DNMT3B Alleviates Liver Steatosis Induced by Chronic Low-grade LPS via Inhibiting CIDEA Expression.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is the most prevalent chronic liver disease and threats to human health. Gut dysbiosis caused by lipopolysaccharide (LPS) leakage has been strongly related to nonalcoholic fatty liver disease progression, although the underlying mechanisms remain unclear. METHODS: Previous studies have shown that low-grade LPS administration to mice on a standard, low-fat chow diet is sufficient to induce symptoms of fatty liver. This study confirmed these findings and supported LPS as a lipid metabolism regulator in the liver. RESULTS: Mechanically, LPS induced dysregulated lipid metabolism by inhibiting the expression of DNA methyltransferases 3B (DNMT3B). Genetic overexpression of DNMT3B alleviated LPS-induced lipid accumulation, whereas its knockdown increased steatosis in mice and human hepatocytes. LPS-induced lower expression of DNMT3B led to hypomethylation in promoter region of CIDEA, resulting in increased binding of SREBP-1c to its promoter and activated CIDEA expression. Hepatic interference of CIDEA reversed the effect of LPS on lipogenesis. These effects were independent of a high-fat diet or high fatty acid action. CONCLUSIONS: Overall, these findings sustain the conclusion that LPS is a lipogenic factor and could be involved in hepatic steatosis progression.

SUMO-specific protease 2 regulates lipid droplet size through ERRα-mediated CIDEA expression in adipocytes.

Lipid droplets are not only lipid storage sites but also are closely related to lipid metabolism. Lipid droplet growth increases lipid storage capacity and suppresses lipolysis via lipase associated with the lipid droplet surface. The cell death-inducing DFF45-like effector (CIDE) family of proteins mediates lipid droplet fusion, which mainly contributes to lipid droplet growth. We previously demonstrated small ubiquitin-like modifier (SUMO)-specific protease 2 (SENP2) plays important roles in lipid metabolism and induction/maintenance of adipogenesis. In this study, we determined whether SENP2 regulates lipid droplet size in adipocytes. Overexpression of SENP2 increased lipid droplet size in differentiated 3T3-L1 adipocytes and facilitated CIDEA transcription. We found SENP2 increased CIDEA expression mainly through desumoylation of estrogen-related receptor α (ERRα), which acted in coordination with peroxisome proliferator-activated receptor γ-coactivator α. In addition, palmitate treatment increased SENP2 and CIDEA mRNA levels. Specific small interfering RNA-mediated knockdown of SENP2, as well as ERRα knockdown, eliminated palmitate-induced CIDEA expression. These results suggest SENP2 enhances CIDEA expression by modulating ERRα when SENP2 is upregulated, such as after palmitate treatment, to increase lipid droplet size in adipocytes.

Use of CIDEA Reporter Mouse Model for Screening Thermogenic Fat-Activating Drugs.

Excessive fat accumulation is a risk factor for metabolic diseases. Activating non-shivering thermogenesis in adipose tissue increases energy expenditure and potentially reverses obesity-related metabolic dysfunctions. While brown/beige adipocytes specialize in non-shivering thermogenesis and catabolic lipid metabolism, thermogenic stimuli and pharmacological intervention can induce the recruitment and metabolic activation of these cell types in adipose tissue. Thus, these adipocytes are attractive therapeutic targets to combat obesity, and there is an increasing need for efficient screening strategies for thermogenic drugs. Cell death-inducing DNA fragmentation factor-like effector A (CIDEA) is a well-known marker of the thermogenic capacity of brown and beige adipocytes. We recently developed a CIDEA reporter mouse model that expresses multicistronic mRNAs encoding CIDEA, luciferase 2, and tdTomato proteins under endogenous Cidea promoter control. Here, we introduce the CIDEA reporter model system as a tool for in vitro and in vivo screening of drug candidate molecules with thermogenic effects and provide a detailed protocol to monitor CIDEA reporter expression.

Dysregulation of Lipid Droplet Protein Expression in Adipose Tissues and Association with Metabolic Risk Factors in Adult Females with Obesity and Type 2 Diabetes.

BACKGROUND: Adipocyte dysregulation of lipid droplet (LD) metabolism caused by altered expression of LD proteins contributes to obesity-related metabolic diseases. OBJECTIVES: We aimed to investigate whether expression levels of PLIN1, CIDEA, and CIDEC were altered in adipose tissues of women with obesity and type 2 diabetes and whether their alterations were associated with metabolic risk factors. METHODS: Normal-weight (NW; 18.5 kg/m2 < BMI ≤ 25 kg/m2; n = 43), nondiabetic obese (OB; BMI > 30 kg/m2; n = 38), and diabetic obese (OB/DM; BMI > 30 kg/m2, fasting glucose ≥ 126 mg/dL, HbA1c ≥ 6.5%; n = 22) women were recruited. Metabolic parameters were measured, and expressions of PLIN1, CIDEA, CIDEC, and obesity-related genes were quantified in abdominal subcutaneous (SAT) and visceral adipose tissues (VAT). Effects of proinflammatory cytokines, endoplasmic reticulum (ER) stress inducers, and metabolic improvement agents on LD protein gene expressions were investigated in human adipocytes. RESULTS: PLIN1, CIDEA, and CIDEC expressions were lower in SAT and higher in VAT in OB subjects relative to NW subjects; however, they were suppressed in both fat depots in OB/DM subjects relative to OB (P < 0.05). Across the entire cohort, whereas VAT PLIN1 (r = 0.349) and CIDEC expressions (r = 0.282) were positively associated with BMI (P < 0.05), SAT PLIN1 (r = -0.390) and CIDEA expressions (r = -0.565) were inversely associated. After adjustment for BMI, some or all of the adipose LD protein gene expressions were negatively associated with fasting glucose (r = -0.259 or higher) and triglyceride levels (r = -0.284 or higher) and positively associated with UCP1 expression (r = 0.353 or higher) (P < 0.05). In adipocytes, LD protein gene expressions were 55-70% downregulated by increased proinflammatory cytokines and ER stress but 2-4-fold upregulated by the metabolic improvement agents exendin-4 and dapagliflozin (P < 0.05). CONCLUSIONS: The findings suggest that reduction of adipose LD protein expression is involved in the pathogenesis of metabolic disorders in women with obesity and type 2 diabetes and that increasing LD protein expression in adipocytes could control development of metabolic disorders.

METTL16-mediated translation of CIDEA promotes non-alcoholic fatty liver disease progression via m6A-dependent manner.

Background: As the most prevalent chemical modifications on eukaryotic mRNAs, N6-methyladenosine (m6A) methylation was reported to participate in the regulation of various metabolic diseases. This study aimed to investigate the roles of m6A methylation and methyltransferase-like16 (METTL16) in non-alcoholic fatty liver disease (NAFLD). Methods: In this study, we used a model of diet-induced NAFLD, maintaining six male C57BL/6J mice on high-fat diet (HFD) to generate hepatic steatosis. The high-throughput sequencing and RNA sequencing were performed to identify the m6A methylation patterns and differentially expressed mRNAs in HFD mice livers. Furthermore, we detected the expression levels of m6A modify enzymes by qRT-PCR in liver tissues, and further investigated the potential role of METTL16 in NAFLD through constructing overexpression and a knockdown model of METTL16 in HepG2 cells. Results: In total, we confirmed 15,999 m6A recurrent peaks in HFD mice and 12,322 in the control. Genes with differentially methylated m6A peaks were significantly associated with the dysregulated glucolipid metabolism and aggravated hepatic inflammatory response. In addition, we identified five genes (CIDEA, THRSP, OSBPL3, GDF15 and LGALS1) that played important roles in NAFLD progression after analyzing the differentially expressed genes containing differentially methylated m6A peaks. Intriguingly, we found that the expression levels of METTL16 were substantially increased in the NAFLD model in vivo and in vitro, and further confirmed that METTL16 upregulated the expression level of lipogenic genes CIDEA in HepG2 cells. Conclusions: These results indicate the critical roles of m6A methylation and METTL16 in HFD-induced mice and cell NAFLD models, which broaden people's perspectives on potential m6A-related treatments and biomarkers for NAFLD.

CIDEA Regulates De Novo Fatty Acid Synthesis in Bovine Mammary Epithelial Cells by Targeting the AMPK/PPARγ Axis and Regulating SREBP1.

Cell-death-inducing DNA fragmentation factor-α-like effector A (CIDEA) is a lipid-droplet-associated protein that helps to promote lipid metabolism in adipocytes of mice and humans. However, studies on the regulatory mechanism of CIDEA on lipid metabolism in the mammary glands of dairy cows are rare. Therefore, the role of CIDEA in bovine mammary epithelial cells (bMECs) was investigated in this study. The CIDEA expression levels in the mammary glands of high-fat-milk-producing cows were significantly higher compared to those in low-fat-milk-producing cows. Results of in vitro studies in bMECs showed that the inhibition of CIDEA inhibited the expression of fatty acid synthesis-related genes and triglyceride (TAG) synthesis-related genes. Conversely, the overexpression of CIDEA leads to an increase in the content of TAG and fatty acid. The results of mechanistic studies indicated that the overexpression of CIDEA inhibits AMP-activated protein kinase (AMPK) activity, which enhances the expression of peroxisome proliferator-activated receptor-γ (PPARγ) and consequently increases the TAG content. Furthermore, the overexpression of CIDEA promoted the nuclear translocation of sterol regulatory element-binding protein 1 (SREBP1). Therefore, a theoretical framework is provided by this study for the regulation of lipid metabolism in dairy cows by means of nutrition and the hormone targeting of CIDEA.

Ban-xia-xie-xin-tang ameliorates hepatic steatosis by regulating Cidea and Cidec expression in HFD-fed mice.

BACKGROUND: Ban-xia-xie-xin-tang (BXXXT) has been applied in treating metabolic diseases, such as nonalcohol fatty liver disease, diabetes mellitus, and obesity. However, the underlying molecular mechanism of BXXXT in treating diabetes mellitus is unknown. PURPOSE: To clarify the underlying molecular mechanism of BXXXT in alleviating hepatic steatosis in high-fat diet (HFD)-fed mice. METHODS: After 12 weeks of HFD treatment, mice were administered BXXXT for 4 weeks. The main chemical components of BXXXT were identified by UPLC-TQ-MS/MS. Indicators associated with insulin resistance and lipid metabolism were detected. The effect of improving glucose and lipid metabolism between BXXXT and the different components was compared. Differentially expressed genes (DEGs) were identified by hepatic transcriptomics. Key DEGs and proteins were further detected by real-time quantitative polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence staining. LDs and mitochondria were detected by transmission electron microscopy. RESULTS: First of all, our data demonstrated that the capacity to improve glucose and lipid metabolism for BXXXT was significantly superior to different components of BXXXT. BXXXT was found to improve HFD-induced insulin resistance. Moreover, BXXXT decreased weight, serum/hepatic triglycerides, total cholesterol, and FFAs to alleviate HFD-induced hepatic steatosis. According to the results of the hepatic transcription, Cidea and Cidec were identified as critical DEGs for promoting LD fusion and reducing FFAs ß-oxidation in mitochondria and peroxisome resulting in hepatic steatosis, which was reversed by BXXXT. CONCLUSION: BXXXT ameliorates HFD-induced hepatic steatosis and insulin resistance by increasing Cidea and Cidec-mediated mitochondrial and peroxisomal fatty acid oxidation, which may provide a potential strategy for therapy of NAFLD and T2DM.

Severe Hyperprolactinemia Promotes Brown Adipose Tissue Whitening and Aggravates High Fat Diet Induced Metabolic Imbalance.

Background: The association of high serum prolactin and increased body weight is positive but controversial, therefore we hypothesized that additional factors such as diets and the impact of prolactin on brown adipose tissue may condition its metabolic effects. Methods: We used LacDrd2KO females with lifelong severe hyperprolactinemia due dopamine-D2 receptor deletion from lactotropes, and slow onset of metabolic disturbances, and compared them to their respective controls (Drd2 loxP/loxP ). Food intake, and binge eating was evaluated. We then challenged mice with a High Fat (HFD) or a Control Diet (CD) for 8 weeks, beginning at 3 months of age, when no differences in body weight are found between genotypes. At the end of the protocol brown and white adipose tissues were weighed, and thermogenic and lipogenic markers studied, using real time PCR (Ucp1, Cidea, Pgc1a, Lpl, adiponectin, Prlr) or immunohistochemistry (UCP1). Histochemical analysis of brown adipose tissue, and glucose tolerance tests were performed. Results: Hyperprolactinemic mice had increased food intake and binge eating behavior. Metabolic effects induced by a HFD were exacerbated in lacDrd2KO mice. Hyperprolactinemia aggravated HFD-induced body weight gain and glucose intolerance. In brown adipose tissue pronounced cellular whitening as well as decreased expression of the thermogenic markers Ucp1 and Pgc1a were observed in response to high prolactin levels, regardless of the diet, and furthermore, hyperprolactinemia potentiated the decrease in Cidea mRNA expression induced by HFD. In subcutaneous white adipose tissue hyperprolactinemia synergistically increased tissue weight, while decreasing Prlr, Adiponectin and Lpl mRNA levels regardless of the diet. Conclusions: Pathological hyperprolactinemia has a strong impact in brown adipose tissue, lowering thermogenic markers and evoking tissue whitening. Furthermore, it modifies lipogenic markers in subcutaneous white adipose, and aggravates HFD-induced glucose intolerance and Cidea decrease. Therefore, severe high prolactin levels may target BAT function, and furthermore represent an adjuvant player in the development of obesity induced by high fat diets.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

Fsp27 plays a crucial role in muscle performance.

Fsp27 was previously identified as a lipid droplet-associated protein in adipocytes. Various studies have shown that it plays a role in the regulation of lipid homeostasis in adipose tissue and liver. However, its function in muscle, which also accumulate and metabolize fat, remains completely unknown. Our present study identifies a novel role of Fsp27 in muscle performance. Here, we demonstrate that Fsp27-/- and Fsp27+/- mice, both males and females, had severely impaired muscle endurance and exercise capacity compared with wild-type controls. Liver and muscle glycogen stores were similar among all groups fed or fasted, and before or after exercise. Reduced muscle performance in Fsp27-/- and Fsp27+/- mice was associated with severely decreased fat content in the muscle. Furthermore, results in heterozygous Fsp27+/- mice indicate that Fsp27 haploinsufficiency undermines muscle performance in both males and females. In summary, our physiological findings reveal that Fsp27 plays a critical role in muscular fat storage, muscle endurance, and muscle strength.NEW & NOTEWORTHY This is the first study identifying Fsp27 as a novel protein associated with muscle metabolism. The Fsp27-knockout model shows that Fsp27 plays a role in muscular-fat storage, muscle endurance, and muscle strength, which ultimately impacts limb movement. In addition, our study suggests a potential metabolic paradox in which FSP27-knockout mice presumed to be metabolically healthy based on glucose utilization and oxidative metabolism are unhealthy in terms of exercise capacity and muscular performance.

Effects of voluntary exercise on the expression of browning markers in visceral and subcutaneous fat tissue of normotensive and spontaneously hypertensive rats.

High physical activity is important to optimize the function of adipose tissue. Dysfunctional adipose tissue contributes to the development of metabolic stress, chronic inflammation, and hypertension. To improve our current understanding of the interaction between physical exercise and adipose tissue, we analyzed the effect of 10 months voluntary running wheel activity of rats on uncoupling protein (UCP) 1 negative white adipose tissue (visceral and subcutaneous adipose tissue, VWAT and SWAT). Analysis was performed via RT-PCR and immunoblot from adipose tissues depicted from adult normotensive and spontaneously hypertensive female rats. UCP1 negative VWAT differed from UCP1 positive WAT and brown adipose tissue (BAT) from interscapular fat depots, by lacking the expression of UCP1 and low expression of Cidea, a transcriptional co-activator of UCP1. High physical activity affected the expression of five genes in SWAT (Visfatin (up), RBP5, adiponectin, Cidea, and Nrg4 (all down)) but only one gene (Visfatin, up) in VWAT. Furthermore, the expression of these genes is differentially regulated in VWAT and SWAT of normotensive and spontaneously hypertensive rats (SHR) under sedentary conditions (UCP2) and exercise (Visfatin, Cidea, Nrg4). Keeping the animals after 6 months of voluntary exercise under observation for an additional period of 4 months without running wheels, Visfatin, Cidea, and Nrg4 were stronger expressed in VWAT of SHRs than in sedentary control rats. In summary, our study shows that SWAT is more responsible to exercise than VWAT.

In silico interactions of statins with cell death-inducing DNA fragmentation factor-like effector A (CIDEA).

Statins are the low-density lipoproteins (LDL)-cholesterol-lowering drugs of first choice and are used to prevent the increased risk of cardiovascular and cerebrovascular diseases. Although some of their effects are well known, little is known about their ability to regulate other lipid-related proteins which control apoptotic mechanisms. The aim of this study was to explore whether statins can bind to cell death-inducing DNA fragmentation factor-like effector A (CIDEA), which might be a possible pleiotropic mechanism of action of these drugs on the modulation of apoptosis and lipid metabolism. The structures of statins were subjected to molecular docking and dynamics with the human CIDEA protein to investigate the interaction pattern and identify which residues are important. The docking results indicated that atorvastatin and rosuvastatin showed the best interaction energy (-8.51 and -8.04 kcal/mol, respectively) followed by fluvastatin (-7.39), pitavastatin (-6.5), lovastatin (-6.23), pravastatin (-6.04) and simvastatin (-5.29). Atorvastatin and rosuvastatin were further subjected to molecular dynamics at 50 ns with CIDEA and the results suggested that rosuvastatin-CIDEA complex had lower root-mean square deviation and root-mean square fluctuation when compared with atorvastatin-CIDEA. Since two arginine residues -ARG19 and ARG22-were identified to be common for the interaction with CIDEA, a single-point mutation was induced in these residues to determine whether they are important for binding interaction. Mutation of these two residues seemed to affect mostly the interaction of atorvastatin with CIDEA, suggesting that they are important for the binding and therefore indicate another possible metabolic mechanism of the pleiotropic effects of this statin.

Low expression of brown and beige fat genes in subcutaneous tissues in obese patients.

INTRODUCTION: The molecular mechanisms behind obesity pathogenesis remain largely undefined. Impairment in the browning process of subcutaneous tissues proposed to contribute to obesity pathogenesis. In the current study, we aimed to assess whether the expression of brown fat genes in subcutaneous tissues in obese patients is altered as compared to non-obese patients. MATERIAL AND METHODS: Participants were recruited from patients undergoing general surgeries. At the same site of surgery, biopsies were taken from the abdominal subcutaneous tissues from each participant, along with a venous blood sample. The expression of BAT genes was measured using a real-time PCR method. Serum FGF21 was measured using an ELISA kit, and the serum blood lipid profile was measured using the Dimension VistaTM 1500 System. RESULTS: A total of 58 surgical patients was involved. A low expression of BAT genes was observed in the groups with higher body mass index (BMI) (< 30 kg/m2) as compared to the groups with lower BMI (> 30 kg/m2). The expression of CIDEA and CITED1 was significantly higher in the patients with normal weight as compared to obese (p = 0.01 and p = 0.02, respectively). A significant negative correlation was found between the expression of BAT genes and BMI in patients with BMI < 35 kg/m2. However, the strongest negative correlation was observed in the expression of CIDEA (r = -0.5, p = 0.004), followed by TBX1 (r = -0.4, p = 0.01), CITED1, and ZIC1 (r = -0.4, p = 0.03). Whereas the correlation of UCP1 with BMI remained insignificant (r = -0.29, p = 0.08). When including patients with BMI > 35 kg/m2, the correlation decreased and became insignificant (p = 0.08). No significant correlation was found between the expression of BAT genes and blood lipid profiles (p > 0.05). Serum FGF21 was positively and significantly correlated to the expression of UCP1 (r = 0.56, p = 0.02) and TBX1 (r = 0.62, p = 0.01), however, this correlation was missing in patients with severe obesity. CONCLUSIONS: Our data suggested that brown and beige genes expression in abdominal subcutaneous tissues is dysregulated in patients with obesity. Further studies are needed to investigate the role of browning of subcutaneous tissues in regulating body weight and metabolism in human.

Small molecules for fat combustion: targeting obesity.

Obesity is increasing in an alarming rate worldwide, which causes higher risks of some diseases, such as type 2 diabetes, cardiovascular diseases, and cancer. Current therapeutic approaches, either pancreatic lipase inhibitors or appetite suppressors, are generally of limited effectiveness. Brown adipose tissue (BAT) and beige cells dissipate fatty acids as heat to maintain body temperature, termed non-shivering thermogenesis; the activity and mass of BAT and beige cells are negatively correlated with overweight and obesity. The existence of BAT and beige cells in human adults provides an effective weight reduction therapy, a process likely to be amenable to pharmacological intervention. Herein, we combed through the physiology of thermogenesis and the role of BAT and beige cells in combating with obesity. We summarized the thermogenic regulators identified in the past decades, targeting G protein-coupled receptors, transient receptor potential channels, nuclear receptors and miscellaneous pathways. Advances in clinical trials were also presented. The main purpose of this review is to provide a comprehensive and up-to-date knowledge from the biological importance of thermogenesis in energy homeostasis to the representative thermogenic regulators for treating obesity. Thermogenic regulators might have a large potential for further investigations to be developed as lead compounds in fighting 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.

Effects of a high energy and low protein diet on hepatic and plasma characteristics and Cidea and Cidec mRNA expression in liver and adipose tissue of laying hens with fatty liver hemorrhagic syndrome.

Cidea and Cidec are two members of Cell death-inducing DNA fragmentation factor-alpha-like effector family proteins, which could be involved in lipid or fat metabolism. To better understand the roles of Cidea and Cidec in fatty liver hemorrhagic syndrome (FLHS), 150 healthy 155-day-old Hyline Brown laying hens were randomly divided into control group (fed with basic diet) and experimental group (fed with high-energy low-protein [HELP] diet). Analysis of the liver by tissue sectioning and hematoxylin and eosin staining showed that the HELP diet induced micro-vesicular steatosis in laying hens. Subsequently, based on the liver color scores and the range of lipid accumulation observed in histological examination, we classified livers with <50% vacuolization as mild FLHS and >50% as severe FLHS. The results showed that the levels of Cidea and Cidec mRNA expression were markedly elevated in the liver and adipose tissues with FLHS and the levels of Cidea and Cidec mRNA expression in the liver with severe FLHS were significantly higher than that in the liver with mild FLHS. Thus, the present study revealed that the Cidea and Cidec genes may be involved in pathways of FLHS formation.

High expression of cell death-inducing DFFA-like effector a (CIDEA) promotes milk fat content in dairy cows with clinical ketosis.

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis represent a source of fatty acids for milk fat synthesis and explain the increase in milk fat content in ketotic cows. Cell death-inducing DFFA-like effector a (CIDEA) is a lipid droplet coat protein with important roles in the regulation of milk fat synthesis and secretion in mice. Whether ketosis alters the expression of CIDEA in mammary gland tissue and the extent to which it may contribute to regulation of milk fat synthesis and secretion are unknown. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Mammary epithelial cells isolated from cows were infected with CIDEA overexpression adenovirus for 48 h, treated with 0, 0.3, 0.6, or 1.2 mM NEFA for 24 h, or infected with CIDEA-silencing adenovirus for 48 h and treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and ß-hydroxybutyrate were greater in cows with clinical ketosis, and milk production and dry matter intake were lower in cows with clinical ketosis. However, compared with healthy cows, the content of milk fat of cows with clinical ketosis was greater. Compared with healthy cows, abundance of mRNA and protein of CIDEA, fatty acid synthase (FASN), acetyl-coA carboxylase 1 (ACACA), butyrophilin (BTN1A1), and xanthine dehydrogenase (XDH) was greater in mammary tissue of cows with clinical ketosis. Overexpression of CIDEA in cultured mammary epithelial cells increased the abundance of FASN, ACACA, XDH, and BTN1A1, and increased triacylglycerol (TAG) content in mammary epithelial cells. Exogenous NEFA increased the abundance of CIDEA, FASN, ACACA, XDH, and BTN1A1, and increased TAG content in mammary epithelial cells. Importantly, knockdown of CIDEA reversed the upregulation of FASN, ACACA, XDH, and BTN1A1 abundance and TAG content induced by NEFA treatment. Overall, these data suggest that high levels of NEFA stimulate the expression of CIDEA and enhance de novo fatty acid synthesis and milk fat secretion. As such, these mechanisms explain in part the elevation of milk fat content in dairy cows with clinical ketosis.

Identification of gene products that control lipid droplet size in yeast using a high-throughput quantitative image analysis.

Lipid droplets (LDs) are important organelles involved in energy storage and expenditure. LD dynamics has been investigated using genome-wide image screening methods in yeast and other model organisms. For most studies, genes were identified using two-dimensional images with LD enlargement as readout. Due to imaging limitation, reduction of LD size is seldom explored. Here, we aim to set up a screen that specifically utilizes reduced LD size as the readout. To achieve this, a novel yeast screen is set up to quantitatively and systematically identify genes that regulate LD size through a three-dimensional imaging-based approach. Cidea which promotes LD fusion and growth in mammalian cells was overexpressed in a yeast knockout library to induce large LD formation. Next, an automated, high-throughput image analysis method that monitors LD size was utilized. With this screen, we identified twelve genes that reduced LD size when deleted. The effects of eight of these genes on LD size were further validated in fld1 null strain background. In addition, six genes were previously identified as LD-regulating genes. To conclude, this methodology represents a promising strategy to screen for players in LD size control in both yeast and mammalian cells to aid in the investigation of LD-associated metabolic diseases.

Distribution and quantitative analysis of CIDEa and CIDEc in broiler chickens: accounting for differential fat deposition between strains.

1. Differences in the expression of CIDEa and CIDEc in 20 different tissues were examined. Both CIDEa and CIDEc mRNA transcripts were predominantly but variably expressed in white adipose tissue (WAT) but were also expressed at moderate levels in the kidney and liver and at lower levels in the ovary. Interestingly, among WAT types, both CIDEa and CIDEc were expressed at the lowest levels in heart coronary WAT. 2. To better understand the roles of CIDEa and CIDEc in the fat deposition of broiler chickens, the differences in lipid droplet (LD) size and mRNA levels of CIDEa and CIDEc between lean-type and fat-type broiler chicken lines were studied. LD sizes were larger in fat-type broiler lines, and CIDEa and CIDEc mRNA levels in white adipose, kidney and liver tissues were significantly higher in fat-type broiler lines than in their lean counterparts. 3. Developmental expression patterns of CIDEa and CIDEc mRNA were analysed in different tissue types (WAT, liver and kidney) in Arbor Acres broiler chickens, and CIDEa and CIDEc mRNA expression levels increased during sequential developmental stages, achieving peak expression levels at week 6. 4. These observations suggest that the functions of CIDEa and CIDEc reflect inherent characteristics of lipid metabolism that contribute to the differences in fat deposition between strains. The results in this study contribute to a more robust understanding of the tissue distribution and expression patterns of CIDEa and CIDEc mRNA and facilitate further research concerning the molecular mechanism underlying fat deposition in broiler chickens.

CIDE Family-Mediated Unique Lipid Droplet Morphology in White Adipose Tissue and Brown Adipose Tissue Determines the Adipocyte Energy Metabolism.

White adipose tissue (WAT) stores energy as triacylglycerol in preparation for fasting state. In contrast, brown adipose tissue (BAT) consumes energy and produces heat in a cold environment. One of the major differences between these two adipose tissues is the morphology of the intracellular lipid droplet (LD), which is large and unilocular in WAT and small and multilocular in BAT. Although the fat-specific protein 27 alpha (FSP27α), belonging to the cell death-inducing DNA fragmentation factor A (DFFA)-like effector (Cide) family, was known to be indispensable for large unilocular LD formation in WAT, the mechanism that regulated small multilocular LD formation in BAT remained unknown. We recently uncovered that FSP27ß, a novel isoform of FSP27 abundantly expressed in BAT, plays a crucial role in small multilocular LD formation by inhibiting the homodimerization of CideA in BAT. We speculate that unilocular LD formation is ideal for efficient lipid storage in WAT because lipolysis from the LD surface is restricted due to the minimum LD surface area. In addition, hydrolyzed free fatty acid (FFA) and glycerol can efficiently flow out into the circulation from the cell surface. In contrast, small multilocular LD formation is ideal for efficient intracellular lipolysis from the LD surface and the subsequent facilitation of FFA transport to mitochondria that are adjacent to LDs for ß-oxidation in BAT. Thus, intracellular LD morphology is closely related to the functions and characteristics of adipose tissues. Given that the browning of adipose tissue leads to enhanced energy expenditure and the prevention of obesity, clarification of the mechanism with respect to intracellular LD formation is very meaningful.