2.1. The Role of The Hypothalamus in Stimulating Appetite

NPY is part of the pancreatic polypeptide (PP-)fold peptide family (NPY, polypeptide YY (PYY), PP). The medial arcuate nucleus contains the NPY neurons which project to the paraventricular nucleus, hypothalamic nucleus, lateral hypothalamic area, and other hypothalamic sites. NPY synthesis and release are regulated by leptin and insulin (both inhibitory), and glucocorticoids and ghrelin (both stimulatory), among many other factors. The most noticeable physiological response to central administration of NPY is the stimulation of feeding [19]. NPY initiates appetite drive through the NPY G-protein coupled receptors (primarily Y1 and Y5). NPY also represses the anorexigenic effect of melanocortin signaling in the arcuate. In the hypothalamus, NPY is one of the most abundant peptides and one of the most potent orexigenic factors.

AgRP is produced by neurons located within the medial arcuate nucleus that coexpress NPY. AgRP in humans has sequence similarity to the agouti signaling protein in mice. The agouti protein is a paracrine-signaling molecule produced normally in the skin that inhibits the effect of -melanocyte stimulating hormone ( -MSH) on the melanocortin-1 (MC-1) receptor [20]. Overexpression of agouti signaling protein in mice leads to yellow coat color by blocking -MSH at the MC-1 receptor. These mice are also obese, insulin resistant, hyperglycemic, and have increased body length [21]. This is because AgRP, an endogenous antagonist (and inverse agonist) of melanocortin-3 and melanocortin-4 receptors, is implicated in control of energy balance [22]. Blockage of these receptors leads to stimulation of feeding.

2.2. The Peripheral Peptide That Stimulates Appetite

Circulating peptides also play important roles in appetitive behaviors. Of these, ghrelin, or growth hormone (GH)-releasing peptide, is the only known circulating orexigen, or appetite stimulant. It is mainly produced by the endocrine cells of the gastric mucosa of the fundus, but is also found in much smaller amounts in other tissues, including the small intestine, pituitary gland, hypothalamus, pancreas, lung, immune cells, placenta, ovary, testis, and kidney. Ghrelin levels rise prior to meals, then fall quickly after ingestion of nutrients [23]. Thus it is postulated that one primary role of ghrelin is to act as a meal initiator.

The ghrelin receptor, GH secretagogue receptor type 1a (GHS-R1a), is a G-protein coupled receptor that is widely expressed. Within the CNS, it is found in areas involved in the regulation of appetite and energy balance, including the hypothalamic nuclei, dorsal vagal complex, and mesolimbic dopaminergic system. Ghrelin has multiple effects, including stimulation of GH, ACTH, cortisol, aldosterone, catecholamine, and prolactin secretion. Exogenous ghrelin administration has also been found to affect glucose homeostasis, gut motility, pancreatic exocrine secretion, cardiovascular function, immunity, and inflammation [24]. Intracerebroventricular administration of ghrelin in rats leads to increased food intake, excess weight gain, and adiposity [25]. Similarly, administration of ghrelin to obese and lean human subjects leads to increased food intake [26]. Ghrelin leads to this increase of food intake and body weight in part by stimulating the production of NPY and AgRP in the arcuate nucleus [27]. Ghrelin may also alter energy balance by stimulating adipogenesis, inhibiting apoptosis, transitioning from fatty acid oxidation to glycolysis for energy expenditure, and inhibiting sympathetic nervous system activity [28–31].

Globally, ghrelin levels reflect nutritional status and body fat stores. Ghrelin levels in humans are inversely correlated with adiposity, being low in obese subjects, higher in lean subjects, and markedly elevated in subjects with cachexia due to cancer and chronic cardiac failure, as well as those in starvation states such as anorexia nervosa [32–37]. An exception to this is Prader-Willi syndrome, where, despite obesity, affected individuals have high levels of fasting and postprandial ghrelin [38]. Ghrelin treatment in rats has been shown to improve weight gain and lean body mass retention in cancer cachexia and chronic kidney disease [39, 40], offering a potential therapy for cachexia in humans. Alternatively, future studies may examine ghrelin antagonists as a therapeutic option for obesity.

3.1. The Role of The Hypothalamus in Regulating Appetite

The hypothalamus is also the master regulator of satiety, via production of POMC and CART. The POMC gene is expressed by multiple tissues, including the skin and immune system, as well as the pituitary gland and the arcuate nucleus of the hypothalamus. POMC undergoes tissue-specific post-translational cleavage, with the product depending on the endoproteases expressed in that tissue. For example, in the anterior pituitary gland, POMC is primarily converted to ACTH by prohormone convertase 1. In mammals other than primates, prohormone convertase 2 in the intermediate pituitary cleaves ACTH to yield -melanocyte stimulating hormone ( -MSH) that is involved in the control of coat/skin color. With respect to the hypothalamus in humans, leptin (a peptide produced by adipose tissue) is thought to stimulate POMC conversion into -MSH in the arcuate nucleus. The neurotransmitter in turn binds to the melanocortin-4 receptor (MC4R), a key receptor involved in appetite control and energy homeostasis, in the paraventricular nucleus and in numerous other sites throughout the brain.Intracerebroventricular administration of -MSH in rodents inhibits feeding and reduces body weight. As previously mentioned, AgRP is an antagonist of MC4R. Thus, mice overexpressing AgRP or MC4R knockout mice are hyperphagic and obese [41] and are insensitive to -MSH.

MC4R mutations have been found in up to 5.8% of adults with severe childhood-onset obesity [42]. POMC deficiency also leads to obesity (due to lack of binding at MC4R), hypocortisolism (due to lack of binding of ACTH to the MC2R in the adrenal gland), and alteration of pigment (due to lack of binding at MC1R in the skin). This syndrome is defined by severe early onset obesity, adrenal insufficiency, and red hair [43]. Accordingly, in rodent models of cancer and renal failure, MC4R receptor antagonists attenuate symptoms of cachexia by maintaining appetite, lean body mass, and basal energy expenditure [44]. Thus, MC4R antagonists may be a useful clinical treatment of cachexia [45], while agonists are being developed to treat obesity.

Another important satiety regulator in the hypothalamus is cocaine- and amphetamine-regulated transcript (CART), which is coexpressed with POMC in arcuate neurons in animal models and somewhat paradoxically with AgRP and NPY in humans [46]. Similar to POMC neurons, CART neurons are directly stimulated by leptin [47]. CART neurons target areas throughout the hypothalamus and are associated with reinforcement and reward [48], sensory processing, and stress and endocrine regulation [47, 49]. Animals deprived of food have decreased the expression of CART mRNA [47]. Along those same lines, blocking CART with an antiserum increases feeding in normal rats [50]. Intracerebroventricular administration of CART in rats inhibits normal and starvation-induced feeding, as well as blocking the NPY feeding response [47, 50]. At this point, we are not aware of any clinical trials utilizing CART agonists or antagonists for weight regulation perhaps due to the significant nonappetite effects associated with CART.

3.2. Peripheral Peptides Known to Control Satiety

In contrast to ghrelin, the single peripheral peptide known to stimulate hunger, there are many peripheral peptides that are associated with satiety. Various organs secrete these hormones, including the gastrointestinal tract, pancreas, and adipose tissue. The list of satiety hormones is far too extensive to discuss in this review. We will, therefore, focus on the key players starting with cholecystokinin (CCK), the first discovered satiety hormone.

Cholecystokinin (CCK) was initially discovered in 1928 and was one of the first peptides to be found in the gut [51]. In addition to inhibiting food intake, CCK stimulates pancreatic secretion, gall bladder contraction, intestinal motility, and inhibition of gastric mobility. Administration of CCK to rats inhibits food intake by reducing meal size and duration [52], which is enhanced by gastric distention [53]. The half-life of CCK is only 1-2 minutes, therefore it is not effective at reducing meal size if administered more than 15 minutes before a meal [52].

CCK is synthesized throughout the gastrointestinal tract, but mainly in the duodenum and jejunum. Multiple bioactive forms are derived from the same gene product by posttranslational or extracellular processing. CCK is rapidly released locally and into the circulation in response to nutrients in the gut, especially fat and protein, with a gradual increase in levels over 10–30 minutes after meal initiation, remaining elevated for up to 5 hours [54].

CCK-sensitive brain sites include the lateral hypothalamus, medial pons, and lateral medulla. These areas are involved in reward behavior, memory and anxiety, as well as satiety [55, 56]. There are two types of G-protein coupled CCK receptors: CCK-A and CCK-B [57]. CCK-A is involved in satiety whereas CCK-B is not. CCK-A is found on the afferent vagal neurons that have a direct effect on food intake. Rats deficient in CCK-A (Otsuka Long Evans Tokushima Fatty (OLETF) rats) are hyperphagic, obese, and develop diabetes mellitus type 2 [58].

Another satiety peptide, peptide YY (PYY), is part of the pancreatic polypeptide (PP-) fold peptide family (NPY, PYY, PP), all of which have 36 amino acids, contain several tyrosine residues, and require C-terminal amidation for biologic activity. The PP-fold family exerts their effects via the Y family of G-protein coupled receptors (Y1, Y2, Y4, Y5) that are expressed in the hypothalamus. PYY is produced by the intestinal L cells of the ileum, colon, and rectum. Following food intake, PYY is released into the circulation and peaks 1-2 hours postprandially [59]. PYY concentrations are proportional to meal energy content and are therefore higher after fat intake compared to carbohydrates and proteins [60]. Circulating PYY exists in two forms: and . is the peripherally active anorectic signal and is created by cleavage of the N-terminal Tyr-Pro residues by dipeptidyl peptidase IV (DPP-IV) [61]. binds to Y2 receptors leading to inhibition of NPY neurons and stimulation of POMC neurons.

Administration of PYY delays gastric emptying, inhibits secretions from the pancreas and stomach, inhibits gallbladder contraction, and increases the absorption of fluid and electrolytes from the ileum [62]. In rodents, the administration of PYY decreases food intake and reduces weight gain [63], as well as, improvesglycemic control in rodent models of diabetes [64]. PYY-deficient mice are resistant to satiety and develop marked obesity, which is reversed by exogenous PYY administration [65]. In contrast, intracerebroventricular administration of full length PYY stimulates food intake. This is thought to be via action on Y1 and Y5 receptors in the paraventricular nucleus, the neurons targeted by the orexigenic arcuate nucleus NPY neurons.

In obese and lean humans, administration of decreases food intake with a significant decrease in the cumulative 24 hour caloric intake [66]. Obese subjects, however, have a lower endogenous PYY response at each meal compared to normal weight volunteers [67]. This relative PYY deficiency may reduce satiety and could thus reinforce obesity. Obese patients treated by jejunoileal bypass surgery [68] or vertical-banded gastroplasty [69] have elevated PYY levels, which may contribute to their appetite loss.

Pancreatic polypeptide (PP), another member of the PP-fold peptide family, is produced largely in the endocrine pancreas, and also in the exocrine pancreas, colon, and rectum. PP is also released in response to a meal, in proportion to caloric load, and inhibits appetite [70]. PP release is stimulated by ghrelin, as well as motilin (a peptide secreted by the small intestine that enhances gastrointestinal motility) and secretin (a peptide secreted by the duodenum that stimulates gastric acid secretion), whereas somatostatin (a hormone that decreases the rate of gastric emptying, and reduces smooth muscle contraction and blood flow within the intestine) and its analogs significantly reduce PP secretion. PP binds with greatest affinity to the Y4 and Y5 receptors [71]. Peripheral administration of PP in normal mice reduces food intake, gastric emptying, and gastric expression of ghrelin, while it increases vagal tone [72]. Similar to PYY, injection of PP into the third ventricle stimulates daytime food intake [73]. Interestingly, patients with Prader-Willi syndrome have suppressed basal and postprandial PP levels [74]. Administration of PP in Prader-Willi patients leads to reduced food intake [75].

Incretins are hormones released from the gastrointestinal tract into the circulation in response to nutrient ingestion. Incretins enhance glucose-stimulated insulin secretion. Preproglucagon is expressed in the cells of the endocrine pancreas, L cells of the intestine (distal ileum and colon), and neurons located in the caudal brainstem and hypothalamus. It is cleaved into multiple different products, including glucagon and two of the incretins, oxyntomodulin and glucagon-like peptide-1 (GLP-1). Oxyntomodulin and GLP-1 are released from L cells in the distal ileum and colon in response to ingestion of nutrients. Oxyntomodulin binds the GLP-1 receptor that is expressed in the nucleus of the solitary tract in the brainstem and in the arcuate nucleus. Oxyntomodulin inhibits gastric acid secretion, decreases gastric emptying, and decreases pancreatic enzyme secretion which is likely related to decreased gastric output [76]. Administration of oxyntomodulin in humans has been found to suppress ghrelin levels [77], decrease body weight and appetite, decrease leptin, and increase adiponectin levels presumably secondary to loss of adipose tissue [78].

GLP-1 leads to delay in gastric emptying, stimulation of glucose-dependent insulin secretion, inhibition of glucagon secretion, and stimulation of somatostatin secretion. GLP-1 binds to its receptor, a G-protein coupled receptor that belongs to the class B family, including receptors for glucagon and GIP [79]. The GLP-1 receptor is expressed in a wide range of tissues, including the pancreatic islet cells, lung, heart, kidney, stomach, intestine, pituitary, skin, vagus nerve, and several regions of the CNS including the hypothalamus and brainstem. Peripheral and central GLP-1 administration activates neurons in the arcuate and paraventricular nuclei, nucleus of the solitary tract, and area postrema [80] leading to decreased appetite. GLP-1 administration promotes satiety and has beneficial effects on glucose homeostasis.

The properties of GLP-1 have made it a useful drug target. GLP-1 is released rapidly into the circulation after oral nutrient ingestion, and its secretion occurs in a biphasic pattern starting with an early (within 10–15 minutes) phase that is followed by a longer (30–60 minutes) phase [81]. The half-life of GLP-1 is less than 2 minutes owing to rapid inactivation by the enzyme DPP-IV, which also cleaves PYY. This is the basis for the development of exenatide (Byetta), a subcutaneously administered DPP-IV-resistant GLP-1 receptor agonist.

Glucose-dependent insulinotropic polypeptide (GIP) is another incretin that is secreted by the stomach and K cells in the duodenum and jejunum in response to nutrient ingestion. The half-life of GIP is 7 minutes in healthy individuals and 5 minutes in patients with type 2 diabetes [82]. GIP is also inactivated by DPP-IV [82, 83]. The GIP receptor gene is expressed in the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, pituitary, heart, testis, endothelial cells, bone, trachea, spleen, thymus, lung, kidney, thyroid, and several regions in the CNS. GIP leads to glucose-dependent insulin secretion, induction of cell proliferation, promotion of energy storage via direct actions on adipose tissue, and enhancement of bone formation via stimulation of osteoblast proliferation and inhibition of apoptosis. In the CNS, GIP is expressed in the hippocampus and GIP receptor expression is detected in the cerebral cortex, hippocampus, and olfactory bulb. GIP action in the CNS may play a role in neural progenitor cell proliferation and behavior modification [84].

Insulin is another hormonal regulator of appetite. Insulin levels increase rapidly after a meal and vary directly with changes in adiposity. Insulin penetrates the blood-brain barrier via a saturatable, receptor-mediated process at levels proportional to the circulating insulin [85]. Insulin receptors are widely distributed in the brain with highest concentrations found in the olfactory bulbs and arcuate nucleus. Once insulin enters the brain, it acts as an anorexigenic signal [86]. Mice with a neuron-specific disruption of the insulin receptor gene have increased food intake, obesity with increased body fat, and plasma leptin levels, and impaired spermatogenesis and ovarian follicle maturation [87]. There are several insulin receptor substrates (IRS) that are activated by phosphorylation by the insulin receptor on their tyrosine residues [88]. IRS-1 and IRS-2 have been identified in neurons. IRS-2 knockout mice have been found to have increased food intake, increased fat stores, and infertility [89].

Leptin, also termed OB protein, is another important appetite regulator. It is produced by the white and brown adipose tissue, stomach, placenta, mammary gland, ovarian follicles, and certain fetal organs such as heart, bone or cartilage, and perhaps the brain. The ob gene is expressed in all adipose tissue, but to a greater degree in the subcutaneous adipose tissue than the omental fat. Leptin levels are positively correlated with the amount of body fat mass. Leptin secretion does not appear to be driven by meal patterns. Instead, the circadian pattern is characterized by high levels between midnight and early morning hours and a nadir around noon to midafternoon [90]. Leptin is secreted in a pulsatile fashion with 32 peaks per 24-hour period and a pulse duration of 32.8 minutes [91]. This implies that neural and neurohormonal components in the brain may regulate leptin secretion from adipocytes.

Leptin receptors belong to the cytokine receptor superfamily, which uses the Janus activating kinase (JAK-) signal transducer and activator of transcription (STAT) pathway of signal transduction. Leptin receptors have multiple different splice variants. Ob-Rb is the long form of the receptor and has a long intracellular domain, which is necessary for the action of leptin on appetite. Ob-Rb is expressed in multiple different sites within the hypothalamus including the arcuate nucleus, paraventricular nucleus, dorsomedial hypothalamic nucleus, and lateral hypothalamic area. Short forms of the Ob receptor may play a role in the transport of leptin across the blood-brain barrier [92]. After binding the receptor, leptin stimulates a specific signaling cascade that results in inhibition of orexigenic peptides (NPY and AgRP) [93, 94], while stimulating anorectic peptides (including POMC and CART) [93, 95]. Ultimately, this leads to decreased appetite and increased energy expenditure. db/db mice have a mutation in the intracellular portion of Ob-Rb and therefore are unable to respond to the leptin signal and as a result develop profound obesity [96]. Additionally, it has been found that up to 3% of individuals with severe early onset obesity have pathogenic mutations in the leptin-receptor gene [97].

Ob-deficient mice have an absence of circulating leptin and develop severe obesity due to both increased food intake and decreased energy expenditure [98], both of which can be normalized by the administration of leptin [99]. The absence of leptin in humans leads to severe obesity, hypogonadism, and impaired T cell mediated immunity, which are remediable with administration of recombinant leptin [100]. About 5% of obese populations are "relatively" leptin deficient and it is possible that these individuals could benefit from leptin therapy [101].

Finally, the hormone adiponectin is secreted by the mature adipocyte. Adiponectin receptors are expressed in the brain, particularly in the paraventricular nucleus, amygdala, area postrema, and diffusely in the periventricular areas. For reasons that are unclear, adiponectin's concentration in the blood stream is extremely high, approximately 1000 times higher than that of other polypeptide hormones. Its structure closely resembles C1q and types VIII and X collagen. Generally, adiponectin self associates to form homotrimers that then dimerize to yield hexamers. Increased adiponectin levels in rodents appear to decrease body fat mass by stimulation of fatty acid oxidation in muscle [102]. Adiponectin also decreases food intake and obesity in obese rats [103], and improves insulin sensitivity by decreasing hepatic glucose output [104]. In humans, high molecular weight adiponectin (which is thought to be the active form) is reduced in patients with type 2 diabetes, and increasing the proportion of high molecular weight adiponectin by weight loss and treatment with thiazolinediones leads to improved insulin sensitivity [105].