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

Abstract

Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute Ͻ20% 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 Ͼ99% 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 ␤-adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat ␤1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of ␤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 ␤-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 ...

Thematic review series: Adipocyte Biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis

“…2); large perinuclear lipid droplets fragment into myriad perilipin A-coated microlipid droplets that disperse throughout the cytoplasm (72). Similar fragmentation of large lipid droplets has been observed in adipocytes in tissue dissected from rats after systemic infusion of b 3 -adrenergic receptor agonists or after electroporation of b 1 -adrenergic receptors into the fat pads of live rats (71).…”

Perilipin Targets a Novel Pool of Lipid Droplets for Lipolytic Attack by Hormone-sensitive Lipase

“…Rabbit anti-Plin and rabbit anti-HSL antibodies were kindly provided by Dr. A. Chaudhry (Pfizer, Ann Arbor, MI). Plin antibody was developed against the N-terminal 15 amino acids of rat Plin (12) and the HSL antibody was that described by Syu and Saltiel (15). The specificities of these antibodies were confirmed using 293T cells transfected with plasmids encoding Plin or HSL; in each case, the antibodies produced a positive immunofluorescence signal only from cells had been transfected with the appropriate construct (data not shown).…”

“…In Vivo Adiporation-Adiporation was performed according to our published procedure (12). Briefly, 3-month-old male 129/S1SvImJ mice were anesthetized with Avertin, and skin in the interscapular region was removed of fur and disinfected with alcohol.…”

“…Furthermore, cell shape is known to have important effects on phenotypic gene expression and the organization of signaling pathways in adipocytes (11). In this study, we have therefore investigated the organization of lipolytic signaling in unilocular mature adipocytes using a novel, in vivo "adiporation" and imaging techniques (12). In addition, we employed 3T3-L1 adipocytes that were supported in a three-dimensional matrix.…”

Historical perspectives in fat cell biology: the fat cell as a model for the investigation of hormonal and metabolic pathways

“…Mature adipocytes have been shown to be susceptible to electroporation. A selective electroporation of adipocytes, directly within adipose tissue, has also been described (138).…”