Relationship between the Cell Death-Inducing DNA Fragmentation Factor 45-Like Effector Protein Family and the Risk of Dyslipidemia

According to the research, obesity is associated with hyperlipidemia, hypertension, and type 2 diabetes mellitus, which are grouped as metabolic syndrome. Notably, under the obese status, the adipocyte could accumulate excessive lipid as lipid droplets (LDs), leading the dysfunctional fat mass. Recently, emerging evidence has shown that the cell death-inducing DNA fragmentation factor 45-like effector protein (CIDE) family played an important role in regulating lipid metabolism. In addition, diverse CIDE proteins were also confirmed to influence the intracellular lipid metabolism, such as within adipocyte, hepatocyte, and macrophage. Nevertheless, the results which showed the regulatory influence of CIDE proteins are significantly contradictory from in vitro experiments and in vivo clinical studies. Similarly, 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. However, the underlying mechanisms by which the diverse CIDE proteins alter lipid metabolism are not elucidated. In the current review, the understandings of CIDE proteins in lipid catabolism were well-summarized. On the other hand, the relatively mechanisms were also proposed for the further understandings of the CIDE protein family.


Introduction
Obesity is correlated with dyslipidemia, hypertension, and type 2 diabetes mellitus, posing serious risks to the health of general population [1].One of the hallmarks of obesity is excessive lipid accumulation in the adipocyte and the dysfunctional alterations of fat mass [2].Under the obese/overweight conditions, the hypertrophic fat cells could synthesis and produce excessive contents of inflammatory-related adipokines, such as interlukin-1 (IL-1), interlukin-6 (IL-6) and tumor necrosis factor-α (TNFα), resulting in systemic inflammatory response and dyslipidemia that is characterized by increased levels of lowdensity lipoprotein cholesterol (LDL-C) and triglyceride (TG) [3].Otherwise, the fat tissue contains abundant adipose-derived mesenchymal stem cells (AMSCs), which could further differentiate into the mature adipocyte by imbalance between energy intake and expenditure [4].It is note-worthy that excessive adipogenesis of AMSCs might induce dysfunctional adipocyte, promoting the pathogenic development of obese related metabolic diseases.However, the mechanisms are unclarified.
Several years after their discovery and classification as pro-apoptotic proteins, the cell death-inducing DNA fragmentation factor 45-like effector protein (CIDE) family was unexpectedly identified as lipid droplet-associated proteins.Notably, the CIDE protein family, including CIDE-A/B/C, has confirmed to embrace a vital role in lipid metabolic pathways, including adipocyte, macrophage, and hepatocyte [5].Also, this relationship was a direct physical interaction with the surface of the lipid droplets (LDs), as well as with other LDs proteins, such as perilipin [6].Furthermore, the different tissue expression of each member is closely related with the LDs [7].Although emerging results has shown the functions of diverse CIDE proteins, the results remain contradictory in diverse studies.Otherwise, the mechanisms by which the CIDE proteins influence the lipid catabolism are not clarified.In the current review, the knowledges on CIDE proteins and the regulatory functions in lipid catabolism were well-summarized.Moreover, the mechanisms were also be proposed for the further understanding of the CIDE protein family.

Basic Structural of Diverse CIDE Proteins
The CIDE family proteins, including CIDE-A, CIDE-B, and CIDE-C (also named FSP-27 in mice) are enriched on the sites of LDs [8].Given that the enrichment of several LD-related proteins, such as the perilipin-ATGLadipophilin (PAT) family proteins, was not found at this specific sub-organelle sites, the LD-LD connected sites are currently and relatively unique to the CIDE family proteins [9].Notably, multiple reports have shown that the target of different CIDE protein to LDs is dependent on the hydrophobic region which is close to their C-terminus [10].The amphipathic helical region on the CIDE-C domain, in-cluding conserved basic lysine or arginine residues, played an important role in the process of LDs fusion, which may occur via the interaction with the phosphatidic acid on the membrane surface of LDs [11].
As published in previous studies, the LDs fusion and the lipid transferring process induced by CIDE family proteins are considered as a unique intra-cellular progression given that it is distinct from several other kinds of membrane fusion, such as vesicle fusion, mitochondria fusion, and endoplasmic reticulum fusion [12].With in-depth research, the vesicle fusion has been verified to be a relatively fast process that contains multiple subsequent steps, including the formation of the fusion of lipid bilayers, the formation of the interactive membrane structure, and the formation of a temporary fusion pore during a short period [13].Nevertheless, the process of LDs fusion induced by CIDE family protein is a slow process, which could be influenced by both the size of pore and the size of LDs [14].Concerning on this notion, the future research might focus on the relationship between the structure of CIDE proteins with their functions in modulating lipid metabolism.

Relationship between Single Nucleotide Polymorphisms (SNPs) of CIDE Genes and Lipid Metabolism
The extensive sequencing of gene interval in humans has established a close association between chromosome 18 (18p11) with dyslipidemia and its related cardio-metabolic diseases.For instance, it has been shown that several important obesity-related candidate genes were located in the 18p11 region, a finding which has been replicated by another independent study, suggesting that the chromosome 18p11 region had a positive correlation with the prevalence rate of obesity [15,16].Interestingly, it has been shown that the human CIDE-A gene was located in the 18p11 region with 5 exons, and approximately 66 SNPs in CIDE-A gene were confirmed to be associated with human diseases in patients from different countries.For example, a study comprising 1563 Swedish individuals that were categorized into non-obese and obese populations demonstrated that a CIDE-A gene SNP, namely V115F, had a strong association with the occurrence of obesity.However, after analysis, the V115F SNP revealed six different polymorphic sites in obese patients compared with those in the non-obese individuals, and only the C.19878G>T SNP in the polymorphic sites' region was responsible for coding the V115F mutation [17].These results strongly support that the CIDE-A gene alleles are positively and significantly associated with the pathogenesis of obesity.An additional independent study conducted by Zhang et al. [18] which comprised 351 patients, who have evaluated at the Cardiac Clinic in Xuanwu Hospital for metabolic syndrome risks, clarified a positive association of the V115F gene of CIDE-A gene with the risk of metabolic syndrome.After multiple logistic regression analysis, the odds ratios (OR) for metabolic syn-drome, central obesity, and dyslipidemia in the Guanine-Thymine (GT) and Thymine-Thymine (TT) genotypes were all higher compared with the referent Guanine-Guanine (GG) genotype, indicating that the T allele in the V115F SNP of CIDE-A gene is a risk factor for metabolic syndrome in Chinese population [18].Consistently, another cross-sectional study including 270 Japanese patients also determined the modulatory effect of V115F SNP on the phenotypes and the prevalence of metabolic syndrome [19].The authors concluded that the V115F SNP was associated with waist circumference; additionally, the patients who carried the VF (valine, V; phenylanaline, F) + FF had higher fasting serum concentrations of glucose (OR = 2.81) and higher prevalence rate for abdominal obesity (OR = 1.89) and metabolic syndrome (OR = 3.15), indicating that the F allele of the CIDE-A gene may serve as a risk factor for phenotypes associated to metabolic syndrome in Japanese men [19].
Recently, a case-control study investigated the association of five tag-SNPs and haplotype structures of the CIDE-A gene SNPs, including V115F/rs1154588, rs4796955, rs8092502, rs12962340, and rs7230480, with the prevalence rate of obesity in the Han Chinese population.Importantly, the results showed that genotypes of V115F/TT, rs4796955/GG, and rs8092502/CC of CIDE-A gene were identified as risk factors for obesity.The interaction between rs4796955 and V115F SNP embraced a joint role in the development of obesity and its related diseases [20].Furthermore, in 2019, an independent study conducted by Ramshanker et al. [21] revealed an insulinsensitivity-independent association between CIDE-A genes SNPs with the increased serum concentrations of Free fatty acids (FFAs) and the visceral adipose tissue.By contrast, patients who had more lipolysis-increasing alleles exhibited the lower visceral fat mass [21].Overall, these studies unequivocally identified the positive association of CIDE-A gene SNPs with the healthy metabolic phenotypes in humans, and the interaction between SNP1 and V115F of CIDE-A gene SNPs could play a joint role in the pathological process of obesity.Similar to CIDE-A gene, Yu et al. [22] found that the 414Arg SNP of CIDE-B gene was significantly correlated with the fat content in the Berkshire pigs.Additionally, the CIDE-B gene expression was significantly upregulated during the early stage of adipogenesis, suggesting that CIDE-B may contribute towards initiation of adipogenesis [22].Otherwise, a recently discovered SNPs in CIDE-C gene have been determined to correlate with metabolic syndrome.It has been determined since the early 21st century that the SNP/G>T of CIDE-C gene could induce a non-sense mutation, namely E186X SNP, which disrupted the CIDE-C domain and was closely associated with the lipodystrophy in female patients [23].Additionally, this finding has been replicated in a study which enrolled the Asian participants.A study enrolling totally 1064 patients with metabolic syndrome and 1099 healthy controls of Chinese Han nationality investigated the association between rs1053239 SNP with rs2479 SNP of CIDE-C gene with the risk of metabolic syndrome and its components.Interestingly, they found that the patients who carried rs2479 SNP presented higher serum concentrations of fasting glucose and TG compared to those in non-carriers; moreover, patients who carried rs1053239 SNP also showed the aggravation of both systolic and diastolic blood pressures, suggesting that individuals who carry rs2479 SNP or rs1053239 SNP are more predisposed to metabolic syndrome with the higher risk factors including hypertension, dyslipidemia, and hyperglycemia [24].Given the lack of research focusing on the SNP of CIDE-B gene and the function in modulating lipid metabolism, the future clinical trial might aim at explore the relationship mentioned above.

Roles of CIDE-A in Modulating the Whole-Body Energy Homeostasis
CIDE-A, as the first discovered protein of the CIDE family, is confirmed by several research as abundantly expressed in brown adipocytes (BAT) and in human white adipocytes (WAT) in mammal animals [25].Currently, CIDE-A has been considered and used as an alternative brown adipocyte-specific marker in addition to uncoupling protein 1 (UCP1) [26].As reported, CIDE-A is abundantly and specifically expressed in murine brown adipocytes.Compared with the wild mouse models, the CIDE-A genedeficient mice by a high-calorie diet (HCD) could significantly preferred lower lipid storage and up-regulated uptake rate of serum glucose.Meanwhile, these genedeficient mice also presented enhance lipolysis rate and an up-regulated whole-body metabolic rate; moreover, these mice also had reduced plasms concentrations of triacylglycerols and free fatty acids.On the other hand, the CIDE-A gene-deficient adipocytes in human also markedly elevates lipolysis.In details, the results about the effect of CIDE-A on the whole-body energy homeostasis are almost similar between mice and humans.Mice with CIDE-A genedeficiency exhibited less body weight, higher body temperature, and enhanced lipolysis rate in BAT compared with wild mice.Additionally, these mice also had enhanced glucose uptake accompanied with decreased serum concentrations of TG and free fatty acids (FFAs), which could be resistant to the high fat diet (HFD)-induced over-weight or obesity.Notably, the LD sizes in CIDE-A gene-deficient brown adipocytes were significantly decreased [27].Likewise, using human adipocytes with CIDE-A gene knockdown, the authors found that the intra-cellular lipolysis rate was markedly elevated with smaller LDs in these cells [28].On the contrary, the mice with over-expressed of human CIDE-A gene presented a higher body mass index (BMI) compared with the control littermates [29].Me-chanically, CIDE-A is confirmed as a mitochondrial protein which could inhibit the expression levels of UCP1 gene, a brown adipose tissue (BAT)-special gene and could modulate lipolysis in adipocytes isolated from mice.After knocking down CIDE-A gene, the quantity of LDs in those brown adipocytes significantly reduced and the expression of UCP-1 gene was stimulated by cold exposure [30].Similar relationship could also be found in other studies [31,32].On the other hand, it has been shown in another study by Qi et al. [33] that mice with CIDE-A gene-deficiency exhibited enhanced Adenosine Monophosphate (AMP)-activated protein kinase (AMPK) signaling activity in BAT with higher energy consumption in brown adipocytes.Consistent with this finding, Cheng et al. [34] also confirmed that the over-expression of CIDEA gene could inhibit AMPK activity.With in-depth research, the authors also found that CIDE-A could was co-localized with AMPK protein in the endoplasmic reticulum and subsequently stimulated the ubiquitination-mediated degradation of AMPK protein [33].Within the BAT isolated from CIDE-A gene-deficient mice, the protein levels and the enzymatic activity of βsubunit of AMPK were increased, strongly revealing that CIDE-A modulated the whole-body energy homeostasis through, at least partly, suppressing the AMPK signal pathway [33].Likewise, in obese people, the expression levels of CIDE-A gene were negatively correlated with the wholebody metabolic disorders, such as insulin resistance and pre-diabetes [35,36].Furthermore, up-regulated expression of CIDE-A gene were also observed in the liver isolated from patients under the condition of non-alcoholic steatohepatitis (NASH) [37].More recently, an independent study conducted by Li et al. [38] Lipopolysaccharide (LPS) found that the lipopolysaccharide-induced lower expression of DNA methyltransferases 3B (DNMT3B) could lead to hypomethylation in promoter region of CIDE-A gene, which subsequently reversed the effect of lipopolysaccharide on lipogenesis.Xia et al. [39] used the traditional Chinese medicine, named Ban-xia-xie-xin-tang, and confirmed that this medicine could significantly ameliorate hepatic steatosis by regulating CIDE-A gene expression, suggesting a vital role of CIDE-A in the whole-body energy homeostasis in human beings.
On the other hand, since the important roles of CIDE-A in regulating the whole-body energy homeostasis has been the subject of several research, the additional functions of CIDE-A in other tissues or organs have been recently begun to be explored.As reported, the tissue-special knockdown of CIDE-A gene in the mammary gland induces the accumulation of smaller cytosolic LDs [40].Furthermore, Schneider [41] reported that CIDE-A gene expressed in the sebaceous glands of both the skin and the Meibomian glands of the eyelid.Overall, these results shed light on that CIDE-A plays a vital role in modulating the whole-body lipid storage and energy homeostasis.

Roles of CIDE-B in Modulating the Whole-Body Energy Homeostasis
There is a limited number of research focusing on the relationship between CIDE-B and the whole-body energy homeostasis.Apart from mainly expressed in the livers and kidneys, CIDE-B is also expressed with less extent in the small intestines and pancreatic-β cells, which suggests that CIDE-B embraces vital function in energy metabolism.Notably, the animal studies have demonstrated that in CIDE-B gene-deficient mice, the adiposity was decreased and the rate of body metabolic was increased; meanwhile, the insulin resistance was also improved obviously.Furthermore, these mice exhibited significantly reduced biogenesis of nascent very low-density lipoprotein (VLDL) particles and decreased serum concentrations of TG, hand in hand with a lean phenotype after feeding with a high-fat diet [42].With in-depth investigations, CIDE-B was verified to be localized on the surfaces of LDs, which could further modulate the VLDL maturation and chylomicrons lipidation by interacting with Apolipoprotein-B within the liver and the small intestine, respectively.By silencing the CIDE-B gene expression in hepatocytes, the authors reported that the hepatocytes contained smaller LDs and had enhanced hepatic fatty acid oxidation, indicating that CIDE-B could affect the secretion of VLDL particles in the liver, which in turns regulated the serum concentrations of lipid profiles [43].
On the other hand, it is worth noting that in CIDE-B gene-deficient mice, the expression levels of sterol regulatory element binding protein 1C (SREBP-1C), as a crucial adipogenic-related factor, was significantly reduced compared to the control mice, which consequently lead to the down-regulation of fatty acid synthesis gene and the decreased intracellular lipid storage [44].These results indicated that CIDE-B had a negative impact on metabolic homeostasis in mice and provided a molecular explanation for the lean phenotype and increased energy expenditure.Nevertheless, the effect of CIDE-B gene over-expression on the whole-body energy homeostasis is still not elucidated, especially in humans.Thus, it is still required to conduct more studies to further illuminate the relationship and function of CIDE-B in influencing the body energy production and consumption.

Roles of CIDE-C in Modulating the Whole-Body Energy Homeostasis
Owing to the research advances, the eye-catching breakthroughs were also made to elucidate the relationship between the CIDE-C protein and the whole-body energy homeostasis.As reported, CIDE-C or FSP-27, which was initially discovered two decades ago as an adipocytespecific gene and as one of the LDs-related proteins in 3T3-L1 adipocytes, is recently confirmed to be pre-dominantly expressed in either white adipocyte or brown adipocyte and to play a pivotal role in lipid metabolism in adipose tissue [45].Nevertheless, similar to CIDE-A, the mice models used for researching CIDE-C/FSP-27 have given compound responses.Notably, the whole body FSP-27deficient mice displayed lean phenotype with lower concentrations of TG and were resistant to High fat diet (HFD)induced obesity with obviously less BMI compared with those in control mice [46].Lower extent of lipid accumulation in white adipose tissue (WAT) were also seen in those FSP-27-deficient mice, leading to obvious small and multilocular LDs.Moreover, the insulin sensitivity was also improved in these mice [47].Similar with these results, using anti-sense oligonucleotides to silence FSP-27 expression in HFD-fed mice, Langhi et al. [48] found decreased visceral adiposity and improved body glycemic control.Recently, it was confirmed that the CIDE-A/FSP-27 genedouble knockout (CIDE-A/FSP-27-DKO) mice displayed a drastic reduced lipid accumulation capabilities that was characterized by smaller LDs in white adipocytes; nevertheless, these mice did not exhibited improved insulin sensitivity [29].These interesting findings were also replicated by another research conducted by other two independent research [49,50].
On the contrary, mice with over-expressed FSP-27 exhibited larger unilocular LDs in white adipocyte and were prone to high fat diet-induced obesity.The lipolysis mediated by isoproterenol was also suppressed by over-expressed FSP-27 gene due to the down-regulation of multiple genes involved in mitochondrial oxidative metabolism, such as UCP1 gene, diacylglycerol acyltransferase 2 (DGAT2) gene, and adipose triacyl-glyceride lipase (ATGL) gene [51,52].These results shed light on that the CIDE-C/FSP-27 protein participates in the enlargement of LDs, and in particular, CIDE-C/FSP-27 is essential for the formation of unilocular LDs of white adipocyte at least in mice.
On the other hand, it is still unclarified of the CIDE-C function in regulating energy metabolism in humans.For instance, the results reported showed that the serum concentrations of CIDE-C was positively associated with the insulin sensitivity in obese patients, and the adipose-special CIDE-C is supposed to improve insulin sensitivity by increasing lipid accumulation in adipocytes, which consequently reducing ectopic TG accumulation in other insulinsensitive cells such as hepatocytes, suggesting that CIDE-C could protect humans from metabolic syndrome and could be considered as a potential therapeutic application to obese individuals [53].Recently, the relationship of CIDE-C and the risk of diabetes induced by growth hormone in humans was being illuminated recently [54].In details, the authors confirmed that the process of growth hormoneinduced lipolysis was probably mediated by the activation of mitogen-activated protein kinase/extracellular signalregulated kinase (MEK/ERK), which could suppress the transcriptional activity of peroxisome proliferator-activated receptor γ (PPAR-γ) [54].Given that PPAR-γ functions importantly during the pathogenesis of obesity, these results suggested that the mechanism by which CIDE-C affected the insulin sensitivity and the whole-body energy homeostasis in human potentially via influencing the MEK/ERK and PPAR-γ signaling pathway [55].However, further research is still needed to deeply explore the accurate mechanisms whereby CIDE-C modulates the whole-body energy homeostasis.
In conclusions, these results indicate that the CIDE protein family plays a vital function in whole-body energy homeostasis in mammals and in the enlargement of LDs at least in mice.

Roles of CIDE Proteins in Modulating the Formation of LDs
As described, since the energy accumulation within LDs is considered as a fundamental need both in humans and animals, and the efficient sequestration of TG could inhibit fatty acid overload in multiple metabolically active tissues involving in the pathological development of obesity, several studies have focused on the effect of CIDE proteins on affecting the formation of LDs.According to the reports, the capacity of lipid accumulation in LDs is dependent on the sizes of the LDs in multiple cell types, such as the hepatocyte and the adipocyte, due to the LDs have been considered as the subcellular organelles responsible for lipid storage [56].Meanwhile, the sizes of LDs are also influenced by lipolysis within the hepatocyte and the adipocyte in turns [57].
It has been investigated since the early 20th century that several different CIDE proteins have respective functions to modulate the inter-cellular LDs formation, a finding which has been replicated in many other studies from diverse countries.For instance, Toh and colleagues [47] isolated the embryonic fibroblasts from the wild-type mice and the FSP-27 gene-deficient mice, followed by their differentiation into mature adipocytes.Interestingly, the authors demonstrated that the expression levels of FSP-27 gene were significantly increased in the white adipocyte and the hepatocyte isolated from the LEPTIN gene-deficient ob/ob mice [47].Furthermore, the FSP-27 gene-deficient mice were confirmed to resistant to obesity after feeding highfat diet which also exhibited the increased insulin sensitiv-ity; however, the serum concentrations of TG in the FSP-27 gene-deficient mice were significantly lower compared with those in the control mice [47].With in-depth investigation, the FSP-27 gene-deficient mice also presented reduced lipid accumulation and smaller LDs within the white adipocyte, suggesting that the expression levels of FSP-27 gene could significantly affect the growth of LDs and the differentiation of pre-adipocytes [58].Additionally, the authors further found that the expression levels of key brown adipocyte-specific genes, such as the FoxC2 gene and the PPAR gene, were all markedly up-regulated in FSP-27 gene-deficient mice.Nevertheless, the expression levels of genes inhibiting the differentiation of brown adipocyte, such as the RIP140 gene, were significantly lower in the white adipocyte compared with those in the control mice [47].Thus, we could make a reasonable speculation that aside from the function in modulating LDs formation, the FSP-27 proteins could also act as a novel regulator in controlling differentiation of pre-adipocytes.
Consistent with this notion, Sun et al. [59] recently used the human AMSCs and demonstrated that during the adipogenic differentiation process of AMSCs, the expression levels of CIDE-C gene were increased observably.Furthermore, the authors also confirmed that intervention by Apolipoprotein A5 (ApoA5) could reduce the intracellular accumulation of LDs and down-regulate the gene expression levels of adipogenic related factors, including CCAAT enhancer binding proteins α/β (C/EBPα/β), FAS, and fatty acid-binding protein 4 (FABP4).It is further demonstrated that the suppression of adipogenic differentiation process of AMSCs by ApoA5 treatment was mediated through the inhibition of CIDE-C gene expression [59].Nevertheless, over-expressing intra-cellular CIDE-C gene could lead to the loss-of-function of ApoA5 in inhibiting AMSCs adipogenesis [59].Thereby, our results also shed light on the important function of CIDE-C protein in affecting the metabolism of human adipocytes.
On the other hand, increasing evidence showed that the LD sizes were verified to be smaller in brown adipocytes within CIDE-A gene-knockdown adipocytes in mice, hand in hand with a higher metabolic rate and increased lipolysis in brown adipocyte when subjected to cold environment, indicating a role for CIDE-A in regulating energy balance and adiposity [25].Otherwise, another study showed that the hepatocytes with CIDE-B gene deficiency also contained smaller LDs.Of note, Li et al. [60] demonstrated that the liver-specific knockdown of PERILIPIN gene in mice with CIDE-B gene deficiency could induce the significantly reduced accumulation of hepatic TG and increased VLDL-TG secretion.Similar results could be observed in another research which revealed that CIDE-B played an essential role in controlling VLDL lipidation and hepatic lipid homeostasis [61].Nonetheless, growing evidence has shown that the ectopic over-expression of CIDE proteins could result in excessive accumulation of large LDs both in humans and mice [62,63].The summary of roles of diverse CIDE proteins in different tissues was shown in Fig. 1.Therefore, taken together, the findings mentioned above indicated that the CIDE proteins embraced critical roles in controlling the formation of LDs within the adipocyte and the hepatocyte.

Roles of CIDE Proteins in Modulating the Lipidation and Maturation of VLDL Particles
The assembly and maturation of VLDL particles within the hepatocyte has been shown to be generally via the following two steps.In details, the first step of VLDL lipidation occurs in the endoplasmic reticulum with the formation of lipid poor lipoprotein particles, as the pre-VLDL particle, which is dependent on the microsomal triglyceride transporter to transfer the locally synthesized TG to ApoB-100 [64].By this method, the pre-VLDL particles are subsequently lipidated to generate the mature VLDL particles for released into circulation [65].The sub-cellular location of mature VLDL particles, the source of the lipids, and the factors which facilitate VLDL assembly and lipidation are still remained further investigated.
Recently, two hypotheses concerning on the source of lipids involved in VLDL lipidation have been proposed.On one hand, TG is directly transferred via the fusion of the endoplasmic reticulum-lumenal LDs to pre-VLDL particles [66]; on the other hand, TG from cytosolic LDs could be hydrolyzed to yield FFAs which are subsequently re-esterified on the lumenal side of secretory apparatuses and generates lipid-rich VLDL particles.The fusion of endoplasmic reticulum-lumenal LDs within the VLDL particles during VLDL lipidation and maturation is proposed to be mediated by Apolipoprotein-C3 (ApoC3) [67].Other factors, such as phosphoinositide-3 kinase and coat protein complex-II, have also been shown to be involved in VLDL assembly and secretion in recent years [68].In addition, another recent research has demonstrated that the synthesis of phosphatidyl-choline and phosphatidylethanolamine, and their ratio also played an important role in regulating the VLDL assembly and secretion [69].
Emerging evidence showed the vital modulatory roles of CIDE-B in the lipidation and maturation of VLDL particles.As reported, the VLDL particles isolated from either the CIDE-B gene deficient mice or the CIDE-B gene knockout hepatocytes exhibited significantly lower intracellular TG contents whereas had similar levels of ApoB-100 or ApoB-48, revealing an impairment in VLDL lipidation in CIDE-B gene-deficient hepatocytes [70].Furthermore, CIDE-B has been verified to be localized to LDs and the endoplasmic reticulum which could interact with ApoB-100 or ApoB-48.The inter-action between CIDE-B with ApoB-100 is required for VLDL assembly and maturation within hepatocytes [71].Another research conducted by Singaravelu et al. [72] demonstrated an association between CIDE-B concentrations and LD size in human serum intervened with Huh-7.5 cells.Through over-expression of CIDE-B gene, it has been shown that CIDE-B could downregulate the protein levels of adipose differentiation-related protein (ADRP); by contrast, silencing the CIDE-B gene could dynamically increase the sizes of LDs, highlighting a function of CIDE-B in regulating the metabolism of VLDL particles [72].Another research recently reported that the hepatic expression of CIDE-A gene and FSP-27 gene was similarly up-regulated in mouse models of obesity-induced hepatic steatosis [73].On the contrary, the expression levels of CIDE-A gene decreased significantly, whereas the expression levels of FSP-27 gene strongly increased in a mouse model of obesity-induced steatohepatitis [74].With in-depth investigations, the inverse expression pattern of CIDE-A gene and FSP-27 gene was amplified with the severity of the hepatic inflammatory response and injury.Otherwise, in obese patients, the hepatic expression contents of CIDE-C gene were shown to be strongly associated with the activity score and liver injury under the status of non-alcohol fatty liver disease (NAFLD) [75].On the other hand, the expression levels of CIDE-A gene tended to be up-regulated with the pathological development of obe-sity; notably, the expression levels decreased significantly with the progression of NAFLD [37].Using the hepatic cell lines isolated from mice, the authors found that the downregulation of FSP-27 gene resulted in the fractionation of LDs, whereas over-expression of FSP-27 gene decreased the gene expression of the anti-apoptotic marker which was named as B-cell lymphoma-2 (BCL-2) [70].This, in turn, sensitized cells to apoptosis in response to TNF-α and saturated fatty acid [70].The roles of diverse CIDE proteins in modulating different lipid profiles was listed in Table 1.Considered together of the findings mentioned above, it is proposed that differential expression of CIDE-A and CIDE-C is related to the lipidation and maturation of VLDL particles and the NAFLD progression measured by liver injury.Nevertheless, the mechanisms of each CIDE proteinmediated VLDL lipidation and maturation remain to be elucidated.

Roles of CIDE Proteins in the Process of Apoptosis
Aside from the established role of diverse CIDE proteins in modulating lipid metabolism, the emerging functions of CIDE-A, CIDE-B, and CIDE-C in the process of apoptosis have been also considered in the past several decades.In details, the recent results demonstrated that diverse CIDE proteins could be considered as apoptotic proteins through a homology independent signaling way targeting the DFF-45 fragment.It is also worth-noting that the apoptotic signaling way was significantly modu-lated and the prevention was linked to diverse conditions of chronic diseases, such as tumor or systemic lupus erythematosus (SLE) [60].For instance, the apoptotic modulatory roles of diverse CIDE proteins were significantly independent via caspase stimulation.However, in contrast, other studies conducted by Liu et al. [76] found that the apoptotic modulatory role was dependent on different caspase proteins, including caspase-3 and caspase-9, and the release of cytochrome.A basic research using the mice model and demonstrated that FSP-27 could form a homodimer via the interaction with the CIDE-N and CIDE-C domains.On the other hand, the CIDE-domain including the proteins were found to the formation of head-to-tail helical oligomers, which was important for the apoptotic modulatory roles [77].Notably, concerning on the results mentioned above, both the progression of apoptotic process and the serum concentrations of CIDE-A gene expression were significantly up-regulated in skeletal muscle cell following an induced burn injury using the mouse models.On the other hand, the domain of CIDE-B has been firmly established interact with the non-structural protein 2 (NS2) in the hepatocyte which subsequently could prevent the process of apoptosis, and the region of genome including FSP-27 was mutated in different forms of human tumors.However, for some CIDE proteins, the most important process of to induce apoptosis is the co-location of the CIDE proteins fragment with the mitochondria [78].On the contrary, concerning on this notion, the results were challenged in another research conducted by Puri and the colleagues [79].In details, the authors found that FSP-27 and CIDE-A were significantly up-regulated in adipose tissue where the FSP-27 and CIDE-A were localized with LDs.On the other hand, the authors also confirmed that these proteins were also not localized with the surface of mitochondria, whereas they were correlated with LDs and that the endogenous proteins embraced a vital function in lipid catabolism and homeostasis [79].Taken together of the results mentioned above, the different CIDE proteins could be identified as LD-associated proteins and played an important role of modulating lipid metabolism.Now that it has been firmly established that diverse CIDE proteins is associated with the process of apoptosis, focus is shifting towards composing and elucidating underlying mechanisms.For instance, a research found that the fat specific protein (FSP)-27 might not localize with the surface of mitochondria in different cell types.Via overexpressing FSP-27 gene ectopically in 3T3-L1 cells isolated from the mouse model, the authors confirmed that FSP-27 could induce the process of apoptosis independent by a cellular morphology pathway.Nevertheless, there was a lack of DNA fragmentation in 3T3-L1 cells isolated from the mouse model, proposing that the presence of LDs machinery could inhibit the apoptotic interaction of FSP-27 [80].Moreover, by using the HeLa cells which were treated with oleic acid, the authors verified that the ectopic FSP-27 could stimulate the formation of LDs whereas could not promote the process of apoptosis [76].Concerning on the findings mentioned above, the functions of diverse CIDE proteins in regulating the process of apoptosis have not been fully illustrated, whereas the required factors have been partly researched.Overall, it is reasonably speculated that diverse CIDE proteins are participated in DNA formation of the apoptosis.Since the findings of considering CIDE proteins as LD-associated proteins, the future research regarding the proteins might be shifted towards researching the modulatory functions of lipid catabolism.

Conclusions and Future Perspectives
Considerable evidence demonstrated a strong correlation between genetic variability at the diverse CIDE gene loci with the formation of LDs and the pathological development of dyslipidemia and its related atherosclerotic cardiovascular diseases.Additionally, it is well-established to date that different SNPs of CIDE genes embraced unequivocal functions in regulating serum concentrations of lipid profiles and the risk of cardio-metabolic disorder diseases.All of CIDE family proteins, including CIDE-A, CIDE-B, and CIDE-C, has been currently considered as the vital modulatory factors of lipid catabolism within various cells, such as the hepatocyte and the adipocyte.The effect of CIDE protein on hepatic lipid metabolism seems to be dependent on the intra-cellular concentration of ApoB-100.Whereas, it is worth noting that conflicting results have also been reported concerning on the role of diverse CIDE proteins in regulating hepatic and serum lipid homeostasis.Thus, the precise role of the CIDE protein family in the hepatocyte remains to be determined.In the adipocyte, CIDE-C could promote the adipogenic differentiation process of pre-adipocytes both in humans and mice via, at least partly, stimulating the expression of several vital adipogenic related genes, including the C/EBPβ gene, C/EBPα gene, and FABP4 gene.By this vital method, CIDE-C could promote the increased inter-cellular concentrations of TG which resultantly facilitate the formation of mature adipocytes and up-regulate the risk of obesity and its related metabolic syndrome.
Nevertheless, despite emerging gains in understanding and knowledge of the function and molecular mechanism of diverse CIDE proteins in regulating serum or intracellular lipid metabolism and homeostasis, some questions remain to be solved in the future research.For instance, what is the precise biochemical composition and structure of the CIDE-mediated fusion complex?What are the upstream signals regulating this fusion and growth process?What is the molecular correlation between CIDE-mediated LD fusion and lipolysis?Might CIDE-mediated lipid storage play a role in other diseases such as cancer and neurodegenerative diseases?Developing a deeper understanding of the role of CIDE in controlling lipid metabolism may aid in the future discovery of novel therapies to treat obesity, fatty liver and diabetes CIDE proteins are crucial regulators of several pathways of lipid metabolism, including LD fusion and growth, lipid storage, lipid secretion and lipolysis.They are closely linked to the development of many metabolic diseases including obesity, diabetes and fatty liver disease.Recent studies have revealed that CIDE proteins are localized at LDs and enriched at the lipid droplet contact site, where they promote a unique LD fusion and growth process.CIDE proteins control LD fusion by recruiting other regulatory factors to the lipid droplet contact site (LDCS).As a conclusion, the CIDE family proteins are crucial modulators of multiple lipid metabolic pathways, such as intracellular LD fusion and growth, intra-cellular or serum lipid accumulation, and the process of lipolysis.Future research is still required to further explore and illuminate the mechanism whereby the expression of different CIDE protein facilitates the regulation of lipid metabolism and insulin sensitivity, and to examine their function as a potential treatment in cardio-metabolic disorder disease in the daily clinical practice.

Fig. 1 .
Fig. 1.Tissue-specific functions of CIDE proteins.As shown, diverse members of CIDE protein might have different role in regulating several lipid metabolic related progressions.CIDE-A and CIDE-C might induce the lipid accumulation in hepatocytes.In similar, CIDE-C could induce TG production in hepatocytes which subsequently influence the serum levels of TG.Otherwise, CIDE-A and CIDE-C could influence the lipid metabolism in adipocytes.Abbreviations: CIDE, cell death-inducing DNA fragmentation factor 45-like effector protein; TG, triglyceride; LD, lipid droplet; VLDL, very low-density lipoprotein; FSP-27, fat specific protein-27.