Antioxidant treatment with N-acetylcysteine regulates mammalian skeletal muscle Na+-K+-ATPase ∝ gene expression during repeated contractions

Exercise increases Na+–K+ pump isoform gene expression and elevates muscle reactive oxygen species (ROS). We investigated whether enhanced ROS scavenging induced with the antioxidant N-acetylcysteine (NAC) blunted the increase in Na+–K+ pump mRNA during repeated contractions in human and rat muscle. In experiment 1, well-trained subjects received saline or NAC intravenously prior to and during 45 min cycling. Vastus lateralis muscle biopsies were taken pre-infusion and following exercise. In experiment 2, isolated rat extensor digitorum longus muscles were pre-incubated without or with 10 mm NAC and then rested or stimulated electrically at 60 Hz for 90 s. After 3 h recovery, muscles were frozen. In both experiments, the muscles were analysed for Na+–K+ pump α1, α2, α3, β1, β2 and β3 mRNA. In experiment 1, exercise increased α2 mRNA by 1.0-fold (P = 0.03), but α2 mRNA was reduced by 0.40-fold with NAC (P = 0.03). Exercise increased α3, β1 and β2 mRNA by 2.0- to 3.4-fold (P < 0.05), but these were not affected by NAC (P > 0.32). Neither exercise nor NAC altered α1 or β3 mRNA (P > 0.31). In experiment 2, electrical stimulation increased α1, α2 and α3 mRNA by 2.3- to 17.4-fold (P < 0.05), but these changes were abolished by NAC (P > 0.07). Electrical stimulation almost completely reduced β1 mRNA but only in the presence of NAC (P < 0.01). Neither electrical stimulation nor NAC altered β2 or β3 mRNA (P > 0.09). In conclusion, NAC attenuated the increase in Na+–K+ pump α2 mRNA with exercise in human muscle and all α isoforms with electrical stimulation in rat muscle. This indicates a regulatory role for ROS in Na+–K+ pump α isoform mRNA in mammalian muscle during repeated contractions.

Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc

Human telomerase reverse transcriptase (hTERT) underlies cancer cell immortalization, and the expression of hTERT is regulated strictly at the gene transcription. Here, we report that transcription factor Ets2 is required for hTERT gene expression and breast cancer cell proliferation. Silencing Ets2 induces a decrease of hTERT gene expression and increase in human breast cancer cell death. Reconstitution with recombinant hTERT rescues the apoptosis induced by Ets2 depression. In vitro and in vivo analyses show that Ets2 binds to the EtsA and EtsB DNA motifs on the hTERT gene promoter. Mutation of either Ets2 binding site reduces the hTERT promoter transcriptional activity. Moreover, Ets2 forms a complex with c-Myc as demonstrated by co-immunoprecipitation and glutathione S-transferase pulldown assays. Immunological depletion of Ets2, or mutation of the EtsA DNA motif, disables c-Myc binding to the E-box, whereas removal of c-Myc or mutation of the E-box also compromises Ets2 binding to EtsA. Thus, hTERT gene expression is maintained by a mechanism involving Ets2 interactions with the c-Myc transcription factor and the hTERT gene promoter, a protein-DNA complex critical for hTERT gene expression and breast cancer cell proliferation.

Adiponectin decreases pyruvate dehydrogenase kinase 4 gene expression in obese- and diabetic derived

Aim: To investigate the effects of globular adiponectin (gAd) on gene expression and whether these effects are mediated through 3',5'-cyclic monophosphate-activated protein kinase in skeletal muscle myotubes obtained from lean, obese and obese diabetic individuals.

Methods: Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Three distinct primary cell culture groups were established (lean, obese and obese diabetic; n = 7 in each group). Once differentiated, these cultures were then exposed to gAd or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 6 h.

Results: Stimulation with gAd decreased pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the obese and diabetic samples (p ≤ 0.05) and increased cytochrome c oxidase (COX) subunit 4 (COXIV) gene expression in the myotubes derived from lean individuals only (p < 0.05). AICAR treatment also decreased PDK4 gene expression in the obese- and diabetic-derived myotubes (p ≤ 0.05) and increased the gene expression of the mitochondrial gene, COXIII, in the lean-derived samples only (p < 0.05).

Conclusions: This study demonstrated distinct disparity between myotubes derived from lean compared with obese and obese diabetic individuals following gAd and AICAR treatment. Further understanding of the regulation of PDK4 in obese and diabetic skeletal muscle and its interaction with adiponectin signalling is required as this appears to be an important early molecular event in these disease states that may improve blood glucose control and metabolic flux.

Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet

The mechanisms of how tea and epigallocatechin-3-gallate (EGCG) lower body fat are not completely understood. This study investigated long-term administration of green tea (GT), black tea (BT), or isolated EGCG (1 mg/kg per day) on body composition, glucose tolerance, and gene expression related to energy metabolism and lipid homeostasis; it was hypothesized that all treatments would improve the indicators of metabolic syndrome. Rats were fed a 15% fat diet for 6 months from 4 weeks of age and were supplied GT, BT, EGCG, or water. GT and BT reduced body fat, whereas GT and EGCG increased lean mass. At 16 weeks GT, BT, and EGCG improved glucose tolerance. In the liver, GT and BT increased the expression of genes involved in fatty acid synthesis (SREBP-1c, FAS, MCD, ACC) and oxidation (PPAR-α, CPT-1, ACO); however, EGCG had no effect. In perirenal fat, genes that mediate adipocyte differentiation were suppressed by GT (Pref-1, C/EBP-β, and PPAR-γ) and BT (C/EBP-β), while decreasing LPL, HSL, and UCP-2 expression; EGCG increased expression of UCP-2 and PPAR-γ genes. Liver triacylglycerol content was unchanged. The results suggest that GT and BT suppressed adipocyte differentiation and fatty acid uptake into adipose tissue, while increasing fat synthesis and oxidation by the liver, without inducing hepatic fat accumulation. In contrast, EGCG increased markers of thermogenesis and differentiation in adipose tissue, while having no effect on liver or muscle tissues at this dose. These results show novel and separate mechanisms by which tea and EGCG may improve glucose tolerance and support a role for these compounds in obesity prevention.

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-? 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.

Histone modifications and skeletal muscle metabolic gene expression

1.      Skeletal muscle oxidative function and metabolic gene expression are co-ordinately downregulated in metabolic diseases such as insulin resistance, obesity and Type 2 diabetes. Altering skeletal muscle metabolic gene expression to favour enhanced energy expenditure is considered a potential therapy to combat these diseases.

2.      Histone deacetylases (HDACs) are chromatin-remodelling enzymes that repress gene expression. It has been shown that HDAC4 and 5 co-operatively regulate a number of genes involved in various aspects of metabolism. Understanding how HDACs are regulated provides insights into the mechanisms regulating skeletal muscle metabolic gene expression.

3.      Multiple kinases control phosphorylation-dependent nuclear export of HDACs, rendering them unable to repress transcription. We have found a major role for the AMP-activated protein kinase (AMPK) in response to energetic stress, yet metabolic gene expression is maintained in the absence of AMPK activity. Preliminary evidence suggests a potential role for protein kinase D, also a Class IIa HDAC kinase, in this response.

4.      The HDACs are also regulated by ubiquitin-mediated proteasomal degradation, although the exact mediators of this process have not been identified.

5.      Because HDACs appear to be critical regulators of skeletal muscle metabolic gene expression, HDAC inhibition could be an effective therapy to treat metabolic diseases.

6.      Together, these data show that HDAC4 and 5 are critical regulators of metabolic gene expression and that understanding their regulation could provide a number of points of intervention for therapies designed to treat metabolic diseases, such as insulin resistance, obesity and Type 2 diabetes.

Differential gene expression in the skeletal muscle of Psammomys obesis

Focuses on discovering and investigating altered gene expression in the skeletal muscle of Psammomys obesus which is a unique model of obesity and Type II diabetes in which its development is similar to that of the human population. Defects in the skeletal muscle are pivotal to the development of Type II diabetes. Using the latest techniques in molecular biology the regulation of a number of genes was confirmed to be altered in obese or diabetic animals compared to lean. This indicates that changes to gene expression contribute to the metabolic disturbances associated with obesity and Type II diabetes.

Effects of vegetarianism and exercise on muscle creatine loading and gene expression

The results of this thesis indicate that vegetarianism and prior exercise can increase skeletal muscle creatine loading. The mechanisms by which these phenomena occur remain unknown but may be related to changes in the gene expression of a cellular pump within the muscle known as the "creatine transporter".

A gene expression signature for insulin resistance

Insulin resistance is a heterogeneous disorder caused by a range of genetic and environmental factors, and we hypothesize that its aetiology varies considerably between individuals. This heterogeneity provides significant challenges to the development of effective therapeutic regimes for long-term management of type 2 diabetes. We describe a novel strategy, using large-scale gene expression profiling, to develop a Gene Expression Signature (GES) that reflects the overall state of insulin resistance in cells and patients. The GES was developed from 3T3-L1 adipocytes that were made ‘insulin resistant’ by treatment with tumour necrosis factor-alpha (TNFα) and then reversed with aspirin and troglitazone (‘re-sensitized’). The GES consisted of five genes whose expression levels best discriminated between the insulin resistant and insulin re-sensitized states. We then used this GES to screen a compound library for agents that affected the GES genes in 3T3- L1 adipocytes in a way that most closely resembled the changes seen when insulin resistance was successfully reversed using aspirin and troglitazone. This screen identified both known and new insulin sensitizing compounds including non-steroidal anti inflammatory agents, β-adrenergic antagonists, beta-lactams and sodium channel blockers. We tested the biological relevance of this GES in participants in the San Antonio Family Heart Study (n = 1,240) and showed that patients with the lowest GES scores were more insulin resistant (according to HOMA_IR and fasting plasma insulin levels, P < 0.001). These findings show that GES technology can be used for both the discovery of insulin sensitizing compounds and the characterization of patients into subtypes of insulin resistance according to GES scores, opening the possibility of developing a personalized medicine approach to type 2 diabetes.

Docosapentaenoic acid : its metabolism and effect on lipogenic gene expression

This thesis found that the omega-3 fatty acid, docosapentaenoic acid (DPA) down-regulates the expression levels of key lipogenic genes and proteins in vitro. In vivo studies with labelled DPA showed that, like docosahexaenoic acid, DPA is more conserved from oxidation compared with eicosapentaenoic acid.