Insulin

Insulin is a small acidic protein formed from the larger proinsulin precursor. Proinsulin is synthesized and packaged for secretion with trypsin-like proteases in the β cells of the islets of Langerhans. The precursor protein is proteolyzed within the secretory granule to form insulin via cleavage of a sequence of amino acids referred to as the C (connecting) peptide. Insulin is complexed with Zn⁺⁺ within the granule. The active insulin protein is composed of an A peptide (21 amino acids), which has an intramolecular disulfide bond, and a B peptide (30 amino acids) that are covalently joined by two disulfide bonds (Fig. 43-2). Approximately equimolar amounts of insulin and C peptide are stored in and released from the granule, along with a much smaller amount of proinsulin.

FIGURE 43–2 Primary structures of insulin and glucagon. The amino acid sequences of the 21-amino acid A chain and the 30-amino acid B chain of human insulin, and the 29 amino acids in human glucagon are shown. The single letter code for the amino acids is consistent with accepted nomenclature. The two interchain disulfide bridges and the intrachain disulfide in the A chain of insulin are indicated. Residues in beef or pork insulin that differ from those in human insulin are shown above or below the A and B chain sequences. Also depicted are the amino acids in glargine insulin, lispro insulin, and aspart insulin that differ from those in unmodified insulin.

Insulin release is modulated by many factors (Box 43-1) but is controlled primarily by glucose. When blood glucose levels increase, glucose is taken up and metabolized by the pancreatic β cell, generating adenosine triphosphate (ATP) (Fig. 43-3). The increase in the ATP/ADP ratio promotes closure of the inward rectifying potassium channel 6.2 subunit (K_ir6.2) of the ATP-sensitive potassium channel (also referred to as the SUR1/K_ir6.2 channel). The decreased permeability of potassium ions partially depolarizes the cell membrane, promoting calcium uptake via activation of its voltage-gated channels. The elevated intracellular calcium stimulates exocytosis of the granules, releasing insulin and other components into the circulation.

FIGURE 43–3 Stimulation of insulin secretion by glucose and oral hypoglycemic agents involves partial depolarization of the β cell plasma membrane. After an increase in blood glucose (Glc), more Glc enters the β cell via the glucose transporter, GLUT2. The Glc is phosphorylated by glucokinase to glucose 6-phosphate (G6P), which is subsequently metabolized via glycolysis and the TCA cycle, increasing intracellular ATP and decreasing ADP. The resulting increase in the ATP/ADP ratio results in inhibition of the inwardly rectifying K⁺ channel, Kir6.2, resulting in partial membrane depolarization, triggering activation of voltage-gated Ca⁺⁺ channels. The rise in intracellular Ca⁺⁺ stimulates exocytosis of secretory granules containing insulin. The sulfonylureas and meglitinides bind to sites on the ATP-sensitive K⁺ channel to inhibit channel conductance, resulting in membrane depolarization, and Ca⁺⁺ entry, promoting insulin release.

Measurement of the C peptide can be used clinically to estimate insulin secretion. If circulating insulin levels are appropriate, hepatic glucose output is suppressed, and glucose uptake by skeletal muscle and adipocytes is stimulated, resulting in only a transient increase in blood glucose levels. When administered as a drug, insulin lowers blood glucose by mimicking the effects of the endogenous hormone. The disappearance of insulin is regulated by proteolytic systems or insulinase activity in a variety of tissues, with the liver being the most prominent. Almost half of the insulin released by the pancreas into the portal vein is destroyed by hepatic degradation.

The cellular effects of insulin are initiated after insulin binds to a plasma membrane receptor (Fig. 43-4). The insulin receptor is composed of two α-subunits and two β-subunits. The interaction of insulin with the α-subunit results in changes in the β-subunit configuration, leading to activation of the tyrosine protein kinase that resides in the β-subunit. The phosphorylation of peptide substrates by the insulin receptor tyrosine kinase leads to activation of various anabolic pathways, inhibition of catabolic processes, and subsequently modulation of gene expression. The major peptide substrates for the insulin receptor are insulin receptor substrate (IRS)-1 and IRS-2. Phosphotyrosine residues in the IRS proteins serve as binding sites for intermediaries that trigger signal transduction pathways. The best defined of these pathways involves the small guanosine triphosphate (GTP)-binding protein Ras and leads to cell growth, differentiation, or both. However, the most important acute metabolic effects of insulin are mediated by phosphatidylinositol 3-kinase (PI 3-kinase), which is activated upon binding to phosphorylated IRS proteins. The phospholipid products generated by PI 3-kinase promote activation of several protein kinases including protein kinase B(Akt) and protein kinase C (PKC). These kinases phosphorylate effectors that activate glucose transporters (GLUT) and glycogen synthase (GS) and increase the synthesis of triglyceride and protein. Phosphotyrosine phosphatases are also activated that limit the duration of effects promoted by phosphotyrosines.

FIGURE 43–4 Major signal transduction pathways that mediate insulin action. Insulin (I) binding to its receptor results in increased tyrosine (Y) phosphorylation of the adapter proteins, with src humology sequences (Shc) and insulin receptor substrate (IRS) proteins 1 and 2. This creates docking sites for downstream effectors, which bind to phosphotyrosine-containing sites. One such effector is the Grb-2/mSOS complex, which is activated when it binds to either Shc or IRS proteins. Grb-2/mSOS activates Ras, which triggers the sequential phosphorylation and activation of the following kinases: Raf, MEK, mitogen-activated protein kinase (MAPK), and Rsk. This pathway is involved in the proliferative response of cells to insulin and several growth factors. The MAPK pathway is also activated by the protein tyrosine phosphatase SHP-2, which is recruited to IRS proteins in response to insulin. PI 3-kinase uses two SH domains in its p85 regulatory subunit to bind phosphorylated IRS proteins. Binding activates the p110 catalytic subunit of PI 3-kinase, generating phospholipid products that activate several downstream protein kinases, including Akt and isoforms of protein kinase C (PKC). These downstream kinases phosphorylate regulatory proteins in the cell, leading to the activation of glucose transport and to increases in the rates of synthesis of glycogen, lipid, and protein.

Stimulation of glycogen synthesis is very important in the action of insulin to lower blood glucose concentrations. Glucose taken up in response to insulin is deposited as glycogen in liver and skeletal muscle. Activation of glucose transport and GS contributes to the overall effects of insulin on glycogen synthesis. Increasing glucose transport allows more glucose to enter the muscle fiber, and activation of GS converts the glucose into glycogen. The control of GS activity is very important in this process and involves phosphorylation/dephosphorylation mechanisms (Fig. 43-5). GS is inactivated upon phosphorylation by the kinase GSK-3 and activated upon dephosphorylation by the protein phosphatase PP1G. In addition, phosphorylation of GSK-3 by Akt inactivates the kinase. Thus activation of Akt by insulin (see Fig. 43-4) leads to the inactivation of GSK-3, thereby tipping the balance to favor activation of GS by PP1G. In addition, by stimulating glucose transport via GLUT4, insulin increases the concentration of intracellular glucose 6-phosphate, an allosteric activator of GS. Insulin stimulates glucose transport in skeletal muscle and adipocytes by causing GLUT4 to translocate from intracellular vesicular compartments to the plasma membrane, an action promoted by signals from arising from Akt, PKC, or both (see Fig. 43-5). Increasing the number of glucose transporters at the cell surface increases glucose uptake into the cell. Interruption of any of these processes can be associated with genomic-associated insulin resistance.

FIGURE 43–5 Stimulation of glycogen (Gly) synthesis by insulin involves activation of both glucose transport and glycogen synthase (GS). Insulin stimulates glucose transport by promoting movement of glucose transporter (GLUT4)-containing vesicles (GTV) to the plasma membrane. Translocation occurs in response to activation of Akt, PKC, or both. After vesicle fusion, the number of GLUT4 molecules at the cell surface is increased. Glucose (Glc) is then able to cross the membrane more rapidly and is phosphorylated in the cell to glucose 6-phosphate. A portion of the glucose 6-phosphate is metabolized by the hexose monophosphate pathway (HMP). This pathway, which is stimulated by insulin, generates pentoses for nucleotide synthesis and reducing equivalents for fatty acid synthesis. Another portion of glucose 6-phosphate enters the glycolytic pathway. Pyruvate generated from glycolysis enters the tricarboxylic acid (TCA) cycle and is metabolized to CO₂, resulting in the generation of ATP. The bulk of the glucose that enters a muscle fiber in response to insulin is converted to glycogen (Gly). The glucose 6-phosphate is isomerized to glucose 1-phosphate, which is converted to uridine diphosphoglucose (UDPG), the substrate for glycogen synthase (GS), the enzyme that synthesizes glycogen. Insulin activates GS by increasing the activity of Akt, which phosphorylates and inactivates GSK-3. This allows the protein phosphatase, PP1G, to predominate, thereby increasing the fraction of GS that is in the dephosphorylated, active form.