Faculty Bibliography

High plasma lipid/lipoprotein levels are risk factors for various metabolic diseases. We previously showed that circadian rhythms regulate plasma lipids, and deregulation of these rhythms cause hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific Bmal1-deficient mice maintained on a chow or a Western diet developed hyperlipidemia, denoted by the presence of higher amounts of triglyceride- and ApoAIV-rich larger chylomicron and very-low-density lipoprotein, due to overproduction. Bmal1 deficiency decreased Shp and increased MTP, a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates Crebh to modulate ApoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh- and ApoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on ApoAIV expression and lipoprotein size. These studies ind15icate that regulation of ApoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and Rev-erbα interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and ApoAIV, via two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia, and avoiding disruptions in circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.

High plasma lipid levels have been demonstrated to increase cardiovascular disease risk. Despite advances in treatments to decrease plasma lipids, additional therapeutics are still needed because many people are intolerant or nonresponsive to these therapies. We previously showed that increasing cellular levels of microRNA-30c (miR-30c) using viral vectors or liposomes reduces plasma lipids and atherosclerosis. In this study, we aimed to synthesize potent miR-30c analogs that can be delivered to hepatoma cells without the aid of viral vectors and lipid emulsions. We hypothesized that modification of the passenger strand of miR-30c would increase the stability of miR-30c and augment its delivery to liver cells. Here, we report the successful synthesis of a series of miR-30c analogs by using different chemically modified nucleosides. In these analogs, we left the active sense strand untouched so that its biological activity remained unaltered, and we modified the passenger strand of miR-30c to enhance the stability and uptake of miR-30c by hepatoma cells through phosphorothiorate linkages and the addition of GalNAc. We show that these analogs significantly reduced apolipoprotein B secretion in Huh-7 human hepatoma cells and human primary hepatocytes without affecting apolipoprotein A1 secretion and cellular lipid levels. Our results provide a proof of concept that the passenger strand of miR-30c can be modified to increase its stability and delivery to cells while retaining the potency of the sense strand. We anticipate these miR-30c analogs will be useful in the development of more efficacious analogs for the treatment of hyperlipidemias and cardiovascular diseases.

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.

Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

Microsomal triglyceride transfer protein (MTTP) is an endoplasmic reticulum resident protein that is essential for the assembly and secretion of triglyceride (TG)-rich, apoB-containing lipoproteins. Although the function and structure of mammalian MTTP have been extensively studied, how exactly MTTP transfers lipids to lipid acceptors and whether there are other biomolecules involved in MTTP-mediated lipid transport remain elusive. Here we identify a role in this process for the poorly characterized protein PRAP1. We report that PRAP1 and MTTP are partially colocalized in the endoplasmic reticulum. We observe that PRAP1 directly binds to TG and facilitates MTTP-mediated lipid transfer. A single amino acid mutation at position 85 (E85V) impairs PRAP1's ability to form a ternary complex with TG and MTTP, as well as impairs its ability to facilitate MTTP-mediated apoB-containing lipoprotein assembly and secretion, suggesting that the ternary complex formation is required for PRAP1 to facilitate MTTP-mediated lipid transport. PRAP1 is detectable in chylomicron/VLDL-rich plasma fractions, suggesting that MTTP recognizes PRAP1-bound TG as a cargo and transfers TG along with PRAP1 to lipid acceptors. Both PRAP1-deficient and E85V knock-in mutant mice fed a chow diet manifested an increase in the length of their small intestines, likely to compensate for challenges in absorbing lipid. Interestingly, both genetically modified mice gained significantly less body weight and fat mass when on high-fat diets compared with littermate controls and were prevented from hepatosteatosis. Together, this study provides evidence that PRAP1 plays an important role in MTTP-mediated lipid transport and lipid absorption.

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.