In mitochondria, ATP synthesis is coupled to oxygen consumption by the proton electrochemical gradient established across the mitochondrial inner membrane in a process termed oxidative phosphorylation. It has long been known from stoichiometric studies that ATP synthesis is not perfectly coupled to oxygen consumption. The major inef®ciency in the system is leakage of protons across the mitochondrial inner membrane driven by the proton electrochemical gradient. The kinetics of the proton leak can be determined indirectly, by measuring the oxygen consumption of mitochondria under non-phosphorylating conditions (plus oligomycin) as a function of the proton electrochemical gradient. This experimental system provides a convenient means to investigate inner membrane permeability to protons and the effect of factors that may effect that permeability. In this paper we review some results from our laboratory of indirect measurement of mitochondrial proton leak and how it has been applied to investigate the effect of aging, obesity and thyroid status on proton leak. The results show that (i) proton leak in isolated liver mitochondria is not signi®cantly different in a comparison of young and old rats, in contrast (ii) there is an apparent increase in proton leak in in situ mitochondria in hepatocytes from old rats when compared to those from young rats, (iii) proton leak in neuronal mitochondria in situ in synaptosomes is not signi®cantly different in young and old rats, (iv) proton leak is greater in isolated liver mitochondria from obaob mice compared to lean controls, (v) acute leptin (OB protein) administration restores the increased leak rate in isolated liver mitochondria from obaob mice to that of lean controls, (vi) administration of thyroid hormone (T 3 ) increases proton leak in rat muscle mitochondria, and (vii) proton leak in muscle mitochondria is insensitive to the presence of GDP. It is proposed that the experimental system described here for measuring proton leak, is an ideal functional assay for determining whether the novel uncoupling proteins increase inner membrane permeability to protons.
Cellular phenotyping of human dermal fibroblasts (HDFs) from patients with inherited diseases provides invaluable information for diagnosis, disease aetiology, prognosis and assessing of treatment options. Here we present a cell phenotyping protocol using image cytometry that combines measurements of crucial cellular and mitochondrial parameters: (1) cell number and viability, (2) thiol redox status (TRS), (3) mitochondrial membrane potential (MMP) and (4) mitochondrial superoxide levels (MSLs). With our protocol, cell viability, TRS and MMP can be measured in one small cell sample and MSL on a parallel one. We analysed HDFs from healthy individuals after treatment with various concentrations of hydrogen peroxide (H2O2) for different intervals, to mimic the physiological effects of oxidative stress. Our results show that cell number, viability, TRS and MMP decreased, while MSL increased both in a time- and concentration-dependent manner. To assess the use of our protocol for analysis of HDFs from patients with inherited diseases, we analysed HDFs from two patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD), one with a severe clinical phenotype and one with a mild one. HDFs from both patients displayed increased MSL without H2O2 treatment. Treatment with H2O2 revealed significant differences in MMP and MSL between HDFs from the mild and the severe patient. Our results establish the capacity of our protocol for fast analysis of cellular and mitochondrial parameters by image cytometry in HDFs from patients with inherited metabolic diseases.
Objective Low-energy extracorporeal shockwave therapy (LE-ESWT) has been shown to induce organ repair and neo-vascularization. The ability of LE-ESWT to improve erectile function in rodents as measured by improvements in intracavernosal pressure is well-established in various pathological situations. The underlying molecular mechanism are unclear and likely vary between different disorders, making rational drug design for synergetic effects with LE-ESWT difficult, without further research. In this placebo-controlled study, we aim to establish whether LE-ESWT can activate neovascularization biomarkers in diabetic tissues. Material and Methods Forty Wistar rats, aged 8 weeks, were randomly divided into 4 groups: 8 untreated controls, 12 controls that underwent LE-ESWT treatment, 8 controls with induced diabetes mellitus (DM) and 12 with DM underwent LE-ESWT treatment. DM was induced by streptozotocin. LE-ESWT treatment was performed with a Duolith SD1 machine (Storz), with a total amount of energy of 6.4 J per treatment. The rats received a total of three LE-ESWT treatments with 2-week intervals between treatments. Results Diabetic rats had significantly elevated blood glucose concentrations compared to control rats (P < 0.001) and experienced significant weight loss compared to controls (P < 0.001). Diabetic rats had elevated creatinine and urea and lower albumin (P < 0.001). Histologic analysis of penile tissue showed significant levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) expression in the LE-ESWT groups compared to controls (P< 0.01). Conclusion LE-ESWT induces neo-angiogenesis as expressed by VEGF and FGF in erectile tissue in normal and diabetic rats.
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