Phlorotannins from Ecklonia cava Attenuates Palmitate-Induced Endoplasmic Reticulum Stress and Leptin Resistance in Hypothalamic Neurons

Abstract

Leptin resistance in the hypothalamus has an essential role in obesity. Saturated fatty acids such as palmitate bind to Toll-like receptor 4 (TLR4) and lead to endoplasmic reticulum (ER) stress and leptin resistance. In this study, we evaluated whether extracts of Ecklonia cava would attenuate the ER stress induced by palmitate and reduce leptin resistance in hypothalamic neurons and microglia. We added palmitate to these cells to mimic the environment induced by high-fat diet in the hypothalamus and evaluated which of the E. cava phlorotannins-dieckol (DK), 2,7-phloroglucinol-6,6-bieckol (PHB), pyrogallol-phloroglucinol-6,6-bieckol (PPB), or phlorofucofuroeckol-A (PFFA)-had the most potent effect on attenuating leptin resistance. TLR4 and NF-κB expression induced by palmitate was attenuated most effectively by PPB in both hypothalamic neurons and microglia. ER stress markers were increased by palmitate and were attenuated by PPB in both hypothalamic neurons and microglia. Leptin resistance, which was evaluated as an increase in SOCS3 and a decrease in STAT3 with leptin receptor expression, was increased by palmitate and was decreased by PPB in hypothalamic neurons. The culture medium from palmitate-treated microglia increased leptin resistance in hypothalamic neurons and this resistance was attenuated by PPB. In conclusion, PPB attenuated leptin resistance by decreasing ER stress in both hypothalamic neurons and microglia.

Keywords: Ecklonia cava; endoplasmic reticulum stress; hypothalamic neurons; leptin resistance; microglia; obesity; phlorotannins; toll-like receptor 4.

Figure 1

Effects of E. cava extract on TLR4 expression and cell death in palmitate-treated hypothalamic neurons and microglia. The hypothalamic neurons and microglia were exposed to palmitate in phosphate buffered saline (PBS) or in different concentrations of E. cava extract (ECE). (A) In hypothalamic neurons, TLR4 mRNA levels were increased by palmitate in PBS. Addition of ECE decreased TLR4 mRNA levels in a concentration-dependent manner, with significant effects at 25 μg/mL and 50 μg/mL ECE. (B) In microglia, TLR4 expression was increased by palmitate, but was significantly decreased at 50 μg/mL ECE. (C,D) The survival ratios of hypothalamic neurons and microglia were measured using a cell survival assay after incubation with palmitate in PBS or different concentrations of ECE. Cell survival ratio was decreased by palmitate and was significantly increased by ECE at 50 μg/mL. Data are means ± SD. Values with different letters are significantly different at p < 0.05. TLR4, Toll-like receptor 4.

Figure 2

Effects of phlorotannins on TLR4 and NF-κB expression in palmitate-treated hypothalamic neurons and microglia (A,B) Schematic illustration of the treatment. Hypothalamic neurons and microglia were treated not only with palmitate but also with phlorotannins. (C) In hypothalamic neurons, the TLR4 mRNA level increased by palmitate treatment was decreased significantly by DK and PPB. (D) In microglia, the TLR4 mRNA level increased by palmitate was decreased significantly by all 4 phlorotannins; PPB had the most significant effect. (E) In hypothalamic neurons, the mRNA level of NF-κB was increased by palmitate treatment and was significantly decreased when palmitate was supplemented with DK or PPB. (F) In microglia, the mRNA level of NF-κB was increased by palmitate and was significantly decreased when palmitate was supplemented with any of the 4 phlorotannins; the strongest effect was observed with PPB. Data are means ± SD. Values with different letters are significantly different at p < 0.05. TLR4, Toll-like receptor 4; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B; PBS, phosphate-buffered saline; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.

Figure 3

Effects of phlorotannins on ER stress in palmitate-treated hypothalamic neurons and microglia. (A–D) In hypothalamic neurons, the mRNA levels of the ER stress markers (PERK, eIF2α, IRE1, and Xbp1) were significantly increased by palmitate and were significantly decreased when palmitate was supplemented with any of the 4 phlorotannins—DK and PPB had the most significant effects. (E–H) In microglia, the mRNA levels of the same markers were significantly increased by palmitate and were significantly decreased when palmitate was supplemented with any of the 4 phlorotannins—PPB had the most significant effect. Data are as means ± SD. Values with different letters are significantly different at p < 0.05. ER, endoplasmic reticulum; ECE, E. cava extract; PERK, PKR-like ER protein kinase; eIF2α, eukaryotic initiation factor 2 alpha; IRE1, inositol-requiring-enzyme-1; Xbp1, X-box-binding protein 1; PBS, phosphate-buffered saline; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.

Figure 4

Effects of phlorotannins on the expression of factors involved in leptin resistance in hypothalamic neurons and the effect of microglia (A) Expression of SOCS3 in hypothalamic neurons was increased by palmitate. The SOCS3 mRNA level increased by palmitate was significantly decreased by the 4 phlorotannins; DK and PPB had the most significant effects. (B,C) STAT3 and ObR mRNA levels were decreased by palmitate. In the presence of palmitate, they were significantly increased by the 4 phlorotannins; DK and PPB had the most significant effects. (D) Illustration explaining processes of how to make cell samples. Microglia was treated not only with palmitate but also with phlorotannins and then, the microglia supernatant was incubated with hypothalamic neurons. (E) In hypothalamic neurons incubated with microglial supernatant, SOCS3 expression was increased by supernatant from microglia treated with palmitate. The SOCS3 mRNA level increased by palmitate was significantly decreased by the 4 phlorotannins; PPB had the most significant effect. (F,G) In hypothalamic neurons treated with microglial supernatant, STAT3 and ObR expression was significantly decreased by palmitate. In the presence of palmitate, it was significantly increased by phlorotannins; PPB had the most significant effect. Data are means ± SD. Values with different letters are significantly different at p < 0.05. SOCS3, suppressor of cytokine signaling 3; ObR, leptin receptor; PBS, phosphate-buffered saline; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.

Figure 5

The attenuating effects of PPB on cell death in hypothalamic neuron cell affected by microglia. (A) TNF-α expression in microglia was increased by palmitate, and the effect of palmitate was decreased by phlorotannins, most strongly by PPB and DK. Data are means ± SD. Values with different letters are significantly different at p < 0.05. (B) The design of the experiment was as in Figure 4D. TUNEL stain showing that microglia supernatant incubated apoptotic hypothalamic neurons (pink) were increased by palmitate and decreased by PPB. All cell nuclei were detected by DAPI (blue). TNF-α, tumor necrosis factor alpha; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PBS, phosphate-buffered saline; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.

Figure 6

Summary of the findings of this study. Palmitate induced ER stress in hypothalamic neurons via binding to TLR4 and an increase in NF-κB expression. The increased NF-κB and ER stress lead to leptin resistance in hypothalamic neurons, whereas PPB decreased ER stress and leptin resistance. ER stress and NF-κB expression were also increased by palmitate in microglia, leading to an increase in TNF-α expression. TNF-α might induce leptin resistance in hypothalamic neurons. PPB attenuated ER stress, NF-κB expression, and TNF-α expression in microglia and decreased leptin resistance in hypothalamic neurons. ER, endoplasmic reticulum; TLR4, toll-like receptor 4; NF-κB, nuclear factor kappa-light chain enhancer of activated B cells; TNF-α, tumor necrosis factor alpha; ECE, E. cava extract; DK, dieckol; PHB, 2,7-phloroglucinol-6,6-bieckol; PPB, pyrogallol-phloroglucinol-6,6-bieckol; PFFA, phlorofucofuroeckol A.