Low protein diet changes the energetic balance and sympathetic activity in brown adipose tissue of growing rats

Objective: The aim of this study was to assess the effects of protein restriction in growing rats. Methods: Rats (approximate weight, 100 g) were maintained with low-protein (LP; 6%) or normo-proteic (control; 17%) diets, and at the end of the 15th day, hormonal and biochemistry parameters and energetic balance were evaluated. Data were analyzed using Student`s t test (with statistical significance set at P <= .05). Results: LP animals were hyperphagic and showed increased energetic gain (24%) and energy expenditure (EE) compared with controls. The increase in EE was followed by increased sympathetic activity in brown adipose tissue, evidenced by increased norepinephrine turnover, suggesting increased thermogenesis. In spite of hyperphagia, protein ingestion in LP animals was lower than that of controls (P < 0.01). The LP diet impaired body growth and caused deep alterations in body chemical composition, with an increase in carcass lipid content (64%) and reductions of protein and water. In LP animals, postprandial glycemia was unchanged, and insulinemia was lower than in controls (P <= .01). Reduction in fasting glycemia without changes in insulinemia also was detected (P < .01), suggesting increased insulin sensitivity. The LP diet caused a 100% increase in serum leptin (P < .01). Conclusions: Protein restriction led to an increase in EE, with probable activation of thermogenesis in brown adipose tissue, evidenced by an increase in catecholamines levels. Despite the higher EE, energetic gain and lipids increased. The high level of leptin associated with hyperphagia led to the supposition that these animals are leptin resistant, and the increase in insulin sensitivity, suggested by the relation between insulin and glycemia in fasting and fed animals, might contribute to lipid accumulation. (C) 2009 Elsevier Inc. All rights reserved.

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-(14)C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-(14)C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria.

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.

The Growth of Brown Adipose Tissue in Cold-acclimatized Rats after Depletion of Mast Cell Histamine by Compound 48/80

Cold acclimatization (4-5°C) is accompanied by 2-3 fold increase of brown adipose tissue (BAT). This rapid growth of interscapular BAT was studied after histamine depletion. In control rats maintained at room temperature (28 ± 2°C) the BAT histamine content was 23.4 ± 5.9 (mean ± SD) µg/g of tissue and cold acclimatization (5±1°C) produced a significant increase of BAT weight, but reduced the histamine content to 8.4 ± 1.9 µg/g. The total weight of BAT after 20 days of acclimatization was unaffected by depletion of histamine due to compound 48/80. The low level of histamine in BAT of cold acclimatized rats could be due to a fast rate of amine utilization; alternatively an altered synthesis or storage process may occur during acclimatization.

Cell autonomous lipin 1 function is essential for development and maintenance of white and brown adipose tissue.

Through analysis of mice with spatially and temporally restricted inactivation of Lpin1, we characterized its cell autonomous function in both white (WAT) and brown (BAT) adipocyte development and maintenance. We observed that the lipin 1 inactivation in adipocytes of aP2(Cre/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice resulted in lipodystrophy and the presence of adipocytes with multilocular lipid droplets. We further showed that time-specific loss of lipin 1 in mature adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice led to their replacement by newly formed Lpin1-positive adipocytes, thus establishing a role for lipin 1 in mature adipocyte maintenance. Importantly, we observed that the presence of newly formed Lpin1-positive adipocytes in aP2(Cre-ERT2/+)/Lp(fEx2)(-)(3/fEx2)(-)(3) mice protected these animals against WAT inflammation and hepatic steatosis induced by a high-fat diet. Loss of lipin 1 also affected BAT development and function, as revealed by histological changes, defects in the expression of peroxisome proliferator-activated receptor alpha (PPARα), PGC-1α, and UCP1, and functionally by altered cold sensitivity. Finally, our data indicate that phosphatidic acid, which accumulates in WAT of animals lacking lipin 1 function, specifically inhibits differentiation of preadipocytes. Together, these observations firmly demonstrate a cell autonomous role of lipin 1 in WAT and BAT biology and indicate its potential as a therapeutical target for the treatment of obesity.

Stimulation by leptin of 3H GDP binding to brown adipose tissue of fasted but not fed rats.

OBJECTIVES: To assess the effects of intracerebroventricular (i.c.v.) leptin administration on rats fed ad libitum or fasted on 3H GDP binding to brown adipose tissue (BAT). SUBJECTS: Groups of 5-6 ten-week-old male Wistar rats. EXPERIMENTAL DESIGN: An i.c.v. cannula was inserted and unilateral denervation of interscapular brown adipose tissue (BAT) was performed 5 d before each study. Thereafter, leptin was infused i.c.v. during 72 h while rats were fed ad libitum or fasted. Vehicle-infused, pair-fed or fasted rats were used as controls. MEASUREMENTS: 3H GDP binding to innervated and denervated BAT mitochondria. RESULTS: 3H GDP binding to innervated or denervated BAT of rats fed ab libitum compared to vehicle-infused, pair-fed rats was not increased by i.c.v. leptin. 3H GDP binding was lower in fasted than in fed rats, and the difference was larger in innervated than denervated BAT. I.c.v. leptin increased 3H GDP binding by 30% in innervated, and by 51% in denervated BAT (P < 0.05) in fasted rats. CONCLUSIONS: I.c.v. leptin does not increase 3H GDP binding to BAT of rats fed ad libitum compared to pair-fed (food-restricted) rats. In contrast, i.c.v. leptin produces a mild stimulation of 3H GDP binding to BAT of fasted rats. This effect is not mediated by the sympathetic nervous system, because it is observed in both innervated and denervated BAT. These results are compatible with the concept that, in fasting rats, the decrease in leptin secretion contributes to the reduction in 3H GDP binding to BAT mitochondria.

Characterisation of Functional Brown Adipose Tissue In Adult Humans

The white adipose tissue mainly serves the purpose of energy storage, while brown adipose tissue (BAT) has the capacity to generate heat under cold conditions in mammals and in human infants. BAT is controlled by the central nervous system, and BAT function is accompanied by increased energy expenditure. However, it was not previously certain whether adult humans also have functional BAT. The aim of this doctoral work was to identify functional BAT in adult humans and to characterise its glucose uptake and blood flow under cold and insulin stimulation conditions in lean and in obese humans, by using positron emission tomography. Further, the impact of weight loss on BAT glucose uptake was assessed. Cerebral glucose uptake was also studied in relation to BAT function and cold exposure. The results showed that healthy adult humans have functional BAT, as assessed by the intense cold-induced glucose uptake and by biopsies. BAT was also found to be a highly insulinsensitive tissue in lean humans, but the effects of insulin and cold exposure were attenuated in obese humans, although the glucose uptake capacity of cold-activated BAT might be increased by weight loss. Blood flow in the BAT of lean humans was associated with whole-body energy expenditure. The presence of cold-activated BAT was related to lower body mass index and higher insulin sensitivity. Finally, BAT activation was linked to the activity of the cerebellum, the thalamus and certain neocortical regions. The cold-induced cerebral glucose uptake was also lower in obese than in lean adult humans.

Gut microbiota modulate the metabolism of brown adipose tissue in mice

A two by two experimental study has been designed to determine the effect of gut microbiota on energy metabolism in mouse models. The metabolic phenotype of germ-free (GF, n = 20) and conventional (n = 20) mice was characterized using a NMR spectroscopy-based metabolic profiling approach, with a focus on sexual dimorphism (20 males, 20 females) and energy metabolism in urine, plasma, liver, and brown adipose tissue (BAT). Physiological data of age-matched GF and conventional mice showed that male animals had a higher weight than females in both groups. In addition, conventional males had a significantly higher total body fat content (TBFC) compared to conventional females, whereas this sexual dimorphism disappeared in GF animals (i.e., male GF mice had a TBFC similar to those of conventional and GF females). Profiling of BAT hydrophilic extracts revealed that sexual dimorphism in normal mice was absent in GF animals, which also displayed lower BAT lactate levels and higher levels of (D)-3-hydroxybutyrate in liver, plasma, and BAT, together with lower circulating levels of VLDL. These data indicate that the gut microbiota modulate the lipid metabolism in BAT, as the absence of gut microbiota stimulated both hepatic and BAT lipolysis while inhibiting lipogenesis. We also demonstrated that (1)H NMR metabolic profiles of BAT were excellent predictors of BW and TBFC, indicating the potential of BAT to fight against obesity.

Orexin restores aging-related brown adipose tissue dysfunction in male mice

The aging process causes an increase in percent body fat, but the mechanism remains unclear. In the present study we examined the impact of aging on brown adipose tissue (BAT) thermogenic activity as potential cause for the increase in adiposity. We show that aging is associated with interscapular BAT morphologic abnormalities and thermogenic dysfunction. In vitro experiments revealed that brown adipocyte differentiation is defective in aged mice. Interscapular brown tissue in aged mice is progressively populated by adipocytes bearing white morphologic characteristics. Aged mice fail to mobilize intracellular fuel reserves from brown adipocytes and exhibit deficiency in homeothermy. Our results suggest a role for orexin (OX) signaling in the regulation of thermogenesis during aging. Brown fat dysfunction and age-related assimilation of fat mass were accelerated in mice in which OX-producing neurons were ablated. Conversely, OX injections in old mice increased multilocular morphology, increased core body temperature, improved cold tolerance, and reduced adiposity. These results argue that BAT can be targeted for interventions to reverse age-associated increase in fat mass.

Increased Glyceride-Glycerol Synthesis in Liver and Brown Adipose Tissue of Rat: In-Vivo Contribution of Glycolysis and Glyceroneogenesis

We have previously shown that a high-protein, carbohydrate-free diet can decrease the production of glycerol-3-phosphate (G3P) from glucose and increase glyceroneogenesis in both brown (BAT) and epididymal (EAT) adipose tissue. Here, we utilized an in-vivo approach to examine the hypothesis that there is reciprocal regulation in the G3P synthesis from glucose (via glycolysis) and glyceroneogenesis in BAT, EAT and liver of fasted rats and cafeteria diet-fed rats. Glyceroneogenesis played a prominent role in the generation of G3P in the liver (similar to 70 %) as well as in BAT and EAT (similar to 80 %) in controls rats. The cafeteria diet induced an increase in the total glyceride-glycerol synthesis and G3P synthesis from glucose and a decrease in glyceroneogenesis in BAT; this diet did not affect either the total glyceride-glycerol synthesis or G3P generation from glyceroneogenesis or glycolysis in the liver or EAT. Fasting induced an increase in total glyceride-glycerol synthesis and glyceroneogenesis and a decrease in G3P synthesis from glucose in the liver but did not affect either the total glyceride-glycerol synthesis or G3P synthesis from glyceroneogenesis in BAT and EAT, despite a reduction in glycolysis in these tissues. These data demonstrate that reciprocal changes in the G3P generation from glucose and from glyceroneogenesis in the rat liver and BAT occur only when the synthesis of glycerides-glycerol is increased. Further, our data suggest that this increase may be essential for the systemic recycling of fatty acids by the liver from fasted rats and for the maintenance of the thermogenic capacity of BAT from cafeteria diet-fed rats.

PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2

Festuccia WT, Blanchard PG, Oliveira TB, Magdalon J, Paschoal VA, Richard D, Deshaies Y. PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2. Am J Physiol Regul Integr Comp Physiol 303: R1277-R1285, 2012. First published October 24, 2012; doi:10.1152/ajpregu.00299.2012.-Here, we investigated whether pharmacological PPAR gamma activation modulates key early events in brown adipose tissue (BAT) recruitment induced by acute cold exposure with the aim of unraveling the interrelationships between sympathetic and PPAR gamma signaling. Sprague-Dawley rats treated or not with the PPAR gamma ligand rosiglitazone (15 mg.kg(-1).day(-1), 7 days) were kept at 23 degrees C or exposed to cold (5 degrees C) for 24 h and evaluated for BAT gene expression, sympathetic activity, thyroid status, and adrenergic signaling. Rosiglitazone did not affect the reduction in body weight gain and the increase in feed efficiency, VO2, and BAT sympathetic activity induced by 24-h cold exposure. Rosiglitazone strongly attenuated the increase in serum total and free T4 and T3 levels and BAT iodothyronine deiodinase type 2 (D2) and PGC-1 alpha mRNA levels and potentiated the reduction in BAT thyroid hormone receptor (THR) beta mRNA levels induced by cold. Administration of T3 to rosiglitazone-treated rats exacerbated the cold-induced increase in energy expenditure but did not restore a proper activation of D2 and PGC-1 alpha, nor further increased uncoupling protein 1 expression. Regarding adrenergic signaling, rosiglitazone did not affect the changes in BAT cAMP content and PKA activity induced by cold. Rosiglitazone alone or in combination with cold increased CREB binding to DNA, but it markedly reduced the expression of one of its major coactivators, CREB binding protein. In conclusion, pharmacological PPAR gamma activation impairs short-term cold elicitation of BAT adrenergic and thyroid signaling, which may result in abnormal tissue recruitment and thermogenic activity.

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca2+-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis. (C) 2011 Elsevier Ltd. All rights reserved.

Oleoylethanolamide enhances β-adrenergic-mediated thermogenesis and white-to-brown adipocyte phenotype in epididymal white adipose tissue in rat.

β-adrenergic receptor activation promotes brown adipose tissue (BAT) β-oxidation and thermogenesis by burning fatty acids during uncoupling respiration. Oleoylethanolamide (OEA) can inhibit feeding and stimulate lipolysis by activating peroxisome proliferator-activating receptor-α (PPARα) in white adipose tissue (WAT). Here we explore whether PPARα activation potentiates the effect of β3-adrenergic stimulation on energy balance mediated by the respective agonists OEA and CL316243. The effect of this pharmacological association on feeding, thermogenesis, β-oxidation, and lipid and cholesterol metabolism in epididymal (e)WAT was monitored. CL316243 (1 mg/kg) and OEA (5 mg/kg) co-administration over 6 days enhanced the reduction of both food intake and body weight gain, increased the energy expenditure and reduced the respiratory quotient (VCO2/VO2). This negative energy balance agreed with decreased fat mass and increased BAT weight and temperature, as well as with lowered plasma levels of triglycerides, cholesterol, nonessential fatty acids (NEFAs), and the adipokines leptin and TNF-α. Regarding eWAT, CL316243 and OEA treatment elevated levels of the thermogenic factors PPARα and UCP1, reduced p38-MAPK phosphorylation, and promoted brown-like features in the white adipocytes: the mitochondrial (Cox4i1, Cox4i2) and BAT (Fgf21, Prdm16) genes were overexpressed in eWAT. The enhancement of the fatty-acid β-oxidation factors Cpt1b and Acox1 in eWAT was accompanied by an upregulation of de novo lipogenesis and reduced expression of the unsaturated-fatty-acid-synthesis enzyme gene, Scd1. We propose that the combination of β-adrenergic and PPARα receptor agonists promotes therapeutic adipocyte remodelling in eWAT, and therefore has a potential clinical utility in the treatment of obesity.

Thermogenic and metabolic consequences of thyroid hormone treatment in brown and white adipose tissue

Male rats were treated with triiodothyronine in the drinking water for 12 days. In vitro rates of isoprenaline stimulated lipolysis were significantly greater in brown but not white adipose tissue. Rates of [14C]glucose incorporation into triacylglycerols were significantly reduced in BAT (brown adipose tissue) and WAT (white adipose tissue) under basal and isoprenaline stimulated conditions. In a second experiment, hyperthyroid animals showed impaired weight gain, despite increased food intake during 19 days' treatment. Energy expenditure on days 5 and 12, and BAT core temperature differences (TBAT - TCORE) on day 19, were significantly greater than in control animals. Epididymal white fat pad weight was reduced and interscapular brown fat pad weight increased by triiodothyronine treatment.