Inflammatory Microenvironment and Adipogenic Differentiation in Obesity: The Inhibitory Effect of Theobromine in a Model of Human Obesity In Vitro

Abstract

Objective: Obesity is considered a clinic condition characterized by a state of chronic low-grade inflammation. The role of macrophages and adipocytokines in adipose tissue inflammation is in growing investigation. The physiopathological mechanisms involved in inflammatory state in obesity are not fully understood though the adipocytokines seem to characterize the biochemical link between obesity and inflammation. The aim of this work is to analyze the effect of theobromine, a methylxanthine present in the cocoa, on adipogenesis and on proinflammatory cytokines evaluated in a model of fat tissue inflammation in vitro.

Methods: In order to mimic in vitro this inflammatory condition, we investigated the interactions between human-like macrophages U937 and human adipocyte cell lines SGBS. The effect of theobromine on in vitro cell growth, cell cycle, adipogenesis, and cytokines release in the supernatants has been evaluated.

Results: Theobromine significantly inhibits the differentiation of preadipocytes in mature adipocytes and reduces the levels of proinflammatory cytokines as MCP-1 and IL-1β in the supernatants obtained by the mature adipocytes and macrophages interaction.

Conclusion: Theobromine reduces adipogenesis and proinflammatory cytokines; these data suggest its potential therapeutic effect for treating obesity by control of macrophages infiltration in adipose tissue and inflammation.

Figure 1

Human preadipocytes SGBS cells: in panel (a) cytometric profile of cell cycle and in panel (b) optical photography. Differentiated human SGBS cells: in panel (c) cytometric profile of cell cycle and in panel (d) optical photography after stained with Oil Red O solution to display the accumulated lipid droplets. The acquired FACS data were analyzed by ModFit LT software.

Figure 2

(a) Preadipocytes SGBS were treated with indicated concentration of TB (200 μg/ml and 100 μg/ml) for 48 hrs. Cell proliferation was evaluated by MTT assay. The results are expressed as the percentage of the cell growth inhibition obtained by four experiments (preformed in triplicate). Bar represent the error standard of the percentage mean (^∗p < 0.05). (b) Regulatory effects of TB on cell cycle progression in SGBS preadipocytes cultured in a medium containing 100 and 200 μg/ml TB for 24 h. The cells were stained with a PI solution and analyzed by flow cytometry. The acquired FACS data were analyzed by ModFit LT software.

Figure 3

Inhibitory effect of TB on adipogenic differentiation in preadipocytes SGBS. (a) Inhibitory effect of TB on adipogenic differentiation in preadipocytes SGBS. The cells were cultured in a differentiation medium (DM) containing TB (100 μg/ml) added on day 8 from the start of the differentiation process. The cells were stained with Oil Red and were subject to a quantitative analysis of intracellular lipidic accumulation by an ELISA reader (λ = 490). The results are expressed as the percentage of the inhibition obtained by four experiments (preformed in triplicate). Bar represent the error standard of the percentage mean (p < 0.01). (b) Inhibitory effect of TB on adipogenic differentiation. The SGBS control (A) and the SGBS cultured in a differentiation medium (DM) containing TB (100 μg/ml) added on day 8 from the start of the differentiation process (B). Optical photography.

Figure 4

MCP-1, IL-1β, and Il-6 accumulation (pg/ml) in the supernatant of coculture of dSGBS and dU937 untreated (column C) or treated for 48 h with TB (100 μg/ml, column D) measured by ELISA. CTR 1 is the supernatant of the SGBS on day 8 of the differentiation process. CTR2 is the supernatant of dU937. The results are expressed as the mean obtained by four experiments (preformed in triplicate). Bar represent the error standard of the mean (^∗p < 0.01, ^∗∗p < 0.05).

Figure 5

MCP-1, IL-1β, and Il-6 accumulation (pg/ml) in the supernatant of dSGBS cells cultured with 100% MacCM (column A) for 48 h alone or in the presence of TB (100 μg/ml, column B) measured by ELISA. CTR 1 is the supernatant of the SGBS on day 8 of the differentiation process. CTR2 is the supernatant of dU937. The results are expressed as the mean obtained by four experiments (preformed in triplicate). Bar represent the error standard of the mean (^∗p < 0.01).

Figure 6

IL-1β accumulation (pg/ml) in the supernatant of macrophages dU937 stimulated ApoMC alone (column A) or in combination with TB (100 μg/ml for 48 h, column B) by ELISA. CTR 1 is IL-1β accumulation (pg/ml) in the supernatant of the SGBS on day 8 of the differentiation process. CTR2 is IL-1β accumulation (pg/ml) in the supernatant of dU937. The results are expressed as the mean obtained by four experiments (preformed in triplicate). Bar represent the error standard of the mean (^∗p < 0.01).