Increased pyruvate dehydrogenase in response to sepsis
kinase activity
THOMAS C. VARY Department of Cellular and Molecular Physiology, Milton S. Hershey Pennsylvania State University, Hershey, Pennsylvania 17033
VARY, THOMAS C. Increased pyruvate dehydrogenase kinase activity in response to sepsis. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E669-E674, 1991.-The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-l (r = 0.993; P < 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-l; r = 0.999; P < 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-’ (r = 0.987; P < 0.001) (P < 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity. kinase activator tal muscle
protein;
dichloroacetate;
mitochondria;
skele-
CHRONIC SEPSIS produces physiological and metabolic pertubations resulting in a hyperdynamic cardiovascular response, net whole body protein catabolism, and increased glucose turnover (12, 19, 20, 27, 30). Hyperglycemia and hyperlactatemia are frequent manifestations of the metabolic response to sepsis(l&19,26,30). Whole body studies utilizing tracer methodologies have suggested that a diversion of glucose carbon away from oxidation leads to increased plasma lactate concentrations in sepsis (12, 19, 20, 27, 30). The activity of the pyruvate dehydrogenase (PDH) complex is a key mechanism in the control of carbohydrate oxidation and conservation of whole body glucose. The PDH complex is the first irreversible step in the oxidative pathway of pyruvate in mitochondria and is tightly regulated by both end product inhibition and reversible phosphorylation (13-14, 21). Phosphorylation (inactivation) is catalyzed by a PDH kinase, and dephosphorylation (reactivation) is catalyzed by a mito0193~1849/91
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chondrial PDH phosphatase (13-14,21). Interconversion of phosphorylated and dephosphorylated forms of the PDH complex plays a major role in the regulation of glucose oxidation under a variety of conditions in animals and humans (for review see Ref. 16). The PDH kinase and phosphatase reactions operate simultaneously, and the proportion of the PDH complex in the active (dephosphorylated) form depends upon the relative rates of these two reactions. The PDH phosphatase reaction requires Mg”+ and, in the presence of Mg”+, is activated by increasing the Ca2+ concentration (17). The PDH kinase reaction is accelerated by increasing intramitochondrial concentration (square brackets) ratios of [ATP] to [ADP], [NADH] to [NAD], and [acetylCoA] to [CoA] (9, 11). Changes in these concentration ratios appear to be the principal mechanism responsible for changes in the proportion of active PDH complex induced by oxidation of fatty acids or ketone bodies or by changes in work in isolated tissues (for review see Ref. 16). There also appears to be another longer-term mechanism that increases PDH kinase activity. The mechanism of PDH kinase activation is independent of mitochondrial oxidation of fatty acids or ketone bodies and is not mediated by changes in the metabolite effector concentrations of the PDH kinase (4-6,8-11). Activation of the PDH kinase (up to 3-fold) during starvation and diabetes in mitochondrial extracts from heart and liver has been suggested to be due to a protein factor termed kinase activator protein (4-5, 8, 10). Previous studies from this laboratory have demonstrated that sepsis decreases the proportion of active PDH complex in skeletal muscle without any alteration in the total amount of PDH complex activity (25-27). The effect of sepsis to reduce PDH complex activity is proposed to be mediated by an increased PDH kinase activity secondary to an increased [acetyl-CoA] -to- [CoA] ratio (25). However, other mechanisms may be involved. After intraperitoneal injection of dichloroacetate, the proportion of active PDH complex in septic animals is significantly less than the full activation of the PDH complex in nonseptic animals (26). Similar observations regarding the inability of dichloroacetate to fully activate PDH complex in starvation and diabetes have been associated with an increase in PDH kinase activity (6, 11). Stimulation of the PDH kinase reaction by the kinase activator protein has been proposed to account for relative refractory effects to dichloroacetate in star-
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vation and diabetes. Evidence is presented in the present study suggesting that the effects of sepsis to lower the proportion of active PDH complex are consistent with a stable increase in PDH kinase activity.
N,N,N’,N’-tetraacetic acid (EGTA), 5 KH2P04, pH 7.41 in Eppendorf microcentrifuge tubes with further additions as given in Tables 1 and 2. For estimation of total PDH complex and PDH kinase activity, mitochondria were incubated for 15 min in buffer containing 10 PM carbonyl cyanide m-chlorophenylhydrazone without respiratory substrates that would effect conversion of inacMATERIALS AND METHODS tive complex into active complex (5-6). Mitochondria Intra-abdominal abscess model. Chronic sepsis was inwere separated by centrifugation (40s Eppendorf 5214 duced in male Sprague-Dawley rats (Charles River centrifuge), the supernatants were aspirated, and the Breeding, Wilmington, MA) (250-300 g) by the intraperpellets were frozen in liquid N2. Mitochondrial extracts itoneal introduction of a fecal-agar pellet (1.5 ml) inoc- for assay of PDH complex activity and citrate synthase ulated with 10” colony-forming units (CFU) Escherichia activity were prepared from the frozen mitochondrial coli (Serotype 018 ac) plus 10” CFU Bacteroides fragilis pellets by ultrasonic disintegration as described by Ker(ATCC no. 23745) (24-27). The sterile inflammatory bey et al. (11). For assay of PDH kinase activity, mitoabscesswas generated by replacing the bacteria with an chondrial extracts of the frozen mitochondrial pellets equal volume of sterile physiological saline. Fed rats that were prepared by alternate thawing (3O”C), dispersing were not subjected to the surgical procedures served as (Hamilton syringe aspiration), and freezing (liquid Na) control animals. After recovery from implantation of the (two times) into buffer containing 30 mM KHaP04, 5 fecal-agar pellet, all animals were allowed free access to mM EGTA, 5 mM dithiothreitol, 1% (wt/vol) fatty acidrat chow (Purina) and water, and the intra-abdominal free bovine serum albumin, 1 mM tosyl-lysyl-chloroabscesswas allowed to form for 5-7 days. Previous stud- methyl ketone, 25 pg/ml oligomycin ,& pH 7.0 (5-6, 8). ies have demonstrated that septic rats consume as much To verify conversion of inactive to active PDH complex, rat chow as control or sterile inflammatory animals on mitochondrial extracts (40 ~1~1) were incubated in buffer day 2 to 7 postimplantation of fecal-agar pellets (27). containing 75 mM Tris, pH 7.0, with partially purified Changes in the proportion of active PDH complex in PDH phosphatase (0.5 U/ml), 0.2 mg fatty acid-free septic animals are not significantly different 5 or 7 days bovine serum albumin, 10 mM MgC12, and 4 mM CaC12 after the introduction of the fecal-agar pellet (25-26), for 15 min at 30°C (11). PDH activity was then reassayed All animals that survived the peritonitis stage (100% in spectrophotometrically. PDH phosphatase was partially sterile inflammatory, 65% of septic animals) formed an purified from beef heart as described by Coore et al. (3). intra-abdominal abscess(25-27). Assays. PDH complex activity was assayed spectroSkeletal muscle mitochondria isolation. Five to seven photometrically by coupling the formation of acetyl-CoA days after the implantation of the fecal-agar pellet, anifrom pyruvate to acylation of p-( p-aminophenylazo)mals were anesthetized (Nembutal, 50 mg/kg body wt benzenesulfphonic acid using arylamine acetyltransferip). All hindlimb muscles (-25 g) from fed animals in ase as described by Coore et al. (3). Citrate synthase was each group were excised, trimmed of fat and connective assayed by the method of Srere (18) as modified by Coore tissue, minced finely, and homogenized (Waring Blender, et al. (3). All assays were performed in duplicate. One 12.5 s) in 180 ml of isolation buffer [(in mM) 100 unit of PDH complex activity converts 1 pmol of subtris(hydroxymethyl)aminomethane (Tris) . HCl, 10 strate into product per minute at 30°C. EDTA, 210 mannitol, 70 sucrose, 0.1% (wt/vol) bovine PDH kinase activity was assayed in mitochondrial serum albumin, pH 7.41 at 0°C (6). The homogenate was diluted with another 100 ml of isolation buffer, and the extracts by the rate of ATP-dependent inactivation of homogenization procedure was repeated. The homoge- the PDH complex (5, 6, 8). Mitochondrial extracts connate was incubated with stirring on ice with 50 mg of taining -30-35 mU of PDH complex were warmed to porcine trypsin (Sigma type IX) for 12.5 min and was 30°C (2 min). ATP was added to a final concentration of 0.3 mM Mg”+-ATP, and samples were taken for assay of filtered through a plastic sieve. The filtrate was centriPDH complex activity between 0.25 and 6 min. The fuged (10 min, 500 g), and the supernatant was filtered. exact times depended upon the observed rate of ATPMitochondria were sedimented (8 min, 10,000 g) from the filtered supernatant and washed twice by resuspen- dependent inactivation determined in pilot experiments. Time 0 activity was determined on a separate sample of sion and centrifugation (8 min, 10,000 g) in isolation extract. Mitochondria were also incubated (6 min) in the buffer (as above except 10 mM Tris . HCl). After the final absence of ATP to ensure that there was no appreciable centrifugation, mitochondria were suspended in isolation loss of PDH activity over the course of the incubations. buffer (containing 10 mM Tris HCl) at a concentration of lo-20 mg mitochondrial protein/ml. The yield of Inactivation of the PDH complex by ATP was calculated without ATP). mitochondria was -1 mg of protein/g fresh wt of muscle. as 100 (activity PDH with ATP)/(activity Protein concentrations of mitochondrial suspensions The PDH kinase activity was calculated as the apparent were determined by the method of Gornall et al. (7) with pseudo-first-order rate constant for the ATP-dependent inactivation of the PDH complex as determined from a crystalline serum albumin as the standard. least-squares linear regression analysis of the natural log Incubation and extraction of mitochondria. Mitochondria (0.5-l mg of protein) were incubated at 30°C in of inactivation by ATP against time of incubation (10). 0.5 ml of incubation (KCl) medium [(in mM) 20 Tris . This gives a negative rate constant. In all cases,the plots HCl, 120 KCl, 2 ethylene glycol-bis(/3-aminoethyl ether)were linear with r > 0.95, and the time 0 intercept was Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (129.100.058.076) on September 23, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
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close to 100% of PDH complex activity in the absence of ATP. The experimental data for each condition are summarized as means t SE for 4-14 mitochondrial preparations in each group of animals. Statistical evaluation of the data was performed using the Student’s t test for unpaired comparisons. For PDH kinase activity, significance between differences among the different groups was determined by analysis of covariance of the slopes for each group by Scheffe method. Differences among means were considered significant when P < 0.05 (28). RESULTS
Effect of respiratory substrates. In the work described below the total PDH complex activity was assayed in mitochondria incubated for 15 min without respiratory substrates to convert inactive to fully active PDH complex. To validate this approach in mitochondria isolated from hindlimb of sterile inflammatory or septic rats, extracts prepared from mitochondria previously incubated for 15 min without respiratory substrate were incubated with PDH phosphatase. In control rats, total PDH complex activity was 74 t 7 mU/mg mitochondrial protein (n = 6), which was not significantly different from the values obtained after incubation of mitochondria without respiratory substrates (Table 1). Total concentration of PDH complex in skeletal muscle mitochondrial extracts from sterile inflammatory (78 t 3 mU/mg mitochondrial protein; n = 4) or septic (78 t 5 mU/mg mitochondrial protein; n = 5) animals, as estimated after incubation with PDH phosphatase, was not significantly different from control. Total PDH complex activity in mitochondria from sterile inflammatory or septic rats incubated without respiratory substrates was 92 and 96%, respectively, of the value obtained in mitochondrial extracts incubated with PDH phosphatase (Table 1). To assess whether the yield of mitochondria from sterile inflammatory or septic animals might influence the concentration of total PDH complex, citrate synthase
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activity was estimated. There were no significant decreases in the concentration of citrate synthase activity (mU/mg mitochondrial protein) in sterile inflammation (934 t 85; n = 7) or sepsis (750 t 107; n = 5) compared with control (1,083 t 122; n = 10). These results confirm our previous observations using whole tissue extracts that showed the changes in proportion of active PDH complex in septic animals were not due to a decreased concentration of total enzyme activities (2526). The concentration of active PDH complex in mitochondria freshly prepared (not incubated) from fed control animals was 42 t 6%. This value is about twofold higher than estimates of the proportion of active PDH complex (20%) in skeletal muscle in vivo (25-26) and most likely is due to partial activation of PDH complex during the isolation procedure. Similar observations have been described in heart mitochondria (11). Incubation with respiratory substrates (a-ketoglutarate and malate) decreased the proportion of active PDH complex to 28 t 2%. Inclusion of either pyruvate or dichloroacetate, both inhibitors of PDH kinase, in the incubation buffer increased the proportion of active PDH complex -2-fold and M-fold, respectively, in mitochondria from control rats. Similar results were obtained with mitochondria from skeletal muscle of sterile inflammatory rats isolated and incubated under similar conditions (Table 1). These changes in the proportion of active PDH complex in mitochondria from sterile inflammatory animals under the various incubation conditions were not significantly different compared with control animals. In contrast to sterile inflammation, the concentration of active PDH complex activity in mitochondria freshly prepared (not incubated) from septic animals was significantly (P < 0.005) reduced to 45% of values obtained from control animals. This relative change is approximately equal to the difference in the proportion of active PDH complex estimated in situ between control (20%) and septic (10%) rats (26). Incubations with respiratory substrates diminished the proportion of active PDH in mitochondria isolated from septic animals, and the con-
1. Effect of respiratory substrates on concentration of active pyruvate dehydrogenase complex in skeletal muscle from control, sterile inflammatory, and septic rats
TABLE
Active Control mU/mg
No substrate Not incubated (as made) 5 mM a-Ketoglutarate and 0.5 mM malate 5 mM cu-Ketoglutarate, 0.5 mM malate, and 3 mM pyruvate 5 mM cu-Ketoglutarate, 0.5 mM malate, and 50 PM dichloroacetate
protein
PDH Sterile
n
%Total complex
mU/mg
protein
Complex
Activity
inflammation
Septic %Total complex
n
72+4 31+5 20t2
12 8 18
98k6 4226 28rt2
70t4 28-+4 17t4
14 9 6
35k5
8
49t7
23klO
54t5
8
75t7
38+5 -
mU/mg
protein
abscess n
%Total complex
9225 41t6 251k6
74t7 14t3* 9+4$
11 9 4
96k9 18t5t 11t5”
6
43t5
17+4$
5
21+4-f -
6
60t9
28e3$
4
44+12$
Results are means & SE; n, no. of mitochondrial preparations. Mitochondria isolated from skeletal muscle were incubated in KC1 medium for 15 min without respiratory substrates (no substrate) or for 5 min in KC1 medium with additions as shown. Mitochondria were separated by centrifugation and frozen in liquid N,. Extracts were prepared using ultrasonic dispersion and assayed for active pyruvate dehydrogenase (PDH) complex activity. Total PDH complex (sum of active and inactive forms) was assayed after conversion of inactive into active complex after addition of partially purified PDH phosphatase to mitochondrial extracts previously incubated without respiratory substrates. Otherwise, assume the value for %total complex was based on values for mU PDH/mg mitochondrial protein derived from no substrate incubations. All assays were performed in duplicate. * P < 0.01, “r P < 0.005, $ P < 0.05 vs. control.
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centration of active PDH was significantly decreased 60% relative to control animals. Hence, the decreased proportion of active PDH complex activity in skeletal muscle from septic animals observed in situ persists during isolation and incubation of mitochondria prepared from these animals. Furthermore, the ability of pyruvate or dichloroacetate to effect conversion of PDH into its active form was significantly decreased by 55 and 37%) respectively, in mitochondria from skeletal muscle of septic animals compared with control. PDH kinase actiuity. Previous studies in heart mitochondria from diabetic rats have suggested that the decreased responsiveness to the effects of pyruvate and dichloroacetate on the proportion of active PDH complex may be due to increased PDH kinase activity (8, 10). As shown in Fig. IA, the proportion of active PDH complex remained essentially as the initial (time 0) PDH activity after 6 min of incubation when ATP was omitted. Addition of ATP resulted in the inactivation of PDH complex such that inactivation of PDH complex was essentially complete (>90%) within 6 min of the addition of ATP. The apparent first-order rate constant for inactivation of the PDH complex by ATP in mitochondrial extracts from fed control animals was -0.42 min-’ (Table 2), which is comparable to values reported by Fuller and Randle (6). The rate of inactivation in mitochondrial extracts from skeletal muscle of sterile inflammatory animals was not significantly different from control (Fig. lA, Table 2). However, as can be seen in Fig. IA, the rate of inactivation of PDH complex was markedly faster after addition of ATP in extracts of skeletal muscle mitochondria from septic animals. This resulted in an over 2.5-fold increase (P < 0.001) in PDH kinase activity in mitochondrial extracts from septic animals compared with either control or sterile inflammation (Table 2).
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The PDH kinase reaction is inhibited by pyruvate, and pyruvate increases the proportion of active PDH complex in mitochondria from heart (11) and skeletal muscle (6). Inclusion of 2.5 mM pyruvate in the incubation medium inhibited the PDH kinase reaction in extracts of mitochondria from control animals (compare Fig. 1, A and B). No differences in the extent of inhibition of PDH kinase by pyruvate were observed between control and sterile inflammation. However, the extent of inactivation of PDH complex was significantly greater in extracts from mitochondria of septic animals compared with control or sterile inflammation. Therefore the enhanced activity of the PDH kinase reaction (relative to controls) in extracts of mitochondria from septic rats was retained even when 2.5 mM pyruvate was present. Dichloroacetate is an uncompetitive inhibitor of PDH kinase reaction; and it increases the proportion of active PDH in mitochondria isolated from heart and skeletal muscle of normal animals (7, 12, 30). Inclusion of 1 mM dichloroacetate in the incubation medium essentially completely inhibited the PDH kinase reaction in extracts of mitochondria from control animals (compare Fig. 1, A and C). The concentration of dichloroacetate giving a 50% inhibition of the rate constant was -25 PM (data not shown). This is in good agreement with the value of 16 PM reported by Fuller and Randle (6) in intact mitochondrial incubations. As was observed with pyruvate, no differences in the extent of inhibition of PDH kinase by dichloroacetate were observed between control and sterile inflammation. However, the extent of inactivation of PDH complex after incubation with 1 mM dichloroacetate was greater at each time point in extracts from mitochondria of septic animals compared with control or sterile inflammation. Hence, enhanced activity of the PDH kinase reaction (relative to controls) in extracts of C
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FIG. 1. Effects of sterile inflammation or sepsis on activity of pyruvate dehydrogenase (PDH) kinase in extracts of mitochondria from skeletal muscle. Extracts were prepared from mitochondria obtained from control (o), sterile inflammatory (A), and septic rats (6) in which inactive PDH complex had been converted into active complex by incubation in KC1 medium without respiratory substrates for 15 min at 30°C. PDH kinase reaction was initiated by addition of ATP to final concentration a 0.3 mM. In some experiments, ATP was omitted from incubation mixture (+). Details of incubation are as above except that sodium pyruvate (2.5 mM, B) or potassium dichloroacetate (1 mM, C) were present throughout 2-min preincubation and incubation with ATP. Samples were taken for assay of PDH complex at times shown. Each point is mean of 6 or more observations from 3-8 mitochondrial preparations for each group. Initial concentrations of PDH complex (mu/ml) in mitochondrial extracts were 176 t 15 (SE) (control, n = 5), 187 t 19 (sterile inflammation, n = 4), and 182 t 16 (septic abscess, n = 3). B: *P < 0.05 vs. control; C: *P < 0.005 vs. control; **P < 0.001 vs. control.
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TABLE 2. Apparent first-order rate constant for ATP-dependent inactivation of PDH complex in mitochondrial extracts
skeletal muscle. This difference between fed control and sepsis was most obvious when mitochondria were incubated in the presence of inhibitors of PDH kinase, pyruvate, and dichloroacetate. The effects of sepsis on the PDH Kinase Activity, proportion of active PDH complex in the presence of Condition n min-’ inhibitors of PDH kinase were similar to those observed Cont,rol 5 -0.42 in mitochondria isolated from heart (10) or skeletal Sterile inflammation 5 -0.43 muscle (6) of starved or diabetic rats. This suggests that Sepsis 3 -1.08* similar mechanisms may be operating in sepsis, starvaData were obtained by calculating slope of plot of natural log of tion, and diabetes to lower the proportion of active PDH percent initial PDH complex activity vs. time of incubation using data complex. However, unlike starvation or diabetes, the presented in Fig. IA by a least-squares linear regression analysis. plasma insulin concentrations in septic animals were not Correlation coefficients were 0.993 (P < O.OOl), 0.999 (P < O.OOl), and 0.987 (P < 0.001) in mitochondrial extracts prepared from control, decreased (24). sterile inflammatory, and septic animals, respectively. Significance The mechanism responsible for decreased PDH activbetween differences in PDH kinase activity was determined by analysis ity in sepsis may be due to either an increased PDH of covariance of slopes for each group by Scheffi! method (F,l,l:i = 681.7, kinase activity, a decreased PDH phosphatase activity, P < 0.001). * P < 0.001 vs. control or sterile inflammation. or both. Because the effects of starvation and diabetes mitochondria from septic rats was retained at a concen- appear kinase mediated, the effect of sepsis on PDH tration of dichloroacetate that was approximately 40- kinase activity was determined. The activity of the PDH fold higher than the inhibitory constant for control ani- kinase can be followed in extracts of mitochondria by mals. following the inactivation of PDH complex by ATP or by incorporation of [32P]ATP into PDH complex (8). Under the incubation conditions employed, the concenDISCUSSION trations of the metabolite activators of PDH kinase are The molecular basis for the inactivation of the PDH too low to have a stimulatory effect, and the phosphatase complex through increased activity of PDH kinase from is inoperative since EGTA is present (8). Using this various rat tissues appears to result from two mecha- approach, the PDH kinase reaction is M-fold more nisms. Oxidation of fatty acids and/or ketone bodies may active in mitochondria prepared from septic animals increase PDH kinase activity by increasing the mitocompared with either control or sterile inflammation. chondrial concentration ratios of [acetyl-CoA] to [CoA] Furthermore the effects of sepsis to accelerate PDH and [NADH] to [NAD] (9). This shorter-term mecha- kinase activity were not abolished by inhibiting the PDH nism can be rapidly reversed in heart muscle or skeletal kinase using either pyruvate or dichloroacetate. muscle in vitro by withdrawal of fatty acids or in vivo in The mechanism for enhanced PDH kinase activity in heart and kidney by administration of inhibitors of fatty mitochondria isolated from skeletal muscle of septic anacid oxidation such as Z-tetradecylglycidate (1). In ad- imals is currently unknown. The findings of the present dition to the acute effects of fatty acids, there appears to study suggest the mechanism must involve a rather stable be a longer-term mechanism involving activation of the change, since increased PDH kinase activity persists PDH kinase. For example, complete reactivation of PDH through isolation, incubation, and extraction of mitocomplex activity in cardiac or skeletal muscle is not chondria. Several potential mechanisms can be proposed observed for 4-24 h after refeeding (10). The increased including 1) more favorable binding of PDH kinase to PDH kinase activity is stable, since it persists during PDH complex, 2) increased reduction and acetylation of isolation, incubation, and extraction of mitochondria. It lipoyl residues of dihydrolipoamide acetyltransferase (8)) has been suggestedelsewhere that a factor, termed kinase 3) increased concentration of PDH kinase, and 4) anactivator protein, increases the PDH kinase activity in other protein factor that activates the PDH kinase. Acetstarvation and diabetes (4-5, LO). ylation or reduction of the lipoyl moieties of the dihydroSepsis is also associated with a decrease in the proporlipoamide acetyltransferase of the PDH complex seems tion of active PDH complex in skeletal muscle (25-27). unlikely, since addition of CoA and NAD to the PDH The decreased respiratory quotient observed in septic kinase assays did not reverse the increased PDH kinase patients suggests an increased dependence on fatty acids activity in extracts of heart mitochondria from diabetic as a respiratory fuel (15). Furthermore, previous studies animals (8). Studies are currently underway to determine have shown a threefold increase in the [acetyl-CoA]-tothe nature of the enhanced PDH kinase and the failure [CoA] concentration ratio in skeletal muscle from septic of pyruvate and dichloroacetate to inhibit the PDH kianimals compared with control (25). Hence, the effects nase. of sepsisto lower the proportion of active PDH complex In summary, increased PDH kinase activity may be of could simply be a consequence of increased ,&oxidation fundamental importance in the long-term regulation of of fatty acids as is observed in obese mice (2). In obese glucose oxidation and in the conservation of glucose in animals, inactivation of PDH complex in heart is not sepsis as well as other conditions (cf. Ref. 22). In fact, mediated by a stable activation of the PDH kinase. studies have demonstrated that activation of PDH comHowever, evidence presented in this study suggests that plex by dichloroacetate was associated with a normaliincreased PDH kinase activity occurs in sepsis. zation of the glucose metabolic clearance rate and lactate The effect of sepsisto decrease the proportion of active concentrations in sepsis, demonstrating the importance PDH complex persisted in mitochondria prepared from of PDH complex in whole body glucose homeostasis (26).
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The author acknowledges the assistance of Drs. J. G. Morris and B. D. Tall in preparing the bacterial inoculations for the septic animal model and the technical assistance of D. Jawor, J. Smith, and J. Arnold in conducting these experiments. This work was supported in part by National Institute of General Medical Sciences Grants GM-36139 and GM-39277. T. C. Vary is the recipient of Research Career Development Award K04 GM-00570. Address for reprint requests: T. C. Vary, Dept. of Physiology, Milton S. Hershey Medical Center, Pennsylvania State Univ., PO Box 850, Hershey, PA 17033. Received
26 October
1989; accepted
in final
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19 December
1990.
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REFERENCES 1. CATERSON, I. D., S. J. FULLER, AND P. J. RANDLE. Effect of fatty acid inhibitor 2-tetradecylglycidic acid on pyruvate dehydrogenase complex activity in starved and alloxan-diabetic rats. Biochem. J. 208: 53-60, 1982. 2. CATTERSON, I. D., A. L. KERBEY, G. J. COONEY, R. FRANKLAND, G. S. DENYER, J. NICKS, AND P. F. WILLIAMS. Inactivation of pyruvate dehydrogenase complex in heart muscle mitochondria of gold-thioglucose-induced obese mice is not due to a stable increase in activity of pyruvate dehydrogenase kinase. Biochem. J. 253: 291294, 1988. B. R. MARTIN, AND P. J. RANDLE. 3. COORE, H. G., R. M. DENTON, Regulation of adipose tissue pyruvate dehydrogenase by insulin and other hormones. Biochem. J. 125: 115-127, 1971. 4. DENYER, G. S., A. L. KERBEY, AND P. J. RANDLE. Kinase activator protein mediates longer-term effects of starvation on activity of pyruvate dehydrogenase kinase in rat liver mitochondria. Biochem. J. 239: 347-354, 1986. H. R., T. C. VARY, AND P. J. RANDLE. Modulation of 5. FANTANIA, pyruvate dehydrogenase kinase activity in cultured hepatocytes by glucagon and n-octanoate. Biochem. J. 234: 233-236, 1986. 6. FULLER, S. J., AND P. J. RANDLE. Reversible phosphorylation of pyruvate dehydrogenase in skeletal muscle mitochondria. Biochem. J. 219: 635-646, 1984. GORNALL, A. G., C. J. BARDAWILL, AND M. M. DAVID. Determination of serum proteins by means of biuret reaction. J. Biol. Chem. 177: 751-766, 1949. HUTSON, N. J., AND P. J. RANDLE. Enhanced activity of pyruvate dehydrogenase kinase in rat heart mitochondria in alloxan diabetes or starvation. FEBS Lett. 92: 73-76, 1978. KERBEY, A. L., P. M. RADCLIFFE, AND P. J. RANDLE. Diabetes and control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of ATP/ADP, reduced/oxidized nictotinamide adenine dinucletoide and of acetyl-coenzyme A/coenzyme A. Biochem. J. 164: 509-519, 1977. 10. KERBEY, A. L., AND P. J. RANDLE. Pyruvate dehydrogenase kinase/ activator in rat heart mitochondria. Biochem. J. 206: 103-111, 1982. 11. KERBEY, A. L., P. J. RANDLE, R. H. COOPER, S. WHITEHOUSE, H. T. PASK, AND R. M. DENTON. Regulation of pyruvate dehydrogenase in rat heart. Biochem. J. 154: 327-348, 1976. 12. LANG, C. H., G. J, BAGBY, AND J. J. SPITZER. Carbohydrate dynamics in hypermetabolic septic rats. Metab. Clin. Exp. 33: 959963, 1984. 13. LINN, T. C., F. H. PETTIT, F. HUCHO, AND L. J. REED. Comparative studies of regulatory properties of pyruvate dehydrogenase com-
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plexes from kidney, heart and liver mitochondria. Proc. Natl. Acad. Sci. USA 64: 227-234, 1969. LINN, T. C., F. H. PETTIT, AND L. J. REED. cu-Ketoacid dehydrogenase complexes. X. Regulation of activity of pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation. Proc. Natl. Acad. Sci. USA 62: 234-241, 1969. NANNI, G., J. H. SIEGEL, B. COLEMAN, P. FADER, AND R. CASTIGLIONE. Increased lipid fuel dependence in the critically ill septic patient. J. Trauma 24: 14-30, 1984. RANDLE, P. J. Fuel selection in animals. Biochem. Sot. Trans. 14: 799-806, 1986. SEVERSON, D. L., R. M. DENTON, H. T. PASK, AND P. J. RANDLE. Calcium and magnesium ions as effecters of adipose tissue pyruvate dehydrogenase phosphate phosphatase. Biochem. J. 140: 225-237, 1974. SRERE, P. A. Citrate synthase. Methods Enzymol. 13: 3-11, 1969. SHAW, J. H. F., S. KLEIN, AND R. R. WOLFE. Assessment of alanine, urea, and glucose interrelationships in normal subjects and in patients with sepsis with stable isotope tracers. Surgery St. Louis 97: 557-567, 1985. SIEGEL, J. H., AND T. C. VARY. Sepsis abnormal metabolic control and the multiple organ failure syndrome. In: Trauma: Emergency Surgery and CriticaL Care, edited by J. H. Siegel. London: Livingstone, 1987, p. 411-502. STEPP, L. R., F. H. PETTIT, S. J. YEAMAN, AND L. J. REED. Purification and properties of pyruvate dehydrogenase kinase from bovine kidney. J. Biol. Chem. 253: 9454-9458, 1983. SUGDEN, M. C., AND M. J. HOLNESS. Effects of refeeding after prolonged starvation on pyruvate dehydrogenase activities in heart, diaphragm and selected skeletal muscles of the rat. Biochem. J. 262: 669-672, 1989. SUGDEN, P. H., N. J. HUTSON, A. L. KERBEY, AND P. J. RANDLE. Phosphorylation of additional sites on pyruvate dehydrogenase inhibits its reactivation by pyruvate dehydrogenase phosphate phosphatase. Biochem. J. 69: 433-435, 1978. VARY, T. C., AND J. M. MURPHY. Role of extra-splanchnic organs in the metabolic response to sepsis: effect of insulin. Circ. Shock 29: 41-57, 1989. VARY, T. C., J. H. SIEGEL, T. NAKATANI, T. SATO, AND H. AOYAMA. Effect of sepsis on activity of pyruvate dehydrogenase complex in skeletal muscle and liver. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E634-E640, 1986. VARY, T. C., J. H. SIEGEL, B. D. TALL, AND J. G. MORRIS. Metabolic effects of partial reversal of pyruvate dehydrogenase activity by dichloroacetate in sepsis. Circ. Shock 24: 3-18, 1988. VARY, T. C., J. H. SIEGEL, A. ZECHNICH, B. D. TALL, J. G. MORRIS, R. PLACKO, AND D. JAWOR. Pharmacological reversal of abnormal glucose regulation, BCAA utilization and muscle catabolism in sepsis by dichloroacetate. J. Trauma 28: 1301-1312, 1988. WALLENSTEIN, S., C. L. ZUCKER, AND J. F. FLEISS. Some statistical methods useful in circulation research. Circ. Shock 8: 503517,198l. WHITEHOUSE, S., R. H. COOPER, AND P. J. RANDLE. Mechanism of action of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem. J. 141: 761-774, 1974. WOLFE, R. R., AND J. F. BURKE. Glucose and lactate metabolism in experimental septic shock. Am. J. Physiol. 235 (ReguZatory Integrative Comp. Physiol. 4): R219-R227, 1978.
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kinase activity
THOMAS C. VARY Department of Cellular and Molecular Physiology, Milton S. Hershey Pennsylvania State University, Hershey, Pennsylvania 17033
VARY, THOMAS C. Increased pyruvate dehydrogenase kinase activity in response to sepsis. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E669-E674, 1991.-The effect of sterile inflammation and sepsis on the proportion of active pyruvate dehydrogenase complex (PDH) in mitochondria isolated from skeletal muscle has been investigated. The proportion of active PDH in mitochondria isolated from septic animals was significantly reduced compared with control under all incubation conditions examined, even in the presence of inhibitors of the PDH kinase. There was no significant difference between control and sterile inflammation in any of the incubations examined. The rate constant for ATP-dependent inactivation of the PDH complex in mitochondrial extracts from control animals was -0.42 min-l (r = 0.993; P < 0.001) and was not altered in mitochondrial extracts from sterile inflammatory animals (-0.43 min-l; r = 0.999; P < 0.001). However, rate constants for inactivation in septic animals was significantly increased over twofold to -1.08 min-’ (r = 0.987; P < 0.001) (P < 0.001 vs. control or sterile inflammation). In the presence of inhibitors of the PDH kinase reaction (2.5 mM pyruvate or 1 mM dichloroacetate), inactivation of PDH after addition of ATP was significantly greater in mitochondrial extracts from septic than either control or sterile inflammatory animals. These results suggest that sepsis, but not sterile inflammation, induces a stable factor in skeletal muscle mitochondria that increased PDH kinase activity. kinase activator tal muscle
protein;
dichloroacetate;
mitochondria;
skele-
CHRONIC SEPSIS produces physiological and metabolic pertubations resulting in a hyperdynamic cardiovascular response, net whole body protein catabolism, and increased glucose turnover (12, 19, 20, 27, 30). Hyperglycemia and hyperlactatemia are frequent manifestations of the metabolic response to sepsis(l&19,26,30). Whole body studies utilizing tracer methodologies have suggested that a diversion of glucose carbon away from oxidation leads to increased plasma lactate concentrations in sepsis (12, 19, 20, 27, 30). The activity of the pyruvate dehydrogenase (PDH) complex is a key mechanism in the control of carbohydrate oxidation and conservation of whole body glucose. The PDH complex is the first irreversible step in the oxidative pathway of pyruvate in mitochondria and is tightly regulated by both end product inhibition and reversible phosphorylation (13-14, 21). Phosphorylation (inactivation) is catalyzed by a PDH kinase, and dephosphorylation (reactivation) is catalyzed by a mito0193~1849/91
$1.50 Copyright
Medical
Center,
chondrial PDH phosphatase (13-14,21). Interconversion of phosphorylated and dephosphorylated forms of the PDH complex plays a major role in the regulation of glucose oxidation under a variety of conditions in animals and humans (for review see Ref. 16). The PDH kinase and phosphatase reactions operate simultaneously, and the proportion of the PDH complex in the active (dephosphorylated) form depends upon the relative rates of these two reactions. The PDH phosphatase reaction requires Mg”+ and, in the presence of Mg”+, is activated by increasing the Ca2+ concentration (17). The PDH kinase reaction is accelerated by increasing intramitochondrial concentration (square brackets) ratios of [ATP] to [ADP], [NADH] to [NAD], and [acetylCoA] to [CoA] (9, 11). Changes in these concentration ratios appear to be the principal mechanism responsible for changes in the proportion of active PDH complex induced by oxidation of fatty acids or ketone bodies or by changes in work in isolated tissues (for review see Ref. 16). There also appears to be another longer-term mechanism that increases PDH kinase activity. The mechanism of PDH kinase activation is independent of mitochondrial oxidation of fatty acids or ketone bodies and is not mediated by changes in the metabolite effector concentrations of the PDH kinase (4-6,8-11). Activation of the PDH kinase (up to 3-fold) during starvation and diabetes in mitochondrial extracts from heart and liver has been suggested to be due to a protein factor termed kinase activator protein (4-5, 8, 10). Previous studies from this laboratory have demonstrated that sepsis decreases the proportion of active PDH complex in skeletal muscle without any alteration in the total amount of PDH complex activity (25-27). The effect of sepsis to reduce PDH complex activity is proposed to be mediated by an increased PDH kinase activity secondary to an increased [acetyl-CoA] -to- [CoA] ratio (25). However, other mechanisms may be involved. After intraperitoneal injection of dichloroacetate, the proportion of active PDH complex in septic animals is significantly less than the full activation of the PDH complex in nonseptic animals (26). Similar observations regarding the inability of dichloroacetate to fully activate PDH complex in starvation and diabetes have been associated with an increase in PDH kinase activity (6, 11). Stimulation of the PDH kinase reaction by the kinase activator protein has been proposed to account for relative refractory effects to dichloroacetate in star-
0 1991 the American
Physiological
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vation and diabetes. Evidence is presented in the present study suggesting that the effects of sepsis to lower the proportion of active PDH complex are consistent with a stable increase in PDH kinase activity.
N,N,N’,N’-tetraacetic acid (EGTA), 5 KH2P04, pH 7.41 in Eppendorf microcentrifuge tubes with further additions as given in Tables 1 and 2. For estimation of total PDH complex and PDH kinase activity, mitochondria were incubated for 15 min in buffer containing 10 PM carbonyl cyanide m-chlorophenylhydrazone without respiratory substrates that would effect conversion of inacMATERIALS AND METHODS tive complex into active complex (5-6). Mitochondria Intra-abdominal abscess model. Chronic sepsis was inwere separated by centrifugation (40s Eppendorf 5214 duced in male Sprague-Dawley rats (Charles River centrifuge), the supernatants were aspirated, and the Breeding, Wilmington, MA) (250-300 g) by the intraperpellets were frozen in liquid N2. Mitochondrial extracts itoneal introduction of a fecal-agar pellet (1.5 ml) inoc- for assay of PDH complex activity and citrate synthase ulated with 10” colony-forming units (CFU) Escherichia activity were prepared from the frozen mitochondrial coli (Serotype 018 ac) plus 10” CFU Bacteroides fragilis pellets by ultrasonic disintegration as described by Ker(ATCC no. 23745) (24-27). The sterile inflammatory bey et al. (11). For assay of PDH kinase activity, mitoabscesswas generated by replacing the bacteria with an chondrial extracts of the frozen mitochondrial pellets equal volume of sterile physiological saline. Fed rats that were prepared by alternate thawing (3O”C), dispersing were not subjected to the surgical procedures served as (Hamilton syringe aspiration), and freezing (liquid Na) control animals. After recovery from implantation of the (two times) into buffer containing 30 mM KHaP04, 5 fecal-agar pellet, all animals were allowed free access to mM EGTA, 5 mM dithiothreitol, 1% (wt/vol) fatty acidrat chow (Purina) and water, and the intra-abdominal free bovine serum albumin, 1 mM tosyl-lysyl-chloroabscesswas allowed to form for 5-7 days. Previous stud- methyl ketone, 25 pg/ml oligomycin ,& pH 7.0 (5-6, 8). ies have demonstrated that septic rats consume as much To verify conversion of inactive to active PDH complex, rat chow as control or sterile inflammatory animals on mitochondrial extracts (40 ~1~1) were incubated in buffer day 2 to 7 postimplantation of fecal-agar pellets (27). containing 75 mM Tris, pH 7.0, with partially purified Changes in the proportion of active PDH complex in PDH phosphatase (0.5 U/ml), 0.2 mg fatty acid-free septic animals are not significantly different 5 or 7 days bovine serum albumin, 10 mM MgC12, and 4 mM CaC12 after the introduction of the fecal-agar pellet (25-26), for 15 min at 30°C (11). PDH activity was then reassayed All animals that survived the peritonitis stage (100% in spectrophotometrically. PDH phosphatase was partially sterile inflammatory, 65% of septic animals) formed an purified from beef heart as described by Coore et al. (3). intra-abdominal abscess(25-27). Assays. PDH complex activity was assayed spectroSkeletal muscle mitochondria isolation. Five to seven photometrically by coupling the formation of acetyl-CoA days after the implantation of the fecal-agar pellet, anifrom pyruvate to acylation of p-( p-aminophenylazo)mals were anesthetized (Nembutal, 50 mg/kg body wt benzenesulfphonic acid using arylamine acetyltransferip). All hindlimb muscles (-25 g) from fed animals in ase as described by Coore et al. (3). Citrate synthase was each group were excised, trimmed of fat and connective assayed by the method of Srere (18) as modified by Coore tissue, minced finely, and homogenized (Waring Blender, et al. (3). All assays were performed in duplicate. One 12.5 s) in 180 ml of isolation buffer [(in mM) 100 unit of PDH complex activity converts 1 pmol of subtris(hydroxymethyl)aminomethane (Tris) . HCl, 10 strate into product per minute at 30°C. EDTA, 210 mannitol, 70 sucrose, 0.1% (wt/vol) bovine PDH kinase activity was assayed in mitochondrial serum albumin, pH 7.41 at 0°C (6). The homogenate was diluted with another 100 ml of isolation buffer, and the extracts by the rate of ATP-dependent inactivation of homogenization procedure was repeated. The homoge- the PDH complex (5, 6, 8). Mitochondrial extracts connate was incubated with stirring on ice with 50 mg of taining -30-35 mU of PDH complex were warmed to porcine trypsin (Sigma type IX) for 12.5 min and was 30°C (2 min). ATP was added to a final concentration of 0.3 mM Mg”+-ATP, and samples were taken for assay of filtered through a plastic sieve. The filtrate was centriPDH complex activity between 0.25 and 6 min. The fuged (10 min, 500 g), and the supernatant was filtered. exact times depended upon the observed rate of ATPMitochondria were sedimented (8 min, 10,000 g) from the filtered supernatant and washed twice by resuspen- dependent inactivation determined in pilot experiments. Time 0 activity was determined on a separate sample of sion and centrifugation (8 min, 10,000 g) in isolation extract. Mitochondria were also incubated (6 min) in the buffer (as above except 10 mM Tris . HCl). After the final absence of ATP to ensure that there was no appreciable centrifugation, mitochondria were suspended in isolation loss of PDH activity over the course of the incubations. buffer (containing 10 mM Tris HCl) at a concentration of lo-20 mg mitochondrial protein/ml. The yield of Inactivation of the PDH complex by ATP was calculated without ATP). mitochondria was -1 mg of protein/g fresh wt of muscle. as 100 (activity PDH with ATP)/(activity Protein concentrations of mitochondrial suspensions The PDH kinase activity was calculated as the apparent were determined by the method of Gornall et al. (7) with pseudo-first-order rate constant for the ATP-dependent inactivation of the PDH complex as determined from a crystalline serum albumin as the standard. least-squares linear regression analysis of the natural log Incubation and extraction of mitochondria. Mitochondria (0.5-l mg of protein) were incubated at 30°C in of inactivation by ATP against time of incubation (10). 0.5 ml of incubation (KCl) medium [(in mM) 20 Tris . This gives a negative rate constant. In all cases,the plots HCl, 120 KCl, 2 ethylene glycol-bis(/3-aminoethyl ether)were linear with r > 0.95, and the time 0 intercept was Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (129.100.058.076) on September 23, 2018. Copyright © 1991 American Physiological Society. All rights reserved.
PDH
KINASE
close to 100% of PDH complex activity in the absence of ATP. The experimental data for each condition are summarized as means t SE for 4-14 mitochondrial preparations in each group of animals. Statistical evaluation of the data was performed using the Student’s t test for unpaired comparisons. For PDH kinase activity, significance between differences among the different groups was determined by analysis of covariance of the slopes for each group by Scheffe method. Differences among means were considered significant when P < 0.05 (28). RESULTS
Effect of respiratory substrates. In the work described below the total PDH complex activity was assayed in mitochondria incubated for 15 min without respiratory substrates to convert inactive to fully active PDH complex. To validate this approach in mitochondria isolated from hindlimb of sterile inflammatory or septic rats, extracts prepared from mitochondria previously incubated for 15 min without respiratory substrate were incubated with PDH phosphatase. In control rats, total PDH complex activity was 74 t 7 mU/mg mitochondrial protein (n = 6), which was not significantly different from the values obtained after incubation of mitochondria without respiratory substrates (Table 1). Total concentration of PDH complex in skeletal muscle mitochondrial extracts from sterile inflammatory (78 t 3 mU/mg mitochondrial protein; n = 4) or septic (78 t 5 mU/mg mitochondrial protein; n = 5) animals, as estimated after incubation with PDH phosphatase, was not significantly different from control. Total PDH complex activity in mitochondria from sterile inflammatory or septic rats incubated without respiratory substrates was 92 and 96%, respectively, of the value obtained in mitochondrial extracts incubated with PDH phosphatase (Table 1). To assess whether the yield of mitochondria from sterile inflammatory or septic animals might influence the concentration of total PDH complex, citrate synthase
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activity was estimated. There were no significant decreases in the concentration of citrate synthase activity (mU/mg mitochondrial protein) in sterile inflammation (934 t 85; n = 7) or sepsis (750 t 107; n = 5) compared with control (1,083 t 122; n = 10). These results confirm our previous observations using whole tissue extracts that showed the changes in proportion of active PDH complex in septic animals were not due to a decreased concentration of total enzyme activities (2526). The concentration of active PDH complex in mitochondria freshly prepared (not incubated) from fed control animals was 42 t 6%. This value is about twofold higher than estimates of the proportion of active PDH complex (20%) in skeletal muscle in vivo (25-26) and most likely is due to partial activation of PDH complex during the isolation procedure. Similar observations have been described in heart mitochondria (11). Incubation with respiratory substrates (a-ketoglutarate and malate) decreased the proportion of active PDH complex to 28 t 2%. Inclusion of either pyruvate or dichloroacetate, both inhibitors of PDH kinase, in the incubation buffer increased the proportion of active PDH complex -2-fold and M-fold, respectively, in mitochondria from control rats. Similar results were obtained with mitochondria from skeletal muscle of sterile inflammatory rats isolated and incubated under similar conditions (Table 1). These changes in the proportion of active PDH complex in mitochondria from sterile inflammatory animals under the various incubation conditions were not significantly different compared with control animals. In contrast to sterile inflammation, the concentration of active PDH complex activity in mitochondria freshly prepared (not incubated) from septic animals was significantly (P < 0.005) reduced to 45% of values obtained from control animals. This relative change is approximately equal to the difference in the proportion of active PDH complex estimated in situ between control (20%) and septic (10%) rats (26). Incubations with respiratory substrates diminished the proportion of active PDH in mitochondria isolated from septic animals, and the con-
1. Effect of respiratory substrates on concentration of active pyruvate dehydrogenase complex in skeletal muscle from control, sterile inflammatory, and septic rats
TABLE
Active Control mU/mg
No substrate Not incubated (as made) 5 mM a-Ketoglutarate and 0.5 mM malate 5 mM cu-Ketoglutarate, 0.5 mM malate, and 3 mM pyruvate 5 mM cu-Ketoglutarate, 0.5 mM malate, and 50 PM dichloroacetate
protein
PDH Sterile
n
%Total complex
mU/mg
protein
Complex
Activity
inflammation
Septic %Total complex
n
72+4 31+5 20t2
12 8 18
98k6 4226 28rt2
70t4 28-+4 17t4
14 9 6
35k5
8
49t7
23klO
54t5
8
75t7
38+5 -
mU/mg
protein
abscess n
%Total complex
9225 41t6 251k6
74t7 14t3* 9+4$
11 9 4
96k9 18t5t 11t5”
6
43t5
17+4$
5
21+4-f -
6
60t9
28e3$
4
44+12$
Results are means & SE; n, no. of mitochondrial preparations. Mitochondria isolated from skeletal muscle were incubated in KC1 medium for 15 min without respiratory substrates (no substrate) or for 5 min in KC1 medium with additions as shown. Mitochondria were separated by centrifugation and frozen in liquid N,. Extracts were prepared using ultrasonic dispersion and assayed for active pyruvate dehydrogenase (PDH) complex activity. Total PDH complex (sum of active and inactive forms) was assayed after conversion of inactive into active complex after addition of partially purified PDH phosphatase to mitochondrial extracts previously incubated without respiratory substrates. Otherwise, assume the value for %total complex was based on values for mU PDH/mg mitochondrial protein derived from no substrate incubations. All assays were performed in duplicate. * P < 0.01, “r P < 0.005, $ P < 0.05 vs. control.
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centration of active PDH was significantly decreased 60% relative to control animals. Hence, the decreased proportion of active PDH complex activity in skeletal muscle from septic animals observed in situ persists during isolation and incubation of mitochondria prepared from these animals. Furthermore, the ability of pyruvate or dichloroacetate to effect conversion of PDH into its active form was significantly decreased by 55 and 37%) respectively, in mitochondria from skeletal muscle of septic animals compared with control. PDH kinase actiuity. Previous studies in heart mitochondria from diabetic rats have suggested that the decreased responsiveness to the effects of pyruvate and dichloroacetate on the proportion of active PDH complex may be due to increased PDH kinase activity (8, 10). As shown in Fig. IA, the proportion of active PDH complex remained essentially as the initial (time 0) PDH activity after 6 min of incubation when ATP was omitted. Addition of ATP resulted in the inactivation of PDH complex such that inactivation of PDH complex was essentially complete (>90%) within 6 min of the addition of ATP. The apparent first-order rate constant for inactivation of the PDH complex by ATP in mitochondrial extracts from fed control animals was -0.42 min-’ (Table 2), which is comparable to values reported by Fuller and Randle (6). The rate of inactivation in mitochondrial extracts from skeletal muscle of sterile inflammatory animals was not significantly different from control (Fig. lA, Table 2). However, as can be seen in Fig. IA, the rate of inactivation of PDH complex was markedly faster after addition of ATP in extracts of skeletal muscle mitochondria from septic animals. This resulted in an over 2.5-fold increase (P < 0.001) in PDH kinase activity in mitochondrial extracts from septic animals compared with either control or sterile inflammation (Table 2).
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The PDH kinase reaction is inhibited by pyruvate, and pyruvate increases the proportion of active PDH complex in mitochondria from heart (11) and skeletal muscle (6). Inclusion of 2.5 mM pyruvate in the incubation medium inhibited the PDH kinase reaction in extracts of mitochondria from control animals (compare Fig. 1, A and B). No differences in the extent of inhibition of PDH kinase by pyruvate were observed between control and sterile inflammation. However, the extent of inactivation of PDH complex was significantly greater in extracts from mitochondria of septic animals compared with control or sterile inflammation. Therefore the enhanced activity of the PDH kinase reaction (relative to controls) in extracts of mitochondria from septic rats was retained even when 2.5 mM pyruvate was present. Dichloroacetate is an uncompetitive inhibitor of PDH kinase reaction; and it increases the proportion of active PDH in mitochondria isolated from heart and skeletal muscle of normal animals (7, 12, 30). Inclusion of 1 mM dichloroacetate in the incubation medium essentially completely inhibited the PDH kinase reaction in extracts of mitochondria from control animals (compare Fig. 1, A and C). The concentration of dichloroacetate giving a 50% inhibition of the rate constant was -25 PM (data not shown). This is in good agreement with the value of 16 PM reported by Fuller and Randle (6) in intact mitochondrial incubations. As was observed with pyruvate, no differences in the extent of inhibition of PDH kinase by dichloroacetate were observed between control and sterile inflammation. However, the extent of inactivation of PDH complex after incubation with 1 mM dichloroacetate was greater at each time point in extracts from mitochondria of septic animals compared with control or sterile inflammation. Hence, enhanced activity of the PDH kinase reaction (relative to controls) in extracts of C
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FIG. 1. Effects of sterile inflammation or sepsis on activity of pyruvate dehydrogenase (PDH) kinase in extracts of mitochondria from skeletal muscle. Extracts were prepared from mitochondria obtained from control (o), sterile inflammatory (A), and septic rats (6) in which inactive PDH complex had been converted into active complex by incubation in KC1 medium without respiratory substrates for 15 min at 30°C. PDH kinase reaction was initiated by addition of ATP to final concentration a 0.3 mM. In some experiments, ATP was omitted from incubation mixture (+). Details of incubation are as above except that sodium pyruvate (2.5 mM, B) or potassium dichloroacetate (1 mM, C) were present throughout 2-min preincubation and incubation with ATP. Samples were taken for assay of PDH complex at times shown. Each point is mean of 6 or more observations from 3-8 mitochondrial preparations for each group. Initial concentrations of PDH complex (mu/ml) in mitochondrial extracts were 176 t 15 (SE) (control, n = 5), 187 t 19 (sterile inflammation, n = 4), and 182 t 16 (septic abscess, n = 3). B: *P < 0.05 vs. control; C: *P < 0.005 vs. control; **P < 0.001 vs. control.
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TABLE 2. Apparent first-order rate constant for ATP-dependent inactivation of PDH complex in mitochondrial extracts
skeletal muscle. This difference between fed control and sepsis was most obvious when mitochondria were incubated in the presence of inhibitors of PDH kinase, pyruvate, and dichloroacetate. The effects of sepsis on the PDH Kinase Activity, proportion of active PDH complex in the presence of Condition n min-’ inhibitors of PDH kinase were similar to those observed Cont,rol 5 -0.42 in mitochondria isolated from heart (10) or skeletal Sterile inflammation 5 -0.43 muscle (6) of starved or diabetic rats. This suggests that Sepsis 3 -1.08* similar mechanisms may be operating in sepsis, starvaData were obtained by calculating slope of plot of natural log of tion, and diabetes to lower the proportion of active PDH percent initial PDH complex activity vs. time of incubation using data complex. However, unlike starvation or diabetes, the presented in Fig. IA by a least-squares linear regression analysis. plasma insulin concentrations in septic animals were not Correlation coefficients were 0.993 (P < O.OOl), 0.999 (P < O.OOl), and 0.987 (P < 0.001) in mitochondrial extracts prepared from control, decreased (24). sterile inflammatory, and septic animals, respectively. Significance The mechanism responsible for decreased PDH activbetween differences in PDH kinase activity was determined by analysis ity in sepsis may be due to either an increased PDH of covariance of slopes for each group by Scheffi! method (F,l,l:i = 681.7, kinase activity, a decreased PDH phosphatase activity, P < 0.001). * P < 0.001 vs. control or sterile inflammation. or both. Because the effects of starvation and diabetes mitochondria from septic rats was retained at a concen- appear kinase mediated, the effect of sepsis on PDH tration of dichloroacetate that was approximately 40- kinase activity was determined. The activity of the PDH fold higher than the inhibitory constant for control ani- kinase can be followed in extracts of mitochondria by mals. following the inactivation of PDH complex by ATP or by incorporation of [32P]ATP into PDH complex (8). Under the incubation conditions employed, the concenDISCUSSION trations of the metabolite activators of PDH kinase are The molecular basis for the inactivation of the PDH too low to have a stimulatory effect, and the phosphatase complex through increased activity of PDH kinase from is inoperative since EGTA is present (8). Using this various rat tissues appears to result from two mecha- approach, the PDH kinase reaction is M-fold more nisms. Oxidation of fatty acids and/or ketone bodies may active in mitochondria prepared from septic animals increase PDH kinase activity by increasing the mitocompared with either control or sterile inflammation. chondrial concentration ratios of [acetyl-CoA] to [CoA] Furthermore the effects of sepsis to accelerate PDH and [NADH] to [NAD] (9). This shorter-term mecha- kinase activity were not abolished by inhibiting the PDH nism can be rapidly reversed in heart muscle or skeletal kinase using either pyruvate or dichloroacetate. muscle in vitro by withdrawal of fatty acids or in vivo in The mechanism for enhanced PDH kinase activity in heart and kidney by administration of inhibitors of fatty mitochondria isolated from skeletal muscle of septic anacid oxidation such as Z-tetradecylglycidate (1). In ad- imals is currently unknown. The findings of the present dition to the acute effects of fatty acids, there appears to study suggest the mechanism must involve a rather stable be a longer-term mechanism involving activation of the change, since increased PDH kinase activity persists PDH kinase. For example, complete reactivation of PDH through isolation, incubation, and extraction of mitocomplex activity in cardiac or skeletal muscle is not chondria. Several potential mechanisms can be proposed observed for 4-24 h after refeeding (10). The increased including 1) more favorable binding of PDH kinase to PDH kinase activity is stable, since it persists during PDH complex, 2) increased reduction and acetylation of isolation, incubation, and extraction of mitochondria. It lipoyl residues of dihydrolipoamide acetyltransferase (8)) has been suggestedelsewhere that a factor, termed kinase 3) increased concentration of PDH kinase, and 4) anactivator protein, increases the PDH kinase activity in other protein factor that activates the PDH kinase. Acetstarvation and diabetes (4-5, LO). ylation or reduction of the lipoyl moieties of the dihydroSepsis is also associated with a decrease in the proporlipoamide acetyltransferase of the PDH complex seems tion of active PDH complex in skeletal muscle (25-27). unlikely, since addition of CoA and NAD to the PDH The decreased respiratory quotient observed in septic kinase assays did not reverse the increased PDH kinase patients suggests an increased dependence on fatty acids activity in extracts of heart mitochondria from diabetic as a respiratory fuel (15). Furthermore, previous studies animals (8). Studies are currently underway to determine have shown a threefold increase in the [acetyl-CoA]-tothe nature of the enhanced PDH kinase and the failure [CoA] concentration ratio in skeletal muscle from septic of pyruvate and dichloroacetate to inhibit the PDH kianimals compared with control (25). Hence, the effects nase. of sepsisto lower the proportion of active PDH complex In summary, increased PDH kinase activity may be of could simply be a consequence of increased ,&oxidation fundamental importance in the long-term regulation of of fatty acids as is observed in obese mice (2). In obese glucose oxidation and in the conservation of glucose in animals, inactivation of PDH complex in heart is not sepsis as well as other conditions (cf. Ref. 22). In fact, mediated by a stable activation of the PDH kinase. studies have demonstrated that activation of PDH comHowever, evidence presented in this study suggests that plex by dichloroacetate was associated with a normaliincreased PDH kinase activity occurs in sepsis. zation of the glucose metabolic clearance rate and lactate The effect of sepsisto decrease the proportion of active concentrations in sepsis, demonstrating the importance PDH complex persisted in mitochondria prepared from of PDH complex in whole body glucose homeostasis (26).
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The author acknowledges the assistance of Drs. J. G. Morris and B. D. Tall in preparing the bacterial inoculations for the septic animal model and the technical assistance of D. Jawor, J. Smith, and J. Arnold in conducting these experiments. This work was supported in part by National Institute of General Medical Sciences Grants GM-36139 and GM-39277. T. C. Vary is the recipient of Research Career Development Award K04 GM-00570. Address for reprint requests: T. C. Vary, Dept. of Physiology, Milton S. Hershey Medical Center, Pennsylvania State Univ., PO Box 850, Hershey, PA 17033. Received
26 October
1989; accepted
in final
form
19 December
1990.
IN
14.
15.
16. 17.
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