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Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes.

Chu M, Sampath H, Cahana DY, Kahl CA, Somwar R, Cornea A, Roberts CT, Varlamov O - Mol. Biol. Cell (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Clinical Nutrition, Department of Medicine, Portland, OR 97239.

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Lipid exchange between mLDs and cLDs in unilocular adipocytes. (A–D) Visceral WAT explants were incubated for 2 h under basal conditions or in the presence of 10 nM insulin, labeled with BODIPY-C12 for 15 min, chased in BODIPY-free basal or insulin-containing medium for indicated periods of time, fixed, and examined by confocal microscopy. Bar, 50 μm. (B) Quantification of basal and insulin-stimulated BODIPY-C12 uptake immediately after a 15-min pulse. Error bars represent SEM, n = 10. **p < 0.01, t test. (C) Time course of fluorescence intensities associated with mLDs in insulin-stimulated adipocytes (representative images are shown in A). Error bars represent SEM, n = 7–10. **p < 0.01, one-way ANOVA followed by t test. (D) Time-lapse microscopy of living unilocular adipocytes (Supplemental Video S2). Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, rinsed in warm medium, and placed on a confocal stage equilibrated to 37°C. Images were recorded in the xyzt-mode every 5 min and quantified as described in Materials and Methods. Error bars represent SEM, n = 10 cells. This experiment was repeated twice with similar results. (E) Dynamics of mLDs. Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, chased in label-free medium for 30 min, and studied by time-lapse confocal microscopy. Images were recorded in the xyzt-mode every 20 s and quantified as described in Materials and Methods. Representative snapshot of a BODIPY-C12–labeled adipocyte shows docked mLDs discharging their fluorescent content (circle) or remaining static (square) during a 10-min imaging interval. (F) Two examples of fluorescence traces showing active mLDs (solid lines) and two examples of static mLDs (dashed lines). (G) The average fluorescence trace of active mLDs. Error bars represent SEM, n = 10.
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Figure 4: Lipid exchange between mLDs and cLDs in unilocular adipocytes. (A–D) Visceral WAT explants were incubated for 2 h under basal conditions or in the presence of 10 nM insulin, labeled with BODIPY-C12 for 15 min, chased in BODIPY-free basal or insulin-containing medium for indicated periods of time, fixed, and examined by confocal microscopy. Bar, 50 μm. (B) Quantification of basal and insulin-stimulated BODIPY-C12 uptake immediately after a 15-min pulse. Error bars represent SEM, n = 10. **p < 0.01, t test. (C) Time course of fluorescence intensities associated with mLDs in insulin-stimulated adipocytes (representative images are shown in A). Error bars represent SEM, n = 7–10. **p < 0.01, one-way ANOVA followed by t test. (D) Time-lapse microscopy of living unilocular adipocytes (Supplemental Video S2). Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, rinsed in warm medium, and placed on a confocal stage equilibrated to 37°C. Images were recorded in the xyzt-mode every 5 min and quantified as described in Materials and Methods. Error bars represent SEM, n = 10 cells. This experiment was repeated twice with similar results. (E) Dynamics of mLDs. Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, chased in label-free medium for 30 min, and studied by time-lapse confocal microscopy. Images were recorded in the xyzt-mode every 20 s and quantified as described in Materials and Methods. Representative snapshot of a BODIPY-C12–labeled adipocyte shows docked mLDs discharging their fluorescent content (circle) or remaining static (square) during a 10-min imaging interval. (F) Two examples of fluorescence traces showing active mLDs (solid lines) and two examples of static mLDs (dashed lines). (G) The average fluorescence trace of active mLDs. Error bars represent SEM, n = 10.

Mentions: A close association of mLDs with the cLD and the ER suggests that mLDs may represent intermediary compartments mediating the packaging of newly synthesized TG into the cLD. To verify this hypothesis, we followed the time course of intracellular BODIPY-C12 transport by confocal microscopy. Control (basal) or insulin-treated WAT explants were pulsed with BODIPY-C12 for 15 min and then chased in label-free medium for different time periods. Insulin significantly stimulated the uptake of BODIPY-C12 into adipocytes (Figure 4, A and B). Immediately after an insulin pulse, fluorescence appeared in mLDs scattered around the cLD (Figure 4A, Ins 0h). After an insulin chase, mLD-associated fluorescence gradually decreased, whereas cLD-associated fluorescence concomitantly increased (Figure 4, A and C). Live-cell imaging of BODIPY-C12–labeled WAT explants verified that the disappearance of fluorescence from mLDs is concurrent with the appearance of fluorescence in cLDs (Figure 4D and Supplemental Video S2). Imaging BODIPY-C12–labeled WAT explants at higher spatiotemporal resolution revealed that individual mLDs discharge their fluorescent content with a half-time on the order of 50 s (Figure 4, E, circle, F, solid lines, and G, and Supplemental Video S3). Many mLDs remained static over a 10-min imaging interval (Figure 4, E, square, and F, dotted lines), suggesting that the observed disappearance of mLDs has a probabilistic nature. BODIPY-labeled palmitic acid (BODIPY-C16) and BODIPY-C12 showed similar kinetics of transport in unilocular adipocytes (unpublished data). Collectively, these results confirm a unidirectional transport of BODIPY-labeled lipids between mLDs and cLDs.


Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes.

Chu M, Sampath H, Cahana DY, Kahl CA, Somwar R, Cornea A, Roberts CT, Varlamov O - Mol. Biol. Cell (2014)

Lipid exchange between mLDs and cLDs in unilocular adipocytes. (A–D) Visceral WAT explants were incubated for 2 h under basal conditions or in the presence of 10 nM insulin, labeled with BODIPY-C12 for 15 min, chased in BODIPY-free basal or insulin-containing medium for indicated periods of time, fixed, and examined by confocal microscopy. Bar, 50 μm. (B) Quantification of basal and insulin-stimulated BODIPY-C12 uptake immediately after a 15-min pulse. Error bars represent SEM, n = 10. **p < 0.01, t test. (C) Time course of fluorescence intensities associated with mLDs in insulin-stimulated adipocytes (representative images are shown in A). Error bars represent SEM, n = 7–10. **p < 0.01, one-way ANOVA followed by t test. (D) Time-lapse microscopy of living unilocular adipocytes (Supplemental Video S2). Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, rinsed in warm medium, and placed on a confocal stage equilibrated to 37°C. Images were recorded in the xyzt-mode every 5 min and quantified as described in Materials and Methods. Error bars represent SEM, n = 10 cells. This experiment was repeated twice with similar results. (E) Dynamics of mLDs. Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, chased in label-free medium for 30 min, and studied by time-lapse confocal microscopy. Images were recorded in the xyzt-mode every 20 s and quantified as described in Materials and Methods. Representative snapshot of a BODIPY-C12–labeled adipocyte shows docked mLDs discharging their fluorescent content (circle) or remaining static (square) during a 10-min imaging interval. (F) Two examples of fluorescence traces showing active mLDs (solid lines) and two examples of static mLDs (dashed lines). (G) The average fluorescence trace of active mLDs. Error bars represent SEM, n = 10.
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Figure 4: Lipid exchange between mLDs and cLDs in unilocular adipocytes. (A–D) Visceral WAT explants were incubated for 2 h under basal conditions or in the presence of 10 nM insulin, labeled with BODIPY-C12 for 15 min, chased in BODIPY-free basal or insulin-containing medium for indicated periods of time, fixed, and examined by confocal microscopy. Bar, 50 μm. (B) Quantification of basal and insulin-stimulated BODIPY-C12 uptake immediately after a 15-min pulse. Error bars represent SEM, n = 10. **p < 0.01, t test. (C) Time course of fluorescence intensities associated with mLDs in insulin-stimulated adipocytes (representative images are shown in A). Error bars represent SEM, n = 7–10. **p < 0.01, one-way ANOVA followed by t test. (D) Time-lapse microscopy of living unilocular adipocytes (Supplemental Video S2). Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, rinsed in warm medium, and placed on a confocal stage equilibrated to 37°C. Images were recorded in the xyzt-mode every 5 min and quantified as described in Materials and Methods. Error bars represent SEM, n = 10 cells. This experiment was repeated twice with similar results. (E) Dynamics of mLDs. Insulin-stimulated WAT explants were labeled with BODIPY-C12 for 15 min, chased in label-free medium for 30 min, and studied by time-lapse confocal microscopy. Images were recorded in the xyzt-mode every 20 s and quantified as described in Materials and Methods. Representative snapshot of a BODIPY-C12–labeled adipocyte shows docked mLDs discharging their fluorescent content (circle) or remaining static (square) during a 10-min imaging interval. (F) Two examples of fluorescence traces showing active mLDs (solid lines) and two examples of static mLDs (dashed lines). (G) The average fluorescence trace of active mLDs. Error bars represent SEM, n = 10.
Mentions: A close association of mLDs with the cLD and the ER suggests that mLDs may represent intermediary compartments mediating the packaging of newly synthesized TG into the cLD. To verify this hypothesis, we followed the time course of intracellular BODIPY-C12 transport by confocal microscopy. Control (basal) or insulin-treated WAT explants were pulsed with BODIPY-C12 for 15 min and then chased in label-free medium for different time periods. Insulin significantly stimulated the uptake of BODIPY-C12 into adipocytes (Figure 4, A and B). Immediately after an insulin pulse, fluorescence appeared in mLDs scattered around the cLD (Figure 4A, Ins 0h). After an insulin chase, mLD-associated fluorescence gradually decreased, whereas cLD-associated fluorescence concomitantly increased (Figure 4, A and C). Live-cell imaging of BODIPY-C12–labeled WAT explants verified that the disappearance of fluorescence from mLDs is concurrent with the appearance of fluorescence in cLDs (Figure 4D and Supplemental Video S2). Imaging BODIPY-C12–labeled WAT explants at higher spatiotemporal resolution revealed that individual mLDs discharge their fluorescent content with a half-time on the order of 50 s (Figure 4, E, circle, F, solid lines, and G, and Supplemental Video S3). Many mLDs remained static over a 10-min imaging interval (Figure 4, E, square, and F, dotted lines), suggesting that the observed disappearance of mLDs has a probabilistic nature. BODIPY-labeled palmitic acid (BODIPY-C16) and BODIPY-C12 showed similar kinetics of transport in unilocular adipocytes (unpublished data). Collectively, these results confirm a unidirectional transport of BODIPY-labeled lipids between mLDs and cLDs.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Division of Endocrinology, Diabetes, and Clinical Nutrition, Department of Medicine, Portland, OR 97239.

Show MeSH
Related in: MedlinePlus