In-depth exploration of the high and normal pH beef proteome: First insights emphasizing the dynamic protein changes in Longissimus thoracis muscle from pasture-finished Nellore bulls over different postmortem times.

This study aimed to evaluate for the first time the temporal dynamic changes in early postmortem proteome of normal and high ultimate pH (pHu) beef samples from the same cattle using a shotgun proteomics approach. Ten selected carcasses classified as normal (pHu < 5.8; n = 5) or high (pHu ≥ 6.2; n = 5) pHu beef from pasture-finished Nellore (Bos taurus indicus) bulls were sampled from Longissimus thoracis muscle at 30 min, 9 h and 44 h postmortem for proteome comparison. The temporal proteomics profiling quantified 863 proteins, from which 251 were differentially abundant (DAPs) between high and normal pHu at 30 min (n = 33), 9 h (n = 181) and 44 h (n = 37). Among the myriad interconnected pathways regulating pH decline during postmortem metabolism, this study revealed the pivotal role of energy metabolism, cellular response to stress, oxidoreductase activity and muscle system process pathways throughout the early postmortem. Twenty-three proteins overlap among postmortem times and may be suggested as candidate biomarkers to the dark-cutting condition development. The study further evidenced for the first time the central role of ribosomal proteins and histones in the first minutes after animal bleeding. Moreover, this study revealed the disparity in the mechanisms underpinning the development of dark-cutting beef condition among postmortem times, emphasizing multiple dynamic changes in the muscle proteome. Therefore, this study revealed important insights regarding the temporal dynamic changes that occur in early postmortem of high and normal muscle pHu beef, proposing specific pathways to determine the biological mechanisms behind dark-cutting determination.

Exploratory lipidome and metabolome profiling contributes to understanding differences in high and normal ultimate pH beef.

The aim of this work was to compare the lipidome and metabolome profiling in the Longissimus thoracis muscle early and late postmortem from high and normal ultimate pH (pHu) beef. Lipid profiling discriminated between high and normal pHu beef based on fatty acid metabolism and mitochondrial beta-oxidation of long chain saturated fatty acids at 30 min postmortem, and phospholipid biosynthesis at 44 h postmortem. Metabolite profiling also discriminated between high and normal pHu beef, mainly through glutathione, purine, arginine and proline, and glycine, serine and threonine metabolisms at 30 min postmortem, and glycolysis, TCA cycle, glutathione, tyrosine, and pyruvate metabolisms at 44 h postmortem. Lipid and metabolite profiles showed reduced glycolysis and increased use of alternative energy metabolic processes that were central to differentiating high and normal pHu beef. Phospholipid biosynthesis modification suggested high pHu beef experienced greater oxidative stress.

Resistance to pH decline and slower calpain-1 autolysis are associated with higher energy availability early postmortem in Bos taurus indicus cattle.

Beef from Bos taurus indicus is associated with toughness compared to Bos taurus taurus, suggesting there is antagonism between adaptability to heat and beef quality. Resistance to cellular stress in muscle may be protective postmortem, thereby delaying its conversion to meat. Therefore, our objective was to determine pH decline, calpain-1 and caspase 3 activation, and proteolysis in different biological cattle types. Angus, Brangus, and Brahman steers (n = 18) were harvested, and Longissimus lumborum were assessed postmortem for pH decline, ATP content, protease activation, and calpastatin content; and myofibrillar protein degradation was evaluated in beef aged to 14d. Brahman Longissimus lumborum exhibited resistance to pH decline, greater ATP content at 1 h, and delayed calpain-1 autolysis. Although content of caspase-3 zymogen was lower in Brahman, there was no evidence of caspase-3 mediated proteolysis. Greater resistance to energetic and pH changes early postmortem in Brahman Longissimus lumborum are associated with calpain-1 autolysis but not mitochondria mediated apoptosis.

Effects of growth hormone on insulin signal transduction in rat adipose tissue maintained in vitro.

Growth hormone treatment (GH) decreases adipose tissue sensitivity to insulin. However, the exact molecular mechanism(s) involved remains unclear. In the present study, we have evaluated the chronic effects of GH on adipose tissue explants cultured in a defined media. The objective was to determine the effects of GH treatment for 24 and 48 hours on the early steps of the insulin signal transduction, including IRS-3. The 24-hour culture media contained no hormones or 100 ng/ml GH. The 48-hour culture media contained insulin and dexamethasone supplemented with or without 100 ng/ml of GH. Results demonstrated a reduction in the cellular concentration of IRS-1 by around 30% when adipose tissue was chronically treated with growth hormone for either 24 or 48 hours. IRS-3 protein levels were also decreased by 15% after the 24-hour treatment, and by 27% after culture with GH for 48 hours in the presence of insulin and dexamethasone. PI 3-kinase concentrations were also reduced by GH in both experiments by around 25%. At the end of the 24-hour culture with GH adipose explants were stimulated with insulin in a short-term incubation, after which phosphorylation and association of the IRSs with PI 3-kinase were evaluated. After the insulin stimulus, the association of PI 3-kinase with IRS-1 and IRS-3 were decreased in explants chronically cultured with GH by 44 and 28%, respectively. After this short-term insulin stimulus, the IRS-3 phosphorylation was also lowered in GH-treated explants. The results with chronic cultures of adipose presented here are consistent with similar changes in IRS-1 and IRS-2 concentration and phosphorylation observed for liver and muscle after long-term (3-5 days) in vivo treatment with GH. The data suggest that chronic GH treatment alters the early steps of the insulin signal transduction pathway, and may explain the changes in adipose tissue sensitivity to insulin.

Effect of growth hormone on fatty acid synthase gene expression in porcine adipose tissue cultures

We describe an efficient in vitro assay to test growth hormone effects on mRNA levels and fatty acid synthase (FAS, EC. 2.3.1.85) activity. Swine adipose tissue explants were long-term cultured in medium containing growth hormone and FAS mRNA levels and enzyme activity were measured. We quantified FAS transcripts by competitive reverse transcriptase PCR (RT-PCR) using total RNA from cultured adipose tissue explants and RT-PCR standard-curves were constructed using a cloned 307 bp segment of native FAS cDNA and a shorter fragment from which a 64 bp (competitor, 243 bp) internal sequence had been deleted. A known amount of competitor was added to each PCR as an internal control and æ-actin transcripts were also measured to correct for differences in total RNA extraction and reverse transcription efficiency. In cultures with added growth hormone FAS mRNA levels decreased 70 percent (p < 0.01) and FAS enzyme activity decreased 22 percent (p < 0.05). These in vitro growth hormone effects were consistent with those observed in vivo, showing that in vitro adipose tissue culture combined with RT-PCR is a useful and accurate tool for studying growth hormone modulation of adipose tissue metabolism. This technique allowed the diagnosis of lower levels of FAS mRNA in the presence of growth hormone and these low levels were associated with decreased FAS activity in the adipose tissue explants.