Overexpression of Insig-1 protects β cell against glucolipotoxicity via SREBP-1c

Background High glucose induced lipid synthesis leads to β cell glucolipotoxicity. Sterol regulatory element binding protein-1c (SREBP-1c) is reported to be partially involved in this process. Insulin induced gene-1 (Insig-1) is an important upstream regulator of Insig-1-SREBPs cleavage activating protein (SCAP)-SREBP-1c pathway. Insig-1 effectively blocks the transcription of SREBP-1c, preventing the activation of the genes for lipid biosynthesis. In this study, we aimed to investigate whether Insig-1 protects β cells against glucolipotoxicity. Methods An Insig-1 stable cell line was generated by overexpression of Insig-1 in INS-1 cells. The expression of Insig-1 was evaluated by RT-PCR and Western blotting, then, cells were then treated with standard (11.2 mM) or high (25.0 mM) glucose for 0 h, 24 h and 72 h. Cell viability, apoptosis, glucose stimulated insulin secretion (GSIS), lipid metabolism and mRNA expression of insulin secretion relevant genes such as IRS-2, PDX-1, GLUT-2, Insulin and UCP-2 were evaluated. Results We found that Insig-1 suppressed the high glucose induced SREBP-1c mRNA and protein expression. Our results also showed that Insig-1 overexpression protected β cells from ER stress-induced apoptosis by regulating the proteins expressed in the IRE1α pathway, such as p-IRE1α, p-JNK, CHOP and BCL-2. In addition, Insig-1 up-regulated the expression of IRS-2, PDX-1, GLUT-2 and Insulin, down-regulated the expression of UCP-2 and improved glucose stimulated insulin secretion (GSIS). Finally, we found that Insig-1 inhibited the lipid accumulation and free fatty acid (FFA) synthesis in a time-dependent manner. Conclusions There results suggest that Insig-1 may play a critical role in protecting β cells against glucolipotoxicity by regulating the expression of SREBP-1c.


Background
Pancreatic β cell dysfunction is a crucial pathological contributor to the development of type 2 diabetes. The effects of glucose and free fatty acid (FFA) on β cell dysfunction have been extensively studied [1][2][3][4]. Chronic exposure to high glucose or high lipid leads to "glucotoxicity" or "lipotoxicity" [5]. The term "glucolipotoxicity" is now widely accepted to describe the combined effects of high glucose and high lipid on β cell dysfunction [6]. Apoptosis, impaired glucose stimulated insulin secretion (GSIS) [7] and lipid accumulation [8] are critical components involved in glucolipotoxicity.
Insulin induced gene-1 (Insig-1), an ER-resident protein that contains six transmembrane segments, negatively regulates SREBPs and HMG-CoA reductase, and plays a critical role in the feedback control of lipid synthesis. Sterol-stimulated binding of Insig-1 to SREBPs cleavage activating protein (SCAP) facilitates the retention of SCAP/SREBP-1c complex in the ER, prevents SREBP-1c from entering into the Golgi apparatus, decreases production of the nuclear forms of SREBPs (nSREBPs) and limits transcription of SREBP-1c target genes [21]. Therefore, Insig-1 is an important upstream factor, which regulates lipid synthesis via Insig-1-SCAP-SREBP pathway. Overexpression of Insig-1 has been found to inhibit lipid synthesis both in vitro and in vivo experiments [22], however, it is still unclear whether Insig-1 regulates β cell function at SREBP-1c level.
The present study was performed to comprehensively investigate whether overexpression of Insig-1 protects β cells against glucolipotoxicity via SREBP-1c. Insig-1 stable transfected INS-1 cells were generated and the cell viability, apoptosis, insulin secretion, lipid metabolism and mRNA expression of relevant genes such as IRS-2, PDX-1, GLUT-2, Insulin and UCP-2 were evaluated.
To generate an Insig-1 overexpression INS-1 cell line (INS-1-Insig-1), INS-1 cells were transfected with pcDNA3.1(+)-Insig-1. In brief, INS-1 cells were plated in a six-well plate and grown overnight to approximately 60% confluency. Cells were transfected using Lipofectamine 2000 (Invitrogen, USA) and 1.6 μg DNA per well in serum-free medium according to the manufacturer's instructions. After 48 h, cells were subjected to 100 μg/ ml G418 selection for 30 days. Stable transfection cell line was derived from a single stable clone. Insig-1 gene and protein expression were confirmed by RT-PCR and Western blot analysis, respectively.
To study the effects of Insig-1 on glucose-induced glucolipotoxicity on β cell, INS-1-Insig-1 cells and control INS-1 cells were incubated in RPMI medium at 11.2 mM or 25.0 mM glucose for 0, 24 and 72 h.

Detection of apoptotic cells
For flow cytometric analysis, cells were collected by trypsinization and washed with cold phosphate-buffered saline and incubated for 10 min with Annexin V (Sigma, USA) for 15 min, then stained with propidium iodide (PI) (Sigma, USA). The analysis was performed with a FACScan flow cytometer (BD Biosciences, USA) using the CellQuest software (BD Biosciences). Cells that are in early apoptosis are Annexin V positive and PI negative.

Glucose stimulated insulin secretion (GSIS)
After exposure to the indicated glucose (11.2 mM or 25.0 mM) for 0, 24 and 72 h, two groups of cells were washed twice with PBS, followed by pre-incubation in Krebs-Ringer bicarbonate HEPES buffer (KRBH buffer: 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 25 mM HEPES, 0.5% BSA, pH 7.4) containing 3 mM glucose at 37°C for 30 min. The buffer was removed completely and collected for basal insulin secretion measurement. Fresh KRBH buffer containing 20 mM glucose was then added to the cells and cells were incubated for an additional 30 min as glucose stimulated insulin secretion. Insulin concentration was measured using an insulin radioimmunoassay (RIA) kit (Beijing Atom HighTech, China) according to the manufacture's protocol.
Total RNA preparation and real-time PCR Total RNA was extracted and reverse transcribed as described above. Real-time PCR was performed using QuantiTect SYBR green PCR master mix kit (ToYoBo, Japan) following the manufacturer's instructions on a mastercycler EP realplex RT-PCR instrument (Eppendorf, Germany). The reaction volume was 10 μL and contained 5 μL QuantiTect SYBR green PCR master mix, 0.5 μmol/L primers and 100 ng cDNA and RNasefree water. Primer sequences used in the PCR are provided in Table 1. The PCR conditions were: an initial denaturation at 95°C for 1 min, followed by 40 PCR cycles. Each cycle consisted of 15 s at 95°C, 30 s at 60°C and 30 s at 72°C. All quantifications were performed with rat GAPDH as an internal standard. The relative amount of all mRNA was calculated using the 2-ΔΔCT method.

Oil Red O staining and measurement of FFA content
Cells were fixed with 10% (v/v) formalin and were stained with Oil Red O as described by Kuri-Harcuch and Green [25]. Lipid droplets were observed and photographed under a microscope (TE2000-E; Nikon). At the end of 0, 24 and 72 h incubation, media were collected and FFA content of each sample was

Statistical analysis
Data shown are means ± SE. Statistical significance of differences between two groups was determined using the Student's t-test. Groups of three or more were analyzed by one-way ANOVA. P value of less than 0.05 was considered significant.

mRNA and protein expression of Insig-1 in INS-1 cells overexpressed with Insig-1
To determine the transfection efficiency, we analyzed protein and mRNA expression of mouse Insig-1 in INS-1-Insig-1 cells. The Insig-1 primary antibody was mouse specific and did not cross react with rat Insig-1 protein.
The Western blot results showed that Insig-1 protein was not expressed in control INS-1 cells, while mouse Insig-1 was highly expressed in two randomly selected stable cell lines with Insig-1 overexpression (sample 1 and 2), and the expression was especially high in sample 2 ( Figure 1A, B). Similarly, mouse Insig-1 mRNA was not expressed in control INS-1 cells, but the 819 bp mouse Insig-1 fragment was detected in INS-1-Insig-1 stable cell lines (especially in sample 2) which was in accordance with our original design ( Figure 1C). Sample 2 was thus selected as our INS-1-Insig-1 stable cell line for further experiment.

Insig-1 increases cell viability and decreases apoptosis against glucolipotoxicity
To determine the effect of Insig-1 overexpression on cell viability and apoptosis, INS-1 cells and INS-1-Insig-1 cells were exposed to 11.   Figure 2B), suggesting that Insig-1 overexpression protected β cell viability and apoptosis against chronic high glucose induced glucolipotoxicity.

Insig-1 suppresses nSREBP-1c expression and decreases ER stress through IRE1a pathway
To further investigate the underlying molecular mechanisms by which Insig-1 prevents β cell apoptosis, we examined the expression of nSREBP-1c, a nuclear active form of SREBP-1c. Cell total nuclear protein was extracted and SREBP-1c protein expression was evaluated by Western bolt analysis. The results showed that nSREBP-1c protein expression was significantly less upregulated in control INS-1 cells exposed to 25.0 mM glucose for 24 h and 72 h, while in INS-1-Insig-1 cells, nSREBP-1c was significantly down-regulated when exposed to standard (11.2 mM) or high (25.0 mM) glucose compared to control INS-1 cells at all time points. We also examined ER stress related protein expression   Figure 3A, B). These results suggested that overexpression of Insig-1 down regulated the expression of nSREBP-1c, suppressed the IRE1α pathway of ER stress and prevented β cells from apoptosis.
Although insulin mRNA expression was not changed by incubation with 11.2 mM glucose, it revealed a 1.59 and 1.82 fold increase with 25.0 mM glucose for 24 h and 72 h, respectively ( Figure 5E). The mRNA level of UCP-2, a negative modulator of insulin secretion, decreased 49% -70% ( Figure 5F). FAS is one of the most important lipid synthesis genes, the mRNA level of FAS decreased by 21% -65% in INS-1-Insig-1 cells ( Figure 5G). These results implicated that Insig-1 regulated insulin secretion and lipid genes expression through SREBP-1c.

Insig-1 inhibits intracellular accumulation of lipid droplets and reduces FFA synthesis
We further assessed whether Insig-1 prevented lipid accumulation and FFA synthesis.  Figure 6B). These results indicated that high glucose induced FFA production, which leads to glucolipotoxicity in INS-1 cells, was attenuated by Insig-1 overexpression.

Discussion
In the present study, we overexpressed Insig-1 in INS-1 cells and investigated the role of Insig-1 in glucolipotoxicity and demonstrated that Insig-1 partially improved β cell dysfunction induced by high glucose. Glucolipotoxicity is an important factor in β cell dysfunction [26]. SREBP-1c is a critical component involved in this process and has been examined extensively. We hypothesized that the potential mechanism is that high glucose induces lipid accumulation through SREBP-1c and leads to β cell dysfunction, which is accompanied by apoptosis, impaired GSIS and lipid accumulation [9]. Overexpression of SREBP-1c has been found to induce glucolipotoxicity in insulinoma cells (INS-1 and MIN6) and isolated rat islets [27]. Insig-1 acts as an upstream regulator of SREBP-1c and regulates lipid metabolism through the Insig-1-SCAP-SREBP1-c pathway. In vitro experiments showed that overexpression of Insig-1 prevented lipogenesis and inhibited differentiation of preadipocyte 3T3-L1 cells [28]. Furthermore, an in vivo study using recombinant adenovirus containing mouse Insig-1 cDNA transfected Zucker diabetic fatty (ZDF) (fa/fa) rats resulted in a striking reduction of lipid synthesis in liver [29].
To investigate the underlying molecular mechanism of Insig-1 in preventing β cell apoptosis, we examined the expression of SREBP-1c mRNA and protein in control  [27,28]. This suggests that overexpression of Insig-1 strongly suppresses SREBP-1c expression. We also assessed ER stress related protein expression in p-IRE1α pathway. ER stress plays an important role in β cell apoptosis. Wang et al. observed that the treatment of isolated rat islets with high glucose or ER stress inducers, drastically increased SREBP-1c activity, and the induction of a dominant negative mutant of SREBP-1c prevented high glucose induced ER stress [23]. Several studies have shown the p-IRE1α pathway which involves p-JNK activation, CHOP up-regulation and BCL-2 down-regulation in the process of apoptosis [29,30]. We observed that after exposing to   concentrations could produce FFA to switch on the IRE1α pathway at this time.

INS-1 cells and INS-1-Insig-1 cells under different
SREBP-1c participates in multiple regulatory mechanisms of GSIS. In vitro, overexpression of SREBP-1c results in impaired insulin secretion in isolated rat islets [31]. In vivo, similar results were also observed by Takahashi et al. by using transgenic mice overexpressing the active form of SREBP-1c [32]. In addition, SREBP-1c knockout mice had increased basal and high glucose stimulated insulin secretion [33]. SREBP-1c also regulates the expression of insulin genes [34]. Recently, it was demonstrated that IRS-2, a gene plays an important role in β cell growth and survival, participated in GSIS through direct binding of SREBP-1c to its promoter [35]. Furthermore, both in vitro and in vivo experiments showed that overexpression of SREBP-1c could suppress the expression of PDX-1, a crucial transcription factor of insulin secretion [36]. It was also observed that high nutrition could accumulate the expression of UCP-2, a regulator of cytoplasmic ATP/ADP ratio in the process of GSIS, through SREBP-1c [34]. GLUT-2 is known as a transporter of glucose, and is activated upon binding with SREBP-1c [37]. Our results showed that in INS-1-Insig-1 cells, IRS-2, PDX-1 and GLUT-2 mRNA expressions were increased to various degrees compared with control INS-1 cells. The change in UCP-2 mRNA expression was similar to and in accordance with that of SREBP-1c, and although drastically increased at 25.0 mM glucose. Insulin mRNA expression did not increase when exposed to standard 11.2 mM glucose stimulation. These results are consistent with our mRNA study indicating that overexpression of Insig-1 decreased insulin secretion only after exposure to high glucose and we therefore postulated that other mechanisms might also be involved.
We measured the FFA concentration to further elucidate whether Insig-1 could suppress the lipid accumulation and FFA synthesis. Wang et al. [26] confirmed that chronic exposure to high glucose for 72 h induced lipid accumulation in INS-1 cells by increasing lipogenic gene expression, while chronic incubation with 1.5 mM FFA (2:1 oleate/palmitate) did not obtain the same results [38]. Our results showed a significantly increase of lipid droplets accumulation and FFA concentration after exposing to 25.0 mM glucose for 24 h and 72 h. We also verified that chronic high glucose induced FFA synthesis participated in β cell glucolipotoxicity, and this process was partially prevented by overexpression of Insig-1. FFA concentration and lipid accumulation in INS-1-Insig-1 cell were significantly decreased compare to that of control INS-1 cells.
We observed more drastically effect when two groups cultured for 72 h than 24 h, especially when INS-1 cells exposed to 25.0 mM glucose, for example, enhanced early cell apoptosis rate, an ER stress response, an inhibition of GSIS, an accumulation of intracellular lipid droplets and so on. We therefore postulate that high glucose stimulates more FFA, leads to lipotoxicity, and subsequently induces the impairment of beta cells.

Conclusion
In summary, our results demonstrated that β cell dysfunction through chronic exposure of high glucose could be inhibited by Insig-1. The process may involve reducing cell apoptosis and increasing cell viability, preventing lipid accumulation and improving impaired GSIS. One possible molecular mechanism is that Insig-1 suppresses high glucose induced SREBP-1c transcription, which leads to reduced FFA and other lipid production, decreases expression of ER stress pathway protein and up-regulates the expression of insulin secretion genes. In conclusion, overexpression of Insig-1 protects β cells against glucolipotoxicity via SREBP-1c, thus improvement of Insig-1 activity should be considered as a therapy aimed at β cell protection.