Fig 2: Restriction of the glycolytic phosphoenolpyruvate (PEP) supply reveals the importance of PEP carboxykinase (PCK2) for cytosolic ATP/ADP.Average ß-cell ATP/ADPc in islets from PCK2-ßKO (A, D, G), PKm1-ßKO (B, F, H), and PKm2-ßKO (C, F, I) mice in response to glucose in the presence of 1 mM leucine (A–C) or mixed amino acids (AA) provided at three times their physiological concentrations (×1=Q, 0.6 mM; L, 0.5 mM; R, 0.2 mM; A, 2.1 mM) in the presence of 2.7 mM glucose (2.7G) to remove the enolase contribution to cytosolic PEP (D–I). Pyruvate kinase activator (PKa) (10 µM TEPP-46) was present in G, H, and I. ATP/ADPc is quantified as area under the curve from PCK2-ßKO (A, n=65 islets from 3 mice; D, n=73 islets from 3 mice; G, n=71 islets from 3 mice) and littermate controls (A, n=63 islets from 3 mice; D, n=90 islets from 3 mice; G, n=77 islets from 3 mice); PKm1-ßKO (B, n=88 islets from 3 mice; E, n=66 islets from 3 mice; H, n=72 islets from 3 mice) and littermate controls (B, n=92 islets from 3 mice; E, n=69 islets from 6 mice; H, n=69 islets from 3 mice); and PKm2-ßKO (C, n=86 islets from 3 mice; F, n=99 islets from 3 mice; I, n=89 islets from 3 mice) and littermate controls (C, n=90 islets from 3 mice; F, n=100 islets from 6 mice; I, n=85 islets from 3 mice). Scale bars: 0.025 Perceval-HR excitation ratio for A–C and 0.1 Perceval-HR excitation ratio for D–I (R500/430). See Figure 4—source data 1 for source data. Data are shown as mean ± SEM. *p<0.05, ****p<0.0001 by t-test. Figure 4—source data 1.Source data for beta cell ATP/ADP measurements in Figure 4.

Fig 3: Expression of proteins related to the insulin signaling pathway in mice in different groups. (A) Representative western blotting images of proteins in the insulin signaling pathway. (B) PI3K; (C) p-PI3K/PI3K; (D) AKT; (E) p-AKT/AKT; (F) Socs3; (G) PEPCK. Data are presented as the mean ± SD (n=3). *P<0.05 vs. CON and #P<0.05 vs. HFD. CON, control; HFD, high-fat diet; MET, metformin; p-, phosphorylated; SOCS3, suppressor of cytokine signaling 3; PEPCK, phosphoenolpyruvate carboxykinase.

Fig 4: AIF deficient mice exhibit aberrant 1-C metabolism. (A) Proteomic profiling using isobaric tags (TMT/iTRAQ) was performed in quadriceps muscle tissues from 6 month-old Aifm1 (R200del) KI mice and littermates (n = 3 per genotype). Volcano plot shows the median log2 ratio against –log10 (p-value). Dots represent individual proteins. Black dots are not significant; colored dots indicate differently expressed proteins. (B) Table of proteins differently expressed in KI mice compared to littermates. (C) Schematic representation of folate-driven 1C metabolism in mammalian cells. In the mitochondria, the reversible conversion of 5,10-methylene-THF to 10-formyl-THF is mediated by MTHFD2 (red) in a NAD(P)+-dependent manner. Enzymes involved in serine metabolism are in black solid box, whereas other enzymes analyzed in this study are in black dashed box. (D) RT-PCR analyses showing Mthfd2 expression levels in muscle and cerebellar tissues from 3 to 6 month-old mice (mean ± SEM, Student's t-test, n = 3–5 per genotype, **p < 0.01, *p < 0.05 compared to littermates). (E) Immunoblot analysis for MTHFD2 and Diablo/SMAC protein expression levels in muscles from 6 month-old animals. (F) RT-PCR analyses for Phgdh, Psat1, and Shmt2 expression in muscle samples from 6 month-old Aifm1 (R200 del) KI and Hq mice (mean ± SEM, Student's t-test, n = 4–6 per genotype, ***p < 0.001, *p < 0.05 compared to littermates). (G) RT-PCR showing expression levels of Fgf21 in Aifm1 (R200 del) KI and Hq mice compared to littermate controls. Quadriceps muscle tissues from 3 to 6 month-old animals were used (mean ± SEM, Student's t-test, n > 3 per genotype, *p < 0.05). (H) RT-PCR analyses for the ATF4 target genes Gdf15, Asns, and Psph in quadriceps muscle and cerebellar tissues from 6 month-old Aifm1 (R200 del) KI and Hq mice compared to littermate controls (mean ± SEM, Student's t-test, n = 4–6 per genotype, ***p < 0.001, **p < 0.01). (I) Immunoblot analysis of muscles from 6 month-old Aifm1 (R200 del) KI and Hq mice showing expression levels of the following enzymes: PEPCK-C, PEPCK-M, PKM1, PKM2, and GSR (densitometry is mean ± SEM, Student's t-test, n = 4–7 per genotype, *p < 0.05 compared to littermate controls). GAPDH was used as internal loading control. Color code is: black = wt (for KI); red = KI; dark grey = wt (for Hq); green = Hq.

Fig 5: Pyruvate tolerance test and hepatic PEPCK expression(A) Pyruvate tolerance test of the AS160-knockout and wild-type male mice at 9–10 weeks of age. After deprivation of food for 16 h (overnight), the mice were intraperitoneally injected with a bolus of pyruvate (2 mg/g of body weight). At zero time, and at the indicated times after injection, blood glucose levels were determined with a Breeze 2 glucometer (Bayer). The incremental area under the curve (IncAUC) was calculated. Data are given as the means±S.E.M. (n=4 for each genotype). *P<0.05. (B–D) PEPCK protein expression in liver from the AS160-knockout and wild-type mice. Liver lysates (40 µg) were separated by SDS/PAGE and subject to Western blot analysis. The levels of cytosolic (anti-PCK-1) and mitochondrial (anti-PCK-2) isoforms of PEPCK, FBP-1, glycogen synthase (GS), glycogen phosphorylase (GP) and GAPDH were measured using the antibodies indicated. The PEPCK signals were quantified and normalized with GAPDH. Data are given as means±S.E.M. (n=4). *P<0.05. A.U., arbitrary unit; KO, knockout; WT, wild-type.