Faculty Bibliography

Lysosomal phospholipid degradation produces two types of metabolites, either 2-lysophospholipids with saturated fatty acids in sn-1 position or 1-lysophospholipids with unsaturated fatty acids in sn-2 position. They may either be degraded further or re-used for phospholipid synthesis. We found that LPLAT7 (LPGAT1), an acyltransferase of the endoplasmic reticulum, re-acylates specifically lysosome-derived 1-lysophospholipids that carry an unsaturated chain. The enzymatic activity of LPLAT7 was specific for stearoyl-CoA and 1-lyso-2-acyl positional isomers of unsaturated lysophospholipids. In Huh7 cells, Lplat7 knockout prevented the reacylation of 1-lysophospholipids generated by the lysosomal degradation of exogenous ²H-phosphatidylcholine. Inhibition of lysosomal phospholipid degradation reduced the abundance of 1-stearoyl-2-unsaturated PC in Huh7 cells. Lplat7 knockout blunted the loss of unsaturated lysophosphatidylcholine (LPC) in response to lysosomal inhibition, suggesting that LPLAT7 consumes unsaturated LPC formed by lysosomes. In mice, Lplat7 knockout increased the concentration of unsaturated lysophospholipids, reduced the abundance of 1-stearoyl-2-unsaturated species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and inhibited the regeneration of cellular membranes. It also triggered the accumulation of triglycerides, confirming earlier reports that unsaturated lysophospholipids induce lipid droplet formation. Thus, by re-acylating unsaturated 1-lysophospholipids, LPLAT7 shifts lipid metabolism from the biogenesis of lipid droplets to the biogenesis of membranes.

Apolipoprotein B (APOB) containing lipoproteins contribute to atherosclerosis by entering the arterial wall through the endothelial cell (EC) surface receptors scavenger receptor-BI (SR-BI) and activin receptor-like kinase 1 (ALK1). We used N-terminal fragments of APOB, molecular modeling, and site-directed mutagenesis to identify and block the binding of chylomicrons and LDL to these receptors in cells and mice. We discovered that different APOB regions interact with SR-BI and ALK1 expressed on ECs APOB48 lipoproteins were only internalized by SR-BI. A fragment of APOB, comprising 18% of the N-terminal sequence, APOB18, reduced the uptake and transport of both chylomicrons and LDL by ECs, whereas a shorter fragment, APOB12, only blocked ALK1 mediated uptake of APOB100 containing lipoproteins. Importantly, overexpressing APOB18 decreased atherosclerosis in hypercholesterolemic mice. These findings identify the N-terminal region of APOB as the cause of atherosclerosis and illustrate an approach to treating or preventing vascular disease.

High plasma cholesterol levels substantially contribute to cardiovascular disease. Hepatic delivery of the microRNA-30c analog C2 decreased plasma cholesterol in apoB-containing lipoproteins in hypercholesterolemic C57BL/6 mice, in African green monkeys that spontaneously developed diabetes and hyperlipidemia and prevented diet-induced hypercholesterolemia in mice with humanized livers. Furthermore, C2 significantly reduced plasma cholesterol and atherosclerosis in LDL receptor knockout mice. C2 did not affect hepatic triglyceride and cholesterol, plasma ALT, AST, CK-MB, ALP, IL-6, TNF-α, and INF-ϒ, thus indicating an absence of tissue lipid accumulation and inflammatory response. In contrast, MTP inhibitor lomitapide significantly reduced plasma lipids and caused hepatic steatosis. Mechanistic studies revealed that C2 reduced hepatic microsomal triglyceride transfer protein expression, secretion of apolipoprotein B-containing lipoproteins and FA synthesis and increased hepatic FA oxidation, plasma bile acids and fecal cholesterol excretion. C2 is a first-in-class microRNA therapeutic that decreases plasma cholesterol and atherosclerosis, without causing hepatic injury and inflammatory response.

BACKGROUND & AIMS/OBJECTIVE:Lipoprotein assembly in the small intestine and liver is critical for the transport of dietary and endogenous lipids. Pla2g12b has recently been shown to play a role in lipoprotein assembly in mice livers and zebrafish larvae. Pla2g12b knockout and mutant (MUT) mice with the C129Y missense mutation have low plasma cholesterol levels. However, the role of Pla2g12b in the intestine and the reason why C129Y mutation decreases plasma lipids are unknown. METHODS:) and WT control mice were used in parallel to study plasma lipids and lipoproteins, lipid absorption and hepatic lipoprotein production studies. Transmission electron microscopy was used to visualize lipid transit through enterocytes. RESULTS:We observed that Pla2g12b expression was the highest in the duodenum. Furthermore, male and female chow fed 3-month-old MUT mice and wildtype (WT) mice expressed similar amounts of Pla2g12b protein and several genes in lipid metabolism. Nonetheless, the MUT mice had significantly lower plasma triglyceride (TG), cholesterol, HDL-C, LDL-C, apoB48, and apoB100 levels than WT mice. Several mechanisms for lower plasma lipids and lipoproteins in MUT mice were investigated. C129Y mutation had no effect on the expression of Pla2g12b and several other proteins necessary for lipid transport. Therefore, the low plasma lipid levels in MUT mice were neither due to the absence of Pla2g12b protein nor due to reductions in critical proteins in lipid transport. Next, we addressed the role of Pla2g12b in hepatic lipid mobilization and intestinal lipid absorption. MUT livers exhibited normal TG synthesis, defective TG secretion, and enhanced fat accumulation. MUT mice also showed defective intestinal TG absorption, intracellular lipid accumulation, and elevated TG excretion in the feces. CONCLUSIONS:We propose that C129 in Pla2g12b is critical for the assembly and secretion of lipoproteins by the liver and intestine.

BACKGROUND & AIM/UNASSIGNED:Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing health issue. Identifying factors that prevent hepatic lipid accumulation could inform new MASLD prevention or treatment strategies. We previously demonstrated that adipocyte microsomal triglyceride transfer protein (MTP) regulates intracellular lipolysis by inhibiting adipose triglyceride lipase activity. The aim of this study was to investigate the impact of adipose MTP deficiency on MASLD. METHODS/UNASSIGNED: RESULTS/UNASSIGNED: CONCLUSION/UNASSIGNED:These findings highlight the importance of regulated FA flux from adipose tissue to the liver and the liver's adaptive capacity to utilize adipose-derived FAs in maintaining hepatic health. Modulation of adipocyte FA release may represent a therapeutic strategy to reduce hepatic steatosis. IMPACT AND IMPLICATIONS/UNASSIGNED:This study provides significant insights into the role of adipose-specific microsomal triglyceride transfer protein in regulating hepatic lipid metabolism and its potential implications for treating metabolic dysfunction-associated steatotic liver disease. By demonstrating that microsomal triglyceride transfer protein deficiency in adipose tissue leads to increased fatty acid oxidation and reduced hepatic steatosis through enhanced PPARα activation, the research underscores the importance of adipose-liver crosstalk in maintaining liver health. These findings suggest that targeting adipocyte fatty acid release could be a promising therapeutic strategy to mitigate hepatic lipid accumulation and combat metabolic dysfunction-associated steatotic liver disease, offering a novel approach to addressing this growing health issue.

OBJECTIVES/OBJECTIVE:Triglycerides (TGs) associate with apolipoprotein B100 (apoB100) to form very low density lipoproteins (VLDLs) in the liver. The repertoire of factors that facilitate this association is incompletely understood. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytosolic lipid droplet (LD) biogenesis 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. METHODS:Experiments 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, stimulated Raman scattering (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. MAIN FINDINGS/RESULTS:1) 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 low density lipoprotein (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 CONCLUSIONS/CONCLUSIONS:The 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 an important factor in the partitioning of TG between cytosolic LDs and VLDL particles.

BACKGROUND AND AIMS/OBJECTIVE:The assembly and secretion of VLDL from the liver, a pathway that affects hepatic and plasma lipids, remains incompletely understood. We set out to identify players in the VLDL biogenesis pathway by identifying genes that are co-expressed with the MTTP gene that encodes for microsomal triglyceride transfer protein, key to the lipidation of apolipoprotein B, the core protein of VLDL. Using human and murine transcriptomic data sets, we identified small leucine-rich protein 1 (SMLR1), encoding for small leucine-rich protein 1, a protein of unknown function that is exclusively expressed in liver and small intestine. APPROACH AND RESULTS/RESULTS:To assess the role of SMLR1 in the liver, we used somatic CRISPR/CRISPR-associated protein 9 gene editing to silence murine Smlr1 in hepatocytes (Smlr1-LKO). When fed a chow diet, male and female mice show hepatic steatosis, reduced plasma apolipoprotein B and triglycerides, and reduced VLDL secretion without affecting microsomal triglyceride transfer protein activity. Immunofluorescence studies show that SMLR1 is in the endoplasmic reticulum and Cis-Golgi complex. The loss of hepatic SMLR1 in female mice protects against diet-induced hyperlipidemia and atherosclerosis but causes NASH. On a high-fat, high-cholesterol diet, insulin and glucose tolerance tests did not reveal differences in male Smlr1-LKO mice versus controls. CONCLUSIONS:We propose a role for SMLR1 in the trafficking of VLDL from the endoplasmic reticulum to the Cis-Golgi complex. While this study uncovers SMLR1 as a player in the VLDL assembly, trafficking, and secretion pathway, it also shows that NASH can occur with undisturbed glucose homeostasis and atheroprotection.

BACKGROUND:The triglyceride (TG) transfer activity of microsomal triglyceride transfer protein (MTP) is essential for lipoprotein assembly in the liver and intestine; however, its function in adipose tissue, which does not assemble lipoproteins, is unknown. Here we have elucidated the function of MTP in adipocytes. APPROACH AND RESULTS/RESULTS:mice maintained higher body temperature by mobilizing more fatty acids. Biochemical studies indicated that MTP deficiency de-repressed adipose triglyceride lipase (ATGL) activity and increased TG lipolysis. Both wild type MTP and mutant MTP deficient in TG transfer activity interacted with and inhibited ATGL activity. Thus, the TG transfer activity of MTP is not required for ATGL inhibition. C-terminally truncated ATGL that retains its lipase activity interacted less efficiently than full-length ATGL. CONCLUSION/CONCLUSIONS:Our findings demonstrate that adipose-specific MTP deficiency increases ATGL-mediated TG lipolysis and enhances energy expenditure, thereby resisting diet-induced obesity. We speculate that the regulatory function of MTP involving protein-protein interactions might have evolved before the acquisition of TG transfer activity in vertebrates. Adipose-specific inhibition of MTP-ATGL interactions may ameliorate obesity while avoiding the adverse effects associated with inhibition of the lipid transfer activity of MTP.

Mammalian spermatogenesis is associated with the transient appearance of condensed mitochondria, a singularity of germ cells with unknown function. Using proteomic analysis, respirometry, and electron microscopy with tomography, we studied the development of condensed mitochondria. Condensed mitochondria arose from orthodox mitochondria during meiosis by progressive contraction of the matrix space, which was accompanied by an initial expansion and a subsequent reduction of the surface area of the inner membrane. Compared to orthodox mitochondria, condensed mitochondria respired more actively, had a higher concentration of respiratory enzymes and supercomplexes, and contained more proteins involved in protein import and expression. After the completion of meiosis, the abundance of condensed mitochondria declined, which coincided with the onset of the biogenesis of acrosomes. Immuno-electron microscopy and the analysis of sub-cellular fractions suggested that condensed mitochondria or their fragments were translocated into the lumen of the acrosome. Thus, it seems condensed mitochondria are formed from orthodox mitochondria by extensive transformations in order to support the formation of the acrosomal matrix.

Most mammalian phospholipids contain a saturated fatty acid at the sn-1 carbon atom and an unsaturated fatty acid at the sn-2 carbon atom of the glycerol backbone group. While the sn-2 linked chains undergo extensive remodeling by deacylation and reacylation (Lands cycle), it is not known how the composition of saturated fatty acids is controlled at the sn-1 position. Here, we demonstrate that lysophosphatidylglycerol acyltransferase 1 (LPGAT1) is an sn-1 specific acyltransferase that controls the stearate/palmitate ratio of phosphatidylethanolamine (PE) and phosphatidylcholine. Bacterially expressed murine LPGAT1 transferred saturated acyl-CoAs specifically into the sn-1 position of lysophosphatidylethanolamine (LPE) rather than lysophosphatidylglycerol and preferred stearoyl-CoA over palmitoyl-CoA as the substrate. In addition, genetic ablation of LPGAT1 in mice abolished 1-LPE:stearoyl-CoA acyltransferase activity and caused a shift from stearate to palmitate species in PE, dimethyl-PE, and phosphatidylcholine. Lysophosphatidylglycerol acyltransferase 1 KO mice were leaner and had a shorter life span than their littermate controls. Finally, we show that total lipid synthesis was reduced in isolated hepatocytes of LPGAT1 knockout mice. Thus, we conclude that LPGAT1 is an sn-1 specific LPE acyltransferase that controls the stearate/palmitate homeostasis of PE and the metabolites of the PE methylation pathway and that LPGAT1 plays a central role in the regulation of lipid biosynthesis with implications for body fat content and longevity.