Cookies help us deliver our services. By using our services, you agree to our use of cookies. More information

Template:MitoPedia: FAT4BRAIN

From Bioblast

MitoPedia: FAT4BRAIN

»FAT4BRAIN«
TermAbbreviationDescription
2-mercaptoacetate2-mercaptoacetate is an inhibitor of medium-chain acyl-CoA dehydrogenase, MCAD, the rate-limiting enzyme of octanoylcarnitine oxidation. 2-mercaptoacetate has been used as an inhibitor of fatty acid oxidation (F-pathway control state). In permeabilized rat soleus muscle fibers, pre-incubation with 1 mM 2-mercaptoacetate for 45 min resulted in 58% inhibition of MCAD and decreased octanoylcarnitine&malate stimulated respiration by approximately 60% (Osiki 2016 FASEB J).
Acetyl-CoA
acetyl-CoA
Acetyl-CoA, C23H38N7O17P3S, is a central piece in metabolism involved in several biological processes, but its main role is to deliver the acetyl group into the TCA cycle for its oxidation. It can be synthesized in different pathways: (i) in glycolysis from pyruvate, by pyruvate dehydrogenase, which also forms NADH; (ii) from fatty acids β-oxidation, which releases one acetyl-CoA each round; (iii) in the catabolism of some amino acids such as leucine, lysine, phenylalanine, tyrosine and tryptophan.
In the mitochondrial matrix, acetyl-CoA is condensed with oxaloacetate to form citrate through the action of citrate synthase in the tricarboxylic acid cycle. Acetyl-CoA cannot cross the mitochondrial inner membrane but citrate can be transported out of the mitochondria. In the cytosol, citrate can be converted to acetyl-CoA and be used in the synthesis of fatty acid, cholesterol, ketone bodies, acetylcholine, and other processes.
AcylcarnitineACAcylcarnitines are esters derivative of carnitine and fatty acids, involved in the metabolism of fatty acids. Long-chain acylcarnitines such as palmitoylcarnitine must be transported in this form, conjugated to carnitine, into the mitochondria to deliver fatty acids for fatty acid oxidation and energy production. Medium-chain acylcarnitines such as octanoylcarnitine are also frequently used for high-resolution respirometry.
CarnitineCarCarnitine is an important factor for the transport of long-chain fatty acids bound to carnitine (carnitine acyltransferase) into the mitochondrial matrix for subsequent β-oxidation. There are two enantiomers: D- and L-carnitine. Only the L-isomer is physiologically active.
Carnitine acyltransferaseCarnitine acyltransferases mediate the transport of long-chain fatty acids across the inner mt-membrane by binding them to carnitine. First, long-chain fatty acids are activated by an energy-requiring step in which the fatty acid ester of CoA is formed enzymatically at the expense of ATP. The fatty acids then pass through the inner mt-membrane and enter the mitochondria as carnitine esters (acylcarnitines). The fatty acyl group is then transferred from carnitine to intramitochondrial CoA and the resulting fatty acyl CoA is used as a substrate in the fatty acid oxidation (FAO) cycle in the mt-matrix.
Carnitine palmitoyltransferase ICPT-ICarnitine palmitoyltransferase I (CPT-I, also known as carnitine acyltransferase I) is a regulatory enzyme in mitochondrial long-chain acyl-CoA uptake and further oxidation. CPT-I is associated with the mt-outer membrane mtOM and catalyses the formation of acylcarnitines from acyl-CoA and L-carnitine. In the next step, acyl-carnitines are transported to the mitochondrial matrix via carnitine-acylcarnitine translocase in exchange for free carnitine. In the inner side of the mtIM carnitine palmitoyltransferase II converts the acyl-carnitines to carnitine and acyl-CoAs. There are three enzyme isoforms: CPT-1A (liver type), CPT-1B (muscle type), CPT-1C (brain type). Isoforms have significantly different kinetic and regulatory properties. Malonyl-CoA is an endogenous inhibitor of CPT-I.
Carnitine palmitoyltransferase IICPT-IICarnitine palmitoyltransferase II (CPT-II, also known as carnitine acyltransferase II) is part of the carnitine shuttle which is responsible for the mitochondrial transport of long-chain fatty acids. CPT-II is located on the inner side of the mtIM and converts the acylcarnitines (produced in the reaction catalyzed by carnitine palmitoyltransferase I) to carnitine and acyl-CoAs, which undergo ß-oxidation in the mitochondrial matrix. Free carnitines are transported out of the mitochondrial matrix in exchange for acyl-carnitines via an integral mtIM protein carnitine-acylcarnitine translocase (CACT). Short- and medium-chain fatty acids do not require the carnitine shuttle for mitochondrial transport.
Carnitine-acylcarnitine translocaseCACTCarnitine-acylcarnitine translocase (CACT) is part of the carnitine shuttle which mediates the mitochondrial transport of long-chain fatty acids where the fatty acid oxidation occurs. CACT is an internal mt-IM protein and transports acylcarnitines into the mitochondrial matrix in exchange for free carnitine.
Complex II ambiguitiesCII ambiguities
CII-ambiguities Graphical abstract.png
The current narrative that the reduced coenzymes NADH and FADH2 feed electrons from the tricarboxylic acid (TCA) cycle into the mitochondrial electron transfer system can create ambiguities around respiratory Complex CII. Succinate dehydrogenase or CII reduces FAD to FADH2 in the canonical forward TCA cycle. However, some graphical representations of the membrane-bound electron transfer system (ETS) depict CII as the site of oxidation of FADH2. This leads to the false believe that FADH2 generated by electron transferring flavoprotein (CETF) in fatty acid oxidation and mitochondrial glycerophosphate dehydrogenase (CGpDH) feeds electrons into the ETS through CII. In reality, NADH and succinate produced in the TCA cycle are the substrates of Complexes CI and CII, respectively, and the reduced flavin groups FMNH2 and FADH2 are downstream products of CI and CII, respectively, carrying electrons from CI and CII into the Q-junction. Similarly, CETF and CGpDH feed electrons into the Q-junction but not through CII. The ambiguities surrounding Complex II in the literature call for quality control, to secure scientific standards in current communications on bioenergetics and support adequate clinical applications.
EtomoxirEtoEtomoxir (Eto; 2[6(4-chlorophenoxy)hexyl]oxirane-2-carboxylate) is an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I) on the outer face of the mitochondrial inner membrane. Eto inhibits fatty acid oxidation by blocking the formation of acyl carnitines from long-chain fatty acids which require the carnitine shuttle for transport into mitochondria. In contrast to long-chain fatty acids, the transport of short- and medium-chain fatty acids is carnitine-independent.
F-junction
F-junction
The F-junction is a junction for convergent electron flow in the electron transfer pathway (ET-pathway) from fatty acids through fatty acyl CoA dehydrogenase (reduced form FADH2) to electron transferring flavoprotein (CETF), and further transfer through the Q-junction to Complex III (CIII). The concept of the F-junction and N-junction provides a basis for defining categories of SUIT protocols. Fatty acid oxidation, in the F-pathway control state, not only depends on electron transfer through the F-junction (which is typically rate-limiting) but simultaneously generates NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the N-junction substrate malate is required as a co-substrate for FAO in mt-preparations, since accumulation of AcetylCoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of AcetylCoA into the TCA cycle catalyzed by citrate synthase.
Fatty acidFAFatty acids are carboxylic acids with a carbon aliphatic chain. The fatty acids can be divided by the length of this chain, being considered as short-chain (1–6 carbons), medium-chain (7–12 carbons) and long-chain and very long-chain fatty acids (>12 carbons).

Long-chain fatty acids must be bound to carnitine to enter the mitochondrial matrix, in a reaction that can be catalysed by carnitine acyltransferase. For this reason, long-chain fatty acids, such as palmitate (16 carbons) is frequently supplied to mt-preparations in the activated form of palmitoylcarnitine. Fatty acids with shorter chains, as octanoate (8 carbons) may enter the mitochondrial matrix, however, in HRR they are more frequently supplied also in the activated form, such as octanoylcarnitine.

Once in the mitochondrial matrix, the fatty acid oxidation (FAO) occurs, generating acetyl-CoA, NADH and FADH2. In the fatty acid oxidation pathway control state electrons are fed into the F-junction involving the electron transferring flavoprotein (CETF). FAO cannot proceed without a substrate combination of fatty acids & malate, and inhibition of CI blocks FAO. Low concentration of malate, typically 0.1 mM, does not saturate the N-pathway; but saturates the F-pathway.
Fatty acid oxidationFAOFatty acid oxidation is a multi-step process by which fatty acids are broken down in β-oxidation to generate acetyl-CoA, NADH and FADH2 for further electron transfer to CoQ. Whereas NADH is the substrate of CI, FADH2 is the substrate of electron-transferring flavoprotein complex (CETF) which is localized on the matrix face of the mtIM, and supplies electrons from FADH2 to CoQ. Before the ß-oxidation in the mitochondrial matrix, fatty acids (short-chain with 1-6, medium-chain with 7–12, long-chain with >12 carbon atoms) are activated by fatty acyl-CoA synthases (thiokinases) in the cytosol. For the mitochondrial transport of long-chain fatty acids the mtOM-enzyme carnitine palmitoyltransferase I (CPT-1; considered as a rate-limiting step in FAO) is required which generates an acyl-carnitine intermediate from acyl-CoA and carnitine. In the next step, an integral mtIM protein carnitine-acylcarnitine translocase (CACT) catalyzes the entrance of acyl-carnitines into the mitochondrial matrix in exchange for free carnitines. In the inner side of the mtIM, another enzyme carnitine palmitoyltransferase 2 (CPT-2) converts the acyl-carnitines to carnitine and acyl-CoAs, which undergo ß-oxidation in the mitochondrial matrix. Short- and medium-chain fatty acids do not require the carnitine shuttle for mitochondrial transport. Octanoate, but not palmitate, (eight- and 16-carbon saturated fatty acids) may pass the mt-membranes, but both are frequently supplied to mt-preparations in the activated form of octanoylcarnitine or palmitoylcarnitine.
Fatty acid oxidation pathway control stateF, FAO
F-junction
In the fatty acid oxidation pathway control state (F- or FAO-pathway), one or several fatty acids are supplied to feed electrons into the F-junction through fatty acyl CoA dehydrogenase (reduced form FADH2), to electron transferring flavoprotein (CETF), and further through the Q-junction to Complex III (CIII). FAO not only depends on electron transfer through the F-junction (which is typically rate-limiting relative to the N-pathway branch), but simultaneously generates FADH2 and NADH and thus depends on N-junction throughput. Hence FAO can be inhibited completely by inhibition of Complex I (CI). In addition and independent of this source of NADH, the type N substrate malate is required at low concentration (0.1 mM) as a co-substrate for FAO in mt-preparations, since accumulation of Acetyl-CoA inhibits FAO in the absence of malate. Malate is oxidized in a reaction catalyzed by malate dehydrogenase to oxaloacetate (yielding NADH), which then stimulates the entry of Acetyl-CoA into the TCA cycle catalyzed by citrate synthase. Peroxysomal β-oxidation carries out few β-oxidation cycles, thus shortening very-long-chain fatty acids (>C20) for entry into mitochondrial β-oxidation. Oxygen consumption by peroxisomal acyl-CoA oxidase is considered as residual oxygen consumption rather than cell respiration.
Flavin adenine dinucleotideFAD, FADH2Flavin adenine dinucleotide, FAD and FADH2, is an oxidation-reduction prosthetic group (redox cofactor; compare NADH). FMN and FAD are the prosthetic groups of flavoproteins (flavin dehydrogenases). Type F substrates (fatty acids) generate FADH2, the substrate of electron transferring flavoprotein (CETF). Thus FADH2 forms a junction or funnel of electron transfer to CETF, the F-junction (compare N-junction, Q-junction), in the F-pathway control state. In contrast, FADH2 is not the substrate but the internal product of succinate dehydrogenase (CII). FAD is the oxidized (quinone) form, which is reduced to FADH2 (hydroquinone form) by accepting two electrons and two protons.
MalateM
Malic acid

Malic acid, C4H6O5, occurs under physiological conditions as the anion malate2-, M, with pKa1 = 3.40 and pKa2 = 5.20. L-Malate is formed from fumarate in the TCA cycle in the mitochondrial matrix, where it is the substrate of malate dehydrogenase oxidized to oxaloacetate. Malate is also formed in the cytosol. It cannot permeate through the lipid bilayer of membranes and hence requires a carrier (dicarboxylate carrier, tricarboxylate carrier and 2-oxoglutarate carrier). Malate alone cannot support respiration of mt-preparations from most tissues, since oxaloacetate accumulates in the absence of pyruvate or glutamate.

Malate is a type N substrate (N) required for the FAO-pathway. In the presence of anaplerotic pathways (e.g., mitochondrial malic enzyme, mtME) the capacity of the FAO-pathway can be overestimated due to a contribution of NADH-linked respiration, F(N) (see SUIT-002).
OctanoateOcaOctanoate (octanoic acid). C8H16O2 Common name: Caprylic acid.
OctanoylcarnitineOctOctanoylcarnitine is a medium-chain fatty acid (octanoic acid: eight-carbon saturated fatty acid) covalently linked to carnitine, frequently applied as a substrate for fatty acid oxidation (FAO) in mitochondrial preparations.
PalmitatePaaPalmitate is a term for the salts and esters of palmitic acid (CH3(CH2)14COOH). Palmitic acid is the first fatty acid produced during fatty acid synthesis and the precursor to longer fatty acids. Palmitate negatively feeds back on acetyl-CoA carboxylase (ACC), which is responsible for converting acetyl-CoA to malonyl-CoA, which in turn is used to add to the growing acyl chain, thus preventing further palmitate generation. In order to dissolve the water-insoluble sodium palmitate, BSA is needed to form the water-soluble compound called palmitate:BSA.
Palmitoyl-CoAPa-CoAPalmitoyl-CoA is a coenzyme A derivative of palmitate formed by acyl-CoA synthase. In contrast to medium- and short-chain acyl-CoA, palmitoyl-CoA cannot freely diffuse into the mitochondrial matrix. Formation of palmitoylcarnitine by CPTI is necessary prior to transfer into mitochondria for further fatty acid oxidation (β-oxidation). To study Fatty acid oxidation using Palmitoyl-CoA, Carnitine and low amount of malate is needed on mitochondrial preparations.
PalmitoylcarnitinePalPalmitoylcarnitine is an ester derivative of carnitine (long-chain acylcarnitine) involved in the metabolism of fatty acids. Within the cell, palmitoylcarnitine is transported into the mitochondria to deliver palmitate for fatty acid oxidation and energy production.
Flag yellow low.jpg
FAT4BRAIN
The project FAT4BRAIN has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 857394


Questions.jpg


Click to expand or collaps


Pages to be created:
SCAD
MCAD
VLCAD (or just one for the acyl-CoA dehydrogenase)
L-hydroxyacyl-CoA dehydrogenase
Enoyl-CoA hydratase
Tiolase
Acyl-CoA

SUIT protocols

TermAbbreviationDescription
SUIT-036 O2 mt D089FAO(Pal) & M kinetics1D;2M.1;3Pal;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama.png
SUIT-037 O2 mt D090FAO(Oct) & M kinetics1D;2M.1;3Oct;3c;4M.2;4M.5;4M1;4M2;5P;6G;7S10;7S50;8Gp;9U;10Rot;11Ama.png
SUIT-038 O2 mt D091FAO control & M kinetics1D;2M.1;2H2O;2c;3M.2;3M.5;3M1;3M2;4P;5G;6S10;6S50;7Gp;8U;9Rot;10Ama.png
SUIT-039 O2 mt D092FAO and NS-pathways1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama.png
SUIT-039 O2 pfi D093FAO and NS-pathways1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8U;9Rot;10Ama.png
SUIT-040 O2 mt D094FAO and NSGp-pathways1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama.png
SUIT-040 O2 pfi D095FAO and NSGp-pathways1D;2M.1;3Pal;3c;4M2;5P;6G;7S;8Gp;9U;10Rot;11Ama.png
SUIT-041 O2 mt D096Optimum [acylcarnitine] test1D;2M.1;3AC;3c;4M2;5P;6S;7Rot;8Ama.png

FAT4BRAIN FAO-mito-CNS platform