Effect of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C_T) values were established for ß-actin, ß2-microglobulin (ß₂M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C_T values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C_T values and the linear value of 2^-C_T, respectively. Interassay variability was 2.3% for raw C_T values and 34% for the linear value of 2^-C_T. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C_T values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas ß-actin, ß₂M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for ß₂M with more stable expressions for both ß-actin and CYC. We conclude that, using real-time RT-PCR, ß-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

Effect of elevated lipid concentrations on human skeletal muscle gene expression

Dietary fatty acids regulate the abundance and activity of various proteins involved in the regulation of fat oxidation by functioning as regulators of gene transcription. To determine whether the transcription of key lipid metabolic proteins necessary for fat metabolism within human skeletal muscle are regulated by acute elevations in circulating free fatty acid (FFA) concentrations, 7 healthy men underwent 3 randomized resting infusions of Intralipid (20%) with heparin sodium, saline and heparin sodium, or saline only for 5 hours. These infusions significantly elevated plasma FFA concentrations by 15-fold (to 1.67 ± 0.13 mmol/L) in the Intralipid infusion trial, with modest elevations observed in the saline and heparin sodium and saline alone infusion groups (0.67 ± 0.09 and 0.49 ± 0.087 mmol/L, P < .01 both vs Intralipid infusion). Analysis of messenger RNA (mRNA) concentration demonstrated that pyruvate dehydrogenase kinase isoform 4 (PDK4) mRNA, a key negative regulator of glucose oxidation, was increased in all trials with a 24-fold response after Intralipid infusion, 15-fold after saline and heparin infusion, and 9-fold after saline alone. The PDK4 increases were not significantly different between the 3 trials. The mRNA concentration of the major uncoupling protein within skeletal muscle, uncoupling protein 3, was not elevated in parallel to the increased plasma FFA as similar (not, vert, similar2-fold) increases were evident in all trials. Additional genes involved in lipid transport (fatty acid translocase/CD36), oxidation (carnitine palmitoyltransferase I), and metabolism (1-acylglycerol-3-phosphate O-acyltransferase 1, hormone-sensitive lipase, and peroxisomal proliferator-activated receptor-γ coactivator-1α) were not altered by increased circulating FFA concentrations. The present data demonstrate that of the genes analyzed that encode proteins that are key regulators of lipid homeostasis within skeletal muscle, only the PDK4 gene is uniquely sensitive to increasing FFA concentrations after increased plasma FFA achieved by intravenous lipid infusion.

Glucose ingestion during exercise blunts exercise-induced gene expression of skeletal muscle fat oxidative genes.

Ingestion of carbohydrate during exercise may blunt the stimulation of fat oxidative pathways by raising plasma insulin and glucose concentrations and lowering plasma free fatty acid (FFA) levels, thereby causing a marked shift in substrate oxidation. We investigated the effects of a single 2-h bout of moderate-intensity exercise on the expression of key genes involved in fat and carbohydrate metabolism with or without glucose ingestion in seven healthy untrained men (22.7 ± 0.6 yr; body mass index: 23.8 ± 1.0 kg/m²; maximal O2 consumption: 3.85 ± 0.21 l/min). Plasma FFA concentration increased during exercise (P < 0.01) in the fasted state but remained unchanged after glucose ingestion, whereas fat oxidation (indirect calorimetry) was higher in the fasted state vs. glucose feeding (P < 0.05). Except for a significant decrease in the expression of pyruvate dehydrogenase kinase-4 (P < 0.05), glucose ingestion during exercise produced minimal effects on the expression of genes involved in carbohydrate utilization. However, glucose ingestion resulted in a decrease in the expression of genes involved in fatty acid transport and oxidation (CD36, carnitine palmitoyltransferase-1, uncoupling protein 3, and 5'-AMP-activated protein kinase-α2; P < 0.05). In conclusion, glucose ingestion during exercise decreases the expression of genes involved in lipid metabolism rather than increasing genes involved in carbohydrate metabolism.

Decreased expression of adipogenic genes in obese subjects with type 2 diabetes

Objective: Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.

Research Methods and Procedures: We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [ CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPdelta, GATA domain-binding protein 3 (GATA3), C/EBPbeta, peroxisome proliferator-activated receptor (PPAR) gamma2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor alpha, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARitalic gamma coactivator 1alpha, PPARitalic gamma coactivator 1beta, and PPARdelta] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.

Results: Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 plusminus 1.9 vs. 7.5 plusminus 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 plusminus 0.26 vs. 0.78 plusminus 0.17 mum; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARitalic gamma2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPbeta only tended to be lower (p = 0.07).

Discussion: This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.

Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1α and PPARß/δ gene expression upon rosiglitazone treatment in obese patients with type 2 diabetes mellitus

Objective: To examine whether rosiglitazone alters gene expression of some key genes involved in mitochondrial biogenesis and oxidative capacity in skeletal muscle of type 2 diabetic patients, and whether this is associated with alterations in skeletal muscle oxidative capacity and lipid content.

Design: Skeletal muscle gene expression, mitochondrial protein content, oxidative capacity and lipid accumulation were measured in muscle biopsies obtained from diabetic patients, before and after 8 weeks of rosiglitazone treatment, and matched controls. Furthermore, whole-body insulin sensitivity and substrate utilization were assessed.

Subjects: Ten obese type 2 diabetic patients and 10 obese normoglycemic controls matched for age and BMI.

Methods: Gene expression and mitochondrial protein content of complexes I–V of the respiratory chain were measured by quantitative polymerase chain reaction and Western blotting, respectively. Histochemical staining was used to quantify lipid accumulation and complex II succinate dehydrogenase (SDH) activity. Insulin sensitivity and substrate utilization were measured during a hyperinsulinemic–euglycemic clamp with indirect calorimetry.

Results: Skeletal-muscle mRNA of PGC-1a and PPARb/d – but not of other genes involved in glucose, fat and oxidative metabolism – was significantly lower in diabetic patients (Po0.01). Rosiglitazone significantly increased PGC-1a (B2.2-fold, Po0.01) and PPARb/d (B2.6-fold, Po0.01), in parallel with an increase in insulin sensitivity, SDH activity and metabolic flexibility (Po0.01). Surprisingly, none of the measured mitochondrial proteins was reduced in type 2 diabetic patients, nor affected by rosiglitazone treatment. No alterations were seen in muscular fat accumulation upon treatment.

Conclusion: These results suggest that the insulin-sensitizing effect of rosiglitazone may involve an effect on muscular oxidative capacity, via PGC-1a and PPARb/d, independent of mitochondrial protein content and/or changes in intramyocellular lipid.

Effects of cannabinoid receptors on skeletal muscle oxidative pathways

The endocannabinoids, a recently discovered endogenous, lipid derived, signaling system regulating energy metabolism, have effects on central and peripheral energy metabolism predominantly via the cannabinoid receptor type 1 (CB1). CB1 is expressed centrally in the hypothalamus and nucleus accumbens and peripherally in adipocytes and skeletal muscle. This study determined the effect of endocannabinoids on the expression of genes regulating energy metabolism in human skeletal muscle. Primary cultures of myotubes (lean and obese; n = 3/group) were treated with the cannabinoid receptor agonist, anandamide (AEA) (0.2 and 5 μM) and the CB1 specific antagonist AM251 (0.2 and 5 μM) separately and in combination for 24 h. The expression of mRNA for AMP-activated protein kinase (AMPK) alpha 1 (α1) and alpha 2 (α2), pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) were determined using ‘Real Time’ RT-PCR. AMPKα1 mRNA increased in lean and obese myotubes in response to AM251 (P < 0.05). AEA inhibited the effect of AM251 on AMPKα1 mRNA levels in myotubes from lean and obese subjects (P < 0.05); the dose–response curve was shifted to the left in the obese. In response to AM251, irrespective of the presence of AEA, PDK4 expression was decreased in lean and obese myotubes (P < 0.05). Taken together these data suggest that endocannabinoids regulate pathways affecting skeletal muscle oxidation, effects particularly evident in myotubes from obese individuals.

No effect of mild heat stress on the regulation of carbohydrate metabolism at the onset of exercise

To investigate the influence of heat stress on the regulation of skeletal muscle carbohydrate metabolism, six active, but not specifically trained, men performed 5 min of cycling at a power output eliciting 70% maximal O(2) uptake in either 20 degrees C (Con) or 40 degrees C (Heat) after 20 min of passive exposure to either environmental condition. Although muscle temperature (T(mu)) was similar at rest when comparing trials, 20 min of passive exposure and 5 min of exercise increased (P < 0.05) T(mu) in Heat compared with Con (37.5 +/- 0.1 vs. 36.9 +/- 0.1 degrees C at 5 min for Heat and Con, respectively). Rectal temperature and plasma epinephrine were not different at rest, preexercise, or 5 min of exercise between trials. Although intramuscular glycogen phosphorylase and pyruvate dehydrogenase activity increased (P < 0.05) at the onset of exercise, there were no differences in the activities of these regulatory enzymes when comparing Heat with Con. Accordingly, glycogen use in the first 5 min of exercise was not different when comparing Heat with Con. Similarly, no differences in intramuscular concentrations of glucose 6-phosphate, lactate, pyruvate, acetyl-CoA, creatine, phosphocreatine, or ATP were observed at any time point when comparing Heat with Con. These results demonstrate that, whereas mild heat stress results in a small difference in contracting T(mu), it does not alter the activities of the key regulatory enzymes for carbohydrate metabolism or glycogen use at the onset of exercise, when plasma epinephrine levels are unaltered.

Carbohydrate ingestion reduces skeletal muscle acetylcarnitine availability but has no effect on substrate phosphorylation at the onset of exercise in man

This study investigated the effect of reduced acetylcarnitine availability on oxidative metabolism during the transition from rest to steady-state exercise. Eight male subjects completed two randomised exercise trials at 68 % of the peak rate of O2 uptake (V̇O2,peak). On one occasion subjects ingested 1 g (kg body mass)−1 glucose 75 min prior to exercise (CHO), whereas the other trial acted as a control (CON). Muscle samples were obtained pre- and 75 min post-ingestion, and following 1 and 10 min of exercise. Plasma glucose and insulin were elevated (P < 0.05), and plasma free fatty acids (FFA) were lower at the onset of exercise in CHO. Acetylcarnitine (CON, 4.8 ± 1.8; CHO, 1.5 ± 0.9 mmol (kg dry mass (d.m.))−1, P < 0.05) and acetyl CoA (CON, 13.2 ± 2.3; CHO, 6.3 ± 0.6 μmol (kg d.m.)−1, P < 0.05) were lower at rest, whereas pyruvate dehydrogenase activation (PDHa) was greater in CHO compared with CON (CON, 0.78 ± 0.07; CHO, 1.44 ± 0.19 mmol min−1 (kg wet mass (w.m.))−1). Respiratory exchange ratio (RER) was significantly elevated during exercise in CHO. The acetyl groups increased at similar rates at the onset of exercise (1 min) and there was no difference in substrate phosphorylation as determined from lactate accumulation and phosphocreatine degradation between trials. Subsequently, oxidative metabolism during the transition from rest to steady-state exercise was not affected by prior carbohydrate ingestion. Although exercise resulted in the rapid activation of PDH in both trials, PDHa was greater at 1 min in CHO (CON, 2.36 ± 0.22; CHO, 2.91 ± 0.18 mmol min−1 (kg w.m.)−1). No differences in muscle metabolite levels and PDHa were observed after 10 min of moderate exercise between trials. In summary, at rest, carbohydrate ingestion induced multiple metabolic changes which included decreased acetylcarnitine availability and small increases in PDHa. The prior changes in PDHa and acetylcarnitine availability had no effect on substrate phosphorylation and oxidative metabolism at the onset of exercise. These data suggest that acetylcarnitine availability is unlikely to be the site of metabolic inertia during the transition from rest to steady-state moderate intensity exercise.

17beta-estradiol upregulates the expression of peroxisome proliferator-activated receptor alpha and lipid oxidative genes in skeletal muscle

This study examined the actions of 17β-estradiol (E2) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically,
carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (β-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague–Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both hormones in combination (E+P). Additionally,
sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and protein content (P<0·05), compared with Ovx treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P<0·05). PPARγ mRNA expression was also increased (P<0·05) by E2 treatment, although protein content remained unaltered. These data
demonstrate the novel regulation of E2 on PPARα and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.

Regulation of metabolic genes in human skeletal muscle by short-term exercise and diet manipulation

Changes in dietary macronutrient intake alter muscle and blood substrate availability and are important for regulating gene expression. However, few studies have examined the effects of diet manipulation on gene expression in human skeletal muscle. The aim of this study was to quantify the extent to which altering substrate availability impacts on subsequent mRNA abundance of a subset of carbohydrate (CHO)- and fat-related genes. Seven subjects consumed either a low- (LOW; 0.7 g/kg body mass CHO) or high- (HIGH; 10 g/kg body mass CHO) CHO diet for 48 h after performing an exhaustive exercise bout to deplete muscle glycogen stores. After intervention, resting muscle and blood samples were taken. Muscle was analyzed for the gene abundances of GLUT4, glycogenin, pyruvate dehydrogenase kinase-4 (PDK-4), fatty acid translocase (FAT/CD36), carnitine palmitoyltransferase I (CPT I), hormone-sensitive lipase (HSL), β-hydroxyacyl-CoA dehydrogenase (΄β-HAD), and uncoupling binding protein-3 (UCP3), and blood samples for glucose, insulin, and free fatty acid (FFA) concentrations. Glycogen-depleting exercise and HIGH-CHO resulted in a 300% increase in muscle glycogen content (P < 0.001) relative to the LOW-CHO condition. FFA concentrations were twofold higher after LOW- vs. HIGH-CHO (P < 0.05). The exercise-diet manipulation exerted a significant effect on transcription of all carbohydrate-related genes, with an increase in GLUT4 and glycogenin mRNA abundance and a reduction in PDK-4 transcription after HIGH-CHO (all P < 0.05). FAT/CD36 (P < 0.05) and UCP3 (P < 0.01) gene transcriptions were increased following LOW-CHO. We conclude that 1) there was a rapid capacity for a short-term exercise and diet intervention to exert coordinated changes in the mRNA transcription of metabolic related genes, and 2) genes involved in glucose regulation are increased following a high-carbohydrate diet.

Real time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either a interval-type cycle ergometry workout to exhaustion (~75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise and 3 h and 48 h following exercise. Real-time RT-PCR was performed on ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ~65% of O2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, from muscle biopsy samples taken immediately postexercise. We showed that CYC increased 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01), and 28S rRNA increased immediately following END exercise (2-fold; P = 0.02). ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4-fold; P = 0.067). In contrast, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise.

Creatine transporters: a reappraisal

Creatine (Cr) plays a key role in cellular energy metabolism and is found at high concentrations in metabolically active cells such as skeletal muscle and neurons. These, and a variety of other cells, take up Cr from the extra cellular fluid by a high affinity Na⁺/Cl^--dependent creatine transporter (CrT). Mutations in the crt gene, found in several patients, lead to severe retardation of speech and mental development, accompanied by the absence of Cr in the brain.
In order to characterize CrT protein(s) on a biochemical level, antibodies were raised against synthetic peptides derived from the N- and C-terminal cDNA sequences of the putative CrT-1 protein. In total homogenates of various tissues, both antibodies, directed against these different epitopes, recognize the same two major polypetides on Western blots with apparent Mr of 70 and 55 kDa. The C-terminal CrT antibody (α-CrTCOOH) immunologically reacts with proteins located at the inner membrane of mitochondria as determined by immuno-electron microscopy, as well as by subfractionation of mitochondria. Cr-uptake experiments with isolated mitochondria showed these organelles were able to transport Cr via a sulfhydryl-reagent-sensitive transporter that could be blocked by anti-CrT antibodies when the outer mitochondrial membrane was permeabilized. We concluded that mitochondria are able to specifically take-up Cr from the cytosol, via a low-affinity CrT, and that the above polypeptides would likely represent mitochondrial CrT(s). However, by mass spectrometry techniques, the immunologically reactive proteins, detected by our anti-CrT antibodies, were identified as E2 components of the agr-keto acid dehydrogenase multi enzyme complexes, namely pyruvate dehydrogenase (PDH), branched chain keto acid dehydrogenase (BC-KADH) and α-ketoglutarate dehydrogenase (α-KGDH). The E2 components of PDH are membrane associated, whilst it would be expected that a mitochondrial CrT would be a transmembrane protein. Results of phase partitioning by Triton X-114, as well as washing of mitochondrial membranes at basic pH, support that these immunologically cross-reactive proteins are, as expected for E2 components, membrane associated rather than transmembrane. On the other hand, the fact that mitochondrial Cr uptake into intact mitoplast could be blocked by our α-CrTCOOH antibodies, indicate that our antisera contain antibodies reactive to proteins involved in mitochondrial transport of Cr. The presence of specific antibodies against CrT is also supported by results from plasma membrane vesicles isolated from human and rat skeletal muscle, where both 55 and 70 kDa polypeptides disappeared and a single polypeptide with an apparent electrophoretic mobility of ~ 60 kDa was enriched This latter is most likely representing the genuine plasma membrane CrT.
Due to the fact that all anti-CrT antibodies that were independently prepared by several laboratories seem to cross-react with non-CrT polypeptides, specifically with E2 components of mitochondrial dehydrogenases, further research is required to characterise on a biochemical/biophysical level the CrT polypeptides, e.g. to determine whether the ~ 60 kDa polypeptide is indeed a bona-fide CrT and to identify the mitochondrial transporter that is able to facilitate Cr-uptake into these organelles. Therefore, the anti-CrT antibodies available so far should only be used with these precautions in mind. This holds especially true for quantitation of CrT polypeptides by Western blots, e.g. when trying to answer whether CrT's are up- or down-regulated by certain experimental interventions or under pathological conditions.
In conclusion, we still hold to the scheme that besides the high-affinity and high-efficiency plasmalemma CrT there exists an additional low affinity high Km Cr uptake mechanism in mitochondria. However, the exact biochemical nature of this mitochondrial creatine transport, still remains elusive. Finally, similar to the creatine kinase (CK) isoenzymes, which are specifically located at different cellular compartments, also the substrates of CK are compartmentalized in cytosolic and mitochondrial pools. This is in line with ¹⁴C-Cr-isotope tracer studies and a number of [³¹P]-NMR magnetization transfer studies, as well as with recent [¹H]-NMR spectroscopy data.

Interaction of contractile activity and training history on mRNA abundance in skeletal muscle from trained athletes

Skeletal muscle displays enormous plasticity to respond to contractile activity with muscle from strength- (ST) and endurance-trained (ET) athletes representing diverse states of the adaptation continuum. Training adaptation can be viewed as the accumulation of specific proteins. Hence, the altered gene expression that allows for changes in protein concentration is of major importance for any training adaptation. Accordingly, the aim of the present study was to quantify acute subcellular responses in muscle to habitual and unfamiliar exercise. After 24-h diet/exercise control, 13 male subjects (7 ST and 6 ET) performed a random order of either resistance (8 x 5 maximal leg extensions) or endurance exercise (1 h of cycling at 70% peak O2 uptake). Muscle biopsies were taken from vastus lateralis at rest and 3 h after exercise. Gene expression was analyzed using real-time PCR with changes normalized relative to preexercise values. After cycling exercise, peroxisome proliferator-activated receptor- coactivator-1 (ET 8.5-fold, ST 10-fold, P < 0.001), pyruvate dehydrogenase kinase-4 (PDK-4; ET 26-fold, ST 39-fold), vascular endothelial growth factor (VEGF; ET 4.5-fold, ST 4-fold), and muscle atrophy F-box protein (MAFbx) (ET 2-fold, ST 0.4-fold) mRNA increased in both groups, whereas MyoD (3-fold), myogenin (0.9-fold), and myostatin (2-fold) mRNA increased in ET but not in ST (P < 0.05). After resistance exercise PDK-4 (7-fold, P < 0.01) and MyoD (0.7-fold) increased, whereas MAFbx (0.7-fold) and myostatin (0.6-fold) decreased in ET but not in ST. We conclude that prior training history can modify the acute gene responses in skeletal muscle to subsequent exercise.

Skeletal muscle htererogeneity in fasting-induced upregulation of genes encoding UCP2, UCP3, PPARĂ and key enzymes of lipid oxidation

The uncoupling protein homologs UCP2 and UCP3 have been proposed as candidate genes for the regulation of lipid metabolism. Within the context of this hypothesis, we have compared, from fed and fasted rats, changes in gene expression of skeletal muscle UCP2 and UCP3 with those of carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase, two key enzymes regulating lipid flux across the mitochondrial #-oxidation pathway. In addition, changes in gene expression of peroxisome proliferator-activated receptor gamma, a nuclear transcription factor implicated in lipid metabolism, were also investigated. The results indicate that in response to fasting, the mRNA levels of UCP2, UCP3, carnitine palmitoyltransferase I and medium-chain acyl-CoA dehydrogenase are markedly increased, by three- to sevenfold, in the gastrocnemius and tibialis anterior (fast-twitch muscles, predominantly glycolytic or oxidative-glycolytic), but only mildly increased, by less than twofold, in the soleus (slow-twitch muscle, predominantly oxidative). Furthermore, such muscle-type dependency in fasting-induced transcriptional changes in UCP2, UCP3, carnitine palmitoyltransferase and medium-chain acyl-CoA dehydrogenase persists when the increase in circulating levels of free fatty acids during fasting is abolished by the anti-lipolytic agent nicotinic acid - with blunted responses only in the slow-twitch muscle contrasting with unabated increases in fast-twitch muscles. Independently of muscle type, however, the mRNA levels of peroxisome proliferator-activated receptor gamma are not altered during fasting. Taken together, these studies indicate a close association between fasting-induced changes in UCP2 and UCP3 gene expression with those of key regulators of lipid oxidation, and are hence consistent with the hypothesis that these UCP homologs may be involved in the regulation of lipid metabolism. Furthermore, they suggest that in response to fasting, neither the surge of free fatty acids in the circulation nor induction of the peroxisome proliferator-activated receptor gamma gene may be required for the marked upregulation of genes encoding the UCP homologs and key enzymes regulating lipid oxidation in fast-twitch muscles.

Insights into exazaphosphorine resistance and possible approaches to its circumvention

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.