Chapter 18
Phosphoinositide 3-Kinases, Protein Kinase B, and Signalling through the Insulin Receptor
In a viva voce examination at one of the more august universities, a student was being questioned about insulin and how it works. After some embarrassed hesitation, he assured the examiners that he did know how it works, but unfortunately he had now forgotten. ‘What a pity’, came the response. ‘That means that now, nobody knows.’
Insulin receptor signalling; it took a little time to work out the details
As should be abundantly clear from Chapter 1, a lack of knowledge about how a particular drug or therapy works has never been an impediment to doctors working in the clinic. Indeed, for most practitioners, it is generally
a very secondary consideration. A well-known case in point is aspirin, first introduced by the Bayer Company in 1898. With the wholehearted encouragement of the medical profession, it has been consumed by the
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Signal Transduction
The many abbreviations used in this chapter are collected together at the end of the chapter.
tonne, and with good effect too. Yet it was more than 80 years before its most sensitive target, cyclo-oxygenase, was identified as responsible for the synthesis of prostaglandins.1 Another example is the treatment of childhood (type I) diabetes with insulin.2,3
Several glycogenolytic hormones (adrenaline, glucagon, vasopressin, growth hormone) act to mobilize the metabolic stores of carbohydrate and fat, to maintain the concentration of blood glucose, but insulin uniquely has the reverse effect. It increases the net uptake of glucose from the blood into the tissues and increases its conversion to glycogen and triglyceride, at the same time inhibiting their breakdown. Since the acute effects of the glycogenolytic hormones all require elevations of cAMP or Ca2 , it follows that the actions of insulin must be mediated through a third route, one that escapes the attentions of either PKA or PKC. Only in 1980 did it become apparent that the mechanism involves autophosphorylation of the receptor.4 The discovery of protein kinase B (also known as Akt) and the 3-phosphorylated inositol lipids eventually provided the key.4
The association of diabetes mellitus with the pancreas was established by the work of Oscar Minkowski.5 A dog from which he had removed the pancreas began to suffer an uncontrollable polyuria. The following day, there being no cage large enough to accommodate the animal, which had been well house-trained, it was kept tied up in the laboratory. According to the story (subsequently denied by Minkowski), the assistant noticed that flies settled wherever it had passed urine. Regardless of this, the animal passed 12% of sugar and was suffering from diabetes mellitus.
It is customary to credit the Canadians Banting and Best with the discovery of insulin, their colleagues McLeod and Collip somehow standing close by (or not quite so close by in the case of McLeod, who may have spent some of the critical months on a fishing holiday on the Isle of Skye). For sure, were it not for Banting’s certainty and indeed obsession, it is unlikely that patients would have been successfully treated within a few months of the commencement of the experimental trials. However, it is legitimate to ask whether the Canadians were actually the first discoverers of insulin. Here, we are not considering
the widely canvassed claim of Nicholas Paulescu,2,3 who was certainly in the race at about the same time, but that of Eugene Gley who was working 25 years earlier. Sadly, his right to scientific immortality fails on two counts. First, he was never in a position to apply his preparation in the treatment of patients. This is the importance of Banting and Best’s contribution. Whether
he had the means to make the repeated and rapid analyses of blood glucose concentration, necessary to monitor its antiglycosuric effect, is uncertain. More likely, he just lacked the audacity to inject his preparation into people, a restraint to which Banting was quite insensitive. Anyway, Gley was working in 1895 and all this had to wait for another quarter century, but we do know
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Phosphoinositide 3-Kinases, Protein Kinase B, and Signalling through Insulin Receptor
from his report that there can be little doubt that he had successfully isolated an antiglycosuric agent from extracts of pancreas. He provides sufficient detail, rare in his day, for the modern reader to have some confidence that his preparation was as good as he claimed.
But where was Gley’s report all this time? Instead of going public, he sealed it in an envelope which he placed into the hands of the secretary of the Société de Biologie de Paris, with the firm instruction that it was not to be opened until directed by him.6 With this bizarre act he waived all claims to be credited as the discoverer of insulin, and so it lay hidden until word came through from Canada in 1921. The application of insulin was certainly one of the key milestones of modern medical practice, yet its mechanism of action as a glucose-lowering hormone has been widely regarded as a ‘mystery’, even into the last decade of the 20th century.7,8
Signalling through phosphoinositides
In Chapter 5 we discussed the effector, phospholipase C, that utilizes the minority phospholipid, phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2), as its substrate. Cleavage of the phospholipid head group produces the soluble second messenger inositol trisphosphate (IP3), which releases Ca2 from intracellular stores into the cytosol. The other product is the lipid, diacylglycerol (DAG) which activates PKC. PI(4,5)P2 is present in unstimulated cells and its level in cell plasma membranes was originally considered to be under homeostatic control; it was therefore not classed as a signalling molecule. However, this is an oversimplification. Quite apart from being a substrate for PLC, the head group of PI(4,5)P2 provides an important anchoring point on membranes for many cytosolic signalling proteins. (Principally those with phosphoinositide-binding domains; see page 774.) Furthermore, this phospholipid also exists in other cellular membranes, such as those of the Golgi apparatus, and even in the plasma membrane it is distributed unevenly, tending to segregate into dynamic microdomains rich in cholesterol and sphingolipids, commonly referred to as lipid rafts (see page 522). Exactly how the levels of PI(4,5)P2 in these
different pools are regulated is unclear, but there is evidence for local control through the phosphatidylinositol phosphate kinases (PIPkins) that convert phosphatidylinositol-4-phosphate and phosphatidylinositol-5-phosphate (PI(4)P and PI(5)P) to PI(4,5)P2. For example, PIPKinI (also known as PI(4)P 5- kinase) functions downstream of the small GTPases Rho9, Rac,10 and Arf.11
In addition to all this, PI(4,5)P2 is just one of many phosphoinositides that exist in cell membranes. They are formed by a series of reversible phosphorylations and dephosphorylations of the head group. An array of kinases and phosphatases regulate their interconversion. The metabolism
of the seven principal phosphoinositides is outlined in Figure 18.1. Although
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