Chronic exposure to elevated norepinephrine suppresses insulin secretion in fetal sheep with placental insufficiency and intrauterine growth restriction

Abstract

In this study, we examined chronic norepinephrine suppression of insulin secretion in sheep fetuses with placental insufficiency-induced intrauterine growth restriction (IUGR). Glucose-stimulated insulin secretion (GSIS) was measured with a square-wave hyperglycemic clamp in the presence or absence of adrenergic receptor antagonists phentolamine (alpha) and propranolol (beta). IUGR fetuses were hypoglycemic and hypoxemic and had lower GSIS responsiveness (P < or = 0.05) than control fetuses. IUGR fetuses also had elevated plasma norepinephrine (3,264 +/- 614 vs. 570 +/- 86 pg/ml; P < or = 0.05) and epinephrine (164 +/- 32 vs. 60 +/- 12 pg/ml; P < or = 0.05) concentrations. In control fetuses, adrenergic inhibition increased baseline plasma insulin concentrations (1.7-fold, P < or = 0.05), whereas during hyperglycemia insulin was not different. A greater (P < or = 0.05) response to adrenergic inhibition was found in IUGR fetuses, and the average plasma insulin concentrations increased 4.9-fold at baseline and 7.1-fold with hyperglycemia. Unlike controls, basal plasma glucose concentrations fell (P < or = 0.05) with adrenergic antagonists. GSIS responsiveness, measured by the change in insulin, was higher (8.9-fold, P < or = 0.05) in IUGR fetuses with adrenergic inhibition than controls (1.8-fold, not significant), showing that norepinephrine suppresses insulin secretion in IUGR fetuses. Strikingly, in IUGR fetuses, adrenergic inhibition resulted in a greater GSIS responsiveness, because beta-cell mass was 56% lower and the maximal stimulatory insulin response tended (P < 0.1) to be higher than controls. This persistent norepinephrine suppression appears to be partially explained by higher mRNA concentrations of adrenergic receptors alpha(1D), alpha(2A), and alpha(2B) in a cohort of fetuses that were naïve to the antagonists. Therefore, norepinephrine suppression of insulin secretion was maintained, in part, by upregulating adrenergic receptor expression, but the beta-cells also appeared to compensate with enhanced GSIS. These findings may begin to explain why IUGR infants have a propensity for increased glucose requirements if norepinephrine is suddenly decreased after birth.

Fig. 1.

Fetal glucose concentrations during glucose-stimulated insulin secretion (GSIS) clamp periods. Steady-state glucose concentrations for control (n = 9) and intrauterine growth-restricted (IUGR) (n = 5) fetuses during the Saline and adrenergic inhibition (Block) GSIS studies are presented (means ± SE). Time relative to the start of the exogenous dextrose bolus, time 0 min, is presented on the x-axis. *In IUGR fetuses, basal mean glucose concentrations were significantly less than in controls for the respective GSIS study. #Adrenergic blockade decreased (P < 0.05) basal glucose concentrations in IUGR fetuses. No differences in mean glucose concentrations were found during the hyperglycemic steady-state clamp.

Fig. 2.

Fetal insulin concentrations during GSIS steady-state periods. Mean plasma insulin concentrations are presented for control (n = 9) and IUGR (n = 5) fetuses (treatment groups) during basal and hyperglycemic steady-state periods of the Saline or Block (adrenergic inhibited) GSIS study. Differences between means within the steady-state period were determined independently (statistical models 1 and 2). The first tier of P values indicates comparisons between GSIS studies (Saline vs. Block) within a treatment group. The second tier of P values indicates comparisons between treatment groups for differences between GSIS studies.

Fig. 3.

GSIS responsiveness. Mean change in insulin is presented for control and IUGR treatments during the Saline or adrenergic inhibition (Block) GSIS study. Linear contrasts for Saline and Block GSIS studies within treatment groups were determined, and P values are presented in the 1st row above the bars being compared. The P value for the difference between treatments in response to adrenergic inhibition (Block − Saline-GSIS) is presented in the 2nd row.

Fig. 4.

Adrenergic receptor expression in fetal sheep islets. Adrenergic receptors (ADR) were cloned from sheep tissues, and their expression was determined for fetal sheep islets isolated from control or IUGR islets at 135 dGA. PCR products were separated by agarose gel electrophoresis and stained with ethidium bromide. Lanes for each gel include a negative control (no cDNA; −) and positive control (adipose; +) alongside 2 representative samples from islet mRNA extracted from control or IUGR islets. Quantitative PCR was performed (n = 6 controls and n = 7 IUGRs). ΔC_T ± SE are presented for control and IUGR islets. Fold change (fold Δ) in IUGR islets from control islets was calculated by the 2^−ΔΔC_T method for all adrenergic receptor isoforms, and results are presented. Statistical analysis was performed on ΔC_T values with ribosomal protein S15 as the reference gene (*P < 0.05 between treatment groups).