Glucagon-like peptide-1 receptor agonists improve cholesterol metabolism by inhibiting SREBP-2 via SIRT6-AMPK pathway in HepG2 cells treated with palmitic acid

Beneficial effects of GLP-1RAs

Article information

Cardiovasc Prev Pharmacother. 2025;7(3):61-72

Publication date (electronic) : 2025 July 24

doi : https://doi.org/10.36011/cpp.2025.7.e11

Jinmi Lee ¹^,^*

, Eun-Jung Rhee ²^,^*

, Yu-Mi Lim ¹

, Seok-Woo Hong^,¹

, Won-Young Lee^,²

¹Institute of Medical Research, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea

²Division of Endocrinology and Metabolism, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, Korea

Correspondence to Seok-Woo Hong, PhD Institute of Medical Research, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, 29 Saemunan-ro, Jongno-gu, Seoul 03181, Korea Email: hardy98@hanmail.net

Won-Young Lee, MD Division of Endocrinology and Metabolism, Department of Internal Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, 29 Saemunan-ro, Jongno-gu, Seoul 03181, Korea Email: wonyoung2.lee@samsung.com

*Jinmi Lee and Eun-Jung Rhee contributed equally to this work as co-first authors.

Received 2025 March 24; Revised 2025 April 14; Accepted 2025 April 16.

Abstract

Background

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) exhibit not only hypoglycemic effects but also protective effects against nonalcoholic fatty liver disease and cardiovascular diseases, conditions that are associated with dyslipidemia.

Methods

To evaluate the beneficial effects of GLP-1RAs on hepatic cholesterol metabolism, HepG2 cells were exposed to palmitic acid (PA) and subsequently treated with or without the GLP-1RAs, exendin-4 and liraglutide. Cholesterol levels and the expression of cholesterol metabolism-related factors were measured.

Results

Exendin-4 and liraglutide reduced cholesterol levels in both cell lysates and culture media of PA-treated HepG2 cells. They also decreased the expression of genes involved in cholesterol synthesis (ACAT1, SREBP-2, HMGCR, and SQLE), bile acid synthesis (LXRα and CYP7A1), and PCSK9, while increasing the expression of genes involved in the reverse cholesterol transport pathway (ABCA1 and SR-B1) and low-density lipoprotein cholesterol uptake (LDLR). SREBP-2 inhibition by small interfering RNA in GLP-1RAs treated cells amplified the reduction in the expression of HMGCR, SQLE, LXRα, CYP7A1, and PCSK9 genes and HMGCR protein, as well as the increase in expression of the LDLR gene. However, inhibition of SIRT6 and AMPK, which were increased by GLP-1RAs, reversed the suppression of SREBP-2 and its downstream factor genes and amplified the increase in expression of the LDLR gene in PA-treated HepG2 cells.

Conclusions

These findings demonstrate that GLP-1RAs improve cholesterol metabolism through activation of the SIRT6-AMPK pathway, resulting in the inhibition of SREBP-2 in PA-treated HepG2 cells. Moreover, the upregulation of LDLR gene expression in cells treated with GLP-1RAs occurs through both SREBP-2-dependent and SREBP-2-independent pathways.

Keywords: Glucagon-like peptide-1 receptor agonist; Hepatic cholesterol; Sterol regulatory element-binding protein 2; AMP-activated protein kinase; SIRT6

INTRODUCTION

Cholesterol is essential for maintaining cell structure as a key component of cellular membranes and is involved in synthesizing various hormones [1]. A healthy adult synthesizes approximately 70% of their daily cholesterol requirement in the liver, with the remaining 30% provided by dietary intake. Cholesterol homeostasis is regulated through balanced processes including cholesterol biosynthesis, uptake, efflux, reverse cholesterol transport, metabolism, and excretion via bile acids [2,3]. However, elevated cholesterol levels contribute to nonalcoholic fatty liver disease (NAFLD) and are known risk factors for cardiovascular disease (CVD) [4]. Increased cholesterol biosynthesis and uptake, along with reduced cholesterol export and excretion, have been observed in conditions such as fatty liver disease, liver cancer, and hepatocellular carcinoma [5].

Sterol regulatory element-binding protein 2 (SREBP-2), one of the two members of the SREBP family, serves as a primary transcription factor controlling cholesterol synthesis [6]. Acetyl coenzyme A (Acetyl-CoA), produced through the metabolism of glucose, fatty acids, and amino acids [7], is utilized in cholesterol synthesis, a process regulated by SREBP-2 through the enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis. Alternatively, acetyl-CoA is directed towards triglyceride (TG) synthesis via fatty acid synthase and acetyl-CoA carboxylase, regulated by SREBP-1 [8]. SREBP-2 additionally influences low-density lipoprotein cholesterol (LDL-C) uptake by upregulating LDL receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) gene expression. However, increased PCSK9 expression impairs LDL-C uptake by promoting LDLR degradation [9].

Adenosine monophosphate-activated protein kinase (AMPK) is a critical regulator of energy metabolism across various pathways including lipid, glucose, and cholesterol metabolism [10]. AMPK suppresses cholesterol and TG synthesis by inhibiting HMGCR [11]. Reverse cholesterol transport (RCT), a mechanism by which excess cholesterol is transported from peripheral tissues back to the liver, is enhanced by AMPK activators. These activators increase the expression of ATP-binding cassette transporter subfamily A member 1 (ABCA1) and ATP-binding cassette transporter subfamily G member 1 (ABCG1) in peritoneal macrophages, as well as scavenger receptor class B member 1 (SR-BI) and lecithin-cholesterol acyltransferase (LCAT) expression in the liver of apoE^−/− mice, thereby reducing inflammatory cytokine production (tumor necrosis factor α, monocyte chemoattractant protein-1, andinterleukin-6) and atherosclerotic plaque formation [12].

Histone deacetylase sirtuin 6 (SIRT6), a member of the sirtuin family, plays critical roles in metabolic regulation, apoptosis, cell survival, development, inflammation, and aging [13]. Hepatic overexpression of SIRT6 inhibits intestinal cholesterol and fat absorption and reduces very low-density lipoprotein secretion, providing protection against steatohepatitis and atherosclerosis induced by a Western diet in Ldlr^−/− mice [14].

Glucagon-like peptide-1 (GLP-1) has emerged as a therapeutic agent for metabolic diseases such as obesity, NAFLD, and atherosclerosis, largely by improving lipid, glucose, and cholesterol metabolism [15]. Liraglutide, a GLP-1 receptor agonist (GLP-1RA), was shown to reduce serum total cholesterol, LDL-C, high-density lipoprotein cholesterol (HDL-C), and TG levels by decreasing hepatic LDLR and PCSK9 expression in db/db mice, but not in wild-type mice [16]. Previous studies from our group demonstrated that GLP-1RAs provide protection against steatohepatitis in high-fat diet (HFD)-induced obese mice [17], and against high phosphate-induced vascular calcification—a hallmark of atherosclerosis—through AMPK activation [18].

In this study, we investigated the protective effects and regulatory mechanisms by which GLP-1RA corrects cholesterol homeostasis dysregulation in hepatocytes treated with palmitic acid (PA).

METHODS

Ethics statement

Ethics approval was not required for this in vitro study, as it did not involve human participants, identifiable human data, or animal subjects.

Cell culture

The human hepatoma cell line, HepG2, was purchased from the American Type Culture Collection. Cells were cultured in Dulbecco’s modified Eagle’s medium high glucose (4.5 g/L; Welgene) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin/streptomycin (Gibco). Cultures were maintained in an incubator at 37 °C with a humidified atmosphere containing 5% CO₂. Saturated free fatty acids are known to induce hepatic steatosis and promote cholesterol biosynthesis [19–21]. To investigate the regulatory effects of GLP-1RAs on cholesterol metabolism in hepatocytes treated with saturated free fatty acids, cells were pretreated with 400 μM PA (Sigma-Aldrich) for 24 hours, followed by treatment with 100 nM exendin-4 (Sigma-Aldrich) or liraglutide (MedChemExpress) for an additional 24 hours. To activate AMPK, 2 mM of 5-aminoimidazole-4-carboxamide riboside (AICAR; Sigma-Aldrich) was used, while 0.5 μM of the endoplasmic reticulum stress inducer thapsigargin (Sigma-Aldrich) was applied to activate SREBP-2, thereby upregulating genes involved in cholesterol biosynthesis and uptake [22].

Cholesterol assay

Cholesterol was extracted from cultured cells using a chloroform:isopropanol:NP-40 mixture (7:11:0.1). Samples were centrifuged at 13,000 × g for 10 minutes, and the organic phase was evaporated under vacuum. Dried lipid pellets were dissolved in 200 μL of cholesterol assay buffer. Equal volumes of cell culture media were used for cholesterol analysis. Cholesterol content was quantified using an Amplex Red cholesterol assay kit (A12216, Invitrogen), following the manufacturer’s instructions. Total cholesterol measurements included cholesteryl esters and free cholesterol, and cholesterol levels in cell lysates were normalized to protein concentration.

Quantitative real-time polymerase chain reaction

Total RNA from cultured HepG2 cells was isolated using TRIzol reagent (Invitrogen). RNA concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The complementary DNA synthesis was conducted from 2 µg RNA via reverse transcription using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (qRT-PCR) for target gene expression was performed using the SensiFAST SYBR No-ROX Kit (Bioline). Forward and reverse primer pairs for qRT-PCR were purchased from Bioneer, and the primer sequences are listed in Tables S1 and S2. ACTB was used as an internal control gene, and the expression levels of the target genes were calculated using the 2^−ΔΔCt method.

Western blotting

Cultured HepG2 cells were washed with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology) containing Halt Protease and Phosphatase Inhibitor Cocktail (100×; Thermo Fisher Scientific). Protein concentrations were measured using the Bradford assay (Bio-Rad). Protein samples (20 µg) were prepared by adding lithium dodecyl sulfate (LDS) sample buffer (4×) and reducing sample agent (10×). Samples were denatured by boiling at 95 °C for 10 minutes, loaded onto a 4% to 12% gradient gel (Thermo Fisher Scientific), and separated by electrophoresis. Proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane using an iBlot2 PVDF stack (Invitrogen) and blocked with 5% bovine serum albumin in tris-buffered saline with 0.1% Tween-20 (TBST) for 1 hour at room temperature. Primary antibodies targeting specific proteins (Table S1) were incubated with membranes overnight at 4 °C on a shaker. After washing five times with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence reagent (Bio-Rad) on a Vilber Fusion FX imaging system (Vilber).

Transient siRNA transfection

HepG2 cells were transfected with 50 nM AMPK, SIRT6, or SREBP-2 small interfering RNA (siRNA; Bioneer) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) for 24 hours, following the manufacturer’s instructions. Scrambled siRNA was employed as a negative control. Cells were then pretreated with 400 μM PA for 24 hours, followed by treatment with 100 nM exendin-4 or liraglutide for 24 hours and subsequently harvested for further analysis.

Statistical analysis

All data are expressed as mean±standard error. Statistical analyses were performed using SPSS ver. 12.0 (SPSS Inc). Differences between two groups were evaluated using t-tests, and comparisons among multiple groups were performed using one-way analysis of variance with Bonferroni post hoc test. Statistical significance was set at P<0.05.

RESULTS

GLP-1RAs decrease the cholesterol level and regulate the expression of cholesterol metabolism-associated genes in HepG2 cells treated with PA

In HepG2 cells, treatment with PA significantly increased cholesterol levels in both cell lysates and culture supernatants. In contrast, the GLP-1RAs exendin-4 and liraglutide reduced these cholesterol levels (Fig. 1). PA treatment elevated the expression of genes associated with cholesterol synthesis, such as acyl-CoA C-acyltransferase 1 gene (ACAT1), SREBP-2, HMGCR, and squalene monooxygenase gene (SQLE), as well as genes related to bile acid synthesis, including liver X receptor α gene (LXRα) and cholesterol 7-alpha-hydroxylase gene (CYP7A1). However, treatment with exendin-4 and liraglutide reversed these PA-induced gene expression changes in HepG2 cells (Fig. 2A–F). Additionally, GLP-1RAs increased the expression levels of genes involved in cholesterol uptake, such as LDLR, scavenger receptor class B type I gene (SR-B1; an HDL-C receptor), and ABCA1, a critical factor in HDL-C biogenesis. Conversely, GLP-1RAs decreased the expression of PCSK9, which negatively regulates LDLR activity (Fig. 2G–J). Collectively, these results indicate that GLP-1RAs contribute to maintaining cholesterol homeostasis by inhibiting cholesterol synthesis and enhancing cholesterol uptake in hepatocytes experiencing PA-induced dyslipidemia.

Fig. 1.

Exendin-4 and liraglutide reduced cholesterol levels in palmitic acid (PA)-treated HepG2 cells. Cells were pretreated with 400 μM PA, followed by treatment with or without 100 nM exendin-4 and liraglutide for 24 hours. (A) Cholesterol content in cell lysates. (B) Cell culture supernatants. *P<0.05 and **P<0.01 when compared with the control cells; ^†P<0.05 when compared with the PA-treated cells.

Fig. 2.

Exendin-4 and liraglutide reduced the expression of genes involved in cholesterol synthesis and bile acid synthesis and increased the expression of genes involved in low-density lipoprotein cholesterol uptake and reverse cholesterol transport in palmitic acid (PA)-treated HepG2 cells. (A–D) Cells were pretreated with 400 μM PA for 24 hours, followed by treatment with or without 100 nM exendin-4 and liraglutide for 24 hours. The messenger RNA expression levels of the (A) ACAT1, (B) SREBP-2, (C) HMGCR, (D) SQLE, (E) LXRα, (F) CYP7A1, (G) LDLR, (H) PCSK9, (I) SR-B1, and (J) ABCA1 genes were analyzed by quantitative real-time polymerase chain reaction and normalized to that of the ACTB gene. ^*P<0.05 and ^**P<0.01 when compared with the control cells; ^†P<0.05 and ^††P<0.01 when compared with the PA-treated cells.

GLP-1RAs reduce the expression of cholesterol synthesis– and bile acid synthesis–associated genes by inhibiting SREBP-2 in HepG2 cells treated with PA

To investigate whether SREBP-2 inhibition mediated by GLP-1RAs affects the expression of cholesterol metabolism-related genes, HepG2 cells were transfected with SREBP-2 siRNA for 24 hours, followed by PA and GLP-1RA treatments. SREBP-2 silencing significantly decreased the expression of HMGCR, SQLE, LXRα, CYP7A1, and PCSK9 genes, while increasing LDLR gene expression compared to nontransfected control cells (Fig. 3A–H). Consistent with these gene expression changes, SREBP-2 siRNA transfection reduced HMGCR protein expression compared with un-transfected groups (Fig. 3I–K). ABCA1 gene expression remained unchanged regardless of SREBP-2 silencing. These findings suggest that hepatic SREBP-2 significantly contributes to cholesterol synthesis, cholesterol uptake, and bile acid synthesis regulation, and that its inhibition by GLP-1RAs is essential for improving cholesterol metabolism in PA-treated HepG2 cells.

Fig. 3.

The decreased expression of genes involved in cholesterol synthesis and bile acid synthesis and the increased expression of genes involved in low-density lipoprotein cholesterol uptake by exendin-4 and liraglutide were mediated by the inhibition of SREBP-2 in palmitic acid (PA)-treated cells. HepG2 cells, transfected with 50 nM SREBP-2 small interfering RNA (siRNA) or scrambled (Scr) siRNA for 24 hours, were treated with 400 μM PA, followed by treatment with or without 100 nM exendin-4 and liraglutide for 24 hours. The messenger RNA expression levels of the (A) SREBP-2, (B) ACAT1, (C) HMGCR, (D) SQLE, (E) LXRα, (F) CYP7A1, (G) LDLR, and (H) PCSK9 genes were analyzed with quantitative real-time polymerase chain reaction and normalized to that of the ACTB gene. (I–K) Expression levels of SREBP-2 and HMGCR were analyzed using Western blotting, and β-actin was used as the loading control. ^*P<0.05 and ^**P<0.01 when compared with the control cells; ^†P<0.05 and ^††P<0.01 when compared with the PA-treated cells; ^‡P<0.05 and ^‡‡P<0.01 when compared with the un-transfected cells.

GLP-1RAs increase the expression of AMPK and SIRT6 and inhibit the expression of SREBP-2 through activation of AMPK and SIRT6 in HepG2 cells treated with PA

GLP-1RAs increased the expression levels of AMPK and SIRT6 genes and their corresponding proteins in PA-treated HepG2 cells (Fig. 4A–D). To clarify whether elevated AMPK and SIRT6 expression following GLP-1RA treatment influenced SREBP-2 expression, HepG2 cells were pre-transfected with AMPK or SIRT6 siRNA for 24 hours, followed by PA and GLP-1RA treatments. In GLP-1RA-treated cells, SIRT6 inhibition reduced AMPK expression slightly, but not significantly, whereas AMPK inhibition did not affect SIRT6 expression. Notably, siRNA-mediated inhibition of both AMPK and SIRT6 reversed the GLP-1RA-induced suppression of SREBP-2 expression in PA-treated cells (Fig. 4E–M). Similarly, inhibition of AMPK and SIRT6 reversed the GLP-1RA-induced decrease in expression of the HMGCR, SQLE, LXRα, CYP7A1, and PCSK9 genes. Interestingly, AMPK inhibition—but not SIRT6 inhibition—further upregulated LDLR gene expression in GLP-1RA-treated cells. Additionally, LDLR gene expression was independently elevated by thapsigargin, an endoplasmic reticulum stress inducer known to activate SREBP-2, and AICAR, an AMPK agonist (Fig. S1). These results suggest that GLP-1RAs regulate SREBP-2 and its downstream targets through increased AMPK and SIRT6 expression in PA-treated HepG2 cells, and that SIRT6 may partially function as an upstream regulator of AMPK. Furthermore, LDLR gene expression can be increased by both SREBP-2-dependent and AMPK-dependent mechanisms.

Fig. 4.

SREBP inhibition by exendin-4 and liraglutide was mediated by activated AMPK and SIRT6 in palmitic acid (PA)-treated HepG2 cells. (A–D) Cells were pretreated with 400 μM PA, followed by treatment with or without 100 nM exendin-4 and liraglutide for 24 hours. Expression levels of the (A) AMPK and (B) SIRT6 genes and their associated proteins were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, and β-actin was used as the loading control. (B) HepG2 cells, transfected with 50 nM AMPK, SIRT6, or scrambled (Scr) small interfering RNA (siRNA) for 24 hours, were treated with 400 μM PA, followed by treatment with or without 100 nM exendin-4 and liraglutide for 24 hours. The messenger RNA expression levels of the (E) AMPK, (F) SIRT6, (G) SREBP-2, (H) HMGCR, (I) SQLE, (J) LXRα, (K) CYP7A1, (L) LDLR, and (M) PCSK9 genes were analyzed using quantitative RT-PCR and normalized to that of the ACTB gene. ^*P<0.05 and ^**P<00.01 when compared with the control cells; ^†P<0.05 and ^††P<0.01 when compared with the PA-treated cells; ^‡P<0.05 and ^‡‡P<0.01 when compared with the un-transfected cells.

DISCUSSION

In this study, we demonstrated that GLP-1RAs beneficially maintain cholesterol homeostasis by activating the SIRT6-AMPK pathway in PA-treated HepG2 cells. The GLP-1RAs, exendin-4 and liraglutide, decreased cholesterol levels in cell lysates and culture media. Exendin-4 and liraglutide reduced the expression of genes involved in cholesterol synthesis (ACAT1, SREBP-2, HMGCR, and SQLE), bile acid synthesis (LXRα and CYP7A1), and the PCSK9 gene, while increasing the expression of genes associated with the RCT pathway (ABCA1 and SR-B1) and LDL-C uptake (LDLR). SREBP-2 inhibition via siRNA lowered the expression of HMGCR, SQLE, LXRα, CYP7A1, and PCSK9 genes as well as HMGCR protein, and increased LDLR gene expression. However, inhibition of AMPK and SIRT6, which were upregulated by GLP-1RAs, reversed the suppression of SREBP-2 and its downstream genes.

GLP-1RAs, which are antidiabetic drugs, improve NAFLD and CVD in patients with type 2 diabetes mellitus by activating AMPK and SIRT6, key regulators of lipid, glucose, and cholesterol metabolism pathways [23–26]. GLP-1RAs such as CNTO3649 and exendin-4 reduced hepatic steatosis and lowered hepatic triglyceride, cholesterol, and phospholipid contents in HFD-fed APOE*3-Leiden transgenic mice [27]. Additionally, GLP-1RAs protect against CVD by improving endothelial dysfunction, an early phase of atherosclerosis [28,29], triggered by high blood pressure, diabetes, and elevated cholesterol [30,31]. Our findings reveal that exendin-4 and liraglutide significantly reduce hepatic cholesterol accumulation and suppress SREBP-2 expression, a crucial regulator of cholesterol metabolism, via activation of the SIRT6-AMPK pathway in PA-treated HepG2 cells. These results suggest that the protective effects of GLP-1RAs against NAFLD and CVD might be mediated by the regulation of hepatic cholesterol metabolism.

SREBP-2 not only upregulates genes involved in cholesterol synthesis from acetyl-CoA, such as HMGCR and SQLE, but also modulates bile acid synthesis by regulating transcription of related genes. Bile acids are synthesized from cholesterol in the liver, promoting biliary lipid secretion to maintain cholesterol homeostasis [32]. CYP7A1 is the rate-limiting enzyme in converting cholesterol to bile acids, and LXRs, which function as cholesterol sensors, increase Cyp7a1 expression in response to acute cholesterol intake in mice [2,33]. Among the two LXR isoforms, LXRα and LXRβ, hepatic LXRα plays a pivotal role in maintaining systemic cholesterol balance [34]. However, the regulatory effects of GLP-1RAs on bile acid synthesis remain unclear. Exendin-4 reduced Cyp7a1 expression without altering Lxr expression in Ldlr^–/– mice [35], whereas liraglutide increased Cyp7a1 expression in a mouse model of nonalcoholic steatohepatitis [36]. In the current study, we observed that expression of LXRα and CYP7A1 was increased by PA and reversed by exendin-4 and liraglutide treatment. Moreover, SREBP-2 inhibition by siRNA further reduced LXRα and CYP7A1 expression in GLP-1RA–treated cells. These results suggest that GLP-1RAs inhibit bile acid synthesis through SREBP-2 suppression in PA-treated cells, possibly linked to reduced hepatic cholesterol levels following GLP-1RA treatment.

The RCT pathway is a process by which the body removes excess cholesterol from peripheral tissues back to the liver for excretion, representing a crucial cardioprotective mechanism. ABCA1 is pivotal for HDL-C biogenesis and cholesterol efflux, whereas SR-B1 mediates uptake of HDL cholesteryl esters [37,38]. Exendin-4, a GLP-1RA, upregulated hepatic ABCA1 via the CaMKK/CaMKIV/PREB pathway and alleviated fatty liver in HFD-fed mice [39]. Li et al. [40] reported that exendin-4 increased LXR-mediated ABCA1 expression through AMPK activation, improving cholesterol-induced toxicity in INS-1 cells. In apoE^–/– mice, AMPK activation enhanced expression of ABCA1 and ABCG1 in peritoneal macrophages without affecting LXRα expression, while increasing SR-B1 expression in the liver [12]. Our results similarly indicate that GLP-1RAs increase ABCA1 and SR-B1 expression in PA-treated HepG2 cells, independent of LXR-mediated regulation of ABCA1.

Reducing LDL production and enhancing LDL clearance from circulation lowers the levels of atherogenic LDL-C, thereby reducing CVD risk [41]. Enhanced LDLR-mediated LDL-C uptake in the liver effectively lowers blood cholesterol. Dysregulated LDLR pathways in ApoE knockout mice exacerbate NAFLD and atherosclerosis progression under inflammatory conditions [42]. PCSK9 inhibition, a posttranslational regulator of LDLR degradation, improves hepatic steatosis, inflammation, and CVD outcomes in mice and humans [43,44]. Previous studies reported transcriptional upregulation of LDLR and PCSK9 by SREBP-2 [45,46]. However, our results demonstrate that GLP-1RAs reduce cholesterol levels in culture media, decrease PCSK9 gene expression, and elevate LDLR gene expression in PA-treated HepG2 cells. PCSK9 expression was reduced by siRNA-mediated SREBP-2 inhibition and increased by AMPK inhibition, which elevated SREBP-2. Interestingly, LDLR expression increased following inhibition of SREBP-2 or AMPK by siRNA, and also following treatment with thapsigargin, an endoplasmic reticulum stress inducer activating SREBP-2, or AICAR, an AMPK agonist. Zhang et al. [47] reported similar findings, showing that the AMPK activator metformin increased LDLR expression in HepG2 cells through both SREBP-2-dependent and independent mechanisms. Thus, our data suggest GLP-1RAs may enhance LDL-C clearance by increasing LDLR expression, potentially improving CVD outcomes, with LDLR regulation occurring through both SREBP-2-dependent and SREBP-2-independent pathways.

In conclusion, this study demonstrates that GLP-1RAs exert direct protective effects against cholesterol metabolism dysregulation in PA-treated hepatocytes by inhibiting SREBP-2. Specifically, GLP-1RAs reduce SREBP-2 expression via activation of the SIRT6-AMPK pathway, resulting in decreased expression of cholesterol and bile acid synthesis genes, and increased expression of genes associated with RCT and LDL-C uptake. Activation of the SIRT6-AMPK signaling pathway by GLP-1RAs may therefore represent an attractive therapeutic strategy to prevent the development and progression of NAFLD and related cardiovascular diseases by modulating cholesterol metabolism. Further animal studies are necessary to clarify the regulatory factors and mechanisms involved in LDLR transcription.

Notes

Author contributions

Conceptualization: all authors; Data curation: all authors; Formal analysis: JL, EJR; Investigation: JL, SWH; Methodology: all authors; Project administration: all authors; Software: JL; Supervision: SWH, WYL; Validation: JL, EJR, YML; Visualization: JL, EJR, YML; Writing–original draft: JL, EJR, SWH, WYL; Writing–review & editing: JL, EJR, SWH, WYL. All authors read and approved the final manuscript.

Conflicts of interest

Won-Young Lee and Eun-Jung Rhee are co-editors-in-chief of this journal, but were not involved in the peer reviewer selection, evaluation, or decision process of this article. The authors have no other conflicts of interest to declare.

Funding

The authors received no financial support for this study.

Supplementary materials

Table S1. List of primers used in this study

cpp-2025-7-e11-Table-S1.pdf

Table S2. List of primary antibodies used in this study

cpp-2025-7-e11-Table-S2.pdf

Fig. S1. The expression of the LDLR gene was increased by AICAR and thapsigargin in HepG2 cells.

cpp-2025-7-e11-Fig-S1.pdf

Supplementary materials are available from https://doi.org/10.36011/cpp.2025.7.e11.

References

1. Zampelas A, Magriplis E. New insights into cholesterol functions: a friend or an enemy? Nutrients 2019;11:1645. 10.3390/nu11071645. 31323871.

2. Zhao C, Dahlman-Wright K. Liver X receptor in cholesterol metabolism. J Endocrinol 2010;204:233–40. 10.1677/joe-09-0271. 19837721.

3. Duan Y, Gong K, Xu S, Zhang F, Meng X, Han J. Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics. Signal Transduct Target Ther 2022;7:265. 10.1038/s41392-022-01125-5. 35918332.

4. Kim EJ, Kim BH, Seo HS, Lee YJ, Kim HH, Son HH, et al. Cholesterol-induced non-alcoholic fatty liver disease and atherosclerosis aggravated by systemic inflammation. PLoS One 2014;9e97841. 10.1371/journal.pone.0097841. 24901254.

5. Min HK, Kapoor A, Fuchs M, Mirshahi F, Zhou H, Maher J, et al. Increased hepatic synthesis and dysregulation of cholesterol metabolism is associated with the severity of nonalcoholic fatty liver disease. Cell Metab 2012;15:665–74. 10.1016/j.cmet.2012.04.004. 22560219.

6. Madison BB. Srebp2: a master regulator of sterol and fatty acid synthesis. J Lipid Res 2016;57:333–5. 10.1194/jlr.c066712. 26798145.

7. Shi L, Tu BP. Acetyl-CoA and the regulation of metabolism: mechanisms and consequences. Curr Opin Cell Biol 2015;33:125–31. 10.1016/j.ceb.2015.02.003. 25703630.

8. Horton JD, Goldstein JL, Brown MS. SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125–31. 10.1172/jci15593. 11994399.

9. Deprince A, Haas JT, Staels B. Dysregulated lipid metabolism links NAFLD to cardiovascular disease. Mol Metab 2020;42:101092. 10.1016/j.molmet.2020.101092. 33010471.

10. Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Rev 2009;89:1025–78. 10.1152/physrev.00011.2008. 19584320.

11. Loh K, Tam S, Murray-Segal L, Huynh K, Meikle PJ, Scott JW, et al. Inhibition of adenosine monophosphate-activated protein kinase-3-hydroxy-3-methylglutaryl coenzyme A reductase signaling leads to hypercholesterolemia and promotes hepatic steatosis and insulin resistance. Hepatol Commun 2018;3:84–98. 10.1002/hep4.1279. 30619997.

12. Ma A, Wang J, Yang L, An Y, Zhu H. AMPK activation enhances the anti-atherogenic effects of high density lipoproteins in apoE-/- mice. J Lipid Res 2017;58:1536–47. 10.1194/jlr.m073270. 28611100.

13. Alageel A, Tomasi J, Tersigni C, Brietzke E, Zuckerman H, Subramaniapillai M, et al. Evidence supporting a mechanistic role of sirtuins in mood and metabolic disorders. Prog Neuropsychopharmacol Biol Psychiatry 2018;86:95–101. 10.1016/j.pnpbp.2018.05.017. 29802856.

14. Zhu Y, Hu S, Pan X, Gopoju R, Cassim Bawa FN, Yin L, et al. Hepatocyte sirtuin 6 protects against atherosclerosis and steatohepatitis by regulating lipid homeostasis. Cells 2023;12:2009. 10.3390/cells12152009. 37566087.

15. Bu T, Sun Z, Pan Y, Deng X, Yuan G. Glucagon-like peptide-1: new regulator in lipid metabolism. Diabetes Metab J 2024;48:354–72. 10.4093/dmj.2023.0277.

16. Yang SH, Xu RX, Cui CJ, Wang Y, Du Y, Chen ZG, et al. Liraglutide downregulates hepatic LDL receptor and PCSK9 expression in HepG2 cells and db/db mice through a HNF-1a dependent mechanism. Cardiovasc Diabetol 2018;17:48. 10.1186/s12933-018-0689-9. 29618348.

17. Lee J, Hong SW, Chae SW, Kim DH, Choi JH, Bae JC, et al. Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice. PLoS One 2012;7e31394. 10.1371/journal.pone.0031394.

18. Lee J, Hong SW, Kim MJ, Kwon H, Park SE, Rhee EJ, et al. Metformin, resveratrol, and exendin-4 inhibit high phosphate-induced vascular calcification via AMPK-RANKL signaling. Biochem Biophys Res Commun 2020;530:374–80. 10.1016/j.bbrc.2020.07.136. 32800550.

19. Dietary fats, fatty acids, and their effects on lipoproteins. Curr Atheroscler Rep 2006;8:466–71. 10.1007/s11883-006-0021-0. 17045072.

20. Fernandez ML, West KL. Mechanisms by which dietary fatty acids modulate plasma lipids. J Nutr 2005;135:2075–8. 10.1093/jn/135.9.2075. 16140878.

21. Lee J, Hong SW, Kim MJ, Moon SJ, Kwon H, Park SE, et al. Dulaglutide ameliorates palmitic acid-induced hepatic steatosis by activating FAM3A signaling pathway. Endocrinol Metab (Seoul) 2022;37:74–83. 10.3803/enm.2021.1293. 35144334.

22. Colgan SM, Tang D, Werstuck GH, Austin RC. Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2. Int J Biochem Cell Biol 2007;39:1843–51. 10.1016/j.biocel.2007.05.002. 17604677.

23. Napoli R, Avogaro A, Formoso G, Piro S, Purrello F, Targher G, et al. Beneficial effects of glucagon-like peptide 1 receptor agonists on glucose control, cardiovascular risk profile, and non-alcoholic fatty liver disease: an expert opinion of the Italian diabetes society. Nutr Metab Cardiovasc Dis 2021;31:3257–70. 10.1016/j.numecd.2021.08.039. 34627692.

24. Zhou R, Lin C, Cheng Y, Zhuo X, Li Q, Xu W, et al. Liraglutide alleviates hepatic steatosis and liver injury in T2MD rats via a GLP-1R dependent AMPK pathway. Front Pharmacol 2021;11:600175. 10.3389/fphar.2020.600175. 33746742.

25. Dong XC. Sirtuin 6: a key regulator of hepatic lipid metabolism and liver health. Cells 2023;12:663. 10.3390/cells12040663. 36831330.

26. Balestrieri ML, Rizzo MR, Barbieri M, Paolisso P, D’Onofrio N, Giovane A, et al. Sirtuin 6 expression and inflammatory activity in diabetic atherosclerotic plaques: effects of incretin treatment. Diabetes 2015;64:1395–406.

27. Parlevliet ET, Wang Y, Geerling JJ, Schroder-Van der Elst JP, Picha K, O’Neil K, et al. GLP-1 receptor activation inhibits VLDL production and reverses hepatic steatosis by decreasing hepatic lipogenesis in high-fat-fed APOE*3-Leiden mice. PLoS One 2012;7e49152. 10.1371/journal.pone.0049152. 23133675.

28. Koska J, Sands M, Burciu C, D’Souza KM, Raravikar K, Liu J, et al. Exenatide protects against glucose- and lipid-induced endothelial dysfunction: evidence for direct vasodilation effect of GLP-1 receptor agonists in humans. Diabetes 2015;64:2624–35. 10.2337/db14-0976.

29. Ribeiro-Silva JC, Tavares CA, Girardi AC. The blood pressure lowering effects of glucagon-like peptide-1 receptor agonists: a mini-review of the potential mechanisms. Curr Opin Pharmacol 2023;69:102355. 10.1016/j.coph.2023.102355. 36857807.

30. Medina-Leyte DJ, Zepeda-Garcia O, Dominguez-Perez M, Gonzalez-Garrido A, Villarreal-Molina T, Jacobo-Albavera L. Endothelial dysfunction, inflammation and coronary artery disease: potential biomarkers and promising therapeutical approaches. Int J Mol Sci 2021;22:3850. 10.3390/ijms22083850. 33917744.

31. Steinberg HO, Bayazeed B, Hook G, Johnson A, Cronin J, Baron AD. Endothelial dysfunction is associated with cholesterol levels in the high normal range in humans. Circulation 1997;96:3287–93. 10.1161/01.cir.96.10.3287. 9396418.

32. Hofmann AF. The continuing importance of bile acids in liver and intestinal disease. Arch Intern Med 1999;159:2647–58. 10.1001/archinte.159.22.2647. 10597755.

33. Gupta S, Pandak WM, Hylemon PB. LXR alpha is the dominant regulator of CYP7A1 transcription. Biochem Biophys Res Commun 2002;293:338–43. 10.1016/s0006-291x(02)00229-2. 12054605.

34. Zhang Y, Breevoort SR, Angdisen J, Fu M, Schmidt DR, Holmstrom SR, et al. Liver LXRα expression is crucial for whole body cholesterol homeostasis and reverse cholesterol transport in mice. J Clin Invest 2012;122:1688–99. 10.1172/jci59817. 22484817.

35. Hori M, Hasegawa Y, Hayashi Y, Nakagami T, Harada-Shiba M. Acute cholesterol-lowering effect of exendin-4 in Ldlr-/- and C57BL/6J mice. J Atheroscler Thromb 2023;30:74–86. 10.5551/jat.60921. 35314564.

36. Choi YJ, Johnson JD, Lee JJ, Song J, Matthews M, Hellerstein MK, et al. Seladelpar combined with complementary therapies improves fibrosis, inflammation, and liver injury in a mouse model of nonalcoholic steatohepatitis. Am J Physiol Gastrointest Liver Physiol 2024;326:G120–32. 10.1152/ajpgi.00158.2023. 38014444.

37. Phillips MC. Is ABCA1 a lipid transfer protein? J Lipid Res 2018;59:749–63. 10.1194/jlr.r082313. 29305383.

38. Ji A, Wroblewski JM, Cai L, de Beer MC, Webb NR, ; van der Westhuyzen DR. Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI. J Lipid Res 2012;53:446–55. 10.1194/jlr.m017079. 22190590.

39. Lyu J, Imachi H, Fukunaga K, Sato S, Kobayashi T, Dong T, et al. Role of ATP-binding cassette transporter A1 in suppressing lipid accumulation by glucagon-like peptide-1 agonist in hepatocytes. Mol Metab 2020;34:16–26. 10.1016/j.molmet.2019.12.015. 32180556.

40. Li R, Sun X, Li P, Li W, Zhao L, Zhu L, et al. GLP-1-induced AMPK activation inhibits PARP-1 and promotes LXR-mediated ABCA1 expression to protect pancreatic β-cells against cholesterol-induced toxicity through cholesterol efflux. Front Cell Dev Biol 2021;9:646113. 10.3389/fcell.2021.646113. 34307343.

41. Srivastava RA. A review of progress on targeting LDL receptor-dependent and -independent pathways for the treatment of hypercholesterolemia, a major risk factor of ASCVD. Cells 2023;12:1648. 10.3390/cells12121648. 37371118.

42. Zhang Y, Ma KL, Ruan XZ, Liu BC. Dysregulation of the low-density lipoprotein receptor pathway is involved in lipid disorder-mediated organ injury. Int J Biol Sci 2016;12:569–79. 10.7150/ijbs.14027.

43. Momtazi-Borojeni AA, Banach M, Ruscica M, Sahebkar A. The role of PCSK9 in NAFLD/NASH and therapeutic implications of PCSK9 inhibition. Expert Rev Clin Pharmacol 2022;15:1199–208. 10.1080/17512433.2022.2132229.

44. Zhang PY. PCSK9 as a therapeutic target for cardiovascular disease. Exp Ther Med 2017;13:810–4. 10.3892/etm.2017.4055. 28450903.

45. Park SW, Moon YA, Horton JD. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver. J Biol Chem 2004;279:50630–8. 10.1074/jbc.m410077200. 15385538.

46. Jeong HJ, Lee HS, Kim KS, Kim YK, Yoon D, Park SW. Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9 expression by sterol-regulatory element binding protein-2. J Lipid Res 2008;49:399–409. 10.1194/jlr.m700443-jlr200. 17921436.

47. Zhang F, Sun W, Chen J, Jiang L, Yang P, Huang Y, et al. SREBP-2, a new target of metformin? Drug Des Devel Ther 2018;12:4163–70. 10.2147/dddt.s190094.

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