Báo cáo khoa học: Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells

Diverging regulation of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells Emily L. Abbot1, James G. McCormack2, Christine Reynet2, David G. Hassall3, Kevin W. Buchan3,* and Stephen J. Yeaman1

1 Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, UK 2 Prosidion Ltd, Oxford, UK 3 GlaxoSmithKline, Stevenage, UK

Keywords gene regulation; mitochondria; peroxisome proliferator-activated receptor; pyruvate dehydrogenase kinase; skeletal muscle

Correspondence S.J. Yeaman, The Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK Fax: +44 191 222 7424 Tel: +44 191 222 7433 E-mail: s.j.yeaman@ncl.ac.uk

*Present address GE Healthcare, Amersham, UK

(Received 7 January 2005, revised 21 March 2005, accepted 8 April 2005)

doi:10.1111/j.1742-4658.2005.04713.x

The pyruvate dehydrogenase complex occupies a central and strategic posi- tion in muscle intermediary metabolism and is primarily regulated by phos- phorylation ⁄ dephosphorylation. The identification of multiple isoforms of pyruvate dehydrogenase kinase (PDK1–4) and pyruvate dehydrogenase phosphatase (PDP1–2) has raised intriguing new possibilities for chronic pyruvate dehydrogenase complex control. Experiments to date suggest that PDK4 is the major isoenzyme responsible for changes in pyruvate dehy- drogenase complex activity in response to various different metabolic con- ditions. Using a cultured human skeletal muscle cell model system, we found that expression of both PDK2 and PDK4 mRNA is upregulated in response to glucose deprivation and fatty acid supplementation, the effects of which are reversed by insulin treatment. In addition, insulin directly downregulates PDK2 and PDK4 mRNA transcript abundance via a phos- phatidylinositol 3-kinase-dependent pathway, which may involve glycogen synthase kinase-3 but does not utilize the mammalian target of rapamycin or mitogen-activated protein kinase signalling pathways. In order to further elucidate the regulation of PDK, the role of the peroxisome proliferators- activated receptors (PPAR) was investigated using highly potent subtype selective agonists. PPARa and PPARd agonists were found to specifically upregulate PDK4 mRNA expression, whereas PPARc activation selectively decreased PDK2 mRNA transcript abundance. PDP1 mRNA expression was unaffected by all conditions analysed. These results suggest that in human muscle, hormonal and nutritional conditions may control PDK2 and PDK4 mRNA expression via a common signalling mechanism. In addition, PPARs appear to independently regulate specific PDK isoform transcipt levels, which are likely to impart important metabolic mediation of fuel utilization by the muscle.

mammals, there is no pathway for the net conversion of acetyl-CoA to pyruvate and thus the catalytic activ- ity of PDC represents the irreversible utilization of

The pyruvate dehydrogenase complex (PDC) oxida- tively decarboxylates pyruvate to acetyl-CoA and CO2, coupled with the reduction of NAD+ to NADH. In

Abbreviations BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GSK3, glycogen synthase kinase-3; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyan-4-one; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MEM, minimal essential medium; mTOR, mammalian target of rapamycin; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; PtdIns3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PPAR, peroxisome proliferator-activated receptor; ZDF, Zucker diabetic fatty rat.

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carbohydrate fuels. The predominant chronic control mechanism used to regulate PDC activity is a reversi- ble phosphorylation ⁄ dephosphorylation cycle [1]. Phos- phorylation of three serine residues on the E1a subunit, by pyruvate dehydrogenase kinase (PDK), causes inactivation of the complex [2]. Such inhibition can be reversed only by dephosphorylation catalysed by pyruvate dehydrogenase phosphatase (PDP).

suggests

To date, four isoforms of PDK (PDK1–4) and two isoforms of PDP (PDP1–2) have been identified in humans [3–5]. These isoforms display unique tissue dis- tribution [3–5] and varied kinetic and regulatory prop- erties [3,5,6] suggesting that the activity of PDC in any given tissue reflects the relative abundance of each PDK ⁄ PDP isoform, their specific activities and their sensitivity to allosteric regulators.

insulin-stimulated glucose disposal

Investigations into the role of PPARa and PPARd in regulating PDK expression have been performed in human skeletal muscle cells [17,18]. In human myo- tubes, activation of either PPARa or PPARd receptors (by the agonists GW7647 and GW0742, respectively) resulted in a significant increase in the rate of fatty acid oxidation. In addition, both agonists caused a marked increase in the levels of PDK4 transcript abun- dance without any effect on PDK2 mRNA expression [17,18]. Treatment of Zucker diabetic fatty (ZDF) rats with the PPARc agonist GW1929 for 7 days resulted in a 7.5-fold decrease in PDK4 mRNA expression in muscle [19]. This decrease in PDK4 mRNA expression associated with GW1929 treatment that PDK4 repression may be an important mechanism by which PPARc agonists enhance glucose utilization in muscle [19]. However, such effects in muscle may be via additional regulatory pathways, which along with the major alterations in adipoctye gene expression, lead to changes in plasma lipid levels.

Collectively, these investigations suggest that chan- ges in the concentration of free fatty acids and insulin are important in regulating the expression of PDK iso- forms, either directly or indirectly. Alterations in these factors, induced by starvation, high-fat feeding, and diabetes, result in an imbalance in PDK ⁄ PDP activity and thus in hyperphosphorylation and inactivation of PDC.

Skeletal muscle, by virtue of its relative mass, is the major site of in mammals, a process impaired in type 2 diabetes melli- tus and obesity, and has thus been the focus of several investigations into PDC regulation. The Pima Indians have one of the highest known prevalences of type 2 diabetes mellitus in the world [7]. In this group, levels of PDK2 and PDK4 skeletal muscle mRNA tran- scripts were found to be positively correlated with fast- ing plasma insulin concentrations as well as percentage body fat, and negatively correlated with insulin-medi- ated glucose uptake rates [8]. During a hyperinsulinae- mic–euglycaemic clamp, levels of both transcripts decreased in response to insulin, suggesting that the transcription of both PDK2 and PDK4 are regulated by a common mechanism in humans [8]. In addition, skeletal muscle from obese patients with raised fatty acids has a reduced oxidative capacity, with reduction in type 1 fibres, similar to that seen in rodents fed a high fat diet [9,10]. Under these conditions of modified tissue delivery, changes in PDK4 have been observed [11].

Most studies to date have utilized animal models or animal-derived cell lines to investigate chronic changes in PDK ⁄ PDP isoform expression. However, little work has been done in human systems. Data from our laboratory suggest that cultured human muscle cells represent a valuable system for metabolic studies [20–24]. This study examines the effects of different hormonal, nutritional, and pharmacological conditions on the mRNA expression of the two main isoforms expressed in human muscle, namely PDK2 and PDK4 [3–5]. It also confirms the significant contribution made to muscle metabolism by PPAR modulation and highlights the importance of PPARd in these regula- tory mechanisms.

In rat gastrocnemius muscle, starvation has been reported to specifically upregulate PDK4 expression [12–14]. In contrast, the administration of a high-fat diet for 28 days was associated with significant increa- ses in PDK2 and PDK4 protein expression in rat muscle [15].

Results

Identification of PDK1–4 and PDP1 isoforms in human myoblasts

Primers designed to amplify specifically human PDK1–4 and PDP1 were used in PCR and products were identified by gel electrophoresis (data not shown). Molecular cloning of each PDK or PDP isoform was confirmed by sequence comparison of each clone with

Elevated plasma free fatty acids are a common char- acteristic of high-fat feeding, starvation and diabetes. Numerous fatty acids and their derivatives serve as lig- ands for the peroxisome proliferator-activated recep- tors (PPARs), thus these receptors are thought to play a key role in sensing nutrient levels and modulating metabolism accordingly [16] and could be linked to changes in expression of metabolic genes, by their influence as transcriptional activators.

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Table 1. The effects of glucose ± insulin on PDK2 and PDK4 tran- script abundance expressed as a percentage of basal (5 mM) glu- cose levels. Results are the means ± SEM of n ¼ 3, from cells prepared from three different subjects and values are expressed as a percentage of basal (100%, 5 mM glucose, minus insulin). Statisti- cal significance compared with basal untreated levels (P < 0.05) is indicated by *, or statistical significance as compared to no glucose values (P < 0.05 and < 0.001) are represented by (cid:1) and (cid:1)(cid:1). Results are expressed against the 5 mM glucose control values (see Experi- mental procedures).

the previously reported DNA sequences [3–5]. This verified that all the selected primer pairs were specific for their designated isoform. Although mRNAs for all four PDK isoforms were detected in our muscle cell culture system, previous studies have reported PDK2 and PDK4 to be the predominant isoforms expressed in mature human muscle [3–5], and therefore subse- quent semi-quantitative RT–PCR experiments in this study focused on changes in mRNA expression of these isoforms.

No glucose

25 mM glucose No glucose + insulin (1 lM)

PDK2 172.0 ± 25.5* 101.1 ± 25.0 97.9 ± 18.9 PDK4 205.5 ± 35.4*

83.7 ± 4.3(cid:1)(cid:1) 80.7 ± 12.8(cid:1)

The regulatory influence of glucose, fatty acids and insulin on PDK2 and PDK4 mRNA expression

We examined the effects of the two predominant meta- bolic fuels in muscle, namely glucose and fatty acids, on PDK2 and PDK4 mRNA expression in human myoblasts. Cells were incubated for 5 h in the presence of different glucose concentrations. Depriving the cells of glucose significantly increased PDK2 and PDK4 mRNA expression above basal (5 mm) values (Fig. 1A and Table 1). In contrast, incubating the cells in a high glucose medium (25 mm) had no significant effect on the expression of either isoform compared with basal levels. Insulin (1 lm) was found to markedly reverse the effect of glucose deprivation on PDK2 and PDK4 transcript abundance by returning the transcript levels to approximate basal values (5 mm glucose, minus insulin) (Table 1).

Myoblasts were also incubated for 18 h in SF Ham’s F10 media in the presence of saturated (palmitate, 100 lm), unsaturated (oleate, 100 lm) or both fatty acids combined (100 lm of each). Each fatty acid, sin- gularly or combined, significantly increased PDK2 or PDK4 mRNA levels above basal (minus fatty acids) values (Fig. 1B and Table 2). The effects on PDK2 and PDK4 transcript levels appeared maximal at 100 lm of each fatty acid (data not shown) and no fur- ther effects were observed in the presence of both fatty acids (Table 2). Insulin (1 lm) reversed the effect of the fatty acids (100 lm of each) on PDK mRNA expression by returning transcript abundance of PDK2 and PDK4 to (minus fatty acids, minus insulin) values (Table 2).

A

B

Fig. 1. Semi-quantitative RT-PCR showing the effect of glucose and fatty acids on PDK2 and PDK4 mRNA expression. (A) Myoblasts were incubated for 5 h in SF DMEM plus 0.2% (w ⁄ v) BSA under glu- cose deprivation conditions (NG), 5 mM glucose (5 mM) and 25 mM glucose (25 mM). A typical experiment representing amplification of b-actin, PDK2 and PDK4 (amplified with full-length primers) is shown; quantitative data is given in Table 1. (B) Myoblasts were incubated in SF Ham’s F10 for 18 h in basal conditions [plus 0.12% (w ⁄ v) BSA; B], supplemented with 100 lM palmitate (P), supplemented with 100 lM oleate (O), supplemented with 100 lM of palmitate and ole- representing amplification of ate (BOTH). A typical experiment b-actin, PDK2 and PDK4 (amplified with full-length primers) is shown; quantitative data is given in Table 2.

The ability of insulin alone to regulate PDK2 and PDK4 transcript abundance was also investigated (Fig. 2). Myoblasts were incubated for 5 h in the pres- ence or absence of insulin (1 lm). Insulin markedly decreased PDK2 and PDK4 mRNA levels below basal values. In order to investigate the mechanisms by which insulin regulates PDK mRNA expression, select- ive inhibitors of signalling pathways known to be activated by insulin were used. Two distinct phosphati- dylinositol 3-kinase (PtdIns3K) inhibitors, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyan-4-one (LY294002) [25,26], were used to examine the role of PtdIns3K in regulating PDK transcript abundance in response to insulin (Fig. 2). Incubation with either LY294002 (50 lm) or wortmannin (100 nm) signifi- cantly inhibited the effects of insulin on PDK2 and PDK4 mRNA expression by returning transcript abundance to approximately basal levels (Fig. 2A,B). Downstream targets of PtdIns3K include glycogen syn- thase kinase-3 (GSK3) and the mammalian target of rapamycin (mTOR). GSK3 is inactivated in response to insulin via a PtdIns3K ⁄ protein kinase B (PKB)- dependent pathway [27,28]. Involvement of GSK3 in

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Table 2. The effects of fatty acids ± insulin on PDK2 and PDK4 transcript abundance expressed as a percentage of basal levels. Results are the means ± SEM of n ¼ 3, from cells prepared from three different subjects and values are expressed as a percentage of basal (100%, minus fatty acids and insulin) levels. Statistical significance as compared to basal (minus fatty acid) values (P < 0.05, < 0.001 and < 0.0001) is indicated by *, ** and ***, respectively, or statistical significance as compared to palmitate plus oleate values (statistical significance P < 0.05) is indicated by (cid:1). Results are expressed against basal (minus fatty acid) control value (see Experimental procedures).

Palmitate (100 lM)

Oleate (100 lM)

Palmitate and Oleate (100 lM)

Palmitate and Oleate (100 lM) and Insulin (1 lM)

182.4 ± 18.1* 152.8 ± 11.6*

180.7 ± 15.2** 153.5 ± 6.3**

163.5 ± 3.0*** 148.0 ± 9.5**

PDK2 PDK4

82.1 ± 10.3(cid:1) 97.1 ± 10.8(cid:1)

U0126 to inhibit insulin-stimulated phosphorylation of MAPK in our cell system was confirmed by immuno- blotting with phospho-MAPK after the 5 h incubation period (data not shown).

Identification of PPAR isoforms in human myotubes

Prior to investigating the effects of PPAR agonists on PDK mRNA expression, it was first necessary to con- firm expression of each receptor in human myotubes. Total RNA was isolated from 7-day differentiated myotubes and subsequently used as a template for full- length, first-strand cDNA synthesis. Primers were designed to specifically amplify human PPAR a, d and c1 and PCR products were identified by gel elec- trophoresis (data not shown). Molecular cloning of each isoform was confirmed by sequence analysis and comparison of each clone with the reported DNA sequence of human PPAR a, d and c1 [34–36]. This confirmed that all three receptors are expressed in dif- ferentiated myotubes.

The effects of PPAR agonists on PDK2 and PDK4 mRNA expression

the insulin-induced downregulation of PDK mRNA expression was assessed using lithium, an allosteric inhibitor of GSK3 [29] (Fig. 2C). LiCl (50 mm) mim- icked the effects of insulin on PDK transcript abun- dance by significantly reducing PDK2 and PDK4 mRNA expression below basal (minus insulin and lith- ium) values. mTOR is important in regulating several components of the protein translational machinery and has been established as an insulin-sensitive target pro- tein [30]. Incubation of myoblasts with the mTOR- selective inhibitor rapamycin (100 nm) for 5 h was employed to further elucidate the insulin-to-PDK path- way downstream of PtdIns3K. In contrast to the results obtained with the PtdIns3K inhibitors, rapa- mycin (100 nm) did not reverse the effects of insulin (1 lm) on PDK mRNA expression (Fig. 2D). How- ever, incubation with rapamycin alone significantly reduced PDK2 (77.1 ± 5.3, n ¼ 3; P < 0.05) and PDK4 (73.2 ± 7.6, n ¼ 3, P < 0.05) mRNA levels below basal values (100%, minus rapamycin), suggest- ing that inhibition of basal mTOR activity affects PDK mRNA expression (data not shown). As p70S6K is a downstream target of mTOR, the ability of rapa- mycin to inhibit insulin-stimulated phosphorylation of p70S6K, by immunoblotting with phospho-p70S6K, con- firmed that this inhibitor was still operating after the 5 h incubation period (data not shown).

PPARd

concurrent with lower band corresponds

suggesting that

(Fig. 2D),

The effects of PPARa (GW7647), PPARd (GW0742) and PPARc (GW7845) specific agonists, at several different concentrations, on the mRNA expression of PDK2 and PDK4 were studied in human myotubes (Fig. 3). Incubation (24 h) with the PPARd agonist significantly augmented PDK4 transcript abundance in a concentration-dependent manner, at nanomolar concentrations affinity to PDK4 the (Fig. 3A; mRNA amplification as this band is of the correct Mr, the larger band was an unidentified product). Incubation (24 h) with the PPARa agonist also signi- ficantly upregulated PDK4 mRNA expression at 10 and 100 nm (Fig. 3B). However, at the lower concen- trations no effect on PDK4 mRNA expression was

Insulin stimulation of the mitogen-acitvated protein kinase (MAPK) pathway results in the phosphoryla- tion of transcription factors in the nucleus, leading to cellular proliferation and differentiation [31]. This pathway is selectively inhibited by the mitogen-activa- ted protein kinase kinase (MEK) inhibitor, U0126 [32,33]. Therefore, the role of the MAPK pathway in regulating PDK mRNA expression was investigated by incubating myoblasts for 5 h in the presence of insulin (1 lm) and U0126 (100 lm). U0126 failed to reverse the effects of insulin on PDK2 and PDK4 transcript the MAPK abundance signalling cascade is not involved in transducing the insulin-to-PDK transcriptional signal. The ability of

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A

B

subjects. Statistical

C

Fig. 2. Semi-quantitative RT-PCR showing the effects of insulin and LY294002, wortmannin, or LiCl on PDK2 and PDK4 mRNA expres- sion. (A–C) Myoblasts were incubated in SF Ham’s F10 for 5 h in basal conditions (B), plus 1 lM insulin (I), 1 lM insulin plus 50 lM LY294002 (I + LY) ⁄ 100 nM wortmannin (I + Wt) ⁄ 50 mM LiCl (I + LiCl) or 50 lM LY294002 alone (LY) ⁄ 100 nM wortmannin alone (Wt) ⁄ 50 mM LiCl alone (LiCl). Typical experiments representing amplification of b-actin, PDK2 and PDK4 (A, B amplified with full- length primers; C, amplified with short primers) are shown. (D) Results are expressed as a percentage of basal (minus insulin) from cells pre- levels and are the means ± SEM of n ¼ 3, pared from three different significance (P < 0.05, < 0.001 and < 0.0001) compared with basal untreated values is indicated by *, ** and ***, respectively, or statistical sig- nificance compared with insulin values (P < 0.05 and < 0.0001) are represented by (cid:1), or (cid:1)(cid:1)(cid:1), respectively.

D

A

B

C

Fig. 3. Semiquantitative RT-PCR showing the effects of the PPARd agonist (GW0742), PPARa agonist (GW7647) or PPARc agonist (GW7845) on PDK2 and PDK4 mRNA expression. Myotubes were incubated for 24 h in a-MEM plus 2% FBS under basal conditions (plus 0.01% DMSO, B), or plus indicated concentrations in nM of (A) GW0742 (B) GW7647 (10 nM GW0742 was included as a posit- ive control) (C) GW7845. Typical experiments representing amplifi- cation of b-actin, PDK2 and PDK4 (amplified with short primers) are shown; quantitative data is given in Table 3.

Discussion

Numerous investigations have focused on the effects of starvation, high-fat feeding and chemically induced diabetes on the levels of PDK expression [11]. In sum- mary, these studies have generally observed a selective increase in PDK4 mRNA and protein expression in response to various metabolic challenges. Although the majority of these investigations have observed coordi- nated regulation of mRNA and protein expression, an increase in PDK4 protein abundance independent of

induced. Neither the PPARa nor PPARd agonists affected PDK2 mRNA expression (Fig. 3A,B). In incubation (24 h) with the PPARc agonist contrast, selectively downregulated PDK2 transcript abundance (Fig. 3C). This effect was evident at agonist concen- trations of 1, 10 and 100 nm. However, treatment with this agonist had no effect on PDK4 mRNA transcript abundance (Fig. 3C). This data is summar- ized in Table 3.

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levels. Table 3. The effects of PPAR a, d and c agonists on PDK2 and PDK4 transcript abundance expressed as a percentage of basal Results are the means ± SEM of cell preparations from three different subjects. Values are expressed as a percentage of basal (100%, minus agonist) levels and statistical significance (P < 0.05, < 0.001 and < 0.0001) compared with basal untreated values is indicated by *, ** and ***, respectively.

PPARd PDK2 PDK4(s)

PPARa PDK2 PDK4(s)

PPARc PDK2 PDK4(s)

0.01 nM 126.9 ± 13.8 133.7 ± 5.3*** 0.1 nM 98.3 ± 7.8 102.7 ± 10.7 0.1 nM 97.0 ± 4.1 89.5 ± 11.0

0.1 nM 92.7 ± 3.5 194.7 ± 10.4*** 1 nM 91.3 ± 6.4 109.9 ± 18.0 1 nM 84.0 ± 6.4* 84.4 ± 14.3

1 nM 104.2 ± 8.8 237.3 ± 13.3*** 10 nM 99.1 ± 5.7 166.4 ± 16.5** 10 nM 75.9 ± 3.9*** 89.4 ± 21.9

10 nM 86.4 ± 3.0 247.1 ± 6.2*** 100 nM 99.8 ± 20.9 179.0 ± 10.5** 100 nM 61.4 ± 5.4*** 114.9 ± 12.7

rat

This result is in partial contrast to findings from biop- sies of the vastus lateralis muscle of subjects exposed to a 3-day low-carbohydrate ⁄ high-fat diet (5% carbo- hydrate, 73% fat, 22% protein) [42]. These authors reported a specific upregulation of PDK4 mRNA levels, without affecting PDK2 transcript abundance. Insulin reversed the effects of glucose deprivation or fatty-acid-supplemented medium, by returning PDK2 and PDK4 mRNA transcript levels to control (minus insulin) values (Tables 1 and 2). In addition, insulin alone significantly reduced PDK2 and PDK4 transcript abundance below basal values (Fig. 2). Thus, the activ- ity of PDC is regulated independently by the main fuel sources in muscle and by insulin through directly altering the expression of the human PDK2 and PDK4 isoforms.

changes in mRNA levels has been reported [37]. Such a result suggests the importance of both mRNA and protein analyses when investigating chronic PDK regu- lation. Majer et al. [8] reported that rabbit antiserum developed against recombinant PDK2 protein cross-reacted with the purified human recombinant PDK4 protein in western blot analyses. We observed similar cross-reactivity with both rat PDK2 and PDK4 antiserum against human recombinant PDK1–4 pro- teins (unpublished observation). Using short peptides representing human PDK2 and PDK4 amino acid sequences, antibodies specific for human PDK2 and PDK4 were successfully generated. However, due to poor antibody sensitivity and low levels of PDK pro- tein expression in cultured cells, changing levels of PDK protein expression could not be analysed in this study.

Glucose deprivation (5 h)

insulin-to-PDK transcription

the

elicited a significant increase in PDK2 and PDK4 mRNA levels when com- pared with controls in complete medium (Fig. 1A), consistent with previous findings in a human rhabdo- myosarcoma cell line (20-h glucose deprivation) and in rat liver, kidney, white adipose tissue, and lactating mammary gland in vivo after 48 h starvation [8,38]. However, investigations in rat heart and skeletal mus- cle have reported a selective increase in PDK4 mRNA after fasting (except in fast-oxidative muscle fibres in which an increase in both PDK2 and PDK4 mRNA was observed) [12–14,39,40]. Further work is needed to determine the mechanism by which glucose deprivation elicits these changes in expression. A recent study by Furuyama et al. [41] suggests that upregulation of PDK4 mRNA expression in C2C12 cells may be induced by the starvation-responsive forkhead-homo- logue in rhabdomyosarcoma (FKHR) transcription factor.

Incubating myoblasts for 18 h in the presence of fatty acids (saturated and unsaturated) also enhanced the expression of PDK2 and PDK4 mRNA (Fig. 1B).

In addition to our findings, insulin has been shown to decrease the mRNA for PDK2 and PDK4 in 7800C1 hepatoma cells, human rhabdomyosarcoma cells and whole skeletal muscle biopsies from nondia- betic Pima Indians [8,43]. However, the insulin signal- ling pathway utilized to relay this signal remains relatively uncharacterized. Figure 2 demonstrates that the two PtdIns3K inhibitors, LY294002 and wortman- nin, prevented insulin-induced downregulation of PDK2 and PDK4 mRNA, returning transcript abun- dance to control levels. However, neither mTOR nor MAPK activation appeared to be necessary for trans- signal ducing (Fig. 2D). Yet in contrast, inhibition of GSK3 by lith- ium mimicked the effects of insulin on PDK mRNA expression by reducing PDK2 and PDK4 transcript transcription factors, abundance (Fig. 2C). Several including c-Jun, c-Myc and CREB have been identified as potential substrates for GSK3 phosphorylation [44]. Therefore, insulin-mediated phosphorylation and thus inhibition of GSK3 may prevent the subsequent phos- phorylation and activation of transcription factors which are involved in transcribing PDK mRNA. In

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factors

in

addition, the importance of PKB-alpha and the FOXO transcription glucocorticoid-stimulated human PDK4 gene expression has recently been dem- onstrated [45].

GW1929 reduced the expression of PDK4 mRNA in muscle biopsies of ZDF rats, but PDK2 transcript abundance was not analysed [19]. Thus, the PDK iso- form regulated in response to PPARc activation appears to differ between rat and human tissues. A selective increase in PDK4 expression in response to PPARa and d activation renders the tissue relatively insensitive to changes in the concentrations of acute effector molecules, such as pyruvate. Therefore, by specifically reducing PDK2 mRNA expression, this method of ensuring chronic regulation in response to PPARc activation is maintained, as the pyruvate- unresponsive isoform remains predominantly expre- ssed. Our study suggests that direct effects of PPARc are present in human muscle, and thus the anti-dia- betic efficacy of the TZDs may not be solely the con- sequence of adipocyte-specific effects. The effects of PPARc activation in muscle are consistent with a decreased reliance on lipids and an enhanced depend- ence on glucose as a source of energy. Thus inhibition of PDK2 expression may represent an important mechanism by which PPARc agonists enhance glucose utilization in muscle.

The effects of PPARa activation, using GW7647, in upregulating PDK4 transcript abundance have been reported previously in primary cultures of human muscle cells [17,18]. However, in these investigations GW7647 was used at a concentration of 1 lm [17,18]. The EC50 values of GW7647 for a, d and c receptors are 0.0061, 1 and 8 lm, respectively [46], and thus at a 1 lm concentration GW7647 may have been activating both PPARa and PPARd receptors. Therefore, in this study, a concentration range of GW7647 (0.1, 1, 10 and 100 nm) was used to characterize specifically the effects of PPARa activation in human myotubes. Figure 3 shows that activating the PPARa receptor with agonist concentrations of 10 and 100 nm selec- tively increases PDK4 mRNA transcript abundance. In similar experiments the effects of PPARd activation using the selective agonist GW0742 (EC50 of 1.2, 0.0001, 4.1 lm for a, d and c receptors, respectively; K Buchan, unpublished) was determined. It is evident (Fig. 3) that PPARd activation also markedly stimu- lates PDK4 mRNA expression, even at a concentra- tion of 0.01 nm. Figure 1 demonstrates that fatty acids regulate the mRNA expression of both PDK2 and PDK4. However, in contrast, PPARa and d activation selectively increase the levels of PDK4 mRNA without affecting PDK2 expression. This observation suggests that PPARa or d target directly the PDK4 transcrip- tional machinery, whereas fatty acids augment PDK2 and PDK4 transcript abundance via an indirect mech- anism.

PDP1 mRNA expression appeared to be unaffected by all the conditions analysed in this investigation (data not shown). This is consistent with the findings of Huang et al. [50] who reported no change in PDP1 mRNA and protein expression in response to starva- tion and streptozotocin-induced diabetes in rat heart and kidney. There is a limited amount of evidence to suggest that PDP2 levels may change [50], but overall the work to date suggests that control of expression of PDK isoforms is the major mechanism for chronic regulation of the activity state of PDC.

In conclusion,

support

role

the

Recent observations in transgenic mouse models overexpressing PPARd in skeletal muscle have shown adaptive re-modelling of the muscle, leading to fibre- type switching and improvements in exercise endurance [47,48]. These observations for PPARd as an important transcriptional regulator, not only for PDK4 but also in the coordinated responses of muscle metabolism and phenotype re-modelling. In addition, the left shift in the dose–response curve for PDK4 upregulation with GW0742 (compared with PPARa GW7647) suggests a more significant role for PPARd than PPARa in modulating these events.

two significant aspects of

The effects of the PPARc agonist GW7845 (EC50 of 3.5 lm, inactive at 10 lm, 0.00071 lm for a, d and c receptors, respectively) [49] was also analysed and shown to selectively regulate PDK2 mRNA expres- sion by decreasing transcript abundance in a dose- responsive manner but was without effect of PDK4. It has previously been reported that treatment with

in response to various nutritional conditions (glucose and fatty acid) and hormonal con- ditions (insulin) the expression of PDK2 and PDK4 appeared to be regulated in concert. This suggests that the human PDK isoenzymes may be regulated by these metabolic factors by relatively general mechanisms, and our data using inhibitors strongly implicates the PtdIns3K and GSK3 signalling pathways. In contrast, PPAR agonists appeared to regulate PDK2 and PDK4 in an isoform specific manner, suggesting that these agonists are directly targeting specific human PDK genes and support the observations in vivo that the nuclear hormone PPARd is a key player in fatty acid utilization in skeletal muscle. In addition, the coordi- nated regulation of glucose and fatty acid metabolism by PPARs, in both adipose tissue and muscle, place them as central players in obesity and insulin resist- ance, the metabolic syn- drome.

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Experimental procedures

Materials

> 90%. Myoblast differentiation was carried out on cells which had reached 90–100% confluence. Differentiation was induced by incubating the cells in a-minimal essential media (a-MEM) containing 2% FBS, 100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomycin for a minimum of 7 days. For glucose-deprivation experiments, cells were incubated in Dulbecco’s modified Eagle’s medium (DMEM) minus glucose or DMEM supplemented with 5 or 25 mm d-glu- cose (BDH, Poole, UK). Prior to acute treatments, cells were incubated in serum-free media containing 0.2% (w ⁄ v) bovine serum albumin (BSA) for a minimum of 4 h.

Molecular cloning

reactions were prepared without General laboratory reagents were supplied by Sigma (Poole, UK) with the following exceptions. Tissue culture flasks and plates were supplied by Greiner (Stonehouse, UK), all media, fetal bovine serum (FBS), trypsin ⁄ EDTA and peni- cillin ⁄ streptomycin were from Invitrogen (Paisley, UK). Chick embryo extract was obtained from Sera Laboratories International (Salisbury, UK). Actrapid insulin was from Novo Nordisk (Copenhagen, Denmark). The PtdIns3K inhibitors LY294002 and wortmannin were from Alexis Corporation (Nottingham, UK) and Sigma, respectively. The mTOR inhibitor, rapamycin, was purchased from Sig- ma and the MEK inhibitor, U0126, was from Promega (Southampton, UK). The PPAR agonists; GW7845, GW7647 and GW0742 were kindly supplied by Glaxo- SmithKline Pharmaceuticals (Stevenage, UK).

Cell culture

Table 4. Primer sequences designed to specifically amplify full length PDK1–4, a short fragment of PDK4, PDP1 and PPARa, d and c1 iso- forms from human muscle cell cDNA.

Primer

Sequence

PDK1F PDK1R PDK2F PDK2R PDK3F PDK3R PDK4F PDK4R PDK4F (short) PDK4R (short) PDP1F PDP1R PPARaF PPARaR PPARdF PPARdR PPARcF PPARcR

5¢-TGGCCCATGGTTCCGGGCCCAGGTGGAGTTCTACGCG-3¢ 5¢-CGCGCTCGAGGGCACTGCGGAACGTCGTCATGTCTTTGG-3¢ 5¢-TGGCGAATTCGGCCCAAGTACATAGAGCACTTCAGCAAGTTC-3¢ 5¢-CGCGAAGCTTCGTGACGCGGTACGTGGACGTGTTCTTGG-3¢ 5¢-CGCGGAGCTCGGCCCAAGCAGATCGAGCGCTACTCG-3¢ 5¢-CGCGCTCGAGCTGTTTTGCTTTTGCTTTGTATTTTGAAGCATCC-3¢ 5¢-CGCGCCATGGTCAAGATGAAGGCGGCCCGCTTCGTGCTGCGC-3¢ 5¢-CGCGCTCGAGGTCCTGAGTGTCCCTCTTCACATGGCCAC-3¢ 5¢-GAGCCTGATGGATTTGGT-3¢ 5¢-GTTGCCCGCATTGCATTC-3¢ 5¢-GGCCAAAGGAGAACTGGTGGCAGTACACCC-3¢ 5¢-GGCATCAGCAAGCCAAGCAGCCGATCC-3¢ 5¢-CGCAATCCATCGGCGAGGATAGTTCTG-3¢ 5¢-GGCCACCAGCGTCTTCTCAGC-3¢ 5¢-CGGGAAGAGGAGGAGAAAGAG-3¢ 5¢-CACGCTGATCTCCTTGTAGGG-3¢ 5¢-GTGGAGCCTGCATCTCCACC-3¢ 5¢-CTCCTGCAGGGGGGTGATGTG-3¢

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Isolation of RNA from muscle cells was performed using TRI Reagent (Sigma). RNA (5 lg) was used to synthesize cDNA with a dT15 oligonucleotide and Superscript II (Invi- the trogen). Control addition of reverse transcriptase. The gene-specific oligo- nucleotide primers for PCR were designed according to the nucleotide sequences available on EMBL DNA database and are shown in Table 4. PCR was performed using 50 pmol of each gene-specific primer, 1 ng of double-stran- ded cDNA, dNTPs (200 lm), buffers and 0.5 U of Expand High Fidelity Polymerase (Roche Diagnostics Ltd, Lewes, UK) in a final volume of 100 lL. Ten PCR cycles were car- ried out using 15 s at 94 (cid:1)C (denaturing), 30 s at 45 (cid:1)C (annealing) and 2 min at 72 (cid:1)C (extension). Twenty cycles were subsequently performed using 94 (cid:1)C for 15 s (denatur- ing), 45 (cid:1)C for 30 s (annealing), 72 (cid:1)C for 2 min (extension) and cycle elongation of 5 s for each cycle. In order to verify primer specificity, the product of each reaction was cloned Human myoblasts were grown from needle biopsy samples taken from the gastrocnemius muscle of healthy subjects with no family history of type 2 diabetes and with normal glucose tolerance and insulin sensitivity, as assessed using insulin tolerance test. Myoblasts were main- the short tained in growth medium consisting of Ham’s F10 nutrient mixture supplemented with 20% FBS, 1% chick embryo extract, 100 UÆmL)1 penicillin and 100 lgÆmL)1 streptomy- cin. Experiments were performed using myoblast cells between the 5th and 15th passage at a confluence of

E. L. Abbot et al.

Regulation of hPDK2 and hPDK4 gene expression

2 Yeaman SJ, Hutcheson ET, Roche TE, Pettit FH,

into the pET21(d) vector (CN Sciences, Nottingham, UK) and the fidelity of each construct confirmed by DNA sequen- cing (Molecular Biology Unit, University of Newcastle upon Tyne, UK). Brown JR, Reed LJ, Watson DC & Dixon GH (1978) Sites of phosphorylation on pyruvate dehydrogenase from bovine kidney and heart. Biochemistry 17, 2364– 2370. 3 Gudi R, Bowker-Kinley MM, Kedishvili NY, Zhao Y

Semi-quantitative RT-PCR

& Popov KM (1995) Diversity of the pyruvate dehydro- genase kinase gene family in humans. J Biol Chem 270, 28989–28994. 4 Rowles J, Scherer SW, Xi T, Majer M, Nickle DC,

Rommens JM, Popov KM, Harris RA, Riebow NL, Xia J et al. (1996) Cloning and characterization of PDK4 on 7q21.3 encoding a fourth pyruvate dehydro- genase kinase isoenzyme in human. J Biol Chem 271, 22376–22382. 5 Huang B, Gudi R, Wu P, Harris RA, Hamilton J &

Popov KM (1998) Isoenzymes of pyruvate dehydrogen- ase phosphatase. DNA-derived amino acid sequences, expression and regulation. J Biol Chem 273, 17680– 17688. 6 Bowker-Kinley MM, Davis WI, Wu P, Harris RA &

Popov KM (1998) Evidence for existence of tissue-speci- fic regulation of the mammalian pyruvate dehydrogen- ase complex. Biochem J 329, 191–196. 7 Bennett PH, Burch TA & Miller M (1971) Diabetes mellitus in American (Pima) Indians. Lancet 2, 125–128.

PCR amplification was performed using Taq DNA poly- merase (Sigma). Each reaction mixture contained 25 pmol of each primer, 1 ng of double-stranded tcDNA and dNTPs (200 lm) in a final volume of 50 lL. Samples were initially heated for 5min at 95 (cid:1)C before 2.5 U of Taq DNA polymerase was added. Thirty amplification cycles were performed with the following parameters: 92 (cid:1)C for 1 min (denaturing), 55 (cid:1)C for 1 min (annealing) and 72 (cid:1)C for 1.5 min (elongation). b-Actin transcript abundance, amplified with primers (5¢-TCCACGAACTACCTTCAAC- 3¢ and 5¢-TTTAGGATGGCAAGGGAC-3¢), was used to standardize the amount of cDNA added to each reaction. Products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. Quantification of transcript abundance was performed using tina (v. 2.09d). In order to confirm that amplification was not saturated after 30 PCR cycles, b-actin cDNA abundance was ana- lysed after 10, 20, 30 and 40 PCR cycles. Amplification continued to increase up to 40 cycles verifying that at the cDNA concentrations and PCR parameters employed, mRNA abundance will not be saturated, allowing detection of changes in their levels.

8 Majer M, Popov KM, Harris RA, Bogardus C & Pro- chazka M (1998) Insulin downregulates pyruvate dehy- drogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resis- tant subjects. Mol Genet Metab 65, 181–186.

Statistical analysis

9 Abou MJ, Yakubu F, Lin. D, Peters JC, Atkinson JB

& Hill JO (1992) Skeletal muscle composition in dietary obesity-susceptible and dietary obesity-resistant rats. Am J Physiol 262, R684–R688. 10 Hickey MS, Carey JO, Azevedo JL, Houmard JA,

Data were analysed by Student’s t-test (unpaired) using graph pad prism (v. 3.0) and presented as means ± SEM with the number of different cell lines in parenthesis. Tests were analysed using the raw data (arbitrary units from gel scans) and are given with respect to control values which were normalized to 100%.

Pories WJ, Israel RG & Dohm GL (1995) Skeletal mus- cle fiber composition is related to adiposity and in vitro glucose transport rate in humans. Am J Physiol 268, E543–E547.

Acknowledgements

11 Sugden MC & Holness MJ (2003) Recent advances in

mechanisms regulating glucose oxidation at the level of the pyruvate dehydrogenase complex by PDKs. Am J Physiol Endocrinol Metab 284, E855–E862.

ELA was supported by a Biotechnology and Biologi- cal Sciences Research Council CASE studentship in collaboration with Novo Nordisk. We wish to thank Mrs Dorothy Fittes for her excellent technical assist- ance.

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