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.

Weight-dependent changes of immune system in adipose tissue: importance of leptin.

Ancestral lymphoid cells reside in adipose tissues, and their numbers are highly altered in obesity. Leptin, production of which is correlated to fat mass, is strongly involved in the relationships between adipose tissues and immune system. We investigated in epididymal (EPI) and inguinal (ING) fat pads to determine whether 1) lymphocyte phenotypes were correlated to the tissue weight and 2) leptin was involved in such relationships. Immunohistological analyses revealed a tight relationship between the T and NK lymphocytes of the stromal vascular fraction and adipocytes. We identified a significant negative and positive correlation between EPI weight and the percentage of NK and total T cells respectively by cytofluorometric analyses. The NK and ancestral gammadelta T cell contents were directly dependent of leptin since they increased significantly in high-fat (HF) diet mice but not in leptin-deficient (ob/ob) mice as compared to control. By contrast, the alphabeta T cell content seemed independent of leptin because their percentages increased significantly with the EPI weight whatever the type of mice (control, HF, ob/ob). The present study suggests that adipose tissues present, according to their localization, different immunological mechanisms that might be involved in the regulation of adipose cells functions and proliferations.

Adipose tissues as an ancestral immune organ: site-specific change in obesity.

Close relationships have been demonstrated between adipose tissue and the inflammatory/immune system. Furthermore, obesity is increasingly considered as a state of chronic inflammation. Cytofluorometric analysis reveals the presence of significant levels of lymphocytes in the stroma-vascular fraction of white adipose tissues. In epididymal (EPI) fat, lymphocytes display an "ancestral" immune system phenotype (up to 70% of natural killer (NK), gammadelta+ T and NKT cells among all lymphocytes) whereas the inguinal (ING) immune system presents more adaptive characteristics (high levels of alphabeta+ T and B cells). The percentage of NK cells in EPI fat was decreased in obese mice fed with a high-fat diet, whereas gammadelta positive cells were significantly increased in ING fat. These data support the notion that adipose tissue may elaborate immunological mechanisms to regulate its functions which might be altered in obesity.

Fenofibrate prevents Rosiglitazone-induced body weight gain in ob/ob mice.

AIMS/HYPOTHESIS: Fibrates and thiazolidinediones are commonly used for the treatment of dyslipidemia and type 2 diabetes, respectively. The aim of this study was to investigate the effects on body weight as well as on glucose and lipid homeostasis of ligands for PPARalpha and PPARgamma, Fenofibrate and Rosiglitazone, alone or in association. METHODS: Ob/ob mice were divided into four groups: control, and mice daily injected (intraperitoneally), either with 10 mg/kg Rosiglitazone, 100 mg/kg Fenofibrate or both molecules. Body weight and food intake were monitored daily. After 13 days of treatment, mice were killed, and blood samples were collected for posterior metabolite quantification. The liver and adipose tissues were dissected and weighed. RESULTS: Body weight was significantly reduced or increased by Fenofibrate and Rosiglitazone, respectively. The effect of Rosiglitazone was prevented by coadministration of Fenofibrate. This was accompanied by a normalization of the daily food efficiency. Compared to those treated with Rosiglitazone, animals treated with Fenofibrate alone or in combination presented a decreased white adipose tissue mass. Fenofibrate or Rosiglitazone alone significantly reduced the levels of plasma lipid parameters. Surprisingly, Fenofibrate also decreased blood glucose levels in ob/ob mice, despite having no effect on insulin levels. By contrast, both glucose and insulin levels were decreased by Rosiglitazone treatment. Coadministration of both drugs improved all parameters as with Rosiglitazone. Fenofibrate restored almost normal hepatocyte morphology and significantly reduced the triglyceride content of the liver. This was accompanied by an increase in fatty acid oxidation in the liver in all groups receiving Fenofibrate. CONCLUSION/INTERPRETATION: These biological effects suggest that combined therapy with a PPARalpha and a PPARgamma ligand is more effective in ameliorating, specifically, lipid homeostasis than in activating any of this receptor separately. Furthermore, Fenofibrate prevents one of the most undesirable effects of Rosiglitazone, namely increased adiposity and body weight gain.

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.

Cloning of BUG demonstrates the existence of a brown preadipocyte distinct from a white one.

BACKGROUND: Several indirect arguments agree with the existence of a brown preadipocyte distinct from a white one. Nevertheless, to date, no molecular marker has been available to directly in vivo demonstrate this hypothesis. OBJECTIVE: The aim of this study was to find a gene expressed in brown preadipocyte but not in white and to use it as a molecular marker to analyse brown preadipocyte recruitment in different physiological and physiopathological situations. METHOD: Differential display was performed on stromal-vascular and adipocyte fractions of white and brown adipose tissues in rat. RESULTS: We identified a new gene, BUG, preferentially expressed in the stromal-vascular fraction of brown fat vs other adipose tissues fractions in adult rat. This RNA is also highly expressed in heart and, to a lesser extent, in other tissues such as kidney and brain. The BUG transcript is detected by in situ hybridization in putative preadipocytes within brown adipose tissue. Its level is transiently and specifically up-regulated during early stages of brown preadipocyte differentiation in a primary culture system, before the acquisition of late brown adipocyte phenotype. During development, BUG can be detected before the emergence of UCP-1 expression. In adult rats, BUG expression is inversely associated to brown adipose tissue (BAT) activation during cold exposure as well as in obese animals. CONCLUSIONS: The pattern of BUG expression agrees with an early divergence between brown and white adipocyte lineages. It also reveals the existence of a pool of committed brown preadipocytes within BAT that are recruited during cold exposure. BUG expression is increased in obese animals, suggesting that an early defect in brown preadipocyte differentiation could account for impaired BAT function in genetically obese rats.

Brown fat UCP1 is specifically expressed in uterine longitudinal smooth muscle cells.

Until now, uncoupling protein 1 (UCP1) was considered as unique to brown adipocytes. It supports a highly regulated uncoupling of oxidative phosphorylation that is associated with diet as well as with non-shivering thermogenesis. Here we report that UCP1 is not specific to brown adipocytes and can be expressed in longitudinal smooth muscle layers. In the uterus, this conclusion was drawn from different convergent data. A specific antibody against mouse UCP1 revealed, in mitochondrial fractions, a protein with the same molecular weight as brown fat UCP1. Sensitive and specific reverse transcriptase-polymerase chain reaction detected a mRNA whose sequence was totally homologous to that of brown fat UCP1 mRNA. Antibody against UCP1 as well as a UCP1 antisense probe specifically stained uterine longitudinal smooth muscles. UCP1 was also expressed in longitudinal smooth muscle of digestive and male reproductive tracts but was never expressed in other types of smooth muscle, including those of arterial vessels. In uterine tract, UCP1 content was increased after cold exposure or beta-adrenergic agonist treatment. It was also up-regulated during the postovulatory period after sexual cycle synchronization. Its content transiently increased during gestation and decreased markedly after birth. These regulations strongly argue about a role for UCP1 in thermogenesis as well as in relaxation of longitudinal smooth muscle layers.

Emergence during development of the white-adipocyte cell phenotype is independent of the brown-adipocyte cell phenotype.

In mammals, two types of adipose tissue are present, brown and white. They develop sequentially, as brown fat occurs during late gestation whereas white fat grows mainly after birth. However, both tissues have been shown to have great plasticity. Thus an apparent transformation of brown fat into white fat takes place during post-natal development. This observation raises questions about a possible conversion of brown into white adipocytes during development, although indirect data argue against this hypothesis. To investigate such questions in vivo, we generated two types of transgenic line. The first carried a transgene expressing Cre recombinase specifically in brown adipocytes under the control of the rat UCP1 promoter. The second corresponded to an inactive lacZ gene under the control of the human cytomegalovirus promoter. This dormant gene is inducible by Cre because it contains a Stop sequence between two loxP sequences, separating the promoter from the coding sequence. Adipose tissues of progeny derived by crossing independent lines established from both constructs were investigated. LacZ mRNA corresponding to the activated reporter gene was easily detected in brown fat and not typically in white fat, even by reverse transcriptase PCR experiments. These data represent the first direct experimental proof that, during normal development, most white adipocytes do not derive from brown adipocytes.

Fast decline of hematopoiesis and uncoupling protein 2 content in human liver after birth: location of the protein in Kupffer cells.

Hepatic hematopoiesis is prominent during fetal life and ceases around birth. In rodent liver, the decline of the hepatic hematopoiesis starts abruptly at birth being accompanied by a decrease of mitochondrial uncoupling protein 2 (UCP2) expression in monocytes/macrophages, whereas hepatocytes may express UCP2 only under pathologic situations. The goals of this study were to characterize hepatic hematopoiesis in humans around birth, and to identify cells expressing UCP2. Hematopoiesis was evaluated histologically in the liver of 22 newborns (mostly very premature neonates), who died between 45 min and 140 d after birth, and one fetus. UCP2 expression was characterized by Northern blots, immunoblotting, immunohistochemistry, and by in situ hybridization. The number of hematopoietic cells started to decrease rapidly at birth, irrespectively of the gestational age (23-40 wk) of neonates. A similar decline was observed for UCP2 expression, which was relatively high in fetal liver. UCP2 was detected only in myeloid cells (mainly in Kupffer cells), but not in hepatocytes, although sepsis or other pathologies occurred in the critically ill newborns. Kupffer cells represent the major site of mitochondrial UCP2 expression in the human newborn. UCP2 may be essential for the differentiation and function of macrophages and serve as a marker for these cells in human liver during the perinatal period.

High expression of uncoupling protein 2 in foetal liver.

To assess the putative role of mitochondrial uncoupling protein 2 (UCP2) during perinatal development, its expression was analysed in mice and rats. Expression was detected in a large range of foetal tissues. A unique developmental pattern of UCP2 expression was found in liver, where the level of UCP2 mRNA was about 30-fold higher in foetuses than in adults (mice data), and started to decline immediately after birth. Neither UCP1 nor UCP3 mRNA was expressed in foetal liver. As in adult liver, immunohistochemical analysis suggested exclusive localisation of UCP2 in the monocyte/macrophage cells. Our results indicate a role of UCP2 in haematopoietic system development.

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

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

Kupffer cells are a dominant site of uncoupling protein 2 expression in rat liver.

The mechanisms underlying thermogenesis in liver are not well understood. They may involve proteins related to the mitochondrial uncoupling protein (UCP1) of brown adipocytes. In this paper, it is demonstrated that UCP1 is not expressed in any liver cell type of rat while UCP2, a recently cloned homologue of UCP1, is expressed at a very high level in Kupffer cells but not in hepatocytes. This high level of expression of UCP2 in Kupffer cells allowed cross immunoreactivity with antibodies directed against UCP1. This cross reactivity was confirmed by the detection of UCP2 with anti-UCP1 antibody, in western blotting analysis of transfected yeasts expressing rat UCP2. The high level expression of UCP2 in Kupffer cells suggests a particular function of UCP2 in macrophages.

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.

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.

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.

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.

Dietary restriction amplifies the metabolic disturbances of very-low-density lipoproteins in cholesterol-fed rabbits.

The effect of dietary restriction (half of the control ration) on VLDL turnover was investigated in cholesterol-fed rabbits. Rabbits on standard, cholesterol and restricted cholesterol diets were injected with homologous 125I-labelled VLDL. Accompanying the amplification of hypercholesterolemia, additional disturbances of VLDL turnover were observed when cholesterol feeding was associated with dietary restriction. Cholesterol-fed rabbits with normal caloric ration exhibited delayed clearance of 125I-labelled apolipoprotein B component of VLDL compared to control rabbits. This was markedly accentuated in underfed rabbits, indicating further down-regulation of apolipoprotein B,E receptors in these animals. Furthermore, a reduced proportion of radiolabelled apolipoprotein B was converted from VLDL to intermediate-density lipoprotein (IDL) and LDL in both groups receiving cholesterol-rich diets. Thus, the combination of further impairment in plasma clearance of VLDL and the poor conversion into IDL and LDL could account for the massive increase of beta-VLDL in underfed animals on cholesterol-rich diets.

Beneficial effect of wheat germ on circulating lipoproteins and tissue lipids in rats fed a high fat, cholesterol-containing diet.

Adult male rats were fed for 7 wk either a low fat diet (3% fat) or a high fat-cholesterol diet (20% fat, 0.5% cholesterol) containing 7% wheat germ or not. Body weights and food intakes were unchanged by adding wheat germ to the control low fat or high fat diets. Adding wheat germ to the high fat-cholesterol diet significantly increased high density lipoprotein (HDL) cholesterol and the HDL-serum cholesterol ratio and lowered the very low density lipoprotein (VLDL) triglycerides. Thus the lipoprotein pattern was comparable to that obtained with the low fat diet, but the VLDL lipid composition remained altered. At the same time, triglyceride and cholesterol accumulation in the liver and the triglyceride content in skin were significantly decreased. When wheat germ was added to the low fat diet, cholesterol and triglycerides were not significantly modified. No adaptative change in lipase and colipase contents was observed in the pancreas of rats fed the wheat germ-supplemented diets, whereas the high fat diet increased these values. The results show a beneficial effect of wheat germ added to a high fat-cholesterol diet on the lipid status of rats; the implicated mechanisms are yet to be elucidated.

Effects of a low-energy diet associated with egg supplementation on plasma cholesterol and lipoprotein levels in normal subjects: results of a cross-over study.

1. The influence of a low-energy diet when associated with high-cholesterol intake was investigated in seventeen normal men during an 8-week cross-over study. The subjects were given a daily supplement of two whole eggs and two egg yolks (approximately 1 g cholesterol) either with their usual diet for 4 weeks or with a low-energy diet for 4 weeks. Each subject took part randomly in both dietary periods. 2. During the first part of the study, no changes occurred in the plasma cholesterol of the subjects with egg supplementation of the usual diet. 3. In contrast, the low-energy diet and associated weight loss markedly decreased tolerance to high-cholesterol intake resulting in increased plasma cholesterol. The mean rise was 22.7% but with wide individual variations in the response. This was almost completely normalized when the subjects returned to their usual energy intake indicating the involvement of weight reduction in the increase observed. 4. Changes in low-density-lipoprotein (LDL) cholesterol were parallel to those of total plasma cholesterol with an increase following the low-energy diet and normalization after body-weight recovery. 5. The opposite effect was shown with the low-energy diet after previous adaptation to the consumption of four eggs per day. This dietary regimen resulted in a decrease in plasma cholesterol although it was not significant. Moreover, the lipoprotein profile was improved with a decrease in very-low-density-lipoprotein (VLDL) cholesterol and an increase in high-density-lipoprotein (HDL) cholesterol. 6. High-cholesterol intake induced significant changes in lipoprotein composition whatever the energy ration. LDL and HDL were enriched in cholesterol esters as early as the 1st month of egg supplementation of the diet. 7. Taken together, the results emphasize the possible adverse effect of slimming diets when associated with high-cholesterol intake. The existence of 'high-responders' to these dietary conditions calls for special attention to be paid to the cholesterol content of restricted diets.

The effect of cholestyramine on apolipoproteins in cholesterol-fed rabbits.

The changes in apolipoproteins on cholestyramine therapy associated with cholesterol feeding were compared to those observed in cholesterol-fed rabbits. Only one molecular species of apo B, identified as apo B-100, was present whatever the dietary or pharmacological treatment indicating the hepatic origin of apo B-containing lipoproteins in these conditions. Cholestyramine has a clear preventive effect on the rise of apo B and apo E which appear following cholesterol feeding. These changes are highly correlated with those of the plasma cholesterol level. In addition, cholestyramine induced a 50% increase in apo AI compared with normal rabbits. These data strongly confirm the beneficial effects of cholestyramine in the treatment of hypercholesterolemia.