Munc18-dependent and -independent clustering of syntaxin in the plasma membrane of cultured endocrine cells.

Syntaxin helps in catalyzing membrane fusion during exocytosis. It also forms clusters in the plasma membrane, where both its transmembrane and SNARE domains are thought to homo-oligomerize. To study syntaxin clustering in live PC12 cells, we labeled granules with neuropeptide-Y-mCherry and syntaxin clusters with syntaxin-1a green fluorescent protein (GFP). Abundant clusters appeared under total internal reflection (TIRF) illumination, and some of them associated with granules ("on-granule clusters"). Syntaxin-1a-GFP or its mutants were expressed at low levels and competed with an excess of endogenous syntaxin for inclusion into clusters. On-granule inclusion was diminished by mutations known to inhibit binding to Munc18-1 in vitro. Knock-down of Munc18-1 revealed Munc18-dependent and -independent on-granule clustering. Clustering was inhibited by mutations expected to break salt bridges between syntaxin's Hb and SNARE domains and was rescued by additional mutations expected to restore them. Most likely, syntaxin is in a closed conformation when it clusters on granules, and its SNARE and Hb domains approach to within atomic distances. Pairwise replacements of Munc18-contacting residues with alanines had only modest effects, except that the pair R114A/I115A essentially abolished on-granule clustering. In summary, an on-granule cluster arises from the specific interaction between a granule and a dense cluster of syntaxin-Munc18-1 complexes. Off-granule clusters, by contrast, were resistant to even the strongest mutations we tried and required neither Munc18-1 nor the presence of a SNARE domain. They may well form through the nonstoichiometric interactions with membrane lipids that others have observed in cell-free systems.

Expression of Transcription Factor CREM in Human Tissues.

Cyclic AMP element modulator (CREM) is a transcription factor best known for its intricate involvement in spermatogenesis. The CREM gene encodes for multiple protein isoforms, which can enhance or repress transcription of target genes. Recent studies have identified fusion genes, with CREM as a partner gene in many neoplastic diseases. EWSR1-CREM fusion genes have been found in several mesenchymal tumors and in salivary gland carcinoma. These genes encode fusion proteins that include the C-terminal DNA-binding domain of CREM. We used a transcriptomic approach and immunohistochemistry to study the expression of CREM isoforms that include DNA-binding domains across human tissues. We found that CREM protein is widely expressed in almost all normal human tissues. A transcriptomic analysis of normal tissues and cancer showed that transcription of CREM can be altered in tumors, suggesting that also wild-type CREM may be involved in cancer biology. The wide expression of CREM protein in normal human tissues and cancer may limit the utility of immunohistochemistry for identification of tumors with CREM fusions.

GWAS of allometric body-shape indices in UK Biobank identifies loci suggesting associations with morphogenesis, organogenesis, adrenal cell renewal and cancer.

Genetic studies have examined body-shape measures adjusted for body mass index (BMI), while allometric indices are additionally adjusted for height. We performed the first genome-wide association study of A Body Shape Index (ABSI), Hip Index (HI) and the new Waist-to-Hip Index and compared these with traditional indices, using data from the UK Biobank Resource for 219,872 women and 186,825 men with white British ancestry and Bayesian linear mixed-models (BOLT-LMM). One to two thirds of the loci identified for allometric body-shape indices were novel. Most prominent was rs72959041 variant in RSPO3 gene, expressed in visceral adipose tissue and regulating adrenal cell renewal. Highly ranked were genes related to morphogenesis and organogenesis, previously additionally linked to cancer development and progression. Genetic associations were fewer in men compared to women. Prominent region-specific associations showed variants in loci VEGFA and HMGA1 for ABSI and KLF14 for HI in women, and C5orf67 and HOXC4/5 for ABSI and RSPO3, VEGFA and SLC30A10 for HI in men. Although more variants were associated with waist and hip circumference adjusted for BMI compared to ABSI and HI, associations with height had previously been reported for many of the additional variants, illustrating the importance of adjusting correctly for height.

Loss of CREB Coactivator CRTC1 in SF1 Cells Leads to Hyperphagia and Obesity by High-fat Diet But Not Normal Chow Diet.

Cyclic adenosine monophosphate responsive element-binding protein-1-regulated transcription coactivator-1 (CRTC1) is a cytoplasmic coactivator that translocates to the nucleus in response to cyclic adenosine monophosphate. Whole-body knockdown of Crtc1 causes obesity, resulting in increased food intake and reduced energy expenditure. CRTC1 is highly expressed in the brain; therefore, it might play an important role in energy metabolism via the neuronal pathway. However, the precise mechanism by which CRTC1 regulates energy metabolism remains unknown. Here, we showed that mice lacking CRTC1, specifically in steroidogenic factor-1 expressing cells (SF1 cells), were sensitive to high-fat diet (HFD)-induced obesity, exhibiting hyperphagia and increased body weight gain. The loss of CRTC1 in SF1 cells impaired glucose metabolism. Unlike whole-body CRTC1 knockout mice, SF1 cell-specific CRTC1 deletion did not affect body weight gain or food intake in normal chow feeding. Thus, CRTC1 in SF1 cells is required for normal appetite regulation in HFD-fed mice. CRTC1 is primarily expressed in the brain. Within the hypothalamus, which plays an important role for appetite regulation, SF1 cells are only found in ventromedial hypothalamus. RNA sequencing analysis of microdissected ventromedial hypothalamus samples revealed that the loss of CRTC1 significantly changed the expression levels of certain genes. Our results revealed the important protective role of CRTC1 in SF1 cells against dietary metabolic imbalance.

Adrenal gland fine needle aspiration: a multi-institutional analysis of 139 cases.

INTRODUCTION: Adrenal gland lesions span a range of entities from benign and malignant primary neoplasms to metastatic tumors. Fine-needle aspiration (FNA) provides a minimally invasive diagnostic tool to stage patients with known malignancy and procure material for molecular testing. This study characterizes the clinicopathologic associations of patients with adrenal gland FNA from 2 large hospitals. MATERIALS AND METHODS: FNAs were identified by query of electronic medical record from 2002-2019. Clinical and pathological information was collated and correlated with corresponding surgical diagnosis when available. RESULTS: Of 139 cases, the majority (n = 127, 91%) were adequate computed tomography-guided FNAs and included the following diagnoses: positive for malignancy (n = 77, 55%), negative for malignancy (n = 32, 23%), neoplastic cells present (n = 16, 12%), nondiagnostic (n = 12, 9%), atypical (n = 1, 1%), and suspicious for carcinoma (n = 1, 1%). The majority (94%, n = 72 of 77) of malignancies were metastatic tumors, most frequently carcinoma (n = 53 of 72, 74%), followed by melanoma (n = 11 of 72, 15%), lymphoma (n = 4 of 72, 6%), and sarcoma (n = 4 of 72, 6%). Metastatic carcinomas included lung (n = 21 of 72, 29%), genitourinary (n = 12 of 72, 17%), and hepatobiliary or gastrointestinal tract (n = 11 of 72, 15%) primaries. Primary adrenal neoplasms (n = 23) included adenomas (n = 11 of 23, 48%), pheochromocytomas (n = 4 of 23, 17%), and myelolipomas (n = 3 of 23, 13%). Thirty-two patients with metastases died of disease after median follow-up of 8 months. CONCLUSIONS: High specimen adequacy (n = 127, 91%) and low indeterminate rates (n = 2, 2%) are achieved with adrenal FNA. Most aspirated lesions represent metastases from primary lung carcinomas, but other primary sites including those below the diaphragm are part of the diagnostic differential. Adrenal metastasis was associated with a poor prognosis, with median survival of 8 months.

The receptor tyrosine kinase EPHB6 regulates catecholamine exocytosis in adrenal gland chromaffin cells.

The erythropoietin-producing human hepatocellular receptor EPH receptor B6 (EPHB6) is a receptor tyrosine kinase that has been shown previously to control catecholamine synthesis in the adrenal gland chromaffin cells (AGCCs) in a testosterone-dependent fashion. EPHB6 also has a role in regulating blood pressure, but several facets of this regulation remain unclear. Using amperometry recordings, we now found that catecholamine secretion by AGCCs is compromised in the absence of EPHB6. AGCCs from male knockout (KO) mice displayed reduced cortical F-actin disassembly, accompanied by decreased catecholamine secretion through exocytosis. This phenotype was not observed in AGCCs from female KO mice, suggesting that testosterone, but not estrogen, contributes to this phenotype. Of note, reverse signaling from EPHB6 to ephrin B1 (EFNB1) and a 7-amino acid-long segment in the EFNB1 intracellular tail were essential for the regulation of catecholamine secretion. Further downstream, the Ras homolog family member A (RHOA) and FYN proto-oncogene Src family tyrosine kinase (FYN)-proto-oncogene c-ABL-microtubule-associated monooxygenase calponin and LIM domain containing 1 (MICAL-1) pathways mediated the signaling from EFNB1 to the defective F-actin disassembly. We discuss the implications of EPHB6's effect on catecholamine exocytosis and secretion for blood pressure regulation.

The Potential Role of Aldosterone-Producing Cell Clusters in Adrenal Disease.

Primary aldosteronism (PA) is the most common cause of secondary hypertension. The hallmark of PA is adrenal production of aldosterone under suppressed renin conditions. PA subtypes include adrenal unilateral and bilateral hyperaldosteronism. Considerable progress has been made in defining the role for somatic gene mutations in aldosterone-producing adenomas (APA) as the primary cause of unilateral PA. This includes the use of next-generation sequencing (NGS) to define recurrent somatic mutations in APA that disrupt calcium signaling, increase aldosterone synthase (CYP11B2) expression, and aldosterone production. The use of CYP11B2 immunohistochemistry on adrenal glands from normal subjects, patients with unilateral and bilateral PA has allowed the identification of CYP11B2-positive cell foci, termed aldosterone-producing cell clusters (APCC). APCC lie beneath the adrenal capsule and like APA, many APCC harbor somatic gene mutations known to increase aldosterone production. These findings suggest that APCC may play a role in pathologic progression of PA. Herein, we provide an update on recent research directed at characterizing APCC and also discuss the unanswered questions related to the role of APCC in PA.

Chromaffin Cells of the Adrenal Medulla: Physiology, Pharmacology, and Disease.

Chromaffin cells (CCs) of the adrenal gland and the sympathetic nervous system produce the catecholamines (epinephrine and norepinephrine; EPI and NE) needed to coordinate the bodily "fight-or-flight" response to fear, stress, exercise, or conflict. EPI and NE release from CCs is regulated both neurogenically by splanchnic nerve fibers and nonneurogenically by hormones (histamine, corticosteroids, angiotensin, and others) and paracrine messengers [EPI, NE, adenosine triphosphate, opioids, γ-aminobutyric acid (GABA), etc.]. The "stimulus-secretion" coupling of CCs is a Ca2+ -dependent process regulated by Ca2+ entry through voltage-gated Ca2+ channels, Ca2+ pumps, and exchangers and intracellular organelles (RE and mitochondria) and diffusible buffers that provide both Ca2+ -homeostasis and Ca2+ -signaling that ultimately trigger exocytosis. CCs also express Na+ and K+ channels and ionotropic (nAChR and GABAA ) and metabotropic receptors (mACh, PACAP, ß-AR, 5-HT, histamine, angiotensin, and others) that make CCs excitable and responsive to autocrine and paracrine stimuli. To maintain high rates of E/NE secretion during stressful conditions, CCs possess a large number of secretory chromaffin granules (CGs) and members of the soluble NSF-attachment receptor complex protein family that allow docking, fusion, and exocytosis of CGs at the cell membrane, and their recycling. This article attempts to provide an updated account of well-established features of the molecular processes regulating CC function, and a survey of the as-yet-unsolved but important questions relating to CC function and dysfunction that have been the subject of intense research over the past 15 years. Examples of CCs as a model system to understand the molecular mechanisms associated with neurodegenerative diseases are also provided. Published 2019. Compr Physiol 9:1443-1502, 2019.

Vesicular Transmitter Content in Chromaffin Cells Can Be Regulated via Extracellular ATP.

The energy carrying molecule adenosine triphosphate (ATP) has been implicated for its role in modulation of chemical signaling for some time. Despite this, the precise effects and mechanisms of action of ATP on secretory cells are not well-known. Here, bovine chromaffin cells have been used as a model system to study the effects of extracellular ATP in combination with the catecholamine transmitter norepinephrine (NE). Both transmitter storage and exocytotic release were quantified using complementary amperometric techniques. Although incubation with NE alone did not cause any changes to either transmitter storage or release, coincubation with NE and ATP resulted in a significant increase that was concentration dependent. To probe the potential mechanisms of action, a slowly hydrolyzable version of ATP, ATP-γ-S, was used either alone or together with NE. The result implicates two different behaviors of ATP acting on both the purinergic autoreceptors and as a source of the energy needed to load chromaffin cell vesicles.

EPHB6 controls catecholamine biosynthesis by up-regulating tyrosine hydroxylase transcription in adrenal gland chromaffin cells.

EPHB6 is a member of the erythropoietin-producing hepatocellular kinase (EPH) family and a receptor tyrosine kinase with a dead kinase domain. It is involved in blood pressure regulation and adrenal gland catecholamine (CAT) secretion, but several facets of EPHB6-mediated CAT regulation are unclear. In this study, using biochemical, quantitative RT-PCR, immunoblotting, and gene microarray assays, we found that EPHB6 up-regulates CAT biosynthesis in adrenal gland chromaffin cells (AGCCs). We observed that epinephrine content is reduced in the AGCCs from male Ephb6-KO mice, caused by decreased expression of tyrosine hydroxylase, the rate-limiting enzyme in CAT biosynthesis. We demonstrate that the signaling pathway from EPHB6 to tyrosine hydroxylase expression in AGCCs involves Rac family small GTPase 1 (RAC1), MAP kinase kinase 7 (MKK7), c-Jun N-terminal kinase (JNK), proto-oncogene c-Jun, activator protein 1 (AP1), and early growth response 1 (EGR1). On the other hand, signaling via extracellular signal-regulated kinase (ERK1/2), p38 mitogen-activated protein kinase, and ELK1, ETS transcription factor (ELK1) was not affected by EPHB6 deletion. We further report that EPHB6's effect on AGCCs was via reverse signaling through ephrin B1 and that EPHB6 acted in concert with the nongenomic effect of testosterone to control CAT biosynthesis. Our findings elucidate the mechanisms by which EPHB6 modulates CAT biosynthesis and identify potential therapeutic targets for diseases, such as hypertension, caused by dysfunctional CAT biosynthesis.

Novel methods in adrenal research: a metabolomics approach.

Metabolic alterations have implications in a spectrum of tissue functions and disease. Aided by novel molecular biological and computational tools, our understanding of physiological and pathological processes underpinning endocrine and endocrine-related disease has significantly expanded over the last decade. Herein, we focus on novel metabolomics-related methodologies in adrenal research: in situ metabolomics by mass spectrometry imaging, steroid metabolomics by gas and liquid chromatography-mass spectrometry, energy pathway metabologenomics by liquid chromatography-mass spectrometry-based metabolomics of Krebs cycle intermediates, and cellular reprogramming to generate functional steroidogenic cells and hence to modulate the steroid metabolome. All four techniques to assess and/or modulate the metabolome in biological systems provide tremendous opportunities to manage neoplastic and non-neoplastic disease of the adrenal glands in the era of precision medicine. In this context, we discuss emerging clinical applications and/or promising metabolic-driven research towards diagnostic, prognostic, predictive and therapeutic biomarkers in tumours arising from the adrenal gland and extra-adrenal paraganglia as well as modern approaches to delineate and reprogram adrenal metabolism.

Paracrine Regulation of Aldosterone Secretion in Physiological and Pathophysiological Conditions.

Aldosterone secretion by the zona glomerulosa of the adrenal cortex is controlled by circulating factors including the renin angiotensin system (RAS) and potassium. Mineralocorticoid production is also regulated through an autocrine/paracrine mechanism by a wide variety of bioactive signals released in the vicinity of adrenocortical cells by chromaffin cells, nerve endings, cells of the immune system, endothelial cells and adipocytes. These regulatory factors include conventional neurotransmitters and neuropeptides. Their physiological role in the control of aldosterone secretion is not fully understood, but it is likely that they participate in the RAS-independent regulation of zona glomerulosa cells. Interestingly, recent observations indicate that autocrine/paracrine processes are involved in the pathophysiology of primary aldosteronism. The intraadrenal regulatory systems observed in aldosterone-producing adenomas (APA), although globally similar to those occurring in the normal adrenal gland, harbor alterations at different levels, which tend to strengthen the potency of paracrine signals to activate aldosterone secretion. Enhancement of paracrine stimulatory tone may participate to APA expansion and aldosterone hypersecretion together with somatic mutations of driver genes which activate the calcium signaling pathway and subsequently aldosterone synthase expression. Intraadrenal regulatory mechanisms represent thus promising pharmacological targets for the treatment of primary aldosteronism.

Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

The adrenal gland is a multiendocrine organ with a steroidogenic mesenchymal cortex and an inner catecholamine-producing medulla of neuroendocrine origin. After embryonic development, this plastic organ undergoes a functional postnatal remodeling. Elucidating these complex processes is pivotal for understanding the early bases of functional endocrine disorders and tumors affecting the mature gland. We developed an in vitro human adrenal cell model derived from fetal adrenal specimens at different gestational ages, consisting of neuroendocrine and cortical components and expressing the zona and functional markers of the original fetal organ. These cortical and neuroendocrine progenitor cells retain in vitro an intrinsic gestational-age-related differentiation and functional program. In vitro these cells spontaneously form 3-dimensional structure organoids with a structure similar to the fetal gland. The organoids show morphofunctional features and adrenal steroidogenic factor, steroid acute regulatory, cytochrome-P450-17A1, dosage-sensitive, sex-reversal, adrenal hypoplasia-critical region on chromosome X protein , NOTCH1, and nephroblastoma overexpressed/cysteine-rich protein 61/connective tissue growth factor/nephroblastoma overexpressed gene-3; stem (BMI1, nestin); and chromaffin (chromogranin A, tyrosine hydroxylase) markers similar to those of the populations of origin. This in vitro human adrenal system represents a unique but preliminar model for investigating the pathophysiological processes underlying physiologic adrenal remodeling and pathologic alterations involved in organ hypo- and hyperplasia and cancer.-Poli, G., Sarchielli, E., Guasti, D., Benvenuti, S., Ballerini, L., Mazzanti, B., Armignacco, R., Cantini, G., Lulli, M., Chortis, V., Arlt, W., Romagnoli, P., Vannelli, G. B., Mannelli, M., Luconi, M. Human fetal adrenal cells retain age-related stem- and endocrine-differentiation potential in culture.

Involvement of DHH and GLI1 in adrenocortical autograft regeneration in rats.

Bilateral adrenalectomy forces the patient to undergo glucocorticoid replacement therapy and bear a lifetime risk of adrenal crisis. Adrenal autotransplantation is considered useful to avoid adrenal crisis and glucocorticoid replacement therapy. However, the basic process of regeneration in adrenal autografts is poorly understood. Here, we investigated the essential regeneration factors in rat adrenocortical autografts, with a focus on the factors involved in adrenal development and steroidogenesis, such as Hh signalling. A remarkable renewal in cell proliferation and increase in Cyp11b1, which encodes 11-beta-hydroxylase, occurred in adrenocortical autografts from 2-3 weeks after autotransplantation. Serum corticosterone and adrenocorticotropic hormone levels were almost recovered to sham level at 4 weeks after autotransplantation. The adrenocortical autografts showed increased Dhh expression at 3 weeks after autotransplantation, but not Shh, which is the only Hh family member to have been reported to be expressed in the adrenal gland. Increased Gli1 expression was also found in the regenerated capsule at 3 weeks after autotransplantation. Dhh and Gli1 might function in concert to regenerate adrenocortical autografts. This is the first report to clearly show Dhh expression and its elevation in the adrenal gland.

Autogenous transplants of adrenal fragments in an animal model.

Introduction Adrenal insufficiency is a typical complication after surgical treatment of adrenal tumors, especially after the removal of both adrenal glands. Human beings are not able to survive without adrenal glands and without proper hormonal substitution. Autotransplantation of a fragment of the adrenal gland may prevent this complication. This can be done by transplanting the entire adrenal glands or its fragment, such as the adrenal cortex cells. In the case of adrenal tumors, the entire adrenal gland can not be transplanted. However, it is possible to transplant cells from the tumor-free part. Succesful adrenal autografts may result in a new treatment of adrenal insufficiency. MATERIALS AND METHODS: Autograft transplantation was performed on 3 groups of Sprague Dawley rats. In the first group, physiological corticosterone concentrations were determined. These animals were not operated. In the second group, both adrenal glands were removed. Corticosterone concentrations were determined after bilateral adrenalectomy. The third group was divided into two parts. In the first subgroup, bilateral adrenalectomy was performed simultaneosly with adrenal transplant into the omentum. In the second subgroup, right adrenalectomy was performed simultaneosly with and adrenal transplant into the omentum followed a month later by left adrenalectomy. During the experiment, corticosterone concentrations were measured at 4 time points. RESULTS: The statistical difference between corticosterone concentrations in rats after two timed adrenalectomies and rats after bilateral adrenalectomy was statistically different, but these results were far from physiological concentrations.

Generation of Adrenal Chromaffin-like Cells from Human Pluripotent Stem Cells.

Adrenomedullary chromaffin cells are catecholamine (CA)-producing cells originating from trunk neural crest (NC) via sympathoadrenal progenitors (SAPs). We generated NC and SAPs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in vitro via BMP2/FGF2 exposure, ascertained by qPCR and immunoexpression of SOX10, ASCL1, TFAP2α, and PHOX2B, and by fluorescence-activated cell sorting selection for p75NTR and GD2, and confirmed their trunk-like HOX gene expression. We showed that continuing BMP4 and curtailing FGF2 in vitro, augmented with corticosteroid mimetic, induced these cells to upregulate the chromaffin cell-specific marker PNMT and other CA synthesis and storage markers, and we demonstrated noradrenaline and adrenaline by Faglu and high-performance liquid chromatography. We showed these human cells' SAP-like property of migration and differentiation into cells expressing chromaffin cell markers by implanting them into avian embryos in vivo and in chorio-allantoic membrane grafts. These cells have the potential for investigating differentiation of human chromaffin cells and for modeling diseases involving this cell type.

Aldosterone-Producing Cell Clusters in Normal and Pathological States.

Primary aldosteronism (PA) significantly increases the risk of cardiovascular complications, and early diagnosis and targeted treatment based on its pathophysiology is warranted. Next-generation sequencing (NGS) has revealed recurrent somatic mutations in aldosterone-driving genes in aldosterone-producing adenoma (APA). By applying CYP11B2 (aldosterone synthase) immunohistochemistry and NGS to adrenal glands from normal subjects and PA patients, we and others have shown that CYP11B2-positive cells make small clusters, termed aldosterone-producing cell clusters (APCC), beneath the adrenal capsule, and that APCC harbor somatic mutations in genes mutated in APA. We have shown that APCC are increased in CT-negative PA adrenals, while others showed potential progression from APCC to micro APA through mutations. These results suggest that APCC are a key factor for understanding the origin of PA, and further investigation on the relation between APCC and PA is highly needed.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.

Metabolic stability of cells for extended metabolomical measurements using NMR. A comparison between lysed and additionally heat inactivated cells.

NMR measurements for metabolic characterization of biological samples like cells, biopsies or plasma, may take several hours for advanced methods. Preanalytical issues, such as sample preparation and stability over the measurement time, may have a high impact on metabolite content, and potentially lead to misinterpretation. The aim of this study was therefore to investigate by 1H HR-MAS NMR the impact of different cell handling preparation protocols on the stability of the cell metabolite content over the measurement time. For this purpose, the metabolite content of fibroblasts and adrenal cells were measured at different time points after lysis and after additional heating. Interestingly the results showed similar metabolite concentrations between lysed and lysed-heated cells at the beginning of the measurement, but increasing differences after some hours of measurement. In lysed cells, metabolism was ongoing, producing metabolite changes over time, contrary to a stable metabolite content of the lysed-heated cells. These results were confirmed in both fibroblasts and adrenal cells. Therefore, in order to minimize metabolite content modifications over the measurement time, it is suggested to use cell lysis in combination with heat inactivation for extended HR-MAS NMR measurements.