Increased mitochondrial pyruvate metabolism promotes gluconeogenesis via Pyruvate Carboxylase to suppress protein synthesis. a. Schematic illustration of the 13C-glucose tracing strategy used to measure the activity of Pyruvate Dehydrogenase (PDH) and Pyruvate Carboxylase (PC). TCA metabolites labeled with two heavy carbons (13C or M+2 TCA pool) result from PDH activity, whereas M+3 TCA metabolites result from PC activity. PDH is inhibited by PDK-mediated phosphorylation. DCA and AZD7545 are inhibitors of PDK. b. Fractional enrichment of M+2 succinate in empty vector (EV; red) and MPC expressing (MPC+; blue) HepG2 cells at the indicated times after 13C-glucose tracing. MPC expression was induced for 24 hours by treatment with 1 µg/ml doxycycline and media was changed to 12C-glucose. The change in M+2 succinate is significant (by two-way ANOVA test) at one hour after 13C glucose incubation. c. Fractional enrichment of M+3 succinate in EV (red) and MPC+ (blue) HepG2 at the indicated times after 13C-glucose tracing. MPC expression was induced for 24 hours by treatment with 1 μg/ml doxycycline. The change in M+3 succinate is significant (by two-way ANOVA test) at four hours after 13C-glucose incubation. d. Rate of oxygen consumption (OCR) in EV and MPC+ HepG2 cells. e-f. e) Representative images of phalloidin- and DAPI-stained fat body cells. Arrows indicate GFP-positive clones with MPC expression (MPC+), Pcb knock down with MPC expression (MPC+, Pcb-KD) or Pcb overexpression (Pcb+). The scale bar represents 20 µm. f) Quantification of the area of GFP-positive clones with control, MPC+, Pcb over-expression (Pcb+), Pcb and MPC co-expression (MPC+, Pcb+), Pcb knock down (Pcb-KD) and Pcb knock down with MPC expression (MPC+, Pcb-KD) shown as mean ± s.d. of five biological replicates, with each group representing the analysis of 20 the indicated clonal cells. g-h. Concentration of glucose in the fat body (g) and hemolymph (h) of larva with fat body-specific expression MPC or control. Data is presented as mean ± s.d. of three biological replicates analyzed by unpaired t-tests. i-k. i) Schematic illustration of the strategy to analyze gluconeogenesis from 13C-lactate. Cells convert 13C-lactate into 13C-pyruvate, which is transported into mitochondria by the MPC. PC converts 13C-pyruvate (M+3) into oxaloacetate (M+3). PEPCK2 converts oxaloacetate (M+3) into phosphoenolpyruvate (M+3), which is converted into M+6 glucose and excreted from cells. j) Relative abundances of M+3 phosphoenolpyruvate (PEP) in EV and MPC+ HepG2 cells and k) M+6 glucose in their respective media following treatment with 20 mM 13C-lactate for four hours. Data is presented as mean ± s.d. of three biological replicates, each with an average of three technical replicates. l-m. l) Representative brightfield images of EV, MPC+, and PC knockout (KO) or PEPCK2 KO with or without MPC expression HepG2 spheroids. The scale bar represents 200 µm. m) Quantification of spheroid area is presented as mean ± s.d. of 30 technical replicates. n-o. n) Representative images of phalloidin- and DAPI-stained of fat body cells. Arrows indicate GFP-positive clones with MPC expression (MPC+), and Pepck2 knockdown with MPC expression (MPC+, Pepck2-KD). The scale bar represents 20 µm. o) Quantification of the area of GFP-positive clones with MPC+, Pepck2 knock down (Pepck2-KD), Pepck2 knock down with MPC+ (MPC+, Pepck2-KD), Fbp knock down (Fbp-KD), Fbp knock down with MPC+ (MPC+, Fbp-KD). Data is presented as mean ± s.d. of five biological replicates, with each group analyzing 20 clonal cells of the mentioned genetic manipulations. n-o. n) Representative images of fat body clones stained with OPP (red). Arrows indicate GFP-positive clones with MPC expression (MPC+), Pcb knockdown with MPC expression (MPC+, Pcb-KD), or Pepck2 knockdown with MPC expression (MPC+, Pepck2-KD). The scale bar represents 20 µm. o) Quantification of OPP intensity in the indicated clones compared with adjacent wild-type cells. Data is presented as mean ± s.d. Unpaired t-tests, one-way ANOVA tests, or two-way ANOVA tests were performed to evaluate the statistical significance of the data, with p-values mentioned in the graph if significance is noted. Panel a was created using BioRender.com. Panel i was created using BioRender.com.