Jorge A. Rodriguez

Enhanced susceptibility to pancreatitis in severe hypertriglyceridaemic lipoprotein lipase-deficient mice and agonist-like function of pancreatic lipase in pancreatic cells

Background and aims: Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia in patients with various gene mutations in lipoprotein lipase (LPL) or apolipoprotein CII. However, the exact pathogenetic mechanism has not yet been defined. Methods: Susceptibility to pancreatitis in LPL-deficient mice was compared with that of wild-type mice after intraperitoneal injections of caerulein by determination of amylase release and pancreatic pathological scores. The effect of chylomicrons and fatty acids on enzyme release, Ca2+ signalling and cell injury in isolated pancreatic acinar cells from wild-type and LPL-deficient mice was investigated. Results: Caerulein induced higher levels of serum amylase and more severe inflammation in the pancreas of LPL-deficient mice than in wild-type mice. Addition of free fatty acids or chylomicrons to isolated pancreatic acinar cells led to the release of amylase and caused cell injury at higher concentrations. The effect of chylomicrons was partially blocked by orlistat, an inhibitor of pancreatic lipase. These results suggest that increased concentrations of free fatty acids from chylomicron hydrolysis by pancreatic lipase can induce acinar cell injury. Surprisingly, pancreatic lipase, whether in its active or inactive state could act like an agonist by inducing amylase secretion without cell injury. It caused an increase in cGMP levels and conversion of cell-damaging sustained elevations of [Ca2+] to normal Ca2+ oscillations. Conclusions: LPL-deficient mice with severe hypertriglyceridaemia display enhanced susceptibility to acute pancreatitis. High levels of chylomicrons and free fatty acids result in pancreatic cell injury. Pancreatic lipase has a dual effect: generating free fatty acids from chylomicrons and preventing Ca2+ overload in pancreatic acinar cells.

The role of free fatty acids, pancreatic lipase and Ca2+ signalling in injury of isolated acinar cells and pancreatitis model in lipoprotein lipase-deficient mice

Aim and methods:  Recurrent pancreatitis is a common complication of severe hypertriglyceridaemia (HTG) often seen in patients carrying various gene mutations in lipoprotein lipase (LPL). This study investigates a possible pathogenic mechanism of cell damage in isolated mouse pancreatic acinar cells and of pancreatitis in LPL‐deficient and in wild type mice.

Lid opening and unfolding in human pancreatic lipase at low pH revealed by site-directed spin labeling EPR and FTIR spectroscopy.

The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.