Characterization of the two PPAR target genes FIAF (Fasting-Induced Adipose Factor) and G0S2 (G0/G1 switch gene 2)

The prevalence of obesity has increased dramatically over the last decades. Obesity, defined as excess body fat, develops if energy expenditure is lower than its intake and if the surplus energy is stored in adipose tissue as fat. Excess adipose tissue, especially around the waist, is associated with an increased risk for diseases such as type 2 diabetes and atherosclerosis.These disorders aremajor causes of death from cardiovascular disease in the Western world.Common features of obesity, atherosclerosis and diabetes are insulin resistance and elevated plasma levels of triglycerides (TG) and low-density lipoprotein (LDL) cholesterol, whereas high-density lipoprotein (HDL) cholesterol is decreased.A number of the pharmacological interventions to treat early stages of atherosclerosis and type 2 diabetes target the peroxisome proliferator-activated receptors (PPARs). Activation of these transcription factors results in the expression of a variety of target genes, many of which play important roles in lipid metabolism. There are three PPAR isoforms: PPARa, PPARbor PPARd,and PPARg. Synthetic ligands for PPARaand forPPARgdecrease plasma TG levels and lower the concentration of LDL-cholesterol in blood whereas they elevate plasma HDL-cholesterol levels. Linked to their hypolipidaemic effect, they may also have hypoglycaemic effects, reducing chronically elevated insulin signalling and associated insulin resistance, which predisposes to the development of type 2 diabetes.In an effort to gain more insight into the relationship between PPAR target gene expression and its beneficial effect on lipid metabolism with regard to atherosclerosis and type 2 diabetes, the expression of genes in liver of wild-type mice and mice that lack functional PPARα was compared during fasting. Amongthe genes that were found to be differentially regulated in the wild-type and the PPARα mice, wereboththefasting-induced adipose factor (FIAF) and the G 0 /G 1 switch gene 2 (G0S2) strongly up-regulated in the wild-type mice during fasting.The research described in this thesis focuses on the characterization and elucidation of the function of these two genes and their protein products. FIAF belongs tothe family of fibrinogen/angiopoietin-like proteins and was previously found to be highly expressed in adipose tissue and to be up-regulated in response to fasting, hence its name. For G0S2, which was also highly expressed in adipose tissue and which we found to be localized to the endoplasmic reticulum (ER), no homologous genes could be found. During adipogenesis, the differentiation of pre-adipocytes into fully differentiated adipocytes, the levels of mRNA and protein for FIAF and G0S2 were greatly up-regulated.Subsequent experiments indicated that G0S2 is a direct PPARγ and probable PPARα target gene with a functional PPRE (PPAR-responsive element) in its promoter. Using the same approach, a functional PPRE was found within intron 3 of the FIAF gene, establishing FIAF as being a direct PPAR target gene too.The up-regulation of G0S2 mRNA during the differentiation of adipocytes seemed to be specific for adipogenesis, no up-regulation of G0S2 mRNA was observed during osteogenesis or myogenesis.Furthermore, G0S2 expression was associated with cell cycle arrest in3T3-L1pre-adipocytes, which is required for the differentiation of these cells into adipocytes.This indicates that G0S2 may be involved in adipocyte differentiation.Further investigation showed thatFIAF was present as the native protein and in truncated forms in bothmouse and human blood plasma. Interestingly,truncated FIAF was produced by human liver and treatment with PPARα agonist markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. The levels of both truncated and native FIAF showed marked inter-individual variation but were not associated with body mass index and were not influenced by prolonged semistarvation.To determine the physiological role of FIAF, we studied the effect of FIAF overexpression in a transgenic mouse model (FIAF-tg mice). Thetransgenic mice had markedly reduced adipose tissue stores compared to their wild-type littermates, despite similar food intake. The FIAF-tg mice also had elevated plasma levels of TG, glycerol, free fatty acids (FFA), and HDL as well as very low-density (VLDL) cholesterol. The increase of plasma TG levels was attributable to elevated VLDL levels. Oral lipid loading showed that the FIAF-tg mice had severely impaired plasma TG clearance. The effects on plasma TG levels are most likely the result of FIAF-mediated inhibition of the activity of lipoprotein lipase (LPL), a key regulator of plasma TG clearance. The elevated levels of FFA and glycerol are indicative of increased lipolysis, a notion supported by theincreased expression level of adipose triglyceride lipase (ATGL) in the adipose tissue of FIAF-tg mice. Additional genes that were differentially expressed are involved in oxidative metabolism and uncoupling, which might explain the decreased weight of the FIAF-tg mice while their food intake was similar to that of their wild-type littermates.The elevated HDL levels might be the result of FIAF-mediated inhibition of other lipases in addition to LPL, e.g.endothelial and hepatic lipase (EL/HL).Interestingly, after fractionation of mouse plasma by FPLC, the full length form of FIAF was present specifically in the HDL-containing fractions, whereas the truncated form of FIAF was specifically present in the LDL-containing fractions.In human plasma, both full length and truncated FIAF were only present in the HDL-containing fractions. In addition, the levels of truncated FIAF and HDL-cholesterol in human plasma correlated positively. Combined with our earlier finding that treatment with synthetic PPARaligandincreased the plasma levels of truncated FIAF in humans, this raises the possibility that FIAF might be involved in the mechanism by which PPARaligand treatment increases HDL-cholesterol levels in humans, resulting in a protective effect on atherosclerosis.The up-regulation of FIAF during fasting and the ability to inhibit plasma TG clearance indicate that FIAF might play an important role in repartitioning TG from adipose tissue to other tissues under circumstances of energy shortage. In addition, alterations in FIAF signalling might be involved in dyslipidemia, the presence of abnormal lipid levels in the blood. FIAF thus forms an interesting candidate for therapeutic targeting of dyslipidemia.