Brown Adipocytes Research Articles

The rs1421085 variant within FTO promotes brown fat thermogenesis.

One lead genetic risk signal of obesity-the rs1421085 T>C variant within the FTO gene-is reported to be functional in vitro but lacks evidence at an organism level. Here we recapitulate the homologous human variant in mice with global and brown adipocyte-specific variant knock-in and reveal that mice carrying the C-allele show increased brown fat thermogenic capacity and resistance to high-fat diet-induced adiposity, whereas the obesity-related phenotypic changes are blunted at thermoneutrality. Both in vivo and in vitro data reveal that the C-allele in brown adipocytes enhances the transcription of the Fto gene, which is associated with stronger chromatin looping linking the enhancer region and Fto promoter. Moreover, FTO knockdown or inhibition effectively eliminates the increased thermogenic ability of brown adipocytes carrying the C-allele. Taken together, these findings identify rs1421085 T>C as a functional variant promoting brown fat thermogenesis.

Albizia julibrissin Exerts Anti-Obesity Effects by Inducing the Browning of 3T3L1 White Adipocytes.

This study investigated the effects of the Albizia julibrissin Leaf extracts (AJLE) on adipocytes using 3T3-L1 cells. AJLE inhibited adipogenesis by reducing the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding proteins (C/EBPs) that regulate enzymes involved in fat synthesis and storage, and subsequently reduced intracellular lipid droplets, glycerol-3-phosphate dehydrogenase (GPDH), and triglyceride (TG). AJLE also increased the expression of brown adipocyte markers, such as uncoupling protein-1 (UCP-1), PR/SET domain 16 (PRDM16), and bone morphogenetic protein 7 (BMP7) by inducing the differentiation of brown adipocytes, as shown by a decrease in the lipid droplet sizes and increasing mitochondrial mass. AJLE increased the expression of transcription factor A, mitochondrial (TFAM), mitochondrial DNA (mtDNA) copy number, and UCP-1 protein expression, all of which are key factors in regulating mitochondrial biogenesis. AJLE-induced browning was shown to be regulated by the coordination of AMPK, p38, and SIRT1 signaling pathways. The ability of AJLE to inhibit adipogenesis and induce brown adipocyte differentiation may help treat obesity and related diseases.

Leptin deficiency impairs adipogenesis and browning response in mouse mesenchymal progenitors

Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning.

Mitochondrial respiratory complex I deficiency inhibits brown adipogenesis by limiting heme regulation of histone demethylation

Mitochondrial functions play a crucial role in determining the metabolic and thermogenic status of brown adipocytes. Increasing evidence reveals that the mitochondrial oxidative phosphorylation (OXPHOS) system plays an important role in brown adipogenesis, but the mechanistic insights are limited. Herein, we explored the potential metabolic mechanisms leading to OXPHOS regulation of brown adipogenesis in pharmacological and genetic models of mitochondrial respiratory complex I deficiency. OXPHOS deficiency inhibits brown adipogenesis through disruption of the brown adipogenic transcription circuit without affecting ATP levels. Neither blockage of calcium signaling nor antioxidant treatment can rescue the suppressed brown adipogenesis. Metabolomics analysis revealed a decrease in levels of tricarboxylic acid cycle intermediates and heme. Heme supplementation specifically enhances respiratory complex I activity without affecting complex II and partially reverses the inhibited brown adipogenesis by OXPHOS deficiency. Moreover, the regulation of brown adipogenesis by the OXPHOS-heme axis may be due to the suppressed histone methylation status by increasing histone demethylation. In summary, our findings identified a heme-sensing retrograde signaling pathway that connects mitochondrial OXPHOS to the regulation of brown adipocyte differentiation and metabolic functions.

Dietary Apigenin Relieves Body Weight and Glycolipid Metabolic Disturbance via Pro-Browning of White Adipose Mediated by Autophagy Inhibition.

Apigenin (AP) has many pharmacological activities, including anti-inflammation, hyperlipidemia-lowering, and so on. Previous studies show that AP can reduce lipid accumulation in adipocytes in vitro. However, it remains unclear whether and how AP can promote fat-browning. Therefore, mouse obesity model and preadipocyte induction model in vitro are used to investigate the effects of AP on glycolipid metabolism, browning and autophagy as well as the possible mechanisms. The obese mice are intragastrically administrated with AP (0.1mgg-1 d-1 ) for 4 weeks; meanwhile, the differentiating preadipocytes are respectively treated with the indicated concentrations of AP for 48h. Metabolic phenotype, lipid accumulation, and fat-browning are respectively evaluated by morphological, functional, and specific markers analysis. The results show that AP treatment alleviates the body weight, glycolipid metabolic disorder, and insulin resistance in the obese mice , which is contributed to the pro-browning effects of AP in vivo and in vitro. Moreover, the study finds that the pro-browning effect of AP is accomplished through autophagy inhibition mediated by the activation of PI3K-Akt-mTORpathway. The findings highlight that autophagy inhibition promotes the browning of white adipocytes and suggest that AP would prevent and treat obesity and the associated metabolic disorders.

Acteoside improves adipocyte browning by CDK6-mediated mTORC1-TFEB pathway

Adipocyte browning increases energy expenditure by thermogenesis, which has been considered a potential strategy against obesity and its related metabolic diseases. Phytochemicals derived from natural products with the ability to improve adipocyte thermogenesis have aroused extensive attention. Acteoside (Act), a phenylethanoid glycoside, exists in various medicinal or edible plants and has been shown to regulate metabolic disorders. Here, the browning effect of Act was evaluated by stimulating beige cell differentiation from the stromal vascular fraction (SVF) in the inguinal white adipose tissue (iWAT) and 3T3-L1 preadipocytes, and by converting the iWAT-SVF derived mature white adipocytes. Act improves adipocyte browning by differentiation of the stem/progenitors into beige cells and by direct conversion of mature white adipocytes into beige cells. Mechanistically, Act inhibited CDK6 and mTOR, and consequently relieved phosphorylation of the transcription factor EB (TFEB) and increased its nuclear retention, leading to induction of PGC-1α, a driver of mitochondrial biogenesis, and UCP1-dependent browning. These data thus unveil a CDK6-mTORC1-TFEB pathway that regulates Act-induced adipocyte browning.

Applicability and Limitations of Fluorescence Intensity-Based Thermometry Using a Palette of Organelle Thermometers

Fluorescence thermometry is a microscopy technique in which a fluorescent temperature sensor records temperature changes as alterations of fluorescence signals. Fluorescence lifetime imaging (FLIM) is a promising method for quantitative analysis of intracellular temperature. Recently, we developed small-molecule thermometers, termed Organelle Thermo Greens, that target various organelles and achieved quantitative temperature mapping using FLIM. Despite its highly quantitative nature, FLIM-based thermometry cannot be used widely due to expensive instrumentation. Here, we investigated the applicability and limitations of fluorescence intensity (FI)-based analysis, which is more commonly used than FLIM-based thermometry. Temperature gradients generated by artificial heat sources and physiological heat produced by brown adipocytes were visualized using FI- and FLIM-based thermometry. By comparing the two thermometry techniques, we examined how the shapes of organelles and cells affect the accuracy of the temperature measurements. Based on the results, we concluded that FI-based thermometry could be used for “qualitative”, rather than quantitative, thermometry under the limited condition that the shape change and the dye leakage from the target organelle were not critical.

Molecular and cellular regulation of thermogenic fat.

Thermogenic fat, consisting of brown and beige adipocytes, dissipates energy in the form of heat, in contrast to the characteristics of white adipocytes that store energy. Increasing energy expenditure by activating brown adipocytes or inducing beige adipocytes is a potential therapeutic strategy for treating obesity and type 2 diabetes. Thus, a better understanding of the underlying mechanisms of thermogenesis provides novel therapeutic interventions for metabolic diseases. In this review, we summarize the recent advances in the molecular regulation of thermogenesis, focusing on transcription factors, epigenetic regulators, metabolites, and non-coding RNAs. We further discuss the intercellular and inter-organ crosstalk that regulate thermogenesis, considering the heterogeneity and complex tissue microenvironment of thermogenic fat.

HIGD1A links SIRT1 activity to adipose browning by inhibiting the ROS/DNA damage pathway

Energy-dissipating adipocytes have the potential to improve metabolic health. Here, we identify hypoxia-induced gene domain protein-1a (HIGD1A), a mitochondrial inner membrane protein, as a positive regulator of adipose browning. HIGD1A is induced in thermogenic fats by cold exposure. Peroxisome proliferator-activated receptor gamma (PPARγ) transactivates HIGD1A expression synergistically with peroxisome proliferators-activated receptor γ coactivator α (PGC1α). HIGD1A knockdown inhibits adipocyte browning, whereas HIGD1A upregulation promotes the browning process. Mechanistically, HIGD1A deficiency impairs mitochondrial respiration to increase reactive oxygen species (ROS) level. This increases NAD+ consumption for DNA damage repair and curtails the NAD+/NADH ratio, which inhibits sirtuin1 (SIRT1) activity, thereby compromising adipocyte browning. Conversely, overexpression of HIGD1A blunts the above process to promote adaptive thermogenesis. Furthermore, mice with HIGD1A knockdown in inguinal and brown fat have impaired thermogenesis and are prone to diet-induced obesity (DIO). Overexpression of HIGD1A favors adipose tissue browning, ultimately preventing DIO and metabolic disorders. Thus, the mitochondrial protein HIGD1A links SIRT1 activity to adipocyte browning by inhibiting ROS levels.

UNCOUPLING PROTEIN UCP1 EXPRESSION DYNAMICS IN ADIPOSE TISSUES OF THE OUTBRED ICR MICE IN POSTNATAL ONTOGENESIS

Uncoupling protein (UCP1) uncouples mitochondrial respiration from ATP synthesis, resulting in heat production in brown and beige adipocytes. The presence of adipocytes with UCP1 expression in fat depots has been shown to promote metabolic health and provide protection against metabolic disorders. It stimulates interest in studying the age dynamics of UCP1 expression. There are few data available, mainly obtained on the C57Bl/6J mouse line predisposed to obesity and cover either early or late ontogenesis. In our study, for the first time, the expression of the UCP1 protein in the adipose tissues of male ICR mice was studied from the weaning to old age. Interscapular brown adipose tissue (BAT), inguinal and perigonadal white adipose tissue (IWAT and GWAT) of 20-day, 1.5, 6, 18 months mice were collected. UCP1 levels were detected by western-blotting. IWAT UCP1 expression decreased by 2 times between 20 days and 1.5 months. No UCP1 bands on blots from mice older than 1.5 months were observed. In gonadal depot UCP1 was detected only in 30% of the samples from 1.5- and 6‑months old mice, and UCP1 expression level was ten times lower in compare to inguinal depot. No statistically significant changes in UCP1 protein expression were detected in brown adipose tissue. The physiological role of UCP1-expressing cells in GWAT is discussed, as well as a possible relationship between the timing and rate of UCP1 expression decrease during the growth and maturation of reproductive function with the activation of lipogenesis in inguinal adipose tissue.

Transcriptomic Analysis Reveals Fibroblast Growth Factor 11 (FGF11) Role in Brown Adipocytes in Thermogenic Regulation of Goats.

Brown adipose tissue (BAT) is the main site of adaptive thermogenesis, generates heat to maintain body temperature upon cold exposure, and protects against obesity by promoting energy expenditure. RNA-seq analysis revealed that FGF11 is enriched in BAT. However, the functions and regulatory mechanisms of FGF11 in BAT thermogenesis are still limited. In this study, we found that FGF11 was significantly enriched in goat BAT compared with white adipose tissue (WAT). Gain- and loss-of-function experiments revealed that FGF11 promoted differentiation and thermogenesis in brown adipocytes. However, FGF11 had no effect on white adipocyte differentiation. Furthermore, FGF11 promoted the expression of the UCP1 protein and an EBF2 element was responsible for UCP1 promoter activity. Additionally, FGF11 induced UCP1 gene expression through promoting EBF2 binding to the UCP1 promoter. These results revealed that FGF11 promotes differentiation and thermogenesis in brown adipocytes but not in white adipocytes of goats. These findings provide evidence for FGF11 and transcription factor regulatory functions in controlling brown adipose thermogenesis of goats.

Glibenclamide promotes FGF21 secretion in interscapular BAT and attenuates depression-like behaviors in male mice with HFD-induced obesity

AimsEpidemiological evidence suggests that comorbidity of obesity and depression is extremely common and continues to grow in prevalence. However, the mechanisms connecting these two conditions are unknown. In this study, we explored how treatment with KATP channel blocker glibenclamide (GB) or the well-known metabolic regulator FGF21 impact male mice with high-fat diet (HFD)-induced obesity and depressive-like behaviors. Materials and methodsMice were fed with HFD for 12 weeks and then treated with recombinant FGF21 protein by infusion for 2 weeks, followed by intraperitoneal injection of 3 mg/kg recombinant FGF21 once per day for 4 days. Measurements were made of catecholamine levels, energy expenditure, biochemical endpoints and behavior tests, including sucrose preference and forced swim tests were. Alternatively, animals were infused with GB into brown adipose tissue (BAT). The WT-1 brown adipocyte cell line was used for molecular studies. Key findingsCompared to HFD controls, HFD + FGF21 mice exhibited less severe metabolic disorder symptoms, improved depressive-like behaviors, and more extensive mesolimbic dopamine projections. FGF21 treatment also rescued HFD-induced dysregulation of FGF21 receptors (FGFR1 and co-receptor β-klotho) in the ventral tegmental area (VTA), and it altered dopaminergic neuron activity and morphology in HFD-fed mice. Importantly, we also found that FGF21 mRNA level and FGF21 release were increased in BAT after administration of GB, and GB treatment to BAT reversed HFD-induced dysregulation of FGF21 receptors in the VTA. SignificanceGB administration to BAT stimulates FGF21 production in BAT, corrects HFD-induced dysregulation of FGF21 receptor dimers in VTA dopaminergic neurons, and attenuates depression-like symptoms.

Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

AbstractAdipocytes thermogenesis is an important mechanism for increasing energy expenditure. Here, screening of thermogenic activators (Sirt1 and Ucp1) investigated in C3H10T1/2 cells revealed ethanol extract of lotus leaf (LLE) may exert higher activity compared with that of the other 11 ingredients. UPLC‐MS/MS analysis showed that miquelianin was identified as the main flavonoid compounds in LLE (12.8%) and therefore deserves further investigation. The results in C3H10T1/2 cells revealed miquelianin promoted lipolysis, enhanced thermogenic programming (Sirt1, Ppargc1a, Prdm16, Cox7a, Nrf1, Cox2, and Ucp1), and induced brown‐like adipocyte formation (Fgf21, Cd137, Tmem26, Tbx1, Cd40, and Cited1). Moreover, miquelianin increased mitochondrial abundance, mitochondrial membrane potential, and NAD+/NADH ratio, suggesting its potential in mitochondria activity. We also observed that miquelianin improved mitochondrial quality by the establishment of mitochondrial dynamics (Mfn1, Mfn2, Opa1, Drp1, and Fis1) and subsequent suppression of autophagy. Chemical inhibition experiment revealed miquelianin may trigger browning via p38‐PINK1‐PARKIN‐mediated mitophagy. Furthermore, in ML385‐treated cells, NRF2, HMOX1, TFAM, and UCP1 were compromised by miquelianin treatment, suggesting that NRF2 signaling is necessary during miquelianin‐induced mitochondria biogenesis. Together, our findings demonstrate that miquelianin may induce browning and mitochondria thermogenic programing by suppressing mitophagy via p38‐PINK1‐PARKIN and mimicking NRF2 signaling.

Differences in the metabolic profiles of brown and white adipocytes based on secretomics

Brown adipocytes and white adipocytes play important roles in systemic metabolism and energy homeostasis. Recent studies have demonstrated that white adipocytes and brown adipocytes secrete numerous adipokines and thus act as endocrine cells. However, differences in the metabolites secreted from white adipocytes and brown adipocytes have never been reported. In this study, we assessed the metabolites secreted from white adipocytes and brown adipocytes. In total, the levels of 47 metabolites in brown adipocytes were significantly different from those in white adipocytes, with 31 high and 16 low in brown adipocytes as compared with those in white adipocytes. We classified these secreted metabolites as amino acids and peptides, fatty acids, and conjugates, glycerophosphocholines, furanones, and trichloroacetic acids. In addition, we identified the glycerophospholipid metabolism activated in white adipocytes, and these differentially expressed metabolites were associated with the mitogen-activated protein kinase pathway and Janus kinase-signal transducer and activator of transcription signaling pathway according to the Ingenuity Pathway Analysis (IPA) software analysis. This study revealed novel metabolites secreted from brown adipocytes and white adipocytes, and these metabolites from adipocytes may perform specific biological functions based on the type of adipocyte that secretes them, and this forms the material basis of the interaction between adipocytes and other cells.

Mitochondrial matrix protein LETMD1 maintains thermogenic capacity of brown adipose tissue in male mice

Brown adipose tissue (BAT) has abundant mitochondria with the unique capability of generating heat via uncoupled respiration. Mitochondrial uncoupling protein 1 (UCP1) is activated in BAT during cold stress and dissipates mitochondrial proton motive force generated by the electron transport chain to generate heat. However, other mitochondrial factors required for brown adipocyte respiration and thermogenesis under cold stress are largely unknown. Here, we show LETM1 domain-containing protein 1 (LETMD1) is a BAT-enriched and cold-induced protein required for cold-stimulated respiration and thermogenesis of BAT. Proximity labeling studies reveal that LETMD1 is a mitochondrial matrix protein. Letmd1 knockout male mice display aberrant BAT mitochondria and fail to carry out adaptive thermogenesis under cold stress. Letmd1 knockout BAT is deficient in oxidative phosphorylation (OXPHOS) complex proteins and has impaired mitochondrial respiration. In addition, BAT-specific Letmd1 deficient mice exhibit phenotypes identical to those observed in Letmd1 knockout mice. Collectively, we demonstrate that the BAT-enriched mitochondrial matrix protein LETMD1 plays a tissue-autonomous role that is essential for BAT mitochondrial function and thermogenesis.

Type II Transmembrane Serine Proteases as Modulators in Adipose Tissue Phenotype and Function.

Adipose tissue is a crucial organ in energy metabolism and thermoregulation. Adipose tissue phenotype is controlled by various signaling mechanisms under pathophysiological conditions. Type II transmembrane serine proteases (TTSPs) are a group of trypsin-like enzymes anchoring on the cell surface. These proteases act in diverse tissues to regulate physiological processes, such as food digestion, salt-water balance, iron metabolism, epithelial integrity, and auditory nerve development. More recently, several members of the TTSP family, namely, hepsin, matriptase-2, and corin, have been shown to play a role in regulating lipid metabolism, adipose tissue phenotype, and thermogenesis, via direct growth factor activation or indirect hormonal mechanisms. In mice, hepsin deficiency increases adipose browning and protects from high-fat diet-induced hyperglycemia, hyperlipidemia, and obesity. Similarly, matriptase-2 deficiency increases fat lipolysis and reduces obesity and hepatic steatosis in high-fat diet-fed mice. In contrast, corin deficiency increases white adipose weights and cell sizes, suppresses adipocyte browning and thermogenic responses, and causes cold intolerance in mice. These findings highlight an important role of TTSPs in modifying cellular phenotype and function in adipose tissue. In this review, we provide a brief description about TTSPs and discuss recent findings regarding the role of hepsin, matriptase-2, and corin in regulating adipose tissue phenotype, energy metabolism, and thermogenic responses.

LncDGAT2 is a novel positive regulator of the goat adipocyte thermogenic gene program

Brown and beige adipose thermogenesis are important for newborn mammals to maintain their body temperature. In addition, these thermogenic fats are regulated by multiple molecular interactions. How the long non-coding RNAs (lncRNAs) regulate adipose thermogenesis in newborn mammals upon cold exposure remains unexplored. Here, we identified lncRNAs induced by cold exposure in brown adipose tissue (BAT) of newborn goats and found that lncDGAT2 was enriched in BAT after cold exposure. Functional studies revealed that lncDGAT2 promoted brown and white adipocyte differentiation as well as thermogenic gene expression. Additionally, PRDM4 directly bound the lncDGAT2 promoter to activate the transcription of lncDGAT2 and the PRDM4-lncDGAT2 axis was essential for the brown adipocyte thermogenic gene program. These findings provide evidence for lncRNA and transcription factor regulatory functions in controlling adipose thermogenesis and energy metabolism of newborn goats.

1670-P: Sex-Dependent Effects of PERK Deletion in Thermogenic Adipocytes

Brown and beige adipocytes dissipate chemical energy as heat through thermogenic respiration, thereby playing important roles in adaptive thermogenesis and energy homeostasis in mice. The Protein Kinase R (PKR)-Like Endoplasmic Reticulum Kinase (PERK) has been shown to be required for mitochondrial thermogenesis in cultured brown adipocytes, and in mice lacking PERK in white and brown adipose tissues. Here, we tested the hypothesis that PERK expression specifically in thermogenic adipocytes is required for adaptive thermogenesis and to regulate energy homeostasis in mice, by selectively deleting PERK in Ucp1-expressing cells (brown and beige adipocytes) of male and female mice. At baseline conditions, mitochondria oxygen consumption rates (OCR) were significantly reduced in brown adipocytes isolated from male, but not from female KO mice. After being housed at thermoneutrality for 7 days (30 °C), wild type (WT) and KO mice were exposed to 4 °C for 3 days. Surprisingly, both male and female KO mice were able to properly adapt to cold exposure, as demonstrated by preserved core body temperatures relative to WT mice. Interestingly, age-induced weight gain, fat mass expansion and impairments in glucose homeostasis were all attenuated in KO males, while weight gain was exacerbated in KO females. This correlated with increased energy expenditure (EE) in male mice, while EE was reduced in female mice. We, next, tested the hypothesis that male KO mice would be resistant to diet-induced obesity. After 12 weeks of high-fat feeding (60% calories from fat), male KO mice became as obese, glucose intolerant and insulin resistant as WT mice. In conclusion, our data suggest that PERK expression in thermogenic adipocytes reduces mitochondrial OCR in male mice, but is dispensable for thermoregulation after short-term cold exposure in both male and female mice. Furthermore, our data reveal a sex-dependent effect of PERK in thermogenic adipocytes in the regulation of energy homeostasis, which is dependent on changes in EE. Disclosure J.Jena: None. L.M.García-peña: None. R.Pereira: None. Funding National Institutes of Health (DK125405, 2T32DK112751-06)

1506-P: Effect of Beta-Agonist Treatment on Selenium Metabolism in Differentiated Brown Adipocytes

Selenium (Se) is an essential trace element important to several body processes such as metabolism, thyroid function, and curbing of oxidative stress. Se is utilized as the amino acid known selenocysteine (Sec), decomposed by the enzyme selenocysteine lyase (Scly). Selenoproteins have a broad range of functions, including protecting against lipid peroxidation and apoptosis, metabolic dysregulation such as glucose intolerance, and other immune responses. In brown adipocytes, the selenoprotein glutathione peroxidase 4 (GPX4) plays a key role in lipid peroxidation which in turn allows for proper thermogenic responses triggered by adrenergic signals. GPX4 removes toxic lipid peroxides by converting them into non-toxic alcohol and water. When Se levels are insufficient in brown adipocytes, the selenoprotein GPX4 may be downregulated, leading cells to accumulate a lethal amount of lipid peroxidases and triggering ferroptosis, a type of cell death. Ferroptosis activation, in turn, negatively impact pathways dependent on adrenergic responses in brown adipocytes, such as thermogenesis activation. To determine how Se levels regulate the expression of Scly and GPX4 upon treatment with adrenergic agonist in differentiated PAZ6 brown adipocytes. Human PAZ6 pre-adipocytes were differentiated for 21 days, then treated with insufficient (20 nM), adequate (80 nM), and high sodium selenite (160 nM) levels for 48h. Differentiated brown adipocytes were also treated with 1 μM CL-316243, a β3-adrenergic receptor agonist, for 24h after selenite treatment. Cells were then harvested for qPCR and Western blot analysis. Differentiated PAZ6 cells had an increase in UCP1 expression, a marker of brown adipocytes. Upon Se treatment, both Scly and GPX4 expression were unchanged in PAZ6 cells. Treatment with β3-adrenergic receptor agonist also did not affect the expression levels of both proteins. In differentiated brown adipocytes, expression of Scly and GPX4 is not dependent on Se nor β-agonist signals. Disclosure S.Swanson: None. Funding National Institutes of Health (R01DK128390)

1501-P: Deletion of miR-494 Induces Adipocyte Browning and Slows Aging in Mice

Adipocyte browning is a potent therapeutic strategy for obesity and metabolic disorders. We previously reported that miR-494 served as a negative regulator of mitochondrial biogenesis during browning in cultured adipocytes. However, the in vivo evidence has not been provided. In this study, we examined the role of miR-494 in adipocyte browning and aging using miR-494 knockout (KO) mice. KO mice were generated using CRISPR/Cas9 system. KO mice had equivalent energy expenditure, physical activity, food intake and body weight compared to wild-type (WT) mice; had lower resting respiratory quotient (0.95±0.006 vs 0.91±0.004), less visceral fat (16.3±1.08 vs 13.4±0.96 g/kg body weight) and 3.7-fold increased expression of Ucp1 in subcutaneous fat. In addition, KO mice showed cold tolerance during 3 hours exposure to 4ºC and increased oxygen consumption than WT mice after administration of a β3 agonist in thermoneutral condition, indicating an increase in non-shivering thermogenesis. In adipose tissues of KO mice, increased expression of Pgc-1α, a target gene of miR-494, and tendency to increase in mitochondrial DNA were observed. Primary adipocytes derived from KO mice showed enhanced mitochondrial respiration driven by fatty acids as determined by extracellular flux analysis. Finally, KO mice showed an extended median lifespan 29% relative to WT mice (from 113.7 to 146.7 weeks). Aged KO mice (≥ 2 years old) showed reduced age-related changes, such as hair graying and low physical activity. In conclusion, deletion of miR-494 demonstrated increased adipocyte browning and survival advantage in mice. Although there are numerous studies that focused on promoting adipocyte browning, its long-term effect has not been well evaluated. Thus, our data showing the association among miR-494, adipocyte browning and longevity provide a novel insight into adipocyte biology and aging study. The gene sequence of miR-494 and its binding site to Pgc-1α are conserved from mice to humans; it is expected to be applied to human research. Disclosure L.Sugawara: None. K.Morino: Research Support; AstraZeneca, Astellas Pharma Inc., Sanwa Kagaku Kenkyusho, Ono Pharmaceutical Co., Ltd. H.Iwasaki: None. S.Ida: None. T.Yanagimachi: None. M.Lemecha: None. H.Maegawa: None. Y.Fujita: Research Support; Terumo Corporation, Boehringer Ingelheim Japan, Inc., Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Sumitomo Dainippon Pharma Co., Ltd. S.Kume: Research Support; Boehringer Ingelheim International GmbH, Japan Society for the Promotion of Science. Funding Grants-in-Aid for Scientific Research (22K08650)