First FHM3 mouse model shows spontaneous cortical spreading depolarizations.

Here we show, for the first time, spontaneous cortical spreading depolarization (CSD) events - the electrophysiological correlate of the migraine aura - in animals by using the first generated familial hemiplegic migraine type 3 (FHM3) transgenic mouse model. The mutant mice express L263V-mutated α1 subunits in voltage-gated NaV 1.1 sodium channels (Scn1aL263V ). CSDs consistently propagated from visual to motor cortex, recapitulating what has been shown in patients with migraine with aura. This model may be valuable for the preclinical study of migraine with aura and other diseases in which spreading depolarization is a prominent feature.

Coffin-Siris syndrome and the BAF complex: genotype-phenotype study in 63 patients.

De novo germline variants in several components of the SWI/SNF-like BAF complex can cause Coffin-Siris syndrome (CSS), Nicolaides-Baraitser syndrome (NCBRS), and nonsyndromic intellectual disability. We screened 63 patients with a clinical diagnosis of CSS for these genes (ARID1A, ARID1B, SMARCA2, SMARCA4, SMARCB1, and SMARCE1) and identified pathogenic variants in 45 (71%) patients. We found a high proportion of variants in ARID1B (68%). All four pathogenic variants in ARID1A appeared to be mosaic. By using all variants from the Exome Variant Server as test data, we were able to classify variants in ARID1A, ARID1B, and SMARCB1 reliably as being pathogenic or nonpathogenic. For SMARCA2, SMARCA4, and SMARCE1 several variants in the EVS remained unclassified, underlining the importance of parental testing. We have entered all variant and clinical information in LOVD-powered databases to facilitate further genotype-phenotype correlations, as these will become increasingly important because of the uptake of targeted and untargeted next generation sequencing in diagnostics. The emerging phenotype-genotype correlation is that SMARCB1 patients have the most marked physical phenotype and severe cognitive and growth delay. The variability in phenotype seems most marked in ARID1A and ARID1B patients. Distal limbs anomalies are most marked in ARID1A patients and least in SMARCB1 patients. Numbers are small however, and larger series are needed to confirm this correlation.

Glucocorticoid treatment impairs microvascular function in healthy men in association with its adverse effects on glucose metabolism and blood pressure: a randomised controlled trial.

AIMS/HYPOTHESIS: Glucocorticoids (GCs) are widely used anti-inflammatory agents that frequently induce side effects, including insulin resistance, diabetes and hypertension. Here, we investigated the contribution of microvascular dysfunction to the development of these adverse effects in healthy men. METHODS: In a randomised, placebo-controlled, dose-response intervention study, 32 healthy normoglycaemic men (age: 21 ± 2 years; BMI: 21.9 ± 1.7 kg/m(2)) were allocated to receive prednisolone 30 mg once daily (n = 12), prednisolone 7.5 mg once daily (n = 12) or placebo (n = 8) for 2 weeks using block randomisation. A central office performed the treatment allocation, and medication was dispersed by the hospital pharmacy that was also blinded. Treatment allocation was kept in concealed envelopes. Participants, study personnel conducting the measures and assessing the outcome were blinded to group assignment. The study was conducted at a university hospital. Primary endpoint was prednisolone-induced changes in microvascular function, which was assessed by capillary microscopy. Insulin sensitivity was determined by hyperinsulinaemic-euglycaemic clamp and postprandial glycaemic excursions by standardised meal tests. RESULTS: Compared with placebo, prednisolone 7.5 mg and 30 mg decreased insulin-stimulated capillary recruitment by 9 ± 4% and 17 ± 3%, respectively (p < 0.01). In addition, prednisolone 7.5 mg and 30 mg reduced insulin sensitivity (M value) by -11.4 ± 4.5 µmol kg(-1) min(-1) and -25.1 ± 4.1 µmol kg(-1) min(-1) (p < 0.001) and increased postprandial glucose levels by 11 ± 5% and 27 ± 9% (p < 0.001), respectively. Only high-dose prednisolone increased systolic blood pressure (6 ± 1.2 mmHg, p = 0.006). Prednisolone-induced changes in insulin-stimulated capillary recruitment were associated with insulin sensitivity (r = +0.76; p < 0.001), postprandial glucose concentrations (r = -0.52; p < 0.03) and systolic blood pressure (r = -0.62; p < 0.001). Prednisolone increased resistin concentrations, which were negatively related to insulin-stimulated capillary recruitment (r = -0.40; p = 0.03). No effects were noted on adiponectin and leptin concentrations. Prednisolone treatment was well tolerated; none of the participants left the study. CONCLUSIONS/INTERPRETATION: Prednisolone-induced impairment of insulin-stimulated capillary recruitment was paralleled by insulin resistance, increased postprandial glucose levels, hypertension and increased circulating resistin concentrations in healthy men. We propose that GC-induced impairments of microvascular function may contribute to the adverse effects of GC treatment on glucose metabolism and blood pressure. TRIAL REGISTRATION: isrctn.org ISRTCN 78149983. FUNDING: The study was funded by the Dutch Top Institute Pharma T1-106.

Proline-rich Akt substrate of 40-kDa contains a nuclear export signal.

The proline-rich Akt substrate of 40-kDa (PRAS40) has been linked to the regulation of the activity of the mammalian target of rapamycin complex 1 as well as insulin action. Despite these cytosolic functions, PRAS40 was originally identified as nuclear phosphoprotein in Hela cells. This study aimed to detail mechanisms and consequences of the nucleocytosolic trafficking of PRAS40. Sequence analysis identified a potential leucine-rich nuclear export signal (NES) within PRAS40. Incubation of A14 fibroblasts overexpressing human PRAS40 (hPRAS40) resulted in nuclear accumulation of the protein. Furthermore, mutation of the NES mimicked the effects of leptomycin B, a specific inhibitor of nuclear export, on the subcellular localization of hPRAS40. Finally, A14 cells expressing the NES-mutant showed impaired activation of components of the Akt-pathway as well as of the mTORC1 substrate p70 S6 kinase after insulin stimulation. This impaired insulin signaling could be ascribed to reduced protein levels of insulin receptor substrate 1 in cells expressing mutant NES. In conclusion, PRAS40 contains a functional nuclear export signal. Furthermore, enforced nuclear accumulation of PRAS40 impairs insulin action, thereby substantiating the function of this protein in the regulation of insulin sensitivity.

Prednisolone induces the Wnt signalling pathway in 3T3-L1 adipocytes.

Synthetic glucocorticoids are potent anti-inflammatory drugs but show dose-dependent metabolic side effects such as the development of insulin resistance and obesity. The precise mechanisms involved in these glucocorticoid-induced side effects, and especially the participation of adipose tissue in this are not completely understood. We used a combination of transcriptomics, antibody arrays and bioinformatics approaches to characterize prednisolone-induced alterations in gene expression and adipokine secretion, which could underlie metabolic dysfunction in 3T3-L1 adipocytes. Several pathways, including cytokine signalling, Akt signalling, and Wnt signalling were found to be regulated at multiple levels, showing that these processes are targeted by prednisolone. These results suggest that mechanisms by which prednisolone induce insulin resistance include dysregulation of wnt signalling and immune response processes. These pathways may provide interesting targets for the development of improved glucocorticoids.

Prednisolone-induced beta cell dysfunction is associated with impaired endoplasmic reticulum homeostasis in INS-1E cells.

Glucocorticoids (GCs), such as prednisolone (PRED), are widely prescribed anti-inflammatory drugs, but their use may induce glucose intolerance and diabetes. GC-induced beta cell dysfunction contributes to these diabetogenic effects through mechanisms that remain to be elucidated. In this study, we hypothesized that activation of the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress could be one of the underlying mechanisms involved in GC-induced beta cell dysfunction. We report here that PRED did not affect basal insulin release but time-dependently inhibited glucose-stimulated insulin secretion in INS-1E cells. PRED treatment also decreased both PDX1 and insulin expression, leading to a marked reduction in cellular insulin content. These PRED-induced detrimental effects were found to be prevented by prior treatment with the glucocorticoid receptor (GR) antagonist RU486 and associated with activation of two of the three branches of the UPR. Indeed, PRED induced a GR-mediated activation of both ATF6 and IRE1/XBP1 pathways but was found to reduce the phosphorylation of PERK and its downstream substrate eIF2α. These modulations of ER stress pathways were accompanied by upregulation of calpain 10 and increased cleaved caspase 3, indicating that long term exposure to PRED ultimately promotes apoptosis. Taken together, our data suggest that the inhibition of insulin biosynthesis by PRED in the insulin-secreting INS-1E cells results, at least in part, from a GR-mediated impairment in ER homeostasis which may lead to apoptotic cell death.

Glucagon-like peptide-1 receptor agonist treatment prevents glucocorticoid-induced glucose intolerance and islet-cell dysfunction in humans.

OBJECTIVE: Glucocorticoids (GCs) are regarded as diabetogenic because they impair insulin sensitivity and islet-cell function. This study assessed whether treatment with the glucagon-like peptide receptor agonist (GLP-1 RA) exenatide (EXE) could prevent GC-induced glucose intolerance. RESEARCH DESIGN AND METHODS: A randomized, placebo-controlled, double-blind, crossover study in eight healthy men (age: 23.5 [20.0-28.3] years; BMI: 26.4 [24.3-28.0] kg/m(2)) was conducted. Participants received three therapeutic regimens for 2 consecutive days: 1) 80 mg of oral prednisolone (PRED) every day (q.d.) and intravenous (IV) EXE infusion (PRED+EXE); 2) 80 mg of oral PRED q.d. and IV saline infusion (PRED+SAL); and 3) oral placebo-PRED q.d. and intravenous saline infusion (PLB+SAL). On day 1, glucose tolerance was assessed during a meal challenge test. On day 2, participants underwent a clamp procedure to measure insulin secretion and insulin sensitivity. RESULTS: PRED+SAL treatment increased postprandial glucose levels (vs. PLB+SAL, P = 0.012), which was prevented by concomitant EXE (vs. PLB+SAL, P = NS). EXE reduced PRED-induced hyperglucagonemia during the meal challenge (P = 0.018) and decreased gastric emptying (vs. PRED+SAL, P = 0.028; vs. PLB+SAL, P = 0.046). PRED+SAL decreased first-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL, P = 0.017 and P = 0.05, respectively), whereas PRED+EXE improved first- and second-phase glucose- and arginine-stimulated C-peptide secretion (vs. PLB+SAL; P = 0.017, 0.012, and 0.093, respectively). CONCLUSIONS: The GLP-1 RA EXE prevented PRED-induced glucose intolerance and islet-cell dysfunction in healthy humans. Incretin-based therapies should be explored as a potential strategy to prevent steroid diabetes.