Prenatal Oxygen and Glucose Therapy Normalizes Insulin Secretion and Action in Growth-Restricted Fetal Sheep

Abstract

Placental insufficiency (PI) lowers fetal oxygen and glucose concentrations, which disrupts glucose-insulin homeostasis and promotes fetal growth restriction (FGR). To date, prenatal treatments for FGR have not attempted to correct the oxygen and glucose supply simultaneously. Therefore, we investigated whether a 5-day correction of oxygen and glucose concentrations in PI-FGR fetuses would normalize insulin secretion and glucose metabolism. Experiments were performed in near-term FGR fetal sheep with maternal hyperthermia-induced PI. Fetal arterial oxygen tension was increased to normal levels by increasing the maternal inspired oxygen fraction and glucose was infused into FGR fetuses (FGR-OG). FGR-OG fetuses were compared with maternal air insufflated, saline-infused fetuses (FGR-AS) and control fetuses. Prior to treatment, FGR fetuses were hypoxemic and hypoglycemic and had reduced glucose-stimulated insulin secretion (GSIS). During treatment, oxygen, glucose, and insulin concentrations increased, and norepinephrine concentrations decreased in FGR-OG fetuses, whereas FGR-AS fetuses were unaffected. On treatment day 4, glucose fluxes were measured with euglycemic and hyperinsulinemic-euglycemic clamps. During both clamps, rates of glucose utilization and production were greater in FGR-AS than FGR-OG fetuses, while glucose fluxes in FGR-OG fetuses were not different than control rates. After 5 days of treatment, GSIS increased in FGR-OG fetuses to control levels and their ex vivo islet GSIS was greater than FGR-AS islets. Despite normalization in fetal characteristics, GSIS, and glucose fluxes, FGR-OG and FGR-AS fetuses weighed less than controls. These findings show that sustained, simultaneous correction of oxygen and glucose normalized GSIS and whole-body glucose fluxes in PI-FGR fetuses after the onset of FGR.

Keywords: fetal therapy; glucose homeostasis; intrauterine intervention; islets of Langerhans; placental insufficiency.

Figure 1.

Experimental design and flow diagram. Panel A. Experimental design and timeline for fetal surgery, treatment, physiological studies, and islet isolation. Prior to starting the treatment (day 0), GSIS was determined in each FGR fetus with a square-wave hyperglycemic clamp. On treatment day 4, whole-body glucose flux measurements were performed with radiolabeled tracers to determine glucose- and insulin-stimulated glucose disposal rates. On treatment day 5, a second GSIS study was performed in the FGR groups. Panel B. Consort diagram for animal used in the experimental procedures.

Figure 2.

Fetal therapy improves endocrine parameters in FGR-OG fetuses. Fetal blood samples were measured daily. Administration of oxygen and glucose was initiated after sample collection on day 0 in fetuses with FGR. The maternal insufflation of oxygen and fetal intravenous infusion of glucose was sustained for 5 days (x axis; Day of Treatment). Mean values are presented for each experimental group FGR-AS (FGR fetuses receiving air and saline; n = 9), FGR-OG (FGR fetuses receiving oxygen and glucose; n = 9), and a representative control group (CON, n = 10). Daily averages are presented for the partial pressure of oxygen (PO₂, panel A) and blood oxygen content (panel B). Plasma glucose (C), insulin (D), norepinephrine (E), and cortisol (F) concentrations are presented. Differences were determined with a mixed-model ANOVA and post hoc Tukey-Kramer test. *Indicates no difference between FGR-AS vs FGR-OG groups but that both FGR groups were different (P < 0.05) from controls. †Identifies differences (P < 0.05) between FGR-OG and FGR-AS groups. ‡Identifies differences (P < 0.05) between FGR-AS and CON.

Figure 3.

Fetal therapy improves GSIS in FGR-OG fetuses. GSIS studies were performed prior to treatment (day 0; A and B) and on day 5 of treatment (C and D) in FGR-AS and FGR-OG fetuses. The bars represent group means (±SEM) for plasma glucose (A and C) and insulin (B and D) concentrations at basal and hyperglycemic periods of the GSIS study. Panel E shows the hyperglycemic minus basal insulin difference (Δ insulin concentrations) for the pre- and posttreatment GSIS studies (group × study interaction P < 0.01). Individual means are shown for each fetus within their experimental group. The GSIS study prior to treatment included FGR-AS fetuses (n = 9) and FGR-OG fetuses (n = 9), whereas the GSIS study on day 5 of treatment had 1 less FGR-AS. GSIS studies in control fetuses (CON; n = 10) were included in the analysis as a reference. Comparisons were made with an ANOVA for group at glucose level. Differences (P < 0.05) between groups were determined with a Tukey-Kramer test. *Indicates FGR groups differ from controls. †Identifies differences between FGR-OG and FGR-AS groups. ‡Identifies differences (P < 0.05) between FGR-AS and CON groups.

Figure 4.

Oxygen and glucose therapy normalized glucose fluxes in FGR fetuses. Weight-specific rates for glucose entry (GER, umbilical + exogenous infusion), glucose utilization (GUR), glucose production (GPR) and glucose oxidation (GOR) were measured during the euglycemic clamp (EC; panel A) and hyperinsulinemic-euglycemic clamp (HEC; panel B). Individual means are shown for each fetus with their experimental group, and the groups include control fetuses (CON, n = 10), FGR fetuses treated with air and saline (FGR-AS, n = 7), and FGR fetuses treated with oxygen and glucose (FGR-OG, n = 8). The bars represent the group mean ± SEM. Groups were analyzed with an ANOVA and difference (P < 0.05) were identified with Tukey-Kramer test. †Denotes differences between FGR-OG and FGR-AS groups. *Denotes differences between FGR-AS and CON groups.

Figure 5.

Fetal therapy restores GSIS in FGR islets. GSIS was measured in islets isolated from FGR-AS (n = 7), FGR-OG (n = 8), and CON (n = 8) fetuses. Static islet incubation (n ≥ 3 technical replicates) were performed in KRB/BSA media supplemented with low (0.5 mM) and high (11.1 mM) glucose. Insulin concentrations (ng of released insulin) per 10 islets are presented and the difference (Δ) between high and low glucose was calculated. Individual means of the technical replicates are shown for each fetus with their experimental group. The bars and error bars represent the group mean ± SEM. Groups were analyzed with an ANOVA and group mean differences (P < 0.05) were identified with a post hoc Tukey-Kramer test. †Indicates differences between FGR-OG and FGR-AS groups and an *Indicates differences between FGR-AS and CON groups.