The discovery of peptides used for the treatment of diabetes/obesity: A personal journey.

The aim of this personal reminiscence is to acquaint the reader with seminal workwork carried out in 1960 s and 1970 s that made possible the subsequent development of highly effective long-acting GLP-1R agonists and GLP-1R/GIPR co-agonists that are now in clinical practice for the treatment of Type 2 diabetes and obesity. The article highlights the particular contributions of the author's collaborators Ellis Samols and Desmond Turner in elucidating the nature and significance of gut glucagon-like immunoreactivity (enteroglucagon) and GIP. The potent incretin GLP-1(7-36)amide identified in the 1980 s met the criteria for a glucagon-like-substance with incretin like properties postulated to exist by Samols and others in 1966.

The early history of GIP 1969-2000: From enterogastrone to major metabolic hormone.

This paper describes the early history of Gastric Inhibitory Polypeptide, better referred to simply as GIP, from its isolation by purification from a crude preparation of CCK-PZ (cholecystokinin/pancreozymin) to its recognition as a key play in the pathogenesis of obesity and other metabolic disorders far removed from the enterogastrone properties by which it was originally identified. Augmentation of glucose mediated insulin release, the incretin effect, was discovered soon after GIP was first isolated and only much later was its important role in the pathogenesis of obesity, through mechanism other than its insulin secretion, appreciated. Immunoassay - the method by which the concentration of GIP was measured in plasma until quite recently - was found to be flawed and to depend upon which specific epitope of the hormone an assay detected. This was especially true if it was an amino-acid sequence specific to porcine rather than human GIP. A further confounder was the discovery that much of the GIP measured by immunoassay was its biological antagonist produced by cleavage of its two N-terminal amino-acids in the circulation by the same dipeptidyl-peptidase as de-activates GLP-1. Potential use of synthetic agonistic and antagonistic GIP analogues in therapeutics was barely alluded to before year 2000.

The early history of GIP 1969-2000: From enterogastrone to major metabolic hormone.

This paper describes the early history of Gastric Inhibitory Polypeptide, better referred to simply as GIP, from its isolation by purification from a crude preparation of CCK-PZ (cholecystokinin/pancreozymin) to its recognition as a key player in the pathogenesis of obesity and other metabolic disorders far removed from the enterogastrone properties by which it was originally identified. Augmentation of glucose mediated insulin release, the incretin effect, was discovered soon after GIP was first isolated and only much later was its important role in the pathogenesis of obesity, through mechanism other than insulin secretion, appreciated. Immunoassay - the only method by which the concentration of GIP was measured in plasma until quite recently - was found to be flawed and to depend upon which specific epitope of the hormone an assay detected. This was especially true if it was an amino-acid sequence specific to porcine rather than human GIP. A further confounder was the discovery that much of the GIP measured by immunoassay was its biological antagonist produced by cleavage of its two N-terminal amino-acids in the circulation by the same dipeptidyl-peptidase as de-activates GLP-1. Potential use of synthetic agonistic and antagonistic GIP analogues in therapeutics was barely alluded to before year 2000.

Rebirth of the Incretin Concept: Its conception and early development.

This paper describes the resurrection of the Incretin Concept in the early 1960s. It began with the more or less simultaneous discovery by three groups working independently in London. Dupre demonstrated that secretin given intravenously with glucose increased its rate of disappearance from the blood, McIntyre and co-workers established that hyperglycaemia evoked by oral glucose stimulated more insulin secretion than comparable hyperglycaemia produced by intravenous glucose and Marks and Samols established the insulinotropic properties of glucagon. The concept evolved with the discovery by Samols and co-workers that oral glucose stimulated the release of immunoreactive glucagon-like substances from the gut mucosa and the subsequent isolation of glucagon immunoreactive compounds, most notably oxyntomodulin and glicentin, and of gastic inhibitory polypetide (GIP). It concluded with the isolation and characterisation of glucagon-like peptide 1 (7-36) amide.

Hypoglycaemia and automatism.

Cognitive impairment unattended by subjective symptoms or objective signs is an uncommon but important consequence of hypoglycaemia. It can lead to a condition in which a patient behaves as an automaton in a manner totally alien to their usual personality and of which they have no recollection when their blood glucose level is restored to normal. It can cause a wide range of criminal behaviour, although the commonest offences relate to a loss of control, for example driving offences. Determination of criminal responsibility is extremely difficult and relies very heavily upon the quality of the medical evidence and interpretation of the law, which is out of step with current medical science.

Insulin and the law.

Hypoglycaemia, if it can be proved, may be used as a defence against almost any criminal charge provided it can be established that the perpetrator was in a state of neuroglycopenic (hypoglycaemic) automatism at the time of the offence. Hypoglycaemia produced by exogenous insulin can also be used as a suicidal or homicidal weapon. This paper discusses some of the pitfalls confronting the investigator of suspected insulin misuse including problems arising from the increasing prevalence of insulin analogues and the unreliability of immunoassays for their detection and measurement in the forensic context.

Forensic aspects of insulin.

Insulin or, more appropriately, hypoglycaemia gives rise to a wide variety of interactions with the law. In most cases its role is not seriously open to question occasionally however, it is. This is especially true of situations in which insulin is suspected of having been used inappropriately or maliciously. The major differences between investigation of hypoglycaemia in clinical and forensic situation are that in the latter the history is often unreliable, appropriate samples for analysis were not collected, preserved or labelled correctly and analytical results are likely to be challenged on grounds of specificity, accuracy and interpretation. Immunoassay remains the mainstay of clinical investigation of hypoglycaemia but likely to become displaced by mass-spectrometry in the forensic situation especially now that human insulin is being replaced by synthetic insulin analogues for the treatment of diabetes.

Murder by insulin: suspected, purported and proven-a review.

Murder by insulin-whether attempted, suspected or proven-is rare. Only 66 cases worldwide could be found for this review. A conviction was secured in 31 cases and additional weapon was employed in 11. Differentiation of attempted homicide from Munchausen syndrome by proxy in the young and from 'mercy killing' in the elderly was not attempted. Most perpetrators were close relatives and most victims were alive when discovered and responded to treatment. Hypoglycaemia is the first clue to homicidal insulin use in living subjects and requires the demonstration of a plasma insulin concentration of generally more than 1000 pmol/L and undetectable plasma C-peptide concentration to establish the diagnosis. Serum glucose measurements are valueless in victims found dead. The presence near the body of insulin vials, syringes or needles, loose talk by the suspected perpetrator or their ready access to insulin may be the only clue. The demonstration of insulin in tissue around an injection site by immunohistopathology or by measuring it in an extract clinches the diagnosis. Immunoassays suitable for clinical use to detect and measure insulin and C-peptide are subject to random errors and cannot be relied upon unless special precautions including separation by gel filtration or HPLC are undertaken prior to analysis. They do not detect or measure accurately a new generation of synthetic insulin analogues. Mass spectrometry will be required to do this and to validate clinical immunoassays, upon which convictions have always had to rely in the past.

Human obesity: its hormonal basis and the role of gastric inhibitory polypeptide.

Obesity is an abnormal expansion of the adipose organ and is a pathophysiological response to an imbalance between energy intake and energy expenditure. It is the result of a large number of diverse factors involving heritable and environmental characteristics. A simple definition of obesity is difficult and unsatisfactory and its age dependency has largely been ignored. Differentiation between healthy, age-related plumpness and obesity is often blurred and responsible for overdiagnosis of obesity in the developed world. In the past, epidemiological studies have often ignored the different prognostic significance of the two major phenotypes of human obesity making their conclusions of limited value. The role of heritable factors in determining both the propensity to develop obesity under favourable environmental conditions, including inactivity and unlimited access to fat-rich foods, and the phenotype it assumes received an enormous fillip from experiments involving genetically modified animals. The most important of these have demonstrated the key role played by a number of newly discovered or recently resurrected polypeptide hormones that are released from the intestine in response to food. Molecular manipulation of these hormones, especially of glucose-dependent insulin-stimulatory polypeptide offers a new therapeutic approach.

The metabolic syndrome.

Prevention of disease rather than its alleviation or cure has always been the aim of healthcare professionals. While this goal has been achieved for many infectious diseases through improvements in hygiene and vaccination, as well as for acute deficiency diseases such as scurvy, less progress has been made with chronic diseases. These are increasing as the population ages. Chief among the chronic diseases on the increase are: Type 2 diabetes. Hypertension and cardiovascular disease, which are responsible for over 40 per cent of all deaths in the UK (DoH 1999). Obesity, which is a major cause of morbidity and predisposes to diabetes, cardiovascular disease and other chronic illnesses. Cancer. This article describes the nature, aetiology and prevention of the metabolic syndrome. Although not a disease, the metabolic syndrome is like smoking in that it strongly predisposes to disease and is preventable.

False-positive immunoassay results: a multicenter survey of erroneous immunoassay results from assays of 74 analytes in 10 donors from 66 laboratories in seven countries.

BACKGROUND: Analytical interference in immunoassays can produce serious errors, but it is generally considered rare with modern analytical systems. METHOD: Blood was collected from 10 donors with illnesses known to be associated with rheumatoid factor. Immunoassays for 74 analytes were performed in 66 clinical laboratories. Each sample was measured in duplicate, and again in duplicate after the addition of a proprietary heterophil blocking reagent, with the laboratory's routinely used reagents and equipment. Reagents were typically supplied by the manufacturers of the closed analytical systems. Both competitive and sandwich immunoassays were investigated. RESULTS: Overall approximately 8.7% of the 3445 results were considered to be "false positive", many of them seriously so. Twenty-one percent of the erroneously high results (1.8% of all results) were potentially misleading and were corrected by blocking reagent, although 49% of such seriously high results (4.2% of all results) were not. A further 39% of the false-positive results (2.6% of all results) would not necessarily have appeared likely to produce adverse clinical consequences but were substantially lowered by the addition of the blocking reagent. CONCLUSIONS: Random errors can occur with all types of immunoassays tested and can be difficult to identify even when repeated in another laboratory. Clinicians need to be aware of these limitations.