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 AAALAC International. Experimental details are reported in compliance with the ARRIVE 2.0 guidelines ^{Reference Percie du Sert, Hurst and Ahluwalia26} .

Studies were conducted in pregnant late gestation Columbia-Rambouillet mixed-breed sheep (Nebeker Ranch, Lancaster, CA & University of Arizona Sheep Unit, Tucson, AZ) carrying a singleton fetus (n = 12; 7 males and 5 females). All ewes were derived from the same flock and were part of a synchronized breeding system. Ewes were acclimatized to the facility for 21 ± 1 days and then fasted for 24 hours and thirsted for 12 hours before surgery. Surgeries were performed at 122 ± 1 days gestational age (dGA; term = 147–152 dGA, average 149 dGA) to place indwelling polyvinyl catheters as previously described ^{Reference Hay, Sparks, Quissell, Battaglia and Meschia27–Reference Boehmer, Brown, Wesolowski, Hay and Rozance31} . Briefly, 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 units intramuscularly) prior to surgery. A midline incision was made along the linea alba and the uterine horn containing the fetus was exposed. A hysterotomy was performed and the fetal hindlimbs were exteriorized. Polyvinyl catheters were placed 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 prior to 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 post-operatively. Animals were allowed to recover for a minimum of five days following surgery prior to initiating experimental infusions.

Experimental design

Ten of the twelve animals underwent the following protocol, as the additional two animals were included for islet studies only. Fetal sheep received an intravenous infusion of either IGF-1 LR3 (IGF-1) (GroPep Bioreagents, Thebarton, ASTL) at 6.6 μg·kg⁻¹·hr⁻¹ based on an estimated fetal weight of 3.5 kg or a vehicle control (CON) infusion (saline with 0.5% bovine serum albumin, BSA) at 0.2 ml·hr^-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 ^{Reference Sundgren, Giraud and Schultz32} . The dose was selected based on previous direct infusion studies into fetal sheep resulting in increased organ weights and lower insulin concentrations ^{Reference Lok, Owens, Mundy, Robinson and Owens13–Reference Stremming, Heard and White15,Reference White, Stremming and Boehmer23,Reference Sundgren, Giraud and Schultz32} . Each animal received both infusions spaced 2–3 days apart. The initial fetal intravenous experimental infusion (either IGF-1 or CON) was alternated such that five animals received IGF-1 first, and five received CON first. Therefore, animals that received IGF-1 for the initial infusion received CON for the second infusion, while animals that received CON first received IGF-1 for the second infusion. This methodology was utilized to limit the potential effects of animal variability, increasing gestational age, and exposure priming. The first infusion was performed at 130 ± 1 dGA, and the second infusion was performed at 132 ± 1 dGA. Fetal blood was sampled for pre-infusion blood gas, hormone, and nutrient concentrations immediately before the experimental infusions began. To measure the response to the experimental infusion and to determine insulin and glucose concentrations prior to the hyperglycemic clamp, fetal blood was then sampled 70 and 80 min after the experimental infusion began, averaged, and noted as Post-75 min ^{Reference Boehmer, Baker Ii, Brown, Wesolowski and Rozance33} . After 90 min of experimental infusion, fetal GSIS was measured using a primed, continuous, variable-rate hyperglycemic clamp ^{Reference White, Stremming and Boehmer23,Reference Boehmer, Baker Ii, Brown, Wesolowski and Rozance33–Reference Gadhia, Maliszewski and O’Meara35} . 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 this breed of fetal sheep ^{Reference Green, Macko and Rozance36} . 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 infusions (IGF-1 or CON) were continued throughout the GSIS studies. All infusions were stopped after minute 105.

Biochemical analysis

Biochemical analyses were performed as previously described ^{Reference Culpepper, Wesolowski and Benjamin30,Reference Boehmer, Baker Ii, Brown, Wesolowski and Rozance33} . 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, USA). 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, USA; intra- and inter-assay coefficients of variation (CVs) = 5.6% and 4.7%, respectively; sensitivity = 0.14 ng·ml^-1; IGF-1: ALPCO; intra- and inter-assay CVs, 3.1% and 5.6%, respectively; sensitivity, 0.09 ng·ml^-1; cortisol: ALPCO; intra- and inter-assay CVs = 4.6% and 5.8%, respectively; sensitivity = 1.0 ng·ml^-1] and norepinephrine by high-performance liquid chromatography (HPLC; Model 2475, Waters, Milford, MA, USA; intra- and inter-assay CVs = 9.2% and 9.0%, respectively; sensitivity = 170 pg·ml^-1). Amino acid concentrations were measured using a Dionex 300 model 4500 amino acid analyzer (Thermo Fisher Scientific, Waltham, MA, USA) after deproteinization with sulfosalicyclic acid. Any results below the lower limit of detection were as assumed to be half of the lower limit of detection value. We were unable to directly measure circulating IGF-1 LR3 concentrations.

Fetal pancreatic islet isolation

Prior to necropsy, the above ten fetuses received a third and final IGF-1 or CON infusion at 138 ± 1 dGA, which was the same infusate as received for the second infusion, but this time without the GSIS study. The goal of this study was to test the impact of in vivo IGF-1 LR3 infusion on isolated pancreatic islet insulin secretion. One additional animal was included to receive IGF-1, and another was included to receive CON infusion at time of necropsy to increase the power to detect differences in islet insulin secretion. Neither of these animals received the prior GSIS studies. Necropsies were performed at the 90-min mark of the final experimental infusion while the infusion was still running. Pancreases were left in situ for perfusion with a collagenase solution and islet isolation ^{Reference White, Stremming and Boehmer23,Reference Limesand, Rozance, Zerbe, Hutton and Hay37} . Islet isolation and in vitro insulin secretion studies were performed as previously described (IGF-1, n = 6; CON, n = 6) ^{Reference White, Stremming and Boehmer23,Reference Limesand, Rozance, Zerbe, Hutton and Hay37,Reference Benjamin, Culpepper and Brown38} . Islets were isolated from the fetal pancreas after collagenase perfusion and digestion and then purified. They were then cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Waltham, MA) containing 2.8 mmol·L^-1 glucose supplemented with 1% fetal bovine serum and 1X Penicillin-Streptomycin-Amphotericin B solution (Sigma-Aldrich, St. Louis, MO) to provide adequate nutrients and prevent antimicrobial growth. They were then incubated overnight at 37 °C in 95% O₂ and 5% CO₂ to allow recovery from the isolation process ^{Reference White, Stremming and Boehmer23} .

Static islet incubation

Islet insulin secretion was assayed in static incubations of 10 islets ^{Reference White, Stremming and Boehmer23,Reference Benjamin, Culpepper and Brown38} . Each in vitro incubation condition for each animal was tested in four replicates. After overnight incubation, the islets were washed twice in Krebs-Ringer buffer (KRB) with 0.5% BSA (wt:vol) media and once in KRB-BSA media supplemented with 10 μmol·L^-1 forskolin (Sigma-Aldrich). Forskolin was added to the incubation media to activate adenylate cyclase and augment insulin release. Following this step, 10 islets were handpicked and placed into a 1.7 mL tube, and static incubations were performed for 30 min in KRB-BSA-forskolin media to allow for equilibration. An aliquot of this media was removed after 30 min to determine basal insulin secretion in the absence of glucose or potassium chloride (KCl) test media. Supplemental glucose or KCl was then added to each tube to reach final supplement concentrations of 1.1, 2.7, or 11 mmol·L⁻¹ glucose or 30 mmol·L⁻¹ KCl. Islets were incubated in these conditions at 37 °C for 1 hour and then placed on ice and pelleted at 2400 × g for 5 min at 4 °C. Medium was then aspirated and frozen for eventual determination of insulin concentration. The islet pellet was frozen at −80 °C until insulin was acid-ethanol extracted with 1 mol·L⁻¹ HCl in 70% ethanol at −20 °C for 24 h. Cellular debris was removed by centrifugation (30 min at 15,000 × g) and the supernatant was saved for determination of insulin concentration. Insulin concentrations were measured by ELISA (ALPCO, Salem, NH) on dilutions of the basal media, test media, and acid-ethanol extract to determine insulin released (test media minus basal media) and total islet insulin content (test media plus islet extract). Insulin secretion was calculated as the fraction of the total islet insulin content released in response to glucose or KCl stimulation ^{Reference White, Stremming and Boehmer23,Reference Rozance, Limesand and Hay24} . All KRB media used was equilibrated to 37 °C and 95% O₂/5% CO₂.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 9 (San Diego, CA). Results were analyzed in a mixed models ANOVA with terms to account for repeated measures within the same fetus and based on infusion received. Time, experimental infusion (IGF-1 or CON), 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. For in vitro insulin secretion, the terms in the mixed models ANOVA were incubation condition (1.1, 2.7, or 11 mmol·L⁻¹ glucose or 30 mmol·L⁻¹ KCl), in vivo experimental infusion (IGF-1 or CON), and their interaction. Measurements made once to compare IGF-1 and CON infusions were analyzed by Student’s t-test. Results are expressed as means ± SEM. P values of ≤ 0.05 were accepted as significant. One KCl test media static islet incubation replicate out of four total from an animal that received IGF-1 infusion at time of necropsy was deemed an outlier based on a physiologically improbable value (80% secretion) and excluded from analysis; all other data points were included in the analyses.