Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition

by Colin T. Shearn, Aimee L. Anderson, Michael W. Devereaux, Karim C. El Kasmi, David J. Orlicky, Ronald J. Sokol

We have developed a mouse model of Parenteral Nutrition Associated Cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of bile acid and sterol signaling and transport. In the liver, the master circadian gene regulators Bmal/Arntl and Clock drive circadian modulation of hepatic functions, including bile acid synthesis. Once activated, Bmal and Clock are downregulated by several transcription factors including Reverbα (Nr1d1), Dbp (Dbp), Dec1/2 (Bhlhe40/41), Cry1/2 (Cry1/2) and Per1/2 (Per1/2). The aim of this study was to examine the effects of PN on expression of hepatic circadian rhythm (CR) regulatory genes in mice.

WT, IL1^KO or TNFR^KO mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN) and the expression of key CR regulatory transcription factors evaluated. Animals were NPO on a 14 hr light-dark cycle and were administered PN continuously over 24 hrs. Mice were sacrificed, and hepatic tissue obtained at 9-10AM (Zeitgeber Z+3/Z+4 hrs). PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and total bilirubin and the effect of i.p. injection of recombinant IL-1β (200ng/mouse) or TNFα (200ng/mouse) on CR expression was examined after 4 hrs.

In the PNAC model, DSS-PN increased serum biomarkers of hepatic injury (ALT, AST, serum bile acids) which was suppressed in both DSS-PN IL1^KO and DSS-PN TNFR^KO mice. In WT DSS-PN, mRNA expression of Arntl and Dec1 was suppressed corresponding to increased Nr1d1, Per2, Dbp and Dec2. These effects were ameliorated in both DSS-PN IL1^KO and DSS-PN TNFR^KO groups. Western analysis of the circadian transcription factor network revealed in WT mice DSS-PN significantly suppressed Reverbα, Bmal, Dbp, Per2 and Mtnr1b. With the exception of Dbp, DSS-PN mediated suppression was ameliorated by both IL1^KO and TNFR^KO. Intraperitoneal injection of IL-1β or TNFα into WT mice increased serum AST and ALT and suppressed mRNA expression of Nr1d1, Arntl and Clock and increased Dbp and Per2.

Altered expression of CR-dependent regulatory genes during PNAC accompanies cholestasis and is, in part, due to increased cytokine (IL-1β and TNFα) production. Evaluation of the effects of modulating CR in PNAC thus deserves further investigation.