Consumption of Alcopops During Brain Maturation Period: Higher Impact of Fructose Than Ethanol on Brain Metabolism.

Alcopops are flavored alcoholic beverages sweetened by sodas, known to contain fructose. These drinks have the goal of democratizing alcohol among young consumers (12-17 years old) and in the past few years have been considered as fashionable amongst teenagers. Adolescence, however, is a key period for brain maturation, occurring in the prefrontal cortex and limbic system until 21 years old. Therefore, this drinking behavior has become a public health concern. Despite the extensive literature concerning the respective impacts of either fructose or ethanol on brain, the effects following joint consumption of these substrates remains unknown. Our objective was to study the early brain modifications induced by a combined diet of high fructose (20%) and moderate amount of alcohol in young rats by 13C Nuclear Magnetic Resonance (NMR) spectroscopy. Wistar rats had isocaloric pair-fed diets containing fructose (HF, 20%), ethanol (Et, 0.5 g/day/kg) or both substrates at the same time (HFEt). After 6 weeks of diet, the rats were infused with 13C-glucose and brain perchloric acid extracts were analyzed by NMR spectroscopy (1H and 13C). Surprisingly, the most important modifications of brain metabolism were observed under fructose diet. Alterations, observed after only 6 weeks of diet, show that the brain is vulnerable at the metabolic level to fructose consumption during late-adolescence throughout adulthood in rats. The main result was an increase in oxidative metabolism compared to glycolysis, which may impact lactate levels in the brain and may, at least partially, explain memory impairment in teenagers consuming alcopops.

Time-dependent effect of ethanol force-feeding on glycogen repletion: NMR evidence of a link with ATP turnover in rat liver.

The purpose was to study the hepatic effects of low-dose ethanol on the links between ATP and glycogen production. Fasted male Wistar rats received a single force-feeding of glucose plus ethanol or isocaloric glucose. At different times after force-feeding (0-10 h), glycogen repletion and ATP characteristics (content, apparent catalytic time constant, mitochondrial turnover) were monitored by (13)C- or (31)P-nuclear magnetic resonance (NMR) in perfused and isolated liver. In vivo glycogen repletion after force-feeding was slower after glucose plus ethanol vs. glucose (12.04 ± 0.68 and 8.50 ± 0.86 µmol/h/g liver wet weight [ww], respectively), reaching a maximum at the 6th hour. From the 3rd to the 8th hour, glycogen content was lower after glucose plus ethanol vs. glucose. After glucose plus ethanol, the correlation between glycogen and ATP contents presented two linear steps: before and after the 3rd hour (30 and 102 µmol glycogen/g ww per µmol ATP/g ww, respectively, the latter being near the single step measured in glucose). After glucose plus ethanol, ATP turnover remained stable for 2 h, was 3-fold higher from the 3rd hour to the 8th hour, and was higher than after glucose (2.59 ± 0.45 and 1.39 ± 0.19 µmol/min/g ww, respectively). In the 1st hour, glucose plus ethanol induced a transient acidosis and an increase in the phosphomonoesters signal. In conclusion, after ethanol consumption, a large part of the ATP production was diverted to redox re-equilibrium during the first 2 h, thereby reducing the glycogen synthesis. Thereafter, the maintenance of a large oxidative phosphorylation allowed the stimulation of glycogen synthesis requiring ATP.

Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using (1)H- and (13)C-NMR spectroscopy.

The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-(13)C]glucose (Glc) or [2,4-(13)C2]ß-hydroxybutyrate (ß-HB). Specific enrichments of amino acids (AAs) measured by (1)H- and (13)C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-(13)C]Glc infusion whereas they were higher in KD rats after [2,4-(13)C2]ß-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.

Energy restriction does not prevent insulin resistance but does prevent liver steatosis in aging rats on a Western-style diet.

OBJECTIVE: The aim of this study was to evaluate the effects of long-term energy restriction (ER) on plasma, liver, and skeletal muscle metabolite profiles in aging rats fed a Western-style diet. METHODS: Three groups of male Sprague-Dawley rats were studied. Group 1 consisted of 2 mo old rats fed ad libitum; group 2 were 19 mo old rats also fed ad libitum; and group 3 were 19 mo old rats subjected to 40% ER for the last 11.5 mo. To imitate a Western-style diet, all rats were given a high-sucrose, very low ω-3 polyunsaturated fatty acid (PUFA) diet. High-resolution magic angle spinning-(1)H nuclear magnetic resonance spectroscopy was used for hepatic and skeletal muscle metabolite determination, and fatty acid profiles were measured by capillary gas chromatography on plasma, liver, and skeletal muscle. RESULTS: ER coupled with a Western-style diet did not prevent age-induced insulin resistance or the increase in triacylglycerol content in plasma and skeletal muscle associated with aging. However, in the liver, ER did prevent steatosis and increased the percent of saturated and monounsaturated fatty acids relative to ω-6 and ω-3 PUFA. CONCLUSIONS: Although steatosis was reduced, the beneficial effects of ER on systemic insulin resistance and plasma and skeletal muscle metabolites observed elsewhere with a balanced diet seem to be compromised by high-sucrose and low ω-3 PUFA intake.

Resveratrol mainly stimulates the glycolytic ATP synthesis flux and not the mitochondrial one: a saturation transfer NMR study in perfused and isolated rat liver.

Our aim was to monitor the effects of resveratrol (RSV) on the respective contribution of glycolysis and oxidative phosphorylation on the unidirectional flux of ATP synthesis in whole isolated rat liver perfused with Krebs-Henseleit Buffer (KHB). The rate of tissular ATP supply was measured directly by monitoring the chemical exchange Pi toward ATP with saturation transfer (ST) (31)P nuclear magnetic resonance, a method applied for the first time for studying the effects of RSV. ST allows the measurement of the total cellular Pi→ATP chemical exchange; after specific inhibition of glycolysis with iodacetate, ST could provide the Pi→ATP flux issued from mitochondria. This latter was compared to mitochondrial ATP turn-over evaluated after chemical ischemia (CI), performed with specific inhibition (KCN) of oxidative phosphorylation, and measured by standard (31)P NMR spectroscopy. In controls (KHB alone), the apparent time constant (ks) of Pi exchange toward ATP as measured by ST was 0.48±0.04s(-1) leading to a total ATP synthesis rate of 37±3.9µmolmin(-1)g(-1). KHB+RSV perfusion increased ks (+52%; p=0.0009 vs. KHB) leading to an enhanced rate of total ATP synthesis (+52%; p=0.01 vs. KHB). When glycolysis was previously inhibited in KHB, both ks and ATP synthesis flux dramatically decreased (-87% and -86%, respectively, p<0.0001 vs. KHB without inhibition), evidencing a collapse of Pi-to-ATP exchange. However, glycolysis inhibition in KHB+RSV reduced to less extent ks (-41%, p=0.0005 vs. KHB+RSV without inhibition) and ATP synthesis flux (-18%). Using the CI method in KHB and KHB+RSV, KCN addition after glycolysis inhibition induced a rapid fall to zero of the ATP content. The mitochondrial ATP turnover R(t0) and its time constant kd mito were similar in KHB (1.18±0.19µmolmin(-1)g(-1) and 0.91±0.13min(-1)) and KHB+RSV (1.36±0.26µmolmin(-1)g(-1) and 0.77±0.18min(-1)). Since mitochondrial ATP turnover was not increased by RSV, the stimulation of Pi-to-ATP exchange by RSV mainly reflected an increase in glycolytic ATP synthesis flux. Moreover, the maintenance by RSV of a high level of Pi-to-ATP exchange after glycolysis inhibition evidenced a protective effect of the polyphenol, in agreement with our previous hypothesis of a stimulation of substrate flux throughout the glycolysis 3-carbon step.

Long-term calorie restriction has minimal impact on brain metabolite and fatty acid profiles in aged rats on a Western-style diet.

The effect of long-term calorie restriction (CR) on metabolites, fatty acid profiles and energy substrate transporter expression in the brain was assessed in aged rats. Three groups of male Sprague-Dawley rats were studied: (i) a 2 month old ad libitum-fed (2AL group), (ii) a 19 month old ad libitum-fed (19AL group), and (iii) a 19 month old group subjected to 40% CR from the age of 7.5 to 19 months (19CR group). The diet contained high sucrose and low n-3 polyunsaturated fatty acids (PUFA) so as to imitate a Western-style diet. High resolution magic angle spinning-(1)H NMR showed an effect of aging on brain cortex metabolites compared to 2AL rats, the largest differences being for myo-inositol (+251% and +181%), lactate (+203% and +188%), ß-hydroxybutyrate (+176% and +618%) and choline (+148% and +120%), in 19AL and 19 CR rats, respectively. However, brain metabolites did not differ between the 19AL and 19CR groups. Cortex fatty acid profiles showed that n-3 PUFA were 35-47% lower but monounsaturated fatty acids were 40-52% higher in 19AL and 19CR rats compared to 2AL rats. Brain microvessel glucose transporter (GLUT1) was 68% higher in 19AL rats than in 2AL rats, while the monocarboxylate transporter, MCT1, was 61% lower in 19CR rats compared to 19AL rats. We conclude that on a high-sucrose, low n-3 PUFA diet, the brain of aged AL rats had higher metabolites and microvessel GLUT1 expression compared to 2AL rats. However, long-term CR in aged rats did not markedly change brain metabolite or fatty acid profile, but did reduce brain microvessel MCT1 expression.

Resveratrol plus ethanol counteract the ethanol-induced impairment of energy metabolism: ³¹P NMR study of ATP and sn-glycerol-3-phosphate on isolated and perfused rat liver.

The effects of trans-resveratrol (RSV) combined with ethanol (EtOH) were evaluated by (31)P NMR on total ATP and sn-glycerol-3-phosphate (sn-G3P) contents measured in real time in isolated and perfused whole liver of the rat. Mitochondrial ATP turnover was assessed by using specific inhibitors of glycolytic and mitochondrial ATP supply (iodacetate and KCN, respectively). In RSV alone, the slight decrease in ATP content (-14±5% of the initial content), sn-G3P content and ATP turnover were similar to those in the Krebs-Henseleit buffer control. Compared to control, EtOH alone (14 or 70 mmol/L) induced a decrease in ATP content (-24.95±2.95% of initial content, p<0.05) and an increase in sn-G3P (+158±22%), whereas ATP turnover tended to be increased. RSV (20 µmol/L) combined with EtOH, (i) maintained ATP content near 100%, (ii) induced a 1.6-fold increase in mitochondrial ATP turnover (p=0.049 and p=0.004 vs EtOH 14 and 70 mmol/L alone, respectively) and (iii) led to an increase in sn-G3P (+49±9% and +81±6% for 14 and 70 mmol/L EtOH, respectively). These improvements were obtained only when glycolysis was efficient at the time of addition of EtOH+RSV. Glycolysis inhibition by iodacetate (IAA) evidenced an almost 21% contribution of this pathway to ATP content. RSV alone or RSV+EtOH prevented the ATP decrease induced by IAA addition (p<0.05 vs control). This is the first demonstration of the combined effects of RSV and EtOH on liver energy metabolism. RSV increased (i) the flux of substrates through ATP producing pathways (glycolysis and phosphorylative oxidation) probably via the activation of AMPkinase, and (ii) maintained the glycolysis deviation to sn-G3P linked to NADH+H⁺ re-oxidation occurring during EtOH detoxication, thus reducing the energy cost due to the latter.

A metabolic link between mitochondrial ATP synthesis and liver glycogen metabolism: NMR study in rats re-fed with butyrate and/or glucose.

BACKGROUND: Butyrate, end-product of intestinal fermentation, is known to impair oxidative phosphorylation in rat liver and could disturb glycogen synthesis depending on the ATP supplied by mitochondrial oxidative phosphorylation and cytosolic glycolysis. METHODS: In 48 hr-fasting rats, hepatic changes of glycogen and total ATP contents and unidirectional flux of mitochondrial ATP synthesis were evaluated by ex vivo 31P NMR immediately after perfusion and isolation of liver, from 0 to 10 hours after force-feeding with (butyrate 1.90 mg + glucose 14.0 mg.g-1 body weight) or isocaloric glucose (18.2 mg.g-1 bw); measurements reflected in vivo situation at each time of liver excision. The contribution of energetic metabolism to glycogen metabolism was estimated. RESULTS: A net linear flux of glycogen synthesis (~11.10 ± 0.60 µmol glucosyl units.h-1.g-1 liver wet weight) occurred until the 6th hr post-feeding in both groups, whereas butyrate delayed it until the 8th hr. A linear correlation between total ATP and glycogen contents was obtained (r2 = 0.99) only during net glycogen synthesis. Mitochondrial ATP turnover, calculated after specific inhibition of glycolysis, was stable (~0.70 ± 0.25 µmol.min-1.g-1 liver ww) during the first two hr whatever the force-feeding, and increased transiently about two-fold at the 3rd hr in glucose. Butyrate delayed the transient increase (1.80 ± 0.33 µmol.min-1.g-1 liver ww) to the 6th hr post-feeding. Net glycogenolysis always appeared after the 8th hr, whereas flux of mitochondrial ATP synthesis returned to near basal level (0.91 ± 0.19 µmol.min-1.g-1 liver ww). CONCLUSION: In liver from 48 hr-starved rats, the energy need for net glycogen synthesis from exogenous glucose corresponds to ~50% of basal mitochondrial ATP turnover. The evidence of a late and transient increase in mitochondrial ATP turnover reflects an energetic need, probably linked to a glycogen cycling. Butyrate, known to reduce oxidative phosphorylation yield and to induce a glucose-sparing effect, delayed the transient increase in mitochondrial ATP turnover and hence energy contribution to glycogen metabolism.

Vitamin A deficiency in rats induces anatomic and metabolic changes comparable with those of neurodegenerative disorders.

Anatomic and metabolic changes in central nervous system induced by 14 wk of vitamin A deprivation (VAD) were monitored and quantified in rats. In vivo brain magnetic resonance imaging (4.7T) was performed at 5, 7, 9, 11, and 14 wk of each diet after weaning in the following: 1) VAD group; 2) control pair-fed group; and 3) control group that consumed the diet ad libitum (1.15 microg retinol/g diet). After 14 wk, high-resolution magic angle spinning proton NMR spectroscopy (11.7T) was performed on small samples of cortex, hippocampus, and striatum. Serum retinol concentrations remained stable and cerebral volume (CV) increased as a linear function of body weight in the ad libitum group (R(2) = 0.78; P = 0.047) and pair-fed controls (R(2) = 0.78; P = 0.046). In VAD rats, retinol decreased from the onset of deprivation (2.2 +/- 0.14 micromol/L) to reach 0.3 +/- 0.13 micromol/L at wk 5, followed by a stopping of body weight gain from wk 7. In VAD rats, the CV decreased from wk 5 and reached a value 11% lower than that of the control group (P < 0.001) at wk 14 and was correlated with retinol status (R(2) = 0.99; P = 0.002). The VAD hippocampal volume decreased beginning at wk 9 and was 22% lower than that of the control group at wk 14 (P < 0.001). Compared with the control, VAD led to lower N acetyl aspartate:creatine+phosphocreatine (Cr) in cortex (-36%), striatum (-22%), and hippocampus (-19%) and higher myoinositol:Cr in cortex (+127%) and striatum (+150%). VAD induced anatomic and metabolic changes comparable to those associated with neurodegenerative disorders. By wk 7 of deprivation, the slowing in cerebral growth that correlated with the retinol level could be considered as a predictive marker of brain disorders, confirmed by metabolic data from VAD rats after 14 wk.

Butyrate ingestion improves hepatic glycogen storage in the re-fed rat.

BACKGROUND: Butyrate naturally produced by intestinal fiber fermentation is the main nutrient for colonocytes, but the metabolic effect of the fraction reaching the liver is not totally known. After glycogen hepatic depletion in the 48-hour fasting rat, we monitored the effect of (butyrate 1.90 mg + glucose 14.0 mg)/g body weight versus isocaloric (glucose 18.2 mg/g) or isoglucidic (glucose 14.0 mg/g) control force-feeding on in vivo changes in hepatic glycogen and ATP contents evaluated ex vivo by NMR in the isolated and perfused liver. RESULTS: The change in glycogen was biphasic with (i) an initial linear period where presence of butyrate in the diet increased (P = 0.05) the net synthesis rate (0.20 +/- 0.01 micromol/min.g(-1) liver wet weight, n = 15) versus glucose 14.0 mg/g only (0.16 +/- 0.01 micromol/min.g(-1) liver ww, n = 14), and (ii) a plateau of glycogen store followed by a depletion. Butyrate delayed the establishment of the equilibrium between glycogenosynthetic and glycogenolytic fluxes from the 6th to 8th hour post-feeding. The maximal glycogen content was then 97.27 +/- 10.59 micromol/g liver ww (n = 7) at the 8th hour, which was significantly higher than with the isocaloric control diet (64.34 +/- 8.49 micromol/g, n = 12, P = 0.03) and the isoglucidic control one (49.11 +/- 6.35 micromol/g liver ww, n = 6, P = 0.003). After butyrate ingestion, ATP content increased from 0.95 +/- 0.29 to a plateau of 2.14 +/- 0.23 micromol/g liver ww at the 8th hour post-feeding (n = 8) [P = 0.04 versus isoglucidic control diet (1.45 +/- 0.19 micromol/g, n = 8) but was not different from the isocaloric control diet (1.70 +/- 0.18 micromol/g, n = 12)]. CONCLUSION: The main hepatic effect of butyrate is a sparing effect on glycogen storage explained (i) by competition between butyrate and glucose oxidation, glucose being preferentially directed to glycogenosynthesis during the post-prandial state; and (ii) by a likely reduced glycogenolysis from the newly synthesized glycogen. This first demonstration of the improvement of liver glycogen storage by acute butyrate supply may be an important contribution to explaining the beneficial effects on glucose homeostasis of nutritional supply increasing butyrate amount such as fiber diets.

Decrease in oxidative phosphorylation yield in presence of butyrate in perfused liver isolated from fed rats.

BACKGROUND: Butyrate is the main nutrient for the colonocytes but the effect of the fraction reaching the liver is not totally known. A decrease in tissue ATP content and increase in respiration was previously demonstrated when livers were perfused with short-chain fatty acids (SCFA) such as butyrate, or octanoate. In fed rats the oxidative phosphorylation yield was determined on the whole isolated liver perfused with butyrate in comparison with acetate and octoanoate (3 mmol/L). The rate of ATP synthesis was determined in the steady state by monitoring the rate of ATP loss after inhibition of (i) cytochrome oxidase (oxidative phosphorylation) with KCN (2.5 mmol/L) and (ii) glyceraldehyde 3-phosphate dehydrogenase (glycolysis) with IAA (0.5 mmol/L). The ATP flux, estimated by 31P Nuclear Magnetic Resonance, and the measured liver respiration allowed the ATP/O ratio to be determined. RESULTS: ATP turnover was significantly lower in the presence of butyrate (0.40 +/- 0.10 micromoles/min.g, p = 0.001, n = 7) and octanoate (0.56 +/- 0.10 micromoles/min.g, p = 0.01, n = 5) than in control (1.09 +/- 0.13 micromoles/min.g, n = 7), whereas perfusion with acetate induced no significant decrease (0.76 +/- 0.10 micromoles/min.g, n = 7). Mitochondrial oxygen consumption was unchanged in the presence of acetate (1.92 +/- 0.16 vs 1.86 +/- 0.16 for control) and significantly increased in the presence of butyrate (p = 0.02) and octanoate (p = 0.0004) (2.54 +/- 0.18 and 3.04 +/- 0.15 micromoles/min.g, respectively). The oxidative phosphorylation yield (ATP/O ratio) calculated in the whole liver was significantly lower with butyrate (0.07 +/- 0.02, p = 0.0006) and octanoate (0.09 +/- 0.02, p = 0.005) than in control (0.30 +/- 0.05), whereas there was no significant change with acetate (0.20 +/- 0.02). CONCLUSION: Butyrate or octanoate decrease rather than increase the rate of ATP synthesis, resulting in a decrease in the apparent ATP/O ratio. Butyrate as a nutrient has the same effect as longer chain FA. An effect on the hepatic metabolism should be taken into account when large quantities of SCFA are directly used or obtained during therapeutic or nutritional strategies.

Effects of hyperglycemia on glucose metabolism before and after oral glucose ingestion in normal men.

The plasma glucose excursion may influence the metabolic responses after oral glucose ingestion. Although previous studies addressed the effects of hyperglycemia in conditions of hyperinsulinemia, it has not been evaluated whether the route of glucose administration (oral vs. intravenous) plays a role. Our aim was to determine the effects of moderately controlled hyperglycemia on glucose metabolism before and after oral glucose ingestion. Eight normal men underwent two oral glucose clamps at 6 and 10 mmol/l plasma glucose. Glucose turnover and cycling rates were measured by infusion of [2H7]glucose. The oral glucose load was labeled by D-[6,6-2H2]glucose to monitor exogenous glucose appearance, and respiratory exchanges were measured by indirect calorimetry. Sixty percent of the oral glucose load appeared in the systemic circulation during both the 6 and 10 mmol/l plasma glucose tests, although less endogenous glucose appeared during the 10 mmol/l tests before glucose ingestion (P < 0.05). This inhibitory effect of hyperglycemia was not detectable after oral glucose ingestion, although glucose utilization was increased (+28%, P < 0.05) due to increased nonoxidative glucose disposal [10 vs. 6 mmol/l: +20%, not significant (NS) before oral glucose ingestion; +40%, P < 0.05 after oral glucose ingestion]. Glucose cycling rates were increased by hyperglycemia (+13% before oral glucose ingestion, P < 0.001; +31% after oral glucose ingestion, P < 0.05) and oral glucose ingestion during both the 6 (+10%, P < 0.05) and 10 mmol/l (+26%, P < 0.005) tests. A moderate hyperglycemia inhibits endogenous glucose production and contributes to glucose tolerance by enhancing nonoxidative glucose disposal. Hyperglycemia and oral glucose ingestion both stimulate glucose cycling.

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.

BACKGROUND: There 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. RESULTS: Liver 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 % x min(-1) and ATP content decreased at a rate of -0.28 +/- 0.029 % x 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: flux(glycogen) = 72.543(fluxATP) + 172.08, R2 = 0.98. CONCLUSION: Only 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.

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

BACKGROUND: In 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. RESULTS: Ethanol (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. CONCLUSION: Various 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.

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.