Kailash Gulshan

Ceramide as a Mediator of Non-Alcoholic Fatty Liver Disease and Associated Atherosclerosis

Cardiovascular disease (CVD) is a serious comorbidity in nonalcoholic fatty liver disease (NAFLD). Since plasma ceramides are increased in NAFLD and sphingomyelin, a ceramide metabolite, is an independent risk factor for CVD, the role of ceramides in dyslipidemia was assessed using LDLR-/- mice, a diet-induced model of NAFLD and atherosclerosis. Mice were fed a standard or Western diet (WD), with or without myriocin, an inhibitor of ceramide synthesis. Hepatic and plasma ceramides were profiled and lipid and lipoprotein kinetics were quantified. Hepatic and intestinal expression of genes and proteins involved in insulin, lipid and lipoprotein metabolism were also determined. WD caused hepatic oxidative stress, inflammation, apoptosis, increased hepatic long-chain ceramides associated with apoptosis (C16 and C18) and decreased very-long-chain ceramide C24 involved in insulin signaling. The plasma ratio of ApoB/ApoA1 (proteins of VLDL/LDL and HDL) was increased 2-fold due to increased ApoB production. Myriocin reduced hepatic and plasma ceramides and sphingomyelin, and decreased atherosclerosis, hepatic steatosis, fibrosis, and apoptosis without any effect on oxidative stress. These changes were associated with decreased lipogenesis, ApoB production and increased HDL turnover. Thus, modulation of ceramide synthesis may lead to the development of novel strategies for the treatment of both NAFLD and its associated atherosclerosis.

ABCA1 Mediates Unfolding of Apolipoprotein AI N Terminus on the Cell Surface Before Lipidation and Release of Nascent High-Density Lipoprotein

Objective—To gain insight into the mechanism by which ABCA1 generates nascent high-density lipoprotein. Approach and Results—HEK293 cells were stably transfected with ABCA1 vectors, encoding wild type, and the W590S and C1477R Tangier disease mutation isoforms, along with the K939M ATP-binding domain mutant. Apolipoprotein AI (ApoAI) binding, plasma membrane remodeling, cholesterol efflux, apoAI cell surface unfolding, and apoAI cell surface lipidation were determined, the latter 2 measured using novel fluorescent apoAI indicators. The W590S isoform had decreased plasma membrane remodeling and lipid efflux activities, and the C1477R isoform had decreased apoAI binding, and lipid efflux activities, whereas the K939M isoform did not bind apoAI, remodel the membrane, or efflux cholesterol. However, all ABCA1 isoforms led to apoAI unfolding at the cell surface, which was higher for the isoforms that increased apoAI binding. ApoAI lipidation was not detected on ABCA1-expressing cells, only in the conditioned medium, consistent with rapid release of nascent high-density lipoprotein from ABCA1-expressing cells. Conclusions—We identified a third activity of ABCA1, the ability to unfold the N terminus of apoAI on the cell surface. Our results support a model in which unfolded apoAI on the cell surface is an intermediate in its lipidation and that, once apoAI is lipidated, it forms an unstable structure that is rapidly released from the cells to generate high-density lipoprotein.

ORMDL orosomucoid-like proteins are degraded by free-cholesterol-loading–induced autophagy

Significance Accumulation of toxic lipids in macrophages or human plaques leads to endoplasmic reticulum (ER) stress and induction of autophagy. However, the mechanism by which ER stress and autophagy help in fine tuning cellular lipid homeostasis to counter free-cholesterol toxicity is not clear. Our studies demonstrate that cholesterol induces the translocation of ORMDL orosomucoid-like proteins out of the ER and targets them to autophagosomes, thereby relieving their negative regulation on cellular de novo sphingolipid synthesis. In addition, ORMDL3 has been recently implicated in childhood asthma and in eosinophil trafficking and activation. Thus, our finding of increased turnover of ORMDL proteins by free cholesterol is relevant not only in context to atherosclerotic disease progression but may also shed new insights on lipid homeostasis in other human diseases. Eukaryotic cells have evolved robust mechanisms to counter excess cholesterol including redistribution of lipids into different compartments and compensatory up-regulation of phospholipid biosynthesis. We demonstrate here that excess cellular cholesterol increased the activity of the endoplasmic reticulum (ER) enzyme serine palmitoyl-CoA transferase (SPT), the rate-limiting enzyme in sphingomyelin synthesis. This increased SPT activity was not due to altered levels of SPTLC1 or SPTLC2, the major subunits of SPT. Instead, cholesterol loading decreased the levels of ORMDL1, a negative regulator of SPT activity, due to its increased turnover. Several lines of evidence demonstrated that free-cholesterol–induced autophagy, which led to increased turnover of ORMDL1. Cholesterol loading induced ORMDL1 redistribution from the ER to cytoplasmic p62 positive autophagosomes. Coimmunoprecipitation analysis of cholesterol-loaded cells showed increased association between ORMDL1 and p62. The lysosomal inhibitor chloroquine or siRNA knockdown of Atg7 inhibited ORMDL1 degradation by cholesterol, whereas proteasome inhibitors showed no effect. ORMDL1 degradation was specific to free-cholesterol loading as autophagy induced by serum starvation or general ER stress did not lead to ORMDL1 degradation. ORMDL proteins are thus previously unidentified responders to excess cholesterol, exiting the ER to activate SPT and increase sphingomyelin biosynthesis, which may buffer excess cellular cholesterol.

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

Background: Phosphatidylserine floppase activity of ABCA1 is required for optimal cholesterol efflux, as demonstrated via a floppase-impaired ABCA1 mutation. Results: Sphingomyelin depletion compensates for floppase-impaired ABCA1 and increases cell surface phosphatidylserine. Conclusion: Sphingomyelin depletion inhibits flip of anionic phospholipids and thus promotes cholesterol efflux. Significance: Flippase inhibition may serve as a novel drug target to increase cholesterol efflux. The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.

PI(4,5)P2 Is Translocated by ABCA1 to the Cell Surface Where It Mediates Apolipoprotein A1 Binding and Nascent HDL Assembly

RATIONALE The molecular mechanism by which ATP-binding cassette transporter A1 (ABCA1) mediates cellular binding of apolipoprotein A-I (apoA1) and nascent high-density lipoprotein (HDL) assembly is not well understood. OBJECTIVE To determine the cell surface lipid that mediates apoA1 binding to ABCA1-expressing cells and the role it plays in nascent HDL assembly. METHODS AND RESULTS Using multiple biochemical and biophysical methods, we found that apoA1 binds specifically to phosphatidylinositol (4,5) bis-phosphate (PIP2). Flow cytometry and PIP2 reporter-binding assays demonstrated that ABCA1 led to PIP2 redistribution from the inner to the outer leaflet of the plasma membrane. Enzymatic cleavage of cell surface PIP2 or decreased cellular PIP2 by knockdown of phosphatidylinositol-5-phosphate 4-kinase impaired apoA1 binding and cholesterol efflux to apoA1. PIP2 also increased the spontaneous solubilization of phospholipid liposomes by apoA1. Using site-directed mutagenesis, we found that ABCA1s PIP2 and phosphatidylserine translocase activities are independent from each other. Furthermore, we discovered that PIP2 is effluxed from cells to apoA1, where it is associated with HDL in plasma, and that PIP2 on HDL is taken up by target cells in a scavenger receptor-BI-dependent manner. Mouse plasma PIP2 levels are apoA1 gene dosage-dependent and are >1 μM in apoA1 transgenic mice. CONCLUSIONS ABCA1 has PIP2 floppase activity, which increases cell surface PIP2 levels that mediate apoA1 binding and lipid efflux during nascent HDL assembly. We found that PIP2 itself is effluxed to apoA1 and it circulates on plasma HDL, where it can be taken up via the HDL receptor scavenger receptor-BI.

Free-cholesterol-mediated autophagy of ORMDL1 stimulates sphingomyelin biosynthesis

Cholesterol confers unique biophysical properties to the plasma membrane bilayer that are essential for maintaining optimal membrane fluidity, which in turn regulate multiple physiological functions required to promote cellular integrity and viability. Conversely, excessive cholesterol causes pathological conditions such as atherosclerosis that can lead to heart attacks. Human atheroma macrophages carry a large burden of free cholesterol (FC) in addition to cholesterol esters. It is recognized that sterols can modulate the levels of other lipids to attain lipid homeostasis; thus, excess FC may play a role in modulating compensatory sphingolipid pathways. Recent studies have shown that excess lipids can cause ER stress and apoptosis. In contrast, autophagy may play a protective role by clearing excess lipids from macrophage foam cell lipid droplets. Interestingly, a macrophage study using a TLR4-specifc agonist showed that de novo sphingolipid biosynthesis is essential for autophagy induction, suggesting links between sphingolipid biosynthesis and autophagy. While the role of autophagy in removing excess lipids has been the focus of many studies, its role in fine-tuning cellular lipid homeostasis remains largely unexplored.

Sphingomyelin regulation of plasma membrane asymmetry, efflux and reverse cholesterol transport

Abstract Sphingomyelin (SM) is a major component of the plasma membrane in mammalian cells, and a major component of plasma lipids, which are packaged in lipoprotein particles of varying densities. SM can be synthesized de novo from serine and palmitoyl-CoA, catalyzed by the rate-limiting enzyme serine palmitoyltransferase. The antibiotic myriocin potently inhibits serine palmitoyltransferase and decreases the accumulation of SM and other sphingolipids. Sphingolipid depletion leads to reduced dietary cholesterol absorption and increased reverse cholesterol transport. Recently, we demonstrated that SM depletion increases cholesterol efflux in both ABCA1-dependent and -independent manners, which was accompanied by increased cell surface phosphatidylserine and the reduced inward translocation of phosphatidylserine. This review will discuss SM’s role in maintaining plasma membrane asymmetry and lipid raft domains, HDL metabolism, reverse cholesterol transport, and how SM depletion alters plasma membrane structure and function, leading to modulation of cholesterol extractability by lipoproteins and other acceptors.

Miltefosine increased cholesterol efflux, induced autophagy and inhibited NLRP3-inflammasome assembly and IL-1β release.

In advanced human plaques and aged patients, athero-protective pathways such as autophagy and reverse cholesterol transport (RCT) become dysfunctional, while atherogenic pathways such as NLRP3 inflammasome and TLR2/4 are induced. Here, we report that Miltefosine, an FDA approved drug for treating leishmaniasis, increased cholesterol efflux, induced membrane remodeling, and induced autophagy in macrophages. Macrophages treated with Miltefosine exhibited markedly increased ABCA1 mediated cholesterol efflux and decreased phosphatidylserine flip from the cell-surface. Miltefosine treatment of macrophages led to redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from plasma membrane to actin rich regions of the cell. RAW264.7 macrophages treated with Miltefosine showed marked increase in p62 and LC3 puncta staining vs. control cells. The Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester: free cholesterol) ratio. The TLR4 signaling pathway in LPS primed bone marrow derived macrophages was blunted by Miltefosine treatment, leading to ~75% reduction in pro-IL-1β mRNA levels. Miltefosine pretreatment of macrophages potently inhibited NLRP3 inflammasome assembly induced by LPS/ATP treatment, exhibiting ~70% reduction in ASC1 speck forming cells. Gasdermin D mediated release of mature IL-1β was reduced by ~80% in Miltefosine treated vs. control cells. The qRT-PCR and western blot analysis showed no changes in basal or LPS induced levels of inflammasome components (NLRP3, ASC1, procaspase1), while the LPS mediated induction in ROS levels was significantly blunted in Miltefosine treated vs. control macrophages. Miltefosine did not alter the AIM2 inflammasome activity indicating specific targeting of Nlrp3 inflammasome pathway. Overall, this study showed that Miltefosine targets lipid trafficking, cell migration, autophagy, and Nlrp3 inflammasome activity in macrophages. Significance Statement Atherosclerosis is a progressive inflammatory disease. In advanced human plaques, the athero-protective pathways such as reverse cholesterol transport (RCT) and autophagy become increasingly dysfunctional while atherogenic pathways such as NLRP3 inflammasome are aberrantly induced. The cholesterol efflux via RCT prevents atherosclerosis and inflammation by reducing lipid loads in foam cells. Autophagy, in addition to playing a pivotal role in removing stored cholesterol from macrophages, promotes removal of inflammasome activating stimuli from cytosol (such as damaged mitochondria), thus helping in sequestering IL-1β. Failure of foam cells to egress from plaques and prolific engulfment of modified lipids results in formation of cholesterol crystals, leading to induction of NLRP3 inflammasome assembly. The release of IL-1β from foam cells further amplify the inflammation and promote further infiltration of immune cells to plaque. How RCT, autophagy, and inflammation pathways coordinate with each other to maintain cellular homeostasis is not clear; thus, the interplay between these pathways needs to be investigated thoroughly. Increased cholesterol efflux and induced autophagy with simultaneous dampening of Nlrp3 inflammasome can prevent atherosclerosis. Here, we show that Miltefosine can target multiple pathways involved in lipid homeostasis and inflammation. The detailed investigation of mechanisms involved in Miltefosine’s action may led to novel therapeutic targets for preventing and treating atherosclerosis and CVD.