Seeing the trees in the forest: selective electroporation of adipocytes within adipose tissue

James G. Granneman, Pipeng Li, Yuyan Lu, and Jacqueline Tilak

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute <20% of cells residing in adipose tissue, we hypothesized that fat cells might be susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. Results demonstrate the feasibility of electroporating DNA into mature fat cells with >99% selectivity over other cells in the tissue. Further experiments used the "adiporation" technique to image the subcellular targeting of fluorescent bioreporter molecules to the nucleus, mitochondria, and lipid droplets of adipocytes within intact adipose tissue. Finally, we utilized fluorescent bioreporters to examine the effects of constitutive activation of the {beta}-adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat {beta}1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of {beta}3-adrenergic receptor agonists. Hallmarks of the altered morphology include pronounced fragmentation of the single lipid droplet, repositioning of the nucleus, and induction of mitochondrial biogenesis. These results indicate that activation of {beta}-adrenergic signaling within adipocytes is sufficient to induce a phenotype that resembles typical brown adipocytes and suggest that in vivo electroporation will allow molecular dissection of the mechanisms involved.

lipid droplet; morphology; perilipin; subcellular targeting; plasticity; mitochondrial biogenesis; gene transfer; biological imaging


WORK OVER THE LAST DECADE has established the significance of adipocytes in the regulation of body energy homeostasis (7). Not only are adipocytes the principal energy reservoir in mammals, but these cells also regulate various physiological functions through the secretion of numerous adipokines, like leptin and adiponectin. Analysis of fat cell function by acute genetic manipulation has relied on use of primary cell culture and established cell lines. Although use of cultured cells has provided invaluable information regarding fat cell differentiation and metabolism, these in vitro models have several limitations compared with adipocytes in intact tissue, including low phenotypic gene expression of continuous cell lines and rapid dedifferentiation of freshly isolated cells (4, 12, 15). These and other data indicate that full phenotypic expression and morphology require important interactions with the extracellular matrix of the intact tissue that are difficult, if not impossible, to replicate in vitro and point to the need for developing techniques for adipocyte-specific gene transfer in vivo.

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 40–80 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.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Animals. Experiments were performed with C57/Bl6 mice that were obtained from Jackson Labs and bred at Wayne State University (WSU). Animals of either sex were used at 2–4 mo of age. Animal use was approved by the Wayne State University Institutional Animal Care and Use Committee.

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 1–2 mm. The needle was first inserted to a length of 5–7 mm, and injection was made while the needle was slowly withdrawn. Immediately after injection, the injection site was flanked (1–2 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 {Omega}. 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 {beta}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 {beta}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 3–7 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 200–800 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).


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Adipose tissue contains a complex mixture of mature fat cells and stromal cells that are more numerous but much smaller. Adipose tissue appears, in standard 6-µm-thick paraffin sections (Fig. 1A), to be relatively homogeneous, consisting mainly of mature adipocytes. However, because the average diameter of the mature adipocytes is >10 times greater than the nucleus, it is clear that most of the nuclei stained in each section do not belong to fat cells. We calculate, on the basis of the cell size, that mature fat cells account for only 15.6 ± 1.3% of the tissue nuclei. Endothelial cells, which can be readily identified by elongated nuclei and association with capillaries, constitute 45%, with the remainder of cells classified as stromal cells. Because the cytoplasm of stromal cells is very scant and poorly stained by eosin, it is difficult to differentiate the border between these cells and the mature fat cells with which they are closely associated.



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Fig. 1. Cellular complexity of adipose tissue. A: 6-µm-thick paraffin section of epididymal adipose tissue stained with hematoxylin and eosin. B: 30-µm-thick paraffin section stained with Hoescht 33258. Image shows several fluorescent nuclei of stromal cells surrounding each nonfluorescent adipocyte. C: intact suprascapular white adipose tissue (SSWAT) stained with concanavalin A-conjugated Alexa 488. Concanavalin A preferentially labeled numerous stromal cells in adipose tissue. D: x600 magnification of intact SSWAT stained with concanavalin A-conjugated Alexa 488. Shown is the close apposition of several concanavalin-labeled stromal cells (arrows) with a single adipocyte. Bar, 25 µm. Wide-field fluorescence images of B–D were deconvolved and rendered as detailed in MATERIALS AND METHODS.

 
The complexity of adipose tissue can be clearly shown using fluorescence microscopic analysis of 30-µm-thick paraffin sections (Fig. 1B) or intact tissue (Fig. 1, C and D). Staining of nuclei with Hoechst 33258 illustrates that 4–8 stromal/vascular cells surround each fat cell (Fig. 1B). To visualize stromal/vascular cells in situ, fixed intact tissue was stained with Alexa 488-conjugated concanavalin A, and images were collected using wide-field fluorescence microscopy. Three-dimensional rendering of the z-axis images identified numerous labeled stromal cells that were closely apposed to mature adipocytes within adipose tissue (Fig. 1, C and D). Although these cells are the most abundant cell type in the tissue, their diameters and surface areas are, respectively, nearly one and two orders of magnitude smaller than those of typical mature adipocytes.

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 1–3 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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Fig. 2. Selective electroporation of enhanced green fluorescent protein (eGFP) and DsRed plasmids into mature adipocytes within SSWAT. A: wide-field fluorescence image of SSWAT transfected with eGFP showing numerous transfected adipocytes, each with a single labeled nucleus. Bar, 50 µm. B: expression of DsRed, illustrating the cytoplasmic distribution and the presence of numerous small lipid droplets. Bar, 25 µm. C: fluorescence image of electroporated SSWAT in which nuclei of all cells are stained with Hoescht 33258. D: eGFP fluorescence of same field as C, illustrating the specificity of electroporation for mature adipocytes (arrows). Bar, 25 µm.

 
Several variables contribute to the numbers of cells transfected using electroporation, including the amount of DNA injected, charge density, and pulse duration and number. The area of successful electrotransfer is limited to the area between the electrode surfaces; thus the number of cells transfected is strongly influenced by the electrode configuration. Although electrodes with larger conductive surfaces can transfect hundreds of cells per electroporation, their use can be unwieldy. By contrast, fine bipolar forceps like those used here transfect somewhat fewer cells (typically 20–60 cells) in a highly defined region. Because relatively low charge densities are required to transfect, little cellular damage was noted, as evidenced by the uptake of Mitotraker Red in adjacent nontransfected cells (not shown).

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 (40–80 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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Fig. 3. Cellular distribution of eGFP-perilipin in adipocytes tranfected in vivo. A and B: sequential confocal optical slices of eGFP-perilipin fluorescence in typical unilocular fat cells. Slice A is superficial to B and reveals numerous lipid droplets at the the fat cell surface. Slice B is through the central portion of the major lipid storage droplet (LSD). Note the absence of fluorescence beneath the area occupied by the nucleus (A) and within the major lipid droplet (B). C: 3-dimensional rendering of a series of z-axis confocal slices of adipocytes expressing eGFP-perilipin. eGFP-perilipin is strongly targeted to small lipid droplets that are not uniformly distributed over the surface of the major LSD. D: immunohistochemical detection of endogenous perilipin in control (nonelectroporated) WAT.

 
Nrbf1 is a novel 2-enoyl-CoA reductase that contains a consensus sequence for mitochondrial targeting (13). As expected, the NH2-terminal 42 amino acids of Nrbf1 targeted eGFP to mitochondria (Fig. 4), as confirmed by double-labeling mitochondria with streptavidin Alexa 555 (Fig. 4C). Deconvolution and three-dimensional rendering of wide-field adipocyte images clearly illustrate the nonuniform distribution of adipocyte mitochondria (Fig. 4, B–D). Numerous mitochondria surrounded the eccentric nucleus, which was labeled by cotransfection with DsRed-HNF4 fusion protein (Fig. 4D). Although some mitochondria seem to be randomly distributed in the thin cytoplasm, many of the mitochondria that do not surround the nucleus appear to cluster along string-like structures that radiate along the surface of the intracellular lipid droplet.



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Fig. 4. Fluorescence imaging of mitochondria in electroporated adipocytes. A: low-magnification image showing numerous mature adipocytes expressing Nrbf1-eGFP fusion protein. Bar, 75 µm. B: high-magnification image of A, illustrating the nonuniform distribution of Nrbf1-eGFP fluorescence, including perinuclear accumulation. Bar, 25 µm. C: mitochondria labeled with Nrbf1-eGFP (top) and strepavidin-Alexa 555 (bottom). Bar, 25 µm. D: merged pseudocolor images of an adipocyte electroporated in vivo with Nrbf1-eGFP and hepatocyte nuclear factor (HNF)4-DsRed plasmids labeling mitochondria (green) and the nucleus (red). Note perinuclear localization of mitochondria. Bar, 25 µm.

 
Effects of {beta}-adrenergic receptor activation on adipocyte morphology and distribution and expression of mitochondria within adipocytes in vivo. Recent work has demonstrated that chronic activation of adipocyte {beta}3-adrenergic receptors induces cellular plasticity within adipose tissue, including the appearance of multilocular adipocytes that contain numerous mitochondria and resemble brown adipocytes (5, 9). The origin and function of these cells have been controversial. Himms-Hagen et al. (9) suggested that these relatively small multilocular adipocytes are derived from large, mature adipocytes and that the induction of mitochondrial biogenesis reflects a novel metabolic plasticity of mature white adipocytes. On the other hand, the expression of UCP1 in adipose tissue of animals treated with {beta}3-adrenergic receptor agonists raises the possibility that some of the small multilocular cells represent newly recruited brown fat cells derived from stromal progenitors within typical white fat depots (8). Traditional histological approaches are static measures that cannot address track cell fate over time. In contrast, electroporation is highly selective for mature fat cells and offers a means of tracking the fate of these cells.

Three groups of animals were used to evaluate adipocyte fate during {beta}-adrenergic receptor activation. The SSWAT of all mice was electroporated with an expression plasmid encoding eGFP. One group of mice was infused with the {beta}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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Fig. 5. {beta}-Adrenergic receptor activity alters fat cell morphology. Adipose tissue was electroporated with eGFP bioreporter plasmid. Control mice received vehicle infusion, whereas CL mice were infused with 3 nmol/h CL-316234 for 3 days. A 3rd group of mice was cotransfected with an expression vector encoding the rat {beta}1-adrenergic receptor ({beta}1AR). Top: representative fluorescence micrographs. Bottom: quantitation of cells exhibiting a multilocular phenotype after 3 days.

 
One prominent feature of chronic pharmacological activation of {beta}3-adrenergic receptors is massive generation of mitochondria within adipose tissue (2, 5, 9). It is not known whether this mitochondrial biogenesis involves the direct activation of adipocytes or indirect systemic effects of the agonist. In addition, it has been debated whether the cells undergoing mitochondrial biogenesis are mature adipocytes or newly recruited brown adipocytes. The observations above indicate that the combined use of fluorescent bioreporter molecules and constitutively active signaling molecules might offer a technical approach to address these questions. First, directing constitutively active molecules to adipocytes avoids systemic effects that are often produced by pharmacological probes. Furthermore, because the electrotransfer protocol is highly selective for mature adipocytes, it can be used to trace the fate of mature fat cells during {beta}-adrenergic receptor activation and, using mitochondrially targeted eGFP, evaluate whether {beta}-receptor activation is sufficient to affect the distribution and abundance of mitochondria in white adipocytes.

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 {beta}-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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Fig. 6. Expression of Adrb1 alters subcellular distribution of mitochondria. Shown are wide-field fluorescence images of Nrbf1-eGFP. Left: control cell transfected with Nrbf1-eGFP. Right: adipocyte cotransfected with Adrb1 and Nrbf1-eGFP. Bars, 25 µm.

 


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Fig. 7. Constitutive activation of {beta}-adrenergic receptors alters cellular morphology and induces mitochondrial biogenesis. A: Nrbf1-eGFP fluorescence of control adipocytes cotransfected with empty vector. B: streptavidin-Alexa 555 fluorescence of cells in A captured at same charge-coupled device settings used in D. C: Nrbf1-eGFP fluorescence of adipocyte cotransfected with rat Adrb1 and Nrbf1-eGFP. D: streptavidin-Alexa 555 fluorescence of C. E and G: Nrbf1-eGFP fluorescence of cells cotransfected with Adrb1. F and H: corresponding streptavidin-Alexa 555 fluorescence. Bars, 25 µm.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In vivo electrotransfer is a relatively new technique that has numerous applications and certain advantages over other methods of somatic cell gene transfer (16). Adipose tissue contains a complex mixture of cell types of which relatively few cells are mature adipocytes. Nevertheless, the present study demonstrates that electrotransfer in SSWAT achieves highly selective transfection of mature adipocytes. Although the mechanisms for selective transfection of adipocytes cannot be stated with certainty, it is likely that the well-established inverse relation between cell size and critical voltage for generating electropores plays an important role. It is also possible that the much greater cell surface of adipocytes (up to 100-fold greater than stromal cells) contributes to selectivity, owing to greater cellular access to injected DNA. Regardless of mechanism, the ability to genetically manipulate adipocytes in vivo has numerous applications.

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 10–50 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 {beta}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 {beta}-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 {beta}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.


    GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-062292 and DK-066505, and Michigan Economic Development Corporation Grant MLSC-27. Use of the Microscopy and Imaging Resources Laboratory at WSU was supported by National Institutes of Health Grants P30-ES-06639 and P30-CA-22453.


    ACKNOWLEDGMENTS
 
We thank Drs. Todd Leff and Robert MacKenzie for helpful comments, Dr. A. Chaudhry for access to the confocal microscope, and Rae Granneman for graphics assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Granneman, CIMER/WSU School of Medicine, Scott Hall, Detroit, MI 48201 (E-mail:jgranne{at}med.wayne.edu).

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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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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