To determine the role of nutrient status on ghrelin regulation of insulin secretion. We hypothesize that ghrelin and glucagon-like peptide-1 (GLP-1)(both are hormones made in the gut,) have differential effects on β-cell function in the fed state. We will compare insulin secretion and glucose turnover during meal ingestion using a dual glucose tracer and mixed meal protocol in subjects receiving ghrelin or saline. We will also determine the role of ghrelin-stimulated GLP-1 levels in this process using the GLP-1 receptor (GLP-1R) antagonist Exendin(9-39) (Ex-9).
We plan to study 20 healthy subjects on 4 occasions where they will receive ghrelin, ghrelin+Ex-9, Ex-9 or saline infusion after an overnight fast in a randomized order; Ex-9 will be used to block GLP-1 action. A 240-minute meal tolerance test (MTT) using a dual glucose tracer method will serve as the foundation of each study visit. One tracer, \[6,6-2H2\]glucose will be infused intravenously before and during the test meal to quantify fasting endogenous glucose production (EGP), and glucose disappearance during the meal. A second tracer, \[U-13C\]glucose, will be included in the meal to trace the appearance of oral glucose. The systemic appearance rates of both ingested tracer and total (i.e., ingested and endogenously produced) glucose will be calculated. Using this protocol, we will be able to evaluate a) insulin secretion in response to mixed-meal ingestion, b) glucose appearance and glucose disappearance during meal ingestion, c) the ghrelin effect on these parameters without GLP-1, and d) the effect of GLP-1 in the response based on the effects with and without Ex-9. This dual-tracer method has been used to assess the ability of an individual to dispose of an oral glucose load, and accurately fractionates the appearance of ingested glucose in plasma (Ra meal), EGP, and peripheral glucose disposal (Rd) in this setting 41-42. The \[6,6-2H2\]glucose and \[U-13C\]glucose are stable-isotope tracers and are different from radioactive-isotope tracers in that they do not emit radiation. All procedures will be performed at the CTRC at the Cincinnati Children's Hospital Medical Center (CCHMC).
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
BASIC_SCIENCE
Masking
SINGLE
Enrollment
30
University of Cincinnati
Cincinnati, Ohio, United States
RECRUITINGCincinnati Children's Hospital and Medical Center
Cincinnati, Ohio, United States
RECRUITINGpost-prandial insulin secretion
Postprandial insulin secretion (ISR-meal) will be derived from plasma C-peptide concentrations during MTT using deconvolution with population estimates of C-peptide clearance.
Time frame: 1 year
endogenous GLP-1 contribution to postprandial insulin secretion
The endogenous GLP-1 contribution to postprandial insulin secretion (GLP-1 effect) will be calculated as the difference in ISR-meal with and without Ex-9.
Time frame: 1 year
β-cell response to glucose
An index of β-cell response to glucose will be calculated as the incremental insulin/glucose (I/G) AUC (ΔAUCI/G).
Time frame: 1 year
insulin sensitivity
Whole body insulin sensitivity will be estimated using the Matsuda Index that has been well validated in large cohort studies and has demonstrated a good correlation with IVGTT or hyperinsulinemic-euglycemic clamp derived measures of insulin sensitivity. β-cell function (DI-meal) will be calculated as ΔAUCI/G x Matsuda Index
Time frame: 1 year
fasting EGP
Fasting EGP will be calculated as the ratio of 6,6-\[2H2\]glucose infusion rate to plasma tracer enrichment (tracer-to-tracee ratio \[TTR\]6,6 from measurements obtained in the last 20 min of the basal tracer equilibration period, when plasma glucose concentration and 6,6-\[2H2\]glucose enrichment are stable).
Time frame: 1 year
glucose appearance
Total rates of glucose appearance after meal ingestion (total Ra) will be calculated by modeling 6,6-\[2H2\] enrichment (\[TTR\]6,6) using both a two-compartment model and Steele's equation42. Meal glucose appearance will be determined from the analysis of \[U-13C\]-glucose fluxes. The meal will be labeled to 2.66% with U-13C\]-glucose (TTRmeal). From the measurement of plasma \[TTR\]13C, we will calculate the exogenous (meal) glucose concentrations, \[Gmeal\] , from total glucose concentrations, \[Gtot\] , using the formula \[Gmeal\] = \[Gtot\] x \[TTR\]13C / TTRmeal as previously described 42,45. Endogenous glucose concentration \[Gend\] will be calculated as \[Gend\] = \[Gtot\] - \[Gmeal\].
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Time frame: 1 year
EGP during MTT
EGP during MTT will be calculated using model analysis of TTR of endogenous glucose (\[TTR\]end) and calculated as \[TTR\]end = \[TTR\]6,6 x \[Gtot\] / \[Gend\]
Time frame: 1 year
exogenous glucose rate of appearance
Exogenous glucose rate of appearance, Ra meal, will be calculated by subtracting EGP during meal from total Ra (Ra meal = total Ra - EGP).
Time frame: 1 year
metabolic glucose clearance
As for indexes of peripheral insulin sensitivity, we will calculate metabolic glucose clearance during MTT using peripheral glucose disposal (Rd) divided by glucose concentration (expressed as ml/min/kg) where Rd is calculated by subtracting the rate of change of plasma glucose mass from total Ra.
Time frame: 1 year
area under the curve
AUC for glucagon, GLP-1, GIP, and free fatty acids (FFA), a measure of lipolysis.
Time frame: 1 year
ghrelin measures
AG and total ghrelin levels will be measured during clamp-MTT.
Time frame: 1 year
gastric emptying
Gastric emptying estimated by the gastric half-emptying time calculated from the emptying curve as the time when 50% of the total cumulated dose of acetaminophen has been eliminated46.
Time frame: 1 year