Chronic intermittent psychological stress promotes macrophage reverse cholesterol transport by impairing bile acid absorption in mice

Abstract

Psychological stress is a risk factor for atherosclerosis, yet the pathophysiological mechanisms involved remain elusive. The transfer of cholesterol from macrophage foam cells to liver and feces (the macrophage-specific reverse cholesterol transport, m-RCT) is an important antiatherogenic pathway. Because exposure of mice to physical restraint, a model of psychological stress, increases serum levels of corticosterone, and as bile acid homeostasis is disrupted in glucocorticoid-treated animals, we investigated if chronic intermittent restraint stress would modify m-RCT by altering the enterohepatic circulation of bile acids. C57Bl/6J mice exposed to intermittent stress for 5 days exhibited increased transit through the large intestine and enhanced fecal bile acid excretion. Of the transcription factors and transporters that regulate bile acid homeostasis, the mRNA expression levels of the hepatic farnesoid X receptor (FXR), the bile salt export pump (BSEP), and the intestinal fibroblast growth factor 15 (FGF15) were reduced, whereas those of the ileal apical sodium-dependent bile acid transporter (ASBT), responsible for active bile acid absorption, remained unchanged. Neither did the hepatic expression of cholesterol 7α-hydroxylase (CYP7A1), the key enzyme regulating bile acid synthesis, change in the stressed mice. Evaluation of the functionality of the m-RCT pathway revealed increased fecal excretion of bile acids that had been synthesized from macrophage-derived cholesterol. Overall, our study reveals that chronic intermittent stress in mice accelerates m-RCT specifically by increasing fecal excretion of bile acids. This novel mechanism of m-RCT induction could have antiatherogenic potential under conditions of chronic stress.

Keywords: Bile acids; physical restraint; psychological stress; reverse cholesterol transport.

Figure 1

Schematic illustration of the chronic intermittent stress regime. The 5-day stress regime included a total of 14 h of restraint stress divided in 2-h episodes separated by sedentary periods. The regime was terminated on day 4 (after a 4-h sedentary period) or on day 5 (immediately after stress). Feces were collected for 24 or 48 h, depending on the assay. Mice were euthanized immediately after the final stress episode.

Figure 2

Chronic intermittent stress modulates serum corticosterone, body weight, and fecal pellet output. (A) Concentration of corticosterone (ng/mL) in serum obtained from mice subjected to repeated restraint stress for five (N = 15 mice/group) or 14 days (N = 5 mice/group) and from respective control mice. Blood samples were drawn immediately after the final stress episode of the regimes. (B) Food consumption (g/mouse) during the final 24 h of the 5-day stress regime (N = 5 mice/group). (C) Body weight (g) at the start (initial) and at the end (final) of the 5-day stress regime (N = 10 mice/group). (D) Fecal pellet output during a 3-h stress episode at the end of the 5-day stress regime (N = 5 mice/group). (E) Fecal pellet output during the two final days of the 5-day stress regime (N = 10 mice/group). **P < 0.01; ***P < 0.001. All data are presented as mean + SD.

Figure 3

Chronic intermittent stress stimulates m-RCT without affecting cholesterol efflux or the expression of hepatic cholesterol transporters. (A) Mice were injected intraperitoneally with [³H]cholesterol-loaded macrophages. ³H-radioactivity (as % of injected dose) in fecal cholesterol (CHOL) and bile acid (BA) fractions, in total serum and HDL fraction, and in the liver was determined 24 h after the injection (N = 8–10 mice/group). (B) The capacity of serum (2.5% v/v in medium) to stimulate cholesterol efflux from J774A macrophage foam cells was measured in vitro (serum pools from 5 mice/group). (C) mRNA levels (relative units) of hepatic cholesterol transporters and transcription factors (N = 5–10 mice/group). (D) Hepatic scavenger receptor BI (SR-BI) protein level (as optical density, OD, normalized to that of β-actin) (N = 5 mice/group). *P < 0.05. All data are presented as mean + SD.

Figure 4

Chronic intermittent stress does not alter intestinal cholesterol absorption but it affects mRNA expression of intestinal cholesterol transporters and stimulates large intestinal transit. (A) Fractional cholesterol absorption efficiency (%) was measured by the fecal dual isotope method over 24 h (N = 6–10 mice/group). (B) Intestinal and fecal [¹⁴C]cholesterol/[³H]sitostanol ratios and the serum [¹⁴C]cholesterol content were measured 3 h after an oral gavage of [¹⁴C]cholesterol and [³H]sitostanol (N = 5 mice/group). (C) mRNA levels (relative units) of intestinal transcription factors and cholesterol transporters regulating cholesterol absorption (N = 5–15 mice/group). (D) [³H]sitostanol levels (as % of gavaged dose) in the gastrointestinal tract of control and repeatedly stressed mice 3 h after oral dosing (N = 10). *P < 0.05, **P < 0.01. All data are presented as mean + SD.

Figure 5

Chronic intermittent stress alters the intestinal distribution of [³H]taurocholate and stimulates the total fecal excretion of bile acids. (A) Distribution of orally gavaged [³H]taurocholate (as % of dose normalized to the recovery of an internal standard) in serum and liver, gallbladder, small and large intestine, and feces 24 h (or 3 h in the case of the first blood sample) after administration (N = 10 mice/group). (B) Total fecal bile acid (BA) and cholesterol (CHOL) excretion rate (μmol per day per gram of body weight, BW) in repeatedly stressed and control mice during the final 48 h of the 5-day stress regime (N = 5 mice/group). (C) Composition of the bile acid pool (N = 5 mice/group) and neutral sterol pool (N = 3 mice/group) in feces excreted over 24 h on day 4 of the 5-day stress regime. CA = cholic acid, MCA = muricholic acid, CDCA = chenodeoxycholic acid, LCA = litocholic acid, DCA = deoxycholic acid. The cholesterol-derived neutral sterols included cholesterol and coprostanol, and the plant-derived neutral sterols and stanols included campesterol, campestanol, stigmasterol, sitosterol, sitostanol, and avenesterol. *P < 0.05. All data are presented as mean + SD.

Figure 6

Effect of chronic intermittent stress on the intestinal and hepatic expression of transcription factors, enzymes, and transporters that govern the enterohepatic circulation of bile acids. (A) mRNA levels (relative units) of key bile acid transporters and the transcription factor FXR in the small intestine (N = 5–10 mice/group). (B) Ileal ASBT protein (as optical density, OD, normalized to that of β-actin) (N = 5 mice/group). (C) Hepatic mRNA levels (relative units) of the key bile acid transporters, enzymes of synthesis pathways, and regulating transcription factors (N = 5–15 mice/group). (D) Hepatic CYP7A1 protein expression (as optical density, OD, normalized to that of β-actin) (N = 5 mice/group). *P < 0.05, **P < 0.01, ***P < 0.001. All data are presented as mean + SD.

Figure 7

Gallbladder bile acid levels and protein expression of CYP7A1 and ASBT in repeatedly stressed and control mice immediately after stress and during a 4-h sedentary period. Gallbladder bile was collected immediately (day 5) or 4 h after stress (sedentary period, day 4) from control and stressed mice. All mice were fasted for 2 h before sample collection. (A) Bile acid (BA), cholesterol (CHOL), and phospholipid (PL) concentrations and (B) the bile acid/cholesterol (BA/CHOL) and bile acid/phospholipid (BA/PL) ratios were measured in the collected bile (N = 4–8 mice/group). (C) Hepatic CYP7A1 and ileal ASBT protein expression (as optical density, OD, normalized to that of β-actin) in control and repeatedly stressed mice after a 4-h sedentary period (N = 5 mice/group). *P < 0.05. All data are presented as mean + SD.