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 () 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 of (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.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the α-subunit of HIF-1 (HIF-1α), which occurs in response to hypoxia or activation of growth factor pathways, is associated with poor prognosis in many types of cancer. Therefore, down-regulation of HIF-1α protein by RNA antagonists may control cancer growth. EZN-2968 is a RNA antagonist composed of third-generation oligonucleotide, locked nucleic acid, technology that specifically binds and inhibits the expression of HIF-1α mRNA. In vitro, in human prostate (15PC3, PC3, and DU145) and glioblastoma (U373) cells, EZN-2968 induced a potent, selective, and durable antagonism of HIF-1 mRNA and protein expression (IC₅₀, 1-5 nmol/L) under normoxic and hypoxic conditions associated with inhibition of tumor cell growth. Additionally, down-regulation of HIF-1α protein by EZN-2968 led to reduction of its transcriptional targets and of human umbilical vein endothelial cell tube formation. In vivo, administration of EZN-2968 to normal mice led to specific, dose-dependent, and highly potent down-regulation of endogenous HIF-1α and vascular endothelial growth factor in the liver. The effect can last for days after administration of single dose of EZN-2968 and is associated with long residence time of locked nucleic acid in certain tissues. In efficacy studies, tumor reduction was found in nude mice implanted with DU145 cells treated with EZN-2968. Ongoing phase I studies of EZN-2968 in patients with advanced malignancies will determine optimal dose and schedule for the phase II program. [Mol Cancer Ther 2008;7(11):3598–608]

To gain further insight into the mechanisms underlying muscle insulin resistance, the influence of obesity and type 2 diabetes on GLUT4 immunoreactivity in slow and fast skeletal muscle fibers was studied. Through a newly developed, very sensitive method using immunohistochemistry combined with morphometry, GLUT4 density was found to be significantly higher in slow compared with fast fibers in biopsy specimens from lean and obese subjects. In contrast, in type 2 diabetic subjects, GLUT4 density was significantly lower in slow compared with fast fibers. GLUT4 density in slow fibers from diabetic patients was reduced by 9% compared with the weight-matched obese subjects and by 18% compared with the lean control group. The slow-fiber fraction was reduced to 86% in the obese subjects and to 75% in the diabetic subjects compared with the control group. Estimated GLUT4 contribution from slow fibers was reduced to 77% in the obese subjects and to 61% in type 2 diabetic patients compared with the control subjects. We propose that a reduction in the fraction of slow-twitch fibers, combined with a reduction in GLUT4 expression in slow fibers, may reduce the insulin-sensitive GLUT4 pool in type 2 diabetes and thus contribute to skeletal muscle insulin resistance.

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25–100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25–40% to 80–100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.