isolation-adipogenic-fibro-inflammatory-stromal-cell-subpopulations

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

University of Texas Southwestern Medical Center

Hypoxia Inducible Factor 1 (HIF-1) promotes fibrosis and inflammation in adipose tissues, while estrogens and Estrogen Receptor α (ERα) have the opposite effect. Here we identify an Estrogen Response Element (ERE) in the promoter of Phd3, which is a negative regulatory enzyme of HIF-1, and we demonstrate HIF-1α is ubiquitinated following 17-β estradiol (E2)/ERα mediated Phd3 transcription. Manipulating ERα in vivo increases Phd3 transcription and reduces HIF-1 activity, while addition of PHD3 ameliorates adipose tissue fibrosis and inflammation. Our findings outline a novel regulatory relationship between E2/ERα, PHD3 and HIF-1 in adipose tissues, providing a mechanistic explanation for the protective effect of E2/ERα in adipose tissue. 

ERα upregulates Phd3 to ameliorate HIF-1 induced fibrosis and inflammation in adipose tissue.

Roux-en-Y gastric bypass (RYGB) is an effective method of weight loss and remediation of type-2 diabetes; however, the mechanisms leading to these improvements are unclear. Additionally, adipocytes within white adipose tissue (WAT) depots can manifest characteristics of brown adipocytes. These 'BRITE/beige' adipocytes express uncoupling protein 1 (UCP1) and are associated with improvements in glucose homeostasis and protection from obesity. Interestingly, atrial and B-type natriuretic peptides (NPs) promote BRITE/beige adipocyte enrichment of WAT depots, an effect known as "browning." Here, we investigate the effect of RYGB surgery on NP, NP receptors, and browning in the gonadal adipose tissues of female mice. We propose that such changes may lead to improvements in metabolic homeostasis commonly observed following RYGB.

Activation of natriuretic peptides and the sympathetic nervous system following Roux-en-Y gastric bypass is associated with gonadal adipose tissues browning.

The expansion of white adipose tissue (WAT) in obesity involves de novo differentiation of new adipocytes; however, the cellular origin of these cells remains unclear. Here, we utilize Zfp423(GFP) reporter mice to characterize adipose mural (Pdgfrβ(+)) cells with varying levels of the preadipocyte commitment factor Zfp423. We find that adipose tissue contains distinct mural populations, with levels of Zfp423 distinguishing adipogenic from inflammatory-like mural cells. Using our "MuralChaser" lineage tracking system, we uncover adipose perivascular cells as developmental precursors of adipocytes formed in obesity, with adipogenesis and precursor abundance regulated in a depot-dependent manner. Interestingly, Pdgfrβ(+) cells do not significantly contribute to the initial cold-induced recruitment of beige adipocytes in WAT; it is only after prolonged cold exposure that these cells differentiate into beige adipocytes. These results provide genetic evidence for a mural cell origin of white adipocytes in obesity and suggest that beige adipogenesis may originate from multiple sources. 

Pdgfrβ+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice.

The selective estrogen receptor modulator tamoxifen, in combination with the Cre-ER(T2) fusion protein, has been one of the mainstream methods to induce genetic recombination and has found widespread application in lineage tracing studies.

Impact of tamoxifen on adipocyte lineage tracing: Inducer of adipogenesis and prolonged nuclear translocation of Cre recombinase.

The transcriptional regulators Ebf2 and Prdm16 establish and maintain the brown and/or beige fat cell identity. However, the mechanisms operating in white adipocytes to suppress the thermogenic gene program and maintain an energy-storing phenotype are less understood. Here, we report that the transcriptional regulator Zfp423 is critical for maintaining white adipocyte identity through suppression of the thermogenic gene program. Zfp423 expression is enriched in white versus brown adipocytes and suppressed upon cold exposure. Doxycycline-inducible inactivation of Zfp423 in mature adipocytes, combined with β-adrenergic stimulation, triggers a conversion of differentiated adiponectin-expressing inguinal and gonadal adipocytes into beige-like adipocytes; this reprogramming event is sufficient to prevent and reverse diet-induced obesity and insulin resistance. Mechanistically, Zfp423 acts in adipocytes to inhibit the activity of Ebf2 and suppress Prdm16 activation. These data identify Zfp423 as a molecular brake on adipocyte thermogenesis and suggest a therapeutic strategy to unlock the thermogenic potential of white adipocytes in obesity. 

Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program.

 is a multi zinc-finger transcription factor expressed in preadipocytes and mature adipocytes . Our recent work has revealed a critical role for  in maintaining the fate of white adipocytes in adult mice through suppression of the beige cell thermogenic gene program; loss of  in mature adipocytes of adult mice results in a white-to-beige phenotypic switch. However, the exact requirements of  in the fetal stages of early adipose development  have not been clarified.

Fetal development of subcutaneous white adipose tissue is dependent on Zfp423.

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

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

Gupta and colleagues highlight recent work, including their own, that suggested based on lineage tracing that mural cells are adipogenic, contrasting with the conclusions of a recent Cell Stem Cell paper.

Do Adipocytes Emerge from Mural Progenitors?

Visceral adiposity confers significant risk for developing metabolic disease in obesity whereas preferential expansion of subcutaneous white adipose tissue (WAT) appears protective. Unlike subcutaneous WAT, visceral WAT is resistant to adopting a protective thermogenic phenotype characterized by the accumulation of Ucp1 beige/BRITE adipocytes (termed 'browning'). In this study, we investigated the physiological consequences of browning murine visceral WAT by selective genetic ablation of , a transcriptional suppressor of the adipocyte thermogenic program.  deletion in fetal visceral adipose precursors (; ), or adult visceral white adipose precursors (; ), results in the accumulation of beige-like thermogenic adipocytes within multiple visceral adipose depots. Thermogenic visceral WAT improves cold tolerance and prevents and reverses insulin resistance in obesity. These data indicate that beneficial visceral WAT browning can be engineered by directing visceral white adipocyte precursors to a thermogenic adipocyte fate, and suggest a novel strategy to combat insulin resistance in obesity.

Directing visceral white adipocyte precursors to a thermogenic adipocyte fate improves insulin sensitivity in obese mice.

Pathologic expansion of white adipose tissue (WAT) in obesity is characterized by adipocyte hypertrophy, inflammation, and fibrosis; however, factors triggering this maladaptive remodeling are largely unknown. Here, we test the hypothesis that the potential to recruit new adipocytes from Pdgfrβ preadipocytes determines visceral WAT health in obesity. We manipulate levels of Pparg, the master regulator of adipogenesis, in Pdgfrβ precursors of adult mice. Increasing the adipogenic capacity of Pdgfrβ precursors through Pparg overexpression results in healthy visceral WAT expansion in obesity and adiponectin-dependent improvements in glucose homeostasis. Loss of mural cell Pparg triggers pathologic visceral WAT expansion upon high-fat diet feeding. Moreover, the ability of the TZD class of anti-diabetic drugs to promote healthy visceral WAT remodeling is dependent on mural cell Pparg. These data highlight the protective effects of de novo visceral adipocyte differentiation in these settings, and suggest Pdgfrβ adipocyte precursors as targets for therapeutic intervention in diabetes.

De novo adipocyte differentiation from Pdgfrβ preadipocytes protects against pathologic visceral adipose expansion in obesity.

Fat grafting has been used extensively in plastic surgery in the past two decades. Here, the authors report the retrospective comparison of patients who underwent fractionated fat injection to blend the lid-cheek junction with those who had regular fat injection.

Role of Fractionated Fat in Blending the Lid-Cheek Junction.

Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. The fate of the original adipocytes during lactation and the developmental origin of the re-appearing adipocyte post involution are unclear. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα preadipocyte- and fibroblast-like cells during pregnancy and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. This cycle occurs over multiple pregnancies. These observations reveal the potential of terminally differentiated adipocytes to undergo repeated cycles of de-differentiation and re-differentiation in a physiological setting.

Reversible De-differentiation of Mature White Adipocytes into Preadipocyte-like Precursors during Lactation.

White adipose tissue (WAT) remodeling is dictated by coordinated interactions between adipocytes and resident stromal-vascular cells; however, the functional heterogeneity of adipose stromal cells has remained unresolved. We combined single-cell RNA-sequencing and FACS to identify and isolate functionally distinct subpopulations of PDGFRβ+ stromal cells within visceral WAT of adult mice. LY6C- CD9- PDGFRβ+ cells represent highly adipogenic visceral adipocyte precursor cells ('APCs'), whereas LY6C+ PDGFRβ+ cells represent fibro-inflammatory progenitors ('FIPs'). FIPs lack adipogenic capacity, display pro-fibrogenic/pro-inflammatory phenotypes, and can exert an anti-adipogenic effect on APCs. The pro-inflammatory phenotype of PDGFRβ+ cells is regulated, at least in part, by NR4A nuclear receptors. These data highlight the functional heterogeneity of visceral WAT perivascular cells, and provide insight into potential cell-cell interactions impacting adipogenesis and inflammation. These improved strategies to isolate FIPs and APCs from visceral WAT will facilitate the study of physiological WAT remodeling and mechanisms leading to metabolic dysfunction.

Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice.

Dermal adipose tissue (also known as dermal white adipose tissue and herein referred to as dWAT) has been the focus of much discussion in recent years. However, dWAT remains poorly characterized. The fate of the mature dermal adipocytes and the origin of the rapidly reappearing dermal adipocytes at different stages remain unclear. Here, we isolated dermal adipocytes and characterized dermal fat at the cellular and molecular level. Together with dWAT's dynamic responses to external stimuli, we established that dermal adipocytes are a distinct class of white adipocytes with high plasticity. By combining pulse-chase lineage tracing and single-cell RNA sequencing, we observed that mature dermal adipocytes undergo dedifferentiation and redifferentiation under physiological and pathophysiological conditions. Upon various challenges, the dedifferentiated cells proliferate and redifferentiate into adipocytes. In addition, manipulation of dWAT highlighted an important role for mature dermal adipocytes for hair cycling and wound healing. Altogether, these observations unravel a surprising plasticity of dermal adipocytes and provide an explanation for the dynamic changes in dWAT mass that occur under physiological and pathophysiological conditions, and highlight the important contributions of dWAT toward maintaining skin homeostasis.

Dermal adipose tissue has high plasticity and undergoes reversible dedifferentiation in mice.

Activated beige adipocytes have therapeutic potential due to their ability to improve glucose and lipid homeostasis. To date, the origin of beige adipocytes remains enigmatic. Whether beige cells arise through de novo differentiation from resident precursors or through reprogramming of mature white adipocytes has been a topic of intense discussion. Here, we offer our perspective on the natural origin of beige adipocytes in mice. In particular, we revisit recent lineage-tracing studies that shed light on this issue and offer new insight into how environmental housing temperatures early in life influence the mode of beige adipocyte biogenesis upon cold exposure later in life. We suggest a unified model in which beige adipocytes (UCP1 multilocular cells) in rodents initially arise predominantly from progenitors (i.e., de novo beige adipogenesis) upon the first exposure to cold temperatures and then interconvert between "dormant beige" and "active beige" phenotypes (i.e., beige cell activation) upon subsequent changes in environmental temperature. Importantly, we highlight experimental considerations needed to visualize de novo adipogenesis versus beige cell activation in mice. A precise understanding of the cellular origins of beige adipocytes emanating in response to physiological and pharmacological stimuli may better inform therapeutic strategies to recruit beige adipocytes in vivo.

Article	Year
ERα upregulates Phd3 to ameliorate HIF-1 induced fibrosis and inflammation in adipose tissue. Molecular metabolism\| PubMed ID: 25161887	2014
Activation of natriuretic peptides and the sympathetic nervous system following Roux-en-Y gastric bypass is associated with gonadal adipose tissues browning. Molecular metabolism\| PubMed ID: 25973390	2015
Pdgfrβ+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice. Cell metabolism\| PubMed ID: 26626462	2016
Impact of tamoxifen on adipocyte lineage tracing: Inducer of adipogenesis and prolonged nuclear translocation of Cre recombinase. Molecular metabolism\| PubMed ID: 26629402	2015
Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program. Cell metabolism\| PubMed ID: 27238639	2016
Fetal development of subcutaneous white adipose tissue is dependent on Zfp423. Molecular metabolism\| PubMed ID: 28123942	2017
Sorting out adipocyte precursors and their role in physiology and disease. Genes & development\| PubMed ID: 28202540	2017
Do Adipocytes Emerge from Mural Progenitors? Cell stem cell\| PubMed ID: 28475882	2017
Directing visceral white adipocyte precursors to a thermogenic adipocyte fate improves insulin sensitivity in obese mice. eLife\| PubMed ID: 28722653	2017
De novo adipocyte differentiation from Pdgfrβ preadipocytes protects against pathologic visceral adipose expansion in obesity. Nature communications\| PubMed ID: 29497032	2018
Role of Fractionated Fat in Blending the Lid-Cheek Junction. Plastic and reconstructive surgery\| PubMed ID: 29878987	2018
Reversible De-differentiation of Mature White Adipocytes into Preadipocyte-like Precursors during Lactation. Cell metabolism\| PubMed ID: 29909970	2018
Identification of functionally distinct fibro-inflammatory and adipogenic stromal subpopulations in visceral adipose tissue of adult mice. eLife\| PubMed ID: 30265241	2018
Dermal adipose tissue has high plasticity and undergoes reversible dedifferentiation in mice. The Journal of clinical investigation\| PubMed ID: 31503545	2019
Cellular Origins of Beige Fat Cells Revisited. Diabetes\| PubMed ID: 31540940	2019

Lavanya Vishvanath

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