Plasma S1P and Sphingosine are not Different Prior to Pre-Eclampsia in Women at High Risk of Developing the Disease.

Sphingolipids like sphingosine-1-phosphate (S1P) have been implicated in the pathophysiology of pre-eclampsia. We hypothesized that plasma S1P would be increased in women at high risk of developing pre-eclampsia who subsequently develop the disease. Low circulating placental growth factor (PlGF) is known to be associated with development of pre-eclampsia; so further, we hypothesized that increased S1P would be associated with concurrently low PlGF. This was a case-control study using stored maternal blood samples from 14 to 24 weeks of pregnancy, collected from 95 women at increased risk of pre-eclampsia. Pregnancy outcome was classified as uncomplicated, preterm pre-eclampsia (<37 weeks), or term pre-eclampsia. Plasma lipids were extracted and analyzed by ultraperformance liquid chromatography coupled to electrospray ionization MS/MS to determine concentrations of S1P and sphingosine. Median plasma S1P was 0.339 nmol/ml, and median sphingosine was 6.77 nmol/l. There were no differences in the plasma concentrations of S1P or sphingosine in women who subsequently developed pre-eclampsia, no effect of gestational age, fetal sex, ethnicity, or the presence of pre-existing hypertension. There was a correlation between S1P and sphingosine plasma concentration (P < 0.0001). There was no relationship between S1P or sphingosine with PlGF. Previous studies have suggested that plasma S1P may be a biomarker of pre-eclampsia. In our larger study, we failed to demonstrate there are women at high risk of developing the disease. We did not show a relationship with known biomarkers of the disease, suggesting that S1P is unlikely to be a useful predictor of the development of pre-eclampsia later in pregnancy.

Menopause induces changes to the stratum corneum ceramide profile, which are prevented by hormone replacement therapy.

The menopause can lead to epidermal changes that are alleviated by hormone replacement therapy (HRT). We hypothesise that these changes could relate to altered ceramide production, and that oestrogen may have a role in keratinocyte ceramide metabolism. White Caucasian women were recruited into three groups: pre-menopausal (n = 7), post-menopausal (n = 11) and post-menopausal taking HRT (n = 10). Blood samples were assessed for hormone levels, transepidermal water loss was measured to assess skin barrier function, and stratum corneum lipids were sampled from photoprotected buttock skin. Ceramides and sphingomyelins were analysed by ultraperformance liquid chromatography with electrospray ionisation and tandem mass spectrometry. Post-menopausal stratum corneum contained lower levels of ceramides, with shorter average length; changes that were not evident in the HRT group. Serum oestradiol correlated with ceramide abundance and length. Ceramides had shorter sphingoid bases, indicating altered de novo ceramide biosynthesis. Additionally, post-menopausal women had higher sphingomyelin levels, suggesting a possible effect on the hydrolysis pathway. Treatment of primary human keratinocytes with oestradiol (10 nM) increased production of CER[NS] and CER[NDS] ceramides, confirming an effect of oestrogen on cutaneous ceramide metabolism. Taken together, these data show perturbed stratum corneum lipids post-menopause, and a role for oestrogen in ceramide production.

Chronic glucocorticoid treatment induces hepatic lipid accumulation and hyperinsulinaemia in part through actions on AgRP neurons.

Glucocorticoids (GCs) are widely prescribed anti-inflammatory medicines, but their use can lead to metabolic side-effects. These may occur through direct actions of GCs on peripheral organs, but could also be mediated by the hypothalamic AgRP neurons, which can increase food intake and modify peripheral metabolism. Therefore, the aim of this study was to examine the metabolic effects of chronic treatment with the GC corticosterone (Cort, 75 µg/ml in drinking water) in mice lacking the glucocorticoid receptor (GR) on AgRP neurons. Female AgRP-GR KO mice had delayed onset of Cort-induced hyperphagia. However, AgRP-GR KO had little impact on the increased body weight or adiposity seen with 3 weeks Cort treatment. Cort caused hepatic steatosis in control mice, but in Cort treated female AgRP-GR KO mice there was a 25% reduction in liver lipid content and lower plasma triglycerides. Additionally, Cort treatment led to hyperinsulinaemia, but compared to controls, Cort-treated AgRP-GR KO mice had both lower fasting insulin levels and lower insulin levels during a glucose tolerance test. In conclusion, these data indicate that GCs do act through AgRP neurons to contribute, at least in part, to the adverse metabolic consequences of chronic GC treatment.

Global transcriptomic analysis of the arcuate nucleus following chronic glucocorticoid treatment.

OBJECTIVE: Glucocorticoids (GCs) are widely prescribed medications that are well recognized to cause adverse metabolic effects including hyperphagia, obesity, and hyperglycemia. These effects have been recapitulated in a murine model of GC excess, and we hypothesize that they are mediated, in part, through central mechanisms. This study aimed to identify genes in the hypothalamic arcuate nucleus (ARC) that are altered with GC treatment and evaluate their contribution to GC-induced metabolic abnormalities. METHODS: Corticosterone (Cort; 75 µg/ml) was administered in the drinking water to male C57Bl/6J mice for 2 days or 4 weeks. Phenotypic analysis of each group was undertaken and central and peripheral tissues were collected for biochemical and mRNA analyses. Arcuate nuclei were isolated by laser capture microdissection and tissue analyzed by RNA-seq. RESULTS: RNA-seq analysis of ARC tissue from 4 week Cort treated mice revealed 21 upregulated and 22 downregulated genes at a time when mice had increased food intake, expansion of adipose tissue mass, and insulin resistance. In comparison, after 2 days Cort treatment, when the main phenotypic change was increased food intake, RNA-seq identified 30 upregulated and 16 downregulated genes. Within the genes altered at 2 days were a range of novel genes but also those known to be regulated by GCs, including Fkbp5, Mt2, Fam107a, as well as some involved in the control of energy balance, such as Agrp, Sepp1, Dio2, and Nmb. Of the candidate genes identified by RNA-seq, type-II iodothyronine deiodinase (Dio2) was chosen for further investigation as it was increased (2-fold) with Cort, and has been implicated in the control of energy balance via the modulation of hypothalamic thyroid hormone availability. Targeted knockdown of Dio2 in the MBH using AAV-mediated CRISPR-Cas9 produced a mild attenuation in GC-induced brown adipose tissue weight gain, as well as a 56% reduction in the GC-induced increase in Agrp. However, this conferred no protection from GC-induced hyperphagia, obesity, or hyperglycemia. CONCLUSIONS: This study identified a comprehensive set of genes altered by GCs in the ARC and enabled the selection of key candidate genes. Targeted knockdown of hypothalamic Dio2 revealed that it did not mediate the chronic GC effects on hyperphagia and hyperglycemia.

Metabolic Abnormalities of Chronic High-Dose Glucocorticoids Are Not Mediated by Hypothalamic AgRP in Male Mice.

Glucocorticoids are potent and widely used medicines but often cause metabolic side effects. A murine model of corticosterone treatment resulted in increased hypothalamic expression of the melanocortin antagonist AgRP in parallel with obesity and hyperglycemia. We investigated how these adverse effects develop over time, with particular emphasis on hypothalamic involvement. Wild-type and Agrp-/- male mice were treated with corticosterone for 3 weeks. Phenotypic, biochemical, protein, and mRNA analyses were undertaken on central and peripheral tissues, including white and brown adipose tissue, liver, and muscle, to determine the metabolic consequences. Corticosterone treatment induced hyperphagia within 1 day in wild-type mice, which persisted for 3 weeks. Despite this early increase in food intake, the body weight only started to increase after 10 days. Hyperinsulinemia occurred at day 1. Also, although after 2 days, alterations were present in the genes often associated with insulin resistance in several peripheral tissues, hyperglycemia only developed at 3 weeks. Throughout, sustained elevation in hypothalamic Agrp expression was present. Mice with Agrp deleted [using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, Agrp-/-] were partially protected against corticosterone-induced hyperphagia. However, Agrp-/- mice still had corticosterone-induced increases in body weight and adiposity similar to those of the Agrp+/+ mice. Loss of Agrp did not diminish corticosterone-induced hyperinsulinemia or correct changes in hepatic gluconeogenic genes. Chronic glucocorticoid treatment in mice mimics many of the metabolic side effects seen in patients and leads to a robust increase in Agrp. However, AgRP does not appear to be responsible for most of the glucocorticoid-induced adverse metabolic effects.

Elevated Hypothalamic Glucocorticoid Levels Are Associated With Obesity and Hyperphagia in Male Mice.

Glucocorticoid (Gc) excess, from endogenous overproduction in disorders of the hypothalamic-pituitary-adrenal axis or exogenous medical therapy, is recognized to cause adverse metabolic side effects. The Gc receptor (GR) is widely expressed throughout the body, including brain regions such as the hypothalamus. However, the extent to which chronic Gcs affect Gc concentrations in the hypothalamus and impact on GR and target genes is unknown. To investigate this, we used a murine model of corticosterone (Cort)-induced obesity and analyzed Cort levels in the hypothalamus and expression of genes relevant to Gc action. Mice were administered Cort (75 µg/mL) or ethanol (1%, vehicle) in drinking water for 4 weeks. Cort-treated mice had increased body weight, food intake, and adiposity. As expected, Cort increased plasma Cort levels at both zeitgeber time 1 and zeitgeber time 13, ablating the diurnal rhythm. Liquid chromatography dual tandem mass spectrometry revealed a 4-fold increase in hypothalamic Cort, which correlated with circulating levels and concentrations of Cort in other brain regions. This occurred despite decreased 11ß-hydroxysteroid dehydrogenase (Hsd11b1) expression, the gene encoding the enzyme that regenerates active Gcs, whereas efflux transporter Abcb1 mRNA was unaltered. In addition, although Cort decreased hypothalamic GR (Nr3c1) expression 2-fold, the Gc-induced leucine zipper (Tsc22d3) mRNA increased, which indicated elevated GR activation. In keeping with the development of hyperphagia and obesity, Cort increased Agrp, but there were no changes in Pomc, Npy, or Cart mRNA in the hypothalamus. In summary, chronic Cort treatment causes chronic increases in hypothalamic Cort levels and a persistent elevation in Agrp, a mediator in the development of metabolic disturbances.