Keywords: fetal growth restriction, fetal pancreas, insulin, insulin-like growth factor-1
Abstract
Insulin-like growth factor-1 (IGF-1) and insulin are important fetal anabolic hormones. Complications of pregnancy, such as placental insufficiency, can lead to fetal growth restriction (FGR) with low-circulating IGF-1 and insulin concentrations and attenuated glucose-stimulated insulin secretion (GSIS), which likely contribute to neonatal glucose dysregulation. We previously demonstrated that a 1-wk infusion of IGF-1 LR3, an IGF-1 analog with low affinity for IGF-binding proteins and high affinity for the IGF-1 receptor, at 6.6 μg·kg−1·h−1 into normal fetal sheep increased body weight but lowered insulin concentrations and GSIS. In this study, FGR fetal sheep received either IGF-1 LR3 treatment at 1.17 ± 0.12 μg·kg−1·h−1 (LR3; n = 7) or vehicle (VEH; n = 7) for 1 wk. Plasma insulin, glucose, oxygen, and amino acids were measured before starting treatment and at the end of the treatment period. GSIS was measured on the final treatment day. Fetal body weights, insulin, glucose, oxygen, and GSIS were not different between groups. Amino acid concentrations decreased in LR3 (baseline vs. final individual means comparison P = 0.0232) but not in VEH (P = 0.3866). In summary, a 1-wk IGF-1 LR3 treatment did not improve growth in FGR fetuses. Insulin concentrations and GSIS were not attenuated by IGF-1 LR3, yet circulating amino acids decreased, which could reflect increased amino acid utilization. We speculate that maintaining amino acid concentrations or raising insulin, glucose, and/or oxygen concentrations to values consistent with normally growing fetuses during IGF-1 LR3 treatment may be necessary to increase fetal growth in the setting of placental insufficiency and FGR.
NEW & NOTEWORTHY IGF-1 LR3 treatment administered directly into growth-restricted fetal sheep circulation did not improve fetal growth or attenuate circulating insulin or fetal GSIS. Importantly, IGF-1 LR3 treatment reduced circulating amino acids, notably branched-chain amino acids, which have been shown to potentiate GSIS and protein accretion supporting fetal growth.
INTRODUCTION
Pregnancies complicated by placental insufficiency are characterized by decreased transfer of nutrients and oxygen to the fetus resulting in fetal growth restriction (FGR) and lower concentrations of fetal anabolic growth factors (1–3). FGR human fetuses are characterized by lower cord blood concentrations of insulin and insulin-like growth factor 1 (IGF-1) as compared with appropriately grown fetuses (1–3). Furthermore, animal models of FGR fetuses have smaller pancreatic islets, lower β-cell mass, and impaired glucose-stimulated insulin secretion (GSIS), all of which contribute to decreased insulin concentrations and thus provide insight into the etiology of decreased insulin concentrations in FGR (4–6). Interestingly, despite lower overall fetal insulin secretion in FGR, isolated FGR pancreatic islets have higher fractional insulin release in response to nutrient stimulation suggesting other factors in utero, notably high catecholamines and low oxygen content, suppress fetal insulin secretion (7). Therefore, once removed from the hostile intrauterine environment and with exposure to plentiful nutrients postnatally, the FGR pancreas must secrete more insulin per fewer β-cells to maintain glucose homeostasis over time. This increased demand on the pancreas may help explain why formerly FGR adults are at an increased risk of type 2 diabetes later in life, a disease characterized by premature β-cell failure.
Insulin and IGF-1 are critical fetal growth hormones that have overlapping anabolic actions (3, 8, 9). Concentrations of these hormones are impacted by the health and nutritional status of the fetus, especially in the third trimester of gestation. Because IGF-1 is more strongly correlated with fetal weight than insulin (1, 10, 11), it has been studied in sheep as a fetal intervention for growth restriction (12–14). Additional studies in human islets, rat islets, and transgenic mouse models have shown that IGF-1 preserves pancreatic β-cell mass and protects against β-cell damage (15–17), which could provide an added benefit of improving long-term metabolic outcomes. However, experimentally increasing IGF-1 can also acutely inhibit insulin secretion, which may limit its potential to consistently promote fetal growth and organ development (18–20).
Pregnant sheep have been used to test the basic physiological role of IGF-1 in fetal growth and development (9, 21). IGF-1 is synthesized by the fetus and is present in most fetal tissues (22, 23). In circulation, endogenous IGF-1 is largely bound to one of six major IGF-binding proteins that regulate bioavailability. To isolate the effects of IGF-1 on target tissues without the influence of IGF-binding proteins, recombinant long arginine 3 IGF-1 (IGF-1 LR3) is commonly used in animal models and has been shown to increase growth in fetal sheep (24–26). We previously infused IGF-1 LR3 into normally grown late gestation fetal sheep for 1 wk and found that experimental IGF-1 LR3 infusion at 6.6 μg·kg−1·h−1 resulted in lower fetal plasma insulin concentrations and attenuated fetal GSIS, which persisted in isolated fetal pancreatic islets (20). Although fetal weight increased by ∼15%, demonstrating growth-promoting effects, plasma glucose and amino acid concentrations were decreased in IGF-1 LR3 fetuses by the end of infusion (20, 25, 26). Notably, there were no changes in β-cell mass, but pancreatic insulin content was significantly higher after IGF-1 LR3 infusion, suggesting that insulin was being produced but not secreted (25). This was possibly related to supraphysiological (pharmacological) circulating IGF-1 concentrations in an otherwise normally growing fetus, resulting in direct inhibition of insulin secretion due to receptor cross talk. It is also possible that there was indirect inhibition of insulin secretion from concomitant low fetal plasma glucose and amino acid concentrations.
Although we previously demonstrated the effects of pharmacological IGF-1 on insulin secretion in normally grown, healthy late-gestation fetuses, the impact of a continuous intravenous IGF-1 treatment on fetal growth and insulin secretion in established FGR has not been determined. Therefore, we hypothesized that a 1-wk treatment dose of IGF-1 LR3 into FGR fetal sheep produced using a model of severe placental insufficiency that replicates features of human placental insufficiency and FGR (6, 7, 27) would improve growth without further lowering insulin concentrations or GSIS. The treatment dose was selected to restore IGF-1 in FGR and avoid pharmacological administration.
MATERIALS AND METHODS
Animal Preparation
All experiments were conducted at the Perinatal Research Center, University of Colorado School of Medicine, with the approval of the Institutional Animal Care and Use Committee. This center is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Experimental details are reported in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines (28).
Studies were conducted in pregnant Columbia-Rambouillet mixed-breed sheep (University of Arizona Sheep Unit, Tucson, AZ) carrying twin fetuses. All ewes were derived from the same flock and were part of a synchronized breeding system. Pregnant sheep (n = 18) were exposed to elevated ambient temperatures (40°C for 12 h; 35°C for 12 h) and moderate humidity (dew point at 22°C) from 40 ± 1 days gestational age (dGA) until 96 ± 2 dGA to produce placental insufficiency and FGR (5, 29–31). Ewes were then transported to the Perinatal Research Center and acclimatized to the facility for 14 ± 2 days before surgery. Surgeries were performed at 118 ± 1 dGA (term = 149 dGA) to place indwelling polyvinyl catheters as previously described (32–37). Briefly, after being fasted for 12–24 h and thirsted for 12 h, pregnant sheep were given diazepam (0.2 mg·kg−1) and ketamine (20 mg·kg−1) through a superficial vein to induce anesthesia and then were maintained on isoflurane inhalation anesthesia (2–4%) for the duration of the surgical procedure. Depth of anesthesia was determined and maintained in response to maternal corneal reflex, toe pinch, assessment of jaw tone, continuous pulse oximetry, heart rate monitoring, and exhaled CO2 monitoring; anesthetic effect in the fetus was assessed by muscle tone. Sheep were given penicillin G procaine (600,000 U im) before surgery. A midline incision was made along the linea alba and the uterus was exposed. A hysterotomy was performed for each twin and the fetal hindlimbs were exteriorized. Polyvinyl catheters were placed in each fetus in the bilateral fetal pedal arteries with the tip positioned in the external iliac artery and bilateral saphenous veins with the tip positioned in the common femoral vein. Ampicillin (500 mg) was injected into the amniotic fluid before surgical closure of the abdomen. Catheters were then placed in the maternal femoral artery and vein. Maternal and fetal catheters were tunneled subcutaneously to the maternal flank. Before skin closure at maternal midline, Marcaine was applied for local analgesic (3 mL, 5 mg·mL−1 Marcaine, 0.5% bupivacaine hydrochloride). Sheep received flunixin meglumine on the day of surgery (2.2 mg·kg−1 divided twice per day intramuscularly) and for the two following days (2.2 mg·kg−1 per day intramuscularly). The sheep also received Probios (10 g by mouth twice per day) for 2 days postoperatively. Animals were allowed to recover for a minimum of 5 days following surgery before initiating experimental treatments.
Experimental Design
Fetal intravenous treatments were initiated at 125 ± 1 dGA. One twin was randomly assigned to receive IGF-1 LR3 (LR3) (GroPep Bioreagents, Thebarton, ASTL) at 1.17 ± 0.12 μg·kg−1·h−1 (based on an average actual final fetal weight of 2.27 ± 0.41 kg), and the other twin received vehicle control (VEH) (saline with 0.5% bovine serum albumin, BSA) at 0.2 mL·h−1 to match the delivery rate of IGF-1 LR3. IGF-1 LR3, an IGF-1 analog with very low affinity for IGF-binding proteins and high affinity for the IGF-1 receptor, was used to isolate the actions of IGF-1 on its target tissues (38). We did not stratify randomization based on fetal sex or size, as these were not evaluated intraoperatively. Previous IGF-1 LR3 direct infusion studies into normally grown fetal sheep showed an infusion rate of 6.6 μg·kg−1·h−1 based on an estimated fetal weight of 3.5 kg resulting in increased organ weights and lower insulin concentrations (20, 24–26, 38). Therefore, the IGF-1 LR3 treatment in this study was decreased by ∼80% with the goal to gradually increase circulating IGF-1 concentrations without achieving pharmacological dosing. Our original study design included using increasing doses of IGF-1 LR3 treatment. However, because we experienced fetal demise with our first attempt at increased treatment dosing, we elected to maintain treatment at the lowest dose for the duration of the study. Fetuses received treatment for 7.6 ± 0.3 days. Fetal blood gas analysis, glucose, and lactate concentrations were obtained before starting and at the end of the treatment period. Fetal arterial plasma IGF-1, insulin, glucagon, cortisol, and amino acid concentrations were also measured before beginning treatment and before testing in vivo GSIS.
Eighteen ewes underwent surgery to place vascular catheters. Three of these 18 ewes were found to have at least one deceased fetus during surgery. Two more ewes had a fetal demise before treatment initiation. Six of the remaining 13 ewes had a demise of one (n = 4 ewes) or two (n = 2 ewes) fetuses after initiation of treatment. Of the four fetal demises in which the other twin survived, all four animals that died were receiving LR3. There were no fetal demises in the remaining seven ewes and fetuses from these seven were included in the final analysis.
Biochemical Analysis
Biochemical analyses were performed as previously described (35, 37, 39). Blood was immediately analyzed for hematocrit, pH, PaCO2, PaO2, and O2 saturation using an ABL 825 blood gas analyzer (Radiometer, Copenhagen, Denmark). O2 content of the blood was calculated by the ABL 825 analyzer. Glucose and lactate were immediately measured from plasma samples (Yellow Springs Instrument 2900, Yellow Springs, OH). Additional arterial plasma samples were frozen at −80°C for measurement of hormone and amino acid concentrations. Insulin, endogenous IGF-1, and cortisol were measured by an enzyme-linked immunosorbent assay [ELISA; Insulin: ALPCO, Salem, NH; intra- and interassay coefficients of variation (CVs) = 5.6% and 4.7%, respectively; sensitivity = 0.14 ng·mL−1; IGF-1: ALPCO; intra- and interassay CVs, 3.1% and 5.6%, respectively; sensitivity, 0.09 ng·mL−1; cortisol: ALPCO; intra- and interassay CVs = 4.6% and 5.8%, respectively; sensitivity = 1.0 ng·mL−1]. Fetal arterial plasma glucagon was measured by radioimmunoassay (Millipore, Billerica, MA; intra- and interassay CVs: 4.8% and 11.7%; sensitivity = 18.5 pg·mL−1). Amino acid concentrations were measured using a Dionex 300 model 4500 amino acid analyzer (Thermo Fisher Scientific, Waltham, MA) after deproteinization with sulfosalicyclic acid. We were unable to directly measure circulating IGF-1 LR3 concentrations due to a lack of available assays and therefore only measured endogenous IGF-1. Cross-reactivity between IGF-1 LR3 and endogenous IGF-1 has not been reported by us or any other group for the IGF-1 assay used. In prior studies, IGF-1 measured by the assay decreased in IGF-1 LR3-infused normally grown sheep fetuses, consistent with a lack of cross-reactivity of this assay for IGF-1 LR3 (20, 26).
In Vivo Insulin Secretion Studies
On the final day of treatment (132 ± 1 dGA), fetal GSIS was measured using a primed, continuous, variable-rate hyperglycemic clamp (20, 37, 39–41). Baseline plasma insulin and glucose concentrations were obtained at −60, −45, −30, and −15 min. At time 0, the hyperglycemic clamp was initiated with a 33% dextrose (wt:vol in saline) bolus (825 mg glucose) into the fetus. This was followed by a constant infusion of 33% dextrose, titrated to keep the plasma glucose concentration near 2.5 mmol·L−1, which elicits 90% maximal insulin concentrations in vivo in normally grown singletons in this breed of fetal sheep as determined using a stepwise increase in plasma glucose concentrations starting from baseline euglycemia to maximal measured insulin secretion (42). The dextrose infusion rate was held constant from minute 45 until the completion of the GSIS study. Fetal arterial samples were collected for measurement of glucose and insulin concentrations at 5, 10, 15, 20, 30, 45, 60, 75, 90, and 105 min. Experimental treatments (LR3 or VEH) were continued throughout the GSIS studies and through necropsy.
Pancreas Collection
At the conclusion of the study, animals were euthanized with an intravenous overdose of a pentobarbital euthanasia solution. Fetal sex and weights were recorded. The pancreases were weighed and dissected into the splenic portion, processed for histological evaluation, and the hepatic portion, which was snap frozen in liquid nitrogen and transferred to −80°C for mRNA extraction.
mRNA Analysis
mRNA was extracted from pulverized −80°C pancreas (100 mg), reverse transcribed into complimentary DNA (cDNA), and measured by quantitative PCR analysis for INS (insulin), GCG (glucagon), PPY (pancreatic polypeptide), SST (somatostatin), PDX1 (pancreatic and duodenal homeobox-1), GCK (glucokinase), SLC2A2 [glucose transporter-2 (GLUT-2)], IGF1 (insulin-like growth factor-1), IGF2 (insulin-like growth factor-2), VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), MLF2 (myeloid leukemia factor 2), and S15 (ribosomal protein S15) as previously described (LR3, n = 7; VEH, n = 7) (25, 41, 43–46). The total volume of each reaction was 10 μL: 4 μL cDNA, 0.5 μL for each primer (0.5 mol/L), and 5 μL Faststart Universal SYBR Green MM (Roche, San Francisco, CA). The temperature cycles were 5 min at 95°C to activate the enzyme; then 40 cycles of 30 s at 60°C, 30 s at 72°C, and 10 s at 96°C. Melt curve analysis was used to ensure a single peak. The cDNA samples were analyzed in triplicate and the standard curve method of relative quantification was used. Genes of interest were normalized to the reference genes, MLF2 and S15, which were not different between groups. Results are presented relative to vehicle controls.
Histology of the Fetal Pancreas
Histological evaluation of the fetal pancreas (LR3, n = 7; VEH, n = 7) was based on our previous protocols (25, 43, 47) with minor modifications. Frozen 5-μm sections were adapted to room temperature for 30 min before three 5 min washes in deionized water. Sections were washed three times in phosphate-buffered saline (PBS) for 5 min then blocked for 60 min in 5% BSA in PBS, followed by immunolabeling for insulin with guinea pig anti-swine insulin (1:250; Dako, Carpinteria, CA) and griffonia simplicifolia lectin 1 agglutinin-FITC conjugated (green, 15 μg/mL; Vector Laboratories, Burlingame, CA). Insulin was detected with donkey anti-guinea pig Alexa Fluor 680 (1:500).
Pancreatic tissue sections stained for insulin and lectin were scanned using an Olympus VS120 Whole Slide Scanning System (VS120-S6-W; Olympus America Inc., Center Valley, PA) at ×40 magnification.
Pancreatic Vascularity and β-Cell Quantifications
Files of the scanned tissue sections in “.Versus” format were imported into the Visiopharm platform (Visiopharm, Hoersholm, Denmark). The scanned sections were analyzed through a series of two custom-made (SIA) Analysis Protocol Packages (APPs) using Visiopharm software to calculate total pancreatic tissue areas, insulin and lectin positive areas, and total insulin (or lectin) per tissue area percent positive. Pancreatic tissues were detected and segmented from the background using Decision Forest (90% accuracy) machine learning method using FITC channel [mean filter; 3x3; at ×1 magnification] as a feature. Four out of 14 sections were randomly selected and used to train the algorithm. Tissue areas smaller than 200,000 μm2 were excluded. Subsequently, total section areas were calculated for each sample. FITC and CY5 channels were used to calculate lectin-positive and insulin-positive areas, respectively, via thresholding. The resulting total areas were classified and their percent positive area from the total tissue area was calculated for each section. Manual inspection for quality control of automated image analyses of all slides was performed. Insulin-positive area was used to determine the β-cell mass by multiplying the pancreas weight by the percent insulin-positive in total pancreas area for each animal.
Statistical Analysis
Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA). Results were analyzed in a mixed models ANOVA with a term to account for paired fetuses from the same pregnancy and a term to account for repeated measures within the same fetus when appropriate. Time, experimental treatment (LR3 or VEH), and their interaction were included as fixed terms. If interaction terms in the overall ANOVA were P < 0.1, then protected Fisher’s least significant difference was used for individual means comparisons. Measurements made once to compare LR3 and VEH groups were analyzed by Student’s t test. Results are expressed as means ± SE. P values of ≤ 0.05 were accepted as significant.
RESULTS
IGF-1 LR3 treatment was associated with fetal demise in four animals. All four of these fetuses were males (weight range 0.85–3.25 kg) and displayed hypoglycemia and hypoxemia in the setting of IGF-1 LR3 treatment. Glucose decreased by 42% (Time × Treatment P = 0.0319; individual means comparisons LR3 Baseline vs. Final P = 0.0027 and VEH Baseline vs. Final P = 0.1060) and Pao2 decreased by 30% during LR3 treatment (Time × Treatment P = 0.0300; individual means comparisons LR3 Baseline vs. Final P = 0.0032 and VEH Baseline vs. Final P = 0.1710). Three of these four fetuses underwent a demise in the final 1–2 days of the planned treatment duration. These animals were receiving a final IGF-1 LR3 dose of 1.12 ± 0.30 μg·kg−1·h−1 (based on an average final fetal weight of 2.21 ± 0.61 kg). The other one of these four fetuses underwent a demise within the first 2 days of treatment. This animal was receiving a final IGF-1 LR3 infusion rate of 6.78 μg·kg−1·h−1 (based on final fetal weight of 0.85 kg). Animals that survived to study completion (n = 7) were receiving a final IGF-1 LR3 dose of 1.17 ± 0.12 μg·kg−1·h−1 (based on an average final fetal weight of 2.27 ± 0.41 kg).
Although we were not powered to detect sex differences in this study, we have included different symbols for males versus females in our figures. None of our findings suggest obvious sex differences.
Fetal Biochemistry and Hormones
Fetal arterial biochemistry and hormones following vehicle versus IGF-1 LR3 treatment are shown in Table 1. Fetal arterial blood gas values and lactate did not differ based on treatment. Plasma cortisol and glucagon concentrations were also similar.
Table 1.
Fetal arterial biochemistry and plasma hormones
Biochemistry VEH (n = 7) LR3 (n = 7) P Value pH 7.34 ± 0.01 7.33 ± 0.01 0.8219 PaCO2, mmHg 50.5 ± 1.2 52.3 ± 2.0 0.4530 PaO2, mmHg 17.7 ± 1.2 16.1 ± 1.6 0.4511 SaO2, % 40.0 ± 4.9 32.6 ± 6.2 0.3668 O2 content, mmol·L−1 2.8 ± 0.4 2.4 ± 0.5 0.5335 Hgb, g·dL−1 7.4 ± 0.4 7.6 ± 0.4 0.6557 Lactate, mmol·L−1 1.8 ± 0.1 1.6 ± 0.3 0.4862 Plasma hormones Cortisol, ng·mL−1 54.3 ± 9.1 75.7 ± 36.5 0.5350# Glucagon, pg·mL−1 125.5 ± 34.4 86.2 ± 7.5 0.5350#
Values are means ± SE. Measurements were made at the end of treatment, just before the fetal in vivo insulin secretion study. VEH, vehicle fetuses; LR3, IGF-1 LR3 fetuses. Statistical analysis was performed by Student’s t test or Mann–Whitney test; #Mann–Whitney test.
Baseline and final fetal plasma insulin, glucose, oxygen content, and endogenous IGF-1 concentrations are displayed in Fig. 1. Insulin, glucose, oxygen content, and endogenous IGF-1 concentrations were not different between groups nor did they change within groups over time.
Figure 1.
Plasma insulin, glucose, O2 content, and endogenous IGF-1 did not change in either group. Fetal plasma insulin (A), glucose (B), O2 content (C), and endogenous IGF-1 concentrations (D) did not change over time based on treatment. VEH, vehicle (male, open circles; female, open triangles; n = 7 fetuses); LR3, IGF-1 LR3 (male, closed circles; female, closed triangles; n = 7 fetuses). Means ± SE are shown. Statistical analysis was performed by mixed-model ANOVA; protected Fisher’s least significant difference test was performed for individual means comparisons if interaction P < 0.1 and labeled if P < 0.05.
Fetal Amino Acids
Baseline and final summed amino acid concentrations are shown in Fig. 2, and final individual fetal amino acids are shown in Table 2. Summed total amino acid concentrations decreased over time in LR3 (Baseline 3,269 ± 241 μmol·L−1 vs. Final 2,888 ± 301 μmol·L−1, individual means comparison P = 0.0232) but not in VEH (Baseline 3,361 ± 210 μmol·L−1 vs. Final 3,493 ± 202 μmol·L−1, individual means comparison P = 0.3866). Summed nonessential amino acid concentrations decreased over time with LR3 treatment (individual means comparison P = 0.0470). Summed essential amino acid concentrations decreased over time with LR3 (individual means comparison P = 0.0253) and were lower than VEH at the end of treatment (individual means comparison P = 0.0392). When comparing individual amino acid concentrations at the end of treatment, the branched-chain amino acids (BCAA), leucine and valine, were lower at the end of LR3 treatment as compared with vehicle treatment (Table 2; Student’s t test P < 0.05). In fact, summed BCAA were 21% lower at the end of treatment in LR3 as compared with VEH (LR3 427.6 ± 39.3 μmol·L−1 vs. VEH 539.5 ± 21.6 μmol·L−1, P = 0.0280).
Figure 2.
Plasma amino acids decreased with IGF-1 LR3 treatment. Summed total amino acids (A), summed nonessential amino acids (B), and summed essential amino acids (C) decreased in fetuses receiving IGF-1 LR3 but not in those receiving vehicle. VEH, vehicle (male, open circles; female, open triangles; n = 7 fetuses); LR3, IGF-1 LR3 (male, closed circles; female, closed triangles; n = 7 fetuses). Means ± SE are shown. Statistical analysis was performed by mixed-model ANOVA; protected Fisher’s least significant difference test was performed for individual means comparisons if interaction P < 0.1 and labeled if P < 0.05.
Table 2.
Fetal plasma amino acids
VEH (n = 7) LR3 (n = 7) P Value Essential, μmol·L−1 Histidine 53.2 ± 7.8 40.5 ± 4.9 0.1905 Isoleucine 78.5 ± 7.5 63.8 ± 10.2 0.2672 Leucine 122.2 ± 5.8 92.1 ± 10.4 0.0267 Lysine 95.9 ± 12.3 73.5 ± 11.7 0.2115 Methionine 86.4 ± 6.4 77.5 ± 7.3 0.3787 Phenylalanine 81.9 ± 7.1 91.3 ± 16.8 0.6148 Threonine 317.2 ± 33.7 236.0 ± 40.8 0.1510 Tryptophan 45.6 ± 2.8 42.0 ± 2.6 0.3621 Valine 338.8 ± 18.4 271.7 ± 24.3 0.0474 Nonessential, μmol·L−1 Alanine 257.7 ± 27.4 222.1 ± 48.7 0.5365 Arginine 110.6 ± 14.4 113.5 ± 16.4 0.8947 Asparagine 45.9 ± 6.4 31.1 ± 3.5 0.0650 Aspartate 26.5 ± 2.1 21.8 ± 2.4 0.1699 Cysteine 23.2 ± 2.6 18.5 ± 1.4 0.1367 Glutamate 38.6 ± 5.0 40.0 ± 5.8 0.8527 Glutamine 431.9 ± 39.3 338.8 ± 38.6 0.1170 Glycine 380.2 ± 46.2 337.6 ± 43.7 0.5156 Ornithine 67.3 ± 9.0 51.8 ± 12.2 0.3241 Proline 179.2 ± 25.1 121.3 ± 16.2 0.0766 Serine 446.8 ± 65.0 410.8 ± 85.4 0.7428 Taurine 159.4 ± 36.5 88.1 ± 30.9 0.1616 Tyrosine 105.7 ± 7.4 104.1 ± 21.0 0.9422
Values are means ± SE. Measurements were made at the end of treatment, just before the fetal in vivo insulin secretion study. VEH, vehicle fetuses; LR3, IGF-1 LR3 fetuses. Statistical analysis was performed by Student’s t test. Significant P values where P < 0.05 are bolded.
Fetal In Vivo Glucose-Stimulated Insulin Secretion
Fetal glucose-stimulated insulin secretion was measured in vivo with a primed, continuous, variable-rate hyperglycemic clamp. Baseline steady-state glucose (LR3 0.54 ± 0.11 mmol·L−1 vs. VEH 0.62 ± 0.10 mmol·L−1, P = 0.5944) and insulin (LR3 0.16 ± 0.07 ng·mL−1 vs. VEH 0.17 ± 0.06 ng·mL−1, P = 0.9085) concentrations were similar between groups. During the GSIS study, glucose concentrations remained similar, as designed and expected (Fig. 3A; clamp steady-state LR3 2.91 ± 0.32 mmol·L−1 vs. VEH 2.80 ± 0.28 mmol·L−1, P = 0.7988). Plasma insulin concentrations were similar between groups (Fig. 3B; clamp steady-state LR3 0.82 ± 0.37 ng·mL−1 vs. VEH 0.83 ± 0.22 ng·mL−1, P = 0.9855).
Figure 3.
Glucose-stimulated insulin secretion (GSIS) is not different based on treatment. Fetal plasma glucose (A) and insulin (B) during measurement of GSIS are plotted relative to the start of the hyperglycemic clamp at time 0. VEH, vehicle (open circles, n = 7 fetuses); LR3, IGF-1 LR3 (closed squares, n = 7 fetuses). Means ± SE are shown. Statistical analysis was performed by mixed-model ANOVA.
Fetal Characteristics
Fetal characteristics are shown in Table 3. Fetal body weights (LR3 2.27 ± 0.41 kg vs. VEH 1.99 ± 0.36 kg; P = 0.6154) and hindlimb lengths (LR3 28.6 ± 2.2 cm vs. VEH 27.4 ± 2.4 cm; P = 0.7319) were not different between groups. Adrenal glands were significantly larger in animals treated with LR3 as compared with VEH. All other organ weights were not different between groups.
Table 3.
Fetal characteristics at necropsy
VEH (n = 7) LR3 (n = 7) P Value % male 71% 71% Fetal weight, kg 1.99 ± 0.36 2.27 ± 0.41 0.6154 Crown rump length, cm 41.0 ± 3.0 41.8 ± 2.3 0.8327 Hindlimb length, cm 27.4 ± 2.4 28.6 ± 2.2 0.7319 Brain, g 48.1 ± 2.9 46.4 ± 2.8 0.6941 Per fetal weight, g·kg−1 28.3 ± 4.1 23.6 ± 2.9 0.3672 Heart, g 15.9 ± 2.2 15.1 ± 2.3 0.8071 Per fetal weight, g·kg−1 8.6 ± 0.9 7.4 ± 0.9 0.3397 Lungs, g 73.2 ± 15.3 79.9 ± 15.3 0.7605 Per fetal weight, g·kg−1 36.5 ± 2.9 34.7 ± 1.3 0.5884 Liver, g 51.4 ± 11.0 57.5 ± 12.3 0.7186 Per fetal weight, g·kg−1 25.0 ± 1.4 24.6 ± 1.4 0.8351 Kidneys, g 14.0 ± 2.8 17.0 ± 2.4 0.4416 Per fetal weight, g·kg−1 7.1 ± 0.4 8.0 ± 0.8 0.2985 Adrenal glands, g 0.30 ± 0.05 0.44 ± 0.04 0.0436 Per fetal weight, g·kg−1 0.16 ± 0.03 0.26 ± 0.05 0.1732 Spleen, g 4.4 ± 0.9 5.4 ± 1.3 0.5465 Per fetal weight, g·kg−1 2.2 ± 0.3 2.3 ± 0.3 0.7649
Values are means ± SE. VEH, vehicle fetuses; LR3, IGF-1 LR3 fetuses. Statistical analysis was performed by Student’s t test. Significant P values where P < 0.05 are bolded.
Fetal Pancreas Characteristics
Fetal pancreatic weights were not different between groups (Table 4). There were no differences between groups in the proportion of the pancreatic sections staining insulin-positive or in calculated β-cell mass. There also were no differences in total pancreatic vascularity between groups. There were no differences in pancreatic insulin mRNA expression or the expression of the other major pancreatic endocrine hormones or paracrine growth factors (Table 5).
Table 4.
Histological characteristics of the fetal pancreas
VEH (n = 7) LR3 (n = 7) P Value Pancreas weight, g 2.18 ± 0.45 2.35 ± 0.41 0.7789 Insulin positive area, % 1.06 ± 0.23 0.94 ± 0.22 0.7222 β-cell mass, mg 22.93 ± 5.55 18.54 ± 5.00 0.5677 Pancreatic vessel density, % 3.56 ± 0.59 3.37 ± 0.22 0.7657
Values are means ± SE. VEH, vehicle fetal pancreases; LR3, IGF-1 LR3 fetal pancreases. Statistical analysis was performed by Student’s t test.
Table 5.
Fetal pancreatic gene expression
Pancreatic mRNA VEH (n = 7) LR3 (n = 7) P Value Insulin 1.00 ± 0.33 1.22 ± 0.30 0.6292 Glucagon 1.00 ± 0.23 0.76 ± 0.12 0.3633 Pancreatic polypeptide 1.00 ± 0.15 1.10 ± 0.20 0.7071 Somatostatin 1.00 ± 0.22 0.76 ± 0.07 0.3129 PDX1 1.00 ± 0.10 1.15 ± 0.10 0.3021 Glucokinase 1.00 ± 0.13 1.25 ± 0.17 0.2719 GLUT2 1.00 ± 0.18 1.17 ± 0.18 0.5126 IGF-1 1.00 ± 0.13 1.21 ± 0.10 0.2314 IGF-2 1.00 ± 0.09 1.23 ± 0.14 0.1838 VEGF 1.00 ± 0.07 1.08 ± 0.13 0.6326 HGF 1.00 ± 0.09 1.14 ± 0.19 0.5082
Values are means ± SE. VEH, vehicle fetal pancreases; LR3, IGF-1 LR3 fetal pancreases. Statistical analysis was performed by Student’s t test. Pancreatic mRNA expression is presented as fold change (arbitrary units) relative to VEH.
DISCUSSION
IGF-1 LR3 treatment at a dose of 1.17 ± 0.12 μg·kg−1·h−1 directly into growth-restricted fetal sheep circulation did not improve fetal growth, insulin secretion, or β-cell mass. It also did not attenuate circulating insulin or fetal GSIS. Although fetal glucose and oxygen concentrations remained similar between groups, IGF-1 LR3 treatment reduced circulating amino acids, notably BCAA, which have been shown to promote GSIS and protein accretion supporting fetal growth. There were no differences in morphological or biochemical characteristics of the pancreas. These results differ from our findings using a higher experimental IGF-1 LR3 dose of 6.6 μg·kg−1·h−1 into normally grown fetal sheep that showed increased fetal growth despite decreased fetal insulin secretion and plasma glucose and amino acid concentrations, suggesting more efficient utilization of available nutrients to support organ-specific growth (20, 26). Thus, given a limited baseline nutrient supply in FGR, we conclude that maintaining circulating amino acid concentrations or raising insulin concentrations and oxygen content to values consistent with normally growing fetuses during a low-dose IGF-1 LR3 treatment may be necessary to increase fetal growth in the setting of placental insufficiency and FGR. Alternatively, a higher IGF-1 LR3 dose may be required.
Several prior studies using single hormones or nutrient treatments did not demonstrate benefit to the FGR fetus, and some have even shown adverse effects. Administration of a continuous insulin infusion for 2 wk directly into FGR fetal circulation resulted in increased circulating insulin and IGF-1 concentrations but did not improve fetal growth or pancreatic β-cell mass and decreased fetal Pao2 and BCAA concentrations (45). A 2-wk-long direct fetal glucose infusion did not improve fetal insulin concentrations or β-cell mass but rather resulted in progressive fetal hypoxia and acidosis (48). In addition, while a direct leucine infusion into normally grown late-gestation fetal sheep potentiated insulin secretion and resulted in increased β-cell proliferation, β-cell mass, islet size, and pancreatic insulin content (36), these findings were absent in FGR fetal sheep and their islets in response to a fetal leucine infusion (36, 49). Lack of an increase in fetal growth or insulin secretion from IGF-1 LR3 treatment in this study further supports the fact that a multipronged approach combining nutrient supplements with anabolic growth factors and oxygen is likely necessary, especially to support glucose oxidation and insulin secretion in the setting of placental insufficiency (50).
Interestingly, even at a dose that did not change circulating insulin, glucose, or oxygen concentrations, IGF-1 LR3 treatment reduced circulating summed amino acids, which could be the result of either reduced fetal amino acid uptake from the placenta or increased fetal amino acid utilization. In previous studies experimentally infusing IGF-1 LR3 into normally grown fetuses, we found that IGF-1 LR3 either decreased or did not affect umbilical amino acid uptake rates (26, 51). Because FGR fetuses are known to have lower umbilical uptake of amino acids at baseline (52), decreasing uptake even more or not increasing uptake to meet demands, may negatively affect baseline insulin secretion. Chronic supplementation of amino acids for 10–12 days directly into FGR fetal sheep circulation during late-gestation results in increased GSIS, increased pancreatic insulin area, and increased β-cell mass as compared with saline-infused FGR fetuses (44). Therefore, relatively lower plasma concentrations of amino acids, even in the setting of increased growth-promoting circulating IGF-1, may limit improvements in insulin secretion thereby limiting growth promotion.
We previously reported that a 1-wk, higher experimental dose of IGF-1 LR3 into normally grown late-gestation fetal sheep increased pancreatic islet vascularity and pancreatic VEGF, HGF, IGF1, and IGF2 mRNA expressions while decreasing insulin secretion (25). There may be several reasons why these findings were absent in the current study. First, FGR fetal islets may be resistant to those changes at this point in gestation. In prior studies, we have shown that while pulsatile hyperglycemia and chronic leucine infusions, for example, promote increased β-cell mass and insulin secretion in normally grown late-gestation fetuses, these benefits are notably absent in FGR fetuses. Although the mechanisms that underlie resistance to improvement require further investigation, it is possible that the IGF-1 LR3 unresponsiveness in established FGR is due to other factors characteristic of FGR fetuses including hypoxemia, prolonged glucose and amino acid deprivation, or elevated cortisol and catecholamines, all factors that can suppress insulin secretion (5) and therefore the positive growth effects of increased circulating IGF-1 concentrations. In addition, intrinsic β-cell and islet adaptations to chronic placental insufficiency may prevent any improvements in islet structure and function in response to hormone or nutrient supplementation.
We may not have delivered a sufficient dose of IGF-1 LR3 to observe improvement in islet function and increased fetal growth. Our prior study infusing IGF-1 LR3 into normally grown late-gestation fetal sheep that resulted in increased fetal growth but decreased fetal insulin secretion used a dose of 6.6 μg·kg−1·h−1 based on an estimated fetal weight of 3.5 kg (final average LR3 dose 5.23 μg·kg−1·h−1 based on average actual fetal weight of 4.42 ± 0.14 kg) (25). Although not statistically significant, in the current study, LR3 fetuses were 14% heavier than VEH. In addition, adrenal glands in LR3 were significantly heavier as compared with VEH, a finding consistent with the effects of IGF-1 LR3 infusion in normally grown fetuses. These observations suggest similar effects with the dosing used in the current FGR study compared with higher relative dosing in normally grown fetuses. However, without knowing whether the treatment dose used in the current study would promote growth in normal, healthy fetuses, it is not possible to conclude whether the lack of statistically significant growth we observed was dose-related or due to nutrient and oxygen supply constraints in our placental insufficiency model of FGR. We were also unable to measure fetal growth rates in utero. It is possible that a higher dose in this study may have improved growth without causing adverse consequences. Unfortunately, when we infused IGF-1 LR3 at a dose of 6.78 μg·kg−1·h−1 (based on fetal weight of 0.852 kg at the time of necropsy), we observed fetal demise within 48 h of initiating treatment. This happened by chance to be a severely growth-restricted fetus, so it is difficult to directly compare results with larger fetuses. Notably, in evaluating the three other IGF-1 LR3 fetal losses, we found progressive hypoxia and hypoglycemia in response to IGF-1 LR3 treatment, ultimately reaching the threshold of fetal demise in the final 1–2 days of treatment. Given the variability of our FGR model, as well as variability in human FGR, more targeted and personalized dosing titrations based on acute physiological responses to treatment may be required. Unfortunately, we are unable to measure plasma IGF-1 LR3 thus limiting our ability to gradually titrate to a desired free IGF-1 circulating concentration available for receptor interaction. Furthermore, our previous study in normally grown late-gestation fetal sheep only used singleton pregnancies. Differences between twin and singleton fetuses should be considered when comparing our current results to previous studies.
Finally, as noted above, IGF-1 LR3 may need to be combined with nutrient and oxygen supplementation (50) and/or insulin infusion to exhibit desired effects. In a recent study infusing IGF-1 LR3 for 1 wk with insulin and glucose to maintain euinsulinemia and euglycemia, normally growing fetuses displayed increased whole body growth despite lower plasma amino acid and oxygen concentrations and elevated lactate. Umbilical uptake rates of oxygen increased over time, and final umbilical uptake of essential amino acids was increased compared with control infusions (51). These findings suggest that IGF-1 coinfusion with insulin and glucose into the normally grown fetus increases utilization and extraction rates of amino acids and oxygen to support faster growth rates. Whether the same holds true in FGR fetuses is critical to evaluate and is currently under investigation in our laboratory.
In conclusion, this study demonstrates a lack of response in the late-gestation FGR fetus to IGF-1 treatment with respect to either promoting growth or inhibiting insulin secretion. Although it is possible that a higher dose of IGF-1 LR3 may be required to see the primary responses we interrogated, any plan to use a higher dose must also consider our finding that IGF-1 LR3 treatment may have been associated with increased fetal demise. The use of twin fetuses in the current study as opposed to singleton fetuses in prior studies may also be relevant. The implications of this are important when considering strategies to improve fetal growth and pancreatic development. We speculate that therapeutic interventions for FGR aimed at improving fetal growth, islet function, and β-cell mass, thereby promoting improved long-term metabolic outcomes should include a combination of nutrient supplementation and oxygen, in conjunction with anabolic endocrine factors.
DATA AVAILABILITY
Data will be made available upon reasonable request.
GRANTS
This work was supported by the National Institute of Health Grants R01DK088139 (to P.J.R.), R01HD093701 (to P.J.R.), R01DK108910 (to S.R.W.), R01HD079404 (to L.D.B.), and S10OD023553.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
A.W., J.S., S.R.W., S.I.A., E.D., S.W.L., L.D.B., and P.J.R. conceived and designed research; A.W. performed experiments; A.W., J.S., S.R.W., S.I.A., E.D., S.W.L., L.D.B., and P.J.R. analyzed data; A.W., J.S., S.R.W., S.I.A., E.D., S.W.L., L.D.B., and P.J.R. interpreted results of experiments; A.W. prepared figures; A.W. drafted manuscript; A.W., J.S., S.R.W., S.I.A., E.D., S.W.L., L.D.B., and P.J.R. edited and revised manuscript; A.W., J.S., S.R.W., S.I.A., E.D., S.W.L., L.D.B., and P.J.R. approved final version of manuscript.
ACKNOWLEDGMENTS
The authors thank David Caprio, Dan LoTurco, Gates Roe, Larry Toft, and Karen Trembler for their technical support. Graphical abstract created in BioRender. White, A. (2024) https://BioRender.com/z59u205.
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Data Availability Statement
Data will be made available upon reasonable request.