- Open Access
Overexpression of Insig-1 protects β cell against glucolipotoxicity via SREBP-1c
© Chen et al; licensee BioMed Central Ltd. 2011
- Received: 15 February 2011
- Accepted: 16 August 2011
- Published: 16 August 2011
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.
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.
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.
There results suggest that Insig-1 may play a critical role in protecting β cells against glucolipotoxicity by regulating the expression of SREBP-1c.
- Free Fatty Acid
- Glucose Stimulate Insulin Secretion
- Free Fatty Acid Concentration
- SREBPs Cleavage Activate Protein
- Stable Transfection Cell Line
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 [1–4]. Chronic exposure to high glucose or high lipid leads to "glucotoxicity" or "lipotoxicity" . The term "glucolipotoxicity" is now widely accepted to describe the combined effects of high glucose and high lipid on β cell dysfunction . Apoptosis, impaired glucose stimulated insulin secretion (GSIS)  and lipid accumulation  are critical components involved in glucolipotoxicity.
Sterol regulatory element binding protein-1c (SREBP-1c), a lipogenic transcription factor, has been found to play a critical role in the development of β cell dysfunction caused by elevated glucose and FFA . SREBP-1c is a membrane-bound transcription factor from the basic helix-loop-helix (bHLH) leucine zipper family and has been described as a regulator of lipogenic enzymes in liver, adipocytes, myocytes and β cells . Overexpression of SREBP-1c induced β cell dysfunction, such as apoptosis, GSIS and lipid accumulation [9, 11]. SREBP-1c preferentially activates genes involved in FFA and triglyceride synthesis, like fatty acid synthesis (FAS), elongation of very long-chain fatty acids (ELOVL), and Δ5-desaturase (DSR5) [12, 13]. First, high glucose up-regulates the synthesis of FFA and leads to β cell apoptosis. Several mechanisms are implicated in this process, such as ER stress, oxidative stress, ceramide formation and modulation of microRNAs pathways [14–17]. ER stress mechanism is When FFA activates misfolded proteins in the ER lumen, Igheavy chain binding protein (BIP) dissociates from ER stress transducers. BIP then leads to an unfolded protein respond (UPR), including inositol requiring ER-to-nucleus signal kinase 1α (IRE1α), activating transcription factor (ATF6), and PKR-like ER kinase (PERK), the UPR is activated. The IRE1α then activates c-Jun N-terminal kinase (JNK), C/EBP homologous protein (CHOP), inhibits BCL-2 and results in apoptosis . Second, increased FFA synthesis results in impaired GSIS. SREBP-1c is implicated to be involved through regulation of insulin receptor substrate 2 (IRS-2), pancreatic duodenal homeobox factor-1 (PDX-1) and uncoupling protein-2 (UCP-2) . SREBP-1c directly regulates the transcription of the genes mentioned above and glucose transporter isoform-2 (GLUT-2) through sterol response element (SRE). GLUT-2 takes up glucose thereby increases ATP and ultimately up-regulates the expression of insulin . Third, SREBP-1c regulates lipid synthesis in β cell. High glucose both acutely and chronically induces de novo lipogenesis, and activates SREBP-1c transcription of lipid synthesis .
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 . 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 , 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.
The mouse Insig-1 cDNA fragment was generated by RT-PCR. Total RNA was extracted from mouse 3T3-L1 cells using TRIzol Reagent (Invitrogen, USA) following manufacture's instructions. Total RNA was reverse transcribed using moloney murine leukemia virus reverse transcriptase (Fermentas, USA). Fragments of mouse Insig-1 (NM-153526.5) cDNA were amplified by PCR using the following primers: forward5'-GAAGCTT ATGCCCAGGCTGC-3' and reverse 5'-GAATTC TTCCACTCTGAACCATGT-3'. Hind III and EcoR I restriction sites were included (underlined) to facilitate the ligation of cDNA product into pcDNA3.1(+) vector (Invitrogen, USA). The mouse Insig-1 primers used in this study were tested using Blast software (http://blast.ncbi.nlm.nih.gov/Blast.cgi), and did not recognize the rat Insig-1 mRNA sequence. The sequence of pcDNA3.1(+)-Insig-1 was verified by DNA sequencing.
Cell culture and generation of stable transfection cell line
Rat pancreatic INS-1 cells were maintained in RPMI 1640 medium (Gibco-BRL, U.K.) with 11.2 mM glucose supplemented with 10% heat-inactivated fetal calf serum, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 μΜ β-mercaptoethanol, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C in a humidified atmosphere (5% CO2 and 95% air).
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.
Cell viability was measured by adding 200 g/ml 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) (Dingguo, China) to INS-1 cells and INS-1-Insig-1 cells, and incubated for 3 h at 37°C. The reaction was stopped and the purple formazan precipitate formed was dissolved using dimethyl sulfoxide (DMSO) and the color intensity was measured at 550 nm using a multiwell spectrophotometer (Thermo Labsystems, USA).
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.
Western blot analysis
Proteins were extracted as previously described [23, 24]. Briefly, cells were washed twice with PBS, and protein was extracted using lysis buffer (Sigma, USA). The supernatant was obtained by centrifugation at 4°C, 12,000 rmp for 10 minutes. The nuclear protein was extracted using a nuclear protein extraction kit (Generay, China) according to the manufacture's protocol. Proteins were resolved by SDS-PAGE and were transferred to nitrocellulose membranes. After incubation with primary antibody and secondary antibody conjugated to horseradish peroxidase, the bands were detected with the enhanced chemiluminescence system (Amersham Bioscience, USA). Immunoblots were scanned and quantified using Scion Image software (Scion Corporation, USA). The following primary antibodies (Santa Cruz Biotechnology or Novus Biologicals, USA) and dilution were used: Insig-1 (sc-51102, 1:500), nSREBP-1 (sc-8984, 1:300), p-JNK(sc-6254, 1:100), CHOP (sc-575, 1:500), BCL-2 (sc-7382, 1:200), GAPDH (sc-47724, 1:200) and p-IRE1α (NB100-2323, 1:500).
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
Sequence information on the primers used for real-time PCR
Sequences for forward and reverse primers (5'-3')
Gene Bank accession number
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 . 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 determined using an ELISA kit (Uscnlife, China) according to the manufacturer's protocol.
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
Insig-1 increases cell viability and decreases apoptosis against glucolipotoxicity
Insig-1 suppresses nSREBP-1c expression and decreases ER stress through IRE1α pathway
Insig-1 improved the GSIS in INS-1 cells
Insig-1 regulates the expression of insulin secretion genes and FAS via SREBP-1c
Insig-1 inhibits intracellular accumulation of lipid droplets and reduces FFA synthesis
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 . 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 . Overexpression of SREBP-1c has been found to induce glucolipotoxicity in insulinoma cells (INS-1 and MIN6) and isolated rat islets . 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 . 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 .
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 INS-1 cells and INS-1-Insig-1 cells under different incubation conditions. Both mRNA and protein expression of SREBP-1c were increased after 25.0 mM glucose stimulation in INS-1 and INS-1-Insig-1 cells, while INS-1-Insig-1 cells showed less SREBP-1c expression at all time points compared to control INS-1 cells, which is consistent with previous findings by others [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 . 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 high glucose, p-IRE1α, p-JNK and CHOP were markedly up-regulated, while BCL-2 were down-regulated in both INS-1 and INS-1-Insig-1 cells and Insig-1 overexpression cells showed less change compared with INS-1 cells. Thus, our study showed that overexpression of Insig-1 could strongly suppress p-IRE1α, p-JNK and CHOP protein expression after exposure to high glucose. At standard glucose level(11.2 mM), p-IRE1α and p-JNK protein expression were both down-regulated in INS-1-Insig-1 cells compared with INS-1 cells at all time points, while the protein expression of BCL-2 in INS-1-Insig-1 cells showed up-regulation compared to control INS-1 cells, though there was no difference in CHOP expression. We therefore postulated that even normal glucose concentrations could produce FFA to switch on the IRE1α pathway at this time.
SREBP-1c participates in multiple regulatory mechanisms of GSIS. In vitro, overexpression of SREBP-1c results in impaired insulin secretion in isolated rat islets . In vivo, similar results were also observed by Takahashi et al. by using transgenic mice overexpressing the active form of SREBP-1c . In addition, SREBP-1c knockout mice had increased basal and high glucose stimulated insulin secretion . SREBP-1c also regulates the expression of insulin genes . 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 . 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 . 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 . GLUT-2 is known as a transporter of glucose, and is activated upon binding with SREBP-1c . 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.  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 . 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.
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.
We thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript.
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