Mitochondrial Oxidative Metabolism Regulates Maternal-Fetal Metabolic Communication

Macronutrient metabolism includes the suite of chemical transformations that sustain cells and allow for organismal growth and development. Mammalian pregnancy is perhaps the most nutritionally sensitive stage in life as all nutrients for fetal growth are provided by the mother. Glucose and oxygen are two of the most important molecules transferred to the fetus, and their metabolic fates converge at the metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex that consists of two essential components, MPC1 and MPC2. Here we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. While Mpc1 deletion was lethal during mid-gestation, a hypomorphic knock-in (KI) allele of Mpc1 resulted in perinatal lethality. Late-gestation Mpc1 KI fetuses were smaller than littermates with tissue-specific compensatory changes in lipid and amino acid metabolism. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be partially mitigated by alternative anaplerotic substrates in utero. To further probe the capacity for metabolic plasticity in this model, late-gestation dams were fasted for 24 hours. Maternal fasting increased serum lipid metabolites, promoted fetal liver triglyceride accumulation, and stunted fetal growth. To determine the contribution of maternally-derived lipids to the fetal fasting response, we used two genetic models of impaired fatty acid oxidation: (1) liver-specific loss of mitochondrial β-oxidation of long-chain fatty acids via carnitine palmitoyltransferase 2 (Cpt2) and (2) genetic loss of a transcriptional regulator of lipid oxidative metabolism, PPARα. Upon fasting, these mice exhibit differing degrees of hepatic lipid accumulation and impaired ketogenesis. The fetal response to maternal fasting was determined by liver transcriptional program and steady-state metabolite measurements. The maternal fasting response is a better indicator of fetal outcome than is fetal genotype, suggesting that maternally-derived factors dominate this communication. Furthermore, maternal effects persist into the early postnatal period, highlighting the importance of maternal lipid metabolism during gestation and lactation. The use of genetic models and biochemical approaches to obtain a greater mechanistic understanding of maternal-fetal metabolic communication may inform interventions for conditions such as gestational diabetes

The mitochondrial Ca2+ channel MCU is critical for tumor growth by supporting cell cycle progression and proliferation

Introduction: The mitochondrial uniporter (MCU) Ca2+ ion channel represents the primary means for Ca2+ uptake by mitochondria. Mitochondrial matrix Ca2+ plays critical roles in mitochondrial bioenergetics by impinging upon respiration, energy production and flux of biochemical intermediates through the TCA cycle. Inhibition of MCU in oncogenic cell lines results in an energetic crisis and reduced cell proliferation unless media is supplemented with nucleosides, pyruvate or α-KG. Nevertheless, the roles of MCU-mediated Ca2+ influx in cancer cells remain unclear, in part because of a lack of genetic models.Methods: MCU was genetically deleted in transformed murine fibroblasts for study in vitro and in vivo. Tumor formation and growth were studied in murine xenograft models. Proliferation, cell invasion, spheroid formation and cell cycle progression were measured in vitro. The effects of MCU deletion on survival and cell-death were determined by probing for live/death markers. Mitochondrial bioenergetics were studied by measuring mitochondrial matrix Ca2+ concentration, membrane potential, global dehydrogenase activity, respiration, ROS production and inactivating-phosphorylation of pyruvate dehydrogenase. The effects of MCU rescue on metabolism were examined by tracing of glucose and glutamine utilization for fueling of mitochondrial respiration.Results: Transformation of primary fibroblasts in vitro was associated with increased MCU expression, enhanced MCU-mediated Ca2+ uptake, altered mitochondrial matrix Ca2+ concentration responses to agonist stimulation, suppression of inactivating-phosphorylation of pyruvate dehydrogenase and a modest increase of mitochondrial respiration. Genetic MCU deletion inhibited growth of HEK293T cells and transformed fibroblasts in mouse xenograft models, associated with reduced proliferation and delayed cell-cycle progression. MCU deletion inhibited cancer stem cell-like spheroid formation and cell invasion in vitro, both predictors of metastatic potential. Surprisingly, mitochondrial matrix [Ca2+], membrane potential, global dehydrogenase activity, respiration and ROS production were unaffected. In contrast, MCU deletion elevated glycolysis and glutaminolysis, strongly sensitized cell proliferation to glucose and glutamine limitation, and altered agonist-induced cytoplasmic Ca2+ signals.Conclusion: Our results reveal a dependence of tumorigenesis on MCU, mediated by a reliance on MCU for cell metabolism and Ca2+ dynamics necessary for cell-cycle progression and cell proliferation

Mitochondrial Oxidative Metabolism Regulates Maternal-Fetal Metabolic Communication

Macronutrient metabolism includes the suite of chemical transformations that sustain cells and allow for organismal growth and development. Mammalian pregnancy is perhaps the most nutritionally sensitive stage in life as all nutrients for fetal growth are provided by the mother. Glucose and oxygen are two of the most important molecules transferred to the fetus, and their metabolic fates converge at the metabolism of pyruvate in mitochondria. Pyruvate enters the mitochondrial matrix through the mitochondrial pyruvate carrier (MPC), a complex that consists of two essential components, MPC1 and MPC2. Here we define the requirement for mitochondrial pyruvate metabolism during development with a progressive allelic series of Mpc1 deficiency in mouse. While Mpc1 deletion was lethal during mid-gestation, a hypomorphic knock-in (KI) allele of Mpc1 resulted in perinatal lethality. Late-gestation Mpc1 KI fetuses were smaller than littermates with tissue-specific compensatory changes in lipid and amino acid metabolism. These data show that impaired mitochondrial pyruvate transport results in biosynthetic deficiencies that can be partially mitigated by alternative anaplerotic substrates in utero. To further probe the capacity for metabolic plasticity in this model, late-gestation dams were fasted for 24 hours. Maternal fasting increased serum lipid metabolites, promoted fetal liver triglyceride accumulation, and stunted fetal growth. To determine the contribution of maternally-derived lipids to the fetal fasting response, we used two genetic models of impaired fatty acid oxidation: (1) liver-specific loss of mitochondrial β-oxidation of long-chain fatty acids via carnitine palmitoyltransferase 2 (Cpt2) and (2) genetic loss of a transcriptional regulator of lipid oxidative metabolism, PPARα. Upon fasting, these mice exhibit differing degrees of hepatic lipid accumulation and impaired ketogenesis. The fetal response to maternal fasting was determined by liver transcriptional program and steady-state metabolite measurements. The maternal fasting response is a better indicator of fetal outcome than is fetal genotype, suggesting that maternally-derived factors dominate this communication. Furthermore, maternal effects persist into the early postnatal period, highlighting the importance of maternal lipid metabolism during gestation and lactation. The use of genetic models and biochemical approaches to obtain a greater mechanistic understanding of maternal-fetal metabolic communication may inform interventions for conditions such as gestational diabetes

Metabolic and Tissue-Specific Regulation of Acyl-CoA Metabolism

<div><p>Acyl-CoA formation initiates cellular fatty acid metabolism. Acyl-CoAs are generated by the ligation of a fatty acid to Coenzyme A mediated by a large family of acyl-CoA synthetases (ACS). Conversely, acyl-CoAs can be hydrolyzed by a family of acyl-CoA thioesterases (ACOT). Here, we have determined the transcriptional regulation of all ACS and ACOT enzymes across tissues and in response to metabolic perturbations. We find patterns of coordinated regulation within and between these gene families as well as distinct regulation occurring in a tissue- and physiologically-dependent manner. Due to observed changes in long-chain ACOT mRNA and protein abundance in liver and adipose tissue, we determined the consequence of increasing cytosolic long-chain thioesterase activity on fatty acid metabolism in these tissues by generating transgenic mice overexpressing a hyperactive mutant of Acot7 in the liver or adipose tissue. Doubling cytosolic acyl-CoA thioesterase activity failed to protect mice from diet-induced obesity, fatty liver or insulin resistance, however, overexpression of Acot7 in adipocytes rendered mice cold intolerant. Together, these data suggest distinct modes of regulation of the ACS and ACOT enzymes and that these enzymes act in a coordinated fashion to control fatty acid metabolism in a tissue-dependent manner.</p></div

Nutritional perturbation in mice.

<p><b>(A</b>) Body weights of male C57Bl/6J mice for 12 weeks on either control diet (CD), high-fat diet (HFD), or ketogenic diet (KD) n = 15–30. (<b>B</b>) Serum non-esterified free fatty acids (NEFA), (<b>C</b>) beta-hydroxybutyrate (β-OH), and (<b>D</b>) blood glucose in CD, HFD, KD, Fasted (Fast), Fasting-Refed (FR), and cold exposed mice, n = 8–10. <b>(E</b>) Whole body, <b>(F</b>) liver, (<b>G</b>) gonadal white adipose, and (<b>H</b>) inguinal white adipose weight in CD, HFD, KD, Fast, FR, and cold exposed mice, n = 10–15. Data represent mean ± SEM, * represents p≤0.05 by Student’s t-test relative to the CD group. Significant differences among group means are represented by letters and were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA.</p

Development of a transgenic mouse model with a conditional tissue-specific and cytoplasmically targeted long-chain acyl-CoA thioesterase.

<p>(<b>A</b>) Transgenic construct schematic and representative western blot confirming Acot7HA-FLAG overexpression in liver. (<b>B</b>) Thioesterase activity for oleoyl-CoA in liver lysate from control and Acot7HA-Liv transgenic mice, n = 5–7. Overnight fasted control and Acot7HA-Liv liver slice rates of (<b>C</b>) 14C-oleate oxidation, (<b>D</b>) 14C-oleate incorporation into complex lipids, and (<b>E</b>) 3H-acetate incorporation into lipids, n = 5–7. Data represent mean ± SEM, * represent p≤0.05 by Student’s t-test.</p

Doubling of hepatic cytoplasmic long-chain acyl-CoA thioesterase activity does not alter liver fatty acid metabolism.

<p>Control and Acot7HA-Liv (<b>A</b>) weight gain, (<b>B</b>) response to glucose tolerance test, (<b>C</b>) liver weight, and (<b>D</b>) liver triacylglycerol (TAG) in response to high-fat diet feeding for 11 weeks, n = 8–12. Control and Acot7HA-Liv (<b>E</b>) liver weight, (<b>F</b>) liver TAG, (<b>G</b>) serum non-esterified fatty acids (NEFA), (<b>H</b>) serum β-hydroxybutyrate, (<b>I</b>) blood glucose, and (<b>J</b>) liver mRNA abundance of gluconeogenic genes in response to overnight fasting (18 hours), n = 7–11. Data represent mean ± SEM.</p

Tissue-specific posttranscriptional regulation of Acot1 and Acot7.

<p>Protein abundance for (<b>A</b>) Acot1 and (<b>F</b>) Acot7 across tissues expressed as percent of total protein visualized, n = 3. Gene mRNA and protein abundance across dietary conditions, relative to control diet group, in (<b>B,G</b>) liver, (<b>C</b>,<b>H</b>) heart, (<b>D</b>,<b>I</b>) gonadal white adipose tissues (gWAT), and (<b>E</b>,<b>J</b>) inguinal white adipose tissue (iWAT) for Acot1 and Acot7, respectively, n = 5–6. Significant differences were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA. Images of blots are provided in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116587#pone.0116587.s003" target="_blank">S3 Fig.</a></b></p

Nutritional modulation of ACS enzymes.

<p>Fold-change of tissue mRNA abundance for each gene, relative to control diet, for high-fat diet (HFD), ketogenic diet (KD), overnight fasted (Fast), overnight fasted followed by 12-hour refeeding (FR), or cold exposed (Cold) mice (n = 6–8). ND indicates not detectable. Significant differences between CD and all other groups represented in <i>yellow</i> and were determined by Tukey multiple comparison tests (p<0.05) after one-way ANOVA except for cold treatment which was analyzed by Student’s t-test. Complete statistical analysis via one-way ANOVA is provided in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116587#pone.0116587.s002" target="_blank">S2 Fig.</a></b></p