Possibility of uncoupling protein 1 expression in bovine fast-twitch muscle fibers

Abstract

Uncoupling protein 1 (UCP1) is responsible for non-shivering thermogenesis in brown/beige adipocytes in humans and rodents. Previously, we showed unexpected expression of UCP1 in bovine skeletal muscles. Here we evaluated Ucp1 mRNA levels in the muscle tissue of Japanese Black steers. Expression of Ucp1 was higher in 30-month-old cattle than in 26-month-old cattle. Levels of myosin heavy chain (Myh)1, an MYH predominantly expressed in fast-twitch muscles, were also significantly higher in cattle aged 30 months. A similar tendency was observed in the expression of other Myhs that are highly expressed in fast-twitch muscles, Myh2 and Myh4. Ucp1 expression was positively correlated with expression of Myh1, Myh2, and Myh4. Our results indicate the possibility of Ucp1 expression in fast-twitch muscle fibers.

Brown/beige adipocytes expediate chemical energy as heat [6, 13]. Uncoupling protein 1 (UCP1) is the principal molecule responsible for non-shivering thermogenesis in brown/beige adipocytes through the uncoupling of electron transfer with ATP production [6, 13]. Energy expenditure in brown/beige adipocytes is preferable for preventing and treating obesity in humans. However, the presence of brown/beige adipocytes is likely to be undesirable in a beef cattle because it leads to decreased fattening efficiency. In fact, our previous study indicated that feed efficiency was low in beef cattle expressing significantly high Ucp1 mRNA levels in subcutaneous and mesenteric fat [8].

We also demonstrated an unexpected expression of UCP1 in bovine skeletal muscle tissues [1]; at present, to best of our knowledge, no information is available on the muscular expression of UCP1 in other animals without introducing exogenous Ucp1. Although regulation of Ucp1 expression is well characterized in brown/beige adipocytes of rodents [6], it may not be applicable to the regulation of bovine muscles. Our previous in vitro study using bovine myosatellite cells revealed that Ucp1 expression did not increase in response to several factors known to stimulate Ucp1 transcription in rodents [6]; forskolin (a protein kinase A activator) and retinoic acid did not increase Ucp1 expression in bovine myosatellite cells and myotubes [2]. In addition, the bone morphogenetic protein (BMP) pathway increased Ucp1 expression through stimulation of murine brown adipogenesis [20], but inhibition of the BMP pathway increased Ucp1 expression without affecting myogenesis in bovine muscular cells [2]. These inconsistent regulations between rodent adipocytes and bovine muscular cells in cell culture systems suggest the necessity of evaluating regulatory Ucp1 expression in bovine skeletal muscle tissue.

The skeletal muscle consists of two types of muscle fibers (fast and slow)- the slow muscle predominantly generates ATP through aerobic metabolism, while ATP is mainly generated through anaerobic metabolism in the fast muscle. Myosin heavy chain (MYH)1, MYH2, and MYH4 are categorized as fast-twitch MYH, whereas MYH7 is a slow-twitch MYH [19]. As a first step to clarify the regulatory expression of muscular Ucp1, the present study aimed to evaluate the relationship between Ucp1 expression and muscle fiber type.

The animal care and experiments were approved by the Animal Care Committee of Kyoto University (30-21). All animal experiments were conducted in accordance with approved guidelines. Eight Japanese Black steers were reared at Kyoto University Livestock Farm. The animals were slaughtered at 26 (n=4) or 30 (n=4) months of age. During the fattening period (9–26 months or 11–30 months), they were fed roughage and formula feed mainly consisting of barley and corn grains on an ad libitum basis throughout the study, and were allowed free access to drinking water and a mineral block (KNZ Ammonium Chloride Lick, Akzo Nobel Functional Chemicals, Arnhem, Netherlands). The steers were slaughtered at Kyoto commercially. The specific date of slaughter and the temperature at Kyoto is shown in Supplementary Table 1. Cold exposure is well-known to induce Ucp1 expression in brown/beige adipocytes in humans and rodents [6, 13]. In view of the temperature, we do not consider that the sampling date affected muscular Ucp1 expression in this study. The musculus longissimus cervicis was obtained immediately after slaughter to measure the gene expression levels. Total RNA isolation and real-time RT-qPCR were performed as previously described [15]. The dissociation (melting) curve of the PCR products was examined by changes in the ramp temperature from 60°C to 95°C. Each sample showed a single peak, suggesting the expected PCR product. The nucleotide sequences of qPCR primers are provided in Supplementary Table 2. The threshold cycle (Ct) values for each gene are shown in Supplementary Table 3. The ΔΔCt method was used to normalize the levels of target transcripts to those of hypoxanthine phosphoribosyltransferase (Hprt) 1. Data are expressed as mean ± SEM. Gene expression data were log-transformed to provide an approximation of normal distribution before analysis. Statistical analyses were performed using SAS statistical software (version 9.1; SAS Inst. Inc., Cary, NC, USA). Data were analyzed using one-way analysis of variance (ANOVA). The relationship between the expression levels of the genes was examined using the Pearson correlation coefficient. Differences at P<0.05 were considered significant, and those 0.05≤P<0.10 were considered to be tendency.

The muscular expression of total Ucp1 was higher in 30-month-old cattle than in 26-month-old cattle (Fig. 1A). We recently identified four variants of bovine Ucp1 [12]. We examined the levels of Ucp1 variants using PCR primers to specifically detect each variant [12]. The levels of muscular Ucp1 variants 2–4 were below detection limits. The Ct value for Ucp1 variant 1 was around 37 (Supplementary Table 3). Although more than Ct value of 35 is generally believed to lack linear amplification with the cycle of PCR, we verified the linear amplification of Ucp1 variant 1 by 44 cycles of PCR (data not shown). Therefore, we consider that expression levels of Ucp1 variant 1 could be semi-quantified in bovine muscles. The levels of muscular Ucp1 variant 1 were higher in cattle aged 30 months than in those aged 26 months (Fig. 1A). We also evaluated the expression of Ucp3, a UCP1-related molecule that is highly expressed in the skeletal muscle in addition to brown adipose tissue and heart [18] (Fig. 1B). Expression of Ucp3 was not affected by the age of cattle. The PR/SET domain (Prdm) 16, peroxisome proliferator-activated receptor γ coactivator-1α (Ppargc1a), cell death inducing DFFA-like effector A (Cidea), and deiodinase (Dio) 2 are highly expressed in brown/beige adipocytes, and are involved in brown adipogenesis [13, 20]. Unlike the regulatory expression of Ucp1 with age, expression levels of Prdm16, Ppargc1a, Cidea, and Dio2 were not affected by the age of cattle (Fig. 1B).

Fig. 1.

Expression of Ucp1 and its variants as well as Ucp3, brown/beige adipocyte-related genes, and Myh in the skeletal muscle of Japanese Black fattening cattle. Japanese black steers were slaughtered at 26 or 30 months. Expression levels of total Ucp1 and Ucp1 variant 1 (A), Ucp3 and brown/beige adipocyte-related genes (B) and Myh (C) in the musculus longissimus cervicis of fattening cattle aged 26 or 30 months were quantified using RT-qPCR. The expression in fattening cattle aged 26 months was set to 1. Data are presented as mean ± SE (n=4). *: P<0.05.

We further evaluated the expression of Myhs (Fig. 1C). The levels of Myh1 were significantly higher in the skeletal muscle of cattle aged 30 months than in those of cattle aged 26 months. In addition, the muscular expression of Myh2 and Myh4 tended to be higher in 30-month-old cattle than in 26-month-old cattle. However, the expression of Myh7 was comparable between cattle aged 26 months and those aged 30 months.

As expected, the levels of Myh1, Myh2, and Myh4 were positively interrelated (Fig. 2) because these MYHs are highly expressed in fast-twitch muscle fibers. In contrast, Myh7 expression was not correlated to Myh1, Myh2, or Myh4 expression. The expression of Ucp1 was positively correlated with those of Myh1, Myh2, or Myh4 but not Myh7. Ucp1 expression was not related to Prdm16 expression, either. The expression of Ucp3 was closely related to that of Ucp1 and Prdm16 as well as Myh1, Myh2 or Myh4.

Fig. 2.

Relationship between expression levels of muscular Ucps, Prdm16, and Myhs. The relationship between the expression levels of Ucp1, Ucp3, Prdm16, Myh1, Myh2, Myh4, and Myh7 was evaluated using Pearson’s correlation coefficient. Upper: correlation coefficient; Lower: P value. The relationship is also shown in the graphs. Circle: 26-month-old cattle; Triangle: 30-month-old cattle. The solid line indicates a significant regression line (P<0.05).

The present study showed that the mRNA levels of Myhs expressed in fast-twitch muscles were generally higher in cattle aged 30 months than those in cattle aged 26 months. Fast-twitch muscle fibers may be formed more efficiently than slow-twitch muscle fibers during the later phase of growth. Ozawa et al. [17] indicated a positive relationship between age and fast-twitch muscle fibers in the longissimus thoracis of Japanese Black steers within the age range of 24 to 32 months. The present results are consistent with these results.

We previously identified UCP1 expression in bovine skeletal muscle [1], but the detailed localization and role of UCP1 remained unclear; immunoreactive UCP1 protein was not detected in adipocytes in the skeletal muscle of fattening cattle [1]. The present study showing that age of cattle affected expression of Ucp1 but not brown adipocyte-related genes such as Prdm16, Ppargc1a, Cidea, and Dio2 are consistent with the previous results [1]. In fact, expression level of Ucp1 was not related to that of Prdm16 that is a gene responsible for the conversion of muscular cells to brown adipocytes [13].

The present study revealed higher expression of muscular Ucp1 in cattle aged 30 months than in cattle aged 26 months, and positive relationships between the expression levels of Ucp1 and those of fast-twitch Myhs, that is, Myh1, Myh2, and Myh4. These results suggest that Ucp1 is expressed in the fast-twitch muscle fibers of beef cattle. Previously, we showed that treatment with A-83-01 (an inhibitor of transforming growth factor-β type I receptor) increased the expression of Myh1 and Myh2 but not Myh7 during myogenesis in bovine myosatellite cells [2]. In addition, Ucp1 levels were numerically increased in myogenic cells treated with A-83-01 [2], suggesting that myotubes with the characteristics of fast-twitch myofibers express Ucp1, and that the results obtained in vitro are consistent with the present results in beef cattle. Ectopic expression of UCP1 in murine skeletal muscle modified the population of MYHs; the percentage of MYH1 and MYH2 was greatly increased, but that of MYH4 was decreased, which may reflect preferential expression of UCP1 in particular myofibers [11].

Recently, we revealed the presence of four variants of Ucp1 resulting from the alternative splicing of exons 3 and 5 [12] in bovine brown adipose tissue. Bovine Ucp1 variant 1, expected to encode proteins with uncoupling activity, was the one mainly expressed among these variants; the mRNA levels of Ucp1 variants 2 and 4 were below the detection limits, and UCP1 variant 2–4 proteins were easily degraded through the proteasomal system [12]. Thus, the mRNA levels of Ucp1 variant 1 might substantially reflect the function of UCP1. In view of the similarity of age-related changes in expression levels between total Ucp1 and Ucp1 variant 1, Ucp1 variant 1 may be the main variant in bovine skeletal muscle.

The role of UCP1 in bovine skeletal muscle remains unclear. Ectopic expression of UCP1 in the skeletal muscle led to less weight gain irrespective of the increase in food intake compared with that in control mice [11]. These results suggest that, similar to adipose UCP1, muscular UCP1 leads to decreased feed efficiency resulting from thermogenesis. In addition to shivering thermogenesis, non-shivering thermogenesis in skeletal muscle contributes to the total energy expenditure [4], and sarcolipin-mediated regulation of sarco/endoplasmic reticulum Ca²⁺-ATPase is responsible for non-shivering thermogenesis in mice [4]. UCP1-mediated uncoupling of mitochondrial electron transfer with ATP production may also be involved in non-shivering thermogenesis in bovine skeletal muscle.

UCP1 in bovine fast-twitch myofibers may facilitate reactive oxygen species removal. Anderson and Neufer [3] showed that free radical leakage, that is, the ratio of produced hydroxy peroxide to consumed oxygen, was higher in fast-twitch muscle than in slow-twitch muscle under basal conditions in rats. Furthermore, although statistical analyses were not performed, the lipid hydroperoxide content, a marker of oxidative stress, was 27% higher in the extensor digitorum longus muscle than in the soleus muscle in rats [5]; and the extensor digitorum longus and soleus muscles mainly consist of fast-twitch myofibers and slow-twitch myofibers, respectively [10]. It is still debated and not convincingly resolved, but mitochondrial UCP1 in brown adipose tissue and thymus has been suggested to be involved in defense against oxidative stress [9, 16]. Forced expression of UCP1 in skeletal muscle induces the antioxidant defense system in mice [14].

Although age of cattle did not affect the expression of muscular Ucp3 (Fig. 1C), the expression of Ucp3 was closely related to that of Ucp1 as well as the fast-twitch Myh (Fig. 2). UCP3 has been suggested to have a role in protection against oxidative stress [7], but the precise role of UCP3 is not definitely determined yet [18]. It is possible that muscular UCP1 promotes defense against oxidative stress in concert with UCP3.

In summary, we showed that muscular expression of Ucp1 is related to fast-twitch Myh in beef cattle. The localization of UCP1 and its relation to muscle fibers characterized by histochemical analysis should be examined in the future. In addition, regulatory expression of Ucp1 in the skeletal muscle other than the musculus longissimus cervicis should be evaluated. Furthermore, studies should be directed to clarify the regulatory expression of Ucp1 variants and the physiological significance of muscular UCP1 in cattle.

CONFLICT OF INTEREST

All authors declare that they have no conflicts of interest.