Understanding Insulin: A Primer

Abstract

Insulin is an essential protein hormone secreted by the beta cells of the islet of Langerhans in the pancreas which is involved in glucose homeostasis, cell metabolism and mitogenesis. It is essential that healthcare providers are conversant with the normal physiology of this protein in the human body, to aid them in clinical decision-making when processes pertaining to this crucial substance go aberrant in the “corpus”. Healthcare providers will then be able to better appreciate the pathophysiology of disease states pertaining to this hormone most importantly diabetes which is a great cause of global morbidity and mortality. Healthcare providers should be comfortable especially in recognizing these disease states clinically and instituting the most appropriate form of management in-line with the most recent evidence-based medicine to improve patient outcomes.

1. Introduction

Insulin (from the Latin word insula, meaning island) is an essential protein hormone secreted by the beta cells of the islet of Langerhans in the pancreas which is involved in glucose utilization, cell metabolism and mitogenesis [1, 2]. It is essential that healthcare providers are conversant with the normal physiology of this protein in the human body, to aid them in clinical decision-making when processes pertaining to this crucial substance go aberrant in the “corpus”.

Diabetes mellitus (DM) needs no introduction as a significant cause of morbidity and mortality. In 2015, it was estimated that 415 million people were afflicted by diabetes, this number is expected to rise to 642 million in 2040, with an increasing disease burden being shared by people in the developing world. The microvascular and macrovascular complications of this disease have plagued man for a long time. These negative consequences include nephropathy, neuropathy, retinopathy, stroke, renovascular disease, limb ischemia and the dreaded amputation of the limb [3, 4]. The earliest references to this disease in written literature go as far back as 1550 BCE [5]. Despite great progress in our understanding of diabetes and our ever growing arsenal of therapeutic agents used to combat the disease, this old foe of man remains very much alive.

Currently, diabetic kidney disease (DKD) is the leading cause of end-stage renal failure in the world. Approximately, 30% of individuals affected by type 1 DM develop diabetic nephropathy, this increases to 40% in persons affected by type 2 DM [6]. This situation has been compounded by the ever increasing rates of obesity in the world [7]. In 2013 pertaining to the United States of America (USA), the age-adjusted prevalence of obesity was 35% in men and 40% in women representing an upward trajectory of this statistic when juxtaposed to the year 2000 where the overall prevalence of obesity in that country stood at 31% [6, 8]. The connection between obesity and type 2 DM is corroborated by many a research. This only strengthens calls for healthcare professionals involved in the management of this canker to spell out clearly to their clients the gargantuan benefits of lifestyle interventions in the management of DM. Insulin therapy has been a cornerstone in the medical management of type 1 DM. It is also used in the treatment of type 2 DM being necessitated by beta cell exhaustion [9].

This review will attempt to feebly summarize the advances we have made over the years in understanding insulin pertaining to its structure, effects on the various tissues and the development of analogues.

2. An important protein

Insulin’s significance to medical science cannot be overemphasized. This small yet mighty peptide hormone together with other counter-regulatory hormones notably glucagon and epinephrine is important in glucose homeostasis in the body. These hormones are important in mediating the switch between anabolic and catabolic phases of the human body enabling it to respond adequately to various stressors such as major burns, sepsis, major surgery and other possibly injurious states. Glucose is the chief fuel used by many cells in the body, typified by neurons and erythrocytes, in cellular respiration to release adenosine triphosphate (ATP) [1, 2].

ATP provides the energy to power life processes of the cell. This is needed to maintain the physiologic milieu of the organism. Too much insulin which is more common in diabetic patients on insulin and insulin secretagogues such as the sulfonylureas and meglitinides may tip the individual into life-threatening hypoglycemia which may be manifested by neurogenic and neuroglycopenic symptoms. Neurogenic symptoms are said to be a response of the autonomic nervous, principally the adrenergic division, to the dip in glucose and are characterized by tremor, anxiety, palpitation, paresthesia, diaphoresis and sensation of hunger. Although, this can be compounded by hypoglycemia unawareness where these symptoms may be blunted and the individual is not able to tell that they are hypoglycemic. Hypoglycemia unawareness affects about 40% of people with type 1 DM. It affects a lesser number of individuals with type 2 DM. Repeated bouts of hypoglycemia lower the level of glucose at which the human body may respond, whiles chronically elevated levels of this may raise this threshold. Neuroglycopenic symptoms which typically occur at lower glucose levels are the result of brain neuronal deprivation of glucose and include confusion, dizziness, headaches, seizures, coma among other symptoms [10, 11].

Perturbation about inducing iatrogenic hypoglycemia presents a hindrance in achieving glycemic targets [10]. Too little insulin for a prolonged period or a reduced action of the hormone may manifest acutely as a decompensated diabetic state such as diabetic ketoacidosis and hyperglycemic hyperosmolar state. Regrettably, the incidence of these two hyperglycemic crises has been on the increase. In the USA, for example, there were 220,340 hospital admissions for DKA in 2017 compared to 168,000 in 2014. Mortality from these conditions though is subsiding. Mortality rate was estimated to be under 1% for DKA, while it can get as high as 20% for HHS. Advanced age, comorbidity and severe dehydration contributed to higher fatality rates in HHS. There is the need for healthcare practitioners and their clients to be more proactive in preventing these dangerous states which can impact a significant economic toll. DKA is the cause of more than 500,000 hospital days per year in the USA according to the Centers for Disease Control and Prevention (CDC). The cost of inpatient care for DKA stood at approximately 6.76 billion US dollars in 2014; in 2017 it was 5.1 billion US dollars [6].

2.1 Discovery

More than a century has elapsed since the crude “isolation” and discovery of insulin by Canadian surgeon Banting and American-Canadian medical student Best with aid from Scottish professor Macleod and Canadian biochemist Collip at the University of Toronto, Canada. Their method was to try to obtain a purified insulin preparation from dog pancreases (later on, rabbits). Their persistence finally paid off in 1921 when their preparation proved capable of normalizing hyperglycemia and glycosuria in dog subjects. That monumental year of 1921 ushered medicine in into a new era. Indeed, insulin has been a pioneer protein for medical research in many regards [12].

In the wake of Paul Langerhans description of islets in the pancreas in 1869, which now eponymously bear his name, scientists had found out that a substance in the pancreas if deficient was implicated in the pathogenesis of diabetes. They had tried to purify this substance from the pancreas for a possible transition from bench-to-beside in the treatment of diabetes. However, despite some strides no one had been able to isolate this substance and demonstrate its reproducible use in man convincingly without toxic reactions up until 1921. In 1889, Minkowksi and von Mering found out that severe diabetes mellitus could be induced in dogs by carrying out a total pancreatectomy leading them to the realization that a substance in the pancreas was needed in the control of blood glucose level. In 1909, Belgian de Meyer suggested the name “insuline” for this substance. Scientific thinking evolved over the years localizing this substance to the islets of Langerhans in the pancreas, thus setting the stage for the work of Dr. Banting and his colleagues. By 1909, Eugene L. Opie had demonstrated histological evidence of hyaline degenerative changes in the islets of Langerhans of individuals with diabetes mellitus [2, 7, 12].

Doctors at Toronto General Hospital led by Walter Campbell were finally able to inject a young boy in 1923 with success to their delight. Fourteen-year old Leonard Thompson showed marked clinical and laboratorial amelioration after he received a second trial of more purified insulin. Testament to its significance, Banting and Macleod received the Nobel Prize in Medicine or Physiology in 1923. Hitherto insulin’s discovery, a diagnosis of diabetes mellitus had largely been a death sentence [5].

Of interesting note was the fact that very little was known of insulin’s structure at the time of its discovery. To buttress this, Macleod did not think insulin was even a protein. It would take painstaking work of another- Fred Sanger- to elucidate the polypeptide structure of the hormone using 1-fluoro-2,4-dinitrobenzene (DNFB) (Sanger’s reagent) among other techniques. He surmised that bovine insulin must be made of two polypeptide chains and deduced their amino acid sequences. Indeed, three different scientific groups set out to confirm Sanger’s proposition about insulin’s structure with the aid of protein chemical synthesis in the 1960s. They confirmed his earlier findings about insulin’s structure by showing that the synthesized protein had biological activity. For his efforts, Sanger received the Nobel Prize much earlier in 1958. X-ray crystallography was used to determine the three-dimensional structure of insulin by Hodgkin, Blundell and Dodson. They published their results in 1969. Yalow and Berson invented radioimmunoassay which proved capable of quantifying insulin levels in the blood in 1959. For this work Yalow received the Nobel prize in 1977, after Berson had died [12].

Again, with the advent of recombinant DNA technology in the 1970s, the gauntlet fell to insulin. It became the second protein after somatostatin to be synthesized by these methods forever revolutionizing the care of individuals with DM. It was the first therapeutic protein engineered by recombinant DNA technology to be approved for human use by the US FDA in 1982. Prior to this the food industry supplied animal pancreases for the extraction of porcine and cattle insulin which both differ from human insulin by 1 (B30) and 3 amino acids (B30, A8 &A10) positions respectively. Recombinant DNA techniques as well as the elucidation of the hormone’s structure introduced new paradigms in the treatment of diabetes that allowed mass production of better quality insulin as well as the modification of its chains to produce analogues with desirable properties that more closely mimicked insulin’s famous natural biphasic secretion pattern (Figure 1) [12, 13].

Figure 1.

Primary structure of human insulin.

2.2 Structure and secretion

Insulin is produced by beta cells of the islet of Langerhans in the pancreas. Recent findings suggest that some cells in the brain may also be capable of synthesizing insulin. The constituents of the islets of Langerhans include alpha cells which produce glucagon, beta cells which produce insulin, delta cells which produce somatostatin and pancreatic polypeptide (PP) cells which produce pancreatic polypeptide. Beta cells make up the majority of cells in the islets of Langerhans and are found in the centre of the islets compared to the α cells which tend to be found in the periphery. There human pancreas contains over a million islets of Langerhans. They make up about 1–2% of the weight of the pancreas, although they receive about 10% of the organ’s blood supply. These are scattered groups of endocrine cells within the exocrine acinar cells of the pancreas. The pancreas, just like the liver is both an endocrine and exocrine gland with important roles in digestion and assimilation of food substances. The acinar cells are responsible for manufacture of various enzymes (trypsinogen, chymotrypsinogen, procarboxypeptidase, deoxyribonuclease, ribonuclease, pancreatic lipase and pancreatic amylase) that are important components of the succus entericus which is essential in the hydrolysis of peptides, nucleic acids, lipids and carbohydrates. These are first secreted as zymogens which later undergo activation to function. The exocrine cells in response to secretin produced by enteroendocrine cells in the duodenal mucosa also secrete sodium bicarbonate which serves to neutralize acidic chyme from the stomach among other functions [14, 15].

Insulin is a peptide hormone made up of 51 amino acids, with a molecular weight of 5802 and its isoelectric point at pH 5.5. It is composed of two chains: a B chain with 30 amino acids and an A chain with 21 amino acids. These chains are joined together by two interchain disulphide bonds connected via cysteine residues. The A chain also has an intrachain disulphide bond. The gene for insulin’s synthesis is located on chromosome 11p [2].

Insulin is first synthesized as 110-amino acid chain known as preproinsulin by polysomes. Preproinsulin consists of an N-terminal signal peptide, a B chain, a connecting peptide (C-peptide) and a carboxyl-terminal A chain. The N-terminal signal peptide directs insulin to the endoplasmic reticulum where it is cleaved off by an endopeptidase. The protein undergoes further repackaging in the Golgi bodies and is transmitted through the trans-Golgi network. It is incorporated into secretory vesicles from which it can be released from beta cells by exocytosis for example upon glucose stimulation [16]. Glucose is transported into human beta cells by glucose transporters (GLUT) 1 and 3; GLUT2 is currently thought to be more important in this function in rodents. Although, the question about the functions of GLUTs in the human β cell is still not completely answered. Upon cell entry, glucose is phosphorylated by glucokinase to form glucose 6-phosphate [1]. Eventually, ATP is formed by oxidative processes in the cell. ATP-sensitive K+ channels in the cell membrane of the beta cell subsequently close as the ratio of ATP/ADP molecules in the β cell increases. This causes depolarization of the cell and resultant movement of Ca²⁺ ions into the cell via voltage dependent Ca²⁺ channels. These ions are important in causing glucose stimulated insulin secretion (GSIS) into the bloodstream. The mechanism of action of sulphonylureas which are a drug class used in used in the treatment of type 2 DM also involves closure of ATP-sensitive K⁺ channels independent of blood glucose levels by their binding to the sulphonylurea receptor of these particular channels. GSIS occurs by both ATP-regulated K⁺dependent and ATP-regulated K⁺ independent pathways. Insulin secretion in response to glucose is classically said to be biphasic. In that there is an initial rapid phase lasting a few minutes in response to postprandial hyperglycemia, then a more sustained second phase. The increase in intracellular calcium ions causes exocytosis of secretory granules in the readily realizable pool (RRP). Typically, blood glucose concentrations above 5 mmol/l are necessary to cause the initial surge in insulin secretion. Lower levels of between 2 and 4 mmol/l, however are enough to cause synthesis of insulin into secretory granules. This is necessary to ensure that β cells can respond rapidly to the body’s metabolic demands [1, 2]. Insulin exists as a hexamer complexed with zinc ions in the secretory granules.

Nutrient in the gastrointestinal tract are able to cause insulin secretion which is not dependent on glucose. The incretin hormones, that is gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP1), are implicated in this process. While nutrients and non-nutrients can cause the initial phase, the more sustained phase is mediated primarily by nutrients. Amino acids like alanine cause an increase in insulin secretion. Non-nutrient secretagogues are also able to cause an increase in insulin secretion. There are endocrine, autocrine, paracrine and neural factors that affect insulin release. Somatostatin inhibits both insulin and glucagon secretion. While insulin downregulates glucagon secretion, glucagon upregulates insulin secretion. Insulin is thought to have positive effects on its own secretion. Cholinergic stimulation increases insulin secretion, while activation of α2 receptors by sympathetic nervous system inhibits insulin secretion. Vasoactive intestinal peptide from peptidergic fibers also cause an increase insulin secretion [1, 2].

2.3 Insulin’s effect on tissues and mechanism of action

The insulin receptor (IR) responsible for mediating insulin’s effects is a tyrosine kinase with a heterotetrameric structure consisting of 2 alpha and 2 beta subunits and is located on the cell surface membrane. The gene for the insulin receptor is 150-kb long, has 22 exons and is on human chromosome 19p13.3–p13.2. Insulin binds to the extracellular alpha units, with the intracellular part of the beta subunit involved in recruitment of several adaptor proteins including the insulin receptor substrates (IRS) which mediate insulin’s intracellular actions via phosphorylation. There are two sites in the extracellular portion of the receptor with different affinities where insulin is capable of binding to. The IR is said to exhibit negative cooperative binding. There are two known isoforms of the receptor: IRA and IRB. IRA has fewer amino acids due to alternative splicing of exon 11. IRB is more specific for insulin than IRA. Three regions in the beta subunit of note undergo trans-autophosphorylation: one in the juxtamembrane domain (Y₉₆₅ and Y₉₇₂), one in the activation loop (Y₁₁₆₂, Y₁₁₅₈ and Y₁₁₆₃) and another in the C-terminal portion of the receptor (Y₁₃₂₈ and Y₁₃₃₄) after insulin binds to the extracellular portion to cause a transformational change in shape. IRS 1 and 2 are more widely distributed in the tissues. Upon activation of the IR, after phosphorylation of NPEpY732 in the juxtamembrane zone of the receptor, IRS-1 is recruited to this site. IRS-1 has amino-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) regions which are necessary for membrane and receptor association. Also, it has an unstructured part consisting of 14 tyrosine phosphorylation sites which follow these two other regions and undergo phosphorylation when the IRS-1 is activated. After phosphorylation, two YMPM motifs in IRS-1 bind src-homology 2 (SH2) binding sites of p85α which is a regulatory subunit of phosphatidylinositol-3-kinase (PI3K). The catalytic portion PI3K then becomes able to phosphorylate phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) which is responsible for some of insulin’s downstream actions including the activation of phosphoinositide – dependent protein kinase 1 (PDK-1). PDK-1 activates AKT. AKT2 mainly mediates insulin’s metabolic functions in skeletal muscle, adipose tissue, hepatic tissue and other bodily tissues. These processes will be discussed in more elaborate detail in the text below. It is now known that insulin, as well as the insulin-like growth factors (IGFs) 1 and 2, has important roles in other tissues of the body apart from the striated muscle, fat and the liver mediated by various receptors [1, 2, 7, 17].

Insulin just like IGF-1 has mitogenic actions. Insulin plays various roles in cell growth, division, differentiation and survival mainly via mitogen activated protein kinase pathway (MAPK). The insulin concentration needed for its mitogenic functions is greater than that for its metabolic activities. This involves the binding of IRS-1 to growth factor receptor-bound protein 2 (Grb2) which occurs after insulin signaling via the insulin receptor. Son of sevenless Ras/Rac guanine nucleotide exchange factor 1 (SOS1) is recruited by Grb2 leading to the activation of the rat sarcoma protein (RAS) via GTP-GDP exchange. RAS then involves c-Raf which phosphorylates MAPK/extracellular-signal regulated kinase (MERK), which then activates extracellular-signal regulated kinase (Erk). Translocation of Erk to the nucleus leads to the transcription of nuclear factors important in mitogenesis [1, 2, 17].

2.3.1 Skeletal muscle

The skeletal muscle is the primary site of postprandial glucose uptake. Insulin acting on the IR causes the translocation of GLUT4 receptor to the surface membrane. This effect is mediated via the AKT pathway. This involves the phosphorylation of Cbl-associated protein (CAP) and CAP: CBL: CRKII complex formation. GLUT4 shuttles glucose molecules into the cell by facilitated diffusion. The translocation of GLUT4 is reversible as insulin concentrations dwindle. Insulin is thought to also influence GLUT4 gene expression in muscle and adipose tissue. Once inside the cell, glucose can be used for energy release or stored as glycogen. Insulin upregulates glycogenesis n skeletal muscle and inhibits glycogenolysis [1, 17, 18].

2.3.2 Adipose tissue

Insulin stimulates white adipose tissue to undergo lipogenesis and inhibits lipolysis. Glucose is converted to triacylglycerol in the fed state. The functions of adipose tissue, especially visceral fat is of special interest in the pathogenesis of hyperinsulinemia, insulin resistance, obesity and type 2 DM. Insulin resistance is when the response of target cells to insulin is lower than it should be. In cases of nutrient excess, when the storage capacity of fat tissue is exceeded, lipases hydrolyse triacylglycerol into free fatty acids (FFA) and glycerol which enter the circulation. Insulin is unable to inhibit lipolysis, and this may lead to “lipotoxicity”. Ectopic accumulation of lipids occurs in skeletal muscle and hepatocytes. This encourages the development of non-alcoholic fatty liver disease (NAFLD). In skeletal muscle, this intramyocellular accumulation sometimes is associated with the development insulin resistance [1, 17, 18].

2.3.3 Liver

The liver has a special role in the regulation of glucose homeostasis. The liver is capable of converting non-carbohydate sources like alanine, lactate and pyruvate into glucose in a process known as gluconeogenesis. This is upregulated by glucagon and inhibited by insulin and alcohol. Insulin also inhibits glycogenolysis but upregulates glycogenesis. The liver typically holds about 100 g of glycogen, with about 500 g in skeletal muscle. This helps to explain partly the tendency of patients with liver cirrhosis to suffer hypoglycemia. GLUT 2 receptor is bidirectional as it is involved in both glucose uptake and release into the blood [1, 7, 18].

When there is lipotoxicity, hyperinsulinemia is unable to suppress the release of non-esterified fatty acids (NEFA) by adipose tissue. The liver uses some of these excess fatty acids to synthesize VLDL leading to hypertriglyceridemia. Also, individual are prone to suffering non-alcoholic steatohepatosis (NASH) and non-alcoholic fatty liver disease (NAFLD). Infection with hepatitis C virus is thought to also potentiate such pathways. Insulin-resistance may lead to uncontrolled gluconeogenesis with attendant chronic hyperglycemia precipitating end-organ damage. Dysregulation of forkhead box O family (FOXO) of transcription factors by defective insulin PI3K/AKT signaling allows the unrestrained translocation of these factors to the nucleus to promote gluconeogenesis [17, 18].

2.3.4 Brain

The brain is selective for glucose as its principal fuel, though it is capable of switching in lean times. GLUT1 receptors at the blood-brain barrier are capable of extracting glucose from the blood in an insulin-independent fashion. Recent findings though suggest that certain areas in the brain such as the pineal gland choroid plexus and the pituitary as well as the spinal cord obtain glucose in an insulin-dependent fashion. Studies show that insulin may improve cognition and memory in individuals with Alzheimer’s disease (AD). Besides this, patients with AD are more prone to developing type 2 DM. This link has generated much excitement in medical circles with some practitioners labeling AD as “type 3 diabetes”. Intranasal insulin is being tried in this respect [1, 19].

2.3.5 Endothelial tissue

Insulin acts via the insulin receptor on endothelial tissues. Aberrant insulin signaling associated with selective insulin resistance may lead reduced nitric oxide production, endothelial dysfunction, poor wound-healing, atherogenesis, pro-thrombotic and pro-inflammatory states with an increased risk of hypertension and other cardiovascular morbidity and mortality. Novel targeted therapies are needed to attenuate these effects [1].

2.4 Insulin today

When insulin was first made available clinically, large volumes of the product had to be given which often led to local site infections and abscesses. These problems have reduced for today’s diabetic patient. Also, increased ease of delivery of insulin and glucose monitoring have improved the quality of life of people living with diabetes. Infusion pumps and insulin pens have allowed more efficient drug administration. Blood glucose monitoring with capillary glucose tests, continuous glucose monitoring and widespread availability of HbA1C test have allowed more precise glycemic control and attenuated the risks of diabetic complications [9].

Today’s insulin’s preparations have been upgraded with refinements in their peak action times and duration of action, although they are still a poor substitute for insulin’s complex physiology with its feedback systems. They have facilitated the use of today’s basal bolus regimen in the management of diabetes mellitus.

There are rapid-acting, short-acting, intermediate-acting and long-acting insulin analogs. Rapid-acting insulin analogs include lispro, aspart and glulisine which have on the average an onset of action of about 5–15 min, a usual peak action time of about 30–60 min and duration of 2–5 h. Short-acting insulin analogs such as regular human insulin typically have an onset of action of time of about 30–60 min, a usual peak action time of about 1–3 h and duration of 4–8 h. Intermediate-acting insulin analogs include Neutral Protamine Hagedorn (NPH) on the average have an onset of action of time about 1–2 h, a usual peak action time of about 4–8 h and duration of 8–12 h. Long-acting analogs including detemir, and glargine have on the average an onset of action of time of about 30–60 min, with no peak action time, last about 16–24 h and can generally be dosed as a single shot per day. There are also premixed analog formulations available which combine different types of analogs [9, 20]. Concerns have been raised about the theoretical risk of analogs to cause cancer through insulin’s mitogenic pathways. Evidence to support this stance though remains paltry, and more research work will need to be done in this regard [21]. Economic barriers remain in acquiring these novel therapies perhaps in ironic contrast to this saying attributed to Banting, “insulin does not belong to me, it belongs to the world [5].”

3. Conclusion

It goes without saying that all healthcare professionals especially doctors should be abreast of the ever-changing landscape in the management of DM. The pervasive nature of this malady in surgery, internal medicine, child health and obstetrics & gynecology make this imperative. This of course will be a nine-day wonder without an understanding of insulin’s mechanisms not only in type 1 DM, but also in other forms of DM. This in the long run will boost patient outcomes. Medical research must also continue with necessary funding. Additionally, it will be of great benefit for pharmaceutical companies to consider the costs of drugs so as to make them accessible to society’s most vulnerable. As history has shown us, it is only on the intrepid steps of alacrity and sometimes good old serendipity will other scientific breakthroughs be made in the treatment of DM. As the torch is passed on to this generation of medical practitioners in the next century after insulin’s discovery we owe a lot to the prescience of the men who have contributed to our understanding of this interesting peptide hormone. It is only befitting tribute to their memories that we expand their work and finally “defeat” DM together.

Conflict of interest

The author declares no conflict of interest.