Perilipin-2 (PLIN2) is a gene that codes for the protein adipophilin, which is responsible for lipid storage in tissues and is associated with obesity and other metabolic diseases including fatty liver. It is generally conserved among primates, including humans. Recent studies show that savanna monkeys (Chlorocebus spp.) in South Africa, which moved to a colder climate, have both increased body mass and a duplication of the PLIN2 gene, presumably in response to colder temperatures. This project investigates variation and selection in the PLIN2 gene in silico among 73 wild savanna monkeys using an established bioinformatics pipeline consisting of command line tools, R packages, and Linux-based programs. We found significant genetic differentiation in the PLIN2 gene region at the taxonomic and population level. There are 45 SNPs outside of Hardy-Weinberg equilibrium, one being a missense variant. Our Tajima’s D results suggest balancing selection in two 1kb regions (12:60604000-60605000, D ~ 2.2, Intron 7-8 and 12:60606000-60607000, D ~ 2.4, 3’UTR). While we did not see clear evidence of any positive selective sweeps, we found 11 SNPs with integrated haplotype scores (iHS) that reach the p<0.05 threshold. Out of these, five are also out of Hardy-Weinberg equilibrium, and eight show an association with ecological variables like insolation and temperature. Humans and savanna monkeys develop obesity and other fatty diseases similarly, and the PLIN2 gene may, in part, be implicated in these diseases. A better understanding of the variation in the PLIN2 gene could provide better insights into metabolic disorders in humans.

Vitamin D has been connected with increased intramyocellular lipid (IMCL) mitochondrial function in skeletal muscle. It is also shown to prevent lipotoxicity in several tissues, but this has not yet been examined in skeletal muscle. Perilipin 2 (PLIN2), a lipid droplet protein upregulated with vitamin D treatment, is integral to managing IMCL capacity and lipid oxidation in skeletal muscle. Increased lipid storage and oxidation is associated with increased tolerance to a hyperlipidic environment and resistance to lipotoxicity. Therefore, I hypothesized that vitamin D increases β-oxidation and lipid turnover though a PLIN2 mediated mechanism, thereby preventing lipotoxicity. This hypothesis was divided into two specific aims: 1) Characterize the effect of vitamin D and PLIN2 on lipid turnover and β-oxidation in mature myotubes, and 2) Determine the role of vitamin D and PLIN2 in regulating key markers of lipotoxicity. To address these aims, cells were treated with or without vitamin D, palmitate, and PLIN2 siRNA in an eight group, 2x2x2 design. Key experiments included quantitative real time polymerase chain reaction for markers of lipid accumulation, lipolysis, and lipotoxicity; Seahorse oxygen consumption assay; 14C-palmitate oxidation assay; and analyses of lipid accumulation and profile. Failure of the palmitate treatment to produce a reliable model for lipotoxicity resulted in negative data for Aim 2 of this dissertation and a focus on vitamin D and PLIN2 knockdown treatments as a four group, 2x2 model. Aim 1 showed that vitamin D reliably increases markers of lipolysis and lipid accumulation. Most of these markers were in turn decreased after PLIN2 knockdown, and DGAT2 exhibited an interaction effect between the two treatments. Contrary to our hypothesis and some published research, PLIN2 knockdown did not prevent lipid accumulation. Vitamin D increased oxygen consumption, especially consumption driven by mitochondrial complex II. PLIN2 knockdown decreased oxygen consumption and demonstrated an interaction effect specific to mitochondrial complex II. Data in this dissertation show that vitamin D increases mitochondrial function, and these effects are at least in part accomplished through a PLIN2 mediated mechanism. However, this work lacks the data required to make specific claims regarding β-oxidation and lipid turnover. This research is some of the first to show that PLIN2 knockdown carries negative impacts for skeletal muscle mitochondria and makes valuable contributions to general knowledge of how vitamin D and lipid storage impact muscle health and function. This ultimately provides additional evidence to advocate for vitamin D supplementation as a means of improving musculoskeletal health and function. Future research should investigate how vitamin D and PLIN2 impact markers of lipotoxicity in skeletal muscle.

CYP2A5 is a major enzyme responsible for nicotine and cotinine metabolism in mice. Nicotine and cotinine enhance alcoholic fatty liver in wild type (WT) mice but not in CYP2A5 knockout (KO) mice, and reactive oxygen species (ROS) generated during the CYP2A5-mediated metabolism contributes to the enhancing effect. In combination with ethanol, nicotine and cotinine increased lipid peroxidation end product 4-hydroxynonenal (HNE) in WT mice but not in KO mice. In ethanol-fed KO mice, only 5 and 10 genes were regulated by nicotine and cotinine, respectively. However, in ethanol-fed WT mice, 59 and 104 genes were regulated by nicotine and cotinine, respectively, and 7 genes were up-regulated by both nicotine and cotinine. Plin 2 and Cdkn1a are among the 7 genes. Plin2 encodes adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, which was confirmed to be increased by nicotine and cotinine in WT mice but not in KO mice. Cdkn1a encodes P21 and elevated P21 in nuclei was also confirmed. HNE can increase P21 and P21 inhibit cell proliferation. Consistently, hepatocyte proliferation markers proliferating cell nuclear antigen (PCNA) and Ki67 were decreased in WT mice but not in KO mice by nicotine/ethanol and cotinine/ethanol, respectively. These results suggest that inhibition of liver proliferation via a ROS-HNE-P21 pathway is involved in nicotine- and cotinine-enhanced alcoholic fatty liver.