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Available online at www.sciencedirect.com

Metabolism www.metabolismjournal.com

Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress Hiroto Hayashi, Ren Yamada, Siddhartha Shankar Das, Taiki Sato, Aki Takahashi, Masahiro Hiratsuka, Noriyasu Hirasawa⁎ Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan

A R T I C LE I N FO Article history:

AB S T R A C T Objects. Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-

Received 10 September 2013

stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined

Accepted 17 February 2014

hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a highfat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic

Keywords: ER stress

reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model. Methods. To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate.

GLP-1

Results. Palmitate, but not oleate caused ER stress and decreased the protein levels of

PC1/3

prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same

GLUTag

phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells. Conclusions/Interpretation. These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production. © 2014 Elsevier Inc. All rights reserved.

1.

Introduction

Glucagon-like peptide-1 (GLP-1) is a peptide hormone made from proglucagon with prohormone convertase 1/3 (PC1/3) [1] and is secreted from intestinal L cells. GLP-1 enhances glucose-stimulated insulin secretion and protects pancreas β cells from endoplasmic reticulum (ER) stress-induced apoptosis [2,3]. Moreover, a previous study showed that GLP-1

increased the β cell mass in rats by stimulating GLP-1 receptor [4]. Thus, GLP-1 is used as a therapeutic hormone for the treatment of type 2 diabetes. Hyperlipidemic conditions are well known to induce ER stress. Palmitate, a saturated fatty acid, was experimentally shown to promote ER stress [5,6]. On the other hand, in humans, serum levels of free fatty acids (FFAs) were elevated in obese subjects [7] and have been used as an independent predictor of future type 2

Abbreviations: GLP-1, glucagon-like peptide-1; PC1/3, prohormone convertase 1/3; FFAs, free fatty acids; ER stress, endoplasmic reticulum stress; XBP1, X-box binding protein 1; CHOP, C/EBP homologous protein. ⁎ Corresponding author at: Noriyasu Hirasawa, Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences Tohoku University, Sendai, Japan. Tel.: +81 22 795 5915; fax: + 81 22 795 5504. E-mail address: [email protected] (N. Hirasawa). http://dx.doi.org/10.1016/j.metabol.2014.02.012 0026-0495/© 2014 Elsevier Inc. All rights reserved.

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diabetes [8]. Furthermore, a high intake of saturated fatty acids has been linked to a higher risk of type 2 diabetes [9]. ER stress promotes the unfolded protein response (UPR) via transmembrane sensors that detect unfolded proteins in the ER. These sensor proteins include protein kinase activated by doublestranded RNA-like endoplasmic reticulum-regulated kinase (PERK), inositol-requiring protein 1 alpha (IRE1α), and activating transcription factor 6 (ATF6). The activation of these proteins led to the expression of spliced X-box binding protein 1 (XBP1) [10] and C/EBP homologous protein (CHOP) [11]. An increase in CHOP was shown to induce a loss in glucose homeostasis due to a decrease in insulin secretion and insulin resistance. Previous studies in humans showed an inverse correlation between obesity and GLP-1 secretion [12,13]. A high-fat diet also caused a reduction in active GLP-1 levels in the portal vein of a mouse model of obesity after glucose intake [14]. Therefore, we hypothesized that overnutrition conditions may cause ER stress in L cells and affect their functions. However, few studies have examined hypernutrition stress in L cells and its effects on GLP-1 secretion. In this study, we showed that hyperlipidemic conditions caused ER stress and reduced GLP-1 production in the intestinal L cell line, GLUTag cells [15].

2.

Experimental procedures

2.1.

Materials

Dulbecco’s modified Eagle’s medium was purchased from Nissui Seiyaku (Tokyo, Japan). Penicillin G and streptomycin were purchased from Meiji Seika Pharma (Tokyo, Japan). FBS was purchased from Biowest (Miami, Florida). Trypsin was purchased from Life Technologies (Carlsbad, California). Thapsigargin, forskolin, and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sodium palmitate (palmitate) was purchased from Chem Service (West Chester, USA). Sodium oleate (oleate) was purchased from Sigma-Aldrich (St. Louis, USA).

2.2.

Mouse experiments

Five-week-old C57BL/6 male mice were obtained from SLC (Shizuoka, Japan). All mice were housed in specific pathogenfree barrier facilities, maintained under a 12 h light/dark cycle, fed a standard rodent food diet (STD) consisting of 4.6% fat (CLEA, Tokyo, Japan) or a high-fat diet (HFD) consisting of 60% fat (Research Diets, New Brunswick, NJ). All animal procedures were performed in the Institutional Animal Care and Use Committee of Tohoku University.

2.3.

Glucose-stimulated insulin and GLP-1 secretion in vivo

The glucose-stimulated insulin and GLP-1 secretion in vivo study was performed as previously reported with modifications [16,17]. Experiments were performed on 13-week-old mice fed STD or HFD for 8 weeks. Mice were fasted for 12 h and administrated 20 mg/kg KR62436 (DPP-IV inhibitor; Sigma) per oral. Thirty minutes later, 100 μL blood was collected from awake mice via the tail vein with haematokrit-kapillaren® (Hirschmann-Laborgerate, Neckartenzlingen, German) into a diprotin A (1 mmol/L) for initial

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GLP-1 measurements, and mice were dosed by gavage with 1.5 g/kg glucose solution. After fifteen minutes, blood was collected via the tail vein to measure glucose-induced insulin and GLP-1 secretion. Blood samples were centrifuged and plasma was placed on ice before ELISA.

2.4.

Measurement of biochemical parameters

Plasma glucose levels, blood insulin, and active GLP-1 levels were determined with an Ascensia (Bayer HealthCare), insulin ELISA kit (Morinaga, Yokohama, Japan), and active GLP-1 ELISA kit (Shibayagi, Gunma, Japan), respectively.

2.5.

Determination of ER stress in the intestine

To detect ER stress in the intestine, 2 cm of the intestine just close to the junction regions to cecum was collected in a sample tube for mRNA analysis via quantitative real time PCR (described below).

2.6.

Cell culture and treatment

GLUTag cells were cultured in Dulbecco’s modified Eagle’s medium (5.6 mmol/L glucose), supplemented with 10% (v/v) FBS, 15 μg/mL penicillin G, and 50 μg/mL streptomycin. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2, with medium changes every 3 days. All studies were conducted using 80%–90% confluent cells, which were treated with the indicated concentrations of FFAs or thapsigargin for 24 h. FFAs were prepared in Dulbecco’s modified Eagle’s medium containing 2% (w/v) fatty acid-free bovine serum albumin (Sigma-Aldrich).

2.7. Cell harvest and measurement of prohormone convertase 1/3 activity After the various treatments, cells were washed with ice-cold phosphate-buffered saline and lysed in buffer containing 25 mmol/L PIPES, 120 mmol/L NaCl, 5 mmol/L KCl, 0.4 mmol/L MgCl2-6H2O, 40 mmol/L NaOH, and 1 mmol/L CaCl2 (pH 7.2, PIPES-NaOH buffer). After sonication with a Handy Sonic (Tomy Seiko, Tokyo, Japan), lysates were centrifuged to remove insoluble materials. PC1/3 activity was measured as previously reported with modifications [18,19]. Briefly, proteins (20 μg) in the supernatants were added to the reaction mixture. The reaction mixture contained PIPES-NaOH buffer, 0.1% (w/v) BSA, and 0.4 mmol/L Boc-Arg-Val-Arg-Arg-MCA (Peptide Institute, Osaka, Japan) in a total reaction volume of 200 μL. The reaction was performed at 37 °C for 30 min, and stopped by adding 0.8 mL of 1 mol/L acetate buffer (pH 4.8). The fluorescence intensity of the solution was read at an emission of 460 nm with excitation at 380 nm with a fluorescence spectrophotometer (F-2000; Hitachi, Ibaragi, Japan).

2.8.

GLP-1 secretion in vitro

The GLP-1 secretion in vitro study was performed according to the methods reported by Reimann and Gribble [20] with slight modifications. GLUTag cells were plated in 24-well culture plates and allowed to reach 80%–90% confluence. Cells were washed with 500 μl of glucose-free Krebs–Ringer medium

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containing 120 mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 22 mmol/L NaHCO3, 0.1 mmol/L diprotin A, and 0.5% (w/v) BSA, and then treated with 10 μmol/L forskolin, 20 μmol/L IBMX, and 25 mmol/L glucose in 500 μl of Krebs– Ringer for 2 h at 37 °C, 5% CO2. After the incubation, the medium was collected and centrifuged to remove any floating cells. GLP-1 in the supernatant was assayed by ELISA as described above.

2.9.

Quantitative real-time PCR

Total RNA was extracted from cultured GLUTag cells or tissues using an RNAiso plus (TaKaRa Bio, Shiga, Japan), according to the manufacturer’s protocol. cDNA was synthesized from total RNA using PrimeScript® RT Master Mix (TaKaRa Bio, Shiga, Japan). The primers used were shown in supplementary Table 1. Quantitative real time PCR was performed using SYBR Green reagent (TaKaRa Bio, Shiga, Japan), on a Takara Thermal Cycler Dice® (TP800). The PCR conditions were one cycle at 95 °C for 30 s, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was determined using the ΔΔCT method, using GAPDH or 18 s rRNA as the internal control. Primer specificity was confirmed by melting curve analysis.

2.11.

Results

3.1.

HFD impaired glucose-stimulated GLP-1 secretion in mice

We first examined the effect of HFD-induced obesity on GLP-1 secretion. HFD was given for 8 weeks, which resulted in an increase in body weight (Fig. 1A). Blood glucose levels before and 15 min after the glucose treatments were higher in HFD-treated mice than in STD-fed mice (Fig. 1B). In addition, insulin levels before and 15 min after the glucose treatment were higher in HFD-treated mice than those in STD-fed mice (Fig. 1C), which indicated that HFD induced insulin resistance and hyperinsulinemia. Interestingly, although the glucose treatment induced an apparent increase in blood GLP-1 levels at 15 min in normal mice, this response was significantly attenuated in HFD-treated mice (Fig. 1D).

3.2.

HFD induced ER stress in the intestine

HFD was previously shown to induce ER stress in some tissues [23,24]. Thus, we investigated whether the chronic treatment with HFD induced the expression of the ER stress maker, C/EBP homologous protein (CHOP), in the intestine where L cells are distributed. As shown in Fig. 2A, HFD for 8 weeks induced the expression of CHOP in the intestine. However, proglucagon mRNA levels were not reduced by HFD (Fig. 2B).

3.3.

Palmitate, but not oleate induced ER stress in GLUTag cells

To clarify the effects of ER stress on GLP-1 production and/or secretion in L cells, GLUTag cells were treated with palmitate. Incubation with 0.5 mmol/L palmitate induced the expression of CHOP at 12 and 24 h (Fig. 3A) and BiP, another ER stress marker, (Fig. 3C), and phosphorylation of PERK (Fig. 3E) at 24 h. In contrast, oleate at the same concentration as palmitate did not affect these ER stress markers (Fig. 3B, D and E). We confirmed that the treatment with palmitate, but not oleate slightly reduced the viability of GLUTag cells by the MTT assay (data not shown).

RT-PCR analysis

RT-PCR analysis was performed according to the previous study with slight modifications [23]. The XBP1 cDNA was then amplified by PCR using the primers, (sense) ACCTGAGCCCGGAGGAGAAA and (antisense) GTCCAGAATGCCCAAAAGGA. GAPDH cDNA was amplified using the primers, (sense) ACCACAGTCCATGCCATCAC and (antisense) TCCACCACCCTGTTGCTGTA. PCR products were separated on a 3.5% (w/v) or 2% (w/v) agarose gel and detected by Light-Capture.

2.12.

3.

Western blotting

Western blotting was carried out as described previously [21,22]. Cells or tissues were lysed in cold buffer containing 20 mmol/L HEPES, 50 mmol/L NaF, 2.5 mmol/L p-nitrophenylphosphate, 20 μmol/L leupeptin, 10% glycerol, 1% Triton X-100, 1 mmol/L sodium orthovanadate, 57 μmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L EDTA. After sonication, lysates were centrifuged to remove insoluble materials. The proteins in supernatants were separated by SDS-PAGE, and PC1/3, p-PERK, proglucagon and actin were detected by Western blotting using anti-PC1/3 (Abcam, Cambridge, MA), anti-p-PERK (Cell Signaling Technology, Danvers, MA), anti-proglucagon (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). Signals were detected with a chemiluminescent detection system (Western Lightning PlusECL; PerkinElmer Life Sciences, Boston, MA, USA) using a CCD camera system (Light-Capture; ATTO, Tokyo, Japan). Immunoblots were quantified using ImageJ software.

2.10.

Data involving more than two groups were assessed by a oneway analysis with the Bonferroni/Dunnett post hoc test of variance. All calculations were performed with SPSS (version 16.0 for Windows; SPSS Chicago, IL).

Statistical analysis

All values are given as means ± S.E.M. Differences between two groups were assessed using unpaired, two-tailed t tests.

3.4. Palmitate reduced PC1/3 activity and PC1/3 protein levels in GLUTag cells We examined the effects of palmitate and oleate on the activity and expression of PC1/3, the processing enzyme of proglucagon to GLP-1, in GLUTag cells. Cell lysates were prepared from cells treated with palmitate or oleate at 0.5 and 1.0 mmol/L for 24 h. Palmitate (Fig. 4A), but not oleate (Fig. 4B) decreased the activity of PC1/3 in a concentration-dependent manner. Consistent with the reduction in its activity, PC1/3 protein levels were also decreased by the treatment with 0.5 mmol/L palmitate for 24 h (Fig. 4C) whereas PC1/3 mRNA levels were not (Fig. 4E). Oleate affected neither PC1/3 activity nor the expression of PC1/3 (Fig. 4D and F).

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Fig. 1 – GLP-1 secretion was impaired in HFD-fed mice. A, Changes in the body weights of mice fed a high fat diet (HFD) or normal diet (STD) were monitored for 8 weeks. Blood glucose levels (B), insulin levels (C), and active GLP-1 levels in the plasma (D) were determined before (0 min) and 15 min after the 1.5 g/kg glucose treatment in mice fed HFD (n = 8) or STD (n = 7) for 8 weeks by oral gavage. All data show the mean ± S.E.M. and statistical significance; * p < .05, ** p < .01, *** p < .001 vs. STD mice at the corresponding times. †† p < .01, ††† p < .001 vs. the corresponding 0 min group.

3.5. Oleate cancelled palmitate-induced ER stress and the reduction in PC1/3 activity Oleate was previously shown to cancel the effect of palmitate on ER stress [23,25]. To clarify that the palmitate-induced responses

described above were mediated by ER stress, cells were cotreated with oleate and palmitate at the same concentrations. Palmitate induced expression of CHOP (Fig. 5A), splicing of XBP1 mRNA (Fig. 5B), reduction in PC1/3 activity (Fig. 5C) and PC1/3 protein level (Fig. 5D), and increase in proglucagon protein level

Fig. 2 – HFD induced ER stress in the intestine. The expression of mRNA for CHOP (A) and proglucagon (B) in the intestines of mice fed HFD and STD for 8 weeks was detected with real-time PCR. The ratio of each mRNA level to 18 s rRNA was calculated and the values of STD mice were set to 1.0. All data show the mean ± S.E.M. (n = 7–8) and significance; * p < .05 vs. the STD-fed group.

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Fig. 3 – Palmitate induced ER stress in GLUTag cells. GLUTag cells were treated for the indicated periods with 0.5 mmol/L palmitate (A and C) or oleate (B and D). The expression of mRNA for CHOP and BiP was detected with real-time PCR. The ratio of CHOP and BiP mRNA levels to 18 s rRNA was calculated and the value of time 0 was set to 1.0. E, GLUTag cells were treated with 0.5 mmol/L palmitate or oleate for 24 h. PERK phosphorylation and actin were detected with western blot. All data show the mean ± S.E.M. (n = 3) and significance; * p < .05 vs. time 0.

(Fig. 5E), and these were all recovered by oleate. The level of mRNA for proglucagone was not increased by palmitate (data not shown). Furthermore, palmitate attenuated stimulantinduced GLP-1 secretion, while oleate recovered it (Fig. 5F).

3.6.

sion of mRNA for CHOP and BiP, with maximum expression being observed at 4 h and 12 h, respectively (Fig. 6).

3.7. Thapsigargin reduced the activity and protein levels of PC1/3

Thapsigargin induced ER stress in GLUTag cells

Thapsigargin is a well-used inducer of ER stress [26–28]. We confirmed that thapsigargin could induce ER stress in GLUTag cells. GLUTag cells were treated with 1, 3, and 10 nmol/L thapsigargin for 12 h and the expression of CHOP and BiP was determined. Thapsigargin at 10 nmol/L induced the expres-

Since thapsigargin induced ER stress in GLUTag cells similar to palmitate, we examined the effects of thapsigargin on the activity and expression of PC1/3. PC1/3 activity was significantly reduced in the cell lysate prepared from cells treated with thapsigargin at 10 nmol/L for 24 h (Fig. 7A). Consistent with this activity, PC1/3 protein levels were also decreased

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Fig. 4 – PC1/3 activity was impaired by palmitate, but not oleate. A and B, GLUTag cells were treated with 0.5 and 1 mmol/L of palmitate (A) or oleate (B) for 24 h. PC1/3 activity was determined in the cells as described in the Methods. C–F, GLUTag cells were treated with 0.5 mmol/L palmitate (C and E) or oleate (D and F) for 24 h (C and D) or the indicated periods (E and F). PC1/3 and actin protein levels (C and D), and the expression of mRNA for PC1/3 (E and F) were detected. The ratios of PC1/3 levels to actin, and of each mRNA level to 18 s rRNA were calculated and the values of the control were set to 1.0. All data show the mean ± S.E.M. (n = 3) and significance; *** p < .001 vs control.

with 10 nmol/L thapsigargin (Fig. 7B) whereas PC1/3 mRNA levels were not (Fig. 7C). Furthermore, proglucagon protein levels were increased with 10 nmol/L thapsigargin (Fig. 7B)

although mRNA level was not changed (data not shown). In addition, stimulant-induced GLP-1 secretion was decreased in 10 nmol/L thapsigargin-treated cells (Fig. 7D).

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Fig. 5 – Oleate blocked palmitate-induced ER stress and the impairment in PC1/3 activity. GLUTag cells were treated with 0.5 mmol/ L palmitate in the presence or absence of 0.5 mmol/L oleate for 12 h (A), 24 h (B–D) or 48 h (E). The levels of mRNA for CHOP (A), splicing of XBP1 mRNA (B), PC1/3 activity (C), and protein levels of PC1/3 (D) and proglucagon (E) were determined. The ratio of each mRNA level to 18 s rRNA was calculated and the values of the control were set to 1.0. All data show the mean ± S.E.M. (n = 3). F, Cells were stimulated with glucose, IBMX and forskolin for 2 h and active GLP-1 released was determined (n = 3). Significance; ** p < .01, *** p < .001 vs the untreated group, and ††† p < .001 vs 0.5 mmol/L palmitate alone.

4.

Discussion

In this study, we found that HFD induced ER stress in the intestine and impaired glucose-induced secretion of GLP-1. In addition, we demonstrated that excessive palmitate, as well as thapsigargin, induced ER stress in GLUTag cells, which caused a decrease in PC1/3 activity, resulting in impairment in GLP-1 production and secretion.

We showed that glucose-stimulated GLP-1 secretion was impaired in mice fed HFD for 8 weeks (Fig. 1D), whereas glucose-stimulated insulin secretion was not (Fig. 1C). These results indicated that GLP-1 production and/or secretion was impaired at the early stage of HFD-induced obesity when insulin secretion had not yet been impaired. HFD also caused CHOP expression in the intestine (Fig. 2A), which indicated that ER stress was elicited in the cells of the intestine. These findings prompted us to study the

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Fig. 6 – Thapsigargin induced ER stress in GLUTag cells. GLUTag cells were treated for 12 h with indicated concentrations of thapsigargin (A and B) or the indicated period with 10 nmol/L thapsigargin (C and D). mRNA levels of CHOP (A and C) and BiP (B and D) were determined with quantitative real-time PCR. The ratio of each mRNA level to 18 s rRNA was calculated and the value of the control was set to 1.0. All data show the mean ± S.E.M. (n = 3). Significance; *** p < .001 vs the control.

effects of HFD-induced ER stress on GLP-1 production or secretion in L cells. HFD-induced ER stress is known to impair glucose homeostasis by inducing insulin resistance or an impairment in insulin secretion. Hyperlipidemic conditions were shown to reduce autophagy and proteasome activity in β cells and hepatocytes [29–31], which play important roles in the quality control of proteins and contribute to intracellular homeostasis [32,33]. In addition, a long-term elevation in FFA levels was shown to delay the processing of proinsulin and decreased PC2 and PC3 protein levels in the MIN6 cell line [34]. Palmitate also induced ER stress in pancreatic β cells, hepatocytes, and skeletal muscle in vitro [23,35,36]. Palmitate was shown to slow ER-to-Golgi protein trafficking and, as a result, induce ER stress [37]. Proteins levels in the ER gradually increase, which finally leads to ER stress. We confirmed that palmitate induced ER stress in GLUTag cells,

too (Fig. 3A and C). Interestingly, we found that palmitate induced a reduction in the activity and protein levels of PC1/3 (Fig. 4A and C) without affecting mRNA levels (Fig. 4E). The reduction in PC1/3 activity resulted in reduced GLP-1 production from proglucagon, which led to an impairment in the stimulant-induced release of GLP-1 in palmitate-treated GLUTag cells (Fig. 5). We concluded that the palmitateinduced reduction in PC1/3 protein levels was caused by the ER stress-induced impairment of protein homeostasis for the following reasons. First, palmitate-induced reduction in the activity and protein levels of PC1/3, and increase in proglucagon protein levels, as well as the expression of CHOP and spliced XBP1, were all recovered by the co-treatment with oleate, which cancelled palmitate-induced ER stress [23] (Fig. 5). Moreover, the impairment in GLP-1 secretion by palmitate was also cancelled by oleate (Fig. 5F). Second, thapsigargin, a strong ER inducer via the inhibition of sarco/

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Fig. 7 – Thapsigargin reduced the activity of PC1/3. GLUTag cells were treated for 12 h (C) or 24 h (A, B and D) with the indicated concentrations of thapsigargin. PC1/3 activity (A), the protein levels of PC1/3 proglucagon and actin (B), and the expression levels of mRNA for PC1/3 (C) were determined. The ratios of PC1/3 levels to actin, proglucagon levels to actin (B) and of each mRNA level to 18 s rRNA of the control (C) were calculated, and the values of the control were set to 1.0. D, Cells were stimulated with glucose, IBMX, and forskolin for 2 h and active GLP-1 released was determined. All data show the mean ± S.E.M. (n = 3) and significance; ** p < .01, *** p < .001 vs the control and † p < .05 vs the non-treated group.

endoplasmic reticulum Ca2 +-ATPase [38], also induced CHOP expression and a reduction in the activity and protein levels of PC1/3 (Figs. 6 and 7). It was unlikely that palmitate caused dysfunction in L cells via stimulating toll like receptor 4 (TLR4) [39,40]. However, although GLUTag cells express TLR4 (data not shown), lipopolysaccharide (LPS), a TLR4 ligand, did not affect the

activity or protein levels of PC1/3 (Supplementary Fig. 1A–D). Additionally, GLP-1 secretion was not changed by the LPS treatment (Supplementary Fig. 1E). Palmitate is also known to produce reactive oxygen species (ROS) via β-oxidation in mitochondria [41,42]. ROS affects various cells functions, including insulin resistance, and cardiac hypertrophy [43,44]. Recently, Kappe et al.

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showed palmitate-induced apoptosis of GLUTag cells via the induction of ROS [45]. However, the involvement of ROS in palmitate-induced reductions in PC1/3 was unlikely because 2-bromohexadecanoic acid, which is a chemically-modified palmitate that does not lead to ROS production [43,46], reduced PC1/3 activity as well as palmitate (Supplementary Fig. 2A) and N-acetylcysteine, an antioxidant, did not cancel the palmitate-induced suppression of PC1/3 activity (Supplementary Fig. 2B). On the other hand, ROS produces oxidized low-density lipoprotein (ox-LDL), and GLUTag cells and intestinal cells express CD36, which is the scavenger receptor of ox-LDL [47]. We also confirmed GLUTag cells express CD36 (data not shown). Thus, ox-LDL induces lipotoxicity in the macrophage via CD36 [48], it is possible that ox-LDL also induced lipotoxicity to GLUTag cells through CD36. Palmitate is reported to induce a mitochondrial energetic defect in the MIN6 cells [49]. We treated with cyclosporine A to investigate a mitochondrial energetic defect possibility. However, cyclosporine A did not affect palmitate-induced ER stress (data not shown). These results suggested that mitochondrial energetic defect was not the main cause of ER stress by palmitate. Our results suggested that free fatty acids induce lipotoxicity in the intestinal L cells through ER stress. However, it needs to be further studied about circulating levels of free fatty acids to enough of an extent as to make them susceptible to lipotoxicity. Free fatty acid level in serum in db/db mice, which are mouse model of type 2 diabetes, is about 1.5 mmol/ L [50]. A recent study showed that the infusion of ethylpalmitate, which increased ethylpalmitate level in serum to about 0.5 mmol/L, induced inflammation and reduces pancreatic β cells in mice [51]. These findings suggested that circulating free fatty acids attained to the level to induce lipotoxicity in intestinal L cells. Loss of function mutations in PC1/3 resulted in monogenic obesity and impaired glucose tolerance in humans [52,53]. Moreover, variants of the PC1/3 gene have been shown to be consistently associated with an obesity risk [54]. These findings suggest that a reduction in PC1/3 activity leads to obesity and diabetes. Although hyperglycemia was not shown to predict the decrease in GLP-1 secretion [55], GLP-1 levels have been negatively associated with obesity [12,13]. Our findings, which showed that HFD caused ER stress in L cells and reduced PC1/3 activity, suggest one reason why obese patients exhibit reduced levels of GLP-1 and are at higher risk of developing diabetes. Further investigations are needed to reveal GLP-1 levels and type 2 diabetes. In conclusion, our results suggest that FFAs elicited ER stress in intestine L cells, which resulted in an impairment in GLP-1 production. Identifying and improving of ER stress may be a possible therapeutic approach for type 2 diabetes.

Contribution statement H.H. researched the data, contributed to discussion, and wrote the manuscript. R.Y. researched the data, and contributed to discussion. S.S.D., T.S., A.T., and M.H. contributed to discus-

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sion. N.H. conceived and designed the experiments, contributed to discussion, wrote the manuscript, and reviewed and edited the manuscript.

Funding No specific funding was used for this study.

Acknowledgments We thank Dr. Daniel J. Drucker of University of Toronto, Dr. Akira Hirasawa of University of Kyoto, and Dr. Tohru Hira and Dr. Hiroshi Hara of Hokkaido University for the GLUTag cell line.

Conflict of interest There are no conflicts of interest or other current overlapping publications. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.metabol.2014.02.012.

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Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress.

Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. ...
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