Hormone-sensitive Lipase Is a Cholesterol Esterase of the Intestinal Mucosa
2003; Elsevier BV; Volume: 278; Issue: 8 Linguagem: Inglês
10.1074/jbc.m208513200
ISSN1083-351X
AutoresJacques Grober, Stéphanie Lucas, Maria Sörhede‐Winzell, Isabelle Zaghini, Aline Mairal, Juan-Antonio Contreras, Philippe Besnard, Cecilia Holm, Dominique Langin,
Tópico(s)Enzyme Catalysis and Immobilization
ResumoThe identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5′-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine. The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5′-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine. Hormone-sensitive lipase (HSL) 1The abbreviations used are: HSL, hormone-sensitive lipase; ABC, ATP-binding cassette; ALBP, adipocyte lipid-binding protein; I-FABP, intestinal fatty acid-binding protein; RT, reverse transcription 1The abbreviations used are: HSL, hormone-sensitive lipase; ABC, ATP-binding cassette; ALBP, adipocyte lipid-binding protein; I-FABP, intestinal fatty acid-binding protein; RT, reverse transcription is a multifunctional enzyme with broad substrate specificity (1Holm C. Østerlund T. Laurell H. Contreras J.-A. Annu. Rev. Nutr. 2000; 20: 365-393Google Scholar). It hydrolyzes tri-, di-, and monoacylglycerols, cholesteryl esters, and retinyl esters. The activity against diacylglycerol is higher than the activity toward tri- and monoacylglycerols. The enzyme also exhibits cholesterol esterase activity, which is almost twice the activity toward triacylglycerols. Much has been learned in the recent years about the domain structure of HSL. Sequence comparisons revealed that HSL belongs to a family of esterases which is mainly represented by prokaryotic enzymes (2Langin D. Holm C. Trends Biochem. Sci. 1993; 18: 466-467Google Scholar, 3Hemilä H. Koivula T.T. Palva I. Biochim. Biophys. Acta. 1994; 1210: 249-253Google Scholar). From a structural point of view, HSL is the most complex protein of the family. Sequence alignments together with biochemical experiments suggest that adipocyte HSL is composed of two structural domains (4Contreras J.-A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Google Scholar,5Østerlund T. Danielsson B. Degerman E. Contreras J.-A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Google Scholar). The first 315 amino acids make up the N-terminal domain, which shows very little sequence similarities to other known proteins. The region responsible for the interaction with adipocyte lipid-binding protein (ALBP) was mapped to this domain (6Shen W.J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Google Scholar, 7Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Sridhar K. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Google Scholar). In adipose tissue, ALBP could increase the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and through specific protein-protein interactions. The C-terminal domain is divided in two functional parts, a catalytic core and a regulatory module. The latter is composed of 150 amino acids, including all of the known phosphorylation sites of HSL. Unlike other known mammalian triacylglycerol lipases, the activity of HSL is regulated by phosphorylation. The phosphorylation sites of protein kinase A, extracellular signal-regulated kinase, and AMP-dependent protein kinase have been mapped (8Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Google Scholar, 9Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Google Scholar, 10Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Google Scholar). The catalytic core is the region that shows homology with the other members of the family. Modeling of the part revealed that it adopts an α/β-hydrolase fold that harbors the catalytic triad constituted by Ser423, Asp703, and His733 (4Contreras J.-A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Google Scholar,11Wei Y. Contreras J.-A. Sheffield P. Østerlund T. Derewenda U. Kneusel R.E. Matern U. Holm C. Derewenda S. Nature Struct. Biol. 1999; 6: 340-345Google Scholar).Several forms of HSL transcripts and the exon-intron organization of the HSL gene have been characterized in humans. The 88-kDa adipocyte HSL is translated from a 2.8-kb mRNA and encoded by 9 exons (12Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4897-4901Google Scholar,13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). The transcription start site was mapped in a short noncoding exon called exon B. In the adenocarcinoma cell line HT29, two mRNA species are found, the adipocyte HSL mRNA and a mRNA with a different 5′-end transcribed from exon A. Two testicular forms of HSL have been characterized. The 3.9-kb mRNA encodes a 120-kDa protein that contains a unique N-terminal region encoded by exon T1, a region that presumably forms a third structural domain in this isoform (14Stenson Holst L. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser H.W. Edgren G. Holm C. Genomics. 1996; 35: 441-447Google Scholar). The 3.3-kb mRNA encodes a protein that is identical to the adipocyte HSL form (15Mairal A. Melaine N. Laurell H. Grober J. Stenson Holst L. Guillaudeux T. Holm C. Jégou B. Langin D. Biochem. Biophys. Res. Commun. 2002; 291: 286-290Google Scholar). However, the mRNA species differ in their 5′-ends. Exon usage is mutually exclusive, exon T2 being only transcribed in testis and exon B being transcribed in adipose tissue.The nature and role of lipases and esterases participating in the digestion of dietary lipids in the lumen of the gastrointestinal tract are well established. In addition to the enzymes in the lumen, there is evidence of lipase activity in enterocytes (16Shen H. Howles P. Tso P. Adv. Drug Delivery Rev. 2001; 50: S103-S125Google Scholar). The presence of cholesterol esterase activity is more elusive. The exact identity of the enzymes responsible for the hydrolysis of intracellular acylglycerols and cholesteryl esters is still unclear. Pancreatic triacylglycerol lipase, microsomal triacylglycerol hydrolase, and pancreatic cholesterol esterase have all been suggested to be responsible for the hydrolytic activities (17Field F.J. J. Lipid Res. 1984; 25: 389-399Google Scholar, 18Dolinsky V.W. Sipione S. Lehner R. Vance D.E. Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar, 19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Pancreatic triacylglycerol lipase may be synthesized by the small intestine and accounts for the alkaline lipase activity of the enterocytes (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Microsomal triacylglycerol hydrolase could also be involved (18Dolinsky V.W. Sipione S. Lehner R. Vance D.E. Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar). However, it has been shown that most of the lipase activity is cytosolic (20Rao R.H. Mansbach II, C.M. Arch. Biochem. Biophys. 1993; 304: 483-489Google Scholar). Pancreatic cholesterol esterase, also called bile salt-stimulated lipase, is able to hydrolyze cholesteryl esters and triacylglycerols. In the absence of bile salts, the contribution of this enzyme is presumably minor (21Holm C. Olivecrona G. Ottosson M. Methods Mol. Biol. 2001; 155: 97-119Google Scholar). Because HSL is a cytosolic enzyme with a wide range of hydrolytic activities, the purpose of the present paper was to determine whether HSL was expressed in the intestine and could contribute to the hydrolysis of intracellular lipids.DISCUSSIONHere we have found that HSL contributes to lipase activity and is the major cholesterol esterase in the intestinal mucosa. HSL mRNA and protein were readily detected along the small intestine. Immunohistochemistry data revealed that the enzyme is expressed preferentially in differentiated cells of the villi. Data from HSL-null mice showed that HSL does not account for a significant part of total esterase activity. However, the enzyme is responsible for all neutral cholesteryl ester hydrolase activity both in the cytosolic and particulate fractions. It also contributes between one-third and one-half of diacylglycerol lipase activity in the jejunum.The size of intestinal HSL mRNA and protein corresponds to those of mouse adipocyte HSL, i.e. a 2.6–2.8-kb mRNA and an 82-kDa protein. The 5′-ends of the mRNA species are transcribed from two exons that correspond to human exons A and B. Exon A is located in the mouse gene ≈ 7 kb upstream of exon B (25Laurin N.N. Wang S.P. Mitchell G.A. Mamm. Genome. 2000; 11: 972-978Google Scholar). Use of the two exons is mutually exclusive. Interestingly, we have shown previously that HSL is expressed in the human adenocarcinoma cell line of intestinal origin HT29 (26Remaury A. Laurell H. Grober J. Reynisdottir S. Dauzats M. Holm C. Langin D. Biochem. Biophys. Res. Commun. 1995; 207: 175-182Google Scholar). The two mRNA 5′-ends were found in HT29 HSL mRNA (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). In human and mouse adipose tissue, the main transcription start site is located in exon B. Exon A-containing transcripts are found at very low levels in humans (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar) and are much less abundant than mRNA with exon B in mice ((25) and present work). To date, there is little information on the mechanisms controlling tissue-specific expression of HSL. We have shown that the first 95 bp of human exon T1 5′-flanking region conferred expression of a reporter gene exclusively in testis of transgenic mice (27Blaise R. Guillaudeux T. Tavernier G. Daegelen D. Evrard B. Mairal A. Holm C. Jégou B. Langin D. J. Biol. Chem. 2001; 276: 5109-5115Google Scholar). Exon B 5′-flanking region contains an active promoter with an E-box and two GC-boxes as functional cis-acting elements (28Smih F. Rouet P. Lucas S. Mairal A. Sengenes C. Lafontan M. Vaulont S. Casado M. Langin D. Diabetes. 2002; 51: 293-300Google Scholar). In adipocytes, the E-box mediates the glucose-mediated induction of HSL gene expression. However, the sequences responsible for the adipose tissue-specific expression of HSL are not present in this region because the pattern of promoter activity up to 2.4 kb was similar in adipocytes and in HeLa cells that do not express HSL (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). No data are yet available for the exon A 5′-flanking region. In the enterocytes, exons A and B are represented in equal amounts, suggesting that two alternative promoters control HSL gene expression providing the possibility of distinct transcriptional regulation.Lipid processing through the intestine is a complex pathway with multiple control steps. The intestine is unable to transport neutral lipids into the lymph at the rate with which they are absorbed, especially at high input rates. Nearly half of the triacylglycerol mass infused into rat intestine does not appear in the lymph (29Mansbach C.M. II Arnold A. Cox M.A. Am. J. Physiol. 1985; 249: G642-G648Google Scholar). It is unlikely that the lipids are oxidized because β-oxidation of lipid entering the mucosa from the lumen is limited. There is no evidence that triacylglycerols are transported via the portal vein (30Sabesin S.M. Frase S. J. Lipid Res. 1977; 18: 496-511Google Scholar). These studies suggest that some triacylglycerols in the enterocyte are undergoing hydrolysis. In support of this concept, a mucosal triacylglycerol pool distinct from the chylomicron triacylglycerol precursor pool has been characterized (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipolysis of the mucosal pool has been shown both in vitro and in vivo. Both acidic and alkaline lipase activities have been described in the mucosa (20Rao R.H. Mansbach II, C.M. Arch. Biochem. Biophys. 1993; 304: 483-489Google Scholar, 32Rao R.H. Mansbach II, C.M. Biochim. Biophys. Acta. 1990; 1043: 273-280Google Scholar). Because most of the lipolytic activity was found at neutral or basic pH, the physiological importance of the acidic lipase is unclear. Here, we confirm that significant neutral lipase activity is found in the enterocyte. This activity was inhibited by diethyl-p-nitrophenyl phosphate as shown previously for mucosal lipolysis in triolein-infused rats (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipase activity was found in the different parts of the small intestine. Data from HSL-null mice show that HSL contributes to lipase activity in the distal section but not in the first part of the small intestine. Recently, Mansbach and colleagues (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar) showed that pancreatic lipase was expressed in the intestine with most of the enzyme detected in the first quarter (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Altogether, the data suggest that the hydrolysis of mucosal triacylglycerols is caused by pancreatic lipase in the proximal part of the small intestine and HSL in the more distal parts.The nature of the enzyme responsible for the hydrolysis of cholesteryl esters in the intestine has remained unclear. Pancreatic cholesterol esterase (bile salt-stimulated lipase) is internalized upon binding to the surface of enterocytes (33Bruneau N. Lombardo D. Bendayan M. J. Cell Sci. 1998; 111: 2665-2679Google Scholar). The esterase could hydrolyze intracellular cholesteryl esters or conversely participate, at acid pH, in the esterification of cholesterol (17Field F.J. J. Lipid Res. 1984; 25: 389-399Google Scholar, 34Ponz de Leon M. Carrubi F. Di Donato P. Carulli N. Digest. Dis. Sci. 1985; 30: 1053-1064Google Scholar). However, the intracellular esterase activity in the absence of cofactors such as bile salts may be very low. Contribution to cholesterol esterification is also unlikely because studies on knockout mice revealed that the enzyme is responsible for mediating intestinal absorption of cholesteryl esters but does not influence free cholesterol absorption (35Howles P.N. Carter C.P. Hui D.Y. J. Biol. Chem. 1996; 271: 7196-7202Google Scholar). In contrast, acyl-CoA:cholesterol acyltransferase 2-deficient mice are resistant to diet-induced hypercholesterolemia (36Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V. Nat. Med. 2000; 6: 1341-1347Google Scholar). Localization of pancreatic cholesterol esterase in intestinal epithelium may therefore not be related to intracellular metabolism. There is evidence that the enzyme, via an apical-to-basolateral transcytotic pathway, is released at the basolateral membrane level and may contribute to serum pancreatic cholesterol esterase activity (37Bruneau N. Nganga A. Bendayan M. Lombardo D. Exp. Cell Res. 2001; 271: 94-108Google Scholar). Our data reveal that HSL and not pancreatic cholesterol esterase accounts for neutral cholesterol esterase activity in the small intestine.The expression of HSL in the enterocytes may open new paths in our understanding of cholesterol intestinal absorption and metabolism. HSL-mediated hydrolysis of the intracellular pool of cholesteryl esters may contribute together with the esterification process mediated by acyl-CoA:cholesterol acyltransferase-2 and cholesterol transport mediated by ATP-binding cassette (ABC) transporters to the control of cholesterol homeostasis. Several transporters are expressed in the intestinal epithelium. ABCA1 is expressed in the small intestine and may modulate cholesterol absorption. However, data from ABCA1-deficient mice are conflicting (38McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Google Scholar, 39Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Buchler C. Fung-Leung W.P. Von Bergmann K. Schmitz G. Gastroenterology. 2001; 120: 1203-1211Google Scholar). Studies in patients with sitosterolemia (40Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Google Scholar, 41Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Nat. Genet. 2001; 27: 79-83Google Scholar) and in transgenic mice overexpressing ABCG5 and ABCG8 (42Yu L. Li Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Google Scholar) suggest that the half-transporters participate in cholesterol efflux. Hydrolysis of cholesteryl esters by HSL may produce free cholesterol for export through ABC transporters into the lumen. Because of the unique properties of HSL, the present work paves the way for future studies on lipid metabolism in the enterocyte. Hormone-sensitive lipase (HSL) 1The abbreviations used are: HSL, hormone-sensitive lipase; ABC, ATP-binding cassette; ALBP, adipocyte lipid-binding protein; I-FABP, intestinal fatty acid-binding protein; RT, reverse transcription 1The abbreviations used are: HSL, hormone-sensitive lipase; ABC, ATP-binding cassette; ALBP, adipocyte lipid-binding protein; I-FABP, intestinal fatty acid-binding protein; RT, reverse transcription is a multifunctional enzyme with broad substrate specificity (1Holm C. Østerlund T. Laurell H. Contreras J.-A. Annu. Rev. Nutr. 2000; 20: 365-393Google Scholar). It hydrolyzes tri-, di-, and monoacylglycerols, cholesteryl esters, and retinyl esters. The activity against diacylglycerol is higher than the activity toward tri- and monoacylglycerols. The enzyme also exhibits cholesterol esterase activity, which is almost twice the activity toward triacylglycerols. Much has been learned in the recent years about the domain structure of HSL. Sequence comparisons revealed that HSL belongs to a family of esterases which is mainly represented by prokaryotic enzymes (2Langin D. Holm C. Trends Biochem. Sci. 1993; 18: 466-467Google Scholar, 3Hemilä H. Koivula T.T. Palva I. Biochim. Biophys. Acta. 1994; 1210: 249-253Google Scholar). From a structural point of view, HSL is the most complex protein of the family. Sequence alignments together with biochemical experiments suggest that adipocyte HSL is composed of two structural domains (4Contreras J.-A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Google Scholar,5Østerlund T. Danielsson B. Degerman E. Contreras J.-A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Google Scholar). The first 315 amino acids make up the N-terminal domain, which shows very little sequence similarities to other known proteins. The region responsible for the interaction with adipocyte lipid-binding protein (ALBP) was mapped to this domain (6Shen W.J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Google Scholar, 7Shen W.-J. Liang Y. Hong R. Patel S. Natu V. Sridhar K. Jenkins A. Bernlohr D.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 49443-49448Google Scholar). In adipose tissue, ALBP could increase the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and through specific protein-protein interactions. The C-terminal domain is divided in two functional parts, a catalytic core and a regulatory module. The latter is composed of 150 amino acids, including all of the known phosphorylation sites of HSL. Unlike other known mammalian triacylglycerol lipases, the activity of HSL is regulated by phosphorylation. The phosphorylation sites of protein kinase A, extracellular signal-regulated kinase, and AMP-dependent protein kinase have been mapped (8Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Google Scholar, 9Greenberg A.S. Shen W.J. Muliro K. Patel S. Souza S.C. Roth R.A. Kraemer F.B. J. Biol. Chem. 2001; 276: 45456-45461Google Scholar, 10Garton A.J. Campbell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 179: 249-254Google Scholar). The catalytic core is the region that shows homology with the other members of the family. Modeling of the part revealed that it adopts an α/β-hydrolase fold that harbors the catalytic triad constituted by Ser423, Asp703, and His733 (4Contreras J.-A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Google Scholar,11Wei Y. Contreras J.-A. Sheffield P. Østerlund T. Derewenda U. Kneusel R.E. Matern U. Holm C. Derewenda S. Nature Struct. Biol. 1999; 6: 340-345Google Scholar). Several forms of HSL transcripts and the exon-intron organization of the HSL gene have been characterized in humans. The 88-kDa adipocyte HSL is translated from a 2.8-kb mRNA and encoded by 9 exons (12Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4897-4901Google Scholar,13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). The transcription start site was mapped in a short noncoding exon called exon B. In the adenocarcinoma cell line HT29, two mRNA species are found, the adipocyte HSL mRNA and a mRNA with a different 5′-end transcribed from exon A. Two testicular forms of HSL have been characterized. The 3.9-kb mRNA encodes a 120-kDa protein that contains a unique N-terminal region encoded by exon T1, a region that presumably forms a third structural domain in this isoform (14Stenson Holst L. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser H.W. Edgren G. Holm C. Genomics. 1996; 35: 441-447Google Scholar). The 3.3-kb mRNA encodes a protein that is identical to the adipocyte HSL form (15Mairal A. Melaine N. Laurell H. Grober J. Stenson Holst L. Guillaudeux T. Holm C. Jégou B. Langin D. Biochem. Biophys. Res. Commun. 2002; 291: 286-290Google Scholar). However, the mRNA species differ in their 5′-ends. Exon usage is mutually exclusive, exon T2 being only transcribed in testis and exon B being transcribed in adipose tissue. The nature and role of lipases and esterases participating in the digestion of dietary lipids in the lumen of the gastrointestinal tract are well established. In addition to the enzymes in the lumen, there is evidence of lipase activity in enterocytes (16Shen H. Howles P. Tso P. Adv. Drug Delivery Rev. 2001; 50: S103-S125Google Scholar). The presence of cholesterol esterase activity is more elusive. The exact identity of the enzymes responsible for the hydrolysis of intracellular acylglycerols and cholesteryl esters is still unclear. Pancreatic triacylglycerol lipase, microsomal triacylglycerol hydrolase, and pancreatic cholesterol esterase have all been suggested to be responsible for the hydrolytic activities (17Field F.J. J. Lipid Res. 1984; 25: 389-399Google Scholar, 18Dolinsky V.W. Sipione S. Lehner R. Vance D.E. Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar, 19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Pancreatic triacylglycerol lipase may be synthesized by the small intestine and accounts for the alkaline lipase activity of the enterocytes (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Microsomal triacylglycerol hydrolase could also be involved (18Dolinsky V.W. Sipione S. Lehner R. Vance D.E. Biochim. Biophys. Acta. 2001; 1532: 162-172Google Scholar). However, it has been shown that most of the lipase activity is cytosolic (20Rao R.H. Mansbach II, C.M. Arch. Biochem. Biophys. 1993; 304: 483-489Google Scholar). Pancreatic cholesterol esterase, also called bile salt-stimulated lipase, is able to hydrolyze cholesteryl esters and triacylglycerols. In the absence of bile salts, the contribution of this enzyme is presumably minor (21Holm C. Olivecrona G. Ottosson M. Methods Mol. Biol. 2001; 155: 97-119Google Scholar). Because HSL is a cytosolic enzyme with a wide range of hydrolytic activities, the purpose of the present paper was to determine whether HSL was expressed in the intestine and could contribute to the hydrolysis of intracellular lipids. DISCUSSIONHere we have found that HSL contributes to lipase activity and is the major cholesterol esterase in the intestinal mucosa. HSL mRNA and protein were readily detected along the small intestine. Immunohistochemistry data revealed that the enzyme is expressed preferentially in differentiated cells of the villi. Data from HSL-null mice showed that HSL does not account for a significant part of total esterase activity. However, the enzyme is responsible for all neutral cholesteryl ester hydrolase activity both in the cytosolic and particulate fractions. It also contributes between one-third and one-half of diacylglycerol lipase activity in the jejunum.The size of intestinal HSL mRNA and protein corresponds to those of mouse adipocyte HSL, i.e. a 2.6–2.8-kb mRNA and an 82-kDa protein. The 5′-ends of the mRNA species are transcribed from two exons that correspond to human exons A and B. Exon A is located in the mouse gene ≈ 7 kb upstream of exon B (25Laurin N.N. Wang S.P. Mitchell G.A. Mamm. Genome. 2000; 11: 972-978Google Scholar). Use of the two exons is mutually exclusive. Interestingly, we have shown previously that HSL is expressed in the human adenocarcinoma cell line of intestinal origin HT29 (26Remaury A. Laurell H. Grober J. Reynisdottir S. Dauzats M. Holm C. Langin D. Biochem. Biophys. Res. Commun. 1995; 207: 175-182Google Scholar). The two mRNA 5′-ends were found in HT29 HSL mRNA (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). In human and mouse adipose tissue, the main transcription start site is located in exon B. Exon A-containing transcripts are found at very low levels in humans (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar) and are much less abundant than mRNA with exon B in mice ((25) and present work). To date, there is little information on the mechanisms controlling tissue-specific expression of HSL. We have shown that the first 95 bp of human exon T1 5′-flanking region conferred expression of a reporter gene exclusively in testis of transgenic mice (27Blaise R. Guillaudeux T. Tavernier G. Daegelen D. Evrard B. Mairal A. Holm C. Jégou B. Langin D. J. Biol. Chem. 2001; 276: 5109-5115Google Scholar). Exon B 5′-flanking region contains an active promoter with an E-box and two GC-boxes as functional cis-acting elements (28Smih F. Rouet P. Lucas S. Mairal A. Sengenes C. Lafontan M. Vaulont S. Casado M. Langin D. Diabetes. 2002; 51: 293-300Google Scholar). In adipocytes, the E-box mediates the glucose-mediated induction of HSL gene expression. However, the sequences responsible for the adipose tissue-specific expression of HSL are not present in this region because the pattern of promoter activity up to 2.4 kb was similar in adipocytes and in HeLa cells that do not express HSL (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). No data are yet available for the exon A 5′-flanking region. In the enterocytes, exons A and B are represented in equal amounts, suggesting that two alternative promoters control HSL gene expression providing the possibility of distinct transcriptional regulation.Lipid processing through the intestine is a complex pathway with multiple control steps. The intestine is unable to transport neutral lipids into the lymph at the rate with which they are absorbed, especially at high input rates. Nearly half of the triacylglycerol mass infused into rat intestine does not appear in the lymph (29Mansbach C.M. II Arnold A. Cox M.A. Am. J. Physiol. 1985; 249: G642-G648Google Scholar). It is unlikely that the lipids are oxidized because β-oxidation of lipid entering the mucosa from the lumen is limited. There is no evidence that triacylglycerols are transported via the portal vein (30Sabesin S.M. Frase S. J. Lipid Res. 1977; 18: 496-511Google Scholar). These studies suggest that some triacylglycerols in the enterocyte are undergoing hydrolysis. In support of this concept, a mucosal triacylglycerol pool distinct from the chylomicron triacylglycerol precursor pool has been characterized (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipolysis of the mucosal pool has been shown both in vitro and in vivo. Both acidic and alkaline lipase activities have been described in the mucosa (20Rao R.H. Mansbach II, C.M. Arch. Biochem. Biophys. 1993; 304: 483-489Google Scholar, 32Rao R.H. Mansbach II, C.M. Biochim. Biophys. Acta. 1990; 1043: 273-280Google Scholar). Because most of the lipolytic activity was found at neutral or basic pH, the physiological importance of the acidic lipase is unclear. Here, we confirm that significant neutral lipase activity is found in the enterocyte. This activity was inhibited by diethyl-p-nitrophenyl phosphate as shown previously for mucosal lipolysis in triolein-infused rats (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipase activity was found in the different parts of the small intestine. Data from HSL-null mice show that HSL contributes to lipase activity in the distal section but not in the first part of the small intestine. Recently, Mansbach and colleagues (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar) showed that pancreatic lipase was expressed in the intestine with most of the enzyme detected in the first quarter (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Altogether, the data suggest that the hydrolysis of mucosal triacylglycerols is caused by pancreatic lipase in the proximal part of the small intestine and HSL in the more distal parts.The nature of the enzyme responsible for the hydrolysis of cholesteryl esters in the intestine has remained unclear. Pancreatic cholesterol esterase (bile salt-stimulated lipase) is internalized upon binding to the surface of enterocytes (33Bruneau N. Lombardo D. Bendayan M. J. Cell Sci. 1998; 111: 2665-2679Google Scholar). The esterase could hydrolyze intracellular cholesteryl esters or conversely participate, at acid pH, in the esterification of cholesterol (17Field F.J. J. Lipid Res. 1984; 25: 389-399Google Scholar, 34Ponz de Leon M. Carrubi F. Di Donato P. Carulli N. Digest. Dis. Sci. 1985; 30: 1053-1064Google Scholar). However, the intracellular esterase activity in the absence of cofactors such as bile salts may be very low. Contribution to cholesterol esterification is also unlikely because studies on knockout mice revealed that the enzyme is responsible for mediating intestinal absorption of cholesteryl esters but does not influence free cholesterol absorption (35Howles P.N. Carter C.P. Hui D.Y. J. Biol. Chem. 1996; 271: 7196-7202Google Scholar). In contrast, acyl-CoA:cholesterol acyltransferase 2-deficient mice are resistant to diet-induced hypercholesterolemia (36Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V. Nat. Med. 2000; 6: 1341-1347Google Scholar). Localization of pancreatic cholesterol esterase in intestinal epithelium may therefore not be related to intracellular metabolism. There is evidence that the enzyme, via an apical-to-basolateral transcytotic pathway, is released at the basolateral membrane level and may contribute to serum pancreatic cholesterol esterase activity (37Bruneau N. Nganga A. Bendayan M. Lombardo D. Exp. Cell Res. 2001; 271: 94-108Google Scholar). Our data reveal that HSL and not pancreatic cholesterol esterase accounts for neutral cholesterol esterase activity in the small intestine.The expression of HSL in the enterocytes may open new paths in our understanding of cholesterol intestinal absorption and metabolism. HSL-mediated hydrolysis of the intracellular pool of cholesteryl esters may contribute together with the esterification process mediated by acyl-CoA:cholesterol acyltransferase-2 and cholesterol transport mediated by ATP-binding cassette (ABC) transporters to the control of cholesterol homeostasis. Several transporters are expressed in the intestinal epithelium. ABCA1 is expressed in the small intestine and may modulate cholesterol absorption. However, data from ABCA1-deficient mice are conflicting (38McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Google Scholar, 39Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Buchler C. Fung-Leung W.P. Von Bergmann K. Schmitz G. Gastroenterology. 2001; 120: 1203-1211Google Scholar). Studies in patients with sitosterolemia (40Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Google Scholar, 41Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Nat. Genet. 2001; 27: 79-83Google Scholar) and in transgenic mice overexpressing ABCG5 and ABCG8 (42Yu L. Li Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Google Scholar) suggest that the half-transporters participate in cholesterol efflux. Hydrolysis of cholesteryl esters by HSL may produce free cholesterol for export through ABC transporters into the lumen. Because of the unique properties of HSL, the present work paves the way for future studies on lipid metabolism in the enterocyte. Here we have found that HSL contributes to lipase activity and is the major cholesterol esterase in the intestinal mucosa. HSL mRNA and protein were readily detected along the small intestine. Immunohistochemistry data revealed that the enzyme is expressed preferentially in differentiated cells of the villi. Data from HSL-null mice showed that HSL does not account for a significant part of total esterase activity. However, the enzyme is responsible for all neutral cholesteryl ester hydrolase activity both in the cytosolic and particulate fractions. It also contributes between one-third and one-half of diacylglycerol lipase activity in the jejunum. The size of intestinal HSL mRNA and protein corresponds to those of mouse adipocyte HSL, i.e. a 2.6–2.8-kb mRNA and an 82-kDa protein. The 5′-ends of the mRNA species are transcribed from two exons that correspond to human exons A and B. Exon A is located in the mouse gene ≈ 7 kb upstream of exon B (25Laurin N.N. Wang S.P. Mitchell G.A. Mamm. Genome. 2000; 11: 972-978Google Scholar). Use of the two exons is mutually exclusive. Interestingly, we have shown previously that HSL is expressed in the human adenocarcinoma cell line of intestinal origin HT29 (26Remaury A. Laurell H. Grober J. Reynisdottir S. Dauzats M. Holm C. Langin D. Biochem. Biophys. Res. Commun. 1995; 207: 175-182Google Scholar). The two mRNA 5′-ends were found in HT29 HSL mRNA (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). In human and mouse adipose tissue, the main transcription start site is located in exon B. Exon A-containing transcripts are found at very low levels in humans (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar) and are much less abundant than mRNA with exon B in mice ((25) and present work). To date, there is little information on the mechanisms controlling tissue-specific expression of HSL. We have shown that the first 95 bp of human exon T1 5′-flanking region conferred expression of a reporter gene exclusively in testis of transgenic mice (27Blaise R. Guillaudeux T. Tavernier G. Daegelen D. Evrard B. Mairal A. Holm C. Jégou B. Langin D. J. Biol. Chem. 2001; 276: 5109-5115Google Scholar). Exon B 5′-flanking region contains an active promoter with an E-box and two GC-boxes as functional cis-acting elements (28Smih F. Rouet P. Lucas S. Mairal A. Sengenes C. Lafontan M. Vaulont S. Casado M. Langin D. Diabetes. 2002; 51: 293-300Google Scholar). In adipocytes, the E-box mediates the glucose-mediated induction of HSL gene expression. However, the sequences responsible for the adipose tissue-specific expression of HSL are not present in this region because the pattern of promoter activity up to 2.4 kb was similar in adipocytes and in HeLa cells that do not express HSL (13Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar). No data are yet available for the exon A 5′-flanking region. In the enterocytes, exons A and B are represented in equal amounts, suggesting that two alternative promoters control HSL gene expression providing the possibility of distinct transcriptional regulation. Lipid processing through the intestine is a complex pathway with multiple control steps. The intestine is unable to transport neutral lipids into the lymph at the rate with which they are absorbed, especially at high input rates. Nearly half of the triacylglycerol mass infused into rat intestine does not appear in the lymph (29Mansbach C.M. II Arnold A. Cox M.A. Am. J. Physiol. 1985; 249: G642-G648Google Scholar). It is unlikely that the lipids are oxidized because β-oxidation of lipid entering the mucosa from the lumen is limited. There is no evidence that triacylglycerols are transported via the portal vein (30Sabesin S.M. Frase S. J. Lipid Res. 1977; 18: 496-511Google Scholar). These studies suggest that some triacylglycerols in the enterocyte are undergoing hydrolysis. In support of this concept, a mucosal triacylglycerol pool distinct from the chylomicron triacylglycerol precursor pool has been characterized (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipolysis of the mucosal pool has been shown both in vitro and in vivo. Both acidic and alkaline lipase activities have been described in the mucosa (20Rao R.H. Mansbach II, C.M. Arch. Biochem. Biophys. 1993; 304: 483-489Google Scholar, 32Rao R.H. Mansbach II, C.M. Biochim. Biophys. Acta. 1990; 1043: 273-280Google Scholar). Because most of the lipolytic activity was found at neutral or basic pH, the physiological importance of the acidic lipase is unclear. Here, we confirm that significant neutral lipase activity is found in the enterocyte. This activity was inhibited by diethyl-p-nitrophenyl phosphate as shown previously for mucosal lipolysis in triolein-infused rats (31Tipton A.D. Frase S. Mansbach C.M. Am. J. Physiol. 1989; 257: G871-G878Google Scholar). Lipase activity was found in the different parts of the small intestine. Data from HSL-null mice show that HSL contributes to lipase activity in the distal section but not in the first part of the small intestine. Recently, Mansbach and colleagues (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar) showed that pancreatic lipase was expressed in the intestine with most of the enzyme detected in the first quarter (19Mahan J.T. Hada G.D. Rao R.H. Mansbach II, C.M. Am. J. Physiol. 2001; 280: G1187-G1196Google Scholar). Altogether, the data suggest that the hydrolysis of mucosal triacylglycerols is caused by pancreatic lipase in the proximal part of the small intestine and HSL in the more distal parts. The nature of the enzyme responsible for the hydrolysis of cholesteryl esters in the intestine has remained unclear. Pancreatic cholesterol esterase (bile salt-stimulated lipase) is internalized upon binding to the surface of enterocytes (33Bruneau N. Lombardo D. Bendayan M. J. Cell Sci. 1998; 111: 2665-2679Google Scholar). The esterase could hydrolyze intracellular cholesteryl esters or conversely participate, at acid pH, in the esterification of cholesterol (17Field F.J. J. Lipid Res. 1984; 25: 389-399Google Scholar, 34Ponz de Leon M. Carrubi F. Di Donato P. Carulli N. Digest. Dis. Sci. 1985; 30: 1053-1064Google Scholar). However, the intracellular esterase activity in the absence of cofactors such as bile salts may be very low. Contribution to cholesterol esterification is also unlikely because studies on knockout mice revealed that the enzyme is responsible for mediating intestinal absorption of cholesteryl esters but does not influence free cholesterol absorption (35Howles P.N. Carter C.P. Hui D.Y. J. Biol. Chem. 1996; 271: 7196-7202Google Scholar). In contrast, acyl-CoA:cholesterol acyltransferase 2-deficient mice are resistant to diet-induced hypercholesterolemia (36Buhman K.K. Accad M. Novak S. Choi R.S. Wong J.S. Hamilton R.L. Turley S. Farese R.V. Nat. Med. 2000; 6: 1341-1347Google Scholar). Localization of pancreatic cholesterol esterase in intestinal epithelium may therefore not be related to intracellular metabolism. There is evidence that the enzyme, via an apical-to-basolateral transcytotic pathway, is released at the basolateral membrane level and may contribute to serum pancreatic cholesterol esterase activity (37Bruneau N. Nganga A. Bendayan M. Lombardo D. Exp. Cell Res. 2001; 271: 94-108Google Scholar). Our data reveal that HSL and not pancreatic cholesterol esterase accounts for neutral cholesterol esterase activity in the small intestine. The expression of HSL in the enterocytes may open new paths in our understanding of cholesterol intestinal absorption and metabolism. HSL-mediated hydrolysis of the intracellular pool of cholesteryl esters may contribute together with the esterification process mediated by acyl-CoA:cholesterol acyltransferase-2 and cholesterol transport mediated by ATP-binding cassette (ABC) transporters to the control of cholesterol homeostasis. Several transporters are expressed in the intestinal epithelium. ABCA1 is expressed in the small intestine and may modulate cholesterol absorption. However, data from ABCA1-deficient mice are conflicting (38McNeish J. Aiello R.J. Guyot D. Turi T. Gabel C. Aldinger C. Hoppe K.L. Roach M.L. Royer L.J. de Wet J. Broccardo C. Chimini G. Francone O.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4245-4250Google Scholar, 39Drobnik W. Lindenthal B. Lieser B. Ritter M. Christiansen Weber T. Liebisch G. Giesa U. Igel M. Borsukova H. Buchler C. Fung-Leung W.P. Von Bergmann K. Schmitz G. Gastroenterology. 2001; 120: 1203-1211Google Scholar). Studies in patients with sitosterolemia (40Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Science. 2000; 290: 1771-1775Google Scholar, 41Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Nat. Genet. 2001; 27: 79-83Google Scholar) and in transgenic mice overexpressing ABCG5 and ABCG8 (42Yu L. Li Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. J. Clin. Invest. 2002; 110: 671-680Google Scholar) suggest that the half-transporters participate in cholesterol efflux. Hydrolysis of cholesteryl esters by HSL may produce free cholesterol for export through ABC transporters into the lumen. Because of the unique properties of HSL, the present work paves the way for future studies on lipid metabolism in the enterocyte. We acknowledge gratefully the contribution of the staff of the Louis Bugnard Institute Animal Care facility. We thank Birgitta Danielsson (Lund University) for technical assistance and Drs. Pascal and Patricia Degrace (Université de Bourgogne) for help with immunocytochemistry experiments.
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