UCP1-independent thermogenesis in white adipose tissue of cold-acclimated Ucp1-/- mice

Apart from UCP1-based nonshivering thermogenesis in brown adipocytes, the identity of thermogenic mechanisms that can be activated to reduce a positive energy balance is largely unknown. To identify potentially useful mechanisms, we have analyzed physiological and molecular mechanisms that enable mice, genetically deficient in UCP1 and sensitive to acute exposure to the cold at 4 degrees C, to adapt to long term exposure at 4 degrees C. UCP1-deficient mice that can adapt to the cold have increased oxygen consumption and show increased oxidation of both fat and glucose as indicated from serum metabolite levels and liver glycogen content. Enhanced energy metabolism in inguinal fat was also indicated by increased oxygen consumption and fat oxidation in tissue suspensions and increased AMP kinase activity in dissected tissues. Analysis of gene expression in skeletal muscle showed surprisingly little change between cold-adapted Ucp1+/+ and Ucp1-/- mice, whereas in inguinal fat a robust induction occurred for type 2 deiodinase, sarcoendoplasmic reticulum Ca2+-ATPase, mitochondrial glycerol 3-phosphate dehydrogenase, PGC1alpha, CoxII, and mitochondrial DNA content. Western blot analysis showed an induction of total phospholamban and its phosphorylated form in inguinal fat and other white fat depots, but no induction was apparent in muscle. We conclude that alternative thermogenic mechanisms, based in part upon the enhanced capacity for ion and substrate cycling associated with brown adipocytes in white fat depots, are induced in UCP1-deficient mice by gradual cold adaptation.     

Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.     

White adipose tissue contributes to UCP1-independent thermogenesis

Beta3-adrenergic receptors (AR) are nearly exclusively expressed in brown and white adipose tissues, and chronic activation of these receptors by selective agonists has profound anti-diabetes and anti-obesity effects. This study examined metabolic responses to acute and chronic beta3-AR activation in wild-type C57Bl/6 mice and congenic mice lacking functional uncoupling protein (UCP)1, the molecular effector of brown adipose tissue (BAT) thermogenesis. Acute activation of beta3-AR doubled metabolic rate in wild-type mice and sharply elevated body temperature and BAT blood flow, as determined by laser Doppler flowmetry. In contrast, beta3-AR activation did not increase BAT blood flow in mice lacking UCP1 (UCP1 KO). Nonetheless, beta3-AR activation significantly increased metabolic rate and body temperature in UCP1 KO mice, demonstrating the presence of UCP1-independent thermogenesis. Daily treatment with the beta3-AR agonist CL-316243 (CL) for 6 days increased basal and CL-induced thermogenesis compared with naive mice. This expansion of basal and CL-induced metabolic rate did not require UCP1 expression. Chronic CL treatment of UCP1 KO mice increased basal and CL-stimulated metabolic rate of epididymal white adipose tissue (EWAT) fourfold but did not alter BAT thermogenesis. After chronic CL treatment, CL-stimulated thermogenesis of EWAT equaled that of interscapular BAT per tissue mass. The elevation of EWAT metabolism was accompanied by mitochondrial biogenesis and the induction of genes involved in lipid oxidation. These observations indicate that chronic beta3-AR activation induces metabolic adaptation in WAT that contributes to beta3-AR-mediated thermogenesis. This adaptation involves lipid oxidation in situ and does not require UCP1 expression.     

Role of IL-6 in Exercise Training- and Cold-Induced UCP1 Expression in Subcutaneous White Adipose Tissue

Expression of brown adipose tissue (BAT) associated proteins like uncoupling protein 1 (UCP1) in inguinal WAT (iWAT) has been suggested to alter iWAT metabolism. The aim of this study was to investigate the role of interleukin-6 (IL-6) in exercise training and cold exposure-induced iWAT UCP1 expression. The effect of daily intraperitoneal injections of IL-6 (3 ng/g) in C57BL/6 mice for 7 days on iWAT UCP1 expression was examined. In addition, the expression of UCP1 in iWAT was determined in response to 3 days of cold exposure (4°C) and 5 weeks of exercise training in wild type (WT) and whole body IL-6 knockout (KO) mice. Repeated injections of IL-6 in C57BL/6 mice increased UCP1 mRNA but not UCP1 protein content in iWAT. Cold exposure increased iWAT UCP1 mRNA content similarly in IL-6 KO and WT mice, while exercise training increased iWAT UCP1 mRNA in WT mice but not in IL-6 KO mice. Additionally, a cold exposure-induced increase in iWAT UCP1 protein content was blunted in IL-6 KO mice, while UCP1 protein content in iWAT was lower in both untrained and exercise trained IL-6 KO mice than in WT mice. In conclusion, repeated daily increases in plasma IL-6 can increase iWAT UCP1 mRNA content and IL-6 is required for an exercise training-induced increase in iWAT UCP1 mRNA content. In addition IL-6 is required for a full induction of UCP1 protein expression in response to cold exposure and influences the UCP1 protein content iWAT of both untrained and exercise trained animals.     

Convertible adipose tissue in mice

Summary Ability to express uncoupling protein (UCP) and establish UCP-dependent thermogenesis was analyzed in anatomical areas of mice that are generally considered to be white adipose tissue: mesenterial, perimetral, epididymal, inguinal, and superficial layer of interscapular white adipose tissue. The mice were acclimatized for 1 week to 4° C; the following week they were exposed to cold stress (1 h at-20° C, 2–3 times daily). In such conditions in inguinal adipose tissue, slot-blot analysis detected significant amount of UCP mRNA and lipoprotein lipase mRNA. Immuno-electron-microscopic localization of UCP showed that developed mitochondria of cold-stressed inguinal adipocytes contained UCP in the same amount as uncoupled (UC)-mitochondria of brown adipocytes. Morphological and morphometrical analysis showed that such inguinal adipose tissue appeared as brown adipose tissue. Since in control mice, inguinal adipose tissue was UCP-negative and tissue appeared as white adipose tissue, the duration of this white-to-brown adipose tissue conversion was analyzed. Mice, cold stressed for 1 week, were rewarmed at 28° C and their inguinal adipose tissue was analyzed in comparison with interscapular brown adipose tissue and epididymal white adipose tissue for another 37 days. During that time inguinal adipocytes ceased expressing UCP mRNA; UC-mitochondria in inguinal adipocytes were destroyed and replaced with common, C-mitochondria; and UCP was undetectable immunohistochemically. Adipocytes accumulated lipids, and the tissue morphologically once again resembled white adipose tissue. Described changes showed that besides typical brown and white adipose tissue in mice, there existed a third type of adipose tissue described as convertible adipose tissue.     

UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background

The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.

Methodology/Principal Findings

Here we report that cyclooxygenase (COX) activity and prostaglandin E₂ (PGE₂) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE₂ receptor antagonists implicated EP₄ as a main PGE₂ receptor, and injection of the stable PGE₂ analog (EP_3/4 agonist) 16,16 dm PGE₂ induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.

Conclusions/Significance

Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.     

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca²⁺‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca²⁺ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.     

Central leptin regulates the UCP1 and ob genes in brown and white adipose tissue via different beta-adrenoceptor subtypes

The three known subtypes of beta-adrenoreceptors (beta(1)-AR, beta(2)-AR, and beta(3)-AR) are differentially expressed in brown and white adipose tissue and mediate peripheral responses to central modulation of sympathetic outflow by leptin. To assess the relative roles of the beta-AR subtypes in mediating leptin's effects on adipocyte gene expression, mice with a targeted disruption of the beta(3)-adrenoreceptor gene (beta(3)-AR KO) were treated with vehicle or the beta(1)/beta(2)-AR selective antagonist, propranolol (20 microgram/g body weight/day) prior to intracerebroventricular (ICV) injections of leptin (0.1 microgram/g body weight/day). Leptin produced a 3-fold increase in UCP1 mRNA in brown adipose tissue of wild type (FVB/NJ) and beta(3)-AR KO mice. The response was unaltered by propranolol in wild type mice, but was completely blocked by this antagonist in beta(3)-AR KO mice. In contrast, ICV leptin had no effect on leptin mRNA in either epididymal or retroperitoneal white adipose tissue (WAT) from beta(3)-AR KOs. Moreover, propranolol did not block the ability of exogenous leptin to reduce leptin mRNA in either WAT depot site of wild type mice. These results demonstrate that the beta(3)-AR is required for leptin-mediated regulation of ob mRNA expression in WAT, but is interchangeable with the beta(1)/beta(2)-ARs in mediating leptin's effect on UCP1 mRNA expression in brown adipose tissue.     

Acylation-stimulating protein (ASP)/complement C3adesArg deficiency results in increased energy expenditure in mice

Acylation-stimulating protein (ASP) acts as a paracrine signal to increase triglyceride synthesis in adipocytes. In mice, C3 (the precursor to ASP) knock-out (KO) results in ASP deficiency and leads to reduced body fat and leptin levels yet they are hyperphagic. In the present study, we investigated the mechanism for this energy repartitioning. Compared with wild-type (WT) mice, male and female C3(-/-) ASP-deficient mice had elevated oxygen consumption (VO2) in both the active (dark) and resting (light) phases of the diurnal cycle: +8.9% males (p < 0.05) +9.4% females (p < 0.05). Increased physical activity (movement) was observed during the dark phase in female but not in male KO animals. Female WT mice moved 16.9 +/- 2.4 m whereas KO mice moved 30.1 +/- 5.4 m, over 12 h, +78.4%, p < 0.05). In contrast, there was no difference in physical activity in male mice, but a repartitioning of dietary fat following intragastric fat administration was noted. This was reflected by increased fatty acid oxidation in liver and muscle in KO mice, with increased UCP2 (inguinal fat) and UCP3 (muscle) mRNA expression (p = 0.005 and 0.036, respectively). Fatty acid uptake into brown adipose tissue (BAT) and white adipose tissue (WAT) was reduced as reflected by a decrease in the fatty acid incorporation into lipids (BAT -68%, WAT -29%. The decrease of FA incorporation was normalized by intraperitoneal administration of ASP at the time of oral fat administration. These results suggest that ASP deficiency results in energy repartitioning through different mechanisms in male and female mice.     

CL316,243 and cold stress induce heterogeneous expression of UCP1 mRNA and protein in rodent brown adipocytes.

Uncoupling protein 1 (UCP1), the mammalian thermogenic mitochondrial protein, is found only in brown adipocytes, but its expression by immunohistochemistry is not homogeneous. Here we present evidence that the non-homogeneous pattern of immunostaining for UCP1 (referred to as the "Harlequin phenomenon") is particularly evident after acute and chronic cold (4C) stimulus and after administration of a specific beta(3)-adrenoceptor agonist (CL316,243). Accordingly, mRNA in situ expression confirmed the UCP1 non-homogeneous pattern of gene activation under conditions of adrenergic stimulus. Furthermore, morphometric analysis of immunogold-stained thin sections showed that UCP1-gold particle density was different among neighboring brown adipocytes with mitochondria of the same size and density. When the adrenergic stimulus was reduced in warm-acclimated animals (28C), UCP1 protein and mRNA expression was reduced and consequently the Harlequin phenomenon was barely visible. These data suggest the existence of an alternative and controlled functional recruitment of brown adipocytes in acute adrenergically stressed animals, possibly to avoid heat and metabolic damage in thermogenically active cells. Of note, the heat shock protein heme oxygenase 1 (HO1) is heterogeneously expressed in adrenergically stimulated brown adipose tissue and, specifically, cells expressing strong immunoreactivity for UCP1 also strongly express HO1.     

ERRγ enhances UCP1 expression and fatty acid oxidation in brown adipocytes

Objective:

Estrogen‐related receptors (ERRs) are important regulators of energy metabolism. Here we investigated the hypothesis that ERRγ impacts on differentiation and function of brown adipocytes.

Design and Methods:

We characterize the expression of ERRγ in adipose tissues and cell models and investigate the effects of modulating ERR? activity on UCP1 gene expression and metabolic features of brown and white adipocytes.

Results:

ERRγ was preferentially expressed in brown compared to white fat depots, and ERRγ was induced during cold‐induced browning of subcutaneous white adipose tissue and brown adipogenesis. Overexpression of ERRγ positively regulated uncoupling protein 1 (UCP1) expression levels during brown adipogenesis. This ERRγ‐induced augmentation of UCP1 expression was independent of the presence of peroxisome proliferator‐activated receptor coactivator‐1 (PGC‐1α) but was associated with increased rates of fatty acid oxidation in adrenergically stimulated cells. ERR? did not influence mitochondrial biogenesis, and its reduced expression in white adipocytes could not explain their low expression level of UCP1.

Conclusions:

Through its augmenting effect on expression of UCP1, ERRγ may physiologically be involved in increasing the potential for energy expenditure in brown adipocytes, a function that is becoming of therapeutic interest.

     

Zic1 mRNA is transiently upregulated in subcutaneous fat of acutely cold-exposed mice

In the mammalian adipose organ cold exposure not only activates typical brown adipose tissue, but also induces browning, that is the formation of thermogenic multilocular adipocytes in white, or predominantly white, adipose depots such as subcutaneous fat. Unlike typical brown adipocytes, newly formed thermogenic adipocytes have been reported not to express the gene zinc finger of the cerebellum 1 (Zic1). Here, a time course approach enabled us to document a significant increase in Zic1 messenger RNA in inguinal subcutaneous fat from acutely (24 hr) cold-exposed mice, which was paralleled by an increase in multilocular and paucilocular uncoupling protein 1-positive adipocytes and in parenchymal noradrenergic innervation. This transient, depot-specific molecular signature was associated not to Zic1 promoter demethylation, but to chromatin remodeling through an H3K9me3 histone modification. These findings challenge the notion that Zic1 is exclusively expressed by typical brown adipocytes and suggest its involvement in brown adipocyte precursor differentiation and/or white-to-brown adipocyte transdifferentiation.     

Impaired response of UCP family to cold exposure in diabetic (db/db) mice

Impaired activity of the uncoupling protein (UCP) family has been proposed to promote obesity development. The present study examined differences in UCP responses to cold exposure between leptin-resistance obese (db/db) mice and their lean (C57Ksj) littermates. Basal UCP1 and UCP3 mRNA expression in brown adipose tissue was lower in obese mice compared with lean mice, but UCP2 expression in white adipose tissue (WAT) was higher. Basal skeletal muscle UCP3 did not change remarkably. The UCP family mRNAs, which were upregulated 12 and 24 h after cold exposure (4 degrees C), were returned to prior levels 12 h after rewarming exposure (21 degrees C) in lean mice. The accelerating effects of cold exposure on the UCP family were impaired in db/db obese mice. Together with these changes, WAT lipoprotein lipase mRNA was downregulated, and the concentration of serum free fatty acid was increased in response to cold exposure in the lean mice but not in db/db obese littermates. The impaired function of the UCP family and diminished lipolysis in response to cold exposure indicate that the reduced lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.     

Multilocular fat cells in WAT of CL-316243-treated rats derive directly from white adipocytes

Multilocular,mitochondria-rich adipocytes appear in white adipose tissue (WAT) ofrats treated with the 3-adrenoceptor agonist, CL-316243 (CL).Objectives were to determine whether these multilocular adipocytesderived from cells that already existed in the WAT or fromproliferation of precursor cells and whether new mitochondria containedin them were typical brown adipocyte mitochondria. Use of5-bromodeoxyuridine to identify cells that had undergone mitosis duringthe CL treatment showed that most multilocular cells derived from cellsalready present in the WAT. Morphological techniques showed that atleast a subpopulation of unilocular adipocytes underwent conversion tomultilocular mitochondria-rich adipocytes. A small proportion ofmultilocular adipocytes (~8%) was positive for UCP1 byimmunohistochemistry. Biochemical techniques showed that mitochondrialprotein recovered from WAT increased 10-fold and protein isolated frombrown adipose tissue (BAT) doubled in CL-treated rats. Stained gelsshowed a different protein composition of new mitochondria isolatedfrom WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a muchlower concentration than in BAT mitochondria, and UCP3, at a higherconcentration than that in BAT mitochondria. We hypothesize thatmultilocular adipocytes present at 7 days of CL treatment have twoorigins. First, most come from convertible unilocular adipocytes thatbecome multilocular and make many mitochondria that contain UCP3.Second, some come from a cell that gives rise to more typical brownadipocytes that express UCP1.

     

FABP3 and brown adipocyte-characteristic mitochondrial fatty acid oxidation enzymes are induced in beige cells in a different pathway from UCP1

Cold exposure and β₃-adrenergic receptor agonist (CL316,243) treatment induce the production of beige cells, which express brown adipocytes(BA)-specific UCP1 protein, in white adipose tissue (WAT). It remains unclear whether the beige cells, which have different gene expression patterns from BA, express BA-characteristic fatty acid oxidation (FAO) proteins. Here we found that 5 day cold exposure and CL316,243 treatment of WAT, but not CL316,243 treatment of primary adipocytes of C57BL/6J mice, increased mRNA levels of BA-characteristic FAO proteins. These results suggest that BA-characteristic FAO proteins are induced in beige cells in a different pathway from UCP1.     

Browning attenuates murine white adipose tissue expansion during postnatal development

During postnatal development of mice distinct white adipose tissue depots display a transient appearance of brown-like adipocytes. These brite (brown in white) adipocytes share characteristics with classical brown adipocytes including a multilocular appearance and the expression of the thermogenic protein uncoupling protein 1. In this study, we compared two inbred mouse strains 129S6sv/ev and C57BL6/N known for their different propensity to diet-induced obesity. We observed transient browning in retroperitoneal and inguinal adipose tissue depots of these two strains. From postnatal day 10 to 20 the increase in the abundance of multilocular adipocytes and uncoupling protein 1 expression was higher in 129S6sv/ev than in C57BL6/N pups. The parallel increase in the mass of the two fat depots was attenuated during this browning period. Conversely, epididymal white and interscapular brown adipose tissue displayed a steady increase in mass during the first 30 days of life. In this period, 129S6sv/ev mice developed a significantly higher total body fat mass than C57BL6/N. Thus, while on a local depot level a high number of brite cells is associated with the attenuation of adipose tissue expansion the strain comparison reveals no support for a systemic impact on energy balance. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.