Julie Brossaud

Retinoids and glucocorticoids target common genes in hippocampal HT22 cells.

Vitamin A metabolite retinoic acid (RA) plays a major role in the aging adult brain plasticity. Conversely, chronic excess of glucocorticoids (GC) elicits some deleterious effects in the hippocampus. We questioned here the involvement of RA and GC in the expression of target proteins in hippocampal neurons. We investigated proteins involved either in the signaling pathways [RA receptor β (RARβ) and glucocorticoid receptor (GR)] or in neuron differentiation and plasticity [tissue transglutaminase 2 (tTG) and brain‐derived neurotrophic factor (BDNF)] in a hippocampal cell line, HT22. We applied RA and/or dexamethasone (Dex) as activators of the pathways and investigated mRNA and protein expression of their receptors and of tTG and BDNF as well as tTG activity and BDNF secretion. Our results confirm the involvement of RA‐ and GC‐dependent pathways and their interaction in our neuronal cell model. First, both pathways regulate the transcription and expression of own and reciprocal receptors: RA and Dex increased RARβ and decreased GR expressions. Second, Dex reduces the expression of tTG when associated with RA despite stimulating its expression when used alone. Importantly, when they are combined, RA counteracts the deleterious effect of glucocorticoids on BDNF regulation and thus may improve neuronal plasticity under stress conditions. In conclusion, GC and RA both interact through regulations of the two receptors, RARβ and GR. Furthermore, they both act, synergistically or oppositely, on other target proteins critical for neuronal plasticity, tTG and BDNF.

Urinary glucocorticoid metabolites: biomarkers to classify adrenal incidentalomas?

Total urinary cortisol metabolites represent cortisol production and metabolism. We hypothesized that to assay metabolites could add some information to the one provided by a sole cortisol assay.

Aldosterone determination: Comparison of a RIA assay and a CLIA assay

OBJECTIVE To extend knowledge about the clinical performances of a new chemiluminescent immunoassay (CLIA) for aldosterone set up in available analysers. DESIGN AND PATIENTS We compared the results of a RIA assay to those of a CLIA assay in 198 serum and 80 urine samples from patients in endocrine and hypertension departments. Furthermore, for serum samples the concordance of results for postural tests was analysed. RESULTS RIA and CLIA aldosterone serum concentration was linearly correlated with a slope of 0.988 and an intercept of 70.4pmol/L. The variations of aldosterone serum concentration obtained with the two assays during postural tests were very consistent. There was no significant difference of aldosterone concentrations after thawing with the CLIA assay. RIA and CLIA aldosterone urine concentrations were linearly correlated with a slope of 0.787 and an intercept of -2.64nmol/L. Omitting the preservative boric acid from urine samples did not modify aldosterone concentration at least up to 48h after collection. CONCLUSION The RIA and CLIA assays were well correlated for the most useful serum samples. It is well suited to circumvent isotopic assays with the throughput of available analysers.

Use of an automated ACTH assay for the diagnosis of pituitary and adrenal-related diseases ☆

OBJECTIVE To evaluate Liaison Diasorins automated ACTH assay. DESIGN We investigated the limit of quantification (LOQ) and simulated the usage of the analyzer using our ACTH results database. RESULTS The LOQ was close to the cut-off determining Cushings syndrome ACTH dependency. 25% concentrations of normal subjects were lower than the LOQ. Although biased, the results were concordant with those of an IRMA assay. CONCLUSION This assay is not sensitive enough to diagnose ACTH-independent Cushings syndrome.

Nocturnal activity of 11β-hydroxy steroid dehydrogenase type 1 is increased in type 1 diabetic children

AIM The objective of this study was to investigate low-grade inflammation in children with type 1 diabetes (T1D) and its association with cortisol levels as well as its bioavailability through 11β-hydroxy steroid dehydrogenase type 1 (11β-HSD1) activity. METHODS Children with T1D (n=45) and their non-diabetic siblings (n=28) participated in the study. Interleukin-6 (IL-6) and high-sensitivity C-reactive protein (CRPhs) were measured between 1400 and 1800h. Glucocorticoid metabolites were measured in the first morning urine on clinic day and 11β-HSD1 activity was estimated by tetrahydrocortisol/tetrahydrocortisone (THF/THE) ratio. RESULTS Diabetic patients presented with an increased THF/THE ratio compared with controls (median: 0.68 [range: 0.45-1.18] vs 0.45 [0.27-0.98], respectively; P<10(-3)). There was no difference between diabetic patients and controls for IL-6 (0.6ng/mL [0.6-6.8] vs 0.6 [0.6-2.2], respectively; P=0.43) and CRPhs (0.4mg/L [0-7.4] vs 0.3 [0-8.2]; P=0.26, respectively). When adjusted for age, gender and BMI, the THF/THE ratio was significantly associated with CRPhs (β=0.32, P=0.02) in diabetic patients, but not in controls. CONCLUSION Low-grade inflammation assessed by plasma CRPhs and IL-6 concentrations was not detectable in our cohort of T1D children. Nocturnal 11β-HSD1 activity was increased and associated with plasma CRPhs concentration in diabetic patients. These results may be explained by either a direct or inflammation-mediated effect of the relative hepatic lack of insulin due to subcutaneous insulin therapy.

Different methods to estimate serum free cortisol: a comparison during cortisol tetracosactide testing.

Abstract Background: Serum cortisol is routinely quantified by immunoassays. In intensive care units serum free cortisol (FC) determination has been described as a better indicator of survival than total cortisol (TC). To estimate FC different methods are available including saliva sampling. We compared five methods to estimate FC, before and after an ACTH stimulating test in patients suspected of adrenal insufficiency. Method: Serum and saliva was collected from 130 patients from the Endocrine Department of a university hospital before and after tetracosactide injection for TC determination. FC was estimated: after serum ultrafiltration, quadratic (Coolens’) or cubic (Dorin’s) equations, using TC/cortisol-binding globulin concentrations ratio or using cortisol concentration determination in saliva. Results: FC concentrations obtained by different techniques were significantly correlated and Passing-Bablok regressions showed no deviation from linearity between salFC and filtFC or quadFC. Using the routine assumption that the patients were correctly diagnosed using a post-tetracosactide TC threshold of 550 nmol/L the FC methods generating the best ROC curves were salFC and filtFC or cubFC 30 min after tetracosactide injection. Conclusions: FC concentrations obtained by different techniques are significantly but not similarly correlated with TC. As, salFC and filtFC are more convenient to perform than methods involving CBG assays and are better correlated to TC during tetracosactide tests they may be preferred as FC surrogate assays.

Retinoic acid increases glucocorticoid receptor phosphorylation via cyclin-dependent kinase 5

Abstract Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long‐term glucocorticoid exposure. A 4‐day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)‐induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter‐acted the effect of RA on GR sensitive target proteins such as BDNF or tissue‐transglutaminase. These data help understanding the interaction between RA‐ and glucocorticoid‐signalling pathways, both of which have strong influences on the adult brain. HighlightsWe investigated the retinoic acid and glucocorticoid pathways in the brain.We hypothesised that retinoic acid modulate the glucocorticoid receptor activity.Retinoic acid potentiated glucocorticoid receptor phosphorylation in the HT22 cells.This phosphorylation is dependent on cyclin‐dependent kinase 5 and p35/25 ratio.

Phasing-in plasma metanephrines determination

OBJECTIVES We set up plasma normetanephrine (pNMA) and metanephrine (pMA) assays as they demonstrated their usefulness for diagnosing phaeochromocytomas. Our scope is to describe some practical laboratory aspects and the clinical relevance of these assays in our endocrinological or cardiological departments. METHODS We retrospectively reviewed the results of MA from a population of in- and outpatients over a 7-year period. Subjects (n=2536) from endocrinological or cardiological departments were investigated (66 phaeochromocytomas). Urinary NMA (uNMA) and pNMA, and urinary MA (uMA) and pMA were assayed by HPLC with electrochemical detection. RESULTS pNMA and pMA assays are now more frequently requested than uNMA and uMA. This changed our laboratory work load with improved delivery, sensitivity and reliability of plasma assays as well as reduced apparatus maintenance time. The pNMA and pMA upper reference limits (URLs) of subjects with no phaeochromocytoma were 1040 and 430 pmol/l respectively. Sensitivity and specificity based on receiver operating characteristic curves optimal points were 83 and 93% for pNMA at 972 pmol/l and 67 and 98% for pMA at 638 pmol/l. Sensitivity and specificity of paired tests of pMA (positive test: at least one analyte above its URLs) were 100 and 91% respectively. CONCLUSION The very low concentration of analytes requires a sustained very good apparatus analytical sensitivity. This can be obtained in an up-to-date laboratory. In terms of clinical performances, assays in plasma or urine are equivalent. Depending on local preferences, populations, strategies or departments, requests for one or the other assay may sustain the need for specifically defined reference ranges.

Inflammatory, endocrine and metabolic correlates of fatigue in obese children

Alterations in endocrine functions and low-grade systemic inflammation represent fundamental characteristics of obesity. These biological systems have been repeatedly linked to fatigue symptoms. The aim of the study was to assess the relationship between fatigue dimensions and metabolic/inflammatory markers in a sample of non-diabetic obese children. The possibility that inflammation-induced alterations in tryptophan metabolism relates to specific dimensions of fatigue was also investigated in a subsample of patients. The study was conducted in 41 obese children, median aged 12 [9-15] years, recruited in a pediatric tertiary center. Three dimensions of fatigue (e.g., general fatigue, sleep/rest, cognitive fatigue) were assessed using the Pediatric Quality of Life Inventory Multidimentional Fatigue Scale. In addition, a principal component analysis was performed to identify fatigue dimensions that were specific to the population under study. This analysis extracted five relevant dimensions corresponding respectively to concentration, energy, self-perceived cognitive efficiency, sleep/rest and motivation/anhedonia. Blood samples were collected for the measurement of inflammatory and metabolic markers, including high sensitivity C-reactive protein (hs-CRP), insulin, uricemia and glycaemia. Tryptophan, kynurenine and neopterin levels were also determined in a subsample of 17 patients. In the whole population under study, cognitive fatigue and reduced motivation/anhedonia were associated with BMI, independently of sex and age. The dimension of reduced motivation/anhedonia was associated with insulin resistance and inflammatory biomarkers. The association with insulin resistance persisted when the extent of fat mass (BMI-SDS) was taken into account. No association was found between tryptophan metabolism and specific dimensions of fatigue, but kynurenine and the kynurenine/tryptophan ratio correlated with insulin and HOMA-IR. These data indicate that insulin resistance in non diabetic obese children is associated with both cognitive fatigue and reduced motivation/anhedonia and with alterations in tryptophan metabolism. Further investigations are needed to determine whether inflammation-induced alterations in tryptophan metabolism is directly or indirectly implicated in insulin resistance and related fatigue.

Rebaudioside A and cortisol metabolism: sweet news for consumers.

Extracts of Stevia rebaudiana Bertoni are traditional food sweeteners in South America. Stevioside and other sweet-tasting compounds such as steviobioside and rebaudiosides were then isolated from Stevia leaves. Subsequently, rebaudioside A has been authorised in many countries as a sweetener [1]. Reversible oedema, hypokalaemia and elevated blood pressure were reported in a patient who ingested large amounts of Stevia extracts [2]. This was synchronous to a modification of the cortisol/cortisone ratio in urine. Stevia extract was presumed guilty through modification of the activity of 11β-hydroxysteroiddehydrogenase type 2 (HSD2): the enzyme implicated with HSD1 in the inactivation/reactivation shuttle of cortisol in various tissues including abdominal fat. Considering the worldwide prevalence of diabetes and the great number of diabetics using sweeteners, such an enzyme inhibition by rebaudioside would have dire consequences. We thus investigated the specific effects of rebaudioside on the excretion of cortisol metabolites. Healthy subjects (n = 23) ingested 4 pellets/meal of a commercially available sweetener containing rebaudioside A (Canderel “green”, total of 12 pellets in a day). Nocturnal urine was collected the mornings before and after ingestion of the sweetener. The subjects did not ingest any sweetener at least a month before the study. No adverse events were reported. Themetabolic consequences of this sweetener ingestion on HSD2 and HSD1 activities was investigated through the excretions of cortisol, cortisone, tetrahydrocortisol and allo-tetrahydrocortisol (THFs), and tetrahydrocortisone (THE) using cortisol/cortisone and THFs/THE ratios, respectively [3,4]. Intraassay variabilities were b8%. Age and BMI of the subjects (10 M/14 F) were 34 [23;50] yr and 22 [17;27] kg/m (median [range]). Cortisol/cortisone and THFs/THE basal ratios were: 0.60 [0.33;1.26] and 0.62 [0.49;0.99] (Fig. 1). Interindividual coefficients of variation of these ratios were 32 and 21%, respectively. We found no significant modification of the ratios after rebaudioside ingestion (paired t-test, Fig. 1). Intraindividual variations of the ratios were: 8 [3;49] and 9 [0;26]%. This absence of significant effect of rebaudioside on HSD1 or HSD2 activities is reassuring although it is not definite proof of the absence of consequences for a long-term intake. However, prior publications about rebaudioside reported no modification of blood pressure or ion urinary excretion [5]. Our study has limits: the absence of diabetics in this population. Hypothetically, enzyme activities may be modified by diabetes and superimposed rebaudioside could act differently in such