Are cultured human myotubes far from home?

Satellite cells can be isolated from skeletal muscle biopsies, activated to proliferating myoblasts and differentiated into multinuclear myotubes in culture. These cell cultures represent a model system for intact human skeletal muscle and can be modulated ex vivo. The advantages of this system are that the most relevant genetic background is available for the investigation of human disease (as opposed to rodent cell cultures), the extracellular environment can be precisely controlled and the cells are not immortalized, thereby offering the possibility of studying innate characteristics of the donor. Limitations in differentiation status (fiber type) of the cells and energy metabolism can be improved by proper treatment, such as electrical pulse stimulation to mimic exercise. This review focuses on the way that human myotubes can be employed as a tool for studying metabolism in skeletal muscles, with special attention to changes in muscle energy metabolism in obesity and type 2 diabetes.     

Eicosapentaenoic acid (20:5 n-3) increases fatty acid and glucose uptake in cultured human skeletal muscle cells

This study was conducted to evaluate the chronic effects of eicosapentaenoic acid (EPA) on fatty acid and glucose metabolism in human skeletal muscle cells. Uptake of [14C]oleate was increased >2-fold after preincubation of myotubes with 0.6 mM EPA for 24 h, and incorporation into various lipid classes showed that cellular triacylgycerol (TAG) and phospholipids were increased 2- to 3-fold compared with control cells. After exposure to oleic acid (OA), TAG was increased 2-fold. Insulin (100 nM) further increased the incorporation of [14C]oleate into all lipid classes for EPA-treated myotubes. Fatty acid beta-oxidation was unchanged, and complete oxidation (CO2) decreased in EPA-treated cells. Basal glucose transport and oxidation (CO2) were increased 2-fold after EPA, and insulin (100 nM) stimulated glucose transport and oxidation similarly in control and EPA-treated myotubes, whereas these responses to insulin were abolished after OA treatment. Lower concentrations of EPA (0.1 mM) also increased fatty acid and glucose uptake. CD36/FAT (fatty acid transporter) mRNA expression was increased after EPA and OA treatment compared with control cells. Moreover, GLUT1 expression was increased 2.5-fold by EPA, whereas GLUT4 expression was unchanged, and activities of the mitogen-activated protein kinase p38 and extracellular signal-regulated kinase were decreased after treatment with OA compared with EPA. Together, our data show that chronic exposure of myotubes to EPA promotes increased uptake and oxidation of glucose despite a markedly increased fatty acid uptake and synthesis of complex lipids.     

Identifying the drivers of multidrug-resistant Klebsiella pneumoniae at a European level

Beta-lactam- and in particular carbapenem-resistant Enterobacteriaceae represent a major public health threat. Despite strong variation of resistance across geographical settings, there is limited understanding of the underlying drivers. To assess these drivers, we developed a transmission model of cephalosporin- and carbapenem-resistant Klebsiella pneumoniae. The model is parameterized using antibiotic consumption and demographic data from eleven European countries and fitted to the resistance rates for Klebsiella pneumoniae for these settings. The impact of potential drivers of resistance is then assessed in counterfactual analyses. Based on reported consumption data, the model could simultaneously fit the prevalence of extended-spectrum beta-lactamase-producing and carbapenem-resistant Klebsiella pneumoniae (ESBL and CRK) across eleven European countries over eleven years. The fit could explain the large between-country variability of resistance in terms of consumption patterns and fitted differences in hospital transmission rates. Based on this fit, a counterfactual analysis found that reducing nosocomial transmission and antibiotic consumption in the hospital had the strongest impact on ESBL and CRK prevalence. Antibiotic consumption in the community also affected ESBL prevalence but its relative impact was weaker than inpatient consumption. Finally, we used the model to estimate a moderate fitness cost of CRK and ESBL at the population level. This work highlights the disproportionate role of antibiotic consumption in the hospital and of nosocomial transmission for resistance in gram-negative bacteria at a European level. This indicates that infection control and antibiotic stewardship measures should play a major role in limiting resistance even at the national or regional level.     

Myotubes from Severely Obese Type 2 Diabetic Subjects Accumulate Less Lipids and Show Higher Lipolytic Rate than Myotubes from Severely Obese Non-Diabetic Subjects

About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several severely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to fundamental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexibility were examined in human myotubes derived from severely obese subjects without (BMI 44±7 kg/m²) and with type 2 diabetes (BMI 43±6 kg/m²). Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to severely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also similar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher lipolysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes.     

Characterization of a Novel Flavivirus from Mosquitoes in Northern Europe That Is Related to Mosquito-Borne Flaviviruses of the Tropics

A novel flavivirus was isolated from mosquitoes in Finland, representing the first mosquito-borne flavivirus from Northern Europe. The isolate, designated Lammi virus (LAMV), was antigenically cross-reactive with other flaviviruses and exhibited typical flavivirus morphology as determined by electron microscopy. The genomic sequence of LAMV was highly divergent from the recognized flaviviruses, and yet the polyprotein properties resembled those of mosquito-borne flaviviruses. Phylogenetic analysis of the complete coding sequence showed that LAMV represented a distinct lineage related to the Aedes sp.-transmitted human pathogenic flaviviruses, similarly to the newly described Nounané virus (NOUV), a flavivirus from Africa (S. Junglen et al., J. Virol. 83:4462-4468, 2009). Despite the low sequence homology, LAMV and NOUV were phylogenetically grouped closely, likely representing separate species of a novel group of flaviviruses. Despite the biological properties preferring replication in mosquito cells, the genetic relatedness of LAMV to viruses associated with vertebrate hosts warrants a search for disease associations.The genus Flavivirus in the family Flaviviridae consists of 53 recognized virus species that are enveloped, positive-sense single-stranded RNA viruses. The virion consists of three structural proteins: capsid (C), membrane (M), and envelope (E). In addition, seven nonstructural proteins are present in infected cells (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). Based on their antigenic properties and vector associations, flaviviruses have been grouped into mosquito-borne, tick-borne, and no-known-vector viruses and have been isolated from vertebrates, bats, and rodents (15, 25). The grouping of flaviviruses according to their transmission mode is strongly supported by phylogenetic analyses of their genomic sequences (9, 18, 31).Mosquito-borne flaviviruses are a large and divergent group of viruses that can be differentiated phylogenetically into those associated either with encephalitic disease and transmission by Culex spp. mosquitoes or with diseases with hemorrhagic complications and transmission by Aedes spp. (18). Seven groups of mosquito-borne flaviviruses, namely, the Aroa, dengue, Japanese encephalitis, Kokobera, Ntaya, Spondweni, and yellow fever virus groups are recognized (15, 25). These groups include important animal and human pathogens such as dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV).Unclassified insect flaviviruses that have no recognized association with vertebrates have been isolated from a variety of mosquito species and also from mosquito cell lines. These insect flaviviruses do not appear to infect vertebrate cells and are not associated with human or animal disease. The cell fusing agent virus (CFAV), a tentative species in the genus Flavivirus, was the first of these insect viruses to be characterized (5, 40), Although CFAV was originally identified in cultured mosquito cells, it was later isolated from mosquitoes collected in Puerto Rico (7). This as-yet-unclassified insect flavivirus group now also includes Kamiti river virus (KRV) isolated in Kenya (10, 38) and a virus isolated from Culex spp. in Japan, designated culex flavivirus (22). In addition, related viral sequences or isolates have been recently reported from mosquitoes in Spain (1), the United States and Trinidad (26), and Mexico (14). Moreover, the identification of flaviviruslike sequences integrated within the genomes of Aedes mosquitoes further complicates the evolutionary history of the flaviviruses. These sequences, currently referred to as cell silent agent are genetically most closely related to CFAV and possibly share common evolutionary origin (11). Phylogenetically, the insect viruses form a divergent outgroup that may represent a primordial flavivirus lineage. Apart from the insect flaviviruses, the other recently discovered novel flaviviruses represent highly divergent lineages, such as Tamana bat virus (13), and Ngoye virus (20). Recently, a novel flavivirus, Nounané virus (NOUV) was isolated from a novel mosquito vector species, Uranotaenia mashonaensis in Côte d''Ivoire (23), and was shown to be phylogenetically related to the human pathogenic mosquito-borne flaviviruses.Several arboviruses have been reported from Northern Europe including the flavivirus tick-borne encephalitis virus (24, 36) but, to date, no mosquito-borne flaviviruses have been isolated. Our aim was to screen for arboviruses in Finland by studying mosquitoes using virus isolation and subsequent arbovirus antigen detection, which resulted in the identification of a novel flavivirus. We present here the isolation and characterization of this isolate, designated Lammi virus (LAMV), and discuss the implications of our findings.     

Cost-Effectiveness of “Golden Mustard” for Treating Vitamin A Deficiency in India

Background

Vitamin A deficiency (VAD) is an important nutritional problem in India, resulting in an increased risk of severe morbidity and mortality. Periodic, high-dose vitamin A supplementation is the WHO-recommended method to prevent VAD, since a single dose can compensate for reduced dietary intake or increased need over a period of several months. However, in India only 34 percent of targeted children currently receive the two doses per year, and new strategies are urgently needed.

Methodology

Recent advancements in biotechnology permit alternative strategies for increasing the vitamin A content of common foods. Mustard (Brassica juncea), which is consumed widely in the form of oil by VAD populations, can be genetically modified to express high levels of beta-carotene, a precursor to vitamin A. Using estimates for consumption, we compare predicted costs and benefits of genetically modified (GM) fortification of mustard seed with high-dose vitamin A supplementation and industrial fortification of mustard oil during processing to alleviate VAD by calculating the avertable health burden in terms of disability-adjusted life years (DALY).

Principal Findings

We found that all three interventions potentially avert significant numbers of DALYs and deaths. Expanding vitamin A supplementation to all areas was the least costly intervention, at $23–$50 per DALY averted and $1,000–$6,100 per death averted, though cost-effectiveness varied with prevailing health subcenter coverage. GM fortification could avert 5 million–6 million more DALYs and 8,000–46,000 more deaths, mainly because it would benefit the entire population and not just children. However, the costs associated with GM fortification were nearly five times those of supplementation. Industrial fortification was dominated by both GM fortification and supplementation. The cost-effectiveness ratio of each intervention decreased with the prevalence of VAD and was sensitive to the efficacy rate of averted mortality.

Conclusions

Although supplementation is the least costly intervention, our findings also indicate that GM fortification could reduce the VAD disease burden to a substantially greater degree because of its wider reach. Given the difficulties in expanding supplementation to areas without health subcenters, GM fortification of mustard seed is an attractive alternative, and further exploration of this technology is warranted.     

Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1^gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1^gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1^gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1^gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage.KEY WORDS: NCU-G1, Lysosome, Fibrosis     

Influenza A H1N1 Pandemic Strain Evolution – Divergence and the Potential for Antigenic Drift Variants

The emergence of a novel A(H1N1) strain in 2009 was the first influenza pandemic of the genomic age, and unprecedented surveillance of the virus provides the opportunity to better understand the evolution of influenza. We examined changes in the nucleotide coding regions and the amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) segments of the A(H1N1)pdm09 strain using publicly available data. We calculated the nucleotide and amino acid hamming distance from the vaccine strain A/California/07/2009 for each sequence. We also estimated P_epitope–a measure of antigenic diversity based on changes in the epitope regions–for each isolate. Finally, we compared our results to A(H3N2) strains collected over the same period. Our analysis found that the mean hamming distance for the HA protein of the A(H1N1)pdm09 strain increased from 3.6 (standard deviation [SD]: 1.3) in 2009 to 11.7 (SD: 1.0) in 2013, while the mean hamming distance in the coding region increased from 7.4 (SD: 2.2) in 2009 to 28.3 (SD: 2.1) in 2013. These trends are broadly similar to the rate of mutation in H3N2 over the same time period. However, in contrast to H3N2 strains, the rate of mutation accumulation has slowed in recent years. Our results are notable because, over the course of the study, mutation rates in H3N2 similar to that seen with A(H1N1)pdm09 led to the emergence of two antigenic drift variants. However, while there has been an H1N1 epidemic in North America this season, evidence to date indicates the vaccine is still effective, suggesting the epidemic is not due to the emergence of an antigenic drift variant. Our results suggest that more research is needed to understand how viral mutations are related to vaccine effectiveness so that future vaccine choices and development can be more predictive.     

22-Hydroxycholesterols regulate lipid metabolism differently than T0901317 in human myotubes

The nuclear liver X receptors (LXRalpha and beta) are regulators of lipid and cholesterol metabolism. Oxysterols are known LXR ligands, but the functional role of hydroxycholesterols is at present unknown. In human myotubes, chronic exposure to the LXR ligand T0901317 promoted formation of diacylglycerol (DAG) and triacylglycerol (TAG), 22-R-hydroxycholesterol (22-R-HC) had no effect, and 22-S-hydroxycholesterol (22-S-HC) reduced the formation. In accordance with this, 22-HC and T0901317 regulated the expression of fatty acid transporter CD36, stearoyl-CoA desaturase-1, acyl-CoA synthetase long chain family member 1 and fatty acid synthase (FAS) differently; all genes were increased by T0901317, 22-R-HC did not change their expression level, while 22-S-HC reduced it. Transfection studies confirmed that the FAS promoter was activated by T0901317 and repressed by 22-S-HC through an LXR response element in the promoter. Both 22-R-HC and T0901317 increased gene expression of LXRalpha, sterol regulatory element-binding protein 1c and ATP-binding cassette transporter A1, while 22-S-HC had little effect. In summary, 22-R-HC regulated lipid metabolism and mRNA expression of some LXR target genes in human myotubes differently than T0901317. Moreover, 22-S-HC did not behave like an inactive ligand; it reduced synthesis of complex lipids and repressed certain genes involved in lipogenesis and lipid handling.     

Remodeling of Oxidative Energy Metabolism by Galactose Improves Glucose Handling and Metabolic Switching in Human Skeletal Muscle Cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [¹⁴C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [¹⁴C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments.