Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB

Abstract

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

Figure 1

Expression of mRNA of MCP-1 (a), TLR-4 (b), GPR120 (c), and β-Arrestin 2 (d) in undifferentiated versus differentiated 3T3-L1 cells. UDC and DC were obtained as described under Materials and Methods. The results are expressed as relative mRNA levels (fold stimulation over UDC set to 1) and are the means ± SEM of 3 independent experiments. Data were analyzed by t-test for unique sample; ^∗ p < 0.05 and ^∗∗∗ p < 0.005 versus UDC.

Figure 2

mRNA expression of MCP-1 (a) and MCP-1 protein secretion (b) in differentiated 3T3-L1 cells treated with LPS in the presence or absence of FK. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 10 µM FK, 1 µg/mL LPS, and LPS + FK. (a) The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and (b) secreted MCP-1 protein levels are the means ± SEM of 3 independent experiments. Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05 versus CTL; ^# p < 0.05 versus LPS.

Figure 3

mRNA expression of TLR-4 in differentiated 3T3-L1 cells treated with LPS in the presence or absence of FK. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 10 µM FK, 1 µg/mL LPS, and LPS + FK. The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and are the means ± SEM of 3 independent experiments. Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05 versus CTL; ^# p < 0.05 versus LPS.

Figure 4

mRNA expression of GPR120 in differentiated 3T3-L1 cells treated with LPS in the presence or absence of FK. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 10 µM FK, 1 µg/mL LPS, and LPS + FK. The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and are the means ± SEM of 3 independent experiments. Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05 versus CTL; ^## p < 0.01 versus LPS.

Figure 5

mRNA expression of β-Arrestin 2 in differentiated 3T3-L1 cells treated with LPS in the presence or absence of FK. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 10 µM FK, 1 µg/mL LPS, and LPS + FK. The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and are the means ± SEM of 3 independent experiments. Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05 versus CTL.

Figure 6

NFκB1 mRNA and IκBα protein levels in differentiated 3T3-L1 cells treated with LPS in the absence or presence of FK. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 10 µM FK, 1 µg/mL LPS, and LPS + FK. (a) The results are expressed as mRNA levels (fold stimulation over CTL set to 1). (b) Phospho-IκBα and total IκBα protein levels were determined in DC by Western blot analysis. (c) Semiquantitative determination of protein expression was performed as described under Materials and Methods. The results are expressed as protein expression (ratio of total IκBα or P-IκBα band density over β-actin band density) expressed as percent of the CTL value (CTL set to 100%) and are the mean ± SEM (n = 3) (in % of CTL). Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05 versus CTL; ^# p < 0.05 versus LPS.

Figure 7

mRNA expression of MCP-1 (a), TLR-4 (b), GPR120 (c), and NFκB1 (d) in differentiated 3T3-L1 cells treated with LPS in the presence or absence of inhibitors. Differentiated 3T3-L1 cells were treated as described in Materials and Methods under the following conditions: CTL, 1 µg/mL LPS, 3 µM CLI-095 + 1 µg/mL LPS (CLI + LPS), and 10 µM of BAY11-7082 + 1 µg/mL LPS (BAY + LPS). The results are expressed as mRNA levels (fold stimulation over CTL set to 1) and are the means ± SEM of 3 independent experiments. Data were analyzed using t-test for unique sample and paired t-test; ^∗ p < 0.05; ^∗∗ p < 0.01 versus CTL; ^# p < 0.05; ^## p < 0.01 versus LPS.

Figure 8

Data summary. (a) When adipocytes are stimulated with LPS, GPR120 mRNA levels are decreased, while MCP-1 mRNA and protein levels are upregulated via TLR-4 signaling pathway and NFκB activation. (b) When adipocytes are treated with LPS in the presence of FK, the LPS-induced effects on both MCP-1 and GPR120 are inhibited via PKA and CREB activation necessitating CBP binding. AC: adenylyl cyclase; ATP: adenosine triphosphate; cAMP: cyclic adenosine monophosphate; CBP: CREB-binding protein; CREB: cAMP response element-binding protein; FFA: free fatty acids; FK: Forskolin; G-3-P: glycerol-3-phosphate; GK: glycerol kinase; GPR120: G-protein coupled receptor 120; H: catecholamines or hormones; HSL: hormone sensitive lipase; IκBα: inhibitor of kappa B; LPS: lipopolysaccharide; MCP-1: monocyte chemotactic protein-1; NFκB: nuclear factor-κB; p: phosphorylated; PKA: protein kinase A; TAG: triacylglycerol; TLR-4: Toll-like receptor 4.