Role of small proliferative adipocytes: possible beige cell progenitors

Figures

• 

  Figure 1. Preparation of SPA. (A) Method for preparation of SPA rich fraction (fraction B), stromal vascular cells (SVC), and mature white adipocytes (MWA). The fraction combined fraction B and C was regarded as fraction D. (B) Morphology of fraction B and SVC cultured for 1 day (left), 5 days (middle), and 10 days (right). (C) Expression of leptin and adiponectin in cultured fraction B and SVC. (D and E) Protein levels of PPARγ, adiponectin, and leptin in SVC, fraction B, and MWA were evaluated by immunoblot analysis. Typical immunoblots (D) and quantified results (E) are shown. Each value shows the average of the relative protein levels (PPARγ white, adiponectin: gray, leptin: black) (n = 3) (MWA as 1). (F) Incorporation of EdU into cells in fraction B and SVC. Control: EdU was not added to the medium before visualization. EdU: EdU was added to the medium before visualization. EdU + Adiponectin: EdU was added to the medium before visualization and subsequent immunostaining was performed with anti-adiponectin antibody. Red: EdU, Green: adiponectin. Scale: 50 µm (G) Percentage of EdU-positive cells/total cells in SVC and fraction B (n = 3).

• 

  Figure 2. Gene expression of SVC, SPA, and MWA. Results of real-time PCR are demonstrated. (A) Pparg2, (B) Adipoq, (C) Lep, (D) Retn, (E) Plin1, (F) Plin4, (G) Fabp4, (H) Adrb3, (I) Klb, (J) Aqp7, (K) Srebp1, (L) Fas, (M) Sncg, (N) Gabrg2, (O) Plf, (P) Dlk1, (Q) Pdgfra, (R) Pdgfrb, (S) Ly6c1, (T) Cd9, (U) Myf5, (V) Zic1, (W), Cd137, and (X) Tbx1. All values represent mean ± s.e. (n = 5) of relative expression levels of mRNA (SVC as 1). Relative mRNA levels in brown adipose tissue (BAT) were added in U–X.

• 

  Figure 3. Expression of neuronal proteins and proliferin in SPA and adipose tissue. (A) Expression of SNCG and adiponectin in SPA. (B) Expression of PLF and adiponectin in SPA. (C, D, E, and F) Flow cytogram of AQP7+, ADRB3+ cells in fraction D isolated from epididymal adipose tissue. (C) Negative control, cells were incubated with the secondary antibodies. (D) Representative result cells were incubated with anti-AQP7 antibody and anti-ADRB3 antibody followed by incubated with secondary antibody. AQP7+, ADRB3+: red spots, AQP7+, ADRB3+: green spots (E) Distribution of AQP7+, ADRB3+ cells, and AQP-, ADRB3+ cells in SS/LS plot (F) Distribution of AQP7+, ADRB3+ cells and AQP-, ADRB3+ cells in cell diameter (G) Expression of SNCG and adiponectin in epididymal fat. (H) Expression of PLF and adiponectin in epididymal fat. Scale: 50 µm. (I, J, K, L, M, N, O, P, Q, R, S, T, and U) Comparison of gene expression levels of Pparg2 (I), Adipoq (J), Lpl (K), Gabrg2 (L), Sncg (M), Pdgfra (N), Tbx1 (O), Cd137 (P), Plf (Q), Pdgfrb (R), Ly6c1 (S), Cd9 (T), and Wt1 (U) mRNA between epididymal SPA and inguinal SPA. All values represent mean ± s.e. (n = 4) of relative expression levels of mRNA (Epididymal as 1).

• 

  Figure 4. Differentiation of SPA into mature adipocytes. (A) SPA were treated without (control) or with 100 nM insulin (Ins), 1 µM dexamethasone (Dexa), 10 µM pioglitazone (Pio), differentiation medium (Ins/Dexa/IBMX) containing 200 nM insulin/1 µM dexamethasone/0.5 mM IBMX or 10 µM pioglitazone and differentiation medium (Ins/Dexa/IBMX/Pio) for 5 days. Differentiation was evaluated by accumulation of lipid stained with LipiDye (green). (B) Quantification of the results shown in (A). Percentages of lipid-laden cells in SPA (open column) and SVC (closed column). The P values are for each control value (n = 4). (C, D, and E) Effects of treatment with differentiation medium for 5 days in SVC and SPA on the expression of Pparg2 (C), Adipoq (D), and Sncg (E). Open column: control cells, Solid column: treated cells, (n = 4). (F and G) Comparison of ability to differentiate into lipid-laden cells between epididymal and inguinal SPA/fraction D. Epididymal and inguinal SPA/fraction D were incubated with the differentiation medium for 5 days. Image of typical lipid-laden cells in differentiated SPA (F) and quantified results (G) were shown. Open column: SPA, Solid column: SVC, Gray column: fraction…

• 

  Figure 5. Expression of PLF, UCP1, and mitochondria-related genes in differentiated SPA and differentiated SVC. (A, B, C, and D) Results of real-time PCR were demonstrated. (A) Ucp1, (B) Pgc1a, (C) Ppara, and (D) Adrb3. All values represent the mean ± s.e. (n = 8) of relative expression levels of mRNA (SPA, control as 1). To compare the expression levels of these genes, the relative expression levels in BAT were added. Open column represents control cells and solid column represents differentiated cells with the differentiation medium and 10 µM pioglitazone for 5 days (n = 4). (E) Protocol for treatment with pioglitazone (Pio) and CL316243. (F) Expression of PLF and UCP1 in SPA treated with Pio and CL316243. Scale: 50 µm. (G-I) Relative expression levels of Ucp1 (G), Adipoq (H), and Plf (I) in SPA treated with or without Pio and CL316243 are shown. Open column represents results in the absence of CL316243 and solid column represents those in the presence of CL316243. All values represent the mean ± s.e. (n = 4) of relative expression levels of mRNA (control without CL316423 as 1). (J) Effect of treatment with CL316243 and Pio on cell growth in SPA. Percent of EdU-positive cells was shown.…

• 

  Figure 6. Expression of UCP1 and PLF in beige cells. (A) Beige cells were generated by the daily peritoneal injection of CL316243 to C57/BL mice for 7 days of a total 21 days. HE staining of specimens obtained from epididymal and inguinal fat isolated from control or treated mice (n = 4) is shown. (B and C) Expression of Ucp1(B) and Plf (C) mRNA in epididymal (open) and inguinal (solid) fat is shown (control in epididymal adipose tissue as 1, n = 4). (D) Expression of UCP1 and PLF in epididymal and inguinal fat isolated from control or treated mice with CL316243. Scale: 50 µm. (E) A model for differentiation of SPA and adipose progenitor cells (APC) in visceral and subcutaneous adipose tissues.