden increase in hepatic glucose output (33). This hypothesis is consistent with our findings of low levels of insulin and glucagon at birth, but a normal decrease in hepatic glycogen content in the first day of life in TgPWS mice. Because of the rapid depletion of glycogen, liver glycogenolysis can support glucose homeostasis for only a short period of time, and maintenance of the newborns blood glucose levels requires active liver gluconeogenesis. TgPWS mice develop severe hypoglycemia by d 2 of life that is likely due to impaired gluconeogenesis. Phosphoenolpyruvate carboxykinase, the rate-limiting enzyme of gluconeogenesis, is not turned on until several hours after birth and is activated by a decrease in the insulin to glucagon ratio (fall in insulin and rise in glucagon) and an increase in glucocorticoid levels. In response to hypoglycemia, corticosterone levels were appropriately higher in TgPWS mice, but the mutant mice were unable to increase glucose production. The low levels of glucagon and limited substrate supply are likely to be among the primary causes of the inability of the TgPWS mouse to mount an adequate gluconeogenic response. It will be important in future studies to examine the expression levels of genes encoding critical gluconeogenic enzymes or the levels and/or activity of phosphoenolpyruvate carboxykinase or pyruvate carboxylase, for example, that could be altered in the TgPWS mouse. The low levels of glucagon may also explain the relatively low levels of triglycerides and free fatty acids observed in P1 TgPWS pups. The surge of glucagon that normally occurs after birth induces mobilization of lipids from peripheral tissues. After birth, milk provides a relatively high-fat diet, and lipids are the main energy source of the newborn. However, due to severe failure to thrive, TgPWS mice are not able to suckle normally and lipid levels continue to be low. Because ketone concentrations are actually higher in TgPWS mice, it is unlikely that these mice have fatty acid oxidation defects. Fetal TgPWS mice are significantly growth retarded, and growth rates remain abnormally low after birth. PWS patients also have low birth weight and reduced weight gain in infancy (34, 35, 36). Placental insufficiency cannot be implicated because placental weights are normal for TgPWS animals. The growth restriction in TgPWS mice starts in fetal life and is not due to GH deficiency that occurs postnatally in PWS patients (3, 8, 9) because GH does not regulate fetal growth (37, 38). Additionally, levels of hepatic Igf1 and Igfbp1 mRNA were normal in TgPWS at E18, so these are unlikely to play a role in growth retardation of the TgPWS fetus. However, we cannot completely rule out a causative role for the IGF axis because we did not measure Igf1 receptor levels. In contrast, because insulin is significantly reduced in the TgPWS fetus, decreased activity of insulin signaling pathways (39) may be responsible for the growth restriction observed in fetal and newborn TgPWS mice. Nevertheless, because PWS patients have obesity-independent GH and IGF-I deficiencies (3, 4, 5, 6, 7, 8, 9, 10, 28), a pituitary defect could contribute to or underlie postnatal growth retardation of PWS infants and children. Analysis of hepatic gene expression revealed significantly deregulated Igf1 and Igfbp1 expression but normal Igfbp3 expression in TgPWS newborn mice. These changes correlate with known hormonal regulation of these genes. For example, whereas Igfbp3 production is primarily regulated by GH (29), insulin and/or corticosterone are implicated in the hepatic transcriptional regulation of Igfbp1 and Igf1 (40, 41, 42, 43), and these genes are therefore deregulated in the expected manner in hypoinsulinemic TgPWS mice at P1 and P5. Indeed, by P5 there is a dramatic increase in Igfbp1 mRNA in TgPWS on progression of failure to thrive. Because hepatic overexpression of Igfbp1 in two mouse models have been correlated with postnatal growth retardation (44, 45), this may contribute to failure to thrive in the TgPWS mouse. Nevertheless, despite the known alteration in insulin, IGF-I and IGFBP-1 and -3 in individuals with PWS (3, 8, 28, 29), the hormonal and metabolic status of newborns with PWS is unknown, and thus, it is not clear what role the insulin-IGF axis might play in the failure-to-thrive component of PWS in the human, but it is expected that this will be revealed from retrospective or prospective patient studies. Body composition measurements revealed that postnatal TgPWS mice maintain the same proportion of whole-body water and protein content as do their WT littermates, but whole-body fat (triglyceride) levels decrease after P2, a pattern similar to that seen in animals under conditions of energy restriction (46). This is not unexpected for TgPWS pups, given their hypotonia and failure to thrive. The absence of dehydration in TgPWS mice is consistent with the presence of milk in their stomachs at each age (21), although the amount is usually visibly less than for WT littermates. Nevertheless, intake and nutrient uptake have as yet not been examined in TgPWS mice. Although the milk intake is sufficient to prevent dehydration, a reduced intake may explain the decrease in liver lipids, plasma triglycerides, and plasma free fatty acids as well as whole body fat. Another model generated by gene targeting of the imprinting center (Fig. 1) is PWS ID mice, which disrupts a genetic element controlling imprinting and active expression of all the paternally expressed genes in the PWS domain (20). Recently it was found that these PWS ID mice generally survive on an FVB strain but not other genetic backgrounds, with surviving mice being small and not displaying hyperphagia or obesity (47). However, if the PWS hyperphagia and obesity phase results from a compensatory attempt to overcome a metabolic basis for neonatal failure to thrive, as we propose, the lack of the latter phenotype in the PWS ID mice on the FVB strain (47) leads to the prediction that these mice would not develop hyperphagia and obesity. In contrast, TgPWS deletion mice did not survive on the FVB background, even with removal of most WT littermates (Ohta, T., and R. D. Nicholls, unpublished data). An increase in plasma ghrelin levels occurs in postnatal TgPWS mice and appears to begin at the onset of severe hypoglycemia but is not directly coincident with hypoinsulinemia. These findings are consistent with known regulators of ghrelin expression and secretion because both glucose and insulin have been shown to suppress ghrelin levels (48). By P5, ghrelin levels in TgPWS mice are approximately 3-fold higher than in WT littermates, suggesting that high ghrelin levels in TgPWS might be a physiological adaptive mechanism in an attempt to increase feeding via its actions on the arcuate nucleus (49) to ameliorate the rapidly worsening failure to thrive. However, either this signal is unrecognized due to an unknown mechanism, or it may be too late to elicit a physiological response. The finding of high ghrelin levels in TgPWS mice echoes observations in PWS children and adults, in whom plasma ghrelin levels are 2.5- to 4.5-fold higher than those in normal lean and obese controls (13, 14, 15). Although these studies suggested that this orexigenic hormone could be a mediator for the hyperphagia observed in PWS, our mouse data are consistent with an alternative hypothesis that PWS patients metabolically do not sense the degree of adipose tissue, and hence their lean body mass is in a starvation state that induces ghrelin production (50, 51). In accordance with severe hypoglycemia and hypoinsulinemia, postnatal TgPWS pups show evidence of increased fat oxidation, compared with their WT littermates, as revealed by lower plasma levels of triglycerides and free fatty acids as well as increased ketogenesis. Consistent with this, hepatic energy status (ATP content and phosphorylation potential) was higher in TgPWS vs. WT at P2 and P3, possibly as a result of compensatory ί-oxidation due to impaired glucose homeostasis. In adults, it has been proposed that low liver ATP levels stimulates food intake through signals to the central nervous system via vagal sensory neurons (52, 53). Because a decrease in liver ATP in rats is associated with increased eating (24, 54), it is possible that a negative feedback signal of elevated hepatic ATP in TgPWS pups may contribute to a decrease in feeding behavior as part of the failure to thrive process. At least in the newborn rat, it appears that the vagal afferent connections are intact and sufficiently mature (55, 56). In contrast, leptin neural pathways are not mature until closer to the time of weaning (57, 58), suggesting that leptin signaling abnormalities do not underlie the TgPWS phenotype. In conclusion, we characterized the endocrine and metabolic profile of a TgPWS deletion mouse model with fetal growth retardation and neonatal failure to thrive. Most surprising among the deficiencies found in TgPWS mice were fetal insulin and glucagon insufficiency, suggestive of a primary pancreatic defect. It will be important to further examine the mechanisms that might contribute to the phenotype in TgPWS mice and PWS newborns and children. Furthermore, many of the findings in TgPWS mice, such as hypoinsulinemia, low glucose and IGF-I, and high IGFBP-1 and ketones, are also consistently seen in small-for-gestational-age infants in the human (59). The TgPWS mouse therefore serves as a useful PWS and small-for-gestational-age animal model for further investigation, and additional studies on animal models and similar studies on PWS newborns will shed new understanding on the underlying pathophysiological basis for this syndrome. J Clin Endocrinol Metab. 2005 May. The cause of the unique elevation in fasting plasma levels of the orexigenic gastric hormone ghrelin in many patients with Prader-Willi syndrome (PWS) is unclear. We measured fasting and postprandial plasma ghrelin in nonobese (n = 16 fasting and n = 8 postprandial) and obese non-PWS adults (n = 16 and 9), adults with genetically confirmed PWS (n = 26 and 10), and patients with hypothalamic obesity from craniopharyngioma tumors (n = 9 and 6). We show that 1) plasma ghrelin levels decline normally after food consumption in PWS, but there is still fasting and postprandial hyperghrelinemia relative to the patient's obesity (2.0-fold higher fasting ghrelin, 1.8-fold higher postprandial ghrelin, adjusting for percentage of body fat); 2) the fasting and postprandial hyperghrelinemia in PWS appears to be at least partially, but possibly not solely, explained by the concurrent relative hypoinsulinemia and preserved insulin sensitivity for the patient's obesity (residual 1.3- to 1.6-fold higher fasting ghrelin, 1.2- to 1.5-fold higher postprandial ghrelin in PWS, adjusting for insulin levels or homeostasis model assessment of insulin resistance); 3) hyperghrelinemia and hypoinsulinemia are not found in craniopharyngioma patients with hypothalamic obesity, and indeed, these patients have relative hyperinsulinemia for their obesity; and 4) there is no deficiency of the anorexigenic intestinal hormone peptide YY(3-36) in PWS contributing to their hyperghrelinemia. Excerpts from the full text article Introduction Prader-Willi syndrome (PWS) is a genetic cause of hyperphagia and obesity thought to arise from developmental defects in the brain, including the hypothalamus (1). Many PWS subjects have elevated fasting plasma levels of the stomach-derived GH secretagogue ghrelin, especially when assessed relative to their body mass index (BMI) or total adiposity, with obesity itself normally associated with reduced plasma ghrelin (2, 3, 4, 5, 6). Given the orexigenic and metabolic actions of ghrelin, it has been suggested that this chronic elevation in ghrelin levels could contribute to phenotypes such as hyperphagia, GH deficiency, or sleep disturbance in some PWS subjects, although this currently remains unproven (7, 8, 9). The cause of hyperghrelinemia in PWS relative to their obesity is unknown. In a previous study it appeared partly explicable by relative hypoinsulinemia and preserved insulin sensitivity in PWS, which in itself may reflect reduced visceral adiposity (5, 10, 11), but it does not appear to be related to the concurrent GH deficiency seen in PWS (12, 13). A lack of the normal postprandial suppression of plasma ghrelin has also been reported in PWS adults, which could theoretically contribute to early return of hunger after a meal (3). However, studies in children with PWS have shown normal postmeal ghrelin suppression (14, 15). Obesity and hyperphagia are common sequelae to intracranial tumors involving the hypothalamus, such as craniopharyngioma (16). Vagally mediated hyperinsulinemia and autonomic imbalance are also thought to contribute to hypothalamic obesity from craniopharyngioma (17). Such obesity may respond to somatostatin analogs, perhaps through reductions in insulin secretion (18). Somatostatin and its analogs also reduce ghrelin secretion in non-PWS and PWS subjects (14, 19, 41). Peptide YY336 (PYY) is an anorexigenic hormone secreted postprandially from the distal intestine that reduces plasma ghrelin (20). Reduced PYY secretion in obesity and increased PYY secretion after gastric bypass surgery to treat obesity may play pathogenic roles in alterations in appetite and food intake (20, 21). We therefore hypothesized that 1) hyperghrelinemia might also be seen in hypothalamic obesity due to craniopharyngioma; 2) hyperghrelinemia in PWS is caused by PYY deficiency, which could also contribute to obesity in PWS; and 3) there is abnormal suppression of plasma ghrelin after meals in PWS adults. We therefore measured fasting and postprandial plasma ghrelin and PYY in control, PWS, and craniopharyngioma adults with and without hypothalamic obesity. Subjects and Methods Recruitment ... All subjects were over 18 yr of age and were not known to be diabetic, none had a fasting glucose level greater than 6.0 mmol/liter (108 mg/dl), and in the postprandial study all had a peak glucose level less than 9.8 mmol/liter (176 mg/dl) and a 2-h postprandial (77 g carbohydrate) glucose level less than 8.3 mmol/liter (149 mg/dl). Non-PWS and noncraniopharyngioma females were premenopausal. All PWS subjects had positive genetic testing: fasting study: eight exact molecular class unknown (e.g. only methylation pattern studied), 10 ch15q11-q13 deletion, five maternal uniparental disomy (UPD), two UPD or imprinting center defect, and one unbalanced chromosomal translocation (46,XYt15:Y); postprandial study: three exact molecular class unknown, four ch15q11-q13 deletion, two UPD, and one UPD or imprinting center defect. PWS subjects had not had GH stimulation testing or GH day profiles measured, but IGF-I levels were available for all PWS subjects in the postprandial study. Of these, 10% had IGF-I levels less than 2 SD below the age-related median reference value (<120126 ng/ml), 40% between 2 and 1 SD (<155173 ng/ml), 20% between 1 SD and the median (<189220 ng/ml), and 30% between the median and 1 SD (<260294 ng/ml). [...] Results Fasting study Clinical information for those subjects who had only fasting blood sampling is given in Table 1. In view of significant differences between some comparison groups, adjustment was made for age, sex, percentage of body fat, fasting insulin, or HOMA-IR by multiple linear regression analysis when comparing plasma ghrelin between groups. OB, CRHO, and PWS subjects had significantly greater percentage of body fat than NO subjects, and although percentage of body fat was similar in PWS and CRHO subjects, it was slightly lower in PWS than OB subjects (Table 1 and Fig. 1A). However, although OB and CRHO subjects both had significantly higher fasting insulin and HOMA-IR than NO subjects, values were similar in NO and PWS subjects (Table 1 and Fig. 1B). Fasting insulin and HOMA-IR levels were lower in PWS than in OB or CRHO subjects (Table 1 and Fig. 1B), and this remained significant when adjusting for age, sex, and percentage of body fat (all P < 0.001). Plasma ghrelin was negatively correlated with percentage of body fat in NO and OB subjects combined (r = 0.68; P < 0.001), and all subjects with craniopharyngioma (CR and CRHO; r = 0.53; P < 0.05), but not in PWS subjects (r = 0.04; P = 0.84; Fig. 1D). Plasma ghrelin was negatively correlated with fasting insulin (r = 0.53, P = 0.002; r = 0.52, P < 0.05; and r = 0.53, P = 0.006, respectively), and HOMA-IR (r = 0.52, P = 0.003; r = 0.47, P = 0.08; and r = 0.49, P = 0.01, respectively) in all three groups (Fig. 1E). In NO and OB subjects, the negative correlation of fasting ghrelin with percentage of body fat remained significant when including fasting insulin as a variable in multiple regression analysis (P = 0.003). Fasting plasma ghrelin was lower in OB and CRHO subjects compared with NO subjects, but was similar in NO and PWS subjects (Table 1 and Fig. 1C). Fasting plasma ghrelin was higher in PWS than in OB or CRHO subjects (Table 1 and Fig. 1C), and this remained significant (2.2- and 2.9-fold higher, respectively) when correcting for age, sex, and percentage of body fat (Table 2). In expanded datasets, fasting plasma ghrelin was 2.0-fold higher in PWS subjects compared with either NO and OB subjects combined or all non-PWS subjects, adjusting for age, sex, and percentage of body fat (Table 2 and Fig. 1D). There was no significant correlation of fasting plasma PYY with percentage of body fat in NO and OB (r = 0.05; P = 0.80), PWS (r = 0.23; P = 0.28), CR (r = 0.17; P = 0.75), and CRHO (r = 0.14; P = 0.71) subjects; all craniopharyngioma subjects (r = 0.02; P = 0.95), or all subjects (r = 0.07; P = 0.571). Fasting PYY in PWS was not significantly different from that in OB or CRHO subjects (Table 1) or when adjusted for age, sex, and percentage of body fat (both P = 0.6), fasting insulin (P = 0.80.9), or HOMA-IR (both P = 0.7). Postprandial study Clinical information for those subjects who had postprandial blood sampling is given in Table 3. There was no significant difference in percentage of body fat between OB, CRHO, and PWS subjects (P = 0.60.9). Fasting insulin and FHOMA-IR were lower in PWS than in either OB or CRHO subjects, whereas postprandial AUC insulin or PHOMA-IR in PWS subjects were lower than those in CRHO subjects and tended to be lower than those in OB subjects (P = 0.08; Table 3 and Fig. 2B). Indeed, peak postprandial insulin, postprandial insulin AUC, and PHOMA-IR were higher in CRHO than in OB subjects (Table 3 and Fig. 2B). In NO and OB subjects combined, fasting, postprandial trough, and AUC plasma ghrelin levels were negatively correlated with percentage of body fat (r = 0.83, P < 0.001; r = 0.77, P < 0.001; and r = 0.79, P < 0.001, respectively). There was a stronger negative correlation of fasting ghrelin with postprandial insulin AUC than with fasting insulin or FHOMA-IR in NO and OB subjects combined (Table 4). Similarly, there was a stronger negative correlation of postprandial ghrelin levels with postprandial insulin AUC levels or PHOMA-IR than with either fasting insulin or FHOMA-IR (Table 4). Fasting and postprandial plasma ghrelin levels in PWS subjects were higher than those in OB and CRHO subjects, but not significantly different from those in NO subjects (Table 3 and Fig. 2A). PWS subjects had 2.0-fold higher fasting ghrelin and 1.8-fold higher postprandial ghrelin levels compared with NO and OB subjects combined, after adjusting for age, sex, and percentage of body fat (Table 5). When adjusting for insulin or HOMA-IR measurements (instead of percentage of body fat), fasting ghrelin levels were significantly higher (1.6-fold) in PWS when adjusting for postprandial insulin levels or PHOMA-IR (Table 5). However, the increase (1.3- to 1.6-fold) in fasting ghrelin in PWS did not quite reach statistical significance when adjusting for fasting insulin or FHOMA-IR (Table 5). Similarly, postprandial ghrelin levels were significantly higher (1.4- to 1.5-fold) in PWS than NO and OB subjects after adjusting for postprandial insulin levels or PHOMA-IR, but the increase (1.2-fold) was not significant when adjusting for fasting insulin or FHOMA-IR (Table 5). This is consistent with postprandial insulin and PHOMA-IR having stronger correlations than fasting insulin and FHOMA-IR with ghrelin levels in non-PWS subjects, as noted above (Table 4). PWS subjects also had higher plasma ghrelin compared with CRHO subjects, after being adjusted for age, sex, and percentage of body fat: 2.4-fold higher fasting ghrelin (1.24.9; P = 0.04), 2.4-fold higher postprandial trough ghrelin (1.25.2; P = 0.04), and 2.1-fold higher postprandial AUC ghrelin (1.04.3; P = 0.05). However, fasting and postprandial ghrelin levels were not significantly different between PWS and CRHO subjects after adjusting for age, sex, and fasting or postprandial insulin levels or HOMA-IR (P = 0.20.7). Mean ghrelin levels were similar in OB compared with CRHO subjects (Table 3) and after adjusting for age, sex, and percentage of body fat (P = 0.20.7). The postprandial fall in ghrelin was significant in all groups (Table 3, basal vs. trough: NO, P = 0.002; OB, P = 0.001; CRHO, P = 0.02; PWS, P < 0.001; by paired t test). The maximum percent postprandial fall in ghrelin was less in OB than NO subjects (Table 3). In NO and OB subjects combined, the maximum percent postprandial fall in ghrelin was positively correlated to basal ghrelin (r = 0.73; P = 0.001) and tended to be negatively correlated with fasting insulin (r = 0.42; P = 0.10) and FHOMA-IR (r = 0.42; P = 0.07), but not with postprandial insulin AUC (r = 0.19; P = 0.48), PHOMA-IR (r = 0.13; P = 0.63), maximum percent postprandial increase in insulin (r = 0.27; P = 0.30), or maximum percent postprandial increase in PYY (r = 0.15; P = 0.56). Similarly, there was no significant correlation between the maximum absolute postprandial change or postprandial incremental AUC for ghrelin and insulin or PYY (P = 0.3). In PWS subjects, ghrelin fell postprandially by a similar percentage as that in NO subjects (P = 0.7), but by a greater percentage than that in OB subjects (Table 3). The maximum percent fall in ghrelin in PWS subjects was not significantly different from that in NO and OB subjects combined after adjusting for age, sex, baseline ghrelin, fasting or postprandial insulin, or HOMA-IR (P = 0.51.0). There was no significant difference in fasting, postprandial peak, or AUC plasma PYY levels between PWS and other groups (Table 3) or after adjusting for age, sex, and percentage of body fat (P = 0.10.9). Discussion Elevated plasma ghrelin after fasting and before meals may play a role in meal initiation (24), and its decline after food intake may act together with increased secretion of anorexigenic gut hormones, such as PYY, to limit subsequent food intake (20). Ghrelin secretion is inhibited by insulin and PYY (20, 25, 26, 27). Obesity is usually associated with reduced fasting ghrelin, probably through chronic hyperinsulinemia (5, 28, 29). In PWS subjects, there is fasting hyperghrelinemia relative to the degree of obesity, although there is not always significant hyperghrelinemia when comparing absolute levels with nonobese control subjects (2, 3, 4, 5, 6). In this study we show that 1) plasma ghrelin levels decline normally after food intake in PWS subjects, but there fasting and postprandial hyperghrelinemia still exists relative to their obesity; 2) the fasting and postprandial hyperghrelinemia in PWS appear to be at least partially, but possibly not solely, explained by their concurrent relative hypoinsulinemia and preserved insulin sensitivity; 3) fasting or postprandial hyperghrelinemia and hypoinsulinemia are not found in craniopharyngioma patients with hypothalamic obesity, and indeed, they have relative hyperinsulinemia; and 4) there is no PYY deficiency in PWS subjects contributing to the hyperghrelinemia. Obesity and insulin resistance in PWS and craniopharyngioma Obesity is usually associated with the metabolic syndrome, consisting of a spectrum of detrimental phenotypes, including insulin resistance and hypertriglyceridemia, with increased risk of diabetes mellitus and cardiovascular disease, particularly mediated by increased visceral adiposity (30). We found lower fasting insulin levels and FHOMA-IR in PWS compared with OB subjects and a tendency for lower postprandial insulin and PHOMA-IR. This confirms the findings of several other studies that the metabolic complications of obesity are surprisingly reduced or absent in PWS adults and children, with preservation of insulin sensitivity (5, 10, 31, 32, 33). Possible explanations include childhood-onset GH deficiency and/or a selective reduction in visceral adiposity in PWS adults (10, 11, 31). This preserved insulin sensitivity was not seen in subjects with CRHO, and indeed, postprandial insulin levels and PHOMA-IR were even higher in CRHO patients than in similarly obese OB and PWS subjects, consistent with previous reports (17). Hyperghrelinemia and adiposity in PWS A negative relationship between fasting ghrelin levels and overall adiposity was seen in non-PWS, but not PWS, subjects, in agreement with our earlier study, which also showed a significant negative correlation with magnetic resonance imaging (MRI)-determined visceral adiposity in both non-PWS and PWS subjects (5). This is explicable by the unusual relationship between visceral adiposity and overall adiposity in PWS adults (5, 10, 11). Fasting ghrelin levels in PWS adults were 2.0-fold higher than those in non-PWS adults after correcting for total adiposity, consistent with earlier studies (2, 3, 4, 5), and our study also found postprandial ghrelin levels to be 1.8-fold higher in PWS patients after correcting for total adiposity. A potential criticism of this analysis is our use of bioimpedance analysis (BIA) to measure total adiposity in PWS subjects, because there may be changes in the compart-mentalization of body water in disease states such as GH deficiency. However, we found that there are excellent and parallel correlations between percentage of body fat measurements determined by BIA and whole body MRI in both non-PWS women (n = 44; r = 0.93; P < 0.001) and PWS women (n = 13; r = 0.93; P < 0.001) (Goldstone, A. P., E. L. Thomas, A. E. Brynes, G. Frost, J. D. Bell, unpublished observations). BIA did, however, slightly underestimate MRI-determined percentage of body fat in PWS compared with non-PWS women by an absolute value of 3.2 ± 1.1% (P < 0.005). This underestimate of overall adiposity by BIA in PWS subjects would therefore have, if anything, tended to underestimate the degree of hyperghrelinemia in PWS subjects relative to overall adiposity. This may have contributed to the finding of a lower degree of hyperghrelinemia in PWS subjects relative to adiposity seen in the current study using BIA compared with our previous study in which fasting ghrelin levels were increased 3.4- to 3.6-fold relative to MRI-determined total adiposity (5). The use of different ghrelin assays in these two studies is a potential additional factor. Hyperghrelinemia and preserved insulin sensitivity in PWS Furthermore, our previous study and others have found stronger negative correlations of fasting ghrelin levels with insulin levels or insulin resistance than with overall adiposity in non-PWS subjects (5, 28, 29). However, unlike overall adiposity, there was a significant negative correlation of fasting ghrelin with fasting insulin levels and HOMA-IR in both non-PWS and PWS subjects in this and our previous study (5). Adjustment for differences in insulin levels or insulin resistance levels in comparison of ghrelin levels between groups therefore circumvents any confounding factors introduced by the use of BIA for body composition analysis. Postprandial ghrelin levels were also negatively correlated with insulin levels and HOMA-IR in non-PWS subjects. Interestingly, we found a stronger negative correlation between fasting or postprandial ghrelin levels and postprandial than fasting insulin in non-PWS subjects. This difference may be related to the repeated measurements of postprandial insulin values reducing statistical variability compared with fasting values, or postprandial insulin levels giving a better indication of the prevailing chronic hyperinsulinemic environment. Postprandial hyperinsulinemia is also a better predictor than fasting hyperinsulinemia of the risk for metabolic syndrome and coronary artery disease (34). We found that at least part, but perhaps not all, of the explanation for both the fasting and postprandial hyperghrelinemia in PWS may be these patients relative hypoinsulinemia and preserved insulin sensitivity (5, 10, 11). Thus, fasting ghrelin levels were 1.3- to 1.8-fold higher in PWS, adjusting for fasting insulin or HOMA-IR, although this did not always reach statistical significance, probably as a result of the smaller sample numbers in some datasets. When adjusting for postprandial insulin or HOMA-IR, fasting ghrelin levels were 1.6-fold higher, and postprandial ghrelin levels were 1.4- to 1.5-fold higher in PWS. These results suggest a lower degree of hyperghrelinemia than in our earlier study (5), in which fasting ghrelin levels were 3.0-fold higher in PWS after adjusting for fasting insulin or HOMA-IR, which could reflect the use of different ghrelin and insulin assays in these two studies. Nevertheless, the available evidence of persistent hyperghrelinemia in PWS even when adjusting for simultaneous differences in insulin levels or sensitivity from these two studies does suggest that an additional cause(s) may be present, although the effect may be smaller than previously considered and before adjustment for the hypoinsulinemia. This conclusion that factors additional to hypoinsulinemia contribute to hyperghrelinemia in PWS is also suggested by other studies in children. Two studies have shown that mean fasting ghrelin levels in PWS children tend to be higher than those in lean non-PWS children despite the PWS children having higher mean fasting insulin levels than these less obese non-PWS subjects, although this interpretation is complicated by the lack of formal covariate analysis and, in one study, genetic confirmation of PWS (4, 15). Measurement of ghrelin levels in PWS children at different stages of development and in larger numbers of PWS adults after correction for prevailing insulin levels will be needed to confirm that there are factors additional to hypoinsulinemia that cause hyperghrelinemia in PWS. Additional problems in this interpretation are 1) the use of surrogate markers of total insulin secretion and insulin sensitivity (fasting or postprandial plasma insulin or HOMA-IR) in our study; and 2) the fact that other unidentified circulating factors that are normally associated with insulin resistance, such as adipocytokines, could contribute to low ghrelin concentrations in obesity, with low insulin levels and HOMA-IR merely a marker of another regulatory factor that is abnormal in PWS, resulting in both improved insulin sensitivity and hyperghrelinemia (29, 35). Assessment of the relationship between other measures of insulin sensitivity and adipocytokines with ghrelin in PWS will therefore be of interest. Intact regulatory influences on ghrelin secretion in PWS Plasma ghrelin levels decrease postprandially by a smaller amount in OB than NO non-PWS subjects, in agreement with other studies (36, 37). In PWS subjects, plasma ghrelin fell postprandially by 32%, which appeared appropriate for their fasting ghrelin levels. This normal postprandial fall of ghrelin in PWS adults is in agreement with recent studies of PWS children, but contradicts a single study in PWS adults that only examined one postprandial time point (3, 14, 15). It suggests a normal response of ghrelin-secreting cells to hormonal or neural mediators in the postprandial state (38). Interestingly, the mediator does not appear to be postprandial secretion of insulin itself, although insulin may provide a permissive environment for the postprandial fall (39, 40). This is supported by the lack of any significant positive correlation between the postprandial fall in ghrelin and the postprandial increase in insulin in non-PWS subjects in our study. Combined with the 1) normal negative correlation of plasma ghrelin with visceral adiposity and insulin levels in PWS in this and our earlier study (5), and 2) similar falls in plasma ghrelin after somatostatin or octreotide therapy in PWS (14, 41) as in other studies of non-PWS subjects (19, 42), this suggests that the cause of hyperghrelinemia in PWS is not an intrinsic primary abnormality of ghrelin-secreting cells, but, more likely, the loss of an inhibitory, or excess of a stimulatory, neural or hormonal input. Increased nongastric expression of ghrelin in PWS remains another possibility requiring investigation (43). Hyperghrelinemia and PYY secretion in PWS Although the anorexigenic intestinal hormone PYY acutely reduces fasting and postprandial ghrelin levels in non-PWS subjects (20), the normal fasting and postprandial plasma levels of PYY in PWS in our study have excluded PYY deficiency as contributing to hyperghrelinemia in PWS. PYY is secreted from the gut in proportion to calories consumed. Although there was a vigorous postprandial elevation in PYY levels in PWS subjects after eating a much larger meal (mean ± SEM, 1737 ± 538 kcal) in our recent study (41), the absence of any comparison with a control group means that we cannot definitively exclude the possibility that impaired PYY release contributes to delayed satiety and earlier return of hunger in PWS after larger meals (44). The lack of any significant correlation between the postprandial fall in ghrelin and the postprandial increase in PYY in non-PWS subjects in the current study suggests that the release of PYY may not be responsible for the postprandial fall in ghrelin secretion, at least with the size and macronutrient nature of the meal used in our study (20). Other possible contributions to hyperghrelinemia in PWS It remains possible that other factors contribute to the residual elevation of ghrelin levels in PWS compared with control subjects in addition to differences in insulin levels. These include 1) changes in other gut hormones that are known to alter ghrelin secretion (26, 27, 41, 45); 2) congenital GH or IGF-I deficiency, which has been associated with hyperghrelinemia in examples other than PWS (6, 46, 47); and 3) defects in neural inputs regulating ghrelin secretion from the stomach, because abnormal cardiac, pupillary, and pancreatic autonomic innervation have been suggested by some, but not all, studies in PWS (48, 49, 50, 51, 52), although the presence and nature of any autonomic control of ghrelin secretion in humans is unclear (53, 54, 55, 56, 57). Hormonal differences between PWS and craniopharyngioma The finding that hyperghrelinemia and relative hypoinsulinemia are seen in patients with PWS, but not CRHO, suggests significantly different pathophysiologies. These hormonal differences may be related to CRHO subjects: 1) lacking the factors preserving insulin sensitivity and reducing visceral adiposity in PWS (10, 11, 31); 2) having altered autonomic innervation of pancreatic ί-cells and gastric and other peripheral tissues as a result of different hypothalamic defects or a lack of the other neural defects seen in PWS; 3) having a different balance between parasympathetic and sympathetic nervous activity (1, 17); 4) having damage primarily to the basal hypothalamus, because tumor arises from the suprasellar region, akin to that in ventromedial hypothalamus-lesioned rodents (16, 17), whereas in PWS the basal infundibular nucleus appears normal, but there are abnormalities in the more dorsal paraventricular nucleus with reduced total and reduced oxytocin cell number (1, 58); 5) having nonphysiological cortisol dynamics contributing to postbreakfast hyperinsulinemia given the pharmacokinetics of oral glucocorticoid replacement for ACTH deficiency; and 6) having a later age of onset of GH deficiency than PWS subjects, in whom GH deficiency appears to be present from early infancy (59), because the average age of craniopharyngioma diagnosis or initial treatment was between 22 and 30 yr in our CRHO patients. The hormonal differences between CRHO and PWS (relative hyperinsulinemia and hypoghrelinemia in CRHO vs. relative hypoinsulinemia and hyperghrelinemia in PWS) also have implications for the potential treatment of hypothalamic obesity with somatostatin analogs (14, 18). Somatostatin analogs may benefit CRHO patients by reducing hyperinsulinemia and, hence, insulin-mediated adipogenesis, but this may be less effective in PWS, because there is already relative hypoinsulinemia. Interestingly hypoghrelinemia and relative hyperinsulinemia are also seen in subjects with mutations in the melanocortin-4 receptor located in the hypothalamus and other brain regions, distinguishing this monogenic cause of human obesity from PWS (2, 4, 60). Hyperghelinemia and hyperphagia in PWS Increasing ghrelin levels by 2- to 4-fold in non-PWS subjects increases acute food intake by about 30% (7, 8). Mean ghrelin levels may be elevated by a similar amount in PWS subjects relative to their obesity (2, 3, 4, 5, 6), which may contribute to inappropriate hyperphagia despite obesity. However, an exclusive or even major role for hyperghrelinemia in causing hyperphagia in PWS is questioned by the 1) lower degree of hyperghrelinemia in PWS when correcting for insulin levels, 2) frequent absence of significant elevations in mean ghrelin levels in PWS vs. non-PWS NO subjects, 3) frequent overlap between ghrelin levels between individual PWS and non-PWS subjects over the range of obesity and insulin sensitivity, and 4) the magnitude and near universal presence of hyperphagia in PWS (61, 62). Furthermore, a preliminary study has failed to show any acute anorexigenic effect of normalizing ghrelin levels with a somatostatin infusion in four PWS male adults, although this was complicated by a simultaneous reduction in PYY secretion (41). The orexigenic effect of brain, particularly hypothalamic, and/or other hormonal abnormalities in PWS may override changes in ghrelin (1). Although neuropeptide Y and agouti-related protein neurons, vital hypothalamic targets for the orexigenic action of ghrelin, appear normal in PWS, it is unknown if other defects in PWS make brain appetite pathways hypo-, hyper-, or normosensitive to changes in circulating ghrelin (1, 58, 63, 64). Chronic studies of somatostatin analogs in PWS and particularly the development of ghrelin antagonists will be necessary to definitively investigate any role for hyperghrelinemia in the hyperphagia of PWS and other phenotypes, such as GH deficiency and sleep disturbance (1, 9). Conclusion Fasting and postprandial hyperghrelinemia relative to the degree of obesity is a feature of PWS adults, but not of patients with hypothalamic obesity due to craniopharyngioma. Ghrelin levels fall postprandially by an amount appropriate for their baseline levels in PWS adults. Relative hypoinsulinemia and preserved insulin sensitivity are also features of PWS, but not of craniopharyngioma (where there is, in fact, relative hyperinsulinemia), and this may explain at least some, but perhaps not all, of the hyperghrelinemia in PWS. There is no evidence that impaired secretion of PYY contributes to the hyperghrelinemia in PWS, or that hyperghrelinemia contributes to obesity resulting from hypothalamic damage in craniopharyngioma. Int J Mol Med. 2005 Apr. Ghrelin and peptide YY (PYY) are peptides generally produced by the gastrointestinal organs which are involved in appetite regulation via highly specialized centers in the brain. Abnormal plasma ghrelin and PYY levels compared with controls have been reported for subjects with Prader-Willi syndrome (PWS) which is characterized by infantile hypotonia, poor suck reflex and failure to thrive followed by hyperphagia and marked obesity in early childhood. We studied gene expression of ghrelin, peptide YY, and their receptors (i.e., GHS-R1a, GHS-R1b, and NPY2R) in six different brain regions (frontal cortex, temporal cortex, visual cortex, pons, medulla, and hypothalamus) obtained from three subjects with PWS, two individuals with Angelman syndrome, and six controls to determine if expression of these genes is detectable in different regions of the brain in subjects with and without PWS. In general, expression of these genes using RT-PCR was detected in all subjects and no obvious differences were seen in their pattern of expression between subjects with or without PWS. Additional studies including quantitative gene expression measurements will be required to further evaluate the role of these genes in the eating disorder seen in PWS. J Pediatr Endocrinol Metab. 2004 Sep. An insatiable appetite is a cardinal feature of Prader-Willi syndrome (PWS) with stomach rupturing as a reported consequence. Peptide YY, secreted by the intestine and released post-prandially, inhibits appetite, while ghrelin, secreted by the stomach during mealtime hunger, stimulates appetite. Both peptide YY and ghrelin act at the brain level, particularly the hypothalamus. Recently, plasma ghrelin levels were reported to be elevated in children and adults with PWS but peptide YY levels have not been studied in this syndrome or ghrelin in infants with PWS. To further address the abnormal eating behavior in PWS, we obtained fasting plasma peptide YY and ghrelin levels in 12 infants and children with PWS ranging in age from 2.5 months to 13.3 years and compared them with values from normal populations reported in the literature. Plasma ghrelin levels in our patients with PWS were similar to those of other children with PWS and were significantly higher than those reported in obese children without PWS. Our infants with PWS had similar plasma ghrelin levels compared with our children with PWS but peptide YY levels in our children and infants with PWS were lower than reported in similarly aged individuals without PWS. In addition, we performed preliminary gene expression analysis of ghrelin and peptide YY and their receptors in patients with PWS using established lymphoblastoid cell lines but gene expression did not correlate with plasma ghrelin or peptide YY levels. Horm Metab Res. 2004 Mar. Ghrelin is a 28-amino acid peptide recently identified in the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a). Ghrelin is a potent stimulator of GH secretion. It was recently shown that circulating ghrelin levels in humans rise shortly before and fall shortly after every meal, and that ghrelin administration increases voluntary food intake. The hypothesis that ghrelin hypersecretion might contribute to genetic obesity has never been investigated. In this context, Prader-Willi syndrome is the most common form of human syndromic obesity. As ghrelin affects appetite as well as GH secretion and both are abnormal in PWS, it has been surmised that these alterations might be due to ghrelin dysregulation. The aim of the study was to investigate whether ghrelin is suppressed by the meals differently in PWS children than in PWS adults. Overnight circulating fasting ghrelin levels and ghrelin levels 120 min after breakfast were assayed in 7 PWS children (10.2 +/- 1.7 yr), 7 subjects with morbid obesity (10.3 +/- 1.3 yr), and 5 normal controls (8.4 +/- 1.4 yr). Because of the data spread, no statistical difference was observed in fasting ghrelin levels between PWS and control children (p = NS); anyway, fasting ghrelin levels were significantly lower in obese children than in the other groups (p < 0.05 vs. control and PWS children). Ghrelin levels were slightly suppressed by the meal in control subjects (mean fasting ghrelin: 160.2 +/- 82 pg/ml; after the meal, 141.2 +/- 57 pg/ml, p = NS); the meal failed to suppress ghrelin levels in obese children (mean fasting ghrelin: 126.4 +/- 8.5 pg/ml; after the meal, 119.1 +/- 8.3 pg/ml, p = NS). Interestingly, the meal markedly suppressed ghrelin levels in PWS children (mean fasting ghrelin: 229.5 +/- 70.4 pg/ml; after the meal, 155.8 +/- 34.2 pg/ml, p < 0.01). In conclusion, since a lack of decrease in circulating ghrelin induced by the meal was previously reported in PWS adults, the finding of a meal-induced decrease in ghrelin levels in our population of young PWS would imply that the regulation of the ghrelin system involved in the orexigenic effects of the peptide is operative during childhood, although it progressively deteriorates and is absent in adulthood when hyperphagia and obesity progressively worsen. Growth Horm IGF Res. 2004 Feb. Prader-Willi syndrome (PWS) is a genetic disorder characterized by mild mental retardation, short stature, abnormal body composition, muscular hypotonia and distinctive behavioural features. Excessive eating causes progressive obesity with increased cardiovascular morbidity and mortality. In the PWS genotype loss of one or more normally active paternal genes in region q11-13 on chromosome 15 is seen. It is supposed that the genetic alteration leads to dysfunction of several hypothalamic centres and growth hormone (GH) deficiency (GHD) is common. PWS is well described in children, in whom GH treatment improves body composition, linear growth, physical strength and agility. Few studies have focused on adults. We examined a cohort of 19 young adults with clinical PWS (13 with positive genotype) and mean BMI of 35 kg/m2. At baseline the activity of the GH-insulin-like growth factor-I (IGF-I) system was impaired with low GH values, low total IGF-I and in relation to the obesity low levels of free IGF-I and non-suppressed IGF-binding-protein-1 (IGFBP-1). 2/3 were hypogonadal. Bone mineral density (BMD) was low. Four patients had impaired glucose tolerance and nine patients high homeostasis model assessment (HOMA) index, indicating insulin resistance. Seven patients had a moderate dyslipidemia. The 13 patients with the PWS genotype were shorter and had significantly lower IGF-I. Seventeen (9 men and 8 women), subsequently completed a 12 months GH treatment trial, and GH had beneficial effects on body composition without significant adverse effects. The effects were more pronounced in the patients with the PWS genotype. Analysis of peptides involved in appetite regulation showed that leptin levels were high reflecting obesity and as a consequence NPY levels were low. In relation to the patients obesity circulating oxytocin levels were abnormally low and ghrelin levels abnormally high. Thus, oxytocin and ghrelin might be involved in the hyperphagia. NPY, leptin and ghrelin did not change during GH treatment. In conclusion this pilot study showed that adults with PWS have a partial GH deficiency, and GH treatment has beneficial effects on body composition in adult PWS without significant side-effects. Larger and longer term studies on the effect of GH replacement in adult PWS are encouraged. Horm Res. 2004. Background: Elevated plasma ghrelin levels have recentl |