Cinnamaldehyde potentially attenuates gestational hyperglycemia in rats through modulation of PPARγ, proinflammatory cytokines and oxidative stress

Biomedicine & Pharmacotherapy 88 (2017) 52–60

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Original article

Cinnamaldehyde potentially attenuates gestational hyperglycemia in rats through modulation of PPARg, proinflammatory cytokines and oxidative stress Ahmed A. Hosnia , A. Adel Abdel-Moneima,* , Eman S. Abdel-Reheima , Samah M. Mohamedb , Hamdi Helmyc a

Molecular Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, 62511 Beni-Suef, Egypt Biochemistry Department, Theodor Bilharz Research Institute, 12411 Giza, Egypt c Clinical Pathology Department, Faculty of Veterinary Medicine, Beni-Suef University, 62511 Beni-Suef, Egypt b

A R T I C L E I N F O

Article history: Received 6 November 2016 Received in revised form 6 January 2017 Accepted 9 January 2017 Keywords: Cinnamaldehyde Cinnamomum zeylanicum Gestational diabetes Pro-inflammatory cytokines Oxidative stress Maternal outcome

A B S T R A C T

Cinnamon has a history of use for medicinal purposes and its major benefits have been linked to cinnamaldehyde. The present study aimed to investigate the hypoglycemic action of cinnamaldehyde against fatty-sucrosed diet/streptozotocin (FSD/STZ)-rat model of gestational diabetes. Female albino rats were divided into three groups. Group I fed with normal diet (ND) while group II and III were fed with FSD for eight weeks (five weeks pre-gestational and three weeks gestational). Rats of group III were administered with a daily oral dose of 20 mg/kg cinnamaldehyde one week before mating onward. At the 7th day of gestation, FSD-fed rats were injected intraperitoneally with STZ (25 mg/kg b.wt.) to induce gestational diabetes. Pre-mating treatment of cinnamaldehyde controls hyperphagia and glucose intolerance during the gestational period than in diabetic rats. It also reduced levels of fructosamine, total cholesterols, triglycerides, leptin, tumor necrosis factor-alpha (TNF-a), malondialdehyde (MDA) and nitric oxide (NO), while it significantly increased levels of high-density lipoprotein (HDL)-cholesterol, adiponectin, liver glycogen, reduced glutathione (GSH) and catalase activity at term pregnancy. In addition, cinnamaldehyde administration up-regulated the mRNA expression of peroxisome proliferated activated receptor-gamma (PPARg) and also ameliorated the number of viable fetuses, implantation loss sites, fetal glucose and insulin levels. In conclusion, cinnamaldehyde has safe hypoglycemic action on gestational diabetes by potentiating insulin secretion and sensitivity through activating the antioxidant defense system, suppressing pro-inflammatory cytokines production, upregulating PPARg gene expression and alleviating the reproductive performance. © 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction Diabetes mellitus (DM) is a complex, chronic illness requiring continuous medical care with multifactorial risk-reduction strategies beyond glycemic control [1]. According to the International Diabetes Federation (IDF), 415 million people worldwide, or 8.8% of adults aged 20–79, are estimated to have diabetes. An additional 21 million cases of high blood glucose in pregnancy are estimated to contribute to the global burden of diabetes [2]. With the rapid global rise in the prevalence of DM, the incidence of the gestational diabetes mellitus (GDM) increased in the developing countries

* Corresponding author. E-mail addresses: [email protected], [email protected] (A. A. Abdel-Moneim). http://dx.doi.org/10.1016/j.biopha.2017.01.054 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

from 2.9% to 8.8% over the last twenty years [3] constituting a health threat to the upcoming generations. GDM is defined as glucose intolerance of variable severity with onset or first recognition during pregnancy, irrespective of the glycemic status after delivery [4]. Insulin resistance and b-cell dysfunction are thought to be major determinants of its development. GDM expose the affected women to higher risk for subsequent development of type 2 diabetes (T2D) and cardiovascular diseases later in life, and their infants revealed a greater incidence of obesity and T2D in adulthood [5]. Peroxisome proliferator activated receptors (PPARs) are a family of ligand activated transcription factors belonging to the nuclear hormone receptor superfamily. PPARs regulate the expression of multiple genes involved in metabolic, anti-inflammatory, developmental processes and are involved in the maternal adaptational

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dynamics during pregnancy to serve the requirements of the growing fetus [6]. Three PPAR isotypes have been identified in mammals termed PPARa, PPARb and PPARg [7]. PPARa exists at the highest level in the liver and is believed to play a critical role in the regulation of fatty acids metabolism, PPARb is ubiquitously distributed with a higher expression in the digestive tract, while PPARg is mostly expressed in the adipose tissue, placenta and immune system. Moreover, PPARg is a key regulator of glucose and lipid metabolism, promotes preadipocyte differentiation, stimulates the storage of fatty acids in adipocytes and enhances insulin sensitivity [8]. In general, medications preferred to be avoided during pregnancy, but for women with chronic conditions in pregnancy such as epilepsy, psychiatric disorders and diabetes mellitus, the use of medication may be required and discontinuation of treatment is not always an option. Historically, insulin has been the therapeutic agent of choice for controlling hyperglycemia in pregnant women. Difficulty in insulin administration with multiple daily injections, potential for hypoglycemia that occurs in approximately 71% of women who take insulin at some times during their pregnancy and the increase in appetite and weight makes this therapeutic option cumbersome for many pregnant patients [9]. Although the use of the synthetic oral hypoglycemic agents (SOHAs; metformin and glyburide) in pregnancy has opened new vistas for GDM management, they have dermatological and gastrointestinal adverse effects including diarrhea, flatulence, nausea and vomiting with an incidence rate 63% [10] rather than being expensive. Medicinal plants provide valuable and safe therapeutic agents, both in modern medicine and in the traditional system. Cinnamomum zeylanicum (cinnamon) has a history of use for medicinal purposes as far back as in China and ancient Egypt. Nonmedical reports claimed that cinnamon administration at large doses during pregnancy was implicated in triggering uterine contractions and stimulating preterm labor. Coumarin (naturally occurring constituent of cinnamon) and its derivatives may be involved in that case [11]. Cinnamon bark contains a wide range of essential oils, mainly trans-cinnamaldehyde. Biological activities of cinnamaldehyde (Ci) mainly include antioxidant [12] and antiinflammatory [13] properties. In addition, Subash-Babu et al. [14] reported the antihyperglycemic effect of cinnamaldehyde in streptozotocin (STZ)-induced diabetic rats with a recommended dose of 20 mg/kg b.wt. Although the safety evaluation of cinnamaldehyde administration during pregnancy revealed non-embryotoxic effects [15], no data are available about its role in controlling the elevated blood

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glucose level during pregnancy and its effect on maternal outcome and the fetal glycemic state. This study was designed to investigate the hypoglycemic action of cinnamaldehyde against FSD/STZ-rat model that might act through up-regulating PPARg expression which targeting both TNF-a and adiponectin; vital factors affecting insulin sensitivity and blood glucose level. 2. Material and methods 2.1. Chemicals Ethyl acetate, n-hexane, streptozotocin, reduced glutathione (GSH), 5,50 -dithiobis-2-nitrobenzoic acid (DTNB, Ellman’s reagent), thiobarbituric acid (TBA), 1,1,3,3-tetramethoxypropane and RNA later were purchased from Sigma Chemical Co. (USA). Metaphosphoric acid and pyrogallol were purchased from Fluka analytical (Germany). All other chemicals were of analytical grade and obtained from standard commercial supplies. 2.2. Plant material and extract preparation The bark of Cinnamomum zeylanicum Blume was collected from the botanical garden of the Faculty of Agriculture, Cairo University, Egypt. The samples were identified by Dr. M. Omar, Taxonomist, Botany Department, Faculty of Science, Beni-Suef University, Egypt. Voucher specimens of Cinnamomum zeylanicum Blume (no. BuPD 36) were deposited in Pharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, Egypt by Dr. A. Ismail. Fresh bark of Cinnamomum zeylanicum Blume (0.5 kg) was subjected to hydro-distillation in a Clevenger-type apparatus for 4 h. The yield (v/w) of volatile oil was 0.118%; 3.1 g. According to Subash-Babu et al. [14], biologically guided the phytochemical investigation of the resulted oil led to the isolation of a bioactive compound; cinnamaldehyde. 2.3. Animals and dietary formulas Female albino rats (Rattus norvegicus) weighing about 100  10 g, 60 days old were obtained from the animal house of Helwan town, Cairo, Egypt. The animals were housed individually in standard polypropylene cages and maintained in an airconditioned atmosphere, at a temperature of 25  C with alternatively 12 h light and dark cycles for one week before the onset of the experiment to be acclimatized. Rats had free access to water and to two dietary regimens by feeding either the normal diet (ND) or fatty-sucrosed diet (FSD).

Fig. 1. Experimental design. ND, normal diet. FSD, fatty-sucrosed diet. STZ, streptozotocin. Ci, cinnamaldehyde. NP, normal pregnant. GD, gestational diabetic. GD + Ci, gestational diabetic pre-treated with cinnamaldehyde (20 mg/kg b.wt.).

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The composition and preparation of both diets were described elsewhere [16]. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (NIH), that was recommended by the animal ethical committee of the Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef, Egypt. 2.4. Experimental design The experimental design was summarized in Fig. 1. A total of 45 animals were randomly divided into three groups (15 animal/ group) as follows:  Group I: feds on the normal diet (ND) during the entire study.  Group II: feds on the fatty-sucrosed diet (FSD) during the entire study. After four weeks of dietary manipulation, rats were orally given a vehicle (0.5% dimethyl sulfoxide; DMSO) daily to the end of the experiment.  Group III: feds on FSD during the entire study. After four weeks of dietary manipulation, rats were orally treated with cinnamaldehyde (20 mg/kg b.wt. dissolved in 0.5% DMSO) daily to the end of the experiment. The dosage was adjusted weekly according to changes in body weight to standardize the dosages over the duration of the study. Five weeks from the beginning of the experiment, females of all groups were time mated overnight with males and the presence of sperms in the vaginal smear checked in the morning was considered the zero day of pregnancy (females with negative detected sperms in the vaginal smear were excluded). The time before mating referred to the pre-gestational period while that after mating referred to gestational period. At the 7th day of pregnancy, rats fasted for 16 h and those of groups II and III were injected intraperitoneally (i.p.) with a low dose of STZ (25 mg/kg b.wt. in citrate buffer; pH 4.5) [16], while rats of group I was i.p. injected with the buffer only.

Blood samples were collected at the 6th day of gestation (6th week; pre-STZ), the 13th day (7th week; post-STZ) and the 20th day (8th week; post-STZ) from the tail veins of each rat for estimation of blood glucose and insulin levels to assess the development of gestational diabetes. At the 21st day of pregnancy, overnight fasted dams were sacrificed under complete anesthesia (sodium pentobarbital, 50 mg/kg body weight, i.p.) where blood samples and all measured parameters were referred to main three groups (n = 10, each): - Normal pregnant (NP) group: received ND. - Gestational diabetic (GD) group: received FSD/minimal dose of STZ. - Gestational diabetic group pre-treated with cinnamaldehyde (GD + Ci): received FSD/minimal dose of STZ and pre-treated with cinnamaldehyde (20 mg/kg b.wt.).

2.5. Determination of food intake and body weight changes The food was provided in standard stainless steel hoppers. Food intake was calculated daily at the same time by subtracting the amount of food left over in the cage of each rat from the measured amount of food provided at the previous day. The mean of food consumption per each rat was considered by dividing the amount of food eaten in a week by 7. The average of food consumptions was represented in g/day/rat. Body weight for each rat was determined once a week (g) during the entire study. 2.6. Blood and tissue samples collection Table 1 indicated the time point of samples collection for all measurements and assays. Maternal blood samples were collected from the axillary vein of each dam, allowed coagulating, centrifuged and sera were kept at 24  C for subsequent analyses. After dissection of the sacrificed rats, visceral adipose tissue from each dam was immediately kept in RNA later and stored at 24  C

Table 1 Time point of samples collection for all measurements and assays. Experimental period

Time

Sample collected

Parameter measured

Pre-gestational period

Zero week 1st week 2nd week 3rd week 4th week 5th week

– – – – – 0.5 ml blood adjusted with fasting glucose and insulin, not body weight, food intake

body weight. body weight, food intake. body weight, food intake. body weight, food intake. body weight, food intake. body weight, food intake, fasting glucose, insulin and leptin.

Gestational period

6th week (6th day of gestation; Pre-STZ) 7th week (13th day of gestation; Post-STZ) 8th week (20th day of gestation; Post-STZ)

0.5 ml blood adjusted with fasting glucose and insulin, not body weight, food intake

body weight, food intake, fasting glucose and insulin.

0.5 ml blood adjusted with fasting glucose and insulin, not body weight, food intake

body weight, food intake, fasting glucose and insulin.

Sacrification

1 ml blood adjusted with fasting glucose and insulin, not body weight, food intake, body weight, food intake fasting glucose and insulin, OGTT.

End of the 8th week 6–8 ml blood collected from the axillary vein of each rat. (21st day of gestation; Visceral adipose tissue of each rat Post-STZ) Liver of each rat 0.3 ml blood pool collected from the axillary vein of fetuses of each rat

serum fructosamine, triglycerides total cholesterols, HDLcholesterol, leptin, TNF-a and adiponectin. PPARg RT-PCR. glycogen content, oxidative stress and antioxidant parameters. fetal glucose, insulin, number of live fetuses/uterus of each rat, uterine implantation loss sites, fetal body weight.

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for gene expression analysis. Fresh maternal liver samples were collected for determination of glycogen content, oxidative stress and antioxidant parameters. In addition, each rat uterine horns were exposed to count viable fetuses and implantation loss sites. Fetuses were delivered and weighed. Blood pool was collected from the axillary vein of fetuses of each dam and sera were separated for the immediate measurement of serum glucose and insulin levels. 2.7. Determination of biochemical analyses Oral glucose tolerance test (OGTT) was performed for all dams at the 20th day of pregnancy. Blood samples were obtained from lateral tail veins of fasted animals (6–8 h). Successive blood samples were then taken at 30, 60, 90 and 120 min following the administration of glucose solution (3 g/kg b.wt.) by gastric intubation. Blood samples were centrifuged and sera were obtained quickly for determination of glucose concentration. Glucose concentrations were determined according to the method of Trinder [17], using reagent kit obtained from Spinreact Co. (Spain), while insulin was assayed by sandwich ELISA using reagent

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kit purchased from BioSource Europe S.A. (Belgium), according to the method of Templer et al. [18]. Glycogen content was determined in the liver tissue homogenate of each dam according to the method of Seifter et al. [19], while serum fructosamine, triglycerides and cholesterol levels were determined according to methods of Baker et al. [20], Buccolo and David [21], and Allain et al. [22], respectively, using reagent kits purchased from Spinreact Co. (Spain). Pro-inflammatory cytokines (leptin and TNF-a) and anti-inflammatory cytokine (adiponectin) were assayed by the sandwich ELISA method according to methods of Maffei et al. [23] Brouckaert et al. [24] and Hotta et al. [25], respectively, using rat reagent kits procured from RayBiotech, Inc. (USA). Malondialdehyde (MDA; lipid peroxidation marker) and nitrite (nitric oxide “NO” marker) concentrations were determined in the liver tissue homogenate according to the method of Yagi [26] and the NO-reagent kit (Biodiagnostic, Egypt), respectively, as main oxidative stress biomarkers. On the other hand, glutathione (nonenzymatic antioxidant) concentration and catalase (CAT; the antioxidant enzyme) activity were determined by methods of Beutler et al. [27] and Sinha [28], respectively.

Fig. 2. (A) Represented the body weight changes during the pre-gestational and gestational periods. Two-way ANOVA for group  time interaction indicated: FCalculated = 3.775, P < 0.001 at the pre-gestational period and FCalculated = 27.707, P < 0.001 at the gestational period. (B) represented the food intake changes during the pregestational and gestational periods. Two-way ANOVA for group  time interaction indicated: FCalculated = 14.006, P < 0.001 at the pre-gestational period and FCalculated = 10.559, P < 0.001 at the gestational period. (C) & (D) represented the fasting serum glucose and insulin levels during the gestational period, respectively. Two-way ANOVA for group  time interaction indicated: FCalculated = 117.08, P < 0.001 for fasting glucose and FCalculated = 29.062, P < 0.001 for fasting insulin. One-way ANOVA followed by Tukey– Kramer test for post hoc analysis indicated: a P < 0.05 versus group I, b P < 0.05 versus group II; compared to the respective time. Results are presented as means  SEM (n = 15 animals during the pre-gestational period and n = 10 during the gestational period in each group).

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2.8. RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was extracted from the visceral adipose tissue of each dam using TriFastTM reagent (PeQlab, Germany). RNA was purified and spectrophotometrically quantified. cDNA was synthesized from 2 mg RNA by using RT-PCR kit (promega, USA). The RTproduct (cDNA) was used in 25 ml PCR amplification reaction for the detection of PPARg using primer synthesized by Midland Certified Reagent Company Inc. (USA) with a forward fragment 50 CCCTGGCAAAGCATTTGTAT-30 , a reverse fragment 50 -ACTGGCACCCTTGAAAAATG-30 and an extension fragment of 222 bp. b-actin was used as the internal control with primer have a forward fragment of 50 -TGGGACGATATGGAGAAGAT-30 , a reverse fragment of 50 -ATTGCCGATAGTGATGACCT-30 and an extension fragment of 522 bp. PCR was performed using Green master mix (Promega, USA) and T100TM thermal cycler (Bio-Rad Laboratories, USA) under the following conditions: initial denaturation at 95  C for 5 min, 35 cycles set at 94  C (1 min) for denaturation, 55  C (1 min) for annealing and 72  C (1 min) for extension, and finally at 72  C (5 min) to complete the extension reaction. PCR products were subjected to electrophoresis on 1.5% agarose gels containing ethidium bromide [29]. Images from electrophoresed gels were captured by a camera in a computer assisted gel documentation system. Relative band intensities of each sample were calculated after being normalized with the band intensity of b-actin using Phoretix 1-D densitometry software v.11 (TotalLab Ltd., UK) and values presented as% mRNA relative to control. 2.9. Statistical analysis Data are presented as means  SEM. Results of maternal body weight, food intake, serum glucose and insulin levels and OGTT were analyzed using the two-way analysis of variance (ANOVA) where the fixed factors were defined as ‘group’ and ‘time’. The twoway ANOVA indicated interactions between ‘group’ and ‘time’ (‘group  time’, P < 0.05, n = 15 animals during the pre-gestational period and n = 10 during the gestational period in each group). One-way ANOVA test followed by Tukey–Kramer test for post hoc analysis were performed to further analyze the significance differences in the means between the groups. Other measured variables were analyzed using one-way ANOVA test followed by Tukey–Kramer test for post hoc analysis. P-values < 0.05 were considered statistically significant. All statistical analyses were performed using the SPSS v.22 software (SPSS Inc., Chicago, USA).

Table 2 Serum glucose, insulin and leptin levels at the end of the pre-gestational period. Group

Glucose (mg/dL)

Insulin (mIU/ml)

Leptin (pg/ml)

I II III

88.23  2.06 118.49  5.32a 106.00  3.14a,b

16.03  0.84 20.61  2.01a 18.92  1.00a

503.28  21.20 767.30  8.63a 712.96  11.11a,b

a b

P < 0.05 versus group I. P < 0.05 versus group II. Results are presented as means  SEM (n = 15).

3.2. Effect of Ci on glucose and insulin levels At the end of the pre-gestational period (after one week of cinnamaldehyde administration), rats of group III showed a notable decrease in their fasting glucose but without a significant decrease in their insulin levels as compared to those of group II (Table 2). Fig. 2C and D depicts the potent protective effect of cinnamaldehyde administration against STZ-induced hyperglycemia that injected at the mid gestation. Both fasting serum glucose and insulin levels of cinnamaldehyde administrating rats appeared near normal ones at the late gestation. 3.3. Effect of Ci on oral glucose tolerance curve Fig. 3 shows that the cinnamaldehyde administration at a dose of 20 mg/kg b.wt. for about 30 days caused a significant antihyperglycemic effect when compared to the respective hyperglycemic GD-group. Glucose-lowering effect of Ci at fasting and after 30, 60, 90 and 120 min of an oral glucose load of 3 g/kg b.wt. at the 20th day of gestation were around 65%, 44%, 56%, 59% and 62%, respectively. 3.4. Effect of Ci on liver glycogen content, serum fructosamine, triglycerides and cholesterols levels As compared to NP-dams, a significant decrease (P < 0.05) in liver glycogen content and serum HDL-cholesterol with a subsequent increase in serum fructosamine, triglycerides and total cholesterols were clearly represented in fasted GD-rats. Oral

3. Results 3.1. Body weight and food intake changes during the pre- and gestational periods Following FSD-feeding, rats of group II and III showed an obvious increase in their body weight up to the 4th weeks; this accompanied with a marked decrease in their food intake onward as compared to ND-fed rats (Fig. 2A and B). Oral administration of cinnamaldehyde to groups III from the beginning of the 5th week controls the further increase in the body weight. At the early pregnancy (week 6; pre-STZ injection) rats of group II showed a significant increase in their body weight as compared to those of group I and III. This increase is no longer continuing after injection of STZ at the 7th day of gestation that caused an obvious decrease in their body weights at the mid and late gestation (week 7 and 8). Besides, rats of group II showed a state of hyperphagia after STZ injection mainly at late gestation. Cinnamaldehyde administration to groups III offers protection from the body weight loss and justify their food intake like the normal group.

Fig. 3. OGTT at the 20th day of gestation of normal pregnant (NP), gestational diabetic (GD) and gestational diabetic dams pre-treated with cinnamaldehyde (GD + Ci). Two-way ANOVA for group  sampling time interaction indicated: FCalculated = 7.936, P < 0.001. One-way ANOVA followed by Tukey–Kramer test for post hoc analysis indicated: a P < 0.05 versus NP, b P < 0.05 versus GD; compared to the respective time. Results are presented as means  SEM (n = 10).

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Table 3 Effect of cinnamaldehyde on liver glycogen content, serum fructosamine, triglycerides and cholesterols levels at term pregnancy. Group

Liver glycogen (mg/g tissue)

Fructosamine (mmol/L)

Triglycerides (mg/dL)

Total cholesterols (mg/dL)

HDL-cholesterol (mg/dL)

NP GD GD + Ci

12.69  0.57 3.55  0.19a 10.18  0.27a,b

45.39  3.05 185.70  5.25a 75.04  3.06a,b

58.72  1.44 189.64  1.34a 60.90  3.70b

81.12  1.45 185.13  2.51a 130.08  0.88a,b

58.06  1.54 52.52  1.18a 89.76  1.12a,b

a b

P < 0.05 versus NP. P < 0.05 versus GD. Results are presented as means  SEM (n = 10).

Table 4 Effect of cinnamaldehyde on serum leptin, TNF-a and adiponectin at term pregnancy. Group

Leptin (pg/ml)

TNF-a (pg/ml)

Adiponectin (pg/ml)

NP GD GD + Ci

574.65  46.38 1265.40  42.15a 842.80  30.80a,b

210.77  4.42 374.40  7.19a 215.12  4.13b

946.68  4.54 693.95  16.73a 919.94  8.27b

a b

normal pregnant ones. No significance was observed in the fetal weights among experimental groups. Gestational diabetes elevated the fetal glucose and insulin serum levels significantly as compared to those of fetuses of NP-rats. Interestingly, oral administration of cinnamaldehyde revealed profound amelioration of the maternal reproductive performance and control the fetal glycemia (Table 6). 3.8. Effect of Ci on adipose tissue PPARg mRNA expression

P < 0.05 versus NP. P < 0.05 versus GD. Results are presented as means  SEM (n = 10).

administration of Ci potentially alleviated these parameters with an interest to its ability to increase HDL-cholesterol level than normal group (Table 3). 3.5. Effect of Ci on serum leptin, TNF-a and adiponectin levels

As shown in Fig. 4, gel image analysis revealed a significant decrease in PPARg mRNA expression in gestational diabetic rats as compared to normal control ones. On the other hand, the treatment with cinnamaldehyde induced significant amelioration in the tested gene expression. 4. Discussion

The induction of diabetes in pregnant rats increased serum leptin and TNF-a concentration reaching 2.2- and 1.8-fold, respectively compared to the normal pregnant group (Table 4). Their secretions were markedly inhibited as a result of cinnamaldehyde administration. Adiponectin exhibited an opposite behavioral pattern; its concentration was declined markedly in diabetic rats as compared to normal ones, and cinnamaldehyde administration nearly normalized it. 3.6. Effect of Ci on liver oxidative stress and antioxidant biomarkers Table 5 illustrates the effect of cinnamaldehyde on liver lipid peroxidation, NO, GSH and activity of catalase. The results revealed significant elevation in MDA and nitrite levels in GD-group when compared to NP-one. Notably, cinnamaldehyde attenuated both MDA and nitrite production. GSH concentration and catalase activity were reduced in diabetic rats significantly when compared to normal rats. Cinnamaldehyde treated rats demonstrated a profound increase in GSH concentration and catalase activity compared to the diabetic group. 3.7. Effect of Ci on maternal reproductive outcome and fetal glycemic state A lower mean number of live fetuses and a higher implantation loss sites were observed in the diabetic dams as compared to the

In the current study, consumption of the FSD during the pregestational period facilitates the development of a positive energy balance leading to an increase in the visceral fat deposition. This increase in the body weight was accompanied by a significant decrease in the food intake secondary to an increase in serum leptin level that set the received calorie by the rats [16]. In addition, FSD-feeding induced a state of glucose intolerance that confirmed by an increase in serum insulin release. Marked elevation in blood glucose with diminished serum insulin level was observed in diabetic rats. Prolonged FSD-feeding induces insulin resistance mainly through glucose-fatty acid cycle [30]. Briefly, the presence of high level of triglycerides due to excess fat intake could constitute a source of increased fatty acid availability and oxidation. The preferential use of increased fatty acids for oxidation blunts the insulin-mediated reduction of hepatic glucose output and reduces the glucose uptake or utilization in skeletal muscle, which are common features of insulin resistance. Moreover, injection of the low dose of STZ caused a moderate destruction of pancreatic b-cells through the generation of reactive oxygen species (ROS) that induce apoptosis and suppression of insulin biosynthesis [31]. All of these divert the body toward the catabolism of fats and proteins resulting in body weight loss and subsequent hyperphagia which are main symptoms of diabetes mellitus. Cinnamaldehyde administration one week before mating and during the gestation potentially

Table 5 Effect of cinnamaldehyde on liver oxidative stress and antioxidant defense system parameters at term pregnancy. Group

MDA (nmol/g tissue)

NO (nmol/g tissue)

GSH (nmol/100 mg tissue)

CAT (K  102)

NP GD GD + Ci

54.63  1.64 120.93  2.18a , 80.85  2.4a b

82.36  0.88 92.52  1.33a 83.25  0.95b

450.38  13.72 237.18  9.94a 422.31  23.12b

63.61  1.11 31.62  1.01a , 50.92  0.75a b

a b

P < 0.05 versus NP. P < 0.05 versus GD. Results are presented as means  SEM (n = 10).

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Table 6 Effect of cinnamaldehyde on maternal reproductive outcome and fetal glycemic state. Group

Live fetuses

Implantation loss

Fetal weight (g)

Fetal glucose (mg/dL)

Fetal insulin (mIU/ml)

NP GD GD + Ci

8.75  0.52 4.25  0.22a 8.00  0.27b

3.50  0.20 9.56  0.38a 3.53  0.19b

3.08  0.03 3.40  0.10 3.18  0.41

42.94  1.46 161.50  4.12a 52.10  2.18a,b

3.60  0.14 6.20  0.16a 4.01  0.19a,b

a b

P < 0.05 versus NP. P < 0.05 versus GD. Results are presented as means  SEM (n = 10).

protects the treated-rats from the body weight loss through its better control of glucose level, stimulating insulin release and making rats sensitive to the caloric properties of the diet to set their food intake. Our results show an enormous depletion in hepatic glycogen content of GD-rats. Glycogen considered the primary intracellular storable form of glucose and its level in various tissues especially in the liver indicates a direct reflection of insulin activity since it regulates glycogen deposition by stimulating glycogen synthase system [32]. Oral administration of cinnamaldehyde significantly increases hepatic glycogen level secondary to the reactivation of the glycogen synthase system as a result of increased insulin secretion. Elevated blood glucose level over a period of time makes glucose molecules permanently combine with blood proteins in a process called glycation [33], so serum fructosamine (a putative measure of glycated proteins) found to be particularly useful in monitoring the effectiveness of therapies in diabetes. In the present study, serum fructosamine was profoundly increased in diabetic dams as compared with normal ones. On the other hand, cinnamaldehyde administration induced an obvious decrease of elevated fructosamine level that possibly by the increased insulin secretion and sensitivity. Subtle alterations in lipid metabolism have been reported in GDM. Our data revealed marked increase in triglycerides (TG) and total cholesterols with a reduction in HDL-cholesterol in FSD/STZdiabetic rats. The hypertriglyceridemia and hypercholesterolemia may be due to increasing dietary triglycerides and cholesterol absorption from the small intestine following the intake of FSD. In addition, it has been reported that dysfunction of lipoprotein lipase (LPL) in insulin deficient state contributes to hypertriglyceridemia due to impaired catabolism of triglyceride-rich particles and decreased TG uptake in peripheral tissues [34]. The significant

amelioration of the serum lipid variables in cinnamaldehyde treated-rats might have been due to its insulinotrophic and insulin sensitizing actions [14] and may through its activation of LPL which increases TG clearance. Interestingly, cinnamaldehyde elevated serum HDL-cholesterol that potentially prevent the development of atherosclerosis and coronary heart disease; commonly secondary complications of GDM. The liver is the focal organ of oxidative and detoxifying processes as well as free radical reactions and the biomarkers of oxidative stress are elevated in the liver at an early stage in many diseases, including DM. Insulin insufficiency and sustained hyperglycemia that results from b-cell dysfunction considered as a principle mediator of increased ROS generation that augments liver damage in diabetes [35]. Moreover, ROS have the ability to oxidize polyunsaturated fatty acids of hepatocellular membranes and initiate lipid peroxidation. Our study demonstrates a significant increase in MDA levels (the marker of lipid peroxidation) in the liver of GD-rats. Lipid peroxidation changes the fluidity of cell membranes, reduces the capacity to maintain an equilibrated gradient of concentration inactivates some of membrane-bound enzyme and increases membrane permeability leading to cell death. Besides, liver NO is markedly elevated in diabetic dams. NO is a free radical produced in mammalian cells and is involved in the regulation of various physiological processes but its excessive production is associated with several diseases. Under aerobic conditions, NO is unstable and can react with O2 to produce the nitrite and the very reactive oxidant; peroxynitrite (ONOO). Peroxynitrite can react with tyrosine residues of the insulin receptor (IR) and prevents its phosphorylation by protein tyrosine kinases causing disruption of insulin signaling pathways and insulin resistance [36]. The current study hypothesized that the oral administration of cinnamaldehyde significantly protects against the formation of lipid peroxides through its potent free

Fig. 4. RT-PCR analysis of visceral adipose tissue PPARg and b-actin expression in NP, GD and GD-dams pre-treated with cinnamaldehyde (GD + Ci). (a) Gel photograph depicting representative PCR products (M, DNA marker). (b) Corresponding densitometric analysis of PCR products. Results are expressed as mean  SEM. a P < 0.05 versus NP, b P < 0.05 versus GD.

A.A. Hosni et al. / Biomedicine & Pharmacotherapy 88 (2017) 52–60

radical-scavenging activity, and its capability to compete with oxygen to react with NO to suppress nitrite and peroxynitrite production [12]. In contrast, the liver of GD-dams showed a marked reduction of GSH content with a concomitant reduced activity of the antioxidant enzyme catalase. GSH constitutes the first line of defense against free radicals and cosubstrate for glutathione peroxidase (GPx). Regeneration of GSH from its oxidized form (GSSG) required NADPH that necessitates an increase in glucose oxidation via the pentose phosphate cycle [37]. As a result of insulin deficiency in the diabetic dams, the level of intracellular NADPH is decreased and thereby the level of GSH is decreased provoking the susceptibility to oxidative stress. In addition, persistent hyperglycemia during pregnancy causes glycation of the catalase (the primary scavenger of H2O2; one of the reactive non-radicals) making it inactive. The antioxidant activity of cinnamaldehyde referred to its ability to control the glycemic state and inhibiting the glycation of the antioxidant enzymes. Our study revealed a significant decrease in PPARg mRNA expression level in FSD/STZ-gestational diabetic group in comparison with that of normal pregnant and cinnamaldehyde treated groups. PPARg alleviates insulin sensitivity in Ci-treated rats through its ability to channel FFAs into adipose tissue, lowering the hepatic triglyceride content and activating hepatic glucokinase expression [8]. In addition, PPARg is a key regulator of adipocyte hormones, cytokines and proteins that are involved in insulin resistance. Indeed, PPARg appeared to downregulates the expression of genes encoding TNF-a and upregulates adiponectin expression, which increases fatty acid oxidation by activation of AMP-activated protein kinase pathway. GDM is a common pathologic state that contributed to inflammatory processes. Our findings showed significantly increase in circulatory levels of leptin and TNF-a which are strongly correlated with insulin resistance in GD-rats. Leptin is an antiobesity hormone supposed to regulate body weight through a negative feedback signal between adipose tissue and the hypothalamic center of satiety causing a decrease in food intake and an increase in energy expenditure. It has shown that leptin and TNF-a are the strongest predictors of pregnancy-associated insulin resistance, far greater than previously suggested for gestational hormones, including human placental lactogen and steroids [38]. Moreover, the elevated leptin concentrations may actually represent a state of hyperleptinemia and leptin resistance which directly implicated in insulin resistance through a number of mechanisms involving impairment of IR-tyrosine phosphorylation, increasing peroxynitrite-mediated oxidative stress and stimulating immune cells-proliferation that control release of TNF-a [39]. TNF-a has been shown to mediates lipolysis, increases circulating free fatty acids (FFAs) and stimulates IR serine phosphorylation which collectively contributes to the pathogenesis of insulin resistance [40]. In contrast, serum adiponectin level was reported to be in agreement with insulin sensitivity and its reduced level in GD-rats is associated with the insulin resistance state. Adiponectin regulates glucose metabolism through stimulation of adenine monophosphate-activated protein kinase (AMPK), decreases expression of gluconeogenic enzymes, suppresses the hepatic glucose production and enhances fatty acid oxidation and energy dissipation causing a decrease in tissue TG accumulation and increase in insulin sensitivity [41]. Treatment with cinnamaldehyde markedly decreased serum levels of leptin and TNF-a and increased circulating adiponectin confirming its anti-inflammatory efficacy [13]. Concerning the maternal outcome, we observed a reduced number of viable fetuses and, consequently an increased loss rate after embryonic implantation, reflecting the hyperglycemiainduced reproductive disturbances [16]. As the placental transfer

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of glucose is carried out by facilitated diffusion according to concentration-dependent kinetics [42], fetuses of GD-rats showed elevated serum glucose level which stimulates the fetal pancreatic beta-cell hyperplasia and hyperinsulinemia. In contrast to clinical studies of human gestational diabetes, we did not obtain macrosomic fetuses from GD-rats. This is explained by the fact that fat accumulation takes place intra-uterus in humans, whereas in rats it occurs after birth as a result of their short pregnancy period [43]. Oral administration of cinnamaldehyde protects the fetuses from diabetes-induced damages and alleviates the maternal reproductive performance. 5. Conclusion The present study is the first report that demonstrates the safe hypoglycemic action of cinnamaldehyde on gestational diabetes. Cinnamaldehyde effectively improved glucose tolerance of GD-rats by potentiating insulin secretion, sensitivity and action mainly through attenuation of oxidative stress, enhancement of the cellular antioxidant defense status, upregulation of PPARg gene expression and diminishing the proinflammatory cytokines. Also, cinnamaldehyde controls fetal glycemia and alleviates the reproductive performance. Conflict of interest The authors declare that there is no conflict of interest. Funding This work was supported by the Scientific Research Development Unit, Beni-Suef University, Egypt. Acknowledgements We would like to thank Professor Gamal Morsy, Faculty of Science, Cairo University, Egypt for his fruitful directions in statistical analysis, and also appreciate Mr. Ahmed Ismail, Pharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, Egypt for his assist in extract preparation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. biopha.2017.01.054. References [1] American diabetes association, Diagnosis and classification of diabetes mellitus, Diabetes Care 36 (2013) S67–S74. [2] International Diabetes Federation, Diabetes Atlas, seventh ed., International Diabetes Federation, Brussels, Belgium, 2015. [3] F.A. Habib, Evaluation of periodontal status among Saudi females with gestational diabetes and its relation to glucose and lipid homeostasis in Ohud Hospital, Al Madina Al-Munwarrah, Int. J. Health Sci. 3 (2009) 143–154. [4] E.R. Wendland, M.R. Torloni, M. Falavigna, J. Trujillo, M.A. Dode, M.A. Campos, B.B. Duncan, M.I. Schmidt, Gestational diabetes and pregnancy outcomes-a systematic review of the World Health Organization (WHO) and the International Association of Diabetes in Pregnancy Study Groups (IADPSG) diagnostic criteria, BMC Pregnancy Childb. 31 (2012) 12–23. [5] M. Hod, L.G. Jovanovic, G.C. Di-Renzo, A. De-Leiva, O. Langer, Textbook of Diabetes and Pregnancy, second ed., Informa Health Care, London, 2008, pp. 330–333. [6] F. Wieser, L. Waite, C. Depoix, R.N. Taylor, PPAR action in human placental development and pregnancy and its complications, PPAR Res. 2008 (2008), doi:http://dx.doi.org/10.1155/2008/527048 ID 527048, 14 pages. [7] S. Kersten, B. Desvergne, W. Wahli, Roles of PPARS in health and disease, Nature 405 (2000) 421–424. [8] J.N. Feige, L. Gelman, L. Michalik, B. Desvergne, W. Wahli, From molecular action to physiological outputs: peroxisome proliferator-activated receptors

60

[9] [10]

[11]

[12]

[13]

[14] [15] [16]

[17] [18] [19] [20]

[21] [22] [23]

[24]

[25]

A.A. Hosni et al. / Biomedicine & Pharmacotherapy 88 (2017) 52–60 are nuclear receptors at the crossroads of key cellular functions, Prog. Lipid Res. 45 (2006) 120–159. N. Magon, V. Seshiah, Gestational diabetes mellitus: non-insulin management, Indian J. Endocrinol. Metab. 15 (2011) 284–293. S. Bolen, L. Feldman, J. Vassy, L. Wilson, H.C. Yeh, S. Marinopoulos, C. Wiley, E. Selvin, R. Wilson, E.B. Bass, F.L. Brancati, Systematic review: comparative effectiveness and safety of oral medications for type 2 diabetes mellitus, Ann. Intern. Med. 147 (2007) 386–399. G.G. Briggs, R.K. Freeman, S.J. Yaffe, Drugs in Pregnancy and Lactation: A Reference Guide to Fetal and Neonatal Risk, ninth ed., Lippincott Williams & Wilkins, Philadelphia, 2011. P. Subash-Babu, A.A. Alshatwi, S. Ignacimuthu, Beneficial antioxidative and antiperoxidative effect of cinnamaldehyde protect streptozotocin-induced pancreatic b-cells damage in Wistar rats, Biomol. Ther. 22 (2014) 47–54. L.K. Chao, K.F. Hua, H.Y. Hsu, S.S. Cheng, I.F. Lin, C.J. Chen, S.T. Chen, S.T. Chang, Cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling, Food Chem. Toxicol. 46 (2008) 220–231. P. Subash-Babu, S. Prabuseenivasan, S. Ignacimuthu, Cinnamaldehyde – a potential antidiabetic agent, Phytomedicine 14 (2007) 15–22. A.N. Zaitsev, N.B. Maganova, Embryotoxic effects of some aromatizers for food products, Vopr. Pitan. 3 (1975) 64–68. E.S. Abdel-Reheim, A. Adel Abd-Elmoneim, A.A. Hosni, Fatty-Sucrosed diet/ minimal dose of streptozotocin-treated rat: a novel model of gestational diabetes mellitus, metabolic and inflammatory insight, J. Diabetes Metab. 5 (2014) 430, doi:http://dx.doi.org/10.4172/2155-6156.1000430. P.A. Trinder, Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen, J. Clin. Pathol. 22 (1969) 158–161. R.C. Templer, P.M. Clark, C.N. Hales, Measurement of insulin secretion in type 2 diabetes: problems and pitfalls, Diabet. Med. 9 (1992) 503–512. S. Seifter, S. Dayton, B. Novic, E. Muntwyler, The estimation of glycogen with anthrone reagent, Arch. Biochem. 25 (1950) 191–200. J.R. Baker, P.A. Metcalf, R. Johnson, D. Newman, P. Rietz, Use of protein-based standards in automated colorimetric determinations of fructosamine in serum, Clin. Chem. 31 (1985) 1550–1554. G. Buccolo, H. David, Quantitative determination of serum triglycerides by use of enzymes, Clin. Chem. 19 (1973) 476–482. C.C. Allain, L.S. Poon, W. Richmond, P.C. Fu, Enzymatic determination of total serum cholesterol, Clin. Chem. 20 (1974) 470–475. M. Maffei, J. Halaas, E. Ravussin, R.E. Pratley, G.H. Lee, Y. Zhang, H. Fei, S. Kim, R. Lallone, S. Ranganathan, P.A. Kern, J.M. Friedman, Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight reduced subjects, Nat. Med. 1 (1995) 1155–1161. P. Brouckaert, C. Libert, B. Everaerdt, N. Takahashi, A. Cauwels, W. Fiers, Tumor necrosis factor, its receptors and the connection with interleukin 1 and interleukin 6, Immunobiology 187 (1993) 317–329. K. Hotta, T. Funahashi, Y. Arita, M. Takahashi, M. Matsuda, Y. Okamoto, H. Iwahashi, H. Kuriyama, N. Ouchi, K. Maeda, M. Nishida, S. Kihara, N. Sakai, T. Nakajima, K. Hasegawa, M. Muraguchi, Y. Ohmoto, T. Nakamura, S. Yamashita, T. Hanafusa, Y. Matsuzawa, Plasma concentration of a novel adipose-specific protein, adiponectin, in type2 diabetic patients, Arterioscler. Thromb. Vasc. Biol. 20 (2000) 1595–1599.

[26] K. Yagi, Lipid peroxides and human diseases, Chem. Phys. Lipids 45 (1987) 337– 351. [27] E. Beutler, O. Duron, B. Mikus-Kelly, Improved method for determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [28] A. Sinha, Colorimetric assay of catalase, Anal. Biochem. 47 (1972) 389–394. [29] J. Sambrook, D.W. Russell, Molecular Cloning, A Laboratory Manual, vol. I, II, III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001. [30] F. Zhang, C. Ye, G. Li, W. Ding, W. Zhou, H. Zhu, G. Chen, T. Luo, M. Guang, Y. Liu, D. Zhang, S. Zheng, J. Yang, Y. Gu, X. Xie, M. Luo, The rat model of type 2 diabetic mellitus and its glycometabolism characters, Exp. Anim. 52 (2003) 401–407. [31] S. Lenzen, The mechanisms of alloxan- and streptozotocin-induced diabetes, Diabetologia 51 (2008) 216–226. [32] V. Vats, S. Yadav, J. Grover, Ethanolic extract of Ocimum sanctum leaves partially attenuates streptozotocin-induced alterations in glycogen content and carbohydrate metabolism in rats, J. Ethnopharmacol. 90 (2004) 155–160. [33] S. Asgary, G. Naderi, S. Zadegan, R. Vakili, The inhibitory effects of pure flavonoids on in vitro protein glycosylation, J. Herb. Pharmacother. 2 (2002) 47–55. [34] I. Raz, R. Eldor, S. Cernea, E. Shafrir, Diabetes: insulin resistance and derangements in lipid metabolism. Cure through intervention in fat transport and storage, Diabetes Metab. Res. 21 (2005) 3–14. [35] R. Schmatz, L.B. Perreira, N. Stefanello, C. Mazzanti, R. Spanevello, J. Gutierres, M. Bagatini, C.C. Martins, F.H. Abdalla, J. Daci da Silva Serres, D. Zanini, J.M. Vieira, A.M. Cardoso, M.R. Schetinger, V.M. Morsch, Effects of resveratrol on biomarkers of oxidative stress and on the activity of delta aminolevulinic acid dehydratase in liver and kidney of streptozotocin-induced diabetic rats, Biochimie 94 (2012) 374–383. [36] A.M. Distasi, C. Mallozzi, G. Macchia, T.C. Petrucci, M. Minetti, Peroxynitrite induces tyrosine nitration and modulates tyrosine phosphorylation of synaptic proteins, J. Neurochem. 73 (1999) 727–735. [37] M. Brownlee, Biochemistry and molecular cell biology of diabetic complications, Nature 13 (1993) 813–820. [38] J.P. Kirwan, S. Hauguel-De Mouzon, J. Lepercq, J.C. Challier, L. Huston-Presley, J. F. Friedman, S.C. Kalhan, P.M. Catalano, TNF-a is a predictor of insulin resistance in human pregnancy, Diabetes 51 (2002) 2207–2213. [39] S. Chatterjee, D. Ganini, E.J. Tokar, A. Kumar, S. Das, J. Corbett, M.B. Kadiiska, M. P. Waalkes, A.M. Diehl, R.P. Mason, Leptin is key to peroxynitrite-mediated oxidative stress and Kupffer cell activation in experimental non-alcoholic steatohepatitis, J. Hepatol. 58 (2013) 778–784. [40] L. Rui, V. Aguirre, J.K. Kim, G.I. Shulman, A. Lee, A. Corbould, A. Dunaif, M.F. White, Insulin/IGF-1 and TNF-alpha stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways, J. Clin. Invest. 107 (2001) 181–189. [41] N. Stefan, B. Vozarova, T. Funahashi, Y. Matsuzawa, C. Weyer, R.S. Lindsay, J.F. Youngren, P.J. Havel, R.E. Pratley, C. Bogardus, P.A. Tataranni, Plasma adiponectin concentration is associated with skeletal muscle insulin receptor tyrosine phosphorylation, and low plasma concentration precedes a decrease in whole-body insulin sensitivity in humans, Diabetes 51 (2002) 1884–1888. [42] P. Day, J. Cleal, E. Lofthouse, M. Hanson, R. Lewis, What factors determine placental glucose transfer kinetics? Placenta 34 (2013) 953–958. [43] B. Dallaqua, F.H. Saito, T. Rodrigues, I.M. Calderon, M.V. Rudge, E. Herrera, D.C. Damasceno, Treatment with Azadirachta indica in diabetic pregnant rats: negative effects on maternal outcome, J. Ethnopharmacol. 143 (2012) 805–811.