# Diabetes Alters Contraction-Induced Mitogen   Activated Protein Kinase Activation in the Rat Soleus and Plantaris

Authors: A. Katta; D. L. Preston; S. K. Karkala; S. Asano; S. Meduru; S. P. Mupparaju; E. Yokochi; Kevin M. Rice; D. H. Desai; E. R. Blough
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

The prescription of anaerobic exercise has recently been advocated for the management of diabetes; however exercise-induced signaling in diabetic muscle remains largely unexplored. Evidence from exercise studies in nondiabetics suggests that the extracellular-signal-regulated kinases (Erk1/2), p38, and c-JUN NH2-terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs) are important regulators of muscle adaptation. Here, we compare the basal and the in situ contraction-induced phosphorylation of Erk1/2- p38- and Jnk-MAPK and their downstream targets (p90rsk and MAPKAP-K2) in the plantaris and soleus muscles of normal and obese (fa/fa) Zucker rats. Compared to lean animals, the time course and magnitude of Erk1/2, p90rsk and p38 phosphorylation to a single bout of contractile stimuli were greater in the plantaris of obese animals.  Jnk phosphorylation in response to contractile stimuli was muscle-type dependent with greater increases in the plantaris than the soleus. These results suggest that diabetes alters intramuscular signaling processes in response to a contractile stimulus.


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## Body

## 1. Introduction

Exercise has long been recognized to have important health benefits for people with type
II diabetes. The molecular events underlying exercise-induced adaptations in
diabetic muscle remain largely undefined. It is thought that beneficial effects
of exercise on structural and functional adaptations of muscle are mediated
through the activation of various signaling molecules, which activate signaling
cascades involved in regulating changes in gene expression. Similar to that
seen with aerobic exercise modalities [1–3], recent data has suggested
that anaerobic exercise may also be beneficial in the treatment of diabetes [4–11]. For example, progressive resistance training
has been found to improve glycemic control, increase skeletal muscle
size and strength, and positively change body composition by increasing lean
body mass and decreasing visceral and total body fat [12–15]. Whether diabetes is
characterized by differences in how muscle responds to resistance training is
not well understood.Although not fully delineated, it has been postulated that the mitogen activated protein
kinase (MAPK) signaling cascade may be particularly important in mediating the
effects of altered contractile activity. Indeed, muscle contraction and
load-induced muscle hypertrophy in humans and rodents have been found to be associated with the
activation of the extracellular signal-regulated kinases 1/2 (Erk1/2), p38, and
c-Jun NH2-terminal kinase (Jnk) MAPK proteins [16–20]. Several
downstream substrates of MAPK signaling have also been identified, including the
Erk1/2-dependent protein MAPK-activated proteinkinase-1 (MAPKAP-K1), also
called p90 ribosomal S6kinase (p90rsk) [21] and the p38-dependent protein
MAPK-activated protein kinase-2 (MAPKAP-K2) [22, 23]. It
is thought that the phosphorylation (activation) of MAPK proteins and their
substrates is important in regulating such diverse processes as gene
expression, glucose uptake, cell replication, and protein synthesis [16, 24, 25]. To our knowledge, the effect
of diabetes on contraction-induced MAPK signalingin muscle has not been investigated.The purpose of this study was to examine whether diabetes alters MAPK signaling after an acute bout of contractile
activity. We hypothesized that contraction induced MAPK signaling would differ between normal and diabetic muscles. To
test this hypothesis, the phosphorylation status of Erk1/2-, p38-, and Jnk-MAPK
and their downstream kinases were analyzed in the plantaris and soleus muscles
of the normal and diabetic rats immediately after and during the recovery phase
of an acute bout of high-frequency electrical stimulation. Taken together, our
data suggests that diabetes alters contraction-induced MAPK phosphorylation in
skeletal muscle.


## 2. Materials and Methods

### 2.1. Animal Care

Young (10 weeks,n = 12) male lean
normal Zucker (LNZ) and young (10 weeks, n = 12) male obese syndrome-X Zucker (OSXZ) rats were
obtained from the Charles River Laboratories. Rats were housed to a cage in an
AAALAC approved vivarium. Housing conditions consisted of a 12H: 12H dark-light
cycle and temperature was maintained at 22° ± 2°C. Animals were provided food
and water ad libitum and allowed to recover from shipment for at least two
weeks before experimentation. During this time, the animals were carefully
observed and weighed weekly to ensure none exhibited signs of failure to
thrive, such as precipitous weight loss, disinterest in the environment, or
unexpected gait alterations. All procedures were performed as outlined in the
Guide for the Care and Use of Laboratory Animals as approved by the Council of
the American Physiological Society and the institutional Animal Use Review
Board.


### 2.2. Materials

Anti-Erk1/2 (#9102), phospho-Erk1/2Thr202/Tyr204 (#9106), p38 (#9212), phospho-p38Thr180/Tyr182 (#9216), SAPK/Jnk
(#9252), phospho-SAPK/JnkThr183/Tyr185 (#9251), p90rsk (#9334), phospho-p90rsk (Ser380#9335),
MAPKAP-K2 (#3042), phospho-MAPKAP-K2Thr334 (#3007), Mouse IgG, and Rabbit IgG antibodies were purchased from
Cell Signaling Technology (Beverly, Mass, USA). Enhanced chemiluminescence (ECL)
western blotting detection reagent was from Amersham Biosciences (Piscataway, NJ, USA).
Restore western blot stripping buffer was obtained from Pierce (Rockford, Ill, USA), and 3T3
cell lysates were from Santa Cruz Biotechnology (Santa Cruz, Calif, USA).
Antirabbit and antimouse fluorescein antibodies and VectaSheild Hardset Mounting
Medium with DAPI was purchased from Vector Laboratories (Burlingame, Calif, USA).
All other chemicals were purchased from Sigma (St. Louis,
Mo, USA) or Fisher Scientific (Hanover, Ill, USA).


### 2.3. Contractile Stimulation of Skeletal Muscles

The high-frequency electrical
stimulation (HFES) model has been previously described [26] and was chosen on the basis
of its efficacy in stimulating protein translation and muscle
hypertrophy in vivo [27]. By directly stimulating the
motor nerve, this model elicits a maximal contraction that when repeated over
time produces a muscle adaptation that closely mimics that seen in humans
following a program of resistance training.
HFES loading of the rat hindlimb was performed using a voltage of 5–7 V (1-millisecond pulses, 100 Hz) as described previously [27]. These conditions have been
shown to produce a maximal contraction. Muscle contractions lasted 3 seconds
and were followed by a 10-second rest. Ten sets of 6 repetitions were performed
with each set being separated by a 50-second rest period. This protocol results in concentric
(shortening) contraction of the plantaris and soleus. The fast-twitch
plantaris (PLA) and slow-twitch soleus (Sol) muscles were chosen on the basis
of previous studies demonstrating that HFES induces MAPK phosphorylation in
these muscles [17, 28–30]. Animals were killed
by drug overdose at baseline, immediately following, 1hour or 3 hours (n = 6 normal, n = 6 diabetic for 0, 1, and 3 hours) after HFES. Once excised,
muscles were blotted dry, trimmed of visible fat and tendon projections,
weighed, immediately frozen in liquid nitrogen, and stored at −80°C.


### 2.4. Preparation of Protein Isolates and Immunoblotting

Muscles were pulverized in liquid nitrogen using a mortar and pestle
until a fine powder was obtained. After washing with ice cold PBS, pellets were
lysed on ice in for 15 minutes in T-PER (2 mL/1 g tissue weight) (Pierce, Rockford, Ill, USA)
and centrifuged for 10 minutes at 2000 X g to pellet particulate matter. This
process was repeated twice and the supernants combined for protein
concentration determination using the Bradford method (Pierce, Rockford, Ill, USA).
Samples were diluted to a concentration of 3μg/μL in SDS loading buffer,
boiled for 5 minutes, and 60 μg of protein were separated using 10% SDS-PAGE
gels. Transfer of protein onto nitrocellulose membranes, verification of
transfer, and determination of equal loading between lanes and membranes was
determined as outlined previously [31]. Protein immunodetection was
performed as outlined by the antibody manufacturer while immunoreactive bands
were visualized with ECL (Amersham Biosciences, NJ, USA). Exposure time was adjusted
to keep the integrated optical densities within a linear and nonsaturated range.
Band signal intensity was normalized to β-actin by densitometry using a flatbed
scanner (Epson Perfection 3200 PHOTO) and imaging software (AlphaEaseFC). Molecular
weight markers (Cell Signaling) were used as molecular mass standards, and NIH
3T3 cell lysates were included as positive controls. To allow direct
comparisons to be made between the concentration levels of different signaling
molecules, immunoblots were stripped and re-probed with Restore western blot
stripping buffer as detailed by the manufacturer (Pierce, Rockford, Ill, USA).


### 2.5. Statistical Analysis

Results are presented as mean ±SEM. Data were analyzed by using the Sigma Stat 3.0
statistical program. Effects of exercise on type II diabetes mellitus in the
presence of metabolic syndrome were analyzed using a two-way ANOVA followed by
the Student-Newman-Keuls post hoc testing when appropriate. AP value <.05 was considered to be
statistically significant.


## 2.1. Animal Care

Young (10 weeks,n = 12) male lean
normal Zucker (LNZ) and young (10 weeks, n = 12) male obese syndrome-X Zucker (OSXZ) rats were
obtained from the Charles River Laboratories. Rats were housed to a cage in an
AAALAC approved vivarium. Housing conditions consisted of a 12H: 12H dark-light
cycle and temperature was maintained at 22° ± 2°C. Animals were provided food
and water ad libitum and allowed to recover from shipment for at least two
weeks before experimentation. During this time, the animals were carefully
observed and weighed weekly to ensure none exhibited signs of failure to
thrive, such as precipitous weight loss, disinterest in the environment, or
unexpected gait alterations. All procedures were performed as outlined in the
Guide for the Care and Use of Laboratory Animals as approved by the Council of
the American Physiological Society and the institutional Animal Use Review
Board.


## 2.2. Materials

Anti-Erk1/2 (#9102), phospho-Erk1/2Thr202/Tyr204 (#9106), p38 (#9212), phospho-p38Thr180/Tyr182 (#9216), SAPK/Jnk
(#9252), phospho-SAPK/JnkThr183/Tyr185 (#9251), p90rsk (#9334), phospho-p90rsk (Ser380#9335),
MAPKAP-K2 (#3042), phospho-MAPKAP-K2Thr334 (#3007), Mouse IgG, and Rabbit IgG antibodies were purchased from
Cell Signaling Technology (Beverly, Mass, USA). Enhanced chemiluminescence (ECL)
western blotting detection reagent was from Amersham Biosciences (Piscataway, NJ, USA).
Restore western blot stripping buffer was obtained from Pierce (Rockford, Ill, USA), and 3T3
cell lysates were from Santa Cruz Biotechnology (Santa Cruz, Calif, USA).
Antirabbit and antimouse fluorescein antibodies and VectaSheild Hardset Mounting
Medium with DAPI was purchased from Vector Laboratories (Burlingame, Calif, USA).
All other chemicals were purchased from Sigma (St. Louis,
Mo, USA) or Fisher Scientific (Hanover, Ill, USA).


## 2.3. Contractile Stimulation of Skeletal Muscles

The high-frequency electrical
stimulation (HFES) model has been previously described [26] and was chosen on the basis
of its efficacy in stimulating protein translation and muscle
hypertrophy in vivo [27]. By directly stimulating the
motor nerve, this model elicits a maximal contraction that when repeated over
time produces a muscle adaptation that closely mimics that seen in humans
following a program of resistance training.
HFES loading of the rat hindlimb was performed using a voltage of 5–7 V (1-millisecond pulses, 100 Hz) as described previously [27]. These conditions have been
shown to produce a maximal contraction. Muscle contractions lasted 3 seconds
and were followed by a 10-second rest. Ten sets of 6 repetitions were performed
with each set being separated by a 50-second rest period. This protocol results in concentric
(shortening) contraction of the plantaris and soleus. The fast-twitch
plantaris (PLA) and slow-twitch soleus (Sol) muscles were chosen on the basis
of previous studies demonstrating that HFES induces MAPK phosphorylation in
these muscles [17, 28–30]. Animals were killed
by drug overdose at baseline, immediately following, 1hour or 3 hours (n = 6 normal, n = 6 diabetic for 0, 1, and 3 hours) after HFES. Once excised,
muscles were blotted dry, trimmed of visible fat and tendon projections,
weighed, immediately frozen in liquid nitrogen, and stored at −80°C.


## 2.4. Preparation of Protein Isolates and Immunoblotting

Muscles were pulverized in liquid nitrogen using a mortar and pestle
until a fine powder was obtained. After washing with ice cold PBS, pellets were
lysed on ice in for 15 minutes in T-PER (2 mL/1 g tissue weight) (Pierce, Rockford, Ill, USA)
and centrifuged for 10 minutes at 2000 X g to pellet particulate matter. This
process was repeated twice and the supernants combined for protein
concentration determination using the Bradford method (Pierce, Rockford, Ill, USA).
Samples were diluted to a concentration of 3μg/μL in SDS loading buffer,
boiled for 5 minutes, and 60 μg of protein were separated using 10% SDS-PAGE
gels. Transfer of protein onto nitrocellulose membranes, verification of
transfer, and determination of equal loading between lanes and membranes was
determined as outlined previously [31]. Protein immunodetection was
performed as outlined by the antibody manufacturer while immunoreactive bands
were visualized with ECL (Amersham Biosciences, NJ, USA). Exposure time was adjusted
to keep the integrated optical densities within a linear and nonsaturated range.
Band signal intensity was normalized to β-actin by densitometry using a flatbed
scanner (Epson Perfection 3200 PHOTO) and imaging software (AlphaEaseFC). Molecular
weight markers (Cell Signaling) were used as molecular mass standards, and NIH
3T3 cell lysates were included as positive controls. To allow direct
comparisons to be made between the concentration levels of different signaling
molecules, immunoblots were stripped and re-probed with Restore western blot
stripping buffer as detailed by the manufacturer (Pierce, Rockford, Ill, USA).


## 2.5. Statistical Analysis

Results are presented as mean ±SEM. Data were analyzed by using the Sigma Stat 3.0
statistical program. Effects of exercise on type II diabetes mellitus in the
presence of metabolic syndrome were analyzed using a two-way ANOVA followed by
the Student-Newman-Keuls post hoc testing when appropriate. AP value <.05 was considered to be
statistically significant.


## 3. Results

Average body mass of obese Zucker rats was∼82% higher than lean counterparts (P<.05) (see Table 1). Plantaris and solues muscle mass in obese Zuckers, as a percentage of body
weight, was only ∼34% and ∼58%, respectively, of muscle mass in lean Zuckers
(P<.05) (see Table 1).Table 1
Body weight and muscle mass of lean and obese (fa/fa) Zucker rats. An asterisk (*)
indicates significant differences (P<.05) from the lean Zucker value.
Lean ZuckerObese ZuckerBody mass, g328 ± 12.2597 ± 21.7*PLA mass, mg373 ± .009244 ± .007*PLA mass/Body Wt (mg/g)1.14 ± .430.41 ± .12*SOL mass, mg143 ± .005146 ± .008SOL mass/Body Wt (mg/g)0.44 ± .130.24 ± .12*


### 3.1. MAPK Protein Levels and Phosphorylation Status Are Altered in The Diabetic Soleus and Plantaris

Immunoblotting analysis of proteins normalized toβ-actin indicated that there were no significant differences
in total p38 MAPK content in plantaris and solues in the OSXZ compared to the LNZ
(P<.05) (see Figure 1). Plantaris Erk1/2 (p44/p42) content was
not significantly different between the groups, while Erk1/2(p44/p42) content
in the OSXZ soleus was 17.0±4.8% and 20.3±6.9% lower (P<.05) than
that observed in their lean counterparts (see Figure 1). Jnk1 levels were 46.1±10.2% higher in OSXZ plantaris and 22.2±7.2% higher in the OSXZ soleus,
compared to LNZ (P<.05) (see Figure 1). Jnk2 expression was 29.0±9.34% higher in OSXZ plantaris as compared to lean counterparts (P<.05), with no significant difference in the OSXZ soleus muscle (see Figure 1). There was no detectable expression of Jnk3 in the plantaris,
while Jnk3 content did not differ between LNZ and OSXZ solei. There were
no significant differences between OSXZ and LNZ in  p90rsk, or MAPKAP-K2 protein levels for either
the plantaris or soleus (data not shown).Type 2 diabetes is associated with alterations in skeletal muscle MAPK proteins. Plantaris
and soleus muscles from LNZ and obese (fa/fa) Zuckers (OSXZ) were analyzed by Western blot analysis for diabetes-related
changes in total Erk1/2-, p38 MAPK, and Jnk protein expression. Protein
quantification was normalization to the abundance ofβ-actin. Results are expressed as a percent of the normal, LNZ value.
An asterisk (*) indicates significant differences (P<.05) from the
lean Zucker value.
(a)(b)Basal levels of phosphorylated forms
of MAPK proteins were measured to determine if diabetes altered MAPK activation.
Immunoblot analysis using phospho-specific antibodies indicated that the basal
level of phospho-Erk1/2 was41.2±13.3% and 45.1±13.4% lower (P<.05), respectively, in OSXZ soleus compared to LNZ, with no significant
difference in the plantaris (see Figure 2). The basal level of p38 phosphorylation
was unchanged in the plantaris and was
24.9±8.7%
lower in the OSXZ versus the LNZ soleus (P<.05) (see Figure 3). There
was no difference in the basal expression of any of the phosphorylated Jnk
isoforms between LNZ and OSXZ muscles (see Figure 4).  In the plantaris, the basal
phosphorylation level of p90s6k was 128.3±19.28% higher OSXZ compared to the
LNZ rats (P<.05) while the basal phosphorylation of MAPKAP-K2 was
unchanged (see Figure 5).Contraction-induced Erk1/2-MAPK
phosphorylation is altered with type II diabetes. Plantaris and soleus muscles from lean (LNZ) and obese
Zucker (OSXZ) rats were obtained immediately after, 1 and 3 hours after a bout
of HFES. Alterations in Erk1 (a),(b) and Erk2 (c),(d)-MAPK
phosphorylation with exercise were determined by immunoblotting.
An asterisk (*) indicates significant difference (P<.05) from the
control within animal model, and a cross (†) indicates significant difference (P<.05) at corresponding time points across animal models.
(a)(b)(c)(d)Effects of concentric, maximal muscle
contraction in situ on phosphorylation of p38-MAPK. The basal (control) and contraction-induced
phosphorylation of the Jnk in plantaris and soleus muscles from lean (LNZ) and obese
Zucker (OSXZ) rats at 0, 1, and 3 hours after HFES. Phosphorylation
of p38-MAPK was determined by immunodetection of phosphorylation on Thr180 and
Tyr182 (phospho-p38α MAPK). An asterisk (*) indicates significant difference (P<.05)
from the control time point within animal model, and a cross (†)
indicates significant difference (P<.05) at corresponding time
points across animal models.
(a)(b)Figure 4
Effects of concentric, maximal muscle contraction in situon phosphorylation of Jnk. The basal (control) and contraction-induced
phosphorylation of the Jnk1 (d),(e), Jnk2 (a),(b), Jnk3-MAPK (c) in plantaris
and soleus muscles from lean and diabetic Zucker rats
at 0, 1, and 3 hours after contractile stimulus. Jnk phosphorylation was
determined by Western analysis and immunodetection for Jnk phosphorylation on Thr183
and Tyr185. An asterisk (*) indicates significant difference (P<.05)
from the control within animal model, and a cross (†) indicates significant
difference (P<.05)
at corresponding time points across animal models.Contraction-induced p90rsk (MAPKAP-K1) and MAPKAPK-2 phosphorylation are
altered with type II diabetes. Plantaris muscles from lean (LNZ) and obese
Zucker (OSXZ) rats were obtained immediately after, 1 and 3 hours after a bout
of HFES. Alterations in p90rsk (a) and MAPKAPK-2 (b)
phosphorylation with exercise were determined by immunoblotting. An asterisk
(*) indicates significant difference (P<.05) from the control
within animal model, and a cross (†) indicates significant
difference (P<.05) at corresponding time points across animal models.
(a)(b)


### 3.2. MAPK Phosphorylation in Response to Contractile Stimulus Is Altered by Diabetes

Phosphorylation of p38, Erk1/2, and Jnk-MAPK was
determined at 0, 1, and 3 hours after a bout of HFES for comparison with
control values. In the case of each molecule examined, significant differences
existed between lean and obese Zucker rat models. Differences also were present
between the predominately fast twitch plantaris and slow twitch soleus (see Figures2–4). LNZ plantaris muscles showed no differences in p38 phosphorylation
status post exercise. Conversely, the OSXZ plantaris exhibited a 38.5±33.8% increase in phosphorylation from
baseline at 3-hours post exercise (P<.05) (see Figure 3). The
LNZ soleus had a 33.5±9.7% and 27.2±8.0% decrease in phosphorylation of
p38 at 1 and 3 hours post exercise, respectively, while OSXZ soleus muscles showed
an increase of 18.8±10.7% at 3 hours post exercise from the baseline (P<.05) (see Figure 3).Both Erk1/2 MAP kinases showed significant increases in
phosphorylation levels from baseline in response to the contractile stimulus.
In the plantaris muscle, LNZ animals had an increase in phosphorylation of Erk1
(p44) of106.1±13.8%, 59.8±10.9%, and 215.9±29.5% at 0, 1, and
3hours, respectively. OSXZ animals showed an increase of 244.3±24.7%, 239.3±13.9%, and 200.3±28.7% at 0, 1, and 3 hours, respectively, (P<.05)  (see Figure 2). The slow twitch soleus
muscle of LNZ had an increase in the phosphorylation level of p44 by 51.3±9.3
%, 90.3±14.9%, and 36.8±13.7% from baseline at 0, 1, and 3 hours,
respectively, (P<.05). The soleus muscles of OSXZ animals
responded differently with increases in p44 phosphorylation of 124.7±19.7% and
78.5±25.9% at 0 and 3 hours, respectively, (P<.05)  (see Figure 2). In LNZ animals, the plantaris
exhibited increases of Erk2 (p-42) phosphorylation of 368.5±16.6%,
244.2±29.8%, and 364.1±22.3%, while
contraction in OSXZ elicited increases in p-42 phosphorylation of 582.7±55.2%, 679.0±48.2%, and 716.6±9.2% at 0, 1, and 3 hours,
respectively, (P<.05)  (see Figure
2). LNZ soleus
muscles increased p42 phosphorylation by 24.8±55.2%, 43.3±55.2%, and 15.5±55.2% above baseline at 0, 1, and 3 hours, respectively, (P<.05). This response appeared
greater in OSXZ solei as soleus contraction in these animals increased the
phosphorylation of p42 by 120.9±55.2%, 30.5±55.2%, and 122.0±55.2% at
0, 1, and 3 hours, respectively, (P<.05)  (see Figure 2).Similar to ERK1/2 and p38, muscle contraction also increased the phosphorylation of Jnk
isoforms. In the LNZ plantaris, Jnk1 phosphorylation increased115.2±31.9%,
140.0±27.9%, and 105.4±34.5%, and Jnk2 by 354.2±140.3%, 472.8±49.7%,
and 153.0±70.0% from baseline at 0, 1, and 3 hours, respectively, (P<.05). In obese
animals, Jnk1 phosphorylation with contraction was increased by 65.2±35.3%, 92.7±36.5%, and 52.7±27.1%,
and Jnk2 by 300.4±97.8%, 417.8±72.2% and 174.5±58.2%  from baseline at 0, 1, and 3 hours,
respectively, (P<.05) (see Figure 4). In LNZ soleus muscles, HFES did not change the phosphorylation
level of Jnk1 and Jnk2 from that seen in control animals,  however the phosphorylation level of Jnk3
increased 36.7±7.2% from baseline immediately after exercise (P<.05).
HFES did not alter Jnk1, Jnk2, or Jnk3 phosphorylation in the OSXZ soleus (see Figure
4).To determine whether differences
in MAPK signaling with type II diabetes were associated with changes in the
regulation of potential downstream MAPK targets, we examined the regulation of
p90RSK and MAPKKAP-K2. Contraction induced significant increases in phosphorylation
levels of p90s6k (downstream target of Erk1/2) from baseline in the plantaris
muscles of both normal and diabetic animals. In the LNZ animals, HFES increased
the phosphorylation level of p90s6k by382.2±22.3%, 304.4±17.4%, and
387.8±24.7% from baseline at 0, 1, and 3 hours, respectively. In a similar
fashion, plantaris muscles from obese animals demonstrated increases in
phosphorylation of p90s6k of 134.0±7.6
%, 135.5±6.7%, and 144.5±9.5% from baseline at 0, 1, and 3 hours,
respectively (P<.05)  (see Figure 5). Conversely, the plantaris muscles of LNZ had a decrease in phosphorylation
level of MAPKAP-K2 (downstream target of p38) by 14.5±4.6%, 14.3±4.1%,
and 8.1±6.0% from baseline at 0, 1, and 3 hours, respectively. Plantaris
muscles of OSXZ animals responded in a similar way  and exhibited decreases in MAPKAP-K2
phosphorylation of 27.5±4.2%, 31.1±4.6%,
and 40.0±5.6%  at 0, 1, and 3 hours,
respectively, (P<.05)  (see Figure
5). No evidence of p90RSK or MAPKAP-K2 phosphorylation in any of the soleus
muscles examined (data not shown).


## 3.1. MAPK Protein Levels and Phosphorylation Status Are Altered in The Diabetic Soleus and Plantaris

Immunoblotting analysis of proteins normalized toβ-actin indicated that there were no significant differences
in total p38 MAPK content in plantaris and solues in the OSXZ compared to the LNZ
(P<.05) (see Figure 1). Plantaris Erk1/2 (p44/p42) content was
not significantly different between the groups, while Erk1/2(p44/p42) content
in the OSXZ soleus was 17.0±4.8% and 20.3±6.9% lower (P<.05) than
that observed in their lean counterparts (see Figure 1). Jnk1 levels were 46.1±10.2% higher in OSXZ plantaris and 22.2±7.2% higher in the OSXZ soleus,
compared to LNZ (P<.05) (see Figure 1). Jnk2 expression was 29.0±9.34% higher in OSXZ plantaris as compared to lean counterparts (P<.05), with no significant difference in the OSXZ soleus muscle (see Figure 1). There was no detectable expression of Jnk3 in the plantaris,
while Jnk3 content did not differ between LNZ and OSXZ solei. There were
no significant differences between OSXZ and LNZ in  p90rsk, or MAPKAP-K2 protein levels for either
the plantaris or soleus (data not shown).Type 2 diabetes is associated with alterations in skeletal muscle MAPK proteins. Plantaris
and soleus muscles from LNZ and obese (fa/fa) Zuckers (OSXZ) were analyzed by Western blot analysis for diabetes-related
changes in total Erk1/2-, p38 MAPK, and Jnk protein expression. Protein
quantification was normalization to the abundance ofβ-actin. Results are expressed as a percent of the normal, LNZ value.
An asterisk (*) indicates significant differences (P<.05) from the
lean Zucker value.
(a)(b)Basal levels of phosphorylated forms
of MAPK proteins were measured to determine if diabetes altered MAPK activation.
Immunoblot analysis using phospho-specific antibodies indicated that the basal
level of phospho-Erk1/2 was41.2±13.3% and 45.1±13.4% lower (P<.05), respectively, in OSXZ soleus compared to LNZ, with no significant
difference in the plantaris (see Figure 2). The basal level of p38 phosphorylation
was unchanged in the plantaris and was
24.9±8.7%
lower in the OSXZ versus the LNZ soleus (P<.05) (see Figure 3). There
was no difference in the basal expression of any of the phosphorylated Jnk
isoforms between LNZ and OSXZ muscles (see Figure 4).  In the plantaris, the basal
phosphorylation level of p90s6k was 128.3±19.28% higher OSXZ compared to the
LNZ rats (P<.05) while the basal phosphorylation of MAPKAP-K2 was
unchanged (see Figure 5).Contraction-induced Erk1/2-MAPK
phosphorylation is altered with type II diabetes. Plantaris and soleus muscles from lean (LNZ) and obese
Zucker (OSXZ) rats were obtained immediately after, 1 and 3 hours after a bout
of HFES. Alterations in Erk1 (a),(b) and Erk2 (c),(d)-MAPK
phosphorylation with exercise were determined by immunoblotting.
An asterisk (*) indicates significant difference (P<.05) from the
control within animal model, and a cross (†) indicates significant difference (P<.05) at corresponding time points across animal models.
(a)(b)(c)(d)Effects of concentric, maximal muscle
contraction in situ on phosphorylation of p38-MAPK. The basal (control) and contraction-induced
phosphorylation of the Jnk in plantaris and soleus muscles from lean (LNZ) and obese
Zucker (OSXZ) rats at 0, 1, and 3 hours after HFES. Phosphorylation
of p38-MAPK was determined by immunodetection of phosphorylation on Thr180 and
Tyr182 (phospho-p38α MAPK). An asterisk (*) indicates significant difference (P<.05)
from the control time point within animal model, and a cross (†)
indicates significant difference (P<.05) at corresponding time
points across animal models.
(a)(b)Figure 4
Effects of concentric, maximal muscle contraction in situon phosphorylation of Jnk. The basal (control) and contraction-induced
phosphorylation of the Jnk1 (d),(e), Jnk2 (a),(b), Jnk3-MAPK (c) in plantaris
and soleus muscles from lean and diabetic Zucker rats
at 0, 1, and 3 hours after contractile stimulus. Jnk phosphorylation was
determined by Western analysis and immunodetection for Jnk phosphorylation on Thr183
and Tyr185. An asterisk (*) indicates significant difference (P<.05)
from the control within animal model, and a cross (†) indicates significant
difference (P<.05)
at corresponding time points across animal models.Contraction-induced p90rsk (MAPKAP-K1) and MAPKAPK-2 phosphorylation are
altered with type II diabetes. Plantaris muscles from lean (LNZ) and obese
Zucker (OSXZ) rats were obtained immediately after, 1 and 3 hours after a bout
of HFES. Alterations in p90rsk (a) and MAPKAPK-2 (b)
phosphorylation with exercise were determined by immunoblotting. An asterisk
(*) indicates significant difference (P<.05) from the control
within animal model, and a cross (†) indicates significant
difference (P<.05) at corresponding time points across animal models.
(a)(b)


## 3.2. MAPK Phosphorylation in Response to Contractile Stimulus Is Altered by Diabetes

Phosphorylation of p38, Erk1/2, and Jnk-MAPK was
determined at 0, 1, and 3 hours after a bout of HFES for comparison with
control values. In the case of each molecule examined, significant differences
existed between lean and obese Zucker rat models. Differences also were present
between the predominately fast twitch plantaris and slow twitch soleus (see Figures2–4). LNZ plantaris muscles showed no differences in p38 phosphorylation
status post exercise. Conversely, the OSXZ plantaris exhibited a 38.5±33.8% increase in phosphorylation from
baseline at 3-hours post exercise (P<.05) (see Figure 3). The
LNZ soleus had a 33.5±9.7% and 27.2±8.0% decrease in phosphorylation of
p38 at 1 and 3 hours post exercise, respectively, while OSXZ soleus muscles showed
an increase of 18.8±10.7% at 3 hours post exercise from the baseline (P<.05) (see Figure 3).Both Erk1/2 MAP kinases showed significant increases in
phosphorylation levels from baseline in response to the contractile stimulus.
In the plantaris muscle, LNZ animals had an increase in phosphorylation of Erk1
(p44) of106.1±13.8%, 59.8±10.9%, and 215.9±29.5% at 0, 1, and
3hours, respectively. OSXZ animals showed an increase of 244.3±24.7%, 239.3±13.9%, and 200.3±28.7% at 0, 1, and 3 hours, respectively, (P<.05)  (see Figure 2). The slow twitch soleus
muscle of LNZ had an increase in the phosphorylation level of p44 by 51.3±9.3
%, 90.3±14.9%, and 36.8±13.7% from baseline at 0, 1, and 3 hours,
respectively, (P<.05). The soleus muscles of OSXZ animals
responded differently with increases in p44 phosphorylation of 124.7±19.7% and
78.5±25.9% at 0 and 3 hours, respectively, (P<.05)  (see Figure 2). In LNZ animals, the plantaris
exhibited increases of Erk2 (p-42) phosphorylation of 368.5±16.6%,
244.2±29.8%, and 364.1±22.3%, while
contraction in OSXZ elicited increases in p-42 phosphorylation of 582.7±55.2%, 679.0±48.2%, and 716.6±9.2% at 0, 1, and 3 hours,
respectively, (P<.05)  (see Figure
2). LNZ soleus
muscles increased p42 phosphorylation by 24.8±55.2%, 43.3±55.2%, and 15.5±55.2% above baseline at 0, 1, and 3 hours, respectively, (P<.05). This response appeared
greater in OSXZ solei as soleus contraction in these animals increased the
phosphorylation of p42 by 120.9±55.2%, 30.5±55.2%, and 122.0±55.2% at
0, 1, and 3 hours, respectively, (P<.05)  (see Figure 2).Similar to ERK1/2 and p38, muscle contraction also increased the phosphorylation of Jnk
isoforms. In the LNZ plantaris, Jnk1 phosphorylation increased115.2±31.9%,
140.0±27.9%, and 105.4±34.5%, and Jnk2 by 354.2±140.3%, 472.8±49.7%,
and 153.0±70.0% from baseline at 0, 1, and 3 hours, respectively, (P<.05). In obese
animals, Jnk1 phosphorylation with contraction was increased by 65.2±35.3%, 92.7±36.5%, and 52.7±27.1%,
and Jnk2 by 300.4±97.8%, 417.8±72.2% and 174.5±58.2%  from baseline at 0, 1, and 3 hours,
respectively, (P<.05) (see Figure 4). In LNZ soleus muscles, HFES did not change the phosphorylation
level of Jnk1 and Jnk2 from that seen in control animals,  however the phosphorylation level of Jnk3
increased 36.7±7.2% from baseline immediately after exercise (P<.05).
HFES did not alter Jnk1, Jnk2, or Jnk3 phosphorylation in the OSXZ soleus (see Figure
4).To determine whether differences
in MAPK signaling with type II diabetes were associated with changes in the
regulation of potential downstream MAPK targets, we examined the regulation of
p90RSK and MAPKKAP-K2. Contraction induced significant increases in phosphorylation
levels of p90s6k (downstream target of Erk1/2) from baseline in the plantaris
muscles of both normal and diabetic animals. In the LNZ animals, HFES increased
the phosphorylation level of p90s6k by382.2±22.3%, 304.4±17.4%, and
387.8±24.7% from baseline at 0, 1, and 3 hours, respectively. In a similar
fashion, plantaris muscles from obese animals demonstrated increases in
phosphorylation of p90s6k of 134.0±7.6
%, 135.5±6.7%, and 144.5±9.5% from baseline at 0, 1, and 3 hours,
respectively (P<.05)  (see Figure 5). Conversely, the plantaris muscles of LNZ had a decrease in phosphorylation
level of MAPKAP-K2 (downstream target of p38) by 14.5±4.6%, 14.3±4.1%,
and 8.1±6.0% from baseline at 0, 1, and 3 hours, respectively. Plantaris
muscles of OSXZ animals responded in a similar way  and exhibited decreases in MAPKAP-K2
phosphorylation of 27.5±4.2%, 31.1±4.6%,
and 40.0±5.6%  at 0, 1, and 3 hours,
respectively, (P<.05)  (see Figure
5). No evidence of p90RSK or MAPKAP-K2 phosphorylation in any of the soleus
muscles examined (data not shown).


## 4. Discussion

The obese Zucker (fa/fa)
rat is an animal model of severe skeletal muscle insulin resistance that is
also characterized bymarked hyperinsulinemia and glucose
intolerance, dyslipidemia, and central adiposity and therefore is a
suitable animal model of the insulin resistance syndrome [32]. It is thought that the mechanisms underlying the
progression of insulin resistance in these animals closely mimic that seen in human
type II diabetes [33].  The objective of this study was to
examine how type II diabetes affects how skeletal muscle responds to a
contractile stimulus. Our findings suggest that diabetes is associated with
alterations in how muscle contraction regulates MAPK signaling and are
consistent with the notion that diabetes may affect the regulation of these
proteins differently depending upon fiber type.


## 5. Type II Diabetes Associated Alteration in MAPK Protein Content and Basal Phosphorylation Levels

In mammalian cells, the Erk1/2-, p38-, and Jnk-MAPK pathways are thought to be
the three major signaling cascades comprising the MAPK signaling system [34]. When compared to lean animals, the
diabetic plantaris and soleus muscles exhibited increased amounts of Jnk-1
while in the diabetic soleus, the amount of Erk1/2 and phosphorylated Erk1/2 was
diminished (see Figures 1 and 2). Previous studies examining Jnk levels in human
skeletal muscle [35] and Erk1/2 signaling in the obese
Zucker gastrocnemius [36] have yielded similar results. It is
unclear why diabetes may induce differences in the way different muscle types
might regulate the amount and basal activation of these proteins. Indeed,
previous studies investigating the effect of increased glucose or the influence
of diabetes on MAPK protein levels and phosphorylation state have been
equivocal. For example, some studies have demonstrated no effect [37] while others have suggested that
altered glucose levels can increase [38] or diminish the phoshorylation of
these proteins [39]. The factor(s) responsible for
differences between studies are unknown but could be related to cell
type-specific responsesor to differences in the concentrations of
glucose tested or in the severity of hyperglycemia observed in the experimental
preparation. Similarly, diabetes-associated changes in muscle fiber type may
also be involved. For example, Punkt and colleagues have recently demonstrated
that slow oxidative fiber fraction was reduced by 16% with diabetes while the
fast glycolytic fiber fraction was increased by 49% in vastus lateralis of type
II diabetic patients [40]. Given that the regulation of MAPK
proteins may differ between fiber types [41, 42], it is possible that fiber type
transitions associated with diabetes may be involved. Finally, recent data has
suggested that fast- and slow-twitch muscles may rely on different signaling
mechanisms to mediate glucose uptake [43, 44]. Whether these differences explain
some or all of the alterations we observe in the present investigation will require
further study.


## 6. Exercise-Induced Signal Transduction Is Altered in Type II Diabetic Muscle

A major finding of this investigation is that the phosphorylation (activation) of MAPK proteins in
response to a maximal contractile stimulus appears to be altered in diabetic
muscle. In addition, our data suggest that diabetes may alter this response
differently in different muscle types. For example, in the lean plantaris,
Erk1/2 activation typically did not reach maximal levels until three hours
after cessation of exercise. Conversely, in the obese plantaris, Erk1/2
activation was maximal immediately following exercise and remained high through
three hours post exercise (see Figure2). Interestingly, this response appeared
to be reversed in the lean and obese solei (see Figure 2). Similar to what we observed
for the Erk1/2-MAPK proteins, the contraction-induced phosphorylation of p38-
and Jnk-MAPK appeared to differ between the lean and obese animals. Unlike the
regulation of the Erk1/2-MAPK proteins, differences between groups in the
phosphorylation response of p38- and Jnk-MAPK appeared to manifest themselves
during the later stages of the recovery period. For example, while contraction
failed to increase p38α phosphorylation in the lean plantaris, p38α
phosphorylation levels in the obese
plantaris muscles were dramatically increased at the
three hour post exercise time point (see Figure 3). Similarly, the degree of Jnk
phosphorylation was significantly elevated in the obese, compared to lean
plantaris muscles at the one hour post exercise time point (see Figure 4). To
our knowledge, these findings have not been previously reported.The time course and magnitude of increased MAPK phosphorylation following muscle
contraction we observe in the
lean animals are similar to that which have been
reported previously following various modes of exercise in animal models as
well as humans [45–51]. These responses appear to be physiologically relevant, as they were
associated with the exercise-induced activation of p90rsk, a known downstream
target of Erk1/2 [21]. Similar to what was observed with Erk1/2, the basal and contraction-induced phosphorylation levels of p90rsk were
significantly higher in the obese, compared
to lean plantaris at all time points. How Erk1/2 and p90rsk may function in the
diabetic muscle or in producing exercise-induced muscle adaptation is not well
understood. The p90RSK has been proven to be a ubiquitous and versatile
mediator of Erk 1/2 signal transduction with the activation of p90RSK being
thought to be involved in the regulation of nuclear factor-κB
and in the phosphorylation of transcription factors, including
c-Fos, Nur77, and cAMP response element-bindingprotein
[52]. The physiological
significance of the apparent enhancement in Erk1/2 signaling we observe in
diabetic muscle awaits further clarification. Nonetheless, in the light of previous data suggesting that MAPK and
MAPK-associated signaling may be important upstream regulators of load-induced
intracellular signaling events the results of the present study are consistent
with previous work suggesting that diabetes may be associated by alterations in
the ability of skeletal muscle to adapt to an exercise stimulus [7, 9, 53–56]. The extent to which the
combined changes in tissue content and action of the Erk 1/2 proteins may play
a role in the contracting diabetic muscle cannot be accurately assessed without
further studies designed to evaluate the experimental manipulation of these
proteins. Future studies employing
methods to directly test the effects of altered Erk1/2 levels will with no
doubt add to our understanding.Why diabetes might be associated with differences in the
contraction-induced activation of MAPK proteins is not readily apparent.  It is thought that the MAPK proteins may play a key role in
regulating muscle glucose uptake, and it has been hypothesized that the MAPK
pathway may be a potential target for therapeutic strategies to restore the
metabolic balance to type II diabetic patients. The differences in MAPK activation between normal and
diabetic muscles we observed in the present study may be a compensatory
mechanism to bypass defects in insulin dependent signaling [57].
Given the plethora of factors thought to be involved in regulating MAPK
activation (e.g., mechanical stretch, muscle hypoxia, reactive oxygen levels,
muscle damage, local release of growth factors, and cytokines, etc.) it is
possible that the differences we observe between models could be due to several
factors [24].
Because we examined the regulation of MAPK signaling following a series of in vivo contractions as opposed to
using an in vitro experimental preparation it is unclear whether the alterations we found between models are a
by product of differences in systemic environment of the exercising muscle or
due to intrinsic alterations to the muscles themselves. Further research,
perhaps using other experimental designs, will be with no doubt added to our understanding.In conclusion, the results of this study have
shown that contraction-induced regulation of MAPK signaling may be altered in
the skeletal muscles of the obese Zucker rat. Because this model exhibits many
of the same physiological characteristics seen in type II diabetic humans,
these data suggest that diabetes may be associated with differences in how
skeletal muscle “sense” and “respond” to a contractile stimulus. Whether
similar findings would be observed in other types of diabetes (e.g., type I) or
in humans remains to be determined. Given the potential of resistance training to improve the
diabetic condition and the importance of MAPK signaling in regulating muscle
adaptation, the findings of the current study may help to reveal new approaches
for treating metabolic diseases and have applicability for the prescription of
exercise programs aimed at improving muscle function in diabetic individuals.



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*Source: 738101-2008-06-04.xml*