Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo−/−) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo−/− mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species. Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo−/−) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo−/− mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species. The increased low-density lipoprotein cholesterol (LDL-cholesterol or LDL) levels in plasma is a pivotal risk factor for the initiation of cardiovascular disease, the leading cause of death in all countries (1Hunt K.J. Resendez R.G. Williams K. Haffner S.M. Stern M.P. San Antonio Heart S San Antonio Heart StudyNational Cholesterol Education Program versus World Health Organization metabolic syndrome in relation to all-cause and cardiovascular mortality in the San Antonio Heart Study.Circulation. 2004; 110 (15326061): 1251-125710.1161/01.CIR.0000140762.04598.F9Crossref PubMed Scopus (538) Google Scholar). LDL receptor (LDLR) plays a critical role in anti-atherosclerosis by regulating LDL catabolism. The majority of excess LDL can be cleared by hepatic LDLR (2Kong W.J. Liu J. Jiang J.D. Human low-density lipoprotein receptor gene and its regulation.J. Mol. Med. (Berl.)). 2006; 84 (16292665): 29-3610.1007/s00109-005-0717-6Crossref PubMed Scopus (46) Google Scholar). LDLR binds LDL to mediate LDL endocytosis and degradation in lysosomes, and then recycles itself to the cell surface (3Goldstein J.L. Brown M.S. The LDL receptor.Arterioscler. Thromb. Vasc. Biol. 2009; 29 (19299327): 431-43810.1161/ATVBAHA.108.179564Crossref PubMed Scopus (719) Google Scholar). Therefore, mutations of LDLR, either heterozygous or homozygous, result in familial hypercholesterolemia (2Kong W.J. Liu J. Jiang J.D. Human low-density lipoprotein receptor gene and its regulation.J. Mol. Med. (Berl.)). 2006; 84 (16292665): 29-3610.1007/s00109-005-0717-6Crossref PubMed Scopus (46) Google Scholar). It is effective to lower plasma LDL levels by enhancing hepatic LDLR expression. At the transcriptional level, LDLR expression is predominantly activated by sterol regulatory element-binding protein 2 (SREBP2), which binds to the sterol regulatory element (SRE) in the promoter of LDLR to trigger its transcription (4Brown M.S. Goldstein J.L. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell. 1997; 89 (9150132): 331-34010.1016/S0092-8674(00)80213-5Abstract Full Text Full Text PDF PubMed Scopus (2781) Google Scholar). In addition to SREBP2, proprotein convertase subtilisin/kexin 9 (PCSK9) regulates LDLR expression at the post-translational level (5Poirier S. Mayer G. The biology of PCSK9 from the endoplasmic reticulum to lysosomes: new and emerging therapeutics to control low-density lipoprotein cholesterol.Drug Des. Dev. Ther. 2013; 7 (24115837): 1135-114810.2147/DDDT.S36984PubMed Google Scholar). PCSK9 can bind LDLR to lead LDLR degradation intracellularly, resulting in suppressed LDL clearance from the circulation (6Park S.W. Moon Y.A. Horton J.D. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.J. Biol. Chem. 2004; 279 (15385538): 50630-5063810.1074/jbc.M410077200Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Accordingly, reduction of PCSK9 decreases plasma LDL levels efficiently. PCSK9 is mainly synthesized and secreted from the liver, and the circulating PCSK9 has become an important target for treatment of cardiovascular diseases. PCSK9 expression can be regulated by several transcription factors, such as hepatocyte nuclear 1α (HNF-1α), peroxisome proliferator-activated receptor γ (PPARγ), SREBP2 and forkhead box O3 (FoxO3a) (7Li H. Dong B. Park S.W. Lee H.S. Chen W. Liu J. Hepatocyte nuclear factor 1alpha plays a critical role in PCSK9 gene transcription and regulation by the natural hypocholesterolemic compound berberine.J. Biol. Chem. 2009; 284 (19687008): 28885-2889510.1074/jbc.M109.052407Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 8Duan Y. Chen Y. Hu W. Li X. Yang X. Zhou X. Yin Z. Kong D. Yao Z. Hajjar D.P. Liu L. Liu Q. Han J. Peroxisome proliferator-activated receptor γ activation by ligands and dephosphorylation induces proprotein convertase subtilisin kexin type 9 and low density lipoprotein receptor expression.J. Biol. Chem. 2012; 287 (22593575): 23667-2367710.1074/jbc.M112.350181Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 9Tao R. Xiong X. DePinho R.A. Deng C.X. Dong X.C. FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J. Biol. Chem. 2013; 288 (23974119): 29252-2925910.1074/jbc.M113.481473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). SREBP2 or HNF-1α binds to SRE or HNF1 motif to activate PCSK9 transcription (10Dong B. Wu M. Li H. Kraemer F.B. Adeli K. Seidah N.G. Park S.W. Liu J. Strong induction of PCSK9 gene expression through HNF1α and SREBP2: mechanism for the resistance to LDL-cholesterol lowering effect of statins in dyslipidemic hamsters.J. Lipid Res. 2010; 51 (20048381): 1486-149510.1194/jlr.M003566Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). FoxO3a protein can interact with the insulin response element (IRE), which is overlapped by HNF1α binding motif in PCSK9 promoter, so FoxO3a blocks the binding of HNF-1α to the PCSK9 promoter, thereby resulting in inhibition of PCSK9 expression (9Tao R. Xiong X. DePinho R.A. Deng C.X. Dong X.C. FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J. Biol. Chem. 2013; 288 (23974119): 29252-2925910.1074/jbc.M113.481473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Reciprocally, activated PCSK9 expression is observed in FoxO3a liver-specific deficient mice. Moreover, FoxO3a recruits Sirt6 to the proximal region of PCSK9 promoter, which also suppresses PCSK9 transcription (9Tao R. Xiong X. DePinho R.A. Deng C.X. Dong X.C. FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J. Biol. Chem. 2013; 288 (23974119): 29252-2925910.1074/jbc.M113.481473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Ascorbic acid (also known as vitamin C) is a water-soluble antioxidant that is naturally present in some kinds of food. Due to its capability to scavenge free radicals, the beneficial effects of ascorbic acid on lipid profiles have been demonstrated (11Abdollahzad H. Eghtesadi S. Nourmohammadi I. Khadem-Ansari M. Nejad-Gashti H. Esmaillzadeh A. Effect of vitamin C supplementation on oxidative stress and lipid profiles in hemodialysis patients.Int. J. Vitam. Nutr. Res. 2009; 79 (20533214): 281-28710.1024/0300-9831.79.56.281Crossref PubMed Google Scholar). It differs from mice, humans, and guinea pigs are prone to develop ascorbic acid deficiency as the consequence of mutation in l-gulono-γ-lactone oxidase (Gulo), the enzyme is required for the last step of ascorbic acid biosynthesis (12Linster C.L. Van Schaftingen E. Vitamin C: biosynthesis, recycling and degradation in mammals.FEBS J. 2007; 274 (17222174): 1-2210.1111/j.1742-4658.2006.05607.xCrossref PubMed Scopus (440) Google Scholar). Therefore, maintaining adequate levels of ascorbic acid supplement is essential to achieve normal body functions and optimal health (13Michels A.J. Hagen T.M. Frei B. Human genetic variation influences vitamin C homeostasis by altering vitamin C transport and antioxidant enzyme function.Annu. Rev. Nutr. 2013; 33 (23642198): 45-7010.1146/annurev-nutr-071812-161246Crossref PubMed Scopus (67) Google Scholar). Most studies have found that chronic ascorbic acid deficiency in guinea pigs leads to increased cholesterol synthesis and attenuated cholesterol conversion into bile acids (14Frikke-Schmidt H. Lykkesfeldt J. Role of marginal vitamin C deficiency in atherogenesis: in vivo models and clinical studies.Basic Clin. Pharmacol. Toxicol. 2009; 104 (19489786): 419-43310.1111/j.1742-7843.2009.00420.xCrossref PubMed Scopus (46) Google Scholar, 15Björkhem I. Kallner A. Hepatic 7α-hydroxylation of cholesterol in ascorbate-deficient and ascorbate-supplemented guinea pigs.J. Lipid Res. 1976; 17 (950498): 360-365Abstract Full Text PDF PubMed Google Scholar). In addition, in Gulo and ApoE double-deficient (Gulo−/−Apoe−/−) mice, the total cholesterol (CHO) levels is decreased by increased ascorbic acid intake (16Nakata Y. Maeda N. Vulnerable atherosclerotic plaque morphology in apolipoprotein E-deficient mice unable to make ascorbic acid.Circulation. 2002; 105: 1485-149010.1161/01.CIR.0000012142.69612.25Crossref PubMed Scopus (70) Google Scholar). These findings suggest that ascorbic acid supplement can influence LDL metabolism. However, the precise actions and the involved mechanisms by ascorbic acid remain unknown. In this study, we investigated if ascorbic acid can affect LDL levels and the action is completed by regulating PCSK9 and LDLR expression. PCSK9 is mainly present in hepatocytes as a ∼72 kDa precursor and secreted from cells after autocleaved as a mature form with a molecular mass of ∼62 kDa (9Tao R. Xiong X. DePinho R.A. Deng C.X. Dong X.C. FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J. Biol. Chem. 2013; 288 (23974119): 29252-2925910.1074/jbc.M113.481473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 17Zaid A. Roubtsova A. Essalmani R. Marcinkiewicz J. Chamberland A. Hamelin J. Tremblay M. Jacques H. Jin W. Davignon J. Seidah N.G. Prat A. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.Hepatology. 2008; 48 (18666258): 646-65410.1002/hep.22354Crossref PubMed Scopus (262) Google Scholar). We first assessed the effects of ascorbic acid on PCSK9 expression in human hepatic cell lines, HepG2 and Huh7 cells. As shown in Fig. 1A, ascorbic acid down-regulated both PCSK9 precursor and mature form in HepG2 cell in concentration- and time-dependent manners. To determine whether inhibition of PCSK9 protein expression by ascorbic acid is associated with reduced PCSK9 mRNA expression, total cellular RNA was extracted after treatment and used to determine PCSK9 mRNA levels by qRT-PCR. Consistently, ascorbic acid reduced PCSK9 mRNA expression in the similar patterns to protein (Fig. 1B), indicating reduction of PCSK9 expression by ascorbic acid can be at a transcriptional level. Similar to HepG2 cells, ascorbic acid also reduced PCSK9 protein and mRNA expression in Huh7 cells, another human hepatic cell line (Fig. 1, C and D). Inhibition of PCSK9 expression can result in increased LDLR protein levels (18Tavori H. Giunzioni I. Fazio S. PCSK9 inhibition to reduce cardiovascular disease risk: recent findings from the biology of PCSK9.Curr. Opin. Endocrinol. Diabetes Obes. 2015; 22 (25692926): 126-13210.1097/MED.0000000000000137Crossref PubMed Scopus (23) Google Scholar). To detect whether ascorbic acid-reduced PCSK9 can increase LDLR protein expression, we evaluated LDLR protein levels by Western blotting, and found LDLR expression in HepG2 cells was increased by ascorbic acid in a concentration-dependent manner (Fig. 2A). In addition, ascorbic acid increased LDLR mRNA expression (Fig. 2B), indicating it may enhance LDLR levels at both transcriptional and translational levels. Meanwhile, we found a significant increase of Dil-LDL uptake by HepG2 cell in response to ascorbic acid treatment (Fig. 2C), suggesting the enhanced LDLR activity by ascorbic acid. In parallel, ascorbic acid induced LDLR protein and mRNA expression, as well as uptake of Dil-LDL by Huh7 cells (Fig. 2, D–F). Furthermore, we treated primary hepatocytes isolated from C57BL/6J mice with ascorbic acid. Similarly, ascorbic acid increased LDLR although decreased PCSK9 expression in murine primary hepatocytes (Fig. 2G), which indicates that regulation of PCSK9/LDLR expression by ascorbic acid is not species-dependent. To identify the molecular mechanism by which ascorbic acid inhibits PCSK9 expression in hepatocytes, we detected effects of ascorbic acid on transcription factors responsible for PCSK9 expression, such as PPARγ and FoxO3a. Although it has been reported that ascorbic acid deficiency up-regulates PPARγ in the liver of senescence marker protein 30-deficient mice (19Park J.K. Ki M.R. Lee H.R. Hong I.H. Ji A.R. Ishigami A. Park S.I. Kim J.M. Chung H.Y. Yoo S.E. Jeong K.S. Vitamin C deficiency attenuates liver fibrosis by way of up-regulated peroxisome proliferator-activated receptor-γ expression in senescence marker protein 30 knockout mice.Hepatology. 2010; 51 (20162732): 1766-177710.1002/hep.23499Crossref PubMed Scopus (41) Google Scholar), we observed that both PPARγ protein and mRNA levels were not altered by ascorbic acid (Fig. 3, A and B). In contrast, ascorbic acid clearly increased FoxO3a protein and mRNA expression (Fig. 3, A and B). We further examined the transcriptional activity of PPARγ and FoxO3a. Consistently, mRNA levels of CD36 and fatty acid-binding protein 4 (FABP4), the downstream genes of PPARγ (20Briot A. Decaunes P. Volat F. Belles C. Coupaye M. Ledoux S. Bouloumié A. Senescence alters PPARγ (peroxisome proliferator-activated receptor γ)-dependent fatty acid handling in human adipose tissue microvascular endothelial cells and favors inflammation.Arterioscler. Thromb. Vasc. Biol. 2018; 38 (29545239): 1134-114610.1161/ATVBAHA.118.310797Crossref PubMed Scopus (20) Google Scholar), were not affected by ascorbic acid either (Fig. 3C). In contrast, mRNA levels of superoxide dismutase 1 (SOD1) and catalase (CAT), the downstream genes of FoxO3a (21Fasano C. Disciglio V. Bertora S. Lepore Signorile M. Simone C. FOXO3a from the nucleus to the mitochondria: A round trip in cellular stress response.Cells. 2019; 8: 111010.3390/cells8091110Crossref Scopus (32) Google Scholar), were obviously enhanced by ascorbic acid (Fig. 3D). Furthermore, increased FoxO3a nuclear translocation was determined in cells treated with ascorbic acid (Fig. 3, E and F). To investigate if ascorbic acid can decrease FoxO3a phosphorylation to enhance its nuclear translocation because the phosphorylated FoxO3a retains in the cytoplasm (22Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.Cell. 1999; 96 (10102273): 857-86810.1016/S0092-8674(00)80595-4Abstract Full Text Full Text PDF PubMed Scopus (5132) Google Scholar), we determined total and phosphorylated FoxO3a levels, and found ascorbic acid increased total FoxO3a but decreased phosphorylated FoxO3a levels (Fig. 3G). To further determine the role of FoxO3a in ascorbic acid-inhibited PCSK9 expression, we used FoxO3a siRNA to inhibit FoxO3a protein and mRNA expression (Fig. 4, A and B). As expected, inhibition of FoxO3a expression not only increased PCSK9 expression, but also abolished the effect of ascorbic acid on PCSK9 expression (Fig. 4, A and B). Moreover, FoxO3a siRNA attenuated ascorbic acid-induced LDLR expression (Fig. 4A), which should be due to the failure of PCSK9 inhibition in the presence of FoxO3a siRNA. It has been reported that PCSK9 promoter contains a FoxO3a binding motif, the IRE, with a conserved sequence of TGTTTA (from −381 to −376) (9Tao R. Xiong X. DePinho R.A. Deng C.X. Dong X.C. FoxO3 transcription factor and Sirt6 deacetylase regulate low density lipoprotein (LDL)-cholesterol homeostasis via control of the proprotein convertase subtilisin/kexin type 9 (Pcsk9) gene expression.J. Biol. Chem. 2013; 288 (23974119): 29252-2925910.1074/jbc.M113.481473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To clarify the role of FoxO3a in regulation of PCSK9 transcription by ascorbic acid, we constructed a normal human PCSK9 promoter and the promoter with IRE mutation (-381CGTCTT-376, the underlined letters are mutated nucleotides). We found that ascorbic acid reduced PCSK9 promoter activity in a concentration-dependent manner (Fig. 4C). However, mutation in IRE attenuated the inhibitory effect of ascorbic acid on PCSK9 transcription. In addition, FoxO3a siRNA abolished ascorbic acid-inhibited PCSK9 promoter activity (Fig. 4D). These results collectively suggest that FoxO3a plays a central role in suppressing PCSK9 transcription by ascorbic acid. Although inhibition of PCSK9 resulted in increased LDLR protein levels, we also observed induction of LDLR mRNA by ascorbic acid at high concentrations (Fig. 2, B and F). Thus, in addition to the regulation by PCSK9, ascorbic acid may activate LDLR expression by other pathways. SREBP2 is the transcription factor activating LDLR expression at the transcriptional level. We initially determined the effect of ascorbic acid on SREBP2 expression and maturation, and observed that ascorbic acid enhanced SREBP2 expression and maturation (Fig. 4E). To clarify the role of SREBP2 in regulating LDLR transcription by ascorbic acid, we used SREBP2 siRNA to inhibit SREBP2 expression. As expected, inhibition of SREBP2 expression abolished the enhancement of LDLR protein and mRNA expression by ascorbic acid (Fig. 4, E and F). Correspondingly, we found ascorbic acid enhanced LDLR promoter activity at a high concentration (Fig. 4G, left). However, the activation was abolished when SRE in LDLR promoter was mutated (Fig. 4G, right). Moreover, SREBP2 siRNA attenuated ascorbic acid-enhanced LDLR promoter activity (Fig. 4H). In addition, we determined the effect of ascorbic acid on stability of LDLR mRNA. Treatment of HepG2 cells with actinomycin D inhibited RNA transcription, therefore, reduced LDLR mRNA levels were determined with time of actinomycin D treatment due to the mRNA degradation. In contrast, compared with actinomycin D alone, co-treatment with ascorbic acid increased LDLR mRNA levels (Fig. 4I), suggesting ascorbic acid reduces LDLR mRNA degradation or increases its stability. Therefore, the data above suggest that in addition to activation by reduced PCSK9, ascorbic acid also activates LDLR expression by enhancing its stability and SREBP2 transcriptional activity. To determine the physiological relevance of ascorbic acid on PCSK9/LDLR expression, C57BL/6J mice fed either normal chow or a high-fat diet (HFD, containing 21% fat and 0.5% cholesterol) were i.p. injected ascorbic acid solution or vehicle (saline) daily for 1 week. Although WT mice do not depend on exogenous ascorbic acid supplement, as shown in Fig. 5, A and F, injection with ascorbic acid for 1 week still moderately increased ascorbic acid levels in the liver. Compared with control mice, ascorbic acid treatment did not cause differences to the animals, such as the ratio of liver weight to body weight and lipid profiles (Fig. 5, B and G). Similar to results in in vitro studies, PCSK9 levels in the liver of mice fed normal chow were decreased by ascorbic acid (Fig. 5, C and E). PCSK9 is mainly synthesized in the liver and is rapidly secreted into plasma after its maturation through a self-engaged autocatalytic cleavage (17Zaid A. Roubtsova A. Essalmani R. Marcinkiewicz J. Chamberland A. Hamelin J. Tremblay M. Jacques H. Jin W. Davignon J. Seidah N.G. Prat A. Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific low-density lipoprotein receptor degradation and critical role in mouse liver regeneration.Hepatology. 2008; 48 (18666258): 646-65410.1002/hep.22354Crossref PubMed Scopus (262) Google Scholar). Thus, we evaluated the secretion of PCSK9 using an ELISA kit, and found ascorbic acid also significantly reduced circulating PCSK9 levels compared with control mice (27 ± 3 versus 41 ± 3 ng/ml, Fig. 5D). Consistently, ascorbic acid inhibited PCSK9 expression and secretion in HFD-fed mice (Fig. 5, H and I). Consequently, ascorbic acid treatment significantly enhanced LDLR expression in the liver (Fig. 5, C, E, H, and J). In addition to LDLR, associated with changes of FoxO3a and SREBP2 expression by ascorbic acid, expression of the target genes of FoxO3a (SOD1 and CAT) and SREBP2 (HMG-CoA reductase (HMGCR), HMG-CoA synthase (HMGCS)) was also activated (Fig. 5, E and J). Naturally, C57BL/6J mice are able to synthesize ascorbic acid. They are not a hyperlipidemic model even though they are fed HFD. Therefore, technically it is not easy to manipulate ascorbic acid levels in vivo to influence LDL levels in mice naturally. To further clarify the effect of ascorbic acid on cholesterol metabolism in vivo, we investigated the effect of ascorbic acid deficiency on regulation of the LDL metabolism in Gulo−/− mice, the animals are fully dependent on exogenous ascorbic acid supplement. After removal of ascorbic acid from the drinking water for 3 weeks, compared with WT mice, Gulo deficiency reduced ascorbic acid more than 60% in the liver (Fig. 6A). Associated with reduced ascorbic acid, serum PCSK9 levels and hepatic PCSK9 expression were increased (Fig. 6, B-D), which resulted in reduced hepatic LDLR expression in Gulo−/− mice (Fig. 6, C and D). Correspondingly, serum LDL levels were significantly increased, whereas HDL levels were decreased, thereby resulting in decreased ratio of HDL to LDL (Fig. 6E). In contrast to ascorbic acid deficiency, injection of HFD-fed Gulo−/− mice with ascorbic acid solution for 2 weeks substantially increased ascorbic acid levels in the liver (Fig. 6F). Associated with increased hepatic ascorbic acid levels, serum PCSK9 levels and hepatic PCSK9 expression were reduced (Fig. 6, G-I). Meanwhile, hepatic LDLR expression was activated (Fig. 6, H and I). Consequently, serum LDL levels were reduced, whereas HDL levels and the ratio of HDL to LDL were increased by exogenous ascorbic acid supplement in Gulo−/− mice (Fig. 6J). In addition, we observed reduced SREBP2 and FoxO3a activity (decreased HMGCR, HMGCS, SOD1, and CAT, Fig. 6D) by ascorbic acid deficiency, and activated both (activated HMGCR, HMGCS, SOD1 and CAT, Fig. 6I) by ascorbic acid supplement in Gulo−/− mice fed HFD. Taken together, these results suggest the importance of hepatic ascorbic acid in maintaining cholesterol homeostasis. The correlation between PCSK9 and circulating cholesterol levels has been well established (11Abdollahzad H. Eghtesadi S. Nourmohammadi I. Khadem-Ansari M. Nejad-Gashti H. Esmaillzadeh A. Effect of vitamin C supplementation on oxidative stress and lipid profiles in hemodialysis patients.Int. J. Vitam. Nutr. Res. 2009; 79 (20533214): 281-28710.1024/0300-9831.79.56.281Crossref PubMed Google Scholar, 23Abifadel M. Varret M. Rabès J.P. Allard D. Ouguerram K. Devillers M. Cruaud C. Benjannet S. Wickham L. Erlich D. Derré A. Villeger L. Farnier M. Beucler I. Bruckert E. et al.Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.Nat. Genet. 2003; 34 (12730697): 154-15610.1038/ng1161Crossref PubMed Scopus (1923) Google Scholar, 24Abifadel M. Rabès J.P. Devillers M. Munnich A. Erlich D. Junien C. Varret M. Boileau C. Mutations and polymorphisms in the proprotein convertase subtilisin kexin 9 (PCSK9) gene in cholesterol metabolism and disease.Hum. Mutat. 2009; 30 (19191301): 520-52910.1002/humu.20882Crossref PubMed Scopus (190) Google Scholar). Our results above suggest that ascorbic acid deficiency increased PCSK9 expression, which can make a substantial contribution to increased LDL levels in Gulo−/− mice. Similar to guinea pigs or Gulo−/− mice, human beings are not able to produce ascorbic acid either, and our life solely depends on the exogenous ascorbic acid supplement. Therefore, we speculated that ascorbic acid levels in humans could be one of determinants for circulating PCSK9, LDL and total CHO levels. To determine it, we collected serum samples form 24 volunteers and determined levels of CHO, LDL, PCSK9, and ascorbic acid. Consistent with the previous study, serum CHO and LDL levels were positively correlated to PCSK9 levels (data not shown). Interestingly, we observed that PCSK9 levels were negatively correlated to ascorbic acid levels in human serum (Fig. 6K). Furthermore, serum ascorbic acid levels were negatively correlated to serum CHO and LDL levels (Fig. 6, L and M), suggesting the role of ascorbic acid in regulating PCSK9 expression and cholesterol metabolism in humans. As a water-soluble antioxidant, ascorbic acid has multiple biological functions. It is essential for collagen synthesis and enzyme activity maintenance (13Michels A.J. Hagen T.M. Frei B. Human genetic variation influences vitamin C homeostasis by altering vitamin C transport and antioxidant enzyme function.Annu. Rev. Nutr. 2013; 33 (23642198): 45-7010.1146/annurev-nutr-071812-161246Crossref PubMed Scopus (67) Google Scholar, 25Sotiriou S. Gispert S. Cheng J. Wang Y. Chen A. Hoogstraten-Miller S. Miller G.F. Kwon O. Levine M. Guttentag S.H. Nussbaum R.L. Ascorbic-acid transporter Slc23a1 is essential for vitamin C transport into the brain and for perinatal survival.Nat. Med. 2002; 8 (11984597): 514-51710.1038/0502-514Crossref PubMed Scopus (270) Google Scholar). Besides scurvy, many other pathogenesis of ascorbic acid deficiency have been observed. Lack of ascorbic acid production in mice exacerbates diabetic kidney injury, suggesting that compensation for ascorbic acid loss is an effective treatment for diabetic kidney injury (26Ji X. Hu X. Zou C. Ruan H. Fan X. Tang C. Shi W. Mei L. Zhu H. Hussain M. Zeng L. Zhang X. Wu X. Vitamin C deficiency exacerbates diabetic glomerular injury through activation of transforming growth factor-β signaling.Biochim. Biophys. 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