Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, including visceral fat accumulation, dyslipidemia, insulin resistance and hypertension that significantly increase the risk for cardiometabolic disease. Males and females differ in body fat content, basal metabolic rate, and metabolic fuel utilization, which lead to sex differences in the prevalence and progression of MetS. In addition to the recognized roles of sex hormones, we have identified the sex chromosome complement (XX or XY) is an additional determinant of sex differences in adiposity and related metabolic traits. Specifically, the presence of a second X chromosome in the XX karyotype compared to the XY karyotype promotes increases fat mass accumulation. We hypothesized that XX chromosome dosage promotes fat accumulation through the action of specific X chromosome genes that are differentially expressed between XX and XY cells due to their escape from X-chromosome inactivation. We show that the dosage of the X-linked Kdm5c gene, which has consistently higher expression in XX (female) compared to XY (male) cells, contributes to the sex difference in adiposity observed between XX and XY animals. In vivo, female mice with global Kdm5c haploinsufficiency in all tissues have reduced adipose tissue compared to wild-type littermates. In vitro, Kdm5c knockdown impairs differentiation of cultured adipocytes. Generation of male and female mice with reduced or absent Kdm5c gene dosage selectively in preadipocytes leads to reduced white adipose tissue mass, increased energy expenditure, increased mitochondrial content, and improved tolerance to acute cold exposure. Consistent with Kdm5c function as a histone demethylase, adipose tissues and cultured adipocytes with reduced Kdm5c gene dosage have altered chromatin structure and gene expression levels. In particular, Kdm5c-deficient adipose tissue is characterized by activation of transcription programs that enhance thermogenesis and energy dissipation, and by altered histone methylation at the promoters of key genes for mitochondrial metabolism. In summary, this project elucidates physiological and molecular mechanisms that influence sex differences in adiposity and metabolic disease. We describe how intrinsic genetic differences that exist between XX and XY cells lead to altered epigenetic regulation of gene expression in preadipocytes, with critical effects on whole body energy balance. We postulate that this same mechanism will act across additional tissues and contribute to sex differences in many phenotypes in normo- and pathophysiology.

Lipin-2 (encoded by the LPIN2 gene) is an enzyme that acts in the synthesis of glycerolipids. LPIN2 mutations have been implicated in two distinct autoinflammatory diseases--Majeed syndrome and Familial Mediterranean Fever (FMF)--but its role in these diseases has not been described. I hypothesized that lipin-2 has a role in modulating inflammation in macrophages, and that mutations in LPIN2 may occur in FMF in Mediterranean populations, where FMF is most prevalent.

The effect of inflammatory (endotoxin) and lipotoxic (fatty acid) stimuli was assessed in peritoneal macrophages from wild-type and lipin-2-deficient mice. Differences in the induction of pro-inflammatory cytokines were observed between the two genotypes in response to endotoxin. LPIN2 exon sequencing in MEFV heterozygous FMF patients from Israel identified four LPIN2 variants, but these are not likely to be pathogenic. This suggests that mutations in LPIN2 are not prevalent in FMF in the Israeli population.

Lipins are a conserved family of phosphatidate phosphatase (PAP) enzymes that catalyze a critical step in glycerolipid biosynthesis, namely the removal of the phosphate group from phosphatidic acid to form diacylglycerol. While most lower organisms possess a single lipin gene, mammalian genomes contain genes for three lipin family members--lipin-1, lipin-2, and lipin-3--which presumably arose through gene duplication. Distinct tissue expression patterns, as well as unique disease phenotypes associated with different lipin deficiencies, suggest that each family member has a unique role in vivo. Lipin-1 deficiency causes lipodystrophy in the mouse, a consequence of its vital role in adipogenesis and triglyceride storage in adipose tissue. Lipin-2 deficiency affects lipid homeostasis in non-adipose tissues, including liver and brain, and causes a cyclical fever disease in humans called Majeed Syndrome. Little is known about the physiological function of lipin-3, expressed mainly in kidney, intestine, adipose tissue, and liver.

We are interested in teasing apart the unique physiological roles of each lipin family member. A simple working hypothesis is that each lipin is responsible for providing PAP activity in a unique but potentially overlapping set of tissues. In this dissertation, we expand on that model with the discovery of a complex system of cooperative relationships between lipin family members. We demonstrate that lipins work together to maintain lipid homeostasis in diverse tissues, from liver and brain (lipin-1 and lipin-2) to adipose tissue (lipin-1 and lipin-3) to intestine (lipin-2 and lipin-3). We also explore further the established roles of lipin-1 in adipogenesis and lipin-2 in Majeed Syndrome. Together, the findings in this dissertation build upon our previous model of lipin family function and demonstrate that the three mammalian lipins exist not only as separate enzymes specialized for distinct tissues. Instead, they should also be viewed as a cohesive family unit, responding to and functionally interacting with one another to achieve a common end of lipid homeostasis. The combination of individual and cooperative roles is customized for each tissue to create a sophisticated mammalian organism.

The Diet1 gene was identified in an inbred mouse strain that is resistant to diet-induced elevations in plasma cholesterol and atherosclerosis. Diet1 (also known as Malrd1: MAM and LDL Receptor Class A Domain Containing 1) is expressed nearly exclusively in two tissues, the small intestine and kidney. Previous studies identified a role for Diet1 in the regulation of an intestinal hormone that restrains cholesterol conversion to bile acids in the liver. This dissertation further elucidates the role of Diet1 in the absorptive epithelium of the small intestine and provides the initial characterization of Diet1 function in kidney proximal tubules. Following an introductory chapter, Chapter 2 reports the parallels between Diet1-deficiency in the mouse and human primary bile acid diarrhea. These include elevated bile acid levels, increased gastrointestinal motility, and reduced levels of the intestinal hormone fibroblast growth factor 15 (FGF19 in humans). This work also identifies a genetic variant in the coding sequence of human DIET1 that regulates FGF15/19 secretion and is associated with bile acid diarrhea. Chapter 3 further explores the role of Diet1 in intestinal lipid homeostasis and identifies a role for Diet1 in the adaptation to the increased demands for nutrient absorption that occur in mice fed a lipid-enriched diet. Chapter 4 describes the first studies of Diet1 function in the kidney and identifies a role in proximal tubule epithelial cell function. Following the identification of genetic variants in human DIET1 that are associated with albuminuria, we determined that Diet1-deficient mice develop albuminuria due to proximal tubular endocytic dysfunction with intact glomerular function. Transcriptome profiling of primary proximal tubule cells revealed that Diet1 influences the expression of genes associated with extracellular matrix, focal adhesion, and tight junction complexes, many of which are substrates for the meprin metalloproteases. We demonstrated that Diet1 and meprin proteins interact, and that Diet1 deficiency alters meprin protein levels and subcellular localization, which likely contributes to impaired proximal tubule barrier function and paracellular flux of proteins such as albumin in Diet1-deficient mice. The concluding chapter synthesizes our findings that establish a role for Diet1 in the absorptive properties and barrier function of epithelial cells of the small intestine and kidney proximal tubule.

Obesity is associated with numerous metabolic disorders, including hypertension, hyperlipidemia, insulin resistance, and atherosclerosis. In addition, obesity is a risk factor for cardiovascular disease and cancer, the leading causes of mortality. It is thus critical to understand the environmental and genetic components that contribute to the development of obesity, one of which is sex.

The sexual dimorphism in fat accumulation and distribution has been well established for a few decades. Premenopausal women tend to gain fat near the hips and thighs, while men and postmenopausal women tend to accumulate fat in the abdominal cavity. These sex differences have often been attributed to sex hormones, but sex differences in metabolic traits remain long after gonadal hormones have declined. This suggests that factors other than sex hormones could contribute to sex differences in metabolism.

The sex chromosome complement is the fundamental difference between male and female cells. Because XX chromosomes often coexist with ovaries and XY chromosomes are coupled with testes, the distinct effects of gonadal type and sex chromosomes are difficult to distinguish. A unique mouse model called the Four Core Genotypes (FCG) generates XX female, XX male, XY female, and XY male mice, and is an innovative tool to study effects of the sex chromosome complement (XX male/female vs. XY male/female) independently from effects of gonadal hormones (XX/XY male vs. XX/XY female).

We used FCG mice to study sex differences in obesity and related metabolic disorders. We compared gonadally intact mice with gonadectomized mice to assess acute effects of gonadal hormones, and we nutritionally challenged mice with a high fat diet and a high cholesterol diet to identify diet-sex interactions. Male and female mice with two X chromosomes gain more weight, have increased adiposity, and accumulate more hepatic lipid compared to XY mice. In addition, XX mice have increased HDL cholesterol levels, regardless of the dietary or hormone milieu. Sex chromosome differences were also detected at the molecular level, modulating mRNA and microRNA expression.

Our studies demonstrate that the sex chromosome complement is a major determinant of body weight, adiposity, and associated metabolic traits such as hepatic lipid and plasma lipid levels. The key findings from this dissertation will shed light on sex differences in obesity and are important for improving prevention and treatment of metabolic disease.

Members of the lipin protein family -- lipin-1, lipin-2 and lipin-3 -- catalyze the penultimate step in triacylglycerol (TAG) synthesis, the dephosphorylation of phosphatidic acid (PA) to form diacylglycerol. The founding member of the family, lipin-1, has been studied most extensively and has been implicated in metabolic diseases that include lipodystrophy, obesity and insulin resistance. The three lipin proteins exhibit distinct tissue distributions and therefore likely have unique physiological roles, which at present are not well understood. The focus of this dissertation has been to augment our understanding of the physiological and molecular roles of lipin family members through the use of various genetic and molecular tools. We generated lipin-2-deficient mice and determined that loss of lipin-2 causes hepatic and neurological defects in mice. These symptoms were associated with impaired TAG homeostasis in liver, and the accumulation of the lipin enzyme substrate, PA, in cerebellum. Interestingly, in both tissues, we uncovered relationships between lipin-1 and lipin-2 activities, which were particularly important in maintaining hepatic lipid homeostasis under conditions of metabolic stress such as a high fat diet, and in cerebellar function during aging. In addition to the functional interactions between lipin-1 and lipin-2, we determined that the two proteins interact in a protein complex in liver. Furthermore, lipin-1 and lipin-2 influence the protein levels of one another, and this requires a sequence motif in lipin-1 that has been shown to promote nuclear localization and PA binding. Analysis of lipin-2 KO mice also led to the discovery of a role for lipin proteins in bone homeostasis, with lipin-2 deficiency causing altered trabecular bone architecture, which was exacerbated on a high fat diet. Finally, an initial characterization of lipin-3 deficiency in mice is presented. Overall, these studies show that lipin protein family members cooperate to maintain normal glycerolipid homeostasis in vivo. The further elucidation of the regulation and function of lipin protein interactions will contribute to our understanding of lipid homeostasis in normal and diseased states.