Fatty acyl translocation

Fatty acid utilized by muscle may arise from storage triglycerides from either adipose tissue depot or from lipid stores within the muscle itself. Lipolysis of adipose triglyceride in response to hormonal stimulation liberates free fatty acids (see Section 9.6.2) which are transported through the bloodstream to the muscle bound to albumin. Because the enzymes of fatty acid oxidation are located within subcellular organelles (peroxisomes and mitochondria), there is also need for transport of the fatty acid within the muscle cell this is achieved by fatty acid binding proteins (FABPs). Finally, the fatty acid molecules must be translocated across the mitochondrial membranes into the matrix where their catabolism occurs. To achieve this transfer, the fatty acids must first be activated by formation of a coenzyme A derivative, fatty acyl CoA, in a reaction catalysed by acyl CoA synthetase. 

Once the secondary fatty acyl residue(s), VLCFA, has been added to the Kd02lipid-IVA, a number of the core glycosyl residues are added and then, presumably, the resulting COS-LA molecule is flipped to the periplasmic side of the inner membrane after which the OPS is ligated to the COS-LA which is then translocated to the outer leaflet of the outer membrane. The enzymes which modify the... 

Figure 22.24. Reactions of Fatty Acid Synthase. Translocations of the elongating fatty acyl chain between the cysteine...

Synthesis of phosphatidylcholine. The rate-limiting reaction is that catalyzed by cytidylyltransferase (reaction 2) which appears to be active only when attached to the endoplasmic reticulum, although it is also found free in the cytosol. Cytidylyltransferase is inactivated by a cAMP-dependent protein kinase and activated by a phosphatase. Translocation to the endoplasmic reticulum can be stimulated by substrates such as fatty acyl Coenzyme A (CoA). Choline deficiency can result in deposition of triacylglycerol in liver and reduced phospholipid synthesis. Enzymes (1) choline kinase ... 

The answer is a. (Murray, pp 258-297. Scriver, pp 2705-2716. Sack, pp 121—138. Wilson, pp 362—367.) Under conditions of active synthesis of fatty acids in the cytosol of hepatocytes, levels of malonyl CoA are high. Malonyl CoA is the activated source of two carbon units for fatty acid synthesis. Malonyl CoA inhibits carnitine acyltransferase 1, which is located on the cytosolic face of the inner mitochondrial membrane. Thus, long-chain fatty acyl CoA units cannot be transported into mitochondria where p oxidation occurs, and translocation from cytosol to mitochondrial matrix is prevented. In this situation compartmentalization of membranes as well as inhibition of enzymes comes into play. 

The answer is b. (Murray, pp 505-626. Scriver, pp 4029-4240. Sack, pp 121-138. Wilson, pp 287-320.) A deficiency in carnitine, carnitine acyl-transferase 1, carnitine acyltransferase 11, or acylcarnitine translocase can lead to an inability to oxidize long-chain fatty acids. This occurs because all of these components are needed to translocate activated long-chain (>10 carbons long) fatty acyl CoA across mitochondrial inner membrane into the matrix where P oxidation takes place. Once long-chain fatty acids are coupled to the sulfur atom of CoA on the outer mitochondrial membrane, they can be transferred to carnitine by the enzyme carnitine acyltransferase I, which is located on the cytosolic side of the inner mitochondrial membrane. Acyl carnitine is transferred across the inner membrane to the matrix surface by translocase. At this point the acyl group is reattached to a CoA sulfhydryl by the carnitine acyltransferase 11 located on the matrix face of the inner mitochondrial membrane. 

In muscle, most of the fatty acids undergoing beta oxidation are completely oxidized to C02 and water. In liver, however, there is another major fate for fatty acids this is the formation of ketone bodies, namely acetoacetate and b-hydroxybutyrate. The fatty acids must be transported into the mitochondrion for normal beta oxidation. This may be a limiting factor for beta oxidation in many tissues and ketone-body formation in the liver. The extramitochondrial fatty-acyl portion of fatty-acyl CoA can be transferred across the outer mitochondrial membrane to carnitine by carnitine palmitoyltransferase I (CPTI). This enzyme is located on the inner side of the outer mitochondrial membrane. The acylcarnitine is now located in mitochondrial intermembrane space. The fatty-acid portion of acylcarnitine is then transported across the inner mitochondrial membrane to coenzyme A to form fatty-acyl CoA in the mitochondrial matrix. This translocation is catalyzed by carnitine palmitoyltransferase II (CPTII Fig. 14.1), located on the inner side of the inner membrane. This later translocation is also facilitated by camitine-acylcamitine translocase, located in the inner mitochondrial membrane. The CPTI is inhibited by malonyl CoA, an intermediate of fatty-acid synthesis (see Chapter 15). This inhibition occurs in all tissues that oxidize fatty acids. The level of malonyl CoA varies among tissues and with various nutritional and hormonal conditions. The sensitivity of CPTI to malonyl CoA also varies among tissues and with nutritional and hormonal conditions, even within a given tissue. Thus, fatty-acid oxidation may be controlled by the activity and relative inhibition of CPTI. 

Fatty acyl-CoA thioesters that are formed at the outer mitochondrial membrane cannot directly enter the mitochondrial matrix where the enzymes of P-oxidation are located, because CoA and its derivatives are unable to pass rapidly through the inner mitochondrial membrane. Instead, carnitine carries the acyl residues of acyl-CoA thioesters across the inner mitochondrial membrane. The carnitine-dependent translocation of fatty acids across the inner mitochondrial membrane is schematically shown in Fig. 1 [8]. The reversible... 

Fig. 23.5. Transport of long-chain fatty acids into mitochondria. The fatty acyl CoA crosses the outer mitochondrial membrane. Carnitine pahnitoyl transferase I in the outer mitochondrial membrane transfers the fatty acyl group to carnitine and releases Co ASH. The fatty acyl carnitine is translocated into the mitochondrial matrix as carnitine moves out. Carnitine palmitoyl transferase II on the inner mitochondrial membrane transfers the fatty acyl group back to CoASH, to form fatty acyl CoA in the matrix.

Fig. 1 Oxidative metabolism and energy production by mitochondria. The oxidation of pyruvate and free fatty acids (FFA) inside mitochondria produces NADH and FADH2, which transfer their electrons to the mitochondrial respiratory chain. The flow of electrons in mitochondrial complexes I, III, and IV is coupled with the extrusion of protons from the mitochondrial matrix into the intermembrane space. When energy is needed, these protons reenter the matrix through ATP synthase, to generate ATP from ADP. The adenine nucleotide translocator (ANT) then exchanges the formed ATP for cytosolic ADP. G-6-P Glucose 6-phosphate, PDH pyruvate dehydrogenase, LCFA-CoA long-chain fatty acyl-CoA, CPTI carnitine palmitoyltransferase I, TCA cycle tricarboxylic acid cycle, c cytochrome c...

It has been proposed that fatty acyl-CoA esters, which are potent inhibitors of ADP transport in isolated mitochondria, also control flux through the translocator in vivo and hence the production of ATP (see [4] for literature). However, as pointed out by Stubbs [7], such inhibition makes little physiological sense in situations like starvation or long-term exercise, when fatty acids are an important fuel especially under the latter conditions the translocator must operate at high capacity to provide the cytosol with ATP. Indeed, in isolated hepatocytes incubated with various concentrations of fatty acids no inhibition of ATP transport by fatty acyl-CoA could be observed [71]. Possibly this inhibition is prevented by a low-molecular weight cytosolic protein with a high affinity for fatty acyl-CoA [72]. 

Mitochondrial P-oxidation of long-chain fatty acids is the major source of energy production in man. The mitochondrial inner membrane is impermeable to long chain fatty acids or their CoA esters whereas acylcamitines are transported. Three different gene products are involved in this carnitine dependent transport shuttle carnitine palmi-toyl transferase I (CPT I), carnitine acyl-camitine carrier (CAC) and carnitine palmitoyl transferase II (CPT II). The first enzyme (CPT I) converts fatty acyl-CoA esters to their carnitine esters which are subsequently translocated across the mitochondrial inner membrane in exchange for free carnitine by the action of the carnitine acyl-camitine carrier (CAC). Once inside the mitochondrion, CPT II reconverts the carnitine ester back to the CoA ester which can then serve as a substrate for the P-oxidation spiral. 

Fatty acid oxidation occurs in mitochondria, and therefore the fatty acid substrate (in the form of fatty acyl CoA) needs to be transported across the mitochondrial membranes. Short- and medium-chain fatty acids can readily penetrate mitochondria. Long-chain acyl-CoA are able to cross the mitochondrial outer membrane, but cannot penetrate the inner membrane. Translocation of these is a carnitine-dependent process involving the coordinate action of isoforms of carnitine palmitoyl transferase on the mitochondrial outer and inner membranes (see Gurr et aL, 2002). Fatty acid oxidation itself involves the progressive removal of 2-carbon units, as acetyl-CoA, from the carboxyl end of the acyl-CoA (Gurr et aL, 2002). It is often termed the p-oxidation spiral since... 

Carnitine-acylcarnitine translocase (CACT), located in the inner mitochondrial membrane, carries the fatty acyl-carnitine inside the mitochondrion in exchange for a free carnitine molecule. CPT2, located inside the mitochondrion, then catalyzes the reversal of the CPTl reaction. Thus, the concerted actions of CPTl, CACT, and CPT2 effectively translocate fatty acyl-CoA across the inner mitochondrial membrane. 

It is generally accepted that a set of desaturases which is located in the plastids introduces double bonds into the acyl moieties bound as thioesters to acyl carrier protein or as oxoesters in monogalactosyl diacylglycerols. Both saturated and unsaturated fatty acids are translocated as acyl-CoAs from the plastids to the endoplasmic reticulum. Acyl chains of these activated fatty acids are elongated or incorporated into phosphatidylchohnes and other polar lipids II (cf. Fig. 1), and partly modified, for example, by desaturation. Further modification of acyl moieties leads to hydroxy-, epoxy-, and cyclic fatty acids. 

Carnitine serves as a cofactor for several enzymes, including carnitine translo-case and acyl carnitine transferases I and II, which are essential for the movement of activated long-chain fatty acids from the cytoplasm into the mitochondria (Figure 11.2). The translocation of fatty acids (FAs) is critical for the genaation of adenosine triphosphate (ATP) within skeletal muscle, via 3-oxidation. These activated FAs become esterified to acylcamitines with carnitine via camitine-acyl-transferase I (CAT I) in the outer mitochondrial membrane. Acylcamitines can easily permeate the membrane of the mitochondria and are translocated across the membrane by carnitine translocase. Carnitine s actions are not yet complete because the mitochondrion has two membranes to cross thus, through the action of CAT II, the acylcar-nitines are converted back to acyl-CoA and carnitine. Acyl-CoA can be used to generate ATP via 3-oxidation, Krebs cycle, and the electron transport chain. Carnitine is recycled to the cytoplasm for fumre use. 

Localization of radioactivity following a dose of [1,6- C]lipoate was greatest in liver, intestinal contents, and muscle. Retention of the label was approximately twice as high after per os administration than i.p. injection at earlier (4-hour) and later (24-hour) periods. The uptake of lipoate by liver mitochondria and subsequent 3-oxidation to yield CO2 was found to be similar to shorter-chain fatty acids, e.g. octanoate, rather than longer-chain ones, e.g. palmitate. Lipoate and octanoate were relatively insensitive to additions of d7-carnitine to supply the 7-substrate for the long-chain acyl transferase upon which mitochondrial translocation of palmitate is dependent. Moreover, (+)-decanoylcarnitine inhibited the latter but had no effect on the short-chain compounds. 

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