Parametrial fat tissue from high fat diet-treated SKH-1 mice stimulates transformation of mouse epidermal JB6 cells

Abstract

Our previous studies indicated that decreasing visceral adipose tissue by surgical removal of the parametrial fat pads inhibited UVB-induced carcinogenesis in SKH-1 mice fed a high fat diet (HFD), but not a low fat diet (LFD) indicating that the parametrial fat tissue from mice fed a HFD played a role in skin carcinogenesis.

Objective: In the present study, we sought to investigate how a HFD may influence the intrinsic properties of the parametrial fat tissue to influence UVB-induced skin tumor formation.

Methods and results: Immunohistochemical staining, adipokine array, and flow cytometry showed that parametrial fat tissue from mice fed a HFD had a higher density of macrophage-fused dead adipocytes (crown-like structures), more adipokines, and stimulated the production of more reactive oxygen species compared with parametrial fat tissue from mice fed a LFD. These differences between parametrial fat tissue from mice fed a HFD and LFD were associated with their effect on the in vitro transformation of mouse epidermal JB6 cells. Our results indicated that fat tissue filtrate (an aqueous filtrate made from the parametrial fat pad) from mice fed a HFD enhanced the conversion of JB6 cells from an epithelial-like morphology to cells with a fibroblast-like morphology to a greater extent than fat tissue filtrate from mice fed a LFD. Studies indicated that the fibroblast-like cells had decreased levels of E-cadherin, increased levels of Twist as assayed by western blot. Fat tissue filtrate made from the parametrial fat tissue of mice fed a HFD had 160% more transforming activity than that from mice fed a LFD and formed malignant mesenchymal tumors in vivo.

Conclusion: These studies provide the first in vitro demonstration of a parametrial fat tissue-induced transformation of an epidermal cell.

Keywords: JB6 cell transformation; high fat diet; parametrial fat tissue.

Figure 1. Effect of high fat diet feeding on parametrial fat pad size and crown-like structure density

A. Picture demonstrating parametrial fat pad (fat tissue) size after 4 months of LFD or HFD feeding. B. Parametrial fat was fixed in formalin and immunohistochemically stained with F4/80 for CLS. Pictures were taken under 100× microscopic magnification with a light microscope and are representative of data obtained from more than three mice. Parametrial fat from mice fed a HFD demonstrated larger adipocytes and a higher density of CLS (88.8 ± 8.0 mm²; dark brown stain) compared with mice fed a LFD (0.9 ± 0.2 mm²).

Figure 2. Effect of high fat diet feeding on adipokine production and reactive oxygen species stimulation

A. Parametrial fat pad homogenate (0.5 g fat) from female SKH-1 mice that had been fed a 60% kcal high fat diet (HFD) or low fat chow diet (LFD) for 4 months was placed in 1.0 mL of MEM on a hanging well insert. Cytokines and other fat constituents passed through a polyethylene terephthalate membrane with 0.4 μm pores in the insert for 4 hours to provide fat tissue filtrate in MEM. B. Fat was isolated and fat tissue filtrate was made as described in (A). Graph demonstrates the amount of protein in MEM following culture with parametrial fat tissue homogenate or parametrial fat pad secretions from an intact piece of fat. C. Elevated amounts of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), leptin, Serpin E1/plasminogen activator inhibitor-1 (PAI-1) and intracellular adhesion molecule-1 (ICAM-1) were released from the parametrial fat of mice fed a HFD compared with a LFD. D. Mean fluorescence intensity of the 2′, 7′-dichlorofluorescein (DCF) (excitation 488 nm; fluorescence 530 nm) +/− 15 of 5,000 events was measured. Fat tissue filtrate (50 μg protein/mL), or TPA (10 ng/mL) induced ROS in JB6 cells. HFD fat tissue filtrate treatment stimulated the formation of ROS 48% more than LFD fat tissue filtrate as measured by DCF fluorescence at 8 hours **P<0.01. All data are mean ± SEM, Student t test (unpaired, two-tailed).

Figure 3. Fat tissue filtrate from mice fed a high fat diet stimulates the proliferation of JB6 cells growing in liquid medium

A. JB6 cells were treated with 1% FBS, 5% FBS or fat tissue filtrate (50 μg protein/mL) from mice fed a LFD or HFD for 4 months and were counted at 1, 3, 5, and 7 days. The fat tissue filtrate from mice fed a HFD stimulated JB6 cell proliferation to about the same extent as 5% FBS and significantly more than that from mice fed a LFD (* P<0.05). One well = 9.6 cm². B. JB6 cells were seeded at 100 cells per well in a 6-well plate. Cells were treated with 5% FBS or fat tissue filtrate from mice fed a HFD for 7 days and stained with Giemsa. Cloning efficiency, as measured by the number of Giemsa-stained clones, was significantly increased to a greater extent in JB6 cells cultured with fat tissue filtrate from mice fed a HFD for 4 months compared with cells cultured with fat tissue filtrate from mice fed a LFD for 4 months (*P<0.05). Arrows indicate % increase in cloning efficiency at the different doses. One well = 9.6 cm². Data are mean ± SEM, Student t test (unpaired, two-tailed).

Figure 4. Fat tissue filtrate from mice fed a high fat diet stimulates the formation of a fibroblast-like morphology of JB6 cells growing in liquid medium

A. JB6 cells were seeded at 100 cells per well in a 6-well plate. Cells were treated with 5% FBS or fat tissue filtrate from mice fed a HFD for 7 days and stained with Giemsa. The picture shows a comparison of epithelial-like morphology and fibroblast-like morphology of JB6 cells (magnification = 400×). The fibroblast-like clones pictured were characterized as being either early-(A2), middle-(A3), or late-stage (A3) depending upon the size of the foci formed. B. JB6 cells were treated for 7 days with 5% FBS or fat tissue filtrate (50 μg protein/mL) from mice fed a HFD. E-cadherin, Twist and β-Actin levels were determined in the epithelial-like and fibroblast-like clones by western blotting. C. JB6 cells were seeded and treated as described in (A). JB6 cells treated with fat tissue filtrate (50 or 100 μg protein/mL) from mice fed a HFD had a greater ratio of clones with fibroblast-like to epithelial-like cells when compared with cells cultured with fat tissue filtrate from mice fed a LFD (*P<0.05). Arrows indicate % increase in the ratio of fibroblast-like to epithelial-like clones at the different doses. One well = 9.6 cm². All data are mean ± SEM, Student t test (unpaired, two-tailed).

Figure 5. Fat tissue filtrate stimulates the transformation of JB6 cells

A. JB6 cells were treated with 5% FBS or fat tissue filtrate (50 μg protein/mL) for 7 days, and then 10⁴ cells were placed in soft agar for 14 days. The picture shows JB6 cells growing in soft agar that were cultured with fat tissue filtrate from mice fed either LFD or HFD (magnification = 400×). B. Cells were treated as described in (A). Fat tissue filtrate from mice fed a HFD stimulated colony formation in soft agar 160% more than fat pad filtrate from mice fed a LFD (*P<0.05). Data was obtained from 3 separate clones per treatment group. One dish = 20 cm². All data are mean ± SEM, Student t test (unpaired, two-tailed). C. Mice were injected with 2 × 10⁶ JB6 cells subcutaneously in the right and left flank of a SCID mouse. Mice were injected with cells treated with fat tissue filtrate as described in Figure 2. Data shows picture of tumor formation in SCID mice 10 days after injection. D. Immunohistochemistry of tumor in SCID mice injected with JB6 cells treated with HFD fat tissue filtrate demonstrates a fibrosarcoma at 40× and 200×magnification.