The Concentration of Phosphatidylethanolamine in Mitochondria Can Modulate ATP Production and Glucose Metabolism in Mice
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Figures
- Figure 1
Reduced glucose production from pyruvate in Pemt−/− mice. Insulin (A), glucose (B), pyruvate (C), and glycerol (D) tolerance tests were performed in Pemt+/+ mice and Pemt−/− mice. Values are means ± SEM (n = 4–5 per group for insulin and glucose tolerance tests, and n = 9–10 per group for pyruvate and glycerol tolerance tests). *P < 0.05.
- Figure 2
Reduced hepatic glycogen levels and glucose uptake in Pemt−/− mice. A: Hepatic glycogen levels in Pemt+/+ mice (black bars) and Pemt−/− mice (white bars) after an overnight fast. B–D: Glucose uptake by liver (B), muscle (C), and white adipose tissue (D) of Pemt+/+ mice and Pemt−/− mice, measured 15 min after injection of 2-[3H]deoxy-d-glucose i.v. Values are means ± SEM (n = 6 per group). *P < 0.05.
- Figure 3
Hormonal or transcriptional activation of gluconeogenesis is not altered by PEMT deficiency. A: Glucagon tolerance test. Blood glucose levels were monitored after injection of glucagon (140 µg/kg body wt i.p.) into nonfasted Pemt+/+ mice and Pemt−/− mice. B: Gluconeogenic gene expression in livers of Pemt+/+ and Pemt−/− mice was determined using quantitative PCR. Data are relative to cyclophilin mRNA. C: Protein expression of PEPCK and PGC1α in livers of Pemt+/+ mice and Pemt−/− mice. Protein disulfide isomerase (PDI) was used as a loading control. D: Quantification of protein levels from C. Values are means ± SEM (n = 6 per group).
- Figure 4
PEMT deficiency alters hepatic mitochondrial phospholipid composition and morphology. A–C: Levels of PC, PE, and the PC-to-PE ratio were determined in mitochondria from livers of Pemt+/+ mice and Pemt−/− mice (n = 6/group). D: Mitochondrial copy number in livers of Pemt+/+ mice and Pemt−/− mice were determined by the ratio of mitochondrial DNA to nuclear DNA (n = 6/group). E: Transmission electron microscope images of mitochondria in liver. White arrows indicate mitochondria. LD, lipid droplet; N, nucleus. The size (F) and circularity (G) of individual mitochondria (n = 150–350/group) were quantified using ImageJ software. H: Protein expression of mitofusin Mfn1 and -2 and Opa1 in mitochondria from livers of Pemt+/+ mice and Pemt−/− mice. Voltage-dependent anion channel (VDAC) was used as a loading control. I: Quantification of protein levels from H. Values are means ± SEM. *P < 0.05.
- Figure 5
Increased oxidative phosphorylation and ATP production in Pemt−/− mice. A: mRNA expression of genes involved in the TCA cycle in livers of Pemt+/+ mice and Pemt−/− mice was quantified using quantitative PCR. Data are relative to cyclophilin mRNA. Values are means ± SEM (n = 6/group). B: ATP levels in primary hepatocytes isolated from Pemt+/+ mice and Pemt−/− mice. Values are means ± SEM of four independent experiments. C–E: Activities of complexes I, II, and IV in mitochondria isolated from livers of Pemt+/+ mice and Pemt−/−. Values are means ± SEM (n = 6/group). F: Hepatic PDH activity in Pemt+/+ mice and Pemt−/− mice after an overnight fast. Values are means ± SEM (n = 12/group). G: Protein expression of oxidative phosphorylation complexes I–IV in mitochondria from Pemt+/+ mice and Pemt−/− livers. Expression was quantified using ImageJ software (H). Values are means ± SEM (n = 4/group). *P < 0.05.
- Figure 6
Increased respiration in hepatoma cells lacking PEMT. A: PEMT activity in McArdle-RH7777 cells without (pCI) or with (p38) PEMT expression compared with normal mouse liver homogenate. B: Basal rate of O2 consumption in intact cells from endogenous substrates (intact) and substrate-driven O2 consumption in digitonin-permeabilized cells supplied with substrates: pyr/mal, pyruvate plus malate (complex I); Suc, succinate (complex II) plus rotenone (inhibitor of complex I). Values are means ± SEM of four independent experiments, each performed in duplicate. C: Protein expression of oxidative phosphorylation complexes I–IV in mitochondria from pCI and p38 McArdle-RH7777 cells. Expression was quantified using ImageJ software (D). *P < 0.05.
- Figure 7
Metabolism of mitochondrial PC and PE in hepatocytes. A: Decay of radiolabeled PE in mitochondria in primary hepatocytes from Pemt+/+ and Pemt−/− mice. Cells were pulse labeled with [3H]serine for 1 h, followed by a 4-h chase period. B: Incorporation of [3H]serine into the choline head group of PC in Pemt+/+ and Pemt−/− hepatocytes. Values are means ± SEM of three independent experiments.
- Figure 8
ATP levels correlate with mitochondrial PE content of hepatoma cells. Correlation of cellular ATP concentration with PEMT activity (A), mitochondrial PC-to-PE ratio (B), mitochondrial PE (C), and mitochondrial PC (D) in McArdle-RH7777 hepatoma cells that express different amounts of PEMT. Correlations were determined by Pearson correlation. Values are means ± SEM of four independent experiments, each performed in triplicate.
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