Daily injection of the β2 adrenergic agonist clenbuterol improved poor muscle growth and body composition in lambs following heat stress-induced intrauterine growth restriction

Abstract

Background: Intrauterine growth restriction (IUGR) is associated with reduced β2 adrenergic sensitivity, which contributes to poor postnatal muscle growth. The objective of this study was to determine if stimulating β2 adrenergic activity postnatal would rescue deficits in muscle growth, body composition, and indicators of metabolic homeostasis in IUGR offspring. Methods: Time-mated ewes were housed at 40°C from day 40 to 95 of gestation to produce IUGR lambs. From birth, IUGR lambs received daily IM injections of 0.8 μg/kg clenbuterol HCl (IUGR+CLEN; n = 11) or saline placebo (IUGR; n = 12). Placebo-injected controls (n = 13) were born to pair-fed thermoneutral ewes. Biometrics were assessed weekly and body composition was estimated by ultrasound and bioelectrical impedance analysis (BIA). Lambs were necropsied at 60 days of age. Results: Bodyweights were lighter (p ≤ 0.05) for IUGR and IUGR+CLEN lambs than for controls at birth, day 30, and day 60. Average daily gain was less (p ≤ 0.05) for IUGR lambs than controls and was intermediate for IUGR+CLEN lambs. At day 58, BIA-estimated whole-body fat-free mass and ultrasound-estimated loin eye area were less (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. At necropsy, loin eye area and flexor digitorum superficialis muscles were smaller (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Longissimus dorsi protein content was less (p ≤ 0.05) and fat-to-protein ratio was greater (p ≤ 0.05) for IUGR but not IUGR+CLEN lambs than for controls. Semitendinosus from IUGR lambs had less (p ≤ 0.05) β2 adrenoreceptor content, fewer (p ≤ 0.05) proliferating myoblasts, tended to have fewer (p = 0.08) differentiated myoblasts, and had smaller (p ≤ 0.05) muscle fibers than controls. Proliferating myoblasts and fiber size were recovered (p ≤ 0.05) in IUGR+CLEN lambs compared to IUGR lambs, but β2 adrenoreceptor content and differentiated myoblasts were not recovered. Semitendinosus lipid droplets were smaller (p ≤ 0.05) in size for IUGR lambs than for controls and were further reduced (p ≤ 0.05) in size for IUGR+CLEN lambs. Conclusion: These findings show that clenbuterol improved IUGR deficits in muscle growth and some metabolic parameters even without recovering the deficit in β2 adrenoreceptor content. We conclude that IUGR muscle remained responsive to β2 adrenergic stimulation postnatal, which may be a strategic target for improving muscle growth and body composition in IUGR-born offspring.

Keywords: DOHaD; developmental origins of health and disease; fetal growth restriction; fetal programming; growth efficiency; low birthweight.

FIGURE 1

Growth in IUGR-born lambs administered daily injectable clenbuterol. Fasted weights were assessed in controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) at birth, 30 days of age, and 60 days of age. Data are presented for bodyweight (A), average daily gain from birth to 60 days of age for each experimental group (B), and average daily gain from birth to 30 days of age and 30–60 days of age for all lambs (C). Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and noted where significant (p < 0.05). ^{a, b} Means with different superscripts differ (p < 0.05).

FIGURE 2

Estimated whole-body fat-to-protein ratios for IUGR-born lambs administered daily injectable clenbuterol. Bioelectrical impedance analysis (BIA) was performed in controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11). Data are presented for BIA-estimated fat-to-protein ratios in the live lambs at 30 days (A) and 58 days of age (B) and the carcass following necropsy at 60 days of age (C). Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and noted where significant (p < 0.05). ^{a, b} Means with different superscripts differ (p < 0.05).

FIGURE 3

Estimated carcass traits for IUGR-born lambs administered daily injectable clenbuterol. Ultrasonic measurements were performed in controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) at 58 days of age. Data are presented for estimated back fat thickness (A), estimated loin depth (B), and estimated loin-eye area (C) as well as actual loin-eye area measured in the chilled carcass (D) following necropsy at 60 days of age. Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and noted where significant (p < 0.05). ^{a, b, c} Means with different superscripts differ (p < 0.05).

FIGURE 4

Circulating glucose and insulin for IUGR-born lambs administered daily injectable clenbuterol. Arterial blood samples were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) from the 55th to 60th day of age. Data are presented for mean daily blood glucose (A), mean daily plasma insulin (B), and mean daily glucose-to-insulin ratios (C). Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and noted where significant (p < 0.05). ^{a, b, c} Means with different superscripts differ (p < 0.05).

FIGURE 5

Blood metabolites for IUGR-born lambs administered daily injectable clenbuterol. Arterial blood samples were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) from the 55th to 60th day of age. Data are presented for mean daily plasma triglycerides (A), plasma HDL-C (B), plasma non-esterified fatty acids (NEFA) (C) and blood plasma urea nitrogen (BUN) concentrations (D). Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and are noted where significant (p < 0.05). ^{a, b, c} Means with different superscripts differ (p < 0.05).

FIGURE 6

Blood gas parameters for IUGR-born lambs administered daily injectable clenbuterol. Arterial blood samples were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) from the 55th to 60th day of age. Data are presented for mean daily blood pH (A), blood partial pressure of CO₂ (B), blood oxyhemoglobin concentrations (C) and blood carboxyhemoglobin concentrations (D). Effects of experimental group (GRP), day (DAY), and the interaction (G*D) were evaluated and are noted where significant (p < 0.05). ^{a, b, c} Means with different superscripts differ (p < 0.05).

FIGURE 7

Myoblast profiles in skeletal muscle from IUGR-born lambs administered daily injectable clenbuterol. Semitendinosus, biceps femoris, and longissimus dorsi cross-sections were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) at 60 days of age. Data are presented for percentages of nuclei expressing pax7 (i.e., myoblasts) (A), percentages of pax7⁺ nuclei co-expressing PCNA (i.e., proliferating myoblasts) (B), and percentages of nuclei expressing myogenin (i.e., differentiated myoblasts) (C) averaged from 800 nuclei across 3 non-overlapping fields of view. Effects of experimental group (GRP) were evaluated and noted where significant (p < 0.05) or tending toward significant (p < 0.10). ^{a, b, c} Means with different superscripts differ (p < 0.05). ^{x, y, z} Means with different superscripts tend to differ (p < 0.10).

FIGURE 8

Myofiber size in skeletal muscle from IUGR-born lambs administered daily injectable clenbuterol. Semitendinosus and longissimus dorsi cross-sections were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) at 60 days of age. Data are presented for mean fiber size averaged from a minimum of 100 fibers across 3 non-overlapping fields of view. Effects of experimental group (GRP) were evaluated and noted where significant (p < 0.05). ^{a, b} Means with different superscripts differ (p < 0.05).

FIGURE 9

Skeletal muscle protein expression for IUGR-born lambs administered daily injectable clenbuterol. Semitendinosus muscle samples were collected from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11) at 60 days of age. Data are presented for skeletal muscle β2 adrenoreceptor content determined by protein immunoblot. Effects of experimental group (GRP) were evaluated and noted where significant (p < 0.05) or tending towards significant (p < 0.10). ^{x, y} Means with different superscripts differ (p < 0.10). Representative gel micrographs are presented in the lower pane.

FIGURE 10

Skeletal muscle lipid droplet profiles for IUGR-born lambs administered daily injectable clenbuterol. Lipid droplets were assessed in cross-sections of semitendinosus collected at 60 days of age from controls (n = 13), IUGR lambs (n = 12), and IUGR+CLEN lambs (n = 11). Data are presented for average lipid droplet size (A) and lipid droplet size distribution (B). Effects of experimental group (GRP) were evaluated and noted where significant (p < 0.05). ^{a, b, c} Means with different superscripts differ (p < 0.05). * Counts differ (p < 0.05) among experimental groups with the size category.