GNIP1 E3 ubiquitin ligase is a novel player in regulating glycogen metabolism in skeletal muscle.

BACKGROUND: Glycogenin-interacting protein 1 (GNIP1) is a tripartite motif (TRIM) protein with E3 ubiquitin ligase activity that interacts with glycogenin. These data suggest that GNIP1 could play a major role in the control of glycogen metabolism. However, direct evidence based on functional analysis remains to be obtained. OBJECTIVES: The aim of this study was 1) to define the expression pattern of glycogenin-interacting protein/Tripartite motif containing protein 7 (GNIP/TRIM7) isoforms in humans, 2) to test their ubiquitin E3 ligase activity, and 3) to analyze the functional effects of GNIP1 on muscle glucose/glycogen metabolism both in human cultured cells and in vivo in mice. RESULTS: We show that GNIP1 was the most abundant GNIP/TRIM7 isoform in human skeletal muscle, whereas in cardiac muscle only TRIM7 was expressed. GNIP1 and TRIM7 had autoubiquitination activity in vitro and were localized in the Golgi apparatus and cytosol respectively in LHCN-M2 myoblasts. GNIP1 overexpression increased glucose uptake in LHCN-M2 myotubes. Overexpression of GNIP1 in mouse muscle in vivo increased glycogen content, glycogen synthase (GS) activity and phospho-GSK-3α/ß (Ser21/9) and phospho-Akt (Ser473) content, whereas decreased GS phosphorylation in Ser640. These modifications led to decreased blood glucose levels, lactate levels and body weight, without changing whole-body insulin or glucose tolerance in mouse. CONCLUSION: GNIP1 is an ubiquitin ligase with a markedly glycogenic effect in skeletal muscle.

Double deficiency of Trex2 and DNase1L2 nucleases leads to accumulation of DNA in lingual cornifying keratinocytes without activating inflammatory responses.

The cornification of keratinocytes on the surface of skin and oral epithelia is associated with the degradation of nuclear DNA. The endonuclease DNase1L2 and the exonuclease Trex2 are expressed specifically in cornifying keratinocytes. Deletion of DNase1L2 causes retention of nuclear DNA in the tongue epithelium but not in the skin. Here we report that lack of Trex2 results in the accumulation of DNA fragments in the cytoplasm of cornifying lingual keratinocytes and co-deletion of DNase1L2 and Trex2 causes massive accumulation of DNA fragments throughout the cornified layers of the tongue epithelium. By contrast, cornification-associated DNA breakdown was not compromised in the epidermis. Aberrant retention of DNA in the tongue epithelium was associated neither with enhanced expression of DNA-driven response genes, such as Ifnb, Irf7 and Cxcl10, nor with inflammation. Of note, the expression of Tlr9, Aim2 and Tmem173, key DNA sensor genes, was markedly lower in keratinocytes and keratinocyte-built tissues than in macrophages and immune tissues, and DNA-driven response genes were not induced by introduction of DNA in keratinocytes. Altogether, our results indicate that DNase1L2 and Trex2 cooperate in the breakdown and degradation of DNA during cornification of lingual keratinocytes and aberrant DNA retention is tolerated in the oral epithelium.

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.

Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells.

BACKGROUND: 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/G(M). RESULTS: PPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than G(M). PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and G(M) (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6- and G(M)-derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane- and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of ß-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location. CONCLUSIONS: PPP1R6 exerts a powerful glycogenic effect in cultured muscle cells, more than G(M) and less than PTG. PPP1R6 protein translocates from a Golgi to cytosolic location in response to glucose. The molecular size and subcellular location of myotube glycogen particles is determined by the PPP1R6, PTG and G(M) scaffolding.

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.

UCP3 overexpression neutralizes oxidative stress rather than nitrosative stress in mouse myotubes.

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.

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.

The use of voriconazole for the treatment of aspergillosis in falcons (Falco species).

To evaluate the clinical efficacy and safety of voriconazole for the treatment of aspergillosis in falcons, 20 falcons with aspergillosis admitted to the Dubai Falcon Hospital from August 2003 to May 2006 were treated with voriconazole. These falcons included 6 gyrfalcons (Falco rusticolus), 10 gyrfalcon hybrids, 1 lanner (Falco biarmicus), 1 saker (Falco cherrug), and 2 peregrine falcons (Falco peregrinus). Clinical signs were weight loss, inappetence, dyspnea, inspiratory stridor, tachypnea, and biliverdinuria. Aspergillosis was diagnosed from clinical signs, hematologic results, radiographic abnormalities, endoscopic examination of the lower respiratory tract, cytologic examination of biopsy samples from air sacs, and fungal cultures. Birds treated with voriconazole administered by crop gavage were divided into 2 groups: in group 1, birds were treated with 12.5 mg/kg q12 h for 3 days (loading dose), then q24h for an additional 18 to 87 days; in group 2, birds were treated with 12.5 mg/kg ql2h for the full period of 44 to 100 days. Treatment with voriconazole resulted in a successful clinical response in most cases, an acceptable survival rate, and few adverse effects. Complete clinical resolution occurred in 14 birds (70%), a partial response in 5 birds (25%), and 1 bird (5%) died during treatment. From these results, voriconazole appeared to be effective and safe for the treatment of aspergillosis in some species of falcons.

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.

The AP-1/CJUN signaling cascade is involved in muscle differentiation: implications in muscle wasting during cancer cachexia.

The aim of the present study was to investigate a possible role of the AP-1 signaling cascade in the process of wasting associated with cancer cachexia at the level of skeletal muscle. The injection of virus containing the TAM67 protein (a blocker of the AP-1 protein) to the gastrocnemius muscle of tumour-bearing rats resulted in a significant recovery of the muscle mass (which is dramatically reduced as a result of tumour burden), therefore suggesting that AP-1 is certainly involved in the signaling associated with muscle protein accretion. In conclusion, the gene therapy approach presented here clearly suggests an important role for AP-1 in muscle signaling during catabolic states.

Overexpression of UCP3 in both murine and human myotubes is linked with the activation of proteolytic systems: a role in muscle wasting?

Overexpression of the UCP3 gene in both murine and human myotube cell cultures leads to a significant activation of the different proteolytic systems involved in muscle myofibrillar protein breakdown. Thus, lysosomal (cathepsin B) and non-lysosomal (m-calpain and ubiquitin-proteasome) mRNA content was significantly increased in the different cell culture systems used. Interestingly, the overexpression of the UCP3 gene was not associated with any changes in apoptosis. Although the function of the UCP3 protein is not completely understood (uncoupling, oxidative stress), these results suggest a possible relation between these main mechanisms involved in muscle wasting during cancer.

Mediators involved in the cancer anorexia-cachexia syndrome: past, present, and future.

The cachectic syndrome, characterized by a marked weight loss, anorexia, asthenia, and anemia is invariably associated with the presence and growth of the tumor and leads to a malnutrition status due to the induction of anorexia or decreased food intake. In addition, the competition for nutrients between the tumor and the host leads to an accelerated starvation state, which promotes severe metabolic disturbances in the host, including hypermetabolism, which leads to an increased energetic inefficiency. Although the search for the cachectic factor(s) started a long time ago, and although many scientific and economic efforts have been devoted to its discovery, we are still a long way from knowing the whole truth. Present investigation is devoted to revealing the different signaling pathways, in particular transcriptional factors involved in muscle wasting. The main aim of the present review is to summarize and evaluate the different molecular mechanisms and catabolic mediators (both humoral and tumoral) involved in cancer cachexia since they may represent targets for future promising clinical investigations.

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.

Experiencia preventiva del médico y la enfermera en el contingente "Flor Crombet" / An experience of the role played by doctor and nurse on health prevention at the Folr Crobet contingent

Se realiza un estudio con 100 trabajadores del Contingente "Flor Crombet" del municipio Yateras, provincia de Guantánamo. De los trabajadores encuestados (96 hombres y 4 mujeres), todos poseen carné de salud actualizado y a la mayoría de ellos se los han realizado el médico y la enfermera, lo que evita su traslado al policlínico y la pérdida de tiempo de su actividad laboral. Al realizar visitas a los puestos de trabajo, se observa que los obreros que no emplean medios de protección tienen conciencia de la importancia de su uso; se orienta que laboren en lugares con menos riesgo - medida cumplida de inmediato-, pues no existe falta de gestión, sino carencia de estos medios de protección en la empresa. En cuanto a la educación sanitaria la participación de los obreros es del 100 por ciento (AU)

Experiencia preventiva del médico y la enfermera en el contingente "Flor Crombet" / An experience of the role played by doctor and nurse on health prevention at the Folr Crombet contingent

Se realiza un estudio con 100 trabajadores del Contingente "Flor Crombet" del municipio Yateras, provincia de Guantánamo. De los trabajadores encuestados (96 hombres y 4 mujeres), todos poseen carné de salud actualizado y a la mayoría de ellos se los han realizado el médico y la enfermera, lo que evita su traslado al policlínico y la pérdida de tiempo de su actividad laboral. Al realizar visitas a los puestos de trabajo, se observa que los obreros que no emplean medios de protección tienen conciencia de la importancia de su uso; se orienta que laboren en lugares con menos riesgo - medida cumplida de inmediato-, pues no existe falta de gestión, sino carencia de estos medios de protección en la empresa. En cuanto a la educación sanitaria la participación de los obreros es del 100 por ciento