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Introduction

Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). The production of glucose from other metabolites is necessary for use as a fuel source by the brain, testes, erythrocytes and kidney medulla since glucose is the sole energy source for these organs. During starvation, however, the brain can derive energy from ketone bodies which are converted to acetyl-CoA.
Synthesis of glucose from three and four carbon precursors is essentially a reversal of glycolysis. The relevant features of the pathway of gluconeogenesis are diagrammed below.

The relevant reactions of gluconeogenesis are depicted. The enzymes of the 3 bypass steps are indicated in green along with phosphoglycerate kinase. This latter enzyme is included since when functioning in the gluconeogenic direction the reaction consumes energy. Gluconeogenesis from 2 moles of pyruvate to 2 moles of 1,3-bisphosphoglycerate consumes 6 moles of ATP. This makes the process of gluconeogenesis very costly from an energy standpoint considering that glycolysis to 2 moles of pyruvate only yields 2 moles of ATP. Note that several steps are required in going from 2 moles of 1,3-bisphosphoglycerate to 1 mole of fructose-1,6-bisphosphate. First there is a reversal of the glyceraldehyde-3-phosphate dehydrogenase reaction which requires a supply of NADH. When lactate is the gluconeogenic substrate the NADH is supplied by the lactate dehydrogenase reaction, and it is supplied by the malate dehydrogenase reaction when pyruvate is the substrate. Secondly, 1 mole of glyceraldehyde-3-phosphate must be isomerized to DHAP and then a mole of DHAP can be condensed to a mole of glyceraldehyde-3-phosphate to form 1 mole of fructose-1,6-bisphosphate in a reversal of the aldolase reaction. Most non-hepatic tissues lack glucose-6-phosphatase and so the glucose-6-phosphate generated in these tissues would be a substrate for glycogen synthesis. In hepatocytes the glucose-6-phosphatase reactions allows the liver to supply the blood with free glucose. Remember that due to the high Km of liver glucokinase most of the glucose will not be phosphorylated and will flow down its' concentration gradient out of hepatocytes and into the blood.
The three reactions of glycolysis that proceed with a large negative free energy change are bypassed during gluconeogenesis by using different enzymes. These three are the pyruvate kinase, phosphofructokinase-1(PFK-1) and hexokinase/glucokinase catalyzed reactions. In the liver or kidney cortex and in some cases skeletal muscle, the glucose-6-phosphate (G6P) produced by gluconeogenesis can be incorporated into glycogen. In this case the third bypass occurs at the glycogen phosphorylase catalyzed reaction. Since skeletal muscle lacks glucose-6-phosphatase it cannot deliver free glucose to the blood and undergoes gluconeogenesis exclusively as a mechanism to generate glucose for storage as glycogen.
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Pyruvate to Phosphoenolpyruvate (PEP), Bypass 1

Conversion of pyruvate to PEP requires the action of two mitochondrial enzymes. The first is an ATP-requiring reaction catalyzed by pyruvate carboxylase, (PC). As the name of the enzyme implies, pyruvate is carboxylated to form oxaloacetate (OAA). The CO2 in this reaction is in the form of bicarbonate (HCO3-) . This reaction is an anaplerotic reaction since it can be used to fill-up the TCA cycle. The second enzyme in the conversion of pyruvate to PEP is PEP carboxykinase (PEPCK). PEPCK requires GTP in the decarboxylation of OAA to yield PEP. Since PC incorporated CO2 into pyruvate and it is subsequently released in the PEPCK reaction, no net fixation of carbon occurs. Human cells contain almost equal amounts of mitochondrial and cytosolic PEPCK so this second reaction can occur in either cellular compartment.
For gluconeogenesis to proceed, the OAA produced by PC needs to be transported to the cytosol. However, no transport mechanism exist for its' direct transfer and OAA will not freely diffuse. Mitochondrial OAA can become cytosolic via three pathways, conversion to PEP (as indicated above through the action of the mitochondrial PEPCK), transamination to aspartate or reduction to malate, all of which are transported to the cytosol.
If OAA is converted to PEP by mitochondrial PEPCK, it is transported to the cytosol where it is a direct substrate for gluconeogenesis and nothing further is required. Transamination of OAA to aspartate allows the aspartate to be transported to the cytosol where the reverse transamination occurs yielding cytosolic OAA. This transamination reaction requires continuous transport of glutamate into, and a-ketoglutarate out of, the mitochondrion. Therefore, this process is limited by the availability of these other substrates. Either of these latter two reactions will predominate when the substrate for gluconeogenesis is lactate. Whether mitochondrial decarboxylation or transamination occurs is a function of the availability of PEPCK or transamination intermediates.
Mitochondrial OAA can also be reduced to malate in a reversal of the TCA cycle reaction catalyzed by malate dehydrogenase (MDH). The reduction of OAA to malate requires NADH, which will be accumulating in the mitochondrion as the energy charge increases. The increased energy charge will allow cells to carry out the ATP costly process of gluconeogenesis. The resultant malate is transported to the cytosol where it is oxidized to OAA by cytosolic MDH which requires NAD+ and yields NADH. The NADH produced during the cytosolic oxidation of malate to OAA is utilized during the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. The coupling of these two oxidation-reduction reactions is required to keep gluconeogenesis functional when pyruvate is the principal source of carbon atoms. The conversion of OAA to malate predominates when pyruvate (derived from glycolysis or amino acid catabolism) is the source of carbon atoms for gluconeogenesis. When in the cytoplasm, OAA is converted to PEP by the cytosolic version of PEPCK. Hormonal signals control the level of PEPCK protein as a means to regulate the flux through gluconeogenesis (see below).
The net result of the PC and PEPCK reactions is:

Pyruvate + ATP + GTP + H2O ---> PEP + ADP + GDP + Pi + 2H+

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Fructose-1,6-bisphosphate to Fructose-6-phosphate, Bypass 2

Fructose-1,6-bisphosphate (F1,6BP) conversion to fructose-6-phosphate (F6P) is the reverse of the rate limiting step of glycolysis. The reaction, a simple hydrolysis, is catalyzed by fructose-1,6-bisphosphatase (F1,6BPase). Like the regulation of glycolysis occurring at the PFK-1 reaction, the F1,6BPase reaction is a major point of control of gluconeogenesis (see below).
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Glucose-6-phosphate (G6P) to Glucose (or Glycogen), Bypass 3

G6P is converted to glucose through the action of glucose-6-phosphatase G6Pase). This reaction is also a simple hydrolysis reaction like that of F1,6BPase. Since the brain and skeletal muscle, as well as most non-hepatic tissues, lack G6Pase activity, any gluconeogenesis that occurs in these tissues is not utilized for blood glucose supply. In the kidney, muscle and especially the liver, G6P can be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis are an alternate bypass 3 series of reactions.
Phosphorolysis of glycogen is carried out by glycogen phosphorylase, whereas, glycogen synthesis is catalyzed by glycogen synthase. The G6P produced from gluconeogenesis can be converted to glucose-1-phosphate (G1P) by phosphoglucose mutase (PGM). G1P is then converted to UDP-glucose (the substrate for glycogen synthase) by UDP-glucose pyrophosphorylase, a reaction requiring hydrolysis of UTP.
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Substrates for Gluconeogenesis

Lactate:

Lactate is a predominate source of carbon atoms for glucose synthesis by gluconeogenesis. During anaerobic glycolysis in skeletal muscle, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). This reaction serves two critical functions during anaerobic glycolysis. First, in the direction of lactate formation the LDH reaction requires NADH and yields NAD+ which is then available for use by the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis. These two reaction are, therefore, intimately coupled during anaerobic glycolysis. Secondly, the lactate produced by the LDH reaction is released to the blood stream and transported to the liver where it is converted to glucose. The glucose is then returned to the blood for use by muscle as an energy source and to replenish glycogen stores. This cycle is termed the Cori cycle.
The Cori cycle invloves the utilization of lactate, produced by glycolysis in non-hepatic tissues, (such as muscle and erythrocytes) as a carbon source for hepatic gluconeogenesis. In this way the liver can convert the anaerobic byproduct of glycolysis, lactate, back into more glucose for reuse by non-hepatic tissues. Note that the gluconeogenic leg of the cycle (on its own) is a net consumer of energy, costing the body 4 moles of ATP more than are produced during glycolysis. Therefore, the cycle cannot be sustained indefinitely.

Pyruvate:

Pyruvate, generated in muscle and other peripheral tissues, can be transaminated to alanine which is returned to the liver for gluconeogenesis. The transamination reaction requires an a-amino acid as donor of the amino group, generating an a-keto acid in the process. This pathway is termed the glucose-alanine cycle. Although the majority of amino acids are degraded in the liver some are deaminated in muscle. The glucose-alanine cycle is, therefore, an indirect mechanism for muscle to eliminate nitrogen while replenishing its energy supply. However, the major function of the glucose-alanine cycle is to allow non-hepatic tissues to deliver the amino portion of catabolized amino acids to the liver for excretion as urea. Within the liver the alanine is converted back to pyruvate and used as a gluconeogenic substrate (if that is the hepatic requirement) or oxidized in the TCA cycle. The amino nitrogen is converted to urea in the urea cycle and excreted by the kidneys.

Amino Acids:

All 20 of the amino acids, excepting leucine and lysine, can be degraded to TCA cycle intermediates as discussed in the metabolism of amino acids. This allows the carbon skeletons of the amino acids to be converted to those in oxaloacetate and subsequently into pyruvate. The pyruvate thus formed can be utilized by the gluconeogenic pathway. When glycogen stores are depleted, in muscle during exertion and liver during fasting, catabolism of muscle proteins to amino acids contributes the major source of carbon for maintenance of blood glucose levels.

Glycerol:

Oxidation of fatty acids yields enormous amounts of energy on a molar basis, however, the carbons of the fatty acids cannot be utilized for net synthesis of glucose. The two carbon unit of acetyl-CoA derived from b-oxidation of fatty acids can be incorporated into the TCA cycle, however, during the TCA cycle two carbons are lost as CO2. Thus, explaining why fatty acids do not undergo net conversion to carbohydrate.
The glycerol backbone of lipids can be used for gluconeogenesis. This requires phosphorylation to glycerol-3-phosphate by glycerol kinase and dehydrogenation to dihydroxyacetone phosphate (DHAP) by glyceraldehyde-3-phosphate dehydrogenase(G3PDH). The G3PDH reaction is the same as that used in the transport of cytosolic reducing equivalents into the mitochondrion for use in oxidative phosphorylation. This transport pathway is called the glycerol-phosphate shuttle. The glycerol backbone of adipose tissue stored triacylgycerols is ensured of being used as a gluconeogenic substrate since adipose cells lack glycerol kinase. In fact adipocytes require a basal level of glycolysis in order to provide them with DHAP as an intermediate in the synthesis of triacyglycerols.

Propionate:

Oxidation of fatty acids with an odd number of carbon atoms and the oxidation of some amino acids generates as the terminal oxidation product, propionyl-CoA. Propionyl-CoA is converted to the TCA intermediate, succinyl-CoA. This conversion is carried out by the ATP-requiring enzyme, propionyl-CoA carboxylase then methylmalonyl-CoA epimerase and finally the vitamin B12 requiring enzyme, methylmalonyl-CoA mutase. The utilization of propionate in gluconeogenesis only has quantitative significance in ruminants.
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Regulation of Gluconeogenesis

Obviously the regulation of gluconeogenesis will be in direct contrast to the regulation of glycolysis. In general, negative effectors of glycolysis are positive effectors of gluconeogenesis. Regulation of the activity of PFK-1 and F1,6BPase is the most significant site for controlling the flux toward glucose oxidation or glucose synthesis. As described in control of glycolysis, this is predominantly controlled by fructose-2,6-bisphosphate, F2,6BP which is a powerful negative allosteric effector of F1,6Bpase activity.
Regulation of glycolysis and gluconeogenesis by fructose 2,6-bisphosphate (F2,6BP). The major sites for regulation of glycolysis and gluconeogenesis are the phosphofructokinase-1 (PFK-1) and fructose-1,6-bisphosphatase (F-1,6-BPase) catalyzed reactions. PFK-2 is the kinase activity and F-2,6-BPase is the phosphatase activity of the bi-functional regulatory enzyme, phosphofructokinase-2/fructose-2,6-bisphosphatase. PKA is cAMP-dependent protein kinase which phosphorylates PFK-2/F-2,6-BPase turning on the phosphatase activity. (+ve) and (-ve) refer to positive and negative activities, respectively.

The level of F2,6BP will decline in hepatocytes in response to glucagon stimulation as well as stimulation by catecholamines. Each of these signals is elicited through activation of cAMP-dependent protein kinase (PKA). One substrate for PKA is PFK-2, the bifunctional enzyme responsible for the synthesis and hydrolysis of F2,6BP. When PFK-2 is phosphorylated by PKA it acts as a phosphatase leading to the dephosphorylation of F2,6BP with a concomitant increase in F1,6Bpase activity and a decrease in PFK-1 activity. Secondarily, F1,6Bpase activity is regulated by the ATP/ADP ratio. When this is high, gluconeogenesis can proceed maximally.
Gluconeogenesis is also controlled at the level of the pyruvate to PEP bypass. The hepatic signals elicited by glucagon or epinephrine lead to phosphorylation and inactivation of pyruvate kinase (PK) which will allow for an increase in the flux through gluconeogenesis. PK is also allosterically inhibited by ATP and alanine. The former signals adequate energy and the latter that sufficient substrates for gluconeogenesis are available. Conversely, a reduction in energy levels as evidenced by increasing concentrations of ADP lead to inhibition of both PC and PEPCK. Allosteric activation of PC occurs through acetyl-CoA. Each of these regulations occurs on a short time scale, whereas long-term regulation can be effected at the level of PEPCK. The amount of this enzyme increases in response to prolonged glucagon stimulation. This situation would occur in a starving individual or someone with an inadequate diet.
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This article has been modified by Dr. M. Javed Abbas.
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20:39 21/12/2002