# From Genetic Findings to new Intestinal Molecular Targets in Lipid Metabolism

Cédric Le May, Simon Ducheix, Bertrand Cariou, Antoine Rimbert

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Cédric Le May, Simon Ducheix, Bertrand Cariou, Antoine Rimbert. From Genetic Findings to new Intestinal Molecular Targets in Lipid Metabolism. Current Atherosclerosis Reports, 2025, 27 (1), pp.26. 10.1007/s11883-024-01264-w . hal-05361443

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<table><tr><td>ArticleTitle</td><td colspan="2">From Genetic Findings to new Intestinal Molecular Targets in Lipid Metabolism</td></tr><tr><td>Article Sub-Title</td><td colspan="2"></td></tr><tr><td>Article CopyRight</td><td colspan="2">The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature<br/>(This will be the copyright line in the final PDF)</td></tr><tr><td>Journal Name</td><td colspan="2">Current Atherosclerosis Reports</td></tr><tr><td>Corresponding Author</td><td>FamilyName<br/>Particle<br/>Given Name<br/>Suffix<br/>Division<br/>Organization<br/>Address<br/>Phone<br/>Fax<br/>Email<br/>URL<br/>ORCID</td><td><b>Rimbert</b><br/><br/><b>Antoine</b><br/><br/>CHU Nantes, CNRS, Inserm<br/>Nantes Université, l'institut du thorax<br/>F-44000, Nantes, France<br/><br/>antoine.rimbert@univ-nantes.fr</td></tr><tr><td>Author</td><td>FamilyName<br/>Particle<br/>Given Name<br/>Suffix<br/>Division<br/>Organization<br/>Address<br/>Phone<br/>Fax<br/>Email<br/>URL<br/>ORCID</td><td><b>May</b><br/><br/><b>Cédric Le</b><br/><br/>CHU Nantes, CNRS, Inserm<br/>Nantes Université, l'institut du thorax<br/>F-44000, Nantes, France</td></tr><tr><td>Author</td><td>FamilyName<br/>Particle<br/>Given Name<br/>Suffix<br/>Division<br/>Organization<br/>Address<br/>Phone<br/>Fax<br/>Email<br/>URL<br/>ORCID</td><td><b>Ducheix</b><br/><br/><b>Simon</b><br/><br/>CHU Nantes, CNRS, Inserm<br/>Nantes Université, l'institut du thorax<br/>F-44000, Nantes, France</td></tr><tr><td>Author</td><td>FamilyName<br/>Particle<br/>Given Name<br/>Suffix<br/>Division</td><td><b>Cariou</b><br/><br/><b>Bertrand</b><br/>CHU Nantes, CNRS, Inserm</td></tr></table>
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Abstract

Purpose of Review:

While lipid-lowering therapies demonstrate efficacy, many patients still contend with significant residual risk of atherosclerotic cardiovascular diseases (ASCVD). The intestine plays a pivotal role in regulating circulating lipoproteins levels, thereby exerting influence on ASCVD pathogenesis. This review underscores recent genetic findings from the last six years that delineate new biological pathways and actors in the intestine which regulate lipid-related ASCVD risk.

Recent Findings:

Specifically, we detail the role of LIMA1 in cholesterol absorption within enterocytes, the function of PLA2G12B in the expansion and lipidation of chylomicrons, the involvement of SURF4 in lipoprotein secretion, and the discovery of a gut-derived hormone named CHOLESMAN that modulates cholesterol homeostasis through GPR146 via a gut-liver crosstalk. We further discuss the potential of these newly identified genes and pathways as novel targets for pharmaceutical intervention.

Summary:

Newly identified genetic and intestinal molecular mechanisms offer promising opportunities for preventing and treating ASCVD, but careful evaluation and further research are needed to optimize their clinical application.

Keywords (separated by ' - ') Lipid metabolism - Atherosclerotic cardiovascular diseases - Intestine - Genetics - Therapeutic targets

Footnote Information
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REVIEW

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# From Genetic Findings to new Intestinal Molecular Targets in Lipid Metabolism

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Cédric Le May¹ · Simon Ducheix¹ · Bertrand Cariou¹ · Antoine Rimbert¹

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Accepted: 3 December 2024

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© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024

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## Abstract

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**Purpose of Review** While lipid-lowering therapies demonstrate efficacy, many patients still contend with significant residual risk of atherosclerotic cardiovascular diseases (ASCVD). The intestine plays a pivotal role in regulating circulating lipoproteins levels, thereby exerting influence on ASCVD pathogenesis. This review underscores recent genetic findings from the last six years that delineate new biological pathways and actors in the intestine which regulate lipid-related ASCVD risk.

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**Recent Findings** Specifically, we detail the role of LIMA1 in cholesterol absorption within enterocytes, the function of PLA2G12B in the expansion and lipidation of chylomicrons, the involvement of SURF4 in lipoprotein secretion, and the discovery of a gut-derived hormone named CHOLESNIN that modulates cholesterol homeostasis through GPR146 *via* a gut-liver crosstalk. We further discuss the potential of these newly identified genes and pathways as novel targets for pharmaceutical intervention.

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**Keywords** Lipid metabolism · Atherosclerotic cardiovascular diseases · Intestine · Genetics · Therapeutic targets

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## Introduction

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Despite significant reductions in atherosclerotic cardiovascular diseases (ASCVD) over recent decades, ASCVD remains the leading global cause of death worldwide [1]. Dyslipidemia, characterized by abnormal plasma lipid levels, is a major risk factor for ASCVD. Elevated low-density lipoprotein cholesterol (LDL-c) in plasma is the primary modifiable risk factor and target for ASCVD management [2, 3]. Although effective drugs are available and recent additions have expanded the therapeutic options for lowering LDL-c, many patients still experience substantial ‘residual’ lipid-related ASCVD-risk even after achieving LDL-c lowering goals [4]. This indicates a need for novel lipid-lowering therapies to further reduce ASCVD risk [5]. Recent evidences have highlighted the role of elevated triglyceride-rich lipoproteins (TRLs) in ASCVD risk [6]. This residual risk is particularly evident in patients with

metabolic syndrome, characterized by visceral obesity, insulin resistance, and combined dyslipidemia, who present with elevated circulating TRLs levels [6, 7]. TRLs include liver-derived very low-density lipoproteins (VLDL) containing Apolipoprotein-B-100 (ApoB100), and intestinal-derived chylomicrons containing Apolipoprotein-B-48 (ApoB48). It is the cholesterol component of TRLs, known as remnant cholesterol (RC), rather than triglycerides, that increases the risk of ASCVD [8].

Cholesterol in the body originates either from *de novo* biosynthesis, primarily in the liver, or from dietary absorption in the intestines. Enterocytes absorb cholesterol from the food bolus and subsequently secrete it into the lymphatic/blood circulation as chylomicrons (described in Fig. 1). Cholesterol can then be excreted *via* the hepatobiliary pathway, where it may be reabsorbed by the intestine or eliminated in feces, or *via* transintestinal cholesterol excretion (TICE), where the intestine directly secretes cholesterol into the intestinal lumen for fecal elimination (Fig. 1).

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Higher dietary cholesterol intake is significantly associated with an increased risk of ASCVD [9]. Additionally, increased secretion of chylomicrons, which is twice as high

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Antoine Rimbert

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antoine.rimbert@univ-nantes.fr

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CHU Nantes, CNRS, Inserm, Nantes Université, l'institut du thorax, F-44000 Nantes, France

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**Fig. 1** In enterocytes, Niemann-Pick C1 like 1 (NPC1L1), is required for intestinal cholesterol absorption [27]. After emulsification of dietary lipids by bile salts NPC1L1 takes up dietary cholesterol through vesicular endocytosis [28]. The initiation of NPC1L1 endocytosis starts with the formation of microdomains composed of NPC1L1 and flotillins [29]. The generation of clathrin-coated vesicles is permitted by the NUMB protein [30]. The vesicular trafficking of NPC1L1 carries a large amount of cholesterol to the endocytic recycling compartment (ERC). When the cholesterol level in ERC drops, NPC1L1 interacts with LIMA1 and is recycled back to the plasma membrane via the association with myosinVb and the actin cytoskeleton [31]. After being taken up by enterocytes, cholesterol is re-esterified by the acetyl-CoA acetyltransferase 2 (ACAT2) [32, 33]. Recently, Aster-B and C have been shown to be responsible for direct transport of cholesterol from ERC to endoplasmic reticulum (ER) [34], a mechanism which is facilitated by the intervention of bile acids [35]. In the ER lipids are incorporated into ApoB48 containing chylomicrons. Microsomal triglyceride transfer protein (MTP) facilitates the co-translational lipidation of ApoB48 in the endoplasmic reticulum (ER) together with PLA2G12B [36], producing dense ApoB48 containing lipoprotein particles [37]. These particles then merge with ApoB48-free lipid droplets in the ER lumen to form pre-chylomicrons (pre-CMs) together with ApoAIV [38]. Pre-CMs are too large to exit the ER via classic coat protein complex II (COPII) vesicles. Instead, specialized pre-CM transport vesicles (PCTV) transport pre-CMs from the ER to the Golgi apparatus, where they are processed into mature chylomicrons and subsequently released from the basolateral side of enterocytes. The transit of pre-chylomicron from the ER to the Golgi is the rate limiting governing the movement of absorbed lipids across

the enterocytes to the lymph [39]. The process of PCTV budding from the ER is facilitated by the proteins Vesicle-associated membrane protein 7, CD36 and Fatty Acid-Binding Protein 1 (FABP1) [39, 40]. FABP1 is released from a multiprotein cytosolic complex via the phosphorylation of the secretion-associated RAS-related GTPase 1B (SAR1B) [41]. Homozygous variants in the SAR1B gene have been linked to the Chylomicron retention disease, also called the Anderson's disease (OMIM#246700) [42]. The protein SURF4 has been found to interact with SAR1B and that together are involved in pre-chylomicrons' trafficking [43, 44]. After PTCV budding, the protein complex SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) facilitates the targeting and fusion of a specific vesicle with the Golgi membrane [45]. Little is known about the exit of chylomicrons from the Golgi, but the vesicles fuse with the basolateral membrane, and the chylomicrons secreted are released into the lymph [46]. Reverse cholesterol transport (RCT) is generally defined as the efflux of cholesterol from peripheral tissues, transportation to the liver and subsequent hepatobiliary secretion into the feces. However, a second pathway contributing to fecal cholesterol excretion has also been implicated in RCT: the direct transintestinal cholesterol excretion (TICE) of plasma-derived cholesterol by enterocytes into the lumen of the small intestine. In this process, plasma cholesterol is taken-up from lipoproteins at the basolateral membrane of enterocytes via lipoprotein receptors (mechanism not fully defined) and secreted into the intestinal lumen via the heterodimer, ATP binding cassette subfamily G member (ABCG) 5 and 8 and the transporter, ATP binding cassette subfamily G member (ABCB) 1, at the apical side of the enterocytes [13, 14].

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in patients with diabetes, significantly contributes to residual ASCVD risk [10].

Postprandial lipid uptake primarily occurs in the intestinal lumen, where lipids are emulsified by bile acids and hydrolyzed by various lipases. Most absorbed lipids are then esterified and packaged into chylomicrons in the enterocytes. These chylomicrons are released from the basolateral side of enterocytes, transported to the lymphatic system, and enter the circulation through the thoracic duct to be delivered to peripheral tissues (described in Fig. 1 legend). The absorption of dietary lipids, their secretion into the bloodstream, and their elimination in the intestine are regulated by various mechanisms as depicted and described in Fig. 1 and recently reviewed by [11, 12]. In addition to its role in regulating

cholesterol absorption, the intestine also plays a major role in its elimination. In particular, it is involved in the hepatobiliary cholesterol excretion pathway and the enterohepatic bile acid cycle, cholesterol can also be eliminated directly by the intestine *via* the transintestinal cholesterol efflux pathway (TICE) through enterocytes [13, 14].

Given its pivotal role in the transcriptional regulation of above-mentioned intestinal pathways, the nuclear liver X receptor (LXR) has emerged as a prime target for cholesterol level control and atherosclerosis treatment. Intestinal LXR negatively regulates the expression of NPC1L1 [15] and ACAT2 [16] and positively induces ABCG5/G8 expression [17], thereby limiting intestinal absorption. Moreover, intestinal LXR promotes reverse cholesterol transport (RCT) by

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inducing intestinal and hepatic pre-beta HDL secretion *via* increased ABCA1 expression [16, 18, 19], and the non-biliary cholesterol excretion pathway or TICE (transintestinal cholesterol efflux) [20]. However, the clinical use of LXR agonists to regulate cholesterol homeostasis has been hampered by the fact that pharmacological activation of LXR also promotes hepatic lipogenesis, resulting in steatosis and hypertriglyceridemia [21, 22]. While tissue-specific or partial LXR agonists have shown promising results [23, 24], the path to clinical application remains challenging. Consequently, there is a pressing need to identify novel intestinal players able to regulate cholesterol levels, for which genetic approaches are particularly well-suited.

The identification of genetic factors involved in dyslipidemias has in fact greatly advanced our understanding of the genetic architecture and metabolism of circulating lipids. Discovering the genetic origins of familial dyslipidemias has led to major scientific breakthroughs and provided key targets for pharmaceutical intervention against ASCVD [25]. Additionally, genome-wide association studies (GWAS) have identified many independent variants associated with plasma lipid levels in the general population [26]. The majority of these variants, approximately 85%, are located in non-coding regions, making it challenging to identify causally related genes and biological mechanisms. However, advancements in technology and methodology, such as denser genotyping arrays, widespread use of next-generation sequencing, integration of genetic, epigenetic, transcriptomic, and proteomic tissue-specific data, and improved imputation methods, along with multi-ancestry studies, have refined associated loci and identified novel genes involved in regulating plasma lipid levels. Overall, genetic findings have significantly contributed to identifying biological mechanisms involved in intestinal lipid metabolism and potential therapeutic targets for lipid-related ASCVD.

This review focuses on recent genetic findings of the genes *LIMA1*, *PLA2G12B*, *SURF4*, and *C7ORF50*, which were recently discovered to play a role in the regulation of plasma cholesterol levels. We describe the new insights provided by these key studies and discuss the potential of the newly identified targets for pharmaceutical intervention to lower lipid levels and ASCVD risk (blue circles in Fig. 1).

## Genetic Variants Associated with Cholesterol Absorption and the Processes of Endocytic Recycling

In 2018, Zhang et al. conducted a Cardiovascular Risk survey in western China and discovered a three-generation Chinese Kazakh family with inherited low levels of total and LDL-c in plasma [31]. Through whole exome sequencing, they identified a rare variant, *LIMA1*-p.K306fs, which demonstrated autosomal dominant transmission and co-segregated

with hypocholesterolemia in the family. Additionally, three other families were found to carry the *LIMA1*-p.L25I variant, with carriers also exhibiting hypocholesterolemia.

In the initial family, carriers of the *LIMA1* variant exhibited reduced cholesterol absorption. The *LIMA1* gene encodes the LIM Domain and Actin Binding 1 protein, which is primarily expressed in the small intestine and moderately in the liver. Intracellularly, it is located on the brush border of enterocytes. Consistent with human observations, the study showed that intestinal-specific (*I-LIMA1*⁻¹) or full-body knockout mice exhibited hypocholesterolemia and impaired dietary cholesterol absorption. When subjected to a high cholesterol diet, *I-LIMA1*⁻¹ mice showed a 29% reduction in plasma cholesterol and a 58% reduction in enterocytes cholesterol content.

As depicted in Fig. 1, NPC1L1 (Niemann-Pick C1 like 1) is responsible for approximately 70% of cholesterol absorption in mice and 54% in humans [27]. Notably, patients with variants at the glycosylation sites exhibit defects in cholesterol absorption, which provides protection against ASCVD [47]. Furthermore, the pharmacological inhibition of NPC1L1 using Ezetimibe lowers LDL-c levels and improves cardiovascular outcomes, as demonstrated in the IMPROVE-IT trial (NCT00202878) [48]. As for *Npc1l1* deficient mice, *I-LIMA1*⁻¹ mice harbor an increased hepatic cholesterol synthesis [27]. Notably, *I-LIMA1*⁻¹ mice treated with ezetimibe did not show a further reduction in cholesterol absorption compared to controls, and their absorption capacity was comparable to *Npc1l1*⁻¹ mice. These findings indicate that the effects of LIMA1 depletion on intestinal cholesterol absorption are dependent on NPC1L1. Further, the authors demonstrated that LIMA1, bridges NPC1L1 and the myosinVb and that this triplex is involved in the internalization of NPC1L1 in the endocytic recycling compartment and its transportation at the plasma membrane (Fig. 1).

Recently, it has been demonstrated that the E3 ubiquitin ligase RNF40 may directly target LIMA1, promoting its degradation and thereby suppress LIMA1-mediated lipid accumulation in liver cells [49]. This study, along with another showing a connection between LIMA1-enriched liver-derived extracellular vesicles and the development of liver fibrosis in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) [50], supports the involvement of LIMA1 in liver pathophysiology.

Beyond offering significant biological insights, this study underscores the value of conducting genetic research on populations with non-European ancestry, to uncover new genetic origins of dyslipidemia and provide key information for a proper personalized medical care [51, 52]. As far as we know, no other genetic variants were identified in familial forms of hypocholesterolemia in the *LIMA1* gene worldwide. This led the Medical Advisory Panel of the Abetalipoproteinemia and Related Disorders Foundation to not report

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LIMA1 as a genes responsible for familial forms of familial hypocholesterolemia [53].

Taken together, one could think that LIMA1 may not be an ideal therapeutic target since it relies on NPC1L1, which is already targeted by ezetimibe. Also, the modulation of cytoskeleton dynamics plays a pivotal role in several hallmark processes of cancer such as tumor growth invasion, metastasis and treatment resistance [54]. Via its role in cytoskeleton regulation, LIMA1-downregulation has been associated with the progression of several cancers while overexpression decreases tumor growth and invasiveness [55, 56], reviewed by [57].

## PLA2G12B: A Novel Participant in Chylomicrons Lipidation

Several common genetic variants in the locus 10q22.1 have been linked to plasma lipid levels, including triglycerides, total, LDL, and HDL-cholesterol, as well as coronary artery disease [58, 59] (Table 1). These variants are located in intronic regions or near the gene PLA2G12B, in a variety of open chromatin marks and predicted transcription factor binding sites [36]. PLA2G12B encodes the Phospholipase A2 group 12B which is primarily expressed in the small intestine and the liver. In 2019, Thierer et al. demonstrated in a zebrafish model that the invalidation of the PLA2G12B ortholog gene resulted in a significant accumulation of small TRLs and a depletion of larger particle classes in the plasma [60].

More recently, studies in zebrafish and mice have shown that PLA2G12B is involved in TRL expansion [36]. The authors found in zebrafish, that PLA2G12B acts downstream of Microsomal triglyceride transfer protein (MTP) in the TRLs biogenesis pathway without affecting their secretion (Fig. 1). Accordingly, the silencing of PLA2G12B in zebrafish led to intra-cellular lipid accumulation in the liver and intestine. Notably, zebrafish lacks ApoB48, making it difficult to distinguish the liver or intestine-specific actions

of PLA2G12B. Further, in vitro approaches using HepG2 and Caco2 liver and intestinal cell lines demonstrated that TRL production (VLDL and chylomicrons) is impaired when PLA2G12B is inactivated.

These findings suggest that PLA2G12B is a key mediator of lipid partitioning between nascent TRLs and luminal lipid droplets in both fish and humans. Moreover, PLA2G12B is retained intracellularly and closely associates with endoplasmic reticulum (ER) membranes. In mice, genetic inhibition of PLA2G12B led to a significant decrease in VLDL secretion, with these particles showing over an 80% reduction in triglyceride content. Finally, in a murine model of atherosclerosis, genetic inhibition of PLA2G12B resulted in profound hypolipidemia and resistance to atherosclerosis [36].

In conclusion, while targeting PLA2G12B shows significant potential in addressing cardiovascular pathologies via a strong reduction of TRLs it is noteworthy that non-secreted lipoproteins tend to accumulate in the liver and intestine, leading to steatosis in these tissues. In fact, the variant rs3829126 is significantly associated with circulating alanine aminotransferase [58, 61], a marker of liver damage. Therefore, while PLA2G12B remains a promising target for the treatment of lipid disorders and the prevention of cardiovascular diseases, careful consideration and further research are needed to reduce the risks of lipid accumulation in liver and intestinal tissues.

## SURF4 is Involved in the Pre-chylomicrons' Trafficking

The chylomicron retention disease (CMRD), also called the Anderson's disease (OMIM#246700), is a rare disease characterized by the inability to secrete chylomicrons from the enterocytes [62]. In 2003, homozygous variants in the SAR1B gene have been linked to the CMRD [42]. SAR1B encodes the secretion associated ras related GTPase 1B protein, a small GTPase, that acts as an essential homodimeric factor of the coat protein complex II (COPII),

<table><caption>**Table 1** This data is mandatory, please provide</caption><thead><tr><th>Gene</th><th>Locus / Genetic variant</th><th>Phenotype</th><th>References</th></tr></thead><tbody><tr><td>LIMA1</td><td>12q13.12<br/>Rare variants in familial cases.<br/>LIMA1 p.K306fs<br/>3 families with LIMA1 p.L25I</td><td>Chinese Kazakh family. Lower total cholesterol and LDL-c plasma levels.<br/>Reduced intestinal cholesterol absorption.</td><td>[31]</td></tr><tr><td>PLA2G12B</td><td>10q22.1<br/>(rs3829126)</td><td>Significantly associated with:<br/>plasma triglycerides, total, LDL and HDL cholesterol, and risk of coronary artery disease.</td><td>[26, 36, 80]</td></tr><tr><td>SURF4</td><td>9q34.2<br/>(rs3758348)</td><td>Significantly associated with LDL and total cholesterol plasma levels, risk for coronary heart disease and myocardial infarction.</td><td>[26, 63]</td></tr><tr><td>C7ORF50<br/>(Cholesin)</td><td>7p22.3<br/>(rs1007765)</td><td>Significantly associated with: total cholesterol, LDL, HDL, ApoB.</td><td>[26, 64, 72, 81, 82]</td></tr></tbody></table>

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protein C7orf50, which the authors renamed "Cholesin." Plasma levels of Cholesin are also elevated in the postprandial state in humans. Cholesin is encoded by the human gene C7ORF50 and is predominantly expressed in the stomach, small intestine, and colon.

Through refined GWAS on plasma lipids, the authors identified a common variant, rs1007765, located approximately 4 kb downstream of the 3' UTR of C7ORF50, significantly associated with total, LDL, and HDL cholesterol plasma levels (Table 1). They demonstrated that the C-allele of rs1007765 is associated with increased C7ORF50 gene expression and is dose-dependently linked to elevated Cholesin plasma levels. Furthermore, Cholesin plasma levels are inversely correlated with total, LDL-c, and ApoB plasma levels in humans.

To further investigate the physiological functions of Cholesin, they generated and thoroughly studied mice with intestine-specific Cholesin knockout (Cholesin iKO). In a series of in vivo experiments, the study convincingly showed that intestinal inhibition of Cholesin in mice induces a 20% increase in cholesterol plasma levels across all lipoprotein fractions (VLDL, LDL, HDL). This increase is attributed to changes in hepatic cholesterol synthesis and VLDL secretion, with no changes in intestinal absorption, chylomicron catabolism, or biliary excretion of bile acids and cholesterol.

Conversely, LDL-receptor invalidated mice injected with recombinant Cholesin exhibited a significant reduction in plasma cholesterol levels (mainly due to decreased hepatic cholesterol synthesis) and were protected against atherosclerosis. The authors then screened for Cholesin target organs, finding stronger binding in the liver, adipose tissues, kidney, and skeletal muscle. To identify the Cholesin receptor, they performed a genome-wide silencing screen. Using Cas9-expressing MCF7 cells infected with a lentiviral sgRNA library, they incubated the cells with tagged Cholesin and selected, via FACS, those unable to bind Cholesin. Sequencing revealed that cells unable to bind GST-Cholesin were enriched for knockouts of the gene GPR146.

GPR146 is a G-protein coupled receptor that promotes hepatic sterol regulatory element-binding protein 2 (SREBP2) activity through the activation of the extracellular signal-regulated kinase (ERK) signaling pathway (recently reviewed in [73, 74]). GPR146 deficiency or silencing has been shown to reduce circulating total, LDL, and HDL cholesterol levels in both humans and mice [75-77]. Notably, GPR146 deficiency also significantly reduces cholesterol plasma levels and atherosclerotic lesions independently of the LDL-receptor.

Until the study by Hu et al. [72] the ligand of GPR146 was unknown. To establish the direct link between Cholesin and GPR146, they demonstrated that Cholesin-related cholesterol-lowering effects were abrogated in GPR146-liver-specific knockout mice (Fig. 1).

Overall, the study by Hu et al. is groundbreaking as it reveals for the first time the inter-organ effect of the newly discovered hormone Cholesin as a sensor of intestinal cholesterol absorption that regulates cholesterol homeostasis in the liver and circulation, independent of plasma cholesterol levels. The identification of the regulatory axis between Cholesin and GPR146 offers promising potential for developing lipid-lowering strategies to prevent atherosclerosis in patients who do not meet clinical targets. This is particularly valuable in patients who do not respond well to existing treatments such as patients with homozygous familial hypercholesterolemia, intolerant to existing treatments or in secondary prevention without reaching low enough LDL-c plasma levels. Despite strong arguments showing the reduction of atherosclerosis in mice injected with recombinant Cholesin in plasma, the effects on ASCVD remain to be shown in human by genetic approaches, mendelian randomization studies or in clinical trials.

Nevertheless, further research is required to gain a deeper understanding of this physiological axis. The mechanisms by which intestinal cholesterol stimulates Cholesin transcription and secretion are not fully understood. Although cholesterol absorption by NPC1L1 is known to be involved, the precise mechanisms by which cholesterol stimulates the transcription and secretion of Cholesin in the intestine remain unclear. This knowledge gap could hinder the development of effective and predictable therapies targeting this pathway and warrants further research.

When compared to previous candidates, and based on previous studies, it seems that targeting the axis cholesin - GPR146 might have beneficial effects not only on lipid related ASCVD but also on metabolic traits such as glycemia, adiposity and liver function [72, 75, 77, 78] which reinforces the strong interest for such a target and reinsure about potential unwanted potential side effects. However, GPR146, receptor of cholesin is ubiquitously expressed. This widespread expression warrants attention about potential off-target effects and systemic side effects when manipulating this pathway. Also, despite these promising results, the effects of pharmacological modulation of the Cholesin-GPR146 axis in humans still has to be demonstrated and human physiology might respond differently.

When thinking of way to target the cholesin-gpr146 pathway, one could think of injecting a recombinant protein (as shown in mice [72]), but other strategies could be considered such as the use of compounds which would increase the expression of cholesin in the enterocytes or silence GPR146 in the liver using advanced gene-silencing technologies [79].

In summary, even though significant challenges and unknowns must be addressed before it can be considered a viable therapeutic option, targeting the Cholesin-GPR146 axis presents a promising new approach to managing cholesterol levels and preventing atherosclerosis.

<figure><img src="image_7.png" /></figure>
473 **Conclusion / Discussion**

Despite the current difficulties in experimentally exploring the functionality of molecular targets in the gut, recent genetic discoveries have unveiled novel intestinal mechanisms regulating plasma lipids and ASCVD risk. These findings highlight the value of human genetics in identifying unexpected actors in lipid metabolism. This progress has been achieved through multiple complementary approaches: investigations of familial dyslipidemia cases; advancements in GWAS-loci refinement methodologies, incorporating multi-omic approaches such as transcriptomics, proteomics, and epigenomics; and expansion of genetic studies to include populations with diverse ancestries [26].

As a result, recent studies have identified new functional targets which have enhanced our understanding of the intestinal role in regulating plasma lipid levels notably by impacting: -the endocytic recycling compartment (LIMA1); - the co-translational lipidation of ApoB48 with MTP in the endoplasmic reticulum (PLA2G12B); -the vesicular transport of pre- and mature chylomicrons from the ER to the Golgi and their subsequent release at the basolateral side of enterocytes (SURF4): -the interorgan communication between the intestine and liver (the CHOLESN-GPR146 axis).

While rodent studies show promising effects on plasma cholesterol levels and atherosclerosis development, human validation is needed to further confirm these promises. Potential therapeutic targets must be carefully evaluated due to their pleiotropic effects and expression in multiple tissues. Challenges include lipid-lowering potency and redundancy with existing treatments, potential side effects like hepatic steatosis, and undesirable effects on developmental.

As illustrated by the newly discovered Cholesin-GPR146 axis, such intestinal targets offer potential for therapeutic development but require thorough investigation to optimize their clinical application and mitigate associated risks.

508 **Key References**

509 ● Graham SE, Clarke SL, Wu KHH, Kanoni S, Zajac GJM, Ramdas S, et al. The power of genetic diversity in genome-wide association studies of lipids. Nature. 2021 Dec;600(7890):675-9.

513 ○ The most extensive multi-ancestry meta-analysis examining the relationship between genetic variants and plasma lipid levels published so far.

516 ●● Zhang YY, Fu ZY, Wei J, Qi W, Baituola G, Luo J, et al. A LIMA1 variant promotes low plasma LDL cholesterol and decreases intestinal cholesterol absorption. Science. 2018 Jun 8;360(6393):1087-92.

○ Princeps paper showing the involvement of LIMA1 as a key protein regulating intestinal cholesterol absorption.

●● Thierer JH, Foresti O, Yadav PK, Wilson MH, Moll TOC, Shen MC, et al. PLA2G12b drives expansion of triglyceride-rich lipoproteins. Nat Commun. 2024 Mar 7;15 (1):2095.

○ First study linking genetic association with a role of PLA2G12B as a key driver of triglyceride incorporation into lipoproteins.

● Tao G, Wang H, Shen Y, Zhai L, Liu B, Wang B, et al. Surf4 (Surfeit Locus Protein 4) Deficiency Reduces Intestinal Lipid Absorption and Secretion and Decreases Metabolism in Mice. Arterioscler Thromb Vasc Biol. 2023 Apr;43 (4):562-80.

○ Study which specifically investigates the role of SURF4 in the intestine.

●● Wang X, Wang H, Xu B, Huang D, Nie C, Pu L, et al. Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans. Cell Metab. 2021 Feb 2;33 (2):350-366.e7.

○ Princeps paper showing the interaction of SURF4 with SAR1B and its link with lipid-related ASCVD.

●● Hu X, Chen F, Jia L, Long A, Peng Y, Li X, et al. A gut-derived hormone regulates cholesterol metabolism. Cell. 2024 Mar 28;187 (7):1685-1700.e18.

○ Groundbreaking study which demonstrates that the Cholesin-GPR146 axis mediates the inhibitory effect of intestinal cholesterol absorption on hepatic cholesterol synthesis.

**Author Contributions** CLM, SD, BC and AR wrote the main manuscript and reviewed the manuscript. AR prepared figures.

**Funding** This work was supported by the GENESIS project, funded by the "Agence Nationale de la Recherche" (ANR-21-CE14-0051) and the 'Region Pays de la Loire' (Grant 2023_06536).

**Data Availability** No datasets were generated or analysed during the current study.

**Declarations**
The figure was originally generated with Biorender (agreement number: WP275AIQB9).

**Competing interests** The authors declare no competing interests.

**Human and Animal Rights and Informed Consent** No animal or human subjects were used in this study.
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