The small intestine generally transports dietary fats to circulation in triglyceride (TG)-rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low-density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco-2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco-2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono-olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco-2 cells contained significant amount of TGs and ApoB-48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco-2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80-200 nm.

Non-alcoholic fatty liver disease (NAFLD), described by the build-up of excess fat in the liver, is the most prevalent chronic liver condition globally. One of the essential metabolic functions of the liver is the production of ketone bodies, a process called, ketogenesis. Ketone bodies serve as alternative fat-derived sources of fuel for tissues under conditions of nutrient deficit (i.e., fasting). Interestingly, recent studies have found that ketogenesis is dysregulated in NAFLD patients. Similarly, we also found that high-fat diet-induced NAFLD mice exhibited diminished fasting-induced ketogenesis with reduced expression of liver Hmgcs2, the rate-limiting enzyme of ketogenesis. To understand the role of ketogenesis in NAFLD pathogenesis and treatment, we generated mouse models of ketogenic insufficiency and activation through Hmgcs2 loss- and gain-of-function, respectively. Notably, a change in dietary environment rescued the fatty liver phenotype of Hmgcs2 knockout mice and increased ketogenic function through HMGCS2 overexpression improved NAFLD-associated metabolic dysfunction and hepatosteatosis in adult mice. Furthermore, an untargeted metabolomics approach provided a comprehensive metabolic view underlying HMGCS2 overexpression-mediated NAFLD improvement, suggesting that hepatic ketogenesis impacts liver metabolism via regulation of other metabolic pathways. Together, our study adds new knowledge to the field of ketone body metabolism and suggests a viable therapeutic strategy involving ketogenesis activation in the prevention and treatment of NAFLD.

Indiana University-Purdue University Indianapolis (IUPUI) / The cell membrane serves as a barrier between the interior and exterior of a living cell. Its main structural component is the lipid bilayer, which is composed of various kinds of lipids that segregate into domains. These lipid domains, distinguished in composition and physical properties from the bulk lipids that surround them, are believed to modulate the function of resident proteins by providing an appropriate lipid environment. Polyunsaturated fatty acids (PUFA) are a type of fatty acid that contain multiple C=C double bonds. They have a lot of health benefits, which may originate in part due to their incorporation into lipids in the plasma membrane. Hypotheses that PUFA-containing lipids laterally separate into domains and/or modulate the structure of existing domains have been raised to explain the fundamental role played by PUFA. In our research, we use molecular dynamics (MD) simulations to simulate model membranes composed of PUFA-containing phospholipids and to investigate their interaction with cholesterol and vitamin E that are influential membrane constituents. The presumptive function for vitamin E in membranes is to protect PUFA against oxidation. Although the chemistry of the process is well established, the role played by the molecular structure that we address with atomistic molecular dynamics (MD) simulations remains controversial. We compared the behavior of vitamin E in lipid bilayers composed of 1-stearoyl-2-docosahexaenoylphosphatidylcholine (SDPC, 18:0-22-6PC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) via all-atom MD simulations at 37° C. SDPC represents a PUFA-containing lipid, and SOPC serves as monounsaturated control. From the calculation of van der Waals energy of interaction between vitamin E and fatty acid (FA) chains, we found higher probability that the PUFA chains surround the chromanol head group on vitamin E. This is further demonstrated by probability density maps of acyl chains around vitamin E molecules. Also, an ability to more easily penetrate deep into the PUFA containing bilayer of vitamin E is detected by faster flip-flop rate of vitamin E observed in the SDPC bilayers. These results showed that the high disorder of polyunsaturated docosahexaenoic acid (DHA) chains allows vitamin E to easily tunnel down into the bilayer and often brings the PUFA chains up to the surface of the bilayer, improving the likelihood that the reactive (hydroxyl) group on vitamin E would encounter a lipid peroxyl radical and terminate the oxidation process. Thus, the simulations indicate that the molecular structure of vitamin E supports its role as an antioxidant in a PUFA-containing membrane. A subsequent study on the partitioning of vitamin E into PUFA-containing lipids was done by analyzing the binding energy of vitamin E in the corresponding lipid bilayer. The binding energy is obtained from the potential of mean force (PMF) profile of vitamin E alone the membrane normal direction (z), which is calculated from umbrella sampling MD simulations. We found the binding in SDPC is smaller in SOPC, indicating that vitamin E does not prefer PUFA-containing phospholipids. The flip-flop rate was also estimated from the PMF profile, confirming that vitamin E flip-flops across the SDPC bilayer more easily than the SOPC bilayer. From the simulations it was noted that the membrane deforms as vitamin E is pulled out, which suggests interactions between the phospholipids contribute to the binding energy of the vitamin E. In a final study, a comparison was made between the effect on membrane organization of the three types of long chain omega-3 (n-3) PUFA found in fish oils: eicosapentaenoic acid (EPA, 20:5), DHA (22:6) and docosapentaenoic acid (DPA, 22:5). MD simulations were run on lipid bilayers composed of 1-stearoyl-2-eicosapentaenoylphosphatidylcholine (EPA-PC, 18:0-20:5PC), 1-stearoyl-2-docosapentaenoylphosphatidylcholine (DPA-PC, 18:0-22:5PC), SDPC (DHA-PC, 18:0-22:6PC) and, as a monounsaturated control, SOPC (OA-PC, 18:0-18:1PC) in the absence and presence of cholesterol. By analyzing the physical properties such as membrane order and thickness, we found all three n-3 PUFAs disorder the membrane. The disordering is greatest with EPA and least with DPA. Unique among the n-3 PUFA-containing membranes, there is region of high order in the upper portion of the DPA chain. The PUFA-containing lipids were found to less favorably interact with cholesterol compared to the OA-containing lipid, which is caused by their disorder. We speculate that differences between DPA, DHA and EPA might potentially modulate their effect on lipid domain formation.

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No description available.

Lipid oxidation degrades the quality and decreases the shelf-stability of muscle foods. The depletion of phosphates prior to cooking may be a major factor in this undesirable reaction. Thus, the effects on lipid oxidation with the use of an encapsulate to protect the phosphates during raw storage was investigated. Unencapsulated and encapsulated sodium tripolyphosphate (STP) and sodium acid pyrophosphate (SAPP), at a level of 0.5%, were compared to control samples in cooked, ground beef patties at 0 and 6 days. The unencapsulated and encapsulated treated samples were different (P<0.05) from the controls with an 81.1% to 89.7% improvement in the reduction of lipid oxidation. However, encapsulated phosphates did not decrease the level of oxidation beyond the unencapsulated treatment. This observation was attributable to the lack of a storage time prior to evaluating rancidity. Therefore, with an increase of precooked storage time, the 0.10% active encapsulated STP was essentially as effective as 0.20% unencapsulated STP for both 3 and 11 days. Unencapsulated STP (0.3% or 0.5%), encapsulated STP (0.3% or 0.5% active), a blend of unencapsulated (0.3%) and encapsulated (0.2% active) STP, and a control treatment was incorporated in ground turkey breast and stored at 3°C for 0, 5, and 10 days. The treated samples were cooked to two different endpoint temperatures (74°C and 79°C) and stored at 3°C (4 and 24 hr) before cooking. An improvement of 77% and 80% was found in the reduction of Thiobarbituric Acid Reactive Substances (TBARS) with the 0.3% and 0.5% encapsulated STP, respectively, in comparison to the unencapsulated STP. The best results were seen with a shorter storage time (4 hr) prior to cooking and a higher endpoint temperature (79°C). The unencapsulated and encapsulated STP were compared to commercial antioxidant blends, Lemo-fos and Freez-Gard FP 15, at a level of 0.5%, to determine differences in their capabilities of lipid oxidation reduction. The encapsulated phosphate was lower (P<0.05) in TBARS (3.5 mg/kg) in comparison to the treatments which ranged from 15.6 to 20.4 mg/kg. However, the CIE a* values were higher in the encapsulated samples due to the decrease in lipid oxidation. The effect of liquid nitrogen on TBARS values was investigated to identify a means of analyzing a large quantity of samples. The use of cryogenic freezing was not significantly different in TBARS in comparison with a fresh, unfrozen control. Raw and cooked ground turkey samples were submerged into liquid nitrogen and stored intact or immediately reduced in particle size to compare particle reduction effects on TBARS. The different particle reduction methods were not significantly different, although, the immediately reduced sample was more efficient in TBARS determination. The samples stored in an ultralow freezer (-80°C) for 14 and 33 days were not different (P>0.05). Overall, when encapsulated STP is used with sufficient pre-cook storage time, lipid oxidation can be more effectively reduced than with the use of unencapsulated phosphates. The use of cryogenic freezing and ultralow temperature storage can also aid in the determination of lipid oxidation in large sample quantities due to the stability of TBARS values. / Master of Science

The goal of the present study is to elucidate the modulatory effect of lipid compositions on phospholipase A2 (PLA2) activity. Sphingomyelin (SM) incorporation inhibited catalytic activity and membrane-damaging activity of native and mutated PLA2 toward egg yolk phosphatidylcholine (EYPC) vesicles. The inhibitory effects were through the reduction of membrane fluidity and modulation of the mode of membrane binding of PLA2 at water/lipid interface. The modulated effect of SM depended on inherent structural elements of PLA2. Moreover, cholesterol (Chol) incorporation into EYPC/egg yolk sphingomyelin (EYSM) vesicles relieved the inhibitory effect of sphingomyelin on PLA2 activity via lipid domain formation by SM and Chol. The effects on the interactive mode of PLA2 with phospholipids induced by the physical state changes of membrane bilayers abolished the inhibition of SM on catalytic activity and membrane-damaging activity of PLA2. Additionally, quercetin incorporation increased PLA2 activity and membrane-damaging activity toward EYPC/SM vesicles via its raft-making effect. Quercetin incorporation reduced PLA2 activity and membrane-damaging activity toward EYPC/SM/Chol vesicles via its raft-breaking effect. Membrane-inserted quercetin affected on membrane structure and membrane-bound mode of PLA2 to modulate PLA2 interfacial activity and membrane-damaging activity. Finally, studies on the effects of phosphatidylserine (PS) content on the sensitivity of lipid vesicles mimicking inner and outer plasma membrane toward PLA2 activity revealed that the membrane-binding mode adopted by PLA2 depended on the lipid composition. The effects of PS content on the extent of lipid domain formation and the conformation of PLA2 adopted at water-lipid interface modulate PLA2 catalytic activity. Collectively, these results indicate that lipid composition modulates PLA2 activity via its effects on membrane structure and membrane-bound mode of PLA2

Most gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in beta-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of corp plants.

<p>Biological membranes play host to a number of processes essential for cellular function and are the most important biological interface. The structurally complex and highly dynamic nature of the membrane poses significant measurement challenges, requiring an experimental technique capable of accessing very short, nanometer length scales, and fast, micro-pico second time scales.</p> <p>The experimental work presented in this thesis uses a variety of neutron scattering techniques to study the structure and dynamics of biologically relevant model membrane systems. The main body of this work can be sub-divided into two distinct topics: (1) lateral diffusion of lipid molecules in a bilayer; and (2) the measurement of domains in the membrane.</p> <p>Diffusion is the fundamental mechanism for lipids and proteins to move throughout the lipid matrix of a biological membrane. Despite a strong effort to model lipid diffusion, there is still no coherent model which describes the motion of lipid molecules from less than a lipid-lipid distance to macroscopic length scales. The experiments presented on this topic attempt to extend the range over which diffusion is typically measured by neutron scattering, to initiate the development of a more complete lipid diffusion model.</p> <p>Lipid domains and rafts are thought be platforms for many cellular functions; however, their small size and transient nature makes them notoriously difficult to observe. The penultimate chapter of this thesis provides evidence supporting the existence of domains in a model lipid/cholesterol system by probing of the dynamics of the system. The challenge of observing these structures directly was addressed by modifying the traditional neutron triple-axis spectrometry setup to increase its sensitivity to systems with short-range order. This technique was employed to examine the coexistence of fluid and gel domains in a single-component lipid bilayer system, as well as the presence of highly ordered lipid domains in a model membrane containing cholesterol.</p> / Doctor of Philosophy (PhD)