Faculty Bibliography

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.

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.

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.

Lipases constitute an important class of water-soluble enzymes that catalyze the hydrolysis of hydrophobic triacylglycerol (TAG). Their enzymatic activity is typically measured using multistep procedures involving isolation and quantification of the hydrolyzed products. We report here a new fluorescence method to measure lipase activity in real time that does not require the separation of substrates from products. We developed this method using adipose triglyceride lipase (ATGL) and lipoprotein lipase (LpL) as model lipases. We first incubated a source of ATGL or LpL with substrate vesicles containing nitrobenzoxadiazole (NBD)-labeled TAG, then measured increases in NBD fluorescence, and calculated enzyme activities. Incorporation of NBD-TAG into phosphatidylcholine (PC) vesicles resulted in some hydrolysis; however, incorporation of phosphatidylinositol into these NBD-TAG/PC vesicles and increasing the ratio of NBD-TAG to PC greatly enhanced substrate hydrolysis. This assay was also useful in measuring the activity of pancreatic lipase and hormone-sensitive lipase. Next, we tested several small-molecule lipase inhibitors and found that orlistat inhibits all lipases, indicating that it is a pan-lipase inhibitor. In short, we describe a simple, rapid, fluorescence-based triacylglycerol hydrolysis assay to assess four major TAG hydrolases: intracellular ATGL and hormone-sensitive lipase, LpL localized at the extracellular endothelium, and pancreatic lipase present in the intestinal lumen. The major advantages of this method are its speed, simplicity, and elimination of product isolation. This assay is potentially applicable to a wide range of lipases, is amenable to high-throughput screening to discover novel modulators of triacylglycerol hydrolases, and can be used for diagnostic purposes.

Infantile hemangioma is a vascular tumor characterized by the rapid growth of disorganized blood vessels followed by slow spontaneous involution. The underlying molecular mechanisms that regulate hemangioma proliferation and involution still are not well elucidated. Our previous studies reported that NOGOB receptor (NGBR), a transmembrane protein, is required for the translocation of prenylated RAS from the cytosol to the plasma membrane and promotes RAS activation. Here, we show that NGBR was highly expressed in the proliferating phase of infantile hemangioma, but its expression decreased in the involuting phase, suggesting that NGBR may have been involved in regulating the growth of proliferating hemangioma. Moreover, we demonstrate that NGBR knockdown in hemangioma stem cells (HemSCs) attenuated growth factor-stimulated RAS activation and diminished the migration and proliferation of HemSCs, which is consistent with the effects of RAS knockdown in HemSCs. In vivo differentiation assay further shows that NGBR knockdown inhibited blood vessel formation and adipocyte differentiation of HemSCs in immunodeficient mice. Our data suggest that NGBR served as a RAS modulator in controlling the growth and differentiation of HemSCs.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly and secretion of apolipoprotein B-containing lipoproteins. MTP transfers diverse lipids such as triacylglycerol (TAG) and phospholipids (PLs) between vesicles in vitro. Previously, we described methods to measure these transfer activities using N-7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled lipids. The NBD-TAG transfer assay is sensitive and can measure MTP activity in cell and tissue homogenates. In contrast, the NBD-PL transfer assay shows high background and is less sensitive; therefore, purified MTP is required to measure its PL transfer activity. Here, we optimized the assay to measure also the PL transfer activity of MTP in cell and tissue homogenates. We found that donor vesicles containing dioleoylphosphoethanolamine and palmitoyloleoylphosphoethanolamine result in a low background signal and are suitable to assay the PL transfer activity of MTP. This assay was capable of measuring protein-dependent and substrate-dependent saturation kinetics. Furthermore, the MTP inhibitor lomitapide blocked this transfer activity. One drawback of the PL transfer assay is that it is less sensitive at physiological temperature than at room temperature, and it requires longer incubation times than the TAG transfer assay. Nevertheless, this significantly improved sensitive assay is simple and easy to perform, involves few steps, can be conducted at room temperature, and is suitable for high-throughput screening to identify inhibitors. This assay can be adapted to measure other PL transfer proteins and to address biological and physiological importance of these activities.

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.