Maternofetal inflammation induced for 2 wk in late gestation reduced birth weight and impaired neonatal growth and skeletal muscle glucose metabolism in lambs

Abstract

Intrauterine stress impairs growth and metabolism in the fetus and offspring. We recently found that sustained maternofetal inflammation resulted in intrauterine growth-restricted (MI-IUGR) fetuses with asymmetric body composition, impaired muscle glucose metabolism, and β-cell dysfunction near term. These fetuses also exhibited heightened inflammatory tone, which we postulated was a fetal programming mechanism for the IUGR phenotype. Thus, the objective of this study was to determine whether poor growth and metabolism persisted in MI-IUGR lambs after birth. Polypay ewes received serial lipopolysaccharide or saline injections in the first 2 wk of the third trimester of pregnancy to produce MI-IUGR (n = 13) and control (n = 12) lambs, respectively. Lambs were catheterized at 25 d of age. β-Cell function was assessed at 29 d, hindlimb glucose metabolism at 30 d, and daily blood parameters from day 26 to 31. Glucose metabolism was also assessed in flexor digitorum superficialis (FDS) muscle isolated at necropsy on day 31. Asymmetric body composition persisted in MI-IUGR neonates, as these lambs were lighter (P < 0.05) than controls at birth and 31 d, but body and cannon bone lengths did not differ at either age. FDS muscles from MI-IUGR lambs were smaller (P < 0.05) and exhibited reduced (P < 0.05) glucose oxidation and Akt phosphorylation but similar glucose uptake compared with controls when incubated in basal or insulin-spiked media. Similarly, hindlimb glucose oxidation was reduced (P < 0.05) in MI-IUGR lambs under basal and hyperinsulinemic conditions, but hindlimb glucose utilization did not differ from controls. Circulating urea nitrogen and cholesterol were reduced (P < 0.05), and triglycerides, high-density lipoprotein cholesterol, and glucose-to-insulin ratios were increased (P < 0.05) in MI-IUGR lambs. Glucose and insulin concentrations did not differ between groups during basal or hyperglycemic conditions. Although circulating monocyte and granulocyte concentrations were greater (P < 0.05) in MI-IUGR lambs, plasma tumor necrosis factor α (TNFα) was reduced (P < 0.05). FDS muscle contained greater (P < 0.05) TNF receptor 1 (TNFR1) and IκBα protein content. These findings indicate that maternofetal inflammation in late pregnancy results in fetal programming that impairs growth capacity, muscle glucose oxidation, and lipid homeostasis in offspring. Inflammatory indicators measured in this study appear to reflect heightened cytokine sensitivity in muscle and compensatory systemic responses to it.

Introduction

Intrauterine growth restriction (IUGR) leads to lifelong deficits in growth, body composition, and metabolic function in offspring (Yates et al., 2018). These pathologies increase the risk for metabolic disorders in humans (Hales and Barker, 2013) and reduce growth efficiency and carcass merit in livestock (Greenwood et al., 2005). Studies in sheep have demonstrated that chronic intrauterine stress results in fetal programming that impairs skeletal muscle growth, glucose oxidative metabolism, pancreatic β-cell function, and insulin sensitivity (Brown et al., 2015; Camacho et al., 2017; Yates et al., 2019). The mechanisms for these pathologies are poorly understood, but we recently found evidence of enhanced inflammatory cytokine signaling in skeletal muscle from maternofetal inflammation-induced IUGR (MI-IUGR) fetuses (Cadaret et al., 2019a, 2019b). These changes were observed well after the induction of inflammation was discontinued (9 d in fetal rats and 13 d in fetal sheep) and coincided with impaired muscle growth and glucose metabolism. Similar evidence of enhanced cytokine signaling was observed in muscle and myoblasts from IUGR fetuses produced by hyperthermia-induced placental insufficiency (i.e., hyperthermic PI-IUGR; Posont et al., 2018). Skeletal muscle growth is highly responsive to cytokine signaling (Spangenburg and Booth, 2003; Al-Shanti et al., 2008; Muñoz-Cánoves et al., 2013), and we previously reported that cytokines affect skeletal muscle insulin signaling and glucose metabolism (Cadaret et al., 2017). TNFα is a particularly potent regulator of skeletal muscle via its ubiquitous receptor, TNFR1 (Webster et al., 2020), and heightened TNFα activity has been linked to muscle wasting disorders (Akash et al., 2018). Moreover, chronic inflammation and stress are implicated factors in a number of metabolic disease pathologies (Cohen et al., 2012; Turner et al., 2014; Liu et al., 2017). Coincidentally, advances in technologies such as proteomics have begun to allow for the rapid assessment of cytokines and other protein biomarkers to support more traditional diagnostic techniques and better inform therapeutic approaches (Demkow, 2010). However, these advances will require an accurate characterization of the expression profiles of such proteins. Thus, we postulated that programmed changes in cytokine responsiveness are a key mechanism underlying poor muscle growth and glucose metabolism in IUGR fetuses and offspring. The first step in elucidating their mechanistic role was to determine whether they manifest as programmed changes. Based on this, our objective for this study was to determine if the growth and metabolic deficits observed in near-term MI-IUGR fetuses persisted in MI-IUGR neonates due to the inflammatory programming of skeletal muscle.

Materials and Methods

Animals and experimental design

This study was approved by the Institutional Animal Care and Use Committee at the University of Nebraska–Lincoln, which is accredited by AAALAC International. Twenty commercial Polypay ewes purchased from a single source were timed-mated to a single sire at 24 to 36 mo of age. Ewes were housed in individual pens and nutritional management was practiced as previously described (Cadaret et al., 2019a). Ewes were assigned via simple randomization to produce control (n = 12 lambs from 10 different ewes, 5 singleton-born and 7 twin-born, 5 males and 7 females) or MI-IUGR (n = 13 lambs from 10 different ewes, 7 singleton-born and 6 twin-born, 8 males and 5 females) lambs. To produce MI-IUGR lambs, ewes were injected with bacterial lipopolysaccharide (LPS) endotoxin (100 ng/kg body weight in 0.6 mL saline; Escherichia coli O55:B5; Sigma-Aldrich, St. Louis, MO) IV every 72 h from 100 to 112 d of gestational age. Control lambs were produced from ewes that were injected with saline carrier and pair fed to the average of the MI-IUGR group. Maternal blood samples were collected at 0, 3, 6, 12, 24, and 48 h after each injection, and differential white blood cell concentrations were determined. Lambs were weaned at birth, fed pooled colostrum for the first 36 h, and then reared on commercial milk replacer (Milk Specialities Co., Dundee, IL) offered ad libitum as previously described (Yates et al., 2019). Body metrics were measured at birth and again at 30 d of age. Surgical hindlimb catheterizations were performed at 25 ± 1 d of age, square-wave hyperglycemic clamps were performed at 29 ± 1 d of age, and hyperinsulinemic–euglycemic clamps (HEC) were performed at 30 ± 1 d of age. Arterial blood samples were collected daily from day 26 to 31. Lambs were euthanized via barbiturate overdose at 31 ± 1 d of age to determine organ masses and to collect primary skeletal muscle for ex vivo metabolic studies.

Surgical preparation

Surgical hindlimb preparations were performed as previously described (Cadaret et al., 2019a; Yates et al., 2019), with some modifications. Briefly, lambs were induced with ketamine, intubated, and maintained under anesthesia with isoflurane gas. Indwelling Tygon catheters (US Plastics, Lima, OH) filled with heparinized saline were placed in the descending aorta for arterial blood sampling and inferior vena cava for infusions via the femoral artery and vein, respectively. A catheter was also placed in the contralateral external iliac vein via the distal femoral vein for venous blood sampling. The deep circumflex iliac artery and vein of this hindlimb were ligated and severed, and a Precision S-series Flow Probe (Transonic Systems, Inc., Ithaca, NY) was placed around the external iliac artery. Catheters and flow probe cables were tunneled subcutaneously to the flank, exteriorized, and kept in a plastic mesh pouch sutured to the skin. At surgery, lambs received 6,600 U/kg penicillin G procaine, 2.2 mg/kg ketofen, 10 mg/kg phenylbutazone, and 3 mg/kg atropine. Postoperative phenylbutazone was continued for at least 48 h after surgery, and catheters were flushed daily with heparinized saline.

Glucose-stimulated insulin secretion

Glucose-stimulated insulin secretion (GSIS) was estimated from circulating insulin concentrations under basal and steady-state hyperglycemic periods of a square-wave hyperglycemic clamp as previously described (Yates et al., 2012, 2019). Briefly, unfasted lambs were placed in Panepinto slings with access to milk. Basal blood glucose and plasma insulin concentrations were determined from the average of three arterial blood samples collected in 5-min intervals at the lamb’s natural resting (i.e., basal) glycemic conditions. Hyperglycemia (2 × basal blood glucose concentration) was then initiated with an intravenous bolus and variable-rate infusion of 33% dextrose solution. Beginning 20 min after steady-state hyperglycemia was achieved, three additional blood samples were collected at 5-min intervals to determine second-phase plasma insulin concentrations. Arterial catheters failed in 1 MI-IUGR lamb prior to the day of study, and thus GSIS was measured in 12 controls and 12 MI-IUGR lambs. Studies were performed simultaneously on pairs of lambs to avoid isolation.

Hindlimb glucose metabolism

Hindlimb-specific glucose metabolic rates were determined under basal and hyperinsulinemic conditions during a HEC as previously described (Cadaret et al., 2019a; Yates et al., 2019) with some modifications. Unfasted lambs were placed in Panepinto slings, bolused, and infused with [¹⁴C(U)]-d-glucose (37.2 μCi/mL; PerkinElmer, Boston, MA) at a constant rate of 2 mL/h. After 40 min, four sets of simultaneously drawn arterial and venous blood samples were collected at 10-min intervals. To induce hyperinsulinemia, lambs were bolused and infused with insulin (250 mU/kg; HumulinR; Lilly; Indianapolis, IN) at a constant rate of 4 mU/min/kg. Steady-state euglycemia (i.e., basal blood glucose ± 10%) was maintained with a concurrent variable-rate infusion of 33% dextrose. Beginning 60 min after hyperinsulinemia and euglycemia were initiated, four additional sets of simultaneously drawn arterial and venous blood samples were collected at 10-min intervals. Hindlimb blood flow was estimated from blood flow rates measured in the exterior iliac artery. Catheters or blood flow probes failed in 1 control and 2 MI-IUGR lambs prior to the day of study, and thus hindlimb glucose metabolism was measured in 11 controls and 11 MI-IUGR lambs.

The Fick principle, which incorporates arteriovenous differences in substrate concentrations and arterial blood flow rates, was used to estimate hindlimb-specific metabolic fluxes for glucose (Camacho et al., 2017; Rozance et al., 2018; Cadaret et al., 2019a). Glucose utilization rates by the tissues of the hindlimb were estimated from the differences in glucose concentrations between each set of simultaneously collected arterial and venous samples, normalized to the average blood flow rate, during the study period and hindlimb mass. Glucose oxidation rates were estimated from the arteriovenous difference in radiolabeled CO₂, which was determined from three technical replicates of each arterial and venous sample. Briefly, whole blood aliquots were added to micro-centrifuge tubes containing 2M HCl and suspended over a pool of 1M NaOH inside sealed scintillation vials. Acidification of the blood facilitated the release of CO₂, which was subsequently captured by the NaOH at the bottom of the scintillation vial. After 24-h incubation at room temperature, the centrifuge tube was removed, UltimaGold scintillation fluid (PerkinElmer, Inc., Waltham, MA) was added to the scintillation vial, and captured ¹⁴CO₂ was determined with a Beckman-Coulter 1900 TA LC counter (Beckman-Coulter, Fullerton, CA). The difference between venous and arterial ¹⁴C-specific activities, which represents the amount of CO₂ produced from radiolabeled glucose, was normalized to the ¹⁴C-specific activity of the radiolabeled glucose infusate to determine nmol of glucose oxidized. This was then normalized to blood flow rate and hindlimb mass.

Blood sample analyses

Blood samples were collected from ewes via jugular venipuncture and from lambs via catheters into EDTA-treated and heparinized syringes. The total blood volume collected did not exceed 20 mL in any 24-h period. Hematology was performed on EDTA-treated blood using a HemaTrue Veterinary Hematology Analyzer (Heska, Loveland, CO) with ovine software specifications as previously described (Barnes et al., 2019; Swanson et al., 2020) to determine whole-blood concentrations of total white blood cells, lymphocytes, monocytes, granulocytes, hematocrit, hemoglobin, red blood cells, and platelets, as well as mean red blood cell volume, mean corpuscular hemoglobin concentration, red blood cell distribution width, and mean platelet volume. An ABL90 FLEX blood gas analyzer (Radiometer, Brea, CA) was used to determine whole blood concentrations of glucose, lactate, and HCO₃ as well as blood pH, base excess of HCO₃, partial pressures of O₂ (pO₂) and CO₂ (pCO₂), and fractions of oxyhemoglobin and carboxyhemoglobin in heparinized blood as previously described (Cadaret et al., 2019a). After blood plasma was isolated by centrifugation (14,000 × g for 5 min) and stored at −80 °C, commercial enzyme-linked immunoassay (ELISA) kits were used to determine circulating concentrations of insulin (Ovine Insulin, Alpco Diagnostics, Windham, NH) and TNFα (Wuhan Fine Biotech Co., Ltd., Wuhan, China) from duplicate aliquots as previously described (Cadaret et al., 2019a; Swanson et al., 2020). Intra-assay and inter-assay coefficients of variance were less than 10%. Plasma concentrations of urea nitrogen, cholesterol, high-density lipoprotein cholesterol (HDLC), and triglycerides were determined using a Vitros-250 Chemistry Analyzer (Ortho Clinical Diagnostics, Linden, NJ) by the University of Nebraska Biomedical and Obesity Research Core as previously described (Swanson et al., 2020).

Skeletal muscle metabolic responsiveness

The flexor digitorum superficialis (FDS) muscle from each hindlimb was isolated at necropsy and separated into longitudinal strips (873 ± 18 mg) to determine ex vivo glucose uptake and oxidation rates as previously described (Cadaret et al., 2017, 2019a). Briefly, muscle strips were washed in ice-cold phosphate-buffered saline and preincubated for 1 h at 37 °C in Krebs–Henseleit bicarbonate buffer (KHB) containing 0.1% bovine serum albumin, 5 mM d-glucose (Millipore Sigma), and either no additive (basal), 5 mU/mL insulin, or 20 ng/mL TNFα (Sigma-Aldrich, St. Louis, MO). Muscle strips were then washed in glucose-free KHB with the respective additive for 20 min at 37 °C. All KHB media were O₂-saturated by aerating with 95% O₂, 5% CO₂ for 1 min just before use. Glucose uptake was estimated by incubating muscle strips with radiolabeled 2-deoxyglucose, which is taken up by muscle but not metabolized, and then determining its intracellular accumulation. After preincubation and wash steps, these muscle strips were incubated at 37 °C in KHB containing the respective additive as well as 1 mM [³H]2-deoxyglucose (300 µCi/mmol; PerkinElmer) and 39 mM [1-¹⁴C] mannitol (1.25 µCi/mmol; PerkinElmer). After 20 min, they were removed from the media, washed and weighed, and then lysed in 2M NaOH (Sigma-Aldrich) at 37 °C. UltimaGold scintillation fluid was added to vortexed lysates, and specific activities for ³H and ¹⁴C were determined by liquid scintillation. Specific activities for ³H and ¹⁴C were also determined from triplicate 10-µL aliquots of media. Mannitol concentrations in the lysates were used to estimate extracellular fluid volume, as it is not taken up by muscle tissues. Intracellular accumulation of 2-deoxyglucose in mmol was determined by normalizing to the specific activity of the media. Glucose oxidation was estimated by incubating muscle strips with radiolabeled d-glucose. These muscle strips were individually placed on one side of a sealed dual-well chamber and incubated for 2 h at 37 °C in KHB containing the respective additive along with 5 mM [¹⁴C-U]-d-glucose (0.25 µCi/mmol). The adjacent well contained 2M NaOH to capture the CO₂ produced by the muscle strip. Following the incubation, chambers were cooled at −20 °C for 2 min. The media was acidified by injecting 2M HCl through a rubber seal, which caused the release of the media-bound CO₂. Chambers were incubated for an additional 1 h at 4 °C, and muscle strips were then removed, dried, and weighed. The NaOH from the chamber was mixed with UltimaGold scintillation fluid, and the specific activity of ¹⁴CO₂ was determined via liquid scintillation. Glucose oxidation in pmol was calculated by normalizing to the specific activity of the media.

Protein immunoblot

Protein concentrations were determined in total protein isolates from FDS muscle strips incubated in basal, insulin-spiked, and/or TNFα-spiked KHB media for 20 min at 37 °C as previously described (Barnes et al., 2019; Cadaret et al., 2019a). After incubation, muscle strips were dried and weighed before being snap-frozen and homogenized in RIPA buffer containing manufacturer-recommended concentrations of protease and phosphatase inhibitors (Thermo Fisher). Each homogenate was sonicated for 15 s and centrifuged (14,000 × g, 5 min, 4 °C). Total supernatant protein concentration was measured with a Pierce BCA Protein Assay Kit (Thermo Fisher). About 30 μg of protein was combined with BioRad 4x Laemmli Sample Buffer to make a 1x solution. This solution was then heated for 5 min at 95 °C and separated by SDS-PAGE. The gels were transferred to poly-vinylidene fluoride low-fluorescent membranes (BioRad Laboratories, Hercules, CA) and incubated in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature before being washed with 1x TBS-T (20 mM Tris-HCl + 150 mM NaCl + 0.01% Tween 20). Membranes were incubated with rabbit antiserum raised against Akt (1:1,000; Cell Signaling Technologies, Danvers, MA), phosphorylated Akt (Ser⁴⁷³; 1:2,000; Cell Signaling), IκBα (1:200; Santa Cruz, Dallas, TX), or TNFR1 (1:1,000; Cell Signaling) diluted in Odyssey blocking buffer at room temperature for 1 h (Akt) or 2 h (IκBα), or at 4 °C overnight (phosphorylated Akt and TNFR1). Membranes were then incubated with goat anti-rabbit IR800 IgG secondary antiserum (LI-COR Biosciences) diluted in Odyssey blocking buffer at room temperature for 1 h. Blots were scanned with the Odyssey Infrared Imaging System and analyzed with Image Studio Lite Software Ver 5.2 (LI-COR Biosciences).

Statistical analysis

All data were analyzed using SAS 9.4 (SAS Institute, Cary, NC). Hematology and blood gas/oximetry variables were analyzed by analysis of variance (ANOVA) using the mixed procedure with repeated measures to analyze the effects of the experimental group, day (or time from initial LPS injection for maternal samples), and the interaction. Appropriate covariance structures were selected based on best-fit statistics. Sex of the lamb was included as a random variable but not as a main effect due to insufficient power as previously described (Cadaret et al., 2019a; Yates et al., 2019). Data from in vivo and ex vivo metabolic studies were likewise analyzed using the mixed procedure, with study period and incubation condition as the respective repeated measures. Where necessary, means were separated with Fisher’s LSD test. The four samples collected within each period of the in vivo metabolic studies were averaged and the mean is reported. Similarly, the four technical replications for each incubation condition of the ex vivo metabolic studies were averaged. Biometric data were analyzed by one-way ANOVA. Ewe was considered the experimental unit for maternal outputs. Lambs were individually reared and studied and thus were considered the experimental unit for offspring outputs. The threshold for significance was a P-value of ≤ 0.05, and P-values of ≤ 0.10 were considered tendencies. All data are presented as the mean ± SE.

Results

Maternal blood parameters during serial LPS injections

Experimental group × hour (from initial LPS injection) interactions were observed (P < 0.05) for maternal concentrations of total white blood cells, lymphocytes, monocytes, granulocytes, red blood cells, hematocrit, and hemoglobin but not for the other maternal blood components. Maternal total white blood cell concentrations were reduced (P < 0.05) in LPS-injected compared with saline-injected ewes at 3 and 6 h after each injection but elevated (P < 0.05) at 24 and 48 h after the first injection, at 24 h after the second injection, and at 12 and 24 h after the third, fourth, and fifth injections (Supplementary Figure 1A). Maternal lymphocyte concentrations were reduced (P < 0.05) in LPS-injected compared with saline-injected ewes at 3 and 6 h after every injection, at 12 h after the first injection, and at 12 and 48 h after the second injection (Supplementary Figure 1B). Maternal monocyte concentrations were reduced (P < 0.05) in LPS-injected ewes at 3 and 6 h after each injection but were elevated (P < 0.05) at 24 and 48 h after the first injection, at 48 h after the second injection, at 12 and 24 h after the third and fourth injections, and at 12, 24, and 48 h after the fifth injection (Supplementary Figure 1C). Maternal granulocyte concentrations were reduced (P < 0.05) in LPS-injected ewes at 3 h after the first, second, and fifth injections and were elevated (P < 0.05) at 48 h after the first injection and at 12 and 24 h after all injections (Supplementary Figure 1D). Maternal platelet concentrations did not differ between saline-injected and LPS-injected ewes (Supplementary Figure 1E). Red blood cell concentrations were greater (P < 0.05) in LPS-injected ewes at 3 and 6 h after every injection and were also greater (P < 0.05) at 24 and 48 after the second injection, at 12 and 48 h after the third injection, and at 24 h after the fourth injection (Supplementary Figure 1F). Hematocrit was greater (P < 0.05) in LPS-injected ewes at 3 and 6 h after every injection except the first, where it was only greater (P < 0.05) at 6 h (Supplementary Figure 1G). Hematocrit was also greater (P < 0.05) at 48 h after the third injection. Hemoglobin concentrations were greater (P < 0.05) in LPS-injected ewes at 3 and 6 h after every injection and were also greater (P < 0.05) at 24 h after the second injection, at 12 and 48 h after the third injection, and at 48 h after the fourth injection (Supplementary Figure 1H). Maternal values did not differ between saline-injected and LPS-injected ewes for mean red blood cell volume (34.9 ± 0.5 vs. 33.9 ± 0.6 fL, respectively), red blood cell distribution width (21.6 ± 0.2 vs. 21.7 ± 0.3 %, respectively), mean corpuscular hemoglobin concentration (36.8 ± 0.7 vs. 36.9 ± 0.3 %, respectively), and mean platelet volume (5.3 ± 0.1 vs. 5.3 ± 0.1 fL, respectively).

Growth and biometry of lambs

Body mass at birth, body mass at 30 d of age, and average daily gain were all less (P < 0.05) for MI-IUGR lambs than for controls (Table 1). At birth, crown circumference tended to be less (P = 0.08) for MI-IUGR lambs than for controls, but body length, abdominal circumference, and hindlimb cannon bone length did not differ between experimental groups. At 30 d of age, body length, crown circumference, abdominal circumference, and cannon bone length did not differ between experimental groups. The fractional change in body mass between birth and 30 d of age was less (P < 0.05) for MI-IUGR lambs than for controls, but fractional changes in body length, crown circumference, abdominal circumference, and cannon bone length did not differ between experimental groups.

Table 1.

Biometry of MI-IUGR lambs at birth and 30 d of age¹

Variable	Control	MI-IUGR	P-value
n	12	13
Sex ratio, M:F	5:7	8:5
Birth
Body mass, kg	4.42 ± 0.15	3.61 ± 0.27	0.01
Body length, cm	61.17 ± 0.91	59.42 ± 1.73	ND3
Crown circumference, cm	29.17 ± 0.72	27.42 ± 0.64	0.08
Abdominal circumference, cm	39.01 ± 1.01	37.64 ± 1.58	ND
Cannon bone length, cm	17.12 ± 0.30	17.04 ± 0.45	ND
30 d of age
Body mass, kg	14.01 ± 0.37	11.58 ± 0.73	<0.01
Body length, cm	83.21 ± 0.96	79.76 ± 1.68	ND
Crown circumference, cm	35.38 ± 0.83	34.39 ± 0.46	ND
Abdominal circumference, cm	58.33 ± 1.56	56.64 ± 0.78	ND
Cannon bone length, cm	34.78 ± 0.69	34.33 ± 0.47	ND
Average daily gain, kg/d	0.32 ± 0.01	0.24 ± 0.02	<0.01
Fractional ∆  2, Birth to 30 d
Body mass	3.18 ± 0.09	2.79 ± 0.14	0.02
Body length	1.36 ± 0.03	1.35 ± 0.03	ND
Crown circumference	1.23 ± 0.02	1.25 ± 0.03	ND
Abdominal circumference	1.50 ± 0.03	1.53 ± 0.06	ND
Cannon bone length	1.28 ± 0.02	1.24 ± 0.03	ND

¹Values are expressed as means ± SE.

²Fractional change (∆), value at 30 d/value at birth.

³ND, not different (i.e., P > 0.10).

At necropsy, the combined mass of both FDS muscles was less (P < 0.05) in MI-IUGR lambs than in controls (Table 2). Heart mass, lung mass, and kidney mass were less (P < 0.05) and liver mass tended to be less (P = 0.08) for MI-IUGR lambs than for controls, but brain mass did not differ between experimental groups. Liver-to-body mass and brain-to-body mass ratios were greater (P < 0.05) for MI-IUGR lambs than for controls, but heart-to-body mass, lung-to-body mass, and kidney-to-body mass ratios did not differ between experimental groups.

Table 2.

Organ mass in MI-IUGR lambs at 30 d of age¹

Variable	Control	MI-IUGR	P-value
Organ mass, g
Heart	132 ± 7	101 ± 7	0.01
Lungs	274 ± 12	236 ± 13	0.02
Liver	434 ± 16	396 ± 19	0.08
Kidneys	99 ± 5	83 ± 4	0.02
Brain	80 ± 1	82 ± 1	ND2
FDS	26.2 ± 1.7	30.2 ± 1.7	0.02
Organ mass/body mass, g/kg
Heart	9.90 ± 0.50	9.09 ± 0.51	ND
Lungs	19.41 ± 0.72	20.02 ± 0.66	ND
Liver	29.70 ± 0.93	33.06 ± 1.44	0.03
Kidneys	6.79 ± 0.32	6.78 ± 0.23	ND
Brain	5.88 ± 0.15	6.89 ± 0.36	0.01
FDS	2.33 ± 0.03	2.24 ± 0.12	ND

¹Values are expressed as means ± SE.

²ND, not different (i.e., P > 0.10).

Daily blood parameters in lambs

Experimental group × day interactions were observed (P < 0.05) for pCO₂, HCO₃, and plasma triglyceride concentrations and tended to be observed (P < 0.07) for pO₂, oxyhemoglobin, and base excess HCO₃ but were not observed for any other blood parameter. Plasma TNFα concentrations were less (P < 0.05) in MI-IUGR lambs (110 ± 26 ng/mL) than in controls (257 ± 38 ng/mL), regardless of the day. Circulating concentrations of total white blood cells (Figure 1A) and lymphocytes (Figure 1B) did not differ between experimental groups, but concentrations of monocytes (Figure 1C), granulocytes (Figure 1D), and platelets (Figure 1E) tended to be greater (P < 0.09) for MI-IUGR lambs than for controls, regardless of the day. Red blood cell concentrations did not differ between experimental groups (Figure 1F). Blood pO₂ tended to be greater (P = 0.07) for MI-IUGR lambs than for controls on day 26 but tended to be less (P = 0.07) on days 28 and 30 (Figure 2A). Blood pCO₂ was greater (P < 0.05) for MI-IUGR lambs on days 26 and 31 (Figure 2B). Oxyhemoglobin (i.e., the fraction of hemoglobin bound to O₂) was less (P < 0.05) for MI-IUGR lambs on day 28 and tended to be less (P = 0.08) on day 31 (Figure 2C). Carboxyhemoglobin (i.e., the fraction of hemoglobin bound to CO) tended to be less (P = 0.09) in MI-IUGR lambs, regardless of the day (Figure 2D). Blood HCO₃ concentrations were greater (P < 0.05) for MI-IUGR lambs on days 26 and 31 but less (P < 0.05) on days 27, 28, and 29 (Figure 2E). Blood pH did not differ between experimental groups (Figure 2F) nor did hematocrit (Supplementary Figure 2A), mean red blood cell volume (Supplementary Figure 2B), red blood cell distribution width (Supplementary Figure 2C), hemoglobin concentration (Supplementary Figure 2D), mean corpuscular hemoglobin concentration (Supplementary Figure 2E), blood glucose concentration (Supplementary Figure 2F), blood lactate concentration (Supplementary Figure 2G), or mean platelet volume (5.08 vs. 5.03 fL, respectively). Base excess HCO₃ was less (P < 0.05) for MI-IUGR lambs than controls on day 28 but greater (P < 0.05) on day 31 (Supplementary Figure 2H). Blood plasma urea nitrogen (Figure 3A) and cholesterol (Figure 3B) concentrations were less (P ≤ 0.05) in MI-IUGR lambs than controls, regardless of the day. Blood plasma triglyceride concentrations were greater (P < 0.05) in MI-IUGR lambs than controls on days 30 and 31 (Figure 3C). Blood plasma HDLC concentrations were greater (P < 0.05) in MI-IUGR lambs than controls, regardless of the day (Figure 3D).

Figure 1.

Circulating blood cells in MI-IUGR neonatal lambs. Arterial blood samples from control (n = 12) and MI-IUGR lambs (n = 13) were collected daily from day 26 to 31 of age. Data are shown for whole-blood concentrations of total white blood cells (A), lymphocytes (B), monocytes (C), granulocytes (D), platelets (E), and red blood cells (F). Effects of the experimental group (GRP), day (DAY), and the interaction (G * D) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10).

Figure 2.

Blood gases and pH in MI-IUGR neonatal lambs. Arterial blood samples from control (n = 12) and MI-IUGR lambs (n = 13) were collected daily from day 26 to 31 of age. Data are shown for whole-blood partial pressure of O₂ (A), partial pressure of CO₂ (B), hemoglobin-bound O₂ (C), hemoglobin-bound CO₂ (D), HCO₃ concentrations (E), and pH (F). Effects of the experimental group (GRP), day, and the interaction (G * D) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10). *Means within the same day differ (P < 0.05). ^#Means within the same day tend to differ (P < 0.10).

Figure 3.

Blood urea nitrogen and lipid concentrations in MI-IUGR neonatal lambs. Plasma separated from arterial blood samples from control (n = 12) and MI-IUGR lambs (n = 13) was collected daily from day 26 to 31 of age. Data are shown for concentrations of urea nitrogen (A), total cholesterol (B), total triglycerides (C), and HDLC (D). Effects of the experimental group (GRP), day, and the interaction (G * D) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10). *Means within the same day differ (P < 0.05).

In vivo metabolism in lambs

Glucose-stimulated insulin secretion

Blood glucose concentrations did not differ between groups during the basal period (i.e., under resting conditions) of the GSIS study (Figure 4A). By design, blood glucose concentrations did not differ between groups during the square-wave hyperglycemic clamp and were increased (P < 0.05) from basal by an average of 2.3-fold in all lambs. Plasma insulin concentrations did not differ between groups under basal or hyperglycemic conditions (Figure 4B). Likewise, values did not differ between controls and MI-IUGR lambs for GSIS (i.e., the difference in insulin concentrations between basal and hyperglycemic periods) and glucose-to-insulin ratios under basal (3.5 ± 0.7 vs. 4.2 ± 0.8, respectively) and hyperglycemic (1.2 ± 0.3 vs. 1.0 ± 0.2, respectively) conditions.

Figure 4.

GSIS in MI-IUGR neonatal lambs. Arterial blood samples were collected from control (n = 12) and MI-IUGR lambs (n = 12) during basal (i.e., resting) glycemia and during steady-state hyperglycemia (2.5 × basal). Data are shown for blood glucose concentrations (A) and plasma insulin concentrations (B). ∆, the difference in insulin concentrations between basal conditions and hyperglycemia. Effects of the experimental group (GRP), period, and the interaction (G * P) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10).

Hindlimb-specific glucose metabolism

Hindlimb glucose utilization rates did not differ between controls and MI-IUGR lambs under basal or hyperinsulinemic conditions (Figure 5A), and insulin-stimulated glucose utilization (i.e., the difference in glucose utilization rates between basal and hyperinsulinemic periods) likewise did not differ. Hindlimb glucose oxidation rates were less (P < 0.05) for MI-IUGR lambs than for controls under both basal and hyperinsulinemic conditions (Figure 5B), and insulin-stimulated glucose oxidation (i.e., the difference in glucose oxidation rates between basal and hyperinsulinemic periods) was likewise reduced (P < 0.05) for MI-IUGR lambs.

Figure 5.

Hindlimb glucose metabolism in MI-IUGR neonatal lambs. Arterial and venous blood samples were collected simultaneously from control (n = 11) and MI-IUGR lambs (n = 11) during basal (i.e., resting) conditions and during a steady-state HEC. Data are shown for hindlimb-specific rates of glucose uptake (A) and glucose oxidation (B). ∆, the difference in rates between basal and HEC periods. Effects of the experimental group (GRP), period, and the interaction (G * P) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10).*Means within the same period differ (P < 0.05).

Blood parameters during hyperinsulinemia

Blood glucose (Supplementary Figure 3A) and plasma insulin concentrations (Supplementary Figure 3B) did not differ between experimental groups under basal or hyperinsulinemic conditions. Glucose-to-insulin ratios did not differ between groups under basal conditions but were greater (P < 0.05) for MI-IUGR lambs than for controls under hyperinsulinemic conditions (Supplementary Figure 3C). Insulin sensitivity for glucose utilization (i.e., glucose utilization rate/insulin concentration) did not differ between controls and MI-IUGR lambs under basal (7.6 ± 1.8 vs. 9.5 ± 2.7, respectively) or hyperinsulinemic (2.2 ± 0.3 vs. 3.2 ± 0.7, respectively) conditions. Insulin sensitivity for glucose oxidation (i.e., glucose oxidation rate/insulin concentration) was less (P < 0.05) for MI-IUGR lambs than for controls under basal (1.2 ± 0.6 vs. 2.8 ± 0.6, respectively) and hyperinsulinemic (0.9 ± 0.1 vs. 0.5 ± 0.1, respectively) conditions. Blood plasma urea nitrogen (Supplementary Figure 4A) and cholesterol (Supplementary Figure 4B) concentrations were less (P < 0.05) in MI-IUGR lambs than controls, regardless of period. Blood plasma triglyceride concentrations did not differ between groups or periods (Supplementary Figure 4C). Blood plasma HDLC was less (P < 0.05) in MI-IUGR lambs than controls, regardless of period (Supplementary Figure 4D).

Ex vivo skeletal muscle glucose metabolism and protein analyses

An experimental group × culture media interaction tended to be observed (P = 0.07) for skeletal muscle TNFR1 concentration but not for any other ex vivo measurements. Glucose uptake rates did not differ between muscle strips from control and MI-IUGR lambs but were greater (P < 0.05) in strips incubated in insulin-spiked media compared with those incubated in basal media, regardless of the experimental group (Figure 6A). Glucose oxidation rates were less (P < 0.05) for muscle strips from MI-IUGR lambs than for those from controls, regardless of media, and were greater (P < 0.05) for muscle strips incubated in insulin-spiked media than those incubated in basal media, regardless of the experimental group (Figure 6B). Representative gels for all immunoblots are depicted in Supplementary Figure 5. The proportion of phosphorylated-to-total Akt protein concentrations tended to be less (P = 0.07) for muscle strips from MI-IUGR lambs than for those from controls, regardless of media conditions (Figure 6D). Protein concentrations of IκBα were greater (P < 0.05) in muscle from MI-IUGR lambs than controls, regardless of culture conditions (Figure 7A). Protein concentrations of TNFR1 tended to be greater (P = 0.07) in muscle from MI-IUGR lambs than controls when incubated in basal media but did not differ between groups when incubated in TNFα-spiked media (Figure 7B).

Figure 6.

Ex vivo skeletal muscle glucose metabolism in MI-IUGR neonatal lambs. FDS muscle strips were collected from control (n = 12) and MI-IUGR lambs (n = 13) and incubated in basal and insulin-spiked (5 mU/mL) media. Data are shown for rates of muscle-specific glucose uptake (A), glucose oxidation (B), and Akt phosphorylation (C). Effects of the experimental group (GRP), media, and the interaction (G * M) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10).

Figure 7.

Skeletal muscle cytokine pathway components in MI-IUGR neonatal lambs. FDS muscle strips were collected from control (n = 12) and MI-IUGR lambs (n = 13) and incubated in basal and TNFα-spiked (20 pg/mL) media. Data are shown for muscle protein concentrations of IκBα (A) and TNFR1 (B). Effects of the experimental group (GRP), media, and the interaction (G * M) were evaluated and are noted where significant (P < 0.05) or tending toward significance (P < 0.10). ^#Means within the same day tend to differ (P < 0.10).

Discussion

In a previous study, we found that maternofetal inflammation during the early third trimester of gestation restricted subsequent fetal growth, impaired muscle glucose metabolism, and led to an IUGR phenotype near term (Cadaret et al., 2019a). In the present study, these deficits persisted in the neonate despite fewer indicators of systemic inflammation, which demonstrates the fetal programming effect of maternofetal inflammation on postnatal growth and metabolism. In addition to being smaller at birth, MI-IUGR lambs grew slower than controls, and morphometric assessments showed that both prenatal and neonatal growth was asymmetric. Similar to the fetal stage, MI-IUGR neonates were deficient in skeletal muscle glucose oxidation despite normal glucose uptake. Unlike in the fetal stage, however, these lambs exhibited normal GSIS, which indicated that their pancreatic islets were adequately functional after birth. Additionally, indicators of skeletal muscle insulin resistance were less profound in the MI-IUGR neonate than in the fetus, although evidence of whole-body insulin resistance was observed. Whole-body lipid homeostasis was also disrupted in MI-IUGR lambs, as circulating cholesterol levels were reduced but HDLC and triglyceride levels were increased. Growth and metabolic deficits coincided with changes in systemic and muscle-specific components of inflammatory regulation that appeared to represent a combination of enhanced inflammatory tone and compensatory responses to it. For example, skeletal muscle TNFR1 was increased, but circulating TNFα concentrations were reduced. These findings lead us to conclude that sustained maternofetal inflammation restricts neonatal growth capacity and impairs metabolic function by modifying developmental programming. Together, these changes yield a neonatal phenotype similar to that observed in hyperthermic PI-IUGR lambs and other IUGR animal models as well as IUGR humans (Yates et al., 2018, 2019; Beede et al., 2019).

Maternofetal inflammation reduced prenatal growth and impaired postnatal growth capacity. At birth, MI-IUGR lambs were about 19% lighter than controls, which is comparable to the reduction in mass that we observed in near-term MI-IUGR fetal sheep (Cadaret et al., 2019a) and rats (Cadaret et al., 2019b). This reduced birthweight in the presence of normal skeletal growth indicators such as body and cannon bone length reflects asymmetric fetal growth restriction, which is a hallmark of fetuses afflicted with placental insufficiency (Limesand and Rozance, 2017; Yates et al., 2018). Although placental function was not measured in the present study, fetal hypoxemia and greater maternofetal O₂ gradients in our previous study were indicative of placental insufficiency (Cadaret et al., 2019a). In addition to poor prenatal growth, postnatal morphometric assessments show that MI-IUGR lambs maintained their growth deficits and asymmetric body composition during the neonatal stage. Indeed, the fractional changes in body mass from birth to 1 mo of age were diminished, but fractional changes in skeletal measurements were not. The failure of these lambs to exhibit meaningful catch-up growth is noteworthy, as their 25% reduction in average daily gain resulted in 18% lighter body mass at 1 mo of age. Adipose-driven catch-up growth is a common characteristic of IUGR-born humans (Ong et al., 2000) and animals (De Almeida Silva et al., 2020). However, our previous studies in hyperthermic PI-IUGR lambs indicated that greater adiposity was not present in neonates and in fact did not occur until the juvenile stage (Cadaret et al., 2019c; Gibbs et al., 2020). Thus, reduced body mass together with normal structural growth in our MI-IUGR neonates demonstrates that asymmetric growth patterns programmed by maternofetal inflammation conditions were not concomitant with greater adiposity. In addition to preserving structural growth, MI-IUGR lambs exhibited preservation of brain and liver growth, as these organs were equal in mass and greater in fraction of body weight than for controls. Brain sparing was not surprising, as protection of neural growth and development has been shown to be a priority even in severe IUGR (Hunter et al., 2017). The presence of mild liver sparing and the absence of heart sparing were somewhat unexpected based on previous findings in IUGR fetal sheep (Limesand et al., 2006; Davis et al., 2015; Cadaret et al., 2019a), but neither was unprecedented (Leos et al., 2010; Yates et al., 2019). Although more in-depth analyses of body composition are warranted, reduced FDS muscle mass in MI-IUGR lambs is consistent with disproportional growth restriction of skeletal muscle, which was present in MI-IUGR fetal sheep and rats (Cadaret et al., 2019a, 2019b) and in hyperthermic PI-IUGR lambs (Gibbs et al., 2019, 2020).

Poor glucose oxidation persisted in MI-IUGR skeletal muscle after birth, presumably as a consequence of fetal programming aimed at metabolic thrift (Posont and Yates, 2019; Reynolds et al., 2019). Moreover, increased circulating glucose-to-insulin ratios in our MI-IUGR lambs were consistent with whole-body insulin resistance that typically characterizes IUGR-born offspring (Dunlop et al., 2015). Despite these findings, our direct evidence for muscle-specific insulin resistance at the neonatal stage was quite limited. Indeed, skeletal muscle glucose uptake and hindlimb insulin sensitivity for glucose utilization were normal in MI-IUGR lambs, and hindlimb glucose oxidation was diminished under both basal and hyperinsulinemic conditions. Moreover, ex vivo skeletal muscle glucose oxidation and Akt phosphorylation were diminished by similar magnitudes whether incubated in basal or insulin-spiked media, just as they were in MI-IUGR fetuses (Cadaret et al., 2019a). Previous studies in hyperthermic PI-IUGR fetuses (Limesand et al., 2007; Brown et al., 2015) and lambs (Yates et al., 2019) also showed that glucose oxidation deficits were not necessarily dependent upon impaired insulin signaling, even when considerable insulin resistance indicators were present.

Pancreatic β-cell dysfunction was not apparent in MI-IUGR neonates. In fact, normal insulin secretion and stimulus–secretion coupling indicate that the previously observed hypoinsulinemia in MI-IUGR fetuses (Cadaret et al., 2019a) was a transient response to the intrauterine conditions and not a product of developmental programming. The preservation of β-cell function was surprising, as impaired insulin secretion is a frequent outcome of IUGR (Gatford and Simmons, 2013; Boehmer et al., 2017) and was present in our hyperthermic PI-IUGR neonates (Cadaret et al., 2019c). However, there is growing evidence that developmental pathologies in IUGR islets are primarily associated with adrenergic adaptations (Limesand and Rozance, 2017). Although not measured in the present study, we found little evidence for adrenergic changes in MI-IUGR rats (Cadaret et al., 2019b). Furthermore, catecholamine-stimulating hypoxemia was mild in MI-IUGR fetuses compared with hyperthermic PI-IUGR fetuses (Macko et al., 2016; Cadaret et al., 2019a). Thus, the transient reduction of insulin secretion in our MI-IUGR model may have been associated with circulating TNFα concentration (Zhang and Kim, 1995) rather than adrenergic effects, as the former were elevated in the fetus but not in the neonate.

In earlier studies, we found that several indicators of systemic inflammation and muscle-specific cytokine sensitivity remained increased in MI-IUGR fetal sheep and rats well after the induction of inflammation was discontinued (Cadaret et al., 2019a, 2019b). We postulated that this enhanced inflammatory tone was a potential programming mechanism for growth and metabolic deficits associated with IUGR. In our present study, FDS muscle isolated from MI-IUGR lambs contained about 40% more of the ubiquitous TNFα receptor TNFR1. This along with greater circulating monocyte and granulocyte populations would be consistent with heightened sensitivity to inflammatory mediators. Surprisingly, however, circulating TNFα concentrations were consistently reduced in MI-IUGR lambs despite being elevated in the MI-IUGR fetus (Cadaret et al., 2019a). Although unexpected, it is perhaps reasonable to postulate that reduced circulating TNFα was a compensatory response to the apparent increase in inflammatory responsiveness, which was indicated by greater blood triglycerides, lower blood cholesterol, and increased glucose-to-insulin ratios in addition to the greater skeletal muscle TNFR1. The same may be true for the increase in skeletal muscle IκBα, which is the arrest protein for the inflammatory transcription factor NFκB.

From these findings, we can conclude that sustained maternofetal inflammation in late gestation results in tissue-specific fetal programming that alters neonatal growth rates, body composition, and metabolism. Like fetal growth, neonatal growth in MI-IUGR lambs was restricted in an asymmetrical fashion, which was at least partially due to disproportional reduction in skeletal muscle growth. Our findings show that muscle was also a key target for metabolic programming, which manifested in reduced glucose oxidation that appeared to be independent of insulin sensitivity. Systemic lipid homeostasis was also disrupted in MI-IUGR lambs, although this study did not address the contribution of skeletal muscle to this condition. Despite the subsidence of high circulating TNFα after birth, greater skeletal muscle TNFR1 was indicative of enhanced sensitivity to the inflammatory cytokine, which likely contributed to greater triglycerides, increased glucose-to-insulin ratios, and other parameters consistent with greater inflammatory tone. In fact, reduced circulating TNFα may be a potential clinical biomarker of this condition in afflicted neonates, especially in the context of muscle TNFR1 content and other parameters. Conversely, the transient nature of reduced insulin secretion and stimulus–secretion coupling indicates that maternofetal inflammation conditions did not induce programmed changes in pancreatic β-cell function observed in hyperthermic PI-IUGR lambs. This finding was surprising and underscores the importance of examining IUGR programming in a tissue-specific fashion. Nevertheless, the MI-IUGR lambs in this study exhibited growth and metabolic phenotypes consistent with the general phenotype of IUGR-born humans and animals, which supports the hypothesized role for inflammation-induced programming as an underlying mechanism for IUGR pathologies.

Glossary

Abbreviations

FDS

flexor digitorum superficialis

GSIS

glucose-stimulated insulin secretion

HDLC

high-density lipoprotein cholesterol

HEC

hyperinsulinemic–euglycemic clamp

KHB

Krebs–Henseleit bicarbonate buffer

LPS

lipopolysaccharide

MI-IUGR

maternofetal inflammation-induced intrauterine growth restriction

pCO₂

partial pressure of CO₂

PI-IUGR

placental insufficiency-induced intrauterine growth restriction

pO₂

partial pressure of O₂

Conflict of interest statement

The authors have no conflicts of interest to disclose.