Glucose and fructose have sugar-specific effects in both liver and skeletal muscle in vivo: a role for liver fructokinase.

We examined glucose and fructose effects on serine phosphorylation levels of a range of proteins in rat liver and muscle cells. For this, healthy adult rats were subjected to either oral glucose or fructose loads. A mini-array system was utilized to determine serine phosphorylation levels of liver and skeletal muscle proteins. A glucose oral load of 125 mg/100 g body weight (G 1/2) did not induce changes in phosphorylated serines of the proteins studied. Loading with 250 mg/100 g body weight of fructose (Fr), which induced similar glycemia levels as G 1/2, significantly increased serine phosphorylation of liver cyclin D3, PI3 kinase/p85, ERK-2, PTP2 and clusterin. The G 1/2 increased serine levels of the skeletal muscle proteins cyclin H, Cdk2, IRAK, total PKC, PTP1B, c-Raf 1, Ras and the ß-subunit of the insulin receptor. The Fr induced a significant increase only in muscle serine phosphorylation of PI3 kinase/p85. The incubation of isolated rat hepatocytes with 10 mM glucose for 5 min significantly increased serine phosphorylation of 31 proteins. In contrast, incubation with 10 mM fructose produced less intense effects. Incubation with 10 mM glucose plus 75 µM fructose counteracted the effects of the incubation with glucose alone, except those on Raf-1 and Ras. Less marked effects were detected in cultured muscle cells incubated with 10 mM glucose or 10 mM glucose plus 75 µM fructose. Our results suggest that glucose and fructose act as specific functional modulators through a general mechanism that involves liver-generated signals, like micromolar fructosemia, which would inform peripheral tissues of the presence of either glucose- or fructose-derived metabolites.

Fatty acid transport protein 1 (FATP1) localizes in mitochondria in mouse skeletal muscle and regulates lipid and ketone body disposal.

FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and ß-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and ß-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from ß-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.

A new spiral microelectrode assembly for electroporation and impedance measurements of adherent cell monolayers.

In this study, a new microelectrode assembly based on spiral geometry applicable to in situ electroporation of adherent cell monolayers on standard multiwell plates is presented. Furthermore, the structure is specially conceived to perform electrical impedance spectroscopy (EIS) measurements during electroporation. Its performance for cell membrane permeabilization is tested with a fluorescent probe. Gene electrotransfer is also assayed using a plasmid DNA encoding GFP in four different cell lines (CHO, HEK293, 3T3-L1 and FTO2B). Additionally, siRNA α-GFP electrotransfection is tested in GFP gene-expressing CHO cells. Our data show considerable differences between permeabilization and gene transfer results and cell line dependence on gene expression rates. Successful siRNA electro-mediated delivery is also achieved. We demonstrate the applicability of our device for electroporation-mediated gene transfer of adherent cells in standard laboratory conditions. Finally, electrical impedance measurements during electroporation of CHO and 3T3-L1 cells are also given.

Gene expression profiling identifies molecular pathways associated with collagen VI deficiency and provides novel therapeutic targets.

Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.

Glucose dependence of glycogen synthase activity regulation by GSK3 and MEK/ERK inhibitors and angiotensin-(1-7) action on these pathways in cultured human myotubes.

Glycogen synthase (GS) is activated by glucose/glycogen depletion in skeletal muscle cells, but the contributing signaling pathways, including the chief GS regulator GSK3, have not been fully defined. The MEK/ERK pathway is known to regulate GSK3 and respond to glucose. The aim of this study was to elucidate the GSK3 and MEK/ERK pathway contribution to GS activation by glucose deprivation in cultured human myotubes. Moreover, we tested the glucose-dependence of GSK3 and MEK/ERK effects on GS and angiotensin (1-7) actions on these pathways. We show that glucose deprivation activated GS, but did not change phospho-GS (Ser640/1), GSK3ß activity or activity-activating phosphorylation of ERK1/2. We then treated glucose-replete and -depleted cells with SB415286, U0126, LY294 and rapamycin to inhibit GSK3, MEK1/2, PI3K and mTOR, respectively. SB415286 activated GS and decreased the relative phospho-GS (Ser640/1) level, more in glucose-depleted than -replete cells. U0126 activated GS and reduced the phospho-GS (Ser640/1) content significantly in glucose-depleted cells, while GSK3ß activity tended to increase. LY294 inactivated GS in glucose-depleted cells only, without affecting relative phospho-GS (Ser640/1) level. Rapamycin had no effect on GS activation. Angiotensin-(1-7) raised phospho-ERK1/2 but not phospho-GSK3ß (Ser9) content, while it inactivated GS and increased GS phosphorylation on Ser640/1, in glucose-replete cells. In glucose-depleted cells, angiotensin-(1-7) effects on ERK1/2 and GS were reverted, while relative phospho-GSK3ß (Ser9) content decreased. In conclusion, activation of GS by glucose deprivation is not due to GS Ser640/1 dephosphorylation, GSK3ß or ERK1/2 regulation in cultured myotubes. However, glucose depletion enhances GS activation/Ser640/1 dephosphorylation due to both GSK3 and MEK/ERK inhibition. Angiotensin-(1-7) inactivates GS in glucose-replete cells in association with ERK1/2 activation, not with GSK3 regulation, and glucose deprivation reverts both hormone effects. Thus, the ERK1/2 pathway negatively regulates GS activity in myotubes, without involving GSK3 regulation, and as a function of the presence of glucose.

Design and implementation of a microelectrode assembly for use on noncontact in situ electroporation of adherent cells.

In situ electroporation of adherent cells provides significant advantages with respect to electroporation systems for suspension cells, such as causing minimal stress to cultured cells and simplifying and saving several steps within the process. In this study, a new electrode assembly design is shown and applied to in situ electroporate adherent cell lines growing in standard multiwell plates. We designed an interdigitated array of electrodes patterned on copper with printed circuit board technology and covered with nickel/gold. Small interelectrode distances were used to achieve effective electroporation with low voltages. Epoxy-based microseparators were constructed to avoid direct contact with the cells and to create more uniform electric fields. The device was successful in the electropermeabilization of two different adherent cell lines, C2C12 and HEK 293, as assessed by the intracellular delivery of the fluorescent dextran FD20S. Additionally, as a collateral effect, we observed cell electrofusion in HEK 293 cells, thus making this device also useful for performing cell fusion. In summary, we show the effectiveness of this minimally invasive device for electroporation of adherent cells cultured in standard multiwell plates. The cheap technologies used in the fabrication process of the electrode assembly indicate potential use as a low-cost, disposable device.

PGC-1α induces mitochondrial and myokine transcriptional programs and lipid droplet and glycogen accumulation in cultured human skeletal muscle cells.

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a chief activator of mitochondrial and metabolic programs and protects against atrophy in skeletal muscle (skm). Here we tested whether PGC-1α overexpression could restructure the transcriptome and metabolism of primary cultured human skm cells, which display a phenotype that resembles the atrophic phenotype. An oligonucleotide microarray analysis was used to reveal the effects of PGC-1α on the whole transcriptome. Fifty-three different genes showed altered expression in response to PGC-1α: 42 upregulated and 11 downregulated. The main gene ontologies (GO) associated with the upregulated genes were mitochondrial components and processes and this was linked with an increase in COX activity, an indicator of mitochondrial content. Furthermore, PGC-1α enhanced mitochondrial oxidation of palmitate and lactate to CO(2), but not glucose oxidation. The other most significantly associated GOs for the upregulated genes were chemotaxis and cytokine activity, and several cytokines, including IL-8/CXCL8, CXCL6, CCL5 and CCL8, were within the most highly induced genes. Indeed, PGC-1α highly increased IL-8 cell protein content. The most upregulated gene was PVALB, which is related to calcium signaling. Potential metabolic regulators of fatty acid and glucose storage were among mainly regulated genes. The mRNA and protein level of FITM1/FIT1, which enhances the formation of lipid droplets, was raised by PGC-1α, while in oleate-incubated cells PGC-1α increased the number of smaller lipid droplets and modestly triglyceride levels, compared to controls. CALM1, the calcium-modulated δ subunit of phosphorylase kinase, was downregulated by PGC-1α, while glycogen phosphorylase was inactivated and glycogen storage was increased by PGC-1α. In conclusion, of the metabolic transcriptome deficiencies of cultured skm cells, PGC-1α rescued the expression of genes encoding mitochondrial proteins and FITM1. Several myokine genes, including IL-8 and CCL5, which are known to be constitutively expressed in human skm cells, were induced by PGC-1α.

Expression of glycogen phosphorylase isoforms in cultured muscle from patients with McArdle's disease carrying the p.R771PfsX33 PYGM mutation.

BACKGROUND: Mutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle's disease. Previous studies in muscle biopsies and cultured muscle cells from McArdle patients have shown that PYGM mutations abolish GP activity in skeletal muscle, but that the enzyme activity reappears when muscle cells are in culture. The identification of the GP isoenzyme that accounts for this activity remains controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this study we present two related patients harbouring a novel PYGM mutation, p.R771PfsX33. In the patients' skeletal muscle biopsies, PYGM mRNA levels were ∼60% lower than those observed in two matched healthy controls; biochemical analysis of a patient muscle biopsy resulted in undetectable GP protein and GP activity. A strong reduction of the PYGM mRNA was observed in cultured muscle cells from patients and controls, as compared to the levels observed in muscle tissue. In cultured cells, PYGM mRNA levels were negligible regardless of the differentiation stage. After a 12 day period of differentiation similar expression of the brain and liver isoforms were observed at the mRNA level in cells from patients and controls. Total GP activity (measured with AMP) was not different either; however, the active GP activity and immunoreactive GP protein levels were lower in patients' cell cultures. GP immunoreactivity was mainly due to brain and liver GP but muscle GP seemed to be responsible for the differences. CONCLUSIONS/SIGNIFICANCE: These results indicate that in both patients' and controls' cell cultures, unlike in skeletal muscle tissue, most of the protein and GP activities result from the expression of brain GP and liver GP genes, although there is still some activity resulting from the expression of the muscle GP gene. More research is necessary to clarify the differential mechanisms of metabolic adaptations that McArdle cultures undergo in vitro.

Comparative gene expression profiling between human cultured myotubes and skeletal muscle tissue.

BACKGROUND: A high-sensitivity DNA microarray platform requiring nanograms of RNA input facilitates the application of transcriptome analysis to individual skeletal muscle (SM) tissue samples. Culturing myotubes from SM-biopsies enables investigating transcriptional defects and assaying therapeutic strategies. This study compares the transcriptome of aneurally cultured human SM cells versus that of tissue biopsies. RESULTS: We used the Illumina expression BeadChips to determine the transcriptomic differences between tissue and cultured SM samples from five individuals. Changes in the expression of several genes were confirmed by QuantiGene Plex assay or reverse transcription real-time PCR. In cultured myotubes compared to the tissue, 1216 genes were regulated: 583 down and 633 up. Gene ontology analysis showed that downregulated genes were mainly associated with cytoplasm, particularly mitochondria, and involved in metabolism and the muscle-system/contraction process. Upregulated genes were predominantly related to cytoplasm, endoplasmic reticulum, and extracellular matrix. The most significantly regulated pathway was mitochondrial dysfunction. Apoptosis genes were also modulated. Among the most downregulated genes detected in this study were genes encoding metabolic proteins AMPD1, PYGM, CPT1B and UCP3, muscle-system proteins TMOD4, MYBPC1, MYOZ1 and XIRP2, the proteolytic CAPN3 and the myogenic regulator MYF6. Coordinated reduced expression of five members of the GIMAP gene family, which form a cluster on chromosome 7, was shown, and the GIMAP4-reduction was validated. Within the most upregulated group were genes encoding senescence/apoptosis-related proteins CDKN1A and KIAA1199 and potential regulatory factors HIF1A, TOP2A and CCDC80. CONCLUSIONS: Cultured muscle cells display reductive metabolic and muscle-system transcriptome adaptations as observed in muscle atrophy and they activate tissue-remodeling and senescence/apoptosis processes.

Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

Effects of aging and calorie restriction on rat skeletal muscle glycogen synthase and glycogen phosphorylase.

Calorie restriction's (CR) effects on age-associated changes in glycogen-metabolizing enzymes were studied in rat soleus (SOL) and tibialis anterior (TA) muscles. Old (24 months) compared to young (6 months) rats maintained ad libitum on a standard diet had reduced glycogen synthase (GS) activity, lower muscle GS protein levels, increased phosphorylation of GS at site 3a with less activation in SOL. Age-associated impairments in GS protein and activation-phosphorylation were also shown in TA. There was an age-associated reduction in glycogen phosphorylase (GP) activity level in SOL, while brain/muscle isoforms (B/M) of GP protein levels were higher. GP activity and protein levels were preserved, but GP was inactivated in TA with age. Glycogen content was unchanged in both muscles. CR did not alter GS or GP activity/protein levels in young rats. CR hindered age-related decreases in GS activity/protein, unrelated to GS mRNA levels, and GS inactivation-phosphorylation; not on GP. In older rats, CR enhanced glycogen accumulation in SOL. Short-term fasting did not recapitulate CR effects in old rats. Thus, the predominant age-associated impairments on skeletal muscle GS and GP activities occur in the oxidative SOL muscle of rats, and CR can attenuate the loss of GS activity/activation and stimulate glycogen accumulation.

FATP1 localizes to mitochondria and enhances pyruvate dehydrogenase activity in skeletal myotubes.

Fatty acid transport protein 1 (FATP1) has been previously immunolocalized in intracellular compartments. Here we show that FATP1 localizes to the mitochondria in cultured myotubes, by immunoblots of subcellular fractions and immunocytology of the fusion protein FATP1-GFP. FATP1 strongly stimulates CO(2) production from glucose whereas nonmitochondrial metabolism of glucose is only slightly enhanced. FATP1 raises the activity and activates the pyruvate dehydrogenase (PDH) complex and the pyruvate decarboxylase PDH-E1 catalytic subunit, without changing E2, E3BP or E1alpha and increasing E1beta protein content. These data reveals the localization and points to a regulatory function of FATP1 in myotube mitochondria.

Expression and glycogenic effect of glycogen-targeting protein phosphatase 1 regulatory subunit GL in cultured human muscle.

Glycogen-targeting PP1 (protein phosphatase 1) subunit G(L) (coded for by the PPP1R3B gene) is expressed in human, but not rodent, skeletal muscle. Its effects on muscle glycogen metabolism are unknown. We show that G(L) mRNA levels in primary cultured human myotubes are similar to those in freshly excised muscle, unlike subunits G(M) (gene PPP1R3A) or PTG (protein targeting to glycogen; gene PPP1R3C), which decrease strikingly. In cultured myotubes, expression of the genes coding for G(L), G(M) and PTG is not regulated by glucose or insulin. Overexpression of G(L) activates myotube GS (glycogen synthase), glycogenesis in glucose-replete and -depleted cells and glycogen accumulation. Compared with overexpressed G(M), G(L) has a more potent activating effect on glycogenesis, while marked enhancement of their combined action is only observed in glucose-replete cells. G(L) does not affect GP (glycogen phosphorylase) activity, while co-overexpression with muscle GP impairs G(L) activation of GS in glucose-replete cells. G(L) enhances long-term glycogenesis additively to glucose depletion and insulin, although G(L) does not change the phosphorylation of GSK3 (GS kinase 3) on Ser9 or its upstream regulator kinase Akt/protein kinase B on Ser473, nor its response to insulin. In conclusion, in cultured human myotubes, the G(L) gene is expressed as in muscle tissue and is unresponsive to glucose or insulin, as are G(M) and PTG genes. G(L) activates GS regardless of glucose, does not regulate GP and stimulates glycogenesis in combination with insulin and glucose depletion.

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.

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.

Fiber type- and fatty acid composition-dependent effects of high-fat diets on rat muscle triacylglyceride and fatty acid transporter protein-1 content.

Intramuscular triacylglyceride (TAG) is considered an independent marker of insulin resistance in humans. Here, we examined the effect of high-fat diets, based on distinct fatty acid compositions (saturated, monounsaturated or n-6 polyunsaturated), on TAG levels and fatty acid transporter protein (FATP-1) expression in 2 rat muscles that differ in their fiber type, soleus, and gastrocnemius; the relationship to whole body glucose intolerance was also studied. Compared with carbohydrate-fed rats, the groups subjected to any one of the high-fat diets consistently exhibited enhanced body weight gain and adiposity, elevated plasma free fatty acids and TAG in the fed condition, hyperinsulinemia, and glucose intolerance. TAG content was consistently higher in soleus than in gastrocnemius, but was only significantly elevated by the n-6 polyunsaturated-based diet. FATP-1 levels in soleus were double those in gastrocnemius muscle in carbohydrate-fed animals. High-fat diets caused an elevation in FATP-1 protein content in soleus, but a reduction in gastrocnemius. In conclusion, the hyperinsulinemic hyperlipidemic condition upregulates FATP-1 expression in soleus and downregulates that of gastrocnemius. Hypercaloric saturated, monounsaturated, or n-6 polyunsaturated lipid diets cause equivalent whole body insulin resistance in rats, but only an n-6 polyunsaturated acid-based diet triggers intramuscular TAG accumulation.

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.

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.