miR-1275 Inhibits Human Omental Adipose-Derived Stem Cells Differentiation Toward the Beige Phenotype via PRDM16.

Beige adipocytes have recently attracted attention for their potential as new therapeutic targets in the management of obesity and related metabolic disorders. MicroRNAs (miRNAs) have been reported as transcriptional regulators or biomarkers of brown and beige adipogenesis. Nevertheless, the effects of miRNAs involved in beige differentiation of human visceral adipocytes remain to be investigated. In this study, microarray screening showed that miR-1275 was significantly decreased during the differentiation of beige adipocytes induced by human omental adipose-derived stem cells (hASCs). Overexpression of miR-1275 suppressed the "brown-like" differentiation of hASCs by inhibiting the key transcriptional factor PR domain containing 16 (PRDM16) without affecting the proliferation. Adipogenesis and mitochondrial biogenesis of beige adipocytes derived from hASCs were impaired by miR-1275 overexpression. The regulatory effect of miR-1275 was determined by direct binding to the 3'-untranslated region of PRDM16, which was demonstrated by a dual-luciferase assay. Taken together, this study identified miR-1275 as a negative regulator of beige cell development in hASCs by inhibiting PRDM16. Thus, miR-1275 might be a potential target in the management of visceral obesity and related metabolic diseases.

The Effect of TLR Agonists and Myokines on Secretory Activity of Adipogenically Differentiated MSC Cultures.

We studied the effect of bacterial pathogen-associated molecular patterns and myokines on the secretion of adipokines by mesenchymal stem cells (MSC) and products of their adipogenic differentiation. The secretion of adiponectin, adipsin, leptin, and insulin by adipogenically differentiated cell cultures was quantitatively determined using multiplex ELISA. MSC obtained from the stromal vascular fraction of human subcutaneous adipose tissue were shown to secrete a known adipokine adipsin. The ability of white adipocytes to secrete significant amounts of insulin (in vitro) has been shown for the first time. Control cultures of white adipocytes secreted much higher levels of adiponectin, leptin, and insulin when compared to other adipocytes cultures. On the other hand, beige and brown adipocyte cultures secreted more adipsin than white adipocyte cultures. The influence of myokine ß-aminoisobutyric acid on the secretion of adipsin in MSC, white, beige, and brown adipocytes was also studied.

Recruitment of Thermogenic Fat: Trigger of Fat Burning.

Brown and beige adipose tissues possess the remarkable capacity to convert energy into heat, which potentially opens novel therapeutic perspectives targeting the epidemic of metabolic syndromes such as obesity and type 2 diabetes. These thermogenic fats implement mitochondrial oxidative phosphorylation and uncouple respiration to catabolize fatty acids and glucose, which leads to an increase in energy expenditure. In particular, beige adipocytes that arise in white adipose tissue display their thermogenic capacity through various noncanonical mechanisms. This review aims to summarize the general overview of thermogenic fat, especially including the UCP1-independent adaptive thermogenesis and the emerging mechanisms of "beiging", which may provide more evidence of targeting thermogenic fat to counteract obesity and other metabolic disorders in humans.

Beige adipocytes mediate the neuroprotective and anti-inflammatory effects of subcutaneous fat in obese mice.

Visceral obesity increases risk of cognitive decline in humans, but subcutaneous adiposity does not. Here, we report that beige adipocytes are indispensable for the neuroprotective and anti-inflammatory effects of subcutaneous fat. Mice lacking functional beige fat exhibit accelerated cognitive dysfunction and microglial activation with dietary obesity. Subcutaneous fat transplantation also protects against chronic obesity in wildtype mice via beige fat-dependent mechanisms. Beige adipocytes restore hippocampal synaptic plasticity following transplantation, and these effects require the anti-inflammatory cytokine interleukin-4 (IL4). After observing beige fat-mediated induction of IL4 in meningeal T-cells, we investigated the contributions of peripheral lymphocytes in donor fat. There was no sign of donor-derived lymphocyte trafficking between fat and brain, but recipient-derived lymphocytes were required for the effects of transplantation on cognition and microglial morphology. These findings indicate that beige adipocytes oppose obesity-induced cognitive impairment, with a potential role for IL4 in the relationship between beige fat and brain function.

Beiging Modulates Inflammatory Adipogenesis in Salt-Treated and MEK6-Transfected Adipocytes.

To investigate whether the beiging process changes the interactive effects of salt and MEK6 gene on inflammatory adipogenesis, the salt treatment (NaCl 50 mM) and MEK6 transfection of Tg(+/+) cells were performed with white adipocytes (WAT) and beige-like-adipocytes (BLA). BLA induced by T3 were confirmed by UCP-1 expression and the MEK6 protein was 3.5 times higher in MEK6 transfected WAT than the control. The adipogenic genes, PPAR-γ and C/EBP-α, were 1.5 times more highly expressed in the salt-treated groups than the non-salt-treated groups, and adipogenesis was greatly increased in Tg(+/+) WAT compared to non-transfected Tg(-/-). The adipogenesis induced by salt treatment and MEK6 transfection was significantly reduced in BLA. The inflammatory adipocytokines, TNF-α, IL-1ß, and IL-6, were increased in the salt-treated Tg(+/+) WAT, but an anti-inflammation biomarker, the adiponectin/leptin ratio, was reduced in Tg(+/+), to tenth of that in Tg(-/-). However, the production of adipocytokines in WAT was strongly weakened in BLA, although a combination of salt and MEK6 transfection had the most significant effects on inflammation in both WAT and BLA. Oxygen consumption in mitochondria was maximized in salt-treated and MEK6 transfected WAT, but it was decreased by 50% in BLA. In conclusion, beiging controls the synergistic effects of salt and MEK6 on adipogenesis, inflammation, and energy expenditure.

Ellagic acid induces beige remodeling of white adipose tissue by controlling mitochondrial dynamics and SIRT3.

To determine whether ellagic acid (EA) induces the "beige remodeling" of white adipose tissue (WAT), we treated cold-exposed mice and mouse stromal vascular fraction (SVF) cells with EA, a phytochemical abundant in fruits and vegetables, in particular berries. We then investigated the mechanism of EA in beige remodeling with a particular focus on DRP1-mediated mitochondrial fission and SIRT3. EA induced the trans-differentiation of white adipocytes to beige adipocytes by promoting the expression of UCP1 and other brown and beige adipocytes/fat factors (PRDM16, UCP1, PGC1α, CD137, and TBX1) and mitochondrial dynamics-related factors (SIRT3, NRF1, CPT1ß, DRP1, and FIS1) in 3T3-L1/SVF cells, and these were confirmed in the inguinal WAT of a cold-exposed mouse model. The browning effect of EA was abolished by a potent DRP1 inhibitor Mdivi-1 or SIRT3 knockdown, suggesting that EA induces beige remodeling of WAT by regulating the mitochondrial dynamics and SIRT3.

Glucose-dependent insulinotropic polypeptide modifies adipose plasticity and promotes beige adipogenesis of human omental adipose-derived stem cells.

The adipocyte precursors (APs) located in white adipose tissue (WAT) are functionally significant in adipose plasticity and browning. Modifying adipogenesis or WAT browning targeted on APs is a promising mechanism for anti-obesity drug. We herein explored the in vitro actions and mechanisms of glucose-dependent insulinotropic polypeptide (GIP), a gut-derived peptide, in human adipose-derived mesenchymal stem cells (hADSCs) isolated from omentum. The hADSCs were cotreated with 100 nM GIP with or without equimolar concentration of GIP3-42 (a GIP receptor antagonist), and subsequently examined in vitro. CCK-8, EdU incorporation, and flow cytometry assays were used to assess cellular proliferation. Annexin V FTIC/PI double stain, TUNEL staining, and Western blot were applied for apoptosis evaluation. Adipogenesis was reflected by Western blot, real-time PCR, Oil Red O staining, mitochondrial staining, and mitochondrial DNA analysis. Results showed that GIP promoted proliferation and inhibited apoptosis of hADSCs via pleiotropic effects. Besides, GIP facilitated de novo beige adipogenesis, by accelerating mitotic clonal expansion (MCE), upregulating core adipogenic regulators (C/EBPα and PPARγ), augmenting beige-related genes (UCP1, PGC1α, and PRDM16), increasing mitochondrial content and improving beige adipocyte functionalities. Above all, our study expands knowledge on the mechanisms of GIP modifying adipogenesis especially in inducing beige adipogenesis, and thus provides a theoretical support for clinical usage of GIP on obesity treatment.

Asc-1 regulates white versus beige adipocyte fate in a subcutaneous stromal cell population.

Adipose tissue expansion, as seen in obesity, is often metabolically detrimental causing insulin resistance and the metabolic syndrome. However, white adipose tissue expansion at early ages is essential to establish a functional metabolism. To understand the differences between adolescent and adult adipose tissue expansion, we studied the cellular composition of the stromal vascular fraction of subcutaneous adipose tissue of two and eight weeks old mice using single cell RNA sequencing. We identified a subset of adolescent preadipocytes expressing the mature white adipocyte marker Asc-1 that showed a low ability to differentiate into beige adipocytes compared to Asc-1 negative cells in vitro. Loss of Asc-1 in subcutaneous preadipocytes resulted in spontaneous differentiation of beige adipocytes in vitro and in vivo. Mechanistically, this was mediated by a function of the amino acid transporter ASC-1 specifically in proliferating preadipocytes involving the intracellular accumulation of the ASC-1 cargo D-serine.

The natural compound rutaecarpine promotes white adipocyte browning through activation of the AMPK-PRDM16 axis.

The prevalence of obesity is increasing globally and is associated with many metabolic disorders, such as type 2 diabetes and cardiovascular diseases. In recent years, a number of studies suggest that promotion of white adipose browning represents a promising strategy to combat obesity and its related metabolic disorders. The aim of this study was to identify compounds that induce adipocyte browning and elucidate their mechanism of action. Among the 500 natural compounds screened, a small molecule named Rutaecarpine, was identified as a positive regulator of adipocyte browning both in vitro and in vivo. KEGG pathway analysis from RNA-seq data suggested that the AMPK signaling pathway was regulated by Rutaecarpine, which was validated by Western blot analysis. Furthermore, inhibition of AMPK signaling mitigated the browning effect of Rutaecaripine. The effect of Rutaecaripine on adipocyte browning was also abolished upon deletion of Prdm16, a downstream target of AMPK pathway. In collusion, Rutaecarpine is a potent chemical agent to induce adipocyte browning and may serve as a potential drug candidate to treat obesity.

Histone H3 methyltransferase Ezh2 promotes white adipocytes but inhibits brown and beige adipocyte differentiation in mice.

Obesity is a disease characterized by imbalance between energy intake and expenditure, excessive energy store in white adipocytes, but brown and beige adipocytes consume energy to relieve obesity. In this study, we want to explore the role of the histone H3 methyltransferase Ezh2 in the differentiation of white, brown and beige adipocytes with Ezh2 conditional knockout mice (Ezh2flox/floxPrx1-cre) and mouse embryonic fibroblasts (MEFs). The results showed that Ezh2-deficient mice have a leaner phenotype and less white adipose tissues. The morphological changes in the adipose tissue included smaller white adipose tissue depots, white adipocytes with smaller diameter, smaller lipid droplets inside the brown adipocytes and more beige adipocytes in the Ezh2-deficient mice compared with the control. The differentiation markers of white adipocytes in Ezh2 knockout mice decreased; Ucp1 and other browning markers increased in brown and beige adipocytes. The Ezh2 knockout mice could better tolerate cold stimulation, and they can also resist obesity and insulin resistance induced by a high-fat diet. The Ezh2 inhibitor GSK126 could inhibit the differentiation of MEFs into white adipocytes but promote their differentiation into brown/beige adipocytes. The H3K27me3 demethylase Jmjd3/UTX inhibitor GSKJ4 inhibited MEFs' differentiation into brown/beige adipocytes. These results showed that Ezh2 promotes the differentiation of white adipocytes and inhibits the differentiation of brown and beige adipocytes in vivo and in vitro through its methylase activity and this may represent new knowledge for obesity therapeutic strategy.

Reduction in endoplasmic reticulum stress activates beige adipocytes differentiation and alleviates high fat diet-induced metabolic phenotypes.

Endoplasmic reticulum (ER) stress is closely associated with various metabolic diseases, such as obesity and diabetes. Development of beige/brite adipocytes increases thermogenesis and helps to reduce obesity. Although the relationship between ER stress and white adipocytes has been studied considerably, the possible role of ER stress and the unfolded protein response (UPR) induction in beige adipocytes differentiation remain to be investigated. In this study we investigated how ER stress affected beige adipocytes differentiation both in vitro and in vivo. Phosphorylation of eIF2α was transiently decreased in the early phase (day 2), whereas it was induced at the late phase with concomitant induction of C/EBP homologous protein (CHOP) during beige adipocytes differentiation. Forced expression of CHOP inhibited the expression of beige adipocytes markers, including Ucp1, Cox8b, Cidea, Prdm16, and Pgc-1α, following the induction of beige adipocytes differentiation. When ER stress was reduced by the chemical chaperone tauroursodeoxycholic acid (TUDCA), the expression of the beige adipocytes marker uncoupling protein 1 (UCP1) was significantly enhanced in inguinal white adipose tissue (iWAT) and high fat diet (HFD)-induced abnormal metabolic phenotype was improved. In summary, we found that ER stress and the UPR induction were closely involved in beige adipogenesis. These results suggest that modulating ER stress could be a potential therapeutic intervention against metabolic dysfunctions via activation of iWAT browning.

The colorful versatility of adipocytes: white-to-brown transdifferentiation and its therapeutic potential in humans.

Brown and brite adipocytes contribute to energy expenditure through nonshivering thermogenesis. Though these cell types are thought to arise primarily from the de novo differentiation of precursor cells, their abundance is also controlled through the transdifferentiation of mature white adipocytes. Here, we review recent advances in our understanding of the regulation of white-to-brown transdifferentiation, as well as the conversion of brown and brite adipocytes to dormant, white-like fat cells. Converting mature white adipocytes into brite cells or reactivating dormant brown and brite adipocytes has emerged as a strategy to ameliorate human metabolic disorders. We analyze the evidence of learning from mice and how they translate to humans to ultimately scrutinize the relevance of this concept. Moreover, we estimate that converting a small percentage of existing white fat mass in obese subjects into active brite adipocytes could be sufficient to achieve meaningful benefits in metabolism. In conclusion, novel browning agents have to be identified before adipocyte transdifferentiation can be realized as a safe and efficacious therapy.

Lactate fluxes mediated by the monocarboxylate transporter-1 are key determinants of the metabolic activity of beige adipocytes.

Activation of energy-dissipating brown/beige adipocytes represents an attractive therapeutic strategy against metabolic disorders. While lactate is known to induce beiging through the regulation of Ucp1 gene expression, the role of lactate transporters on beige adipocytes' ongoing metabolic activity remains poorly understood. To explore the function of the lactate-transporting monocarboxylate transporters (MCTs), we used a combination of primary cell culture studies, 13C isotopic tracing, laser microdissection experiments, and in situ immunofluorescence of murine adipose fat pads. Dissecting white adipose tissue heterogeneity revealed that the MCT1 is expressed in inducible beige adipocytes as the emergence of uncoupling protein 1 after cold exposure was restricted to a subpopulation of MCT1-expressing adipocytes suggesting MCT1 as a marker of inducible beige adipocytes. We also observed that MCT1 mediates bidirectional and simultaneous inward and outward lactate fluxes, which were required for efficient utilization of glucose by beige adipocytes activated by the canonical ß3-adrenergic signaling pathway. Finally, we demonstrated that significant lactate import through MCT1 occurs even when glucose is not limiting, which feeds the oxidative metabolism of beige adipocytes. These data highlight the key role of lactate fluxes in finely tuning the metabolic activity of beige adipocytes according to extracellular metabolic conditions and reinforce the emerging role of lactate metabolism in the control of energy homeostasis.

CD38 downregulation modulates NAD+ and NADP(H) levels in thermogenic adipose tissues.

Different strategies to boost NAD+ levels are considered promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme involved in the regulation of different cell functions. In the context of systemic energy metabolism, it has been demonstrated that brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis. In this study we aim to understand the functional relevance of CD38 for NAD+ and energy metabolism in BAT and WAT, also using a CD38-/- mouse model. During cold exposure, an increase in NAD+ levels occurred in BAT of wild type mice, together with a marked downregulation of CD38, as detected at the mRNA and protein level. CD38 downregulation was observed also in WAT of cold-exposed mice, where it was accompanied by a strong increase in NADP(H) levels. Accordingly, NAD kinase and glucose-6-phosphate dehydrogenase activities were enhanced in WAT (but not in BAT). Increased NAD+ levels were observed in BAT/WAT from CD38-/- compared with wild type mice, in line with CD38 being a major NAD+-consumer in AT. CD38-/- mice kept at 6 °C had higher levels of Ucp1 and Pgc-1α in BAT and WAT, and increased levels of phosphorylated hormone-sensitive lipase in BAT, compared with wild type mice. These results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT.

Cold-induced beigeing of stem cell-derived adipocytes is not fully reversible after return to normothermia.

Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid-laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige-like morphology and exhibited an intermediate gene expression profile, with both beige-like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post-transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide-4-phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC-derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown-like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.

NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation.

The transcription factor nuclear factor I-A (NFIA) is a regulator of brown adipocyte differentiation. Here we show that the C-terminal 17 amino acid residues of NFIA (which we call pro#3 domain) are required for the transcriptional activity of NFIA. Full-length NFIA-but not deletion mutant lacking pro#3 domain-rescued impaired expression of PPARγ, the master transcriptional regulator of adipogenesis and impaired adipocyte differentiation in NFIA-knockout cells. Mechanistically, the ability of NFIA to penetrate chromatin and bind to the crucial Pparg enhancer is mediated through pro#3 domain. However, the deletion mutant still binds to Myod1 enhancer to repress expression of MyoD, the master transcriptional regulator of myogenesis as well as proximally transcribed non-coding RNA called DRReRNA, via competition with KLF5 in terms of enhancer binding, leading to suppression of myogenic gene program. Therefore, the negative effect of NFIA on the myogenic gene program is, at least partly, independent of the positive effect on PPARγ expression and its downstream adipogenic gene program. These results uncover multiple ways of action of NFIA to ensure optimal regulation of brown and beige adipocyte differentiation.

Stiffness of the extracellular matrix regulates differentiation into beige adipocytes.

Beige/brite adipocytes, which express high levels of uncoupling protein 1 (UCP1) to generate heat using stored triglycerides, are induced under specific stimuli such as cold exposure in inguinal white adipose tissue (iWAT). Although extracellular microenvironments such as extracellular matrix (ECM) stiffness are known to regulate cell behaviors, including cell differentiation into adipocytes, the effect on iWAT cells is unknown. In this study, we show that rigid ECM promotes the cell spreading of iWAT-derived preadipocytes. Furthermore, the expression of UCP1 and other thermogenic genes in iWAT cells is promoted when the cells are cultured on rigid ECM. The expression of mTOR, a kinase known to regulate the differentiation to beige adipocytes, is decreased on rigid substrates. These results suggest that ECM stiffness plays an important role in the differentiation to beige adipocytes.

TC-E 5003, a protein methyltransferase 1 inhibitor, activates the PKA-dependent thermogenic pathway in primary murine and human subcutaneous adipocytes.

We previously reported the involvement of protein arginine methyltransferase 1 (PRMT1) in adipocyte thermogenesis. Here, we investigate the effects of PRMT1 inhibitors on thermogenesis. Unexpectedly, we find that the PRMT1 inhibitor TC-E 5003 (TC-E) induces the thermogenic properties of primary murine and human subcutaneous adipocytes. TC-E treatment upregulates the expression of Ucp1 and Fgf21 significantly and activates protein kinase A signaling and lipolysis in primary subcutaneous adipocytes from both mouse and humans. We further find that the thermogenic effects of TC-E are independent of PRMT1 and beta-adrenergic receptors. Our data indicate that TC-E exerts strong effects on murine and human subcutaneous adipocytes by activating beige adipocytes via PKA signaling.

Transcriptional Factors of Thermogenic Adipocyte Development and Generation of Brown and Beige Adipocytes From Stem Cells.

Brown and beige adipocytes have been widely known for their potential to dissipate excessive energy into heat form, resulting in an alleviation of obesity and other overweight-related conditions. This review highlights the origins, characteristics, and functions of the various kinds of adipocytes, as well as their anatomic distribution inside the human body. This review mainly focuses on various essential transcriptional factors such as PRDM16, FGF21, PPARα, PPARγ and PGC-1α, which exert their effects on the development and activation of thermogenic adipocytes via important pathways such as JAK-STAT, cAMP-PKA and PI3K-AKT signaling pathways. Additionally, this review will underline promising strategies to generate an unexhausted source of thermogenic adipocytes differentiated from human stem cells. These exogenous thermogenic adipocytes offer therapeutic potential for improvement of metabolic disorders via application as single cell or whole tissue transplantation. Graphical abstract Caption is required. Please provide.

Adipogenic commitment induced by green tea polyphenols remodel adipocytes to a thermogenic phenotype.

The potential contribution of green tea (GT) to the development of thermogenic/beige cells have been scarcely investigated. Here we investigated if the beneficial effects of GT in the induction of thermogenic/beige adipocytes results from an initial cell commitment during adipogenesis. Male C57Bl/6 mice (3 months) were divided into 3 groups: Control (chow diet), Obese (cafeteria diet), and Obese + GT. Mice received GT gavage (500 mg/kg of BW) over 12 weeks (5 days/week), after 4 weeks of diet, totalizing 16 weeks of experimentation. GT treatment increased energy expenditure (EE) in mice fed with cafeteria-diet leading to reduced BW gain, decreased adiposity, reduced inflammation, and improving insulin sensitivity. Those phenotypes were associated with enhanced expression of oxidative, thermogenic and beige genes. GT induced a futile cycle through de novo lipogenesis activating the thermogenic pathway. Induction of beige phenotype occurs autonomously in adipocytes and involves the PPARγ/FGF21/AMPK/UCP1 pathway. Our study identified that metabolic changes caused by GT may involve the temporal expression of PPARγ promoting the induction of thermogenic cells by reprogramming initial steps of adipocyte commitment.