RGS7 silence protects palmitic acid-induced pancreatic β-cell injury by inactivating the chemokine signaling pathway

Abstract Impaired insulin secretion due to pancreatic β-cell injury is an important cause of type 2 diabetes (T2D). Regulators of guanine nucleotide binding protein (G protein) signaling proteins played a key role in regulating insulin sensitivity in vivo. To explore the role of RGS7 on palmitic acid-induced pancreatic β-cell injury, pancreatic β-cells Beta-TC-6 and Min6 were treated with palmitic acid (PA) to similar type 2 diabetes (T2D) injury in vitro. The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT), 5-ethynyl-2′-deoxyuridine (EdU), and flow cytometry were used to analyze cell viability, proliferation, and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) kits were used to analyze the changes of inflammation-related cytokines. The expression of gene and protein was measured by quantitative real-time PCR (qRT-PCR) and western blot. PA modeling induced apoptosis, increased levels of inflammation-related cytokines, and suppressed cell viability and proliferation of pancreatic β-cells. RGS7 silence markedly alleviated the cell injury induced by PA. RGS7 overexpression further aggravated apoptosis and inflammatory response in PA-induced pancreatic β-cells and inhibited cell viability and proliferation. It is worth noting that RGS7 activated the chemokine signaling pathway. Silence of the key gene of the chemokine signaling pathway could eliminate the negative effect of RGS7 on PA-induced pancreatic β-cells. RGS7 silence protects pancreatic β-cells from PA-induced injury by inactivating the chemokine signaling pathway.


Introduction
Diabetes is a chronic hyperglycemia caused by pancreatic β-cell damage, deficiency of its action, or deficiency of insulin secretion caused by both [1,2].Type 2 diabetes (T2D) is characterized by the dysfunction of pancreatic β-cell in elevating blood glucose levels, impaired insulin secretion, and failure of peripheral tissue response to physiological insulin levels [3,4].T2D has spread rapidly around the world due to the combination of genetic and environmental factors [5,6] and has affected about 500 million people [7,8].In addition, T2D increases the risk of a variety of diseases and results in increased morbidity and economic burden [9].In terms of treatment, the results of lifestyle-oriented and drug intervention results for T2D are often disappointing [10].Therefore, finding the effective targets for protecting pancreatic β-cells becomes an important therapeutic strategy for T2D.
Regulators of guanine nucleotide binding protein (G protein) signaling (RGSs) are originally identified as guanosine triphosphate (GTP)-activating proteins of Gα subunit of heterotrimer G protein [11].Presently, more than 30 RGS proteins have been identified [12].Of these, RGS7 is evolutionarily conservative in all animals and plays a key role in many processes and organ systems [13,14].More importantly, RGS proteins were reported to play a key role in regulating insulin sensitivity in vivo [11,[15][16][17].RGS16 reconstitution substantially exacerbated insulin resistance in mice [18].RGS7 is highly expressed in the brain, especially in the hypothalamus, and may be involved in regulating the hypothalamic-pituitary-adrenal axis in response to different stresses and stimuli [19].Previous studies have provided evidence that RGS7 may constitute an obesity locus in humans [19,20].Based on these findings, we speculated that RGS proteins may provide a new therapeutic opportunity for T2D.
Prolonged exposure of β-cells to high fatty acid concentrations results in increased insulin-secreting cell death [21].Palmitic acid (PA) is the most abundant long-chain saturated free fatty acid physiologically.In insulin-secreting cells, such as pancreatic β-cells, excess fatty acid accumulation leads to cellular dysfunction and damage [22].In this study, we stimulated pancreatic β-cells with PA to similar T2D injury to investigate the effect of RGS7 in T2D.We hypothesize that RGS7 silence could alleviate PA-induced injury in pancreatic β-cells.

Cell culture and treatment
Mice islet cells Beta-TC-6, NIT-1 (Procell, Wuhan, China), Min6 (BeNa Culture Collection, Beijing, China), and hamster islet cells HIT-T15 (Procell, Wuhan, China) were cultured in 90% RPMI-1640 supplemented with 10% fetal bovine serum (FBS; endotoxin levels < 5 EU/mL), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.05 mM β-mercaptoethanol medium (culture temperature, 37 °C; gas atmosphere, 5% CO 2 + 95% air).When the cell density reaches 80%-90%, palmitic acid (PA, Sigma-Aldrich, St. Louis, MO, USA) was conjugated with fatty-acid-free bull serum albumin (BSA) prior to addition to cell culture.PA was dissolved in 99% ethanol to a concentration of 100 mM, and then mixed with 10% BSA in serum-free high glucose Dulbecco's modified eagle medium (DMEM) to make a 4 mM PA stock solution.The BSA-conjugated PA was added to cells at a final concentration of 0.4 mM.Cells treated with 0.4 mM PA for 48 h were used as PA group.Cells treated with ethanol and fatty-acid free BSA without PA were used as control group.All experiments were biologically repeated three times.

Bioinformatics analysis
The data downloaded from GEO (GEO accession: GSE118230) [23] was used to analyze RGS7 expression in normal and PA-treated human islet.Gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Gene and Genome (KEGG) were used to evaluate the potential mechanism underlying the involvement of RGS7 in T2D based on GEO data (GEO accession: GSE118230).

Cell viability analysis
The cell viability was analyzed by using the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay (C0009S, Beyotime).Beta-TC-6 cells and Min6 cells were seeded in 96-well plates (5 × 10 4 cells/well) and incubated overnight.Forty-eight hours after PA or transfection treatment, 10 μL of MTT solution was added to each well, and the cells were incubated for 4 h in a cell incubator.Add 100 μL of Formazan lysis solution to each well, mix properly, and incubate in a cell incubator until formazan is completely dissolved.The absorbance was measured at 570 nm using a microplate reader.All the experiments were performed in triplicate.

Flow cytometry analysis
The cell apoptosis was analyzed by the Annexin V-FITC Apoptosis Detection Kit (C1062S, Beyotime).Beta-TC-6 and Min6 cells were washed with phosphate buffer saline (PBS) and digested using trypsin cell digestion solution until the cells were blown down by gentle blowing, and the trypsin cell digestion solution was aspirated.Cells were then centrifuged at 1000 g for 5 min, resuspended with PBS and counted.The resuspended cells (5 × 10 4 ) were centrifuged at 1000 g for 5 min and then discarded the supernatant.Briefly, 195 μL of Annexin V-FITC binding buffer was added to resuspend the cells.Subsequently, 5 μL of Annexin V-FITC and 10 μL propidium iodide staining solution were added and incubated for 10-20 min at room temperature in the dark.The apoptosis cells were detected by flow cytometry.The sum of the proportions of early (AnnexinV + / PI -) and late (AnnexinV + /PI + ) apoptotic cells is the apoptosis rate.All the experiments were performed in triplicate.

5-Ethynyl-2′-deoxyuridine analysis
The cell proliferation was analyzed by using the 5-ethynyl-2′deoxyuridine (EdU) Cell Proliferation Kit (C0078S, Beyotime).Beta-TC-6 and Min6 cells (2 × 10 4 cells/well) were cultured in 24-well plates overnight.Cells were incubated with EdU solution (20 μM) for 2 h.Subsequently, 4% paraformaldehyde was added for 15 min at room temperature.After incubating with PBS for 10-15 min at room temperature, cells were incubated with endogenous peroxidase blocking solution for 20 min to inactivate endogenous peroxidase.After three times wash with PBS, 4′,6-diamidino-2-phenylindole (DAPI) was added and incubated for 10 min in a dark room at room temperature to stain cell nuclei.Finally, EdU-positive cells were observed and counted with a fluorescence microscope.All the experiments were performed in triplicate.

Western blot analysis
The protein was extracted form Beta-TC-6 and Min6 cells using the RIPA buffer, followed by separating by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to the polyvinylidene fluoride (PVDF) membranes.

Statistical analysis
All the data were presented as mean ± SD and analyzed by using GraphPad Prism 7.0 software (GraphPad Software Inc., San Diego, CA).The differences between two groups were analyzed by the Student's t test.The differences among multiple groups were analyzed by one-way ANOVA followed by post-hoc test.p < 0.05 was considered statistically different.

RGS7 is highly expressed in PA-induced pancreatic β-cells
The analysis of GEO dataset GSE118230 showed the level of R7 family members (Figure 1A).The top three members of the expression level are RGS4, RGS2, and RGS7 (Figure 1A).The role of RGS4 and RGS2 in pancreatic β-cells has been reported previously [24][25][26][27].Thus, RGS7 was selected for subsequent study.GEO dataset GSE118230 showed that the expression level of RGS7 was significantly increased after 7 days of treatment with PA (Figure 1B).Then, total of four pancreatic β-cell lines (Beta-TC-6, NIT-1, Min6, and HIT-T15) were employed to measure RGS7 expression.As shown in Figure 1C,D, RGS7 expression was the highest in Beta-TC-6 cells and the lowest in Min6 cells.Therefore, Beta-TC-6 and Min6 cells were selected for our study.We then treated the pancreatic β-cells Beta-TC-6 and Min6 cells, and measured the change of RGS7 expression.The results showed that the levels of RGS7 mRNA and protein in PA group were obviously upregulated compared with control group (Figure 1E,F).These results suggested that RGS7 might play a vital role in PA-induced pancreatic β-cells.

RGS7 regulates proliferation and apoptosis of PA-induced pancreatic β-cells
To investigate the effect of RGS7 in PA-induced pancreatic β-cells, RGS7 siRNAs were transfected into Beta-TC-6 cells.As shown in Figure 2A, RGS7 expression was obviously suppressed by RGS7 siRNAs.PA treatment markedly increased RGS7 expression compared with control group.CCK-8 assay showed no significant difference between si-NC group and si-RGS7 groups (Figure 2B).Compared with si-NC group, RGS7 expression was significantly decreased in PA + si-RGS7 groups (Figure 2C).Then, MTT and EdU assays were performed to detect cell viability and proliferation of pancreatic β-cells.Compared with control group, PA modeling markedly inhibited cell viability and decreased EdU-positive cells of pancreatic β-cells.RGS7 silence obviously increased the cell viability and proliferation (Figure 2D,E).In addition, cell apoptosis were measured by flow cytometry.PA modeling accelerated apoptosis, while RGS7 silence partly inhibited the increase of apoptosis induced by PA (Figure 2F).Then, RGS7 overexpressed plasmid was transfected into Min6 cells to further explore the effect of RGS7 on PA-induced pancreatic β-cells.As shown in Figure 3A, RGS7 expression was markedly increased by RGS7 overexpression.Compared with PA + Vector group, RGS7 expression in PA + RGS7 group was further increased (Figure 3B).We also analyzed the effect of RGS7 overexpression on cell viability, proliferation, and apoptosis.The results showed that RGS7 overexpression obviously decreased cell viability, proliferation whereas increased apoptosis of PA-induced pancreatic β-cells in comparison with PA + Vector group (Figure 3C-E).To confirm the increase in apoptosis upon RGS7 overexpression, overexpression of RGS7 and its mutant were transferred into Min6 cells.The expression of RGS7 was detected by qRT-PCR and western blot.The results showed that both RGS7 and RGS7-mutant significantly increased RGS7 expression (Figure 3F).The apoptosis was detected by flow cytometry.Notably, RGS7-mutant has no significant effect on apoptosis (Figure 3G).

RGS7 regulates cytokines levels of PA-induced pancreatic β-cells
Then, we analyzed the changes of inflammation-related cytokines after transfection with RGS7 siRNAs or RGS7 overexpressed plasmid.PA modeling markedly increased the levels of TNF-α, IL-1β, IL-6, and IL-18 of Beta-TC-6 and Min6 cells.Compared with PA + si-NC group, RGS7 silence significantly reduced the levels of these inflammation-related cytokines in Beta-TC-6 cells (Figure 4A).Oppositely, RGS7 overexpression further increased the levels of TNF-α, IL-1β, IL-6, and IL-18 in Min6 cells compared with PA + Vector group (Figure 4B).

RGS7 activates the chemokine signaling pathway in PA-induced pancreatic β-cells
To investigate the mechanism of RGS7 in PA-induced pancreatic β-cells, KEGG enrichment analysis was performed using GSEA.The analysis result showed that RGS7 activated the chemokine signaling pathway (Figure 5A).The genes enriched in the chemokine signaling pathway were shown as the Blue-Pink O' Gram (Figure 5B).Then, we measured the expression of chemokine signaling pathway-related proteins RAP1A, AKT3, PIK3R3, XCL2, and XCL1.PA treatment remarkably enhanced the expression of RAP1A, XCL2, and XCL1, and reduced the expression of AKT3 and PIK3R3 (Figure 3C,D).RGS7 silence markedly decreased the levels of RAP1A, XCL2, and XCL1 and increased the levels of AKT3 and PIK3R3 in Beta-TC-6 cells (Figure 5E).Oppositely, RGS7 overexpression markedly increased the levels of RAP1A, XCL2, and XCL1 whereas suppressed the levels of AKT3 and PIK3R3 in Min6 cells (Figure 5F).

RGS7 overexpression eliminates the effect of RAP1A silence on PA-induced pancreatic β-cells
To confirm whether RGS7 functions in PA-induced pancreatic β-cells by regulating signaling pathways, RGS7 overexpression vector and RAP1A siRNA were co-transfected into Min6 cells.The data in Figure 6A showed that RAP1A expression was obviously suppressed by RAP1A siRNAs, especially si-RAP1A no. 2. Thus, si-RAP1A no. 2 was used for follow-up experiments.The MTT and EdU assays showed that RGS7 overexpression suppressed cell viability and proliferation of PA-induced pancreatic β-cells.However, the inhibitory effect of RGS7 overexpression was partly eliminated by RAP1A silence (Figure 6B,C).The flow cytometry data showed that RGS7 overexpression accelerated apoptosis of PA-induced pancreatic β-cells and the acceleration was eliminated by RAP1A silence (Figure 6D).Similarly, the increase of inflammation-related cytokines levels induced by RGS7 overexpression was eliminated by RAP1A silence in PA-induced pancreatic β-cells (Figure 6E).

Discussion
RGS proteins are important physiologic regulators of Gαi/o and Gαq/11 signaling and have been implicated in the regulation of pancreatic function [25,28].In present study, we found that RGS7 silence could promote β-cell activity and proliferation, and inhibit apoptosis and inflammatory responses of PA-treated pancreatic β-cells by regulating the chemokine signaling pathway.The findings may offer a novel therapeutic approach for T2D.
Inflammation is one of the common features of diabetes [29,30].Inhibition of inflammatory response could alleviate the diabetes symptoms in vivo and in vitro [29,31,32].Suppression of pancreatic islet inflammation attenuates the dysfunction of pancreatic β-cells induced by lipotoxicity [33].Exacerbated islet inflammation leads to increase of β-cell apoptosis [34].In this study, RGS7 silence markedly enhanced cell viability and proliferation ability while decreased apoptosis of pancreatic β-cells.Studies have reported the changes of IL-1β, IL-6, IL-18, and TNF-α level in pancreatic [35][36][37].We found that RGS7 silence obviously reduced the levels of TNF-α, IL-1β, IL-6, and IL-18 in pancreatic β-cells.The contrary results were obtained in RGS7 overexpressed pancreatic β-cells.These data were consistent with previous study, suggesting that RGS7 silence may protect pancreatic β-cells from PA by reducing the levels of inflammation-related cytokines and decreasing apoptosis.
Chemokines can attract immune cells to inflammation sites due to their ability to induce directed chemotaxis of   nearby responding cells Chemokines signaling participated in many physiological processes, such as cell migration [40][41][42], angiogenesis [43][44][45], cancer dissemination [46][47][48], and inflammatory response [49,50].XCL1 and XCL2 are members of the C-chemokine subfamily, which promote inflammatory progression and are involved in a variety of inflammatory diseases [51,52].Ras-associated protein 1A (Rap1A) is a type of small GTP-binding protein that belongs to the Ras subfamily [53], and plays an essential regulatory role in a variety of cellular processes, including proliferation [54].PIK3R3 is one of the regulatory subunits of phosphoinositide 3-kinase, which notably affects cell genesis, proliferation, and apoptosis [55][56][57].In this study, RGS7 activated the chemokine signaling pathway, showing as increased levels of RAP1A, XCL2, and XCL1, and decreased the levels of AKT3 and PIK3R3 in pancreatic β-cells.Furthermore, inhibition of RAP1A, the key gene of the chemokine signaling pathway, markedly eliminated the negative effect of RGS7 overexpression on pancreatic β-cells.These above data suggested that RGS7 silence protected pancreatic β-cells from PA by inactivating the chemokine signaling pathway.
Our findings suggested that RGS7 silence suppressed inflammatory response and apoptosis of PA-induced pancreatic β-cells.RGS7 activated the chemokine signaling pathways.RGS7 silence inhibited PA-induced inflammatory response by inactivating the chemokine signaling pathway, thereby reducing apoptosis of pancreatic β-cells.
However, there are several limitations in the present study.It is more convincing to perform RGS7 silencing and overexpression experiments in Beta-TC-6 and Min6 cells simultaneously.The regulatory effect of RGS7 should be explored in vivo.Besides, the role of chemokine signaling pathway should also be further explored in future study.Anyway, more in-depth studies are needed to explore the mechanism RGS7 in diabetes.

Figure 1 .
Figure 1.RGS7 is highly expressed in PA-induced pancreatic β-cells.A: Data of GSE118230 showed the expression of R7 family members.B: Data of GSE118230 showed that RGS7 was upregulated in PA-induced pancreatic β-cells.C and D: qRt-PCR and western blot showed the expression of RGS7 in four pancreatic β-cell lines.E and F: qRt-PCR and western blot showed that RGS7 was upregulated in PA-induced pancreatic β-cells.All the experiments were performed triplicate.*p < 0.05 vs.Control group.

Figure 5 .
Figure 5. RGS7 activates the chemokine signaling pathway in PA-induced pancreatic β-cells.A: KEGG analysis showed that RGS7 activated the chemokine signaling pathway in PA-induced pancreatic β-cells.B: RGS7 silence decreased RAP1A expression and increased the expression of AKt3 and PiK3R3 in PA-induced pancreatic β-cells.*p < 0.05 vs. si-nC group.C: RGS7 overexpression increased RAP1A expression and decreased the expression of AKt3 and PiK3R3 in PA-induced pancreatic β-cells.*p < 0.05 vs.Control group.All the experiments were performed triplicate.

Figure 6 .
Figure 6.RGS7 overexpression eliminates the effect of RAPiA silence on PA-induced pancreatic β-cells.A: RAP1A siRnAs decreased RAP1A expression in pancreatic β-cells.*p < 0.05 vs. si-nC group according to one-way AnoVA.B: insulin content in PA-induced pancreatic β-cells.C: Cell viability of PA-induced pancreatic β-cells.D: the proliferation of PA-induced pancreatic β-cells.E: the apoptosis rate of PA-induced pancreatic β-cells.F: the inflammation levels of PA-induced pancreatic β-cells.*p < 0.05 vs. Vector group, #p < 0.05 vs. RGS7 + si-nC group.All the experiments were performed triplicate.