Molecular and Cellular Endocrinology 412 (2015) 1–11

Contents lists available at ScienceDirect

Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Fetuin A promotes lipotoxicity in β cells through the TLR4 signaling pathway and the role of pioglitazone in anti-lipotoxicity Ximei Shen, Liyong Yang *, Sunjie Yan, Huanhuan Zheng, Liyu Liang, Xiuhui Cai, Meng Liao Endocrinology Department, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China

A R T I C L E

I N F O

Article history: Received 28 December 2014 Received in revised form 10 May 2015 Accepted 12 May 2015 Available online 15 May 2015 Keywords: Fetuin A Palmitic acid Toll-like receptor 4 JNK Apoptosis G-protein-coupled receptor 40

A B S T R A C T

Objective: Fetuin A (FetA), a secreted glycoprotein, is known to affect inflammation and insulin resistance (IR) in obese humans and animals. Lipotoxicity from chronic hyperlipidemia damages pancreatic β cells, hastening the onset of diabetes. We sought to determine whether FetA promotes lipotoxicity through modulation of the toll-like receptor 4 (TLR4) inflammatory signaling pathway as well as the protective effect of pioglitazone(PIO) on lipotoxicity. Methods: βTC6, a glucose-sensitive mouse pancreatic β cell line, and Sprague–Dawley rats with dietinduced obesity, were used to investigate FetA-mediated lipotoxicity. Protein expression/activation were measured by Western blotting. Small interfering (si)RNAs for TLR4 were used. Cell apoptosis was quantified by TUNEL analysis or flow cytometry, respectively. Insulin release was assessed with an insulin ELISA. Results: FetA dose-dependently aggravated palmitic acid (PA)-induced βTC6 cell apoptosis, insulin secretion impairment, and inhibition of the expression of G-protein-coupled receptor 40 (GPR40) and pancreatic duodenal homeobox-1(PDX-1). Combined FetA + PA induced TLR4 expression, and subsequent inhibition of TLR4 signaling or expression was shown to prevent the strengthening effect of FetA on PA-induced lipotoxicity in βTC6 cells. FetA + PA induced p-JNK and nuclear factor-κB (NF-κB) subunit P65 expression, and inhibition of this activity reduced PA+ FetA lipotoxicity in βTC6 cells. PIO could ameliorate PA+ FetA-induced damage to βTC6 cells. Similarly, PIO improved insulin secretion disorder, reduced apoptosis, decreased FetA, TLR4, p-JNK, NF-κB subunit P65 and cleaved caspase 3 expression, and increased GPR40 and PDX-1 expression in islet β cells of diet-induced obese rats. The correlative bivariate analysis showed that increases in Fetuin A were directly proportional to the development of β cell injury. Conclusions: FetA can promote lipotoxicity in β cells through the TLR4-JNK-NF-κB signaling pathway. The protective effects of PIO on lipotoxicity in β cells may involve the inhibition of the activation of the FetA and TLR4 signaling pathway. © 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Increasing evidence demonstrates that inflammation contributes to lipotoxicity in β cells (Hotamisligil, 2006; Tang et al., 2013);

Abbreviations: ERS, endoplasmic reticulum stress; FetA, Fetuin A; FFA, free fatty acids; GPR40, G-protein-coupled receptor 40; GSIS, glucose-stimulated insulin secretion; HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; IR, insulin resistance; ITT, insulin tolerance test; JNK, c-Jun NH2-terminal kinase; KRBB, Krebs– Ringer bicarbonate buffer; LPS, lipopolysaccharide; NF-κB, nuclear factor κ-B; PA, palmitic acid; PDX-1, pancreatic duodenal homeobox-1; PIO, pioglitazone; p-JNK, phosphorylated c-Jun NH2-terminal kinase; PPARγ, peroxisome proliferatoractivated receptor γ; SD, Sprague–Dawley; TLR4, toll-like receptor 4. Co-first Author: Sunjie Yan. * Corresponding author. Endocrinology Department, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China. Tel.: +86 130 0381 6668; fax: +86 591 8331 8716. E-mail address: [email protected] (L. Yang). http://dx.doi.org/10.1016/j.mce.2015.05.014 0303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.

however, the mechanism underlying the inflammatory response mediated by FFA-induced β cell dysfunction is not clear. TLR4 plays a key role in signal transduction pathways involved in the activation of the innate immune system, and it is widely suspected in the pathogenesis of type 2 diabetes mellitus. Recent research has shown that FFA can stimulate adipose tissue to produce inflammatory cytokines through the pattern recognition receptor TLR4, resulting in insulin resistance (Kim, 2006; Yan et al., 2010). However, FFA does not directly bind to TLR4, and thus the interaction between FFA and TLR4 is probably mediated by an endogenous ligand (Erridge and Samani, 2009; Schaeffler et al., 2009). Due to the absence of a direct association between FFA and TLR4, the mechanism underlying the activation of TLR4 in β cells by FFA remains unresolved. Therefore, identifying the endogenous TLR4 ligand that controls crosstalk between FFA and TLR4 would greatly enhance our understanding of the FFA–TLR4 regulating signaling pathway, and would help us elucidate the mechanism underlying FFA-induced inflammatory injury in β cells.

2

X. Shen et al./Molecular and Cellular Endocrinology 412 (2015) 1–11

FetA, a liver secretory glycoprotein, is one of the carrier proteins of FFA in the circulation (Cayatte et al., 1990; Pal et al., 2012). It stimulates the production of inflammatory cytokines from adipocytes and macrophages, and is therefore considered to be a biomarker for chronic inflammatory diseases (Siegel-Axel et al., 2014). FetA is also associated with obesity-related diseases (Ley et al., 2014; Sun et al., 2013). It has been reported that FetA- or TLR4knockout mice are protected from high-fat diet (HFD)-induced insulin resistance (Pal et al., 2012). But it remains unclear whether FetA can mediate FFA–TLR4 crosstalk resulting in lipotoxicity-mediated inflammatory injury in β cells. It is well known that pioglitazone (PIO), a peroxisome proliferator activated receptor gamma (PPARg) agonist, confers resistance to lipotoxicity on beta-cells. Our previous studies have shown that PIO can reduce β-cell apoptosis (Shen et al., 2014). Recent studies have reported that PIO might exert an anti-inflammatory response by decreasing the expression of TLR4 in renal tissue, and regulating the balance between proinflammatory and anti-inflammatory signals (Yu et al., 2014). The present study aims to assess whether FetA can enhance lipotoxicity induced by FFA in β cells, and to analyze the relationship between this FetA-mediated effect and the signal pathway of TLR4-JNK-NF-κB, as well as the possible protective effect of PIO. 2. Materials and methods 2.1. βTC6 cell culture The mouse islet cell line βTC6 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured with complete medium: high-glucose Dulbecco’s modified Eagle’s medium (HDMEM, 25 mmol/L glucose), supplemented with 10% fetal bovine serum (FBS) and 2 mmol/L L-glutamine at 37 °C and 5% CO2. All reagents were purchased from Gibco (Carlsbad, CA, USA). Cells at passages 26–35 were used for all experiments. 2.2. TLR4 small interfering (si) RNA construct and permanent transfection An siRNA targeting mouse TLR4 mRNA (GenBank Accession Number: 021297.2; CCACCUCUCUACCUUAAUA) was designed and synthesized by GenePharma Co, Ltd. (Shanghai, China). RNAi lentiviral vector was prepared and packaged by GenechemCo, Shanghai, China. The RNAi lentiviral vector was diluted with Enhanced infection Solution (ENi.S., GenePharma Co, Ltd.) to 1 × 108 IU/mL, and then was diluted directly into HDMEM + 10% FBS with 2 mmol/L L-glutamine under the multiplicity of infection 50. After 8–10 h of incubation, the transfection medium (enhanced infection solution + HDMEM + 10%FBS + 2 mmol/L L-glutamine) was replaced with fresh complete medium (HDMEM + 10% FBS + 2 mmol/L L-glutamine) and the cells were allowed to incubate for 72 h. TLR4 protein expression was analyzed by Western blotting. 2.3. Animals Five-to-six weeks’ old, SPF-grade, male Sprague–Dawley (SD) rats (220 ± 18 g) were obtained from the laboratory animal facility at Fujian Medical University. After a 1-week acclimation period, the animals were weighed and measured, and randomly divided into two groups: those receiving normal control diet (n = 20), and those receiving a high-fat diet (HFD, n = 20). Control rats were subdivided into two groups, half of which were given PIO [10 μg/kg body weight (b.w.), gavage, per day, n = 10], while the other half remained untreated (n = 10). All the control rats were fed a normal pellet diet for 16 weeks. HFD rats received a HFD (Research Diets; D12492; 60% calories from fat, 20% calories from protein, 20%

calories from carbohydrate, ingredient see Supplementary material 1) for 8 weeks, to induce obesity. This test group was then subdivided into two groups, half of which were given PIO [10 μg/kg body weight (b.w.), gavage, per day, n = 10], while the other half remained untreated (n = 10). Diet-induced obesity was defined as an average increase, above control, in weight and Lee’s index (Wei et al., 2011) [Lee’s index = body weight (g)1/3 × 103/ body length (cm)] of 15% and 1.5%, respectively. Animals were housed, five rats per cage, in a temperature- and humiditycontrolled room (20 ± 26 °C, 40–60% humidity), with a set 12-hour light–dark cycle. The body weight and length were recorded once/ week for the duration of the study. The pancreatic β-cell function was assessed by intraperitoneal glucose tolerance test and HOMAIR, β and IS (see Supplementary material 2). This study was approved by the Ethics Committee of Biomedical Research of the First Affiliated Hospital of Fujian Medical University. 2.4. Serum insulin, FetA and biochemical indicators measurements Rats were fasted overnight for 8 hours and were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.03 ml/kg), and abdominal aorta blood sampling was collected, then centrifuged at 3000 rpm for 10 min. Serum was separated and stored at −20 °C for further analysis. Blood was tested for the measurement of fasting plasma glucose, fasting plasma insulin, FetA, triglycerides (TG), total cholesterol (TC), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C) and VLDL-cholesterol (VLDL-C). The ELISA kit (Uscn Life Science Inc., China, SEA178Ra, detection range 0.625–40 ng/mL, the sensitivity: 0.297 ng/mL) was used to detected the serum level of FetA. The Rat insulin ELISA kit (Cusabio, Rat Insulin, INS ELISA Kit,, CSBE05070r) had intra- and interassay coefficients of variation of