Abstract
AimsIn vitro, beta cells immediately secrete stored but readily releasable insulin in response to a rise of glucose. During a prolonged insulin response, this is followed by newly synthesized insulin. Our aim was to develop an in vivo test to determine the ratio between readily available and newly synthesized insulin after a stimulus in humans by labelling newly synthesized insulin.MethodsA stable isotope tracer of 1.0 g 13C leucine with C-peptide as target peptide was administered 45 min prior to 75 g glucose load of a frequently blood sampled 210-min oral glucose tolerance test (OGTT). Our OGTT also encompassed collection of urine, which has a high content of C-peptide. Prior, the optimal conditions under which the tracer 13C leucine was administered for enrichment of (pre) proinsulin were established. Also, techniques to obtain urinary C-peptide under highly purified circumstances were set up. Our main outcome measure was the stable isotope enrichment of de novo C-peptide, which we related to early plasma insulin and glucose AUC. Twelve healthy Caucasian individuals (M4F8, age 41.8 ± 2.3, BMI 28.3 ± 1.7) with normal glucose tolerance underwent our OGTT.ResultsWe found that during a 75-g OGTT, newly synthesized insulin contributed approximately 20 % of total insulin secretion. The pattern of isotope enrichment obtained by collecting multiple urine voids was suggestive that the newly synthesized insulin contributes to the late phase of insulin secretion. De novo C-peptide correlated negatively with both early plasma insulin AUC (r = −0.629, P = 0.028) and early plasma glucose AUC (r = −0.605, P = 0.037).ConclusionsWith stable isotope technique added to OGTT, we were able to measure newly synthesized insulin in healthy individuals. This new technique holds the promise that it is feasible to develop a direct in vivo beta cell function test.Electronic supplementary materialThe online version of this article (doi:10.1007/s00592-016-0896-3) contains supplementary material, which is available to authorized users.
Highlights
Abnormal function of the pancreatic beta cells is crucial to the development of type 2 diabetes (T2D) [1]
To test whether 1.0 g 13C leucine and timing of administration would result in enough precursor enrichment, we examined isotope enrichment in the extracellular fluid and isotope enrichment in the intracellular fluid (KIC mole per cent excess (MPE)) in both plasma and saliva in non-diabetics during oral glucose tolerance test (OGTT) (n = 6, M4F2, age 35.5 ± 17.4 BMI 23.9 ± 3.3)
No significant difference in the amount of average leucine MPE and ketoisocaproic acid (KIC) MPE/min between plasma and saliva was observed after 13C leucine administration; equilibrium with KIC MPE as valid surrogate marker was assumed with high precursor enrichment
Summary
Abnormal function of the pancreatic beta cells is crucial to the development of type 2 diabetes (T2D) [1]. We explored whether it is feasible to develop a test with a stable isotope tracer to quantify the newly synthesized insulin. Following an acute rise of glucose concentrations, a biphasic insulin secretion response occurs [4,5,6]. This results from the glucose transport into beta cells through the glucose transporter 2 (GLUT2) [7], which activates calcium-dependent triggering as well as calcium-independent amplifying pathways [8]. After an in vitro glucose stimulus, rat pancreatic islets have a biphasic insulin response and synthesize de novo proinsulin, which is stored in newly synthesized granules and subsequently secreted after 1 h [17, 18]. The dynamics of newly synthesized insulin and granular secretion of (de novo) insulin have not yet been investigated in humans in vivo
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