Possible mechanisms of weight loss of Siberian hamsters (Phodopus sungorus sungorus) exposed to short photoperiod.

Several weeks of short day photoperiod (SD) exposure promote a dramatic decrease of white adipose tissue (WAT) mass in Siberian hamsters(Phodopus sungorus sungorus). This slimming effect is accompanied by changes in the adipocyte responsiveness to adrenergic stimulation that are still under debate. We investigated whether possible changes in the antilipolytic responses, and/or lipogenic activities could be involved in such lipid deposition/mobilisation imbalance. Male Siberian hamsters were exposed for 11 weeks to SD or long day photoperiod and basal or stimulated lipolytic and lipogenic activities were measured on white adipocytes. As expected, the body mass of SD-animals was decreased. Besides a slight reduction in the basal lipolysis and in the maximal response to dibutyryl-cAMP, the responses to adrenergic and non-adrenergic lipolytic agents (forskolin, adenosine deaminase) were similar in both groups. Fat mass loss was likely not resulting from changes in the lipolytic responses of adipocytes to biogenic amines (e.g. octopamine), which were unaltered, or to a direct lipolytic stimulation by melatonin or histamine, which were inactive. Antilipolytic responses to insulin or tyramine were slightly decreased in SD-adipocytes. Basal or insulin-stimulated lipid accumulation in WAT, measured by glucose incorporation into lipids, did not change after SD-exposure. However, a significant decrease in the lipoprotein lipase activity was observed in the WAT of SDanimals. Despite the observed changes, the weight loss of SD-exposed Siberian hamsters was likely not resulting only from impaired antilipolytic orde novo lipogenic activities in white adipocytes, but either from other dramatic changes occurring during seasonal photoperiod-sensitive body weight regulation.

Local sympathetic denervation of white adipose tissue in rats induces preadipocyte proliferation without noticeable changes in metabolism.

The direct influence of the sympathetic nervous system on white adipose tissue was studied by performing a unilateral surgical denervation of the retroperitoneal fat pad in rats, the contralateral pad being used as a control. One week after surgery, the weight of the denervated pad was significantly higher than that of the intact pad. In vivo, glucose utilization was not altered by denervation. The expression of GLUT4 as well as the expression and activity of fatty acid synthase, lipoprotein lipase, and hormone-sensitive triglyceride lipase were similar in the two pads. Lipolysis in response to norepinephrine, determined in vitro, was not modified by denervation although the ratio between alpha 2- and beta-adrenergic receptors was changed. Denervation induced an increase in DNA content without change in the number of mature adipocytes. The expression of A2COL6/pOb24, a marker of the early step of adipocyte differentiation, was significantly enhanced in the denervated pad, suggesting an increased number of preadipocytes. This was confirmed by an increased cell number observed in the denervated fat pad 1 month after surgery. In conclusion, surgical denervation of the white fat pad does not alter the glucose and lipid metabolisms. By contrast, it accelerated adipocyte differentiation and led to the recruitment of new precursors.

Characterization of a melatonin binding site in Siberian hamster brown adipose tissue.

Melatonin has been shown, in various rodent species, to mediate photoperiodic effects on body weight and, consequently, fat mass. Pharmacological investigations indicated that the brown adipose tissue of Siberian hamsters possesses a melatonin binding site with a dissociation constant of 570+/-300 pM and a density of 3.2+/-1.8 fmol/mg protein. This binding site can also be detected on mature brown adipocyte membranes. The rank order of potency of a variety of drugs to displace 2-[125I]iodomelatonin from binding sites on Siberian hamster brown adipose tissue was as follows: 2-iodomelatonin > melatonin = prazosin > GR135531 (5-methoxycarbonylamino-N-acetyltryptamine) > N-acetylserotonin > 6-chloromelatonin > S20304 (N-(2-(1-naphthyl)ethyl)cyclobutanecarboxamide) >> methoxamine, phenylephrine, serotonin. Mel(1a) mRNA was not detected by RT-PCR (reverse transcription-polymerase chain reaction) in brown adipose tissue. Melatonin had no effect on either basal or stimulated lipolysis. Moreover, melatonin did not modify intracellular cAMP accumulation or inositol phosphate content. Together, these results suggest that the melatonin binding site characterized in brown adipose tissue is clearly different from the Mel(1) cloned subtype and has some features different from those of the Mel2 subtype.

Sympathoadrenal activity and hypoglycemia in the hibernating garden dormouse.

The hibernating garden dormouse is spontaneously hypophagic during the prehibernating period at which time we found a low peripheral sympathetic activity (S.A.). The aim of this work was to investigate the link between dietary intake and S.A. The S.A. was evaluated by measurement of catecholamines in both plasma and adrenal glands by HPLC. Food intake, body weight, energy expenditure and plasma glucose were measured during the reentry phase of the hibernating period. The following results were obtained: the energy intake in pretorpid animals (55 to 83 kJ/24 h/100 g body weight) was less than energy expenditure which was between 145 and 97 kJ/24 h/100 g. The energy deficit induces marked hypoglycemia immediately before the onset of hypothermia (117 mg/dl vs. 76 mg/dl) and leads to a drastic drop in the peripheral sympathetic system. This, in turn, reduced energy production, causing the hypothermia.

Non-shivering thermogenesis activation and maintenance in the aging gray mouse lemur (Microcebus murinus).

The cold-induced enhancement of non-shivering thermogenesis (NST), involving brown-adipose tissue (BAT) metabolism, could participate to impair energy balance in the aged gray mouse lemur (Microcebus murinus). We first investigated the age-related modulations of cold-stimulated BAT cell morphology and contents. Then, NST was pharmacologically stimulated to assess whether aging impaired NST activation in the mouse lemur. In reference conditions, the ability to activate NST was preserved during aging in the mouse lemur as BAT morphology and UCP-1 presence did not differ between adult and aged mouse lemurs. Also, the pharmacological activation of NST revealed similar increased levels of O(2) consumption in adult and aged animals, confirming that no age effect could be evidenced on NST activation at 25 degrees C. However, preliminary histological data revealed a lack of lipid resources in one aged individual during cold exposure. Surprisingly, the pharmacological activation of NST revealed an impaired evacuation of the excess body heat in aged animals, associated with increased energy expenditure. Thus, aging seems to be related to decreased capacities in the maintenance of NST rather than in its activation. Energy mobilization could be impaired in the aging mouse lemur but remains to be demonstrated.

[Lethargic hypothermia induced by a protein free diet in a hibernating rodent, the dormouse (Eliomys quercinus L.)]. / Phases de léthargie hypothermique provoquées par une ration alimentaire dépourvue de protéines chez un rongeur hibernat, le Lérot (Eliomys quercinus L.).

In garden dormouse protein deficiency leads to reversible hypothermic torpor, comparable with that provoked by starvation or occuring naturally during hibernation, whether the diet consists wholly of apples or of synthetic protein-free food. Torpor induced by protein deficiency occurs even though the energy requirements of the animal are amply satisfied. These phases of lethargy occur after a certain delay and with a variable frequency, both of which vary with the ambient temperature.

[Tissue lactates, pH and blood gasses in hibernating dormouse: changes during periodic arousals]. / Lactates tissulaires, pH et gaz du sang chez le lérot hibernant : évolution au cours des réveils périodiques

Periodic arousal in the garden dormouse is accompagnied by a rise in plasma and muscle lactate levels and a diminution of muscle glycogen. In addition, the decrease of arterial blood pH and O2 concentration agrees well with these results. All of which are in good agreement with the general stimulation of adrenergic activity observed at the onset of rewarming.

Predominance of beta 3-adrenergic component in catecholamine activation of lipolysis in garden dormouse adipocytes.

The beta 3-adrenoceptor (AR) agonists are potent activators of lipolysis in white adipose tissue. beta-AR agonists were tested here on the lipolytic activity of a hibernator, the garden dormouse (Eliomys quercinus L.). All the agonists exhibited full intrinsic activity; the most potent was the beta 3-AR agonist BRL-37344 [half-maximal effective concentration (EC50) = 0.8 nM]. The beta-antagonist idocyanopindolol (ICYP) also stimulated lipolysis of white adipocytes with the same potency and intrinsic activity as BRL-37344. The blockade of lipolytic effects of epinephrine or norepinephrine was similar to that of BRL-37344: the beta 1- and the beta 2-antagonists were quite ineffective. Total blockade occurred only with 100 microM bupranolol whatever the beta-agonist tested. This argues for the presence of a beta 3-component in the adrenergic-induced lipolysis. (-)-[125I]ICYP and (-)-[3H]CGP-12177 both labeled two populations of binding sites. On adipocyte membranes, binding of 0.6 nM (-)-[3H]CGP-12177 was inhibited with the following order of potency: isoproterenol > BRL-37344 > epinephrine. This order was modified at 20 nM, arguing for the beta-atypical nature of the low-affinity sites. Thus garden dormouse adipocytes possess beta 3-ARs, which are involved to an important degree in the adrenergic activation of lipolysis.

[Annual cycle of oxygen consumption (QO 2) in a hibernating animalthe garden dormouse: reduction of the QO 2 and hypothermia following fasting]. / Cycle annuel de la consommation d'oxygène (QO2) chez un hibernant, le lérot: diminution de la QO2 et hypothermie entraînées par le jeûne

A circannual cycle of oxygen consumption (QO2) was observed in active garden dormice maintained during 19 months under external conditions for temperature and lighting. QO2 was minimum in october and maximum in may. In the same animals deprivation of food induced hypothermia and a 23% decrease in QO2 which always preceeded hypothermia. No circannual rhythm was noted in the amplitude of this reduction.

[Role of the season and of the nutritonnal state on the distribution of tissular fatty acids in the hibernating dormouse (Eliomys quercinus L.)]. / Rôles de la saison et de l'état nutritionnel sur la répartition des acides gras tissulaires chez le lérot hibernant (Eliomys quercinus L.)

Seasonal changes were observed in fatty acid composition of plasma, liver and cardiac muscle. During hibernation unsaturated fatty acids levels increase in plasma cholesterol esters (CE), glycerides (GL) and phospholipids. After spring arousal, the oleic acid content decreases in total fatty acids of liver and cardiac muscle. In summer fasting induces lipid changes similar to that occuring in natural hibernation. Desaturation observed in winter can be explained by the accumulation of unsaturated GL and CE in the tissues.

Liver and brown fat mitochondrial response to cold in the garden dormouse (Eliomys quercinus).

The involvement of two organs, i.e. the liver and the brown adipose tissue (BAT) in response to cold in a hibernating species such as the garden dormouse has been studied. 2. In animals living in the cold, mitochondrial respiratory rates significantly increased (with respect to those living at 28 degrees C) in both organs with a larger increase in the BAT (+152% in the BAT and 67% in the liver). 3. The increase in BAT activity was obtained by a concomitant increase in: (a) the BAT mass (+30%), (b) the total mitochondrial mass (+20%), and (c) the mitochondrial respiratory rate (+64%). In the liver the increase was due only to an augmentation in mitochondrial mass and activity. 4. These results indicate that: (a) the BAT exerts a pre-eminent role in the physiological response to cold of garden dormouse, (b) a certain non-shivering thermogenesis (NST) is present in the liver of such species. In addition we suggest that a local thermoregulatory response would take place in a metabolically important organ such as the liver.

Brown fat and nonshivering thermogenesis in the gray mouse lemur (Microcebus murinus).

The gray mouse lemur Microcebus murinus is a rare example of a primate exhibiting daily torpor. In captive animals, we examined the metabolic rate during arousal from torpor and showed that this process involved nonshivering thermogenesis (NST). Under thermoneutrality (28 degrees C), warming-up from daily torpor (body temperature <33 degrees C) involved a rapid (<5 min) increase of O(2) consumption that was proportional to the depth of torpor (n = 8). The injection of a beta-adrenergic agonist (isoproterenol) known to elicit NST induced a dose-dependent increase in metabolic rate (n = 8). Moreover, maximum thermogenesis was increased by cold exposure. For the first time in this species, anatomic and histological examination using an antibody against uncoupling protein (UCP) specifically demonstrated the presence of brown fat. With the use of Western blotting with the same antibody, we showed a likely increase in UCP expression after cold exposure, suggesting that NST is also used to survive low ambient temperatures in this tropical species.

Increased GDP binding and thermogenic activity in brown adipose tissue mitochondria during arousal of the hibernating garden dormouse (Eliomys quercinus L.).

1. The thermogenic activity of brown adipose tissue in hibernating garden dormice during hypothermic torpor and at different states of arousal were studied. High levels of GDP binding were observed on isolated brown fat mitochondria, indicating that the thermogenic proton conductance pathway is very active in brown fat during arousal. 2. In order to investigate this phenomenon, the uncoupling protein was assessed by immunological assay and the mRNA for UCP was analysed. 3. Animals during arousal exhibited neither increase in UCPmRNA nor an increase in UCP. 4. Our results suggest that during the rewarming of garden dormice there is an acute unmasking of GDP binding sites on the protein.

Evidence for a direct effect of melatonin on mitochondrial genome expression of Siberian hamster brown adipocytes.

Photoperiod variations are known to participate in the regulation of energy balance in different rodent species via melatonin, a neurosecretory product synthesized by the pineal gland during the night. A direct effect of melatonin on adipose tissue has been suggested since binding sites for the indole have been described on brown adipocytes. The aim of this study was to investigate a genetic effect of melatonin on isolated Siberian hamster brown adipocytes using differential display RT-PCR (DDRT-PCR). Brown adipose cells were isolated from brown adipose tissue and treated for 3 hr with 0.1 and 10 microM melatonin. Total RNA was extracted and DDRT-PCR experiments were performed. A differential band, which disappeared after melatonin treatment, was detected. After confirmation and cloning, the corresponding cDNA fragment B18 was sequenced. B18 had 85 and 81% similarity with a portion of rat and mouse cytochrome b mRNA, respectively, suggesting that B18 corresponds to hamster cytochrome b. This hypothesis was confirmed by the close parallel between the changes in mRNA content, detected by B18, and by cytochrome b mRNA content, detected by a rat probe. Cytochrome b mRNA is encoded by the mitochondrial genome, suggesting a similar effect of melatonin on the whole mitochondrial transcripts. Indeed, 3 hr of treatment with melatonin (10 nM and 0.1 microM) decreased by 44% mitochondrial transcript contents. This work constitutes the first evidence of a direct biological effect of melatonin on Siberian hamster brown adipocytes.

Cellular changes during cold acclimatation in adipose tissues.

Cold exposure is a well-known physiological stimulus that activates the sympathetic nervous system and induces brown adipose tissue (BAT) hyperplasia. The effects of cold exposure or cold acclimatation have been extensively studied in interscapular BAT (IBAT). However, it has been recently shown that studied adipocytes are present in adipose deposits considered as white fat such as periovarian (PO) fat pad. We have investigated the kinetic of brown precursor recruitment in adipose tissues using DNA measurement and specific marker expression. In IBAT, cold exposure induces proliferation of precursor cells and differentiation into preadipocytes characterized by the expression of A2COL6, a marker specific to early steps of the differentiation process. A chronic stimulation of the tissue is necessary to observe the full effect. In PO fat pad, no proliferation can be detected, whereas differentiation of brown preadipocytes and maybe phenotypic conversion of white adipocytes seems to be promoted. In conclusion, these data demonstrated that 1) the same stimulus (cold exposure) does not induce the same response in terms of preadipocyte proliferation and differentiation in periovarian and brown adipose tissues, although both contain brown adipocytes, and 2) preadipocyte recruitment in adipose tissues after cold exposure depends on the predominant type of fat cells.

Antilipolytic effects of alpha 2-adrenergic agonists, neuropeptide Y, adenosine, and PGE1 in mammal adipocytes.

Lipolysis is stimulated by different hormones, depending on the species, but is also regulated by antilipolytic modulators, such as catecholamines (alpha 2-agonists), neuropeptide Y (NPY), adenosine, and prostaglandin E1 (PGE1), for which species-specific variations are poorly described. Comparison of the efficiency of these antilipolytic systems showed that PGE1 or phenylisopropyladenosine was able to totally inhibit lipolysis activation in nine mammalian species. However, the antilipolytic responses to clonidine or UK-14304 were fully developed in hamster and rabbit but blunted in the adipocytes of jerboa, rat, guinea pig, garden dormouse, and dormouse. A powerful antilipolytic effect of NPY was found only in the garden dormouse. Only human and dog adipocytes exhibited antilipolytic responses to alpha 2-adrenergic and NPY stimulation. These observations were explained by differences in alpha 2-adrenergic and NPY/peptide YY receptor number. Thus, inhibitory regulation of lipolysis in white adipocytes seems to be composed of two systems: a constitutive one that is related to paracrine mediators (adenosine, prostaglandins) and a regulatory one including neuroendocrine messengers such as catecholamines and NPY.

Lipolytic and antilipolytic responses of the Siberian hamster (Phodopus sungorus sungorus) white adipocytes after weight loss induced by short photoperiod exposure.

Short day photoperiod promotes thermogenesis and extensive weight loss in Siberian hamsters (Phodopus sungorus sungorus). To determine whether a change in hormone-sensitive lipolysis occurs after short-photoperiod exposure, some lipolytic responses were measured on white adipocytes isolated from animals exposed in warm conditions to short or Long daylight photoperiod. The body mass of male Siberian hamsters exposed during 11 weeks to short days (SD; light: dark, 6:18 hr) reached only 50% of those kept in long days (LD; 16: 8 hr). In SD-hamsters, adipose depot mass also represented approximately 50% of the LD group. A lower DNA content was observed in intra-abdominal fat pads of SD-hamsters. Lipolytic responses to noradrenaline, adrenaline, isoproterenol and ACTH were unchanged. However, sensitivity to the beta-3 adrenergic agonist, BRL 37344, was moderately increased. The major component of the adrenergic control of lipolysis was mediated by beta-3 adrenoceptors in both LD- and SD-Siberian hamsters. The limited antilipolytic effect of alpha-2 adrenergic agonists, PYY or insulin was rather surprising in Siberian hamsters since these inhibitory systems are efficient in hibernants and other photoperiod-sensitive rodents. Our results show that, after short photoperiod exposure, white adipose tissue mass and DNA content are reduced, especially in the epididymal fat pad, with only minor changes in the adipocyte sensitivity to lipolytic hormones.

Effects of both a melatonin agonist and antagonist on seasonal changes in body mass and energy intake in the garden dormouse.

BACKGROUND: Many physiological functions including nycthemeral rhythm, reproductive cycles, body temperature and body mass are controlled by photoperiodic changes in different species. In the hibernating garden dormouse, both energy intake and body mass increase with the duration of the night. This seasonal mass gain is spontaneous and reversible. AIM: We have studied the occurrence of the increase of body mass by taking into account the endogenous variations of melatonin due to changing photoperiod or to pharmacological treatment. RESULTS: A single daily administration of either a melatonin agonist or antagonist just before night mimics the short day and long day effects, respectively. Compared to the control animals (natural photoperiod), the mass gain was greater and occurred earlier in animals under short days (6 h light (L)/18 h dark (D)) and in those receiving the melatonin agonist (S 20304). The animals treated with the antagonist (S 20928) during the same period exhibited no mass gain and their response was similar to that of the long-day group (16L/8D). Solely agonist treatment acted on metabolic rate. CONCLUSION: These results demonstrate that the duration of melatonin-receptor exposure per day determines the onset of seasonal obesity in garden dormice and, on the other hand, that restriction of melatonin-receptor exposure by pharmacological treatment prevents it.