Cèlia García-Martínez

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 β-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 β-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 β-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.

PPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/GM.

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.

The deleterious effects of oxidants on proteins may be modified by overexpression of uncoupling protein 3 (UCP3) in skeletal muscle cells exposed to hyperoxia or H ₂ O ₂ . UCP3 overexpression significantly attenuated the increase in protein carbonylation in response to hyperoxia and H ₂ O ₂ exposures. However, antioxidant enzyme content and activity (superoxide dismutases, peroxiredoxins, glutathione peroxidase‐I, and catalase) were reduced or not modified in UCP3‐overexpressing myotubes exposed to oxidants. Protein nitration increased in UCP3‐overexpressing cells exposed to hyperoxia, but not to H ₂ O ₂ . We conclude that protein oxidation rather than nitration is neutralized by UPC3 overexpression in mouse myotubes exposed to abundant reactive oxygen species.

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.