Márta Korbonits

Ghrelin--a hormone with multiple functions.

The growth hormone secretagogue ghrelin is in the centre of interest since its discovery in 1999. It stimulates growth hormone, corticotropic hormone and prolactin secretion, but also plays an important role in the regulation of appetite, carbohydrate- and lipid metabolism and possibly on gastric acid secretion, gastric motility, heart function and as well as immune functions and cell proliferation. Ghrelin was originally identified from the stomach but it is also present in all tissue among others in: hypothalamus, pituitary, pancreas, lung, immune cells, placenta, ovary, testis, kidney and in different tumours including pituitary adenoma, neuroendocrine tumours, thyroid carcinomas, endocrine tumours of the pancreas and lung. The gene structure and its receptor are similar to motilin, they are both synthesized in the upper gastrointestinal tract and both have prokinetic activity on gut motility. The ghrelin receptor (growth hormone secretagogue receptor) is a member of G protein-coupled seven transmembrane domain receptor. The receptor is localised in the central nervous system, kidney, thyroid, pancreas, myocardium and spleen. Starvation and low body mass index decrease, while food intake, hyperglycaemia, elevated insulin levels and high body mass index increase the endogenous ghrelin levels. Although we know much about the ghrelin, number of questions remain unanswered, such as the effects of the locally-produced ghrelin or its role in the cell metabolism.

Metformin—mode of action and clinical implications for diabetes and cancer

Metformin has been the mainstay of therapy for diabetes mellitus for many years; however, the mechanistic aspects of metformin action remained ill-defined. Recent advances revealed that this drug, in addition to its glucose-lowering action, might be promising for specifically targeting metabolic differences between normal and abnormal metabolic signalling. The knowledge gained from dissecting the principal mechanisms by which metformin works can help us to develop novel treatments. The centre of metformins mechanism of action is the alteration of the energy metabolism of the cell. Metformin exerts its prevailing, glucose-lowering effect by inhibiting hepatic gluconeogenesis and opposing the action of glucagon. The inhibition of mitochondrial complex I results in defective cAMP and protein kinase A signalling in response to glucagon. Stimulation of 5′-AMP-activated protein kinase, although dispensable for the glucose-lowering effect of metformin, confers insulin sensitivity, mainly by modulating lipid metabolism. Metformin might influence tumourigenesis, both indirectly, through the systemic reduction of insulin levels, and directly, via the induction of energetic stress; however, these effects require further investigation. Here, we discuss the updated understanding of the antigluconeogenic action of metformin in the liver and the implications of the discoveries of metformin targets for the treatment of diabetes mellitus and cancer.

Imprinting of the Gsα gene GNAS1 in the pathogenesis of acromegaly

Approximately 40% of growth hormone-secreting pituitary adenomas have somatic mutations in the GNAS1 gene (the so-called gsp oncogene). These mutations at codon 201 or codon 227 constitutively activate the alpha subunit of the adenylate cyclase-stimulating G protein G(s). GNAS1 is subject to a complex pattern of genomic imprinting, its various promoters directing the production of maternally, paternally, and biallelically derived gene products. Transcripts encoding G(s)alpha are biallelically derived in most human tissues. Despite this, we show here that in 21 out of 22 gsp-positive somatotroph adenomas, the mutation had occurred on the maternal allele. To investigate the reason for this allelic bias, we also analyzed GNAS1 imprinting in the normal adult pituitary and found that G(s)alpha is monoallelically expressed from the maternal allele in this tissue. We further show that this monoallelic expression of G(s)alpha is frequently relaxed in somatotroph tumors, both in those that have gsp mutations and in those that do not. These findings imply a possible role for loss of G(s)alpha imprinting during pituitary somatotroph tumorigenesis and also suggest that G(s)alpha imprinting is regulated separately from that of the other GNAS1 products, NESP55 and XLalphas, imprinting of which is retained in these tumors.

The Role of the Aryl Hydrocarbon Receptor-Interacting Protein Gene in Familial and Sporadic Pituitary Adenomas

CONTEXT Mutations have been identified in the aryl hydrocarbon receptor-interacting protein (AIP) gene in familial isolated pituitary adenomas (FIPA). It is not clear, however, how this molecular chaperone is involved in tumorigenesis. OBJECTIVE AIP sequence changes and expression were studied in FIPA and sporadic adenomas. The function of normal and mutated AIP molecules was studied on cell proliferation and protein-protein interaction. Cellular and ultrastructural AIP localization was determined in pituitary cells. PATIENTS Twenty-six FIPA kindreds and 85 sporadic pituitary adenoma patients were included in the study. RESULTS Nine families harbored AIP mutations. Overexpression of wild-type AIP in TIG3 and HEK293 human fibroblast and GH3 pituitary cell lines dramatically reduced cell proliferation, whereas mutant AIP lost this ability. All the mutations led to a disruption of the protein-protein interaction between AIP and phosphodiesterase-4A5. In normal pituitary, AIP colocalizes exclusively with GH and prolactin, and it is found in association with the secretory vesicle, as shown by double-immunofluorescence and electron microscopy staining. In sporadic pituitary adenomas, however, AIP is expressed in all tumor types. In addition, whereas AIP is expressed in the secretory vesicle in GH-secreting tumors, similar to normal GH-secreting cells, in lactotroph, corticotroph, and nonfunctioning adenomas, it is localized to the cytoplasm and not in the secretory vesicles. CONCLUSIONS Our functional evaluation of AIP mutations is consistent with a tumor-suppressor role for AIP and its involvement in familial acromegaly. The abnormal expression and subcellular localization of AIP in sporadic pituitary adenomas indicate deranged regulation of this protein during tumorigenesis.

The Orexigenic Effect of Ghrelin Is Mediated through Central Activation of the Endogenous Cannabinoid System

Introduction Ghrelin and cannabinoids stimulate appetite, this effect possibly being mediated by the activation of hypothalamic AMP-activated protein kinase (AMPK), a key enzyme in appetite and metabolism regulation. The cannabinoid receptor type 1 (CB1) antagonist rimonabant can block the orexigenic effect of ghrelin. In this study, we have elucidated the mechanism of the putative ghrelin-cannabinoid interaction. Methods The effects of ghrelin and CB1 antagonist rimonabant in wild-type mice, and the effect of ghrelin in CB1-knockout animals, were studied on food intake, hypothalamic AMPK activity and endogenous cannabinoid content. In patch-clamp electrophysiology experiments the effect of ghrelin was assessed on the synaptic inputs in parvocellular neurons of the hypothalamic paraventricular nucleus, with or without the pre-administration of a CB1 antagonist or of cannabinoid synthesis inhibitors. Results and Conclusions Ghrelin did not induce an orexigenic effect in CB1-knockout mice. Correspondingly, both the genetic lack of CB1 and the pharmacological blockade of CB1 inhibited the effect of ghrelin on AMPK activity. Ghrelin increased the endocannabinoid content of the hypothalamus in wild-type mice and this effect was abolished by rimonabant pre-treatment, while no effect was observed in CB1-KO animals. Electrophysiology studies showed that ghrelin can inhibit the excitatory inputs on the parvocellular neurons of the paraventricular nucleus, and that this effect is abolished by administration of a CB1 antagonist or an inhibitor of the DAG lipase, the enzyme responsible for 2-AG synthesis. The effect is also lost in the presence of BAPTA, an intracellular calcium chelator, which inhibits endocannabinoid synthesis in the recorded parvocellular neuron and therefore blocks the retrograde signaling exerted by endocannabinoids. In summary, an intact cannabinoid signaling pathway is necessary for the stimulatory effects of ghrelin on AMPK activity and food intake, and for the inhibitory effect of ghrelin on paraventricular neurons.

AMPK as a mediator of hormonal signalling

AMP-activated protein kinase (AMPK) is a key molecular player in energy homeostasis at both cellular and whole-body levels. AMPK has been shown to mediate the metabolic effects of hormones such as leptin, ghrelin, adiponectin, glucocorticoids and insulin as well as cannabinoids. Generally, activated AMPK stimulates catabolic pathways (glycolysis, fatty acid oxidation and mitochondrial biogenesis) and inhibits anabolic pathways (gluconeogenesis, glycogen, fatty acid and protein synthesis), and has a direct appetite-regulating effect in the hypothalamus. Drugs that activate AMPK, namely metformin and thiazolidinediones, are often used to treat metabolic disorders. Thus, AMPK is now recognised as a potential target for the treatment of obesity and associated co-morbidities.

The cannabinoid CB1 receptor antagonist SR141716 blocks the orexigenic effects of intrahypothalamic ghrelin

The paraventricular nucleus (PVN) of the hypothalamus plays a key role in the control of appetite and energy balance. Both ghrelin and cannabinoid receptor agonists increase food intake when administered into this nucleus: this study investigated possible interactions between the two systems in relation to eating. The orexigenic effect of ghrelin (100 pmol) when infused in to the PVN was reversed by a small, systemic dose of the CB1 cannabinoid receptor antagonist SR141716 (1 mg kg−1). This is the first demonstration of a functional relationship between brain ghrelin and endocannabinoid systems, and, although it needs to be further investigated, the effect of ghrelin on food intake when injected into the PVN seems to be mediated by stimulation of cannabinoid release.

Presence of ghrelin in normal and adenomatous human pituitary

Recently, an endogenous ligand has been described for the growth hormone secretagogue receptor (GHS-R), named ghrelin. It was originally isolated from the stomach, but it is also present in the hypothalamus, where the highest concentration of GHS-R has been detected. It is well established that synthetic GHSs exert their effects on the growth hormone (GH) axis principally via the hypothalamus, although they are also able to stimulate GH release directly from the pituitary. We have previously demonstrated the presence of GHS-R mRNA expression in normal and abnormal human pituitary. We have therefore now investigated the expression of the newly recognized endogenous ligand in rat as well as in human pituitary. We readily detected ghrelin mRNA message in normal rat pituitary using reverse transcriptase polymerase chain reaction with published primers. We then designed primers to the corresponding region on the human ghrelin sequence and successfully detected mRNA message in normal human pituitary, as well as in somatotroph, lactotroph, corticotroph, thyrotroph, and nonfunctioning adenomas. We confirmed the expected polymerase chain reaction product by direct sequencing. In conclusion, we suggest that in addition to the probable hypothalamic effects of ghrelin, the peptide is synthesized locally within the pituitary gland, where it may influence the release of GH in an autocrine or paracrine manner.

Leptin levels do not change acutely with food administration in normal or obese subjects, but are negatively correlated with pituitary‐adrenal activity

Leptin is a peptide secreted by white adipose tissue which has been shown to have a major influence on body weight regulation, while animal studies have revealed widespread interconnections between leptin and other endocrine systems, especially with insulin. However, its acute regulation has been little studied in the human. We have therefore investigated the effect of a 1000 kcal meal and fasting on the levels of leptin, insulin and cortisol, in both normal and obese subjects.

AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing’s syndrome

Chronic exposure to glucocorticoid hormones, resulting from either drug treatment or Cushings syndrome, results in insulin resistance, central obesity, and symptoms similar to the metabolic syndrome. We hypothesized that the major metabolic effects of corticosteroids are mediated by changes in the key metabolic enzyme adenosine monophosphate‐activated protein kinase (AMPK) activity. Activation of AMPK is known to stimulate appetite in the hypothalamus and stimulate catabolic processes in the periphery. We assessed AMPK activity and the expression of several metabolic enzymes in the hypothalamus, liver, adipose tissue, and heart of a rat glucocorticoid‐excess model as well as in in vitro studies using primary human adipose and primary rat hypothalamic cell cultures, and a human hepatoma cell line treated with dexamethasone and metformin. Glucocorticoid treatment inhibited AMPK activity in rat adipose tissue and heart, while stimulating it in the liver and hypothalamus. Similar data were observed in vitro in the primary adipose and hypothalamic cells and in the liver cell line. Metformin, a known AMPK regulator, prevented the corticosteroidinduced effects on AMPK in human adipocytes and rat hypothalamic neurons. Our data suggest that glucocorticoid‐induced changes in AMPK constitute a novel mechanism that could explain the increase in appetite, the deposition of lipids in visceral adipose and hepatic tissue, as well as the cardiac changes that are all characteristic of glucocorticoid excess. Our data suggest that metformin treatment could be effective in preventing the metabolic complications of chronic glucocorticoid excess.— Christ‐Crain M., Kola, B., Lolli F., Fekete, C., Seboek, D., Wittmann, G., Feltrin, D., Igreja, S. C., Ajodha, S., Harvey‐White, J., Kunos, G., Müller B., Pralong, F., Aubert, G., Arnaldi, G., Giacchetti, G., Boscaro, M., Grossman, A. B., Korbonits M. AMP‐activated protein kinase mediates glucocorticoidinduced metabolic changes: a novel mechanism in Cushings syndrome. FASEB J. 22, 1672–1683 (2008)