Regulation of fatty acid metabolism by mTOR in adult murine hearts occurs independently of changes in PGC-1α

Abstract

Mechanistic target of rapamycin (mTOR) is essential for cardiac development, growth, and function, but the role of mTOR in the regulation of cardiac metabolism and mitochondrial respiration is not well established. This study sought to determine cardiac metabolism and mitochondrial bioenergetics in mice with inducible deletion of mTOR in the adult heart. Doxycycline-inducible and cardiac-specific mTOR-deficient mice were generated by crossing cardiac-specific doxycycline-inducible tetO-Cre mice with mice harboring mTOR floxed alleles. Deletion of mTOR reduced mTORC1 and mTORC2 signaling after in vivo insulin stimulation. Maximum and minimum dP/dt measured by cardiac catheterization in vivo under anesthesia and cardiac output, cardiac power, and aortic pressure in ex vivo working hearts were unchanged, suggesting preserved cardiac function 4 wk after doxycycline treatment. However, myocardial palmitate oxidation was impaired, whereas glucose oxidation was increased. Consistent with reduced palmitate oxidation, expression of fatty acid metabolism genes fatty acid-binding protein 3, medium-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein)-α and -β was reduced, and carnitine palmitoyl transferase-1 and -2 enzymatic activity was decreased. Mitochondrial palmitoyl carnitine respiration was diminished. However, mRNA for peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1β, protein levels of PGC-1α, and electron transport chain subunits, mitochondrial DNA, and morphology were unchanged. Also, pyruvate-supported and FCCP-stimulated respirations were unchanged, suggesting that mTOR deletion induces a specific defect in fatty acid utilization. In conclusion, mTOR regulates mitochondrial fatty acid utilization but not glucose utilization in the heart via mechanisms that are independent of changes in PGC expression.

Keywords: cardiac substrate metabolism; mechanistic target of rapamycin; mitochondrial respiration; peroxisome proliferator-activated receptor-γ coactivator-1α.

Fig. 1.

Generation and verification of mechanistic target of rapamycin (mTOR) deletion in mTOR knockout (KO) mice. A: Western blots of mTOR, raptor, and rictor in the heart and Western blots of mTOR in the liver and skeletal muscle from control and mTOR KO mice. B: quantification of mTOR protein levels in control and mTOR KO hearts. n = 4. C: mTOR mRNA measured by quantitative RT-PCR in control and mTOR KO cardiac tissue. n = 6. D: basal and insulin-stimulated Akt, S6 (Ser235/236), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation in control and mTOR KO hearts. There were three bands for 4E-BP1. The bottom band is nonphosphorylated 4E-BP1, the middle band is phosphorylated 4E-BP1, and the top band is hyperphosphorylated 4E-BP1. Phosphorylation of 4E-BP1 by insulin leads to a shift from the bottom to top band. E: basal and insulin-stimulated glycogen synthase kinase (GSK)-3β (Ser9) and tuberous sclerosis complex subunit 2 (TSC2) (Thr1462) phosphorylation in control and mTOR KO hearts. **P < 0.01.

Fig. 2.

Cardiac substrate oxidation in mTOR KO hearts measured in ex vivo working hearts. A: cardiac output, cardiac power, and aortic pressure measured in working hearts. n = 5–8. B: palmitate oxidation, O₂ consumption, and cardiac efficiency measured in working hearts. n = 5–8. C: glucose oxidation measured in working hearts. n = 5–7. D: free glucose, glucose-6-phosphate, lactic acid, 2-phosphoglycerate, and 3-phosphoglycerate in control and mTOR KO hearts measured by gas chromatography-mass spectrometry. n = 6–8. AU, arbitrary units. *P < 0.05; **P < 0.01.

Fig. 3.

Genes and proteins involved in fatty acid utilization and glucose utilization. A: expression of genes in fatty acid transport and fatty acid metabolism measured by quantitative RT-PCR in control and mTOR KO hearts. n = 6. B: carnitine palmitoyl transferase (CPT)-1 and CPT-2 enzymatic activity in isolated mitochondria. n = 4. C: 4-hydroxynonenal (4-HNE) Western blots of immunocaptured CPT-1b protein. Quantification is shown on the right of the blots; n = 4 for quantification. D: peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and PGC-1β gene expression measured by quantitative RT-PCR in control and mTOR KO hearts. n = 6. PGC-1α protein levels in control and mTOR KO hearts were detected by Western blot analysis. The western blot was done with the same set of samples used in Fig. 1A. The same tubulin blot was used as a loading control. E: expression of glucose transporter (GLUT)1 and GLUT4 measured by quantitative RT-PCR. n = 6. GLUT1 and GLUT4 protein levels were detected by Western blot analysis. F: glycogen content in control and mTOR KO hearts. n = 5. G: four cardiac pyruvate dehydrogenase kinase (PDK) isoforms, phosphorylated pyruvated dehydrogenase (PDH), and total PDH protein levels detected by Western blot analysis. The Western blot was done with the same set of samples used in Fig. 3E. The same tubulin blot was used as a loading control. H: PDH activity of control and mTOR KO hearts. n = 7–8. *P < 0.05; **P < 0.01.

Fig. 4.

Mitochondrial respiration supplemented with palmitoyl-carnitine (A) or pyruvate (B). V₀ is basal respiration, V_ADP is respiration after the addition of ADP, V_FCCP is respiration after the addition of FCCP, and V_Oligo is respiration after the addition of oligomycin. ATP is ATP generation from fibers after the addition of ADP when supplemented with the indicated substrate, and the ATP-to-O ratio (ATP/O) is ATP generation normalized by V_ADP, which is an indicator of mitochondrial coupling. *P < 0.05.

Fig. 5.

Mitochondrial (mt)DNA content, citrate synthase (CS) activity, mitochondrial electron transport complex subunit proteins, and mitochondrial morphology. A: mtDNA content measured by quantitative RT-PCR in control and mTOR KO hearts. n = 8. B: mitochondrial CS activity measured in control and mTOR KO hearts. n = 5–6. C: representative mitochondrial complex subunit proteins detected by Western blot analysis. OxPhos, oxidative phosphorylation. D: mitochondrial ultrastructure revealed by electron microscopy.

Fig. 6.

Development of cardiac dysfunction and lethality in mTOR KO mice. A: serial echocardiography of control and mTOR KO mice. n = 5–8. B: heart weight of control and mTOR KO mice normalized to body weight 4 wk after doxycycline (DOX) treatment. n = 7–10. C: cardiac hemodynamic parameters measured by catheterization 4 wk after DOX treatment. n = 6–7. D: survival curve of mTOR KO mice after DOX treatment. n = 9–10. E, top: four-champer view of a control heart and an mTOR KO heart 6 wk after DOX treatment stained with hematoxylin and eosin. Scale bar = 1 mm. Bottom, trichrome staining of histological sections from control and mTOR KO hearts. Scale bar = 50 μm. F: mitochondrial respiration of cardiac fibers from control and mTOR hearts 6 wk after DOX treatment supplemented with palmitoyl-carnitine or pyruvate. G: Western blots of autophagy-related proteins in control and mTOR KO hearts 4 wk after DOX treatment. The Western blot was done with the same set of samples used in Fig. 1A. The same tubulin blot was used as a loading control. *P < 0.05; **P < 0.01.