Antidiabetic and cardiovascular beneficial effects of a liver-localized mitochondrial uncoupler.

Inducing mitochondrial uncoupling (mUncoupling) is an attractive therapeutic strategy for treating metabolic diseases because it leads to calorie-wasting by reducing the efficiency of oxidative phosphorylation (OXPHOS) in mitochondria. Here we report a safe mUncoupler, OPC-163493, which has unique pharmacokinetic characteristics. OPC-163493 shows a good bioavailability upon oral administration and primarily distributed to specific organs: the liver and kidneys, avoiding systemic toxicities. It exhibits insulin-independent antidiabetic effects in multiple animal models of type I and type II diabetes and antisteatotic effects in fatty liver models. These beneficial effects can be explained by the improvement of glucose metabolism and enhancement of energy expenditure by OPC-163493 in the liver. Moreover, OPC-163493 treatment lowered blood pressure, extended survival, and improved renal function in the rat model of stroke/hypertension, possibly by enhancing NO bioavailability in blood vessels and reducing mitochondrial ROS production. OPC-163493 is a liver-localized/targeted mUncoupler that ameliorates various complications of diabetes.

Can thermogenic adipocytes protect from obesity?

The role of brown adipocytes and adipocytes of a new beige type in the energy metabolism of a healthy person and in the pathogenesis of obesity has extensively been discussed in recent years. The interest to these cells has been stimulated owing to the application of new noninvasive methods for studying the metabolic activity of tissues. Using these methods, the presence of thermogenically active adipocytes in adults and their reactivity to cold stimuli have been proved. These data, together with the results of animal experiments support the idea of thermogenic fat being a direct regulator of the energy balance of man. However, for several reasons there are some objections to this viewpoint. The main objection is that the total activity of the human thermogenic adipocytes is about 100 kJ/day, i.e., it is negligible. In addition, the burn of excessive nutrients is biologically inappropriate for an organism. Therefore, the idea that obesity is caused by the decreased activity of thermogenic adipocytes is erroneous. The statement that the causes of obesity are associated with the increased efficiency of energy-dependent processes seems more reasonable. The consequence is a reduction in energy expenditure to perform a unit of biological work. This results in excess of nutrients deposited in the form of fat

The interactions of cetyltrimethylammonium with mitochondria: an uncoupler or a detergent?

The interactions of cetyltrimethylammonium (CTA) with mitochondria have been investigated. We confirm, as already observed in a previous paper, that this compound behaves as proton carrier (or uncoupler) of the oxidative phosphorylation, but evidences suggest that this compound enhances the membrane permeability to many other compounds such as sucrose. We conclude therefore that CTA as a detergent enhances membrane permeability to all ions including protons. Some evidences are also given that the inhibitory effect of CTA on the mitochondrial respiratory chain is a consequence of the swelling induced.

Uncoupling effect of mercuric chloride on mitochondria isolated from an hepatic cell line.

A human fetal hepatic cell line (WRL-68) was used as a model to study the damage produced by mercury. The Hg(II) uptake by WRL-68 cells was found to be in a biphasic manner with a rapid initial uptake phase lasting about 5 min, followed by a sustained phase of slower accumulation. Distribution of mercury was studied and mitochondria were found to be the major target for mercury in this cell line (48%), followed by nuclei (38%), cytosol (8%) and microsomes (7%). Mitochondrial morphological damage after mercury treatment was observed by transmission electron microscopy. To determine if the toxic effect of mercury on mitochondrial bioenergetics was direct or indirect, mitochondria were isolated from WRL-68 cells after 1 h of pre-incubation with 0.5 microM HgCl(2). Oxygen consumption was quantified in two sets of experiments: in the presence of classical mitochondrial respiratory inhibitors; and in the presence of oligomycin. No significant difference was found in respiration with classical inhibitors, indicating that mercury does not affect directly the mitochondrial respiratory chain. However, mitochondria of Hg-treated cells were not inhibited when oligomycin was added, probably due to an uncoupling effect. This effect was prevented with dithiothreitol (DTT) treatment. A possible explanation for mercury's effect on mitochondria and its relation with oxidative stress is presented.

Analysis of the toxic effects of linoleic acid, 12,13-cis-epoxyoctadecenoic acid, and 12,13-dihydroxyoctadecenoic acid in rabbit renal cortical mitochondria.

P450 epoxidation of linoleic acid has been associated with many pathological conditions that often lead to acute renal failure. However, there is only suggestive evidence that linoleic acid monoepoxides and/or linoleic diols directly induce mitochondrial dysfunction. Using isolated rabbit renal cortical mitochondria (RCM), we found that linoleic acid (50 microM) and the linoleic acid monoepoxide, cis-12,13-epoxy-9-octadecenoic acid (12,13-EOA, 50 microM) increased state 4 and oligomycin-insensitive respiration and reduced state 3 and oligomycin-sensitive respiration. Concomitant with these effects, linoleic acid and 12,13-EOA decreased mitochondrial membrane potential (DeltaPsi). In contrast, the hydrolyzed product of 12,13-EOA, 12,13-dihydroxyoctadecenoic acid (12,13-DHOA, 50 microM), had no effect on state 3, state 4, oligomycin-sensitive, and oligomycin-insensitive respiration, and DeltaPsi. Neither linoleic acid or its metabolites altered uncoupled respiration, which suggests that these compounds have no affect on electron transport chain in RCM. Nucleotides such as ATP (0.5 mM) and GDP (0.5 mM) partially prevented the decrease in DeltaPsi but did not attenuate the increase in oligomycin-insensitive respiration after exposure to linoleic acid (50 microM) and 12,13-EOA (50 microM). These results demonstrate that linoleic acid metabolism to the 12,13-DHOA is a detoxification pathway that prevents mitochondrial dysfunction in RCM. The increase in state 4 respiration concomitant with decreases in state 3 respiration and DeltaPsi suggest that, in addition to uncoupling effects, linoleic acid and 12,13-EOA may have other effects, such as alterations of mitochondrial membranes. The inability of ATP and GDP to fully attenuate the uncoupling effects of linoleic acid and 12,13-EOA suggests that these effects are mediated through a nucleotide-independent mechanism.

Interactions of trialkyllead compounds with rat liver mitochondria.

The interactions of two trialkyllead (TAL) compounds, (trimethyl)Pb-Cl and (tributyl)Pb-Cl, with mitochodria from rat liver have been studied. A stimulation of the respiratory rate induced by the trialkyllead compounds added at low doses was observed which was not dependent on the presence of chloride in the medium. In contrast with the major current view, we propose that trialkyllead compounds behave as uncouplers of the oxidative phosphorylation and not (or not only) as Cl-/OH- exchangers. In fact the present results suggest that the TAL compounds enter the mitochondria as (alkyl)3Pb+ cations and are extruded as electroneutral (alkyl)3 Pb-OH compounds, the overall result being the transport of a proton through the membrane as in the case of classical uncouplers. The uncoupling effect could explain the toxicity of the compounds as a result of the decrease in the energy level of the cell. Furthermore, such a mechanism, in which the uptake of TAL compounds is supposed to be driven by a negative potential, could explain their preferential toxicity for neuronal cells, which maintain a higher negative-inside potential than most other cell types.

Uptake of N,N-dimethyl-2-phenylethylene HCl into acini cells removed from rabbit lacrimal glands.

Acini cells were obtained from the lacrimal gland of the white New Zealand rabbit. Following isolation and purification, the cells were used to study the uptake of N,N'-dimethyl-2-phenylethylamine HCl (AF2975), which was found to be sodium- and proton-independent, but energy-dependent. Uptake was mainly accomplished via a carrier-mediated transport system for which a Km of 8.72+/-0.96 mM, a Vmax of 602.6+/-41.3 nmol/mg of protein/min, and an exponential coefficient of 2.55+/-0.46 were obtained following a least squares nonlinear fit to the Hill equation. With the addition of the metabolic inhibitors, sodium azide or 2,4-dinitrophenol, the initial uptake rates were reduced from the control experiments by 35.7% and 26.2%, respectively.

In vitro percutaneous absorption of model compounds glyphosate and malathion from cotton fabric into and through human skin.

Chemicals are introduced to fabric at many steps during manufacture and use. Fabrics containing chemicals can cause medical problems such as dermatitis and death. Insecticides impregnated into uniforms worn by "Desert Storm" personnel are implicated in "Gulf War Syndrome'. These chemicals must get from fabric into and through skin to cause toxic effects. The objective of the present study was to determine in vitro percutaneous absorption of model chemicals glyphosate (water soluble) and malathion (relative water insoluble) from cotton fabric into and through human skin. The percutaneous absorption of glyphosate from water solution was 1.42 +/- 0.25% dose. This decreased to 0.74 +/- 0.26% for glyphosate added to cotton sheets and immediately put onto skin. If the cotton sheets were dried for 1 or 2 days, then applied to skin, absorption was 0.08 +/- 0.02% and 0.08 +/- 0.01% respectively. However, wetting the 2-day dried cotton sheet with water to simulate sweating or wet conditions increased absorption to 0.36 +/- 0.07%. Similar results were found for malathion. Absorption of malathion from aqueous ethanol solution was 8.77 +/- 1.43%. This decreased to 3.92 +/- 0.49%, 0.62 +/- 0.11% and 0.60 +/- 0.14% for 0, 1- and 2-day-treated cotton sheets. However, malathion absorption from 2-day treated/dried cotton sheets increased to 7.34 +/- 0.61% when wetted with aqueous ethanol. These results show that chemicals in fabric (clothing, rug, upholstery, etc.) can transfer from fabric into and through human skin to cause toxic effects.