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Am J Physiol Endocrinol Metab 292: E571-E576, 2007. First published October 3, 2006; doi:10.1152/ajpendo.00327.2006
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Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Paul J. LeBlanc,1 Robert A. Harris,2 and Sandra J. Peters1

1Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada; and 2Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana

Submitted 15 July 2006 ; accepted in final form 29 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Fiber type specificity of pyruvate dehydrogenase (PDH) phosphatase (PDP) was determined in fed (CON) and 48-h food-deprived (FD) rats. PDP activity and isoform protein content were determined in soleus (slow-twitch oxidative), red gastrocnemius (RG; fast-twitch oxidative glycolytic), and white gastrocnemius (WG; fast-twitch glycolytic) muscles. When normalized for mitochondrial volume, there was no difference in PDP activity between muscle types or CON and FD. When expressed per gram wet tissue weight, PDP activity was higher in RG compared with soleus and WG in both CON and FD rats. PDP activities from CON muscles were 1.48 ± 0.19, 2.68 ± 0.65, and 1.20 ± 0.33 nmol·min–1·g wet tissue wt–1 in soleus, RG, and WG, respectively, and decreased in FD muscles (1.22 ± 0.22, 2.00 ± 0.57, and 0.84 ± 0.18 nmol·min–1·g wet tissue wt–1). This correlated with increased PDP2 protein, however, only in RG, as PDP2 was not detectable in soleus or WG. PDP1 protein was not responsive to food deprivation in all fiber types. In conclusion, PDP activity and protein content were higher in fast-twitch oxidative glycolytic muscles from CON and FD rats, identifying a unique inter- and intramuscular distribution. FD induced a small but significant decrease in PDP activity that was partially due to decreases in PDP2 protein. As a result, coordinate changes to PDP activity opposite to those of the other regulatory enzyme, PDH kinase, during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply.

pyruvate dehydrogenase phosphatase-1; pyruvate dehydrogenase phosphatase-2; starvation; carbohydrate oxidation

PYRUVATE DEHYDROGENASE (PDH) is a key component of glucose homeostasis, as it regulates mitochondrial carbohydrate oxidation. The activation of PDH (PDHa), via dephosphorylation by PDH phosphatase (PDP), promotes carbohydrate oxidation. During periods of carbohydrate conservation (e.g., food deprivation), PDH is deactivated via phosphorylation by PDH kinase (PDK; as reviewed in Refs. 10 and 38). The complexity of PDH control by PDK and PDP is enhanced by the presence of multiple isoforms, four PDK [PDK1–4 (5)] and two PDP [PDP1 and -2 (18)], with PDK2 and -4 (5) and PDP1 (18) predominantly expressed in skeletal muscle. Intercomparison of both PDK and PDP isoforms reveals differences in specific activities and kinetic properties, tissue-specific mRNA, and protein content and responsiveness to different allosteric regulators, resulting in unique responses to certain metabolic demands. Alterations in activities of either PDK or PDP can occur due either to altered total activity or to modulation through intramitochondrial effectors. Thus, the ratio of PDK and PDP activities, with changes to either covalent modulator, alters the activation state of PDH.

Food deprivation is characterized by a metabolic shift to increased fat utilization, resulting in a conservation of endogenous carbohydrate to be used by brain and nervous tissue. A key enzyme that facilitates this shift from carbohydrates to fat utilization, especially in skeletal muscle, is PDH (35). Numerous studies have demonstrated that 12–48 h of food deprivation result in a significant decrease in skeletal muscle PDH activity (11, 13, 14, 17, 36). This appears to be mediated, in part, through increased PDK activity, predominantly in oxidative muscle (28, 33, 36, 39). These changes in PDK activity are mainly due to an altered expression of the PDK4 isoform (28, 36, 39). Previous studies have reported that food deprivation decreases PDP activity in rat mammary gland (7) and in kidney and heart tissues (19). These changes in PDP activity are due mainly to a decreased expression of the PDP2 isoform (19). This raises the possibility that changes in PDP opposite to those changes in PDK may occur to maximize the inactivation of PDH in skeletal muscle during altered nutritional states.

To date, only one study has examined PDP activity in skeletal muscle, demonstrating no change with 48 h of food deprivation or alloxan-diabetic rats (13). This study was conducted using whole hindlimb skeletal muscle preparation, which does not distinguish relative fiber type responses. No studies have been conducted to examine the relative expression and fiber type distribution of PDP1 and -2 in mammalian skeletal muscle. In addition, alterations in skeletal muscle PDP activity and isoform expression have not been examined with altered nutritional status. Thus the purposes of this study were to 1) measure PDP activity and isoform expression in different skeletal muscle fiber types and 2) comprehensively examine the changes in PDP activity and relative isoform in response to 48 h of food deprivation. We hypothesized that PDP activity and PDP2 isoform expression would decrease with food deprivation and to a greater extent in the more oxidative skeletal muscle fibers, with no change in PDP1. To represent the three major fiber types, slow-twitch oxidative soleus (Sol, 84% type I, 7% type IIA, 0% type IIB), fast-twitch oxidative glycolytic red gastrocnemius (RG, 30–51% type I, 35–62% type IIA, 1–8% type IIB), and fast-twitch glycolytic white gastrocnemius (WG, 0% type I, 0% type IIA, 92% type IIB) muscles were used (8).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing on average 295 ± 8 g were used in the experiment. The animals were housed in a controlled environment with a 12:12-h light-dark cycle (lights on at 7 AM) and were fed standard rat chow (27% protein, 11% fat, 63% carbohydrate; 5012 Rat Diet, Lab Diet, Oakville, ON, Canada) ad libitum until food withdrawal. The study was approved by the Brock University Animal Care and Utilization Committee (AUP no. 010501).

Study design. To determine the effects of food deprivation, food, but not water, was withheld from a group of rats for 48 h (FD; n = 10). A separate control group of rats (CON; n = 10) were fed ad libitum throughout the time period of the study. Due to the potential diurnal effects on blood and muscle measurements (34), rats were sampled in pairs (1 CON and 1 FD) at the same time each day (10 AM, 3 h into light cycle) exactly 48 h after the commencement of food deprivation. Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt), and selected hindlimb skeletal muscles (Sol, RG, and WG) were extracted as quickly as possible. 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.

Blood analysis. An aliquot of whole blood was deproteinized 1:5 with 6% perchloric acid for analysis of beta-hydroxybutyrate (beta-HB), glucose, lactate, and glycerol (4). A second aliquot was centrifuged at 15,900 g for 2 min to isolate plasma for analysis of free fatty acids (FFA; Wako Chemicals, Richmond, VA). Insulin was determined on the remaining plasma by use of a sensitive rat insulin RIA kit (Linco Research, St. Charles, MO).

Muscle analysis. Intact mitochondria were extracted from fresh muscle (26, 30), and citrate synthase (CS) activities were measured to assess mitochondrial recovery and quality (28). An aliquot of extracted mitochondria was used to determine PDP activity by utilizing the nonradioactive phosphatase assay system (Promega, Madison, WI) (19), with the exception that the assay was conducted at 37°C and the synthesized polypeptide substrate (27) was synthesized and purified by New England Peptide (Gardner, MA). PDP activity is expressed as nanomoles of phosphate released per minute per milligram of extracted mitochondrial protein. On the basis of mitochondrial recoveries for each skeletal muscle fiber type, PDP activity is also expressed per gram wet tissue weight. A small 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. Standard SDS-PAGE and Western blotting were performed as previously described (24, 29). Monoclonal antibodies against PDP isoforms (PDP1 and -2; Kamiya Biomedical, Seattle, WA) were used. Due to the relatively high abundance of PDP1 and -2 in rat heart and kidney (19), extracts of these tissues were used as positive controls. The remainder of the frozen muscle was used for the determination of PDHa activity (32).

Statistical analysis. All data are presented as means ± SE. For blood, plasma, and muscle PDHa and PDP2 protein data, unpaired t-tests were used to establish differences between treatments. For CS and PDP activities and PDP1 protein muscle data, a two-way analysis of variance with repeated measures was used to establish differences between treatment and skeletal muscle fiber types. 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, square root, and inverse square root) were used when appropriate to meet the above assumptions.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Mitochondrial preparation and CS activities. Mitochondrial recoveries were similar between treatment groups for soleus (CON, 14 ± 5%; FD, 19 ± 4%), RG (CON, 18 ± 4%; FD, 16 ± 2%), and WG (CON, 9 ± 3%; FD, 10 ± 2%) and were similar between fiber types. Mitochondrial quality was also similar between treatment groups for soleus (CON, 69 ± 6%; FD, 76 ± 4%), RG (CON, 67 ± 3%; FD, 68 ± 7%), and WG (CON, 60 ± 8%; FD, 60 ± 4%) and were similar between fiber types.

There were no significant differences between whole muscle homogenate CS activities between treatment groups for soleus (CON, 22.2 ± 1.7; FD, 22.0 ± 2.2 µmol·min–1·g wet tissue wt–1), RG (CON, 27.0 ± 1.2; FD, 28.3 ± 2.0 µmol·min–1·g wet tissue wt–1), and WG (CON, 12.6 ± 1.2; FD, 15.1 ± 4.0 µmol·min–1·g wet tissue wt–1), with soleus CS activity being significantly lower than RG but significantly higher than WG.

Blood. beta-HB and FFA were significantly elevated, and insulin was significantly lower after 48 h of food deprivation (Table 1). Glucose levels remained unchanged.


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  		Table 1. Plasma insulin and FFA concentrations and whole blood beta-HB and glucose concentrations in fed and food-deprived rats

 
PDH and PDP activity. Food deprivation significantly decreased PDHa activity in all skeletal muscle fiber types (Table 2). PDP activities, expressed per milligram of extracted mitochondrial protein or gram wet tissue weight, were significantly lower (main effect) in FD animals compared with CON irrespective of skeletal muscle type (Table 3 and Fig. 1). PDP activities per gram wet tissue weight were significantly higher (main effect) in RG compared with soleus and WG, irrespective of treatment (Fig. 1).


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  		Table 2. PDHa activity for the 3 skeletal muscle types in fed and food-deprived rats

 

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  		Table 3. PDP activity per mg of extracted mitochondrial protein for the 3 skeletal muscle types in fed and food-deprived rats

 

Figure 1
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  		Fig. 1. Pyruvate dehydrogenase phosphatase (PDP) activity per g wet tissue wt for the 3 skeletal muscle types in fed and food-deprived (FD) rats. Values are means ± SE for tissue samples obtained from 8–10 animals in each group. CON, control; RG, red gastrocnemius; WG, white gastrocnemius. Two-way ANOVA revealed a significant main effect for treatment ({dagger}P < 0.001) and skeletal muscle types ({ddagger}P = 0.016).

 
PDP isoform protein. There was significantly more PDP1 protein in RG compared with soleus and WG (Fig. 2). There was no effect of food deprivation on PDP1 protein in any of the skeletal muscle fiber types. PDP2 protein was not detectable in soleus and WG (Fig. 3). Food deprivation significantly decreased PDP2 protein in RG.


Figure 2
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  		Fig. 2. PDP1 isoform protein content in the 3 skeletal muscle types in fed and FD rats. Values are means ± SE for tissue samples obtained from 6–7 animals in each group. {ddagger}Two-way ANOVA revealed a significant main effect for skeletal muscle types (P < 0.001).

 

Figure 3
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  		Fig. 3. PDP2 isoform protein content in the 3 skeletal muscle types in fed and FD rats. Values are means ± SE for tissue samples obtained from 6–7 animals in each group. nd, not detectable. *Significance from CON (P < 0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Extensive work has been done on PDK activity and protein isoform expression in various tissues under various physiological conditions; however, the understanding of PDP expression and regulation to date is limited. This represents the first study to examine PDP activity and both known PDP isoforms (PDP1 and -2) in three of the major skeletal muscle fiber types represented by slow-twitch oxidative soleus, fast-twitch oxidative glycolytic RG, and fast-twitch glycolytic WG. This study also describes fiber type variations in PDP activity and protein in response to 48 h of food deprivation. The major findings from the present study were that 1) PDP activity is fiber-type specific, with higher activity in fast-twitch oxidative glycolytic skeletal muscle fibers (RG); 2) PDP1 protein is ubiquitous in all skeletal muscle fiber types, whereas PDP2 protein is detectable only in RG; and 3) 48 h of food deprivation led to a significant decrease in PDP activity (main effect) and PDP2 protein, with no effect on PDP1 protein.

PDP activity. To our knowledge, the present study is only the second to measure PDP activity in mammalian skeletal muscle and the first to examine PDP activity in representative skeletal muscle fiber types. The only other study to measure PDP activity in mammalian skeletal muscle utilized a mixed hindlimb preparation (13). Comparatively, PDP activities in all fiber types of the current study were slightly higher compared with mixed skeletal muscle (13). This difference is possibly due to the methodology of the assay used in the current study, with higher assay temperature (37 as opposed to 30°C) and a sensitive and specific substrate (synthesized amino acid sequence surrounding phosphorylation sites 1 and 2 of PDH E1{alpha} subunit as opposed to 32P-phosphorylated pig heart PDH complex).

In response to food deprivation, PDP activity decreased in all skeletal muscle types, a result similar to previous studies in nonskeletal muscle tissues (3, 7, 19), thus supporting the concept of a coordinated regulation of PDH (for review see Ref. 15). In addition, the present study demonstrates the importance of examining individual skeletal muscle types which may, in part, help explain the earlier results from Fuller and Randle (13), demonstrating no change in mixed skeletal muscle PDP activity with food deprivation.

When expressed per milligram of extracted mitochondrial protein, there are no differences across skeletal muscle fiber types examined, indicating a relationship between PDP activity and mitochondrial density. The PDH complex, along with PDP, is found within the mitochondrial matrix. As such, it may be expected that highly oxidative tissues with more mitochondria would have higher PDP activity. When expressed per gram wet tissue weight, PDP activity is highest in RG [predominantly type I and IIA (8)] and lowest in WG [predominantly type IIB (8)], similar to skeletal muscle fiber type-specific oxidative capacity, represented by CS activity demonstrated in the current study, and PDK activity (28). In fact, PDK correlates positively with the oxidative capacity of differing skeletal muscle fiber types (28). However, when the two highly oxidative skeletal muscle fiber types examined in the current study are compared, PDP activity in soleus [predominantly type I (8)] is about two times lower than RG. As a result, it appears that the skeletal muscle distribution of PDP activity is fiber type specific and not solely associated with mitochondrial density or oxidative capacity. Similar results have been seen in other processes responsible for oxidative glucose disposal in fast oxidative glycolytic vs. slow oxidative fibers. Insulin sensitivity, glucose uptake, and GLUT4 protein content are highest in skeletal muscles that have predominantly type IIA fibers compared with type I (16).

PDP protein. These changes in total PDP activity 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+, and insulin (9). It is important to note that the mitochondrial isolation procedures are rigorous, and the resulting dilutions during the isolation and enzymatic assay methodologies very likely lower the concentration of potential allosteric effectors enough to prevent their effects, as previously determined (20). Thus, the decreased PDP activities with food deprivation that persisted after the mitochondrial preparation indicate that these changes are stable rather than dynamic regulatory mechanisms.

The unique properties of PDP1 and -2 reflect their relative importance in the tissue of interest. PDP1 is stimulated by Ca2+ (18, 21) and preferentially expressed in Ca2+-sensitive tissues [e.g., contractile tissue, brain, testes (18, 19)], whereas PDP2 is relatively unaffected by the absence or presence of Ca2+ (18, 21) but is stimulated by insulin (6) and found more abundantly in liver and adipose tissue (18, 19). Until now, the skeletal muscle fiber type distribution of PDP isoforms was unknown, as previous studies reported relative abundance of PDP1 and -2 in mixed rat hindlimb (1, 18), which would exhibit the combined properties of all fiber types.

The abundance of Ca2+-sensitive PDP1 protein and mRNA in contractile tissues (18, 19) identifies the need for rapidly increased PDH activation during contraction. PDP1 protein was the dominant isoform in all skeletal muscle fiber types examined, in good agreement with previous mixed skeletal muscle data of rat hindlimb (19). This supports the concept that total PDP activity in skeletal muscle primarily reflects the activity of PDP1. When compared against fiber types, PDP1 protein mirrored PDP activity, with the highest found in RG. In response to food deprivation, there is a lack of evidence in the literature to suggest changes in skeletal muscle Ca2+ flux that may impact PDP1 protein expression. Thus it is not surprising that PDP1 protein did not change with food deprivation, a result that has been demonstrated in rat kidney and heart (19).

The skeletal muscle fiber type distribution of PDP2 differs from that of PDP1, with no detectable protein in soleus or WG. These findings suggest that PDP2 distribution does not appear to be related to oxidative capacity or fiber type. Similar to our findings in RG, Huang et al. (18) demonstrated low but detectable PDP2 protein in mixed skeletal muscle of rats. The identification of the fiber type-specific location of PDP2 appears to be restricted to type IIA fibers and requires further work to explain the significance of this localization.

In response to food deprivation, insulin-sensitive PDP2 may be the target for decreased skeletal muscle PDP activity, as insulin decreases during food deprivation. The effects of insulin on PDH complex activity, purportedly through changes in PDP activity (31), have been demonstrated in various mammalian tissues (2, 19, 31), including skeletal muscle (11, 25). Decreased PDP activity after 48 h of food deprivation may be attributed, in part, to a decreased PDP2 protein, which was seen in RG. The PDH-E1{alpha} subunit possesses three serine residues that, when phosphorylated, determine the activation state of PDH. Phosphorylation of the first site is sufficient for more than 98% inactivation of the enzyme complex (23). It has been proposed that phosphorylation of the other two sites is required to regulate the dephosphorylation and reactivation of the PDH complex by PDP (37). The site-specific dephosphorylation displayed by the PDP2 isoform (21, 22) augments the importance of its downregulation with food deprivation.

In addition to insulin, changes in circulating plasma fatty acids, which increase with food deprivation, may impact skeletal muscle PDP activity. A previous study demonstrated a negative correlation between plasma FFA and the abundance of PDP1 mRNA (PDP2 was not measured) in a rodent model of spontaneous development of type 2 diabetes (1), suggesting that the activation of fat-dependent promoters (e.g., peroxisome proliferator-activated receptor-{alpha}) may play a role in decreasing PDP expression. There are numerous metabolic and hormonal factors (12) that could contribute to altered PDP expression in response to food deprivation. Future research will need to focus on regulatory parameters linked to PDP transcription and translation.

In summary, the present study clearly demonstrates that PDP activity is highest in a representative fast-twitch oxidative glycolytic muscle compared with both slow-twitch oxidative and fast-twitch glycolytic muscles. There is a unique inter- and intramuscular distribution of PDP isoforms, with PDP1 protein highest in predominantly type IIA muscle compared with types I and IIB muscles and PDP2 protein detected exclusively in predominantly type IIA muscles. In response to 48 h of food deprivation, PDP activity decreased in all muscle types, PDP2 decreased in fast-twitch oxidative glycolytic muscle, and PDP1 was unaltered. As a result, coordinate changes to PDP activity opposite to those of PDK activity during food deprivation would maximize the inactivation of skeletal muscle PDH and enhance carbohydrate conservation during periods of limited carbohydrate supply (15).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Natural Sciences and Engineering Research Council of Canada (S. J. Peters) and grants from National Institute of Diabetes and Digestive and Kidney Diseases (DK-47844, R. A. Harris) and the Grace M. Showalter Residuary Trust (R. A. Harris). P. J. LeBlanc was supported by a postdoctoral fellowship from the Natural Sciences and Engineering Research Council of Canada.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. LeBlanc, Faculty of Applied Health Sciences, Brock University, 500 Glenridge Ave., St. Catharines, ON, Canada L2S 3A1 (e-mail: pleblanc{at}brocku.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
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 DISCUSSION
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N. S. Bradley, G. J. F. Heigenhauser, B. D. Roy, E. M. Staples, J. G. Inglis, P. J. LeBlanc, and S. J. Peters
The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity
J Appl Physiol, January 1, 2008; 104(1): 1 - 9.
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