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Limited survival and impaired hepatic fasting metabolism of glucose, amino acids and lipids in mice with constitutive Rag GTPase signaling

ABSTRACT: The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we have rescued neonatal lethality in RagAGTP mice and found morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice revealed a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects were partially recapitulated by restricting activation of RagA to hepatocytes, and reverted by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling did not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.

INSTRUMENT(S): Liquid Chromatography MS - alternating - hilic

SUBMITTER: Ivan Nemazanyy

PROVIDER: MTBLS2397 | MetaboLights | 2021-05-11

REPOSITORIES: MetaboLights

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Project description: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 20-22Â°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 -80Â°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 LabChipÂ® 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 Î¼g 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.

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Limited survival and impaired hepatic fasting metabolism of glucose, amino acids and lipids in mice with constitutive Rag GTPase signaling

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