Glycolysis & Metabolism Key Steps

• Gluconeogenesis is the synthesis of glucose or glycogen from non-carbohydrate compounds like lactate, glycerol, and amino acids (excluding leucine and lysine).
• Occurs mainly in the liver and kidneys, inside the cytoplasm and mitochondria.
• The liver contributes 90% of gluconeogenesis during an overnight fast, while the kidneys contribute 10%.
• During prolonged fasting, the kidneys become major glucose-producing organs, contributing 40% of the total.
• Gluconeogenesis is important for organs that require a continuous glucose supply, such as the brain, red blood cells, kidneys, and exercising muscle.
• The process involves glycolysis, citric acid cycle, and some special reactions.
• Glycolysis and gluconeogenesis share the same pathway but in opposite directions and are reciprocally regulated.
• Three irreversible reactions in glycolysis prevent simple reversal for gluconeogenesis and these include: glucokinase, phosphofructokinase-1, and pyruvate kinase
• The major fuel for most tissues is glucose derived from diet.
• The pathway of glycolysis further converts glucose to pyruvate aerobically or lactate anaerobically.
• Glucose can be reduced to acetyl-CoA, is a precursor of fatty acids and cholesterol.

Gluconeogenic Precursors

• Gluconeogenic precursors are molecules used for net glucose synthesis.
• The most important gluconeogenic precursors include glycerol, lactate, and α-keto acids which are derived from glucogenic amino acids.
• Almost all amino acids are glucogenic except leucine and lysine.
• Glycerol released during triacylglycerol hydrolysis in adipose tissue, is delivered to the liver via the blood, and becomes glucose.
• Glycerol is phosphorylated to glycerol 3-phosphate by glycerol kinase using 1 ATP.
• Glycerol 3-phosphate, oxidized by glycerol 3-phosphate dehydrogenase, results in dihydroxyacetone phosphate (DHAP), an intermediate for both glycolysis and gluconeogenesis.
• DHAP then becomes fructose 1,6-bisphosphate, converted to fructose phosphate via fructose 1,6 bisphosphatase enzyme.

Metabolic Processes

• Acetyl-CoA enters the citric acid cycle, which is important for ATP generation in oxidative phosphorylation.
• Glycogenesis: the process may also undergo glycogen synthesis and acts as a storage polymer in the skeletal muscle and liver
• The pentose phosphate pathway provides reducing equivalents (NADPH) for fatty acid synthesis, and ribose for nucleotide and nucleic acid synthesis.
• Pyruvate and citric acid cycle intermediates provide skeletons for amino acid synthesis.

Cori Cycle

• The Cori Cycle involves glucose uptake in tissues, conversion to lactate via glycolysis, lactate release into circulation, lactate uptake by the liver, and conversion back to glucose via gluconeogenesis.

Glucose-Alanine Cycle

• Occurs during fasting and maintains blood glucose levels.
• Provides an indirect mechanism for utilizing muscle glycogen to sustain blood glucose during fasting.
• During fasting, skeletal muscles release excess alanine, formed by transamination of pyruvate from muscle glycogen glycolysis and is exported to the liver.
• In the liver, alanine undergoes transamination back to pyruvate, serving as a substrate for gluconeogenesis, where amino acids provide carbon.
• Nitrogen from amino acids is converted to urea.
• Alanine, the major gluconeogenic amino acid, and the ATP needed for hepatic gluconeogenesis, is derived from fatty acid oxidation.

Amino Acids

• Amino acids, from tissue proteins, are major sources of glucose during fasting.
• Their breakdown produces α-keto acids like pyruvate (converted into glucose), and α-ketoglutarate which enters the TCA cycle to form oxaloacetate (OAA).
• OAA is a key precursor for phosphoenolpyruvate (PEP) in glucose production.
• Acetyl CoA and compounds giving rise only to acetyl CoA cannot give rise to a net glucose synthesis due to the irreversible nature of pyruvate dehydrogenase complex
• The pyruvate dehydrogenase complex, converts pyruvate to acetyl CoA
• They instead give rise to ketone bodies (ketogenic), like acetoacetate, lysine, and leucine

Regulation of Gluconeogenesis

• Fructose 2,6-bisphosphate is elevated when glucose is elevated, therefore regulates glycolysis and gluconeogenesis in the liver
• Fructose 2,6-bisphosphate is a potent positive allosteric activator/stimulator of phosphofructokinase-1(PFK-1) in the liver, also the most potent inhibitor of fructose 1,6 bisphosphate in the liver, and relieves inhibition of phosphofructokinase-1 by ATP
• Concentration is under both substrate (allosteric) and hormonal control (covalent modification).
• Phosphofructokinase-2 functions include two activities: the kinase activity phosphorylates fructose 6-phosphate forming Fructose 2,6-bisphosphate, and the phosphatase activity breaks down Fructose 2,6-bisphosphate back to fructose 6-phosphate.
• Both functions are controlled by fructose-6-phosphate, stimulating the kinase and inhibiting the phosphatase.
• Concentration is under both substrate (allosteric) and from hormonal control from covalent modification.
• During a the feeding state (when glucose is abundant) the concentration of fructose 2,6-bisphosphate increases, stimulating glycolysis by activating phosphofructokinase-1 and inhibiting fructose 1,6-bisphosphatase.
• Fructose 1,6-bisphosphatase inhibited by a compound of AMP, that activates phosphofructokinase-1, which results in reciprocal regulation of glycolysis and gluconeogenesis.
• In the the fasting state, glucagon stimulates cAMP production, activating cAMP-dependent protein kinase.
• cAMP-dependent protein kinase inactivates phosphofructokinase-2 and activates fructose 2-6 bisphosphate by phosphorylation.
• Gluconeogenesis is stimulated by a decrease in fructose 2,6-bisphosphate concentration.
• If glucose level decreases, fructose 2,6-bisphosphate also decreases, so PFK-1 will not be stimulated, which results in gluconeogenesis.

Glycolysis and Gluconeogenesis

• Gluconeogenesis in the liver and kidney uses reversible glycolysis reactions, along with additional reactions to bypass non-equilibrium steps.
• Non-equilibrium reactions are steps 1, 3, and 10, catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase, preventing simple glycolysis reversal for glucose synthesis.

Triglycerides/Triacylglycerol

• Lipolysis breaks down triacylglycerol into its two main components: glycerol and fatty acids.

Glycolysis Steps

• Step 1: Glycerol phosphorylation by Glycerol kinase converts Glycerol into Glycerol-3-phosphate
• Step 2: Glycerol-3-phosphate oxidation by Glycerol-3-phosphate dehydrogenase converts Glycerol-3-phosphate into Dihydroxyacetone phosphate (DHAP)
• Step 3: Dihydroxyacetone phosphate isomerization by Triosephosphate isomerase converts Dihydroxyacetone phosphate into Glycerol-3-phosphate (G3P)
• Step 4: Aldolase reaction by Aldolase converts 2 molecules of G3P into Fructose-1,6-bisphosphate (F1,6BP)
• Step 5: Dephosphorylation using Fructose-1,6-bisphosphatase converts Fructose-1,6-bisphosphate into Fructose-6-phosphate
• Step 6: Isomerization using Phosphoglucose isomerase converts Fructose-6-phosphate into Glucose-6-phosphatase
• Step 7: Dephosphorylation using Glucose-6-phosphatase converts Glucose-6-phosphate into Glucose
• The reaction occurs in the endoplasmic reticulum, where glucose-6-phosphatase dephosphorylates G6P to yield glucose and phosphate

Citric Acid Cycle

Oxidation of Lactate to Pyruvate

• Lactate, converted to pyruvate, moves from the cytosol to mitochondria.
• This is done via a specific transporter, which is known as mitochondrial pyruvate carrier (MPC).
• Oxidation using Lactate Dehydrogenase converts Lactate into Pyruvate

Carboxylation of Pyruvate to Oxaloacetate

• Carboxylation using Pyruvate carboxylase converts Pyruvate into Oxaloacetate Oxidative Decarboxylation of Pyruvate to Acetyl-CoA
• For fatty acid synthesis to occur, pyruvate must be changed into acetyl-CoA.
• The step above is a tie in between the citric acid cycle and fatty acid synthesis

Additional Steps

• Oxidative Decarboxylation of Pyruvate using Pyruvate dehydrogenase converts Pyruvate into Acetyl-CoA
• Condensation using Citrate synthase converts Oxaloacetate and Acetyl-CoA into Citrate
• Isomerization using Aconitase converts Citrate into Isocitrate
• Oxidative Decarboxylation using Isocitrate dehydrogenase converts Isocitrate into Alpha-ketoglutarate. The decarboxylation requires Mg2+ or Mn2+ ions
• Oxidative Decarboxylation using Alpha-ketoglutarate dehydrogenase complex converts Alpha-ketoglutarate into Succinyl-CoA
• Hydrolysis using Succinate thiokinase converts Succinyl-CoA into Succinate. This is the only example of substrate level phosphorylation in the citric acid cycle
• Dehydrogenation using Succinate dehydrogenase converts Succinate into Fumarate. The enzyme has FAD and iron-sulfur protein, and directly reduces ubiquinone in the electron transport chain
• Hydration using Fumarase converts Fumarate into Malate
• Oxidation using Malate dehydrogenase converts Malate into Oxaloacetate (OAA)

Protein Catabolism to Glucose

• Glucogenic Amino Acids can be converted to form glucose
• However Ketogenic Amino acids cannot be converted to form glucose, due to them forming ketones
• Ketogenic Amino Acids (Leucine and Lysine) enter Pyruvate Dehydrogenase Complex (PDHC) to be converted to Acetyl Co- A.
• Instead the proteins are converted to pyruvate, oxaloacetate, alpha-ketoglutarate, and Fumarate in the Krebs cycle.

Role of Fatty Acids

• The role of fatty acids in gluconeogenesis depends on even or odd chain fatty acids.
• Oxidation provides the ATP needed for the process to occur, and FAs are oxidized to acetyl-CoA in the mitochondria.
• Acetyl-CoA is not converted to pyruvate as pyruvate dehydrogenase reaction is irreversible
• With odd-chain fatty acids: the three carbons at the carbonyl-end of an odd-chain fatty acid are converted to Propionate, entering the TCA cycle as succinyl-CoA, which forms malate (an intermediate in glucose formation).
• Methylmalonyl CoA racemase rearranges D-Methylmalonyl CoA to L-Methylmalonyl CoA
• Methylmalonyl CoA mutase synthesis L-Methylmalonyl Coa into Succinyl Coa

Energy Requirements

• From Pyruvate: Conversion of pyruvate to oxaloacetate by pyruvate carboxylase requires one ATP and Conversion of oxaloacetate to phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxykinase (PEPCK) requires one GTP
• From Glycerol: Conversion of glycerol to glycerol-3-phosphate, which is oxidized to DHAP, requires one ATP and since 2 moles of glycerol are required to form I mole of glucose, 2 moles of high-energy phosphate are required for the synthesis of I mole of glucose

Additional Notes

• Muscles, pyruvate (produced from glycolysis) is converted to alanine via the enzyme alanine transaminase (ALT)