Fig 1.
Summary of circadian regulatory feedback loops.
In the liver, diurnal expression of metabolic pathways is controlled by light/dark as well as fast/feed cycles. Critical in this regulation is the transcription factor Bmal (Arntl). In the liver, Bmal regulates daily rhythmic expression of bile acid as well as lipid synthesis linking nutrition to homeostasis. Regulation of Bmal occurs via a network of nuclear receptor (Green) and transcription factors (orange) which form multiple feedback loops (Yellow) ensuring diurnal regulation of bile acid and lipid metabolism.
Table 1.
Primers used for qRT-PCR in this study.
Table 2.
Antibodies used for Western analysis in this study.
Fig 2.
Parenteral nutrition dysregulates circadian regulatory expression in mice.
mRNA expression of circadian transcription factors in hepatic tissue isolated from Chow and DSS-PN mice (Z+2/Z+3). Expression was normalized against HPRT. N = 4 per condition. Values are Mean± SEM. *p<0.05. **p<0.01.
Fig 3.
Genetic inhibition of IL-1β or TNFα signaling ameliorates DSS-PN induced liver injury.
Wild-type (WT) or (A) IL1KO or (B)TNFRKO mice were treated with chow or DSS-PN and euthanized after 14 days [4, 11]. Liver injury was assessed using serum (Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total serum bile acids). N = 3–9 per condition. Values are Mean± SEM, ***p<0.001. ****p<0.0001. C. Hematoxylin and Eosin (H&E) staining of liver sections isolated from indicated conditions. D. Immunohistochemical analysis of F4/80+ macrophages in liver sections isolated from indicated conditions, N = 3 per condition, 200X, PT, Portal triad; CV, Central vein. E. Quantification of F4/80 positive staining. Values are Mean± SEM, ****p<0.0001. F. Immunohistochemical analysis of Ki67 positive nuclei in liver sections isolated from indicated conditions, N = 3 per condition, 200X. G. Immunohistochemical analysis of Cytokeratin 7 (CK7) staining liver sections isolated from indicated conditions, N = 2–3 per condition, 200X. H. Quantification of Ki67 positive staining/100X field, values are Mean± SEM. I. Quantification of CK7 staining, values are Mean± SEM.
Fig 4.
Administration of recombinant IL-1β and TNFα increases liver injury and rapidly alters transcription of the circadian machinery.
8–10 week old C57BL6 mice were injected with recombinant IL-1β (200ng/mouse/i.p.) or TNFα (200ng/mouse/i.p.) and sacrificed after 4hr. Serum and liver tissue was harvested, and A. Hematoxylin and Eosin staining of liver sections isolated from each condition. B. Liver injury assessed by serum ALT, AST and alkaline phosphatase. C. mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. Values are Mean± SEM. *p<0.05. **p<0.01, ***p<0.001, ****p<0.0001, N = 3–4 per condition.
Fig 5.
Deletion of IL-1β signaling ameliorates expression of hepatic circadian transcription factors following DSS-PN.
8–10 week old IL1KO mice were subjected to Chow feeding or DSS-PN. A. Liver tissue was harvested, and mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. Values are Mean± SEM, N = at least 3/condition. *p<0.05. **p<0.01, ***P<0.001, ****P<0.0001. B. Western analysis of circadian regulatory proteins. Expression was normalized using Gapdh expression for each blot. Values are Mean± SEM, N = 3/condition, a = significantly different from respective chow control, b = significantly different compared to WT DSS-PN.
Fig 6.
Genetic inhibition of TNFα signaling normalizes expression of hepatic circadian transcription factors following DSS-PN.
8–10 week old TNFRKO mice were subjected to Chow feeding or DSS-PN. A. Liver tissue was harvested, and mRNA expression of transcription factors regulating hepatic CR analyzed by qRT-PCR. Expression was normalized against HPRT. N = 4 per condition. Values are Mean± SEM. *p<0.05. **p<0.01, ***p<0.001. B. Western analysis of circadian regulatory proteins. Expression was normalized using Gapdh expression for each blot. Values are Mean± SEM, N = 3/condition, a = significantly different from respective chow control, b = significantly different compared to WT DSS-PN.