Intestinal Monoacylglycerol Metabolism
2007; Elsevier BV; Volume: 282; Issue: 46 Linguagem: Inglês
10.1074/jbc.m706994200
ISSN1083-351X
AutoresSu‐Hyoun Chon, Yin Zhou, Joseph L. Dixon, Judith Storch,
Tópico(s)Alcohol Consumption and Health Effects
ResumoIntestinal monoacylglycerol (MG) metabolism is well known to involve its anabolic reesterification to triacylglycerol (TG). We recently provided evidence for enterocyte MG hydrolysis and demonstrated expression of the monoacylglycerol lipase (MGL) gene in human intestinal Caco-2 cells and rodent small intestinal mucosa. Despite the large quantities of MG derived from dietary TG, the regulation of MG metabolism in the intestine has not been previously explored. In the present studies, we examined the mRNA expression, protein expression, and activities of the two known MG-metabolizing enzymes, MGL and MGAT2, in C57BL/6 mouse small intestine, as well as liver and adipose tissues, during development and under nutritional modifications. Results demonstrate that MG metabolism undergoes tissue-specific changes during development. Marked induction of small intestinal MGAT2 protein expression and activity were found during suckling. Moreover, while substantial levels of MGL protein and activity were detected in adult intestine, its regulation during ontogeny was complex, suggesting post-transcriptional regulation of expression. In addition, during the suckling period MG hydrolytic activity is likely to derive from carboxyl ester lipase rather than MGL. In contrast to intestinal MGL, liver MGL mRNA, protein and activity all increased 5–10-fold during development, suggesting that transcriptional regulation is the primary mechanism for hepatic MGL expression. Three weeks of high fat feeding (40% kcal) significantly induced MGL expression and activity in small intestine relative to low fat feeding (10% kcal), but little change was observed upon starvation, suggesting a role for MGL in dietary lipid assimilation following a high fat intake. Intestinal monoacylglycerol (MG) metabolism is well known to involve its anabolic reesterification to triacylglycerol (TG). We recently provided evidence for enterocyte MG hydrolysis and demonstrated expression of the monoacylglycerol lipase (MGL) gene in human intestinal Caco-2 cells and rodent small intestinal mucosa. Despite the large quantities of MG derived from dietary TG, the regulation of MG metabolism in the intestine has not been previously explored. In the present studies, we examined the mRNA expression, protein expression, and activities of the two known MG-metabolizing enzymes, MGL and MGAT2, in C57BL/6 mouse small intestine, as well as liver and adipose tissues, during development and under nutritional modifications. Results demonstrate that MG metabolism undergoes tissue-specific changes during development. Marked induction of small intestinal MGAT2 protein expression and activity were found during suckling. Moreover, while substantial levels of MGL protein and activity were detected in adult intestine, its regulation during ontogeny was complex, suggesting post-transcriptional regulation of expression. In addition, during the suckling period MG hydrolytic activity is likely to derive from carboxyl ester lipase rather than MGL. In contrast to intestinal MGL, liver MGL mRNA, protein and activity all increased 5–10-fold during development, suggesting that transcriptional regulation is the primary mechanism for hepatic MGL expression. Three weeks of high fat feeding (40% kcal) significantly induced MGL expression and activity in small intestine relative to low fat feeding (10% kcal), but little change was observed upon starvation, suggesting a role for MGL in dietary lipid assimilation following a high fat intake. sn-2-Monoacylglycerol (MG) 2The abbreviations used are: MG, monoacylglycerol; MGAT, monoacylglycerol acyltransferase; MGL, monoacylglycerol lipase; TG, triacylglycerol; DG, diacylglycerol; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; G3P, glycerol-3-phosphate; CEL, carboxyl ester lipase (also known as BSL, bile salt-stimulated lipase); GPAT, glycerol-3-phosphate acyltransferase; VLDL, very low density lipoprotein; FAS, fatty acid synthase; CB, cannabinoid receptor. 2The abbreviations used are: MG, monoacylglycerol; MGAT, monoacylglycerol acyltransferase; MGL, monoacylglycerol lipase; TG, triacylglycerol; DG, diacylglycerol; DGAT, diacylglycerol acyltransferase; ER, endoplasmic reticulum; G3P, glycerol-3-phosphate; CEL, carboxyl ester lipase (also known as BSL, bile salt-stimulated lipase); GPAT, glycerol-3-phosphate acyltransferase; VLDL, very low density lipoprotein; FAS, fatty acid synthase; CB, cannabinoid receptor. is one of the major digestive products of dietary triacylglycerol (TG). Along with fatty acid, it is formed by the action of pancreatic triacylglycerol lipase (PTL) in the intestinal lumen, because PTL preferentially cleaves the sn-1 and 3 positions of TG (1Tso P. Crissinger K. Stipanuk M.H. Biochemical and Physiological Aspects of Human Nutrition. W.B. Saunder Co., Philadelphia2002: 125-141Google Scholar). Both hydrolysis products are absorbed as monomers across the apical membrane of the intestinal epithelial cell (1Tso P. Crissinger K. Stipanuk M.H. Biochemical and Physiological Aspects of Human Nutrition. W.B. Saunder Co., Philadelphia2002: 125-141Google Scholar, 2Ho S.-Y. Storch J. Am. J. Physiol. 2001; 281: C1106-C1117Crossref PubMed Google Scholar). The mechanism of sn-2-MG uptake into the enterocyte has been demonstrated to be a saturable function of the monomer concentration of sn-2-MG at both apical and basal lateral surfaces of the cell, suggesting carrier-mediated uptake (2Ho S.-Y. Storch J. Am. J. Physiol. 2001; 281: C1106-C1117Crossref PubMed Google Scholar, 3Murota K. Storch J. J. Nutr. 2005; 135: 1626-1630Crossref PubMed Scopus (50) Google Scholar). At higher concentrations, a diffusional uptake pathway is also apparent (2Ho S.-Y. Storch J. Am. J. Physiol. 2001; 281: C1106-C1117Crossref PubMed Google Scholar, 3Murota K. Storch J. J. Nutr. 2005; 135: 1626-1630Crossref PubMed Scopus (50) Google Scholar). After absorption, sn-2-MG is rapidly reincorporated into TG in the endoplasmic reticulum (ER) via the so-called monoacylglycerol acyltransferase (MGAT) pathway, which is catalyzed by two enzymes, MGAT2 and diacylglycerol acyltransferase (DGAT). Two DGAT isoforms (DGAT1 and 2Ho S.-Y. Storch J. Am. J. Physiol. 2001; 281: C1106-C1117Crossref PubMed Google Scholar) have been identified, and both are expressed in small intestine (4Cases S. Smith S.J. Zheng Y.W. Myers H.M. Lear S.R. Sande E. Novak S. Collins C. Welch C.B. Lusis A.J. Erickson S.K. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13018-13023Crossref PubMed Scopus (869) Google Scholar, 5Cases S. Stone S.J. Zhou P. Yen E. Tow B. Lardizabal K.D. Voelker T. Farese Jr., R.V. J. Biol. Chem. 2001; 276: 38870-38876Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). In addition to the MG pathway, the intestine can also synthesize TG via the glycerol-3-phosphate (G3P) pathway, which is the dominant TG synthetic pathway in other tissues such as adipose and liver (1Tso P. Crissinger K. Stipanuk M.H. Biochemical and Physiological Aspects of Human Nutrition. W.B. Saunder Co., Philadelphia2002: 125-141Google Scholar). In the intestine, however, more than 75% of postprandial TG resynthesis is catalyzed by the MGAT pathway (6Johnston J.M. Rao G.A. Lowe P.A. Biochim. Biophys. Acta. 1967; 137: 578-580Crossref PubMed Scopus (41) Google Scholar, 7Kayden H.J. Senior J.R. Mattson F.H. J. Clin. Investig. 1967; 46: 1695-1703Crossref PubMed Scopus (83) Google Scholar). Reesterified TG and apolipoproteins are assembled into chylomicron particles, which are then secreted into the lymphatic circulation. Intestinal MG metabolism has generally been thought to involve only an anabolic pathway, the reesterification to TG via the MGAT pathway. Nevertheless, the presence of a MG hydrolytic activity in small intestine was noted several decades ago (8Pope J.L. McPherson J.C. Tidwell H.C. J. Biol. Chem. 1966; 241: 2306-2310Abstract Full Text PDF PubMed Google Scholar) and the partial purification of MG lipase (MGL; EC 3.1.1.23) activity from rat intestinal mucosa was reported (9De Jong B.J. Kalkman C. Halsmann W.C. Biochim. Biophys. Acta. 1978; 1393: 119-127Google Scholar). Recently, additional insight into intestinal MG metabolism was obtained. After incubation of Caco-2 cells with sn-2-[3H]MG at either the apical or basal lateral surface, a substantial amount of radioactivity was recovered in the unesterified fatty acid fraction (10Ho S.-Y. Delgado L. Storch J. J. Biol. Chem. 2002; 277: 1816-1823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In addition, human MG lipase mRNA expression was detected in Caco-2 cells, and the murine MG lipase gene (11Karlsson M. Contreras J.A. Hellman U. Tornqvist H. Holm C. J. Biol. Chem. 1997; 272: 27218-27223Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar) was also shown to be expressed in rodent small intestine (10Ho S.-Y. Delgado L. Storch J. J. Biol. Chem. 2002; 277: 1816-1823Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These results suggested that catabolic processing of sn-2-MG may occur in intestinal mucosa, in addition to the well known anabolic processing. The regulation and function of this MG hydrolytic activity in the enterocyte are at present entirely unknown. On a daily basis, the human small intestine metabolizes an estimated average of 100 g of dietary fat which is composed of more than 90% TG (1Tso P. Crissinger K. Stipanuk M.H. Biochemical and Physiological Aspects of Human Nutrition. W.B. Saunder Co., Philadelphia2002: 125-141Google Scholar). Therefore, the mechanism by which the enterocyte metabolizes sn-2-MG, which is one of the major products of luminal TG hydrolysis and a backbone for TG reesterification, is of great importance for dietary lipid assimilation. Despite this physiological significance, surprisingly little is known about the regulation of intestinal MG metabolism. In the liver, MG metabolism has been shown to be developmentally regulated, with hepatic MGAT activity dramatically higher in the suckling period than in adult rat liver (12Coleman R.A. Haynes E.B. J. Biol. Chem. 1984; 259: 8934-8938Abstract Full Text PDF PubMed Google Scholar). At present, nothing is known about the developmental expression of hepatic MGL, nor about the developmental expression of either MGL or MGAT2 in the intestine. Therefore, in the present studies, we examined the expression and activity of the two MG metabolizing enzymes, MGAT and MGL, in intestine as well as liver during ontogeny. Cao et al. (13Cao J. Hawkins E. Brozinick J. Liu X. Zhang H. Burn P. Shi Y. J. Biol. Chem. 2004; 279: 18878-18886Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) reported that a high fat diet induced MGAT2 expression and activity in small intestine, indicating a functional role for this enzyme in dietary fat absorption. It is known that the expression of other genes involved in intestinal lipid metabolism are also altered upon a change in nutritional status. For example, the induction of small intestinal liver fatty acid-binding protein (LFABP) expression by high fat feeding has been reported (14Lin M.C. Arbeeny C. Bergquist K. Kienzle B. Gordon D.A. Wetterau J.R. J. Biol. Chem. 1994; 269: 29138-29145Abstract Full Text PDF PubMed Google Scholar, 15Poirier H. Niot I. Degrace P. Monnot M. Bernard A. Besnard P. Am. J. Physiol. 1997; 273: G289-G295Crossref PubMed Google Scholar), as has the expression of microsomal triacylglycerol transfer protein (MTP), an essential protein for chylomicron assembly (14Lin M.C. Arbeeny C. Bergquist K. Kienzle B. Gordon D.A. Wetterau J.R. J. Biol. Chem. 1994; 269: 29138-29145Abstract Full Text PDF PubMed Google Scholar). Furthermore, it is well known that a standard fasting and refeeding regime markedly alters hepatic lipid metabolism (16Sul H.S. Wang D. Annu. Rev. Nutr. 1998; 18: 331-351Crossref PubMed Scopus (236) Google Scholar, 17Coleman R.A. Lewin T.M. Muoio D.M. Ann. Rev. Nutr. 2000; 20: 77-103Crossref PubMed Scopus (260) Google Scholar), but the influence on intestinal lipid metabolism is less clear. Thus, the effects of nutritional status on intestinal MG metabolism, in comparison with other tissues, were also investigated in the present studies. The results show that MG metabolism is dramatically altered in a tissue-specific manner in both intestine and liver during development and following a high fat diet. Intestinal MGL expression and activity are increased by high fat feeding whereas little or no changes are found in the fasted state, suggesting a potential role for MGL in dietary lipid assimilation. Materials—sn-2-[14C]Monoolein (oleoyl-1-[14C], 55 mCi/mmol) was purchased from American Radiolabeled Chemicals, Inc. [14C]Oleoyl-CoA (oleoyl-1-[14C], 57 mCi/mmol) was purchased from PerkinElmer Life Sciences. Unlabeled sn-2-monoolein was obtained from Doosan Serdary Research Laboratories (Toronto, Canada). The 3% borate impregnated thin layer chromatography (TLC) plates were purchased from Analtech (Newark, DE). Silica gel G TLC plates were obtained from Sigma. Mouse MGL cDNA was a generous gift from Dr. Cecilia Holm (Lund University), and mouse MGAT1 and 2 cDNAs were generously provided by Dr. Robert Farese (UCSF). Antibodies against mouse MGL and MGAT2 sequences were generous gifts from Dr. Daniele Piomelli (UC Irvine) and Dr. Yuguang Shi (Lilly Research Laboratory), respectively. The anit-CEL antibodies were generously provided by Dr. David Hui (University of Cincinnati). β-Actin antibody was purchased from Sigma, and mouse cyclophilin A antibody and cDNA were obtained from Ambion (Austin, TX). Animals, Diets, and Tissue Collection—For each developmental regulation study, wild-type mice (C57BL/6, n = 3 at each age) from ages 6 days before birth to 3-months old were reared in the animal facility at Rutgers University. Prenatal mice were obtained from pregnant dams. The day when the vaginal plug was seen was considered as gestational day 0, with embryonic days counted thereafter. Litter size was consistent at 7–10. Animals were maintained on a 12 h light and dark cycle and fed regular chow diet (Purina Mouse Chow 5015, Purina Co., St. Louis, MO) ad libitum after weaning. For the high fat feeding studies, 3-month-old male mice (C57BL/6) were divided into two groups (n = 8 per group). Purified rodent diet (10% fat by calories, from soybean oil) was given to the control group and the high fat group was fed a 40% kcal fat diet containing the additional 30% kcal from coconut oil, rich in short chain saturated fatty acids (D12327 and D12325, respectively, Research Diets, New Brunswick, NJ) for 3 weeks ad libitum. In a second high fat feeding protocol, female mice were divided into three groups (n = 8) and fed with either a 10% kcal fat diet, or high fat diets rich in saturated fat from lard (45 or 60% kcal) for 3 months. (D12450B, D12451, and D12492, respectively, Research Diets). For the starvation and refeeding trial, 3-month-old male mice were divided into 4 groups (n = 7 per group) as follows: fed (Purina Mouse Chow 5015, Purina Co.), starvation for 12 h, starvation for 24 h, and refeeding with a high sucrose diet (D11725, Research Diet) after a 24-h starvation (18Lewin T.M. Granger D.A. Kim J.H. Coleman R.A. Arch. Biochem. Biophys. 2001; 396: 119-127Crossref PubMed Scopus (63) Google Scholar, 19Kim T.-S. Freake H.C. J. Nutr. 1996; 126: 611-617Crossref PubMed Scopus (58) Google Scholar). Animals were sacrificed using CO2, and the entire small intestine from pylorus to cecum was immediately excised. For the nutritional regulation studies, the intestine was rinsed twice with saline and then the mucosa was harvested by scraping. For developmental studies, whole intestine was collected followed by rinsing. Samples were immediately frozen on dry ice and kept at –70 °C. Liver and adipose tissue (peri-renal and epididymal fat) were also collected, snap frozen on dry ice, and stored at –70 °C. Northern Blot Analysis of MGL and MGAT1 and 2 mRNA Expression—Tissues were homogenized in Solution D (4 m guanidinium thiocyanate, 25 mm sodium citrate, 0.1 m 2-mercaptoethanol) using several strokes of a Polytron. Total RNA was further purified by phenol extraction. For detecting MGAT2 transcript in liver, poly(A)+ RNA was prepared using a Qiagen mRNA extraction kit. 20–40 μg of total RNA or 2 μgof poly(A)+ RNA were loaded onto 1% agarose gels, separated by electrophoresis, and transferred onto nylon membranes (PerkinElmer Life Sciences). Full-length coding regions of MGL, MGAT1, and MGAT2 cDNA were labeled with 32P (PerkinElmer Life Sciences) using the Random Prime labeling system (GE Healthcare, Piscataway, NJ). Membranes were prehybridized for 1 h and hybridized for 2–3 h at 68 °C using Quik-hybridization solution (Stratagene, La Jolla, CA). Blots were washed twice at room temperature with 2× SSC, 0.1% SDS for 15 min. An additional high temperature (65 °C) wash with 0.1× SSC, 0.1% SDS for 30 min was completed before exposing the blots to a PhosphorImager screen. Quantification was done using the Molecular Dynamics STORM scanner and Image-QuaNT software (Molecular Dynamics, Sunnyvale, CA). Blots were stripped and reprobed with 18S rRNA cDNA or mouse cyclophilin A cDNA for internal loading controls. Quantitative RT-PCR for Intestinal MGL mRNA Expression—Relative MGL mRNA expression in small intestine was analyzed by quantitative RT-PCR (SYBR Green method). Total RNA was extracted as described above and further purified using the RNeasy clean up kit (Qiagen, Valencia, CA) along with DNase 1 treatment to minimize genomic DNA contamination. Reverse transcription was performed using 1 μg of total RNA, random primer, RNase inhibitor and AMV reverse transcriptase (Promega Madison, WI) in a total volume of 25 μl. Primer sequences for MGL and β-actin (endogenous control) were retrieved from Primer Bank (Harvard Medical School QPCR primer data base), as follows: MGL: forward 5′-CAGAGAGGCCAACCTACTTTTC-3′, reverse 5′-ATGCGCCCCAAGGTCATATTT-3′; β-actin: forward 5′-GGCTGTATTCCCCTCCATCG-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′. Efficiencies of PCR amplification for both primers were tested during preliminary experiments and similar PCR efficiencies were confirmed. Real time PCR reactions were performed in triplicate using an Applied Biosystems 7300 instrument. Each reaction contained 80 ng of cDNA, 250 nm of each primer, and 12.5 μl of SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in a total volume of 25 μl. Relative quantification of MGL expression was calculated using the comparative Ct method normalized to β-actin. Western Blot Analysis of Protein Expression—Tissues were homogenized in 5–10 volumes of homogenization buffer on ice for 30 s. using a Wheaton tissue homogenizer (Wheaton Science, Millville, NJ), and crude tissue homogenates were centrifuged at 600 × g for 10 min at 4 °C to remove unbroken cell debris. Homogenization buffer contained 50 mm Tri-HCl and 0.32 m sucrose (pH 8) with 0.5% (v/v) protease inhibitors (Sigma 8340). For MGAT2 detection, a total membrane fraction was obtained by further ultracentrifugation (100,000 × g, 1 h at 4 °C). Protein concentration was determined by the Bradford assay (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214515) Google Scholar). 30–50 μg of total cell protein or 5–10 μg of membrane protein were loaded onto 12% polyacrylamide gels and separated by SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride membranes using a semi-dry transfer system (BioRad) for 1 h at 20V. All membranes were incubated in a 5% nonfat dry milk blocking solution overnight at 4 °C and then probed with primary antibody for 1 h. Dilution of the anti-mMGL antibody was 1:5,000–10,000, and for anti-mMGAT2 dilution was 1:1,000. Dilution of the anti-CEL antibody was 1:1,000. After washing three times, blots were incubated with anti-rabbit IgG-horseradish peroxidase conjugate at 1:10,000 dilution for 1 h and then developed by chemiluminescence (ECL reagent, GE Healthcare, Piscataway, NJ). Blots were stripped and reprobed with mouse β-actin (Sigma) or cyclophilin A antibody (Ambion) as indicated to check the integrity of the sample and as loading controls. Quantification of protein bands was conducted using Image J software (NIH). In Vitro MGL Assay—The MGL assay was established in the laboratory based on previous reports (9De Jong B.J. Kalkman C. Halsmann W.C. Biochim. Biophys. Acta. 1978; 1393: 119-127Google Scholar, 21Dinh T.P. Carpenter D. Leslie F.M. Freund T.F. Katona I. Sensi S.L. Kathuria S. Piomelli D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10819-10824Crossref PubMed Scopus (1118) Google Scholar), and measures the release of fatty acid from sn-2-MG. The purity of sn-2-[14C]monoolein (labeled on the fatty acyl chain, 55 mCi/mmol, American Radiolabeled Chemicals) was checked using 3% borate-impregnated TLC plates and a solvent system of CHCl3/acetone/methanol/acetic acid (90:5:2:0.5, v/v). The sn-2-[14C]monoolein was >90% pure. 14C-radiolabeled sn-2-monoolein was mixed with unlabeled sn-2-monoolein to obtain the desired concentrations. The mixture of 14C-radiolabeled and unlabeled sn-2-monoolein was dried under N2 gas, and 0.125 m Tris-HCl buffer containing 1.25% bovine serum albumin was added. Substrate emulsions were prepared by brief sonication (1 min) on ice. 0.4 ml of substrate emulsion containing sn-2-monoolein was allocated in each reaction tube. Samples containing 100–200 μg of protein were introduced as an enzyme source. Reaction conditions for each tissue were optimized during preliminary experiments to ensure linearity with time and protein concentration. 2.5 mmsn-2-monoolein was used for liver and adipose tissue MGL assays, and 25 μm was used for intestine samples. The reaction was initiated by adding tissue homogenate. Liver and adipose tissue samples were incubated for 5 min and intestinal samples for 10 min at 23 °C. Lipids were extracted using chloroform/methanol (2:1, v/v), and the organic phase was subjected to TLC analysis. To monitor isomerization of substrate, 3% borate-impregnated TLC plates (Analtech, Newark, DE) were used for separation of lipids. Spontaneous isomerization to the sn-1 isomer (20–30%) was always accounted for when determining the enzyme activity. All reaction times and temperatures were optimized to minimize spontaneous isomerization during the assay. Quantification of the specific activity of 14C-labeled end products separated by TLC was done using the Molecular Dynamics STORM scanner and ImageQuaNT software. On each plate, known amounts of specific radioactivity of [14C]oleate (PerkinElmer Life Sciences) were spotted and used for formulating standard curves to calculate enzyme activities for each sample. In Vitro MGAT Assay—The MGAT assay followed a well established protocol based on the method of Coleman and Haynes (12Coleman R.A. Haynes E.B. J. Biol. Chem. 1984; 259: 8934-8938Abstract Full Text PDF PubMed Google Scholar), with slight modifications. Activity was measured as the incorporation of [14C]oleoyl-CoA into diacylglycerol (DG). 25 μm oleoyl-CoA and 250 μmsn-2-monoolein (Doosan Serdary Research) were used as substrates. [14C]Oleoyl-CoA was purchased from PerkinElmer Life Sciences (57 mCi/mmol) and cold oleoyl-CoA was purchased from Sigma. The assay buffer contained 100 mm Tris-HCl, 4 mm MgCl2, 1 mg/ml bovine serum albumin, and 100 μm each of phosphatidylcholine and phosphatidylserine. 5 or 10 μg of membrane fraction protein, prepared by ultracentrifugation at 100,000 × g for 1 h at 4 °C, were used as the enzyme source. The enzyme assay was initiated by adding [14C]oleoyl-CoA, and incubation was for 5 min at 25 °C. Lipids were extracted using chloroform/methanol (2:1, v/v) and the organic phase was subjected to TLC analysis using standard silica gel plates (Sigma) and a solvent system of hexane/ethyl ether/acetic acid, 70:30:1, v/v. The plates were exposed to a PhosphorImager screen to visualize incorporation of [14C]oleoyl-CoA into neutral lipids. Specific activities found in the DG fraction plus half of the TG fractions were considered as MGAT activity (12Coleman R.A. Haynes E.B. J. Biol. Chem. 1984; 259: 8934-8938Abstract Full Text PDF PubMed Google Scholar, 22Yen C.-L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8512-8517Crossref PubMed Scopus (150) Google Scholar). Quantification of each lipid fraction was analyzed using ImageQuaNT software. Developmental Regulation of Intestinal MGL and MGAT2—Changes in mRNA and protein expression of the two MG-metabolizing enzymes, as well as both enzyme activities were determined over the course of intestinal ontogeny. The results showed a relatively abundant expression of MGL mRNA at early developmental stages, declining thereafter to a lower but detectable level (Fig. 1A). The expression pattern for MGL protein over the same time period was not consistent with the changes in its mRNA expression. MGL protein was detected in prenatal and adult intestine but not in the suckling period (Fig. 1B). MGL activity was measured as the release of radiolabeled fatty acid from sn-2-[14C]monoolein, and it was found that activity increased throughout intestinal development, plateauing at a relatively high level after day 6 (Fig. 1C). Thus, MGL activity shows discrepancies with the expression of both mRNA and protein levels (Fig. 1D), particularly during the suckling period. We hypothesized that carboxyl ester lipase (CEL; also known as bile salt-stimulated lipase), another enzyme with known sn-2-monoacylglycerol hydrolytic activity, might be present in suckling intestine. The results in Fig. 1E show that CEL protein was detected in neonatal intestine at approximately equivalent levels to those in adult intestine. For intestinal MGAT2, mRNA levels were very low at day –6 but then rose and remained relatively constant from day –3 to day +90 (Fig. 2A). Protein and activity levels were also very low at day –6 and increased thereafter; both appeared to be up-regulated during the early suckling period, and declined thereafter (Fig. 2, B and C). Developmental Regulation of Hepatic MGL and MGAT2—In marked contrast to intestinal MGL, similar patterns were observed for liver MGL mRNA, protein, and activity levels. All were increased 5–10-fold during development (Fig. 3D), suggesting that transcriptional regulation is the primary mechanism of hepatic MGL expression (Fig. 3, A, B, and C). It has been reported that the MGAT1 transcript was detected in adult mouse liver by Northern analysis (22Yen C.-L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8512-8517Crossref PubMed Scopus (150) Google Scholar); however, we were unable to detect MGAT1 in the present studies. The reason for the discrepancy is unknown. As described below, the MGAT1 transcript was detected in adipose tissue, in agreement with Yen et al. (22Yen C.-L. Stone S.J. Cases S. Zhou P. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8512-8517Crossref PubMed Scopus (150) Google Scholar). The MGAT2 transcript was readily detected in 2 μg of liver mRNA, consistent with the report of Cao et al. (23Cao J. Lockwood J. Burn P. Shi Y. J. Biol. Chem. 2003; 278: 13860-13866Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Thus, the present results for mRNA regulation in liver represent changes in the MGAT2 transcript during ontogeny. In contrast to the developmental pattern found for hepatic MGL, hepatic MGAT2 mRNA was detected during the pre- and post-natal stages but declined after day 6 (Fig. 4A). MGAT activity levels were generally consistent with this pattern of mRNA expression (Fig. 4B), suggesting that, as for hepatic MGL, transcription is the predominant mechanism of MGAT regulation in the liver during development. Overall, an inverse regulation of MGAT and MGL during ontogeny was found in liver (Figs. 3 and 4); however such reciprocal regulation of the two MG-metabolizing enzymes was not found in small intestine (Figs. 1 and 2). Because liver TG content has been reported to decline to low adult levels during development in the rat (24Waterman I.J. Price N.T. Zammit V.A. J. Lipid Res. 2002; 43: 1555-1562Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 25Jamdar S.C. Moon M. Bow S. Fallon H.J. J. Lipid Res. 1978; 19: 763-770Abstract Full Text PDF PubMed Google Scholar), we wanted to determine whether a similar decline is found in mouse liver. The results in Fig. 4D show that TG levels in adult mouse liver are 5-fold lower than those found in day 0 liver. Nutritional Control of Intestinal MGL and MGAT2—To examine the effect of an increased substrate supply on intestinal MGL, 3-month-old C57BL/6 male mice were fed either a high fat (40% kcal) or standard fat diet (10% kcal) for 3 weeks. Body weight was significantly elevated in the high fat fed group, along with a marked increase in total fat pad weight, nearly 4-fold greater than control (Fig. 5, A and B). Interestingly, we found that intestinal MGL protein expression increased 2–3-fold, and significant elevations in MG hydrolysis in small intestine were observed following high fat feeding (Fig. 5C). Relative mRNA levels of MGL were determined by quantitative RT-PCR, and also showed a consistent induction although it did not reach statistical significance. A similar result was observed in a relatively long term, 3-month high fat feeding study (10, 45, or 60% kcal), though the response was somewhat blunted compared with results from the short term feeding study (results not shown). In contrast to this induction by a high fat challenge, starvation up to 24 h did not significantly alter MGL expression and activity (Fig. 5E). These results show that upon increased lipid flux to the enterocyte, MG catabolism was stimulated, but fasting did not
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