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PCSK9 promotes intestinal overproduction of apolipoprotein-b lipoproteins through ldl-receptor dependent and independent mechanisms
Abstracts / Atherosclerosis 235 (2014) e84–e191
Objectives: Familial combined hyperlipidemia (FCH) is the most frequent lipid genetic disorder in developed countries and is associated with a high incidence of premature cardiovascular disease. FCH is also associated with insulin resistance, abdominal obesity, fatty liver disease, endothelial dysfunction, low-grade inflammation and with the classic lipid phenotype of T2DM, presenting high triglyceride plasma level, decreased high density lipoprotein (HDL) cholesterol and increased amounts of small, dense LDL particles. Additionaly, impaired incretin secretion has been observed in diabetic individuals, and in other populations caraterized as insulin resistance, such as patients with metabolic syndrome and impaired glucose tolerance. The aim of this study was to investigate the relationship between insulin resistance and the pattern of incretin secretion in patients with FCH. Methods: We conducted a case-control study, enrolling 30 patients with known FCH and 14 healthy controls. After the extraction of a basal blood sample, we administered an oral mixed (glucose and lipids) load to both groups, and measured insulin concentrations at 30 minutes intervals up to 2 hours after the oral load. Insulin resistance was measured by HOMA test, and insulin sensitivity was measured by Matsuda index. Results: There were no statistical differences in age and anthropometric characteristics between both groups. Absolute insulin secretion in response to the oral mixed load, calculated as area under the 120 minutes secretion curve, was significantly higher in patients with FCH than in control subjects. We also found significant differences in insulin resistance and sensitivity between groups: mean HOMA test value was higher in patients with FCH, and mean Matsuda Index value was lower in the same patients, when compared with control group. Increase in GIP secretion was significantly related to insulin resistance but not with GLP-1 and GLP-2. Conclusion: Insulin resistance is an important feature in patients with FCH and is associated with high GIP secretion levels. 34 - Intestinal and biliary lipid metabolism EAS-1096. SIMVASTATIN AND EZETIMIBE HAVE OPPOSING REGULATION ON INTESTINAL CHOLESTEROL ABSORPTION AND TRANS INTESTINAL CHOLESTEROL EXCRETION (TICE) DURING METABOLIC SYNDROME R. Mangat, F. Borthwick, S. Warnakula, M. Jacome-Sosa, D. Vine, S. Proctor Alberta Institute for Human Nutrition, University of Alberta, Edmonton, Canada Objectives: Recent results from the Copenhagen Heart Study have demonstrated a role for non-fasting remnant cholesterol as a causal risk factor for cardiovascular disease (CVD). Evidence in humans, rats and hamsters also show that insulin resistance stimulates overproduction of intestinal derived remnant lipoproteins and may further exacerbate CVD risk. We have recently demonstrated that statins can increase intestinal remnant cholesterol in a rat model of the Metabolic Syndrome (MetS). Here we proposed to evaluate the impact of combining simvastatin (SV) with an intestinal cholesterol absorption inhibitor (ezetimibe [EZ]) on intestinal cholesterol secretion and/or Trans Intestinal Cholesterol Excretion (TICE) in a rodent model of MetS. Methods: JCR:LA-cp control and MetS rats were fed a diet supplemented with 1% w/w cholesterol with/without SV (0.01% w/w), EZ (0.01% w/w) or SV+EZ for 8 weeks. Mesenteric lymph was collected in fasted rats following intralipid infusion via lymphatic fistulae procedure and assessed for cholesterol and particle number. TICE was measured in jejunal segments as flux of H3-cholesterol via Using chamber the technique Results: MetS rats had increased cholesterol per particle (160%) compared to control. SV treatment (but not EZ) significantly increased the ratio of cholesterol per particle (600%). Interestingly, the combination of SV+EZ significantly decreased (43%) cholesterol per particle compared to EZ only group.
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TICE was significantly suppressed (45%) in MetS rats compared to control. EZ significantly upregulated (153%) TICE, while SV had no additional effect either alone or in combination with EZ. Conclusion: The addition of EZ (dual therapy) to SV monotherapy can ameliorate statin-induced up-regulation of cholesterol absorption. EZ stimulates TICE in a model of the MetS while SV has no effect. The combination of EZ+SV beneficially modulates intestinal lipid secretion and TICE. 34 - Intestinal and biliary lipid metabolism EAS-0040. PCSK9 PROMOTES INTESTINAL OVERPRODUCTION OF APOLIPOPROTEIN-B LIPOPROTEINS THROUGH LDL-RECEPTOR DEPENDENT AND INDEPENDENT MECHANISMS S. Rashid MD a, H. Tavori MD b, I. Guinzioni MD b, S. Fazio MD b a
Pharmacology, Dalhousie University, Saint John, Canada; Vanderbilt University, Nashville, USA
b
Medicine,
Objectives: Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its deficiency in humans results in low plasma LDL-cholesterol and protection against coronary heart disease (CHD). Here we studied the effects of PCSK9 on the intestinal metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins (TRL), another important CHD risk factor, and elucidated for the first time the cellular and molecular mechanisms involved. Methods: Cultured human enterocytes (CaCo-2 cells) were treated with either recombinant human PCSK9 (10 mg/mL, 24 hours) or PCSK9 siRNA (15 nmol/L, 48 hours). Proteins and gene expression pathways in the cellular TRL production pathway were then assessed by Western blot and real-time qPCR, respectively and confirmed in vivo in human PCSK9 transgenic mice. Results: PCSK9 treatment increased the secretion of apoB48 and apoB100 in CaCo-2 cells by 40-55% each (p<0.01 vs. untreated cells), whereas PCSK9 gene knockdown substantially reversed this effect. PCSK9 stimulation of apoB was due to: (1) a 1.5-fold increase in apoB mRNA (p<0.01); and (2) enhanced apoB protein stability through both LDLR-dependent and LDLR-independent mechanisms. PCSK9 decreased LDLR protein (p<0.01) and increased cellular apoB stability via activation of microsomal triglyceride transfer protein (MTP). In mice, human PCSK9 expression increased intestinal MTP activity regardless of LDLR expression. Conclusion: PCSK9 increases intestinal TRL apoB production at the transcriptional and post-transcriptional levels mediated in part by the LDLR and by the newly identified PCSK9 target, MTP. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia. 34 - Intestinal and biliary lipid metabolism EAS-0258. WESTERN DIET ENHANCES DPP-IV ACTIVITY IN RAT MESENTERIC LYMPH M. Toyozakia, M. Osakaa, K. Kondob, M. Yoshidaa a
Department of Life Science and Bioethics, Tokyo Medical and Dental University, Tokyo, Japan; b Institute of Environmental Science for Human Life, Ochanomizu University, Tokyo, Japan Objectives: DPP-IV (dipeptydil peptidase-IV; CD26) has been known of for its role in regulation of GLP-1 (glucagon like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide), gut hormones important in insulin metabolism. Recent clinical studies indicated the efficacy of its inhibitor in the treatment of diabetes. Though causative role of DPP-IV in diabetic condition was extensively studied, their role in dyslipidemia is not well understood. In this study, we utilized an intestinal
Objectives: Familial combined hyperlipidemia (FCH) is the most frequent lipid genetic disorder in developed countries and is associated with a high incidence of premature cardiovascular disease. FCH is also associated with insulin resistance, abdominal obesity, fatty liver disease, endothelial dysfunction, low-grade inflammation and with the classic lipid phenotype of T2DM, presenting high triglyceride plasma level, decreased high density lipoprotein (HDL) cholesterol and increased amounts of small, dense LDL particles. Additionaly, impaired incretin secretion has been observed in diabetic individuals, and in other populations caraterized as insulin resistance, such as patients with metabolic syndrome and impaired glucose tolerance. The aim of this study was to investigate the relationship between insulin resistance and the pattern of incretin secretion in patients with FCH. Methods: We conducted a case-control study, enrolling 30 patients with known FCH and 14 healthy controls. After the extraction of a basal blood sample, we administered an oral mixed (glucose and lipids) load to both groups, and measured insulin concentrations at 30 minutes intervals up to 2 hours after the oral load. Insulin resistance was measured by HOMA test, and insulin sensitivity was measured by Matsuda index. Results: There were no statistical differences in age and anthropometric characteristics between both groups. Absolute insulin secretion in response to the oral mixed load, calculated as area under the 120 minutes secretion curve, was significantly higher in patients with FCH than in control subjects. We also found significant differences in insulin resistance and sensitivity between groups: mean HOMA test value was higher in patients with FCH, and mean Matsuda Index value was lower in the same patients, when compared with control group. Increase in GIP secretion was significantly related to insulin resistance but not with GLP-1 and GLP-2. Conclusion: Insulin resistance is an important feature in patients with FCH and is associated with high GIP secretion levels. 34 - Intestinal and biliary lipid metabolism EAS-1096. SIMVASTATIN AND EZETIMIBE HAVE OPPOSING REGULATION ON INTESTINAL CHOLESTEROL ABSORPTION AND TRANS INTESTINAL CHOLESTEROL EXCRETION (TICE) DURING METABOLIC SYNDROME R. Mangat, F. Borthwick, S. Warnakula, M. Jacome-Sosa, D. Vine, S. Proctor Alberta Institute for Human Nutrition, University of Alberta, Edmonton, Canada Objectives: Recent results from the Copenhagen Heart Study have demonstrated a role for non-fasting remnant cholesterol as a causal risk factor for cardiovascular disease (CVD). Evidence in humans, rats and hamsters also show that insulin resistance stimulates overproduction of intestinal derived remnant lipoproteins and may further exacerbate CVD risk. We have recently demonstrated that statins can increase intestinal remnant cholesterol in a rat model of the Metabolic Syndrome (MetS). Here we proposed to evaluate the impact of combining simvastatin (SV) with an intestinal cholesterol absorption inhibitor (ezetimibe [EZ]) on intestinal cholesterol secretion and/or Trans Intestinal Cholesterol Excretion (TICE) in a rodent model of MetS. Methods: JCR:LA-cp control and MetS rats were fed a diet supplemented with 1% w/w cholesterol with/without SV (0.01% w/w), EZ (0.01% w/w) or SV+EZ for 8 weeks. Mesenteric lymph was collected in fasted rats following intralipid infusion via lymphatic fistulae procedure and assessed for cholesterol and particle number. TICE was measured in jejunal segments as flux of H3-cholesterol via Using chamber the technique Results: MetS rats had increased cholesterol per particle (160%) compared to control. SV treatment (but not EZ) significantly increased the ratio of cholesterol per particle (600%). Interestingly, the combination of SV+EZ significantly decreased (43%) cholesterol per particle compared to EZ only group.
e185
TICE was significantly suppressed (45%) in MetS rats compared to control. EZ significantly upregulated (153%) TICE, while SV had no additional effect either alone or in combination with EZ. Conclusion: The addition of EZ (dual therapy) to SV monotherapy can ameliorate statin-induced up-regulation of cholesterol absorption. EZ stimulates TICE in a model of the MetS while SV has no effect. The combination of EZ+SV beneficially modulates intestinal lipid secretion and TICE. 34 - Intestinal and biliary lipid metabolism EAS-0040. PCSK9 PROMOTES INTESTINAL OVERPRODUCTION OF APOLIPOPROTEIN-B LIPOPROTEINS THROUGH LDL-RECEPTOR DEPENDENT AND INDEPENDENT MECHANISMS S. Rashid MD a, H. Tavori MD b, I. Guinzioni MD b, S. Fazio MD b a
Pharmacology, Dalhousie University, Saint John, Canada; Vanderbilt University, Nashville, USA
b
Medicine,
Objectives: Proprotein convertase subtilisin kexin type 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its deficiency in humans results in low plasma LDL-cholesterol and protection against coronary heart disease (CHD). Here we studied the effects of PCSK9 on the intestinal metabolism of triglyceride-rich apolipoprotein B (apoB) lipoproteins (TRL), another important CHD risk factor, and elucidated for the first time the cellular and molecular mechanisms involved. Methods: Cultured human enterocytes (CaCo-2 cells) were treated with either recombinant human PCSK9 (10 mg/mL, 24 hours) or PCSK9 siRNA (15 nmol/L, 48 hours). Proteins and gene expression pathways in the cellular TRL production pathway were then assessed by Western blot and real-time qPCR, respectively and confirmed in vivo in human PCSK9 transgenic mice. Results: PCSK9 treatment increased the secretion of apoB48 and apoB100 in CaCo-2 cells by 40-55% each (p<0.01 vs. untreated cells), whereas PCSK9 gene knockdown substantially reversed this effect. PCSK9 stimulation of apoB was due to: (1) a 1.5-fold increase in apoB mRNA (p<0.01); and (2) enhanced apoB protein stability through both LDLR-dependent and LDLR-independent mechanisms. PCSK9 decreased LDLR protein (p<0.01) and increased cellular apoB stability via activation of microsomal triglyceride transfer protein (MTP). In mice, human PCSK9 expression increased intestinal MTP activity regardless of LDLR expression. Conclusion: PCSK9 increases intestinal TRL apoB production at the transcriptional and post-transcriptional levels mediated in part by the LDLR and by the newly identified PCSK9 target, MTP. These findings indicate that targeted PCSK9-based therapies may also be effective in the management of postprandial hypertriglyceridemia. 34 - Intestinal and biliary lipid metabolism EAS-0258. WESTERN DIET ENHANCES DPP-IV ACTIVITY IN RAT MESENTERIC LYMPH M. Toyozakia, M. Osakaa, K. Kondob, M. Yoshidaa a
Department of Life Science and Bioethics, Tokyo Medical and Dental University, Tokyo, Japan; b Institute of Environmental Science for Human Life, Ochanomizu University, Tokyo, Japan Objectives: DPP-IV (dipeptydil peptidase-IV; CD26) has been known of for its role in regulation of GLP-1 (glucagon like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide), gut hormones important in insulin metabolism. Recent clinical studies indicated the efficacy of its inhibitor in the treatment of diabetes. Though causative role of DPP-IV in diabetic condition was extensively studied, their role in dyslipidemia is not well understood. In this study, we utilized an intestinal