- Email: [email protected]
Induction of NPC1L1 membrane translocation and cholesterol absorption by cholecystokinin
Abstracts / Atherosclerosis 235 (2014) e27–e83
a
Centre for Vascular Research, University of New South Wales, Sydney, Australia; b Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Vanderbilt, USA; c Department of Surgery, Salford Royal Hospital, Salford, United Kingdom; d Cardiovascular Trials Unit, University Department of Medicine, Manchester, United Kingdom Objectives: We have recently demonstrated that high-density lipoproteins (HDL) transport microRNAs (miRNAs) in the plasma. Here we aim to investigate how HDL-associated miRNAs are regulated in morbidly obese subjects and more importantly, how weight loss after bariatric surgery affected HDL-miRNAs in these subjects. Methods: Plasma HDL were isolated from morbidly obese subjects (n¼23) before, 6 months and 12 months after bariatric surgery by immunoprecipitation using goat anti-human apoA-I microbeads. We also isolated HDL from 12 normal subjects using the same methods. miRNAs were extracted from isolated HDL and levels of miR-223, miR-24 and miR-126 on HDL were determined by TaqMan miRNA assays. Results: miR-223, miR-24 and miR-126 were present on HDL from obese subjects before and after bariatric surgery. Levels of miR-223, miR-24 and miR-126 on HDL from morbidly obese subjects were significantly higher than the levels on HDL from normal subjects. At 12 months after bariatric surgery, miR-223, miR-24 and miR-126 levels on HDL from obese subjects were significantly increased by 2.9-Fold (p<0.0), 2.3-Fold (p<0.05) and 2.7-Fold (p<0.05), respectively, compared to before surgery. Conclusion: HDL-associated miRNAs are differentially regulated in healthy versus morbidly obese subjects. miR-223, miR-24 and miR-126 are sensitive to weight loss induced by bariatric surgery. This may potentially identify novel treatment targets for obesity and related metabolic disorders. 07 - Role of the intestine for lipid homeostasis and atherosclerosis EAS-0703. LIVER X RECEPTOR ACTIVATION DECREASES CHYLOMICRON ASSEMBLY AND IMPROVES POST-PRANDIAL TRIGLYCERIDEMIA VIA INTESTINAL SR-BI DOWNREGULATION O. Brianda, V. Touchea, S. Colina, C. Dugardina, A. Davalosb, A. Tailleuxa, A. Moschettac, G. Brufaud, A.K. Groend, V. Claveya, B. Staelsa, S. Lestavela a Récepteurs Nucléaires Maladies Cardiovasculaires et Diabète, Université Lille 2 F-59000 Lille France Inserm U1011 F-59000 Lille France EGID F-59000 Lille France Institut Pasteur de Lille F-59019, LILLE, France; b CEI UAM+CSIC 28049, IMDEA Food Institute, Madrid, Spain; c IRCCS Oncologico Giovanni Paolo II, National Cancer Research Center, Bari, Italy; d Departments of Pediatrics and Laboratory Medicine, Center for Liver Digestive and Metabolic Diseases University Medical Center Groningen University of Groningen, Groningen, Netherlands
Objectives: Atherogenic postprandial lipoprotein management is on of the promising strategies for the treatment of CV risk associated with type 2 diabetes and obesity. Initially described as an HDL receptor, SR-BI is also an apical membrane protein expressed by enterocytes. During the postprandial period, SR-BI interacts with dietary micelles and activates sensing events leading to chylomicron assembly and lipid absorption. The nuclear receptor LXR (Liver X receptor) controls expression of genes involved in cholesterol and fatty acid metabolism, however its role in post-prandial triglyceridemia (PPTG) has never been documented. Methods: In vivo murine models were either fed a cholesterol-enriched diet or treated with the LXR agonist GW3965. PPTG after an oral fat load challenge was evaluated and immuno-histochemistry was used to localise SR-BI. Synthetic and natural LXR agonist-treated Caco-2/TC7 cells were then used to decipher the molecular mechanisms of SR-BI regulation. Results: GW3965 and a cholesterol-enriched diet both activate LXR in the intestine and reduce PPTG in mice. Both in vivo and in vitro LXR activation leads to a post-transcriptional down-regulation of intestinal SR-BI protein and to its redistribution from microvillosities towards intracellular organelles including early endosomes and lysosomes. In vitro, SR-BI mediated signal transduction induced by lipid micelles and apoB-lipoprotein
e33
secretion are decreased. Based on reporter assays using miRNA-responsiveelement-rich regions, we show that LXR activation decreases the activity of the 3'UTR of both SR-BI and PDZK1. By using the SR-BI 3'UTR in a miRNA sponge approach, we demonstrate that LXR induces miRNAs triggering SRBI and PDZK1 post-transcriptional downregulation in enterocytes. Conclusion: In vitro and in vivo LXR activation leads to a decrease of luminal SR-BI in the intestine. Moreover, intestinal LXR activation decreases PPTG by inducing a desensitization process of enterocytes to dietary lipid micelles. 07 - Role of the intestine for lipid homeostasis and atherosclerosis EAS-0252. INDUCTION OF NPC1L1 MEMBRANE TRANSLOCATION CHOLESTEROL ABSORPTION BY CHOLECYSTOKININ
AND
H. Yanga, L. Zhoua, E. Okoroa, Z.M. Guoa a
Physiology, Meharry Medical College, Nashville, USA
Objectives: Cholecystokinin (CCK) has been extensively studied as a gastrointestinal hormone and neuropeptide. We previously reported that intravenous injection of CCK increased mouse plasma cholesterol. The goal of the present study is to investigate the effect of CCK on cholesterol absorption in Caco-2 cells and mouse models. Methods: Intestinal absorption of 3[H]-cholesterol was determined in wildtype (WT) and low-density lipoprotein receptor knockout (LDLR-/-) mice after intravenously injected with [Thr28, Nle31]-CCK. Transcellular cholesterol transport, Niemann-Pick C1 Like 1 (NPC1L1) translocation and related gene expression were determined in Caco-2 cells treated with Thr28, Nle31]-CCK.Intestinal absorption of 3[H]-cholesterol was determined in wild-type (WT) and low-density lipoprotein receptor knockout (LDLR-/-) mice after intravenously injected with [Thr28, Nle31]-CCK. Transcellular cholesterol transport, Niemann-Pick C1 Like 1 (NPC1L1) translocation and related gene expression were determined in Caco-2 cells treated with Thr28, Nle31]-CCK. Results: CCK increased intestinal absorption of 3[H]-cholesterol in both WT and LDLR-/- mice, and increased cholesterol absorption in Caco-2 cells. Antagonists and siRNA specific for CCK receptor-1 (CCK1R) and CCK2R blocked CCK-induced cholesterol absorption. In addition, CCK increased cell surface NPC1L1 transporter but not its total protein level. Inhibition of NPC1L1 translocation by ezetimibe attenuated CCK-induced absorption. Moreover, CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation, and augmented the interaction of NPC1L1 and Rab11. Knockdown CCK1R and 2R inhibited CCK-induced PI3K phosphorylation. Inhibition of PI3K reduced CCK-induced NPC1L1-Rab11 interaction and cholesterol absorption. Knockdown of Rab11 inhibited CCK-induced NPC1L1 translocation and cholesterol absorption. Conclusion: These data, together with the known regulatory role of PI3KAkt-Rab11a pathway on NPLC1lL1, suggest that activation of both CCK1R and 2R triggers the PI3K-Akt- Rab11a pathway, leading to NPC1L1 translocation to the cell surface, which in turn increase cholesterol absorption. 08 - Adipose tissue biology EAS-0656. LINKING WHITE-TO-BROWN FAT REMODELING TO FATTY ACID AND LIPOPROTEIN METABOLISM L. Schejaa, A. Bartelta, C. Johna, L. Cherradia, C. Schleina, A. Worthmanna, F. Rinningerb, J. Heerena a Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; b Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Objectives: Brown adipose tissue (BAT) activation lowers plasma triglyceride-rich lipoprotein levels through the action of lipoprotein lipase (LPL). In contrast, excess white adipose tissue (WAT) in obese diabetics is associated with high triglycerides and low HDL cholesterol – so called diabetic
a
Centre for Vascular Research, University of New South Wales, Sydney, Australia; b Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Vanderbilt, USA; c Department of Surgery, Salford Royal Hospital, Salford, United Kingdom; d Cardiovascular Trials Unit, University Department of Medicine, Manchester, United Kingdom Objectives: We have recently demonstrated that high-density lipoproteins (HDL) transport microRNAs (miRNAs) in the plasma. Here we aim to investigate how HDL-associated miRNAs are regulated in morbidly obese subjects and more importantly, how weight loss after bariatric surgery affected HDL-miRNAs in these subjects. Methods: Plasma HDL were isolated from morbidly obese subjects (n¼23) before, 6 months and 12 months after bariatric surgery by immunoprecipitation using goat anti-human apoA-I microbeads. We also isolated HDL from 12 normal subjects using the same methods. miRNAs were extracted from isolated HDL and levels of miR-223, miR-24 and miR-126 on HDL were determined by TaqMan miRNA assays. Results: miR-223, miR-24 and miR-126 were present on HDL from obese subjects before and after bariatric surgery. Levels of miR-223, miR-24 and miR-126 on HDL from morbidly obese subjects were significantly higher than the levels on HDL from normal subjects. At 12 months after bariatric surgery, miR-223, miR-24 and miR-126 levels on HDL from obese subjects were significantly increased by 2.9-Fold (p<0.0), 2.3-Fold (p<0.05) and 2.7-Fold (p<0.05), respectively, compared to before surgery. Conclusion: HDL-associated miRNAs are differentially regulated in healthy versus morbidly obese subjects. miR-223, miR-24 and miR-126 are sensitive to weight loss induced by bariatric surgery. This may potentially identify novel treatment targets for obesity and related metabolic disorders. 07 - Role of the intestine for lipid homeostasis and atherosclerosis EAS-0703. LIVER X RECEPTOR ACTIVATION DECREASES CHYLOMICRON ASSEMBLY AND IMPROVES POST-PRANDIAL TRIGLYCERIDEMIA VIA INTESTINAL SR-BI DOWNREGULATION O. Brianda, V. Touchea, S. Colina, C. Dugardina, A. Davalosb, A. Tailleuxa, A. Moschettac, G. Brufaud, A.K. Groend, V. Claveya, B. Staelsa, S. Lestavela a Récepteurs Nucléaires Maladies Cardiovasculaires et Diabète, Université Lille 2 F-59000 Lille France Inserm U1011 F-59000 Lille France EGID F-59000 Lille France Institut Pasteur de Lille F-59019, LILLE, France; b CEI UAM+CSIC 28049, IMDEA Food Institute, Madrid, Spain; c IRCCS Oncologico Giovanni Paolo II, National Cancer Research Center, Bari, Italy; d Departments of Pediatrics and Laboratory Medicine, Center for Liver Digestive and Metabolic Diseases University Medical Center Groningen University of Groningen, Groningen, Netherlands
Objectives: Atherogenic postprandial lipoprotein management is on of the promising strategies for the treatment of CV risk associated with type 2 diabetes and obesity. Initially described as an HDL receptor, SR-BI is also an apical membrane protein expressed by enterocytes. During the postprandial period, SR-BI interacts with dietary micelles and activates sensing events leading to chylomicron assembly and lipid absorption. The nuclear receptor LXR (Liver X receptor) controls expression of genes involved in cholesterol and fatty acid metabolism, however its role in post-prandial triglyceridemia (PPTG) has never been documented. Methods: In vivo murine models were either fed a cholesterol-enriched diet or treated with the LXR agonist GW3965. PPTG after an oral fat load challenge was evaluated and immuno-histochemistry was used to localise SR-BI. Synthetic and natural LXR agonist-treated Caco-2/TC7 cells were then used to decipher the molecular mechanisms of SR-BI regulation. Results: GW3965 and a cholesterol-enriched diet both activate LXR in the intestine and reduce PPTG in mice. Both in vivo and in vitro LXR activation leads to a post-transcriptional down-regulation of intestinal SR-BI protein and to its redistribution from microvillosities towards intracellular organelles including early endosomes and lysosomes. In vitro, SR-BI mediated signal transduction induced by lipid micelles and apoB-lipoprotein
e33
secretion are decreased. Based on reporter assays using miRNA-responsiveelement-rich regions, we show that LXR activation decreases the activity of the 3'UTR of both SR-BI and PDZK1. By using the SR-BI 3'UTR in a miRNA sponge approach, we demonstrate that LXR induces miRNAs triggering SRBI and PDZK1 post-transcriptional downregulation in enterocytes. Conclusion: In vitro and in vivo LXR activation leads to a decrease of luminal SR-BI in the intestine. Moreover, intestinal LXR activation decreases PPTG by inducing a desensitization process of enterocytes to dietary lipid micelles. 07 - Role of the intestine for lipid homeostasis and atherosclerosis EAS-0252. INDUCTION OF NPC1L1 MEMBRANE TRANSLOCATION CHOLESTEROL ABSORPTION BY CHOLECYSTOKININ
AND
H. Yanga, L. Zhoua, E. Okoroa, Z.M. Guoa a
Physiology, Meharry Medical College, Nashville, USA
Objectives: Cholecystokinin (CCK) has been extensively studied as a gastrointestinal hormone and neuropeptide. We previously reported that intravenous injection of CCK increased mouse plasma cholesterol. The goal of the present study is to investigate the effect of CCK on cholesterol absorption in Caco-2 cells and mouse models. Methods: Intestinal absorption of 3[H]-cholesterol was determined in wildtype (WT) and low-density lipoprotein receptor knockout (LDLR-/-) mice after intravenously injected with [Thr28, Nle31]-CCK. Transcellular cholesterol transport, Niemann-Pick C1 Like 1 (NPC1L1) translocation and related gene expression were determined in Caco-2 cells treated with Thr28, Nle31]-CCK.Intestinal absorption of 3[H]-cholesterol was determined in wild-type (WT) and low-density lipoprotein receptor knockout (LDLR-/-) mice after intravenously injected with [Thr28, Nle31]-CCK. Transcellular cholesterol transport, Niemann-Pick C1 Like 1 (NPC1L1) translocation and related gene expression were determined in Caco-2 cells treated with Thr28, Nle31]-CCK. Results: CCK increased intestinal absorption of 3[H]-cholesterol in both WT and LDLR-/- mice, and increased cholesterol absorption in Caco-2 cells. Antagonists and siRNA specific for CCK receptor-1 (CCK1R) and CCK2R blocked CCK-induced cholesterol absorption. In addition, CCK increased cell surface NPC1L1 transporter but not its total protein level. Inhibition of NPC1L1 translocation by ezetimibe attenuated CCK-induced absorption. Moreover, CCK enhanced phosphatidylinositide 3-kinase (PI3K) and Akt phosphorylation, and augmented the interaction of NPC1L1 and Rab11. Knockdown CCK1R and 2R inhibited CCK-induced PI3K phosphorylation. Inhibition of PI3K reduced CCK-induced NPC1L1-Rab11 interaction and cholesterol absorption. Knockdown of Rab11 inhibited CCK-induced NPC1L1 translocation and cholesterol absorption. Conclusion: These data, together with the known regulatory role of PI3KAkt-Rab11a pathway on NPLC1lL1, suggest that activation of both CCK1R and 2R triggers the PI3K-Akt- Rab11a pathway, leading to NPC1L1 translocation to the cell surface, which in turn increase cholesterol absorption. 08 - Adipose tissue biology EAS-0656. LINKING WHITE-TO-BROWN FAT REMODELING TO FATTY ACID AND LIPOPROTEIN METABOLISM L. Schejaa, A. Bartelta, C. Johna, L. Cherradia, C. Schleina, A. Worthmanna, F. Rinningerb, J. Heerena a Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; b Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Objectives: Brown adipose tissue (BAT) activation lowers plasma triglyceride-rich lipoprotein levels through the action of lipoprotein lipase (LPL). In contrast, excess white adipose tissue (WAT) in obese diabetics is associated with high triglycerides and low HDL cholesterol – so called diabetic