endocrinology 3

The study of ghrelin, a key endocrine hormone involved in various physiological processes, originates from extensive research into growth hormone (GH) regulation. The human endocrine system encounters numerous challenges including energy imbalance, growth regulation, and metabolic processes, where ghrelin acts as a critical player in maintaining homeostasis. As a primary regulator of appetite and energy balance, ghrelin plays significant roles not only in growth processes but also in metabolic pathways affecting overall health.

The Classical Growth Hormone Axis

Control of growth hormone secretion involves an intricate and highly regulated interplay between the hypothalamus and the pituitary gland. Specifically, two primary hypothalamic factors regulate the synthesis and release of GH from the pituitary:

1. GHRH (Growth Hormone Releasing Hormone / GRF): This hormone is produced in the Arcuate Nucleus of the hypothalamus and exerts a positive effect on GH secretion by stimulating target cells in the anterior pituitary gland.

2. Somatostatin (SRIF): Synthesized in the Periventricular Nucleus, somatostatin serves as a negative regulator of GH secretion, limiting its release during periods when GH is not required for growth stimulation.

Once GH is released into the bloodstream, it stimulates the production of IGF-1 (Insulin-like Growth Factor 1) mainly from the liver and various tissues. This IGF-1 serves as a mediator of many growth-related actions of GH. Importantly, both GH and IGF-1 provide feedback inhibition on the hypothalamus and the pituitary, thus regulating their own secretion through a negative feedback mechanism.

Discovery of Growth Hormone Secretagogues (GHSs)

In the 1980s, scientists aimed to uncover synthetic compounds capable of mimicking or enhancing GH secretion. These biologically active substances are categorized as Growth Hormone Secretagogues (GHSs), and research has elucidated their mechanisms of action.

Early Compounds

• Met-enkephalin: Identified as an endogenous opioid peptide, met-enkephalin features the sequence $ext{Tyr-Gly-Gly-Phe-Met}$, and has been shown to have a stimulating effect on GH release.

• GHRP-6: A synthetic hexapeptide secretagogue with the sequence $ext{His-D-Trp-Ala-Trp-D-Phe-Lys-NH}_2$, GHRP-6 has demonstrated significant GH-releasing properties in both animal models and human studies.

Initial studies involving lambs and calves underscored the effectiveness of various GHSs in stimulating GH secretion, marking a crucial advancement in endocrinology (Bowers CY et al., 1984).

Mechanism of Action

Research conducted by Dickson SL et al. (1995) established that GHSs predominantly act through the arcuate nucleus GRF neurones. The administration of GHRP-6 has been shown to provoke an increase in the expression of Fos, a marker indicative of neuronal activation, within the arcuate nucleus. Notably, this activation occurs irrespective of whether GHRP-6 is administered intracerebroventricularly (ICV) or intravenously (IV), while control substances do not elicit a similar response, emphasizing the specificity of GHS action.

The GHS Receptor ($ext{GHS-R1a}$)

GHSs do not interact with the typical GHRH receptor; instead, they target a specific G-protein-coupled receptor (GPCR) known as GHS-R1a.

Characteristics of $ext{GHS-R1a}$

• Cloning: The receptor was first cloned in 1996 by Howard AD et al. (Science 273, 974), paving the way for further exploration of ghrelin's physiological roles.

• Expression Sites: High levels of $ext{GHS-R1a}$ mRNA are notably found in the Hypothalamus and the Pituitary (Guan X et al., 1997), underscoring its significance in endocrine signaling.

• Signaling Pathway: The activation of $ext{GHS-R1a}$ triggers the $ext{G}_q$ signal transduction pathway: Phospholipase C (PLC) acts on $ext{PIP}_2$ to produce $ext{IP}_3$ and DAG, with $ext{IP}_3$ binding to receptors on the Endoplasmic Reticulum, leading to the release of intracellular Calcium ($ext{Ca}^{2+}$), which plays a crucial role in numerous cellular processes.

• Variants: A truncated, non-functional splice variant known as GHS-R1b exists, which does not contribute to GH signaling.

• Pharmaceutical Application: MK0677, developed as an orally active GHS, demonstrates promising results as a "pill for growth," capable of inducing GH release in humans and suggesting therapeutic applications in growth hormone deficiencies (Jacks T et al., 1996).

The Discovery and Structure of Ghrelin

The discovery of ghrelin was achieved through a process termed "Reverse Pharmacology," where researchers had identified the receptor ($ext{GHS-R1a}$) but were not initially aware of its endogenous ligand.

Reverse Pharmacology Process

Kojima M et al. (Nature, 1999) executed a cellular screening approach utilizing a CHO-GHSR62 cell line (Chinese Hamster Ovary cells expressing the GHS receptor). Using a fluorescent calcium indicator (Fluo4), the team detected $ext{Ca}^{2+}$ mobilization when various tissue extracts were introduced.

• The screening revealed that a specific fraction from the stomach resulted in considerable fluorescence, suggesting a biologically active compound associated with ghrelin signaling.

• Upon further purification of this fraction, researchers identified the compound and named it "Ghrelin."

Molecular Structure of Ghrelin

Ghrelin is characterized as a 28-amino acid peptide that undergoes a distinctive post-translational modification essential for its biological activity.

• Octanoylation: Critical for its functionality, an n-octanoyl group (C8:0) is attached to the third amino acid (Serine 3, Ser3), facilitating its binding to GHS-R1a.

• Enzyme Action: The enzyme GOAT (ghrelin O-acyltransferase) catalyzes the transformation of UAG (Unacylated Ghrelin) into its active acylated form (Yang J et al., 2008).

• Activity: This octanoylation is paramount for the hormone's ability to effectively bind to $ext{GHS-R1a}$ and exert its biological effects.

• Circulating Forms: Despite the biological importance of acylated ghrelin in GH release, the major circulating form of the hormone is Des-octanoyl ghrelin (UAG), which has different biological roles compared to acylated ghrelin (Hosoda H et al., 2000).

Distribution and Regulation of Ghrelin

Sites of Production

1. Stomach: The primary site of ghrelin production, extensive research employing Northern blot techniques, in-situ hybridization, and immunocytochemistry demonstrate elevated mRNA and protein levels of ghrelin specifically in gastric tissues (Kojima M et al., 1999).

2. Brain: A network of ghrelinergic neurones can be found in the hypothalamus, particularly concentrated in the Arcuate Nucleus and the Periventricular Nucleus (Cowley MA et al., 2003), suggesting its dual role in energy balance and growth regulation.

Secretion Regulation

Ghrelin levels are acutely responsive to nutritional status and other physiological factors:

• Fasting: Extended periods of fasting lead to a significant rise in circulating ghrelin levels, which stimulate appetite to encourage food intake.

• Refeeding: Following the consumption of a meal, ghrelin levels experience a rapid suppression, reflecting its adaptive role in energy homeostasis.

• Intraprandial Peaks: Studies reveal that circulating ghrelin levels peak prior to meals in both humans and rats, suggesting its critical involvement in meal initiation and appetite stimulation (Cummings DE et al., 2001; Bodosi B et al., 2004).

Physiological Actions of Ghrelin

1. Growth Hormone and Gonadotropin Secretion

• Acute GH Stimulation: Ghrelin portrayed as a significant stimulant of GH secretion, particularly enhancing release during fasting states through activation of GRF neurones within the arcuate nucleus.

• Chronic Suppression: However, prolonged exposure to ghrelin or GHSs may lead to a state referred to as "growth fatigue," where continuous infusion can suppress GH secretion. This suggests a complex regulatory role where pulsed release of ghrelin is necessary for promoting growth and body weight gain (Thompson NM et al., 2003).

• Reproductive Axis: Ghrelin's action extends to the endocrine control of reproduction as it can suppress the secretion of Luteinizing Hormone (LH) and Follicle-Stimulating Hormone (FSH) in the reproductive axis (Martini AC et al., 2006).

2. Orexigenesis (Feeding Behavior)

Ghrelin is recognized as a potent orexigenic peptide that drives positive energy balance by stimulating appetite.

• Mechanism: It exerts its orexigenic effects through the activation of NPY (Neuropeptide Y) neurones located in the arcuate nucleus. This results in increased spontaneous electrical activity in these neurones, as well as elevated Fos expression.

• Pattern Independence: Importantly, ghrelin's influence on feeding behavior does not depend on the timing or pattern; both continuous and pulsed administration effectively stimulate food intake (Thompson NM et al., 2004).

3. Fat Deposition and Adipogenesis

Ghrelin's role in energy regulation includes effects on fat storage and adipogenesis; however, sensitivity to this hormone varies based on the fat depot in the body.

• Adipogenesis: The adipogenic effects of ghrelin are observed in bone marrow, with unacylated ghrelin (UAG) mimicking these effects, suggesting the involvement of a $ext{GHS-R1a}$-independent pathway.

• Abdominal Fat Storage: The accumulation of intra-abdominal fat is found to be specifically mediated through the $ext{GHS-R1a}$ receptor (Davies JS et al., 2009), highlighting the complexity of ghrelin’s endocrine influence on fat distribution.

Physiological Significance: Lessons from Knockout (KO) Models

Surprisingly, studies conducted on mice with a deletion of the ghrelin gene revealed that these models exhibit relatively normal phenotypic attributes under standard living conditions (Sun Y et al., 2003):

• Growth: These knockout mice demonstrate normal rates of skeletal growth despite the absence of ghrelin.

• Food Intake: Cumulative food intake over time remains consistent with that of wild-type animals, indicating potential compensatory mechanisms within energy balance regulation.

• Body Composition: The overall body composition, including fat deposition, appears comparable to wild-type controls.

These findings suggest that while exogenous ghrelin can stimulate growth and feeding behaviors when administered in pharmacological contexts, it may not be strictly essential for these processes to occur naturally under physiological conditions, presumably due to compensatory adaptations.

Summary of Ghrelin's Multisystem Functions

Ghrelin functions fundamentally as an "energy deficit signal," acting in opposition to Leptin, which signals satiety and sufficiency within the body (Zigman JM et al., 2003).

Major Functions (Pradhan G et al., 2013):

• Metabolic: Ghrelin decreases insulin secretion (which may involve modulations by $ext{GHS-R}$ and receptor interactions with somatostatin receptor 5); stimulates glucagon secretion; promotes adipogenesis; reduces lipolysis within adipose tissues.

• Energy Expenditure: It decreases thermogenesis, suggesting a conserving energy mechanism during fasting states.

• Skeletal System: Ghrelin stimulates osteoblast proliferation and differentiation while also playing a role in the regulation of osteoclastogenesis (effects vary across age groups).

• Cardiovascular System: The hormone lowers sympathetic nerve activity and arrhythmia and has been linked to enhanced survival rates following Myocardial Infarctions (MI) and Cardiopulmonary Bypass (CPB).

• Musculoskeletal System: Ghrelin promotes muscle differentiation and fusion and can protect against muscle atrophy, offering potential therapeutic implications in cachexia and sarcopenia.

• Cancer Pathology: Variants of ghrelin, such as In1-ghrelin, are frequently detected within tumors, including human breast cancer, indicating its potential role in cancer biology and the metabolic syndrome associated with malignancies.