Human monocytes accelerate proliferation and blunt differentiation of preadipocytes in association with suppression of C/EBPΑ mRNA

Abstract

Obesity, type 2 diabetes, and HIV-associated lipodystrophy are associated with abnormalities in adipocyte growth and differentiation. In persons with these conditions, adipose depots contain increased numbers of macrophages, but the origins of these cells and their specific effects are uncertain. Peripheral blood mononuclear cells (PBMC)-derived monocytes, but not T cells, cocultured via transwells with primary subcutaneous preadipocytes, increased proliferation (approximately twofold) and reduced differentiation (~50%) of preadipocytes. Gene expression analyses in proliferating preadipocytes (i.e., prior to hormonal induction of terminal differentiation) revealed that monocytes down-regulated mRNA levels of CCAAT/enhancer binding protein, alpha (C/EBPα) and up-regulated mRNA levels of G0/G1 switch 2 (G0S2) message, genes important for the regulation of adipogenesis and the cell cycle. These data indicate that circulating peripheral blood monocytes can disrupt adipogenesis by interfering with a critical step in C/EBPα and G0S2 transcription required for preadipocytes to make the transition from proliferation to differentiation. Interactions between preadipocytes and monocytes also increased the inflammatory cytokines IL-6 and IL-8, as well as a novel chemotactic cytokine, CXCL1. Additionally, the levels of both IL-6 and CXCL1 were highest when preadipocytes and monocytes were cultured together, compared to each cell in culture alone. Such cross-talk amplifies the production of mediators of tissue inflammation.

Figure 1

Cell cycle and proliferation of human subcutaneous preadipocytes during coculture with PBMCs, T cells, or monocytes. Preadipocyte cell cycle was measured by flow cytometry after staining with a DNA dye, and the G₁, S, and G₂/M phases were delineated using FlowJo analysis software. (a) Representative cell cycle histograms of preadipocytes after 3 days of culture with control media, PBMCs, T cells, or monocytes. (b) Proliferation of preadipocytes after 3 days of coculture with increasing numbers of PBMCs, T cells, or monocytes. Proliferation was determined by addition of the S and G₂/M phases of the cell cycle. N = 3, error bars are means ± s.d., *P < 0.05 compared to control media. (c) Proliferation of preadipocytes during coculture with PBMCs, T cells, or monocytes for up to 6 days at a 50:1 leukocyte:preadipocyte ratio. N = 3, error bars are means ± s.d., *P < 0.05 compared to control media. (d) Representative phase contrast microscopy images (×40 magnification) of preadipocyte confluency after 6 days of coculture with PBMCs, T cells, or monocytes. PBMC, peripheral blood mononuclear cell.

Figure 2

Differentiation of human subcutaneous preadipocytes for 2 weeks following 6 days of coculture with PBMCs, T cells, or monocytes (i.e., after 6 days of coculture, PBMCs, T cells, or monocytes were removed and preadipocytes were differentiated in the absence of these cells). (a) Quantification of preadipocyte differentiation after 2 weeks following hormonal induction. Adipocyte lipids were stained with Oil Red O and four random fields of observation from each condition were recorded. Pixel values of Oil Red O-stained areas were calculated with Adobe Photoshop software and averaged. Normal differentiation with control media was established as 100%. N = 3, error bars are means ± s.d. (b) Representative phase contrast microscopy images (×40) of Oil Red O-stained preadipocytes after 2 weeks of differentiation. PBMC, peripheral blood mononuclear cell.

Figure 3

Expression of key adipogenic and cell cycle (C/EBPα and G0S2) proteins in proliferating human subcutaneous preadipocytes during coculture with monocytes and subsequent terminal differentiation. (a) Gene expression of C/EBPβ, C/EBPα, PPARγ, and G0S2 by real-time PCR in preadipocytes during coculture with monocytes for up to 5 days. The fold change expression of each gene in preadipocytes compares coculture with monocytes to culture with control media after normalization to an internal control (GAPDH) using the 2^−ΔΔCt calculation for relative expression. Data are from one experiment done in duplicate representative of three independent experiments, error bars are means ± s.e.m. (b) Western blot of C/EBPβ, C/EBPα, and G0S2 in preadipocytes at the 5-day time point during coculture with monocytes. The two bands for C/EBPβ represent the two isoforms for this protein at ~36 kDa. PPARγ was probed for but not detected. Densitometry of blots was normalized to actin loading control. (c) Representative phase contrast microscopy images (×40) of preadipocyte confluency after 5 days of coculture with monocytes just prior to hormonal induction of differentiation. (d) Representative phase contrast microscopy images (×40) of Oil Red O-stained adipocytes after 2 weeks of preadipocyte differentiation following 5 days of preadipocyte coculture with monocytes (i.e., after 5 days of coculture with preadipocytes, monocytes were removed and preadipocytes were differentiated in the absence of monocytes). Error bars are means ± s.d. of pixel intensity quantification of four images per condition from one experiment representative of three. *P < 0.05 compared to control media. C/EBPβ, CCAAT/enhancer binding protein (C/EBP), beta; PPARγ, peroxisome proliferator-activated receptor gamma 2; C/EBPα, CCAAT/enhancer binding protein (C/EBP), alpha; G0S2, G0/G1 switch 2.

Figure 4

Production of inflammatory cytokines during coculture of human subcutaneous preadipocytes with monocytes, and preadipocyte proliferation and differentiation after exogenous treatment with inflammatory cytokines. (a) The inflammatory cytokines IL-6, IL-8, and TNFα were measured in supernatants using a flow cytometry multiplex assay, and CXCL1 was measured with an ELISA kit. Data are from one experiment done in triplicate that is representative of three independent experiments, error bars are means ± s.d. For IL-6 and CXCL1, asterisks indicate P < 0.05 comparing the “preadipocytes + monocytes” condition to either the “preadipocytes + media” or “monocytes + media” condition. (b) Preadipocyte cell cycle after treatment with 100 ng/ml recombinant IL-6, IL-8, TNFα, or CXCL1 for 3 days. CXCL1 is compared with its own media control since that treatment experiment was conducted in a separate experiment with a different lot of preadipocytes. Histograms are representative of two separate experiments. (c) Preadipocyte differentiation after treatment with 100 ng/ml recombinant IL-6, IL-8, TNFα, or CXCL1. Shown are representative images (×40) of Oil Red O-stained adipocytes after 2 weeks of differentiation. ELISA, enzyme-linked immunosorbent assay.