Mol. Cells 2021; 44(2): 116-125
Published online February 28, 2021
https://doi.org/10.14348/molcells.2021.2147
© The Korean Society for Molecular and Cellular Biology
Correspondence to : jean-mathieu.berger@utsouthwestern.edu (JMB); yamoon15@inha.ac.kr (YAM)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Cardiovascular diseases (CVDs) are the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD) and dyslipidemia is considered at least partially responsible for the increased CVD risk in NAFLD patients. The aim of the present study is to understand how hepatic de novo lipogenesis influences hepatic cholesterol content as well as its effects on the plasma lipid levels. Hepatic lipogenesis was induced in mice by feeding a fat-free/high-sucrose (FF/HS) diet and the metabolic pathways associated with cholesterol were then analyzed. Both liver triglyceride and cholesterol contents were significantly increased in mice fed an FF/HS diet. Activation of fatty acid synthesis driven by the activation of sterol regulatory element binding protein (SREBP)-1c resulted in the increased liver triglycerides. The augmented cholesterol content in the liver could not be explained by an increased cholesterol synthesis, which was decreased by the FF/HS diet. HMGCoA reductase protein level was decreased in mice fed an FF/HS diet. We found that the liver retained more cholesterol through a reduced excretion of bile acids, a reduced fecal cholesterol excretion, and an increased cholesterol uptake from plasma lipoproteins. Very low-density lipoproteintriglyceride and -cholesterol secretion were increased in mice fed an FF/HS diet, which led to hypertriglyceridemia and hypercholesterolemia in Ldlr-/- mice, a model that exhibits a more human like lipoprotein profile. These findings suggest that dietary cholesterol intake and cholesterol synthesis rates cannot only explain the hypercholesterolemia associated with NAFLD, and that the control of fatty acid synthesis should be considered for the management of dyslipidemia.
Keywords cholesterol, dyslipidemia, fatty liver, lipogenesis
Nonalcoholic fatty liver disease (NAFLD) is the most common liver condition with a prevalence of ~33% among the adult population in the US (Browning et al., 2004). NAFLD is associated with an increased incidence of cardiovascular diseases (CVDs) (Anstee et al., 2013; Targher et al., 2010) and patients with NAFLD are more likely to die from CVD than liver disease (Chatrath et al., 2012; Cohen and Fisher, 2013). Atherogenic dyslipidemia, which is characterized by an increased level of plasma triglycerides, decreased level of high-density lipoprotein (HDL), and the presence of small and dense low-density lipoprotein (LDL) particles, is considered at least partially responsible for the increased CVD risk in NAFLD patients (Cohen and Fisher, 2013). NAFLD is strongly associated with obesity and insulin resistance, which are also known risk factors for dyslipidemia (Angulo, 2002). However, hepatic steatosis was independently associated with atherogenic dyslipidemia after adjustment for obesity, physical activity, hyperglycemia, and systemic inflammation (Makadia et al., 2013). The pathogenesis of dyslipidemia in NAFLD is not completely understood, but hepatic overproduction of very low-density lipoprotein (VLDL), alteration in lipoprotein lipase activity, and dysregulated clearance of lipoproteins are considered major contributors (Chatrath et al., 2012; Howard, 1987; Semenkovich, 2006). Understanding the pathophysiology of the dyslipidemia associated with NAFLD would guide better strategies to reduce morbidity and mortality in patients with NAFLD.
In the liver, triglycerides come from the diet,
The role of SREBP-1c activation in the development of hypertriglyceridemia has been studied in different animal models (Horton et al., 1999; Moon et al., 2012; Okazaki et al., 2010). In hamsters, activation of SREBP-1 by a high sucrose diet induced hypertriglyceridemia (Moon et al., 2012). They also exhibited hypercholesterolemia, characterized by a substantial increase in VLDL-cholesterol levels. In a mouse model with a
In the present study, we determined how hepatic
Males C57BL/6J wild-type and
Blood was obtained from the tail vein of restrained animals in heparin-coated tubes and plasma was separated and stored at –80°C. Plasma cholesterol and triglyceride concentrations were determined using Infinity Cholesterol Liquid Stable Reagent and Infinity Triglycerides Liquid Stable Reagent (Thermo Fisher Scientific, USA), respectively. Plasma lipoprotein profiles were obtained for each group by pooling equal amounts of plasma from each mouse. After filtration (Costar; Corning, USA), 20 μl of each plasma sample was pooled to a total of 200 μl per group, and the pooled plasma sample was injected into the fast protein liquid chromatography (FPLC) system. FPLC analysis was performed at 4°C using a Superose 6 10/300 GL exclusion column at a flow rate of 0.5 ml/min. Cholesterol and triglyceride concentrations in the collected fractions were then determined. Plasma Apo-CIII concentration was measured by the APOC3 (mouse) ELISA Kit (Abnova, USA). Bile was collected from the gallbladder after 3 h of fasting, and bile acid concentrations were measured using the Colorimetric Total Bile Acid Assay Kit from Diazyme Laboratories (USA).
Human LDL (1.019-1.063 g/ml) and HDL (1.125-1.215 g/ml) were prepared as described previously (Goldstein et al., 1983). The purity of each lipoprotein was validated by FPLC. Human LDL or HDL was labeled with cholesteryl oleate [cholesteryl-1,2-3H(N)] (Perkin Elmer, USA) as described previously (May et al., 2013).
Mice were anesthetized using pentobarbital (80 mg/kg of body weight) and 3H-cholesteryl oleate-labeled LDL or HDL (106 cpm/mouse) was injected via the penile vein. Blood was collected before injection (0 min) and at 2, 15, 30, and 60 min after injection. Plasma was separated, and triglyceride and cholesterol concentrations were measured as described above. Radioactivity in 20 μl of the plasma was measured. One hour after injection, mice were euthanized with isoflurane and perfused with saline. The liver was collected, washed with saline, and the whole liver was dissolved in 3 ml of 50% KOH in ethanol. The 3H-labelled cholesterol content of the liver was measured.
Tyloxapol was purchased from Sigma-Aldrich (USA); 10% tyloxapol solution was prepared using saline. Mice were fasted for 6 h and injected with tyloxapol (500 mg/kg) via the penile vein. Blood was collected from the tail vein before injection (0 min) and at 15, 30, 60, 90, 120, and 180 min after injection. Plasma was separated, and triglyceride and cholesterol concentrations were determined. Triglyceride and cholesterol secretion rates were calculated from the linear regression analysis of time versus concentration.
The synthesis rates of sterol and fatty acids in the liver were determined in mice using 3H-labeled water as described previously (Shimano et al., 1996).
Hepatocytes were isolated from C57BL/6J wild-type mice and cultured as described previously (Kim et al., 2020; Matsuda et al., 2001). After a 3-h attachment period, the cells were incubated with 0.5 mM 14C-sodium acetate for 4 h. Lipids were extracted, and then, fatty acids, cholesterol, cholesteryl ester, and triglycerides were separated by TLC as described previously (Matsuda et al., 2001; Moon et al., 2009). Incorporation of 14C-acetate into newly synthesized lipids was then determined.
The amount of cholesterol in feces was measured as described previously, with slight modifications (Turley et al., 1996). Mice were housed in individual cages a week before the experiment, and feces were collected every 24 h for three days. Feces were dried completely, ground, and resuspended in water (100 μg/2 ml). Lipids were extracted in chloroform/methanol (2:1) and resuspended in isopropanol. Cholesterol concentrations in the extracts were measured using Infinity Cholesterol Liquid Stable Reagent (Thermo Fisher Scientific).
Hepatocytes isolated from C57BL/6J wild-type mice were seeded at a density of 1 × 106 cells in a 60-mm dish. After a 2-h attachment period, the cells were washed and incubated with 1 μg/ml of BODIPY 493/503 (Thermo Fisher Scientific) and 15 μg/ml of dehydroergosterol (DHE; Sigma-Aldrich) in either the presence or absence of 20 μM oleic acid for 24 h. After incubation, the cells were imaged with a ZEISS LSM 780 confocal microscope (Zeiss, USA).
Membrane and nuclear proteins were prepared individually from frozen livers, and equal amounts of protein from each mouse of the same group were pooled as previously described (Engelking et al., 2004). Aliquots of pooled proteins were subjected to SDS-PAGE on 8% gels and transferred to a nitrocellulose membrane (Bio-Rad, USA). Immunoblot analyses were performed using polyclonal anti-mouse SREBP-1, SREBP-2, and HMG-CoA reductase antibodies as previously described (Rong et al., 2017). Anti-Scavenger receptor class B type 1 (SR-BI) antibody was obtained from Abcam (USA) and anti-cluster of differentiation 36 (CD36) antibody was obtained from R&D Systems (USA). Anti-mouse cAMP response element binding protein (CREB; Invitrogen, USA) and anti-dog calnexin (Enzo Life Science, USA) antibodies were used as loading controls for nuclear and membrane proteins, respectively. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate System (Thermo Fisher Scientific) and visualized with Odyssey CLx Imaging system and LI-COR Image StudioTM software (LI-COR, USA).
Total RNA was prepared from mouse livers with an RNA STAT-60 kit (Tel-Test, USA), and qPCR was performed as previously described (Rong et al., 2017). All reactions were performed in triplicate, and the relative amount of all mRNAs was calculated using the comparative threshold cycle (C
All results are reported as the mean ± SE. Statistical significance was analyzed using a Student’s
FF/HS diets have been previously used to induce hepatic
Cholesterol content was also significantly increased in the livers of mice fed an FF/HS diet (Fig. 2A). All genes encoding enzymes responsible for cholesterol synthesis are regulated by SREBP-2 (Horton et al., 2002); therefore, SREBP-2 and its target genes were analyzed. While the precursor form of SREBP-2 appeared slightly increased, the amount of the transcriptionally active nuclear form was not altered by the FF/HS diet (Fig. 2B). The mRNA expression levels of neither SREBP-2 (
To determine why the liver cholesterol content was increased despite the reduced cholesterol synthesis in the mice fed an FF/HS diet, additional metabolic pathways involved in cholesterol homeostasis were examined. In the liver, cholesterol can be excreted into the bile directly as free cholesterol or after its conversion into bile acids. Feces contains cholesterol from bile, diet, and transintestinal cholesterol excretion. Both diets used in this study are cholesterol-free; therefore, the cholesterol found in the feces must be endogenous. Feces of mice fed a chow or an FF/HS diet were collected for 3 days at the end of the diet feeding period, and fecal cholesterol contents were measured. Fecal cholesterol content of mice fed an FF/HS diet was reduced by 73% compared to mice fed a chow diet (Fig. 3A). Bile acid concentrations were also significantly reduced in the bile of mice fed the FF/HS diet, compared to the mice fed the chow (Fig. 3B). These results suggest that cholesterol excretion was reduced in the mice fed an FF/HS diet and may partly explain why the liver retained more cholesterol despite a reduced hepatic cholesterol synthesis. mRNA expression of genes involved in bile acid synthesis (
Hepatocytes can acquire cholesterol from the uptake of plasma LDL and HDL. Therefore, we examined whether hepatic cholesterol uptake was altered in the liver of mice fed an FF/HS diet. Mice were injected with LDL labeled with 3H-cholesteryl oleate, then hepatic content of 3H-cholesteryl oleate was measured 1 h after injection. As shown in Fig. 4A, the hepatic content of 3H-cholesterol from LDL was significantly increased in mice fed the FF/HS diet. mRNA expression of the LDL receptor was not changed compared to the mice fed a chow (Fig. 4B).
Next, the hepatic uptake of HDL-cholesterol was analyzed by injecting HDL labeled with 3H-cholesteryl oleate. We observed that the hepatic content of 3H-cholesteryl oleate from HDL was significantly increased in the mice fed an FF/HS diet (Fig. 4C). SR-BI is one of the major proteins responsible for the uptake of cholesterol from plasma HDL into the liver (Acton et al., 1996; Connelly and Williams, 2004). Liver mRNA expression level of SR-BI (
Formation of cholesteryl esters, the storage form of cholesterol, was compared using primary hepatocytes isolated from mice fed a chow or an FF/HS diet. Hepatocytes were incubated with 14C-acetate, and its incorporation into fatty acids, triglycerides, cholesterol, and cholesteryl esters was measured (Fig. 5A). Consistent with the
To examine whether fatty acids in hepatocytes can affect the localization of cholesterol in lipid droplets, primary hepatocytes were isolated from C57BL/6J mice and incubated with fluorescence-labeled DHE, a tracer of cholesterol, in either the presence or absence of oleic acid. BODIPY 493/503 was used to stain neutral lipid droplets (Fig. 5B). When the cells were incubated with DHE in the presence of oleic acid, a strong fluorescence from DHE was detected within the cytosolic lipid droplets, while fluorescence from DHE was dispersed and weak in the absence of oleic acid (Fig. 5B). Results in Fig. 5 suggest that increased fatty acids in hepatocytes facilitates the incorporation of cholesterol into lipid droplets, mainly as cholesteryl esters. Increased cholesteryl ester formation and facilitated incorporation into lipid droplets may further explain the increased hepatic cholesterol content in mice with high
Lipids stored in cytoplasmic lipid droplets are mobilized through lipid droplet-associated proteins for the lipidation of apoB to form VLDL (Ye et al., 2009; Zhang et al., 2017). To determine whether fatty liver induced by the FF/HS diet secretes more VLDL-triglyceride and -cholesterol, plasma triglyceride and cholesterol concentrations were measured at various time points after injection of tyloxapol, an inhibitor of lipoprotein lipase. Mice fed an FF/HS diet secreted more VLDL-triglyceride and -cholesterol into the plasma compared to the chow-fed group (Fig. 6A). The expression levels of genes that are essential for VLDL assembly, such as
To determine the effects of an FF/HS diet on dyslipidemia,
SREBP-1c activation leads to hepatic triglyceride accumulation and hypertriglyceridemia in different animal models including hamsters fed a high sucrose diet (Moon et al., 2012), Tg-SREBP1a;
The chow diet and the FF/HS diet used in this study were of different nature and it has been shown that dietary fiber contents might influence the rate of cholesterol excretion into the feces. We used diets with similar total dietary fiber contents for this study as the chow diet contains 17% fiber and the FF/HS diet contains 16.5% fiber. However, there is still a possibility that the difference in the nature of the diets might affect the cholesterol retention in the body.
The availability of fatty acids facilitates the conversion of cholesterol to cholesteryl esters (Xie et al., 2002), which could be readily incorporated into lipid droplets in hepatocytes, as presented in Fig. 5. Fatty acids not only drove cholesterol esterification, but also enhanced secretion of cholesteryl esters into the plasma (Xie et al., 2002). Acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2), the enzyme responsible for cholesteryl ester formation in the liver, prefers oleic acid as a substrate, and its activity is primarily regulated by the availability of fatty acid substrates (Xie et al., 2002). Upon activation of fatty acid synthesis by SREBP-1c in the liver, oleic acid (C18:1, n-9) is the fatty acid that is the most increased (Moon et al., 2014; Shimomura et al., 1998). Although ACAT2 mRNA expression levels were not significantly different between mice fed a chow and an FF/HS diet (Supplementary Fig. S1C), our findings suggest that the increased fatty acid availability from
Triglycerides and cholesteryl esters are packed into VLDL in hepatocytes. SREBP-1c increases PLTP expression as well as genes required for fatty acid synthesis. PLTP plays a major role in the incorporation of phospholipids to growing VLDLs to expand the particle and its lipidation, resulting in bigger VLDL particles rich in triglycerides (Okazaki et al., 2010; Yazdanyar and Jiang, 2012). The FF/HS diet induced PLTP expression as well as other targets of SREBP-1c, leading to an increased VLDL-triglyceride and -cholesterol secretion and subsequent development of hypercholesterolemia and hypertriglyceridemia in
In humans, while fatty acids released from adipose tissue are the major source of fat in livers of individuals with NAFLD (Donnelly et al., 2005),
This work was supported by grants from the National Research Foundation of Korea funded by the Korean government (2018R1A2B6007576), the National Institutes of Health (HL-20948), and the Leducq Foundation (5200829301).
The authors thank Sijeong Bae (Department of Molecular Medicine, Inha University College of Medicine) Angel Loza Valdes, Ajit Kumar Koduri, Tuyet Dang, Judith Sanchez, Norma Anderson, and Lisa Beatty (Department of Molecular Genetics, UT Southwestern Medical Center), and Abhijit Bugde (the Live Cell Imaging Core Facility, UT Southwestern Medical Center) for their technical assistance. The authors also thank Dr. Youngah Jo for providing the anti-HMG CoA-R antibody and Dr. Jay Horton for scientific advice.
J.M.B. and Y.A.M. conceived and performed experiments, analyzed the data, and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2021; 44(2): 116-125
Published online February 28, 2021 https://doi.org/10.14348/molcells.2021.2147
Copyright © The Korean Society for Molecular and Cellular Biology.
Jean-Mathieu Berger1,* and Young-Ah Moon2,*
1Departments of Internal Medicine and Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA, 2Department of Molecular Medicine, Inha University College of Medicine, Incheon 22212, Korea
Correspondence to:jean-mathieu.berger@utsouthwestern.edu (JMB); yamoon15@inha.ac.kr (YAM)
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
Cardiovascular diseases (CVDs) are the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD) and dyslipidemia is considered at least partially responsible for the increased CVD risk in NAFLD patients. The aim of the present study is to understand how hepatic de novo lipogenesis influences hepatic cholesterol content as well as its effects on the plasma lipid levels. Hepatic lipogenesis was induced in mice by feeding a fat-free/high-sucrose (FF/HS) diet and the metabolic pathways associated with cholesterol were then analyzed. Both liver triglyceride and cholesterol contents were significantly increased in mice fed an FF/HS diet. Activation of fatty acid synthesis driven by the activation of sterol regulatory element binding protein (SREBP)-1c resulted in the increased liver triglycerides. The augmented cholesterol content in the liver could not be explained by an increased cholesterol synthesis, which was decreased by the FF/HS diet. HMGCoA reductase protein level was decreased in mice fed an FF/HS diet. We found that the liver retained more cholesterol through a reduced excretion of bile acids, a reduced fecal cholesterol excretion, and an increased cholesterol uptake from plasma lipoproteins. Very low-density lipoproteintriglyceride and -cholesterol secretion were increased in mice fed an FF/HS diet, which led to hypertriglyceridemia and hypercholesterolemia in Ldlr-/- mice, a model that exhibits a more human like lipoprotein profile. These findings suggest that dietary cholesterol intake and cholesterol synthesis rates cannot only explain the hypercholesterolemia associated with NAFLD, and that the control of fatty acid synthesis should be considered for the management of dyslipidemia.
Keywords: cholesterol, dyslipidemia, fatty liver, lipogenesis
Nonalcoholic fatty liver disease (NAFLD) is the most common liver condition with a prevalence of ~33% among the adult population in the US (Browning et al., 2004). NAFLD is associated with an increased incidence of cardiovascular diseases (CVDs) (Anstee et al., 2013; Targher et al., 2010) and patients with NAFLD are more likely to die from CVD than liver disease (Chatrath et al., 2012; Cohen and Fisher, 2013). Atherogenic dyslipidemia, which is characterized by an increased level of plasma triglycerides, decreased level of high-density lipoprotein (HDL), and the presence of small and dense low-density lipoprotein (LDL) particles, is considered at least partially responsible for the increased CVD risk in NAFLD patients (Cohen and Fisher, 2013). NAFLD is strongly associated with obesity and insulin resistance, which are also known risk factors for dyslipidemia (Angulo, 2002). However, hepatic steatosis was independently associated with atherogenic dyslipidemia after adjustment for obesity, physical activity, hyperglycemia, and systemic inflammation (Makadia et al., 2013). The pathogenesis of dyslipidemia in NAFLD is not completely understood, but hepatic overproduction of very low-density lipoprotein (VLDL), alteration in lipoprotein lipase activity, and dysregulated clearance of lipoproteins are considered major contributors (Chatrath et al., 2012; Howard, 1987; Semenkovich, 2006). Understanding the pathophysiology of the dyslipidemia associated with NAFLD would guide better strategies to reduce morbidity and mortality in patients with NAFLD.
In the liver, triglycerides come from the diet,
The role of SREBP-1c activation in the development of hypertriglyceridemia has been studied in different animal models (Horton et al., 1999; Moon et al., 2012; Okazaki et al., 2010). In hamsters, activation of SREBP-1 by a high sucrose diet induced hypertriglyceridemia (Moon et al., 2012). They also exhibited hypercholesterolemia, characterized by a substantial increase in VLDL-cholesterol levels. In a mouse model with a
In the present study, we determined how hepatic
Males C57BL/6J wild-type and
Blood was obtained from the tail vein of restrained animals in heparin-coated tubes and plasma was separated and stored at –80°C. Plasma cholesterol and triglyceride concentrations were determined using Infinity Cholesterol Liquid Stable Reagent and Infinity Triglycerides Liquid Stable Reagent (Thermo Fisher Scientific, USA), respectively. Plasma lipoprotein profiles were obtained for each group by pooling equal amounts of plasma from each mouse. After filtration (Costar; Corning, USA), 20 μl of each plasma sample was pooled to a total of 200 μl per group, and the pooled plasma sample was injected into the fast protein liquid chromatography (FPLC) system. FPLC analysis was performed at 4°C using a Superose 6 10/300 GL exclusion column at a flow rate of 0.5 ml/min. Cholesterol and triglyceride concentrations in the collected fractions were then determined. Plasma Apo-CIII concentration was measured by the APOC3 (mouse) ELISA Kit (Abnova, USA). Bile was collected from the gallbladder after 3 h of fasting, and bile acid concentrations were measured using the Colorimetric Total Bile Acid Assay Kit from Diazyme Laboratories (USA).
Human LDL (1.019-1.063 g/ml) and HDL (1.125-1.215 g/ml) were prepared as described previously (Goldstein et al., 1983). The purity of each lipoprotein was validated by FPLC. Human LDL or HDL was labeled with cholesteryl oleate [cholesteryl-1,2-3H(N)] (Perkin Elmer, USA) as described previously (May et al., 2013).
Mice were anesthetized using pentobarbital (80 mg/kg of body weight) and 3H-cholesteryl oleate-labeled LDL or HDL (106 cpm/mouse) was injected via the penile vein. Blood was collected before injection (0 min) and at 2, 15, 30, and 60 min after injection. Plasma was separated, and triglyceride and cholesterol concentrations were measured as described above. Radioactivity in 20 μl of the plasma was measured. One hour after injection, mice were euthanized with isoflurane and perfused with saline. The liver was collected, washed with saline, and the whole liver was dissolved in 3 ml of 50% KOH in ethanol. The 3H-labelled cholesterol content of the liver was measured.
Tyloxapol was purchased from Sigma-Aldrich (USA); 10% tyloxapol solution was prepared using saline. Mice were fasted for 6 h and injected with tyloxapol (500 mg/kg) via the penile vein. Blood was collected from the tail vein before injection (0 min) and at 15, 30, 60, 90, 120, and 180 min after injection. Plasma was separated, and triglyceride and cholesterol concentrations were determined. Triglyceride and cholesterol secretion rates were calculated from the linear regression analysis of time versus concentration.
The synthesis rates of sterol and fatty acids in the liver were determined in mice using 3H-labeled water as described previously (Shimano et al., 1996).
Hepatocytes were isolated from C57BL/6J wild-type mice and cultured as described previously (Kim et al., 2020; Matsuda et al., 2001). After a 3-h attachment period, the cells were incubated with 0.5 mM 14C-sodium acetate for 4 h. Lipids were extracted, and then, fatty acids, cholesterol, cholesteryl ester, and triglycerides were separated by TLC as described previously (Matsuda et al., 2001; Moon et al., 2009). Incorporation of 14C-acetate into newly synthesized lipids was then determined.
The amount of cholesterol in feces was measured as described previously, with slight modifications (Turley et al., 1996). Mice were housed in individual cages a week before the experiment, and feces were collected every 24 h for three days. Feces were dried completely, ground, and resuspended in water (100 μg/2 ml). Lipids were extracted in chloroform/methanol (2:1) and resuspended in isopropanol. Cholesterol concentrations in the extracts were measured using Infinity Cholesterol Liquid Stable Reagent (Thermo Fisher Scientific).
Hepatocytes isolated from C57BL/6J wild-type mice were seeded at a density of 1 × 106 cells in a 60-mm dish. After a 2-h attachment period, the cells were washed and incubated with 1 μg/ml of BODIPY 493/503 (Thermo Fisher Scientific) and 15 μg/ml of dehydroergosterol (DHE; Sigma-Aldrich) in either the presence or absence of 20 μM oleic acid for 24 h. After incubation, the cells were imaged with a ZEISS LSM 780 confocal microscope (Zeiss, USA).
Membrane and nuclear proteins were prepared individually from frozen livers, and equal amounts of protein from each mouse of the same group were pooled as previously described (Engelking et al., 2004). Aliquots of pooled proteins were subjected to SDS-PAGE on 8% gels and transferred to a nitrocellulose membrane (Bio-Rad, USA). Immunoblot analyses were performed using polyclonal anti-mouse SREBP-1, SREBP-2, and HMG-CoA reductase antibodies as previously described (Rong et al., 2017). Anti-Scavenger receptor class B type 1 (SR-BI) antibody was obtained from Abcam (USA) and anti-cluster of differentiation 36 (CD36) antibody was obtained from R&D Systems (USA). Anti-mouse cAMP response element binding protein (CREB; Invitrogen, USA) and anti-dog calnexin (Enzo Life Science, USA) antibodies were used as loading controls for nuclear and membrane proteins, respectively. Signals were detected using the SuperSignal West Pico Chemiluminescent Substrate System (Thermo Fisher Scientific) and visualized with Odyssey CLx Imaging system and LI-COR Image StudioTM software (LI-COR, USA).
Total RNA was prepared from mouse livers with an RNA STAT-60 kit (Tel-Test, USA), and qPCR was performed as previously described (Rong et al., 2017). All reactions were performed in triplicate, and the relative amount of all mRNAs was calculated using the comparative threshold cycle (C
All results are reported as the mean ± SE. Statistical significance was analyzed using a Student’s
FF/HS diets have been previously used to induce hepatic
Cholesterol content was also significantly increased in the livers of mice fed an FF/HS diet (Fig. 2A). All genes encoding enzymes responsible for cholesterol synthesis are regulated by SREBP-2 (Horton et al., 2002); therefore, SREBP-2 and its target genes were analyzed. While the precursor form of SREBP-2 appeared slightly increased, the amount of the transcriptionally active nuclear form was not altered by the FF/HS diet (Fig. 2B). The mRNA expression levels of neither SREBP-2 (
To determine why the liver cholesterol content was increased despite the reduced cholesterol synthesis in the mice fed an FF/HS diet, additional metabolic pathways involved in cholesterol homeostasis were examined. In the liver, cholesterol can be excreted into the bile directly as free cholesterol or after its conversion into bile acids. Feces contains cholesterol from bile, diet, and transintestinal cholesterol excretion. Both diets used in this study are cholesterol-free; therefore, the cholesterol found in the feces must be endogenous. Feces of mice fed a chow or an FF/HS diet were collected for 3 days at the end of the diet feeding period, and fecal cholesterol contents were measured. Fecal cholesterol content of mice fed an FF/HS diet was reduced by 73% compared to mice fed a chow diet (Fig. 3A). Bile acid concentrations were also significantly reduced in the bile of mice fed the FF/HS diet, compared to the mice fed the chow (Fig. 3B). These results suggest that cholesterol excretion was reduced in the mice fed an FF/HS diet and may partly explain why the liver retained more cholesterol despite a reduced hepatic cholesterol synthesis. mRNA expression of genes involved in bile acid synthesis (
Hepatocytes can acquire cholesterol from the uptake of plasma LDL and HDL. Therefore, we examined whether hepatic cholesterol uptake was altered in the liver of mice fed an FF/HS diet. Mice were injected with LDL labeled with 3H-cholesteryl oleate, then hepatic content of 3H-cholesteryl oleate was measured 1 h after injection. As shown in Fig. 4A, the hepatic content of 3H-cholesterol from LDL was significantly increased in mice fed the FF/HS diet. mRNA expression of the LDL receptor was not changed compared to the mice fed a chow (Fig. 4B).
Next, the hepatic uptake of HDL-cholesterol was analyzed by injecting HDL labeled with 3H-cholesteryl oleate. We observed that the hepatic content of 3H-cholesteryl oleate from HDL was significantly increased in the mice fed an FF/HS diet (Fig. 4C). SR-BI is one of the major proteins responsible for the uptake of cholesterol from plasma HDL into the liver (Acton et al., 1996; Connelly and Williams, 2004). Liver mRNA expression level of SR-BI (
Formation of cholesteryl esters, the storage form of cholesterol, was compared using primary hepatocytes isolated from mice fed a chow or an FF/HS diet. Hepatocytes were incubated with 14C-acetate, and its incorporation into fatty acids, triglycerides, cholesterol, and cholesteryl esters was measured (Fig. 5A). Consistent with the
To examine whether fatty acids in hepatocytes can affect the localization of cholesterol in lipid droplets, primary hepatocytes were isolated from C57BL/6J mice and incubated with fluorescence-labeled DHE, a tracer of cholesterol, in either the presence or absence of oleic acid. BODIPY 493/503 was used to stain neutral lipid droplets (Fig. 5B). When the cells were incubated with DHE in the presence of oleic acid, a strong fluorescence from DHE was detected within the cytosolic lipid droplets, while fluorescence from DHE was dispersed and weak in the absence of oleic acid (Fig. 5B). Results in Fig. 5 suggest that increased fatty acids in hepatocytes facilitates the incorporation of cholesterol into lipid droplets, mainly as cholesteryl esters. Increased cholesteryl ester formation and facilitated incorporation into lipid droplets may further explain the increased hepatic cholesterol content in mice with high
Lipids stored in cytoplasmic lipid droplets are mobilized through lipid droplet-associated proteins for the lipidation of apoB to form VLDL (Ye et al., 2009; Zhang et al., 2017). To determine whether fatty liver induced by the FF/HS diet secretes more VLDL-triglyceride and -cholesterol, plasma triglyceride and cholesterol concentrations were measured at various time points after injection of tyloxapol, an inhibitor of lipoprotein lipase. Mice fed an FF/HS diet secreted more VLDL-triglyceride and -cholesterol into the plasma compared to the chow-fed group (Fig. 6A). The expression levels of genes that are essential for VLDL assembly, such as
To determine the effects of an FF/HS diet on dyslipidemia,
SREBP-1c activation leads to hepatic triglyceride accumulation and hypertriglyceridemia in different animal models including hamsters fed a high sucrose diet (Moon et al., 2012), Tg-SREBP1a;
The chow diet and the FF/HS diet used in this study were of different nature and it has been shown that dietary fiber contents might influence the rate of cholesterol excretion into the feces. We used diets with similar total dietary fiber contents for this study as the chow diet contains 17% fiber and the FF/HS diet contains 16.5% fiber. However, there is still a possibility that the difference in the nature of the diets might affect the cholesterol retention in the body.
The availability of fatty acids facilitates the conversion of cholesterol to cholesteryl esters (Xie et al., 2002), which could be readily incorporated into lipid droplets in hepatocytes, as presented in Fig. 5. Fatty acids not only drove cholesterol esterification, but also enhanced secretion of cholesteryl esters into the plasma (Xie et al., 2002). Acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2), the enzyme responsible for cholesteryl ester formation in the liver, prefers oleic acid as a substrate, and its activity is primarily regulated by the availability of fatty acid substrates (Xie et al., 2002). Upon activation of fatty acid synthesis by SREBP-1c in the liver, oleic acid (C18:1, n-9) is the fatty acid that is the most increased (Moon et al., 2014; Shimomura et al., 1998). Although ACAT2 mRNA expression levels were not significantly different between mice fed a chow and an FF/HS diet (Supplementary Fig. S1C), our findings suggest that the increased fatty acid availability from
Triglycerides and cholesteryl esters are packed into VLDL in hepatocytes. SREBP-1c increases PLTP expression as well as genes required for fatty acid synthesis. PLTP plays a major role in the incorporation of phospholipids to growing VLDLs to expand the particle and its lipidation, resulting in bigger VLDL particles rich in triglycerides (Okazaki et al., 2010; Yazdanyar and Jiang, 2012). The FF/HS diet induced PLTP expression as well as other targets of SREBP-1c, leading to an increased VLDL-triglyceride and -cholesterol secretion and subsequent development of hypercholesterolemia and hypertriglyceridemia in
In humans, while fatty acids released from adipose tissue are the major source of fat in livers of individuals with NAFLD (Donnelly et al., 2005),
This work was supported by grants from the National Research Foundation of Korea funded by the Korean government (2018R1A2B6007576), the National Institutes of Health (HL-20948), and the Leducq Foundation (5200829301).
The authors thank Sijeong Bae (Department of Molecular Medicine, Inha University College of Medicine) Angel Loza Valdes, Ajit Kumar Koduri, Tuyet Dang, Judith Sanchez, Norma Anderson, and Lisa Beatty (Department of Molecular Genetics, UT Southwestern Medical Center), and Abhijit Bugde (the Live Cell Imaging Core Facility, UT Southwestern Medical Center) for their technical assistance. The authors also thank Dr. Youngah Jo for providing the anti-HMG CoA-R antibody and Dr. Jay Horton for scientific advice.
J.M.B. and Y.A.M. conceived and performed experiments, analyzed the data, and wrote the manuscript.
The authors have no potential conflicts of interest to disclose.
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