Abstract Potential implications of gut hormones in bodymass and torpor and behavioral pattern changes induced byan incremental (40 and 80%) calorie restriction (CR) inlong-days (LD, summer) and short-days (SD, winter) wereinvestigated in gray mouse lemurs. Only 80% food-deprived LD and SD animals showed a continuous massloss resulting in a 10 and 15% mass reduction, respectively.Ghrelin levels of all food-deprived groups increased by 2.6-fold on average and remained high after re-feeding whilepeptide YY (PYY) levels increased by 3.8-fold only in LDanimals under 80% CR. In the re-fed SD group, body masswas positively associated with ghrelin and negatively asso-ciated with PYY, while no correlations were noted in there-fed LD animals. Plasma glucagon-like peptide-1 (GLP-1) increased by 2.9-fold only in LD food-restricted mouselemurs and was negatively associated with the minimalbody temperature. No signiWcant correlations were reportedin food-deprived SD animals. These results suggest thatghrelin, PYY and GLP-1 may be related to pre-winteringfattening mechanisms and to the modulation of torporexpression, respectively. Such observation clearly warrantsfurther investigations, but it opens an interesting area ofresearch in torpor regulation.
Keywords GLP-1 · Ghrelin · PYY · Body temperature · Photoperiod
AbbreviationsAUC Area under curveCR Calorie restrictionCRi Calorie restriction intensityGIP Glucose-dependant insulinotropic polypeptideGLP-1 Glucagon-like peptide 1LD Long-daysLD40 Animals exposed to long-days under
40% calorie restrictionLD80 Animals exposed to long-days under
80% calorie restrictionNPY Neuropeptide YPP Peptide PPYY Peptide YYSD Short-daysSD40 Animals exposed to short-days under
40% calorie restrictionSD80 Animals exposed to short-days under
80% calorie restrictionTb Body temperature
Introduction
Torpor represents an outstanding ability of energy savingfor survival when faced with unfavorable external chal-lenges. Torpor is essentially described in small endotherms(<10 Kg), and involves a periodic and facultative loweringof body thermostat resulting in a hypometabolic state forenergy and water economy. This unique strategy can beobserved from the arctic region to tropics in some birds andmammals, and at least six orders are concerned among
Communicated by G. Heldmaier.
S. Giroud · M. PerretMécanismes Adaptatifs et Evolution, UMR 7179 CNRS, Muséum National d’Histoire Naturelle, 4 Avenue du Petit Château, 91800 Brunoy, France
S. Giroud · Y. Le Maho · I. Momken · C. Gilbert · S. Blanc (&)Département d’Ecologie, Physiologie, Ethologie, Institut Pluridisciplinaire Hubert Curien, UMR 7178 CNRS, Université Louis Pasteur, 23 rue Becquerel, 67087 Strasbourg, Francee-mail: [email protected]
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mammalians: Monotremata, Marsupialia, Insectivora, Chi-roptera, Rodentia and even Primates (Geiser and Ruf 1995;Lyman 1982).
Seasonal torpor is essentially driven by environmentalfactors such as photoperiod, ambient temperature and foodavailability/quality. As described in the Siberian hamster(Stamper et al. 1999) and the deer mouse (Geiser et al.2007), torpor is to a large extent controlled by photoperiodand coincides with the seasonal rarefaction of resources inthe wild. Faced with cold environments and/or a food-deprived period, the Siberian hamster and the elephantshrew show increases in torpor frequency and depth (Elliottet al. 1987; Ruf et al. 1991, 1993). Among environmentalfactors, diet quality was also shown to modulate torporexpression (Geiser 1991; Geiser and Heldmaier 1995;Geiser and Kenagy 1987, 1993; Geiser et al. 1997). How-ever, although the control of torpor is an active researcharea, the underlying physiological mechanisms are not yetfully characterized.
Torpor expression is modulated by several intrinsic fac-tors such as the sympathetic nervous system, metabolitesand hormones. A speciWc activation of white adipose tissueby the sympathetic nervous system, mediated through �3-adrenergic receptors during fasting, generates favorableconditions for the initiation of torpor in mice (Swoap et al.2006). Metabolites are also involved in the modulation oftorpor bouts as suggested by a reduced glucose availabilityreported before entrance into torpor in the Siberian hamster(Dark et al. 1994, 1996) and in dormice (Atgie et al. 1990).Among hormones, leptin was shown to inhibit daily torporin the marsupial Sminthopsis macroura, thus increasing itsenergy expenditure by 9% (Geiser et al. 1998), and a drasticreduction in the leptin level was shown to be permissive todisplay a torpor bout in the Siberian hamster (Freemanet al. 2004). Recently, it has been demonstrated that periph-eral administration of ghrelin, an orexigenic hormone, dur-ing fasting signiWcantly deepened torpor bouts in mice(Gluck et al. 2006) and induced a phase-advance of loco-motor activity rhythm after 30 h of food deprivation inmice (Yannielli et al. 2007).
The large family of gut-derived peptidic hormones, towhich belongs the stomach-produced ghrelin, are involvedon numerous aspects of fuel homeostasis such as foodintake, energy expenditure and behavior in diverse animalspecies (Murphy and Bloom 2006). Peripheral administra-tion of ghrelin caused body mass gain by reducing fat utili-zation in mice and rats and an intra-cerebro-ventricularinjection generated a dose-dependant increase in foodintake (Tschop et al. 2000). Another gut-derived hormone,the glucagon-like peptide-1 (GLP-1), synthesized by L-cells throughout the length of the gut, was shown to signiW-cantly decrease body temperature and gross locomotoractivity in Japanese quail (Shousha et al. 2007), to suppress
food intake (Turton et al. 1996) and to reduce energyexpenditure in humans (Flint et al. 2000). The L-cells alsosynthesize the peptide YY (PYY), an anorexigenic hor-mone belonging to the PP-fold family, that decreasesenergy expenditure and reduces respiratory quotient inmice, i.e., promotes fat use (Adams et al. 2006). Con-versely, another member of the PP-fold family, the pancre-atic polypeptide (PP) deriving from the endocrine pancreas,increases satiety and energy expenditure, thus leading tobody mass loss (Kojima et al. 2007). Lastly, the glucose-dependant insulinotropic polypeptide (GIP) was shown toinduce an increased exploratory behavior and performancein some motor function tests in a transgenic mouse thatover-expressed GIP (Ding et al. 2006). Taken together, allthese data demonstrate that gut-derived hormones havecomplex synergistic and antagonistic actions on the physio-logical and behavioral regulation of fuel homeostasis.
Based on this growing body of data, it is likely that thesehormones may play a role in the control of energy homeo-stasis in heterothermic species, which reduce both bodytemperature and metabolic rates during their torpor phase.The energy-regulatory mechanism of torpor was exten-sively studied in a small Malagasy mouse lemur (Microce-bus murinus) that is unique among primates by its markedseasonal rhythms easily reproducible in captivity (Aujardet al. 1998; Genin and Perret 2000; Perret 1992). Short-daysexposure (SD < 12 h of daylight) triggers a resting state, afattening and an increase in torpor depth and durationwhereas long-days exposure (LD > 12 h of daylight) leadsto an increase in behavioral activities, to a reduction of bodymass and to a decrease in the ability to display torpor. Tor-por patterns in the gray mouse lemur are further modulatedby environmental cues, such as experimental food short-ages, and these changes diVer between seasons. Short-termfood restriction and/or cold exposure studies (Genin andPerret 2003; Seguy and Perret 2005) on mouse lemursrevealed that animals both in summer and winter displaychanges in torpor expression by advancing the entry intotorpor, and increasing length and duration of torpor bouts.These body temperature adjustments occur in a greatermagnitude in animals expressing their winter phenotype.
This study was conducted to investigate time-courses ofgut hormones and their possible relation with body massand torpor pattern changes during a long-term energyrestriction and a re-feeding in Microcebus murinus.
Material and methods
Animals
The 24 male gray mouse lemurs (Microcebus murinus,Cheirogaleidae, Primates) used in this study were adults
(2–5 years old) and born in the laboratory-breeding colonyof Brunoy (UMR7179 CNRS/MNHN, France; EuropeanInstitutions Agreement # 962773) from a stock originallycaught 40 years ago along the southwestern coast of Mada-gascar. Seasonal Malagasy rhythms of M. murinus werereproduced by an alternation of 6 months of a long photo-period (L:D 14:10) and 6 months of a short photoperiod(L:D 10:14). To minimize social inXuences, the animalswere housed individually in cages (50 £ 40 £ 30 cm) andvisually separated from each other. Whereas relativehumidity in animal rooms was maintained constant (55%),individuals in the summer and winter were kept at ambientroom temperatures of 30 and 25°C, respectively, to mimicnatural seasonal variations.
Energy intake during the control period and calculation of calorie restriction
Before calorie restriction (CR), individual energy require-ment was measured during a 10-day control period in orderto determine food-restricted allotments. In ad libitum con-ditions, animals were fed on fresh banana and a standard-ized homemade mixture containing baby cereals, spicebread, egg, concentrated milk, white cheese, vitamins anddietary minerals (Vitapaulia/MR, Intervet, France and Toi-son d’orR, Clément Thékan, France). Since isolated ani-mals, and particularly those under winter phenotype, tendto overfeed and gain mass during the control period, energyintake was clamped to the level required to stabilize theirbody masses. Such procedure was required to avoid asigniWcant underestimation of the CR intensities. Eachindividual was initially fed ad-libitum with banana and thehomemade mixture and progressively, daily energy intakewas narrowed according to the body mass evolution.Patterns of body temperature (Tb) and activity were notmodiWed during the control period and none of the animalslost weight.
Half of the animals in each photoperiod were then pro-vided 60% (=40% CR) or 20% (=80% CR) of these individ-ually derived energy requirements. Food-restrictedindividuals were fed every day with the reference mixtureat the very onset of the dark phase. Water was always pro-vided ad-libitum. Daily food intake was calculated from thediVerence between provided and remaining food massesand was corrected for water evaporation. Grams of foodintake were converted to kJ using equivalents of 3.7 kJ/gfor the banana and 4.6 kJ/g for the mixture. Over the5 weeks of the intervention, the 40% CR received anenergy allotment of 57 § 2 and 52 § 6 kJ/day in long-days(LD) and short-days (SD) groups, designated LD40 andSD40, respectively. The 80% CR corresponded to anenergy allotment of 17 § 0 and 19 § 2 kJ/day in LD andSD groups (LD80 and SD80, respectively).
Following the 5-week food deprivation, animals were re-fed during a period of 2 weeks with the bananas and thehomemade mixture. For each animal, energy allotment dur-ing this period was that estimated during the control periodwhile energy intake was clamped to the level required tostabilize the animal body mass. During the re-feedingperiod, SD mouse lemurs received an energy allotment of88 § 7 kJ/day and LD animals 86 § 1 kJ/day.
Body mass
During the control period, body masses of SD and LDmouse lemurs were 108 § 4 and 84 § 1 g, respectively.Through CR and re-feeding periods, the body mass of eachanimal was measured every 2 days. For ethical reasons,special attention was paid to the body mass time-course ofthe LD80 group during the CR period, because of theirleanness at inclusion. Through the time of lowered foodsupply, animals were excluded from the study when bodymasses reached the lowest value reported in the colony forthis photoperiod (Perret and Aujard 2001). Practically thisconcerned only one animal that was excluded at the end ofthe fourth week of CR, and entered the 2-week re-feedingphase 1 week in advance compared to the other animals.
Tb and locomotor activity recording
A telemetric transmitter (TA10TA-F20, 3.2 g, Data ScienceCo., Minnesota, USA) was implanted into the abdominalcavity, under general anesthesia (pre-anesthesia: Valium10 mg, 2 mg/100 g IM; anesthesia: Ketamine Imalgene500 mg, 10 mg/100 g IM) as routinely done in the labora-tory (Seguy and Perret 2005). Animals were included in theexperimental protocol one month after surgery. Thereceiver board (RPC-1, Data Science Co., Minnesota,USA) positioned in front of the nest-box, collected theradio frequency signals. Tb was recorded for 10 s every5 min. Locomotor activity was recorded continuously andthe sum-up of recorded values in arbitrary unit (a.u.) wasreported every 5 min. Data were analyzed by the softwareDataquest (LabPro Data Science Co., Minnesota, USA).After the study, transmitters were removed by surgery andanimals returned to their breeding groups.
Hormonal sampling and assays
Blood sampling was carried out the last day of the controlperiod, after 1, 3 and 5 weeks of CR, and at the end of the 2-week re-feeding period. Blood collections were taken viathe saphenous vein of the animals, without anesthesia, at theend of their resting phase, before the food allotment becameavailable. Blood collections were of a volume of 150 and100 �L on animals under control and food-restricted condi-
tion, respectively, and represented less than 0.1% of theblood volume of SD and LD animals. Blood samples werecentrifuged at 3,500 rpm at 5°C during 10 min. Plasma wasstored at ¡30°C according to the assay procedure.
Levels of active ghrelin, GIP, PP, PYY and GLP-1 weremeasured in duplicate using the human gut hormones mul-tiplex panel (LincoplexTM Multiplex Assays, Bioscience)and Luminex technology at the core facility of the SaintAntoine hospital in Paris dedicated to micro-assays in smallanimals (IRSSA, Inserm IFR65). Test accuracies were 85%for active ghrelin, 89% for GIP, 83% for GLP-1, 88% forPP and 107% for PYY. Inter-assay and intra-assay preci-sions of tests were <19 and <11%, respectively. HumanLinco antibodies for gut peptides have been validated indiVerent species and non-human primates (Angeloni et al.2004; Nieminen et al. 2007). As no studies were reportedfor Microcebus murinus, the peptide assay was validatedsuch that serial dilutions of the Microcebus murinus plasmashowed linear changes in sample values that were parallelwith standard curves produced with standards of the manu-facturer. Recovery of known quantity of standard peptidesadded to mouse lemur plasma was close to 100% (data notdetailed). Plasma values of active ghrelin observed in thisnon-human primate were in the low range of what has beenso far observed in humans (Bribiescas et al. 2007; Katsukiet al. 2004; Kawamata et al. 2007) and the range of stan-dards originally from 13.9 pg/ml to 11.4 ng/ml was reducedfrom 1.8 pg/ml to 1–1.1 ng/ml. The number of individualsin each group for hormonal level data was therefore: 6 inSD40, 6 in SD80, 6 in LD40 and 5 in LD80.
Telemetry data
Due to unexpected transmitter failure, three individuals(one in SD40 and two in LD80) were excluded from thetelemetry data analysis. The number of individuals in eachgroup for telemetry data was therefore: 5 in SD40, 6 inSD80, 6 in LD40 and 4 in LD80.
Rhythms of body temperature (Tb) and locomotor activity
The gray mouse lemur is a nocturnal primate that showshigh levels of locomotor activity and normothermic Tb dur-ing the night. Just before the onset of the light phase, themouse lemur enters in a resting state and decreases its bodytemperature until the following dark phase, when Tbreturns to normothermic values.
Parameters studied
Four parameters were deWned in order to characterize bodytemperature (Tb) and locomotor activity patterns. The hypo-thermia phase started when the Tb dropped below the aver-
age Tb of the active period and continued to decrease at aconstant rate, and lasted until the Tb reached the average Tbof the following night. Because a substantial drop in bodytemperature is associated with a reduction in metabolic rate(Geiser 2004), the use of the hypothermia phase as abovedeWned was considered to be appropriate to account foroverall energy savings. We then calculated the hypothermiaduration which the reported values during the control periodwere 655.0 § 9.3 min for SD animals and 865 § 9.3 min forLD ones. Negative number in minutes, accounted from theonset of the dark phase, indicated the time of entry in hypo-thermia from which Tb dropped below the average Tb of theactive period and continues to decrease at a constant rate,until the minimal Tb is reached. Therefore, the more nega-tive the time of entry in hypothermia was, the more phase-advanced was the onset of hypothermia, which showedbaseline values in SD and LD groups of ¡98.2 § 17.5 and¡28.5 § 4.2 min, respectively. We also reported the mini-mal Tb, which characterized the depth of the hypothermiabout and that corresponded to values of 34.6 § 0.6°C in SDanimals and 35.3 § 0.1°C in LD ones, during the controlperiod. Finally, locomotor activity levels, expressed in arbi-trary unit, were calculated over the nycthemere, i.e., for thewhole 24 h-period. Baseline values of this activity parame-ter in SD and LD groups were 1828.4 § 170 and2,743 § 137 a.u., respectively.
Statistical analyses
Normality of telemetry parameters and hormonal concen-trations were checked and parametric statistical tests wereused. To determine the time-course of each parameter oftelemetry data and hormone levels during the 5 weeks offood deprivation, an analysis of variance with repeatedmeasures was used, with the photoperiod (LD vs. SD) andcalorie restriction intensities (40 vs. 80%) as the main fac-tors, and time as the repeated measure. In addition, we cal-culated the incremental area under the proWle curves (netAUC) for each time-course of hormones over 5 weeks ofcalorie restriction. A factorial analysis of variance was thenperformed, with photoperiod (LD vs. SD) and calorierestriction intensities (40 vs. 80%) as the main factors.Fisher’s least signiWcant diVerence (LSD) tests were carriedout for post-hoc analysis. The re-feeding period and weeksof calorie restriction were compared with a paired Student’st test. Observed P values were adjusted (Padj) using theBonferroni method (Bland and Altman 1995) according tothe number of comparisons made. Correlation analysis wasperformed between hormonal levels and telemetry parame-ters at a given week. All reported values are means § SEMand P < 0.05 was considered signiWcant. All statisticalcomputations were performed by Statistica (V7.1.515.0,Statsoft France Paris).
Seasonal diVerences in body mass and telemetry-derivedparameters have been previously well-described by ourteam and will not be further developed (Genin and Perret2000). Hormonal levels during the control period did notdiVer between SD and LD animals (Table 1).
Body mass
Both SD80 and LD80 animals displayed a continuous lossin body mass resulting in a 10 and 15% mass reduction,respectively, on the Wfth week of food deprivation (Fig. 1).Conversely, animals under 40% CR, whatever the season,did not show signiWcant changes in body mass. After2 weeks of re-feeding, body masses of LD40, SD40 andSD80 groups did not show signiWcant diVerent values com-pared to the control period but remained 22% lower inLD80 mouse lemurs.
Parameters of body temperature pattern and locomotor activity
Time of entry in hypothermia (Fig. 2a)
The LD40 mouse lemurs did not show any phase-advanceof the entry into torpor during the 5 weeks of CR. LD80animals advanced their entry into torpor by 170 min in thethird week of CR. To a lower extent compared to the LD80group, both SD40 and SD80 animals signiWcantly phase-advanced their entry into torpor. This corresponded to a 2.7and 2.2-fold phase-advance of entry into torpor in SD40and SD80 groups, respectively from the third week of low-ered food supply compared to the control values (SD40:¡217 § 31 vs. ¡81 § 14 min, P = 0.016; SD80: ¡253 §58 vs. ¡113 § 30 min, P = 0.007). After a 2-week re-feeding,
mouse lemurs under both winter and summer phenotypesshowed times of entry into torpor similar to those observedin the control period.
Hypothermia duration (Fig. 2b)
Except for the LD40 group that did not modify their hypo-thermia duration, the three other groups of animals showeda signiWcant mean increase of 153 § 8.8 min of the hypo-thermia duration from the third week until the end of theCR period (1,018 § 64 vs. 849 § 4 min, P = 0.007;747 § 46 vs. 596 § 33 min, P < 0.001; 843 § 68 vs.704 § 29 min, P = 0.007 in LD80, SD40 and SD80 groups,respectively). After 2 weeks of re-feeding, values of hypo-thermia duration of all groups of animals were similar tothose reported during the control period.
Minimal Tb
Among mouse lemurs under long-days exposure, onlythose facing an 80% CR displayed a decrease in minimalTb that reached signiWcance in the third week of loweredfood supply (Fig. 2c). Both SD40 and SD80 animalsincreased their torpor depth from the Wrst week of CR.Until the end of the CR period, both groups showed adecrease of 8°C in their minimal Tb. After 2-weeks of re-feeding, minimal Tb returned to baseline values in allgroups of animals, except the SD40 group (31.5 § 1.0 vs.34.4 § 0.9°C, t = ¡7.20, P < 0.01).
PP pancreatic polypeptide, GIP glucose-dependant insulinotropicpolypeptide, PYY peptide YY, GLP-1 glucagon-like peptide 1.Between photoperiod-comparisons were realized by a t test
Long-days n = 11
Short-days n = 12
t P
Ghrelin 3.0 § 0.1 3.0 § 0.2 0.29 0.78
PP 46.1 § 12.7 52.1 § 13.8 ¡0.32 0.75
GIP 47.0 § 10.7 31.3 § 8.4 1.16 0.26
PYY 33.5 § 7.7 24.8 § 5.6 0.92 0.36
GLP-1 21.9 § 3.0 18.5 § 2.4 0.89 0.38
Fig. 1 Body mass. LD40 (n = 5) and LD80 (n = 5) correspond tolong-days acclimated animals under 40 and 80% calorie restriction,respectively. SD40 (n = 6) and SD80 (n = 6) were short-days accli-mated animals under 40 and 80% calorie restriction, respectively. Therepeated measure ANOVA was conducted with time (P < 0.001) as therepeated measure and photoperiod (LD vs. SD; P < 0.001) and calorierestriction intensities (40 vs. 80%; P = 0.14) as the main factors. Onlythe time £ intensity of restriction interaction appeared signiWcant(P < 0.001). PLSD Fisher’s test results: ** P < 0.01 vs. control. Pairedt test results: # P < 0.05 vs. control
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Daily locomotor activity level (Fig. 3)
No signiWcant overall changes were reported on the time-course of the locomotor activity level during the 5 weeks offood restriction in SD40, SD80 and LD40. Only LD80 mouselemurs signiWcantly increased their locomotor activity level by2.2-fold in the third week of CR (6,056 § 2,067 vs. 2,761 §352 a.u., P < 0.001). After a 2-week re-feeding period, the
locomotor activity level of all animal groups showed similarvalues compared to those of the control period.
Gut hormonal patterns
Ghrelin (Fig. 4a)
LD40, SD40 and SD80 mouse lemurs displayed signiWcant2.3-fold mean increases in ghrelin levels that reached a pla-teau at a value of 6.6 § 0.8 pg/ml, in the third week of CR.Moreover, ghrelin levels of LD80 animals graduallyincreased until the Wfth week of CR to result in a 7.2-foldincrease (11.9 § 1.9 vs. 3.0 § 0.3 pg/ml, P < 0.001). TheAUC (area under curve) supported this observation byshowing a twofold greater cumulative secretion of ghrelin,although non-signiWcant, over 35 days of CR, in LD80 ani-mals compared to the other groups. After a 2-week period ofre-feeding, none of the animal groups returned to baseline.
PP
A signiWcant overall threefold reduction in PP levels duringthe 5 weeks of food deprivation was observed (Fig. 4b).Post-hoc analysis revealed, however, that LD40 and SD40animals did not show a signiWcant decrease in plasma PPlevels during the overall CR trial. PP levels of the two othergroups of animals, LD80 and SD80, were reduced by four-
Fig. 2 Torpor parameters. LD40 (n = 6) and LD80 (n = 4) correspondto long-days acclimated animals under 40 and 80% calorie restriction,respectively. SD40 (n = 5) and SD80 (n = 6) were short-days accli-mated animals under 40 and 80% calorie restriction, respectively. aTime of entry in hypothermia. The repeated measure ANOVA wasconducted with time (P < 0.001) as the repeated measure and photope-riod (LD vs. SD; P < 0.01) and calorie restriction intensities (40 vs.80%; P < 0.05) as the main factors. Only the time*intensity of restric-tion interaction appeared signiWcant (P < 0.01). b Hypothermia dura-tion. A time (P < 0.001), photoperiod (LD vs. SD; P < 0.001) andcalorie restriction intensities (40 vs. 80%; P < 0.05) eVects were notedalong with a time £ intensity of restriction interaction (P < 0.01). cMinimal body temperature. The eVect of time (P < 0.001) and photo-period (LD vs. SD; P < 0.001) but not calorie restriction intensities (40vs. 80%; P < 0.17) appeared signiWcant. A time £ intensity of restric-tion interaction was also noted (P < 0.01). For all panels: PLSD Fish-er’s test results, *P < 0.05 vs. control, **P < 0.01 vs. control. Paired ttest results, # P < 0.05 vs. control
Fig. 3 Nycthemeral locomotor activity. LD40 (n = 6) and LD80(n = 4) correspond to long-days acclimated animals under 40 and 80%calorie restriction, respectively. SD40 (n = 5) and SD80 (n = 6) wereshort-days acclimated animals under 40 and 80% calorie restriction,respectively. The repeated measure ANOVA was conducted with time(P = 0.34) as the repeated measure and photoperiod (LD vs. SD;P < 0.001) and calorie restriction intensities (40 vs. 80%; P = 0.08) asmain factors. Only the time £ photoperiod interaction appeared sig-niWcant (P < 0.05). PLSD Fisher’s test results: **P < 0.01 vs. control
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fold at the end of the food-deprived period, showing a meanvalue of 18.4 § 3.9 pg/ml in the Wfth week of CR. PP secre-tions (AUC) of LD80 and SD80 animals were higher com-pared to those of LD40 and SD40 groups, although the onlytrends were noted. After 2 weeks of re-feeding, both LDand SD mouse lemurs, whatever the CR intensity, did notmodify their PP levels, showing similar values from thoseobserved on the Wfth week of CR.
GLP-1
Plasma levels of GLP-1 of SD mouse lemurs were not modi-Wed along the 5 weeks of CR (Fig. 4c). LD80 animalsshowed a signiWcant 3.3 and 8-fold increases in GLP-1 lev-els, compared to baseline values, in the third (57.9 § 12.6 vs.17.7 § 3.4 pg/ml, P = 0.002) and Wfth week of CR(141.6 § 18.7 vs. 17.7 § 3.4 pg/ml, P < 0.001), respectively.Conversely, only a signiWcant 2.4-fold rise in GLP-1 levels inthe third week of CR was reported in LD40 animals. GLP-1levels in the Wfth week returned to baseline value. OverallLD80 mouse lemurs displayed signiWcant greater values ofcumulative GLP-1 levels than the three other groups of ani-mals (AUC). After a 2-week re-feeding period, all animalgroups showed GLP-1 levels similar to baseline values.
PYY
Overall PYY increased by 26 and 147% in SD and LDmouse lemurs, respectively (Fig. 4d). Post-hoc analysis,however, revealed a signiWcant increase in PYY levels onlyin the LD80 group, in the Wfth week of CR (73.7 § 8.1 vs.19.5 § 4.8 pg/ml, P < 0.001). After 2 weeks of re-feeding,none of the animal groups changed their PYY level com-pared to the last week of CR.
GIP
Whatever the intensity of food restriction, LD mouselemurs displayed a signiWcant 75% reduction of GIP levelsat the Wfth week of CR compared to baseline (Fig. 4e). Thesame response was observed in the SD80 group whereasonly a tendency to reduction of GIP levels was observed inSD40 animals, compared to baseline. Re-feeding did notchange CR levels of GIP.
Hormonal determinants of body mass
SigniWcant correlations between hormones and body masswere reported only in SD group, after 2 weeks of re-feeding.In re-fed SD animals, body mass was correlated positivelywith ghrelin levels (Fig. 5a) and negatively with PYY levels
Fig. 4 Gut-derived hormone levels. LD40 (n = 6) and LD80 (n = 5)correspond to long-days acclimated animals under 40% and 80% calo-rie restriction, respectively. SD40 (n = 5) and SD80 (n = 6) were short-days acclimated animals under 40 and 80% calorie restriction, respec-tively. a Active ghrelin. The repeated measure ANOVA only revealeda global time eVect (P < 0.0001) for the active ghrelin response. NodiVerences were noted on the AUC cumulated over the 35 days of cal-orie restriction. b Pancreatic polypeptide (PP). Only a global timeeVect (P < 0.0001) was noted on the kinetics of PP. AUC showed atrend towards signiWcance for the intensity of calorie restriction(P = 0.07). c Glucagon-like peptide-1 (GLP-1). The kinetics of GLP-1varied signiWcantly according to photoperiod (P < 0.001), intensity ofrestriction (P < 0.01) and time (P < 0.001) and the AUC showed a sig-niWcant photoperiod*intensity of restriction interaction (P < 0.05). dPeptide YY (PYY). Globally PYY varied according to photoperiod(P < 0.01) and time (P < 0.05). e Glucose-dependant insulinotropicpolypeptide (GIP). Only a time eVect was observed (P < 0.001) andAUC did not further reveal photoperiod or the intensity of restrictioneVects. For all panels: PLSD Fisher’s test results, *P < 0.05 vs. control,**P < 0.01 vs. control
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(Fig. 5c). No other signiWcant correlations in this group werereported during the control and food-deprived periods. Con-cerning the LD group, no signiWcant correlations were high-lighted, at any time, between hormonal levels and body mass,as shown for ghrelin or PYY levels and body mass (Fig. 5b,d) during the control, food-deprived and re-feeding periods.
Hormonal determinants of torpor
In the LD group, GLP-1 levels correlated negatively with theminimal Tb (Fig. 6a). These correlations suggest that, asGLP-1 levels increased, mouse lemurs displayed deeper tor-por bout. We considered the calorie restriction intensity(CRi) as a continuum in energy balance in this analysis andthis is why the 40 and 80% food-restricted groups werepooled, according to photoperiod. We were concerned how-ever, that the observed relationships were driven by theLD80 animals that showed marked changes. In fact, an anal-
ysis of covariance revealed that the slope of the relationshipbetween GLP-1 and minimal Tb did not diVer between LD40and LD80 groups (CRi £ GLP-1, P = 0.851). No other cor-relations between hormonal levels and torpor parameters, inthe LD group, were noticed during the control, food-deprivedand re-feeding periods. In SD animals, no signiWcant correla-tions between hormonal levels and torpor parameters werereported at any periods, as shown between GLP-1 levels andthe minimal Tb (Fig. 6b; Tables 2, 3).
Discussion
Active ghrelin and PYY may be related to wintering mechanisms
To cope with the seasonal Malagasy lowering of food sup-ply, the gray mouse lemur sets up strong modiWcations in
Fig. 5 Correlations between body mass and active ghrelin or PYY in short (SD) and long-days (LD) animals, after a 2-week re-feeding
Fig. 6 Correlation between minimal body temperature and GLP-1 in long- (LD) and short-days (SD) animals, after the Wfth week of calorie restriction
123
J Comp Physiol B
energy metabolism in forecast of the winter season. Underwinter-like short photoperiod, mouse lemurs increase theirbody mass in parallel to an increase in calorie intake whiletheir resting metabolic rate remains constant, accountingfor early body mass changes (Genin and Perret 2000).Then, animals enter a resting state, lengthen their torporbouts, improve their thermoregulatory eYciency andincrease the frequency of deep torpor. All these seasonalmechanisms in winter mouse lemurs contribute to fat depo-sition through important reduction in energy expenditure(Aujard et al. 1998; Ortmann et al. 1997; Perret et al. 1998;
Schmid 1999). Genin and co-workers (Genin and Perret2000) have also suggested that gastrointestinal satiety pep-tides are likely to contribute to the regulation of body massin mouse lemurs, as evidenced in seasonal mammals (Mer-cer 1998; Morgan and Mercer 2001).
The underlying mechanisms by which body massincreases in re-fed mouse lemurs exposed to winter-likeshort-days would match those of fattening processesreported by previously cited studies on M. murinus. Areduced fat utilization and thus body mass gain have beenreported in mice and rats, after peripheral daily administra-tion of ghrelin and, food intake and body mass increase, ina dose dependant manner, after intra-cerebro-ventricularadministration of ghrelin (Tschop et al. 2000). Based onthese results, authors suggested a role for ghrelin in induc-ing adiposity in rodents. Moreover, plasma ghrelin concen-trations in humans and rodents increase rapidly duringfasting and decrease (within 12 h) with re-feeding (Tschopet al. 2000). The higher levels of active ghrelin reported inall our animal groups, after a 2-week re-feeding, can beexplained by ghrelin actions on food intake and lipolysis,overall contributing to fat accumulation (Tschop et al.2000) until those animals fully regain their appropriate sea-sonal body mass. After the re-feeding period, most bodymass of the animals remained lower compared to what wasexpected from their seasonal mass, i.e., 128 and 87 g in SDand LD group, respectively. As reported in the groundsquirrel and Siberian hamster during re-feeding, animalsregain mass until their seasonal set point, which corre-sponds to the body mass value they should display at thecorresponding time in their seasonal cycle (Karmann et al.1994; Steinlechner et al. 1983). Therefore, the existence, inseasonal mammals, of a body mass set point likely explainsthe respective higher or lower hormonal levels of the re-fedanimals. In the Weld, mouse lemurs display a body mass30% lower compared to captive animals. Therefore, guthormonal responses in wild mouse lemurs during food-restriction and re-feeding should be as signiWcant as, if notmore, than those observed in captive animals. Nevertheless,as gut hormones are largely implied in the regulation ofenergy balance and numerous studies on M. murinusshowed similarity between mechanisms of fuel homeostasisin wild and captive conditions in this species (Perret 1998;Genin et al. 2005), it is tempting to suggest that gut hor-monal patterns during food restriction and re-feeding, andoverall underlying mechanisms of body mass recovery,may be similar between captive and Weld mouse lemurs. Ithas also been reported that PYY administration caused adecrease in food intake, and an increase in thermogenesisand lipolysis in rats and humans (Adams et al. 2006; Batter-ham et al. 2002; Sloth et al. 2007), that would support thenegative correlation of PYY with body mass in re-fed ani-mals in winter. Therefore, our results are in agreement with
Table 2 Summary of telemetry parameters and hormonal levelschanges in long-days (summer) and short-days (winter) acclimatedanimals, during calorie restriction
LD40 animals exposed to long-days under a 40% calorie restriction,LD80 animals exposed to long-days under an 80% calorie restric-tion, SD40 animals exposed to short-days under a 40% calorierestriction, SD80 animals exposed to short-days under an 80% calorierestriction
Table 3 Summary of telemetry parameters and hormonal levelschanges in long-days (summer) and short-days (winter) acclimatedanimals, after re-feeding
LD40 animals exposed to long-days under a 40% calorie restriction,LD80 animals exposed to long-days under an 80% calorie restriction,SD40 animals exposed to short-days under a 40% calorie restric-tion, SD80: animals exposed to short-days under an 80% calorierestriction, B baseline, W5 Wfth week of calorie restriction
these Wndings and suggest that these two gut-produced hor-mones (ghrelin and PYY) were related to underlying mech-anisms of body mass regain only in re-fed mouse lemursunder winter phenotype.
Many studies reported excessive fat deposition duringbody mass recovery after mass loss. This phenomenonresults from a disproportionately faster rate to regain bodyfat rather than lean tissue leading to an accelerated fatrecovery or “catch-up fat” (Dulloo et al. 2002). This pro-cess of fat deposition shares common feature with fatteningmechanisms of M. murinus and has survival value becauseit enables the rapid replenishment of fat stores under condi-tions of intermittent periods of food shortage, as thosefaced by the gray mouse lemur in its natural habitat. Indeed,mouse lemurs show a seasonal cycle of body mass that ischaracterized by a succession of fattening and slimmingprocesses, allowing animals to cope with environmentalXuctuations, especially with periods of food scarcity in win-ter. As winter survival of M. murinus mainly depends onthis anticipatory fattening process, it appears essential forre-fed animals in winter, unlike those in summer, to regainsuYcient body mass, i.e., amount of fat mass, to cope withenvironmental challenges during the scarce season.
Putative relation of gut-derived hormones with regulatory-mechanisms of energy balance in summer animals
GLP-1 reduces food intake and decreases body temperaturein mammals and birds as reported in rat and Japanese quail(Shousha et al. 2007; Turton et al. 1996). In these two stud-ies, GLP-1 injected either peripherally or centrally caused atransient and dose-dependant decrease in body temperature,which likely aVects energy expenditure. It has been sug-gested by Shousha and coworkers (Shousha et al. 2007)that the mechanism underlying this transient thermalchange in Japanese quail might be due to metabolic change,because the thermoregulatory center is located close to thefeeding regulatory area in the hypothalamus. This suggeststhat regulatory mechanisms for temperature and feedingmay be linked. Then, these results suggest a putative rela-tion of GLP-1 with the control of torpor expression in M.murinus under the summer phenotype, as its level increasedduring calorie restriction and it was negatively correlatedwith the hypothermia depth in long-days animals.
It has been reported, by Gluck and co-workers, thatperipheral ghrelin administration signiWcantly deepens tor-por bout in fasting mice (Gluck et al. 2006) but the authorssuggested that ghrelin might not play a role in animals thatdepend largely on photoperiod changes for a signal for tor-por bout. The Siberian hamster can enter torpor when main-tained for extended periods of time in a shortenedphotoperiod (Freeman et al. 2004; Paul et al. 2005),
although entry into torpor occurs only after a signiWcant fatmass loss (Ruf et al. 1993). Circulating ghrelin levels ofSiberian hamsters are relatively unchanged between longphotoperiods (non torpor season) vs. short photoperiods(torpor season) (Tups et al. 2004). Our results support theabove suggestion of Gluck and co-workers (Gluck et al.2006) since active ghrelin levels of mouse lemurs did notdiVer between seasons and no signiWcant correlation wasreported between ghrelin levels and the torpor depth of ani-mals in both seasons.
Furthermore, it has been demonstrated that the ghrelin-induced deepening of torpor bouts in fasting mice was med-iated through NPY neurons in the arcuate nucleus (Glucket al. 2006). Another study reported no change in NPYmRNA in the arcuate nucleus when GLP-1 was intra-cere-bro-ventricularly injected (Turton et al. 1996), suggestingthat GLP-1 does not act by altering hypothalamic NPY syn-thesis. Taken together, these Wndings suggest that GLP-1modulated torpor expression in food-deprived mouselemurs, through a mechanism that may not necessarilyimply NPY, as ghrelin does in fasting mice (Gluck et al.2006).
In addition to its regulatory eVect on body temperature,it has been reported that peripheral GLP-1 decreases carbo-hydrate oxidation in humans (Flint et al. 2000). In the sameway, PYY has been shown to decrease the respiratory quo-tient, i.e., increase fat use, to help meet energy requirementin obese mice (Adams et al. 2006). Paradoxically, intra-cerebro-ventricular administration of PYY stimulates feed-ing and locomotor activity in mice, leading to increasedenergy expenditure (Nakajima et al. 1993). Our Wndings donot support these results since no association between loco-motor activity and levels of gut hormones, as PYY, wasreported in mouse lemurs in the summer and in winter.During the torpor phase (low metabolic rate), heterothermicmammals shift the source of substrate use, from carbohy-drate to fat stores (Dark 2005). We then postulate that theincrease in PYY levels of mouse lemurs during food depri-vation would participate to the process of changes in sub-strate utilization and promote fat oxidation during torporbout, and by extension during food restriction, acting in thesame way as GLP-1 that decreases carbohydrate use.Because under low food supply, carbohydrate stores arerapidly exhausted, food-deprived mouse lemurs wouldpreferentially use fat stores as the main source of energyand then would spare their protein mass. This is supportedby the role of GLP-1 in decreasing whole protein break-down in healthy humans (Shalev et al. 1997). Further stud-ies are clearly needed to determine the putativeimplications of GLP-1 and PYY in the substrate-type oxi-dation during torpor in food-deprived mouse lemurs.
In summer, the gray mouse lemur is under an activestate and the survival of the species depends on its repro-
ductive success. On Madagascar, the possible occurrenceof the El-Niño phenomenon in summer triggers a drasticfood shortage, the intensity of which can be similar tothat of the severe (80%) experimental food restriction inour study. Such unpredictable lowering of food supplyleads to an unusual pressure on the gray mouse lemurunder reproductive states. In these conditions, food-deprived mouse lemurs would remain suYciently activeto ensure a high reproductive success in their natural hab-itat. Therefore, maintaining their lean body mass by spar-ing proteins appears to be of great importance in suchcontext.
Lack of GLP-1 implication in changes of torpor patternsin winter animal
In our study, food restriction induced much more changesin torpor expression in mouse lemurs under winter pheno-type than those in summer. Potential relation of GLP-1with torpor pattern changes was only highlighted in food-deprived animals under summer phenotype. Therefore, wepostulated that winter mouse lemurs set up other seasonalenergy-regulatory mechanisms that would proportionallyreduce the importance of gut hormone implications inchanges of torpor patterns. As described above, mouselemurs in winter show marked seasonal adaptations char-acterized not only by an increased ability to display torporbut also by large changes in body mass. In seasonal mam-mals, sensitivity to gastro-intestinal peptides variesaccording to the photoperiodic state of the animal. Sibe-rian hamsters display higher sensitivity to the anorexigeniceVect of the cholecystokinin-8, when they expressed awinter phenotype compared to a summer state (Bartnesset al. 1986). This peptide is also linked to other satiety hor-mones implied in the regulation of energy homeostasis.Leptin acts, in part, by inXuencing the eYcacy of meal-generated satiety peptides, as cholecystokinin which theanorexigenic eVect is enhanced by leptin administration(Matson et al. 1997). Administration of exogenous leptinhas also been shown to induce loss of adipose tissue, insummer hamsters that normally display low leptin concen-trations. By contrast, hamsters in winter with high adiposetissue reserves are refractory to the eVects of leptin (Klin-genspor et al. 2000). This phenomenon of seasonal leptinresistance appears to be a general feature of seasonallybreeding mammals that have to enter an energy-economystate, as torpor bout during the scarce season. Therefore,the lack of a GLP-1 putative contribution in torpor patternchanges in winter mouse lemurs may be due to a reducedsensibility of mechanisms leading to GLP-1 secretion and/or to an increased GLP-1 resistance in M. murinus in win-ter under food restriction.
Conclusion
In this study, we reported a potential relation of two gut-produced hormones, active ghrelin and PYY, with the sea-sonal mechanism of fattening. Conversely, the putativerelation of GLP-1 with the energy-regulatory process (tor-por phase), was likely masked when M. murinus fullyexpressed its winter phenotype but clearly emerged in thegray mouse lemur in summer. Studies manipulating levelsof gut hormones, such as ghrelin, PYY and GLP-1, onenergy-saving processes are required on hibernating animalmodels (Siberian hamsters, ground squirrels), other than theendangered species of mouse lemurs. Particularly, as GLP-1 is also implied in the substrate-type oxidation in mam-mals, concerns are also focusing on the shift in fuel utiliza-tion and sparing in mouse lemur under food restriction, inboth summer and winter states. In this context, further stud-ies are required to determine modiWcations of the relativeamount of fat mass and fat-free mass in food-deprived-mouse lemurs and the potential role of GLP-1 and othergut-derived hormones in this process. Such studies wouldbring new information and insights in the Weld of theenergy homeostasis regulated by gut-produced hormones.
Acknowledgments S. Giroud is supported by an MNRT fellowship.The study was supported by an ATIP from the CNRS (S. Blanc), theBettencourt Schueller Fondation (Y. Le Maho), the GIS Longévité (S.Blanc) and the ANR Alimentation & Nutrition Humaine (M Perret, SBlanc). This protocol received all ethic authorizations and was con-ducted under the authorization number 67-223 (CNRS).
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