Algae Undaria pinnatifida Protects Hypothalamic Neurons against Endoplasmic Reticulum Stress through Akt/mTOR Signaling

Increased endoplasmic reticulum (ER) stress is known to be one of the causes of hypothalamic neuronal damage, as well as a cause of metabolic disorders such as obesity and diabetes. Recent evidence has suggested that Undaria pinnatifida (UP), an edible brown algae, has antioxidant activity. However, the neuroprotective effect of UP has yet to be examined. In this study, to investigate the neuroprotective effect of UP on ER stress-induced neuronal damage in mouse hypothalamic neurons, mice immortal hypothalamic neurons (GT1-7) were incubated with extract of UP. ER stress was induced by treating with tunicamycin. Tunicamycin induced apoptotic cell death was compared with the vehicle treatment through excessive ER stress. However UP protected GT1-7 cells from cell death, occurring after treatment with tunicamycin by reducing ER stress. Treatment with UP resulted in reduced increment of ATF6 and CHOP, and recovered the decrease of phosphorylation of Akt/mTOR by tunicamycin and the increment of autophagy. These results show that UP protects GT1-7 cells from ER stress induced cell death through the Akt/mTOR pathway. The current study suggests that UP may have a beneficial effect on cerebral neuronal degeneration in metabolic diseases with elevated ER stress.


Introduction
The endoplasmic reticulum (ER) provides a unique environment for the synthesis, folding, and maturation of secreted and transmembrane proteins. The ER also plays a critical role toward maintenance of cellular calcium homeostasis. ER stress is an emerging important mechanism induced from imbalance between the protein folding capacity of the ER and the newly synthesized protein load, resulting in accumulation of misfolded protein [1]. To counteract ER stress, the cell activates the unfolded protein response (UPR) to restore cellular homeostasis by clearing the misfolded protein within the ER lumen. Nonetheless, under severe and prolonged ER stress, when homeostasis cannot be restored, UPR activates unique pathways that lead to cell death through apoptosis [2]. Disruption of these physiological functions by ER stress has been implicated in a wide variety of human diseases, including Alzheimer's disease, Parkinson's disease, neuronal damage by ischemia, prion Molecules 2015, 20, 20998-21009; doi:10.3390/molecules201219744 www.mdpi.com/journal/molecules disease, cystic fibrosis, obesity, and diabetes mellitus [1,3]. In particular, hypothalamic ER stress has been suggested to cause feeding behavior disorder and glucose dysregulation as for obesity and diabetes [4][5][6][7]. The edible brown algae Undaria pinnatifida (UP) is widely distributed in Northeast Asia (China, Japan, and Korea). Fucoidan, a class of fucose-enriched sulfated polysaccharides, derived from UP has been reported to have various biological activities, including anti-tumor, anti-oxidative, anti-inflammatory, anti-viral, and anti-angiogenic properties [8,9]. Recently, UP was shown to attract development and complexity of neurons [10,11]. However, no study to elucidate the direct effect of UP on ER stress and ER stress-induced cell death in hypothalamic neurons has been reported. Therefore, since hypothalamic ER stress had been widely considered as an approach toward prevention of obesity and diabetes, we were interested in whether UP also has a protective effect from ER stress-induced neuronal cell death. In this study, we demonstrated the suppression effect of ethanol extract of UP on ER stress-induced cell death on GT1-7 cells, and attempted to examine the mechanism by which UP has anti-apoptotic activity against ER stress.

UP Has a Protective Effect in Tunicamycin-Induced Cell Death
To evaluate the effect of tunicamycin, ER stress inducer induced cell death in GT1-7 cells, GT1-1 cells were exposed to various concentrations of tunicamycin for 24 h resulted in decreased cell viability in a concentration-dependent manner ( Figure 1A). Subsequently, to determine the role of UP in tunicamycin-induced cell death, cells were exposed to various concentrations of UP with tunicamycin. Simultaneous treatment with UP (5-40 µg/mL) and tunicamycin (5 µg/mL) for 24 h resulted in attenuation of tunicamycin-induced cell death ( Figure 1B). In addition, no effects on cell viability were observed at the concentrations of UP used in the current study ( Figure 1C). These results raised the possibility that UP has a beneficial effect against tunicamycin-induced cell death in GT1-7 cells.
The edible brown algae Undaria pinnatifida (UP) is widely distributed in Northeast Asia (China, Japan, and Korea). Fucoidan, a class of fucose-enriched sulfated polysaccharides, derived from UP has been reported to have various biological activities, including anti-tumor, anti-oxidative, anti-inflammatory, anti-viral, and anti-angiogenic properties [8,9]. Recently, UP was shown to attract development and complexity of neurons [10,11]. However, no study to elucidate the direct effect of UP on ER stress and ER stress-induced cell death in hypothalamic neurons has been reported. Therefore, since hypothalamic ER stress had been widely considered as an approach toward prevention of obesity and diabetes, we were interested in whether UP also has a protective effect from ER stress-induced neuronal cell death. In this study, we demonstrated the suppression effect of ethanol extract of UP on ER stress-induced cell death on GT1-7 cells, and attempted to examine the mechanism by which UP has anti-apoptotic activity against ER stress.

UP Has a Protective Effect in Tunicamycin-Induced Cell Death
To evaluate the effect of tunicamycin, ER stress inducer induced cell death in GT1-7 cells, GT1-1 cells were exposed to various concentrations of tunicamycin for 24 h resulted in decreased cell viability in a concentration-dependent manner ( Figure 1A). Subsequently, to determine the role of UP in tunicamycin-induced cell death, cells were exposed to various concentrations of UP with tunicamycin. Simultaneous treatment with UP (5-40 μg/mL) and tunicamycin (5 μg/mL) for 24 h resulted in attenuation of tunicamycin-induced cell death ( Figure 1B). In addition, no effects on cell viability were observed at the concentrations of UP used in the current study ( Figure 1C). These results raised the possibility that UP has a beneficial effect against tunicamycin-induced cell death in GT1-7 cells.

UP Attenuates ER Stress
In previous works, tunicamycin increased ER stress markers, such as CHOP, ATF6, ATF4, and XBP-1 splicing in the hypothalamus, often leading to cell death [12][13][14]. Therefore we investigated the expression levels of ER stress-induced CHOP, ATF6, and ATF4 in GT1-7 cells (Figure 2A). Tunicamycin treatment increased levels of CHOP, ATF6, and ATF4 in a concentration-dependent manner up to 2-10 fold. We next determined alternative XBP-1 mRNA splicing, part of the endoplasmic reticulum stress response, using RT-PCR. Exposure to tunicamycin increased alternative XBP-1 mRNA splicing ( Figure 2B). Because UP reliably increased the cell viability against tunicamycin induced cell death, we performed additional studies to determine whether UP reduces tunicamycin induced ER stress. As shown in Figure 2C, treatment with UP reduced expression level of tunicamycin induced

UP Attenuates ER Stress
In previous works, tunicamycin increased ER stress markers, such as CHOP, ATF6, ATF4, and XBP-1 splicing in the hypothalamus, often leading to cell death [12][13][14]. Therefore we investigated the expression levels of ER stress-induced CHOP, ATF6, and ATF4 in GT1-7 cells (Figure 2A). Tunicamycin treatment increased levels of CHOP, ATF6, and ATF4 in a concentration-dependent manner up to 2-10 fold. We next determined alternative XBP-1 mRNA splicing, part of the endoplasmic reticulum stress response, using RT-PCR. Exposure to tunicamycin increased alternative XBP-1 mRNA splicing ( Figure 2B). Because UP reliably increased the cell viability against tunicamycin induced cell death, we performed additional studies to determine whether UP reduces tunicamycin induced ER stress. As shown in Figure 2C, treatment with UP reduced expression level of tunicamycin induced ER stress marker, specifically CHOP and ATF6, but ATF4 expression was not significantly changed (data not shown).
Molecules 2015, 20, page-page 3 ER stress marker, specifically CHOP and ATF6, but ATF4 expression was not significantly changed (data not shown).

UP Attenuates ER Stress Induced Apoptotic Cell Death
Interference with ER function leads to accumulation and aggregation of unfolded proteins and initiation of the unfolded protein response (UPR) to restore ER function. However, if the stress is prolonged, apoptotic cell death ensues [14,15]. After determining that UP attenuates tunicamycininduced ER stress, we hypothesized that UP may regulate apoptotic cell death induced by ER stress. We determined whether tunicamycin induced-apoptosis is modulated by UP treatment using apoptotic markers, such as cleaved-PARP and cleaved-Caspase-3. As shown in Figure 3, the levels of cleaved-PARP and cleaved-Caspase-3 were significantly increased in a concentration ( Figure 3A) and time ( Figure 3B) dependent manner after treatment with tunicamycin. However these apoptotic signals

UP Attenuates ER Stress Induced Apoptotic Cell Death
Interference with ER function leads to accumulation and aggregation of unfolded proteins and initiation of the unfolded protein response (UPR) to restore ER function. However, if the stress is prolonged, apoptotic cell death ensues [14,15]. After determining that UP attenuates tunicamycin-induced ER stress, we hypothesized that UP may regulate apoptotic cell death induced by ER stress. We determined whether tunicamycin induced-apoptosis is modulated by UP treatment using apoptotic markers, such as cleaved-PARP and cleaved-Caspase-3. As shown in Figure 3, the levels of cleaved-PARP and cleaved-Caspase-3 were significantly increased in a concentration ( Figure 3A) and time ( Figure 3B) dependent manner after treatment with tunicamycin. However these apoptotic signals were decreased with UP treatment ( Figure 3C). On the basis of our results, UP seems to alleviate the pro-apoptotic signals through the reduction of ER stress.
Molecules 2015, 20, page-page 4 were decreased with UP treatment ( Figure 3C). On the basis of our results, UP seems to alleviate the pro-apoptotic signals through the reduction of ER stress.

UP Increases Cell Viability via Akt/mTOR Signaling
Akt/mTOR pathway is well established as a cell survival signal [16]. Crosstalk between the Akt/mTOR and ER stress pathway has emerged [17,18]. Because Akt/mTOR operates both upstream and downstream of ER stress signals, we investigated the effect of tunicamycin on Akt and S6K1 (a marker of mTOR activity) phosphorylation. Tunicamycin treatment decreased phosphorylation of Akt and S6K1 in a concentration dependent manner, which resulted in increase of autophagy ( Figure 4A). However, phosphorylation of Akt and S6K1 was increased by UP treatment suggesting that UP can attenuate ER stress-induced apoptotic cell death by increasing Akt/mTOR signaling ( Figure 4B). After determining the roles of UP in Akt/S6K1 phosphorylation, we examined whether UP can restore the Akt/S6K1 phosphorylation reduced by tunicamycin. As shown in Figure 4C, UP certainly restored Akt/S6K1 phosphorylation, which resulted in a decrease of autophagy. After determining that UP increases Akt and S6K1 phosphorylation, we hypothesized that UP may regulate cell viability through Akt/mTOR signaling. To determine whether UP can increase the cell viability through Akt/mTOR signaling, we examined cell viability after treatment with the Akt inhibitor and knock-down ( Figure 5A-C). As a result, tunicamycin treatment with Akt inhibitor and knock-down strongly suppressed cell viability and the UP effect reducing tunicmycin-induced cell death was abolished with Akt inhibitor and knock-down. Furthermore, in the presence of mTOR inhibitor, rapamycin, drastic decrement of UP induced cell survival was shown under ER stress condition ( Figure 5E). These results suggest that Akt/mTOR signaling was necessary for neuroprotective effect of UP against tunicamycin induced cell death.

UP Increases Cell Viability via Akt/mTOR Signaling
Akt/mTOR pathway is well established as a cell survival signal [16]. Crosstalk between the Akt/mTOR and ER stress pathway has emerged [17,18]. Because Akt/mTOR operates both upstream and downstream of ER stress signals, we investigated the effect of tunicamycin on Akt and S6K1 (a marker of mTOR activity) phosphorylation. Tunicamycin treatment decreased phosphorylation of Akt and S6K1 in a concentration dependent manner, which resulted in increase of autophagy ( Figure 4A). However, phosphorylation of Akt and S6K1 was increased by UP treatment suggesting that UP can attenuate ER stress-induced apoptotic cell death by increasing Akt/mTOR signaling ( Figure 4B). After determining the roles of UP in Akt/S6K1 phosphorylation, we examined whether UP can restore the Akt/S6K1 phosphorylation reduced by tunicamycin. As shown in Figure 4C, UP certainly restored Akt/S6K1 phosphorylation, which resulted in a decrease of autophagy. After determining that UP increases Akt and S6K1 phosphorylation, we hypothesized that UP may regulate cell viability through Akt/mTOR signaling. To determine whether UP can increase the cell viability through Akt/mTOR signaling, we examined cell viability after treatment with the Akt inhibitor and knock-down ( Figure 5A-C). As a result, tunicamycin treatment with Akt inhibitor and knock-down strongly suppressed cell viability and the UP effect reducing tunicmycin-induced cell death was abolished with Akt inhibitor and knock-down. Furthermore, in the presence of mTOR inhibitor, rapamycin, drastic decrement of UP induced cell survival was shown under ER stress condition ( Figure 5E). These results suggest that Akt/mTOR signaling was necessary for neuroprotective effect of UP against tunicamycin induced cell death.

Discussion
Hypothalamic ER stress is known to be increased in metabolic diseases such as obesity and diabetes. Pharmacologic or genetic induction of ER stress in the hypothalamus caused central leptin and insulin resistance, resulting in increased food intake, glucose intolerance, and hypertension, whereas reduction of ER stress significantly alleviated these metabolic derangements [19,20]. Previous reports have implicated ER stress-induced cellular dysfunction and cell death are major contributors in many diseases. Therefore making modulators of the ER stress pathway is a potential target for therapeutic strategies [14]. It is well known that prolonged ER stress can lead to cell apoptosis. Several pathways have been identified to explain how cells trigger apoptotic cell death when faced with ER stress. However, the molecular mechanisms underlying ER stress-induced apoptotic cell death in hypothalamic neuronal cells are just emerging.
The findings have shown that Akt, a serine/threonine protein kinase, is a mediator of growth factor-induced cell survival and suppresses apoptotic death [21,22]. Increasing evidence suggests that the PI3K/Akt pathway is involved in ER stress. Activation of Akt in response to ER stress in different cell types has been reported [21]. However, Akt activation was regulated dually on the ER stress according to the ER stress exposure time. Akt was activated on short-term exposure to ER stress but down-regulated on long-term exposure to ER stress [23]. mTOR is essential for Akt-mediated survival signaling [24,25]. That the ER stress can also act upstream of mTORC1 adds a further layer of complexity. Pharmacological induction of UPR leads to rapid activation of the PI3K-Akt-mTORC1 signaling axis [26,27] which depends on the ATF6α branch of the UPR [28]. Prolonged treatment with ER-stress-inducing agents, however, inhibits Akt and mTORC1, which has been attributed to the mTORC1-S6K1-IRS1 negative feedback loop [23,[26][27][28][29][30][31]. Suppression of Akt following an extended period of ER stress apparently plays a central role in activation of Ire1α-ASK1-JNK downstream of mTORC1, possibly by derepression of the ASK1 adapt protein TRAF2 [18]. Increasing evidence has shown that the Akt/mTOR pathway is a mediator of growth factor induced cell survival and suppresses apoptotic cell death [21,22,26,27].
On the contrary, in ER stress, downregulation of Akt/mTOR pathway has been reported to be a cytoprotective response to the overload of misfolded or unfolded proteins through autophagy [32]. The previous report supported that PI3K/Akt/mTOR pathway promotes necrotic cell death via suppression of autophagy [33]. Conversely, autophagy induced by ER stress in a normal human colon cell line and in non-transformed murine embryonic fibroblasts contributes to cell death rather than protection. Therefore, the relevance of autophagy in cell survival is not always clear. The impact of autophagy on cell survival during ER stress, whether cytoprotective or proapototic, is likely contingent on the status of cells [34]. Our results reflect its role in promoting cell death. As shown in Figures 1C and 4C, UP can attenuate ER stress-induced cell death through reducing autophagy through upregulation of the Akt/mTOR pathway. These results suggest that autophagy can contribute to ER stress-induced cell death, which may depend on cellular status. However, the exact mechanisms of Akt/mTOR in ER stress-induced cellular dysfunction and cell death are far from fully elucidated [35][36][37].
In summary, our most significant finding is that UP, an ethanol extract of seaweed, shows a neuroprotective effect against ER stress through upregulation of the Akt/mTOR pathway leading to a decrease of autophagy ( Figure 5F). Considering the neuroprotective effect of UP on hypothalamic ER stress, our observations suggest that UP may be effective as an anti-obesity and anti-diabetic agent. However, further studies are warranted to clarify which ingredients of UP have those effects as well as to provide strong evidence of the functional roles of UP with Akt/mTOR pathways on hypothalamic ER stress-induced metabolic disease conditions such as obesity and diabetes mellitus.

Preparation of Undaria pinnatifida Extract
Powdered Undaria pinnatifida (UP) was extracted by pouring 95% ethanol into a conical flask containing 5 g of Undaria pinnatifida powder at a ratio of 50:1 (v/w). The mixture was incubated at RT for 24 h under dark conditions. After incubating, the supernatant was filtered and completely dried under a stream of nitrogen gas. The extract was dissolved in dimethyl sulfoxide (DMSO) to make an aliquot (5 mg/mL) and stored at´20˝C for further experiments [10,38].

Cell Viability Test
GT1-7 cells (1ˆ10 4 /well in 96-well plates) were cultured, and then cell viability was determined by a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. GT1-7 cells were seeded in triplicate at a density of 1ˆ10 4 cells per well on a 96-well plate. At the end of the treatment, the culture media were removed and MTT (0.5 mg/mL) was added, followed by incubation at 37˝C for 2 h in a CO 2 incubator. After dissolving the insoluble crystals that formed in DMSO, absorbance was measured at 570 nm using a microplate reader (Anthos Labtec Instruments, Wals-Siezenheim, Salzburg, Austria).

Transient Transfection with siRNA
Desalted and pre-annealed siRNA duplexes were purchased from Genolution Pharmaceuticals (Seoul, Korea). The siRNAs were designed using a proprietary algorithm devised by Genolution Pharmaceuticals (Table 1). To knockdown specific gene expression, GT1-7 cells were transfected with siRNAs at a final concentration of 10 nM using LipofectAMINE 2000 (Invitrogen), according to the manufacturer's instructions. Cells were harvested 24-48 h after transfection, and the potencies of siRNAs to silence gene expression were measured by RT-PCR. A control random sequence siRNA was also purchased from Genolution Pharmaceuticals and used as negative control.

Statistical Analysis
Unless stated otherwise, results are presented as the mean˘SD of three or more independent experiments. The student's ttest or one-way ANOVA with Dunnett's multiple-comparison test was used for comparison of treatments. SPSS version 19.0K (SPSS Inc., Chicago, IL, USA) was used for the analysis, and p value differences of <0.05 were considered statistically significant.

Conclusions
UP, an ethanol extract of Undaria pinnatifida, shows a neuroprotective effect against ER stress through upregulation of the Akt/mTOR pathway leading to a decrease of autophagy. Our observations suggest that UP may represent a viable therapeutic option as an anti-obesity and anti-diabetic agent.