International Journal of Obesity (2015) 39, 321–330 © 2015 Macmillan Publishers Limited All rights reserved 0307-0565/15 www.nature.com/ijo ORIGINAL ARTICLE Changes in morphology and function of adrenal cortex in mice fed a high-fat diet MM Swierczynska1,2,4, I Mateska1,2, M Peitzsch3, SR Bornstein2, T Chavakis2, G Eisenhofer3, V Lamounier-Zepter2,5 and S Eaton1,5 BACKGROUND/OBJECTIVES: Obesity is a major risk factor for the development of type 2 diabetes and other debilitating diseases. Obesity and diabetes are intimately linked with altered levels of adrenal steroids. Elevated levels of these hormones induce insulin resistance and cause cardiovascular diseases. The mechanisms underlying obesity-related alterations in adrenal steroids are still not well understood. Here, we investigated how diet-induced obesity affects the morphology and function of the mouse adrenal cortex. METHODS: We fed animals either a high-fat diet (HFD) or a normal diet (60% kcal from fat or 10% kcal from fat, respectively) for 18 weeks. We then assessed various aspects of adrenal gland morphology and function, as well as basal plasma concentrations of steroid hormones and ACTH. RESULTS: We show that adrenal glands of mice fed a HFD release more corticosterone and aldosterone, resulting in higher plasma levels. This increase is driven by adrenal cortical hyperplasia, and by increased expression of multiple genes involved in steroidogenesis. We demonstrate that diet-induced obesity elevates Sonic hedgehog signaling in Gli1-positive progenitors, which populate the adrenal capsule and give rise to the steroidogenic cells of the adrenal cortex. Feeding animals with a HFD depletes Gli1-positive progenitors, as the adrenal cortex expands. CONCLUSIONS: This work provides insight into how diet-induced obesity changes the biology of the adrenal gland. The association of these changes with increased Shh signaling suggests possible therapeutic strategies for obesity-related steroid hormone dysfunction. International Journal of Obesity (2015) 39, 321–330; doi:10.1038/ijo.2014.102 INTRODUCTION with corticosterone suffices to increase adiposity, a phenotype 17 Obesity constitutes a major risk factor for the development of type that resolves after cessation of treatment. Thus, abnormal levels 2 diabetes, cardiovascular diseases, non-alcoholic steatohepatitis of adrenal steroids may contribute to the development of obesity and liver failure, certain forms of cancer and sleep-breathing and its comorbidities. However, the mechanisms that underlie disorders.1 It has become an epidemic in western countries, and elevated levels of adrenal steroids in obesity are still not well its prevalence is continuously increasing.2 Thus, obesity is understood. currently one of the most important public health problems. Aldosterone synthesis is controlled mostly by systemically 6 Obesity is associated with alterations in plasma cortisol and circulating angiotensin II. On the other hand, secretion of aldosterone levels.3,4 Cortisol and other glucocorticoids promote glucocorticoids, and to lesser extent aldosterone, is controlled differentiation of pre-adipocytes to adipocytes, and may con- mainly by the hypothalamic–pituitary–adrenal (HPA) axis, and tribute to increased body fat mass. Moreover, they inhibit glucose directly induced by the pituitary hormone ACTH.18,19 Increased uptake by peripheral tissues, and stimulate gluconeogenesis in the steroid hormone levels in obesity result, at least in part, from liver.5 Aldosterone, on the other hand, is the major regulator of hyperactivity of the HPA axis at the central level.20 Obese people blood volume and blood pressure homeostasis.6 Obese people also appear to have higher adrenal sensitivity to ACTH.8 However, tend to have elevated levels of aldosterone and increased rates synthesis of steroid hormones in obese patients might also be of glucocorticoid production and clearance.6–9 Similarly, animal affected by other systemic and paracrine factors.18,19,21 models of obesity have elevated levels of aldosterone and The adrenal gland undergoes remodeling in response to chronic corticosterone, the major glucocorticoid in rodents.10–14 Interest- changes in physiological demand for steroid hormones.22 This ingly, increased levels of cortisol and aldosterone have been process might involve the Gli1-positive progenitor cells that linked to insulin resistance and development of cardiovascular populate the adrenal capsule and can give rise to all steroidogenic – diseases.6,15 A potential causal link between elevated cortisol and lineages.23 26 These Gli1-positive cells respond to Sonic hedgehog aldosterone levels and obesity-related diseases may be inferred (Shh) secreted by cells residing in peripheral cortex. Therefore, it from observations that patients with disorders primarily caused by has been proposed that Shh signaling might have a role in increased secretion of adrenal steroids are more prone to develop adrenocortical maintenance and remodeling in response to obesity and its comorbidities.16 Accordingly, treatment of mice physiological stimuli.27,28 1Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; 2Department of Internal Medicine III, University Hospital Carl Gustav Carus, Dresden University of Technology, Dresden, Germany and 3Department of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Dresden University of Technology, Dresden, Germany. Correspondence: Dr S Eaton, Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. E-mail: [email protected] 4Current address: Biozentrum, University of Basel, Basel, Switzerland. 5These authors contributed equally to this work. Received 7 January 2014; revised 9 May 2014; accepted 15 May 2014; accepted article preview online 12 June 2014; advance online publication, 8 July 2014 Adrenal cortex in mice fed a high-fat diet MM Swierczynska et al 322 The ob/ob mouse and the Zucker (fa/fa) rat, which become Biochemical analysis obese due to the loss of leptin signaling, have been used to study Plasma cholesterol, triglycerides and ACTH levels were analyzed using dysregulation of steroid hormones in obesity. In these models, Cholesterol Quantitation Kit (Sigma-Aldrich), EnzyChrom Triglyceride Assay increased plasma steroid hormone levels are associated with Kit (Bioassays, Hayward, CA, USA), and ACTH (Mouse/Rat) ELISA (Abnova increased conversion of 11-dehydrocorticosterone to cortico- GMBH, Heidelberg, Germany) as instructed by the manufacturers. sterone in peripheral tissues, increased sensitivity to angiotensin II, hyperactivity of the HPA axis, enlargement of the adrenal cortex Steroid hormones measurements and increased release of steroid hormones by the adrenal Aldosterone, corticosterone and their precursors were measured in mouse gland.10–12,29,30 However, as leptin directly inhibits secretion of plasma and supernatants of organ cultures by liquid chromatography- steroid hormones by adrenal glands31,32 these monogenic models tandem mass spectrometry. An AB Sciex QTRAP 5500, equipped with may not accurately reflect the changes that occur in diet-induced atmospheric pressure chemical ionization source, coupled to the Acquity obesity. Feeding C57BL/6 mice with a high-fat diet (HFD) has been UPLC system (Waters, Milford, MA, USA) was used. Samples were prepared by offline solid phase extraction supported by positive pressure as shown to accurately reflect various aspects of obesity in 34 14,33 described elsewhere. Analytes were detected by the triple quadrupole humans. However, the effects of diet-induced obesity on mass spectrometer in multiple reaction-monitoring scan mode using adrenal function/morphology have not been studied in detail. positive atmospheric pressure chemical ionization. Analyte concentrations Here, we investigated the effect of diet-induced obesity on were quantified using ratios of analyte peak area to the respective internal various aspects of adrenal morphology and function, including the standard peak area for which deuterated analogs were used. activity of Shh pathway and its impact on Gli1-positive capsular progenitor cells. We show that feeding animals a HFD elevates Immunofluorescence adrenal production of aldosterone, corticosterone and their PFA-fixed glands were cryopreserved in 30% sucrose (AppliChem GMBH, precursors, thereby elevating their levels in plasma. We demon- Darmstadt, Germany)/phosphate-buffered saline overnight at 4 °C, strate that increased steroidogenic output in obese animals results embedded in TFM medium (Triangle Biomedical Sciences, Durham, NC, from adrenal cortex hyperplasia, and from increased expression of USA), and frozen at − 80 °C. Each adrenal was cut into 8-μm-thick serial genes controlling various aspects of steroidogenesis. Finally, we sections. Special care was taken to process every gland in a uniform show that the expansion of the adrenal gland in obese animals is fashion. accompanied by depletion of Gli1-positive capsular progenitor Adrenal cryosections were incubated overnight at 4 °C with appropriate cells, with a concomitant upregulation of the Shh signaling primary antibodies, washed and incubated for 1 h at room temperature pathway in individual cells. Our work provides insight into how with the respective secondary antibody together with DAPI (1:5000; Roche, Penzberg, Germany). Antibodies used are listed in Supplementary Table 1. diet-induced obesity changes the biology of the adrenal gland, and suggests that Shh pathway