Expression of glucose transporters SLC2A1, SLC2A8, and SLC2A12 in different chicken muscles during ontogenesis

INTRODUCTION

Glucose transport into cells is the first limiting step in the regulation of glucose utilization. It is mediated by a family of facilitative glucose transporters (GLUTs; encoded by SLC2A* genes; Augustin, 2010; Mueckler and Thorens, 2013). GLUT4 has been one of the most intensively studied in mammals (Huang and Czech, 2007) because it is responsible for the insulin-mediated increase in glucose uptake that occurs in response to elevated plasma glucose and insulin levels in the postprandial state. This pathway dominates in skeletal muscle, contributing largely to glucose homeostasis (Scheepers et al., 2004) although recent findings suggest that other isoforms such as GLUT1, GLUT12 (Rogers et al., 2002; Stuart et al., 2009; Purcell et al., 2011), and GLUT8 (de Laat et al., 2015) might also participate in glucose uptake in insulin-sensitive tissues. Phylogeny and synteny analyses confirmed the lack of GLUT4 in chickens (Coudert et al., 2015). Nevertheless, the immunoneutralization of insulin in young chickens induces an increase in plasma glucose levels (Dupont et al., 2008) and glucose transport increases in chicken skeletal muscle in response to exogenous insulin (Tokushima et al., 2005), which suggests that alternative mechanisms of insulin-responsive glucose transport exist in this species. Among the GLUTs reported in chickens, only GLUT1 (Kono et al., 2005), GLUT8 (Seki et al., 2003), and GLUT12 (Coudert et al., 2015) are expressed in skeletal muscles, but the understanding of their contribution and regulation remains incomplete in this species. The first aim of the present study was to compare the expression of these three major GLUTs during the early phase of chick development, e.g., when intense changes in energy requirements and supply occur. The second aim was to determine the effects of the muscle type and function (i.e., glycolytic vs. oxidative; fast vs. slow contraction) as well as the nutritional or insulin status of the animal on their pattern of expression.

MATERIALS AND METHODS

RESULTS

DISCUSSION

As in mammals, the striated skeletal muscle in birds is a major glucose-utilizing tissue. However, chicken glucose metabolism presents some particular features such as high glycemia, a relative insensitivity to exogenous insulin (Simon, 1989; Akiba et al., 1999; Braun and Sweazea, 2008) and the absence of GLUT4 (Carver et al., 2001; Seki et al., 2003; Coudert et al., 2015). In mammals, the tissue expression of each GLUT varies throughout development (Santalucia et al., 1992; Macheda et al., 2002). At the mRNA and/or protein level, GLUT1 has been shown to be the primary GLUT expressed in the rat heart and skeletal muscle, with levels declining after birth, while GLUT4 expression increases during late gestation (Santalucia et al., 1992). The expression of GLUT12 also increases during late gestation in rat fetal insulin-sensitive tissues, including skeletal muscles (Macheda et al., 2002). It was suggested that GLUT12 could act as a “substitute” for GLUT4 in fetal tissues and allow the increased uptake of the glucose into the cells in the presence of insulin. In chickens, the skeletal muscle expression of each GLUT studied here also varied during development. Indeed, SLC2A1 mRNA expression was high during mid-embryogenesis (E12) then was lower at E19 before increasing to reach similar levels to those observed at E12 after hatch. Carver et al. (2001) similarly reported that the mRNA level of SLC2A1 decreased progressively from day 10 to day 19 during chick embryogenesis. Interestingly, our result pointed out that this trend is only observed in the glycolytic (PM) muscle but not in the mixed oxidoglycolytic SART muscle, where the mRNA expression of the SLC2A1 gene did not change during muscle ontogenesis. Originally, the present study demonstrated that the expression of SLC2A12 increased continuously during embryogenesis and after hatching, this trend being observed whatever the muscle type.

The mRNA expression SLC2A8 showed a peak at hatching, at least in the fast-twitch glycolytic PM muscle, suggesting a particular role for this transporter at this stage of development which represents a high-consuming energy period for chicks to achieve pipping and hatch. Unlike other transporters such as GLUT1 that resides in the cell or GLUT4 that is shuttled to the plasma membrane, GLUT8 appears to be mainly found intracellularly (Augustin et al., 2005). GLUT8 may play a role in the hexose transport, facilitating the recycling of sugars during processes requiring an abundance of energy (Schmidt et al., 2009; Adastra et al., 2012). Because of the limited carbohydrate content in the egg, maintenance of glucose homeostasis during late-term embryonic development depends on the amount of glucose kept in reserve, primarily as glycogen in the liver, and glucose generated by gluconeogenesis from proteins (proteolysis and amino acid metabolism) mobilized first from albumen and then from muscles. At the end of incubation, the glycerol issued from the hepatic metabolism of triglycerides and originating from egg yolk is the predominant substrate for the synthesis of glycogen in the liver and muscle (Sunny and Bequette, 2011). Between 15 and 19 d of incubation, active metabolism takes place in the liver to transfer the glucose and fatty acids to the cervical pipping muscle that is progressively enriched in glucose, glycogen, and protein to sustain hatching activities (Pulikanti et al., 2010). Insufficient energy supply from glycogen or albumen means that the embryo mobilizes more muscle protein for gluconeogenesis, which can explain why during late embryogenesis the PM muscle yield significantly decreased (Guernec et al., 2003). Moreover, the increased expression of the atrophy-related genes, atrogin-1 and MuRF1, observed between E18 and hatching (Everaert et al., 2013) is consistent with the negative allometric development of the PM muscle. Chicken GLUT8 could therefore act as a lysosomal hexose transporter, as already suggested in mammals (Adastra et al., 2012), to provide sufficient carbohydrate supply to the chick’s skeletal muscle during the hatching process that requires a high level of energy. The rapid decrease in SLC2A8 expression observed in PM muscle after hatching supports this hypothesis. In contrast, the expression of both SLC2A1 and SLC2A12 clearly increased after hatching at least in the PM muscle. During this period, there is a transition of nutrient supply in newly hatched chicks from residual yolk to exogenous feeding, corresponding to chick’s ingestion of carbohydrate-rich diets. Glucose becomes the main energy source for chicks that need to reconstitute their glycogen store and initiate their growth. In the absence of GLUT4 (Coudert et al., 2015), it is likely that the chicken GLUT12 may have a major role in providing energy to muscle cells. Indeed, a compensation process has already been described in genetically altered mice lacking GLUT4 in which an increase in the expression of GLUT12 was reported (Aerni-Flessner et al., 2012). Only GLUT4 shows increased expression after birth in mammalian skeletal muscle since GLUT1 is mainly expressed in fetal muscle but progressively decreases after birth (Santalucia et al., 1992). In mammals, the only condition in which expression of GLUT1 can be induced again in skeletal muscle fibers is the muscle regeneration process (Gaster et al., 2000a). The reason why mRNA expression of chicken GLUT1 increased in the PM muscle after hatching remains to be investigated, but these results may highlight a specific role of this transporter in the chicken different from that described in mammals.

Our study clearly indicates that the regulation of GLUT mRNA expression during early development (ontogenesis) and according to the physiological status of chickens is muscle type-dependent. The expression of all GLUTs (1, 8, 12) was regulated during ontogenesis and by fasting or immunoneutralization in the PM muscle, whereas GLUT1 and GLUT8 expressions were not affected by any of these treatments in the leg muscles. Such tissue-specific physiological regulation (fasting, diet, insulin, etc.) has previously been reported but with some variations due to the muscle features linked to the species. Indeed, changes in GLUT1 and GLUT4 mRNA expressions were observed in red but not in white muscles in fish (Capilla et al., 2002), whereas changes in GLUT4 mRNA expression were reported in white but not in red muscles in rats (Camps et al., 1992). In comparison with GLUT1 and 8, the mRNA expression of GLUT12 was regulated during ontogenesis or in relation to nutritional or hormonal (insulin) status in both breast and leg muscles. The PM muscle has been described as a pure glycolytic white muscle that is composed exclusively of type IIB fibers, poor in mitochondria content and enzymes involved in oxidative pathways, like CS and HAD, but rich in glycogen and enzymes involved in glycolytic activity, like LDH (Rémignon et al., 1994). This muscle mainly depends on glucose provision by glucose transport via GLUTs after a meal or by glycogenolysis in the fasted state for functioning. Leg muscles, such as the SART muscle, are generally described as oxidoglycolytic type muscles and are composed of type IIB, IIA with or without I fibers, these two last type of fibers being richer in mitochondria and oxidative enzymes but poorer in glycolytic enzymes. This typology makes these muscles more adapted to oxidize glucose and fatty acids to produce ATP than glycolytic muscles. The greater sensitivity of GLUTs to nutritional, hormonal, and more generally physiological states during ontogenesis of the breast PM muscle is therefore consistent with its predominant glucose-dependence to optimize glucose uptake and stimulate glycogen storage compared to leg muscles.

The impact of muscle type on GLUT expression was further explored in a wide panel of muscle types. The three genes encoding GLUTs (GLUT1, GLUT8, GLUT12) were all expressed in the different types of muscle. However, there was a gradient of expression for both SLC2A1 and SLC2A8 that were more highly expressed in slow oxidative than in fast glycolytic muscles, oxidoglycolytic muscles generally showing intermediate expression levels. These results are consistent with previous findings obtained in human muscle showing that GLUT1 is predominantly expressed in type I slow oxidative fibers (Stuart et al., 2006). Regarding GLUT12 (as for GLUT4 in mammals), the link between its mRNA expression and muscle fiber typology depends on the species. Indeed, GLUT4 and GLUT12 are predominantly expressed in type I slow oxidative fibers in human muscle (Gaster et al., 2000b; Stuart et al., 2006). Expression of GLUT4 was also found to be greater in oxidative than in glycolytic muscles in the rodent (James et al., 1989; Henriksen et al., 1990; Marette et al., 1992) but not in bovine and goat, in which GLUT4 shows higher expressions in glycolytic and oxidoglycolytic than in oxidative muscles (Hocquette et al., 1995). Our study indicated a more complex relationship between GLUT12 expression and muscle typology in the chicken. For instance, expression of SLC2A12 was five times greater in the PM than in the PLD muscles, which are both glycolytic muscles, despite differing in MyHC isoform expression (fast-twitch and mixed muscles, respectively). The expression of SLC2A12 was almost eight times greater in SART and ILIO than in ADP muscles, all classified as oxidoglycolytic muscles.

In avian species, at least three slow and seven fast myosin heavy chain genes have been identified (Bandam and Rosser, 2000), which cannot be unambiguously assigned to the different subtypes of slow or fast fibers. Depending on the muscle, fast fibers of adult muscles can express one of the embryonic, neonatal, or adult fast myosin heavy chain genes. In fact, the persistence of embryonic or neonatal isoforms and the expansion of adult isoforms during late development differ according to muscles even belonging to the same type according to the classical nomenclature (Barnard et al., 1982). Indeed, the embryonic and neonatal myosin forms have been reported to be replaced by the adult form more rapidly in the PM than in the PLD muscle in the chicken in which both embryonic and neonatal myosin forms were still expressed up to at least 60 d posthatching (Gauthier and Orfanos, 1993). It has been shown that differences in contractile velocity even exist within avian fast myosin heavy chain isoforms, the embryonic form having the lowest velocity and the adult the highest (Lowey et al., 1993a, Reiser et al., 1996). The diversity of myosin isoforms in developing and adult muscle fibers provides the continuum of shortening velocities needed to meet changing functional demands (Lowey et al., 1993a). The mRNA expression of GLUT12 in the chicken could therefore be closely related to the myosin expression pattern within muscle fibers that itself is adapted to several specialized functions including locomotion, maintenance, and posture. Among fast-twitch glycolytic muscles, the very high expression of GLUT12 in the PM muscle is consistent with its high velocity rate, which requires rapid energy provision, and its primordial role in wing flapping, thermogenesis, and protein reserve functions compared to the back PLD muscle that is mostly solicited for wing flapping, a function barely required in modern commercial broiler lines. It could be also hypothesized from our results that according to its low GLUT12 expression the ADP would be less often solicited than other oxidoglycolytic leg muscles studied here (SART, ILIO).

This study showed for the first time in the chicken the differential regulation of the expression of three major GLUTs during early muscle development and in response to nutritional or insulin status. It also highlighted a specificity of GLUT expression and regulation according to the muscle type or function, with some specific features related to the species, thus providing new insights to improve understanding regarding the role of GLUT transporters in the fine regulation of glucose utilization in the chicken muscle.