## 4 Discussion
The reason for the discordant results is not obvious. In the current study, the mRNA expression of HMGCR was associated with relative abundance of several gut bacteria, including a positive association with the _Murbaculaceae_, _Alloprevtotalla_, _Alistipes_ and _Lachospiraceae_, _NKA136_group_ and inversely association with _Faecalibaculum_. In particular, the mRNA expression of HMGCR was inversely associated with the relative abundance of _Lactococcus_, which was lower in both NR-supplemented groups than the CD and HFD groups without supplementation. These findings indicated that NR supplementation may alter the gene expression of HMGCR via changes in the gut microbiota. PCSK9 has been reported to inhibit the LDL receptor, resulting in lower cholesterol uptake by the liver and subsequent higher cholesterol concentrations in the circulation [38]. Intestinal ABCA1 promotes the efflux of free cholesterol from cells to HDL, which contributes to reverse cholesterol transport [39]. Based on our data, NR supplementation resulted in the lower mRNA expression of hepatic PCSK9 and higher mRNA expression of intestinal ABCA1 in ApoE/- mice fed with HFD than mice without supplementation. These results indicated that NR supplementation may promote cholesterol uptake and efflux via regulating the expression of genes involved in these pathways, although these alterations did not affect circulating cholesterol concentrations. In addition, the relative abundance of _Coriobacteriaceae_, _UCG-002_, which was lower following NR supplementation in ApoE/- mice fed with HFD than mice without supplementation, had a positive correlation with the hepatic mRNA expression of PCSK9 and an inverse correlation with the intestinal mRNA expression of ABCA1. Therefore, NR supplementation may regulate the gene expression of PCSK9 and ABCA1 via changes in the gut microbiota, which requires further investigation. CYP7A1 is a key enzyme in the conversion of cholesterol to bile acids and plays an important role in maintaining the stability of cholesterol concentrations in the circulation [40]. Previous studies have reported that RYR and extracts upregulate the mRNA expression of CYP7A1 [13; 20], which may contribute to the decrease of circulating cholesterol concentrations via promoting the conversion of cholesterol to bile acids. Our data were consistent with these findings, although we did not observe subsequent changes in plasma cholesterol concentrations after NR supplementation. The relative abundance of _Lactococcus_, which was lower in CD- or HFD-fed mice supplemented with NR in comparison to non-supplemented mice, had an inverse correlation with the hepatic mRNA expression of CYP7A1. Prior to our study, the effect of supplementation with natto or RYR or NR on the relative abundance of _Lactococcus_ has not been reported. Further studies are required to explore whether NR supplementation could regulate the gene expression of CYP7A1 via changes in the gut microbiota. The majority of previous studies focus on the effect of natto or RYR on lipid and lipoprotein profiles, and less attention has been paid to the glucose metabolism. The current study observed that NR supplementation resulted in lower fasting blood glucose concentration in ApoE/- mice fed with HFD, which was consistent with some prior reports with regard to RYR supplementation [41; 42]. Recent studies have demonstrated the role of gut microbiota and related metabolites in the maintenance of glucose homeostasis [43]. Data from the current study also demonstrated that the relative abundance of the _Muribaculaceae_, _Alistipes_ and _Lachospiraceae_\(\_\)_NKA136_group_, which were lower in both HFD and HFD + NR groups compared to CD and CD + NR groups, had inverse correlations with the fasting plasma concentrations of glucose. The relative abundance of the _[Eubacterium]_\(\_\)coprostanoligenes_\(\_\)group_, which was higher in both the HFD and HFD + NR groups compared to the CD and CD + NR groups, was positively correlated with fasting blood glucose concentrations.