Jean-Louis Gallis

Freshwater adaptation in the euryhaline teleost, Chelon labrosus. I. Effects of adaptation, prolactin, cortisol and actinomycin D on plasma osmotic balance and (Na+-K+)ATPase in gill and kidney.

Abstract Microsomal (Na + ue5f8K + )ATPase activities in gills and kidneys of thick-lipped mullet, Chelon labrosus , which had been gradually acclimated to fresh water were studied using initial velocity measurements. In mullet transferred to fresh water, the branchial ATPase activity rises gradually during a 2-week period (long-term fresh water adaptation: L-TFW). The renal ATPase activity rises rapidly after the transfer to fresh water (FW). In the L-TFW mullet and in seawater (SW) mullet, cortisol injections cause no, or only slight, stimulation of renal or branchial ATPase activity; on the other hand, cortisol causes a marked decrease in ATPase activity in mullet adapted to FW for only a short period of time (S-TFW). Only branchial ATPase, and not kidney ATPase, is inhibited by prolactin, and this only in S-TFW and SW mullet. Actinomycin D does not inhibit renal ATPase activity, but the response of branchial ATPase to this antibiotic depends on the environment; SW-acclimated mullet are sensitive to actinomycin D but the longer mullet are acclimated to FW the more insensitive they become. These findings may indicate that more than one functional form of gill (Na + +K + )ATPase exists, and that these vary in activity with external salinity.

Osmoregulation and differential penetration of two grey mullets, Chelon labrosus (Risso) and Liza ramada (Risso) in estuarine fish ponds

Abstract Grey mullet ponds situated in Arcachon Bay on the western Atlantic coast of France, display contrasting mixohaline habitats. The pattern of osmoregulation of Chelon labrosus and Liza ramada exhibiting differential penetration of fish ponds has been investigated. Key variables of salinity and temperature have been considered. The tolerances to low salinities and to fresh water have been determined. The dynamics of variation in plasma osmotic pressure and major plasma electrolytes (Na + , Cl − , K + ) were studied in terms of: (1) gradual adaptation to a decreasing salinity gradient; (2) effects of a long-term adaptation in fresh water; (3) effects of low temperatures on sea water adapted fish. Both species can adapt to a wide range of salinities; however, while L. ramada show a good hypoosmotic regulation, with no alteration in plasma concentration over a wide range of salinity and after a long stay (5 months) in fresh water, C. labrosus cannot live for long periods of time in a wholly fresh water habitat. Both field samples obtained from thick-lipped grey mullets forced to stay in a freshwater pond and experimentally adapted fish showed, after 3 months, an unequivoqual decrease in plasma sodium and chloride, and plasma osmolality decreased to the same extent; this pattern of no compensation is bound, sooner or later, to end in death. The inability of C. labrosus to develop a long-term hypoosmoregulation in fresh water is referred to salinity preferences which probably limit the upstream penetration of this species. Plasma osmolality and major electrolytes (sodium and chloride) increased in cold (4 °C) sea water. This response might be due to a reduced ability of grey mullets to osmoregulate in cold waters. In the poly-mesohaline ponds, Winter drop in temperature (4–6 °C) causes grey mullets to migrate to the meso-oligohaline ponds where the mean temperature is more elevated (8 °C) and temperature changes less rapid (6–9 °C).

Phosphorus-31 nuclear magnetic resonance of isolated rat liver during hypothermic ischemia and subsequent normothermic perfusion

The effects of prolonged hypothermic ischemia and subsequent normothermic perfusion on the energetic metabolism and intracellular pH (pHin) of isolated rat livers were studied by phosphorus-31 nuclear magnetic resonance spectroscopy. Nucleoside triphosphate (NTP) depletion and intracellular pH were studied within an 18-h-storage phase, by using the following preservation media: Eurocollins (EC), UW Lactobionate (UW) and Bretschneiders solution (HTK). Values obtained after 8-h ischemia were chosen to estimate the performance of the various media: NTP levels were 37 +/- 7%, 10 +/- 5% and 0% of control levels, respectively, in livers stored in UW, HTK and EC solutions. pHin reached values of 7.15 +/- 0.10 in UW and HTK, and 6.96 +/- 0.10 in EC-stored livers. Ischemic damage was assessed by reperfusing the stored organ with Krebs medium: NTP recovery was around 70 +/- 20% for the three solutions used. Recovery of pHin was near the control value (7.23 +/- 0.08), except for EC solution (7.05 +/- 0.20). The main results are that (i) the rates of NTP and pHin decrease are strongly dependent on the nature of the preservation solution, whereas (ii) NTP recovery is not significantly different during post-ischemic reperfusion. With regard to animal survival, UW solution is at present considered largely superior to EC medium for liver preservation. Thus, our data suggest that the rates of NTP depletion and pHin fall during cold preservation could be both considered as better indicators assessing liver injury than the post-ischemic NTP recovery.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus ☆: II. Effects of continuance of adaptation, cortisol treatment, and environmental calcium on water influx in isolated gill

Abstract The effects of freshwater adaptation duration, cortisol, and environmental calcium on water influx in isolated gill of the thick-lipped gray mullet, Chelon labrosus , were studied using an in vitro technique. An estimation of the water permeability and the osmotic permeability coefficient in this euryhaline fish was made from the rate of weight increase in isolated gills immersed in deionized water after preincubation in saline solution. The water influx in the isolated gills and/or permeability is reduced progressively with the adaptation to fresh water in C. labrosus . Cortisol increases the water influx and/or water permeability only in seawater-adapted fish. An increased calcium content of fresh water reduces the weight increase of isolated gill. These results are in accord with the osmoregulatory performance characteristic of this fish.

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats.

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria. The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O(2) Clark electrodes. The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86+/-0.02 micromol/min g wet weight (min g ww)] than in control [1.44+/-0.18 micromol/min g ww]. However, mitochondrial respiration remained unchanged [2.20+/-0.13 micromol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33+/-0.035 (control) to 0.42+/-0.015 (ethanol). The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.

Insulin induces a positive relationship between the rates of ATP and glycogen changes in isolated rat liver in presence of glucose; a 31P and 13C NMR study.

BackgroundThere is an emerging theory suggesting that insulin, which is known to be the predominant postprandial anabolic hormone, is also a major regulator of mitochondrial oxidative phosphorylation in human skeletal muscle. However, little is known about its effects in the liver. Since there is a theoretical relationship between glycogen metabolism and energy status, a simultaneous and continuous investigation of hepatic ATP and glycogen content was performed in intact and isolated perfused liver by 31P and 13C nuclear magnetic resonance (NMR) The hepatic rates of ATP and glycogen changes were evaluated with different concentrations of insulin and glucose during continuous and short-term supply.ResultsLiver from rats fed ad libitum were perfused with Krebs-Henseleit Buffer (KHB)(controls) or KHB containing 6 mM glucose, 30 mM glucose, insulin alone, insulin + 6 mM glucose, insulin + 30 mM glucose. In the control, glycogenolysis occurred at a rate of -0.53 ± 0.021 %·min-1 and ATP content decreased at a rate of -0.28 ± 0.029 %·min-1. In the absence of insulin, there was a close proportional relationship between the glycogen flux and the glucose concentration, whereas ATP rates never varied. With insulin + glucose, both glycogen and ATP rates were strongly related to the glucose concentration; the magnitude of net glycogen flux was linearly correlated to the magnitude of net ATP flux: fluxglycogen = 72.543(fluxATP) + 172.08, R2 = 0.98.ConclusionOnly the co-infusion of 30 mM glucose and insulin led to (i) a net glycogen synthesis, (ii) the maintenance of the hepatic ATP content, and a strong positive correlation between their net fluxes. This has never previously been reported. The specific effect of insulin on ATP change is likely related to a rapid stimulation of the hepatic mitochondrial oxidative phosphorylation. We propose that variations in the correlation between rates of ATP and glycogen changes could be a probe for insulin resistance due to the action of substrates, drugs or pathologic situations. Consequently, any work evaluating insulin resistance on isolated organs or in vivo should determine both ATP and glycogen fluxes.

Freshwater adaptation in the euryhaline teleost, Chelon labrosus—IV. Changes of DNA and protein contents, RNA/DNA ratio and acid phosphatase activity in the branchial tissue

Abstract 1. 1. Acid phosphatase activity, as an index of lysosomal activity, of the cytoplasmic fraction of gill tissue transiently increases during the FW adaptation of the mullet, Chelon labrosus . 2. 2. A concomitant and unexpected DNA decrease occurs. Moreover, RNA/DNA ratio in the nucleimitochondria fraction increases with the continuance of FW adaptation, while the DNA renewal of gill tissue is enhanced in the fully adapted FW mullet. 3. 3. The use of histologic techniques indicates that chloride cells are probably concerned with AcPase and DNA changes. 4. 4. These results are discussed with regard to the renewal and differentiation of two chloride cell populations. In addition, a model for the control of chloride cell populations in gill epithelium of C. labrosus , is suggested.

Some processes of energy saving and expenditure occurring during ethanol perfusion in the isolated liver of fed rats; a Nuclear Magnetic Resonance study.

BackgroundIn the isolated liver of fed rats, a 10 mM ethanol perfusion rapidly induced a rapid 25% decrease in the total ATP content, the new steady state resulting from both synthesis and consumption. The in situ rate of mitochondrial ATP synthesis without activation of the respiration was increased by 27%, implying an increased energy demand. An attempt to identify the ethanol-induced ATP-consuming pathways was performed using 31P and 13C Nuclear Magnetic Resonance.ResultsEthanol (i) transiently increased sn-glycerol-3-phosphate formation whereas glycogenolysis was continuously maintained; (ii) decreased the glycolytic ATP supply and (iii) diminished the intracellular pH in a dose-dependent manner in a slight extend. Although the cytosolic oxidation of ethanol largely generated H+ (and NADH), intracellular pHi was maintained by (i) the large and passive excretion of cellular acetic acid arising from ethanol oxidation (evidenced by exogenous acetate administration), without energetic cost or (ii) proton extrusion via the Na+-HCO3- symport (implying the indirect activation of the Na+-K+-ATPase pump and thus an energy use), demonstrated during the addition of their specific inhibitors SITS and ouabaïn, respectively.ConclusionVarious cellular mechanisms diminish the cytosolic concentration of H+ and NADH produced by ethanol oxidation, such as (i) the large but transient contribution of the dihydroxyacetone phosphate / sn- glycerol-3-phosphate shuttle between cytosol and mitochondria, mainly implicated in the redox state and (ii) the major participation of acetic acid in passive proton extrusion out of the cell. These processes are not ATP-consuming and the latter is a cellular way to save some energy. Their starting in conjunction with the increase in mitochondrial ATP synthesis in ethanol-perfused whole liver was however insufficient to alleviate either the inhibition of glycolytic ATP synthesis and/or the implication of Na+-HCO3- symport and Na+-K+-ATPase in the pHi homeostasis, energy-consuming carriers.

Fresh water adaptation in the euryhaline teleost, Chelon labrosus—III Biochemical characterization and increase in the acid phosphatase activity in gill

Abstract Abstrac 1. 1. In the gill of the euryhaline teleost, Chelon labrosus , the basic characteristics of acid phosphatase activity, a “lysosomal” enzyme, are similar to those of the other vertebrates. 2. 2. The pH optimum is 4.75; the enzyme activity reaches a plateau from pH 3.8 to pH 5.0; at higher pH levels, enzyme activity rapidly declines. 3. 3. Normal kinetic patterns of enzyme activity occur when substrate, enzyme concentration and time of incubation in enzyme assays are varied. The ions (Na + K + , Cl − , Ca 2+ and Mg 2+ ) and chelators (EGTA, EDTA). do not affect the enzyme activity. Decrease (12% to 25%) in in vitro enzyme activity occurs after one day at 0°-4°C. 4. 4. Acid phosphatase activity, found above all 50,000 g × 1 hr fraction and moved in slighter subcellular fraction after in vitro osmotic shock, is probably localyzed within the lysosomes. 5. 5. The specific activity increases when fish is transferred from sea water to freshwater.

Glucose flux in controlled hyperglycaemia before and after oral glucose ingestion in men with mild type 2 diabetes.

AIMSnThis study aimed to determine how insufficiently suppressed endogenous glucose production vs. reduced peripheral glucose uptake contribute to postprandial hyperglycaemia in type 2 diabetes (T2D).nnnMETHODSnEight men with T2D (age: 52+/-7 years; BMI: 26.6+/-2.3 kg/m(2); fasting glycaemia: 7.1+/-1.5 mmol/L) were compared with eight non-diabetic controls (age: 51+/-5 years; BMI: 24.6+/-2.9 kg/m(2); fasting glycaemia: 4.9+/-0.4 mmol/L). Their glucose turnover rates and hepatic glucose cycles were measured by monitoring [2H7]glucose infusion, with m+7 and m+6 enrichment, 3 h before and 4 h after the ingestion of [6,6-2H2]-labelled glucose, while maintaining glycaemia at 10 mmol/L using the pancreatic clamp technique.nnnRESULTSnOf the 700 mg/kg oral glucose load, 71% appeared in the systemic circulation of the T2D patients vs. 63% in the controls (NS). Endogenous glucose production and hepatic glucose cycles did not differ from normal either before or after oral glucose ingestion, while peripheral glucose uptake was reduced by 40% in the T2D group both before (P<0.01) and after (P<0.05) ingestion of oral glucose.nnnCONCLUSIONnWhen T2D patients were compared with non-diabetic subjects with similarly controlled levels of hyperglycaemia after oral glucose ingestion, they essentially differed only in peripheral glucose uptake, whereas endogenous glucose production was apparently unaltered.