Blood flow and muscle bio-energetics by 31P-nuclear magnetic resonance after local cold acclimation.

To clarify the origin of local cold adaptation and to define precisely its influence on muscle bio-energetics during local exercise, five subjects were subjected to repeated 5 degrees C cold water immersion of the right hand and forearm. The first aim of our investigation was therefore carried out by measuring local skin temperatures and peripheral blood flow during a cold hand test (5 degrees C, 5 min) followed by a 10-min recovery period. The 31P by nuclear magnetic resonance (31PNMR) muscle bio-energetic changes, indicating possible heat production changes, were measured during the recovery period. The second aim of our investigation was carried out by measuring 31PNMR muscle bioenergetics during handgrip exercise (10% of the maximal voluntary contraction for 5 min followed by a 10-min recovery period) performed both at a comfortable ambient temperature (22 degrees C; E) and after a cold hand test (EC), before and after local cold adaptation. Local cold adaptation, confirmed by warmer skin temperatures of the extremities (+30%, P less than 0.05), was related more to an increased peripheral blood flow, as shown by the smaller decrease in systolic peak [-245 (SEM 30) Hz vs -382 (SEM 95) Hz, P less than 0.05] than to a change in local heat production, because muscle bioenergetics did not vary. Acute local cold immersion decreased the inorganic phosphate:phosphocreatine (PC) ratio during EC compared to E [+0.006 (SEM 0.010) vs +0.078 (SEM 0.002) before acclimation and +0.029 (SEM 0.002) vs +0.090 (SEM 0.002) after acclimation respectively, P less than 0.05] without significant change in the PC:beta-adenosine triphosphate ratio and pH. Local adaptation did not modify these results statistically. The recovery of PC during E increased after acclimation [9.0 (SEM 0.2) min vs 3.0 (SEM 0.4) min, P less than 0.05]. These results suggested that local cold adaptation is related more to peripheral blood flow changes than to increased metabolic heat production in the muscle.     

Targeting Bcl-2/Bcl-XL Induces Antitumor Activity in Uveal Melanoma Patient-Derived Xenografts

Purpose

Uveal melanoma (UM) is associated with a high risk of metastases and lack of efficient therapies. Reduced capacity for apoptosis induction by chemotherapies is one obstacle to efficient treatments. Human UM is characterized by high expression of the anti-apoptotic protein Bcl-2. Consequently, regulators of apoptosis such as Bcl-2 family inhibitors may constitute an attractive approach to UM therapeutics. In this aim, we have investigated the efficacy of the Bcl-2/Bcl-X_L inhibitor S44563 on 4 UM Patient-Derived Xenografts (PDXs) and derived-cell lines.

Experimental Design

Four well characterized UM PDXs were used for in vivo experiments. S44563 was administered alone or combined with fotemustine either concomitantly or after the alkylating agent. Bcl-2, Bcl-X_L, and Mcl-1 expressions after S44563 administration were evaluated by immunohistochemistry (IHC).

Results

S44563 administered alone by at 50 and 100 mg/kg i.p. induced a significant tumour growth inhibition in only one xenograft model with a clear dose effect. However, when S44563 was concomitantly administered with fotemustine, we observed a synergistic activity in 3 out of the 4 tested models. In addition, S44563 administered after fotemustine induced a tumour growth delay in 2 out of 3 tested xenografts. Finally, IHC analyses showed that Bcl-2, Bcl-X_L, and Mcl-1 expression were not modified after S44563 administration.

Conclusion

The novel anti-apoptotic experimental compound S44563, despite a relative low efficacy when administered alone, increased the efficacy of fotemustine in either concomitant or sequential combinations or indeed subsequent to fotemustine. These data support further exploration of potential therapeutic effect of Bcl-2/Bcl-xl inhibition in human UM.     

Implication of mitochondrial dysfunction and cell death in cold preservation--warm reperfusion-induced hepatocyte injury

Cold ischemia--warm reperfusion (CI/WR) injury of liver transplantation involves hepatocyte cell death, the nature and underlying mechanisms of which remain unclear. Isolated hepatocytes and isolated perfused livers were used to determine the prevalence of necrosis and apoptosis as well as mitochondrial dysfunction. In isolated cells, propidium iodide and Hoechst 33342 staining showed a cold-storage, time-dependent increase in necrosis, whereas apoptosis was minimal even after 48 h of hypothermia. Nonetheless, a progressive loss of mitochondrial membrane potential was observed. Translocation of mitochondrial cytochrome c toward microsomes occurred within 24 h of CI/WR, with cytochrome c reaching the cytosol later. Mitochondria isolated from whole livers subjected to CI/WR also display reduced metabolic parameters and increased susceptibility to swelling. These events are associated with increased activity of major initiator (caspase 9) and effector (caspase 3) caspases. The results demonstrate that CI/WR induces mitochondrial dysfunction in isolated cells and in the whole organ; only in the latter is that sufficient to trigger the classical mitochondrial pathway of apoptosis. Our study also provides evidence for the involvement of endoplasmic reticulum stress in CI/WR hepatocyte injury. Combined protection of mitochondria and endoplasmic reticulum may thus represent an innovative therapeutic avenue to enhance liver graft viability and functional integrity.     

Determination of heat debt in the cold: partitional calorimetry vs. conventional methods.

Measurements of core temperature (Tc) at different sites produce on some occasions different cooling curves in cold-exposed humans, suggesting that the corresponding thermometric heat debts (HD) could be equally different when calculated by conventional methods [via the change in either Tc or mean body temperature (Tb)]. The present study also compared these thermometric HD values with the calorimetric HD obtained by partitional calorimetry (S). Nine subjects who showed similar initial but different final Tc [rectal (Tre) and auditory canal temperatures (Tac)] during nude cold exposure (2 h at 1 degrees C at rest) were used. Tc-derived HD corresponded to a heat gain of 12 +/- 21 kJ and an HD of 78 +/- 20 kJ with use of Tre and Tac, respectively, whereas the Tb-derived HD varied from 266 +/- 35 to less than or equal to 1,479 +/- 71 kJ with the use of various well-known Tb weighing coefficients. In contrast, S corresponded to 504 +/- 79 kJ, a level that could have been obtained only if the thermoneutral/cold Tb weighing coefficients had been 0.818/0.818 for Tre and 0.865/0.865 for Tac. The results demonstrate that calculation by conventional methods can markedly overestimate or underestimate HD. These differences could not be explained by the site chosen to represent Tc, inasmuch as about the same effect was observed with use of either Tre or Tac. It is concluded that the thermometric value of HD in the cold is not, at least under the present conditions, as accurate and reliable as S.     

General and local cold adaptation after a ski journey in a severe arctic environment.

It is hypothesized that some of the variability in the conclusions of several human cold adaptation studies could be explained if not only were the changes in core and shell temperatures taken into account, before and after cold adaptation, but also the absolute temperatures and metabolic rate in both thermally neutral environments and in the cold. Such an approach was used in a group of volunteers before and after a ski journey (3 weeks at -20 to -30 degrees C) across Greenland. Eight subjects were submitted to cold tests (Tdb = 1 degree C, r.h. = 40%, wind speed = 0.8 m.s-1) for 2 hours. Thermoregulatory changes were also monitored in a neutral environment (Tdb = 30 degrees C). In the neutral environment, the arctic journey increased metabolic rate (11.2%; P less than 0.05) and mean skin temperature [Tsk: 33.5 (SEM 0.2) degrees C vs 32.9 (SEM 0.2) degrees C, P less than 0.05]. During the cold test, the arctic journey was associated with a lower final rectal temperature [36.8 (SEM 0.2) degrees C vs 37.3 (SEM 0.2) degrees C, P less than 0.01], a lower final Tsk [20.7 (SEM 0.4) degrees C vs 21.2 (SEM 0.3) degrees C, P less than 0.01] with no change in metabolic heat production. These observations are indicative of an hypothermic insulative isometabolic general cold adaptation, which was associated with a local cold adaptation of the extremities, as shown by warmer foot temperatures [12.3 (SEM 0.9) degrees C vs 9.8 (SEM 0.9) degrees C, P less than 0.001].     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

Mechanism of enhanced cold tolerance by an ephedrine-caffeine mixture in humans

The influence of a thermogenic mixture of ephedrine- (1 mg/kg) caffeine (2.5 mg/kg) on cold tolerance was investigated in nine healthy young male subjects during two seminude exposures to cold air (3 h at 10 degrees C). The drug ingestion reduced the total drop in core, mean skin, and mean body temperatures (P less than 0.01), thus producing significantly warmer final core, mean skin, and mean body temperatures compared with the placebo ingestion. The drug ingestion increased the total 3-h energy expenditure by 18.6% compared with that of the placebo ingestion in the cold (P less than 0.01). By means of the nonprotein respiratory exchange ratio to calculate the rates of substrate oxidation, it was found that the drug ingestion increased carbohydrate oxidation by as much as 41.7% above that of the placebo (P less than 0.05). In contrast, the drug mixture had no significant influence on lipid or protein metabolism. The results demonstrate that the ingestion of an ephedrine-caffeine mixture improves cold tolerance in humans by significantly increasing body temperatures in the cold. These improvements were not caused by an increased conservation of heat but by a greater energy expenditure, which appears to be dependent on an enhanced carbohydrate utilization.     

Influence of caffeine on metabolic responses of men at rest in 28 and 5 degrees C

Cold stress and caffeine ingestion are each reported to increase plasma catecholamines, free fatty acid (FFA) concentrations, and energy metabolism. This study examined the possible interaction of these two metabolic challenges in four double-blind counterbalanced trials. Young adult men (n = 6) ingested caffeine (5 mg/kg) or placebo (dextrose, 5 mg/kg) and rested for 2 h in 28 or 5 degrees C air. Cold stress alone elevated (P less than 0.05) plasma norepinephrine, metabolism (O2 consumption, VO2), and respiratory exchange ratio (RER). Caffeine alone increased (P less than 0.05) plasma epinephrine and FFA but not RER. When the two challenges were combined (caffeine plus 5 degrees C for 2 h) norepinephrine and epinephrine were increased (P less than 0.05) as was FFA. However, VO2, RER, and skin and rectal temperatures were not different from the responses observed at 5 degrees C after placebo ingestion. The data suggest that caffeine selectively increases plasma epinephrine, whereas cold air increases norepinephrine. During the cold exposure, increasing epinephrine and FFA above normal levels did not appear to influence the metabolic or thermal responses to the cold stress. In fact the increase in RER suggested a greater carbohydrate oxidation.     

Rates of energy substrates utilization during human cold exposure

Although it is well established in animals that acute cold exposure markedly increases the oxidation of energy substrates, the absolute quality and quantity of substrate oxidation is poorly understood in humans. This study compared the rates of substrate utilization in seven healthy young men exposed to both the warm (control exposure at 29 degrees C; semi-nude, 14 h fasted) and to the cold for 2 h (10 degrees C, 1 m.s-1 wind velocity). Substrate utilization was calculated using indirect calorimetry and the nonprotein respiratory exchange ratio, which was derived from the urinary urea nitrogen output. Cold exposure induced a 3.1 +/- 0.2 degrees C drop in mean body temperature and a body heat debt of 825.9 +/- 63.3 kJ (p less than 0.01). These parameters remained essentially unchanged in the warm. Cold exposure elevated the 2 h energy expenditure 2.46-fold in comparison to the warm (p less than 0.01). This cold-induced thermogenesis was accompanied by increases of 588% in carbohydrate oxidation (p less than 0.01) and 63% in fat oxidation (p less than 0.05), whereas protein oxidation remained unchanged. Although the greatest proportion of the energy expenditure in the warm was derived from lipid (59%), carbohydrate oxidation represented the major fuel for thermogenesis in the cold, since it accounted for 51% of the corresponding total energy expenditure. The results demonstrate that cold exposure causes a much greater increase in the utilization of carbohydrate than lipid. It is suggested that these substrates are directly utilized for thermogenesis in the shivering skeletal muscles.     

Plasma glucose and insulin responses to oral and intravenous glucose in cold-exposed humans

Although glucose tolerance and skeletal muscle glucose uptake are markedly improved by cold exposure in animals, little is known about such responses in humans. This study used two variations of a glucose tolerance test (GTT) to investigate changes in carbohydrate metabolism in healthy males during nude exposure to cold. In experiment 1, an oral GTT was performed in the cold and in the warm (3 h at 10 or 29 degrees C). To bypass the gastrointestinal tract, and to suppress hepatic glucose output, a second experiment was carried out as described above, using an intravenous GTT. Even though cold exposure raised metabolic rate greater than 2.5 times, plasma glucose and insulin responses to an oral GTT remained unaltered. In contrast, cold exposure reduced the entire plasma glucose profile as a function of time during the intravenous GTT (P less than 0.05), as plasma glucose was returned to basal levels within 1 h in comparison to a full 2 h in the warm, despite low insulin levels. The results of the intravenous GTT demonstrate that even with low insulin levels, carbohydrate metabolism is increased in cold-exposed males. This effect could be masked in the oral GTT by gastrointestinal factors and a high hepatic glucose output. Cold exposure may enhance insulin sensitivity and/or responsiveness for glucose uptake, mainly in shivering skeletal muscles.