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Temporal changes in glycogenolytic enzyme mRNAs during myogenesis of primary porcine satellite cells P.R. Henckel *, P.K. Theil, I.L. Sørensen, N. Oksbjerg Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark The objective was to study the regulation of glycogenolytic enzyme mRNAs in porcine satellite cells during proliferation and differ- entiation. Beyond 80% confluence, cells were grown in absence or presence of 1 lM insulin. The observed increases in abundance ofmRNA for glycogenin, glycogen synthase, phosphorylase kinase, phosphorylase and glycogen debranching enzyme, and no alterationsof the transporter molecule GLUT4, clearly indicate that glycogenolytic enzymes of potential importance to meat quality developmentare regulated at the gene level during myogenesis, and are heavily involved in muscle cell and muscle fibre development. The genes, how-ever, are not influenced by insulin, and the lack of response to insulin of expression of gene-encoding enzymes involved in the formationand degradation of glycogen may question the applicability of porcine cell culture systems, like the one applied, as a model to study theregulation and regulatory mechanism of energy metabolism in muscles.
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Keywords: Cell culture; Gene expression; Glycogenolytic enzymes; Pig; Satellite cells & Petersen, 2002; Lindahl, Henckel, Karlsson, & Andersen,2006; Scha¨efer, Rosenvold, Purslow, Andersen, & Henckel, To evaluate the quality of meat, whether from a con- 2002), but to study regulatory and adaptational mecha- sumer point of view or from the industry, three traits are nisms in energy conversion in muscle in response to individ- considered of uppermost importance, namely drip loss, ulti- ual stressors, more simple model systems like muscle cell mate pH and colour. All of these traits are highly influenced cultures can be useful to provide knowledge that will enable by the rate and the extent of pH development postmortem us in the future to target more precise investigations in live (Bendall & Swatland, 1989; Briskey, 1964). pH develop- animals with the aim of improving meat quality. The impor- ment is the result of the genetic prerequisites of the muscles tance of insulin in maintaining glucose homeostasis and reg- for energy production and how these are affected by the ulating carbohydrate metabolism is well recognised and environmental factors, to which animals can be exposed described (Saltiel & Kahn, 2001). Insulin in supraphysiolog- in connection with slaughter, such as handling procedures, ical concentrations is in the present investigation used pri- transport conditions, lairage conditions, stunning method marily as an enhancing agent to stimulate differentiation and killing procedure. To control all these factors is very and fusion (Ørtenblad, Young, Oksbjerg, Nielsen, & Lam- difficult, even under experimental conditions. Nevertheless, bert, 2003). As high levels of insulin are known to alter the the development and the application of whole animal mod- sensitivity of myoblasts to insulin by reducing the rate of els have given us a deeper insight into the importance of glucose uptake by the GLUT4 transporter and by reducing energy metabolism (Henckel, Karlsson, Jensen, Oksbjerg, activation of the insulin signalling cascade (Huang et al.,2002), factors which also precede the development of non- insulin-dependent diabetes mellitus, we were interested in Corresponding author. Tel.: +45 8999 1239; fax: +45 8999 1564.
E-mail address: (P.R. Henckel).
clarifying whether insulin at the concentrations used in the present experiment has any effects on energy metabo- 2.0 g of tissue were equally distributed among wells in two lism of cultured cells during cell proliferation and differen- 24-well plates (or seeded at a density of %30,000 viable cells tiation. Insulin in supraphysiological concentrations has per cm2). The two plates were seeded to study transcription also been reported to cause a decrease in protein degrada- levels in the absence and presence of insulin, respectively.
tion as well as an increase in protein synthesis in myotubes When the cells had grown to near confluence (approxi- derived from porcine myoblasts (Hembree, Hathaway, & mately 80%), cell differentiation was induced by switching Dayton, 1991). Low concentrations of insulin have been the medium to Dulbecco’s modified Eagle’s medium shown to cause an increase in Ca2+-activated proteinase (DMEM) with 10% foetal calf serum (FCS) for 24 h, and indicating an influence on the calpain system (Brooks, Goll, then to DMEM containing 5% FCS. From 80% conflu- Peng, Greweling, & Hennecke, 1983), which has recently ence, the cells were grown either in the absence or presence been supported by the observations of Theil, Sørensen, of 1 lM of porcine insulin, and cytosine arabinosid was Therkildsen, and Oksbjerg (2006a). Insulin also exerts an added to inhibit DNA synthesis. The cells were harvested influence on myogenic factors (Theil, Sørensen, Nissen, & from four wells per animal at approximately 50% and 80% confluence (50cf and 80cf, respectively), and after 0, The transition from satellite cell over proliferation and 1, 2 or 3 days of induction of differentiation (d0, d1, d2 differentiation to fused myotubes is interesting in itself, and d3, respectively). Within a plate, all four wells in a col- and cell cultures offer ideal opportunities for studying reg- umn were harvested at the same stage.
ulatory mechanisms responsible for this development.
However, most likely the transition also requires a com- plete change in energy metabolism. When satellite cells fuseinto myotubes, the requirements for energy shift from a Sample preparation, RNA extraction, cDNA synthesis survival-/proliferative-related type to an activity-related and real time RT-PCR are described in detail by Theil type, which in addition to the necessary functions for sur- et al. (2006a). In short, cells harvested during proliferation vival requires huge amounts of energy for muscle contrac- (50cf and 80cf) were pooled from four wells, while cells har- tion. We were thus interested in clarifying how this change vested during differentiation were pooled from two wells, in energy metabolism takes place by studying the transcrip- centrifuged at 1000g for 10 min and stored at À80 °C until tion of a glucose transporter protein (GLUT4) and glycog- analysis. The RNA was extracted using the RNeasy mini enin and glycogen synthase, enzymes involved in synthesis kit (Qiagen, Albertslund, Denmark), and the total RNA of glycogen, and transcription of phosphorylase, phos- was assessed after dilution by measuring the absorbance phorylase kinase and glycogen debranching enzymes, which are involved in the degradation of glycogen. It was Purified RNA was reversely transcribed with oligo-dT our intention to use muscle cell cultures as a model for primers and Superscript II RNase H reverse transcriptase studying energy metabolism in muscles of farm animals kit (Invitrogen, Taastrup, Denmark). Reversely transcribed and considering the short lifespan of these cell cultures, material (1 ll) was amplified with TaqMan Universal PCR we were interested in clarifying when one might expect Master Mix and detected quantitatively by fluorescent changes to be caused by growth rather than development.
MGB probes using the ABI 7900 HT detection system Furthermore, any additional effects of administering exog- (Applied Biosystems, Stockholm, Sweden). Primers and enous insulin on transcription of glycogenolytic genes were probes were designed specifically for each gene by using evaluated. To fulfill these purposes, primary porcine satel- Primer Express 2.0 software (Applied Biosystems, Stock- lite cells, isolated from M. semimembranosus, were cultured holm, Sweden). Details of primer/probe design and runs in vitro and harvested at various developmental stages.
of real time RT-PCR are given in Table 1. Amplicon lengthwas tested after real time RT-PCR analysis on a 2% aga- rose gel, and the single l amplicon length agreed with thepredicted length based on the nucleotide sequences (data not shown) analysed in duplicate using the ABI 7900HTdetection system (Applied Biosystems, Stockholm, Swe- Cells were isolated from M. semimembranosus in three den). Expression of target genes was normalised according female pigs at 6 weeks of age, and a culture of satellite cells to GAPDH and b-actin (housekeeping genes) (Theil, Lab- was established from each pig. The procedures for isolation ouriau, Sejrsen, Thomsen, & Sørensen, 2005). The and culturing of cells were described in detail by Theil et al.
sequences of forward primers, MGB probes and reverse (2006b). Briefly, muscle tissue was excised, stripped of fat and connective tissue, finely chopped with a pair of scissorsand digested in a Ca2+-free phosphate-buffered saline solu- Glut 4: 50-TGTGGGTGGCATGTTCTCT-30, 50-GCA- tion (PBS) containing glucose, collagenase II, trypsin and GGAAG-30, 50-CAGCATTGCCTTCTTCCTTC-30.
DNAse. Percoll gradients (20% Percoll) were used to enrich the relative proportion of satellite cells in the cell suspen- sion (Ørtenblad et al., 2003). Suspended cells isolated from Table 1Accession numbers, amplicon location, amplicon length, range of Ct values in samples and slope of standard curve of the analysed genes Glycogen synthase: 50-CCGGCTTCGGCTGCTT-30, 50- CGCAGACCCCTCGGCTTACGGTATC-30, 50-CCG-CCGGTCCAGAATG-30.
3.1. Myoblast fusion and effect of insulin Glycogen debranching enzyme: 50-TGTTCTTTCTCGA-CATTATGTTCATCT-30, 50-AGCGATCCCCTTGGA- Myoblasts were grown in growth medium, then induced to differentiate by a change to fusion medium (10% FCS) either in the presence or absence of insulin, as described Phosphorylase kinase: 50- CATCCTGCGCAAGGTCT- in Section 2. At d1 the cells were confluent, and alignment of cells was initiated. The medium was changed at d1 to 5% FCS, and extensive fusion to multinucleated myotubes was visualised during the following 48 h, whereas no changes Glycogen phosphorylase: 50- GTGGACACACAGGTG- were visualised after that point (i.e. from d3 to d4).
(Fig. 1a) was neither effected by insulin nor by stage,and remained surprisingly constant throughout the per-iod. Glycogenin transcription (Fig. 1b), on the other hand, displayed a steady and significant increase inexpression until an apparent plateau was reached at d3 The RNA concentration was calculated as: RNA con- (P < 0.001). Insulin treatment had no effect on glycogenin centration (lg/ml) = 40 · A260 · dilution factor. The exper- expression (P = 0.76). Transcription of glycogen synthase iment was regarded as a split-plot design, and mRNA (Fig. 1c), which encodes the enzyme responsible for the quantities were analysed using the MIXED procedure of elongation of the glucose string, was also increased during SAS (SAS Inst., Inc., Cary, NC) as described by Littell, proliferation and differentiation (P < 0.001), but levelled Milliken, Stroup, and Wolfinger (1996). Effects of develop- off a day earlier. As for the glycogenin gene no effect of mental stage, insulin and stage · insulin interaction were insulin was observed (P = 0.47). A similar response was tested, and repeated measurements made on cells originat- observed for the expression of the glycogen debranching ing from the same pig were accounted for by incorporating enzyme (Fig. 1d). Phosphorylase kinase (Fig. 1e) activates a random effect of pig nested within stage and insulin level.
glycogen phosphorylase, and is, as such, part of the regu- To evaluate mRNA quantities, data were obtained as Ct latory system. The transcription peaked at day 2 with an values (the cycle number at which logarithmic plots crosses almost 10-fold increment and then decreased significantly a calculated threshold line) according to the manufacturer’s throughout the ‘‘stable’’ period (P < 0.01). The expression guidelines, and were used to determine DCt values (DCt = of glycogen phosphorylase (Fig. 1f) displayed the most Ct of the target gene À Ct of the housekeeping gene). When dramatic changes. At the third day after stimulation of testing transcription of b-actin and GAPDH (house keeping differentiation, an almost 50-fold increase in expression genes), the RNA concentration (lg/ml) was incorporated as had taken place, but there was no significant difference a covariate. To exclude potential bias because of averaging between the values observed at day 2, 3 and 4. As for data that had been transformed through the equation the genes involved in the synthesis of glycogen, transcrip- 2ÀDDCt, all statistics were performed at the DCt stage.
tion of genes involved in degradation was not influenced LSMEANS of DCt values of target genes were normalised to the level observed at 50% confluence by calculating theDDCt values (DCt observed at a given stage À DCt observed at 50% confluence), and the relative mRNA quantity wascalculated by using the formula: Relative quantity = Transcription of b-actin was slightly but significantly 2ÀDDCt. However, the base number of 2 was changed influenced by development (P < 0.022), but not by insulin, accordingly, if the PCR efficiency was below 100%.
whereas GAPDH was affected both by insulin (P < 0.001) GLUT4 (arb. scale)
Developmental stage
Insulin NSStage P<0,0001Insulin*stage NS Glycogenin (arb. scale)
Developmental Stage
Glycogen Synthase (arb, scale
Developmental stage
Fig. 1. (a)–(f) Changes during the development in the transcriptional level (expressed relative to the level observed at 50% confluence) of the substancesusing a log scale on the y-axis. Significant effects of insulin and developmental stage as well as their interactions are indicated in the individual figures.
and by development (P < 0.001) as well as displaying an insulin depended on the choice of housekeeping gene insulin * development (P < 0.04).
applied. Furthermore, the changes of transcription of However, no conclusions regarding effects of stage and housekeeping genes were much lower than the changes Insulin NSStage P<0,001Insulin*stage NS Glycogen phosphorylase (arb. scale)
Developmental stage
Insulin NSStage P<0,001Insulin*stage NS Phosphorylase kinase (arb. scale)
Developmental stage
Glycogen debranching enzyme (log scale
Developmental stage
observed for the target genes. Hence to obtain a more transcription of target genes as was done for suckled robust normalisation, the mean Ct values of GAPDH and non-suckled mammary glands of lactating sows (Theil and b-actin were calculated and employed to normalise insulinemia was not caused by alterations in GLUT4 at theprotein level, but was rather attributed to alterations both It is customary to view development of myoblast cell in transcriptional and activity level of proteins involved in cultures as a two-stage process, which includes prolifera- the insulin-signalling system. In L6 myotubes, a 24 h pre- tion and differentiation. An alternative proposal for subdi- treatment with high insulin and high glucose level also vision of these stages has been put forward by Steenstrup implied a decrease in insulin-stimulated GLUT4 transloca- and Hannon (2000), who suggested that differentiation tion together with a reduced activation of the insulin-sig- actually should be subdivided into two phases: exit from nalling cascade and also a concomitant 40% increase in the cell cycle and expression of muscle-specific genes, and basal glucose uptake, which was considered to be an adap- fusion into multinucleated fibres or myotubes. As we are tive response to the treatment (Huang et al., 2002). The fact dealing with developmental changes the three-stage model that we observed no influence on the transcriptional level appears in our view to be more appropriate as it allows of GLUT4 (Fig. 1a) agrees with these results. It should for a more detailed description that relates to all the impor- be noted that long-term exposure to insulin has also been tant events in a cell transforming from one function to a shown to induce an increase in GLUT4 mRNA in human totally different function. However, given the main purpose of this investigation, it may not be of any significant impor- It is obvious that the type of cell culture used is of signif- tance in this context. According to Florini (1989), the dif- icant importance for the interpretation of the data, as to a ferent stages of development can be identified by the level certain extent it is a phenotypic response that is displayed of activity of creatine kinase (CK), and also the level of by the cells. GLUT4 transporter content has been shown myogenin has been suggested as an indicator of develop- to vary considerably from muscle to muscle, and oxidative mental stage (Florini, Ewton, & Roof, 1991). CK values muscles display higher contents of GLUT4 than glycolytic and transcriptional level of myogenin at the different muscles, and differences between bovine and porcine mus- stages, using the same cell cultures as used in the present cles have also been shown (Duhlmeier, Hacker, Widdel, study, are given by Theil et al. (2006b). Based on these von Engelhardt, & Sallmann, 2005). In our case the initial results, which showed that both CK activity and myogenin cell culture was produced from the semimembranosus mus- expression peaked at day 2, although the shape of the curve cle, which is extremely glycolytic. Consequently, we may differed, and on a visual examination of the cell cultures, expect a low content of GLUT4 in these cell cultures, one might suggest that the time from 50% to 80% conflu- and this may partly explain the lack of response to insulin.
ence is exclusively proliferative. The time from 80% to When looking at development, the results clearly indicate day 1 is a time of exit from the cell cycle and of initiating that there is no change in the expression of GLUT4. The expression of muscle-specific genes, and the time from potential capacity for transmembrane glucose transport is day 1 to day 2 is a period of fusion, and thereon observed thus unaltered during all three stages of myogenesis.
changes are primarily caused by growth of muscle fibres.
Glycogenin is the protein precurser of glycogen. It is By visual examination, however, one might expect the characterised by autocatalytic activity enabling it to add fusion to be terminated at day three, which points to a several glucose units from UDP-glucose to its active Tyr- slight discrepancy in the evaluation of the stages between 194 site (Campbell & Cohen, 1989; Smythe, Caudwell, Fer- the methods. We can be certain, however, that changes guson, & Cohen, 1988), before it becomes an integrated part observed from day 3 to day 4 can be attributed to growth of glycogen synthase for further formation of glycogen, or treatment only and not to development.
catalysed by glycogen synthase and glycogen branching During normal conditions in vivo, insulin facilitates enzyme. Glycogenin and glycogen synthase have been muscle glycogen synthesis through action on both glucose shown to exist in a 1:1 molar ratio (Pitcher, Smythe, Camp- transport and glycogen synthase activity. The effect on glu- bell, & Cohen, 1987). As the amount of glycogenin will have cose transport is mediated via insulin receptors. When insu- an influence on the storage capacity of glycogen in muscles, lin levels are increased, the subsequent activation of the it has been suggested that the production of the active glyc- intracellular signalling system via the receptors results ulti- ogenin primer has at least the potential to be an overall rate- mately in translocation of GLUT4 to the cell membrane. In limiting process in the formation of glycogen, even more normal, rested condition, GLUT4 is sequestered into spec- important than the phosphorylation and dephosphoryla- ialised storage vesicles within the muscle fibres. By declin- tion processes involved in the regulation of glycogen syn- ing insulin levels, the reverse process is initiated by thase and phosphorylase (Alonzo, Lomako, Lomako, & endocytosis of GLUT4 on the membrane surface (Watson Whelan, 1995). More recent data, however, does not sup- & Pessin, 2001). A review of the signals participating in port this concept. Hansen, Derave, Jensen, and Richter GLUT4 translocation is given by Patel, Huang, and Klip (2000) showed that both the protein level and the activity (2006). In vivo, chronic hyperinsulinemia has been shown of glycogenin were poorly correlated to maximal attainable to imply a reduction in insulin-mediated glycogen synthesis glycogen content and suggested glycogen concentration as a in muscles (Del Prato et al., 1994). Bertacca et al. (2005) possible candidate for regulation. This hypothesis has showed using human myoblasts (no fusion) that this recently been supported by Jensen et al. (2006), who showed reduced glycogen synthesis by exposure to moderate hyper- that high glycogen levels implied a reduction in glycogen synthase activity, but detailed information on how this chain to the main chain, and then the remaining glucose activity is regulated and its importance to glycogen levels units from the short chain are liberated as free glucose remains to be elucidated. We observed a two-fold increase (Bates, Heaton, Taylor, Kernohan, & Cohen, 1975) in con- in mRNA for glycogenin (Fig. 1b) during the proliferative trast to the glucose liberated by glycogen phosphorylase stage and a further 1.5-fold increase until day 3. These which is phosphorylated (glucose-1-P). Glycogen phos- results agree with those of Pak, Sangaralingham, and Pang phorylase exists in an active and an inactive form, and (1999), who showed that high levels of glycogenin appeared activity is initiated by phosphorylation of the enzyme by to correlate to the proliferative state of cardiac myocyte phosphorylase kinase. Apart from this main function phos- growth and reduced levels of glycogenin correlated to the phorylase kinase may also be part of the regulation of gly- postmitotic period. The role of glycogenin if any in regulat- cogen synthase as it has been shown to be the likely ing the ratio of proglycogen to macroglycogen both during physiological kinase for ser-7, an active site at the N-termi- synthesis and degradation is still an open question.
The synthesis of glycogen is primarily controlled through We observed an almost 50-fold increase in transcrip- regulation of glycogen synthase. The enzyme is allosteri- tional level for glycogen phosphorylase (Fig. 1f) from 50% cally regulated by glucose-6-phosphate and covalently by confluence to day 3, a 9.4-fold increase in phosphorylase reversible phosphorylation at nine known sites (Wilson kinase (Fig. 1e) from 50% confluence to day 2 and a 13-fold et al., 2005). The enzyme is inactivated by phosphorylation, increase in mRNA for glycogen debranching enzyme and full activity can be restored by the presence of glucose- (Fig. 1d) over the same time period. The development from 6-phosphate. Insulin may stimulate glycogen synthase by satellite cell to fused myotubes thus implies a complete several signalling transduction pathways, but it is currently change in capacity for glycogen metabolism, a transition believed to be mainly mediated via the phosphatidyl-inosi- that favours the capacity for glycogen degradation, which tol 3-kinase/protein kinase B pathway. This leads to inacti- is to be expected. The large increase in capacity for glycogen vation of glycogen synthase kinase-3 (Gaster et al., 2004), degradation enables the myotubes to produce large amounts which implies dephosphorylation and activation of glyco- of energy (ATP) locally by glycolysis. This is a characteristic gen synthase, as already shown by Cohen (1993). Also, of most muscles in pigs, but in particular the one from which the level of glycogen has been shown to exert a strong influ- the cell cultures were originally derived. The lack of effect of ence on the activity of glycogen synthase (Danforth, 1965; insulin on these parameters indicates that caution should be Halse, Bonavaud, Armstrong, McCormack, & Yeaman, taken when comparing such results to live conditions. It would be worthwhile to investigate whether this apparent We observed a two-fold increase in mRNA for glycogen insulin insensitivity is due to the fact that we worked on cell synthase (Fig. 1b) initially during proliferation and a fur- cultures or it can be attributed to muscle type and hence is a ther 2-fold increase during the time of exit from the cell more or less general feature of pig muscles.
cycle and initiation of expression of muscle-specific genes, Most effects of development indicate that fusion is ter- peaking at day 2 after a further 1.5-fold increase during minated at day 2. However, both the results of transcrip- the period of fusion into multinucleated fibres or myotu- bes. This was followed by a stable or slight decrease in examination of the cell cultures indicates that adaptational expression throughout the rest of the experimental period.
changes may still occur from day 2 to day 3. We are conse- Insulin had no effect on the transcriptional level of glyco- quently left with only 1 or 2 days in which we can perform gen synthase nor on any of the other enzymes investigated, investigation on the effect on energy metabolism of other which supports the concept that the effect of insulin is pri- stressors like hypoxia, electrical stimulation or exposure marily mediated via posttranscriptional control rather than to gasses. The model used here thus offers opportunities regulated at the transcriptional level.
to study the effects of short-term stressors and rapid recov- We did, however, observe a significant increase in the ery from such stressors, whereas other models should be transcriptional level of GAPDH, implying a possible chosen for studying the effects of longer-term stress and increase in activity of the glycolytic pathway for energy slow recovery. The apparent insensitivity to insulin, how- or ATP production as an effect of insulin. GAPDH has ever, should be clarified, before valid comparisons to live previously been used histochemically as an indicator of the glycolytic capacity of muscle fibres.
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