White adipocytes in subcutaneous fat depots require KLF15 for maintenance in preclinical models.

Healthy adipose tissue is essential for normal physiology. There are 2 broad types of adipose tissue depots: brown adipose tissue (BAT), which contains adipocytes poised to burn energy through thermogenesis, and white adipose tissue (WAT), which contains adipocytes that store lipids. However, within those types of adipose, adipocytes possess depot and cell-specific properties that have important implications. For example, the subcutaneous and visceral WAT confers divergent risk for metabolic disease. Further, within a depot, different adipocytes can have distinct properties; subcutaneous WAT can contain adipocytes with either white or brown-like (beige) adipocyte properties. However, the pathways that regulate and maintain this cell and depot-specificity are incompletely understood. Here, we found that the transcription factor KLF15 is required for maintaining white adipocyte properties selectively within the subcutaneous WAT. We revealed that deletion of Klf15 is sufficient to induce beige adipocyte properties and that KLF15's direct regulation of Adrb1 is a critical molecular mechanism for this process. We uncovered that this activity is cell autonomous but has systemic implications in mouse models and is conserved in primary human adipose cells. Our results elucidate a pathway for depot-specific maintenance of white adipocyte properties that could enable the development of therapies for obesity and associated diseases.

Molecular Regulation of Thermogenic Mechanisms in Beige Adipocytes.

Adipose tissue is conventionally recognized as a metabolic organ responsible for storing energy. However, a proportion of adipose tissue also functions as a thermogenic organ, contributing to the inhibition of weight gain and prevention of metabolic diseases. In recent years, there has been significant progress in the study of thermogenic fats, particularly brown adipose tissue (BAT). Despite this progress, the mechanism underlying thermogenesis in beige adipose tissue remains highly controversial. It is widely acknowledged that beige adipose tissue has three additional thermogenic mechanisms in addition to the conventional UCP1-dependent thermogenesis: Ca2+ cycling thermogenesis, creatine substrate cycling thermogenesis, and triacylglycerol/fatty acid cycling thermogenesis. This paper delves into these three mechanisms and reviews the latest advancements in the molecular regulation of thermogenesis from the molecular genetic perspective. The objective of this review is to provide readers with a foundation of knowledge regarding the beige fats and a foundation for future research into the mechanisms of this process, which may lead to the development of new strategies for maintaining human health.

Macrophage-derived chemokine CCL22 establishes local LN-mediated adaptive thermogenesis and energy expenditure.

There is a regional preference around lymph nodes (LNs) for adipose beiging. Here, we show that local LN removal within inguinal white adipose tissue (iWAT) greatly impairs cold-induced beiging, and this impairment can be restored by injecting M2 macrophages or macrophage-derived C-C motif chemokine (CCL22) into iWAT. CCL22 injection into iWAT effectively promotes iWAT beiging, while blocking CCL22 with antibodies can prevent it. Mechanistically, the CCL22 receptor, C-C motif chemokine receptor 4 (CCR4), within eosinophils and its downstream focal adhesion kinase/p65/interleukin-4 signaling are essential for CCL22-mediated beige adipocyte formation. Moreover, CCL22 levels are inversely correlated with body weight and fat mass in mice and humans. Acute elevation of CCL22 levels effectively prevents diet-induced body weight and fat gain by enhancing adipose beiging. Together, our data identify the CCL22-CCR4 axis as an essential mediator for LN-controlled adaptive thermogenesis and highlight its potential to combat obesity and its associated complications.

Estrogen counteracts age-related decline in beige adipogenesis through the NAMPT-regulated ER stress response.

Thermogenic beige adipocytes are recognized as potential therapeutic targets for combating metabolic diseases. However, the metabolic advantages that they offer are compromised with aging. Here we show that treating mice with estrogen (E2), a hormone that decreases with age, can counteract the age-related decline in beige adipogenesis when exposed to cold temperature while concurrently enhancing energy expenditure and improving glucose tolerance in mice. Mechanistically, we found that nicotinamide phosphoribosyl transferase (NAMPT) plays a pivotal role in facilitating the formation of E2-induced beige adipocytes, which subsequently suppresses the onset of age-related endoplasmic reticulum (ER) stress. Furthermore, we found that targeting NAMPT signaling, either genetically or pharmacologically, can restore the formation of beige adipocytes by increasing the number of perivascular adipocyte progenitor cells. Conversely, the absence of NAMPT signaling prevents this process. Together, our findings shed light on the mechanisms regulating the age-dependent impairment of beige adipocyte formation and underscore the E2-NAMPT-controlled ER stress pathway as a key regulator of this process.

The browning and mobilization of subcutaneous white adipose tissue supports efficient skin repair.

Adipocytes in dermis are considered to be important participants in skin repair and regeneration, but the role of subcutaneous white adipose tissue (sWAT) in skin repair is poorly understood. Here, we revealed the dynamic changes of sWAT during wound healing process. Lineage-tracing mouse studies revealed that sWAT would enter into the large wound bed and participate in the formation of granulation tissue. Moreover, sWAT undergoes beiging after skin injury. Inhibition of sWAT beiging by genetically silencing PRDM16, a key regulator to beiging, hindered wound healing process. The transcriptomics results suggested that beige adipocytes in sWAT abundantly express neuregulin 4 (NRG4), which regulated macrophage polarization and the function of myofibroblasts. In diabetic wounds, the beiging of sWAT was significantly suppressed. Thus, adipocytes from sWAT regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes.

Alternative isoform expression of key thermogenic genes in human beige adipocytes.

Background: The beneficial effect of thermogenic adipocytes in maintaining body weight and protecting against metabolic disorders has raised interest in understanding the regulatory mechanisms defining white and beige adipocyte identity. Although alternative splicing has been shown to propagate adipose browning signals in mice, this has yet to be thoroughly investigated in human adipocytes. Methods: We performed parallel white and beige adipogenic differentiation using primary adipose stem cells from 6 unrelated healthy subjects and assessed differential gene and isoform expression in mature adipocytes by RNA sequencing. Results: We find 777 exon junctions with robust differential usage between white and beige adipocytes in all 6 subjects, mapping to 562 genes. Importantly, only 10% of these differentially spliced genes are also differentially expressed, indicating that alternative splicing constitutes an additional layer of gene expression regulation during beige adipocyte differentiation. Functional classification of alternative isoforms points to a gain of function for key thermogenic transcription factors such as PPARG and CITED1, and enzymes such as PEMT, or LPIN1. We find that a large majority of the splice variants arise from differential TSS usage, with beige-specific TSSs being enriched for PPARγ and MED1 binding compared to white-specific TSSs. Finally, we validate beige specific isoform expression at the protein level for two thermogenic regulators, PPARγ and PEMT. Discussion: These results suggest that differential isoform expression through alternative TSS usage is an important regulatory mechanism for human adipocyte thermogenic specification.

The histone methyltransferase SUV420H2 regulates brown and beige adipocyte thermogenesis.

Activation of brown adipose tissue (BAT) thermogenesis increases energy expenditure and alleviates obesity. Here we discover that histone methyltransferase suppressor of variegation 4-20 homolog 2 (Suv420h2) expression parallels that of Ucp1 in brown and beige adipocytes and that Suv420h2 knockdown significantly reduces - whereas Suv420h2 overexpression significantly increases - Ucp1 levels in brown adipocytes. Suv420h2 knockout (H2KO) mice exhibit impaired cold-induced thermogenesis and are prone to diet-induced obesity. In contrast, mice with specific overexpression of Suv420h2 in adipocytes display enhanced cold-induced thermogenesis and are resistant to diet-induced obesity. Further study shows that Suv420h2 catalyzes H4K20 trimethylation at eukaryotic translation initiation factor 4E-binding protein 1 (4e-bp1) promoter, leading to downregulated expression of 4e-bp1, a negative regulator of the translation initiation complex. This in turn upregulates PGC1α protein levels, and this upregulation is associated with increased expression of thermogenic program. We conclude that Suv420h2 is a key regulator of brown/beige adipocyte development and thermogenesis.

Aging impairs cold-induced beige adipogenesis and adipocyte metabolic reprogramming.

The energy-burning capability of beige adipose tissue is a potential therapeutic tool for reducing obesity and metabolic disease, but this capacity is decreased by aging. Here, we evaluate the impact of aging on the profile and activity of adipocyte stem and progenitor cells (ASPCs) and adipocytes during the beiging process in mice. We found that aging increases the expression of Cd9 and other fibro-inflammatory genes in fibroblastic ASPCs and blocks their differentiation into beige adipocytes. Fibroblastic ASPC populations from young and aged mice were equally competent for beige differentiation in vitro, suggesting that environmental factors suppress adipogenesis in vivo. Examination of adipocytes by single nucleus RNA-sequencing identified compositional and transcriptional differences in adipocyte populations with aging and cold exposure. Notably, cold exposure induced an adipocyte population expressing high levels of de novo lipogenesis (DNL) genes, and this response was severely blunted in aged animals. We further identified Npr3, which encodes the natriuretic peptide clearance receptor, as a marker gene for a subset of white adipocytes and an aging-upregulated gene in adipocytes. In summary, this study indicates that aging blocks beige adipogenesis and dysregulates adipocyte responses to cold exposure and provides a resource for identifying cold and aging-regulated pathways in adipose tissue.

Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

The Role of ß3-Adrenergic Receptors in Cold-Induced Beige Adipocyte Production in Pigs.

After exposure to cold stress, animals enhance the production of beige adipocytes and expedite thermogenesis, leading to improved metabolic health. Although brown adipose tissue in rodents is primarily induced by ß3-adrenergic receptor (ADRB3) stimulation, the activation of major ß-adrenergic receptors (ADRBs) in pigs has been a topic of debate. To address this, we developed overexpression vectors for ADRB1, ADRB2, and ADRB3 and silenced the expression of these receptors to observe their effects on the adipogenic differentiation stages of porcine preadipocytes. Our investigation revealed that cold stress triggers the transformation of subcutaneous white adipose tissue to beige adipose tissue in pigs by modulating adrenergic receptor levels. Meanwhile, we found that ADRB3 promotes the transformation of white adipocytes into beige adipocytes. Notably, ADRB3 enhances the expression of beige adipose tissue marker genes, consequently influencing cellular respiration and metabolism by regulating lipolysis and mitochondrial expression. Therefore, ADRB3 may serve as a pivotal gene in animal husbandry and contribute to the improvement of cold intolerance in piglets.

GPCR-mediated regulation of beige adipocyte formation: Implications for obesity and metabolic health.

Obesity and its associated complications pose a significant burden on health. The non-shivering thermogenesis (NST) and metabolic capacity properties of brown adipose tissue (BAT), which are distinct from those of white adipose tissue (WAT), in combating obesity and its related metabolic diseases has been well documented. However, beige adipose tissue, the third and relatively novel type of adipose tissue, which emerges in extensive presence of WAT and shares similar favorable metabolic properties with BAT, has garnered considerable attention in recent years. In this review, we focused on the role of G protein-coupled receptors (GPCRs), the largest receptor family and the most successful class of drug targets in humans, in the induction of beige adipocytes. More importantly, we highlight researchers' clinical treatment attempts to ameliorate obesity and other related metabolic diseases through the formation and activation of beige adipose tissue. In summary, this review provides valuable insights into the formation of beige adipose tissue and the involvement of GPCRs, based on the latest advancements in scientific research.

BDE-99 stimulates generation of aberrant brown/beige adipocytes.

Adipose tissue compromises one of the principal depots where brominated flame retardants (BFR) accumulate in vivo, yet whether BFR disturb thermogenic brown/beige adipocytes is still not referred to date. Herein, effects of BDE-99, a major congener of polybrominated diphenyl ethers (PBDEs) detected in humans, on brown/beige adipocytes were explored for the first time, aiming to provide new knowledge evaluating the obesogenic and metabolic disrupting effects of BFR. Our results firstly demonstrated that exposure to BDE-99 during the lineage commitment period significantly promoted C3H10T1/2 MSCs differentiating into brown/beige adipocytes, evidenced by the increase of brown/beige adipocyte marker UCP1, Cidea as well as mitochondrial membrane potential and basal respiration rate, which was similar to pharmacological PPARγ agonist rosiglitazone. Unexpectedly, the mitochondrial maximal respiration rate of BDE-99 stimulated brown/beige adipocytes was not synchronously enhanced and resulted in a significant reduction of mitochondrial spare respiration capacity (SRC) compared to control or rosiglitazone stimulated adipocytes, indicating a deficient energy-dissipating capacity of BDE-99 stimulated thermogenic adipocytes. Consistently with compromised mitochondrial SRC, lipidomic analysis further revealed that the lipids profile of mitochondria derived from BDE-99 stimulated brown/beige adipocytes were quite different from control or rosiglitazone stimulated cells. In detail, BDE-99 group contains more free fatty acid (FFA) and lyso-PE in mitochondria. In addition to energy metabolism, our results also demonstrated that BDE-99 stimulated brown/beige adipocytes were deficient in endocrine, which secreted more adverse adipokine named resistin, coinciding with comparable beneficial adipokine adiponectin compared with that of rosiglitazone. Taken together, our results showed for the first time that BDE-99 stimulated brown/beige adipocytes were aberrant in energy metabolism and endocrine, which strongly suggests that BDE-99 accumulated in human adipose tissue could interfere with brown/beige adipocytes to contribute to the occurrence of obesity and relevant metabolic disorders.

Nanomodulator-Mediated Restructuring of Adipose Tissue Immune Microenvironments for Antiobesity Treatment.

In obesity, the interactions between proinflammatory macrophages and adipocytes in white adipose tissues are known to play a crucial role in disease progression by providing inflammatory microenvironments. Here, we report that the functional nanoparticle-mediated modulation of crosstalk between adipocytes and macrophages can remodel adipocyte immune microenvironments. As a functional nanomodulator, we designed antivascular cell adhesion molecule (VCAM)-1 antibody-conjugated and amlexanox-loaded polydopamine nanoparticles (VAPN). Amlexanox was used as a model drug to increase energy expenditure. Compared to nanoparticles lacking antibody modification or amlexanox, VAPN showed significantly greater binding to VCAM-1-expressing adipocytes and lowered the interaction of adipocytes with macrophages. In high fat diet-fed mice, repeated subcutaneous administration of VAPN increased the populations of beige adipocytes and ameliorated inflammation in white adipose tissues. Moreover, the localized application of VAPN in vivo exerted a systemic metabolic effect and reduced metabolic disorders, including insulin tolerance and liver steatosis. These findings suggested that VAPN had potential to modulate the immune microenvironments of adipose tissues for the immunologic treatment of obesity. Although we used amlexanox as a model drug and anti-VCAM-1 antibody in VAPN, the concept of immune nanomodulators can be widely applied to the immunological treatment of obesity.

M2 macrophages independently promote beige adipogenesis via blocking adipocyte Ets1.

Adipose tissue macrophages can promote beige adipose thermogenesis by altering local sympathetic activity. Here, we perform sympathectomy in mice and further eradicate subcutaneous adipose macrophages and discover that these macrophages have a direct beige-promoting function that is independent of sympathetic system. We further identify adipocyte Ets1 as a vital mediator in this process. The anti-inflammatory M2 macrophages suppress Ets1 expression in adipocytes, transcriptionally activate mitochondrial biogenesis, as well as suppress mitochondrial clearance, thereby increasing the mitochondrial numbers and promoting the beiging process. Male adipocyte Ets1 knock-in mice are completely cold intolerant, whereas male mice lacking Ets1 in adipocytes show enhanced energy expenditure and are resistant to metabolic disorders caused by high-fat-diet. Our findings elucidate a direct communication between M2 macrophages and adipocytes, and uncover a function for Ets1 in responding to macrophages and negatively governing mitochondrial content and beige adipocyte formation.

PPARα/γ synergism activates UCP1-dependent and -independent thermogenesis and improves mitochondrial dynamics in the beige adipocytes of high-fat fed mice.

OBJECTIVE: The aim of this study was to investigate the role of peroxisome proliferator-activated receptor (PPAR) activation (single PPARα or PPARγ, and dual PPARα/γ) on UCP1-dependent and -independent thermogenic pathways and mitochondrial metabolism in the subcutaneous white adipose tissue of mice fed a high-fat diet. METHODS: Male C57BL/6 mice received either a control diet (10% lipids) or a high-fat diet (HF; 50% lipids) for 12 wk. The HF group was divided to receive the treatments for 4 wk: HFγ (pioglitazone, 10 mg/kg), HFα (WY-14643, 3.5 mg/kg), and HFα/γ (tesaglitazar, 4 mg/kg). RESULTS: The HF group was overweight, insulin resistant, and had subcutaneous white adipocyte dysfunction. Treatment with PPARα and PPARα/γ reduced body mass, mitigated insulin resistance, and induced browning with increased UCP1-dependent and -independent thermogenesis activation and improved mitochondrial metabolism to support the beige adipocyte phenotype. CONCLUSION: PPARα and dual PPARα/γ activation recruited UCP1+ beige adipocytes and favored UCP1-independent thermogenesis, yielding body mass and insulin sensitivity normalization. Preserved mitochondrial metabolism emerges as a potential target for obesity treatment using PPAR agonists, with possible clinical applications.

Dietary Ferulic Acid-Mediated Suppression of Fat Deposits Is Associated with Induction of Beige Adipocyte Formation and Thermogenesis in Inguinal White Adipose Tissue in Mice.

Ferulic acid (FA) is the most abundant phenolic acid in wheat grains. Recent studies have reported that FA intake significantly suppresses body weight gain and accumulation of fat deposits in mice. However, the mechanism by which FA intake affects body fat accumulation remains unclear. We hypothesized that dietary FA induces the formation of beige adipocytes and contributes to the suppression of body fat accumulation. In this study, we investigated whether dietary FA significantly induces beige adipocyte formation and thermogenesis in mice. We found that intake of dietary FA (control diet supplemented with 10 g of FA/kg diet) for 4 wk significantly decreased white adipose tissue (WAT) deposits and body weight gain and significantly induced beige adipocyte formation in inguinal WAT (iWAT) in mice. Furthermore, dietary FA specifically induced thermogenesis in iWAT, dependent upon the significant induction of uncoupling protein 1 expression. These findings suggest that the dietary FA-mediated reduction of WAT accumulation and body weight gain is associated with the induction of beige adipocyte formation and thermogenesis in iWAT, which increases energy expenditure. Our study presents a novel example of dietary FA intake-mediated bioactivity as a functional food-derived factor.

Lactate Conversion by Lactate Dehydrogenase B Is Involved in Beige Adipocyte Differentiation and Thermogenesis in Mice.

Adipose tissue (AT) is the primary reservoir of lipid, the major thermogenesis organ during cold exposure, and an important site for lactate production. However, the utilization of lactate as a metabolic substrate by adipocytes, as well as its potential involvement in the regulation of adipocyte thermogenesis, remain unappreciated. In vitro experiments using primary stromal vascular fraction preadipocytes isolated from mouse inguinal white adipose tissue (iWAT) revealed that lactate dehydrogenase B (LDHB), the key glycolytic enzyme that catalyzes the conversion of lactate to pyruvate, is upregulated during adipocyte differentiation, downregulated upon chronic cold stimulation, and regained after prolonged cold exposure. In addition, the global knockout of Ldhb significantly reduced the masses of iWAT and epididymal WAT (eWAT) and impeded the utilization of iWAT during cold exposure. In addition, Ldhb loss of function impaired the mitochondrial function of iWAT under cold conditions. Together, these findings uncover the involvement of LDHB in adipocyte differentiation and thermogenesis.

HDAC1 inhibits beige adipocyte-mediated thermogenesis through histone crotonylation of Pgc1a/Ucp1.

Obesity, one of the most serious public health issues, is caused by the imbalance of energy intake and energy expenditure. Increasing energy expenditure via induction of adipose tissue browning has become an appealing strategy to treat obesity and associated metabolic complications. Although histone modifications have been confirmed to regulate cellular energy metabolism, the involved biochemical mechanism of thermogenesis in adipose tissue is not completely understood. Herein, we report that class I histone deacetylases (HDAC) inhibitor MS275 increased PGC1α/UCP1 protein levels in inguinal white adipose tissue (iWAT) concomitant with elevated energy expenditure, reduced obesity and ameliorated glucose tolerance compared to control littermates. H3K18cr and H3K18ac levels were elevated after MS275 treatment. MS275 also promoted the transcription of Pgc1α and Ucp1 by enhancing the enrichment of H3K18cr and H3K18ac in the Pgc1α/Ucp1 enhancer and promoter, with a notable increase in H3K18cr. Mechanistically, the deletion of Hdac1 in beige adipocyte increases H3K18cr levels in enhancers and promoters of Pgc1α and Ucp1 genes, regulated the chromosomal state, thereby affecting the transcription of Pgc1α/Ucp1. Taken together, HDAC1 inhibits beige adipocyte-mediated thermogenesis through histone crotonylation of Pgc1a/Ucp1. This finding may provide a therapeutic strategy through increasing energy expenditure in obesity and related metabolic disorders.

Echinacoside Induces UCP1- and ATP-Dependent Thermogenesis in Beige Adipocytes via the Activation of Dopaminergic Receptors.

Echinacoside (ECH) is a naturally occurring phenylethanoid glycoside, isolated from Echinacea angustifolia, and this study aimed to analyze its effect on thermogenesis and its interaction with dopaminergic receptors 1 and 5 (DRD1 and DRD5) in 3T3-L1 white adipocytes and mice models. We employed RT-PCR, immunoblot, immunofluorescence, a staining method, and an assay kit to determine its impact. ECH showed a substantial increase in browning signals in vitro and a decrease in adipogenic signals in vivo. Additionally, analysis of the iWAT showed that the key genes involved in beiging, mitochondrial biogenesis, and ATP-dependent thermogenesis were upregulated while adipogenesis and lipogenesis genes were downregulated. OXPHOS complexes, Ca2+ signaling proteins as well as intracellular Ca2+ levels were also upregulated in 3T3-L1 adipocytes following ECH treatment. This was collectively explained by mechanistic studies which showed that ECH mediated the beiging process via the DRD1/5-cAMP-PKA and subsequent downstream molecules, whereas it co-mediated the α1-AR-signaling thermogenesis via the DRD1/5/SERCA2b/RyR2/CKmt pathway in 3T3-L1 adipocytes. Animal experiments revealed that there was a 12.28% reduction in body weight gain after the ECH treatment for six weeks. The effects of ECH treatment on adipose tissue can offer more insights into the treatment of obesity and metabolic syndrome.