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15.3 Glucagon and Epinephrine Signaling

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    In the previous sections, we’ve discussed insulin signaling and the process of building glycogen (glycogenesis) in detail. Now let’s take a look at the other side of the homeostatic balance which begins with glucagon signaling. During hypoglycemia (or low blood glucose levels), pancreatic alpha (α) cells release the hormone peptide, glucagon, which stimulates gluconeogenesis (the formation of glucose) and glycogenolysis (the breakdown of glycogen) in the liver, resulting in the release of glucose to the plasma, and the raising of blood glucose levels (Figure 15.3.1).

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    Figure 15.3.1 Summary of Glucagon Signaling During Low Blood Glucose Levels. Image from Hædersdal, S., et al (2018) Mayo Clinic Proceedings 93(2):217-239

    Let’s review a few terms before we begin. In the last section we were introduced to glycogenesis, or the synthesis of glycogen. We saw that this pathway was activated during insulin signaling. In glucagon signaling, this pathway is inhibited and the opposite pathway, glycogenolysis (glycogen breakdown) is activated. Glucagon signaling in the liver also down regulates glycolysis (the utilization of glucose for energy production), as the liver is trying to use glucose to maintain blood glucose levels. It doesn’t utilize it for its own energy needs during this time. Instead, lipids can be used by liver cells to generate ATP energy, and in fact, glucagon signaling increases lipolysis or the breakdown of lipids. Finally, glucagon also up regulates the process of gluconeogenesis or the generation of glucose from non-sugar metabolites. We will address the mechanisms of glycolysis and gluconeogenesis regulation in a later section. Here we will only take a cursory look at these pathways, and will focus more on the process of glycogenolysis.

    Overview of Glucagon Signaling

    Glucagon signaling begins when the hormone binds with its receptor on liver cells (Figure 15.3.2) Glucagon receptors are not widespread within the body, like insulin receptors have evolved. Since the purpose of this hormone is to cause the release of glucose back into the blood stream, this process is highly controlled and only the liver can deliver glucose back into the blood stream to maintain homeostasis. Thus, other target tissues such as skeletal muscle do not need to have these receptors expressed and are not sensitive to glucagon signaling.

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    Figure 15.3.2 Overview of Glucagon Signaling Cascade

    The glucagon receptor is a G-protein-coupled receptor and is also referred to as a 7TM receptor (as it contains 7 transmembrane domains that span the plasma membrane). This family of receptors is widespread throughout the body and responsible for many of the pharmaceutical mechanisms of action seen in our treatment of different disease conditions. With regards to this pathway, once glucagon binds to the receptor, the receptor moves laterally in the plasma membrane and binds with a G-protein that is stationed as a peripheral protein to the plasma membrane. (click) The G-protein contains three major domains, the alpha, the beta, and the gamma domain. The alpha domain is capable of binding to the GDP/GTP cofactor. When the G-protein is inactive, all three subunits stay together and the alpha subunit remains inactive and bound to GDP.

    When the G-protein associates with an activated receptor, the alpha subunit exchanges GTP for the bound GDP cofactor and the gamma and beta subunits dissociate into the cytoplasm. The activated alpha subunit moves laterally on the periphery of the plasma membrane until it contacts the adenylyl cyclase enzyme (also called adenylate cyclase). This activates the adenylyl cyclase that converts ATP into cyclic AMP (cAMP). cAMP production is an amplification step within this pathway. That means that more cAMP is produced that G-proteins are activated.

    After a period of time, a G-protein hydrolase will cause the hydrolysis of the GTP to GDP and inactivate the G-protein. At this point, the G-protein will associate with the gamma and beta subunits reforming its inactive state. Another glucagon signaling event will be required to reactivate the process. The cyclic AMP produced in the process serves as a second messenger in the process and activates a myriad of downstream targets. We will focus on two of the major targets.

    The first is Protein Kinase A, it becomes activated upon binding with cAMP. The second target is a cAMP Response Element-Binding Protein (CREB). The CREB protein is also activated when bound to the cAMP molecule. This causes the CREB protein to translocate from the cytosol into the mitochondria and into the nucleus. In both of these locations, the activated CREB binds to specific response element sequences in the DNA and activates the transcription of genes that are involved in gluconeogenesis. These genes and their encoded proteins have been discussed in more detail in chapter 14. What is important to note now, is that glucagon signaling in the liver results in the upregulation of glucose production de novo from non-carbohydrate precursors. This is NOT a favored pathway in the body. It is expensive energetically for the liver to manufacture glucose. In fact, more expensive in the cost of ATP than can be produced from the newly formed molecule. However, organs like the brain can only utilize free glucose as an energy resource. Thus, the liver will engage in this energy deficit to build glucose for use by the brain and other cellular targets.

    Glucagon signaling also leads to the downregulation of glycolysis, which we will cover in more depth in section 15.4 and glycogenesis. It also leads to an increase in glycogenolysis, or the breakdown of glycogen. Let's take a further look at the regulation of both of these processes.

    Down Regulation of Glycogenesis

    Since glycogen synthase (GS) is the primary enzyme required for glycogenisis, it is also the primary target for the regulation of this pathway. Recall that GS is active in the dephosphorylated state. Thus, PKA down regulates the activity of this enzyme through the phosphorylation of GS (Figure 15.3.3). Phosphorylation of GS causes it to shift into its inactive conformation and inhibits glycogenesis.

    clipboard_eca88dedb866aecdd639f5f8c01fd24bd.png
    Figure 15.3.3 Inactivation of Glycogen Synthase through Phosphorylation. When bound to cAMP, protein kinase A (PKA) phosphorylates and inactivates the glycogen synthase enzyme. The activity of protein phosphorylase 1 (PP1) is required to dephosphorylate GS and restore its activity. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

    In addition, activated PKA also phosphorylates the protein phosphatase 1 (PP1) enzyme leading to the inactivation of the phosphatase. PP1 normally dephosphorylates GS, helping to retain the active conformation of GS. Thus, phosphorylation of PP1 by PKA, helps to maintain the GS in the phosphorylated, inactive state. The inhibition of PP1, is actually quite complicated (Figure 15.3.4). PP1 contains a regulatory domain and a catalytic domain. Normally the regulatory domain of PP1 binds with glycogen, keeping the molecule close to the location where GS will be present. Thus, when GS is near its substrate in can also bind with PP1 and be dephosphorylated into its active state. This is more efficient that having to diffuse around the cell trying to find the PP1 randomly. When PKA phosphorylates the regulatory domain of PP1, it dissociates from the catalytic domain, causing the catalytic domain to float away from the glycogen molecule. This makes PP1 less efficient at dephosphorylating GS because it is harder for the molecules to randomly come into contact with one another. Thus, PP1 is less active. PKA reduces this activity even further, by phosphorylating an allosteric inhibitor (I) of PP1. In the phosphorylated state, the inhibitor can bind to PP1 fully inactivating the phosphatase. Both phosphorylation events need to be reversed to regain full PP1 activity.

    clipboard_ea0329fb8de712608f10982c52314d94f.png
    Figure 15.3.4 Inactivation of Phosphorylase 1 (PP1) by Protein Kinase A (PKA). PKA phosphorylates PP1 causing it to dissociate from glycogen and become less active. PKA also phosphorylates an allosteric inhibitor (I) of PP1 which increases its binding affinity for PP1. The phosphorylated inhibitor maintains PP1 in an inactive conformation. Image Modified from: Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Servier Medical Art

    In summary, glucagon signaling in the liver downregulates glycogenesis through the activation of PKA. PKA phosphorylates GS directly, inactivating the enzyme, and maintains it in the inactive state by also inhibiting the PP1 responsible for dephosphorylating GS.

    Activation of Glycogenolysis

    In addition to phosphorylating GS and PP1 during the inactivation of glycogenesis, PKA also phosphorylates the Phosphorylase Kinase Enzyme, which is upstream of Glycogen Phosphorylase, the primary enzyme involved in glycogen breakdown. The subsequent phosphorylation of Glycogen Phosphorylase leads to increased activity of the protein and the breakdown of glycogen. Figure 15.3.5 details the phosphorylation cascade required for glycogen phosphorylase activation.

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    Figure 15.3.5 Activation of Glycogen Phosphorylase by Protein Kinase A (PKA) Signaling. PKA upregulates the activity of Phosphorylase Kinase through direct phosphorylation. The activated kinase enzyme phosphorylates its downstream target, Glycogen Phosphorylase (GP). In the phosphorylated state, GP is more active. Image modified from Yan, A., et al (2016) In J Biol Sci 12(12):1544-1554 and Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

    The phosphorylase kinase enzyme is a heterotetramer that is primarily regulated by phosphorylation through the PKA pathway as shown in Figure 15.3.5. However, this enzyme is also regulated by the allosteric binding of calcium ions. Calcium may be present within cellular targets due to nerve impulse firing, muscle contraction, or through hormone signaling. The presence of calcium in the cell generally indicates that there is high energy demand on the cell at that time, and that energy production is needed. Thus, calcium binding to the phosphorylase kinase is a positive effector of the enzyme and upregulates activity. Maximal activity of the enzyme is achieved through combined phosphorylation and calcium binding. Thus, phosphorylase kinase can exist in 4 different states of activity as shown in Figure 15.3.6.

    clipboard_e6d7e0e93d92fab7b454253493e37fec4.png
    Figure 15.3.6 Activation States of Phosphorylase Kinase (PK). In the left hand diagram, PK is in the inactive state, with the kinase-containing alpha domains shown in red. The upper diagram shows the activation of PK through phosphorylation by Protein Kinase A during hormone signaling. This leads to a partially active enzyme. Similarly, calcium binding, shown in the lower diagram, also results in a partially active enzyme. Calcium plays a particularly important role in the activation of this enzyme in skeletal muscle. The process of muscle contraction causes the release of high levels of calcium into the cytoplasm. Thus, the presence of calcium within the cytoplasm of muscle cells indicates high energy demand, as the muscle is being called into action. This activates PK and stimulates the breakdown of glycogen within muscle tissue to help meet energy demands. Maximal activity is obtained with both calcium binding and phosphorylation.

    We have been primarily discussing the regulation of glycogenolysis in liver. However, in considering the activity of PK and its reactivity with Ca2+ ions, we should also consider the activation of glycogenolysis in skeletal muscle, as well. Of note, glycogenolysis in liver tissue and skeletal muscle has many differences. First, the G-protein coupled pathway is activated by different hormones. Liver tissue is responsive to Glucagon stimulation, as well as stimulation through the Epinephrine hormone signaling pathway. Glycogenolysis in skeletal muscle tissue, on the other hand, is only activated by the Epinephrine signaling pathway, but not by glucagon. This is because the liver is the primary organ responsible for regulating blood glucose levels. Thus, pancreatic signaling due to low blood glucose levels primarily targets glycogenolysis within the liver tissue. Both systems are responsive to Epinephrine, which is described in more detail below.

    Epinephrine Signaling

    Epinephrine is a small amino acid-derived hormone (can you guess the amino acid?? Yes it is Tyrosine!!) (Figure 15.3.7). It is also called adrenaline, as it is secreted from the adrenal glands located just above the kidneys, during the flight or fight response. It is also secreted during heavy or sustained exercise. Epinephrine has pleiotropic responses in the body, which include the activation of glycogenolysis in the liver and skeletal muscles. Epinephrine also promotes fat breakdown in adipose tissue, which releases this energy reserve into the blood stream for utilization by muscle tissue. It also causes the relaxation of smooth muscles in the lungs and respiratory track enabling better oxygen absorption. Cardiac contractility is also increased to increase blood flow to skeletal muscles. This supports the generation of ATP from glucose and fatty acids for sustained muscle utilization. It also reduces blood flow to the skin and causes the contraction of smooth muscles in the skin causing goosebumps.

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    Figure 15.3.7: Structure of Epinephrine

    Within the liver and skeletal muscle, the epinephrine signaling pathway overlaps with the glucagon signaling pathway seen in the liver. The epinephrine receptor is also a G-protein coupled receptor related to the glucagon receptor. However, it is specific for epinephrine and cannot bind with glucagon. It does activate the same G-protein pathway leading to Protein Kinase A activation (Figure 15.3.8). The body is very efficient at reusing machinery in different parts of the body, in this case, it does so under different regulatory parameters.

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    Figure 15.3.8 Similarities of Glucagon and Epinephrine Signaling Pathways

    The Glycogen Phosphoyrlase enzymes are also encoded by different genes within the Liver and Skeletal Muscle. These are known as isozymes. Recall, that isozymes have the same biological function, but since they are expressed from different genes, they have different enzyme kinetics and they are regulated in different and unique ways within each tissue.

    The liver and skeletal muscle forms of Glycogen Phosphorylase share approximately 90% sequence identity. Both isozymes can exist in two major conformations, the a-form and the b-form. The protein adopts the a-form when it is phosphorylated at a serine residue by phosphorylase kinase (Figure 15.3.9). The Glycogen Phosphorylase enzyme can also be in two different states, the relaxed, flexible state which is the active form of the enzyme, and the tense or rigid state that is inactive. When the protein is in the a-conformation, it favors the relaxed and active state of the protein. Therefore, phosphorylation of Glycogen Phosphorylase leads to an increase in the activity of the enzyme. This is depicted in the following diagram.

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    Figure 15.3.9 Enzymatic States of Liver and Skeletal Muscle Glycogen Phosphorylase. Both isozymes of Glycogen Phosphorylase are responsive to phosphorylation by phosphorylase kinase. Phorsphorylation of Glycogen Phosphorylase causes it to shift from the b-form to the a-form of the protein. The a-form of the protein favors the relaxed, active state of the protein, whereas the b-form of the protein favors the tense and inactive state of the protein. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

    The different isozymes of the Glycogen Phosphorylase enzyme are also regulated by different, tissue-specific allosteric effectors. Within the liver, glucose is a negative regulator of Glycogen Phosporylase, which makes sense, as the role of this pathway in liver tissue is to promote the release of glucose into the blood stream. The presence of free glucose in the cytoplasm of the liver, would indicate either the fed-state when blood glucose levels are high, or that high levels of glycogenolysis have released substantial glucose. Within liver tissue, the presence of free glucose will cause the a-form of Glycogen Phosphorylase to shift to the Tense state, reducing the activity of the enzyme. This is shown in Figure 5.3.10.

    clipboard_ede9343f83a16c8338ad0bf0c9b6d6e91.png
    Figure 5.3.10 Regulation of Liver Glycogen Phosphorylase by Free Glucose. When the liver isoform of Glycogen Phosphorylase is in the a-form (phosphorylated), it can be shifted into the Tense state in the presence of high levels of free glucose. This blocks the glycogen binding site of the enzyme, essentially serving as a competitive inhibitor of the enzyme. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

    Skeletal muscle glycogen phosphorylase (or Myophosphorylase, as it is sometimes called), is more responsive to allosteric effectors that indicate the energy state of the cell. This makes sense, as the main purpose of glycogen breakdown in muscle tissue is to fuel the energy demand for the muscle tissue. Thus, the energy housed in glucose will be used to produce ATP within these cells. The presence of either Glucose 6-phosphate or ATP within skeletal muscle indicates high levels of energy are present. Thus, glycogen breakdown will be inhibited. The presence of AMP, on the other hand, indicates a low energy state and is an activator of Glycogen Phosphorylase. This is shown in Figure 5.3.11 - Figure 5.3.13.

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    Figure 5.3.11 Regulation of Skeletal Muscle Glycogen Phosphorylase by Glucose 6-phoshate. Muscle Glycogen Phosphorylase is negatively regulated by the presence of high levels of Glucose 6-phosphate. Regardless of form (a or b) the enzyme is shifted into the Tense state. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919
    clipboard_e29b341d6e22171e1e1740f1a8e1e28c1.png
    Figure 5.3.12 Regulation of Skeletal Muscle Glycogen Phosphorylase by ATP. In the presence of high levels of ATP, GP is converted to the Tense state, showing the inhibition of the enzyme in the presence of high levels of energy. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919
    clipboard_ea9b68f9c0f3b2cc5701f4b1c00a72037.png
    Figure 5.3.13 Regulation of Skeletal Muscle Glycogen Phosphorylase by AMP. In the presence of low energy indicators, such as AMP, the glycogen phosphorylase enzyme is activated, even in the absence of phosphorylation. Thus, when AMP is bound, the b-form of glycogen phosphorylase is converted into the relaxed and active state. Image modified from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919
    McArdle's Disease

    McArdle's Disease, also referred to as myophosphorylase deficiency or type V glycogen storage disease, is a recessive inherited disorder characterized by an inability to metabolize glycogen due to the absence of a functional myophosphorylase (PYGM). In Figure 5.3.14, the normal functional pathway is shown in blue, on the left, while the mutant pathway is shown on the right in red. Patients with this disease lack sufficient glucose-1-phosphate (G1P) monomers needed for glycolysis and the hexosamine biosynthetic pathway (HBP). This results in lower ATP and, consequently, lower muscle contraction, as well as in lower post-translational modifications by O-GlcNAcylation in comparison to normal conditions. This is especially pronounced during extended or heavy workouts, where people with McArdle’s Disease will sustain painful cramping of their muscle tissue during workouts, can have dark red/brown urine, and can easily tire during activity. Some patients also note a second wind phenomena occur during workouts as the body shifts from carbohydrates to lipids as a primary energy source. The dark red/brown color in the urine happens if muscle tissue is damaged during the workout. The damaged muscle releases the protein myoglobin into the bloodstream. This is filtered out by the kidneys and excreted in the urine, causing the color change. Severe uncontrolled disease can cause life threatening kidney problems.

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    Figure 5.3.14 McArdle's Disease. Left hand side (blue) represents the normal pathway, whereas the right hand side (red) notes the deficiency of muscle glycogen phosphorylase. Image from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

    Figure 5.3.15 presents an overview of glucose metabolism in skeletal muscle. . Both glucose-1-phosphate (G1P) released from the intracellular glycogen stores by glycogen phosphorylase (GP), as well as the glucose introduced into the cell through glucose transporters (GLUT) are converted to glucose-6-phosphate (G6P) by phosphoglucomutase (PGM) and hexokinase (HK), respectively. The G6P can be directed to different destinations. One of them is the pentose phosphate pathway for the formation of nucleic acid building blocks (ribose and deoxyribose). Another destination is in the formation of energy (ATP). Here G6P enters the metabolic pathway of glycolysis. The glycolytic reactions will culminate in the production of pyruvate and adenosine triphosphate (ATP). Pyruvate can be fermented in lactate by the catalysis of the lactate dehydrogenase (LDH), as happens during anaerobic muscle exercise. On the other hand, pyruvate can be used to obtain ATP through full oxidation in the Kreb Cycle. In total, oxidative phosphorylation produces 36 molecules of ATP, 6 molecules of carbon dioxide (CO2), and 6 molecules of water (H2O) from 1 glucose molecule. Whereas, glycolysis alone, only produces two net ATP molecules per glucose. Glucose, moreover, in addition to being the fuel of the cell’s energy metabolism, is also used by the cellular machinery as a vitally important substrate for the production of key intermediaries of the hexosamine biosynthetic pathway (HBP) forming O-GlcNAc, β-linked N-acetylglucosamine. And finally, in times of plenty, glucose will by utilized by glycogen synthase (GS) to make glycogen.

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    Figure 5.3.15 Summary of Glucose Metabolism in Skeletal Muscle. Image from Llavero, F., et al (2019) Int. J. Mol. Sci. 20(23):5919

    15.3 Glucagon and Epinephrine Signaling is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski and Patricia Flatt.