ABSTRACT: Background; In the postabsorptive state, the portal-drained viscera are a major energy consumer, but a comprehensive overview of the adaptive fasting response in the liver is lacking. Hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after 0, 12, 24, and 72 hours of fasting. Results; Liver weight has fallen to 50% of control after three days of fasting, hepatocyte size was reduced with no apparent increase in apoptosis, while the basic liver structure and metabolic zonnation were preserved. Expression profiling and pathway analysis depicted four master processes, all with response peaking at 24 hours, and all but one decreasing towards 72h. Changes in gene expression were compatible with cellular energy deficiency, and metabolic adaptations were directed at enhanced glucose production, stimulation of lipid catabolism and ketone body synthesis, and strongly augmented ATP production. Consequently, the expression of genes involved in oxidative and endoplasmic reticulum stress response was increased. In addition, urea cycle genes were strongly upregulated during whole duration of fasting, in spite no obvious increase in amino-acid catabolism. Major physiological change upon continued fasting was restoration of glycogen reserves, but with reverse localization, demonstrating utilization of different glycogenic precursor compared to the fed controls. Conclusion; The changes in liver gene expression indicate a rapid onset of generating glucose and ketone bodies during short, and a return to near normal situation in prolonged fasting. The reason apparently lies in glycogen repletion, due to late fasting glucose production by (gut and) kidney. Experiment Overall Design: Male FVB mice (Charles River, Maastricht, The Netherlands) were housed at 20-22°C, 50-60% humidity, a 12 hours light/dark cycle, and food and water ad libitum. At 6 weeks of age, mice were fasted by removing chow for up to 72 hours before sacrifice (n = 6 per group). All animals were sacrificed between 9:00 and 10:00 a.m. by cervical dislocation. The liver was removed quickly, weighed, snap-frozen in liquid N2, and stored at -80°C. Total intestinal RNA was extracted from frozen tissue using TRIzol (Invitrogen), followed by a cleanup through a RNeasy column (Qiagen). The quality of RNA was assessed with the RNA 6000 Nano LabChip® Kit in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, USA). The 60-mer Mouse Development (22K) Oligo Microarray G4120A (Agilent) was used. Three arrays per experimental condition were used. Per microarray, 20 μg mRNA, pooled from 2 livers, was reverse transcribed with Cy3-labelled dCTP (Perkin Elmer, Boston, USA), using the Agilent Fluorescent Direct Label Kit. Cy5-labeled cDNA produced from RNA pooled from livers of 6 fed animals served as the common reference across all arrays. Hybridized cDNAs were detected with Agilentâ??s dual-laser microarray slide scanner. The data were retrieved with Agilentâ??s Feature Extraction software 6.1.1.
