Crocetin upregulated the electron transport chain to yield more energy in the brain of treated old mice
To identify the significant pathways affected by crocetin, we used the GO and KEGG pathway analysis (Supplementary Spreadsheet SS2). Most of the significantly upregulated pathways in the hippocampi of crocetin-treated mice were related to energy metabolism. GO dot plots depicting BP, MF, and CC identified energy metabolism pathways located in the mitochondria being the main target of crocetin. The BP related to the ETC were particularly found to be upregulated in crocetin-treated mice (Figure 3A). Further, CC analysis also showed high expression of genes belonging to various mitochondrial complexes involved in processing energy precursors (Figure 3A). The MF analysis further revealed the upregulated activity of mitochondrial membrane transporters and enzymes such as NADH dehydrogenase involved in the production of energy metabolites (Figure 3A). Additionally, the downregulated genes did not involve in any GO term at adjusted p value< 0.05.
KEGG pathway analysis of upregulated genes revealed oxidative phosphorylation as the main target of crocetin (Figure 3B). Thus, confirming the effect of crocetin on energy metabolism in the hippocampi. Similar to GO analysis, there were no pathways found to be downregulated in KEGG pathway analysis at adjusted p value< 0.05.
We further compared the levels of different energy metabolites in the whole brain samples of control and crocetin groups. The average NAD+ levels were found to be about 50% higher in mice treated with crocetin than in the control group (Figure 3C). However, no significant change was observed in the levels of NADH between the two groups (Figure 3C). The ATP levels in the crocetin group were almost two times higher than the control group, though the levels of AMP were not changed significantly (Figure 3C). Further to know if the increased levels of NAD+ and ATP were due to higher levels of glucose present in the brain, we analyzed its level in the brain through LCMS. Interestingly, crocetin-treated mice had two times higher concentrations of glucose in the brain than the untreated control group (Figure 3C). However, no increase in the plasma glucose level in the crocetin treated group was observed compared to the control group (Supplementary Table ST3).
Further to know if crocetin impact on the mitochondrial genome stability to cause its anti-aging effect, we compared the mitochondrial genome of old control mice with the crocetin group for the possible change in SNPs or InDels by using the GATK4 pipeline. However, our data showed that crocetin treatment did not alter the type of SNPs or InDels usually found in the aged mitochondrial genome (Supplementary Table ST4).