The exchangeable apolipoproteins in lipid metabolism and obesity

Clinica Chimica Acta 503 (2020) 128–135

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Review

The exchangeable apolipoproteins in lipid metabolism and obesity Xin Su, Daoquan Peng

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T

Department of Cardiovascular Medicine, the Second Xiangya Hospital of Central South University, Changsha, Hunan, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Exchangeable apolipoproteins Adipocyte Hepatocyte Dyslipidemia Obesity

Dyslipidemia, characterized by increased plasma levels of low-density lipoprotein cholesterol (LDL-C), very lowdensity lipoprotein cholesterol (VLDL-C), triglyceride (TG), and reduced plasma levels of high-density lipoprotein cholesterol (HDL-C), is confirmed as a hallmark of obesity and cardiovascular diseases (CVD), posing serious risks to the future health of humans. Thus, it is important to understand the molecular metabolism of dyslipidemia, which could help reduce the morbidity and mortality of obesity and CVD. Currently, several exchangeable apolipoproteins, such as apolipoprotein A1 (ApoA1), apolipoprotein A5 (ApoA5), apolipoprotein E (ApoE), and apolipoprotein C3 (ApoC3), have been verified to exert vital effects on modulating lipid metabolism and homeostasis both in plasma and in cells, which consequently affect dyslipidemia. In the present review, we summarize the findings of the effect of exchangeable apolipoproteins on affecting lipid metabolism in adipocytes and hepatocytes. Furthermore, we also provide new insights into the mechanisms by which the exchangeable apolipoproteins influence the pathogenesis of dyslipidemia and its related cardio-metabolic disorders.

1. Introduction Dyslipidemia, characterized by increased plasma levels of lowdensity lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol (VLDL-C), triglyceride (TG), and reduced plasma levels of high-density lipoprotein cholesterol (HDL-C), is considered as a key factor associated with the occurrence of a series of health problems which are always grouped together as metabolic syndrome, posing serious risks to the future health of humans [1]. Currently, it has been shown that the hallmark of dyslipidemia is dysfunctional changes in adipocytes and hepatocytes [2]. Rather than simply being a reservoir for lipid storage, the adipocyte could also function as endocrine cell, secreting multiple hormones and molecules, namely adipokines [3]. Under obese status, the hypertrophic adipocytes produce higher levels of pro-inflammatory adipokines and free fatty acid (FFA), resulting in inflammation, dyslipidemia and ectopic fat accumulation [4]. In addition, the excessive plasma LDL-C could be phagocytosed by macrophages and intruded into sub-endothelium, which is involved in the cascade to atherosclerosis, suggesting that dyslipidemia is a key risk factor of cardiovascular diseases (CVD) [5]. Consistently, the prevalence rate of atherosclerosis increases with the elevated BMI, while

patients using lipid-lowering therapy display improvement in dyslipidemia and its related cardio-metabolic disorders [6]. Thereby, it is conceivable that dyslipidemia has a significant impact on the pathogenesis of obesity and CVD. Exchangeable apolipoproteins, such as apolipoprotein A1 (ApoA1), apolipoprotein A5 (ApoA5), apolipoprotein C3 (ApoC3), and apolipoprotein E (ApoE), are predominantly synthesized and secreted by hepatocytes or adipocytes [7]. It has been shown that these exchangeable apolipoproteins possess a common unique structure as the presence of tandem 11- or 22-mer domains which forms an amphipathic α-helices structure, and the functional exchangeability of those apolipoproteins is dependent on the quantity of this structure [8]. Importantly, aside from their established extracellular role in regulating serum levels of lipid profiles, the exchangeable apolipoproteins have been also considered as modulators in lipid metabolism in adipocytes and hepatocytes. However, the underlying mechanisms are still remain unclear. In this review, we summarize the findings of the role of important exchangeable apolipoproteins in lipid metabolism and provide new insights into exchangeable apolipoproteins which function as a molecular link between adipocytes (Fig. 1) and hepatocytes (Fig. 2) in terms of obesity and cardio-metabolic disorders.

Abbreviations: LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low-density lipoprotein cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; CVD, cardiovascular diseases; ApoA1, apolipoprotein A1; ApoA1, apolipoprotein A5; ApoE, apolipoprotein E; ApoC3, apolipoprotein C3; FFA, free fatty acid; RCT, reverse cholesterol transport; HSL, hormone-sensitive lipase; UCP1, uncoupling protein 1; ACS, acute coronary syndromes ⁎ Corresponding author at: Department of Cardiovascular Medicine, the Second Xiangya Hospital of Central South University, No. 139 Middle Renmin Road, Changsha 410012, Hunan, China. E-mail address: [email protected] (D. Peng). https://doi.org/10.1016/j.cca.2020.01.015 Received 15 November 2019; Received in revised form 15 January 2020; Accepted 17 January 2020 Available online 22 January 2020 0009-8981/ © 2020 Elsevier B.V. All rights reserved.

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Fig. 1. Functions of exchangeable apolipoproteins in lipid homeostasis in adipocytes. The big arrows depict the direction of lipid flow. The thickness of arrows represents quantity and extent. In adipocytes, ApoE could maintain TG concentration by regulating lipid uptake, TG synthesis and hydrolysis as well as FFA oxidation. ApoA1, ApoA5, and ApoO could modulate TG storage in adipocytes. Under normal status, energy balance is obtained by disciplinary lipid delivery; however, under obese status, hypertrophic adipocytes release higher levels of FFA to the circulation. Abbreviation: ApoA5, apolipoprotein A5; ApoO, apolipoprotein O; ApoE, apolipoprotein E; LDs, lipid droplets; LDLR, low density lipoprotein receptor; FFA, free fatty acid.

Fig. 2. Functions of exchangeable apolipoproteins in lipid homeostasis in hepatocytes. The big arrows depict the direction of lipid flow. The thickness of arrows represents quantity and extent. In hepatocytes, ApoA1, ApoA4, ApoA5, ApoC3, and ApoE are involved in ApoB lipidation at different stages in ER and in Golgi apparatus, promoting the synthesis and secretion of VLDL. Under normal status, energy balance is obtained by disciplinary lipid delivery; however, under obese status, increased lipid uptake in liver induce the energy imbalance. Abbreviation: ApoA1, apolipoprotein A1; ApoA4, apolipoprotein A4; ApoA5, apolipoprotein A5; ApoC3, apolipoprotein C3; ApoE, apolipoprotein E; LD, lipid droplet; LLD, lumenal lipid droplet; LDLR, low density lipoprotein receptor; FFA, free fatty acid; VLDL, very low density lipoprotein.

2. Effect of exchangeable apolipoproteins on lipid metabolism in adipocytes

in modulating the energy homeostasis in adipocytes in addition to its athero-protective effect. For instance, mice with APOA1-deficiency gained more body weight and fat mass compared with wild type (WT) mice [12]. In contrast, mice with over-expressed APOA1 gene exhibited increased levels of lipolytic enzymes, such as hormone-sensitive lipase (HSL), uncoupling protein 1 (UCP1), and UCP2, reflecting enhanced TG hydrolysis and FFA oxidation within adipose tissue [13]. Moreover, the adipocytic ApoA1 could facilitate the catecholamine-induced lipolysis and modulated lipid metabolism through a direct receptor-mediated mechanism [14]. Alternatively, concerning the beneficial effect of ApoA1 on affecting energy homeostasis in adipocytes, researchers recently focused on the mimetic peptide and the mutant of ApoA1, such as D-4F and ApoA1-

2.1. ApoA1 Human APOA1/C3/A4/A5 gene clusters are located on chromosome 11q23. As the most abundant protein constituent of HDL, ApoA1 has been confirmed to have anti-atherogenic effects in past several decades. The reduced plasma level of ApoA1 has been considered as the risk factor for CVD [9], and the mechanisms whereby ApoA1 protects CVD mainly underline in ApoA1-induced reverse cholesterol transport (RCT) [10] and anti-inflammatory effect [11]. Recently, numerous studies have discovered the functions of ApoA1 129

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Thereby, the results indicate that ApoA5 modulates the intracellular lipid metabolism in adipocytes via, at least partly, affecting the expression of CIDE-C and other adipogenic-related factors [27]. On the other hand, Zheng et al. found that the internalized ApoA5 could further enhance lipolysis in adipocytes by upregulating gene expression of UCP1, revealing indirectly that ApoA5 could induce the phenotypic transformation of white adipocytes into brown adipocytes and consequently increasing the energy consumption [27]. Our recent study explored the effect of ApoA5 on human AMSCs adipogenesis. After analysis, we observed that during the adipogenic process of AMSCs, ApoA5 reduced the intracellular LDs accumulation and the TG levels; meanwhile, ApoA5 down-regulated the expression level of several important adipogenic-related factors, including C/EBP α/β, FAS, and fatty acid-binding protein 4 (FABP4). Furthermore, the suppression of adipogenesis by ApoA5 was also mediated through the inhibition of CIDE-C expression during the adipogenic process of AMSCs. However, over-expressing intracellular expression of CIDE-C could lead to the loss-of-function of ApoA5 in inhibiting AMSCs adipogenesis. Thus, we could make a conclusion that ApoA5 could inhibit human AMSCs adipogenesis through, at least partly, down-regulating CIDE-C expression [28]. The relationship of serum levels of ApoA5 and the prevalence of obesity is being elucidated, and the function of ApoA5 in pre- or mature adipocytes seems to be clarified, however, we still need more studies to explore the underlying molecular mechanisms.

Milano, respectively, and demonstrated that the both the mimetic peptide and the mutant of ApoA1 exhibited the positive effect on lipid metabolism. Ruan et al. [13] and Peterson et al. [15] showed that D-4F treatment could upregulate UCP1 gene expression via stimulation of adenosine monophosphate activated protein kinase (AMPK) signaling pathway and promote the uncoupling respiration in brown adipocytes, providing a novel insight that the activated AMPK signaling pathway could be the underlying mechanism accounting for D-4F-induced UCP1 gene upregulation. On the other hand, the ApoA1-Milano has been also confirmed to modulate lipid metabolism in adipocytes. The ApoA1Milano treated mice displayed a rapid weight loss and reduced adipose tissue mass [16]. Furthermore, the ApoA1-Milano could stimulate cholesterol efflux and increase the glycerol release which is independent on the β-adrenergic stimulation in primary rat adipocytes. Nevertheless, the pharmacological inhibition or siRNA-induced silencing of ATP-binding cassette transporter A1 (ABCA1) could not diminish the ApoA1-Milano-stimulated lipolysis, suggesting that the decreased adipose tissue mass and increased lipolysis following ApoA1Milano treatment could be explained, at least partly, by the stimulation of lipolysis independently on the canonical cAMP signaling pathway [14]. Given that ApoA1 reduces the weight of adipose tissue in mice, we could speculate that ApoA1 also interacts with the adipose-derived mesenchymal stem cells (AMSCs) that are abundant in adipose tissue. Notably, the important role of ApoA1 in AMSCs has gained appreciation. It is worth noting that the expression of β-ATPase increased remarkably during the 3T3-L1 pre-adipocytes adipogenesis [17], which could further identify the α-helices structure of ApoA1 and combine to ApoA1 [18,19]. Thus, we could infer from these results that the combination of ApoA1 and β-ATPase could subsequently influence the preadipocytes adipogenesis process. However, due to the limited studies, we could not elucidate the function of ApoA1 in affecting lipid metabolism and adipogenesis of pre-adipocytes. Further studies are still needed.

2.3. ApoE ApoE is predominantly synthesized in liver and is considered as a surface component of chylomicrons (CM), VLDL, and HDL [29]. It has been pointed out that ApoE is highly expressed in adipocytes during the adipogenesis and has close relationship with adipose tissue and body fat mass [30]. Indeed, the human AMSCs with APOE-knockdown has been verified to have markedly less intracellular TG concentration compared to that in control cells. Moreover, the gene expression levels of multiple adipogenic related markers, such as adiponectin (ADIPOQ) gene and fatty acid binding protein 4 (FABP4) gene, were reduced during the process of adipogenesis in APOE-deficient AMSCs [31]. Thus, the important role of ApoE in regulating lipid metabolism in adipocytes has gain much appreciation. Early in 1991, Zechner et al. firstly observed that the adipocytederived ApoE was exactly paralleled with LDs formation during adipogenesis [32]. Afterward, Huang et al. also unraveled that the epididymal WAT isolated from the APOE-knockout mice with high fat diet (HFD) presented lower intracellular TG concentration compared with those in WT mice, implying a vital role for ApoE in affecting the adipocytic lipid metabolism [33]. Additionally, Takazawa et al. investigated the function of peroxisome proliferator-activated receptor (PPAR) gamma agonist, such as rosiglitazone, on regulation of ApoE and its receptor, VLDL receptor (VLDLR), expression both in WAT of obese mice and in cultured adipocytes. As described, rosiglitazone increased the expression of VLDLR in cultured adipocytes. However, in APOE-deficient mice, rosiglitazone ameliorated insulin sensitivity, signifying that rosiglitazone directly increases VLDLR expression and consequently enhances the ApoE-VLDLR-dependent lipid accumulation in adipocytes [34]. Several mechanisms could account for the role of ApoE on lipid metabolism in adipocytes. One of the viewpoints concerns the accumulation of TG which occurs in response to the interaction with TG-rich lipoproteins (TRLs). It has been shown that the levels of ApoE-recognizing receptors, such as VLDLR and LRP, were decreased or redistributed away from the membrane surface of adipocytes in APOEdeficient mice, which leads to an impaired acquisition of TG from TRLs in adipocytes [35]. Given that reduced VLDLR could inhibit TG storage in adipocytes and protect mice from obesity, we could infer from these results that ApoE exhibited the regulatory function in TG storage in adipocytes potentially through the impaired receptor-mediated

2.2. ApoA5 ApoA5 is a novel apolipoprotein which has been shown as a potent regulator of plasma TG metabolism [20]. Increasing evidence indicates that the single nucleotide polymorphisms (SNPs) of APOA5 gene in human, such as APOA5 −1131T > C and c.56C > G, not only alter plasma TG levels but also influence the prevalence rate of obesity [21,22]. With the in-depth research, the subjects carrying the homozygous of −1131T > C major allele presented an increased BMI, significantly higher serum levels of TG and lower serum levels of HDLC, compared with those in the non-carriers. Additionally, the plasma levels of ApoA5 were lower in obese patients from different countries and were verified to be inversely correlated with BMI [23]. Whereas a portion of hepatic-derived ApoA5 is secreted into plasma which facilitates lipoprotein lipase (LPL)-mediated TG hydrolysis, another portion of ApoA5 protein is unraveled to be co-localized with lipid droplets (LDs) within adipocytes [1]. Since adipocytes are the largest storage depot for energy in form of TG within LDs, we speculate that ApoA5 may also target to adipocytes. Consistently, Zheng et al. confirmed that ApoA5 could be internalized into human mature adipocytes and be co-localized with perilipin through binding to LDL receptor (LDLR) family members such as LDLR-related protein 1 (LRP1); meanwhile, the internalized ApoA5 could significantly reduce the intracellular TG concentration. Additionally, they also demonstrated that ApoA5 suppressed the expression level of cell death-inducing DNA fragmentation factor-α-like effector C (CIDE-C) and several important adipogenic-related factor, including CCAAT/enhancer binding protein α and β (C/EBP α/β), fatty acid synthetase (FAS) [24]. As verified in previous studies, CIDE-C is a new LDs-associated protein which participates in lipid accumulation in white adipose tissue (WAT) by promoting the formation of unilocular LDs and restricting lipolysis [25,26]. 130

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F442A pre-adipocytes; meanwhile, with confocal immunofluorescence microscopy, the authors also revealed that the intracellular ApoO was co-localized with perilipins in the LDs, showing a close relationship of ApoO with lipid metabolism in adipocytes. On the other hand, in the human diabetic cardiomyocytes, the expression level of ApoO gene is also significantly up-regulated [52]. These findings provided the elementary mechanisms whereby ApoO modulated the lipid homeostasis in adipocytes, however, we still need further investigations to confirm the detailed mechanisms.

endocytosis pathway induced by absence of VLDLR. Interestingly, another possible mechanism implicating the role of ApoE in regulating lipid metabolism in adipocytes focus on caveolin-1, which is encoded by CAV1 gene and is a major functional protein of caveolae specialized in membrane invagination and associated to LDs in adipocyte. The expression level of CAV1 gene is dramatically elevated during adipogenesis [36] and is associated with the formation of LDs in 3T3-L1 adipocytes [37]. Moreover, the secretion of caveolin-1 is also evident in adipocytes and is substantially promoted in HFD-fed mice. Under obese status, the hypertrophied adipocytes could produce excessive caveolin-1, suggesting a close relationship between adipocytes and caveolin-1 [38]. Currently, evidence showed that ApoE was colocalized with caveolin-1 at the plasma membrane of adipocytes during adipogenesis [39]. However, the expression of caveolin-1 were significantly suppressed in adipocytes isolated from APOE-null mice, which further caused the impaired intracellular transportation of FFA and the resultant excessive TG synthesis [35,40,41]. Notably, these defects could be reversed by increasing CAV1 gene expression [35]. Additionally, functions of ApoE are shown to be isoforms-specificity in humans. There are three gene alleles, APOE-ε2 (Cys-112 and Cys158) gene, APOE-ε3 (Cys-112 and Arg-158) gene, and APOE-ε4 (Arg112 and Arg-158) gene, which encodes the isoform namely ApoE2, ApoE3, and ApoE4 that has different binding inclination for corresponding receptors, respectively [42,43]. To date, several epidemiological studies pointed out an association of ApoE isoforms with the prevalence rate of cardio-metabolic disorders. For instance, Zeljko et al. confirmed that the APOE-ε2 gene allele was positively associated with BMI [44]. Likewise, Afroze et al. investigated the association between ApoE isoforms and atherosclerosis in Kashmiri population. They indicated that the patients carrying APOE-ε4 gene allele had significantly higher serum levels of TC and LDL-C, indicating a significant association of ApoE ε4 allele with the risk of atherosclerosis [45]. With indepth investigations, mice carrying APOE-ε2 gene allele in adipocytes presented an increase in ApoE synthesis with increased size of gonadal fat mass. In isolated adipocytes, the ApoE2 isoform could produce lipogenesis and increase TG hydrolysis [46]. On the other hand, mice with APOE-ε4 gene allele presented a lower BMI and a severely blunted adipogenesis [47]. These results shed light on the impact of ApoE on the metabolic disorders which could be dependent on the specific isoforms of ApoE in humans.

3. Effect of exchangeable apolipoproteins on lipid metabolism in hepatocytes 3.1. ApoA1 In addition to its anti-atherogenic role, studies have pointed out that ApoA1 could reduce hepatic lipid accumulation and inhibit the inflammatory process. The BEL-7402 hepatocytes with over-expressed APOA1 gene presented increased cholesterol efflux and decreased TG concentration. However, this effect could be reversed by siRNA-induced silencing ABCA1 gene [53]. Another study demonstrated that the BEL7402 hepatocytes with over-expressed APOA1 gene manifested the decreased reactive oxygen species (ROS) as well as the cyclooxygenase2, a vital promoter of intracellular inflammation, providing a novel mechanism whereby ApoA1 inhibits cellular inflammation [54]. On the other hand, using the QSG-7701 hepatocytes with over-expressed APOA1 gene and ABCA1 gene, Ma et al. pointed out that in addition to the results mentioned above, ApoA1 also caused reduced 27-hydroxycholesterol and FAS levels, indicating another potential mechanism by which ApoA1 affects hepatic lipid accumulation [55]. To date, it has been shown that the excessive intracellular lipid storage could induce endoplasmic reticulum (ER) stress in hepatocytes, which could further dysregulate the endogenous sterol response pathway, leading to increased hepatic uptake of fatty acids with ensuing TG-synthesis and promoting the pathological development of liver diseases, such as non-alcohol fatty liver diseases (NAFLD) and nonalcoholic steatohepatitis (NASH) [56]. Given that ApoA1 could modulate intracellular TG concentration, the effect of ApoA1 on ER stress has been given substantial attention in recent years. Importantly, the first experimental evidence concerning the relationship between ER stress and hepatic ApoA1 was provided by Naem et al. who demonstrated that ER stress could decrease ApoA1 in HepG2 cells [57], suggesting that the function of ApoA1 in affecting lipid accumulation and metabolism in hepatocytes could be influenced by ER stress. Consistent with this hypothesis, Guo et al. recently unraveled that the HepG2 cells with over-expressed APOA1 gene presented lower TG concentration and decreased levels of several lipogenic related genes such as FAS gene [58]. In addition, some of the genes related to ER stress-induced apoptosis, such as glucose regulated protein 78 (GRP78) gene and sterol regulatory element binding protein-1 (SREBP-1) gene, were also affected by the expression of ApoA1 [58], indicating that ApoA1 could reduce hepatic lipid storage by suppressing ER stress.

2.4. ApoO ApoO is another novel apolipoprotein containing a 198-amino acid protein and was first discovered in the dog’s hearts [48]. The mean plasma levels of ApoO in healthy individuals is around 2.21 µg/ml, whereas it is around 4.94 µg/ml in patients with acute coronary syndromes (ACS) [49]. Thus, the elevated serum levels of ApoO is now considered as an independent risk predictor of ACS. With in-depth research, ApoO is found to contain a chondroitin sulfate chain which mainly exists in HDL particles. Mice with over-expressed APOO gene by recombinant adenovirus presented a substantial increase in plasma ApoO levels as well as in the diameters of HDL particles; however, the plasma levels of TG, TC, HDL-C, and the HDL functionality did not change significantly [50], suggesting that the alteration of serum levels of ApoO showed no functions in modulating plasma lipid profiles. Recently, the biological role of ApoO is increasingly illuminated. An SNP of APOO gene in chromosome Xp22.11 is proposed to be involved in increasing the suicidal ideation formation during anti-depressant treatment of depression patient [51], revealing a function of ApoO within the local tissues in central nervous system of humans. Additionally, increasing evidence reveals that ApoO could exert vital effect on modulating lipid metabolism and cardio-metabolic disorders in mammals since it is expressed at various levels in adipocytes, hepatocytes, and cardiomyocytes. Lamant et al. found that the ApoO levels were increased significantly during adipogenesis of 3T3-

3.2. ApoA4 As mentioned above, the human APOA1/C3/A4/A5 gene clusters are located on chromosome 11q23. Earlier animal and cell culture studies established that APOC3 enhancer acted as a common regulatory sequence to direct hepatic and intestinal APOA4 gene expression [59]. With the in-depth investigation, ApoA4 has been verified as a lipidbinding protein, which is primarily synthesized in the enterocytes of small intestine, packaged into CM, and secreted into intestinal lymph circulation as a major part of HDL during fat absorption [60]. Early in 2006, Klos et al. have already identified that the common SNPs in APOA1/C3/A4/A5 gene loci played a vital role in the RCT pathway and resultantly influenced the plasma levels of TC, HDL-C, LDL-C, and TG 131

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expression, whereas the diameter of VLDL2 particle could not be affected by ApoA5 [74]. Therefore, the ApoA5-involved decreased of hepatic TG secretion could be considered to be induced by the attenuation of second-step maturation of VLDL particles. More recently, Lin et al. demonstrated that metformin could dosedependently ameliorate hepatic steatosis in ob/ob mice, with a reduction in TG levels and ApoA5 expression through increasing AMPK signaling pathway [75]. Taken together, these results indicated that ApoA5 play an essential role in regulating the intracellular TG metabolism in hepatocytes.

[61]. Afterwards, multiple functions, including anti-inflammatory response [62] and regulating lipid metabolism [63], have been ascribed to the level of ApoA4 expression in different experiments. Mice with APOA4-deficiency exhibited a higher plasma TG concentration and greater inflammatory response, which could be improved via exogenous administration of ApoA4 protein [64]. By contrast, mice with over-expressed APOA4 gene presented a reduced secretion of pro-inflammatory cytokines after LPS administration, revealing an important role of ApoA4 in modulating systemic inflammation and circulating TG levels [65]. Although increasing evidence supports a role of apoA4 in plasma lipid metabolism, its function in hepatic lipid metabolism still remains unclarified. Notably, Gallagher et al. have proposed a novel potential mechanism of protein-protein interaction between ApoA4 and ApoB which could consequently facilitate VLDL formation [66]. Consistent with this notion, Weinberg et al. used the McA-RH7777 rat hepatoma cells cotransfecting with human APOA4 gene and showed that ApoA4 could induce an approximately 20–35% increase in TG secretion accompanied by an approximately 55% increase in secretion of ApoB-100 containing VLDL particles. This effect was further confirmed to be independent of microsomal triglyceride transfer protein (MTP) activity [67]. Likewise, VerHague et al. also discovered a positive linear correlation between hepatic TG content and APOA4 mRNA abundance in mice. Furthermore, the APOA4-deficient mice displayed an approximately 24% reduction in hepatic TG secretion rate and an approximately 33% decrease in hepatic VLDL particles secretion. In contrast, mice infected with a recombinant human ApoA4 protein presented increased hepatic TG secretion rate and reduced hepatic TG content, indicating that the hepatic steatosis in mice could lead to ApoA4 expression in hepatocytes, which in turn facilitates the VLDL particles formation and decreases the hepatic lipid accumulation [68]. Similar results could be observed in the study conducted by Cheng and colleagues [69]. Herein, we could make a conclusion that ApoA4 could regulate the VLDL particle formation and secretion by influencing the nascent ApoB-containing particles, thereby enhancing TG secretion and modulating the plasma lipid levels.

3.4. ApoC3 ApoC3 is a small protein with 79 amino acids. Ever since its discovery, ApoC3 has been considered to be essential in lipid metabolism. A major proportion of ApoC3 is produced in liver while a minor proportion of ApoC3 is synthesized in intestine [76]. Importantly, it has been shown that reduced plasma ApoC3 levels were closely associated with decreased risk of CVD. Recently, a nonsense mutation of APOC3 gene, R19X, was verified in a large-scale study [77] and was shown to be associated with an approximately 50% reduction of serum ApoC3 levels. Subjects carrying the rare variant R19X had lower prevalence of CVD [78]. Moreover, the effect of R19X and other three rare variants, two splice site mutations (IVS2 + 1G > A; IVS3 + 1G > T) and one missense mutation (A43T) in APOC3 gene were also verified. The heterozygous carriers of at least one of those four mutations presented reduced circulating levels of ApoC3 compared to those in non-carriers, and the risk of CVD in subjects carrying any rare APOC3 gene mutation was significantly lower than non-carriers [77]. The similar conclusions were also demonstrated in a cohort of 75,725 participants, which showed that the prevalence of ischemic heart disease (IHD) was decreased obviously in heterozygotes for loss-of-function mutations in APOC3 gene (R19X or A43T or IVS2 + 1 G > A), with corresponding risk reductions of 36% [77]. Historically, the expression of APOC3 gene could be regulated by hepatocyte nuclear factor 4 alpha (HNF4α) gene and chicken ovalbumin upstream-transcription factor II (COUP-TFII) gene [79]. Currently, a study showed that a natural compound, alantolactone (ALA), could inhibit the promoter activity and the expression levels of APOC3 gene in L02 cells via inhibiting the tyrosine phosphorylation (Tyr705pho) of STAT3. Consistent with this notion, over-expression of STAT3 gene could upregulate the APOC3 gene expression, which further increased the intracellular TG contents in L02 cells. These results provide novel regulators of APOC3 gene and the resultant influence of hepatic lipid accumulation [80]. In addition to the APOC3 gene, several studies have focused on the relationship between ApoC3 protein with the lipid metabolism in hepatocytes. For instance, Sundaram et al. found that under lipid-rich condition, McA-RH7777 cells with over-expressed APOC3 gene presented enhanced secretion of VLDL1 particles; meanwhile, an increase in de novo lipogenesis (DNL) was also observed. The hepatocytes isolated from mice with over-expressed APOC3 gene displayed increased lipogenic genes expression, indicating that ApoC3 could stimulate VLDL1 production in hepatocytes through enhanced DNL [81]. Furthermore, Qin et al. demonstrated that the APOC3-deficient mice infected with recombinant human ApoC3 protein presented increased VLDL1 production after fed with a HFD, signifying that exogenous supplement of ApoC3 could also induce the production of VLDL1 and increase the intracellular TG concentration. Mechanically, with metabolic labelling experiments, the authors revealed that ApoC3 could regulate the late stage of VLDL maturation by facilitating the formation of microsomal associated lumenal LDs [82]. Similar results could be observed in the study conducted by Yao et al. who found that feeding APOC3-deficient mice with palm oil gavage failed to stimulate VLDL1 synthesis; however, reconstitution of APOC3 gene expression led to a robust production of VLDL1 particles [82]. Intriguingly, Yao et al. also

3.3. ApoA5 ApoA5, secreted solely by the liver, is a low abundance protein which could strongly influence the plasma TG levels. The relationship of the APOA5 gene SNPs and the alterations of serum lipid profiles was mentioned above. Due to the technological advances, major breakthroughs have been made to explain the association between ApoA5 and lipid metabolism in hepatocytes, which is also of significance to modulate dyslipidemia and its related cardio-metabolic disorders. It has been well demonstrated that ApoA5 could be largely retained in hepatocytes and be associated with cytosolic LDs [70], suggesting that ApoA5 modulated intracellular TG storage pools. Consistent with this hypothesis, Shu et al. showed that the hepatocytes isolated from the APOA5 gene transgenic mice exhibited higher levels of TG [70]. Additionally, Ress et al. found that in obese subjects, the hepatic APOA5 gene expression decreased significantly after treatments of weight loss and improvements in hepatic steatosis. However, silencing APOA5 gene expression in HepG2 cells could induce lower intracellular TG content [71], signifying an important intracellular regulatory role of ApoA5 in lipid accumulation. Currently, though the mechanisms whereby ApoA5 affects hepatic TG storage and metabolism have not been fully elucidated, several studies have provided evidence for a modulatory role of ApoA5 in hepatic VLDL assembly and secretion. Either the McA-RH7777 cells or the HepG2 cells with over-expressed APOA5 gene displayed reduced TG secretion without obvious alterations in ApoB secretion [72,73], suggesting that the intrahepatic ApoA5 protein impaired ApoB lipidation and further suppressed VLDL synthesis. Consistently, the diameter of VLDL1 particle reduced obviously under the status of APOA5 gene over132

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not be simply explained by different cell types or animal models in the experiments. Of note, there are yet several possible mechanisms which account for the functional heterogeneity. One of the viewpoint is that autophagy, which is considered as a pathway process of intracellular component degradation within lysosomes, has been shown to have an opposite effect on modulating TG metabolism in adipocytes and hepatocytes. For instance, autophagy is required for cell differentiation and LDs formation during adipogenesis [93,94]; however, in hepatocytes, autophagy inhibit the TG storage in cytoplasmic LDs via the specific lipid-autophagy pathway [95]. Furthermore, the activation of autophagy could facilitate VLDL production in hepatocytes. Blockage of autophagy could decrease VLDL secretion and synergistically increase hepatic TG concentration [96]. Thus, we could infer from these results that the converse effects on lipid homeostasis in adipocytes and hepatocytes could induce the functional heterogeneity. Secondly, the different sources of a specific apolipoprotein could present multiple functions in lipid metabolism. For example, experiments concerning the effect of ApoE in adipocytes may overwhelmingly pay attention to the endogenously expressed ApoE. By contrast, since the triglyceride-rich lipoprotein (TRL)-derived ApoE could escape from degradation in hepatocytes, experiments concerning the effect of hepatic ApoE may focus on the exogenous ApoE. In addition, the generation of different apolipoproteins in different experiments may also cause the function heterogeneity. In terms of ApoA5, the recombinant ApoA5 protein used by Zheng et al. was generated by recombinant technology and purified from virus inclusions, while the ApoA5 protein used in other experiments was produced in eukaryotic cells. These different source may induce the alterations in bioactive properties of ApoA5 and further influence its regulatory effect in adipocytes. It is worth noting that there are multiple genetic diversities between adipocytes and hepatocytes which could contribute to the heterogeneity. As known, adipocytes provide the largest depot for TG, whereas hepatocytes could not serve as a depot for lipid. Thus, the concentration of hepatic TG is lower under physiological status. This discrepancy in TG levels may induce the lipid metabolism in hepatocytes different from that in adipocytes. Actually, several important lipogenic related genes, such as SREBP-1c, were upregulated only during hepatocyte differentiation but not during adipogenesis. Taken together, the genetic diversities between adipocytes and hepatocytes may be the potential mechanism of functional heterogeneity of exchangeable apolipoproteins.

demonstrated that the subjects with two loss-of-function APOC3 variants, Ala23Thr and Lys58Glu, presented significantly lower TG concentration compared with the control individuals. The hepatocytes isolated from those patients showed entirely abolished function of ApoC3 in promoting VLDL1 secretion [83]. 3.5. ApoE It has been firstly shown since 20th century that the McA-RH7777 cells with over-expressed APOE gene displayed activated lipogenesis, however, no obvious effect on HDL particles was observed [84,85]. Afterwards, Mensenkamp et al. demonstrated that the hepatocytes isolated from APOE-deficient mice exhibited excessive accumulation of TG in ER and ER-derived vesicles, while no aberrant TG storage were observed in Golgi apparatus [86], indicating indirectly that ApoE may influence the early stage of VLDL assembly in ER rather than VLDL maturation during transportation from ER into Golgi apparatus. These findings were replicated in the experiment conducted by Gusarova and colleagues who revealed that the VLDL formation in the Golgi apparatus was not dependent on ApoE [87]. Furthermore, the APOE-null mice showed a decreased in VLDL production and the average size of VLDL particles in hepatocytes [88]. Recently, a novel mice model carrying the homozygous floxed APOE gene alleles as tissue-specific APOE-deficiency were used to discover the functions of hepatocyte-derived ApoE separately. Mice lacking ApoE only in hepatocytes (APOEΔHep) exhibited higher body weights with a Western type diet. Additionally, the extent of hepatic steatosis and the markers of inflammation were more pronounced in APOEΔHep mice. Dyslipidemia induced by impaired uptake of VLDL particles and reduced TRLs remnant clearance was also observed in APOEΔHep mice [89]. These findings, in conclusion, established the role of ApoE in the regulation of hepatic VLDL production. The function of ApoE on facilitating hepatic VLDL-TG production and secretion by an isoform-independent manner has been given substantial attention in recent years. However, limited studies demonstrated the effect of ApoE isoforms on VLDL secretion. Tsukamoto and colleagues demonstrated that the APOE-knockout mice with liver-specific over-expressed APOE-ε2 gene, APOE-ε3 gene, and APOE-ε4 gene could increase VLDL-TG secretion [90]. However, using hepatocytes isolated from mice with over-expressed those three genes, Mensenkamp et al. unraveled that only the APOE-ε3 gene could induce increased VLDL particle secretion, and the APOE-ε3-leiden mice also presented the most severe hypertriglyceridemia [86]. Moreover, the APOE-ε4 gene was confirmed to induce severe dyslipidemia and atherosclerosis in streptozotocin-induced diabetes mice. Livers isolated from these mice with over-expressed APOE-ε4 gene presented reduced FFA oxidation and excessive TG accumulation [91]. Nevertheless, it is necessary to conduct more studies to further clarify whether the regulatory effect of ApoE on TG metabolism in hepatocytes is isoform-specific.

5. Conclusions Growing evidence proposes a vital intracellular function of exchangeable apolipoproteins in both adipocytes and hepatocytes, which are two major lipogenic cell types participating in lipid homeostasis in humans. Intracellular exchangeable apolipoproteins not only modulate the lipid metabolism but also induce the crosstalk of cardio-metabolic disorders between adipose tissues and livers, resultantly affecting the pathogenesis of obesity, steatosis, and CVD. Furthermore, the genetic diversities and the different sources of exchangeable apolipoproteins also induce the alterations of bioactive properties and lead to the functional heterogeneity of different apolipoproteins.

4. Mechanisms of functional heterogeneity in exchangeable apolipoproteins The regulatory effects of different exchangeable apolipoproteins on lipid metabolism in adipose tissues are seemingly contradictory to livers, and this functional heterogeneity is predominantly found in ApoE and ApoA5. For instance, ApoE could facilitate the pre-adipocytes adipogenesis and TG accumulation in adipocytes [92], while it could promote TG secretion in hepatocytes [33]. On the other hand, ApoA5 significantly decreased TG storage in human mature adipocytes through downregulating the expression levels of CIDE-C and other important adipogenic-related factors [24], whereas it could attenuate TG secretion and induce excessive TG accumulation in hepatocytes, inducing steatohepatitis and fatty liver disease [20]. The mechanism of this functional heterogeneity is barely expounded in current studies and could

Author contributions X.S. and D.Q.P. contributed to the study design; X.S. wrote the manuscript. All authors reviewed drafts and approved the final version of the manuscript. Ethical approval This article does not contain any studies with human participants performed by any of the authors. 133

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Funding

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