Human skeletal muscle PDH kinase activity and isoform expression during a 3-day high-fat/low-carbohydrate diet

Sandra J. Peters1, Robert A. Harris3, Pengfei Wu3, Tanya L. Pehleman1, George J. F. Heigenhauser2, and Lawrence L. Spriet1

1 Department of Human Biology and Nutritional Sciences, University of Guelph, Guelph, Ontario N1G 2W1; 2 Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 3Z5; and 3 Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The increase in skeletal muscle pyruvate dehydrogenase kinase (PDK) activity was measured in skeletal muscle of six healthy males after a eucaloric high-fat/low-carbohydrate (HF/LC; 5% carbohydrate, 73% fat, and 22% protein of total energy intake) diet compared with a standardized prediet (50% carbohdyrate, 30% fat, and 21% protein). Biopsies were obtained from the vastus lateralis muscle after 3 days on the prediet (day 0) and after 1, 2, and 3 days of the HF/LC diet. Intact mitchondria were extracted from fresh muscle and analyzed for PDK activity and Western blotting of PDK2 and PDK4 protein. A second biopsy was taken at each time point and frozen for Northern blot analysis of PDK2 and PDK4 mRNAs. PDK activity increased in a linear fashion over the 3-day HF/LC diet and was significantly higher than control by 1 day. PDK activity was 0.09 ± 0.03, 0.18 ± 0.05, 0.30 ± 0.07, and 0.37 ± 0.09 min-1 at 0, 1, 2, and 3 days, respectively. PDK4 protein and mRNA increased maximally by day 1, and PDK2 protein and mRNA were unaffected by the HF/LC diet. Resting respiratory exchange ratios decreased after 1 day of the HF/LC diet (from 0.79 ± 0.02 to 0.72 ± 0.02) and remained depressed throughout the 3-day dietary intervention (0.68 ± 0.01). The immediate shift to fat utilization was accompanied by increased blood glycerol, beta -hydroxybutyrate, and plasma free fatty acid concentrations. These results suggest that the continuing increase in PDK activity over the 3-day HF/LC diet is not due to increasing PDK protein beyond 1 day. This could be due to the contribution of another isoform to the total PDK activity or to a continual increase in PDK4 or PDK2 specific activity.

pyruvate dehydrogenase kinase 2; pyruvate dehydrogenase kinase 4; respiratory exchange ratio; pyruvate dehydrogenase activity; mitochondria


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PYRUVATE DEHYDROGENASE (PDH) is a multienzyme complex that catalyzes the conversion of pyruvate to acetyl-coenzyme A. The reaction is the first irreversible step in the oxidation of carbohydrate-derived carbon and regulates the entry of carbohydrate into the tricarboxylic acid cycle. PDH activity is tightly regulated by reversible phosphorylation and dephosphorylation reactions catalyzed by an intrinsic kinase and phosphatase (22, 23, 35, 40). Phosphorylation of E1 catalytic subunits of PDH by PDH kinase (PDK) causes inactivation of the enzyme, whereas PDH phosphatase removes phosphate and returns the enzyme to its active form (PDHa) (22, 23). The relative activities of the phosphatase and PDK determine the proportion of the complex in the active form.

During rest, PDH is acutely regulated by the allosteric regulation of the kinase and phosphatase by intramitochondrial effectors. High ATP-to-ADP, NADH-to-NAD+, and acetyl-CoA-to-CoASH ratios and low pyruvate concentrations favor PDK activation and decreased PDH activity (9, 12, 35, 40). During exercise, PDH phosphatase is accelerated by increasing Ca2+ concentrations, allowing for activation of the PDH complex (32).

In addition to this acute regulation, rodent studies have demonstrated an adaptive increase in PDK activity after prolonged periods of carbohydrate restriction (e.g., starvation, diabetes) (16, 19, 34) or increased dietary fat (27). The increase in PDK persists even after a rigorous mitochondrial preparation, indicating that enhanced activity was not simply the result of effector accumulation (the result of increased reliance on fat fuel) but reflected a stable adaptation or covalent modification.

By virtue of its size, skeletal muscle is the major site of whole body glucose disposal. Overall regulation of carbohydrate disposal by skeletal muscle is regulated by a combination of glucose uptake and phosphorylation, storage as glycogen, and oxidation through PDH. With starvation and a prolonged high-fat diet, decreased flux through PDH serves a protective role, helping to preserve scant carbohydrate stores. However, in diabetes, the decrease in carbohydrate oxidation due to increased PDK activity can exacerbate the clinical dilemma of decreased skeletal muscle glucose disposal.

A recent study from our laboratory (29) demonstrated that human skeletal muscle PDK activity increased three- to fivefold when subjects consumed a high fat/low carbohydrate (HF/LC) diet. Increased activity was observed as early as 3 days on the diet, and the effect persisted until 6 days. That study did not investigate whether the increase in PDK activity occurred before 3 days or whether it was the result of increased protein synthesis or a stable increase in the specific activity.

There are currently four known isoforms of PDK (13, 31), which differ significantly in their tissue distribution (6, 13) as well as in their specific activity and sensitivity to effectors (6). Therefore, the relative concentrations of the various isoforms at any given time will affect the way in which the kinase responds to acute changes in intramitochondrial concentrations of effectors and determine the proportion of the complex that is active. The tissue distribution of the four isoforms is also different between rats and humans, with all four isoforms present in human skeletal muscle but only PDK1, PDK2, and PDK4 detected in rat skeletal muscle (6, 13). Early studies in rodent models indicated that PDK4 was the only isoform contributing to increased activity in heart (42) and skeletal muscle (41) with diabetes and starvation. However, more recently, an increase in PDK2 was observed in mixed fast-twitch muscle after a prolonged, 48-h fast and a 28-day HF diet (14, 36). Only one study has examined PDK isoform expression in human skeletal muscle in response to increased reliance on fat fuel, associated with insulin resistance in the Pima Indians of Arizona (1, 20). Those subjects had higher levels of both PDK2 and PDK4 mRNA in skeletal muscle of insulin-resistant individuals compared with their nondiabetic counterparts (25). However, no studies to date have investigated the early response to an increased reliance on fat fuel through dietary manipulation, and currently there are no human studies that link PDK activity to isoform protein and mRNA.

The initial purpose of this study was to examine the time course of the increase in PDK activity during the first 3 days of a HF/LC diet. We hypothesized that there would be a gradual increase in PDK activity over the 3 days, significantly different by 1 or 2 days. The second purpose was to characterize the changes in PDK protein and mRNA of the most abundant isoforms in human skeletal muscle (PDK2 and PDK4). Our goal was to determine whether the increase in PDK activity over 3 days on a HF/LC diet was the result of an increase in abundance of one or both of the PDK isoforms. We hypothesized that PDK4 expression would increase in concert with the increases in PDK activity and that PDK2 expression would be unaltered during the short-term dietary intervention.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Six male university students volunteered for this study. They were in good health with a mean age of 22.0 ± 0.4 yr (range 19-25 yr), weight of 82.5 ± 6.6 kg (range 70-105 kg), and height of 187 ± 5 cm (range 173-201 cm). Three of the subjects were very active (aerobic activity >8 h/wk), two were moderately active (aerobic activity ~4 h/wk), and one was less active (~1 h/wk), as determined by activity questionnaires. The mean relative maximal oxygen uptake (VO2 max) was 51 ± 4 ml · kg-1 · min-1 (range 32-57.8 ml · kg-1 · min-1). Three additional subjects volunteered for a less invasive study of the changes in the respiratory exchange ratio (RER). Similar to the other subjects, all were recreationally active and participated in some form of aerobic activity >= 3 times/wk. These three subjects were 22 yr of age, with an average weight of 72.3 kg (68-80 kg) and height of 180 cm (174-187 cm). Subjects were informed of the risks of the procedures before giving informed consent, and the study was approved by the ethics committees of the University of Guelph and McMaster University.

Study design. Subjects provided a 3-day diet record of their regular (REG) diet. An individualized eucaloric prediet [PRE; only minor changes were made to achieve 50% carbohydrate (CHO), 30% fat, and 20% protein] and a HF/LC diet (5% CHO, 73% fat, and 22% protein) were designed and prescribed for each subject (Fig. 1). The eucaloric HF/LC diet was designed without including fish or flaxseed products, which would be high in n-3 fatty acids, because these have been shown to influence PDK gene expression in animal tissue (11). The dietary regimens were outlined in detail for the subjects, and they were given instructions concerning the importance of consuming the diets as prescribed and recording everything they ate or drank. Food was purchased and packaged for each subject. Before the actual study, the subjects reported to the lab to determine VO2 max. The subjects then reported to the laboratory four times. They began the PRE diet 3 days before reporting to the laboratory for the first time (day 0). In every case, the subjects arrived in the laboratory 1-1.5 h after a breakfast that adhered to the diet they were consuming the night before (PRE or HF/LC diet). On day 0, two biopsies were taken from the vastus lateralis muscle as previously described (3). They immediately began the HF/LC diet. Subjects continued with their normal daily activities but refrained from formal exercise sessions during the diet intervention. They returned to the laboratory on days 1, 2, and 3 of the HF/LC diet for two biopsies on each day.


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Fig. 1.   Study design. Subjects consumed a prescribed prediet (PRE), which closely resembled a normal Western diet consisting of 50% carbohydrate, 30% fat, and 20% protein of the total energy intake. They reported to the laboratory on day 0 for blood samples and two muscle biopsies before beginning a high-fat/low carbohydrate (HF/LC) diet consisting of 5% carbohydrate, 73% fat, and 22% protein for 3 days. Sampling of blood and muscle occurred on days 1, 2, and 3 of the diet intervention.

In addition, on each of days 0, 1, 2, and 3, ~3 ml of blood were drawn from the antecubital vein into heparinized tubes. One portion of whole blood was deproteinized 1:2 with 6% (wt/vol) perchloric acid for analysis of beta -hydroxybutyrate, glucose, lactate, and glycerol (2). A second portion of whole blood was centrifuged, and a 400-µl aliquot of plasma was removed, added to 100 µl of 6 M sodium chloride, and incubated for 30 min at 56°C to inactivate lipoprotein lipase. This plasma supernatant was analyzed for free fatty acids (FFA) using a Wako NEFA C test kit (Wako Chemicals, Richmond, VA). Insulin was measured on an aliquot of the remaining plasma by means of a Coat-a-Count insulin test kit (Diagnostics Products, Los Angeles, CA).

Processing of muscle samples. Intact mitochondria were extracted from fresh muscle (~75-80 mg) for the analysis of PDK activity and for Western blotting for the PDK isoforms. A second biopsy (~50-100 mg) was frozen in liquid nitrogen for Northern blotting for the mRNA of the PDK isoforms.

Mitochondrial extraction. Intact mitochondria were extracted by differential centrifugation as previously described (17, 26, 29). Briefly, minced muscle was homogenized using a glass-on-glass Potter homogenizer in 20 volumes of a buffer containing (in mM) 100 KCl, 40 Tris · HCl, 10 Tris base, 5 magnesium sulfate, 1 EDTA, and 1 ATP (pH 7.5). The supernatant was retained after centrifugation at 700 g for 10 min, and a crude mitochondrial pellet was extracted with centrifugation at 14,000 g (10 min). The pellet was washed, resuspended, and pelleted twice (7,000 g, 10 min) in 10 volumes of (in mM) 100 KCl, 40 Tris · HCl, 10 Tris base, 1 magnesium sulfate, 0.1 EDTA, and 0.25 ATP (pH 7.5). The first wash buffer included 1% (wt/vol) bovine serum albumin, and the second was protein free. The final mitochondrial pellet was resuspended in a volume corresponding to 1 µl/mg fresh muscle extracted. The final buffer contained (in mM) 220 sucrose, 70 mannitol, 10 Tris · HCl, and 1 EDTA (pH 7.4). All procedures were carried out at 0-4°C. Unless specifically stated, all chemicals were obtained from Sigma Chemical (St. Louis, MO).

Incubation of mitochondria for PDK activity. The final mitochondrial suspension (50 µl) was diluted with 250 µl of buffer containing 10 µM carbonyl cyanide m-chlorophenylhydrazone, 20 mM Tris · HCl, 120 mM KCl, 2 mM EGTA, and 5 mM potassium (pH 7.4) and incubated for 20 min at 30°C, driving ATP concentration to zero and causing complete conversion of PDH to the active form as previously described (10). Mitochondria were pelleted at 7,000 g for 10 min and stored in liquid nitrogen for later analysis of PDK.

PDK activity. PDK activity was determined as previously described (29). Briefly, the mitochondrial pellet was resuspended in ~300 µl of a buffer containing 30 mM KH2PO4, 5 mM EGTA, 5 mM dithiothreitol, 25 µg/ml oligomycin B, 1.0 mM tosyl-lysyl chloromethyl ketone, 0.1% (wt/vol) Triton X-100, and 1% (wt/vol) bovine serum albumin (pH 7.0), and freeze thawed twice to ensure that all mitochondria were broken. The suspension was warmed to 30°C, and two aliquots of the suspension were diluted 1:1 in a buffer containing (in mM) 200 sucrose, 50 potassium chloride, 5 magnesium chloride, 5 EGTA, 50 Tris · HCl, 50 sodium fluoride, and 5 dichloroacetate and 0.1% (wt/vol) Triton X-100 (pH 7.8) for later analysis of PDH activity. This point represents "zero-time" or "total PDH." Magnesium ATP was added to the remaining suspension to bring the concentration to 0.3 mM, and timed samples were taken every 20-30 s for 3-5 min (depending on PDK activity) as previously described (10, 29, 38). For our method, however, the samples were diluted 1:1 in the previously described sodium fluoride-dichloroacetate buffer to lock the PDHa activity through inhibition of the phosphatase and kinase, respectively. The samples were stored on ice for analysis of PDHa activity by radioisotopic measurement as described previously (8, 30). PDK activity is reported as the apparent first-order rate constant of the inactivation of PDH (min-1), or the slope of ln {%[PDHa activity (with ATP addition)]/total PDH (without ATP addition)} vs. time (10, 38). There was no appreciable loss of activity in the absence of ATP over the 3- to 5-min experiment.

Calculation of recovery and quality of mitochondrial preparation. Citrate synthase (CS) activities on the total muscle homogenate (CShomog) and mitochondrial suspensions were measured as previously described (29, 33). Briefly, a small volume of the mitochondrial suspension was diluted 20-fold with the final sucrose and mannitol buffer and divided into two fractions. Extramitochondrial CS (CSem) was measured in the intact mitochondrial preparation, and CS activity in the total suspension (CSts) was measured after the preparation was frozen and thawed twice to open mitochondria. Triton (0.1%) was included in the cuvette for measurement of CSts and CShomog.

Recovery of intact mitochondria was calculated as
%fractional recovery<IT>=</IT>100<IT>×</IT>(CS<SUB>ts</SUB><IT>−</IT>CS<SUB>em</SUB>)<IT>/</IT>CS<SUB>homog</SUB>
and averaged 31 ± 2% for this study. As with previous work (4, 29), the quality of the preparation (88 ± 1% for this study) was checked with each extraction and was calculated as the percentage of intact mitochondria
%intact mitochondria<IT>=</IT>100<IT>×</IT>(CS<SUB>ts</SUB><IT>−</IT>CS<SUB>em</SUB>)<IT>/</IT>CS<SUB>ts</SUB>

Western blotting. Mitochondria were diluted to a final protein concentration of 1 µg/µl in 50 mM Tris · HCl, pH 6.8, containing 2% (wt/vol) SDS, 0.1 M dithiothreitol, 0.1% (wt/vol) bromophenol blue, 10% (vol/vol) glycerol, 1 mM benzamidine, 0.1 mg/ml trypsin inhibitor, 1 µg/ml aprotinin, 0.1 mM tosyl-lysyl phenylmethyl ketone, 1 µM leupeptin, and 1 µM pepstatin A. 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 10% separating gel (5 µg mitochondrial protein/lane). Electroblotting and immunodetection were performed as previously described (42). Polyclonal antisera against recombinant PDK2 and PDK4 had previously been tested for cross-reactivity (42). Antibody-antigen complexes were visualized with 125I-labeled protein A (ICN Pharmaceuticals, Irvine, CA) followed by autoradiography. Relative densities were quantified using Northern Eclipse from Empix Imaging (Mississauga, ON, Canada), and results were normalized to the day 0 (or control) value for each subject.

Northern blotting. Total RNA was extracted from frozen muscle by use of a BIO101 FastRNA Kit-Green (La Jolla, CA). Total RNA (6-8 µg) was loaded onto 1% denaturing agarose gel and run with 10 mM MOPS, 4 mM sodium acetate, and 0.5 mM EDTA buffer (pH 7.0) at 75 V for 4 h. Gels were visualized under ultraviolet light, and then RNA was blotted using capillary action with a 20× standard sodium citrate solution overnight onto a Nytran supercharged membrane (Schleicher & Schuell, Keene, NH). cDNAs for PDK2 and PDK4 have been described previously (6, 42). The individual cDNAs were labeled using [32P]ATP and [32P]CTP (Amersham Pharmacia Biotech, Piscataway, NJ) and the random primed DNA labeling kit (Boehringer Mannheim, Montreal, QC, Canada). Stratagene QuickHyb solution and protocol were used for hybridization with the labeled probe, except that hybridization was performed for 2 h at 68°C (Stratagene Cloning Systems, La Jolla, CA). After blots were washed according to the method prescribed for Statagene QuickHyb, autoradiographs were developed using Kodak X-omat film (Rochester, NY). Relative densities were quantified using Northern Eclipse from Empix Imaging.

Resting RER. The three participants underwent the diets as described for the major study and reported to the laboratory at least 4 h postprandial on days 0, 1, 2, and 3 of the HF/LC diet. Blood was sampled on days 0 andas previously described. Subjects were asked to lie in a supine position in a quiet, darkened room for 30 min. At this time, their expired gases were analyzed for volume and oxygen and carbon dioxide concentration for 15 min (model 2900, SensorMedics, Yorba Linda, CA). These measurements were used to calculate oxygen consumption (VO2) and carbon dioxide production (VCO2) and the average resting RER (VCO2/VO2).

Diet analysis and statistics. REG, PRE, and HF/LC diets were analyzed using Nutripro (West Publishing, Salem, OR) and then reported as the mean analysis for 3 days for REG, PRE, and HF/LC diets.

Results were analyzed using a one-way ANOVA (time) with repeated measures. A Fisher protected least significant difference post hoc test was used to compare means. Significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Diet analysis. The results of the diet analysis for the REG, PRE, and HF/LC diets demonstrated that the eucaloric diets were maintained. Total energy intakes were 13,180 ± 1,151, 13,560 ± 1,053, and 13,308 ± 974 kJ for the REG, PRE, and HF/LC diets, respectively. The PRE diet adhered closely to the target values of an average North American mixed diet (49.3 ± 0.5% CHO, 29.8 ± 0.5% fat, and 20.8 ± 0.4% protein) and did not vary greatly from the REG diet. The HF/LC diet was severely restricted in CHO (4.9 ± 0.2%) and very high in fat (73.2 ± 0.9%) but had similar protein content compared with the PRE diet (22.5 ± 0.3%).

Blood measurements. Plasma FFA and whole blood beta -hydroxybutyrate were significantly elevated after 1 day on the HF/LC diet and remained elevated at days 2 and 3 (Table 1). Blood glycerol levels were significantly elevated on days 2 and 3 on the HF/LC diet. Plasma insulin decreased by ~55% on days 1-3, but the high day 0 variability precluded statistical significance. Blood lactate and glucose were unchanged by the 3-day HF/LC diet.

                              
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Table 1.   Time course (day 0-day 3) for plasma insulin, FFA, and whole blood glycerol, beta -hydroxybutyrate, and glucose concentrations

For the three subjects participating in the RER study, blood results were similar to those in the main study, and respective values for days 0 and 3 of the HF/LC diet were, respectively: FFA, 0.2 ± 0.1 vs. 1.0 ± 0.4; insulin, 14 ± 4 vs. 8.1 ± 0.5 mIU/l; glycerol, 0.05 ± 0.01 vs. 0.09 ± 0.02 mM; beta -hydroxybutyrate, 0.07 ± 0.01 vs. 0.28 ± 0.03 mM; lactate, 0.8 ± 0.2 vs. 0.7 ± 0.1 mM; and glucose, 4.7 ± 0.3 vs. 4.8 ± 0.1 mM. Glycerol and beta -hydroxybutyrate concentrations were significantly elevated by day 3 in this subject subset.

Resting RER. Resting RER decreased from 0.79 ± 0.02 on day 0 to 0.72 ± 0.02 on day 1 and remained depressed on days 2 and 3 (0.68 ± 0.01) on the HF/LC diet (Fig. 2).


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Fig. 2.   Resting respiratory exchange ratio (RER) during 3 days of a HF/LC diet. Three additional subjects followed the exact protocol for the major study, except that only resting RER was measured on each of the 3 days [O2 uptake/CO2 production (VCO2/VO2)]. aSignificantly different from day 0.

PDK activity and isoforms. Mean PDK activity was elevated as early as 1 day on the HF/LC diet and continued to increase up to 3 days in a linear fashion (Fig. 3A). The individual data (Fig. 3B) showed that PDK increased in all subjects, with a large variability between subjects.


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Fig. 3.   Pyruvate dehydrogenase kinase (PDK) activity during 3 days of a HF/LC diet. A: mean PDK activity. aSignificantly different from day 0; bsignificantly different from day 1. B: individual subject data.

Mean changes in PDK2 and PDK4 protein are represented in Fig. 4. PDK2 was unaltered by the HF/LC diet, whereas PDK4 increased an average of 70% (range 20-280%) by day 1, remaining high throughout the dietary intervention.


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Fig. 4.   Mitochondrial protein concentrations of PDK2 (A) and PDK4 (B), assessed from Western blotting. Films from blots were integrated, and intensities of the bands were normalized to day 0 for each subject. aSignificantly different from day 0.

PDK4 mRNA increased an average of ~30-fold by day 1 on the HF/LC diet, remaining high throughout the 3-day dietary intervention (Fig. 5). PDK2 mRNA was unaltered by the diet (Fig. 5). Representative blots for isoform protein and mRNA are depicted in Fig. 6.


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Fig. 5.   PDK2 (A) and PDK4 (B) mRNA concentrations, assessed from Northern blotting. Films from blots were integrated, and intensities of the bands were normalized to day 0 for each subject. aSignificantly different from day 0.



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Fig. 6.   Representative blots of PDK4 (A) and PDK2 (B) isoform protein and mRNA. Western blotting was performed using standard SDS-PAGE techniques with a 4% stacking and 10% separating gel. Mitochondrial protein was loaded (5 µg/lane), and visualization of the blots for PDK2 and PDK4 was achieved using 125I-labeled protein A. For Northern blotting, 6-8 µg of total RNA were loaded onto 1% denaturing agarose gel. For further details, see METHODS.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A major finding of this study was that PDK activity increased as early as 1 day on a HF/LC diet. The increase in PDK activity was linear throughout the 3 days, gradually increasing in all subjects. The increase in PDK activity was accompanied by an increase in PDK4 protein and mRNA, but PDK2 protein and mRNA were unchanged throughout the 3 days. The increase in PDK4 protein and mRNA was maximal after 1 day. Therefore, the continuing increase in activity could be due to the contribution of another isoform or to changes in the specific activity of PDK2 or PDK4. This provides evidence of a dissociation of the protein and mRNA concentration of PDK4 from the total enzyme activity and underscores the need for measurement of PDK activity, because increased mRNA levels alone are not adequate to quantitatively predict changes in all situations. Although a few studies report changes in PDK isoform mRNA, this study and our previous study (29) are the only reported measures of human skeletal muscle PDK activity.

Fuel utilization in response to HF/LC diet. The experimental diet chosen in this study was extreme, with both a very high fat content (73 vs. 30%) and a very low CHO content (5 vs. 50%) compared with the average Western diet. The decrease in resting RER by 1 day indicated a rapid shift in fuel utilization from a mix of CHO and fat to almost complete fat oxidation. This was consistent with data from a previous study that used a similar HF/LC diet (72% of energy provided by fat) for 5 days and demonstrated a decrease in resting RER to 0.71 (18). However, the previous work did not examine the time course during the 5-day diet to determine how rapidly such a shift would occur. Our data do not preclude the possibility that the fuel switch happened even earlier, within the first 24 h.

The shift to reliance on fat metabolism was correlated with the increased plasma FFA, glycerol, and beta -hydroxybutyrate throughout the 3-day HF/LC diet intervention. Plasma insulin decreased ~55%, but this was not significant in the 3-day period. Lactate and glucose were unchanged. These data were consistent with the blood parameters from our previous study (29). FFA, glycerol, beta -hydroxybutyrate, and insulin changed by an intermediate amount in the first 3 days of the HF/LC diet compared with the 6-day data previously documented (29). After 6 days of HF/LC diet intervention, insulin significantly decreased (~67%), a trend which is evident from the 3-day data (29).

Time course of increased PDK activity and PDK isoform expression. PDK activity increased rapidly during the HF/LC diet and was significantly different from control by day 1. This correlated with the change in RER and the shift to fat metabolism. Although PDK2 protein and mRNA were unaltered throughout the diet, PDK4 protein and mRNA were also increased maximally in 1 day. However, PDK activity continued to increase gradually in a linear fashion over the 3-day period. When the data from the present study and a previous study (29) from this laboratory were combined, PDK activity increased linearly throughout the first 6 days of the HF/LC diet (Fig. 7). This suggests that either another PDK isoform was contributing to the continuous increase in PDK activity or there was an increase in PDK specific activity. The binding of the kinase to the PDH complex has been shown to stimulate activity; therefore, one possibility is that the specific activity of newly synthesized PDK4 (or loosely associated PDK2) could be gradually enhanced as it is assembled and bound into the complex (24, 43). The concept of gradual incorporation of PDK is supported by a recent study on PDK activity in mitochondria from 5-day septic rats (39). That work examined the increase in PDK activity in both the complex-bound fraction and the loosely associated "free" PDK fraction. Those results demonstrated that PDK activity was significantly increased in both the fractions, threefold in the PDK fraction that was intrinsic to the complex and almost twofold in the free PDK fraction (39). Although protein was not measured, it was clear that not all of the PDK protein was immediately incorporated into the complex. Therefore, this could account for the discrepancy in the present study between the time course of increased total mitochondrial PDK4 protein and the total PDK activity.


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Fig. 7.   PDK activity in human skeletal muscle during 6 days of a HF/LC diet. Combined data from the current study (days 0, 1, 2, and 3) and from Peters et al. (29) (days 0, 3, and 6). No. of observations for each time point: n = 14, 6, 6, 11, and 8 for days 0, 1, 2, 3, and 6, respectively.

Rate of increase of PDK activity and the role of insulin sensitivity. Although PDK activity increased in all six subjects, there was considerable variability in the rate of increase during the first 3 days of the HF/LC diet. The subjects with the greatest increase in PDK activity in the first 3 days were those individuals who were more physically active (as suggested by their prestudy activity questionnaire). There was a strong correlation between a subject's physical activity level and the change in (Delta )PDK activity in the first 3 days of the dietary intervention (Fig. 8). However, this correlation disappeared by 6 days (Fig. 8). This suggests that the early rate of response to the diet is sensitive to a subject's aerobic capacity but that the overall change in PDK activity over 6 days was similar for all individuals, regardless of training status. The mechanism for this is unclear. Decreasing insulin levels (or a decreased response to insulin) and increased skeletal muscle fat metabolism have both been implicated as important regulators of PDK activity and expression (7, 25, 27, 28, 37). It has been suggested previously that there is an increase in insulin sensitivity with training through some postreceptor event (5). Because there was no correlation between a subject's activity level and the decrease in plasma insulin by 1 or 3 days of this study, it is possible that trained individuals were more sensitive to the similar decrease in circulating insulin concentration, resulting in a larger early increase in PDK activity.


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Fig. 8.   Correlation between the relative maximal O2 uptake (VO2 max) of the individual and the change in (Delta )PDK activity after either 3 (A) or 6 days (B) on a HF/LC diet. Data are combined from Peters et al. (29) and the present study. The correlation with relative VO2 max is significant for the increase in PDK activity during the first 3 days (r2 = 0.82, P = 0.00014) but not after 6 days of the dietary intervention (r2 = 0.016, P = 0.76).

Rate of increase of PDK activity and the role of increased fat metabolism. Training also increases skeletal muscle fat metabolism; therefore, it is also possible that the increased FFA metabolism in the active individuals stimulated the more rapid increase in PDK activity. The family of peroxisomal proliferator-activated receptors (PPARs) has evoked considerable interest as the possible link between FFAs, metabolic regulation, and gene expression. Rat work has demonstrated increased skeletal muscle PDK4 expression with PPARalpha agonist, and it is possible that PPARalpha may participate as a link between increased fat metabolism and decreased carbohydrate oxidation by downregulating PDH (41). PPARalpha is similarly implicated in the upregulation of many fat-metabolizing enzymes and could serve as a generalized coordinator of metabolism (21). Recent work has demonstrated that PPARalpha increases with exercise training, highlighting the possibility that increased concentrations of PPARalpha could contribute to the rapid increase in PDK activity observed in fit individuals (15). It is clear that these possibilities require further investigation.

In summary, this study describes the time course of the increase in human skeletal muscle PDK activity and PDK2 and PDK4 expression during the first 3 days of a HF/LC diet. PDK activity increased in a linear fashion throughout the 3 days and was significantly increased after only 1 day on the diet. This does not preclude the possibility that PDK activity is elevated within the first 24 h. PDK2 protein and mRNA were unaltered by the 3-day dietary intervention. In contrast, PDK4 protein and mRNA were maximally increased by 1 day. These results suggest that the increase in PDK4 protein was responsible for the increase in PDK activity during the 1st day of the HF/LC diet. However, the continuing increase in PDK activity from days 1 to 3 was not due to further increases in PDK protein. It could be the result of increased concentration of another PDK isoenzyme, and/or to a gradual increase in PDK2 or PDK4 specific activity throughout the 3 days.


    ACKNOWLEDGEMENTS

The technical assistance of Linda Van Ostaaijen is gratefully acknowledged.


    FOOTNOTES

This study was supported by operating grants from the Natural Sciences and Engineering Research, and Medical Research Councils of Canada, and the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47844). S. J. Peters was supported by an Ontario Graduate Scholarship and the Gatorade Sport Science Institute.

Address for reprint requests and other correspondence: S. J. Peters, Dept. of Physical Education, Faculty of Applied Health Sciences, Brock University, St. Catharines, ON, Canada L2S 3A1 (E-mail: speters{at}arnie.pec.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.

Received 3 April 2001; accepted in final form 19 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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