5.1: Gluconeogenesis and glycogenolysis

Gluconeogenesis and glycogenolysis are the two pathways essential for glucose homeostasis. Figure 5.1 illustrates the time frame and overlap of glycogenolysis and gluconeogenesis. These pathways are activated nearly simultaneously when the insulin to glucagon ratio becomes sufficiently reduced. Over time, the reliance on the pathways changes.

Figure 5.1: Glucose production by glycogenolysis and gluconeogenesis.

Gluconeogenesis (GNG) is an anabolic pathway that produces glucose from lactate, glycerol, or glucogenic amino acids. This pathway is activated primarily in the liver during fasting and is coordinated with the catabolic pathways of \(\beta\)-oxidation and protein catabolism. The pathway follows the reverse of glycolysis with the exception of four unique enzymes, which overcome the irreversible steps of glycolysis (figure 5.2).

Figure 5.2: Comparison of glycolysis and gluconeogenesis.

Substrates for GNG

Amino acids

The primary substrates for GNG are derived from glucogenic amino acids released through cortisol-mediated protein catabolism. In the fasted state, cortisol is elevated, and it supports fasted state pathways through the activation of protein catabolism — in the skeletal muscle — and by increasing the transcription of enzymes needed for gluconeogenesis (specifically phosphoenol carboxykinase (PEPCK)). As amino acids are released from the skeletal muscle, primarily as glutamine and alanine, they are taken up by the liver. In order to be used for glucose synthesis, they undergo transamination to generate a useful intermediate of the TCA cycle, predominantly \(\alpha\)-ketoglutarate and pyruvate (see figures 5.3 and 5.10) . In the case of alanine, this can be transaminated to generate pyruvate. Glutamine will first be deaminated by glutaminase, and the remaining glutamate will be transaminated to form \(\alpha\)-ketoglutarate (see figure 5.11). Both pyruvate and \(\alpha\)-ketoglutarate will increase substrates in the TCA cycle, ultimately increasing the pool of available malate to be shuttled out of the mitochondria. It is through this process of protein catabolism and transamination that glucogenic amino acids contribute to the synthesis of oxaloacetate (OAA) needed for gluconeogenesis.

Figure 5.3: Locations of amino acid and lactate entering gluconeogenesis as substrates for the pathway.

Lactate

Lactate is primarily produced through the Cori cycle or from anaerobic glucose oxidation. (Note: The Cori cycle, or lactic acid cycle, refers to the metabolic pathway in which lactate produced by anaerobic glycolysis in the muscle or RBC travels to the liver and is converted to glucose. The glucose returns to the peripheral tissues and is metabolized back to lactate.) Once in the liver, lactate can be oxidized back to pyruvate through the reverse reaction catalyzed by lactate dehydrogenase (figure 5.3).

Glycerol

When lipolysis is stimulated by epinephrine or glucagon, activation of hormone-sensitive lipase in the adipose allows for the hydrolysis of triacylglycerol into three free fatty acid chains and glycerol. The glycerol released into circulation will be taken up by the liver. Once in the liver it can be converted into dihydroxyacetone phosphate (DHAP), a glycolytic intermediate. This is an additional way in which carbons can be obtained for glucose synthesis (figure 5.4).

Figure 5.4: Glycerol as a substrate for gluconeogenesis; after phosphorylation to glycerol 3-phosphate it can be converted to DHAP, which can enter directly into glycolysis.

Interconnection of GNG and other metabolic pathways

Gluconeogenesis is heavily reliant on support from other pathways. It requires amino acids for carbon substrates from cortisol-mediated protein catabolism. The ability of those amino acids to be deaminated relies on the ability of the urea cycle to remove ammonia in the form of nontoxic urea, and perhaps most importantly, gluconeogenesis relies on the process of \(\beta\)-oxidation.

Figure 5.5: Reaction catalyzed by pyruvate carboxylase; this allows the bypass of the irreversible step catalyzed by pyruvate kinase.

\(\beta\)-oxidation

The process of \(\beta\)-oxidation supports gluconeogenesis in two major ways:

1. The NADH and FADH₂ generated from \(\beta\)-oxidation is oxidized in the electron transport chain to produce ATP. This ATP provides the needed energy for glucose synthesis. It also supplies energy to the urea cycle for nitrogen disposal.
2. \(\beta\)-oxidation also produces acetyl-CoA. This compound is needed to allosterically activate pyruvate carboxylase (figure 5.5).

Acetyl-CoA produced from \(\beta\)-oxidation itself is not a substrate for gluconeogenesis, rather it is required for allosteric activation of pyruvate carboxylase, which is the first step in GNG. Again, acetyl-CoA is not a substrate for this process; it is fully oxidized in the TCA cycle and provides no additional carbons to be exported from the TCA cycle as malate. Therefore the cell has to rely on amino acid carbon skeletons, glycerol, and lactate as substrates for glucose production (section 5.2).

Regulation of gluconeogenesis

Pyruvate carboxylase and phosphoenol carboxykinase (PEPCK)

Gluconeogenesis is essentially the reverse of glycolysis with four key regulatory steps that allow the bypass of the three irreversible steps of glycolysis (figure 5.2). This initial step of GNG starts in the mitochondria using pyruvate carboxylase (figure 5.5). This enzyme converts pyruvate in the mitochondria to oxaloacetate and requires biotin as a cofactor. This enzyme is allosterically activated by acetyl-CoA. The OAA produced is reduced to malate, which is shuttled out of the mitochondria using the malate-aspartate shuttle. Once in the cytosol, the malate is oxidized back to OAA and decarboxylated by the enzyme phosphoenol carboxykinase (PEPCK) to generate phosphoenol pyruvate (figure 5.3). The combination of these two enzymes, pyruvate carboxylase and PEPCK, allows the cell to bypass the irreversible step catalyzed by pyruvate kinase.

Once phosphoenol pyruvate (PEP) is synthesized, it will continue through the reverse process using the glycolytic enzymes until it reaches its next irreversible conversion.

Fructose 1,6-bisphosphatase (FBP1)

As PEP continues through the reverse of glycolysis, fructose 1,6-bisphosphate is generated. To bypass the irreversible step catalyzed by phosphofructokinase 1 (PFK1) in glycolysis, the enzyme fructose 1,6-bisphosphatase (FBP1) is present and dephosphorylates fructose 1,6-bisphosphate to produce fructose 6-phosphate. This enzyme, FBP1, is inhibited by AMP and fructose 2,6-bisphosphate (figure 5.2).

Like glycolysis, there is an additional regulation here by the bifunctional enzyme phosphofructokinase 2 (PFK2)/fructose 2,6-bisphosphatase (figure 4.1). This bifunctional enzyme functions as a kinase in the fed state (PFK2) and generates fructose 2,6-bisphosphate that allosterically activates PFK1. In the fasted state the enzyme is phosphorylated by glucagon-activated protein kinase A, and this actives the phosphatase activity of the enzyme. The enzyme dephosphorylates fructose 2,6-bisphosphate and therefore reduces the allosteric activation of PFK1 facilitating the reverse reaction by fructose 1,6-bisphosphatase (figure 5.2).

Glucose 6-phosphatase

Finally, glucose 6-phosphatase is required to dephosphorylate glucose 6-phosphate so it can be released from the liver. This is a key step for both glycogenolysis and gluconeogenesis, and deficiencies in this enzyme can lead to severe bouts of fasting hypoglycemia.

Glycogenolysis

In contrast to glycogen synthesis, glycogenolysis is the release of glucose 6-phosphate from glycogen stores. It can occur in both the liver and the skeletal muscle but under two different conditions (figures 5.6 and 5.7). As noted above, this is a pathway active in the fasted state.

• In the liver, glycogenolysis is the initial source of glucose for the maintenance of blood glucose levels when glucagon levels start to increase. The glucose 6-phosphate generated from liver glycogenolysis is dephosphorylated and released into the blood stream.
• In skeletal muscle, glycogenolysis provides glucose only for the skeletal muscle, and this fuel is not released into the blood stream as skeletal muscle lacks glucose 6-phosphatase, the enzyme needed to dephosphorylate glucose. Therefore, skeletal muscle glycogen is primarily used under anaerobic exercise conditions when oxidizing fatty acids is not rapid enough to produce ATP for the exercising tissue.

Regulation of glycogenolysis

Hepatic glycogenolysis

In the liver, glucagon will initiate glycogenolysis through a GPCR-mediated signaling cascade. This leads to the activation of adenylyl cyclase and an increase in cAMP. cAMP activates protein kinase A, which phosphorylates and activates glycogen phosphorylase. Glycogen phosphorylase will initiate glycogen degradation. Also under these conditions, using the same mechanism, glycogen synthase will be phosphorylated and inactivated, ensuring glycogen synthesis is not occurring at the same time (figure 5.6).

Figure 5.6: Hepatic glycogenolysis by epinephrine.

Epinephrine can also enhance hepatic glycogenolysis by binding an \(\alpha\)-agonist receptor. This initiates the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol 1,4,5-triphosphate (IP₃) and diacylglyerol (DAG) by phospholipase C. IP₃ stimulates \(\ce{Ca}^{2+}\) release from endoplasmic reticulum and results in both:

1. phosphorylation and activation of glycogen phosphorylase and
2. phosphorylation and inactivation of glycogen synthase.

In all cases, the glucose 6-phosphate released from glycogen stores is dephosphorylated by glucose 6-phosphatase and released from the liver.

Skeletal muscle glycogenolysis

Skeletal muscle glycogen is not impacted by glucagon but responds to AMP, \(\ce{Ca}^{2+}\), and epinephrine (figure 5.7).

Figure 5.7: Skeletal muscle glycogenolysis.

• The primary regulator of this process is AMP. Elevated AMP will allosterically activate glycogen phosphorylase independent of phosphorylation.
• Next, glycogen phosphorylase can be activated by \(\ce{Ca}^{2+}\). Similar to the above cascade, calcium will activate the \(\ce{Ca}^{2+}\) calmodulin complex, which will in turn activate phosphorylase kinase, ultimately leading to the phosphorylation and activation of glycogen phosphorylase.
• Finally, epinephrine can also stimulate skeletal muscle glycogenolysis through an increase in cAMP (the cascade of events is the same as glucagon-stimulated hepatic glycogenolysis).

Summary of pathway regulation

Metabolic pathway	Major regulatory enzyme(s)	Allosteric effectors	Hormonal effects
Gluconeogenesis	Fructose 1,6-bisphosphatase (FBP1)	Citrate (+) Fructose 2,6-BP, AMP(-)	Glucagon \(\uparrow\) decreases F 2,6-BP by reducing activation of PFK1
	Pyruvate carboxylase Phosphoenolpyruvate carboxykinase	Acetyl-CoA (+)	Cortisol-mediated enhanced transcription
Glycogenolysis	Glycogen phosphorylase	AMP (+) muscle \(\ce{Ca}^{2+}\) (+) in muscle	Glucagon \(\uparrow\)­ (liver) Epi \(\uparrow\)­ (muscle)

Table 5.1: Summary of pathway regulation.

References and resources

Text

Ferrier, D. R., ed. Lippincott Illustrated Reviews: Biochemistry, 7th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2017, Chapter 10: Gluconeogenesis: Section II, III, IV, Chapter 11: Glycogen Metabolism: Section V, VI, Chapter 16: Fatty Acid Ketone Body and TAG Metabolism: Section III, IV, V, Chapter 19: Removal of Nitrogen from Amino Acids: Section V, VI, Chapter 23: Metabolic Effect of Insulin and Glucagon, Chapter 25: Diabetes Mellitus.

Le, T., and V. Bhushan. First Aid for the USMLE Step 1, 29th ed. New York: McGraw Hill Education, 2018, 78, 82, 86, 89–90.

Lieberman, M., and A. Peet, eds. Marks' Basic Medical Biochemistry: A Clinical Approach, 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2018, Chapter 3: The Fasted State, Chapter 19: Basic Concepts in Regulation, Chapter 24: Oxidative Phosphorylation and the ETC, Chapter 26: Formation of Glycogen, Chapter 28: Gluconeogenesis, Chapter 30: Oxidation of Fatty Acids, Chapter 34: Integration of Carbohydrate and Lipid Metabolism, Chapter 36: Fate of Amino Acids Nitrogen: Urea Cycle.

Figures

Ferrier D. Figure 5.1 Glucose production by glycogenolysis and gluconeogenesis. Adapted under Fair Use from Lippincott Illustrated Reviews Biochemistry. 7th Ed. pp 329. Figure 24.11 Sources of blood glucose after ingestion of 100 g of glucose. 2017.

Grey, Kindred, Figure 5.2 Comparison of glycolysis and gluconeogenesis. 2021. https://archive.org/details/5.2-new. CC BY 4.0.

Grey, Kindred, Figure 5.3 Locations of amino acid and lactate entering gluconeogenesis as substrates for the pathway. 2021. https://archive.org/details/5.3_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.4 Glycerol as a substrate for gluconeogenesis, after phosphorylation to glycerol 3 -phosphate it can be converted to DHAP which can enter directly into glycolysis. 2021. https://archive.org/details/5.4_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.5 Reaction catalyzed by pyruvate carboxylase, this allows the by pass of the irreversible step catalyzed by pyruvate kinase. 2021. https://archive.org/details/5.5_20210924. CC BY 4.0.

Grey, Kindred, Figure 5.7 Skeletal muscle glycogenolysis. 2021. https://archive.org/details/5.7_20210924. CC BY 4.0. Added Muscle by Pascal Heß from the Noun Project.

Lieberman M, Peet A. Figure 5.6 Hepatic glycogenolysis by epinephrine. Adapted under Fair Use from Marks' Basic Medical Biochemistry. 5th Ed. pp 534. Figure 26.7 Regulation of glycogen synthesis and degradation in the liver. 2017. Added ion channel by Léa Lortal from the Noun Project.