​Recent Advance in The Bioanalytical Approaches in The Detection of Incretins

Will ingestion of a sweet candy containing a large amount of glucose versus direct I.V injection of glucose have the same effect on insulin secretion in the human body? If your answer is yes, you should read this article to understand, the silent yet strange, effect of “incretin” hormones in the human body.

The mechanism of insulin secretion is a well-studied process that has proven to be far more complicated than what was once thought. It all starts by ingesting some sort of carbohydrate that fuels into the necessary energy production for human activity. However, a rapid spike of blood glucose level is not a very well tolerated outcome in the body, so it utilizes a variety of biochemical processes to control and reduce high sugar levels in the blood stream. Insulin is the primary tool to effectively control and reduce blood sugar levels within safe margins. It is generally secreted from pancreas in response to different endogenous molecules in the systemic circulation. While you may think insulin is the savior that keeps a healthy balance of blood glucose level, you should also be conscious of the disadvantages associated with high insulin levels. Imbalanced levels of insulin could be alarming and indicative of metabolic disease or abnormalities. Hence understanding its effects, stimuli and physiological consequences is of great scientific importance.

There are differences in insulin response depending on the route of stimulant entrance into the human body. For example, glucose administered orally causes secretion of a unique cocktail of hormones, along with insulin, to control safe levels in systemic circulation. The same concentration of glucose ingested orally brings a different mix of cocktail that contains different molecules and hormones in it. The longevity of the body’s response to orally ingested glucose is much higher than that of injected. This means that insulin concentration in the systemic circulation is much longer in response to eating glucose, which has concerning health consequences over time. This leaves a gap to develop accurate and reliable tools that identify and quantify constituents of hormonal cocktail for healthcare purposes.

One effect observed from physiological exposure to insulin is called incremental insulin secretion, and the biomarkers that contribute to it are known as incretins. The two most important hormones within this family are Glucagon-like peptide (GLP) and (GLP-1) that are secreted from the gastrointestinal tract. GLP-1 is best known for its glucose-dependent insulinotropic effects and plays an important role for postprandial glucose regulation by potentiating glucose-induced insulin secretion. It is secreted in its intact form, referred to as GLP-1 7-36NH2 (active), but is rapidly degraded by the dipeptidyl peptidase 4 (DPP-4) enzyme, converting >90% to the primary metabolite, GLP-1 (9-36NH2) before reaching the targets via the systemic circulation. This suggests that activation of vagal sensory neurons before its degradation by DPP-4 contributes to the postprandial effects of GLP-1. Quantification in biological fluids of not only GLP-1 7-36NH2, but also GLP-1 9- 36NH2, is of great physiological interest. However, direct quantification of GLP-1 9-36NH2 has been hampered by lack of suitable assays. Plasma levels have been estimated using a subtraction approach whereby the difference between concentrations of ‘total GLP-1’ measured using a C-terminally directed assay (x-36NH2) and intact GLP-17-36NH2 (measured using a sandwich assay) has been assumed to equal the concentration of GLP-1 9-36NH2.

While this approach may seem reasonable given that GLP-1 9-36NH2 is the major circulating amidated GLP-1 isoform, studies indicate that GLP-1 is also a reasonable substrate for the enzyme neural endopeptidase (NEP) and degraded rapidly. Moreover, the subtraction-based estimation has an inherent weakness since it relies on the analytical precision and accuracy of not only one, but two assays.

The assessment of ‘total’ GLP-1 plasma levels often relies on assays employing antibodies directed towards the amidated C-terminus of the molecule. There are two possible outcomes associated with this quantification. It could either overestimate the numerical results because of cross reactivity with other GLP-1 isoforms such as the N-terminally extended GLP-1 1-36NH2 from the pancreas. In another case, it could underestimate because of lack of reactivity with any C-terminally glycine-extended forms such as GLP-1 7-37.

To address concerns and to accurately measure metabolite, much effort has been focused on development of chromatographic, mass spectroscopic or immunoassay approaches for effective quantification of total GLP-1.

Among the tools available in the analytical toolbox, immunoassays are the most convenient approach for the bioanalytical community. IBL has comprehensive incretin assay kits and improved the sensitivity of our kit for measuring rodents samples and low concentration of incretin in fasting. Majority of our incretin assay kits are designed using specific monoclonal antibodies that were internally developed from selecting sequence of detecting region for immunogen. Selection of sequence for the immunogen is critical part for stablishing high specific monoclonal antibodies. IBL has a confident for the specificity and sensitivity for detecting targeted protein (GLP-1 and GIP). Scientists at have developed and validated sandwich ELISA’s, which recognize GLP-1 9-36NH2 as well as non-amidated GLP-1 9-37. With this assay, they measured circulating levels of the metabolite under physiological conditions in humans. The kit also investigates the validity of the results obtained by comparison of results from the subtraction-based methods, and studies to what extent inhibition of the DPP-4 enzyme in patients with type 2 diabetes attenuates formation of the N-terminally truncated metabolite.

While ELISA analysis is very effective and cheap, they still require laborious sample preparation. Some experiments give rise to artificially high values, when the plasma is analyzed directly by radioimmunoassay, particularly in the fasting state when incretin (intact) concentrations are low, and result in a high degree of variability because of considerable intrasubject differences. The introduction of an extraction step has proven to be effective to reduce the nonspecific interference, leading to greater reproducibility as well as reducing the limit of quantification, below which concentrations cannot reliably be differentiated from the background. Although the use of two-site sandwich ELISAs using high-affinity monoclonal antibodies had already provided reasonable solution to avoiding the extraction steps in earlier studies.

Conquering diabetes relies on several factors, including but not limited to, role of incretin and their relationship with insulin secretion. The use of incretin hormones in diabetes therapy are old concepts with roots dating back to the second half of the nineteenth century, but it is only in the recent years that a clear image of their importance has been revealed by the grace of bioanalytical tools. Immunological methods that determine incretin hormone concentrations are rapidly developing to deal with obstacles of stability and degradation of analytes. There is a plethora of related peptides which can cross-react to varying degrees with assays.

Learn more about the comprehensive Incretin related ELISA kit offerings from IBL-America.

References:

Bak, M. J., Wewer Albrechtsen, N. J., Pedersen, J., Knop, F. K., Vilsbøll, T., Jørgensen, N. B., . . . Holst, J. J. (2014). Specificity and sensitivity of commercially available assays FOR glucagon-like peptide-1 (GLP-1): Implications For GLP-1 measurements in clinical studies. Diabetes, Obesity and Metabolism, 16(11), 1155-1164. doi:10.1111/dom.12352

Deacon, C. F., & Holst, J. J. (2009). Immunoassays for THE INCRETIN Hormones Gip AND GLP-1. Best Practice & Research Clinical Endocrinology & Metabolism, 23(4), 425-432. doi:10.1016/j.beem.2009.03.006

Osborne, R. I., Ming, W., Troutt, J. S., Siegel, R. W., & Konrad, R. J. (2016). A DUAL-MONOCLONAL, sandwich IMMUNOASSAY specific For Glucagon like peptide-19–36/7 (GLP-19–36/7). Clinical Biochemistry, 49(12), 897-902. doi:10.1016/j.clinbiochem.2016.03.014

Rehfeld, J. F. (2018). The origin and understanding of the incretin concept. Frontiers in Endocrinology, 9. doi:10.3389/fendo.2018.00387

Wewer Albrechtsen, N. J., Asmar, A., Jensen, F., Törang, S., Simonsen, L., Kuhre, R. E., . . . Hartmann, B. (2017). A sandwich Elisa for measurement of the primary glucagon-like Peptide-1 metabolite. American Journal of Physiology-Endocrinology and Metabolism, 313(3). doi:10.1152/ajpendo.00005.2017

Windeløv, J. A., Wewer Albrechtsen, N. J., Kuhre, R. E., Jepsen, S. L., Hornburg, D., Pedersen, J., . . . Holst, J. J. (2017). Why is it so difficult to measure glucagon-like peptide-1 in a mouse? Diabetologia, 60(10), 2066-2075. doi:10.1007/s00125-017-4347-7