Changes of peroxisomal fatty acid metabolism during cold acclimatization in hibernating jerboa (Jaculus orientalis)

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Changes of peroxisomal fatty acid metabolism during cold acclimatization in hibernating jerboa (Jaculus orientalis)

Mostafa Kabineâ,b, Marie-Claude Clémencetâ, Jacqueline Brideâ,1, M’hammed Saïd El Kebbaj^b, Norbert Latruffeâ, Mustapha Cherkaoui-Malkiâ,*

aBMC (GDR-CNRS n° 2583), Faculté des Sciences Gabriel, LBMC – Université de Bourgogne, 6, boulevard Gabriel, 21000 Dijon, France

bLaboratoire de Biochimie, Faculté des Sciences-Aïn Chock, Université Hassan II-Aïn chock, Casablanca, Morocco Received 19 December 2002; accepted 30 May 2003

Abstract

Jerboa (Jaculus orientalis) is a deep hibernator originating from sub-desert highlands and represents an excellent model to help to understand the incidence of seasonal variations of food intake and of body as well as environmental temperatures on lipid metabolism. In jerboa, hibernation processes are characterized by changes in the size of mitochondria, the number of peroxisomes in liver and in the expression of enzymes linked to fatty acid metabolism. In liver and kidney, cold acclimatization shows an opposite effect on the activities of the mitochondrial acyl-CoA dehydrogenase (–50%) and the peroxisomal acyl-CoA oxidase (AOX) (+50%), while in brown and white adipose tissues, both activities are decreased down to 85%. These enzymes activities are subject to a strong induction in brown and in white adipose tissue (3.4- to 7.5-fold, respectively) during the hibernation period which is characterized by a low body temperature (around 10 °C) and by starvation. Expression level of AOX mRNA and protein are increased during both pre-hibernation and hibernation periods. Unexpectedly, treatment with ciprofibrate, a hypolipemic agent, deeply affects lipolysis in brown adipose tissue by increasing acyl-CoA dehydrogenase activity (3.4–fold), both AOX activity and mRNA levels (2.8- and 3.8-fold, respectively) during pre-hibernation. Therefore, during pre- hibernation acclimatization, there is a negative regulation of fatty acid degradation allowing to accumulate a lipid stock which is later degraded during the hibernation period (starvation) due to a positive regulation of enzymes providing the required energy for animal survival.

Keywords: Acyl-CoA-oxidase; Cold adaptation; Hibernation; Jerboa; Jaculus orientalis; Mitochondria; Peroxisomes;b-Oxidation; Lipid metabolism

1. Introduction

Hibernation is a process adopted by several species to survive in the cold environment. This phenomenon requires a special adaptation of cell metabolism with physiological changes that especially necessitate a strict control of lipid metabolism, since lipids play a critical role in supplying the energy requirements during hibernation.

Among the hibernators, jerboa (Jaculus orientalis), of the family of dipodidea, is a rodent of rat size living in the sub-desert highlands. It is mainly a nocturnal and saltatorial

herbivore[1]. Jerboa is considered as a deep hibernator[1], increasing its fat content in autumn, corresponding to the pre-hibernating period, and consuming this fat during hibernation and arousal. After cold acclimatization at +6 °C[1], rectal temperature of jerboa was not significantly different from control (36 ± 1.5 °C). After hibernation induced by food deprivation, animals are characterized by the crouched posi- tion and breathing pattern: alternating deep respiration and apnea[1,2]. Rectal temperature dropped to 9.8 ± 0.7 °C and the mean heart frequency stabilized to 9.3 ± 1.5 beat/min, compared to active animal with 37 °C body temperature and 300 beat/min, respectively[1].

It is well known that all fatty acid oxidation is performed in mitochondria and in peroxisomes [3]. This b-oxidation occurs by two distinct pathways in each of the above cited organelles as shown by Hryb and Hogg[4]. From yeasts to mammals, there is now an evidence of the presence of a peroxisomalb-oxidation system distinct from the mitochon-

Abbreviations: AOX, acyl-CoA oxidase.

* Corresponding author.

E-mail address: malki@u-bourgogne.fr (M. Cherkaoui-Malki).

1 Present address: Laboratoire de Biologie cutanée, Université de Franche-Comté, Besançon, France.

www.elsevier.com/locate/biochi

doi:10.1016/S0300-9084(03)00117-2

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drial one[3]. These twob-oxidation machineries act in liver cells as shown by Hryb and Hogg[4], where mitochondrial b-oxidation is more active towards short and medium chains acyl-CoA, but the peroxisomal system[3,5]acts primarily on long- and very long-chain acyl-CoAs. We have shown that jerboa liver exhibits higher palmitoyl-CoA oxidase activity than rat liver[6].

Brown adipose tissue and white adipose tissue play a central role during pre-hibernating fattening and provide further metabolic energy during the hibernation period.

White adipose tissue is specialized in storing triglycerides during adipogenesis and releases fatty acids as energetic fuel, while brown adipose tissue is involved in thermogenesis via the generation of heat. Current research using gene targeting, knock-out or transgenic experiments bring a new light on the role of brown adipose tissue thermogenesis in the regulation of body weight and the prevention of obesity[7–9]. Further- more, the brain controls adipose tissue proliferation and thermogenesis through the sympathetic nervous system by releasing epinephrine and norepinephrine. Such signaling molecules are ligands of adrenergic receptors which control the expression of several transcription factors (c-FOS, C/EBP) which regulate brown adipose tissue hypertrophy and hyperplasia[10].

Previously, we have shown in preliminary results that cold exposure and hibernation affected homogeneity of liver peroxisomes in jerboa [11]. In fact, we observed variations characterized by a decrease in peroxisomal protein yield and by a differential change in peroxisomal protein pattern in sodium dodecyl sulfate polyacrylamide gel electrophoresis, during pre-hibernation or hibernation states [11]. Also, we have shown that liver of active jerboa exhibits peroxisome proliferation after treatment with a hypolipidaemic agent; i.e.

ciprofibrate[6]. In addition, physiological plasmatic param- eters, such as glucose,D-3-hydroxybutyrate, cholesterol and triglycerides levels are affected during first days of hibernation[11]decrease of glucose andD-3-hydroxybutyrate level, increase of cholesterol and increase of triglyceride.

To help to understand the incidence of seasonal variations of food intake and of body as well as environmental temperatures on lipid metabolism in a rodent hibernator model, i.e.

jerboa, we focused our investigations on the morphological aspect of liver mitochondria and peroxisomes and on the expression of acyl-CoA oxidase (AOX) enzyme activities, protein and mRNA levels under different conditions (euthermic, pre-hibernation and hibernation states).

2. Materials and methods

Male young adult jerboa (J. orientalis) between 4 and 6 month old (110–140 g body weight) were captured in the sub-desert of eastern Morocco and transferred to France.

They were pre-acclimatized in the laboratory for 3 weeks at 22 ± 2 °C with food (rabbit diet; Aliments UAR- Villemoisson, Orge, France) and salad. The circadian rhythm was 10 h light and 14 h dark. For pre-hibernating and hiber-

nating states, a group of animals (3–4 per cage) was kept with food in a cold room (6 °C) for 3 weeks. This group was called the pre-hibernator group (PH). A second group was kept under the same conditions as PH, except that the food con- tained ciprofibrate at a dosage of 3 mg/kg body weight/d, and was called the ciprofibrate-treated pre-hibernator group (PHC). The third group was housed as PH, and at the end of 3 weeks of the pre-hibernation period, the food was removed leading to hibernation after 24–36 h, so called group (H).

These animals were sacrificed on the fourth or the sixth day of hibernation. The reference groups correspond to euthermic active animals (A), and euthermic active ciprofibrate- treated animals (AC), received the same dosage as PHC, i.e.

3 mg/kg body weight/d.

2.1. Electron microscopy

The method used for the detection of peroxisomes is based on the visualization of the peroxidatic activity of catalase with diaminobenzidine (DAB) which produces a black stain [12]. Samples of median liver lobe removed immediately after sacrifice were sliced using a razor blade. Tissue slices were fixed 1 h in a fixative containing 4% paraformaldehyde, 2% glutaldehyde and 0.03% CaCl₂ in 0.1 M cacodylate buffer at pH 7.4. Then the tissues were processed for cytochemical staining and for electron microscopy as described previously[13]. Ultrathin sections of Epon-embedded mate- rial were contrasted with lead citrate observed in a Hitachi 600 electron microscope (Centre de Microscopie Appliquée à la Biologie, Université de Bourgogne). In each experimen- tal group of animals, the number of peroxisomes per cellular area unit (250 µm²) was counted in 15 electron micrographs randomly taken at an original magnification of 5000×.

2.2. Enzyme assays

Crude tissue homogenates were prepared as described by Kabine et al.[11]. Cyanide insensitive AOX activity using palmitoyl-CoA was performed as described by Lazarow and de Duve[14]. Protein content was estimated with the Bio- Rad assay according to Bradford[15].

2.3. Immunoblotting

Proteins from purified peroxisomal fractions[16]by 30%

Nycodenz (Nycomed Pharma AS, Oslo, Norway) step gradi- ent centrifugation were separated by SDS-PAGE (10% W/V gel). The gel was stained with Coomassie Blue to compare protein patterns between different samples. Electrotransfer and immunostaining were performed on nitrocellulose membrane (Millipore) with semidry multigel electroblotter[17].

The membrane was incubated with polyclonal antibody raised against rat AOX (the 72 kDa native form), a generous gift of Dr. Dariush Fahimi, Heidelberg, Germany, as described by Pacot and Latruffe[16]. Detection was done with alkaline phosphatase conjugated anti rabbit IgG from goat.

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2.4. Northern blot analysis

Total RNAs were obtained from interaxillary BAT and liver tissues previously frozen in liquid nitrogen and stored at –80 °C using the LiCl method described by Auffray and Rougeon[17]. Northern blots were performed as described by Cherkaoui-Malki and Caira[18]. Hybridization was done at 42 °C overnight in 50% formamide, 5× Denhardt’s, 5×

SSC and 0.1% SDS. The filters were washed twice at 42 °C for 30 min in 2× SSC, 0.5% SDS and once at 65 °C for 30 min in 1× SSC, 0.1% SDS. cDNA probe were generously given by Dr. Takahashi Osumi for rat AOX (Kamigori, Japan).

Northern blots were normalized with the 18S ribosomal RNAs.

2.5. Densitometry analysis

Densitometry scanning was realized using the Alphaim- ager™ 1220 (Alphainnotech).

2.6. Statistical analysis

Values are reported as mean ± standard deviations. P values were calculated by ANOVA.

3. Results

3.1. Morphology and numerical density of peroxisomes

The structural analysis of the liver lobule during different physiological stages of hibernation of jerboa exhibits marked morphological alterations in the aspect and numerical density of peroxisomes compartment.Fig. 1ashows a hepato- cyte containing intense DAB stained rod-shaped peroxisomes that appear round to oval. They exhibit a diameter between 0.1 and 0.4 µm. Ciprofibrate treatment induces peroxisome proliferation in active jerboa (Fig. 1b) and increases the numerical density by 51% (Fig. 1f). A typical needle- and rod-shaped peroxisome often occur in clusters with increas- ingsize of peroxisomes.

Compared to active jerboa (Fig. 1a) pre-hibernating animals reveal a remarkable augmentation in number, size and shape of peroxisomes in cluster around a lipid droplet (Fig. 1c). This peroxisomes proliferation is indicated by 60.7% increase in their numerical density (Fig. 1f) with a heterogeneous aspect in the DAB reaction deposit. Ciprofi- brate treatment during cold adaptation does not provoke additional peroxisome proliferation (Fig. 1d). A similar increase, 51% of the numerical density, was recorded (Fig. 1f).

Peroxisomes have the same aspect in the active ciprofibrate- treated jerboa (with some large-size peroxisome) as those seen in the cold-adapted jerboa.

During hibernation rod-shaped peroxisomes exhibit an increased size and irregularly DAB-stained matrices in hepa- tocyte (Fig. 1e). Hibernating jerboa shows a diminution of 47% of the number of peroxisomes compared to pre-

hibernating animals and only 15% compared to active ones (Fig. 1f). Concerning mitochondria where theb-oxidation is carnitine-dependent[19], we observed a remarkable increase of their size particularly during the hibernation period.

3.2. Measurement of enzymatic activities

The activity of AOX was measured in liver tissue (Fig. 2).

We observed a significant augmentation of AOX activity in liver (×1.6). In the opposite way, a decrease of AOX activity was seen in brown adipose tissue (×0.7) and white adipose tissue (×0.3) (data not shown). The passage from the pre- hibernating (PH) to hibernating (H) state strongly increases the AOX activity in kidney (×1.9), in brown adipose tissue (×6) and in white adipose tissue (×3.4), whereas no change of this activity was observed in liver tissue. The jerboa in pre- hibernating state treated with ciprofibrate (PHC) shows an increase of AOX activity in liver (×3.8). Moreover, the im- pact of different physiological states on the level of AOX protein was investigated in liver tissue (Fig. 3). The immunoblotting protein pattern shows the three major bands (72, 52 and 19 kDa) of AOX resulting from peroxisomal post- translational processing of 72 kDa as described for rat AOX (24). Considering the native 72 kDa band of AOX (Fig. 3), there is an increase in the amount of AOX during pre- hibernation by twofold (Fig. 3, PH). This augmentation was more important (×2.9) after treatment with ciprofibrate (Fig. 3, PHC). The amount of AOX in liver of hibernating jerboa increases, respectively, by 2.5-fold at the fourth day (H4) and by 2.1-fold at the sixth day (H6) of hibernation (Fig. 3, H4 and H6). A similar augmentation was found for 52 and 19 kDa subunits.

Northern blotting analysis of AOX mRNA was performed using liver total RNA. The results demonstrate that during jerboa cold exposure in the liver tissue there is a slight change in AOX mRNA level (×1.2). Hibernating jerboas show an induction (×1.4) of liver tissue AOX mRNA (Fig. 4, lane 3).

Ciprofibrate treatment provokes an increase of AOX mRNA levels in liver by 3.5-fold (Fig. 4, lane 4).

4. Discussion

Morphological analysis of liver peroxisomes by electron microscopy reveals a strong alteration in shape and size of this compartment. Cold exposure of jerboa results in peroxisome proliferation with an increase in their number and size.

During hibernation only the size becomes large, forming giant peroxisomes with a decrease of their number in hepatocytes.

We have reported previously[11]that the protein yield of the purified peroxisomal fraction per gram of liver decreased by 60% in both pre-hibernating and hibernating jerboa. Tak- ing into account the observed increase in size of peroxisomes in these two physiological states, decrease in the protein yield might be explained by a shift in peroxisome density that implies the modification of the sedimenting properties. A

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similar explanation was reported regarding cold adaptation in rat[20].

Concerning mitochondria, cold exposure (PH) did not induce changes in their size or number, whereas in our previous results[11]we obtained an increase (×1.47) of the protein of purified mitochondrial fraction. This augmentation seems to be a result of a shift, as above, of peroxisomes density, becoming similar to the mitochondrial density.

During hibernation, the great majority of mitochondria exhibit an increase in their size, which may correspond to an enhancement of their density, while there is no change in the yield of purified mitochondrial fraction per gram of liver [11]. Taking into account the morphological observation concerning mitochondria, this unchanged yield could be explained by both the shift of large amount of mitochondria to a higher density and the compensation of this amount by a

Fig. 1.Comparative morphology of hepatic peroxisomes in jerboa by electron microscopy, using catalase cytochemistry (see Section 2), during the successive stages of hibernation in the control jerboa (a, c and e) or in the ciprofibrate-treated jerboa (b and d). (a) Euthermic active jerboa (A), (b) Active ciprofibrate-treated jerboa (AC), (c) Pre-hibernating jerboa (PH), (d) Pre-hibernating ciprofibrate-treated jerboa (PHC), (e) Hibernating jerboa (H), (f) Numerical density of peroxisome per 250 µm²of cellular area. Bar = 1 µM, 8750×.

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fraction of heavier peroxisomes. It has previously been reported that the size and the number of mitochondria in heart and skeletal muscles of ground squirrels increase during the hibernating season [21,22], and that liver mitochondria of hibernating ground squirrels are in a condensed state that seems to cause inhibition of electron transport in the respira- tory chain[23]. Such changes have not been observed in BAT and myocardium of hedgehogs[24].

In the light of these morphological observations and inter- pretations above reported, we propose a scheme showing different populations of mitochondria and peroxisomes in terms of size and density (Scheme 1). We have reported in

this scheme the already published yield of purified fractions of mitochondria and peroxisomes and the projection of suggested populations of mitochondria and peroxisomes appear- ing from different physiological states of jerboa. Each popu- lation would sediment at the corresponding density during a differential centrifugation experiment (Scheme 1).

The present work reports a regulation of fatty acid degradation in both mitochondria and peroxisomes. Concerning mitochondria, we observed (results not shown) in all tissues an inhibition of fatty acid degradation during pre-hibernation due to the diminution of the transport of fatty acids (depressed carnitine acyl-transferase activity) and fatty acid oxidation by acyl-CoA dehydroganse. Also in the pre- hibernating period, the degradation of fatty acids is strongly depressed (ADH and AOX activities) in both brown and white adipose tissues. The level of peroxisomal fatty acid degradation is slightly enhanced in brain and kidney, and significantly induced in liver (induction of AOX activity).

The induction in liver of peroxisomal AOX activity is related to the increase of both AOX protein synthesis and AOX mRNA level as demonstrated by immunoblotting and North- ern blotting hybridization. It has been reported that white adipose tissue may participate in selective retention of essential fatty acids during the pre-hibernating period[25]. Ned- ergaard et al. [25] reported a 10-fold increase of brown adipose tissue peroxisomalb-oxidation in cold adapted rat and calculated up to 10% of total brown fat proteins to be peroxisomal. However, recent experiments on rat after 4 weeks cold exposure[26]show neither significant increase in brown adipose tissue peroxisome proliferation, nor induction of peroxisomal enzymes during the first 2 weeks of cold adaptation, corresponding to the initial proliferative phase of brown adipose tissue. In the same experiment a sevenfold increase in AOX content and peroxisome volume was observed in rat liver[27].

All of our results show that the general depression of fatty acid catabolism is in accordance with the fattening period of cold acclimatization. In results not shown the liver acetyl- CoA carboxylase mRNA level, is increased during pre-

Fig. 2.Specific activities of AOX during different periods of hibernation and effect of ciprofibrate. Activities AOX were measured in liver of euthermic active (A), pre-hibernating (PH), pre-hibernating ciprofibrate-treated (PHC) or hibernating jerboa (H). Histograms represent values ± standard deviations in percent of the control (active jerboa A) of at least three different animals at

* * P < 0.01 and * P < 0.05 as compared to the active (A) or at °° P < 0.01 as compared to the PH calculated by ANOVA. The control value of AOX in active jerboa was 0.08 ± 0.003 nmol palmitoyl-CoA/min/mg protein of homogenate.

Fig. 3.Western blotting analysis of AOX in liver of euthermic active (A), pre-hibernating (PH), pre-hibernating ciprofibrate-treated (PHC) or hibernating jerboa (H4 = 4 d of hibernation and H6 = 6 d of hibernation). The molecular weight in kDa of the three AOX polypeptides (72, 52 and 19 kDa) are indicated. (a) Relative fold induction, of native 72 kDa, as compared to active animal (A) value calculated by densitometry.

Fig. 4.Northern blot analysis of AOX, mRNAs in liver and brown adipose tissue of euthermic active (A), pre-hibernating (PH), pre-hibernating cipro- fibratetreated (PHC) or hibernating jerboa (H). 18S represent the 18S ribosomal RNAs. (a–c) represents, respectively, the relative fold induction of AOX mRNAs obtained by densitometry and normalized to 18S ribosomal RNAs.

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hibernation. An increase of acyl-CoA carboxylase level was reported in brown adipose tissue after 48 h of exposure of rat at cold temperature [28]. Similar results concerning Acyl- CoA synthetase (another enzyme of lipolysis) were observed for both ground squirrel (Spermophilus lateralis) [29]and marmot (Marmota flaviventris)[30].

During hibernation, compared to the pre-hibernation state, jerboa consumes its lipid stock by degrading fatty acids as indicated by stimulation of activities of acyl-CoA dehydroganse (not shown) and AOX in the main tested tissues, except for AOX in brain and liver. In the brain there is no change of AOX activity probably because of the role of this tissue, considered to be a consumer which should maintain its steady state energy level. As for liver tissue, the unchanged AOX activity during hibernation suggests that fatty acids accumulated during pre-hibernation period are mainly represented by medium chain fatty acids, which are prefer- entially degraded in mitochondria as underlined by the increased mitochondrial acyl-CoA dehydrogenase activity (not shown). This is in accordance with the protein level as shown by Western blotting experiment where the amount of AOX seems not significantly changed compared to the pre- hibernating state. This result is confirmed by the level of AOX mRNA. Furthermore, starvation after cold exposure did not provoke a change of AOX expression in terms of activity or protein level. Compared to the active state, hibernation appears to have similar effects compared to the pre- hibernation state.

The decrease of acyl-CoA carboxylase mRNA during the hibernation period (not shown) indicated a low level of fatty acid synthesis. Consequently, this suggests a stimulation of fatty acid degradation that is related to an increased activity of mitochondrial acyl-CoA dehydrogenase in accordance with the possible accumulation of medium fatty acid chain as suggested above. A decrease of acyl-CoA carboxylase activ-

ity was signaled in the heart of ground squirrel during hibernation concomitantly with a decrease of the expression of the 280 kDa isoform of acyl-CoA carboxylase (which predomi- nates in cardiac muscle)[31].

Hibernation process is characterized by a strong degradation of peroxisomal fatty acids due to transcriptional or post-transcriptional regulation of AOX gene in both tissues (liver for pre-hibernation and brown adipose tissue for hibernation). In liver, this regulation is induced by cold adaptation, while in brown adipose tissue it is caused by starvation in cold conditions. This is in accordance with the nature of each tissue function, i.e. liver is the site of lipid metabolism, which should adapt to changes in physiological conditions; whereas brown adipose tissue has a central role in thermogenesis.

This thermogenic role is essentially induced during hibernation when the body needs an energy supply. Indeed, the induction of UCP1 during the pre-hibernation state seems to participate in maintaining body temperature during cold exposure. In addition, uncoupling protein 1 participates during hibernation in the production of heat supplied by fatty acid degradation. The increase of uncoupling protein 1 mRNA expression in brown adipose tissue was also observed after cold exposure in rat[27,32]and squirrel[33].

Ciprofibrate treatment of jerboa during pre-hibernation increases fatty acid catabolism in all tissues as indicated by the stimulation of mitochondrial acyl-CoA dehydrogenase (not shown) and peroxisomal AOX. This is corroborated by the enhancement of the expression rate of AOX protein and mRNA in liver and AOX mRNA in brown adipose tissue.

Ciprofibrate treatment similarly regulates AOX in both tissues leading to strong fatty acid degradation. The latter implies additional heat production leading to dissipation of energy during cold exposure as shown by a strong increase of uncoupling protein 1 mRNA.

Scheme 1.Possible different classes of peroxisomes and mitochondria during the jerboa hibernating process.

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Food deprivation of ciprofibrate-treated pre-hibernating animals (PHC) causes the death of all jerboas. This death could be related to the strong hypoglycemia and high ketonemia as previously reported by our group in the hibernation state[11]. Indeed, in these conditions high ketonemia leads to a toxic acidonemia and the lack of neoglucogenesis from fatty acids, which is essential for glucose catabolism.

Ciprofibrate induces a strong breakdown of fatty acid stock, as indicated by increased activities of acyl-CoA dehydrogenase (not shown) and AOX, leading to high production of acetyl-CoA-generating ketone bodies.

In conclusion, in this work we have investigated different stages of hibernation of a rodent jerboa (J. orientalis) in view of morphological and molecular aspects. Morphological analysis has shown variations in mitochondria and peroxisomes shape and size. The lipid metabolism is subject to opposing control: negative regulation during pre-hibernation (lipid accumulation) and positive regulation during the hibernation period (starvation and fatty acid degradation). This double regulation seems to be necessary to progress from cold adaptation (negative regulation) to hibernation (positive regulation). Thus, all those variations are necessary to maintain a sufficient energy level to permit jerboa to survive in inhospitable cold conditions.

Acknowledgements

We thank Mrs. J. Relot for performing electron microscopy analysis at the SERCOBIO of the Université de Bour- gogne, Dr. William S. Cook for a valuable discussion. We also acknowledge Mrs. N. Bancod for typing the manuscript.

This work has been supported by the regional council of Burgundy and IFR n° 92, and by the “programme d’appui à la recherche scientifique-Morocco, Biologie n° 134”, the Ac- tion Intégrée Franco-Marocaine AI-MA/01/22 and by the CNRS GDR n° 2583.

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