Key points• Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects.• The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein, and complex I-IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres).• Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content.• mtDNA was found to be a poor biomarker of mitochondrial content.• Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.Abstract Skeletal muscle mitochondrial content varies extensively between human subjects. Biochemical measures of mitochondrial proteins, enzyme activities and lipids are often used as markers of mitochondrial content and muscle oxidative capacity (OXPHOS). The purpose of this study was to determine how closely associated these commonly used biochemical measures are to muscle mitochondrial content and OXPHOS. Sixteen young healthy male subjects were recruited for this study. Subjects completed a graded exercise test to determine maximal oxygen uptake (V O 2 peak ) and muscle biopsies were obtained from the vastus lateralis. Mitochondrial content was determined using transmission electron microscopy imaging and OXPHOS was determined as the maximal coupled respiration in permeabilized fibres. Biomarkers of interest were citrate synthase (CS) activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I-V protein content, and complex I-IV activity. Spearman correlation coefficient tests and Lin's concordance tests were applied to assess the absolute and relative association between the markers and mitochondrial content or OXPHOS. Subjects had a large range ofV O 2 peak (range 29.9-71.6 ml min −1 kg −1 ) and mitochondrial content (4-15% of cell volume). Cardiolipin content showed the strongest association with mitochondrial content followed by CS and complex I activities. mtDNA was not related to mitochondrial content. Complex IV activity showed the strongest association with muscle oxidative capacity followed by complex II activity. We conclude that cardiolipin content, and CS and complex I activities are the biomarkers that exhibit the strongest association with mitochondrial content, while complex IV activity is strongly associated with OXPHOS capacity in human skeletal muscle.

Adipose tissue exerts important endocrine and metabolic functions in health and disease. Yet the bioenergetics of this tissue is not characterized in humans and possible regional differences are not elucidated. Using high resolution respirometry, mitochondrial respiration was quantified in human abdominal subcutaneous and intra-abdominal visceral (omentum majus) adipose tissue from biopsies obtained in 20 obese patients undergoing bariatric surgery. Mitochondrial DNA (mtDNA) and genomic DNA (gDNA) were determined by the PCR technique for estimation of mitochondrial density. Adipose tissue samples were permeabilized and respirometric measurements were performed in duplicate at 37• C. Substrates (glutamate (G) + malate (M) + octanoyl carnitine (O) + succinate (S)) were added sequentially to provide electrons to complex I + II. ADP ( D ) for state 3 respiration was added after GM. Uncoupled respiration was measured after addition of FCCP. Visceral fat contained more mitochondria per milligram of tissue than subcutaneous fat, but the cells were smaller. Robust, stable oxygen fluxes were found in both tissues, and coupled state 3 (GMOS D ) and uncoupled respiration were significantly (P < 0.05) higher in visceral (0.95 ± 0.05 and 1.15 ± 0.06 pmol O 2 s −1 mg −1 , respectively) compared with subcutaneous (0.76 ± 0.04 and 0.98 ± 0.05 pmol O 2 s −1 mg −1 , respectively) adipose tissue. Expressed per mtDNA, visceral adipose tissue had significantly (P < 0.05) lower mitochondrial respiration. Substrate control ratios were higher and uncoupling control ratio lower (P < 0.05) in visceral compared with subcutaneous adipose tissue. We conclude that visceral fat is bioenergetically more active and more sensitive to mitochondrial substrate supply than subcutaneous fat. Oxidative phosphorylation has a higher relative activity in visceral compared with subcutaneous adipose tissue.

No abstract

Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4-11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.

Aims/hypothesis Mitochondrial respiration has been linked to insulin resistance. We studied mitochondrial respiratory capacity and substrate sensitivity in patients with type 2 diabetes (patients), and obese and lean control participants.

These simvastatin-treated patients were glucose intolerant. A decreased Q(10) content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment.

Aims/hypothesis The glucose-lowering drug metformin has been shown to inhibit complex I of the mitochondrial electron transport chain in skeletal muscle. To investigate this effect in vivo we studied skeletal muscle mitochondrial respiratory capacity and content from patients with type 2 diabetes treated with metformin (n=14) or sulfonylurea (n= 8) and healthy control (n=18) participants. Methods Mitochondrial respiratory capacity was measured ex vivo in permeabilised muscle fibres obtained from the vastus lateralis muscle of all participants. The respiratory response to in vitro titration with metformin was measured in controls. Citrate synthase (CS) activity, and fasting plasma glucose, insulin and HbA 1c levels were measured and body composition was determined. Results Participants were matched for age, BMI and percentage body fat. Fasting plasma glucose concentrations were higher (p<0.05) in those treated with sulfonylureas and metformin than in controls. CS activity was comparable between metformin-treated and control participants, but tended to be lower in those receiving sulfonylureas. Mitochondrial respiratory capacity with substrates for complex I and complex I and II was comparable in the groups, both when estimated per mg of tissue and when normalised to CS activity. In vitro metformin titration demonstrated a dose-dependent inhibitory effect on complex I and II in human skeletal muscle at suprapharmacological concentrations. Conclusions/interpretation Metformin treatment does not inhibit mitochondrial complex I respiration in the electron transport chain in human skeletal muscle of patients with type 2 diabetes when measured ex vivo. Inhibition of complex I and II respiration in controls was demonstrated by metformin titration in vitro at doses well above those observed during metformin treatment.

Persistent infection with high-risk human papillomavirus (HPV) and expression of the proteins E6 and E7 is a prerequisite for development of cervical cancer. The distal non-coding part of E6/E7 messengers from several HPV types is able to downregulate synthesis of a reporter gene through mechanisms with involvement of cytoplasmic polyadenylation elements (CPEs) in the messengers. We here show that the mRNA levels of one of the four known CPE-binding proteins (CPEBs), the CPEB3, were downregulated in HPV-positive cervical cancers, whereas in ovarian cancer the CPEB1 mRNA level was downregulated. In addition, we showed that the RNA levels of the widely used reference marker GAPDH were upregulated in both cancer forms, and the level of the reference marker U6snRNA was upregulated in cervical cancers. Moreover, a possible correlation between the degree of U6snRNA upregulation and cervical cancer propagation was shown. These changes observed in CPEB1 and CPEB3 might indicate regulatory functions of CPEBs in cancer development of HPV-positive and HPV-negative tumors, respectively, and the U6snRNA, GAPDH mRNA and CPEB1 mRNA levels may be useful as tumor markers for genital cancers although further investigations are needed.