Reduction of hyperglycemia in STZ-induced diabetic mice by prophylactic treatment with heat-killed : possible effects on glucose utilization, mitochondrial uncoupling, and oxidative stress in liver and skeletal muscle.

Background: In addition to conventional treatment and modifications in physical activity and diet, alternative strategies have been investigated to manage, prevent, or delay diabetes in humans. In this regard, one strategy has relied on the immunomodulatory properties of mycobacteria, whereby Bacillus Calmette-Guerin, an attenuated live strain of , has been shown to improve glycemic control in patients with diabetes and to alleviate hyperglycemia in selected murine models of diabetes. A novel heat-killed (HK) whole-cell preparation of () is currently under development as a potential food supplement; nevertheless, its potential bioactivity remains largely unknown.

Mitochondrial bioenergetics, uncoupling protein-2 activity, and reactive oxygen species production in the small intestine of a TNBS-induced colitis rat model.

Inflammatory bowel disease (IBD) is often associated with a decrease in energy-dependent nutrient uptake across the jejunum that serves as the main site for absorption in the small intestine. This association has prompted us to investigate the bioenergetics underlying the alterations in jejunal absorption in 2,4,6-trinitrobenzenesulfonic acid-induced colitis in rats. We have found that mitochondrial oxygen consumption did not change in state 2 and state 3 respirations but showed an increase in state 4 respiration indicating a decrease in the respiratory control ratio of jejunal mitochondria during the peak of inflammation.

Uncoupling proteins: Martin Klingenberg's contributions for 40 years.

The uncoupling protein (UCP1) is a proton (H) transporter in the mitochondrial inner membrane. By dissipating the electrochemical H gradient, UCP1 uncouples respiration from ATP synthesis, which drives an increase in substrate oxidation via the TCA cycle flux that generates more heat. The mitochondrial uncoupling-mediated non-shivering thermogenesis in brown adipose tissue is vital primarily to mammals, such as rodents and new-born humans, but more recently additional functions in adult humans have been described.

Absence of effect of the antiretrovirals Duovir and Viraday on mitochondrial bioenergetics.

Most toxicity associated with antiretroviral drugs is thought to result from disruption of mitochondrial function. Unfortunately, there are no validated laboratory markers for clinically assessing the onset of mitochondrial toxicity associated with antiretroviral therapy. In a previous study on mitochondrial hepatocytes, the protease inhibitor lopimune was shown to induce mitochondrial toxicity by increasing reactive oxygen species (ROS) production and decreasing respiratory control ratio (RCR) reflecting compromised mitochondrial efficiency in adenosine triphosphate production.

Differential regulation of surface receptor expression, proliferation, and apoptosis in HaCaT cells stimulated with interferon-γ, interleukin-4, tumor necrosis factor-α, or muramyl dipeptide.

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively.

Fragrance chemicals lyral and lilial decrease viability of HaCat cells' by increasing free radical production and lowering intracellular ATP level: protection by antioxidants.

We investigate in this study the biochemical effects on cells in culture of two commonly used fragrance chemicals: lyral and lilial. Whereas both chemicals exerted a significant effect on primary keratinocyte(s), HaCat cells, no effect was obtained with any of HepG2, Hek293, Caco2, NIH3T3, and MCF7 cells. Lyral and lilial: (a) decreased the viability of HaCat cells with a 50% cell death at 100 and 60 nM respectively; (b) decreased significantly in a dose dependant manner the intracellular ATP level following 12-h of treatment; (c) inhibited complexes I and II of electron transport chain in liver sub-mitochondrial particles; and (d) increased reactive oxygen species generation that was reversed by N-acetyl cysteine and trolox and the natural antioxidant lipoic acid, without influencing the level of free and/or oxidized glutathione.

Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein 2 and the production of reactive oxygen and reactive nitrogen species.

The synthetic immunomodulator muramyl dipeptide (MDP) has been shown to induce, in vivo, mitochondrial proton leak. In the present work, we extended these findings to the cellular level and confirmed the effects of MDP in vitro on murine macrophages. The macrophage system was then used to analyse the mechanism of the MDP-induced mitochondrial proton leak.

Mitochondrial uncoupling proteins--what is their physiological role?

The physiological functions of the mitochondrial uncoupling proteins (UCP2 and UCP3) are still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, they are not crucial for nonshivering thermogenesis and do not catalyze the basal proton conductance of mitochondria. Rather, there is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues.

4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production.

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane.

Hydroxynonenal and uncoupling proteins: a model for protection against oxidative damage.

In this mini review we summarize recent studies from our laboratory that show the involvement of superoxide and the lipid peroxidation product 4-hydroxynonenal in the regulation of mitochondrial uncoupling. Superoxide produced during mitochondrial respiration is a major cause of the cellular oxidative damage that may underlie degenerative diseases and ageing. Superoxide production is very sensitive to the magnitude of the mitochondrial protonmotive force, so can be strongly decreased by mild uncoupling.

Mitochondrial superoxide and aging: uncoupling-protein activity and superoxide production.

Mitochondria are a major source of superoxide, formed by the one-electron reduction of oxygen during electron transport. Superoxide initiates oxidative damage to phospholipids, proteins and nucleic acids. This damage may be a major cause of degenerative disease and aging.

Molecular properties of purified human uncoupling protein 2 refolded from bacterial inclusion bodies.

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2).

Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria.

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63-65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria.

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone.

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components.

A signalling role for 4-hydroxy-2-nonenal in regulation of mitochondrial uncoupling.

Oxidative stress and mitochondrial dysfunction are associated with disease and aging. Oxidative stress results from overproduction of reactive oxygen species (ROS), often leading to peroxidation of membrane phospholipids and production of reactive aldehydes, particularly 4-hydroxy-2-nonenal. Mild uncoupling of oxidative phosphorylation protects by decreasing mitochondrial ROS production.

Superoxide stimulates a proton leak in potato mitochondria that is related to the activity of uncoupling protein.

The ability of plant mitochondrial uncoupling proteins to catalyze a significant proton conductance in situ is controversial. We have re-examined conditions that lead to uncoupling of mitochondria isolated from the tubers of potato (Solanum tuberosum). Specifically, we have investigated the effect of superoxide.

Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al.

Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies.

Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx.

Superoxide activates mitochondrial uncoupling proteins.

Uncoupling protein 1 (UCP1) diverts energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue by catalysing a regulated leak of protons across the inner membrane. The functions of its homologues, UCP2 and UCP3, in other tissues are debated. UCP2 and UCP3 are present at much lower abundance than UCP1, and the uncoupling with which they are associated is not significantly thermogenic.

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value.