Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes.
Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG.This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte.This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.
Affiliation: Division of Endocrinology, Diabetes, and Clinical Nutrition, Department of Medicine, Portland, OR 97239.
- Lipid Metabolism*
- Adipose Tissue, White/cytology
- Cells, Cultured
- Lipid Droplets/metabolism
- Macaca mulatta
- Tissue Culture Techniques
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Figure 5: Metabolism and intracellular transport of BODIPY-C12 in unilocular adipocytes. (A) Visceral WAT explants were incubated for 2 h in basal medium (Bas) or with 10 nM insulin (Ins), pulsed with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 0 (lanes 1 and 2), 30 (lanes 3 and 4), or 120 (lanes 5 and 6) min. Lipids were extracted and analyzed by TLC, as described in Materials and Methods. The experiment was repeated twice using duplicate WAT explants from the same animal and further verified in WAT from a different animal. Asterisk and bracket indicate the position of BODIPY-labeled products formed in the presence of insulin. (B) WAT explants were incubated for 2 h in control insulin-containing medium alone (C) or in the presence of inhibitors (10 μM triacsin C [TC] or 30 μM etomoxir [EX]), pulsed with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 120 min with insulin alone or with addition of inhibitors. The experiment was repeated three times with similar results. (C–E) A short time course of BODIPY-C12 metabolism and transport in adipocytes. Two sets of WAT explants were incubated for 2 h in basal medium or with 10 nM insulin, pulsed with BODIPY-C12 for indicated periods of time, and analyzed by TLC (C) or confocal microscopy (D, E). (D) Quantification of mLD-associated fluorescence during a continuous 10-min pulse. Error bars represent SEM, n = 20. (E) Representative images showing the progressive accumulation of fluorescence in mLDs (green) in relation to the ER (red) in adipocytes incubated under basal (left) or insulin-stimulated conditions (middle and right). Right, enlargements of the middle. Asterisks, mLDs; arrowheads, BODIPY-positive fluorescent speckles can be seen at earlier incubation times. All images were autoscaled to allow better visualization. Green fluorescence was quantified in D. (F, G) The effect of triacsin C on BODIPY-C12 incorporation in adipocytes. WAT explants were labeled with BODIPY-C12 and chased for 0 or 5 h, as described in Figure 4A, in insulin-containing medium alone or in the presence of10 μM triacsin C. (G) Quantification of intracellular fluorescence. Error bars represent SEM, n = 10. **p < 0.001, t test; A.U., arbitrary units.
To verify that BODIPY-C12 is readily metabolized into BODIPY-TG, we pretreated WAT explants with basal or insulin-containing media, pulsed them for 15 min with BODIPY-C12, chased them in label-free media, and analyzed FFA metabolites by thin-layer chromatography (TLC). Under basal “no-chase” conditions, BODIPY-C12 migrated primarily as FFA, with only a minor fraction of total fluorescence incorporated into BODIPY-TG (Figure 5A, lane 1). After a chase in basal medium, the BODIPY-TG/BODIPY-C12 ratio increased, consistent with the slow basal rate of TG synthesis. In addition to apparent BODIPY-TG synthesis, there was a time-dependent accumulation of additional BODIPY-labeled species (Figure 5A, asterisk and bracket). Insulin treatment of WAT explants increased the BODIPY-TG/BODIPY-C12 ratio and stimulated the formation of additional BODIPY-labeled species in comparison with basal conditions (Figure 5A, compare lanes 2 and 1). After an insulin chase, BODIPY-C12 was progressively converted into TG (Figure 5A, compare lanes 2, 4, and 6).