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Insulin Synthesis, Release, and Degradation
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The process involved in the synthesis and release of insulin, a polypeptide hormone, by the β-cells of the pancreas is similar to that of other peptide hormones, as discussed in Chapter 1 (Figure 1–2). Preproinsulin undergoes cleavage of its signal peptide during insertion into the endoplasmic reticulum, generating proinsulin (Figure 7–1). Proinsulin consists of an amino-terminal β-chain, a carboxy-terminal α-chain, and a connecting peptide; known as the C-peptide, that links the α- and β-chains. Linking of the 2 chains allows proper folding of the molecule and the formation of disulfide bonds between the 2 chains. In the endoplasmic reticulum, proinsulin is processed by specific endopeptidases, which cleave the C-peptide exposing the end of the insulin chain that interacts with the insulin receptor, generating the mature form of insulin. Insulin and the free C-peptide are packaged into secretory granules in the Golgi. These secretory granules accumulate in the cytoplasm in 2 pools; a readily releasable (5%) and a reserve pool of the granules (more than 95%). On stimulation, the β-cell releases insulin in a biphasic pattern; initially from the readily releasable pool followed by the reserve pool of granules. Only a small proportion of the cellular stores of insulin are released even under maximal stimulatory conditions. Insulin circulates in its free form, has a half-life of 3–8 minutes, and is degraded predominantly by the liver, with more than 50% of insulin degraded during its first pass. Additional degradation of insulin occurs in the kidneys as well as at target tissues by insulin proteases following endocytosis of the receptor-bound hormone.
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Exocytosis of secretory granule content results in the release of equal amounts of insulin and C-peptide into the portal circulation. The importance of C-peptide is that unlike insulin, it is not readily degraded in the liver. Thus, the relatively long half-life of the peptide (35 minutes) allows its release to be used as an index of the secretory capacity of the endocrine pancreas. C-peptide, may have some biologic action as recent evidence indicates that replacement of C-peptide improves renal function and nerve dysfunction in patients with type 1 diabetes. The receptor and signaling mechanisms involved in mediating these responses are still under investigation.
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The amino acid sequence of insulin is highly conserved among species. In the past, porcine and bovine insulin were used to treat patients with diabetes. Currently, human recombinant insulin is available and has replaced animal-derived insulin, avoiding problems such as the development of antibodies to nonhuman insulin.
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Regulation of Insulin Release
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The pancreatic β-cell functions as a neuroendocrine integrator that responds to changes in plasma levels of energy substrates (glucose and amino acids), hormones (insulin, glucagon-like peptide I, somatostatin, and epinephrine), and neurotransmitters (norepinephrine and acetylcholine) by increasing or decreasing insulin release (
Figure 7–2). Glucose is the principal stimulus for insulin release from the pancreatic β-cells. In addition, glucose exerts a permissive effect for the other modulators of insulin secretion.
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The glucose-induced stimulation of insulin release is the result of glucose metabolism by the β-cell (see
Figure 7–2). Glucose enters the β-cell through a membrane-bound glucose transporter 2 (GLUT 2) and undergoes immediate phosphorylation by glucokinase in the initial step of glycolysis, leading eventually to the generation of adenosine triphosphate (ATP) by the Krebs cycle. The resulting increase in intracellular ATP to adenosine diphosphate ratio inhibits (closes) the ATP-sensitive K
+ channels (K
ATP) in the β-cell, reducing the efflux of K
+. Decreased K
+ efflux results in membrane depolarization; activation (opening) of voltage-dependent Ca
2+ channels, and increased Ca
2+ influx. The increase in intracellular Ca
2+ concentrations triggers the exocytosis of insulin secretory granules and the release of insulin into the extracellular space and into the circulation. It is important to note that the regulation of K
+ channels by ATP is mediated by the sulfonylurea receptor. This is the basis for the therapeutic use of sulfonylurea drugs in the treatment of diabetes.
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The β-cell Ca2+ concentrations can also be elevated by amino acids through their metabolism and ATP generation, or by direct depolarization of the plasma membrane. Other factors (shown in Figure 7–2) that amplify the glucose-induced release of insulin from the β-cell include acetylcholine; cholecystokinin; gastrointestinal peptide, also known as glucose-dependent insulinotropic polypeptide; and glucagon-like peptide 1 (GLP 1). These substances all bind to cell surface receptors and trigger downstream signaling mechanisms controlling insulin release. Acetylcholine and cholecystokinin promote phosphoinositide breakdown, with a consequent mobilization of Ca2+ from intracellular stores, Ca2+ influx across the cell membrane, and activation of protein kinase C. GLP 1 increases levels of cyclic 3′,5′-adenosine monophosphate (cAMP) and activates cAMP-dependent protein kinase A. The generation of cAMP, inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonic acid and the activation of protein kinase C amplify the Ca2+ signal by decreasing Ca2+ uptake by cellular stores and promoting both the phosphorylation and activation of proteins that trigger the exocytosis of insulin. Catecholamines and somatostatin inhibit insulin secretion through G protein–coupled receptor mechanisms, inhibition of adenylate cyclase, and modification of Ca2+ and K+ channel gating.
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The short-term regulation of insulin release is mediated through modification of proinsulin mRNA translation. Over longer periods, glucose also increases proinsulin mRNA content by both stimulating proinsulin gene transcription and stabilizing the mRNA. As mentioned above, the release of insulin in response to glucose is biphasic, with an initial rapid release of preformed insulin followed by a more sustained release of newly synthesized insulin. This biphasic response to glucose is a major characteristic of glucose-stimulated insulin secretion. The first phase occurs over a period of minutes, the second over an hour or more. Several hypotheses have been proposed to explain the biphasic nature of insulin secretion; including the involvement of 2 separate pools of insulin granules.
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The release of insulin throughout the day is pulsatile and rhythmic in nature (see Figure 7–1). The pulsatile release of insulin is important for achieving maximal physiologic effects. In particular, it appears to be critical in the suppression of liver glucose production and in insulin-mediated glucose disposal by adipose tissue. Insulin release increases after a meal in response to the increases in plasma levels of glucose and amino acids. Secretion is the result of a combination of an increase in the total amount of insulin released in each secretory burst and an increased pulse frequency of a similar magnitude (see Figure 7–1). The synchronized increase in insulin release is thought to be the result of recruitment of β-cells to release insulin. Although it is not clear how the β-cells communicate with each other to synchronize the release of insulin, some of the proposed mechanisms include gap junctions allowing the passage of ions and small molecules; and propagation of membrane depolarization, aiding the synchronization between the cells. In addition, intra-pancreatic neural, hormonal, and substrate factors have been shown to play an important role in the pulsatile pattern of insulin release.
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Physiologic Effects of Insulin
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Insulin produces a wide variety of effects that range from immediate (within seconds), such as the modulation of ion (K+) and glucose transport into the cell; early (within minutes), such as the regulation of metabolic enzyme activity; moderate (within minutes to hours), such as the modulation of enzyme synthesis; to delayed (within hours to days), such as the effects on growth and cellular differentiation. Overall, the actions of insulin at target organs are anabolic and promote the synthesis of carbohydrate, fat, and protein, and these effects are mediated through binding to the insulin receptor (Table 7–1).
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The insulin receptor is part of the insulin-receptor family, which includes the insulin-like growth-factor receptor (Figure 7–3). The insulin receptor is a heterotetrameric glycoprotein membrane receptor composed of 2 α- and 2 β-subunits, linked by disulfide bonds. The extracellular α-chain is the site for insulin binding. The intracellular segment of the β-chain has intrinsic tyrosine kinase activity, which on insulin binding, undergoes autophosphorylation on tyrosine residues. The activated receptor phosphorylates tyrosine residues of several proteins known as insulin receptor substrates 1 through 4 (IRS-1–4); facilitating the interaction of the insulin receptor with intracellular substrates. The result is the coupling of insulin receptor activation to signaling pathways, mainly the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) pathways (see Figure 7–3).
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The PI3K pathway involves phosphorylation of inositol phospholipids and the generation of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate. These products, in turn, attract serine kinases to the plasma membrane, including the phosphoinositide-dependent kinase and different isoforms of protein kinase B, which, when activated, catalyze some of the cellular effects of insulin. The PI3K pathway is involved predominantly in mediating the metabolic effects of the hormone, including glucose transport, glycolysis, and glycogen synthesis, and plays a crucial role in the regulation of protein synthesis by insulin. Moreover, this pathway is involved in cell growth and transmits a strong antiapoptotic signal, promoting cell survival. The other main signaling pathway that is activated by insulin binding to its receptor is the MAPK pathway. Although signaling cascades in this pathway do not appear to play a significant role in the metabolic effects of insulin, they are involved in mediating the proliferative and differentiation effects elicited by insulin.
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Signal transduction by the insulin receptor is not limited to its activation at the cell surface. The activated ligand-receptor complex is internalized into endosomes. Endocytosis of activated receptors is thought to enhance the insulin receptor tyrosine kinase activity on substrates that are distant from those readily accessible at the plasma membrane. Following acidification of the endosomal lumen, insulin dissociates from its receptor, ending the insulin receptor-mediated phosphorylation events, and promoting the degradation of insulin by proteases such as the acidic insulinase. The insulin receptor can then be recycled into the cell surface, where it becomes available for insulin binding again.
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The number of available insulin receptors is modulated by exercise, diet, insulin, and other hormones. Chronic exposure to high insulin levels, obesity, and excess growth hormone all lead to a downregulation of insulin receptors. In contrast, exercise and fasting upregulate the number of receptors, improving insulin responsiveness.
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Insulin Effects at Target Organs
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Early Effects
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Although the expression of insulin receptors is widespread, the specific effects of insulin on skeletal muscle glucose utilization dominate insulin action. Insulin mediates approximately 40% of glucose disposal by the body, the great majority (80%–90%) of which occurs in skeletal muscle. The movement of glucose into the cell is mediated by glucose transporters, with their own unique tissue distribution, summarized in Table 7–2.
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Insulin-stimulated glucose transport is mediated through GLUT 4, most of which is sequestered intracellularly in the absence of insulin or other stimuli such as exercise. Insulin binding to its receptor results in increased GLUT 4 translocation through targeted exocytosis and decreased rate of its endocytosis. This is the underlying mechanism by which insulin stimulates glucose transport into fat and muscle cells.
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Intermediate Effects
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The intermediate effects of insulin are mediated by modulation of protein phosphorylation of enzymes involved in metabolic processes in muscle, fat, and liver (Figure 7–4). In fat, insulin inhibits lipolysis and ketogenesis by triggering the dephosphorylation of hormone- sensitive lipase and stimulates lipogenesis by activating acetylcoenzyme A (acetyl-CoA) carboxylase. Dephosphorylation of hormone-sensitive lipase inhibits the breakdown of triglycerides to fatty acids and glycerol, the rate-limiting step in the release of free fatty acids mediated by lipolysis. This process thereby reduces the amount of substrate that is available for ketogenesis. Insulin antagonizes catecholamine-induced lipolysis through the phosphorylation and activation of phosphodiesterase, leading to a decrease in intracellular cAMP levels and a concomitant decrease in protein kinase A activity.
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In the liver, insulin stimulates the gene expression of enzymes involved in glucose utilization (eg, glucokinase, pyruvate kinase) and lipogenic enzymes and inhibits the gene expression of enzymes involved in glucose production (eg, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) (see Figure 7–4). Insulin stimulates glycogen synthesis by increasing phosphatase activity, leading to the dephosphorylation of glycogen phosphorylase and glycogen synthase. In addition, insulin-mediated dephosphorylation of inhibitory sites on hepatic acetyl-CoA carboxylase increases the production of malonylcoenzyme A (malonyl-CoA) and simultaneously reduces the rate at which fatty acids can enter hepatic mitochondria for oxidation and ketone body production.
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In muscle, insulin stimulates glucose uptake and favors protein synthesis though phosphorylation of a serine/threonine protein kinase known as mammalian target of rapamycin (mTOR). In addition, insulin favors lipid storage in muscle as well as in adipose tissue. As discussed later, insulin deficiency leads to glucose accumulation in blood, a decrease in lipid storage, and protein loss, resulting in negative nitrogen balance and muscle wasting.
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Sustained insulin stimulation enhances the synthesis of lipogenic enzymes and the repression of gluconeogenic enzymes. The growth-promoting and mitogenic effects of insulin are long-term responses mediated through the MAPK pathway. Both MAPK and particularly, the chronic activation of extracellular receptor kinase by insulin-receptor binding, lead to excessive cell growth. Although this pathway of insulin action is not as well elucidated as the effects that are mediated through the activation of IRS-PI3K, evidence suggests its involvement in the pathophysiologic consequences of chronic insulin elevations as those that occur in insulin resistant individuals.
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Insulin levels are high (reflecting insulin resistance) during the development and early stages of type 2 diabetes. Chronic hyperinsulinemia has been linked to increased risk of cancers including endometrium, postmenopausal breast, colon, and kidney. Conditions that cause elevated insulin levels include high waist circumference, excess visceral fat, high waist-to-hip ratio, high body mass index, sedentary lifestyle, and high energy intake. In addition, the proliferative effects of chronic hyperinsulinemia influence vascular smooth muscle cells, which are responsible for the maintenance of vascular tone. These cells play an important role in the pathogenesis of several diseases, including hypertension, atherosclerosis, cardiovascular disease, and dyslipidemia, all of which are closely associated with insulin resistance and hyperinsulinemia. The molecular basis of insulin’s effect on vascular smooth muscle cell growth and its association with hypertension are currently unclear.
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Glucagon, is a 29–amino acid polypeptide hormone secreted by the α-cells of the islets of Langerhans, that plays an important role in the regulation of glucose homeostasis by producing antagonistic effects on insulin action. The primary sequence of glucagon is almost perfectly conserved among vertebrates, and it is structurally related to the secretin family of peptide hormones. Glucagon is synthesized as proglucagon and then proteolytically processed to yield glucagon. The prohormone proglucagon is expressed in the pancreas, and also in other tissues, such as enteroendocrine cells in the intestinal tract and in the brain. However, the processing of the prohormone differs among tissues. The 2 main products of proglucagon processing are glucagon in the α-cells of the pancreas and GLP 1 in the intestinal cells. GLP 1 is produced in response to a high concentration of glucose in the intestinal lumen. GLP 1 is known as an incretin, a mediator that amplifies insulin release from the β-cell in response to a glucose load. Glucagon has a short half-life (5–10 minutes) and is degraded mostly in the liver.
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Regulation of Glucagon Release
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The mechanisms involved in the regulation and stimulus-secretion coupling of glucagon release are not as well understood as those for insulin. Glucagon release is inhibited by hyperglycemia (high blood-glucose levels) and stimulated by hypoglycemia (low blood-glucose levels). A meal rich in carbohydrates suppresses glucagon release and stimulates insulin release from the β-cells through intestinal release of GLP 1. Somatostatin also inhibits glucagon release. High amino acid levels following an amino acid–rich meal stimulate glucagon release. Epinephrine stimulates release of glucagon through a β2-adrenergic mechanism (whereas it suppresses insulin release from β-cells through an α2-adrenergic mechanism). Vagal (parasympathetic) stimulation increases glucagon release.
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Physiologic Effects of Glucagon
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The principal target tissue for glucagon is the liver. Glucagon’s main physiologic effect is to increase plasma glucose concentrations by stimulating de novo hepatic glucose production through gluconeogenesis and glycogen breakdown; overall, these actions counteract the effects of insulin (Figure 7–5).
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Glucagon mediates its effects by binding to the glucagon Gαs protein–coupled receptor. The glucagon and GLP 1 peptide receptors belong to a family of G protein–coupled receptors that include those for secretin, calcitonin, vasoactive intestinal polypeptide, parathyroid hormone, and growth hormone-releasing factor. The glucagon receptor is expressed in liver, pancreatic β-cells, kidney, adipose tissue, heart, and vascular tissues, as well as in some regions of the brain, stomach, and adrenal glands. Glucagon binding activates adenylate cyclase and results in intracellular accumulation of cAMP, mobilization of intracellular Ca2+, protein kinase A activation, and phosphorylation of effector proteins. The glucagon-receptor complex undergoes endocytosis into intracellular vesicles, where glucagon is degraded. The role of glucagon receptors in tissues other than the liver is still unclear.
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Glucagon Effects at Target Organs
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Glucagon stimulates hepatic glucose output by stimulating glycogen breakdown and gluconeogenesis and decreasing glycolysis (see
Figure 7–5). The key enzymatic steps regulated by glucagon that mediate the stimulation of hepatic glucose output are summarized in
Table 7–3. Glucagon has effects on adipose tissue that are relevant primarily during periods of stress or food deprivation, particularly when insulin release is suppressed.
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In the adipocyte, glucagon stimulates protein kinase A–mediated phosphorylation (activation) of hormone-sensitive lipase, the enzyme that breaks down triglycerides (stored fat) into diacylglycerol and free fatty acids, releasing them into the circulation. Glycerol released into the circulation can be utilized in the liver for gluconeogenesis or for reesterification. Free fatty acids are used as fuel by most tissues, predominantly skeletal muscle and liver. In the liver, free fatty acids are used for reesterification, or they undergo β-oxidation and conversion into ketone bodies (
Figure 7–6). Thus, ketogenesis is regulated by the balance between the effects of glucagon and insulin at their target organs. The importance of this balance is evident during insulin deficiency and glucagon excess, as seen in uncontrolled diabetes (discussed later).
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Somatostatin is a 14–amino acid peptide hormone produced by the δ-cells of the pancreas. Its release is stimulated by high-fat, high-carbohydrate, and particularly protein-rich meals, and is inhibited by insulin. Somatostatin has a generalized inhibitory effect on virtually all gastrointestinal and pancreatic exocrine and endocrine functions. The regulation of its release is not well studied because of the difficulty in analyzing the small number of islet cells that produce this hormone. Further, the importance of endogenous paracrine inhibition of insulin and glucagon release is not well established. Because δ-cells are located in the periphery of the β-cells and because blood flows from the center of the islets of Langerhans toward the periphery, pancreatic somatostatin may have a limited contribution toward physiologic control of insulin and glucagon release. However, exogenous administration of somatostatin does suppress the release of both insulin and glucose and is used in the clinical setting for the management of insulin or glucagon producing tumors.
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Pancreatic Polypeptide
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Pancreatic polypeptide is a 36–amino acid peptide hormone that belongs to a peptide family including neuropeptide Y and peptide YY. It is produced in the endocrine type F cells located in the periphery of pancreatic islets and is released into the circulation after a meal, exercise, and vagal stimulation. The effects of pancreatic polypeptide include inhibition of pancreatic exocrine secretion, gallbladder contraction, modulation of gastric acid secretion, and gastrointestinal motility. Pancreatic polypeptide crosses the blood-brain barrier and has been postulated to play a role in regulating feeding behavior.
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Amylin, or islet amyloid polypeptide, is a 37–amino acid peptide hormone that belongs to the calcitonin family (calcitonin, calcitonin gene-related peptide, and adrenomedullin). Amylin is synthesized as a small precursor, undergoes posttranslational modification (amidation), is stored in β-granules, and is released along with insulin and C-peptide. Plasma amylin concentrations increase after a meal or glucose infusion. Amylin appears to work with insulin to regulate plasma glucose concentrations in the bloodstream, suppressing the postprandial secretion of glucagon and slowing gastric emptying. In muscle, amylin opposes glycogen synthesis and activates glycogenolysis and glycolysis, thereby increasing lactate production. Circulating amylin is increased in obesity, hypertension, and gestational diabetes; it is low or absent in type 1 diabetes mellitus. Amylin is the main component of pancreatic islet amyloid, found in the vast majority of patients with non-insulin-dependent (type 2) diabetes mellitus, and is thought to contribute to destruction of the pancreatic β-cell. Amylin binds to a variant of the calcitonin G protein–coupled receptor. The modified calcitonin receptor has higher affinity for amylin, an effect mediated by transmembrane proteins known as receptor activity modifying proteins.