PCSK9 and triglyceride-rich lipoprotein metabolism

Irena Druce,Hussein Abujrad,Teik Chye Ooi,2,?

1Clinical Research Laboratory,Division of Endocrinology and Metabolism,Department of Medicine,University of Ottawa, Ottawa,Ontario K1H 8L6,Canada;

2Chronic Disease Program,Ottawa Hospital Research Institute,The Ottawa Hospital,Ottawa,Ontario K1H 7W9,Canada.

PCSK9 and triglyceride-rich lipoprotein metabolism

Irena Druce1,Hussein Abujrad1,Teik Chye Ooi1,2,?

1Clinical Research Laboratory,Division of Endocrinology and Metabolism,Department of Medicine,University of Ottawa, Ottawa,Ontario K1H 8L6,Canada;

2Chronic Disease Program,Ottawa Hospital Research Institute,The Ottawa Hospital,Ottawa,Ontario K1H 7W9,Canada.

Pro-protein convertase subtilisin-kexin 9(PCSK9)is known to affect low-density lipoprotein(LDL)metabolism, butthere are indications from severallines of research thatitmay also influence the metabolism of other lipoproteins,especially triglyceride-rich lipoproteins(TRL).This review summarizes the currentdata on this possible role of PCSK9.A link between PCSK9 and TRL has been suggested through the demonstration of(1)a correlation between plasma PCSK9 and triglyceride(TG)levels in health and disease,(2)a correlation between plasma PCSK9 and markers of carbohydrate metabolism,which is closely related to TG metabolism,(3)an effectof TG-lowering fibrate therapy on plasma PCSK9 levels,(4)an effectof PCSK9 on postprandiallipemia,(5)an effect of PCSK9 on adipose tissue biology,(6)an effectof PCSK9 on apolipoprotein B production from the liver and intestines,(7)an effectof PCSK9 on receptors otherthan low density lipoprotein receptor(LDLR)thatare involved in TRL metabolism,and(8)an effectofanti-PCSK9 therapy on serum TG levels.The underlying mechanisms are unclear butstarting to emerge.

hyperlipidemia,hypercholesterolemia,molecular biology

Introduction

Pro-protein convertase subtilisin-kexin 9(PCSK9) belongs to a family of intracellular enzymes that convert inactive precursor proteins to active products. These products comprise a broad array of molecules, including hormones,receptors,growth factors and even otherenzymes[1].Yet,PCSK9 itselfis notknown to have an enzymatic role,otherthan to auto-catalytically cleave a pro-domain offitself.PCSK9 playsa role in liverregeneration[2],viralinfection[3]and neuronaldifferentiation[2], but since its discovery in 2003[2]and the demonstration of familialhypercholesterolemia(FH)in subjects with gain-of-function(GOF)PCSK9 mutations[4],the majority ofresearch has been done to elucidate its role in lipoprotein metabolism.

PCSK9 ismosthighly expressed in the liver,intestine, brain and kidneys[2].Itis a secreted protein,mostly from the liver,and its levels are measurable in human plasma or serum[5-8].After reaching the circulation,itis delivered to thecellsurfaceoftissues,notably theliver,whereitbinds to the low density lipoprotein receptor(LDLR),prevents itsrecycling and instead promotesits lysosomaldegradation[9-11].Thisresultsin diminished uptakeofLDLparticles and elevation in LDL cholesterol(LDLC)levels.Thus, PCSK9 can be seen asa pro-atherogenic molecule.

The PCSK9 precursorprotein hasa molecularweight of-74 kDa.Autocatalytic cleavage in the endoplasmicreticulum separates a-14 kDa prodomain from the-60 kDa mature PCSK9 protein.The prodomain remains associated with the rest of PCSK9 by non-covalent bonds and is essential for its role as a modulator of lipoprotein metabolism[12].The cleaved complex is transported through the Golgiapparatus and secreted[2]. Circulating PCSK9 binds to the EGF-A-like domain of hepatic LDLR[10]and escorts it from the endosomal recycling pathway to the lysosome for degradation[9], independent of its catalytic activity[13].There is also evidence for furin cleavage of a-7k Da segment, Ser153-Arg218,from the N-terminus of the catalytic domain.The-53k Da furin-cleaved PCSK9 has less LDLR degradation activity,possibly because of the loss of a region of PCSK9 required for LDLR binding as well as the concomitant loss of the-14k Da prodomain[14].Another study,however,has shown that furin cleavage did not result in dissociation of the pro-domain[15].

The effecton the LDLR is the bestknown action of PCSK9,butthere are indications from severallines of research that PCSK9 may have other effects on lipoprotein metabolism.Here,we provide a review ofthe positive as well as contradictory data on the possible involvementof PCSK9 in the metabolism of triglyceride-rich lipoproteins(TRL),namely chylomicrons (CM),very low density lipoproteins(VLDL)and their remnants.

Data on a possible role of PCSK9 in TRL metabolism

Link between plasma PCSK9 and parameters of triglyceride metabolism

One of the first hints at a link between PCSK9 and TRL metabolism was the demonstration of a positive correlation between plasma PCSK9 and plasma triglyceride(TG)levels[16-23].The correlation demonstrated in moststudies is modest(correlation coefficients of-0.25-0.3).This,however,is akin to the earlier finding of a correlation between plasma PCSK9 and LDLC levels,which prompted the subsequentelucidation of a mechanism for PCSK9's role in regulating LDLR-mediated clearance of LDL.Regarding the link to TG, investigation into possible mechanisms for PCSK9's role is currently underway.A cohesive model has not yetbeen arrived at.Itis noteworthy thatthere are some studies thatdo notshow this correlation[7,24].The reason for the discrepancy among studies is unclear.

An importantrecentstudy wentbeyond TG levels to look atthe relationship between plasma PCSK9 and sub-fractions of VLDL and LDL.Plasma PCSK9 wasshown to predominantly relate to intermediate density lipoproteins(IDL),a TG-rich sub-fraction,which represents primarily VLDL remnants.Thisfinding suggests the possibility of PCSK9 having an effect on plasma TG via effects on the metabolism of VLDL and their remnants[22].

Plasma PCSK9 concentration has also been evaluated via VLDL-TG kinetic studies using stable isotopes in non-diabetic butobese subjects.Here,the results provide no indication that PCSK9 has an impacton TRL metabolism as plasma PCSK9 concentrations did not correlate with VLDL-TG secretion or clearance[25].

Link between plasma PCSK9 and parameters of triglyceride metabolism in disease states

The positive correlation between plasma PCSK9 and TG levels has been found notjustin the generalpopulation but also in some specific disease states.

In patients with proteinuria and chronic kidney disease(CKD)stages 2 and 3[26],and in those with CKD 5 on hemodialysis[27],PCSK9 levels correlate positively with TG levels[26].

An intriguing observation regarding the relationship of PCSK9 and TG was the finding thatin members of four British families with the D374Y gain-of-function (GOF)PCSK9 mutation,which is linked to FH,TG levels were significantly higher(but still within reference range),as compared to controls[28].This suggests that a PCSK9 perturbation may have an impact on TRL metabolism.However,in a study on heterozygous FH(HeFH)and homozygous FH(HoFH)with identified LDLR mutations,TG levels were similar to those in controls[29].This discrepancy in findings may be due to a difference in underlying genetic defect and to the factthatplasma PCSK9 levels in the D374Y patients are low[30],while those in patients with LDLR mutations are high[29].

Link between PCSK9 and diabetes and glucose metabolism

Given the close association between glucose and TG metabolism,furthersupportfora link between PCSK9 and TG metabolism is emerging in the form of evidence that PCSK9 may have a role in glucose homeostasis.In one of the largestcohorts with plasma PCSK9 measurement,in addition to a positive correlation between plasma PCSK9 and TG,Lakoski etal.identified a significantcorrelation between plasma PCSK9 and fasting glucose,insulin and homeostasis model assessmentofinsulin resistance(HOMA-IR),a marker ofinsulin sensitivity[23].The same correlationswere found in a healthy adultpopulation[6],and a cohortof childrenand adolescents aged 9-16 years in a French Canadian population[17].Anothervery large population also demonstrated an association between plasma PCSK9 and plasma glucose and insulin levels[21].

The mechanism of how PCSK9 affects glucose metabolism is still unclear.One study found that rat hepatocytes incubated with insulin for 24 hours had an increase in PCSK9 messenger RNA(mRNA)[31]. In human studies,a 24-hourinsulin infusion in a hyperinsulinemic glucose clamp experimentin healthy and diabetic(type 2)individuals had no effect on plasma PCSK9 levels[32],while a 3-hour euglycemic-hyperinsulinemic clamp study in non-diabetic postmenopausal obese patients actually decreased plasma concentrations of PCSK9[33].In addition,human liver-derived cell lines produced less PCSK9 protein and PCSK9 mRNA and secreted less PCSK9 into theirculture medium in response to the presence of insulin[33].

Otherresearch found thatplasma PCSK9 levels were not different among patients with normal glucose metabolism,impaired glucose metabolism and type-2 diabetes mellitus[34].Also,no association was found between plasma PCSK9 levels and body mass index (BMI),waistcircumference,fatmass and fat-free mass, orvisceraland subcutaneousadipose tissue measured by computed tomography in abdominally obese men[16].

Thus,there are differing data on the effectof insulin on the regulation of PCSK9 and on how insulin resistance affects PCSK9 status.Insulin action has major effectson TRL metabolism and more studies are needed to clearly determine its effect on PCSK9.It is possible that the putative link between PCSK9 and TG is the resultof the influence of insulin action on PCSK9.

Conversely,the impactof PCSK9 deficiency on glucose metabolism also remains uncertain.In a study comparing wild-typ e to PCSK9 knockout mice, Mbikay etal.showed that knockoutmice had higher fasting plasma glucose,lower plasma insulin and higher glycemia on an oral glucose tolerance test[35]. However,another study failed to detect any alteration in glucose homeostasis in PCSK9-deficientmice[36].

PCSK9 and fibrate therapy

Fibrates are a class of medications used to treat hypertriglyceridemia,and they exert their action via the peroxisome proliferator-activated receptorα (PPAR-α)to increase peripheral TRL lipolysis via lipoprotein lipase,decrease intracellular lipolysis in adipose tissue and decrease secretion of VLDL[37].The demonstration of fibrate therapy having an effect on plasma PCSK9 levels raises the question of whether PCSK9 mediates some of the effects of fibrates on TRL metabolism.The picture,however,is unclear. Some studies have demonstrated a decrease in PCSK9 with fibrate use[7,38]while others have demonstrated an increase[8,39-41].A recentmeta-analysis looked at 6 studies and a total of 218 patients treated with fibrates for a period of 6 to 24 weeks.The conclusion was thatfibrate treatment,especially when compared to controls,significantly raised PCSK9 levels[42].In cell culture experiments,various fibrates were shown to repress PCSK9 expression in immortalized human hepatocytes[43].Beyond that,itis notknown how the fibrate-induced changes in PCSK9 influence TRL metabolism.It is also plausible,but unproven,that plasma PCSK9 changes are secondary to fibrateinduced changes in TRL metabolism.Further research is needed to clearly elucidate the link between fibrate treatmentand plasma PCSK9;the implications are relevantand interesting.

PCSK9 and postprandial lipemia

Postprandial lipemia(PPL)refers to the status of lipids and lipoproteins in blood following a fat load or a meal.Le May and his colleagues fed PCSK9 knockoutmice and theirwild-type littermates,a bolus of olive-oil.Attime zero,levels of TG were comparable;however,after 2 hours,the knockoutmice showed a significantly attenuated TG response.In addition,this study demonstrated that PCSK9 was highly expressed throughout the digestive tract and colon at levels equivalentto thatin the liver.Intestinallymph analysis revealed reduction in apoB,butnotTGoutput,resulting in larger TG-rich CM in knockoutmice compared to wild-type littermates.In addition,kinetic studies showed that PCSK9-deficient mice had an increased ability to clear CM compared to wild-type littermates. The study further demonstrated that the difference in observed TG levels in the knockoutmice was notdue to alterations in fat absorption,gastric emptying or intestinal transit[36].

A few studies have looked at PCSK9’s relation to PPL in humans.The first,by Cariou et al.,found no change in PCSK9 levels following an oral fat load in a small10-patientsample.They also demonstrated that postprandialtriglyceride excursion was notaltered in 2 carriers of a PCSK9 loss-of-function(LOF)mutation compared with non-carriers[19].The second by Chan etal.looked at17 obese subjects who were given an oralfatload.They found thatin the postprandialperiod (totalof 24 hours post),PCSK9 was significantly associated with the area-under-the-curve for apoB-48 and inversely with the TG-apo B-48 fractional catabolic rate.They interpreted their findings as indicating thatcatabolism of TG and apoB-48-containing CM may be coordinated by PCSK9 in the postprandial state in obese individuals[20].

Overall,data on the effectof PCSK9 on PPL,and of PPL on PCSK9,are very limited.Since the postprandialperiod is when TRL metabolism is most active, a demonstration of an effect of PCSK9 on indices of PPL would be an indication of an involvement of PCSK9 in TRL metabolism.

PCSK9 and fat deposition

Animalstudies have shown a link between PCSK9 and fat-deposition.Roubtsova and colleagues demonstrated that PCSK9 knockout mice accumulated 80% more visceralbody fat(perigonadaland perirenaldeposits)than wild-type mice.They subsequently showed that this was the result of adipocyte hypertrophy and increased fatuptake into adipose tissue via highercellsurface levels of very-low-density lipoprotein receptor (VLDLR;see below).Further ex-vivo models showed thatadipose,muscle and livertissue from knockoutmice had a higher rate of TG synthesis.Finally,they demonstrated the same effectin mice thatwere knockouts for both PCSK9 and LDLR,demonstrating that the effect they observed was LDLR-independent[44].

A recentstudy by Mbikay etal.supported these findings;they also found that PCSK9 knockoutmice accumulated more perigonadalfat.Interestingly,they noted a greater effect in female mice fed a‘Western’high fatdiet[45].

These findings provide evidence for a role o f PCSK9 in adipose tissue biology,which in turn could have a strong influence on overall TRL metabolism.

PCSK9 effect on apolipoprotein B

Effect of PCSK9 on ApoB in the liver

Various cellculture and animalstudies have demonstrated a link between PCSK9 and apolipoprotein B (apo B)secretion from the liver.Rat hepatoma cells (McArdle-7777)overexpressing the GOF D374YPCSK9 mutant demonstrated increased secretion of apoB-containing lipoproteins[46].Similarly,mice overexpressing PCSK9 from a transgenic vectorhad higher plasma levels of both apoB100 and apoB48,and TG, and the effect was independent of the LDLR[47]. Interestingly,this study also provided data on the lack of effect of PCSK9 expression on genes governing cholesterol and TG biosynthesis[47],suggesting that PCSK9’s effecton TRL metabolism may be mediated mainly through an effecton apoB.Furthermore,studies showed that apo B100 secretion from primary hepatocytes of PCSK9 knockoutmice was reduced[48].

There have also been some studies in human subjects on the effect of PCSK9 on apoB.It was shown in a stable isotope kinetic study that FH patients carrying the GOF S127R mutation in PCSK9 over-produce apo B-100(3-fold)along with overproduction of VLDL(3-fold),IDL(3-fold),and LDL(5-fold)[49]. Furthermore,Chernogubova et al.looked ata sample ofalmost6000 middle-aged subjects and found a significantcorrelation between serum PCSK9 and apoB[21].

The link between PCSK9 and secretion of TRL has also been studied,although data are limited.Mice overexpressing PCSK9 were found to have increased hepatic VLDL production during fasting[50]and increased secretion of TRL into the serum[51].

A possible mechanism by which PCSK9 may regulate apoB levelsis via the LDLR.Ithas been shown that intracellular LDLR protein can bind nascentapoB-containing lipoproteins and direct them for degradation[52]. It is therefore possible that when LDLR is depleted through the action ofPCSK9,apo-B isdegraded ata lesser rate.

Other research has suggested that PCSK9 may affect apoB by stabilizing it and preventing its degradation. Using pulse chase experiments on primary hepatocytes from these mice over-expressing apoB,Sun etal.showed thatPCSK9 bindsto apoB via its N-terminusand protects it from undergoing autophagy.This effect was found to be independentofthe LDLR[47].Autophagy hasbeen previously demonstrated to be a means ofapoB degradation in hepatocytes,in addition to the lysosome pathway[47].

Effect of PCSK9 on ApoB in the intestine

Several studies have shown that human enterocytes treated with recombinanthuman PCSK9 demonstrated increased cellular and secreted apo B-48 and apoB-100[36,53,54].Levy etal.demonstrated in a human enterocyte cell line(Caco2/15 cells)that GOF D374YPCSK9 enhanced cholesterol uptake was associated with increased expression of cholesterol transporters NPC1L1 and CD36,and increased CM secretion through increased lipid and apo B48 biogenesis. PCSK9 silencing had the opposite effects.These responses were independent of the LDLR[53].

Rashid etal.showed thatthe same cells,treated with recombinant human PCSK9,had increased production of TRL through both LDLR-dependent and LDLR-independe n t mecha n ism.Cellular and secreted apoB48 and apo B100 were enhanced.The increase in apoB was due to increased apoB mRNA transcription and enhanced apoB stability.In line with findings thatintracellular neutrallipids inhibitdegradation and enhance stability of apo B,they demonstrated thatPCSK9 treatment increased cellular neutral lipids via augmentation of levels of lipid-generating enzymes. They also demonstrated in mice that the levels and activity of intestinal microsomal triglyceride transfer protein(MTP),which transfers neutral lipids to apo B,is increased by PCSK9,again resulting in greater apoB stability.All of the demonstrated effects were reversed by short-term inhibition of PSCK9 via smallinterfering RNA(siRNA)[54].

Taken together,these findings indicate that PCSK9 may be linked with production of apoB48 necessary for intestinal CM assembly.

PCSK9 effect on other receptors in the LDLR family

Effect of PCSK9 on VLDLR,apoE2 receptor and LRP-1

Otherresearch has suggested thatPCSK9 may affect TRL metabolism via its effects on receptors other than LDLR.It has been shown that PCSK9 binds to 3 other members of the LDLR family;the VLDL receptor(VLDLR),apo E2 receptor(apoER2)[12,44,55],and LRP-1[56].These receptors show high homology with the LDLR;the VLDLR has 59%identity,and the apoE2 receptor has 46%identity with the LDLR[55]and the LRP-1,40%[14,56].When HEK293 cells(human embryonic kidney cell line)expressing VLDLR and apoER2 were incubated with conditioned media containing PCSK9,cellular levels of these receptors were decreased[55].Thus VLDLR and apoER2 are downregulated by PCSK9 and the mechanism is the same as for the LDLR,namely,redirection of receptors to the lysosomalcompartment for degradation[55].

Both the VLDLR and apoER2 bind VLDL,but whether these receptors play a significant role in TG metabolism in humans is still unclear.Homozygous VLDLR knockoutmice had normalplasma lipoproteins but they had lower body weight and adipose tissue mass,indicating a possible role for VLDLR in storage of lipids in adipose tissue[57].This was further supported by the finding that when PCSK9 was added back to the knockoutmice,the expression of VLDLR protein in adipose tissue significantly decreased[44].

LRP-1,on the otherhand,is known to play a major role in clearance of CM and VLDL remnants.While early studies suggested little link between LRP1 and PCSK9[9,14],subsequent research by Canuel et al. showed that PCSK9 could degrade LRP-1 and thatit competeswith the LDLR forPCSK9 activity[56].Apositive effect of PCSK9 on LRP-1 function would lend furthersupportfora role of PCSK9 in TRL metabolism. Effect of PCSK9 on CD36

Finally,there is preliminary evidence that PCSK9 may target CD36,a scavenger receptor with multiple ligands and cellular functions,including facilitating cellular uptake of free fatty acids,though the results are contrasting.Roubtsova and her colleagues noted thatin PCSK9 knockoutmice,there was no effecton CD36 expression in perigonadalfat deposits of male mice,but that in females CD36 mRNA expression was increased by almost 80%in response to the loss of PCSK9[44].Similarly,Levy atal.showed thathuman enterocytes made to overexpress normal PCSK9,and the D374Y-PCSK9 GOF mutant,produce increased CD36[53].On the other hand,recent research by Demers noted that overexpression of PCSK9 led to the downregulation of CD36 in several cell lines, including adipoctyes[58].Whether PCSK9 targets CD36 for degradation or regulates it via a different mechanism requires further study;however,as CD36 has been linked to CM remnant clearance[59],its link to PCSK9 is relevantto further support PCSK9’s role in TRL metabolism.

Effect of pharmacologic targeting of PCSK9 on TG

LDL cholesterolis a well-established risk factor for cardiovascular disease and it is a primary target in lipid-lowering therapy.TRL have also been implicated in atherosclerosis[60].

In light of the discovery of PCSK9’s effect on LDLC,ithas recently become a targetfor pharmaceutical intervention.Several different approaches have been explored to inhibit or reduce PCSK9 action.In human clinicaltrials,there has been most interest in antibodies against PCSK9 and two have been extensively studied;evolocumab and alirocumab are fully human monoclonal antibodies against PCSK9.The trials were designed to examine the effects of these novelmedications on LDLC,butseveralof them have reported on the effecton TG and VLDL.

Trials have been done on patients withoutany known genetic predisposition towards dyslipidemia[61-69],and those with FH,both HeFH[70,71]and Ho FH[72-74].The reported effects on TRL have been mixed;severaltrials report no change in serum TG or VLDL cholesterol (VLDLC)levelwith treatment[61,65-68,70,72,73],while others demonstrate a significantlowering of these measured parameters[62-64,69,71,74].Interestingly,the majority of the positive trials tended to be larger,theirstudy population being generally between 300 and 900 patients[62-64,69,71,74], while the negative ones were smaller,with patientnumbers ranging from eightto 183[61,65,67,68,70,72,73],suggestingthatperhaps the negative trials lacked powerto demonstrate an effect.

Stein etal.recently published a pooled analysis ofa totalof 1359 patients from 4 of the above-discussed trials and they showed that treatment with anti-PCSK9 antibodies sig nificantly lowered TG[75]. Overall,these findings further suggesta link between PCSK9 and TRL metabolism and hintatthe possibility of a novel therapy againsthypertriglyceridemia.

Conclusions

The effect of PCSK9 on LDL metabolism through promotion of LDLR degradation is well described. As more data are gathered,there are indications that PCSK9 may have additionalroles in other aspects of lipoprotein metabolism.This review focuses specifically on TRL metabolism.The pieces of evidence available are mostly observational and piecemeal.In some cases,the data are contradictory and uncertain. The available data seem also to suggestthatthe effect of PCSK9 on TRL metabolism is modest.No cohesive mechanistic model has been thus far been described. However,the existing data for a putative role of PCSK9 in TRL metabolism should be recognized and pursued further.It is encouraging that evidence to support a mechanism for the effect of PCSK9 on TRL metabolism is starting to emerge,especially in the area of PCSK9’s effect on intestinal production of TRL.As with the currentdevelopmentof treatment for elevated LDLC with anti-PCSK9 agents,itis possible that similar PCSK9-related strategies could be found for the treatment of hypertriglyceridemia.

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?Corresponding author:Dr.T C Ooi,The Ottawa Hospital-Riverside Campus,1967 Riverside Drive,Ottawa,Ontario K1H 7W9,Canada, Tel:(613)738 8400 Ext.81950,E-mail:tcooi@toh.on.ca.

Received 30 March 2015,Accepted 01 July 2015,Epub 20 July 2015

R589.2,Document code:A

The authors reported no conflict of interests.

?2015 by the Journal of Biomedical Research.All rights reserved.

10.7555/JBR.29.20150052