REVERBa couples the circadian clock to hepatic glucocorticoid action.

The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism

Metabolic voxel-based analysis of the complete human brain using fast 3D-MRSI: Proof of concept in multiple sclerosis

International audiencePURPOSE: To detect local metabolic abnormalities over the complete human brain in multiple sclerosis (MS) patients, we used optimized fast volumic echo planar spectroscopic imaging (3D-EPSI). MATERIALS AND METHODS: Weighted mean combination of two 3D-EPSI covering the whole brain acquired at 3T in AC-PC and AC-PC+15° axial planes was performed to obtain high-quality metabolite maps for five metabolites: N-acetyl aspartate (NAA), glutamate+glutamine (Glx), choline (Cho), myo-inositol (m-Ins), and creatine+phosphocreatine (tCr). After spatial normalization, maps from 19 patients suffering from relapsing-remitting MS were compared to 19 matched controls using statistical mapping analyses to determine the topography of metabolic abnormalities. Probabilistic white matter (WM) T2 lesion maps and gray matter (GM) atrophy maps were also generated. RESULTS: Two-group analysis of variance (ANOVA) (SPM8, P \textless 0.005, false discovery rate [FDR]-corrected P \textless 0.05 at the cluster level with age and sex as confounding covariates) comparing patients and controls matched for age and sex showed clusters of abnormal metabolite levels with 1) decreased NAA (around -15%) and Glx (around 20%) predominantly in GM within prefrontal cortices, motor cortices, bilateral thalami, and mesial temporal cortices in line with neuronal/neuro-astrocytic dysfunction; 2) increased m-Ins (around + 20%) inside WM T2 lesions and in the normal-appearing WM of temporal-occipital lobes, suggesting glial activation. CONCLUSION: We demonstrate the ability to noninvasively map over the complete brain-from vertex to cerebellum-with a validated sequence, the metabolic abnormalities associated with MS, for characterizing the topography of pathological processes affecting widespread areas of WM and GM and its functional impact. J. Magn. Reson. Imaging 2016;44:411-419

Compounds have insignificant activity in IRE1 branch.

<p>HT1080 cells pretransduced with XBP-1 splicing reporter gene were treated with compounds at different concentrations for 3 h at 37°C and 5% CO₂. Luciferase activity was measured with Steady Glo reagents after the treatment. Data were average of 8 repeats.</p

Compounds increase Heme Oxygenase-1 (HO-1) expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM of compound for 1 h or 3 μM of compound for 24 h at 37°C and 5% CO₂, respectively. Tg (0.5 μM) and hemin (3 μM) served as controls. HO-1 protein was then determined. (B) NHBE cells were transfected with 25 nM non-target siRNA, PERK siRNA or NRF2 siRNA for 48 h, then treated with compounds at 3 μM for 24 h. HO-1 mRNA level was then analyzed. (C) NHBE cells transfected with non-targeting, NRF2 or PERK siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B, C or hemin for 24 h. HO-1 protein level was determined. Data was representative of 3 experiments.</p

LanthaScreen assay signal was PERK dependent.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam under the optimized conditions for overnight. Cells were then trypsinized and plated into a 384 well plate. Different doses of a specific PERK inhibitor GSK2606414 (A) or an inactive analog (B) was incubated with cells for 30 min at 37°C and 5% CO₂. DMSO, 1 μM Tg or 5 μM Tn was then added and incubated for 2 h at 37°C 5% CO₂. Phosphorylation of GFP-eIF2α was analyzed with LanthaScreen assay. Data was the average of 2 repeats.</p

Robustness features of the LanthaScreen assay.

<p>U-2 OS cells were transduced with GFP-eIF2α BacMam for overnight in flasks. Cells were then trypsinized, washed and resuspended for plating into 384 well plates for compound treatment. The low control was DMSO only, the high control was 1 μM Tg. All the conditions were the optimized final conditions as described in results.</p><p>Robustness features of the LanthaScreen assay.</p

Compounds increase NQO-1 protein expression in a NRF2 dependent pattern.

<p>(A) NHBE cells were treated with 10 μM compound for 1 h or 3 μM compound for 24 h at 37°C and 5% CO₂. NQO-1 protein level was determined by ELISA. Stars indicate p value ≤0.001 (n = 3). (B) NHBE cells were transfected 25nM siRNA for 48 h, treated with DMSO, 0.5μTg, 3 μM compound B or C for 24 h. NQO-1 mRNA level was then determined by Real-Time PCR and was normalized with DMSO control (n = 3). (C) NHBEs were transfected 25 nM siRNA for 48 h, treated with DMSO, 0.5 μM Tg, 3 μM compound B or C for 24 h. NQO-1 protein level was determined by Western blot. Data was representative of 3 experiments.</p

Titration of GFP-eIF2α BacMam.

<p>(A) U-2 OS cells were mixed with different amount of GFP-eIF2α BacMam and plated to a 384 well culture plate for overnight. Cells were then treated with either DMSO or 2 μM Tg for 1 h at 37°C 5% CO₂. LanthaScreen was performed to detect eIF2α phosphorylation. Data was the average of 4 repeats. (B) U-2 OS cells were transduced with GFP-eIF2α BacMam in a culture flask with the optimized MOT for overnight. After trypsinized and washed, cells were resuspended to different cell density and plated into a 384 well plate. After the treatment with Tg for 2.5 h at 37°C and 5% CO₂, LanthaScreen assay was performed to detect the phosphorylation of GFP-eIF2α. Data was the average of 3 repeats.</p