Research For LifeReports of research work funded by grants prior to 2016
University of Otago Wellington
Active GLP-1 Analysis by ELISA and Magnetic Bead Multiplex Assay
B Corley, J Krebs, R Hall, R Carroll, M Weatherall, D McCarthy, J Clapham
Centre for Endocrine Diabetes and Obesity Research.
In the late 1990’s Walter Pories, a bariatric surgeon published his observations on the disappearance of diabetes following bariatric surgery. That diabetes once established could be reversed was a welcome discovery and a multitude of studies since the late 1990’s has confirmed Pories observations and attempted to determine the means by which this remission of diabetes occurs.
There are several hypotheses relating to diabetes remission, of which caloric restriction, gut peptides, bile acids and gut microbiota are but a few. Apart from caloric restriction none of the other hypotheses have clearly been demonstrated independently to induce diabetes remission. This in part relates to the challenge of defining diabetes remission, replicating the complex physiologic changes that occur before, during and at various time-points after bariatric surgery, and the limitations of the tools to study potential mechanisms of diabetes remission.
Gut peptides are biochemical messengers released from endocrine cells in the bowel. They include peptide YY (PYY), Glucagon-Like Peptide-1 (GLP-1), GLP-2, Gastric Inhibitory Polypeptide (GIP) as well as Ghrelin and Amylin. While these peptides have in common their origin from the gut and their peptide structure, they regulate a wide variety of physiological responses such as appetite, satiety and insulin secretion. There is clear evidence that GLP-1, GLP-2 & GIP are increased following bariatric surgery, and these hormones are known to play a role in glucose regulation. It is unsurprising then that a number of authors have proposed a central role for these hormones in sustained diabetes remission following bariatric surgery. However these assertions are tempered by some key considerations; the definition of a clinically relevant GLP-1 or GLP-2 level, a study that demonstrates GLP-1 or GLP-2 alone can induce diabetes remission; the absence of an incompletely defined mechanism of action of GLP-1 & GLP-2 in the post bariatric gut; lack of a standardised approach across studies to the stimulus used to provoke a gut peptide response; failure to adequately control for caloric restriction and, the use of assays of uncertain validity and precision in a small number of studies.
Our research group has previously used a magnetic bead assay to study a large number of gut peptides. This assay has the chief advantage of being able to analyse a large number of gut peptides simultaneously in a cost effective manner. While the magnet bead technique is extremely innovative, there are concerns in relation to the specificity of the assay particularly with regard to glucagon like peptide-1 which resembles several other peptides, most notably glucagon, as the name suggests.
We had initially planned to study gut peptide responses following bariatric surgery. However, during data analysis of a pilot low volume meal test developed by our group it became clear that the GLP-1 assay readings for participants in the fasted state varied widely with readings several fold higher in some fasted participants as compared with others. On the basis of these results we revised our study plan and performed a GLP-1 ELISA on the same blood samples used previously. The Millipore ELISA has previously been independently validated and has been shown to possess excellent sensitivity and specificity.
In conjunction with our collaborators at the ESR, we have recently completed analysing these samples. While data analysis is still underway, there is evidence of significant discrepancies between the ELISA and the Multiplex assay. We are completing analysis of this data currently to determine if this can be adequately explained by poor specificity of the magnetic bead assay or whether there is an alternate explanation. If the specificity of the magnetic bead assay is unreliable, despite optimal testing conditions and specimen handling, it will not be used for further studies. As a consequence, the cost of multiple gut peptide assays to evaluate gut peptide responses following bariatric surgery would be prohibitive.
