Insulin

Insulin ( ; from Latin insula 'island') is a peptide hormone synthesized by the beta cells within the pancreatic islets, with its production in humans encoded by the insulin (INS) gene. It functions as the body's principal anabolic hormone. Its primary role involves regulating the metabolism of carbohydrates, lipids, and proteins by facilitating glucose absorption from the bloodstream into hepatic, adipose, and skeletal muscle cells. Within these tissues, the assimilated glucose is transformed into either glycogen through glycogenesis or triglycerides via lipogenesis; notably, in the liver, both conversions occur. Elevated concentrations of insulin in the blood significantly inhibit hepatic glucose production and secretion. Furthermore, circulating insulin influences protein synthesis across a diverse range of tissues. Therefore, it acts as an anabolic hormone, fostering the conversion of smaller circulating molecules into larger cellular macromolecules. Conversely, diminished blood insulin levels induce widespread catabolism, particularly impacting stored body fat.

Insulin ( ; from Latin insula 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (INS) gene. It is the main anabolic hormone of the body. It regulates the metabolism of carbohydrates, fats, and protein by promoting the absorption of glucose from the blood into cells of the liver, fat, and skeletal muscles. In these tissues the absorbed glucose is converted into either glycogen, via glycogenesis, or fats (triglycerides), via lipogenesis; in the liver, glucose is converted into both. Glucose production and secretion by the liver are strongly inhibited by high concentrations of insulin in the blood. Circulating insulin also affects the synthesis of proteins in a wide variety of tissues. It is thus an anabolic hormone, promoting the conversion of small molecules in the blood into large molecules in the cells. Low insulin in the blood has the opposite effect, promoting widespread catabolism, especially of reserve body fat.

Beta cells exhibit sensitivity to blood glucose concentrations, releasing insulin into the bloodstream in response to hyperglycemia and suppressing its secretion when glucose levels are low. Insulin synthesis is also modulated by glucose; elevated glucose concentrations stimulate insulin production, whereas reduced levels result in decreased synthesis. Insulin facilitates cellular glucose uptake and metabolism, consequently lowering blood glucose. Conversely, adjacent alpha cells, influenced by beta cell activity, secrete glucagon into the blood in an inverse pattern: secretion increases during hypoglycemia and decreases when glucose concentrations are high. Glucagon elevates blood glucose by stimulating hepatic glycogenolysis and gluconeogenesis. The coordinated secretion of insulin and glucagon into the bloodstream, in response to blood glucose concentration, constitutes the principal mechanism of glucose homeostasis.

Insufficient or absent insulin activity leads to diabetes, a medical condition characterized by elevated blood glucose levels (hyperglycemia). This disease manifests in two primary forms. In type 1 diabetes, an autoimmune reaction causes the destruction of beta cells, thereby preventing insulin synthesis and secretion into the bloodstream. In contrast, type 2 diabetes involves less severe beta cell destruction compared to type 1, and this is not attributed to an autoimmune process. Instead, amyloid accumulates within the pancreatic islets, which is presumed to impair their anatomical structure and physiological function. While the precise pathogenesis of type 2 diabetes remains incompletely elucidated, contributing factors are recognized to include a diminished population of islet beta-cells, impaired secretory function of surviving islet beta-cells, and peripheral tissue insulin resistance. Type 2 diabetes is further characterized by heightened glucagon secretion, which remains unaffected by and unresponsive to blood glucose concentrations. However, insulin continues to be secreted into the blood in response to blood glucose. Consequently, glucose accumulates in the bloodstream.

Human insulin protein comprises 51 amino acids and possesses a molecular mass of 5808 Da. It exists as a heterodimer, consisting of an A-chain and a B-chain, which are covalently linked by disulfide bonds. The structural configuration of insulin exhibits minor variations across different animal species. Due to these interspecies differences, insulin derived from non-human animal sources demonstrates somewhat varied efficacy in carbohydrate metabolism compared to human insulin. Porcine insulin, being particularly similar to its human counterpart, was extensively utilized for treating individuals with type 1 diabetes prior to the large-scale production of human insulin via recombinant DNA technologies.

Insulin holds the distinction of being the inaugural peptide hormone identified. In 1921, Frederick Banting and Charles Best, conducting research in John Macleod's laboratory at the University of Toronto, successfully isolated insulin from canine pancreatic tissue. Frederick Sanger subsequently elucidated its complete amino acid sequence in 1951, marking insulin as the first protein to be fully sequenced. Dorothy Hodgkin determined the crystal structure of solid-state insulin in 1969. Furthermore, insulin was the first protein to be both chemically synthesized and manufactured using recombinant DNA technology. It is included in the WHO Model List of Essential Medicines, signifying its importance as a fundamental medication within basic healthcare systems.

Evolutionary Trajectory and Interspecies Distribution

Insulin's molecular origins likely extend over a billion years, tracing back to the earliest unicellular eukaryotes. Beyond the animal kingdom, insulin-like proteins have been identified in fungi and protists.

In most vertebrates, insulin is synthesized by the beta cells within the pancreatic islets, while certain teleost fish produce it in the Brockmann body. Notably, venomous sea snails, specifically Conus geographus and Conus tulipa, employ modified insulin forms within their venom cocktails to incapacitate small fish. This insulin toxin, structurally more akin to fish insulin than to the snails' endogenous insulin, reduces the prey's blood glucose levels, thereby slowing them.

Production

In mammals, insulin is exclusively generated by the beta cells of the pancreatic islets, whereas in some fish, it originates from the Brockmann body. Human insulin synthesis is directed by the INS gene, situated on chromosome 11. Rodents possess two functional insulin genes: one is a homolog of most mammalian genes (Ins2), and the other is a retroposed copy, containing a promoter sequence but lacking an intron (Ins1). Insulin gene transcription escalates in response to elevated blood glucose, a process primarily regulated by transcription factors that bind to enhancer sequences approximately 400 base pairs upstream of the gene's transcription start site.

Key transcription factors that regulate insulin secretion include PDX1, NeuroD1, and MafA.

In conditions of low glucose, PDX1 (pancreatic and duodenal homeobox protein 1) resides in the nuclear periphery due to its interaction with HDAC1 and HDAC2, leading to a downregulation of insulin secretion. Conversely, elevated blood glucose levels induce PDX1 phosphorylation, prompting its nuclear translocation and subsequent binding to the A3 element within the insulin promoter. Following translocation, PDX1 engages with coactivators HAT p300 and SETD7. PDX1 influences histone modifications through acetylation, deacetylation, and methylation, and is also reported to suppress glucagon.

NeuroD1, also identified as β2, governs insulin exocytosis in pancreatic β cells by directly stimulating the expression of genes crucial for this process. While typically cytosolic, β2 undergoes glycosylation by OGT and/or phosphorylation by ERK in response to high glucose, triggering its nuclear translocation. Within the nucleus, β2 heterodimerizes with E47, binds to the E1 element of the insulin promoter, and recruits the co-activator p300, which acetylates β2. Furthermore, β2 can interact with other transcription factors to activate the insulin gene.

MafA undergoes proteasomal degradation when blood glucose levels are low. Elevated glucose concentrations lead to the glycosylation of an unidentified protein, which then functions as a transcription factor for MafA through an unknown mechanism, causing MafA to be transported out of the cell. Subsequently, MafA translocates back into the nucleus, where it binds to the C1 element of the insulin promoter.

These transcription factors operate synergistically within an intricate regulatory network. Prolonged hyperglycemia can impair the binding capacities of these proteins, consequently diminishing insulin secretion and contributing to diabetes. This reduction in binding activity may be mediated by glucose-induced oxidative stress, and antioxidants are reported to mitigate decreased insulin secretion in glucotoxic pancreatic β cells. Furthermore, stress signaling molecules and reactive oxygen species inhibit the insulin gene by interfering with both the transcription factors themselves and their binding cofactors.

The promoter region of the human insulin gene contains several regulatory sequences that bind to specific transcription factors. Generally, A-boxes interact with Pdx1 factors, E-boxes with NeuroD, C-boxes with MafA, and cAMP response elements with CREB. Additionally, silencer elements are present, which inhibit transcription.

Synthesis

Insulin is synthesized initially as preproinsulin, an inactive precursor protein comprising 110 amino acids. This preproinsulin undergoes direct translation within the rough endoplasmic reticulum (RER), where signal peptidase subsequently excises its signal peptide, yielding proinsulin. During the folding process of proinsulin, its opposing termini, designated as the "A-chain" and "B-chain," become covalently linked by three disulfide bonds. Subsequently, the folded proinsulin traverses the Golgi apparatus and is encapsulated within specialized secretory vesicles, also known as granules. Within these granules, proinsulin undergoes proteolytic cleavage by proprotein convertase 1/3 and proprotein convertase 2, which excises the central segment of the protein, termed the "C-peptide." Ultimately, carboxypeptidase E facilitates the removal of two amino acid pairs from the protein's termini, culminating in the formation of active insulin, characterized by its A- and B-chains now interconnected by two disulfide bonds.

The mature insulin thus formed is stored within mature granules, awaiting specific metabolic stimuli (e.g., leucine, arginine, glucose, and mannose) and vagal nerve activation to trigger its exocytosis from the cell into the systemic circulation.

Research indicates that insulin and its associated proteins are synthesized within the brain, and diminished concentrations of these proteins are correlated with Alzheimer's disease.

Insulin secretion is further stimulated by beta-2 receptor activation and suppressed by alpha-1 receptor activation. Furthermore, during periods of physiological stress, cortisol, glucagon, and growth hormone exert antagonistic effects on insulin's actions. Additionally, insulin suppresses the release of fatty acids mediated by hormone-sensitive lipase within adipose tissue.

Structure

Initially, hormones were presumed to be relatively small chemical entities; however, insulin, as the first peptide hormone whose structure was elucidated, proved to be considerably larger. A monomeric unit of human insulin comprises 51 amino acids and possesses a molecular mass of 5808 Da. Its molecular formula is C₂₅₇H₃₈₃N₆₅O₇₇S§8_{9§. Structurally, it is a dimer composed of two peptide chains, designated the A-chain and the B-chain, which are interconnected by two disulfide bonds. The A-chain contains 21 amino acids, whereas the B-chain comprises 30 residues. These interchain disulfide bonds are established between cysteine residues at positions A7-B7 and A20-B19. An additional intrachain disulfide bond exists within the A-chain, linking cysteine residues at positions A6 and A11. The A-chain displays two antiparallel α-helical regions spanning A1-A8 and A12-A19. In contrast, the B-chain features a central α-helix (encompassing residues B9-B19), flanked by the disulfide bonds, and two β-sheets (covering B7-B10 and B20-B23).}

The amino acid sequence of insulin demonstrates remarkable conservation, exhibiting only minor variations across different species. For instance, bovine insulin diverges from human insulin by merely three amino acid residues, while porcine insulin differs by only one. Notably, insulin derived from certain fish species possesses sufficient structural similarity to human insulin to elicit clinical efficacy in human subjects. Furthermore, insulin found in some invertebrates exhibits considerable sequence similarity to human insulin and elicits comparable physiological responses. This pronounced homology across diverse species' insulin sequences indicates its significant evolutionary conservation throughout animal phylogeny. Conversely, the C-peptide of proinsulin displays considerably greater interspecies variability; although it also functions as a hormone, its role is considered secondary.

Within the body, insulin is synthesized and stored as a hexamer, comprising six insulin molecules, although its biologically active form is the monomer. This hexameric complex has an approximate molecular mass of 36000 Da. The six constituent molecules are arranged into three dimeric units, forming a symmetrical structure. A critical structural characteristic is the presence of zinc atoms (Zn²⁺) situated along the axis of symmetry, each coordinated by three water molecules and three histidine residues at position B10.

The insulin hexamer represents an inactive, stable form that safeguards the highly reactive insulin while maintaining its availability. The transformation between hexamer and monomer is a critical consideration in the development of insulin formulations for injection. While the hexamer exhibits superior stability, which is advantageous for practical applications, the monomer acts as a significantly faster-acting therapeutic agent due to the inverse relationship between diffusion rate and particle size. The rapid action of the monomer allows insulin injections to be administered closer to mealtimes, thereby enhancing scheduling flexibility for individuals with diabetes. Furthermore, insulin is susceptible to aggregation, forming fibrillar interdigitated beta-sheets, which can lead to injection amyloidosis and impede long-term storage.

Function

Secretion

Beta cells within the islets of Langerhans secrete insulin through a biphasic process. The initial phase of release is rapidly initiated by elevated blood glucose concentrations and typically persists for approximately 10 minutes. The subsequent second phase involves a sustained, gradual release of newly synthesized vesicles, which is triggered independently of glucose levels and reaches its peak within 2 to 3 hours. The existence of these two distinct phases of insulin secretion implies the presence of diverse populations or "pools" of insulin granules. During the first phase of insulin exocytosis, the majority of granules primed for release are discharged following calcium internalization. This specific collection of granules is termed the Readily Releasable Pool (RRP). RRP granules constitute 0.3-0.7% of the total insulin-containing granule population and are situated in close proximity to the plasma membrane. In contrast, the second phase of exocytosis necessitates the mobilization of granules to the plasma membrane and their prior preparation for release. Consequently, the rate of the second phase of insulin secretion is dictated by the speed at which these granules become ready for discharge. This pool is referred to as the Reserve Pool (RP). The RP exhibits a slower release rate compared to the RRP, with observed rates of 18 granules/min for RRP and 6 granules/min for RP. A diminished first-phase insulin release may represent the earliest identifiable beta cell dysfunction indicative of type 2 diabetes onset. Both first-phase release and insulin sensitivity function as independent prognostic indicators for diabetes.

The initial phase of insulin release is characterized by the following sequence of events:

• Glucose permeates β-cells via glucose transporters, specifically GLUT 2. When blood glucose levels are low, minimal glucose enters the β-cells; conversely, elevated blood glucose concentrations facilitate the entry of substantial quantities of glucose into these cells.
• Upon entering the β-cell, glucose undergoes phosphorylation to glucose-6-phosphate (G-6-P) catalyzed by glucokinase (hexokinase IV). Unlike hexokinases I-III found in other tissues, glucokinase is not subject to product inhibition by G-6-P. This characteristic ensures that the intracellular G-6-P concentration directly correlates with the prevailing blood glucose concentration.
• Glucose-6-phosphate subsequently enters the glycolytic pathway and then, through the pyruvate dehydrogenase reaction, proceeds into the Krebs cycle. Within the Krebs cycle, the oxidation of acetyl CoA, which serves as the cycle's substrate, generates numerous high-energy ATP molecules, consequently elevating the intracellular ATP:ADP ratio.
• An elevated intracellular ATP:ADP ratio induces the closure of the ATP-sensitive SUR1/Kir6.2 potassium channel. This action impedes the efflux of potassium ions (K⁺) from the cell via facilitated diffusion, resulting in an accumulation of intracellular potassium ions. Consequently, the internal cellular environment becomes less negatively charged relative to the exterior, culminating in the depolarization of the cell surface membrane.
• Subsequent to depolarization, voltage-gated calcium ion (Ca²⁺) channels activate, enabling the influx of calcium ions into the cell through facilitated diffusion.
• The concentration of cytosolic calcium ions can also be augmented by the release of calcium from intracellular stores, a process mediated by the activation of ryanodine receptors.
• The calcium ion concentration in the cytosol of beta cells can also be elevated through the activation of phospholipase C, triggered by the binding of an extracellular ligand (e.g., hormone or neurotransmitter) to a G protein-coupled membrane receptor. This enzyme cleaves the membrane phospholipid phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Subsequently, IP3 binds to receptor proteins located on the endoplasmic reticulum (ER) membrane. This binding facilitates the release of Ca²⁺ ions from the ER through IP3-gated channels, thereby increasing the cytosolic calcium concentration irrespective of elevated blood glucose levels. Parasympathetic stimulation of pancreatic islets utilizes this pathway to augment insulin secretion into the bloodstream.
• This substantial elevation in cytosolic calcium ion concentration triggers the exocytosis of pre-synthesized insulin, which is stored within intracellular secretory vesicles, into the bloodstream.

This pathway represents the principal mechanism for insulin secretion. Additional substances recognized for stimulating insulin release encompass the amino acids arginine and leucine, acetylcholine released through parasympathetic stimulation (operating via the phospholipase C pathway), sulfonylurea, cholecystokinin (CCK), which also acts through phospholipase C, and gastrointestinally derived incretins, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP).

Insulin secretion is significantly suppressed by norepinephrine (noradrenaline), contributing to elevated blood glucose concentrations during periods of stress. Catecholamine release from the sympathetic nervous system exerts contradictory effects on beta-cell insulin secretion, as it is inhibited by α₂-adrenergic receptors but stimulated by β₂-adrenergic receptors. Consequently, the overall impact of norepinephrine from sympathetic innervation and epinephrine from the adrenal glands on insulin release is inhibitory, primarily due to the predominance of α-adrenergic receptor activation.

As blood glucose levels return to their physiological baseline, insulin secretion from β-cells decelerates or ceases. Should blood glucose concentrations fall below this threshold, particularly to critically low levels, hyperglycemic hormones (predominantly glucagon from pancreatic islet alpha cells) induce the release of glucose into the bloodstream from hepatic glycogen reserves, with gluconeogenesis supplementing this process if glycogen stores are exhausted. Through the elevation of blood glucose, these hyperglycemic hormones effectively prevent or rectify potentially life-threatening hypoglycemia.

Impaired first-phase insulin release is observable during a glucose tolerance test, characterized by a significantly elevated blood glucose level 30 minutes post-ingestion of a glucose load (75 or 100 g), followed by a gradual decline over the subsequent 100 minutes, yet remaining above 120 mg/100 mL two hours after the test's commencement. Conversely, in healthy individuals, blood glucose levels are normalized (and may even exhibit slight over-correction) by the test's conclusion. An initial insulin spike represents a 'first response' to elevated blood glucose; this response is individual and dose-specific, despite prior assumptions that it was solely food-type specific.

Oscillations

During digestion, typically one to two hours post-prandially, pancreatic insulin secretion is not continuous but oscillates with a periodicity of 3–6 minutes, fluctuating between blood insulin concentrations exceeding approximately 800 pmol/L and falling below 100 pmol/L (as observed in rats). This oscillatory pattern is hypothesized to prevent the downregulation of insulin receptors in target cells and to facilitate hepatic insulin extraction from the bloodstream. Consequently, this pulsatile secretion is a crucial consideration for insulin-stimulating medication administration, as an oscillating blood insulin concentration, rather than a constant high level, is the ideal therapeutic objective. Such a rhythmic delivery may be accomplished through pulsatile insulin administration to the portal vein, light-activated delivery systems, or islet cell transplantation into the liver.

Blood insulin level

Blood insulin concentrations can be quantified using international units (e.g., μIU/mL) or molar concentrations (e.g., pmol/L), with 1 μIU/mL equivalent to 6.945 pmol/L. Inter-meal blood insulin levels typically range from 8–11 μIU/mL (57–79 pmol/L).

Signal transduction

Insulin's physiological effects commence upon its binding to the insulin receptor (IR), an integral membrane protein located on the cell surface. This receptor comprises both alpha (α) and beta (β) subunits, which typically associate to form a homodimer. Insulin specifically binds to the extracellular α-subunits of this homodimeric complex, activating the intrinsic tyrosine kinase activity of the β-subunits. This activation leads to the autophosphorylation of the β-subunits, followed by the phosphorylation of intracellular proteins termed insulin receptor substrates (IRS). The phosphorylation of IRS proteins initiates a complex signal transduction cascade, culminating in the activation of various kinases and transcription factors that orchestrate insulin's intracellular actions.

A key signaling cascade, initiated by IRS-1 activation of phosphoinositol 3-kinase (PI3K), mediates several critical metabolic processes. These include the translocation of GLUT4 glucose transporters to the cell membranes of muscle and adipose cells, the synthesis of glycogen in hepatic and muscular tissues, and the conversion of glucose into triglycerides within the liver, adipose tissue, and lactating mammary glands. PI3K catalyzes the phosphorylation of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3), which subsequently activates protein kinase B (PKB). Activated PKB promotes the fusion of GLUT4-containing endosomes with the plasma membrane, thereby increasing the density of GLUT4 transporters on the cell surface. Furthermore, PKB phosphorylates and inactivates glycogen synthase kinase (GSK). Consequently, its substrate, glycogen synthase (GS), remains dephosphorylated and thus active. Active glycogen synthase (GS) is crucial for the rate-limiting step in glycogen synthesis from glucose. Analogous dephosphorylation events influence enzymes regulating glycolysis, which leads to fat synthesis via malonyl-CoA in triglyceride-producing tissues, and also enzymes governing hepatic gluconeogenesis. The cumulative outcome of these enzymatic dephosphorylations is the stimulation of glycogen and fat synthesis from glucose in relevant tissues, alongside the inhibition of hepatic glucose production through glycogenolysis and gluconeogenesis. Additionally, the lipolysis of triglycerides into free fatty acids and glycerol in adipose tissue is suppressed.

Following the generation of intracellular signals initiated by insulin-receptor binding, signal termination mechanisms become essential. A primary mechanism for signal termination involves the endocytosis and subsequent degradation of the insulin-receptor complex. Furthermore, the signaling pathway is attenuated by the dephosphorylation of tyrosine residues within various signaling components, mediated by tyrosine phosphatases. Serine/threonine kinases are also recognized for their role in reducing insulin activity.

The structural elucidation of the insulin–insulin receptor complex has been achieved through X-ray crystallography.

Physiological effects

The actions of insulin on global human metabolism include:

• Enhanced cellular uptake of specific substances, particularly glucose in muscle and adipose tissues, which constitute approximately two-thirds of the body's cells.
• Promotion of DNA replication and protein synthesis, mediated by the regulation of amino acid uptake.
• Modification of the activity of numerous enzymes.

The cellular actions of insulin (both indirect and direct) encompass:

• Stimulates Glucose Uptake: Insulin reduces blood glucose levels by promoting cellular glucose absorption. This effect is primarily achieved through insulin's induction of GLUT4 transporter insertion into the cell membranes of muscle and adipose tissues, thereby facilitating glucose entry into these cells.
• Enhanced Fat Synthesis: Insulin compels adipocytes to absorb blood glucose, which is subsequently converted into triglycerides; a reduction in insulin levels elicits the opposite effect.
• Elevated Fatty Acid Esterification: Insulin stimulates adipose tissue to synthesize neutral fats (triglycerides) from fatty acids; conversely, diminished insulin levels reverse this process.
• Reduced Lipolysis: Insulin inhibits the conversion of adipocyte lipid stores into circulating free fatty acids and glycerol; a decrease in insulin levels promotes the inverse reaction.
• Elevated glucose concentrations stimulate insulin to induce glycogen formation through the activation of hexokinase, an enzyme that phosphorylates glucose, thereby trapping it within the cell. Concurrently, insulin inhibits glucose-6-phosphatase, which typically removes this phosphate group. Both enzymes are crucial for glycogen synthesis, a process further supported by insulin's activation of phosphofructokinase and glycogen synthase.
• Insulin significantly reduces gluconeogenesis and glycogenolysis, thereby decreasing the hepatic production of glucose from non-carbohydrate precursors; the majority of endogenous insulin reaching the liver remains within this organ. Conversely, insufficient insulin levels lead to increased hepatic glucose production from various substrates.
• Insulin decreases proteolysis, which is the breakdown of proteins.
• Autophagy, the degradation of damaged organelles, is diminished by insulin, with postprandial insulin levels completely inhibiting this process.
• Insulin promotes cellular absorption of circulating amino acids; a reduction in insulin levels consequently inhibits this uptake.
• Insulin induces relaxation of arterial wall muscles, particularly in microarteries, leading to increased blood flow; a deficiency in insulin causes these muscles to contract, thereby reducing flow.
• Insulin enhances the secretion of hydrochloric acid by gastric parietal cells.
• Insulin stimulates potassium uptake by cells synthesizing glycogen—a highly hydrated substance that increases intracellular water content and associated K⁺ ions—from extracellular fluids; a lack of insulin impedes this absorption. This increase in cellular potassium uptake by insulin consequently lowers potassium concentrations in blood plasma, potentially through insulin-mediated translocation of the Na⁺/K⁺-ATPase to the surface of skeletal muscle cells.
• Insulin reduces renal sodium excretion.
• In hepatocytes, acute insulin binding activates protein phosphatase 2A (PP2A), which dephosphorylates the bifunctional enzyme fructose bisphosphatase-2 (PFKB1), thereby activating the phosphofructokinase-2 (PFK-2) active site. PFK-2 subsequently increases the production of fructose 2,6-bisphosphate, which allosterically activates PFK-1, favoring glycolysis over gluconeogenesis. Enhanced glycolysis leads to increased formation of malonyl-CoA, a molecule that can be directed towards lipogenesis and allosterically inhibits carnitine palmitoyltransferase I (CPT1), a mitochondrial enzyme essential for translocating fatty acids into the mitochondrial intermembrane space for metabolism.

Insulin also exerts influence on various other physiological functions, including vascular compliance and cognitive processes. Upon entering the human brain, insulin improves learning and memory, with a particular benefit to verbal memory. Furthermore, enhancing brain insulin signaling via intranasal insulin administration augments the acute thermoregulatory and glucoregulatory responses to food intake, suggesting that central nervous system insulin coordinates a broad spectrum of homeostatic and regulatory processes. Insulin additionally stimulates gonadotropin-releasing hormone release from the hypothalamus, thereby promoting fertility.

Anabolic vs. Reponic Classification

Historically, insulin has been broadly categorized as an anabolic hormone due to its role in stimulating the synthesis of complex molecules. However, contemporary medical literature proposes reclassifying insulin specifically as a reponic hormone (derived from the Latin repono, meaning "to store") to more accurately differentiate its metabolic function from that of classic anabolic hormones such as testosterone or growth hormone. While classic anabolic hormones drive energy-intensive processes to construct metabolically active lean tissue, the primary physiological role of a reponic hormone is to conserve and store systemic energy. Insulin achieves this by facilitating the cellular uptake of glucose and lipids for storage (lipogenesis and glycogenesis) while simultaneously inhibiting the breakdown of stored energy (lipolysis and glycogenolysis). Researchers contend that this distinction offers a clearer framework for comprehending why hyperinsulinemia is uniquely characterized by central adiposity and metabolic syndrome, rather than by lean tissue growth.

Degradation

After an insulin molecule binds to its receptor and exerts its physiological effect, it can either be released back into the extracellular space or undergo cellular degradation. The liver and kidneys constitute the primary organs responsible for insulin clearance. Insulin is catabolized by protein-disulfide reductase (glutathione), an enzyme that cleaves the disulfide bonds connecting the A and B chains. The liver primarily clears insulin during its initial passage, while the kidneys are responsible for clearing the majority of insulin circulating systemically. Typically, degradation involves the endocytosis of the insulin-receptor complex, subsequently followed by the action of insulin-degrading enzyme. Endogenously produced insulin from beta cells is estimated to be degraded approximately one hour after its initial release into circulation, with an observed half-life of about 4–6 minutes.

Regulator of Endocannabinoid Metabolism

Insulin functions as a significant regulator of endocannabinoid (EC) metabolism; therapeutic insulin administration has been demonstrated to decrease intracellular levels of ECs, specifically 2-arachidonoylglycerol (2-AG) and anandamide (AEA). These reductions correlate with insulin-sensitive alterations in the expression of enzymes involved in EC metabolism. Within insulin-resistant adipocytes, the typical patterns of insulin-induced enzyme expression are disrupted, leading to increased EC synthesis and diminished EC degradation. Research indicates that insulin-resistant adipocytes are unable to properly regulate EC metabolism and reduce intracellular EC levels following insulin stimulation, resulting in elevated EC concentrations in obese, insulin-resistant individuals. This metabolic dysregulation contributes to excessive visceral fat accumulation, decreased adiponectin secretion from abdominal adipose tissue, and the subsequent development of various cardiometabolic risk factors associated with obesity and type 2 diabetes.

Hypoglycemia

Hypoglycemia, commonly referred to as "low blood sugar," occurs when blood glucose levels fall below the physiological normal range. This condition can manifest with diverse symptoms, such as impaired coordination, speech difficulties, disorientation, loss of consciousness, seizures, or even mortality. Additional manifestations may include sensations of hunger, diaphoresis, tremors, and asthenia. The onset of these symptoms is typically rapid.

The predominant etiology of hypoglycemia involves pharmacological agents employed in diabetes management, specifically insulin and sulfonylureas. The risk is elevated in diabetic individuals who have reduced their typical food intake, increased physical activity, or consumed alcohol. Other contributing factors to hypoglycemia encompass renal insufficiency, specific neoplastic conditions like insulinoma, hepatic dysfunction, hypothyroidism, prolonged fasting, inherited metabolic disorders, severe infections, reactive hypoglycemia, and various pharmaceutical agents, including ethanol. Furthermore, normoglycemic infants may experience low blood sugar if they have not fed for several hours.

Diseases and Syndromes

Pathological disturbances in insulin function are associated with several medical conditions:

• Diabetes is a broad designation encompassing all physiological states characterized by hyperglycemia. This condition manifests in various forms, including:
  • Type 1 diabetes, which involves the autoimmune-mediated destruction of pancreatic insulin-producing β-cells, leading to an absolute deficiency of insulin.
  • Type 2 diabetes is characterized by either insufficient insulin production from β-cells, insulin resistance, or a combination of both, stemming from etiologies that are not yet fully elucidated.
    • Correlations have been observed with dietary patterns, sedentary lifestyles, obesity, advanced age, and metabolic syndrome. Causal relationships have been established in various model organisms, such as mice and monkeys. Notably, Type 2 diabetes can also affect non-obese individuals due to dietary factors, sedentary habits, and unidentified risk factors, although the precise causal link in these cases may require further investigation.
    • It is posited that a genetic predisposition contributes to the development of Type 2 diabetes when individuals are exposed to specific environmental conditions.
  • Other types of impaired glucose tolerance.
• Insulinoma is a tumor originating from pancreatic beta cells, resulting in excessive insulin production or reactive hypoglycemia.
• The metabolic syndrome, initially termed "syndrome X" by Gerald Reaven, represents a condition that remains incompletely understood. Its etiology is ambiguous, with ongoing debate regarding whether it stems from a singular, treatable cause or arises as a consequence of physiological alterations that predispose individuals to type 2 diabetes. Key diagnostic criteria include elevated blood pressure, dyslipidemia (defined as abnormalities in blood cholesterol fractions and other circulating lipids), and an increased waist circumference, particularly observed in populations within developed nations. A fundamental underlying mechanism is potentially insulin resistance, a precursor to type 2 diabetes, characterized by a reduced responsiveness to insulin in specific tissues, such as muscle and adipose tissue. The development of associated comorbidities, including essential hypertension, obesity, type 2 diabetes, and cardiovascular disease (CVD), is frequently observed.
• Polycystic ovary syndrome (PCOS) is a multifaceted condition affecting women during their reproductive years, typically manifesting with anovulation and androgen excess, often evident as hirsutism. Insulin resistance is a prevalent feature in numerous cases of PCOS.

Medical Applications

Biosynthetic human insulin (insulin human rDNA, INN) intended for clinical application is produced through recombinant DNA technology. This biosynthetic form exhibits superior purity compared to insulin extracted from animal sources, with its enhanced purity contributing to a reduction in antibody formation. Furthermore, researchers have successfully integrated the human insulin gene into plants, specifically safflower, establishing an alternative production method termed "biopharming." This innovative technique is projected to lower manufacturing expenses.

Multiple human insulin analogs are currently accessible. These analogs, structurally similar to human insulin, have been engineered to address specific requirements for glycemic management, offering both rapid-acting (prandial) and long-acting (basal) profiles. Humalog (insulin lispro) was the inaugural biosynthetic insulin analog developed for clinical application as a mealtime (prandial) insulin, demonstrating faster subcutaneous absorption and an onset of action within 15 minutes post-injection compared to regular insulin. Other rapid-acting analogs, such as NovoRapid and Apidra, exhibit comparable pharmacokinetic profiles. Their rapid absorption is attributed to specific amino acid sequences that inhibit the formation of dimers and hexamers, as monomeric insulin forms are absorbed more quickly. Unlike human and animal insulins, rapid-acting formulations eliminate the need for a pre-meal injection interval. The second category comprises long-acting insulins, with Lantus (insulin glargine) being the first introduced. These provide a consistent therapeutic effect over an extended duration, typically 18 to 24 hours. Similarly, Levemir, another protracted insulin analog, utilizes a fatty acid acylation strategy. The attachment of a myristic acid molecule to this analog facilitates its association with abundant serum albumin, thereby prolonging its action and mitigating the risk of hypoglycemia. Both protracted analogs are administered once daily and serve as basal insulin for individuals with type 1 diabetes. Furthermore, combination formulations of rapid-acting and protracted insulins are available, enabling patients to achieve an insulin profile that more closely emulates endogenous insulin secretion. Beyond therapeutic applications, insulin is employed in various cell lines, including CHO-s, HEK 293, and Sf9, for the production of monoclonal antibodies, viral vaccines, and gene therapy products.

Insulin is typically administered via subcutaneous injection using single-use syringes with needles, an insulin pump, or reusable insulin pens equipped with disposable needles. Inhaled insulin is also commercially available within the United States market.

The Dispovan Single-Use Pen Needle, manufactured by HMD, was introduced as the first insulin pen needle in India designed to facilitate self-administration. These pen needles incorporate extra-thin walls and a multi-bevel tapered point, features intended to enhance patient comfort by reducing pain and optimizing medication delivery. The product's distribution strategy focuses on making pen needles accessible and affordable in developing regions of the country. Furthermore, their universal design ensures compatibility with various insulin pens.

Unlike numerous pharmaceutical agents, insulin cannot be administered orally because, similar to most proteins introduced into the gastrointestinal tract, it undergoes proteolytic degradation into inactive fragments. Consequently, research efforts have focused on developing methods to shield insulin from digestive enzymes, thereby enabling oral or sublingual administration.

In 2021, the World Health Organization incorporated insulin into its model list of essential medicines.

In the United Kingdom, the National Health Service provides insulin and all other necessary medications to individuals with diabetes without charge.

Historical Investigations

Initial Discovery

In 1869, Paul Langerhans, a medical student in Berlin, observed previously unidentified tissue clusters dispersed within the pancreatic structure during microscopic examination. The precise function of these "little heaps of cells," subsequently designated as the islets of Langerhans, was initially undetermined. However, Édouard Laguesse later proposed that these structures might generate secretions crucial for regulating digestion. Archibald Langerhans, Paul's son, also contributed to elucidating this regulatory function.

In 1889, physician Oskar Minkowski, collaborating with Joseph von Mering, extirpated the pancreas from a healthy canine to investigate its presumed digestive function. Subsequent urinalysis revealed the presence of sugar, thereby establishing the inaugural link between the pancreas and diabetes. A significant advancement occurred in 1901 when American physician and scientist Eugene Lindsay Opie localized the pancreas's role to the islets of Langerhans, stating: "Diabetes mellitus when the result of a lesion of the pancreas is caused by destruction of the islets of Langerhans and occurs only when these bodies are in part or wholly destroyed".

During the subsequent two decades, researchers undertook multiple endeavors to isolate the secretions from the islets. In 1906, George Ludwig Zuelzer attained limited success in canine treatment using a pancreatic extract, though he could not sustain his research. From 1911 to 1912, E.L. Scott at the University of Chicago experimented with aqueous pancreatic extracts, observing "a slight diminution of glycosuria"; however, his director remained unconvinced of the research's merit, leading to its termination. Israel Kleiner demonstrated comparable effects at Rockefeller University in 1915, but World War I disrupted his efforts, preventing his return to the project.

In 1916, Nicolae Paulescu formulated an aqueous pancreatic extract that, upon administration to a diabetic canine, exhibited a normalizing influence on blood glucose concentrations. His experimental work was interrupted by World War I. In 1921, he authored four papers detailing his research conducted in Bucharest and his trials involving a diabetic dog. Subsequently, in the same year, he published "Research on the Role of the Pancreas in Food Assimilation".

The term "insulin" was introduced by Edward Albert Sharpey-Schafer in 1916 to denote a hypothetical molecule, theorized to be synthesized by the pancreatic islets of Langerhans (derived from the Latin insula, meaning islet or island), responsible for regulating glucose metabolism. Unbeknownst to Sharpey-Schafer, Jean de Meyer had previously proposed the highly similar term "insuline" in 1909 for the identical molecule.

Extraction and Purification

In October 1920, Canadian researcher Frederick Banting hypothesized that the digestive secretions previously investigated by Minkowski were degrading the islet secretion, thus impeding successful extraction. As a trained surgeon, Banting understood that pancreatic duct ligation would induce atrophy in the majority of the pancreas while preserving the islets of Langerhans. He posited that a comparatively pure extract could be obtained from the islets once the bulk of the pancreatic tissue had degenerated. He recorded a personal note: "Ligate pancreatic ducts of dog. Keep dogs alive till acini degenerate leaving Islets. Try to isolate the internal secretion of these + relieve glycosurea[sic]."

In the spring of 1921, Banting journeyed to Toronto to present his concept to John Macleod, Professor of Physiology at the University of Toronto. Macleod initially expressed skepticism, given Banting's lack of research experience and unfamiliarity with contemporary literature; nevertheless, he consented to provide laboratory facilities for Banting to investigate his hypotheses. Macleod also arranged for two undergraduate students to serve as Banting's laboratory assistants for the summer, though Banting ultimately required only one. Charles Best and Clark Noble decided by a coin toss, with Best winning and commencing the initial shift. This outcome proved disadvantageous for Noble, as Banting retained Best for the entire summer and subsequently shared half of his Nobel Prize monetary award and recognition for the discovery with Best. On July 30, 1921, Banting and Best successfully isolated an extract, termed "isletin," from the islets of a duct-ligated canine. Upon injection into a diabetic dog, this extract reduced its blood glucose levels by 40% within one hour.

Upon Macleod's return to Toronto in the autumn of 1921, Banting and Best presented their findings; however, Macleod identified deficiencies in the experimental design and recommended a replication of the experiments utilizing an increased number of canines and enhanced instrumentation. Subsequently, he relocated Banting and Best to a superior laboratory facility and commenced remunerating Banting from his allocated research grants. Weeks thereafter, the subsequent experimental phase also proved successful, leading Macleod to facilitate the private publication of their results in Toronto during November of that year. Confronted by the laborious process of ligating canine pancreatic ducts and enduring several weeks for insulin extraction, Banting conceived the innovative approach of isolating insulin from fetal calf pancreases, which lack developed digestive glands. By December, they had additionally achieved success in extracting insulin from mature bovine pancreases. Macleod subsequently ceased all other research endeavors within his laboratory to dedicate efforts exclusively to insulin purification. He extended an invitation to biochemist James Collip to assist with this undertaking, and the team anticipated readiness for clinical trials within a month.

On January 11, 1922, Leonard Thompson, a 14-year-old diabetic patient in critical condition at the Toronto General Hospital, received the inaugural insulin injection. Nevertheless, the extract's significant impurity precipitated a severe allergic reaction in Thompson, leading to the cancellation of subsequent injections. For the ensuing 12 days, Collip diligently labored to refine the bovine pancreatic extract. A second administration occurred on January 23, effectively resolving the glycosuria characteristic of diabetes without inducing discernible adverse effects. Elizabeth Hughes, daughter of U.S. Secretary of State Charles Evans Hughes, became the first American patient. The initial patient treated within the United States was James D. Havens, who later became a prominent woodcut artist; John Ralston Williams facilitated this by importing insulin from Toronto to Rochester, New York, for Havens' treatment.

Banting and Best consistently experienced difficulties collaborating with Collip, perceiving him as an intrusive presence, which led to Collip's prompt departure from the project. Throughout the spring of 1922, Best successfully refined his methodologies, enabling the extraction of substantial insulin quantities as required, though the preparation retained impurities. Eli Lilly and Company, a pharmaceutical firm, had extended an offer of assistance shortly after the initial publications in 1921, an offer which was accepted in April. By November, George B. Walden, Lilly's chief chemist, identified isoelectric precipitation, thereby enabling the production of significant volumes of highly purified insulin. Subsequently, insulin became commercially available to the public.

Patent Considerations

By late January 1922, escalating tensions among the four "co-discoverers" of insulin led Collip to briefly contemplate independently patenting his purification methodology. Consequently, John G. FitzGerald, who directed the non-commercial public health entity Connaught Laboratories, intervened to mediate the dispute. The subsequent agreement, formalized on January 25, 1922, stipulated two primary conditions: 1) the collaborators were to execute a contract precluding them from securing a patent with any commercial pharmaceutical company during an initial collaborative period with Connaught; and 2) any modifications to research policy necessitated prior consultation among FitzGerald and the four collaborators. This accord served to mitigate disagreements and aligned the research with Connaught's public health mission.

Initially, Macleod and Banting expressed significant reservations about patenting their insulin production process, citing medical ethics. Nevertheless, apprehension persisted regarding the potential for a private third party to appropriate and monopolize the research (a possibility hinted at by Eli Lilly and Company), and the challenge of ensuring safe distribution without adequate quality control mechanisms. In response, Edward Calvin Kendall provided crucial counsel. Kendall had previously isolated thyroxin at the Mayo Clinic in 1914, subsequently patenting the process via an agreement involving himself, the Mayo brothers, and the University of Minnesota, ultimately transferring the patent to the public university. On April 12, Banting, Best, Collip, Macleod, and FitzGerald collectively addressed a letter to the President of the University of Toronto, proposing an analogous arrangement to assign a patent to the university's Board of Governors. The correspondence underscored the following:

The patent's sole purpose would be to preclude other entities from obtaining a proprietary patent. Upon publication of the detailed preparation methodology, any individual would be at liberty to produce the extract, yet no party could establish a lucrative monopoly.

The transfer of the insulin patent to the University of Toronto Board of Governors was finalized on January 15, 1923, for a nominal fee of $1.00. This arrangement was lauded in The World's Work in 1923 as "a step forward in medical ethics." In the 2010s, it garnered significant media scrutiny concerning healthcare and pharmaceutical affordability.

Subsequent to concerns about Eli Lilly's efforts to secure separate patents for specific components of the insulin manufacturing process, Robert Defries, Connaught's Assistant Director and Head of the Insulin Division, instituted a patent pooling policy. This policy mandated that all producers openly share any advancements in the manufacturing process, thereby ensuring continued affordability.

Structural Analysis and Synthesis

Initially, purified animal-derived insulin constituted the sole form accessible for both experimental research and therapeutic use in diabetic patients. John Jacob Abel achieved the first crystallization of insulin in 1926. The proteinaceous nature of insulin was initially suggested by Michael Somogyi, Edward A. Doisy, and Philip A. Shaffer in 1924, and definitively confirmed in 1935 when Hans Jensen and Earl A. Evans Jr. successfully isolated the amino acids phenylalanine and proline.

Frederick Sanger first elucidated the amino acid sequence of insulin in 1951. Concurrently, in the mid-1960s, the initial synthetic insulin was developed in the laboratories of Panayotis Katsoyannis at the University of Pittsburgh and Helmut Zahn at RWTH Aachen University. Chinese researchers successfully synthesized crystalline bovine insulin in 1965. The comprehensive three-dimensional structure of insulin was subsequently resolved through X-ray crystallography in Dorothy Hodgkin's laboratory in 1969.

Hans E. Weber identified preproinsulin in 1974 while serving as a research fellow at the University of California, Los Angeles. During 1973–1974, Weber acquired proficiency in techniques for messenger RNA isolation, purification, and translation. To advance insulin research, he procured pancreatic tissues, initially from a Los Angeles slaughterhouse and subsequently from UCLA's animal stock. He successfully isolated and purified total messenger RNA from pancreatic islet cells, which was then translated within Xenopus laevis oocytes and subsequently precipitated using anti-insulin antibodies. Analysis of the total translated protein via SDS-polyacrylamide gel electrophoresis and sucrose gradient centrifugation revealed distinct peaks corresponding to insulin and proinsulin. Unexpectedly, Weber also isolated a third peak, indicative of a molecule larger than proinsulin. Repeated experimentation consistently showed this prominent peak preceding proinsulin, leading him to conclude it represented a larger precursor molecule upstream of proinsulin. In May 1975, at the American Diabetes Association meeting in New York, Weber formally presented his findings, becoming the first to designate this precursor molecule "preproinsulin." Following this presentation, Donald Steiner, a researcher known for his contributions to proinsulin characterization, invited Weber to discuss his research. In April 1976, a year later, Steiner further characterized and sequenced this molecule, explicitly acknowledging Weber's pioneering work and discovery. Preproinsulin subsequently emerged as a crucial molecule for investigating the mechanisms of transcription and translation.

The inaugural genetically engineered (recombinant) synthetic human insulin was synthesized in 1978 using E. coli by Arthur Riggs and Keiichi Itakura at the Beckman Research Institute of the City of Hope, in collaboration with Herbert Boyer at Genentech. Genentech, established by Swanson, Boyer, and Eli Lilly and Company, subsequently introduced Humulin, the first commercially available biosynthetic human insulin, in 1982. Currently, the predominant form of insulin utilized globally is biosynthetic recombinant human insulin or its analogues. More recently, an innovative recombinant strategy has been employed by a Canadian research team, leveraging an easily cultivated safflower plant to produce significantly more affordable insulin.

Recombinant insulin is synthesized using either yeast, typically Saccharomyces cerevisiae, or E. coli. Within yeast, insulin can be engineered as a single-chain protein incorporating a KexII endoprotease site, which is a yeast homolog of PCI/PCII, designed to cleave the insulin A chain from a C-terminally truncated insulin B chain. Subsequently, a chemically synthesized C-terminal tail, which includes the absent threonine, is attached to the insulin molecule via reverse proteolysis, employing the cost-effective protease trypsin. Generally, the lysine residue on this C-terminal tail is safeguarded with a chemical protecting group to inhibit unintended proteolysis. The simplicity of modular synthesis and the comparative safety of modifications within this specific region explain the prevalence of insulin analogs featuring C-terminal alterations, such as lispro, aspart, and glulisine. Conversely, methods like the Genentech synthesis and entirely chemical syntheses, exemplified by Bruce Merrifield's approach, are less favored. This preference stems from the low efficiency of recombining the two insulin chains, largely attributable to competitive precipitation of the insulin B chain.

Nobel Prizes

In 1923, the Nobel Prize committee recognized a team at the University of Toronto for the practical extraction of insulin, awarding the Nobel Prize to Frederick Banting and John Macleod. They received the Nobel Prize in Physiology or Medicine in 1923 for their discovery of insulin. Banting, expressing indignation over Best's omission, subsequently shared his prize with Best, while Macleod promptly shared his with James Collip. The patent rights for insulin were subsequently transferred to the University of Toronto for a nominal sum of one dollar.

Two additional Nobel Prizes have been conferred for research pertaining to insulin. Frederick Sanger, a British molecular biologist, received the 1958 Nobel Prize in Chemistry for his determination of insulin's primary structure in 1955. Rosalyn Sussman Yalow was awarded the 1977 Nobel Prize in Medicine for her pioneering work in developing the radioimmunoassay for insulin.

Several other Nobel Prizes also exhibit an indirect association with insulin. George Minot, who co-received the 1934 Nobel Prize for developing the initial effective treatment for pernicious anemia, was himself a diabetic. William Castle noted that the 1921 discovery of insulin, by sustaining Minot's life, consequently facilitated the discovery of a cure for pernicious anemia. Dorothy Hodgkin received a Nobel Prize in Chemistry in 1964 for her advancements in crystallography, a technique she subsequently employed to elucidate the complete molecular structure of insulin in 1969.

Controversy

The research published by Banting, Best, Collip, and Macleod detailed the preparation of a purified insulin extract deemed suitable for human therapeutic application. While Paulescu had identified the fundamental principles of the treatment, his saline extract was unsuitable for human administration, leading to his omission from the 1923 Nobel Prize recognition. Ian Murray actively campaigned to rectify what he termed "the historical wrong" concerning Nicolae Paulescu. Murray held positions as a professor of physiology at the Anderson College of Medicine in Glasgow, Scotland, head of the Department of Metabolic Diseases at a prominent Glasgow hospital, vice-president of the British Association of Diabetes, and a founding member of the International Diabetes Federation. Murray articulated:

Insufficient recognition has been given to Paulescu, the distinguished Romanian scientist, who at the time when the Toronto team were commencing their research had already succeeded in extracting the antidiabetic hormone of the pancreas and proving its efficacy in reducing the hyperglycaemia in diabetic dogs.

In a private communication, Arne Tiselius, the former head of the Nobel Institute, conveyed his personal conviction that Paulescu merited equal recognition for the 1923 award.

References

• University of Toronto Libraries Collection: The Discovery and Initial Development of Insulin, 1920–1925.
• CBC Digital Archives: Banting, Best, Macleod, Collip: The Pursuit of a Diabetes Cure.
• Animations illustrating insulin's physiological action (archived 9 March 2011).
• Comprehensive structural data for UniProt: P01308 (Insulin) available via PDBe-KB.