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Diosgenin ameliorates gestational diabetes through inhibition of sterol regulatory element-binding protein-1
Biomedicine & Pharmacotherapy 84 (2016) 1460–1465
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Original article
Diosgenin ameliorates gestational diabetes through inhibition of sterol regulatory element-binding protein-1 Shaofang Huaa,* , Yueqin Lib , Lijun Sua , Xiajun Liua a b
Department of Obstetrics, Second Hospital of Tianjin Medical University, Tianjin 300211, China Neonatal Department, Second Hospital of Tianjin Medical University, Tianjin 300211, China
A R T I C L E I N F O
Article history: Received 20 August 2016 Received in revised form 27 September 2016 Accepted 17 October 2016 Keywords: Gestational diabetes Diosgenin Oxidative stress Lipid profile SREBP-1
A B S T R A C T
Gestational diabetes (GD) is a pathological condition, affecting 2–5% of pregnant women. Diosgenin (DSG) possesses a variety of biological activities. The present study was designed to examine the effect of DSG on GD and to investigate the possible mechanism in C57BL/KsJ-Lepdb/+ (db/+) mice. We found that DSG could remarkably ameliorated GD in pregnant db/+ mice, as reflected by the improvement of glucose and insulin intolerance, and the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content. The results showed that DSG could inhibit oxidative stress in pregnant db/+ mice, as evidenced by decrease of thiobarbituric acid reactive substances (TBARS) content, increase of glutathione (GSH) level and superoxide dismutase (SOD) and catalase (CAT) activities. DSG could also attenuate the abnormal changes of lipid profiles, including TC, TG and LDL, in pregnant db/+ mice. The increase of the expression of sterol regulatory element-binding transcription factor-1 (SREBP-1) and its target genes, including fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl coenzyme A carboxylase (ACC), was inhibited by DSG in pregnant db/+ mice. Moreover, overexpression of SREBP-1 by LV-SREBP-1 injection could markedly inhibit the protective effect of DSG against disorder of glucose and lipid metabolism and oxidative stress in GD mice. The data demonstrated that SREBP-1 may be of major target of DSG that mediated its anti-diabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Gestational diabetes (GD) is a pathological condition, affecting 2–5% of pregnant women [1,2]. GD is characterized by glucose intolerance or hyperglycemia resulted from defects in insulin secretion and action [3]. GD could both induce diabetic complications in maternal bodies and result in abnormality in fetal development [4]. Part of GD patients may even develop long-term diabetes after pregnancy [5]. Thus, development of effective treatments for GD is urgent. Trigonellafoenum-graecum (fenugreek), a traditional plant in India and North Africa, is used for cooking and traditional herbal medicine in the treatment of various pathological conditions, including diabetes and hyperlipidemia [6,7]. Diosgenin (DSG) is the main biologically active steroid sapogenin present in fenugreek that possesses a variety of biological activities including anti-
* Corresponding author. E-mail address: [email protected] (S. Hua). http://dx.doi.org/10.1016/j.biopha.2016.10.049 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
diabetic, anti-tumor, anti-oxidative and anti-inflammatory effects [8–12]. For instance, Hirai et al. showed that DSG attenuated inflammatory changes in the interaction between adipocytes and macrophages [13]. Yamada et al. discovered that dietary DSG attenuated subacute intestinal inflammation associated with indomethacin in rats [12]. Kalailingam et al. found that DSG protected against cardiovascular risk, insulin secretion, and beta cells in streptozotocin (STZ)-induced diabetic rats [10]. Moreover, Pari et al. showed that DSG inhibited oxidative stress in aorta of STZ-induced diabetic rats [11]. However, whether DSG exhibited beneficial effects against GD is still not clear. The present study was designed to examine the effect of DSG on GD and to investigate the possible mechanism in C57BL/KsJLepdb/+ (db/+) mice. The db/+ mice, harboring a heterozygous mutation in the leptin receptor gene Lepr [14], represent similar GD symptoms with human patients. The mice exhibit normal glucose and insulin tolerance at non-pregnant state [15,16]. After gravidity, the db/+ females would develop hyperglycemia, insulin resistance and obesity. Moreover, fetal development was also
S. Hua et al. / Biomedicine & Pharmacotherapy 84 (2016) 1460–1465
adversely affected, with increased body weight of offspring at birth [17,18]. The results showed that DSG decreased sterol regulatory element-binding transcription factor-1 (SREBP-1) expression, improved the abnormal changes of lipid profiles and oxidative stress, and ameliorated GD in pregnant db/+ mice. SREBP-1 may be of major target of DSG that mediated its antidiabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. 2. Materials and methods 2.1. Chemicals and materials Diosgenin (Purity 98%) was provided by Santa Cruz Biotechnology (Santa Cruz, CA). b-Actin, SREBP-1 antibodies were obtained from Santa Cruz Biotechnology. All other reagents used in the experiments were of analytical grade and of highest purity. 2.2. Animals and treatment Care and use of the animals in this study were approved by Animal Care and Use Committee of the Second Hospital of Tianjin Medical University (SHTMN-2015-10-11) and abided the guidelines for the care and use of laboratory animals published by the National Institute of Health. All efforts were exhausted to minimize unnecessary suffering of experimental mice. C57BL/ KsJ+/+ (wild type, homozygous) and C57BL/KsJdb/+ (db/+, heterozygous) mice (6–8 week old, 18–22 g) were housed in a room with controlled temperature (22 2 C), humidity (40–60%) and light cycle (12/12 h light/dark) and fed with the chow diet and water. Protocol 1: Female mice were randomly divided into four groups (n = 15 per group): Wild type, ad libitum-fed; GD, db/+ mice (pair-fed), fed the same amount of food with wild-type; low dose of DSG (L-DSG), db/+ mice (pair-fed) were administered orally with DSG (10 mg/kg b.w.) using an intra-gastric tube; high dose of DSG (H-DSG), db/+ mice (pair-fed) were administered orally with DSG (20 mg/kg b.w.). DSG was dissolved in DMSO and diluted in saline solution. The doses of DSG were selected based on previous studies [19]. Mice in Wild type and GD groups were orally gavaged with vehicle (0.1% DMSO in saline) only daily throughout the entire span of the study. The model of GD mice was established as previously described [20]. In brief, female mice were individually mated with males of the same genotype at 10–12 weeks of age. The presence of a copulatory plugon the next morning was observed to confirm the mating, and the day was designated gestation day 0. More than 15 females were used to ensure at least 15 pregnant females each experimental group. The experimental period is 20 days. Protocol 2: Female mice were randomly divided into three groups (n = 15 per group): GD group; DSG group, db/+ mice were administered orally with DSG (20 mg/kg b.w.) using an intragastric tube; DSG + LV-SREBP-1 group, db/+ mice were pre-injected with SREBP-1 lentivirus through caudal vein. Mice in GD and DSG groups were injected with control lentivirus. The procedures were carried out as protocol 1.
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2.4. Intraperitoneal glucose/insulin tolerance test Intraperitoneal glucose/insulin tolerance test (IPGTT/IPITT) was conducted on gestation day 10. For IPGTT, mice were fasted for 6 h and injected intraperitoneally with 2 g/kg glucose. Blood samples were collected from the tail using capillary tubes at 0, 30, 60 and 120 min after glucose administration. For IPITT, mice were fasted 6 h and injected intraperitoneally with insulin at 0.75 U/kg body weight. Blood samples were collected from the tail using capillary tubes at 0, 30, 60 and 120 min after insulin administration. 2.5. Measurement of hepatic glycogen content Liver tissues were homogenated and glycogen content was measured by a commercial assay kit (BioVision) according to the manufacture’s protocols. Hepatic glycogen content was expressed as mg per g protein. 2.6. Evaluation of oxidative stress Liver tissues were homogenated and oxidative stress was examined by the determination of TBARS and GSH level and SOD and CAT activities using commercial kits (Nanjing Jiancheng, China) according to the manufacture’s instructions. 2.7. RNA isolation and real-time PCR Total RNA was isolated from liver samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 500 ng RNA was used for reverse transcription. The real-time PCR assay was performed with Quantitect SYBR Green PCR kit (Qiagen, USA). The PCR protocol was performed according to the manufacture’s instructions as described: initial denaturation at 95 C for 10 min followed by 30 cycles at 95 C for 1 min, annealing at 53 C for 1 min, extension at 72 C for 1 min, and final extension at 72 C for 5 min. Real-time PCR assays was performed using a real-time PCR system (Bio-Rad, USA). The expression of target gene transcripts was related to b-actin. Results were shown as folds of control. 2.8. Western blot Total protein was extracted from liver tissues and the loading sample was separated by 10% SDS-PAGE and then were transferred to an Immobilon-P membrane (Millipore). After blocking, the membrane was incubated with primary antibodies and then with a secondary antibody. 2.9. Statistical analysis The data were presented as mean SEM. Data were assessed by ANOVA with a post hoc Tukey-Kramer or Dunnet’s multiple comparison test. Differences were considered significant at P < 0.05. 3. Results
2.3. Measurement of serum glucose, insulin and lipids
3.1. DSG ameliorated GD in db/+ mice
Serum glucose, insulin and lipid levels were measured at gestation day 20. Fasting blood samples were obtained via tail venipuncture, and serum glucose level was determined by glucometer (LifescanSurestep). Plasma insulin levels were quantified by a Mouse Insulin ELISA kit (Thermo Scientific). Plasma and liver TC, TG, LDL and HDL levels were determined using commercial kits (Cayman Chemical, USA).
In the current study, we first examined the effect of DSG on GD in pregnant db/+ mice. On gestation day 10, glucose and insulin tolerance was examined by intraperitoneally glucose/insulin tolerance test (IPGTT/IPITT). In Fig. 1A, we showed that the blood glucose level in pregnant db/+ mice was significantly higher than that of wild type mice in response to glucose injection. DSG administration could notably inhibited the increase of blood
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Fig. 1. Effect of DSG on GD in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 10, glucose (A) and insulin (B) tolerance were evaluated. On gestation day 20, serum and livers were collected. Fasting blood glucose (C) and insulin (D) level was measured and hepatic glycogen content (E) was determined. *p<0.05, vs control. #p<0.05, vs GD.
glucose level in IPGTT test (Fig. 1A). Moreover, in response to insulin injection, the blood glucose levels in pregnant db/+ mice at different time points were significantly higher than that of wild type mice (Fig. 1B). The administration of DSG notably inhibited the increase of blood glucose levels in IPITT test (Fig. 1B). The results indicated that DSG could ameliorate glucose and insulin intolerance in GD mice.
On gestation day 20, fasting blood glucose level and insulin level were determined. The results showed that the levels of fasting blood glucose and insulin were significantly higher than that of wild type mice (Fig. 1C and D). DSG notably inhibited the increase of fasting blood glucose and insulin levels in GD mice (Fig. 1C and D). Moreover, hepatic glycogen content was significantly decreased in pregnant db/+ mice, compared with that of wild type mice (Fig. 1E). The administration of DSG
Fig. 2. Effect of DSG on oxidative stress in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 20, livers were collected and homogenated. Levels of TBARS (A) and GSH (B) and SOD (C) and CAT (D) activities were determined using commercial kits. *p<0.05, vs control. #p<0.05, vs GD.
S. Hua et al. / Biomedicine & Pharmacotherapy 84 (2016) 1460–1465 Table 1 Effect of Diosgenin on lipid profiles in GD mice. Parameters
Control
GD
L-DSG
H-DSG
Plasma TC(mg/ml) Liver TC(mg/g) Plasma TG(mg/ml) Liver TG(mg/g) Plasma LDL(mg/ml) Plasma HDL(mg/ml)
0.84 0.07 9.95 0.23 0.56 0.09 8,97 0.62 0.19 0.04 0.31 0.03
1.68 0.18* 16.82 1.03* 1.78 0.12* 15.68 1.13* 0.32 0.03* 0.32 0.07
1.34 0.09# 12.32 0.86# 1.21 0.11# 12.79 0.36# 0.26 0.03# 0.33 0.06
1.02 0.11# 10.69 0.65# 0.86 0.08# 11.02 0.63# 0.21 0.04# 0.32 0.08
* #
P < 0.05, vs control. P < 0.05, vs GD.
ameliorated the decrease of hepatic glycogen content. These data suggested that DSG could ameliorate GD in pregnant db/+ mice. 3.2. DSG attenuated oxidative stress in db/+ mice Oxidative stress is closely associated with the pathogenesis of GD. In the study, we examined the effect of DSG on oxidative stress in pregnant db/+ mice. The results showed that thiobarbituric acid reactive substances (TBARS) level in pregnant db/+ mice was significantly higher than that of wild type mice (Fig. 2A). The treatment of DSG markedly inhibited the increase of TBARS level (Fig. 2A). Glutathione (GSH) content in pregnant db/+ mice was notably lower than that of wild type mice (Fig. 2B). DSG and markedly inhibited the decrease of GSH in pregnant db/+ mice (Fig. 2B). Moreover, superoxide dismutase (SOD) and catalase (CAT) activities were markedly decreased in pregnant db/+ mice, compared with that of wild type mice (Fig. 2C). DSG markedly inhibited the decrease of SOD and CAT activities in pregnant db/+ mice (Fig. 2C). The results indicated that DSG inhibited oxidative stress under the GD pathological condition in pregnant db/+ mice. 3.3. DSG attenuated abnormal changes of lipids in db/+ mice Disorder of lipid metabolism is closely associated with oxidative stress and a major contributor to diabetic condition. In the next step, we examined the changes of lipid profiles. In Table 1, we showed that in pregnant db/+ mice, the content of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) were significantly higher than that of wild type mice. The
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administration of DSG prevented that increase of TC, TG and LDL in pregnant db/+ mice (Table 1). The results indicated that DSG could attenuated the abnormal changes of lipid profiles under GD pathological condition.
3.4. DSG inhibited SREBP-1 and its target gene expression in db/+ mice Sterol regulatory element-binding transcription factor-1 (SREBP-1) is a crucial regulator of lipid synthesis and accumulation. To examine the possible mechanism of the protective effect of DSG against GD, the expression of SREBP-1 was determined. As shown in Fig. 3A, mRNA expression of SREBP-1 was significantly increased in pregnant db/+ mice, compared with that of wild type mice. DSG could notably inhibited the increase of SREBP-1 mRNA expression (Fig. 3A). In pregnant db/+ mice, protein expression of precursor and mature SREBP-1 was significantly increased, compared with that of wild type mice (Fig. 3B). The increase of protein expression of precursor and mature SREBP-1 in pregnant db/+ mice was notably inhibited DSG which was (Fig. 3B). Moreover, the mRNA expression of SREBP-1 target genes was examined, including fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl coenzyme A carboxylase (ACC). mRNA expression of FAS, SCD-1 and ACC was significantly increased in pregnant db/+ mice compared with that of wild type mice (Fig. 3C–E). DSG notably inhibited the increase of FAS, SCD-1 and ACC mRNA expression in pregnant db/+ mice. 3.5. Downregulation of SREBP-1 was involved in the effect of DSG on GD in db/+ mice To examine the role of downregulation of SREBP-1 in the effect of DSG on GD, the mice were pre-injected with SREBP-1 lentivirus to enhance the expression of SREBP-1. The results showed that the injection of LV-SREBP-1 significantly inhibited DSG-induced decrease of TC, TG, and LDL in pregnant db/+ mice (Table 2). Moreover, DSG-induced decrease of blood glucose level at different time points in IPGTT and IPITT test was markedly inhibited by LVSREBP-1 (Fig. 4A and B). LV-SREBP-1 also suppressed the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content induced by DSG in pregnant db/+ mice
Fig. 3. Effect of DSG on the expression of SREBP-1 and its target genes in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 20, livers were collected and total proteins were extracted. mRNA expression of SREBP-1 (A) and protein expression of precursor and mature SREBP-1 (B) were measured. mRNA expression of FAS (C), SCD-1 (D) and ACC (E) was examined. *p<0.05, vs control. #p<0.05, vs GD.
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Table 2 Role of decrease of SREBP-1 in the effect of Diosgenin on lipid profiles in GD mice. Parameters
GD
DSG
DSG + LV-SREBP-1
Plasma TC(mg/ml) Liver TC(mg/g) Plasma TG(mg/ml) Liver TG(mg/g) Plasma LDL(mg/ml) Plasma HDL(mg/ml)
1.72 0.15 16.67 1.15 1.88 0.23 15.54 1.28 0.35 0.05 0.31 0.05
1.08 0.16* 9.52 0.8* 0.85 0.18* 10.43 0.78* 0.22 0.03* 0.30 0.07
1.48 0.11# 13.49 1.15# 1.37 0.21# 13.27 0.45# 0.27 0.02# 0.32 0.07
* #
P < 0.05, vs GD. P < 0.05, vs DSG.
(Fig. 4C–E). Pre-injection of LV-SREBP-1 also suppressed DSGinduced decrease of TBARS content in pregnant db/+ mice (Fig. 4F). The results indicated that downregulation of SREBP-1 was involved in DSG-induced amelioration of abnormal changes of lipid profiles and oxidative stress, and the final improvement of GD in pregnant db/+ mice. 4. Discussion In the current study, we examined the effect of DSG on GD and explored the possible mechanisms using pregnant db/+ mice. We found that DSG could remarkably ameliorated GD in pregnant db/+ mice, as reflected by the improvement of glucose and insulin intolerance, and the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content. There are literatures reporting that DSG exhibited anti-diabetic activities [6,21–25]. For instance, Naidu et al. reported that DSG reorganized hyperglycaemia and distorted tissue lipid profile in high-fat dietstreptozotocin-induced diabetic rats [22]. Roghani-Dehkordi et al. [21] found that DSG mitigated diabetes-induced vascular dysfunction of the rat aorta. Based on the previous discoveries, our results demonstrated that DSG functioned as a potential anti-diabetic
agent both under general diabetic condition but also under gestation diabetic condition. Previous studies have shown that DSG exhibited antioxidant activities which may be involved in the anti-diabetic effects [11]. In the current study, we also assessed the effect of DSG on oxidative stress under GD condition. We found that DSG could inhibit oxidative stress in pregnant db/+ mice, as evidenced by decrease of TBARS content, increase of GSH level and SOD and CAT activities. The results indicated that the antioxidant activities of DSG may be involved in the anti-diabetic effect of DSG in GD mice. It has been also reported that DSG exhibited lipid-lowering effects under different pathological conditions [23,26]. Abnormal changes of lipid profiles are closely associated with oxidative stress [27,28] and the development of GD [29,30]. In our study, we also examined the effect of DSG on lipid profiles. We discovered that DSG could attenuated the abnormal changes of lipid profiles in pregnant db/+ mice, as illustrated by decrease of TC, TG and LDL. The results indicated that attenuation of abnormal changes of lipid profiles may participate in the anti-diabetic effects of DSG in GD mice. To further explore the mechanism of DSG-induced improvement of lipid profiles in GD mice, we examined the changes of SREBP-1 and its target gene expression. SREBP-1 is a well-known nuclear transcription factors involved in the biosynthesis of cholesterol, fatty acid, and triglyceride in mammals [31] through regulating its target genes involved in fatty acid biosynthesis, including FAS, SDC-1, ACC [32]. Abnormal expression of SREBP-1 is associated with the pathogenesis of diabetes [33]. It has been reported that Yamogenin, a diastereomer of diosgenin in fenugreek, inhibited lipid accumulation through the suppression of SREBP-1c in hepatocytes [34]. In the study, we found that DSG significantly decreased SREBP-1 and FAS, SDC-1, and ACC expression in pregnant db/+ mice. Moreover, overexpression of SREBP-1 by LV-SREBP-1 injection could markedly inhibit the protective
Fig. 4. Role of downregulation of SREBP-1 in the protective effect of DSG against GD. Pregnant db/+ mice were pre-injected with LV-SREBP-1 and administrated with DSG. On gestation day 10, glucose (A) and insulin (B) tolerance were evaluated. On gestation day 20, serum and livers were collected. Fasting blood glucose (C) and insulin (D) level was measured and hepatic glycogen content (E) was determined. Levels of TBARS were determined using commercial kits (F). *p<0.05, vs GD. #p<0.05, vs DSG.
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effect of DSG against disorder of glucose and lipid metabolism and oxidative stress in GD mice. The data demonstrated that downregulation of SREBP-1 was involved in the anti-diabetic effect of DSG in GD mice. In conclusion, we found that DSG decreased SREBP-1 expression, improved the abnormal changes of lipid profiles and oxidative stress, and ameliorated GD in pregnant db/+ mice. SREBP-1 may be of major target of DSG that mediated its anti-diabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. References [1] D.R. Coustan, Gestational diabetes mellitus, Clin. Chem. 59 (9) (2013) 1310– 1321. [2] A.B. Gilmartin, S.H. Ural, J.T. Repke, Gestational diabetes mellitus, Rev. Obstet. Gynecol. 1 (3) (2008) 129–134. [3] A.D. Association, Gestational diabetes mellitus, Diabetes Care 27 (Suppl. 1) (2004) S88–90. [4] J. Gardosi, A. Francis, A customized standard to assess fetal growth in a US population, Am. J. Obstet. Gynecol. 201 (1) (2009) 25.e1–25.e7. [5] G.E. Tutino, W.H. Tam, X. Yang, J.C. Chan, T.T. Lao, R.C. Ma, Diabetes and pregnancy: perspectives from Asia, Diabetic Med.: J. Br. Diabetic Assoc. 31 (3) (2014) 302–318. [6] S. Fuller, J.M. Stephens, Diosgenin, 4-hydroxyisoleucine and fiber from fenugreek: mechanisms of actions and potential effects on metabolic syndrome, Adv. Nutr. (Bethesda, Md.) 6 (2) (2015) 189–197. [7] F. Gao, X.Z. Shen, F. Jiang, Y. Wu, C. Han, DNA-guided genome editing using the Natronobacterium gregoryi argonaute, Nat. Biotechnol. 34 (7) (2016) 768–773. [8] E. Wang, J. Wylie-Rosett, Review of selected Chinese herbal medicines in the treatment of type 2 diabetes, Diabetes Educ. 34 (4) (2008) 645–654. [9] S. Das, K.K. Dey, G. Dey, I. Pal, A. Majumder, S. MaitiChoudhury, S.C. kundu, M. Mandal, Antineoplastic and apoptotic potential of traditional medicines thymoquinone and diosgenin in squamous cell carcinoma, PLoS One 7 (10) (2012) e46641. [10] P. Kalailingam, B. Kannaian, E. Tamilmani, R. Kaliaperumal, Efficacy of natural diosgenin on cardiovascular risk, insulin secretion, and beta cells in streptozotocin (STZ)-induced diabetic rats, Phytomed.: Int. J. Phytother. Phytopharm. 21 (10) (2014) 1154–1161. [11] L. Pari, P. Monisha, A. Mohamed Jalaludeen, Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats, Eur. J. Pharmacol. 691 (1–3) (2012) 143–150. [12] T. Yamada, M. Hoshino, T. Hayakawa, H. Ohhara, H. Yamada, T. Nakazawa, T. Inagaki, M. Iida, T. Ogasawara, A. Uchida, C. Hasegawa, G. Murasaki, M. Miyaji, A. Hirata, T. Takeuchi, Dietary diosgenin attenuates subacute intestinal inflammation associated with indomethacin in rats, Am. J. Physiol. 273 (2 Pt. 1) (1997) G355–G364. [13] S. Hirai, T. Uemura, N. Mizoguchi, J.Y. Lee, K. Taketani, Y. Nakano, S. Hoshino, N. Tsuge, T. Narukami, R. Yu, N. Takahashi, T. Kawada, Diosgenin attenuates inflammatory changes in the interaction between adipocytes and macrophages, Mol. Nutr. Food Res. 54 (6) (2010) 797–804. [14] R.C. Kaufmann, K.S. Amankwah, G. Dunaway, L. Maroun, J. Arbuthnot, J.W. Roddick Jr., An animal model of gestational diabetes, Am. J. Obstet. Gynecol. 141 (5) (1981) 479–482. [15] T. Ishizuka, P. Klepcyk, S. Liu, L. Panko, S. Liu, E.M. Gibbs, J.E. Friedman, Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/+) mice, Diabetes 48 (5) (1999) 1061–1069. [16] S. Lambin, R. van Bree, S. Caluwaerts, L. Vercruysse, I. Vergote, J. Verhaeghe, Adipose tissue in offspring of Lepr(db/+) mice: early-life environment vs. genotype, Am. J. Physiol. Endocrinol. Metab. 292 (1) (2007) E262–E271.
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Original article
Diosgenin ameliorates gestational diabetes through inhibition of sterol regulatory element-binding protein-1 Shaofang Huaa,* , Yueqin Lib , Lijun Sua , Xiajun Liua a b
Department of Obstetrics, Second Hospital of Tianjin Medical University, Tianjin 300211, China Neonatal Department, Second Hospital of Tianjin Medical University, Tianjin 300211, China
A R T I C L E I N F O
Article history: Received 20 August 2016 Received in revised form 27 September 2016 Accepted 17 October 2016 Keywords: Gestational diabetes Diosgenin Oxidative stress Lipid profile SREBP-1
A B S T R A C T
Gestational diabetes (GD) is a pathological condition, affecting 2–5% of pregnant women. Diosgenin (DSG) possesses a variety of biological activities. The present study was designed to examine the effect of DSG on GD and to investigate the possible mechanism in C57BL/KsJ-Lepdb/+ (db/+) mice. We found that DSG could remarkably ameliorated GD in pregnant db/+ mice, as reflected by the improvement of glucose and insulin intolerance, and the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content. The results showed that DSG could inhibit oxidative stress in pregnant db/+ mice, as evidenced by decrease of thiobarbituric acid reactive substances (TBARS) content, increase of glutathione (GSH) level and superoxide dismutase (SOD) and catalase (CAT) activities. DSG could also attenuate the abnormal changes of lipid profiles, including TC, TG and LDL, in pregnant db/+ mice. The increase of the expression of sterol regulatory element-binding transcription factor-1 (SREBP-1) and its target genes, including fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl coenzyme A carboxylase (ACC), was inhibited by DSG in pregnant db/+ mice. Moreover, overexpression of SREBP-1 by LV-SREBP-1 injection could markedly inhibit the protective effect of DSG against disorder of glucose and lipid metabolism and oxidative stress in GD mice. The data demonstrated that SREBP-1 may be of major target of DSG that mediated its anti-diabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Gestational diabetes (GD) is a pathological condition, affecting 2–5% of pregnant women [1,2]. GD is characterized by glucose intolerance or hyperglycemia resulted from defects in insulin secretion and action [3]. GD could both induce diabetic complications in maternal bodies and result in abnormality in fetal development [4]. Part of GD patients may even develop long-term diabetes after pregnancy [5]. Thus, development of effective treatments for GD is urgent. Trigonellafoenum-graecum (fenugreek), a traditional plant in India and North Africa, is used for cooking and traditional herbal medicine in the treatment of various pathological conditions, including diabetes and hyperlipidemia [6,7]. Diosgenin (DSG) is the main biologically active steroid sapogenin present in fenugreek that possesses a variety of biological activities including anti-
* Corresponding author. E-mail address: [email protected] (S. Hua). http://dx.doi.org/10.1016/j.biopha.2016.10.049 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
diabetic, anti-tumor, anti-oxidative and anti-inflammatory effects [8–12]. For instance, Hirai et al. showed that DSG attenuated inflammatory changes in the interaction between adipocytes and macrophages [13]. Yamada et al. discovered that dietary DSG attenuated subacute intestinal inflammation associated with indomethacin in rats [12]. Kalailingam et al. found that DSG protected against cardiovascular risk, insulin secretion, and beta cells in streptozotocin (STZ)-induced diabetic rats [10]. Moreover, Pari et al. showed that DSG inhibited oxidative stress in aorta of STZ-induced diabetic rats [11]. However, whether DSG exhibited beneficial effects against GD is still not clear. The present study was designed to examine the effect of DSG on GD and to investigate the possible mechanism in C57BL/KsJLepdb/+ (db/+) mice. The db/+ mice, harboring a heterozygous mutation in the leptin receptor gene Lepr [14], represent similar GD symptoms with human patients. The mice exhibit normal glucose and insulin tolerance at non-pregnant state [15,16]. After gravidity, the db/+ females would develop hyperglycemia, insulin resistance and obesity. Moreover, fetal development was also
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adversely affected, with increased body weight of offspring at birth [17,18]. The results showed that DSG decreased sterol regulatory element-binding transcription factor-1 (SREBP-1) expression, improved the abnormal changes of lipid profiles and oxidative stress, and ameliorated GD in pregnant db/+ mice. SREBP-1 may be of major target of DSG that mediated its antidiabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. 2. Materials and methods 2.1. Chemicals and materials Diosgenin (Purity 98%) was provided by Santa Cruz Biotechnology (Santa Cruz, CA). b-Actin, SREBP-1 antibodies were obtained from Santa Cruz Biotechnology. All other reagents used in the experiments were of analytical grade and of highest purity. 2.2. Animals and treatment Care and use of the animals in this study were approved by Animal Care and Use Committee of the Second Hospital of Tianjin Medical University (SHTMN-2015-10-11) and abided the guidelines for the care and use of laboratory animals published by the National Institute of Health. All efforts were exhausted to minimize unnecessary suffering of experimental mice. C57BL/ KsJ+/+ (wild type, homozygous) and C57BL/KsJdb/+ (db/+, heterozygous) mice (6–8 week old, 18–22 g) were housed in a room with controlled temperature (22 2 C), humidity (40–60%) and light cycle (12/12 h light/dark) and fed with the chow diet and water. Protocol 1: Female mice were randomly divided into four groups (n = 15 per group): Wild type, ad libitum-fed; GD, db/+ mice (pair-fed), fed the same amount of food with wild-type; low dose of DSG (L-DSG), db/+ mice (pair-fed) were administered orally with DSG (10 mg/kg b.w.) using an intra-gastric tube; high dose of DSG (H-DSG), db/+ mice (pair-fed) were administered orally with DSG (20 mg/kg b.w.). DSG was dissolved in DMSO and diluted in saline solution. The doses of DSG were selected based on previous studies [19]. Mice in Wild type and GD groups were orally gavaged with vehicle (0.1% DMSO in saline) only daily throughout the entire span of the study. The model of GD mice was established as previously described [20]. In brief, female mice were individually mated with males of the same genotype at 10–12 weeks of age. The presence of a copulatory plugon the next morning was observed to confirm the mating, and the day was designated gestation day 0. More than 15 females were used to ensure at least 15 pregnant females each experimental group. The experimental period is 20 days. Protocol 2: Female mice were randomly divided into three groups (n = 15 per group): GD group; DSG group, db/+ mice were administered orally with DSG (20 mg/kg b.w.) using an intragastric tube; DSG + LV-SREBP-1 group, db/+ mice were pre-injected with SREBP-1 lentivirus through caudal vein. Mice in GD and DSG groups were injected with control lentivirus. The procedures were carried out as protocol 1.
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2.4. Intraperitoneal glucose/insulin tolerance test Intraperitoneal glucose/insulin tolerance test (IPGTT/IPITT) was conducted on gestation day 10. For IPGTT, mice were fasted for 6 h and injected intraperitoneally with 2 g/kg glucose. Blood samples were collected from the tail using capillary tubes at 0, 30, 60 and 120 min after glucose administration. For IPITT, mice were fasted 6 h and injected intraperitoneally with insulin at 0.75 U/kg body weight. Blood samples were collected from the tail using capillary tubes at 0, 30, 60 and 120 min after insulin administration. 2.5. Measurement of hepatic glycogen content Liver tissues were homogenated and glycogen content was measured by a commercial assay kit (BioVision) according to the manufacture’s protocols. Hepatic glycogen content was expressed as mg per g protein. 2.6. Evaluation of oxidative stress Liver tissues were homogenated and oxidative stress was examined by the determination of TBARS and GSH level and SOD and CAT activities using commercial kits (Nanjing Jiancheng, China) according to the manufacture’s instructions. 2.7. RNA isolation and real-time PCR Total RNA was isolated from liver samples using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. 500 ng RNA was used for reverse transcription. The real-time PCR assay was performed with Quantitect SYBR Green PCR kit (Qiagen, USA). The PCR protocol was performed according to the manufacture’s instructions as described: initial denaturation at 95 C for 10 min followed by 30 cycles at 95 C for 1 min, annealing at 53 C for 1 min, extension at 72 C for 1 min, and final extension at 72 C for 5 min. Real-time PCR assays was performed using a real-time PCR system (Bio-Rad, USA). The expression of target gene transcripts was related to b-actin. Results were shown as folds of control. 2.8. Western blot Total protein was extracted from liver tissues and the loading sample was separated by 10% SDS-PAGE and then were transferred to an Immobilon-P membrane (Millipore). After blocking, the membrane was incubated with primary antibodies and then with a secondary antibody. 2.9. Statistical analysis The data were presented as mean SEM. Data were assessed by ANOVA with a post hoc Tukey-Kramer or Dunnet’s multiple comparison test. Differences were considered significant at P < 0.05. 3. Results
2.3. Measurement of serum glucose, insulin and lipids
3.1. DSG ameliorated GD in db/+ mice
Serum glucose, insulin and lipid levels were measured at gestation day 20. Fasting blood samples were obtained via tail venipuncture, and serum glucose level was determined by glucometer (LifescanSurestep). Plasma insulin levels were quantified by a Mouse Insulin ELISA kit (Thermo Scientific). Plasma and liver TC, TG, LDL and HDL levels were determined using commercial kits (Cayman Chemical, USA).
In the current study, we first examined the effect of DSG on GD in pregnant db/+ mice. On gestation day 10, glucose and insulin tolerance was examined by intraperitoneally glucose/insulin tolerance test (IPGTT/IPITT). In Fig. 1A, we showed that the blood glucose level in pregnant db/+ mice was significantly higher than that of wild type mice in response to glucose injection. DSG administration could notably inhibited the increase of blood
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Fig. 1. Effect of DSG on GD in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 10, glucose (A) and insulin (B) tolerance were evaluated. On gestation day 20, serum and livers were collected. Fasting blood glucose (C) and insulin (D) level was measured and hepatic glycogen content (E) was determined. *p<0.05, vs control. #p<0.05, vs GD.
glucose level in IPGTT test (Fig. 1A). Moreover, in response to insulin injection, the blood glucose levels in pregnant db/+ mice at different time points were significantly higher than that of wild type mice (Fig. 1B). The administration of DSG notably inhibited the increase of blood glucose levels in IPITT test (Fig. 1B). The results indicated that DSG could ameliorate glucose and insulin intolerance in GD mice.
On gestation day 20, fasting blood glucose level and insulin level were determined. The results showed that the levels of fasting blood glucose and insulin were significantly higher than that of wild type mice (Fig. 1C and D). DSG notably inhibited the increase of fasting blood glucose and insulin levels in GD mice (Fig. 1C and D). Moreover, hepatic glycogen content was significantly decreased in pregnant db/+ mice, compared with that of wild type mice (Fig. 1E). The administration of DSG
Fig. 2. Effect of DSG on oxidative stress in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 20, livers were collected and homogenated. Levels of TBARS (A) and GSH (B) and SOD (C) and CAT (D) activities were determined using commercial kits. *p<0.05, vs control. #p<0.05, vs GD.
S. Hua et al. / Biomedicine & Pharmacotherapy 84 (2016) 1460–1465 Table 1 Effect of Diosgenin on lipid profiles in GD mice. Parameters
Control
GD
L-DSG
H-DSG
Plasma TC(mg/ml) Liver TC(mg/g) Plasma TG(mg/ml) Liver TG(mg/g) Plasma LDL(mg/ml) Plasma HDL(mg/ml)
0.84 0.07 9.95 0.23 0.56 0.09 8,97 0.62 0.19 0.04 0.31 0.03
1.68 0.18* 16.82 1.03* 1.78 0.12* 15.68 1.13* 0.32 0.03* 0.32 0.07
1.34 0.09# 12.32 0.86# 1.21 0.11# 12.79 0.36# 0.26 0.03# 0.33 0.06
1.02 0.11# 10.69 0.65# 0.86 0.08# 11.02 0.63# 0.21 0.04# 0.32 0.08
* #
P < 0.05, vs control. P < 0.05, vs GD.
ameliorated the decrease of hepatic glycogen content. These data suggested that DSG could ameliorate GD in pregnant db/+ mice. 3.2. DSG attenuated oxidative stress in db/+ mice Oxidative stress is closely associated with the pathogenesis of GD. In the study, we examined the effect of DSG on oxidative stress in pregnant db/+ mice. The results showed that thiobarbituric acid reactive substances (TBARS) level in pregnant db/+ mice was significantly higher than that of wild type mice (Fig. 2A). The treatment of DSG markedly inhibited the increase of TBARS level (Fig. 2A). Glutathione (GSH) content in pregnant db/+ mice was notably lower than that of wild type mice (Fig. 2B). DSG and markedly inhibited the decrease of GSH in pregnant db/+ mice (Fig. 2B). Moreover, superoxide dismutase (SOD) and catalase (CAT) activities were markedly decreased in pregnant db/+ mice, compared with that of wild type mice (Fig. 2C). DSG markedly inhibited the decrease of SOD and CAT activities in pregnant db/+ mice (Fig. 2C). The results indicated that DSG inhibited oxidative stress under the GD pathological condition in pregnant db/+ mice. 3.3. DSG attenuated abnormal changes of lipids in db/+ mice Disorder of lipid metabolism is closely associated with oxidative stress and a major contributor to diabetic condition. In the next step, we examined the changes of lipid profiles. In Table 1, we showed that in pregnant db/+ mice, the content of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) were significantly higher than that of wild type mice. The
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administration of DSG prevented that increase of TC, TG and LDL in pregnant db/+ mice (Table 1). The results indicated that DSG could attenuated the abnormal changes of lipid profiles under GD pathological condition.
3.4. DSG inhibited SREBP-1 and its target gene expression in db/+ mice Sterol regulatory element-binding transcription factor-1 (SREBP-1) is a crucial regulator of lipid synthesis and accumulation. To examine the possible mechanism of the protective effect of DSG against GD, the expression of SREBP-1 was determined. As shown in Fig. 3A, mRNA expression of SREBP-1 was significantly increased in pregnant db/+ mice, compared with that of wild type mice. DSG could notably inhibited the increase of SREBP-1 mRNA expression (Fig. 3A). In pregnant db/+ mice, protein expression of precursor and mature SREBP-1 was significantly increased, compared with that of wild type mice (Fig. 3B). The increase of protein expression of precursor and mature SREBP-1 in pregnant db/+ mice was notably inhibited DSG which was (Fig. 3B). Moreover, the mRNA expression of SREBP-1 target genes was examined, including fatty acid synthase (FAS), stearoyl-CoA desaturase-1 (SCD-1), and acetyl coenzyme A carboxylase (ACC). mRNA expression of FAS, SCD-1 and ACC was significantly increased in pregnant db/+ mice compared with that of wild type mice (Fig. 3C–E). DSG notably inhibited the increase of FAS, SCD-1 and ACC mRNA expression in pregnant db/+ mice. 3.5. Downregulation of SREBP-1 was involved in the effect of DSG on GD in db/+ mice To examine the role of downregulation of SREBP-1 in the effect of DSG on GD, the mice were pre-injected with SREBP-1 lentivirus to enhance the expression of SREBP-1. The results showed that the injection of LV-SREBP-1 significantly inhibited DSG-induced decrease of TC, TG, and LDL in pregnant db/+ mice (Table 2). Moreover, DSG-induced decrease of blood glucose level at different time points in IPGTT and IPITT test was markedly inhibited by LVSREBP-1 (Fig. 4A and B). LV-SREBP-1 also suppressed the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content induced by DSG in pregnant db/+ mice
Fig. 3. Effect of DSG on the expression of SREBP-1 and its target genes in db/+ mice. Pregnant db/+ mice were administrated with DSG. On gestation day 20, livers were collected and total proteins were extracted. mRNA expression of SREBP-1 (A) and protein expression of precursor and mature SREBP-1 (B) were measured. mRNA expression of FAS (C), SCD-1 (D) and ACC (E) was examined. *p<0.05, vs control. #p<0.05, vs GD.
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Table 2 Role of decrease of SREBP-1 in the effect of Diosgenin on lipid profiles in GD mice. Parameters
GD
DSG
DSG + LV-SREBP-1
Plasma TC(mg/ml) Liver TC(mg/g) Plasma TG(mg/ml) Liver TG(mg/g) Plasma LDL(mg/ml) Plasma HDL(mg/ml)
1.72 0.15 16.67 1.15 1.88 0.23 15.54 1.28 0.35 0.05 0.31 0.05
1.08 0.16* 9.52 0.8* 0.85 0.18* 10.43 0.78* 0.22 0.03* 0.30 0.07
1.48 0.11# 13.49 1.15# 1.37 0.21# 13.27 0.45# 0.27 0.02# 0.32 0.07
* #
P < 0.05, vs GD. P < 0.05, vs DSG.
(Fig. 4C–E). Pre-injection of LV-SREBP-1 also suppressed DSGinduced decrease of TBARS content in pregnant db/+ mice (Fig. 4F). The results indicated that downregulation of SREBP-1 was involved in DSG-induced amelioration of abnormal changes of lipid profiles and oxidative stress, and the final improvement of GD in pregnant db/+ mice. 4. Discussion In the current study, we examined the effect of DSG on GD and explored the possible mechanisms using pregnant db/+ mice. We found that DSG could remarkably ameliorated GD in pregnant db/+ mice, as reflected by the improvement of glucose and insulin intolerance, and the decrease of fasting blood glucose and insulin level and the increase of hepatic glycogen content. There are literatures reporting that DSG exhibited anti-diabetic activities [6,21–25]. For instance, Naidu et al. reported that DSG reorganized hyperglycaemia and distorted tissue lipid profile in high-fat dietstreptozotocin-induced diabetic rats [22]. Roghani-Dehkordi et al. [21] found that DSG mitigated diabetes-induced vascular dysfunction of the rat aorta. Based on the previous discoveries, our results demonstrated that DSG functioned as a potential anti-diabetic
agent both under general diabetic condition but also under gestation diabetic condition. Previous studies have shown that DSG exhibited antioxidant activities which may be involved in the anti-diabetic effects [11]. In the current study, we also assessed the effect of DSG on oxidative stress under GD condition. We found that DSG could inhibit oxidative stress in pregnant db/+ mice, as evidenced by decrease of TBARS content, increase of GSH level and SOD and CAT activities. The results indicated that the antioxidant activities of DSG may be involved in the anti-diabetic effect of DSG in GD mice. It has been also reported that DSG exhibited lipid-lowering effects under different pathological conditions [23,26]. Abnormal changes of lipid profiles are closely associated with oxidative stress [27,28] and the development of GD [29,30]. In our study, we also examined the effect of DSG on lipid profiles. We discovered that DSG could attenuated the abnormal changes of lipid profiles in pregnant db/+ mice, as illustrated by decrease of TC, TG and LDL. The results indicated that attenuation of abnormal changes of lipid profiles may participate in the anti-diabetic effects of DSG in GD mice. To further explore the mechanism of DSG-induced improvement of lipid profiles in GD mice, we examined the changes of SREBP-1 and its target gene expression. SREBP-1 is a well-known nuclear transcription factors involved in the biosynthesis of cholesterol, fatty acid, and triglyceride in mammals [31] through regulating its target genes involved in fatty acid biosynthesis, including FAS, SDC-1, ACC [32]. Abnormal expression of SREBP-1 is associated with the pathogenesis of diabetes [33]. It has been reported that Yamogenin, a diastereomer of diosgenin in fenugreek, inhibited lipid accumulation through the suppression of SREBP-1c in hepatocytes [34]. In the study, we found that DSG significantly decreased SREBP-1 and FAS, SDC-1, and ACC expression in pregnant db/+ mice. Moreover, overexpression of SREBP-1 by LV-SREBP-1 injection could markedly inhibit the protective
Fig. 4. Role of downregulation of SREBP-1 in the protective effect of DSG against GD. Pregnant db/+ mice were pre-injected with LV-SREBP-1 and administrated with DSG. On gestation day 10, glucose (A) and insulin (B) tolerance were evaluated. On gestation day 20, serum and livers were collected. Fasting blood glucose (C) and insulin (D) level was measured and hepatic glycogen content (E) was determined. Levels of TBARS were determined using commercial kits (F). *p<0.05, vs GD. #p<0.05, vs DSG.
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effect of DSG against disorder of glucose and lipid metabolism and oxidative stress in GD mice. The data demonstrated that downregulation of SREBP-1 was involved in the anti-diabetic effect of DSG in GD mice. In conclusion, we found that DSG decreased SREBP-1 expression, improved the abnormal changes of lipid profiles and oxidative stress, and ameliorated GD in pregnant db/+ mice. SREBP-1 may be of major target of DSG that mediated its anti-diabetic activities in GD. The data provide novel insights into the biological activities of DSG and pave way for the investigation of the anti-diabetic activities against GD. References [1] D.R. Coustan, Gestational diabetes mellitus, Clin. Chem. 59 (9) (2013) 1310– 1321. [2] A.B. Gilmartin, S.H. Ural, J.T. Repke, Gestational diabetes mellitus, Rev. Obstet. Gynecol. 1 (3) (2008) 129–134. [3] A.D. Association, Gestational diabetes mellitus, Diabetes Care 27 (Suppl. 1) (2004) S88–90. [4] J. Gardosi, A. Francis, A customized standard to assess fetal growth in a US population, Am. J. Obstet. Gynecol. 201 (1) (2009) 25.e1–25.e7. [5] G.E. Tutino, W.H. Tam, X. Yang, J.C. Chan, T.T. Lao, R.C. Ma, Diabetes and pregnancy: perspectives from Asia, Diabetic Med.: J. Br. Diabetic Assoc. 31 (3) (2014) 302–318. [6] S. Fuller, J.M. Stephens, Diosgenin, 4-hydroxyisoleucine and fiber from fenugreek: mechanisms of actions and potential effects on metabolic syndrome, Adv. Nutr. (Bethesda, Md.) 6 (2) (2015) 189–197. [7] F. Gao, X.Z. Shen, F. Jiang, Y. Wu, C. Han, DNA-guided genome editing using the Natronobacterium gregoryi argonaute, Nat. Biotechnol. 34 (7) (2016) 768–773. [8] E. Wang, J. Wylie-Rosett, Review of selected Chinese herbal medicines in the treatment of type 2 diabetes, Diabetes Educ. 34 (4) (2008) 645–654. [9] S. Das, K.K. Dey, G. Dey, I. Pal, A. Majumder, S. MaitiChoudhury, S.C. kundu, M. Mandal, Antineoplastic and apoptotic potential of traditional medicines thymoquinone and diosgenin in squamous cell carcinoma, PLoS One 7 (10) (2012) e46641. [10] P. Kalailingam, B. Kannaian, E. Tamilmani, R. Kaliaperumal, Efficacy of natural diosgenin on cardiovascular risk, insulin secretion, and beta cells in streptozotocin (STZ)-induced diabetic rats, Phytomed.: Int. J. Phytother. Phytopharm. 21 (10) (2014) 1154–1161. [11] L. Pari, P. Monisha, A. Mohamed Jalaludeen, Beneficial role of diosgenin on oxidative stress in aorta of streptozotocin induced diabetic rats, Eur. J. Pharmacol. 691 (1–3) (2012) 143–150. [12] T. Yamada, M. Hoshino, T. Hayakawa, H. Ohhara, H. Yamada, T. Nakazawa, T. Inagaki, M. Iida, T. Ogasawara, A. Uchida, C. Hasegawa, G. Murasaki, M. Miyaji, A. Hirata, T. Takeuchi, Dietary diosgenin attenuates subacute intestinal inflammation associated with indomethacin in rats, Am. J. Physiol. 273 (2 Pt. 1) (1997) G355–G364. [13] S. Hirai, T. Uemura, N. Mizoguchi, J.Y. Lee, K. Taketani, Y. Nakano, S. Hoshino, N. Tsuge, T. Narukami, R. Yu, N. Takahashi, T. Kawada, Diosgenin attenuates inflammatory changes in the interaction between adipocytes and macrophages, Mol. Nutr. Food Res. 54 (6) (2010) 797–804. [14] R.C. Kaufmann, K.S. Amankwah, G. Dunaway, L. Maroun, J. Arbuthnot, J.W. Roddick Jr., An animal model of gestational diabetes, Am. J. Obstet. Gynecol. 141 (5) (1981) 479–482. [15] T. Ishizuka, P. Klepcyk, S. Liu, L. Panko, S. Liu, E.M. Gibbs, J.E. Friedman, Effects of overexpression of human GLUT4 gene on maternal diabetes and fetal growth in spontaneous gestational diabetic C57BLKS/J Lepr(db/+) mice, Diabetes 48 (5) (1999) 1061–1069. [16] S. Lambin, R. van Bree, S. Caluwaerts, L. Vercruysse, I. Vergote, J. Verhaeghe, Adipose tissue in offspring of Lepr(db/+) mice: early-life environment vs. genotype, Am. J. Physiol. Endocrinol. Metab. 292 (1) (2007) E262–E271.
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