The following is a tutorial essay written for the Cellular Biochemistry module – the writing style is pitched at university-level but can also be followed by A Level biology students.
If you’re curious about the intricacies of glucose synthesis in the liver or just want to see what an Oxford biochemical tutorial essay looks like – read on!
The glucose-alanine cycle, also known as the Cahill cycle, describes the metabolic pathway in which extrahepatic tissue (notably, the skeletal muscle) exports alanine to the liver, alanine is used as a substrate in gluconeogenesis, and nascent glucose is exported back from the liver to the other tissues.
Alanine (an amino acid) from broken down protein in our muscles is transported to the liver to be converted into glucose (gluconeogenesis) and recirculated to the muscles to fuel exercise 
The gluconeogenic pathway of the Cahill Cycle with irreversible, regulated steps highlighted 
Cortisol, glucagon, and insulin regulate the gluconeogenesis pathway
ALANINE –> PYRUVATE
Under extreme catabolic conditions, such as fasting, intracellular protein hydrolysis rate exceeds the rate of its resynthesis – resulting in the liberation of free amino acids which can either be oxidised for energy (in the case of the branched chain amino acids, leucine, isoleucine, and valine) or transported out of skeletal muscle into the bloodstream for gluconeogenesis in the liver.
In the liver, alanine is first deaminated in a transamination reaction, catalysed by hepatic alanine aminotransferase, in which α-ketoglutarate acts as an amino group acceptor. Overall, this reaction is
Alanine + α-Ketoglutarate ⇄ Glutamate + Pyruvate.
Aminotransferases are cytosolic enzymes, particularly abundant in liver cells, which require the coenzyme pyridoxal phosphate (PLP) to be bound to the active site. Transamination of alanine produces pyruvate and glutamate. Glutamate can then be deaminated by glutamate dehydrogenase to reform α-ketoglutarate which can enter the Krebs cycle; this anaplerotic reaction also forms NH4+ which enters the urea cycle to be safely excreted.
PYRUVATE –> OXALOACETATE
The pyruvate can leave the Cahill cycle at this point and be oxidised for ATP production; in periods of fasting, however, it is energetically favourable to remain in the cycle. The conversion of pyruvate into oxaloacetate, catalysed by pyruvate carboxylase, is the next step in the Cahill cycle. The metabolic pathway encounters a physical barrier here since the necessary enzyme is localised in the mitochondrial matrix. The mitochondrial pyruvate carrier (MPC) complex, consisting of dimerised subunits MPC-1 and MPC-2, translocates pyruvate into the mitochondria.
Expression of MPC is under hormonal regulation; increase in circulating pancreatic glucagon during fasting upregulates expression of MPC on mitochondrial outer membranes. Glucagon binds to G protein-coupled receptors and activates adenylate cyclase which increases cAMP production. cAMP phosphorylates PKA which in turn activates the cAMP responsive element binding protein (CREB). CREB, a transcription factor, directly stimulates gluconeogenesis by binding the promoter of the MPC1 and MPC2 genes as well as the promoters of important enzymes involved later in the glucose-alanine cycle. This long-term regulation promotes gluconeogenesis during extended periods of fasting in which blood glucagon concentration is consistently high.
Once in the mitochondria, pyruvate is converted to oxaloacetate by pyruvate carboxylase. The enzyme requires a covalently-bonded prosthetic group, biotin, to act as a carrier of activated CO2. Pyruvate carboxylase consists of four identical subunits each made of four domains: a biotin carboxylase (BC) domain, a biotin carboxyl carrier protein (BCCP), a pyruvate carboxylase (PC) domain, and a PT domain which stabilises the tetramer
First, carbonic acid reacts with ATP to produce activated CO2. This CO2 is then transferred to the biotin attached to the BCCP subunit, releasing a orthophosphate molecule. Finally, the CO2 is transferred to a pyruvate molecule (catalysed in the active site of the PC subunit) to form oxaloacetate.
This is the first irreversible, committed step of the gluconeogenesis pathway and, hence, is a key step for regulation. The energy charge of the cell plays a crucial role in activating pyruvate carboxylase – biotin cannot be carboxylated unless the allosteric regulator acetyl CoA is bound to the enzyme. If the energy charge in the liver cell is low, acetyl CoA concentration will be low and pyruvate will instead be decarboxylated to enter the TCA cycle, inhibiting gluconeogenesis. If acetyl CoA concentration is high, phosphate dehydrogenase (a glycolytic enzyme) is inhibited and pyruvate carboxylase activity is stimulated.
OXALOACETATE –> PHOSPHOENOLPYRUVATE (PEP)
The enzyme that catalyses the conversion of oxaloacetate to PEP is only present in the cytoplasm so the tricarboxylic acid must first be transported out of the mitochondria. The inner mitochondrial membrane is impermeable to oxaloacetate but not to malate; reduction to malate via malate dehydrogenase allows the molecule to be transported by the malate- α-ketoglutarate transporter. In the cytoplasm, malate is then oxidised by cytosolic malate dehydrogenase to reform oxaloacetate.
The malate shuttle serves another important purpose as it enables the transport of reducing equivalents across the outer mitochondrial membrane (which is impermeable to NADH). When malate is converted back to oxaloacetate, cytoplasmic NADH is formed which reduces substrates later in the gluconeogenic pathway.
Oxaloacetate is simultaneously decarboxylated and phosphorylated (using GTP) by phosphoenolpyruvate carboxykinase (PEPCK) to produce PEP. Metal ions, Mn2+ and Mg2+ act as cofactors and stabilise the several negative charges interacting in this reaction.
Cytosolic PEPCK gene expression is hormonally regulated by glucagon via cAMP (as described earlier) as well as by cortisol. This hormone diffuses directly through the phospholipid bilayer of hepatic cells and binds to the glucocorticoid receptor (GR) to form a cortisol-GR complex. The complex then translocates into the nucleus and binds the Glucocorticoid Response Element upstream of the PEPCK gene – activating it. Insulin, on the other hand, downregulates PEPCK expression – inhibiting gluconeogenesis.
By carboxylating and subsequently decarboxylating pyruvate, the phosphorylation of the substrate (typically a very endergonic reaction) is made more favourable. Direct addition of a phosphoryl group has a high ΔG ̊of +31 kJmol-1, making the reverse dephosphorylation reaction irreversible. Decarboxylation drives the gluconeogenic pathway forwards by bypassing this unfavourable step with a much lower ΔG ̊of +0.8 kJmol-1.
PEP à FRUCTOSE-1,6-BISPHOSPHATE
The six following reactions are an exact reversal of the glycolytic pathway and are catalysed by glycolysis enzymes; this is possible as all these reactions are reversible and near equilibrium in hepatic cells – if the metabolite concentrations favour gluconeogenesis, PEP will be converted to fructose-1,6-bisphosphate via:
Phosphoenolpyruvate + H2O ⇌ 2-phosphoglycerate
2-Phosphoglycerate ⇌ 3-phosphoglycerate
3-Phosphoglycerate + ATP ⇌ 1,3-bisphosphoglycerate + ADP
1,3-Bisphosphoglycerate + NADH + H+ ⇌ glyceraldehyde 3-phosphate + NAD+ + Pi
Glyceraldehyde 3-phosphate ⇌ dihydroxyacetone phosphate
Glyceraldehyde 3-phosphate + dihydroxyacetone phosphate ⇌ fructose 1,6-bisphosphate
FRUCTOSE-1,6-BISPHOSPHATE –> FRUCTOSE-6-PHOSPHATE
The dephosphorylation of F16BP to F6P, mediated by fructose-1,6-phosphatase (FBPase) and Mg2+ ion cofactors, bypasses another irreversible step of the glycolytic pathway and is a key point of regulation in the gluconeogenic pathway. FBPase is a tetrameric enzyme with an AMP binding site as well as an active site; fructose-2,6-phosphate competitively inhibits FBPase by binding the active site and sterically inhibiting the substrate F16BP from binding whereas AMP inhibits FBPase by binding an allosteric site.
F26BP is an allosteric effector molecule that is not an intermediate of either pathway but regulates both glycolysis and gluconeogenesis. Intracellular concentration of F26BP is under hormonal control; glucagon decreases the concentration whereas insulin increases it. Glucagon stimulates cAMP which activates PKA which phosphorylates a serine residue in the active site of the bi-enzyme PFK-2/FBPase-2. Phosphorylation increases the phosphatase activity of the enzyme, converting F26BP to F6P, so the effector is unable to bind and inhibit FBPase – stimulating gluconeogenesis. Insulin, on the other hand, binds to specific membrane receptors and activates phosphoprotein phosphatase 2A (PP2A) which catalyses the dephosphorylation of PFK-2/FBPase-2. This allows the bi-enzyme to act as a kinase and increase F26BP concentration – inhibiting gluconeogenesis.
AMP acts as a non-competitive inhibitor; upon AMP ligation, hydrogen bonds holding the FBPase tetramer together are disrupted causing a 15 ̊ to 17 ̊ rotation of the upper dimer relative to the lower dimer which converts the enzyme from the R state to the inactive T state.
FBPase regulation plays a crucial role in survival for obligate hibernators. During hibernation, glycolysis must be prioritised so hepatic gluconeogenesis is inhibited. Respiration rate dramatically decreases, creating relatively anoxic conditions in liver tissue which inhibits FBPase by making it more sensitive to allosteric inhibitors (ex. AMP, ADP, Pi) and F26BP. FBPase affinity for its substrate also decreases through covalent modification; for instance, hibernating bats show a 75% decrease in KM compared to euthermic bats.
GLUCOSE-6-PHOSPHATE –> GLUCOSE
Fructose-6-phosphate is readily converted to glucose-6-phosphate by the same glycolytic enzyme that catalyses the reverse reaction since the reaction is maintained close to equilibrium.
Standard gluconeogenesis terminate here in most tissues since glucose-6-phosphate is unable to readily diffuse out of cells and can be converted to glycogen for faster mobilisation during aerobic respiration. However, the interorgan glucose-alanine cycle is unique in its production of free glucose which is transported out of the liver cells and recirculated to the skeletal muscle. This dephosphorylation requires a third bypass of an irreversible step of the glycolytic pathway; glucose-6-phosphatase, a protein complex embedded in the ER, catalyses the reaction instead of hexokinase/glucokinase. Only tissues that maintain blood-glucose homeostasis, such as the liver and kidney, contain these G6Pase complexes.
SUMMARY
The overall reaction for the conversion of alanine to glucose is:
2 Alanine + 10 ATP + CO2 ⇌ Glucose + urea + 10 ADP + 10 Pi
The alanine-glucose Cahill cycle repurposes carbon skeletons of the liberated amino acid and provides a means of safely exporting and processing amino groups (via the linked Urea cycle) whilst ensuring that the liver bears the energetic burden of gluconeogenesis instead of actively respiring skeletal muscle tissue.
The three main points of regulation in gluconeogenesis:
- pyruvate –> oxaloacetate
- oxaloacetate –> PEP
- F16BP –> F6P
correspond with irreversible steps in the glycolytic pathway. The main forms of regulation are hormonal control via antagonistic glucagon/cortisol and insulin as well as allosteric regulation by acetyl CoA and AMP.
BIBLIOGRAPHY
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Curiosity Killed The Cation 