Hoppel 2017 MiP2017

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Charles Hoppel
Pathways of mitochondrial fatty acid oxidation. Hoppel_Presentation

Link: MiP2017

Hoppel CL (2017)

Event: MiP2017

COST Action MITOEAGLE
Long chain fatty acids are oxidized in the mitochondrial matrix via β-oxidation. They are activated on the cytosolic side of the mitochondrial outer membrane (mtOM) by long-chain acyl-CoA synthetase (ACSL). The activated fatty acids as well as other substrates, ions, and nucleotides, cross the mitochondrial outer membrane (mtOM) through the voltage-dependent anion channel (VDAC). In a study of the effects of exposure to the 22 kDa polyanion (PA22), rat liver mitochondrial ADP-stimulated glutamate, succinate (+rotenone), and palmitoylcarnitine (+malate) oxidation rates were not affected. However, oxidation of palmitate (+ATP, Mg2+, CoASH, carnitine, and malate) and palmitoyl-CoA (+carnitine and malate) were inhibited at 1-2 nmol PA22 per mg mitochondrial protein. These data indicate that PA22 is selective for the first two steps in fatty acid oxidation. Additionally, we showed PA22 interacts with VDAC and inhibits binding of hexokinase to rat liver mitochondria; VDAC was initially identified as a hexokinase-binding protein. The next step is the carnitine-dependent transport of activated fatty acids, catalyzed by carnitine palmitoyltransferase 1a (CPT1a, an integral outer membrane protein), which converts fatty acyl-CoAs into acylcarnitines.

To examine the state of CPT1a in the mtOM, an approach was used that combined sizing by mass and isolation by immunological methods. Blue native electrophoresis of purified rat liver mtOM extracts yielded several high molecular weight bands containing CPT1a, ACSL, and VDAC; IP of mtOM extracts with CPT1a antibodies or antisera against ACSL and VDAC revealed strong interactions between these 3 proteins. This strongly suggests that CPT1a forms hetero-oligomeric complexes with ACSL and VDAC to transfer fatty acids across the outer membrane.

In similar studies using rat liver contact sites, in addition to the mtOM proteins described above, mitochondrial inner membrane (mtIM) proteins, the very-long chain acyl-CoA dehydrogenase, the phosphate carrier, and the adenine nucleotide translocase 2 were present in association with CPT1a, ACSL, and VDAC. The 960 kDa to 600 kDa bands also contained the mtIM trifunctional protein, but the carnitine-acylcarnitine translocase was found only in lighter bands.

The control of palmitoylcarnitine oxidation in normal liver mitochondria was investigated. The main product of liver mitochondrial palmitoylcarnitine oxidation was either citrate or acetoacetate, depending of the availability of a source of oxaloacetate. The conversion of one equivalent of palmitate to carbon dioxide and water requires 46 equivalents of atomic oxygen. When citrate is the end product 22 units of atomic oxygen will be used per unit of palmitate. When only acetoacetate is being formed, oxygen consumption is linked only to the dehydrogenations in β-oxidation and 14 oxygen atoms will be used. In the presence of rotenone to inhibit NADH oxidation and in the presence of oxaloacetate, palmitate oxidation is limited to the acyl-CoA dehydrogenase step and only 7 equivalents of atomic oxygen are used. In rat liver mitochondria, the rate of utilization of palmitoylcarnitine (4.3-4.4 nmoles/min/mg) was not affected by the kind of product to which it was converted. In contrast, heart mitochondria oxidized palmitoylcarnitine past citrate, while skeletal muscle have complete oxidation using 46 equivalents of atomic oxygen.


Bioblast editor: Kandolf G O2k-Network Lab: US OH Cleveland Hoppel CL


Labels: MiParea: Respiration 


Organism: Rat  Tissue;cell: Liver 


Coupling state: OXPHOS  Pathway: F, N, S  HRR: Oxygraph-2k 


Affiliations

Center Mitochondrial Diseases; Dep7 Pharmacology Medicine; Case Western Reserve Univ School Medicine, Cleveland, Ohio, USA. - charles.hoppel@case.edu

References

  1. Lee K, Kerner J, Hoppel CL (2011) Mitochondrial carnitine palmitoyltransferase 1a is part of an outer membrane fatty acid transfer complex J Biol Chem 286:25655-62.
  2. Hoppel CL, DiMarco J, Tandler B (1979) Riboflavin and rat hepatic cell structure and function. Mitochondrial oxidative metabolism in deficiency states. J Biol Chem 254:4164-70.
  3. Bremer J, Davis EJ (1972) Phosphorylation coupled to acyl-coenzme A dehydrogenase-linked oxidation of fatty acids by liver and heart mitochondria. BBA 275:298-301.