Serum Visfatin does not seem to be a Useful Marker to Guide Glucocorticoid Substitution in Adrenal Insufficiency.

Primary adrenal insufficiency (Addison's disease, AD) requires lifelong steroid substitution. Excess exogenous glucocorticoids promote abdominal obesity, insulin-glucose imbalance, and hypertension. Reliable markers of the adequate glucocorticoid replacement are lacking. Visfatin is a pro-inflammatory adipokine, with enzymatic activity of nicotinamide phosphoribosyltransferase. It enhances leukocyte function and synthesis of tumour necrosis factor α (TNFα) and interleukin-6 (IL-6). Serum visfatin is elevated in autoimmunity, but also in obesity, insulin resistance, and metabolic syndrome. This study was aimed to investigate whether serum visfatin could guide the glucocorticoid substitution in AD. Biochemical analyses were performed in 96 patients with AD (mean age 43.3±14.9 years) and 91 controls (43.5±12.5 years). Visfatin level was significantly elevated in patients with AD compared to controls (p<0.0001). Higher circulating IL-6 was also detected among subjects with AD (p=0.006). In AD, visfatin level was positively correlated with IL-6 (p=0.014), TNFα (p=0.001), body mass (p=0.015), fasting insulin (p=0.001) and HOMA-IR (p=0.001). No relationship was noticed with daily hydrocortisone (p=0.096) and urinary free cortisol excretion (p=0.499). Only the correlations with IL-6 and fasting insulin survived multiple regression analysis (p=0.049 and p=0.005, respectively). Additionally, positive correlation between visfatin and autoantibodies to 21-hydroxylase was noted (p=0.005). In the control group serum visfatin was correlated with IL-6 (p=0.009) and TNFα (p=0.0002). The current study reveals elevated serum visfatin in autoimmune AD. Visfatin does not seem a useful marker of the glucocorticoid replacement, although it correlates with fasting insulin and pro-inflammatory molecules. Further functional analyses are warranted to elucidate the role of visfatin in autoimmunity.

Arylacetamide deacetylase knockout mice are sensitive to ketoconazole-induced hepatotoxicity and adrenal insufficiency.

Orally administered ketoconazole may rarely induce liver injury and adrenal insufficiency. A metabolite formed by arylacetamide deacetylase (AADAC)-mediated hydrolysis has been observed in cellulo studies, and it is relevant to ketoconazole-induced cytotoxicity. This study tried to examine the significance of AADAC in ketoconazole-induced toxicity in vivo using Aadac knockout mice. Oral administration of 150 mg/kg ketoconazole resulted in the area under the plasma concentration-time curve values of ketoconazole and N-deacetylketoconazole, a hydrolyzed metabolite of ketoconazole, in Aadac knockout mice being significantly higher and lower than those in wild-type mice, respectively. With the administration of ketoconazole (300 mg/kg/day) for 7 days, Aadac knockout mice showed higher mortality (100%) than wild-type mice (42.9%), and they also showed significantly higher plasma alanine transaminase and lower corticosterone levels, thus representing liver injury and steroidogenesis inhibition, respectively. It was suggested that a higher plasma ketoconazole concentration likely accounts for the inhibition of the synthesis of corticosterone, which has anti-inflammatory effects, in the adrenal gland in Aadac KO mice. In Aadac knockout mice, hepatic mRNA levels of immune- and inflammation-related factors were increased by the administration of 300 mg/kg ketoconazole, and the increase was restored by the replenishment of corticosterone (40 mg/kg, s.c.) along with recoveries of plasma alanine transaminase levels. In conclusion, Aadac defects exacerbate ketoconazole-induced liver injury by inhibiting glucocorticoid synthesis and enhancing the inflammatory response. This in vivo study revealed that the hydrolysis of ketoconazole by AADAC can mitigate ketoconazole-induced toxicities.

Characterization of the CYP11A1 Nonsynonymous Variant p.E314K in Children Presenting With Adrenal Insufficiency.

Context: Cholesterol side-chain cleavage enzyme (P450scc), encoded by CYP11A1, catalyzes the first step of steroidogenesis. Complete P450scc deficiency leads to primary adrenal insufficiency (PAI) and 46,XY disordered sexual development. Partial impairment can cause variable adrenal and gonadal dysfunction. Objective: Our aim was to evaluate the effects of the CYP11A1 variant p.E314K, identified in patients with PAI, specifically on P450scc enzyme stability and function. Patients and Methods: We studied four boys from two unrelated families presenting with PAI during childhood (3.6 to 9 years old). All patients were compound heterozygous for c.940G>A (p.E314K), a CYP11A1 nonsynonymous variant likely to be pathogenic by some but not all in silico prediction models, and c.835delA (p.I79Yfs*10), a known pathogenic variant. HEK293T cells were transfected with wild type (WT) and p.E314K mutant vectors, and a cycloheximide chase assay was performed to analyze protein stability. Pregnenolone production was assayed from cells expressing WT and p.E314K-F2 fusion proteins. Results: Two boys experienced spontaneous puberty but then developed evidence of primary gonadal failure at 14 and 18 years old. Two boys had testicular adrenal rest tumor (TART), detected by ultrasound at ages 8.6 and 16 years. Compared with WT, mutant protein synthesis was reduced (P = 0.0006) with increased protein turnover, and mutant P450scc half-life was decreased by ~50%. p.E314K mutant P450scc retained 60% of WT enzymatic activity (P = 0.007). Conclusions: The CYP11A1 p.E314K variant impairs P450scc stability and is a possible cause of PAI in childhood. Pathogenic CYP11A1 variants potentially affect both adrenal and gonadal function, and male patients may develop TART.

Unexplained death in patients with NGLY1 mutations may be explained by adrenal insufficiency.

Homozygous mutations in NGLY1 were recently found to cause a condition characterized by a complex neurological syndrome, hypo- or alacrimia, and elevated liver transaminases. For yet unknown reasons, mortality is increased in patients with this condition. NGLY1 encodes the cytosolic enzyme N-glycanase 1, which is responsible for the deglycosylation of misfolded N-glycosylated proteins. Disruption of this process is hypothesized to lead to an accumulation of misfolded proteins in the cytosol. Here, we describe the disease course of a girl with a homozygous mutation in NGLY1, namely c.1837del (p.Gln613 fs). In addition to the previously described symptoms, at the age of 8 she presented with recurrent infections and hyperpigmentation, and, subsequently, a diagnosis of primary adrenal insufficiency was made. There are no previous reports describing adrenal insufficiency in such patients. We postulate that patients with NGLY1 deficiency may develop adrenal insufficiency as a consequence of impaired proteostasis, and the accompanying proteotoxic stress-induced cell death, through defective Nrf1 function. We recommend an annual evaluation of adrenal function in all patients with NGLY1 mutations in order to prevent unnecessary deaths.

Acute adrenal crisis and hypercalcemia in a patient assuming high liquorice doses.

Two months after monolateral adrenalectomy, a 47-year-old woman stopped taking corticosteroid replacement therapy in the first 15 days of therapy. She was admitted to the Department of Internal Medicine because of hypertension, severe hypercalcemia, uncompensated metabolic alkalosis and clinical symptoms of acute adrenal insufficiency. The presence of hypokalemia and hypernatremia precluded a diagnosis of hypocortisolism, therefore no corticosteroids were given during the time required to investigate the cause of hypercalcemia, which resulted negative. Administration of intravenous saline infusion produced no improvement in her clinical condition. Despite electrolyte alterations, hydrocortison (100 mg i.v.) and zoledronate (4 mg i.v.) were also administered, leading to a rapid and marked improvement in her clinical picture within a few hours, with normalization of the calcemia and the other electrolytic disturbances. After her neurological condition had fully normalized, the patient admitted she had been assuming large amounts of liquorice as a laxative for many years; this compound very likely compensated the adrenal insufficiency by inhibiting 11 b steroid-dehydrogenase and disguised the clinical presentation at the time of admission. This case report confirms that, though rare, hypercalcemia may be a finding in acute adrenal insufficiency and can be rapidly corrected by corticosteroid administration. Furthermore, excessive liquorice intake can induce a clinical picture resembling that of primary hyperaldosteronism. In patients with adrenal insufficiency, it can, at least in part, disguise its metabolic effects and delay diagnosis and treatment.

Use of a combination of routine hematologic and biochemical test results in a logistic regression model as a diagnostic aid for the diagnosis of hypoadrenocorticism in dogs.

OBJECTIVE To assess the discriminatory value for corticosteroid-induced alkaline phosphatase (CiALP) activity and other variables that can be measured routinely on a CBC and biochemical analysis for the diagnosis of hypoadrenocorticism in dogs. SAMPLE Medical records of 57 dogs with confirmed hypoadrenocorticism and 57 control dogs in which hypoadrenocorticism was suspected but ruled out. PROCEDURES A retrospective case-control study was conducted. Dogs were included if a CBC and complete biochemical analysis had been performed. Dogs with iatrogenic hypoadrenocorticism and dogs treated previously with glucocorticoids were excluded. Cortisol concentration for dogs with hypoadrenocorticism was ≤ 2 µg/dL both before and after ACTH administration. Cortisol concentration for control dogs was > 4 µg/dL before or after ACTH administration. RESULTS Area under the receiver operating characteristic (ROC) curve for CiALP activity was low (0.646; 95% confidence interval, 0.494 to 0.798). Area under the ROC curve for a model that combined the CiALP activity, Na-to-K ratio, eosinophil count, activity of creatine kinase, and concentrations of SUN and albumin was high (0.994; 95% confidence interval, 0.982 to 1.000). Results for this model could be used to correctly classify all dogs, except for 1 dog with hypoadrenocorticism and no electrolyte abnormalities. CONCLUSIONS AND CLINICAL RELEVANCE CiALP activity alone cannot be used as a reliable diagnostic test for hypoadrenocorticism in dogs. Combined results for CiALP activity, Na-to-K ratio, eosinophil count, creatine kinase activity, and concentrations of SUN and albumin provided an excellent means to discriminate between hypoadrenocorticism and diseases that mimic hypoadrenocorticism.

Sphingosine-1-phosphate lyase mutations cause primary adrenal insufficiency and steroid-resistant nephrotic syndrome.

Primary adrenal insufficiency is life threatening and can present alone or in combination with other comorbidities. Here, we have described a primary adrenal insufficiency syndrome and steroid-resistant nephrotic syndrome caused by loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1). SGPL1 executes the final decisive step of the sphingolipid breakdown pathway, mediating the irreversible cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P). Mutations in other upstream components of the pathway lead to harmful accumulation of lysosomal sphingolipid species, which are associated with a series of conditions known as the sphingolipidoses. In this work, we have identified 4 different homozygous mutations, c.665G>A (p.R222Q), c.1633_1635delTTC (p.F545del), c.261+1G>A (p.S65Rfs*6), and c.7dupA (p.S3Kfs*11), in 5 families with the condition. In total, 8 patients were investigated, some of whom also manifested other features, including ichthyosis, primary hypothyroidism, neurological symptoms, and cryptorchidism. Sgpl1-/- mice recapitulated the main characteristics of the human disease with abnormal adrenal and renal morphology. Sgpl1-/- mice displayed disrupted adrenocortical zonation and defective expression of steroidogenic enzymes as well as renal histology in keeping with a glomerular phenotype. In summary, we have identified SGPL1 mutations in humans that perhaps represent a distinct multisystemic disorder of sphingolipid metabolism.

Pretreatment with low dose etomidate prevents etomidate-induced rat adrenal insufficiency by regulating oxidative stress-related MAPKs and apoptosis.

Etomidate is frequently used as an anesthetic and sedation agent in the clinic setting. This study determined that a low-dose pre-infusion followed by a continuous dose infusion of etomidate could reduce etomidate-induced adrenal gland insufficiency. Sixty adult male Wistar rats were used, with six rats per group. Based on preliminary experiments, 0.6mg/kg etomidate was selected as the low dose for this study. Oxidative stress and apoptosis-related proteins in the adrenal glands were assayed using Western blot, and serum levels of CORT and 11ß-hydroxylase were detected using ELISA. Pretreatment with a single bolus of low dose etomidate significantly increased the levels of CORT and 11ß-hydroxylase as well as the activities of superoxide dismutase (SOD), catalase (CAT) and glutathioneperoxidase (GPx) in the adrenal glands, but reduced nitric oxide (NO) production when compared to the positive group. Furthermore, Western blot data showed that pretreatment with low dose etomidate increased extracellular signal-regulated kinase1/2 (ERK1/2), CREB and bcl-2 activation, but suppressed the p-p38, c-JunN-terminal kinase (JNK), inducible NO synthase (iNOS), cleaved-caspase3, cleaved-poly-ADP-ribose polymerase (PARP), bax, and AKT activation. The ERK inhibitor PD98059 and the p38MAPK inhibitor SB203580 abolished the protective effect of low dose etomidate pretreatment. These data demonstrated that pretreatment with low dose etomidate attenuated etomidate-induced adrenal insufficiency to rat adrenal glands. Oxidative stress-related MAPKs and apoptosis proteins might be responsible for mediating the etomidate preconditioning effect in rats.

The nucleoporin ALADIN regulates Aurora A localization to ensure robust mitotic spindle formation.

The formation of the mitotic spindle is a complex process that requires massive cellular reorganization. Regulation by mitotic kinases controls this entire process. One of these mitotic controllers is Aurora A kinase, which is itself highly regulated. In this study, we show that the nuclear pore protein ALADIN is a novel spatial regulator of Aurora A. Without ALADIN, Aurora A spreads from centrosomes onto spindle microtubules, which affects the distribution of a subset of microtubule regulators and slows spindle assembly and chromosome alignment. ALADIN interacts with inactive Aurora A and is recruited to the spindle pole after Aurora A inhibition. Of interest, mutations in ALADIN cause triple A syndrome. We find that some of the mitotic phenotypes that we observe after ALADIN depletion also occur in cells from triple A syndrome patients, which raises the possibility that mitotic errors may underlie part of the etiology of this syndrome.

"Masked" 21-hydroxylase deficiency of the adrenal presenting with gynecomastia and bilateral testicular masses.

An infertile 27 year old man with precocious puberty is described. He presented in adulthood with unilateral and then bilateral gynecomastia, and subsequently testicular tumors developed. An early diagnosis of congenital adrenal hyperplasia would have avoided unnecessary surgery. Initial detailed metabolic evaluation led to the erroneous diagnosis of 11-hydroxylase deficiency because of the presence of an unusual steroid (21-desoxycortisol) in serum which was falsely reported as an increased 11-desoxycortisol (compound S). The observed low urinary pregnanetriol measurements would have supported this diagnosis. Subsequent specific measurements of 21-desoxycortisol established its presence in the serum and its major metabolite, tetrahydro-21-desoxycortisol, in the urine. The unique features in this case of 21-hydroxylase deficiency alert the physician to its unusual clinical presentation and the pitfalls that may be encountered when evaluating adrenal steroidogenesis.

Fine mapping of glycerol kinase deficiency and congenital adrenal hypoplasia within Xp21 on the short arm of the human X chromosome.

We have studied patients with Duchenne muscular dystrophy (DMD), DMD together with glycerol kinase (GK) deficiency, or DMD together with both GK deficiency and congenital adrenal hypoplasia (AHC). Analysis of deletions in these patients allows the mapping of these mutations in Xp21. The following order is proposed: Xpter - L1 - AHC - GK - DMD - Xcen. One of the boys with DMD, GK, and AHC is shown by pulsed-field-gel electrophoresis to have a deletion which has a proximal endpoint at least 500 kb distal from the pERT87 (DXS164) locus.

N6-phenylisopropyladenosine stimulates in normal and inhibits in adrenalectomized rats the low KM cyclic AMP phosphodiesterase in the brain.

In crude rat brain membranes, apparent adenylate cyclase activity tested in the absence of phosphodiesterase inhibitors was dose-dependently inhibited by the adenosine 'R-site' agonist N6-phenylisopropyladenosine (N6-PIA). In membranes from adrenalectomized rats, however, N6-PIA induced, under the same conditions, an activation. However, in the presence of the phosphodiesterase inhibitor Ro-20-1724, brain adenylate cyclase responsiveness to N6-PIA resulted in a dose-dependent inhibition in both sham-operated and adrenalectomized rats. Thus, the low KM cyclic AMP phosphodiesterase activity present in these brain membranes was investigated. Although this activity (tested in the presence of GTP) was unaltered by adrenalectomy, the dose-response curve of this enzyme to low concentrations of N6-PIA showed an activation in sham-operated and an inhibition in adrenalectomized rats, two effects which were suppressed by sodium (80 mM). These results showing that N6-PIA modulates both adenylate cyclase and cyclic AMP phosphodiesterase in the brain, provide an additional argument for a potential role of adenosine in the regulation of cyclic AMP metabolism in normal as well as in pathological brain.

Familial adrenal insufficiency, achalasia, alacrima, peripheral neuropathy, microcephaly, normal plasma very long chain fatty acids, and normal muscle mitochondrial respiratory chain enzymes.

Adrenal insufficiency has been associated with adrenoleukodystrophy and adrenomyeloneuropathy. In these diseases, plasma very long chain fatty acids are elevated. Peripheral neuropathy is frequently seen in adults with adrenomyeloneuropathy. We encountered two first cousins with adrenal insufficiency, who also developed peripheral neuropathy, achalasia, alacrima, and microcephaly. However, plasma very long chain fatty acids, pipecolic acid, phytanic acid, and cranial computed tomographic scan were normal. Muscle mitochondrial respiratory chain enzymes were also normal. This syndrome of adrenal insufficiency, achalasia, alacrima, microcephaly, and peripheral neuropathy is different from either adrenomyeloneuropathy or adrenoleukodystrophy.

[Effect of vibration on gamma-aminobutyric acid metabolism in the brain in various functional conditions of the adrenal cortex]. / Vliianie vibratsii na obmen gamma-aminomaslianoi kisloty mozga pri razlichnykh funktsional'nykh sostoianiiakh kory nadpochechnikov.

The low-frequency vibration during 30 min (20 Hz, A = 0.4 mm) has been studied for its influence on the level of components of the GABA system and dicarbonic ++amino acids in male rats at hypo- and hyperfunction of the adrenal cortex. It is shown that under these conditions of the experiment the GABA level and glutamate-decarboxylase activity increase. Hyperfunction of the adrenal cortex against the background of vibration causes a relatively less pronounced increase in the GABA content, than the vibration alone or against the background of inhibition of adrenocortical function in the organism.

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency.

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.

Hypocortisolism in the South African angora goat: the role of 3betaHSD.

South African Angora goats are susceptible to cold stress, due to their inability to produce sufficient levels of cortisol. During adrenal steroidogenesis the production of cortisol relies on the activity of two key enzymes, namely cytochrome P450 17alpha-hydroxylase and 3beta-hydroxysteroid dehydrogenase. Cytochrome P450 17alpha-hydroxylase has previously been identified as a factor contributing to hypocortisolism in the South African Angora goat. In this comparative study, the catalytic activity of Angora and ovine 3beta-hydroxysteroid dehydrogenase, which differ by five amino acid residues, was characterized. The conversion of 17-hydroxypregnenolone and dehydroepiandosterone to their corresponding products, 17-hydroxyprogesterone and androstenedione, by the two enzymes differed significantly. The enzymes were subsequently co-expressed with Angora P450 17alpha-hydroxylase. Major differences were observed in pregnenolone metabolism with a significant reduction in the formation of the cortisol precursor, 17-hydroxyprogesterone, by cells expressing Angora 3beta-hydroxysteroid dehydrogenase, implicating 3beta-hydroxysteroid dehydrogenase as an additional factor contributing to hypocortisolism in the South African Angora goat.

Intracellular ROS level is increased in fibroblasts of triple A syndrome patients.

Triple A syndrome is named after the main symptoms of alacrima, achalasia, and adrenal insufficiency but also presents with a variety of neurological impairments. To investigate the causes of progressive neurodegeneration, we examined the oxidative status of fibroblast cultures derived from triple A syndrome patients in comparison to control cells. Patient cells showed a 2.1-fold increased basal level of reactive oxygen species (ROS) and a massive boost after induction of artificial oxidative stress by paraquat. We examined the expression of the ROS-detoxifying enzymes superoxide dismutase 1 and 2 (SOD1, SOD2), catalase, and glutathione reductase. The basal expression of SOD1 was significantly (1.3-fold) increased, and the expression of catalase was 0.7-fold decreased in patient cells after induction of artificial oxidative stress. We show that the mitochondrial network is 1.8-fold more extensive in patient cells compared to control fibroblasts although the maximal ATP synthesis was unchanged. Despite having the same energy potential as the controls, the patient cells showed a 1.4-fold increase in doubling time. We conclude that fibroblasts of triple A patients have a higher basal ROS level and an increased response to artificially induced oxidative stress and undergo "stress-induced premature senescence". The increased sensitivity to oxidative stress may be a major mechanism for the neurodegeneration in triple A syndrome.