Differential transport pathways of saturated and unsaturated fatty acid esters in male mouse hepatocytes

Chen, Fengwu; Yang, Aizhen; Lu, Yue; Zhang, Yuxin; Zhang, Jingyu; Bu, Jianan; Guo, Runlin; Han, Yue; Wu, Depei; Wu, Yi

doi:10.1038/s41467-025-56620-4

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Published: 04 February 2025

Differential transport pathways of saturated and unsaturated fatty acid esters in male mouse hepatocytes

Nature Communications volume 16, Article number: 1344 (2025) Cite this article

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Abstract

Saturated fatty acid (SFA) and unsaturated fatty acid (UFA) have distinct impacts on health. Whether SFA and UFA are differentially transported in liver remains elusive. Here, we find the secretion of UFA but not SFA esters is retarded in a male mouse hepatic endoplasmic reticulum (ER) stress model. Among 13 members of protein disulfide isomerase (PDI) family, only PDIA1 (PDI) deficiency leads to hepatosteatosis and hypolipidemia. In PDI-deficient male mouse liver, there is a severe accumulation but secretory blockade of UFA esters, whereas the accumulation and secretion of SFA esters remain normal. PDI catalyzes the oxidative folding of microsomal triglyceride transfer protein (MTP). In addition, PDI deficiency in hepatocytes abolishes Apolipoprotein B-100 (ApoB-100) very low-density lipoprotein (VLDL) secretion while maintaining partial ApoB-48 VLDL secretion. In summary, we find that the secretion of UFA esters is PDI-MTP indispensable, while SFA esters could be transferred out of liver via ApoB-48 VLDL through a PDI-MTP-independent pathway.

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Introduction

UFA and SFA have distinct impacts on health. Consuming high amounts of SFA is widely considered to increase plasma low-density lipoprotein (LDL) cholesterol, subsequently elevating the risk of cardiovascular disease¹. In contrast, high consumption of UFA primarily leads to increases in body weight², hyperglycemia², and liver damage^3,4. Many studies have advocated for the replacement of dietary SFAs with UFAs to reduce cardiovascular risk^5,6,7,8,9,10. However, some studies indicate that the suitability of such dietary replacements for everyone is controversial^11,12.

Numerous lines of evidence suggest that there could be different mechanisms involved in the secretion and accumulation of SFA and UFA esters in cells. Abdel-Fattah et al. fed guinea pigs with corn oil, lard oil, and palm kernel oil and found that dietary fat chain length and saturation have specific effects on VLDL secretion¹³. Furthermore, many studies have shown that UFA promotes the formation of triglyceride (TG)-enriched lipid droplets (LDs) while SFA is poorly converted into TG-enriched LDs in bovine oocytes¹⁴, DAOY cancer cells¹⁵, microglia¹⁶, renal proximal tubular cells¹⁷, HepG2 cells^18,19, and primary mouse hepatocytes¹⁸. However, little is known about the different transport mechanisms of UFA and SFA esters in cells.

It’s well known that endoplasmic reticulum (ER) stress causes hepatic steatosis^20,21,22, and the main metabolic pathway affected by ER stress is thought to be lipogenesis^23,24. However, whether ER stress affects the process of lipids release from liver, and whether ER stress is associated with the differential transport mechanism of SFA and UFA remain unknown.

Protein disulfide isomerase (PDI) family is a group of thiol oxidoreductases in ER that catalyze posttranslational disulfide exchange necessary for proper protein folding in ER²⁵. PDI is the prototypical member of this family and plays an important role in VLDL assembly. PDI catalyzes the oxidative folding of ApoB-100²⁶. More importantly, PDI and MTP forms a heterodimer, which plays a vital role in VLDL assembly via transferring neutral lipids to the newly synthesized ApoB protein²⁷. MTP has lipid-binding and lipid-transfer activity, while PDI is thought to maintain MTP in a soluble form²⁸ and ensure MTP in the ER via its KDEL motif^29,30. Although the redox activity of PDI is critical for various biological processes^31,32,33, it was proposed to be unnecessary for the function of MTP^34,35,36. However, this conclusion has never been verified in a genetic model. Moreover, the PDI family has more than 20 members, most of which show very similar sequences, structures, and functions with PDI. For example, PDI, ERp57, and ERp72 contain the same CGHC motif and play similar roles in thrombosis and hemostasis^{33,37,38,39,40}. Although PDI is obligatory for MTP activity, whether the other PDI paralogs (PDIs) play a specific or redundant role in maintaining intrahepatic and plasma lipid homeostasis remain elusive.

In this study, based on the observation that ER stress is associated with the different transport mechanisms of SFA and UFA, we screened 13 members of the PDI family using hepatocyte-specific and whole-body gene-deficient mouse models, and identified that only PDI plays an indispensable and irreplaceable role in regulating intrahepatic and intravascular lipid homeostasis. Further study revealed that PDI is vital for transferring UFA esters out of liver, but not SFA esters. In addition, PDI catalyzes the oxidative folding of MTP, is responsible for the formation of at least 3 disulfide bonds that are very important for the structural stability of MTP. PDI deficiency in hepatocytes abolishes ApoB-100 VLDL secretion while maintaining partial ApoB-48 VLDL secretion. These findings advance the understanding of the principle of fatty acid transport, and may facilitate the development of a novel therapeutic strategy for the treatment of hepatic steatosis, hypolipidemia, and obesity.

Results

ER stress is associated with differential transport of UFA and SFA esters

To determine whether ER stress affects the transfer of different FA esters out of liver, we constructed a tunicamycin (TM)-induced in vivo ER stress model (Fig. 1a). TM caused significantly increased expression of Bip, p-PERK, p-IRE1α, XBP1, and ATF6, the marker proteins of ER stress, in the liver (Fig. 1b and Supplementary Fig. 1), and obvious hepatic steatosis (Fig. 1c–e). Numerous TG and cholesterol (CHOL) were accumulated (Fig. 1f, g) in the livers of TM-treated mice. To assess the secretion of lipids from the liver, both TM- and dextrose-treated mice were injected with tyloxapol, a compound that blocks plasma lipolytic activity and thus the breakdown of TG-rich lipoproteins. Before and 2-h after tyloxapol injection, mouse plasma was isolated and the levels of TG, LDL, and CHOL were detected. The results of blood biochemical showed that ER stress significantly restrained the secretion of TG and CHOL, but not LDL (Fig. 1h, j). To determine whether ER stress has any influence on the transport of SFA and UFA, the levels of long-chain fatty acids and fatty acid esters in the plasma acquired 2 h after tyloxapol injection were quantified by ultra-high performance liquid chromatography- triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) (Supplementary Data 1). As shown in Fig. 1k, l, ER stress reduces the secretion of UFA, especially palmitoleic acid (FA 16:1n7c), oleic acid (FA 18:1n9c), linoleic acid (FA 18:2n6c), and arachidonic acid (FA 20:4n6c).

**Fig. 1: ER stress is associated with the differential transport of UFA and SFA esters.**

Deficiency of hepatic PDI results in hypolipidemia and hepatosteatosis

The PDI family is involved in the refolding of misfolded proteins, thus alleviating ER stress. To determine whether the PDI family is responsible for the differential secretion of UFA and SFA in the TM-treated ER stress model, we generated hepatocyte-specific or whole-body knockout mice deficient in 13 members of this family (Supplementary Figs. 2 and 3a). As shown by the results of plasma biochemical test, Pdia2^−/−, Alb-cre/Pdia3^fl/fl, Pdia4^−/−, Pdia5^−/−, Pdia8^−/−, Pdia9^−/−, Pdia11^−/−, Pdia13^−/−, Pdia14^−/−, Pdia15^−/−, CAG-cre/Pdia16^fl/fl, and Pdia19^−/− mice had comparable levels of plasma TG and CHOL with their littermate controls (Fig. 2a, b). In contrast, both TG and CHOL levels in plasma were significantly decreased in Alb-cre/Pdi^fl/fl mice (Fig. 2a, b). These results indicate that PDI plays an indispensable and irreplaceable role in regulating intravascular lipid homeostasis. Further analysis of the plasma metabolic profile indicated that plasma LDL, HDL, and glucose levels were also decreased in the Alb-cre/Pdi^fl/fl mice, while other biochemical parameters remained normal (Supplementary Table 1).

We further evaluated the effects of PDI deficiency on hepatic lipid homeostasis. In the liver of Alb-cre/Pdi^fl/fl mice, endogenous PDI and MTP was almost depleted, and the expression of PDI family members PDIA3 and PDIA4, as well as ER stress marker proteins Bip, ATF6, and XBP1, showed varying degrees of increases (Supplementary Fig. 3b–f). Alb-cre/Pdi^fl/fl mice at 12 weeks of age did not show any apparent abnormalities including body weight (Supplementary Fig. 3g). However, the livers of the Alb-cre/Pdi^fl/fl mice appeared enlarged and whitish compared with those of the littermate Pdi^fl/fl mice (Fig. 2c), and the ratio of liver weight to body weight was significantly greater in the Alb-cre/Pdi^fl/fl mice than that of the Pdi^fl/fl mice (Fig. 2d). Alb-cre/Pdi^fl/fl mice exhibited a 3-fold increase in hepatic TG content compared with that of their littermate controls (Fig. 2e), although the hepatic CHOL levels of Alb-cre/Pdi^fl/fl mice did not increase (Fig. 2f). The free fatty acid level in the livers of the PDI-deficient mice also increased (Fig. 2g). As shown by hematoxylin and eosin (H&E) and Oil Red O staining, the livers of the PDI-deficient mice exhibited severe lipid accumulation (Fig. 2h, i). Meanwhile, the expression of fatty acid synthase and desaturase, such as ACC, FASN1, and SCD1, in liver are all decreased at both mRNA and protein levels (Supplementary Fig. 3h, i). The ratio of inguinal fat pad weight to body weight showed no difference (Supplementary Fig. 3k). The finding that PDI-deficient mice suffer from hypolipidemia and hepatosteatosis indicated that PDI is essential for lipid transfer out of the liver.

PDI deficiency in the liver leads to severe accumulation but secretory blockade of UFA esters

As detected by transmission electron microscopy (TEM), the color of lipid droplets (LDs) in the livers of PDI-deficient mice was different from that in the liver of control mice (Fig. 3a), suggesting that the constituents of accumulated fatty acid esters in each lipid droplet may be different. Drops of unsaturated lipids were brown-colored and saturated lipids were white-colored in Toluidine blue staining^41,42. The large number of brown-colored lipid droplets observed via toluidine blue staining of PDI-deficient mouse liver semithin sections (Fig. 3b) indicated that the accumulated fatty acids might be UFAs. Thus, to compare the abundance of UFA and SFA esters in the livers of PDI-deficient and control mice, medium- and long-chain fatty acid and fatty acid esters were quantified by UHPLC-MS/MS. The results showed that the PDI-deficient liver accumulated more abundance of UFA esters than control liver, whereas the levels of SFA esters were comparable (Fig. 3c, d).

**Fig. 3: PDI deficiency in liver leads to severe accumulation but secretory blockade of UFA esters.**

To compare the secretion of UFA and SFA ester in the livers of PDI-deficient and control mice, PDI-deficient and control mice were fasted for 16 h and then injected with tyloxapol at 500 mg/kg body weight, 2 h later the venous blood was collected and plasma was produced. The medium and long-chain fatty acid esters in plasma were quantified using the UHPLC-MS/MS. The levels of UFA esters in PDI-deficient plasma markedly decreased while the levels of SFA esters remained comparable with control mice (Fig. 3e, f). These results indicate that PDI plays a vital role in transferring UFA out of the liver.

PDI catalyzes the oxidative folding of MTP

We further studied the potential mechanism underlying the role of PDI in the secretion of UFA ester. A proteomics analysis with quantitative tandem mass tag mass spectrometry (TMT-MS) was performed to screen for altered hepatocyte proteins in the livers of PDI-deficient versus control mice, and we analyzed the expression of proteins that are involved in the composition and assembly of VLDL. The expression levels of ApoB, ApoC1, ApoC3, and ApoE proteins, which compose VLDL, as well as ApoE, ApoC3, CideB, and ADRP proteins, which regulate VLDL assembly³⁶, in the livers of Alb-cre/Pdi^fl/fl mice were comparable to or increased compared with those in the livers of control mice (Supplementary Fig. 4a). These TMT-MS results were verified by Western blotting (Fig. 4a, b). Only ApoB-100 and MTP were significantly reduced (Fig. 4a, b). In contrast to the decrease in MTP protein expression, MTP mRNA expression was increased in the livers of Alb-cre/Pdi^fl/fl mice (Fig. 4c), suggesting that PDI deficiency may result in the degradation of the newly synthesized MTP protein, leading to an increase in MTP mRNA transcription as a feedback mechanism.

To determine whether PDI deficiency is associated with degradation of the MTP protein through a proteasome-dependent or autophagy-dependent pathway, the effects of the proteasome inhibitor MG132 and the autophagy inhibitor 3-methyladenine (3-MA) on hepatocyte MTP expression were tested. Primary hepatocytes from Alb-cre/Pdi^fl/fl mice and Pdi^fl/fl mice were cultured and treated with MG132 or 3-MA at 37 °C for 20 h^43,44, after which the cells were collected for Western blotting analysis. Treatment with MG132 and 3-MA increased the expression of the MTP protein in PDI-deficient hepatocytes (Fig. 4d, e), indicating that the decreased MTP expression induced by PDI deficiency is associated with proteasome- and autophagy-dependent degradation of the MTP protein. Neither MG132 nor 3-MA completely rescued the expression of MTP in cultural PDI-deficient hepatocytes. It might be caused by the rapid decrease in MTP mRNA in the cultured primary hepatocytes (Supplementary Fig. 4b–e).

The degradation of the MTP protein with increased mRNA expression in the PDI-deficient hepatocytes suggested that PDI may be required for the correct folding of MTP. To test this hypothesis, the free thiols of proteins in the lysates of livers from Pdi^fl/fl and Alb-cre/Pdi^fl/fl mice were labeled with 3-(N-maleimido-propionyl)-biocytin (MPB), and the MPB-labeled proteins were pulled down by streptavidin beads. The immunoblotting results revealed that, although the total MTP protein concentration was markedly lower, the free-thiol form of MTP was more abundant in the PDI-deficient liver lysate compared to the normal liver lysate (Fig. 4f). The normalization of the MPB-labeled MTP to the total MTP showed that PDI deficiency increased the amount of free thiol groups in MTP by 6-fold (Fig. 4g), suggesting that PDI is responsible for the oxidative folding of MTP. In contrast, PDI deficiency did not change the amount of free thiol groups in the ApoB-48 protein (Fig. 4g).

Each disulfide bond in MTP folded by PDI is important for the structural stability of MTP

The mature human MTP protein contains 10 cysteines, 8 of which form 4 disulfide bonds: Cys174-Cys194, Cys298-Cys301, Cys440-Cys445, and Cys827-Cys878²⁷(Fig. 5a). These 4 pairs of disulfide bonds are identical to those in mice. To identify the disulfide bonds of MTP that are catalyzed by PDI, seven peptides containing one or two cysteines of MTP and one peptide with no cysteine were synthesized (Fig. 5b) and used for differential cysteine alkylation and PRM-MS analysis (Fig. 5c). The synthesized peptides were alkylated with iodoacetamide (IAM) and analyzed by data-independent acquisition mass spectrometry (DDA-MS) to generate the reference spectral library for PRM-MS (Fig. 5c and Supplementary Table 2). All the targeted peptides in the liver lysates of Pdi^fl/fl and Alb-cre/Pdi^fl/fl mice were quantified via PRM-MS (Supplementary Fig. 5). The ratio of the reduced form of each cysteine was calculated by comparing the peak area of the native alkylated peptide to that of the fully alkylated peptide. The reduced ratio of almost all the targeted peptides increased in the Alb-cre/Pdi^fl/fl lysate (Fig. 5d), indicating the crucial biological function of PDI in the oxidative folding of MTP. In the absence of PDI, the reduced ratio of 3 disulfide bonds in MTP, Cys174-Cys194, Cys440-Cys445, and Cys827-Cys878, significantly increased, suggesting that PDI catalyzes the formation of at least these three disulfide bonds in MTP. The reduced ratio of Cys301 in the Alb-cre/Pdi^fl/fl lysate was decreased (Fig. 5d), which was probably due to the low amount of the peptide in the Pdi^fl/fl and Alb-cre/Pdi^fl/fl liver lysates (Supplementary Fig. 5d), which is near the detection threshold boundary and may cause edge effects.

To map the functional disulfide bonds of MTP for protein expression, HepG2 cells were transfected with plasmids expressing human wild-type MTP or with mutants of cysteine to alanine in Cys174-Cys194, Cys298-Cys301, Cys440-Cys445, Cys827-Cys878, Cys397, and Cys866, respectively. Compared with those of the wild-type MTP protein, the expression levels of the mutants including MTP/C174A-C194A, MTP/C298A-C301A, MTP/C440A-C445A, and MTP/C827A-C878A were reduced to various extents; in particular, the C827A-C878A mutation almost completely eliminated MTP protein expression (Fig. 5e, f). These results indicated that these four disulfide bonds are important for the structural stability of MTP. The mutations of C397A and C886A did not affect the expression (Fig. 5e, f), suggesting that these two free thiols are not involved in thiol-disulfide exchange during MTP folding. The changes in Gibbs free energy (ΔΔG) were calculated with FoldX 3.0 software to evaluate the structural stability of the MTP mutants. As shown in Supplementary Fig. 6a, the ΔΔG values for these mutants were consistent with the MTP expression results: the higher the ΔΔG value was for one mutant, the lower the expression. The structural changes in the oxidized and reduced states of MTP were examined by molecular dynamics (MD) simulations^45,46 based on the crystal structure of intact MTP (PDB identifier 6I7S)²⁷. Ablation of the disulfide bonds Cys174-Cys194, C298-C301, Cys440-Cys445, or Cys827-Cys878 resulted in clearly different conformational distributions from those of the oxidized MTP protein (Supplementary Fig. 6b–f). The results of the ΔΔG calculations and MD simulations further support the data of the cellular expressions of these cysteine mutants, suggesting that the disulfide bonds, whose formations are catalyzed by PDI, are all important for the structural stability of MTP.

PDI deficiency in hepatocytes abolishes ApoB-100 VLDL secretion while maintaining partial ApoB-48 VLDL secretion

To evaluate the role of PDI in VLDL secretion, PDI-deficient and control mice were fasted for 16 h and then injected with tyloxapol at 500 mg/kg body weight. Blood was collected before and 2- and 4 h after injection, and plasma was separated (Supplementary Fig. 7a). Alb-cre/Pdi^fl/fl mice presented significant decreases in plasma TG, CHOL, and HDL levels compared with that of control mice at each time point, while plasma LDL level exhibited no significant difference (Fig. 6a and Supplementary Fig. 7b–d). Fractionation of the pooled plasma of the same genotype by size exclusion chromatography (FPLC) further confirmed the reduction of TG in VLDL and CHOL in LDL and HDL fractions upon hepatic PDI deficiency (Fig. 6b and Supplementary Fig. 7e). In the fragments corresponding to VLDL⁴⁷, expression of ApoB-100 in PDI-deficient group was nearly depleted, while the expression of ApoB-48 was normal, compared with the controls (Fig. 6c). Given that enterocytes also express and secrete ApoB-48, we cannot completely exclude the intestinal origin of plasma ApoB-48 even after fasting mice for 16 h. The observation that plasma ApoB-48 levels in liver-specific PDI knockout mice are comparable to those in control mice after fasting does not necessarily indicate that PDI deficiency does not affect hepatic ApoB-48 secretion. To determine whether PDI deficiency affects hepatic ApoB-48 secretion, we examined the ability of primary hepatocytes from Alb-cre/Pdi^fl/fl mice to secrete ApoB. The results showed that PDI-deficient hepatocytes do not express or secrete ApoB-100, but normally express and partially secrete ApoB-48 (Supplementary Fig. 8). These findings suggest that in PDI-deficient hepatocytes, fatty acid esters are exclusively secreted through ApoB-48 VLDL.

**Fig. 6: PDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL.**

Fatty acid composition analysis was performed on VLDL from PDI knockout and TM-treated mice (Fig. 6d, e). Compared to the control, the total release of SFA in the PDI knockout and TM-treated groups slightly decreased, while the total release of UFA was significantly decreased (Fig. 6f, g). The proportion of SFA esters in these two types of VLDL markedly increased, while the proportion of UFA esters notably decreased (Fig. 6h, i). These results further validate the vital role of MTP-PDI in the transport of UFA esters. A decrease in total SFA secretion was observed in the VLDL results, whereas the plasma results showed no reduction in total SFA secretion. We speculate that this discrepancy may be attributed to the presence of small amounts of chylomicrons or free fatty acids in the plasma, which could have masked the relatively subtle differences.

PDI is indispensable for the export of UFA but not SFA esters from liver

To account for the phenotype that TM-induced ER stress leads to blockage of UFA secretion, we checked the expression of PDI, MTP, ApoB-48, and ApoB-100 in the TM-treated liver. The expression of PDI and MTP are normal in TM-treated liver, while the expression of ApoB-48 and ApoB-100 are significantly decreased, compared with the control (Fig. 7a, b). Meanwhile, both the PDI reductase activity and MTP activity decreased (Fig. 7c, d), which may be responsible for the decrease in UFA secretion.

**Fig. 7: PDI is indispensable for exporting UFA but not SFA esters from liver.**

To further clarify the role of MTP in the differential transport of SFA and UFA, we examined the activity of PDI-MTP transporting SFA esters (1,3-distearin, 2-NBD-X glycerol ester, DSG-NBD) and UFA esters (1,3-diolein, 2-NBD-X glycerol ester, DOG-NBD). The results showed that the activity of transporting DSG-NBD was reduced by 50% in liver lysates lacking PDI-MTP (Fig. 7e), while the activity of transporting DOG-NBD was almost lost (Fig. 7f). These results suggested that there may be an PDI-MTP independent pathway to transfer SFA, while PDI-MTP is indispensable for the transfer of UFA.

In summary, we propose that there are two pathways for esterified fatty acids transferring out of hepatocytes. The first is PDI-MTP dependent, where PDI-MTP shuttles SFA esters and UFA esters to ApoB proteins (ApoB-100 and ApoB-48) to form VLDL. The second pathway is PDI-MTP independent, where SFA esters could be preferentially transferred to ApoB-48 and ApoB-100 VLDL. In PDI-deficient hepatocytes, MTP and ApoB-100 are absent, and the PDI-MTP dependent pathway is disrupted, while the PDI-MTP independent pathway remains functional, allowing for partial secretion of SFA-rich ApoB-48 VLDL. This results in the phenotype of hepatosteatosis and hypolipidemia, as well as differential transport of fatty acids esters (Fig. 7g).

Discussion

In the present study, we found that ER stress is associated with the different transfer mechanisms of SFAs and UFAs. PDI plays an indispensable role in secretion of UFA esters, but not SFA esters. Whereas the other 12 PDI paralogs have limited effects on VLDL secretion. In addition, PDI catalyzes the oxidative folding of MTP, and is responsible for the formation of at least 3 disulfide bonds in MTP. Furthermore, PDI-deficient hepatocytes do not express or secrete ApoB-100, but normally express and partially secrete ApoB-48.

Previous studies have shown that ER stress leads to a reduction in VLDL secretion⁴⁸. In the tunicamycin-induced in vivo ER stress model, many researchers have observed the accumulation of hepatic lipids and the decrease of VLDL secretion^49,50,51. Based on this model, we injected these fasting mice with tyloxapol and identified these newly secreted fatty acid esters using UHPLC-MS/MS, and finally identified the decreased FA content caused by ER stress mainly as UFAs. Lipogenesis is often considered the primary cause of ER stress-induced hepatic steatosis^23,24. However, expressions of fatty acid synthases are decreased in PDI-deficient liver, we speculated that the hepatic steatosis in PDI-deficient mice was mainly attributed to the hinders of VLDL secretion. Since SCD1 is much less reduced than FASN (Supplementary Fig. 3h, i), and the presence of other fatty acid desaturases, thus imported FAs, such as those provided by the adipose tissue, could be adequately desaturated to form UFAs. However, the transfer of UFAs into VLDL is severely impaired in PDI-deficient hepatocyte, leading to the accumulation of UFA esters in the liver. Alternatively, it is possible that desaturated FAs are preferentially imported into the liver under conditions of VLDL secretion deficiency, but further evidence is needed to support this hypothesis.

Apolipoprotein B is a crucial component of lipoprotein particles and exists in two main isoforms: ApoB-48 and ApoB-100, ApoB-48 containing only 48% of the ApoB-100 sequence. In human body, ApoB-48 is exclusively synthesized in the intestine and serves as the main structural protein of chylomicrons, while ApoB-100 is synthesized in the liver and a major protein component of VLDL and LDL. Unlike human liver, murine liver expresses both ApoB-48 and ApoB100, which both can independently form VLDL. Albumin plays a critical role in the transport of free fatty acids in the bloodstream; however, it doesn’t transfer fatty acid esters in the process of VLDL assembly and secretion. The phenotype that PDI-MTP complex-deficient liver can normally secrete lipoprotein particles containing ApoB-48 has been discovered by Dr. Stephen G. Young as early as 1999⁵². Dr. Stephen G. Young called these particles “apo B-48–containing HDL”, “apo B-48–containing HDL-sized particles”, or “small and dense apo B-48 particles”⁵². We just performed fractionation of the newly released lipoproteins by size exclusion chromatography (FPLC) and found that ApoB-48 was located mainly in the VLDL fractions, thus confirming that these particles are ApoB-48 VLDL. In the VLDL secretion assay, we observed that the “secretion” of LDL appeared to be unaffected by the deficiency of PDI-MTP (Fig. 6d). We speculate that these LDL may originate from the ApoB-48 VLDLs, as although tyloxapol inhibits the hydrolysis of TG, it does not completely prevent the hydrolysis of cholesterol esters, hence leading to a slight generation of these LDL. Furthermore, the finding that the assembly of ApoB-48 VLDL could be PDI-MTP-independent challenges the view that the PDI-MTP complex is required for the first step of ApoB lipoprotein assembly, although this view has already been controversial^{52,53,54,55,56,57,58}.

This study provides the first genetic evidence revealing an essential role of PDI in oxidative folding of MTP. For a long time, PDI was thought to serve as a chaperone to maintain MTP in a soluble form and ensure MTP residing in ER^28,29,30, and little is known about the oxidative folding of MTP. In the cells transfected with inactive enzymatic mutant PDI (mPDI), MTP was fully functional in promoting ApoB and triglyceride secretion, based on which the redox activity of PDI was proposed unnecessary for the function of MTP^34,35,36. The conclusion drawn from these cell line-based studies is different from our observation in PDI-deficient mice. We speculate that these differences may be attributed to the varying amount of the remaining PDI protein in PDI-knockdown cells and PDI-deficient livers. In PDI-knockdown cells, about 20% of the remaining endogenous PDI²⁶ is sufficient for catalyzing the correct folding of MTP, and the transfected mPDI could still form a complex with the correctly folded MTP and keep it in a soluble form. In the hepatocytes of Alb-cre/Pdi^fl/fl mice, endogenous PDI was almost depleted (Supplementary Fig. 3b–e). The remaining endogenous PDI in PDI-knockdown cells also causes other different phenotypes compared with that of PDI-deficient mice. For example, there was only about a 20% decrease of MTP protein level in PDI-knockdown cells, and without any changes in TG level²⁶, however, in the PDI-deficient liver, PDI deficiency decreased MTP expression by more than 80% and caused obvious TG accumulation in the liver (Supplementary Fig. 3f).

A very recent study similar to ours was reported by another group⁵⁹. The main phenotypes presented in both studies are consistent, namely that PDI deficiency leads to the loss of MTP and ApoB-100, as well as a dramatic reduction in VLDL secretion. However, we also observed some different phenotypes. For instance, in their study, ApoB-48 was not secreted into the plasma in hepatic PDI-knockout mice, whereas it was in our study. We speculate that this discrepancy may be due to the differences in the recombinase activity of Alb-cre in the mice we used, resulting in different degrees of PDI knockout. Additionally, in their study, MG132 failed to rescue the expression of MTP in the PDI deficient primary hepatocyte, whereas it did in our study. We speculate that this may be due to the different concentration of MG132 used in their study (20 μM) compared to ours (30 μM), and differences in the activity of MG132. Additionally, it should be noted that in this experiment, the primary hepatocytes should be processed from isolation to lysis for Western blotting as quickly as possible. This is because the level of MTTP mRNA in hepatocytes decreases sharply with increased culture time (Supplementary Fig. 4c).

To our knowledge, this study first employed the method of PRM-MS to study the redox states of the disulfide bonds of interest. Currently, a non-targeted DDA based isotopic labeling MS approach is commonly used for resolving and quantifying the redox states of targeted disulfide bonds^45,46. However, in our preliminary experiment, only 3 cysteine-containing peptides in MTP protein were identified via TMT-LC-MS/MS analysis and none cysteine-containing peptide was identified via label-free MS analysis. Since the non-targeted MS approaches, such as TMT and label-free MS, are aimed at obtaining a global quantitative proteomic profile, whereas targeted MS approaches, such as PRM-MS, where fragmentation is forced upon selected precursor masses, thereby certainly increasing the sensitivity for selected target masses excluding all others, may better meet our requirements. Furthermore, PRM-MS has been successfully applied in various studies of posttranslational modifications (PTMs)⁶⁰. As a result, the PRM-MS remarkably improved the coverage, and 9 cysteines in MTP were quantified (Supplementary Table 2), except the Cys298 was missed, which is located in a peptide containing only three amino acids acquired by trypsin digestion, too short to be detected by MS. Therefore, PRM-MS has huge advantages in improving coverage and reproducibility, and is a practical method for targeting the redox states of disulfide bonds in proteins of interest.

The differential transport pathways may explain why SFAs and UFAs have entirely different effects on health. The secretion of UFA esters requires the assistance of PDI-MTP complex, of which the lipid transfer activity could be affected by various conditions, such as ER stress, leading to more possibilities of secretory blockade and then accumulation of UFA esters in the liver. Therefore, for people who suffer from hepatic steatosis or other conditions that arouse high level of liver ER stress, such as taking drug and choric hepatitis, should consider carefully to choose the replacing dietary SFAs with UFAs. Whereas SFAs and cholesterol esters could be transferred to ApoB-48 VLDL via a PDI-MTP independent pathway, they are more readily secreted and delivered into blood circulation, subsequently raising the level of LDL-C, and the risk of cardiovascular disease.

There are some interesting questions remain unresolved in this study, and await further investigation. First, what’s the PDI-MTP independent pathway? Second, because PDI-MTP complex is indispensable for the transport of UFA but not SFA esters, so, does PDI-MTP complex have a preference in transferring UFA esters? And, does MTP have a preference to add lipids to ApoB-48 or ApoB-100? Dr. M. Mahmood Hussain has reported that the triglyceride and phospholipid transfer activities of MTP could be decoupled by a point mutation⁶¹, which indicated that MTP could choose lipids to transfer at the molecular level. Therefore, the hypothesis that PDI-MTP complex preferentially transfers UFA esters may be valid. Furthermore, given the indispensable role of PDI in UFA ester transfer, targeting PDI in drug design for the treatment of lipid metabolism-related diseases might have promising prospects.

Male mice were used in all experiments, as the previous study suggested that sex hormones play a crucial role in determining plasma lipid profiles⁶². In fact, we measured the levels of plasma TG and CHOL in female Alb-cre/Pdi^fl/fl mice, which were also significantly lower than those in control mice. However, we did not analyze the composition of fatty acid in the liver, plasma, and VLDL of female mice.

In summary, our study reveals distinct mechanisms for the intracellular transport of SFA esters and UFA esters during VLDL secretion. PDI is indispensable for the secretion of UFA, while it is not for SFA. The impairment of PDI activity may hinder UFA secretion. These discoveries could potentially provide new insights for the development of therapeutic agents targeting hepatic and plasma lipid regulation.

Methods

Mouse models

Experiments with mice were performed in accordance with institutional guidelines and with the approval of the Institutional Animal Care and Use Committees of Soochow University (Suzhou, China), under the ethics approval number SUDA20230727A04. The overall development and health of the animals were monitored according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. Mice were housed in a specific pathogen-free (SPF) animal facility at Soochow University under a 12-h light/dark cycle, a temperature range of 24 ± 2 °C, and a humidity of 55 ± 5%. All mice were fed with acidified water and standard LabDiet® 5K52 diet (19% protein, 6% fat, 3.2 kcal/g metabolizable energy density). Male mice of 12-week-old were used in all the experiments, unless otherwise specified. Littermates were used as controls in experiments unless specified. Alb^CrePDI^fl/fl mice and Alb^CrePdia3^fl/fl mice were generated by mating albumin-Cre (Alb^cre) mice with Pdi^fl/fl mice and Pdia3^fl/fl mice³⁹. The embryonic stem cells for PDI-floxed mice (clone number: EPD0317_6_D10)⁶³, PDIA2 (PDIp) KO first (Pdia2^−/−) mice (clone number: EPD0753_5_D11), PDIA5 (PDIr) KO first (Pdia5^−/−) mice (clone number: DEPD00576_3_G10), PDIA8 (ERp27) KO first (Pdia8^−/−) mice (clone number: EPD0688_4_C05), PDIA9 (ERp29) KO first (Pdia9^−/−) mice (clone number: EPD0667_5_E05), PDIA13 (TMX3) KO first (Pdia13^−/−) mice (clone number: HEPD0721_1_D05), PDIA14 (TMX4) KO first (Pdia14^−/−) mice (clone number: EPD0684_1_C05), PDIA15 (ERp46) KO first (Pdi15^−/−) mice (clone number: HEPD0564_9_A11), and PDIA19 (ERdj5) KO first (Pdia19^−/−) mice (clone number: HEPD0721_1_D05) were generated by the International Knockout Mouse Consortium (IKMC) at the Cambridge-Suda Genomic Resource Center. After passing production quality control, the ES cells were injected into murine blastocysts and transferred to pseudopregnant female mice to generate the target KO first mice. PDIA16 (ERp18) whole-body knockout mice (CAG^crePdia16^fl/fl) were generated by mating CAG^cre mice with Pdia16^fl/fl mice. Pdia16^fl/fl mice were produced by Cyagen Biosciences Inc. by a CRISPR/Cas9-based protocol. Briefly, the gRNA to mouse Pdia16 gene, the donor vector containing loxP sites, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted conditional knockout offspring. ERp72(PDIA4) KO first (Pdia4^−/−) mice⁴⁰, and TMX1(PDIA11) KO first (Pdia11^−/−) mice⁶⁴ were generated and characterized as previously described. Genotyping of mice was performed by PCR analysis of tail DNA, and the gene expression was confirmed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) or Western blotting. Primers used for genotyping and semiquantitative RT-PCR were listed in Table S3.

Cell lines

HepG2 (SCSP-510) and Hepa1-6 (SCSP-512) cells were obtained from the National Collection of Authenticated Cell Cultures (NCACC) in China. All cells were cultured in a Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C in the presence of 5% CO₂.

Quantitative and semiquantitative RT-PCR

Mouse liver (20–50 mg) or hepatic cells (10⁶ cells) were homogenized in 1 ml TRIzol reagent (Invitrogen, USA), and total RNA was extracted. First-strand cDNA synthesis was performed using the 1st Strand cDNA Synthesis kit (Vazyme) according to the manufacturer’s instructions. RT-qPCR was performed on a 7500 Real-time PCR system (Applied Biosystems) using a SYBR qPCR Master Mix (Vazyme). For semiquantitative RT-PCR, PCR products were analyzed in 1.5% agarose gels, stained with GelRed (Biotium), and photographed under ultraviolet light. The primers are listed in Table S3.

Western blotting and immunoprecipitation

Mice were euthanized and the liver was perfused with phosphate-buffered saline (PBS). The livers or hepatic cells were lysed with lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Triton X-100, 1 mM EDTA, 0.5% sodium deoxycholate, and protease inhibitor cocktail form Roche) and centrifuged to discard the cell debris. Protein concentration was measured using a BCA Protein Assay Kit (Beyotime). For blotting of ApoB-100, the liver lysates or FPLC fragments were boiled with 2× Laemmli sample buffer containing 5% β-mercaptoethanol for 10 min at 70 °C, for other proteins, the temperature was set at 100 °C. Equal amounts of liver or cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked by 5% skimmed milk for 1 h at room temperature and probed with various antibodies as indicated. Actin or GAPDH was used as loading control. For immunoprecipitation, the antibody was added into liver or cell lysate and incubated for 2 h at 4 °C, followed by incubation with protein G beads for 1 h. The beads were washed with lysis buffer 4 times and boiled with 2× Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol for 5 min at 100 °C. The samples were analyzed by SDS-PAGE and Western blotting. The intensity of each band was quantitated using ImageJ software and normalized to the loading control.

Plasma biochemistry parameters and hepatic lipid analysis

Mouse blood was collected by venous puncture using EDTA anticoagulant and centrifuged at 2000 × g for 15 min to obtain plasma. Plasma levels of ALT, AST, TP, ALB, BILT, GLU, BUN, LDH, TG, CHOL, HDL, and LDL were measured using a fully automatic biochemical analyzer (Hitachi 7100). Hepatic levels of TG and CHOL were determined using the assay kits from EnzyChrom^TM (Bioassay, Hayward, CA). Briefly, 400 mg of liver was homogenized in 4 ml of lipid extraction buffer (n-heptane/isopropanol = 4/7, v/v). After centrifugation, the supernatant was measured for the contents of TG and CHOL in livers using the assay kits according to the manufacturer’s instructions.

Histology and transmission electron microscopy

For histological analysis, liver tissues were excised and fixed in 4% paraformaldehyde. After paraffin embedding, the fixed specimens were sectioned at 5 μm and mounted on glass slides. The sections were then deparaffined, hydrated and stained with hematoxylin and eosin (H&E). For Oil Red O staining, the fixed livers were embedded in OCT compound followed by snap freezing. Frozen tissues were sectioned at 8 μm and stained with Oil Red O and hematoxylin according to the manufacturer’s instructions. For Toluidine blue staining, the fresh liver samples were quickly cut into blocks of 1 mm³ and fixed in 2.5% glutaraldehyde + 2.5% paraformaldehyde in 0.1MPBS pH 7.4, for 4 h at 4 °C, rinsed, post-fixed with 1% osmium tetroxide at 4 °C for 2 h. After wash with cold PBS, samples were dehydrated in graded concentrations of alcohol, and embedded in Epon812 (55 °C, 48 h). Semithin sections (1.5 μm) were cut stained with 1% Toluidine Blue^41,42. Images were acquired using an orthostatic optical microscope (Nikon Eclipse E100) with an imaging system (NIKON DS-U3).

For transmission electron microscopy, mice were euthanized and perfused with PBS. A small piece of liver less than 1 mm³ was cut-off quickly and immersed in a fixative solution for TEM (Servicebio) at 4 °C for 4 h. After dehydrating in different concentrations of ethanol solution, the liver was permeated in acetone for 30 min, in a 1:1 mixture of acetone and 812 embedding (Embed-812) for agents 3 h, in a 2:1 mixture of acetone and Embed-812 overnight, and in pure EMBed-812 for 6 h. Embedding was performed at 60 °C for 48 h, and ultrathin sections at 60 nm was cut using an ultramicrotome. Sections were stained with uranyl acetate in pure ethanol for 15 min, and leas citrate for 15 min, then dried overnight at room temperature. The images were collected and analyzed using a transmission electron microscope (HITACHI HT7700).

VLDL secretion assay and fast-protein liquid chromatography (FPLC) analysis

Mice fasted for 16 h were anesthetized and injected i.v. with tyloxapol (150 mg/ml suspension in saline) at the dose of 500 mg/kg body weight. EDTA anticoagulated blood samples were collected at indicated timepoint (0, 2, and 4 h after tyloxapol injection). Plasma was obtained by centrifugation of 2000 × g for 15 min at room temperature, and levels of plasma TG, CHOL, LDL and HDL were measured using a fully automatic biochemical analyzer as described above. For some experiments, the plasma samples were used for quantification of fatty acid esters by UHPLC-MS/MS, for other experiments, pooled plasma samples were used for fast-protein liquid chromatography (FPLC) analysis.

Quantification of fatty acid esters by UHPLC-MS/MS

The total contents of long chain fatty acids and fatty acid esters in liver and plasma were quantified based on UHPLC-MS/MS platform. For liver sample, 20 mg of flash-frozen liver spiked with 400 μL water and deuterated fatty acid internal standard solution (Cayman Chemical) was homogenized using a tissuelyser with beads at 40 Hz for 4 min. The homogenates were mixed with 400 μL methanol and 800 μL chloroform, followed by vortex and centrifugation at 3500 × g for 10 min. The chloroform phase was evaporated to dryness, redissolved in 500 μL of 75% ethanol and 0.5 M KOH and incubated in water bath at 80 °C for 1 h. After cooling, 100 μL formic acid and 600 μL hexane were added, vortexed, and centrifugated for 10 min. The hexane phase was diluted 40-fold and 20 μL was dried under nitrogen gas. The dried residues were reconstituted in 20 μL 1-hydroxybenzotriazole (HoBt), 40 μL cholamine and 20 μL 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), and the mixture was incubated at room temperature for 30 min. After derivatization, 120 μL acetonitrile was added.

The UHPLC-MS/MS analysis was performed on an Agilent 1290 Infinity II UHPLC system coupled to a 6470A Triple Quadrupole mass spectrometry. Samples were injected onto a Hypersil™ GOLD C18 HPLC column (100 mm × 2.1 mm, 1.9 μm, Thermo Scientific) at a flow rate of 0.3 mL/min. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. The chromatographic separation was conducted by a gradient elution program as follows: 0 min, 10% B; 4 min, 30% B; 8 min, 45% B; 11 min, 50% B; 14 min, 70% B; 15 min, 90% B; 15.5–18.0 min, 100% B; 18.1–20 min, 10% B. The eluted analysts were ionized in an electro spray ionization source in positive mode (ESI⁺). The temperatures of source drying gas and sheath gas were 300 °C. The flow rates of source drying gas and sheath gas were 5 and 11 L/min, respectively. The pressure of nebulizer was 40 psi, and capillary voltage was 4000 V. The dynamic multiple reaction monitoring (dMRM) was used to acquire data in optimized MRM transition.

The raw data were processed by MassHunter Workstation Software (version B.08.00, Agilent). The peak areas of each compound in all samples were integrated. Calibration curves of ten-point were constructed by plotting the peak area ratio of each compound to internal standard against concentration of each compound. The concentrations (C, ng/mL) of fatty acids in prepared sample (for determination) were quantified automatically, and finally output for quantitative calculation of milligram liver samples of mouse in Excel with the following formula:

$${{{\rm{Content}}}}({{{\rm{ng}}}}/{{{\rm{mg}}}}\; {{{\rm{sample}}}})=10 \times 40 \times 0.6 \times C/m$$

where C is the concentration quantified in prepared sample (ng/mL), m is the weight of tissue sample (mg).

For plasma sample, 20 μL of mouse plasma was spiked with 220 μL of 0.1% formic acid in water, then the homogenates were mixed with 240 μL methanol and 480 μL chloroform, followed by vortex and centrifugation at 3500 × g for 10 min, and the chloroform phase was evaporated to dryness. The following operation of derivatization and UHPLC-MS/MS analysis were undertaken as same as liver sample. The formula for quantitative calculation of milliliter plasma samples of mouse was as follows:

$${{{\rm{Content}}}}({{{\rm{\mu }}}}{{{\rm{g}}}}/{{{\rm{mL}}}}\; {{{\rm{plasma}}}})=10 \times 30 \times C$$

where C is the quantified concentration in prepared sample (μg/mL).

Isolation and culture of primary hepatocytes

Mice fasted for 12 h were anesthetized and the peritoneal cavity was opened. The liver was perfused in situ through the portal veins at 37 °C with 25 ml of Hank’s buffer containing 0.5 mM EGTA to remove the blood, and the perfusion was continued with 25 ml of digestion buffer (0.5 mg/ml collagenase IV and 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in DMEM-low glucose medium) for 5 min. The liver was then excised and placed into a 10 cm dish containing isolation buffer (DMEM/F-12 containing 10% FBS). The lobes of the liver were teared apart, and the remaining section of the liver was shaken gently to free residual cells. After filtering through a 70-micron membrane, the cell suspension was washed four times with isolation buffer by centrifugation. The hepatocytes were suspended in isolation buffer and plated onto a type I collagen-coated 6-well plates (600,000 cells each well). After incubation for 1 h, the culture medium was replaced by DMEM low glucose medium with 10% FBS. Four hours later, the medium was replaced with a fresh DMEM-low glucose without serum. All the hepatocyte-based studies were completed within 48 h post-plating.

MPB labeling and pull-down assay

The liver tissues isolated from Alb-cre/Pdi^fl/fl and Pdi^fl/fl mice were solubilized using lysis buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4, 1% Triton X-100, 1 mM EDTA, 0.5% sodium deoxycholate, and protease inhibitor cocktail) and centrifuged to discard cell debris. Mouse hepatocytes lysates (2–3 mg/mL, 200 μL) were incubated with 25 μM MPB for 30 min at room temperature, followed by addition of 100 μM GSH to quench the labeling reaction. The lysates were incubated with streptavidin agarose beads with rotation overnight at 4 °C. After washing with lysis buffer, the beads were boiled with 80 μL of 2× Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol for 5 min. The samples were analyzed by SDS-PAGE and Western blotting.

Expression of the recombinant MTP protein in HepG2 cells

The plasmid expressing full-length human MTP cDNA in a pCMV3 vector was purchased from Sino Biological Inc. FLAG-tag sequence DYKDDDDK was subcloned into the plasmid using an Ultra One Step Cloning Kit (Vazyme) for expressing the MTP protein at the C-terminus. The MTP mutants including C174A-C194A, C298A-C301A, C440A-C445A, C827A-C878A, C397A, and C866A were constructed using a Fast Mutagenesis Kit V2 (Vazyme). The primers used for homologous recombination and site-directed mutagenesis are shown in Table S3. Wild-type or mutant MTP plasmids were transfected into HepG2 cells using the Lipofectamine 3000 according to the manufacturer’s instructions. The cells were harvested for 48 h after transfection, and the expressions of wild-type MTP and the variants were detected by Western blotting with anti-FLAG antibody.

Plasma characterization and FPLC analysis

After 16 h of fasting, 12-week-old Pdi^fl/fl and Alb-cre/Pdi^fl/fl male mice were injected i.v. with tyloxapol at a dose of 500 mg/kg body weight. Two hours later, blood was collected with EDTA as an anticoagulant, and plasma was isolated. A total volume of 300 μL pooled plasma samples of the same genotype were subjected to FPLC by using the Superose 6 Increase 10/300 GL columns at 0.5 mL/min flow rate. The plasma samples were fractionated into 40 tubes, each containing 300 μL of volume^65,66. Total triglycerides and cholesterol levels of each fraction were measured by commercial kits (100000220 and 100000180 from Zhongsheng Beikong, respectively) according to the manufacturer’s protocol. The level of ApoB protein in each fraction was detected by Western blotting.

PDI reductase activity assay and MTP activity assay

Fifty milligrams of liver tissue were homogenized in 1 mL of 10 mM Tris-HCl, pH 7.4 buffer containing 150 mM NaCl, 1 mM EDTA, 0.5 mM PMSF and 20 μg/ml leupeptin and then sonicated on ice. After centrifugation at 10,000 × g for 10 min at 4 °C, the intermediate layer of the supernatants was collected. Repeat the above steps twice. After protein quantification, 100 μg of liver lysates were used for PDI activity assay and MTP activity assay according to the instructions provided by the manufacturers (ENZ-51024 and Roar#RB-MTP, respectively)^26,36,67,68.

Triglyceride transfer assays

Triglyceride transfer activity was measured in vitro using an established method^69,70 with slight adjustment. Briefly, reconstitute the lipids (30 mM phosphatidylcholine, PC, Sigma #131601P; 0.2 mM 1,3-diolein, 2-NBD-X glycerol ester, DOG-NBD, Setareh Biotech #6285; 0.2 mM 1,3-distearin, 2-NBD-X glycerol ester, DSG-NBD, synthesized by AHJYHX, Hefei, China) with chloroform in glass vials and cover it with aluminum foil to protect the contents from light. Mass spectrometry identification results for DSG-NBD are provided in Supplementary Data 2. For donor vesicles (DVs), 450 nmol of PC and 14 nmol of DOG-NBD or DSG-NBD per milliliter were used; for acceptor vesicles (AVs), 2400 nmol of PC per milliliter was used. Evaporate chloroform completely in a hood under nitrogen and add 1 mL of vesicle buffer (15 mM Tris-HCl, pH 7.4, 40 mM NaCl, 1 mM EDTA, and 0.02% NaN3) to the vial. Vortex hard until all the lipids are suspended in the solution. The lipids in the glass vials were sonicated on ice with 40% amplitude (Sonics VCX130) until the turbid solution became clear. To remove invisible particles, each lipid samples were centrifuged at 4 °C for 1 h using a SW55 Ti rotor at 250,000 × g which was followed by storing clear supernatants in new glass vials. The vesicles were stored at 4 °C, protected from light, and use within a week.

Liver samples (about 20 mg) were suspended in 1 mL of buffer K (10 mM Tris-Cl, 150 mM NaCl, 1 mM MgCl2, and 1 mM EGTA, pH 7.4) containing 10 μl/mL of protease inhibitor cocktail (Thermo, #78410) and homogenized 20 s on ice, and then sonicated on ice with 35% amplitude, 2 s pulse on and 1 s off, for 90 s. The liver lysates were centrifuged at 13,500 × g for 10 min at 4 °C to remove unbroken cells and cell debris. The clear cell lysates were collected and subjected to immunoprecipitation with anti-PDI antibody to deplete any remaining PDI protein. Then, protein concentration was estimated and 50 μg of protein was used for triglyceride transfer assays. Five μl of AV, 5 μl of DV, and 10 μl of 1% BSA were added to the wells of a 96-well black round bottom microtiter plate, and then 80 μl of liver lysate was added in triplicate. For blank controls, only 80 μl of buffer K was added to vesicles. After brief mixing by aspiration, the plate was transferred to a pre-warmed plate reader set to 37 °C and fluorescence was quantified every minute for 2 h at λex of 463 nm and λem of 536 nm.

Quantification and statistical analysis

No statistical method was used to predetermine the sample size. Specifically, at least triplicates were included for any statistical analysis. Data analysis was performed using the GraphPad Prism 8 software. For parametric comparison, one-way ANOVA for multiple groups and Student’s t test for two groups were used, and the values were expressed as the mean ± SEM. p < 0.05 was considered statistically significant. Asterisks denote corresponding statistical significance *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t test).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data of fatty acid targeted quantitative mass spectrometry generated in this study have been deposited in the MassIVE database under accession code MSV000096686 [https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=172d7183e3074dcea96d82890fba2532]. The data of TMT-based quantitative proteome are available via ProteomeXchange with identifier PXD055737, and the data of parallel reaction monitoring-mass spectrometry (PRM-MS) are available via ProteomeXchange with identifier PXD055650. The remaining data are available within the Article, Supplementary Information, or Source data file. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81970128, 82170129, and 82470132 to Y.W., 31970890 and 82270136 to A.Y., 8217011021 and 82020108003 to D.W.), the Translational Research Grant of NCRCH (2020ZKPA02 to Y.W., 2020WSA04 to A.Y.), the collaboration fund from State Key Laboratory of Radiation Medicine and Protection (GZN1201802 to Y.W.), the Suzhou Science and Technology Development Project (SKJY2021043 to A.Y.), the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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These authors contributed equally: Fengwu Chen, Aizhen Yang, Yue Lu.

Authors and Affiliations

National Clinical Research Center for Hematologic Diseases, Cyrus Tang Medical Institute, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou, 215123, China
Fengwu Chen, Aizhen Yang, Yue Lu, Yuxin Zhang, Jianan Bu, Runlin Guo & Yi Wu
The State Key Laboratory of Membrane Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, 100084, Beijing, China
Fengwu Chen
Department of Hematology, Key Laboratory of Hematology of Hebei Province, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, China
Yuxin Zhang & Jingyu Zhang
National Clinical Research Center for Hematologic Diseases, Institute of Blood and Marrow Transplantation, Collaborative Innovation Center of Hematology, First Affiliated Hospital of Soochow University, Suzhou, 215006, China
Yue Han, Depei Wu & Yi Wu

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Fengwu Chen
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Aizhen Yang
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Yue Lu
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Yuxin Zhang
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Jingyu Zhang
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Jianan Bu
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Runlin Guo
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Depei Wu
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Yi Wu
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Contributions

F.C. and Y.W. conceived and designed the study. F.C., A.Y. and Y.L. performed the experiments. Y.Z., J.B., R.G. and J.Z. assisted with experiments. Y.H. and D.W. contributed critical reagents and support. F.C., D.W. and Y.W. supervised the investigation and wrote the manuscript.

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Correspondence to Fengwu Chen, Depei Wu or Yi Wu.

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Chen, F., Yang, A., Lu, Y. et al. Differential transport pathways of saturated and unsaturated fatty acid esters in male mouse hepatocytes. Nat Commun 16, 1344 (2025). https://doi.org/10.1038/s41467-025-56620-4

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Received: 25 January 2024
Accepted: 20 January 2025
Published: 04 February 2025
DOI: https://doi.org/10.1038/s41467-025-56620-4