Diet-induced hepatic steatosis abrogates cell-surface LDLR by inducing de novo PCSK9 expression in mice

The worldwide prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing rapidly. Although this condition is generally benign, accumulating evidence now suggests that patients with NAFLD are also at increased risk of cardiovascular disease (CVD); the leading cause of death in developed nations. Despite the well-established role of the liver as a central regulator of circulating low-density lipoprotein (LDL) cholesterol levels, a known driver of CVD, the mechanism(s) by which hepatic steatosis contributes to CVD remains elusive. Interestingly, a recent study has shown that circulating proprotein convertase subtilisin/kexin type 9 (PCSK9) levels correlate positively with liver steatosis grade. Given that PCSK9 degrades the LDL receptor (LDLR) and prevents the removal of LDL from the blood into the liver, in the present study we examined the effect of hepatic steatosis on LDLR expression and circulating LDL cholesterol levels. We now report that in a manner consistent with findings in patients, diet-induced steatosis increases circulating PCSK9 levels as a result of de novo expression in mice. We also report the finding that steatosis abrogates hepatic LDLR expression and increases circulating LDL levels in a PCSK9-dependent manner. These findings provide important mechanistic insights as to how hepatic steatosis modulates lipid regulatory genes, including PCSK9 and the LDLR, and also highlights a novel mechanism by which liver disease may contribute to CVD.

[1]  A. Baranova,et al.  Systematic review: the epidemiology and natural history of non‐alcoholic fatty liver disease and non‐alcoholic steatohepatitis in adults , 2011, Alimentary pharmacology & therapeutics.

[2]  Xuchen Zhang,et al.  Non-alcoholic fatty liver disease: An expanded review , 2017, World journal of hepatology.

[3]  Hong Chen,et al.  Accumulation of endoplasmic reticulum stress and lipogenesis in the liver through generational effects of high fat diets. , 2012, Journal of hepatology.

[4]  J. Krepinsky,et al.  GDF10 blocks hepatic PPARγ activation to protect against diet-induced liver injury , 2019, Molecular metabolism.

[5]  Jingwen Liu,et al.  Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine* , 2009, The Journal of Biological Chemistry.

[6]  J. Górski,et al.  The effect of high fat diet and metformin treatment on liver lipids accumulation and their impact on insulin action , 2018, Scientific Reports.

[7]  J. Breslow,et al.  Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Bernier,et al.  Novel loss-of-function PCSK9 variant is associated with low plasma LDL cholesterol in a French-Canadian family and with impaired processing and secretion in cell culture. , 2011, Clinical chemistry.

[9]  A. Prat,et al.  Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism* , 2016, The Journal of Biological Chemistry.

[10]  Qifu Li,et al.  Metformin Ameliorates Hepatic Steatosis and Inflammation without Altering Adipose Phenotype in Diet-Induced Obesity , 2014, PloS one.

[11]  M. Svendsen,et al.  Effect of low carbohydrate high fat diet on LDL cholesterol and gene expression in normal-weight, young adults: A randomized controlled study. , 2018, Atherosclerosis.

[12]  S. Choi,et al.  Palmitate induces ER calcium depletion and apoptosis in mouse podocytes subsequent to mitochondrial oxidative stress , 2015, Cell Death and Disease.

[13]  A. Depaoli-Roach,et al.  Sterol Regulatory Element-binding Protein-1 (SREBP-1) Is Required to Regulate Glycogen Synthesis and Gluconeogenic Gene Expression in Mouse Liver* , 2014, The Journal of Biological Chemistry.

[14]  Gang Wu,et al.  Chlorogenic acid against palmitic acid in endoplasmic reticulum stress-mediated apoptosis resulting in protective effect of primary rat hepatocytes , 2018, Lipids in Health and Disease.

[15]  Jonathan C. Cohen,et al.  Genetic and metabolic determinants of plasma PCSK9 levels. , 2009, The Journal of clinical endocrinology and metabolism.

[16]  Mats Fredrikson,et al.  Fibrosis stage is the strongest predictor for disease‐specific mortality in NAFLD after up to 33 years of follow‐up , 2015, Hepatology.

[17]  Arya M. Sharma,et al.  The chemical chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the unfolded protein response[S] , 2009, Journal of Lipid Research.

[18]  Juan Chen,et al.  Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway , 2012, Molecular and Cellular Biochemistry.

[19]  Seong Ho Park,et al.  Natural history of hepatic steatosis: observed outcomes for subsequent liver and cardiovascular complications. , 2014, AJR. American journal of roentgenology.

[20]  L. V. Van Gaal,et al.  Circulating PCSK9 levels are not associated with the severity of hepatic steatosis and NASH in a high-risk population. , 2018, Atherosclerosis.

[21]  S. Bartz,et al.  Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR, and plasma lipids in mouse and rhesus monkey[S] , 2016, Journal of Lipid Research.

[22]  I. Zineh,et al.  High-dose atorvastatin causes a rapid sustained increase in human serum PCSK9 and disrupts its correlation with LDL cholesterol , 2010, Journal of Lipid Research.

[23]  A. Prat,et al.  Pcsk9 knockout exacerbates diet-induced non-alcoholic steatohepatitis, fibrosis and liver injury in mice , 2019, JHEP reports.

[24]  PCSK9 induces a pro-inflammatory response in macrophages , 2018, Scientific Reports.

[25]  G. Koob,et al.  PCSK9 inhibition as a novel therapeutic target for alcoholic liver disease , 2019, Scientific Reports.

[26]  Jonathan C. Cohen,et al.  Molecular characterization of proprotein convertase subtilisin/kexin type 9-mediated degradation of the LDLR , 2012, Journal of Lipid Research.

[27]  A. Prat,et al.  NARC-1/PCSK9 and Its Natural Mutants , 2004, Journal of Biological Chemistry.

[28]  Annik Prat,et al.  Proprotein convertase subtilisin/kexin type 9 (PCSK9): Hepatocyte‐specific low‐density lipoprotein receptor degradation and critical role in mouse liver regeneration , 2008, Hepatology.

[29]  Jiandie D. Lin,et al.  SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion , 2013, eLife.

[30]  J. Rodés,et al.  Endoplasmic reticulum stress inhibition protects steatotic and non-steatotic livers in partial hepatectomy under ischemia–reperfusion , 2010, Cell Death and Disease.

[31]  Hongyang Shi,et al.  Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells , 2012, Lipids in Health and Disease.

[32]  Wenfeng Zhao,et al.  Lunasin functionally enhances LDL uptake via inhibiting PCSK9 and enhancing LDLR expression in vitro and in vivo , 2017, Oncotarget.

[33]  P. Magni,et al.  Liver fat accumulation is associated with circulating PCSK9 , 2016, Annals of medicine.

[34]  S. Koo,et al.  Bax Inhibitor-1 regulates hepatic lipid accumulation via ApoB secretion , 2016, Scientific Reports.

[35]  J. Weissenbach,et al.  Mutations in PCSK9 cause autosomal dominant hypercholesterolemia , 2003, Nature Genetics.

[36]  P. Ridker,et al.  Posttranslational modification of proprotein convertase subtilisin/kexin type 9 is differentially regulated in response to distinct cardiometabolic treatments as revealed by targeted proteomics. , 2018, Journal of clinical lipidology.

[37]  Hong-Bo Xiao,et al.  Naringin Activates AMPK Resulting in Altered Expression of SREBPs, PCSK9, and LDLR To Reduce Body Weight in Obese C57BL/6J Mice. , 2018, Journal of agricultural and food chemistry.

[38]  N. Seidah,et al.  Loss-of-function PCSK9 mutants evade the unfolded protein response sensor GRP78 and fail to induce endoplasmic reticulum stress when retained , 2018, The Journal of Biological Chemistry.

[39]  P. R. Sharma,et al.  Chemical chaperone 4-phenyl butyric acid (4-PBA) reduces hepatocellular lipid accumulation and lipotoxicity through induction of autophagy[S] , 2017, Journal of Lipid Research.

[40]  L. Henry,et al.  Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in the United States and the Rest of the World. , 2016, Clinics in liver disease.

[41]  N. Kaplowitz,et al.  The contribution of endoplasmic reticulum stress to liver diseases , 2011, Hepatology.

[42]  A. Keech,et al.  Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease , 2017, The New England journal of medicine.

[43]  Greger Lindberg,et al.  Decreased survival of subjects with elevated liver function tests during a 28‐year follow‐up , 2010, Hepatology.

[44]  L. Adams,et al.  International Journal of Molecular Sciences the Natural Course of Non-alcoholic Fatty Liver Disease , 2022 .

[45]  R. Austin,et al.  Endoplasmic reticulum stress and lipid dysregulation , 2011, Expert Reviews in Molecular Medicine.

[46]  R. A. Palozi,et al.  Development of a Predictive Model to Induce Atherogenesis and Hepato-Renal Impairment in Female Rats , 2019, Biomolecules.

[47]  S. Francque,et al.  Non-alcoholic fatty liver disease and cardiovascular risk: Pathophysiological mechanisms and implications. , 2016, Journal of hepatology.

[48]  A. Gingras,et al.  The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9 , 2018, bioRxiv.

[49]  Jay D. Horton,et al.  Increased Levels of Nuclear SREBP-1c Associated with Fatty Livers in Two Mouse Models of Diabetes Mellitus* , 1999, The Journal of Biological Chemistry.

[50]  D. Tang,et al.  Endoplasmic reticulum stress causes the activation of sterol regulatory element binding protein-2. , 2007, The international journal of biochemistry & cell biology.

[51]  G. Sood,et al.  Non-alcoholic fatty liver disease and cardiovascular risk , 2017, World journal of gastrointestinal pathophysiology.

[52]  A. Khera,et al.  Effects of niacin, statin, and fenofibrate on circulating proprotein convertase subtilisin/kexin type 9 levels in patients with dyslipidemia. , 2015, The American journal of cardiology.

[53]  M. Adorni,et al.  PCSK9 induces a pro-inflammatory response in macrophages , 2018, Scientific Reports.

[54]  H. Jo,et al.  Accelerated atherosclerosis development in C57Bl6 mice by overexpressing AAV-mediated PCSK9 and partial carotid ligation , 2017, Laboratory investigation; a journal of technical methods and pathology.

[55]  P. Tontonoz,et al.  LXR Regulates Cholesterol Uptake Through Idol-Dependent Ubiquitination of the LDL Receptor , 2009, Science.

[56]  W. Shao,et al.  Sterol Regulatory Element-binding Protein (SREBP) Cleavage Regulates Golgi-to-Endoplasmic Reticulum Recycling of SREBP Cleavage-activating Protein (SCAP)* , 2014, The Journal of Biological Chemistry.

[57]  Q. Ji,et al.  Free Fatty Acid Induces Endoplasmic Reticulum Stress and Apoptosis of β-cells by Ca2+/Calpain-2 Pathways , 2013, PloS one.

[58]  J. Mayne,et al.  Lipids in Health and Disease BioMed Central , 2008 .

[59]  A. Prat,et al.  The Proprotein Convertases in Hypercholesterolemia and Cardiovascular Diseases: Emphasis on Proprotein Convertase Subtilisin/Kexin 9 , 2017, Pharmacological Reviews.

[60]  H. Tavori,et al.  Vascular Health and Risk Management Dovepress New Developments in Atherosclerosis: Clinical Potential of Pcsk9 Inhibition , 2022 .

[61]  A. Prat,et al.  Gene Inactivation of Proprotein Convertase Subtilisin/Kexin Type 9 Reduces Atherosclerosis in Mice , 2012, Circulation.

[62]  Dylan L. Steen,et al.  A review of low-density lipoprotein cholesterol, treatment strategies, and its impact on cardiovascular disease morbidity and mortality. , 2016, Journal of clinical lipidology.

[63]  N. Seidah The PCSK9 revolution and the potential of PCSK9-based therapies to reduce LDL-cholesterol , 2015, Global cardiology science & practice.

[64]  H. Gerstein,et al.  Metformin and salicylate synergistically activate liver AMPK, inhibit lipogenesis and improve insulin sensitivity. , 2015, The Biochemical journal.

[65]  S. Banni,et al.  Palmitic Acid: Physiological Role, Metabolism and Nutritional Implications , 2017, Front. Physiol..

[66]  Do Yeon Kim,et al.  Eugenol ameliorates hepatic steatosis and fibrosis by down-regulating SREBP1 gene expression via AMPK-mTOR-p70S6K signaling pathway. , 2014, Biological & pharmaceutical bulletin.