Biology subjects

Goldberg, I. J.

Publications and source records attributed to Goldberg, I. J..

5 recordsLinked to original sources

bioRxiv2025

Secretion and transfer of adipose lipoprotein lipase utilizes neutral sphingomyelinase 2 generated exosomes

Lipoprotein lipase (LPL) is critical for clearance of circulating triglycerides and for tissue fatty acid supply. LPL is primarily synthesized and secreted by adipocytes into the interstitium and must traffic from there to the abluminal/basolateral side of capillary endothelial cells. There, LPL binds glycosylphosphatidylinositol-anchored protein 1, GPIHBP1, which stabilizes the protein and facilitates its movement across the endothelial cells to the luminal side where it functions in hydrolysis of lipoprotein triglycerides. Importance of LPL traffic is supported by findings that rare mutations in GPIHBP1 cause hypertriglyceridemia. However our understanding of how LPL is secreted by adipocytes and traffics to endothelial cells is incomplete. Here we examined the possibility that secretion and traffic of adipocyte LPL might involve generation of small extracellular vesicles (sEVs/exosomes) which often mediate cell-cell communication. Proteomic analysis of sEVs secreted by adipocytes showed them enriched in LPL. To study LPL secretion and transfer we generated human derived pre-adipocytes (HPA) that stably express tagged LPL (FLAG and His epitopes). LPL pulldown and sEV isolation from HPA conditioned media documented that greater than 70% of secreted LPL is present in sEVs. The mechanism for LPL secretion in sEVs was found to involve the ESCRT-independent neutral sphingomyelinase 2 (nSMase2) pathway, as treatment with the nSMase2 inhibitor GW4869 reduced secretion by 80%. The above observations were reproduced using highly sensitive nanoparticle flow cytometry. The sEV associated LPL has lipolytic activity and it is released by heparin addition indicating it is on the sEV surface. In addition, using human derived microvascular endothelial cells with stable lentiviral expression of GPIHBP1 we show that LPL positive sEVs transfer LPL to these cells, but not to control cells without GPIHBP1. Our findings suggest that sEV formation by nSMase2 controls adipocyte LPL secretion and traffic, that sEVs protect LPL activity and facilitate LPL transfer to GPIHBP1 on endothelial cells.

cell biology↗

bioRxiv2025

Endothelial lipase facilitates low-density lipoprotein (LDL) uptake in LDL receptor deficiency by a heparan sulfate proteoglycan-dependent mechanism.

Current lipid-lowering drugs are relatively ineffective in reducing low-density lipoprotein (LDL) cholesterol in patients with Familial Hypercholesterolemia (FH) due to a dysfunctional LDL receptor (LDLR). However, LDL cholesterol reductions have been achieved in FH patients using angiopoietin-like 3 (ANGPTL3) inhibitors, which act through an uncharacterized, LDLR-independent pathway that requires endothelial lipase (EL). Here, we aim to investigate ELs direct role in LDLR-independent uptake of LDL in hepatocytes. Control and LDLR-KO HepG2 cells were transfected with an empty plasmid or a plasmid encoding the human LIPG gene, and the cellular uptake of fluorescent human LDL was measured by FACS. To test the contribution of heparan sulfate proteoglycans (HSPG), LDL uptake was assessed with and without the pre-incubation with heparin or a heparinase cocktail. Finally, tetrahydrolipstatin was used to inhibit EL enzymatic activity in uptake studies. Unsurprisingly, LDLR-KO HepG2 cells showed an 80% reduction in LDL uptake compared to controls (p<0.001). Remarkably, EL overexpression almost fully rescued LDL uptake in LDLR-KO cells (p<0.001), without effects in control cells. EL-mediated LDL uptake was completely blocked by heparinases and heparin in LDLR-KO cells, suggesting a crucial role of HSPG in EL-mediated LDL uptake. Notably, treatment with tetrahydrolipstatin reduced LDL uptake in EL-overexpressing LDLR-KO cells (p=0.0015). Our data reveals that EL facilitates the uptake of LDL in hepatocytes through an LDLR-independent, HSPG-dependent pathway that involves ELs enzymatic activity. This pathway provides an additional mechanism to explain the reduction of LDL cholesterol induced by ANGPTL3 inhibitors and represents a potential druggable target to treat FH.

physiology↗

bioRxiv2025

Extracellular vesicles from chylomicron-treated endothelial cells drive macrophage inflammation

BackgroundMovement of circulating lipids into tissues and arteries requires transfer across the endothelial cell barrier. This process allows the heart to obtain fatty acids (FAs), its chief source of energy and apolipoprotein B (apoB)-containing lipoproteins to cross the arterial endothelial barrier leading to cholesterol accumulation in the subendothelial space. Multiple studies have established elevated postprandial triglyceride-rich lipoproteins (TRLs) as an independent risk factor for cardiovascular disease (CVD). We explored how chylomicrons affect ECs and transfer their FAs across the EC barrier. MethodsWe had reported that media from chylomicron-treated ECs leads to lipid droplet (LD) formation in macrophages. To determine the responsible component of this media, we assessed whether removing the extracellular vesicles (EVs) would obviate this effect. EVs from control and treated cells were then characterized by protein, lipid and microRNA (miR) content. We also studied the EV-induced transcription changes in macrophages and ECs and whether knockdown of scavenger receptor-BI (SR-BI) altered these responses. In addition, using chylomicrons labeled with [13C]oleate, we studied the uptake and release of this labed by ECs. ResultsChylomicron treatment of ECs led to an inflammatory response that included production of EVs that drove macrophage LD accumulation. The EVs contained little free fatty acids and triglyceride, but abundant phospholipids and diacylglycerols. In concert with this, [13]C labeled chylomicron triglycerides exited ECs primarily in phospholipids. EVs from chylomicron treated versus untreated ECs were larger, more abundant, and contained specific miRs. Treatment of macrophages and naive ECs with media from chylomicron-treated ECs increased expression of inflammatory genes. ConclusionsEC chylomicron metabolism produces EVs that increase macrophage inflammation and create LDs. Media containing these EVs also increases EC inflammation, illustrating an autocrine inflammatory process. FAs within chylomicron triglycerides are converted to phospholipids within EVs. Thus, EC uptake of chylomicrons constitutes an important pathway for vascular inflammation and tissue lipid acquisition.

cell biology↗

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↗

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↗