Increased expression of thyroid hormone responsive protein (THRSP) is the result but not the cause of higher intramuscular fat content in cattle

Abstract

Thyroid hormone responsive protein (THRSP) is known to be involved in lipogenic processes in rodents. In cattle, THRSP could be a potential molecular marker for intramuscular fat (IMF) deposition since mRNA abundance was frequently found to be increased in skeletal muscle with high IMF content compared to those with low IMF. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of THRSP as candidate for increased IMF content in cattle. By combination of mRNA and protein analyses, we could demonstrate that THRSP is present mainly in nuclei of adipose tissue, in intramuscular fat cells and associated cells, and in cells of the portal triad of liver, whereas muscle cells did not express THRSP. Cell culture analyses revealed furthermore that THRSP is expressed in mature adipocytes rather than in early stages of adipogenesis. Collectively, our data support the putative role of THRSP as transcriptional regulator and demonstrate that an increased expression of THRSP in M. longissimus is a consequence of but not the reason for a higher number of intramuscular adipocytes in cattle with enhanced IMF deposition.

Keywords: THRSP; cattle.; intramuscular fat.

Figure 1

mRNA-expression of THRSP in different bovine tissues (n = 10 per group) Expression was normalized to UXT and B2M (subcutaneous fat (SCF)), UXT and RPS9 (liver), and B2M and TOP2B (M. longissimus dorsi (MLD)). Graphs show group means (duplicate measurements) with standard errors calculated by REST software .

Figure 2

Structure of the bovine THRSP locus Primer positions are marked by arrows. Exons are given as grey boxes, introns and intergenic sequences are white. Numbers refer to nucleotide positions relative to the start of the mRNA. Single nucleotide polymorphisms are marked above the graph.

Figure 3

Sections of M. longissimus dorsi (MLD) used for laser capture microdissection (LCM) Adipocytes (a) and myocytes (b) were marked (green lines) and sampled by LCM.

Figure 4

Cellular localization of THRSP in bovine tissues Immuno-histochemical localization of THRSP was performed in cross-sections of subcutaneous fat (SCF) (a), liver (c) and M. longissimus dorsi (MLD) (e). THRSP was detected with a specific bovine antibody and the secondary antibody Alexa Fluor® 488 goat anti-rabbit IgG. It was found in the nuclei of fat cells, interstitial cells and cells of the portal triad. Identical images of each tissue presented with nuclear staining permit a direct comparison (b, d, f). The yellow arrows point to THRSP located in nuclei and adipocytes without THRSP signal are marked by a yellow box.

Figure 5

Western blot with fractionated proteins of bovine liver and subcutaneous fat (SCF) THRSP could be detected in the nuclear (NF) but not cytoplasmic (CF) fraction of liver and SCF. THRSP could also be detected in the total protein extract (TP) from SCF. MW - molecular weight marker.

Figure 6

THRSP protein expression in different bovine tissues of F₂-bulls (n = 10 per group) Representative protein bands from the total protein fraction of subcutaneous fat (SCF) (a). The dilution of the antibody was 1:500. Comparison of THRSP protein amount in bovine tissues from bulls with high and low intramuscular fat content (IMF-content) in M. longissimus dorsi (MLD) (b). H - high IMF-content, L - low IMF-content, NF - nuclear fraction, TP - total protein

Figure 7

(a) Oil Red O staining of 3T3-L1 cells at different time points of the differentiation to mature adipocytes. (b) Relative mRNA expression of different genes in 3T3-L1 cells. The expression values of the target genes (duplicate analyses) were normalized with B2m and Hprt. The analysis and the comparison of the relative gene expression were conducted with the 2^-ΔCt method for normalized individual data points recommended by Schmittgen & Livak .

Figure 8

Relative mRNA expression of different genes in bovine primary stromal vascular cells (n = 3 per time point) The expression of target genes was normalized to B2M and UXT. The analysis and the comparison of relative gene expression was done as described by Schmittgen & Livak .