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J. Biol. Chem., Vol. 279, Issue 3, 2038-2045, January 16, 2004
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From the
Touchstone Center for Diabetes Research and the Departments of Physiology and ||Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-8854
Received for publication, July 14, 2003 , and in revised form, October 10, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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angiopoietin-related, fasting induced adipose factor, and hepatic fibrinogen/angiopoietin-related protein, recombinant Angptl4 causes increase of plasma very low density lipoprotein levels by inhibition of lipoprotein lipase activity. Similar to angiopoietins and other angiopoietin-like proteins, Angptl4 contains an amino-terminal coiled-coil domain and a carboxyl-terminal fibrinogen-like domain. We report here that Angptl4 is evolutionarily conserved among several mammalian species and that full-length Angptl4 protein is an oligomer containing intermolecular disulfide bonds. Oligomerized Angptl4 undergoes proteolytic processing to release its carboxyl fibrinogen-like domain, which circulates as a monomer. Angptl4's N-terminal coiled-coil domain mediates its oligomerization, which by itself is sufficient to form higher order oligomeric structure. Adenovirus-mediated overexpression of Angptl4 in 293 cells shows that conversion of full-length, oligomerized Angptl4 is mediated by a cell-associated protease activity induced by serum. These findings demonstrate a novel property of angiopoietin-like proteins and suggest that oligomerization and proteolytic processing of Angptl4 may regulate its biological activities in vivo. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One of the recently identified secreted factors from adipose tissue is angiopoietin-like protein 4 (Angptl4)1, also named PPAR angiopoietin related, fasting induced adipose factor, or hepatic fibrinogen/angiopoietin-related protein. It is a secreted protein selectively expressed in adipose tissue, liver, and placenta (5–7). Mouse Angptl4 is composed of 410 amino acids that include an N-terminal signal sequence, a coiled-coil domain, and a fibrinogen-like motif at the carboxyl terminus. Such domain organization is preserved in both angiopoietins and angiopoietin-like proteins.
Transcriptional regulation of Angptl4 is rather complex. Several transcription factors have been identified as having a role in Angptl4 transcription, including two members of the peroxisome proliferator-activated receptor family of transcription factors, PPAR
and PPAR (6, 7), as well as hypoxia-inducible factor 1 (8). As a transcriptional target of PPAR, Angptl4 has been hypothesized to play a role in the modulation of adipogenesis, insulin sensitivity, or energy metabolism (7). Mice heterozygous for PPAR knockout have reduced Angptl4 mRNA expression in adipose tissue, where PPAR is normally abundantly expressed. Mice with PPAR deficiency also exhibit undetectable Angptl4 mRNA levels in liver, where PPAR is normally abundantly expressed. Conversely, when mice are treated with a potent agonist of PPAR, WY14643, Angptl4 transcription is increased (6). In vitro, treatment of NIH 3T3 cells stably expressing PPAR with its agonist, pioglitazone, caused an increase of at least 10-fold in Angptl4 mRNA levels in the absence of protein synthesis (7). PPAR and hypoxia-inducible factor 1 also cause the synergistic activation of Angptl4 transcription in cardiomyocytes (8). In a recent report, Angptl4 was found to be one of nine genes silenced by methylation in human gastric cancers (9). In aggregate, these data suggest that Angptl4 may play a variety of roles in vivo, ranging from adipogenesis and angiogenesis to carcinogenesis (10). Because both ischemic tissue and solid tumor tissue are in a hypoxic state, Angptl4 could be playing a shared role of modulating angiogenesis under both conditions.
The expression of angpt4 is also under nutritional and hormonal control. Under fasting conditions, transcription of Angptl4 in both liver and adipose tissue is induced independently of the presence of PPAR (6). Circulating levels of Angptl4 are also increased in genetically obese mice (ob/ob or db/db) that lack leptin signaling (7). However, in rodents fed a high-fat diet, circulating levels of Angptl4 are reduced, providing further evidence that Angptl4 may play a role in response to the availability of nutrients (6).
The function of Angptl4 is not completely understood. Recently, it was reported that Angptl4 is a potent hyperlipidemia-inducing factor and an inhibitor of lipoprotein lipase (11). To obtain better insight on the structural basis of Angptl4 function, we have now determined that full-length Angptl4 is an oligomer, similar to angiopoietin 1 (12). In contrast to angiopoietin 1, however, Angptl4 undergoes regulated proteolysis to release its carboxyl-terminal fibrinogen-like domain both in vitro and in vivo, the first example of proteolysis-mediated processing of a member of this family of proteins.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cloning of Rat Angptl4 and Construction of Adenoviral Expression Vectors—To amplify cDNA encoding full-length Angptl4 from rat adipose tissue, first-strand cDNA was generated by reverse transcription of total RNA using SuperScript II RNase H– reverse transcriptase. Both full-length Angptl4 and the amino-terminal coiled-coil domain of Angptl4 were amplified using the same 5' primer, CL307. It contains an XhoI site (underlined) for in-frame subcloning into the mammalian expression vector pcDNA3.1 (-)/Myc-HisA and has the following sequences: 5'-CCGCTCGAGAATCATGCGCTGCGCTCCG-3'; the 3' ends of the full-length and the amino-terminal coiled-coil domain of Angptl4 were amplified by primers CL379 and CL380, each containing a HindIII site (underlined) for in-frame subcloning into the same vector. The sequences are: CL379, 5'-CCC AAG CTT AGA GGC TGC TGT AGC CTC CAT G-3'; CL380, 5'-CCC AAG CTT CTG GGT CAT CTT GGG AAG-3'. Clones containing inserts from primer pairs CL307-CL379 and CL307-CL380 were verified by sequencing analysis and transient transfection.
To generate adenoviruses encoding the N-terminal coiled-coil domain (Ad-Angptl4(S)-myc) or full-length (Ad-Angptl4(L)-myc) rat Angptl4, the AdEasy system was used, with a slight modification (13). Briefly, to facilitate detection of virally produced Angptl4 proteins, inserts in the pcDNA3.1(–)/Myc-His vector were removed along with the carboxyl-terminal c-myc tag as a PmeI fragment and ligated to the EcoRV site of pShuttle-CMV. The resulting plasmid was then linearized with PmeI, purified using the MinElute Kit (QIAGEN), and co-electroporated with pAdEasy1into BJ5183 cells to generate recombinant viral DNA. Recombinants were screened by running an aliquot of undigested miniprep DNA of 2-ml cultures from KanR colonies. PacI digestion was then performed to confirm the presence of inserts. Typically, this procedure yields 
50% recombinants. Viral DNA was transformed into DH5 cells and propagated before being used for transfection into low passage 293 cells as described previously (14).
Purification of Angptl4 Protein—Conditioned media with or without serum from 293 cells infected wirh Ad-Angptl4(L)-myc virus were collected and filtered through a 0.22-µm filter. Buffering capacity and salt concentrations were adjusted to 20 mM Tris-HCl, pH 8.0, and 300 mM NaCl by diluting conditioned media with 1/10 volume of 200 mM Tris-HCl, pH 8.0, and 3 M NaCl. 5 µl of TALON Metal Affinity resin was pre-equilibrated with 50 ml of wash buffer (20 mM Tris-HCl, pH 8.0, and 300 mM NaCl), before 1000 ml of conditioned medium was applied to the resin. Medium and resin were rocked gently at 4 °C overnight. Resin was washed with 3 x 50 ml of wash buffer, and bound proteins were eluted with 6 x 5 ml of wash buffer containing 250 mM imidazole. Fractions containing Angptl4 protein were pooled, concentrated using Centriprep concentrators (Amicon Inc, Beverly, MA), and further purified by size exclusion chromatography on a Superdex 200 column (10/30). Fractions at each stage of purification were analyzed by SDS-PAGE, followed by Coomassie Blue staining and/or Western blotting to confirm the presence of Angptl4 protein.
Generation of Polyclonal Antibodies against Angptl4 —Recombinant Angptl4 was prepared from mammalian cells (antigen 1) or bacteria (antigens 2 and 3) for generation of rabbit polyclonal antibodies using different expression systems. Antigen 1 was purified from processed carboxyl terminus of Angptl4 from conditioned medium containing 5% FBS of Ad-Angptl4(L)-myc virus-infected 293 cells as described above. Partially purified Angptl4 protein was separated on preparative 12% SDS-PAGE, and the Angptl4 was band cut out after staining with Coomassie brilliant blue. Gel slice was homogenized and injected into female New Zealand White rabbits for antibody production. Antigen 2 was generated using a pET expression system (Novagen). A 512-bp cDNA sequence encoding the carboxyl fibrinogen-like domain of Angptl4 was PCR-amplified and cloned into the SmaI site of pET-44b vector (Novagen). Fusion protein was expressed in bacteria and purified according to the manufacturer's instructions. The purified protein was also used for injection into rabbit. Antigen 3 was generated using a glutathione S-transferase fusion protein of Angptl4 as follows. Full-length Angptl4 cDNA (minus the signal peptide) was fused to the carboxyl terminus of glutathione S-transferase and expressed in bacteria, following standard methods. Fusion protein was run on preparative SDS-PAGE and cut out after staining with Coomassie blue. Gel slice was homogenized and injected into female New Zealand White rabbits for antibody production. In all cases, specificity of the antibodies was determined by serial dilution of antigen, by comparison with the signal recognized by the c-myc monoclonal antibody, and by comparison with pre-immune serum. All blots shown are recombinant Angptl4 expressed after transient transfection or adenoviral infection.
Expression and Detection of Angptl4 in 293 Cells—293 cells were cultured in 6-well plates until 80–90% confluent. Culture medium was replaced with fresh Dulbecco's modified Eagle's medium with or without 5% FBS before adenoviruses encoding Angptl4 were added at a multiplicity of infection of 100. Multiplicity of infection was determined based on the virus titer, obtained by serial dilution of viruses and infections of individual wells of 96-well plates. Typically, titer of purified viruses ranges from 109 to 1010 plaque-forming units per milliliter. Forty-eight hours after virus infection, conditioned medium was collected and centrifuged to obtain supernatant. 200 µl of radioimmunoprecipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) was added to the remaining cells, and 20 µl lysate was mixed with 2x reducing SDS-PAGE loading buffer (containing 0.2% 
-mercaptoethanol) for analysis. Unless otherwise indicated, no protease inhibitors were used during purification of Angptl4 protein from the supernatant of 293 cells. -Mercaptoethanol was added in SDS-PAGE loading buffer in all blots except for Figs. 2B and 3C, where lanes are labeled "non-reduced." For Western blotting, samples were incubated with SDS loading buffer either with (reduced) or without (nonreduced) 0.1% -mercaptoethanol and subjected to 12% SDS-PAGE. After electrophoresis, samples were transferred to nitrocellulose membrane (Schleicher & Schüll). Membrane was blocked with 5% non-fat dry milk in Tris-buffered saline/Tween 20 (20 mM Tris, pH 7.5, 0.9% NaCl, and 0.05% Tween 20). Angptl4 protein was detected with a c-myc-HRP antibody (Roche Diagnostic, Inc.) or polyclonal antibodies as described above using the ECL-Western blotting kit from Amersham Biosciences. After detection, membrane was exposed to Blue Bio film (Denville Scientific Inc., Metuchen, NJ) for about 30 s.
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0.5 µg of Angptl4 protein) was then applied to the same column. Because of the lower expression levels, conditioned media containing recombinant Angptl4(S)-myc was first concentrated 5-fold using Centriprep (Amicon) before column loading. Fractions of 300 µl were collected, and each fraction was analyzed by dot blotting or Western blotting, using c-myc-HRP for detection. To detect recombinant Angptl4 by dot blotting, membrane containing applied samples from each fraction was first treated with PBS containing 0.25% H2O2 for 30 min to render peroxidase inactive in samples. The filter was then washed with PBS 3 times for 5 min each. Blocking and antibody incubation were then carried out as for Western blotting. Adenovirus Infusion into Mice to Overexpress Angptl4 in Vivo—Adenoviruses encoding Angptl4 (Ad-Angptl4(L)-myc) or green fluorescent protein (Ad-GFP) were infused via the jugular vein at a dosage of 1 x 1011 virus particles/mouse, as described previously (14, 15). Four days after virus injection, blood samples were taken from the tail vein to detect the levels of virally produced Angptl4 protein by Western blotting analysis. Mice were anesthetized and sacrificed for tissue removal and characterization. Animal use was approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.
Time Course of Serum-induced Angptl4 Processing—293 cells were cultured in 6-well plates until 80–90% confluent. Cells were then washed once with PBS and medium was replaced with 2 ml of fresh Dulbecco's modified Eagle's medium without serum; adenoviruses encoding full-length Angptl4 were added to cells at a multiplicity of infection of 100. Twenty-four hours after virus infection, FBS was added to cells to a final concentration of 5%. FBS was applied to all wells except one, which was used as control. Conditioned medium was collected at different time points from 0 to 48 h after addition of FBS and used directly for Western blotting to detect recombinant Angptl4. No protease inhibitors were added to media at any stages of Angptl4 expression.
To determine whether serum-activated proteolytic activity is released into conditioned media and whether adenovirus infection per se causes release of nonspecific proteolytic activity, supernatant was collected from 293 cells cultured in serum-containing media, with or without infection by the control adenovirus Ad-GFP. About 0.1 µg of Angptl4 protein was then incubated with collected media at 37 °C for 24 h and used for Western blotting with c-myc monoclonal antibody. Cells infected with Ad-Angptl4(L)-myc were also cultured in serum-containing media in the absence or presence of complete protease inhibitor mixture to determine whether processing of recombinant Angptl4 might be inhibited.
Protein Deglycosylation—Angptl4 protein (about 1 µg) was denatured in 1x glycoprotein denaturing buffer (0.5% SDS and 1% -mercaptoethanol) at 100 °C for 10 min. One unit of PNGase F was added and incubated at 37 °C for 2 h in 1x G7 Reaction Buffer (50 mM sodium phosphate, pH 7.5), supplemented with 1% Nonidet P-40. After treatment of the samples with PNGase F, Angptl4 protein was detected by SDS-PAGE and Western blotting.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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74 kDa and a doublet of 50 kDa. Further analysis demonstrated that the 74-kDa band represents full-length Angptl4 protein, whereas the 50-kDa doublet is the proteolytic product of Angptl4, corresponding to its carboxyl fibrinogen-like domain. The amino acid sequence boundaries of the 50-kDa doublets are not known because the N termini of both bands are blocked when subjected to sequencing analysis. One explanation for the appearance of the doublet band is the additional cleavage of primary proteolysis product, because the doublet is detected by the same carboxyl-terminal antibodies recognizing fibrinogen-like domain or c-myc tag of Angptl4.
The same supernatant containing recombinant Angptl4 was loaded onto a Superdex 200 column, and fractions were collected and analyzed by Western blotting to determine the elution profile of Angptl4 protein. Fig. 2A shows that Angptl4 first appears in an early fraction, near the void volume of the column, which excludes globular proteins of 600 kDa and larger. It continues to elute over a broad range to a position that corresponds to a protein of 100 kDa. Its broad range of mobility in a gel filtration column suggests that Angptl4, similar to angiopoietin-1 (12), also forms variable-sized multimers. We also performed sucrose density gradient sedimentation experiments, with results similar to those obtained by gel filtration (data not shown)
Interestingly, the doublet band of recombinant Angptl4 from total starting supernatant, a proteolytic product of full-length Angptl4, eluted as an apparent monomer from the same Superdex 200 column. This doublet is recognized by both a c-myc monoclonal antibody, which recognizes the c-myc tag fused to the carboxyl terminus of Angptl4, as well as a polyclonal antibody generated in rabbit using the carboxyl fibrinogen-like domain of Angptl4 as antigen (data not shown). These results demonstrate that the monomeric carboxyl fibrinogen-like domain is converted from oligomerized, full-length Angptl4. Consequently, the carboxyl fibrinogen-like domain of Angptl4 loses the ability to form higher order structure.
Formation of high molecular weight protein complexes frequently involves disulfide bond formation between protein monomers, such as adiponectin/ACRP30 (16). We asked whether the oligomerization of full-length Angptl4 also requires disulfide bond formation between individual Angptl4 molecules. Fig. 2B shows that when denatured full-length Angptl4 was treated with SDS-PAGE sample buffer containing -mercaptoethanol or not, the presence of -mercaptoethanol caused Angptl4 protein to migrate at a size predicted of a monomer; in the absence of -mercaptoethanol, bands that migrate at the size of a dimer and higher molecular weight complexes also form. Because the sizes of large proteins in a nonreducing gel are difficult to estimate, it is impossible to accurately determine the number of Angptl4 monomers that participate in disulfide bond formation. The results from this experiment are consistent with the broad elution profile of full-length Angptl4 protein during size exclusion chromatography (Fig. 2A).
Coiled-coil Domain of Angptl4 Is Sufficient to Form Higher Order Oligomers—The elution profile of the carboxyl fibrinogen-like domain of Angptl4 in FPLC suggests that this domain is insufficient to form an oligomer. To determine whether the N-terminal coiled-coil domain, in the absence of fibrinogen-like domain, is sufficient to mediate Angptl4 oligomerization, we generated adenoviral vectors expressing only the N-terminal coiled-coil domain (Fig. 3A). Both the full-length and the coiledcoil domain of Angptl4 were tagged with c-myc and polyhistidine epitopes at the carboxyl terminus to facilitate detection of recombinant proteins. When supernatant of 293 cells infected with viruses encoding full-length Angptl4 (Ad-Angptl4(L)-myc) or N-terminal coiled-coil domain of Angptl4 (Ad-Angptl4(S)-myc) was run on a Superdex 200 column and detected with the c-myc monoclonal antibody, both proteins eluted at similar positions as very large high molecular weight complexes (Fig. 3B). These results demonstrate that the N-terminal coiled-coil domain is sufficient to form high-order oligomeric structures. This conclusion is also supported by the observation that under nonreducing conditions, both full-length and N-terminal coiledcoil domain of Angptl4 migrates as larger molecular weight species (Fig. 3C).
Serum-Dependent Processing of Full-length Angptl4 —We proceeded to determine how processing of full-length, oligomerized Angptl4 is regulated. Ad-Angptl4(L)-myc was used to infect 293 cells again and cells were maintained in the presence or absence of serum to determine whether the presence of serum is necessary for Angptl4 processing. Fig. 4A shows that when 293 cells were infected with adenoviruses encoding Angptl4 or GFP and maintained in media without serum, Angptl4 is secreted predominantly as a full-length protein of 74 kDa (Fig. 4A, long arrow). In the presence of 5% serum, Angptl4 protein detected in the supernatant exists as either a full-length protein as that secreted into serum-free media or as a doublet of 50 kDa (Fig. 4A, short arrow). Supernatant or pellet samples of cells infected with Ad-GFP showed no signal. Both the full-length and the carboxyl fibrinogen-like domain of Angptl4 were detected by the c-myc antibody, which recognizes the carboxyl terminus of Angptl4 (Fig. 4A) and polyclonal antibodies against Angptl4 (data not shown). No processed Angptl4 fragment was detected in total cell lysate when cells were cultured in either serum-free or serum-containing media, suggesting that cleavage of full-length did not occur during the transport of Angptl4 to plasma membrane. The amino acid boundaries at the N terminus of the doublet are not known because sequencing analysis revealed that the N termini of both bands are blocked (data not shown).
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To gain a better understanding of the cleavage process of full-length Angptl4, we expressed Angptl4 in the presence of protease inhibitors. Recombinant Angptl4 secreted into the media under either serum-free or serum-containing conditions remained mostly intact in the presence of complete protease inhibitor mixture for up to 2 days (Fig. 5A). We then determined whether the conversion of full-length, oligomerized Angptl4 to monomeric form is mediated by a protease activity present in serum or that induced in the presence of serum. Incubation of full-length Angptl4 protein with conditioned media from cells cultured in the presence of serum, with or without infection by control Ad-GFP virus shows that the protease is not released into media (Fig. 5B, lane 3), nor was processing a nonspecific effect caused by adenovirus infection (Fig. 5B, lane 4). These results demonstrate that proteolytic processing of full-length, oligomerized Angptl4 requires the presence of serum as well as molecules from cells. The time course of protease induction is shown in Fig. 5C. Angptl4 was first allowed to express in serum-free media for 24 h before serum was added. 24 to 48 h after serum addition, the appearance of the 50-kDa doublet reaches peak levels, suggesting full induction of proteolytic activity. In aggregate, these results demonstrate that the proteolytic processing of Angptl4 occurs before its release into media, mediated by a cell-associated protease activity that is activated by serum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The processing of oligomerized, full-length Angptl4 is reminiscent of that of adiponectin/ACRP30. In both cases, proteolytic conversion is associated with changes of the properties of full-length proteins. In the case of adiponectin/ACRP30, processing of full-length adiponectin/ACRP30 causes enhanced effect on muscle fat oxidation and glucose transport (17, 18). The formation of high molecular weight complex of adiponectin/ACRP30 is regulated by insulin and dependent on Cys-39 of adiponectin/ACRP30. Its plasma levels are also under complex hormonal control and may play a key role in determining systemic insulin sensitivity under the respective conditions (19, 20). Whereas cysteine is required for dimer formation of Angptl4, the key residue involved in disulfide formation and the regulation of Angptl4 processing from high molecular weight complexes remain to be determined.
An earlier report indicates that Angptl4 causes hyperlipidemia by inhibiting lipoprotein lipase activity (11). Another circulating protein structurally related to Angptl4, angiopoietin-like protein 3 (Angptl3), also causes elevation of plasma very low density lipoprotein elevation by inhibition of LPL (21). Analogous to Angptl3, Angptl4 may thus be another circulating regulator of plasma lipid homeostasis. The similarities between Angptl3 and Angptl4 also extend to their regulation by nuclear hormone receptors; Angptl3 is a direct target of liver X receptor (22, 23), whereas Angptl4 is a target of PPAR and PPAR. Because both PPARs and liver X receptor dimerizes with retinoid X receptor for full biological activity, it is conceivable that both Angptl3 and -4 are also regulated by the same factor(s) in vivo. In addition, Angptl3 has also been reported to activate lipolysis by directly binding to adipocytes to stimulate the release of FFA and glycerol (24). However, Angptl3 is expressed exclusively by liver, whereas Angptl4 is expressed selectively by adipose tissue, liver, and placenta, and potentially other tissues. In addition, the levels of Angptl3 expression are not regulated by nutritional status. The differences in sites and regulation of expression could thus confer tissue specific actions of Angptl3 and -4.
In summary, we show that Angptl4, a circulating protein primarily expressed by adipose tissue and liver, resembles angiopoietin 1 in forming a higher order structure consisting of variable-sized multimers (12). In contrast to angiopoietin 1, however, oligomerized full-length Angptl4 undergoes regulated proteolysis into an amino-terminal coiled-coil domain and a carboxyl fibrinogen-like domain, the first example among all angiopoietins and angiopoietin-like proteins. An exciting possibility is that proteolytic processing of full-length, oligomerized Angptl4 could serve as a mechanism of activation, with each released domain having distinct roles. One such consequence is the hyperlipidemic effect of Angptl4 (11), which could be mediated by either domain. It is intriguing to speculate that inappropriate proteolytic conversion of Angptl4 in vivo could be associated with certain disorders of altered plasma lipid homeostasis as well as possibly other pathological conditions.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK60137. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY393999
[GenBank]
. 
¶ Present address: Shanghai Haojia Technology Development Co. Ltd., Shanghai 200235, China.
Recipient of a Career Development Award from the American Diabetes Association.
** To whom reprint requests should be addressed: 5323 Harry Hines Blvd., Dallas, TX 75390-8854. Tel.: 214-648-3340; Fax: 214-648-9191; E-mail: cai.li{at}utsouthwestern.edu.
1 The abbreviations used are: Angptl, angiopoietin-like protein; PPAR, peroxisome proliferator-activated receptor; FBS, fetal bovine serum; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; GFP, green fluorescent protein; PNGase, peptide N-glycosidase F.
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ACKNOWLEDGMENTS |
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REFERENCES |
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