Background and AimReceptor‐interacting serine/threonine kinase 3 and mixed lineage kinase domain‐like pseudokinase (MLKL) have gained attention as apoptosis alternate cell death signaling molecules. We aimed to evaluate the role of MLKL in non‐alcoholic fatty liver disease (NAFLD).MethodsHepatic tissue MLKL expression was compared between NAFLD patients and healthy controls. High‐fat diet was fed to wild‐type and MLKL‐knockout (KO) mice for 12 weeks. Brown adipose fat tissue was measured by [18F]‐fluorodeoxyglucose positron emission tomography. Energy expenditure was measured by indirect calorimetry. Anti‐MLKL effects were also evaluated in in vitro setting using U937 and HepG2 cells.ResultsHepatic tissue MLKL expression increased in NAFLD patients compared with healthy controls. MLKL expression increased according to the degree of steatosis, ballooning, and inflammation. High‐fat diet‐fed MLKL‐KO mice displayed decreased alanine aminotransferase, triglycerides, liver weight, NAFLD activity score (6.3 vs 3.5, P < 0.001), steatosis score (3.0 vs 1.8, P < 0.001), inflammation, and ballooning degeneration compared with wild‐type mice. SREBP1c, fatty acid synthase, and SCD‐1 expressions decreased in MLKL‐KO mice. Adipose tissue F4/80‐positive crown‐like structures were also reduced in MLKL‐KO mice. HepG2 cells treated with necrosulfonamide (an MLKL inhibitor) showed reduced Nile red staining and reduced SREBP1c and SCD‐1 expressions. Stimulation of necroptosis using lipopolysaccharide + caspase inhibitor (zVAD) increased CXCL1/2 expressions in U937 monocyte cells. Lipopolysaccharide + zVAD‐induced increased expressions of CXCL1/2 were reduced with necrosulfonamide treatment.ConclusionsMixed lineage kinase domain‐like pseudokinase inhibition has protective effects in non‐alcoholic steatohepatitis by decreasing hepatic de novo fat synthesis and chemokine (C‐X‐C motif) ligand expressions.

BackgroundPrevious studies have demonstrated protective effects of anti-receptor interacting protein kinase 1 (RIP1), a key necroptosis molecule. However, it is uncertain whether necroptosis has a crucial role in hepatic IR injury. Therefore, we evaluated the role of necroptosis in hepatic IR injury.MethodThe IR mice underwent 70% segmental IR injury induced by the clamping of the hepatic artery and portal vein for 1 hr followed by reperfusion for 4 hr. The key necroptosis molecules (RIP1, RIP3, and MLKL) and other key molecules of regulated necrosis (PGAM5 and caspase-1) were evaluated in the warm IR injury model. A RIP1 inhibitor (necrostain-1s) and/or an anti-mitochondrial permeability transition (MPT)-mediated necrosis mediator (cyclosporine A, CyA) were administered before clamping. Necrotic injury was quantified using Suzuki’s scoring system. qRT-PCR and western blot were performed to evaluate RIP1, RIP3, MLKL and PGAM5 expressions.ResultsRIP1, RIP3, MLKL and PGAM5 expression did not change in the hepatic IR injury model. Moreover, Nec1s pretreatment did not improve histology or biochemical markers. The overall Suzuki score (cytoplasmic vacuolization, sinusoidal congestion and hepatocytes necrosis) was increased in the RIP3(-/-) mice compared to the IR group (3.5 vs. 5, p = 0.026). CyA pretreatment and/or RIP3(-/-) mice decreased Bax/Bcl2 expression; however, it did lead to an overall change in the levels of AST, ALT and LDH or necrotic injury. The Bax/Bcl2 ratio and the expression of caspase-1 and caspase-3 did not increase in our hepatic IR injury model.ConclusionKey necroptosis molecules did not increase in the necrosis-dominant hepatic IR injury model. Anti-necroptosis and/or cyclosporine-A treatment did not have an overall protective effect on necrosis-dominant hepatic IR injury.

Background Glutamate chemical exchange saturation transfer (GluCEST) imaging has been widely used in brain psychiatric disorders. Glutamate signal changes may help to evaluate the sleep‐related disorders, and could be useful in diagnosis. Purpose To evaluate signal changes in the hippocampus and cortex of a rat model of stress‐induced sleep disturbance using GluCEST. Study Type Prospective animal study. Animal Model Fourteen male Sprague–Dawley rats. Field Strength/Sequence 7.0T small bore MRI / fat‐suppressed, turbo‐rapid acquisition with relaxation enhancement (RARE) for CEST, and spin‐echo, point‐resolved proton MR spectroscopy (1H MRS). Assessment Rats were divided into two groups: the stress‐induced sleep‐disturbance group (SSD, n = 7) and the control group (CTRL, n = 7), to evaluate and compare the cerebral glutamate signal changes. GluCEST data were quantified using a conventional magnetization transfer ratio asymmetry in the left‐ and right‐side hippocampus and cortex. The correlation between GluCEST signal and glutamate concentrations, derived from 1H MRS, was evaluated. Statistical Analysis Wilcoxon rank‐sum test between CEST signals and multiparametric MR signals, Wilcoxon signed‐rank test between CEST signals on the left and right hemispheres, and a correlation test between CEST signals and glutamate concentrations derived from 1H MRS. Results Measured GluCEST signals showed significant differences between the two groups (left hippocampus; 4.23 ± 0.27% / 5.27 ± 0.42% [SSD / CTRL, P = 0.002], right hippocampus; 4.50 ± 0.44% / 5.04 ± 0.34% [P = 0.035], left cortex; 2.81 ± 0.38% / 3.56 ± 0.41% [P = 0.004], and right cortex; 2.95 ± 0.47% / 3.82 ± 0.26% [P = 0.003]). GluCEST signals showed positive correlation with glutamate concentrations (R2 = 0.312; P = 0.038). Data Conclusion GluCEST allowed the visualization of cerebral glutamate changes in rats subjected to sleep disturbance, and may yield valuable insights for interpreting alterations in cerebral biochemical information. Level of Evidence: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:1866–1872.

AIMTo validate the effects of receptor interacting protein kinase-3 (RIP3) deletion in non-alcoholic fatty liver disease (NAFLD) and to clarify the mechanism of action.METHODSWild-type (WT) and RIP3 knockout (KO) mice were fed normal chow and high fat (HF) diets for 12 wk. The body weight was assessed once weekly. After 12 wk, the liver and serum samples were extracted. The liver tissue expression levels of RIP3, microsomal triglyceride transfer protein, protein disulfide isomerase, apolipoprotein-B, X-box binding protein-1, sterol regulatory element-binding protein-1c, fatty acid synthase, cluster of differentiation-36, diglyceride acyltransferase, peroxisome proliferator-activated receptor alpha, tumor necrosis factor-alpha (TNF-α), and interleukin-6 were assessed. Oleic acid treated primary hepatocytes from WT and RIP3KO mice were stained with Nile red. The expression of inflammatory cytokines, including chemokine (C-X-C motif) ligand (CXCL) 1, CXCL2, and TNF-α, in monocytes was evaluated.RESULTSRIP3KO HF diet fed mice showed a significant gain in body weight, and liver weight, liver to body weight ratio, and liver triglycerides were increased in HF diet fed RIP3KO mice compared to HF diet fed WT mice. RIP3KO primary hepatocytes also had increased intracellular fat droplets compared to WT primary hepatocytes after oleic acid treatment. RIP3 overexpression decreased hepatic fat content. Quantitative real-time polymerase chain reaction analysis showed that the expression of very-low-density lipoproteins secretion markers (microsomal triglyceride transfer protein, protein disulfide isomerase, and apolipoprotein-B) was significantly suppressed in RIP3KO mice. The overall NAFLD Activity Score was the same between WT and RIP3KO mice; however, RIP3KO mice had increased fatty change and decreased lobular inflammation compared to WT mice. Inflammatory signals (CXCL1/2, TNF-α, and interleukin-6) increased after lipopolysaccharide and pan-caspase inhibitor (necroptotic condition) treatment in monocytes. Neutrophil chemokines (CXCL1, and CXCL2) were decreased, and TNF-α was increased after RIP3 inhibitor treatment in monocytes.CONCLUSIONRIP3 deletion exacerbates steatosis, and partially inhibits inflammation in the HF diet induced NAFLD model.