Characterization of ANGPTL3 deficiency and molecular mechanisms of ANGPTL3 deficiency induced hypolipidemia

Anna Tikka

Research output: ThesisDoctoral ThesisCollection of Articles

Abstract

Introduction and aims: ANGPTL3 is a hepatokine and a regulator of lipid metabolism primarily via its inhibitory effect on lipoprotein lipase (LPL), an enzyme responsible for hydrolysing triglycerides (TGs) in lipoproteins. Genetic loss-of-function (LOF) mutations in ANGPTL3 cause a distinct phenotype in humans: low levels of triglycerides, total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels in the circulation (familial combined hypolipidemia, FHBL2). In the first study, ANGPTL3 was silenced in human hepatocytes to investigate intracellular mechanisms of ANGPTL3 deficiency. The study was repeated in human lipoprotein-producing enterocytes. In the second study, carriers of ANGPTL3 LOF s17* were challenged with a high-fat meal to clinically characterize the effects of ANGPTL3 deficiency on postprandial lipid metabolism. In the third study, data from two large health surveys were utilized to investigate whether sequence variants in ANGPTL3 contribute to dyslipidemias in Finns and if ANGPTL3 serum levels correlate with lipid metabolic biomarkers in hypolipidemic or hyperlipidemic subjects. Methods and results: ANGPTL3 was silenced in human hepatocytes and enterocytes using lentiviral shRNA vector. Substrate uptake, their conversion into TG, and TG/apoB secretion was measured in cells and cell culture media by enzymatic assays, ELISA assays, and labeled fatty acid and glucose substrates. As a result, we demonstrated that ANGPTL3 expression and secretion are regulated by insulin, LXR agonists, and PPARy agonist rosiglitazone and that in hepatocytes, silencing affects the lipidation of lipoprotein particles upon insulin stimulation and increases glucose uptake in these cells. Changes in lipoprotein secretion were not seen in human enterocytes. Silencing of ANGPTL3 decreased CD36 fatty acid transporter expression in both cell lines and phospholipid transfer protein PLTP expression in hepatocytes. S17* ANGPTL3 LOF carriers and control subjects were challenged with a high-fat meal followed by blood withdrawal during fasting and at 2, 4, and 6 hours after a meal. Plasma lipids, lipoproteins, and key regulatory proteins were measured in the plasma samples by enzymatic assays and ELISA assays. As a result, TG values in homozygous carriers remained low throughout, whereas heterozygotes and control subjects showed a typical elevated postprandial response in TG levels. Homozygous ANGPTL3 LOF carriers had lower fasting non-esterified fatty acid (NEFA) values, which may result in decreased supply of FA in the liver. In the Finnish population study, one rare ANGPTL3 variant and one carrier with abnormally high TC and LDL-C levels was identified. ANGPTL3 protein levels did not correlate with lipids or lipoprotein levels in hypolipidemic or hyperlipidemic subjects, but ANGPTL3 did positively correlate with PLTP and CETP activities, and age. Conclusions: The cell culture experiments with ANGPTL3-silenced hepatocytes indicate that ANGPTL3 not only affects the clearance of lipoprotein particles in the bloodstream via accelerated activity of LPL, but may also alter hepatic glucose and fatty acid substrate utilization, VLDL lipidation, and PLTP expression. Homozygous ANGPTL3 LOF carriers manifest hypolipidemia in postprandial phase, which can be considered anti-atherogenic and protective against the development of atherosclerotic cardiovascular disease. Based on the genetic data, genetic variants in ANGPTL3 are rare and do not contribute to dyslipidemia on a population level. ANGPTL3 protein levels in hyperlipidemic or hypolipidemic subjects had no predictive biomarker value for plasma lipid levels.
Original languageEnglish
Supervisors/Advisors
  • Jauhiainen, Matti, Supervisor, External person
Award date19 Jan 2018
Place of PublicationHelsinki
Publisher
Print ISBNs978-951-51-3906-1
Electronic ISBNs978-951-51-3907-8
Publication statusPublished - 2018
MoE publication typeG5 Doctoral dissertation (article)

Fields of Science

  • Lipid Metabolism
  • Angiopoietin-like Proteins
  • +deficiency
  • +genetics
  • +metabolism
  • Cholesterol
  • Hepatocytes
  • Enterocytes
  • Glucose
  • Insulin
  • Lipids
  • Lipase
  • Lipoprotein Lipase
  • Phospholipid Transfer Proteins
  • Liver
  • Triglycerides
  • RNA
  • Lipoproteins, HDL
  • Lipoproteins, LDL
  • Lipoproteins, VLDL
  • Dyslipidemias
  • Loss of Function Mutation
  • Gene Expression Profiling
  • 3121 Internal medicine

Cite this

@phdthesis{9c0f3c1bad594a0ca3434e8e7111f8b3,
title = "Characterization of ANGPTL3 deficiency and molecular mechanisms of ANGPTL3 deficiency induced hypolipidemia",
abstract = "Introduction and aims: ANGPTL3 is a hepatokine and a regulator of lipid metabolism primarily via its inhibitory effect on lipoprotein lipase (LPL), an enzyme responsible for hydrolysing triglycerides (TGs) in lipoproteins. Genetic loss-of-function (LOF) mutations in ANGPTL3 cause a distinct phenotype in humans: low levels of triglycerides, total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels in the circulation (familial combined hypolipidemia, FHBL2). In the first study, ANGPTL3 was silenced in human hepatocytes to investigate intracellular mechanisms of ANGPTL3 deficiency. The study was repeated in human lipoprotein-producing enterocytes. In the second study, carriers of ANGPTL3 LOF s17* were challenged with a high-fat meal to clinically characterize the effects of ANGPTL3 deficiency on postprandial lipid metabolism. In the third study, data from two large health surveys were utilized to investigate whether sequence variants in ANGPTL3 contribute to dyslipidemias in Finns and if ANGPTL3 serum levels correlate with lipid metabolic biomarkers in hypolipidemic or hyperlipidemic subjects. Methods and results: ANGPTL3 was silenced in human hepatocytes and enterocytes using lentiviral shRNA vector. Substrate uptake, their conversion into TG, and TG/apoB secretion was measured in cells and cell culture media by enzymatic assays, ELISA assays, and labeled fatty acid and glucose substrates. As a result, we demonstrated that ANGPTL3 expression and secretion are regulated by insulin, LXR agonists, and PPARy agonist rosiglitazone and that in hepatocytes, silencing affects the lipidation of lipoprotein particles upon insulin stimulation and increases glucose uptake in these cells. Changes in lipoprotein secretion were not seen in human enterocytes. Silencing of ANGPTL3 decreased CD36 fatty acid transporter expression in both cell lines and phospholipid transfer protein PLTP expression in hepatocytes. S17* ANGPTL3 LOF carriers and control subjects were challenged with a high-fat meal followed by blood withdrawal during fasting and at 2, 4, and 6 hours after a meal. Plasma lipids, lipoproteins, and key regulatory proteins were measured in the plasma samples by enzymatic assays and ELISA assays. As a result, TG values in homozygous carriers remained low throughout, whereas heterozygotes and control subjects showed a typical elevated postprandial response in TG levels. Homozygous ANGPTL3 LOF carriers had lower fasting non-esterified fatty acid (NEFA) values, which may result in decreased supply of FA in the liver. In the Finnish population study, one rare ANGPTL3 variant and one carrier with abnormally high TC and LDL-C levels was identified. ANGPTL3 protein levels did not correlate with lipids or lipoprotein levels in hypolipidemic or hyperlipidemic subjects, but ANGPTL3 did positively correlate with PLTP and CETP activities, and age. Conclusions: The cell culture experiments with ANGPTL3-silenced hepatocytes indicate that ANGPTL3 not only affects the clearance of lipoprotein particles in the bloodstream via accelerated activity of LPL, but may also alter hepatic glucose and fatty acid substrate utilization, VLDL lipidation, and PLTP expression. Homozygous ANGPTL3 LOF carriers manifest hypolipidemia in postprandial phase, which can be considered anti-atherogenic and protective against the development of atherosclerotic cardiovascular disease. Based on the genetic data, genetic variants in ANGPTL3 are rare and do not contribute to dyslipidemia on a population level. ANGPTL3 protein levels in hyperlipidemic or hypolipidemic subjects had no predictive biomarker value for plasma lipid levels.",
keywords = "Lipid Metabolism, Angiopoietin-like Proteins, +deficiency, +genetics, +metabolism, Cholesterol, Hepatocytes, Enterocytes, Glucose, Insulin, Lipids, Lipase, Lipoprotein Lipase, Phospholipid Transfer Proteins, Liver, Triglycerides, RNA, Lipoproteins, HDL, Lipoproteins, LDL, Lipoproteins, VLDL, Dyslipidemias, Loss of Function Mutation, Gene Expression Profiling, 3121 Internal medicine",
author = "Anna Tikka",
note = "M1 - 39 s. + liitteet",
year = "2018",
language = "English",
isbn = "978-951-51-3906-1",
series = "Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis",
publisher = "Helsingin yliopisto",
number = "2/2018",
address = "Finland",

}

Characterization of ANGPTL3 deficiency and molecular mechanisms of ANGPTL3 deficiency induced hypolipidemia. / Tikka, Anna.

Helsinki : Helsingin yliopisto, 2018. 39 p.

Research output: ThesisDoctoral ThesisCollection of Articles

TY - THES

T1 - Characterization of ANGPTL3 deficiency and molecular mechanisms of ANGPTL3 deficiency induced hypolipidemia

AU - Tikka, Anna

N1 - M1 - 39 s. + liitteet

PY - 2018

Y1 - 2018

N2 - Introduction and aims: ANGPTL3 is a hepatokine and a regulator of lipid metabolism primarily via its inhibitory effect on lipoprotein lipase (LPL), an enzyme responsible for hydrolysing triglycerides (TGs) in lipoproteins. Genetic loss-of-function (LOF) mutations in ANGPTL3 cause a distinct phenotype in humans: low levels of triglycerides, total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels in the circulation (familial combined hypolipidemia, FHBL2). In the first study, ANGPTL3 was silenced in human hepatocytes to investigate intracellular mechanisms of ANGPTL3 deficiency. The study was repeated in human lipoprotein-producing enterocytes. In the second study, carriers of ANGPTL3 LOF s17* were challenged with a high-fat meal to clinically characterize the effects of ANGPTL3 deficiency on postprandial lipid metabolism. In the third study, data from two large health surveys were utilized to investigate whether sequence variants in ANGPTL3 contribute to dyslipidemias in Finns and if ANGPTL3 serum levels correlate with lipid metabolic biomarkers in hypolipidemic or hyperlipidemic subjects. Methods and results: ANGPTL3 was silenced in human hepatocytes and enterocytes using lentiviral shRNA vector. Substrate uptake, their conversion into TG, and TG/apoB secretion was measured in cells and cell culture media by enzymatic assays, ELISA assays, and labeled fatty acid and glucose substrates. As a result, we demonstrated that ANGPTL3 expression and secretion are regulated by insulin, LXR agonists, and PPARy agonist rosiglitazone and that in hepatocytes, silencing affects the lipidation of lipoprotein particles upon insulin stimulation and increases glucose uptake in these cells. Changes in lipoprotein secretion were not seen in human enterocytes. Silencing of ANGPTL3 decreased CD36 fatty acid transporter expression in both cell lines and phospholipid transfer protein PLTP expression in hepatocytes. S17* ANGPTL3 LOF carriers and control subjects were challenged with a high-fat meal followed by blood withdrawal during fasting and at 2, 4, and 6 hours after a meal. Plasma lipids, lipoproteins, and key regulatory proteins were measured in the plasma samples by enzymatic assays and ELISA assays. As a result, TG values in homozygous carriers remained low throughout, whereas heterozygotes and control subjects showed a typical elevated postprandial response in TG levels. Homozygous ANGPTL3 LOF carriers had lower fasting non-esterified fatty acid (NEFA) values, which may result in decreased supply of FA in the liver. In the Finnish population study, one rare ANGPTL3 variant and one carrier with abnormally high TC and LDL-C levels was identified. ANGPTL3 protein levels did not correlate with lipids or lipoprotein levels in hypolipidemic or hyperlipidemic subjects, but ANGPTL3 did positively correlate with PLTP and CETP activities, and age. Conclusions: The cell culture experiments with ANGPTL3-silenced hepatocytes indicate that ANGPTL3 not only affects the clearance of lipoprotein particles in the bloodstream via accelerated activity of LPL, but may also alter hepatic glucose and fatty acid substrate utilization, VLDL lipidation, and PLTP expression. Homozygous ANGPTL3 LOF carriers manifest hypolipidemia in postprandial phase, which can be considered anti-atherogenic and protective against the development of atherosclerotic cardiovascular disease. Based on the genetic data, genetic variants in ANGPTL3 are rare and do not contribute to dyslipidemia on a population level. ANGPTL3 protein levels in hyperlipidemic or hypolipidemic subjects had no predictive biomarker value for plasma lipid levels.

AB - Introduction and aims: ANGPTL3 is a hepatokine and a regulator of lipid metabolism primarily via its inhibitory effect on lipoprotein lipase (LPL), an enzyme responsible for hydrolysing triglycerides (TGs) in lipoproteins. Genetic loss-of-function (LOF) mutations in ANGPTL3 cause a distinct phenotype in humans: low levels of triglycerides, total cholesterol (TC), low density lipoprotein (LDL) and high density lipoprotein (HDL) levels in the circulation (familial combined hypolipidemia, FHBL2). In the first study, ANGPTL3 was silenced in human hepatocytes to investigate intracellular mechanisms of ANGPTL3 deficiency. The study was repeated in human lipoprotein-producing enterocytes. In the second study, carriers of ANGPTL3 LOF s17* were challenged with a high-fat meal to clinically characterize the effects of ANGPTL3 deficiency on postprandial lipid metabolism. In the third study, data from two large health surveys were utilized to investigate whether sequence variants in ANGPTL3 contribute to dyslipidemias in Finns and if ANGPTL3 serum levels correlate with lipid metabolic biomarkers in hypolipidemic or hyperlipidemic subjects. Methods and results: ANGPTL3 was silenced in human hepatocytes and enterocytes using lentiviral shRNA vector. Substrate uptake, their conversion into TG, and TG/apoB secretion was measured in cells and cell culture media by enzymatic assays, ELISA assays, and labeled fatty acid and glucose substrates. As a result, we demonstrated that ANGPTL3 expression and secretion are regulated by insulin, LXR agonists, and PPARy agonist rosiglitazone and that in hepatocytes, silencing affects the lipidation of lipoprotein particles upon insulin stimulation and increases glucose uptake in these cells. Changes in lipoprotein secretion were not seen in human enterocytes. Silencing of ANGPTL3 decreased CD36 fatty acid transporter expression in both cell lines and phospholipid transfer protein PLTP expression in hepatocytes. S17* ANGPTL3 LOF carriers and control subjects were challenged with a high-fat meal followed by blood withdrawal during fasting and at 2, 4, and 6 hours after a meal. Plasma lipids, lipoproteins, and key regulatory proteins were measured in the plasma samples by enzymatic assays and ELISA assays. As a result, TG values in homozygous carriers remained low throughout, whereas heterozygotes and control subjects showed a typical elevated postprandial response in TG levels. Homozygous ANGPTL3 LOF carriers had lower fasting non-esterified fatty acid (NEFA) values, which may result in decreased supply of FA in the liver. In the Finnish population study, one rare ANGPTL3 variant and one carrier with abnormally high TC and LDL-C levels was identified. ANGPTL3 protein levels did not correlate with lipids or lipoprotein levels in hypolipidemic or hyperlipidemic subjects, but ANGPTL3 did positively correlate with PLTP and CETP activities, and age. Conclusions: The cell culture experiments with ANGPTL3-silenced hepatocytes indicate that ANGPTL3 not only affects the clearance of lipoprotein particles in the bloodstream via accelerated activity of LPL, but may also alter hepatic glucose and fatty acid substrate utilization, VLDL lipidation, and PLTP expression. Homozygous ANGPTL3 LOF carriers manifest hypolipidemia in postprandial phase, which can be considered anti-atherogenic and protective against the development of atherosclerotic cardiovascular disease. Based on the genetic data, genetic variants in ANGPTL3 are rare and do not contribute to dyslipidemia on a population level. ANGPTL3 protein levels in hyperlipidemic or hypolipidemic subjects had no predictive biomarker value for plasma lipid levels.

KW - Lipid Metabolism

KW - Angiopoietin-like Proteins

KW - +deficiency

KW - +genetics

KW - +metabolism

KW - Cholesterol

KW - Hepatocytes

KW - Enterocytes

KW - Glucose

KW - Insulin

KW - Lipids

KW - Lipase

KW - Lipoprotein Lipase

KW - Phospholipid Transfer Proteins

KW - Liver

KW - Triglycerides

KW - RNA

KW - Lipoproteins, HDL

KW - Lipoproteins, LDL

KW - Lipoproteins, VLDL

KW - Dyslipidemias

KW - Loss of Function Mutation

KW - Gene Expression Profiling

KW - 3121 Internal medicine

M3 - Doctoral Thesis

SN - 978-951-51-3906-1

T3 - Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis

PB - Helsingin yliopisto

CY - Helsinki

ER -

Tikka A. Characterization of ANGPTL3 deficiency and molecular mechanisms of ANGPTL3 deficiency induced hypolipidemia. Helsinki : Helsingin yliopisto, 2018. 39 p. (Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis; 2/2018).