Center for Integrative Metabolic and Endocrine Research, Departments of Pathology and Psychiatry, Wayne State University School of Medicine, Detroit, Michigan 48201
Submitted 12 December 2003 ; accepted in final form 30 April 2004
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ABSTRACT |
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lipid droplet; morphology; perilipin; subcellular targeting; plasticity; mitochondrial biogenesis; gene transfer; biological imaging
Adipose tissue is a complex tissue that contains several cell types in addition to lipid-laden adipocytes, including those that constitute the vasculature (endothelial and vascular smooth muscle) and adipocyte progenitors. Of the cells residing in adipose tissue, mature adipocytes are by far the largest, with diameters averaging 4080 µm, depending on the age of the animal. Thus typical mature adipocytes have diameters that are 10-fold greater, and cell surface areas 100-fold greater, than those of other closely-associated cells in the tissue.
Electroporation has been recently adapted as a technique for introducing foreign macromolecules, like DNA, into cells in vivo (16). The technique offers certain advantages over other somatic gene transfer techniques, such as viral vectors, pressure injection, and particle bombardment, and has numerous applications, including cell lineage tracing and analysis of signaling pathways in vivo. The factors affecting successful electroporation of cell suspensions have been studied extensively, and one parameter that has a major influence on the formation of membrane electropores is cell size: all other things being equal, larger cells are electroporated at lower critical field strengths (3). This well-established relationship in vitro suggested the possibility of selectively introducing DNA into mature adipocytes within adipose tissue in vivo. Below, we describe a technique for introducing DNA expression vectors into adipocytes within adipose tissue with greater than 99% selectivity. The technique has numerous applications, including visualizing three-dimensional cellular morphology in situ, lineage tracing, and functional analysis.
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MATERIALS AND METHODS |
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Standard electroporation protocol. Mice were anesthetized with Avertin, and skin in the interscapular region was removed of fur and disinfected with alcohol. A 15-mm incision was made along the midline, and the skin overlying suprascapular white adipose tissue (SSWAT) was gently dissected so that an approach to this subcutaneous pad could be easily made on either side.
Plasmid DNA was reconstituted in sterile water at a total concentration of 1 µg/µl, and injections (7 µl) were made into the SSWAT pad with a 26-gauge needle and a Hamilton microliter syringe. Injections were made parallel to the surface of the pad at a depth of 12 mm. The needle was first inserted to a length of 57 mm, and injection was made while the needle was slowly withdrawn. Immediately after injection, the injection site was flanked (12 mm on either side) with Gerald bipolar forceps (1.5 x 8 mm, model ES-300C; Anthony Products, Indianapolis, IN) and gently gathered to a gap width of 1.0 mm.
Electroporation was accomplished using a Grass S88 stimulator (Natick, MA). A range of electrical parameters was evaluated in preliminary experiments. From these experiments, a standard DNA electroporation protocol was devised in which seven square-wave pulses were delivered over 3 s. Each pulse was 50 V and lasted 20 ms, with the internal resistance of the stimulator set at 250 . Usually two electroporations were made in each (right and left) SSWAT pad. In this way, control and experimental plasmids could be tested in contralateral pads of the same animal.
In one experiment, mice were implanted at the time of electrotransfer with osmotic minipumps (Alzet) that delivered the 3-adrenergic receptor agonist CL-316243 at a rate of 3 nmol/h. In parallel experiments, mice were electroporated with an expression plasmid encoding the rat
1-adrenergic receptor (Adrb1) along with a fluorescent reporter gene [enhanced green fluorescent protein (eGFP) or Nrbf1-eGFP fusion] at a 4:1 ratio. Tissue was harvested 3 days after electrotransfer. Preliminary experiments with cotranfections of eGFP and DsRed plasmids mixed, respectively, at a 4:1 ratio demonstrated that all DsRed-positive cells were also positive for eGFP.
SSWAT was dissected 37 days after electrotransfer and fixed in 4% paraformaldehyde. The region of electroporation occurred along the injection site, between the 1-mm electrode gap. When eGFP reporters were used, this region could be readily identified by fluorescence microscopy owing to the shallow depth at which the injections were made and the translucency of the tissue. Underlying interscapular brown adipose tissue, which was not targeted, did not contain transfected cells. Brown fat was readily distinguished from white fat by its intense FAD autofluorescence and the presence of multilocular cells with diameters smaller than 20 µm. The suprascapular region was carefully dissected, placed between coverslips in a Leiden chamber, and visualized directly without further processing with wide-field or confocal fluorescent microscopy.
Perilipin immunohistochemistry was performed on fixed tissue cut to a thickness of 200800 µm. Tissue sections were blocked with 0.2 M glycine in phosphate-buffered saline (PBS) containing 100 ng/ml saponin and then incubated overnight at 4°C with perilipin antisera diluted 1:1,000 in PBS containing saponin and 5% normal goat serum. Perilipin antiseum was provided by Dr. A. Chaudhry (Parke-Davis, Ann Arbor, MI). Binding of the primary antibodies was detected by incubating sections with goat anti-rabbit antibodies (1 µg/ml) conjugated to Alexa 555 (Molecular Probes, Eugene, OR).
Microscopy and image analysis. Wide-field microscopy was performed using an Olympus IX81 microscope with x20 (0.7 NA), x40 (0.85 NA), and x60 (0.90 NA) objective lenses. Images were captured with a Retiga 1300 cooled charge-coupled device camera. Wide field z-axis images were deconvolved using the point spread function algorithm and rendered in three dimensions with Microtome and VoxBlast imaging software (Vaytek, Fairfield, IA). Confocal images were collected on an Olympus Fluoview (x100, 1.40 NA objective) or a Zeiss LSM 310 (x63, 1.25 NA objective), as previously described (5). Postcapture image processing was limited to contrast and brightness adjustments performed using Image Pro Plus (Media Cybernetics) or Photoshop (Adobe). Cell diameters were determined using Image Pro Plus software.
Plasmids and fluorescent probes. Expression vector for eGFP was obtained from Clontech, and COOH-terminal fusion of eGFP with full-length mouse perilipin was obtained from Dr. A. Chaudhry (Parke-Davis) and hepatocyte nuclear factor-4 (HNF4)-DsRed fusion protein from Dr. U. Varanasi (WSU). A mitochondrially targeted eGFP was created by fusing the NH2-terminal 42 amino acids of mouse Nrbf1 (GenBank accession no. NM_025297) to eGFP (Granneman JG, in preparation). Expression plasmid containing rat Adrb1 has been described (6).
Hoechst 33258 (3 µg/ml) was used to stain cell nuclei within tissue. Stromal cells were stained by incubating fixed tissue with Alexa 488-conjugated concanavalin-A (12 µg/ml, Molecular Probes) dissolved in PBS containing 0.1% Triton X-100. Mitochondria were visualized via the binding of streptavidin-conjugated Alexa 555 (1 µg/ml dissolved in PBS containing 0.1% Triton X-100, Molecular Probes) to mitochondrial biotin-containing proteins (10).
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RESULTS |
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In vivo electroporation selectively transfects adipocytes within adipose tissue. The critical electric-field strength for generating membrane electopores is inversely proportional to cell size (3). Given the large size of mature fat cells relative to other cells in the tissue, we reasoned that it might be possible to selectively electroporate differentiated fat cells in adipose tissue containing a complex mixture of cell types. SSWAT was electroporated with 7 µg of an expression vector encoding eGFP or DsRed, as detailed in MATERIALS AND METHODS, and tissue was examined 13 days later by fluorescence microscopy. Numerous eGFP- and DsRed-positive adipocytes were observed in low-magnification images (Fig. 2, A and B). eGFP was largely concentrated in the cell nucleus, whereas DsRed was more uniformly distributed around the thin cytoplasm that surrounds the major lipid storage droplet. Staining the intact tissue with Hoechst 33258 illustrates the cellular complexity of the tissue (Fig. 2C), whereas eGFP fluorescence images of the same field demonstrate the selective transfection of mature adipocytes (Fig. 2D). To quantitate the selectivity of the electrotransfer to mature fat cells, eGFP-positive cells were sampled from eight independent electrotransfer experiments. Of the 1,801 eGFP-positive cells encountered, 1,789 were identified as mature adipocytes on the basis of morphology and illumination of a single nucleus per cell. Thus the electroporation protocol detailed above transfects mature adipocytes with >99% selectivity.
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Assuming that all cells residing between the electrode surfaces are available for electrotransfer, we calculate that <1% of available adipocytes are transfected under the conditions described. Because cell size is an important variable, we examined whether successful electroporation might be biased toward larger fat cells by comparing the diameters of eGFP-positive cells with those of immediately surrounding eGFP-negative adipocytes. In three independent experiments, the mean diameter of transfected adipocytes was 101.5 ± 3.6% of adjacent nontransfected cells. Thus, although the electrotransfer technique was highly selective for adipocytes, it did not appear to be biased toward fat cells of a certain size.
Subcellular targeting of fluorescent reporter molecules in transfected adipocytes in vivo. Because of their large size, analysis of adipocyte morphology and imaging of the subcellular distribution of organelles and proteins are difficult, if not impossible, using traditional histological approaches. Thus relatively little is known about the subcellular distribution of organelles and proteins in fat cells within adipose tissue. Work in the neural tissue, however, has demonstrated the utility of fluorescence imaging techniques to "illuminate" three-dimensional structures, like neuronal arbors, in relatively thick, complex biological samples. To demonstrate the feasibility of this approach, adipocytes within SSWAT were electroporated with expression vectors designed to target eGFP to lipid droplets (eGFP-perilipin) or mitochondria (Nrbf1-eGFP). We also examined the targeting of DsRed fused to HNF4, a nuclear transcription factor. Three days after electrotransfer, cells were imaged using wide-field or confocal fluorescence microscopy.
A major function of white adipocytes is the sequestration and storage of neutral lipids that occur in intracellular lipid storage droplets (LSDs). Very little is known about the structure or dynamics of LSDs in adipocytes of adipose tissue. One means of visualizing LSDs in adipocytes is through the use of fluorescent proteins fused to proteins, like perilipin, that coat LSDs (14). Electroporation of eGFP-perilipin in vivo revealed a heterogeneity of lipid droplet structures not predicted by standard histological approaches. Optical sectioning of adipose tissue by confocal microscopy clearly revealed numerous small lipid droplets interposed between the surface of the major LSD and the plasma membrane (Fig. 3). Figure 3, A and B, shows sequential confocal slices illustrating fluorescence along the major LSD as well as strong targeting to numerous small lipid droplets. The small lipid droplets were not uniformly distributed and were excluded from the area between the nucleus and major storage droplet. Figure 3C is a three-dimensional projection of a series of z-axis confocal slices indicating that eGFP-perilipin is strongly targeted to small lipid droplets that are not uniformly distributed over the surface of the major LSD. The subcellular targeting pattern of eGFP-perilipin was highly similar to that of endogenous perilipin, detected by immunohistochemistry, in nonelectroporated adipose tissue (Fig. 3D). These images suggest that adipocytes contain two pools of triglyceride: triglyceride contained in the large (4080 µm) "unilocular" LSD and triglyceride present in the numerous small surface droplets that interface between the major droplet and the plasma membrane.
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Three groups of animals were used to evaluate adipocyte fate during -adrenergic receptor activation. The SSWAT of all mice was electroporated with an expression plasmid encoding eGFP. One group of mice was infused with the
3-receptor agonist CL-316243, whereas another group received vehicle infusion. A third group of mice was cotransfected with an expression plasmid encoding the rat Adrb1, which exhibits constitutive activity when overexpressed in vitro (Granneman JG, unpublished observations), along with the eGFP tracer plasmid. Three days later, adipose tissue was dissected, and cell morphology of eGFP-positive cells was evaluated using fluorescence microscopy.
As shown in Fig. 5, 85% of eGFP-positive cells were typical fat cells containing a single large LSD. After treatment with CL-316243 or transfection with the rat Adrb1, the percentage of cells exhibiting a unilocular phenotype dramatically decreased, whereas those with a multilocular phenotype increased more than fivefold (P < 0.001). It should be noted that the lipid droplets of multilocular cells detected by eGFP at low magnification are distinct from the small surface droplets that are targeted by eGFP-perilipin. Image analysis of adipocytes indicated that expression of rat Adrb1 decreased cell diameter by 18 ± 5% in 3 days (P < 0.001), translating to a 45% decline in cell volume. It is noteworthy that, as cells lost lipid, the eccentric nucleus became positioned near the center of the cell and began to resemble a typical brown adipocyte.
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As shown in Fig. 6, expression of Adrb1 had marked effects on the subcellular distribution and apparent abundance of mitochondria in white adipocytes. In control cells, mitochondria were sparsely distributed on the surface of the lipid droplet and in the perinuclear area (Fig. 6, left, and Fig. 4). Chronic activation of -adrenergic signaling altered the subcellular distribution of Nrbf1-eGFP fluorescence such that labeled mitochondria nearly completely surrounded fragmented lipid droplets. To determine whether the elevation of Nrbf1-eGFP fluorescence might represent mitochondrial biogenesis, tissue mitochondria were stained with fluorescent streptavidin (Fig. 7). By considering only cells that were eGFP positive, it is clear that expression of Adrb1 strongly enhanced streptavidin staining (compare Fig. 7, B with D). Cotransfected cells also had much greater streptavidin staining compared with all neighboring eGFP-negative cells that retained a unilocular appearance (Fig. 7, D and F). Occasionally, elevated streptavidin fluorescence was observed in multilocular eGFP-negative cells that were near cells cotransfected with Adrb1 (Fig. 7H). Such cells were not observed in control tissue. Noting that Adrb1 was transfected at a 4:1 excess over eGFP, it is likely that these cells expressed Adrb1 (but not eGFP) not only because of the intense mitochondrial staining but also because of the multilocular phenotype.
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DISCUSSION |
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One application is the ability to view the three-dimensional distribution of fluorescent reporter molecules in adipocytes in their native environment. Traditional histological approaches to adipocyte morphology employ physical sectioning of tissue; however, standard sections, which are usually less than one-tenth of a fat cell diameter, are ill suited for appreciating the three-dimensional structure of fat cells, whereas sections thick enough to accommodate the large size of fat cells are difficult to interpret, owing to cellular complexity of the tissue. In contrast, fluorescent bioreporter molecules essentially "illuminate" individual adipocytes in situ, thereby eliminating interfering cellular complexity, whereas confocal or wide-field/deconvolution microscopy allows optical sectioning and three-dimensional rendering.
The present study demonstrates the utility of this approach using eGFP fusion proteins targeted to distinct subcellular domains. Visualization of eGFP-perilipin fluorescence demonstrated complex lipid droplet structures in mature adipocytes. Standard histology of adipose tissue, which involves lipid extraction by organic solvents, indicates the presence of a single LSD in mature adipocytes. In contrast, fluorescence imaging indicates the presence of numerous small lipid droplets between the major storage droplet and the plasma membrane. The heterogeneity of these surface droplets was confirmed by immunohistochemistry with perilipin antibodies and thus does not appear to be an artifact of electroporation. The small surface lipid droplets observed in the present study are strikingly similar to those defined with antibodies to cannexin, a marker of the endoplasmic reticulum (1). It is thought that the nascent LSD is formed as lipid accumulates within the lumen of the endoplasmic reticulum (1). The surface-to-volume ratio of the surface droplets is 1050 times greater than the major LSD, and recent proteomic analysis in Chinese hamster ovary cells suggests involvement of small droplets in transport and metabolism (11). It is thus tempting to speculate that these structures represent a dynamic interface that regulates triglyceride flux and that eGFP-perilipin might be useful in monitoring this process. More generally, several eGFP-based fusion proteins have been used to evaluate subcellular targeting in cultured cell models. The present technique allows in vivo confirmation of in vitro results and offers the possibility of examining subcellular targeting and translocation in vivo (or ex vivo) under physiological and pathophysiological conditions.
We and others have reported that chronic stimulation of 3-adrenergic receptors induces novel cellular plasticity in adipose tissue involving mitochondrial biogenesis and induction of genes of lipid oxidation (5, 9). The present study applied selective electroporation to the study of cellular plasticity of white adipocytes and demonstrates that chronic activation of
-adrenergic receptor signaling in mature adipocytes is sufficient to induce a cellular phenotype that resembles brown adipocytes. Features of the phenotype include multiple lipid droplets, numerous mitochondria, and a central nucleus. The ability to induce phenotypic conversion in individual transfected cells indicates that, although
3-receptor agonists can have multiple systemic effects in vivo, these effects are not required for phenotypic transformation. Studies are underway to further dissect the pathways involved using constitutively active and dominant negative constructs. In this regard, electrotransfer of plasmid-based short interfering RNA vectors should greatly expand the ability to evaluate signaling pathways of adipocytes in vivo.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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