The role of ADP-ribose metabolism in metabolic regulation, adipose tissue differentiation, and metabolism.

Poly(ADP-ribose) polymerases (PARPs or ARTDs), originally described as DNA repair factors, have metabolic regulatory roles. PARP1, PARP2, PARP7, PARP10, and PARP14 regulate central and peripheral carbohydrate and lipid metabolism and often channel pathological disruptive metabolic signals. PARP1 and PARP2 are crucial for adipocyte differentiation, including the commitment toward white, brown, or beige adipose tissue lineages, as well as the regulation of lipid accumulation. Through regulating adipocyte function and organismal energy balance, PARPs play a role in obesity and the consequences of obesity. These findings can be translated into humans, as evidenced by studies on identical twins and SNPs affecting PARP activity.

Asparagine reinforces mTORC1 signaling to boost thermogenesis and glycolysis in adipose tissues.

Brown and beige fat are specialized for energy expenditure by dissipating energy from glucose and fatty acid oxidation as heat. While glucose and fatty acid metabolism have been extensively studied in thermogenic adipose tissues, the involvement of amino acids in regulating adaptive thermogenesis remains little studied. Here, we report that asparagine supplementation in brown and beige adipocytes drastically upregulated the thermogenic transcriptional program and lipogenic gene expression, so that asparagine-fed mice showed better cold tolerance. In mice with diet-induced obesity, the asparagine-fed group was more responsive to ß3-adrenergic receptor agonists, manifesting in blunted body weight gain and improved glucose tolerance. Metabolomics and 13 C-glucose flux analysis revealed that asparagine supplement spurred glycolysis to fuel thermogenesis and lipogenesis in adipocytes. Mechanistically, asparagine stimulated the mTORC1 pathway, which promoted expression of thermogenic genes and key enzymes in glycolysis. These findings show that asparagine bioavailability affects glycolytic and thermogenic activities in adipose tissues, providing a possible nutritional strategy for improving systemic energy homeostasis.

Sorting out adipocyte precursors and their role in physiology and disease.

The ability to maintain and expand the pool of adipocytes in adults is integral to the regulation of energy balance, tissue/stem cell homeostasis, and disease pathogenesis. For decades, our knowledge of adipocyte precursors has relied on cellular models. The identity of native adipocyte precursors has remained unclear. Recent studies have identified distinct adipocyte precursor populations that are physiologically regulated and contribute to the development, maintenance, and expansion of adipocyte pools in mice. With new tools available, the properties of adipocyte precursors can now be defined, and the regulation and function of adipose plasticity in development and physiology can be explored.

Brown Adipocyte ADRB3 Mediates Cardioprotection via Suppressing Exosomal iNOS.

BACKGROUND: The ADRB3 (ß3-adrenergic receptors), which is predominantly expressed in brown adipose tissue (BAT), can activate BAT and improve metabolic health. Previous studies indicate that the endocrine function of BAT is associated with cardiac homeostasis and diseases. Here, we investigate the role of ADRB3 activation-mediated BAT function in cardiac remodeling. METHODS: BKO (brown adipocyte-specific ADRB3 knockout) and littermate control mice were subjected to Ang II (angiotensin II) for 28 days. Exosomes from ADRB3 antagonist SR59230A (SR-exo) or agonist mirabegron (MR-exo) treated brown adipocytes were intravenously injected to Ang II-infused mice. RESULTS: BKO markedly accelerated cardiac hypertrophy and fibrosis compared with control mice after Ang II infusion. In vitro, ADRB3 KO rather than control brown adipocytes aggravated expression of fibrotic genes in cardiac fibroblasts, and this difference was not detected after exosome inhibitor treatment. Consistently, BKO brown adipocyte-derived exosomes accelerated Ang II-induced cardiac fibroblast dysfunction compared with control exosomes. Furthermore, SR-exo significantly aggravated Ang II-induced cardiac remodeling, whereas MR-exo attenuated cardiac dysfunction. Mechanistically, ADRB3 KO or SR59230A treatment in brown adipocytes resulted an increase of iNOS (inducible nitric oxide synthase) in exosomes. Knockdown of iNOS in brown adipocytes reversed SR-exo-aggravated cardiac remodeling. CONCLUSIONS: Our data illustrated a new endocrine pattern of BAT in regulating cardiac remodeling, suggesting that activation of ADRB3 in brown adipocytes offers cardiac protection through suppressing exosomal iNOS.

A modified system using macrophage-conditioned medium revealed that the indirect effects of anti-inflammatory food-derived compounds improve inflammation-induced suppression of UCP-1 mRNA expression in 10T1/2 adipocytes.

Recently, it has been suggested that brown and beige adipocytes may ameliorate obesity because these adipocytes express uncoupling protein-1 (UCP-1), which generates heat by consuming lipid. However, obesity-induced inflammation suppresses the expression of UCP-1. To improve such conditions, food components with anti-inflammatory properties are attracting attention. In this study, we developed a modified system to evaluate only the indirect effects of anti-inflammatory food-derived compounds by optimizing the conventional experimental system using conditioned medium. We validated this new system using 6-shogaol and 6-gingerol, which have been reported to show the anti-inflammatory effects and to increase the basal expression of UCP-1 mRNA. In addition, we found that the acetone extract of Sarcodon aspratus, an edible mushroom, showed anti-inflammatory effects and rescued the inflammation-induced suppression of UCP-1 mRNA expression. These findings indicate that the system with conditioned medium is valuable for evaluation of food-derived compounds with anti-inflammatory effects on the inflammation-induced thermogenic adipocyte dysfunction.

ß-Adrenergic Signal and Epigenomic Regulatory Process for Adaptive Thermogenesis.

Activation of ß-adrenergic (ß-AR) signaling induces fight-or-flight stress responses which include enhancement of cardiopulmonary function, metabolic regulation, and muscle contraction. Classical dogma for ß-AR signaling has dictated that the receptor-mediated response results in an acute and transient signal. However, more recent studies support more wide-ranging roles for ß-AR signaling with long-term effects including cell differentiation that requires precisely timed and coordinated integration of many signaling pathways that culminate in precise epigenomic chromatin modifications. In this chapter, we discuss cold stress/ß-AR signaling-induced epigenomic changes in adipose tissues that influence adaptive thermogenesis. We highlight recent studies showing dual roles for the histone demethylase JMJD1A as a mediator of both acute and chronic thermogenic responses to cold stress, in two distinct thermogenic tissues, and through two distinct molecular mechanisms. ß-AR signaling not only functions through transient signals during acute stress responses but also relays a more sustained signal to long-term adaptation to environmental changes.

Transcriptomics profiling reveal the heterogeneity of white and brown adipocyte.

The marker genes associated with white adipocytes and brown adipocytes have been previously identified; however, these markers have not been updated in several years, and the differentiation process of preadipocytes remains relatively fixed. Consequently, there has been a lack of exploration into alternative differentiation schemes. In this particular study, we present a transcriptional signature specific to brown adipocytes and white adipocytes. Notably, our findings reveal that ZNF497, ZIC1, ZFY, UTY, USP9Y, TXLNGY, TTTY14, TNNT3, TNNT2, TNNT1, TNNI1, TNNC1, TDRD15, SOX11, SLN, SFRP2, PRKY, PAX3KLHL40, PAX3, INKA2-AS1, SOX11, and TDRD15 exhibit high expression levels in brown adipocytes. XIST, HOXA10, PCAT19, HOXA7, PLSCR3, and AVPR1A exhibited high expression levels in white adipocytes, suggesting their potential as novel marker genes for the transition from white to brown adipocytes. Furthermore, our analysis revealed the coordinated activation of several pathways, including the PPAR signaling pathway, focal adhesion, retrograde endocannabinoid signaling, oxidative phosphorylation, PI3K-Akt signaling pathway, and thermogenesis pathways, in brown adipocytes. Moreover, in contrast to prevailing culture techniques, we conducted a comparative analysis of the differentiation protocols for white preadipocytes and brown preadipocytes, revealing that the differentiation outcome remained unaffected by the diverse culture schemes employed. However, the expression levels of certain marker genes in both adipocyte types were found to be altered. This investigation not only identified potential novel marker genes for adipocytes but also examined the impact of different differentiation methods on preadipocyte maturation. Consequently, these findings offer significant insights for further research on the differentiation processes of diverse adipocyte subtypes.

The Role of Thermogenic Fat Tissue in Energy Consumption.

Mammalian adipose tissues are broadly divided into white adipose tissue (WAT) and thermogenic fat tissue (brown adipose tissue and beige adipose tissue). Uncoupling protein 1 (UCP1) is the central protein in thermogenesis, and cells that exhibit induced UCP1 expression and appear scattered throughout WAT are called beige adipocytes, and their induction in WAT is referred to as "beiging". Beige adipocytes can differentiate from preadipocytes or convert from mature adipocytes. UCP1 was thought to contribute to non-shivering thermogenesis; however, recent studies demonstrated the presence of UCP1-independent thermogenic mechanisms. There is evidence that thermogenic fat tissue contributes to systemic energy expenditure even in human beings. This review discusses the roles that thermogenic fat tissue plays in energy consumption and offers insight into the possibility and challenges associated with its application in the treatment of obesity and type 2 diabetes.

An indispensable role for dynamin-related protein 1 in beige and brown adipogenesis.

Emerging evidence indicates that proper mitochondrial dynamics are critical for adipocyte differentiation and functional thermogenic capacity. We found that the mitochondrial fission protein dynamin-related protein 1 (DRP1, also known as DNML1) is highly expressed in brown adipose tissue compared to expression in white adipose tissue, and these expression levels increase during brown adipocyte differentiation. Our results reveal that the inhibition of DRP1 using mdivi-1 mitigates beige adipocyte differentiation and differentiation-associated mitochondrial biogenesis. We found that DRP1 is essential for the induction of the early-phase beige adipogenic transcriptional program. Intriguingly, inhibition of DRP1 is dispensable following the induction of beige adipogenesis and adipogenesis-associated mitochondrial biogenesis. Altogether, we demonstrate that DRP1 in preadipocytes plays an essential role in beige and brown adipogenesis.This article has an associated First Person interview with the first author of the paper.

Stimulation of uncoupling protein 1 expression by ß-alanine in brown adipocytes.

Carnosine, which is abundant in meat, is a dipeptide composed of ß-alanine and histidine, known to afford various health benefits. It has been suggested that carnosine can elicit an anti-obesity effect via induction and activation of brown/beige adipocytes responsible for non-shivering thermogenesis. However, the relationship between carnosine and brown/beige adipocytes has not been comprehensively elucidated. We hypothesized that ß-alanine directly modulates brown/beige adipogenesis and performed an in vitro assessment to test this hypothesis. HB2 brown preadipocytes were differentiated using insulin from day 0. Cells were treated with various concentrations of ß-alanine (12.5-100 µM) during adipogenesis (days 0-8) and differentiation (days 8-10). Then, cells were further stimulated with or without forskolin, an activator of the cAMP-dependent protein kinase pathway, on day 8 or day 10 for 4 h before harvesting. We observed that HB2 cells expressed molecules related to the transport and signal transduction of ß-alanine. Treatment with ß-alanine during brown adipogenesis dose-dependently enhanced forskolin-induced Ucp1 expression; this was not observed in differentiated brown adipocytes. Consistent with these findings, treatment with ß-alanine during days 0-8 increased phosphorylation levels of CREB in forskolin-treated HB2 cells. In addition, ß-alanine treatment during brown adipogenesis increased the expression of Pparα, known to induce brown/beige adipogenesis, in a dose-dependent manner. These findings revealed that ß-alanine could target HB2 adipogenic cells and enhance forskolin-induced Ucp1 expression during brown adipogenesis, possibly by accelerating phosphorylation and activation of CREB. Thus, ß-alanine, a carnosine-constituting amino acid, might directly act on brown adipogenic cells to stimulate energy expenditure.

Pyruvate carboxylase supports basal ATP-linked respiration in human pluripotent stem cell-derived brown adipocytes.

Brown adipocytes (BA) are a specialized fat cell which possesses a high capacity for fuel oxidation combined with heat production. The maintenance of high metabolic activity in BA requires elevated oxidation of fuel through the tricarboxylic acid cycle. Pyruvate carboxylase (PC) was previously proposed to be essential for coordination between fuel oxidation and thermogenesis. By differentiating human pluripotent stem cells to mature BA in vitro, we showed that ablation of PC gene by CRISPR Cas9 genome engineering did not impair the ability of stem cells to generate mature BA. However, brown adipocytes deficient for PC expression displayed a 35% reduction in ATP-linked respiration, but not thermogenesis under both basal and isoproterenol-stimulated conditions. This relatively mild impairment of ATP-link respiration in PC knockout BA was protected by increased spare mitochondrial respiratory capacity. Taken together, this study highlights the role of PC in supporting fuel oxidation rather than thermogenesis in human BA.

Identification of two natural coumarin enantiomers for selective inhibition of TRPV2 channels.

Thermosensitive transient receptor potential vanilloid 2 (thermoTRPV2) is a nonselective Ca2+ -permeable cation channel broadly expressed, and is implicated in the pathology of diseases such as diabetes and pancreatitis. However, the physiological and pharmacological functions of TRPV2 channels have not been extensively investigated because of the absence of specific modulators. In this study, we report a pair of natural coumarin derivative enantiomers (-)-murraxocin (B304-1) and (+)-murraxocin (B304-2) from Murraya exotica for their selective inhibition of TRPV2 channels expressed in HEK293 cells and native TRPV2 currents in differentiated brown adipocytes. Whole-cell patch clamp recordings confirmed the enantiomers B304-1 and B304-2 could selectively inhibit the agonist mediated activation of TRPV2 current with IC50 values of 22.2 ± 7.8 µM and 3.7 ± 0.7 µM, respectively. Molecular docking and site-directed mutagenesis revealed a key residue I600 of TRPV2 critical for the binding of the enantiomers. Furthermore, B304-1 and B304-2 significantly reversed TRPV2 agonist-induced inhibition of mouse brown adipocyte differentiation. Taken together, our identification of two natural coumarin enantiomers provides valuable tools and chemical leads for further elucidation of TRPV2 channel function, and pharmacological modulation of thermoTRPV2 in brown adipocytes may represent a new therapeutic strategy for treatment of energy imbalance or metabolic disorders.

Resveratrol and HIV-protease inhibitors control UCP1 expression through opposite effects on p38 MAPK phosphorylation in human adipocytes.

Brown and brown-like adipocytes (BBAs) control thermogenesis and are detected in adult humans. They express UCP1, which transforms energy into heat. They appear as promising cells to fight obesity. Deciphering the molecular mechanisms leading to the browning of human white adipocytes or the whitening of BBAs represents a goal to properly and safely control the pathways involved in these processes. Here, we analyzed how drugs endowed with therapeutic potential affect the differentiation of human adipose progenitor-cells into BBAs and/or their phenotype. We showed that HIV-protease inhibitors (PI) reduced UCP1 expression in BBAs modifying their metabolic profile and the mitochondria functionality. Lopinavir (LPV) was more potent than darunavir (DRV), a last PI generation. PPARγ and PGC-1α were decreased in a PI or cell-specific manner, thus altering UCP1's constitutive expression. In addition, LPV altered p38 MAPK phosphorylation, blunting then the ß-adrenergic responses. In contrast, low doses of resveratrol stimulated the activatable expression of UCP1 in a p38 MAPK-dependent manner and counteracted the LPV induced loss of UCP1. This effect was independent of the resveratrol-induced sirtuin-1 expression. Altogether our results uncover how drugs impact crucial components of the networks regulating the expression of the thermogenic signature. They provide important information to control the relevant pathways involved in energy expenditure.

MiR-92a regulates brown adipocytes differentiation, mitochondrial oxidative respiration, and heat generation by targeting SMAD7.

Brown adipocytes are rich in mitochondria and linked to the body's blood fat levels and obesity. MiR-92a is negatively correlated with the activity of brown adipocytes. This study aimed to explore the mechanism of miR-92a on brown adipocytes. The expression of miR-92a in C2C12 cell was detected by a quantitative real-time-polymerase chain reaction (qRT-PCR). C2C12 cells were induced to brown adipocytes. The direct target gene of miR-92a was determined using the dual-luciferase reporter assay. Brown adipocytes were treated with isoprenaline (Iso) and transfected by miR-92a inhibitor and siSMAD7. The expression of heat-producing genes and adipose differentiation genes related to brown adipocytes were detected by qRT-PCR and Western blot analysis. The expression of SMAD7, p-SMAD2, and p-SMAD3 were detected using Western blot analysis. The mitochondrial content was measured by mitotracker fluorescent staining. MiR-92a inhibitor significantly decreased the expression of miR-92a in C2C12 cells. MiR-92a inhibitor could upregulate the expression of Ucp1, Cox7a1, Elovl3, Ppargc1α, PPARγ, and FABP4, and its effect on Ucp1 was increased after the treatment of isoprenaline. Moreover, miR-92a inhibitor increased mitochondrial content, oxygen consumption rate (OCR) and the expression of SMAD7 and suppressed the expressions of p-SMAD2 and p-SMAD3, whereas miR-92a directly targeted SMAD7 to exert its inhibitory effects. SiSMAD7 reversed the effects of the inhibitor on heat-producing genes, mitochondrial content, OCR and the expressions of SMAD7, p-SMAD2, and p-SMAD3 in brown adipocytes. Blocking miR-92a might promote brown adipocytes differentiation, mitochondrial oxidative respiration, and thermogenesis by targeting SMAD7 to inhibit the expressions of p-SMAD2 and p-SMAD3.

Brown adipocyte-derived exosomal miR-132-3p suppress hepatic Srebf1 expression and thereby attenuate expression of lipogenic genes.

Recent evidence has revealed a novel signaling mechanism through which brown adipose tissue (BAT)-derived exosomal microRNAs (miRNAs) influence hepatic gene expression. Here, we uncover neuronal control of these miRNAs and identify exosomal miR-132-3p as a regulator of hepatic lipogenesis under cold stress conditions. Norepinephrine, a sympathetic nervous system neurotransmitter mediating cold-induced BAT activation, altered the composition of brown adipocyte (BAC)-derived exosomal miRNAs; among them, miR-132-3p was significantly induced. The isolated BAC-derived exosomes suppressed expression of hepatic Srebf1, a predicted target of miR-132-3p. In an indirect co-culture system, BACs suppressed expression of hepatic Srebf1 and its target lipogenic genes; this effect was not seen with miR-132-3p-inhibited BACs. Srebf1 was experimentally validated as an miR-132-3p target. Cold stimuli consistently induced miR-132-3p expression in BAT and attenuated Srebf1 expression in the liver. Our results suggest that BAT-derived exosomal miR-132-3p acts as an endocrine factor that regulates hepatic lipogenesis for cold adaptation.

Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the ß3 adrenergic stimulation.

Bone marrow provides progenitors of several types of cells, including muscle and white adipocytes, ensuring peripheral tissue homeostasis. However, the role of bone marrow-derived cells (BMCs) in induction of thermogenic adipocytes is unresolved. The purpose of this study is to examine whether BMCs are involved in the emergence of thermogenic adipocytes through adrenergic activation. Irradiation of mice with 8 Gy of X-ray-depleted BMCs and peripheral blood mononucleated cells (PBMCs), which in turn impaired induction of uncoupling protein 1 (UCP1) through administration of ß3 adrenergic receptor agonist, CL 316,243 (CL), in inguinal white adipose tissue (iWAT). In contrast, CL-induced UCP1 induction in brown adipose tissue was unaffected by BMC depletion. Transplantation of normal BMCs into mice depleted of BMCs recovered PBMC levels and rescued the ability of iWAT browning by CL. Furthermore, analyses of mice transplanted with green fluorescent protein (GFP)-labeled BMCs revealed that the number of GFP-positive BMCs and PBMCs were significantly decreased by CL and that GFP-positive stromal cells and GFP-positive UCP1-expressing multilocular adipocytes appeared in iWAT after CL administration, demonstrating differentiation of BMC-derived preadipocytes into UCP1-expressing thermogenic adipocytes. These results unveiled a crucial role of the BMC as a nonresident origin for a subset of thermogenic adipocytes, contributing to browning of white adipose tissue.-Yoneshiro, T., Shin, W., Machida, K., Fukano, K., Tsubota, A., Chen, Y., Yasui, H., Inanami, O., Okamatsu-Ogura, Y., Kimura, K. Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the ß3 adrenergic stimulation.

Macrophages and brown adipocytes cross-communicate to modulate a thermogenic program following methamphetamine exposure.

Hyperthermia is a potentially lethal side-effect of Methamphetamine (Meth), a stimulant drug. Activation of non-shivering thermogenesis in brown adipose tissue (BAT) is partly responsible for Meth-induced rise in temperature, with contributing sympathetic neurotransmitters, such as norepinephrine (NE), and reactive oxygen species (ROS). However, the mechanisms controlling the development of a molecular thermogenic program in brown adipocytes (BA) following Meth are unknown. We hypothesize that Meth and NE affect BAT cells, BA and macrophages, to modify their physiology and interactions, with consequences to thermogenic genes. We also hypothesize that ROS play a critical role in signaling transcription of thermogenic genes and their regulatory components. Using primary BA and macrophage cultures, we measured Meth and NE interference with physiological and phenotypic measures that are relevant to thermogenesis in BAT. Meth caused both BA and macrophages to decrease mitochondrial maximal capacity and increase ROS. In BA, the thermogenic genes UCP1, PPARγ, PGC1α and GADD45γ were transcriptionally increased by Meth in a ROS-dependent manner. In macrophages, Meth increased oxidative stress response and caused a predominance of M2 subset markers. BA transcriptional changes in response to Meth and NE were significantly controlled by macrophages. The results suggest that BA and macrophages respond to Meth and NE, with effects on mitochondrial functions and transcription of genes involved in thermogenesis. ROS-dependent signals in BA and cellular interactions between BA and macrophages synergize to regulate the BAT environment and control critical pathways leading to Meth-hyperthermia.

Comparative Secretome Analyses of Primary Murine White and Brown Adipocytes Reveal Novel Adipokines.

The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that carbohydrate-metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete a large variety of proteins. Upon NE stimulation, an increased secretion of known adipokines is favored by white adipocytes while brown adipocytes secreted higher amounts of novel adipokines. Furthermore, the secretory response between NE-stimulated and basal state was multifaceted addressing lipid and glucose metabolism, adipogenesis, and antioxidative reactions. Intriguingly, NE stimulation drastically changed the secretome in brown adipocytes. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE. Given the beneficial effects of brown adipose tissue activation on its endocrine function and systemic metabolism, this study provides an archive of novel batokine candidates and biomarkers for activated brown adipose tissue.

Isoliquiritigenin Enhances the Beige Adipocyte Potential of Adipose-Derived Stem Cells by JNK Inhibition.

Human adipose-derived stem cells (hASCs) can be isolated from fat tissue and have attracted interest for their potential therapeutic applications in metabolic disease. hASCs can be induced to undergo adipogenic differentiation in vitro by exposure to chemical agents or inductive growth factors. We investigated the effects and mechanism of differentiating hASC-derived white adipocytes into functional beige and brown adipocytes with isoliquiritigenin (ILG) treatment. Here, we showed that hASC-derived white adipocytes could promote brown adipogenesis by expressing both uncoupling protein 1 (UCP1) and PR/SET Domain 16 (PRDM16) following low-dose ILG treatments. ILG treatment of white adipocytes enhanced the expression of brown fat-specific markers, while the expression levels of c-Jun N-terminal kinase (JNK) signaling pathway proteins were downregulated. Furthermore, we showed that the inhibition of JNK phosphorylation contributed to white adipocyte differentiation into beige adipocytes, which was validated by the use of SP600125. We identified distinct regulatory effects of ILG dose responses and suggested that low-dose ILG induced the beige adipocyte potential of hASCs via JNK inhibition.

Jmjd maintains the morphology and function of lipid droplets and mitochondria of brown adipocytes in mice. / Jmjd3ç»´æŒå°é¼ æ£•è‰²è„‚è‚ªç»†èƒžè„‚æ»´åŠçº¿ç²’ä½“çš„å½¢æ€å’ŒåŠŸèƒ½.

OBJECTIVES: Jmjd3 can promote the differentiation of brown adipocytes, but its role in the ultrastructure of lipid droplets and mitochondria, the two key thermogenic organelles, is still unclear. The aim of this study is to investigate the effects of histone H3K27me3 demethylase Jmjd3 deletion on lipid droplets and mitochondria in brown adipocytes in mice. METHODS: Jmjd3 general knockout (Jmjd3-/-) mice and adipose tissue specific conditional knockout (Jmjd3F/FFabp4-Cre+) mice were constructed.Brown adipose tissue (BAT) was taken from the back of Jmjd3-/- and their littermates (Jmjd3+/+) at the 19.5 days of embryo (E19.5). BAT was also taken from the back of 6-week-old Jmjd3F/FFabp4-Cre+ and their littermates (Jmjd3flox/+or Jmjd3+/+Fabp4-Cre+). The morphology of brown adipocytes was observed by light microscopy after HE staining. The ultrastructure of lipid droplets and mitochondria was observed by electron microscopy. Western blotting was used to detect the expression of H3K27me3, Jmjd3, and uncouple protein 1 (UCP1) in BAT. RT-qPCR was used to detect the expression of Jmjd3, peroxisome proliferator-activated receptor γ (PPARγ), fatty acid binding protein 4 (Fabp4), UCP1, PR domain-containing 16 (PRDM16), cell death-inducing DFFA-like effector a (CIDEa), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), mitochondrial cytochrome c oxidase subunit I (COX1), and cytochrome c (Cyt c). ATP content in the BAT was tested. The body temperature of Jmjd3F/FFabp4-Cre+ and their control mice during cold stimulation was detected. RESULTS: Two types of Jmjd3 knockout (Jmjd3-/- and Jmjd3F/FFabp4-Cre+) mice were smaller than their littermates. The expression of Jmjd3 mRNA (P<0.05) and protein in BAT was significantly decreased, and the protein expression of H3K27me3 was increased, indicating that the knockout mice were successfully constructed. HE staining showed that lipid droplets in BAT of Jmjd3-/- mice were significantly less than those in the control group. The results of electron microscopy showed that the area of brown adipocytes in Jmjd3-/- mice was smaller (P<0.05), the number of lipid droplets was less (P<0.01), and the size of lipid droplets was not significantly different (P>0.05). Mitochondrial edema and cristae rupture were observed in the Jmjd3-/- group.The number and area of mitochondria were not affected (both P>0.05), the number of mitochondrial cristae was significantly less than that of the control group (P<0.05). The protein expression of UCP1 of Jmjd3-/- mice was decreased. The mRNA expression of UCP1, CIDEa, and PGC-1α in BAT of Jmjd3-/- mice was significantly less than that of the control group (all P<0.05). ATP content in BAT of Jmjd3-/- mice was decreased (P<0.05). HE staining showed that lipid droplets in BAT of Jmjd3F/FFabp4-Cre+ mice were larger than those in the control group. Electron microscopy showed that there was no significant difference in size of adipocytes between the 2 groups (P>0.05). In the Jmjd3F/FFabp4-Cre+ mice, lipid droplets were less (P<0.05), but their diameters were larger (P<0.001). Mitochondrial edema and cristae rupture were observed in the Jmjd3F/FFabp4-Cre+ mice. The number of mitochondrial cristae in the Jmjd3F/FFabp4-Cre+ mice was significantly less than that in the control group (P<0.05). The protein expression of UCP1 in Jmjd3F/FFabp4-Cre+ mice was decreased. The mRNA expression of UCP1, PRDM16, CIDEa, COX1, PGC-1α in BAT of Jmjd3F/FFabp4-Cre+ mice was significantly less than that of the control group (all P<0.05). ATP content in BAT of Jmjd3F/FFabp4-Cre+ mice was decreased (P<0.05). In the Jmjd3F/FFabp4-Cre+ mice, the body temperature was decreased more and the cold tolerance was poor (P<0.05). CONCLUSIONS: Jmjd3 promotes the differentiation of brown adipocytes by enhancing the formation of lipid droplets in mouse brown adipocytes and maintaining the normal morphology and function of mitochondria.