The involvement of INSIGs, DGAT1 and APOB in human lipid metabolism

Abstract: Atherosclerosis is a complex pathophysiological condition characterized by lipid accumulation in the vascular wall. These lipids are derived from the lipoproteins present in the circulation. Two major plasma lipoprotein fractions, low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs), play significant roles in the atherosclerotic process, whereas the relevance of triglyceride-rich lipoproteins (TRLs) remains less clear. However, recent epidemiological studies demonstrated a high predictive value of the plasma TG concentration for coronary heart disease (CHD). The overall aim of this thesis was to identify and characterise major regulatory proteins involved in the control of secretion of TRLs by the liver and the regulation of the plasma triglyceride concentration. To this end, the impact of two critical proteins regulating the synthesis of triglycerides and other lipids was analysed using a combination of in vitro techniques and population studies. In subsequent studies, the relevance of two additional proteins with putative roles in the regulation of TRL secretion was evaluated. The insulin-induced genes (INSIG) -1 and -2 code for proteins known to regulate the rate-limiting step in the sterol regulatory element binding protein (SREBP) pathway, a key metabolic pathway regulating triglyceride and cholesterol metabolism. INSIG1 and INSIG2 were screened for genetic variants and functional polymorphisms were identified in the promoters of INSIG1 (-169C>T) and INSIG2 (-102G>A). The associations of these polymorphisms with biochemical and anthropometric traits related to lipid and glucose metabolism were analysed in cohorts of healthy, middle-aged subjects. No relationships between the promoter polymorphisms and plasma lipid levels were observed. However, significant associations were found between the INSIG1 -169C>T polymorphism and parameters related to glucose metabolism and between the INSIG2 -102G>A polymorphism and parameters related to body weight. The unique roles of INSIG1 in hepatic metabolism and of INSIG2 in adipose tissue were corroborated by the results from expression studies in liver and adipocyte cell-lines/tissues. Factors regulating the secretion of TRLs were studied using as a model system the human hepatoma Huh7 cell-line in combination with the small interfering RNA (siRNA) inhibition technique. The impact of siRNA inhibition of apolipoprotein B (APOB), the major structural protein of triglyceride-rich lipoprotein particles, was analysed in order to elucidate the physiological significance of variation in APOB mRNA level for the rate of secretion of TRLs. Positive relationships between the APOB mRNA level and the rate of secretion of TRLs were observed under various experimental conditions. A similar positive relationship was observed in human samples between the hepatic APOB mRNA level and plasma LDL concentration, the ultimate product of intravascular remodelling of TRLs secreted by the liver. The data from these in vitro and in vivo studies thus provide further evidence for an important role of transcriptional regulation of APOB for lipid metabolism. Subsequently, we analyzed the significance of hepatic nuclear factor (HNF) 4A, a transcription factor involved in lipid and glucose homeostasis, for the regulation of secretion of TRLs. It was found that siRNA inhibition of HNF4A leads to a marked decrease in the secretion of TRLs, a phenomenon that is associated with a decreased expression of the diacylglycerol acyltransferase (DGAT) 1 gene. Functional studies substantiated the role of DGAT1 in the secretion of TRLs. These studies thus demonstrated that DGAT1 mediates the effect of HNF4A on hepatic secretion of TRLs.