Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex

Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex

Molecular and Cellular Endocrinology 144 (1998) 139 – 147 Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruva...

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Molecular and Cellular Endocrinology 144 (1998) 139 – 147

Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex Mohamed Amessou a, Franc¸oise Fouque a, Neirouz Soussi a, Bernard Desbuquois a, Isabelle Hainaut b, Jean Girard c, Chantal Benelli a,* a

INSERM U 30, Hoˆpital des Enfants Malades, Tour La6oisier 6 e`me e´tage, 149, rue de Se`6res 75743, Paris, France b INSERM U.465, Institut Biome´dical des Cordeliers, Paris, France c Centre de Recherches sur l’Endocrinologie Mole´culaire et le De´6eloppement (CNRS), Meudon-Belle6ue, France Received 6 May 1998; received in revised form 26 June 1998; accepted 26 June 1998

Abstract The tissue-specific expression of the mitochondrial pyruvate dehydrogenase complex (PDHc) has been studied in an animal model of obesity with hyperinsulinemia, the obese (fa/fa) Zucker rat. Liver and heart were obtained from 4 and 8 week-old obese rats and age-matched lean animals, and in each tissue the following parameters were analyzed: (1) total activity of the mitochondrial PDHc; (2) abundance of the mitochondrial PDHc subunits on Western blots; and (3) abundance of the E1a and E1b subunit mRNAs on Northern blots and semi-quantitative RT-PCR. Regardless of age, obese rats showed an increase in liver total PDHc activity and a coordinate increase in liver E1a and E1b PDHc subunit abundance. At 4 weeks, obese rats also showed an increase in liver PDH E1a mRNA level, but regardless of age E1b mRNA level was unchanged. In contrast, neither total PDHc activity nor the concentration of its protein subunits were increased in heart of obese rats. Thus, obese Zucker rats display a liver-specific early increase in PDHc which results from a selective up-regulation of the E1a gene expression. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Zucker rat; Pyruvate dehydrogenase complex (PDHc); Ela gene; Regulation

1. Introduction The pyruvate dehydrogenase complex (PDHc) is a mitochondrial multienzymatic complex which plays a key role in intermediary metabolism. It consists of three catalytic components: pyruvate dehydrogenase (subunits E1a and E1b) (E.C 1.2.4.1), dihydrolipoamide acetyltransferase (subunit E2) (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (subunit E3) (EC 1.8.1.4). Other components of the complex include a tightly-associated protein of unknown function, protein X, and two enzymes which regulate the phosphorylation – dephosphorylation of serine residues on the E1a * Corresponding author. Tel.: +33 144 494332; fax: + 33 142 733081; e-mail: [email protected]

subunit, pyruvate dehydrogenase kinase (PDK) and pyruvate deshydrogenase phosphatase (Patel and Roche, 1990; Behal et al., 1993). E1, the first catalytic component of the PDHc, catalyzes the irreversible decarboxylation of pyruvate, which is the rate-limiting step in the overall reaction of the complex. In vivo, the proportion of PDHc in the active (dephosphorylated) state varies among individual tissues, being higher in heart, kidney and brain than in liver and adipose tissue (Behal et al., 1993). In addition, it is under short- and long-term regulation by nutritional, metabolic, developmental and hormonal factors (Wieland, 1983; Kilgour and Vernon, 1987; Macaulay and Larkins, 1988; Feldhoff et al., 1993; Patel et al., 1996). For instance, starvation and insulinopenic diabetes result in a decrease in PDHc activity which is

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reversed by refeeding and insulin treatment, respectively (Wieland et al., 1971). Both PDK and PDH phosphatase activities determine the activation (phosphorylation) state of the PDHc, and in many circumstances changes in the activation state of the PDHc directly correlate with the activity of PDK (Sugden and Holness, 1994). In the short term, PDK is reversibly regulated by the intramitochondrial ratios of metabolite and cofactor pairs acetyl-CoA/CoA, NADH/NAD + and ATP/ ADP, and the phosphatase is activated by insulin and Ca2 + (Patel and Roche, 1990; Behal et al., 1993). In the long term, a stable enhancement of PDK activity, attributed to increased fatty acid oxidation, increased tissue cAMP level and/or decreased plasma insulin, occurs in conditions associated with a decreased PDHc activity such as starvation, low carbohydrate/ high fat diet and insulinopenic diabetes (Sugden and Holness, 1996). Furthemore, of the four PDK isoenzymes differentially expressed in rat tissues (BowkerKinley et al., 1998), at least two, PDK 2 in liver (Sugden et al., 1998) and PDK 4 in heart (Wu et al., 1998), have been shown to be overexpressed in the latter conditions, indicating a pretranslational level of regulation. Although generally unaffected by most physiopathological states, total PDH activity has been reported to be altered in some conditions including very long-term high fat (Stansbie et al., 1976) and high carbohydrate (Da Silva et al., 1993) feeding, development (Malloch et al., 1986) and suckling – weaning transition (Maury et al., 1995). Interestingly, the increase in PDH activity which occurs in white adipose tissue upon weaning is accompanied by a parallel increase in the amount of E1a, E1b and E2 subunit protein and in their respective mRNAs, indicating a pretranslational level of regulation (Maury et al., 1995). This could result from the increase in plasma glucose and/or plasma insulin triggered by dietary carbohydrates. That glucose is a potential regulator of the PDHc genes is suggested by its ability to increase the level of E1a mRNA in cultured pancreatic islets of adult rats (MacDonald et al., 1991). The present study was conducted to test the hypothesis that PDH activity is regulated at a translational and/or pretranslational site in genetically obese Zucker rats, a rodent model of obesity with hyperinsulinemia (Jeanrenaud, 1988; Shafrir, 1992). To address this question, liver and heart, two oxidative tissues, were obtained from 4 to 8 week-old genetically obese Zucker rats and their lean littermates, and in each tissue the activity of PDHc and the abundance of the PDHc subunits and their corresponding mRNAs were analyzed in detail.

2. Materials and methods

2.1. Chemicals Carbonyl cyanide m-chlorophenylhydrazone (CCCP); benzamidine; Triton X-100; phenylmethylsulfonyl fluoride (PMSF); dithiothreitol; sodium fluoride; Tween-20; bovine serum albumin (BSA); cocarboxylase; NAD, and benzethonium hydroxide were purchased from Sigma (St Louis, MO). All reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad (Richmond, CA). Sodium dichloroacetate and N-tosyl-Llysylchloromethane hydrochloride (TLCK) were from Merck (Darmstadt, Germany). Nitrocellulose membranes were from Schleicher and Schuell (Dassel, Germany). Chemicals for enzyme assays were from Boehringer Mannhein (Meylan, France). [1-14C]-pyruvic acid (9.8 mCi/mmol) was from New England Nuclear (Boston, MA). The ECL detection system was from Amersham (Little Chalfont, UK). [a-32P]dATP (specific activity, 3000 Ci/mmol) and autoradiography films were from Amersham (Bucks, UK). The antiserum against purified pig PDH was raised as described by Lindsay (De Marcucci and Lindsay, 1985). This antiserum recognizes all subunits of the PDH complex except the E3 subunit which is poorly immunoreactive (De Marcucci and Lindsay, 1985).

2.2. Animals Animal studies were conducted according to the French guidlines for the care and use of experimental animals. Four to 8 week-old obese Zucker rats (fa/fa) and their lean littermates (Fa/?) were bred at INSERM U465, Paris. Preobese and obese rats were distinguished from lean animals by the ratio of inguinal adipose tissue weight to body weight up to the age of 1 month (Lavau and Bazin, 1982) and by body weight and gross morphological appearance after 5 weeks. The animals were maintained under a constant light–dark cycle (light from 07:00–19:00 h) and given free access to food and water. Obese and lean age-matched animals were starved 16 h before sacrifice by decapitation. Blood samples were taken from all the animals. Plasma was separated by rapid centrifugation in a microfuge and frozen for subsequent assay of glucose, insulin and corticosterone. Heart and liver were collected between 10:00 and 12:00 h and immediatly homogenized for preparation of mitochondria for the assay of PDHc and citrate synthase (CS) activities. Some characteristics of the animals are summarized in Table 1. Relative to age-matched lean rats, 4 and 8 week-old obese rats showed a significant increase in body and liver weights (PB 0.01) and in plasma insulin (PB 0.01), and 4 week-old obese rats also showed a

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Table 1 Body, liver and heart weights, plasma glucose, plasma insulin, and plasma corticosterone in genetically obese (fa/fa) Zucker rats and their lean littermates (Fa/?) Age (weeks) 4

Body weight (g) Liver weight (g) Heart weight (g) Plasma glucose (mmol) Plasma insulin (ng/ml) Plasma corticosterone (ng/ml)

8

Fa/?

fa/fa

Fa/?

fa/fa

64.1 9 2.4 3.49 9 0.08 0.27 9 0.01 6.70 9 0.59 0.25 9 0.04 86 9 28

78.4 9 1.4*** 4.39 90.08*** 0.33 90.01*** 6.39 90.21 0.3890.04* 106 9 22*

205 9 7 9.28 9 0.38 0.74 9 0.03 8.28 9 0.18 0.43 9 0.05 282 9 169

227 94** 11.28 90.28** 0.71 90.02 8.69 90.40 1.02 9007** 464 9 140

Results are expressed as the mean 9 SEM of 5–10 determinations in separate rats. Asterisks indicate a statistical difference between obsese and lean rats: *PB0.05; **PB0.01; ***PB0.001.

significant increase in heart weight (P B 0.001) and plasma corticosterone (P B 0.05). Regardless of age, no change in plasma glucose was observed.

2.3. Mitochondria preparations Exised tissues were immediately homogenized in icecold 5 mM Tris–HCl buffer, pH 7.4, containing 250 mM sucrose and 2 mM EGTA as described by Denyer et al. (1989). After centrifugation of the homogenate at 800× g for 10 min, the supernatant was decanted and centrifuged at 10000× g for 10 min to pellet the mitochondrial fraction. Prior to measurement of total PDH activity, depletion of intramitochondrial ATP was achieved using an uncoupler of oxidative phosphorylation CCCP. The buffer used for activation contained 20 mM Tris–HCl, pH 7.4, 120 mM KCl, 5 mM potassium phosphate, 2 mM EGTA and 10 mM CCCP. Activation was allowed to proceed for 30 min at 30°C, as previously described (Denyer et al., 1989). In order to maintain PDH in its active, dephosphorylated form, the mitochondrial pellet was stored in the presence of 10 mM DCA, which inhibits PDH kinase. Mitochondria for Western blotting experiments were prepared by differential centrifugation as described above. They were extracted in a medium containing 50 mM potassium phosphate buffer, pH 7.0, 10 mM EGTA, 2 mM dithiothreitol, 1 mM benzamidine, 1 mM PMSF, 0.3 mM TLCK, 2.5% Triton X-100 by repeated (three times) thawing (30°C) and freezing in liquid nitrogen. Mitochondria were stored at − 80°C until used.

2.4. Enzyme assays and protein determination Mitochondria prepared from heart and liver were extracted for enzyme assay by repeated thawing and freezing in extraction buffer (50 mM potassium phosphate, pH 7.0, 10 mM EGTA, 2 mM dithiothreitol) as previously reported (Clot et al., 1988).

PDH activity was assayed as the release of [14C] CO2 from [1-14C] pyruvic acid (Clot et al., 1988). Incubations were performed in a final volume of 125 ml. Total activity (PDHt) was estimated by preincubating samples with 0.5 mM Ca2 + and 10 mM Mg2 + for 10 min at 37°C to activate endogenous PDH phosphatase. One unit of enzyme activity was defined as the amount that converts 1 mmol of pyruvate into acetyl-CoA/min at 30°C. Citrate synthase (CS) activity was also measured (Denyer et al., 1989) and PDHc activity was expressed per unit of CS activity to correct for any difference in the recovery or purity of the mitochondrial extracts. Protein content was determined by the Bradford method (Bradford, 1976) using bovine serum albumin as a standard.

2.5. SDS-PAGE and Western blot analysis of PDHc subunits Heart and liver mitochondria (0.5–1 mg protein) were subjected to a 10% SDS-PAGE under reducing conditions according to Laemmli (1970). Proteins were electrotransferred onto nitrocellulose membranes at 100 V for 2 h in 25 mM Tris, 192 mM glycine buffer, pH 8.3. Apparent Mr values of transferred proteins were calculated with reference to the mobility of the following proteins: phosphorylase (Mr 97400), bovine serum albumin (Mr 69000), ovalbumin (Mr 46000), and carbonic anhydrase (Mr 30000). Membranes were incubated for 1 h in PBS containing 2% non fat dry milk, 0.02% thimerosal, washed in PBS, 0.1% Tween-20 and incubated overnight at 4°C with rabbit polyclonal antiPDHc diluted (1: 3000) in PBS, 1% BSA, 0.05% Tween20 and 0.02% thimerosal. After extensive washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase conjugated goat anti-rabbit IgG (1:3000). Immunocomplexes were detected by the Amersham ECL detection reagents and quantified by densitometry with a phosphorImager system

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(IMSTAR, France). Results were expressed per unit of CS activity to correct for any difference in the quantity of different mitochondrial extracts subjected to electrophoresis. Antibodies against the E1a, E1b and E2 subunits of the PDHc did not crossreact with the E1 and E2 subunits of the two other a-oxoacid dehydrogenase complexes (Wexler et al., 1988; Fischer et al., 1989; Nakano et al., 1991).

2.6. Northern blot analysis of the mRNAs coding for PDHc E1a and b subunits Total liver RNA was isolated by the single step guanidium isothiocyanate method (Chomczynski and Sacchi, 1987). RNA samples (40 mg) were size-fractionated by agarose gel electrophresis and transferred onto nylon Hybond membranes. Transferred RNA was hybridized with cDNA probes coding for the human PDH E1a and E1b subunits (a kind gift from Dr B.H. Robinson, the Hospital of Sick Children, Toronto, Canada), labelled with [a-32P]dATP by the randompriming method. Hybridization was carried out according to Coupe´ et al. (1990) except for a 24 h prehybridization. Blots were subjected to autoradiography and hybridization signals were quantified by scanning densitometry. To correct for variations in the amount of RNA analysed, results were normalized to hybridization signals obtained using 32P-labeled probes specific for rRNA 18S, b-actin and poly A binding protein. A RNA ladder (Boehringer-Mannheim) was used as the size marker. There was no cross-reactivity of the PDHc E1a and E1b cDNA probes with the E1a and b subunits mRNAs of the two other a-oxoacid dehydrogenase complexes (Wexler et al., 1988; Fischer et al., 1989; Nakano et al., 1991).

and an antisense–primer ‘CA’ (position 230–248, sequence: 5%-cgtgtgaagtcaccaccct-3%. Two microliters of the cDNA reaction were subjected to PCR amplification in a final volume of 50 ml, containing 1X TAQ buffer (10 mM Tris–HCl pH 8.8, 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100), 1 mM PDS1, 1 mM PDA1, 0.5 mM CS, 0.5 mM CA, 0.1 mM each dATP, dGTP, dTTP, 0.06 mCi of [a32 P]dCTP and 0.2 U/ml HI-TAQ DNA polymerase (Bioprobe Systems). The PCR profile consisted of 5 min at 95°C followed by 26 cycles of 60 s at 94°C, 60 s at 57°C and 80 s at 72°C (DNA Thermal Cycler, Perkin Elmer/Cetus, France). Ten microliters of the PCR reactions were analysed by separation on a 8% polyacrylamide gel and visualised by autoradiography. The PCR products were quantified by direct measurement of radioactivity on the dried gel using a PhosphorImager (molecular dynamics).

2.8. Plasma glucose and hormone determinations Plasma glucose was measured by the glucose–oxidase method using an analyzer CX3 (Beckman). Plasma insulin was measured by radioimmunoassay (Kevran et al., 1976). For rat corticosterone assay, a commercial kit was used (Amersham): inter and intra assay coefficients of variation are 4.8 and 5%, respectively.

2.9. Statistical analysis Results were expressed as mean 9 SEM for the indicated number of independently performed experiments. Statistical comparisons were made using the ANOVA and Student’s t-tests. Probability values of PB0.05 were considered statistically significant.

2.7. Semi quantitati6e RT-PCR assay

3. Results

One microgram of total RNA was reverse transcribed (RT) into a single strand cDNA by 1 U/ml of AMV reverse transcriptase (Ozyme) using 90 mM/ml of random hexamer primers. The RT reaction was carried out for 2 h at 42°C in a volume of 20 ml containing 50 mM Tris–HCl pH 8.3, 60 mM KCL, 10 mM MgCl2, 1 mM DTT, 1 U/ml RNase inhibitor (BoehringerMannheim) and dNTP (each 0.5 mM). Incubations in the absence of reverse transcriptase were used as negative controls. Two synthetic oligodeoxyribonucleotide primers were used to amplify the PDH E1a cDNA: a sense primer ‘PDS1’ (5%-tcaagtactacaggatgatg-3%) corresponding to nucleotides 223 – 242 and an antisense primer ‘PDA1’ (5%-ggcgtacatgtgcattgatc-3%) complementary to nucleotides 490 – 509. Cyclophylin (used as an internal standard) was coamplified with PDH E1a cDNA using two specific primers: a sense primer ‘CS’ (position 43–62, sequence: 5%-atggtcaaccccaccgtgtt-3%)

3.1. PDHc acti6ity in heart and li6er mitochondria from lean and obese Zucker rats Total PDH activity was measured in freshly extracted mitochondria of heart and liver from 4 and 8 week-old lean and obese Zucker rats (Table 2). In 4 week-old lean rats, PDH activity was higher in liver than in heart (12.69 1.1 and 4.7 9 0.4 mU/U citrate synthase, respectively). At 8 weeks, PDH activities were at least 2-fold higher than at 4 weeks (PB 0.01), concomitantly with a slight increase in insulinemia. In obese rats, liver PDH activity was significantly increased (by about 70 to 120%) relative to lean age-matched animals as early as 4 weeks and all over the study period, but heart PDH activity was unchanged. Similar differences in liver PDH activity between lean and obese rats were observed in the fed state at 4 and 8 weeks (results not shown).

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Table 2 Pyruvate dehydrogenase complex (PDHc) activity in liver and heart tissues in 4- and 8 week-old obese (fa/fa) zucker rats and their lean littermates (Fa/?) Liver

Heart

Age (weeks)

4

8

4

8

Lean (Fa/?) Obese (fa/fa)

12.5691.11 (6) 22.1992.22 (6)**

32.95 9 1.07 (11) 52.12 9 2.07 (12)**

4.72 9 0.37 (6) 4.99 9 0.17 (6)

9.08 9 0.53(11) 8.04 9 0.32 (10)

Liver mitochondrial extracts of age-matched lean and obese rats were prepared and assayed for PDHc activity as described in Section 2, with results expressed per unit of citrate synthase (CS) activity. Results are the mean 9SEM (the number of animals tested in each group is given in parenthesis). Asterisks indicate, in obese rats, results statistically significant from lean rats; **PB0.01.

Activities of citrate synthase in 4 week-old lean rats were 3.39 and 0.395 U/mg mitochondrial protein in heart and liver, respectively. Comparable activities were observed in 8 week-old lean rats and in 4- and 8 week-old obese rats, indicating that the recovery of mitochondrial enzyme was reproducible in these studies. The values of PDH activity observed in liver and heart of Zucker rats were somewhat lower than those found in tissues of normal Wistar rats in two previous reports (Conney et al., 1993; Bryson et al., 1995). This may reflect differences in the methods used to disrupt mitochondria and to assay PDH activity.

3.2. Analysis and quantitation of PDHc subunits in heart and li6er mitochondria from lean and obese Zucker rats To determine whether the increase in PDHc activity reflects an increase in PDHc concentration, a Western blot analysis was performed to analyse and quantitate mitochondrial PDHc subunits from heart and liver. To this aim, we used an antibody raised against the PDH complex of bovine heart, which recognizes the E1a, E1b, E2, and X subunits. Regardless of obesity status and age, the immunoblot showed four major bands with estimated Mr values of 70, 51, 40.5, and 35 kDa, which correspond to the E2, X, E1a and E1b subunits of the complex, respectively (Fig. 1A). In 4 week-old lean rats, the respective proportions of E2, X, E1a and E1b subunits were about 50, 20, 15 and 16% of total subunits for liver and heart; a similar pattern of distribution was observed in 4- and 8 week-old rats (results not shown). Within each group of age, liver mitochondria from obese rats showed a significant increase in abundance of E1a and E1b subunits relative to mitochondria from lean rats. This change was readily detectable at 4 weeks (40 – 80% increase, P B 0.01) and achieved 110–140% at 8 weeks (P B 0.002) (Fig. 1B). In contrast, heart mitochondria from obese rats showed no significant changes in the abundance of E1a and E1b subunits relative to lean rats.

3.3. Analysis and quantitation of li6er E1a and E1b mRNAs To assess whether the increased abundance of E1a and E1b subunits in liver mitochondria from obese rats was linked to a pretranslational event, we quantitated the corresponding mRNAs by Northern blot analysis. As probes, we used human cDNAs coding for the most conserved regions of mammalian PDH E1a and EIb (Dahl et al., 1987), in particular the region of the E1a subunit which contains the three serine phosphorylation sites (De Meirleir et al., 1988); the E1a cDNA probe has previously been shown to hybridize to two mRNA species of 3.5 and 1.6 kb in human tissues (Dahl et al., 1987; De Meirleir et al., 1988; Wexler et al., 1988). As shown on Fig. 2A, Northern blot analysis of liver RNA using the E1a cDNA probe revealed a single mRNA species of 3.5 kb. At the age of 4 weeks, the abundance of the 3.5 kb mRNA species in liver was about three times higher in obese rats than in lean rats, whether signals were normalized to b-actin, PABP and rRNA 18 S signals (Fig. 2B). In contrast, at the age of 8 weeks the abundance of the E1a was similar in obese and lean rats. Like in human heart (Ho et al., 1988), the human E1b probe detected in livers of lean and obese Zucker rats one major mRNA species of 1.6 kb (Fig. 2A). Neither the age nor the obesity status affected the abundance of this species (Fig. 2B).

3.4. Semi-quantitati6e RT-PCR analysis of li6er E1a mRNA To confirm the overexpression of E1a mRNA detected on Northern blots, we developed a semi quantitative RT-PCR assay using specific PDH E1a primers PDS1 and PDA1, which generated a fragment of 268 bp (Fig. 3A). To correct for any difference in the recovery of the amplification product, we used cyclophilin as an internal standard. Only 4 week-old obese Zucker rats displayed a significant rise (twofold) in the level of liver E1a mRNA (Fig. 3B), thus confirming the results obtained by Northern-blot analysis.

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Fig. 1. Western-blot analysis of PDHc subunit proteins of liver and heart mitochondria from lean and obese Zucker rats. Mitochondrial extracts from 4- 8 week-old obese Zucker rats and their lean littermates were subjected to Western immunoblotting as described in Section 2. The immunoreactive proteins were visualized by ECL detection reagents and quantitated by scanning densitometry. A, representative western blot showing the PDH subunit components in liver and heart mitochondria of 4 and 8 week lean (L) and obese (Ob) rats. The amount of mitochondrial protein used was 1 and 0.5 mg for liver and heart, respectively, corresponding to 1 and 2 mU citrate synthase; B, concentrations of immunoreactive E1a and E1b subunits in liver and heart mitochondria of 4–8 week old lean ( ) and obese ( ) rats. The results are expressed in arbitrary unit per unit of citrate synthase, mean 9SEM of 3–7 determinations in each group. Asterisks indicate results statistically different from lean rats: *PB 0.05, **PB0.01, ***PB0.001.

4. Discussion The present study shows that PDHc activity is increased in liver mitochondria from 4- to 8 week-old obese Zucker rats relative to age-matched lean littermates. The values of PDH activity were associated with a coordinate increase in the amount of E1a and E1b PDH subunits, and in 4 week-old rats, by an increase in PDH E1a mRNA level. In contrast, neither total PDHc activity and nor the amount of PDH subunits were modified in heart of obese rats. These data indicate an early, liver-specific increase in PDHc which results from a selective up-regulation of E1a gene expression. A liver-specific regulation of total PDH activity is not unique to obese Zucker rats since it has been shown to also occur in rats subjected to nutritional manipulations. For instance, high sucrose feeding induces an

increase in total PDH activity in liver but not in heart and skeletal muscle (Da Silva et al., 1992). Such a tissue-specific regulation of the expression of a metabolic enzyme having a ubiquitous function is a more common phenomenon than anticipated. For instance, the ubiquitously expressed enzyme cytosolic aspartate aminotransferase (cAspAT) is under hormonal control solely in liver where it is involved in gluconeogenesis, a tissue-specific function (Barouki et al., 1989; Aggerbeck et al., 1993). Indeed, in the latter tissue, cAspAT expression is stimulated by cAMP and glucocorticoids whereas it is inhibited by insulin (Barouki et al., 1989). Like cAspAT, PDH is an ubiquitously expressed enzyme that appears to be controlled by insulin, but with a large degree of tissue heterogeneity, probably related to glucose oxidation or conversion to lipid capacity (Conney et al., 1993). Whether PDH, like

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cAspAT, is involved in a tissue-specific metabolic pathway in liver is at present unknown. The parallel increase in the concentrations of E1a and E1b subunits which occurs in liver of 4- and 8 week-old obese rats is consistent with the observation that these two subunits are present in a 1:1 molar ratio in the PDH heterotetramer. An increase in the content of the X protein was also observed in liver of 8 week-

Fig. 2. Northern-blot analysis of PDH E1 a and b subunit mRNA of livers of 4- and 8 week-old lean and obese Zucker rats. Total liver RNA (40 mg) was subjected to Northern blot analysis as described in Section 2. Blots were hybridized with 32P-labeled cDNA probes coding for PDH E1a and E1b subunit, b-actin, rRNA 18S and PABP, followed by radioautography. (A) representative autoradiograms showing the hybridization signals obtained with the four labeled probes. (B) concentrations of PDH E1a and E1b mRNAs in obese Zucker rats ( ), with results corrected for 18s RNA recovery and expressed as the percentage of mean values of age matched lean rats ( ). Values in lean rats (arbitrary units, mean9 SEM of the number of determinations indicated in parenthesis) were respectively E1a/18S: 1.02 9 0.30 (11), E1b/18S: 0.909 0.16 (9) in 4 week-old rats and E1a/18S: 1.13 90.11 (11), E1b/18S: 1.00 9 0.26 (10) in 8 weekold rats.

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old obese rats. A close correlation between the changes in total PDH activity and the changes in enzyme content measured by western blotting has already been observed in rats fed with a high-fat or a high-sucrose diets (Da Silva et al., 1993), or during rat brain development (Malloch et al., 1986). The concomittant increase in E1a protein and E1a mRNA abundance in liver of 4 week-old obese rats suggests an increase in either the transcription rate of the E1a gene and/or the stability of the mRNA. Surprisingly, despite an increase in E1a protein abundance, E1a mRNA abundance was unchanged in 8 week-old obese rats, and whatever age and obesity status, E1b mRNA abundance was also unchanged. These findings suggest the implication of other regulatory mechanisms, probably at translational and/or post-translational steps. The non coordinate expression of the E1a and E1b genes in obese Zucker rats is consistent with the different organization of their respective promoters (Chang et al., 1993; Madhusudhan et al., 1995). Indeed, the E1a promoter contains a putative insulin responsive-element (IRE) which is also found in the promoter of other insulin-regulated genes such as phosphoenolpyruvate carboxykinase (O’Brien et al., 1990). In contrast, the E1b promoter represents an unusual variation of a housekeeping gene with a unique combination of initiation sites and Sp1 sites together with sites that bind proteins expressed in a tissue-specific manner. While insulin has clearly both rapid and long-term effects on the state of phosphorylation and activity of the PDHc in liver and adipose tissue cells, its ability to regulate the total amount of the complex in these tissues is not well established. In the present study, the increase in liver PDHc activity in obese rats was shown to coexist with hyperinsulinemia and the abundance of the E1a protein was correlated with plasma insulin, suggesting a link between PDHc expression and hyperinsulinemia. An increase in plasma insulin triggered by dietary carbohydrates has also been implicated in the increase in PDH activity and PDH subunit concentrations which occurs in white adipose tissue during the suckling-weaning transition (Maury et al., 1995). However, these observations by no means establish that insulin directly affects the expression of the PDHc. In addition, besides hyperinsulinemia, a number of other hormonal abnormalities may, directly or not, contribute to the changes in PDH expression in genetically obese Zucker rats. For instance, decreased plasma levels of thyroid hormones (Chomard et al., 1994) and growth hormone (Finkelstein et al., 1986) and conversely an increased level of corticosterone (Dubuc et al., 1975) have been reported in these rats. However, acute GH treatment has been shown to increase basal but not total PDH activity (Clot et al., 1988) and dexamethasone treatment decreased PDH flux with pyruvate as substrate in subsequently isolated hepato-

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Fig. 3. Semi-quantitative RT-PCR analysis of PDH E1a mRNA in liver of 4- and 8 week-old lean and obese Zucker rats.Total RNA was prepared from livers of 4 and 8 week-old lean and obese Zucker rats. PDH E1a mRNA was subjected to semi-quantitative radioactive RT-PCR analysis as described in Section 2. (A) representative autoradiograms of the products of amplification of PDH E1a and cyclophylin cDNA (used as internal standard) after resolution by nondenaturing polyacrylamide gel electrophoresis. (B) Quantification of PDH E1a mRNA with results corrected for cyclophylin mRNA (internal standard) in lean ( ) and obese ( ) Zucker rats.

cytes (Jones et al., 1993), making the involvment of GH and glucocorticoid hormones unlikely. In summary, these results suggest a liver-specific regulation of PDHc expression in genetically obese and hyperinsulinemic Zucker rats, which affects the E1a gene at transcriptional and/or post-transcriptional steps. Further studies are needed to identify the hormonal and/or metabolic determinants implicated in the tissue-specific regulation of the PDH genes in this animal model.

Acknowledgements We thank gratefully Dr C. Forest for helpful discussions and his critical review of this manuscript. We thank Professor J.P. Clot (University Rene´ Descartes, Paris) for performing the densitometric analysis of Western blot experiments and Dr J.G. Lindsay (University of Glasgow, Glasgow) for providing antibodies against pyruvate dehydrogenase complex purified from pig heart. This work was supported by the Association Franc¸aise contre les Myopathies (AFM). A preliminary report of this sudy was presented at the VIe International Symposium on Insulin Receptors & Insulin Action, Copenhagen Denmark, 1996.

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