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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.
Ó 2006 Elsevier Ltd. All rights reserved.
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: email@example.com (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
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-
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)
Insulin NSStage P<0,0001Insulin*stage NS
Glycogenin (arb. scale)
Glycogen Synthase (arb, scale
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)
Insulin NSStage P<0,001Insulin*stage NS
Phosphorylase kinase (arb. scale)
Glycogen debranching enzyme (log scale
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.
enzymes: glycogen phosphorylase and glycogen debran-ching enzyme. Glycogen phosphorylase catalyses the
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Revista HISTEDBR On-line Artigo LA EDUCACION: ESTRATEGIA DE RESISTENCIA Y RECONSTRUCCIÓN ORGANIZACIONAL CAMPESINA COOPERATIVA.1 Doctor en Educaçao pela Universidade Estadual de Campinas Profesor do Departamento de Educaçao da Universidad De La Frontera, Temuco, Chile . RESUMO: El Articulo Analiza la funcion de los Procesos Educacionales en la Resistencia y Reorganizac