The hibernation cycle and related changes in the brown fat tissue of Citellus lateralis

The hibernating habits of Citellus lateralis under standard laboratory conditions are described. It has been shown that it is possible to use a biopsy technique to investigate the brown fat tissues without serious disturbance to the hibernation pattern. This technique was used to sample brown and white fat tissues in the same animal at various seasons of the year. Histological studies did not reveal any changes in morphology or lipid composition which could be related purely to the hibernating season, however, during the short periods of arousal a large proportion of the brown fat cells appeared to be partially depleted of neutral fat. At this time a large increase in Luxol fast blue staining was always observed, which may be due to unmasking of tissue phosphatides.     

Role of the season and of the nutritonnal state on the distribution of tissular fatty acids in the hibernating dormouse (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.     

Seasonal changes in critical enzymes of lipogenesis and triacylglycerol synthesis in the marmot (Marmota flaviventris)

Fatty acid metabolism and triacylglycerol synthesis are critical processes for the survival of hibernating mammals that undergo a prolonged fasting period. Fatty acid synthase, fatty-acid-CoA ligase, diacylglycerol acyltransferase, and monoacylglycerol acyltransferase activities were measured in liver and in white and brown adipose tissue, in order to determine whether enzymes of lipogenesis and triacylglycerol synthesis vary seasonally during hibernation in the yellow-bellied marmot (Marmota flaviventris). Compared with mid-winter hibernation, fatty acid synthase activity was higher in all three tissues during early spring when marmots emerged from hibernation and in mid-summer when they were feeding, consistent with the synthesis of fatty acids from the carbohydrate-rich summer diet. Fatty-acid-CoA ligase and diacylglycerol acyltransferase activities were highest in summer in white adipose tissue when triacylglycerol synthesis would be expected to be high; diacylglycerol acyltransferase activity was also high in brown adipose tissue during spring and summer. In liver, however, diacylglycerol acyltransferase specific activity was highest during hibernation, suggesting that triacylglycerol synthesis may be prominent in liver in winter. Monoacylglycerol acyltransferase activity, which may aid in the retention of essential fatty-acids, was 80-fold higher in liver than in white or brown adipose tissue, but did not vary seasonally. Its dependence on palmitoyl-CoA suggests that a divalent cation might play a role in enzyme activation. The high hepatic diacylglycerol acyltransferase activity during hibernation suggests that the metabolism of very low density lipoprotein may be important in the movement of adipose fatty acids to brown adipose tissue and muscle during the rewarming that occurs periodically during hibernation. These studies suggest that enzymes of lipid metabolism vary seasonally in the marmot, consistent with requirements of this hibernator for triacylglycerol synthesis and metabolism.Abbreviations BAT brown adipose tissue - DGAT diacylglycerol acyltransferase - FAS fatty acid synthase - K _m Michaelis constant - MGAT monoacylglycerol acyltransferase - RQ respiratory quotiant - VLDL very low density lipoprotein - WAT white adipose tissue     

White adipose tissue composition in the free-ranging fat-tailed dwarf lemur (Cheirogaleus medius; Primates), a tropical hibernator

In temperate species, hibernation is enhanced by high levels of essential fatty acids in white adipose tissue. Essential fatty acids cannot be synthesized by mammals, thus nutritional ecology should play a key role in physiological adaptations to hibernation. Tropical hibernators are exposed to different physiological demands than hibernators in temperate regions and are expected to be subject to different constraints. The aims of this study were to assess whether or not the tropical hibernator Cheirogaleus medius shows biochemical changes in its white adipose tissue before and during hibernation. A capture-recapture study was combined with feeding observations in western Madagascar. Before and after hibernation, 77 samples of white adipose tissue from 57 individuals of C. medius, as well as dietary items eaten during pre-hibernation fattening, were sampled and analyzed for their fatty acid composition. In contrast to temperate hibernators, C. medius exhibits extremely low essential fatty acid concentrations in its white adipose tissue (2.5%) prior to hibernation. The fatty acid pattern of the white adipose tissue did not change during pre-hibernation fattening and did not reflect dietary fatty acid composition. During hibernation, fat stores showed only minor but significant compositional changes. Because of its prevalence, the main fuel during hibernation was the monounsaturated oleic acid, which seemed to be preferentially synthesized from dietary carbohydrates. Results suggest that essential fatty acids do not represent an ecological limitation for hibernation in the tropics, at least not in the fat-tailed dwarf lemur.     

Hibernation activates glyoxylate cycle and gluconeogenesis in black bear brown adipose tissue

Biochemical studies on brown adipose tissue removed from a hibernating black bear and a non-hibernating control animal demonstrate that this tissue: (1) can carry out cyanide-insensitive fatty acid oxidation, and (2) possesses catalase activity and the enzyme activities unique to the glyoxylate cycle, isocitrate lyase and malate synthase. These activities are all markedly increased in brown fat obtained from the hibernating animal. Additionally, hibernation enhances the ability of the tissue to synthesize glycogen in the presence of a fatty acid substrate. The glyoxylate cycle enzymes and the ability to convert fatty acid carbons to glucose have been generally regarded as being absent from vertebrate cells and tissues.     

Dissociation of Large and Small Bovine Casein Micelles by Dialysis

The effects of dietary fat types on the thermogenic activity of brown adipocytes isolated from rat were examined. When beef tallow (saturated fatty acids + oleic) and safflower oil (linoleic) were the dietary fats, the respiration rates of brown adipocytes activated either by norepinephrine or an uncoupler of mitochondrial respiration (carbonylcyanide-m-chlorophenylhydrazone) were both slightly higher in rats fed the polyunsaturated fat. When the effects of safflower oil and evening primrose oil (linoleīc + γ-linolenic) were compared, the activated respiration rate tended to be higher in the latter. The respiratory responses to varying concentrations of norepinephrine were apparently dependent on the dietary fat types. Triglyceride stored in interscapular brown adipose tissue appeared to be modified by dietary fat types. Dietary fat also characteristically modified the fatty acid compositions of interscapular brown and epididymal white adipose tissues. Thus, the type of dietary fat caused an alteration to the thermogenesis of brown adipose tissue at the cellular level.     

The influence of hibernation patterns on the critical enzymes of lipogenesis and lipolysis in prairie dogs

White-tailed prairie dogs (Cynomys leucurus) are spontaneous hibernators that enter torpor each fall, whereas black-tailed prairie dogs (C. ludovicianus) hibernate facultatively only when food- or water-stressed during the winter. The body masses of both species greatly increase during the fall feeding period, with most of this gain in the form of depot fat. Body fat is utilized during winter fasting and/or hibernation. We measured the activities of fatty acid synthase (FAS), ATP-citrate lyase (ACL), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), and hormone-sensitive lipase (HSL) in the tissues of both C.leucurus (hibernating and euthermic) and C. ludovicianus (euthermic only) under controlled conditions. The activities of FAS, ACL, and G6PDH in the liver all decreased during hibernation. The activities of ME and G6PDH in white adipose tissue (WAT) were also reduced during hibernation. Euthermic C. leucurus and euthermic C. ludovicianus differed only in brown adipose (BAT) ACL and WAT G6PDH activities. No significant differences in HSL activities were found between these two species or between euthermic and hibernating animals. These results suggest that this seasonal body fat cycle is due, at least in part, to seasonal variations in the activities of FAS, ME, ACL, and G6PDH that affect the rate of fatty acid synthesis. This study also demonstrates that spontaneous hibernators do not have a greater capacity to synthesize fatty acids during the fall than facultative hibernators, as previously suggested.     

Effects of high fat diets on hibernation and adipose tissue in Turkish hamsters

Summary The effects of dietary fat saturation and fat content on hibernation and several properties of white and brown adipose tissue (WAT and BAT, respectively) were investigated in Turkish hamsters (Mesocricetus brandti). Male hamsters were housed in a long photoperiod (LD 16:8) at 23°C and fed one of three diets: (1) chow (6.5% fat per weight), (2) chow+13.5% vegetable oil (OIL, 20% fat per weight [largely unsaturated fat]) and (3) chow+13.5% vegetable shortening [SHORTENING, 20% fat per weight (largely saturated fat)]. Five weeks later body weights had stabilized and the animals were transferred to a short photoperiod (LD 8:16) at 3°C. At the peak of the hibernation season (17 weeks) the animals were sacrificed within 24 h of arousal. Chow-fed hamsters had the greatest percentage of animals hibernating and days found torpid compared with the two fat-fed groups, with no differences found between the latter two groups for these measures. There were no differences between hibernating (HIB) and nonhibernating (NON-HIB) hamsters across or within the diet groups for any of the BAT measures [uncoupling protein content, mitochondrial mass, lipoprotein lipase (LPL) activity, and in vivo lipogenesis], nor were there significant effects of the diet on these measures. CHOW-and OIL-fed HIB hamsters showed decreases in body weight. All HIB groups had decreases in each carcass component, several fat pad weights, testes weight, and food intake. No consistent differences in WAT LPL activity or in vivo lipogenesis were found between HIB and NON-HIB hamsters. Feeding saturated high fat diets inhibits hibernation in some species; however, in the present experiment, feeding of both saturated and unsaturated fat-laden diets inhibited hibernation to a similar degree.Abbreviations BAT brown adipose tissue - COA cytochrome-c oxidase - DS dorsal subcutaneus - DSWAT dorsal subcutaneous white adipose tissue - E epididymal - EWAT epididymal white adipose tissue - FFDM fat-free dry mass - HIB hibernating - I interscapular - IBAT intercapsular brown adipose tissue - IS inguinal subeutaneus - ISWAT inguinal subcutaneous white adipose tissue - LPL lipoprotein lipase - NON-HIB non-hibernating - R retroperitoneal - RWAT retroperitoneal white adipose tissue - SDS sodium dodecyl sul - UCP uncoupling protein - WAT white adipose tissue     

Dietary gamma-linolenic acid in the form of borage oil causes less body fat accumulation accompanying an increase in uncoupling protein 1 mRNA level in brown adipose tissue

Rats were fed a low-fat diet containing 2% safflower oil or 20% fat diets containing either safflower oil rich in linoleic acid, borage oil containing 25% gamma (gamma)-linolenic acid or enzymatically prepared gamma-linolenic acid enriched borage oil containing 47% gamma-linolenic acid for 14 days. Energy intake and growth of animals were the same among groups. A high safflower oil diet compared with a low-fat diet caused significant increases in both epididymal and perirenal white adipose tissue weights. However, high-fat diets rich in gamma-linolenic acid failed to do so. Compared with a low-fat diet, all the high-fat diets increased mRNA levels of uncoupling protein 1 and lipoprotein lipase in brown adipose tissue. The extents of the increase were greater with high-fat diets rich in gamma-linolenic acid. Various high-fat diets, compared with a low-fat diet, decreased glucose transporter 4 mRNA in white adipose tissue to the same levels. The amount and types of dietary fat did not affect the leptin mRNA level in epididymal white adipose tissue. However, a high safflower oil diet, but not high-fat diets rich in gamma-linolenic acid relative to a low-fat diet, increased perirenal white adipose tissue leptin mRNA levels. All high-fat diets, relative to a low-fat diet, increased the hepatic mitochondrial fatty acid oxidation rate and fatty acid oxidation enzyme mRNA abundances to the same levels. High-fat diets also increased these parameters in the peroxisomal pathway, and the increases were greater with high-fat diets rich in gamma-linolenic acid. The physiological activity in increasing brown adipose tissue gene expression and peroxisomal fatty acid oxidation was similar between the two types of borage oil differing in gamma-linolenic acid content. It was suggested that dietary gamma-linolenic acid attenuates body fat accumulation through the increase in gene expressions of uncoupling protein 1 in brown adipose tissue. An increase in hepatic peroxisomal fatty acid oxidation may also contribute to the physiological activity of gamma-linolenic acid in decreasing body fat mass.     

Experimental Coxsackie B-3 virus infection in Citellus lateralis

Coxsackie B-3 virus produces a mild infection which passes unnoticed in the non-hibernating Citellus lateralis. A severe infection occurs in the hibernating animal. The progress of infection in the hiberating animal is related to the number of arousal hours and quite independent of the number of infected days. Nevertheless, it has been shown that the low-temperature hibernation phase has a potentiating effect upon subsequent viral production. Infective virus has not been found in the inoculated brown fat pads during the hibernation phase but very high virus titers are recorded 48 hours after arousal. This results in an earlier viraemia and earlier and higher titers in other organs of the body. A similar effect is noted when virus is inoculated at the very beginning of the arousal phase. While most animals recover with subsequent antibody development, a few succumb. Antibody development is slow and related only to the number of arousal hours experienced. Particular note is made of the fact that an animal may successfully resume hibernation despite complete loss of the axillary brown fat pads.     

The optimal depot fat composition for hibernation by golden-mantled ground squirrels (Spermophilus lateralis)

Golden-mantled ground squirrels (Spermophilus lateralis) are herbivores that hibernate during winter. Although little is known about the nutritional/physiological constraints on hibernation, numerous studies have demonstrated that increasing the amount of linoleic acid (a polyunsaturated fatty acid) in the diet enhances hibernation. This is probably because high linoleic acid diets reduce the melting points of the depot fats produced for hibernation which makes them more metabolizable at low body temperatures. This suggests that a major limitation on hibernation may be obtaining enough linoleic acid in the diet for proper hibernation. In all previous studies, however, the amount of linoleic acid in the diets of free-ranging animals was either not considered, or the range of dietary linoleic acid contents in the experiments was less than that of natural diets. It is thus not known whether the amount of linoleic acid available to hibernators under natural conditions actually limits their torpor patterns. A series of laboratory feeding and hibernation experiments were conducted with S. lateralis and artificial diets with different linoleic acid contents that were either below or above the linoleic acid content of the natural diet. The results demonstrated that when dietary linoleic acid contents are either below or above natural levels, hibernation ability is greatly reduced. Hibernation ability was reduced when the squirrels were maintained on a high linoleic acid diet probably by the production of toxic lipid peroxides in brown adipose tissues. The results indicate that there is an optimal level of dietary linoleic acid for proper hibernation, and this is equal to that of the natural diet. The amount of linoleic acid available in the diet thus does not limit hibernation under normal natural conditions.Abbreviations BAT brown adipose tissue - bm body mass - FA fatty acid - PUFA polyunsaturated fatty acid - T _a ambient temperature - T _b body temperature - WAT white adipose tissue     

Hormonal changes and energy substrate availability during the hibernation cycle of Syrian hamsters

Animals have to adapt to seasonal variations in food resources and temperature. Hibernation is one of the most efficient means used by animals to cope with harsh winter conditions, wherein survival is achieved through a significant decrease in energy expenditure. The hibernation period is constituted by a succession of torpor bouts (hypometabolism and decrease in body temperature) and periodic arousals (eumetabolism and euthermia). Some species feed during these periodic arousals, and thus show different metabolic adaptations to fat-storing species that fast throughout the hibernation period. Our study aims to define these metabolic adaptations, including hormone (insulin, glucagon, leptin, adiponectin, GLP-1, GiP) and metabolite (glucose, free fatty acids, triglycerides, urea) profiles together with body composition adjustments. Syrian hamsters were exposed to varied photoperiod and temperature conditions mimicking different phases of the hibernation cycle: a long photoperiod at 20 °C (LP20 group), a short photoperiod at 20 °C (SP20 group), and a short photoperiod at 8 °C (SP8). SP8 animals were sampled either at the beginning of a torpor bout (Torpor group) or at the beginning of a periodic arousal (Arousal group). We show that fat store mobilization in hamsters during torpor bouts is associated with decreased circulating levels of glucagon, insulin, leptin, and an increase in adiponectin. Refeeding during periodic arousals results in a decreased free fatty acid plasma concentration and an increase in glycemia and plasma incretin concentrations. Reduced incretin and increased adiponectin levels are therefore in accordance with the changes in nutrient availability and feeding behavior observed during the hibernation cycle of Syrian hamsters.     

Effect of high-fat diet on the expression of proteins in muscle, adipose tissues, and liver of C57BL/6 mice

In the present study, the effect of a high fat diet on the expression of proteins in insulin target tissues was analyzed using a proteomic approach. Gastrocnemius muscle, white and brown adipose tissue, and liver were taken from C57BL/6 mice either fed on a high-fat or a chow diet. Expression levels of approximately 10 000 polypeptides for all the four tissues were assessed by two-dimensional gel electrophoresis (2-DE). Computer-assisted image analysis allowed the detection of 50 significantly (p < 0.05) differentially expressed proteins between obese and lean mice. Interestingly, more than half of these proteins were detected in the brown adipose tissue. The differentially expressed proteins were identified by tandem mass spectrometry. Several stress and redox proteins were modulated in response to the high-fat diet. A key glycolytic enzyme was found to be downregulated in adipose tissues and muscle, suggesting that at elevated plasma fatty acid concentrations, fatty acids compete with glucose as an oxidative fuel source. Furthermore, in brown adipose tissue there were significant changes in mitochondrial enzymes involved in the Krebs tricarboxylic acid (TCA) cycle and in the respiratory chain in response to the high-fat diet. The brown adipose tissue is an energy-dissipating tissue. Our data suggest that the high-fat diet treated mice were increasing energy expenditure to defend against weight gain.     

Fine structural modifications of liver, pancreas and brown adipose tissue mitochondria from hibernating, arousing and euthermic dormice

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia-hibernation-arousal cycle.     

ELOVL3 is an important component for early onset of lipid recruitment in brown adipose tissue

During the recruitment process of brown adipose tissue, the mRNA level of the fatty acyl chain elongase Elovl3 is elevated more than 200-fold in cold-stressed mice. We have obtained Elovl3-ablated mice and report here that, although cold-acclimated Elovl3-ablated mice experienced an increased heat loss due to impaired skin barrier, they were unable to hyperrecruit their brown adipose tissue. Instead, they used muscle shivering in order to maintain body temperature. Lack of Elovl3 resulted in a transient decrease in the capacity to elongate saturated fatty acyl-CoAs into very long chain fatty acids, concomitantly with the occurrence of reduced levels of arachidic acid (C20:0) and behenic acid (C22:0) in brown adipose tissue during the initial cold stress. This effect on very long chain fatty acid synthesis could be illustrated as a decrease in the condensation activity of the elongation enzyme. In addition, warm-acclimated Elovl3-ablated mice showed diminished ability to accumulate fat and reduced metabolic capacity within the brown fat cells. This points to ELOVL3 as an important regulator of endogenous synthesis of saturated very long chain fatty acids and triglyceride formation in brown adipose tissue during the early phase of the tissue recruitment.     

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

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 degrees 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.     

Dietary fats and body lipid composition in relation to hibernation in free-ranging echidnas

Laboratory studies have shown that high levels of dietary unsaturated fatty acids prolong torpor and lower body temperatures in hibernating herbivorous rodents, which may in turn improve winter survival. The importance of nutritional ecology in relation to hibernation in insectivorous hibernators is unknown. We therefore studied fatty acid composition of dietary insects and the depot fat of echidnas Tachyglossus aculeatus (Monotremata) during the pre-hibernation season and compared depot fat fatty acid composition before and after hibernation. Echidna depot fat fatty acid composition during the pre-hibernation season was almost identical to that of the most abundant prey species, the ant Iridomyrmex sp. Oleic acid (C18:1) was by far the most common fatty acid in both Iridomyrmex sp. (60%) and echidna depot fat (62%). After about 5 months of hibernation and an 18% loss of body mass, echidna fatty acid composition had changed significantly. The percentage of the monounsaturated oleic acid (C18:1) and palmitoleic acid (C16:1) had declined, whereas that of the saturated fatty acids (C12:0, C16:0, C18:0) and the polyunsaturated linoleic acid (C18:2) had increased. Our study suggests that, unlike herbivorous rodent hibernators, echidnas rely to a large extent on monounsaturated fatty acids as fuel for hibernation, reflecting the most common fatty acid in their food. Moreover, it appears that the high concentration of monounsaturated fatty acids compensates for the moderate availability of polyunsaturates and enables them to hibernate at low body temperatures.     

Recruitment of brown fat and conversion of white into brown adipocytes: Strategies to fight the metabolic complications of obesity?

The role of white and brown adipose tissues in energy metabolism is well established. However, the existence of brown fat in adult humans was until very recently a matter of debate, and the molecular mechanisms underlying brown adipocyte development remained largely unknown. In 2009, several studies brought direct evidence for functional brown adipose tissue in adults. New factors involved in brown fat cell differentiation have been identified. Moreover, work on the origin of fat cells took an unexpected path with the recognition of different populations of brown fat cell precursors according to the anatomical location of the fat depots: a precursor common to skeletal muscle cells and brown adipocytes from brown fat depots, and a progenitor cell common to white adipocytes and brown adipocytes that appear in certain conditions in white fat depots. There is also mounting evidence that mature white adipocytes, including human fat cells, can be converted into brown fat-like adipocytes, and that the typical fatty acid storage phenotype of white adipocyte can be altered towards a fat utilization phenotype. These data open up new opportunities for the development of drugs for obesity and its metabolic and cardiovascular complications.