Daily Injection of the β2 Adrenergic Agonist Clenbuterol Improved Muscle Glucose Metabolism, Glucose-Stimulated Insulin Secretion, and Hyperlipidemia in Juvenile Lambs Following Heat-Stress-Induced Intrauterine Growth Restriction

Abstract

Stress-induced fetal programming diminishes β2 adrenergic tone, which coincides with intrauterine growth restriction (IUGR) and lifelong metabolic dysfunction. We determined if stimulating β2 adrenergic activity in IUGR-born lambs would improve metabolic outcomes. IUGR lambs that received daily injections of saline or the β2 agonist clenbuterol from birth to 60 days were compared with controls from pair-fed thermoneutral pregnancies. As juveniles, IUGR lambs exhibited systemic inflammation and robust metabolic dysfunction, including greater (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, increased (p < 0.05) intramuscular glycogen, reduced (p < 0.05) circulating IGF-1, hindlimb blood flow, glucose-stimulated insulin secretion, and muscle glucose oxidation. Daily clenbuterol fully recovered (p < 0.05) circulating TNFα, IL-6, and non-esterified fatty acids, hindlimb blood flow, muscle glucose oxidation, and intramuscular glycogen. Glucose-stimulated insulin secretion was partially recovered (p < 0.05) in clenbuterol-treated IUGR lambs, but circulating IGF-1 was not improved. Circulating triglycerides and HDL cholesterol were elevated (p < 0.05) in clenbuterol-treated IUGR lambs, despite being normal in untreated IUGR lambs. We conclude that deficient β2 adrenergic regulation is a primary mechanism for several components of metabolic dysfunction in IUGR-born offspring and thus represents a potential therapeutic target for improving metabolic outcomes. Moreover, benefits from the β2 agonist were likely complemented by its suppression of IUGR-associated inflammation.

Daily Eicosapentaenoic Acid Infusion in IUGR Fetal Lambs Reduced Systemic Inflammation, Increased Muscle ADRβ2 Content, and Improved Myoblast Function and Muscle Growth

“…Fetal hypoxemia is resolved by birth, and circulating monocyte numbers consequently return to normal in IUGR offspring [24]. However, we recently found that blood cytokine concentrations remained elevated well after birth [24,25], which is consistent with the programming of a more inflammatory phenotype in IUGR monocytes [59]. In the present study, systemic inflammation in IUGR fetuses also coincided with a 42% reduction in circulating concentrations of the anti-inflammatory ω-3 PUFA eicosapentaenoic acid, which was presumably a product of stress-impaired ∆5 and ∆6 desaturase activity, as previously observed [60][61][62][63].…”

“…Experiments were performed at the University of Nebraska-Lincoln, which is accredited by AAALAC International. After a 7-day acclimation period, Polypay ewes (2 to 4 years of age; 72.7 ± 0.4 kg; 2.5 to 3 body condition score) were timed-mated to a single Polypay male and heat-stressed to produce placental insufficiency-induced IUGR fetuses, as previously described [ 25 , 42 ]. Briefly, ewes carrying singleton or twin pregnancies were housed under ambient conditions of 40 °C and 35% relative humidity (86 temperature–humidity index per [ 43 ]) from the 40th to the 95th day of gestational age (dGA) and were then returned to thermoneutral conditions (25 °C, 35% relative humidity, 70 temperature–humidity index) for the duration of the study.…”

“…Daily fetal blood samples were also collected into EDTA syringes and analyzed with a HemaTrue veterinary hematology analyzer (Heska Corp., Loveland, CO, USA) for concentrations of total white blood cells, granulocytes, monocytes, lymphocytes, platelets, and red blood cells, as well as hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean packed cell volume, and red blood cell distribution width. Blood plasma was separated from EDTA-treated whole blood by centrifugation (14,000× g, 2 min) and stored at −80 • C. Commercial ELISA kits were used to determine plasma concentrations of TNFα (Wuhan Biotech, Wuhan, China) and eicosapentaenoic acid (MyBioSource, Inc., San Diego, CA, USA) in duplicate, as previously described [25,46]. Coefficients of variance (inter-and intra-assay) were less than 15% for both ELISAs.…”

“…Total protein isolated from snap-frozen semitendinosus was used to determine the content of β2 adrenoceptor and interleukin-6 receptor (IL6R), as previously described [24,25]. Briefly, muscle samples were homogenized by sonication in lower salt extraction buffer containing 20 mM TRIS, 80 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 1 mM EDTA, 0.1% SDS, 1% Triton-X 100, 10% glycerol, 2.5% protease, and 2.5% phosphatase inhibitor, and total protein was isolated by 5 min centrifugation at 14,000× g. Supernatant protein concentrations were determined using Piece BCA Assay (Thermo Fisher, Waltham, MA, USA), and 50-µg aliquots were mixed with 4x Laemmli buffer (Bio-Rad Laboratories, Hercules, CA, USA).…”

“…β2 adrenergic pathways increase nutrient uptake, protein synthesis, and metabolic rates in skeletal muscle [2,[16][17][18][19][20] and thus are critical to efficient lean muscle growth [12,[21][22][23]. Recent studies have confirmed the role of β2 adrenergic deficits in poor growth and metabolic function of IUGR muscle [24,25]. Conversely, heightened inflammatory activity disrupts the function of myoblasts (i.e., muscle stem cells) [26][27][28][29][30] and, in turn, impairs their ability to facilitate hypertrophic muscle growth [31][32][33].…”