Francesca Lolli

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.

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)

Changes in Adenosine 5-Monophosphate-Activated Protein Kinase as a Mechanism of Visceral Obesity in Cushing's Syndrome

OBJECTIVE Features of the metabolic syndrome such as central obesity with insulin resistance and dyslipidemia are typical signs of Cushings syndrome and common side effects of prolonged glucocorticoid treatment. AMP-activated protein kinase (AMPK), a key regulatory enzyme of lipid and carbohydrate metabolism as well as appetite, is involved in the development of the deleterious metabolic effects of excess glucocorticoids, but no data are available in humans. In the current study, we demonstrate the effect of high glucocorticoid levels on AMPK activity of human adipose tissue samples from patients with Cushings syndrome. METHODS AMPK activity and mRNA expression of genes involved in lipid metabolism were assessed in visceral adipose tissue removed at abdominal surgery of 11 patients with Cushings syndrome, nine sex-, age-, and weight-matched patients with adrenal incidentalomas, and in visceral adipose tissue from four patients with non-endocrine-related abdominal surgery. RESULTS The patients with Cushings syndrome exhibited a 70% lower AMPK activity in visceral adipose tissue as compared with both incidentalomas and control patients (P = 0.007 and P < 0.001, respectively). Downstream targets of AMPK fatty acid synthase and phosphoenol-pyruvate carboxykinase were up-regulated in patients with Cushings syndrome. AMPK activity was inversely correlated with 0900 h serum cortisol and with urinary free cortisol. CONCLUSIONS Our data suggest that glucocorticoids inhibit AMPK activity in adipose tissue, suggesting a novel mechanism to explain the deposition of visceral adipose tissue and the consequent central obesity observed in patients with iatrogenic or endogenous Cushings syndrome.

The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release

This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1‐knockout (KO) and/or wild‐type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1‐KO or CB1 antagonist‐treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1‐KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1‐KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin‐cannabinoid–CB1–AMPK pathway.—Kola, B., Wittman, G., Bodnár, I., Amin, F., Lim, C. T., Oláh, M., Christ‐Crain, M., Lolli, F., van Thuijl, H., Leontiou, C. A., Füzesi, T., Dalino, P., Isidori, A. M., Harvey‐White, J., Kunos, G., Nagy, G. M., Grossman, A. B., Fekete, C., Korbonits, M., The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release. FASEB J. 27, 5112–5121 (2013). www.fasebj.org

Androgen modulation of pro-inflammatory and anti-inflammatory cytokines during preadipocyte differentiation.

Abstract Background: Macrophages and adipocytes contribute to release of cytokines resulting in the chronic inflammatory profile of the metabolic syndrome. The local increase of proinflammatory cytokines impairs adipogenesis, resulting in formation of dysfunctional adipocytes that are unable to store and handle lipids. The altered lipid fluxes in/from adipocytes affect whole-body metabolism. We investigated the role of androgens on adipocyte-derived proinflammatory and anti-inflammatory cytokines during preadipocyte differentiation. Materials and methods: Various differentiation methods were used to obtain full conversion of 3T3-L1 into mature adipocytes. The degree of adipocyte conversion in the presence/absence of dihydrotestosterone (DHT) was analyzed by measuring intracellular triglycerides (Oil Red O staining). The effects of DHT administration on interleukin 1β (IL-1β), IL-2, IL-6, IL-10, IL-12, interferon γ (IFNγ) and tumor necrosis factor α (TNFα) secretion was measured at days 0, 4, 6 and 8 of differentiation using the SearchLight multiplex protein array. Results: DHT regulates a number of cytokines in committed and mature 3T3-L1 adipocytes. IL-1β and TNFα were readily suppressed at the very early stages of differentiation. IFNγ release was inhibited at day 4, but the effect was no longer detectable on day 8. IL-6 and IL-12 were significantly reduced at day 8 of differentiation. Conversely, the differentiation-dependent increase of IL-2 and IL-10 was further stimulated by DHT since day 0. Conclusions: We provide evidence that androgens promote an anti-inflammatory profile that parallels the acquisition of a functional adipocyte phenotype. The crosstalk between androgens, adipocyte-derived mediators of inflammation and intracellular lipid fluxes could have profound implications on metabolism of men with obesity and metabolic syndrome.

AIP, a Protein Mutated in Familial Acromegaly, Plays a Role in the Regulation of Cell Proliferation and Shows Cell-Type Specific Subcellular Localisation

a Endocrinology, b Histopathology, and c Cardiac, Vascular & Inflammation Research, Barts and the London Medical School, d Division of Molecular and Cellular Neuroscience, Institute of Ophthalmology, University College London, London, e Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford , UK, and f Section of Endocrinology, University of Illinois at Chicago, Chicago, Ill. , USA