Pyruvate Dehydrogenase Kinase 4 mRNA Research Articles

Induction of PDK4 in the heart muscle of JVS mice, an animal model of systemic carnitine deficiency, does not appear to reduce glucose utilization by the heart

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA has been reported as an up-regulated gene in the heart and skeletal muscle of carnitine-deficient juvenile visceral steatosis (JVS) mice under fed conditions. PDK4 plays an important role in the inhibition of glucose oxidation via the phosphorylation of pyruvate dehydrogenase complex (PDC). This study evaluated the meaning of increased PDK4 mRNA in glucose metabolism by investigating PDK4 protein levels, PDC activity and glucose uptake by the heart and skeletal muscle of JVS mice. PDK4 protein levels in the heart and skeletal muscle of fed JVS mice were increased in accordance with mRNA levels, and protein was enriched in the mitochondria. PDK4 protein was co-fractionated with PDC in sucrose density gradient centrifugation, like PDK2 protein; however, the activities of the pyruvate dehydrogenase complex (PDC) active form in the heart and skeletal muscle of fed JVS mice were similar to those in fed control mice. Fed JVS mice showed significantly higher glucose uptake in the heart and similar uptake in the skeletal muscle compared with fed control mice. Thus, in carnitine deficiency under fed conditions, glucose was preferentially utilized in the heart as an energy source despite increased PDK4 protein levels in the mitochondria. The preferred glucose utilization may be involved in developing cardiac hypertrophy from carnitine deficiency in fatty acid oxidation abnormality.

Mechanistic population modeling of diabetes disease progression in Goto‐Kakizaki rat muscle

Pyruvate dehydrogenase kinase 4 (PDK4) is a lipid status responsive gene involved in muscle fuel selection. Evidence is mounting in support of the therapeutic potential of PDK4 inhibitors to treat diabetes. Factors that regulate PDK4 mRNA expression include plasma corticosterone, insulin and free fatty acids. The objective was to determine the impact of those plasma factors on PDK4 mRNA and to develop and validate a population mathematical model to differentiate aging, diet and disease effects on muscle PDK4 expression. The Goto-Kakizaki (GK) rat, a polygenic non-obese model of type 2 diabetes, was used as the diabetic animal model. Muscle PDK4 mRNA expression was examined by real-time QRTPCR. Groups of GK rats along with controls fed with either a normal or high fat diet were killed at 4, 8, 12, 16 and 20 weeks of age. Plasma corticosterone, insulin and free fatty acids were measured. The proposed mechanism-based model successfully described the age, disease and diet effects and the relative contribution of these plasma regulators on PDK4 mRNA expression. Muscle growth reduced the PDK4 mRNA production rate by 14% per gram increase. The high fat diet increased the initial production rate constant in GK rats by 2.19-fold. The model indicated that corticosterone had a moderate effect and PDK4 was more sensitive to free fatty acid than insulin fluxes, which was in good agreement with the literature data.

Epinephrine-mediated regulation of PDK4 mRNA in rat adipose tissue

Fatty acid reesterification in adipose tissue is dependent on the generation of glycerol 3-phosphate, and, at least in rodent adipose tissue, this appears to occur primarily through glyceroneogenesis. A key enzyme in this process is pyruvate dehydrogenase kinase 4 (PDK4). PDK4 is induced in white adipose tissue by thiazolidinediones (TZDs) and the inhibition or knockdown of PDK4 inhibits TZD-induced increases in glyceroneogenesis. Since TZDs have many unwanted side effects, we were interested in identifying alternative mechanisms that could regulate PDK4 mRNA expression in white adipose tissue. In this regard we hypothesized that exercise, fasting, and epinephrine would increase PDK4 mRNA levels in rat epididymal adipose tissue. We further postulated that the p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) signaling pathways would control PDK4 mRNA expression in cultured adipose tissue. Exercise, fasting, and in or ex vivo epinephrine treatment increased PDK4 mRNA levels. These perturbations did not increase the expression of PDK1, -2, or -3. Pyruvate dehydrogenase phosphorylation was increased after an overnight fast and 4 h after the cessation of exercise. In cultured adipose tissue, epinephrine increased p38 and AMPK signaling; however, the direct activation of AMPK by AICAR or metformin led to reductions in PDK4 mRNA levels. The p38 inhibitor SB202190 reduced epinephrine-mediated increases in p38 MAPK activation without altering hormone-sensitive lipase or AMPK phosphorylation or attenuating epinephrine-induced increases in lipolysis. Reductions in p38 MAPK signaling were associated with decreases in PDK4 mRNA expression. The inhibition of peroxisome proliferator-activated receptor-γ (PPARγ) also attenuated the induction of PDK4. Our results are the very first to demonstrate an epinephrine-mediated regulation of PDK4 mRNA levels in white adipose tissue and suggest that p38 MAPK and PPARγ could be involved in this pathway.

Oxidative Gene Expression in Single Muscle Fibres after Continuous and Intermittent Exercise

Fibre‐type heterogeneity is a potential problem in the interpretation of gene expression in mixed muscle. The current study aimed to investigate the fibre‐type variation of master genes involved in oxidative metabolism and signaling pathway as well as the influence of exercise. Untrained subjects (n = 8) cycled for 90 min either at a constant 60 % of VO2max (CE) or at an intensity alternating between 120 % (12 s) and 20 % of VO2max (18 s) (IE). Muscle biopsies were obtained before (Pre) and 3 h after (Post) exercise. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) and pyruvate dehydrogenase kinase 4 (PDK4) genes were measured with quantitative real‐time PCR in freeze‐dried muscle single type I or type II fibres. PGC‐1α mRNA showed large variation within the same fibre type (> 3‐fold). PGC‐1α and PDK4 mRNA was 2–3‐fold higher in type I fibres (P < 0.05 vs. type II) at Pre, but at Post the difference was reduced (IE) or abolished (CE). Acute cycling increased PGC‐1α and PDK4 mRNA in both fibre types. Exercise‐induced increases in mRNA of PGC‐1α and PDK4 were negatively correlated with the basal level. We conclude that human skeletal muscle has higher baseline mRNA level of oxidative genes in type I than in type II fibres, but this difference is reduced after exercise. This may in part be explained by the negative correlation between the basal mRNA level and the exercise‐induced increase in mRNA. This work was supported by grants from the Swedish Research Council (23998‐50466), GIH and the National Centre for Research in Sports (CIF).

PDC deletion: the way to a man's heart disease

the heart has continuous high-energy demands required to sustain efficient contraction. This is met by the metabolism of major circulating substrates (e.g., glucose, lactate, or lipids), according to availability, since the heart has a limited capacity for nutrient storage (reviewed in Ref. [16][1

Interaction between dietary fructose and protein: marginal protein deficiency induces pyruvate dehydrogenase kinase 4 (PDK4) and glucose transporter 4 (GLUT4) genes in rat muscle

In animal models, marginally protein deficient diets increase adiposity, while high fructose diets cause hyperlipidemia and hypertension. The purpose of this study was to evaluate the interactive effects of dietary protein and fructose on metabolic syndrome. Male Sprague‐Dawley rats (n=24) were fed one of 6 diets varying in macronutrients for 30 days. Diets were based on a 2x3 factorial with 2 levels of protein (10% (P10) and 20% of energy), and 3 levels of fructose (0% (F0), 20%, and 40% of energy, replacing glucose). Baseline and final body composition were measured by dual energy X‐ray absorptiometry. Final blood pressure was measured by tail cuff. While fructose tended to increase blood pressure (p=0.06), it was P10‐F0 that had the highest percent body fat (p<0.005) and cumulative food intake (p<0.05). In P10 compared to P20 groups, lean body mass was lower (p=0.008), while muscle PDK4 mRNA was higher (p=0.04), suggesting decreased muscle protein synthesis accompanied by decreased glucose oxidation. Unexpectedly, muscle GLUT4 mRNA was higher in P10 groups (p=0.01), whereas there was no difference in plasma insulin between any groups. In conclusion, 1) no significant interaction between dietary fructose and protein was observed, and 2) marginal protein deficiency may shift glucose metabolism toward anaerobic glycolysis.

Transcriptional Regulation of Metabolic Switching PDK4 Gene under Various Physiological Conditions

Pyruvate dehydrogenase kinase 4 (PDK4) phosphorylates and inactivates the pyruvate dehydrogenase complex to respond to physiologic conditions. This response switches the energy source from glucose to fatty acids to maintain blood glucose levels. Transcription of the PDK4 gene is activated by fasting or by the administration of a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand in a tissue-specific manner. However, the two mechanisms to induce PDK4 mRNA as well as the relationship between the two have not been studied in detail. In this study, we show that the two mechanisms are independent, at least in the mouse skeletal muscle, and that estrogen-related receptor alpha (ERRalpha) is directly involved in the PPARalpha-independent transcriptional activation of the PDK4 gene with peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) as a specific partner. The latter conclusion is based on the following evidence: 1) Deletion and point mutation analyses of the cloned mouse PDK4 gene promoter sequence identified an exact possible ERRalpha-binding motif as the PGC-1alpha responsive element. 2) The overexpression of ERRalpha by cotransfection enhanced, and the knocking down of it by specific shRNAs diminished, the PGC-1alpha-dependent activation. 3) Specific binding of ERRalpha to the identified PGC-1alpha-responsive sequence of the mouse PDK4 promoter was confirmed in the electrophoresis mobility shift assay using anti-ERRalpha antibodies. These results suggest that PGC-1alpha plays an essential role not only in regulating the amounts of energy creating enzymes, but also at the step of metabolic switching with unevenly distributed tissue transcription factors such as ERRalpha in the skeletal muscle, thus harmonizing tissue-specific functions and energy metabolism.

Insulin, but not lipid availability, regulates calpain‐10 at the transcriptional level in human skeletal muscle

We investigated the molecular alterations underlying the reduction in carbohydrate (CHO) utilization with increased lipid availability in human skeletal muscle. On 2 occasions, 10 healthy young men [age 22 ± 1 (mean ± SEM), BMI 24 ± 1] underwent hyperinsulinemic (50 mU/m2/min) euglycemic (4.5 mM) clamps for 6h with (LIPID) and without (CON) intravenous intralipid (20% at a rate of 90 ml/h) plus heparin (200 U prime + 600 U/h) infusion. CHO and fat oxidation rates were determined by indirect calorimetry. Muscle biopsies were obtained before and after 3 and 6h of infusion for the determination of hexokinase II (HKII), pyruvate dehydrogenase kinase 4 (PDK4) and the diabetes linked gene calpain-10 (CAPN10) mRNA levels by real-time PCR. Lipid infusion increased fat oxidation after 90 min (P<0.05) and decreased CHO oxidation after 150 min (P<0.05) and glucose disposal after 210 min (P<0.05). At the end of the clamp, CHO disposal was ~50% lower in the LIPID compared to the CON trial (14.5 ± 0.7 vs 6.9 ± 0.8 mg/min/kg lean mass, P<0.001). CAPN10 mRNA levels decreased with insulin in both clamps (P<0.01) and were not affected by lipid infusion. HKII mRNA was upregulated 2-fold (P<0.01) in the CON trial, but this was impaired in the LIPID trial. PDK4 mRNA content was downregulated 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. These results show that lipid-induced insulin resistance is associated with significant changes in genes regulating CHO utilisation. In contrast, it appears that insulin, and not lipid, regulates CAPN10 at the transcriptional level.

PGC-1α Coactivates PDK4 Gene Expression via the Orphan Nuclear Receptor ERRα: a Mechanism for Transcriptional Control of Muscle Glucose Metabolism

The transcriptional coactivator PGC-1alpha is a key regulator of energy metabolism, yet little is known about its role in control of substrate selection. We found that physiological stimuli known to induce PGC-1alpha expression in skeletal muscle coordinately upregulate the expression of pyruvate dehydrogenase kinase 4 (PDK4), a negative regulator of glucose oxidation. Forced expression of PGC-1alpha in C(2)C(12) myotubes induced PDK4 mRNA and protein expression. PGC-1alpha-mediated activation of PDK4 expression was shown to occur at the transcriptional level and was mapped to a putative nuclear receptor binding site. Gel shift assays demonstrated that the PGC-1alpha-responsive element bound the estrogen-related receptor alpha (ERRalpha), a recently identified component of the PGC-1alpha signaling pathway. In addition, PGC-1alpha was shown to activate ERRalpha expression. Chromatin immunoprecipitation assays confirmed that PGC-1alpha and ERRalpha occupied the mPDK4 promoter in C(2)C(12) myotubes. Additionally, transfection studies using ERRalpha-null primary fibroblasts demonstrated that ERRalpha is required for PGC-1alpha-mediated activation of the mPDK4 promoter. As predicted by the effects of PGC-1alpha on PDK4 gene transcription, overexpression of PGC-1alpha in C(2)C(12) myotubes decreased glucose oxidation rates. These results identify the PDK4 gene as a new PGC-1alpha/ERRalpha target and suggest a mechanism whereby PGC-1alpha exerts reciprocal inhibitory influences on glucose catabolism while increasing alternate mitochondrial oxidative pathways in skeletal muscle.

Effect of carbohydrate ingestion on exercise-induced alterations in metabolic gene expression

Skeletal muscle possesses a high degree of plasticity and can adapt to both the physical and metabolic challenges that it faces. An acute bout of exercise is sufficient to induce the expression of a variety of metabolic genes, such as GLUT4, pyruvate dehydrogenase kinase 4 (PDK-4), uncoupling protein-3 (UCP3), and peroxisome proliferator-activated receptor-gamma coactivator 1 (PGC-1). Reducing muscle glycogen levels before exercise potentiates the effect of exercise on many genes. Similarly, altered substrate availability induces transcription of many of these genes. The purpose of this study was to determine whether glucose ingestion attenuates the exercise-induced increase in a variety of exercise-responsive genes. Six male subjects (28 +/- 7 yr; 83 +/- 3 kg; peak pulmonary oxygen uptake = 46 +/- 6 ml.kg(-1).min(-1)) performed 60 min of cycling at 74 +/- 2% of peak pulmonary oxygen uptake on two separate occasions. On one occasion, subjects ingested a 6% carbohydrate drink. On the other occasion, subjects ingested an equal volume of a sweet placebo. Muscle samples were obtained from vastus lateralis at rest, immediately after exercise, and 3 h after exercise. PDK-4, UCP3, PGC-1, and GLUT4 mRNA levels were measured on these samples using real-time RT-PCR. Glucose ingestion attenuated (P < 0.05) the exercise-induced increase in PDK-4 and UCP3 mRNA. A similar trend (P = 0.09) was observed for GLUT4 mRNA. In contrast, PGC-1 mRNA increased following exercise to the same extent in both conditions. These data suggest that glucose availability can modulate the effect of exercise on metabolic gene expression.

Protein Kinase B-α Inhibits Human Pyruvate Dehydrogenase Kinase-4 Gene Induction by Dexamethasone Through Inactivation of FOXO Transcription Factors

Starvation and diabetes increase pyruvate dehydrogenase kinase-4 (PDK4) expression, which conserves gluconeogenic substrates by inactivating the pyruvate dehydrogenase complex. Mechanisms that regulate PDK4 gene expression, previously established to be increased by glucocorticoids and decreased by insulin, were studied. Treatment of HepG2 cells with dexamethasone increases the relative abundance of PDK4 mRNA, and insulin blocks this effect. Dexamethasone also increases human PDK4 (hPDK4) promoter activity in HepG2 cells, and insulin partially inhibits this effect. Expression of constitutively active PKB alpha abrogates dexamethasone stimulation of hPDK4 promoter activity, while coexpression of constitutively active FOXO1a or FOXO3a, which are mutated to alanine at the three phosphorylation sites for protein kinase B (PKB), disrupts the ability of PKB alpha to inhibit promoter activity. A glucocorticoid response element for glucocorticoid receptor (GR) binding and three insulin response sequences (IRSs) that bind FOXO1a and FOXO3a are identified in the hPDK4 promoter. Mutation of the IRSs reduces the ability of glucocorticoids to stimulate PDK4 transcription. Transfection studies with E1A, which binds to and inactivates p300/CBP, suggest that interactions between p300/CBP and GR as well as FOXO factors are important for glucocorticoid-stimulated hPDK4 expression. Insulin suppresses the hPDK4 induction by glucocorticoids through inactivation of the FOXO factors.

Impaired expression of NADH dehydrogenase subunit 1 and PPARγ coactivator-1 in skeletal muscle of ZDF rats: restoration by troglitazone

Type 2 diabetes has been related to a decrease of mitochondrial DNA (mtDNA) content. In this study, we show increased expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) and its target genes involved in fatty acid metabolism in skeletal muscle of Zucker Diabetic Fatty (ZDF) (fa/fa) rats. In contrast, the mRNA levels of genes involved in glucose transport and utilization (GLUT4 and phosphofructokinase) were decreased, whereas the expression of pyruvate dehydrogenase kinase 4 (PDK-4), which suppresses glucose oxidation, was increased. The shift from glucose to fatty acids as the source of energy in skeletal muscle of ZDF rats was accompanied by a reduction of subunit 1 of complex I (NADH dehydrogenase subunit 1, ND1) and subunit II of complex IV (cytochrome c oxidase II, COII), two genes of the electronic transport chain encoded by mtDNA. The transcript levels of PPARgamma Coactivator 1 (PGC-1) showed a significant reduction. Treatment with troglitazone (30 mg/kg/day) for 15 days reduced insulin values and reversed the increase in PDK-4 mRNA levels, suggesting improved insulin sensitivity. In addition, troglitazone treatment restored ND1 and PGC-1 expression in skeletal muscle. These results suggest that troglitazone may avoid mitochondrial metabolic derangement during the development of diabetes mellitus 2 in skeletal muscle.

Fibrates and statins rapidly and synergistically induce pyruvate dehydrogenase kinase 4 mRNA in the liver and muscles of mice.

We previously identified pyruvate dehydrogenase kinase 4 (PDK4) mRNA as a most rapidly induced mRNA by fibrates and suggested the possibility that the coupled induction of PDK4 and reduction of serum triglyceride and fatty acid levels can cause protein degradation in muscles. To investigate whether the drugs that are known to have a risk of rhabdomyolysis induce PDK4 mRNA in skeletal muscle, the effects of statins and new quinolon anti-bacterial drugs on the expression levels of the mRNA were examined using mice and cultured cells. Several statins and new quinolon anti-bacterial drugs solely induced PDK4 mRNA in the muscle as efficiently as fibrates and at least some combinations were synergistic. The present results suggest that induction of PDK4 mRNA is involved in the drug induced acute rhabdomyolysis when the muscle is restricted to use fatty acids as a major energy source.

A metabolic switching hypothesis for the first step in the hypolipidemic effects of fibrates.

Pyruvate dehydrogenase kinase 4 (PDK4) mRNA was identified as a transcript that was rapidly and generally induced by fibrates in various tissues of the mouse. The time course of induction was much faster than those of typical peroxisome proliferator-activated receptor alpha (PPAR alpha)-regulated peroxisomal mRNAs. Rapid and efficient induction of PDK4 mRNA in various tissues would inactivate pyruvate dehydrogenase and stimulate fatty acid oxidation at the whole body level, leading to enhanced utilization of serum fatty acids and triglycerides. This metabolic switching mechanism can explain the early phase of the fibrate-induced decrease in the serum levels of triglycerides. Furthermore, the muscle in the fibrate-induced early state with limited activity of pyruvate dehydrogenase and low levels of fatty acids will utilize proteins as an energy source and extensive degradation of proteins may lead to myopathy or rhabdomyolysis.

Hypoxia in Vivo Decreases Peroxisome Proliferator-Activated Receptor α-Regulated Gene Expression in Rat Heart

We tested the hypothesis that hypoxia decreases PPARα-regulated gene expression in heart muscle in vivo. In two rat models of systemic hypoxia (cobalt chloride treatment and iso-volemic hemodilution), transcript levels of PPARα and PPARα-regulated genes (pyruvate dehydrogenase kinase 4 (PDK4), muscle carnitine palmitoyltransferase-I (mCPT-I), and malonyl-CoA decarboxylase (MCD)) were measured using real-time quantitative RT-PCR. Data were normalized to the housekeeping gene β-actin. Atrial natriuretic factor (ANF) and pyruvate dehydrogenase kinase 2 (PDK2), which are not regulated by PPARα, served as controls. CoCl2 treatment decreased PPARα, PDK4, mCPT-I, and MCD mRNA levels. Iso-volemic anemia also caused a significant decrease in PPARα, PDK4, and MCD mRNA levels. Transcript levels of mCPT-I showed a slight, but not significant decrease (P = 0.08). Gene expression of β-actin, ANF, and PDK2 did not change with either CoCl2 treatment nor with anemia. Myocardial PPARα-regulated gene expression is decreased in two models of hypoxia in vivo. These results suggest a transcriptional mechanism for decreased fatty oxidation and increased reliance of the heart for glucose during hypoxia.

Pyruvate dehydrogenase kinase 4 mRNA is increased in the hypertrophied ventricles of carnitine-deficient juvenile visceral steatosis (JVS) mice.

We isolated a mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up-regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency. The PDK4 mRNA level was 5 times higher in JVS mice than in control mice under fed conditions. After 24 h starvation, this level increased to 20 times in JVS and 7 times in control, compared with the control fed level. On the other hand, carnitine administration reduced the high level of PDK4 mRNA in JVS mice to the control fed level. In control mice, the change in PDK4 mRNA was inversely correlated with the change in PDH activity. In JVS mice, however, the PDK4 mRNA level was not always correlated with the active-form PDH level.

Protective effects of C2-ceramide against glutamate-induced neurotoxicity in cultured cortical neurons.

Pyruvate dehydrogenase kinase 4 (PDK4), a mammalian mitochondrial serine kinase has emerged as an interesting candidate for diabetes therapy. Due to the high prevalence of this disease especially type 2 diabetes (T2D) and the health complications associated with it, there is extensive effort to find the appropriate treatment. Understanding the regulation of PDK4 activity would therefore contribute significantly to the development of therapeutic agents. This research outlines the utilization of bioinformatics tools such as Interweaver, ClustalW and Protein Structure Visualizer, in order to predict proteins that potentially interact with PDK4 and possibly regulate its activity. Interweaver database identified 96 proteins that have possible interaction sites for PDK4. Protein p100/p49, containing a death domain that is known to have a role in suppressing apoptosis, was identified as a potential partner for PDK4. The alignment between p100/p49 primary sequence and that of PDK4 using ClustalW demonstrated sequence similarity between the two proteins. Swiss PDB Viewer then located the positions of the amino acids that are in the hypothetical protein binding motif of p100/p49 within the 3D structure of hPDK4. These amino acids were found to be located in the region of PDK4 which is known to bind protein substrates of PDK4 and may be accessible to other proteins as well. These findings were very interesting as PDK4 has not previously been associated with apoptosis and this could be the link between apoptosis and insulin resistance. Cell biology studies were then performed to verify the relationship between PDK4 and apoptosis. In this regard, HeLa and HepG2 cells were treated with apoptosis-inducing agents such as TNFα, C2-ceramide, and linoleic acid. These cells were then monitored for apoptosis and PDK4 mRNA expression using a DNA laddering assay as well as Real Time PCR. The results showed that these factors induced apoptosis in a concentration dependent manner and suppressed PDK4 mRNA levels. These findings suggested a relationship between PDK4 and apoptosis.

Mechanisms of dopamine D2-induced neuroprotection against glutamate neurotoxicity in rat mesencephalon

Pyruvate dehydrogenase kinase 4 (PDK4), a mammalian mitochondrial serine kinase has emerged as an interesting candidate for diabetes therapy. Due to the high prevalence of this disease especially type 2 diabetes (T2D) and the health complications associated with it, there is extensive effort to find the appropriate treatment. Understanding the regulation of PDK4 activity would therefore contribute significantly to the development of therapeutic agents. This research outlines the utilization of bioinformatics tools such as Interweaver, ClustalW and Protein Structure Visualizer, in order to predict proteins that potentially interact with PDK4 and possibly regulate its activity. Interweaver database identified 96 proteins that have possible interaction sites for PDK4. Protein p100/p49, containing a death domain that is known to have a role in suppressing apoptosis, was identified as a potential partner for PDK4. The alignment between p100/p49 primary sequence and that of PDK4 using ClustalW demonstrated sequence similarity between the two proteins. Swiss PDB Viewer then located the positions of the amino acids that are in the hypothetical protein binding motif of p100/p49 within the 3D structure of hPDK4. These amino acids were found to be located in the region of PDK4 which is known to bind protein substrates of PDK4 and may be accessible to other proteins as well. These findings were very interesting as PDK4 has not previously been associated with apoptosis and this could be the link between apoptosis and insulin resistance. Cell biology studies were then performed to verify the relationship between PDK4 and apoptosis. In this regard, HeLa and HepG2 cells were treated with apoptosis-inducing agents such as TNFα, C2-ceramide, and linoleic acid. These cells were then monitored for apoptosis and PDK4 mRNA expression using a DNA laddering assay as well as Real Time PCR. The results showed that these factors induced apoptosis in a concentration dependent manner and suppressed PDK4 mRNA levels. These findings suggested a relationship between PDK4 and apoptosis.