Fig 1.
Aberrant morphology of adipose tissue in Plin1-/- mice.
(A) Hematoxylin and eosin (HE) staining of adipose tissues in Plin1-/- and Plin1+/+ male mice at 8 and 25 weeks old. The boxed fields (100×) were showed underneath at high magnification (400×). (B) Cumulative adipocyte frequency from epididymal adipose tissues of Plin1-/- and Plin1+/+ mice (n = 3 for each genotype) at age of 25 weeks. For each mice, 10~12 fields of vision at 100 × magnification from different segments of fat tissue were randomly selected for analysis. A total 6,265 of Plin1+/+ unilocular adipocytes (circles) and 11,471 Plin1-/- unilocular (squares) and 1,881 Plin1-/- multilocular (triangles) adipocytes were counted. Cell area was measured by use of NIH Image-J software. The y-axis values represent the cumulative cell percentage for adipocytes at and below the corresponding sizes on the x-axis. The lines labeled as 50th percentile intersect the curves at the median cell sizes (boxes) below which 50% of the adipocytes in each population were distributed. The leftward shift of the curves indicates that the adipocyte population in Plin1-/- mice tend toward smaller cell area as compared with that in Plin1+/+ mice. The inset panel shows the relative (non-cumulative) cell frequency versus cell area, and demonstrates that Plin1-/- mice have a greater proportion of small adipocytes than Plin1+/+ mice.
Fig 2.
Dysregulation of adipogenic signaling in adipose tissues in Plin1-/- mice.
Adipose tissue isolated from Plin1-/- and Plin+/+ mice was utilized for the following examinations. (A) Relative levels of target mRNA detected by real-time PCR. *, P < 0.05 compared with Plin1+/+ mice. (B and C) Immunoblotting.
Fig 3.
Flow cytometric analysis of SVC cells.
The stromal vascular cells (SVCs) were isolated from epididymal fat of Plin1+/+ and Plin1-/- mice and underwent analysis. Adipocyte progenitors in the SVC fraction were immunostained simultaneously with antibodies to CD34 (Alexa Fluor 647), CD29 (FITC), Sca-1 (PE), and CD24 (PerCP-Cy5.5). Cells were incubated with fluorescent isotype-matched IgG as negative controls and single-color positive cells were stained with each antibody alone. The CD29+:CD34+ adipocyte progenitor population was gated (in box) and further separated for the subpopulation coexpressing Sca-1 and CD24 (CD29+:CD34+:Sca-1+:CD24+). The percentage of adipocyte progenitor population in SVC cells was denoted in box.
Fig 4.
Plin1 defect attenuates the SVC differentiation into adipocytes.
(A) Effect of Plin1 gene dosage on adipocyte differentiation. SVCs were isolated from epididymal fat tissue in Plin1+/+, heterozygous Plin1+/-, and Plin1-/- mice, epididymal fat tissue were differentiated into adipocytes. At day 5, intracellular lipid droplets were stained with Oil-red O (red bright-field) and Nile red (red fluorescence). Nuclei (blue) were stained with Hoechst 33258. (B) Dynamic changes of the lipid droplets (red) in Plin1-/- and Plin1+/+ SVCs at day 0, 0.5, 1, 2, 3 and 4 of differentiation.
Fig 5.
Dysregulation of adipogenic signaling in differentiating Plin1-/- SVCs.
The SVC cells isolated from adipose tissues in Plin1+/+, Plin1+/-, and Plin1-/- mice were differentiated. (A) Relative mRNA expression of adipogenic genes at day 5 of differentiation.*, P < 0.05 compared with Plin1+/+ cells. (B) Immunoblotting. Protein expression of adipogenic modulators at day 5 of differentiation in the SVCs from epididymal (Epi) and inguinal (Ing) fat depots in Plin1+/+, Plin1+/-, and Plin1-/- mice. (C) Immunoblotting. Protein expression of adipogenic modulators in the SVCs differentiated for 0, 1, and 5 days. (D) Immunofluorescence of Plin2 and Plin1 switch on lipid droplets. Plin1-/- and Plin1+/+ SVCs were differentiated for 0, 1, 2, and 5 days and immunostained with primary antibody against Plin1 (upper 2 rows) and Plin2 (lower 2 rows) and FITC-conjugated lgG. Nuclei (blue) were stained with Hoechst 33258. Triangles at the central of lipid droplets denote the large droplets disassociated from Plin2 in a fully-differentiated Plin1+/+ adipocyte, compared to the smaller droplets associated with Plin2 in ill-differentiated Plin1-/- SVCs.
Fig 6.
Exogenous Plin1 expression restores Plin1-/- adipocyte differentiation.
Plin1-/- and Plin+/+ epididymal SVCs were infected with the adenovirus dually expressing green fluorescence protein (GFP) and Plin1 (Ad-Plin1, a−c), with the adenovirus expressing GFP and Plin2 (Ad-Plin2, d−f) as the control. Fluorescence microscopy of cells induced to differentiate into adipocytes for 4 days, then stained with Nile red. Lipid droplets exhibit yellow/gold fluorescence. GFP-positive cells express ectopic Plin1 or Plin2. Well-differentiated Plin1-/- cells with Ad-Plin1 infection (circles with solid line) and poorly-differentiated cells without Ad-Plin1 infection (circles with dashed line) are labeled in the field of vision (b, c).
Fig 7.
Enhanced induction improves Plin1-/- SVC differentiation.
Epididymal SVCs were induced for 2 days (D1~2) in differentiation medium (IDMI) containing 250 μM IBMX, 17 nM insulin, 0.1 μM dexamethasone and 60 μM indomethacin, followed by an additional 2-day (D3~4) treatment with 17 nM insulin. Lipid droplets were stained with Oil-red O. (A) Effect of IBMX dose on lipid-droplet formation and adipocyte differentiation of Plin1+/+ and Plin1-/- SVCs. (B) Effect of replacing indomethacin with 5 μM rosiglitazone (IDMR) on lipid-droplet growth and adipogenesis in SVCs. (C) After day-4 differentiation, SVCs were further stimulated with 17 nM insulin (Ins), 400 μM oleic acid (OA), or both for an additional 2 days (D5~6).