Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses

Research in Veterinary Science 89 (2010) 445–450

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Abundance of some skeletal muscle mitochondrial proteins is associated with increased blood serum insulin in bovine fetuses Beata Pajak a, Patrycja Pawlikowska b, Isabelle Cassar-Malek c, Brigitte Picard c, Jean-François Hocquette c, Arkadiusz Orzechowski a,d,* a

Mossakowski Medical Research Center, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland Cochin Institute, Department of Hematology, INSERM, U567, CNRS, UMR 8104, Paris Decartes University, UMR-S 8104, Paris, F-75014, France INRA, UR1213, Unité de Recherches sur les Herbivores, Equipe Croissance et Métabolisme du Muscle, Theix, 63122 Saint-Genès Champanelle, France d Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Nowoursynowska 159, 02-776 Warsaw, Poland b c

a r t i c l e

i n f o

Article history: Accepted 13 April 2010

Keywords: ATP synthase Bovine skeletal muscles Cytochrome-c oxidase Insulin Myogenesis Pregnancy

a b s t r a c t The aim of this study was to investigate the evolution of the abundance of cytochrome oxidase c subunit IV (NCOIV) and beta subunit of ATP synthase (b-ATP) during the last third of gestation in bovine skeletal muscles. Semitendinosus, longissimus thoracis and rectus abdominis muscles were chosen for the immunoblotting of the respective protein levels. Muscle and blood samples from bovine fetuses of randomly selected breeds were collected at 180, 210, and 260 days post-conception (dpc). The muscle tissue expressions of NCOIV, b-ATP were compared to blood glucose and insulin. At 260 dpc, protein levels of NCOIV raised in skeletal muscles. Additionally, b-ATP in semitendinosus and longissimus thoracis were elevated and paralleled by higher concentrations of blood serum insulin. It corroborates our previous observations indicating that accelerated metabolic differentiation of bovine skeletal muscles is associated with elevated blood insulin and occurs during the last trimester of gestation. Our observations point to the connection between insulin-sensitivity and the molecular mechanisms of mitochondrial contribution to ontogenesis of skeletal muscles. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Muscle ontogenesis leads to molecular changes which determine the fibre typing of skeletal muscles at birth. Adult skeletal muscles are composed of multinucleated fibres with different metabolic properties which are adapted to the energetic requirements of the tissue, although these properties might be also answerable for specific responses to particular stimuli, for example to hormones or exercise. Several studies suggested that the metabolic differentiation of skeletal muscles and heart occurs at the last third of fetal growth (Hocquette et al., 2006; Gagnière et al., 1997, 1999a,b; Picard et al., 1994). It was observed that especially during the last trimester of pregnancy muscle fibre composition undergoes changes. The transition either into more oxidative or more glycolytic muscle fibre phenotype depends of animal species (Gondret et al., 1996; Picard et al., 1994, 1995). In cattle, they are

* Corresponding author at: Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (SGGW), Nowoursynowska 159, 02-776 Warsaw, Poland. Tel.: +48 022 593 62 43; fax: +48 022 847 24 52. E-mail addresses: [email protected], arkadiusz_orzechowski@ sggw.pl (A. Orzechowski). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.04.013

accompanied by the elevated activity of lactate dehydrogenase (LDH), and of isocitrate dehydrogenase and succinate dehydrogenase (ICDH and SDH, respectively), two key mitochondrial enzymes in the tricarboxylic acid cycle and indirect indicators of muscle oxidative capacity (Gagnière et al., 1999b; Hocquette et al., 1998). Overall, such changes suggest the importance of enzymes of mitochondrial electron transport chain (ETC) in the control of the growth and differentiation of muscle cells (Mikula et al., 2005; Rochard et al., 2000; Wrutniak-Cabello et al., 2001). The composition in type I (oxidative) muscle fibres is also associated with higher level of sensitivity to insulin since the increase in mitochondrial protein expression was demonstrated in response to insulin (Boirie et al., 2001). In accordance, skeletal muscle glucose uptake is regulated through insulin-dependent regulation of mitochondrial gene expression (Huang et al., 1999; Lemosquet et al., 2002). In our ‘‘in vitro” studies performed on clonal C2C12 and L6 muscle cell lines we reported that insulin stimulates biogenesis of mitochondria in muscle cells (Pawlikowska et al., 2006). Moreover, the insulin-induced myogenesis was delayed by metabolic inhibitors of ETC. Insulin promoted myogenesis through PI3-Kmediated Akt phosphorylation (Lawlor and Alessi, 2001) and this effect was further accelerated by the MEK inhibitor PD98059 (Kwiecin´ska et al., 2005). Interestingly, mitochondrial GTPase

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2. Materials and methods 2.1. Animals and samples This study was carried out as part of a research program approved by the ‘‘Institut National de la Recherche Agronomique” (INRA, France) Ethical Committee. The study included bovine (Bos taurus) fetuses of randomly selected breeds collected at day 180, 210, and day 260 post-conception as previously described by Cassar-Malek et al. (2007a): 12 samples (n = 12) of each muscle (m. semitendinosus – ST, m. rectus abdominis – RA, and m. longissimus thoracis – LT) were taken from deep freezer ( 80 °C) for further studies. The rationale for sampling these three muscles was that they can be classified from the most slow oxidative to the most fast glycolytic in the following order: RA > LT > ST. Deeply frozen blood serum samples (n = 12) collected from the same fetuses were used to determine fetal concentration of blood plasma glucose and insulin levels.

2.4. Electrophoresis and immunoblotting Frozen tissues were homogenized in a freshly prepared buffer composed of urea (8.3 M), thiourea (2 M), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 2%), dithiotreitol (DTT, 1%), and PMSF (1 mM). Insoluble material was removed by centrifugation at 10,000g for 30 min at 8 °C. Soluble protein concentrations in the supernatant fraction were determined by a specific binding of copper ions to protein (PlusOne 2D Quant Kit, Amersham Biosciences, San Francisco, CA, USA) using bovine serum albumin (BSA) as standard. In brief, protein was precipitated, supernatant discarded, and protein pellet resuspended according to the manufacturer protocol in order to determine protein concentrations. The remainder of the sample was stored at 80 °C. Equal amounts (30 lg) of proteins from the crude preparations were used for Western blot analysis (SDS–PAGE, 12%). Immunoblotting was performed as previously described (Hocquette et al., 1996; Orzechowski et al., 2005) using commercial antibodies at a final dilution ranging from 1:750 to 1:2000 depending on selected protein. A horseradish peroxidase-labeled second antibodies were always used at a final dilution of 1:10,000. Blots were transiently stained with Ponceau S dye to check that equal amounts of protein were loaded in each lane. After washing in TBS containing 0.05% (v/v) Tween 20, the membranes were immunostained by standard methods provided by the manufacturers. The enhanced chemiluminescence (ECL) method was used for antibody–antigen complexes detection (Amersham Int., Aylesbury, UK). Blots were developed on X-ray films. The relative abundance of determined proteins was assayed by densitometric analysis (integrated optical

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2.2. Analytical methods Blood samples were collected into tubes containing heparin at a very low concentration (2 IU/ml) to avoid interference with the insulin radioimmunoassay performed according to Faverdin (1985) as quoted by Lemosquet et al. (2002). Samples were kept on ice and immediately centrifuged at 2500g for 10 min at 4 °C. Plasma was aliquoted and stored at 20 °C. Plasma glucose assay was performed on a Cobas analyzer (Roche, Neuilly-sur Seine, France) using enzymatic kit D-glucose oxidase D-peroxidase (Unimate 7 Gluc PAP 073-6740, Roche, Neuilly, France). Plasma insulin was determined by radioimmunoassay adapted to bovine samples with the INSI-PR kit (Cis Bio-International, Gif sur Yvettes, France). 2.3. Reagents for studies of enzyme proteins All reagents for enzymatic analyses were of high purity, and unless otherwise stated they were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). Primary mouse monoclonal anti-cytochrome-c oxidase subunit IV (NCOIV) and anti-ATP synthase subunit beta (b-ATP synthase) antibodies were obtained from Molecular Probes (Eugene, Oregon, USA). Secondary HRP-conjugated donkey antimouse were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Sodium dodecyl sulfate (SDS) 10% (w/ v), Sequi-Blot PVDF membrane 0.2 lm and all reagents for immunoblotting were obtained from Bio-Rad Laboratories (Hercules, CA, USA).

Plasma glucose Glucose concentration [mM]

mitofusin 2 (Mfn2) seems to play a crucial role for the insulin-induced muscle fibre formation (Pawlikowska et al., 2007). Taken together, these data indicate that higher insulin sensitivity evokes oxidative metabolism, and that muscle development is associated with higher contribution of oxidative-type muscles. Accordingly, mitochondria are the main insulin target to provide energy indispensable for protein synthesis and gene transcription in skeletal muscles (Stump et al., 2003). Consequently, in insulin resistant states this effect is not achieved and one might even predict insulin resistance (Petersen et al., 2004) if biogenesis of mitochondria is impaired since less subsarcolemmal mitochondria were found in obesity and type 2 diabetes (Ritov et al., 2005). Sudden augmentation in insulin blood plasma is observed at the perinatal period, with extreme rise at parturition. In this study, we have examined temporal relationships between insulin blood levels and muscle abundance of mitochondrial proteins in bovine fetuses during the last trimester of gestation.

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Fetal age [dpc] Fig. 1. Vertical scatter plots showing blood serum concentrations of insulin or glucose in the individual blood serum samples obtained from bovine fetuses at increasing age (dpc). Blood serum insulin (A, lU/ml) and glucose (B, mM) average values are evidenced by horizontal lines. Means ± SEM that differs significantly from other means is ticked by asterisk.

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density, IOD) of immunoblots and expressed as OD  mm sity/area).

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Actually, blood serum glucose (mM) ranged from 0.85 to 0.91 between 180 and 260 dpc (Fig. 1A). 3.2. Abundance of mitochondrial NCOIV increases at 260 dpc in the three examined skeletal muscles

2.5. Statistical evaluation Results were statistically evaluated using one way ANOVA and Tukey’s comparison multiple range test by GraphPad Prism™ version 4.0 software (GraphPad Software Inc., San Diego, CA, USA). Results are expressed as mean ± SEM and a value of p < 0.05 was determined to be significant, p < 0.01 as highly significant and p < 0.001 as very highly significant.

3. Results

Analysis of changes in the abundance of NCOIV in every oxidative-glycolytic skeletal muscle type revealed relatively stable expression of the subunit IV of mitochondrial cytochrome oxidase c at 180 and 210 dpc. No differences were observed between these two fetal ages for the average intensity/area ratios (Fig. 2A). In contrast, at 260 dpc the average intensity/area ratios increased significantly (p < 0.05) by 33%, 40%, and 93% for ST, LT, and RA, respectively (Fig. 3A). 3.3. Abundance of mitochondrial b-ATP synthase increases at 260 dpc of bovine fetal age in ST and LT muscles but not in RA muscle

3.1. Dissociation in blood serum levels of glucose and insulin Between 210 and 260 dpc average blood serum insulin raised to 10.15 lU/ml and was significantly higher (p < 0.05) than that at 180 dpc (8.14) or 210 dpc (8.94 lU/ml) (Fig. 1B). Blood serum glucose concentration did not come across elevated insulin level.

Similarly to NCOIV, the abundance of the beta subunit of mitochondrial ATP synthase remained stable between 180 and 210 dpc (Fig. 2B). A significant rise (p < 0.05) in the abundance of this pro-

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Fig. 2. Western blot analysis of muscle sample proteins: (A) nucleus encoded subunit IV of cytochrome-c oxidase (NCOIV). (B) Beta subunit of ATP synthase (b-ATP). From left to right the immunoblots are shown from individual muscle samples of bovine fetuses at 180, 210, and 260 dpc, respectively. Arabic numerals (1–12) stand for triads of samples representing fetuses of increasing age (180 > 210 > 260 dpc). Total protein extracts (30 lg protein per sample) from semitendinosus (ST) or rectus abdominis (RA) or longissiumus thoracis (LD) muscles were resolved by 12% SDS/PAGE followed by immunobloting (see Section 2). Molecular weight is indicated with arrowheads.

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tein was observed at 260 dpc in two (ST and LD) of three examined muscles (Fig. 2B). An increase in the average intensity/area ratio by 45% and 52% was registered in ST and LT, respectively. b-ATP synthase level did not change significantly in RA (Fig. 3B).

4. Discussion During the last few decades muscle development was extensively studied. Generally speaking, prenatal muscle fibres are

formed through the subsequent assembly and fusion of mononucleated myogenic cells called myoblasts. Several steps of muscle fibre formation lead to the generation of primary, secondary and adult muscle fibres. Finally, muscle fibres undergo metabolic differentiation which is featured by changes in the expression of step-limiting enzymes for contractile (e.g. myosin heavy chain, MyHC) and metabolic activities (e.g. lactate, isocitrate or succinate dehydrogenases, LDH, ICDH, and SDH, respectively). It was previously reported that in prenatal FG or FOG muscles (Picard et al., 2002) as well as in the heart (Hocquette et al., 2006), LDH activity

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Fig. 3. Bar charts calculated for blots showing optical density (IOD) expressed per unit area (OD  mm 2). (A) Average values of optical density are shown as index of NCOIV protein expression. (B) Similar layout of bar charts with respect to protein expression of b-ATP synthase. Means ± SEM are present with significant differences between means as indicated by different lowercase letters.

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increases with fetal age in concert with metabolic differentiation. However, it does not necessarily mean that metabolic differentiation is accompanied by rise in the level of respective enzyme, although these process is evolutionary conserved and species specific (Picard et al., 2002). In this experiment we hypothesized that hormonal control exerted by insulin is involved in the shift from glycolytic to more oxidative muscle fibre type. 4.1. Ontogenesis of metabolic enzymes in muscles Abundance of some proteins of the mitochondrial oxidative phosphorylation system (OXPHOS) was evaluated in muscle samples at the beginning (180 dpc) middle (210 dpc), and end (260 dpc) of the third trimester of gestation. The results of this study indicate that prior to parturition there is a substantial increase in the abundance of NCOIV in all three examined skeletal muscles (ST, RA, and LT). Similarly, a rise of b-ATP was observed in ST and LT but not in RA muscle. The lack of elevation of b-ATP is puzzling, although RA is the most oxidative-type muscle in the examined group. In the oxidative-type muscle the low throughput of myosin ATP-ase might explain stable expression of ATP synthase during metabolic differentiation. Additionally, changes in the expression of NCOIV and b-ATP are less likely to occur as a result of specific breed type, since no specific bovine breed was used in this study. In younger fetal age (180 vs. 210 dpc) no changes in the abundance of some OXPHOS enzymes were observed. These findings strongly support general believe that metabolic differentiation of skeletal muscle is triggered at the end of pregnancy but it differs with respect to enzyme and muscle type, but not the type of breed. At that time fetal hormonal profile has to be recalled. Insulin secretion and subsequent rise of blood serum insulin is the physiological reaction to hyperglycemia. In our study this was not the case, since average serum levels of blood glucose remained almost the same regardless of examined fetal age (euglycemia) (Fig. 1A). In contrast, insulin concentration increased at the end of fetal age (Fig. 2B, p < 0.05). The absence of a temporal relationship between serum blood glucose and insulin in the bovine fetuses suggest distinctive mechanism of elevated insulin secretion (release from inhibition?). These results agree with those of Cassar-Malek et al. (2007b) who observed no change in activities of mitochondrial enzymes citrate synthase (CS) and cytochrome-c oxidase (COX) in various muscle types between 180 and 210 dpc but an increase between 210 and 260 dpc, especially in the most oxidative muscles. More generally, Lehnert et al. (2007) observed an increase in the expression of genes encoding metabolic enzymes at 195 dpc compared to 60 or 135 dpc whereas expression of genes encoding collagen types was reduced between 60 and 135 dpc. In addition, the study of Gagnière et al. (1999b) and Cassar-Malek et al. (2007a) at the enzymatic level and also that of Sudre et al. (2003, 2005) at the transcript level both indicated that myogenesis occurs at slightly different rhythms or speeds depending on the muscle despite a common general pattern of muscle differentiation. Although highly regulated before birth, myogenesis at genomic level is also affected by the type of bovine genotype (Sudre et al., 2005) and by major genes such as myostatin both at young (Potts et al., 2003) and old fetal stages (Cassar-Malek et al., 2007a) which determines the final functional characteristics of muscle fibres. In our study, however, we did not observe individual differences between the type of breed in the pattern of elevated immunoreactivity of NCOIV and b-ATP (Fig. 2). The huge changes in metabolic activity which occurs in skeletal muscles before birth were also observed in the bovine heart as shown by an increase in ICDH activity and in GLUT4 expression during the last third of gestation (Hocquette et al., 2006). These metabolic changes prior to birth are hypothesized to render the muscle tissues and the heart (which have a high metabolic activity)

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well adapted to the switch which occurs at birth from carbohydrates to fats as main energy sources. By contrast, the differentiation of adipose tissue (whose function is to store energy rather than to use it) is quite different from that of muscle tissues (Hocquette et al., 2006). Previous studies identified temporal relationships between mitochondrial development in skeletal muscles and thyroid hormone ontogeny suggesting the importance of thyroid hormones in the differentiation of bovine muscles (CassarMalek et al., 2007b). The present study examined the potential involvement of insulin and insulin action in this process. 4.2. Differentiation of the glucose–insulin axis Previous data from the literature confirm these observations but underline that blood glucose concentration is much higher during the first two thirds of gestation (Hocquette et al., 2006). Elevated blood serum levels of insulin prior to calving associated with lower expression of insulin and IGF-I receptors at least in the heart (Hocquette et al., 2006) suggests a regulation of insulin sensitivity throughout gestation. This could be linked to accelerated anabolism in fetal tissues including skeletal muscles. These results recapitulate our former data obtained ‘‘in vitro” using clonal muscle cell lines (L6 rat myoblasts, C2C12 mouse satellite cells). Insulin highly significantly enhanced the expression of NCOIV and b-ATP synthase in myotubes and this effect was in causal relationship with insulin-dependent phosphatidylinositol-3-kinase signaling pathway. Thus, from the present ‘‘ex vivo” study, it is clear that higher levels of insulin parallel the elevated expression of mitochondrial OXPHOS proteins. Insulin targeted several organs including skeletal muscles especially when peripheral blood glucose level got high. Nevertheless, no raise in fetal blood glucose was recorded suggesting that other mechanisms were involved to drive insulin secretion from fetal pancreatic beta islets. We speculate that in end-term fetuses there was a shift to more oxidative metabolism, and that insulin might play a crucial role for this shift. Insulin resistance in peripheral tissues or higher sensitivity of beta-cells in pancreatic islets or both might be an alternative explanation for mild hyperinsulinemia observed at 260 dpc. To shed more light on the metabolic effects of insulin on muscle mitochondria prior to parturition one has to bear in mind that insulin action is markedly impeded by glucocorticoids (Dupont et al., 1999) and affect energy metabolism in mitochondria (Dumas et al., 2003). Obviously, to trigger parturition glucocorticoids must be released from adrenal cortex. Thus, we suggest, that elevated blood serum insulin might counteract the catabolic action of glucocorticoids. More importantly, retrograde (insulin vs. glucocorticoids) regulation is possible, as we provide evidence from previous ‘‘in vitro” studies where insulin-dependent myogenesis did not occur if either, the activity of particular respiratory enzyme complexes (OXPHOS) or mitochondrial membrane potential are declined. Additionally, one has to keep in mind that by the third trimester there is an adaptation of nutrient supplies to the fetus. Glucose is shifted from mother leaving more available for fetal energy metabolism. At the same time higher insulin levels accelerate glucose utilization. Overall, results obtained from this study strongly indicate, that in addition to thyroid hormones, insulin may also contribute to the metabolic differentiation of skeletal muscles by enhancing their mitochondrial biogenesis prior to parturition. Further studies are needed to depict the molecular mechanisms governing the imbalance in glycolytic vs. oxidative-type muscle fibres with respect to insulin. Acknowledgements The authors are grateful to Christiane Barboiron for her help in Western blotting and densitometric analyses. Support for this work

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was partially provided by a grant no. N401 033 32/0759 and grant no. 117/E-385/SPB/COST/P-06/DWM from the Ministry of Science and Higher Education in Poland. This study was performed in the frame of COST 925 Action on ‘‘The importance of prenatal events for postnatal muscle growth in relation to the quality of muscle based foods”.

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