Glycolysis and gluconeogenesis

Glycolysis is the metabolic process by which glucose is broken down, while gluconeogenesis is the metabolic process by which glucose is synthesized. In glycolysis, the breakdown of glucose molecules generates two net adenosine triphosphate (ATP) molecules, which provide a readily available source of energy for various reactions in the cell, and two pyruvate molecules, which can be further converted into lactate (used in gluconeogenesis), acetyl-CoA (used in citric acid cycle), oxaloacetate (used in citric acid cycle), and alanine (used in transamination reactions). Glycolysis, which occurs exclusively in the cytoplasm, is the sole source of ATP in cells that lack mitochondria (e.g., RBCs). In gluconeogenesis, a process that ensures euglycemia during fasting, glucose is synthesized from noncarbohydrate precursors such as glucogenic amino acids (mainly alanine and glutamine), odd-chain fatty acids, glycerol, pyruvate, and lactate. While the glycolytic pathway occurs in all cells, gluconeogenesis occurs almost exclusively in the liver. Phosphofructokinase-1 is the rate-limiting enzyme for glycolysis, while fructose 1,6-bisphosphatase is the rate-limiting enzyme for gluconeogenesis. The metabolism of glucose is mainly controlled by hormones such as insulin, which stimulates glycolysis, and glucagon, which stimulates gluconeogenesis. Glucose can also be shunted to the pentose phosphate pathway (also known as the hexose monophosphate shunt), which is a metabolic pathway that generates nicotinamide adenine dinucleotide phosphate (NADPH) and ribose 5-phosphate from glucose-6-phosphate. The pentose phosphate pathway occurs exclusively in the cytosol and is highly active in the adrenal cortex, liver, and RBCs. Ribose 5-phosphate is required for nucleotide synthesis, while NADPH is required for cholesterol synthesis, steroid synthesis, reduction of glutathione, and respiratory burst. Hemolytic anemia is caused by glucose-6-phosphate dehydrogenase deficiency, which results in insufficient production of NADPH. NADPH is required for the reduction of glutathione, an antioxidant preventing excess hydrogen peroxide and free radicals from damaging RBC membranes.

• Glucose breakdown and synthesis are essential processes in the human body.
• Glucose provides the required substrates for aerobic and anaerobic metabolism. Glycolysis is the main route of metabolism for most carbohydrates (e.g., galactose and fructose).
• RBCs, which lack mitochondria, depend entirely on glucose to function normally.
• The metabolism of glucose is primarily controlled by hormones such as insulin and glucagon. Insulin is released in the postprandial state for anabolic metabolism, in which glucose is broken down to be transformed into storage forms (glycogen and fat).
• Conversely, glucagon predominates in the fasting state for catabolic metabolism, in which stored products (e.g., fats, glycogen, and amino acids) are broken down into glucose to be used as an energy source.

Gluconeogenesis is more than just the reversal of glycolysis. The reactions of the key enzymes of glycolysis are irreversible due to thermodynamics and must, therefore, be reversed by different enzymes that are only active in gluconeogenesis.

• Definition: a metabolic pathway in which glucose is broken down by substrate-level phosphorylation and oxidation, yielding 2 pyruvate molecules and 2 ATP per 1 glucose molecule. Under anaerobic conditions, glycolysis yields 2 lactate molecules and 2 ATP per 1 glucose molecule.
• Location: cytosol
• Enzymes
  • Rate-limiting enzyme: phosphofructokinase-1 (PFK-1)
  • There are three irreversible steps in the pathway of glycolysis, executed by the following enzymes:
    • Glucokinase: liver and β-cells of the pancreas have a lower affinity for glucose than hexokinases of other tissues
      • Low glycemia (portal blood): Glucose is available to extrahepatic tissue.
      • High glycemia (portal blood): Glucose enters the liver and is stored as glycogen.
    • PFK-1
    • Pyruvate kinase
  • PFK-1 and hexokinase/glucokinase require ATP.

Pyruvate kinase deficiency in erythrocytes causes chronic hemolytic anemia due to impaired glycolysis and a lack of ATP in the RBCs.

Glucose is composed of a 6-carbon skeleton (C₆H₁₂O₆). Each glucose molecule produces 2 pyruvate molecules, which are composed of a 3-carbon skeleton.

1. Glucose → glucose-6-phosphate (G6P)
   • Via enzymes:
     • Hexokinase: in all tissues (inhibited by G6P)
     • Glucokinase: in β-cells of the pancreas and liver tissue (inhibited by fructose 6-phosphate)
   • Requires ATP
2. G6P → fructose 6-phosphate (F6P) via G6P isomerase
3. F6P → fructose 1,6-bisphosphate
   • Via enzyme: PFK-1
   • Requires ATP
4. Fructose 1,6-bisphosphate → 2 glyceraldehyde 3-phosphate (GAP) via aldolase
5. 2 GAP → 2 1,3-biphosphoglycerate (1,3-BPG)
   • Via enzyme: GAP dehydrogenase
   • Requires 2 NAD⁺ + inorganic P (P_i)
   • Produces 2 NADH + H⁺
6. 2 1,3-BPG → 2 3-phosphoglycerate
   • Via enzyme: phosphoglycerate kinase
   • Produces 2 ATP
7. 2 3-phosphoglycerate → 2 2-phosphoglycerate via phosphoglycerate mutase
8. 2 2-phosphoglycerate → 2 phosphoenolpyruvate (PEP) via enolase
9. 2 PEP → 2 pyruvate
   • Via enzyme: pyruvate kinase
   • Produces 2 ATP
   • Stimulated by fructose 1,6-bisphosphate
   • Inhibited by ATP and alanine

The net reaction for glycolysis: glucose + 2 P_i + 2 ADP + 2 NAD⁺ → 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2 H2O

In glycolysis, 2 ATP are invested to gain 4 ATP. In total, there is a net gain of 2 ATP per 1 molecule of glucose.

• The regulation of glycolysis is determined by the activity of the enzymes hexokinase (or glucokinase in the liver and β-cells of the pancreas), phosphofructokinase-1, and pyruvate kinase.
• Common characteristics of these regulatory enzymes:
  • They catalyze irreversible reactions in the glycolysis pathway.
  • Their activity can be regulated (i.e., up- or downregulation) by metabolites and other intermediates, which are often part of feedback loops.

Phosphofructokinase-1 is the rate-limiting enzyme in glycolysis.

Fructose 2,6-bisphosphate (feed-forward regulation)

• Function: Fructose 2,6-bisphosphate (Fru-2,6-P2) stimulates glycolysis by increasing PFK-1 activity in states of high blood glucose concentrations.
• Synthesis
  • Phosphofructokinase-2 (PFK-2) and fructose bisphosphatase-2 (FBPase-2) are two domains of the same bifunctional enzyme.
  • Protein kinase A (PKA) can phosphorylate the bifunctional enzyme and thereby determine which of the two domains is active.
    • Dephosphorylated state: ↑ PFK-2 activity results in ↑ fructose 2,6-bisphosphate (conversion of fructose 6-phosphate to fructose 2,6-bisphosphate).
    • Phosphorylated state: ↑ FBPase-2 activity results in ↓ fructose 2,6-bisphosphate (conversion of fructose 2,6-bisphosphate to fructose 6-phosphate).
• Regulation
  • In the liver
    • Postprandial state: ↑ blood glucose → ↑ insulin → ↓ cAMP → ↓ protein kinase A activity → ↓ phosphorylation of PFK-2/FBPase-2 → ↑ PFK-2 activity and ↓ FBPase-2 → ↑ Fru-2,6-P2 → ↑ PFK-1 activity → ↑ glycolysis and ↓ gluconeogenesis
    • Fasting state: ↓ blood glucose → ↑ glucagon → ↑ cAMP → ↑ protein kinase A activity → ↑ phosphorylation of PFK-2/FBPase-2 → ↑ FBPase-2 activity and ↓ PFK-2 activity → ↓ Fru-2,6-P2 → ↓ PFK-1 activity → ↑ gluconeogenesis and ↓ glycolysis
  • In the heart: epinephrine and/or insulin → ↑ PFK-2 activity → ↑ Fru-2,6-P2 → ↑ PFK-1 activity → ↑ glycolysis provides quick energy, e.g., during stress or physical activity
• Inhibitors: Arsenate inhibits glyceraldehyde-3-phosphate (GAP), which leads to a net yield of zero ATP molecules.

The activity of FBPase is increased during fasting: Fasting Boosts its Power.
The activity of PFK is increased after eating: PFK Prefers a Full stomaK.

Pyruvate characteristics

• An alpha-keto acid that is the last product of glycolysis
• An intermediate product that can be converted into several products, including lactate (in anaerobic conditions), acetyl-coenzyme A (acetyl-CoA), oxaloacetate (OAA), and alanine
• The conversion of pyruvate to lactate and pyruvate to alanine is reversible and occurs in the liver.
  • ALT converts alanine to pyruvate for gluconeogenesis (glucose-alanine cycle).
  • Lactate dehydrogenase (LDH) converts lactate to pyruvate for gluconeogenesis or for metabolism to acetyl-CoA (Cori cycle in the liver).

LDH is found in almost every cell of the body. Elevated LDH levels without exercise may indicate cell injury due to cancer (e.g., germ cell tumors), hemolytic anemia, myocardial infarction, infection, kidney, or liver disease.

Pyruvate dehydrogenase complex

• A complex of mitochondrial enzymes that connects glycolysis to the tricarboxylic acid cycle (TCA cycle)
• Oxidative decarboxylation reaction: pyruvate + NAD⁺ + CoA → acetyl-CoA + CO₂ + NADH + H⁺
• Composed of:
  • 3 enzymes
    • Pyruvate dehydrogenase
    • Dihydrolipoyl transacetylase
    • Dihydrolipoyl dehydrogenase
  • 5 cofactors
    • NAD⁺ (vitamin B₃/niacin)
    • FAD (vitamin B₂/riboflavin)
    • CoA (vitamin B₅/pantothenic acid)
    • Lipoic acid
    • Thiamine pyrophosphate (vitamin B₁)
• Active during a well-fed state and inactive during fasting
• Similar in structure to α-ketoglutarate dehydrogenase complex (converts α-ketoglutarate to succinyl-CoA in the TCA cycle)
  • Both enzyme complexes have the same cofactors.
  • Substrates are structurally similar (pyruvate, α-ketoglutarate).
  • Both enzyme complexes add CoA to the substrate (acetyl-CoA, succinyl-CoA).
• See also “Pyruvate dehydrogenase complex deficiency.”

Products of pyruvate metabolism

Tender Loving Care For Nancy: Thiamine, Lipoic acid, CoA, FAD, and NAD⁺ are the five cofactors of the pyruvate dehydrogenase complex.

Arsenic inhibits lipoic acid, which prevents the production of acetyl-CoA and inhibits the TCA cycle.

Pyruvate dehydrogenase complex deficiency results in impaired conversion of pyruvate to acetyl-CoA, reduced production of citrate, and, therefore, impairment of the TCA cycle, leading to severe energy deficits (especially in the CNS). Long-term treatment includes a ketogenic diet (high fat, low carbohydrate) and cofactor supplementation with thiamine and lipoic acid.

Overview

• Definition: a series of metabolic events that allows for the production of glucose from noncarbohydrate precursors
• Purpose: During fasting, gluconeogenesis becomes the main method of glycemic control after glycogen stores are depleted (after 1–3 days of normal activity).
• Cell location: : Responsible enzymes are located in the cytosol and mitochondria.
• Sites of gluconeogenesis
  • Primarily carried out in the liver
  • Renal cortex
  • Intestinal epithelium
  • Skeletal muscle cannot participate in gluconeogenesis because it lacks glucose-6-phosphatase.
• Rate-limiting enzyme: fructose 1,6-bisphosphatase
• Noncarbohydrate precursors: Glucogenic amino acids (mainly alanine and glutamine), lipids, glycerol, pyruvate, and lactate can all be converted to glucose in order to preserve serum glucose levels. These reactions are energy intensive, as they rely on the consumption of high-energy molecules (GTP, ATP).

Primary substrates

• Glucogenic amino acids: Generated from the hydrolysis of protein tissue (typically during fasting), these amino acids are capable of being converted to α-keto acids (e.g., oxaloacetate, α-ketoglutarate) and then to glucose.
• Lactate: Via the Cori cycle, lactate produced from anaerobic glycolysis (mostly in skeletal muscle) is shuttled to the liver, where it is converted to pyruvate, which serves as the first true substrate in gluconeogenesis.
• Propionyl-CoA
  • Formed from odd-chain fatty acid β-oxidation, this molecule can be carboxylated to form succinyl-CoA. Succinyl-CoA can enter the gluconeogenesis pathway after being converted to oxaloacetate via the TCA cycle.
  • Even-chain fatty acids can only be oxidized to acetyl-CoA, so they cannot be used for gluconeogenesis.
• Glycerol 3-phosphate:
  1. Released into the blood during hydrolysis of triacylglycerols in adipose tissue
  2. Phosphorylated by glycerol kinase in the liver
  3. Oxidized by glycerol phosphate dehydrogenase to dihydroxyacetone phosphate (an intermediate of glycolysis)

All amino acids, except for leucine and lysine, can be used as substrates for gluconeogenesis.

Gluconeogenesis reactions and regulation

• Gluconeogenesis is activated when energy is required (i.e., low ATP/AMP ratio) and inhibited if there is an excess of energy available (i.e., large ATP/AMP ratio).
• Gluconeogenesis is also stimulated by glucagon and inhibited by insulin (see “Phosphofructokinase-2” for the mechanism).
• The table below shows the key steps of gluconeogenesis, which are irreversible and can only be bypassed with specific enzymes. The other steps are merely the opposite reactions of glycolysis that are carried out by bidirectional enzymes (see “Glycolysis”).

Overview

• Description: consists of a group of reactions in which G6P is degraded, leading to the formation of NADPH and ribose 5-phosphate
  • It is an alternative metabolic pathway when G6P is abundantly available.
  • The needs of the cell determine which metabolic pathway is taken.
• Function
  • Ribose 5-phosphate is needed for synthesis of nucleotides (e.g., DNA, RNA).
  • NADPH is required for:
    • Synthesis of fatty acid, cholesterol, and steroid hormones (especially in the liver, adrenal cortex, and lactating mammary glands)
    • Reduction of glutathione: Glutathione prevents oxidative damage to the cell membrane of RBCs by reacting with hydrogen peroxide and free radicals.
    • Respiratory burst
• Location: : cytosol of all cells
• Rate-limiting enzyme: glucose-6-phosphate dehydrogenase (G6PD)
  • Stimulated by glucose-6-phosphate, NADP⁺
  • Inhibited by NADPH

In the pentose phosphate pathway, no ATP is produced or used up.

G6PD deficiency is the most common human enzyme deficiency. It results in insufficient NADPH production, which is required for reduction of the antioxidant glutathione to prevent excess hydrogen peroxide and free radicals from damaging RBC membranes (and causing hemolytic anemia).

Sequence of reactions (2 phases)

Oxidative phase (irreversible)

Net reaction in 3 steps: G6P + 2 NADP⁺ + H₂O → ribulose 5-phosphate + 2 NADPH + 2 H⁺ + CO₂

1. G6P → 6-phosphogluconolactone
   • Enzyme: G6PD
   • NADP⁺ is reduced to NADPH + H⁺.
2. 6-phosphogluconolactone → 6-phosphogluconate
   • Enzyme: 6-phosphogluconolactonase
   • Requires 1 H₂O
3. 6-phosphogluconate → ribulose 5-phosphate
   • Enzyme: 6-phosphogluconate dehydrogenase
   • NADP⁺ is reduced to NADPH + H⁺
   • CO₂ is released.

Nonoxidative phase (reversible)

Net reaction: 3 ribulose 5-phosphate ⇄ ribose 5-phosphate + 2 xylulose 5-phosphate ⇄ 2 fructose 6-phosphate + glyceraldehyde 3-phosphate

• Function depends on the cell's needs:
  • Creation of ribose 5-phosphate from ribulose 5-phosphate (used for nucleotide synthesis)
  • Create sugars that can be exchanged between the pentose phosphate pathway and glycolysis
• Main reactions include:
  • Ribulose 5-phosphate ⇄ ribose 5-phosphate (isomerization reaction) via ribose 5-phosphate isomerase
  • Ribulose 5-phosphate ⇄ xylulose 5-phosphate (epimerization reaction) via ribulose 5-phosphate epimerase (phosphopentose epimerase)
  • Ribose 5-phosphate + xylulose 5-phosphate ⇄ fructose 6-phosphate + glyceraldehyde 3-phosphate via transketolase (requires thiamine pyrophosphate) and transaldolase

• Von Gierke disease: inborn deficiency of glucose-6-phosphatase
• Wernicke-Korsakoff syndrome
• Breakdown of ethanol
  • Excess alcohol consumption → alcohol dehydrogenase breaks down ethanol by reducing NAD+ to NADH → buildup of NADH → signals to liver that plenty of energy is available and inhibits the citric acid cycle
    • ↑ Glycolysis, ↓ gluconeogenesis (severe hypoglycemia)
    • ↑ Fat synthesis, ↓ β-oxidation (fatty liver)
    • ↑ Pyruvate converted to lactate (lactic acidosis)
    • See Metabolic consequences of heavy ethanol consumption for more details.