Pancreatic Lipase-related Protein Research Articles

Monitoring galactolipid digestion and simultaneous changes in lipid-bile salt micellar organization by real-time NMR spectroscopy

The use of Nuclear Magnetic Resonance spectroscopy for studying lipid digestion in vitro most often consists of quantifying lipolysis products after they have been extracted from the reaction medium using organic solvents. However, the current sensitivity level of NMR spectrometers makes possible to avoid the extraction step and continuously quantify the lipids directly in the reaction medium. We used real-time 1H NMR spectroscopy and guinea pig pancreatic lipase-related protein 2 (GPLRP2) as biocatalyst to monitor in situ the lipolysis of monogalactosyl diacylglycerol (MGDG) in the form of mixed micelles with the bile salt sodium taurodeoxycholate (NaTDC). Residual substrate and lipolysis products (monogalactosyl monoacylglycerol (MGMG); monogalactosylglycerol (MGG) and octanoic acid (OA) were simultaneously quantified throughout the reaction thanks to specific proton resonances. Lipolysis was complete with the release of all MGDG fatty acids. These results were confirmed by thin layer chromatography (TLC) and densitometry after lipid extraction at different reaction times. Using diffusion-ordered NMR spectroscopy (DOSY), we could also estimate the diffusion coefficients of all the reaction compounds and deduce the hydrodynamic radius of the lipid aggregates in which they were present. It was shown that MGDG-NaTDC mixed micelles with an initial hydrodynamic radius rH of 7.3 ± 0.5 nm were changed into smaller micelles of NaTDC-MGDG-MGMG of 2.3 ± 0.5 nm in the course of the lipolysis reaction, and finally into NaTDC-OA mixed micelles (rH of 2.9 ± 0.5 nm) and water soluble MGG. These results provide a better understanding of the digestion of galactolipids by PLRP2, a process that leads to the complete micellar solubilisation of their fatty acids and renders their intestinal absorption possible.

Pancreatic lipase-related protein 2 is selectively expressed by peritubular myoid cells in the murine testis and sustains long-term spermatogenesis.

Spermatogenesis is a complicated process of germ cell differentiation that occurs within the seminiferous tubule in the testis. Peritubular myoid cells (PTMCs) produce major components of the basement membrane that separates and ensures the structural integrity of seminiferous tubules. These cells secrete niche factors to promote spermatogonial stem cell (SSC) maintenance and mediate androgen signals to direct spermatid development. However, the regulatory mechanisms underlying the identity and function of PTMCs have not been fully elucidated. In the present study, we showed that the expression of pancreatic lipase-related protein 2 (Pnliprp2) was restricted in PTMCs in the testis and that its genetic ablation caused age-dependent defects in spermatogenesis. The fertility of Pnliprp2 knockout animals (Pnliprp2-/-) was normal at a young age but declined sharply beginning at 9months. Pnliprp2 deletion impaired the homeostasis of undifferentiated spermatogonia and severely disrupted the development and function of spermatids. Integrated analyses of single-cell RNA-seq and metabolomics data revealed that glyceride metabolism was changed in PTMCs from Pnliprp2-/- mice. Further analysis found that 60 metabolites were altered in the sperm of the Pnliprp2-/- animals; notably, lipid metabolism was significantly dysregulated. Collectively, these results revealed that Pnliprp2 was exclusively expressed in PTMCs in the testis and played a novel role in supporting continual spermatogenesis in mice. The outcomes of these findings highlight the function of lipid metabolism in reproduction and provide new insights into the regulation of PTMCs in mammals.

In situ monitoring of galactolipid digestion by infrared spectroscopy in both model micelles and spinach chloroplasts

Galactolipids are the main lipids from plant photosynthetic membranes and they can be digested by pancreatic lipase related protein 2 (PLRP2), an enzyme found in the pancreatic secretion in many animal species. Here, we used transmission Fourier-transform infrared spectroscopy (FTIR) to monitor continuously the hydrolysis of galactolipids by PLRP2, in situ and in real time. The method was first developed with a model substrate, a synthetic monogalactosyl diacylglycerol with 8-carbon acyl chains (C8-MGDG), in the form of mixed micelles with a bile salt, sodium taurodeoxycholate (NaTDC). The concentrations of the residual substrate and reaction products (monogalactosylmonoglyceride, MGMG; monogalactosylglycerol, MGG; octanoic acid) were estimated from the carbonyl and carboxylate vibration bands after calibration with reference standards. The results were confirmed by thin layer chromatography analysis (TLC) and specific staining of galactosylated compounds with thymol and sulfuric acid. The method was then applied to the lipolysis of more complex substrates, a natural extract of MGDG with long acyl chains, micellized with NaTDC, and intact chloroplasts isolated from spinach leaves. After a calibration performed with α-linolenic acid, the main fatty acid (FA) found in plant galactolipids, FTIR allowed quantitative measurement of chloroplast lipolysis by PLRP2. A full release of FA from membrane galactolipids was observed, that was not dependent on the presence of bile salts. Nevertheless, the evolution of amide vibration band in FTIR spectra suggested the interaction of membrane proteins with NaTDC and lipolysis products.

Interfacial adsorption and activity of pancreatic lipase-related protein 2 onto heterogeneous plant lipid model membranes

Pancreatic lipase related-protein 2 (PLRP2) exhibits remarkable galactolipase and phospholipase A1 activities, which depend greatly on the supramolecular organization of the substrates and the presence of surfactant molecules such as bile salts. The objective of the study was to understand the modulation of the adsorption mechanisms and enzymatic activity of Guinea pig PLRP2 (gPLRP2), by the physical environment of the enzyme and the physical state of its substrate. Langmuir monolayers were used to reproduce homogeneous and heterogeneous photosynthetic model membranes containing galactolipids (GL), and/or phospholipids (PL), and/or phytosterols (pS), presenting uncharged or charged interfaces. The same lipid mixtures were also used to form micrometric liposomes, and their gPLRP2 catalyzed digestion kinetics were investigated in presence or in absence of bile salts (NaTDC) during static in vitro, so called “bulk”, digestion.The enzymatic activity of gPLRP2 onto the galactolipid-based monolayers was characterized with an optimum activity at 15 mN/m, in the absence of bile salts. gPLRP2 showed enhanced adsorption onto biomimetic model monolayer containing negatively charged lipids. However, the compositional complexity in the heterogeneous uncharged model systems induced a lag phase before the initiation of lipolysis. In bulk, no enzymatic activity could be demonstrated on GL-based liposomes in the absence of bile salts, probably due to the high lateral pressure of the lipid bilayers. In the presence of NaTDC (4 mM), however, gPLRP2 showed both high galactolipase and moderate phospholipase A1 activities on liposomes, probably due to a decrease in packing and lateral pressure upon NaTDC adsorption, and subsequent disruption of liposomes.

Transcriptome analysis of the hepatopancreas in Penaeus vannamei under experimental infection with Enterocytozoon hepatopenaei (EHP)

Enterocytozoon hepatopenaei (EHP) is a specialized intracellular parasite that mainly resides in the hepatopancreas of shrimp, causing significant growth retardation in shrimp. In this study, Penaeus vannamei was infected with EHP through an artificial challenge experiment, and the different genes and pathways in the hepatopancreas between EHP-infected and healthy shrimp were analyzed by transcriptome sequencing. The results showed that a total of 240 significantly differentially expressed genes were obtained, including 99 up-regulated genes and 141 down-regulated genes. Immune-related genes such as Astakine, lysozyme, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3), and macrophage mannose receptor 1-like (MMR) were up-regulated, and the expression levels of lipid metabolism-related genes pancreatic lipase-related protein 2 (PLRP2), lysosomal acid lipase (LIPA), and adiponectin receptor protein (AdipoR) were also increased. However, several genes were down-regulated in carbohydrate and protein metabolism, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), trypsin-1, and delta-1-pyrroline-5-carboxylate synthase (ALDH18A1). The results suggested that EHP infection of shrimps could significantly activate the immune system, but the energy and material metabolism processes were disturbed. This study identified a substantial number of genes and pathways associated with EHP infection, providing a valuable resource for revealing the molecular mechanism of growth retardation in shrimp.

Pancreatic lipase related protein 1 as a potential target in triglyceride breakdown: A molecular docking studies with in vitro appraisal

Targeting obesity by altering the absorption of dietary triglycerides by inhibiting the pancreatic lipase is an interesting exploration. We aimed to understand the role of pancreatic lipase related protein 1 (PLRP1) and to explore whether flavonoids could bind to the protein and provide a treatment option for obesity. The selected 50 known plant-derived flavonoids have been tested for pancreatic lipase inhibition activity. The molecular docking analysis were performed to understand the binding interactions of the protein and molecules that have not been previously reported, along with in vitro pancreatic lipase enzyme activity of four flavonoid molecules to support the docking studies. The reported flavonoid molecules were also screened for parameters such as drug-likeness and oral bioavailability. Amongst the 50 tested flavonoid molecules, 13 molecules showing poor GI-absorption in the study. Of note, from the 13 molecules, (−)-epicatechin, cyanidin, petunidin and peonidin were predicted to exhibit the most potent binding with the protein target involving the Glu102, Ser129, ILE 97 and TRP270 residues to be the hotspots towards protein binding. The in vitro results demonstrated that the compound petunidin was potent inhibitor when compared to the control. Also, the kinetics data for all the compounds showed to possess an uncompetitive type of inhibition. Therefore, the findings of this study will be able to deliver useful insights to researchers in designing interesting weight-control drugs. Also, PLRP1 protein could open us a new insight and as an interesting target approach in the triglyceride breakdown for the management of obesity.

Lipases: it's not just pancreatic lipase!

Lipases are water-soluble enzymes that hydrolyze water-insoluble lipid molecules, such as triglycerides, phospholipids, and galactolipids. They are ubiquitous in nature and are present in humans, animals, insects, plants, fungi, and microorganisms. While we commonly consider pancreatic lipase, this review provides an overview of several lipases that are important for the digestion and metabolism of lipids in veterinary species. All of these enzymes have specific functions but share a common α/β-hydrolase fold and a catalytic triad where substrate hydrolysis occurs. The pancreatic lipase gene family is one of the best characterized lipase gene families and consists of 7 mammalian subfamilies: pancreatic lipase, pancreatic lipase related proteins 1 and 2, hepatic lipase, lipoprotein lipase, endothelial lipase, and phosphatidylserine phospholipase A1. Other mammalian lipases that play integral roles in lipid digestion include carboxyl ester lipase and gastric lipase. Although most enzymes have preferred substrate specificity, much overlap occurs across the plethora of lipases because of the similarities in their structures. This has major implications for the development and clinical utilization of diagnostic assays. These implications are further explored in our companion Currents in One Health article by Lim et al in the August 2022 issue of the Journal of American Veterinary Medical Association, which focuses on pancreatic lipase assays for the diagnosis of pancreatitis.

The suppression of pancreatic lipase-related protein 2 ameliorates experimental hepatic fibrosis in mice

Quiescent hepatic stellate cells (HSCs) store vitamin A as lipid droplets in the cytoplasm. When activated, these cells lose vitamin A and exhibit an increased capacity for proliferation, mobility, contractility, and the synthesis of collagen and other components of the extracellular matrix. Our previous work demonstrated that the lipid hydrolytic gene pancreatic lipase-related protein 2 (mPlrp2) is involved in the hydrolysis of retinyl esters (REs) in the liver. Here, we showed that bile duct ligation (BDL)-induced liver injury triggered the conditional expression of mPlrp2 in livers and describe evidence of a strong relationship between the expression of mPlrp2 and Acta-2, a marker for activated HSCs. RNA interference targeting mPlrp2 inhibited HSC activation and ameliorated hepatic fibrosis induced by BDL in mice. Liver BDL markedly reduced the adenosine level and increased the ratio between S-adenosyl-L-methionine (SAM) and S-adenosyl-L-homocysteine (SAH). Chromatin immunoprecipitation (ChIP) analysis demonstrated an increase in trimethylated histone H3K4 at the mPlrp2 promoter in BDL mice, which was associated with the conditional expression of mPlrp2 in the liver. SAM, a well-known hepatoprotective substance, inhibited mPlrp2 expression and reduced RE hydrolysis in mice with hepatic fibrosis induced by chronic CCl4 treatment. Liver fibrosis induced by CCl4 or BDL was improved in Plrp2−/− mice. Our results reveal that mPlrp2 suppression is a potential approach for treating hepatic fibrosis.

Digestion and Absorption of Milk Phospholipids in Newborns and Adults.

Milk polar lipids provide choline, ethanolamine, and polyunsaturated fatty acids, which are needed for the growth and plasticity of the tissues in a suckling child. They may also inhibit cholesterol absorption by interacting with cholesterol during micelle formation. They may also have beneficial luminal, mucosal, and metabolic effects in both the neonate and the adult. The milk fat globule membrane contains large proportions of sphingomyelin (SM), phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and some phosphatidylserine (PS), phosphatidylinositol (PI), and glycosphingolipids. Large-scale technical procedures are available for the enrichment of milk fat globule membrane (MFGM) in milk replacement formulations and food additives. Pancreatic phospholipase A2 (PLA2) and mucosal phospholipase B digest glycero-phospholipids in the adult. In the neonate, where these enzymes may be poorly expressed, pancreatic lipase-related protein 2 probably has a more important role. Mucosal alkaline SM-ase and ceramidase catalyze the digestion of SM in both the neonate and the adult. In the mucosa, the sphingosine is converted into sphingosine-1-phosphate, which is both an intermediate in the conversion to palmitic acid and a signaling molecule. This reaction sequence also generates ethanolamine. Here, we summarize the pathways by which digestion and absorption may be linked to the biological effects of milk polar lipids. In addition to the inhibition of cholesterol absorption and the generation of lipid signals in the gut, the utilization of absorbed choline and ethanolamine for mucosal and hepatic phospholipid synthesis and the acylation of absorbed lyso-PC with polyunsaturated fatty acids to chylomicron and mucosal phospholipids are important.

Structure and Function of Pancreatic Lipase-Related Protein 2 and Its Relationship With Pathological States.

Pancreatic lipase is critical for the digestion and absorption of dietary fats. The most abundant lipolytic enzymes secreted by the pancreas are pancreatic triglyceride lipase (PTL or PNLIP) and its family members, pancreatic lipase-related protein 1 (PNLIPRP1or PLRP1) and pancreatic lipase-related protein 2 (PNLIPRP2 or PLRP2). Unlike the family’s other members, PNLIPRP2 plays an elemental role in lipid digestion, especially for newborns. Therefore, if genetic factors cause gene mutation, or other factors lead to non-expression, it may have an effect on fat digestion and absorption, on the susceptibility to pancreas and intestinal pathogens. In this review, we will summarize what is known about the structure and function of PNLIPRP2 and the levels of PNLIPRP2 and associated various pathological states.

Quantitative monitoring of galactolipid hydrolysis by pancreatic lipase-related protein 2 using thin layer chromatography and thymol-sulfuric acid derivatization

Galactolipids are the most abundant lipids on earth where they are mainly found in photosynthetic membranes of plant, algae, and cyanobacteria. Pancreatic lipase-related protein 2 (PLRP2) is an enzyme with galactolipase activity allowing mammals, especially herbivores, to digest this important source of fatty acids. We present a method for the quantitative analysis of galactolipids and galactosylated products resulting from their digestion by guinea pig PLRP2 (GPLRP2), using thin-layer-chromatography (TLC), thymol-sulfuric acid as derivatization reagent and scanning densitometry for detection. Thymol-sulfuric acid reagent has been used for the colorimetric detection of carbohydrates. It is shown here that the derivatization of galactosyl group from galactolipids by this reagent is not affected by the bound acyl glycerol, acyl chains length and number of galactose residues in the polar head. This allowed quantifying simultaneously the initial substrate and all galactosylated products generated upon the hydrolysis of monogalactosyl di-octanoylglycerol (C8-MGDG) by GPLRP2 using a single calibration with C8-MGDG as reference standard. The reaction products, monogalactosyl monooctanoyl glycerol (C8-MGMG) and monogalactosyl glycerol (MGG), were identified and quantified, MGG being recovered from the aqueous phase and analyzed by a separate TLC analysis. This method is therefore suitable to quantify the products resulting from the release of both fatty acids present in MGDG and thereby shows that PLRP2 can contribute to the complete digestion of galactolipids and further intestinal absorption of their fatty acids.

Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

A chromosome-level genome assembly of the parasitoid wasp Pteromalus puparum.

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short-read, PacBio long-read and Hi-C scaffolding technologies was used to develop a high-quality chromosome-level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome-level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338Mb with a contig N50 of 38.7kb and a scaffold N50 of 1.16Mb. Hi-C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set, supporting a high-quality assembly and annotation. In total, 40.1% (135.6Mb) of the assembly is composed of repetitive sequences, and 14,946 protein-coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase-related proteins and kynurenine-oxoglutarate transaminases, which have amplified in the P.puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P.puparum. These examples illustrate how chromosome-level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.

Decreased T-cell mediated hepatic injury in concanavalin A-treated PLRP2-deficient mice

Concanavalin A (Con A) activates innate immunity and causes liver damage mediated by cytotoxic T lymphocytes (CTL) in mice. The Pancreatic lipase-related protein 2 (PLRP2) is induced by interleukin (IL)-4 in vitro in CTLs and associated with CTL functions. We examined the role of PLRP2 in a mouse model of Con A-induced T cell-mediated hepatitis. PLRP2-knockout and wild-type (WT) mice were inoculated with 20 mg/kg Con A. Mice lacking PLRP2 reduced Con A-induced hepatitis, which was manifested by a decrease in serum aminotransferase and histopathological assessment. The expression and secretion of cytokines including tumor necrosis factor-alpha (TNF-α), interferon (IFN)-γ, IL-6, and IL-1β were suppressed in Con A-treated PLRP2-knockout mice. In PLRP2 knockout mice, Con A-induced liver chemokines and adhesion molecules (such as MIP-1α, MIP-1β, ICAM-1 and MCP-1) were also down regulated. In the WT liver treated with Con A, the number of T cells (CD4+ and CD8+) and macrophages (CD11b+ F4/80+) increased significantly, while the lack of PLRP2 reduced the number of T cells in the liver, but had no effect on macrophages. The shift of the metabolic profiles was impaired in Con A-treated PLRP2-knockout mice compared to WT mice. In conclusion, these results indicate that PLRP2 deficiency reduces T-cell mediated Con A-induced hepatitis, and suggest PLRP2 is a potential target of anti-inflammatory and immunomodulatory drugs to treat immune-mediated hepatitis.

Spontaneous lid closure and substrate-induced lid opening dynamics of human pancreatic lipase-related protein 2: A computational study

Pancreatic lipases are essential dietary enzymes with a well-conserved catalytic triad covered by a lid. Unlike classical lipases, pancreatic lipase-related proteins 2 (PLRP2) are active in aqueous medium without any colipase. As their substrate accessibility is controlled by the lid in N-terminal domain, we investigated the lid dynamics of human PLRP2 (HPLRP2) in its closed and open conformational states using molecular dynamics (MD) simulation. The free energy landscape constructed from the MD trajectory of HPLRP2 in the open conformation suggests that the well-conserved W254 along with the hydrophobic region G256-V261 on the lid, which differs from the classical lipases, show larger conformational changes during the lid closure pathway of HPLRP2. Further, HPLRP2 in the closed conformation could attain the open state only after introducing a double mutation D251S/E255C and upon the addition of substrate molecules, tributyrin (Tbt). This initially disrupted the stable interactions between the lid and β6 loop, and the lid opening movement was further facilitated by the interactions of Tbt with β8 and β9 loops. Tbt also bound to C-terminal domain and significantly altered its orientation. The lid opening pathway of HPLRP2 was found to be distinct from its closing trajectory and tributyrin could access the active site even when the lid did not attain its fully-extended open conformation. The results indicate that the lid dynamics of HPLRP2 is different from the classical lipases and facilitates its function in the absence of any colipase.

Characterization of all the lipolytic activities in pancreatin and comparison with porcine and human pancreatic juices

Porcine pancreatic extracts (PPE), also named pancreatin, are commonly used as a global source of pancreatic enzymes for enzyme replacement therapy in patients with exocrine pancreatic insufficiency. They are considered as a good substitute of human pancreatic enzymes and they have become a material of choice for in vitro models of digestion. Nevertheless, while the global PPE contents in lipase, protease and amylase activities are well characterized, little is known about individual enzymes. Here we characterized the lipase, phospholipase, cholesterol esterase and galactolipase activities of PPE and compared them with those of porcine (PPJ) and human (HPJ) pancreatic juices. The phospholipase to lipase activity ratio was similar in PPJ and HPJ, but was 4-fold lower in PPE. The galactolipase and cholesterol esterase activities were found at lower levels in PPJ compared to HPJ, and they were further reduced in PPE. The enzymes known to display these activities in HPJ, pancreatic lipase-related protein 2 (PLRP2) and carboxylester hydrolase/bile salt-stimulated lipase (CEH/BSSL), were identified in PPJ using gel filtration experiments, SDS-PAGE and LC-MS/MS analysis. The galactolipase and cholesterol esterase activities of PPE indicated that PLRP2 and CEH/BSSL are still present at low levels in this enzyme preparation, but they were not detected by mass spectrometry. Besides differences between porcine and human enzymes, the lower levels of phospholipase, galactolipase and cholesterol esterase activities in PPE are probably due to some proteolysis occurring during the production process. In conclusion, PPE do not provide a full substitution of the lipolytic enzymes present in HPJ.

Pancreatic lipase-related protein 2 is responsible for the increased hepatic retinyl ester hydrolase activity in vitamin A-deficient mice.

The formation and hydrolysis of hepatic retinyl esters (RE) is a key process inmaintaining serum retinol homeostasis. During vitamin A deficiency, the activity of RE hydrolases (REH) in liver increases to cope with the reduced dietary vitamin A intake. However, it remains unclear which REH is the main enzyme responsible for RE hydrolysis in the liver under physiological conditions. Our previous studies have shown that pancreatic lipase-related protein 2 (PLRP2) is conditionally expressed in the liver and may be involved in the hydrolysis of hepatic RE. In the current study, we generated Plrp2-/- mice using transcription activator-like effector nuclease technology to investigate the role of PLRP2 in the metabolism of hepatic RE. Compared with the mice fed normal diet, the hepatic REH activity of wild-type (WT) mice fed vitamin A-deficient diet (VAD) increased significantly, while this activity did not increase in Plrp2-/- mice fed VAD. Plrp2-/- mice showed higher residual RE content in liver and lower serum retinol level, compared with WT mice fed VAD. Hepatic metabolic profiling from 1 H NMR-based metabolomics suggested that Plrp2-/- mice were more sensitive to VAD. Docking analysis and enzyme activity assay revealed that retinyl palmitate was the substrate with higher affinity for PLRP2. Our results indicate that Plrp2 can be activated in the liver and is responsible for the increased REH activity in the liver of mice fed VAD.

Carboxyl Ester Lipase May Not Mediate Lipotoxic Injury during Severe Acute Pancreatitis

Acute lipolysis of visceral fat or circulating triglycerides may worsen acute pancreatitis (AP)-associated local and systemic injury. The pancreas expresses pancreatic triacylglycerol lipase (PNLIP), pancreatic lipase-related protein 2 (PNLIPRP2), and carboxyl ester lipase (CEL), which may leak into the visceral fat or systemic circulation during pancreatitis. We, thus, aimed to determine the pancreatic lipase(s) regulating lipotoxicity during AP. For this AP, associated fat necrosis was analyzed using Western blot analysis. Bile acid (using liquid chromatography-tandem mass spectrometry) and fatty acid (using gas chromatography) concentrations were measured in human fat necrosis. The fat necrosis milieu was simulated invitro using glyceryl trilinoleate because linoleic acid is increased in fat necrosis. Bile acid requirements to effectively hydrolyze glyceryl trilinoleate were studied using exogenous or overexpressed lipases. The renal cell line (HEK 293) was used to study lipotoxic injury. Because dual pancreatic lipase knockouts are lethal, exocrine parotid acini lacking lipases were used to verify the results. PNLIP, PNLIPRP2, and CEL were increased in fat necrosis. Although PNLIP and PNLIPRP2 were equipotent in inducing lipolysis and lipotoxic injury, CEL required bile acid concentrations higher than in human fat necrosis. The high bile acid requirements for effective lipolysis make CEL an unlikely mediator of lipotoxic injury in AP. It remains to be explored whether PNLIP or PNLIPRP2 worsens AP severity invivo.

In vitro digestion of galactolipids from chloroplast-rich fraction (CRF) of postharvest, pea vine field residue (haulm) and spinach leaves.

The removal of intact chloroplasts from their cell wall confinement offers a novel way to obtain lipophilic nutrients from green biomass, especially carotenoids and galactolipids. These latter are the main membrane lipids in plants and they represent a major source of the essential α-linolenic acid (18:3; ALA). Nevertheless, knowledge on their digestion is still limited. We have developed a physical method of recovering a chloroplast-rich fraction (CRF) from green biomass and tested its digestibility in vitro under simulated gastrointestinal conditions. Using a two-step static model, CRF from both spinach leaves and postharvest, pea vine field residue (haulm) were first exposed to enzymes from rabbit gastric extracts and then either to pancreatic enzymes from human pancreatic juice (HPJ) or to porcine pancreatic extracts (PPE). The lipolysis of monogalactosyldiacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) was monitored by thin layer chromatography and gas chromatography of fatty acid methyl esters. For both CRF preparations, MGDG and DGDG were converted to monogalactosylmonoacylglycerol (MGMG) and digalactosylmonoacylglycerol (DGMG), respectively, during the intestinal phase and ALA was the main fatty acid released. Galactolipids were more effectively hydrolysed by HPJ than by PPE, and PPE showed a higher activity on MGDG than on DGDG. These findings may be explained by the higher levels of galactolipase activity in HPJ compared to PPE, which mainly results from pancreatic lipase-related protein 2. Thus, we showed that CRF galactolipids are well digested by pancreatic enzymes and represent an interesting vehicle for ALA supplementation in human diet.