Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Antioxidant/prooxidant effects of dietary non-flavonoid phenols on the Cu2+-induced oxidation of human low-density lipoprotein (LDL)

A central role in the oxidative development of atherosclerotic lesions has been ascribed to the peroxidation of plasma low-density lipoprotein (LDL). Dietary supplementation with virgin olive oils increases the total plasma antioxidant status and the resistance of low-density lipoprotein to ex vivo oxidation. We have studied the effects of some dietary non-flavonoid phenols from Olea europaea L., both in purified form or in complex mixtures obtained by biotransformation of olive leaf extracts, on the LDL oxidation induced by Cu2+ ions. Cu2+-Induced LDL oxidation is inhibited by oleuropein and hydroxytyrosol in the initiation phase of the reaction at concentrations of phenols higher than that of Cu2+ ions. Interestingly, at lower concentration, both phenols anticipated the initiation process of LDL oxidation, thus exerting prooxidant capacities. Although similar effects are already described for flavonoids, such as quercetin, rutin, and apigenin, it is the first time that a prooxidant effect of dietary non-flavonoid phenols, such as oleuropein and hydroxytyrosol, on the LDL oxidation is reported. Our results show that a net effect of oleuropein and hydroxytyrosol on Cu2+-induced LDL peroxidation is determined by a balance of their pro- and antioxidant capacities. It is worth to underline that, during Cu2+-induced LDL oxidation in the presence of bioreactor eluates, we have evidence of a synergistic effect among phenolic compounds that enhance their antioxidant capacities so avoiding the prooxidant effects.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.     

Effect of pre-cooling, with and without thigh cooling, on strain and endurance exercise performance in the heat

Body cooling before exercise (i.e. pre-cooling) reduces physiological strain in humans during endurance exercise in temperate and warm environments, usually improving performance. This study examined the effectiveness of pre-cooling humans by ice-vest and cold (3 degrees C) air, with (LC) and without (LW) leg cooling, in reducing heat strain and improving endurance performance in the heat (35 degrees C, 60% RH). Nine habitually-active males completed three trials, involving pre-cooling (LC and LW) or no pre-cooling (CON: 34 degrees C air) before 35-min cycle exercise: 20 min at approximately 65% VO2peak then a 15-min work-performance trial. At exercise onset, mean core (Tc, from oesophagus and rectum) and skin temperatures, forearm blood flow (FBF), heart rate (HR), and ratings of exertion, body temperature and thermal discomfort were lower in LW and LC than CON (P<0.05). They remained lower at 20 min [e.g. Tc: CON 38.4+/-0.2 (+/-S.E.), LW 37.9+/-0.1, and LC 37.8+/-0.1 degrees C; HR: 177+/-3, 163+/-3 and 167+/-3 b.p.m.), except that FBF was equivalent (P=0.10) between CON (15.5+/-1.6) and LW (13.6+/-1.0 ml.100 ml tissue(-1) x min(-1)). Subsequent power output was higher in LW (2.95+/-0.24) and LC (2.91+/-0.25) than in CON (2.52+/-0.28 W kg(-1), P=0.00, N=8), yet final Tc remained lower. Pre-cooling by ice-vest and cold air effectively reduced physiological and psychophysical strain and improved endurance performance in the heat, irrespective of whether thighs were warmed or cooled.     

Hemodynamic and autonomic correlates of postexercise hypotension in patients with mild hypertension

We investigated the interplay of neural and hemodynamic mechanisms in postexercise hypotension (PEH) in hypertension. In 15 middle-aged patients with mild essential hypertension, we evaluated blood pressure (BP), cardiac output (CO), total peripheral resistance (TPR), forearm (FVR) and calf vascular resistance (CVR), and autonomic function [by spectral analysis of R-R interval and BP variabilities and spontaneous baroreflex sensitivity (BRS)] before and after maximal exercise. Systolic and diastolic BP, TPR, and CVR were significantly reduced from baseline 60-90 min after exercise. CO, FVR, and HR were unchanged. The low-frequency (LF) component of BP variability increased significantly after exercise, whereas the LF component of R-R interval variability was unchanged. The overall change in BRS was not significant after exercise vs. baseline, although a significant, albeit small, BRS increase occurred in response to hypotensive stimuli. These findings indicate that in hypertensive patients, PEH is mediated mainly by a peripheral vasodilation, which may involve metabolic factors linked to postexercise hyperemia in the active limbs. The vasodilator effect appears to override a concomitant, reflex sympathetic activation selectively directed to the vasculature, possibly aimed to counter excessive BP decreases. The cardiac component of arterial baroreflex is reset during PEH, although the baroreflex mechanisms controlling heart period appear to retain the potential for greater opposition to hypotensive stimuli.     

Biomimetic synthesis of a water soluble conducting molecular complex of polyaniline and lignosulfonate

A new biomimetic route for the synthesis of a conducting molecular complex of polyaniline (Pani) and a natural polyelectrolyte, lignosulfonate (LGS) is presented. A poly(ethylene glycol) modified hematin (PEG-hematin) was used to catalyze the polymerization of aniline in the presence of LGS to form a Pani/LGS complex. UV-vis, FTIR, conductivity and TGA studies for the LGS-polyaniline complex indicate the presence of a thermally stable and electrically conductive form of polyaniline. Also the presence of LGS in this complex, an inexpensive byproduct from pulp processing, provides a unique combination of properties such as electronic conductivity, processability and biodegradability. The use of this conductive complex for corrosion protection is also proposed.     

CD36 deficiency in mice impairs lipoprotein lipase-mediated triglyceride clearance

CD36 is involved in high-affinity peripheral FFA uptake. CD36-deficient (cd36(-)(/)(-)) mice exhibit increased plasma FFA and triglyceride (TG) levels. The aim of the present study was to elucidate the cause of the increased plasma TG levels in cd36(-)(/)(-) mice. cd36(-)(/)(-) mice showed no differences in hepatic VLDL-TG production or intestinal [(3)H]TG uptake compared with wild-type littermates. cd36(-)(/)(-) mice showed a 2-fold enhanced postprandial TG response upon an intragastric fat load (P < 0.05), with a concomitant 2.5-fold increased FFA response (P < 0.05), suggesting that the increased FFA in cd36(-/-) mice may impair LPL-mediated TG hydrolysis. Postheparin LPL levels were not affected. However, the in vitro LPL-mediated TG hydrolysis rate as induced by postheparin plasma of cd36(-)(/)(-) mice in the absence of excess FFA-free BSA was reduced 2-fold compared with wild-type plasma (P < 0.05). This inhibition was relieved upon the addition of excess FFA-free BSA. Likewise, increasing plasma FFA in wild-type mice to the levels observed in cd36(-)(/)(-) mice by infusion prolonged the plasma half-life of glycerol tri[(3)H]oleate-labeled VLDL-like emulsion particles by 2.5-fold (P < 0.05). We conclude that the increased plasma TG levels observed in cd36(-)(/)(-) mice are caused by decreased LPL-mediated hydrolysis of TG-rich lipoproteins resulting from FFA-induced product inhibition of LPL.