Involvement Of AMPK And SIRT-1 In The Influence Of Timed High-fat Diet In The Evening On Hepatic Biological Clock Gene Expressions In Mice

Aim: To investigate whether timed high-fat diet in the evening affects the hepatic circadian clock gene and lipid metabolism-related gene expressions in fatty liver mice and the possible mechanisms.Methods: Kunming male mice were used in this study. A mouse model with hyperlipidemic fatty liver was established by timed high-fat diet in the evening(19:00) and a 12-h light(8:00-20:00) and 12-h dark(20:00-8:00) cycle for 6 weeks, and then mice were sacrificed at the different time-points(8:00, 12:00, 16:00, 20:00, 24:00 and 4:00). The total cholesterol(TC), triglycerides(TG), free fatty acids(FFA) and nicotinamide adenine dinucleotide(NAD+) contents in liver were measured with the colorimetric method, and hepatic morphological changes were examined under a light microscope. The hepatic AMP-activated protein kinase(AMPK) content was measured by ELISA method. The hepatic m RNA expressions of Clock, Bmal1, Per1-2, Cry1-2, peroxisome proliferator-activated receptor α(PPARα), peroxisome proliferator-activated receptor gamma coactivator-1(PGC-1) α, lipoprotein lipase(LPL), diacylglycerol acyltransferase(DGAT) 2, acetyl-Co A carboxylase-1(ACC-1) α, carnitine palmitoyltransferase-1(CPT-1) and nicotinamide phosphoribosyl transferase(NAMPT) were determined by the Real-time PCR method. The hepatic AMPK, phosphorylated-AMPK(p-AMPK), silent information regulator of transcription-1(SIRT-1), Cry2 and Per2 protein expressions were determined by Western blot method.Results: After administration of a timed high-fat diet in the evening for 6 weeks, the numerous hepatocytes filled with fat droplets were seen, and the hepatic TC, TG and FFA contents were also high at all time-points with different levels. Compare with the control mice, the change of hepatic TC content in the high-fat diet-fed mice was gentler, but its amplitude was significantly increased during the light. Also, the hepatic TG level was higher in the high-fat diet-fed mice during the light, and its peak was present at 12:00. The hepatic FFA level had no obvious rhythmic change. At the same time, Real-time PCR assays showed that in the high-fat diet-fed mice, the lipid metabolism-related genes, such as hepatic PPARα, PGC-1α, LPL, DGAT2, ACC-1α and CPT-1 m RNA expressions had significant changes in their rhythms and/or amplitudes, while the hepatic circadian clock genes Clock, Bmal1, Per1-2, and Cry2 m RNA expressions exhibited notable variation as well. Western blot study also indicated that the protein expressions of Per2 and Cry2 had significant changes in their amplitudes.Our further study found that a timed high-fat diet in the evening for 6 weeks might decrease the hepatic AMPK and p-AMPK protein expressions. The peak of AMPK in the high-fat diet-fed mice had a 8-h delay relative to the matched control mice, and the amplitudes between two groups at 12:00 and 20:00 were obvious difference. Compare with the matched control mice,the p-AMPK protein expression in the high-fat diet-fed mice was decreased at 12:00 and increased at 20:00, respectively. Likewise, the hepatic NAMPT m RNA expression, NAD+ content and SIRT-1 protein expression in the high-fat diet-fed mice had obvious changes in their rhythms and/or amplitudes.Conclusion: Timed high-fat diet in the evening could change the hepatic circadian rhythmic expressions of some clock genes and lipid metabolism-related genes, and led to the hepatic lipid accumulation, its mechanisms might be partly related with the reduction of negative regulatory effects of Cry2 and Per2 on Clock/Bmal1 due to their protein degradation caused by the nutrition sensor AMPK and SIRT-1 circadian rhythm changes.