Insulin

Insulin is a hormone that is central to regulating fat and steroids metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle.

Insulin stops the use of fat as an energy source. When insulin is absent, glucose is not taken up by body cells and the body begins to use fat as an energy source, for example, by transfer of lipids from adipose tissue to the liver for mobilization as an energy source. As its level is a central metabolic control mechanism, its status is also used as a control signal to other body systems (such as amino acid uptake by body cells). In addition, it has several other anabolic effects throughout the body.

When control of insulin levels fails, diabetes mellitus will result. As a consequence, insulin is used medically to treat some forms of diabetes mellitus. Patients with Type 1 diabetes mellitus depend on external insulin (most commonly injected subcutaneously) for their survival because the hormone is no longer produced internally. Patients with Type 2 diabetes mellitus are insulin resistant, and because of such resistance, may suffer from a relative insulin deficiency. Some patients with Type 2 diabetes may eventually require insulin if other medications fail to control blood glucose levels adequately, though this is somewhat uncommon.

Insulin also influences other body functions, such as vascular compliance and cognition. Once insulin enters the human brain, it enhances learning and memory and in particular benefits verbal memory. Enhancing brain insulin signaling by means of intranasal insulin administration also enhances the acute thermoregulatory and glucoregulatory response to food intake, suggesting that central nervous insulin contributes to the control of whole-body energy homeostasis in humans.

Insulin is a peptide hormone composed of 51 amino acids and has a molecular weight of 5808 Da. It is produced in the islets of Langerhans in the pancreas. The name comes from the Latin insula for "island". Insulin's structure varies slightly between species of animal. Insulin from animal sources differs somewhat in "strength" (in carbohydrate metabolism control effects) in humans because of those variations. Porcine (pig) insulin is especially close to the human version.

The proinsulin precursor of insulin is encoded by the INS gene.

A variety of mutant alleles with changes in the coding region have been identified. There is a read-through gene, INS-IGF2, which overlaps with this gene at the 5' region and with the IGF2 gene at the 3' region.

There are several regulatory sequences in the promoter region of the human insulin gene, to which transcription factors bind.

In general, the A-boxes bind to Pdx1 factors, E-boxes bind to NeuroD, C-boxes bind to MafA and cAMP response elements to CREB.

There are also silencers that inhibit transcription.

Within vertebrates, the amino acid sequence of insulin is extremely well preserved. Bovine insulin differs from human in only three amino acid residues, and porcine insulin in one. Even insulin from some species of fish is similar enough to human to be clinically effective in humans. Insulin in some invertebrates is quite similar in sequence to human insulin, and has similar physiological effects. The strong homology seen in the insulin sequence of diverse species suggests that it has been conserved across much of animal evolutionary history. The C-peptide of proinsulin (discussed later), however, differs much more amongst species; it is also a hormone, but a secondary one.

Insulin is produced and stored in the body as a hexamer (a unit of six insulin molecules), while the active form is the monomer. The hexamer is an inactive form with long-term stability, which serves as a way to keep the highly reactive insulin protected, yet readily available. The hexamer-monomer conversion is one of the central aspects of insulin formulations for injection. The hexamer is far more stable than the monomer, which is desirable for practical reasons, however the monomer is a much faster reacting drug because diffusion rate is inversely related to particle size. A fast reacting drug means that insulin injections do not have to precede mealtimes by hours, which in turn gives diabetics more flexibility in their daily schedule. Insulin can aggregate and form fibrillar interdigitated beta-sheets. This can cause injection amyloidosis, and prevents the storage of insulin for long periods.

Insulin is produced in the pancreas and released when any of the several stimuli are detected. The stimuli include ingested protein and glucose in the blood produced from digested food. Carbohydrate can be polymers of simple sugars or the simple sugars themselves. If the carbohydrate includes glucose then that glucose will be absorbed into the bloodstream and blood glucose level will begin to rise. In target cells, insulin initiates a signal transduction, which has the effect of increasing glucose uptake and storage. Finally, insulin is degraded, terminating the response.

In mammals, insulin is synthesized in the pancreas within the beta cells (β-cells) of the islets of Langerhans. One million to three million islets of Langerhans (pancreatic islets) form the endocrine part of the pancreas, which is primarily an exocrine gland. The endocrine portion accounts for only 2% of the total mass of the pancreas. Within the islets of Langerhans, beta cells constitute 60–80% of all the cells.

In beta cells, insulin is synthesized from the proinsulin precursor molecule by the action of proteolytic enzymes, known as prohormone convertases (PC1 and PC2), as well as the exoprotease carboxypeptidase E. These modifications of proinsulin remove the center portion of the molecule (i.e., C-peptide), from the C- and N- terminal ends of proinsulin. The remaining polypeptides (51 amino acids in total), the B- and A- chains, are bound together by disulfide bonds/disulphide bonds. Confusingly, the primary sequence of proinsulin goes in the order "B-C-A", since B and A chains were identified on the basis of mass, and the C peptide was discovered after the others.

The endogenous production of insulin is regulated in several steps along the synthesis pathway:

It has been shown that insulin and its related proteins, are also produced inside the brain and that reduced levels of these proteins are linked to Alzheimer's disease.

Beta cells in the islets of Langerhans release insulin in two phases. The first phase insulin release is rapidly triggered in response to increased blood glucose levels. The second phase is a sustained, slow release of newly formed vesicles that are triggered independently of sugar. The description of first phase release is as follows:

This is the main mechanism for release of insulin. In addition some insulin release takes place generally with food intake, not just glucose or carbohydrate intake, and the beta cells are also somewhat influenced by the autonomic nervous system. The signaling mechanisms controlling these linkages are not fully understood.

Other substances known to stimulate insulin release include amino acids from ingested proteins, acetylcholine released from vagus nerve endings (parasympathetic nervous system), gastrointestinal hormones released by enteroendocrine cells of intestinal mucosa and glucose-dependent insulinotropic peptide (GIP). Three amino acids (alanine, glycine and arginine) act similarly to glucose by altering the beta cell's membrane potential. Acetylcholine triggers insulin release through phospholipase C, while the last acts through the mechanism of adenylate cyclase.

The sympathetic nervous system (via α2-adrenergic stimulation as demonstrated by the agonists clonidine or methyldopa) inhibit the release of insulin. However, it is worth noting that circulating adrenaline will activate β2-Receptors on the Beta cells in the pancreatic Islets to promote insulin release. This is important since muscle cannot benefit from the raised blood sugar resulting from adrenergic stimulation (increased gluconeogenesis and glycogenolysis from the low blood insulin: glucagon state) unless insulin is present to allow for GLUT-4 translocation in the tissue. Therefore, beginning with direct innervation, norepinephrine inhibits insulin release via α2-receptors, then subsequently, circulating adrenaline from the adrenal medulla will stimulate β2-receptors thereby promoting insulin release.

When the glucose level comes down to the usual physiologic value, insulin release from the beta cells slows or stops. If blood glucose levels drop lower than this, especially to dangerously low levels, release of hyperglycemic hormones (most prominently glucagon from Islet of Langerhans' alpha cells) forces release of glucose into the blood from cellular stores, primarily liver cell stores of glycogen. By increasing blood glucose, the hyperglycemic hormones prevent or correct life-threatening hypoglycemia. Release of insulin is strongly inhibited by the stress hormone norepinephrine (noradrenaline), which leads to increased blood glucose levels during stress.

Evidence of impaired first phase insulin release can be seen in the glucose tolerance test, demonstrated by a substantially elevated blood glucose level at 30 minutes, a marked drop by 60 minutes, and a steady climb back to baseline levels over the following hourly time points.

Even during digestion, generally one or two hours following a meal, insulin release from pancreas is not continuous, but oscillates with a period of 3–6 minutes, changing from generating a blood insulin concentration more than ~800 pmol/l to less than 100 pmol/l. This is thought to avoid downregulation of insulin receptors in target cells and to assist the liver in extracting insulin from the blood. This oscillation is important to consider when administering insulin-stimulating medication, since it is the oscillating blood concentration of insulin release, which should, ideally, be achieved, not a constant high concentration. This may be achieved by delivering insulin rhythmically to the portal vein or by islet cell transplantation to the liver. Future insulin pumps hope to address this characteristic. (See also Pulsatile Insulin.)

There are special transporter proteins in cell membranes through which glucose from the blood can enter a cell. These transporters are, indirectly, under blood insulin's control in certain body cell types (e.g., muscle cells). Low levels of circulating insulin, or its absence, will prevent glucose from entering those cells (e.g., in Type 1 diabetes). However, more commonly there is a decrease in the sensitivity of cells to insulin (e.g., the reduced insulin sensitivity characteristic of Type 2 diabetes), resulting in decreased glucose absorption. In either case, there is 'cell starvation', weight loss, sometimes extreme. In a few cases, there is a defect in the release of insulin from the pancreas. Either way, the effect is, characteristically, the same: elevated blood glucose levels.

Activation of insulin receptors leads to internal cellular mechanisms that directly affect glucose uptake by regulating the number and operation of protein molecules in the cell membrane that transport glucose into the cell. The genes that specify the proteins that make up the insulin receptor in cell membranes have been identified and the structures of the interior, transmembrane section, and the extra-membrane section of receptor have been solved.

Two types of tissues are most strongly influenced by insulin, as far as the stimulation of glucose uptake is concerned: muscle cells (myocytes) and fat cells (adipocytes). The former are important because of their central role in movement, breathing, circulation, etc., and the latter because they accumulate excess food energy against future needs. Together, they account for about two-thirds of all cells in a typical human body.

According to the study of Raman spectra, a low-frequency wave number of 22 cm−1 has been observed for insulin molecules. Subsequently, it was identified as the accordion-like vibration of the helix (B9-B19) in the B-chain of insulin.

The actions of insulin on the global human metabolism level include:

The actions of insulin on cells include:

Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment, or it may be degraded by the cell. Degradation normally involves endocytosis of the insulin-receptor complex followed by the action of insulin degrading enzyme. Most insulin molecules are degraded by liver cells. It has been estimated that an insulin molecule produced endogenously by the pancreatic beta cells is degraded within approximately one hour after its initial release into circulation (insulin half-life ~ 4–6 minutes).

Although other cells can use other fuels for a while (most prominently fatty acids), neurons depend on glucose as a source of energy in the non-starving human. They do not require insulin to absorb glucose, unlike muscle and adipose tissue, and they have very small internal stores of glycogen. Glycogen stored in liver cells (unlike glycogen stored in muscle cells) can be converted to glucose, and released into the blood, when glucose from digestion is low or absent, and the glycerol backbone in triglycerides can also be used to produce blood glucose.

Sufficient lack of glucose and scarcity of these sources of glucose can dramatically make itself manifest in the impaired functioning of the central nervous system; dizziness, speech problems, and even loss of consciousness, can occur. Low glucose is known as hypoglycemia or, in cases producing unconsciousness, "hypoglycemic coma" (sometimes termed "insulin shock" from the most common causative agent). Endogenous causes of insulin excess (such as an insulinoma) are very rare, and the overwhelming majority of insulin-excess induced hypoglycemia cases are iatrogenic and usually accidental. There have been a few reported cases of murder, attempted murder, or suicide using insulin overdoses, but most insulin shocks appear to be due to errors in dosage of insulin (e.g., 20 units of insulin instead of 2) or other unanticipated factors (didn't eat as much as anticipated, or exercised more than expected, or unpredicted kinetics of the subcutaneously injected insulin itself).

Possible causes of hypoglycemia include:

There are several conditions in which insulin disturbance is pathologic:

Biosynthetic "human" insulin is now manufactured for widespread clinical use using recombinant DNA technology. More recently, researchers have succeeded in introducing the gene for human insulin into plants and in producing insulin in plants, specifically safflower. It is anticipated that this technique will reduce production costs.

Several of these are slightly modified versions of human insulin that, while having a clinical effect on blood glucose levels as though they were exact copies, have been designed to have somewhat different absorption or duration of action characteristics. They are usually referred to as "insulin analogues". For instance, the first available, insulin lispro, does not exhibit a delayed absorption effect found in "regular" insulin, and begins to have effect in as little as 15 minutes. Other rapid acting analogues are NovoRapid and Apidra with similar profiles. All are rapidly absorbed due to a mutation in the sequence that prevents the insulin analogue in forming hexamers. Instead, the insulin molecule is a monomer, which is more rapidly absorbed. Using it, therefore, does not require the pre-planning required for other insulins that begin to take effect much later (up to many hours) after administration. Another type is extended release insulin; the first of these was Lantus (insulin glargine). These have a steady effect for the entire time they are active, without the peak and drop of effect in other insulins; typically, they continue to have an insulin effect for an extended period from 18 to 24 hours. Similar another protracted insulin analogue "Levemir" is based on a fatty acid acylation approach. A fatty acid (myristyric acid) is attached to this analogue, which in turn associates the insulin molecule to the abundant serum albumin. This in turn extends the effect and in addition it reduces the risk of hypoglycemia. Both protracted analogues need to be taken only once-daily and are very much used in the type 1 diabetes market as the basal insulin. A mix combination of a rapid acting and a protracted insulin is also available for the patients making it more likely for the patient to achieve an insulin profile that mimics that of the body´s own insulin release.

Unlike many medicines, insulin currently cannot be taken orally. Like nearly all other proteins introduced into the gastrointestinal tract, it is reduced to fragments (even single amino acid components), whereupon all "insulin activity" is lost. There has been some research into ways to protect insulin from the digestive tract, so that it can be administered orally or sublingually. While experimental, several companies now have various formulations in human clinical trials.

Insulin is usually taken as subcutaneous injections by single-use syringes with needles, an insulin pump, or by repeated-use insulin pens with needles.

In 1869 Paul Langerhans, a medical student in Berlin, was studying the structure of the pancreas under a microscope when he identified some previously un-noticed tissue clumps scattered throughout the bulk of the pancreas. The function of the "little heaps of cells," later known as the Islets of Langerhans, was unknown, but Edouard Laguesse later suggested that they might produce secretions that play a regulatory role in digestion. Paul Langerhans' son, Archibald, also helped to understand this regulatory role. The term insulin origins from insula, the Latin word for islet/island.

In 1889, the Polish-German physician Oscar Minkowski in collaboration with Joseph von Mering removed the pancreas from a healthy dog to test its assumed role in digestion. Several days after the dog's pancreas was removed, Minkowski's animal keeper noticed a swarm of flies feeding on the dog's urine. On testing the urine they found that there was sugar in the dog's urine, establishing for the first time a relationship between the pancreas and diabetes. In 1901, another major step was taken by Eugene Opie, when he clearly established the link between the Islets of Langerhans and diabetes: Diabetes mellitus … is caused by destruction of the islets of Langerhans and occurs only when these bodies are in part or wholly destroyed. Before his work, the link between the pancreas and diabetes was clear, but not the specific role of the islets.

Over the next two decades, several attempts were made to isolate whatever it was the islets produced as a potential treatment. In 1906 George Ludwig Zuelzer was partially successful treating dogs with pancreatic extract but was unable to continue his work. Between 1911 and 1912, E.L. Scott at the University of Chicago used aqueous pancreatic extracts and noted a slight diminution of glycosuria but was unable to convince his director of his work's value; it was shut down. Israel Kleiner demonstrated similar effects at Rockefeller University in 1915, but his work was interrupted by World War I and he did not return to it.

Nicolae Paulescu, a professor of physiology at the University of Medicine and Pharmacy in Bucharest, was the first one to isolate insulin, which he called at that time pancrein, and published in 1921 the work that he had carried out in Bucharest. Use of his techniques was patented in Romania, though no clinical use resulted.

In October 1920 Canadian Frederick Banting was reading one of Minkowski's papers and concluded that it is the very digestive secretions that Minkowski had originally studied that were breaking down the islet secretion(s), thereby making it impossible to extract successfully. He jotted a note to himself: Ligate pancreatic ducts of the dog. Keep dogs alive till acini degenerate leaving islets. Try to isolate internal secretion of these and relieve glycosurea.

The idea was that the pancreas's internal secretion, which supposedly regulates sugar in the bloodstream, might hold the key to the treatment of diabetes. A surgeon by training, Banting knew that certain arteries could be tied off that would lead to atrophy of most of the pancreas while leaving the islets of Langerhans intact. He theorized that a relatively pure extract could be made from the islets once most of the rest of pancreas was gone.

In the Spring of 1921 Banting traveled to Toronto to explain his idea to J.J.R. Macleod who was Professor of Physiology at the University of Toronto, and asked Macleod if he could use his lab space to test the idea. Macleod was initially skeptical, but eventually agreed to let Banting use his lab space while he was on vacation for the summer. He also supplied Banting with ten dogs to experiment on, and two medical students, Charles Best and Clark Noble, to use as lab assistants, before leaving for Scotland. Since Banting required only one lab assistant, Best and Noble flipped a coin to see which would assist Banting for the first half of the summer. Best won the coin toss, and took the first shift as Banting's assistant. Loss of the coin toss may have proved unfortunate for Noble, given that Banting decided to keep Best for the entire summer, and eventually shared half his Nobel Prize money and a large part of the credit for the discovery of insulin with the winner of the toss. Had Noble won the toss, his career might have taken a different path. Banting's method was to tie a ligature (string) around the pancreatic duct, and, when examined several weeks later, the pancreatic digestive cells had died and been absorbed by the immune system, leaving thousands of islets. They then isolated an extract from these islets, producing what they called isletin (what we now know as insulin), and tested this extract on the dogs. Banting and Best were then able to keep a pancreatectomized dog named Alpha alive for the rest of the summer by injecting her with the crude extract they had prepared. Removal of the pancreas in test animals essentially mimics diabetes, leading to elevated blood glucose levels. Alpha was able to remain alive because the extracts, containing isletin, were able to lower her blood glucose levels.

Banting and Best presented their results to Macleod on his return to Toronto in the fall of 1921, but Macleod pointed out flaws with the experimental design, and suggested the experiments be repeated with more dogs and better equipment. He then supplied Banting and Best with a better laboratory, and began paying Banting a salary from his research grants. Several weeks later, it was clear the second round of experiments was also a success; and Macleod helped publish their results privately in Toronto that November. However, they needed six weeks to extract the isletin, which forced considerable delays. Banting suggested that they try to use fetal calf pancreas, which had not yet developed digestive glands; he was relieved to find that this method worked well. With the supply problem solved, the next major effort was to purify the extract. In December 1921, Macleod invited the biochemist James Collip to help with this task, and, within a month, the team felt ready for a clinical test.

On January 11, 1922, Leonard Thompson, a 14-year-old diabetic who lay dying at the Toronto General Hospital, was given the first injection of insulin. However, the extract was so impure that Thompson suffered a severe allergic reaction, and further injections were canceled. Over the next 12 days, Collip worked day and night to improve the ox-pancreas extract, and a second dose was injected on January 23. This was completely successful, not only in having no obvious side-effects but also in completely eliminating the glycosuria sign of diabetes. The first patient in the United States was Elizabeth Hughes Gossett.

Children dying from diabetic keto-acidosis were kept in large wards, often with 50 or more patients in a ward, mostly comatose. Grieving family members were often in attendance, awaiting the (until then, inevitable) death.

In one of medicine's more dramatic moments Banting, Best, and Collip went from bed to bed, injecting an entire ward with the new purified extract. Before they had reached the last dying child, the first few were awakening from their coma, to the joyous exclamations of their families.

Banting and Best never worked well with Collip, regarding him as something of an interloper, and Collip left the project soon after.

Over the spring of 1922, Best managed to improve his techniques to the point where large quantities of insulin could be extracted on demand, but the preparation remained impure. The drug firm Eli Lilly and Company had offered assistance not long after the first publications in 1921, and they took Lilly up on the offer in April. In November, Lilly made a major breakthrough and were able to produce large quantities of highly refined insulin. Insulin was offered for sale shortly thereafter.

Purified animal-sourced insulin was the only type of insulin available to diabetics until genetic breakthroughs occurred later with medical research. The amino-acid structure of insulin was characterized in the 1950s and the first synthetic insulin was produced simultaneously in the labs of Panayotis Katsoyannis at the University of Pittsburgh and Helmut Zahn at RWTH Aachen University in the early 1960s.

The first genetically-engineered, synthetic "human" insulin was produced in a laboratory in 1977 by Herbert Boyer using E. coli. Partnering with Genentech founded by Boyer, Eli Lilly went on in 1982 to sell the first commercially available biosynthetic human insulin under the brand name Humulin. The vast majority of insulin currently used worldwide is now biosynthetic recombinant "human" insulin or its analogs.

The Nobel Prize committee in 1923 credited the practical extraction of insulin to a team at the University of Toronto and awarded the Nobel Prize to two men; Frederick Banting and J.J.R. Macleod. They were awarded the Nobel Prize in Physiology or Medicine in 1923 for the discovery of insulin. Banting, insulted that Best was not mentioned, shared his prize with Best, and Macleod immediately shared his with James Collip. The patent for insulin was sold to the University of Toronto for one dollar.

While Paulescu's pioneering work was surprisingly being completely ignored by the Nobel prize committee, Professor Ian Murray was particularly active in working to correct the historical wrong against Paulescu. Murray was a professor of physiology at the Anderson College of Medicine in Glasgow, Scotland, the head of the department of Metabolic Diseases at a leading Glasgow hospital, vice-president of the British Association of Diabetes, and a founding member of the International Diabetes Federation. In an article for a 1971 issue of the Journal of the History of Medicine and Allied Sciences, Murray wrote:

Furthermore, Murray reported:

The primary structure of insulin was determined by British molecular biologist Frederick Sanger. It was the first protein to have its sequence be determined. He was awarded the 1958 Nobel Prize in Chemistry for this work.

In 1969, after decades of work, Dorothy Crowfoot Hodgkin determined the spatial conformation of the molecule, the so-called tertiary structure, by means of X-ray diffraction studies. She had been awarded a Nobel Prize in Chemistry in 1964 for the development of crystallography.

Rosalyn Sussman Yalow received the 1977 Nobel Prize in Medicine for the development of the radioimmunoassay for insulin.