Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus

Abstract

Green AS, Macko AR, Rozance PJ, Yates DT, Chen X, Hay WW Jr, Limesand SW. Characterization of glucose-insulin responsiveness and impact of fetal number and sex difference on insulin response in the sheep fetus. Am J Physiol Endocrinol Metab 300: E817-E823, 2011. First published February 22, 2011 doi:10.1152/ajpendo.00572.2010.-GSIS is often measured in the sheep fetus by a square-wave hyperglycemic clamp, but maximal ␤-cell responsiveness and effects of fetal number and sex difference have not been fully evaluated. We determined the dose-response curve for GSIS in fetal sheep (0.9 of gestation) by increasing plasma glucose from euglycemia in a stepwise fashion. The glucose-insulin response was best fit by curvilinear third-order polynomial equations for singletons (y ϭ 0.018x 3 Ϫ 0.26x 2 ϩ 1.2x Ϫ 0.64) and twins (y ϭ Ϫ0.012x 3 ϩ 0.043x 2 ϩ 0.40x Ϫ 0.16). In singles, maximal insulin secretion was achieved at 3.4 Ϯ 0.2 mmol/l glucose but began to plateau after 2.4 Ϯ 0.2 mmol/l glucose (90% of maximum), whereas the maximum for twins was reached at 4.8 Ϯ 0.4 mmol/l glucose. In twin (n ϭ 18) and singleton (n ϭ 49) fetuses, GSIS was determined with a square-wave hyperglycemic clamp Ͼ2.4 mmol/l glucose. Twins had a lower basal glucose concentration, and plasma insulin concentrations were 59 (P Ͻ 0.01) and 43% (P Ͻ 0.05) lower in twins than singletons during the euglycemic and hyperglycemic periods, respectively. The basal glucose/insulin ratio was approximately doubled in twins vs. singles (P Ͻ 0.001), indicating greater insulin sensitivity. In a separate cohort of fetuses, twins (n ϭ 8) had lower body weight (P Ͻ 0.05) and ␤-cell mass (P Ͻ 0.01) than singleton fetuses (n ϭ 7) as a result of smaller pancreata (P Ͻ 0.01) and a positive correlation (P Ͻ 0.05) between insulin immunopositive area and fetal weight (P Ͻ 0.05). No effects of sex difference on GSIS or ␤-cell mass were observed. These findings indicate that insulin secretion is less responsive to physiological glucose concentrations in twins, due in part to less ␤-cell mass.␤-cell; glucose-stimulated insulin secretion; pancreas; and twin THE PREGNANT SHEEP IS A VALUABLE MODEL for multiple pathophysiological processes during pregnancy because placental exchange and fetal physiology can be monitored in utero in conscious animals (3). The tolerance of the uterus to surgical manipulation makes the sheep fetus suitable for chronic catheterization during late gestation, and ample fetal blood volume permits repeated sampling during in vivo studies. Furthermore, commonly used sheep breeds usually have one or two offspring per pregnancy in contrast to other animal models, which often have litters.In sheep, glucose-stimulated insulin secretion (GSIS) is present during the second half of gestation (1, 2, 31, 33), and insulin plays a major role in coordinating nutrient availability with fetal growth (11, 18). Although GSIS is routinely measured in the sheep fetus by acute glucose boluses or squarewave hyperglycemic clamps to determine fetal ␤-cell responsiveness, the dyna...

Enhanced insulin secretion responsiveness and islet adrenergic desensitization after chronic norepinephrine suppression is discontinued in fetal sheep

“…Square-wave hyperglycemic clamps were performed as described previously (21). Three baseline blood samples were collected at 5-min intervals before the hyperglycemic clamp was initiated.…”

“…Following the last baseline sample, at time designated 0 min, a glucose bolus (110 mg/kg estimated fetal weight) was given to the fetuses and followed immediately by a continuous infusion of 33% dextrose solution (Abbott Laboratories, Abbott Park, IL) that was varied to maintain fetal glucose concentration at ϳ2.4 mM. This is the recommended value to achieve maximum glucose-stimulated insulin concentrations in singleton sheep fetuses (21). During the hyperglycemic clamp, three fetal arterial plasma samples were collected every 5 min starting from 35 min.…”

Increased amino acid supply potentiates glucose-stimulated insulin secretion but does not increase β-cell mass in fetal sheep

“…Baseline plasma glucose and insulin concentrations were determined at Ϫ60, Ϫ45, Ϫ30, and Ϫ15 min. The hyperglycemic clamp was initiated with a 33% dextrose (wt/vol in saline) bolus (825 mg of glucose) into the fetus, followed by a variable infusion of 33% dextrose (wt/vol in saline) that was adjusted to maintain fetal arterial plasma concentration at 45 mg/dl, which elicits 90% maximal insulin concentrations in fetal sheep (15). The dextrose infusion was held constant beginning at minute 45.…”

“…The dextrose infusion was held constant beginning at minute 45. Fetal arterial plasma samples were collected at 5,10,15,20,30,45,60,75,90, and 105 min for glucose and insulin measurement. To measure glucose-potentiated arginine-stimulated insulin secretion (ASIS), a bolus of arginine (0.5 mmol/kg estimated fetal weight) in 5 ml of 0.4 mol/l sodium acetate and 0.9% NaCl was injected over 4 min into the fetal circulation beginning at 110 min.…”

“…Amino acid or saline infusions were initiated 100 min prior to the square-wave hyperglycemic clamp that was performed as described above. Fetal plasma arterial insulin and glucose were measured 15, 10, and 5 min prior to the initiation of the amino acid or saline infusion, as well as 15, 10, and 5 min prior to the initiation of the glucose clamp, and then at minutes 5,10,15,20,30,45,60,75, and 90 after the start of the glucose clamp. Amino acids also were measured during the basal and hyperaminoacidemic periods.…”