Acyl-ghrelin administration increases food intake, body weight, and blood glucose. In contrast, mice lacking ghrelin or ghrelin receptors (GHSRs) exhibit life-threatening hypoglycemia during starvation-like conditions, but do not consistently exhibit overt metabolic phenotypes when given ad libitum food access. These results, and findings of ghrelin resistance in obese states, imply nutritional state dependence of ghrelin's metabolic actions. Here, we hypothesized that liver-enriched antimicrobial peptide-2 (LEAP2), a recently characterized endogenous GHSR antagonist, blunts ghrelin action during obese states and postprandially. To test this hypothesis, we determined changes in plasma LEAP2 and acyl-ghrelin due to fasting, eating, obesity, Roux-en-Y gastric bypass (RYGB), vertical sleeve gastrectomy (VSG), oral glucose administration, and type 1 diabetes mellitus (T1DM) using humans and/or mice. Our results suggest that plasma LEAP2 is regulated by metabolic status: its levels increased with body mass and blood glucose and decreased with fasting, RYGB, and in postprandial states following VSG. These changes were mostly opposite of those of acyl-ghrelin. Furthermore, using electrophysiology, we showed that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons. We predict that the plasma LEAP2/acyl-ghrelin molar ratio may be a key determinant modulating acyl-ghrelin activity in response to body mass, feeding status, and blood glucose.

Meals inhibited secretion of both ghrelin and des-acyl ghrelin, yet long-term fasting inhibited acylation but not total secretion. Acylation may be regulated independently of secretion by nutrient availability in the gut or by esterases that cleave the acyl group. These studies highlight the importance of stringent conditions for sample collection and evaluation of full-length ghrelin and des-acyl ghrelin using specific two-site assays.

These findings suggest possible mechanisms contributing to the effects of high-protein/low-carbohydrate diets to promote weight loss, and high-fat diets to promote weight gain.

Ghrelin administration and fasting have similar acute stimulatory effects on hedonic responses and the activation of corticolimbic reward-cognitive systems during food evaluations. Similar effects of recurrent or chronic hyperghrelinemia on an anticipatory food reward may contribute to the negative impact of skipping breakfast on dietary habits and body weight and the long-term failure of energy restriction for weight loss.

Phosphoinositide (PI) 3-kinase is involved in insulin-mediated effects on glucose uptake, lipid deposition, and adiponectin secretion from adipocytes. Genetic disruption of the p85␣ regulatory subunit of PI 3-kinase increases insulin sensitivity, whereas elevated p85␣ levels are associated with insulin resistance through PI 3-kinase-dependent and -independent mechanisms. Adipose tissue plays a critical role in the antagonistic effects of growth hormone (GH) on insulin actions on carbohydrate and lipid metabolism through changes in gene transcription. The objective of this study was to assess the role of the p85␣ subunit of PI 3-kinase and PI 3-kinase signaling in GH-mediated insulin resistance in adipose tissue. To do this, p85␣ mRNA and protein expression and insulin receptor substrate (IRS)-1-associated PI 3-kinase activity were measured in white adipose tissue (WAT) of mice with GH excess, deficiency, and sufficiency. Additional studies using 3T3-F442A cells were conducted to confirm direct effects of GH on free p85␣ protein abundance. We found that p85␣ expression 1) is decreased in WAT from mice with isolated GH deficiency, 2) is increased in WAT from mice with chronic GH excess, 3) is acutely upregulated in WAT from GH-deficient and -sufficient mice after GH administration, and 4) is directly upregulated by GH in 3T3-F442A adipocytes. The insulininduced increase in PI 3-kinase activity was robust in mice with GH deficiency, but not in mice with GH excess. In conclusion, GH regulates p85␣ expression and PI 3-kinase activity in WAT and provides a potential explanation for 1) the insulin hypersensitivity and associated obesity and hyperadiponectinemia of GH-deficient mice and 2) the insulin resistance and associated reduced fat mass and hypoadiponectinemia of mice with GH excess. Diabetes

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.

The distribution of alpha 2C-adrenergic receptors (ARs) in rat brain and spinal cord was examined immunohistochemically by using an affinity purified polyclonal antibody. The antibody was directed against a recombinant fusion protein consisting of a 70-amino-acid polypeptide portion of the third intracellular loop of the alpha 2C-AR fused to glutathione-S-transferase. Selectivity and subtype specificity of the antibody were demonstrated by immunoprecipitation of [125I]-photoaffinity-labeled alpha 2-AR and by immunohistochemical labeling of COS cells expressing the individual rat alpha 2-AR subtypes. In both cases the antibody recognized only the alpha 2C-AR subtype, and immunoreactivity was eliminated by preadsorption of the antibody with excess antigen. In rat brain, alpha 2C-AR-like immunoreactivity (alpha 2C-AR-LI) was found primarily in neuronal perikarya, with some labeling of proximal dendrites; analysis by confocal microscopy revealed the intracellular localization of some of the immunoreactivity. Areas of dense immunoreactivity include anterior olfactory nucleus, piriform cortex, septum, diagonal band, pallidum, preoptic areas, supraoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, amygdala, hippocampus (CA1 and dentate gyrus), substantia nigra, ventral tegmental area, raphe (pontine and medullary), motor trigeminal nucleus, facial nucleus, vestibular nucleus, dorsal motor nucleus of the vagus, and hypoglossal nucleus. Labeling was found in specific laminae throughout the cortex, and a sparse distribution of very darkly labeled cells was observed in the striatum. At all levels of the spinal cord there were small numbers of large, darkly labeled cells in layer IX and much smaller cells in layer X. In general, the pattern of alpha 2C-LI throughout the neuraxis is consistent with previously published reports of the distribution of receptor mRNA detected by hybridization histochemistry.

Reductions in levels of the hunger-stimulating hormone ghrelin have been proposed to mediate part of the effects of vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass surgeries for obesity. We studied circulating levels of acyl and desacyl ghrelin in rats after these surgeries. We found that blood levels of ghrelin were reduced after VSG, but not after Roux-en-Y gastric bypass, based on enzyme-linked immunosorbent assay and mass-spectrometry analyses. We compared the effects of VSG in ghrelin-deficient mice and wild-type mice on food intake, body weight, dietary fat preference, and glucose tolerance. We found that VSG produced comparable outcomes in each strain. Reduced ghrelin signaling therefore does not appear to be required for these effects of VSG.