Glycolysis and gluconeogenesis

Glycolysis and gluconeogenesis are metabolic processes responsible for glucose degradation or glucose synthesis respectively. 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., red blood cells). In gluconeogenesis, glucose, which ensures euglycemia during fasting, 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 red blood cells (RBCs). Ribose 5-phosphate is required for nucleotide synthesis, while NADPH is required for cholesterol synthesis, steroid synthesis, reduction of glutathione, and respiratory burst. Glucose 6-phosphate dehydrogenase deficiency results in hemolytic anemia due to insufficient production of NADPH, which is required for the reduction of the antioxidant glutathione to prevent excess hydrogen peroxide and free radicals from damaging RBC membranes.

Glucose breakdown and synthesis are an essential process 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). Red blood cells, which lack mitochondria, even depend entirely on metabolizing glucose for energy and normal function. 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 (e.g., glycogen, fat). Conversely, glucagon predominates in the fasting state for catabolic metabolism, in which stored products are broken down (e.g., fats, amino acids) into glucose to be used as an energy source. The following table provides an overall comparison between glycolysis and gluconeogenesis.

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.

Overview

• Definition: : A metabolic pathway that breaks down glucose by substrate-level phosphorylation and oxidation, yielding two pyruvate molecules and two ATP per one glucose molecule. Under anaerobic conditions glycolysis yields two lactate molecules and two ATP per one glucose molecule.
• Location: cytosol of cells
• Enzymes
  • Rate-limiting enzyme: phosphofructokinase-1 (PFK-1)
  • There are three irreversible steps in the pathway of glycolysis, executed by the following enzymes:
    • Glucokinase (hexokinase IV)
      • The glucokinase of the liver and β-cells of the pancreas has a lower affinity for glucose than hexokinases of other tissues.
      • This causes the liver to store glucose in the form of glycogen only at high glucose concentrations in the portal blood.
      • At low concentrations, the supply of extrahepatic tissue with glucose is guaranteed.
    • PFK-1
    • Pyruvate kinase
• Net reaction: glucose + 2 P_i + 2 ADP + 2 NAD⁺→ 2 pyruvate + 2 ATP + 2 NADH + 2 H⁺ + 2 H₂O

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)
   • Enzyme
     • 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)
   • Enzyme: G6P isomerase
3. F6P → fructose 1,6-biphosphate
   • Enzyme: PFK-1
   • Requires ATP
4. Fructose 1,6-biphosphate → glyceraldehyde 3-phosphate (GAP) (2 molecules)
   • Enzyme: aldolase
5. (2x) GAP → (2x) 1,3-Biphosphoglycerate (1,3-BPG)
   • Enzyme: GAP dehydrogenase
   • Requires (2x) NAD⁺ + inorganic P (P_i)
   • Produces (2x) NADH + H⁺
6. (2x) 1,3-BPG → (2x) 3-phosphoglycerate
   • Enzyme: phosphoglycerate kinase
   • Produces (2x) ATP
7. (2x) 3-phosphoglycerate → (2x) 2-phosphoglycerate
   • Enzyme: phosphoglycerate mutase
8. (2x) 2-phosphoglycerate → (2x) phosphoenolpyruvate (PEP)
   • Enzyme: enolase
9. (2x) PEP → (2x) pyruvate
   • Enzyme: pyruvate kinase
   • Produces (2x) ATP
   • Stimulated by fructose 1,6-biphosphate
   • Inhibited by ATP and alanine

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

In glycolysis, 2 ATP are being invested to gain 4 ATP, so in total, a net gain of 2 ATP per 1 molecule of glucose!

The regulation of glycolysis is determined by the activity of the enzymes hexokinase, phosphofructokinase-1, and pyruvate kinase, which catalyze essentially irreversible reactions in glycolysis and are therefore the main sites of control.

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

• Fructose 2,6-bisphosphate is synthesized by phosphofructokinase-2.
• Phosphofructokinase-2 is a bifunctional enzyme with a phosphatase (FBPase-2) and a kinase (PFK-2) domain
  • Regulation ;:
    • In the liver
      • Fasting state: low blood glucose → increased circulating glucagon levels → increased levels of cAMP → increased protein kinase A (PKA) activity → stimulation of FBPase-2 and inhibition of PFK-2 domain → decreased production of F-2,6-P2 → less glycolysis + more gluconeogenesis
      • Postprandial state: high blood glucose → increased circulating insulin levels (indicate a high abundance of blood glucose available for glycolysis) → decreased levels of cAMP → decreased PKA activity → inhibition of FBPase-2 and stimulation of PFK-2 domain → increased production of F-2,6-P2 → F-2,6-P2 activates PFK-1 → more glycolysis + less gluconeogenesis
    • In the heart: epinephrine and/or insulin → stimulation of PFK-2 domain → increases production of F-2,6-P2 → F-2,6-P2 activates PFK-1 → more glycolysis provides quick energy, e.g., in the case of stress

Pyruvate characteristics

• An alpha-keto acid that is the last product of glycolysis.
• It is an intermediate product that can be converted to several products, including lactate (in anaerobic conditions), acetyl-coenzyme A (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.

Products of pyruvate metabolism

The five cofactors of the pyruvate dehydrogenase complex: Tender (Thiamine) Loving (lipoic acid) Care (CoA) For (FAD) Nancy (NAD+).

Arsenic inhibits lipoic acid, thereby preventing the production of acetyl-CoA and inhibiting the TCA cycle!

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

References:^[1]

Overview

• Definition: A series of metabolic events that allows for the production of glucose from noncarbohydrate precursors.
• Purpose: During fasting, gluconeogenesis becomes the main source of glycemia maintenance after glycogen stores are depleted (after 1–3 days of normal activity)
• Cell location: : Responsible enzymes are located in cytosol and mitochondria.
• Sites of gluconeogenesis
  • Primarily carried out in the liver
  • Renal cortex
  • Intestinal epithelium
  • Skeletal muscle cannot participate in gluconeogenesis due to absent 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 an attempt 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.
• Glycerol-3-phosphate: released during hydrolysis of triacylglycerols in adipose tissue → blood → liver → phosphorylated by glycerol kinase → 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 inhibited when there is an excess of energy available (i.e., large ATP/AMP ration) and activated if energy is required (i.e., low ATP/AMP ratio).
• Gluconeogenesis is also stimulated by glucagon and inhibited by insulin (see phosphofructokinase-2 for the mechanism).
• The following shows the key steps of gluconeogenesis that are irreversible and need to be bypassed with special enzymes. The other steps are merely the opposite reactions of glycolysis that are carried out by bidirectional enzymes (see glycolysis and this illustration ).

Clinical significance

• Von Gierke disease: inborn deficiency of glucose-6-phosphatase
• Breakdown of ethanol
  • Excess alcohol consumption → buildup of NADH (signals to liver that plenty of energy is available)
    • ↑ Glycolysis, ↓ gluconeogenesis (severe hypoglycemia)
    • ↑ Fat synthesis, ↓ β-oxidation (fatty liver)
    • ↑ Pyruvate is converted to lactate (lactic acidosis)

References:^[2]

Overview

• Description: The pentose phosphate pathway consists of a group of reactions in which G6P is degraded, leading to NADPH and ribose 5-phosphate formation. It is an alternative metabolic pathway for glucose. 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
    • Fatty acid synthesis
    • Cholesterol synthesis
    • 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
  • Highest activity in:
    • Liver, adrenal cortex: NADPH is needed for fatty acid, cholesterol, and steroid hormone synthesis.
    • RBCs: NADPH is required for reduction of glutathione, which is needed to prevent oxidative damage to RBCs' cell membrane.
• Rate-limiting enzyme: glucose-6-phosphate dehydrogenase (G6PD)
  • Stimulated by: glucose-6-phosphate, NADP⁺
  • Inhbitited 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 membrane (and causing hemolytic anemia).

Sequence of reactions (two 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⁺
     • Carbon dioxide (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:
  • Create 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)
    • Enzyme: ribose-5-phosphate isomerase
  • Ribulose 5-phosphate ⇄ xylulose 5-phosphate (epimerization reaction)
    • Enzyme: ribulose-5-phosphate epimerase (phosphopentose epimerase)
  • Ribose 5-phosphate + xylulose 5-phosphate ⇄ fructose 6-phosphate + glyceraldehyde 3-phosphate
    • Enzymes: transketolase and transaldolase
      • Transketolase requires thiamine pyrophosphate