Gluconeogenesis oxaloacetate transport

To initiate gluconeogenesis, oxaloacetate is reduced to malate, which is then transported to the cytosol in the reverse of the malate shuttle. 

For PEPCK to function in gluconeogenesis, oxaloacetate produced in the pyruvate carboxylase reaction in the mitochondria, must be transported to the cytoplasm. PEPCK is not under any known allosteric control. Activity of the enzyme is regulated by hormonal control of its transcription. Glucagon stimulates transcription of the structural gene for PEPCK. Insulin inhibits transcription of the enzyme. By inhibiting PEPCK gene transcription, insulin tends to depress gluconeogenesis rates. 

FIGURE 23.5 Pyruvate carboxyl compartmentalized reaction. Pyruva verted to oxaloacetate in the mitoci Because oxaloacetate cannot be trai across the mitochondrial membrant reduced to malate, transported to tl and then oxidized back to oxaloace gluconeogenesis can continue. 

Step 1 of Figure 29.13 Carboxylation Gluconeogenesis begins with the carboxyl-afion of pyruvate to yield oxaloacetate. The reaction is catalyzed by pyruvate carboxylase and requires ATP, bicarbonate ion, and the coenzyme biotin, which acts as a carrier to transport CO2 to the enzyme active site. The mechanism is analogous to that of step 3 in fatty-acid biosynthesis (Figure 29.6), in which acetyl CoA is carboxylated to yield malonyl CoA. 

In pigeon, chicken, and rabbit liver, phospho-enolpymvate carboxykinase is a mitochondrial enzyme, and phosphoenolpyruvate is transported into the cytosol for gluconeogenesis. In the rat and the mouse, the enzyme is cytosolic. Oxaloacetate does not cross the mitochondrial inner membrane it is converted to malate, which is transported into the cytosol, and convetted back to oxaloacetate by cytosolic malate dehydrogenase. In humans, the guinea pig, and the cow, the enzyme is equally disttibuted between mitochondria and cytosol. 

A problem for gluconeogenesis is that pyruvate carboxylase, which produces oxaloacetate from pyravate, is present in the mitochondria but phosphoenolpyruvate carboxylase, at least in human liver, is present in the cytosol. For reasons given in Chapter 9, oxaloacetate cannot cross the mitochondrial membrane and so a transporter is not present in any cells. Hence, oxaloacetate is converted to phosphoenolpyruvate which is transported across the membrane (Figure 6.25). 

C. Oxaloacetate can also be converted to malate and transported to the cytoplasm for gluconeogenesis under fasting conditions (see Chapter 6). 

FIGURE 20-35 Conversion of stored fatty acids to sucrose in germinating seeds. This pathway begins in glyoxysomes. Succinate is produced and exported to mitochondria, where it is converted to oxaloacetate by enzymes of the citric acid cycle. Oxaloacetate enters the cytosol and serves as the starting material for gluconeogenesis and for the synthesis of sucrose, the transport form of carbon in plants. 

Pyruvate carboxylase is a mitochondrial enzyme, vhereas the other enzymes of gluconeogenesis are cytoplasmic. Oxaloacetate, the product of the pyruvate carboxylase reaction, is reduced to malate inside the mitochondrion for transport to the cytosol. The reduction is accomplished by an NADH-linked malate dehydrogenase. When malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD+-linked malate dehydrogenase in the cytosol (Figure 16.28). 

Figure 16.28. Compartmental Cooperation. Oxaloacetate utilized in the cytosol for gluconeogenesis is formed in the mitochondrial matrix by carboxylation of pyruvate. Oxaloacetate leaves the mitochondrion by a specific transport system (not shovm) in the form of malate, -which is reoxidized to oxaloacetate in the cytosol.

Pyruvate carboxylase is a mitochondrial enzyme, whereas the other enzymes of gluconeogenesis are present primarily in the cytoplasm. Oxaloacetate, the product of the pyruvate carboxylase reaction, must thus be transported to the cytoplasm to complete the pathway. Oxaloacetate is transported from a mitochondrion in the form of malate oxaloacetate is reduced to malate inside the mitochondrion by an NADH-linked malate dehydrogenase. After malate has been transported across the mitochondrial membrane, it is reoxidized to oxaloacetate by an NAD -linked malate dehydrogenase in the cytoplasm (Figure 16.26). The formation of oxaloacetate from malate also provides NADH for use in subsequent steps in gluconeogenesis. Finally, oxaloacetate is simultaneously decarboxylated and phospho-ry lated by phosphoenolpyruvate carboxy kinase to generate phosphoenol pyruvate. The phosphoryl donor is GTP. The GO2 that was added to pyruvate by pyruvate carboxylase comes off in this step. 

In tissues other than the RBC, pyruvate has alternative metabolic fates that, depending on the tissue, include gluconeogenesis, conversion to acetyl-CoA by pyruvate dehydrogenase for further metabolism to CO in the tricarboxylic acid (TCA) cycle, transamination to alanine or carboxylation to oxaloacetate by pyruvate carboxylase (Table 23-1). In the RBC, however, the restricted enzymatic endowment precludes all but the conversion to lactate. The pyruvate and lactate produced are end products of RBC glycolysis that are transported out of the RBC to the liver where they can undergo the alternative metabolic conversions described above. 

Pyruvate carboxylase is activated allosterically by acetyl-CoA. The enzyme is a tetrameric protein carrying four molecules of biotin, each bound covalently through an amide bond involving the -amino group of a lysine residue. In animals the reaction catalyzed by pyruvate carboxylase is the most important anaplerotic reaction, particularly in liver and kidney. Pyruvate carboxylase is the only enzyme of gluconeogenesis in the mitochondria, requiring pyruvate and oxaloacetate to be transported across the mitochondrial membrane for gluconeogenesis to occur. 

P-Oxidation supplies NADH and FAD(2H), which are used by the electron transport chain for oxidative phosphorylation. As ATP levels increase, less NADH is oxidized, and the NADH/NAD ratio is increased. (4) Oxaloacetate is converted into malate because of the high NADH levels, and the malate enters the cytoplasm for gluconeogenesis,. (5) Acetyl CoA is diverted from the TCA cycle into ketogenesis, in part because of low oxaloacetate levels, which reduces the rate of the citrate synthase reaction. 

The anabolic reactions of gluconeogenesis take place in the cytosol. Oxaloacetate is not transported across the mitochondrial membrane. Two mechanisms exist for the transfer of molecules needed for gluconeogenesis from mitochondria to the cytosol. One mechanism takes advantage of the fact that phosphoenolpyruvate can be formed from oxaloacetate in the mitochondrial matrix (this reaction is the next step in gluconeogenesis) phosphoenolpyruvate is then transferred to the cytosol, where the remaining reactions take place (Figure 19.12). The other mechanism relies on the fact that malate, which is another intermediate of the citric acid cycle, can be transferred to the cytosol. There is a malate dehydrogenase enzyme in the cytosol as well as in mitochondria, and malate can be converted to oxaloacetate in the cytosol. 

FIGURE 19.12 Transfer of the starting materials of gluconeogenesis from the mitochondrion to the cytosol. Note that phosphoenolpyruvate (PEP) can be transferred from the mitochondrion to the cytosol, as can malate. Oxaloacetate is not transported across the mitochondrial membrane. 

While the citric acid cycle takes place in mitochondria, many anabolic reactions take place in the cytosol. Oxaloacetate, the starting material for gluconeogenesis, is a component of the citric acid cycle. Malate, but not oxaloacetate, can be transported across the mitochondrial membrane. After malate from mitochondria is carried to the cytosol, it can be converted to oxaloacetate by malate dehydrogenase, an enzyme that requires NAD+. Malate, which crosses the mitochondrial membrane, plays a role in lipid anabolism, in a reaction in which malate is oxidatively decarboxylated to pyruvate by an enzyme that requires NADP+, producing NADPH. 

Mitochondrial aspartate efflux is important in processes like urea synthesis [90], gluconeogenesis from lactate and the transport of cytosolic reducing equivalents to the mitochondria via the so-called malate-aspartate shuttle, e.g. during ethanol oxidation and gluconeogenesis from reduced substrates like glycerol, sorbitol, and xylitol [4,5]. During gluconeogenesis from lactate oxaloacetate is transported to the cytosol as aspartate to circumvent the low permeability of the mitochondrial membrane for oxaloacetate. 

THE FED STATE Two to four hours after a meal, the body is in the absorptive (fed) state. This is a period of anabolism, where there is a high insulin gfucagon ratio in the circulation. In the liver, glycolysis is increased, and glycogen synthesis is favoured. There is no gluconeogenesis. Surplus amino acids are used for protein synthesis or deaminated in the liver to pyruvate, acetyl CoA or oxaloacetate, which enter the TCA cycle. FA synthesis is favoured sinc e excess acetyl CoA is converted to malonyl CoA (by acetyl CoA carboxylase). Malonyl CoA inhibits the carnitine shuttle which transports FAs to be oxidized in the mitochondria, therefore they have to be stored as TAG in the periphery. 

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