Zinc finger protein ZNF638 regulates triglyceride metabolism via ANGPTL8 in an estrogen dependent manner.

BACKGROUND AND AIM: Triglyceride (TG) levels are closely related to obesity, fatty liver and cardiovascular diseases, while the regulatory factors and mechanism for triglyceride homeostasis are still largely unknown. Zinc Finger Protein 638 (ZNF638) is a newly discovered member of zinc finger protein family for adipocyte function in vitro. The aim of the present work was to investigate the role of ZNF638 in regulating triglyceride metabolism in mice. METHODS: We generated ZNF638 adipose tissue specific knockout mice (ZNF638 FKO) by cross-breeding ZNF638 flox to Adiponectin-Cre mice and achieved adipose tissue ZNF638 overexpression via adenoviral mediated ZNF638 delivery in inguinal adipose tissue (iWAT) to examined the role and mechanisms of ZNF638 in fat biology and whole-body TG homeostasis. RESULTS: Although ZNF638 FKO mice showed similar body weights, body composition, glucose metabolism and serum parameters compared to wild-type mice under chow diet, serum TG levels in ZNF638 FKO mice were increased dramatically after refeeding compared to wild-type mice, accompanied with decreased endothelial lipoprotein lipase (LPL) activity and increased lipid absorption of the small intestine. Conversely, ZNF638 overexpression in iWAT reduced serum TG levels while enhanced LPL activity after refeeding in female C57BL/6J mice and obese ob/ob mice. Specifically, only female mice exhibited altered TG metabolism upon ZNF638 expression changes in fat. Mechanistically, RNA-sequencing analysis revealed that the TG regulator angiopoietin-like protein 8 (Angptl8) was highly expressed in iWAT of female ZNF638 FKO mice. Neutralizing circulating ANGPTL8 in female ZNF638 FKO mice abolished refeeding-induced TG elevation. Furthermore, we demonstrated that ZNF638 functions as a transcriptional repressor by recruiting HDAC1 for histone deacetylation and broad lipid metabolic gene suppression, including Angptl8 transcription inhibition. Moreover, we showed that the sexual dimorphism is possibly due to estrogen dependent regulation on ZNF638-ANGPTL8 axis. CONCLUSION: We revealed a role of ZNF638 in the regulation of triglyceride metabolism by affecting Angptl8 transcriptional level in adipose tissue with sexual dimorphism.

Browning of adipose tissue and increased thermogenesis induced by Methotrexate.

Methotrexate (MTX) is widely used for the treatment of rheumatoid arthritis due to its well-known anti-inflammatory role in immune cells but its impact on brown and beige adipose tissue biology has not yet been investigated. Here, we present the novel evidence that MTX treatment increases the gene expression of thermogenic genes in brown and beige adipose tissues in a fat cell autonomous manner. Furthermore, we show that treatment of mice with MTX is associated with cold resistance, improved glucose homeostasis, decreased inflammation, and reduced hepatosteatosis in high-fat diet states. Overall, our data provide novel evidence of a role of MTX on thermogenic tissues not previously appreciated.

The forkhead box transcription factor FoxP4 regulates thermogenic programs in adipocytes.

Forkhead box transcription factors have been shown to be involved in various developmental and differentiation processes. In particular, members of the FoxP family have been previously characterized in depth for their participation in the regulation of lung and neuronal cell differentiation and T-cell development and function; however, their role in adipocyte functionality has not yet been investigated. Here, we report for the first time that Forkhead box P4 (FoxP4) is expressed at high levels in subcutaneous fat depots and mature thermogenic adipocytes. Through molecular and gene expression analyses, we revealed that FoxP4 is induced in response to thermogenic stimuli, both in vivo and in isolated cells, and is regulated directly by the heat shock factor protein 1 through a heat shock response element identified in the proximal promoter region of FoxP4. Further detailed analysis involving chromatin immunoprecipitation and luciferase assays demonstrated that FoxP4 directly controls the levels of uncoupling protein 1, a key regulator of thermogenesis that uncouples fatty acid oxidation from ATP production. In addition, through our gain-of-function and loss-of-function studies, we showed that FoxP4 regulates the expression of a number of classic brown and beige fat genes and affects oxygen consumption in isolated adipocytes. Overall, our data demonstrate for the first time the novel role of FoxP4 in the regulation of thermogenic adipocyte functionality.

MicroRNA-33 Inhibits Adaptive Thermogenesis and Adipose Tissue Beiging.

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PPARγ agonists delay age-associated metabolic disease and extend longevity.

Aging leads to a number of disorders caused by cellular senescence, tissue damage, and organ dysfunction. It has been reported that anti-inflammatory and insulin-sensitizing compounds delay, or reverse, the aging process and prevent metabolic disorders, neurodegenerative disease, and muscle atrophy, improving healthspan and extending lifespan. Here we investigated the effects of PPARγ agonists in preventing aging and increasing longevity, given their known properties in lowering inflammation and decreasing glycemia. Our molecular and physiological studies show that long-term treatment of mice at 14 months of age with low doses of the PPARγ ligand rosiglitazone (Rosi) improved glucose metabolism and mitochondrial functionality. These effects were associated with decreased inflammation and reduced tissue atrophy, improved cognitive function, and diminished anxiety- and depression-like conditions, without any adverse effects on cardiac and skeletal functionality. Furthermore, Rosi treatment of mice started when they were 14 months old was associated with lifespan extension. A retrospective analysis of the effects of the PPARγ agonist pioglitazone (Pio) on longevity showed decreased mortality in patients receiving Pio compared to those receiving a PPARγ-independent insulin secretagogue glimepiride. Taken together, these data suggest the possibility of using PPARγ agonists to promote healthy aging and extend lifespan.

The mRNA levels of heat shock factor 1 are regulated by thermogenic signals via the cAMP-dependent transcription factor ATF3.

Heat shock factor 1 (HSF1) regulates cellular adaptation to challenges such as heat shock and oxidative and proteotoxic stresses. We have recently reported a previously unappreciated role for HSF1 in the regulation of energy metabolism in fat tissues; however, whether HSF1 is differentially expressed in adipose depots and how its levels are regulated in fat tissues remain unclear. Here, we show that HSF1 levels are higher in brown and subcutaneous fat tissues than in those in the visceral depot and that HSF1 is more abundant in differentiated, thermogenic adipocytes. Gene expression experiments indicated that HSF1 is transcriptionally regulated in fat by agents that modulate cAMP levels, by cold exposure, and by pharmacological stimulation of ß-adrenergic signaling. An in silico promoter analysis helped identify a putative response element for activating transcription factor 3 (ATF3) at -258 to -250 base pairs from the HSF1 transcriptional start site, and electrophoretic mobility shift and ChIP assays confirmed ATF3 binding to this sequence. Furthermore, functional assays disclosed that ATF3 is necessary and sufficient for HSF1 regulation. Detailed gene expression analysis revealed that ATF3 is one of the most highly induced ATFs in thermogenic tissues of mice exposed to cold temperatures or treated with the ß-adrenergic receptor agonist CL316,243 and that its expression is induced by modulators of cAMP levels in isolated adipocytes. To the best of our knowledge, our results show for the first time that HSF1 is transcriptionally controlled by ATF3 in response to classic stimuli that promote heat generation in thermogenic tissues.

Transcriptional Regulation of ZNF638 in Thermogenic Cells by the cAMP Response Element Binding Protein in Male Mice.

Zinc finger factors are implicated in a variety of cellular processes, including adipose tissue differentiation and thermogenesis. We have previously demonstrated that zinc finger protein 638 (ZNF638) is a transcriptional coactivator acting as an early regulator of adipogenesis in vitro. In this study, we show, to our knowledge for the first time, that, in vivo, ZNF638 abounds selectively in mature brown and subcutaneous fat tissues and in fully differentiated thermogenic adipocytes. Furthermore, gene expression studies revealed that ZNF638 is upregulated by cAMP modulators in vitro and by cold exposure and by pharmacological stimulation of ß-adrenergic signaling in vivo. In silico analysis of the upstream regulatory region of the ZNF638 gene identified two putative cAMP response elements within 500 bp of the ZNF638 transcription start site. Detailed molecular analysis involving EMSA and chromatin immunoprecipitation assays demonstrated that cAMP response element binding protein (CREB) binds to these cAMP response element regions of the ZNF638 promoter, and functional studies revealed that CREB is necessary and sufficient to regulate the levels of ZNF638 transcripts. Taken together, these results demonstrate that ZNF638 is selectively expressed in mature thermogenic adipocytes and tissues and that its induction in response to classic stimuli that promote heat generation is mediated via CREB signaling, pointing to a possible novel role of ZNF638 in brown and beige fat tissues.

Ablation of PPARγ in subcutaneous fat exacerbates age-associated obesity and metabolic decline.

It is well established that aging is associated with metabolic dysfunction such as increased adiposity and impaired energy dissipation; however, the transcriptional mechanisms regulating energy balance during late life stages have not yet been fully elucidated. Here, we show that ablation of the nuclear receptor PPARγ specifically in inguinal fat tissue in aging mice is associated with increased fat tissue expansion and insulin resistance. These metabolic effects are accompanied by decreased thermogenesis, reduced levels of brown fat genes, and browning of subcutaneous adipose tissue. Comparative studies of the effects of PPARγ downregulation in young and mid-aged mice demonstrate a preferential regulation of brown fat gene programs in inguinal fat in an age-dependent manner. In conclusion, our study uncovers an essential role for PPARγ in maintaining energy expenditure during the aging process and suggests the possibility of targeting PPARγ to counteract age-associated metabolic dysfunction.

Browning and Graying: Novel Transcriptional Regulators of Brown and Beige Fat Tissues and Aging.

Obesity represents a major risk factor for the development of a number of metabolic disorders, including cardiovascular disease and type 2 diabetes. Since the discovery that brown and beige fat cells exist in adult humans and contribute to energy expenditure, increasing interest has been devoted to the understanding of the molecular switches turning on calorie utilization. It has been reported that the ability of thermogenic tissues to burn energy declines during aging, possibly contributing to the development of metabolic dysfunction late in life. This review will focus on the recently identified transcriptional modulators of brown and beige cells and will discuss the potential impact of some of these thermogenic factors on age-associated metabolic disorders.

Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue.

Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3's expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids.

Calorie hoarding and thrifting: Foxa3 finds a way.

Obesity and diabetes are major health concerns worldwide. Western diets, often calorically rich, paired with sedentary habits are driving the current worldwide epidemic of pediatric and adult obesity. In addition, age related energy imbalances lead to increased adiposity and metabolic disorders later in life, making the middle aged population particularly susceptible. Here we discuss how Forkhead box A3 (Foxa3), a family member of the forkhead box binding proteins, can potentially contribute to pathology by playing a double role in metabolism. Recent data revealed that Foxa3 favors the selective expansion of visceral depots under high caloric conditions (e.g., high fat diet) and suppresses subcutaneous fat tissue energy expenditure during aging. This evidence suggests that Foxa3 acts to both preserve and conserve calories, by accumulating fat and by reducing metabolic burn. In other words, Foxa3 appears to function to enable energy "hoarding," which may be critical for survival of organisms with intermittent exposure to external caloric sources, but pathologic in circumstances where calories are abundant. Understanding how this "calorie hoarder gene" functions may suggest approaches to combat obesity and associated metabolic disorders.

Celastrol Protects against Obesity and Metabolic Dysfunction through Activation of a HSF1-PGC1α Transcriptional Axis.

Altering the balance between energy intake and expenditure is a potential strategy for treating obesity and metabolic syndrome. Nonetheless, despite years of progress in identifying diverse molecular targets, biological-based therapies are limited. Here we demonstrate that heat shock factor 1 (HSF1) regulates energy expenditure through activation of a PGC1α-dependent metabolic program in adipose tissues and muscle. Genetic modulation of HSF1 levels altered white fat remodeling and thermogenesis, and pharmacological activation of HSF1 via celastrol was associated with enhanced energy expenditure, increased mitochondrial function in fat and muscle and protection against obesity, insulin resistance, and hepatic steatosis during high-fat diet regimens. The beneficial metabolic changes elicited by celastrol were abrogated in HSF1 knockout mice. Overall, our findings identify the temperature sensor HSF1 as a regulator of energy metabolism and demonstrate that augmenting HSF1 via celastrol represents a possible therapeutic strategy to treat obesity and its myriad metabolic consequences.

Role of forkhead box protein A3 in age-associated metabolic decline.

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown. Here we report that during aging, levels of Foxa3 are significantly and selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter. Overall, our data demonstrate a role for Foxa3 in energy expenditure and in age-associated metabolic disorders.

The adipogenic transcriptional cofactor ZNF638 interacts with splicing regulators and influences alternative splicing.

Increasing evidence indicates that transcription and alternative splicing are coordinated processes; however, our knowledge of specific factors implicated in both functions during the process of adipocyte differentiation is limited. We have previously demonstrated that the zinc finger protein ZNF638 plays a role as a transcriptional coregulator of adipocyte differentiation via induction of PPARγ in cooperation with CCAAT/enhancer binding proteins (C/EBPs). Here we provide new evidence that ZNF638 is localized in nuclear bodies enriched with splicing factors, and through biochemical purification of ZNF638's interacting proteins in adipocytes and mass spectrometry analysis, we show that ZNF638 interacts with splicing regulators. Functional analysis of the effects of ectopic ZNF638 expression on a minigene reporter demonstrated that ZNF638 is sufficient to promote alternative splicing, a function enhanced through its recruitment to the minigene promoter at C/EBP responsive elements via C/EBP proteins. Structure-function analysis revealed that the arginine/serine-rich motif and the C-terminal zinc finger domain required for speckle localization are necessary for the adipocyte differentiation function of ZNF638 and for the regulation of the levels of alternatively spliced isoforms of lipin1 and nuclear receptor co-repressor 1. Overall, our data demonstrate that ZNF638 participates in splicing decisions and that it may control adipogenesis through regulation of the relative amounts of differentiation-specific isoforms.

Understanding the variegation of fat: novel regulators of adipocyte differentiation and fat tissue biology.

The differentiation of uncommitted cells into specialized adipocytes occurs through a cascade of transcriptional events culminating in the induction and activation of the nuclear receptor PPARγ, the central coordinator of fat cell function. Since the discovery of PPARγ, two decades ago, our views of how this molecule is activated have been significantly refined. Beyond the cell, we also now know that diverse signals and regulators control PPARγ function in a fat-depot specific manner. The goal of this article is to review the latest in our understanding of the early and late transcriptional events that regulate adipocyte development and their potential impact on energy storage and expenditure in different fat depots. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.

A multifunctional protein, EWS, is essential for early brown fat lineage determination.

The recent surge in obesity has provided an impetus to better understand the mechanisms of adipogenesis, particularly in brown adipose tissue (BAT) because of its potential utilization for antiobesity therapy. Postnatal brown adipocytes arise from early muscle progenitors, but how brown fat lineage is determined is not completely understood. Here, we show that a multifunctional protein, Ewing Sarcoma (EWS), is essential for determining brown fat lineage during development. BATs from Ews null embryos and newborns are developmentally arrested. Ews mutant brown preadipocytes fail to differentiate due to loss of Bmp7 expression, a critical early brown adipogenic factor. We demonstrate that EWS, along with its binding partner Y-box binding protein 1 (YBX1), activates Bmp7 transcription. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Remarkably, Ews null BATs and brown preadipocytes ectopically express myogenic genes. These results demonstrate that EWS is essential for early brown fat lineage determination.

The winged helix transcription factor Foxa3 regulates adipocyte differentiation and depot-selective fat tissue expansion.

Conversion of mesenchymal stem cells into terminally differentiated adipocytes progresses sequentially through regulated transcriptional steps. While it is clear that the late phases of adipocyte maturation are governed by the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), less is known about the transcriptional control of the initial stages of differentiation. To identify early regulators, we performed a small interfering RNA (siRNA) screen of Forkhead-box genes in adipocytes and show here for the first time that the winged helix factor Foxa3 promotes adipocyte differentiation by cooperating with C/EBPß and -δ to transcriptionally induce PPARγ expression. Furthermore, we demonstrate that mice with genetic ablation of Foxa3 have a selective decrease in epididymal fat depot and a cell-autonomous defect to induce PPARγ specifically in their visceral adipocytes. In obese subjects, FOXA3 is differentially expressed in visceral and subcutaneous adipose depots. Overall, our study implicates Foxa3 in the regulation of adipocyte differentiation and depot-selective adipose tissue expansion.

Disrupted erythropoietin signalling promotes obesity and alters hypothalamus proopiomelanocortin production.

Although erythropoietin (Epo) is the cytokine known to regulate erythropoiesis, erythropoietin receptor (EpoR) expression and associated activity beyond haematopoietic tissue remain uncertain. Here we show that mice with EpoR expression restricted to haematopoietic tissues (Tg) develop obesity and insulin resistance. Tg-mice exhibit a decrease in energy expenditure and an increase in white fat mass and adipocyte number. Conversely, Epo treatment of wild-type (WT)-mice increases energy expenditure and reduces food intake and fat mass accumulation but shows no effect in body weight of Tg-mice. EpoR is expressed at a high level in white adipose tissue and in the proopiomelanocortin (POMC) neurons of the hypothalamus. Although Epo treatment in WT-mice induces the expression of the polypeptide hormone precursor, POMC, mice lacking EpoR show reduced levels of POMC in the hypothalamus. This study provides the first evidence that mice lacking EpoR in non-haematopoietic tissue become obese and insulin resistant with loss of Epo regulation of energy homeostasis.

Regulation of adipocyte differentiation by the zinc finger protein ZNF638.

Zinc finger proteins constitute the largest family of transcription regulators in eukaryotes. These factors are involved in diverse processes in many tissues, including development and differentiation. We report here the characterization of the zinc finger protein ZNF638 as a novel regulator of adipogenesis. ZNF638 is induced early during adipocyte differentiation. Ectopic expression of ZNF638 increases adipogenesis in vitro, whereas its knockdown inhibits differentiation and decreases the expression of adipocyte-specific genes. ZNF638 physically interacts and transcriptionally cooperates with CCAAT/enhancer-binding protein (C/EBP) ß and C/EBPδ. This interaction leads to the expression of peroxisome proliferator-activated receptor γ, which is the key regulator of adipocyte differentiation. In summary, ZNF638 is a novel and early regulator of adipogenesis that works as a transcription cofactor of C/EBPs.