Cell Death-inducing DNA Fragmentation Factor Research Articles

Cell-autonomous Action of the FGF19 Promotes UCP1 Expression and Function in Human Brown Adipocytes

Fibroblast growth factor 19 (FGF19) is an intestine-derived hormone that regulates glucose and energy metabolism. Central and systemic administration of human FGF19 in mice promotes increased energy expenditure and body weight loss through mechanisms that are not fully understood. In this study, we investigated whether FGF19 can activate thermogenesis in brown adipocytes (BA) in a cell-autonomous manner. We differentiated immortalized human brown preadipocytes into mature adipocytes with adipogenic induction media in the presence and absence of FGF19 for 18 days to evaluate the effects of FGF19 on adipogenesis and thermogenesis. Through qPCR and RNA-sequencing analyses, we observed that FGF19 receptors were expressed in both brown preadipocytes and mature BA. However, the obligatory FGF19 co-receptor, β-klotho, was not expressed in preadipocytes, suggesting that the effects of FGF19 must occur preferentially in mature BA. Lipid staining with Oil Red O as well as gene expression of the adipogenic markers fatty acid binding protein (FABP4) and peroxisome proliferator activated receptor-γ (PPAR-γ) showed that adipogenesis was not affected by FGF19. We then treated mature BA with FGF19 (100 ng/mL) acutely and chronically to evaluate FGF19-induced changes in signaling, gene transcription, and thermogenic function. FGF19 signaling was assessed by measuring ERK1/2 phosphorylation levels, which increased within 5 min and remained elevated for at least 30 min after treatment. Using Seahorse assays, we observed that 24-hour FGF19 treatment promoted increased basal respiration, ATP production, and proton leak in human BA, indictive of enhanced mitochondrial function. Since proton leak across the inner membrane through the uncoupling protein 1 (UCP1) generates heat, we assessed the expression of UCP1 and other thermogenic genes. Interestingly, FGF19 treatment specifically increased UCP1 gene expression without affecting other classical adrenergic-mediated thermogenic genes, such as deiodinase 2 (DIO2), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α), PR domain containing 16 (PRDM16), and cell death-inducing DNA fragmentation factor A (CIDEA). These findings suggest that FGF19 regulates UCP1 expression through non-canonical pathways. Future work will elucidate the mechanism by which FGF19 promotes UCP1 gene expression in mature BA. Taken together, FGF19 stimulates ERK1/2 phosphorylation and UCP1 expression in human BA, leading to enhanced mitochondrial respiration. These results provide new mechanistic insights into FGF19’s effects on metabolic regulation. Topic subcategories: Adipocyte biology. Funding sources: Sao Paulo Research Foundation (FAPESP): 2022/15148-2 and NIH R01 DK102898. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cotadutide improves brown adipose tissue thermogenesis in obese mice

We studied the effect of cotadutide, a dual agonist glucagon-like peptide 1 (GLP1)/Glucagon, on interscapular brown adipose tissue (iBAT) remodeling and thermogenesis of obese mice. Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Then, animals were redivided, adding cotadutide treatment: C, CC, HF, and HFC for four additional weeks. The multilocular brown adipocyte structure showed fat conversion (whitening), hypertrophy, and structural disarray in the HF group, which was reverted in cotadutide-treated animals. Cotadutide enhances the body temperature, thermogenesis, and sympathetic innervation (peroxisome proliferator-activated receptor-α, β3 adrenergic receptor, interleukin 6, and uncoupled protein 1), reduces pro-inflammatory markers (disintegrin and metallopeptidase domain, morphogenetic protein 8a, and neuregulin 4), and improves angiogenesis (vascular endothelial growth factor A, and perlecan). In addition, cotadutide enhances lipolysis (perilipin and cell death-inducing DNA fragmentation factor α), mitochondrial biogenesis (nuclear respiratory factor 1, transcription factor A mitochondrial, mitochondrial dynamin-like GTPase, and peroxisome proliferator-activated receptor gamma coactivator 1α), and mitochondrial fusion/fission (dynamin-related protein 1, mitochondrial fission protein 1, and parkin RBR E3 ubiquitin protein ligase). Cotadutide reduces endoplasmic reticulum stress (activating transcription factor 4, C/EBP homologous protein, and growth arrest and DNA-damage inducible), and extracellular matrix markers (lysyl oxidase, collagen type I α1, collagen type VI α3, matrix metallopeptidases 2 and 9, and hyaluronan synthases 1 and 2). In conclusion, the experimental evidence is compelling in demonstrating cotadutide's thermogenic effect on obese mice's iBAT, contributing to unraveling its action mechanisms and the possible translational benefits.

Filbertone, (2E)-5-methyl-2-hepten-4-one, regulates thermogenesis and lipid metabolism in skeletal muscle of a high-fat diet fed mice

Filbertone, the principal flavor compound of hazelnuts, is known to have preventive effects against hypothalamic inflammation, obesity and adipocity in vitro and in vivo. However, the effect of filbertone in skeletal muscle remains unknown. In the present study, we determined the effect of filbertone in skeletal muscle of mice fed a high-fat diet (HFD). To identify the underlying molecular and cellular processes of filbertone, we performed whole transcriptome sequencing in skeletal muscle. The muscle transcriptome analysis revealed that the upregulated differentially expressed genes (DEGs) in filbertone-fed mice were substantially associated with several pathways including thermogenesis, fatty acid degradation, oxidative phosphorylation, and branched chain amino acids (BCAAs) degradation. Furthermore, the expression level of thermogenic genes such as uncoupling protein 1 (Ucp1; p < 0.05), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea; p < 0.05), peroxisome proliferator-activated receptor alpha (Ppara; p < 0.05) and lipid droplet-associated protein genes such as Plin3 (p < 0.05), Plin4 (p < 0.05), and Plin5 (p < 0.05) were significantly upregulated in muscle tissue of HFD with filbertone fed mice compared to HFD only fed mice. Filbertone also elevated the protein level of UCP1 (p < 0.05) and PPARα (p < 0.05). In addition, filbertone reduced the accumulation of intracellular lipids in C2C12 myotubes (p < 0.05). On the basis of these results, we suggest that filbertone has a crucial effect in the regulation of muscle lipid metabolism and energy balance.

Creatine ameliorates high-fat diet-induced obesity by regulation of lipolysis and lipophagy in brown adipose tissue and liver

Diet-induced obesity in mice and humans is commonly associated with an imbalance between energy intake and expenditure. Reportedly, creatine can enhance energy expenditure in brown adipose tissue and reduce hepatic triglycerides accumulation; however, the molecular mechanism underlying the role of exogenous creatine supplementation in regulating lipid droplet mobilization remains elusive. Herein, we employed a high-fat diet (HFD)- induced mouse model to investigate the role of creatine in regulating lipolysis and lipophagy in brown adipose tissue and the liver. Exogenous creatine supplementation ameliorated HFD-induced obesity, increased insulin sensitivity and improved glucose homeostasis. Creatine supplementation enhanced the expression of uncoupling protein 1 (UCP1), cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA), and other brown adipose tissue-specific thermogenic genes Cpt1a, Gyk, and Pgc1β in brown adipose tissue. Furthermore, creatine inhibited the expression of CIDEA, which promotes hepatic lipid accumulation. Creatine stimulated the expression of triglyceride lipase adipose triglyceride lipase, and phospho-hormone-sensitive lipase (HSL) induced increased lipolysis in brown adipose tissue and the liver. Meanwhile, reduced LC3B expression was accompanied by an increased level of p62 in HFD-fed mice, indicating diminished basal autophagy in brown adipose tissue and the liver; however, creatine enhanced P62/LC3B induced lipophagy in brown adipose tissue and the liver. Collectively, our results suggest that creatine may function as a brown adipose tissue activator to increase whole-body energy metabolism via coordinated lipolysis and lipophagy in brown adipose tissue and the liver.

Adding fuel to the fire: The lipid droplet and its associated proteins in cancer progression

A lipid droplet (LD) is an organelle that consists of a phospholipid monolayer and a neutral lipid core, with proteins embedded in or attached to its surface. Until recently, cancers had long been regarded as genetic disorders with the abnormal activation of oncogenes and inactivation of tumor suppressor genes before their quality of a metabolic disorder began to be recognized. The last decade has witnessed the recognition of several metabolic characteristics of cancer cells, among which one is the accumulation of lipid droplets; therefore, attention has been given to exploring the role of LDs in carcinomas. In addition, there has been a remarkable expansion in understanding the complexity of LD's function in cellular homeostasis, including but not limited to energy supply, endoplasmic reticulum (ER) stress and oxidative stress management, or lipotoxicity alleviation. Thus, lipid droplet-associated proteins, which to a great extent determine the dynamics of a lipid droplet, have attracted the interest of numerous cancer researchers and their potential as cancer diagnostic biomarkers and therapeutic targets has been affirmed by emerging evidence. In this review, we systematically summarize the critical role of LDs in cancer and then focus on four categories of lipid droplet-associated proteins having the most direct influence on LD biosynthesis (diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2)), degradation (adipose triglyceride lipase (ATGL)), and two renowned protein families on the LD surface (perilipins and cell death-inducing DNA fragmentation factor alpha-like effectors (CIDEs)). In this way, we aim to highlight their important role in tumor progression and their potential in clinical applications.

Small Intestine-specific Knockout of CIDEC Improves Obesity and Hepatic Steatosis by Inhibiting Synthesis of Phosphatidic Acid.

The small intestine is main site of exogenous lipid digestion and absorption, and it is important for lipid metabolic homeostasis. Cell death-inducing DNA fragmentation-factor like effector C (CIDEC) is active in lipid metabolism in tissues other than those in the intestine. We developed small intestine-specific CIDEC (SI-CIDEC-/-) knockout C57BL/6J mice by Cre/LoxP recombination to investigate the in vivo effects of intestinal CIDEC on lipid metabolism. Eight-week-old SI-CIDEC-/- mice fed a high-fat diet for 14 weeks had 15% lower body weight, 30% less body fat mass, and 79% lower liver triglycerides (TG) than wild-type (WT) mice. In addition, hepatic steatosis and fatty liver inflammation were less severe in knockout mice fed a high-fat diet (HFD) compared with wild-type mice fed an HFD. SI-CIDEC-/- mice fed an HFD diet had lower serum TG and higher fecal TG and intestinal lipase activity than wild-type mice. Mechanistic studies showed that CIDEC accelerated phosphatidic acid synthesis by interacting with 1-acylglycerol-3-phosphate-O-acyltransferase to promote TG accumulation. This study identified a new interacting protein and previously unreported CIDEC mechanisms that revealed its activity in lipid metabolism of the small intestine.

CIDEA expression in SAT from adolescent girls with obesity and unfavorable patterns of abdominal fat distribution.

This study investigated whether variations in cell death-inducing DNA fragmentation factor alpha subunit-like effector A (CIDEA) mRNA expression and protein levels are modulated by the pattern of abdominal fat distribution in adolescent girls with obesity. This study recruited 35 adolescent girls with obesity and characterized their abdominal fat distribution by magnetic resonance imaging. Participants had only a periumbilical/abdominal (n = 14) or a paired abdominal and gluteal subcutaneous adipose tissue (SAT) biopsy (n = 21). CIDEA expression was determined by reverse transcription-polymerase chain reaction, CIDEA protein level by Western blot, and the turnover of adipose lipids and adipocytes by 2 H2 O labeling. In six girls, a second abdominal SAT biopsy was performed (after ~34.2 months) to explore the weight gain effect on CIDEA expression in abdominal SAT. CIDEA expression decreased in abdominal SAT from participants with high visceral adipose tissue (VAT)/(VAT+SAT); CIDEA inversely correlated with number of small adipocytes, with the increase in preadipocyte proliferation, and with adipogenesis. A strong inverse correlation was found between CIDEA protein level with the newly synthetized glycerol (r = -0.839, p = 0.0047). Following weight gain, an increase in adipocytes' cell diameter with a decrease in CIDEA expression and RNA-sequencing transcriptomic profile typical of adipocyte dysfunction was observed. Reduced expression of CIDEA in girls with high VAT/(VAT+SAT) is associated with adipocyte hypertrophy and insulin resistance.

Development of an Optimized Culture System for Generating Mouse Alveolar Macrophage-like Cells.

Alveolar macrophages (AMs) play critical roles in maintaining lung homeostasis and orchestrating the immune responses. Although the essential factors known for AM development have been identified, currently an optimal in vitro culture system that can be used for studying the development and functions of AMs is still lacking. In this study, we report the development of an optimized culture system for generating AM-like cells from adult mouse bone marrow and fetal liver cells on in vitro culture in the presence of a combination of GM-CSF, TGF-β, and peroxisome proliferator-activated receptor γ (PPAR-γ) agonist rosiglitazone. These AM-like cells expressed typical AM surface markers sialic acid-binding Ig-like lectin-F (Siglec-F), CD11c, and F4/80, and AM-specific genes, including carbonic anhydrase 4 (Car4), placenta-expressed transcript 1 (Plet1), eosinophil-associated RNase A family member 1 (Ear1), cell death-inducing DNA fragmentation factor A-like effector c (Cidec), and cytokeratin 19 (Krt19). Similar to primary AMs, the AM-like cells expressed alternative macrophage activation signature genes and self-renewal genes. Moreover, this culture system could be used for expansion of bronchoalveolar lavage fluid-derived AMs in vitro. The AM-like cells generated from bone marrow resembled the expanded bronchoalveolar lavage fluid-derived AMs in inflammatory responses and phagocytic activity. More importantly, these AM-like cells could be obtained in sufficient numbers that allowed genetic manipulation and functional analysis in vitro. Taken together, we provide a powerful tool for studying the biology of AMs.

Fish Oil Increases Diet-Induced Thermogenesis in Mice.

Increasing energy expenditure (EE) is beneficial for preventing obesity. Diet-induced thermogenesis (DIT) is one of the components of total EE. Therefore, increasing DIT is effective against obesity. We examined how much fish oil (FO) increased DIT by measuring absolute values of DIT in mice. C57BL/6J male mice were given diets of 30 energy% fat consisting of FO or safflower oil plus butter as control oil (Con). After administration for 9 days, respiration in mice was monitored, and then the data were used to calculate DIT and EE. DIT increased significantly by 1.2-fold in the FO-fed mice compared with the Con-fed mice. Body weight gain was significantly lower in the FO-fed mice. FO increased the levels of uncoupling protein 1 (Ucp1) mRNA and UCP1 protein in brown adipose tissue (BAT) by 1.5- and 1.2-fold, respectively. In subcutaneous white adipose tissue (subWAT), the levels of Ucp1 mRNA and UCP1 protein were increased by 6.3- and 2.7-fold, respectively, by FO administration. FO also significantly increased the expression of markers of browning in subWAT such as fibroblast growth factor 21 and cell death-inducing DNA fragmentation factor α-like effector a. Thus, dietary FO seems to increase DIT in mice via the increased expressions of Ucp1 in BAT and induced browning of subWAT. FO might be a promising dietary fat in the prevention of obesity by upregulation of energy metabolism.

Mangiferin induces the expression of a thermogenic signature via AMPK signaling during brown-adipocyte differentiation

Mangiferin (MF) from Mangifera indica has been serendipitously found to ameliorate obesity and is used as an antioxidant, anti-inflammatory, antimicrobial, and anticancer agent. Nonetheless, the mechanism of MF-induced brown-adipose-tissue activation has not been studied. Therefore, we investigated the effect of MF on thermogenic features during brown-adipocyte differentiation. Treatment with MF improved the expression of a brown-fat signature and of mitochondrial-mass–related genes, thus resulting in UCP1 induction. MF also raised the expression of other thermogenic regulators, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), PR domain–containing protein 16 (PRDM16), and peroxisome proliferator-activated receptors alpha and gamma (PPAR-α and -γ). MF promoted mitochondrial biogenesis, judging by increased expression of cell death–inducing DNA fragmentation factor α–like effector A (CIDEA), mitochondrial transcription factor A (TFAM), iodothyronine deiodinase 2 (DIO2), cytochrome c oxidase subunit 7A (COX7A), cyclooxygenase 2 (COX2), sirtuin 1 (SIRT1), and nuclear respiratory factor 1 (NRF1). MF treatment increased the mitochondrial DNA amount and improved mitochondrial respiratory function by increasing the oxygen consumption rate during brown-adipocyte differentiation. A gene knockdown assay involving small interfering RNA and competitive inhibition with dorsomorphin revealed that MF may promote thermogenesis in brown preadipocytes via activation of AMPK signaling. Collectively, our findings suggest that MF may be a novel pharmaceutical agent that can ameliorate obesity via activation of brown adipose tissue.

Fat-specific protein 27b is regulated by hepatic peroxisome proliferator-activated receptor γ in hepatic steatosis.

The fat-specific protein 27 gene (Fsp27) belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue and fatty liver. In adipocytes, FSP27 localizes to the membrane of lipid droplets and promotes lipid droplet hypertrophy. Recently, FSP27 was shown to consist of two isoforms, FSP27α and FSP27β. Previously, we demonstrated that Fsp27a is directly regulated by peroxisome proliferator-activated receptor γ (PPARγ) in fatty livers of genetically obese leptin deficient ob/ob mice and that Fsp27b may potentially be regulated by different factors transcriptionally as they both have a different promoter region. Thus, the aim of the present study was to elucidate whether Fsp27b is regulated by PPARγ in fatty liver. Fsp27a and Fsp27b were markedly induced in fatty liver of ob/ob mice compared with those in the normal liver. However, both Fsp27a/b were expressed at markedly lower levels in liver-specific PPARγ knockout mice with an ob/ob background. Further, the PPAR response element (PPRE) for the PPARγ-dependent promotion of Fsp27b promotor activity was revealed at position -1,163/-1,151 from the transcriptional start site (+1). Interestingly, the cis-element responsible for the PPARγ-dependent induction of Fsp27b was the same as that responsible for PPARγ-dependent induction of Fsp27a. These results suggest that PPARγ regulates not only Fsp27a but also Fsp27b in fatty liver of ob/ob mice through a common PPRE.

CIDE family proteins control lipid homeostasis and the development of metabolic diseases.

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER- and LD-associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD-LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE-mediated LD fusion and growth.

Comparison of the Effects of Browning-Inducing Capsaicin on Two Murine Adipocyte Models.

The increasing prevalence of obesity and its associated comorbidities has gained attention in developing effective treatments and strategies that promote energy expenditure and the conversion of fat from a white to a brite phenotype. Capsaicin, bioactive component of chili peppers and a transient receptor potential channel vanilloid 1 (TRPV1) agonist, has been known to stimulate the process of thermogenesis. In this study, the effects of capsaicin were assessed on two murine cellular models by quantifying the dynamic of lipid droplets (LDs) and the expression of genes involved in adipocyte browning. Present findings demonstrated that treatment with norepinephrine or capsaicin combined with norepinephrine on 3T3-L1 cells and X9 cells significantly promoted the reduction of LDs area surface and size. The transcription of browning related genes such as uncoupling protein 1 (Ucp1), T-box transcription factor 1 (Tbx1), PR domain containing 16 (Prdm16), peroxisome proliferator-activated receptor γ coactivator 1α (Ppargc1a) and cell death-inducing DNA fragmentation factor A-like effector A (Cidea) was up-regulated by chronic capsaicin treatment on differentiated 3T3-L1 cells. Instead, X9 cells were significantly responsive only to the treatment with norepinephrine, used as positive control.

Thermoneutrality Inhibits Thermogenic Markers and Exacerbates Nonalcoholic Fatty Liver in Mice (P21-068-19)

Abstract Objectives Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease, affecting more than a third of the US population and 25% of the global population. In this study, we investigated whether manipulation of brown adipose tissue (BAT) activity via housing C57BL/6 J mice under two different housing temperatures (22°C vs 27°C) influences the development of NAFLD. Methods Male C57BL/6 J mice were randomly allocated to 4 groups (8 mice/group). Briefly, mice housed at the standard temperature (ST) (22°C) or thermoneutral temperature (TT) (27°C) were fed either a control chow diet (CHD) (Picolab rodent diet 20, LabDiet) with a regular drinking water or an “fast food” diet (FFD) (Research Diets, D12079B) plus fructose 23.1 g/L and glucose 18.9 g/L added to the drinking water for 10 weeks. Results Mice under TT had significant less food intake compared to their counter partners under ST, regardless of diets. Mice exposed to TT and fed FFD had the highest body weight and plasma leptin level among all groups. For mice fed CHD, mice at TT did not present hepatic steatosis although they slightly gained more body weight compared to mice at ST. For mice fed FFD, thermoneutral housing mice had greater liver weight, liver triglyceride, and exacerbated hepatic steatosis compared to the standard housing mice. Moreover, for mice fed FFD, TT compared to ST had significantly elevated expression of fatty acid synthase, sterol regulatory element-binding transcription factor 1, fatty acid translocase, and monocyte chemoattractant protein-1 in liver. By contrast, TT did not change expression of above genes in mice fed CHD. Furthermore, thermoneutrally housed mice displayed a decrease in gene expression of thermogenic markers, such as uncoupling protein 1, elongation of very long chain fatty acids 3, and cell death-inducing DNA fragmentation factor alpha-like effector A in BAT and inguinal white adipose tissue (WAT). Conclusions Thermoneutrality inhibits expression of thermogenic markers in both BAT and WAT, which correlated to exacerbated NAFLD in mice. Our data indicate that activating BAT and/or promoting WAT browning may represent a potential strategy for the management of NAFLD. Funding Sources The College of Human Sciences of Texas Tech University; the National Center for Complementary &amp; Integrative Health.

CIDEA and CIDEC are regulated by CREB and are not induced during fasting in grass carp Ctenopharyngodon idella adipocytes.

Cell death-inducing DNA fragmentation factor 45-like effector family proteins, including CIDEA, CIDEB and CIDEC, play an important role in energy metabolism. In the present study, CIDEA, CIDEB and CIDEC cDNAs were firstly isolated and characterized from grass carp Ctenopharyngodon idella, encoding peptides of 205, 208 and 238 amino acids, respectively. Analysis of the exon-intron structures clarified that grass carp CIDEA, CIDEB and CIDEC consisted of 5 coding exons, 5 coding exons and 6 coding exons, respectively, which is similar with human and mouse. Both CIDE family genes mRNAs were expressed in a wide range of tissues, but the abundance of each CIDE family gene mRNA showed the tissue-dependent expression patterns. Time-course analysis of CIDE family expressions indicated that their expression were enhanced significantly from day 0 to day 8 after differentiation. Forskolin caused an increase in CIDEA and CIDEC expression, and the effects were attenuated by treatment with CREB inhibitor, revealing that CIDEA and CIDEC are regulated by CREB. Further study found that CIDEA and CIDEC mRNA levels did not show significant changes during fasting. These results provide the groundwork to elucidate the gene structure and physiological function of CIDE family in fish.

Beneficial Metabolic Effects of Mirabegron In Vitro and in High-Fat Diet-Induced Obese Mice.

Mirabegron, a β3-adrenergic receptor agonist, has been shown to stimulate the activity of brown fat and increase the resting metabolic rate in humans. However, it is unknown whether mirabegron can reduce body weight and improve metabolic health. We investigated the antiobesity effects of mirabegron using both in vitro and in vivo models. Mouse brown preadipocytes and 3T3-L1 cells were treated with different concentrations of mirabegron (0.03-3 µg/ml), and the expression of brown fat-related genes was measured by quantitative real-time polymerase chain reaction. Furthermore, male C57BL/6J mice were fed a high-fat diet for 10 weeks, and mirabegron (2 mg/kg body weight) or a vehicle control was delivered to the interscapular brown adipose tissue (iBAT) using ALZET osmotic pumps from week 7 to 10. The metabolic parameters and tissues were analyzed. In both mouse brown preadipocytes and 3T3-L1 cells, mirabegron stimulated uncoupling protein 1 (UCP1) expression. In animal studies, mirabegron-treated mice had a lower body weight and adiposity. Lipid droplets in the iBAT of mirabegron-treated mice were fewer and smaller in size compared with those from vehicle-treated mice. H&E staining and immunohistochemistry indicated that mirabegron increased the abundance of beige cells in inguinal white adipose tissue (iWAT). Compared with vehicle-treated mice, mirabegron-treated mice had a higher gene expression of UCP1 (14-fold) and cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) (4-fold) in iWAT. Furthermore, mirabegron-treated mice had improved glucose tolerance and insulin sensitivity. Taken together, mirabegron enhances UCP1 expression and promotes browning of iWAT, which are accompanied by improved glucose tolerance and insulin sensitivity and prevention from high-fat diet-induced obesity.

CIDE Proteins in Human Health and Disease.

Cell death-Inducing DNA Fragmentation Factor Alpha (DFFA)-like Effector (CIDE) proteins have emerged as lipid droplet-associated proteins that regulate fat metabolism. There are three members in the CIDE protein family—CIDEA, CIDEB, and CIDEC (also known as fat-specific protein 27 (FSP27)). CIDEA and FSP27 are primarily expressed in adipose tissue, while CIDEB is expressed in the liver. Originally, based upon their homology with DNA fragmentation factors, these proteins were identified as apoptotic proteins. However, recent studies have changed the perception of these proteins, redefining them as regulators of lipid droplet dynamics and fat metabolism, which contribute to a healthy metabolic phenotype in humans. Despite various studies in humans and gene-targeting studies in mice, the physiological roles of CIDE proteins remains elusive. This review will summarize the known physiological role and metabolic pathways regulated by the CIDE proteins in human health and disease.

Lipid-exchange Rate Assay for Lipid Droplet Fusion in Live Cells.

Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular LD formation in the adipocytes, lipid storage in the liver, milk lipid secretion in the mammary epithelia cells, and lipid secretion in the skin sebocytes. Previous methodology by Gong et al. (2011) first reported a lipid-exchange rate assay to evaluate the fusion ability of each LD pair in the cells mediated by CIDE family proteins and their regulators, but photobleaching issue remains a problem and a detailed procedure was not provided. Here, we provide an improved and detailed protocol for the lipid-exchange rate measurement. The three key steps for this assay are cell preparation, image acquisition, and data analysis. The images of the fluorescence recovery are acquired after photobleaching followed by the measurement of the intensity changes in the LD pair. The difference in fluorescent intensity is used to obtain the lipid exchange rate between the LDs. The accuracy and repetitiveness of the calculated exchange rates are assured with three-cycle of photobleaching process and the linear criteria in data fitting. With this quantitative assay, we are able to identify the functional roles of the key proteins and the effects of their mutants on LD fusion.

Interaction mode of CIDE family proteins in fly: DREP1 and DREP3 acidic surfaces interact with DREP2 and DREP4 basic surfaces.

Cell death-inducing DNA fragmentation factor 45 (DFF45)-like effector (CIDE) domains were initially identified as protein interaction modules in apoptotic nucleases and are now known to form a highly conserved family with diverse functions that range from cell death to lipid homeostasis. In the fly, four CIDE domain-containing proteins (DFF-related protein [DREP]-1–4) and their functions, including interaction relationships, have been identified. In this study, we introduced and investigated acidic side-disrupted mutants of DREP1, DREP2, and DREP3. We discovered that the acidic surface patches of DREP1 and DREP3 are critical for the homo-dimerization. In addition, we found that the acidic surface sides of DREP1 and DREP3 interact with the basic surface sides of DREP2 and DREP4. Our current study provides clear evidence demonstrating the mechanism of the interactions between four DREP proteins in the fly.

Cell death-inducing DNA fragmentation factor A-like effector A and fat-specific protein 27β coordinately control lipid droplet size in brown adipocytes

Adipose tissue stores neutral lipids and is a major metabolic organ involved in regulating whole-body energy homeostasis. Triacylglycerol is stored as unilocular large lipid droplets (LDs) in white adipocytes and as multilocular small LDs in brown adipocytes. Proteins of the cell death-inducing DNA fragmentation factor A-like effector (Cide) family include CideA, CideB, and fat-specific protein of 27 (FSP27). Of these, FSP27 has been shown to play a crucial role in the formation of unilocular large LDs in white adipocytes. However, the mechanisms by which brown adipocytes store small and multilocular LDs remain unclear. An FSP27 isoform, FSP27β, was recently identified. We herein report that CideA and FSP27β are mainly expressed in brown adipose tissue and that FSP27β overexpression inhibits CideA-induced LD enlargements in a dose-dependent manner in COS cells. Furthermore, RNAi-mediated FSP27β depletion resulted in enlarged LDs in HB2 adipocytes, which possess the characteristics of brown adipocytes. Brown adipocytes in FSP27-knock-out mice that express CideA, but not FSP27β, had larger and fewer LDs. Moreover, we confirmed that FSP27β and CideA form a complex in brown adipose tissue. Our results suggest that FSP27β negatively regulates CideA-promoted enlargement of LD size in brown adipocytes. FSP27β appears to be responsible for the formation of small and multilocular LDs in brown adipose tissue, a morphology facilitating free fatty acid transport to mitochondria adjacent to LDs for oxidation in brown adipocytes.