Expression Profiling and Structural Characterization of MicroRNAs in Adipose Tissues of Hibernating Ground Squirrels

Micro RNAs（mi RNAs） are small non-coding RNAs that are important in regulating metabolic stress. In this study, we determined the expression and structural characteristics of 20 mi RNAs in brown（BAT） and white adipose tissue（WAT） during torpor in thirteen-lined ground squirrels. Using a modified stem-loop technique, we found that during torpor, expression of six mi RNAs including let-7a, let-7b, mi R-107, mi R-150, mi R-222 and mi R-31 was significantly downregulated in WAT（P 〈 0.05）, which was 16%–54% of euthermic non-torpid control squirrels,whereas expression of three mi RNAs including mi R-143, mi R-200 a and mi R-519 d was found to be upregulated by 1.32–2.34-fold. Similarly, expression of more mi RNAs was downregulated in BAT during torpor. We detected reduced expression of 6 mi RNAs including mi R-103 a, mi R-107, mi R-125 b, mi R-21, mi R-221 and mi R-31（48%–70% of control）, while only expression of mi R-138 was significantly upregulated（2.91 ± 0.8-fold of the control, P 〈 0.05）. Interestingly,mi RNAs found to be downregulated in WAT during torpor were similar to those dysregulated in obese humans for increased adipogenesis, whereas mi RNAs with altered expression in BAT during torpor were linked to mitochondrial b-oxidation. mi RPath target prediction analysis showed that mi RNAs downregulated in both WAT and BAT were associated with the regulation of mitogen-activated protein kinase（MAPK） signaling, while the mi RNAs upregulated in WAT were linked to transforming growth factor b（TGFb） signaling. Compared to mouse sequences, no unique nucleotide substitutions within the stem-loop region were discovered for the associated pre-mi RNAs for the mi RNAs used in this study, suggesting no structure-influenced changes in pre-mi RNA processing efficiency in the squirrel. As well, the expression of mi RNA processingenzyme Dicer remained unchanged in both tissues during torpor. Overall, our findings suggest that changes of mi RNA expression in adipose tissues may be linked     

Antioxidant defense in hibernation: cloning and expression of peroxiredoxins from hibernating ground squirrels, Spermophilus tridecemlineatus

Mammalian hibernation is characterized by prolonged torpor bouts interspersed by brief arousal periods. Adequate antioxidant defenses are needed both to sustain cell viability over weeks of deep torpor and to defend against high rates of oxyradical formation associated with massive oxygen-based thermogenesis during arousal. The present study shows that up-regulation of peroxiredoxins contributes to antioxidant defense during torpor in thirteen-lined ground squirrels, Spermophilus tridecemlineatus. Expression levels of three isozymes of the 2-Cys peroxiredoxin (Prdx) family were quantified by Western blotting, the results showing 4.0- and 12.9-fold increases in Prdx1 protein in brown adipose tissue (BAT) and heart, respectively, during hibernation compared with euthermia. Comparable increases in Prdx2 were 2.4- and 3.7-fold whereas Prdx3 rose by 3.1-fold in heart of torpid animals. Total 2-Cys peroxiredoxin enzymatic activity also rose during hibernation by 1.5-fold in heart and 3.5-fold in BAT. Furthermore, RT-PCR showed that prdx2 mRNA levels increased by 1.7- and 3.7-fold in BAT and heart, respectively, during hibernation. A partial nucleotide sequence of prdx2 from ground squirrels was obtained by PCR amplification, the deduced amino acid sequence showing 96-97% identity with Prdx2 from other mammals. Some unique amino acid substitutions were identified that might contribute to stabilizing Prdx2 conformation at the near 0 degrees C body temperatures during torpor.     

Role of hormone-sensitive lipase in beta-adrenergic remodeling of white adipose tissue

Free fatty acids (FFA) are important extracellular and intracellular signaling molecules and are thought to be involved in beta-adrenergic-induced remodeling of adipose tissue, which involves a transient inflammatory response followed by mitochondrial biogenesis and increased oxidative capacity. This work examined the role of hormone-sensitive lipase (HSL), a key enzyme of acylglycerol metabolism, in white adipose tissue (WAT) remodeling using genetic inactivation or pharmacological inhibition. Acute treatment with the beta(3)-adrenergic agonist CL-316,243 (CL) induced expression of inflammatory markers and caused extravasation of myeloid cells in WAT of wild-type (WT) mice. HSL-knockout (KO) mice had elevated inflammatory gene expression in the absence of stimulation, and acute injection of CL did not further recruit myeloid cells, nor did it further elevate inflammatory gene expression. Acute pharmacological inhibition of HSL with BAY 59-9435 (BAY) had no effect on inflammatory gene expression in WAT or in cultured 3T3-L1 adipocytes. However, BAY prevented induction of inflammatory cytokines by beta-adrenergic stimulation in WAT in vivo and in cultured 3T3-L1 adipocytes. Chronic CL treatment stimulated mitochondrial biogenesis, expanded oxidative capacity, and increased lipid droplet fragmentation in WT mice, and these effects were significantly impaired in HSL-KO mice. In contrast to HSL-KO mice, mice with defective signaling of Toll-like receptor 4, a putative FFA receptor, showed normal beta-adrenergic-induced remodeling of adipose tissue. Overall, results reveal the importance of HSL activity in WAT metabolic plasticity and inflammation.     

Translocator Protein 18 kDa (TSPO) Is Regulated in White and Brown Adipose Tissue by Obesity

Translocator protein 18 kDa (TSPO) is an outer-mitochondrial membrane transporter which has many functions including participation in the mitochondrial permeability transition pore, regulation of reactive oxygen species (ROS), production of cellular energy, and is the rate-limiting step in the uptake of cholesterol. TSPO expression is dysregulated during disease pathologies involving changes in tissue energy demands such as cancer, and is up-regulated in activated macrophages during the inflammatory response. Obesity is associated with decreased energy expenditure, mitochondrial dysfunction, and chronic low-grade inflammation which collectively contribute to the development of the Metabolic Syndrome. Therefore, we hypothesized that dysregulation of TSPO in adipose tissue may be a feature of disease pathology in obesity. Radioligand binding studies revealed a significant reduction in TSPO ligand binding sites in mitochondrial extracts from both white (WAT) and brown adipose tissue (BAT) in mouse models of obesity (diet-induced and genetic) compared to control animals. We also confirmed a reduction in TSPO gene expression in whole tissue extracts from WAT and BAT. Immunohistochemistry in WAT confirmed TSPO expression in adipocytes but also revealed high-levels of TSPO expression in WAT macrophages in obese animals. No changes in TSPO expression were observed in WAT or BAT after a 17 hour fast or 4 hour cold exposure. Treatment of mice with the TSPO ligand PK11195 resulted in regulation of metabolic genes in WAT. Together, these results suggest a potential role for TSPO in mediating adipose tissue homeostasis.     

A polyphenolic extract from green tea leaves activates fat browning in high-fat-diet-induced obese mice

Fat browning has emerged as an attractive target for the treatment of obesity and related metabolic disorders. Its activation leads to increased energy expenditure and reduced adiposity, thus contributing to a better energy homeostasis. Green tea extracts (GTEs) were shown to attenuate obesity and low-grade inflammation and to induce the lipolytic pathway in the white adipose tissue (WAT) of mice fed a high-fat diet. The aim of the present study was to determine whether the antiobesity effect of an extract from green tea leaves was associated with the activation of browning in the WAT and/or the inhibition of whitening in the brown adipose tissue (BAT) in HF-diet induced obese mice. Mice were fed a control diet or an HF diet supplemented with or without 0.5% polyphenolic GTE for 8 weeks. GTE supplementation significantly reduced HF-induced adiposity (WAT and BAT) and HF-induced inflammation in WAT. Histological analysis revealed that GTE reduced the adipocyte size in the WAT and the lipid droplet size in the BAT. Markers of browning were induced in the WAT upon GTE treatment, whereas markers of HF-induced whitening were reduced in the BAT. These results suggest that browning activation in the WAT and whitening reduction in the BAT by the GTE could participate to the improvement of metabolic and inflammatory disorders mediated by GTE upon HF diet. Our study emphasizes the importance of using GTE as a nutritional tool to activate browning and to decrease fat storage in all adipose tissues, which attenuate obesity.     

Conjugated linoleic acid reduces adiposity and increases markers of browning and inflammation in white adipose tissue of mice

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.     

The impact of cold acclimation and hibernation on antioxidant defenses in the ground squirrel (Spermophilus citellus): An update

Any alteration in oxidative metabolism is coupled with a corresponding response by an antioxidant defense (AD) in appropriate subcellular compartments. Seasonal hibernators pass through circannual metabolic adaptations that allow them to either maintain euthermy (cold acclimation) or enter winter torpor with body temperature falling to low values. The present study aimed to investigate the corresponding pattern of AD enzyme protein expressions associated with these strategies in the main tissues involved in whole animal energy homeostasis: brown and white adipose tissues (BAT and WAT, respectively), liver, and skeletal muscle. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 ± 1 °C) and then divided into two groups: (1) animals fell into torpor (hibernating group) and (2) animals stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We examined the effects of cold acclimation and hibernation on the tissue-dependent protein expression of four enzymes which catalyze the two-step detoxification of superoxide to water: superoxide dismutase 1 and 2 (SOD 1 and 2), catalase (CAT), and glutathione peroxidase (GSH-Px). The results showed that hibernation induced an increase of AD enzyme protein expressions in BAT and skeletal muscle. However, AD enzyme contents in liver were largely unaffected during torpor. Under these conditions, different WAT depots responded by elevating the amounts of specific enzymes, as follows: SOD 1 in retroperitoneal WAT, GSH-Px in gonadal WAT, and CAT in subcutaneous WAT. Similar perturbations of AD enzymes contents were seen in all tissues during cold acclimation, often in a time-dependent manner. It can be concluded that BAT and muscle AD capacity undergo the most dramatic changes during both cold acclimation and hibernation, while liver is relatively unaffected by either condition. Additionally, this study provides a basis for further metabolic study that will illuminate the causes of these tissue-specific AD responses, particularly the novel finding of distinct responses by different WAT depots in hibernators.     

Comparing lipid remodeling of brown adipose tissue,white adipose tissue,and liver after one-week high fat diet intervention with quantitative Raman microscopy

Brown adipose tissue (BAT) consists of highly metabolically active adipocytes that catabolize nutrients to produce heat. Playing an active role in triacylglycerol (TAG) clearance, research has shown that dietary fatty acids can modulate the TAG chemistry deposition in BAT after weeks-long dietary intervention, similar to what has been shown in white adipose tissue (WAT). Our objective was to compare the influence of sustained, nonchronic dietary intervention (a 1-week interval) on WAT and interscapular BAT lipid metabolism and deposition in situ. We use quantitative, label-free chemical microscopy to show that 1 week of high fat diet (HFD) intervention results in dramatically larger lipid droplet (LD) growth in BAT (and liver) compared to LD growth in inguinal WAT (IWAT). Moreover, BAT showed lipid remodeling as increased unsaturated TAGs in LDs, resembling the dietary lipid composition, while WAT (and liver) did not show lipid remodeling on this time scale. Concurrently, expression of genes involved in lipid metabolism, particularly desaturases, was reduced in BAT and liver from HFD-fed mice after 1 week. Our data show that BAT lipid chemistry remodels exceptionally fast to dietary lipid intervention compared WAT, which further points towards a role in TAG clearance.     

瘦素在育肥后达乌尔黄鼠能量平衡及体温调节中的作用

育肥完成后到冬眠前的阶段被认为是贮脂类冬眠动物从体温常态到冬眠之间的过渡阶段。为研究此阶段瘦素对能量平衡和体温调节的作用,将完成育肥的达乌尔黄鼠随机分成3组,分别在侧脑室植入微渗透泵,持续灌注瘦素（0.5μg/day）、瘦素拮抗剂（0.5μg/day瘦素+5μg/day瘦素拮抗剂）以及人工脑脊液（对照组）,为期4周。为了检测瘦素对动物入眠的影响,我们在药物处理最后一周将动物移入低温（5 ^oC±1^oC）、恒黑条件下诱导蛰眠。药物处理过程中测定动物体重、能量摄入、代谢率和体温,药物处理结束后测定身体脂肪重量、褐色脂肪组织中解偶联蛋白1（UCP1）含量以及血清中与能量平衡相关的激素水平。结果发现：育肥后达乌尔黄鼠能量摄入、体重和每日体温自发降低。低温条件下,对照组中50%个体自发进入冬眠状态。瘦素处理和瘦素拮抗剂处理对能量摄入和体重变化没有显著影响。瘦素处理对入眠率没有影响,瘦素拮抗剂处理减少蛰眠表达。瘦素拮抗剂组血清中T₄水平高于瘦素处理组。育肥后期瘦素以及瘦素拮抗剂处理对脂肪重量、代谢率以及UCP1含量没有显著影响。结果表明,瘦素对育肥结束后达乌尔黄鼠的冬眠表达具有一定调节作用。     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells简

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue (WAT) or brown adipose tissue (BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brown-like adipocytes were discovered in WAT. These brown-like adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expression pattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation (adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Leucine deprivation stimulates fat loss via increasing CRH expression in the hypothalamus and activating the sympathetic nervous system

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis.     

Distinction of white,beige and brown adipocytes derived from mesenchymal stem cells

Adipose tissue is a major metabolic organ, and it has been traditionally classified as either white adipose tissue(WAT) or brown adipose tissue(BAT). WAT and BAT are characterized by different anatomical locations, morphological structures, functions, and regulations. WAT and BAT are both involved in energy balance. WAT is mainly involved in the storage and mobilization of energy in the form of triglycerides, whereas BAT specializes in dissipating energy as heat during cold- or diet-induced thermogenesis. Recently, brownlike adipocytes were discovered in WAT. These brownlike adipocytes that appear in WAT are called beige or brite adipocytes. Interestingly, these beige/brite cells resemble white fat cells in the basal state, but they respond to thermogenic stimuli with increased levels of thermogenic genes and increased respiration rates. In addition, beige/brite cells have a gene expressionpattern distinct from that of either white or brown fat cells. The current epidemic of obesity has increased the interest in studying adipocyte formation(adipogenesis), especially in beige/brite cells. This review summarizes the developmental process of adipose tissues that originate from the mesenchymal stem cells and the features of these three different types of adipocytes.     

Brown and beige fat: From molecules to physiology

The recent re-discovery of brown adipose tissue (BAT) and even more recent discovery of the browning of white adipose tissue (WAT) in humans have generated intense scientific interest in the role of adipose tissue as potential target against obesity and its metabolic complications. The purpose of this review is to: i) critically evaluate the current evidence on the physiological significance of BAT and the browning of WAT in metabolic function in humans and ii) discuss factors that have been reported to regulate BAT and/or the browning of WAT in humans. The current literature supports that BAT and the browning of WAT constitute promising emerging targets for interventions aiming to prevent and/or treat of obesity and its metabolic complications. Further research is needed to better understand the physiological significance of BAT and browning of WAT in health and disease along with the factors modulating their metabolic function in humans.