Gluconeogenesis is the biosynthesis of new glucose, (i.e. not glucose from glycogen). This process is frequently referred to as endogenous glucose production (EGP). The production of glucose from other carbon skeletons is necessary since the testes, erythrocytes and kidney medulla exclusively utilize glucose for ATP production. The brain also utilizes large amounts of the daily glucose consumed or produced via gluconeogenesis. However, in addition to glucose, the brain can derive energy from ketone bodies which are converted to acetyl-CoA and shunted into the TCA cycle. The primary carbon skeletons used for gluconeogenesis are derived from pyruvate, lactate, glycerol, and the amino acids alanine and glutamine. The liver is the major site of gluconeogenesis, however, as discussed below, the kidney and the small intestine also have important roles to play in this pathway.
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:
Reactions of Gluconeogenesis: Gluconeogenesis from two moles of pyruvate to two moles of 1,3-bisphosphoglycerate consumes six moles of ATP. This makes the process of gluconeogenesis very costly from an energy standpoint considering that glucose oxidation to two moles of pyruvate yields two moles of ATP. The major hepatic substrates for gluconeogenesis (glycerol, lactate, alanine, and pyruvate) are enclosed in red boxes for highlighting. The reactions that take place in the mitochondria are pyruvate to OAA and OAA to malate. Pyruvate from the cytosol is transported across the inner mitochondrial membrane by the pyruvate transporter. Transport of pyruvate across the plasma membrane is catalyzed by the SLC16A1 protein (also called the monocarboxylic acid transporter 1, MCT1) and transport across the outer mitochondrial membrane involves a voltage-dependent porin transporter. Transport across the inner mitochondrial membrane requires a heterotetrameric transport complex (mitochondrial pyruvate carrier) consisting of the MPC1 gene and MPC2 gene encoded proteins. Following reduction of OAA to malate the malate is transported to the cytosol by the malate transporter (SLC25A11). In the cytosol the malate is oxidized to OAA and the OOA then feeds into the gluconeogenic pathway via conversion to PEP via PEPCK. The PEPCK reaction is another site for consumption of an ATP equivalent (GTP is utilized in the PEPCK reaction). The reversal of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reaction requires a supply of NADH. When lactate is the gluconeogenic substrate the NADH is supplied by the lactate dehydrogenase (LDH) reaction (indicated by the dashes lines), and it is supplied by the malate dehydrogenase reaction when pyruvate and alanine are the substrates. Secondly, one 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. In hepatocytes the glucose-6-phosphatase (G6Pase) reaction 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. ALT: alanine transaminase. PGAM1: phosphoglycerate mutase 1. PGK1: phosphoglycerate kinase 1. TMI: triose isomerase. PGI: glucose-6-phosphate isomerase. GPD1: cytosolic glycerol-3-phosphate dehydrogenase. F1,6BPase: fructose-1,6-bisphosphatase.
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, intestine, or kidney cortex, 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.back to the top
Conversion of pyruvate to PEP requires the action of two enzymes: pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK).
The first reaction of bypass 1 utilizes the ATP and biotin-requiring enzyme pyruvate carboxylase, (PC). PC is referred to as an ABC enzyme due to the role of ATP, Biotin, and CO2 in its catalytic activities. The CO2 utilized in the PC reaction is in the form of bicarbonate (HCO3-) . As the name of the enzyme implies, pyruvate is carboxylated to form oxaloacetate (OAA). PC is a somewhat unique enzyme in that it is one of only two metabolically important enzymes that requires an obligate activator. In the absence of its obligate activator, acetyl-CoA, PC is completely inactive. The primary source of the acetyl-CoA required by PC comes from the oxidation of fatty acids which are being delivered to the liver after release from adipose tissue in response to fasting or stress. Another critical enzyme that functions only in the presence of an obligate activator is carbamoylphosphate synthetase I (CPS I) of the urea cycle.
Although the major function of PC is to drive precursor carbon atoms (from pyruvate, lactate, and alanine) into the generation of endogenous glucose, the production of oxaloacetate is also an important anaplerotic reaction since it can be used to fill-up the TCA cycle. Indeed, within the brain the primary function of PC is to ensure that glial cells have sufficient oxaloacetate to drive the TCA cycle. In these cells, in addition to energy generation, the TCA cycle is vital to the continued generation of 2-oxoglutarate (α-ketoglutarate) which can be siphoned off the TCA cycle and utilized for the synthesis of glutamate as a critical excitatory neurotransmitter.
Like the other biotin-dependent carboxylating enzymes in mammals, PC is multi-functional and contains three distinct enzymatic domains: the biotin carboxylase (BC) domain, the carboxyltransferase (CT) domain, and the biotin carboxyl carrier protein (BCCP) domain. PC is composed of four identical subunits generating an α4 homotetrameric enzyme. The human PC gene is located on chromosome 11q13.2 and contains 19 exons spanning 16 kbp of DNA. The expressed 4.2 kb PC mRNA encodes a protein of 1178 amino acids with a molecular weight of 129.6 kDa.
The reaction catalyzed by PC occurs in a two-step process. The first partial reaction involves the fixation of CO2 to biotin that involves the BC and BCCP domains. During this initial stage of the reaction, biotin is moved to interact with the BC domain forming carboxybiotin. The carboxybiotin is brought into contact with the carboxyltransferase domain resulting in the formation of carboxylated biotin. This biotin carboxylase reaction involves a carboxyphosphate intermediate formed directly from ATP and bicarbonate. During the second step of the overall PC reaction, carboxybiotin is decarboxylated and pyruvate is concurrently carboxylated forming oxaloacetate.
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 (designated PEPCK-m and PEPCK-c, respectively) so this second reaction can occur in either cellular compartment. The PEPCK-c gene (official gene symbol: PCK1) is located on chromosome 20q13.31 and is composed of 10 exons encoding a protein of 622 amino acids. The PEPCK-m gene (official gene symbol: PCK2) is located on chromosome 14q11.2 and is composed of 10 exons encoding a 640 amino acid protein. The PCK2 gene is primarily expressed in the liver, kidney, and intestine as would be expected for a major gluconeogenic enzyme. The liver expresses the PCK1 and PCK2 genes at essentially equivalent levels.
Transcription of the PCK1 and PCK2 genes has been shown to be regulated by insulin, glucagon, and glucocorticoids. Regulation of PEPCK gene expression by glucagon is exerted via the activation of the transcription factor, CREB (cAMP response element-binding protein). When glucagon binds its receptor the result is activation of adenylate cyclase with resultant increases in cAMP production. The increased cAMP in turn activates PKA which, among numerous substrates, phosphorylates CREB. Phosphorylated CREB then migrates to the nucleus where it binds to a cAMP-response element (CRE) in the PEPCK genes activating their rate of transcription. The stress hormone, cortisol, exerts a very similar effect on PEPCK gene expression via binding of the cortisol-activated glucocorticoid receptor to a glucocorticoid-response element (GRE) in the PEPCK genes.
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: (1) conversion to PEP as indicated above through the action of the mitochondrial PEPCK; (2) transamination to aspartate; or (3) reduction to malate, all of which are transported to the cytosol. Transport of mitochondrial PEP to the cytosol is carried out by the tricarboxylate transporter encoded by the SLC25A1 gene. The transport of malate to the cytosol is carried out by the transporter encoded by the SLC25A11 gene. The transport of aspartate to the cytosol is carried out by either of two transporters, one is encoded by the SLC25A12 gene and the other is encoded by the SLC25A13 gene. In the context of the transamination of OAA to aspartate and the reduction of OAA to malate, there is a need for adequate levels of the other intermediates of the malate-aspartate shuttle to ensure these latter two reactions can continue.
The malate-aspartate shuttle. This shuttle is the principal mechanism for the movement of reducing equivalents (in the form of NADH; highlighted in the red boxes) from the cytoplasm to the mitochondria. The glycolytic pathway is a primary source of NADH. Within the mitochondria the electrons of NADH can be coupled to ATP production during the process of oxidative phosphorylation. The electrons are "carried" into the mitochondria in the form of malate. Cytoplasmic malate dehydrogenase (MDH) reduces oxaloacetate (OAA) to malate while oxidizing NADH to NAD+. Malate then enters the mitochondria where the reverse reaction is carried out by mitochondrial MDH. Movement of mitochondrial OAA to the cytoplasm to maintain this cycle requires it be transaminated to aspartate (Asp, D) with the amino group being donated by glutamate (Glu, E). The Asp then leaves the mitochondria and enters the cytoplasm. The deamination of glutamate generates 2-oxoglutarate, 2-OG, (α-ketoglutarate) which leaves the mitochondria for the cytoplasm. All the participants in the cycle are present in the proper cellular compartment for the shuttle to function due to concentration dependent movement. When the energy level of the cell rises the rate of mitochondrial oxidation of NADH to NAD+ declines and therefore, the shuttle slows. GAPDH is glyceraldehyde-3-phosphate dehydrogenase. AST is aspartate transaminase. SLC25A11 is the malate transporter and SLC25A13 is one of the two mitochondrial aspartate/glutamate transporters.
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 2-oxoglutatrate (α-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 gluconeogenesis. 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:
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). The existence of two distinct forms of F1,6BPase was recognized by comparison of the kinetic and regulatory properties of the purified liver and muscle enzymes. In addition, in patients with an inborn error in the gene encoding the liver F1,6BPase isoform, there is no reduction in skeletal muscle F1,6BPase activity. This led to the characterization of two F1,6BPase genes in the human genome. One expresses a liver version of the enzyme (gene symbol: FBP1) and the other a muscle version of the enzyme (gene symbol: FBP2). The FBP1 gene is located on chromosome 9q22.3 and is composed of 8 exons that encode a protein of 338 amino acids. The FBP2 gene is located at the same chromosomal location as the FBP1 gene but is composed of 7 exons that encode a protein of 339 amino acids. The liver and muscle F1,6BPase enzymes share 77% amino acid sequence identity.
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).back to the top
Glucose-6-phosphate 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 might occur in these tissues is not utilized for blood glucose supply. In the kidney, muscle and especially the liver, G6P be shunted toward glycogen if blood glucose levels are adequate. The reactions necessary for glycogen synthesis are an alternate bypass series of reactions.
The glucose-6-phosphatase gene (official gene symbol G6PC; formerly G6PT) is located on chromosome 17q21.31 and is composed of 5 exons that encode a 357 amino acid protein. Only three human tissues express the G6PC gene, liver, kidney, and small intestine. Likewise, these are the only tissues that can contribute to endogenous glucose production. Defects in the G6PC gene are associated with the glycogen storage disease known as von Gierke disease (glycogen storage disease type Ia).
Phosphorolysis of glycogen is carried out by glycogen phosphorylase, whereas, glycogen synthesis is catalyzed by glycogen synthase. The G6P produced from gluconeogenesis can be used used as a substrate for the synthesis of glycogen. In this case the G6P is converted to glucose-1-phosphate (G1P) by phosphoglucomutase (PGM). G1P is then converted to UDP-glucose (the substrate for glycogen synthase) by UDP-glucose pyrophosphorylase, a reaction requiring hydrolysis of UTP.back to the top
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: This cycle involves 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, 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 α-amino acid as donor of the amino group, generating an α-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.
The glucose-alanine cycle: This cycle is used primarily as a mechanism for skeletal muscle to eliminate nitrogen while replenishing its energy supply. Glucose oxidation produces pyruvate which can undergo transamination to alanine. This reaction is catalyzed by alanine transaminase, ALT (ALT used to be referred to a serum glutamate-pyruvate transaminase, SGPT). Additionally, during periods of fasting, skeletal muscle protein is degraded for the energy value of the amino acid carbons and alanine is a major amino acid in protein. The alanine then enters the blood stream and is transported to the liver. Within the liver alanine is converted back to pyruvate which is then a source of carbon atoms for gluconeogenesis. The newly formed glucose can then enter the blood for delivery back to the muscle. The amino group transported from the muscle to the liver in the form of alanine is converted to urea in the urea cycle and excreted.
All of the amino acids present in proteins, 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. Of all the amino acids utilized for gluconeogenesis, glutamine is the most important as this amino acid is critical for glucose production by the kidneys and small intestine.
Glutamine is the sole source of carbon atom for the gluconeogenesis pathway carried out in the kidney and the small intestine. In these two tissues, glutamine is first deaminated to glutamate via the action of glutaminase. The glutamate is then further deaminated, via the action of the enzyme glutamate dehydrogenase, yielding 2-oxoglutarate (α-ketoglutarate). The 2-oxoglutarate can then enter the TCA cycle where it is eventually converted to malate. As described earlier, malate can be transported out of the mitochondria and oxidized to oxaloacetate via the action of cytoplasmic malate dehydrogenase. The oxaloacetate is then converted to PEP via the action of the cytoplasmic version of phosphoenolpyruvate carboxykinase (PEPCK-c). Alternatively, malate can be oxidized to oxaloacetate within the mitochondria then the action of mitochondrial PEPCK (PEPCK-m) can convert the oxaloacetate to PEP. If this pathway is utilized the PEP is transported to the cytosol for gluconeogenesis.
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 β-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. However, the glycerol backbone that is released from adipocytes following hormone-induced triglyceride breakdown can be used for gluconeogenesis. This requires phosphorylation of the glycerol to glycerol-3-phosphate by glycerol kinase within hepatocytes. Following formation of glycerol-3-phosphate it is oxidized to dihydroxyacetone phosphate (DHAP) by cytosolic glycerol-3-phosphate dehydrogenase 1 (GPD1). The glycerol backbone of adipose tissue stored triacylglycerides is ensured of being used as a gluconeogenic substrate by the liver since adipocytes 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 triacylglycerides. The GPD1 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 phosphate shuttle. This shuttle is a secondary mechanism for the transport of electrons from cytosolic NADH to mitochondrial carriers of the oxidative phosphorylation pathway. The primary cytoplasmic NADH electron shuttle is the malate-aspartate shuttle. Two enzymes are involved in this shuttle. One is the cytosolic version of GPD (GPD1) which utilizes NADH as a co-enzyme. The second is the mitochondrial form of the enzyme (GPD2) which utilizes FAD+ as co-enzyme. The net result is that there is a continual conversion of the glycolytic intermediate, DHAP and glycerol-3-phosphate with the concomitant transfer of the electrons from reduced cytosolic NADH to mitochondrial oxidized FAD+. Since the electrons from mitochondrial FADH2 feed into the oxidative phosphorylation pathway at coenzyme Q (as opposed to NADH-ubiquinone oxidoreductase [complex I]) only 2 moles of ATP will be generated from glycolysis. GAPDH is glyceraldehyde-3-phoshate dehydrogenase.
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.
Propionyl-CoA carboxylase functions as a heterododecameric enzyme (subunit composition: α6β6) and the two different subunits are encoded by the PCCA and PCCB genes respectively. The PCCA gene is located on chromosome 13q32 and is composed of 27 exons that generates three alternatively spliced mRNAs. The PCCB gene is located on 3q21–q22 and is composed of 17 exons that generate two alternatively spliced mRNAs. Methylmalonyl-CoA epimerase is encoded by the MCEE gene located on chromosome 2p13.3 and is composed of 4 exons that encode a 176 amino acid protein. Methylmalonyl-CoA mutase is encoded by the MUT gene located on chromosome 6p12.3 and is composed of 13 exons that encode a protein of 750 amino acids. Mutations in the MUT gene are one cause of the methylmalonic acidemias. Mutations in either the PCCA or PCCB gene are associated with propionic acidemia associated with severe ketoacidosis. The original identification of a child suffering from propionyl-CoA deficiency was in 1961. This child suffered frequent episodes of severe ketoacidosis, all of which were precipitated by protein ingestion. Blood and urine analysis demonstrated marked elevations in glycine levels. These initial laboratory studies lead to the disorder being called ketotic hyperglycinemia. However, there is no defect in glycine metabolism with inherited mutations in PCCA or PCCB. The clinical hallmark of the disease is severe ketoacidosis of an episodic nature.back to the top
The gut, in particular the small intestine, plays a critical role in the uptake and delivery of glucose from the diet. As such, the gut plays a central role in the overall regulation of glucose homeostasis. Glucose uptake from the lumen of the gut and trans-epithelial transport to the portal circulation had been shown to occur via action of two distinct glucose transporters. First, glucose is taken up from the intestinal lumen through the action of the sodium-dependent glucose transporter-1 (SGLT-1) then it is transported into the portal blood via the action of the facilitated glucose transporter GLUT2 present in the basolateral membrane. Evidence has also indicated that GLUT2 present in the apical (luminal) membrane of enterocytes was involved in glucose uptake. However, GLUT2 is not present in the apical membrane in the absence of a glucose load. The mechanism of GLUT2 presentation in the apical membrane involves a glucose-induced translocation of GLUT2 to this membrane. Thus, glucose uptake by the small intestine enhances additional uptake by promoting presentation of an additional transporter in the apical membrane.
The small intestine also utilizes glucose, obtained from the diet or from the blood, for energy production. Recently it was shown that the intestine is able to utilize glutamine for energy with the same efficiency as glucose. Indeed, glutamine has been considered to be a major energy substrate for this organ. Of additional, significance, and only recently having been determined, is the role of intestinal gluconeogenesis in overall endogenous glucose production (EGP). A little over 10 years ago, molecular analysis allowed for the characterization of the expression of glucose-6-phosphatase (G6Pase) within enterocytes of the small intestine. Expression of G6Pase thus, confers upon the intestine, the ability to carry out gluconeogenesis. Now it is known that glutamine serves as the major precursor of glucose formed within the small intestine. The genes for both G6Pase and the cytosolic form of phosphoenolpyruvate carboxykinase (PEPCK-c) are controlled by insulin in the small intestine similarly to the regulation of these genes in the liver. The presence of G6Pase within the small intestine also plays a role in the export of glucose to the portal circulation. This can either be dietary glucose, glucose released from intestinal glycogen stores, or glucose produced via gluconeogenesis. This glucose export mechanism is dependent on the previous phosphorylation of glucose by hexokinases followed by G6Pase-mediated dephosphorylation.
Pathways of gluconeogenesis in the small intestine and coupling to gluconeogenic substrate delivery to the liver. Glucose and glutamine arrive in intestinal enterocytes either from the diet or the arterial blood supply as depicted. The carbon atoms of glutamine serve as the major substrate for intestinal gluconeogenesis via the two-step process catalyzed by glutaminase and alanine transaminase (ALT). The resultant 2-oxoglutarate (α-ketoglutarate) is converted to oxaloacetate (OAA) and then to phosphoenolpyruvate (PEP) which is then diverted into the gluconeogenic pathway. Glucose that enters the enterocyte can be oxidized to pyruvate via glycolysis and then the carbons of pyruvate can be reduced to lactate or transaminated to alanine, both of which can serve as major gluconeogenic substrate in the liver following delivery via the portal circulation. The contributions of intestinal glycerol and glucose from glycogen to the role of the intestine ion overall glucose homeostasis is also depicted. GPD is glycerol-3-phosphate dehydrogenase. PGM is phosphoglycerate mutase. G6P is glucose-6-phosphate. G3P is glyceraldehyde-3-phosphate. LDH is lactate dehydrogenase.
The importance of intestinal gluconeogenesis, to overall EGP, has been demonstrated both in experimental animals (mice with specific knockout of PEPCK-c in the liver) and in humans in the anhepatic phase during liver transplantation. In mice without hepatic PEPCK-c there is an efficient adaptation to fasting conditions such that blood glucose levels decrease by only 30%. In addition, in these mice, and humans undergoing liver transplant, there occurs a significant increase in plasma glutamine concentration. These observations stressed the likely role of the kidney and/or intestine in glucose production, because glutamine is a major glucose precursor in the kidney and the small intestine, but not in the liver. The role of the intestine in this glucose control was demonstrated by the fact that in these experimental conditions there is no observable difference in glucose concentration between arterial and portal blood.
During periods of fasting the small intestine accounts for approximately 20% of EGP by 48hrs and up to 35% by 72hrs. However, expression of the key gluconeogenic genes, G6Pase and PEPCK-c, is dependent on plasma insulin concentrations, and these do not change throughout these time frames of fasting. Yet expression of both of these genes is seen to increase within intestinal cells between 24 hrs and 48 hrs of the initiation of fasting. The promoter region of the G6Pase gene (G6PC), containing the presumed TATA box (TATAAAA, located -31 to -25 bp upstream of the transcription start site), also constitutes a putative binding site for transcription factors of the caudal-related homeobox (CDX) family. In adult mammals, the CDX genes are exclusively expressed in the gut, where they are involved in the differentiation of both the crypt-villus and anteroposterior axis. The TATAAAA element in the G6PC gene is indeed responsible for its transactivation by CDX1 (but not CDX2) since disruption of the sequence strongly blunts basal transcription and CDX1 transactivation of the gene.
Protein-rich diets are known to reduce hunger and subsequent food intake in both humans and experimental animals. In addition, protein-rich, carbohydrate-free diets have been shown to strongly induce the expression of G6Pase, PEPCK-c, and glutaminase in the intestine. In addition, the gut releases glucose to the portal circulation following the intake of a protein-rich, carbohydrate-free diet. The rate of glucose release by the gut can be estimated to provide about 15% to 20% of EGP in protein-fed experimental animals. Significantly, this level of glucose release from the gut is sufficient to account for the level of reduction in food intake observed in protein-fed animals, where an equivalent infusion of glucose into the portal vein of the control animals also decreased food intake and by a comparable value. Although this glucose, derived by intestinal gluconeogenesis, does not increase overall EGP this is because the liver adapts by decreasing its own level of gluconeogenesis while also increasing glycogen storage.
That intestinal gluconeogenesis is indeed crucial in the control of food intake by dietary protein was established with the use mice in which expression of the G6Pase gene was specifically and conditionally abolished in the intestine. When these mice are fed a protein-rich, carbohydrate-free diet they do not exhibit a decrease in their level of food intake such as is seen in control mice on the same diet. The same loss of satiety induction by protein-rich diets or portal glucose infusion is seen in animals whose portal vein afferent nerve connections are chemically or surgically destroyed. Afferent nerves send signals from body locations to the brain. These types of studies demonstrate that portal sensing of intestinal gluconeogenesis is a key mechanism in the satiety effect induced by dietary protein. Brain areas involved in the control of feeding behaviors include the brain stem and the hypothalamus. For a detailed discussion of the role of the hypothalamus in the control of feeding behaviors visit the Gut-Brain Interactions page. In experimental animals fed protein-rich diets or who have had glucose infusions into the portal vein, neuronal activation is observed in several hypothalamic nuclei involved in feeding behavior regulation including the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial nucleus (VMN), and paraventricular nucleus (PVN). In similar experiments in animals whose gut afferent circuits have been destroyed, there is no increase in neuronal activity following portal vein glucose infusion or consumption of protein-rich diets. In addition to the effects, on feeding behavior, of intestinal glucose (via gluconeogenesis) delivery to the portal circulation, numerous gut hormones are known to be involved in the control of hunger sensations and do so, in part, via gastrointestinal afferent circuits. The hunger-modulating effects initiated by the release of meal-dependent gut hormones, including cholecystokinin (CKK), glucagon-like peptide-1 (GLP-1), and PYY3-36, are all strongly attenuated by disrupting nerve circuitry between the gastrointestinal and central nervous systems.back to the top
Although the liver has the critical role of maintaining blood glucose homeostasis and therefore, is the major site of gluconeogenesis, the kidney plays an important role. During periods of severe hypoglycemia that occur under conditions of hepatic failure, the kidney can provide glucose to the blood via renal gluconeogenesis. In the renal cortex, glutamine is the preferred substance for gluconeogenesis.
Glutamine is produced in high amounts by skeletal muscle during periods of fasting as a means to export the waste nitrogen resulting from amino acid catabolism. Through the actions of transaminases, a mole of waste ammonia is transferred to 2-oxoglutarate (α-ketoglutarate) via the glutamate dehydrogenase catalyzed reaction yielding glutamate. Glutamate is then a substrate for glutamine synthetase which incorporates another mole of waste ammonia generating glutamine (see the Nitrogen Metabolism page for more details). The glutamine is then transported to the kidneys where the reverse reactions occur liberating the ammonia and producing 2-oxoglutarate which can enter the TCA cycle and the carbon atoms diverted to gluconeogenesis via oxaloacetate. This process serves two important functions. The ammonia (NH3) that is liberated spontaneously ionizes to ammonium ion (NH4+) and is excreted in the urine effectively buffering the acids in the urine. In addition, the glucose that is produced via gluconeogenesis can provide the brain with critically needed energy.back to the top
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. Green arrows indicate positive actions. Red T-lines represent inhibitory actions.
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. Activation of PC occurs through interaction with acetyl-CoA. Indeed, PC is catalytically inactive in the absence of acetyl-CoA. This fact defines the role of acetyl-CoA as an obligate activator of PC. These regulations occur 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.
Whereas glucagon actions results in increased levels of cAMP and subsequent activation of gluconeogenesis, insulin action exerts the opposite effect. The mechanisms by which insulin turns off gluconeogenesis are complex. Reduction in the level of cAMP is exerted via the insulin-mediated activation of phosphodiesterase (PDE3B) which hydrolyzes cAMP to AMP. At the level of the regulation of genes involved in gluconeogenesis, cAMP signaling leads to phosphorylation of the transcription factor CREB at Ser133. When phospho-CREB binds to the cAMP response element (CRE) of a target gene it results in the recruitment of the coactivators CBP and p300 (which are closely related). This complex activates gene expression through their intrinsic histone acetyltransferase activity and through recruitment of other coactivator molecules. The coactivator CBP is a target of insulin-dependent phosphorylation at Ser436. This residue, which is adjacent to the CREB-binding domain (CREB-BD), is phosphorylated by a phosphoinositol-3-kinase (PI3K)−dependent insulin signaling pathway, and is not conserved in the related cofactor p300.
The transcriptional coactivator PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) functions as a central regulator of gluconeogenesis by binding to several factors, including the glucocorticoid receptor, hepatic nuclear factor 4α (HNF4α) and a forkhead transcription factor (FOXO1) upstream of several genes encoding gluconeogenic enzymes. One mechanism by which insulin signaling antagonizes gluconeogenesis is through phosphorylation of FOXO1 and its subsequent exclusion from the nucleus. Expression of PGC-1α, however, is also regulated by cAMP through a well-defined CRE present in the promoter region of the PGC-1α gene.back to the top