Keywords
ApoB100/ApoAI ratio, ApoB100, ApoAI, Hepatic lipase, Glucose
ApoB100/ApoAI ratio, ApoB100, ApoAI, Hepatic lipase, Glucose
The paper was revised in response to the reviewers’ comment. Since it is too early to publish the hepatic lipase (HL) data in this research, all the content related to HL was removed.
See the author's detailed response to the review by Anne Tailleux
See the author's detailed response to the review by Li Lin
Atherosclerotic coronary artery occlusion is the most frequent cause of coronary heart disease (CHD); numerous epidemiologic studies and randomized clinical trials have established that lipoprotein metabolism is a major contributor to CHD.1 Conventionally, it was thought that increases in plasma low-density lipoprotein cholesterol (LDL-C) and decreases in high-density lipoprotein cholesterol (HDL-C) were the major factors causing CHD.2 However, numerous in vivo human cohort studies have suggested that another factor in CHD risk might be the apolipoprotein B100/apolipoprotein AI (ApoB100/ApoAI) ratio.3–7
ApoB100 is a large surface protein present on low-density lipoprotein (LDL) and serves as a ligand for the LDL receptor, which facilitates its clearance from the plasma. Apolipoprotein AI (ApoAI) is the major protein constituent on high-density lipoprotein (HDL) and plays a central role in reverse cholesterol transport by stabilizing the HDL particle, interacting with the ATP-binding cassette transporter I, activating lecithin cholesterol acyl transferase and acting as a ligand for the hepatic scavenger receptor.2
In order to prove those in vivo human cohort findings that ApoB100/ApoAI ratio is a CHD risk factor and understand the molecular mechanism of how ApoB100/ApoAI ratios are modulated in atherosclerosis, an in vitro cell assay model was built in the current study. Extracellular expression of ApoB100 and ApoAI were measured in cultured HepG2 cells in response to different concentrations of glucose. This research may provide the targets of many novel therapeutics and is an area with great potential for the prevention and treatment of CHD.
It has been shown that a high-carbohydrate (high-CHO) diet can reduce the risk of CHD. Further analysis found that this high-CHO diet affects the ApoB100/ApoAI ratio instead of the HDL-C related ratios.6–8 In the current study, various amounts of glucose were used to stimulate the secretion of ApoB100 and ApoAI in cultured HepG2 cells. It was hypothesized that the different concentration of glucose could modulate ApoB100/ApoAI ratio in vitro.
HepG2 cells (American Type Culture Collection, ATCC, Rockville, MD, USA) were routinely cultured in DMEM + 10% FBS (contains 4.5 g/L glucose, normal glucose group) at 37°C in a 5% CO2 incubator up to 70% confluency. For ApoB100 and ApoAI expression assays, cell culture media that contained different concentrations of glucose (1 and 10g/L) were then added to the cultured cells in order to make the low glucose group and high glucose group besides the original normal glucose group. All the cell groups then continued to be cultured for eight hours before the assays.
The culture media of HepG2 cells were collected. ApoB100 and ApoAI expression were measured with commercial sandwich enzyme-linked immunosorbent assay (ELISA) kits (cat. no. H0124 and H0123) from ShangHai MEIXUAN Biological Science and Technology Ltd (Shanghai, China). The microplate provided in this kit had been pre-coated with a monoclonal antibody specific for ApoB100 or ApoAI. Standards or samples were then pipetted into the microplate wells, and ApoB100 or ApoAI present in the samples or standards binds to antibodies adsorbed to the microplate wells. To quantitatively determine the amount of ApoB100 or ApoAI present in the samples, the horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for ApoB100 or ApoAI was added to each well. The microplate was incubated for one hour, and then the wells were thoroughly washed to remove any unbound components. The substrate solutions A and B were respectively added to each well. After the enzyme (HRP) and substrate reacting over a short period, this reaction was stopped by addition of a sulphuric acid solution and the color change is measured at a wavelength of 450 nm. The proportion to amount of ApoB100 or ApoAI bound in the initial step develops in the color change. Color intensities were measured using a MK3 microplate reader (Thermo Fisher, MA, USA).
To explore the impacts of glucose on ApoB100 and ApoAI secretion over different concentrations, HepG2 cells were exposed to low (1 g/L), medium (4.5 g/L) and high (10 g/L) concentrations of glucose. As shown in Table 1, ELISA revealed that with the glucose concentration increase, ApoB100 concentrations were significantly increased (1 g/L < 4.5 g/L < 10 g/L), while ApoAI concentrations were significantly decreased (1 g/L > 4.5 g/L > 10 g/L). ApoB100/ApoAI ratios were significantly increased (1 g/L < 4.5 g/L < 10 g/L). ANOVA showed that the differences are significant (p < 0.001).18
Hepatic lipase activity, ApoB100, ApoAI and ApoB100/ApoAI ratio in HepG2 cell cultured in low, normal and high glucose concentration.
Low glucose concentration (1 g/L) | Normal glucose concentration (4.5 g/L) | High glucose concentration (10 g/L) | F | P | |
---|---|---|---|---|---|
ApoB100 (g/ml) | 131.31 ± 10.39 | 146.77 ± 3.82** | 167.02 ± 10.84**## | 24.052 | <0.001 |
ApoAI (μg/ml) | 12.06 ± 0.47 | 10.31 ± 0.19** | 8.18 ± 0.31**## | 190.710 | <0.001 |
ApoB100/ApoAI (×106) | 10.92 ± 1.15 | 14.24 ± 0.23** | 20.49 ± 1.95**## | 82.211 | <0.001 |
While studying the glucose-induced ApoB100 and ApoAI expression in cultured HepG2 cell in vitro, in the culture media that contained a higher concentration of glucose, ApoB100/ApoAI ratios were found to be significantly increased. In the culture media that contained a lower concentration of glucose, however, ApoB100/ApoAI ratios were found to be significantly decreased. Although previous in vivo human cohort studies indicated that the ApoB100/ApoAI ratios might associated with the CHD risk from statistical analysis,6–8 in the current study, the ApoB100/ApoAI ratios were quantitatively measured in vitro at the first time, which solidly proved that ApoB100/ApoAI ratio is a CHD risk factor.
However, a high-CHO diet was found to have generally favorable effects on the ApoB100/ApoAI ratio, reducing CHD risk, in a previous human cohort study in vivo,6 while the higher concentration of glucose induced an undesirable ApoB100/ApoAI ratio change, increasing CHD risk, in the present in vitro study. We can reach a reasonable explanation to this seemingly contradictory data if we look at what is happening inside the body after the meal; the rich viscous fiber contained in the high-CHO diet we administered in our cohort study has been shown to significantly reduce postprandial glucose excursions,9–11 which means the cells inside the body were actually exposed to a lower level of glucose after the high-CHO diet. Thus, the results we obtained from the lower concentration of glucose in the HepG2 cell culture experiment in vitro should correspond to the results we obtained from the high-CHO diet experimental group in vivo.
Lipoproteins are spherical particles that carry lipids in the body. These particles contain both lipids and proteins. LDL and HDL are the two main types of lipoproteins. LDL delivers fat molecules to cells. A high LDL level means too much LDL cholesterol in the blood. This extra LDL, along with other substances, forms plaque which can build up in the arteries to form atherosclerosis, causing heart disease. HDL-C carries cholesterol from other parts of the body back to the liver. The liver then processes the cholesterol for excretion to reduce the risk of heart disease. It is well known that both LDL and HDL consist of heterogeneous particles of different size and density.12 ApoB100 is the primary protein in LDL and ApoAI is the primary protein in HDL. Normally, it is proteins instead of lipids which perform essential functions within organisms, including catalyzing metabolic reactions, providing structure to cells and organisms, and transporting molecules from one location to another. It is likely that lipoprotein metabolism is regulated by directly modulating their protein components like ApoB100 and ApoAI rather than their lipid components like triglycerides, phospholipids, and cholesterol molecules. Although previous in vivo studies have already indicated this,6–8 further in vitro studies are needed to prove it on a molecular level. The current study is the first to explore this issue in an in vitro cell culture system, and the result positively supports this hypothesis.
For many years, the ApoB100/ApoAI ratio rather than ApoB100 or ApoAI alone has been extensively reported as a risk factor of CHD.13–15 However, no one has understood the mechanism or even made any hypothesis about it. Atherosclerosis, the process involved in LDL oxidization within the walls of arteries, might be due to the more powerful function of ApoB100 to deliver lipids to the cells than ApoAI to transport lipids out of the cells and take them back to the liver. The dynamic equilibrium of these two proteins, which can be described in their ratios, is essential to our health.
This finding confirmed in vitro helps to explain why the existing clinical treatments based on the regulation of lipoproteins themselves cannot achieve the desired results in many clinical cases. For example, although statins have documented efficacy in reducing clinical events and angiographic disease progression in patients with coronary atherosclerosis, the results of subsequent large prospective clinical trials using different types of statins clearly demonstrate that statins do not have a short-to-medium term effect on prevention of restenosis after successful conventional percutaneous transluminal coronary angioplasty.16 Current available data also indicate that increased HDL-C levels do not always correlate with enhanced HDL functions because HDL is highly heterogeneous and there are different HDL subpopulations exist.17
In conclusion, in vitro HepG2 cell culture revealed ApoB100/ApoAI ratios change in response to the fluctuations of glucose concentration. Higher amounts of glucose can induce significantly increased ApoB100/ApoAI ratio, while lower amounts of glucose can induce significantly decreased, ApoB100/ApoAI ratio. The result suggests a new and central cell signal transduction pathway in lipoprotein metabolism and might provide molecular targets for clinical diagnoses and treatments of CHD.
Harvard Dataverse: Replication Data for: Glucose induced ApoB100/ApoAI ratio changes in cultured HepG2 cells in vitro, https://doi.org/10.7910/DVN/AHEAES.18
This project contains the following underlying data:
Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).
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Competing Interests: No competing interests were disclosed.
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cardiometabolic diseases, bile acids and their receptors, NAFLD
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Partly
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Prevention, treatment and pathophysiological study of senile and chronic diseases
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Partly
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
No
Are all the source data underlying the results available to ensure full reproducibility?
Partly
Are the conclusions drawn adequately supported by the results?
Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cardiometabolic diseases, bile acids and their receptors, NAFLD
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Version 1 23 Aug 21 |
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