Biology subjects

Fisher, E. A.

Publications and source records attributed to Fisher, E. A..

6 recordsLinked to original sources

bioRxiv2023

FITM2 deficiency results in ER lipid accumulation, ER stress, reduced apolipoprotein B lipidation, and VLDL triglyceride secretion in vitro and in mouse liver

Structured AbstractO_ST_ABSObjectivesC_ST_ABSTriglyceride (TG) association with apolipoprotein B100 (apoB100) serves to form very low density lipoproteins (VLDL) in the liver. The repertoire of factors that facilitate this association is incompletely defined. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytoplasmic lipid droplets (LDs) in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. MethodsExperiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, simulated Raman spectroscopy (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to LDL density; 3) Both in vitro and in vivo, when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. Principal conclusionsThe results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be a limiting factor that ultimately contributes to non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH).

biochemistry↗

bioRxiv2023

β-carotene accelerates resolution of atherosclerosis by promoting regulatory T cell expansion in the atherosclerotic lesion

{beta}-carotene oxygenase 1 (BCO1) catalyzes the cleavage of {beta}-carotene to form vitamin A. Besides its role in vision, vitamin A regulates the expression of genes involved in lipid metabolism and immune cell differentiation. BCO1 activity is associated with the reduction of plasma cholesterol in humans and mice, while dietary {beta}-carotene reduces hepatic lipid secretion and delays atherosclerosis progression in various experimental models. Here we show that {beta}-carotene also accelerates atherosclerosis resolution in two independent murine models, independently of changes in body weight gain or plasma lipid profile. Experiments in Bco1-/- mice implicate vitamin A production in the effects of {beta}-carotene on atherosclerosis resolution. To explore the direct implication of dietary {beta}-carotene on regulatory T cells (Tregs) differentiation, we utilized anti-CD25 monoclonal antibody infusions. Our data show that {beta}-carotene favors Treg expansion in the plaque, and that the partial inhibition of Tregs mitigates the effect of {beta}-carotene on atherosclerosis resolution. Our data highlight the potential of {beta}-carotene and BCO1 activity in the resolution of atherosclerotic cardiovascular disease.

immunology↗

bioRxiv2022

Loss of PRMT2 in myeloid cells in normoglycemic mice phenocopies impaired regression of atherosclerosis in diabetic mice

Regression of atherosclerosis is impaired in diabetes. However, the factors mediating this effect remain incomplete. We identified Protein Arginine Methyltransferase 2 (PRMT2) as a protein whose expression in macrophages is reduced in hyperglycemia and diabetes. PRMT2 catalyzes arginine methylation to target proteins to modulate gene expression. Because PRMT2 expression is reduced in cells in hyperglycemia, we wanted to determine whether PRMT2 plays a causal role in the impairment of atherosclerosis regression in diabetes. We therefore examined the consequence of deleting PRMT2 in myeloid cells during regression of atherosclerosis in normal and diabetic mice. Remarkably, we found a significant impairment of atherosclerosis regression under normoglycemic conditions in mice lacking PRMT2 (Prmt2-/-) in myeloid cells that mimics the decrease in regression of atherosclerosis in WT mice under diabetic conditions. This was associated with increased plaque macrophage retention, as well as increased apoptosis and necrosis. PRMT2-deficient plaque CD68+ cells under normoglycemic conditions showed increased expression of genes involved in cytokine signaling and inflammation compared to WT. Consistently Prmt2-/- bone marrow derived macrophages (BMDMs) showed an increased response of pro-inflammatory genes to LPS, and a decreased response of inflammation resolving genes to IL-4. This increased response to LPS in Prmt2-/- BMDMs is via enhanced NF-kappa B activity. Thus, the loss of PRMT2 is causally linked to impaired atherosclerosis regression via a heightened inflammatory response in macrophages. That PRMT2 expression was lower in myeloid cells in plaques from human subjects with diabetes supports the relevance of our findings to human atherosclerosis.

genetics↗

bioRxiv2020

Transcriptional regulation of ACSL1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for use in both lipid catabolism and lipid synthesis, including of arachidonic acid mediators that promote inflammation. ACSL1 has also been linked to the pro-atherosclerotic effects of diabetes in mice. ACSL1 expression has been reported to be upregulated in monocytes and macrophages by hyperglycemia, as well as enhanced by inflammatory stimuli, yet surprisingly little is known about the mechanisms underlying its transcriptional regulation. Here we show that increased Acsl1 mRNA expression in mouse macrophages by hyperglycemia is via transcription initiation such that nascent ACSL1 RNA and Acsl1 promoter activity are increased. We further demonstrate that the hyperglycemic-dependent induction of Acsl1 mRNA is governed by the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), since the hyperglycemic upregulation of Acsl1 mRNA is lost in mouse bone marrow derived macrophages (BMDMs) from Chrebp knock out mice. In addition, we show that LPS treatment of mouse BMDMs increased Acsl1 mRNA, and this is attenuated by an NF-kappa B inhibitor that blocks p65 subunit binding to DNA. We further show that LPS treatment increased ACSL1 protein abundance and stimulated ACSL1 protein localization to membranes where it likely exerts its activity. Using an ACSL1 reporter gene containing the promoter and 1.6 Kb of upstream regulatory region, which contain multiple predicted CHREBP and NF-kappa B (RELA) binding sites conserved between the human and mouse ACSL1 gene, we found a synergistic increase of ACSL1 promoter activity when CHREBP and RELA were co-expressed. Thus, we have identified pathways controlling the expression of ACSL1 by hyperglycemia and inflammation through CHREBP and NF-kappa B.

molecular biology↗

bioRxiv2020

Inhibiting LXRα Phosphorylation in Hematopoietic Cells Reduces Inflammation and Attenuates Atherosclerosis and Obesity

Atherosclerosis and obesity share pathological features including inflammation mediated by innate and adaptive immune cells. LXR, a nuclear receptor, plays a central role in the transcription of inflammatory and lipid metabolic genes. LXR is modulated by phosphorylation at serine 196 (LXR pS196), however, the functional consequences of LXR pS196 in hematopoietic cell precursors in atherosclerosis and obesity have not been investigated. To assess the importance of LXR phosphorylation, bone marrow from LXRWT and S196A mice was transplanted into Ldlr-/- mice, which were fed a high fat, high cholesterol diet prior to evaluation of atherosclerosis and obesity. Plaques from S196A mice showed reduced inflammatory monocyte recruitment, lipid accumulation, and macrophage proliferation. Expression profiling of CD68+ cells from S196A mouse plaques revealed downregulation of pro-inflammatory genes and upregulation of mitochondrial genes characteristic of anti-inflammatory macrophages. Furthermore, S196A mice had lower body weight and less visceral adipose tissue; this was associated with transcriptional reprograming of the adipose tissue macrophages and resolution of inflammation resulting in less fat accumulation within adipocytes. Thus, reducing LXR pS196 in hematopoietic cells attenuates atherosclerosis and obesity by reprogramming the transcriptional activity of LXR to an anti-inflammatory phenotype.

molecular biology↗

bioRxiv2018

Phosphorylation of LXRα impacts atherosclerosis regression by modulating monocyte/macrophage trafficking

LXR activation in macrophages enhances regression of atherosclerotic plaques in mice by regulating genes crucial for cholesterol efflux, cell motility and inflammation. Diabetes, however, impairs plaque regression in mice. LXR is phosphorylated at serine 198 (pS198), which affects the expression of genes controlling inflammation, lipid metabolism and cell movement. We hypothesize that LXR function is affected by hyperglycemia through changes in LXR pS198. Indeed, macrophages cultured in diabetes relevant high glucose versus normal glucose display alterations in LXR-dependent gene expression and increased LXR pS198. We therefore examined the consequence of disrupting LXR phosphorylation (S196A in mouse LXR) during regression of atherosclerosis in normal and diabetic mice. We find that phosphorylation deficient LXR S196A reduces macrophage retention in plaques in diabetes, which is predicted to be anti-atherogenic and enhance plaque regression. However, this favorable effect on regression is masked by increased monocyte infiltration in the plaque attributed to leukocytosis in LXR S196A mice. RNA-seq of plaque macrophages from diabetic S196A mice shows increased expression of chemotaxis and decreased expression of cell adhesion genes, consistent with reduced macrophage retention by LXR S196A. Thus, the non-phosphorylated form of LXR precludes macrophage retention in the plaque. Our study provides the first evidence for a physiological role of LXR phosphorylation in modulating atherosclerosis regression. Compounds that prevent LXR phosphorylation or ligands that induce the conformation of non-phosphorylated LXR may selectively enhance macrophage emigration from atherosclerotic plaques.

genetics↗