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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2007-1104
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 10 3967-3972
Copyright © 2007 by The Endocrine Society

Elevated Free Fatty Acids Attenuate the Insulin-Induced Suppression of PDK4 Gene Expression in Human Skeletal Muscle: Potential Role of Intramuscular Long-Chain Acyl-Coenzyme A

Kostas Tsintzas, Kamal Chokkalingam, Kirsty Jewell, Luke Norton, Ian A. Macdonald and Dumitru Constantin-Teodosiu

Centre for Integrated Systems Biology and Medicine, Institute of Clinical Research, School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom

Address all correspondence and requests for reprints to: Kostas Tsintzas, Centre for Integrated Systems Biology and Medicine, Institute of Clinical Research, School of Biomedical Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom. E-mail: kostas.tsintzas{at}nottingham.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Aim: We investigated the effect of elevated plasma free fatty acid and insulin concentrations on PDK4 mRNA transcript and protein content and long-chain acyl-coenzyme A accumulation in human skeletal muscle.

Methods: On two occasions, 10 healthy men underwent hyperinsulinemic-euglycemic clamps for 6 h with (LIPID) and without (CON) iv Intralipid (20% at 90 ml/h) plus heparin (200 U prime + 600 U/h) infusion.

Results: Glucose disposal was approximately 50% lower at the end of the clamp in the LIPID compared with the CON trial (37.8 ± 4.4 and 79.6 ± 4.0 µmol/kg lean mass·min, respectively; P < 0.01). In the LIPID trial, muscle long-chain acyl-coenzyme A concentration increased after 6 h, but not 3 h of lipid infusion (P < 0.01). Muscle PDK4 mRNA, but not protein, was down-regulated by 2-fold within 3 h in both clamps and decreased further (6-fold; P < 0.01) at 6 h in the CON but not the LIPID clamp. The lipid-induced attenuation in the suppression of PDK4 gene expression was not dependent on the activation of the Akt/FOXO3 pathway.

Conclusion: Accumulation of im lipids plays a more important role than impaired activation of Akt-mediated pathways in the regulation of muscle PDK4 gene expression in lipid-induced acute insulin-resistant states.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
IN HEALTHY HUMANS an increase in circulating free fatty acid (FFA) levels during a short-term (4–6 h) physiological hyperinsulinemia, through infusion of lipid emulsions and heparin, induces peripheral insulin resistance (1, 2). Recent studies have suggested direct effects of circulating and im lipids on glucose transport and insulin signaling under those conditions (3, 4, 5, 6). Interestingly, under those conditions a decrease in carbohydrate oxidation (COX) precedes the reduction in insulin-stimulated glucose uptake (1). Thus, a decrease in COX may also contribute to the development of lipid-induced insulin resistance in human skeletal muscle.

It is well known that insulin infusion stimulates muscle pyruvate dehydrogenase complex (PDC), which plays a key role in determining the rate of insulin-stimulated glucose oxidation (2, 7, 8). This is an important aspect of insulin’s action on human skeletal muscle because glucose oxidation accounts for most of glucose disposal at physiological levels of hyperinsulinemia (8). Furthermore, lipid-induced insulin resistance impairs the capacity of insulin to increase muscle glucose uptake and PDC activity (2).

Skeletal muscle PDC activity is reduced by phosphorylation of the E1 component of the complex by pyruvate dehydrogenase kinase (PDK), which plays an important role in the development of insulin resistance in metabolic states characterized by elevated lipid metabolism (9, 10). Four isoforms of PDK (PDK1–4) have been identified (11, 12), but PDK2 and PDK4 are the most relevant to human skeletal muscle. Recent studies from our (7, 13) and other laboratories (14, 15) showed a selective up-regulation of PDK4 gene and protein expression in skeletal muscle of healthy humans in response to starvation (13, 15) and high-fat diet (7, 14). In our previous study, the fat-induced up-regulation of muscle PDK4 protein content was associated with inhibition of PDC activity and the subsequent reduction in COX, and preceded changes in glucose uptake highlighting a key role for PDK4 through its regulation of PDC in substrate metabolism and insulin action in human skeletal muscle (7).

We have recently demonstrated for the first time that insulin can rapidly suppress PDK4 gene expression in skeletal muscle in healthy humans (7). Nevertheless, the inhibitory effect of insulin on skeletal muscle PDK4 mRNA content was attenuated when infused together with Intralipid/heparin in rats, and this was associated with impaired insulin-mediated Akt activation (16), suggesting an impairment of insulin’s action on PDK4 gene expression. However, this has not been demonstrated in humans yet. In contrast to published animal studies (16, 17), Intralipid/heparin infusion alone for 4 h clearly showed an increase of severalfold in PDK4 mRNA content in human skeletal muscle (18). Lipid infusion is also associated with accumulation of im triglycerides and a variety of fatty acid metabolites, including long-chain acyl-coenzyme As (LCACs) (4, 6, 19, 20), which serve as modulators of gene transcription in skeletal muscle (21). This raises the interesting possibility that accumulation of im LCACs may also be involved in the regulation of PDK4 gene expression in lipid-induced insulin-resistant states.

Therefore, the aim of this study was to: 1) examine the effect of elevated circulating FFA levels during short-term physiological hyperinsulinemia on changes in expression of PDK4 in human skeletal muscle and relate those measurements to changes in im LCACs levels, substrate use, and insulin action; and 2) elucidate some of the signaling pathways responsible for the molecular alterations underlying the development of lipid-induced down-regulation of COX under those conditions.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Ten healthy men (age 22 ± 1 yr, body mass 78.0 ± 3.4 kg, body mass index 24 ± 1 kg/m2) participated in this study. All procedures used in this study were performed according to the Declaration of Helsinki and approved by the University of Nottingham Medical School Ethics Committee. Dual-energy x-ray absorptiometry was used to assess body composition and calculate lean body mass, which was used for correction of whole body responsiveness to insulin, as determined from the insulin clamps.

Experimental design

On two randomized occasions (2 wk apart), all subjects underwent a hyperinsulinemic (50 mU/m2·min) euglycemic (4.5 mmol/liter) clamp for 360 min with and without iv lipid (20% Intralipid at a rate of 90 ml/h) plus heparin (200 U prime + 600 U/h) infusion. Muscle biopsy samples were obtained before, and after 3- and 6-h infusion.

We confirmed that these rates of Intralipid plus heparin infusion do not prolong blood-clotting time, as measured by activated partial thromboplastin time (APTT) ratio. We also previously showed that these rates of insulin infusion totally suppress endogenous glucose production as determined by infusion of a glucose stable isotope (7). Furthermore, it was demonstrated previously that when lipid infusion was started at the beginning of the insulin clamp, there was no effect on the capacity of insulin (at levels comparable to the present study) to suppress endogenous glucose production (22).

Experimental protocol

On the day of each study, subjects arrived at the laboratory after an overnight fast (10–12 h) having abstained from caffeine on the day of the study, and from alcohol and heavy exercise for the previous 3 d. Sterile cannulae were inserted into an antecubital vein on one arm for glucose/insulin infusion and on the other arm for Intralipid/heparin or saline infusions (in randomized order), and into a dorsal vein on the nondominant hand for sampling, with the hand placed in a hot-air box maintained at 50–55 C to arterialize the blood. Local anesthetic (1% lidocaine) was injected sc before insertion of the cannulae, and a slow infusion of 0.9% sterile saline was used to keep the sampling line patent. After resting for 15 min, a baseline blood sample was obtained, followed by a muscle sample from the vastus lateralis of one leg.

After this (0 min), infusion of human soluble insulin (Actrapid; Novo Nordisk, Copenhagen, Denmark) commenced at a rate of 50 mU/m2·min, with 20% dextrose infused at a variable rate to maintain blood glucose concentrations. Monitoring of blood glucose was performed every 5 min to ensure it remained at normal fasting levels (4.5 mmol/liter). Subjects remained in a semi-recumbent position for the entire period of infusion (6 h). On one occasion, at 0 min, simultaneous iv infusion of Intralipid (Fresenius Kabi, Bad Hamburg, Germany) (20% at a rate of 90 ml/h) and heparin (given as a prime dose of 200 U, followed by a continuous infusion at 600 U/h) commenced and continued throughout the 6-h clamp (LIPID trial). On the other occasion, 0.9% sterile saline (at a rate of 90 ml/h) was infused instead of lipid/heparin (CON trial).

During each clamp, blood samples were obtained every 5 min for the determination of glucose and every 20 min for the determination of plasma FFA, serum insulin and glucagon. A ventilated canopy system linked to a metabolic cart (GEM; Nutren Technologies, Manchester, UK) was used to measure oxygen consumption and carbon dioxide production for 15 min immediately before and every hour during each clamp. Measurements were made while the subjects were lying supine, undisturbed, and awake. Second and third muscle samples were obtained after 3- and 6-h insulin infusion. All three muscle biopsies were sampled from the same leg. The opposite leg was used in the second trial. After the last muscle biopsy, the insulin and Intralipid/heparin infusions were discontinued, whereas the 20% dextrose infusion was continued to prevent symptoms of hypoglycemia. A carbohydrate-rich meal was then provided after which subjects were allowed to leave the laboratory.

Calculations

Glucose disposal (expressed as µmol of glucose per kg lean body mass per min) was calculated from the rate of glucose infusion averaged over 5-min periods as described by DeFronzo et al. (23). COX and fat oxidation (FOX) rates were calculated from the carbon dioxide and oxygen measurements as described previously (24).

Blood analysis

Blood samples were collected into chilled tubes (2 ml) containing 75 µl 200 mM EGTA and glutathione (for the determination of plasma FFA), or allowed to clot (3 ml) for 1 h (for the determination of serum insulin and glucagon). The aliquot used for the glucagon assay was treated with 100 µl aprotinin containing 1000 kallikrein inactivating units and stored in glass tubes at –80 C until analysis. Blood glucose concentrations were measured immediately after collection using a glucose oxidase method (Yellow Springs Instrument Analyzer, YSI, 2300 STAT PLUS; Yellow Springs, OH). Plasma and serum were separated by low-speed centrifugation (15 min at 3000 x g). Plasma was analyzed for FFA concentrations using a commercially available kit (NEFA-C test; Wako Chemicals, Neuss, Germany). Serum was analyzed for insulin and glucagon concentrations by RIAs (Diagnostics Products Corp., Llanberis, Wales, UK).

Muscle sampling and analysis

All muscle samples were obtained using the percutaneous needle biopsy technique with suction being applied. Five- to 10-mg frozen muscle was used to determine the active form of PDC (PDCa) (25). The time delay between sampling and freezing of the sample in liquid nitrogen (on average 10–12 sec) was unlikely to have resulted in significant changes in PDC activity because the values obtained in the present study were comparable to values previously reported by our laboratory in which samples were snap frozen 2–3 sec after removal from the limb (26, 27). LCACs and acetylcarnitine were determined enzymatically using RIAs (28).

RNA extraction and real-time PCR

Total RNA was extracted from 10- to 15-mg frozen muscle tissue by the method of Chomczynski and Sacchi (29) using TRIzol reagent (Invitrogen, Paisley, UK). Quantification of RNA and its RT was performed as described previously (13). TaqMan primers and probes were designed using Primer Express version 2.0 Software (Applied Biosystems, Warrington, UK). The sequences and PCR methodology were reported previously (13).

Protein extraction and Western blotting

Total protein extracts were prepared from 20- to 30-mg frozen biopsy tissue. Samples were first homogenized using a polytron homogenizer for 30 sec on ice in six volumes of buffer containing 50 mM HEPES, 1 mM EDTA, 10 mM NaF, 1 mM Na3VO4, 10% glycerol, 150 mM NaCl, 1% Triton X-100 (pH 7.5), and 4 µl protease inhibitors cocktail (P-8340; Sigma-Aldrich, St. Louis, MO) per ml of buffer. The homogenates were centrifuged at 10,000 x g for 20 min at 4 C, and the supernatants used for the determination of phospho-Akt serine473 and phospho-FOXO3 serine253 (Cell Signaling Technology, Inc., Danvers, MA) and {alpha}-actin (Sigma-Aldrich). Mitochondria were extracted from 20- to 40-mg fresh muscle tissue exactly as previously described (30) and used for the determination of PDK2 and PDK4 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and cytochrome C (BD Biosciences, Franklin Lakes, NJ). Protein concentrations of mitochondrial suspensions and whole tissue extracts were measured using the BCA method (Pierce, Perbio, Aalst, Belgium). Proteins were separated, blocked, and Western blotted as previously described (13). All immunoreactive proteins were visualized using ECL plus (Amersham Biosciences, Buckinghamshire, UK) and quantified by densitometry using the Quantity One 1-D Analysis Software version 4.5 (Bio-Rad Laboratories, Hercules, CA).

Statistical analysis

ANOVA for repeated measures across time was used to assess differences between trials. When a significant difference was obtained, the Holm-Bonferroni step-wise method was used to locate it. Student’s paired t tests were used for single comparisons. Statistical significance was accepted at a 5% level. Results are presented as means ± SEM.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Blood metabolites

At baseline, before infusions, plasma FFA (CON 0.46 ± 0.07 vs. LIPID 0.63 ± 0.11 mmol/liter), serum insulin (CON 8.7 ± 0.2 vs. LIPID 8.3 ± 0.2 mU/liter), blood glucose (CON 4.6 ± 0.1 vs. LIPID 4.6 ± 0.1 mmol/liter), and serum glucagon (CON 72.0 ± 6.1 vs. LIPID 67.7 ± 6.9 pg/ml) concentrations were not different between trials.

Plasma FFA concentrations were elevated throughout the LIPID trial (2.3 ± 0.3 mmol/liter; P < 0.01 from baseline at all time points) but were completely suppressed in the CON trial (0.2 ± 0.0 mmol/liter; P < 0.01 from baseline at all time points) (Fig. 1AGo). The plasma FFA levels in the LIPID trial were higher than those reported in previous studies using similar infusion rates, possibly due to differences in the lipase inhibitory properties of the substances used when collecting blood samples (1, 4). The average blood glucose (CON 4.5 ± 0.02 vs. LIPID 4.6 ± 0.02 mmol/liter) and serum insulin (CON 89.5 ± 2.7 vs. LIPID 96.5 ± 1.8 mU/liter; Fig. 1BGo) concentrations during the clamp were similar in the two trials. Serum glucagon concentrations declined steadily (P < 0.01) during the CON and LIPID clamps to values of 45.5 ± 2.7 and 43.1 ± 3.9 pg/ml at 6 h, respectively (not significant).


Figure 1
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  		FIG. 1. Plasma FFA (A) and serum insulin (B) concentrations during the CON and LIPID trials. Values are means ± SEM.

 
Whole body substrate oxidation rates and insulin sensitivity

Lipid infusion decreased glucose disposal (an index of insulin sensitivity) after 210 min (P < 0.05; Fig. 2AGo). During the last 30 min of the clamp, glucose disposal was approximately 50% lower in the LIPID compared with the CON trial (37.8 ± 4.4 vs. 79.6 ± 4.0 µmol/kg lean mass·min, respectively; P < 0.01).


Figure 2
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  		FIG. 2. Insulin-mediated whole-body glucose disposal (A), COX rates (B), and FOX rates (C) during the CON and LIPID trials. Values are means ± SEM. *, P < 0.05 and **, P < 0.01 when compared with the CON trial.

 
Baseline (noninsulin stimulated) rates of COX (Fig. 2BGo) and FOX (Fig. 2CGo) were not different between trials. When compared with CON, lipid infusion resulted in higher FOX after 90 min (P < 0.05) and lower COX after 150 min into the clamp (P < 0.05). From these time points onward, both FOX and COX continued to increase and decrease, respectively, until beginning to plateau at the end of the clamp at 360 min. At the end of the clamp, FOX was approximately 2.5-fold higher (LIPID 12.8 ± 0.7 vs. CON 4.9 ± 0.9 µmol/kg lean mass·min; P < 0.01), and COX was approximately 3-fold lower (LIPID 7.6 ± 1.6 vs. CON 23.4 ± 2.4 µmol/kg lean mass·min, respectively; P < 0.01) in the LIPID than the CON trial.

Skeletal muscle PDCa activity and PDK expression

There was no difference in baseline (noninsulin stimulated) muscle PDCa activity between treatments [CON 0.47 ± 0.08 vs. LIPID 0.55 ± 0.9 mmol/min·kg wet weight (ww); Fig. 3AGo]. Muscle PDCa activity increased in both trials (P < 0.01), but the insulin-mediated activation was blunted in the LIPID trial when compared with CON. As a result, PDCa activity was lower (P < 0.05) at the end of the LIPID trial when compared with CON (0.83 ± 0.12 vs. 1.50 ± 0.23 mmol/min·kg ww, respectively; Fig. 3AGo). Although PDCa activity was not different after 3-h infusion between trials, the change in activity from 0–3 h was lower in the LIPID than the CON trial (0.07 ± 0.03 vs. 0.29 ± 0.05 mmol/min·kg ww, respectively; P < 0.05).


Figure 3
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  		FIG. 3. Skeletal muscle PDCa activity (mmol/min·kg ww) (A) and acetylcarnitine concentrations (B) during the CON and LIPID trials. Values are means ± SEM. **, P < 0.01 from preclamp. §, P < 0.05 from CON. #, P < 0.01 from CON. Pre, Preclamp.

 
Muscle PDK4 mRNA content was down-regulated by 2-fold in response to insulin within 3 h in both clamps and decreased further (6-fold; P < 0.01) at 6 h in the CON but not the LIPID clamp (Fig. 4BGo). However, there was no effect of either treatment on protein expression of PDK4. Furthermore, there was no effect of insulin or lipid infusion on skeletal muscle PDK2 mRNA and protein content (data not shown).


Figure 4
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  		FIG. 4. Skeletal muscle LCAC concentration (A) and PDK4 mRNA expression (B) during the CON and LIPID trials. Values are means ± SEM. **, P < 0.01 from preclamp. *, P < 0.05 from preclamp. §, P < 0.05 from CON. #, P < 0.01 from CON. Pre, Preclamp.

 
Skeletal muscle LCAC and acetylcarnitine

Muscle LCAC concentration increased after 6-h but not 3-h lipid infusion (P < 0.01; Fig. 4AGo). Muscle acetylcarnitine concentration increased in the LIPID trial only (P < 0.01) and was higher after both 3 (P < 0.05) and 6 h (P < 0.01) when compared with CON (Fig. 3BGo).

Skeletal muscle insulin signaling proteins

In both trials, insulin infusion resulted in a 4-fold activation of muscle Akt at serine473 (P < 0.01; Fig. 5Go), which did not diminish between 3- and 6-h infusion. However, lipid infusion did not alter the insulin-stimulated phosphorylation of muscle Akt (Fig. 5Go) and had no effect on the phosphorylation state of FOXO3 at serine253 (data not shown).


Figure 5
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  		FIG. 5. Skeletal muscle Akt serine473 phosphorylation during the CON and LIPID trials. Two representative Western blots of pAkt and pFOXO3 protein are also shown. Values are means ± SEM. Pre, Preclamp.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The results from this study showed for the first time in healthy humans that: 1) insulin infusion rapidly regulates PDK4 mRNA content in human skeletal muscle; 2) increasing the circulating FFA levels through lipid infusion during a short-term physiological hyperinsulinemia leads to an attenuation in the insulin-mediated suppression of PDK4 gene expression in skeletal muscle; and 3) the lipid-induced effect on PDK4 gene expression is associated with accumulation of intracellular LCACs but is not dependent on the activation of the Akt/FOXO3 pathway, suggesting that im lipids may regulate PDK4 gene expression in human skeletal muscle under those conditions.

In accordance with previous studies (1, 2), in the present study, the increase in lipid availability resulted in a marked development of insulin resistance, as indicated by an approximate 50% reduction in insulin-stimulated glucose uptake, and a shift in substrate use from carbohydrate to fat. The lipid-induced decrease in COX (observed after 150 min into the clamp) preceded the decrease in glucose uptake (observed after 210 min). The lipid-induced increase in FOX was accompanied by an increase in muscle LCAC concentrations, an index of im lipid accumulation, indicating a progressive imbalance between fatty acid entry into the muscle cell and its oxidation. Lipid infusion also resulted in a progressive increase in muscle acetylcarnitine, an index of muscle acetyl-coenzyme A (CoA) accumulation, indicating an imbalance between lipid-derived acetyl-CoA production and its use by the tricarboxylic acid cycle. Because acetyl-CoA is an allosteric inhibitor of muscle PDC (31), this may explain the observation that insulin-mediated activation of muscle PDCa was blunted in the LIPID trial when compared with CON, leading to reduced muscle glucose oxidation in the former trial. Lipid infusion impaired the capacity of insulin to activate muscle PDC and decrease PDK4 gene expression in the absence of changes in PDK4 protein expression over the course of the present experiment. This observation suggests that lipid overload mediates both allosteric (via acetyl-CoA accumulation) and transcriptional modulations of muscle PDC activity (via changes in PDK4).

Increased circulating FFA levels were thought to be responsible for the up-regulation of skeletal muscle PDK4 induced by starvation and high dietary fat in our previous human studies (7, 13). Indeed, results from in vitro studies in Morris hepatoma cells and human myoblasts suggested that both palmitate and oleate can up-regulate PDK4 gene expression (32, 33). This is in agreement with a recent human study, which also observed an increase in skeletal muscle PDK4 gene expression after 4-h lipid infusion alone (18). Surprisingly, recent studies on rodents have challenged the notion that FFAs are important regulators of PDK4 expression in skeletal muscle (16, 17) because lipid infusion alone failed to increase PDK4 mRNA levels; as a matter of fact, a 25% reduction in PDK4 content was observed with lipid infusion (16). Together, the results from in vitro and human studies suggest that, in contrast to animal studies (16, 17), fatty acids per se can modulate the expression of PDK4 in human skeletal muscle, although the exact mechanism of their action is not clear.

Elevated lipid availability is associated with accumulation of im triglycerides and a variety of fatty acid metabolites, including LCACs, diacylglycerol, and ceramides (4, 6, 19, 20). In particular, LCACs can directly modulate the transcriptional activity of a number of nuclear receptors, such as hepatic nuclear factor-4{alpha} (34), peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) (35), and liver X receptor (36). Furthermore, activation of peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} and nuclear receptors such as PPAR {alpha} and {delta}, hepatic nuclear factor-4{alpha}, estrogen-related receptor {alpha} and {gamma}, and liver X receptor have induced PDK4 gene expression (9, 32, 37, 38, 39). Therefore, it is possible that LCACs regulate PDK4 gene transcription by activating one or more of these factors, in accordance with their role as modulators of gene transcription (21).

However, it cannot be excluded that a lipid-induced decrease in insulin sensitivity rather than lipid accumulation per se may be responsible for the attenuation in the insulin-induced suppression of PDK4 gene expression in the LIPID trial. The findings from this study showed that insulin infusion alone rapidly (within 3 h) down-regulates PDK4 mRNA content in human skeletal muscle. This finding is in agreement with results from in vitro studies that showed that insulin regulates PDK4 gene expression via a PI3K/Akt-mediated pathway (32, 40). Indeed, in human hepatoma cells, insulin suppressed PDK4 expression through Akt-mediated phosphorylation, and thus inactivation, of FOXO transcription factors (in particular FOXO3), which can bind directly to the promoter region of the PDK4 gene (40). In a recent animal study, the insulin’s ability to suppress PDK4 gene content in rat skeletal muscle was impaired by lipid infusion, and this was associated with impaired insulin-stimulated Akt and FOXO1 phosphorylation (16), leading the authors to conclude that insulin resistance per se may regulate PDK4 gene expression. In contrast, the present study showed that lipid infusion in healthy humans did not alter the insulin-stimulated phosphorylation of muscle Akt at serine473 and had no effect on the phosphorylation state of its downstream target FOXO3 at serine253. Although these results do not exclude the possibility that insulin infusion alone may down-regulate human skeletal muscle PDK4 expression through Akt activation, they appear to suggest that Akt/FOXO3-independent mechanisms are involved in lipid regulation of PDK4 mRNA expression under insulin-stimulated conditions.

In summary, the results from the present work support the notion that in healthy humans, the development of lipid-induced insulin resistance is associated with changes in gene expression of skeletal muscle PDK4. In contrast to results from animal studies, we showed that FFAs modulate PDK4 gene expression in human skeletal muscle in a manner independent from the Akt/FOXO3 pathway. In particular, it would appear that accumulation of im fatty acid metabolites plays an important role in the regulation of PDK4 gene expression in lipid-induced insulin-resistant states. Further research is required to elucidate the specific molecular pathway(s) underlying this response in human skeletal muscle.


   Footnotes
 
This work was funded by the Biotechnology and Biological Sciences Research Council of United Kingdom (Grant 42/D1563 and postgraduate studentship no. BBS/S/P/2003/10402 to K.T.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online July 24, 2007

Abbreviations: CoA, Coenzyme A; CON, control trial; COX, carbohydrate oxidation; FFA, free fatty acid; FOX, fat oxidation; LCAC, long-chain acyl-CoA; LIPID, Intralipid trial; PDC, pyruvate dehydrogenase complex; PDCa, active form of PDC; PDK, pyruvate dehydrogenase kinase; ww, wet weight.

Received May 18, 2007.

Accepted July 12, 2007.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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