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TRIB3 Is Implicated in Glucotoxicity- and Oestrogen Receptor-Stress-Induced B-Cell Apoptosis

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TRIB3 Is Implicated in Glucotoxicity- and Oestrogen Receptor-Stress-Induced B-Cell Apoptosis 407 TRIB3 is implicated in glucotoxicity- and oestrogen receptor-stress-induced b-cell apoptosis Bo Qian1,2, Haiyan Wang3, Xiuli Men2, Wenjian Zhang2, Hanqing Cai2, Shiqing Xu2, Yaping Xu2, Liya Ye2, Claes B Wollheim3 and Jinning Lou2 1Graduate School of Peking Union Medical College, Beijing 100730, People’s Republic of China 2Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing 100029, People’s Republic of China 3Department of Cell Physiology and Metabolism, University Medical Center, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland (Correspondence should be addressed to J Lou; Email: [email protected]) Abstract We found that TRIB3, an endogenous inhibitor of Akt confirmed that the DJm of mitochondria was decreased, (PKB), is expressed in pancreatic b-cells. The TRIB3 caspase-3 activity was up-regulated and reactive oxygen expression is significantly increased in islets isolated from species content was increased in TRIB3 overexpressing b cells hyperglycemic Goto–Kakizaki rats compared with normal in high glucose condition. Most interestingly, the oestrogen glycemic controls. In vitro high glucose treatment also resulted receptor (ER) stress inducer, thapsigargin, mimicked the high in increased TRIB3 expression in rat INS1 cells. To glucose effects on up-regulation of TRIB3 and generation of investigate the role of TRIB3 in the regulation of b-cell apoptosis in cultured INS1 cells. These effects were function, we established an INS1 stable cell line allowing specifically prevented by siRNA knock down of TRIB3. inducible expression of TRIB3. We demonstrated that We therefore conclude that TRIB3 is implicated in overexpression of TRIB3 mimicked the glucotoxic effects glucotoxicity- and ER stress-induced b-cell failure. TRIB3 on insulin secretion and cell growth in INS1 cells. Moreover, could be a potential pharmacological target for prevention induction of TRIB3 also synergistically enhanced high- and treatment of type 2 diabetes. glucose-elicited apoptosis in INS1 cells, whereas siRNA Journal of Endocrinology (2008) 199, 407–416 knock-down of TRIB3 showed the opposite effects. We also Introduction cells undergoing apoptosis resulting from either, neurotrophic factor deprivation or disrupted calcium homeostasis Progressive b-cell failure is the precipitating factor for the (Mayumi-Matsuda et al. 1999). It has a kinase-like domain transition from the insulin-resistant state to overt type 2 diabetes but is lacking kinase activity (Mayumi-Matsuda et al. 1999, (Muoio & Newgard 2008). Hyperglycemia and oestrogen Boudeau et al. 2006). TRIB3 has recently attracted interest in receptor (ER) stress have been suggested as causing factors of diabetes research as it has emerged as an endogenous inhibitor b-cell dysfunction (Grill & Bjo¨rklund 2001, Harding et al.2001, of Akt (PKB), which plays a key role in insulin signaling Laybutt et al.2007, Marchetti et al.2007, Eizirik et al.2008). (Du et al. 2003). In addition, the mRNA levels of TRIB3 are However, the underlying mechanisms remain to be elucidated. elevated in the liver of db/db mice (Matsushima et al. 2006). ER stress has been implicated in chronic glucotoxicity- Moreover, adenovirus-mediated overexpression of TRIB3 in induced b-cell apoptosis (Wang et al. 2005, Laybutt et al. mouse liver results in hyperglycemia by inhibition of insulin 2007, Marchetti et al. 2007, Eizirik et al. 2008). It has also action on glycogen synthesis and gluconeogenesis (Du et al. been demonstrated that up-regulation of tribbles homolog 3 2003, Koo et al. 2004). By contrast, knockdown of TRIB3 (Drosophila; TRIB3) is one of the major mechanisms involved in mouse liver or muscle improves insulin action and in ER stress-induced cell death via the ATF4-CHOP glucose tolerance (Koo et al. 2004, Koh et al. 2006). pathway in non-b-cells (Ohoka et al. 2005, Carracedo et al. Therefore, TRIB3 may contribute to the development of 2006). It is, therefore, of interest to study the involvement of insulin resistance. TRIB3 in glucotoxicity- and ER stress-induced b-cell However, nothing is known regarding the role of TRIB3 dysfunction and apoptosis. in the regulation of b-cell function. The present work is TRIB3, a mammalian homolog of Drosophila tribbles, is designed to study the possible involvement of TRIB3 also known as neuronal cell death-inducible putative protein in glucotoxicity and ER stress-evoked b-cell dysfunction kinase, because it is discovered to be expressed in neuronal and apoptosis. Journal of Endocrinology (2008) 199, 407–416 DOI: 10.1677/JOE-08-0331 0022–0795/08/0199–407 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/26/2021 10:44:43AM via free access 408 B QIAN and others . TRIB3 in b cell apoptosis Materials and Methods without 500 ng/ml doxycycline for induction of TRIB3 expression. After 48 h incubation, the cells were fixed in 4% Islet isolation from Goto–Kakizaki (GK) rat paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS containing 0.5% BSA (PBS-BSA) for 30 min. The cells All animal experimental procedures were performed in were then incubated with rabbit anti-TRIB3 polyclonal accordance with China–Japan Friendship Hospital’s Animal antibody (Calbiochem, San Diego, CA, USA) at 1:200 dilutions Studies Committee. Islets were isolated from five hyperglycemic . for 45 min, followed by labeling with FITC-conjugated rat anti- GK rats (mean non-fasting blood glucose 19 54 mmol/l) and rabbit IgG antibody (Sigma) at 1:100 dilutions for 30 min. The five normal glycemic GK rats (mean non-fasting blood glucose cells were viewed under a fluorescent microscope. 4.16 mmol/l; Shanghai Slac Laboratory animal Co. Ltd, Shanghai, China) using the intraductal collagenase digestion technique described previously (Shapiro et al.1996). Briefly, Western blotting 10 ml Hank’s balanced salt solution with 2 mg/ml collagenase For western blotting, cells were cultured in flasks for 24–96 h type IV (Sigma) were injected into the pancreatic duct. After before being treated with indicated concentrations of doxycy- pancreatectomy, the pancreata were digested in a 37 8Cwater cline at the specified time under either standard (11.2 mmol/l) or bath for 19 min. the isolated islets were further purified by Ficoll high (30 mmol/l) glucose conditions. All these cells were (Sigma) gradient centrifugation. The purified islets were washed collected and lysed by sonication. The protein was extracted in thrice and subjected to total RNA extraction. buffer containing 20 mM Tris–HCl, pH 7.4, 2 mM EDTA, 150 mM NaCl, 10 mM NaP, 1% NP-40, and 1 mM phenyl- Total RNA extraction and real-time PCR methylsulfonyl fluoride. Total cellular proteins were fractionated by 10% SDS-PAGE. Immunoblotting was performed using RNeasy mini kit (Qiagen) was used, following manufacturer’s either aforementioned anti-TRIB3 antibody (1:500) or mouse protocol, for total RNA extraction from either INS1-derived cells anti-a-tubulin (Santa Cruz, CA, USA, 1:500) as described or isolated islets. One microgram total RNA was used for reverse previously using enhanced chemiluminescence (Millipore, Bill- transcription using omniscript reverse transcriptase (Qiagen). erica, MA, USA) for detection. Real-time PCR was performed on an Applied Biosystems ABI 7000, using SYBR Green PCR Master Mix (Qiagen) and 1/40 of RT reaction. At the end of each reaction, a melting curve Measurements of insulin secretion and cellular insulin content analysis was performed to confirm the absence of primer Cells were cultured in 24-well plates in 11.2 mmol/l glucose dimmers. Expression levels of TRIB3 gene were normalized to medium with or without 500 ng/ml doxycycline for 48 h. The the expression level of b-actin. The data was analyzed using the cells were then incubated with Krebs–Ringer bicarbonate- K 2 DDCt method. Primer sequences are as follows: HEPES buffer containing 2.5 mmol/l glucose (Basal) or 20 mmol/l glucose (Glucose). The cell supernatant was collected 0 rat TRIB3: forward 5 -TGT CTT CAG CAA CTG TGA after 30 min of stimulation and insulin content was determined 0 GAG GAC GAA G-3 , after extraction with acid ethanol following the procedures 0 reverse 5 -GTA GGA TGG CCG GGA GCT GAG described by Wa ng et al. (2002). Insulin concentration was TAT C-3 0; determined by ELISA kits (Linco, St Charles, MO, USA). rat b-actin: forward 50-GAC ATC CGT AAA GAC CTC TAT GCC-3 0, 0 0 DNA fragmentation reverse 5 -ATAGAG CCACCA ATC CAC ACAGAG-3 . Cells were cultured in medium containing either 11.2mmol/l glucose or 30 mmol/l glucose for 96 h, in the presence or Establishment of INS1 stable cell lines allowing inducible expression absence of 500 ng/ml doxycycline. The cells were washed twice of TRIB3 with PBS, resuspended in the lysis buffer (10 mM Tris–HCl, pH 8.0and10mMEDTA,10mMNaCl,0.5% SDS, 100 mg/ml TheplasmidswereconstructedbysubcloningthecDNAs proteinase K) and stored at 50 8C for 2 h. The crude DNA encoding TRIB3. The stable INS-raˆ cells (also refer to as r9), preparations were further extracted and precipitated. The DNA which carries the reverse tetracycline/doxycycline-dependent pellets were air-dried and resuspended in 100 mlTEbuffer transactivator (Gossen et al. 1995), were used for the secondary (10 mM Tris–HCl, pH 8.0, 1 mM EDTA) containing 100 g/ml stable transfection following the procedures described previously RNase. The concentration of nucleic acid was determined by (Wang & Iynedjian 1997). u.v. absorbance at 260 nm. The same amount of nucleic acids from each sample was found by electrophoresis on a 1.5% agarose gel and visualized by u.v. fluorescence after staining with Immunofluorescence staining ethidium bromide. Gel was visualized and photographed on an The cells were cultured on cover slips in a 24-well plate in a image analyzer instrument (Alphalmager 2000, Alpha Innotech 37 8Cand5%CO2 incubator for 2 days before treatment with or Corp., San Leandro, CA, USA).
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