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.

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.

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.

Genistein-mediated thermogenesis and white-to-beige adipocyte differentiation involve transcriptional activation of cAMP response elements in the Ucp1 promoter.

Genistein is an isoflavone present in soybeans and is considered a bioactive compound due to its widely reported biological activity. We have previously shown that intraperitoneal genistein administration and diet supplementation activates the thermogenic program in rats and mice subcutaneous white adipose tissue (scWAT) under multiple environmental cues, including cold exposure and high-fat diet feeding. However, the mechanistic insights of this process were not previously unveiled. Uncoupling protein 1 (UCP1), a mitochondrial membrane polypeptide responsible for dissipating energy into heat, is considered the most relevant thermogenic marker; thus, we aimed to evaluate whether genistein regulates UCP1 transcription. Here we show that genistein administration to thermoneutral-housed mice leads to the appearance of beige adipocyte markers, including a sharp upregulation of UCP1 expression and protein abundance in scWAT. Reporter assays showed an increase in UCP1 promoter activity after genistein stimulation, and in silico analysis revealed the presence of estrogen (ERE) and cAMP (CRE) response elements as putative candidates of genistein activation. Mutation of the CRE but not the ERE reduced genistein-induced promoter activity by 51%. Additionally, in vitro and in vivo ChIP assays demonstrated the binding of CREB to the UCP1 promoter after acute genistein administration. Taken together, these data elucidate the mechanism of genistein-mediated UCP1 induction and confirm its potential applications in managing 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.

Adipocyte G Protein-Coupled Receptors as Potential Targets for Novel Antidiabetic Drugs.

The functional state of adipocytes plays a central role in regulating numerous important metabolic functions, including energy and glucose homeostasis. While white adipocytes store excess calories as fat (triglycerides) and release free fatty acids as a fuel source in times of need, brown and beige adipocytes (so-called thermogenic adipocytes) convert chemical energy stored in substrates (e.g., fatty acids or glucose) into heat, thus promoting energy expenditure. Like all other cell types, adipocytes express many G protein-coupled receptors (GPCRs) that are linked to four major functional classes of heterotrimeric G proteins (Gs, Gi/o, Gq/11, and G12/13). During the past few years, novel experimental approaches, including the use of chemogenetic strategies, have led to a series of important new findings regarding the metabolic consequences of activating or inhibiting distinct GPCR/G protein signaling pathways in white, brown, and beige adipocytes. This novel information should guide the development of novel drugs capable of modulating the activity of specific adipocyte GPCR signaling pathways for the treatment of obesity, type 2 diabetes, and related metabolic disorders.

Genetically prolonged beige fat in male mice confers long-lasting metabolic health.

A potential therapeutic target to curb obesity and diabetes is thermogenic beige adipocytes. However, beige adipocytes quickly transition into white adipocytes upon removing stimuli. Here, we define the critical role of cyclin dependent kinase inhibitor 2A (Cdkn2a) as a molecular pedal for the beige-to-white transition. Beige adipocytes lacking Cdkn2a exhibit prolonged lifespan, and male mice confer long-term metabolic protection from diet-induced obesity, along with enhanced energy expenditure and improved glucose tolerance. Mechanistically, Cdkn2a promotes the expression and activity of beclin 1 (BECN1) by directly binding to its mRNA and its negative regulator BCL2 like 1 (BCL2L1), activating autophagy and accelerating the beige-to-white transition. Reactivating autophagy by pharmacological or genetic methods abolishes beige adipocyte maintenance induced by Cdkn2a ablation. Furthermore, hyperactive BECN1 alone accelerates the beige-to-white transition in mice and human. Notably, both Cdkn2a and Becn1 exhibit striking positive correlations with adiposity. Hence, blocking Cdkn2a-mediated BECN1 activity holds therapeutic potential to sustain beige adipocytes in treating obesity and related metabolic diseases.

Androgen Receptor is a Negative Regulator of PRDM16 in Beige Adipocyte.

PRDM16 (PR domain containing protein 16) serves as a dominant activator of brown and beige adipocyte. However, mechanisms underlying the regulation of PRDM16 expression are incompletely understood. A Prdm16 luciferase knockin reporter mouse model is generated, enabling high throughput monitoring of Prdm16 transcription. Single clonal analysis reveals high heterogeneity of Prdm16 expression in the inguinal white adipose tissue (iWAT) cells. Amongst all transcription factors, androgen receptor (Ar) shows the strongest negative correlation with Prdm16. A sex dimorphism for PRDM16 mRNA expression is present in human WAT, with female individuals exhibiting increased expression than males. Androgen-AR signaling mobilization suppresses Prdm16 expression, accompanied by attenuated beiging in beige adipocytes, but not in brown adipose tissue. The suppressive effect of androgens on beiging is abolished upon overexpression of Prdm16. Cleavage under targets and tagmentation mapping reveals direct binding of AR within the intronic region of Prdm16 locus, whereas no direct binding is detected on Ucp1 and other browning-related genes. Adipocyte-selective deletion of Ar potentiates beige cell biogenesis whereas adipocyte-specific overexpression of AR attenuates white adipose beiging. This study highlights an essential role of AR in negative regulation of PRDM16 in WAT and provides an explanation for the observed sex difference in adipose beiging.

Thermogenic adipose tissue in energy regulation and metabolic health.

The ability to generate thermogenic fat could be a targeted therapy to thwart obesity and improve metabolic health. Brown and beige adipocytes are two types of thermogenic fat cells that regulate energy balance. Both adipocytes share common morphological, biochemical, and thermogenic properties. Yet, recent evidence suggests unique features exist between brown and beige adipocytes, such as their cellular origin and thermogenic regulatory processes. Beige adipocytes also appear highly plastic, responding to environmental stimuli and interconverting between beige and white adipocyte states. Additionally, beige adipocytes appear to be metabolically heterogenic and have substrate specificity. Nevertheless, obese and aged individuals cannot develop beige adipocytes in response to thermogenic fat-inducers, creating a key clinical hurdle to their therapeutic promise. Thus, elucidating the underlying developmental, molecular, and functional mechanisms that govern thermogenic fat cells will improve our understanding of systemic energy regulation and strive for new targeted therapies to generate thermogenic fat. This review will examine the recent advances in thermogenic fat biogenesis, molecular regulation, and the potential mechanisms for their failure.

Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet-Induced Adiposity.

Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear. Here, the authors screen all Wnt-related paracrine factors in 1994 obese cases and 2161 controls using whole-exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high-fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1 ablation resists HFD-induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4-Wnt/ß-catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4-Wnt/ß-catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain-of-function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator, suppress fat thermogenesis and contribute to obesity in humans.

Gamma-Aminobutyric Acid Promotes Beige Adipocyte Reconstruction by Modulating the Gut Microbiota in Obese Mice.

Given the increasing prevalence of obesity, the white-to-beige adipocyte conversion has attracted interest as a target for obesity treatment. Gamma-aminobutyric acid (GABA) treatment can reduce obesity, but the underlying mechanism remains unclear. Here, we aimed to investigate the mechanism by which GABA triggers weight loss by improving the beiging of inguinal white adipose tissue (iWAT) and the role of gut microbiota in this process. The results showed that GABA reduced body weight and adipose inflammation and promoted the expression of thermogenic genes in the iWAT. The 16S rRNA sequence analysis of gut microbiota showed that GABA treatment increased the relative abundance of Bacteroidetes, Akkermansia, and Romboutsia and reduced that of Firmicutes and Erysipelatoclostridium in obese mice. Additionally, serum metabolomic analysis revealed that GABA treatment increased 3-hydroxybutyrate and reduced oxidized lipid levels in obese mice. Spearman's correlation analysis showed that Akkermansia and Romboutsia were negatively associated with the levels of oxidized lipids. Fecal microbiota transplantation analysis confirmed that the gut microbiota was involved in the white-to-beige adipocyte reconstruction by GABA. Overall, our findings suggest that GABA treatment may promote iWAT beiging through the gut microbiota in obese mice. GABA may be utilized to protect obese people against metabolic abnormalities brought on by obesity and gut dysbiosis.

Regulation of beige adipocyte thermogenesis by the cold-repressed ER protein NNAT.

OBJECTIVE: Cold stimuli trigger the conversion of white adipose tissue into beige adipose tissue, which is capable of non-shivering thermogenesis. However, what process drives this activation of thermogenesis in beige fat is not well understood. Here, we examine the ER protein NNAT as a regulator of thermogenesis in adipose tissue. METHODS: We investigated the regulation of adipose tissue NNAT expression in response to changes in ambient temperature. We also evaluated the functional role of NNAT in thermogenic regulation using Nnat null mice and primary adipocytes that lack or overexpress NNAT. RESULTS: Cold exposure or treatment with a ß3-adrenergic agonist reduces the expression of adipose tissue NNAT in mice. Genetic disruption of Nnat in mice enhances inguinal adipose tissue thermogenesis. Nnat null mice exhibit improved cold tolerance both in the presence and absence of UCP1. Gain-of-function studies indicate that ectopic expression of Nnat abolishes adrenergic receptor-mediated respiration in beige adipocytes. NNAT physically interacts with the ER Ca2+-ATPase (SERCA) in adipocytes and inhibits its activity, impairing Ca2+ transport and heat dissipation. We further demonstrate that NHLRC1, an E3 ubiquitin protein ligase implicated in proteasomal degradation of NNAT, is induced by cold exposure or ß3-adrenergic stimulation, thus providing regulatory control at the protein level. This serves to link cold stimuli to NNAT degradation in adipose tissue, which in turn leads to enhanced SERCA activity. CONCLUSIONS: Our study implicates NNAT in the regulation of adipocyte thermogenesis.

Blockage of PPARγ T166 phosphorylation enhances the inducibility of beige adipocytes and improves metabolic dysfunctions.

Beige adipocytes in mammalian white adipose tissue (WAT) can reinforce fat catabolism and energy expenditure. Promoting beige adipocyte biogenesis is a tantalizing tactic for combating obesity and its associated metabolic disorders. Here, we report that a previously unidentified phosphorylation pattern (Thr166) in the DNA-binding domain of PPARγ regulates the inducibility of beige adipocytes. This unique posttranslational modification (PTM) pattern influences allosteric communication between PPARγ and DNA or coactivators, which impedes the PPARγ-mediated transactivation of beige cell-related gene expression in WAT. The genetic mutation mimicking T166 phosphorylation (p-T166) hinders the inducibility of beige adipocytes. In contrast, genetic or chemical intervention in this PTM pattern favors beige cell formation. Moreover, inhibition of p-T166 attenuates metabolic dysfunction in obese mice. Our results uncover a mechanism involved in beige cell fate determination. Moreover, our discoveries provide a promising strategy for guiding the development of novel PPARγ agonists for the treatment of obesity and related metabolic disorders.

Circadian clock control of MRTF/SRF pathway suppresses beige adipocyte thermogenic recruitment.

The morphological transformation of adipogenic progenitors into mature adipocytes requires dissolution of actin cytoskeleton with loss of myocardin-related transcription factor (MRTF)/serum response factor (SRF) activity. Circadian clock confers temporal control in adipogenic differentiation, while the actin cytoskeleton-MRTF/SRF signaling transduces extracellular physical niche cues. Here, we define a novel circadian transcriptional control involved in actin cytoskeleton-MRTF/SRF signaling cascade that modulates beige fat thermogenic function. Key components of actin dynamic-MRTF/SRF pathway display circadian regulation in beige fat depot. The core clock regulator, brain and muscle arnt-like 1 (Bmal1), exerts direct transcriptional control of genes within the actin dynamic-MRTF/SRF cascade that impacts actin cytoskeleton organization and SRF activity. Employing beige fat-selective gene-targeting models together with pharmacological rescues, we further demonstrate that Bmal1 inhibits beige adipogenesis and thermogenic capacity in vivo via the MRTF/SRF pathway. Selective ablation of Bmal1 induces beigeing with improved glucose homeostasis, whereas its targeted overexpression attenuates thermogenic induction resulting in obesity. Collectively, our findings identify the clock-MRTF/SRF regulatory axis as an inhibitory mechanism of beige fat thermogenic recruitment with significant contribution to systemic metabolic homeostasis.