A second look at leptin and adiponectin actions on the growth of primary porcine myoblasts under serum-free conditions

Cross-talk between adipose tissue and skeletal muscle may be mediated in part by adipokines. This study was conducted to elucidate further aspects of a possible role of recombinant adiponectin and leptin in the in vitro growth of primary porcine skeletal muscle cells cultured in energetically balanced, growth factor-supplemented, serum-free medium (GF-SFM). Therefore, the effects of these adipokines on cell number (DNA content), DNA synthesis rate, cell death and on key intracellular signalling molecules were investigated. Short-term adiponectin and leptin treatment decreased DNA synthesis, measured as [3H]thymidine incorporation, as early as after 4-h exposure (P<0.01), without alterations in DNA content. Both adipokines attenuated the rate of cell death in terms of lactate dehydrogenase (LDH) activity in the culture medium after 48-h treatment (P<0.05). The specific activation of p44/42 MAP kinase (MAPK) was reduced (P<0.05) after 15-min incubation with either adipokine. In conclusion, the early decreases in DNA synthesis of primary porcine myoblasts cultured in GF-SFM in response to adiponectin or leptin are related to p44/42 MAPK signalling and adipokine treatment does not impair cell viability.


Introduction
Today, the view of adipose tissue as an active endocrine organ by its ability to secrete adipokines is well-established (Miner 2004, Hauner 2005, Gimeno & Klaman 2005).Interactions of adipokines with skeletal muscle have been reported and are of scientific interest as this tissue plays a major role in regulating metabolic homeostasis (Bonen 2010).Furthermore, gaining detailed insight into the functional cross-talk between adipocytes and muscle cells is an important aim not only in animal science (Bonnet et al. 2010) but also in human medical research (Fruehbeck 2008).Until now, most of the investigations on adipokines, such as adiponectin and leptin, focused on their function in energy metabolism of several tissues and cell types (Brochu-Gaudreau et al. 2010, Moon et al. 2013).
Concerning adipokine-mediated regulation of intracellular signalling pathways, the AMPactivated protein kinase (AMPK) was shown to be activated by globular and full-length adiponectin treatment in skeletal muscle of rats and humans (Tomas et al. 2002, Tsao et al. 2003, Mullen et al. 2009).Besides AMPK, other kinases, such as protein kinase B (Akt), mammalian target of rapamycin (mTOR) and/or p44/42 mitogen-activated protein kinase (MAPK), known to be involved in growth-mediating intracellular signalling pathways, can be phosphorylated after adiponectin treatment of murine muscle cell lines (Yamauchi et al. 2001, Wang et al. 2007, Fiaschi et al. 2009, Fiaschi et al. 2010) or human embryonic kidney cells (Lee et al. 2008).Similarly, leptin was found to affect skeletal muscle growth of rodents and humans via activation of components of the insulin signalling pathway such as Akt and p44/42 MAPK (Maroni et al. 2005, Maroni et al. 2009) and by the stimulation of AMPK (Minokoshi et al. 2002).As adiponectin and leptin can stimulate antagonistic intracellular pathways, it is difficult to predict the effects of these adipokines on muscle cell growth.This becomes even clearer by looking at inconsistent results of a few studies, most of them performed using rodent muscle, reporting about stimulating (Lamosova and Zeman 2001, Ramsay 2003, Yu et al. 2008, Fiaschi et al. 2010), inhibitory (Arita et al. 2002, Wang et al. 2005) or a lack of effects (Carbo et al. 2000).As previously shown, long-term adiponectin and leptin treatment over 24 or 48 h appeared not to have direct effects on growth and protein metabolism of proliferating porcine myoblasts (Will et al. 2012).However, the presence of growth factors in cell culture medium was suggested to be an important factor for the adipokine effects, especially of adiponectin.To elucidate further aspects of adiponectin and leptin action on the growth of porcine skeletal muscle cells under serum-free culture conditions in growth factor-supplemented serum-free medium (GF-SFM), the effects of these adipokines on cell number (DNA content) and DNA synthesis rate after short-term treatment as well as on cell death and the activation of key signalling molecules involved in energy metabolism and cell growth were investigated.

Muscle cell culture
The right and left semimembranosus (SM) muscles of newborn German Landrace piglets (n=34 males) were taken to establish a pool from all cells isolated during several preparation procedures.To isolate myogenic cells, a Percoll gradient was used as described previously (Mau et al. 2008a).Thereafter, fluorescence analysis of an aliquot of cells immunostained for desmin resulted in 86 % muscle satellite cells in the pool.The flow cytometry step was performed using an EPICS-Elite flow cytometer (Beckman Coulter, Krefeld, Germany) as described before (Mau et al. 2008a, Loehrke et al. 1998).Satellite cell-derived myoblast cultures were used.
The guidelines set by the Animal Care Committee of the State Mecklenburg-Western Pomerania, Germany, based on the German Law of Animal Protection were observed during animal husbandry and slaughter.

Sources of adiponectin and leptin
For this study, recombinant porcine adiponectin produced in E. coli was used (Ledoux et al. 2006).The detailed process of its obtaining was already described by Will et al. (2012) and its biological activity was shown in a former study by inducing periovulatory changes in ovarian follicular cells (Ledoux et al. 2006).Recombinant porcine leptin produced in E. coli was obtained from ProSpec (East Brunswick, NJ, USA).According to the manufacturer, porcine leptin was proved to be biologically active by causing proliferation of BA/F3 cells (murine bone marrow-derived pro-B cell line) stably transfected with the long form of human leptin receptor.

Experiments
For the investigation of the phosphorylation level of AMPKα, Akt, mTOR and p44/42 MAPK in proliferating myoblasts after short-term treatment with adiponectin or leptin (Table 1), satellite cells were seeded in gelatin-coated (0.1 %) 35 mm dishes at a density of 4×10 5 cells per dish.Cells were grown for two days in MEMα and MCDB 110 (ratio 4:1) plus 10 % FBS and 10 % HS plus glutamine and antibiotics as described above.Thereafter, cells were grown in GF-SFM for 24 h.On day 4 of the experiments, cells were treated with adipokines at the same concentrations used for experiments on DNA synthesis rate and cell viability (20 ng/ml leptin, 40 µg/ml adiponectin) for 15 or 30 min.

Protein extraction, SDS-PAGE and Western Blot
The total protein lysate of the cells was extracted and supernatants were collected as described previously (Will et al. 2012).After determining the protein content using a method described by Peterson (1977), aliquots were stored at −80 °C until further analysis.After thawing, the preparation of protein aliquots for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (Will et al. 2012).Before being separated on 10 % SDS-PAGE gels (2-3 h at 125 V), fifty micrograms of protein samples contained in sample buffer (125 mM Tris-HCl, pH 6.8, 4 % SDS, 20 % glycerol, 0.05 % bromophenol blue, 10 % β-mercaptoethanol) were heated for 5 min at 95 °C.After electrophoresis, proteins were transferred to polyvinylidine fluoride (PVDF) membranes (GE Healthcare Europe GmbH, Munich, Germany; 140 mA for 2 blots, 10 V, 1.5 h).Coomassie blue staining of membranes was used to ensure the efficient protein transfer to the membranes.
To verify equal loading of the gels, α tubulin was detected using a mouse monoclonal anti-α tubulin antibody (1:5 000; T5168, Clone B-5-1-2, Sigma-Aldrich).After blocking of the membranes with a solution of 5 % dry milk and Tris-buffered saline for 1 h at room temperature, the incubation with primary antibodies (5 % dry milk and Tris-buffered saline with 0.1 % Tween 20; TBST) was performed overnight at 4 °C.The phosphorylation levels of AMPK (on Thr172, rabbit mAb no.2535), Akt (on Ser473, rabbit mAb no.4060), mTOR (on Ser2448, rabbit mAb no.2971) and p44/42 MAPK (on Thr202/Tyr204, rabbit mAb no.4370) were studied.With the exception of Akt and pAkt antibodies, which were used at dilutions of 1:2 000 and 1:4 000, respectively, all other antibodies were diluted as recommended by the manufacturer.Additionally, all membranes were incubated with antibodies specific for the core proteins (AMPKα no.2603, Akt no.9272, mTOR no.2983, p44/42 MAPK no.4695).All used antibodies were purchased from Cell Signaling Technology (New England Biolabs, Frankfurt, Germany).After treatment with the primary antibody, membranes were washed three times for 10 min in TBST followed by incubation with horseradish peroxidase (HRP)conjugated donkey anti-rabbit IgG (1:5 000; sc-2313; Santa Cruz Biotechnology) or, for α tubulin with HRP-conjugated sheep anti-mouse IgG (1:10 000 or 1:25 000; NA931-100 µl, GE Healthcare), for 1.5 h at room temperature.For the development of the membranes, the ECL Plus Western Blotting Detection System (RPN 2132; GE Healthcare) and a Chemocam HR-16 imager (INTAS, Göttingen, Germany) were used.The LabImage 1D Software (Kapelan Bio-Imaging GmbH, Leipzig, Germany) was utilized to quantify the intensities of the gained protein bands.According to Rehfeldt et al. (2010), signals derived from phosphospecific antibodies were normalized for the expression of their respective core protein to estimate the specific phosphorylation level of the investigated signalling molecules.The analyses were done in triplicates for each protein.

Cell viability
For the experiments on adipokine-mediated effects on cell viability (Table 1), satellite cells were cultured in GF-SFM in 96-well plates as described above.The rates of cell death were determined by measuring lactate dehydrogenase (LDH) activity in cell culture supernatants, as described previously (Mau et al. 2008b), after 10-, 24-and 48-h exposure to leptin (20 ng/ ml) or adiponectin (40 µg/ml).

Statistics
Analysis of variance was performed using the GLM procedure of SAS (Version 9.2; SAS Institute Inc., Cary, NC, USA) with experiment, treatment and respective interactions and side of the plate as fixed factors for experiments on DNA synthesis rate and DNA content.The considered fixed factors for analysis of LDH activity were treatment and side of the plate.
Treatment was the fixed factor for the analysis of the data on specific activation of signalling molecules.Values of P<0.05 and 0.05<P<0.10 were considered to be statistically significant and as a tendency, respectively.

Effects of adiponectin and leptin on DNA synthesis rate and cell viability of porcine skeletal muscle cultures
In addition to 24-h and 48-h exposure to the adipokines (Will et al. 2012), the effects of adiponectin or leptin were examined in proliferating porcine myoblast cultures after shortterm exposure (4 h) in GF-SFM.Both the addition of adiponectin and leptin recombinant proteins led to a significant decrease of DNA synthesis rate (Figure 1A), while the DNA content remained unchanged (Figure 1B).The effects of adiponectin and leptin on cell viability were examined after 10-, 24-, and 48-h exposure in GF-SFM.Treatment of cells with adiponectin or leptin for 48 h decreased LDH activity to approximately 76 % of the control value (Table 2) indicating that myoblast viability was somewhat increased by either adipokine.

Specific activation of signalling molecules
The effects of adiponectin or leptin on the phosphorylation or on their specific activation, respectively, of AMPK, Akt, mTOR and p44/42 MAPK were examined in proliferating porcine myoblast cultures after exposure times of 15 and 30 min (Figure 2).Adiponectin or leptin alone decreased the phosphorylation levels of p44/42 MAPK after 15-min (adiponectin: −39.6 %±0.9 %, P=0.03; leptin: −49.1 %±2.4 % P=0.01), but only numerically after 30-min treatment.No significant changes in the phosphorylation levels of AMPK, Akt or mTOR after myoblast exposure to either adiponectin or leptin were observed compared with untreated controls after 15 min and 30 min (Figure 2).

Discussion
In addition to findings reported in our previous study (Will et al. 2012), it is shown here that short-term treatment of primary porcine skeletal muscle cells with adiponectin or leptin in GF-SFM led to diminished DNA synthesis rate, whereas DNA content remained unaffected.Furthermore, cell viability remained unchanged after 24-h and was increased by adiponectin and leptin treatment after 48-h exposure.We further provide evidence that p44/42 MAPK signalling is involved in mediating the adipokine effects, i.e. proliferation.Not only after 48 h (Will et al. 2012), but already after 4 h, adiponectin treatment of porcine myoblasts resulted in a significant decrease of DNA synthesis rate, which may indicate a delayed onset of proliferation.This is suggested to result from the inhibition of bFGF, which was identified to be the reason for attenuated DNA synthesis of porcine myoblasts after adiponectin long-term treatment (Will et al. 2012).The decrease in the specific activation of p44/42 MAPK after 15-and 30-min exposure to adiponectin, observed in the present study, could also result from an adipokine-mediated attenuation of bFGF action, as p44/42 MAPK is activated by extracellular stimuli like growth factors or hormones (Cobb & Goldsmith 1995).As p44/42 MAPK is an important mediator of cell proliferation, the decline in its specific activation, observed in GF-SFM, may result in a reduced DNA synthesis rate.Further studies on porcine skeletal muscle cells with respect to receptor ligand binding under GF-SFM culture conditions may clarify the adiponectin effects in more detail.Whether the inhibition of p44/42 MAPK in myoblasts by adiponectin is of biological relevance and results in changes in the activities of down-stream targets under the conditions used in this study needs further elucidation.Short-term leptin effects on signalling as well as DNA synthesis rate were similar to those of adiponectin.These effects, however, are considered to be transient, because after long-term treatment with leptin over 24 h or 48 h, any responses of porcine myoblasts in DNA synthesis rate or DNA content were no longer detectable under identical culture conditions (Will et al. 2012).As the event of proliferation occurs as a consequence of DNA synthesis, a decrease in DNA content might be probably not yet apparent after 4-h treatment.Under low serum conditions, the phosphorylation level of p44/42 MAPK behaved differently in response to the adipokines.There was a transient activation of p44/42 MAPK after 15 min, followed by decreases after 60 and 180 min, which was associated with increases in DNA synthesis rate and cell death (Will et al. 2013).
In the long range, the presence of adiponectin or leptin in the culture medium seemed to improve cell viability of the myoblasts as LDH activity was lower in medium samples taken at 48-h, but not after 10-h and 24-h treatments.However, under GF-SFM conditions, the rate of cell death was very low at each of three time points examined, nearly 30-50 % in comparison to low serum (1 % FBS) conditions (Will et al. 2013).In conclusion, adiponectin and leptin treatment will not enhance the rate of cell death in primary porcine myoblast cultures, if growth factors are present in cell culture medium.In contrast, cell viability was transiently decreased by about 30 % in response to adiponectin or leptin after 24-h treatment under low serum conditions (Will et al. 2013).The different responses of porcine myoblasts to the adipokines in GF-SFM compared to low serum conditions suggest that adipokine actions on muscle cells may be modified by endocrine and paracrine actions.
The phosphorylation levels of AMPK, Akt and mTOR were not changed under leptin or adiponectin treatment of cells for 15 or 30 min in GF-SFM, which might be due to a cell typespecific activation of intracellular signalling pathways by adiponectin and leptin.Although various studies have reported on adiponectin-induced activation of AMPK in muscle cells or muscle tissue, this was only in the context of glucose transport or fatty acid oxidation (Yoon et al. 2006).Several other studies showed an activation of AMPK and Akt after leptin treatment (Maroni et al. 2005, Minokoshi et al. 2002).However, nothing is known about intracellular signalling pathways involved in adipokine-mediated effects on cell proliferation of primary porcine myoblasts.Thus, the animal species, the nature of the cells used, culture conditions or the specific preparations of recombinant adiponectin or leptin may be responsible for the lack of activation of these signalling molecules in our study.The growth medium used in this study was energetically balanced.Therefore, adiponectin and leptin might not lead to regulatory cell responses regarding energy metabolism and any short-term effects may be compensated very fast.Moreover, in contrast to other studies using immortalized cell lines, in this study primary skeletal muscle cells were used, which may exhibit different sensitivity to external signals.
In conclusion, short-term adiponectin or leptin treatments resulted in similar decreases in DNA synthesis rate of porcine skeletal muscle cells when cultured under growth factorsupplemented, serum-free conditions.No negative effects were observed on cell viability within 48 h.Phosphorylation levels of AMPK, mTOR and Akt remained unchanged during incubation of primary myoblasts with adiponectin or leptin over a period of 30 min.However, the phosphorylation levels of extracellular-signal regulated kinases-type MAP kinases (p44/42 MAPK) were at least transiently reduced during the initial period of adiponectin or leptin stimulation of cells.Additional research is needed to clarify the lack of effects of adipokine treatment on other key signalling molecules and to identify factors acting as modulators for adipokine action on muscle cell growth. Figure

Figure 2
Figure 2 Activation of various intracellular signalling molecule untreated cultures (control) or after application of 40 ng/ml leptin for 15 or 30 min in growth factor-supple

Figure 2
Figure 2 Activation of various intracellular signalling molecule untreated cultures (control) or after application of 40 ng/ml leptin for 15 or 30 min in growth factor-supple The number of observations per treatment was n=3 for each protein.*P<0.05 vs. control set as 100 %.B: Typical Western Blot of the specific activation of AMPKα.Bands were detected at ~65 kDa.C: Typical Western Blot of the specific activation of Akt.Bands were detected at ~60 kDa.D: Typical Western Blot of the specific activation of mTOR.Bands were detected at ~200 kDa for pmTOR and mTOR, respectively.E: Typical Western Blot of the specific activation of p44/42 MAPK.Bands were detected at ~44 and 42 kDa for pp44/42 MAPK and p44/42 MAPK, respectively The number of observations per treatment was n=3 for each protein.*P<0.05 vs. control set as 100 %.B: Typical Western Blot of the specific activation of AMPKα.Bands were detected at ~65 kDa.C: Typical Western Blot of the specific activation of Akt.Bands were detected at ~60 kDa.D: Typical Western Blot of the specific activation of mTOR.Bands were detected at ~200 kDa for pmTOR and mTOR, respectively.E: Typical Western Blot of the specific activation of p44/42 MAPK.Bands were detected at

Figure 1
Figure 1 DNA synthesis rate (A) and DNA content (B) -taken as equivalent for cell number -of porcine myoblasts measured as incorporation of [³H]-thymidine over 4 h in response to a 4-h exposure to recombinant porcine adiponectin (40 µg/ml) or leptin (20 ng/ml) in growth factor-supplemented, serum-free culture medium

Table 2
Lactate dehydrogenase (LDH) activity in supernatant as a measure for rates of cell death in proliferating porcine myoblast cultures after treatment with adiponectin (40 µg/ml) or leptin (20 ng/ml) for 10, 24 or 48 h in growth factor-supplemented, serum-free medium