Coordinate Regulation of Cholesterol and Bile Acid Metabolism by the Clock Modifier Nobiletin in Metabolically Challenged Old Mice

Abstract

Cholesterol and bile acid (BA) homeostasis plays a central role in systemic metabolism. Accumulating evidence suggests a key regulatory function of the circadian clock, our biological timer, in lipid metabolism, particularly cholesterol and bile acid flux. Previously, we showed that Nobiletin (NOB), a natural compound targeting the ROR (Retinoic acid receptor-related orphan receptor) nuclear receptors in the circadian oscillator, strongly protects lipid homeostasis, including normal serum cholesterol levels in high-fat (HF) fed mice at both young and old ages. In this study, we further examined the role of NOB in cholesterol metabolism in HF-fed aged mice, and found that NOB lowered the serum LDL/VLDL cholesterol levels and consequently the LDL/HDL ratio. BA levels in the serum were markedly reduced in the HF.NOB group, and examination of additional hepatic markers further indicate a protective role of NOB in the liver. At the molecular level, whereas HF feeding downregulated hepatic expression of several ROR target genes involved in bile acid synthesis, NOB treatment (HF.NOB) was able to rescue it. In accordance, fecal BA excretion was enhanced by NOB, and microbial 16S sequencing revealed alteration of several taxa known to be involved in secondary BA production in the gut. Together, these results demonstrate concerted effects of the clock-modulating compound NOB in cholesterol and BA metabolism, suggesting pharmacological manipulation of the clock as a novel therapeutic strategy against metabolic disorders and age-related decline.

Keywords: Nobiletin; aging; cholesterol and bile acid homeostasis; circadian clock modifier; gene expression; gut microbiota; high-fat feeding; liver.

Figure 1

Nobiletin (NOB) improves cholesterol homeostasis in aged mice. (A) Serum total cholesterol levels, HDL cholesterol levels, LDL/VLDL cholesterol levels, and (B) LDL/HDL ratios were measured by colorimetric assays (n = 6–8). RD: regular diet; HF: high-fat diet; HF.NOB: high-fat diet with 0.1% NOB. *p < 0.05, **p < 0.01, ***p < 0.001, One-Way ANOVA; #p < 0.05, t-test. Bar graphs represent Mean ± SEM.

Figure 2

Nobiletin attenuates serum bile acid levels and other hepatic damage markers. (A) Serum total bile acid levels were measured by colorimetric assays (n = 6–8). (B) Serum taurine-conjugated primary bile acid levels were analyzed by metabolomics (n = 7–9). (C) Serum metabolomics analysis of carnitine and acylcarnitine levels (n = 10–12). (D) Serum alanine aminotransferase (ALT) levels were measured by ELISA (n = 11–14). RD: regular diet; HF: high-fat diet; HF.NOB: high-fat diet with 0.1% NOB. *p < 0.05, ** p < 0.01, *** p < 0.001, One-Way ANOVA; #p < 0.05, t-test. Bar graphs represent Mean ± SEM. For box and whisker plots, box edges correspond to 25th and 75th percentiles, lines inside of the box correspond to 50th percentiles and whiskers include extreme data points.

Figure 3

Nobiletin (NOB) reprograms the expression of circadian and lipid regulatory genes. (A) Core clock gene expression levels in the liver were analyzed by qPCR (n = 7–11). (B) Fatty acid and lipid metabolism related genes in the liver were analyzed by qPCR (n = 7–11) (C) Cholesterol biosynthesis related genes in the liver were analyzed by qPCR (n = 7–11). (D) Bile acid synthesis related genes in the liver were analyzed by qPCR (n = 7–11). RD: regular diet; HF: high-fat diet; HF.NOB: high-fat diet with 0.1% NOB. *p < 0.05, ** p < 0.01, *** p < 0.001, One-Way ANOVA; #p < 0.05, t-test. Bar graphs represent Mean ± SEM.

Figure 4

Nobiletin (NOB) enhances fecal bile acid excretion. Fecal taurine (A) and glycine (B) conjugated primary bile acid levels were measured in metabolomics (n = 10–12). RD: regular diet; HF: high-fat diet; HF.NOB: high-fat diet with 0.1% NOB. *p < 0.05, *** p < 0.001, One-Way ANOVA; #p < 0.05, t-test. For the box and whisker plots, box edges correspond to 25th and 75th percentiles, lines inside of the box correspond to 50th percentiles and whiskers include extreme data points.

Figure 5

Nobiletin (NOB) remodels gut microbiota and protects the liver from inflammatory damage. (A) Microbial 16S rRNA sequencing showing the taxa abundance distribution at the genus level. Left top: alpha diversity box plots; right top: Principal component analysis. P value: 0.001; R-squared: 0.142; F-statistic: 3.3. Yellow: HF; blue: HF.NOB. Bottom: top 10 taxa abundance box plot. Panels indicate the comparison between HF and HF.NOB groups. (n = 10–12). (B) Pathway analysis using 16S sequencing data. Data indicate the comparison between HF versus RD and HF.NOB (H.N) vs HF groups. (n = 10–12). (C) Inflammatory cytokine gene expression levels in the liver were analyzed by qPCR (n = 7–11). RD: regular diet; HF: high-fat diet; HF.NOB: high-fat diet with 0.1% NOB. *p < 0.05, ** p < 0.01, One-Way ANOVA; #p < 0.05, t-test. Bar graphs represent the Mean ± SEM.