Identification of the differential transport pathways of saturated and unsaturated fatty acid esters in hepatocytes

Identification of the differential transport pathways of saturated and unsaturated fatty acid esters in hepatocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of the differential transport pathways of saturated and unsaturated fatty acid esters in hepatocytes Fengwu Chen, Aizhen Yang, Yue Lu, Yuxin Zhang, Jianan Bu, Runlin Guo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3861110/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version 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 hepatic endoplasmic reticulum (ER) stress model. Amoug 13 members of protein disulfide isomerase (PDI) family, only PDIA1 (PDI) deficiency leads to hepatosteatosis and hypolipidemia. In PDI-deficient 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 impairs the assembly and secretion of Apolipoprotein B-100 (ApoB-100) very low-density lipoprotein (VLDL) but not ApoB-48 VLDL. 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. Biological sciences/Physiology/Metabolism/Fat metabolism Health sciences/Endocrinology/Endocrine system and metabolic diseases/Dyslipidaemias Biological sciences/Cell biology/Mechanisms of disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 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 disease 1 . In contrast, high consumption of UFA primarily leads to increases in body weight 2 , hyperglycemia 2 , and liver damage 3 , 4 . Many studies have advocated for the replacement of dietary SFAs with UFAs to reduce cardiovascular risk 5 – 10 . However, some research indicates that the suitability of such dietary replacements for everyone is controversial 11 , 12 . Numerous pieces of evidence suggest that there could be different mechanisms involved in the secretion and accumulation of SFA and UFA esters in cells. Ghada 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 secretion 13 . 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 oocytes 14 , DAOY cancer cells 15 , microglia 16 , renal proximal tubular cells 17 , HepG2 cells 18 , 19 , and primary mouse hepatocytes 18 . 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 – 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. PDI plays an important role in VLDL assembly is also well known. PDI was reported to catalyze the oxidative folding of ApoB-100 25 . More importantly, PDI and MTP constitute a heterodimer, which plays a vital role in VLDL assembly via transferring neutral lipids to the newly synthesized ApoB protein 26 – 28 . MTP has lipid-binding and lipid-transfer activity, while PDI is thought to maintain MTP in a soluble form 29 and ensure MTP in the ER via its KDEL motif 30 , 31 . Although the redox activity of PDI is critical for various biological processes 32 – 35 , it was proposed to be unnecessary for the function of MTP 36 – 38 . However, this conclusion has never been verified in a genetic model. PDI is the prototypic molecular of the PDI family that has more than 20 members, and many members show very similar sequences, structures, and functions. For example, PDI, ERp57, and ERp72 play similar roles in thrombosis and hemostasis 35 , 39 – 42 . 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 has remained elusive. In this study, based on the result that ER stress is associated with the different transport mechanisms of SFA and UFA, we screened 13 strains of the PDI family from hepatocyte-specific and whole-body gene-deficient mice, 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 is essential for the assembly of ApoB-100 containing VLDL, but not ApoB-48 containing VLDL. 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. 1 A). TM caused significantly increased expression of Bip, a marker protein of ER stress, in the liver (Fig. 1 B), and obvious hepatic steatosis (Fig. 1 , C-E). Numerous TG and cholesterol (CHOL) were accumulated (Fig. 1 , F and 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-hour 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, but not LDL or CHOL (Fig. 1 , H-J). To determine whether ER stress has any influence on the transport of SFA and UFA, long-chain fatty acid and fatty acid esters in the plasma which were acquired 2 hours after tyloxapol injection were quantified by an ultra-high performance liquid chromatography- triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). As shown in Fig. 1 K and 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). 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 (Figures S1 and S2). 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. 2 , A and B). In contrast, both TG and CHOL levels in plasma were significantly decreased in Alb-cre/Pdi fl/fl mice (Fig. 2 , A and 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 (Table S1). We further evaluated the effects of PDI deficiency on hepatic lipid homeostasis. Alb-cre/Pdi fl/fl mice at 12 weeks of age did not show any apparent abnormalities including body weight (Figure S2G). 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. 2 C), 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. 2 D). Alb-cre/Pdi fl/fl mice exhibited a 3-fold increase in hepatic TG content compared with that of their littermate controls (Fig. 2 E), although the hepatic CHOL levels of Alb-cre/Pdi fl/fl mice did not increase (Fig. 2 F). The free fatty acid level in the livers of the PDI-deficient mice also increased (Fig. 2 G). 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. 2 , H and I). 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. 3 A), 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 43 , 44 . The large number of brown-colored lipid droplets observed via toluidine blue staining of PDI-deficient mouse liver semithin sections (Fig. 3 B) 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 ultra-high performance liquid chromatography- triple quadrupole tandem mass spectrometry (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. 3 , C and D). 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 hours and then injected with tyloxapol at 500 mg/kg body weight, 2 hours 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. 3 , E and 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 level of ApoB, ApoC1, ApoC3, and ApoE proteins, which compose VLDL, as well as the ApoE, ApoC3, CideB, and ADRP proteins, which regulate VLDL assembly 38 in the livers of Alb-cre/Pdi fl/fl mice were comparable to or increased compared with those in the livers of control mice (Fig. 4 A). These TMT-MS results were verified by Western blotting (Fig. 4 , B and C). Only ApoB-100 and MTP were significantly reduced (Fig. 4 , B and C). 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. 4 , D), 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℃ for 20 hours 45 , 46 , after which the cells were collected for Western blot analysis. Treatment with MG132 and 3-MA increased the expression of the MTP protein in PDI-deficient hepatocytes (Fig. 4 , E and F), 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 (Figure S3). The degradation of the MTP protein with increased mRNA expression in the absence of PDI 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 in the PDI-deficient liver lysate than in the normal liver lysate, the free-thiol form of MTP was more abundant in the PDI-deficient liver lysate than in the normal liver lysate (Fig. 4 G). 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. 4 H), suggesting that PDI is responsible for the oxidative folding of MTP. However, PDI deficiency did not change the amount of free thiol groups in the ApoB-48 protein (Fig. 4 H). 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-Cys878 28 ( Fig. 5 A). These 4 pairs of disulfide bonds are also 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. 5 B) and used for differential cysteine alkylation and PRM-MS analysis (Fig. 5 C). 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. 5 C, and Table S2). All the targeted peptides in the liver lysates of Pdi fl/fl and Alb-cre/Pdi fl/fl mice were quantified via PRM-MS (Fig. S4). 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. 5 D), 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. 5 D), which was probably due to the low amount of the peptide in the Pdi fl/fl and Alb-cre/Pdi fl/fl liver lysates (Fig. S4 D), 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. 5 , E and 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. 5 , E and 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 Fig. S5 A, 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 47 , 48 based on the crystal structure of intact MTP (PDB identifier 6I7S) 28 . 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 (Fig. S5, B-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, of which formations are catalyzed by PDI, are all important for the structural stability of MTP. PDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL To evaluate the role of PDI in VLDL secretion, PDI-deficient and control mice were fasted for 16 hours and then injected with tyloxapol at 500 mg/kg body weight. Blood was collected at 0, 2, and 4 hours after injection, and plasma was produced (Fig. 6 A). 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. 6 , B-E). There were significant decreases in TG and CHOL secretion in Alb-cre/Pdi fl/fl mice, despite no significant difference in generation rate of LDL (Fig. 6 , B-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. 6 , F and G). In the fragments corresponding to VLDL 49 , expression of ApoB-100 in PDI-deficient group was nearly depleted, while the expression of ApoB-48 was normal, compared with the controls (Fig. 6 H). These results indicate that PDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL. PDI is vital 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. 7 , A and B). Meanwhile, both the PDI reductase activity and MTP activity decreased (Fig. 7 , C and D), which may be responsible for the decrease in UFA secretion. In summary, we speculated that there are two pathways for fatty acids transferring out of hepatocytes. One is PDI-MTP dependent, in which PDI-MTP shuttles a bulk of neutral lipids (especially UFA esters) to ApoB protein (mainly ApoB-100) to form pre-VLDL and VLDL2, which finally grow into mature VLDL. The other pathway is PDI-MTP independent, in which cholesteryl and SFA esters are transferred to ApoB-48 in a PDI-MTP-independent manner, and finally grow into a small and dense ApoB-48 VLDL. In PDI-deficient hepatocytes, the assembly and secretion of ApoB-100 VLDL are broken down, while the secretion of ApoB-48 VLDL is normal, leading to the phenotype of hepatosteatosis and hypolipidemia (Fig. 7 E). 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-MTP deficiency does not affect the secretion of ApoB-48 VLDL. Prior to this study, Dr. Henry N. Ginsberg’s group and other researchers demonstrated through extensive studies that ER stress leads to a reduction in VLDL secretion 50 . In the tunicamycin-induced in vivo ER stress model, many researchers have observed the accumulation of hepatic lipids and the decrease of VLDL secretion 51 , 52 . 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. 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 1999 53 . Dr. Stephen G. Young called these partocles “apo B-48–containing HDL”, “apo B-48–containing HDL-sized particles”, or “small and dense apo B-48 particles” 53 . 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. 6 D). 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 is 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 27 , 53 – 60 . 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 29 – 31 , 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 36 – 38 . 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 difference in the amount of the remaining PDI protein in PDI-knockdown cells and PDI-deficient livers. In PDI-knockdown cells, about 20% of the remaining endogenous PDI 25 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 (Fig. S2, B-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 level 25 , however, in the PDI-deficient liver, PDI deficiency decreased MTP expression by more than 80% and caused obvious TG accumulation in the liver (Fig. S1 F). To our knowledge, this study first employed the method of PRM-MS to study the redox states of the disulfide bonds of interest. Currently, the DDA model of MS is commonly used for resolving and quantifying the redox states of targeted disulfide bonds 47 , 48 . 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. DDA model of MS is hard to meet our requirements. PRM-MS is a sensitive and efficient quantification technique with quadrupole-Orbitrap hybrid instruments 61 , and has been successfully applied in various studies of posttranslational modifications (PTMs) 62 . As a result, the PRM-MS remarkably improved the coverage, and 9 cysteines in MTP were quantified (Table S2), 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, are worthy of our further investigation. First, 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 mutation 63 , which indicated that MTP could choose lipids to transfer at the atomic level. Therefore, the hypothesis that PDI-MTP complex preferentially transfers UFA esters may be valid. In addition, hepatic PDI-deficient mice exhibit a very low level of plasma HDL (Fig. 6 E), which may be attributed to the significantly reduced ApoA-I level in PDI-deficient liver (data not shown). The phenotype that PDI deficiency affects the expression of ApoA-I is intriguing, and the underlying mechanism remains elusive. 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. 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). 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-hour 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 formulated with 6.2% fat. 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 Cre PDI fl/fl mice and Alb Cre Pdia3 fl/fl mice were generated by mating albumin-Cre ( Alb cre ) mice with Pdi fl/fl mice and Pdia3 fl/fl mice 41 . The embryonic stem cells for PDI-floxed mice (clone number: EPD0317_6_D10), 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 cre Pdia16 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 −/− ) mice 42 , and TMX1(PDIA11) KO first ( Pdia11 −/− ) mice 64 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℃ in the presence of 5% CO 2 . Quantitative and semiquantitative RT-PCR Mouse liver (20–50 mg) or hepatic cells (10 6 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 minutes at 70℃, for other proteins, the temperature was set at 100℃. 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 hour 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 hours at 4℃, followed by incubation with protein G beads for 1 hours. The beads were washed with lysis buffer 4 times and boiled with 2× Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol for 5 minutes at 100℃. 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 2000g for 15 minutes 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™ (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 mm 3 and fixed in 2.5% glutaraldehyde + 2.5% paraformaldehyde in 0.1MPBS pH 7.4, for 4 h at 4℃, 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℃, 48 h). Semithin sections (1.5 µm) were cut stained with 1% Toluidine Blue 43 , 44 . 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 mm 3 was cut-off quickly and immersed in a fixative solution for TEM (Servicebio) at 4℃ for 4 hours. After dehydrating in different concentrations of ethanol solution, the liver was permeated in acetone for 30 minutes, in a 1:1 mixture of acetone and 812 embedding (Embed-812) for agents3 hours, in a 2:1 mixture of acetone and Embed-812 overnight, and in pure EMBed-812 for 6 hours. 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 hours 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 hours after tyloxapol injection). Plasma was obtained by centrifugation of 2000g 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 400µL 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 0.5M 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 (100mm × 2.1mm, 1.9µm, 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; 8min, 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: Content (ng/ mg sample) = 10×40×0.6× C/m where C is the concentration quantified in prepared sample (ng/mL), m is the weight of tissue sample (mg). For plasma sample, 20µL 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: Content (µg/ mL plasma) = 10×30× C where C is the quantified concentration in prepared sample (µg/mL). TMT-LC-MS/MS-based quantitative proteome analysis of mouse liver samples Quantitative proteomics was performed based on tandem mass tag-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (TMT-LC-MS/MS) technology. Protein extraction, in-solution trypsin digestion, HPLC fractionation, and LC-MS/MS analysis were performed as previously described 65 . Briefly, the livers isolated from 3 pairs of Alb-cre/Pdi fl/fl and Pdi fl/fl mice were mixed respectively and grinded in liquid nitrogen. After adding of four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail, 3 µM treostatin A, 50 mM nicotinamide and 2 mM EDTA), the samples were sonicated three times on ice. After centrifugation at 12,000 g for 10 min at 4°C, the supernatant was collected and the protein concentration was measured. The samples were reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. After trypsin digestion, peptide was desalted, vacuum-dried and reconstituted in 0.5 M triethylammonium bicarbonate (TEAB). The Alb-cre/Pdi fl/fl and Pdi fl/fl samples were incubated with TMT6-126 and TMT6-127 for 2 hours at room temperature according to the manufacturer’s protocol, followed by mixture, desalination, and drying by vacuum centrifugation. The labeled peptides were fractionated into 18 fractions by high pH reverse-phase HPLC using an Agilent 300Extend C18 column and dried by vacuum centrifugation. Each fraction was dissolved in 0.1% formic acid and loaded onto a home-made reversed-phase analytical column (15-cm length, 75 µm i.d.) to separate the peptides, which were injected into a nanospray ionization (NSI) source for ionization and analyzed by a Q Exactive™ Plus Mass Spectrometer (Thermo Scientific). The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) that was set to 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Fixed first mass was set as 100 m/z. The MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Isolation and culture of primary hepatocytes Mice fasted for 12 hours 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 minutes. 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 (600000 cells each well). After incubation for 1 hour, 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 hours 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 minutes 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℃. 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 minutes. The samples were analyzed by SDS-PAGE and Western blotting. Parallel reaction monitoring-mass spectrometry (PRM-MS) To generate a spectral library, we synthesized 7 peptides covering 9 of the 10 cysteines in mature MTP protein, and a cysteine-free peptide that was most abundant in liver lysate detectable by DDA mode of mass spectrometry. Because the peptide with Cys298 acquired by trypsin digestion contained only 3 amino acids that was too short to be detected by MS, this peptide was not included. Briefly, 20 µg of synthetic standard peptide dissolved in 200 mM TEAB was reduced by 5 mM of DTT for 1 hour at 37℃, and alkylated with 15 mM iodoacetamide for 1 hour at room temperature in dark. After desalting and vacuum-dry, these peptides was reconstituted in 0.1% formic acid at the concentration of 0.5 µg/µl and analyzed using DDA mode of mass spectrometry. The raw files were searched using a quantitative proteomics software package (MaxQuant 1.6.15.0), and the processed data was imported into Skyline 20.1 software to construct a spectral library (Table S2). Differential alkylation and PRM analysis of liver lysate samples were then performed. Briefly, livers from 3 pairs of Pdi fl/fl and Alb-cre/Pdi fl/fl mice were grinded in liquid nitrogen and lysed with four volumes of lysis buffer (1% SDS containing 1% protease inhibitor cocktail). After removal of the debris by centrifugation at 12,000 g for 10 min at 4°C, the supernatant was collected, and the protein concentration was determined. Equal amounts of total proteins from each sample were taken and divided into two equal aliquots. One aliquot (group 1) was incubated with 15 mM of IAM for 1 hour at room temperature in dark to alkylate the native reduced cysteines that did not form disulfide bond, followed by precipitation with four volumes of precooled acetone at -20℃ for 2 hours. After washing with precooled acetone three times, the protein precipitation was redissolved in lysis buffer and treated with for 1 hour at 37℃ to fully reduce the oxidized disulfide bond. The newly reduced cysteines were alkylated with 15 mM NEM for 1 hour at room temperature in dark. The other aliquot (group 2) was treated with 10 mM TCEP to reduce all the disulfide bond, and all of the cysteines were alkylated with IAM. The alkylated proteins were precipitated and washed with precooled acetone. After the samples were redissolved in 200 mM TEAB and digested overnight using trypsin (1:50), the digested peptides were dissolved in solvent A (0.1% formic acid and 2% acetonitrile in pure water) and separated using an EASY-nLC 1000 UPLC system. The liquid gradient was comprised of an increase from 8–30% solvent B (0.1% formic acid in 90% acetonitrile) over 16 min, 30–40% in 6 min and up to 80% in 4 min then holding at 80% for the last 4 min, at a constant flow rate of 500 nl/min. The separated peptides were injected into a nanospray ionization (NSI) source for ionization and analyzed by a Q Exactive™ Plus mass spectrometer, in which the previously prepared acquisition method has been uploaded. The electrospray voltage was set to 2.1 kV. Precursor peptides and the secondary fragment ions were detected and analyzed by a high-resolution Orbitrap mass spectrometer. The range for full MS scan was set to 350–1405 m/z. The precursor peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE set to 27/35, and the fragments were detected in the Orbitrap at a resolution of 17,500. Automatic gain control (AGC) was set to 3E6 for full MS and 1E5 for MS/MS. The maximum injection time was set at 50 ms for full MS and 200 ms for MS/MS. The isolation window for MS/MS was set at 1.6 m/z. The MS data were processed using the Skyline software. The identities of the peptides (spectral matching) were confirmed by post-acquisition data processing. The traces of the qualified fragment ions of the peptides were extracted and the peak areas were determined and used for quantifying peptide abundance. The percentage of reduced form of each cysteine was calculated by comparing the peak area of matching peptide in group 1 to that in group 2. The peak area of the cysteine-free peptide was used as an internal standard to normalize the quantification of target peptides. 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 hours after transfection, and the expressions of wild-type MTP and the variants were detected by Western blotting with anti-FLAG antibody. FoldX analysis of MTP variants The changes in Gibbs free energy (∆∆G) induced by the MTP mutations at each disulfide bonds were calculated using FoldX 66 . Briefly, the Gibbs free energy (∆G) of wild-type MTP and mutant MTP was calculated based on the sum of the total intermolecular and intramolecular forces, determined by the FoldX forcefield. The relative ∆∆G was obtained by subtracting ∆G mutant from ∆G wild−type of MTP. The “buildmodel” function was used to build the 3D structure of the mutants based on the structure of wild-type MTP complex (PDB identifier 6I7S), and the structures of both wild type and the mutants were added into GeoPPI to obtain the corresponding predicted values 67 . Molecular dynamics (MD) simulations Simulations were prepared from the published MTP crystal structure (PDB identifier 6I7S). All MD simulations of each redox state (Cys174-Cys194, C298-C301, Cys440-Cys445, and Cys827-Cys878 and none of which was reduced) were performed with the GROMACS 2018 software package, using the parameter sets of the GROMOS 54A7 force field. All the simulations were performed under a SPC water model and periodic boundary conditions, allowing for a minimum distance of 1 nm from the protein to any box edge. The workflow of MD simulations includes four steps: energy minimization, heating, equilibration, and production. First, energy minimization was performed using 5000 steps of steepest descent algorithm, and 5000 steps of conjugate gradient algorithm while keeping the protein backbone atoms completely constrained. Next, the minimized structures were thermalized to 300 K over 50 ps. Then, the thermalized systems were simulated under isothermal-isobaric NPT ensemble over 50 ps to ensure equilibration of the periodic box volume. Finally, production dynamics were performed for 100ns in the NPT ensemble with a 2-fs time step and coordinates were saved every 40 ps for analysis. 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 68 , 69 . 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 10000 g for 10 min at 4℃, 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) 25 , 34 , 38 , 70 . 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). Declarations Conflict-of-interest statement The authors declare no competing interests. Author Contributions F.C. and Y.W. conceived and designed the study. F.C., A.Y and Y.L. performed most of the experiments, Y.Z., J.B., R.G., and Y.H. assisted with experiments. F.C. and Y.W. wrote the manuscript. D.W. and Y.W. supervised the investigation and reviewed the manuscript. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (81770138, 81970128, 31970890, 8217011021, 81730003, 82020108003, 82270136), the Translational Research Grant of NCRCH (2020ZKPA02, 2020WSA04), the collaboration fund from State Key Laboratory of Radiation Medicine and Protection (GZN1201802), the Suzhou Science and Technology Development Project (SKJY2021043), the Priority Academic Program Development of Jiangsu Higher Education Institutions. References de Souza RJ et al (2015) Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies. BMJ 351:h3978 DeWitt E, Dysinger I, Larmer Z, Kolwicz SC Jr (2019) High Saturated and Unsaturated Fat Diets Yield Different Disease Phenotypes in Mice. The FASEB Journal 33, 755.753-755.753 Kirpich IA et al (2016) Saturated and Unsaturated Dietary Fats Differentially Modulate Ethanol-Induced Changes in Gut Microbiome and Metabolome in a Mouse Model of Alcoholic Liver Disease. Am J Pathol 186:765–776 Grujic-Milanovic JD et al (2021) Excesive consumption of unsaturated fatty acids leads to oxidative and inflammatory instability in Wistar rats. Biomed Pharmacother 139:111691 Ulven SM et al (2019) Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat-a randomized controlled dietary intervention study. Am J Clin Nutr 109:1239–1250 Weech M et al (2018) Replacement of dietary saturated fat with unsaturated fats increases numbers of circulating endothelial progenitor cells and decreases numbers of microparticles: findings from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study. Am J Clin Nutr 107:876–882 Lian Z et al (2020) Replacing Saturated Fat With Unsaturated Fat in Western Diet Reduces Foamy Monocytes and Atherosclerosis in Male Ldlr(-/-) Mice. Arterioscler Thromb Vasc Biol 40:72–85 Wang DD et al (2016) Association of Specific Dietary Fats With Total and Cause-Specific Mortality. JAMA Intern Med 176:1134–1145 Tindall AM et al (2019) Replacing Saturated Fat With Walnuts or Vegetable Oils Improves Central Blood Pressure and Serum Lipids in Adults at Risk for Cardiovascular Disease: A Randomized Controlled-Feeding Trial. J Am Heart Assoc 8:e011512 Li Y et al (2015) Saturated Fats Compared With Unsaturated Fats and Sources of Carbohydrates in Relation to Risk of Coronary Heart Disease: A Prospective Cohort Study. J Am Coll Cardiol 66:1538–1548 Hannon BA, Thompson SV, An R, Teran-Garcia M (2017) Clinical Outcomes of Dietary Replacement of Saturated Fatty Acids with Unsaturated Fat Sources in Adults with Overweight and Obesity: A Systematic Review and Meta-Analysis of Randomized Control Trials. Ann Nutr Metab 71:107–117 Vafeiadou K et al (2015) Replacement of saturated with unsaturated fats had no impact on vascular function but beneficial effects on lipid biomarkers, E-selectin, and blood pressure: results from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study. Am J Clin Nutr 102:40–48 Abdel-Fattah G, Fernandez ML, McNamara DJ (1995) Regulation of guinea pig very low density lipoprotein secretion rates by dietary fat saturation. J Lipid Res 36:1188–1198 Carro M, Buschiazzo J, Rios GL, Oresti GM, Alberio RH (2013) Linoleic acid stimulates neutral lipid accumulation in lipid droplets of maturing bovine oocytes. Theriogenology 79:687–694 Pan X et al (2013) Increased unsaturation of lipids in cytoplasmic lipid droplets in DAOY cancer cells in response to cisplatin treatment. Metabolomics 9:722–729 Urso CJ, Zhou H (2021) Palmitic Acid Lipotoxicity in Microglia Cells Is Ameliorated by Unsaturated Fatty Acids. Int J Mol Sci 22 Perez-Marti A et al (2022) Reducing lipid bilayer stress by monounsaturated fatty acids protects renal proximal tubules in diabetes. Elife 11 Mei S et al (2011) Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. J Pharmacol Exp Ther 339:487–498 Cheng KP et al (2018) Unsaturated Fatty Acids Increase the Expression of Hepassocin through a Signal Transducer and Activator of Transcription 3-Dependent Pathway in HepG2 Cells. Lipids 53:863–869 Ajoolabady A et al (2023) Endoplasmic reticulum stress in liver diseases. Hepatology 77:619–639 Malhi H, Kaufman RJ (2011) Endoplasmic reticulum stress in liver disease. J Hepatol 54:795–809 Lebeaupin C et al (2018) Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol 69:927–947 Flamment M, Kammoun HL, Hainault I, Ferre P, Foufelle F (2010) Endoplasmic reticulum stress: a new actor in the development of hepatic steatosis. Curr Opin Lipidol 21:239–246 Kim JY et al (2018) ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P. Cell 175:133–145e115 Wang S et al (2015) Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100. Mol Biol Cell 26:594–604 Rustaeus S et al (1999) Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation. J Nutr 129:463S–466S Gordon DA, Jamil H, Gregg RE, Olofsson SO, Boren J (1996) Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J Biol Chem 271:33047–33053 Biterova EI et al (2019) The crystal structure of human microsomal triglyceride transfer protein. Proc Natl Acad Sci U S A 116:17251–17260 Wetterau JR, Combs KA, McLean LR, Spinner SN, Aggerbeck LP (1991) Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. Biochemistry 30:9728–9735 Wetterau JR, Lin MC, Jamil H (1997) Microsomal triglyceride transfer protein. Biochim Biophys Acta 1345:136–150 Banaszak LJ, Ranatunga WK (2008) The assembly of apoB-containing lipoproteins: a structural biology point of view. Ann Med 40:253–267 Jang I et al (2019) PDIA1/P4HB is required for efficient proinsulin maturation and ss cell health in response to diet induced obesity. Elife 8 Bowley SR, Fang C, Merrill-Skoloff G, Furie BC, Furie B (2017) Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation. Nat Commun 8:14151 Kim YM et al (2018) Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence. Cell Rep 23:3565–3578 Zhou J et al (2015) The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis. J Clin Invest 125:4391–4406 Wang L, Fast DG, Attie AD (1997) The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion. J Biol Chem 272:27644–27651 Quan H, Fan G, Wang CC (1995) Independence of the chaperone activity of protein disulfide isomerase from its thioredoxin-like active site. J Biol Chem 270:17078–17080 Wang S et al (2012) IRE1alpha-XBP1s induces PDI expression to increase MTP activity for hepatic VLDL assembly and lipid homeostasis. Cell Metab 16:473–486 Kim K et al (2013) Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice. Blood 122:1052–1061 Wu Y et al (2012) The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis. Blood 119:1737–1746 Wang L et al (2013) Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the alphaIIbbeta3 integrin. Blood 122:3642–3650 Zhou J et al (2017) The disulfide isomerase ERp72 supports arterial thrombosis in mice. Blood 130:817–828 Cimmino F et al (2021) Dietary Micronutrient Management to Treat Mitochondrial Dysfunction in Diet-Induced Obese Mice. Int J Mol Sci 22 Tarantola E et al (2012) Dipeptidylpeptidase–IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats. Eur J Histochem 56:e41 Wang K et al (2016) MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome. Gut 65:1619–1631 Singh R et al (2009) Autophagy regulates lipid metabolism. Nature 458:1131–1135 Pijning AE et al (2021) An alternate covalent form of platelet alphaIIbbeta3 integrin that resides in focal adhesions and has altered function. Blood 138:1359–1372 Passam F et al (2018) Mechano-redox control of integrin de-adhesion. Elife 7 Kasbi Chadli F, Treguier M, Briand F, Sulpice T, Ouguerram K (2020) Ezetimibe Enhances Macrophage-to-Feces Reverse Cholesterol Transport in Golden Syrian Hamsters Fed a High-Cholesterol Diet. J Pharmacol Exp Ther 375:349–356 Ota T, Gayet C, Ginsberg HN (2008) Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents. J Clin Invest 118:316–332 Chen Y et al (2020) Resveratrol Alleviates Endoplasmic Reticulum Stress-Associated Hepatic Steatosis and Injury in Mice Challenged with Tunicamycin. Mol Nutr Food Res 64:e2000105 Kim SH, Seo H, Kwon D, Yuk DY, Jung YS (2022) Taurine Ameliorates Tunicamycin-Induced Liver Injury by Disrupting the Vicious Cycle between Oxidative Stress and Endoplasmic Reticulum Stress. Life (Basel) 12 Raabe M et al (1999) Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J Clin Invest 103:1287–1298 Pan M, Liang Js JS, Fisher EA, Ginsberg HN (2002) The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis. J Biol Chem 277:4413–4421 Kulinski A, Rustaeus S, Vance JE (2002) Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB, as well as for ApoB lipidation. J Biol Chem 277:31516–31525 Mitchell DM et al (1998) Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence. Proc Natl Acad Sci U S A 95:14733–14738 Dashti N, Manchekar M, Liu Y, Sun Z, Segrest JP (2007) Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells. J Biol Chem 282:28597–28608 Manchekar M et al (2004) Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle. J Biol Chem 279:39757–39766 Richardson PE et al (2005) Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle. Biophys J 88:2789–2800 Wang Y, Tran K, Yao Z (1999) The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins. J Biol Chem 274:27793–27800 Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ (2012) Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics 11:1475–1488 Rauniyar N (2015) Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry. Int J Mol Sci 16:28566–28581 Wilson MH et al (2020) A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein. PLoS Genet 16:e1008941 Zhao Z et al (2019) The transmembrane protein disulfide isomerase TMX1 negatively regulates platelet responses. Blood 133:246–251 Gu X et al (2017) Proteome and Acetylome Analysis Identifies Novel Pathways and Targets Regulated by Perifosine in Neuroblastoma. Sci Rep 7:42062 Schymkowitz J et al (2005) The FoldX web server: an online force field. Nucleic Acids Res 33:W382–388 Liu X, Luo Y, Li P, Song S, Peng J (2021) Deep geometric representations for modeling effects of mutations on protein-protein binding affinity. PLoS Comput Biol 17:e1009284 Wang X et al (2021) Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans. Cell Metab 33:350–366e357 Huang D et al (2021) TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis. Cell Metab 33:1655–1670e1658 Wang X, Asghar M (2017) Protein disulfide isomerase regulates renal AT(1) receptor function and blood pressure in rats. Am J Physiol Renal Physiol 313:F461–F466 Additional Declarations There is NO Competing Interest. Supplementary Files Supplemental20240126.docx Dataset 1 Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3861110","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":270907007,"identity":"0f884a11-7d32-4d08-b1cb-ed6ed1132d0a","order_by":0,"name":"Fengwu Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBAC9gYow4CBgfFBQoUNYS08BxBamA0enEkjTQub5MO2Q0RoYe89/Jq3jUHenL33WEUC2wEG/vbuBPxaeM6lWQO1GO7sOZd2I4HnDoPEmbMb8Gqxl8gxM85tY0gwuJFjdiNB4hmDgUQufi088m+gWu6/MStIMDhMhBYJHuPHEFt4zBgSEojRwpNjxvznHIPhhjM5xhIJB9J4CPqFh/2M8ccZZQzyBsfPGH78+c9Gjr+9F78WIGCTYGD4jzCDkHIQYP5AjKpRMApGwSgYwQAAUetGFY0RBqgAAAAASUVORK5CYII=","orcid":"","institution":"Soochow University","correspondingAuthor":true,"prefix":"","firstName":"Fengwu","middleName":"","lastName":"Chen","suffix":""},{"id":270907008,"identity":"952dffd7-4134-45c0-8f87-c17be53f7f64","order_by":1,"name":"Aizhen Yang","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Aizhen","middleName":"","lastName":"Yang","suffix":""},{"id":270907009,"identity":"e8121a4f-3495-45e7-bf6f-276f8457f36d","order_by":2,"name":"Yue Lu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Lu","suffix":""},{"id":270907010,"identity":"1862e60d-9c43-49f3-bfe7-d0cae047298c","order_by":3,"name":"Yuxin Zhang","email":"","orcid":"","institution":"The Second Hospital of Hebei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Zhang","suffix":""},{"id":270907011,"identity":"94dd0498-2799-490c-8baa-5be7ca9f621c","order_by":4,"name":"Jianan Bu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Jianan","middleName":"","lastName":"Bu","suffix":""},{"id":270907012,"identity":"b220347d-6b5b-45c0-8f2a-986b025adcec","order_by":5,"name":"Runlin Guo","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Runlin","middleName":"","lastName":"Guo","suffix":""},{"id":270907013,"identity":"b32731fa-bf50-4cc1-bb67-2e66ac6514bb","order_by":6,"name":"Yue Han","email":"","orcid":"https://orcid.org/0000-0002-7560-7195","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Han","suffix":""},{"id":270907014,"identity":"8988e7aa-4c1e-4806-a20e-c090ebde0a95","order_by":7,"name":"Depei Wu","email":"","orcid":"https://orcid.org/0000-0002-6312-3863","institution":"First Affiliated Hospital of Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Depei","middleName":"","lastName":"Wu","suffix":""},{"id":270907015,"identity":"9ac32871-887a-4813-8722-417c87c2259f","order_by":8,"name":"Yi Wu","email":"","orcid":"","institution":"Soochow University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-01-13 19:50:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3861110/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3861110/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-56620-4","type":"published","date":"2025-02-04T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50718758,"identity":"d3c37079-f625-4458-869f-c35664e10433","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1119990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress is associated with the differential transport of UFA and SFA esters. \u003c/strong\u003e\u0026nbsp;(A)Schematic representation of the experimental procedures used to establish the tunicamycin (TM)-induced \u003cem\u003ein vivo\u003c/em\u003e ER stress model. The mice were fasted, intraperitoneally injected with 1 mg/kg TM in 150 mM dextrose or with an equivalent volume of dextrose solution, intravenously injected with 500 mg/kg tyloxapol, and sampled at the designated times. (B)The expression of Bip in the liver was detected by Western blotting. Representative photos of the livers (C) and the ratio of liver weight to body weight (D) of the mice are shown. (E) Representative micrographs of liver sections stained with H\u0026amp;E and Oil Red O. Scale bars = 100 μm. Levels of hepatic TG (F) and CHOL (G) were measured using the assay kits according to the manufacturer’s instructions. (H-J) Twenty-four hours after injection of tunicamycin or dextrose, the mice were injected with 500 μg/g tyloxapol. Blood was collected before and 2 hours after tyloxapol injection, the levels of TG (H), LDL (I), and CHOL (J) in plasma were detected by a fully automatic biochemical analyzer. The secretion rates of TG, LDL, and CHOL within 2 minutes were calculated. (K)The contents of each medium and long chain fatty acid in plasma were quantified using UHPLC-MS/MS, and (L) the total content of saturated fatty acids and unsaturated fatty acids in plasma was calculated by summing each of the saturated fatty acids and unsaturated fatty acids. Data are presented as mean ± SEM, *P \u0026lt; 0.05, ***P \u0026lt; 0.001. NS, not significant (two-tailed Student’s t-test). NS, not significant (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/332752fe045b1aa22e5ca2fd.jpeg"},{"id":50718753,"identity":"5bc6e0dd-551a-4cbd-8359-d63ed53fa10e","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1429483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeficiency of hepatic PDI results in hypolipidemia and hepatosteatosis. \u0026nbsp;\u003c/strong\u003ePlasma TG (A)\u0026nbsp; and cholesterol \u0026nbsp;(B) levels of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003e(n = 6 for WT, n = 6 for KO), \u003cem\u003ePdia2\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 3 for WT, n = 3 for KO), \u003cem\u003eAlb-cre/Pdia3\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 7 for WT, n = 7 for KO), \u003cem\u003ePdia4\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 5 for WT, n = 5 for KO), \u003cem\u003ePdia5\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 4 for WT, n = 4 for KO), \u003cem\u003ePdia8\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 3 for WT, n = 3 for KO), \u003cem\u003ePdia9\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 3 for WT, n = 3 for KO), \u003cem\u003ePdia11\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 7 for WT, n = 5 for KO), \u003cem\u003ePdia13\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 6 for WT, n = 4 for KO), \u003cem\u003ePdia14\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 9 for WT, n = 8 for KO), \u003cem\u003ePdia15\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 4 for WT, n = 3 for KO), \u003cem\u003eCAG-cre/Pdia16\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003e(n = 3 for WT, n = 3 for KO), \u003cem\u003ePdia19\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e (n = 5 for WT, n = 6 for KO) mice, and corresponding control mice were shown as indicated. (C) Representative photos of livers of the \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice are shown. (D) The ratio of liver weight to body weight of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice (n = 5) and \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice (n = 3). The levels of hepatic TG (E), CHOL (F), and free fatty acid (G) were measured using the assay kits according to the manufacturer’s instructions. n = 3 for the WT and n = 3 for the KO. Representative micrographs of liver sections stained with H\u0026amp;E (H) and Oil Red O (I) are shown. Scale bars = 50 μm (F), 100 μm (G, H left), and 20 μm (H right). Data are presented as mean ± SEM, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001. NS, not significant (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/76e9cff1603d85cd868779d6.jpeg"},{"id":50718754,"identity":"60872f13-be0f-4282-a65b-9d0c1b8f6fbf","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1015897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI deficiency in liver leads to severe accumulation but secretory blockade of UFA esters.\u003c/strong\u003e (A) Liver sections of \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e \u003c/em\u003eand \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were analyzed by TEM. N, nuclear; Nu, nucleolus; M, mitochondrion; RER, rough endoplasmic reticulum; Go, Golgi apparatus; LD, lipid droplet, GL, glycogen; AP, autophagosome. Scale bars, 5 μm. (B) Toluidine blue staining of liver semithin sections. Drops of unsaturated lipids were brown-colored and saturated lipids were white-colored in Toluidine blue staining. (C) The levels of each medium and long chain fatty acid and fatty acid esters in livers from the \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were quantified using the UHPLC-MS/MS. n = 3 for the WT and n = 3 for the KO. (D) Total content of saturated fatty acids and unsaturated fatty acids in the mouse liver was calculated by summing each of the saturated fatty acids and unsaturated fatty acids. (E) PDI deficiency reduces the secretion of unsaturated fatty acid esters in plasma.\u003cem\u003e Alb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were injected with tyloxapol at 500 mg/kg body weight after a 12-hour fasting, blood was collected and the levels of each medium and long chain fatty acid and fatty acid esters in plasma were quantified using the UHPLC-MS/MS. n = 4 for the WT and n = 4 for the KO. (F) Total content of saturated fatty acids and unsaturated fatty acids in plasma was calculated by summing each of the saturated fatty acids and unsaturated fatty acids. Data are presented as mean ± SEM, *P \u0026lt; 0.05, ****P \u0026lt; 0.0001. NS, not significant (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/68dc4e4a063f0726d1dfa6db.jpeg"},{"id":50718757,"identity":"59349a66-a5f8-4cfc-ab8f-a4ad8027714f","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":766289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI catalyzes the oxidative folding of MTP.\u003c/strong\u003e (A) Proteomic analysis with TMT-LC-MS/MS was used to screen for altered hepatocyte proteins in the livers of PDI-deficient mice versus control mice, and a volcano plot analysis was shown. (B) Western blotting was used to verify the results of mass spectrometry with indicated antibodies. (C) The relative abundance of each protein detected by Western blotting was quantitated by the intensity of each band. (D) The mRNA expression levels of genes that encode proteins composing or affecting VLDL assembly were analyzed by qPCR. (E) Hepatocytes isolated from WT and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were plated and treated with DMSO, 30 μM MG132, or 5 mM 3-MA for 20 hours. Then, the cell lysates were analyzed by Western blotting with antibodies against PDI, MTP, and actin. (F) The relative abundance of MTP in each sample was quantitated by the band intensity determined with ImageJ software. (G) Liver lysates from WT and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were labeled with MPB, and the labeled proteins were pulled down by streptavidin beads and analyzed with Western blotting using the indicated antibodies. (H) The relative abundance of reduced protein was calculated by comparing each density of pull-down protein to the density of before pull-down protein, and then the value for the \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003egroup was normalized to 1. Data are presented as mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 and ****P \u0026lt; 0.0001 (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/b80ac40cd1c9e03a939ec961.jpeg"},{"id":50719928,"identity":"09ea6706-e6a8-42b2-99c0-d82f85883c2a","added_by":"auto","created_at":"2024-02-06 09:36:01","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":591793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEach disulfide bond in MTP folded by PDI is important for the structural stability of MTP\u003c/strong\u003e. (A) Positions of 10 cysteines in human MTP protein. (B)Seven peptides containing one or two cysteines of MTP and one peptide with no cysteine were synthesized and used for differential cysteine alkylation and PRM-MS analysis. (C) Schematic of the PRM-MS workflow. (D) Redox state of 9 cysteines in MTP were measured by PRM-MS. (E) The WT and mutant MTP plasmids were transfected into HepG2 cells, and the expressions of WT and mutant MTP proteins were detected by Western blotting with an anti-FLAG antibody. (F) The relative abundance of each MTP protein was quantitated by the intensity of each WB band. Data are presented as mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001 and ****P \u0026lt; 0.0001. NS, not significant (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/f0a18488ca2378c029023589.jpeg"},{"id":50718759,"identity":"3a0ff9ff-915c-4fe9-8a6b-744fb25df095","added_by":"auto","created_at":"2024-02-06 09:28:02","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":542647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL\u003c/strong\u003e. (A) Twelve-week-old \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(n = 5) and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003e(n = 5) male mice were fasted for 16 hours, followed by i.v. injection with tyloxapol at a concentration of 500 mg/kg body weight. Blood was collected before and after injection at indicated time points. (B-E) The levels of TG (B), CHOL (C), LDL (D), and HDL (E) in plasma were detected by a fully automatic biochemical analyzer. The secretion rates of TG, LDL, and CHOL within 2 hours were calculated. (F-H) Size exclusion fractionation of the pooled plasma of the same genotype by size exclusion chromatography (fast-protein liquid chromatography, FPLC), (F)TGs and (G) CHOL in different fractions were measured. Different species of lipoproteins were indicated by arrows: VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein), and HDL (high-density lipoprotein). (H)Size exclusion fractionation of plasma and expressions of ApoB in every other fractions were examined by Western blotting. The blots of plasma from \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003eand \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e\u003csup\u003e \u003c/sup\u003emice were set as control for normalization. Data are presented as mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001. NS, not significant (two-tailed Student’s t-test).\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/d58b1cafbcde15b3c206133e.jpeg"},{"id":50718755,"identity":"3b119846-4840-471d-823e-50330a4ba4d2","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":743888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI is indispensable for exporting UFA but not SFA esters from liver.\u003c/strong\u003e (A-D) Twelve-week-old male mice were injected with 1 mg of tunicamycin or control buffer (150mM dextrose) per kg bodyweight, 16 hours after the liver sample were collected. (A) Liver lysates were examined by Western blotting using the indicated antibodies. (B) The relative abundance of each protein detected by Western blotting was quantitated by the intensity of each band. (C) PDI reductase activity and (D) MTP activity of the liver lysates were measured according to the manufacturer’s instructions. Data are presented as mean ± SEM, *P \u0026lt; 0.05, **P \u0026lt; 0.01, and ***P \u0026lt; 0.001. NS, not significant (two-tailed Student’s t-test). (E) A model for PDI-dependent and PDI-independent pathway for transferring CHOL, UFA, and SFA esters in hepatocytes. CE, cholesterol ester. ER, endoplasmic reticulum. FA, fatty acids. LDL, low-density lipoprotein. LDs, lipid droplets. MTP, microsomal triglyceride transfer protein. PDI, protein disulfide isomerase. SFA, saturated fatty acid. TG, triglyceride. UFA, unsaturated fatty acid. VLDL, very low-density lipoprotein.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/4df4e265f640e27d59623127.jpeg"},{"id":75494640,"identity":"acc903e6-5a27-4ba5-9532-086aee297552","added_by":"auto","created_at":"2025-02-05 08:05:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7644040,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/be355de3-2286-4612-95e2-b795df6972a6.pdf"},{"id":50718750,"identity":"a2704522-e4cf-45fc-8b46-f7a6e33b20e1","added_by":"auto","created_at":"2024-02-06 09:28:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1541143,"visible":true,"origin":"","legend":"Dataset 1","description":"","filename":"Supplemental20240126.docx","url":"https://assets-eu.researchsquare.com/files/rs-3861110/v1/ce1339effac7c6ac09164965.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Identification of the differential transport pathways of saturated and unsaturated fatty acid esters in hepatocytes","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUFA 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 disease\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In contrast, high consumption of UFA primarily leads to increases in body weight\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, hyperglycemia\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and liver damage\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Many studies have advocated for the replacement of dietary SFAs with UFAs to reduce cardiovascular risk\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, some research indicates that the suitability of such dietary replacements for everyone is controversial\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNumerous pieces of evidence suggest that there could be different mechanisms involved in the secretion and accumulation of SFA and UFA esters in cells. Ghada Abdel-Fattah \u003cem\u003eet al.\u003c/em\u003e 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 secretion\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. 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 oocytes\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, DAOY cancer cells\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, microglia\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, renal proximal tubular cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, HepG2 cells\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and primary mouse hepatocytes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eHowever, little is known about the different transport mechanisms of UFA and SFA esters in cells.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eIt's well known that endoplasmic reticulum (ER) stress causes hepatic steatosis \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and the main metabolic pathway affected by ER stress is thought to be lipogenesis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003ePDI plays an important role in VLDL assembly is also well known. PDI was reported to catalyze the oxidative folding of ApoB-100\u003csup\u003e25\u003c/sup\u003e. More importantly, PDI and MTP constitute a heterodimer, which plays a vital role in VLDL assembly via transferring neutral lipids to the newly synthesized ApoB protein\u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. MTP has lipid-binding and lipid-transfer activity, while PDI is thought to maintain MTP in a soluble form\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and ensure MTP in the ER via its KDEL motif\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Although the redox activity of PDI is critical for various biological processes \u003csup\u003e\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, it was proposed to be unnecessary for the function of MTP \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, this conclusion has never been verified in a genetic model. PDI is the prototypic molecular of the PDI family that has more than 20 members, and many members show very similar sequences, structures, and functions. For example, PDI, ERp57, and ERp72 play similar roles in thrombosis and hemostasis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. 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 has remained elusive.\u003c/p\u003e \u003cp\u003eIn this study, based on the result that ER stress is associated with the different transport mechanisms of SFA and UFA, we screened 13 strains of the PDI family from hepatocyte-specific and whole-body gene-deficient mice, 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 is essential for the assembly of ApoB-100 containing VLDL, but not ApoB-48 containing VLDL. 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eER stress is associated with differential transport of UFA and SFA esters.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether ER stress affects the transfer of different FA esters out of liver, we constructed a tunicamycin (TM)-induced \u003cem\u003ein vivo\u003c/em\u003e ER stress model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). TM caused significantly increased expression of Bip, a marker protein of ER stress, in the liver (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and obvious hepatic steatosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, C-E). Numerous TG and cholesterol (CHOL) were accumulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, F and 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-hour 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, but not LDL or CHOL (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, H-J). To determine whether ER stress has any influence on the transport of SFA and UFA, long-chain fatty acid and fatty acid esters in the plasma which were acquired 2 hours after tyloxapol injection were quantified by an ultra-high performance liquid chromatography- triple quadrupole tandem mass spectrometry (UHPLC-MS/MS). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK and 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).\u003c/p\u003e\n\u003ch3\u003eDeficiency of hepatic PDI results in hypolipidemia and hepatosteatosis\u003c/h3\u003e\n\u003cp\u003eThe 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 (Figures S1 and S2). As shown by the results of plasma biochemical test, \u003cem\u003ePdia2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eAlb-cre/Pdia3\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia4\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia8\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia9\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia11\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia13\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia14\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003ePdia15\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eCAG-cre/Pdia16\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003ePdia19\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice had comparable levels of plasma TG and CHOL with their littermate controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, A and B). In contrast, both TG and CHOL levels in plasma were significantly decreased in \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, A and 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 \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, while other biochemical parameters remained normal (Table S1).\u003c/p\u003e\u003cp\u003eWe further evaluated the effects of PDI deficiency on hepatic lipid homeostasis. \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice at 12 weeks of age did not show any apparent abnormalities including body weight (Figure S2G). However, the livers of the \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice appeared enlarged and whitish compared with those of the littermate \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), and the ratio of liver weight to body weight was significantly greater in the \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice than that of the \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice exhibited a 3-fold increase in hepatic TG content compared with that of their littermate controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), although the hepatic CHOL levels of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice did not increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). The free fatty acid level in the livers of the PDI-deficient mice also increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). As shown by hematoxylin and eosin (H\u0026amp;E) and Oil Red O staining, the livers of the PDI-deficient mice exhibited severe lipid accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, H and I). The finding that PDI-deficient mice suffer from hypolipidemia and hepatosteatosis indicated that PDI is essential for lipid transfer out of the liver.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePDI deficiency in the liver leads to severe accumulation but secretory blockade of UFA esters\u003c/h2\u003e \u003cp\u003eAs 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), 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 \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. The large number of brown-colored lipid droplets observed via toluidine blue staining of PDI-deficient mouse liver semithin sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) 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 ultra-high performance liquid chromatography- triple quadrupole tandem mass spectrometry (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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, C and D).\u003c/p\u003e \u003cp\u003eTo 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 hours and then injected with tyloxapol at 500 mg/kg body weight, 2 hours 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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, E and F). These results indicate that PDI plays a vital role in transferring UFA out of the liver\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePDI catalyzes the oxidative folding of MTP\u003c/h2\u003e \u003cp\u003eWe 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 level of ApoB, ApoC1, ApoC3, and ApoE proteins, which compose VLDL, as well as the ApoE, ApoC3, CideB, and ADRP proteins, which regulate VLDL assembly \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e in the livers of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were comparable to or increased compared with those in the livers of control mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These TMT-MS results were verified by Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B and C). Only ApoB-100 and MTP were significantly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B and C). In contrast to the decrease in MTP protein expression, MTP mRNA expression was increased in the livers of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, D), 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.\u003c/p\u003e\u003cp\u003eTo 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 \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were cultured and treated with MG132 or 3-MA at 37℃ for 20 hours \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, after which the cells were collected for Western blot analysis. Treatment with MG132 and 3-MA increased the expression of the MTP protein in PDI-deficient hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, E and F), 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 \u003cem\u003eMTP\u003c/em\u003e mRNA in the cultured primary hepatocytes (Figure S3).\u003c/p\u003e \u003cp\u003eThe degradation of the MTP protein with increased mRNA expression in the absence of PDI 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 \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e 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 in the PDI-deficient liver lysate than in the normal liver lysate, the free-thiol form of MTP was more abundant in the PDI-deficient liver lysate than in the normal liver lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), suggesting that PDI is responsible for the oxidative folding of MTP. However, PDI deficiency did not change the amount of free thiol groups in the ApoB-48 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEach disulfide bond in MTP folded by PDI is important for the structural stability of MTP.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe mature human MTP protein contains 10 cysteines, 8 of which form 4 disulfide bonds: Cys174-Cys194, Cys298-Cys301, Cys440-Cys445, and Cys827-Cys878 \u003csup\u003e28\u003c/sup\u003e( Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). These 4 pairs of disulfide bonds are also 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and used for differential cysteine alkylation and PRM-MS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, and Table S2). All the targeted peptides in the liver lysates of \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were quantified via PRM-MS (Fig. S4). 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 \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e lysate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), 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 \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e lysate was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), which was probably due to the low amount of the peptide in the \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e liver lysates (Fig. S4 D), which is near the detection threshold boundary and may cause edge effects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, E and 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, E and 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 Fig. S5 A, 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 \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e based on the crystal structure of intact MTP (PDB identifier 6I7S)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. 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 (Fig. S5, B-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, of which formations are catalyzed by PDI, are all important for the structural stability of MTP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL\u003c/h2\u003e \u003cp\u003eTo evaluate the role of PDI in VLDL secretion, PDI-deficient and control mice were fasted for 16 hours and then injected with tyloxapol at 500 mg/kg body weight. Blood was collected at 0, 2, and 4 hours after injection, and plasma was produced (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B-E). There were significant decreases in TG and CHOL secretion in \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, despite no significant difference in generation rate of LDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, B-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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, F and G). In the fragments corresponding to VLDL\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, expression of ApoB-100 in PDI-deficient group was nearly depleted, while the expression of ApoB-48 was normal, compared with the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). These results indicate that PDI deficiency impairs the assembly and secretion of ApoB-100 VLDL but not ApoB-48 VLDL.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePDI is vital for the export of UFA but not SFA esters from liver\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, A and B). Meanwhile, both the PDI reductase activity and MTP activity decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, C and D), which may be responsible for the decrease in UFA secretion.\u003c/p\u003e \u003cp\u003eIn summary, we speculated that there are two pathways for fatty acids transferring out of hepatocytes. One is PDI-MTP dependent, in which PDI-MTP shuttles a bulk of neutral lipids (especially UFA esters) to ApoB protein (mainly ApoB-100) to form pre-VLDL and VLDL2, which finally grow into mature VLDL. The other pathway is PDI-MTP independent, in which cholesteryl and SFA esters are transferred to ApoB-48 in a PDI-MTP-independent manner, and finally grow into a small and dense ApoB-48 VLDL. In PDI-deficient hepatocytes, the assembly and secretion of ApoB-100 VLDL are broken down, while the secretion of ApoB-48 VLDL is normal, leading to the phenotype of hepatosteatosis and hypolipidemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn 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-MTP deficiency does not affect the secretion of ApoB-48 VLDL.\u003c/p\u003e \u003cp\u003ePrior to this study, Dr. Henry N. Ginsberg\u0026rsquo;s group and other researchers demonstrated through extensive studies that ER stress leads to a reduction in VLDL secretion\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In the tunicamycin-induced \u003cem\u003ein vivo\u003c/em\u003e ER stress model, many researchers have observed the accumulation of hepatic lipids and the decrease of VLDL secretion\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eThe 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 1999\u003csup\u003e53\u003c/sup\u003e. Dr. Stephen G. Young called these partocles \u0026ldquo;apo B-48\u0026ndash;containing HDL\u0026rdquo;, \u0026ldquo;apo B-48\u0026ndash;containing HDL-sized particles\u0026rdquo;, or \u0026ldquo;small and dense apo B-48 particles\u0026rdquo;\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). 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 is 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 \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR54 CR55 CR56 CR57 CR58 CR59\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis 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 \u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, 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 \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. 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 difference in the amount of the remaining PDI protein in PDI-knockdown cells and PDI-deficient livers. In PDI-knockdown cells, about 20% of the remaining endogenous PDI\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e 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 \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, endogenous PDI was almost depleted (Fig. S2, B-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 level \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, however, in the PDI-deficient liver, PDI deficiency decreased MTP expression by more than 80% and caused obvious TG accumulation in the liver (Fig. S1 F).\u003c/p\u003e \u003cp\u003eTo our knowledge, this study first employed the method of PRM-MS to study the redox states of the disulfide bonds of interest. Currently, the DDA model of MS is commonly used for resolving and quantifying the redox states of targeted disulfide bonds\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. 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. DDA model of MS is hard to meet our requirements. PRM-MS is a sensitive and efficient quantification technique with quadrupole-Orbitrap hybrid instruments\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, and has been successfully applied in various studies of posttranslational modifications (PTMs) \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. As a result, the PRM-MS remarkably improved the coverage, and 9 cysteines in MTP were quantified (Table S2), 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, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePRM-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.\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThe differential transport pathways may explain why SFAs and UFAs have entirely different effects on health.\u003c/span\u003e 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.\u003c/p\u003e \u003cp\u003eThere are some interesting questions remain unresolved in this study, are worthy of our further investigation. First, 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 mutation\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, which indicated that MTP could choose lipids to transfer at the atomic level. Therefore, the hypothesis that PDI-MTP complex preferentially transfers UFA esters may be valid. In addition, hepatic PDI-deficient mice exhibit a very low level of plasma HDL (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), which may be attributed to the significantly reduced ApoA-I level in PDI-deficient liver (data not shown). The phenotype that PDI deficiency affects the expression of ApoA-I is intriguing, and the underlying mechanism remains elusive. 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.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMouse models\u003c/h2\u003e \u003cp\u003e 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). 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-hour light/dark cycle, a temperature range of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, and a humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. All mice were fed with acidified water and standard LabDiet\u0026reg; 5K52 diet formulated with 6.2% fat. Male mice of 12-week-old were used in all the experiments, unless otherwise specified. Littermates were used as controls in experiments unless specified. \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePDI\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003eCre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePdia3\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were generated by mating albumin-Cre (\u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e) mice with \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice and \u003cem\u003ePdia3\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. The embryonic stem cells for PDI-floxed mice (clone number: EPD0317_6_D10), PDIA2 (PDIp) KO first (\u003cem\u003ePdia2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: EPD0753_5_D11), PDIA5 (PDIr) KO first (\u003cem\u003ePdia5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: DEPD00576_3_G10), PDIA8 (ERp27) KO first (\u003cem\u003ePdia8\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: EPD0688_4_C05), PDIA9 (ERp29) KO first (\u003cem\u003ePdia9\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: EPD0667_5_E05), PDIA13 (TMX3) KO first (\u003cem\u003ePdia13\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: HEPD0721_1_D05), PDIA14 (TMX4) KO first (\u003cem\u003ePdia14\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: EPD0684_1_C05), PDIA15 (ERp46) KO first (\u003cem\u003ePdi15\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice (clone number: HEPD0564_9_A11), and PDIA19 (ERdj5) KO first (\u003cem\u003ePdia19\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) 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 (\u003cem\u003eCAG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ePdia16\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e) were generated by mating \u003cem\u003eCAG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e mice with \u003cem\u003ePdia16\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. \u003cem\u003ePdia16\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were produced by Cyagen Biosciences Inc. by a CRISPR/Cas9-based protocol. Briefly, the gRNA to mouse \u003cem\u003ePdia16\u003c/em\u003e gene, the donor vector containing \u003cem\u003eloxP\u003c/em\u003e sites, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted conditional knockout offspring. ERp72(PDIA4) KO first (\u003cem\u003ePdia4\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, and TMX1(PDIA11) KO first (\u003cem\u003ePdia11\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eHepG2 (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\u0026rsquo;s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in the presence of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative and semiquantitative RT-PCR\u003c/h2\u003e \u003cp\u003eMouse liver (20\u0026ndash;50 mg) or hepatic cells (10\u003csup\u003e6\u003c/sup\u003e 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\u0026rsquo;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting and immunoprecipitation\u003c/h2\u003e \u003cp\u003eMice 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\u0026times; Laemmli sample buffer containing 5% β-mercaptoethanol for 10 minutes at 70℃, for other proteins, the temperature was set at 100℃. 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 hour 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 hours at 4℃, followed by incubation with protein G beads for 1 hours. The beads were washed with lysis buffer 4 times and boiled with 2\u0026times; Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol for 5 minutes at 100℃. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlasma biochemistry parameters and hepatic lipid analysis\u003c/h2\u003e \u003cp\u003eMouse blood was collected by venous puncture using EDTA anticoagulant and centrifuged at 2000g for 15 minutes 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\u0026trade; (Bioassay, Hayward, CA). Briefly, 400 mg of liver was homogenized in 4 ml of lipid extraction buffer (n-heptane/isopropanol\u0026thinsp;=\u0026thinsp;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\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eHistology and transmission electron microscopy\u003c/h2\u003e \u003cp\u003eFor histological analysis, liver tissues were excised and fixed in 4% paraformaldehyde. After paraffin embedding, the fixed specimens were sectioned at 5 \u0026micro;m and mounted on glass slides. The sections were then deparaffined, hydrated and stained with hematoxylin and eosin (H\u0026amp;E). For Oil Red O staining, the fixed livers were embedded in OCT compound followed by snap freezing. Frozen tissues were sectioned at 8 \u0026micro;m and stained with Oil Red O and hematoxylin according to the manufacturer\u0026rsquo;s instructions. For Toluidine blue staining, the fresh liver samples were quickly cut into blocks of 1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and fixed in 2.5% glutaraldehyde\u0026thinsp;+\u0026thinsp;2.5% paraformaldehyde in 0.1MPBS pH 7.4, for 4 h at 4℃, rinsed, post-fixed with 1% osmium tetroxide at 4\u0026deg;C for 2 h. After wash with cold PBS, samples were dehydrated in graded concentrations of alcohol, and embedded in Epon812 (55℃, 48 h). Semithin sections (1.5 \u0026micro;m) were cut stained with 1% Toluidine Blue\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Images were acquired using an orthostatic optical microscope (Nikon Eclipse E100) with an imaging system (NIKON DS-U3).\u003c/p\u003e \u003cp\u003eFor transmission electron microscopy, mice were euthanized and perfused with PBS. A small piece of liver less than 1 mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e was cut-off quickly and immersed in a fixative solution for TEM (Servicebio) at 4℃ for 4 hours. After dehydrating in different concentrations of ethanol solution, the liver was permeated in acetone for 30 minutes, in a 1:1 mixture of acetone and 812 embedding (Embed-812) for agents3 hours, in a 2:1 mixture of acetone and Embed-812 overnight, and in pure EMBed-812 for 6 hours. Embedding was performed at 60\u0026deg;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).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eVLDL secretion assay and fast-protein liquid chromatography (FPLC) analysis\u003c/h2\u003e \u003cp\u003eMice fasted for 16 hours 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 hours after tyloxapol injection). Plasma was obtained by centrifugation of 2000g 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of fatty acid esters by UHPLC-MS/MS\u003c/h2\u003e \u003cp\u003eThe 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\u0026micro;L 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 \u0026micro;L methanol and 800 \u0026micro;L chloroform, followed by vortex and centrifugation at 3500 g for 10 min. The chloroform phase was evaporated to dryness, redissolved in 500 \u0026micro;L of 75% ethanol and 0.5M KOH and incubated in water bath at 80\u0026deg;C for 1 h. After cooling, 100 \u0026micro;L formic acid and 600 \u0026micro;L hexane were added, vortexed, and centrifugated for 10 min. The hexane phase was diluted 40-fold and 20 \u0026micro;L was dried under nitrogen gas. The dried residues were reconstituted in 20 \u0026micro;L 1-hydroxybenzotriazole (HoBt), 40 \u0026micro;L cholamine and 20 \u0026micro;L 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 \u0026micro;L acetonitrile was added.\u003c/p\u003e \u003cp\u003eThe 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\u0026trade; GOLD C18 HPLC column (100mm \u0026times; 2.1mm, 1.9\u0026micro;m, 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; 8min, 45% B; 11 min, 50% B; 14 min, 70% B; 15 min, 90% B; 15.5\u0026ndash;18.0 min, 100% B; 18.1\u0026ndash;20 min, 10%B. The eluted analysts were ionized in an electro spray ionization source in positive mode (ESI\u003csup\u003e+\u003c/sup\u003e). The temperatures of source drying gas and sheath gas were 300\u0026deg;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.\u003c/p\u003e \u003cp\u003eThe 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:\u003c/p\u003e \u003cp\u003eContent (ng/ mg sample)\u0026thinsp;=\u0026thinsp;10\u0026times;40\u0026times;0.6\u0026times;\u003cem\u003eC/m\u003c/em\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e is the concentration quantified in prepared sample (ng/mL), \u003cem\u003em\u003c/em\u003e is the weight of tissue sample (mg).\u003c/p\u003e \u003cp\u003eFor plasma sample, 20\u0026micro;L of mouse plasma was spiked with 220 \u0026micro;L of 0.1% formic acid in water, then the homogenates were mixed with 240 \u0026micro;L methanol and 480 \u0026micro;L 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:\u003c/p\u003e \u003cp\u003eContent (\u0026micro;g/ mL plasma)\u0026thinsp;=\u0026thinsp;10\u0026times;30\u0026times;\u003cem\u003eC\u003c/em\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eC\u003c/em\u003e is the quantified concentration in prepared sample (\u0026micro;g/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTMT-LC-MS/MS-based quantitative proteome analysis of mouse liver samples\u003c/h2\u003e \u003cp\u003eQuantitative proteomics was performed based on tandem mass tag-labeling combined with liquid chromatography-mass spectrometry/mass spectrometry (TMT-LC-MS/MS) technology. Protein extraction, in-solution trypsin digestion, HPLC fractionation, and LC-MS/MS analysis were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Briefly, the livers isolated from 3 pairs of \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were mixed respectively and grinded in liquid nitrogen. After adding of four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail, 3 \u0026micro;M treostatin A, 50 mM nicotinamide and 2 mM EDTA), the samples were sonicated three times on ice. After centrifugation at 12,000 g for 10 min at 4\u0026deg;C, the supernatant was collected and the protein concentration was measured. The samples were reduced with 5 mM dithiothreitol for 30 min at 56\u0026deg;C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. After trypsin digestion, peptide was desalted, vacuum-dried and reconstituted in 0.5 M triethylammonium bicarbonate (TEAB). The \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e samples were incubated with TMT6-126 and TMT6-127 for 2 hours at room temperature according to the manufacturer\u0026rsquo;s protocol, followed by mixture, desalination, and drying by vacuum centrifugation. The labeled peptides were fractionated into 18 fractions by high pH reverse-phase HPLC using an Agilent 300Extend C18 column and dried by vacuum centrifugation. Each fraction was dissolved in 0.1% formic acid and loaded onto a home-made reversed-phase analytical column (15-cm length, 75 \u0026micro;m i.d.) to separate the peptides, which were injected into a nanospray ionization (NSI) source for ionization and analyzed by a Q Exactive\u0026trade; Plus Mass Spectrometer (Thermo Scientific). The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) that was set to 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Fixed first mass was set as 100 m/z. The MS/MS data were processed using the Maxquant search engine (v.1.5.2.8).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of primary hepatocytes\u003c/h2\u003e \u003cp\u003eMice fasted for 12 hours were anesthetized and the peritoneal cavity was opened. The liver was perfused \u003cem\u003ein situ\u003c/em\u003e through the portal veins at 37\u0026deg;C with 25 ml of Hank\u0026rsquo;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 minutes. 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 (600000 cells each well). After incubation for 1 hour, 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 hours post-plating.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMPB labeling and pull-down assay\u003c/h2\u003e \u003cp\u003eThe liver tissues isolated from \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e 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\u0026ndash;3 mg/mL, 200 \u0026micro;L) were incubated with 25 \u0026micro;M MPB for 30 minutes at room temperature, followed by addition of 100 \u0026micro;M GSH to quench the labeling reaction. The lysates were incubated with streptavidin agarose beads with rotation overnight at 4℃. After washing with lysis buffer, the beads were boiled with 80 \u0026micro;L of 2\u0026times; Laemmli sample buffer (Bio-Rad) containing 5% β-mercaptoethanol for 5 minutes. The samples were analyzed by SDS-PAGE and Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eParallel reaction monitoring-mass spectrometry (PRM-MS)\u003c/h2\u003e \u003cp\u003eTo generate a spectral library, we synthesized 7 peptides covering 9 of the 10 cysteines in mature MTP protein, and a cysteine-free peptide that was most abundant in liver lysate detectable by DDA mode of mass spectrometry. Because the peptide with Cys298 acquired by trypsin digestion contained only 3 amino acids that was too short to be detected by MS, this peptide was not included. Briefly, 20 \u0026micro;g of synthetic standard peptide dissolved in 200 mM TEAB was reduced by 5 mM of DTT for 1 hour at 37℃, and alkylated with 15 mM iodoacetamide for 1 hour at room temperature in dark. After desalting and vacuum-dry, these peptides was reconstituted in 0.1% formic acid at the concentration of 0.5 \u0026micro;g/\u0026micro;l and analyzed using DDA mode of mass spectrometry. The raw files were searched using a quantitative proteomics software package (MaxQuant 1.6.15.0), and the processed data was imported into Skyline 20.1 software to construct a spectral library (Table S2).\u003c/p\u003e \u003cp\u003eDifferential alkylation and PRM analysis of liver lysate samples were then performed. Briefly, livers from 3 pairs of \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were grinded in liquid nitrogen and lysed with four volumes of lysis buffer (1% SDS containing 1% protease inhibitor cocktail). After removal of the debris by centrifugation at 12,000 g for 10 min at 4\u0026deg;C, the supernatant was collected, and the protein concentration was determined. Equal amounts of total proteins from each sample were taken and divided into two equal aliquots. One aliquot (group 1) was incubated with 15 mM of IAM for 1 hour at room temperature in dark to alkylate the native reduced cysteines that did not form disulfide bond, followed by precipitation with four volumes of precooled acetone at -20℃ for 2 hours. After washing with precooled acetone three times, the protein precipitation was redissolved in lysis buffer and treated with for 1 hour at 37℃ to fully reduce the oxidized disulfide bond. The newly reduced cysteines were alkylated with 15 mM NEM for 1 hour at room temperature in dark. The other aliquot (group 2) was treated with 10 mM TCEP to reduce all the disulfide bond, and all of the cysteines were alkylated with IAM.\u003c/p\u003e \u003cp\u003eThe alkylated proteins were precipitated and washed with precooled acetone. After the samples were redissolved in 200 mM TEAB and digested overnight using trypsin (1:50), the digested peptides were dissolved in solvent A (0.1% formic acid and 2% acetonitrile in pure water) and separated using an EASY-nLC 1000 UPLC system. The liquid gradient was comprised of an increase from 8\u0026ndash;30% solvent B (0.1% formic acid in 90% acetonitrile) over 16 min, 30\u0026ndash;40% in 6 min and up to 80% in 4 min then holding at 80% for the last 4 min, at a constant flow rate of 500 nl/min. The separated peptides were injected into a nanospray ionization (NSI) source for ionization and analyzed by a Q Exactive\u0026trade; Plus mass spectrometer, in which the previously prepared acquisition method has been uploaded. The electrospray voltage was set to 2.1 kV. Precursor peptides and the secondary fragment ions were detected and analyzed by a high-resolution Orbitrap mass spectrometer. The range for full MS scan was set to 350\u0026ndash;1405 m/z. The precursor peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE set to 27/35, and the fragments were detected in the Orbitrap at a resolution of 17,500. Automatic gain control (AGC) was set to 3E6 for full MS and 1E5 for MS/MS. The maximum injection time was set at 50 ms for full MS and 200 ms for MS/MS. The isolation window for MS/MS was set at 1.6 m/z.\u003c/p\u003e \u003cp\u003eThe MS data were processed using the Skyline software. The identities of the peptides (spectral matching) were confirmed by post-acquisition data processing. The traces of the qualified fragment ions of the peptides were extracted and the peak areas were determined and used for quantifying peptide abundance. The percentage of reduced form of each cysteine was calculated by comparing the peak area of matching peptide in group 1 to that in group 2. The peak area of the cysteine-free peptide was used as an internal standard to normalize the quantification of target peptides.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eExpression of the recombinant MTP protein in HepG2 cells\u003c/h2\u003e \u003cp\u003eThe 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\u0026rsquo;s instructions. The cells were harvested for 48 hours after transfection, and the expressions of wild-type MTP and the variants were detected by Western blotting with anti-FLAG antibody.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eFoldX analysis of MTP variants\u003c/h2\u003e \u003cp\u003eThe changes in Gibbs free energy (∆∆G) induced by the MTP mutations at each disulfide bonds were calculated using FoldX \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Briefly, the Gibbs free energy (∆G) of wild-type MTP and mutant MTP was calculated based on the sum of the total intermolecular and intramolecular forces, determined by the FoldX forcefield. The relative ∆∆G was obtained by subtracting ∆G\u003csub\u003emutant\u003c/sub\u003e from ∆G\u003csub\u003ewild\u0026minus;type\u003c/sub\u003e of MTP. The \u0026ldquo;buildmodel\u0026rdquo; function was used to build the 3D structure of the mutants based on the structure of wild-type MTP complex (PDB identifier 6I7S), and the structures of both wild type and the mutants were added into GeoPPI to obtain the corresponding predicted values\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics (MD) simulations\u003c/h2\u003e \u003cp\u003eSimulations were prepared from the published MTP crystal structure (PDB identifier 6I7S). All MD simulations of each redox state (Cys174-Cys194, C298-C301, Cys440-Cys445, and Cys827-Cys878 and none of which was reduced) were performed with the GROMACS 2018 software package, using the parameter sets of the GROMOS 54A7 force field. All the simulations were performed under a SPC water model and periodic boundary conditions, allowing for a minimum distance of 1 nm from the protein to any box edge. The workflow of MD simulations includes four steps: energy minimization, heating, equilibration, and production. First, energy minimization was performed using 5000 steps of steepest descent algorithm, and 5000 steps of conjugate gradient algorithm while keeping the protein backbone atoms completely constrained. Next, the minimized structures were thermalized to 300 K over 50 ps. Then, the thermalized systems were simulated under isothermal-isobaric NPT ensemble over 50 ps to ensure equilibration of the periodic box volume. Finally, production dynamics were performed for 100ns in the NPT ensemble with a 2-fs time step and coordinates were saved every 40 ps for analysis.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003ePlasma characterization and FPLC analysis\u003c/h2\u003e \u003cp\u003eAfter 16 h of fasting, 12-week-old \u003cem\u003ePdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb-cre/Pdi\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e male mice were injected \u003cem\u003ei.v.\u003c/em\u003e 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 \u0026micro;L 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 \u0026micro;L of volume\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Total triglycerides and cholesterol levels of each fraction were measured by commercial kits (100000220 and 100000180 from Zhongsheng Beikong, respectively) according to the manufacturer\u0026rsquo;s protocol. The level of ApoB protein in each fraction was detected by Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003ePDI reductase activity assay and MTP activity assay\u003c/h2\u003e \u003cp\u003eFifty 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 \u0026micro;g/ml leupeptin and then sonicated on ice. After centrifugation at 10000 g for 10 min at 4℃, the intermediate layer of the supernatants was collected. Repeat the above steps twice. After protein quantification, 100 \u0026micro;g 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) \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eQuantification and statistical analysis\u003c/h2\u003e \u003cp\u003eNo 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\u0026rsquo;s t-test for two groups were used, and the values were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Asterisks denote corresponding statistical significance *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (two-tailed Student\u0026rsquo;s t-test).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict-of-interest statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eF.C. and Y.W. conceived and designed the study. F.C., A.Y and Y.L. performed most of the experiments, Y.Z., J.B., R.G., and Y.H. assisted with experiments. F.C. and Y.W. wrote the manuscript. D.W. and Y.W. supervised the investigation and reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81770138, 81970128, 31970890, 8217011021, 81730003, 82020108003, 82270136), the Translational Research Grant of NCRCH (2020ZKPA02, 2020WSA04), the collaboration fund from State Key Laboratory of Radiation Medicine and Protection (GZN1201802), the Suzhou Science and Technology Development Project (SKJY2021043), the Priority Academic Program Development of Jiangsu Higher Education Institutions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Souza RJ et al (2015) Intake of saturated and trans unsaturated fatty acids and risk of all cause mortality, cardiovascular disease, and type 2 diabetes: systematic review and meta-analysis of observational studies. BMJ 351:h3978\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeWitt E, Dysinger I, Larmer Z, Kolwicz SC Jr (2019) High Saturated and Unsaturated Fat Diets Yield Different Disease Phenotypes in Mice. \u003cem\u003eThe FASEB Journal\u003c/em\u003e 33, 755.753-755.753\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKirpich IA et al (2016) Saturated and Unsaturated Dietary Fats Differentially Modulate Ethanol-Induced Changes in Gut Microbiome and Metabolome in a Mouse Model of Alcoholic Liver Disease. Am J Pathol 186:765\u0026ndash;776\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrujic-Milanovic JD et al (2021) Excesive consumption of unsaturated fatty acids leads to oxidative and inflammatory instability in Wistar rats. Biomed Pharmacother 139:111691\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUlven SM et al (2019) Using metabolic profiling and gene expression analyses to explore molecular effects of replacing saturated fat with polyunsaturated fat-a randomized controlled dietary intervention study. Am J Clin Nutr 109:1239\u0026ndash;1250\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeech M et al (2018) Replacement of dietary saturated fat with unsaturated fats increases numbers of circulating endothelial progenitor cells and decreases numbers of microparticles: findings from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study. Am J Clin Nutr 107:876\u0026ndash;882\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLian Z et al (2020) Replacing Saturated Fat With Unsaturated Fat in Western Diet Reduces Foamy Monocytes and Atherosclerosis in Male Ldlr(-/-) Mice. Arterioscler Thromb Vasc Biol 40:72\u0026ndash;85\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang DD et al (2016) Association of Specific Dietary Fats With Total and Cause-Specific Mortality. JAMA Intern Med 176:1134\u0026ndash;1145\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTindall AM et al (2019) Replacing Saturated Fat With Walnuts or Vegetable Oils Improves Central Blood Pressure and Serum Lipids in Adults at Risk for Cardiovascular Disease: A Randomized Controlled-Feeding Trial. J Am Heart Assoc 8:e011512\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y et al (2015) Saturated Fats Compared With Unsaturated Fats and Sources of Carbohydrates in Relation to Risk of Coronary Heart Disease: A Prospective Cohort Study. J Am Coll Cardiol 66:1538\u0026ndash;1548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHannon BA, Thompson SV, An R, Teran-Garcia M (2017) Clinical Outcomes of Dietary Replacement of Saturated Fatty Acids with Unsaturated Fat Sources in Adults with Overweight and Obesity: A Systematic Review and Meta-Analysis of Randomized Control Trials. Ann Nutr Metab 71:107\u0026ndash;117\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVafeiadou K et al (2015) Replacement of saturated with unsaturated fats had no impact on vascular function but beneficial effects on lipid biomarkers, E-selectin, and blood pressure: results from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study. Am J Clin Nutr 102:40\u0026ndash;48\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdel-Fattah G, Fernandez ML, McNamara DJ (1995) Regulation of guinea pig very low density lipoprotein secretion rates by dietary fat saturation. J Lipid Res 36:1188\u0026ndash;1198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarro M, Buschiazzo J, Rios GL, Oresti GM, Alberio RH (2013) Linoleic acid stimulates neutral lipid accumulation in lipid droplets of maturing bovine oocytes. Theriogenology 79:687\u0026ndash;694\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan X et al (2013) Increased unsaturation of lipids in cytoplasmic lipid droplets in DAOY cancer cells in response to cisplatin treatment. Metabolomics 9:722\u0026ndash;729\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrso CJ, Zhou H (2021) Palmitic Acid Lipotoxicity in Microglia Cells Is Ameliorated by Unsaturated Fatty Acids. Int J Mol Sci 22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerez-Marti A et al (2022) Reducing lipid bilayer stress by monounsaturated fatty acids protects renal proximal tubules in diabetes. Elife 11\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei S et al (2011) Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. J Pharmacol Exp Ther 339:487\u0026ndash;498\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng KP et al (2018) Unsaturated Fatty Acids Increase the Expression of Hepassocin through a Signal Transducer and Activator of Transcription 3-Dependent Pathway in HepG2 Cells. Lipids 53:863\u0026ndash;869\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjoolabady A et al (2023) Endoplasmic reticulum stress in liver diseases. Hepatology 77:619\u0026ndash;639\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalhi H, Kaufman RJ (2011) Endoplasmic reticulum stress in liver disease. J Hepatol 54:795\u0026ndash;809\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebeaupin C et al (2018) Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol 69:927\u0026ndash;947\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlamment M, Kammoun HL, Hainault I, Ferre P, Foufelle F (2010) Endoplasmic reticulum stress: a new actor in the development of hepatic steatosis. Curr Opin Lipidol 21:239\u0026ndash;246\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JY et al (2018) ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P. Cell 175:133\u0026ndash;145e115\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S et al (2015) Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100. Mol Biol Cell 26:594\u0026ndash;604\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRustaeus S et al (1999) Assembly of very low density lipoprotein: a two-step process of apolipoprotein B core lipidation. J Nutr 129:463S\u0026ndash;466S\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGordon DA, Jamil H, Gregg RE, Olofsson SO, Boren J (1996) Inhibition of the microsomal triglyceride transfer protein blocks the first step of apolipoprotein B lipoprotein assembly but not the addition of bulk core lipids in the second step. J Biol Chem 271:33047\u0026ndash;33053\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiterova EI et al (2019) The crystal structure of human microsomal triglyceride transfer protein. Proc Natl Acad Sci U S A 116:17251\u0026ndash;17260\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWetterau JR, Combs KA, McLean LR, Spinner SN, Aggerbeck LP (1991) Protein disulfide isomerase appears necessary to maintain the catalytically active structure of the microsomal triglyceride transfer protein. Biochemistry 30:9728\u0026ndash;9735\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWetterau JR, Lin MC, Jamil H (1997) Microsomal triglyceride transfer protein. Biochim Biophys Acta 1345:136\u0026ndash;150\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBanaszak LJ, Ranatunga WK (2008) The assembly of apoB-containing lipoproteins: a structural biology point of view. Ann Med 40:253\u0026ndash;267\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJang I et al (2019) PDIA1/P4HB is required for efficient proinsulin maturation and ss cell health in response to diet induced obesity. Elife 8\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBowley SR, Fang C, Merrill-Skoloff G, Furie BC, Furie B (2017) Protein disulfide isomerase secretion following vascular injury initiates a regulatory pathway for thrombus formation. Nat Commun 8:14151\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim YM et al (2018) Redox Regulation of Mitochondrial Fission Protein Drp1 by Protein Disulfide Isomerase Limits Endothelial Senescence. Cell Rep 23:3565\u0026ndash;3578\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J et al (2015) The C-terminal CGHC motif of protein disulfide isomerase supports thrombosis. J Clin Invest 125:4391\u0026ndash;4406\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Fast DG, Attie AD (1997) The enzymatic and non-enzymatic roles of protein-disulfide isomerase in apolipoprotein B secretion. J Biol Chem 272:27644\u0026ndash;27651\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuan H, Fan G, Wang CC (1995) Independence of the chaperone activity of protein disulfide isomerase from its thioredoxin-like active site. J Biol Chem 270:17078\u0026ndash;17080\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S et al (2012) IRE1alpha-XBP1s induces PDI expression to increase MTP activity for hepatic VLDL assembly and lipid homeostasis. Cell Metab 16:473\u0026ndash;486\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim K et al (2013) Platelet protein disulfide isomerase is required for thrombus formation but not for hemostasis in mice. Blood 122:1052\u0026ndash;1061\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y et al (2012) The disulfide isomerase ERp57 mediates platelet aggregation, hemostasis, and thrombosis. Blood 119:1737\u0026ndash;1746\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L et al (2013) Platelet-derived ERp57 mediates platelet incorporation into a growing thrombus by regulation of the alphaIIbbeta3 integrin. Blood 122:3642\u0026ndash;3650\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou J et al (2017) The disulfide isomerase ERp72 supports arterial thrombosis in mice. Blood 130:817\u0026ndash;828\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCimmino F et al (2021) Dietary Micronutrient Management to Treat Mitochondrial Dysfunction in Diet-Induced Obese Mice. Int J Mol Sci 22\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTarantola E et al (2012) Dipeptidylpeptidase\u0026ndash;IV, a key enzyme for the degradation of incretins and neuropeptides: activity and expression in the liver of lean and obese rats. Eur J Histochem 56:e41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K et al (2016) MDGA2 is a novel tumour suppressor cooperating with DMAP1 in gastric cancer and is associated with disease outcome. Gut 65:1619\u0026ndash;1631\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh R et al (2009) Autophagy regulates lipid metabolism. Nature 458:1131\u0026ndash;1135\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePijning AE et al (2021) An alternate covalent form of platelet alphaIIbbeta3 integrin that resides in focal adhesions and has altered function. Blood 138:1359\u0026ndash;1372\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePassam F et al (2018) Mechano-redox control of integrin de-adhesion. Elife 7\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKasbi Chadli F, Treguier M, Briand F, Sulpice T, Ouguerram K (2020) Ezetimibe Enhances Macrophage-to-Feces Reverse Cholesterol Transport in Golden Syrian Hamsters Fed a High-Cholesterol Diet. J Pharmacol Exp Ther 375:349\u0026ndash;356\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOta T, Gayet C, Ginsberg HN (2008) Inhibition of apolipoprotein B100 secretion by lipid-induced hepatic endoplasmic reticulum stress in rodents. J Clin Invest 118:316\u0026ndash;332\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y et al (2020) Resveratrol Alleviates Endoplasmic Reticulum Stress-Associated Hepatic Steatosis and Injury in Mice Challenged with Tunicamycin. Mol Nutr Food Res 64:e2000105\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SH, Seo H, Kwon D, Yuk DY, Jung YS (2022) Taurine Ameliorates Tunicamycin-Induced Liver Injury by Disrupting the Vicious Cycle between Oxidative Stress and Endoplasmic Reticulum Stress. Life (Basel) 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaabe M et al (1999) Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice. J Clin Invest 103:1287\u0026ndash;1298\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePan M, Liang Js JS, Fisher EA, Ginsberg HN (2002) The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis. J Biol Chem 277:4413\u0026ndash;4421\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKulinski A, Rustaeus S, Vance JE (2002) Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB, as well as for ApoB lipidation. J Biol Chem 277:31516\u0026ndash;31525\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell DM et al (1998) Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence. Proc Natl Acad Sci U S A 95:14733\u0026ndash;14738\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDashti N, Manchekar M, Liu Y, Sun Z, Segrest JP (2007) Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells. J Biol Chem 282:28597\u0026ndash;28608\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManchekar M et al (2004) Apolipoprotein B-containing lipoprotein particle assembly: lipid capacity of the nascent lipoprotein particle. J Biol Chem 279:39757\u0026ndash;39766\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRichardson PE et al (2005) Assembly of lipoprotein particles containing apolipoprotein-B: structural model for the nascent lipoprotein particle. Biophys J 88:2789\u0026ndash;2800\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Tran K, Yao Z (1999) The activity of microsomal triglyceride transfer protein is essential for accumulation of triglyceride within microsomes in McA-RH7777 cells. A unified model for the assembly of very low density lipoproteins. J Biol Chem 274:27793\u0026ndash;27800\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ (2012) Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol Cell Proteomics 11:1475\u0026ndash;1488\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRauniyar N (2015) Parallel Reaction Monitoring: A Targeted Experiment Performed Using High Resolution and High Mass Accuracy Mass Spectrometry. Int J Mol Sci 16:28566\u0026ndash;28581\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilson MH et al (2020) A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein. PLoS Genet 16:e1008941\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Z et al (2019) The transmembrane protein disulfide isomerase TMX1 negatively regulates platelet responses. Blood 133:246\u0026ndash;251\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGu X et al (2017) Proteome and Acetylome Analysis Identifies Novel Pathways and Targets Regulated by Perifosine in Neuroblastoma. Sci Rep 7:42062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchymkowitz J et al (2005) The FoldX web server: an online force field. Nucleic Acids Res 33:W382\u0026ndash;388\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Luo Y, Li P, Song S, Peng J (2021) Deep geometric representations for modeling effects of mutations on protein-protein binding affinity. PLoS Comput Biol 17:e1009284\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X et al (2021) Receptor-Mediated ER Export of Lipoproteins Controls Lipid Homeostasis in Mice and Humans. Cell Metab 33:350\u0026ndash;366e357\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang D et al (2021) TMEM41B acts as an ER scramblase required for lipoprotein biogenesis and lipid homeostasis. Cell Metab 33:1655\u0026ndash;1670e1658\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Asghar M (2017) Protein disulfide isomerase regulates renal AT(1) receptor function and blood pressure in rats. Am J Physiol Renal Physiol 313:F461\u0026ndash;F466\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3861110/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3861110/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSaturated 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 hepatic endoplasmic reticulum (ER) stress model. Amoug 13 members of protein disulfide isomerase (PDI) family, only PDIA1 (PDI) deficiency leads to hepatosteatosis and hypolipidemia. In PDI-deficient 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 impairs the assembly and secretion of Apolipoprotein B-100 (ApoB-100) very low-density lipoprotein (VLDL) but not ApoB-48 VLDL. 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.\u003c/p\u003e","manuscriptTitle":"Identification of the differential transport pathways of saturated and unsaturated fatty acid esters in hepatocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-06 09:27:56","doi":"10.21203/rs.3.rs-3861110/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"812832a7-23ed-4c0a-a58d-1d5b12b2a4cd","owner":[],"postedDate":"February 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28554389,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"},{"id":28554390,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Dyslipidaemias"},{"id":28554391,"name":"Biological sciences/Cell biology/Mechanisms of disease"}],"tags":[],"updatedAt":"2025-02-05T08:05:45+00:00","versionOfRecord":{"articleIdentity":"rs-3861110","link":"https://doi.org/10.1038/s41467-025-56620-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-02-04 05:00:00","publishedOnDateReadable":"February 4th, 2025"},"versionCreatedAt":"2024-02-06 09:27:56","video":"","vorDoi":"10.1038/s41467-025-56620-4","vorDoiUrl":"https://doi.org/10.1038/s41467-025-56620-4","workflowStages":[]},"version":"v1","identity":"rs-3861110","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3861110","identity":"rs-3861110","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}