Galactose Effects on Human Muscle Cell Metabolism Figure 6. Absence of an increase in cytochrome C oxidase activity and AMPK phosphorylation in post-diabetic myotubes differentiated in galactose media. A. Mitochondrial yield measured in post-diabetic myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Mitochondrial yield was determined as mitochondrial protein content per total cellular protein content. Results are presented as means 6 SEM, n = 4. *, p,0.05, LG vs HG, **, p,0.01 LG vs GAL. B. Cytochrome C oxidase activity measured in isolated mitochondria from post-diabetic myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Results are presented as means 6 SEM, n = 4. *, p,0.05, GAL vs HG and LG. C. Top panel: representative Western blot of Complex IV expression in post-diabetic myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Beta-actin was used as a loading control. Bottom panel: quantification by densitometry of complex IV expression. Data are presented normalized to beta-actin expression. Data are shown as mean 6SEM, n = 4. D. Left panel: representative Western blot of P-AMPK expression in post-diabetic myotubes differentiated for 7 days in HG (25 mM glucose), LG (5 mM glucose) or GAL (10 mM galactose). Beta-actin was used as loading controls. Right panels: quantification by densitometry of P-AMPK. Data are presented normalized to beta-actin and AMPKa2 expression. Data are shown as mean 6SEM, n = 4. doi:10.1371/journal.pone.0028536.g006 obese non-diabetic individuals [40]. Despite this decrease oxidative capacity [5,9,40], no difference in OCR was found in the present study between obese non-diabetic and post-diabetic myotubes differentiated in either HG or LG. This is presumably due to the fact that under these conditions, myotubes were highly glycolytic rendering it more difficult to detect a decrease in OCR in post-diabetic compared to obese non-diabetic myotubes. However, the GAL medium was able to identify decreased OCR in post-diabetic myotubes, revealing the utility of this approach to detect mitochondrial dysfunction in vitro. Our results also confirm that post-diabetic myotubes display an abnormal metabolic flexibility when challenged with different substrates (here with galactose) as previously published by others who studied myotubes derived from (post-)diabetic, insulin resistant or obese patients when challenged with high fat levels [9,11,41] or high glucose levels [40]. In the present study, the lack of an increase in OCR in post-diabetic myotubes appears to be related to unaltered COX expression or activity levels or AMPK phosphorylation. Another study showed that myotubes derived from diabetic patients lack the capacity to activate the AMPK pathway (measured as acetyl-CoA carboxylase phosphorylation) in response to of palmitate, leading to an absence of any increase in myotubes palmitate oxidation [9]. It seems that myotubes derived from (post-) diabetic patients have a impaired AMPK activity in response to different AMPK-stimulating agents. Other studies will be needed to determine whether AMPK stimulation is truly impaired in (post-) diabetic muscle and to determine the mechanisms causing AMPK ‘‘dysfunction’’. Interestingly, we also found a significant decrease in non- mitochondrial OCR in post-diabetic myotubes compared to matched obese non-diabetic myotubes, independently of the source of carbohydrates used in the differentiation medium. This result is in accordance with a study from our group showing perturbations in NADPH production due to an impaired glucose- 6-phosphate dehydrogenase in post-diabetic myotubes [42]. This could be due to a defect in the pentose phosphate pathway in post- diabetic myotubes. Another hypothesis explaining the lower non- mitochondrial OCR in post-diabetic myotubes compared to obese control myotubes is a decrease in NADPH oxidase protein content or activity. NADPH oxidase produces superoxide by coupling their electrons to oxygen. Thus this is an enzyme involved in reactive oxygen species production (ROS). Interestingly, the measurement of ROS (by the DCFH-DA assay) in post-diabetic and obese non-diabetic myotubes showed lower ROS content in PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28536