Human adipose tissue conditioned media from lean subjects is protective against H2O2 induced neurotoxicity in human SH-SY5Y neuronal cells

Abstract

Adipose tissue secretes numerous hormone-like factors, which are known as adipokines. Adipokine receptors have been identified in the central nervous system but the potential role of adipokine signaling in neuroprotection is unclear. The aim of this study is to determine (1) Whether adipokines secreted from cultured adipose tissue of lean humans is protective against oxidative stress-induced neurotoxicity in human SH-SY5Y neuronal cells; and (2) To explore potential signaling pathways involved in these processes. Adipose tissue conditioned media (ATCM) from healthy lean subjects completely prevented H2O2 induced neurotoxicity, while this effect is lost after heating ATCM. ATCM activated the phosphorylation of ERK1/2, JNK and Akt at serine 308 in SH-SY5Y cells. PD98059 (25 µM), SP600125 (5 µM) and LY29400 (20 µM) partially blocked the protective effects of ATCM against H2O2 induced neurotoxicity. Findings demonstrate that heat-sensitive factors secreted from human adipose tissue of lean subjects are protective against H2O2 induced neurotoxicity and ERK1/2, JNK, and PI3K signaling pathways are involved in these processes. In conclusion, this study demonstrates preliminary but encouraging data to further support that adipose tissue secreted factors from lean human subjects might possess neuroprotective properties and unravel the specific roles of ERK1/2, JNK and PI3K in these processes.

Figure 1

Heat-sensitive factors secreted from human preperitoneal adipose tissue protect against H₂O₂ induced toxicity in SH-SY5Y neuronal cells. Adipose tissue conditioned media (ATCM) from lean subjects was applied to SH-SY5Y neuronal cells for 24 h in the presence of 800 µM H₂O₂. Control cell cultures were treated with M199 media. (a) ATCM had no direct effects on SH-SY5Y neuronal cell viability assessed by the MTT assay, but lean ATCM completely reversed H₂O₂ induced neurotoxicity; (b) The neuroprotective effect of lean ATCM was abolished when ATCM was heated at 95 °C for 10 min and (c) ATCM from subcutaneous fat of obese subjects demonstrated no effect on H₂O₂ induced neurotoxicity. Data (mean + S.E.M.) are expressed as % viable cells, where 100% values were obtained from SH-SY5Y cells incubated with fresh M199 only. All cell culture experiments were repeated on two separate passages of cells with ATCM from three different subjects (n = 6). * p < 0.05 compared to M199 control.

Figure 2

ERK1/2, JNK and PI3K are involved in the protective effects of lean ATCM against H₂O₂ induced neurotoxicity. (a) Adipose tissue conditioned media (ATCM) from lean subjects was applied to SH-SY5Y neuronal cells for 1 h and the phosphorylation of JNK, Akt at threonine 308, and ERK1/2 was measured by western blotting. Phosphorylated signaling proteins are expressed relative to respective total protein and the ATCM condition was compared to vehicle (veh) treated well and (b) Adipose tissue conditioned media (ATCM) from lean subjects was applied to SH-SY5Y neuronal cells for 24 h in the presence of 800 µM H₂O₂. Chemical inhibition of JNK with SP600125 (5 µM), PI3-K with LY29400 (20 µM) and ERK with PD98059 (25 µM) partially inhibited the neuroprotective effects of ATCM in SH-SY5Y cells under oxidative stress conditions. Representative western blots are shown to the right of the quantified data in panel A. All cell culture experiments were repeated on two separate passages of cells with ATCM from three different subjects (n = 6). * p < 0.05 compared to M199 control.