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Eur Food Res TechnolDOI 10.1007/s00217-014-2322-9

ORIGINAL PAPER

Fenugreek seed extract and its phytocompounds‑ trigonelline and diosgenin arbitrate their hepatoprotective effects through attenuation of endoplasmic reticulum stress and oxidative stress in type 2 diabetic rats

Tharaheswari Mayakrishnan · Jayachandra Reddy Nakkala · Syam Praveen Kumar Jeepipalli · Kumar Raja · Varshney Khub Chandra · Vasanth Kumar Mohan · Sudha Rani Sadras

Received: 16 February 2014 / Revised: 31 August 2014 / Accepted: 1 September 2014 © Springer-Verlag Berlin Heidelberg 2014

FSE, trigonelline, and diosgenin were found to exhibit protec-tive effects individually and brought about significant reduc-tion in serum enzymes, liver TGs, LPO, expression of liver ER stress marker proteins, and significant increase in liver glycogen content and antioxidants. Histological analysis also supported their protective effects suggesting that FSE and its phytoconstituents alleviate T2DM associated liver damage by normalizing ER stress and oxidative stress.

Keywords T2DM · Trigonelline · Diosgenin · ER stress · Hepatotoxicity

AbbreviationsBip Binding immunoglobulin proteinCHOP C/EBP homologous proteineIF2a Eukaryotic initiation factor 2aERO1 Endoplasmic reticulum oxidoreductin 1GADD34 Growth arrest and DNA damage-

inducible protein 34GOD–PAP Glucose oxidase - phenol +

aminophenazoneGPO–PAP–ESPAS Glycerol-3-phosphate oxidase–

Peroxidase and aminoantipyrine–N-ethyl-N-sulfopropyl-n-anisidine

GRP78 Glucose regulated protein 78JNK c-Jun N-terminal kinaseNAFLP Nonalcoholic fatty liverPDI Protein disulfide isomeraseUPR Unfolded protein response

Introduction

Type II diabetes is the commonest form of diabetes constitut-ing 90 % of diabetic population. Recent WHO report states that

Abstract Fenugreek seed and some of its phytoconstituents are known to possess hypoglycemic and hypolipidemic prop-erties but their mode of action in insulin target tissues remains to be elucidated. As hepatic insulin resistance contributes to the development of type 2 diabetes mellitus (T2DM), the pro-tective effects of fenugreek seed extract (FSE) and two of its phytoconstituents-trigonelline and diosgenin were evaluated in T2DM rats with special focus on changes in endoplasmic reticulum (ER) stress and oxidative stress markers in liver. Trigonelline and diosgenin content in FSE were quantified by HPTLC. T2DM was induced in male Sprague–Dawley rats by feeding high-fat diet and administration of low-dose strepto-zotocin. T2DM rats exhibited hyperglycemia and significantly reduced serum C-peptide levels. Liver damage in T2DM rats was marked by significantly elevated levels of liver marker enzymes in serum, liver triglycerides (TGs), and reduced liver glycogen and was further confirmed by histological analysis. T2DM rats also displayed twofold–threefold increase in the levels of ER chaperones Bip, protein disulfide isomerase (PDI) as well as ER stress associated proapoptotic markers CHOP, Caspase12 and Caspase3 in liver along with elevated lipid per-oxidation (LPO) and reduced antioxidant levels. Promisingly,

T. Mayakrishnan · J. R. Nakkala · S. P. K. Jeepipalli · S. R. Sadras (*) Department of Biochemistry and Molecular Biology, School of Life Sciences, Pondicherry University, Kalapet, Puducherry, Indiae-mail: dr.ssrlab@gmail.com; sadrassudha@yahoo.com

K. Raja · V. Khub Chandra Department of Pathology, Rajiv Gandhi College of Veterinary and Animal Sciences, Kurumbapet, Puducherry, India

V. K. Mohan Herbal and Indian Medicine Research Laboratory, Department of Biochemistry, Sri Ramachandra University, Chennai, India

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approximately 364 million people globally suffer from diabe-tes mellitus (DM), and that DM-related deaths would double in 2030 [1]. Several lines of evidences suggest that T2DM is char-acterized by peripheral insulin resistance, pancreatic β-cell dys-function, and decreased β-cells mass due to increased rate of β-cell apoptosis [2, 3]. The sustained peripheral insulin resist-ance in liver, fat, and muscle stimulates the pancreatic β-cells to compensate for hyperglycemia and over stimulation results in β-cell death due to glucotoxicity [4]. Changes in lifestyle such as consumption of high-calorie diet and lack of exercise have increased the global prevalence of obesity, and about 60–90 % of cases of T2DM diabetes are related to obesity [5]. Liver being a major metabolic organ involved in the regulation of glucose homeostasis, hepatic insulin resistance also contribute to a large extent for the onset and progression of T2DM. The contributing factors for β cell failure and progressive β cell loss in T2DM include elevated circulating levels of glucose, free fatty acid (FFA), and islet amyloid polypeptide (IAPP) [6–9]. Other factors that cause β cell loss include endoplasmic reticu-lum (ER) stress and increased reactive oxygen species (ROS). Increased ER stress/unfolded protein response (UPR) mediated signaling and ROS have been implicated in the pathogenesis of T2DM and are associated with increased cell death in liver and pancreas as these organs sustain a high rate of protein synthesis and secretion [10].

Fenugreek (Trigonella foenum graecum), a leguminous plant extensively cultivated in India, Africa, and the Mediterra-nean, has been traditionally used as a medicinal herb and as a spice in India and China. Extracts of seeds and leaves of fenu-greek have been reported to possess hypoglycemic, hypolipi-demic properties in animal models and humans, and also are found to be nontoxic [11–14]. However, detailed studies are required to reveal its mode of action at cellular level.

In the present study, we hypothesized that FSE and its phytoconstituents may bring about their insulin-sensitizing effects through regulation of ER stress/UPR signaling and associated oxidative stress. To validate this, T2DM was induced in male Sprague–Dawley rats by a combination of high-fat diet (HFD) and low-dose streptozotocin (STZ) followed by treatment with FSE, trigonelline and diosgenin individually and their effects against T2DM associated liver damage was analyzed. Apart from analyzing the bio-chemical markers of liver damage, the expression levels of ER stress/UPR pathway proteins and the levels of oxidative stress markers in liver were also assessed in this study.

Materials and methods

Chemicals

Trigonelline, Diosgenin, STZ, β-actin, Caspase 3 and Cas-pase 12 were purchased from Sigma Aldrich, St. Louis,

Mo, USA. Primary antibodies for GADD153/CHOP, Bip, PDI and Horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology and TMB/H2O2 (3, 3′, 5, 5′-Tetramethylbenzidine/hydro-gen peroxide) from MERCK. All other chemicals and sol-vents were of the highest analytical grade.

Preparation of high fat diet

High-fat diet encompassing 58 % fat, 25 % protein, and 17 % carbohydrate which was prepared with the following constituents: powdered normal pellet diet (NPD; 365 g), lard oil (310 g), casein (100 g), yolk(100 g), butter (100), vitamins (10 g) mineral mix (10 g), methionine (3 g), yeast (1 g) & NaC1 (1 g).

Preparation of plant extracts

Dried fenugreek seeds were purchased from supermarket—Nilgris, Puducherry, India. The fenugreek seeds were made powder and defatted using petroleum ether and then sub-jected to methanolic extraction. About 100 g of fenugreek seed powder (FSP) was mixed with 80 % methanol (1:5) and kept at room temperature for 5 days. After 5 days, it was fil-tered and the residue was re-extracted twice under the same condition to ensure the complete extraction, and the com-bined solvent was evaporated by rotary evaporator to get the residue. The residue was lyophilized and stored at −70 °C and later this was used for in vitro and in vivo studies.

HPTLC fingerprinting and quantification

Chromatography was performed on HPTLC plates pre-coated with 0.2 mm layers of silica gel GF254. The plates were pre-washed with methanol and activated at 55 °C for 30 min. Sample was applied on to the plates as 7-mm bands using a Linomat 5 applicator with 100 µL syringe under nitrogen gas flow. The plate was developed for a distance of 70 mm in a vapor saturated twin-trough chamber with mobile phase. Plates were then dried with a stream of hot air. The developed HPTLC plates were scanned by using a TLC scanner 3 with winCATS software. The slit dimen-sion was set at 5.0 × 0.45 mm (Micro), and 20 mm/s scan-ning speed with 100 µL/stop data resolution was used. The maximum absorbance of trigonelline and diosgenin was measured at 254 and 340 nm (deuterium lamb). Quantifi-cation on trigonelline and diosgenin in FSE is achieved by interpolating the peak area in the respective standard graph.

Experimental animals

Male Sprague–Dawley rats of age 8–10 weeks and with body weight (BW) 150–200 g were supplied by National

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Centre for Laboratory Animal Sciences, National Institute of Nutrition, and Hyderabad, India. The animals were main-tained in the animal house facility of Pondicherry Univer-sity, in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Ani-mals (CPCSEA), Govt. of India. The animals were housed in standard polypropylene cages (three rats/cage) and main-tained under controlled room temperature (22 + 2 °C) and humidity (55 + 5 %) with 12:12 h lights and dark cycle. All rats were allowed for acclimatization for 2 weeks. The present work was carried out with the approval from Insti-tutional Animal Ethical Committee, Pondicherry University (Reg. No. 1159/c/07/CPCSEA-PU/IAEC/11/03).

Induction of diabetes in rats

T2DM was induced in male Sprague–Daweley rats by feeding HFD for first 4 weeks and then administering STZ to overnight fasted rats at a dose of 35 mg/kg BW by a single intraperitonial injection (ip) in 0.1 M citrate buffer (pH 4.5). The animals were again continued on HFD for the next 4 weeks. This would mimic natural incidence of the disease as reported earlier [15]. Animals having fasting blood glucose levels more than 300 mg/dL were considered as diabetic.

Experimental design

The animals were divided into five groups of six rats each (n = 6) and the experimental duration was for 8 weeks. Rats in Group 1 were given NPD and they acted as control group; Group 2 rats were given HFD for first 4 weeks fol-lowed by STZ administration (35 mg/kg BW) and contin-ued on HFD for the next 4 weeks; this group served as the T2DM model. Rats in Groups 3, 4 and 5 were induced with T2DM as in Group 2 and were given oral administration of 300 mg FSE/kg BW, 40 mg of trigonelline/kg BW and 60 mg of diosgenin/kg BW, respectively from the 3rd day after STZ administration till the end of the experimental period.

At the end of the experimental period, the rats were euth-anized by cervical dislocation after an overnight fasting and immediately opened surgically. Blood was collected from heart and stored at 4 °C, serum was separated. Blood glu-cose was estimated by GOD–PAP method (AGGAPE-diag-nostics), and the levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transami-nase (SGPT) and alkaline phophatase (ALP) were deter-mined by using diagnostic kits (AGGAPE-diagnostics). Serum C-Peptide level was estimated by using ELISA kit (DiaSource). Liver glycogen and triglyceride (TG) levels were quantified by Plummer method [16] and GPO–PAP–ESPAS method (AGGAPE-diagnostics), respectively.

Estimation of oxidative stress markers

Oxidative stress marker, lipid peroxidation (LPO) [17]; non-enzymatic antioxidant, reduced glutathione (GSH) [18]; and enzymatic antioxidant, superoxide dismutase (SOD) [19], catalase [20], and glutathione peroxidase [21] were analyzed in liver tissue homogenate (10 %) using standard procedures.

Histopathological examination

Liver was collected and fixed in 10 % neutral buffered formalin (NBF). The tissue samples were processed by routine paraffin embedding technique and 5 µm-thick-sections were prepared and stained by haematoxylin and eosin (H&E) [22]. Detailed histopathological examination of the stained sections and the photomicrographic docu-mentations was carried out using Leica DMR research microscope.

Estimation of ER stress markers by Western blot analysis

Twenty milligram of liver tissue was lysed on ice with 200 µL lysis buffer and centrifuged at 13,000×g at 4 °C for 5 min. The protein concentrations in the supernatants were quantified by Lowry’s method. Electrophoresis was performed on a SDS–PAGE electrophoresis using 100 µg of reduced protein extract per lane. Resolved proteins were transferred to nitrocellulose membranes, blocked with 5 % non-fat milk for 1 h at room temperature, finally probed with appropriately diluted primary antibodies at 4 °C over-night. The nitrocellulose membrane was then washed three times with Tris-buffered saline/0.2 % Tween 20 at room temperature and incubated with appropriate secondary anti-body labeled with horseradish peroxidase (goat anti-mouse or anti-rabbit or anti-rat antibody) for 1 h at room tempera-ture. All resolved proteins bands were detected using TMB/H2O2 substrate and quantified with the help of Imagej software.

Statistical analysis

Statistical analysis was performed by one way ANOVA followed by Tukey’s test using SPSS software version 7.5. The significance was accepted at p ≤ 0.05 and 0.001.

Results

This study presents data on T2DM associated pathologi-cal changes in liver and the potential protective effects of fenugreek seed extract (FSE), trigonelline, and diosgenin in T2DM rats.

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HPTLC analysis of fenugreek seed extract

The HPTLC profile of FSE confirmed the presence of trigonelline and diosgenin with Rf value in the range of 0.14–0.28 for trigonelline (Fig. 1a) and 0.80–0.84 for dios-genin (Fig. 1b). The trigonelline content in FSE was quan-tified as 379.2 ng/mg and that of diosgenin was found to be 195.2 ng/mg FSE.

Glucose and C-peptide levels

The serum levels of glucose and C-peptide are presented in Table 1. T2DM rats exhibited significant increase in glu-cose levels along with significant reduction in C-peptide levels as compared to control animals. But encouraging data was obtained in rats treated with FSE, trigonelline, and diosgenin which showed significant reduction in glu-cose levels and increase in C-peptide levels as compared to T2DM group (Table 1). FSE and diosgenin were found to be more effective in regulating the serum glucose and C-peptide levels than trigonelline.

Serum levels of liver marker enzymes

The serum levels of the liver marker enzymes—GOT, GPT, and ALP were found to be significantly elevated (p ≤ 0.001) in T2DM rats while in rats treated with FSE, trigonelline and diosgenin the enzyme levels were signifi-cantly reduced (Table 1). As compared to trigonelline, dios-genin and FSE were found to be more effective in normal-izing the enzyme levels.

Glycogen and triglyceride levels in Liver

The glycogen and TG levels in liver are shown in Table 2. In T2DM rats, the liver TG levels were significantly elevated (p ≤ 0.001) while the glycogen levels were sig-nificantly reduced (p ≤ 0.001) as compared to control animals. On the other hand, treatment with FSE, trigonel-line and diosgenin resulted in significant reduction in TG levels (p ≤ 0.001) while enhancing the glycogen content (p ≤ 0.001) as compared to T2DM group (Table 2).

Lipid peroxidation (LPO) and antioxidant levels in liver

The liver LPO levels in of T2DM rats showed signifi-cant increase (p ≤ 0.001) as compared to control animals (Fig. 2). Associated with increased LPO levels, T2DM rats also displayed significant reduction (p ≤ 0.001) in the levels of GSH and antioxidant enzymes SOD, cata-lase and GPx in liver (Table 3). But encouraging results were obtained in rats treated with FSE, trigonelline and diosgenin which showed significantly reduced LPO levels

(p ≤ 0.001; Fig. 2). FSE, trigonelline and diosgenin treat-ment also resulted in significant increase in the levels of liver antioxidants that were found to be restored to that of control values (Table 3).

Expression of ER stress marker proteins

The expression levels of proteins associated with ER stress/UPR signaling in liver were analyzed by western blotting technique and are shown in Fig. 3a. T2DM rats exhibited significant increase in the expression levels of ER chaperones—Bip and PDI as well as ER stress asso-ciated proapoptotic markers CHOP, Caspase12 and Cas-pase3 as compared to control rats. Bip and PDI showed an increase of about 2.3- and 3.1-fold increases, respectively, while CHOP, Caspase12 and Caspase3 showed an increase of 2.2-, 2.8-, and 3.4-fold, respectively. Conversely, rats treated with FSE, trigonelline, and diosgenin showed sig-nificantly reduced levels of the ER stress markers PDI, Bip, CHOP, Caspase12, and Caspase3 (Fig. 3a) as compared to T2DM rats. The relative expression of ER stress markers in T2DM and treated groups are shown in Fig. 3b.

Histopathological observations of Liver

The histological analysis of control liver revealed normal hepatic parenchyma (Fig. 4a) while in T2DM rats hydropic degeneration to fatty changes were observed (Fig. 4b). On the other hand, the hepatocytes of rats treated with FSE, trigonelline and diosgenin showed marked improvement as compared to T2DM rats. It was interesting to notice that FSE and diosgenin treated rats showed normal histol-ogy (Fig. 4c, e) and trigonelline treated rats displayed focal mononuclear cell infiltration with mild degeneration of hepatocytes (Fig. 4d). These data from histological analy-sis can be correlated with our observations made on serum liver marker enzymes and liver TGs.

Discussion

T2DM results when pancreatic β cells are unable to secrete sufficient insulin to maintain normoglycemia, typically in the context of increasing peripheral insulin resistance in insulin target organs including muscle, fat, and liver. Under a state of hepatic insulin resistance the ability of insulin to shut down glucose production from the liver is diminished which in turn leads to the manifestation of hyperglycemia [23]. Though there are diverse mechanisms involved in the development of hepatic insulin resistance, increased ER stress and an accumulation of lipids within the liver (hepatic steatosis) have been demonstrated to be impor-tant mechanisms [10, 24, 25]. Nonalcoholic fatty liver

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Fig. 1 HPTLC fingerprinting. a, b The HPTLC chromatogram of FSE along with the standards-trigonelline and diosgenin. Chromato-gram of FSE showed nine peak areas of various phytocompounds. a HPTLC chromatogram of FSE, in which the first peak corresponds to

trigonelline and b HPTLC chromatogram of FSE, in which the last peak area corresponds to diosgenin. AU arbitrary unit, Rf retardation factor

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disease (NAFLD) represents a spectrum of chronic diseases ranging from hepatic steatosis to fibrosis or cirrhosis and is linked to obesity and type 2 diabetes [26, 27]. Hepatic steatosis results from increased fatty acid influx, elevated de novo lipogenesis, and reduced fatty acid oxidation. The associations of ER stress signaling and hepatic steatosis has been proven through genetic modulation of the ER proteins in mice models [28]. The present study evaluated the pro-tective effects of FSE and two of its phytocompounds—trigonelline and diosgenin in pure form against hepatic ste-atosis and ER stress mediated liver damage in T2DM rats.

The elevated levels of blood glucose, reduced serum C-peptide, and liver glycogen observed in this study in T2DM rats indicated development of peripheral insulin resistance as reported earlier [15]. Interestingly, rats treated with FSE and its phytoconstituents-trigonelline and dios-genin showed striking reduction in serum glucose level, marked elevation in serum C-peptide level and a significant enhancement of liver glycogen level. These findings are consistent with earlier reports that the antidiabetic effect of fenugreek seeds and the pure compounds trigonelline and diosgenin might be mediated through their ability to improve insulin sensitivity in T2DM models [14, 29–34].

The elevated serum levels of liver marker enzymes such as GOT, GPT, and ALP in T2DM rats observed in the pre-sent study point to T2DM associated liver cell damage. Also, the significant increase in liver TG levels indicated hepatic steatosis which could have caused lipotoxicity and insulin resistance in liver. On the contrary, groups treated

Table 1 Serum levels of Glucose, C-peptide and liver marker enzymes

Results are expressed as mean ± SD from six rats in each group. Values with common superscript letter do not differ significantly

Groups/parameters Glucose (mg/dL) C-Peptide pmol/mL SGOT U/L SGPT U/L ALP U/L

Control 95.87 ± 5.89a 1.38 ± 0.2a 73.19 ± 4.29a 34.19 ± 2.73a 60.2 ± 2.4a

T2DM 506.94 ± 7.06b 0.39 ± .02b 241.45 ± 3.9b 165.65 ± 7.78b 782.74 ± 35.95b

FSE 217.11 ± 2.36c 1.29 ± .09a 98.75 ± 5.07c 63.3 ± 5.99c 141.5 ± 5.6c

Trigonelline 275.13 ± 24.88d 1.13 ± 0.19a 106.16 ± 16.64c 95.29 ± 9.03d 136.82 ± 18.67c

Diosgenin 218.32 ± 37.9c 1.31 ± 0.18a 98.95 ± 4.0c 74.23 ± 5.65c 112.89 ± 3.36d

Table 2 Glycogen and triglyceride levels in liver

Results are expressed as mean ± SD from six rats in each group. Val-ues with common superscript letter do not differ significantly

Groups/Parameters Glycogen (gm/100 gm)

Triglyceride (mg/100 gm tissue)

Control 2.98 ± 0.26a 216.69 ± 9.29a

T2DM 0.47 ± 0.09b 1,778.22 ± 52.75b

FSE 1.98 ± 0.82c 471.78 ± 15.24c

Trigonelline 2.18 ± 0.16c 504.16 ± 21.33c

Diosgenin 2.26 ± 0.14c 495.02 ± 29.14c

Fig. 2 Liver lipid peroxidation (LPO) levels in T2DM and treated groups. Data represent the mean ± SEM of six independent experi-ments with standard errors represented by vertical bars. *Signifi-cantly different from control group, #significantly different from dia-betic group

Table 3 Antioxidant levels in liver

Superoxide dismutase (SOD) activity is expressed as μmole of pyrogallol oxidized/min/mg protein, Catalase activity as μmole of H2O2 degraded/min/mg protein, glutathione peroxidase (GPx) activity as μmole of GSH oxidized/min/mg protein, reduced glutathione (GSH), mg/100 g tissue and thiobarbutric acid (TBARS), nmole of MDA produced/min/mg protein. Results are expressed as mean ± SD from six rats in each group. Values with common superscript letter do not differ significantly

Groups/Parameters SOD Catalase GPx GSH

Control 27.11 ± 1.23a 12.83 ± 1.38a 17.36 ± 1.17a 25.27 ± 1.36a

T2DM 9.08 ± 1.22b 5.30 ± 0.68b 7.18 ± 1.04b 12.33 ± 2.06b

FSE 27.56 ± 2.56a 13.27 ± 1.21a 17.05 ± 1.12a 23.63 ± 2.85a

Trigonelline 23.86 ± 2.51c 12.65 ± 2.05a 16.24 ± 2.94a 22.59 ± 2.08a

Diosgenin 24.73 ± 3.84a 10.17 ± 2.19a 15.75 ± 2.69a 22.89 ± 4.39a

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Fig. 3 Western blot analysis of ER stress marker proteins in liver. a, b Expression levels of liver ER stress marker proteins in T2DM and treated groups. Data represent the mean ± SEM of three independent

experiments with standard errors represented by vertical bars. *Sig-nificantly different from control group, #significantly different from diabetic group

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with FSE, trigonelline and diosgenin showed improve-ment in the serum levels of liver marker enzymes and liver TGs. These observations along with improved blood glu-cose, serum insulin, and liver glycogen levels designated the protective effect of FSE, trigonelline and diosgenin against development of insulin resistance and NAFLD. The observed protective effects of FSE may be attributed to the synergistic action of various phytocompounds including alkaloids, flavanoids, and saponins. Of the two pure com-pounds used in this study, diosgenin showed better activity in regulating the levels of serum glucose, C-peptide, GOT, and GPT than trigonelline.

Endoplasmic reticulum stress has been proposed as one of the molecular mechanisms linking obesity with insulin resistance and might thus be a common molecular pathway for β cell loss [35]. ER stress has been shown to increase in liver under diabetic conditions [36]. As hepatocytes are known to perform a multitude of metabolic functions including plasma protein synthesis, fatty acid synthesis, and cholesterol metabolism, they face the challenge of maintaining ER homeostasis, and a key interconnectedness

between and hepatic steatosis has been recognized by recent studies [37].

In the present study, ER stress response in liver was denoted by increased protein expression of ER chaperones-Bip and PDI in T2DM rats. Our findings corroborate with the earlier observations made in HFD induced obese mice as well as in genetic obesity (ob/ob mice) mice. Male mice fed with HFD for 16 weeks showed increased expression of ER stress markers and they exhibited insulin resistance and T2DM as compared to mice fed on regular diet [38]. HFD and obesity have been shown to induce ER stress response with subsequent activation of JNK pathway [38, 39]. Also, rats fed on high-fructose diet, saturated fatty acids exhib-ited elevated levels of ER stress markers GRP78, CHOP, and Caspase3 [40]. In the present study, the increased expression of pro-apoptotic markers—CHOP, Caspase12 and Caspase3 observed in liver of T2DM rats designated the activation of terminal UPR signaling and provided convincing evidence for the induction of apoptosis in liver. Caspase12 has been recognized to play an important role in ER stress mediated apoptosis in rodents [41]. The

Fig. 4 Histopathology of liver tissue. a Control group showing nor-mal histology (H&E ×400), b T2DM group showing hydropic degen-eration to fatty changes (H&E ×400). c FSE treated group showing normal histology (H&E ×400). d Trigonelline treated group showing

focal mononuclear cell infiltration with mild degeneration of hepato-cytes (H&E ×400). e Diosgenin treated group showing normal histol-ogy (H&E x400)

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histological analysis of liver confirmed the liver cell dam-age in T2DM rats.

There is an intriguing relationship between ER stress and oxidative stress. Increased UPR signaling in the ER can result in increased oxidative stress and it has been estimated that approximately 25 % of cellular ROS origi-nate from the ER through the process of oxidative protein folding [42]. Acting through PDI, the ERO1 oxido-reduc-tase transfers electron from cysteine sulfhydryl groups on ER-translocated proteins to molecular oxygen, gener-ating H2O2 as an ROS byproduct [43]. CHOP activation also causes increased formation of ROS through enhanced expression of GADD34, dephosphorylation of eIF2a, and resumed translation [44]. CHOP deletion, attenu-ation of translation or antioxidant treatment has been shown to reduce oxidative stress and preserve ER func-tion [42–44]. These findings suggest an intimate relation-ship between ER protein misfolding and ROS production. Data obtained in the present study on marked elevation in LPO and significant reduction in the levels of antiox-idants—SOD, catalase, GPx and GSH in liver of T2DM rats suggested a possible relationship between sustained ER stress and increased oxidative stress. GSH is known to protect the cellular system against the toxic effects of LPO [45]. The decline in the reduced glutathione concen-trations in liver of T2DM rats observed in this study may be due to the utilization of non-protein thiols by oxygen free radicals generated in sustained hyperglycemic condi-tions and ER stress. On the other hand, rats treated with FSE, trigonelline and diosgenin showed significant reduc-tion in the levels of ER stress markers—Bip, PDI, CHOP, Caspase12 and Caspase3 in liver. They also showed sub-stantial improvement in the antioxidant status with sig-nificant increase in SOD, catalase, GPx and GSH levels, and reduction in LPO levels in liver. These observations supported the protective effects of FSE, trigonelline and diosgenin against T2DM associated liver damage. The histological analysis of liver also provided convincing evidence for the protective effects of FSE and diosgenin in liver while trigonelline treated group showed mild degenerative changes.

Conclusion

Data from this study designate that the pathological changes observed in liver of T2DM rats result from accu-mulation of TGs, increased LPO, and decreased antioxidant levels along with an increase in ER stress. In a gripping manner, FSE, trigonelline and diosgenin were found to be individually effective in ameliorating ER stress and oxida-tive stress mediated damage in liver.

Acknowledgments The financial support provided by Univer-sity Grants Commission, India, and the equipment support from DST-FIST and DBT-IPLS is gratefully acknowledged. This work is supported by University Grants Commission, India. [MRP F. No. 36-191/2008(SR)].

Conflict of interest We hereby state that we do not have any con-flict of interest in the present work.

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