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J. Biol. Chem., Vol. 278, Issue 51, 51535-51542, December 19, 2003
Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis*![]() ![]() ![]() ![]() ![]() ¶
From the
Received for publication, August 28, 2003
Perilipin (Peri) A is a lipid droplet-associated phosphoprotein that acts dually as a suppressor of basal (constitutive) lipolysis and as an enhancer of cyclic AMP-dependent protein kinase (PKA)-stimulated lipolysis by both hormone-sensitive lipase (HSL) and non-HSL(s). To identify domains of Peri A that mediate these multiple actions, we introduced adenoviruses expressing truncated or mutated Peri A and HSL into NIH 3T3 fibroblasts lacking endogenous perilipins and HSL but overexpressing acyl-CoA synthetase 1 and fatty acid transporter 1. We identified two lipase-selective functional domains: 1) Peri A (amino acids 1300), which inhibits basal lipolysis and promotes PKA-stimulated lipolysis by HSL, and 2) Peri A (amino acids 301517), which inhibits basal lipolysis by non-HSL and promotes PKA-stimulated lipolysis by both HSL and non-HSL. PKA site mutagenesis revealed that PKA-stimulated lipolysis by HSL requires phosphorylation of one or more sites within Peri 1300 (Ser81, Ser222, and Ser276). PKA-stimulated lipolysis by non-HSL additionally requires phosphorylation of one or more PKA sites within Peri 301517 (Ser433, Ser492, and Ser517). Peri 301517 promoted PKA-stimulated lipolysis by HSL yet did not block HSL-mediated basal lipolysis, indicating that an additional region(s) within Peri 301517 promotes hormone-stimulated lipolysis by HSL. These results suggest a model of Peri A function in which 1) lipase-specific "barrier" domains block basal lipolysis by HSL and non-HSL, 2) differential PKA site phosphorylation allows PKA-stimulated lipolysis by HSL and non-HSL, respectively, and 3) additional domains within Peri A further facilitate PKA-stimulated lipolysis, again with lipase selectivity.
Hydrolysis of triacylglycerol (TAG)1 (lipolysis) in adipocytes is a key event that supplies the primary source of energy, free fatty acids, for other tissues. In times of energy need such as fasting (13) and exercise (4, 5), adipocyte lipolysis is regulated by hormones such as catecholamines, which activate cAMP-dependent protein kinase (PKA) (6, 7). Activation of PKA results in a marked increase in lipolysis as compared with spontaneous lipolysis in the absence of hormones (basal or constitutive lipolysis). Thus, basal and PKA-stimulated lipolysis reflect the lipolytic response of adipocytes to the changing energy requirements of the body. In obesity, basal lipolysis is increased, and PKA-stimulated lipolysis is blunted (8). This dysregulation of adipocyte lipolysis is associated with the development of insulin resistance and type 2 diabetes (9).
TAG breakdown (lipolysis) is mediated by lipases, which hydrolyze TAG sequestered in intracellular lipid droplets. Approximately 50% of the neutral triglyceride lipase activity in white adipose tissue is attributable to hormone-sensitive lipase (HSL) (1012). HSL-mediated lipolysis is under the tight control of PKA. In the absence of PKA activation, constitutive HSL activity has been thought to mediate basal lipolysis (13). When PKA is activated, it phosphorylates HSL (13, 14), resulting in enhanced hydrolytic activity (15), translocation of HSL from cytosol to the lipid droplet surface (1618), and enhanced TAG breakdown. HSL was previously considered the rate-limiting enzyme in adipocyte lipolysis. This view, however, has recently been challenged by the following observations: 1) HSL-deficient mice are not obese, suggesting significant activity of lipase(s) other than HSL (10, 12, 19); 2) adipocytes derived from epididymal fat pads or embryonic fibroblasts of HSL null mice retain 50% of basal TAG lipase activity and are responsive to PKA activation (10, 11, 12, 19); and 3) cell lines lacking endogenous HSL, i.e. 3T3-L1 preadipocytes (21), NIH 3T3 fibroblasts (22), and Chinese hamster ovary cells (23), exhibit significant TAG hydrolysis under basal and PKA-stimulated conditions. Thus, both HSL and non-HSL(s) play important roles in basal and PKA-stimulated lipolysis.
The hydrolytic actions of HSL and non-HSLs are regulated at the lipid droplet surface by perilipins, a family of lipid droplet-associated phosphoproteins (24, 25). Although not required for lipid droplet formation and TAG storage, perilipin association with the lipid droplet regulates the magnitude of lipolysis and thus levels of stored TAG (21, 22). Peri A and Peri B are the predominant isoforms of murine adipocytes, with Peri A constituting
How does the dual function of perilipin in regulating lipolyis arise from perilipin protein structure? Recent data have suggested a model of Peri A function in which the phosphorylation of N-terminal PKA sites (PKA1, PKA2, and PKA3; Fig. 1) promotes PKA-stimulated lipolysis (22, 23), and the C terminus of Peri A (aa 406517) acts as a barrier to all lipases (inhibits basal lipolysis) (23). In contrast, novel data in the present study identify multiple, lipase-selective domains within Peri A that function coordinately to regulate basal and stimulated lipolysis. Our data support a model of perilipin function in which 1) discrete lipase-selective protein domains of perilipin act as barriers to HSL and non-HSL(s), respectively, 2) phosphorylation of different PKA sites allows PKA-stimulated lipolysis mediated by HSL and non-HSL(s), respectively, and 3) additional "facilitative" domains within Peri A enhance PKA-stimulated lipolysis, again with lipase selectivity.
Materials A polyclonal antibody (MSM) specific for the N terminus of Peri A was generated using an N-terminal peptide (aa 121, MSMNKGPTLLDGDLPEQENVL). A polyclonal antibody (PREK) specific for the C terminus of Peri A was generated using a C-terminal peptide (aa 483517, PREKPARRVSDSFFRPSVC). A polyclonal anti-HSL antibody was generated using a peptide based upon the rat HSL sequence KDLSFKGNSEPSDSPEM. The antibodies were subsequently affinity-purified and used for Western blotting (1:1000). Tissue culture reagents and media were purchased from Invitrogen. LipofectAMINE PlusTM reagent was purchased from Invitrogen. Horseradish-linked anti-rabbit IgG was from Amersham Biosciences; SuperSignal® Chemiluminescent Substrate was obtained from Pierce. All other chemicals were purchased from Sigma.
Methods Mutagenesis of six putative PKA recognition sites was carried out using Muta-Gene Phagemid in vitro mutagenesis version 2 (Bio-Rad). Mutations of serines to alanines were made in a set of all six sites or in sets of three by grouping the three N-terminal PKA sites as PKA cassette 1 (Ser81, Ser222, and Ser276) and the three C-terminal PKA sites as PKA cassette 2 (Ser433, Ser492, and Ser517). Primers used for mutagenesis were as follows: sequences for Ser81, PKA 1 (5'-GTC CGT CGG CTG GCC ACC CAG TTC ACA GC-3'); Ser222, PKA2 (5'-TTT TGA GGA GGG TCG CCA CCC TGG CCA ACAC TC-3'); Ser276, PKA3 (5'-CCC GGC GGC AGG CTG AGG TGC-3'); Ser433, PKA4 (5'-CAG CAG AGG CGG AGC GCA AAG GGG CCG GGG CGC GG-3'); Ser492, PKA5 (5'-CCT GCG CGC AGA GTC GCC GAC ACG TTC TTC CGG-3'); and Ser517, PKA6 (5'-GCC AGC TGC GCA AGA AGG CCT GAG CAG ACT GCG CC-3'). The identity of each of the truncations and mutated PKA sites was confirmed by sequencing. The cDNAs were used to generate adenoviruses.
Generation of Recombinant AdenovirusesAdenoviruses expressing Adenovirus-mediated Expression of Perilipins and HSLAdenoviruses expressing truncated/mutated Peri A and HSL were expressed in ACS1/FATP1 cells (kindly provided by Dr. Jean E. Schaffer from Washington University, St. Louis, MO). ACS1/FATP1 are NIH 3T3 fibroblasts that are engineered to stably overexpress acyl-CoA synthetase 1 (ACS1) and long chain fatty acid (FA) transport protein 1 (FATP1) (33). These cells were selected for use because 1) they lack endogenous perilipins and HSL, and 2) overexpression of ACS1/FATP1 stimulates FA import and substantial TAG accumulation in lipid droplets (22). These features make ACS1/FATP1 cells powerful tools for elucidating the relative roles of adenovirally expressed perilipins in HSL and non-HSL(s)-mediated lipolysis.
ACS1/FATP1 cells in confluent cultures were transduced with either Ad Control (Ad GFP or Ad FA LoadingTransduced ACS1/FATP1 cells were incubated for 48 h of incubation in Dulbecco's modified Eagle's medium containing 5 mM glucose, 10% calf serum, and 1% fatty acid-free bovine serum albumin bound to palmitic and oleic acid (240 µM each). Incubation was terminated by removing lipid loading medium and washing cells with phosphate-buffered saline. Immunofluorescence MicroscopyImmunofluorescence confocal microscopy was performed (22) to determine the subcellular localization of perilipin truncations. MSM antibody (1:100 dilution) was used to immunolocalize Peri A, Peri B, and Peri 1300. PREK antibody (1:100) was used to localize Peri A, N-terminally deleted Peri A, and PKA site-mutated Peri A. Alexa Fluor 637-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR) was used to visualize perilipin staining. Bodipy 493/503 (Molecular Probes) was used at 10 µg/ml to visualize neutral lipid staining (34). LipolysisAfter transduction and 48 h of incubation with FA, the ACS1/FATP1 cells were washed with phosphate-buffered saline and treated for 4 h with or without a PKA activator, forskolin (20 µm) (35), in Dulbecco's modified Eagle's medium containing 5 mM glucose, 2% FA-free bovine serum albumin. The medium was collected, and glycerol was measured as an index of lipolysis as described (22). Western BlottingThe cellular proteins were extracted and quantified as described (36). The total lysates (10 µg/sample) were subjected to SDS-PAGE and Western blotting (37). Statistical AnalysesThe results are expressed as the mean ± S.E. The data were analyzed by one-way analysis of variance using Graph-Pad InStat Software (version 2.04a, Neoptolemos, University of Birmingham, Birmingham, UK). Multiple comparisons were performed with Tukey's honestly significant differences procedure. p values less than 0.05 were considered statistically significant.
Localization of Truncated and Mutated Perilipins on the Lipid Droplet SurfaceACS1/FATP1 cells were transduced with adenoviruses expressing either Peri A, Peri A truncations, Peri B (a naturally occurring Peri A truncation), or Peri A PKA site mutants. Because association with the lipid droplet membrane is a prerequisite for the ability of perilipin to regulate lipolysis, we initially assessed the ability of these perilipin constructs to target to and associate with lipid droplets. Transduced cells were fixed and prepared for confocal microscopy after incubation with FA for 48 h. Simultaneous fluorescent detection of neutral lipids and perilipins allowed us to correlate immunoreactivity with intracellular structure (Fig. 2). Neutral lipids were stained with Bodipy (red fluorescence). Perilipins were detected with specific antibodies directed against the N or C terminus of Peri A (green fluorescence). Immunostaining for Peri 1300, Peri 142517, Peri 233517, Peri 251517, and Peri 301517 revealed a ring-like pattern that was similarly observed with Peri A and Peri B (Fig. 2). The distinct perilipin rings appear localizing around the perimeters of lipid droplets as shown in the overlay of perlilipin and lipid staining. Coincident staining of perilipin and neutral lipid (yellow color) was also evident in the overlay. These staining patterns are clearly distinct from the diffuse cytoplasmic staining observed with Peri 1200, which serves as a negative control for perilipin association with lipid droplets. Thus, Peri 1300 and N-terminal truncated Peri A retain the ability to target and associate with lipid droplets. Mutation of PKA phosphorylation sites had no effect on the ability of Peri A to target and associate with lipid droplets, because Peri A 123, Peri A 456, and Peri A 16 all localized to the surface of lipid droplets (data not shown). No perilipin immunoreactivity was evident in control cells lacking perilipin expression constructs (data not shown).
Effects of Truncated/Mutated Peri A on Non-HSL-mediated LipolysisWe next investigated the relative roles of truncated and mutated Peri A in lipolysis mediated by non-HSL(s). ACS1/FATP1 cells (which do not express endogenous perilipins or HSL) were transduced with either Ad Control (Ad -gal or Ad GFP) or with adenoviruses expressing Peri A, Peri A truncations, or Peri B. Expression of various perilipins was confirmed with N or C terminus-specific antibodies (see Methods) (Fig. 3, A and B). Transduced cells were incubated for 48 h with FA, followed by FA removal and incubation for 4 h in the presence or absence of forskolin (20 µM), an activator of PKA (35). The extent of lipolysis was then determined by measuring glycerol release.
Compared with cells transduced with Ad Control (control), expression of Peri A lowered basal lipolysis by 60% (p < 0.001) (Fig. 3C). Expression of Peri B and Peri 1300, however, had no significant effect on basal lipolysis (p > 0.05 versus control). In contrast Peri 142517, Peri 201517, Peri 233517, Peri 251517, and Peri 301517 all lowered basal lipolysis to levels at or near that observed with full-length Peri A (p < 0.05 versus control) (Fig. 3C). These results demonstrate that the C-terminal region of Peri A (Peri 301517) inhibits basal lipolysis by non-HSL(s).
In forskolin-treated cells, expression of Peri A enhanced (rather than blocked) lipolysis
Phosphorylation of Peri A PKA sites (Fig. 1) is essential for PKA-stimulated lipolysis (22, 29, 30). The ability of Peri 301517 but not Peri 1300 to promote PKA-stimulated lipolysis (Fig. 3C) led us to hypothesize that 1) phosphorylation of one or more of Peri A PKA sites 46 was both necessary and sufficient to promote PKA-stimulated lipolysis by non-HSL(s), and 2) PKA sites 13 of Peri A, which are present in Peri B and Peri 1300, are not required to promote PKA-stimulated lipolysis by non-HSL(s). To test these hypotheses, we mutated PKA sites 13 (Peri A
As expected, Peri A enhanced lipolysis in forskolin-stimulated cells ( 2-fold) as compared with forskolin-stimulated (control) cells expressing -gal or GFP (p < 0.001) (Fig. 4B). Surprisingly, expression of Peri A 123, Peri A 16, or Peri A 456 failed to enhance lipolysis in forskolin-treated cells (p > 0.05 versus forskolin-treated control). These data strongly suggest that the ability of Peri A to facilitate PKA-stimulated lipolysis by non-HSL requires phosphorylation of one or more of PKA sites 13 in conjunction with phosphorylation of one or more of PKA sites 46. Mutation of PKA sites 13, 46, or 16 did not alter the ability of Peri A to inhibit (basal) lipolysis in the absence of forskolin (p > 0.05 versus Peri A) (Fig. 4B).
Effects of Truncated and Mutated Peri A on HSL-mediated LipolysisHaving identified the domains of Peri A that modulate non-HSL-mediated lipolysis, we examined the effects of truncated and mutated Peri A on HSL-mediated lipolysis. ACS1/FATP1 cells were co-transduced with either Ad control (
Expression of HSL alone increased basal lipolysis by 50% as compared with cells lacking HSL (Fig. 5B). Co-expression of Peri A with HSL reduced lipolysis ( 40%) to levels observed in the absence of HSL (p > 0.05 versus cells lacking HSL), confirming the ability of Peri A to block basal lipolysis mediated by HSL. Peri B and Peri 1300 also decreased basal lipolysis mediated by HSL (p < 0.05 versus HSL alone), although the magnitude of inhibition was approximately one-half that obtained with full-length Peri A (Fig. 5B). Reduced lipolysis in cells co-expressing HSL and either Peri 1300 or Peri B was manifest as increased intracellular TAG storage as compared with cells expressing HSL alone (data not shown). These results suggest that the N-terminal region of Peri A and Peri B (aa 1300) can significantly inhibit constitutive HSL lipolytic actions at the lipid droplet surface. Treatment with forskolin increased lipolysis by 2.5-fold in cells expressing HSL alone (p < 0.001 versus cells lacking HSL). Co-expression of HSL with either Peri A, Peri B, or Peri 1300 in forskolin-stimulated cells enhanced HSL-mediated lipolysis 2-fold (p < 0.001 versus cells expressing HSL alone). These results suggest that aa 1300 within Peri A and Peri B function to promote PKA-stimulated lipolysis by HSL. We next investigated modulation of HSL-mediated lipolysis by N-terminal Peri A truncations. The electrophoretic migration of HSL and N-terminal perilipin truncations on SDS-PAGE are presented in Fig. 6A. As above, HSL expression increased basal lipolysis (p < 0.001), and expression of Peri A abrogated this increase (p < 0.001) (Fig. 6B). Progressive N-terminal truncations of Peri A resulted in progressively reduced ability to inhibit HSL-mediated basal lipolysis, with no inhibition obtained with Peri 301517 (p > 0.05 versus control). This result suggests that a domain within the first 300 amino acids of Peri A is required to block constitutive HSL lipolytic action, a conclusion consistent with our demonstration that Peri 1300 can partially block basal lipolysis mediated by HSL (Fig. 5B).
We also investigated effects of perilipin N-terminal truncations on lipolysis in response to forskolin treatment. Forskolin dramatically increased lipolysis in cells expressing HSL and -gal as compared with cells expressing only -gal (control), presumably reflecting PKA-stimulated enhancement of HSL lipolytic activity (p < 0.001) (Fig. 6B). Peri A expression again resulted in enhanced lipolysis as compared with HSL alone. Similar increases in lipolysis were obtained with Peri 141517, Peri 201517, Peri 233517, Peri 251517, and Peri 301517 (p < 0.001). Thus, progressive N-terminal truncation of Peri A resulted in reduced basal lipolysis but no decrease in PKA-stimulated lipolysis. Considered together, our results indicate that the C-terminal region of Peri A (aa 301517) has no significant effect on HSL-mediated basal lipolysis, but it fully promotes PKA-stimulated lipolysis mediated by HSL.
Our truncation analyses revealed that both the N-terminal region of Peri A (aa 1300) containing PKA sites 13 and the C-terminal region of Peri A (aa 301517) containing PKA sites 46 can independently and fully promote PKA stimulated lipolysis by HSL. We therefore hypothesized that phosphorylation of one or more N-terminal PKA sites (sites 13) or one or more C-terminal PKA sites (sites 46) in Peri A is sufficient to promote PKA-stimulated lipolysis by HSL. Previously we (22) have demonstrated that mutation of PKA sites 13 blocked hormone-stimulated, HSL-mediated lipolysis, but no data are available on whether or not Peri A
As previously observed (Fig. 6B), HSL expression increased basal lipolysis (p < 0.001), and expression of Peri A blocked this increase (p < 0.001) (Fig. 7B). Forskolin increased lipolysis in cells expressing HSL as compared with cells expressing only -gal or GFP (non-HSL control) (p < 0.001), and Peri A expression further enhanced forskolin-stimulated, HSL-mediated lipolysis (> 2-fold) as compared with HSL expression alone (p < 0.001) (Fig. 7B). Notably, mutation of PKA sites 13 (Peri A 123) and PKA sites 16 (Peri A 16) completely abrogated the lipolysis-enhancing effect of Peri A in forskolin-treated cells (Fig. 7B). However, mutation of PKA sites 46 (Peri A 456) failed to diminish the ability of Peri A to promote PKA-stimulated lipolysis mediated by HSL. These results indicate that 1) phosphorylation of one or more of PKA sites 13 is required to facilitate PKA-mediated lipolysis by HSL, and phosphorylation of PKA sites 46, which are present in Peri 301517, is not required. PKA sites appear to play no role in regulating basal lipolysis mediated by HSL, because none of the three Peri A PKA site mutants tested exhibited impaired ability to block HSL-mediated lipolysis in the absence of forskolin (p < 0.001 versus HSL control) (Fig. 7B).
The present study provides a new framework for understanding how perilipins regulate lipid metabolism. In particular, the use of engineered ACS/FATP1 cells enabled us to identify a surprising degree of lipase selectivity among multiple, lipolysis-regulating domains of Peri A. An important strength of our approach was our use of Peri A truncations as well as full-length Peri A PKA site mutants. This approach allowed us to integrate how Peri A regulatory domains and PKA sites functioned together to regulate lipolysis. Our data suggest a model (Fig. 8) in which 1) specific Peri A protein domains selectively act as barriers against HSL and non-HSLs, respectively; 2) phosphorylation of different PKA sites is required for PKA-stimulated lipolysis mediated by HSL and non-HSL(s); and 3) additional domains facilitate PKA-stimulated lipolysis, again with lipase selectivity.
The barrier function of Peri A maps to two lipase-specific domains; Peri 1300 blocks HSL, and Peri 301517 blocks non-HSL(s) (Fig. 8). Thus, it was not totally unexpected that differential patterns of Peri A phosphorylation are required for PKA-stimulated lipolysis mediated by HSL versus non-HSL(s). However, whereas phosphorylation of one or more of PKA sites 13 is required to stimulate HSL-mediated lipolysis, phosphorylation of one or more of PKA sites 46 and 13 in full-length Peri A is required to stimulate non-HSL-mediated lipolysis (Fig. 8). This suggests that HSL and non-HSL(s) interact with the lipid droplet surface and gain access to stored TAG by different means. Intriguingly, Peri 301517 does not contain sites 13, yet it promotes PKA-stimulated lipolysis by non-HSL(s). This observation suggests that 1) non-HSL-mediated, PKA-stimulated lipolysis requires a conformational change in the C terminus of Peri A that is induced by phosphorylation of N-terminal PKA sites 13, and 2) truncation of aa 1300 results in that conformational change. Our data indicate that the domains of Peri A that facilitate PKA-stimulated lipolysis can be separate from the domains that function as barriers to lipases. For example a domain within Peri 301517 distinct from PKA sites 46 can facilitate PKA-stimulated lipolysis by HSL (Fig. 7B), yet Peri 301517 fails to block HSL-mediated basal lipolysis (Fig. 6B). One potential explanation for this observation is that Peri 301517 contains a "docking site" or other facilitory domain for HSL that in full-length Peri A becomes accessible to the lipase only after phosphorylation of PKA site 1, 2, or 3. In contrast to Peri 301517, Peri 1300 functions as both a barrier to and facilitator of HSL actions (Fig. 5B). It is plausible that Peri 1300 also contains an additional domain that becomes accessible to HSL after phosphorylation of PKA sites 13. The present study provides the first demonstration that Peri B is biologically active as a regulator of lipolysis. The HSL selectivity of Peri B reflects the HSL-directed barrier function of aa 1300 (inhibition of basal lipolysis), the role of PKA sites 13 in PKA-stimulated lipolysis, and the lack of C-terminal domains and PKA sites (sites 46) that regulate non-HSL(s). The identity of aa 301405 in Peri A and Peri B argue that the unique C-terminal domain(s) of Peri A that regulates non-HSL(s) is contained within Peri 405517. While our manuscript was in preparation, Tansey et al. (23) reported that the C-terminal region of Peri A inhibits basal lipolysis and that PKA sites 13 promote PKA-stimulated lipolysis (PKA sites 46 were not examined). They also reported that Peri B had no significant effect on either basal or PKA-stimulated lipolysis. These observations into perilipin function were made in Chinese hamster ovary cells, which do not express HSL. Perilipin function requires association with the lipid droplet. Our confocal studies (Fig. 2) demonstrate that neither the perilipin N terminus (aa 1300), C terminus (aa 301517), nor PKA sites are required to target Peri A to lipid droplets. However, Peri 1141 (data not shown) and Peri 1200 failed to localize to the lipid droplet (Fig. 2). Peri 1141 contains the conserved PAT-1 region found in other lipid droplet proteins such as adipose differentiation-related protein and TIP 47 (27). The inability of Peri 1141 and Peri 1200 to localize to the lipid droplet confirms that the PAT-1 region is insufficient to target perilipins to the lipid droplet (34). Considered together, our confocal data support the recent identification of three putative lipid droplet-binding regions of Peri A between aa 233 and 364 (34). The present study elucidates the structural features of perilipin that regulate adipocyte lipolysis, with surprising lipase specificity. A complete understanding of how perilipin regulates the molecular interplay among HSL and non-HSLs remains a challenge for the future. The importance of this challenge is underscored by recent studies linking reduced perilipin expression to dysregulated lipolysis in obese subjects (38, 39) and by the association of increased circulating free fatty acids with the development of insulin resistance and type 2 diabetes (9, 20).
* This work was supported in part by the United States Department of Agriculture under Agreement 581950-9-001, by National Institutes of Health Grant DK 50647 (to A. S. G.), and by research awards from the American Diabetes Association (to A. S. G.). This work was supported in part by the Molecular Biology Core of the Gastroenterology Research on Absorptive and Secretory Processes, Grant P30 DK 34928. Portions of this work were presented at the 63rd (2003) Annual Meeting of the American Diabetes Association. 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. ¶ To whom correspondence should be addressed: JM USDA/HNRCA at Tufts University, Rm. 603, 711 Washington St., Boston, MA 02111. Tel.: 617-556-3144; Fax: 617-556-3224; E-mail: andrew.greenberg{at}tufts.edu.
1 The abbreviations used are: TAG, triacylglycerol; PKA, cAMP-dependent protein kinase; HSL, hormone-sensitive lipase; Peri, perilipin; aa, amino acids; Ad, adenovirus(es) expressing;
We are grateful to Dr. Jean E. Schaffer for providing the ACS1/FATP1 cells.
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