Back fat thickness is one of the major quantitative traits that has an impact on carcass and meat quality in beef cattle and in combination with marbling can be used for stratification of the beef carcasses . The proteome changes that are associated with BF thickness in beef cattle have not yet been well characterized, thus we performed a comparative proteomic analysis of subcutaneous adipose tissues from beef steers with a high and low BF thickness, in order to identify major protein markers associated with fat metabolism in beef cattle.
As shown in Figure 1B and Figure 2B, the identified proteins were significantly (p < 0.05) affected by BF thickness and the host animal. A number of proteins, such as those involved in cellular trafficking (annexin 1, α-SNAP and serotransferrin precursor), were differentially expressed in the CHAR animals, although the genes encoding these proteins had not been previously identified in microarray studies. α-SNAP belongs to the soluble NSF attachment protein receptor (SNARE) protein complex, which are a group of membrane proteins associated with distinct membrane compartments of the secretory and endocytic trafficking pathways, and contribute to the specificity of membrane fusion . These proteins have been shown to function at all sites of constitutive and regulated secretion in eukaryotes . Members of the SNARE complex include two types of cytosolic proteins, NSF and the SNAPs (α-, β- and γ-soluble NSF attachment proteins). Two-dimensional gel analysis of six CHAR animals demonstrated that there is an increased expression of α-SNAP with increased BF thickness in these animals (Table 3), suggesting that membrane secretion plays a role in fat accumulation and might cause the difference in BF thickness. However, the expression of α-SNAP was not significantly higher when a larger numbers of animals (n = 13) were compared (data not shown). Therefore, the function of α-SNAP in fat depot formation needs further validation in CHAR animals.
From both animal treatment groups, annexin 1 was detected in all animals, despite the BF thickness, although it was more highly expressed in the adipose tissues with a higher BF thickness (Table 4). The annexins (or lipocortins) are a family of proteins that are ubiquitously distributed in different tissues and cell types of higher and lower eukaryotes, including mammals, fish, and birds . This protein functions as calcium-dependent phospholipid-binding protein . A recent study by Braun et al.  reported that the annexin family is linked to an inhibition of phospholipase activity, exocytosis and endoctyosis, signal transduction, organization of the extracellular matrix, resistance to reactive oxygen species and DNA replication. Annexin 1 is the first member of the annexin family of proteins that has been characterized to have the ability to bind (i.e. to annex) to cellular membranes in a calcium-dependent manner . The down-regulation of annexin 1 in animals with a higher BF thickness suggests that the cell membrane-binding function of annexin 1 might be important in fat depot formation. This is the first study to report the association of increased annexin 1 expression with increased BF thickness in beef cattle. Further study to identify the role of annexin 1 in adipogenesis will be performed in adipocyte cell lines.
Several proteins in HEAN animals were differentially expressed and associated with differences in BF thickness. A large number of these proteins function in the oxidation-reduction process and carbon metabolism for the transport of energy. For example, glycerol-3-phosphate dehydrogenase (G3PDH), Malic enzyme 1, and isocitrate dehydrogenase were over-expressed in tissues with high BF. Glycerol-3-phosphate dehydrogenase is a marker of triglyceride synthesis, while the other two enzymes are involved in glycerol degradation via the glycerol kinase pathway and catalyze the oxidative decarboxylation of malate to form pyruvate suggesting that these proteins might play major roles in the fat metabolic pathways. Malic enzyme [(S)-malate:NAD(P)+ oxidoreductase (decarboxylating); EC 126.96.36.199-40] catalyzes the oxidative decarboxylation of malate to pyruvate and carbon dioxide, using NAD+ or NADP+ as the electron acceptor [32–34]. The NADP+-dependent isocitrate dehydrogenase IDH (EC 188.8.131.52) which is found in both mitochondria (IDH2) and cytosol (IDH1), catalyzes the decarboxylation of isocitrate to α-ketoglutarate (α-KG) and generates NADPH, which is a primary source of reducing equivalents utilized for fatty acid synthesis in bovine [35, 36].
Host genetics play a role in the differences in carcass traits. The crossbred HEAN have the genetic components of Hereford and Angus, while the CHAR animals have the genetic components of Charolais. Thus, it is not surprising that different proteins were detected for the two animal groups because Hereford and Angus breeds mature and fatten earlier than Charolais . Therefore, the differences in protein expression might point to particular proteins that control fat formation in these two groups of animals. Upon further investigation, the unanalyzed spots might reveal additional proteins that help determine the variation in adipogenesis for the HEAN and CHAR beef cows.
In conclusion, a comparative proteomic analysis of subcutaneous adipose tissues of beef steers with a high and low BF thickness identified a major protein marker (annexin 1) associated with fat accumulation and metabolism. Considering that this adipose depot is the largest and the most energetically inefficient, protein markers such as annexin 1 might be an indicator of BF accumulation, however, for this, annexin 1 levels will have to be determined in young animals in future studies. Furthermore, expression of a protein marker in combination with ultrasound measurement of BF might provide an additional assessment criterion to reduce the impact of the subcutaneous adipose depot on red meat animal production. The protein markers and their associations with the genetic makers could potentially identify animals producing less BF. Further work is in progress to identify proteins that have a consistent differential expression in fat metabolic pathways, and to define the role(s) of annexin 1 in the depot-specific processes of adipogenesis and lipid metabolism.