Lecture 8: Gluconeogenesis in Detail

Learning Outcomes:

• Define the key steps involved in the conversion of 3-carbon intermediates to 6-carbon intermediates in gluconeogenesis

• Review the steps involved in the release of glucose from cytosolic glucose 6-phosphate

• Analyse the effects of the absence of the 2-OH group in glucose and generate rationales for the use of 2-deoxy derivatives in clinical diagnostics

• Predict the flow of glycolysis and gluconeogenesis based on the relative activities of phosphofructokinase and fructose 1,6 bisphosphatase

• Compare the regulators of phosphofructokinase and fructose 1,6 bisphosphatase

• Outline the reasons to produce fructose 2,6 bisphosphate

• Apply knowledge of the effects of fructose 2,6 bisphosphate to the regulation of PFK and F16BPase

• Illustrate the dual identity of PFK/F26BPase through phosphorylation changes

• Outline the consequences of changes in PFK/F26BPase activity

• Compare the conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis to the opposite process in glycolysis

• Predict the effect of fatty acid oxidation on glucose fluxes as a consequence of its effects on pyruvate carboxylase

• Evaluate the importance of anaplerosis of Krebs Cycle intermediates

Getting from 3C to 6C:

• Require phosphatase rather than kinase

• Don’t get the ATP back that we invested – you do in glycolysis

• Phosphatases are named by substrate

  • Fructose 1,6-biphosphatase

  • Glucose 6-phosphatase

  • Phosphatases remove phosphate groups

• Double headed arrow indicate equilibrium reactions – don’t need enzymes to go back either way

Using the ATP:

• Need to bypass glycolytic energy releasing steps

• Need two enzymes to convert pyruvate to phosphophenolpyruvate

• Pyruvate carboxylase (PC) is mitochondrial

• PEP carboxykinase (PEPCK) varies

• Pyruvate → Oxaloacetate (via PC)

• Oxaloacetate → phosphophenolpyruvate (via PEPCK)

• Gluconeogenisis mostly happens in the liver, but these enzymes exist in other tissues as well

• PC exists in most tissues

• Pyruvate is a 3C molecule

• Oxaloacetate is a 4C molecule

  • First carrier in the Krebs cycle with Acetyl-CoA

• PEPCK uses GTP instead of ATP but it does the same thing – gives a phosphate

G6Pase System – Only in Liver:

• In the liver, to get glucose 6-phosphate back to glucose is in the endoplasmic reticulum

• Glucose can make its way out back into the blood stream via GLUT 2 transporters

  • GLUT2 allows equilibrium to be established

• G6P transporter allows G6P into the ER

G6Pase is targeted in diagnostics:

• To diagnose cancer

• Glucose molecule with a radioactive fluorine

• Cells will treat the 2-fluro-deoxyglucose like any other glucose and transport it into the cell

• They will trap it by phosphorylating it with hexokinase

• The molecule can’t continue journey down glycolysis to fructose-6-phosphate

• It gets trapped and accumulates in the cell/cytoplasm

• Cancer cells rely heavily on glucose to lactate metabolism – only getting 2 ATPs

• Radio labelling the glucose can allow screening (full body PET Scan) – high concentration in cancer areas

• Ignore brain (uses a lot of glucose) and bladder (getting rid of unusable glucose)

Reversing the PFK Step of Glycolysis:

• Cells need to decide if they are going to break down or store glucose – complicated

• Cell needs to regulate if they use PFK-1 or FBPase-1

• Controlled through allosteric regulators:

  • Increase ATP negatively regulates PFK-1 to down regulate glycolysis

  • Increase in ADP positively regulates PFK-1 to upregulate glycolysis

  • Increase in AMP can switch on gluconeogenisis and switch ofd PFK-1

  • Increase in citrate negatively regulates PFK-1to down regulate glycolysis

Completely Different Pathway to Glycolysis:

• Fructose 6-phosphate to Fructose 2,6-bisphosphate using PFK-2

• Opposite way using FBPase-2

• Fructose 2,6-bisphosphate (F26BP) is a regulatory molecule and it can only get phosphorylated or dephosphorylated in the loop

• Fructose 2,6-bisphosphate is only formed in small amounts but is more powerful in regulating glycolysis than anything also

• Works at the same step using PFK-1 and FBPase-1

  • Fructose 6-phosphate to Fructose 1,6-biphosphate (and reverse for gluconeogenisis)

• Adding F26BP to the factors that control whether a cell does glycolysis or gluconeogenisis

  • Increase in F26BP upregulates PFK-1and glycolysis

  • Increase in F26BP downregulates FBPase-1 and gluconeogenisis

  • The most powerful regulator

• PFK-2 and FBPase-2 are the same enzyme

  • Swapping from one to another after reversible phosphorylation

  • Insulin and glucagon determine which parts of the enzyme is active

  • Interversion catalysed by Protein Kinase A (PKA)

    • Sensitive to cAMP

    • And therefore, glucagon/insulin

  • PKA can phosphorylate the enzyme in the presence of glucagon and activate the FBPase-2 which inhibits glycolysis and stimulates gluconeogenisis

  • PKA can activate the phosphatase and remove the phosphate group in the presence of insulin to stimulate glycolysis and inhibit gluconeogenisis

When F26BP is high, glycolysis is favours

When F26BP is low, gluconeogenisis is favoured

Summary:

Reversing the PEP to Pyruvate – Early Steps of Gluconeogenisis:

• Requires 2 ATP consuming steps

• Pyruvate carboxylase – stimulated by acetyl-CoA (fatty acid oxidation)

  • Want to create more glucose

  • Also creates oxaloacetate which can be used in the Krebs cycle

• PEPCK (Pepcarboxykinase)

  • Stimulated by increased transcription/translation of the gene

Synthesis of PEP from Pyruvate – reverse last step of glycolysis:

• Bicarbonate + pyruvate

  • Pyruvate carboxylase takes CO2 off of bicarbonate, with ATP as a cofactor and adds it to the pyruvate to make oxaloacetate

• Oxaloacetate - CO2

  • PEP carboxykinase removes the CO2 and adds a phosphate to generate phosphoenolpyruvate (PEP)

    

Fatty Acid Oxidation and Pyruvate Carboxylase:

• Acetyl CoA levels high when beta oxidation is prominent

• This inhibits PDH

  • Prevents wasteful oxidation of glucose

• That same acetyl coa activates pyruvate carboxylase (first step in gluconeogenis)

• This forms more oxaloacetate

  • This can be used in gluconeogenisis or put back into the krebs cycle

• Need to replace oxaloacetate because if we kept using carbons from the kreb cycle for gluconeogenisis, eventually we would run out of carbons and oxaloacetate for the krebs cycle

• Thats why carbon skeletons burned from amino acids

• And why build up of acetyl-CoA doesn’t just turn off pyruvate dehydrogenase (PDH) but also switches on pyruvate carboxylase to turn pyruvate into oxaloacetate

Anaplerosis – replenishing an intermediate that have been extracted for biosynthesis

• Oxaloacetate going back into the krebs cycle is called anaplerotic

• Because we’ve been stealing intermediates from the krebs cycle to make more glucose