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Sylvain Giroud Under the co-supervision of Dr Martine Perret (UMR 7179, Brunoy)

Sylvain Giroud Under the co-supervision of Dr Martine Perret (UMR 7179, Brunoy) Dr Stéphane Blanc (UMR 7178, Strasbourg). Différences saisonnières des mécanismes d’économie d’énergie d’un primate malgache hétérotherme : le Microcèbe.

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Sylvain Giroud Under the co-supervision of Dr Martine Perret (UMR 7179, Brunoy)

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  1. Sylvain Giroud Under the co-supervision of Dr Martine Perret (UMR 7179, Brunoy) Dr Stéphane Blanc (UMR 7178, Strasbourg) Différences saisonnières des mécanismes d’économie d’énergie d’un primate malgache hétérotherme :le Microcèbe Energy Saving Mechanisms In An Heterothermic Malagasy Primate: The Grey Mouse Lemur

  2. Environment with low food availability Food deprivation MASS MASS gain loss Ingested Energy Energie ingérée Ingested Energy Energy Expenditure Physical activity Thermog. Resting metabolic rate ∆ Body Reserves Energy Expenditure Stable Energy Balance Specific Physiological and Behavioral Responses Survival Reproduction Fitness Background - Energy Balance and Global Changes Migration Food storage Huddling Torpor

  3. Background - Torpor Activity Hypo-metabolism Activity 1500 1200 Marmot (Marmota marmota) Metabolic rate (mlO2.kg-1.h-1) 900 1-2% of basal MR 300 5% of basal MR 0 40 30 Arctic ground squirrel Body temperature (°C) 20 “Super-cooling” Minimal Tb ~ -1/-2°C Minimal Tb ~ 6°C 10 0 Time Jan 4th Jan 1st Jan 7th Dec 29th From Ortmann and Heldmaier 2000 Torpor • Strategy used by animals inhabiting from the arctic to the tropics • State of reduced metabolic rate (MR) and lowered body temperature (Tb) Seasonal torpor Predictable 50% of initial body mass

  4. Background - Torpor Hummingbird (Selasphorus rufus) Activity Hypo-metabolism Activity 8 36 Minimal Tb ~ 17°C 32 6 Siberian hamster (Phodopus sungorus) 28 Metabolic rate (mlO2.g-1.h-1) 4 Body temperature (°C) 24 30% of basal MR 20 2 16 12 From Heldmaier et al. 2000 0 6 10 22 18 12 Time of day (hour) Non-seasonal Unpredictable Daily Torpor Torpor = mechanism of energy saving, reached by lowering both metabolic fluxes and body temperature Highly heterogen

  5. Background - Physiological Plasticity Environment With Low Food Availability Food Deprivation Affect MASS gain loss Global Changes Ingested Energy Energie ingérée Energy Expenditure ∆ Body Reserves Energy Saving Mechanisms Survival Reproduction Threatened Biodiversity Fitness Limits? Torpor Plasticity

  6. Background - Critical Hotspots From Myers et al. 2000 • Top-five of critical hotspot for biodiversity conservation (Goodman and Benstead 2005) • High endemism level (Yoder et al. 2000; Wilmé et al. 2006; Tattersall 2007) • Primate diversity: • 21% of primate genera • 36% of primate families • Highly contrasted climate

  7. Background - Fluctuating Malagasy Climate Unpredictable Variability in Trophic Resources El Niño phenomenon 35 120 Rainfall 30 100 Cold and dry season 25 80 Ambient temperature (°C) Minimal T Rainfall (mmH2O) 20 60 15 40 10 1982-83 1994-95 20 Resources El-Niño episodes A S O N D J F M A M J J A S  frequency and intensity Seasonal Variability in Trophic Resources

  8. Background - Torpor in Cheirogaleidae Malagasy environment with marked seasonality and unpredictable events Daily Torpor Season-dependant The Grey Mouse Lemur Less Torpor Torpor Heterothermy Seasonal Fattening Resting Metabolism 140 Body Mass 120 % of Variations 100 80 Cheirogaleus medius Microcebus murinus 60 Season of reproduction Sexual rest Dry season Perret 1998; Perret and Aujard 1999; Schmid 2000

  9. Background - Short-term Food Restriction Studies in Microcebus murinus 80% food restriction Ad-libitum Summer Body temperature (°C) Locomotor’s activity (a.u.) Winter From Genin et al. 2003 Séguy and Perret 2005 • Torpor Ability: Summer << Winter

  10. Background - Distribution Area of M. murinus El Niño Phenomenon Madagascar Repartition Area of M. murinus Dry Deciduous Forests Spiny Desert Forests Wright 1999 Wide distribution of M. murinus in contrasted habitats  Torpor expression of mouse lemurs in summer would be underestimated?  Existence of other efficient mechanisms to survive during unpredictable events in summer?

  11. Objectives Determine the physiological strategies for energy economy in mouse lemurs facing an energy deficit • Specific Aim 1: evaluate the thermoregulatory and locomotor’s activity responses The grey mouse lemur would rely on torpor bouts in winter while would reduce its locomotor’s activities in summer

  12. Experimental Protocol Captive Male Mouse Lemurs Long-days Short-days Winter-like Summer-like Moderate (40%) and Severe (80%) Control Period 35-day food restriction Logger Implanted in the abdominal cavity of each animal • Body Mass • Body Temperature and Locomotor’s Activity (Telemetry)

  13. Material and Methods Daily Rhythm of Body Temperature (Tb) and Locomotor’s Activity (LA) Initiation 100 39 80 36 60 Tb < 33°C Torpor Duration Tb (°c) LA (a.u.) 33 LA Level 40 Minimal Tb 30 20 LA Level 0 27 0:00 4:00 6:00 8:00 2:00 12:00 14:00 16:00 22:00 10:00 12:00 18:00 20:00 Dark = Active Phase Light = Resting Phase

  14. Results - Changes in Torpor Parameters Minimal Body Temperature 0 36 * Summer 40% n = 8 ** 34 -60 Summer 80% n = 3 -120 32 Body Temperature (°c) indicates the time of rupture indicate the time of rupture indicates the time of rupture ** ** -180 30 n = 10 Winter 80% -240 28 Winter 40% n = 8 -300 26 Torpor Duration Torpor Initiation 600 Duration (minutes) ** 480 ** ** ** 360 ** 240 ** 120 0 0 0 5 5 10 10 15 15 20 20 25 25 30 30 Time (days) Time (days) Giroud et al. 2008 Am J Physiol - Regul Integr Comp Physiol Time effect: *p < 0.05 **p < 0.01

  15. Results - Locomotor’s Activity Responses Winter 40% Days of Food Restriction Summer 40% Light Dark Light Dark Light Dark Light Dark 0 5 10 15 20 25 30 16 0 8 0 8 16 0 8 0 8 16 16 0 0 0 5 10 15 20 25 30 Summer 80% Winter 80% Giroud et al. 2008 Am J Physiol - Regul Integr Comp Physiol

  16. Results - Consequences on Body Mass How efficient are these thermoregulatory and behavioral strategies? Winter 40% 120 Winter 80% ** 110 Summer 40% 100 ** Summer 80% 90 Body Mass (g) 80 70 0 5 10 15 20 30 25 Time (Days) 60 50 Giroud et al. 2008 Am J Physiol - Regul Integr Comp Physiol Time effect: **p < 0.01

  17. Conclusions and Question - Thermoregulatory and Locomotor’s Activity Responses • Torpor mechanism is differentially expressed according to season • Increase in torpor expression allows mouse lemurs in winter to face efficiently a moderate food shortage, while the animals under summer phenotype do not rely on torpor How mouse lemurs in summer maintain a stable energy balance without relying on torpor during a moderate food restriction?

  18. Background - Protein and Energy Metabolisms Energy Stores Mobilized During… Hibernating Season Summer Fat Sparing Protein Sparing Ingested Energy MASS MASS gain loss Spermophilus lateralis fat-free mass Spermophilus beldingi Moderate food shortage Stable Energy Balance Under Very-low Calorie Diet (Karmann et al. 1994) Under Food Deprivation (Backman 1994) Energie ingérée Ingested Energy Energy Expenditure Physical activity Physical activity Thermog. Thermog. Resting metabolic rate Resting metabolic rate • Cost of protein turnover ~ 20-40% of basal metabolic rate •  fat-free mass = strategy of significant energy saving Face to a moderate food restriction, mouse lemurs in summer would lower their metabolic costs through a reduction of their fat-free mass

  19. Objectives Determine the physiological strategies for energy economy in mouse lemurs facing an energy deficit • Specific Aim 1: evaluate the thermoregulatory and locomotor’s activity responses • Specific Aim 2: evaluate the changes in • Total Energy Expenditure (doubly-labeled water) • Resting Metabolic Rate (respirometry) • Body Composition (isotope dilution) • Protein Turnover (15N-glycine): protein synthesis/catabolism and nitrogen balance

  20. Results - Body Composition Fat-free Mass nLD40 = 7 nSD40 = 9 nSD80 = 8 100 NS ** ** 80 60 grams Body Mass 40 120 * -12% -13% ** 20 100 ** 80 0 grams Fat Mass 60 40 50 -23% 20 -15% -8% * 40 0 ** AL AL 40% 40% 80% 30 Summer Winter grams 20 NS -36% -23% 10 AL AL 40% 40% 80% 0 Summer Winter Giroud et al. Submitted Am J Physiol - Regul Integr Comp Physiol *p < 0.05 **p < 0.01 vs.AL Summer  fat sparing Winter  protein sparing • Mouse lemurs in summer under 80% reached a survival-threatened body mass at day 22 no energetic data for this animal’s group

  21. Results - Protein Metabolism 16 NS 12 (g.kg-1.day-1) 8 # NS NS NS * 400 n = 8 4 200 80% -35% AL AL 40% 40% n = 8 n = 7 0 0 n = 8 n = 8 n = 7 (mg. kg-1.day-1) Summer -200 16 -400 NS -600 12 ** (g.kg-1.day-1) ** -800 Winter 8 # p =0.08 4 -39% 0 AL AL 40% 40% 80% n = 8 n = 8 n = 7 Summer Winter Protein Synthesis Nitrogen Balance Protein Catabolism Winter: conserves fat-free mass Summer: reduces fat-free mass *p < 0.05 **p < 0.01 vs.AL #p < 0.05 LD vs SD Giroud et al. Submitted Am J Physiol - Regul Integr Comp Physiol

  22. Results - Energy Expenditures 120 Total Energy Expenditure FFM adj NS 80 * kJ.day-1 ** 40 -21% -36% 0 n = 7 n = 8 n = 8 Resting Metabolic Rate FFM adj 60 NS NS ** 40 kJ.day-1 20 -22% 0 AL AL 40% 40% 80% n = 8 n = 7 n = 9 Summer Winter Giroud et al. Submitted Am J Physiol - Regul Integr Comp Physiol *p < 0.05 **p < 0.01 vs.AL Winter: TEE are explained by  in energy saving through torpor Summer:  metabolic costs are explained by  fat-free mass

  23. Conclusions - Protein Metabolism and Energy Expenditures Stable State of Lower Metabolic Demands • FFM Summer Stabilization of Energy Balance Protein Sparing  FFM Keeping High Energetic Costs  Energetic Costs Winter Late  Torpor Expression Summer  Protein Catabolism  Continuous  FFM + Negative Energy Balance  Energetic Costs Winter +  Torpor Expression Moderate Food Restriction • Metabolic Fluxes Torpor Expression Severe Food Restriction

  24. Conclusions and Question - Protein Metabolism and Energy Expenditures • Whatever the intensity of food restriction, torpor episode is immediately increased in mouse lemurs under winter phenotype, while its expression is delayed in animals under summer state What are the mechanisms underlying the expression of torpor, that would explain its differential use according to season?

  25. Background - Underlying Mechanisms of Torpor Social Factors Food Availability Ambient Temperature Food Quality Food Quality Torpor Expression Polyunsaturated Fatty Acids (PUFA) Polyunsaturated Fatty Acids (PUFA) Hormones Testosterone Prolactin Thyroid Leptin Ghrelin … Hypothalamus Photoperiod Autonomic Nervous System Sympathetic and Parasympathetic Ghrelin

  26. Background - Underlying Mechanisms of Torpor numerous aspect of fuel homeostasis in diverse animal species • Ghrelin • Orexigenic and reduces energy expenditure and fat oxidation (Tschop et al. 2000) Increases torpor depth in mice (Gluck et al. 2006) • Peptide YY (PYY):anorexigenic, reduces energy expenditure and promotes fat use (Adams et al. 2006) • Glucagon-like Peptide 1 (GLP-1):anorexigenic, reduces energy expenditure and body temperature (Sousha et al. 2007) ? Torpor expression Ghrelin may regulate ? Other gut hormones Gut-produced Hormones

  27. Objectives Determine the physiological strategies for energy economy in mouse lemurs facing an energy deficit • Specific Aim 1: evaluate the thermoregulatory and locomotor’s activity responses • Specific Aim 2: evaluate the changes in total energy expenditure, resting metabolic rate, body composition and protein turnover • Specific Aim 3: investigate the potential implications of gut hormones in the regulation of torpor expression  Gut Hormones: ghrelin, GLP-1 and PYY (ELISA Multiplex: on a 50L-plasma sample)

  28. Results GLP-1 Area under the curve Calorie Restricted in Summer * 1600 40 1200 30 (pg/ml) x35 days 800 Minimal Tb (°C) 20 r2LD = 0.80 p < 0.001 400 10 0 80% 40% 40% 80% 0 0 50 100 150 200 Summer Winter GLP-1 (pg/ml) Giroud et al. 2008 J Comp Physiol B *p < 0.05 **p < 0.01 vs.AL • GLP-1 would be implied in the regulation of torpor expression through the modulation of the torpor depth • Important implication of GLP-1 in the regulation of torpor in heterothermic species? • No significant correlations were reported between torpor parameters and ghrelin or PYY level

  29. Background - Underlying Mechanisms of Torpor Social Factors Food Availability Ambient Temperature Food Quality Torpor Expression Polyunsaturated Fatty Acids (PUFA) Hormones Testosterone Prolactin Thyroid Leptin Ghrelin … Hypothalamus Photoperiod Autonomic Nervous System Sympathetic and Parasympathetic

  30. Background - Underlying Mechanisms of Torpor Reduction of torpor propensity Torpor Propensity Increase in torpor duration and depth  energy saving (Florant et al. 1993; Geiser and Kenagy 1987, 1993) ? Increase in cellular damages Maintenance of the tissue fluidity and of the physiological function at low body temperatures - + % PUFA In The Diet • susceptibility to peroxidation by free radicals (Hubert 2005) Optimum Lipids • PUFA content in tissues • (Geiser 1991; Falkenstein et al. 2001) Independently of Diet This result suggests a differential partitioning between synthetic and oxidative pathways of lipids in the organism

  31. Background - Underlying Mechanisms of Torpor Trade-off During Torpor (Franck et al. 1998; Munro and Thomas 2004) Maximizing the Differential Lipid-type Oxidation Minimizing Lipid Peroxidation-related Cellular Damages

  32. Objectives Determine the physiological strategies for energy economy in mouse lemurs facing an energy deficit • Specific Aim 1: evaluate the thermoregulatory and locomotor’s activity responses • Specific Aim 2: evaluate the changes in total energy expenditure, resting metabolic rate, body composition and protein turnover • Specific Aim 3: investigate the potential implications of gut hormones in the regulation of torpor expression • Specific Aim 4: determine the seasonal cost-benefit trade-off between • Torpor Optimization • Differential Lipid Oxidation: Saturated (d31-palmitate)vs. Polyunsaturated (3H-linoleate) • Oxidative Stress: hexanoyl-lysine and 8-hydroxydeoxyguanosine

  33. Material and method - Lipid Oxidative Pathways COOH(CD2)14CD3 COOH(CH2)12(CT)4 CH3 D D D D D D D D D D D D D D D3 D D D D D D D D D D D D D D -oxidation d31-palmitate (16:0) 25% 75% 75% T Krebs Cycle T 15% T THO DHO T Sequestration ~ 10% Body water Body Water Urine Sample Urine sample H3-linoleate (18:2 n-6)

  34. Material and Method - Production of Markers of Oxidative Stress Free Radicals O2.,OH., O2H. Lipid Linoleic Acid 18:2 (n-6) Early Stage Auto oxidation Hexanoyl-lysine Hydroperoxyde Hexanoyl Proteins Urine (ELISA) Late Stage DNA 8-hydroxydeoxyguanosine 2-deoxyguanosine

  35. Results - Fatty Acid Oxidations and Consequences on Oxidative Stress Linoleate Oxidation C18:2 n-6 Hexanoyl-lysine * 12 80 * 10 * 60 % Recovery 8 NS * NS nmol.mmol Creatinine-1 40 6 4 20 +160% +40% +84% +48% 2 0 n = 8 n = 10 0 n = 9 n = 10 n = 7 n = 11 8-hydroxydeoxyguanosine Palmitate Oxidation C16:0 8 * 40 * * 6 30 % Recovery * NS NS nmol.mmol Creatinine-1 4 20 +144% +200% 2 10 +43% +41% 0 0 AL AL 40% 40% 80% AL AL 40% 40% 80% n = 7 n = 8 n = 11 n = 7 n = 11 n = 10 Summer Winter Summer Winter *p < 0.05 **p < 0.01 vs.AL Giroud et al. Submitted J Lip Res

  36. Results Torpor Frequency ** R2 = 0.26 p < 0.001 ** R2 = 0.14 p < 0.05 12 HEL (nmol/mmol.creatinine) 10 8 6 NS 4 20 40 60 80 100 20 40 60 80 100 7 TEE (kJ.day-1) FFM-adjusted TEE (kJ.day-1) 2 6 Week 5 Control Control Control Week 5 Week 5 0 Summer AL Summer 40% 5 Summer 40% Winter 80% Winter 40% Winter AL Winter 40% 4 Number of Torpor Per Week Winter 80% 3 2 1 0  These results suggest that high oxidative stress production is associated with torpor expression in mouse lemurs Giroud et al. Submitted J Lip Res

  37. Conclusions • The cost-benefit trade-off between lipid-type oxidation and oxidative stress level is season-dependant • Under a moderate food shortage, only mouse lemurs in winter realize this trade-off • Summering animals increase oxidative stress • Under severe food deprivation, mouse lemurs in winter are not able any more to carry out the cost-benefit trade-off, increasing dramatically oxidative damages

  38. General Conclusion Summer Microcebus Murinus Winter Fat Sparing Protein Sparing vs. Food Shortage LA LA FM oxidative stress FFM FM Limited Torpor Increases Torpor TEE TEE FFM Moderate 40% oxidative stress RMR Survival threatened body mass Severe 80% DNA damage

  39. Perspectives Martine Perret Verena Behringer To determine if other strategies of energy-saving, used in association to torpor, would help M. murinus to stabilize its energy balance during severe food shortage? • Role of social thermoregulation? • Maximal energy benefit of 40% in mouse lemurs in summer as in winter (Perret 1998) • Increases duration of torpor bouts (Séguy and Perret 2005)

  40. Perspectives Night • Role of social thermoregulation? • Role of passive rewarming during torpor episodes? Body T°C Ambient T°C • Wild Microcebus murinus • Reduction of energy costs from the arousal from torpor, by using natural fluctuations of the ambient temperature, before an active rewarming to normothermic level VO2 Modified from Schmid 2000 In the context of global changes?

  41. Acknowledgements I thank the following organizations for their financial support: GIS Longevity Thanks for your attention!!

  42. Acknowledgements Jean-Louis Gendrault Dominique Desplanches Yvon Le Maho Patricia Wright Stéphane Blanc M. et Mrs. Cottet-Emard Brunoy’s lab Nancy’s lab Mrs. Vouillarmet Marie-Laure Jean-Marc Pequignot Mum & Dad Alexandre Zahariev Joëlle Goudable Péguy Guillaume Marie Trabalon DEPE Students François Cédric Sylvie Caroline Thomas Sabrina Jérôme Hélène Damien Nicolas Michaël Cédric isabelle Hugues André Romain Jérémy Audrey Martine Guy Brigitte Thierry Anita Aurélie Marion Family DEPE Peter Stein Iman You got to move it move it… … You got to… MOVE IT!!! Martine Perret

  43. Material and Methods - 15N-glycine and End-products 15N-proteins 15N-proteins Ammoniac 15NH3 Ammoniac 15NH3 15N Catabolism (C) Catabolism (C) Synthesis () Synthesis () NH3 NH3 urine urine 15N-amino-acid pool 15N-amino-acid pool 15N Excretion 15N Excretion Urea CH415N2O Urea CH415N2O 15N-glycine 15N-glycine 15N N (diet) CH4N2O CH4N2O feces feces N N 15N 15N 15N flux = 15N dose/15N excretion 15N 15N 15N N (diet) Protein  Protein  = 15N flux - N excretion (urine + feces) Protein C Protein C = 15N flux - N ingestion (diet) Balance N = Protein S - Protein C

  44. Results Nitrogen Flux 40 NS 30 NS # (mg. kg-1.day-1) x102 20 ** 10 -30% 0 40% 40% AL AL 80% n = 8 n = 7 n = 8 Summer Winter *p < 0.05 **p < 0.01 vs.AL #p < 0.05 LD vs SD • Is associated with the metabolic level on the individual • Ad-libitum: Summer >> Winter • Reduced only in wintering mouse lemurs under moderate food shortage Giroud et al. Submitted Am J Physiol - Regul Integr Comp Physiol

  45. Water Turnover 20 16 ** ** 12 g.day-1 8 ** 4 -37% -32% -63% 0 AL AL 40% 40% 80% n = 8 n = 7 n = 9 Summer Winter

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