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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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mLDs are associated with the ER in unilocular adipocytes. Visceral WAT explants were incubated for 2 h with 10 nM insulin, labeled with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 30 min in the presence of the red fluorescent ER-tracker, as described in Materials and Methods. (A) Representative confocal image of a BODIPY/ER-labeled adipocyte. Bar, 10 μm. (B) The enlarged area of the adipocyte shows the groups of mLDs (green) surrounded by ER patches (red) attached to the surface of the cLD. Arrowheads indicate ER fibers connecting distinct ER patches. Bar, 5 μm. (C) Size distribution of mLDs (n = 100) in the adipocyte (shown in A). Fluorescence intensities of individual mLDs were plotted against their diameters. Horizontal bars are calculated mean intensities of each group of mLDs. The value of 100% on the y-axis represents the intensity of the brightest LD in the image. Similar mLD size distributions were obtained in a WAT adipocytes from a large cohort of animals (n = 38). (D) Example of the large field of mLDs surrounded by highly fenestrated ER. Bar, 5 μm. (E) Optical reconstruction of the mLD-ER patch; left, whole cell; middle, single patch; right, three-dimensional rendering. (F) Perilipin 1a is associated with mLDs. Insulin-treated WAT explants were labeled for 15 min with BODIPY-C12, chased in label-free medium for 30 min, and fixed in formaldehyde. WAT explants were subjected to indirect immunofluorescence analysis, as described in Materials and Methods, using antibodies to perilipin 1a, followed by secondary red fluorescent antibodies. Images represent confocal projections. Bar, 1 μm.
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Figure 2: mLDs are associated with the ER in unilocular adipocytes. Visceral WAT explants were incubated for 2 h with 10 nM insulin, labeled with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 30 min in the presence of the red fluorescent ER-tracker, as described in Materials and Methods. (A) Representative confocal image of a BODIPY/ER-labeled adipocyte. Bar, 10 μm. (B) The enlarged area of the adipocyte shows the groups of mLDs (green) surrounded by ER patches (red) attached to the surface of the cLD. Arrowheads indicate ER fibers connecting distinct ER patches. Bar, 5 μm. (C) Size distribution of mLDs (n = 100) in the adipocyte (shown in A). Fluorescence intensities of individual mLDs were plotted against their diameters. Horizontal bars are calculated mean intensities of each group of mLDs. The value of 100% on the y-axis represents the intensity of the brightest LD in the image. Similar mLD size distributions were obtained in a WAT adipocytes from a large cohort of animals (n = 38). (D) Example of the large field of mLDs surrounded by highly fenestrated ER. Bar, 5 μm. (E) Optical reconstruction of the mLD-ER patch; left, whole cell; middle, single patch; right, three-dimensional rendering. (F) Perilipin 1a is associated with mLDs. Insulin-treated WAT explants were labeled for 15 min with BODIPY-C12, chased in label-free medium for 30 min, and fixed in formaldehyde. WAT explants were subjected to indirect immunofluorescence analysis, as described in Materials and Methods, using antibodies to perilipin 1a, followed by secondary red fluorescent antibodies. Images represent confocal projections. Bar, 1 μm.

Mentions: To elucidate the spatiotemporal dynamics of TG synthesis and packaging in unilocular adipocytes, we labeled WAT explants with the fluorescent FFA analogue BODIPY-C12. Previously, we and others demonstrated that BODIPY-FFA is transported into in vitro–differentiated adipocytes and WAT explants by an insulin-stimulated mechanism (Li et al., 2005; Liao et al., 2005; Wu et al., 2006a, b; Varlamov et al., 2010, 2012, 2013; Somwar et al., 2011) and is esterified to BODIPY-TG in 3T3-L1 adipocytes (Sun et al., 2013). Confocal microscopy analysis unveiled a striking, previously unobserved staining pattern that appeared as clusters of bright fluorescent mLDs scattered around the surface of the cLD (Figure 2, A and D, green). Each group of mLDs was surrounded by a distinct ER patch (Figure 2, red, and Supplemental Video S1). Three-dimensional reconstitution of confocal scans revealed “eggs-in-a-nest”-like structures, with mLDs occupying spherical cavities within each ER patch (Figure 2, B and E).


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)

mLDs are associated with the ER in unilocular adipocytes. Visceral WAT explants were incubated for 2 h with 10 nM insulin, labeled with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 30 min in the presence of the red fluorescent ER-tracker, as described in Materials and Methods. (A) Representative confocal image of a BODIPY/ER-labeled adipocyte. Bar, 10 μm. (B) The enlarged area of the adipocyte shows the groups of mLDs (green) surrounded by ER patches (red) attached to the surface of the cLD. Arrowheads indicate ER fibers connecting distinct ER patches. Bar, 5 μm. (C) Size distribution of mLDs (n = 100) in the adipocyte (shown in A). Fluorescence intensities of individual mLDs were plotted against their diameters. Horizontal bars are calculated mean intensities of each group of mLDs. The value of 100% on the y-axis represents the intensity of the brightest LD in the image. Similar mLD size distributions were obtained in a WAT adipocytes from a large cohort of animals (n = 38). (D) Example of the large field of mLDs surrounded by highly fenestrated ER. Bar, 5 μm. (E) Optical reconstruction of the mLD-ER patch; left, whole cell; middle, single patch; right, three-dimensional rendering. (F) Perilipin 1a is associated with mLDs. Insulin-treated WAT explants were labeled for 15 min with BODIPY-C12, chased in label-free medium for 30 min, and fixed in formaldehyde. WAT explants were subjected to indirect immunofluorescence analysis, as described in Materials and Methods, using antibodies to perilipin 1a, followed by secondary red fluorescent antibodies. Images represent confocal projections. Bar, 1 μm.
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Figure 2: mLDs are associated with the ER in unilocular adipocytes. Visceral WAT explants were incubated for 2 h with 10 nM insulin, labeled with BODIPY-C12 for 15 min, and chased in BODIPY-free medium for 30 min in the presence of the red fluorescent ER-tracker, as described in Materials and Methods. (A) Representative confocal image of a BODIPY/ER-labeled adipocyte. Bar, 10 μm. (B) The enlarged area of the adipocyte shows the groups of mLDs (green) surrounded by ER patches (red) attached to the surface of the cLD. Arrowheads indicate ER fibers connecting distinct ER patches. Bar, 5 μm. (C) Size distribution of mLDs (n = 100) in the adipocyte (shown in A). Fluorescence intensities of individual mLDs were plotted against their diameters. Horizontal bars are calculated mean intensities of each group of mLDs. The value of 100% on the y-axis represents the intensity of the brightest LD in the image. Similar mLD size distributions were obtained in a WAT adipocytes from a large cohort of animals (n = 38). (D) Example of the large field of mLDs surrounded by highly fenestrated ER. Bar, 5 μm. (E) Optical reconstruction of the mLD-ER patch; left, whole cell; middle, single patch; right, three-dimensional rendering. (F) Perilipin 1a is associated with mLDs. Insulin-treated WAT explants were labeled for 15 min with BODIPY-C12, chased in label-free medium for 30 min, and fixed in formaldehyde. WAT explants were subjected to indirect immunofluorescence analysis, as described in Materials and Methods, using antibodies to perilipin 1a, followed by secondary red fluorescent antibodies. Images represent confocal projections. Bar, 1 μm.
Mentions: To elucidate the spatiotemporal dynamics of TG synthesis and packaging in unilocular adipocytes, we labeled WAT explants with the fluorescent FFA analogue BODIPY-C12. Previously, we and others demonstrated that BODIPY-FFA is transported into in vitro–differentiated adipocytes and WAT explants by an insulin-stimulated mechanism (Li et al., 2005; Liao et al., 2005; Wu et al., 2006a, b; Varlamov et al., 2010, 2012, 2013; Somwar et al., 2011) and is esterified to BODIPY-TG in 3T3-L1 adipocytes (Sun et al., 2013). Confocal microscopy analysis unveiled a striking, previously unobserved staining pattern that appeared as clusters of bright fluorescent mLDs scattered around the surface of the cLD (Figure 2, A and D, green). Each group of mLDs was surrounded by a distinct ER patch (Figure 2, red, and Supplemental Video S1). Three-dimensional reconstitution of confocal scans revealed “eggs-in-a-nest”-like structures, with mLDs occupying spherical cavities within each ER patch (Figure 2, B and E).

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