Insulin enhanced leptin-induced STAT3 signaling by inducing GRP78

Leptin, an adipocyte-derived hormone, centrally regulates energy homeostasis. Overlaps in the regulation of glucose and energy homeostasis have been reported between leptin and insulin. However, the effects of insulin on leptin’s actions in the central nervous system (CNS) have not yet been elucidated in detail. In the present study, we found that insulin potentiated leptin’s actions through GRP78 in the neuronal cell line, SH-SY5Y-ObRb. Since insulin induces GRP78, we speculated that it may also enhance leptin’s actions through this induction. We found that insulin enhanced leptin-induced STAT3 phosphorylation and this effect was ameliorated by the knockdown of GRP78. The role of GRP78 in leptin’s actions was also confirmed by impairments in leptin-induced STAT3 phosphorylation in HEK293-ObRb cells in which GRP78 was knocked down. Furthermore, we found that the overexpression of GRP78 enhanced leptin-induced STAT3 phosphorylation. These results suggest that GRP78 plays an important role in leptin’s actions. Furthermore, insulin may enhance the leptin-induced activation of STAT3 by inducing GRP78, which may provide an important connection between insulin and leptin in the CNS.

Scientific RepoRts | 6:34312 | DOI: 10.1038/srep34312 induction of GRP78 by insulin 28 and its critical role in response to ER stress, the aim of the present study was to investigate the function of GRP78 in leptin signaling in insulin-treated neuronal cells.

Results
Insulin induced the phosphorylation of Akt and S6K. PI3K and mammalian target of rapamycin (mTOR) activities are necessary for insulin-induced metabolic pathways 29,30 . The activation of these pathways results in the phosphorylation of its downstream proteins Akt 31 and p70 ribosomal S6 kinase (S6K) 32 . A human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb) was treated with insulin (300 nM, 4 h). As shown in Fig. 1, insulin markedly activated and phosphorylated Akt and S6K in SH-SY5Y-ObRb cells. Thus, insulin signaling was functionally activated in the SH-SY5Y-ObRb cell line.
Additionally, to check that insulin-induced signal would be functional in the ObRb receptor stably transfected SH-SY5Y cells, we analyzed insulin-induced Akt phosphorylation in both SH-SY5Yand SH-SY5Y-ObRb cell lines. SH-SY5Y and SH-SY5Y-ObRb cells were exposed to serum-free medium for 24 h. Insulin (10 and 100 nM) was added during the last 15 min. Insulin-induced Akt activation signal was then analyzed by Western blotting. The result showed that insulin induced Akt phosphorylation in both models ( Supplementary Figs 1 and 2).
Insulin enhanced leptin-induced STAT3 activation. By binding to the long isoform of its receptor, leptin plays a major role in preventing obesity by activating the JAK2-STAT3 signaling pathway 4-6 . Insulin and leptin target the same hypothalamic area in order to suppress eating behavior 11 ; therefore, an investigation of the interaction between leptin and insulin may provide a fundamental understanding of obesity and its related diseases. In the present study, we examined the effects of leptin in the presence or absence of insulin using SH-SY5Y-ObRb cells. In order to achieve this, medium was changed to a serum-free condition 20 h prior to the exposure of cells to insulin (10 and 300 nM, 4 h) and this was followed by a leptin stimulation (0.03 μ g/ml, 15 min). Consistent with previous findings 33 , we showed that the co-stimulation with insulin and leptin enhanced leptin induced-STAT3 more than the leptin treatment alone ( Fig. 2A,B).
At the same time, we also investigated the effect of insulin alone on STAT3 phosphorylation. SH-SY5Y-ObRb cells were exposed to serum-free medium for 24 h. Various concentration of insulin (10, 30, 100 and 300 nM) was added during the last 15 min. As a result, solely treatment with insulin did not induce phosphorylation of STAT3, even at a short time (Fig. 2C).

Involvement of GRP78 in insulin-induced enhancements in leptin's actions. Increasing evidence
supports the overlapping roles of leptin and insulin in signal transduction. Leptin and insulin both activate PI3K 13,34 . The PI3K-mTOR pathway is one of the upstream pathways involved in the induction of GRP78 35 . Of note, insulin was previously reported to increase GRP78 levels via the PI3K-ATF4 pathway 28 . These findings suggest that insulin-induced GRP78 may influence the anti-obesity effects of leptin. Therefore, we examined the induction of GRP78 by insulin under our experimental conditions. SH-SY5Y-ObRb cells were exposed to serum-free medium for 20 h. Insulin (300 nM) was then added for 4 h. In accordance with previous findings 28 , we showed that insulin induced GRP78 in the SH-SY5Y-ObRb cell line (Fig. 3).
We next assessed the influence of insulin-induced GRP78 on leptin signals. We determined whether insulin-induced GRP78 is involved in enhancing leptin-induced STAT3 activation. In order to achieve this, we knocked down GRP78 in insulin-treated cells and analyzed leptin-induced STAT3 phosphorylation. Insulin failed to enhance leptin-induced STAT3 activation in GRP78-knocked down cells (Fig. 4). Taken together, these results suggest that insulin enhances leptin-induced STAT3 phosphorylation via the induction of GRP78.
Influence of GRP78 in leptin-induced STAT3 phosphorylation. We subsequently investigated the role of GRP78 in leptin-induced STAT3 activation. We performed GRP78 knockdown experiments in HEK293-ObRb cells. Cells were transfected with GRP78 siRNA (2 nM) for 72 h. As an indicator of transfection efficacy, the expression of GRP78 was subjected to a Western blotting analysis. We confirmed that GRP78-specific siRNA efficiently inhibited its expression in HEK293-ObRb cells (Fig. 5A,B). Under these conditions, we
analyzed leptin-induced STAT3 signaling. We found that the knockdown of GRP78 significantly reduced the leptin-induced phosphorylation of STAT3 in the HEK293-ObRb cell line (Fig. 5A,B).
In order to elucidate the role of GRP78 in leptin-induced STAT3 signaling, we then overexpressed GRP78 and analyzed leptin-induced signaling. The GRP78 construct was transfected into HEK293-ObRb cells for 48 h and the expression of GRP78 was then analyzed. We confirmed that the transfection of GRP78 increased its levels in HEK293-ObRb cells (Fig. 6). We subsequently stimulated this cellular model with leptin and analyzed STAT3 phosphorylation. As shown in Fig. 6, STAT3 phosphorylation was significantly greater in cells overexpressing GRP78 than in mock-transfected cells. Therefore, these results suggest that GRP78 plays an important role in the activation of leptin-induced STAT3 signaling.

Discussion
The adipocyte-derived hormone, leptin, and pancreatic hormone, insulin, centrally regulate nutrient homeostasis by suppressing feeding desire 7,36 . To date, studies on the relationship between leptin and insulin have been more focused on energy homeostasis 13 . Insulin was previously shown to potentiate leptin-induced STAT3 phosphorylation 33 . Furthermore, it was reported to induce GRP78 and protect against cell death 28 . Although a relationship between insulin and leptin has already been reported 13,37 , the function of GRP78 in leptin signaling has not yet been clarified. In the present study, we investigated the function of GRP78 in leptin-induced signaling, and found that it plays an important role in the leptin-induced phosphorylation of STAT3.
Due to the abundant expression of their receptors in the hypothalamus, leptin and insulin exert parallel effects on reductions in food intake and the activation of adiposity signaling. Leptin and insulin were shown to be secreted in a manner that depended on body fat content 38,39 . Furthermore, the central administration of either peptide is known to reduce appetite 7,40 . Leptin and insulin share the same PI3K molecular pathway. In addition to the JAK2-STAT3 pathway, the PI3K pathway is one of the main pathways involved in the anti-obesity effects of leptin 34,41 . The intracerebroventricular administration of a PI3K inhibitor was previously reported to inhibit the effects of leptin on anorexia 42 . Furthermore, insulin inhibited feeding through IRS-PI3K signaling 9 . Another study demonstrated that insulin up-regulated GRP78 through the PI3K-ATF4 pathway in neuronal cells 28 . In the present study, we found that insulin induced the expression of GRP78, while the inhibition of GRP78 expression ameliorated insulin-induced enhancements in leptin-induced STAT3 phosphorylation. Taken together, these results suggest the following link between insulin and leptin: by inducing GRP78, insulin has the ability to enhance leptin-induced STAT3 signaling. Additionally, we found that the knockdown of GRP78 inhibited leptin-induced STAT3 phosphorylation, and leptin-induced STAT3 phosphorylation was enhanced by its overexpression. Therefore, GRP78 may play an important role in leptin-induced STAT3 phosphorylation. Since our current in vitro results are interesting, further studies should be conducted in more physiological model, i.e. differentiated model of SH-SY5Y or primary hypothalamic neuronal cells.
Resistance to the effects of leptin is a characteristic of obese patients 27 . However, the pathophysiological mechanisms responsible for leptin resistance have not yet been determined. The accumulation of saturated fatty acids in obesity has been shown to cause ER stress, and one of the mechanisms responsible for leptin resistance may be mediated through ER stress 24,26,43 . A previous study reported that insulin contributed to resistance against ER stress 28 . Therefore, it may induce GRP78, thereby ameliorating ER stress-induced leptin resistance in obesity. We previously reported that leptin induced GRP78 expression in SH-SY5Y-ObRb cell line 44 . It is interesting subject to analyze whether leptin will affect insulin signaling through GRP78 expression. Further studies are needed in order to elucidate these mechanisms in more detail.
In conclusion, we herein demonstrated the important role of GRP78 in potentiating leptin signaling in a human neuroblastoma cellular model. Our results highlight the significance of GRP78 and its underlying connection to leptin and insulin. This integration may be advantageous for understanding the regulation of energy homeostasis, which may provide useful information for therapeutic interventions for obesity.

Materials. Human leptin was obtained from Enzo Life Science (NY). Human insulin (Humulin ® R) was purchased from Eli Lilly and Company (IN).
Cell Culture. Human neuroblastoma (SH-SY5Y) and human HEK293 cell lines stably transfected with the Ob-Rb long form of the leptin receptor (SH-SY5Y-ObRb and HEK293-ObRb, respectively) 45 were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37 °C in humidified 5% CO 2 /95% air. All experiments were performed in DMEM.  Plasmid and transfections. pcDNA3.1(+ )-GRP78/Bip was a gift from Richard C. Austin (Addgene plasmid # 32701) 46 . A mock plasmid was used as a negative control. Human HEK293-ObRb cells were transfected using the calcium phosphate transfection method. A total of 0.5 μ g of DNA was used per 35-mm plate. 2xHEPES buffered saline (2xHBS) solution (16.4 g NaCl, 11.9 g HEPES, and 0.54 g Na 2 HPO 4 , per liter, at pH 7.15) was used to form precipitates of calcium phosphate/DNA. Briefly, 3 h prior to transfection, we changed to fresh medium and a solution containing the plasmid and 2.5 mM CaCl 2 was added to 2xHBS solution. Following a 30-min incubation at room temperature, the calcium phosphate-plasmid suspension solution was added dropwise to the culture dish. Eight hours after the incubation, culture medium was changed to DMEM containing antibiotics. Cells were harvested 48 h after transfections.