Gene expression of free fatty acids-sensing G protein-coupled receptors in beef cattle

Abstract

Many physiological functions are regulated by free fatty acids (FFA). Recently, the discovery of FFA-specific G protein-coupled receptors (FFARs) has added to the complexity of their actions at the cellular level. The study of FFAR in cattle is still in its earliest stages focusing mainly on dairy cows. In this study, we set out to map the expression of genes encoding FFARs in 6 tissues of beef cattle. We also investigated the potential effect of dietary forage nature on FFAR gene expression. To this end, 16 purebred Charolais bulls were fed a grass silage ration or a maize silage ration (n = 8/group) with a forage/concentrate ratio close to 60:40 for 196 d. The animals were then slaughtered at 485 ± 42 d and liver, spleen, ileum, rectum, perirenal adipose tissue (PRAT), and Longissimus Thoracis muscle were collected. FFAR gene expression was determined by real-time quantitative PCR. Our results showed that of the five FFARs investigated, FFAR1, FFAR2, FFAR3, and GPR84 are expressed (Ct < 30) in all six tissues, whereas FFAR4 was only expressed (Ct < 30) in PRAT, ileum, and rectum. In addition, our results showed that the nature of the forage, i.e., grass silage or maize silage, had no effect on the relative abundance of FFAR in any of the tissues studied (P value > 0.05). Taken together, these results open new perspectives for studying the physiological role of these receptors in beef cattle, particularly in nutrient partitioning during growth.

Our study shows that free fatty acid receptors are expressed in a wide range of beef cattle tissues. It opens new perspectives to better understand the mode of action of free fatty acids at tissue level in cattle.

Introduction

Fatty acids are a major source of energy for cattle and have a biological effect on the tissues involved in production and reproduction in farm animals (liver, adipose tissue, mammary gland, intestine, muscle, oocyte, immune system, etc.). They are also involved in the regulation of many physiological functions such as ingestion, immunity, reproduction, etc. (reviewed in Bionaz et al., 2020). Given these diverse effects, a major challenge is to gain a better understanding of the mechanisms of action of fatty acids at the cellular level. Until recently, the mechanism of action of free fatty acids (FFA) at the cellular level was described as follows: on entering the cytoplasm, either by passive diffusion or via specific carriers (FATP: fatty acid transporter protein, CD36: cluster of differentiation 36, etc.), FFA bind to nuclear peroxisome proliferator-activated receptor (PPAR) or glucocorticoid receptors and thus activate/inhibit the expression of specific genes involved in lipid metabolism and inflammation. In the mid-2000s, FFAs were shown to be ligands for membrane receptors belonging to the large family of G protein-coupled receptors (GPCRs). FFA-activated GPCRs are called free fatty acid receptors (FFAR). It was shown in humans that long-chain FFA bind to GPR40/FFAR1 and GPR120/FFAR4, short-chain FFA bind to GPR41/FFAR3 and GPR43/FFAR2 and medium-chain FFA bind to GPR84 (Briscoe et al., 2003; Brown et al., 2003; Hirasawa et al., 2005; Wang et al., 2006). Since then, numerous studies in humans have shown that FFARs are expressed in a wide range of tissues leading to regulation of specific physiological responses (immunity, lipolysis, gut hormone secretion, inflammation) through the activation of FFAR (reviewed in Milligan et al., 2017; Kimura et al., 2019). Whether similar FFAR signaling occurs in cattle remains largely unexplored due to the paucity of knowledge on bovine FFAR. In ruminants, the expression of FFAR have been shown in some tissues and their activation by FFA has been implicated in the regulation of the animal’s immune response and reproduction (Mielenz, 2017; Alarcon et al., 2018; Maillard et al., 2018; Olmo et al., 2019) and in milk fat synthesis in the bovine mammary gland (Cheng et al., 2020). These findings suggest that FFAR, as demonstrated in humans, are a core element in mediating the biological role of FFA in the regulation of many physiological functions in cattle. Although some studies have begun to identify tissue expression of FFAR, this tissue profiling is incomplete and mainly focused on FFAR2/GPR43 and FFAR3/GPR41 (reviewed in Mielenz, 2017). In addition, the tissue expression of FFAR has mainly been studied in dairy or mixed cattle (Holstein, Jersey, Angus) with no data available for Bos taurus beef cattle. In parallel with these tissue expression studies, studies have reported the effects of different types of factors such as dietary supplementation with specific fatty acids (propionate, beta-hydroxybutyrate, conjugated linoleic acid and oleic acid; Hosseini et al., 2012; Choi et al., 2014; Chung et al., 2016; Zhang et al., 2018), physiological status (negative energy balance, parturition; Friedrichs et al., 2014, 2016; Agrawal et al., 2017), age (Smith et al., 2012; Choi et al., 2014), and forage/concentrate ratio (Friedrichs et al., 2016) on FFAR gene expression in different bovine tissues. However, these studies did not investigate the expression of FFAR4/GPR120 and GPR84 genes. In addition, the potential effect of forage type on the expression of FFAR genes has not been investigated. We hypothesized that profiling the tissue expression of FFAR in cattle, and in particular in Bos taurus beef cattle, would be the first step in establishing the involvement of FFAR in the growth, maintenance and performance of young cattle reared for meat production. Therefore, the first objective of the present study was to determine the expression of FFAR genes in 6 important tissues involved in cattle physiology and fatty acid metabolism (i.e., liver, spleen, perirenal adipose tissue (PRAT), Longissimus Thoracis (LT) muscle ileum and rectum). Indeed, the liver is a central organ sensing nutrient status and availability. Both liver and muscle are strongly involved in fatty acid uptake and oxidation. Perirenal adipose tissue was selected as one of the major tissue of lipogenesis in ruminant, and thus involved in the uptake and secretion of fatty acids. Among the digestive organs of cattle, the intestine has been selected due to its unique capacity to regulate food intake and insulin release through the secretion of incretins by enteroendocrine cells. A number of studies have demonstrated that fatty acid length and saturation level regulate the release of incretins by enteroendocrine cells (reviewed in Kuhla et al., 2016). The ileum and rectum were selected as the respective representatives of the small intestine and large intestine because they contain the greatest diversity of enteroendocrine cells (Pyarokhil et al., 2012). The second objective of the present study was to investigate the effect of maize-based vs. grass-based diets on FFAR gene expression. These two types of forage were isoproteic but differed in their net energy concentrations, and consequently have induced divergence in dry matter intake, average daily gain, feed conversion efficiency, fat thickness, and adipocytes gain (Guarnido-Lopez et al., 2022). Earlier studies have clearly established a link between the energy content of the diet and the relative expression of genes involved in lipid metabolism in different bovine tissues (Bonnet et al., 2004; Ji et al., 2014; Wærp et al., 2018). However, these studies did not include FFAR genes. Therefore, we can hypothesize that these two contrasting diets may have differentially regulated the FFAR gene expression.

Materials and Methods

This study is part of a larger study by Guarnido-Lopez et al. for which the protocol was approved by the Ethics Committee of the Auvergne-Rhône-Alpes region and the French Ministry of Higher Education, Research and Innovation (Authorization number: APAFIS #16194-2016101016361277 v6 delivered on 14th January 2019) (Guarnido-Lopez et al., 2022).

Animals, housing and experimental design and diet

This study is part of a larger study for which animals, housing and experimental design were previously described by Guarnido-Lopez et al. (2022). Briefly, a total of 50 weaned 9-mo-old purebred Charolais bulls (382 ± 41 kg body weight and 259 ± 42 d old) were used. They were divided into 2 homogeneous groups with respect to age and body weight. Each group was assigned to one of the two experimental diets: a grass-based silage diet or a maize-based silage diet. Concentrates consisted of either wheat grain, beat pulp, and soybean meal (grass silage) or wheat grain and soybean meal (maize silage). Diets were formulated with a forage/concentrate ratio close to 60:40. Both diets were iso-metabolizable protein, but differed in their net energy concentrations (grass silage diet lower than maize silage diet). The grass silage diet was rich in neutral detergent fiber, while the maize silage diet was rich in starch (4.85% for grass silage diet and 32.9% for maize silage diet). Diets were distributed as total mixed rations and animals were fed individually and ad libitum. A detailed composition of the two diets is given in Guarnido-Lopez et al. (2022). Sixteen animals with the most extreme residual feed intake (4 efficient and 4 inefficient for each diet) were used due to the implication of lipid metabolism in feed efficiency (Cantalapiedra-Hijar et al., 2018) with the hypothesis of an association with FFAR gene expression. After 196 d on their respective diets, the animals were slaughtered and their tissues were collected.

Tissue collection and RNA extraction

Liver, ileum, rectum, spleen, LT muscle, and PRAT were collected at slaughter. For LT muscle, whole tissue (including muscle fibers and intramuscular fat) was collected. Tissue samples were immediately snap frozen in liquid nitrogen and stored at −80 °C until RNA extraction. Total RNA was extracted from 60 mg of spleen and liver, 130 mg of ileum and rectum, 450 mg of LT muscle and 1.5 g of PRAT using TRIzol Plus RNA purification kit (Invitrogen) according to the manufacturer’s instructions. After extraction, purified RNA samples were treated with DNAse (RNase-Free DNase set, Qiagen) and washed according to manufacturer’s instructions. Before storage, RNA purity was confirmed using a NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE). RNA samples had an OD_260nm/OD_280nm greater than 1.8. RNA quality was determined using a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Average RIN values were 6.92 ± 0.38 for liver, 6.63 ± 0.96 for ileum, 6.14 ± 0.33 for spleen, 6.66 ± 0.68 for rectum, 7.1 ± 0.41 for LT muscle, and 7.14 ± 0.93 for PRAT.

Real-time Quantitative PCR

Total RNA isolated from the 6 tissue samples was reverse transcribed from 2 µg of purified total RNA using High-Capacity RNA to cDNA kit (ThermoFisher Scientific) according to manufacturer’s instructions. The cDNA has been quantified using NanoDrop spectrophotometer.

Primer sequences for bovine FFAR2 (Friedrichs et al., 2014), bovine FFAR4 (Agrawal et al., 2017), bovine GAPDH (Puech et al., 2015), bovine RPS9 (Choi et al., 2014) and bovine ACTB (Bonnet et al., 2013) were previously published. Primer sequences for bovine FFAR1, bovine FFAR3 and bovine GPR84 were obtained using Primer3 and BLAST software. Primer information and size products are listed in E-supplementary (Supplementary Table S1).

Even though a DNAse treatment step was performed, potential genomic DNA contamination in cDNA samples was checked with a reaction PCR using ACTB primers which have been designed in two different exons. In case of genomic DNA contamination, the PCR will yield a product with a larger size than expected for cDNA. None of the cDNA samples were contaminated with genomic DNA.

Real-time quantitative PCR was performed using 20 ng of cDNA, 1x SYBR Green master mix (Applied Biosystems, CA), 0.5 µM of forward and reverse primers and RNase/DNase-free water in a Mx3000P instrument (Agilent Technologies). Real-time quantitative PCR reactions were performed under the following conditions: 10 min at 95 °C, 35 cycles of 15 s at 95 °C (denaturation), and 45 s at 60 °C (FFAR2, FFAR3, FFAR4, GPR84, GAPDH, RPS9, RPLP0 and ACTB) or 62 °C (FFAR1) (annealing plus extension). Following real-time quantitative PCR reactions, melting curves were generated to ensure reaction specificity (Supplementary Figure S1). Gene expression was normalized to the geometric mean of two housekeeping genes: GAPDH and RPS9 for liver and LT muscle, ACTB and RPS9 for ileum, and PRAT, GAPDH, and ACTB for spleen, and RPS9 and RPLP0 for rectum. Among the four housekeeping genes (ACTB, GAPDH, RPS9, and RPLP0), the combination of the two most stable housekeeping genes was determined by the geNorm algorithm (Vandesompele et al., 2002). Housekeeping gene pairs have an M-value below 0.5 except for retroperitoneal adipose tissue (M-value = 0.801). Genes were considered nonexpressed when Ct > 30. The PCR efficiency of each gene in all tissues examined was calculated according to the LinRegPCR flow chart (Ruijter et al., 2009). PCR efficiencies are listed in the Supplementary data (Supplementary Table S2). Gene expression normalization for each tissue was performed according to geNorm algorithm normalization flow chart. Briefly, Ct were transformed to quantities by using the comparative Ct method. Hence, quantities were divided by the normalization factor calculated by geNorm algorithm.

Statistical analysis

The effect of forage type (maize silage vs. grass silage) on normalized gene expression was evaluated by Mann–Whitney U test using GraphPad Prism (GraphPad Software, Inc.) with differences considered significant if P value < 0.05.

Results

FFAR1, FFAR2, FFAR3, FFAR4, and GPR84 mRNA tissue expression

The real-time quantitative PCR results clearly showed that FFAR1, FFAR2, FFAR3 and GPR84 mRNAs were expressed in the six examined tissues, liver, ileum, spleen, rectum, LT muscle and PRAT. The expression of FFAR4 mRNA was specifically shown in ileum, rectum and PRAT (Figure 1).

Figure 1.

FFAR mRNA tissue abundance in cattle. Each receptor is represented by a colored circle: blue for FFAR1 (long-chain fatty acid receptor), green for FFAR2 (short-chain fatty acid receptor), orange for FFAR3 (short-chain fatty acid receptor), pink for FFAR4 (long-chain fatty acid receptor) and yellow for GPR84 (medium-chain fatty acid receptor). A colored circle signifies that the mRNA of the receptor has been detected. A slashed circle indicates that the mRNA of the receptor has been studied but not detected. Published results: results previously reported. Present study: results from the present study. 1: Friedrichs et al., 2014; 2: Agrawal et al., 2017; 3: Wang et al., 2009

Effect of forage type on FFAR1, FFAR2, FFAR3, FFAR4, and GPR84 mRNA expression

The relative abundance of FFAR1, FFAR2, FFAR3, FFAR4, and GPR84 mRNA was not affected by the type of forage in the diet, i.e., grass silage or maize silage, regardless of the tissue examined (P value > 0.05) (Table 1).

Table 1.

Effect of grass silage or maize silage on Bos taurus beef cattle FFAR1, FFAR2, FFAR3, FFAR4, and GPR84 mRNA relative abundance in liver, ileum, spleen, rectum, LT muscle and PRAT

	FFAR11			FFAR21			FFAR31			FFAR41			GPR841
	Grass	Maize	P	Grass	Maize	P	Grass	Maize	P	Grass	Maize	P	Grass	Maize	P
Liver	0.36 ± 0.24	0.45 ± 0.55	ns	0.55 ± 0.30	0.58 ± 0.61	ns	0.48 ± 0.16	0.58 ± 0.53	ns	NE	NE	ns	0.54 ± 0.30	0.56 ± 0.54	ns
Ileum	0.78 ± 0.45	0.94 ± 0.48	ns	0.56 ± 0.33	0.85 ± 1.30	ns	1.06 ± 0.31	1.12 ± 0.47	ns	1.23 ± 1.23	0.57 ± 0.33	ns	1.02 ± 0.38	1.05 ± 0.45	ns
Spleen	1.07 ± 0.37	1.11 ± 0.61	ns	0.55 ± 0.33	0.39 ± 0.21	ns	1.12 ± 0.4	1.12 ± 0.52	ns	NE	NE	ns	0.59 ± 0.16	0.78 ± 0.43	ns
Rectum	0.73 ± 0.62	0.68 ± 0.58	ns	1.07 ± 0.63	0.88 ± 0.39	ns	1.07 ± 0.75	1.45 ± 0.97	ns	0.99 ± 0.68	1.03 ± 0.64	ns	0.96 ± 0.58	0.96 ± 0.43	ns
LT muscle	0.55 ± 0.71	0.69 ± 0.62	ns	0.82 ± 1.00	1.03 ± 0.88	ns	0.53 ± 0.71	0.6 ± 0.51	ns	NE	NE	ns	0.56 ± 0.64	0.65 ± 0.56	ns
PRAT	4.31 ± 6.9	2.1 ± 2.98	ns	3.79 ± 5.64	2.24 ± 3.25	ns	2.48 ± 3.07	1.64 ± 1.69	ns	0.66 ± 0.39	0.64 ± 0.41	ns	4.28 ± 6.45	2.51 ± 3.25	ns

¹mean ± standard deviation of normalized gene expression of 8 individuals for each diet condition.

P: P value; ns: P value > 0.05, Mann–Whitney U test; NE: not expressed (Ct > 30).

Discussion

Our results established for the first time the expression profile of genes encoding five FFAR in liver, ileum, rectum, spleen, LT muscle, and PRAT of a beef cattle breed, namely the Charolais.

In the liver, our data are similar to previous studies in dairy cattle, showing that all FFAR genes except FFAR4 are expressed (Wang et al., 2009; Friedrichs et al., 2014; Agrawal et al., 2017; Aguinaga Casañas et al., 2017). The lack of expression of FFAR4 in bovine liver is a notable difference from humans. In humans, FFAR4 is expressed in the liver, and activation of FFAR4 by FFA has been shown to play a role in nonalcoholic fatty liver disease as well as reducing inflammation in liver stress (Ulven and Christiansen, 2015). In the present study, we stated that FFAR4 was not expressed, as the mean Ct in this tissue was above the previously established threshold of 30 (32.4 ± 0.90). However, given the proximity of the liver results to the set threshold value, it cannot be excluded that FFAR4 expression in this tissue may be very low. An analysis of the expression of FFAR4 at the protein level would definitively answer the question of the presence of this receptor in this tissue. However, there is currently no commercially available bovine anti-FFAR4 antibody. Previous studies in the bovine liver have reported conflicting results regarding the expression of the genes encoding FFAR1 (FFAR1 present: Friedrichs et al., 2014; FFAR1 absent: Agrawal et al., 2017) and FFAR3 (FFAR3 present: Wang et al., 2009; FFAR3 absent: Friedrichs et al., 2014). These may be attributed to a different Ct threshold for FFAR1 or to a sex effect, i.e., lactating dairy cattle vs. bull for FFAR3. In our study, FFAR1 and FFAR3 genes are both expressed in the liver. In mice, several studies have shown that FFA exerts a beneficial effect by reducing hepatic steatosis through FFAR1-dependent signaling pathways (Ou et al., 2014; Li et al., 2016). In addition, it has been clearly demonstrated in mice that short-chain fatty acids improve hepatic metabolism via FFAR3 (Shimizu et al., 2019). For the latter study, however, it remains to be investigated whether this effect is mediated by FFAR3 expressed by liver cells or by other organs. If these results in mice are confirmed in cattle, they could open up new perspectives for the treatment of hepatic steatosis in beef cattle during the finishing period. Our results report for the first time that diet type (i.e., grass silage vs. corn silage) has no effect on the gene expression of the 5 FFARs studied in the liver of Bos taurus beef cattle. These original results are in agreement with previous data obtained in 2 different beef cattle breeds (Alentejana and Barrosã), showing that the level of either concentrate or maize silage has no effect on the expression of hepatic genes involved in lipid metabolism (FFAR genes were not studied) (da Costa et al., 2014).

We have shown that the five investigated FFAR genes are expressed in both the small intestine (ileum) and in the large intestine (rectum). Previously published results in dairy cattle have shown that FFAR2 and FFAR3 are expressed in colon, duodenum (Wang et al., 2009) and in the ileum (Burakowska et al., 2020). However, the present study reports for the first time the expression of the long-chain fatty acids receptors (FFAR1 and FFAR4) and medium-chain fatty acids receptor GPR84 in the intestine of beef cattle. These results open new perspectives for understanding the mechanisms of action of fatty acids in the intestine of cattle and more specifically their role in satiety control. Indeed, it has been documented that the type of fatty acids that reach the intestine influence food intake (Chilliard, 1993; Allen, 2000). Thus, the length of the carbohydrate chain and its degree of unsaturation modulate food intake by affecting the secretion of incretins (Relling and Reynolds, 2007; Bradford et al., 2008; Hammon et al., 2008; Palmquist and Jenkins, 2017). These hormones are produced by enteroendocrine cells and subsequently released into the plasma. In humans and mice, several studies have shown that FFAR1, FFAR2, and FFAR4 are expressed by enteroendocrine cells and are the key molecules that induce the release of incretins in response to stimulation by FFA (reviewed in Gribble et al., 2017; Lu et al., 2018). Therefore, it would be of interest to investigate the relationship between intestinal abundance of FFAR1, FFAR2 and FFAR4 mRNA and dry matter intake. This type of result would make it possible to manage through FA supplementation the feed intake of the bovine depending on the objective of production. In cattle, GPR84 has been shown to be expressed in blood polymorphonuclear leukocytes, liver, and adipose tissue (Agrawal et al., 2017). In humans, this receptor has been shown to be expressed particularly under inflammatory conditions in adipose tissue, blood polymorphonuclear leukocytes, and macrophages (Nagasaki et al., 2012; Suzuki et al., 2013). The evidence of GPR84 expression in the intestine raises the question of its role in this organ. It remains to be determined whether GPR84, as shown in other tissues, is involved in the activation of the pro-inflammatory response in the intestine. Furthermore, our study is the first to examine the effect of diet forage composition on the expression of FFAR genes at the intestinal level. Our results show no differential effect of diet type on FFAR gene expression. As mentioned in a recent review by Bionaz et al, the effect of diet, and more specifically FA type, on the intestinal transcriptome is still lacking (Bionaz et al., 2020). It has been established in mice that diets that differ in terms of lipid and carbohydrate content profoundly affect the expression of genes involved in lipid metabolism, including FFAR genes (Dantas Machado et al., 2022). This result highlights the need for further research on the effect of diet type on lipid metabolism-related genes (including FFAR genes) in cattle.

Expression of the FFAR genes has already been demonstrated in various adipose tissues: retroperitoneal adipose tissue (Hosseini et al., 2012; Friedrichs et al., 2016), subcutaneous adipose tissue (Hosseini et al., 2012; Friedrichs et al., 2014, 2016; Chung et al., 2016; Agrawal et al., 2017; Westbrook et al., 2021) and intramuscular adipose tissue (Chung et al., 2016; Westbrook et al., 2021), both from dairy breeds or crossbreeds. Our study shows for the first time that all five FFAR are expressed in the PRAT of beef cattle. In cattle, the physiological role of these receptors remains to be determined except for GPR84 for which a mediating role in adipose tissue inflammation during lactation has been suggested (Agrawal et al., 2017). However, several studies in humans and rodents have contributed to elucidate the physiological role of these FFAR in adipose tissue: leptin secretion, adipogenesis, lipolysis, etc (reviewed in Mielenz, 2017). In addition, a recent paper demonstrated both in vivo and in vitro in mouse and human cell lines that long-chain n-3 fatty acids, but not saturated fatty acids, can induce adipogenesis by promoting FFAR4-dependent preadipocyte proliferation (Hilgendorf et al., 2019). This mechanism shifts adipose tissue expansion toward hyperplasia rather than hypertrophy, thereby reducing adipose tissue inflammation. If validated in cattle, this mechanism may pave the way for the development of original feeding strategy during the fattening period to induce expansion of adipocyte numbers and to improve fat deposition while maintaining a low level of inflammation. Furthermore, our results show that there is no effect of forage type on the expression level of FFAR genes in adipose tissue. In contrast, several studies in dairy and beef cattle showed an effect of diet (forage type, energy level, lipid supplementation) on the expression of genes involved in lipid metabolism in different adipose tissues. None of these studies included FFARs among the genes studied (Schmitt et al., 2011; da Costa et al., 2013; Ji et al., 2014; Wærp et al., 2018). The lack of effect of diet type on FFAR gene expression in our study may be explained by the high variability of expression of these genes within adipose tissue despite important fold-change in between the 2 diets. A larger sample size might enable us to detect a significant effect. It would also be interesting to include FFAR genes in the aforementioned studies to determine whether the expression of these genes is sensitive to changes in diet type.

In our experiment, gene expression of all FFAR except FFAR4 was detected in the spleen. FFAR2 and FFAR3 gene expression has been reported in the spleen of dairy cattle (Wang et al., 2009). The contribution of FFAR to bovine spleen physiology has not been determined. However, polyunsaturated fatty acids have been shown to promote neutrophil recruitment and delay neutrophil cell death in mice (Svahn et al., 2019). It has also been shown in mice that both short-chain fatty acids and trans-vaccenic acid are involved in T-cell programming via their respective FFARs (Bachem et al., 2019; Fan et al., 2023). Furthermore, in our experiment, we report for the first time that there was no nutritional effect on the expression of FFAR genes. No published data on the effects of diet on gene expression related to lipid metabolism in the bovine spleen have been already reported to our knowledge. Further studies are needed to confirm or refute this finding in other breeds and at other physiological stages.

In the present study, we have shown that all FFAR genes except FFAR4 are expressed in the LT muscle. Otherwise, FFAR2 and FFAR3 mRNA have been detected in other muscle types (skeletal muscle and semitendinosus muscle) of crossbreed or dairy cattle (Wang et al., 2009; Friedrichs et al., 2014). Further studies would be required to determine whether FFAR are expressed by muscular cell type or by the intramuscular adipocytes. Some studies have shown that FFAR2- or FFAR3-knockout mice have either reduced or increased intramuscular adipose tissue, suggesting a potential role for FFAR in meat marbling (reviewed in Frampton et al., 2020). It has also been demonstrated in rats that FFAR2 plays a key role in the short-chain fatty acids induced proliferation of slow-twitch muscle fibers suggesting a potential role in muscle growth (Maruta and Yamashita, 2020). In addition, our results show that there is no effect of diet type on the expression level of FFAR genes in muscle. This is consistent with previous studies showing that variation in diet composition, whether in energy density or proportion of forage, has no effect on the expression level of genes involved in lipid metabolism in other Bos taurus beef breeds although FFAR genes were not examined in these studies (da Costa et al., 2013; Soret et al., 2016). Conversely, other studies in Bos taurus beef cattle have shown that diet type (presence or absence of forage) modulates the expression of genes related to lipid metabolism in muscle (Teixeira et al., 2017). Whether these discrepancies are due to cattle breed, diet composition, or physiological stage requires further investigation.

Taken together, our results indicate that FFAR-encoding genes are widely expressed in Bos taurus beef breed, suggesting that these receptors may have a function in bovine physiology. In complement, studies to determine the expression profile of FFAR protein in various tissues should be undertaken. However, our results show no effect of diet type on the expression level of FFAR-encoding genes in any of the tissues studied. It is important to note that the study by Guarnido-Lopez et al, from which the samples in the present study were taken, shows contrasting effects of diet type on production efficiency and animal growth. Criteria such as average daily gain, dry matter intake, feed conversion ratio, fat thickness, and adipocyte gain were all affected by diet type. In contrast, forage type had no effect on final animal body weight, adipocyte size, residual feed intake, and carcass composition (Guarnido-Lopez et al., 2022). Thus, more contrasting models with pronounced differences in terms of production performance would provide a clearer indication of the potential role of FFARs in production efficiency and growth of Bos taurus beef breeds.

Glossary

Abbreviations

ACTB

β-actin

CD36

cluster of differentiation 36

FATP

fatty acid transporter protein

FFA

free fatty acids

FFAR

free fatty acid receptors

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GPCR

G protein-coupled receptor

Iso-MP

iso-metabolizable protein

LT muscle

Longissimus Thoracis muscle

PPAR

peroxisome proliferator-activated receptor

PRAT

perirenal adipose tissue

RPLP0

ribosomal protein lateral stalk subunit P0

RPS9

40S ribosomal protein S9

Authors contribution

Guillaume Durand (Conceptualization, validation, formal analysis, visualization, supervision, Writing—original draft, Writing—review and editing),

Pierre Charrier (Investigation and Methodology),

Sébastien Bes (Investigation and Methodology, Validation), Laurence Bernard (Conceptualization, Writing—review and editing), Valérie Lamothe (investigation and methodology), Dominique Gruffat (Conceptualization, Resources, Writing—review and editing), and Muriel Bonnet (Conceptualization, Validation, Resources, supervision, Supervision)

Conflict of interest statement

Authors have no conflict of interest.