|Leucine D7 Labeling of Human C-Peptide for Assessment of Real-Time Insulin Biosynthesis: In Vivo Silac Metabolic Studies|
|Wed 9:00 AM - Session: Metabolomics|
|Insulin biosynthesis is known to be upregulated in vitro by glucose as well as GLP-1. However, there is no clinical data available on rates of insulin biosynthesis in vivo, since the islet tissue itself is not accessible in humans. We have recently validated the use of deuterated leucine combined with Mass Isotopomer Distribution Analysis (MIDA) against the gold standard (3H leucine incorporation) in INS-1 cells for measurement of fractional insulin biosynthetic rate by measuring the incorporation of deuterated and tritiated leucine into newly synthesized proinsulin C-peptide. Fractional insulin biosynthetic rate was monitored in vivo by primed constant infusion of 2H10 or 2H7 leucine into normal subjects during clinical studies. Incorporation of a single or double d-Leucine into C-peptide species was monitored by 2D LC/MS. Fractional synthesis rate (FSR) was calculated by two isotopomer analysis of C-peptide in plasma and urine.
Results: We report for the first time that insulin biosynthesis rates can be measured in vivo by LC/MS-based assay .We found that in vivo insulin biosynthetic rates are responsive to glucose and GLP-1. GLP-1 under fasting conditions is as potent in increasing insulin biosynthesis rates (~two fold) compared to hyperglycemia alone. The time delay for incorporation of leucine tracer into C-peptide is consistent with the known physiology of proinsulin translation, processing and vesicle transport.