Long-term cultured human myotubes decrease contractile gene expression and regulate apoptosis-related genes.

The present study examined time-dependent changes in the gene expression profile of long-term cultured human myotubes. Microarray transcriptional analysis was performed in a primary culture of differentiated myotubes from one subject over seven weeks. This analysis showed a main gradual fall in genes of the contractile apparatus, and a broad upregulation of genes involved in cell development and growth, followed by stress response and signal transduction. Glucose metabolism was also monitored, but no significant alterations in glucose uptake, oxidation or glycogen storage were observed. Mitochondrial membrane potential, or the amount of membrane lipid peroxides, remained similarly unchanged, nor was lactate dehydrogenase leakage observed. Time-dependent changes in eight genes were validated by real-time RT-PCR in primary cultured myotubes from four subjects, of similar age and isolated after equivalent replication cycles in vitro and differentiated over seven weeks. Insulin-like growth factor-binding protein 2 (IGFBP2), a modulator of the IGF signal, was upregulated. The antiapoptotic gene heat-shock 70-kd protein 2 (HSPA2) was induced, whereas the proapoptotic tumor necrosis factor receptor superfamily, member 25 (WSL-1) was suppressed. A decline in the muscle-specific gene M-cadherin and contraction genes, such as slow-twitch troponin I (TNNI1) and myosin heavy chain 2 (MYH2), myosin light chain 1 (MYL1) and myosin-binding protein H (MYBPH), which are expressed in adult fast-twitch muscle, was shown. In summary, these data demonstrate extensive downregulation of contractile genes and modulation of apoptosis-related genes, in favour of cell survival, during maintenance of cultured human myotubes.

Effect of sucrose and saturated-fat diets on mRNA levels of genes limiting muscle fatty acid and glucose supply in rats.

In this study, we examined whether the increased availability of lipids in blood resulting from two types of diet manipulation regulated metabolic gene expression in the skeletal muscle of rats. Feeding for 4 wk on an isocaloric-sucrose or a hypercaloric-fat diet increased plasma TAG in the fed condition by increments of 70 and 40%, respectively, and increased fasting insulinemia (approximately 3-fold) compared with a starch diet. The fat diet impaired glucose tolerance and caused obesity, whereas sucrose-fed rats maintained their normal weight. We analyzed the expression of genes that regulate the exogenous FA supply (LPL, FAT/CD36, FATP1), synthesis (ACC1), glucose (GLUT4, GLUT1, HK2, GFAT1, glycogen phosphorylase) or glycerol (glycerol kinase) provision, or substrate choice for oxidation (PDK4) in gastrocnemius and soleus muscles at the end of the glucose tolerance test. LPL, FAT/CD36, FATP1, PDK4, and GLUT4 mRNA as well as glycogen phosphorylase and glycerol kinase activity levels in both muscles were unchanged by the diets. Increased mRNA levels of GLUT1 (1.6- and 2.6-fold, respectively) and GFAT1 (about 1.7-fold) in gastrocnemius, and of ACC1 (about 1.5-fold) in soleus, were found in both the sucrose and fat groups. In the fat group, HK2 mRNA was also higher (1.8-fold) in the gastrocnemius. Both sucrose and saturated-fat diets prompted hyperinsulinemia and hyperlipemia in rats. These metabolic disturbances did not alter the expression of LPL, FAT/CD36, FATP1, PDK4, and GLUT4 genes or glycogen phosphorylase and glycerol kinase activity levels in either analyzed muscle. Instead, they were linked to the coordinated upregulation in gastrocnemius of genes that govern glucose uptake and the hexosamine pathway, namely, GLUT1 and GFAT1, which might contribute to insulin resistance.

beta3-adrenoceptor agonist prevents alterations of muscle diacylglycerol and adipose tissue phospholipids induced by a cafeteria diet.

BACKGROUND: Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of beta3-adrenoceptor (beta3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective beta3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet. METHODS: Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the beta3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study. RESULTS: In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet. CONCLUSIONS: These results show that activation of the beta3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with beta3-AR activation.

Chicken ovalbumin upstream promoter-transcription factor I represses the transcriptional activity of the human muscle glycogen phosphorylase promoter in C2C12 cells.

The responsiveness of the 1.13 kb proximal human muscle glycogen phosphorylase (MGP) gene promoter to the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) repressor, known to be ablated during muscle cell differentiation, was examined. Constitutive expression of COUP-TFI repressed the activity of the promoter in C2C12 muscle cells and sequential deletion analysis mapped the sensitive region between nucleotides -362 and -185, which included a putative consensus COUP-TF binding half-site at -198/-193. Mutation of this site abolished transcriptional response to COUP-TFI of the -362 construct. A -209/-180 probe bound in vitro to COUP-TFI and to protein extracts from proliferating but not fusing myoblasts. Thus, COUP-TF may be involved in repression of the human MGP gene promoter at the myoblast stage.

A glucose response element from the S. cerevisiae hexose transporter HXT1 gene is sensitive to glucose in human fibroblasts.

Glucose is an essential nutrient, and a regulator of gene expression in eukaryotic cells. Here, a comparative, function-based genomic approach has been used to identify glucose regulatory elements and transduction pathways common to both yeast and mammalian cells. We have isolated a region in the promoter of the Saccharomyces cerevisiae hexose transporter gene HXT1 that conferred glucose sensitivity in yeast, when located upstream of the minimal CYC1 promoter. This element contained binding motifs for Rgt1, a transcriptional modulator involved in the yeast glucose-induction pathway, that were sufficient to elicit glucose responsiveness. The HXT1 regulatory element was then fused to the minimal cytomegalovirus promoter (HXT1-MIN) and inserted into an adenovirus for delivery to human fibroblasts, where it exhibited glucose-dependent transcriptional activation. Glucose action was mimicked by fructose and unrelated to glucose 6-P content, whilst non-metabolizable glucose analogues showed no effect. Activation of AMP kinase by 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranosanide blocked glucose induction, revealing parallels with the yeast glucose-repressing pathway. In contrast, delivery of Rgt1 to fibroblasts did not modify HXT1-MIN responsiveness. Thus, elements of the S.cerevisiae HXT1 gene conserve glucose regulation in human fibroblasts equivalent to the metabolism-dependent, glucose-repressing pathway in yeast. These data suggest that the instructions carried within gene regulatory elements controlling nutrient regulation of gene expression have been conserved throughout evolution.

Mouse PeP: a novel peroxisomal protein linked to myoblast differentiation and development.

The identification of several peroxisomal proteins in the past decade has deepened our understanding of the biology of peroxisomes and their involvement in human disorders. We report the cloning and expression pattern during the mouse development of a cDNA encoding a novel protein, named PeP, and show that its product is imported specifically to the peroxisome matrix in a variety of cell types. We also demonstrate that PeP is imported to the organelle through the PEX5 receptor pathway, which indicates that the C-terminal tripeptide SKI behaves as a type 1 peroxisomal targeting signal (PTS1). PeP expression is tightly regulated, as shown by Northern and in situ hybridization experiments. Thus during embryonic development in the mouse, PeP mRNA is detected almost exclusively in the skeletal muscle, whereas in adult mice, strong expression is also found in the heart and brain. In addition, PeP mRNA accumulation is induced after myoblast differentiation in vitro, when myotube formation is promoted. Sequence analysis reveals that PeP has no significant homology to any known protein, except for a short stretch of amino acids containing the fingerprint of the fibronectin type III superfamily, a domain present in proteins often related to molecular and cellular recognition and binding processes. Thus our data suggest a connection between the function of PeP and murine cell differentiation and development.

Laser scanning cytometry to quantify gene transfer efficiency and transcriptional activity of EGFP constructs.

Enhanced green fluorescent protein (EGFP) is the preferred reporter protein for real-time detection in individual cells, but its usefulness for gene expression quantification is limited by the sensitivity of standard detection techniques. We tested whether the unique feature of single-cell detection and quantification by laser scanning cytometry permits the evaluation of EGFP gene expression in monolayer cultures of kidney epithelial 293 and C2C12 muscle cells. Cells were transfected with plasmids expressing EGFP under the control of either the cytomegalovirus or muscle promoters: namely, muscle creatine kinase (MCK) and muscle glycogen phosphorylase (MGP). Cell monolayers were laser-scanned, fluorescence-imaged, and recorded. A population of fluorescence-emitting cells was discriminated, their contour area was defined, and the integrated fluorescence was estimated. These data were used to assess gene transfer efficiency in cells transfected with CMV-EGFP, which was higher in 293 than in C2C12 cells. Analysis of fluorescence intensity revealed that, as expected, CMV constructs were highly expressed in both cell types, whereas MCK and MGP constructs showed the highest transcriptional activity in C12C12 cells. In summary, we describe the utility of laser scanning cytometry for the automated estimation of gene transfer efficiency and transcriptional activity of EGFP constructs in cell monolayers.