Hugues Oudart

PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation

SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.

Up-regulation of mitochondrial uncoupling protein 3 reveals an early muscular metabolic defect in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting primarily motor neurons. Growing evidence suggests a mitochondrial defect in ALS. The precise molecular mechanisms underlying those defects are unknown. We studied the expression of mitochondrial uncoupling proteins (UCPs), key regulators of mitochondrial functions, in tissues from a mouse model of ALS (SOD1 G86R transgenic mice) and from muscular biopsies of human sporadic ALS. Surprisingly, in SOD1 G86R mice, UCPs, and particularly UCP3, were upregulated in skeletal muscle but not in spinal cord. Consistent with this pattern of expression, ATP levels were selectively depleted in muscle but not in neural tissues 1 month before disease onset and the respiratory control ratio of isolated mitochondria is decreased. UCP3 up‐regulation was not observed in experimentally denervated muscles, suggesting that changes in muscular UCP3 expression are associated with the physiopathological processes of ALS. This is further supported by our observation of increased UCP3 levels in human ALS muscular biopsies. We propose that UCP3 up‐regulation in skeletal muscle contributes to the characteristic mitochondrial damage of ALS and to the onset of the disease. Moreover, since skeletal muscle is a key metabolic tissue, our findings suggest that ALS may not solely arise from neuronal events but also from more generalized metabolic defects.

Mitochondria in amyotrophic lateral sclerosis: a trigger and a target.

Strong evidence shows that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis (ALS), but despite the fact that mitochondria play a central role in excitotoxicity, oxidative stress and apoptosis, the intimate underlying mechanism linking mitochondrial defects to motor neuron degeneration in ALS still remains elusive. Morphological and functional abnormalities occur in mitochondria in ALS patients and related animal models, although their exact nature and extent are controversial. Recent studies postulate that the mislocalization in mitochondria of mutant forms of copper-zinc superoxide dismutase (SOD1), the only well-documented cause of familial ALS, may account for the toxic gain of function of the enzyme, and hence induce motor neuron death. On the other hand, mitochondrial dysfunction in ALS does not seem to be restricted only to motor neurons as it is also present in other tissues, particularly the skeletal muscle. The presence of this ‘systemic’ defect in energy metabolism associated with the disease is supported in skeletal muscle tissue by impaired mitochondrial respiration and overexpression of uncoupling protein 3. In addition, the lifespan of transgenic mutant SOD1 mice is increased by a highly energetic diet compensating both the metabolic defect and the motorneuronal function. In this review, we will focus on the mitochondrial dysfunction linked to ALS and the cause-and-effect relationships between mitochondria and the pathological mechanisms thought to be involved in the disease.

Hypothalamic gene expression in long-term fasted rats: relationship with body fat.

Many hypothalamic neuropeptides are involved in the regulation of energy homeostasis and feeding behavior. We have investigated whether and to what extent neuropeptide Y (NPY), agouti-related protein (AGRP), melanin-concentrating hormone (MCH), and prepro-orexin (prepro-OX) as well as pro-opiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART) mRNA levels are affected in rat hypothalamus. An experimental model of long-term fasting rat characterized by three metabolic phases from changes in lipid and protein utilization was used. Except for prepro-OX and compared to fed group, starvation induced an increase in the orexigenic gene expressions that was much more marked in phase 3 (by 2.5-, 8.1-, and 13.5-fold for MCH, AGRP, and NPY, respectively) than in phase 2 (by about 1.5-2.2-fold as an average) of fasting. AGRP and NPY mRNA levels were inversely related to body fat content. Anorexigenic gene expression was only slightly affected at both fasting stages. We conclude that the regulation of NPY and AGRP gene expression is primarily involved during late fasting and could mediate the concomitant enhanced drive for refeeding.

Brown fat thermogenesis in rats fed high-fat diets enriched with n-3 polyunsaturated fatty acids

OBJECTIVE: To examine the possible involvement of an increase in diet-induced thermogenesis from brown adipose tissue (BAT) in the n-3 polyunsaturated fatty acids (n-3 PUFA) induced limitation of the development of white fat pads during high-fat feeding. DESIGN: Rats fed for four weeks on a low-fat/high-carbohydrate diet (C group) or high-fat diet without n-3 PUFA (REF group), with eicosapentaenoic acid (EPA group), with docosahexaenoic acid (DHA group) or with a mixture of these two fatty acids (MIX group). MEASUREMENTS: Epididymal and retroperitoneal fat pad mass, BAT composition, Guanosine 5’-diphosphate (GDP) binding and uncoupling protein (UCP) content were measured in the five groups of rats. RESULTS: The masses of retroperitoneal and epididymal white fat pads were lower in the groups fed n-3 PUFA than in the C and REF groups. The total BAT GDP binding was 1.6 times higher in the MIX and EPA groups than in the REF group. The BAT from the EPA group presented an enrichment in mitochondria compared to the C and REF groups whereas the BAT from the DHA and REF groups presented a hyperplasia and an increase in thermogenic activity of the mitochondria compared to the C group. The higher thermogenic activity of BAT was observed in the MIX group and is due to hyperplasia and to an increase in thermogenic activity of mitochondria. CONCLUSIONS: n-3 PUFA induce a marked stimulation of BAT thermogenic activity without changes in the UCP content compared to a high-fat diet without n-3 PUFA. The mixture of EPA and DHA has the more pronounced effect while EPA and DHA seem to act in synergy on BAT thermogenesis via different mechanisms.

Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats.

Intestinal hexose absorption and gluconeogenesis have been studied in relation to refeeding after two different fasting phases: a long period of protein sparing during which energy expenditure is derived from lipid oxidation (phase II), and a later phase characterized by a rise in plasma corticosterone triggering protein catabolism (phase III). Such a switch in body fuel uses, leading to changes in body reserves and gluconeogenic precursors, could modulate intestinal gluconeogenesis and glucose transport. The gene and protein levels, and the cellular localization of the sodium–glucose cotransporter SGLT1, and of GLUT5 and GLUT2, as well as that of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (Glc6Pase) were measured. PEPCK and Glc6Pase activities were also determined. In phase III fasted rats, SGLT1 was up‐regulated and intestinal glucose uptake rates were higher than in phase II fasted and fed rats. PEPCK and Glc6Pase mRNA, protein levels and activities also increased in phase III. GLUT5 and GLUT2 were down‐regulated throughout the fast, but increased after refeeding, with GLUT2 recruited to the apical membrane. The increase in SGLT1 expression during phase III may allow glucose absorption at low concentrations as soon as food is available. Furthermore, an increased epithelial permeability due to fasting may induce a paracellular movement of glucose. In the absence of intestinal GLUT2 during fasting, Glc6Pase could be involved in glucose release to the bloodstream via membrane trafficking. Finally, refeeding triggered GLUT2 and GLUT5 synthesis and apical recruitment of GLUT2, to absorb larger amounts of hexoses.

Selectivity of fatty acids on lipid metabolism and gene expression.

Triacylglycerols represent the main form of storage for a wide spectrum of fatty acids. Their utilization first involves mobilization from adipose tissue through lipolysis. The release of individual fatty acids from adipose tissue is selective in vitro and in vivo in animal studies and also in human subjects. Generally, fatty acids are more readily mobilized from fat cells when they are short-chain and unsaturated. This selectivity could affect the storage of individual fatty acids in adipose tissue, and their subsequent supply to tissues. The nature of the dietary fats could affect lipid homeostasis and body fat deposition. Dietary fish oil influences adipose tissue development in a site-specific manner as a function of diet and feeding period. A diet high in n-3 polyunsaturated fatty acids (PUFA) results in a preferential partitioning of ingested energy towards oxidation at the expense of storage. Fatty acids are important mediators of gene expression in the liver. Indeed, genes encoding both glycolytic and lipogenic enzymes and key metabolic enzymes involved in fatty acid oxidation are regulated by dietary PUFA. White adipose tissue could also be a target for PUFA control of gene expression. The treatment of pre-adipose cells by fatty acids induces the expression of numerous genes that encode proteins involved in fatty acid metabolism. The mechanisms of PUFA-mediated repression of gene expression in adipocytes seem to be different, at least partly, from those described in liver. Tissue-specific and site-specific factors are possibly involved in the specific effect of PUFA on gene expression, although other mechanisms cannot be excluded.

Differences in the expression of lipolytic-related genes in rat white adipose tissues

We have investigated in vivo whether the gene expression of the beta3-adrenergic receptor (beta3-AR), perilipin A, hormone-sensitive lipase (HSL), and adipocyte lipid-binding protein (ALBP/aP2) is regulated in a site-specific manner. To induce lipolysis and discriminate between short- and long-term modifications, rats were submitted to an experimental fast for one or five days followed or not by refeeding. The mRNA encoding beta3-AR in retroperitoneal adipose tissue (RP) was significantly increased by one and five days of fasting (4-fold) and then lowered by one day of refeeding (2-fold) compared to fed rats. The reverse trend was observed for perilipin A expression in one day fasted rats. HSL mRNA concentrations were significantly induced (2.2-fold) by five days of fasting relative to fed animals and remained high after refeeding. ALBP/aP2, peroxisome proliferator-activated receptor gamma, and CAAT/enhancer binding protein alpha mRNA levels were essentially unaffected by dietary manipulations. Fasting and/or refeeding were similarly ineffective at regulating gene expression in SC. These data provide a molecular basis for regional differences at different steps of the lipolytic process.

Dietary palmitate and linoleate oxidations, oxidative stress, and DNA damage differ according to season in mouse lemurs exposed to a chronic food deprivation.

This study investigated the extent to which the increase in torpor expression in the grey mouse lemur, due to graded food restriction, is modulated by a trade-off between a whole body sparing of polyunsaturated dietary fatty acids and the related oxidative stress generated during daily torpor. We measured changes in torpor frequency, total energy expenditure (TEE), linoleate (polyunsaturated fatty acid) and palmitate (saturated fatty acid) oxidation, hexanoyl-lysine (HEL; the product of linoleate peroxidation), and 8-hydroxydeoxyguanosine (8OHdG; a marker of DNA damage). Animals under summer-acclimated long days (LD) or winter-acclimated short days (SD) were exposed to a 40% (LD40 and SD40) and 80% (LD80 and SD80) 35-day calorie restriction (CR). During CR, all groups reduced their body mass, but LD80 animals reached survival-threatened levels at day 22 and were then excluded from the CR trial. Only SD mouse lemurs increased their torpor frequency with CR and displayed a decrease in their TEE adjusted for fat-free mass. After CR, SD40 mouse lemurs shifted the dietary fatty acid oxidation toward palmitate and spared linoleate. Such a shift was not observed in LD animals and during severe CR, during which oxidation of both dietary fatty acids was increased. Concomitantly, HEL increased in both LD40 and SD80 groups, whereas DNA damage was only seen in SD80 food-restricted animals. HEL correlated positively with linoleate oxidation confirming in vivo the substrate/product relationship demonstrated in vitro, and negatively with TEE adjusted for fat-free mass, suggesting higher oxidative stress associated with increased torpor expression. This suggests a seasonal-dependant, cost-benefit trade-off between maximizing torpor propensity and minimizing oxidative stress that is associated with a shift toward sparing of dietary polyunsaturated fatty acids that is dependent upon the expression of a winter phenotype.

Clay ingestion enhances intestinal triacylglycerol hydrolysis and non-esterified fatty acid absorption

Consumption by animals and humans of earthy materials such as clay is often related to gut pathologies. Our aim was to determine the impact of kaolinite ingestion on glucose and NEFA transport through the intestinal mucosa. The expression of hexose transporters (Na/glucose co-transporter 1 (SGLT1), GLUT2, GLUT5) and of proteins involved in NEFA absorption (fatty acid transporter/cluster of differentiation 36 (FAT/CD36), fatty acid transport protein 4 (FATP4) and liver fatty acid binding protein (L-FABP)) was measured (1) in rats whose jejunum was perfused with a solution of kaolinite, and (2) in rats who ate spontaneously kaolinite pellets during 7 and 28 d. Also, we determined TAG and glucose absorption in the kaolinite-perfused group, and pancreatic lipase activity, gastric emptying and intestinal transit in rats orally administered with kaolinite. Glucose absorption was not affected by kaolinite perfusion or ingestion. However, kaolinite induced a significant increase in intestinal TAG hydrolysis and NEFA absorption. The cytoplasmic expression of L-FABP and FATP4 also increased due to kaolinite ingestion. NEFA may enter the enterocytes via endocytosis mainly since expression of NEFA transporters in the brush-border membrane was not affected by kaolinite. After uptake, rapid binding of NEFA by L-FABP and FATP4 could act as an intracellular NEFA buffer to prevent NEFA efflux. Increased TAG hydrolysis and NEFA absorption may be due to the adsorption properties of clay and also because kaolinite ingestion caused a slowing down of gastric emptying and intestinal transit.