Figure 6. Model for effect of alcohol on hepatic circadian and metabolic systems.

(a) Alcohol affects the circadian system through three main pathways: 1) alteration of intracellular redox state via NAD/NADH, thereby influencing activity of positive TTFL components. The CAF effect is partly mediated through altering the redox state, and leading to a decrease in the NAD/NADH ratio and in an anti-phase shift in its diurnal profile. The clock is regulated by the NAD/NADH ratio, whereby NADH can enhance DNA-binding activity of NPAS2:BMAL1 heterodimers, and NAD⁺ inhibits DNA-binding activity²³. NAD⁺ regulates the activity of the deacetylase SIRTUIN1, thereby altering CLOCK:BMAL1-dependent transcription²⁸,²⁹. Intersecting feedback loops also result in NAD⁺ being under clock regulation²⁸,²⁹. NAD⁺ also acts as an adenosine diphosphate (ADP) ribose donor for poly-ADP ribose-polymerase-1 (PARP-1), which can bind to the CLOCK:BMAL1 heterodimer and poly (ADP ribosyl)ate CLOCK³⁰. Hence, low NAD/NADH ratio is predicted to cause an increase in CLOCK:BMAL1 on E-box binding and increase transcriptional activity of target clock genes (per2, per3, cry2, Rev-erbα/β) and CCGs (Dbp, Tef, Hes6); 2) reducing heme levels and thus affecting the transcriptional activity of clock components NPAS2 and REV-ERBα/β, which contain heme-binding motifs³⁴,³⁵. Hepatic heme concentration is reduced in rats chronically fed with alcohol³⁷; and 3) cellular energetics via AMP/ATP ratio, in turn affecting CRY1 degradation. CAF has been shown to reduce the activity and phosphorylation of adenosine monophosphate activated protein (AMPK)³⁸. AMPK is under rhythmic regulation and can phosphorylate CRY1, leading to CRY1 degradation³⁹. (b) Downstream clock targets include enzymes involved in FA synthesis, which eventually results in altered regulation of TGs, cholesterol and BA synthesis. Rev-erbα participates in the transcriptional regulation of liver specific miR-122, which was differentially regulated in our study, and which regulates Fas and ACC⁸,²⁵. Additionally, DBP and TEF contribute to the circadian transcription of Acot1⁴⁰. REV-ERBα can also regulate circadian BA and lipid homeostasis via cyclic expression of Insig2 and its subsequent action on SREBP activity⁴¹. CYP7A1 is the rate-limiting enzyme for synthesis of bile acids, and its expression is regulated by DBP and REV-ERBα/β. Rhythmically expressed components are indicated by sinusoidal lines.