The ABA/LANCL1/2 Hormone/Receptor System Controls Adipocyte Browning and Energy Expenditure.

The abscisic acid (ABA)/LANC-like protein 1/2 (LANCL1/2) hormone/receptor system regulates glucose uptake and oxidation, mitochondrial respiration, and proton gradient dissipation in myocytes. Oral ABA increases glucose uptake and the transcription of adipocyte browning-related genes in rodent brown adipose tissue (BAT). The aim of this study was to investigate the role of the ABA/LANCL system in human white and brown adipocyte thermogenesis. Immortalized human white and brown preadipocytes, virally infected to overexpress or silence LANCL1/2, were differentiated in vitro with or without ABA, and transcriptional and metabolic targets critical for thermogenesis were explored. The overexpression of LANCL1/2 increases, and their combined silencing conversely reduces mitochondrial number, basal, and maximal respiration rates; proton gradient dissipation; and the transcription of uncoupling genes and of receptors for thyroid and adrenergic hormones, both in brown and in white adipocytes. The transcriptional enhancement of receptors for browning hormones also occurs in BAT from ABA-treated mice, lacking LANCL2 but overexpressing LANCL1. The signaling pathway downstream of the ABA/LANCL system includes AMPK, PGC-1α, Sirt1, and the transcription factor ERRα. The ABA/LANCL system controls human brown and "beige" adipocyte thermogenesis, acting upstream of a key signaling pathway regulating energy metabolism, mitochondrial function, and thermogenesis.

The autocrine role of FGF21 in cultured adipocytes.

Exposure to cold alters glucose and lipid metabolism of white and brown adipose tissue via activation of ß-adrenergic receptor (ADRB). Fibroblast growth factor 21 (FGF21) has been shown to be locally released from adipose tissue upon activation of ADRBs and FGF21 increases glucose uptake in adipocytes. Therefore, FGF21 may play an autocrine role in inducing glucose uptake after ß-adrenergic stimulation. To determine the putative autocrine role of FGF21, we stimulated three different types of adipocytes in vitro with Isoprenaline (Iso), an ADRB agonist, in the presence or absence of the FGF receptor (FGFR) inhibitor PD 173074. The three cell lines represent white (3T3-L1), beige (ME3) and brown (WT-1) adipocyte phenotypes, respectively. All three cells systems expressed ß-klotho (KLB) and FGFR1 after differentiation and treatment with recombinant FGF21 increased glucose uptake in 3T3-L1 and WT-1 adipocytes, while no significant effect was observed in ME3. Oppositely, all three cell lines responded to Iso treatment and an increase in glucose uptake and lipolysis were observed. Interestingly, in response to the Iso treatment only the WT-1 adipocytes showed an increase in FGF21 in the medium. This was consistent with the observation that PD 173074 decreased Iso-induced glucose uptake in the WT-1 adipocytes. This suggests that FGF21 plays an autocrine role and increases glucose uptake after ß-adrenergic stimulation of cultured brown WT-1 adipocytes.

Restricting glycolysis impairs brown adipocyte glucose and oxygen consumption.

During thermogenic activation, brown adipocytes take up large amounts of glucose. In addition, cold stimulation leads to an upregulation of glycolytic enzymes. Here we have investigated the importance of glycolysis for brown adipocyte glucose consumption and thermogenesis. Using siRNA-mediated knockdown in mature adipocytes, we explored the effect of glucose transporters and glycolytic enzymes on brown adipocyte functions such as consumption of glucose and oxygen. Basal oxygen consumption in brown adipocytes was equally dependent on glucose and fatty acid oxidation, whereas isoproterenol (ISO)-stimulated respiration was fueled mainly by fatty acids, with a significant contribution from glucose oxidation. Knockdown of glucose transporters in brown adipocytes not only impaired ISO-stimulated glycolytic flux but also oxygen consumption. Diminishing glycolytic flux by knockdown of the first and final enzyme of glycolysis, i.e., hexokinase 2 (HK2) and pyruvate kinase M (PKM), respectively, decreased glucose uptake and ISO-stimulated oxygen consumption. HK2 knockdown had a more severe effect, which, in contrast to PKM knockdown, could not be rescued by supplementation with pyruvate. Hence, brown adipocytes rely on glucose consumption and glycolytic flux to achieve maximum thermogenic output, with glycolysis likely supporting thermogenesis not only by pyruvate formation but also by supplying intermediates for efferent metabolic pathways.

Metformin targets brown adipose tissue in vivo and reduces oxygen consumption in vitro.

AIMS: To test the hypothesis that brown adipose tissue (BAT) is a metformin target tissue by investigating in vivo uptake of [11 C]-metformin tracer in mice and studying in vitro effects of metformin on cultured human brown adipocytes. MATERIALS AND METHODS: Tissue-specific uptake of metformin was assessed in mice by PET/CT imaging after injection of [11 C]-metformin. Human brown adipose tissue was obtained from elective neck surgery and metformin transporter expression levels in human and murine BAT were determined by qPCR. Oxygen consumption in metformin-treated human brown adipocyte cell models was assessed by Seahorse XF technology. RESULTS: Injected [11 C]-metformin showed avid uptake in the murine interscapular BAT depot. Metformin exposure in BAT was similar to hepatic exposure. Non-specific inhibition of the organic cation transporter (OCT) protein by cimetidine administration eliminated BAT exposure to metformin, demonstrating OCT-mediated uptake. Gene expression profiles of OCTs in BAT revealed ample OCT3 expression in both human and mouse BAT. Incubation of a human brown adipocyte cell models with metformin reduced cellular oxygen consumption in a dose-dependent manner. CONCLUSION: These results support BAT as a putative metformin target.

p53 regulates expression of uncoupling protein 1 through binding and repression of PPARγ coactivator-1α.

The tumor suppressor p53 (TRP53 in mice) is known for its involvement in carcinogenesis, but work during recent years has underscored the importance of p53 in the regulation of whole body metabolism. A general notion is that p53 is necessary for efficient oxidative metabolism. The importance of UCP1-dependent uncoupled respiration and increased oxidation of glucose and fatty acids in brown or brown-like adipocytes, termed brite or beige, in relation to energy balance and homeostasis has been highlighted recently. UCP1-dependent uncoupled respiration in classic interscapular brown adipose tissue is central to cold-induced thermogenesis, whereas brite/beige adipocytes are of special importance in relation to diet-induced thermogenesis, where the importance of UCP1 is only clearly manifested in mice kept at thermoneutrality. We challenged wild-type and TRP53-deficient mice by high-fat feeding under thermoneutral conditions. Interestingly, mice lacking TRP53 gained less weight compared with their wild-type counterparts. This was related to an increased expression of Ucp1 and other PPARGC1a and PPARGC1b target genes but not Ppargc1a or Ppargc1b in inguinal white adipose tissue of mice lacking TRP53. We show that TRP53, independently of its ability to bind DNA, inhibits the activity of PPARGC1a and PPARGC1b. Collectively, our data show that TRP53 has the ability to regulate the thermogenic capacity of adipocytes through modulation of PPARGC1 activity.

Global gene expression profiling of brown to white adipose tissue transformation in sheep reveals novel transcriptional components linked to adipose remodeling.

BACKGROUND: Large mammals are capable of thermoregulation shortly after birth due to the presence of brown adipose tissue (BAT). The majority of BAT disappears after birth and is replaced by white adipose tissue (WAT). RESULTS: We analyzed the postnatal transformation of adipose in sheep with a time course study of the perirenal adipose depot. We observed changes in tissue morphology, gene expression and metabolism within the first two weeks of postnatal life consistent with the expected transition from BAT to WAT. The transformation was characterized by massively decreased mitochondrial abundance and down-regulation of gene expression related to mitochondrial function and oxidative phosphorylation. Global gene expression profiling demonstrated that the time points grouped into three phases: a brown adipose phase, a transition phase and a white adipose phase. Between the brown adipose and the transition phase 170 genes were differentially expressed, and 717 genes were differentially expressed between the transition and the white adipose phase. Thirty-eight genes were shared among the two sets of differentially expressed genes. We identified a number of regulated transcription factors, including NR1H3, MYC, KLF4, ESR1, RELA and BCL6, which were linked to the overall changes in gene expression during the adipose tissue remodeling. Finally, the perirenal adipose tissue expressed both brown and brite/beige adipocyte marker genes at birth, the expression of which changed substantially over time. CONCLUSIONS: Using global gene expression profiling of the postnatal BAT to WAT transformation in sheep, we provide novel insight into adipose tissue plasticity in a large mammal, including identification of novel transcriptional components linked to adipose tissue remodeling. Moreover, our data set provides a useful resource for further studies in adipose tissue plasticity.

Transcriptome profiling of brown adipose tissue during cold exposure reveals extensive regulation of glucose metabolism.

We applied digital gene expression profiling to determine the transcriptome of brown and white adipose tissues (BAT and WAT, respectively) during cold exposure. Male C57BL/6J mice were exposed to cold for 2 or 4 days. A notable induction of genes related to glucose uptake, glycolysis, glycogen metabolism, and the pentose phosphate pathway was observed in BAT from cold-exposed animals. In addition, glycerol-3-phosphate dehydrogenase 1 expression was induced in BAT from cold-challenged mice, suggesting increased synthesis of glycerol from glucose. Similarly, expression of lactate dehydrogenases was induced by cold in BAT. Pyruvate dehydrogenase kinase 2 (Pdk2) and Pdk4 were expressed at significantly higher levels in BAT than in WAT, and Pdk2 was induced in BAT by cold. Of notice, only a subset of the changes detected in BAT was observed in WAT. Based on changes in gene expression during cold exposure, we propose a model for the intermediary glucose metabolism in activated BAT: 1) fluxes through glycolysis and the pentose phosphate pathway are induced, the latter providing reducing equivalents for de novo fatty acid synthesis; 2) glycerol synthesis from glucose is increased, facilitating triacylglycerol synthesis/fatty acid re-esterification; 3) glycogen turnover and lactate production are increased; and 4) entry of glucose carbon into the tricarboxylic acid cycle is restricted by PDK2 and PDK4. In summary, our results demonstrate extensive and diverse gene expression changes related to glucose handling in activated BAT.

PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation.

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation.

Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes.

BACKGROUND: Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. RESULTS: The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. CONCLUSIONS: UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA.

Full activation of thermogenesis in brown adipocytes requires Basigin action.

Exploring mechanisms responsible for brown adipose tissue's (BAT) high metabolic activity is crucial to exploit its energy-dissipating ability for therapeutic purposes. Basigin (Bsg), a multifunctional highly glycosylated transmembrane protein, was recently proposed as one of the 98 critical markers allowing to distinguish 'white' and 'brown' adipocytes, yet its function in thermogenic brown adipocytes is unknown. Here, we report that Bsg is negatively associated with obesity in mice. By contrast, Bsg expression increased in the mature adipocyte fraction of BAT upon cold acclimation. Additionally, Bsg levels were highly induced during brown adipocyte maturation in vitro and were further increased upon ß-adrenergic stimulation in a HIF-1α-dependent manner. siRNA-mediated Bsg gene silencing in cultured brown adipocytes did not impact adipogenesis nor mitochondrial function. However, a significant decrease in mitochondrial respiration, lipolysis and Ucp1 transcription was observed in adipocytes lacking Bsg, when activated by norepinephrine. Furthermore, using gas chromatography/mass spectrometry-time-of-flight analysis to assess the composition of cellular metabolites, we demonstrate that brown adipocytes lacking Bsg have lower levels of intracellular lactate and acetoacetate. Bsg was additionally required to regulate intracellular AcAc and tricarboxylic acid cycle intermediate levels in NE-stimulated adipocytes. Our study highlights the critical role of Bsg in active brown adipocytes, possibly by controlling cellular metabolism.

An adipose lncRAP2-Igf2bp2 complex enhances adipogenesis and energy expenditure by stabilizing target mRNAs.

lncRAP2 is a conserved cytoplasmic lncRNA enriched in adipose tissue and required for adipogenesis. Using purification and in vivo interactome analyses, we show that lncRAP2 forms complexes with proteins that stabilize mRNAs and modulate translation, among them Igf2bp2. Surveying transcriptome-wide Igf2bp2 client mRNAs in white adipocytes reveals selective binding to mRNAs encoding adipogenic regulators and energy expenditure effectors, including adiponectin. These same target proteins are downregulated when either Igf2bp2 or lncRAP2 is downregulated, hindering adipocyte lipolysis. Proteomics and ribosome profiling show this occurs predominantly through mRNA accumulation, as lncRAP2-Igf2bp2 complex binding does not impact translation efficiency. Phenome-wide association studies reveal specific associations of genetic variants within both lncRAP2 and Igf2bp2 with body mass and type 2 diabetes, and both lncRAP2 and Igf2bp2 are suppressed in adipose depots of obese and diabetic individuals. Thus, the lncRAP2-Igf2bp2 complex potentiates adipose development and energy expenditure and is associated with susceptibility to obesity-linked diabetes.

Lipolysis regulates major transcriptional programs in brown adipocytes.

ß-Adrenergic signaling is a core regulator of brown adipocyte function stimulating both lipolysis and transcription of thermogenic genes, thereby expanding the capacity for oxidative metabolism. We have used pharmacological inhibitors and a direct activator of lipolysis to acutely modulate the activity of lipases, thereby enabling us to uncover lipolysis-dependent signaling pathways downstream of ß-adrenergic signaling in cultured brown adipocytes. Here we show that induction of lipolysis leads to acute induction of several gene programs and is required for transcriptional regulation by ß-adrenergic signals. Using machine-learning algorithms to infer causal transcription factors, we show that PPARs are key mediators of lipolysis-induced activation of genes involved in lipid metabolism and thermogenesis. Importantly, however, lipolysis also activates the unfolded protein response and regulates the core circadian transcriptional machinery independently of PPARs. Our results demonstrate that lipolysis generates important metabolic signals that exert profound pleiotropic effects on transcription and function of cultured brown adipocytes.

Ras signalling regulates differentiation and UCP1 expression in models of brown adipogenesis.

BACKGROUND: The Ras/Raf/MEK/ERK pathway has been recognised as an important signalling module in adipogenesis and adipocyte function, but whether it promotes or inhibits the formation of fat cells has not been reconciled. METHODS: Here we investigate the significance of Ras signalling intensity on two unrelated models of mouse brown adipocyte differentiation. RESULTS: A constitutively active H-Ras mutant (Ras V12) caused a complete block of adipose conversion, as manifested by a lack of both lipid accumulation and induction of adipocyte gene expression. The Ras V12-mediated impediment of differentiation was inefficiently rescued by forced expression of the adipogenic transcription factors C/EBPalpha and PPARgamma. However, the defective differentiation was alleviated by MEK inhibitors, suggesting that the obstruction of differentiation was dependent on activation of ERK. A dominant interfering H-Ras mutant (Ras N17) did not inhibit differentiation, but led to increased expression of genes important for energy dissipation in brown fat cells, including UCP1. GENERAL SIGNIFICANCE: These data suggest that the intensity of Ras signalling is important for differentiation and UCP1 expression in models of brown adipogenesis.

ADD1/SREBP1c activates the PGC1-alpha promoter in brown adipocytes.

Cold adaptation elicits a paradoxical simultaneous induction of fatty acid synthesis and beta-oxidation in brown adipose tissue. We show here that cold exposure coordinately induced liver X receptor alpha (LXRalpha), adipocyte determination and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein-1c (SREBP1c) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC1alpha) in brown and inguinal white adipose tissues, but not in epididymal white adipose tissue. Using in vitro models of white and brown adipocytes we demonstrate that beta-adrenergic stimulation induced expression of LXRalpha, ADD1/SREBP1c and PGC1alpha in cells with a brown-like adipose phenotype. We demonstrate that ADD1/SREBP1c is a powerful inducer of PGC1alpha expression via a conserved E box in the proximal promoter and that beta-adrenergic stimulation led to recruitment of ADD1/SREBP1c to this E box. The ability of ADD1/SREBP1c to activate the PGC1alpha promoter exhibited a striking cell type dependency, suggesting that additional cell type-restricted factors contribute to ADD1/SREBP1c-mediated activation. In conclusion, our data demonstrate a novel role of ADD1/SREBP1c as a regulator of PGC1alpha expression in brown adipose tissue.

Parathyroid hormone receptor stimulation induces human adipocyte lipolysis and browning.

OBJECTIVE: Activation of brown adipose tissue is a promising strategy to treat and prevent obesity and obesity-related disorders. Activation of uncoupling protein 1 (UCP1) leads to uncoupled respiration and dissipation of stored energy as heat. Induction of UCP1-rich adipocytes in white adipose tissue, a process known as 'browning', serves as an alternative strategy to increase whole body uncoupling capacity. Here, we aim to assess the association between parathyroid hormone (PTH) receptor expression and UCP1 expression in human adipose tissues and to study PTH effects on human white and brown adipocyte lipolysis and UCP1 expression. DESIGN: A descriptive study of human neck adipose tissue biopsies substantiated by an interventional study on human neck-derived adipose tissue cell models. METHODS: Thermogenic markers and PTH receptor gene expression are assessed in human neck adipose tissue biopsies and are related to individual health records. PTH-initiated lipolysis and thermogenic gene induction are assessed in cultured human white and brown adipocyte cell models. PTH receptor involvement is investigated by PTH receptor silencing. RESULTS: PTH receptor gene expression correlates with UCP1 gene expression in the deep-neck adipose tissue in humans. In cell models, PTH receptor stimulation increases lipolysis and stimulates gene transcription of multiple thermogenic markers. Silencing of the PTH receptor attenuates the effects of PTH indicating a direct PTH effect via this receptor. CONCLUSION: PTH 1 receptor stimulation by PTH may play a role in human adipose tissue metabolism by affecting lipolysis and thermogenic capacity.

3D Adipose Tissue Culture Links the Organotypic Microenvironment to Improved Adipogenesis.

Obesity and type 2 diabetes are strongly associated with adipose tissue dysfunction and impaired adipogenesis. Understanding the molecular underpinnings that control adipogenesis is thus of fundamental importance for the development of novel therapeutics against metabolic disorders. However, translational approaches are hampered as current models do not accurately recapitulate adipogenesis. Here, a scaffold-free versatile 3D adipocyte culture platform with chemically defined conditions is presented in which primary human preadipocytes accurately recapitulate adipogenesis. Following differentiation, multi-omics profiling and functional tests demonstrate that 3D adipocyte cultures feature mature molecular and cellular phenotypes similar to freshly isolated mature adipocytes. Spheroids exhibit physiologically relevant gene expression signatures with 4704 differentially expressed genes compared to conventional 2D cultures (false discovery rate < 0.05), including the concerted expression of factors shaping the adipogenic niche. Furthermore, lipid profiles of >1000 lipid species closely resemble patterns of the corresponding isogenic mature adipocytes in vivo (R2 = 0.97). Integration of multi-omics signatures with analyses of the activity profiles of 503 transcription factors using global promoter motif inference reveals a complex signaling network, involving YAP, Hedgehog, and TGFß signaling, that links the organotypic microenvironment in 3D culture to the activation and reinforcement of PPARγ and CEBP activity resulting in improved adipogenesis.

The polycomb group protein Suz12 is required for embryonic stem cell differentiation.

Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12(-/-) ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

Pannexin-1 mediated ATP release in adipocytes is sensitive to glucose and insulin and modulates lipolysis and macrophage migration.

AIM: Extracellular ATP signalling is involved in many physiological and pathophysiological processes in several tissues, including adipose tissue. Adipocytes have crucial functions in lipid and glucose metabolism and they express purinergic receptors. However, the sources of extracellular ATP in adipose tissue are not well characterized. In the present study, we investigated the mechanism and regulation of ATP release in white adipocytes, and evaluated the role of extracellular ATP as potential autocrine and paracrine signal. METHODS: Online ATP release was monitored in C3H10T1/2 cells and freshly isolated murine adipocytes. The ATP release mechanism and its regulation were tested in cells exposed to adrenergic agonists, insulin, glucose load and pharmacological inhibitors. Cell metabolism was monitored using Seahorse respirometry and expression analysis of pannexin-1 was performed on pre- and mature adipocytes. The ATP signalling was evaluated in live cell imaging (Ca2+ , pore formation), glycerol release and its effect on macrophages was tested in co-culture and migration assays. RESULTS: Here, we show that upon adrenergic stimulation white murine adipocytes release ATP through the pannexin-1 pore that is regulated by a cAMP-PKA-dependent pathway. The ATP release correlates with increased cell metabolism and is sensitive to glucose. Extracellular ATP induces Ca2+ signalling and lipolysis in adipocytes and promotes macrophage migration. Importantly, ATP release is markedly inhibited by insulin, which operates via the activation of phosphodiesterase 3. CONCLUSIONS: Our findings reveal an insulin-pannexin-1-purinergic signalling crosstalk in adipose tissue and we propose that deregulation of this signalling may contribute to adipose tissue inflammation and type 2 diabetes.

Pyruvate kinase M2 represses thermogenic gene expression in brown adipocytes.

Utilizing the thermogenic capacity of brown adipose tissue is a potential anti-obesity strategy; therefore, the mechanisms controlling expression of thermogenesis-related genes are of interest. Pyruvate kinase (PK) catalyzes the last step of glycolysis and exists as four isoenzymes: PK, liver, PK, red blood cell, PK, muscle (PKM1 and PKM2). PKM2 has both glycolytic and nuclear functions. Here, we report that PKM2 is enriched in brown adipose compared with white adipose tissue. Specific knockdown of PKM2 in mature brown adipocytes demonstrates that silencing of PKM2 does not lead to a decrease in PK activity, but causes a robust upregulation of thermogenic uncoupling protein 1 (Ucp1) and fibroblast growth factor 21 (Fgf21) gene expression. This increase is not mediated by any of the known mechanisms for PKM2-regulated gene expression, thus implying the existence of a novel mechanism for PKM2-dependent effects on gene expression.

Beta-1 and Not Beta-3 Adrenergic Receptors May Be the Primary Regulator of Human Brown Adipocyte Metabolism.

PURPOSE: Brown adipose tissue (BAT) activation in humans has gained interest as a potential target for treatment of obesity and insulin resistance. In rodents, BAT is primarily induced through beta-3 adrenergic receptor (ADRB3) stimulation, whereas the primary beta adrenergic receptors (ADRBs) involved in human BAT activation are debated. We evaluated the importance of different ADRB subtypes for uncoupling protein 1 (UCP1) induction in human brown adipocytes. METHODS: A human BAT cell model (TERT-hBA) was investigated for subtype-specific ADRB agonists and receptor knockdown on UCP1 mRNA levels and lipolysis (glycerol release). In addition, fresh human BAT biopsies and TERT-hBA were evaluated for expression of ADRB1, ADRB2, and ADRB3 using RT-qPCR. RESULTS: The predominant ADRB subtype in TERT-hBA adipocytes and BAT biopsies was ADRB1. In TERT-hBA, UCP1 mRNA expression was stimulated 11.0-fold by dibutyryl cAMP (dbcAMP), 8.0-fold to 8.4-fold by isoproterenol (ISO; a pan-ADRB agonist), and 6.1-fold to 12.7-fold by dobutamine (ADRB1 agonist), whereas neither procaterol (ADRB2 agonist), CL314.432, or Mirabegron (ADRB3 agonists) affected UCP1. Similarly, dbcAMP, ISO, and dobutamine stimulated glycerol release, whereas lipolysis was unaffected by ADRB2 and ADRB3 agonists. Selective knockdown of ADRB1 significantly attenuated ISO-induced UCP1 expression. CONCLUSION: The adrenergic stimulation of UCP1 and lipolysis may mainly be mediated through ADRB1. Moreover, ADRB1 is the predominant ADRB in both TERT-hBA and human BAT biopsies. Thus, UCP1 expression in human BAT may, unlike in rodents, primarily be regulated by ADRB1. These findings may have implications for ADRB agonists as future therapeutic compounds for human BAT activation.