Hepatic PCSK9 Expression Is Regulated by Nutritional Status via Insulin and Sterol Regulatory Element-binding Protein 1c

Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4–5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 μm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia. Familial autosomal dominant hypercholesterolemia is associated with high risk for cardiovascular accidents and is related to mutations in the low density lipoprotein receptor or its ligand apolipoprotein B (apoB). Mutations in a third gene, proprotein convertase subtilisin kexin 9 (PCSK9), were recently associated to this disease. PCSK9 acts as a natural inhibitor of the low density lipoprotein receptor pathway, and both genes are regulated by depletion of cholesterol cell content and statins, via sterol regulatory element-binding protein (SREBP). Here we investigated the regulation of PCSK9 gene expression during nutritional changes. We showed that PCSK9 mRNA quantity is decreased by 73% in mice after 24 h of fasting, leading to a 2-fold decrease in protein level. In contrast PCSK9 expression was restored upon high carbohydrate refeeding. PCSK9 mRNA increased by 4–5-fold in presence of insulin in rodent primary hepatocytes, whereas glucose had no effect. Moreover, insulin up-regulated hepatic PCSK9 expression in vivo during a hyperinsulinemic-euglycemic clamp in mice. Adenoviral mediated overexpression of a dominant or negative form of SREBP-1c confirmed the implication of this transcription factor in insulin-mediated stimulation of PCSK9 expression. Liver X receptor agonist T0901317 also regulated PCSK9 expression via this same pathway (a 2-fold increase in PCSK9 mRNA of primary hepatocytes cultured for 24 h in presence of 1 μm T0901317). As our last investigation, we isolated PCSK9 proximal promoter and verified the functionality of a SREBP-1c responsive element located from 335 bp to 355 bp upstream of the ATG. Together, these results show that PCSK9 expression is regulated by nutritional status and insulinemia. Autosomal dominant hypercholesterolemia is associated with mutations in genes involved in the regulation of LDL 2The abbreviations used are: LDL, low density lipoprotein; LDLr, LDL receptor; apoB, apolipoprotein B; PCSK9, proprotein convertase subtilisin kexin 9; SRE, sterol response element; SREBP, SRE-binding protein, LXR, liver X receptor, GK, glucokinase; MES, 4-morpholineethanesulfonic acid; BisTris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol; EMSA, electrophoretic mobility shift assays. homeostasis. The most common and severe form of monogenic hypercholesterolemia is familial hypercholesterolemia, caused by mutations in the LDL receptor (LDLr) (1Rader D.J. Cohen J. Hobbs H.H. J. Clin. Investig. 2003; 111: 1795-1803Crossref PubMed Scopus (476) Google Scholar). Familial hypercholesterolemia is characterized by elevated plasma LDL-cholesterol levels and premature cardiovascular disease. Another form of this disease, familial defective apolipoprotein (apo)B100, is caused by mutations in the LDLr binding domain of ApoB100 (1Rader D.J. Cohen J. Hobbs H.H. J. Clin. Investig. 2003; 111: 1795-1803Crossref PubMed Scopus (476) Google Scholar). ApoB100 is synthesized by the liver and is the major protein component of very low density lipoprotein and LDL. Recently, proprotein convertase subtilisin kexin 9 (PCSK9) has been identified as the third gene involved in autosomal dominant hypercholesterolemia (5Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1077) Google Scholar). Proprotein convertases are proteolytic enzymes that cleave their substrate, producing a biologically active molecule (2Seidah N.G. Chretien M. Brain Res. 1999; 848: 45-62Crossref PubMed Scopus (690) Google Scholar). We showed that patients mutated in PCSK9 prodomain (mutation S127R) have decreased LDL catabolism and increased very low density lipoprotein, intermediary density lipoprotein, and LDL production (3Ouguerram K. Chetiveaux M. Zair Y. Costet P. Abifadel M. Varret M. Boileau C. Magot T. Krempf M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1448-1453Crossref PubMed Scopus (164) Google Scholar). Studies on knock-out mice and plasma lipid profiles of patients with nonsense mutations, showed that PCSK9 deficiency results in low circulating LDL-cholesterol concentrations (4Brunham L. Clin. Genet. 2005; 67: 476-477Crossref Google Scholar, 5Cohen J. Pertsemlidis A. Kotowski I.K. Graham R. Garcia C.K. Hobbs H.H. Nat. Genet. 2005; 37: 161-165Crossref PubMed Scopus (1077) Google Scholar, 6Rashid S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5374-5379Crossref PubMed Scopus (563) Google Scholar). Although it is established that PCSK9 impairs the LDLr pathway by increasing the degradation of the receptor, its effect on apoB production is still controversial (7Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.C. Hamelin J. Varret M. Allard D. Trillard M. Abifadel M. Tebon A. Attie A.D. Rader D.J. Boileau C. Brissette L. Chretien M. Prat A. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 8Lalanne F. Lambert G. Amar M.J. Chetiveaux M. Zair Y. Jarnoux A.L. Ouguerram K. Friburg J. Seidah N.G. Brewer Jr., H.B. Krempf M. Costet P. J. Lipid Res. 2005; 46: 1312-1319Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Maxwell K.N. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7100-7105Crossref PubMed Scopus (509) Google Scholar, 10Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). 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F. 2004; PubMed Scopus Google Scholar). are SREBP-1a and SREBP-2 are a that is to the it its SREBP-1a and are from gene SREBP-2 and to mRNA In the SREBP-1c is the and the most H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Investig. PubMed Scopus Google Scholar). SREBP-2 genes involved in cholesterol whereas SREBP-1c and genes involved in or carbohydrate or M. C. P. F. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, J.D. Brown M.S. Hammer R.E. Goldstein J.L. H. J. Clin. Investig. 101: PubMed Google Scholar, R. J.L. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. Horton J.D. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Investig. PubMed Scopus Google Scholar, H. Horton J.D. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Investig. PubMed Scopus Google Scholar, H. M. J. Y. F. Y. K. K. T. S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). glucose glucose and SREBP-1c is to have a on gene regulation R. F. F. P. 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Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). was by the of the of Y. A. Wang X. H. A. A. K. 1999; PubMed Scopus Google Scholar). T0901317 was with were by the for the and of mice were with a h h of the mice were on a the and mice were and The was with the and The was 24 h to and the was to with a high for or 24 The high carbohydrate and and glucose concentrations were insulin levels were a The mice were and their livers were and in and mice were with a in the via the and to for the clamp was The mice were for h with after a The and insulin insulin leading to insulin of was a of The and its was to glucose levels to PCSK9 proximal promoter was identified We the of the as to bp was by and the and The was to and mutated was and the and are by from bp to were by The and were verified by the cells were a density of of in glucose of The was with to the and cells were in the same for We used or with expression for active SREBP-1c from H. and The was with the were as P. F. M.C. X. Biol. 2003; PubMed Scopus Google Scholar). SREBP-1c expression was in the or mutated were and and Mutations are as were were with of The were with in SREBP-1c in presence or of a was from is of with a of are as was of were PCSK9 by and upon PCSK9 expression is by mice were to a by with a for and 24 their and were and PCSK9 mRNA and protein levels were 24 h of fasting, PCSK9 mRNA levels to In PCSK9 expression was restored by levels of as as h after mRNA levels to their after 24 h of refeeding. PCSK9 protein and was decreased by after the and restored to after 24 h of refeeding. Although the SREBP-1c content was by fasting, no was for the nuclear However, a increase was after for The LDLr protein quantity during or with has been C. Park C. J. Google Scholar). PCSK9 mRNA in the of in insulin glucose the increased PCSK9 expression in response to we the primary rodent and cells were cultured in the presence of or high of no was in PCSK9 mRNA levels to the by a increase of its mRNA levels that in PCSK9 is regulated by As both SREBP-1c and LDLr mRNA expression in response to high glucose concentrations a glucose PCSK9 mRNA levels increased by in the presence of insulin with cells cultured insulin results were obtained high glucose As mRNA levels for GK, is regulated by insulin via increased by M. C. P. F. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). SREBP-1c mRNA increased in presence of insulin was obtained for the LDLr that PCSK9 response to insulin is we cultured primary hepatocytes in We a increase in PCSK9 and SREBP-1c mRNA levels and no for we verified that insulin PCSK9 expression in vivo by a hyperinsulinemic-euglycemic clamp PCSK9 mRNA levels were increased the of the clamp as as our in PCSK9 is up-regulated by Although was a of SREBP-1c and no in SREBP-2 mRNA SREBP-1c PCSK9 it is that SREBP-1c insulin on gene we PCSK9 expression in response to insulin in primary of hepatocytes by active nuclear truncated SREBP-1c protein a dominant negative form or a The the and SREBP-2 cell Although it that SREBP-2 protein levels are showed that and decreased by 2-fold SREBP-2 mRNA levels We verified the of our by and LDLr mRNA for In with has been in the mRNA increased by cells were with in the of insulin M. C. P. F. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). However, the response to insulin was by overexpressing LDLr mRNA levels a very PCSK9 mRNA quantity was higher in cells with is with insulin PCSK9 response to insulin was by cells were by in no with the in the presence of PCSK9 levels were the gene responsive to insulin mRNA The by with cells to the mRNA in response to insulin for results that insulin PCSK9 via T0901317 PCSK9 via was that T0901317 moderately PCSK9 expression in and it is that SREBP-1c transcription (13Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, G. J. Bashmakov Y. Brown M.S. Goldstein J.L. D.J. PubMed Scopus Google Scholar). we primary hepatocytes to T0901317 for 24 h in the or of with T0901317 1 increased by SREBP-1c whereas had no effect on SREBP-2 mRNA levels T0901317 increased PCSK9 mRNA levels by 2-fold PCSK9 The PCSK9 response was in the presence of that T0901317 PCSK9 via is that binding is a of we its expression in the same of C. J.A. Hobbs H. D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). As for a T0901317 increased mRNA quantity and this response was in the presence of The PCSK9 a the PCSK9 regulation by we a of the of the gene The gene we to the of as As by (11Dubuc G. Chamberland A. Wassef H. Davignon J. Seidah N.G. Bernier L. Prat A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1454-1459Crossref PubMed Scopus (520) Google PCSK9 proximal promoter that is and mice we also from bp to in the SREBP-1c as as we a of this promoter from bp to bp as as a this and from bp to The and mutated and were and In and mutated promoter with a and increase in the of the promoter higher the of the promoter the presence of a element located bp and the activated by we both or and with expression for a active As a SREBP-1c increased and by and and and the in the element this by in the with a very low response to it that the a major in response to that SREBP-1c the PCSK9 proximal promoter we with in SREBP-1c and or mutated was to the in SREBP-1c protein and were to the of this this was with of and its was with of a was obtained by the in SREBP-1c protein with the of the was to to the presence of the by of Together, these results show that the PCSK9 proximal promoter a element is also and activated by that it PCSK9 response to insulin and Here we show that the nutritional status PCSK9 expression and that insulin PCSK9 expression via a pathway we showed that a decreased hepatic PCSK9 mRNA and protein levels in mice and that were restored by refeeding. on for h in a 2-fold increase in hepatic PCSK9 mRNA in vivo the regulation of In primary rodent hepatocytes, PCSK9 was responsive to insulin or to the overexpression of SREBP-1c to The response to insulin was also by a dominant negative agonist also increased PCSK9 mRNA quantity in hepatocytes via we identified the PCSK9 proximal promoter to and isolated a response element that SREBP-1c is to results that PCSK9 responsive to insulin glucose in vitro, in primary of hepatocytes, or in during hyperinsulinemic-euglycemic PCSK9 a gene the to a with and SREBP-1c R. F. F. P. J. C. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, D. D. C. M. P. F. J. PubMed Scopus Google Scholar). In this the decrease of PCSK9 expression during by of nuclear SREBP-1c and that are involved in this The of PCSK9 expression upon is by a increase in nuclear in with J.D. Bashmakov Y. H. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, M. Hammer R.E. Moon Y.A. R. Horton J.D. Goldstein J.L. Brown M.S. PubMed Scopus Google Scholar). Although we that the same involved in this this that SREBP-1c is involved in the expression of PCSK9 during a by of data on the effect of insulin on nuclear we that SREBP-2 in to the PCSK9 response to insulin A. P. P. F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar, J. S. G. H. D. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). we that was as as insulin to PCSK9 and that the response of PCSK9 to The effect of on SREBP-2 mRNA to the PCSK9 response to insulin in this In we were to in protein content these these the that factor involved in PCSK9 regulation in these and that it have effect with SREBP-1c mRNA levels in mice to were PCSK9 and is in with in liver L. J. 2004; PubMed Scopus Google Scholar). that in these SREBP-1c is that the LDLr by sterol depletion and is by a D. PubMed Scopus Google Scholar). PCSK9 is a major of this effect A.D. Seidah N.G. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). that the of the response of LDLr and PCSK9 to insulin and sterol the of are and in the PCSK9 the LDLr pathway. to in the hepatic LDLr protein or the mRNA in the decrease in is during the is that insulin and increase SREBP-1c mRNA and transcription factor M. C. P. F. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, G. J. Bashmakov Y. Brown M.S. Goldstein J.L. D.J. PubMed Scopus Google Scholar, H. A. L. S. Wang S. M. D.J. PubMed Scopus Google Scholar, Bashmakov Y. S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). was recently that insulin the effect of T0901317. T0901317 increased SREBP-1c transcription and insulin SREBP-1c A. P. P. F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: PubMed Scopus Google Scholar). In our a of primary hepatocytes to T0901317 in a 2-fold increase in PCSK9 in with the (13Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (308) Google in vivo in mice. regulation was cells a dominant negative However, the response of a was and was by this dominant negative that PCSK9 via (11Dubuc G. Chamberland A. Wassef H. Davignon J. Seidah N.G. Bernier L. Prat A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1454-1459Crossref PubMed Scopus (520) Google recently showed that a natural increase PCSK9 mRNA levels in HepG2 it increase by the in the of the cells used cell a cell we a effect. SREBP-1c both in HepG2 and cells and the we isolated and is these we the for a regulation H. A. L. S. Wang S. M. D.J. PubMed Scopus Google Scholar). the response of PCSK9 to T0901317 has to SREBP-1c is involved in the of A. T. T. J.A. F. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, H. A. L. S. Wang S. M. D.J. PubMed Scopus Google Scholar). to the of the of PCSK9 in these in in PCSK9 knock-out mice S. Curtis D.E. Garuti R. Anderson N.N. Bashmakov Y. Ho Y.K. Hammer R.E. Moon Y.A. Horton J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5374-5379Crossref PubMed Scopus (563) Google Scholar). for the the PCSK9 promoter and that it a from to bp the ATG. In our results that a is located in a from bp to the is a sterol response element or binding is still to this element the promoter by However, the response to with the effect of the on SREBP-1c binding on the that response element in the Indeed, no binding was SREBP-1c and with as showed with However, of binding to that SREBP-1c the mutated it is high is that are and that transcription that is the for the LDLr H.B. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and is the factor for S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the promoter in primary hepatocytes is upon and both and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and have been in the PCSK9 promoter on a (11Dubuc G. Chamberland A. Wassef H. Davignon J. Seidah N.G. Bernier L. Prat A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1454-1459Crossref PubMed Scopus (520) Google Scholar). to their functionality and to PCSK9 by sterol and insulin in PCSK9 by agonist and by cholesterol cell content is of a gene involved in (13Maxwell K.N. Soccio R.E. Duncan E.M. Sehayek E. Breslow J.L. J. Lipid Res. 2003; 44: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). regulation by SREBP-1c also this D. P. P. F. 2004; PubMed Scopus Google Scholar). and in hepatocytes is by to whereas insulin insulin apoB in K. 2004; PubMed Scopus Google Scholar). The of PCSK9 with apoB production is controversial and by on the of the protein and the of showed that patients of apoB, effect confirmed on in by C.K. D. Genet. 2005; PubMed Scopus Google showed the of this for mutations and (3Ouguerram K. Chetiveaux M. Zair Y. Costet P. Abifadel M. Varret M. Boileau C. Magot T. Krempf M. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1448-1453Crossref PubMed Scopus (164) Google Scholar). In mice or primary hepatocytes, PCSK9 overexpression to apoB (7Benjannet S. Rhainds D. Essalmani R. Mayne J. Wickham L. Jin W. Asselin M.C. Hamelin J. Varret M. Allard D. Trillard M. Abifadel M. Tebon A. Attie A.D. Rader D.J. Boileau C. Brissette L. Chretien M. Prat A. Seidah N.G. J. Biol. Chem. 2004; 279: 48865-48875Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar, 8Lalanne F. Lambert G. Amar M.J. Chetiveaux M. Zair Y. Jarnoux A.L. Ouguerram K. Friburg J. Seidah N.G. Brewer Jr., H.B. Krempf M. Costet P. J. Lipid Res. 2005; 46: 1312-1319Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Maxwell K.N. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7100-7105Crossref PubMed Scopus (509) Google Scholar, 10Park S.W. Moon Y.A. Horton J.D. J. Biol. Chem. 2004; 279: 50630-50638Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). on PCSK9 expression that the nutritional status and insulin concentrations of the PCSK9 and apoB is of increase in apoB production in mice overexpressing PCSK9 and to a nutritional A. L. T. M. C. M. and P. in also that PCSK9 a in in as We C. F. and E. for their and and D. for We also the from the for their and J. for in