Gallstone disease (GSD) is a common biliary system disease composed primarily of cholesterol stones, pigment stones (composed mainly of calcium salts and bile pigments), and mixed stones.¹ Cholecystolithiasis tends to occur in adults with an overall incidence of 10–15%. The incidence increases with age after 40 years, with a higher incidence in women than men.²

Three main types of gallstones exist: cholesterol, pigmented, and mixed.³ Most gallstones are cholesterol gallstones, accounting for approximately 90%.⁴ The symptoms of gallstones are usually caused by cholesterol stones.⁵

Many studies have revealed a close relationship between gallstone formation and human metabolic activity.⁶ The high expression of beta3-adrenergic receptors and HMG-CoA reductase in obese patients⁷ can affect gallbladder contraction and inhibit cholesterol excretion in the gallbladder.^8,9 In metabolic syndrome, the low high-density lipoprotein,¹⁰ elevated triglyceride, and high homocysteine levels lead to a pro-inflammatory state that increases the rate of gallstone formation.^11,12 The abnormal metabolism of bile acids in the human body can also affect cholesterol excretion in the bile.¹³

Gallstone formation is closely related to human metabolism, particularly lipid metabolism. Therefore, studying lipid metabolism will help better understand the mechanism of gallbladder stone formation.

Lipids consist of triglycerides (TGs), phospholipids, and cholesterol. They are important nutrients required by the human body, providing energy and essential fatty acids and participating in cell composition. Fatty acids are the most common form of energy storage in the human body. They are stored mainly in the form of TGs.¹⁴ Phospholipids and cholesterol are important components of the cell membranes.¹⁵

The liver is the main site of lipid synthesis and metabolism.^10,14 The synthesis and metabolism of lipids in the normal human body are balanced. Disruption of this balance leads to various diseases such as obesity, atherosclerosis, and type 2 diabetes.^16,17

1. Cholesterol metabolism

Cholesterol plays an important role in the cell membrane composition, intracellular signaling, and the synthesis of various hormones.¹⁸ Acetyl-CoA is a raw material for cholesterol biosynthesis. It is synthesized in cells through the synergistic action of more than 20 enzymes, e.g., HMG-CoA reductase, exerting its biological functions.^19,20 Cholesterol biosynthesis is related to the levels of foam sterol, cholestanol, and desfilin.^21,22 The liver is the primary site of cholesterol biosynthesis, transmitting endogenous and exogenous cholesterol as extremely low-density lipoproteins (VLDL) into the bloodstream. Excess cholesterol combines with apolipoprotein A-I (apoA-I) to form high-density lipoprotein (HDL).^23,24 Nuclear receptor liver X receptors (LXRs) are important regulatory factors for intracellular cholesterol and lipid homeostasis.^25-27

Cholesterol can produce various oxidative sterols via enzymatic and non-enzymatic pathways.²⁸ Some are further metabolized into bile acids in the liver and discharged into the gallbladder, becoming a component of bile.¹⁹ The stability of the cholesterol metabolism has a regulatory effect on the inflammatory response and tumor microenvironment.^29,30 Moreover, some cholesterol molecules, such as SOAT1, SQLE, and NPC1, have become immunotherapeutic targets for tumors (Fig. 1).^31-33

Figure 1. In vivo synthesis and metabolic pathways of cholesterol.

2. Triglyceride metabolism

TGs are the main source of energy in the human body and are synthesized mainly in the liver via the diglyceride pathway using sugars, glycerol, and fatty acids.^14,34 The liver takes up excess glucose and fructose^35-37 through de novo lipogenesis and converts them into TGs.³⁸ The activation of transcription factor 7-like 2 (TCF7L2)³⁹ and liver cell membrane CD36 increases the intake of fatty acids, promoting de novo lipogenesis.⁴⁰ The action of insulin^41,42 and the secretion of the hepatokine oromucoid II limits the progression of de novo lipogenesis.⁴³ Small intestinal mucosal cells can synthesize TGs using glycerol monoesters and fatty acids absorbed through exogenous digestion.

The lipoprotein lipase protein complex (LPL) determines the breakdown of TGs, and liver receptors (RLDL and LRP-1) are responsible for transporting and clearing the decomposed products.⁴⁴ Dietary habits^45,46 and the size of the TG decomposition products and intermediates,⁴⁷ such as lipid droplets,⁴⁸ can influence the activity of fatty acid TG lipase, affecting TG metabolism.

3. Phospholipid metabolism

Phospholipids are synthesized in the endoplasmic reticulum, where TGs and phosphatidic acids bind to sphingosine to form a phospholipid sphingosine complex. The complex then combines with phosphate and choline to form phospholipid molecules such as lecithin and cephalin.⁴⁹ Some fatty acids, inositol, and enzymes can modify phospholipid molecules to perform various biological functions.⁵⁰ In cells, phospholipids are transported and transmitted through vesicles and cell membranes.⁵¹ The roles of phosphoinositol and phospholipase D in vesicular transport reactions are important.^52-54

Phospholipids can be hydrolyzed into glycerol, fatty acids, phosphate, and various amino alcohols through phospholipase reactions in living organisms. Phospholipases A1 and A2 cleave fatty acids at the 1,2 positions of phospholipid molecules. In addition, the phospholipase D (PLD) enzyme catalyzes the hydrolysis of phosphodiesterase bonds in glycerol phospholipids.^54,55 Sphingosine 1-phosphate (S1P) is a metabolic product of cell membrane sphingolipids, which bind to the S1P receptor (S1PR) coupled with the G protein to regulate embryonic development, organ function, inflammatory response, and multiple system diseases.^56-59 The ABCB4 gene, cleavage of lipoproteins, and structural activity of the endoplasmic reticulum have regulatory effects on the biosynthesis and metabolism of phospholipids (Fig. 2).^60-63

Figure 2. Key enzymes for phospholipid synthesis and metabolism in vivo.

Bile is a three-variable solution; the bile acid, lecithin, and cholesterol concentration ratio in bile affects cholesterol solubility.⁶⁴ Under normal circumstances, bile salts and lecithin can disperse cholesterol to form soluble microcapsules, which are less likely to precipitate and form stones. Bile cholesterol is more likely to become supersaturated when cholesterol secretion in bile increases or the concentration of bile acids and lecithin decreases.¹³

1. Cholesterol metabolism and bile biochemistry

Bile acids mainly pass through cholesterol 7 α-hydroxylase (CYP7A1), which oxidizes cholesterol. The regulatory effect of the nuclear factor farnesoid X receptor (FXR) is a key factor.¹³ FXR downregulates CYP7A1 by inducing a cascade reaction involving human fibroblast growth factor 19-JNK.^65,66 Fibrin can increase the risk of gallstones 1.7-fold.⁶⁷ CYP7A1 polymorphisms also lead to a significantly higher risk of developing gallstones.⁶⁶ Cholesterol precipitates and forms gallstones when cholesterol synthesis is excessively active and cholesterol in the bile is supersaturated.⁹ CYP7A1 is inhibited when FXR is overactive, which affects cholesterol breakdown and metabolism into bile acids, further promoting cholesterol accumulation and precipitation.^66,68

Some receptors on the gallbladder wall regulate cholesterol absorption, such as scavenger receptor SR-BI.⁶⁹ This receptor is related to the occurrence of atherosclerosis, and the supersaturation of bile cholesterol appears to be related to SR-BI receptor inhibition.^70-72

This study suggests that abnormalities in CYP7A1, FXR, and SR-BI can lead to metabolic disorders of cholesterol in bile, accelerating the formation of gallstones. On the other hand, Chen et al. found through Mendelian randomization analysis that the association between the serum total cholesterol (TC) levels and the risk of gallstones was not linear; lower TC levels lead to a higher risk of stone formation.⁷³

2.Triglyceride metabolism and bile biochemistry

Currently, a large number of clinical studies have shown that patients with hyperlipidemia have a higher risk of developing gallstones. Previous studies have shown that knocking out CD36 on the liver cell membrane, which plays a regulatory role in the de novo lipogenesis pathway, significantly reduces the incidence of gallstones in mice.⁷⁴ Elevated levels of very low-density lipoprotein (VLDL) related to TG transport can also accelerate the excretion of bile acids.⁷⁵ Evidence suggests that the gallbladder of high TG patients is less sensitive to CCK. This sensitivity is improved after reversing the high serum TG levels with TG-lowering agents.⁷⁶ These results confirmed the correlation between the increase in TG and gallstones.

3. Phospholipid metabolism and bile biochemistry

Phospholipids are important biochemical components of bile. The solubility of cholesterol in bile salt phospholipid mixtures is much higher than in pure bile salts.⁷⁷ Therefore, phospholipids play an important role in the biochemical balance of bile. Phospholipids are generated mainly in the endoplasmic reticulum, with sphingomyelin synthetase being the key enzyme for their synthesis. Abnormal enzyme levels can cause non-alcoholic fatty liver, atherosclerosis, and diabetes.^78,79 On the other hand, no reports on the relationship between this enzyme abnormality and the occurrence of gallstones have been published.

The most extensively studied phospholipid transport process is the endoplasmic reticulum-mitochondria encounter structure (ERMES) complex between the endoplasmic reticulum and mitochondria, which is a critical pathway for phospholipid transfer between two organelles.⁸⁰ It is unclear if there is a connection between this pathway and gallstone formation. Low phospholipid-associated cholelithiasis (LPAC) is an inherited cholelithiasis associated with low phospholipid levels in the bile, accounting for approximately 0.5–2% of symptomatic cholelithiasis.⁸¹ LPAC is a genetic disease caused by a single gene mutation associated with mutations in ABCB4, which mainly encodes the carrier protein MDR3 in the biliary tract.^61,82 MDR3 exists on the membrane of liver tubules; its main function is to transport phosphatidylcholine from liver cells to the gallbladder. When the MDR3 protein mutates, phospholipid transport disorders lead to easier cholesterol precipitation in bile (Fig. 3).⁸³ In summary, abnormal phospholipid generation and transport caused by various factors can lead to bile cholesterol supersaturation, accelerating gallstone formation.

Figure 3. Effects of MDR3 mutations on phospholipid transport on the membrane of hepatic tubular cells.

4. Lipoproteins and bile biochemistry

Many studies have examined the relationship between the HDL and LDL levels in the serum and bile metabolism, as well as the formation of gallstones. A study on the correlation between serum lipoprotein levels and gallstone formation in Western patients with gallstones found that high LDL levels can increase the cholesterol concentration in bile (p<0.01) and increase the incidence of gallstones (p<0.05).⁸⁴ HDL-C is positively correlated with phospholipids and bile acids in the bile. Hence, high LDL levels can accelerate the formation of gallstones, whereas HDL-C may offer some protection for gallstone formation.⁸⁵

5. Lipid metabolism and gallbladder contraction disorders

Under normal conditions, the gallbladder can drive the intestinal hepatic circulation of bile acids through regular contraction and relaxation.⁸⁶ This process is regulated by cholecystokinin (CCK) secreted by small intestinal cells.⁸⁷ Dietary fat can stimulate CCK secretion to some extent. Active cholesterol synthesis leads to a sustained supersaturation state, resulting in the deposition of cholesterol on the gallbladder wall, COX2-mediated inflammation, and the abnormal contraction and relaxation of the gallbladder.^10,88 Gallbladder contraction disorders can affect the excretion of cholesterol and bile acids, increasing the risk of gallstones.

Omics technology refers to the detection and analysis of data representing the structure and function of a specific biological system, such as genes and proteins, and the acquisition of systematic connections between the data at that level. The technology mainly includes genomics, proteomics, metabolomics, transcriptomics, lipidomics, immunohistochemistry, glycomics, RNA omics, imaging omics, and ultrasound omics.⁸⁹ The integration of multiple omics has become a common trend in constructing comprehensive causal relationships, with genomics, transcriptomics, proteomics, and metabolomics being the most widely used approaches.

Genomic research measures DNA molecules, whereas epigenomics, transcriptomics, proteomics, and metabolomics research measures the chemical states of DNA and its binding proteins, RNA, proteins, and metabolites.⁹⁰ Compared to traditional singularity research, the basic status of genes, RNA, proteins, and metabolites in the target sample can be understood by integrating and analyzing genomic, transcriptomic, proteomic, and metabolomic data, providing a comprehensive interpretation of the biological system.

1. Genomic research on gallstones

Research on the gallbladder genome has found that multiple sets of alleles in the gallbladder tissue of patients with stones have undergone mutations, which have been isolated and localized. These include mutations in the liver cholesterol transporter (ABC transporter) ABCG5/ABCG8,^91,92 CYP7A1, and changes in the multicellular transmembrane protein NPC1L1 (Niemann Pick C1 Like 1) are associated with the intestinal and liver reabsorption of cholesterol.⁹³

ABCG5/ABCG8 are present in intestinal and liver cells to inhibit the intestinal absorption of cholesterol and promote bile secretion.⁹² NAPC1L1 is present in intestinal epithelial and liver cells, promoting cholesterol absorption through endocytosis mediated by grid proteins.⁹⁴ Changes in these genes and proteins promote cholesterol absorption in the human body, inhibit cholesterol transport and bile excretion, and accelerate the nucleation of cholesterol in bile.

The treatment of gallstones is mainly surgical, but early prevention and intervention in gallstones can be achieved by exploring the omics mechanisms of gallstone formation. Currently, in vitro experiments have been conducted to regulate the cholesterol levels in rats through the mediation of NAPC1L1 and ABCG5/ABCG8 proteins by lactic acid bacteria, garlic extract, or Diosgenin.^95-97 In addition, NPC1L1 is the target of the lipid-lowering drug ezetimibe in the intestine,⁹⁸ which may contribute to the early prevention of gallstones.

2. Transcriptomics and Gallstones

Transcriptomic research on gallbladder diseases is progressing rapidly, including the study of genes related to the onset of single diseases such as gallstones, gallbladder polyps, and gallbladder cancer.

In a study of gallstones, a correlation was found between miR-20 (RNA-regulating ABC transporter) and the occurrence of gallstones through the transcriptome analysis of gallbladder tissue.⁹⁹ This confirms the results of the genomic research on ABC transporter proteins. Aquaporin 3 (AQP3) can reduce gallbladder inflammation and inhibit gallstone formation.¹⁰⁰ miR-20 and AQP3 also play important roles in the lipid metabolism. Changes in miR-20 and AQP3 can affect the transportation of cholesterol and TG, promoting the formation of gallstones. A research team from the Netherlands conducted transcriptomic studies on adipose tissue of patients with gallbladder cancer. They revealed a significant increase in apolipoprotein A1 (APOA1) involvement in patients with stones. This can lead to the release of beneficial cholesterol from adipose tissue, resulting in the supersaturation of bile cholesterol.¹⁰¹ Infliximab (IFX) reduces miR-20 expression in children, and research in this area may provide a reference for treating gallstones.¹⁰²

3. Metabolomics and Gallstones

Metabolomic research on gallstones has focused mainly on differential metabolites. By comparing the plasma or bile metabolism of patients with gallstones and normal individuals, some studies have found that cholesterol and various bile acids are significantly elevated in patients with gallstones.^101,103 Relatively little research has been conducted on other metabolites and metabolic pathways related to stone formation.

In an unpublished study, plasma samples were collected from more than 100 patients, and non-targeted metabolomic testing was performed. The detection data were compared with the clinical indicators. The metabolites and metabolic pathways related to GSD generation were obtained using a weighted correlation network analysis (WGCNA) algorithm combined with KEGG pathway enrichment analysis.

The present study found a significant decrease in linoleic acid in the peripheral blood of patients with GSD. Moreover, its corresponding linoleic acid metabolic pathway also showed a significant downregulation trend. The pantothenate and CoA biosynthesis and TCA cycle pathways were significantly upregulated. Linoleic acid is a raw material used to synthesize and convert phospholipids. When the peripheral blood linoleic acid level decreases, there is an obstacle in the production of phospholipids in bile, and the precipitation of cholesterol becomes more pronounced. The pantothenate and CoA bio-synthesis pathways were upregulated in the peripheral blood of patients with GSD. Upregulation of this pathway will increase CoA production, the raw material for cholesterol synthesis, promoting cholesterol production.^23,104 The TCA cycle is the oxidation pathway of nutrients in the body. The strong expression of this pathway can also promote the conversion of carbohydrates to lipids during satiety, increase the burden of lipids in the body, affect the lipid structure of the body, and promote the precipitation of cholesterol in bile (Fig. 4).¹⁰⁵ Based on this study, GSD can be prevented and treated by affecting the linoleic acid cycle, and the effectiveness of drugs, such as ethyl linoleate, may serve as a direction for future research.

Figure 4. Pathway of TCA cycle in the formation of gallstones.

The abnormal metabolism of cholesterol, TGs, and phospholipids plays a significant role in gallstone formation. When FXR is overactivated, or CYP7A1 and SR-BI receptors are inhibited to a certain extent, cholesterol in bile becomes supersaturated, and a COX2-mediated inflammatory response is induced in the gallbladder wall, accelerating gallstone formation. The transport of TGs by VLDL and CD36 molecules in the liver cell membrane is affected, increasing the risk of gallstones. In the phospholipid metabolism, abnormalities in the MDR3 protein that regulates phospholipid transfer cause low phospholipid-related cholelithiasis.

In the genomic and transcriptome analysis of gallbladder tissue, changes in the liver cholesterol transporters ABCG5/ ABCG8 accelerate cholesterol nucleation, while abnormalities in aquaporin 3 (AQP3) and apolipoprotein A1 (APOA1) affect the release and transport of cholesterol and TGs (Fig. 5). Through metabolomic techniques, this study discovered that the linoleic acid metabolism pathway, pantothenic acid and coenzyme A biosynthesis pathways, and the TCA cycle pathway play a role in the generation and balance of phospholipids and cholesterol. These omics-related studies provide a new theoretical basis for preventing and treating gallstones and directions for future research.

Figure 5. Positions and roles of CD36, APOA-1, SR-BI, and CYP7A1 in the KEGG pathway of cholesterol synthesis.

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