Mesenchymal stromal cells as conductors of adipose tissue remodeling.

Adipose tissue exhibits a remarkable capacity to expand, contract, and remodel in response to changes in physiological and environmental conditions. Here, we describe recent advances in our understanding of how functionally distinct tissue-resident mesenchymal stromal cell subpopulations orchestrate several aspects of physiological and pathophysiological adipose tissue remodeling, with a particular focus on the adaptations that occur in response to changes in energy surplus and environmental temperature. The study of adipose tissue remodeling provides a vehicle to understand the functional diversity of stromal cells and offers a lens through which several generalizable aspects of tissue reorganization can be readily observed.

Cold-responsive adipocyte progenitors couple adrenergic signaling to immune cell activation to promote beige adipocyte accrual.

The full array of cold-responsive cell types within white adipose tissue that drive thermogenic beige adipocyte biogenesis remains undefined. We demonstrate that acute cold challenge elicits striking transcriptomic changes specifically within DPP4+ PDGFRß+ adipocyte precursor cells, including a ß-adrenergic receptor CREB-mediated induction in the expression of the prothermogenic cytokine, Il33 Doxycycline-inducible deletion of Il33 in PDGFRß+ cells at the onset of cold exposure attenuates ILC2 accumulation and beige adipocyte accrual. These studies highlight the multifaceted roles for adipocyte progenitors and the ability of select mesenchymal subpopulations to relay neuronal signals to tissue-resident immune cells in order to regulate tissue plasticity.

ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes.

Energy-storing white adipocytes maintain their identity by suppressing the energy-burning thermogenic gene program of brown and beige adipocytes. Here, we reveal that the protein-protein interaction between the transcriptional coregulator ZFP423 and brown fat determination factor EBF2 is essential for restraining the thermogenic phenotype of white adipose tissue (WAT). Disruption of the ZFP423-EBF2 protein interaction through CRISPR-Cas9 gene editing triggers widespread "browning" of WAT in adult mice. Mechanistically, ZFP423 recruits the NuRD corepressor complex to EBF2-bound thermogenic gene enhancers. Loss of adipocyte Zfp423 induces an EBF2 NuRD-to-BAF coregulator switch and a shift in PPARγ occupancy to thermogenic genes. This shift in PPARγ occupancy increases the antidiabetic efficacy of the PPARγ agonist rosiglitazone in obesity while diminishing the unwanted weight-gaining effect of the drug. These data indicate that ZFP423 controls EBF2 coactivator recruitment and PPARγ occupancy to determine the thermogenic plasticity of adipocytes and highlight the potential of therapeutically targeting transcriptional brakes to induce beige adipocyte biogenesis in obesity.

Functional Interplay between Histone H2B ADP-Ribosylation and Phosphorylation Controls Adipogenesis.

Although ADP-ribosylation of histones by PARP-1 has been linked to genotoxic stress responses, its role in physiological processes and gene expression has remained elusive. We found that NAD+-dependent ADP-ribosylation of histone H2B-Glu35 by small nucleolar RNA (snoRNA)-activated PARP-1 inhibits AMP kinase-mediated phosphorylation of adjacent H2B-Ser36, which is required for the proadipogenic gene expression program. The activity of PARP-1 on H2B requires NMNAT-1, a nuclear NAD+ synthase, which directs PARP-1 catalytic activity to Glu and Asp residues. ADP-ribosylation of Glu35 and the subsequent reduction of H2B-Ser36 phosphorylation inhibits the differentiation of adipocyte precursors in cultured cells. Parp1 knockout in preadipocytes in a mouse lineage-tracing genetic model increases adipogenesis, leading to obesity. Collectively, our results demonstrate a functional interplay between H2B-Glu35 ADP-ribosylation and H2B-Ser36 phosphorylation that controls adipogenesis.

TRPV4 is a regulator of adipose oxidative metabolism, inflammation, and energy homeostasis.

PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.

C/EBPß controls exercise-induced cardiac growth and protects against pathological cardiac remodeling.

The heart has the ability to grow in size in response to exercise, but little is known about the transcriptional mechanisms underlying physiological hypertrophy. Adult cardiomyocytes have also recently been proven to hold the potential for proliferation, a process that could be of great importance for regenerative medicine. Using a unique RT-PCR-based screen against all transcriptional components, we showed that C/EBPß was downregulated with exercise, whereas the expression of CITED4 was increased. Reduction of C/EBPß in vitro and in vivo resulted in a phenocopy of endurance exercise with cardiomyocyte hypertrophy and proliferation. This proliferation was mediated, at least in part, by the increased CITED4. Importantly, mice with reduced cardiac C/EBPß levels displayed substantial resistance to cardiac failure upon pressure overload. These data indicate that C/EBPß represses cardiomyocyte growth and proliferation in the adult mammalian heart and that reduction in C/EBPß is a central signal in physiologic hypertrophy and proliferation.

PARP-1 Controls the Adipogenic Transcriptional Program by PARylating C/EBPß and Modulating Its Transcriptional Activity.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by PARP family members, such as PARP-1. Although PARylation has been studied extensively, few examples of definitive biological roles for site-specific PARylation have been reported. Here we show that C/EBPß, a key pro-adipogenic transcription factor, is PARylated by PARP-1 on three amino acids in a conserved regulatory domain. PARylation at these sites inhibits C/EBPß's DNA binding and transcriptional activities and attenuates adipogenesis in various genetic and cell-based models. Interestingly, PARP-1 catalytic activity drops precipitously during the first 48 hr of differentiation, corresponding to a release of C/EBPß from PARylation-mediated inhibition. This promotes the binding of C/EBPß at enhancers controlling the expression of adipogenic target genes and continued differentiation. Depletion or chemical inhibition of PARP-1, or mutation of the PARylation sites on C/EBPß, enhances these early adipogenic events. Collectively, our results provide a clear example of how site-specific PARylation drives biological outcomes.

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

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.

Regulation of cold-induced thermogenesis by the RNA binding protein FAM195A.

Homeothermic vertebrates produce heat in cold environments through thermogenesis, in which brown adipose tissue (BAT) increases mitochondrial oxidation along with uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors regulating the expression of UCP1 and nutrient oxidation genes have been extensively studied, only a few other proteins essential for BAT function have been identified. We describe the discovery of FAM195A, a BAT-enriched RNA binding protein, which is required for cold-dependent thermogenesis in mice. FAM195A knockout (KO) mice display whitening of BAT and an inability to thermoregulate. In BAT of FAM195A KO mice, enzymes involved in branched-chain amino acid (BCAA) metabolism are down-regulated, impairing their response to cold. Knockdown of FAM195A in brown adipocytes in vitro also impairs expression of leucine oxidation enzymes, revealing FAM195A to be a regulator of BCAA metabolism and a potential target for metabolic disorders.

Regulator of Calcineurin 1 helps coordinate whole-body metabolism and thermogenesis.

Increasing non-shivering thermogenesis (NST), which expends calories as heat rather than storing them as fat, is championed as an effective way to combat obesity and metabolic disease. Innate mechanisms constraining the capacity for NST present a fundamental limitation to this approach, yet are not well understood. Here, we provide evidence that Regulator of Calcineurin 1 (RCAN1), a feedback inhibitor of the calcium-activated protein phosphatase calcineurin (CN), acts to suppress two distinctly different mechanisms of non-shivering thermogenesis (NST): one involving the activation of UCP1 expression in white adipose tissue, the other mediated by sarcolipin (SLN) in skeletal muscle. UCP1 generates heat at the expense of reducing ATP production, whereas SLN increases ATP consumption to generate heat. Gene expression profiles demonstrate a high correlation between Rcan1 expression and metabolic syndrome. On an evolutionary timescale, in the context of limited food resources, systemic suppression of prolonged NST by RCAN1 might have been beneficial; however, in the face of caloric abundance, RCAN1-mediated suppression of these adaptive avenues of energy expenditure may now contribute to the growing epidemic of obesity.

Acute loss of adipose tissue-derived adiponectin triggers immediate metabolic deterioration in mice.

AIM/HYPOTHESIS: Adiponectin (APN), a circulating hormone secreted by mature adipocytes, has been extensively studied because it has beneficial metabolic effects. While many studies have focused on the congenital loss of APN and its effects on systemic body glucose and lipid metabolism, little is known about the effects triggered by acute loss of APN in the adult mouse. We anticipated that genetically induced acute depletion of APN in adult mice would have a more profound effect on systemic metabolic health than congenital deletion of Adipoq, the gene encoding APN, with its associated potential for adaptive responses that may mask the phenotypes. METHODS: Mice carrying loxP-flanked regions of Adipoq were generated and bred to the Adipoq (APN) promoter-driven reverse tetracycline-controlled transactivator (rtTA) (APN-rtTA) gene and a tet-responsive Cre line (TRE-Cre) to achieve acute depletion of APN. Upon acute removal of APN in adult mice, systemic glucose and lipid homeostasis were assessed under basal and insulinopenic conditions. RESULTS: The acute depletion of APN results in more severe systemic insulin resistance and hyperlipidaemia than in mice with congenital loss of APN. Furthermore, the acute depletion of APN in adult mice results in a much more dramatic reduction in survival rate, with 50% of inducible knockouts dying in the first 5 days under insulinopenic conditions compared with 0% of congenital Adipoq knockout mice under similar conditions. CONCLUSIONS/INTERPRETATION: Acute systemic removal of APN results in a much more negative metabolic phenotype compared with congenital knockout of Adipoq. Specifically, our data demonstrate that acute depletion of APN is especially detrimental to lipid homeostasis, both under basal and insulinopenic conditions. This suggests that compensatory mechanisms exist in congenital knockout mice that offset some of the metabolic actions covered by APN.

Transcriptional control of preadipocyte determination by Zfp423.

The worldwide epidemic of obesity has increased the urgency to develop a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARgamma and several other transcription factors, but the molecular basis for preadipocyte determination is not understood. Using a new method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of Pparg in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. Short hairpin RNA (shRNA)-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte Pparg expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is markedly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates Pparg expression, in part, through amplification of the BMP signalling pathway, an effect dependent on the SMAD-binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Regulation of early adipose commitment by Zfp521.

While there has been significant progress in determining the transcriptional cascade involved in terminal adipocyte differentiation, less is known about early events leading to lineage commitment and cell fate choice. It has been recently discovered that zinc finger protein 423 (Zfp423) is an early actor in adipose determination. Here, we show that a close paralog of Zfp423, Zfp521, acts as a key regulator of adipose commitment and differentiation in vitro and in vivo. Zfp521 exerts its actions by binding to early B cell factor 1 (Ebf1), a transcription factor required for the generation of adipocyte progenitors, and inhibiting the expression of Zfp423. Overexpression of Zfp521 in cells greatly inhibits adipogenic potential, whereas RNAi-mediated knock-down or genetic ablation of Zfp521 enhances differentiation. In addition, Zfp521⁻/⁻ embryos exhibit increased mass of interscapular brown adipose tissue and subcutaneous white adipocytes, a cell autonomous effect. Finally, Ebf1 participates in a negative feedback loop to repress Zfp521 as differentiation proceeds. Because Zfp521 is known to promote bone development, our results suggest that it acts as a critical switch in the commitment decision between the adipogenic and osteogenic lineages.

Improved methodologies for the study of adipose biology: insights gained and opportunities ahead.

Adipocyte differentiation and function have become areas of intense focus in the field of energy metabolism; however, understanding the role of specific genes in the establishment and maintenance of fat cell function can be challenging and complex. In this review, we offer practical guidelines for the study of adipocyte development and function. We discuss improved cellular and genetic systems for the study of adipose biology and highlight recent insights gained from these new approaches.

Elevated resistin levels induce central leptin resistance and increased atherosclerotic progression in mice.

AIMS/HYPOTHESIS: Resistin was originally identified as an adipocyte-derived factor upregulated during obesity and as a contributor to obesity-associated insulin resistance. Clinically, resistin has also been implicated in cardiovascular disease in a number of different patient populations. Our aim was to simultaneously address these phenomena. METHODS: We generated mice with modest adipocyte-specific resistin overexpression. These mice were crossed with mice deficient in the LDL receptor (Ldlr (-/-)) to probe the physiological role of resistin. Both metabolic and atherosclerotic assessments were performed. RESULTS: Resistin overexpression led to increased atherosclerotic progression in Ldlr (-/-) mice. This was in part related to elevated serum triacylglycerol levels and a reduced ability to clear triacylglycerol upon a challenge. Additional phenotypic changes, such as increased body weight and reduced glucose clearance, independent of the Ldlr (-/-) background, confirmed increased adiposity associated with a more pronounced insulin resistance. A hallmark of elevated resistin was the disproportionate increase in circulating leptin levels. These mice thus recapitulated both the proposed negative cardiovascular correlation and the insulin resistance. A unifying mechanism for this complex phenotype was a resistin-mediated central leptin resistance, which we demonstrate directly both in vivo and in organotypic brain slices. In line with reduced sympathetic nervous system outflow, we found decreased brown adipose tissue (BAT) activity. The resulting elevated triacylglycerol levels provide a likely explanation for accelerated atherosclerosis. CONCLUSIONS/INTERPRETATION: Resistin overexpression leads to a complex metabolic phenotype driven by resistin-mediated central leptin resistance and reduced BAT activity. Hypothalamic leptin resistance thus provides a unifying mechanism for both resistin-mediated insulin resistance and enhanced atherosclerosis.

The Expanding Problem of Regional Adiposity: Revisiting a 1985 Diabetes Classic by Ohlson et al.

Body fat distribution is a predictor of metabolic health in obesity. In this Classics in Diabetes article, we revisit a 1985 Diabetes article by Swedish investigators Ohlson et al. This work was one of the first prospective population-based studies that established a relationship between abdominal adiposity and the risk for developing diabetes. Here, we discuss evolving concepts regarding the link between regional adiposity and diabetes and other chronic disorders. Moreover, we highlight fundamental questions that remain unresolved.

PGC-1alpha regulates a HIF2alpha-dependent switch in skeletal muscle fiber types.

The coactivator peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) coordinates a broad set of transcriptional programs that regulate the response of skeletal muscle to exercise. However, the complete transcriptional network controlled by PGC-1α has not been described. In this study, we used a qPCR-based screen of all known transcriptional components (Quanttrx) to identify transcription factors that are quantitatively regulated by PGC-1α in cultured skeletal muscle cells. This analysis identified hypoxia-inducible factor 2 α (HIF2α) as a major PGC-1α target in skeletal muscle that is positively regulated by both exercise and ß-adrenergic signaling. This transcriptional regulation of HIF2α is completely dependent on the PGC-1α/ERRα complex and is further modulated by the action of SIRT1. Transcriptional profiling of HIF2α target genes in primary myotubes suggested an unexpected role for HIF2α in the regulation of muscle fiber types, specifically enhancing the expression of a slow twitch gene program. The PGC-1α-mediated switch to slow, oxidative fibers in vitro is dependent on HIF2α, and mice with a muscle-specific knockout of HIF2α increase the expression of genes and proteins characteristic of a fast-twitch fiber-type switch. These data indicate that HIF2α acts downstream of PGC-1α as a key regulator of a muscle fiber-type program and the adaptive response to exercise.

Adipose tissue at single-cell resolution.

Adipose tissue exhibits remarkable plasticity with capacity to change in size and cellular composition under physiological and pathophysiological conditions. The emergence of single-cell transcriptomics has rapidly transformed our understanding of the diverse array of cell types and cell states residing in adipose tissues and has provided insight into how transcriptional changes in individual cell types contribute to tissue plasticity. Here, we present a comprehensive overview of the cellular atlas of adipose tissues focusing on the biological insight gained from single-cell and single-nuclei transcriptomics of murine and human adipose tissues. We also offer our perspective on the exciting opportunities for mapping cellular transitions and crosstalk, which have been made possible by single-cell technologies.

Protocol for quantitative proteomic analysis of heterogeneous adipose tissue-residing progenitor subpopulations in mice.

Recent studies have revealed cellular heterogeneity of mesenchymal stromal cells and immune cells in adipose tissue and emphasized the need for quantitative analysis of small numbers of functionally distinct cells using state-of-the-art "omics" technologies. Here, we present an optimized protocol for precise protein quantification from minute amounts of samples. We describe steps for isolation of mouse adipose progenitor cells, proteomics sample preparation, mass spectrometry measurement, and computational analysis. This protocol can be adapted to other samples with limited amounts. For complete details on the use and execution of this protocol, please refer to Shan et al. (2022).1.