Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. ...Herein, we investigated the role of PCSK9 in adipose tissue metabolism.
At 6 months of age, Pcsk9(-/-) mice accumulated ≈80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9(-/-) Ldlr(-/-) mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9(-/-) males and females exhibit 4- and ≈ 40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9(-/-) females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to ≈ 50-fold higher levels of perigonadal VLDLR.
In vivo, endogenous PCSK9 regulates VLDLR protein levels in adipose tissue. This regulation is achieved by circulating PCSK9 that originates entirely in the liver. PCSK9 is thus pivotal in fat metabolism: it maintains high circulating cholesterol levels via hepatic LDLR degradation, but it also limits visceral adipogenesis likely via adipose VLDLR regulation.
The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low‐density lipoprotein receptor (LDLR) and apolipoprotein ...B. PCSK9 enhances LDLR degradation, resulting in low‐density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte‐specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLR‐deficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9‐deficient mice exhibited necrotic lesions, which were prevented by a high‐cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high‐cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646–554.)
The locus of the human proprotein convertase subtilisin–kexin type‐7 (PC7) gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the ...regulation of lipoprotein metabolism. A GWAS reported the association of PCSK7 SNPs with plasma triglyceride (TG), and exome sequencing of African Americans revealed the association of a low‐frequency coding variant of PC7 (R504H; SNP rs142953140) with a ~ 30% TG reduction. Another PCSK7 SNP rs508487 is in linkage disequilibrium with a promoter variant of the liver‐derived apolipoprotein A‐V (apoA‐V), an indirect activator of the lipoprotein lipase (LpL), and is associated with elevated TG levels. We thus hypothesized that PC7 regulates the levels/activity of apoA‐V. Studies in the human hepatic cell line HuH7 revealed that wild‐type (WT) PC7 and its endoplasmic reticulum (ER)‐retained forms bind to and enhance the degradation of human apoA‐V in acidic lysosomes in a nonenzymatic fashion. PC7‐induced degradation of apoA‐V is inhibited by bafilomycin A1 and the alkalinizing agents: chloroquine and NH4Cl. Thus, the PC7‐induced apoA‐V degradation implicates an ER‐lysosomal communication inhibited by bafilomycin A1. In vitro, the natural R504H mutant enhances PC7 Ser505 phosphorylation at the structurally exposed Ser‐X‐Glu507 motif recognized by the secretory kinase Fam20C. Co‐expression of the phosphomimetic PC7‐S505E with apoA‐V resulted in lower degradation compared to WT, suggesting that Ser505 phosphorylation of PC7 lowers TG levels via reduced apoA‐V degradation. In agreement, in Pcsk7−/− mice fed high‐fat diet, plasma apoA‐V levels and adipocyte LpL activity are increased, providing an in vivo mechanistic link for a role of liver PC7 in enhanced TG storage in adipocytes.
The PC7 gene (PCSK7) is on chromosome 11q23.3 close to the gene cluster APOA5/APOA4/APOC3/APOA1, a region implicated in the regulation of lipoprotein metabolism. We propose that PC7 binds apoA‐V directly in the endoplasmic reticulum (ER) and the complex is then sorted to autophagosomes. These autophagosomes then fuse with lysosomes for degradation by macro‐autophagy, which is sensitive to bafilomycin A1.
Proprotein convertase subtilisin kexin type 9 (PCSK9), the last member of the family of Proprotein Convertases related to Subtilisin and Kexin, regulates LDL-cholesterol by promoting the ...endosomal/lysosomal degradation of the LDL receptor (LDLR). Herein, we show that the LDLR cell surface levels dramatically increase in the liver and pancreatic islets of PCSK9 KO male but not female mice. In contrast, in KO female mice, the LDLR is more abundant at the cell surface enterocytes, as is the VLDL receptor (VLDLR) at the cell surface of adipocytes. Ovariectomy of KO female mice led to a typical KO male pattern, whereas 17β-estradiol (E2) treatment restored the female pattern without concomitant changes in LDLR adaptor protein 1 (also known as ARH), disabled-2, or inducible degrader of the LDLR expression levels. We also show that this E2-mediated regulation, which is observed only in the absence of PCSK9, is abolished upon feeding the mice a high-cholesterol diet. The latter dramatically represses PCSK9 expression and leads to high surface levels of the LDLR in the hepatocytes of all sexes and genotypes. In conclusion, the absence of PCSK9 results in a sex- and tissue-specific subcellular distribution of the LDLR and VLDLR, which is determined by E2 levels.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that binds and escorts the low density lipoprotein receptor (LDLR) into the lysosomal degradation pathway. Prescribed ...monoclonal antibodies (mAbs) against PCSK9 prevent its binding to the LDLR, and result in ~60% lower LDL cholesterol (LDLc) levels. Although efficient, mAbs are expensive. Hence other PCSK9 inhibitors are needed. For screening purpose, we developed C57BL/6J mice expressing the human PCSK9 gene under the control of its own promoter, but lacking endogenous mouse PCSK9. All lines recapitulate the endogenous PCSK9 expression pattern. The Tg2 line that expresses physiological levels of human PCSK9 (hPCSK9) was selected to characterize the inhibitory properties of a previously reported single domain antibody (sdAb), PKF8-mFc, which binds the C-terminal domain of PCSK9. Upon intraveinous injection of 10 mg/kg, PKF8-mFc and the mAb evolocumab neutralized ~50% and 100% of the hPCSK9 impact on total cholesterol (TC) levels, respectively, but PKF8-mFc had a more sustained effect. PKF8-mFc barely affected hPCSK9 levels, whereas evolocumab promoted a 4-fold increase 3 days post-injection, suggesting very different inhibitory mechanisms. The present study also shows that the new transgenic mice are well suited to screen a variety of hPCSK9 inhibitors.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes lysosomal degradation of the LDL receptor and is a key regulator of cholesterol metabolism. After the liver, the small intestine is the ...second organ that highly expresses PCSK9. However, the small intestine's ability to secrete PCSK9 remains a matter of debate. While liver-specific PCSK9-deficient mice present no PCSK9 in systemic blood, human intestinal Caco-2 cells can actively secrete PCSK9. This raises the possibility for active intestinal secretion via the portal blood. Here, we aimed to determine whether enterocytes can secrete PCSK9 using in vitro, ex vivo, and in vivo approaches. We first observed that PCSK9 secretion from Caco-2 cells was biphasic and dependent on Caco-2 maturation status. Transcriptional analysis suggested that this transient reduction in PCSK9 secretion might be due to loss of SREBP2-mediated transcription of PCSK9. Consistently, PCSK9 secretion was not detected ex vivo in human or mouse intestinal biopsies mounted in Ussing chambers. Finally, direct comparison of systemic versus portal blood PCSK9 concentrations in WT or liver-specific PCSK9-deficient mice confirmed the inability of the small intestine to secrete PCSK9 into the portal compartment. Altogether, our data demonstrate that mature enterocytes do not secrete PCSK9 and reinforce the central role of the liver in the regulation of the concentration of circulating PCSK9 and consequently of cellular LDL receptors.
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by promoting the degradation of the LDL receptor (LDLR). PCSK9 loss-of-function mutations are ...associated with increased fasting plasma glucose levels and slightly elevated risk of type 2-diabetes. Considering the known detrimental effects of cholesterol accumulation in β-cell, and the widespread use of PCSK9 inhibitors to treat hypercholesterolemia, it is important to gain insight into the role of pancreatic PCSK9 in glucose homeostasis and β-cell function. We generated the first β-cell-specific KO of PCSK9 (βKO). PCSK9 mRNA and protein expression were reduced by 48% and 78% in βKO islets, respectively, indicating that β-cells constitute a major site of PCSK9 expression. In islets, loss of β-cell PCSK9 resulted in unchanged LDLR protein levels, but reduced LDLR mRNA, indicating that cholesterol internalization is enhanced and that β-cell PCSK9 promotes LDLR degradation. In contrast, whole body PCSK9 KO mice exhibited 2-fold higher LDLR protein levels in islets and a stable expression of cholesterogenic genes. Whole body KO and βKO mice presented normal glucose tolerance, insulin release in response to glucose load and insulin sensitivity. Ex vivo glucose-stimulated insulin secretion in presence or absence of fatty acids was similar in WT and KO islets. Like KO mice, individuals carrying loss-of-function PCSK9 variants may be protected from cholesterol-induced toxicity due to reduced circulating cholesterol levels. Using both whole body KO or βKO models, our data demonstrate that PCSK9 deletion in mouse does not have any toxic effect on β-cell function and glucose homeostasis.
•First β-cell-specific KO of PCSK9 (βKO) in mouse showing that pancreatic β-cells express high levels of PCSK9•β-cells, like hepatocytes, secrete PCSK9•βKO islets exhibit reduced levels of cholesterogenic genes, suggesting that LDL internalization is increased•PCSK9 KO and βKO mice exhibit normal glucose tolerance, insulin release, and insulin sensitivity
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