Recent studies have focused on the association between circulating PCSK9 concentrations and metabolic parameters in human[8–11]. Plasma PCSK9 has been found to be consistently associated with LDL-C, and less robustly with TG, fasting plasma glucose and HOMA-IR. The major finding of our studies is that plasma concentrations of PCSK9 were induced in response to short-term HFruc diets by 27 to 93% in healthy volunteers (Figure2B-D). Circulating PCSK9 levels were associated with both whole-body, hepatic insulin resistance, liver steatosis and VLDL-TG (Figure4 and Table1). In accordance with a regulation of PCSK9 by carbohydrate intake, we previously demonstrated that high-carbohydrate refeeding in mice increases hepatic PCSK9 mRNA and protein levels. Our study does not explain whether changes in plasma PCSK9 upon a high-fructose diet are causative of the variations in VLDL-TG and what could be the molecular mechanisms involved, in particular whether PCSK9 acts upon hepatic VLDL production. In humans, using lipoprotein kinetics with stable isotopes, we observed an increase of VLDL production in 2 family members with PCSK9 GOF variant S127R but it is unclear whether this is related to this specific variant or to a general trait of PCSK9 GOF variants. In mice, we showed that PCSK9 overexpression is accompanied with hypertriglyceridemia due to VLDL overproduction. However this phenotype was restricted to fasted mice, and was not observed in fed mice. Interestingly, we showed that PCSK9 is normally decreased during fasting. We hypothesized that VLDL production was increased due to a lack of re-uptake of nascent VLDL by the LDLR (as described by Twisk J. et al.). Indeed, fasting seemed to increase the effect of PCSK9 on the LDLR degradation and these mice had virtually no LDLR in their liver compared with fed mice that overexpressed PCSK9. Fructose inhibits hepatic lipid oxidation and favors VLDL-TG-synthesis and it cannot be excluded that PCSK9 was associated with nascent VLDL particles produced by the liver. However, whether PCSK9 is physically associated with lipoproteins remains a controversial issue[8, 37].
Plasma PCSK9 follows a diurnal rhythm that parallels fluctuations of lathosterol to cholesterol ratio. Cholesterol synthesis is driven by SREBP-2 that translocates to the nucleus in response to lower cholesterol content of the endoplasmic reticulum membrane and activates HMG-Coa reductase. SREBP2 also up-regulates PCSK9[12, 39] and LDLR expression. In order to estimate how the present diets affect cholesterol synthesis, we measured serum ratios of lathosterol to cholesterol as a surrogate marker of cholesterol synthesis. There was an increase upon HF diets, as previously described (Figure3A). This might relate to the non-significant trend towards an increase of plasma PCSK9 we observed. It is possible that the trend would turn out to be significant with more subjects. However, upon HFruc diet, there was no change in cholesterol synthesis (Figure3B), but plasma PCSK9 increased significantly, suggesting that SREBP-2 pathway is not responsible for these changes. We showed that SREBP-1c is able to drive the expression of PCSK9 and that SREBP2 and SREBP1c share the same response element on the promoter of PCSK9[14, 42]. Recent studies in hamsters also support the implication of SREBP-1c in PCSK9 regulation. Diurnal fluctuations of PCSK9 that parallel cholesterol synthesis suggest that SREBP2 pathway is dominant over SREBP-1c activation under non-interventional conditions. Our finding that plasma PCSK9 is increased by a high fructose diet but that cholesterol synthesis is not affected (Figure3B) suggests that PCSK9 regulation is not dependent upon SREBP-2 under this specific diet. Because SREBP-1c is induced by a high fructose diet, it might be responsible for the increase of plasma PCSK9.
Our study underlines the disconnection that might take place between PCSK9 and LDL-C level under specific nutritional conditions. Indeed, the increase of LDL-C (reported in) under a HF diet was not linked to an increase of PCSK9. Conversely, the increase of PCSK9 under HFruc diet was not associated to an increase of LDL-C (Figure2B-D). It is surprising that the large increase of circulating PCSK9 seen under fructose (up to 93% in healthy volunteers in HFruc2) was not associated to an increase of LDL-C (Figure2D). Further studies are needed to unravel the molecular mechanisms involved in this disconnection. It is also unclear why the two studies led to such different magnitude of increase in PCSK9 (23% for the 7 day-long vs 93% for the 6 day-long diet). Subjects had on average similar basal concentrations of PCSK9. It is possible that a peak of concentration occurs at day 6 or before. However, HFruc2 induced a higher hypertriglyceridemia than HFruc1 (+33% in HFruc1 Healthy Patients vs +107% in HFruc2), suggesting a better efficacy of the diet.
Several elements suggested a potential association between PCSK9 and postprandial lipidaemia, in majority represented by chylomicrons and their remnants. First PCSK9 might influence chylomicron clearance by degrading the LDLR, although there is conflicting data in Familial Hypercholesterolemia patients on the role of the LDLR in chylomicrons clearance[44, 45]. Second, we showed that PCSK9-deficiency is associated with reduced postprandial hyperlipidaemia in mice challenged with an olive oil bolus, due to decreased apoB output and a modification of chylomicron size, number and catabolism. Here, we failed to detect any variation in plasma PCSK9 concentrations following the acute oral fat load in healthy volunteers (Figure1B). In addition, we found that 2 subjects with PCSK9 LOF mutation responded in a similar fashion than controls. It is possible that an olive oil load, similar to what we did in PCSK9 knockout mice, would have changed the outcome of the investigation in these 2 subjects. However, these subjects cannot be considered as entirely deficient for PCSK9 because it is unclear how much wild type PCSK9 is present in the cells of these individuals and because some wild type protein is still being secreted for one of them. The R104CV114A variant is not cleaved and not secreted. The variant exerts a dominant negative effect over the wild type protein. Carriers of PCSK9 R104CV114A have different concentrations of plasma PCSK9 despite being both heterozygous for the mono-allelic double mutation. For one of them PCSK9 was virtually absent from the blood, while for the other carrier concentrations were around 100 ng/ml. We hypothesized that this variability is due to the dominant effect of the variant. Because the variant is not secreted, we assume that plasma PCSK9 in these subjects is the wild type protein. If plasma PCSK9 had a role in postprandial lipemia, these two subjects would have had a different response to the oral fat load. Of course, some limitations to our study are to be taken into account, as discussed below. All together, our data suggest that plasma PCSK9 is not associated to postprandial hyperlipidaemia in human.
Recent studies suggest that PCSK9 may interfere with glucose homeostasis, since: i) insulin increases PCSK9 expression in vitro in hepatocytes and in vivo in mice and rats[14, 16]; ii) the expression of PCSK9 is altered in rodent models of diabetes; and iii) circulating PCSK9 concentrations were found to be correlated with the level of insulin sensitivity assessed by the HOMA-R index both in adults and in children and adolescents. A recent phenotyping of PCSK9-deficient mice revealed that they were hypoinsulinemic, hyperglycaemic and glucose intolerant. Our own investigations in mice with a different genetic background didn’t point out any obvious abnormality in terms of glucose homeostasis and pancreatic beta cell function. High fructose intake leads to hypertriglyceridemia and hepatic insulin resistance and obesity. Whether the molecule of fructose itself is responsible for these deleterious effects is not established because high sucrose diets leads to similar defects.
We show here that PCSK9 was only positively associated with both whole-body and hepatic insulin resistance in healthy volunteers (including OffT2D) when they fed a short-term HFruc diet, but not under basal conditions (Figure4 and Table1). Additionally, we found that PCSK9 is associated with liver steatosis upon HFruc diet, without any correlation with IMCL. Previous characterizations of these subjects[23, 24] showed that hepatic steatosis was not accompanied with hepatic insulin resistance when induced by the HF, HFHP diets but that it was under the HFruc diet. Such a positive association between circulating PCSK9 levels and liver TG content assessed by proton magnetic spectroscopy was previously described in the cohort of the Dallas Heart Study, although the level of the correlation coefficient was weak (r = 0.13). In accordance with a potential link between PCSK9 and liver steatosis, we recently described a positive association between PCSK9 and gamma-glutamyl transferase levels, a marker of hepatic steatosis, in type 2 diabetic patients.
Finally, our study had certain limitations. Although our metabolic phenotyping was exhaustive, the number of subjects is small and potentially limited the ability to detect weak correlations. In addition, the duration of each diet is short (≤ 7 days) and additional studies with extended periods of dietary intervention need to be performed to confirm our observations. The variation in the extent of fructose-induced PCSK9 expression between the HFruc1 (+23%) and HFruc2 (+93%) diets is surprising since both diets were similar in term of fructose and energy intake. The only difference was the addition of maltodextrin in HFruc2 diet. In parallel with a higher increase of PCSK9, the hypertriglyceridemia was also more robust in HFruc2 diet. While some of our preliminary observations in a small number of healthy volunteers (n=6) suggest that high glucose diet also increase circulating PCSK9 levels (+47%, p=0.17) (data not shown), it would be interesting to confirm this observation in a larger number of subjects. Although our findings suggest that plasma PCSK9 concentrations do not parallel cholesterol synthesis under a high fructose diet, these studies were not designed to explore these aspects. In particular diurnal rhythms of cholesterol synthesis and plasma PCSK9 concentrations were not determined in these patients. Concerning the effect of PCSK9-deficiency on post-prandial lipid profile, we only investigated two subjects from the same family with the same PCSK9 LOF and that were not from the same gender. It cannot be excluded that different conclusions would emerge from a study with more subjects or with subjects with a different LOF mutation.
In summary, we demonstrated that circulating PCSK9 levels are significantly increased following a short-term high-fructose diet. Under these specific nutritional conditions, PCSK9 concentrations were positively correlated with insulin resistance, liver steatosis and VLDL-TG concentrations.