Aging alone or combined with obesity increases white adipose tissue inflammatory status in male mice.

White adipose tissue (WAT) has been recognized as a fundamental and crucial organ of interest in research focusing on inflammation during obesity or aging. WAT is also proposed as a significant component of cholecalciferol and 25-hydroxyvitamin D (25(OH)D) storage, which participates in the decrease of 25(OH)D plasma levels reported during aging and obesity. In the present study, we evaluated WAT and plasma cholecalciferol and 25(OH)D content together with inflammatory status to highlight the putative relationship between vitamin D status and inflammatory process during aging alone or combined with obesity. Circulating cholecalciferol and 25(OH)D and the stored quantity of cholecalciferol and 25(OH)D in WAT were quantified in young and old mice fed a control or obesogenic diet. The inflammation was assessed by measuring plasma inflammatory cytokines, mRNA, and microRNAs inflammatory-associated in WAT. The combination of aging and obesity decreased 25(OH)D plasma levels but did not modify circulating inflammatory markers. A cumulative effect of aging and obesity was observed in WAT, with rising mRNA inflammatory cytokines, notably Ccl5 and Tnf. Interestingly, aging and obesity-associated were also characterized by increased inflammatory microRNA expression. The inflammatory parameters in WAT were negatively correlated with the plasma 25(OH)D but positively correlated with the quantity of cholecalciferol and 25(OH)D in WAT. These results support the cumulative effect of obesity and aging in aggravation of WAT inflammation and suggest that accumulation of cholecalciferol and 25(OH)D in WAT could constitute a mechanism to counteract WAT inflammation during aging and obesity.

Tomato genotype but not crop water deficit matters for tomato health benefits in diet-induced obesity of C57BL/6JRj male mice.

Several studies have linked the intake of lycopene and/or tomato products with improved metabolic health under obesogenic regime. The aim was to evaluate the differential impact of supplementations with several tomato genotypes differing in carotenoid content and subjected to different irrigation levels on obesity-associated disorders in mice. In this study, 80 male C57BL/6JRj mice were assigned into 8 groups to receive: control diet, high fat diet, high fat diet supplemented at 5 % w/w with 4 tomato powders originating from different tomato genotypes cultivated under control irrigation: H1311, M82, IL6-2, IL12-4. Among the 4 genotypes, 2 were also cultivated under deficit irrigation, reducing the irrigation water supply by 50 % from anthesis to fruit harvest. In controlled irrigation treatment, all genotypes significantly improved fasting glycemia and three of them significantly lowered liver lipids content after 12 weeks of supplementation. In addition, IL6-2 genotype, rich in ß-carotene, significantly limited animal adiposity, body weight gain and improved glucose homeostasis as highlighted in glucose and insulin tolerance tests. No consistent beneficial or detrimental impact of deficit irrigation to tomato promoting health benefits was found. These findings imply that the choice of tomato genotype can significantly alter the composition of fruit carotenoids and phytochemicals, thereby influencing the anti-obesogenic effects of the fruit. In contrast, deficit irrigation appears to have an overall insignificant impact on enhancing the health benefits of tomato powder in this context, particularly when compared to the genotype-related variations in carotenoid content.

The supplementation of female dogs with live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 acts as gut stabilizer at whelping and modulates immunometabolic phenotype of the puppies.

Time around parturition is a stressful period for both bitches and their puppies. The use of probiotics has been proposed, e.g., in pigs, to improve health status of sows, their reproductive performances and in turn, the health and performance of their progeny. The objective of the present study was to evaluate the impact, on both dams and puppies, of a supplementation of bitches with the live yeast Saccharomyces cerevisiae var. boulardii CNCM I-1079 (SB-1079) during the second part of the gestation and the lactation period. A total of 36 bitches of medium and large-sized breeds were enrolled. They were divided into two groups, one of which received 1.3 × 109 colony forming units of live yeast per day. At dam's level, SB-1079 yeast shaped a different microbiota structure between the two groups just after whelping, impacted alpha diversity and some plasma metabolites related to energy metabolism. Regarding reproductive performances, SB-1079 improved gross energy of the colostrum (1.4 vs. 1.2 kcal of ME/g) as well as the concentration of protein in milk at Day 7 after parturition (10.4 vs. 7.6%). SB-1079 also reduced the odds of having low birth weight in the litter. At puppy's level, a modulation of immunometabolic phenotype is suggested by the observation of increased growth rates during the early pediatric period (i.e., between 21 and 56 days of life, 225 vs. 190%) and a decrease of the IL-8:IL-10 ratio after vaccination against rabies (4.2 vs. 16.9). Our findings suggest that SB-1079 supplementation during gestation and lactation has the potential to enhance health of bitches and in turn health of puppies through maternal programming.

Vitamin D metabolism is altered during aging alone or combined with obesity in male mice.

Aging and obesity are associated with a decrease in plasma 25-hydroxyvitamin D (25(OH)D) levels. In the context of a growing aging population and the rising incidence of obesity, we hypothesized that aging process, either independently or in combination with obesity, could influence vitamin D (VD) metabolism, consequently resulting in the reduced 25(OH)D plasma concentrations. C57BL/6JRJ young (6 months) and old (23 months) mice fed with control (CD) or high fat diet (HF) were compared. Plasma and adipose concentration of cholecalciferol and 25(OH)D and mRNA expression of genes coding for the main VD actors were analyzed. Aging was associated with a decrease in plasma 25(OH)D levels, whereas combined effect of obesity and aging did not generate a cumulative effect on plasma 25(OH)D levels. The mRNA expression of Cyp27a1, Cyp3a11, and Cyp2j6 were decreased in the liver during aging. Together, these regulations could explain the reduced 25-hydroxylation. Interestingly, the lack of cumulative reduction of 25(OH)D in aged and obese mice could be related to the strong induction of Cyp2j6. In kidneys, a complex modulation of Cyp27b1 and Cyp24a1 could contribute to the reduced 25-hydroxylation in the liver. In white adipose tissue, an induction of Cyp2r1 was observed during aging and obesity, together with an increase of 25(OH)D quantity, suggesting an exacerbated storage that may participated to the reduced plasma 25(OH)D levels. These findings support the notion that aging alone or combined with obesity, induces regulation of VD metabolism in the organs, beyond the classical reduction of epidermal VD precursor, which may contribute to the decrease in 25(OH)D levels.

Vitamin D Modulates Lipid Composition of Adipocyte-Derived Extracellular Vesicles Under Inflammatory Conditions.

SCOPE: Adipocyte-derived extracellular vesicles (AdEVs) convey lipids that can play a role in the energy homeostasis. Vitamin D (VD) has been shown to limit the metabolic inflammation as it decreases inflammatory markers expression in adipose tissue (AT). However, VD effect on adipocytes-derived EVs has never been investigated. METHODS AND RESULTS: Thus, the aim of this study is to evaluate the AdEVs lipid composition by LC-MS/MS approach in 3T3-L1 cells treated with VD or/and pro-inflammatory factor (tumor necrosis factor α [TNFα]). Among all lipid species, four are highlighted (glycerolipids, phospholipids, lysophospholipids, and sphingolipids) with a differential content between small (sEVs) and large EVs (lEVs). This study also observes that VD alone modulates EV lipid species involved in membrane fluidity and in the budding of membrane. EVs treated with VD under inflammatory conditions have different lipid profiles than the control group, which is more pronounced in lEVs. Indeed, 25 lipid species are significantly modulated in lEVs, compared with only seven lipid species in sEVs. CONCLUSIONS: This study concludes that VD, alone or under inflammatory conditions, is associated with specific lipidomic signature of sEVs and lEVs. These observations reinforce current knowledge on the anti-inflammatory effect of VD.

Maternal vitamin D deficiency and brain functions: a never-ending story.

A large number of observational studies have highlighted the prevalence rates of vitamin D insufficiency and deficiency in many populations including pregnant women. Vitamin D is well known to have a crucial role in differentiation and proliferation, as well as neurotrophic and neuroprotective actions in the brain. It has been observed that this micronutrient can modulate neurotransmission and synaptic plasticity. Recent results from animal and epidemiological studies indicated that maternal vitamin D deficiency is associated with a wide range of neurobiological diseases including autism, schizophrenia, depression, multiple sclerosis and developmental defects. The aim of this review is to summarize the current state of knowledge on the effect of maternal vitamin D deficiency on brain functions and development.

Dielectric and Biological Characterization of Liver Tissue in a High-Fat Diet Mouse Model.

Hepatic steatosis may be caused by type 2 diabetes or obesity and is one of the origins of chronic liver disease. A non-invasive technique based on microwave propagation can be a good solution to monitor hepatic tissue pathologies. The present work is devoted to the dielectric permittivity measurements in healthy and fatty liver in the microwave range. A mouse model following normal and high sugar/glucose (HFS) diets was used. We demonstrated the change in the triglyceride and glucose concentration in the hepatic tissue of HFS diet mice. The difference in the dielectric permittivity of healthy and fatty liver was observed in the range from 100 MHz to 2 GHz. The dielectric permittivity was found to be 42 in the healthy tissue and 31 in the fatty liver tissue at 1 GHz. The obtained results demonstrate that dielectric permittivity can be a sensitive tool to distinguish between healthy and fatty hepatic tissue.

Proposal of a bioinformatics approach to predict molecular mechanisms involved in inflammatory response: case of ATRA and 1,25(OH)2D in adipocytes.

Several inflammatory markers such as cytokines, chemokines, and microRNAs (miRNAs) are well known to be induced during obesity and are strongly linked to their comorbidities. Among many others factors, the micronutrient status is suspected to reduce obesity-associated inflammation via blunting inflammatory signalling pathways. This is notably the case for active forms of vitamin A (all-trans retinoic acid ATRA) and vitamin D (1,25(OH)2D) as previously shown. In the present study, we aimed to implement a new bioinformatics approach to unveil commonly regulated signalling pathways through a combination of gene and miRNA expression sets impacted by ATRA and 1,25(OH)2D in adipocytes. In a first set of experiments, we focused only our attention on ATRA and demonstrated that it reduced LPS-mediated miRNA expression (miR-146a, miR-150, and miR-155) in mouse adipose tissue, in adipocyte cultures, and in adipocyte-derived vesicles. This result was confirmed in TNFα-induced miRNA in human adipocytes. Then, bioinformatic analysis highlighted that both ATRA and 1,25(OH)2D-regulated genes and miRNA converge to the canonical 'nuclear factor Kappa B (NF-κB) signalling pathway.' Altogether, these results showed that ATRA has anti-inflammatory effects on miRNA expression. In addition, the proposed bioinformatic model converges to NF-κB signalling pathway that has been previously demonstrated to be regulated by ATRA and 1,25(OH)2D, thus confirming the interest of such approach.

Vitamin D and Obesity/Adiposity-A Brief Overview of Recent Studies.

Observational studies classically find an inverse relationship between human plasma 25-hydroxyvitamin D concentration and obesity. However, interventional and genetic studies have failed to provide clear conclusions on the causal effect of vitamin D on obesity/adiposity. Likewise, vitamin D supplementation in obese rodents has mostly failed to improve obesity parameters, whereas several lines of evidence in rodents and prospective studies in humans point to a preventive effect of vitamin D supplementation on the onset of obesity. Recent studies investigating the impact of maternal vitamin D deficiency in women and in rodent models on adipose tissue biology programming in offspring further support a preventive metabolically driven effect of vitamin D sufficiency. The aim of this review is to summarize the state of the knowledge on the relationship between vitamin D and obesity/adiposity in humans and in rodents and the impact of maternal vitamin D deficiency on the metabolic trajectory of the offspring.

Recent insights into vitamin D, adipocyte, and adipose tissue biology.

Several studies bring strong evidence for an active role of vitamin D and its metabolites in physiological adipocyte and adipose tissue processes in adulthood. This role includes effects of vitamin D on key adipose tissue and adipocyte biology parameters, including adipogenesis, energy metabolism, and inflammation. Interestingly, recent data also point to a role of maternal vitamin D deficiency in adipocyte and adipose tissue metabolic programming in offspring. This review summarizes the current state of knowledge on the biological effect of vitamin D on adipocyte/adipose tissue physiology.

Leptin Modulates the Expression of miRNAs-Targeting POMC mRNA by the JAK2-STAT3 and PI3K-Akt Pathways.

The central control of energy balance involves a strongly regulated neuronal network within the hypothalamus and the brainstem. In these structures, pro-opiomelanocortin (POMC) neurons are known to decrease food intake and to increase energy expenditure. Thus, leptin, a peripheral signal that relays information regarding body fat content, modulates the activity of POMC neurons. MicroRNAs (miRNAs) are short non-coding RNAs of 22-26 nucleotides that post-transcriptionally interfere with target gene expression by binding to their mRNAs. It has been demonstrated that leptin is able to modulate the expression of miRNAs (miR-383, miR-384-3p, and miR-488) that potentially target POMC mRNA. However, no study has identified the transduction pathways involved in this effect of leptin on miRNA expression. In addition, miRNAs targeting POMC mRNAs are not clearly identified. In this work, using in vitro models, we have identified and confirmed that miR-383, miR-384-3p, and miR-488 physically binds to the 3' untranslated (3'UTR) regions of POMC mRNA. Importantly, we show that leptin inhibits these miRNAs expression by different transduction pathways. Taken together, these results allowed us to highlight the miRNA involvement in the regulation of POMC expression downstream of the leptin signaling and satiety signal integration.

Salivary MicroRNA in Pancreatic Cancer Patients.

BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer death in Western countries, with the lowest 1-year survival rate among commonly diagnosed cancers. Reliable biomarkers for pancreatic cancer diagnosis are lacking and are urgently needed to allow for curative surgery. As microRNA (miRNA) recently emerged as candidate biomarkers for this disease, we explored in the present pilot study the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery, precancerous lesions, inflammatory disease or cancer-free patients as a potential early diagnostic tool. METHODS: Whole saliva samples from patients with pancreatic cancer (n = 7), pancreatitis (n = 4), IPMN (n = 2), or healthy controls (n = 4) were obtained during endoscopic examination. After total RNA isolation, expression of 94 candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm. Human-derived pancreatic cancer cells were xenografted in athymic mice as an experimental model of pancreatic cancer. RESULTS: We identified hsa-miR-21, hsa-miR-23a, hsa-miR-23b and miR-29c as being significantly upregulated in saliva of pancreatic cancer patients compared to control, showing sensitivities of 71.4%, 85.7%, 85,7% and 57%, respectively and excellent specificity (100%). Interestingly, hsa-miR-23a and hsa-miR23b are overexpressed in the saliva of patients with pancreatic cancer precursor lesions. We found that hsa-miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis as compared to the control group, with sensitivity of 100% and 75%, and specificity of 100% and 80%, respectively. Last hsa-miR-216 was upregulated in cancer patients as compared to patients diagnosed with pancreatitis, with sensitivity of 50% and specificity of 100%. In experimental models of PDAC, salivary microRNA detection precedes systemic detection of cancer cells markers. CONCLUSIONS: Our novel findings indicate that salivary miRNA are discriminatory in pancreatic cancer patients that are not eligible for surgery. In addition, we demonstrate in experimental models that salivary miRNA detection precedes systemic detection of cancer cells markers. This study stems for the use of salivary miRNA as biomarker for the early diagnosis of patients with unresectable pancreatic cancer.

3D-fection: cell transfection within 3D scaffolds and hydrogels.

BACKGROUND: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.

Targeting miR-21 for the therapy of pancreatic cancer.

Despite tremendous efforts worldwide from clinicians and cancer scientists, pancreatic ductal adenocarcinoma (PDA) remains a deadly disease for which no cure is available. Recently, microRNAs (miRNAs) have emerged as key actors in carcinogenesis and we demonstrated that microRNA-21 (miR-21), oncomiR is expressed early during PDA. In the present study, we asked whether targeting miR-21 in human PDA-derived cell lines using lentiviral vectors (LVs) may impede tumor growth. We demonstrated that LVs-transduced human PDA efficiently downregulated miR-21 expression, both in vitro and in vivo. Consequently, cell proliferation was strongly inhibited and PDA-derived cell lines died by apoptosis through the mitochondrial pathway. In vivo, miR-21 depletion stopped the progression of a very aggressive model of PDA, to induce cell death by apoptosis; furthermore, combining miR-21 targeting and chemotherapeutic treatment provoked tumor regression. We demonstrate herein for the first time that targeting oncogenic miRNA strongly inhibit pancreatic cancer tumor growth both in vitro and in vivo. Because miR-21 is overexpressed in most human tumors; therapeutic delivery of miR-21 antagonists may still be beneficial for a large number of cancers for which no cure is available.

The rescue of miR-148a expression in pancreatic cancer: an inappropriate therapeutic tool.

MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.

Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer.

BACKGROUND: The multicellular tumor spheroid (MCTS) is an in vitro model associating malignant-cell microenvironment and 3D organization as currently observed in avascular tumors. METHODS: In order to evaluate the relevance of this model for pre-clinical studies of drug combinations, we analyzed the effect of gemcitabine alone and in combination with the CHIR-124 CHK1 inhibitor in a Capan-2 pancreatic cell MCTS model. RESULTS: Compared to monolayer cultures, Capan-2 MCTS exhibited resistance to gemcitabine cytotoxic effect. This resistance was amplified in EGF-deprived quiescent spheroid suggesting that quiescent cells are playing a role in gemcitabine multicellular resistance. After a prolonged incubation with gemcitabine, DNA damages and massive apoptosis were observed throughout the spheroid while cell cycle arrest was restricted to the outer cell layer, indicating that gemcitabine-induced apoptosis is directly correlated to DNA damages. The combination of gemcitabine and CHIR-124 in this MCTS model, enhanced the sensitivity to the gemcitabine antiproliferative effect in correlation with an increase in DNA damage and apoptosis. CONCLUSIONS: These results demonstrate that our pancreatic MCTS model, suitable for both screening and imaging analysis, is a valuable advanced tool for evaluating the spatio-temporal effect of drugs and drug combinations in a chemoresistant and microenvironment-depending tumor model.

Magnetic nanoparticles enhance adenovirus transduction in vitro and in vivo.

PURPOSE: Adenoviruses are among the most powerful gene delivery systems. Even if they present low potential for oncogenesis, there is still a need for minimizing widespread delivery to avoid deleterious reactions. In this study, we investigated Magnetofection efficiency to concentrate and guide vectors for an improved targeted delivery. METHOD: Magnetic nanoparticles formulations were complexed to a replication defective Adenovirus and were used to transduce cells both in vitro and in vivo. A new integrated magnetic procedure for cell sorting and genetic modification (i-MICST) was also investigated. RESULTS: Magnetic nanoparticles enhanced viral transduction efficiency and protein expression in a dose-dependent manner. They accelerated the transduction kinetics and allowed non-permissive cells infection. Magnetofection greatly improved adenovirus-mediated DNA delivery in vivo and provided a magnetic targeting. The i-MICST results established the efficiency of magnetic nanoparticles assisted viral transduction within cell sorting columns. CONCLUSION: The results showed that the combination of Magnetofection and Adenoviruses represents a promising strategy for gene therapy. Recently, a new integrated method to combine clinically approved magnetic cell isolation devices and genetic modification was developed. In this study, we validated that magnetic cell separation and adenoviral transduction can be accomplished in one reliable integrated and safe system.

Genetic and epigenetic alterations in pancreatic carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Despite significant progresses in the last decades, the origin of this cancer remains unclear and no efficient therapy exists. PDAC does not arise de novo: three remarkable different types of pancreatic lesions can evolve towards pancreatic cancer. These precursor lesions include: Pancreatic intraepithelial neoplasia (PanIN) that are microscopic lesions of the pancreas, Intraductal Papillary Mucinous Neoplasms (IPMN) and Mucinous Cystic Neoplasms (MCN) that are both macroscopic lesions. However, the cellular origin of these lesions is still a matter of debate. Classically, neoplasm initiation or progression is driven by several genetic and epigenetic alterations. The aim of this review is to assemble the current information on genetic mutations and epigenetic disorders that affect genes during pancreatic carcinogenesis. We will further discuss the interest of the genetic and epigenetic alterations for the diagnosis and prognosis of PDAC. Large genetic alterations (chromosomal deletion/amplification) and single point mutations are well described for carcinogenesis inducers. Mutations classically occur within key regions of the genome. Consequences are various and include activation of mitogenic pathways or silencing of apoptotic processes. Alterations of K-RAS, P16 and DPC4 genes are frequently observed in PDAC samples and have been described to arise gradually during carcinogenesis. DNA methylation is an epigenetic process involved in imprinting and X chromosome inactivation. Alteration of DNA methylation patterns leads to deregulation of gene expression, in the absence of mutation. Both genetic and epigenetic events influence genes and non-coding RNA expression, with dramatic effects on proliferation, survival and invasion. Besides improvement in our fundamental understanding of PDAC development, highlighting the molecular alterations that occur in pancreatic carcinogenesis could provide new clinical tools for early diagnosis of PDAC and the molecular basis for the development of new effective therapies.

Isolation of neural crest derived chromaffin progenitors from adult adrenal medulla.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

Dehydroepiandrosterone induces a neuroendocrine phenotype in nerve growth factor-stimulated chromaffin pheochromocytoma PC12 cells.

The adrenal androgen dehydroepiandrosterone (DHEA) is produced in the inner zone of the adrenal cortex, which is in direct contact to adrenal medullary cells. Due to their close anatomical proximity and tightly intermingled cell borders, a direct interaction of adrenal cortex and medulla has been postulated. In humans congenital adrenal hyperplasia due to 21-hydroxylase deficiency results in androgen excess accompanied by severe adrenomedullary dysplasia and chromaffin cell dysfunction. Therefore, to define the mechanisms of DHEA action on chromaffin cell function, we investigated its effect on cell survival and differentiation processes on a molecular level in the chromaffin cell line PC12. DHEA lessened the positive effect of NGF on cell survival and neuronal differentiation. Nerve growth factor (NGF)-mediated induction of a neuronal phenotype was inhibited by DHEA as indicated by reduced neurite outgrowth and decreased expression of neuronal marker proteins such as synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein-2. We examined whether DHEA may stimulate the cells toward a neuroendocrine phenotype. DHEA significantly elevated catecholamine release from unstimulated PC12 cells in the presence but not absence of NGF. Accordingly, DHEA enhanced the expression of the neuroendocrine marker protein chromogranin A. Next, we explored the possible molecular mechanisms of DHEA and NGF interaction. We demonstrate that NGF-induced ERK1/2 phosphorylation was reduced by DHEA. In summary, our data show that DHEA influences cell survival and differentiation processes in PC12 cells, possibly by interacting with the ERK1/2 MAPK pathway. DHEA drives NGF-stimulated cells toward a neuroendocrine phenotype, suggesting that the interaction of intraadrenal steroids and growth factors is required for the maintenance of an intact adrenal medulla.