Beta cells occupy the central portion of the islet and are surrounded by a "rind" of alpha and delta cells.

Aside from the insulin, glucagon and somatostatin, a number of other "minor" hormones have been identified as products of pancreatic islets cells.

 

Structure of Insulin Insulin is a rather small protein, with a molecular weight of about 6000 Daltons. It is composed of an A chain and a larger B chain held together by disulfide bonds. The amino acid sequence is highly conserved among vertebrates, and insulin from one mammal almost certainly is biologically active in another. Even today, many diabetic patients are treated with insulin extracted from pig pancreas. Insulin is composed of two peptide chains referred to as the A chain and B chain.A and B chains are linked together by two disulfide bonds, and an additional disulfide is formed within the A chain. In most species, the A chain consists of 21 amino acids and the B chain of 30 amino acids. The molecule viewer below can be used to examine the structure of bovine insulin. Setting the Color parameter to "Chain" will color the A chain green and the B chain red. Sorry: This molecule viewer will not be visible because your browser does not support Java applets Although the amino acid sequence of insulin varies among species, certain segments of the molecule are highly conserved, including the positions of the three disulfide bonds, both ends of the A chain and the C-terminal residues of the B chain. These similarities in the amino acid sequence of insulin lead to a three dimensional conformation of insulin that is very similar among species, and insulin from one animal is very likely biologically active in other species. Indeed, pig insulin has been widely used to treat human patients. Insulin molecules have a tendency to form dimers in solution due to hydrogen-bonding between the C-termini of B chains. Additionally, in the presence of zinc ions, insulin dimers associate into hexamers. These interactions have important clinical ramifications. Monomers and dimers readily diffuse into blood, whereas hexamers diffuse poorly. Hence, absorption of insulin preparations containing a high proportion of hexamers is delayed and somewhat slow. This phenomenon, among others, has stimulated development of a number of recombinant insulin analogs. The first of these molecules to be marketed - called insulin lispro - is engineered such that lysine and proline residues on the C-terminal end of the B chain are reversed; this modification does not alter receptor binding, but minimizes the tendency to form dimers and hexamers.

Biosynthesis of Insulin Insulin is synthesized in significant quantities only in beta cells in the pancreas. The insulin mRNA is translated as a single chain precursor called preproinsulin, and removal of its signal peptide during insertion into the endoplasmic reticulum generates proinsulin. Proinsulin consists of three domains: an amino-terminal B chain, a carboxy-terminal A chain and a connecting peptide in the middle known as the C peptide. Within the endoplasmic reticulum, proinsulin is exposed to several specific endopeptidases which excise the C peptide, thereby generating the mature form of insulin. Insulin and free C peptide are packaged in the Golgi into secretory granules which accumulate in the cytoplasm. When the beta cell is appropriately stimulated, insulin is secreted from the cell by exocytosis and diffuses into islet capillary blood. C peptide is also secreted into blood, but has no known biological activity. Control of Insulin Secretion Insulin is secreted in primarily in response to elevated blood concentrations of glucose. This makes sense because insulin is "in charge" of facilitating glucose entry into cells. Some neural stimuli (e.g. sight and taste of food) and increased blood concentrations of other fuel molecules, including amino acids and fatty acids, also promote insulin secretion. Our understanding of the mechanisms behind insulin secretion remain somewhat fragmentary. Nonetheless, certain features of this process have been clearly and repeatedly demonstrated, yielding the following model: Glucose is transported into the beta cell by facilitated diffusion through a glucose transporter; elevated concentrations of glucose in extracellular fluid lead to elevated concentrations of glucose within the beta cell. Elevated concentrations of glucose within the beta cell ultimately leads to membrane depolarization and an influx of extracellular calcium. The resulting increase in intracellular calcium is thought to be one of the primary triggers for exocytosis of insulin-containing secretory granules. The mechanisms by which elevated glucose levels within the beta cell cause depolarization is not clearly established, but seems to result from metabolism of glucose and other fuel molecules within the cell, perhaps sensed as an alteration of ATP:ADP ratio and transduced into alterations in membrane conductance. Increased levels of glucose within beta cells also appears to activate calcium-independent pathways that participate in insulin secretion. Stimulation of insulin release is readily observed in whole animals or people. The normal fasting blood glucose concentration in humans and most mammals is 80 to 90 mg per 100 ml, associated with very low levels of insulin secretion. The figure to the right depicts the effects on insulin secretion when enough glucose is infused to maintain blood levels two to three times the fasting level for an hour. Almost immediately after the infusion begins, plasma insulin levels increase dramatically. This initial increase is due to secretion of preformed insulin, which is soon significantly depleted. The secondary rise in insulin reflects the considerable amount of newly synthesized insulin that is released immediately. Clearly, elevated glucose not only simulates insulin secretion, but also transcription of the insulin gene and translation of its mRNA.

Adults normally secrete approximately 50 units of insulin each day from the β cells. The rate of insulin secretion is primarily determined by the plasma glucose concentration. Insulin facilitate glucose and potassium entry into adipose and muscle cells; increasing glycogen, protein, and fatty acid synthesis; and decreasing glycogenolysis, gluconeogenesis, ketogenesis, lipolysis, and protein catabolism.

 

Table 34-1 Effects of Insulin.1

Effects on liver Anabolic Promotes glycogenesis Increases synthesis of triglycerides, cholesterol, and VLDL2 Increases protein synthesis Promotes glycolysis Anticatabolic Inhibits glycogenolysis Inhibits ketogenesis Inhibits gluconeogenesis

Effects on muscle Anabolic Increases amino acid transport Increases protein synthesis Anticatabolic Increases glucose transport Enhances activity of glycogen synthetase Inhibits activity of glycogen phosphorylase

Effects on fat Promotes triglyceride storage Induces lipoprotein lipase, making fatty acids available for absorption into fat cells Increases glucose transport into fat cells, thus increasing availability of α-glycerol phosphate for triglyceride synthesis Inhibits intracellular lipolysis

1Reproduced, with permission, from Gardner DG, Shoback D (editors): Greenspan’s Basic & Clinical Endocrinology, 9th edition, McGraw-Hill, 2011.

2VLDL, very low-density lipoprotein.

 

 

 

A. Insulin is a peptide hormone synthesized from preproinsulin. Preproinsulin undergoes posttranslational modification in the endoplasmic reticulum (ER) to form proinsulin. The active form of insulin is produced by modification of proinsulin by cleavage of the C-peptide structure linking the alpha and beta chains. Both insulin and the cleaved C-peptide are packaged in secretory granules and are coreleased in response to glucose stimulation. B. Insulin release occurs in a biphasic mode; from readily releasable secretory granules and from granules that must undergo a series of preparatory reactions including mobilization to the plasma membrane. C. In response to a meal, the increase in insulin release results from a higher frequency and amplitude of pulsatile release. Shown are portal insulin concentrations during basal state (left) and after a meal.

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 inChapter 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.

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.

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.

Regulation of Insulin Release

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.

Regulation of insulin release. Glucose is the principal stimulus for insulin release from the pancreatic β-cell. Glucose enters the β-cell cell by a specific glucose transporter protein (GLUT 2) undergoes glycolysis leading to generation of ATP. The increased ATP/ADP ratio leads to inhibition and closure of the ATP-sensitive K+ channels (the target of sulfonylurea drugs), resulting in plasma membrane depolarization and opening of the voltage-dependent Ca2+ channels. The increased Ca2+ influx coupled with mobilization of Ca2+ from intracellular stores leading to the fusion of insulin-containing secretory granules with the plasma membrane and the release of insulin (and C-peptide) into the circulation. Addition factors can also stimulate insulin release from the β-cell, including hormones (glucagon-like peptide 1) and neurotransmitters (acetylcholine). Glucose synergizes with these mediators and enhances the secretory response of the β-cell to these factors. AC, adenylate cyclase; ADP, adenosine diphosphate; ATP, adenosine triphosphate; CCK, cholecystokinin; GLP 1, glucagon-like peptide-1; PLC, phospholipase C. (Modified, with permission, from Fajans SS, Bell GI, Polonsky KS. Mechanisms of disease: molecular mechanisms and clinical pathophysiology of maturity-onset diabetes of the young. N Engl J Med. 2001;345:971. Copyright © Massachusetts Medical Society. All rights reserved.)

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 ofadenosine triphosphate (ATP) by the Krebs cycle. The resulting increase in intracellular ATP to adenosine diphosphate ratio inhibits (closes) the ATP-sensitive K+ channels (KATP) in the β-cell, reducing the efflux of K+. Decreased K+ efflux results in membrane depolarization; activation (opening) of voltage-dependent Ca2+ channels, and increased Ca2+ influx. The increase in intracellular Ca2+ 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.

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.

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.

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.

Physiologic Effects of Insulin

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).

Insulin Receptor

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).

Insulin receptor signaling. Insulin binding to the receptor activates the intrinsic kinase activity of the intracellular domain of the receptor. This results in downstream activation of cellular events mediated through the phosphorylation of insulin receptor substrates (IRS). Downstream signaling pathways involved in insulin-mediated effects including the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) cascades. Activation of phosphoinositide-3 kinase is a major pathway in the mediation of insulin-stimulated glucose transport and metabolism. Among the immediate effects of insulin is the active recruitment of glucose transporter 4 (GLUT 4), stored in intracellular vesicles to the cell surface. Exercise can also stimulate glucose transport by pathways that are independent of phosphoinositide-3 kinase and thought to involve 5′-adenosine monophosphate (AMP)-activated kinase.

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.

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.

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.

Insulin Effects at Target Organs

Early Effects

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.

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.

Intermediate Effects

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.

Glucagon and insulin effects on hepatic glucose metabolism. Binding of glucagon and insulin to their respective receptors stimulates a cascade of protein phosphorylation steps that activate (or inhibit) key enzymes involved in the regulation of glycogenolysis, gluconeogenesis, and glycolysis. The principal target enzymes for insulin- and glucagon-mediated effects are presented. The overall result is an increase in hepatic glucose output. ADP, adenosine diphosphate; ATP, adenosine triphosphate; G, glucagon; I, insulin; PEP, phosphoenolpyruvate.

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.

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.

Long-Term Effects

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.

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.

Ingested glucose is the primary stimulant of insulin release from the β cells of the pancreas. Insulin’s main action occurs at the three principal tissues of energy storage and metabolism (i.e., liver, adipose tissue, and skeletal muscle). Insulin acts on the liver to facilitate the uptake of glucose and its conversion to glycogen while inhibiting glycogen breakdown (glycogenolysis) and suppressing gluconeogenesis. The net effect of these actions is to promote the storage of glucose in the form of glycogen. Insulin increases lipogenesis in the liver and adipose cells by producing triglycerides from free fatty acids and glycerol while inhibiting the breakdown of triglycerides. Insulin stimulates the uptake of amino acids into muscle cells with subsequent incorporation into muscle protein while preventing the release of amino acids from muscle and hepatic protein sources.

Deficiency in insulin secretion due to loss of islet cell mass is the predominant lesion in diabetes mellitus, and it may be partial or total.