Chronic late gestation fetal hyperglucagonaemia results in lower insulin secretion, pancreatic mass, islet area and beta- and α-cell proliferation

Abstract

Fetal glucagon concentrations are elevated in the presence of a compromised intrauterine environment, as in cases of placental insufficiency and perinatal acidaemia. Our objective was to investigate the impact of late gestation fetal hyperglucagonaemia on in vivo insulin secretion and pancreatic islet structure. Chronically catheterized late gestation fetal sheep received an intravenous infusion of glucagon at low (5 ng/kg/min; GCG-5) or high (50 ng/kg/min; GCG-50) concentrations or a vehicle control (CON) for 8-10 days. Glucose-stimulated fetal insulin secretion (GSIS) was measured following 3 h (acute response) and 8-10 days (chronic response) of experimental infusions. Insulin, glucose and amino acid concentrations were measured longitudinally. The pancreas was collected at the study end for histology and gene expression analysis. Acute exposure (3 h) to GCG-50 induced a 3-fold increase in basal insulin concentrations with greater GSIS. Meanwhile, chronic exposure to both GCG-5 and GCG-50 decreased basal insulin concentrations 2-fold by day 8-10. Chronic GCG-50 also blunted GSIS at the study end. Fetal amino acid concentrations were decreased within 24 h of GCG-5 and GCG-50, while there were no differences in fetal glucose. Histologically, GCG-5 and GCG-50 had lower β- and α-cell proliferation, as well as lower α-cell mass and pancreas weight, while GCG-50 had lower islet area. This study demonstrates that chronic glucagon elevation in late gestation fetuses impairs β-cell proliferation and insulin secretion, which has the potential to contribute to later-life diabetes risk. We speculate that the action of glucagon in lower circulating fetal amino acid concentrations may have a suppressive effect on insulin secretion. KEY POINTS: We have previously demonstrated in a chronically catheterized fetal sheep model that experimentally elevated glucagon in the fetus impairs placental function, reduces fetal protein accretion and lowers fetal weight. In the present study, we further characterized the effects of elevated fetal glucagon on fetal physiology with a focus on pancreatic development and β-cell function. We show that experimentally elevated fetal glucagon results in lower β- and α-cell proliferation, as well as decreased insulin secretion after 8-10 days of glucagon infusion. These results have important implications for β-cell reserve and later-life predisposition to diabetes.

Keywords: alpha‐cell; beta‐cell; fetal pancreas; glucagon; glucose‐stimulated insulin secretion.

Figure 1.. Experimental Protocol.

(A) Experimental timeline with illustration of catheter insertion sites. (B) Acute GSIS using square-wave hyperglycemic clamp (33% dextrose infusion) measuring in vivo beta-cell function performed 3 hours after start of experimental infusions. (C) Chronic GSIS + ASIS using square-wave hyperglycemic clamp with arginine bolus at 110 min measuring in vivo beta-cell function after 8–10 days of experimental infusions. ASIS, glucose-potentiated arginine-stimulated insulin secretion; dga, days gestational age; d, days; GSIS, glucose-stimulated insulin secretion; h, hours; IVC, inferior vena cava; min, minutes; tx, transfusion. Figure created with Biorender.com.

Figure 2.. Experimental Complications, Mortality, and Sample Sizes.

Schematic demonstrating mortality, catheter malfunctions, and sample sizes for the different experiments. Investigations with repeated measures included only animals that completed the entire study (CON n=7, GCG-5 n=7, GCG-50 n=5). Simultaneous immunofluorescent staining of insulin (INS), glucagon (GCG), pancreatic polypeptide (PPY), somatostatin (SST), and endothelial cells (Quad Stain) was insufficient for hormone quantitation in 1 animal from each GCG group precluding islet-specific analysis (CON n=7, GCG-5 n=6, GCG-50 n=4). For these animals lacking optimal Quad Stain, evaluation of insulin and glucagon expression in whole pancreas sections was performed from simultaneous immunofluorescent staining of INS, GCG, Ki-67, and DAPI (Ki67 Stain). Figure adapted from Cilvik et al. (2021) and created with Biorender.com.

Figure 3.. Fetal amino acid and basal insulin concentrations decrease with exposure to hyperglucagonemia.

(A) Fetal glucagon concentrations in vehicle control (CON, white circle, n=7), low-dose glucagon (GCG-5, grey diamond, n=7), or high-dose glucagon (GCG-50, black square, n=5) infusions into late gestation fetal lambs. (B) Fetal glucose, (C) fetal insulin, and (D) fetal cumulative amino acid concentrations over time in response to experimental infusions. Note decrease in basal insulin concentrations after 8–10 days and decrease in cumulative amino acid concentrations after 1 day of glucagon infusion. Results of mixed-effects model indicated on each graph; only animals that survived the full study were included. Individual means were compared for those with interaction (time × treatment) p≤0.1; significant differences between groups at the same timepoint indicated with brackets with p value above the line. Differences compared to treatment group day 0 are highlighted with symbols (#p=0.035, *p=0.024, **p=0.004, ***p=0.002, +p=0.001, $p<0.001).

Figure 4.. Beta-cell response to acute fetal hyperglucagonemia.

Basal (unstimulated, bsl) samples taken 3 hours after start of experimental infusions, followed by square-wave hyperglycemic clamp to measure GSIS in response to acute hyperglucagonemia. (A) Glucose infusion rates (GIR) from minute 60–90 during hyperglycemic clamp. (B) Fetal glucose and (C) insulin concentrations from consolidated basal draws (−20 and −10 min) and GSIS steady state (60, 75, and 90 min). Note higher basal insulin concentrations and enhanced GSIS in GCG-50 fetuses despite similar GIR and fetal glucose concentrations. Results of one- or two-way ANOVA indicated on each graph. Individual means were compared when interaction (time × treatment) p≤0.1; significant differences between groups indicated with brackets with p value above the line. One GCG-50 fetus experienced a transient catheter malfunction preventing blood draws at the time of this GSIS study. CON, white circle, n=12; GCG-5, grey diamond, n=11, GCG-50, black square, n=4).

Figure 5.. Beta-cell response to chronic fetal hyperglucagonemia.

Basal (unstimulated, bsl) plasma samples taken 8–10 days after start of experimental infusions, followed by square-wave hyperglycemic clamp to measure GSIS. (A) Glucose infusion rates (GIR) from minute 60–105 (steady state, SS) during hyperglycemic clamp. (B) GIR normalized to fetal weight at necropsy performed within 24 hours of hyperglycemic clamp. (C) Fetal glucose and (D) insulin concentrations from consolidated basal draws (−45, −30, −15, and 0 min; bsl), GSIS early phase (5, 10, 15, 20, and 30 min; early), and GSIS steady state (60, 75, 90, and 105 min). (E) Insulin to Glucose ratios calculated from bsl, early, and SS phases. Note lower insulin secretion in GCG-50 fetuses. Results of one- or two-way ANOVA indicated on each graph. Individual means were compared when interaction (time × treatment) p≤0.1; significant differences between groups indicated with brackets with p value above the line.

Figure 6.. Pancreatic islet composition and cell proliferation following chronic fetal hyperglucagonemia.

(A) Pancreas weight at necropsy. (B) Percent positive staining of total pancreatic area. (C) Beta- and alpha-cell mass, calculated using percent pancreas area (B) multiplied by pancreas weight (A). (D) Beta- and alpha-cell proliferation, calculated as percentage of Ki67+/insulin+ to total insulin+ cells (beta-cell proliferation) and percentage of Ki67+/glucagon+ cells to total glucagon+ cells (alpha-cell proliferation). (E) Total islet area, and (F) percent positive staining of total islet area. Results of one-way ANOVA as noted. Individual means compared if ANOVA p≤0.05; significant results indicated with brackets and p value within the graph. Each data point represents a single animal (CON, white circle, n=7; GCG-5, grey diamond, n=7 for A-D, n=6 for E-F; GCG-50, black square, n=5 for A-D, n=4 for E-F); individual points in B, D, E, and F represent an average from 2 or more stained sections.

Figure 7.. Pancreatic gene expression following chronic fetal hyperglucagonemia.

Gene expression analysis of pancreatic hormones (INS, GCG, SST, PPY, IGF1, IGF2), glucagon receptor (GCGR), beta-cell markers (PDX, NKX6–1), genes involved in insulin secretion (SLC2A2, GCK, RBP4), alpha-cell marker (PAX6), and vascular markers (VEGFA, HGF). Each gene was normalized to geometric mean of 5 housekeeping genes and expressed as fold-change relative to controls samples. Results of one-way ANOVA noted on graphs. Individual means were compared for those with ANOVA p≤0.05; significant differences indicated with brackets within the graph with p value above the line. CON, white circle, n=7; GCG-5, grey diamond, n=7; GCG-50, black square, n=5.