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Dec 13, 2014 - Research Paper. Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β ...
Journal of Ethnopharmacology 161 (2015) 99–107

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Research Paper

Jinlida granule inhibits palmitic acid induced-intracellular lipid accumulation and enhances autophagy in NIT-1 pancreatic β cells through AMPK activation Dingkun Wang a, Min Tian a, Yuan Qi b, Guang Chen c, Lijun Xu a, Xin Zou a, Kaifu Wang a, Hui Dong a,n, Fuer Lu a,nn a Institute of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China b Shijiazhuang YiLing Pharmaceutical Co., Ltd., Shijiazhuang, Hebei 050035, China c Department of Integrated Traditional Chinese and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

art ic l e i nf o

a b s t r a c t

Article history: Received 20 August 2014 Received in revised form 6 November 2014 Accepted 1 December 2014 Available online 13 December 2014

Ethnopharmacological relevance: Jinlida granule (JLDG), composed of seventeen Chinese medical herbs, is a widely used Chinese herbal prescription for treating diabetes mellitus. However, the mechanism underlying this effect remains unclear. To determine the main components in JLDG and to explore the effect of JLDG on autophagy and lipid accumulation in NIT-1 pancreatic β cells exposed to politic acid (PA) through AMP activated protein kinase (AMPK) signaling pathway. Materials and methods: JLDG was prepared and the main components contained in the granules were identified by ultra performance liquid chromatography (UPLC) fingerprint. Intracellular lipid accumulation in NIT-1 cells was induced by culturing with medium containing PA. Intracellular lipid droplets were observed by Oil Red O staining and triglyceride (TG) content was measured by colorimetric assay. The formation of autophagosomes was observed under transmission electron microscope. The expression of AMPK and phospho-AMPK (pAMPK) proteins as well as its downstream fatty acid metabolism-related proteins (fatty acid synthase, FAS; acetyl-coA carboxylase, ACC; carnitine acyltransferase 1, CPT-1) and autophagy-related genes (mammal target of rapamycin, mTOR; tuberous sclerosis complex 1, TSC1; microtubule-associated protein 1 light chain 3, LC3-II) were determined by Western blot. The expression of sterol regulating element binding protein 1c (SREBP-1c) mRNA was examined by real time PCR (RT-PCR). Results: Our data showed that JLDG could significantly reduce PA-induced intracellular lipid accumulation in NIT-1 pancreatic β cells. This effect was associated with increased protein expression of pAMPK and AMPK in NIT-1 cells. Treatment with JLDG also decreased the expression of AMPK downstream lipogenic genes (SREBP-1c mRNA, FAS and ACC proteins) whereas increased the expression of fatty acid oxidation gene (CPT1 protein). Additionally, JLDG-treated cells displayed a markedly increase in the number of autophagosomes which was accompanied by the down-regulation of mTOR and the up-regulation of TSC1 and LC3-II proteins expression. However, when AMPK phosphorylation was inhibited by Compound C, JLDG supplementation did not exhibit any effect on the expression of these AMPK downstream molecules in NIT-1 cells. Conclusions: The results suggest that JLDG could reduce intracellular lipid accumulation and enhance the autophagy in NIT-1 pancreatic β cells cultured with PA. The mechanism is possibly mediated by AMPK activation. & 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: Jinlida granule Type 2 diabetes Lipid metabolism Autophagy AMP activated protein kinase NIT-1 cells

Abbreviations: JLDG, Jinlida granule; TCM, traditional Chinese medicine; ACC, acetyl-coA carboxylase; FAS, fatty acid synthase; CPT-1, carnitine acyl transferase 1; AMPK, AMP activated protein kinase; pAMPK, phospho-AMP activated protein kinase; SREBP-1c, sterol regulating element binding protein 1c; mTOR, mammal target of rapamycin; TSC1, tuberous sclerosis complex 1; LC3-II, microtubule-associated protein 1 light chain 3; PA, palmitate; T2DM, type 2 diabetes mellitus; RT-PCR, real time PCR; TG, triglyceride; UPLC, ultra performance liquid chromatography; AICAR, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside n Corresponding author. Tel.: þ 86 27 83663660; fax: þ86 27 83663237. nn Corresponding author. Tel./fax: þ86 27 83663237. E-mail addresses: [email protected] (H. Dong), [email protected] (F. Lu). http://dx.doi.org/10.1016/j.jep.2014.12.005 0378-8741/& 2014 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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1. Introduction The prevalence of diabetes mellitus (DM) has continued to increase globally, especially in China. According to the latest report, the prevalence of diabetes was estimated to be 11.6%, representing a total of 113.9 million Chinese adults with diabetes (Xu et al., 2013). Type 2 diabetes mellitus (T2DM) is the most common form which accounts for 90–95% of those with diabetes. In the onset and progression of T2DM, β cell dysfunction caused by elevated levels of glucose and/or free fatty acids (FFAs) play an important role (Marchetti et al., 2010). Chronic exposure to FFAs leads to lipid overload of β cells, impaired insulin secretion and apoptotic cell death (Zhou and Grill, 1994; Shimabukuro et al., 1998; Piro et al., 2002). Therefore the strategy for protecting pancreatic β cells against lipid overload may have a potential for the prevention and treatment of diabetes. Autophagy is a lysosomal degradative pathway that protects cells from death under stressful conditions. This process consists of several sequential steps—sequestration, transport to the lysosome, degradation, and utilization of degradation products (Mizushima, 2007). Through the autophagic process, damaged organelles and proteins are degraded or recycled, which is shown as an adaptive pro-survival response (Ryter and Choi, 2013). Recently, intracellular lipid droplets (LDs) have been also identified as the substrate for autophagy. Autophagy mobilizes lipids from LDs for metabolism through a process termed lipophagy, thereby preventing intracellular lipid overload (Singh et al., 2009; Dong and Czaja, 2011). Defective autophagy has been found to be related to lipid metabolic disorders such as fatty liver and obesity (Singh and Cuervo, 2012). Furthermore, when autophagy is impaired, the architecture and function of beta-cells could not be maintained (Fujitani et al., 2009). However, the relation between autophagy and lipid metabolism in pancreatic β cells under the condition of lipid overload has been rarely reported. DM is referred to as “Xiao Ke” disease in Traditional Chinese Medicine (TCM). “Xiao” means emaciation and “Ke” means thirst. But these two typical symptoms may not be obvious in patients with diabetes. Conversely, some patients have no symptoms except for obesity, which is a condition of excess body fat. According to the theory of TCM, excess body fat is described as excess phlegm and moisture caused by deficiency of spleen. Thus, tonifying spleen may serve as an effective measure against phlegm and moisture overload, thereby protecting cells from lipotoxicity. Jinlida granule (JLDG) is a widely used Chinese herbal prescription for tonifying spleen. It contains seventeen herbs including Radix Ginseng (Panax ginseng C.A.Mey.), Rhizoma Polygonati (Polygonatum kingianum Coll. et Hemal.), Rhizoma Atractylodis (Atractylodes lancea (Thunb.) DC.), Radix Sophorae Flavescentis (Sophora flavescens Ait.), Radix Ophiopogonis (Ophiopogon japonicus (Thunb.) KerGawl.), Radix Rehmanniae (Rehmannia glutinosa Libosch.), Radix Polygoni Multiflori (Polygonum multiflorum Thunb.), Fructus Corni (Cornus officinalis Sieb. et Zucc.), Poria (Poria cocos (Schw.) Wolf.), Herba Eupatorii (Eupatorium fortunei Turcz.), Rhizoma Coptidis (Coptis chinensis Franch.), Rhizoma Anemarrhenae (Anemarrhena asphodeloides Bge.), Herba Epimedii (Epimedium brevicornum Maxim.), Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bge.), Radix Puerariae (Pueraria lobata (Willd.) Ohwi.), Semen Litchi (Litchi chinensis Sonn.) and Cortex Lycii (Lycium chinense Mill.). In modern Chinese medicine practice, JLDG is widely used to treat diabetes mellitus. Clinical trials have also demonstrated that homeostasis model assessment of β-cell function (HOMA-β) score is improved after oral intake of JLDG in diabetic patients (Gao et al., 2010; Yu and Yang, 2013). However, the mechanism underlying this protection of β-cells remains unclear. Recently, few researches have showed that JLDG exerted good protection of β-cells as well as other organs through multi-target activity in diabetic rats

(Shi et al., 2012; Wu et al., 2012; Li and Zhao, 2013). In the present study, we investigated the effect of JLDG on palmitic acid (PA)induced lipid accumulation and autophagy in NIT-1 pancreatic β cells. 2. Material and methods 2.1. Herbal materials and the preparation of JLDG JLDG was supplied by Yiling Pharmaceutical Co. Ltd. (Shijiazhuang, Hebei, China). Seventeen kinds of herbs contained in JLDG were all purchased from Meiwei Traditional Chinese Medicine Company in Hebei Province (Anguo, China). The voucher specimen was deposited at the herbarium of the Pharmacy Faculty at the Beijing University of Chinese Medicine and authenticated by the Department of Pharmacognocy, Beijing University of Chinese Medicine (Beijing, China). Seventeen herbs were crashed into crude grains and extracted by refluxing with boiling water. Then the solution obtained was subsided by ethanol and the extract was concentrated and vacuum-dried to granules. Finally, per gram dry granule obtained is equivalent to 6.69 g raw medical herbs. For the present experiment, suspension solution of the granules was made in 0.5% of carboxymethyl cellulose solution before use. 2.2. Chemicals and reagents Bovine serum albumin (BSA), palmitate (PA) and rapamycin (Rapa) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Pioglitazone (PIO) hydrochloride tablets were purchased from Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) and Compound C were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Roswell Park Memorial Institute-1640 (RPMI-1640) and fetal bovine serum (FBS) were obtained from Hyclone Laboratories Inc. (Logan, UT, USA). Penicillin, streptomycin, trypsin and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit were obtained from Guge biological Technology Co. (Wuhan, Hubei, China). Triglyceride colorimetric assay kit was purchased from Shanghai Mind Bioengineering Co, Ltd (Shanghai, China). Bicinchoninic acid (BCA) protein assay kit and enhanced chemiluminescence (ECL) reagent were obtained from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit antimouse fatty acid synthase (FAS), AMP activated protein kinase (AMPK), acetyl-coA carboxylase (ACC) and carnitine acyl transferase 1 (CPT-1) antibodies were from Abcam Company (Hongkong, China). Rabbit anti-mouse phospho-AMP activated protein kinase (pAMPK) was obtained from Millipore Corporation (Billerica, MA, USA). Rabbit anti-mouse mammal target of rapamycin (mTOR), microtubuleassociated protein 1 light chain 3 (LC3-II) and tuberous sclerosis complex 1 (TSC1) were provided by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Trizol reagent, PrimeScript RT reagent Kit and SYBR Premix Ex Taq were purchased from TaKaRa Bio Inc. (Dalian, Liaoning, China). 2.3. Preparation of JLDG-containing serum The rat dose of JLDG was obtained by the conversion of the human dose to rat equivalent dose based on body surface areas. Male SD rats (200–250 g) were obtained from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Rats were housed under specific pathogen-free conditions and fed a standard rodent laboratory diet from SLAC Laboratory Animal Co., Ltd for two weeks. Then rats were randomly divided into four groups. Rats in the three JLDG groups were orally administrated low (0.75 g/kg), intermediate (1.5 g/kg) and high (3.0 g/kg) doses of JLDG, respectively. Rats in PIO-treated group were orally administrated PIO (25 mg/kg). The corresponding therapy was

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administered twice a day for three days. After 1 h of the last intragastric administration, all rats were anesthetized by intraperitoneal injection of sodium pentobarbital and gathered blood through abdominal aorta under sterile condition. Serum obtained was then sterilized by filtering through a 0.22 mm filter and stored at  20 1C till needed for use. All procedures involving animals complied with the Institutional Animal Care and Use Committees of Huazhong University of science and technology. 2.4. Determinations of the main chemical constituents in JLDG by UPLC analysis Ultra performance liquid chromatography (UPLC) fingerprint was performed to identify the main chemical constituents in JLDG (Jiang et al., 2013). Tanshinol sodium, puerarin, salvianolic acid B, epimendin B, epimendin C, icariin, ginsenoside Rb1, ginsenoside Rc, ginsenoside Rb2, purchased from National Institutes for Food and Drug Control (Beijing, China), were taken as reference standards. UPLC was performed on a Acquity UPLC system (Waters Corporation, Milford, MA, USA) equipped with an Acquity BEH C18 column (2.1 mm  100 mm, 1.7 μm). The mobile phase included acetonitrile (B) and 0.1% phosphate (D) at a flow speed of 0.3 ml/min in the condition of column temperature 40 1C. The gradient elution was as follows: 0–1 min, 4–4%B, 1–3 min, 4–7.5%B, 3–12 min, 7.5–17%B, 12–13 min, 17–20%B, 13–20 min, 20–32%B, 20–21 min, 32–40%B, 21–25 min, 40–56%B, 25–29 min, 56–90%B, 29–35 min, 90–90%B. There were ten batches of JLDG supplied by Yiling Pharmaceutical Co. Ltd to detect the similarity. The similarity of fingerprints was analyzed by professional software named Similarity Evaluation System for Chromatogram Fingerprint of Traditional Chinese Medicine (version of 2004A). 2.5. Cell culture, viability assay and intervention The mouse NIT-1 pancreatic β cells were provided by Department of Immunology, Tongji Medical College, Huazhong University of Science and Technology. The cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 units/ml penicillin and 100 units/ml streptomycin and maintained at 37 1C in a humidified atmosphere of 5% CO2 and 95% air. Mediums containing different types of rat serum (10% low dose JLDG serum, 10% intermediate dose JLDG serum, 10% high dose JLDG serum, 10% PIO serum) were prepared to identify cytotoxicity by MTT assay. NIT-1 cells were seeded at 5  103 cells per well in 96 well-culture plates. After cells have adhered, the medium was replaced by serum containing different dugs. The cell viability in the serum containing different concentration of drugs was evaluated by MTT assay according to manufacturer’s protocol. Under the conditions of this study, 10% intermediate dose JLDG serum and 10% PIO serum had negligible effect on cell viability (data not shown). Approximately, 1  105/ml cells were transferred into two 6-well plates and allowed to grow overnight to 70% confluence. Then all types of culture mediums were respectively replaced according to the different cell groups as follows. The culture medium in control group contained 10% FBS. The medium in model group contained PA (0.25 mmol/l) and 0.5% BSA. The mediums in different treatment groups as below also contained PA(0.25 mmol/l), respectively. Furthermore, the medium in JLDG group contained 10% intermediate dose JLDG serum. The medium in AICAR group contained AICAR (0.5 mmol/l) and 0.5% BSA. The medium in Compound C group contained 10% intermediate dose JLDG and Compound C (10 μmol/l). The medium in PIO group contained 10% PIO serum. For the detection

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of autophagy-related molecules, a RAPA group was also set up and the medium contained Rapa (50 nmol/l) and 0.5% BSA. 2.6. Measurement of intracellular triglyceride content and oil red O staining The content of intracellular TG was measured by a determiner TG kit using colorimetric assay and expressed as micrograms of lipid per milligrams of cell protein (Yang et al., 2012). Oil red O staining was applied to determine the accumulation of lipid as previously described (Lee et al., 2004). Briefly, NIT-1 cells were seeded in 6-well plates at a density of 1  105 cells/well and cultured for 24 h in the presence of PA (0.25 mmol/l). For different treatment groups, the medium also contained corresponding drugs. At the end of the culture, the cells were rinsed with PBS twice and fixed with 10% formalin for half an hour. After twice washing with PBS again, the cells were stained with oil red O for 15 min at room temperature, followed by decolorization with 60% isopropanol. Then the lipid droplets in the cells were observed and the photos were captured under a light microscope (TE2000-E, Nikon). 2.7. Transmission electron microscope The autophagy of NIT-1 cells was evaluated by autophagosome screening under transmission electron microscope (FEI Tecnai G212, Netherlands). At the end of intervention, the cells were digested with 0.25% trypsin and collected in centrifuge tubes. Then the cells were centrifuged at 1500 r/min for 10 min, followed by another centrifugation at a speed of 800 r/min for the same time. The supernatant was discarded and 2.5% glutaraldehyde was added into the tube to fix the cells. After 15 min of fixation, dehydration, embedding, sectioning and staining was performed under general supervision. Then the autophagosomes were observed under a transmission electron microscope and photographed. 2.8. Western blot analysis Cells in each group were lysed with RIPA lysis buffer for extracting cell total protein. The lysates were centrifuged at 12,000  g for 10 min at 4 1C. Then the supernatants were assayed for protein concentration using the BCA method. After solubilized in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) sample buffer and heated in boiling water for 10 min, 100 μg protein was separated on 10% SDS-PAGE and transferred onto nitrocellulose (NC) membranes. Then the membranes were blocked with 5% skimmed milk powder in TBST for 1 h, followed by incubation with primary antibodies (ACC, FAS, AMPK, pAMPK, CPT-1, mTOR, TSC1, LC3-II) for 2 h at room temperature and overnight at 4 1C. Wash the membranes with PBST three times for 10 min each and incubate the membranes with horseradish peroxidase (HRP) conjugated secondary antibodies. After another three washes, the membranes were detected by enhanced chemiluminescence (ECL) Western Blot Detection Kit. Finally, the membranes were exposed to imaging film and quantified using KODAK Digital Imaging System (KODAK, USA). 2.9. RNA extraction and real-time PCR Total RNA from cells in different groups were extracted with Trizol reagent. The purity and concentration of total RNA were measured by a Nucleic Acid/Protein Analyzer (Thermo, Rockford, USA). Then 2 μg of total RNA was reverse-transcribed using a PrimeScript RT reagent Kit in a total reaction volume of 20 μl. cDNA was synthesized in a

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Mastercycler gradient PCR apparatus (Eppendorf Company, Hamburg, Germany). Then 2.0 μl of cDNA was amplified in a 20 μl PCR reaction containing 6.8 μl ddH2O, 0.4 μl forward primer, 0.4 μl reverse primer,

0.4 μl ROX Reference Dye (50  ) and 10.0 μl SYBR premix EX TaqTM (TaKaRa Company, Dalian, China) with an Applied Biosystems StepOne Real-Time PCR System (StepOne, Foster City, USA). The reaction

Fig. 1. UPLC fingerprints of Jinlida granule from ten batches.

Fig. 2. UPLC fingerprint of JLDG (a) and reference standards (b). Peak number and identity: 4: tanshinol; 17: puerarin; 34: salvianolic acid B; 37: epimendin B; 38: epimendin C; 39: icariin; 41: ginsenoside Rb1; 42: ginsenoside Rc; 44: ginsenoside Rb2.

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included stage1, 95 1C for 30 s; stage2, 95 1C for 5 s; stage 3, 60 1C for 30 s. The primer sequences were as follows: SREBP-1c: forward, 50 CCGAGATGTGCGAACTGGA-30 , reverse, 50 -GAAGTCACTGTCTTGGTTGTTGATG-30 ; β-actin: forward, 50 -CATCCGTAAAGACCTCTATGCCAAC-30 , ΔΔ reverse, 50 -ATGGAGCCACCGATCCACA-30 . 2  CT was used to calculate the relative fold-changes of each group. 2.10. Statistical analysis All results were presented as mean 7standard deviation (SD) and analyzed using SPSS 19.0 software. One-way analysis of variance (ANOVA) was used to determine the statistical significance. Based on data assumed equal variances or not assumed equal variances, a LSD or Dunnett’s T3 test was used, respectively. Meanwhile, descriptive and variance homogeneity test were also followed. P o0.05 was considered statistically significant.

3. Results 3.1. UPLC profile of JLDG UPLC fingerprint of JLDG from ten batches was shown in Fig. 1. The result showed that the similarity index of ten batches was up to 0.99 (data not shown). Fifty common peaks were detected (Fig. 2a) and nine of them were identified by comparing the retention time and peak height with reference standard (Fig. 2b). The main constituent of JLDG were as follows: tanshinol, puerarin, salvianolic acid B, epimendin B, epimendin C, icariin, ginsenoside Rb1, ginsenoside Rc and ginsenoside Rb2.

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intracellular lipid accumulation was also found after the intervention with AICAR (P o0.05), which are known activators of AMPK. However, the AMPK inhibitor Compound C blocked the effect of JLDG on decreasing PA-induced intracellular lipid accumulation in NIT-1 cells (P o0.05). 3.3. The effect of JLDG on the expressions of AMPK and its downstream fatty acid metabolism-related genes in NIT-1 pancreatic β cells Since AMPK activation requires phosphorylation of threonine residue (T172) in the catalytic α subunit (Bright et al., 2009), we examined the protein expression of pAMPK and AMPK in NIT-1 cells. As shown in Fig. 5A and B, in our study, treatment with JLDG exhibited a significant increase in the protein expression of pAMPK and AMPK compared with the model group (Po0.01). With regard to the ratio of pAMPK to AMPK (Fig. 5C), it also presented a fold change (Po0.01) of at least 1.5, indicating a significant activation of AMPK after JLDG supplementation. Consistent with AMPK activation, the expression of AMPK downstream lipogenic genes (SREBP1c mRNA, FAS and ACC proteins) was reduced (Po0.05; Po0.01) whereas the expression of fatty acid oxidation gene (CPT-1 protein)

3.2. The effect of JLDG decreased intracellular lipid accumulation in NIT-1 cells To determine the effect of JLDG on lipid accumulation in NIT-1 cells, Oil Red O staining and methods of colorimetric assay were used. As shown in Fig. 3, intracellular lipid droplets in JLDG group were significantly decreased compared with those in model group. Meanwhile, intracellular TG content was also remarkably reduced (P o0.05) in the cells of JLDG group (Fig. 4). This reduction of

Fig. 4. The effect of JLDG on intracellular TG content of NIT-1 pancreatic β cells exposed to PA. JLDG supplementation decreased intracellular TG content in NIT-1 pancreatic β cells. *Po 0.05, significantly different from control. ΔPo 0.05, significantly different from model group. #Po 0.05, significantly different from JLDG group. Each bar represents means 7 SD from three wells.

Fig. 3. The effect of JLDG on PA-induced lipid accumulation in NIT-1 pancreatic β cells. Staining of lipid droplets with Oil Red O showed that PA caused an apparent increase in intracellular lipid accumulation, which was attenuated by the treatment with JLDG.

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Fig. 5. The effect of JLDG on the expressions of AMPK and its downstream fatty acid metabolism-related genes in NIT-1 pancreatic β cells. JLDG supplementation inhibited the expression of lipogenic genes and enhanced the expression of fatty-acid oxidase gene through AMPK activation. Representative gels and protein levels for AMPK (A), pAMPK (B), FAS (D), ACC (E) and CPT-1 (F) measured by Western blot in NIT-1 pancreatic β cells. pAMPK/AMPK ratio (C) and SREBP-1c (G) mRNA levels in NIT-1 cells from control, model, JLDG, AICAR, Compound C and PIO groups. Each bar represents means 7 SD from three wells. nPo 0.05 and nnP o 0.01, significantly different from control. Δ Po 0.05 and ΔΔPo 0.01, significantly different from model group. #Po 0.05 and ##P o 0.01, significantly different from JLDG group.

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Fig. 6. The effect of JLDG on the number of intracellular autophagosomes in NIT-1 pancreatic β cells. JLDG supplementation enhanced the formation of intracellular autophagosomes in NIT-1 cells. Representative transmission electron microscope images from control, model, JLDG, AICAR, Compound C, PIO and Rapa groups. (Arrows are pointing at intracellular autophagosomes).

was enhanced (Po0.01) (Fig. 5D to G). However, when AMPK phosphorylation was inhibited by Compound C, JLDG administration did not exhibit any effect on the expression of these AMPK downstream molecules in NIT-1 cells (Po0.05; Po0.01). These findings indicate that the effect of JLDG on decreasing intracellular TG content may be due to the activation of AMPK.

3.4. The effect of JLDG on PA-induced autophagy and the expression of autophagy-related genes in NIT-1 pancreatic β cells To verify if enhanced autophagy is involved in the effect of JLD on PA-induced lipid accumulation in NIT-1 cells, the formation of autophagosomes was observed and the expression of autophagyrelated genes were examined. As shown in Fig. 6, PA supplementation increased the number of intracellular autophagosomes in NIT-1 cells of model group, accompanied by over-expression (Po0.01) of LC3IIproteins (Fig. 7C), a well-known marker of autophagy. It may serves as an adaptive response under conditions of lipid overload. The cellular autophagy inducer Rapa also markedly enhanced the autophagic vacuoles formation in NIT-1 cells. Compared with the model group, JLDG-treated cells exhibited an obvious increase in the number of intracellular autophagosomes. This increase was associated with decreased expression of mTOR protein (Po0.05) (Fig. 7A) and increased expression of TSC1 and LC3-IIproteins (Po0.05; Po0.01) (Fig. 7B and C). Similar results were also obtained when cells were cultured with AMPK activators (PIO and AICAR) and autophagy inducer (Rapa) (Po0.01). However, supplementation with AMPK inhibitor, Compound C blocked the enhancing effect of JLDG on the autophagy in NIT-1 β cells (Po0.01). Together, these results suggest that JLDG may enhance PA-induced autophagy in β cells via AMPK activation.

4. Discussion JLDG is a Chinese herbal prescription widely used to treat diabetes mellitus in the clinical practice of traditional Chinese medicine. However, the mechanism underlying its anti-hyperglycemic effect is still unknown. In the present study, by using serum pharmacology, we found that JLDG could enhance the autophagy and reduce fat deposition in pancreatic β cells exposed to elevated concentration of FFAs. We also found that both of these actions were inhibited by Compound C, an AMPK inhibitor. Since increased autophagy is considered as a protective mechanism against fatty acid-induced lipotoxicity (Mei et al., 2011), we suppose that the anti-diabetic effect of JLDG might be associated with its protection of pancreatic β cells via AMPK activation. In type 2 diabetes mellitus, a progressive decline of β-cell function and mass is a common feature of the disease (Fonseca, 2009). It has been demonstrated that many factors are related to the reduction of β cell function and mass such as hyperglycemia, elevated FFAs, islet amyloid, cytokines and insulin resistance (Cernea and Dobreanu, 2013). Among these, FFAs have attracted much attention (Shao et al., 2013). Generally, β cells undergo metabolic adaptations including lipid accumulation and insulin release under conditions of high FFAs levels (Vernier et al., 2012). However, when lipid influx exceeds its capacity for lipid storage, lipotoxicity may occur. Elevated FFAs may trigger endoplasmic reticulum (ER) stress followed by the activation of autophagy (Kharroubi et al., 2004; Ogata et al., 2006). Ultrastructural study has also proved that the exposition of β cells to PA, but not to high glucose, is associated with the formation of lipid droplets and autophagolysomes, together with enlarged and prevalently perinuclear cysternae, indicating a remarkable ER stress (Martino et al., 2012). Here, activated autophagy has been proposed as a cell survival

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Fig. 7. The effect of JLDG on the expressions of AMPK downstream antophagy -related genes in NIT-1 pancreatic β cells. JLDG supplementation decreased the expression of mTOR protein and increased the expression of TSC1 and LC3-II. Representative gels and protein levels for mTOR (A), TSC1 (B) and LC3-II (C), measured by Western blot in NIT-1 pancreatic β cells from control, model, JLDG, AICAR, Compound C, PIO and Rapa groups. Each bar represents means7 SD from three wells. nPo 0.05 and nnP o 0.01, significantly different from control. ΔP o0.05 and ΔΔPo 0.01, significantly different from model group. #P o 0.05 and ##Po 0.01, significantly different from JLDG group.

response to lipid overload. On one side, stimulation of autophagy could reduce ER stress associated with the attenuation of apoptosis in β cells (Bachar-Wikstrom et al., 2013). On the other side, autophagy could degrade lipid droplets through a process termed lipophagy (Dong and Czaja, 2011). Thus, activation of autophagy may become a new therapeutic approach for maintenance of normal β cell function and survival. AMPK is known to be a key regulator of cellular lipid metabolism (Hardie, 2008), and is considered as a positive regulator that stimulates autophagy in response to lipid overload. Moreover, AMPK is a nutrient and energy sensor that maintains cellular energy homeostasis (Hardie et al., 2012). The inhibition of AMPK is always associated with the development of fat deposition disorders such as obesity and diabetes. It has been long recognized that AMPK activation could regulate lipid metabolism by stimulating fatty acid oxidation and inhibiting lipogenesis (Hardie, 2004; O’Neill et al., 2013). Therefore the antilipotoxic effect of AMPK activation represents a new therapeutic target for the treatment of obesity and type 2 diabetes. Until now, numerous AMPK activators have been reported. Natural activator (e.g. berberine, resveratrol and capsaicin) and chemical synthesis activator (e.g. AICAR, PIO and metformin) have been proved to be involved in lipid metabolism (Srivastava et al., 2012). However, their effect in the regulation of autophagy has been poorly reported. In the present study, we found

that JLDG, as a new AMPK activator, not only inhibited intracellular fat deposition but also stimulated autophagy in NIT-1 cells. On the one hand, JLDG could directly enhance the lipolysis by up-regulation of CPT-1 and inhibit the lipogenesis by down-regulation of SREBP-1C, ACC and FAS. On the other hand, JLDG could decrease the expression of mTOR protein and increase the expression of TSC1 and LC3-II proteins, leading to the enhancement of autophagy. Based on the concept of lipophagy which connects autophagy and lipid metabolism the stimulation of autophagy by JLDG might also contribute to the reduction of fat deposition within β cells. However, these effects of JLDG can be eliminated in the presence of AMPK inhibitor Compound C. Collectively, it is obvious that Chinese herbal medicine JLDG, via AMPK activation, can directly or indirectly attenuate intracellular lipid overload as discussed above. However, some limitations of our work should be mentioned. In the present study, we did not investigate the effect of JLDG on PA-induced insulin release and apoptosis in NIT-1 cells. Therefore the effect of JLDG on maintaining β-cell function and mass should be further confirmed. Actually, some studies take opposing views on intracellular fat deposition. They even think that the ability of accumulating triglyceride might be able to protect NIT-1 pancreatic β cells from PA-induced lipotoxicity (Cnop et al., 2001; Choi et al., 2011) Furthermore, an over stimulated autophagy might also

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lead to non-apoptotic cell death (Tsujimoto and Shimizu, 2005). Hence, more studies are needed in order to explore the mechanisms underlying the anti-diabetic effect of JLDG. 5. Conclusion JLDG containing serum can reduce intracellular lipid accumulation and enhance the autophagy in NIT-1 pancreatic β cells cultured with PA. The mechanism is possibly mediated by AMPK activation. Acknowledgments This work was supported by the grant from national major technological plan of major drug innovation in the twelfth five years program (2011ZX09401-020) and grants from National Natural Science Foundation of China (Nos. 81173370, 81273683, 81373871). References Bachar-Wikstrom, E., Wikstrom, J.D., Ariav, Y., Tirosh, B., Kaiser, N., Cerasi, E., Leibowitz, G., 2013. Stimulation of autophagy improves endoplasmic reticulum stress-induced diabetes. Diabetes 62, 1227–1237. Bright, N.J., Thornton, C., Carling, D., 2009. The regulation and function of mammalian AMPK-related kinases. Acta Physiologica (Oxford, England) 196, 15–26. Cernea, S., Dobreanu, M., 2013. Diabetes and beta cell function: from mechanisms to evaluation and clinical implications. Biochemia Medica 23, 266–280. Choi, S.E., Jung, I.R., Lee, Y.J., Lee, S.J., Lee, J.H., Kim, Y., Jun, H.S., Lee, K.W., Park, C.B., Kang, Y., 2011. Stimulation of lipogenesis as well as fatty acid oxidation protects against palmitate-induced INS-1 beta-cell death. Endocrinology 152, 816–827. Cnop, M., Hannaert, J.C., Hoorens, A., Eizirik, D.L., Pipeleers, D.G., 2001. Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 50, 1771–1777. Dong, H., Czaja, M.J., 2011. Regulation of lipid droplets by autophagy. Trends in Endocrinology and Metabolism: TEM 22, 234–240. Fonseca, V.A., 2009. Defining and characterizing the progression of type 2 diabetes. Diabetes Care 32 (Suppl. 2), S151–156. Fujitani, Y., Kawamori, R., Watada, H., 2009. The role of autophagy in pancreatic beta-cell and diabetes. Autophagy 5, 280–282. Gao, H., Zhang, J., Wu, Y., Ding, L., Cao, Y., Wang, M., Li, H., Yin, X., 2010. Effects of Jinlida granules on islet β cells in diabetes mellitus. Lishizhen Medicine and Materia Medica Research 21, 1119–1120 (in Chinese). Hardie, D.G., 2004. AMP-activated protein kinase: a master switch in glucose and lipid metabolism. Reviews in Endocrine & Metabolic Disorders 5, 119–125. Hardie, D.G., 2008. AMPK: a key regulator of energy balance in the single cell and the whole organism. International Journal of Obesity 32 (Suppl. 4), S7–12 (2005). Hardie, D.G., Ross, F.A., Hawley, S.A., 2012. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature Reviews Molecular Cell Biology 13, 251–262. Jiang, X., Jia, J., Li, Y., 2013. UPLC Fingerprints of Jinlida Granule. Chinese Journal of Experimental Traditional Medical Formulae 1, 126–129 (in Chinese). Kharroubi, I., Ladriere, L., Cardozo, A.K., Dogusan, Z., Cnop, M., Eizirik, D.L., 2004. Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress. Endocrinology 145, 5087–5096. Lee, K.D., Kuo, T.K., Whang-Peng, J., Chung, Y.F., Lin, C.T., Chou, S.H., Chen, J.R., Chen, Y.P., Lee, O.K., 2004. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40, 1275–1284. Li, F., Zhao, Y., 2013. Protective effect of Jinlida granules on hippocampus of diabetic rats. Academic Journal of Second Military Medical University 34, 137–141 (in Chinese). Marchetti, P., Lupi, R., Del Guerra, S., Bugliani, M., Marselli, L., Boggi, U., 2010. The beta-cell in human type 2 diabetes. Advances in Experimental Medicine and Biology 654, 501–514. Martino, L., Masini, M., Novelli, M., Beffy, P., Bugliani, M., Marselli, L., Masiello, P., Marchetti, P., De Tata, V., 2012. Palmitate activates autophagy in INS-1E betacells and in isolated rat and human pancreatic islets. PLoS One 7, e36188.

107

Mei, S., Ni, H.M., Manley, S., Bockus, A., Kassel, K.M., Luyendyk, J.P., Copple, B.L., Ding, W.X., 2011. Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. The Journal of Pharmacology and Experimental Therapeutics 339, 487–498. Mizushima, N., 2007. Autophagy: process and function. Genes & Development 21, 2861–2873. O’Neill, H.M., Holloway, G.P., Steinberg, G.R., 2013. AMPK regulation of fatty acid metabolism and mitochondrial biogenesis: implications for obesity. Molecular and Cellular Endocrinology 366, 135–151. Ogata, M., Hino, S., Saito, A., Morikawa, K., Kondo, S., Kanemoto, S., Murakami, T., Taniguchi, M., Tanii, I., Yoshinaga, K., Shiosaka, S., Hammarback, J.A., Urano, F., Imaizumi, K., 2006. Autophagy is activated for cell survival after endoplasmic reticulum stress. Molecular and Cellular Biology 26, 9220–9231. Piro, S., Anello, M., Di Pietro, C., Lizzio, M.N., Patane, G., Rabuazzo, A.M., Vigneri, R., Purrello, M., Purrello, F., 2002. Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress. Metabolism: Clinical and Experimental 51, 1340–1347. Ryter, S.W., Choi, A.M., 2013. Autophagy: an integral component of the mammalian stress response. Journal of Biochemical and Pharmacological Research 1, 176–188. Shao, S., Yang, Y., Yuan, G., Zhang, M., Yu, X., 2013. Signaling molecules involved in lipid-induced pancreatic beta-cell dysfunction. DNA and Cell Biology 32, 41–49. Shi, J., Wu, Y., Song, Y., Han, C., Liu, Z., 2012. Protective effect of Jinlida granules on islet β cells in diabetes mellitus rats. Academic Journal of Second Military Medical University 33, 385–389 (in Chinese). Shimabukuro, M., Higa, M., Zhou, Y.T., Wang, M.Y., Newgard, C.B., Unger, R.H., 1998. Lipoapoptosis in beta-cells of obese prediabetic fa/fa rats. Role of serine palmitoyltransferase overexpression. The Journal of Biological Chemistry 273, 32487–32490. Singh, R., Cuervo, A.M., 2012. Lipophagy: connecting autophagy and lipid metabolism. International Journal of Cell Biology 2012, 282041. Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K., Cuervo, A.M., Czaja, M.J., 2009. Autophagy regulates lipid metabolism. Nature 458, 1131–1135. Srivastava, R.A., Pinkosky, S.L., Filippov, S., Hanselman, J.C., Cramer, C.T., Newton, R. S., 2012. AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases. Journal of Lipid Research 53, 2490–2514. Tsujimoto, Y., Shimizu, S., 2005. Another way to die: autophagic programmed cell death. Cell Death and Differentiation 12 (Suppl. 2), 1528–1534. Vernier, S., Chiu, A., Schober, J., Weber, T., Nguyen, P., Luer, M., McPherson, T., Wanda, P.E., Marshall, C.A., Rohatgi, N., McDaniel, M.L., Greenberg, A.S., Kwon, G., 2012. Beta-cell metabolic alterations under chronic nutrient overload in rat and human islets. Islets 4, 379–392. Wu, Y., Shi, J., Song, Y., Han, C., Liang, C., Liu, Z., 2012. Effect of Jinlida granules on renin-angiotensin system in kidney and cardiovascular tissues of diabetic rat. Academic Journal of Second Military Medical University 33, 1065–1069 (in Chinese). Xu, Y., Wang, L., He, J., Bi, Y., Li, M., Wang, T., Jiang, Y., Dai, M., Lu, J., Xu, M., Li, Y., Hu, N., Li, J., Mi, S., Chen, C.S., Li, G., Mu, Y., Zhao, J., Kong, L., Chen, J., Lai, S., Wang, W., Zhao, W., Ning, G., 2013. Prevalence and control of diabetes in Chinese adults. JAMA: The Journal of the American Medical Association, 310; pp. 948–959. Yang, Y., Piao, X., Zhang, M., Wang, X., Xu, B., Zhu, J., Fang, Z., Hou, Y., Lu, Y., Yang, B., 2012. Bioactivity-guided fractionation of the triglyceride-lowering component and in vivo and in vitro evaluation of hypolipidemic effects of Calyx seu Fructus Physalis. Lipids in Health and Disease 11, 38. Yu, L., Yang, J., 2013. Effect of JinLiDa particles on β-cell pancreatic function of the patients with impaired glucose regulation. Journal of Taishan Medical College 34, 513–515 (in Chinese). Zhou, Y.P., Grill, V.E., 1994. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. Journal of Clinical Investigation 93, 870–876.