Inhibition of 3T3-L1 Adipocyte Differentiation by Expression of Acyl-CoA-binding Protein Antisense RNA

Mandrup, Susanne; Sørensen, Rikke Guldberg; Helledie, Torben; Nøhr, Jane; Baldursson, Trausti; Gram, Connie; Knudsen, Jens; Kristiansen, Karsten

doi:10.1074/jbc.273.37.23897

Cited by 52 publications

(36 citation statements)

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“…It has been reported that knockdown of ACBP by small interfering RNA causes growth arrest and lethality in three different mammalian cell lines (26); however, data from our laboratory show that ACBP can be knocked down in many different cell systems without affecting growth and survival (27). 6 Recently, knockdown of ACBP in HepG2 cells was shown to suppress the expression of a number of genes involved in lipid biosynthesis and lead to decreased levels of saturated and monounsaturated fatty acids (28).…”

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“…6 Recently, knockdown of ACBP in HepG2 cells was shown to suppress the expression of a number of genes involved in lipid biosynthesis and lead to decreased levels of saturated and monounsaturated fatty acids (28). In 3T3-L1 preadipocytes, knockdown of ACBP caused a mild impairment of adipocyte differentiation and accumulation of triacylglycerol (TAG) (27), whereas overexpression of ACBP in McA-RH7777 rat hepatoma cells resulted in increased intracellular TAG accumulation (29). Overexpression of ACBP in transgenic mice resulted in accumulation of different lipid classes, including TAG in the liver (30).…”

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Disruption of the Acyl-CoA-binding Protein Gene Delays Hepatic Adaptation to Metabolic Changes at Weaning

Neess¹,

Bloksgaard²,

Bek³

et al. 2011

Journal of Biological Chemistry

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The mouse acyl-CoA-binding protein (ACBP) 5 /diazepam binding inhibitor is a 10-kDa intracellular protein consisting of 86 amino acids. It is highly conserved throughout evolution and expressed in all cell types in the eukaryotes investigated (1, 2). This, together with the characteristics of the ACBP promoter (3, 4), implies a housekeeping function of the gene. However, expression levels vary markedly between tissues (5) and in response to different metabolic stimuli (6 -9), thereby indicating that ACBP might perform more specialized functions in some cell types. The ACBP protein binds C 14 -C 22 acyl-CoA esters with high affinity and specificity (10, 11) and has very little or no affinity toward other ligands (11-13). From in vitro studies, ACBP is known to protect acyl-CoA esters from hydrolysis (14 -16) and to relieve acyl-CoA inhibition of a number of enzymes, including long chain acyl-CoA synthetase, acetyl-CoA carboxylase (ACC), adenine nucleotide translocase, fatty acid synthetase (FAS), carnitine palmitoyltransferase, and acyl-CoA:cholesterol acyltransferase (9, 16 -18). In addition, ACBP is known to donate acyl-CoA esters to phospholipid, glycerolipid, and cholesteryl ester (CE) synthesis (14, 18 -21). Finally, proteolytic products of secreted ACBP have been shown to have signaling functions in Dictyostelium as well as mammalian cells (22). Targeted disruption of the yeast ACBP gene (ACB1) revealed that ACBP deficiency results in increased levels of C18:0 acyl-CoA esters and a decrease in the amount of total C26:0 fatty acids, indicating that transport of FA toward elongation is impaired by lack of ACBP. Furthermore, sphingolipid and ceramide amounts were reduced, membrane structure was altered, and vesicular transport was compromised (23-25).The functions of ACBP in lipid metabolism have been further studied in different mammalian cell culture systems and animal models by both knockdown strategies and overexpression of the protein. It has been reported that knockdown of ACBP by small interfering RNA causes growth arrest and lethality in three different mammalian cell lines (26); however, data from our laboratory show that ACBP can be knocked down in many different cell systems without affecting growth and survival (27). 6 Recently, knockdown of ACBP in HepG2 cells was shown to suppress the expression of a number of genes involved in lipid biosynthesis and lead to decreased levels of saturated and monounsaturated fatty acids (28). In 3T3-L1 preadipocytes, knockdown of ACBP caused a mild impairment of adipocyte differentiation and accumulation of triacylglycerol (TAG) (27), whereas overexpression of ACBP in McA-RH7777 rat hepatoma cells resulted in increased intracellular TAG accumulation (29). Overexpression of ACBP in transgenic mice resulted in accumulation of different lipid classes, including TAG in the liver (30). These results suggest *

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Disruption of the Acyl-CoA-binding Protein Gene Delays Hepatic Adaptation to Metabolic Changes at Weaning

Neess¹,

Bloksgaard²,

Bek³

et al. 2011

Journal of Biological Chemistry

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“…ACBP is also found at abnormally high levels in tumor cells that exhibit very high rates of lipid synthesis as well as proliferation [6,14,16,25,40]. Conversely, ACBP suppression results in reduced cellular growth rates, abnormal fatty acyl-CoA composition, vesicle accumulation, and membrane defects in yeast [13,36]; inhibition of adipocyte differentiation [27]; and lethality in some types of mammalian cells [9].…”

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Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

Petrescu

Huang

Hostetler

et al. 2008

Protein Expression and Purification

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Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Niaffinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the Nterminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (K d =2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (K d =64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP.

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“…Because PPAR␥ has a large hydrophobic ligand binding domain (9) and activation occurs in response to fatty acids (10), endogenous long chain fatty acids or their derivatives have been proposed as natural ligands. These include oleate, linoleate, nitrolinoleate, nitrooleate, 9-hydroxydecaenoic acid, arachidonic acid, and 15-deoxy-prostaglandin J2 (11)(12)(13)(14)(15).…”

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Stearoyl-CoA Desaturase 2 Is Required for Peroxisome Proliferator-activated Receptor γ Expression and Adipogenesis in Cultured 3T3-L1 Cells

Christianson

Nicoloro

Straubhaar

et al. 2008

Journal of Biological Chemistry

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Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferatoractivated receptor ␥ (PPAR␥) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPAR␥ protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPAR␥ protein but decreased the metabolic labeling of PPAR␥ protein using [ 35 S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPAR␥ protein levels and adipogenesis in 3T3-L1 cells.The ability of adipocytes to sense and respond to circulating fatty acid levels is important in maintaining the proper balance between fatty acid storage and fatty acid release for energy utilization. In the case of energy excess, fatty acids are stored in the form of triglyceride, and new adipocytes are generated to efficiently metabolize amino acids, glucose, and fatty acids to triglyceride (1). The key regulator of adipogenesis, the process whereby preadipocytes differentiate into fully mature adipocytes, is the ligand-activated nuclear receptor PPAR␥ 2 (2). Cultured mouse 3T3-L1 preadipocytes are an excellent model system for the study of adipogenesis. These cells differentiate into adipocytes with multilocular lipid droplets through a transcriptional cascade beginning with the rapid and transient expression of C/EBP␤ and C/EBP␦ (3, 4). The up-regulation of these transcription factors precedes the expression of PPAR␥ and C/EBP␣, which are critical for the completion of adipogenesis as well as the maintenance of adipocyte-specific gene expression in fully differentiated cells (3, 4). Other transcription factors have also been shown to play significant roles in adipogenesis and adipocyte biology (for reviews, see Refs. 3, 5, and 6). However, because PPAR␥ controls the expression of large sets of genes required to maintain the adipocyte phen...

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Inhibition of 3T3-L1 Adipocyte Differentiation by Expression of Acyl-CoA-binding Protein Antisense RNA

Cited by 52 publications

References 44 publications

Disruption of the Acyl-CoA-binding Protein Gene Delays Hepatic Adaptation to Metabolic Changes at Weaning

Disruption of the Acyl-CoA-binding Protein Gene Delays Hepatic Adaptation to Metabolic Changes at Weaning

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

Stearoyl-CoA Desaturase 2 Is Required for Peroxisome Proliferator-activated Receptor γ Expression and Adipogenesis in Cultured 3T3-L1 Cells

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