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Biomedical subjects

Anna M Gómez-Foix

Publications and source records attributed to Anna M Gómez-Foix.

10 recordsLinked to original sources
PubMed2006

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.

Adolescent↗
PubMed2006

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.

Animals↗
PubMed2005

Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells.

We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentrations tested. In contrast, long-term CO(2) production was reduced by FATP1 and FAT at all doses of palmitate and at the lower concentrations of oleate. Neither FATP1 nor FAT markedly altered the production of acid-soluble metabolic intermediates from palmitate or oleate. The intracellular localization of fusion constructs of FATP1 and FAT with enhanced green fluorescent protein (EGFP) was examined. Independently of fatty acid treatment, FATPGFP was observed throughout the cytosol in a reticular pattern and concentrated in the perinuclear region, partly overlapping with the Golgi marker GM-130. FATGFP was found in the extracellular membrane and in cytosolic vesicles not coincident with GM-130. Neither FATP1 nor FAT proteins colocalized with lipid droplets in oleate-treated cells. We conclude that whereas FAT is localized on the extracellular membrane, FATP1 is active in the cytosol and imports fatty acids into myotubes. Overall, both FATP1 and FAT stimulated transport and consumption of palmitate and oleate, which they channeled away from complete oxidation and toward TAG synthesis.

Biological Transport, Active↗
PubMed2004

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.

Animals↗
PubMed2004

Regulation of glycogen metabolism in cultured human muscles by the glycogen phosphorylase inhibitor CP-91149.

Pharmacological inhibition of liver GP (glycogen phosphorylase), which is currently being studied as a treatment for Type II (non-insulin-dependent) diabetes, may affect muscle glycogen metabolism. In the present study, we analysed the effects of the GP inhibitor CP-91149 on non-engineered or GP-overexpressing cultured human muscle cells. We found that CP-91149 treatment decreased muscle GP activity by (1) converting the phosphorylated AMP-independent a form into the dephosphorylated AMP-dependent b form and (2) inhibiting GP a activity and AMP-mediated GP b activation. Dephosphorylation of GP was exerted, irrespective of incubation of the cells with glucose, whereas inhibition of its activity was synergic with glucose. As expected, CP-91149 impaired the glycogenolysis induced by glucose deprivation. CP-91149 also promoted the dephosphorylation and activation of GS (glycogen synthase) in non-engineered or GP-overexpressing cultured human muscle cells, but exclusively in glucose-deprived cells. However, this inhibitor did not activate GS in glucose-deprived but glycogen-replete cells overexpressing PTG (protein targeting to glycogen), thus suggesting that glycogen inhibits the CP-91149-mediated activation of GS. Consistently, CP-91149 promoted glycogen resynthesis, but not its overaccumulation. Hence, treatment with CP-91149 impairs muscle glycogen breakdown, but enhances its recovery, which may be useful for the treatment of Type II (insulin-dependent) diabetes.

Amides↗
PubMed2004

Design and performance of an electrical stimulator for long-term contraction of cultured muscle cells.

Excitability in muscle cells manifests itself as contractility and may be evoked by electrical stimulation. Here we describe an electrical stimulator device applicable to cells seeded on standard multiwell plates and demonstrate how it effectively stimulates synchronous contraction of skeletal muscle C2C12 cells without damaging them. The electrical stimulator of cultured cells (ESCC) consists of two connection cards and a network of platinum electrodes positioned in such way that each well in a row is uniformly stimulated. The ESCC may produce a range of outputs based on the stimulation parameters it receives from a commercial pulse generator and can be placed in a standard cell incubator, allowing for long-term stimulation as required for biochemical and molecular biological assays. We show that a 90-min stimulation of C2C12 myotubes at 50 V, 30 ms of pulse duration, and 3 Hz of frequency enhances glucose metabolism and glycogen mobilization while oppositely modulating the activity ratio of glycogen metabolizing enzymes. Thus, we demonstrate that long-term electrical stimulation of C2C12 myotubes with the ESCC results in contractility and metabolic changes, as seen in exercising muscle.

Animals↗
PubMed2003

Regulation and function of the muscle glycogen-targeting subunit of protein phosphatase 1 (GM) in human muscle cells depends on the COOH-terminal region and glycogen content.

G(M), the muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PP1) targeted to the sarcoplasmic reticulum, was proposed to regulate recovery of glycogen in exercised muscle, whereas mutation truncation of its COOH-terminal domain is known to be associated with type 2 diabetes. Here, we demonstrate differential effects of G(M) overexpression in human muscle cells according to glycogen concentration. Adenovirus-mediated delivery of G(M) slightly activated glycogen synthase (GS) and inactivated glycogen phosphorylase (GP) in glycogen-replete cells, causing an overaccumulation of glycogen and impairment of glycogenolysis after glucose deprivation. Differently, in glycogen-depleted cells, G(M) strongly increased GS activation with no further enhancement of early glycogen resynthesis and without affecting GP. Effects of G(M) on GS and GP were abrogated by treatment with dibutyryl cyclic AMP. Expression of a COOH-terminal deleted-mutant (G(M) Delta C), lacking the membrane binding sequence to sarcoplasmic reticulum, failed to activate GS in glycogen-depleted cells, while behaving similar to native G(M) in glycogen-replete cells. This is explained by loss of stability of the G(M) Delta C protein following glycogen-depletion. In summary, G(M) promotes glycogen storage and inversely regulates GS and GP activities, while, specifically, synthase phosphatase activity of G(M)-PP1 is inhibited by glycogen. The conditional loss of function of the COOH-terminal deleted G(M) construct may help to explain the reported association of truncation mutation of G(M) with insulin resistance in human subjects.

Cells, Cultured↗
PubMed2002

Glucokinase gene transfer to skeletal muscle of diabetic Zucker fatty rats improves insulin-sensitive glucose uptake.

Skeletal muscle has a prime role in glucose homeostasis. We have previously demonstrated that adenovirus-mediated glucokinase (GK) gene transfer to skeletal muscle of Wistar rats enhances muscle glucose uptake and whole body glucose disposal under conditions of hyperglycemia and hyperinsulinemia. In this study, we have tested whether GK gene transfer to the muscle of the Zucker Diabetic Fatty rat (ZDF), a genetic model of obesity and type 2 diabetes, could improve glycemic control and prevent the onset of hyperglycemia in obese males. We show that GK delivery results in a doubling of total gastrocnemius muscle glucose phosphorylating activity 9 weeks after gene transfer. GK-treated rats exhibited slightly reduced weight and normal insulin-sensitive glucose uptake, as assessed during an insulin tolerance test, whereas age-matched rats treated with a control virus were clearly insulin resistant. The improved glucose uptake in GK-expressing rats was associated with higher gastrocnemius lactate content, whereas glycogen and triacylglyceride (TAG) levels were unmodified. Remarkably, GK-treated rats showed increased expression of both hexokinase II (HKII) and GLUT4, in accordance with a glucose-dependent regulation of these proteins. Thus, our data show that delivery of GK, despite improving insulin-sensitive glucose disposal in muscle, is not sufficient to prevent or delay the appearance of elevated glucose and insulin levels associated with severe obesity in male ZDF animals.

Animals↗
PubMed2001

Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.

Adenoviridae↗