Fig. 6: TRF upregulated genes associated with AMPK and downstream pathways in the Sk2 model.

a GO analysis and b Reactome pathway analysis of 408 genes that were significantly upregulated under TRF in Sk2 flies. Bar charts represent the -log10 (p-value) of each enriched GO term and pathway. Related pathways are colored similarly. The number of genes identified in each GO term and pathway is shown in parentheses. c Schematic representation of the connection between Gnmt, Ampkα, and downstream of AMPK signaling with important genes (italic bold) encoding enzymes. The color indicates condition (gray WT, Orange HFD, purple Sk2). Up arrows indicate upregulation of gene expression (fold change ≥ 1.25). Asterisks indicate p-value ≤ 0.05. d Expression level of significantly upregulated genes in glycolysis, glycogen metabolism, TCA cycle, and electron transport chain under TRF versus ALF in Sk2 flies. N = 5 time points in WT and Sk2. N = 6 time points in HFD. e, f Flight index of Act88F-Gal4-driven KD of representative genes associated with AMPK downstream pathways at 1 (e) and 3 (f) weeks of age in female flies. Two independent lines per gene were tested. Flight indices were indicated as cohorts of 10–24 flies. Results from independent RNAi lines were indicated with the symbol circle or triangle. Results from Ampkα RNAi #1 are shown in Fig. 6e, f and results from Ampkα RNAi #2 are shown separately in Supplementary Fig. 8g due to varying severity of phenotypes and different KD efficiency verified by  PCR (Supplementary Fig. 8f). # of cohorts per RNAi line per condition = 4–5; # of flies per genotype per age group = 112–174. Details of precise N are in Source Data. Mean ± SEM. One-way ANOVA with Sidak post hoc tests. g Metabolites (fold change ≥ 1.25 under Sk2-TRF versus Sk2-ALF) associated with the AMPK signaling pathway. N = 3 biologically independent replicates. Decreased di- and trisaccharides are potentially broken down in glycogen metabolism. Increased metabolites are related to TCA, and ETC pathways. h Representative western blot of p-AMPKα levels (top) and α-TUBULIN (bottom) from 3-week-old female IFMs in WT, HFD, and Sk2 flies under ALF (A) and TRF (T). Samples were collected at ZT 9. (i) Ratios of p-AMPKα/α-TUBULIN normalized to WT-ALF. N = 3 biological independent samples. Mean ± SEM. Two-way ANOVA with Sidak post hoc tests. j Representative western blot of p-AMPKα levels (top) and α-TUBULIN (bottom) from 3-week-old female thoraces at ZT 3, 9, 15, and 21 in Sk2-ALF and Sk2-TRF flies. k Ratios of p-AMPKα/ α-TUBULIN (normalized to trough of Sk2-ALF value) from 3-week-old female fly thoraces at ZT3, 9, 15, and 21 in Sk2-ALF and Sk2-TRF flies. Source data are provided as a Source Data file.