Insulin: Molecule of the Month for February 2017

I always wrestle with my choice of molecule a few days before my Google Calendar sends me an irritating, but essential reminder to start writing my Molecule of the Month entry. I often set my sights on one molecule and something at the last minute tells me to change tack. This time, I was contemplating an influenza post, but then I read an article about Dorothy Hodgkin (shown left), and realised I have never written about insulin. So, to put this right, here then is my homage to the late, great crystallographer, Dorothy Hodgkin, or rather the molecule that she made her own.
 
With Roy Orbison's classic "Pretty Woman" topping the charts, the Nobel Committee of 1964, announced that Dorothy Crowfoot Hodgkin had been chosen to receive the Nobel Prize in Chemistry, for her work in the field of X-ray crystallography, specifically, the determination of the molecular structure of  Vitamin B₁₂, which at the time represented a major intellectual and technical achievement in the field (RHS). It wasn't until around five years later that the structure of insulin would emerge from Dorothy Hodgkin's laboratory, nearly 35 years after she carried out her first experiments on a molecule that captured her imagination owing to insulin's extraordinary impact on human physiology. Before I launch into the molecule itself, it is worth pointing out that having won the Nobel Prize in 1964, Dorothy Hodgkin received the Order of Merit a year later; only the second woman recipient at the time: the first being Florence Nightingale! However, I do like the fact that alongside her scientific prizes, she was awarded the Lenin Peace Prize. In fact she was awarded the Peace Prize in 1987, just before she opened the Krebs Institute at Sheffield. Since the Prize was discontinued three years later, as the former Soviet Union experienced Perestroika and Glasnost, no other Scientists will ever receive it!

Most of us knows someone who suffers from one of the two forms (Types I or II) of diabetes [a strange word that is derived from the Greek for a syphon: a device that draws off fluid]. Type I diabetes melitus, is an inherited disorder in which the insulin producing, pancreatic beta cells are compromised by an auto-immune attack, leading to a catastrophic loss of insulin and a classically "sweet" tasting blood/urine (hence melitus). Type II diabetes melitus (the melitus is often dropped), arises because the insulin responsive cells have become insulin resistant: so once again the blood is glucose rich, but insulin is still produced. Around 5-8% of the population (1 in 15 adults) have diabetes. However, only 0.5% of these individuals have the Type I form (who require regular insulin top-ups): the remainder generally develop Type II diabetes over the age of 40, and can keep the symptoms at bay by a change of lifestyle (a better diet and more exercise). Thanks to @WillSampsonUW, for providing this nice link, after reading the post.


So what doe insulin "look like" and how did insulin become one of the earliest protein based pharmaceuticals? In fact it was the first synthetic protein to be developed via both Chemistry (in the 1960s) and Biotechnology (in the late 1970s). Human insulin is a relatively small protein, comprising two chains (A and B) each containing 51 amino acids. The chains are held together by disulphide bonds. Insulin is found in all animals and is a highly conserved protein. Until the 1980s, Type I diabetes patients were routinely provided with porcine insulin to manage the symptoms of hyperglycaemia. The early "pharmacological" work of Banting, Macleod, Best and Collip (the first two received the Nobel Prize in Physiology or Medicine in 1923, and famously shared good fortune with the latter two!), identified insulin as the molecular solution to alleviating the symptoms of Type I diabetes. The drug company Eli Lilley began manufacturing, and clinicians administering, porcine insulin in the 1920s/'30s. In 1950, Novo Nordisk began manufacturing a longer lasting form of insulin: the porcine product continued to be used until around 1980, when recombinant insulin became the main form, developed by Genentech in San Francisco using recombinant DNA technology.

I can't possibly leave out Fred Sanger (a role model for many Biochemists of my own and the previous generation of Biochemists) from the insulin story. The primary structure of insulin was determined by Fred Sanger in the early 1950s (earning him his first of two Nobel Prizes: see LHS). This pioneering work led to the determination of the sequences of many subsequent protein structures, which in itself paved the way for our understanding of protein evolution (which was subsequently accelerated by the work, that earned Fred Sanger his second, and perhaps better know Nobel Prize, for developing the principles of nucleic acid sequencing). I include Sanger's work for another reason. The sequences of human and porcine insulin differ by just one amino acid. However, Eli Lilley were producing a protein for injection into humans without any knowledge of its relationship to the endogenous human insulin, missing from diabetics. Today, this would not pass the drug regulatory authorities. However, like the use of penicillin, these ground-breaking biologics (as they are now often called) helped save many thousands of lives and it may be worth discussing this alongside thalidomide in a tutorial on science and ethics.

Returning to the structure of insulin, the human form (often referred to as Lispro) is shown on the right, with the characteristic Zn atom coordinated at the core of the molecule. The circulating form of insulin is generally a hexamer (as shown), but the active species is a monomer, which interacts with the Insulin Receptor, on liver cells, fat cells, skeletal muscle cells etc, to stimulate the assimilation of glucose into glycogen. The converse reaction, glucose release (or glycogen mobilisation) is induced by the yin to insulin's yang, glucagon, which is released from pancreatic alpha cells. The insulin receptor is a membrane bound, Tyrosine Kinase class molecule, which, in response to insulin binding, triggers a sequence of events that leads ultimately to the stimulation of the gluconeogenic pathway, thereby depleting levels of blood glucose. As I mentioned above; when glucose levels are balanced, the release of glucose is stimulated by glucagon. A general scheme is shown below (taken from the nice wiki site on the insulin receptor.

My final comments relate to the conservation of insulin's primary structure, which has emerged from the many animal genome sequences deposited over the last 15 years.The relationship between structure (primary begets tertiary, remember) and function, is a well established concept in chemistry and biochemistry, and I don't wish to suggest otherwise. However, as those of you who have read any of my earlier posts on the ramifications of Anfinsen's law and recent studies on structural equilibria among certain disease related proteins, suggests that primary structure-function relationships are a little more complex than we thought. The logic behind the highly conserved nature of the insulin molecule makes sense and, although this was not a pre-requisite (see above) for the early clinical use of the closely related porcine hormone, it is clear that insulin has many key interactions to make, for such a small protein. This raises an interesting tutorial point: is there any correlation between evolutionary conservation of primary structure and the molecular weight of the protein molecule in question? I hope you agree that insulin is a fascinating molecule, which has been at the heart of Physiology, Biochemistry, Structural Biology, Genetics and disease for over 100 years, and I have no doubt there is more to come...