Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4–2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4–2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training.
- PDP1 and 2
- carbohydrate oxidation
pyruvate dehydrogenase (pdh) is a key component to glucose homeostasis, as it is the first nonreversible step in mitochondrial glucose oxidation. It is a complex enzyme with many copies of the catalytic subunits and proteins (E1α, E1β, E2, E3, and E3 binding protein), as well as its associated regulatory kinase and phophatase enzymes. Decreased carbohydrate flux through PDH is mediated through its phosphorylation and deactivation via PDH kinase (PDK; as reviewed by Refs. 7 and 42). The activation of PDH, via dephosphorylation by PDH phosphatase (PDP), promotes carbohydrate oxidation. The complexity of PDH control by PDK and PDP is enhanced by the presence of multiple isoforms, four PDK [PDK1-4 (4)] and two PDP [PDP1 and 2 (18)]. The two PDP isoforms are unique in their skeletal muscle type distribution and allosteric regulators. PDP1 is the predominant isoform expressed in skeletal muscle, with ∼2.5 times more found in red gastrocnemius (RG) compared with soleus and white gastrocnemius [WG; (26)] and is activated by calcium (18). In contrast, PDP2 is only detectable in RG (26) and is insulin sensitive (18).
Skeletal muscle is the single largest tissue in the body that is responsible for a majority of insulin-mediated glucose disposal, and its health is paramount to an organism's metabolic health. Obesity and its associated metabolic derangements have a large impact on skeletal muscle function (see Refs. 31 and 40 for a review). Of the many known and putative contributors to this obesity-mediated metabolic derangement, PDK has been identified as an important regulatory component (see Ref. 41 for a review). However, no studies have examined the relative role that PDP plays in regulating skeletal muscle PDH activity with obesity.
Repeated bouts of aerobic physical activity (endurance training) play an important role in health, specifically from the perspective of therapy and health maintenance. The regulation of PDH, specifically through PDK, has been identified as one of the important metabolic regulatory points targeted by exercise-induced shifts in skeletal muscle fuel utilization (24, 25, 33). Much like obesity, little evidence exists as to the role of PDP in regulating skeletal muscle PDH with exercise.
There appears to be a clear role of PDK on obesity- and exercise-mediated alterations in skeletal muscle carbohydrate metabolism. However, this represents only half of the potential regulatory picture, as changes to either covalent modulator may alter the activation state of PDH. Coordinate changes in skeletal muscle PDP activity opposite to PDK may result in accentuated regulation of PDH activity. To date, no studies have examined PDP activity and protein expression with obesity and/or endurance training, and this absence of literature warrants examination. Thus, the purpose of this study was to examine the changes in PDP activity and relative isoform content in three skeletal muscle types in a rodent model of obesity, the Zucker rat, before and after 8 wk of endurance training. We hypothesized that total PDP activity and PDP1 protein would 1) decrease with obesity and 2) increase with endurance training, mainly in oxidative skeletal muscle types (soleus and RG) in both lean and obese groups. Although PDP2 exists in much lower concentrations in muscle, and only detectable in RG, we hypothesized that similar changes may be observed in this isoform.
MATERIALS AND METHODS
Obese (fa/fa) and lean Zucker rats were obtained from Charles River Laboratories (Saint-Constant, Quebec, Canada) at 5 wk of age. The animals were housed in a controlled environment with a 12:12-h light-dark cycle and were fed standard rat chow (27% protein, 11% fat, 63% carbohydrate; 5012 Rat Diet, Lab Diet, Oakville, Ontario, Canada) ad libitum. The study and all protocols and procedures were approved by the Brock University Animal Care and Utilization Committee and conformed to all Canadian Council on Animal Care guidelines.
Both obese and lean rats were randomized to train or remain sedentary, with 8 animals assigned to each group. Rats that were trained ran 1 h/day, 5 days/week for 8 wk on a motorized rodent treadmill (Columbus Instruments, Columbus, OH). Training was gradually increased from 10 m/min on a 0% grade to 20 m/min on a 10% grade by the end of 2 wk, which was maintained for the remaining 6 wk. Training volume was similar between exercised groups. During the training sessions, sedentary rats were equally handled and placed on the stationary treadmill to control for handling stress. Body weight of both trained and sedentary rats was taken at the beginning of each training session. To prevent any acute effects of the last training bout, experimental procedures were carried out 48 h after the last training bout. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt), and select hind limb skeletal muscles (soleus, RG, WG) were extracted. Muscles from one leg were immediately frozen in liquid nitrogen, whereas muscle from the other leg was kept fresh at 4°C for isolation of mitochondria. Blood samples (1–2 ml) were collected through intracardiac puncture with a heparinized syringe after muscle excision and placed on ice. Omental and retroperitoneal fat was removed and weighed. Body composition was estimated by bioelectrical impedance analysis (BIA; Quantum II, RJL Systems, Clinton Township, MI), using the regression equation for fat free mass (FFM): FFM (g) = [0.38 × body weight (g)] + [13.8 × length (cm)2 × resistance−1)] + 70.9 (10, 22). In turn, percent fat mass (% FM) was calculated as follows: % FM = [body weight (g)·0.78 − FFM (g)] × body weight (g)−1 × 100 (10).
An aliquot of whole blood was deproteinized 1:5 with 6% perchloric acid for analysis of glucose (2). A second aliquot was centrifuged to isolate plasma for analysis of free fatty acids (Wako Chemicals, Richmond, VA) and insulin (insulin RIA kit, Siemans Healthcare Diagnostics, Tarrytown, NY).
Intact mitochondria (28, 34) were extracted from fresh muscle, and an aliquot was used to determine total PDP activity (19, 26) using a synthesized polypeptide substrate (30), synthesized and purified by New England Peptide (Gardner, MA). Small pieces of frozen muscle were homogenized, and citrate synthase maximal enzyme activity (39) and PDH in the active form (PDHa; 37) were measured.
Western blot analysis.
A piece of frozen muscle was homogenized in 10 volumes of homogenization buffer (pH 6.8) containing 250 mM sucrose, 100 mM KCl, and 2 mM EDTA and resuspended in sample buffer containing 50 mM Tris·HCl (pH 6.8), 2% (wt/vol) sodium dodecyl sulfate, 10% (vol/vol) glycerol, 5% (vol/vol) 2-mercaptoethanol, and 0.1% (wt/vol) bromophenol blue to a final protein concentration of 2 μg/μl (26). Samples were solubilized by boiling for 5 min and then cooled on ice for 5 min. Standard SDS-PAGE was performed with a 4% stacking and 12% separating gel (30 μg protein per lane, within the linearity range of the protein) (23). Electrophoretically separated proteins were transferred onto polyvinylidene fluoride (PVDF; Immobilon-P, Millipore, Bedford, MA) membrane using a Mini Trans-Blot (Bio-Rad Laboratories, Mississauga, Ontario, Canada) with a transfer buffer containing 25 mM Tris pH 8.3, 192 mM glycine, 20% methanol (vol/vol), and 0.1% sodium dodecyl sulfate (wt/vol). Membranes were incubated in Tris-buffered saline-Tween (TBST) buffer [20 mM Tris base, 137 mM NaCl, 0.1% (vol/vol) Tween 20, pH 7.5] with 5% (wt/vol) nonfat dry milk for 1 h to block all nonspecific binding sites. Membranes were then incubated for 1 h in 5% milk-TBST containing monoclonal antibodies against PDP isoforms (PDP1 and 2; Kamiya Biomedical, Seattle, WA; 26). The membranes were washed and then incubated for 1 h in 5% milk-TBST-containing goat antimouse IgG (peroxidase conjugated, Sigma, Ontario, Canada). Membranes were again washed, and antibody-antigen complexes were visualized using Fluorchem 5500 imaging station (Alpha Innotech, San Leandro, CA) after addition of chemiluminescent substrate (ChemiGlow West, Alpha Innotech, San Leandro, CA). Relative densities were quantified using AlphaEase FC chemiluminescent detection software (Alpha Innotech, San Leandro, CA). Blots were washed and stained with DB-71 (16), and total protein per lane was used to normalize loading between lanes on each blot. Data are expressed as the percentage of PDP1 and PDP2 arbitrary density units relative to those in the lean sedentary skeletal muscles (set at 100%). Because of the relatively high abundance of PDP1 and PDP2 in rat heart and kidney (19), extracts of these tissues were used as positive controls.
All data are presented as means ± SE. For all variables measured, a two-way ANOVA was used to establish differences between genotype and training status. Tukey's post hoc test was used to determine significance (P < 0.05). Assumptions for normality and independence were verified by generating appropriate residual plots. Data transformations (log for PDP activity per gram of wet tissue weight of soleus and RG; square root for retroperitoneal fat and glucose; inverse square root for free fatty acids and PDP activity per milligram of mitochondrial protein of RG) were used to meet the above assumptions.
As expected, there was a significant genotype effect on changes in body weight over the 8-wk intervention, with obese being 2 to 2.5 times heavier than lean counterparts (Table 1). After 8 wk of training, only the obese group demonstrated 25% less body weight gain compared with sedentary obese counterparts, while the body weights of lean rats were unaltered with training. There was 4–8 times more omental and retroperitoneal fat in obese compared with lean rats (Table 1). This resulted in ∼4 to 6 times higher % fat mass in obese rats compared with lean (Table 1).
Insulin, FFA, and glucose were significantly elevated in obese compared with lean, but training had no effect (Table 1).
Citrate synthase activity.
There was a significant training effect on all muscle types examined, evident by the 35–50% increase in whole muscle CS activity (Table 2). There were no significant differences between lean or obese rats whether sedentary or trained.
PDP and PDHa activities.
Obesity had no effect on PDP activity expressed per gram of wet tissue weight in all skeletal muscles examined (Table 3). Training in the lean group demonstrated a trending increase in PDP activity in soleus (P = 0.11) and RG (P = 0.13), but these were not significant. In contrast, training did significantly increase PDP activity 1.4–2.2-fold in all skeletal muscles examined in the obese group. When expressed per milligram of extracted mitochondrial protein, obesity impaired PDP activity, but this was only significant in RG (soleus, P = 0.11; WG, P = 0.47), and this effect was reversed by endurance training (Table 4). Resting PDHa activities did not show significance with either obesity or training (Table 5).
PDP isoform protein.
In the sedentary groups, obesity had no effect on PDP1 protein content in all skeletal muscle types examined (Fig. 1, A and B). In contrast, PDP1 protein content was significantly increased with training in soleus of both lean and obese genotypes and RG of the obese rats. PDP2 was not detectable in soleus and WG. Neither obesity (P = 0.51) nor training (P = 0.29) affected PDP2 protein in RG (Fig. 2, A and B).
Relationship between CS and PDP.
Training increased CS activities, a marker of mitochondrial biogenesis, in both groups and in all skeletal muscle types examined by 35–50%. This was matched by comparable increased PDP activities per gram wet tissue weight in soleus and RG of the lean group by 43–51%. In contrast, training in obese Zucker rats increased PDP activities per gram wet tissue weight by 143, 188, and 221% in soleus, RG, and WG, respectively. The relationship between PDP as it relates to a marker of mitochondrial biogenesis (PDP to CS ratio) is shown in Fig. 3.
To the authors' knowledge, this represents the first study to examine PDP activity and PDP isoform expression in three major skeletal muscle types in response to 8 wk of endurance training in both lean and obese Zucker rats. This study represents an integral step in our understanding of PDH regulation in skeletal muscle with obesity-mediated insulin resistance and endurance exercise. The major findings from the present study were 1) obesity resulted in a 46% decrease in red gastrocnemius PDP activity expressed per milligram extracted mitochondrial protein, while PDP isoform content was unchanged; 2) obesity did not affect PDP activity or isoform content in soleus or white gastrocnemius muscles; 3) 8 wk of endurance training led to a significant 1.4–2.2-fold increase in PDP activity of muscle from obese but not lean rats, but the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training.
PDP and obesity.
The current study demonstrates an obesity-mediated impairment to PDH regulation, through PDP, but this was limited to PDP activity, as it is expressed per milligram mitochondrial protein only in RG (46% lower compared with lean counterparts). The generalized lipotoxic effects of obesity on skeletal muscle have been clearly characterized in the literature, which include aberrant intramuscular lipid deposition, insulin resistance, and inflexible metabolic shifts between fat and carbohydrate utilization (see Refs. 31 and 40 for a review). Previous research examining the specific association between obesity/insulin resistance and skeletal muscle PDH regulation has been limited to effects on PDK activity and isoform expression (see Ref. 41 for a review). This association was first demonstrated in a population of Pima Indians that are highly susceptible to obesity and insulin resistance, in which skeletal muscle PDK isoform mRNA expression was negatively correlated with insulin sensitivity (27). This association has also been seen in postbiliopancreatic diversion patients (in which there was a threefold increase in peripheral insulin sensitivity and a 50% decrease in skeletal muscle PDK4 mRNA as body weight was normalized; 38) and a spontaneously diabetic rodent model (which demonstrated markedly increased fasting plasma insulin that correlates with increasing skeletal muscle PDK activity; 1). The end result is an attenuated PDH activity and decreased carbohydrate flux. However, there is evidence in the literature of coordinated regulation of PDH demonstrated in skeletal muscle (26) and nonskeletal muscle (19) tissues. Changes in PDP activity opposite to alterations in PDK activity have been observed presumably as a mechanism to optimize PDH regulation (see Ref. 11 for a review). However, this may only be seen during periods of altered carbohydrate oxidation (i.e., starvation, acute bout of exercise). In the current study, skeletal muscle samples were taken 48 h after the last exercise bout and only after an overnight fast, thus possibly resulting in an inability to detect differences in PDHa activities in lean and obese groups.
Despite a significant obesity-mediated alteration to PDP activity in RG, the implications as it relates to whole organism glucose homeostasis are not as clear. However, the nonsignificant decrease in PDP activity per milligram of mitochondrial protein in soleus and WG does not exclude biological significance. Similar decreases in PDP activity in different skeletal muscle types, significant or not, may collectively result in a decreased ability to control carbohydrate oxidation through PDH. Future studies should focus on fiber-specific PDP responses to determine the relative responses of certain skeletal muscles with varying fiber-type composition.
Changes in PDP activities may be through dynamic (allosteric regulators) or stable (e.g., changes to total enzyme content) mechanisms. Allosteric regulators that enhance PDP activity include Ca2+, Mg2+, insulin (6), and cellular redox potential NADH:NAD+ (35). It is important to note that the mitochondrial isolation procedures are rigorous, and the resulting dilutions during the isolation and enzymatic assay methodologies lower the concentration of potential allosteric effectors enough to prevent their effects, as previously determined (21). Thus, decreased PDP activities with obesity that persisted after the mitochondrial preparation indicate that these changes are stable or “effector-independent” rather than dynamic regulatory mechanisms.
Despite decreased RG PDP activity with obesity, these differences were not demonstrated with PDP1 and PDP2 proteins, identifying a dissociation between protein content and activity. A previous study has shown evidence of elevated PDP activity mediated through phosphorylation of PDP1 in L6 myocytes and PDP2 in liver cells (5). The apparent disconnect between protein content and activity in RG may lie in this regulatory aspect of PDP, in which total PDP protein (PDP1 and PDP2) does not exclusively determine PDP activity. In fact, Caruso et al. (5) demonstrated that insulin stimulation of PDH involves the translocation of protein kinase Cδ to the mitochondria, resulting in phosphorylation and increased activity of PDP, which, in turn, would activate PDH. Thus in a model like the obese Zucker rat, a reduced insulin sensitivity could potentially decrease protein kinase Cδ activity and/or translocation to the mitochondria, resulting in attenuated PDP phosphorylation and activity. Future work is needed to further our understanding of the role PDP phosphorylation plays in skeletal muscle PDH regulation with obesity.
The current study may be the first to report attenuated skeletal muscle PDP activity with obesity, but it is not the first to highlight the importance of this association. Piccinini et al. (36) reported downregulation of lymphocyte PDP activity in obese humans. Also, administration of agents that improve glucose homeostasis and insulin sensitivity also increases hepatic PDP activity in rodent models of type-2 diabetes (8, 9). Despite these findings, these studies did not identify or speculate as to the mode of action in which obesity and/or treatment altered PDP activity. Thus, in addition to the current study, there appears to be growing evidence as to the importance of PDP in obesity-mediated alterations in skeletal muscle carbohydrate metabolism.
PDP and endurance training—lean.
In the current study, training in the lean group had no significant effect on PDP or PDHa activities in all skeletal muscle types examined. Despite a lack of statistical significance, there may be biological significance to the nearly 1.5-fold increase in PDP activity in soleus and RG with training, as there was a concomitant 1.3–1.6-fold increase in PDP1 protein content. These changes in PDP activity and protein reported in both soleus and RG may simply represent a by-product of mitochondrial biogenesis. As demonstrated in Fig. 3, there was no significant difference in PDP activity expressed as a ratio of citrate synthase activity in muscles from lean rats. An important by-product of repeated bouts of aerobic exercise is mitochondrial biogenesis (17), defined by increases in mitochondrial content following training. Training also increases the mitochondrial capacity to produce ATP, resulting in tightly coupled oxidative phosphorylation and higher level of respiratory control, along with an elevation of enzyme systems responsible for fat and carbohydrate metabolism (15). Specifically, 7–8 wk of endurance training results in a greater reliance on fat for ATP synthesis during submaximal exercise (12, 20, 29). This is coupled with increased carbohydrate flux maximal capacity (decreased activation of PDH and increased total PDH activity) and metabolic control sensitivity (increased PDK activity) in both rodent (32) and human (24, 25) mixed muscle. Thus, it appears that, in addition to documented increases in total PDH and PDK activities, an increase in PDP activity with training will enhance maximal capacity and metabolic control sensitivity of carbohydrate flux.
PDP and endurance training—obese.
One of the most widely prescribed modalities to reverse the obesity-mediated metabolic derangement in skeletal muscle is exercise (see Refs. 3 and 13 for a review). To our knowledge, this is the first study to examine the training-induced metabolic alterations in obese skeletal muscle at the level of PDP regulation. Only one other study has examined the outcome of training on obese skeletal muscle PDH, which demonstrated that 9 mo of endurance training in overweight and obese patients doubled protein content of the E1α subunit of PDH (14). This finding was not surprising as a 30% increase in skeletal muscle PDH-E1α protein content with training in lean healthy human subjects has been previously demonstrated (25). Thus, similar to what has been documented with PDH-E1α, it would be expected that a training-induced increase in PDP activity would also change to a greater extent with obesity.
The increased PDP activities in obese skeletal muscle with training did not translate to altered PDHa activities at rest. However, much like obesity and training separately, this response may not be seen until carbohydrate need is altered acutely, such as during starvation or exercise. Also, PDP activities in the obese trained group were greater than what could be accounted for simply by mitochondrial biogenesis, as evident by the PDP:CS ratio (Fig. 3). These responses identify a potential training-induced reversal of the obesity-mediated impairment in skeletal muscle PDP activity. Despite somewhat similar increases in PDP:CS ratios, upon closer examination of PDP activity and PDP isoform content responses, each obese skeletal muscle type examined was unique in its reaction to the training stimulus.
Soleus demonstrated a 2.4-fold increase in PDP activity when expressed per gram wet tissue weight, which was coupled with a 2.5-fold increase in PDP1 protein content. This association is similar to what was seen in the same skeletal muscle type with training in the lean group. Thus, it appears that obesity has no effect on how soleus muscle responds to endurance training.
In contrast to what was seen in soleus, RG demonstrated a disconnect between activity and protein content similar to what was seen in RG of sedentary lean and obese groups. This was evident by a significant 2.9-fold increase in PDP activity and a modest, yet significant, 1.6-fold increase in PDP1 protein content. The effect of training on skeletal muscle carbohydrate utilization is an intricate balance between the metabolic control of carbohydrate utilization, through the activation of PDH, and the maximum potential capacity to utilize carbohydrates, measured as elevated total PDH (25). Thus, an increase in RG PDP1 protein content agrees with a training-induced increase in maximum carbohydrate flux potential, whereas a phosphorylation-mediated regulation of PDP activity would enhance metabolic control sensitivity. One of the many responses of obese RG muscle to a training stimulus may be to enhance insulin sensitivity, which as discussed earlier could elevate PKCδ-mediated increases in PDP activity through phosphorylation (5). However, further studies focused on PDP activity and activation of RG in response to training are required to understand the role of PDP phosphorylation in skeletal muscle PDP activity.
Finally, there was a somewhat unexpected 3.2-fold increase in PDP activity in WG from obese rats in response to training. This training-induced increase in PDP activity occurred without changes to PDP isoform content. Possibly, in much the same way as RG, training-mediated increase in WG PDP activity may be through changes to the phosphorylation state of PDP1. Further studies are required to confirm the existence of this mechanism, to ascertain whether it does take place in WG, and whether any other skeletal muscles containing predominantly type IIB fibers share this similar response.
Perspectives and Significance
The present study clearly demonstrates that obesity and 8 wk of endurance training influence PDH regulation in rodent skeletal muscle. The alterations are evident at the level of PDP activity and, in some cases, PDP1 protein content. As a result, obese skeletal muscle may have impaired carbohydrate flux due to decreased PDP activity, but not decreased PDP isoform protein content. In contrast, endurance-trained skeletal muscle may have an increased exercise-induced response to oxidize carbohydrates, through increased calcium-sensitive PDP1 protein content and PDP activity. Future work needs to establish fiber type-specific alterations in PDP activities and isoform protein content and whether these relationships between obesity, physical activity, and PDH regulation through PDP hold true in human skeletal muscle.
This study was supported by the Natural Sciences and Engineering Research Council of Canada (to S. J. Peters and B. D. Roy). P. J. LeBlanc was supported by a postdoctoral fellowship, and M. Mulligan was supported by a Julie Payette Research Scholarship from the Natural Sciences and Engineering Research Council of Canada. L. MacPherson was supported by a Master's Graduate Training Award from the Canadian Institutes of Health Research.
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- Copyright © 2008 the American Physiological Society