Soliman AM; Mohamed AS; Marie M-AS

Research Article

Austin J Endocrinol Diabetes. 2016; 3(2): 1045.

Effect of Echinochrome on Body Weight, Musculoskeletal System and Lipid Profile of Male Diabetic Rats

Soliman AM, Mohamed AS* and Marie M-AS

Zoology Department, Cairo University, Egypt

*Corresponding author: AMohamed AS, Zoology Department, Faculty of Science, Cairo University, 12613- Egypt- 01275350954, Egypt

Received: June 13, 2016; Accepted: August 01, 2016; Published: August 08, 2016

Abstract

Background: Diabetes mellitus is associated with several musculoskeletal disorders and functional liver abnormalities that affect protein and lipid metabolism.

Objective: The present study was carried out to evaluate effect of echinochrome on musculoskeletal system and metabolism of lipids and proteins in both types of diabetes mellitus.

Methods: Thirty-six male Wistar albino rats were divided into two main groups, type 1 diabetes and type 2 diabetes groups. Each group divided into 3 subgroups (6 rats /subgroup); control, diabetic and echinochrome groups. Diabetic model was induced by single dose of streptozotocin (60 mg/kg, i.p) for type 1 diabetes and by high fat diet for 4 weeks before the injection with streptozotocin (30 mg/kg, i.p) for type 2 diabetes. Diabetic groups were treated orally with echinochrome extract (1mg/kg body weight in 10% DMSO) daily for 4 weeks.

Results:: Diabetes, Sea urchin- Behavior, Insulin resistant, Obesity, Echinochrome Diabetic groups showed significant decrease in the final body weight, time latencies of hot plate and wire suspension tests, total protein, albumin, A/G ratio and HDL-C. However, globulins, TG, TC and LDL-C concentrations increased significantly. On the other hand Ech groups showed significant increase in time latencies hot plate and wire suspension tests, total protein, albumin and A/G ratio. While, globulins, TG, TC and LDL-C concentrations decrease significantly.

Conclusion: The current study demonstrated the potentials of echinochrome in improvement the musculoskeletal system and the metabolism of lipid and protein metabolism in both types of diabetes mellitus.

Keywords: Diabetes, Sea urchin- Behavior, Insulin resistant, Obesity, Echinochrome

Introduction

Diabetes Mellitus (DM) is a chronic disease characterized by hyperglycemia resulting in insulin resistance and/or insulin deficiency caused by the failure of β-pancreatic cells [1]. DM may affect the musculoskeletal system through many ways including glycosylation of proteins, microvascular abnormalities and collagen accumulation [2]. Musculoskeletal complications are most commonly in both types of diabetes [2]. DM is associated with several structural and functional liver abnormalities that affect on protein and lipid metabolism [3]. It is mainly classified into type 1 diabetes and type 2 diabetes, whereas 90,95% of diabetic patients are type 2 [4]. Type Diabetes mellitus (T1DM) is an autoimmune disease, which characterized by loss of insulin producing β-cells and reliance on exogenous insulin for survival [5]. Type 2 Diabetes Mellitus (T2DM) is increasing in prevalence worldwide [6], and it is strongly associated with obesity and insulin resistance [7], as well as defects in pancreatic beta cells function and mass [8].

Streptozotocin (STZ) is an antibiotic produced by the bacterium Streptomyces achromogens and possesses a broad spectrum of antibacterial activities [9]. It is a widely used chemical for the

induction of experimental diabetes model in rodents [10]. T1DM can be induced in rodents by a single dose of STZ injection [11], while type 2 diabetes can be induced by High Fat Diet (HFD) feeding followed by a low-dose STZ injection [12]. In the HFD/STZ rat models, the state of obesity, insulin resistance and/or glucose intolerance in prediabetesis simulated by a period of a HFD.HFD/STZ model in rats is similar to the metabolic profile of type 2 diabetes in humans [12]. Thus, models of STZ-induced diabetes in animals have been very useful in detecting the mechanisms of diabetic pathogenesis and in screening artificial chemicals, natural products, and pharmacological agents that are potentially capable of lowering blood glucose levels [13].

Despite considerable progress in the treatment of diabetes by oral hypoglycemic agents, search for newer drugs continues because the existing synthetic drugs have several limitations, harmful effects and poor effects in relieving clinical symptoms and controlling diabetic complications [14,15]. Sea urchin (P.lividus) is a widespread species in the Atlantic and the Mediterranean coasts and is subjected to intensive commercial fishing in several countries [16]. It has a number of unique substances, such as quinonoid pigments named spinochromes [17,18]. From these compounds, Echinochrome (Ech) which possesses high antioxidant activity and is the most common dark red pigment of sea urchin shells, spines, and eggs [19]. The hypoglycemic activity and the antioxidant role of Echproved by Mohamed et al. [20]. The present study was carried out to evaluate effect of echinochrome on musculoskeletal system and metabolism of lipids and proteins in both types of diabetes mellitus

Materials and Methods

Chemicals and reagents

Streptozotocin and Dimethyl Sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biochemical kits were purchased from the Biodiagnostic Company (El Moror St, Dokki, EGY).

Sea urchin 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 seawater to remove sand and overgrowing organisms at the collection site and transported to the laboratory. The collected specimens were identified by the standard literature of taxonomic guide [21]. The collected specimens were immediately shade dried.

Echinochrome (Ech) extraction

Pigments in the shells and spines were isolated by the Amarowicz method with slight modifications [22,23]. After removal of the internal organs, the shells and spines were washed with a stream of cold water, air-dried at 4°C for 2 days in the dark and then were grounded. The powders (5 g) were dissolved by gradually adding 10 ml of 6 M HCl. The pigments in the solution were extracted 3 times with the same volume of diethyl ether. The ether layer collected was washed with 5% NaCl until the acid was almost removed. The ether solution including the pigments was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure. The extract including the polyhydroxylated naphthoquinone pigment was stored at -30°C in the dark.

Ethical approval

Experimental protocols and procedures used in this study were approved by the Cairo University, Faculty of Science, Institutional Animal Care and Use Committee (IACUC) (Egypt) (CUFS/F/33/14). All the experimental procedures were carried out in accordance with international guidelines for the care and use of laboratory animals.

Experimental animals

Male albino Wistar rats (Rattusnorvegicus) weighing 140 ± 10 gm (6 weeks) for T1DM and 80 ± 10 gm (4 weeks) for T2DMwere used in this study. The rats were obtained from the National Research Center (NRC, Dokki, Giza). Rats were housed in a temperature and humidity controlled environment and given food and water ad libitum.

Induction of type 1 diabetes mellitus (T1DM)

All rats were starved for 12 hrs before the experiment, but were allowed free access to water. T1DM was induced by intraperitoneal injection of 60 mg/kg of Streptozotocin (STZ) dissolved in 0.1mol/l sodium citrate buffer at pH 4.5. Blood glucose levels were measured 72 hr after injection of STZ. Rats were starved, but had access to drinking water for 6 hr before blood glucose measurement. Fasting plasma glucose concentrations ≥ 300mg/100mlwere considered diabetic type 1 in this experiment [24].

Induction of type 2 diabetes mellitus (T2DM)

The rats were fed a high fat diet with energy of 5.3 kcal/g, comprising 60% calories from fat, 35% from protein and 5% from carbohydrate, according to a modification of the protocols of Reed et al. [25-28]. After 4 weeks the rats injected intraperitoneally by a single dose of prepared solution of STZ (30 mg/kg dissolved in 0.1mol/l sodium citrate buffer at pH 4.5). After 72 hours, fasting plasma glucose concentrations ≥ 300mg/100ml were considered diabetic type 2 in this experiment [29].

Experimental design

After one week of acclimatization, 36 rats were assigned into two main groups; T1DM group (18 rats) and T2DM group (18 rats).

T1DM group was divided into 3 subgroups (6rats/subgroup):

Control group: After a single dose of citrate buffer (0.1mol/l, i.p), the rats received 1ml (10% DMSO, orally) daily for 4 weeks.

Diabetes group: After a single dose of STZ (60 mg/kg, i.p), the rats received 1ml (10% DMSO, orally) daily for 4 weeks.

Ech group: After a single dose of STZ (60 mg/kg, i.p), therats received 1mlEch (1mg/kg body weight in 10% DMSO, orally) [30] daily for 4 weeks.

T1DM group was divided also into 3 subgroups (6rats/subgroup):

Control group: After 4 weeks of normal diets feeding, the rats injected with single dose of citrate buffer (0.1mol/l, i.p) then received 1ml of (10% DMSO, orally) daily for 4 weeks.

Diabetes group: After 4 weeks of HFD feeding, the rats injected with single dose of STZ (30 mg/kg, i.p) then received 1ml of (10% DMSO, orally) daily for 4 weeks.

Ech group: After 4 weeks of HFD feeding, the rats injected with single dose of STZ (30 mg/kg, i.p) then received 1mlEch (1mg/kg in 10% DMSO, orally) daily for 4 weeks.

Determination of the physical parameters

Body weight: Body weight measured at the beginning, after administration of HFD and the ending of the experiments.

Hot plate test: The hot plate latency was measured using a modification of the original method of Eddy and Leimbach [31]. Briefly, the modified apparatus consists of an electric cooking plate (Saiso, Japan) with a 1500 Watts stainless steel heating element connected to a thermostat (0-4000C), a thermocouple connects the thermostat to a chrome plated drip pan. The thermocouple together with the thermostat control the temperature of the hot plate within the desired range once set. Pain sensitivity was evaluated by the response latency for paw licking on the hot place. In order to avoid tissue damage, the maximum time the animal could spend on the hot plate was pegged at 60 seconds. Response latencies were measured at 15-minute intervals and the average of the results was taken.

Wire suspension: The wire suspension assay measured muscle strength and the prehensile reflex, an animal’s ability to grasp a taut horizontal wire with its forepaws and to remain suspended. The tail held rats gently and the forepaws were placed on a suspended wire 2 mm in diameter and 62 cm above a cushioned surface. The latency to let go was measured using a stopwatch, with shorter latency to drop indicating reduced strength and/or reflex ability. If a rat did not let go of the wire within 60 s, it was removed from the wire and a latency of 60 s was assigned to that measurement. Each rat was used only once in the wire suspension assay [32].

Animal handling and specimen collection

The rats were fully 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. The blood collected from the rats was separated by centrifugation (3000 rpm, 15 min) to obtain sera, which was stored at -80˚C for the biochemical measurements.

Biochemical analysis

The serum total protein was estimated by the method of Tietz [33], serum albumin [34], serum total lipids [35], serum triglycerides [36], serum total cholesterol [37], serum low density lipoprotein [38] and serum high density lipoprotein [39] were determined according to the manufacturer’s instructions using Spectrum Diagnostics and Bio-diagnostic kits (Giza, Egypt).

Statistical analysis

Values were expressed as means ± SE. The comparisons within groups were evaluated utilizing one-way Analysis Of Variance (ANOVA) with Duncan post hoc test was used to compare the group means and p < 0.05 was considered statistically significant. SPSS, for Windows (version 15.0) was used for the statistical analysis.

Results

Body weight

Data recorded in Table 1 and Figure 1 demonstrated that, final body weight of diabetic rats decreased significantly (P<0.05) at the end of experiment, as compared to the corresponding control groups. On the other hand, a significant increase (P<0.05) in the final body weight was observed after Ech administration, as compared to the corresponding diabetic groups.

Table 1: The curative role of echinochrome on the body weight of diabetic rats.




  
    Item
    T1DM 
  
  
    Intial
    Final
  
  
     
      Groups 
    Control
    140.00±2.89^a 
    211.00±11.99^c 
  
  
    Diabetes
    141.67±1.67^a 
    114.83±4.45^a 
  
  
    Ech
    143.33±3.33^a 
    154.33±7.95^b 
  
  
    Values are means ± se    (n = 6 per group).
      Each value not sharing a common letter    superscript is significantly different (P <0.05).



Table 1: The curative role of echinochrome on the body weight of diabetic rats.

Figure 1: The curative role of Echinochrome (Ech) on body weight of type 2 diabetic rats.

    
    
    Figure 1: The curative role of Echinochrome (Ech) on body weight of type 2
diabetic rats.

Behavioral tests

The obtained data in Figure 2 revealed that T1DM and T2DM rats showed significant decrease (P < 0.05) in time latencies of hot plate and wire suspension tests as compared to control rats. However, the treated rats with Ech showed significant increase in their time latencies as compared to the diabetic groups.

Figure 2: The curative role of Echinochrome (Ech) on musculoskeletal system of diabetic rats. Values are means ± se (n = 6 per group). Each value not sharing a common letter superscript is significantly different (P <0.05).

    
    
    Figure 2: The curative role of Echinochrome (Ech) on musculoskeletal system of diabetic rats. Values are means ± se (n = 6 per group). Each value not sharing a
common letter superscript is significantly different (P <0.05).

Protein profile

The results presented in Table 2 showed that, there was a significant reduction (P <0.05) in the total protein, albumin, and A/G ratio, while globulin concentration increased in the diabetic rats, as compared to control rats. The protein, albumin, and A/G levels was significantly increased, however globulin level decreased significantly after 4 weeks of treatment with Ech, as compared to the corresponding diabetic groups.

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Citation: Soliman AM, Mohamed AS and Marie M-AS. Effect of Echinochrome on Body Weight, Musculoskeletal System and Lipid Profile of Male Diabetic Rats. Austin J Endocrinol Diabetes. 2016; 3(2): 1045. ISSN : 2381-9200

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Parameter	Diabetes type	Groups
Parameter	Diabetes type	Control	Diabetes	Ech
Total protein	T1DM	6.92±0.65^c	3.20±0.17^a	5.75±0.49^b
Total protein	T2DM	5.98±0.27^b	3.15±0.21^a	5.33±0.80^b
Albumin	T1DM	3.24±0.32^b	1.87±0.20^a	3.19±0.20^b
Albumin	T2DM	2.98±0.37^c	0.49±0.06^a	1.65±0.24^b
Globulins	T1DM	1.27±0.09^a	2.37±0.36^b	1.56±0.11^a
Globulins	T2DM	2.63±0.28^a	5.63±0.27^c	4.47±0.26^b
A/G ratio	T1DM	3.59±0.37^c	0.95±0.12^a	2.09±0.16^b
A/G ratio	T2DM	1.25±0.17^c	0.09±0.01^a	0.37±0.05^b
Values are means ± se (n = 6 per group). Each value not sharing a common letter superscript is significantly different (P <0.05).

Parameter	Diabetes type	Groups
Parameter	Diabetes type	Control	Diabetes	Ech
TG	T1DM	157.67±1.31^a	231±3.72^c	183.5±4.14^b
TG	T2DM	129.5±3.66^a	153±4.63^b	133.17±4.75^a
TC	T1DM	165.67±8.67^a	227.17±16.83^b	179.83±5.19^a
TC	T2DM	168.83±11.24^a	211.50±5.79^b	168.33±5.80^a
LDL-C	T1DM	130.67±2.33^a	189.50±8.48^b	132.00±7.36^a
LDL-C	T2DM	119.67±7.02^a	152.67±2.77^b	121.00±4.30^a
HDL-C	T1DM	58.83±2.10^b	42.41±0.28^a	48.90±1.27^a
HDL-C	T2DM	58.04±1.98^b	45.23±1.44^a	49.23±0.79^a
Values are means ± se (n = 6 per group). Each value not sharing a common letter superscript is significantly different (P <0.05).