Fig 2: Intranasal insulin application reduces food intake and weight development in HFD-fed mice. A: Experimental setup of intranasal insulin treatment of 6 week old male C57BL/6N mice feeding a HFD for a total of four days. S = saline, I = insulin. B: Average 9 h food intake per cage after intranasal insulin application of 6 week-old C57BL/6N mice fed a HFD. Two male mice were housed per cage with a total of 16 saline and 16 insulin treated mice. n = 8 (per treatment). C: Average 24 h food intake per cage after intranasal insulin application of mice fed a HFD. Two male mice were housed per cage with a total of 16 saline and 16 insulin treated mice. n = 8 (per treatment). D: Body weight gain of intranasal saline and insulin treated mice fed a HFD for three consecutive days. n = 16 (per treatment). E, F: (D) Blood glucose and (E) plasma insulin concentrations of mice fed a HFD and treated with intranasal saline or insulin for four consecutive days. Each group n = 15–16 (per treatment). G: Average 1 h HFD food intake after overnight fast of mice treated with intranasal saline or insulin. Each group n = 15–16. H: Hypothalamic protein expression of HSP60, ATF4, CHOP, LONP1, and CLPP and densitometric analysis of 6 week-old C57BL/6N mice fed a HFD after intranasal saline or insulin treatment, n = 4–8. β-Actin served as a loading control. two-way ANOVA, insulin's effect. All the data are presented as mean ± SEM. *, P < 0.05, **, P < 0.01, ***, P < 0.001, ****, P < 0.0001. I: Model of hypothalamic insulin regulation of the MSR.

Fig 3: RNAi-mediated disruption of the Drosophila ortholog of IDH2 (CG7176) and other antioxidant enzymes in Drosophila flight muscles impairs OXPHOS assembly.(A) Schematic describing the Gal4-UAS system. (B to D) Mitochondrial preparations from adult flight muscles expressing the UAS-RNAi or UAS-cDNA constructs shown in muscles using Dmef2-Gal4 were analyzed by silver staining of native gels (see Materials and Methods). Samples expressing RNAi constructs for dME3 were analyzed 2 days after eclosure due to early lethality. For some of the dSOD1 and dSOD2 RNAi lines, lethality began by 3 days, but a few flies survived to 5 days. Hence, we dissected flies at 4 days. (E) Total tissue lysates from flight/thoracic muscles isolated from flies with the genotypes listed were analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with anti-dNDUFS2, anti-NDUFS3 (which detects dNDUFS3), anti-dNDUFV3, anti-dUQCRC2, anti-dCOXIV, anti-dCOXII, and anti-dSDHA. (F) Mitochondrial preparations from thoraces of flies with the genotypes listed were analyzed by SDS-PAGE and immunoblotting with anti-dIDH2, anti-dAIF, and anti-Hsp60 (which detects dHsp60). (G) Total tissue lysates from flight/thoracic muscles from flies with the genotypes shown were analyzed by SDS-PAGE and immunoblotting with anti-dIDH2. (H) Mitochondrial preparations from thoraces of flies with the genotypes listed were analyzed by BN-PAGE followed by silver staining (left panel) and Coomassie blue staining (right panel). The asterisk (*) denotes accumulated assembly intermediates (AIs). (I and J) CI, CII, CIV, and CV in-gel activity (IGA) assays. See figs. S1 to S3 for replicates and quantification of immunoblots in (E) to (G). Loading controls used in the western blots are as indicated.

Fig 4: Sleep deprivation resulted in the up-regulation of proteins involved in cellular respiration.(A) Venn diagrams showing the distribution of significantly up-regulated proteins in the CG and CL groups. The overlap of 22 overlapped proteins is detailed in Table 1. (B) All of the complexes in the electron transport chain were influenced by sleep deprivation. Complex I-III had only up-regulated subunits, but Complex IV and V had both up- and down-regulated subunits. Dysregulated proteins are listed in the coloured table. Up-regulated proteins are in red boxes; down-regulated proteins are in green. Double arrows represent changes common to both the CG and CL sample groups. (C) Proteins involved in small molecule metabolism were collectively up-regulated. Metabolic compounds are framed in orange boxes, and connected with one another with solid or dashed lines, which indicate direct or indirect conversions, respectively. Enzymes that are found up-regulated from the CG or the CL groups are enclosed in parentheses or square brackets, respectively, or presented with bold font if increased in both groups. (D) After being synthesized in the cytosol, preproteins enter the mitochondria through the translocase of the outer membrane (TOM) followed by the translocase of the inner membrane (TIM). TIMM9 was up-regulated in both SD groups. During its transit, polypeptide can be bound and stabilized by mtHsp70 (encoded by Hspa9) with the help of Grpel1. Next, the preprotein is handed over to the complex composed of Hsp60 (encoded by Hspd1) and Hsp10 (encoded by Hspe1) and is further assisted with folding. Upon reaching their native states, mature mitochondrial proteins are sorted to their final destinations. Proteins damaged either by ROS attack or misfolding are repaired by entering refolding cycles or are degraded with the assistance of these chaperones. Chaperones that are found up-regulated from the CG or the CL groups are enclosed in parentheses or square brackets, respectively, or presented in bold font if found to be increased in both groups.

Fig 5: mtFoxO3A is involved in cancer cell response to chemotherapeutic agents.a–c HCT116-FoxO3A-/- cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 µM, 24 h). a Upper panel: relative cell viability and relative cell death. Lower panel: immunoblot analysis of total proteins. ß-actin: loading control. b Transcription analysis of selected mitochondrial (ND6 and COX1) and nuclear (BIM) genes by RT-PCR. c HCT116-FoxO3A-/- cells were transfected with the indicated plasmids for 48 h and then treated with irinotecan (CPT-11, 30 µM, 24 h). Relative cell viability and relative cell death were calculated. d Left panel: immunoblot analysis of total and mitochondrial proteins isolated from tumors (n = 7 for each group) derived from HCT116-xenografted nude mice subjected to cisplatin treatment (CDDP, 2 mg/kg, 6 days). ß-actin and HSP60 were used as total lysate and mitochondrial fraction controls, respectively. Right panel: densitometric analysis of full-length and cleaved FoxO3A normalized against the mitochondrial fractionation loading control and the results of the densitometric analysis of the phosphorylated forms of AMPK and ERK normalized against total AMPK and ERK, respectively, and the loading control. e Clonogenic assay on HCT116-FoxO3A+/+ cells treated with increasing concentrations of trametinib (24 h) and/or irinotecan (24 h), as indicated. f Immunoblot analysis of total proteins isolated from HCT116-FoxO3A+/+ cells upon metformin treatment (MET, 10 µM, 72 h). ß-actin: loading control. g HCT116-FoxO3A-/- cells were transfected with the indicated plasmids for 48 h and then treated with metformin (MET, 10 µM, 72 h). Relative cell viability and relative cell death were calculated. h Clonogenic assay on HCT116-FoxO3A+/+ cells treated with increasing concentrations of metformin (24 h) and/or irinotecan (24 h), as indicated. e, h Cell growth percent inhibition at each drug concentration is presented. The data presented are the mean of at least three independent experiments. Where applicable, data are presented as mean ± SEM and significance was calculated with Student’s t test; *p < 0.05, **p < 0.01, and ***p < 0.001