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BackgroundHuman primary myotubes are highly glycolytic when cultured in high glucose medium rendering it difficult to study mitochondrial dysfunction. Galactose is known to enhance mitochondrial metabolism and could be an excellent model to study mitochondrial dysfunction in human primary myotubes. The aim of the present study was to 1) characterize the effect of differentiating healthy human myoblasts in galactose on oxidative metabolism and 2) determine whether galactose can pinpoint a mitochondrial malfunction in post-diabetic myotubes.Methodology/Principal FindingsOxygen consumption rate (OCR), lactate levels, mitochondrial content, citrate synthase and cytochrome C oxidase activities, and AMPK phosphorylation were determined in healthy myotubes differentiated in different sources/concentrations of carbohydrates: 25 mM glucose (high glucose (HG)), 5 mM glucose (low glucose (LG)) or 10 mM galactose (GAL). Effect of carbohydrates on OCR was also determined in myotubes derived from post-diabetic patients and matched obese non-diabetic subjects. OCR was significantly increased whereas anaerobic glycolysis was significantly decreased in GAL myotubes compared to LG or HG myotubes. This increased OCR in GAL myotubes occurred in conjunction with increased cytochrome C oxidase activity and expression, as well as increased AMPK phosphorylation. OCR of post-diabetic myotubes was not different than that of obese non-diabetic myotubes when differentiated in LG or HG. However, whereas GAL increased OCR in obese non-diabetic myotubes, it did not affect OCR in post-diabetic myotubes, leading to a significant difference in OCR between groups. The lack of an increase in OCR in post-diabetic myotubes differentiated in GAL was in relation with unaltered cytochrome C oxidase activity levels or AMPK phosphorylation.Conclusions/SignificanceOur results indicate that differentiating human primary myoblasts in GAL enhances aerobic metabolism. Because this cell culture model elicited an abnormal response in cells from post-diabetic patients, it may be useful in further studies of the molecular mechanisms of mitochondrial dysfunction.

Body weight is a quantitative trait with significant heritability in humans. To identify potential genetic contributors to this phenotype, we resequenced the coding exons and splice junctions of 58 genes in 379 obese and 378 lean individuals. Our 96-Mb survey included 21 genes associated with monogenic forms of obesity in humans or mice, as well as 37 genes that function in body weight-related pathways. We found that the monogenic obesity-associated gene group was enriched for rare nonsynonymous variants unique to the obese population compared with the lean population. In addition, computational analysis predicted a greater fraction of deleterious variants within the obese cohort. Together, these data suggest that multiple rare alleles contribute to obesity in the population and provide a medical sequencing-based approach to detect them.

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Functionally significant heterozygous mutations in the Melanocortin-4 receptor (MC4R) have been implicated in 2.5% of early onset obesity cases in European cohorts. The role of mutations in this gene in severely obese adults, particularly in smaller North American patient cohorts, has been less convincing. More recently, it has been proposed that mutations in a phylogenetically and physiologically related receptor, the Melanocortin-3 receptor (MC3R), could also be a cause of severe human obesity. The objectives of this study were to determine if mutations impairing the function of MC4R or MC3R were associated with severe obesity in North American adults. We studied MC4R and MC3R mutations detected in a total of 1821 adults (889 severely obese and 932 lean controls) from two cohorts. We systematically and comparatively evaluated the functional consequences of all mutations found in both MC4R and MC3R. The total prevalence of rare MC4R variants in severely obese North American adults was 2.25% (CI(95%): 1.44-3.47) compared with 0.64% (CI(95%): 0.26-1.43) in lean controls (P < 0.005). After classification of functional consequence, the prevalence of MC4R mutations with functional alterations was significantly greater when compared with controls (P < 0.005). In contrast, the prevalence of rare MC3R variants was not significantly increased in severely obese adults [0.67% (CI(95%): 0.27-1.50) versus 0.32% (CI(95%): 0.06-0.99)] (P = 0.332). Our results confirm that mutations in MC4R are a significant cause of severe obesity, extending this finding to North American adults. However, our data suggest that MC3R mutations are not associated with severe obesity in this population.