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Immunolocalization, ontogeny and regulation of microsomal transfer protein (MTP) in human fetal intestine
April 2000
AGAA291
1628
1630
LACTASE PROMOTER FRAGMENTS DIRECT DIFFERENTIAL IN VIVO SPATIO-TEMPORAL EXPRESSION PATTERNS IN TRANSGENIC MICE.
THE DISTRIBUTION OF MICROSOMAL TRANSFER PROTEIN (MTP) AND APOLIPOPROTEIN B IN ENDOPLASMIC RETICULUM (ER) AND GOLGI OF INTESTINAL ABSORPTIVE CELLS: POSSIBLE ROLE OF THE GOLGI IN THE ASSEMBLY OF CHYLOMICRONS.
So Young Lee, Chun-ku Lin, Christopher H. Contag, Allen D. Cooper, Eric Sibley, Stanford Univ Sch of Medicine, Palo Alto, CA. Background and Aims: Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. These studies aim to identify regions of the lactase promoter involved in mediating the spatio-temporal expression pattern of the lactase gene and to utilize a novel photonic detection method for in vivo monitoring of promoter-reporter transgene expression in animals. Methods: 2.0- and 0.8kilobase fragments of the 5' flanking region of the rat lactase gene were cloned upstream of a firefly luciferase reporter and transfected into intestinal Caco-2 cells as a culture correlate of promoter activity. Transgenic mice harboring those same lactase promoter-reporter constructs were generated and screened for transgene expression by in vivo photonic detection with an intensified charge-coupled device (ICCD) camera. Luciferase activity was measured quantitatively by luminometer assays in multiple organs, including intestinal segments, harvested from pre and post-weaned mice. Results: Pre-weaned offspring of a 2-kb lactase promoter-reporter transgenic line express high-level luciferase activity in the small intestine (> 8,000 fold over background) with maximal expression in the middle segments. Luciferase expression was barely detectable « 3 fold over background) in colon, stomach, kidney, lung, liver, spleen, and brain. Post-weaned 30-day offspring undergo a region-specific decline in luciferase expression in the small intestine (25% activity in the proximal and middle segments and 1% in the distal segments) relative to pre-weaned levels. In comparison to the full length promoter, the 0.8 -kb promoterreporter construct expressed low-level luciferase activity « 50 fold over background) in multiple organs and throughout the GI tract in transgenic mice. Conclusions: A 2.0-kilobase fragment of the lactase promoter directs region-specific expression in the small intestine of transgenic mice. The pattern of expression mimics that of endogenous lactase with respect to spatial restriction during maturation and differs from that described for related transgenic constructs. Important tissue-specific enhancing elements may reside in the region between 0.8-2.0 kb upstream of the lactase transcription start-site. Photonic detection with the ICCD camera is a sensitive method to rapidly screen for intestinal expression of a luciferase reporter gene in living transgenic mice.
1629 IMMUNOLOCALIZATION, ONTOGENY AND REGULATION OF MICROSOMAL TRANSFER PROTEIN (MTP) IN HUMAN FETAL INTESTINE. Emile Levy, Simona Stan, Carole Garofalo, Edgard E. Delvin, Ernest G. Seidman, Daniel Menard, Univ of Montreal, Montreal, PQ, Canada; Univ of Sherbrooke, Sherbrooke, PQ, Canada. The fetal intestine appears to possess metabolic functions which resemble those of the mature small bowel. We have recently demonstrated that lipid esterification, apolipoprotein biogenesis and lipoprotein assembly occur in the developing human gut. To gain further insight into the multiple stages of lipoprotein packaging during development, we focused on the localization, ontogeny and regulation of MTP, a crucial protein for lipid transport. Using immunofluorescence, MTP was identified in different regions of the human fetal intestine, including the colon. Whereas its distribution was uniform along the crypt-villus axis, it appeared in a gradient fashion decreasing from the proximal small intestine to the distal colon. This was confirmed by immunoblot and by measuring MTP activity (% transfer/ug protein) in jejunum: 16.4 :!: 2.1, ileum: 1O.0:!: 1.9, proximal colon: 1.4 :!: 0.2 and distal colon: 1.3 :!: 0.1. On the other hand, an ascending gradient from crypt-to-villus cells was noted for protein disulfide isomerase (POI,) an obligatory chaperone for MTP function. The activity of MTP in small intestinal explants cultured for different incubation periods (0,4,8,24 h) peaked at 4 h, but remained insensitive to different concentrations of oleic acid. In addition, a trend towards an increase in MTP activity was observed at 20-22 weeks of gestation. The present findings provide the first evidence that the fetal gut is able to express MTP, and emphasize the distinct regional distribution, regulation by fatty acids and ontogeny of MTP. The low MTP activity in the fetal colon explains, in part, the defective synthesis and secretion of lipoproteins compared to fetal small bowel, documented by our group. Supported in part by a grant from the MRC.
Emile Levy, Delvin E. Delvin, Daniel Menard, Carole Garofalo, Isabelle Slight, Ernest G. Seidman, Moise Bendayan, Univ of Montreal, Montreal, PQ, Canada; Univ of Sherbrooke, Sherbrooke, PQ, Canada. Subsequent to intraluminal phases involved in the digestion and solubilization of dietary fat, a complex sequence of events occur within absorptive cells in order to assure the effective transport of lipids to peripheral organs. This intracellular phase, which includes the processing of lipolytic products by enterocytes and their transport by lipoproteins, is an important, yet largely unknown process. Inherited disorders of apo B and MTP deficiency, resulting in abnormalities of lipid transport, have highlighted the critical role of these proteins. MTP appears to reside within the lumen of the endoplasmic reticulum and physically interacts with apo B during lipoprotein assembly. The lipidation of apo B by active MTP complexes to protein disulfide isomerase (POI) protects apo B from early intracellular degradation. Although the importance of MTP has been emphasized in the assembly of nascent apo B-containing lipoproteins, it is not clear how it transfers lipids from their sites of synthesis to apo B, as it is synthesized and translocated across the endoplasmic reticulum membranes. The question as to whether MTP functions in other subcellular organelles and interacts with apo B in Golgi has not been addressed. Expression of apo B, MTP and POI was evaluated by biochemical and morphological techniques in immunoblotting using monoclonal antibodies showed the presence of these three proteins in purified microsomes and Golgi. This was further demonstrated by labeling immunocytochemistry, which confirmed their localization in the endoplasmic reticulum and Golgi of human and rat enterocytes cell lines. In addition, co-localization of apo B and MTP or MTP and POI was observed in these same organelles using double immunogold labeling. Finally, quantitation of transfer activity confirmed the presence of functional MTP complexes in both the endoplasmic reticulum and Golgi. Taken together, these data, demonstrating functional membrane-bound apo B and luminal MTP in the Golgi, suggest an active participation of this organelle in TG-rich lipoprotein assembly. Supported by the Medical Research Council of Canada.
1631 INTESTINAL DEVELOPMENT AND DISTRIBUTION OF THE ASBT AND IBABP IN APOE KNOCKOUT AND C57BUJ6 MICE. Hong Li, Pernilla Hakansson, AstraZeneca Molndal, Molndal, Sweden. Aims: The total plasma cholesterol level in apolipoprotein E (ApoE) knockout (KO) mice is about lO-fold higher than that in its background wildtype mice (C57BLl6J). It was proposed that the .size of the bile acid pool, as well as the functions and the expressions of the ASBT (ileum apical sodium-dependent bile acid transporter) and IBABP (ileum bile acid binding protein) might differ between the two groups of mice. The present study aimed at finding differences in the development and spatial distribution of ASBT and ffiABP between ApoE KO and C57BLl6J mice. Methods: Three ApoE KO or C57BLlJ6 mice were sacrificed on embryonic days 14 and 18 (El4, E18) and on post-natal day I, 5, 13, 22, 28, 42 and 90 (PI,...P90). The whole intestine removed, and was processed for the routine histology and immunohistochemistry. The absorption of bile acid in the adult mice was assessed by using trace amounts of 75Se_ tauroselcholic acid (SeHCAT). Results: A pseudostratified monolayer epithelium of intestine was observed in the mice at E14. At E18, nascent intestinal villi were found. As the intestinal villi developed, nascent crypts of Lieberkuhn were observed on P13, and the number of crypts dramatically were increased over the following 2 weeks. A well developed intestinal mucosa was observed at P28. The immunohistochemical reaction to anti-ASBT-antibody in the intestine was seen already on EI8 in the base of the nascent villi, but disappeared from PI up to P13, and was again found from P22 in the apical membrane of the villi of the distal small intestine. Immunohistochemical reaction to anti-IBABP-antibody in the mice was detected on PI in a low number of scattered enterocytes located in distal intestinal villi, and spread to more enterocytes following the development of the intestine. Most villus-associated enterocytes in the distal small intestine stained with anti-IBABP-antibody from P28 to P90. No significant differences were recorded between ApoE KO and C57BLl6J mice. The daily fecal excretion of the bile acid tracer SeHCAT was on average 30%. This did not significantly differ between ApoE KO and C57BLl6J mice. Conclusions: There are no significant differences between ApoE KO and C57BLl6J mice with regard to expressions of ASBT and IBABP in the ileum, or to the intestinal active absorption of bile acid.
AGAA291
1628
1630
LACTASE PROMOTER FRAGMENTS DIRECT DIFFERENTIAL IN VIVO SPATIO-TEMPORAL EXPRESSION PATTERNS IN TRANSGENIC MICE.
THE DISTRIBUTION OF MICROSOMAL TRANSFER PROTEIN (MTP) AND APOLIPOPROTEIN B IN ENDOPLASMIC RETICULUM (ER) AND GOLGI OF INTESTINAL ABSORPTIVE CELLS: POSSIBLE ROLE OF THE GOLGI IN THE ASSEMBLY OF CHYLOMICRONS.
So Young Lee, Chun-ku Lin, Christopher H. Contag, Allen D. Cooper, Eric Sibley, Stanford Univ Sch of Medicine, Palo Alto, CA. Background and Aims: Lactase gene transcription is spatially restricted to the proximal and middle small intestine of the developing mouse. These studies aim to identify regions of the lactase promoter involved in mediating the spatio-temporal expression pattern of the lactase gene and to utilize a novel photonic detection method for in vivo monitoring of promoter-reporter transgene expression in animals. Methods: 2.0- and 0.8kilobase fragments of the 5' flanking region of the rat lactase gene were cloned upstream of a firefly luciferase reporter and transfected into intestinal Caco-2 cells as a culture correlate of promoter activity. Transgenic mice harboring those same lactase promoter-reporter constructs were generated and screened for transgene expression by in vivo photonic detection with an intensified charge-coupled device (ICCD) camera. Luciferase activity was measured quantitatively by luminometer assays in multiple organs, including intestinal segments, harvested from pre and post-weaned mice. Results: Pre-weaned offspring of a 2-kb lactase promoter-reporter transgenic line express high-level luciferase activity in the small intestine (> 8,000 fold over background) with maximal expression in the middle segments. Luciferase expression was barely detectable « 3 fold over background) in colon, stomach, kidney, lung, liver, spleen, and brain. Post-weaned 30-day offspring undergo a region-specific decline in luciferase expression in the small intestine (25% activity in the proximal and middle segments and 1% in the distal segments) relative to pre-weaned levels. In comparison to the full length promoter, the 0.8 -kb promoterreporter construct expressed low-level luciferase activity « 50 fold over background) in multiple organs and throughout the GI tract in transgenic mice. Conclusions: A 2.0-kilobase fragment of the lactase promoter directs region-specific expression in the small intestine of transgenic mice. The pattern of expression mimics that of endogenous lactase with respect to spatial restriction during maturation and differs from that described for related transgenic constructs. Important tissue-specific enhancing elements may reside in the region between 0.8-2.0 kb upstream of the lactase transcription start-site. Photonic detection with the ICCD camera is a sensitive method to rapidly screen for intestinal expression of a luciferase reporter gene in living transgenic mice.
1629 IMMUNOLOCALIZATION, ONTOGENY AND REGULATION OF MICROSOMAL TRANSFER PROTEIN (MTP) IN HUMAN FETAL INTESTINE. Emile Levy, Simona Stan, Carole Garofalo, Edgard E. Delvin, Ernest G. Seidman, Daniel Menard, Univ of Montreal, Montreal, PQ, Canada; Univ of Sherbrooke, Sherbrooke, PQ, Canada. The fetal intestine appears to possess metabolic functions which resemble those of the mature small bowel. We have recently demonstrated that lipid esterification, apolipoprotein biogenesis and lipoprotein assembly occur in the developing human gut. To gain further insight into the multiple stages of lipoprotein packaging during development, we focused on the localization, ontogeny and regulation of MTP, a crucial protein for lipid transport. Using immunofluorescence, MTP was identified in different regions of the human fetal intestine, including the colon. Whereas its distribution was uniform along the crypt-villus axis, it appeared in a gradient fashion decreasing from the proximal small intestine to the distal colon. This was confirmed by immunoblot and by measuring MTP activity (% transfer/ug protein) in jejunum: 16.4 :!: 2.1, ileum: 1O.0:!: 1.9, proximal colon: 1.4 :!: 0.2 and distal colon: 1.3 :!: 0.1. On the other hand, an ascending gradient from crypt-to-villus cells was noted for protein disulfide isomerase (POI,) an obligatory chaperone for MTP function. The activity of MTP in small intestinal explants cultured for different incubation periods (0,4,8,24 h) peaked at 4 h, but remained insensitive to different concentrations of oleic acid. In addition, a trend towards an increase in MTP activity was observed at 20-22 weeks of gestation. The present findings provide the first evidence that the fetal gut is able to express MTP, and emphasize the distinct regional distribution, regulation by fatty acids and ontogeny of MTP. The low MTP activity in the fetal colon explains, in part, the defective synthesis and secretion of lipoproteins compared to fetal small bowel, documented by our group. Supported in part by a grant from the MRC.
Emile Levy, Delvin E. Delvin, Daniel Menard, Carole Garofalo, Isabelle Slight, Ernest G. Seidman, Moise Bendayan, Univ of Montreal, Montreal, PQ, Canada; Univ of Sherbrooke, Sherbrooke, PQ, Canada. Subsequent to intraluminal phases involved in the digestion and solubilization of dietary fat, a complex sequence of events occur within absorptive cells in order to assure the effective transport of lipids to peripheral organs. This intracellular phase, which includes the processing of lipolytic products by enterocytes and their transport by lipoproteins, is an important, yet largely unknown process. Inherited disorders of apo B and MTP deficiency, resulting in abnormalities of lipid transport, have highlighted the critical role of these proteins. MTP appears to reside within the lumen of the endoplasmic reticulum and physically interacts with apo B during lipoprotein assembly. The lipidation of apo B by active MTP complexes to protein disulfide isomerase (POI) protects apo B from early intracellular degradation. Although the importance of MTP has been emphasized in the assembly of nascent apo B-containing lipoproteins, it is not clear how it transfers lipids from their sites of synthesis to apo B, as it is synthesized and translocated across the endoplasmic reticulum membranes. The question as to whether MTP functions in other subcellular organelles and interacts with apo B in Golgi has not been addressed. Expression of apo B, MTP and POI was evaluated by biochemical and morphological techniques in immunoblotting using monoclonal antibodies showed the presence of these three proteins in purified microsomes and Golgi. This was further demonstrated by labeling immunocytochemistry, which confirmed their localization in the endoplasmic reticulum and Golgi of human and rat enterocytes cell lines. In addition, co-localization of apo B and MTP or MTP and POI was observed in these same organelles using double immunogold labeling. Finally, quantitation of transfer activity confirmed the presence of functional MTP complexes in both the endoplasmic reticulum and Golgi. Taken together, these data, demonstrating functional membrane-bound apo B and luminal MTP in the Golgi, suggest an active participation of this organelle in TG-rich lipoprotein assembly. Supported by the Medical Research Council of Canada.
1631 INTESTINAL DEVELOPMENT AND DISTRIBUTION OF THE ASBT AND IBABP IN APOE KNOCKOUT AND C57BUJ6 MICE. Hong Li, Pernilla Hakansson, AstraZeneca Molndal, Molndal, Sweden. Aims: The total plasma cholesterol level in apolipoprotein E (ApoE) knockout (KO) mice is about lO-fold higher than that in its background wildtype mice (C57BLl6J). It was proposed that the .size of the bile acid pool, as well as the functions and the expressions of the ASBT (ileum apical sodium-dependent bile acid transporter) and IBABP (ileum bile acid binding protein) might differ between the two groups of mice. The present study aimed at finding differences in the development and spatial distribution of ASBT and ffiABP between ApoE KO and C57BLl6J mice. Methods: Three ApoE KO or C57BLlJ6 mice were sacrificed on embryonic days 14 and 18 (El4, E18) and on post-natal day I, 5, 13, 22, 28, 42 and 90 (PI,...P90). The whole intestine removed, and was processed for the routine histology and immunohistochemistry. The absorption of bile acid in the adult mice was assessed by using trace amounts of 75Se_ tauroselcholic acid (SeHCAT). Results: A pseudostratified monolayer epithelium of intestine was observed in the mice at E14. At E18, nascent intestinal villi were found. As the intestinal villi developed, nascent crypts of Lieberkuhn were observed on P13, and the number of crypts dramatically were increased over the following 2 weeks. A well developed intestinal mucosa was observed at P28. The immunohistochemical reaction to anti-ASBT-antibody in the intestine was seen already on EI8 in the base of the nascent villi, but disappeared from PI up to P13, and was again found from P22 in the apical membrane of the villi of the distal small intestine. Immunohistochemical reaction to anti-IBABP-antibody in the mice was detected on PI in a low number of scattered enterocytes located in distal intestinal villi, and spread to more enterocytes following the development of the intestine. Most villus-associated enterocytes in the distal small intestine stained with anti-IBABP-antibody from P28 to P90. No significant differences were recorded between ApoE KO and C57BLl6J mice. The daily fecal excretion of the bile acid tracer SeHCAT was on average 30%. This did not significantly differ between ApoE KO and C57BLl6J mice. Conclusions: There are no significant differences between ApoE KO and C57BLl6J mice with regard to expressions of ASBT and IBABP in the ileum, or to the intestinal active absorption of bile acid.