The DOI matches its Crossref record; 104 of the 104 references it cites were found in a registry or an index; 47 of the 88 sources its claims cite were checked for support.
ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver
Open accessAbstract
Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.
Overview
AI snapshot
Written from the paper’s full text.
TL;DR
A genome-wide CRISPR screen in hepatocytes identifies ABCC4 as a negative regulator of surface LDLR. ABCC4 loss or inhibition increases hepatic LDLR, lowers plasma LDL-C, and acts via intracellular cAMP–Epac2/Rap1a signaling that suppresses PCSK9 protein.
Key findings
- In FACS-based genome-wide CRISPR screens targeting over 18,000 protein-coding genes in AML12 hepatocytes, ABCC4 was the top-ranking putative negative regulator of LDLR; 68 M1 hits were also significant in the independent M2 screen.
- Abcc4/ABCC4 knockout increased cell-surface LDLR and Dil-LDL uptake in murine AML12 and human LO2 hepatocytes without changing Ldlr/LDLR mRNA, total LDLR protein, SREBP2/HMGCR expression, or cellular cholesterol content.
- Liver-specific AAV8_Abcc4_RNAi in male C57BL/6 mice lowered hepatic Abcc4 mRNA by about 44.9% and protein by about 64.7%, increased hepatic membrane LDLR, and decreased serum LDL-C without significant changes in serum TC or TG.
- Ceefourin-1 (10 mg/kg, 4 weeks) reduced serum LDL-C by about 41.1% in NCD-fed mice and 42.2% in HFD-fed mice, increased hepatic surface LDLR, lowered liver TC/TG in HFD-fed mice, and improved glucose tolerance and insulin sensitivity; ABCC4 deficiency lowered intracellular and secreted PCSK9 protein without changing Pcsk9 mRNA, and rhPCSK9 abolished the LDLR/LDL-uptake effect.
Methods
- FACS-based genome-wide CRISPR/Cas9 knockout screens in AML12 cells using a mouse library targeting >18,000 genes; top 10% LDLR-high cells were sorted and analyzed with MAGeCK. Validation used CRISPR knockout in AML12 and LO2 cells, Abcc4/Epac2 and Abcc4/Rap1a double knockouts, RNA-seq, ELISA for cAMP, immunoblotting, flow cytometry, and Dil-LDL uptake assays.
- In vivo studies used 6-week-old male WT C57BL/6 mice. Liver-specific Abcc4 knockdown used AAV8-ApoE/hAAT-Abcc4_RNAi or AAV8_GFP_RNAi (2×10^11 viral genomes per mouse, n=6 per group). Pharmacologic inhibition used Ceefourin-1 (10 mg/kg intraperitoneally three times weekly for 4 weeks) or vehicle under NCD or HFD (n=6 per group).
- Main analyses included serum/liver TC, TG, and LDL-C measurements, PCSK9 and insulin ELISAs, H&E and Oil Red O staining, GTT/ITT, RT-qPCR, immunoblotting of whole and membrane fractions, and public dataset analyses from UK Biobank, Global Lipid Genetics Consortium, and STARNET.
Limitations
- The paper states that the screen failed to prioritize some canonical regulators, possibly due to suboptimal signal-to-noise ratio, stochastic dropout, variable knockout efficiency, or underrepresentation/ineffectiveness of sgRNAs targeting PCSK9 or MYLIP; it also states that the screen focused on genes driving substantial transcriptional changes under homeostatic conditions, and future work should compare transcriptional versus post-transcriptional mechanisms and evaluate sgRNA performance variability.
- The paper states that the liver TC reduction and improved hepatic insulin resistance in HFD-fed mice require further validation in hepatocyte-specific Abcc4 knockout mice under metabolic stress, and that effects on atherosclerotic lesions remain to be clarified.
- The paper states that synergistic effects with statins or PCSK9 inhibitors remain untested. The text also makes evident that in vivo experiments used only male mice and did not include atherosclerosis lesion endpoints.
Model-written summary. Check it against the paper before relying on it.
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Claims the paper makes
47 of 88 cited sources checked · 19 supported · 0 flagged · accuracy not yet measured
- Premisecitation
ABCC4 protein is highly conserved between humans and mice, with 100% sequence identity.
“Notably, ABCC4 protein is highly conserved between humans and mice, with 100% sequence identity, suggesting functional conservation across species39,41,42.”
Quote in the paperSubstantial claim1 of 3 sources checked- cites [39] Zelcer, N. et al. Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4). Biochem J. 371, 361–367 (2003). source
The available copy shows a “Full Text” notice pointing to a PDF and selected references, but no passage comparing human and mouse ABCC4 protein sequences. Searches for “100%,” “sequence identity,” and human–mouse conservation found no support for the claimed 100% identity; the decisive text may be in the unavailable PDF. (LLM, conf=0.73)
Experimental — accuracy not yet measured - cites [41] Denk, G. U. et al. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J. Hepatol. 40, 585–591 (2004). source
- cites [42] Russel, F. G., Koenderink, J. B. & Masereeuw, R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharm. Sci. 29, 200–207 (2008). source
- Premisecitation
ABCC4 is a hepatic efflux transporter of cAMP.
“Functioning as a hepatic efflux transporter of cyclic AMP (cAMP), ABCC4 deficiency led to intracellular accumulation of cAMP, thereby activating the downstream sensor Epac2.”
- Also stated in: § Abstract · plain
“As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway”
Quote in the paperSubstantial claim2 of 5 sources checked- cites [40] Rius, M., Nies, A. T., Hummel-Eisenbeiss, J., Jedlitschky, G. & Keppler, D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38, 374–384 (2003). source
- cites [41] Denk, G. U. et al. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J. Hepatol. 40, 585–591 (2004). source
- cites [42] Russel, F. G., Koenderink, J. B. & Masereeuw, R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharm. Sci. 29, 200–207 (2008). source
- cites [43] Bloch, M., Raj, I., Pape, T. & Taylor, N. M. I. Structural and mechanistic basis of substrate transport by the multidrug transporter MRP4. Structure 31, 1407–1418 (2023). source
The Introduction (page unavailable) identifies MRP4/ABCC4 as an efflux transporter. It does not establish that MRP4 exports cAMP in the liver; the substrate-activity section instead examines PGE2, methotrexate, and topotecan. The hepatic and cAMP-specific parts of the attributed claim are therefore unsupported by our copy. (LLM, conf=0.83)
Experimental — accuracy not yet measured - cites [44] Belleville-Rolland, T. et al. Role of Membrane Lipid Rafts in MRP4 (ABCC4) dependent regulation of the cAMP pathway in blood platelets. Thromb. Haemost. 121, 1628–1636 (2021). source
- Also stated in: § Abstract · plain
- Findingown data
DEGs in Abcc4-KO cells were significantly enriched in cAMP signaling, Rap1 signaling, cGMP-PKG, Phospholipase D, insulin signaling, insulin resistance, Glucagon, and PPAR signaling pathways.
“Moreover, an in-depth analysis of these DEGs revealed significant enrichment in pathways including the cAMP signaling pathway, Rap1 signaling pathway, cGMP-PKG signaling pathway, Phospholipase D signaling pathway, insulin signaling pathway, insulin resistance, Glucagon signaling pathway and PPAR signaling pathway, which were closely implicated in lipid metabolism, as supported by previous studies”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [45] Spolitu, S. et al. Hepatic glucagon signaling regulates PCSK9 and low-density lipoprotein cholesterol. Circ. Res 124, 38–51 (2019). source
The Results section on Gcgr–cAMP, Epac2 and Rap1 links glucagon, cAMP and Rap1 signaling to PCSK9 and cholesterol regulation, supporting the lipid-metabolism relevance of those pathways. The paper does not establish that cGMP-PKG, phospholipase D, insulin signaling, insulin resistance or PPAR signaling are implicated in lipid metabolism. The DEG-enrichment finding is presented as the citing authors’ own analysis, not as a finding attributed to this source. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [71] Shiozaki-Takagi, Y., Ozaki, N. & Toyoda, Y. Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes. J. Diabetes Investig. 15, 429–436 (2024). source
The Introduction and Discussion describe glucagon activating cAMP–Epac2–Rap1 signaling in rat hepatocytes. They do not report Abcc4-KO cells, DEG pathway enrichment, or a link between the listed pathways and lipid metabolism; the Discussion concerns hepatic glucose production instead. No page numbers are available in our copy. (LLM, conf=0.85)
Experimental — accuracy not yet measured
- Findingown data
Transcriptomic profiling revealed a clear separation between Abcc4-KO and control AML12 cell clusters.
“Transcriptomic profiling revealed a clear separation between Abcc4 sgRNA (Abcc4-KO) and Rosa26 sgRNA (control-KO) AML12 cell clusters (Fig. 5a), indicating distinct gene expression landscapes.”
Quote in the paperNot a substantial claim - Findingown data
Differential expression analysis identified 1,290 genes, with 940 (72.87%) significantly upregulated and 350 (27.13%) downregulated in Abcc4-KO cells.
“Differential expression analysis identified 1,290 genes, with 940 (72.87%) significantly upregulated and 350 (27.13%) downregulated in Abcc4-KO cells (P < 0.05; fold change>2.0; Data S5).”
Quote in the paperNot a substantial claim
- Findingown data
Abcc4 knockout significantly increased cell-surface LDLR abundance without altering total LDLR protein levels in murine and human hepatocyte cell lines.
“As expected, Abcc4 knockout significantly increased cell-surface LDLR abundance without altering total LDLR protein levels (Fig. 2c–f; Fig. S2a and S2b).”
- Also stated in: § ABCC4 depletion increases the cell surface LDLR level independent of its gene expression · plain
“This regulatory effect was further validated in human hepatocyte cell line LO2, where similar increases in LDLR surface expression and LDL uptake were observed (Fig. 2k–o; Fig. S2d–S2g).”
Quote in the paperSubstantial claim - Also stated in: § ABCC4 depletion increases the cell surface LDLR level independent of its gene expression · plain
- Findingown data
Abcc4 knockout significantly altered expression patterns of genes related to lipid metabolism and insulin signaling.
“Additionally, Abcc4 knockout significantly altered expression patterns of genes related to lipid metabolism process and insulin signaling pathway (Fig. 5f).”
Quote in the paperNot a substantial claim - Findingown data
Ceefourin-1 significantly potentiated cell surface LDLR abundance in AML12 cells.
“Nevertheless, Ceefourin-1 significantly potentiated cell surface LDLR abundance in AML12 cells by flow cytometry (Fig. 4a, b) and immunoblotting analyses (Fig. 4c, d).”
Quote in the paperSubstantial claim - Findingown data
Ceefourin-1 treatment reduced hepatic lipid accumulation, particularly in HFD-fed mice, as shown by H&E and Oil Red O staining.
“This was further supported by H&E and Oil Red O staining, which revealed a substantial reduction in hepatic lipid accumulation, particularly in the HFD group (Fig. 4l).”
Quote in the paperSubstantial claim - Findingown data
Ceefourin-1 treatment did not significantly change serum total cholesterol or triglyceride levels.
“In contrast, no significant changes were observed in serum TC or TG levels between treated and control groups (Fig. 4h, i).”
Quote in the paperNot a substantial claim - Findingown data
Variants at the ABCC4 gene locus are significantly associated with plasma LDL cholesterol levels in UK Biobank and Global Lipid Genetics Consortium datasets.
“We identified additional variants at the ABCC4 gene locus that were significantly associated with plasma LDL cholesterol levels (Fig. S5a).”
Quote in the paperSubstantial claim - Findingown data
Abcc4 deficiency markedly enhanced LDL uptake in AML12 and LO2 cells.
“By flow cytometry and immunofluorescence analyses, we found that Abcc4 deficiency markedly enhanced LDL uptake (Fig. 2g–j).”
Quote in the paperSubstantial claim - Findingown data
Sixty-eight gene hits from the M1 CRISPR library were also enriched as significant hits in the independent M2 screen.
“Based on two independent screen replicates (M1 and M2), we found 68 gene hits from the M1 library that were also enriched as significant hits in the independent M2 screen.”
Quote in the paperNot a substantial claim - Findingown data
Ceefourin-1 treatment markedly lowered liver total cholesterol and triglyceride contents in HFD-fed mice.
“Interestingly, Ceefourin-1 treatment markedly lowered liver TC and TG contents in HFD-fed mice (Fig. 4j, k).”
Quote in the paperSubstantial claim - Findingown data
Ceefourin-1 treatment reduced serum LDL-C levels by about 41.1% in NCD-fed mice and 42.2% in HFD-fed mice.
“As expected, Ceefourin-1 treatment significantly reduced serum LDL-C levels by about 41.1% in NCD-fed mice and 42.2% in HFD-fed mice (Fig. 4g).”
Quote in the paperSubstantial claim - Findingown data
Abcc4 knockout did not significantly change Ldlr/LDLR mRNA levels.
“No significant differences were observed in Ldlr mRNA expression (Fig. S2c), suggesting a post-transcriptional regulatory mechanism.”
Quote in the paperSubstantial claim - Findingcitation
ABCC4 belongs to a liver gene co-expression network module significantly linked to coronary artery disease and other cardiometabolic outcomes in the STARNET study.
“Moreover, analysis of public data from the STARNET (Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task) study70 revealed that ABCC4 belongs to a liver gene co-expression network module significantly linked to coronary artery disease and other cardiometabolic outcomes (Fig. S5b).”
Quote in the paperNot a substantial claim - Findingown data
GSEA confirmed that Abcc4 deficiency activated genes involved in the cAMP and Rap1 signaling pathways.
“Gene set enrichment analysis (GSEA) further confirmed that Abcc4 deficiency simultaneously activated a broad spectrum of genes involved in the cAMP signaling pathway (Fig. 5d), Rap1 signaling pathway (Fig. 5e).”
Quote in the paperNot a substantial claim - Findingown data
Ceefourin-1 administration significantly increased hepatic surface LDLR protein expression under both NCD and HFD conditions.
“Furthermore, consistent with our in vitro findings, Ceefourin-1 administration significantly increased hepatic surface LDLR protein expression under both NCD and HFD conditions (Fig. 4m–o).”
Quote in the paperSubstantial claim - Findingown data
Double-knockout of Abcc4/Rap1a in AML12 cells depletes LDLR protein expression levels on cell membrane.
“Double-knockout of Abcc4/Rap1a in AML12 cells depleted LDLR protein expression levels on cell membrane, as evidenced by immunoblotting data (Fig. 6h, i).”
Quote in the paperSubstantial claim - Findingown data
HFD feeding significantly elevated fasting blood glucose and serum insulin levels compared with NCD-fed mice.
“As anticipated, HFD feeding significantly elevated fasting blood glucose and serum insulin levels compared with NCD-fed mice (Fig. S4e).”
Quote in the paperNot a substantial claim - Findingown data
RNA-seq data revealed enrichment of glucagon pathway genes in Abcc4-KO cells.
“our RNA-seq data also revealed enrichment of glucagon pathway genes in Abcc4-KO cells (Fig. 5c).”
Quote in the paperSubstantial claim - Findingown data
MAGeCK analysis identified ABCC4 as a top-scoring candidate gene with significant effects on surface LDLR abundance.
“MAGeCK analysis of enriched sgRNAs identified ABCC4 as a top-scoring candidate gene with significant effects on surface LDLR abundance (Fig. 1c).”
Quote in the paperSubstantial claim - Findingown data
Combined treatment with glucagon and Ceefourin-1 further suppresses PCSK9 levels.
“Combined treatment with glucagon and Ceefourin-1 further suppressed PCSK9 levels, suggesting a synergistic effect and reinforcing the mechanistic role of ABCC4 in PCSK9 regulation (Fig. S8a and S8b).”
Quote in the paperNot a substantial claim - Findingown data
Cycloheximide treatment (90 min) leads to a marked reduction in PCSK9 protein levels in Abcc4-KO cells.
“This treatment (90 min) led to a marked reduction in PCSK9 protein levels in Abcc4-KO cells, suggesting increased degradation (Fig. 5q).”
Quote in the paperSubstantial claim - Findingown data
Pharmacological inhibition of ABCC4 with Ceefourin-1 replicates the effects of Abcc4 disruption under both NCD and HFD conditions in vivo.
“Pharmacological inhibition of ABCC4 using the specific inhibitor Ceefourin-1 replicated these effects under both NCD and HFD conditions in vivo.”
Quote in the paperSubstantial claim- Rests on an assumption: Ceefourin-1 specifically inhibits ABCC4.
- Findingown data
In hepatocytes lacking Abcc4, intracellular PCSK9 protein levels decrease.
“As anticipated, we observed a corresponding decrease in both intracellular and secreted PCSK9 protein levels without significant changes in Pcsk9 mRNA expression in hepatocytes lacking Abcc4 (Fig. 5i–l).”
Quote in the paperSubstantial claim - Findingown data
In hepatocytes lacking Abcc4, Pcsk9 mRNA expression does not significantly change.
“As anticipated, we observed a corresponding decrease in both intracellular and secreted PCSK9 protein levels without significant changes in Pcsk9 mRNA expression in hepatocytes lacking Abcc4 (Fig. 5i–l).”
Quote in the paperSubstantial claim - Findingown data
Abcc4 deficiency or inhibition in hepatocytes activates a broad spectrum of genes involved in cAMP and Rap1 signaling pathways.
“In line with this, our sequencing data illustrated that Abcc4 deficiency or inhibition in hepatocytes activated a broad spectrum of genes involved in cAMP and Rap1 signaling pathways (Fig. 5d–f; Fig. S6).”
Quote in the paperSubstantial claim - Findingown data
Lacking both Epac2 and Abcc4 in hepatocytes increases intracellular PCSK9 protein levels.
“Importantly, we also observed that lacking both Epac2 and Abcc4 in hepatocytes led to an increase in intracellular and secreted PCSK9 protein levels, without significant changes in Pcsk9 mRNA expression (Fig. 6e–g).”
Quote in the paperSubstantial claim - Findingown data
Knockout of Abcc4 in AML12 cells inhibits cAMP efflux.
“Obviously, knockout of Abcc4 in AML12 cells effectively inhibited cAMP efflux and promoted activation of the intracellular cAMP signaling pathway, as revealed by RNA-seq analysis.”
Quote in the paperSubstantial claim - Findingown data
Abcc4/Rap1a DKO hepatocytes have increased intracellular PCSK9 protein levels.
“Furthermore, Abcc4/Rap1a DKO hepatocytes displayed a marked increase in both intracellular and secreted PCSK9 protein levels, while Pcsk9 mRNA expression remained unchanged (Fig. 6l–n).”
Quote in the paperSubstantial claim - Findingown data
ABCC4 knockout markedly increases surface LDLR abundance in both murine and human hepatocytes, independent of LDLR transcription.
“Functional validation revealed that ABCC4 knockout markedly increased surface LDLR abundance in both murine and human hepatocytes, independent of LDLR transcription.”
Quote in the paperSubstantial claim - Findingown data
rhPCSK9 treatment completely abolishes the effect of Abcc4 silencing on LDLR surface expression and LDL uptake.
“Remarkably, rhPCSK9 treatment completely abolished the effect of Abcc4 silencing on LDLR surface expression and LDL uptake (Fig. 5r, s)”
Quote in the paperSubstantial claim- Rests on an assumption: Recombinant human PCSK9 protein (rhPCSK9) is functional and mimics endogenous PCSK9.
- Findingown data
In Abcc4/Epac2 DKO AML12 cells, the elevated LDLR surface abundance induced by Abcc4 deficiency is abrogated.
“Interestingly, elevated LDLR surface abundance induced by Abcc4 deficiency was abrogated in Abcc4/Epac2 DKO AML12 cells (Fig. 6b).”
Quote in the paperSubstantial claim - Findingown data
Ceefourin-1 treatment does not significantly change Pcsk9 transcript levels in AML12 cells.
“Importantly, Ceefourin-1 treatment recapitulated these effects on PCSK9 protein, with no significant changes in Pcsk9 transcript levels (Fig. 5m–p).”
Quote in the paperSubstantial claim - Findingown data
Ceefourin-1 treatment alters cAMP distribution in AML12 cells.
“Similarly, Ceefourin-1 treatment altered cAMP distribution in AML12 cells, supporting the role of ABCC4 in cAMP transport (Fig. 5h).”
Quote in the paperNot a substantial claim - Findingown data
Abcc4/Rap1a DKO hepatocytes have increased secreted PCSK9 protein levels.
“Furthermore, Abcc4/Rap1a DKO hepatocytes displayed a marked increase in both intracellular and secreted PCSK9 protein levels, while Pcsk9 mRNA expression remained unchanged (Fig. 6l–n).”
Quote in the paperSubstantial claim - Findingown data
Lacking both Epac2 and Abcc4 in hepatocytes increases secreted PCSK9 protein levels.
“Importantly, we also observed that lacking both Epac2 and Abcc4 in hepatocytes led to an increase in intracellular and secreted PCSK9 protein levels, without significant changes in Pcsk9 mRNA expression (Fig. 6e–g).”
Quote in the paperSubstantial claim - Findingown data
In hepatocytes lacking Abcc4, secreted PCSK9 protein levels decrease.
“As anticipated, we observed a corresponding decrease in both intracellular and secreted PCSK9 protein levels without significant changes in Pcsk9 mRNA expression in hepatocytes lacking Abcc4 (Fig. 5i–l).”
Quote in the paperSubstantial claim - Findingown data
Abcc4/Rap1a DKO cells have impeded LDL uptake by LDLR.
“In line with the observed decline in LDLR surface abundance, Abcc4/Rap1a DKO cells impeded LDL uptake by LDLR (Fig. 6j, k).”
Quote in the paperSubstantial claim - Findingown data
Lacking both Epac2 and Abcc4 in hepatocytes does not significantly change Pcsk9 mRNA expression.
“Importantly, we also observed that lacking both Epac2 and Abcc4 in hepatocytes led to an increase in intracellular and secreted PCSK9 protein levels, without significant changes in Pcsk9 mRNA expression (Fig. 6e–g).”
Quote in the paperNot a substantial claim - Findingown data
Abcc4/Rap1a DKO hepatocytes have unchanged Pcsk9 mRNA expression.
“Furthermore, Abcc4/Rap1a DKO hepatocytes displayed a marked increase in both intracellular and secreted PCSK9 protein levels, while Pcsk9 mRNA expression remained unchanged (Fig. 6l–n).”
Quote in the paperNot a substantial claim - Findingown data
Knockout of Abcc4 in AML12 cells promotes activation of the intracellular cAMP signaling pathway.
“Obviously, knockout of Abcc4 in AML12 cells effectively inhibited cAMP efflux and promoted activation of the intracellular cAMP signaling pathway, as revealed by RNA-seq analysis.”
Quote in the paperSubstantial claim - Interpretationown data
ABCC4 functions as a negative regulator of cell surface LDLR expression.
“Together, these previous studies align with our screening results, suggesting that ABCC4 functions as a negative regulator of cell surface LDLR expression.”
Quote in the paperSubstantial claim - Interpretationreasoning
The reduction in hepatic cholesterol contents may be attributed, at least in part, to suppression of glucose-driven lipogenesis rather than decreased cholesterol uptake alone.
“These findings suggest that the reduction in hepatic cholesterol contents may be attributed, at least in part, to the suppression of glucose-driven lipogenesis rather than decreased cholesterol uptake alone.”
Quote in the paperNot a substantial claim - Interpretationown data
EPAC2 acts as an upstream activator of the small GTPase RAP1.
“EPAC2 protein not only serves as a key intracellular effector of cAMP but also acts as an upstream activator of the small GTPase RAP1, as supported by our RNA sequencing data (Fig. 5).”
Quote in the paperSubstantial claim - Interpretationown data
Increased LDLR surface availability upon Abcc4 deficiency is driven by altered intracellular cAMP distribution and activation of downstream Epac2/Rap1a signaling.
“Mechanistically, increased LDLR surface availability was driven by altered intracellular cAMP distribution and the activation of its downstream Epac2/Rap1a signaling, upon Abcc4 deficiency.”
Quote in the paperSubstantial claim - Interpretationown data
Altered intracellular cAMP distribution in Abcc4-deficient hepatocytes suppresses PCSK9 protein levels via downstream Epac2/Rap1a signals.
“In this context, we propose that altered intracellular cAMP distribution in Abcc4-deficient hepatocytes suppressed PCSK9 protein levels via its downstream Epac2/Rap1a signals.”
Quote in the paperSubstantial claim - Interpretationown data
ABCC4 plays a role in cAMP transport.
“Similarly, Ceefourin-1 treatment altered cAMP distribution in AML12 cells, supporting the role of ABCC4 in cAMP transport (Fig. 5h).”
Quote in the paperSubstantial claim - Interpretationown data
Glucagon and Ceefourin-1 have a synergistic effect on PCSK9 suppression.
“Combined treatment with glucagon and Ceefourin-1 further suppressed PCSK9 levels, suggesting a synergistic effect and reinforcing the mechanistic role of ABCC4 in PCSK9 regulation (Fig. S8a and S8b).”
Quote in the paperNot a substantial claim - Interpretationown data
The ABCC4-cAMP-Epac2/Rap1a signaling pathway plays a pivotal role in modulating surface LDLR availability and LDL-C uptake in hepatocytes.
“Overall, these data highlight the pivotal role of the ABCC4-cAMP-Epac2/Rap1a signaling pathway in modulating surface LDLR availability and LDL-C uptake in hepatocytes.”
Quote in the paperSubstantial claim - Interpretationown data
ABCC4 exerts a posttranscriptional regulatory effect on PCSK9 expression.
“Given that Abcc4 deficiency did not alter Pcsk9 mRNA levels (Fig. 5l), yet resulted in reduced intracellular and secreted PCSK9 levels (Fig. 5i-k), these finding suggest that ABCC4 exerts a posttranscriptional regulatory effect on PCSK9 expression.”
Quote in the paperSubstantial claim - Interpretationown data
Activation of the intracellular cAMP signaling pathway is essential for regulating PCSK9 and LDLR expression in Abcc4-deficiency hepatocytes.
“Together, our findings establish that activation of the intracellular cAMP signaling pathway is essential for regulating PCSK9 and LDLR expression in Abcc4-deficiency hepatocytes.”
Quote in the paperSubstantial claim - Interpretationown data
ABCC4 deficiency increases PCSK9 degradation.
“This treatment (90 min) led to a marked reduction in PCSK9 protein levels in Abcc4-KO cells, suggesting increased degradation (Fig. 5q).”
Quote in the paperSubstantial claim- Rests on an assumption: Cycloheximide treatment blocks protein synthesis and the observed reduction in PCSK9 reflects increased degradation.
- Probably not a substantial claim. 35
- It may state two separate claims as one. 48
- The quote only partly states this claim. 3
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPCSK9 inhibitors prevent the degradation of LDLR in hepatocytes.
“The development of PCSK9 inhibitors (PCSK9i) has manifested their pivotal roles in lipid-lowing therapies by preventing the degradation of LDLR in hepatocytes9,10.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [9] Hummelgaard, S., Vilstrup, J. P., Gustafsen, C., Glerup, S. & Weyer, K. Targeting PCSK9 to tackle cardiovascular disease. Pharm. Ther. 249, 108480 (2023). source
- cites [10] Bao, X. et al. Targeting proprotein convertase subtilisin/kexin type 9 (PCSK9): from bench to bedside. Signal Transduct. Target Ther. 9, 13 (2024). source
It may state two separate claims as one.
PremisecitationLow expression of LDLR in hepatocytes leads to hypercholesterolemia and contributes to atherosclerotic cardiovascular disease.
“Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD).”
Quote in the paperSubstantial claim2 of 4 sources checked- cites [20] Goldstein, J. L. & Brown, M. S. The LDL receptor. Arteriosclerosis, Thrombosis, Vasc. Biol. 29, 431–438 (2019). source
- cites [21] Brown, M. S. & Goldstein, J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 (1986). source
- cites [22] Osono, Y., Woollett, L. A., Herz, J. & Dietschy, J. M. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J. Clin. Invest 95, 1124–1132 (1995). source
The paper’s Abstract (p. 1124) reports that most LDL-receptor-dependent transport occurs in the liver and that deleting LDLR in mice raises plasma LDL cholesterol 14-fold. It does not specifically test low LDLR expression in hepatocytes or establish the claimed progression to atherosclerotic cardiovascular disease. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [23] Wijers, M., Kuivenhoven, J. A. & van de Sluis, B. The life cycle of the low-density lipoprotein receptor: insights from cellular and in-vivo studies. Curr. Opin. Lipido. 26, 82–87 (2015). source
Probably not a substantial claim. It may state two separate claims as one.
PremisecitationLDLR is abundantly expressed on the surface of hepatocytes, where it facilitates LDL cholesterol uptake and maintains lipid homeostasis.
“As a transmembrane glycoprotein, low-density lipoprotein receptor (LDLR) is abundantly expressed on the surface of hepatocytes, where it facilitates the uptake of LDL cholesterol and maintains the homeostasis of lipid metabolism20,21.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [20] Goldstein, J. L. & Brown, M. S. The LDL receptor. Arteriosclerosis, Thrombosis, Vasc. Biol. 29, 431–438 (2019). source
The Regulation of LDL Receptors section (page unavailable) says LDL receptors are displayed on liver-cell membranes, remove LDL from the blood, and help maintain normal liver cholesterol levels. The LDL Receptor Pathway and Cholesterol Homeostasis section describes feedback regulation of cellular cholesterol. Neither passage establishes that LDLR is abundantly expressed on hepatocytes under baseline conditions. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [21] Brown, M. S. & Goldstein, J. L. A receptor-mediated pathway for cholesterol homeostasis. Science 232, 34–47 (1986). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationSREBP2 promotes LDLR synthesis by binding to its promoter region, thereby maintaining normal cellular cholesterol contents.
“Meanwhile, sterol regulatory element-binding protein 2 (SREBP2) promotes LDLR synthesis by binding to its promoter region18,19at the transcriptional level, thereby maintaining normal cellular cholesterol contents.”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites [18] Cao, X. et al. Increased LDL receptor by SREBP2 or SREBP2-induced lncRNA LDLR-AS promotes triglyceride accumulation in fish. iScience 25, 104670 (2022). source
The Results section reports that SREBP2 activates the LDLR promoter and that knocking down SREBP2 inhibits PA-induced LDLR transcription in fish hepatocytes. The paper does not show that this mechanism maintains normal cellular cholesterol content; its Introduction instead notes that excessive LDLR can disrupt hepatic lipid homeostasis. No page numbers were provided in our copy. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [19] Du, Y. et al. Tilianin improves lipid profile and alleviates atherosclerosis in ApoE-/- mice through up-regulation of SREBP2-mediated LDLR expression. Phytomedicine 109, 154577 (2023). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationStatins and NPC1L1 inhibitors enhance LDLR expression in hepatocytes, thereby promoting serum LDL-C clearance.
“Besides PCSK9 antibodies, current clinical therapies including statins13,14 and NPC1L1 (NPC1 like intracellular cholesterol transporter 1) inhibitors15enhance LDLR expression in hepatocytes, thereby promoting serum LDL-C clearance.”
Quote in the paperNot a substantial claim3 of 3 sources checked- cites [13] Liang, X., He, Q. & Zhao, Q. Effect of Stains on LDL reduction and liver safety: a systematic review and meta-analysis. Biomed. Res Int 2018, 7092414 (2018). source
The Introduction states that statins are widely used to reduce LDL, supporting the LDL-lowering part of the statin claim. Our copy does not say that statins increase hepatocyte LDLR expression or that this mechanism promotes serum LDL-C clearance. The NPC1L1-inhibitor claim has a separate citation in the citing sentence and is not attributed to this paper. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [14] Hadjiphilippou, S. & Ray, K. K. Cholesterol-lowering agents. Circn Res. 124, 354–363 (2018). source
The Abstract (page unavailable) says statins reduce LDL-C and identifies ezetimibe as another lipid-modifying therapy. It does not say that either therapy enhances LDLR expression in hepatocytes or that this is how serum LDL-C is cleared. In the citing sentence, the NPC1L1-inhibitor portion has a separate citation. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [15] Jia, L., Betters, J. L. & Yu, L. Niemann-pick C1-like 1 (NPC1L1) protein in intestinal and hepatic cholesterol transport. Annu Rev. Physiol. 73, 239–259 (2011). source
The Introduction says ezetimibe inhibits intestinal cholesterol absorption and lowers blood LDL-C. The section on genetic variation in NPC1L1 says increased absorption downregulates hepatic LDL receptors and reduces LDL clearance. These passages support the proposed pathway, but do not directly establish that NPC1L1 inhibitors enhance LDLR expression in hepatocytes or that this increase causes their LDL-C-lowering effect. (LLM, conf=0.83)
Experimental — accuracy not yet measured
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPCSK9 secreted by the liver binds LDLR, promoting LDLR lysosomal degradation and preventing its recycling back to the cell surface.
“Proprotein convertase subtilisin/kexin type 9 (PCSK9), secreted by the liver, functions as a competitive binder to low-density lipoprotein receptor (LDLR), promoting LDLR lysosomal degradation and preventing its recycling back to the cell surface.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [9] Hummelgaard, S., Vilstrup, J. P., Gustafsen, C., Glerup, S. & Weyer, K. Targeting PCSK9 to tackle cardiovascular disease. Pharm. Ther. 249, 108480 (2023). source
The Figure 3 caption says liver hepatocytes secrete PCSK9, and section 3.2 says PCSK9 binds LDLR, prevents its recycling, and directs the complex to lysosomal degradation. The citing sentence’s additional description of PCSK9 as a “competitive binder” is not established by these passages; sections 3.1 and 3.2 describe different LDLR binding domains for LDL-C and PCSK9. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [10] Bao, X. et al. Targeting proprotein convertase subtilisin/kexin type 9 (PCSK9): from bench to bedside. Signal Transduct. Target Ther. 9, 13 (2024). source
The Background passages on LDLR degradation and the figure description (page not provided) state that hepatocyte-secreted PCSK9 binds LDLR, directs the complex to lysosomes, and prevents LDLR recycling. They do not establish the citing sentence’s additional characterization of PCSK9 as a “competitive binder.” (LLM, conf=0.83)
Experimental — accuracy not yet measured
It may state two separate claims as one.
PremisecitationCeefourin-1 is a highly specific ABCC4 inhibitor that blocks transport of substrates without detectable effects on MRP1, MRP2, MRP3 or MRP5.
“Previous studies have demonstrated that Ceefourin-1 is a highly specific inhibitor of ABCC459–62, effectively blocking the transport of its substrates such as 6-mercaptopurine (6-MP), cAMP, D-luciferin, and estradiol-17β-glucuronide (E217βG), without exerting detectable effects on related transporters MRP1, MRP2, MRP3 or MRP5.”
Quote in the paperSubstantial claim3 of 4 sources checked- cites [59] Cheung, L. et al. High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4). Biochem Pharm. 91, 97–108 (2014). source
- cites [60] Huynh, T. et al. CCI52 sensitizes tumors to 6-mercaptopurine and inhibits MYCN-amplified tumor growth. Biochem Pharm. 172, 113770 (2020). source
Section 3.1 calls Ceefourin-1 a selective MRP4 (ABCC4) inhibitor that blocks a range of substrates. The results also show inhibition of D-luciferin and E217βG transport and selective activity in MRP4-expressing cells after 6-MP exposure. Our copy does not establish inhibition of cAMP transport or report tests showing no detectable effects on MRP1, MRP2, MRP3, or MRP5. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [61] Wolf, R. et al. Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets. Haematologica 107, 2206–2217 (2022). source
The Results section on MRP4-mediated transport reports that Ceefourin-1 inhibits transport of cGMP, thromboxane B2 and S1P. The Discussion describes it as highly selective for MRP4 over several ABC transporters, including MRP1 and MRP3, but does not establish the claimed lack of detectable effects on MRP2 or MRP5. The cited passages also do not demonstrate blocked transport of the sentence’s specifically named substrates: 6-MP, cAMP, D-luciferin and E217βG. Page numbers were unavailable in our copy. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [62] Becerra, E., Berumen, L., Soto-Ontiveros, V. & García-Alcocer, G. Specific MRP4 inhibitor ceefourin-1 enhances apoptosis induced by 6-mercaptopurine in jurkat leukemic cells, but not in normal lymphoblast cell line CRL-1991. Med. (Kaunas.) 58, 695 (2022).
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim.
PremisecitationApproximately 75% of circulating LDL particles are cleared by LDLR through receptor-mediated endocytosis.
“Approximately 75% of circulating LDL particles are cleared by LDLR through receptor-mediated endocytosis22,23.”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [22] Osono, Y., Woollett, L. A., Herz, J. & Dietschy, J. M. Role of the low density lipoprotein receptor in the flux of cholesterol through the plasma and across the tissues of the mouse. J. Clin. Invest 95, 1124–1132 (1995). source
The paper’s opening summary reports that LDLR accounted for 88% of LDL-cholesterol turnover in normal mice, supporting a substantial role for LDLR in clearance. It does not support the stated figure of approximately 75%, an unqualified claim about circulating LDL particles, or the specified endocytosis mechanism. Section and page are not available in our copy. (LLM, conf=0.87)
Experimental — accuracy not yet measured - cites [23] Wijers, M., Kuivenhoven, J. A. & van de Sluis, B. The life cycle of the low-density lipoprotein receptor: insights from cellular and in-vivo studies. Curr. Opin. Lipido. 26, 82–87 (2015). source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationPrevious reports link glucagon signaling to PCSK9 regulation.
“Given previous reports linking glucagon signaling to PCSK9 regulation45,71”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [45] Spolitu, S. et al. Hepatic glucagon signaling regulates PCSK9 and low-density lipoprotein cholesterol. Circ. Res 124, 38–51 (2019). source
- cites [71] Shiozaki-Takagi, Y., Ozaki, N. & Toyoda, Y. Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes. J. Diabetes Investig. 15, 429–436 (2024). source
The Results and Discussion describe glucagon’s effects on hepatic glucose metabolism through Epac signaling, but do not link glucagon signaling to PCSK9 regulation. Searches of our full-text copy for “PCSK9” and “proprotein convertase subtilisin/kexin type 9” found no passage addressing that link. (LLM, conf=0.76)
Experimental — accuracy not yet measured
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationcAMP signaling regulates key physiological processes, including gonadotropin-releasing hormone secretion, insulin granule dynamics, and cardiac function.
“Consistent with earlier studies showing that cAMP signaling regulates key physiological processes, including gonadotropin-releasing hormone secretion, insulin granule dynamics and cardiac function91–94”
Quote in the paperNot a substantial claim4 of 4 sources checked- cites [91] Vitalis, E. A. et al. Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells. Proc. Natl Acad. Sci. USA 97, 1861–1866 (2000). source
- cites [92] Shibasaki, T. et al. Essential role of Epac2/Rap1 signaling in regulation of insulin granule dynamics by cAMP. Proc. Natl Acad. Sci. USA 104, 19333–19338 (2007). source
- cites [93] Wang, Z. et al. A cardiac mitochondrial cAMP signaling pathway regulates calcium accumulation, permeability transition and cell death. Cell Death Dis. 7, e2198 (2016). source
- cites [94] Musheshe, N., Schmidt, M. & Zaccolo, M. cAMP: from long-range second messenger to nanodomain signalling. Trends Pharm. Sci. 39, 209–222 (2018). source
The section ‘The Problem of cAMP-Dependent Hormonal Specificity’ (page unavailable) describes a cAMP-dependent increase in heart contractility, supporting the cardiac-function example. The copy also describes cAMP as regulating a broad range of cellular functions, but the passages found do not address gonadotropin-releasing hormone secretion or insulin granule dynamics. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationABCC4 (MRP4) is an ATP-dependent efflux transporter of the ABC family that exports a broad range of substrates, including anticancer drugs, bile acids, and cyclic nucleotides.
“ABCC4 (MRP4) is an ATP-dependent efflux transporter of the ABC family that exports a broad range of substrates, including anticancer drugs, bile acids, and cyclic nucleotides43,50,84–86.”
Quote in the paperNot a substantial claim2 of 5 sources checked- cites [43] Bloch, M., Raj, I., Pape, T. & Taylor, N. M. I. Structural and mechanistic basis of substrate transport by the multidrug transporter MRP4. Structure 31, 1407–1418 (2023). source
The Introduction identifies MRP4 as an ATP-dependent ABC-family efflux transporter with broad substrate specificity, including chemotherapeutic agents. The References section names cyclic nucleotides and bile acid conjugates as MRP4 substrates, but the paper’s discussion does not establish transport of unconjugated bile acids; that distinction limits support for the claim as worded. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [50] Zhou, S. F. et al. Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development. Curr. Med Chem. 15, 1981–2039 (2008). source
- cites [84] Borst, P., Evers, R., Kool, M. & Wijnholds, J. A family of drug transporters: the multidrug resistance-associated proteins. J. Natl Cancer Inst. 92, 1295–1302 (2000). source
- cites [85] Pourmal, S. et al. Structural basis of prostaglandin efflux by MRP4. Nat. Struct. Mol. Biol. 31, 621–632 (2024). source
The Abstract (page not supplied) identifies MRP4 as an ATP-binding cassette transporter and lists cyclic nucleotides and bile salts among its diverse substrates. Reference 29 (page not supplied) names methotrexate efflux by MRP4, but the paper does not explicitly identify anticancer drugs as a substrate class; that part of the claim is not fully supported by our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [86] Reid, G. et al. The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. Proc. Natl Acad. Sci. USA 100, 9244–9249 (2003). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationcAMP and its downstream signaling pathways regulate PCSK9 homeostasis and LOX-1 expression.
“Furthermore, cAMP and its downstream signaling pathways are known to regulate PCSK9 homeostasis and LOX-1 expression45,46.”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites [45] Spolitu, S. et al. Hepatic glucagon signaling regulates PCSK9 and low-density lipoprotein cholesterol. Circ. Res 124, 38–51 (2019). source
The Results section reports that cAMP signaling through Epac2 and Rap1 regulates PCSK9 protein levels in hepatocytes. The cited paper does not address LOX-1 expression, so it supports only the PCSK9 part of the attributed sentence. (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites [46] Robichaux, W. G. 3rd et al. Epac1 (exchange protein directly activated by camp 1) upregulates LOX-1 (oxidized low-density lipoprotein receptor 1) to promote foam cell formation and atherosclerosis development. Arterioscler Thromb. Vasc. Biol. 40, e322–e335 (2020). source
Probably not a substantial claim.
PremisecitationThe ApoE/hAAT promoter provides effective gene regulation specifically within hepatocytes, as demonstrated in previous studies.
“The ApoE/hAAT promoter is known for its strong and liver-specific expression, which has been demonstrated in previous studies to provide effective gene regulation specifically within hepatocytes56,57,104.”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [56] Tan, J. et al. DRAM1 increases the secretion of PKM2-enriched EVs from hepatocytes to promote macrophage activation and disease progression in ALD. Mol. Ther. Nucleic Acids 27, 375–389 (2021). source
The Materials and methods section identifies the ApoE/hAATp vector used to overexpress DRAM1 in mouse liver cells. The Results section reports overexpression in liver but not other organs, supporting effective liver-specific expression. It does not establish expression specifically within hepatocytes rather than other liver cells, or test the promoter’s strength independently of the AAV construct. Page numbers are unavailable in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [57] Yeh, C. F. et al. Targeting mechanosensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo. Sci. Adv. 8, eabl8096 (2022). source
The Materials and Methods section (‘AAV9-PCSK9 mouse model’; page not provided) identifies HCRApoE/hAAT as a liver-specific promoter used to express mutant PCSK9 in mice. It does not demonstrate strong expression or establish that expression is specifically confined to hepatocytes. (LLM, conf=0.85)
Experimental — accuracy not yet measured - cites [104] Vila-Julià, F. et al. Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides. EBioMedicine 62, 103133 (2020). source
The Methods identify AAV-AAT as using the ApoE-AAT cassette, and the Results describe the tested promoters as liver-specific and AAV-AAT as the most effective at increasing liver TP activity. The Results measure liver tissue, however, not expression specifically within hepatocytes; that cellular specificity is not demonstrated in our copy. No page numbers are available in the indexed passages. (LLM, conf=0.84)
Experimental — accuracy not yet measured
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationEPAC2 plays significant roles in the liver, endocrine glands, and brain.
“Several lines of evidence has conceived that EPAC2 plays significant roles in the liver45,71,96, endocrine glands and brain97–99.”
Quote in the paperNot a substantial claim4 of 6 sources checked- cites [45] Spolitu, S. et al. Hepatic glucagon signaling regulates PCSK9 and low-density lipoprotein cholesterol. Circ. Res 124, 38–51 (2019). source
- cites [71] Shiozaki-Takagi, Y., Ozaki, N. & Toyoda, Y. Epac2 activation mediates glucagon-induced glucogenesis in primary rat hepatocytes. J. Diabetes Investig. 15, 429–436 (2024). source
- cites [96] Ueno, H. et al. Characterization of the gene EPAC2: structure, chromosomal localization, tissue expression, and identification of the liver-specific isoform. Genomics 78, 91–98 (2021). source
- cites [97] Niimura, M. et al. Critical role of the N-terminal cyclic AMP-binding domain of Epac2 in its subcellular localization and function. J. Cell Physiol. 219, 652–658 (2009). source
- cites [98] Sugawara, K., Shibasaki, T., Takahashi, H. & Seino, S. Structure and functional roles of Epac2 (Rapgef4). Gene 575, 577–583 (2016). source
- cites [99] Liu, X. et al. Epac2 in midbrain dopamine neurons contributes to cocaine reinforcement via enhancement of dopamine release. Elife 11, e80747 (2022). source
The Introduction (page not provided) says EPAC2 is predominantly expressed in the brain, and the Results show that it enhances dopamine release there. The paper does not establish a significant role for EPAC2 in endocrine glands. The liver portion of the sentence has separate citations, so it is not assessed against this source. (LLM, conf=0.86)
Experimental — accuracy not yet measured
Probably not a substantial claim.
PremisecitationEPAC1 and EPAC2 contain both a cAMP-binding domain and a GEF domain, enabling activation of RAS superfamily small GTPases Rap1.
“EPAC1 and EPAC2 contain both a cAMP-binding domain and a guanine nucleotide exchange factors (GEFs) domain, enabling activation of RAS superfamily small GTPases Rap172–74,95.”
Quote in the paperSubstantial claim3 of 4 sources checked- cites [72] Cheng, X., Ji, Z., Tsalkova, T. & Mei, F. Epac and PKA: a tale of two intracellular cAMP receptors. Acta Biochim Biophys. Sin. (Shanghai) 40, 651–662 (2018). source
- cites [73] Enserink, J. et al. A novel Epac-specific cAMP analogue demonstrates independent regulation of Rap1 and ERK. Nat. Cell Biol. 4, 901–906 (2002). source
- cites [74] Wang, Z. et al. Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation. Mol. Cell Biol. 26, 2130–2145 (2006). source
- cites [95] Iannucci, L. F. et al. Cyclic AMP induces reversible EPAC1 condensates that regulate histone transcription. Nat. Commun. 14, 5521 (2023). source
It may state two separate claims as one.
PremisecitationThe ApoE/hAAT promoter has strong and liver-specific expression.
“The ApoE/hAAT promoter is known for its strong and liver-specific expression, which has been demonstrated in previous studies to provide effective gene regulation specifically within hepatocytes56,57,104.”
Quote in the paperSubstantial claim3 of 3 sources checked- cites [56] Tan, J. et al. DRAM1 increases the secretion of PKM2-enriched EVs from hepatocytes to promote macrophage activation and disease progression in ALD. Mol. Ther. Nucleic Acids 27, 375–389 (2021). source
The Materials and methods section identifies an ApoE/hAATp-containing AAV vector used to overexpress DRAM1 in mouse liver cells. The Results section reports DRAM1 overexpression in the liver but not other organs, supporting liver-specific expression in this experiment. The paper does not establish that the promoter itself has strong expression or compare its strength with other promoters; it also does not establish expression specifically within hepatocytes rather than liver tissue more broadly. Page numbers are unavailable in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measured - cites [57] Yeh, C. F. et al. Targeting mechanosensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo. Sci. Adv. 8, eabl8096 (2022). source
The Materials and Methods section (‘AAV9-PCSK9 mouse model’; page unavailable) explicitly calls the HCRApoE/hAAT promoter liver-specific and describes its use to express a PCSK9 mutant in mice. It does not establish that expression is strong or demonstrate gene regulation specifically within hepatocytes. (LLM, conf=0.86)
Experimental — accuracy not yet measured - cites [104] Vila-Julià, F. et al. Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides. EBioMedicine 62, 103133 (2020). source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationExperimental evidence has recapitulated key modulators of cellular LDL uptake, as well as posttranscriptional and posttranslational regulators of LDLR.
“Moreover, experimental evidence has recapitulated key modulators of cellular LDL uptake, as well as posttranscriptional and posttranslational regulators of LDLR79–82.”
Quote in the paperNot a substantial claim4 of 4 sources checked- cites [79] Emmer, B. T. et al. Genome-scale CRISPR screening for modifiers of cellular LDL uptake. PLoS Genet 17, e1009285 (2021). source
The Results sections on the primary and secondary CRISPR screens report recovery of known LDL-uptake regulators, including LDLR, SCAP, MBTPS1, MBTPS2, and MYLIP. The LDLR-staining Results and Discussion also support regulation of LDLR protein levels and trafficking, but the passages do not establish that the screen recapitulated posttranscriptional regulators of LDLR. Page numbers are unavailable in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measured - cites [80] Smith, G. A. et al. Cold shock domain-containing protein E1 is a posttranscriptional regulator of the LDL receptor. Sci. Transl. Med 14, eabj8670 (2022). source
- cites [81] Clifford, B. L. et al. RNF130 Regulates LDLR Availability and Plasma LDL Cholesterol Levels. Circ. Res 132, 849–863 (2023). source
- cites [82] Zanoni, P. et al. Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome. Circ. Res 130, 80–95 (2022). source
The Results report that the RNAi screen identified genes affecting cellular LDL uptake, and the Discussion describes experimental validation of U2-spliceosome components as posttranscriptional regulators of LDLR. But the Discussion (p. 9) says the screen missed canonical regulators including MYLIP and PCSK9, so this paper does not support the claim that its experiments recapitulated posttranslational LDLR regulators. (LLM, conf=0.86)
Experimental — accuracy not yet measured
It may state two separate claims as one.
Findingown dataABCC4 barely affected cellular cholesterol contents and did not influence SREBP2-dependent gene expression (Srebp2 and Hmgcr).
“To assess whether Abcc4 deficiency affects intracellular cholesterol levels, we measured cellular cholesterol contents in Abcc4-deficient AML12 cells using ELISA method and found that ABCC4 barely exerted effect on cellular cholesterol contents (Fig. S2h).”
- Also stated in: § ABCC4 depletion increases the cell surface LDLR level independent of its gene expression · plain
“Consistently, the loss of Abcc4 did not influence SREBP2-dependent gene expression (Srebp2 and Hmgcr), as evidenced by RT-qPCR analysis (Fig. S2i).”
Quote in the paperSubstantial claim- Also stated in: § ABCC4 depletion increases the cell surface LDLR level independent of its gene expression · plain
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataReduced hepatic ABCC4 level did not significantly affect body weight in mice.
“Reduced hepatic ABCC4 level did not significantly affect body weight (Fig. S3b).”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataAbcc4 deletion primarily resulted in gene upregulation.
“A circos heatmap of the top 43 DEGs illustrated that Abcc4 deletion primarily resulted in gene upregulation (Fig. 5b).”
Quote in the paperNot a substantial claimIt may state two separate claims as one. The quote only partly states this claim.
Findingown dataCeefourin-1 treatment did not significantly alter ABCC4 protein expression levels in vitro or in vivo.
“In line with previous findings63, Ceefourin-1 treatment exerted little effect on ABCC4 protein expression levels (Fig. S4a and S4b).”
- Also stated in: § Pharmacologic inhibition of ABCC4 by Ceefourin-1 facilitates plasma LDL cholesterol clearance by enhancing surface LDLR protein expression on hepatocytes · plain
“Administration of Ceefourin-1 (10 mg/kg) or vehicle (DMSO) for 4 weeks did not significantly alter body weight (Fig. 4f) or hepatic ABCC4 protein expression levels (Fig. S4c and S4d) in male mice fed with either a normal chow diet (NCD) or high-fat diet (HFD).”
Quote in the paperNot a substantial claim- Also stated in: § Pharmacologic inhibition of ABCC4 by Ceefourin-1 facilitates plasma LDL cholesterol clearance by enhancing surface LDLR protein expression on hepatocytes · plain
It may state two separate claims as one.
Findingown dataBoth genetic ablation and pharmacological inhibition of hepatic ABCC4 elevate hepatic LDLR protein levels and reduce plasma LDL-C levels by approximately 40% in mice.
“Our findings demonstrate that both genetic ablation (Fig. 3) and pharmacological inhibition of hepatic ABCC4 (Fig. 4) effectively elevate hepatic LDLR protein levels and reduce plasma LDL-C levels by approximately 40% in mice.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAAV8_Abcc4_RNAi administration lowered hepatic Abcc4 mRNA by about 44.9% and protein by about 64.7% in wild-type C57BL/6 male mice.
“AAV8_Abcc4_RNAi administration in mice lowered hepatic Abcc4 mRNA expression levels by about 44.9% (Fig. 3b) and protein levels by about 64.7% (Fig. 3c, d).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataGenome-wide CRISPR screening in AML12 hepatocytes identified ABCC4 as a top-ranking negative regulator of LDLR surface expression.
“Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance.”
- Also stated in: § CRISPR screen identifies ABCC4 as a negative regulator of hepatic LDL receptor · hedged
“ABCC4, the top-ranking putative negative regulator of LDLR identified in our screens, is expressed in various tissues, including the liver, brain, kidney, colon, lung and placenta38,39.”
- Also stated in: § Introduction · plain
“Notably, we discovered ABCC4 as a promising hit gene governing LDLR surface abundance.”
Quote in the paperSubstantial claim- Also stated in: § CRISPR screen identifies ABCC4 as a negative regulator of hepatic LDL receptor · hedged
It may state two separate claims as one.
Findingown dataCeefourin-1 treatment improved glucose tolerance and insulin sensitivity in HFD-fed mice.
“Notably, Ceefourin-1 treatment improved glucose tolerance and insulin sensitivity in HFD-fed mice, as demonstrated by glucose tolerance tests (GTT) (Fig. S4f) and insulin tolerance tests (ITT) (Fig. S4g).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataLiver-specific Abcc4 knockdown increased hepatic membrane LDLR protein expression and decreased serum LDL-C levels without significant changes in serum total cholesterol and triglycerides in mice.
“But, it remarkably increased hepatic membrane LDLR protein expression and accordingly decreased serum LDL-C levels, without significant changes in serum total cholesterol (TC) and triglycerides (TG) levels (Fig. 3e-i).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAbcc4 deficiency alters cAMP distribution, thereby activating the intracellular cAMP signaling pathway.
“Regarding to the precise mechanistic explanation for ABCC4, our transcriptomic analysis and cAMP measurements revealed that Abcc4 deficiency altered cAMP distribution, thereby activating the intracellular cAMP signaling pathway.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataCeefourin-1 treatment did not significantly alter body weight in mice fed either NCD or HFD.
“Administration of Ceefourin-1 (10 mg/kg) or vehicle (DMSO) for 4 weeks did not significantly alter body weight (Fig. 4f) or hepatic ABCC4 protein expression levels (Fig. S4c and S4d) in male mice fed with either a normal chow diet (NCD) or high-fat diet (HFD).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataThe CRISPR screen identified previously reported modulators (UROD, CHP1, FASN, TMEM251, CDC42, REPS2) as well as several novel candidate negative regulators of LDLR.
“Notably, we discovered previously reported modulators (UROD31, CHP131, FASN32, TMEM25133, CDC4234,35, REPS236,37), as well as several novel candidate negative regulators of LDLR.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataAbcc4/ABCC4 knockout exerts a regulatory effect on surface LDLR abundance in hepatocytes, promoting LDL uptake.
“Abcc4/ABCC4 knockout exerted a regulatory effect on surface LDLR abundance in hepatocytes, thereby promoting LDL uptake.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Findingown dataThere was a significant decrease in the number of detected sgRNAs in the LDLRhigh subpopulation, indicating a strong selection bias.
“There was a significant decrease in the number of detected sgRNAs in the LDLRhigh subpopulation, indicating a strong selection bias (Table S1 and S2).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataABCC4 emerged as the leading candidate, with its sgRNAs highly enriched in the LDLRhigh subpopulation.
“Among these, ABCC4 emerged as the leading candidate, with its sgRNAs highly enriched in the LDLRhigh subpopulation.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataIn HFD-fed mice, ABCC4 inhibition (Ceefourin-1) reduces liver TC levels and improves hepatic insulin sensitivity/glucose homeostasis.
“Intriguingly, we observed that Ceefourin-1 treatment greatly reduced liver TC levels, improved hepatic insulin sensitivity, and ameliorated glucose homeostasis dysregulation in HFD-fed mice.”
- Also stated in: § Discussion · plain
“However, we observed that ABCC4 inhibition also significantly reduced liver TC levels and improved hepatic insulin resistance in HFD-fed mice.”
Quote in the paperNot a substantial claim- Rests on an assumption: Ceefourin-1 specifically inhibits ABCC4.
- Also stated in: § Discussion · plain
It may state two separate claims as one.
Findingown dataGlucagon treatment significantly reduces both intracellular and secreted PCSK9 levels.
“In follow-up experiments, we indeed found that glucagon (Glu) treatment significantly reduced both intracellular and secreted PCSK9 levels, mimicking the effect of Ceefourin-1.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataDouble-knockout of Abcc4/Epac2 and Abcc4/Rap1a in AML12 cells decreases membrane LDLR protein levels and increases PCSK9 protein levels.
“To gain deeper exploration of ABCC4-cAMP-Epac2/Rap1a signaling in regulating LDLR surface abundance, double-knockout of Abcc4/Epac2 and Abcc4/Rap1a in AML12 cells led to a decrease in membrane LDLR protein levels and an increase in PCSK9 protein levels.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataHepatic-specific disruption of Abcc4 in mice elevates membrane LDLR protein levels in the liver and reduces plasma LDL-C levels.
“Additionally, we showed that hepatic-specific disruption of Abcc4 in mice similarly elevated membrane LDLR protein levels in the liver and reduced plasma LDL-C levels.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAbcc4/Epac2 DKO cells exhibit impaired LDL uptake, in contrast to enhanced uptake in Abcc4-deficient cells.
“Furthermore, when incubated with Dil-LDL particles, Abcc4/Epac2 DKO cells exhibited impaired LDL uptake, in contrast to the enhanced uptake seen in Abcc4-deficient cells (Fig. 6c, d).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Findingown dataCeefourin-1 treatment decreases PCSK9 protein levels in AML12 cells.
“Importantly, Ceefourin-1 treatment recapitulated these effects on PCSK9 protein, with no significant changes in Pcsk9 transcript levels (Fig. 5m–p).”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataABCC4 inhibition increases hepatic membrane LDLR expression and lowers plasma LDL-C levels.
“Accompanied by two individual approaches to silence ABCC4 function in vivo, we further reinforced the idea that ABCC4 inhibition increased hepatic membrane LDLR expression and lowered plasma LDL-C levels.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Findingown dataLDLR, SREBF2, and MBTPS1 were identified as positive hits in the screening data, though not high ranking or statistically significant.
“In addition to novel modifiers, LDLR53,55, SREBF280 and MBTPS183 (membrane-bound transcription factor peptidase, site 1) were identified as positive hits in our screening data, though they did not exhibit high ranking or statistical significance.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Findingown dataAbcc4 deficiency in hepatocytes enhances LDL uptake and accelerates its lysosomal degradation without affecting sterol sensing and cholesterol levels.
“Building on our earlier findings that Abcc4 deficiency in hepatocytes enhances LDL uptake and accelerates its lysosomal degradation, without affecting sterol sensing and cholesterol levels (Fig. 2g–j; Figs. S2c, S2f, S2h-S2j)”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataGenetic deletion of Abcc4/ABCC4 enhances cell surface LDLR protein expression and LDL uptake without affecting sterol sensing, cholesterol synthesis, or LDLR mRNA levels.
“In our study, genetic deletion of Abcc4/ABCC4 enhanced cell surface LDLR protein expression and LDL uptake without affecting sterol sensing, cholesterol synthesis or LDLR mRNA levels.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataIn animal models, both liver-specific disruption and pharmacological inhibition of ABCC4 decrease hepatic PCSK9 protein expression and plasma PCSK9 levels.
“In animal models, both liver-specific disruption and pharmacological inhibition of ABCC4 significantly resulted in decreased hepatic PCSK9 protein expressions and lower plasma PCSK9 levels compared to the control group (Fig. S7a-S7e).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Findingown dataAbcc4-deficient cells have elevated intracellular and reduced extracellular cAMP levels.
“These findings were further validated using ELISA, which demonstrated elevated intracellular and reduced extracellular cAMP levels in Abcc4-deficient cells (Fig. 5g).”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown dataABCC4 silencing alters intracellular cAMP distribution and activates Epac2/Rap1a signaling, which blocks PCSK9 protein expression and prevents lysosomal degradation of LDLR.
“As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR.”
- Also stated in: § Introduction · plain
“Functioning as a hepatic efflux transporter of cyclic AMP (cAMP), ABCC4 deficiency led to intracellular accumulation of cAMP, thereby activating the downstream sensor Epac2. Activated Epac2 subsequently stimulates the small GTPase Rap1 and further suppresses PCSK9 protein expression, thus enhancing LDLR surface availability by preventing its lysosomal degradation.”
Quote in the paperSubstantial claim- Also stated in: § Introduction · plain
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationAfter internalization, the acidic endosome induces conformational changes in LDLR, allowing dissociation from LDL; LDLR recycles to the hepatocyte surface while LDL cholesterol is trafficked to lysosomes for degradation.
“Following internalization, the acidic environment of the endosome induces conformational changes in LDLR, allowing it to dissociate from LDL particles. LDLR is then recycled back to the hepatocyte surface, while LDL cholesterol is trafficked to lysosomes for degradation24–27.”
Quote in the paperNot a substantial claim0 of 4 sources checked- cites [24] Beglova, N. & Blacklow, S. C. The LDL receptor: how acid pulls the trigger. Trends Biochem Sci. 30, 309–317 (2005). source
- cites [25] Ridsdale, A. et al. Cholesterol is required for efficient endoplasmic reticulum-to-Golgi transport of secretory membrane proteins. Mol. Biol. Cell 17, 1593–1605 (2006). source
- cites [26] Brown, M. S., Dana, S. E. & Goldstein, J. L. Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein. Proc. Natl Acad. Sci. USA 72, 2925–2929 (1975). source
- cites [27] He, Y. et al. Neutrophil-to-hepatocyte communication via LDLR-dependent miR-223-enriched extracellular vesicle transfer ameliorates nonalcoholic steatohepatitis. J. Clin. Invest 131, e141513 (2021). source
Checked before — its earlier verdict is held back until a new check runs.
It may state two separate claims as one.
Interpretationown dataBlocking ABCC4 function by a specific inhibitor exerts a protective effect on lipid metabolism by enhancing LDLR availability and mitigating hypercholesterolemia.
“Altogether, blocking ABCC4 function by a specific inhibitor exerts a protective effect on lipid metabolism by enhancing LDLR availability and mitigating hypercholesterolemia, highlighting ABCC4 as a promising therapeutic target.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationLDL-C is a key driver of atherosclerosis by stimulating smooth muscle cell proliferation and progressive luminal narrowing, contributing to plaque expansion.
“Also, LDL-C is a key driver of atherosclerosis by stimulating smooth muscle cell (SMC) proliferation and progressive luminal narrowing, ultimately contributing to plaque expansion6,7.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [6] Chen, Y. et al. Probucol and cilostazol exert a combinatorial anti-atherogenic effect in cholesterol-fed rabbits. Thrombosis Res. 132, 565–571 (2013). source
- cites [7] Chattopadhyay, A. et al. Cholesterol-induced phenotypic modulation of smooth muscle cells to macrophage/fibroblast-like cells is driven by an unfolded protein response. Arterioscler Thromb. Vasc. Biol. 41, 302–316 (2012). source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationIDOL mediates ubiquitination of hepatic LDLR and is a promising therapeutic target for cardiovascular disease.
“Moreover, the inducible degrader of LDLR (IDOL), an E3 ubiquitin ligase that mediates the ubiquitination of hepatic LDLR16,17, has emerged as a promising therapeutic target for cardiovascular disease, similar to PCSK9.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [16] Sorrentino, V. & Zelcer, N. Post-transcriptional regulation of lipoprotein receptors by the E3-ubiquitin ligase inducible degrader of the low-density lipoprotein receptor. Curr. Opin. Lipido. 23, 213–219 (2012). source
- cites [17] Sorrentino, V. et al. Distinct functional domains contribute to degradation of the low density lipoprotein receptor (LDLR) by the E3 ubiquitin ligase inducible Degrader of the LDLR (IDOL). J. Biol. Chem. 286, 30190–30199 (2011). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationRepatha decreases LDL-C levels by 50-70% and lowers the risk of myocardial infarction or stroke.
“Repatha, a marketed PCSK9 monoclonal antibody, has demonstrated the ability to rapidly decrease LDL-C levels by 50–70% and lower the risk of myocardial infarction or stroke in multiple completed and ongoing clinical trials11,12.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [11] Shimada, Y. J. & Cannon, C. P. PCSK9 (Proprotein convertase subtilisin/kexin type 9) inhibitors: past, present, and the future. Eur. heart J. 36, 2415–2424 (2015). source
- cites [12] Jamadade, P., Nupur, N., Maharana, K. C. & Singh, S. Therapeutic monoclonal antibodies for metabolic disorders: major advancements and future perspectives. Curr. Atherosclerosis Rep. 26, 549–571 (2024). source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationLowering serum LDL-C levels reduces the incidence of atherosclerotic disease.
“In recent years, there has been a growing appreciation for the benefit of lowering serum LDL-C levels to reduce the incidence of atherosclerotic disease8.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [8] Paquette, M. et al. Effect of the LDL receptor mutation type on incident major adverse cardiovascular events in familial hypercholesterolaemia. Eur. J. Prev. Cardiol. 29, 2125–2131 (2022). source
It may state two separate claims as one.
Interpretationown dataABCC4 blockade enhances hepatic LDLR surface expression and lipoprotein uptake independent of LDLR gene transcription.
“Taken together, these data indicate that ABCC4 blockade enhances hepatic LDLR surface expression and lipoprotein uptake independent of LDLR gene transcription.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenRepresentative images displayed in figures were selected from at least three biologically independent experiments.
“Representative images displayed in figures were selected from at least three biologically independent experiments.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Interpretationown dataABCC4 plays a critical role in lipid metabolism through cAMP-Epac2/Rap1a signaling, restraining hepatic PCSK9 protein levels, facilitating LDLR surface availability, and promoting clearance of circulating LDL-C.
“Collectively, our results highlight a critical role of ABCC4 in lipid metabolism through cAMP-Epac2/Rap1a signaling, which restrains hepatic PCSK9 protein levels, thereby facilitating LDLR surface availability and promoting clearance of circulating LDL-C.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationABCC4 augments platelet activation by facilitating cAMP efflux.
“Moreover, ABCC4 has been shown to augment the potential for platelet activation by facilitating cAMP efflux, which in turn intensifies cAMP-dependent signal transduction to the nucleus43,44.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [43] Bloch, M., Raj, I., Pape, T. & Taylor, N. M. I. Structural and mechanistic basis of substrate transport by the multidrug transporter MRP4. Structure 31, 1407–1418 (2023). source
Checked before — its earlier verdict is held back until a new check runs.
- cites [44] Belleville-Rolland, T. et al. Role of Membrane Lipid Rafts in MRP4 (ABCC4) dependent regulation of the cAMP pathway in blood platelets. Thromb. Haemost. 121, 1628–1636 (2021). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationABCC4 transports prostaglandins like PGE2 out of the cell, where they can bind to surface receptors and influence LDL-C levels.
“In addition to cAMP substrate, ABCC4 transports prostaglandins like PGE2, out of the cell, where they can bind to surface receptors and influence LDL-C levels47.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [47] Krone, W., Klass, A., Nägele, H., Behnke, B. & Greten, H. Effects of prostaglandins on LDL receptor activity and cholesterol synthesis in freshly isolated human mononuclear leukocytes. J. Lipid Res 29, 1663–1669 (1988). source
It may state two separate claims as one.
Interpretationown dataABCC4 is a key regulator of LDLR protein expression and cholesterol metabolism.
“These findings highlight ABCC4 as a key regulator of LDLR protein expression and cholesterol metabolism, and a promising therapeutic target for hyperlipidemia and the reduction of cardiovascular risk.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationCeefourin-1 inhibits cAMP efflux, thereby reducing platelet adhesion and thrombus formation.
“Ceefourin-1 has been shown to inhibit cAMP efflux, thereby reducing platelet adhesion and thrombus formation61.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [61] Wolf, R. et al. Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets. Haematologica 107, 2206–2217 (2022). source
Checked before — its earlier verdict is held back until a new check runs.
It may state two separate claims as one.
InterpretationreasoningActivation of cAMP signaling pathway due to Abcc4 deficiency suppresses hepatic PCSK9 protein levels, preventing LDLR lysosomal degradation and maintaining systemic LDL-C homeostasis.
“Activation of cAMP signaling pathway due to Abcc4 deficiency suppressed hepatic PCSK9 protein levels, thereby preventing LDLR lysosomal degradation and maintaining systematic LDL-C homeostasis.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationnone givenABCC4 regulates LDLR surface availability in hepatocytes dependent on cAMP-Epac2/Rap1a signaling, contributing to clearance of circulating LDL-C.
“In conclusion, this study provides novel insights into the role of ABCC4 in regulating LDLR surface availability in hepatocytes dependent on cAMP-Epac2/Rap1a signaling, which contributes to the clearance for circulating LDL-C.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown dataABCC4 plays a critical role in modulating PCSK9 levels and consequently influencing LDLR protein expression and LDL-C clearance.
“implicating that ABCC4 plays a critical role in modulating PCSK9 levels and, consequently, influencing LDLR protein expressions and LDL-C clearance.”
Quote in the paperSubstantial claimIt may state two separate claims as one.
Interpretationown dataABCC4 suppresses LDLR availability and inhibits the clearance of plasma LDL cholesterol.
“All the data presented in this study support the conclusion that ABCC4 suppresses LDLR availability and accordingly inhibits the clearance of plasma LDL cholesterol.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductreasoningThe screening failed to prioritize the effects of some canonical regulators.
“although our screening identified novel modifiers of surface LDLR protein expression, it failed to prioritize the effects of some canonical regulators.”
Quote in the paperNot a substantial claimIt may state two separate claims as one.
Study design and conductown dataTwo independent biological replicates of the CRISPR screen demonstrated strong concordance and highlighted top hits that enhanced LDLR surface levels in hepatocytes.
“Two independent biological replicates of this screen demonstrated strong concordance, consistently highlighting top hits that enhanced LDLR surface levels in hepatocytes.”
Quote in the paperSubstantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationABCC4 is localized to the basolateral membrane of hepatocytes, where it transports conjugated substrates into the bloodstream.
“Subcellularly, ABCC4 is localized to the basolateral membrane of hepatocytes, also referred to as the sinusoidal surface, where it transports several conjugated substrates into the bloodstream40–42.”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [40] Rius, M., Nies, A. T., Hummel-Eisenbeiss, J., Jedlitschky, G. & Keppler, D. Cotransport of reduced glutathione with bile salts by MRP4 (ABCC4) localized to the basolateral hepatocyte membrane. Hepatology 38, 374–384 (2003). source
Checked before — its earlier verdict is held back until a new check runs.
- cites [41] Denk, G. U. et al. Multidrug resistance-associated protein 4 is up-regulated in liver but down-regulated in kidney in obstructive cholestasis in the rat. J. Hepatol. 40, 585–591 (2004). source
- cites [42] Russel, F. G., Koenderink, J. B. & Masereeuw, R. Multidrug resistance protein 4 (MRP4/ABCC4): a versatile efflux transporter for drugs and signalling molecules. Trends Pharm. Sci. 29, 200–207 (2008). source
Probably not a substantial claim. The paper’s conclusions may not depend on it.
Study design and conductnone givenThe screening focused primarily on genes driving substantial transcriptional changes under homeostatic conditions.
“Moreover, our screening focused primarily on genes driving substantial transcriptional changes under homeostatic conditions, whereas post-transcriptional regulation also plays an important role in determining protein levels.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
PremisecitationABCC4 is highly expressed in the liver and localized to the basolateral hepatocyte membrane, where it mediates transport of molecules into the bloodstream.
“ABCC4 is highly expressed in the liver and is localized to the basolateral hepatocyte membrane, where it mediates the transport of molecules into the bloodstream51,89,90.”
Quote in the paperNot a substantial claim0 of 3 sources checked- cites [51] Rius, M., Hummel-Eisenbeiss, J., Hofmann, A. F. & Keppler, D. Substrate specificity of human ABCC4 (MRP4)-mediated cotransport of bile acids and reduced glutathione. Am. J. Physiol. Gastrointest. Liver Physiol. 290, G640–G649 (2006). source
- cites [89] Keppler, D. Progress in the molecular characterization of hepatobiliary transporters. Dig. Dis. 35, 197–202 (2017). source
- cites [90] Kurzawski, M. et al. Expression of genes involved in xenobiotic metabolism and transport in end-stage liver disease: up-regulation of ABCC4 and CYP1B1. Pharm. Rep. 64, 927–939 (2012). source
Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.
Study design and conductnone givenRNA quality and integrity was assessed with a Bioanalyzer 2100 (RIN > 7.0) and confirmed by denaturing agarose gel electrophoresis.
“RNA quality and integrity was assessed with a Bioanalyzer 2100 (Agilent, CA, USA; RIN > 7.0) and confirmed by denaturing agarose gel electrophoresis.”
Quote in the paperNot a substantial claimProbably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationABCC4 is expressed in various tissues, including liver, brain, kidney, colon, lung and placenta.
“ABCC4, the top-ranking putative negative regulator of LDLR identified in our screens, is expressed in various tissues, including the liver, brain, kidney, colon, lung and placenta38,39.”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [38] Schuetz, E. G. et al. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J. Biol. Chem. 276, 39411–39418 (2001). source
Checked before — its earlier verdict is held back until a new check runs.
- cites [39] Zelcer, N. et al. Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATP-binding cassette C4). Biochem J. 371, 361–367 (2003). source
Checked before — its earlier verdict is held back until a new check runs.
Probably not a substantial claim. The paper’s conclusions may not depend on it.
PremisecitationLDLR abundance on hepatocyte membranes is inversely correlated with circulating LDL-C levels in both mice and humans.
“This observation aligns with prior findings that LDLR abundance on hepatocyte membranes is inversely correlated with circulating LDL-C levels in both mice and humans58.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [58] Vincent, J. Reducing elevated plasma LDL cholesterol: the central role of the LDL receptor. Clin. Pharm. Ther. 96, 3–7 (2014). source
The paper relies on this without support.
Ceefourin-1 specifically inhibits ABCC4.
“Pharmacological inhibition of ABCC4 using the specific inhibitor Ceefourin-1 replicated these effects under both NCD and HFD conditions in vivo.”
The paper relies on this without support.
Recombinant human PCSK9 protein (rhPCSK9) is functional and mimics endogenous PCSK9.
“we incubated AML12 cells with recombinant human PCSK9 protein (rhPCSK9; 500 ng/ml).”
The paper relies on this without support.
Cycloheximide treatment blocks protein synthesis and the observed reduction in PCSK9 reflects increased degradation.
“we treated cells with cycloheximide (30 min and 90 min) to block protein synthesis.”
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Figure legends use a different significance threshold from Methods
MinorThe Fig. 4, 5, and 6 legends define one star as P ≤ 0.5, whereas Methods defines statistical significance as P < 0.05. The stated one-star threshold would include results the paper otherwise treats as nonsignificant.
“*P ≤ 0.5, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: no significance.”
“Statistical significance was defined ad P values < 0.05.”
LDL uptake duration differs between Methods and figure legends
MinorThe Dil-LDL uptake method specifies three hours at 37 °C, but figure legends describe the uptake assay as one hour. The text does not distinguish these as separate uptake protocols.
“The cells were then washed three times with ice-chilled PBS and shifted to 37 °C for 3 hours to enable uptake.”
“c Flow cytometry analysis of Dil-LDL uptake assay (1 h) in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Epac2 sgRNA).”
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Metadata
- Authors
- Chen, J., Huang, H., Chen, C., Xia, G., Huang, H., Xiong, Y., Luo, P., Chen, Y., Li, J., Wen, L., Li, L., Lin, J., Xu, G., Ji, C., Tian, W., Zhou, J., Wei, P., Shen, C., & Wang, X.
- Affiliations
- University of Electronic Science and Technology of China (CN)
- Shanghai Sixth People's Hospital (CN)
- Sichuan University (CN)
- Shanghai Jiao Tong University (CN)
- Corresponding author
- Zhou, Jin
- Wei, Peng
- Shen, Chengxing
- Wang, Xiaoqing
- ORCID
- Venue
- Communications Biology
- Published
- 2025-10-02
- Volume
- 8
- Issue
- 1
- Pages
- 1414-1414
- ISSN
- 2399-3642 (ISSN-L)
- PMC
- PMC12491593
- OpenAlex
- W4414748510
- Cited by
- 1
- References
- 100
- Citations by year
- 2025: 1
- FWCI
- 0.32
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Communications Biology (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion, cc-by)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- National Natural Science Foundation of China (8217051361)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
- Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipoproteins and Cardiovascular Health
- Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipid metabolism and disorders
- Keywords
- ABCC4, LDL receptor, hypercholesterolemia, atherosclerosis, cAMP efflux, PCSK9, lysosomal degradation, hepatocytes, CRISPR-Cas9 screen
- MeSH terms
- Proprotein Convertase 9, Animals, Humans, Hypercholesterolemia, Cholesterol, LDL, Liver, Male, Mice, Inbred C57BL, Receptors, LDL, Signal Transduction, Hepatocytes, ATP-Binding Cassette, Sub-Family C Proteins, Mice, Hep G2 Cells, CRISPR-Cas Systems
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Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
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