K. J. Thornton

Role of G protein-coupled estrogen receptor-1, matrix metalloproteinases 2 and 9, and heparin binding epidermal growth factor-like growth factor in estradiol-17β-stimulated bovine satellite cell proliferation.

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters. Although the positive effects of E2 on rate and efficiency of bovine muscle growth are well established, the mechanisms involved in these effects are not well understood. Combined E2 and trenbolone acetate implants result in significantly increased muscle satellite cell number in feedlot steers. Additionally, E2 treatment stimulates proliferation of cultured bovine satellite cells (BSC). Studies in nonmuscle cells have shown that binding of E2 to G protein-coupled estrogen receptor (GPER)-1 results in activation of matrix metalloproteinases 2 and 9 (MMP2/9) resulting in proteolytic release of heparin binding epidermal growth factor-like growth factor (hbEGF) from the cell surface. Released hbEGF binds to and activates the epidermal growth factor receptor resulting in increased proliferation. To assess if GPER-1, MMP2/9, and/or hbEGF are involved in the mechanism of E2-stimulated BSC proliferation, we have examined the effects of G36 (a specific inhibitor of GPER-1), CRM197 (a specific inhibitor of hbEGF), and MMP-2/MMP-9 Inhibitor II (an inhibitor of MMP2/9 activity) on E2-stimulated BSC proliferation. Inhibition of GPER-1, MMP2/9, or hbEGF suppresses E2-stimulated BSC proliferation (P < 0.001) suggesting that all these are required in order for E2 to stimulate BSC proliferation. These results strongly suggest that E2 may stimulate BSC proliferation by binding to GPER-1 resulting in MMP2/9-catalyzed release of cell membrane-bound hbEGF and subsequent activation of epidermal growth factor receptor by binding of released hbEGF.

An examination of the association of serum IGF-I concentration, potential candidate genes, and fiber type composition with variation in residual feed intake in progeny of Red Angus sires divergent for maintenance energy EPD1

Investigating the genetic and physiological drivers of postweaning residual feed intake (RFI) and finishing phase feed efficiency (FE) may identify underlying mechanisms that are responsible for the variation in these complex FE traits. The objectives were 1) to evaluate the relationship of serum IGF-I concentration and muscle gene expression with postweaning RFI and sire maintenance energy (MEM) EPD and 2) to determine fiber type composition as it relates to postweaning RFI and finishing phase FE. Results indicate that RFI and serum IGF-I concentration were not associated (P > 0.05); however, negative correlations (P < 0.05) between sire MEM EPD and serum IGF-I concentration were observed. Gene expression differences between high- and low-RFI animals were observed in cohort 1, where IGFBP5 expression was greater (P < 0.05) in high-RFI animals. When animals were grouped according to sire MEM EPD, the low MEM EPD group of cohort 1 showed greater muscle mRNA expression (P < 0.01) of fatty acid synthase (FASN) and marginally (P < 0.10) greater expression of IGFBP5 and C/EBP alpha (C/EBPα) whereas the high MEM EPD group of cohort 2 had greater muscle mRNA expression of IGFBP2 (P < 0.05) and C/EBPα (P ≤ 0.01) and marginally (P < 0.10) greater expression of IGFBP3. Biopsy tissue samples collected at harvest revealed that the percentage of type IIa fibers was lower (P ≤ 0.05) in high-RFI steers, with a similar trend (P < 0.10) being observed in high finishing phase FE steers. The percentage of type IIb fibers was higher (P < 0.05) in high-RFI (and finishing phase FE) steers than in low-RFI (and finishing phase FE) steers. There was a marginal, negative correlation between RFI and type I (r = -0.36, P = 0.08) and IIa (r = -0.37, P = 0.07) fiber percentages and a positive correlation (r = 0.48, P = 0.01) between RFI and type IIb fiber percentage whereas finishing phase FE was negatively correlated (r = -0.43, P = 0.03) with type I fiber percentage and positively correlated (r = 0.44, P = 0.03) with type IIb fiber percentage. Therefore, our data indicate that 1) serum IGF-I (collected at weaning) is not an indicator of postweaning RFI, 2) the GH-IGF axis appears to have some involvement with RFI at the molecular level; however, muscle gene expression results were not consistent across cohorts, and 3) low-RFI animals may have the ability to more efficiently maintain and accrete muscle mass due to their fiber type composition, specifically a greater proportion of type I fibers.

Bovine sire selection based on maintenance energy affects muscle fiber type and meat color of F1 progeny.

A total of 42 F(1) Red Angus progeny from sires divergent in maintenance energy (ME(M)) EPD were analyzed to determine whether selecting for sire ME(M) would alter end-product meat quality. Data from animals were grouped based on the divergence of the ME(M) EPD of their sire from the Red Angus Association-reported breed average and defined as either high or low, the assumption being that high-ME(M) cattle are less efficient because their maintenance requirements represent a larger proportion of their dietary intake. Steer progeny (n = 7) from the high group produced bottom round steaks with a greater a* (redness) color value (P = 0.02) after 5 d in a simulated retail display when compared with bottom round steaks from the low group (n = 18). Bottom round steaks from the high group had a greater b* (yellowness) color value at d 1 (P = 0.03) and d 5 (P = 0.01) of retail display. Samples from the biceps femoris were taken at 12 mo (from both steers and heifers) and 15 mo (from steers only) of age for fiber type proportion analysis. At 12 mo of age, steers from the low group had more type I fibers (P = 0.02), whereas steers from the high group had more type IIb fibers (P = 0.01). Furthermore, samples from steers in the low group at 15 mo had more type I fibers (P = 0.02), and steers from the high group maintained more type IIb fibers (P = 0.02). No changes in fiber type proportions were observed between the high- and low-ME(M) EPD heifers (n = 17). Relative mRNA abundance of genes involved in the synthesis, storage, and breakdown of glycogen were analyzed as a variable important for meat quality, but no statistical differences were observed. At 12 mo age, glycogenin (glyc) was negatively correlated with the proportion of type IIa fibers (r = -0.32 and P = 0.12) as well as with the proportion of type IIb fibers (r = -0.42 and P = 0.03) in the biceps femoris of the steers. In samples taken from the biceps femoris at 15 mo age, glyc was negatively correlated with the proportion of type IIa fibers (r = -0.42 and P = 0.03) in the steers. This indicates that relative mRNA expression of glyc may serve as a marker of muscle glycogen storage capacity in steers. Thus, selection for efficient Red Angus beef cattle based on sire ME(M) EPD does not adversely affect meat quality in F(1) progeny, based on the variables assessed in this study. Furthermore, selection for progeny from low-ME(M) EPD sires may improve fresh meat quality within Red Angus beef cattle.

Analysis of Longissimus thoracis Protein Expression Associated with Variation in Carcass Quality Grade and Marbling of Beef Cattle Raised in the Pacific Northwestern United States

Longissimus thoracis (LD) samples from 500 cattle were screened for protein expression differences relative to carcass quality grade. The LD of the top 5% (low prime and high choice, HQ) and bottom 5% (low select, LQ) carcasses were analyzed using two-dimensional difference gel electrophoresis and Western blot. Following initial screening, 11 candidate proteins were selected for Western blot analyses. Differentially expressed proteins were clustered into four groups: (1) heat shock proteins and oxidative protection, (2) sarcomeric proteins (muscle maturity and fiber type), (3) metabolism and energetics, and (4) miscellaneous proteins. Proteins from groups 1 and 2 were greater in HQ carcasses. Alternatively, increased quantities of proteins from group 3 were observed in LQ carcasses. Proteomic differences provide insights into pathways contributing to carcass quality grade. A deeper understanding of the physiological pathways involved in carcass quality grade development may allow producers to employ production practices that improve quality grade.

Effects of dietary potato by-product and rumen-protected histidine on growth, carcass characteristics and quality attributes of beef

We hypothesized that variable composition in finishing rations, more specifically; the proportion of potato-by-product (PBP) and rumen protected histidine (His) supplementation may influence growth and meat quality attributes. Two different diets were fed (1) finishing ration with corn and barley as grains (CB, n = 20) and (2) substitution of 10% corn, DM basis, with PBP (PBP, n = 20). Additionally, half of each dietary treatment received 50 g/hd/d rumen protected His (HS, n= 20) while the other half received no supplement (NS, n = 20). Inclusion of 10% PBP or HS did not affect growth or carcass traits. Color stability was analyzed using Hunter color values as well as AMSA visual appraisal in both longissimus thoracis (LT) and gluteus medius (GM) muscles. The LT, but not the GM, of CB steers was more color stable over a 9 d simulated retail display compared to those fed a PB diet. Steers receiving HS produced significantly (P < 0.05) more color stable LT and GM steaks.

Role of G protein-coupled estrogen receptor-1 in estradiol 17β-induced alterations in protein synthesis and protein degradation rates in fused bovine satellite cell cultures

In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters in several countries. Treatment with E2 stimulates protein synthesis rate and suppresses protein degradation rate in fused bovine satellite cell (BSC) cultures; however, the mechanisms involved in these effects are not known with certainty. Although the genomic effects of E2 mediated through the classical estrogen receptors have been characterized, recent studies indicate that binding of E2 to the G protein-coupled estrogen receptor (GPER)-1 mediates nongenomic effects of E2 on cellular function. Our current data show that inhibition of GPER-1, matrix metalloproteinases 2 and 9 (MMP2/9), or heparin binding epidermal growth factor-like growth factor (hbEGF) suppresses E2 stimulate protein synthesis rate in cultured BSCs (P < 0.001) suggesting that all of these are required in order for E2 to stimulate protein synthesis in these cultures. In contrast, inhibition of GPER-1, MMP2/9, or hbEGF has no effect on the ability of E2 to suppress protein degradation rates in fused BSC cultures indicating that these factors are not required in order for E2 to suppress protein degradation rate in these cells. Furthermore, treatment of fused BSC cultures with E2 increased (P < 0.05) pAKT levels indicating that the pAKT pathway may play a role in E2-stimulated effects on cultured BSC. In summary, our current data show that active GPER-1, MMP2/9, and hbEGF are necessary for E2-stimulated protein synthesis but not for E2-simulated suppression of protein degradation in cultured BSC. In addition, E2 treatment increases pAKT levels in cultured BSC.

Active G protein–coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate–induced alterations in protein turnover rate of fused bovine satellite cell cultures

Trenbolone acetate (TBA), a testosterone analog, increases protein synthesis and decreases protein degradation in fused bovine satellite cell (BSC) cultures. However, the mechanism through which TBA alters these processes remains unknown. Recent studies indicate that androgens improve rate and extent of muscle growth through a nongenomic mechanism involving G protein-coupled receptors (GPCR), matrix metalloproteinases (MMP), heparin-binding epidermal growth factor (hbEGF), the epidermal growth factor receptor (EGFR), erbB2, and the insulin-like growth factor-1 receptor (IGF-1R). We hypothesized that TBA activates GPCR, resulting in activation of MMP2/9 that releases hbEGF, which activates the EGFR and/or erbB2. To determine whether the proposed nongenomic pathway is involved in TBA-mediated alterations in protein turnover, fused BSC cultures were treated with TBA in the presence or absence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R, and resultant protein synthesis and degradation rates were analyzed. Assays were replicated at least 9 times for each inhibitor experiment utilizing BSC cultures obtained from at least 3 different steers that had no previous exposure to steroid compounds. As expected, fused BSC cultures treated with 10 n TBA exhibited increased ( < 0.05) protein synthesis rates and decreased ( < 0.05) protein degradation rates when compared to control cultures. Treatment of fused BSC cultures with 10 n TBA in the presence of inhibitors for GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R suppressed ( < 0.05) TBA-mediated increases in protein synthesis rate. Alternatively, inhibition of GPCR, MMP2/9, hbEGF, EGFR, erbB2, or IGF-1R in the presence of 10 n TBA each had no ( > 0.05) effect on TBA-mediated decreases in protein degradation. However, inhibition of both EGFR and erbB2 in the presence of 10 n TBA resulted in decreased ( < 0.05) ability of TBA to decrease protein degradation rate. Additionally, fused BSC cultures treated with 10 n TBA exhibit increased ( < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBAs effects on fused BSC cultures.

Does obesity reduce load-induced muscle hypertrophy?

Obesity contributes to the development of insulin resistance, hyperglycaemia and metabolic syndrome (Kahn & Flier, 2000; Kahn et al. 2006). However, effects of obesity on the control of skeletal muscle mass and hypertrophy are not well understood. Muscle hypertrophy is marked by an increase in protein synthesis and addition of contractile filaments to generate muscle force (Glass, 2003). A vast array of signalling networks in response to external stimuli including insulin, insulin growth factor-1 (IGF-1), steroid hormones and also amino acids mediate hypertrophy of skeletal muscle (Glass, 2003). Though the external signals such as insulin and IGF-1 activate their specific signalling, they share common downstream signalling molecules such as PI3-K and Akt. IGF-1/insulin act on their cell surface specific receptors and initiate respective signalling by activation of PI3K followed downstream signalling via activation of Akt (Cheatham & Kahn, 1995; Stitt et al. 2004). The mechanism of distinct actions of these peptide hormones despite activating common signalling molecule: Akt is beginning to be understood. Reported studies suggest that different isoforms of Akt (Akt1, Akt2 and Akt3) confer signal specificity of insulin in glucose metabolism and IGF-1 in growth. Mutational analyses revealed that Akt1-deficient mice have retarded growth and perinatal mortality, where as Akt2 knockout mice have no growth defects, but have impaired glucose metabolism (Chen et al. 2001; Cho et al. 2001a,b;). On the other hand, Akt3 knockout mice had normal body weights and glucose metabolism, but reduced brain sizes (Garofalo et al. 2003). In general, IGF-1 and also insulin to a lesser extent mediate protein synthesis by activating Akt1. The activated Akt1 increases protein synthesis by activating mammalian target of rapamycin (mTOR). mTOR increases protein synthesis by phosphorylating S6 protein kinase (p-S6K), a positive regulator of protein synthesis. mTOR also inhibits the activity of eIF4E binding protein-1 (4E-BP1), a negative regulator of the protein initiation factor, eukaryotic translation initiation factor 4E (eIF-4E) (Proud & Denton, 1997; von Manteuffel et al. 1997). Glycogen synthase kinase 3β (GSK3β) is another substrate of Akt, whose repression is known to induce hypertrophy, and its activity is regulated negatively by the phosphorylation of serine 9 (p-GSK-3β) (Rommel et al. 2001). In a recent issue of The Journal of Physiology, Sitnick et al. (2009) investigated load-induced hypertrophy and underlying molecular mechanisms in response to high-fat diet. The authors investigated whether a high-fat diet (HFD) would alter the ability of skeletal muscle to respond to functional overload (FO). Further, they determined if underlying signalling mechanisms of hypertrophy are compromised in mice fed a high-fat diet. As expected, mice increased their body size in response to HFD. Measurements of fat (epididymal and retroperitoneal fat pads) and muscle (gastrocnemius and soleus) suggested that increases in body size were primarily due to increased fat composition in the body. The authors also determined that mice fed a high-fat diet were hyperinsulinemic, suggesting development of insulin resistance potentially mediated by increased intra-myocellular triglycerides in muscle. On all the tested days there was a significant increase in the mass of pantaris muscle in response to FO mice compared to control. After 14 and 30 days of FO the authors found a 10 and 16% reduction in the growth of the plantaris muscle in the HFD versus the low-fat diet (LFD) mice, suggesting a moderate decrease in the growth of plantaris muscle in response to HFD. The authors noticed that extended HFD nutrition (30 weeks) had a dramatic effect, showing no difference in the mass of plantarius muscle compared to control mice. These observations suggest that a HFD diet has a negative effect on the load-induced hypertrophy. The authors give interesting directions for the research into whether obesity may be a negative factor for load-induced muscular hypertrophy. On a path leading toward understanding the mechanism underlying this apparent relationship, the authors measured general protein translation and also status of the cell signalling molecules such as Akt, S6K1 and GSK3-β. The association of the ribosomal 40S and 60S subunits resulting in a 80S peak is an indicator of active translation process. The analyses demonstrate that the 80S peak in LFD mice was larger than in HFD mice suggesting that the translation process is greater in LFD mice compared to HFD. The authors further suggest that the activity of signalling molecules such as Akt and S6K1 were attenuated, but not for GSK3 β in FO/HFD mice compared FO/LFD mice. Though the analyses provided in the Sitnick et al. paper provide useful information about the status of the signalling molecules in hypertrophy, the data/analyses are not sufficient to provide accurate mechanistic insights. The potential link between obesity and hypertrophy at a mechanistic level may be delineated by further analyses of other important targets including IGF-1 expression and also using appropriate controls in the analyses. For example, the authors observed increased Akt phosphorylation in HFD control mice with apparent insulin resistance. This may be due to higher load on the plantaris muscle in the case of HFD mice due to an increased body mass. The measurement of IGF-1 levels would have been useful for interpreting information. Upon examining the Western blots of Akt presented in the paper, it is clear that total Akt levels increase in response to FO, irrespective of diet; however, the authors did not analyse if the total Akt levels show differences in response to FO in the two diets (LFD vs. HFD). The analyses are presented as a ratio of Akt Ser-373 to total Akt to indicate activity levels of Akt; but these analyses in the context of robust changes in total Akt levels will only provide limited information regarding Akt activity. The analyses would have been improved by probing for a housing-keeping protein such as GAPDH, and normalizing total Akt levels and also phosphorylated Akt levels to GAPDH and making comparisons between diets. The same suggestion applies to the quantification of the expression of S6K1, p-S6K1, GSK-3β and p-GSK-3β. All the molecules chosen in the study are not unique to either insulin or IGF-1, but common to both. It is not clear whether the HFD mice exhibited compromised hypertrophy due to attenuation of insulin signalling or IGF-1 signalling. In this context, future studies directed toward deciphering the specific proximal pathways such as IGF-1 serum levels/muscle expression of IGF-1, IGF-1/insulin receptor activation and also further study of specific Akt isoforms may begin to provide a molecular basis for the apparent relationship between obesity and hypertrophy. On the other hand, it has to be determined if obesity decreases activity of the mice. In summary, the recent paper by Stinick et al. suggests that diet plays a role in the hypertrophy of skeletal muscle, although the exact mechanisms for this process remain unclear. The authors provide a base upon which further investigations can build to elucidate the complex signalling mechanisms of diet and their influence on load-induced hypertrophy.