Dyslipidaemia and Hepatic Steatosis: Distinct Roles of DGAT2 and DGAT1 in Hepatocytes and Enterocytes

Two major determinants of cardiovascular and diabetic risk are the amount of fat (triglyceride) accumulated in the liver (steatosis) and the amount secreted by the liver and the intestine (after a fat-containing meal) into the blood - a mjor determinant of dyslipidaemia (elevated plasma lipids, including triglycerides and cholesterol). It has previously been established that two enzymes that catalyse the reaction dedicated to the formation of triglycerides (DGAT1 and DGAT2) are very important either for survival itself (DGAT2) or for the accumulation of fat in tissues and absorption from the diet (DGAT1). Although the two catalyse the same reaction, they appear to have different functions, and they do not compensate for each other's absence. We want to elucidate the precise function of each of these proteins so as to enable the development of pharmacological strategies aimed at preventing and/or alleviating the effects of the development of steatosis and dyslipidaemia.

Using cellular models of the human liver and small-intestine triglyceride metabolism (the two tissues that are responsible for determining the two potentially pathological states of steatosis and dyslipidaemia) we will use silencing of the genes, and specific inhibitors developed against the human DGAT1 and human DGAT2 proteins to investigate which aspects of triglyceride storage (within cellular lipid droplets) and secretion (within particles released into the blood) are affected. We will test the hypothesis that DGAT2 is specialised for the net synthesis of triglycerides and that it achieves this through its utilization of a specific pool of substrate that is dependent on the synthesis of 'new' fat within the cell. We will also test the hypothesis that DGAT1 plays a role in the remodelling of the structure of triglycerides and their use for the maturation (and growth) of the particles (VLDL and chylomicrons) secreted by the liver and intestine, respectively, thus determining their size, composition and their fate within the circulation. These parameters are highy important in the development of adverse lipid profiles characteristic of (pre)diabetes and heart disease.

We will also use animal models (mice) in which the gene for DGAT1 has been disrupted, to investigate the effect that the absence of this gene has on the formation, remodelling and secretion of triglycerides in vivo. We will also use liver cells isolated from these animals to investigate how the absence of DGAT1 affects the utilization of specific substrates for triglyceride synthesis.

These studies will inform strategies aimed at preventing and treating hepatic steatosis and dyslipidaemia.

Two major risk factors for cardiometabolic risk are the triglyceride content of the liver (steatosis) and dyslipidaemia, particularly hypertriglyceridaemia which is caused primarily by hepatic and intestinal over-secretion of TAG-rich lipoproteins. Post-prandial lipaemia associates particularly strongly with the development and progression of athersclerosis. DGAT1 and DGAT2 belong to different protein families; although they catalyse the identical final and dedicated reaction of triglyceride synthesis from diglyceride, they appear to have different functions, as they do not compensate for each other in knock-out mice. The effects of the absence of DGAT1 from mouse hepatocytes in culture on these parametes will be studied using wild-type and Dgat 1-/- mice. Analysis of the size and triglyceride stereo-isomeric compositon of VLDL particles will be compared with those of cytosolic and endoplasmic reticular lumen lipid droplets. We will use siRNA-mediated gene silencing and selective inhibitors of human DGAT1 and DGAT2 on human-derived cell types (HepG2 and CaCo2 cells) to investigate the effects of the speciic inhibition of the two enzymes on TG synthesis and partitioning between intracellular accumulation and secretion and the interaction with lipogenic and lipase enzyme activities. We will use these models and primary hepatocytes prepared from DGAT1-/- mice to test the hypotheses (i) that DGAT2 acts upstream of DGAT1, (ii) that it is dependent on DAG substrate that incorporates de novo synthesised and desaturated fatty acids, (iii) that DGAT2 is specialised primarily for the net synthesis of triglycerides whereas DGAT1 is involved in the rescue and remodelling of triglyceride molecules, and provision of TAG for the full lipidation and maturation of the nascent VLDL and chylomicron particles, and (iv) that TAG-DAG substrate cycling occurs in both the cytosolic and ER lumenal compartments.

The immediate impact of the proposed work resides in the description of a proposed novel pathway leading from de novo synthesised or preformed fatty acids to secreted triglycerides in hepatocytes and enterocytes, and the distinctive roles that DGAT1 and DGAT2 have in it. This is fundamental knowledge that will elducidate the mechanisms underlying concepts first formulated in 1998 by one of the applicants (VAZ) and developed since.

The long-term impact will be on the ability of the pharma industry and physicians to use the new knowledge to develop strategies to prevent and/or manage steatosis and/or hypertriglyceridaemia simultaneously or separately on an individualised basis for the patient, depending on the type of the dysfunction in lipid metabolism with which they present to clinicians.

The results will rationalise the development of specific agents (not specifically the ones used as tools in the proposed study) by the pharma industry as drugs for the treatment of two important causes of (pre)diabetes and cardiovascular disease, namely fatty liver and hypertriglyceridaemia.

The results will be of fundamental importance for the understanding of (i) the relationships between synthesis, storage and secretion of triglycerides, (ii) the roles of de novo synthesis of fatty acids and their desaturation in triglyceride metabolism in the liver and intestine, and (iii) the proposed importance of DGAT2 as the link between dietary carbohydrate availability and triglyceride synthesis and secretion (dyslipidaemia).