Late gestation fetal hyperglucagonaemia impairs placental function and results in diminished fetal protein accretion and decreased fetal growth

Abstract

Key points: Fetal glucagon concentrations are elevated in the setting of placental insufficiency, hypoxia and elevated stress hormones. Chronically elevated glucagon concentrations in the adult result in profound decreases in amino acid concentrations and lean body mass. Experimental elevation of fetal glucagon concentrations in a late-gestation pregnant sheep results in lower fetal amino acid concentrations, lower protein accretion and lower fetal weight, in addition to decreased placental function. This study demonstrates a negative effect of glucagon on fetal protein accretion and growth, and also provides the first example of a fetal hormone that negatively regulates placental nutrient transport and blood flow.

Abstract: Fetal glucagon concentrations are elevated in the setting of placental insufficiency and fetal stress. Postnatal studies have demonstrated the importance of glucagon in amino acid metabolism, and limited fetal studies have suggested that glucagon inhibits umbilical uptake of certain amino acids. We hypothesized that chronic fetal hyperglucagonaemia would decrease amino acid transfer and increase amino acid oxidation by the fetus. Late gestation singleton fetal sheep received a direct intravenous infusion of glucagon (GCG; 5 or 50 ng/kg/min; n = 7 and 5, respectively) or a vehicle control (n = 10) for 8-10 days. Fetal and maternal nutrient concentrations, uterine and umbilical blood flows, fetal leucine flux, nutrient uptake rates, placental secretion of chorionic somatomammotropin (CSH), and targeted placental gene expression were measured. GCG fetuses had 13% lower fetal weight compared to controls (P = 0.0239) and >28% lower concentrations of 16 out of 21 amino acids (P < 0.02). Additionally, protein synthesis was 49% lower (P = 0.0005), and protein accretion was 92% lower in GCG fetuses (P = 0.0006). Uterine blood flow was 33% lower in ewes with GCG fetuses (P = 0.0154), while umbilical blood flow was similar. Fetal hyperglucagonaemia lowered uterine uptake of 10 amino acids by >48% (P < 0.05) and umbilical uptake of seven amino acids by >29% (P < 0.04). Placental secretion of CSH into maternal circulation was reduced by 80% compared to controls (P = 0.0080). This study demonstrates a negative effect of glucagon on fetal protein accretion and growth. It also demonstrates that glucagon, a hormone of fetal origin, negatively regulates maternal placental nutrient transport function, placental CSH production and uterine blood flow.

Keywords: amino acid; chorionic somatomammotropin; fetal growth; fetal leucine metabolism; fick principle; glucagon; placenta; transplacental diffusion.

Figure 1.. Experimental Protocol.

(A) Experimental timeline. Sample size (n) reported for each group represents total number of fetuses at necropsy; not all animals had complete metabolic studies due to mechanical catheter issues. Lab draw denotes timing of fetal and maternal blood sampling as described in methods. (B) Blood flow and metabolic tracer study protocol. (C) Placenta diagram demonstrating catheter sampling locations on maternal and fetal sides of the placenta. Figure created using BioRender.com.

Figure 2.. Experimental complications and mortality.

Schematic demonstrating animals excluded from analysis due to mortality or catheter malfunction. The 3 control animals that died late in the study (≥ infusion day 7) received limited necropsies and were included in the analysis for nutrient:oxygen quotients over time, but excluded from final biochemical, hormonal, or molecular analysis. γ, umbilical vein catheter; V, uterine vein catheter. Figure created with BioRender.com.

Figure 3.. Late gestation fetal hyperglucagonemia results in lower fetal weight.

(A) Fetal and (B) maternal plasma glucagon concentrations in response to increasing glucagon infusion rates. Significant effect from Kruskal-Wallis test is indicated in panel A; ** p<0.01 vs. CON using multiple comparisons (uncorrected Dunn’s test). (C) Fetal and (D) placental weights at necropsy. CON n=7 (except fetal weight n=10), GCG-5 n=7, GCG-50 n=5. *p<0.05 using unpaired Student’s t-test.

Figure 4.. Lower fetal amino acid, glucose, and lactate concentrations, higher oxygen content following late gestation hyperglucagonemia.

Fetal arterial concentrations of (A) nonessential amino acids, (B) essential amino acids, (C) glucose, (D) oxygen, and (E) lactate at study end. CON n=7, GCG-5 n=7, GCG-50 n=5. * p<0.05, ** p<0.01, + p<0.001 using unpaired Student’s t-test. ALA, alanine; ARG, arginine; ASN, asparagine; ASP, aspartate; CYS, cystine; GLN, glutamine; GLU, glutamate; GLY, glycine; HIS, histidine; ILE, isoleucine; LEU, leucine; LYS, lysine; MET, methionine; PHE, phenylalanine; PRO, proline; SER, serine; TAU, taurine; THR, threonine; TRP, tryptophan; TYR, tyrosine; VAL, valine.

Figure 5.. Similar maternal nutrient concentrations between groups.

Maternal arterial concentrations of (A) nonessential amino acids, (B) essential amino acids, (C) glucose, (D) oxygen, and (E) lactate at study end. CON n=7, GCG-5 n=7, GCG-50 n=5.

Figure 6.. Late gestation fetal hyperglucagonemia results in lower whole body leucine disposal, less leucine flux into fetal tissues, and lower protein synthesis and accretion.

Fetal leucine flux was measured utilizing a L-[1-¹³C]-leucine tracer after 8–10 days of experimental infusions to calculate (A) whole body leucine disposal rate, (B) leucine flux into the placenta, (C) leucine flux into fetal tissue, (D) leucine flux into fetal plasma from the placenta, (E) protein breakdown, (F) leucine oxidation, (G) protein accretion, and (H) protein synthesis. CON n=5, GCG-5 n=6, GCG-50 n=3. * p<0.05, ** p<0.01, + p<0.001 using unpaired Student’s t-test.

Figure 7.. Late gestation fetal hyperglucagonemia results in lower uterine blood flow and less uterine uptake of amino acids, glucose, and oxygen.

(A) Uterine blood flow was measured using a ³H₂O tracer with the transplacental diffusion technique following 8–10 days of experimental infusions. Non-normalized flow (left) was compared to uterine blood flow normalized to placentome weight (middle) or the combined placentome + fetal weight (right). Using the Fick principle and non-normalized uterine blood flow, uterine uptake rates of (B) nonessential amino acids, (C) essential amino acids, (D) total amino acid carbon, (E) total amino acid nitrogen, (F) glucose, and (G) oxygen were calculated. Uterine uptake rates of total amino acid carbon and nitrogen represent global uterine uptake of amino acids. (H) As lactate is produced by the placenta, lactate secretion into the maternal circulation was calculated. CON n=6, GCG-5 n=7, GCG-50 n=5. * p<0.05, ** p<0.01, + p<0.001 using unpaired Student’s t-test. AA, amino acid.

Figure 8.. Lower fetal uptake rates of amino acids with otherwise normal umbilical blood flow following late gestation hyperglucagonemia.

(A) Umbilical blood flow was measured using a ³H₂O tracer with the transplacental diffusion technique following 8–10 days of experimental infusions. Non-normalized flow (left) was compared to umbilical blood flow normalized to fetal weight (right). Using the Fick principle and normalized umbilical blood flow, umbilical uptake rates of (B) nonessential amino acids, (C) essential amino acids, (D) total amino acid carbon, (E) total amino acid nitrogen, (F) glucose, (G) oxygen, and (H) lactate were calculated. Umbilical uptake rates of total amino acid carbon and nitrogen represent global fetal uptake of amino acids. CON n=5, GCG-5 n=6, GCG-50 n=3. * p<0.05, ** p<0.01 using unpaired Student’s t-test.

Figure 9.. Uteroplacental glucose and oxygen utilization rates are lower in hyperglucagonemic fetal lambs.

Following 8–10 days of experimental infusions, uteroplacental utilization rates of amino acids, globally represented by total (A) carbon and (B) nitrogen utilization, as well as uteroplacental utilization of (C) glucose and (D) oxygen were determined by difference between uterine nutrient uptake rates and umbilical nutrient uptake rates. (E) Lactate production by the placenta was calculated by the sum of maternal lactate secretion from placenta and fetal (umbilical) lactate uptake rates. CON n=5, GCG-5 n=6, GCG-50 n=3. * p<0.05, ** p<0.01 using unpaired Student’s t-test.

Figure 10.. Fetal hyperglucagonemia results in lower nutrient:oxygen quotients.

Nutrient:oxygen quotients of (A) glucose, (B) lactate, (C) total amino acids, and (D) the sum of all nutrients were calculated to determine availability of fuel to sustain fetal oxidative metabolism and provide carbon for accretion of new tissue. The 3 control animals who died ≥7 days of infusion were included in this analysis; one control animal was excluded for physiologically inconsistent data. Subscripts beneath bars denote the number of animals sampled at each time point (n). Significant effects in a repeated measures mixed models ANOVA as indicated above each graph; for those with interaction (time x treatment) p≤0.1, significant post-hoc multiple comparison analyses are indicated within the graph (*p<0.05 GCG vs. CON, **p<0.01 GCG vs. CON, ^#p<0.05 GCG day vs. GCG baseline, ^$p<0.05 CON day vs. CON baseline).

Figure 11.. Lower CSH placental gene expression, placental secretion into maternal circulation, and maternal arterial concentrations in pregnancies following 8–10 days of experimental fetal hyperglucagonemia.

(A) Placentome CSH gene expression measured by quantitative reverse-transcriptase PCR and expressed as fold change relative to control average. (B) Placental CSH secretion into maternal circulation at study end, calculated using the Fick principle. (C) Final uterine artery plasma CSH concentrations. CON n=7 (n=6 for CSH secretion due to catheter malfunction), GCG-5 n=7, GCG-50 n=5. **p<0.01, +p<0.001 using unpaired Student’s t-test. CSH, chorionic somatomammotropin.\