Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests.

Project description:In order to analyze the transcriptome characteristics of aldosterone producing cell clusters (APCC) we compared transcript abundances of APCC, zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors. The frozen adrenal glands in O.C.T. compound were cut into 7um sections, and every 10-th section immunostained for aldosterone synthase (CYP11B2). The remaining sections were stained with cresyl violet and used for laser-capture microdissection of tissue to use in the array assays. APCC and ZG samples were captured from CYP11B2 positive regions based on the CYP11B2-stained sections. ZF and ZR were captured from lipid-rich cells in the middle layer and compact cells outside of the medulla, respectively. RNA was isolated using PicoPure RNA isolation kits (Molecular Devices, Sunnyvale, CA). 1-10 ng total RNA was reverse-transcribed and amplified with the Ovation Pico WTA System V2 (NuGEN Technologies, San Carlos, CA). cDNA was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and biotin-labeled using Encore Biotin Module (NuGEN Technologies), followed by hybridization to GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA). Expression values were calculated using the robust multi-array average method (RMA). This resulted in base-2 log-transformed data for each of the 4 tissues from each of the 4 people. In addition to the raw and processed data we also supply a supplementary Excel file holding the data and some statistical analysis, which has features to make simple graphs, and holds probe-set annotation that we used at that time (users may wish to obtain new annotation though). We fit two-way ANOVA models with terms for 4 tissues and 4 people, and compared each probe-set between every pair of tissues using F-tests for pairwise contrasts. We modeled people effects since they were not negligible. The supplement shows how to calculate the tests. Aldosterone producing cell clusters (APCC), zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR), from adrenal glands obtained from 4 kidney transplantation donors, were individually assayed on 16 Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays. Expression values were estimated with the Robust Multi-Arry average (RMA) algorithm, which resulted in log-2 transformed values for each of 54675 probe sets.

Project description:BackgroundThe number of patients with prolonged critical illness (PCI) has been increasing in many countries, and the adrenal gland plays an important role in maintaining homeostasis during PCI. Chronic disease burden is reportedly associated with shorter telomere lengths in human tissues. Telomere shortening in human somatic cells is largely dependent on cell divisions, and critically short telomeres lead to cellular dysfunction and aging. However, the association between PCI and telomere lengths in human adrenal cells is poorly understood. In this study, we investigated this association to assess whether the burden of PCI could accelerate the aging process in adrenal cells.MethodsAdrenocortical tissues from patients who died after PCI usually show a diffuse pattern of intracellular cholesterol ester depletion (i.e., lipid depletion). This study examined near-normal adrenal glands obtained from autopsied patients who died suddenly (control group) and lipid-depleted adrenal glands obtained from autopsied patients who died after PCI (PCI group). The control group included 7 men aged 80 to 94 years (mean age: 85.3 years) and 7 women aged 84 to 94 years (mean age: 87.7 years). The PCI group included 10 men aged 71 to 88 years (mean age: 78.8 years) and 8 women aged 77 to 95 years (mean age: 85.6 years). By using quantitative fluorescence in situ hybridization, relative telomere lengths (RTLs) were determined in the parenchymal cells of the three adrenocortical zones (zona glomerulosa, zona fasciculata, and zona reticularis [ZR]) and in the chromaffin cells of the medulla. The number of adrenal parenchymal cells was determined by immunohistochemistry and digital image analysis.ResultsRTLs in ZR cells were significantly shorter in the PCI group than in the control group for both men and women (P = 0.0001 for men and P = 0.0012 for women). However, RTLs in the remaining three types of adrenal cells did not differ between the control and PCI groups for both men and women. The number of ZR cells was higher in the PCI group than in the control group for both men and women (P < 0.0001 for both men and women). The proportion of the number of ZR cells to the total number of adrenocortical parenchymal cells was also higher in the PCI group than in the control group (P < 0.0001 for both men and women). The Ki-67 proliferation index in ZR cells was higher in the PCI group than in the control group (P = 0.0039 for men and P = 0.0063 for women).ConclusionsThis study demonstrated ZR cell-specific telomere shortening in patients with adrenal lipid depletion who died after PCI. Our results suggest that the reactive proliferation of ZR cells accelerates the telomere shortening and aging process in ZR cells in these patients. The results of our study may contribute to the understanding of adrenal aging during PCI.

Project description:Adropin is a multifunctional peptide hormone encoded by the ENHO (energy homeostasis associated) gene. It plays a role in mechanisms related to increased adiposity, insulin resistance, as well as glucose, and lipid metabolism. The low adropin levels are strongly associated with obesity independent insulin resistance. On the other hand, overexpression or exogenous administration of adropin improves glucose homeostasis. The multidirectional, adropin-related effects associated with the regulation of metabolism in humans also appear to be attributable to the effects of this peptide on the activity of various elements of the endocrine system including adrenal cortex. Therefore, the main purpose of the present study was to investigate the effect of adropin on proliferation and secretory activity in the human HAC15 adrenal carcinoma cell line. In this study, we obtained several highly interesting findings. First, GPR19, the main candidate sensitizer of adrenocortical cells to adropin, was expressed in HAC15 cells. Moreover, GPR19 expression was relatively stable and not regulated by ACTH, forskolin, or adropin itself. Our findings also suggest that adropin has the capacity to decrease expression levels of steroidogenic genes such as steroidogenic acute regulatory protein (StAR) and CYP11A1, which then led to a statistically significant inhibition in cortisol and aldosterone biosynthesis and secretion. Based on whole transcriptome study and research involving transforming growth factor (TGF)-β type I receptor kinase inhibitor we demonstrated that attenuation of steroidogenesis caused by adropin is mediated by the TGF-β signaling pathway likely to act through transactivation mechanism. We found that HAC15 cells treated with adropin presented significantly higher proliferation levels than untreated cells. Using specific intracellular inhibitors, we showed that adropin stimulate proliferation via ERK1/2 and AKT dependent signaling pathways. We have also demonstrated that expression of GPR19 is elevated in adrenocortical carcinoma in relation to normal adrenal glands. High level of GPR19 expression in adrenocortical carcinoma may constitute a negative prognostic factor of disease progression.

Dataset Information

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

Publications

Development of Human Adrenocortical Adenoma (HAA1) Cell Line from Zona Reticularis.

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