miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19.

Fat storage in adult mammals is a highly regulated process that involves the mobilization of adipocyte progenitor cells (APCs) that differentiate to produce new adipocytes. Here we report a role for the broadly conserved miR-26 family of microRNAs (miR-26a-1, miR-26a-2, and miR-26b) as major regulators of APC differentiation and adipose tissue mass. Deletion of all miR-26-encoding loci in mice resulted in a dramatic expansion of adipose tissue in adult animals fed normal chow. Conversely, transgenic overexpression of miR-26a protected mice from high-fat diet-induced obesity. These effects were attributable to a cell-autonomous function of miR-26 as a potent inhibitor of APC differentiation. miR-26 blocks adipogenesis, at least in part, by repressing expression of Fbxl19, a conserved miR-26 target without a previously known role in adipocyte biology that encodes a component of SCF-type E3 ubiquitin ligase complexes. These findings have therefore revealed a novel pathway that plays a critical role in regulating adipose tissue formation in vivo and suggest new potential therapeutic targets for obesity and related disorders.

The developmental origins of adipose tissue.

Adipose tissue is formed at stereotypic times and locations in a diverse array of organisms. Once formed, the tissue is dynamic, responding to homeostatic and external cues and capable of a 15-fold expansion. The formation and maintenance of adipose tissue is essential to many biological processes and when perturbed leads to significant diseases. Despite this basic and clinical significance, understanding of the developmental biology of adipose tissue has languished. In this Review, we highlight recent efforts to unveil adipose developmental cues, adipose stem cell biology and the regulators of adipose tissue homeostasis and dynamism.

Cellular retinoic acid-binding protein 2 inhibits tumor growth by two distinct mechanisms.

Cellular retinoic acid-binding protein 2 (CRABP2) potently suppresses the growth of various carcinomas, but the mechanism(s) that underlies this activity remains incompletely understood. CRABP2 displays two distinct functions. The classical function of this protein is to directly deliver retinoic acid (RA) to RA receptor (RAR), a nuclear receptor activated by this hormone, in turn inducing the expression of multiple antiproliferative genes. The other function of the protein is exerted in the absence of RA and mediated by the RNA-binding and stabilizing protein HuR. CRABP2 directly binds to HuR, markedly strengthens its interactions with target mRNAs, and thus increases their stability and up-regulates their expression. Here we show that the anticarcinogenic activities of CRABP2 are mediated by both of its functions. Transcriptome analyses revealed that, in the absence of RA, a large cohort of transcripts is regulated in common by CRABP2 and HuR, and many of these are involved in regulation of oncogenic properties. Furthermore, both in cultured cells and in vivo, CRABP2 or a CRABP2 mutant defective in its ability to cooperate with RAR but competent in interactions with HuR suppressed carcinoma growth and did so in the absence of RA. Hence, transcript stabilization by the CRABP2-HuR complex significantly contributes to the ability of CRABP2 to inhibit tumorigenesis. Surprisingly, the observations also revealed that HuR regulates the expression of multiple genes involved in nuclear pore formation and is required for nuclear import of CRABP2 and for transcriptional activation by RAR. The data thus point at a novel function for this important protein.

The retinol esterifying enzyme LRAT supports cell signaling by retinol-binding protein and its receptor STRA6.

Vitamin A, retinol, circulates in blood bound to retinol-binding protein (RBP). At some tissues, holo-RBP is recognized by a plasma membrane receptor termed STRA6, which serves a dual role: it mediates transport of retinol from RBP into cells, and it functions as a cytokine receptor that, on binding holo-RBP, activates JAK2/STAT5 signaling. As STAT target genes include SOCS3, an inhibitor of insulin receptor, holo-RBP suppresses insulin responses in STRA6-expressing cells. We have shown previously that the two functions of STRA6 are interdependent. These observations suggest factors that regulate STRA6-mediated retinol transport may also control STRA6-mediated cell signaling. One such factor is retinol metabolism, which enables cellular uptake of retinol by maintaining an inward-directed concentration gradient. We show here that lecithin:retinol acyl transferase (LRAT), which catalyzes esterification of retinol to its storage species retinyl esters, is necessary for activation of the STRA6/JAK2/STAT5 cascade by holo-RBP. In accordance, LRAT-null mice are protected from holo-RBP-induced suppression of insulin responses. Hence, STRA6 signaling, which requires STRA6-mediated retinol transport, is supported by LRAT-catalyzed retinol metabolism. The observations demonstrate that STRA6 regulates key cellular processes by coupling circulating holo-RBP levels and intracellular retinol metabolism to cell signaling.

The STRA6 receptor is essential for retinol-binding protein-induced insulin resistance but not for maintaining vitamin A homeostasis in tissues other than the eye.

The plasma membrane protein STRA6 is thought to mediate uptake of retinol from its blood carrier retinol-binding protein (RBP) into cells and to function as a surface receptor that, upon binding of holo-RBP, activates a JAK/STAT cascade. It was suggested that STRA6 signaling underlies insulin resistance induced by elevated serum levels of RBP in obese animals. To investigate these activities in vivo, we generated and analyzed Stra6-null mice. We show that the contribution of STRA6 to retinol uptake by tissues in vivo is small and that, with the exception of the eye, ablation of Stra6 has only a modest effect on retinoid homeostasis and does not impair physiological functions that critically depend on retinoic acid in the embryo or in the adult. However, ablation of Stra6 effectively protects mice from RBP-induced suppression of insulin signaling. Thus one biological function of STRA6 in tissues other than the eye appears to be the coupling of circulating holo-RBP levels to cell signaling, in turn regulating key processes such as insulin response.

Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses.

It currently is believed that vitamin A, retinol, functions through active metabolites: the visual chromophore 11-cis-retinal, and retinoic acids, which regulate gene transcription. Retinol circulates in blood bound to retinol-binding protein (RBP) and is transported into cells by a membrane protein termed "stimulated by retinoic acid 6" (STRA6). We show here that STRA6 not only is a vitamin A transporter but also is a cell-surface signaling receptor activated by the RBP-retinol complex. Association of RBP-retinol with STRA6 triggers tyrosine phosphorylation, resulting in recruitment and activation of JAK2 and the transcription factor STAT5. The RBP-retinol/STRA6/JAK2/STAT5 signaling cascade induces the expression of STAT target genes, including suppressor of cytokine signaling 3 (SOCS3), which inhibits insulin signaling, and peroxisome proliferator-activated receptor gamma (PPARγ), which enhances lipid accumulation. These observations establish that the parental vitamin A molecule is a transcriptional regulator in its own right, reveal that the scope of biological functions of the vitamin is broader than previously suspected, and provide a rationale for understanding how RBP and retinol regulate energy homeostasis and insulin responses.

Reversing Pdgfrß Signaling Restores Metabolically Active Beige Adipocytes by Alleviating ILC2 Suppression in Aged and Obese Mice.

Objective: Platelet Derived Growth Factor Receptor Beta (Pdgfrß) suppresses the formation of cold temperature-induced beige adipocytes in aged mammals. We aimed to determine if deleting Pdgfrß in aged mice could rejuvenate metabolically active beige adipocytes by activating group 2 innate lymphoid cells (ILC2), and whether this effect could counteract diet-induced obesity-associated beige fat decline. Methods: We employed Pdgfrß gain-of-function and loss-of-function mouse models targeting beige adipocyte progenitor cells (APCs). Our approach included cold exposure, metabolic cage analysis, and age and diet-induced obesity models to examine beige fat development and metabolic function under varied Pdgfrß activity. Results: Acute cold exposure alone enhanced metabolic benefits in aged mice, irrespective of beige fat generation. However, Pdgfrß deletion in aged mice reestablished the formation of metabolically functional beige adipocytes, enhancing metabolism. Conversely, constitutive Pdgfrß activation in young mice stymied beige fat development. Mechanistically, Pdgfrß deletion upregulated IL-33, promoting ILC2 recruitment and activation, whereas Pdgfrß activation reduced IL-33 levels and suppressed ILC2 activity. Notably, diet-induced obesity markedly increased Pdgfrß expression and Stat1 signaling, which inhibited IL-33 induction and ILC2 activation. Genetic deletion of Pdgfrß restored beige fat formation in obese mice, improving whole-body metabolism. Conclusion: This study reveals that cold temperature exposure alone can trigger metabolic activation in aged mammals. However, reversing Pdgfrß signaling in aged and obese mice not only restores beige fat formation but also renews metabolic function and enhances the immunological environment of white adipose tissue (WAT). These findings highlight Pdgfrß as a crucial target for therapeutic strategies aimed at combating age- and obesity-related metabolic decline.

Platelet-derived growth factor receptor beta is required for embryonic specification and confinement of the adult white adipose lineage.

White adipose tissue (WAT) development and adult homeostasis rely on distinct adipocyte progenitor cells (APCs). While adult APCs are defined early during embryogenesis and generate adipocytes after WAT organogenesis, the mechanisms underlying adult adipose lineage determination and preservation remain undefined. Here, we uncover a critical role for platelet-derived growth factor receptor beta (Pdgfrß) in identifying the adult APC lineage. Without Pdgfrß, APCs lose their adipogenic competency to incite fibrotic tissue replacement and inflammation. Through lineage tracing analysis, we reveal that the adult APC lineage is lost and develops into macrophages when Pdgfrß is deleted embryonically. Moreover, to maintain the APC lineage, Pdgfrß activation stimulates p38/MAPK phosphorylation to promote APC proliferation and maintains the APC state by phosphorylating peroxisome proliferator activated receptor gamma (Pparγ) at serine 112. Together, our findings identify a role for Pdgfrß acting as a rheostat for adult adipose lineage confinement to prevent unintended lineage switches.

Smooth muscle cell-derived Cxcl12 directs macrophage accrual and sympathetic innervation to control thermogenic adipose tissue.

Sympathetic innervation of brown adipose tissue (BAT) controls mammalian adaptative thermogenesis. However, the cellular and molecular underpinnings contributing to BAT innervation remain poorly defined. Here, we show that smooth muscle cells (SMCs) support BAT growth, lipid utilization, and thermogenic plasticity. Moreover, we find that BAT SMCs express and control the bioavailability of Cxcl12. SMC deletion of Cxcl12 fosters brown adipocyte lipid accumulation, reduces energy expenditure, and increases susceptibility to diet-induced metabolic dysfunction. Mechanistically, we find that Cxcl12 stimulates CD301+ macrophage recruitment and supports sympathetic neuronal maintenance. Administering recombinant Cxcl12 to obese mice or leptin-deficient (Ob/Ob) mice is sufficient to boost macrophage presence and drive sympathetic innervation to restore BAT morphology and thermogenic responses. Altogether, our data reveal an SMC chemokine-dependent pathway linking immunological infiltration and sympathetic innervation as a rheostat for BAT maintenance and thermogenesis.

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.

Signaling by vitamin A and retinol-binding protein in regulation of insulin responses and lipid homeostasis.

Vitamin A, retinol, circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane protein termed stimulated by retinoic acid 6 (STRA6). It was reported that serum levels of RBP are elevated in obese rodents and humans, and that increased level of RBP in blood causes insulin resistance. A molecular mechanism by which RBP can exert such an effect is suggested by the recent discovery that STRA6 is not only a vitamin A transporter but also functions as a surface signaling receptor. Binding of RBP-ROH to STRA6 induces the phosphorylation of a tyrosine residue in the receptor C-terminus, thereby activating a JAK/STAT signaling cascade. Consequently, in STRA6-expressing cells such as adipocytes, RBP-ROH induces the expression of STAT target genes, including SOCS3, which suppresses insulin signaling, and PPARγ, which enhances lipid accumulation. RBP-retinol thus joins the myriad of cytokines, growth factors and hormones which regulate gene transcription by activating cell surface receptors that signal through activation of Janus kinases and their associated transcription factors STATs. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.

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.

Age-dependent Pdgfrß signaling drives adipocyte progenitor dysfunction to alter the beige adipogenic niche in male mice.

Perivascular adipocyte progenitor cells (APCs) can generate cold temperature-induced thermogenic beige adipocytes within white adipose tissue (WAT), an effect that could counteract excess fat mass and metabolic pathologies. Yet, the ability to generate beige adipocytes declines with age, creating a key challenge for their therapeutic potential. Here we show that ageing beige APCs overexpress platelet derived growth factor receptor beta (Pdgfrß) to prevent beige adipogenesis. We show that genetically deleting Pdgfrß, in adult male mice, restores beige adipocyte generation whereas activating Pdgfrß in juvenile mice blocks beige fat formation. Mechanistically, we find that Stat1 phosphorylation mediates Pdgfrß beige APC signaling to suppress IL-33 induction, which dampens immunological genes such as IL-13 and IL-5. Moreover, pharmacologically targeting Pdgfrß signaling restores beige adipocyte development by rejuvenating the immunological niche. Thus, targeting Pdgfrß signaling could be a strategy to restore WAT immune cell function to stimulate beige fat in adult mammals.

The Regulation of Adipose Tissue Health by Estrogens.

Obesity and its' associated metabolic diseases such as type 2 diabetes and cardiometabolic disorders are significant health problems confronting many countries. A major driver for developing obesity and metabolic dysfunction is the uncontrolled expansion of white adipose tissue (WAT). Specifically, the pathophysiological expansion of visceral WAT is often associated with metabolic dysfunction due to changes in adipokine secretion profiles, reduced vascularization, increased fibrosis, and enrichment of pro-inflammatory immune cells. A critical determinate of body fat distribution and WAT health is the sex steroid estrogen. The bioavailability of estrogen appears to favor metabolically healthy subcutaneous fat over visceral fat growth while protecting against changes in metabolic dysfunction. Our review will focus on the role of estrogen on body fat partitioning, WAT homeostasis, adipogenesis, adipocyte progenitor cell (APC) function, and thermogenesis to control WAT health and systemic metabolism.

Remodeling of gene regulatory networks underlying thermogenic stimuli-induced adipose beiging.

Beige adipocytes are induced by cold temperatures or ß3-adrenergic receptor (Adrb3) agonists. They create heat through glucose and fatty acid (FA) oxidation, conferring metabolic benefits. The distinct and shared mechanisms by which these treatments induce beiging are unknown. Here, we perform single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) on adipose tissue from mice exposed to cold or an Adrb3 agonist to identify cellular and chromatin accessibility dynamics during beiging. Both stimuli induce chromatin remodeling that influence vascularization and inflammation in adipose. Beige adipocytes from cold-exposed mice have increased accessibility at genes regulating glycolytic processes, whereas Adrb3 activation increases cAMP responses. While both thermogenic stimuli increase accessibility at genes regulating thermogenesis, lipogenesis, and beige adipocyte development, the kinetics and magnitudes of the changes are distinct for the stimuli. Accessibility changes at lipogenic genes are linked to functional changes in lipid composition of adipose. Both stimuli tend to decrease the proportion of palmitic acids, a saturated FA in adipose. However, Adrb3 activation increases the proportion of monounsaturated FAs, whereas cold increases the proportion of polyunsaturated FAs. These findings reveal common and distinct mechanisms of cold and Adrb3 induced beige adipocyte biogenesis, and identify unique functional consequences of manipulating these pathways in vivo.

Repression of cellular retinoic acid-binding protein II during adipocyte differentiation.

In preadipocytes, retinoic acid (RA) regulates gene expression by activating the nuclear RA receptor (RAR) and its cognate intracellular lipid-binding protein CRABP-II. It was previously reported that RA inhibits adipocyte differentiation but only when administered early during the differentiation program. The data presented here indicate that the diminished ability of RA to activate RAR following induction of differentiation stems from down-regulation of CRABP-II. The observations show that expression of CRABP-II in preadipocytes is repressed by all three components of the classical hormonal mixture that induces adipocyte differentiation, i.e. isobutylmethylxanthine, insulin, and dexamethasone. Isobutylmethylxanthine-dependent activation of protein kinase A triggered the phosphorylation of the transcription factor cAMP-response element-binding protein, which induced the expression of the cAMP-response element-binding protein family repressor cAMP-response element modulator. In turn, cAMP-response element modulator was found to associate with a cognate response element in the CRABP-II promoter and to repress CRABP-II expression. The data further show that CRABP-II is a direct target gene for the glucocorticoid receptor and that it is subjected to dexamethasone-induced glucocorticoid receptor-mediated repression during adipogenesis. Finally, the observations demonstrate that permanent repression of CRABP-II in mature adipocytes is exerted by the master regulator of adipocyte differentiation CCAAT/enhancer-binding protein alpha and is directly mediated through CCAAT/enhancer-binding protein alpha-response elements in the CRABP-II promoter. Taken together, the observations emphasize the important role of CRABP-II in regulating the transcriptional activity of RA through RAR, and they demonstrate that repression of this gene is critical for allowing adipogenesis to proceed.

Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARbeta/delta to RAR.

Retinoic acid (RA) displays potent anticarcinogenic activities that are mediated by the nuclear retinoic acid receptors (RARs). However, use of RA in oncology is limited by RA resistance acquired during carcinogenesis. Moreover, in some cancers, RA facilitates rather than inhibits growth. A clue to this paradoxical behavior was recently suggested by the findings that RA also activates PPARbeta/delta, a receptor involved in mitogenic and anti-apoptotic activities. The observations that partitioning of RA between its two receptors is regulated by two intracellular lipid-binding proteins-CRABP-II, which targets RA to RAR, and FABP5, which delivers it to PPARbeta/delta-further suggest that RA resistance may stem from the deregulation of the binding proteins, resulting in activation of PPARbeta/delta rather than RAR. Here, we show that, in the RA-resistant mouse model of breast cancer MMTV-neu, RA indeed activates the nonclassical RA receptor PPARbeta/delta. This behavior was traced to an aberrantly high intratumor FABP5/CRABP-II ratio. Decreasing this ratio in mammary tissue diverted RA from PPARbeta/delta to RAR and suppressed tumor growth. The data demonstrate the existence of a mechanism that underlies RA resistance in tumors, indicate that CRABP-II functions as a tumor suppressor, and suggest that the inhibition of FABP5 may comprise a therapeutic strategy for overcoming RA resistance in some tumors.

Progenitor-like characteristics in a subgroup of UCP1+ cells within white adipose tissue.

Thermogenic beige fat found in white adipose tissue is a potential therapeutic target to curb the global obesity and diabetes epidemic. However, these inducible thermogenic beige adipocytes have been thought to be short-lived and to rapidly convert to "white-like" adipocytes after discontinuing stimuli. In this study, using effective labeling techniques and genetic mouse tools, we demonstrate that a subset of UCP1+ cells that exist within white adipose tissue are able to self-divide and contribute to new beige adipocyte recruitment in response to ß3 stimuli. When these cells are depleted or their adipogenic capability is blocked, ß3-induced beige adipocyte formation is impaired. We also identify a cell-cycle machinery of p21 and CDKN2A as a molecular basis of beige adipocyte regulation. Collectively, our findings provide new insights into the cellular and molecular mechanisms of beige adipocyte regulation and potential therapeutic opportunities to induce the beige phenotype and treat metabolic disease.

Dynamic control of adipose tissue development and adult tissue homeostasis by platelet-derived growth factor receptor alpha.

Adipocytes arise from distinct progenitor populations during developmental and adult stages but little is known about how developmental progenitors differ from adult progenitors. Here, we investigate the role of platelet-derived growth factor receptor alpha (PDGFRα) in the divergent regulation of the two different adipose progenitor cells (APCs). Using in vivo adipose lineage tracking and deletion mouse models, we found that developmental PDGFRα+ cells are adipogenic and differentiated into mature adipocytes, and the deletion of Pdgfra in developmental adipose lineage disrupted white adipose tissue (WAT) formation. Interestingly, adult PDGFRα+ cells do not significantly contribute to adult adipogenesis, and deleting Pdgfra in adult adipose lineage did not affect WAT homeostasis. Mechanistically, embryonic APCs require PDGFRα for fate maintenance, and without PDGFRα, they underwent fate change from adipogenic to fibrotic lineage. Collectively, our findings indicate that PDGFRα+ cells and Pdgfra gene itself are differentially required for WAT development and adult WAT homeostasis.

Distinct cellular and molecular mechanisms for ß3 adrenergic receptor-induced beige adipocyte formation.

Beige/brite adipocytes are induced within white adipose tissues (WAT) and, when activated, consume glucose and fatty acids to produce heat. Classically, two stimuli have been used to trigger a beiging response: cold temperatures and ß3-adrenergic receptor (Adrb3) agonists. These two beiging triggers have been used interchangeably but whether these two stimuli may induce beiging differently at cellular and molecular levels remains unclear. Here, we found that cold-induced beige adipocyte formation requires Adrb1, not Adrb3, activation. Adrb1 activation stimulates WAT resident perivascular (Acta2+) cells to form cold-induced beige adipocytes. In contrast, Adrb3 activation stimulates mature white adipocytes to convert into beige adipocytes. Necessity tests, using mature adipocyte-specific Prdm16 deletion strategies, demonstrated that adipocytes are required and are predominant source to generate Adrb3-induced, but not cold-induced, beige adipocytes. Collectively, we identify that cold temperatures and Adrb3 agonists activate distinct cellular populations that express different ß-adrenergic receptors to induce beige adipogenesis.