Lipoprotein lipase activity and interactions studied in human plasma by isothermal titration calorimetry

Abstract

LPL is produced and secreted from parenchymal cells like adipocytes and myocytes for transport to the luminal side of the endothelium via interaction with the glycosylphosphatidylinositol-anchored high density lipoprotein binding protein 1 (GPIHBP1) (8). Several plasma components have been shown to directly or indirectly modulate the activity of LPL. apoC-II and apoA-V increase the activity of LPL, while apoC-I, apoC-III, angiopoietin-like protein (ANGPTL)3, ANGPTL4, and ANGPTL8 decrease the activity (1, 9). The expression of each of these proteins depends on nutritional and hormonal factors, so that lipid uptake in tissues to a large extent is regulated by posttranslational effects on LPL (1, 9). It is possible that the macromolecular environment in plasma itself may be an influence on the interaction of LPL with its ligands. The protein concentration of plasma (80 g/l) has been shown to cause significant crowding effects (10). It is also possible that some plasma regulators of LPL activity have not been identified yet.LPL activity can be measured in vitro by using artificial, usually emulsified, systems of radiolabeled, fluorogenic, or chromogenic substrates, or isolated triglyceride-rich lipoproteins (TRLs). The reaction products are detected at certain time points by chemical quantification or by determination of radioactivity or fluorescence. These methods have been used to unravel important aspects of the action of LPL and also to quantitate the levels of LPL activity in cells and tissues. Only small amounts of LPL activity are normally present in the circulating blood (11). Therefore, intravenous injections of heparin are made to release LPL from its endothelial binding sites. Determination of LPL activity in postheparin plasma, using artificial substrate systems, is considered to give an estimation of the amount of active LPL at the vascular endothelium (12).Lack of a suitable technique for continuous monitoring of triglyceride hydrolysis in plasma has hampered the understanding of the action of LPL under physiological Abstract LPL hydrolyzes triglycerides in plasma lipoproteins. Due to the complex regulation mechanism, it has been difficult to mimic the physiological conditions under which LPL acts in vitro. We demonstrate that isothermal titration calorimetry (ITC), using human plasma as substrate, overcomes several limitations of previously used techniques. The high sensitivity of ITC allows continuous recording of the heat released during hydrolysis. Both initial rates and kinetics for complete hydrolysis of plasma lipids can be studied. The hydrolytic breakdown of plasma triglycerides by LPL at the capillary endothelium is a crucial event that contributes to control of the levels of triglycerides in plasma (1, 2). Many recent studies support the view that an elevated level of triglycerides in plasma is an independent risk factor for development of atherosclerosis (3-5). Therefore, the LPL system is considered to be an interesting target for drug design (6, 7). Education and Research Grant IUT 19-9,...

On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity

“…Our present results, together with our previous observations, demonstrate that the N-terminal ccd of ANGPTL3 is capable of inactivating LPL, even at substoichiometric concentrations, by catalyzing the unfolding of large parts of the catalytic domain (10,33). The levels of ANGPTL3 in human plasma are the highest among all three ANGPTLs studied here, and this level should be high enough to enforce LPL inhibition on its own (33,34). Nonetheless, no correlation between plasma levels of ANGPTL3 and plasma TG or LPL activity in tissues has yet been established.…”

“…Previous studies proposed that ANGPTL3 has weak effects on LPL on its own and that only the complex of ANGPTL3 with ANGPTL8 acts as a regulator of LPL activity (15,17,18). Our present results, together with our previous observations, demonstrate that the N-terminal ccd of ANGPTL3 is capable of inactivating LPL, even at substoichiometric concentrations, by catalyzing the unfolding of large parts of the catalytic domain (10,33). The levels of ANGPTL3 in human plasma are the highest among all three ANGPTLs studied here, and this level should be high enough to enforce LPL inhibition on its own (33,34).…”

“…In the fasted state, liver produces ANGPTL3 and ANGPTL4 that could potentially act on LPL in the bloodstream. However, the average concentration of ANGPTL4 in human plasma is too low to have any effect on the LPL activity in the presence of TG-rich lipoproteins (33,35). In the fed state, the production of ANGPTL4 in WAT is reduced, and ANGPTL4 may form inactive complexes with ANGPTL8, whose expression is greatly increased in the fed state both in WAT and in the liver.…”

Mapping the sites of the lipoprotein lipase (LPL)–angiopoietin-like protein 4 (ANGPTL4) interaction provides mechanistic insight into LPL inhibition

“…In the presence of deoxycholate, nANGPTL4 inhibits LPL by a reversible, noncompetitive mechanism (40,54). Because LPL is known to have enhanced thermal stability within the biological fluids that contain bile acids, like serum and milk, we used them to better mimic physiological conditions in our kinetic assays (58,59).…”

“…Our data suggest nANGPTL4 binds the lid and lid-proximal region of LPL, which may prevent proper lid function and inhibit substrate catalysis. There are conflicting reports whether nANGPTL4 can inhibit LPL in the presence of natural chylomicron or very LDL substrates (58,60). Here, the synthetic substrate DGGR was needed to enable precise measurements of LPL inhibition kinetics.…”