Scholarly article on topic 'Potential impact of Paracentrotus lividus extract on diabetic rat models induced by high fat diet/streptozotocin'

Potential impact of Paracentrotus lividus extract on diabetic rat models induced by high fat diet/streptozotocin Academic research paper on "Biological sciences"

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{T1DM / T2DM / Hyperglycemia / " Paracentrotus lividus extract" / "Lipid profile" / "Liver toxicity" / "Oxidative stress"}

Abstract of research paper on Biological sciences, author of scientific article — Amel M. Soliman

Abstract Antioxidant therapy has been thought to be effectual for the prevention and treatment of various diseases including diabetes. Therefore, the present study was designed to investigate the potency of Paracentrotus lividus extract (PLE) for alleviating the complications that resulted after induction of the diabetic rat models (T1DM and T2DM) using high fat diet (HFD)/streptozotocin (STZ). Thirty six male Wistar albino rats were assigned into normal control, T1DM and T2DM untreated, and PLE treated diabetic rat groups. Induction of T1DM was performed by streptozotocin injection (60mg/kg of dissolved in sodium citrate buffer, 0.1mol/L, i.p). T2DM induction through 4weeks of high fat diet (HFD) intervention was followed by a single low dosage of STZ (30mg/kg dissolved in 0.1mol/L citrate buffer at pH 4.5, i.p). Both diabetic rat models showed a significant increase in serum; levels of fasting glucose, total protein, bilirubin, activities of arginase, transaminases (AST and ALT), alkaline phosphatase (ALP), γ glutamyl transferase (GGT), lipid profile parameters, and liver malondialdehyde (MDA). However, T1DM and T2DM rats have decreased levels of serum insulin, and liver glucose 6 phosphate dehydrogenase (G6PD), glutathione reduced (GSH), nitric oxide (NO), and antioxidant enzymes. Furthermore, the present study showed the hypoglycemic, hypolipidemic, and antioxidant potency of the PLE as confirmed by its ability for ameliorating most of the alterations caused in the studied parameters of diabetic rats. In conclusion, PLE may be useful as therapy against oxidative stress and liver damage in both types of diabetes mellitus and is therefore recommended for further studies.

Academic research paper on topic "Potential impact of Paracentrotus lividus extract on diabetic rat models induced by high fat diet/streptozotocin"

The Journal of Basic & Applied Zoology (2016) 77, 8-20

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The Egyptian German Society for Zoology

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Potential impact of Paracentrotus lividus extract c^Ma* on diabetic rat models induced by high fat diet/streptozotocin

Amel M. Soliman

Zoology Department, Faculty of Science, Cairo University, Egypt

Received 19 November 2015; revised 1 January 2016; accepted 14 January 2016

KEYWORDS

T1DM; T2DM;

Hyperglycemia; Paracentrotus lividus extract; Lipid profile; Liver toxicity; Oxidative stress

Abstract Antioxidant therapy has been thought to be effectual for the prevention and treatment of various diseases including diabetes. Therefore, the present study was designed to investigate the potency of Paracentrotus lividus extract (PLE) for alleviating the complications that resulted after induction of the diabetic rat models (T1DM and T2DM) using high fat diet (HFD)/streptozotocin (STZ). Thirty six male Wistar albino rats were assigned into normal control, T1DM and T2DM untreated, and PLE treated diabetic rat groups. Induction of T1DM was performed by streptozo-tocin injection (60mg/kg of dissolved in sodium citrate buffer, 0.1 mol/L, i.p). T2DM induction through 4 weeks of high fat diet (HFD) intervention was followed by a single low dosage of STZ (30mg/kg dissolved in 0.1 mol/L citrate buffer at pH 4.5, i.p). Both diabetic rat models showed a significant increase in serum; levels of fasting glucose, total protein, bilirubin, activities of argi-nase, transaminases (AST and ALT), alkaline phosphatase (ALP), c glutamyl transferase (GGT), lipid profile parameters, and liver malondialdehyde (MDA). However, T1DM and T2DM rats have decreased levels of serum insulin, and liver glucose 6 phosphate dehydrogenase (G6PD), glutathione reduced (GSH), nitric oxide (NO), and antioxidant enzymes. Furthermore, the present study showed the hypoglycemic, hypolipidemic, and antioxidant potency of the PLE as confirmed by its ability for ameliorating most of the alterations caused in the studied parameters of diabetic rats. In conclusion, PLE may be useful as therapy against oxidative stress and liver damage in both types of diabetes mellitus and is therefore recommended for further studies.

© 2016 The Egyptian German Society for Zoology. Production and hosting by Elsevier B.V. This is an

open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Diabetes is really a devastating epidemic of the 21st century and is becoming the third killer of the health of human beings after cancer, cerebrovascular and cardiovascular diseases. Not

Peer review under responsibility of The Egyptian German Society for Zoology.

only it takes a heavy toll of lives around the world but imposes a serious financial burden on the sufferers and their family members (Bhattacharjee et al., 2014). Diabetes mellitus (DM) is a common metabolic disease with many side effects (Ziaee et al., 2013; Pang et al., 2015). Diabetes is characterized by hyperglycemia resulting in insulin resistance and/or insulin secondary deficiency caused by the failure of beta- (b-) pancreatic cells (Damasceno et al., 2014). Diabetes caused impaired

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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

metabolism of proteins and lipids (Hosseini et al., 2014). Several experimental models of type 1 and type 2 diabetes are available in rats (Fuentes-Antras et al., 2015). Type 1 diabetes mellitus (T1DM) is one of the most prevalent autoimmune diseases in the western world (van den Brandt et al., 2010). Type 1 diabetic patients often present acute symptoms of diabetes and markedly increased glucose levels and in some cases ketoacidosis appears (Damasceno et al., 2014). On the other hand, type 2 diabetes mellitus (T2DM) is characterized by reduced pancreatic beta-cell function and systemic insulin resistance, leading to metabolic dysfunction throughout the body (Onur et al., 2014). The beta cells normally compensate insulin resistance by secreting larger amounts of insulin to maintain the glucose homeostasis (Riguera, 1997). In the course of time, however, this beta cell function gets impaired leading to deterioration in glucose homeostasis and subsequent development of impaired glucose tolerance and frank diabetes (Lebovitz and Banerji, 2004). Type 2 diabetes is frequently not diagnosed until complications appear (Damasceno et al., 2014).

Streptozotocin (STZ) is an antibiotic produced by Strepto-myces achromogenes and is also used as an FDA-approved drug in the metastatic cancer of pancreatic islets cells (Kahraman et al., 2015). It inhibits glucose oxidation and glucose-induced insulin secretion in beta cells via nitric oxide production, alkylation, and DNA fragmentation (Lenzen, 2008). STZ leads to kidney and liver toxicity as well as beta cell damage (Dufrane et al., 2006). STZ is widely used in studies of experimental type-1 diabetes because it selectively destroys pancreatic b cells through the generation of ROS and alkyla-tion of deoxyribonucleic acid (DNA) (Lenzen, 2008). The use of fat-fed/STZ-treated rat models imitates natural disease incidence and metabolic characteristics typical of persons at increased risk of type 2 diabetes because of both insulin resistance and obesity (Srinivasan et al., 2005).

Liver as the major target organ of insulin plays important roles in the development of insulin resistance and type 2 diabetes mellitus, and the underlying mechanisms are still not fully understood (Leng et al., 2014). Previous clinical studies documented liver disease as a major cause of mortality in patients with DM (Clouston and Powell, 2004; Jin et al., 2005). The scope of liver disease in DM includes the nonalcoholic fatty liver disease (NAFLD), characterized by fat accumulation in hepatocytes. Conditions like hypertriglyc-eridemia and hypercholesterolemia are described as a cause ofNAFLD (Al-Jameil et al., 2014). Several organizations, recommend that lifestyle modifications, such as nutrition therapy, has been shown to help some patients to achieve better lipid levels (Jaiswal et al., 2014). In addition, hyperglycemia in diabetic patients is associated with alteration in glucose and lipid metabolism and modification in liver enzyme levels (Jenson et al., 1998).

Hyperglycemia has been identified as a major cause for reactive oxygen species (ROS) generation (Forbes et al., 2008). Oxidative stress is currently suggested as the mechanism underlying diabetes and diabetic complications (Cade, 2008; Lupachyk et al., 2013). Oxidative stress results from an imbalance between radical generating and radical scavenging mechanisms i.e. increased free radical production or abridged activity of antioxidant defenses or both (Ahmed et al., 2014). Szkudelski (2001) reported that oxidative stress is increased in experimental models of streptozotocin (STZ)-induced diabetes mellitus in rats. DM impaired glutathione metabolism,

and caused alterations in the antioxidant enzymes and generation of lipid peroxides (McLennan et al., 1991; Strain, 1991).

Increased oxidative stress has been implicated in the etiology (especially type 1) and pathology (both type 1 and type 2) of diabetic complications (Robertson and Harmon, 2006; Rolo and Palmeira, 2006). Pancreatic beta cells exposed to hyperglycemia and reactive oxygen species displayed reduced insulin secretion and increased insulin resistance (Sakai et al., 2003). It is therefore suggested that suppression of oxidative stress in beta cells may prevent or delay the onset of type 1 and progression of type 2 diabetes and related complications. Several studies have also shown that treatment with antioxidants protects against the onset of diabetes (Kaneto et al., 1999; Yu et al., 2006).

The handling of this disorder requires increased physical activity, healthy eating or diet and administration of anti-diabetic drugs and/or insulin. However, the currently available anti-diabetic drugs are far from being satisfactory. This may partly be attributed to the fact that diabetes is a disorder with multifactorial and heterogeneous etiologies (Erejuwa, 2014). Besides, these agents are costly and, in some cases, not readily available. Although several synthetic hypoglycemics are developed the safe and effective treatment paradigm is yet to be developed (Bhattacharjee et al., 2014). Therefore, a large percentage of the populations are resorting to complementary and alternative medicine (CAM) (Nahas and Moher, 2009). Insulin replacement therapy is the mainstay of treatment in patients with type 1 diabetes while type 2 diabetes should be regarded as a potentially preventable disease (Bastaki, 2005). Consequently, antioxidant therapy has been thought to be effectual for the prevention and treatment of various diseases including diabetes, because oxidative stress plays a key role in the pathogenesis of human diseases (Medina and Moreno-Otero, 2005).

In recent years, great attention has been paid to study the bioactivity of natural products due to their potential pharmacological utilization. However, majority of marine organisms are yet to be screened for discovering useful antibiotics (Bragadeeswaran et al., 2013). Marine organisms represent an excellent source for bioactive compounds (Bickmeyer et al., 2005) and modern technologies have opened vast areas of research for the extraction of biomedical compounds from ocean and seas to treat the deadly diseases (Bragadeeswaran et al., 2013).

Sea urchins are spiny-skinned marine invertebrates with a global distribution (Chung, 2013). It has shown that the use of sea urchin shells confers certain beneficial advantages, including antioxidant and pharmaceutical effects (Kim et al., 2002; Shankarlal et al., 2011). In sea urchin gonads polyhy-doxylated naphthoquinone, echinochrome A, of which potent antioxidant activity has been reported (Lebedev et al., 2001). It was reported that 3-sulfonoquinovosyl-1-monoacylglycerol extracted from sea urchin intestine was effective in suppressing the growth of solid tumors (Sahara et al., 1997). There are much valuable information for new antibiotic discoveries and give new insights into bioactive compounds in sea urchin (Bragadeeswaran et al., 2013). Sea urchins have therefore received increased attention as a possible source of antibiotic replacements (Chung, 2013).

It was proposed here that the hyperglycemia-induced activation of stress pathways plays a key role in the development of not only the late complications in type 1 and type 2 diabetes,

but also the insulin resistance and impaired insulin secretion seen in type 2 diabetes. Therefore, the present study aims to evaluate the hypoglycemic, hypolipidemic and antioxidant effects of the sea urchin (Paracentrotus lividus) soft body methanolic extract on T1DM and T2DM rat models.

Materials and methods

Chemicals and reagents

Streptozotocin, dimethyl sulfoxide (DMSO) and insulin kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Kits for most of the biochemical analysis determinations were purchased from the Biodiagnostic Company (Dokki, Giza, Egypt). The kit for y-glutamyl transferase analysis was purchased from Spectrum Company (Obour City, Cairo, Egypt).

Sea urchins collection

Sea urchins (P. lividus) were collected from the Mediterranean Coast of Alexandria (Egypt) and transported to the laboratory packed in ice. The samples were thoroughly washed with sea water to remove sand and overgrowing organisms at the collection site and transported to the laboratory. The collected specimens identified by the standard literature of taxonomic guide by Clark and Rowe (1971).

Preparation of the P. lividus extracts (PLE)

The sea urchins soft body parts (Gonads, mouth part and gut) were pooled and washed with a stream of cold water, air-dried at 4 0C for 2 days in the dark and then ground. The samples were then stored in the dark at room temperature to avoid photolysis and thermo-degradation of secondary metabolites prior to extraction. Samples (4 g) were homogenized and extracted with 10 volumes (v/w) of 70% (v/v) methanol. The supernatant for each sample was collected by centrifugation (12,000g, 5 min, and 4 0C) and stored at —20 0C. The supernatant of each extract was then filtered through a 0.2 im Mil-lipore filter (Nalge, Rochester, NY, USA).The filtrate was collected immediately, concentrated and lyophilized using lyo-philizer (LABCONCO lyophilizer, shell freeze system, USA).

Experimental animals

The experimental animals used in this study were male Wistar rats (Rattus norvegicus) weighing 150-160 ± 5 g. The animals were obtained from the National Research Center (NRC, Dokki, Giza). Animals were grouped and housed in polyacrylic cages in the well - ventilated animal house of the Zoology Department, Faculty of Science, Cairo University. Animals were given food and water ad libitum. Rats were maintained in a friendly environment of a 12-h/12-h light-dark cycle at room temperature (22-25 0C). Rats were acclimatized to laboratory conditions for 7 days before commencement of the experiment.

Acute oral toxicity study

Acute toxicity studies were performed according to the Organization of Economic Cooperation and Development

(OECD) guideline 425 (OECD, 2001). Ten healthy male albino rats, fasted overnight, were divided into two groups with five animals in each group. The first group received distilled water and served as control. The second group was administered methanol extract of P. lividus (PLE) in suspension at a limit test dosage of 5000 mg/kg body weight. All doses were administered by gastric gavage, and each dose was adjusted to be 2 ml/kg. The animals were observed post dose at 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, and thereafter every day for 14 days.

Induction of type 1 diabetes

All animals were starved for 12 h before the experiment, but were allowed free access to water. Diabetes was induced by intraperitoneal injection of streptozotocin (60 mg/kg dissolved in sodium citrate buffer, 0.1 mol/L). Blood glucose levels were measured 72 h after injection of STZ. Animals were starved, but had access to drinking water for 6 h before blood glucose measurement. Plasma glucose concentrations P300 mg/100 ml were considered diabetic type 1 in this experiment (Chen et al., 2014).

Induction of type 2 diabetes

Following 4 weeks of high fat diet (HFD) (with energy of 5.3 kcal/g, comprising 60% calories from fat, 35% from protein and 5% from carbohydrate) intervention (Reed et al., 2000), the rats were injected intraperitoneally by a single dose of prepared solution of STZ (30 mg/kg suspended in 0.1 mol/L citrate buffer at pH 4.5). If the fasting blood glucose (FBG) was more than 300 mg/100 ml after 72 h of STZ injection, the diabetic type 2 model was successful (Ebaid, 2014).

Experimental design

After one week of acclimatization, Thirty six male Wistar albino rats were assigned into two main experiments:

A. Experiment I (T1DM): Eighteen rats were divided into 2 groups

Group 1: Served as normal control (NC). Six rats received 1 ml (10% DMSO, orally) daily for 15 days after a single dose of citrate buffer (0.1 mol/L, i.p). Group 2: Twelve rats were used for T1DM induction. Then six diabetic rats received 1 ml (10% DMSO, orally) (DC), and the rest of rats received 1 ml PLE dissolved 10% DMSO (500 mg/kg body weight, orally), daily for 15 days.

B. Experiment II (T2DM): Eighteen rats were divided into 2 groups

Group 1: Rats (6 rats) consumed a regular diet without STZ injection. After 4 weeks of normal diets, it received 1 ml (10% DMSO, orally) daily for 15 days after a single dose of citrate buffer (0.1 mol/L, i.p) (NC). Groups 2: Twelve rats were used for T2DM induction. Half of the diabetic rats received 1 ml (10% DMSO, orally) (DC), and the others received 1 ml of PLE (500 mg/kg body weight, orally), daily for 15 days.

Animal handling

At the end of each experiment, the fasted rats were euthanized after being anesthetized with 3% sodium pentobarbital and the chest was opened. A needle was inserted through the diaphragm and into the heart. Negative pressure was gently applied once the heart had been punctured, and the needle was repositioned as required until blood flowed into the syringe. Blood samples were collected in centrifuge tubes for biochemical analysis. Liver and pancreas samples were quickly removed washed with physiological saline to remove traces of blood. Part of the liver was stored at —80 0C for biochemical analysis.

Sample preparation

Serum preparation

Blood samples were collected in centrifuge tubes without anticoagulant, allowed to clot, and centrifuged at 3000 rpm/min for 20 min. The serum obtained was stored at —20 0C until used for biochemical assays.

Liver tissue homogenate preparation

Liver tissue was homogenized (10% w/v) using a Potter Elve-hjem homogenizer (Sigma Aldrich) in ice-cold 0.1 M Tris-HCl buffers (pH 7.4). The homogenate was centrifuged at 860g for 15 min. at 4 0C, and the resultant supernatant was used for different assays.

Biochemical assays

Serum glucose was determined according to Trinder (1969) method, serum arginase (Marsch, 1965), serum insulin (Herbert et al., 1965), aminotransferase enzyme activities (alanine transaminase (ALT) and aspartate transaminase (AST)) (Reitman and Frankel, 1957), alkaline phosphatase (ALP) activity (Belfield and Goldberg, 1971), c-glutamyl transferase (GGt) activity (Szasz, 1974), total protein (Henry, 1964), total and direct bilirubin levels (Walter and Gerade, 1970), total lipids ((Knight et al., 1972), triglycerides (Fossati and Prencipe, 1982), total cholesterol (Allain et al., 1974), high density lipoprotein (HDL) level (Lopez-Virella et al., 1977), and low density lipoprotein (LDH) level (Wieland and Seidel, 1983), liver glucose 6 phosphate dehydrogenase (G6PD) (Kornberg and Horecker, 1955), liver lipid peroxidation (LPO), which was measured by the formation of malondialde-hyde (MDA) (Ohkawa et al., 1979), liver reduced glutathione (GSH) (Aykac et al., 1985), liver nitric oxide (NO) (Montgomery and Dymock, 1961), liver glutathione-S-transferase (GST) (Habig et al., 1974), liver glutathione perox-idase (GPx) (Paglia and Valentine, 1967), liver superoxide dismutase (SOD) (Nishikimi et al., 1972), and liver catalase (CAT) (Aebi, 1984).

Statistical analysis

Results were expressed as mean ± standard error% improvement = treated mean — model mean/control mean x 100%. All data obtained were analyzed by analysis of variance, followed by Student's t test at 95% confidence level. Values of p < 0.05 were considered as statistically significant. All

computations were performed using Statistical Package for Social Sciences version 15.0 software.

Results

Acute oral toxicity test

Single oral administration of P. lividus extract (PLE) (5000 mg/kg body weight) did not show any visible signs of toxicity, abnormal behaviors, or mortality, which indicated that the median lethal dose (LD50) of PLE was higher than 5000 mg/kg body weight. The effective dose (500 mg/kg body weight) was selected based on this proposed LD50.

Diagnostic markers of diabetic

Table 1 showed that serum glucose level and arginase activity of both T1DM and T2DM rats were significantly increased (p < 0.05), as compared to the corresponding control rats. On the other hand, insulin concentration and liver G6PD activity were decreased significantly (p < 0.05), as compared to the corresponding ones of the control groups.

Treatment of T1DM and T2DM rats with PLE (500 mg/kg body weight) significantly (p < 0.05), decreased glucose level and arginase activity as well as increasing the insulin concentration and liver G6PD activity as compared to the corresponding ones of diabetic rats. It was evidenced from the % of improvement calculation that PLE treatment showed highest amelioration regarding T1DM than those in T2DM (Table 1).

Liver function markers

Induction of both types of diabetes caused a significant increase (p < 0.05) in the activities of AST, ALT, ALP, GGT, and levels of total and indirect bilirubin of rats except for the AST of T2DM rats (non significantly changed), as compared to the corresponding control ones (Tables 2 and 3). Significant decrease (p < 0.05) was noticed in the levels of total protein of both T1DM and T2DM rats, as compared to the corresponding control ones (Table 3).

Serum activities of ALT and ALP of both diabetic rat models were significantly decreased (p < 0.05) after 15 days of PLE treatment, as compared to the corresponding DM rats. Moreover, PLE treatment was found more effective to improve the ALT and ALP activities of T1DM rats than T2DM rats (Table 2). Meanwhile, PLE treatment significantly decreased (p < 0.05) the AST activity of T1DM rats and GGT activity of T2DM rats with% of improvement of —225.95 and —2634.73, respectively (Table 2). On the other hand, a significant increase (p < 0.05) was shown in the total protein, total and direct bilirubin levels of T1DM and T2DM rats and indirect bilirubin of T2DM rats only after PLE administration for 15 days, as compared to the corresponding DM groups (Table 3).

Lipid profile markers

Data represented in Table 4 showed that the serum levels of total lipids, total cholesterol, and LDL-cholesterol of both

Table 1 The curative impact of Paracentrotus lividus extract (PLE) on the diabetic markers of the high fat diet/streptozotocin models of diabetes in rats.

Diabetic markers Serum glucose (mg/dl) Serum insulin (lU/ml) Serum arginase (U/L) Liver G6PD (U/min/g protein)

T1DM Control DM model PLE % improvement 89.83 ± 10.14a 368.67 ± 53.27c 138.67 ± 20.13ab -256.03 17.00 ± 0.21d 3.10 ± 0.55a 7.08 ± 0.51b 23.41 106.67 ± 1.58a 1.15 ± 0.07c 195.67 ± 15.70d 0.36 ± 0.08a 151.00 ± 16.11bc 0.66 ± 0.08b -41.87 26.09

T2DM Control DM model PLE % improvement 127.50 ± 6.00ab 315.67 ± 7.20c 180.33 ± 19.12b -106.14 18.00 ± 0.50d 5.99 ± 0.65b 9.67 ± 0.40c 20.44 117.17 ± 3.36a 1.55 ± 0.11d 163.33 ± 6.69c 0.21 ± 0.02a 124.67 ± 8.69ab 0.65 ± 0.16b -32.99 28.39

Values are given as mean ± SEM for 6 rats in each group. Each value not sharing a common letter superscript is significantly different (p < 0.05). DM: diabetes mellitus, G6PD: glucose 6 phosphate dehydrogenase, PLE: Paracentrotus lividus extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus.

Table 2 The curative impact of Paracentrotus lividus extract (PLE) on some serum enzymes (liver function markers) of the high fat diet/streptozotocin models of diabetes in rats.

Liver function markers s.AST (U/ml) s.ALT (U/ml) s.ALP (IU/L) s.GGT (U/L)

T1DM Control DM model PLE % improvement 28.25 ± 1.58ab 92.75 ± 18.17c 28.92 ± 2.15ab -225.95 14.17 ± 3.00a 54.54 ± 6.73c 21.83 ± 2.77a -230.84 88.33 ± 7.06ab 1.16 ± 0.14a 373.33 ± 11.77e 13.74 ± 0.57b 178.33 ± 31.66d 5.32 ± 1.95ab —220.76 —702.59

T2DM Control DM model PLE % improvement 20.17 ± 0.76ab 45.00 ± 1.00b 27.63 ± 1.37ab -86.12 23.58 ± 1.36a 70.83 ± 4.98d 42.17 ± 1.88b —121.54 77.00 ± 13.74a 1.67 ± 0.21a 143.33 ± 6.48cd 46.33 ± 8.91c 92.17 ± 13.84ab 2.33 ± 0.21a —66.44 —2634.73

Values are given as mean ± SEM for 6 rats in each group. Each value not sharing a common letter superscript is significantly different (p < 0.05). s.AST: serum aspartate aminotransaminase, s.ALT: serum alanine aminotransaminase, s.ALP: serum alkaline phosphatase, s.GGT: serum gamma glutamyltransferase, DM: diabetes mellitus, PLE: Paracentrotus lividus extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus.

Table 3 The curative impact of Paracentrotus lividus extract (PLE) on some serum parameters (liver function markers) of the high fat

diet/streptozotocin models of diabetes in rats.

Serum parameters Total protein (g/dl) Total bilirubin (mg/dl) Direct bilirubin (mg/dl) Indirect bilirubin (mg/dl)

Control 6.94 ± 0.75c 5.38 ± 0.34a 4.98 ± 0.19a 0.40 ± 0.16a

DM model 3.22 ± 0.27a 6.15 ± 0.12b 5.20 ± 0.42a 0.95 ± 0.20b

PLE 5.11 ± 0.64b 6.51 ± 0.06c 5.50 ± 0.11b 0.99 ± 0.27b

% improvement 27.23 6.69 6.02 10.00

Control 6.00 ± 0.37b 5.65 ± 0.09a 5.53 ± 0.35a 0.12 ± 0.23a

DM model 3.17 ± 0.31a 6.22 ± 0.09b 5.62 ± 0.40a 0.60 ± 0.14b

PLE 5.67 ± 0.42bc 6.72 ± 0.12c 5.82 ± 0.10b 0.88 ± 0.19c

% improvement 41.67 8.85 3.61 233.33

Values are given as mean ± SEM for 6 rats in each group.

Each value not sharing a common letter superscript is significantly different (p < 0.05). DM: diabetes mellitus, PLE: Paracentrotus lividus

extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus.

diabetic rat models and triglycerides concentration of T2DM rats were significantly increased (p < 0.05). However, a significant decrease (p < 0.05) was noticed in the level of HDL-cholesterol of both diabetic rat models, as compared to the corresponding control rats.

PLE treatment caused a significant decrease (p < 0.05) in the levels of total cholesterol and LDL-cholesterol of both diabetic rat models and only the total lipids level of T1DM as well as the concentration of triglycerides of T2DM rats, as compared to the corresponding DM ones (Table 4). In addition,

Table 4 The curative impact of Paracentrotus lividus extract (PLE) on some serum lipid profile markers of the high fat diet/

streptozotocin models of diabetes in rats.

Lipid profile Total lipids (mg/dl) Triglycerides (mg/dl) Total cholesterol (mg/dl) LDL-cholesterol (mg/dl) HDL-cholesterol (mg/dl)

Control 143.17 ± 6.52a 129.00 ± 2.31a 165.67 ± 8.67a 131.67 ± 3.33ab 60.83 ± 2.20d

DM model 310.17 ± 28.17c 152.50 ± 4.72ab 227.17 ± 16.83b 190.50 ± 15.48c 44.41 ± 0.38a

PLE 109.00 ± 21.40a 142.33 ± 12.33ab 171.67 ± 6.90a 132.00 ± 9.36ab 54.35 ± 4.77bcd

% improvement -140.51 -7.88 -33.50 -44.43 16.34

Control 101.83 ± 15.85a 157.17 ± 9.66bc 168.83 ± 11.24a 120.67 ± 8.02a 60.04 ± 2.08d

DM model 249.50 ± 17.75b 230.50 ± 9.63e 211.50 ± 5.79b 153.67 ± 3.77b 47.23 ± 1.54abc

PLE 230.67 ± 9.28b 173.50 ± 1.73cd 174.50 ± 5.97a 125.33 ± 4.22a 55.32 ± 3.25cd

% improvement -18.49 -36.27 -21.92 -23.49 13.47

Values are given as mean ± SEM for 6 rats in each group.

Each value not sharing a common letter superscript is significantly different (p < 0.05). DM: Diabetes mellitus, PLE: Paracentrotus lividus

extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus.

Table 5 The curative impact of Paracentrotus lividus extract (PLE) on streptozotocin models of diabetes in rats. liver oxidative stress markers of the high fat diet/

Oxidative stress markers MDA (nmol/g protein) GSH (mg/g protein) NO (imol/L)

T1DM Control DM model PLE % improvement 12.90 ± 1.37a 25.47 ± 2.19c 22.77 ± 0.74bc -20.93 7.72 ± 0.64cd 3.12 ± 0.53a 3.91 ± 0.67ab 10.23 37.83 ± 2.40d 22.33 ± 1.20a 28.83 ± 2.76bc 17.18

T2DM Control DM model PLE % improvement 20.00 ± 2.02b 30.33 ± 2.01d 23.17 ± 1.99bc -35.80 11.33 ± 0.67e 3.00 ± 0.77a 8.00 ± 0.37cd 44.13 32.33 ± 3.07bc 28.17 ± 0.31ab 26.67 ± 1.78ab -4.64

Values are given as mean ± SEM for 6 rats in each group. Each value not sharing a common letter superscript is significantly different (p < 0.05). DM: diabetes mellitus, PLE: Paracentrotus lividus extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus, MDA: malondialdehyde, GSH: glutathione reduced, NO: nitric oxide.

Table 6 The curative impact of Paracentrotus lividus extract (PLE) on liver oxidative stress markers of the high fat diet/

streptozotocin models of diabetes in rats.

Oxidative stress markers GST (U/ g protein) GPs (U/g tissue) SOD (U/g protein) CAT (U/g protein)

Control 3.57 ± 0.15cd 5.95 ± 0.90c 78.05 ± 3.16c 1.93 ± 0.02c

DM model 0.90 ± 0.23a 2.60 ± 0.55ab 47.96 ± 7.82b 1.61 ± 0.05a

PLE 2.93 ± 0.19c 5.70 ± 0.60c 69.73 ± 5.11c 1.77 ± 0.05ab

% improvement 56.86 52.10 27.89 8.29

Control 3.97 ± 0.14d 6.13 ± 0.48c 71.17 ± 0.70c 1.86 ± 0.05bc

DM model 1.33 ± 0.40ab 1.33 ± 0.34a 17.00 ± 0.73a 1.68 ± 0.03ab

PLE 3.85 ± 0.63cd 4.36 ± 0.66ab 37.50 ± 4.14b 1.93 ± 0.04c

% improvement 63.48 49.43 28.80 13.44

Values are given as mean ± SEM for 6 rats in each group.

Each value not sharing a common letter superscript is significantly different (p < 0.05). DM: Diabetes mellitus, PLE: Paracentrotus lividus

extract, T1DM: type 1 diabetes mellitus, T2DM: type 2 diabetes mellitus, GST: glutathione S transferase, GPs: glutathione peroxidase, SOD:

superoxide dismutase, CAT: catalase.

PLE treatment significantly increased (p < 0.05) the HDL-cholesterol level of T1DM rats. PLE treatment was found to cause the highest improvement in the total lipids level of T1DM rats by -140.51%.

Liver oxidative stress markers

Experimentally induced T1DM and T2DM caused a significant increase (p < 0.05) in the level of liver MDA (Table 5).

However, a significant decrease (p < 0.05) was noticed in the levels of liver GSH, GST, GPs, and SOD of both diabetic rat models and the levels of liver NO and CAT of T1DM rats only, as compared to the control ones (Tables 5 and 6).

Treatment the T2DM rats with PLE for 15 days significantly decreased (p < 0.05) the MDA level with -35.80% improvement. On the other hand, PLE treatment caused a significant increase (p < 0.05) in the GST and SOD activities of both diabetic rat models (56.86%, 63.48%, 27.89%, 28.80% improvement, respectively), NO level and GPs activity of T1DM rats (17.18, 52.10% improvement, respectively), and GSH level and CAT activity of T2DM rats (44.13%, 13.44% improvement, respectively), as compared to the corresponding DM ones (Tables 5 and 6).

Discussion

Diabetes induced by streptozotocin alters the structure and function of the body including the liver cells (Chalfoun-Mounayar et al., 2012). Diabetes mellitus (DM) is associated with various structural and functional liver abnormalities, including non-alcoholic fatty liver disease (NAFLD) and hepatic glycogenosis (HG). NAFLD represents the most common liver disease associated with DM, especially in patients with type 2 diabetes (T2DM) and metabolic syndrome (Krishnan et al., 2013). Due to the high prevalence of diabetes worldwide, extensive research is still being performed to develop new antidiabetic agents and determine their mechanisms of action, consequently, a number of diabetic animal models have been developed and improved over the years (Islam and Loots, 2009). Although several synthetic antidiabetics are developed but the safe and effective treatment paradigm is yet to be introduced (Bhattacharjee et al., 2014).

The present experimental data showed that a high dose of STZ (60 mg/kg body weight) and a high-fat diet plus low dose of STZ (30 mg/kg body weight) administration can successfully induce T1DM and T2DM rat models, respectively. Type 1 and type 2 diabetes were established in the present study as fasting glucose level increased and insulin level decreased which may indicate impaired glucose tolerance (Bielohuby et al., 2013). In both type 1 and type 2 diabetes, diabetic complications in target organs arise from chronic elevations of glucose (Evans et al., 2003).The significant decrease in serum insulin concentration in the both diabetic rat models was due to the cytotoxic effects of STZ through the induction of free radicals that damaged the pancreatic b cells (Sakai et al., 2003; Aydin and Celik, 2012). On the other hand, PLE administration remarkably attenuated the high blood sugar and increased insulin levels. This ameliorative effect of the current extract may be due to the presence of saponins (Kihara et al., 1985). Saponins, compound with insulin-like properties, stimulate glucose uptake enhancing Glut4 expression, contributing to storage of glucose as glycogen in adipocytes (Elekofehinti et al., 2014). In accord with the present results, Oztiirk et al. (2015) investigated that some substances express anti-diabetic property by influencing cells to stimulate insulin secretion and restore insulin sensitivity.

Arginase is an intracellular enzyme that appears in the plasma only after cell damage or death. The type 1 isoform of arginase predominantly is located in the liver and kidney, while the type 2 isoform is predominantly found in endothelial

cells and can be induced in many cell types by a variety of inflammatory cytokine factors (Morris, 2002). Thus, chronic, low grade inflammation and liver disease are potential sources of elevated arginase activity in type 2 diabetes. In line with the previous investigation, there was an increase in the arginase activity regarding type 2 DM as well as T1DM rat models in the present study. Ramírez-Zamora et al. (2013) and Wei et al. (2013) have inspected the prospective role of arginase (including arginase I and II) in the pathogenesis of DM. Wang et al. (2014a,b) investigated the potential role of arginase I as a diagnostic or prognostic marker for type 2 DM. They added that the diabetic rats exhibited increased levels of arginase, which correlated with the blood glucose level and may contribute to the severity of DM in rats.

Other curative patterns, including aggravating control of hyperglycemia with insulin, should be assessed for their ability to increase arginine bioavailability and inhibited arginase activity (Kashyap et al., 2008). In the current study, administration of PLE for 15 days for both T1DM and T2DM rats caused a significant decrease in the arginase activity. Sarikaphuti et al. (2013) stated that inhibition of arginase has been shown to have a protective effect in DM rat models.

The adverse effect of diabetes mellitus on the liver is still unknown (Orasanu and Plutzky, 2009). The increased activities of serum transaminase enzymes (AST, ALT), alkaline phosphatase (ALP), gama glutamyl transferase (GGT) and total bilirubin levels of both T1DM and T2DM, which were used as markers of liver function, were found to be correlated with the increased arginase activity in the current study. Hepa-tocyte injury caused impairment in the liver cell membrane permeability. As a result, cytoplasmic enzymes such as transaminase (AST and ALT) leakage into the circulation and their activities in serum increase (Arun and Nalini, 2002). In addition, ALP is membrane bound and its alteration is likely to affect the membrane permeability and produce derangement in the transport of metabolites (Mehana et al., 2012). On the other hand, bilirubin value is associated with the function of hepatic cells (Muriel et al., 1992). Bilirubin is generated when the heme part of the hemoglobin has undergone degradation. This results in the formation of biliverdin, which is rapidly converted into bilirubin. In hepatocytes, unconjugated (lipid-soluble) bilirubin is conjugated by uridine diphosphate-glucuronosyl transferase (UDP-GT) to a water-soluble form for excretion. Total bilirubin is the sum of conjugated (direct) and unconjugated (indirect) bilirubin (Hull and Agarwal, 2014). However, unconjugated bilirubin levels have positive correlation with plasma antioxidant capacity, and moderate rises in serum total bilirubin have been linked with reduced capability to several common diseases (Vitek et al., 2002).

Two studies, relating serum ALT content to type 1 diabetes, found elevated levels of the enzyme in 10-35% of type 1 diabetes patients, respectively (West et al., 2006; Leeds et al., 2009). Another study found that ALT, AST, ALP, GGT, and bilirubin were used as indicators of NAFLD and they significantly correlated with insulin resistance in type 1 diabetes patients (Bulum et al., 2011). Furthermore, recognition of the role of the liver in the pathogenesis of type 2 diabetes has been increasing. Nonalcoholic fatty liver disease (NAFLD), characterized by elevated ALT, and GGT is now regarded as the hepatic manifestation of the insulin resistance syndrome (Marchesini and Forlani, 2002). Bell and Allbright (2007)

investigated that the prevalence of hepatobiliary diseases is increased in patients with either type 1 or type 2 diabetes.

The significant decrease of total protein level of diabetic rats in the present study may be due to the anabolic effect of insulin on protein metabolism as it stimulates protein synthesis and retards protein degradation (Murray et al., 2000). Protein synthesis is decreased in all tissues due to decreased ATP production in response to absolute or relative deficiency of insulin and alkaline phosphatase activity (Chatterjee and Rana, 1994). Mahboob et al. (2005) investigated decreased protein content of serum in diabetic patients indicating high rate of the lipid peroxidation process and decreased the antioxidant defensive system.

Treatment of both diabetic rat models with PLE caused a reduction in the activity of the enzymes (AST, ALT, ALP, GGT) and increased total protein level which may be due to the ability of PLE to counteract lipid peroxidation and perhaps heal the damaged cells (Akpan and Ekpo, 2015). Ozturk et al. (2015) results were in accord with the present study. In the present study, PLE treatment caused an unexpected increase in levels of total, direct and indirect bilirubin as compared to the corresponding diabetic rats. Vitek (2012) reported that bilirubin has been recognized as a substance with potent antioxidant properties. Although bilirubin was believed to be only a waste product of the heme catabolic pathway at best, and a potentially toxic compound at worst; recent data have convincingly demonstrated that mildly elevated serum bilirubin levels are strongly associated with a lower prevalence of oxidative stress-mediated diseases (Vitek, 2012). Furthermore, Liu et al. (2015) proved that unconjugated bilirubin mediates heme oxygenase-1 (HMOX1) which is a key antioxi-dant enzyme that has consistently been shown to protect from the development of diabetes (Ndisang, 2010). Therefore, the increased level of bilirubin after PLE treatment may be due to its enhancement effect on HMOX1 via bilirubin.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway (PPP). The major products of the PPP are ribose-5-phosphate, which is required for nucleic acid synthesis, and nicotinamide adenine dinucleotide phosphate (NADPH) generated from NADP by G6PD (Stanton, 2012). There has been a growing understanding of the central importance of G6PD to cellular physiology as it is a major source of NADPH that is required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system, and others. Indeed G6PD is essential for cell survival, since G6PD activity was found to be correlated directly with cell growth. Inhibition of G6PD activity prevented cell growth, and overexpression of G6PD alone stimulated cell growth (Tian et al., 1998). In the present study, liver glucose-6-phosphate dehydrogenase activity of both diabetic rat models decreased significantly. This decrease in liver G6PD activity was consistent with the decreased insulin levels in the current study since; insulin is the key stimulating factor for G6PD which aids in the utilization of glucose via pentose phosphate pathway (Tian et al., 1998). Similarly, significant decreases in G6PD activity were investigated due to hyperglycemia or diabetes in the liver, kidney, pancreas, and other tissues (Diaz-Flores et al., 2006; Zhang et al., 2010). West (2002) proposed that predisposition to diabetes may be due to alterations in gene controlling both insulin secretion and G6PD-mediated antioxidant defenses.

The increased liver G6PD activity subsequent to PLE treatment may be considered as an important therapeutic tactic for the prevention and treatment of diabetic complications (Stanton, 2012). It was concluded that increased G6PD activity was essential for preventing ROS-mediated cell death (Tian et al., 1999).

The liver is an insulin - dependent tissue that plays a significant role in glucose and lipid homeostasis and is severely affected in diabetes (Ozturk et al., 2015). Hyperlipidaemia has been reported to accompany hyperglycemia states (Taskinen, 1996). In the present study diabetic rats (T1DM and T2DM) showed high levels of total lipids, triglycerides (TG), total cholesterol (TC), LDL-cholesterol and a lower level of HDL-cholesterol. High levels of TC; importantly LDL cholesterol is one of the major coronary risk factors (Temme et al., 2002) which is the major cause of morbidity and deaths in diabetic subjects (Baynes, 1991). Similar results were previously reported (Gong et al., 2009; Lu et al., 2010). In addition, Lambert et al. (2013) have proposed that diabetic individuals have reduced synthesis and increased cholesterol absorption. Furthermore, it was concluded that in patients with reduced insulin secretion, the adipocytes may uncouple the adipose tissue from circulating fatty acids so that fatty acids are instead shunted to the liver, resulting in increased synthesis of triglycerides (Nielsen et al., 2014). Ribas et al. (2014) investigated that the increased total cholesterol could result in depletion of mitochondrial glutathione concentrations and oxidative stress.

P. lividus extract treatment to both T1DM and T2DM rats caused alleviations to all the studied lipid profile parameters and confirmed its hypolipidemic potency. This effect may be attributed to the presence of saponins which inhibited cholesterol and/or bile acid absorption (Elekofehinti et al., 2014). The observed hypolipidemic effect of PLE seems to be independent of insulin action and may be through the inhibition of the key enzymes on cholesterol and triglyceride synthesis (Zhang et al., 2002). Nevertheless, it cannot preclude the relation between the observed hypolipidemic effects and diabetes since the major determinant of total cholesterol and triglyceride is glycemic control (Dangi and Mishra, 2010).

Oxidative stress is referred to as a reactive oxygen species (ROS)/antioxidant inequality, occurs when the net amount of ROS exceeds the antioxidant potential. Thus, oxidative stress can occur because of a general increase in ROS generation, a drooping of the antioxidant systems, or both (Frances et al., 2013). It is well established that hyperglycemia excavates an increase in ROS production, which leads to oxidative stress in both type 1 and type 2 diabetes and causes antioxidant defense enzymes and vitamin deficiency (West, 2000). Frances et al. (2010) demonstrated that hyperglycemia increases the hydroxyl radical production in the liver of STZ-induced diabetic rats. In the present study, elevated malondialdehyde (MDA, end product of lipid peroxidation), decreased glutathione reduced (GSH) and nitric oxide (NO), and the inhibition of the antioxidant enzyme activities (glu-tathione S transferase (GST), glutathione peroxidase (GPs), superoxide dismutase (SOD), catalase (CAT)) are evidence of oxidative stress occurrence in the diabetic untreated rats.

Increased levels of liver MDA in diabetics suggest that per-oxidative injury may be involved in the development of liver damage (Srivastava et al., 2001). This marked increase in the lipid peroxidation rates in diabetic liver tissues suggests an

accumulation of oxygen free radicals which can be due to either increased production and/or decreased elimination (Sadi et al., 2013). GSH has free radical scavenging activity and it is involved in the removal of reactive intermediates in the presence of GPx and GSTs. Previous studies have shown that, hepatic GSH concentration of STZ induced diabetic rats significantly decreased (Maritim et al., 2003; Pari and Latha, 2005). NO has been stated to modulate insulin sensitivity and glucose disposal (Kapur et al., 1997), and its activity has been shown to be impaired by hyperglycemia and insulin resistance (Roy et al., 1998). The aforementioned data in the present study revealed that diabetic rats have increased arginase activity which converts L-arginine to urea and ornithine. Since, under conditions of low arginine levels which is the nitric oxide synthase (NOS) substrate, NOS is uncoupled, producing reactive oxygen species and oxidative stress instead of NO (Xia et al., 1996). Furthermore, insulin resistance in type 2 diabetes may contribute to reduced NOS activity by the generation of methylated arginine (Kashyap et al., 2008). The observed inhibition in the liver antioxidant enzyme activities (GST, GPx, SOD, CAT) in the current study may be due to down regulation of their expression genes that resulted from the excessive oxidative stress that occurred in the liver due to diabetes (Raza et al., 2000; Sadi et al., 2008).

Inhibition of oxidative stress with antioxidants might be potent for alleviating the complications of diabetes. Several investigators showed the beneficial effects of antioxidants in the treatment of diabetes (Maritim et al., 2003; Sadi and Guray, 2009). The present study revealed that PLE treatment of diabetic rats (T1DM and T2DM) ameliorated the lipid per-oxidation, which was assessed by the decreased liver MDA level, most probably by decreasing the cellular redox potential (Sadi et al., 2013). Moreover, PLE caused increased levels of liver GSH, NO, and antioxidant enzyme activity (GST, GPx, SOD, CAT) of diabetic rats. These results could confirm the antioxidant potency of PLE which was attributed to the presence of a biologically active pigment known as echino-chrome A, a polyhydroxy naphthoquinone isolated from the sea urchin that has antioxidant properties (Lebedev et al., 2005). Many in vivo and in vitro studies have shown that treatments targeting oxidative stress improve both b-cell function and survival (Wajchenberg, 2007). It has been found that using different antioxidants improve insulin sensitivity in experiments using animal models of diabetes or in vitro studies (Rudich et al., 1999; Maddux et al., 2001). Indeed, PLE and its content from the echinochrome pigment may be able to correct the deficient thiol status of cells by increasing de novo synthesis of GSH and hence increased the liver GSH levels in diabetic animals (Han et al., 1997). Interestingly, since G6PD is the main source of NADPH used by the cellular antioxidant systems, it can be inferred that the enhanced G6PD (aforementioned results) would have a highly beneficial, protective effect by increasing CAT, GPx and SOD activities, as the handling of hydrogen peroxide by either catalase or the glutathione system will involve in cellular defense against a wide variety of free radicals (Medina and Moreno-Otero, 2005; Zhang et al., 2010). In addition, several investigators showed that overexpression of antioxidants as GPx and CAT could protect islets against oxidative stress and provide protection against the deleterious effects of hyperglycemia (Benhamou et al., 1998; Tanaka et al., 2002; Moriscot et al., 2003).

Conclusion

P. lividus extract (PLE) was safe and able to protect diabetic oxidative stress and liver damage because it is rich in diverse antioxidants. The present study confirms that PLE exerted a hypoglycemic effect and improved the lipid profile. PLE efficiency in treating T1DM and T2DM was variable and depending on the estimated parameters. Furthermore, PLE may be useful as therapy against oxidative stress and liver damage in both types of diabetes mellitus and is therefore recommended for further studies. These effects should be studied further in human volunteers and diabetic patients.

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