Malate, fatty acid synthesis

FIGURE 20.23 Export of citrate from mitochondria and cytosolic breakdown produces oxaloacetate and acetyl-CoA. Oxaloacetate is recycled to malate or pyruvate, which re-enters the mitochondria. This cycle provides acetyl-CoA for fatty acid synthesis in the cytosol. 

The acetyl-CoA derived from amino acid degradation is normally insufficient for fatty acid biosynthesis, and the acetyl-CoA produced by pyruvate dehydrogenase and by fatty acid oxidation cannot cross the mitochondrial membrane to participate directly in fatty acid synthesis. Instead, acetyl-CoA is linked with oxaloacetate to form citrate, which is transported from the mitochondrial matrix to the cytosol (Figure 25.1). Here it can be converted back into acetyl-CoA and oxaloacetate by ATP-citrate lyase. In this manner, mitochondrial acetyl-CoA becomes the substrate for cytosolic fatty acid synthesis. (Oxaloacetate returns to the mitochondria in the form of either pyruvate or malate, which is then reconverted to acetyl-CoA and oxaloacetate, respectively.)... 

FIGURE 25.1 The citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents (electrons) for fatty acid synthesis. The shuttle collects carbon substrates, primarily from glycolysis but also from fatty acid oxidation and amino acid catabolism. Most of the reducing equivalents are glycolytic in origin. Pathways that provide carbon for fatty acid synthesis are shown in blue pathways that supply electrons for fatty acid synthesis are shown in red. 

In common with cholesterol synthesis described in the next section, fatty acids are derived from glucose-derived acetyl-CoA. In the fed state when glucose is plentiful and more than sufficient acetyl-CoA is available to supply the TCA cycle, carbon atoms are transported out of the mitochondrion as citrate (Figure 6.8). Once in the cytosol, citrate lyase forms acetyl-CoA and oxaloacetate (OAA) from the citrate. The OAA cannot re-enter the mitochondrion but is converted into malate by cytosolic malate dehydrogenase (cMDH) and then back into OAA by mitochondrial MDH (mMDH) Acetyl-CoA remains in the cytosol and is available for fatty acid synthesis. 

In animals and fungi there is a similar dichotomy. NADPH can be generated by cytosolic malic enzyme which catalyses the reaction malate + NADP+ — pyruvate + COg + NADPH. Cytosolic malate derives from the following successive reactions the pyruvate/ citrate shuttle on the mitochondrial inner membrane takes pyruvate to the mitochondrion in exchange for citrate cytosolic ATP citrate lyase catalyses ATP + citrate + CoA-SH —> acetylCoA (CH3CO-S-C0A) + oxaloacetate and cytosolic malate dehydrogenase, which catalyses NADH + oxaloacetate NAD+ + malate. This scheme provides both acetylCoA and NADPH for subsequent long chain fatty acid synthesis (see section on Fatty acid synthesis ). 

Pyruvate carboxylase is also important in lipogenesis. Citrate is transported out of mitochondria and cleaved in the cytosol to provide acetyl CoA for fatty acid synthesis the resultant oxaloacetate is reduced to malate, which undergoes oxidative decarboxylation to pyruvate, a reaction that provides at least half of the NADPH required for fatty acid synthesis. Pyruvate reenters the mitochondria and is carboxylated to oxaloacetate to maintain the process. 

Oxaloacetate formed in the transfer of acetyl groups to the cytosol must now be returned to the mitochondria The inner mitochondrial membrane is impermeable to oxaloacetate. Hence, a series of bypass reactions are needed. Most important, these reactions generate much of the NADPH needed for fatty acid synthesis. First, oxaloacetate is reduced to malate by NADH. This reaction is catalyzed by a malate dehydrogenase in the cytosol. 

Fatty acid synthesis and degradation. Fatty acids are synthesized in the cytosol by the addition of two-carbon units to a growing chain on an acyl carrier protein. Malonyl CoA, the activated intermediate, is formed by the carboxylation of acetyl CoA. Acetyl groups are carried from mitochondria to the cytosol as citrate by the citrate-malate shuttle. In the cytosol, citrate is cleaved to yield acetyl CoA. In addition to transporting acetyl CoA, citrate in the cytosol stimulates acetyl CoA carboxylase, the enzyme catalyzing the committed step. When ATP and acetyl CoA are abundant, the level of citrate increases, which accelerates the rate of fatty acid synthesis (Figure 30.8). 

The reactions of fatty acid synthesis all take place in the cytosol, but acetyl-CoA is made in the mitochondria and can t cross the inner membrane. The Pyruvate-Malate Cycle (Citrate-Pyruvate Cycle) is used to take acetyl- groups to the cytosol while simultaneously providing a source of NADPH from NADH, and thus, coupling fatty acid synthesis to Glycolysis (Fig. 10.7). Note that the acetyl-CoA is first joined to oxaloacetate to make citrate which is readily transported out of the mitochondria using a co-transporter. The citrate is then cleaved to acetyl-CoA and oxaloacetate, a process requiring ATP to make it favourable (recall the condensation was spontaneous). Acetyl-CoA for fatty acid synthesis is now available in the cytosol, but oxaloacetate must be regenerated for the mitosol. 

The utilization of NAD is illustrated in the sections on glycolysis, the malate-aspartate shuttle, ketone body metabolism, and fatty acid oxidation. The utilization of NADP is illustrated in the sections concerning fatty acid synthesis and the pentose phosphate pathway. 

Thus, citrate not only modulates the rate of fatty acid synthesis but also provides carbon atoms for the synthesis. The oxaloacetate formed from pyruvate may eventually be converted (via malate) to glucose by the gluconeogenic pathway. The glucose oxidized via the pentose phosphate pathway augments fatty acid synthesis by providing NADPH. Pyruvate generated from oxaloacetate can enter mitochondria and be converted to oxaloacetate, which is required for the formation of citrate. 

The regulation of fatty acid synthesis in adipose tissue has been reviewed by Ball, with emphasis on a citrate-malate cycle that provides both extramitochondrial acetyl-CoA and part of the NADPH required for fatty acid synthesis. Further quantitative work on the pathways of glucose and acetate carbon in adipose tissue has also been reported. 

A diet high in D-fructose produces increased concentrations of pyruvate, malate, and acetyl coenzyme A (AcSCoA).294 295 D-Fructose enhances formation of AcSCoA from pyruvate by stimulating pyruvate oxidation.296 As D-fructose causes a fall in the hepatic ATP concentration, pyruvate dehydrogenase (EC 1.2.4.1) is activated, and it produces an increase in AcSCoA formation from pyruvate.297 Thus, dietary sucrose and D-fructose produce higher hepatic fatty acid synthesis than does dietary D-glucose, because of their stimulation of AcSCoA formation. 

When citrate, a citric acid cycle intermediate, moves from the mitochondrial matrix into the cytoplasm, it is cleaved to form acetyl-CoA and oxaloacetate by citrate lyase. The citrate lyase reaction is driven by ATP hydrolysis. Most of the oxaloacetate is reduced to malate by malate dehydrogenase. Malate may then be oxidized to pyruvate and CO, by malic enzyme. The NADPH produced in this reaction is used in cytoplasmic biosynthetic processes, such as fatty acid synthesis. Pyruvate enters the mitochondria, where it may be converted to oxaloacetate or acetyl-CoA. Malate may also reenter the mitochondria, where it is reoxidized to form oxaloacetate. 

The oxaloacetate can be reduced to malate by NADH and then oxidized by malic enzyme to form pyruvate and NADPH. The latter is needed for fatty-acid synthesis. 

The reoxldatlon of NADPH was accomplished by the addition of OAA which was reduced to malate in the stroma. In the experiments of Sauer and Heise (2) all three components of the DHAP shuttle were necessary to observe fatty acid synthesis in the dark. This is rather puzzling since the OAA would be expected to drain off MADPH which one would think is required for the reductive steps of fatty acid synthesis. Perhaps the components of the shuttle have other effects than those outlined above. The results of Browse eta/(JJ) also bear on the question of fatty acid synthesis in the dark. They have reported rates of fatty acid synthesis by leaf discs of spinach kept in darkness which were 12-20% of the rates in the light. 

In tissue culture, cells that are deficient in NADP -linked malate enzyme can be isolated. They exhibit a slightly lower rate of fatty acid synthesis when compared with normal cells. However, cells lacking citrate lyase are very difficult to isolate. Why ... 

Both malate enzyme and citrate lyase are part of the shuttle system that transports two-carbon units from the mitochondrion to the cytosol. Malate enzyme also generates reducing power in the form of NADPH, which is used for fatty acid synthesis however, the pentose phosphate pathway (see the text. Section 20.3) also serves as a source of NADPH, so that fatty acid synthesis can continue even if malate enzyme is deficient. Recall from page 515 of the text that malate can cross the mitochondrial membrane. Citrate lyase is more critical to fatty acid synthesis because it is required to generate acetyl CoA from citrate in the cytosol. Without cytosolic acetyl CoA, fatty acid synthesis cannot take place, and the cells cannot grow and divide. 

The transport of citrate from the mitochondrial matrix into the cytoplasm is linked to the transfer of reducing equivalents yielding NADPH via malate decarboxylation (Fig. 11-21). However, the amount of NADPH produced in this process is insufficient to provide all that is required for fatty acid synthesis. 

Evidence has been presented that citrate is the source of acetyl-CoA in the soybean cotyledon. Nelson and Rinne (1975, 1977a,b) have described the occurrence of citrate lyase in the cytosol of developing soybean seeds. Furthermore, [1,5-1 ] citrate was an effective donor of [ KI ]acetyl-CoA for the synthesis of fatty acids in these extracts. However, Weaire and Kekwick (1975) did not find [l,5- K ]citrate to be an acetyl donor for fatty acid synthesis in avocado extracts. In addition, Yamada and Nakamura (1975) showed with isolated spinach chloroplasts that, whereas pyruvate was effectively incorporated into fatty acid, citrate, malate, and oxeiloacetate were far less active. Apparently different tissues may supply acetyl-CoA from different precursors, i.e., pyruvate, citrate, and acetate. 

Fig. 1. Pathway of fatty acid synthesis from glucose in animal tissues. The key enzymes or enzyme systems involved are (1) pyruvate dehydrogenase, (2) pyruvate carboxylase, (3) citrate synthase, (4) citrate translocation system, (5) citrate cleavage enzyme, (6) acetyl-CoA carboxylase, (7) fatty acid synthetase, (8) 3-phosphoglyceraldehyde dehydrogenase, (9) malate dehydrogenase, (10) malic enzyme, (11) hexose monophosphate shunt.

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