15.1: Insulin Signaling in the Liver

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Glycogen Structure, Function, and Blood Glucose Homeostasis

• Describe the structural features of glycogen — including α1→4 main-chain linkages, α1→6 branch points every 12–15 residues, the central glycogenin dimer, and the abundance of non-reducing ends — and explain how this architecture enables rapid glucose mobilization.
• Explain the physiological importance of hepatic glycogen in maintaining blood glucose homeostasis, with emphasis on the brain's absolute dependence on glucose as its primary fuel source and the metabolic consequences of hypoglycemia.
• Describe the complementary roles of insulin and glucagon in maintaining blood glucose within a narrow homeostatic range, identifying the pancreatic cell types responsible for each hormone, the conditions that trigger their release, and their opposing effects on hepatic glycogen metabolism.

Insulin: Structure, Biosynthesis, and Receptor Activation

• Trace the biosynthetic processing of preproinsulin to mature insulin, identifying the roles of signal peptide cleavage, disulfide bond formation in the ER, C-peptide excision in secretory vesicles, and the diagnostic utility of C-peptide measurements in distinguishing type 1 from type 2 diabetes.
• Describe the structural basis of insulin receptor activation, explaining how insulin binding promotes receptor dimerization, autophosphorylation of tyrosine residues, and the conformational transitions of the intracellular tyrosine kinase domain from an inactive (mobile-loop-occluded) to an active (ATP-accessible) state.

Insulin Signaling Cascade: GLUT4 Translocation and Glycogen Synthesis

• Trace the insulin signaling cascade from receptor autophosphorylation through IRS-1 recruitment, PI3-kinase activation, PIP2 phosphorylation to PIP3, PDK-mediated Akt activation, and AS160 phosphorylation, explaining at each step how the signal is amplified and transduced.
• Explain how activated Akt promotes GLUT4 translocation to the plasma membrane by phosphorylating and inactivating AS160, thereby releasing inhibition of the GTPase Rab10 and allowing GLUT4-containing secretory vesicles to fuse with the plasma membrane — and explain why pre-storing GLUT4 in vesicles enables a faster response than de novo protein synthesis.
• Explain how insulin signaling activates glycogen synthase through a double-negative regulatory mechanism in which Akt phosphorylates and inactivates GSK-3, allowing protein phosphatase 1 (PP1) to dephosphorylate and activate glycogen synthase, and connect this pathway to the broader metabolic goal of glucose storage in the fed state.

Introduction

In this section, we will discuss insulin signaling and glycogen synthesis. Insulin is released in the fed state and promotes glucose uptake, which can be stored as glycogen if not needed to meet energy needs. Recall that glycogen is a large polymer of glucose residues connected in the main chain by α1→4 linkages and with branching side chains about every 12 – 15 residues at the α1→6 positions. The reducing ends of the carbohydrate (two for each polymer) are connected to the glycogenin dimeric protein at the center of the macromolecule (Figure \(\PageIndex{1}\)). Due to the polymer's branching, many non-reducing ends are present, allowing easy access and rapid release of glucose for energy utilization.

Most of the body’s glycogen pools are stored in the liver, with 10% of the liver biomass in glycogen granules, and in the skeletal muscle, with glycogen comprising 2% of the muscle biomass. Each glycogen polymer may have upwards of 30,000 glucose residues, making it visible using standard microscopic techniques. Muscle cells use glycogen stored as a source of energy to fuel muscle contraction. In the liver, glycogen storage serves a different purpose. Glycogen stored at this location helps maintain homeostatic blood glucose levels. The liver is the primary organ that can actively transport glucose into the bloodstream. Our only other major source of glucose within the blood is our diet.

Figure \(\PageIndex{1}\): The Glycogen Polymer. The glycogen-reducing ends of the glycogen polymer are connected with the dimeric glycogenin protein shown at the center of the diagram using the ribbon projection. The polymer is predominantly composed of glucose monomers linked by alpha-1,4 linkages. Alpha 1 --> 6 branches are apparent about every 12 - 15 residues. (Public Domain; Mikael Häggström via Wikipedia.

Blood glucose homeostasis is critical for brain function (Figure \(\PageIndex{2}\)). The brain has a high energy demand but nearly no storage of the key energy molecules required for ATP production. Furthermore, glucose and ketone bodies are the only energy sources that can pass the blood-brain barrier and be utilized by the brain for ATP production. Note that ketone bodies are only produced during starvation or disease states such as diabetes and are not a regular energy source for the brain. Thus, glucose is critical for brain function. The brain uses nearly 10% of the whole body’s energy for nerve impulse transmission. If the blood flow carrying critical oxygen and glucose to the brain is impeded, people will lose consciousness within approximately 20 seconds! Brain death/permanent damage occurs within 4 minutes of blood flow cessation. This exemplifies the liver's role in maintaining blood glucose levels and in oxygen homeostasis.

Figure \(\PageIndex{2}\): Cortical neuron stained with antibody to neurofilament subunit NF-L in green. In red are neuronal stem cells stained with antibodies to alpha-internexin. Image created using antibodies from EnCor Biotechnology Inc. (CC BY-SA 3.0 Unported; GerryShaw via Wikipedia)

Glycogen in the liver or muscle can be broken down into glucose 1-phosphate (Figure \(\PageIndex{3}\)). This can be converted to glucose 6-phosphate, which is readily used in many cellular processes. The glycolysis (or the breakdown of glucose into pyruvate) occurs in all cells and produces a small amount of ATP. Further processing of pyruvate can occur anaerobically (in the absence of oxygen) to produce lactate, or it can continue in the aerobic pathway to complete oxidation to carbon dioxide and water in the Krebs cycle. Note that oxygen from breathing is used to create the water within this pathway. This fuels the process of oxidative phosphorylation within the mitochondria and produces large quantities of ATP (from 30-36 molecules/glucose). Within the liver, glucose can be released from glycogen to maintain homeostatic levels. Glucose 6-phosphate can also serve as a precursor for other major macromolecules, such as ribose and deoxyribose, as well as for the hexosamine compounds commonly found cushioning joints or attached to plasma membrane proteins.

Figure \(\PageIndex{3}\): Pathways of Glycogen Utilization.

In healthy individuals, hormone signaling is critical to maintaining blood glucose homeostasis. Within this system, the hormones glucagon and insulin work together to maintain normal plasma glucose levels ( Figure \(\PageIndex{4}\)). During hyperglycemia, pancreatic beta (β) cells release insulin, which stimulates glucose uptake by energy-consuming cells and glycogen formation in the liver. During hypoglycemia, pancreatic alpha (α) cells release glucagon, which stimulates gluconeogenesis and glycogenolysis in the liver and the release of glucose to the plasma.

Figure \(\PageIndex{4}\): Hormone Signaling Involved in Blood Glucose Homeostasis. The hormones glucagon and insulin, released by the pancreas, are critical for regulating the liver's release or uptake of glucose from the bloodstream. In the upper diagram, beta cells in the pancreas secrete insulin when blood glucose levels rise. Insulin signaling increases liver glucose uptake from the bloodstream and promotes its storage as glycogen. Alternatively, alpha cells in the pancreas release the hormone glucagon when blood glucose levels are low, as shown in the lower diagram. Glucagon signaling in the liver triggers glycogen breakdown and the release of stored glucose into the bloodstream. Hædersdal, S., et al (2018) Mayo Clinic Proceedings 93(2):217-239

The first area we will focus on is the mechanism by which insulin reduces blood glucose levels. Figure \(\PageIndex{5}\) shows the structure of the pancreas and its anatomical relationship with the liver and the stomach. The pancreas is the sensor organ that detects blood glucose levels. It signals the liver to remove or release glucose in response to changing levels. Notably, the pancreas also produces most of the digestive enzymes utilized by the body, including proteases, amylases, and lipases.

Figure \(\PageIndex{5}\): The Anatomy of the Liver and Pancreas. Akinlade, A., et al. (2014) Int. Archives of Med 7(50):28

Figure \(\PageIndex{6}\) shows a light microscope image of the pancreatic islet cells. They are responsible for producing glucagon and insulin. The islets are distinguished from the surrounding tissue by a continuous connective tissue capsule and extensive vascularity.

Figure \(\PageIndex{6}\): Pancreatic Islet Cells. In this diagram, the islet cells that secrete glucagon (known as alpha cells) are stained in red, while the beta cells that produce insulin are stained in blue. Akinlade, A., et al. (2014) Int. Archives of Med 7(50):28

Insulin is a peptide hormone composed of 51 amino acids, as shown in Figure \(\PageIndex{7}\). It is initially synthesized as preproinsulin, which is then converted to proinsulin by removal of the signal peptide.  Two disulfides are made in the ER (catalyzed by protein disulfide isomerase) along with selective proteolytic cleavage to form insulin.  Mature insulin consists of A and B chains that are connected in proinsulin by the C-peptide.  

Figure \(\PageIndex{7}\): Conversion of preproinsulin to mature insulin.  Vasiljević, J. et al. Diabetologia 63, 1981–1989 (2020). https://doi.org/10.1007/s00125-020-05192-7.  http://creativecommons.org/licenses/by/4.0/.

Figure \(\PageIndex{8}\) shows  interactive iCn3D models of human insulin (3I40) and human proinsulin (2KQP).  (Copyright; author via source). Click the image for a popup or use the external link provided:

Human insulin (3I40)	Human Proinsulin (2KQP)
Colored code to show secondary structure External link:  https://structure.ncbi.nlm.nih.gov/i...A3QZdZpfEMxR17	The "future" A chain in mature insulin is shown in magenta, the C-peptide connecting the A and B chains is shown in yellow, and the future B chain is in cyan. External link:  https://structure.ncbi.nlm.nih.gov/i...nQJXhrEfHD1GH9

The maturation of preproinsulin to insulin is shown in more detail in  Figure \(\PageIndex{9}\).  The peptide is first translated on ribosomes linked to the rough endoplasmic reticulum (ER), where a signal peptide docks the peptide to the ER membrane. The proinsulin is folded, and the signal peptide is cleaved. It is transported to the Golgi, where it is further packaged into secretory vesicles. Within secretory vesicles, proinsulin is cleaved to release C-peptide. The A and B peptides are held together by disulfide bridges, forming active insulin.

Figure \(\PageIndex{9}\):  Insulin Production. The process of insulin production includes the translation of the insulin mRNA into the rough endoplasmic reticulum. The peptide is transported through the Golgi and packaged into secretory vesicles, where protease cleavage by carboxypeptidase E produces the mature insulin peptide and peptide C. Image from Fred the Oyster

C-peptide is a bioactive peptide secreted simultaneously with insulin in equal amounts. It also has a longer half-life than insulin and is excreted by the kidneys into the urine, making detection easy. Furthermore, it allows detection of patient-produced insulin, even when patients are receiving insulin injections. Thus, C-peptide detection is often used to distinguish patients with type 1 diabetes from those with type 2 diabetes (or maturity-onset diabetes). Details about the different forms of diabetes will be discussed later.

Once insulin is released from the pancreas, it travels throughout the body and binds to cellular receptors on target cells. The Insulin Receptor is a tyrosine kinase receptor that dimerizes upon insulin binding, as shown in Figure \(\PageIndex{10}\). Insulin receptors are located on most cell types throughout the body, leading to pleiotropic effects during the insulin response. The primary targets of insulin action are the liver, which promotes the uptake of glucose and the production of the glycogen storage molecule, as well as skeletal muscle and fat. The receptor's tyrosine kinase domain, located on the inner surface of the plasma membrane, is quite flexible.

Figure \(\PageIndex{10}\): The Insulin Receptor.  Images from Goodsell, D., et al (2015) RCSB PDB-101 ‘Molecule of the Month’

The left-hand diagram shows a space-filling model of the activated insulin receptor dimer embedded into the plasma membrane (the gray bar). The tyrosine kinase portion of the receptor is shown on the inside of the cell, whereas the insulin binding domain is present on the external side of the plasma membrane. The middle and right-hand diagrams show the inactive (middle) and active forms (far right) of the tyrosine kinase domain of an insulin receptor monomer. When activated, the tyrosine kinase domain binds to ATP (hot pink) and phosphorylates downstream targets, including several of its tyrosine residues (green). In the inactive state (middle), a mobile loop (turquoise) binds to the ATP-binding site and prevents ATP association. When the insulin receptor is activated, the mobile loop opens, allowing for the binding of ATP and self-phosphorylation of tyrosine residues and other signaling proteins (a small peptide from one is shown in light pink).

Figure \(\PageIndex{11}\) below, which shows an interactive iCn3D model of the Full-length mouse insulin receptor bound to four insulins (7SL7).

Figure \(\PageIndex{11}\): Full-length mouse insulin receptor bound to four insulins (7SL7). (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/i...gkTrtvaqQF6o2A

The receptor is a dimer (one monomer gray and the other light brown).  Four insulins are bound in maximally activated insulin receptors.  Two insulins (magenta) are bound at the same respective place in each monomer (site-1, the primary site), and the two others are bound at a second parallel site (site-2).  The full active state is a symmetric T-shape.  Less active receptors have fewer bound insulins, with receptor geometry more asymmetric (one insulin bound at site 1 gives a Γ-shaped conformation, while two produce a Ƭ-shaped conformation as the second insulin binds). When four insulins are bound at both sites, the asymmetric conformation can't be formed.  Although the structure is described as full-length, both monomers end at amino acid 910.  The single membrane-spanning alpha-helix occurs at amino acids 947-967 and is NOT shown in the model.

Activation of the insulin receptor in the liver when insulin is present initiates a phosphorylation signaling cascade, as shown in Figure \(\PageIndex{12}\).  One of the proteins activated is Rab10. Rab 10 promotes the fusion of GLUT4-containing secretory vesicles (GSVs) with the plasma membrane, allowing for increased surface expression of GLUT4. GLUT4 is a glucose transporter protein. Thus, an increased protein concentration at the plasma membrane leads to increased glucose import into the cell. Having GLUT4 proteins stored within secretory vesicles makes them more readily available than having to activate gene transcription pathways and produce the protein de novo. This allows a faster response, helping lower blood glucose levels. The result is increased glucose uptake from the bloodstream into liver cells and other cellular targets, reducing blood glucose levels.

Figure \(\PageIndex{12}\):  Insulin Activation of Liver Cells and Glucose Uptake. Image from Carmichael, R.E., et al (2019) Scientific Reports 9:6477

The insulin receptor is a receptor tyrosine kinase that undergoes dimerization and Tyr autophosphorylation upon insulin binding. The phosphorylated receptor also recruits and phosphorylates the insulin receptor substrate 1 (IRS-1) on tyrosine residues, which then recruits dimeric Phosphoinositol (PI)3-kinase (p85/p110 in the diagram above) and phosphorylates the p85 regulatory subunit. The PI3 kinase catalyzes the phosphorylation of phosphatidylinositol bisphosphate (PIP2) within the plasma membrane to form phosphoinositol, 3,4,5-triphosphate (PIP3). PIP3 recruits PIP3-dependent kinase (PDK), which phosphorylates and activates Akt. Once activated, Akt dissociates from the membrane into the cytosol, where one of its downstream targets is AS160. AS160 is a GTPase that binds typically with Rab10 (a G-protein), causing the cleavage of GTP to GDP. Thus, AS160 downregulates Rab10 activity. In the phosphorylated state, AS160 cannot bind Rab10 or inhibit its activity, allowing Rab10 to release GDP and bind a GTP molecule. In the activated state, Rab10 helps promote the fusion of GLUT4-containing secretory vesicles (GSVs) with the plasma membrane.

Figure \(\PageIndex{13}\) provides a deeper look at some of the initial activation steps in the insulin signaling pathway. This step shows the phosphorylation of Phosphoinositol 4,5-bisphosphate (PIP2) to Phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP2 is a common phospholipid within the lipid bilayer structure. In future lectures, we will see the utilization of this phospholipid in other signaling pathways as well.

Figure \(\PageIndex{13}\):  Phosphorylation of phosphotidylinositol-4,5-bisphosphate During Insulin Signaling.

Once glucose enters a cell, it is rapidly converted to glucose 6-phosphate via the enzyme hexokinase, as shown in Figure \(\PageIndex{14}\) below. This enzyme is covered in more detail in our section on glycolysis. Importantly, phosphorylation traps the glucose inside the cell, preventing its redistribution back into the bloodstream. This helps maintain glucose homeostasis in the bloodstream. In addition, glucose-6-phosphate is the first step in many glucose pathways, including energy production and the formation of building blocks such as ribose and deoxyribose used in RNA and DNA synthesis.

Figure \(\PageIndex{14}\):  Conversion of Glucose to Glucose-6-Phosphate by Hexokinase. Image from Jmun7616

Insulin signaling increases the number of GLUT4 transporters at the plasma membrane, thereby increasing glucose uptake into the cell. If glucose is not required for energy or other metabolic intermediates within liver and muscle tissue, it is then converted to glycogen for storage. The primary enzyme necessary for glycogen synthesis is also activated via insulin signaling, as shown in Figure \(\PageIndex{15}\).  In addition to phosphorylating the AS160 protein, Activated Akt also phosphorylates the Glycogen Synthase Kinase (GSK-3), thereby inactivating it. This allows protein phosphatase 1 (PP1) to dephosphorylate glycogen synthase, shifting it into a more active state and initiating glycogen synthesis.

Figure \(\PageIndex{15}\):  Activation of Glycogen Synthase During Insulin Signaling. Image modified from Carmichael, R.E. et al (2019) Scientific Reports 9:6477

In this section, we have covered two pathways activated by insulin signaling, and I am sure you are feeling a bit overwhelmed by the complexity. However, biological processes are incredibly complex, and signaling pathways have multiple pleiotropic downstream effects. We have only touched the tip of the iceberg for insulin signaling, as evidenced in Figure \(\PageIndex{16}\). This figure gives a more complete representation of the chemical changes induced within a liver cell in response to insulin signaling. For our purposes, we will restrict coverage to the two downstream effects: increased GLUT4 transporters at the plasma membrane and increased glycogen synthase activity.

Figure \(\PageIndex{16}\): Insulin Signaling Pathway. Image by Cell Signaling Technology

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter examines the molecular mechanisms by which insulin coordinates blood glucose uptake and glycogen storage, integrating structural biochemistry, signal transduction, and metabolic regulation into a coherent account of fed-state glucose homeostasis.

Glycogen is the primary storage form of glucose in animals, held predominantly in the liver (up to 10% of liver biomass) and skeletal muscle (~2% of muscle biomass). Its architecture — glucose residues linked by α1→4 bonds in linear chains with α1→6 branch points every 12–15 residues, anchored to a central glycogenin dimer — creates a highly branched structure with numerous non-reducing ends that can be simultaneously acted upon by phosphorylase, enabling rapid glucose mobilization. In the liver, glycogen specifically buffers blood glucose levels; in muscle, it fuels local contraction. Maintaining blood glucose in a narrow range is critical above all for brain function: the brain accounts for approximately 10% of whole-body energy consumption, cannot synthesize or store significant glucose, and loses consciousness within 20 seconds of glucose deprivation, with permanent damage occurring within 4 minutes.

Homeostatic regulation of blood glucose is mediated by the opposing actions of two pancreatic hormones. Insulin, secreted by β cells of the islets of Langerhans in response to hyperglycemia, promotes glucose uptake and glycogen synthesis. Glucagon, secreted by α cells in response to hypoglycemia, promotes glycogenolysis and gluconeogenesis in the liver and the release of glucose into the circulation. This chapter focuses on insulin.

Insulin is a 51-amino-acid peptide hormone synthesized as preproinsulin. After co-translational insertion into the rough ER via its signal peptide, the signal peptide is cleaved to yield proinsulin, in which the A and B chains are connected by the C-peptide. Disulfide bond formation catalyzed by protein disulfide isomerase in the ER establishes the mature disulfide connectivity. The proinsulin is then packaged into Golgi-derived secretory vesicles, where carboxypeptidase E cleaves the C-peptide to generate mature insulin. The C-peptide is co-secreted in equimolar amounts with insulin, has a longer plasma half-life, and is excreted in urine — properties that make its detection clinically valuable for distinguishing endogenous from exogenous insulin and for differentiating type 1 from type 2 diabetes.

The insulin receptor is a disulfide-linked dimeric receptor tyrosine kinase embedded in the plasma membrane. Its extracellular domains contain primary (site-1) and secondary (site-2) insulin binding sites; maximal activation requires four insulin molecules bound symmetrically, producing a T-shaped receptor conformation. Upon insulin binding, the intracellular tyrosine kinase domains undergo autophosphorylation: a mobile activation loop that occupies the ATP binding site in the inactive state repositions upon activation, allowing ATP binding and transphosphorylation of key tyrosine residues. The activated receptor recruits and phosphorylates insulin receptor substrate-1 (IRS-1) on tyrosine residues, which in turn recruits the dimeric PI3-kinase (p85 regulatory/p110 catalytic subunits). PI3-kinase phosphorylates the membrane phospholipid PIP2 to generate PIP3, which recruits the serine/threonine kinase PDK to the membrane. PDK phosphorylates and activates Akt, which then dissociates into the cytosol.

Activated Akt drives two major downstream outcomes. First, Akt phosphorylates AS160, a GTPase-activating protein that normally stimulates GTP hydrolysis in Rab10, holding it in the inactive GDP-bound state. Phosphorylated AS160 cannot bind Rab10, allowing Rab10 to exchange GDP for GTP and become active. Active Rab10 promotes fusion of GLUT4-containing secretory vesicles with the plasma membrane, rapidly increasing surface GLUT4 density and glucose uptake — a faster response than transcriptional induction would allow. Glucose entering the cell is immediately phosphorylated to glucose-6-phosphate by hexokinase, trapping it intracellularly and directing it toward glycolysis, the pentose phosphate pathway, or glycogen synthesis. Second, Akt phosphorylates and inactivates glycogen synthase kinase-3 (GSK-3). Because GSK-3 normally phosphorylates and inactivates glycogen synthase, Akt's inactivation of GSK-3 allows protein phosphatase 1 (PP1) to dephosphorylate glycogen synthase, converting it to its more active form and stimulating glycogen synthesis. This double-negative regulatory mechanism elegantly couples the hormonal signal of insulin abundance to the enzymatic activation of glucose storage.