Adropin: An endocrine link between the biological clock and cholesterol homeostasis

Abstract

Objective: Identify determinants of plasma adropin concentrations, a secreted peptide translated from the Energy Homeostasis Associated (ENHO) gene linked to metabolic control and vascular function.

Methods: Associations between plasma adropin concentrations, demographics (sex, age, BMI) and circulating biomarkers of lipid and glucose metabolism were assessed in plasma obtained after an overnight fast in humans. The regulation of adropin expression was then assessed in silico, in cultured human cells, and in animal models.

Results: In humans, plasma adropin concentrations are inversely related to atherogenic LDL-cholesterol (LDL-C) levels in men (n = 349), but not in women (n = 401). Analysis of hepatic Enho expression in male mice suggests control by the biological clock. Expression is rhythmic, peaking during maximal food consumption in the dark correlating with transcriptional activation by RORα/γ. The nadir in the light phase coincides with the rest phase and repression by Rev-erb. Plasma adropin concentrations in nonhuman primates (rhesus monkeys) also exhibit peaks coinciding with feeding times (07:00 h, 15:00 h). The ROR inverse agonists SR1001 and the 7-oxygenated sterols 7-β-hydroxysterol and 7-ketocholesterol, or the Rev-erb agonist SR9009, suppress ENHO expression in cultured human HepG2 cells. Consumption of high-cholesterol diets suppress expression of the adropin transcript in mouse liver. However, adropin over expression does not prevent hypercholesterolemia resulting from a high cholesterol diet and/or LDL receptor mutations.

Conclusions: In humans, associations between plasma adropin concentrations and LDL-C suggest a link with hepatic lipid metabolism. Mouse studies suggest that the relationship between adropin and cholesterol metabolism is unidirectional, and predominantly involves suppression of adropin expression by cholesterol and 7-oxygenated sterols. Sensing of fatty acids, cholesterol and oxysterols by the RORα/γ ligand-binding domain suggests a plausible functional link between adropin expression and cellular lipid metabolism. Furthermore, the nuclear receptors RORα/γ and Rev-erb may couple adropin synthesis with circadian rhythms in carbohydrate and lipid metabolism.

Keywords: Adropin; Cardiovascular disease; Cholesterol; LDL; Obesity; Sex.

Graphical abstract

Figure 1

Inverse association between plasma adropin concentrations LDL-C levels in data pooled from the RRS, CREG, DRS, IPOP and HERITAGE studies. (A) Box and whisker plot showing distribution and range of plasma adropin concentrations in males and females. Extreme values are shown as circles. Relationship between fasting plasma adropin concentration and LDL-C (Log2-transformed) in males (B) and females (C). In panels B and C shading is used to indicate separation of data into quartiles ranked by plasma adropin concentration. (D) Plasma adropin concentrations in data pooled into quartiles by ranking LDL-C levels low to high (1st through 4th quartile read left to right in the panel). Mean plasma adropin concentrations decline with increasing LDLc in men, but not woman. LDL-C levels in D are estimated marginal means ± SEM adjusted for age and BMI. a p < 0.01 vs. the 4th LDL-C quartile for males, p < 0.05 vs. females all quartiles; b p < 0.05 vs. females in the 1st quartile.

Figure 2

Significantly lower age- and BMI-adjusted levels of atherogenic cholesterol in men with high plasma adropin concentrations. Men were separated into tertiles (n = 116) ranked by BMI (low to high), and then further ranked within BMI tertile into quartiles ranked by plasma adropin concentration. Average concentrations of LDL-C (A), nonHDL-C (C), total cholesterol (E) and HDL-C (G) within adropin quartile (n = 87/group). Relative difference (plus or minus) to the mean of all samples for LDL-C (B), nonHDL-C (D), total cholesterol (F) and HDL-C (H) are shown within groups defined by BMI tertile and adropin quartile (n = 29). *p< 0.05 vs. adropin 1st quartile or groups indicated by line; **p < 0.01 vs. adropin 1st quartile or groups indicated by line. Values are estimated marginal means ± SEM.

Figure 3

An interaction between age and adropin quartile on BMI in men. Men were separated into tertiles (n = 116) ranked by age (youngest to oldest), and then further ranked within age tertile into quartiles ranked by plasma adropin concentration. (A) Average age in years within each age tertile. (B) Average BMI within age tertile demonstrating the predicted increase observed with aging. * all groups are significantly different, 2nd vs 3rd tertile, p < 0.05; p < 0.005 for other comparisons. (C) Average BMI within adropin quartile (n = 87). (D) Average difference from the mean for BMI within groups defined by BMI tertile and adropin quartile (n = 29). The interaction between age tertile and adropin quartile is significant (p < 0.05). Men in the youngest age tertile generally have a lower BMI compared to the mean for all participants. Conversely, men in the oldest age tertile generally have a higher BMI. The largest difference in BMI between age tertile observed in men with higher plasma adropin concentrations. On the other hand, gains in BMI between age groups is modest in men with low plasma adropin concentrations.

Figure 4

Rhythmicity in hepatic Enho expression in mice (A) and in plasma adropin concentration in nonhuman primates (B–E). (A) Liver Enho expression at 4 h intervals over 48 h in AKR/J mice (n = 3/group). Peak expression occurs between ZT20 and ZT4, corresponding to a window 4 h prior to and after lights on at 06:00 h. (B) Averaged plasma adropin concentrations (n = 6/group). (C) Averaged deviation from the 24 h mean. (D) Plasma adropin concentration data shown for the individual monkeys. *, p < 0.05 vs. 12:00 h and 24:00 h; **, p < 0.05 vs. 06:00 h, 12:00 h and 24:00 h. Data shown in panels B-D are double plotted.

Figure 5

Rhythms in adropin expression are regulated by the interaction of RORα/γ and Rev-erb with binding sites upstream of the Enho gene; dietary cholesterol suppresses hepatic Enho expression. (A) An analysis of transcriptional activity of the mouse Enho gene in liver at ZT10 and ZT22 using ChIP-Seq and Gro-SEQ. Histone acetylation is also shown. (B) Liver Enho expression in male B6 mice fed chow (n = 6 for vehicle and SR9009, n = 5 for SR1001) or high cholesterol diet treated with the RORα/γ inverse agonist SR9009 or Rev-erb agonist SR1001 (n = 9/group). *, p < 0.05; ***, p < 0.000. (C) Liver Enho expression in livers of 5 inbred strains used for the Diversity Outbred population maintained on HFCA or control diet (n = 3/group). ENHO expression in HepG2 cells treated with a Rev-erb agonist (D) or RORα/γ inverse agonist (E). (F) Suppression of ENHO expression in HepG2 cells cultured in reduced lipid serum treated with 10 μM 7-beta-hydoxysterol (7β-OHC), 7-Ketocholesterol (7-KC) and cholesterol for 24 h. With the exception of HMGCR, treatment had a significant effect on gene expression (p < 0.005). Columns marked a are significantly different from control, p < 0.005; b from cholesterol (p < 0.05); c the values between all groups were significantly different (p < 0.05 control vs. cholesterol; p < 0.001 for the other groups); d from cholesterol (p < 0.01); e from control (p < 0.05 for cholesterol, p < 0.001 for 7β-OHC and 7-KC).

Figure 6

Adropin overexpression does not reduce cholesterol levels or prevent atherosclerosis in mice. (A) Body weight data for male and female Ldlr−/− or AdrTG;Ldlr−/− mice. (B) Atherosclerotic lesions in Ldlr−/− and AdrTG;Ldlr−/− mice maintained on a high cholesterol (HC) diet are similar. (C) Measurements of plasma cholesterol (total, HDL, LDL) are similar in Ldlr−/− and AdrTG;Ldlr−/− mice maintained on a high cholesterol (HC) diet are similar. (D) Total cholesterol levels and the distribution of cholesterol between VLDL, IDL/LDL and HDL fractions are similar in chow-fed Ldlr−/− and AdrTG;Ldlr−/− mice.

Figure 7

Adropin overexpression does not suppress 3-Hydroxy-3-Methylglutaryl-CoA Reductase (Hmgcr) expression in the mouse liver. Expression of the adropin transcript is significantly increased in mice expressing the adropin transgene (AdrTG). While some genes exhibit a modest reduction in expression, the expression of HMGCR which is rate limiting for cholesterol synthesis is normal. The data shown are estimated marginal means of genes measured using qRT-PCR. *p < 0.05 compared to mice not expressing the adropin transgene. LSS: lanosterol synthase, catalyzes the first step in the biosynthesis of cholesterol. MVK: mevalonate kinase, catalyzes the second step in the biosynthesis of cholesterol. SC4MOL, a sterol-C4-methyl oxidase that is localized to the endoplasmic reticulum membrane thought to function in cholesterol synthesis.