SREBP Activation by Antipsychotic- and Antidepressant-Drugs in Cultured Human Liver Cells: Relevance for Metabolic Side-Effects?

Despite the critical roles of intracellular lipid droplets (LDs) in lipid storage and metabolism, little is known about the molecular mechanisms of their functions. Several protein components associated with the surface of LDs have been identified. A major one is perilipin in adipocytes and steroidogenic cells, whereas ADRP in most other cell types. They are loosely grouped as a small protein family sharing a common N-terminal motif, called the PAT domain. Perilipin regulates the breakdown of triacylglycerol in LDs via its phosphorylation. ADRP is characterized as a fatty acid binding protein and involved in lipid uptake and LD formation. For examining the functions of perilipin and ADRP at the molecular level, we performed yeast two-hybrid screening in this study, to find their functional partners. We identified CGI-58, a product of the causal gene of Chanarin-Dorfman syndrome (CDS), as an interactor for both perilipin and ADRP. Specific interaction between CGI-58 and perilipin was confirmed in a GST-pulldown assay and supported by fluorescence microscopic analyses. We further demonstrated that CGI-58 is principally located at the surface of LDs in 3T3-L1 cells, together with perilipin, and its expression is upregulated upon stimulation for adipocyte differentiation. Other than CGI-58, we also identified in yeast two-hybrid screening HSP86 and D52 tumor proteins as binding partners of perilipin, and IRG-47 of ADRP. These factors might be cooperated with perilipin and ADRP, and hence involved in membrane dynamics of LDs as well as the regulation of lipolysis on the surface of LDs.     

CGI-58在动物脂类代谢中的作用

细胞中脂滴(Lipid droplets, LDs)表面存在多个调控脂肪储存和分解的蛋白, 这些蛋白对机体的脂肪代谢起着很重要的调控作用。CGI-58(Comparative gene identification-58)分布在LDs表面, 属于α/β水解酶折叠家族, 是脂肪甘油三酯脂肪酶(Adipose triglyceride lipase, ATGL)和依赖酰基辅酶A溶血磷脂酸酰基转移酶(Lysophosphatidic acid acyltransferase, LPAAT)的激活剂。在脂肪分解过程中, CGI-58结合PAT蛋白家族成员之一的脂滴包被蛋白(Perlipin)和ATGL, 促进脂肪分解, 同时CGI-58对ATGL的激活功能受脂滴包被蛋白家族成员间蛋白质与蛋白质相互作用的影响。文章结合国内外研究热点, 针对CGI-58在动物脂类代谢中的作用进行了综述。     

CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.     

Perilipin A mediates the reversible binding of CGI-58 to lipid droplets in 3T3-L1 adipocytes

Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the alpha/beta-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin A-coated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.     

Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome

Adipose triglyceride lipase (ATGL) was recently identified as an important triacylglycerol (TG) hydrolase promoting the catabolism of stored fat in adipose and nonadipose tissues. We now demonstrate that efficient ATGL enzyme activity requires activation by CGI-58. Mutations in the human CGI-58 gene are associated with Chanarin-Dorfman Syndrome (CDS), a rare genetic disease where TG accumulates excessively in multiple tissues. CGI-58 interacts with ATGL, stimulating its TG hydrolase activity up to 20-fold. Alleles of CGI-58 carrying point mutations associated with CDS fail to activate ATGL. Moreover, CGI-58/ATGL coexpression attenuates lipid accumulation in COS-7 cells. Antisense RNA-mediated reduction of CGI-58 expression in 3T3-L1 adipocytes inhibits TG mobilization. Finally, expression of functional CGI-58 in CDS fibroblasts restores lipolysis and reverses the abnormal TG accumulation typical for CDS. These data establish an important biochemical function for CGI-58 in the lipolytic degradation of fat, implicating this lipolysis activator in the pathogenesis of CDS.     

Structure of a CGI-58 Motif Provides the Molecular Basis of Lipid Droplet Anchoring

Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10–31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp²¹ and Trp²⁵) and right (harboring Trp²⁹) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp²¹ and Trp²⁵ and two adjacent leucines. Trp²⁹ serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm.     

CGI-58/ABHD5 is phosphorylated on Ser239 by protein kinase A: control of subcellular localization

CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS²³⁹S²⁴⁰, we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno­blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.     

Chanarin–Dorfman syndrome: Deficiency in CGI-58, a lipid droplet-bound coactivator of lipase

Chanarin–Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the α/β hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.     

Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.     

ADRP is dissociated from lipid droplets by ARF1-dependent mechanism

Adipocyte differentiation-related protein (ADRP) is a member of PAT proteins existing in lipid droplets (LDs). By yeast two-hybrid screening, we identified ADP-ribosylation factor 1 (ARF1) as a binding partner of ADRP. The interaction of ADRP and ARF1 was verified by GST pull-down and co-immunoprecipitation experiments. Interestingly, ADRP precipitated the GDP-bound ARF1 preferentially to the GTP-bound ARF1. Consistent with this, either brefeldin A (BFA), a fungal metabolite to inhibit ARF-GEF, or a dominant-negative mutant of ARF1 caused dissociation of ADRP from LD. On the other hand, overexpression of wild-type ARF1 did not promote the ADRP dissociation or new LD formation. By using deletion mutants, a central domain of ADRP, which is dispensable for LD binding, was shown to bind to ARF1. The present study showed that the GDP-bound ARF1 induces dissociation of ADRP from the LD surface, and that LD is a target of BFA action.     

CGI-58/ABHD5 is a coenzyme A-dependent lysophosphatidic acid acyltransferase

Mutations in human CGI-58/ABHD5 cause Chanarin-Dorfman syndrome (CDS), characterized by excessive storage of triacylglycerol in tissues. CGI-58 is an α/β-hydrolase fold enzyme expressed in all vertebrates. The carboxyl terminus includes a highly conserved consensus sequence (HXXXXD) for acyltransferase activity. Mouse CGI-58 was expressed in Escherichia coli as a fusion protein with two amino terminal 6-histidine tags. Recombinant CGI-58 displayed acyl-CoA-dependent acyltransferase activity to lysophosphatidic acid, but not to other lysophospholipid or neutral glycerolipid acceptors. Production of phosphatidic acid increased with time and increasing concentrations of recombinant CGI-58 and was optimal between pH 7.0 and 8.5. The enzyme showed saturation kinetics with respect to 1-oleoyl-lysophosphatidic acid and oleoyl-CoA and preference for arachidonoyl-CoA and oleoyl-CoA. The enzyme showed slight preference for 1-oleoyl lysophosphatidic acid over 1-palmitoyl, 1-stearoyl, or 1-arachidonoyl lysophosphatidic acid. Recombinant CGI-58 showed intrinsic fluorescence for tryptophan that was quenched by the addition of 1-oleoyl-lysophosphatidic acid, oleoyl-CoA, arachidonoyl-CoA, and palmitoyl-CoA, but not by lysophosphatidyl choline. Expression of CGI-58 in fibroblasts from humans with CDS increased the incorporation of radiolabeled fatty acids released from the lipolysis of stored triacylglycerols into phospholipids. CGI-58 is a CoA-dependent lysophosphatidic acid acyltransferase that channels fatty acids released from the hydrolysis of stored triacylglycerols into phospholipids.     

Differential control of ATGL-mediated lipid droplet degradation by CGI-58 and G0S2

Lipid droplets (LDs) are intracellular storage sites for triacylglyerols (TAGs) and steryl esters, and play essential roles in energy metabolism and membrane biosynthesis. Adipose triglyceride lipase (ATGL) is the key enzyme for TAG hydrolysis (lipolysis) in adipocytes and LD degradation in nonadipocyte cells. Lipase activity of ATGL in vivo largely depends on its C-terminal sequence as well as coactivation by CGI-58. Here we demonstrate that the C-terminal hydrophobic domain in ATGL is required for LD targeting and CGI-58-independent LD degradation. Overexpression of wild type ATGL causes a dramatic decrease in LD size and number, whereas a mutant lacking the hydrophobic domain fails to localize to LDs and to affect their morphology. Interestingly, coexpression of CGI-58 is able to promote LD turnover mediated by this ATGL mutant. Recently we have discovered that G0S2 acts as an inhibitor of ATGL activity and ATGL-mediated lipolysis. Here we show that G0S2 binds to ATGL irrelevantly of its activity state or the presence of CGI-58. In G0S2-expressing cells, the combined expression of CGI-58 and ATGL is incapable of stimulating LD turnover. We propose that CGI-58 and G0S2 regulate ATGL via non-competing mechanisms.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor alpha

Cytosolic lipid droplets (LDs) are multifunctional organelles that exist in all types of eukaryotic cells and control lipid homeostasis. In mammalian cells LDs contain a class of proteins in their surface layers that share a homologous sequence called the PAT domain, including perilipin, adipose differentiation-related protein (ADRP), a tail-interacting protein of 47 kDa (TIP47), and S3-12, which are distributed tissue- or cell type-selectively. Expression in some cases is regulated by peroxisome proliferator-activated receptors (PPARs). In this study we identified a new PAT family member named MLDP (myocardial LD protein) in a murine cDNA data base and showed the mRNA and protein to be highly enriched in the heart and also expressed at lower levels in the liver and adrenals. Upon subcellular fractionation, a substantial amount of MLDP was detected in the top fraction enriched with LDs. Furthermore, overexpressed MLDP tagged with green fluorescent protein accumulated at the surfaces of LDs and co-localized with perilipin and ADRP. Deletion analysis demonstrated the N-terminal region containing a PAT-1 domain and the following 33-mer domain to be required for targeting of MLDP to LDs. MLDP was found to be up-regulated at both mRNA and protein levels in the heart and liver by a selective ligand for PPARalpha, Wy14,643, but not in PPARalpha knock-out mice. MLDP expression was also increased upon fasting in parallel with ADRP. These results indicate that MLDP is a bona fide new PAT family member localized in LDs. Its expression depends on the physiological conditions and the action of PPARalpha.     

Reassessing the Potential Activities of Plant CGI-58 Protein

Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.     

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199-1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.     

Identification of a novel splicing isoform of murine CGI-58

The comparative gene identification-58 (CGI-58) gene, mutations of which are linked to Chanarin-Dorfman syndrome, encodes a protein of the α/β hydrolase domain subfamily. We report here a new alternative splicing isoform of the murine CGI-58 gene, termed mCGI-58S. Sequence comparison indicates the lack of second and third exons in this cDNA variant. While the full-length protein displayed perilipin-dependent localization to lipid droplets, mCGI-58S showed a predominant cytoplasmic staining when expressed in cells. mCGI-58S was incapable of activating adipose triglyceride lipase but retained the capacity to acylate lysophosphatidic acid. Overexpression of mCGI-58S failed to promote lipid droplet turnover and loss of intracellular triacylglycerols. These results suggest that this splicing event may be involved in the regulation of lipid homeostasis.     

比较基因识别-58

比较基因识别-58（comparative gene identification-58,CGI-58）属于脂肪酶家族,是脂肪性甘油三酯脂肪酶的激活蛋白,在调控脂肪代谢方面发挥着至关重要的作用,也是调控角质形成细胞的分化过程的重要分子。另外,CGI-58还具有溶血磷脂酸酰基转移酶活性,可将溶血磷脂酸酰化成磷脂酸。若CGI-58的基因发生突变,会引发一种隐性遗传的中性脂肪蓄积病——Chanarin—Dorfman综合征（Chanarin-Dorfman symptom,CDS）,病体表现出鱼鳞病。本文将针对CGI-58调控脂肪代谢以及其它方面的功能,对近些年来CGI-58的研究进展进行综述。