Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Animal Science
Non Technical Summary
Beef cattle deposit large quantities of lipids within their adipose tissue depots even following the age-related decline in lipogenic enzyme activities and expression of genes associated with adipogenesis and de novo fatty acid biosynthesis. We hypothesize that in beef cattle, subcutaneous (s.c.) adipose tissue continues to accumulate lipid by depressing lipolysis via activation of the G-coupled protein receptor 43 (GPR43) by volatile fatty acids (VFA) and oleic acid. Conversely, acetate and potentially other VFA may promote lipolysis in intramuscular (i.m.) adipose, which is strongly antagonized by oleic acid, thereby promoting i.m. adipose tissue development. Oleic acid can be produced either by neighboring adipocytes or by adjacent muscle fibers.Oleic acid appears to act as an autocrine/paracrine factor to promote lipid filling of i.m. adipose tissue. Thus, grain-based diets increase oleic acid in bovine tissues, which in turn may promote additional lipid filling in i.m. adipose tissue. We believe the studies outlined in this proposal will not only provide information on the expression and activation of GPR43 receptors in bovine adipose tissues but also will elucidate a novel mechanism by which GPR43 affects i.m. adipose tissue growth (and thereby increase carcass quality). If GPR43 truly antagonizes the lipolytic effects of acetate in i.m. adipose tissue, then this research could lead to different nutritional strategies that would increase oleic acid intake and/or endogenous synthesis of oleic acid. Alternatively, it may be possible to identify ligands (natural or synthetic) that promote GPR43 activity in i.m. adipose tissue. Thus, an increased understanding of key differences between i.m. and s.c. adipose tissues could allow for specific technologies to be developed that would increase marbling, concomitantly increasing carcass quality and healthfulness of beef and beef products. If we can confirm that i.m. adipose tissue has different responses to VFA and oleic acid than s.c. adipose tissue, then this will allow producers for the first time to specifically target increases in marbling while not exacerbating carcass fatness in beef cattle.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
1. Establish the interactions among individual VFA (acetate, propionate, and butyrate) and between VFA and the LCFA stearic acid (18:0) and oleic acid for the GPR43 receptor in short-term (1 h) ex vivo incubations of bovine i.m. and s.c. adipose tissues.2. Document the interactions among VFA and the LCFA on GPR mRNA and protein expression in a long-term (48 h) bovine i.m. and s.c. ex vivo adipose tissue explant system.3. Establish the effects of VFA and LCFA on AMPKa mRNA and protein expression to provide additional mechanisms by which ligands for the GPR receptors promote TAG accumulation in long-term (48 h) bovine i.m. and s.c. adipose tissue explant cultures.4. Document the effects of VFA and LCFA on cAMP concentrations and lipolysis (glycerol release), lipogenesis (short-term incubations), adipogenic/lipogenic gene expression, and GPR and AMPKa gene and protein expression (long-term incubations) in i.m. and s.c. adipose tissues taken from Angus steers at 12, 16, and 20 mo of age. These ages represent lean, normally-fattened, and overly-fattened beef cattle.
Project Methods
Interactions among VFA and LCFA - Initial Studies. During the first years of the project, we will establish interactions among VFA (acetate, propionate, and butyrate) in the absence and presence of LCFA (oleic and stearic acid) on the depression or stimulation of cAMP production and glycerol release. Adipose tissue samples will be taken from the 5th-8th longissumus thoracis muscle rib section (described below) and used in preliminary experiments to determine interactions among VFA and LCFA on cAMP production and media glycerol. Once dose-response curves have been established for each of the VFA and LCFA independently, experiments will be designed to document the interactions among VFA and LCFA.Source and Production of Steers (Objective 3). Steers for the proposed experiments will be obtained from the TAMU McGregor Research Center. Calves will be weaned at 6 mo of age and will be allowed to forage free choice on native pastures. At 8 mo of age, the steers will be adapted gradually to a corn/milo-based finishing diet (Smith et al., 2012). Steers will be slaughtered at 12, 16, and 20 mo of age (8 for each age group); we estimate the cattle will grade USDA Select, low Choice, and high Choice at 12, 16, and 20 mo of age, respectively. All calves will be adapted to Calan gates and individually fed until their respective sampling time. With this design, animal will be the experimental unit.Obtaining Adipose Tissues from Cattle. Steers will be transported to the TAMU Rosenthal Meat Science and Technology Center and fasted overnight with free access to fresh water. Cattle will be slaughtered by standard industry practices. The 5th-8th longissimus thoracis rib muscle section will be removed immediately following exsanguination by cutting through the hide (approximately 10 min post-exsanguination) (Miller et al., 1989; May et a., 1994).Short-term Incubations. Fresh adipose tissue pieces (estimated to be 50-100 mg) will be transferred immediately to 6-well culture plates containing 3 mL of the KHB/Hepes/5 mM glucose media. Samples will be pre-incubated with 0.5 mM theophylline plus 10 µM forskolin for 30 min in a CO2 incubator, after which increasing concentrations of acetate, propionate, or butyrate in the absence and presence of increasing concentrations stearic or oleic acid will be added to the incubation media. Following an additional 30 min incubation, adipose tissue samples will be transferred to pre-tared tubes containing lysis buffer.Explant Cultures. Fresh i.m. and s.c. adipose tissue explants will be cultured as described previously (Miller et al., 1989; May et al., 1994). Treatment medium will be without or with addition of the optimal concentrations of VFA and LCFA. Adipose tissues will be incubated for 48 h, which we have demonstrated to be sufficient time to elicit changes in GPR expression (Table 1). Adipose tissue pieces will be removed after 48 h and frozen at -80°C until mRNA are extracted for the measurement of gene expression.Measurement of Tissue cAMP and Media Glyerol. Tissue cAMP concentrations in adipose tissue samples will be measured using the Cyclic AMP XP Assay Kit (Cell Signaling Technologies, Danvers, MA). Media glycerol will be measured with the Glycerol Assay Kit (Sigma-Aldrich, St. Louis, MO).RNA Extraction of Adipose Tissues. Total RNA will be extracted with Tri Reagent (Sigma Chemicals, St. Louis, MO), as reported previously (Brooks et al., 2011a). Complementary DNA (cDNA) will be produced from 1 μg RNA using TaqMan Reverse Transcriptase Reagents (Applied Biosystems, Foster City, CA) The cDNA samples produced at TAMU will be shipped on dry ice to TTU for quantitative real-time PCR (qRT-PCR).qRT-PCR. Quantitative RT-PCR will be used to analyze the expression of AMPKa, C/EBPβ, FASN, PPARg, SCD, and GPRs 40, 41, 43, and 120 (Chung et al., 2016). The 40S ribosomal protein S9 (RPS9) will be used as an endogenous gene expression control. The ABI Prism 7000 detection system (Applied Biosystems) will be used to perform the assay utilizing the thermal cycling variables recommended by the manufacturer (50 cycles of 15 s at 95°C and 1 min at 60°C).AMPK expression. Phosphorylation of AMPK will be analyzed from short- and long-term incubations of s.c and i.m. adipose tissues treated with VFA and LCFA. Total protein will be extracted and immunoblotted with anti-phosph-AMP-activated protein kinase (pAMPK) and anti-AMPK.