Short Communication: Beta-adrenergic agonists alter oxidative phosphorylation in primary myoblasts.

Beta-adrenergic agonists (ß-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which ß-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no ß-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by ß-AA treatment in ovine or porcine cells. These findings indicate that ß-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to ß-AA incubation also demonstrates differing physiological responses to ß-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.

Beta-adrenergic agonists (ß-AAs) are supplemented to pigs and cattle to improve growth performance, carcass weight, and loin muscle area. Little is known about the mechanism taking place within individual cells by which ß-AAs achieve this outcome. Previous work reported that ß-AA supplementation improves the efficiency in which cells use glucose as an energy source and alters the expression of genes related to mitochondrial function, a key component of cellular energy production. To further our understanding of the impact of ß-AA supplementation on these cellular functions, our objective was to identify the influence of two ß-AAs used in livestock production, ractopamine HCl and zilpaterol HCl, on the mitochondrial respiratory activity of cells collected from the loin muscle and grown in culture. We isolated cells from cattle, pig, and sheep muscle and measured the oxygen consumption of the cells after treatment with ractopamine HCl, zilpaterol HCl, or with no supplement. We found that both ractopamine HCl and zilpaterol HCl enhance the efficiency of cellular energy production during a state of cellular stress in bovine muscle cells. There was no appreciable effect of the supplement on the energy production of pig or sheep cells. These data indicate that ß-AA supplementation in cattle may increase the muscle cell energy production capacity compared with non-supplemented cells. This study also demonstrates that the efficiency of cell energy production is one plausible mechanism underlying species differences in the response to ß-AA supplementation.

Primary myoblasts from intrauterine growth-restricted fetal sheep exhibit intrinsic dysfunction of proliferation and differentiation that coincides with enrichment of inflammatory cytokine signaling pathways.

Intrauterine growth restriction (IUGR) is linked to lifelong reductions in muscle mass due to intrinsic functional deficits in myoblasts, but the mechanisms underlying these deficits are not known. Our objective was to determine if the deficits were associated with changes in inflammatory and adrenergic regulation of IUGR myoblasts, as was previously observed in IUGR muscle. Primary myoblasts were isolated from IUGR fetal sheep produced by hyperthermia-induced placental insufficiency (PI-IUGR; n = 9) and their controls (n = 9) and from IUGR fetal sheep produced by maternofetal inflammation (MI-IUGR; n = 6) and their controls (n = 7). Proliferation rates were less (P < 0.05) for PI-IUGR myoblasts than their controls and were not affected by incubation with IL-6, TNF-α, norepinephrine, or insulin. IκB kinase inhibition reduced (P < 0.05) proliferation of control myoblasts modestly in basal media but substantially in TNF-α-added media and reduced (P < 0.05) PI-IUGR myoblast proliferation substantially in basal and TNF-α-added media. Proliferation was greater (P < 0.05) for MI-IUGR myoblasts than their controls and was not affected by incubation with TNF-α. Insulin increased (P < 0.05) proliferation in both MI-IUGR and control myoblasts. After 72-h differentiation, fewer (P < 0.05) PI-IUGR myoblasts were myogenin+ than controls in basal and IL-6 added media but not TNF-α-added media. Fewer (P < 0.05) PI-IUGR myoblasts were desmin+ than controls in basal media only. Incubation with norepinephrine did not affect myogenin+ or desmin+ percentages, but insulin increased (P < 0.05) both markers in control and PI-IUGR myoblasts. After 96-h differentiation, fewer (P < 0.05) MI-IUGR myoblasts were myogenin+ and desmin+ than controls regardless of media, although TNF-α reduced (P < 0.05) desmin+ myoblasts for both groups. Differentiated PI-IUGR myoblasts had greater (P < 0.05) TNFR1, ULK2, and TNF-α-stimulated TLR4 gene expression, and PI-IUGR semitendinosus muscle had greater (P < 0.05) TNFR1 and IL6 gene expression, greater (P < 0.05) c-Fos protein, and less (P < 0.05) IκBα protein. Differentiated MI-IUGR myoblasts had greater (P < 0.05) TNFR1 and IL6R gene expression, tended to have greater (P = 0.07) ULK2 gene expression, and had greater (P < 0.05) ß-catenin protein and TNF-α-stimulated phosphorylation of NFκB. We conclude that these enriched components of TNF-α/TNFR1/NFκB and other inflammatory pathways in IUGR myoblasts contribute to their dysfunction and help explain impaired muscle growth in the IUGR fetus.

Myoblasts are stems cells whose functional capacity can limit muscle growth. However, stressful intrauterine conditions cause these cells to be intrinsically dysfunctional. This restricts muscle growth capacity, leading to intrauterine growth restriction (IUGR) of the fetus, low birth weight, and less muscle mass after birth. Consequently, meat yield is reduced in IUGR-born food animals and glucose homeostasis is impaired in IUGR-born humans, which contributes to metabolic dysfunction. Intrinsic dysfunction of IUGR myoblasts has been previously observed, but the fetal programming changes (i.e., permanent changes in the development of cellular mechanisms that explains different functional outcomes) have not been identified. This study shows that one mechanism is the enhancement of signaling pathways for TNF-α and other inflammatory cytokines. These cytokines have roles in stress responses and regulation of muscle growth. Programmed enhancement of these pathways means that IUGR myoblasts are more responsive to even normal amounts of circulating cytokines. Unfortunately, the primary response of myoblasts to cytokines is slower differentiation (i.e., cellular transformation necessary for muscle growth). Programmed enhancement of this response directly impedes myoblast-dependent muscle growth, and the deficit is lifelong. However, identifying this mechanism is a fundamental step for developing strategies to improve muscle growth in low birth weight offspring.

Transcriptome analyses indicate that heat stress-induced inflammation in white adipose tissue and oxidative stress in skeletal muscle is partially moderated by zilpaterol supplementation in beef cattle.

Heat stress (HS) triggers oxidative stress, systemic inflammation, and disrupts growth efficiency of livestock. ß-adrenergic agonists supplemented to ruminant livestock improve growth performance, increase skeletal muscle mass, and decrease carcass fat. The objective of this study was to understand the independent and interacting effects of HS and zilpaterol hydrochloride (ZH) supplementation on the transcriptome of subcutaneous white adipose tissue and the longissimus dorsi muscle in steers. Twenty-four Red Angus-based steers were assigned to thermoneutral (TN; Temperature Humidity Index [THI] = 68) or HS (THI = 73-85) conditions and were not supplemented or supplemented with ZH (8.33 mg/kg/d) for 21 d in a 2 × 2 factorial. Steers in the TN condition were pair-fed to the average daily feed intake of HS steers. RNA was isolated from adipose tissue and skeletal muscle samples collected via biopsy on 3, 10, and 21 d and sequenced using 3' Tag-Seq to an achieved average depth of 3.6 million reads/sample. Transcripts, mapped to ARS-UCD1.2, were quantified. Differential expression (DE) analyses were performed in DESeq2 with a significance threshold for false discovery rate of 0.05. In adipose, 4 loci (MISP3, APOL6, SLC25A4, and S100A12) were DE due to ZH on day 3, and 2 (RRAD, ALB) were DE due to the interaction of HS and ZH on day 10 (Padj < 0.05). In muscle, 40 loci (including TENM4 and OAZ1) were DE due to ZH on day 10, and 6 loci (HIF1A, LOC101903734, PDZD9, HNRNPU, MTUS1, and TMCO6) were DE due to environment on day 21 (Padj < 0.05). To explore biological pathways altered by environment, supplement, and their interaction, loci with DE (Praw < 0.05) were evaluated in Ingenuity Pathway Analysis. In adipose, 509 pathways were predicted to be altered (P < 0.01): 202 due to HS, 126 due to ZH, and 181 due to the interaction; these included inflammatory pathways predicted to be upregulated due to HS but downregulated due to the interaction of HS and ZH. In muscle, 113 pathways were predicted to be altered (P < 0.01): 23 due to HS, 66 due to ZH, and 24 due to the interaction of HS and ZH. Loci and pathway data in muscle suggest HS induced oxidative stress and that the stress response was moderated by ZH. Metabolic pathways were predicted to be altered due to HS, ZH, and their interaction in both tissues. These data provide evidence that HS and ZH interact to alter expression of genes in metabolic and immune function pathways and that ZH moderates some adverse effects of HS.

Heat stress (HS) negatively impacts livestock health and carcass quality. Supplementation of livestock with ß-adrenergic agonists (ß-AA) increases muscle mass and decreases fat deposition. The purpose of this study was to understand how HS and zilpaterol hydrochloride (ZH), a ß-AA, alter gene expression in muscle and in adipose of cattle. Twenty-four steers were assigned to thermoneutral (TN) or HS conditions and were not supplemented (NS) or supplemented with ZH for 21 d. RNA was isolated from muscle and adipose collected on days 3, 10, and 21 to identify changes in gene expression. Several individual loci were differentially expressed (DE) due to HS or ZH in both tissues while the interaction of HS and ZH altered expression in adipose. A less stringent definition of DE used to explore biological pathways predicted that both treatments alter metabolism. Pathway analyses also supported that HS increased inflammation in adipose, but that these inflammatory pathways were downregulated by ZH. HS also was predicted to induce oxidative stress in muscle although ZH moderated this response. This study provides information on how HS and ß-AA act independently and interact to alter physiology, lending insight useful for the development of management and mitigation strategies for stress.

Exogenous administration of lipids to steers alters aspects of the innate immune response to endotoxin challenge.

This study examined the effects of increasing energy availability from both dextrose and lipid treatments on the pro-inflammatory response to LPS in Holstein steers. Steers were randomly assigned to one of three groups: saline at 0.5 ml/kg body weight (Control) or 50% dextrose [0.5 ml/kg body weight (Dextrose) to mimic calm cattle's response to LPS] administered immediately prior to LPS (0.5 µg/kg body weight at 0 h) or continuous lipid emulsion infusion from -1 to 6 h [Intralipid 20% (Baxter, Deerfield, IL USA); 0.5 ml/kg/hr (Lipid) to mimic temperamental cattle]. Concentrations of non-esterified fatty acids (NEFA) were greater in Lipid compared with Control and Dextrose steers. A greater decrease in the change in rectal temperature, relative to baseline, was observed in response to LPS in Dextrose in comparison to control and Lipid steers. Cortisol was greater in Lipid than Dextrose and Control steers from -0.5 to 0 h, yet decreased from 0.5 to 5.5 h relative to LPS challenge. Concentrations of IL-6 were decreased in Lipid steers compared with Dextrose and Control steers, and were decreased in Dextrose compared with Control steers post-LPS challenge. These data suggest that increasing circulating NEFA using an exogenous Lipid emulsion may modulate the pro-inflammatory response in steers.

Evaluation of endocrine and immune responses of steers challenged with infectious bovine rhinotracheitis virus.

OBJECTIVE: To evaluate the endocrine and immune responses of steers challenged with infectious bovine rhinotracheitis virus (IBRV). ANIMALS: 12 crossbred beef steers. PROCEDURES: Steers were randomly assigned to IBRV- (control) or IBRV+ treatment groups. Experimentally challenged steers (IBRV+) received a dose of IBRV intranasally (8.0 50% tissue culture infective doses), IBRV- steers received a saline (0.9% NaCl) solution placebo intranasally, and each group was placed in an isolated paddock. At 72 hours after challenge, all steers were fitted with indwelling jugular catheters and placed into individual stanchions. Blood samples were collected on days 4 through 8. Serum was analyzed for concentrations of cortisol, interleukin-6, interferon-γ, tumor necrosis factor-α, growth hormone, and insulin-like growth factor I. RESULTS: From 72 to 144 hours after challenge inoculation, the IBRV+ group had significantly greater mean rectal temperature, compared with the IBRV- group; the greatest temperatures in both groups were observed at 72 hours. Serum cortisol concentrations were increased in both groups from hours 72 to 136 and serum interferon-γ concentrations were greater in the IBRV+ from 94 to 112 hours after inoculation. Growth hormone concentration was greater in the IBRV+ group at various time points, but no difference in insulin-like growth factor- I concentration was observed. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that IBVR challenge altered growth hormone concentration at some time points but was not associated with large increases in circulating proinflammatory cytokines.