Serum IRAP, a Novel Direct Biomarker of Prediabetes and Type 2 Diabetes?

Abstract

Insulin resistance (IR), currently called prediabetes (PD), affects more than half of the adult population worldwide. Type 2 diabetes (T2D), which often follows in the absence of treatment, affects more than 475 million people and represents 10-20% of the health budget in industrialized countries. A preventive public health policy is urgently needed in order to stop this constantly progressing epidemic. Indeed, early management of prediabetes does not only strongly reduce its evolution toward T2D but also strongly reduces the appearance of cardiovascular comorbidity as well as that of associated cancers. There is however currently no simple and reliable test available for the diagnosis or screening of prediabetes and it is generally estimated that 20-60% of diabetics are not diagnosed. We therefore developed an ELISA for the quantitative determination of serum Insulin-Regulated AminoPeptidase (IRAP). IRAP is associated with and translocated in a stoechiometric fashion to the plasma membrane together with GLUT4 in response to insulin in skeletal muscle and adipose tissue which are the two major glucose storage sites. Its extracellular domain (IRAPs) is subsequently cleaved and secreted in the blood stream. In T2D, IRAP translocation in response to insulin is strongly decreased. Our patented sandwich ELISA is highly sensitive (≥10.000-fold "normal" fasting concentrations) and specific, robust and very cost-effective. Dispersion of fasting plasma concentration values in a healthy population is very low (101.4 ± 15.9 μg/ml) as compared to those of insulin (21-181 pmol/l) and C-peptide (0.4-1.7 nmol/l). Results of pilot studies indicate a clear correlation between IRAPs levels and insulin sensitivity. We therefore think that plasma IRAPs may be a direct marker of insulin sensitivity and that the quantitative determination of its plasma levels should allow large-scale screening of populations at risk for PD and T2D, thereby allow the enforcement of a preventive health policy aiming at efficiently reducing this epidemic.

Keywords: GLUT4; IRAP; biomarker; diabetes; diagnosis; prediabetes; screening.

Figure 1

Insulin-mediated glucose uptake through GLUT4. (A) In the absence of insulin, glucose cannot enter the cell. (B) Secreted insulin binds to its receptors and triggers multiple signaling pathways. (C,D) Among these pathways, insulin triggers the translocation of part of the GLUT4 containing vesicles (GSV) to the plasma membrane. (E) Upon fusion with the plasma membrane (PM), GLUT4 molecules are inserted in the PM and allow glucose to enter the cell. Glucose is rapidly phosphorylated to glucose-6-phosphate (G-6-P) as an intermediate metabolite in glycolysis, pentose pathway and glycogen synthesis. (F) In the fused GSVs, IRAP is inserted the PM together with GLUT4 and its C-terminal extracellular domain is cleaved and secreted in the bloodstream. Subsequently, GSV proteins will be recycled and incorporated in newly formed cytoplasmic GSVs. (G) Part of the GSVs stay in the cytoplasm and are available for future translocation. IRAP molecules are intact.

Figure 2

Structure of the cleaved extracellular domain of IRAP: 3-dimensional model of the cleaved extracellular domain of IRAP. Epitopes recognized by the monoclonal antibodies used in the ELISA are in red.

Figure 3

Immunoblots of the secreted domain of IRAP. (A,B) Samples were submitted to SDS PAGE on 4–15% gradient gels under non-reducing conditions and transferred to PVDF membranes by semidry blotting. Blots were probed resp. with 4G6 mAb (A) and 40C10 mAb (B) and revealed using an anti-mouse-HRP antibody (preadsorbed to human IgG) and luminol. Lanes: 1: molecular weight markers, 2: -, 3: 150 ng recombinant human IRAP secreted domain (rhuIRAP sd), 4: -, 5: 1.5 μL human serum spiked with 150 ng rhuIRAPsd, 6: 1,5 μL fetal bovine serum, 7: 1.5 μL human serum, 8: 0.75 μL human serum. (C) Sera obtained during an insulin-induced hypoglycemia test were submitted to native PAGE on a 4–15% gradient gel under non-reducing conditions and transferred to PVDF membranes by semidry blotting. The blot was probed with 4G6 mAb and revealed using an anti-mouse-HRP antibody (preadsorbed to human IgG) and luminol. Indicated times are in minutes following insulin injection.

Figure 4

Glycemic, insulinemic, and serum IRAP concentration profiles during OGTT. Representative three-time points OGTT profiles in two euglycemic (MB and TD) and two severely insulin-resistant diabetic (KJ and SZ) patients. Glycemia, blue; insulinemia, green; IRAP, red.