TMT-based profiling has become a powerful and useful biochemical tool for protein quantification in recent years7. TMT-based protein quantification explained the relationship between long-term moderate exercise and key neurotrophic signaling pathways in rats9. In 3D rat brain cell cultures, a global view on protein pathways were unveiled using TMT-based protein quantification10. Accordingly, the screening of deregulated proteins might provide a deeper understanding of the molecular mechanisms regulating responses to specific stimuli.
High altitude exposure often triggers hypoxia 11. To the best of our knowledge, no study has previously investigated liver metabolism in rats subjected to acute hypoxia using proteomic techniques. The liver is one of the most important organs for an organism in maintaining homeostasis and regulating metabolism, including lipid, glucose, and amino acid metabolism, as well as synthesizing and redistributing metabolic substances12. Here, we focused on studying the liver of rats under hypoxia exposure and investigated the global metabolic changes by TMT-based protein quantification techniques. We note that metabolic pathways were amongst the most significantly enriched as estimated by GO and KEGG analyses. Accordingly, we discuss our findings by mainly focusing on metabolic-related proteins.
Metabolic changes are a major cause of illness, including for liver and cardiovascular diseases, and hypoxia-related disorders. Lipid metabolism plays a pivotal role throughout the illness process12–14, and is closely associated with acute hypoxia disorders, including lipid digestion, absorption, transport, catabolism and biosynthesis 15. We generated an acute hypoxia rat model and tested several lipid metabolism serum markers, including LDL, HDL, TG, CHO, GLU and ALT. Our results showed that LDL, TG and CHO were significantly elevated in the liver following acute hypoxia. In contrast, HDL levels were significantly decreased in the liver. These results are in accordance with previous studies in mice and rats16, demonstrating that our acute hypoxia model in rats was successfully generated, and indicating a global inhibition of lipid metabolism.
To uncover the intracellular mechanisms that occur during an acute hypoxia challenge, we performed a TMT-based proteomic experiment on liver samples. KEGG analysis of differentially expressed proteins showed metabolism-related pathways were the mostly significantly enriched, especially those associated with lipid metabolism. These included lipid metabolic pathways, steroid hormone biosynthesis, retinol metabolism, chemical carcinogenesis, PPAR signaling pathway and glutathione metabolism.
These two pathways play important roles in homeostasis and control of lipid metabolism by balancing biosynthesis and lipolysis, and controlling biodegradation after oxidative stress17–20. We found the activation of lipid biosynthesis, including the significantly upregulated expression levels of the genes SLC24A1, ACSM2, FADS2, ALOM5, and ANGPTL4, along with the downregulated levels of ACLY and SLC24A1 play an important role in sodium/calcium exchange in retinal rod and cone photoreceptors by mediating the extrusion of one calcium ion and one potassium ion in exchange for four sodium ions. Several diseases have been previously associated with SLC24A1, including night blindness, congenital stationary forms of night blindness, and Type 1 diabetes21. Among the related pathways to SLC24A1are metabolism of fat-soluble vitamins and transport of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds. As for ACSM2, protein is very important for catalyzing fatty acid activation, which is the first step in the metabolism of fatty acids. ACSM2-related pathways include amino acid conjugation and metabolism 22, while gene ontologies-related to this gene include butyrate-CoA ligase activity. FADS2 is a member of the fatty acid desaturase (FADS) gene family that regulates the desaturation of fatty acids through the introduction of double bonds between defined carbons of the fatty acyl chain. Diseases associated with FADS2 include Fanconi anemia complementation group D2 and best vitelliform macular dystrophy (BVMD) 23. Some of the related pathways to this gene encompass alpha-linolenic acid (ALA) metabolism and fatty acid beta-oxidation (peroxisome). In the case of ANGPTL4, this protein is induced by peroxisome proliferation activators and functions as a serum hormone that regulates glucose homeostasis, lipid metabolism, and insulin sensitivity 24. ANGPTL4 can also act as an apoptosis survival factor for vascular endothelial cells and can prevent metastasis by inhibiting vascular growth and tumor cell invasion. Diseases associated with ANGPTL4 include quantitative trait locus (QTL) related to plasma triglyceride levels and gastric antral vascular ectasia (GAVE). Among its related pathways are the regulation of lipid metabolism by peroxisome proliferator-activated receptor alpha (PPARalpha) and developmental processes. Taken together, the upregulation of SLC24A1, ACSM2, FADS2, ALOM5, and ANGPTL4 and the downregulation of ACLY indicate acute hypoxia impacts the activation of lipid biosynthesis.
Besides the activation of lipid biosynthesis, we also found a reduction of lipolysis, including significantly upregulated levels of the genes CYP1A1, CYP1A2, both of which play important roles in lipid metabolism, and encode the cytochrome P450 superfamily of enzymes 25. Cytochrome P450 proteins are monooxygenases that catalyze several reactions in drug metabolism and synthesis of cholesterol, steroids and other lipids. We also found genes associated with oxidative stress-related metabolic activities, including upregulated levels of GSTA3 and downregulation of S100A8 and S100A9. We validated these findings by qPCR analyses (Fig. 6). When compared to the control group, levels of FADS2 (Fig. 6A), ANGPTL4 (Fig. 6B), SLC24A1 (Fig. 6C), ACSM2 (Fig. 6D), CYP1A1 (Fig. 6E) and CYP1A2 (Fig. 6F) in the livers of the rat group submitted to acute hypoxia challenge were in agreement with the proteomic results.
In a global metabolic perspective (Figure.7), we found upregulated levels of PDK4, ND2, and FN3K and downregulated levels of G6PD, UGT21B, ALDH2 and TIMM17A in the livers of rats exposed to acute hypoxia compared to controls. We hypothesize a shift in oxygen delivery given the observed enrichment in the IFG/PI3K/AKT and AMPK pathways based on GO analysis. Finally, the significant upregulation of GIFYF1 and downregulation of ACMSD, ANPEP and SLC38A3 suggest inhibition of amino acid biosynthesis.