Brown Preadipocytes Research Articles

Mangiferin induces the expression of a thermogenic signature via AMPK signaling during brown-adipocyte differentiation

Mangiferin (MF) from Mangifera indica has been serendipitously found to ameliorate obesity and is used as an antioxidant, anti-inflammatory, antimicrobial, and anticancer agent. Nonetheless, the mechanism of MF-induced brown-adipose-tissue activation has not been studied. Therefore, we investigated the effect of MF on thermogenic features during brown-adipocyte differentiation. Treatment with MF improved the expression of a brown-fat signature and of mitochondrial-mass–related genes, thus resulting in UCP1 induction. MF also raised the expression of other thermogenic regulators, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), PR domain–containing protein 16 (PRDM16), and peroxisome proliferator-activated receptors alpha and gamma (PPAR-α and -γ). MF promoted mitochondrial biogenesis, judging by increased expression of cell death–inducing DNA fragmentation factor α–like effector A (CIDEA), mitochondrial transcription factor A (TFAM), iodothyronine deiodinase 2 (DIO2), cytochrome c oxidase subunit 7A (COX7A), cyclooxygenase 2 (COX2), sirtuin 1 (SIRT1), and nuclear respiratory factor 1 (NRF1). MF treatment increased the mitochondrial DNA amount and improved mitochondrial respiratory function by increasing the oxygen consumption rate during brown-adipocyte differentiation. A gene knockdown assay involving small interfering RNA and competitive inhibition with dorsomorphin revealed that MF may promote thermogenesis in brown preadipocytes via activation of AMPK signaling. Collectively, our findings suggest that MF may be a novel pharmaceutical agent that can ameliorate obesity via activation of brown adipose tissue.

Transferrin Receptor 1 Regulates Thermogenic Capacity and Cell Fate in Brown/Beige Adipocytes.

Iron homeostasis is essential for maintaining cellular function in a wide range of cell types. However, whether iron affects the thermogenic properties of adipocytes is currently unknown. Using integrative analyses of multi‐omics data, transferrin receptor 1 (Tfr1) is identified as a candidate for regulating thermogenesis in beige adipocytes. Furthermore, it is shown that mice lacking Tfr1 specifically in adipocytes have impaired thermogenesis, increased insulin resistance, and low‐grade inflammation accompanied by iron deficiency and mitochondrial dysfunction. Mechanistically, the cold treatment in beige adipocytes selectively stabilizes hypoxia‐inducible factor 1‐alpha (HIF1α), upregulating the Tfr1 gene, and thermogenic adipocyte‐specific Hif1α deletion reduces thermogenic gene expression in beige fat without altering core body temperature. Notably, Tfr1 deficiency in interscapular brown adipose tissue (iBAT) leads to the transdifferentiation of brown preadipocytes into white adipocytes and muscle cells; in contrast, long‐term exposure to a low‐iron diet fails to phenocopy the transdifferentiation effect found in Tfr1‐deficient mice. Moreover, mice lacking transmembrane serine protease 6 (Tmprss6) develop iron deficiency in both inguinal white adipose tissue (iWAT) and iBAT, and have impaired cold‐induced beige adipocyte formation and brown fat thermogenesis. Taken together, these findings indicate that Tfr1 plays an essential role in thermogenic adipocytes via both iron‐dependent and iron‐independent mechanisms.

MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

The prevalence of metabolic syndrome (MetS) and obesity is an alarming health issue worldwide. Obesity is characterized by an excessive accumulation of white adipose tissue (WAT), and it is associated with diminished brown adipose tissue (BAT) activity. Twist1 acts as a negative feedback regulator of BAT metabolism. Therefore, targeting Twist1 could become a strategy for obesity and metabolic disease. Here, we have identified miR-337-3p as an upstream regulator of Twist1. Increased miR-337-3p expression paralleled decreased expression of TWIST1 in BAT compared to WAT. Overexpression of miR-337-3p in brown pre-adipocytes provoked a reduction in Twist1 expression that was accompanied by increased expression of brown/mitochondrial markers. Luciferase assays confirmed an interaction between the miR-337 seed sequence and Twist1 3′UTR. The inverse relationship between the expression of TWIST1 and miR-337 was finally validated in adipose tissue samples from non-MetS and MetS subjects that demonstrated a dysregulation of the miR-337-Twist1 molecular axis in MetS. The present study demonstrates that adipocyte miR-337-3p suppresses Twist1 repression and enhances the browning of adipocytes.

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of β1 and β3-adrenoceptor signalling

Objective3′,5′-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. β-adrenergic receptors (β-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes. MethodsWe measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual β-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with β-AR stimulated lipolysis and analysed the expression of β-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots. ResultsUsing pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control β1-AR-induced cAMP. Unexpectedly, β3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by β1-AR associated with PDE3 and PDE4, while another pool is centred around β3-AR and is much less controlled by these PDEs. Functionally, lower control of β3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by β1-AR and is under tight control of PDE3 and PDE4. ConclusionsWe have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of β1-and β3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.

Unmasking cAMP signalling in adipocytes

In recent years, the prevalence of obesity has reached pandemic proportions. In 2016, the World Health Organisation estimated that ca. 3 million people die each year due to complications from being overweight or obese. With more overweight population than ever, finding new targets to counteract obesity has become crucial. Recruitment and activation of brown adipose tissue (BAT) has been postulated as a potential target to increase thermogenic energy expenditure (EE) in order to treat or prevent obesity and its comorbidities.Cyclic adenosine monophosphate (cAMP) is a second messenger important in many biological processes. BAT thermogenesis is primarily mediated by activation of Gs‐coupled receptors such as the beta‐3 adrenergic receptor and the adenosine A2A receptor, which both increase intracellular cAMP. In mature brown adipocytes, cAMP is well known to induce lipolysis and UCP1‐mediated mitochondrial uncoupling through activation of protein kinase A (PKA). However, the precise role of cAMP signaling in adipocyte precursor cells has so far not been well studied. Furthermore, the role of other cAMP effectors (e.g. Exchange proteins activated by cAMP (EPAC)) in adipose tissue metabolism is not clear.Treatment of brown adipocyte precursor cells using analogs specific to different cAMP effectors induce distinct cellular responses. Here, we used genetic and pharmacological approaches to decipher the role of cAMP effectors, focusing on EPAC proteins, in adipocytes. We demonstrate that activation of mature adipocytes and proliferation of brown preadipocytes require the activation of distinct effectors and signaling pathways downstream of cAMP. Furthermore, browning of white adipocytes appears to follow similar patterns. Our data indicate that, depending on the effector, either PKA or EPAC, cAMP regulates browning of white adipocytes by different mechanisms. Moreover, in vivo deletion of EPAC leads to reduced oxygen consumption after high fat diet, demonstrating that the role of cAMP in adipocytes is still not fully understood. In conclusion, we show that the effects of cAMP in BAT and browning of WAT differ vastly depending on both the state of adipocyte differentiation as well as the downstream effector.Support or Funding InformationInstitute of Pharmacology and Toxicology, University of Bonn, 53127 Bonn, GermanySFB1328, Universitätsklinikum Hamburg‐Eppendorf, 20246 Hamburg, Germany

Exendin-4 Improves Diabetic Kidney Disease in C57BL/6 Mice Independent of Brown Adipose Tissue Activation.

Background The role of exendin-4 in brown adipose tissue (BAT) activation was not very clear. This study is to verify the role of BAT involved in renal benefits of exendin-4 in diabetes mellitus (DM). Methods In vivo, C57BL/6 mice were randomly divided into nondiabetic (control) and diabetic groups (DM). The diabetic mice were randomized into a control group (DM-Con), BAT-excision group (DM+Exc), exendin-4-treated group (DM+E4), and BAT-excision plus exendin-4-treated group (DM+Exc+E4). The weight, blood glucose and lipids, 24 h urine albumin and 8-OH-dG, and renal fibrosis were analyzed. In vitro, we investigated the role of exendin-4 in the differentiation process of 3T3-L1 and brown preadipocytes and its effect on the rat mesangial cells induced by oleate. Results The expressions of UCP-1, PGC-1α, ATGL, and CD36 in BAT of DM mice were all downregulated, which could be upregulated by exendin-4 treatment with significant effects on ATGL and CD36. BAT-excision exacerbated high blood glucose (BG) with no significant effect on the serum lipid level. Exendin-4 significantly lowered the level of serum triglycerides (TG) and low-density lipoprotein- (LDL-) c, 24 h urine albumin, and 8-OH-dG; improved renal fibrosis and lipid accumulation; and activated renal AMP-activated protein kinase (AMPK) in diabetic mice regardless of BAT excision. In vitro, there was no significant effect of exendin-4 on brown or white adipogenesis. However, exendin-4 could improve lipid accumulation and myofibroblast-like phenotype transition of mesangial cells induced by oleate via activating the AMPK pathway. Conclusions Exendin-4 could decrease the renal lipid deposit and improve diabetic nephropathy via activating the renal AMPK pathway independent of BAT activation.

MTORC2-AKT signaling to ATP-citrate lyase drives brown adipogenesis and de novo lipogenesis

mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.

Insulin-induced serine 22 phosphorylation of retinoid X receptor alpha is dispensable for adipogenesis in brown adipocytes

ABSTRACT Insulin action initiates a series of phosphorylation events regulating cellular differentiation, growth and metabolism. We have previously discovered, in a mass spectrometry-based phosphoproteomic study, that insulin/IGF-1 signalling induces phosphorylation of retinoid x receptor alpha (RXRα) at S22 in mouse brown pre-adipocytes. Here, we show that insulin induces the phosphorylation of RXRα at S22 in both brown precursor and mature adipocytes through a pathway involving ERK, downstream of IRS-1 and −2. We also found that RXRα S22 phosphorylation is promoted by insulin and upon re-feeding in brown adipose tissue in vivo, and that insulin-stimulated S22 phosphorylation of RXRα is dampened by diet-induced obesity. We used Rxra knockout cells re-expressing wild type (WT) or S22A non-phosphorylatable forms of RXRα to further characterize the role of S22 in brown adipocytes. Knockout of Rxra in brown pre-adipocytes resulted in decreased lipid accumulation and adipogenic gene expression during differentiation, and re-expression of RxraWT alleviated these effects. However, we observed no significant difference in cells re-expressing the RxraS22A mutant as compared with the cells re-expressing RxraWT. Furthermore, comparison of gene expression during adipogenesis in the WT and S22A re-expressing cells by RNA sequencing revealed similar transcriptomic profiles. Thus, our data propose a dispensable role for RXRα S22 phosphorylation in adipogenesis and transcription in differentiating brown pre-adipocytes.

Analyzing Mitochondrial Function in Brown Adipocytes with a Bioenergetic Analyzer.

Brown adipocytes are a cell type with high mitochondrial content and bioenergetic capacity. A critical means to measure mitochondrial function, macromolecule fuel usage, and other important phenotypes is with a bioenergetic analyzer. Here, we describe how to isolate, culture, and differentiate brown preadipocytes into mature adipocytes. We also explain how to perform a mitochondrial (mito) stress test, using the bioenergetic analyzer. The mito stress test is able to give researchers a plethora of insights into mitochondrial function, including basal respiration, proton leak, ATP production, maximal respiration, and reserve capacity, making it a powerful tool for analyzing brown adipocytes.

Conditionally immortalized brown preadipocytes can switch between proliferative and differentiated states

Brown adipose tissue (BAT) is a potential target to treat cardiometabolic disorders because of its capacity to combust glucose and fatty acids for thermoregulation. Its cellular and molecular investigation in humans is hampered by the limited availability of cell material and the heterogeneity of BAT between and within individuals. In this study, monoclonal lines of conditionally immortalized brown preadipocytes (iBPAs) of mouse and human origin were generated. Conditional immortalization was achieved by doxycycline-controlled expression of simian virus 40 large tumor antigen (LT) with a repressor-based Tet-On system. In the presence of doxycycline, both the murine and human cell lines showed long-term proliferation capacity with a population doubling time of ~28 h. After switching off LT expression by doxycycline removal and exposure to adipogenic differentiation medium, cells from both species acquired brown adipocyte properties. This was evidenced by the accumulation of multilocular lipid droplets, the upregulation of brown adipocyte markers including uncoupling protein 1 and an increase in lipolysis and oxygen consumption following adrenergic stimulation. Switching off LT expression before the onset of adipogenic differentiation was only critical for inducing adipogenesis in the human iBPAs, while their murine counterparts showed adipogenesis upon exposure to the adipogenic differentiation cocktail regardless of LT expression. When switched to proliferation medium, cultures of adipogenically differentiated human iBPAs de-differentiated and resumed cell division without losing their adipogenic capacity. We suggest that iBPAs represent an easy-to-use model for fundamental and applied research into BAT offering unique experimental opportunities due to their capacity to switch between proliferative and differentiated states.

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.

1760-P: Tetrahydrobiopterin Regulates Developmental Differentiation of Brown Adipose Tissue

Brown adipose tissue (BAT) is a key organ that produces heat and increases energy expenditure; its regulatory factors could be clinically useful in the treatment of obesity and diabetes. Tetrahydrobiopterin (BH4) is an essential co-factor of tyrosine hydroxylase and nitric oxide synthase. Our group recently showed that BH4 activates BAT function and regulates systemic energy metabolism using Hph-1 mice, a mouse model of BH4 deficiency in which GTP-cyclohydrolase I (GTPCH I), a rate-limiting enzyme of BH4, synthesis is deficient. Interestingly, we found that dysplasia of BAT already has been confirmed in the neonatal period. We investigated the role of BH4 in the development and differentiation of BAT using the mouse model of BH4 deficiency. Compared with control mice, Hph-1 neonatal mice exhibited lower weight of BAT as well as attenuated thermogenesis-related gene expressions (UCP1, Dio2, Pgc1a). We also evaluated the role of BH4 in brown adipocyte differentiation using brown pre-adipocytes isolated from control and Hph-1 mice. Compared with control mice, brown pre-adipocytes from Hph-1 mice reduced differentiation capacity measured by accumulated lipid droplets and thermogenesis-related gene expressions. In addition, inhibition of GTPCH I by 2,4-Diamino-6-Hydroxypyrimidine (DAHP) reduced differentiation capacity in brown pre-adipocytes. These data strongly suggest that BH4 is required for brown adipocyte differentiation. Furthermore, supplementation of BH4 in brown pre-adipocytes from Hph-1 mice dose-dependently increased accumulation of lipid droplets, thermogenesis-related gene expressions, and nitric oxide production. Taken together, BH4 plays an integral role in developmental differentiation of BAT, and has therapeutic potential for metabolic disorders such as obesity and diabetes. Disclosure H. Minamino: None. Y. Fujita: None. Y. Oguri: None. T. Goto: None. A. Ohashi: None. F. Furuya: None. N. Isomura: None. S. Kawarasaki: None. T. Kawada: None. H. Hasegawa: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Japan Tobacco Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

312-OR: Functional Decline in Brown Adipose Progenitors during BAT Remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune-deficient mice. RNA-sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down-regulated, while genes involved in lipid and amino acid metabolism were up-regulated. Single-cell RNA sequencing discovered a unique population of stromal vascular fraction cells specifically expressing genes regulating muscle and mitochondrial function. Knocking down these genes in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans. Disclosure Z. Huang: None. Z. Zhang: None. H. Ruan: None. Funding American Diabetes Association (1-18-IBS-167 to H-B.R.)

Soluble Epoxide Hydrolase Inhibitor t-AUCB Promotes Murine Brown Adipogenesis: Role of PPAR Gamma and PPAR Alpha (P21-069-19)

ObjectivesBrown adipose tissue has recently emerged as a novel target for obesity treatment and prevention. In contrast to the lipid storing function of white adipocytes, brown adipocytes are responsible for dissipating energy as heat, a process involving uncoupling protein 1 (UCP1). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme that converts epoxy fatty acids (EpFAs) into less active diols. By stabilizing endogenous EpFAs, potent small molecule sEH inhibitors have been shown to be beneficial for many chronic diseases. Several recent papers have reported that sEH inhibitors are able to reduce diet-induced obesity, possibly by upregulating UCP1 expression. In the current study, we sought to study the mechanisms by which sEH inhibitor acts on brown preadipocytes. MethodsThe effects of a potent sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), on murine brown adipocyte differentiation were evaluated by lipid accumulation and expression of brown adipocyte marker genes. PPAR alpha and PPAR gamma activation by t-AUCB was measured by their respective transactivation assays. The roles of PPARs were further studied by pharmacological antagonism and knockdown experiments by small RNA interference. ResultsWe report that sEH expression was increased during murine brown adipocyte differentiation. t-AUCB dose-dependently promoted brown adipocyte differentiation. Moreover, we demonstrate that t-AUCB activated PPAR alpha, but not PPAR gamma. t-AUCB-induced upregulation of thermogenic gene Ucp1 and Pgc1 alpha and the general differentiation marker Fabp4 were significantly attenuated by the antagonist of PPAR alpha, GW6471. In contrast, they were only partially attenuated by the antagonist of PPAR gamma, GW9662, and specific knockdown of PPAR gamma. ConclusionsOur findings suggest that sEH may regulate brown adipogenesis and sEH pharmacological inhibition by t-AUCB promotes brown adipogenesis, possibly through activation of PPAR alpha. Funding SourcesThe work is supported by NIH 1R15DK114790-01A1 (to LZ), R00DK100736 (to AB) and R01ES002710 (to BDH).

2044-P: Histone Methyltransferase DOT1L Regulates Brown Adipocyte Differentiation and Thermogenesis

Brown fat has been extensively studied due to its potential in therapies against obesity. Various epigenetic regulators have been shown to modulate brown fat development and function. Here we show that histone methyltransferase DOT1L is a key regulator of brown adipocyte differentiation and thermogenesis. In vitro, we demonstrate that inhibition by DOT1L inhibitor or knockdown of DOT1L markedly promotes brown adipocyte differentiation of C3H10T1/2 cells. Moreover, depletion of DOT1L enhances differentiation and thermogenesis of primary brown adipocytes which separated from DOT1LFlox/Flox mice by transfection with Cre-expressing lentiviruses ex vivo. Mechanistically, knockdown of DOT1L results in increased expression of brown-selective genes, and the expression of brown preadipocyte maker EBF2 is significantly increased due to knockdown of DOT1L at early stage. Furthermore, ChIP assays suggest that DOT1L-mediated H3K79 methylations are negatively associated with transcription of UCP1 and PRDM16. Finally, we observe that the skin surface temperature in interscapular area of DOT1LMyf5 knockout mice is significantly higher. Notably, energy expenditure and the oxygen consumption rate are also increased in knockout mice. These in vivo data suggest that DOT1L depletion leads to enhanced thermogenic function of brown fat. Collectively, our work indicates that DOT1L plays an important role in regulating brown fat differentiation and thermogenesis, and provides another linkage between epigenetics and brown fat development and function. Disclosure L. Shuai: None. J. Li: None. J. Li: None. Funding China Postdoctoral Science Foundation

Berberine promotes the recruitment and activation of brown adipose tissue in mice and humans

Brown adipose tissue (BAT) dissipates metabolic energy and mediates non-shivering thermogenesis, thereby boosting energy expenditure. Increasing BAT mass and activity is expected to be a promising strategy for combating obesity; however, few medications effectively and safely recruit and activate BAT in humans. Berberine (BBR), a natural compound, is commonly used as a nonprescription drug to treat diarrhea. Here, we reported that 1-month BBR intervention increased BAT mass and activity, reduced body weight, and improved insulin sensitivity in mildly overweight patients with non-alcoholic fatty liver disease. Chronic BBR treatment promoted BAT development by stimulating the expression of brown adipogenic genes, enhanced BAT thermogenesis, and global energy expenditure in diet-induced obese mice and chow-fed lean mice, Consistently, BBR facilitated brown adipocyte differentiation in both mouse and human primary brown preadipocytes. We further found that BBR increased the transcription of PRDM16, a master regulator of brown/beige adipogenesis, by inducing the active DNA demethylation of PRDM16 promoter, which might be driven by the activation of AMPK and production of its downstream tricarboxylic acid cycle intermediate α-Ketoglutarate. Moreover, chronic BBR administration had no impact on the BAT thermogenesis in adipose-specific AMPKa1 and AMPKa2 knockout mice. In summary, we found that BBR intervention promoted recruitment and activation of BAT and AMPK–PRDM16 axis was indispensable for the pro-BAT and pro-energy expenditure properties of BBR. Our findings suggest that BBR may be a promising drug for obesity and related metabolic disorders in humans partially through activating BAT.

Beneficial Metabolic Effects of Mirabegron In Vitro and in High-Fat Diet-Induced Obese Mice.

Mirabegron, a β3-adrenergic receptor agonist, has been shown to stimulate the activity of brown fat and increase the resting metabolic rate in humans. However, it is unknown whether mirabegron can reduce body weight and improve metabolic health. We investigated the antiobesity effects of mirabegron using both in vitro and in vivo models. Mouse brown preadipocytes and 3T3-L1 cells were treated with different concentrations of mirabegron (0.03-3 µg/ml), and the expression of brown fat-related genes was measured by quantitative real-time polymerase chain reaction. Furthermore, male C57BL/6J mice were fed a high-fat diet for 10 weeks, and mirabegron (2 mg/kg body weight) or a vehicle control was delivered to the interscapular brown adipose tissue (iBAT) using ALZET osmotic pumps from week 7 to 10. The metabolic parameters and tissues were analyzed. In both mouse brown preadipocytes and 3T3-L1 cells, mirabegron stimulated uncoupling protein 1 (UCP1) expression. In animal studies, mirabegron-treated mice had a lower body weight and adiposity. Lipid droplets in the iBAT of mirabegron-treated mice were fewer and smaller in size compared with those from vehicle-treated mice. H&E staining and immunohistochemistry indicated that mirabegron increased the abundance of beige cells in inguinal white adipose tissue (iWAT). Compared with vehicle-treated mice, mirabegron-treated mice had a higher gene expression of UCP1 (14-fold) and cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) (4-fold) in iWAT. Furthermore, mirabegron-treated mice had improved glucose tolerance and insulin sensitivity. Taken together, mirabegron enhances UCP1 expression and promotes browning of iWAT, which are accompanied by improved glucose tolerance and insulin sensitivity and prevention from high-fat diet-induced obesity.

Functional decline in brown adipose progenitors during BAT remodeling

In humans, there is a striking decrease in the prevalence and activity of brown adipose tissue (BAT) with age and obesity; however, the cellular and molecular mechanisms of BAT remodeling are almost completely unknown. Rodent models have been widely used to study BAT but they possess constitutively active BAT throughout life even at thermoneutrality; therefore, complementary animal models that possess characteristics of human BAT remodeling are needed. Here, we found that BAT progressively whitened in rabbits in a similar manner as in humans. As rabbits mature, preadipocytes in BAT displayed reduced capacity to be differentiated into brown adipocytes, when transplanted into immune‐deficient mice. RNA‐sequencing of brown preadipocytes identified that, during BAT remodeling, genes enriched for muscle contraction and mitochondrial function were down‐regulated, while genes involved in lipid and amino acid metabolism were up‐regulated. Knocking down genes regulating muscle and mitochondrial function in rabbit brown preadipocytes blunted their ability to become mature brown adipocytes in vitro and in vivo. We also identified follistatin like 1 (FSTL1) as a novel “batokine” that is specifically expressed by brown preadipocytes but not mature brown adipocytes. FSTL1 gradually reduced its expression when BAT whitens in rabbits, but was stably expressed in mouse BAT during life. Deleting FSTL1 specifically in the Myf5 lineage in mice reduced BAT size, UCP1 expression, and body temperature in newborns, suggesting the constitutive expression of FSTL1 prevents BAT whitening in mice. Taken together, we identify potential molecular mechanisms underlying functional decline in the browning ability of preadipocytes that contribute to BAT remodeling in rabbits and possibly humans.Support or Funding InformationAmerican Diabetes Association (18‐IBS‐167)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

Transcriptional and epigenetic regulation is fundamentally involved in initiating and maintaining progression of cellular differentiation. The 2 types of thermogenic adipocytes, brown and beige, are thought to be of different origins but share functionally similar phenotypes. Here, we report that lysine-specific demethylase 2 (LSD2) regulates the expression of genes associated with lineage identity during the differentiation of brown and beige adipogenic progenitors in mice. In HB2 mouse brown preadipocytes, short hairpin RNA-mediated knockdown (KD) of LSD2 impaired formation of lipid droplet-containing adipocytes and down-regulated brown adipogenesis-associated genes. Transcriptomic analysis revealed that myogenesis-associated genes were up-regulated in LSD2-KD cells under adipogenic induction. In addition, loss of LSD2 during later phases of differentiation had no obvious influence on adipogenic traits, suggesting that LSD2 functions during earlier phases of brown adipocyte differentiation. Using adipogenic cells from the brown adipose tissues of LSD2-knockout (KO) mice, we found reduced expression of brown adipogenesis genes, whereas myogenesis genes were not affected. In contrast, when LSD2-KO cells from inguinal white adipose tissues were subjected to beige induction, these cells showed a dramatic rise in myogenic gene expression. Collectively, these results suggest that LSD2 regulates distinct sets of genes during brown and beige adipocyte formation.-Takase, R., Hino, S., Nagaoka, K., Anan, K., Kohrogi, K., Araki, H., Hino, Y., Sakamoto, A., Nicholson, T. B., Chen, T., Nakao, M. Lysine-specific demethylase-2 is distinctively involved in brown and beige adipogenic differentiation.

Increased oxidative stress, inflammation and fibrosis in perirenal adipose tissue of patients with cortisol-producing adenoma

ABSTRACT Although much is known about that corticosteroids affect the functions of adipose tissues, little genetic information is available for perirenal adipose tissue (peri-N) from patients with cortisol-producing adenoma (CPA). We conducted microarray analysis of peri-N from patients with CPA by using an Affymetrix human U133 plus 2.0 array. We also analysed the inflammation, fibrosis and oxidative stress in vitro. Compared with normotension (NT) group, CPA group has significantly higher protein levels of TNFα, IL-6, fibronectin (FN) and collagen I (COLI). The protein level of NADPH oxidase 4 (Nox4) significantly increased, while nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) levels were significantly reduced in the CPA group. Dexamethasone markedly induced fibrosis and adipogenesis-related gene expression in predifferentiated stromal vascular fraction (SVF) cells, 3T3-L1 preadipocytes and brown preadipocytes. Chronic exposure to endogenous glucocorticoids due to CPA increases peri-N oxidative stress, inflammation and fibrosis, which may contribute to the metabolic disturbances associated with hypercortisolism in these patients.