Lack of starvation-induced activation of AMP-activated protein kinase in the hypothalamus of the Lou/C rats resistant to obesity.

OBJECTIVE: The AMP-activated protein kinase (AMPK) is involved in the control of food intake by the hypothalamus. The aim of this work was to investigate if modification of hypothalamic AMPK regulation could be related to the spontaneous food restriction of Lou/C rats, a strain resistant to obesity exhibiting a 40% reduction in caloric intake compared with their lean Wistar counterparts. DESIGN: Three-month-old male Lou/C rats were compared with age-matched male Wistar rats in both fed ad libitum and 24-h food deprivation state. MEASUREMENTS AND RESULTS: We first confirmed that starvation activated both isoforms of AMPK catalytic alpha subunits and enhanced the phosphorylation state of its downstream targets acetyl-CoA carboxylase and elongation factor 2 in the hypothalamus of Wistar rats. These changes were not observed in the hypothalamus of Lou/C rats. Interestingly, the starvation-induced changes in hypothalamic mRNA levels of the main orexigenic and anorexigenic neuropeptides were also blunted in the Lou/C rats. Analysis of the concentrations of circulating substrates and hormones known to regulate hypothalamic AMPK indicated that the starvation-induced changes in ghrelin, adiponectin and leptin were not observed in Lou/C rats. Furthermore, an increased phosphorylation state of signal transducer and activator of transcription 3 (STAT3), which admittedly mediates leptin signaling, was evidenced in the hypothalamus of the starved Lou/C rats, as well as modifications of expression of the leptin-sensitive genes suppressor of cytokine signaling-3 and stearoyl-coenzyme A desaturase 1. In addition, despite reduced leptin level in fed Lou/C rats, the phosphorylation state of hypothalamic STAT3 remained similar to that found in fed Wistar rats, an adaptation that could be explained by the concomitant increase in ObRb leptin receptor mRNA expression. CONCLUSION: Activation of hypothalamic AMPK by starvation, which stimulates food intake through changes in (an)orexigenic neuropeptides in the normal rats, was not observed in the spontaneously hypophagic Lou/C rats.

Acute in vivo administration of a fish oil-containing emulsion improves post-ischemic cardiac function in n-3-depleted rats.

A novel i.v. lipid preparation (MCT:FO) containing 80% medium chain-triacylglycerols and 20% fish oil was recently developed to rapidly replenish cell membrane phospholipids with omega 3 (n-3) polyunsaturated fatty acids (PUFA). In regard of this property, we investigated the effect of a single i.v. administration of MCT:FO on the recovery of cardiac function after ischemia in control and n-3-depleted rats. Results were compared with those obtained either with a control preparation, where FO was replaced by triolein (MCT:OO), or with saline. Saline (1 ml) or lipid preparation (also 1 ml) was injected as a bolus via the left saphenous vein. After 60 min the heart was removed and perfused for 20 min in normoxic conditions according to Langendorff. Thereafter, the heart was subjected to a 20 min zero-flow normothermic ischemia, followed by 40 min reperfusion. Cardiac mechanical and metabolic functions were monitored. In control rats, the previous administration of a lipid preparation (MCT:FO or MCT:OO) versus saline improved cardiac function during aerobic reperfusion post-ischemia. N-3-depleted rats showed decreased basal cardiac function and impaired recovery following ischemia. However, the bolus injection of MCT:FO opposed the deleterious effect of long-term n-3-deficiency and, in this respect, was superior to MCT:OO over the first 20 min of reperfusion. This novel approach to rapidly correct n-3 PUFA-deficiency might be clinically relevant and offer interesting perspectives in the management of acute ischemic accidents.

Effect of polyunsaturated fatty acids deficiency on oxidative phosphorylation in rat liver mitochondria.

Liver mitochondria isolated from controls or polyunsaturated fatty acid (PUFA) deficient rats were studied for oxidative phosphorylation. A PUFA-deficient diet led to a dramatic change in the fatty acid composition of mitochondrial lipid content, similar to that reported in the literature. Besides the changes in lipid composition, mitochondrial volume was enlarged (+45% in state 4 and two-fold in state 3). State 4 respiration was increased together with a decrease in protonmotive force. The non-ohmicity of the relationship between non-phosphorylating respiration and protonmotive force was more pronounced in the PUFA-deficient group. State 3 oxygen consumption as well as the rate of ATP synthesis showed no difference between the two groups, whereas the protonmotive force decreased substantially in mitochondria from PUFA-deficient animals. In contrast, ATP/O ratios were decreased in the PUFA-deficient group when determined at subsaturating ADP concentration. Taken together, these results are in agreement with both an increased non-ohmic proton leak and an increased redox slipping. The relative importance of these two effects on the overall efficiency of oxidative phosphorylation depends on both the rate of oxidative phosphorylation and the maintained protonmotive force. Hence, in isolated mitochondria the respective role of each effect may vary between state 4 and state 3.

Kinetic analysis of short-term effects of alpha-agonists on gluconeogenesis in isolated rat hepatocytes.

Isolated hepatocytes from fasted rats were perifused with glycerol as gluconeogenic substrate. Stimulation of gluconeogenesis with phenylephrine (10(-5) M) as alpha-adrenergic agonist consisted of two distinct phases. The first phase was a transient stimulation of gluconeogenesis and was accompanied by transient changes in cytosolic and mitochondrial redox state; this phase was abolished by the transaminase inhibitor aminooxyacetate. The second phase was a stable stimulation of less magnitude, without change in redox state and insensitive to addition of aminooxyacetate. It is concluded that the first phase is due to a transient enhancement of flux through the malate/aspartate shuttle and that the stable phase is probably due to a stimulation of mitochondrial glycerol-3-phosphate dehydrogenase and glycerol kinase.

Control of proteolysis in perifused rat hepatocytes.

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.

Chronic ethanol ingestion increases efficiency of oxidative phosphorylation in rat liver mitochondria.

The efficiency of oxidative phosphorylation was compared between rats chronically fed with ethanol and controls. (i) Results showed that the liver mitochondria state 4 respiratory rate was strongly inhibited, while the corresponding proton-motive force was not affected; (ii) the cytochrome oxidase content and activity were decreased and (iii) the oxidative-phosphorylation yield was increased in the ethanol exposed group. Furthermore, oxidative phosphorylation at coupling site II was not affected by ethanol. Cytochrome oxidase inhibition by sodium-azide mimicked the effects of ethanol intoxication in control mitochondria. This indicates that the decrease in cytochrome oxidase activity induced by ethanol intoxication directly increases the efficiency of oxidative phosphorylation.

Mitochondrial metabolism and type-2 diabetes: a specific target of metformin.

Several links relate mitochondrial metabolism and type 2 diabetes or chronic hyperglycaemia. Among them, ATP synthesis by oxidative phosphorylation and cellular energy metabolism (ATP/ADP ratio), redox status and reactive oxygen species (ROS) production, membrane potential and substrate transport across the mitochondrial membrane are involved at various steps of the very complex network of glucose metabolism. Recently, the following findings (1) mitochondrial ROS production is central in the signalling pathway of harmful effects of hyperglycaemia, (2) AMPK activation is a major regulator of both glucose and lipid metabolism connected with cellular energy status, (3) hyperglycaemia by inhibiting glucose-6-phosphate dehydrogenase (G6PDH) by a cAMP mechanism plays a crucial role in NADPH/NADP ratio and thus in the pro-oxidant/anti-oxidant cellular status, have deeply changed our view of diabetes and related complications. It has been reported that metformin has many different cellular effects according to the experimental models and/or conditions. However, recent important findings may explain its unique efficacy in the treatment of hyperglycaemia- or insulin-resistance related complications. Metformin is a mild inhibitor of respiratory chain complex 1; it activates AMPK in several models, apparently independently of changes in the AMP-to-ATP ratio; it activates G6PDH in a model of high-fat related insulin resistance; and it has antioxidant properties by a mechanism (s), which is (are) not completely elucidated as yet. Although it is clear that metformin has non-mitochondrial effects, since it affects erythrocyte metabolism, the mitochondrial effects of metformin are probably crucial in explaining the various properties of this drug.

Effect of chronic renal failure with metabolic acidosis on alanine metabolism in isolated liver cells.

BACKGROUND & AIMS: Decreased ureagenesis and gluconeogenesis from alanine have been reported during chronic renal failure in rat. This study addressed the respective roles of plasma-membrane transport and intracellular metabolism in these abnormalities of alanine pathways. METHODS: In hepatocytes isolated from uremic and control rats, we investigated: (1) the influence of uremia on gluconeogenesis and ureagenesis during incubations with alanine; (2) the kinetics of alanine plasma-membrane transport; (3) the relationships between intracellular alanine concentrations and its metabolism. Plasma-membrane alanine transport was assessed after addition of alanine (2 mM) by measuring its intracellular accumulation from 0 to 10 min, in the presence of a transaminase inhibitor. Alanine metabolism was studied in perifused hepatocytes by measuring intracellular alanine concentration together with urea, glucose and lactate production in the presence of increasing concentrations of alanine (0-8 mM). RESULTS: Uremic rats showed decreased plasma bicarbonate. Uremia induced (P<0.05) a decrease in both gluconeogenesis (36%) and ureagenesis (22%). Alanine plasma-membrane transport decreased by 20% during uremia. During perifusions, uremia induced a 30-40% decrease in urea, glucose, and lactate production without modifying intracellular alanine concentration. CONCLUSIONS: In uremic rats with acidosis, hepatocyte alanine utilization was impaired at both plasma-membrane transport and intracellular transamination steps.

Cardioprotective effect of spontaneous activity.

In the perspective of giving a better understanding of the cardioprotective effects attributable to the tandem low caloric intake and training, Lou/C rats would be an interesting model since these animals exhibit spontaneously these two characteristics for months, without any dietary manipulations or stressor stimuli. No information was so far available on their cardiac function. Therefore, the aim of this pilot study was (i) to document cardiac function before and after ischemia in this strain, and (ii) to investigate whether spontaneous wheel-running activity can improve the ability of cardiac muscle to recover its function after an ischemic period. Cardiac mechanical and metabolic functions were measured in isolated Langendorff hearts from Wistar sedentary, Lou/C sedentary, and Lou/C wheel-running male rats submitted to a 20-min low-flow ischemia and 20-min reperfusion. In Lou/C sedentary rats, rate-pressure product, an index of cardiac work, was decreased before ischemia as compared to Wistar sedentary animals (- 24 %, p < 0.05). After ischemia, cardiac mechanical function recovery did not significantly differ between these two groups. Nevertheless, flux of non-oxidative glycolysis was lower before and after ischemia in Lou/C sedentary animals than in Wistar sedentary rats. In Lou/C rats, during normoxic perfusion, wheel-running activity significantly decreased heart rate (- 15 %), oxygen consumption (- 2.2 %) and cardiac efficiency (- 37 %), whereas coronary flow and flux of non-oxidative glycolysis were significantly increased (+ 15 % and + 263 %, respectively). After ischemia, recovery of cardiac mechanical function and cardiac efficiency were improved in Lou/C wheel-running rats versus Lou/C sedentary animals (p < 0.05). In conclusion, the impact of ischemia-reperfusion is similar between Lou/C- and Wistar sedentary rats. Spontaneous wheel-running activity decreases cardiac efficiency before ischemia and confers a protection against ischemia- and reperfusion-induced injury in isolated Lou/C rat hearts.

Effect of exogenous adenosine and monensin on glycolytic flux in isolated perfused normoxic rat hearts: role of pyruvate kinase.

We studied the effect of exogenous adenosine in isolated perfused normoxic rat hearts on glycolytic flux through pyruvate kinase (PK). We compared its effect with that of myxothiazol, an inhibitor of mitochondrial ATP production. Moreover, we tested whether an increase of membrane ionic flux with monensin is linked to a stimulation of glycolytic flux through PK. After a 20-min stabilization period adenosine, myxothiazol or monensin were administrated to the perfusate continuously at various concentrations during 10 min. The contraction was monitored and the lactate production in coronary effluents evaluated. The amount of adenine nucleotides and phosphoenolpyruvate was measured in the frozen hearts. Myxothiazol induced a decrease of the left ventricular developed pressure (LVDP : -40%) together with a stimulation of glycolytic flux secondary to PK activation. In contrast, adenosine primarily reduced heart rate (HR: -30%) with only marginal effects on LVDP. This was associated with an inhibition of glycolysis at the level of PK. The Na+ ionophore monensin affected HR (+14%) and LVDP (+25%). This effect was associated with a stimulation of glycolysis secondary to the stimulation of PK. These results provide new information of action of adenosine in the heart and support the concept of a direct coupling between glycolysis and process regulating sarcolemmal ionic fluxes.

Inter-organ substrate exchanges in the critically ill.

Metabolic inter-organ exchange is a major field of research for improving the treatment of the critically ill. Adapting regional blood flows is the first regulatory step, although the relationships between hypoperfusion and metabolic disorders are matter of controversy. Metabolic steady state results from a vast inter-organ interplay and several nutrients or metabolites are signalling molecules in the regulation of gene transcription. Inter- or intra-organ substrate recycling shares or delays the mandatory need for aerobic ATP synthesis in some conditions. Nitrogen metabolism is highly compartmentalised in an inter-organ co-operation and liver, muscle, kidney and gut are the most important organs. By remodelling the amino acid mixture delivered to peripheral cells after intestinal absorption, the liver plays a determinant role in whole body protein synthesis. Albumin turnover increases after brain injury. Since the location of synthesis is different to that of breakdown this turnover can be viewed as an inter-organ exchange. The metabolic side of pH homeostasis is also an inter-organ exchange mainly shared by liver, kidney and muscle.

Energy metabolism in critically ill patients: lactate is a major oxidizable substrate.

The potential role of an energy defect in acute diseases is still in the centre of the pathophysiological understanding of such states and therefore of our attempts to limit or to reverse the possible deleterious consequences of such defect. In fact several recent experimental works have shown that instead of being a negative consequence, the lactate production and the related metabolic acidosis due to the stimulation of anaerobic ATP-production pathway is rather a protective adapted response.

Lactic acidosis. A new insight?

From an intensivist point of view, lactic acid is (i) responsible for metabolic acidosis, (ii) related to anoxia or ischemia and (iii) associated with poor prognosis. Conversely, from a biochemist point of view lactate is a good cellular substrate which can be easily converted to pyruvate and used as gluconeogenic substrate, or oxidised or transaminated into alanine. Hence the main question is not anymore to assess the value of lactate concentration as a marker of severity (it is well established) but rather to understand the metabolic meaning of its increase: is it beneficial or deleterious? In fact several recent experimental works have shown that instead of being a negative consequence, lactate production and related metabolic acidosis due to the stimulation of anaerobic ATP-production pathway could be a protective adapted response.

Role of substrates in the regulation of mitochondrial function in situ.

Investigations of mitochondrial oxidative phosphorylation have been mainly carried out in isolated mitochondria, where the experimental conditions can be precisely set. However, in intact living systems oxidative phosphorylation takes place in a complex environment, whose experimental dissection is a major challenge. It has long been recognized that the efficiency of oxidative phosphorylation depends on the nature of the respiratory substrates, which feed electrons to the respiratory chain at different levels. Yet, the role of substrates in determining mitochondrial function and their response to energetic stress has been largely overlooked. Here we review recent work showing that the nature of the energetic substrates profoundly affects the mitochondrial responses to manipulations of pathophysiological relevance, such as uncoupling and opening of the permeability transition pore (PTP). Uncoupling of intact hepatocytes caused very different metabolic effects depending on whether carbohydrates or lipids were the energy source. With dihydroxyacetone as the substrate dinitrophenol caused a collapse of the mitochondrial membrane potential and of the ATP/ADP ratio, while the respiratory rate was increased only transiently. With octanoate as the substrate, on the other hand, dinitrophenol caused a dramatic stimulation of the respiratory rate, while the mitochondrial membrane potential and ATP/ADP ratio were affected only marginally. We then review results indicating that the activity of complex I directly regulates the PTP, a finding that emphasizes the importance of the respiratory substrates in PTP regulation.

Inhibition of gluconeogenesis in isolated rat hepatocytes after chronic treatment with phenobarbital.

Gluconeogenesis was studied in hepatocytes isolated from phenobarbital-pretreated rats fasted for 24 h. In closed vial incubations, glucose production from lactate (20 mmol/l) and pyruvate (2 mmol/l), alanine (20 mmol/l) or glutamine (20 mmol/l) was suppressed by about 30-45%, although glycerol metabolism was not affected. In hepatocytes perifused with lactate and pyruvate (ratio 10:1), glucose production was inhibited by 50%, even at low gluconeogenic flux. From the determination of gluconeogenic intermediates at several steady states of gluconeogenic flux, we have found a single relationship between phosphoenolpyruvate and the rate of glucose production (Jglucose), and two different curves between cytosolic oxaloacetate and Jglucose in controls and in phenobarbital-pretreated hepatocytes. By using 3-mercaptopicolinate to determine the flux control coefficient of phosphoenolpyruvate carboxykinase we found that phenobarbital pretreatment led to an increase in this coefficient from 0.3 (controls) to 0.8 (phenobarbital group). These observations were confirmed by the finding that the activity of phosphoenolpyruvate carboxykinase was decreased by 50% after phenobarbital treatment. Hence we conclude that the inhibitory effect of phenobarbital on gluconeogenesis is due, at least partly, to a decrease in the flux through phosphoenolpyruvate carboxykinase.

Determination of mitochondrial reactive oxygen species: methodological aspects.

The generation of Reactive Oxygen Species (ROS) as by-products in mitochondria Electron Transport Chain (ETC) has long been admitted as the cost of aerobic energy metabolism with oxidative damages as consequence. The purpose of this methodological review is to present some of the most widespread methods of ROS generation and to underline the limitations as well as some problems, identified with some experiments as examples, in the interpretation of such results. There is now no doubt that besides their pejorative role, ROS are involved in a variety of cellular processes for the continuous adaptation of the cell to its environment. Because ROS metabolism is a complex area (low production, instability of species, efficient antioxidant defense system, several places of production...) bias, variances and limitations in ROS measurements must be recognized in order to avoid artefactual conclusions, and especially to improve our understanding of physiological and pathophysiological mechanisms of such phenomenon.

Metformin decreases gluconeogenesis by enhancing the pyruvate kinase flux in isolated rat hepatocytes.

Metformin (dimethylbiguanide) has been used for more than 30 years as an antihyperglycemic agent in the treatment of diabetes mellitus, but its effect on gluconeogenesis is still controversial. In isolated hepatocytes from fasted rats, a significant inhibition of glucose production from lactate/pyruvate (10:1, mol/mol), fructose, alanine or glutamine, following metformin addition, is observed. Moreover, in hepatocytes perifused with dihydroxyacetone as the gluconeogenic substrate and treated with 0.5 mM metformin, an inhibition of the glucose flux and a simultaneous stimulation of the lactate/pyruvate flux were observed. This enhancement of lactate/pyruvate formation appears to be due to an effect on the pyruvate-kinase enzyme. A direct effect of metformin on pyruvate kinase cannot explain this result, since pyruvate-kinase activity was not affected by metformin at this concentration. In contrast, the addition of metformin caused a significant decrease in the cellular ATP concentration, a known allosteric inhibitor of this enzyme. This could explain the stimulation of pyruvate-kinase activity following metformin addition and thus the inhibition of gluconeogenesis.