INTRODUCTION {#sec1-1}
============

Type 2 diabetes mellitus is a metabolic disease characterized by the presence of chronic hyperglycemia that results from defective or deficient insulin \[[@ref1],[@ref2]\]. It accounts for more than 90% of all diabetic patients \[[@ref3]\]. According to the International Diabetes Federation, the number of patients with diabetes mellitus in 2015 was estimated to be 415 million, and is expected to increase to 642 million by 2040 \[[@ref4]\]. Type 2 diabetes and its complications constitute a major public health problem \[[@ref5]\]. Several lifestyle factors such as physical inactivity \[[@ref6]\], sedentary lifestyle \[[@ref7]\], alcohol consumption \[[@ref8]\] and smoking \[[@ref9]\] are of key importance to the development of Type 2 diabetes. In addition, diet is a modifiable risk factor for Type 2 diabetes. The consumption of fructose has been enormously increased in the last few centuries because of the high increase in using sucrose and high fructose syrup \[[@ref10]\]. Previous studies have demonstrated that high fructose intake is hazardous for human beings and animals \[[@ref11],[@ref12]\], and results in hyperlipidemia, fatty liver, and insulin resistance \[[@ref13]\]. The metabolism of fructose in the liver increases *de novo* lipogenesis \[[@ref14]\], and an increase in high fructose corn syrup consumption has been linked to a rise in obesity and metabolic disorders \[[@ref11]\]. Fructose feeding has also been shown to provoke oxidative damage and exert disturbing effects by diminishing antioxidant defenses, and increasing generation of free radicals \[[@ref15]\]. Thus, the use of antioxidants could offer protection against fructose-induced metabolic alterations.

Currently, there is growing interest in the usefulness of algae for the treatment of diabetes. The cyanobacterium *Spirulina* is gaining a more attention as a nutraceutical and as a source of potential pharmaceutical. Studies have revealed the potential properties of *Spirulina* including antigenotoxic, anti-carcinogenic, immunostimulants, anti-inflammatory, anti-hepatotoxic, anti-diabetic and anti-hypertensive. *Spirulina* is a well-known source of anti-oxidant and anti-inflammatory molecules \[[@ref16]\] such as c-phycocyanin, vitamins, β-carotene, phenolic compounds γ-linolenic acid and minerals \[[@ref17],[@ref18]\]. *Spirulina maxima* (*Arthrospira maxima*), *Spirulina platensis* (*Arthrospira platensis*) and *Spirulina fusiformis* (*Arthrospira fusiformis*) are the most intensively investigated species of *Spirulina* \[[@ref17],[@ref19],[@ref20]\]. Recently, the preliminary anti-diabetic effect of *Spirulina versicolor* was reported in the study of AbouZid *et al*. \[[@ref21]\] in streptozotocin/nicotinamide-induced diabetic mice. The authors reported that *S. versicolor* exerts anti-hyperglycemic effect, depending on assaying fasting and postprandial blood glucose levels in diabetic mice. To the best of our knowledge, nothing has yet been reported on the beneficial effects of *S. versicolor* in fructose-fed rats. Therefore, the current study was undertaken to investigate the anti-hyperglycemic, insulin sensitizing, anti-hyperlipidemic and antioxidant effects of *S. versicolor* in high fructose-fed rats. This investigation could provide an understanding of the anti-diabetic mechanism of *S. versicolor*.

METHODS {#sec1-2}
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Preparation of *S. versicolor* extract {#sec2-1}
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*S. versicolor* was purchased from Harraz medicinal plant company, Cairo, Egypt ([www.harrazegypt.com](http://www.harrazegypt.com)). The algae was ground to a fine powder and extracted by 80% aqueous ethanol. Following filtration, the filtrate was concentrated under reduced pressure in a rotary evaporator and was stored at −20°C until use.

Experimental Animals {#sec2-2}
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Male Wistar rats weighing 130-150 g, obtained from animal house of the National Research Centre (El-Giza, Egypt), were included in the present investigation. The animals were housed in plastic well-aerated cages at a normal atmospheric temperature (25±2°C) and normal 12 h light/dark cycle. Rats had free access to water and were supplied daily with laboratory standard diet of known composition. All animal procedures were undertaken with the approval of Institutional Animal Ethics Committee of Beni-Suef University (Egypt).

Experimental Design {#sec2-3}
-------------------

About 24 rats were allocated into 4 groups, each consisting of six (*n* = 6) animals and were subjected to the following treatments:

Group 1 (Control): Received the vehicle 1% carboxymethylcellulose (CMC) and served as control rats.Group 2 (Control + *S. versicolor*): Received 50 mg/kg b.wt. *S. versicolor* extract suspended in 1% CMC and served as drug control.Group 3 (Diabetic): Received 30% fructose in tap water.Group 4 (Diabetic + *S. versicolor*): Received 30% fructose in tap water and 50 mg/kg b.wt. *S. versicolor* extract suspended in 1% CMC.

Rats were fed 30% fructose solution in drinking water for 4 weeks, and biochemical parameters were estimated. Rats exhibited hyperglycemia and hyperinsulinemia were selected for further subsequent studies. *S. versicolor* extract has been administered by oral gavage for 4 weeks. The doses were balanced consistently as indicated by any change in body weight to keep up the comparable dosage for every kg body weight over the entire period of study.

Samples Preparation {#sec2-4}
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By the end of the experiment, overnight fasted animals were sacrificed, and blood samples were collected, left to coagulate and centrifuged at 3000 rpm for 15 min to separate serum. Liver, kidney, and heart samples were immediately excised and perfused with ice-cold saline. Frozen samples (10% w/v) were homogenized in chilled saline, and the homogenates were centrifuged at 3000 rpm for 10 min. The clear homogenates were collected and used for subsequent assays.

Biochemical Study {#sec2-5}
-----------------

### Oral glucose tolerance test (OGTT) {#sec3-1}

On the day before sacrifice, OGTT was performed using blood samples obtained from lateral tail vein of rats deprived of food overnight. Successive blood samples were then taken at 30, 60, 90 and 120 min following the administration of glucose solution (3 g/kg b.wt.). Blood samples were left to coagulate, centrifuged, and clear sera were obtained for determination of glucose concentration according to the method of Trinder \[[@ref22]\] using reagent kit purchased from Spinreact (Spain).

Determination of Serum Insulin, Adiponectin and Tumor Necrosis Factor Alpha (TNF-α) {#sec2-6}
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Serum levels of insulin, adiponectin and TNF-α were determined using specific ELISA kits (R&D systems) following the manufacturer's instructions. The concentrations of assayed parameters were measured specrophotometrically at 450 nm. Standard curves were constructed by using standard proteins and concentrations of the unknown samples were determined from the standard plots.

Determination of Homeostasis Model of Insulin Resistance (HOMA-IR) {#sec2-7}
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The insulin resistance was evaluated by homeostasis model assessment estimate of insulin resistance (HOMA-IR) \[[@ref23]\] as follows:

![](JIE-5-57-g001.jpg)

Determination of Lipid Profile and Cardiovascular Risk Indices {#sec2-8}
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Serum total cholesterol \[[@ref24]\], triglycerides \[[@ref25]\] and high density lipoprotein (HDL)-cholesterol \[[@ref26]\] were assayed using commercial diagnostic kits (Spinreact, Spain). Serum very low density lipoprotein (vLDL)-cholesterol concentration was calculated according to the following formula \[[@ref27]\]: vLDL-cholesterol = triglycerides/5. Serum LDL-cholesterol level was calculated from the formula \[[@ref28]\]: LDL-cholesterol = Total cholesterol - (\[Triglycerides/5\] + HDL-cholesterol\]. Cardiovascular risk indices were calculated according to Ross \[[@ref29]\] as follows: Cardiovascular risk index 1 = Total cholesterol/HDL-cholesterol and cardiovascular risk index 2 = LDL-cholesterol/HDL-cholesterol. Antiatherogenic index (AAI) was determined according to the following equation \[[@ref30]\]: AAI = HDL-cholesterol × 100/Total cholesterol - HDL-cholesterol.

Assay of Serum Enzymes {#sec2-9}
----------------------

Serum aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK-MB) activities were assayed using reagent kits purchased from Biosystems (Spain) following the methods of Schumann and Klauke \[[@ref31]\], Teitz and Andresen \[[@ref32]\] and Kachmar and Moss \[[@ref33]\], respectively.

Assay of Lipid Peroxidation and Antioxidant Defenses {#sec2-10}
----------------------------------------------------

Lipid peroxidation levels in liver, kidney, and heart homogenates were assayed by measurement of malondialdehyde (MDA) formation according to the method of Preuss *et al*. \[[@ref34]\]. Reduced glutathione (GSH) content and activity of the antioxidant enzymes superoxide dismutase (SOD) and GSH peroxidase (GPx) were measured according to the methods of Beutler *et al*. \[[@ref35]\] Marklund and Marklund \[[@ref36]\] and Matkovics *et al*. \[[@ref37]\], respectively.

Statistical Analysis {#sec2-11}
--------------------

Data were analyzed using Graph Pad Prism 5 software and all statistical comparisons were made by means of the one-way ANOVA test followed by Tukey's test *post hoc* analysis. Results were articulated as mean ± standard error of the mean (SEM) and a *P* value \< 0.05 was considered significant.

RESULTS {#sec1-3}
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*S. versicolor* Represses Hyperglycemia and Insulin Resistance in Fructose-fed Rats {#sec2-12}
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OGTT of the fructose-induced diabetic rats showed significantly (*P* \< 0.001) elevated glucose levels and at all points of the OGTT when compared with the normal control rats \[[Figure 1a](#F1){ref-type="fig"}\]. Oral supplementation of *S. versicolor* extract to fructose-fed rats significantly alleviated the blood glucose levels. The OGTT area under the curve (AUC) showed non-significant (*P* \> 0.05) difference between the control and *S. versicolor* supplemented control rats. On the other hand, fructose-induced diabetic rats exhibited a significant (*P* \< 0.01) increase in AUC when compared with the control rats. Treatment of the diabetic rats with *S. versicolor* markedly (*P* \< 0.01) decreased OGTT AUC when compared with the diabetic control rats, as depicted in [Figure 1b](#F1){ref-type="fig"}.

![Effect of *Spirulina versicolor* on (a and b) glucose tolerance, (c) serum insulin and (d) homeostasis model of insulin resistance. Results are mean ± standard error of the mean (*n* = 6). \*\*\**P* \< 0.001 versus control, and ^\#^*P* \< 0.05, and ^\#\#\#^*P* \< 0.001 versus diabetic group](JIE-5-57-g002){#F1}

Serum insulin level was significantly (*P* \< 0.001) increased in fructose fed rats compared with the control group as depicted in [Figure 1c](#F1){ref-type="fig"}. Oral treatment with *S. versicolor* markedly ameliorated serum insulin levels in the fructose-induced diabetic rats. Similarly, diabetic rats exhibited a significant (*P* \< 0.001) increase in HOMA-IR, an effect that was reversed by oral administration of *S. versicolor* to fructose-induced diabetic rats \[[Figure 1d](#F1){ref-type="fig"}\].

*S. versicolor* Exerts Anti-hyperlipidemic, Cardioprotective and Anti-atherogenic effects in Fructose-fed Rats {#sec2-13}
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Data represented in [Table 1](#T1){ref-type="table"} show the effect of *S. versicolor* on lipid profile, cardiovascular risk indices, heart marker enzymes and antiatherogenic index of control and diabetic rats. Compared with the control group, rats supplemented with *S. versicolor* exhibited non-significant (*P* \> 0.05) changes in all lipid profile parameters. On the other hand, fructose-induced diabetic rats exhibited significant increase in serum total cholesterol (*P* \< 0.001), triglycerides (*P* \< 0.01), LDL-cholesterol (*P* \< 0.001) and vLDL-cholesterol (*P* \< 0.01) when compared with the control group. Serum levels of HDL-cholesterol showed a non-significant (*P* \> 0.05) difference between all studied groups. In addition, diabetic rats showed significantly (*P* \< 0.001) increased HDL-cholesterol/T. cholesterol and LDL-cholesterol/HDL-cholesterol. In addition, the antiatherogenic index was significantly (*P* \< 0.05) declined in diabetic rats. By comparison, the oral supplementation of *S. versicolor* extract to diabetic rats potentially ameliorated the altered serum lipid profile as well as cardiovascular risk indices.

###### 

Effect of *S. versicolor* on serum lipid profile, cardiovascular risk indices and antiatherogenic index in control and fructose-fed rats

![](JIE-5-57-g003)

Serum AST, CK-MB and LDH activities were significantly increased in the fructose-induced diabetic rats when compared with the control group \[[Table 1](#T1){ref-type="table"}\]. Treatment of the diabetic rats with *S. versicolor* extract significantly ameliorated serum activities of AST (*P* \< 0.05), CK-MB (*P* \< 0.05) and LDH (*P* \< 0.001).

*S. versicolor* Increases Circulating Adiponectin and Decreases TNF-α in Fructose-fed Rats {#sec2-14}
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Fructose-fed rats exhibited markedly (*P* \< 0.01) declined serum adiponectin levels when compared with the control group, as represented in [Figure 2a](#F2){ref-type="fig"}. Treatment of the fructose-induced diabetic rats with *S. versicolor* extract significantly (*P* \< 0.01) alleviated serum adiponectin levels. The effect of *S. versicolor* on serum levels of TNF-α in control and fructose-induced diabetic rats showed a significantly (*P* \< 0.01) increased levels of TNF-α (*P* \< 0.001) in diabetic rats and potential (*P* \< 0.05) alleviation following treatment with *S. versicolor* extract \[[Figure 2b](#F2){ref-type="fig"}\].

![Effect of *Spirulina versicolor* on (a) serum adiponectin and (b) tumor necrosis factor alpha. Results are mean ± standard error of the mean (*n* = 6). \*\**P* \< 0.01, and \*\*\**P* \< 0.001 versus control, and ^\#^*P* \< 0.05, and ^\#\#^*P* \< 0.01 versus diabetic group](JIE-5-57-g004){#F2}

*S. versicolor* Attenuates Hyperglycemia-induced Oxidative Stress in Liver, Kidney and Heart of Fructose-fed Rats {#sec2-15}
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Fructose-induced diabetic rats showed significantly increased MDA levels in liver (*P* \< 0.001), kidney (*P* \< 0.01) and heart (*P* \< 0.01) when compared with the control rats \[[Figure 3a](#F3){ref-type="fig"}\]. Treatment of the fructose-induced diabetic rats with *S. versicolor* extract significantly alleviated liver (*P* \< 0.01), kidney (*P* \< 0.001) and heart (*P* \< 0.001) lipid peroxidation levels. Oral supplementation of *S. versicolor* to normal rats produced significant (*P* \< 0.05) decrease in kidney MDA content, with no effect exerted on liver and heart.

![Effect of *Spirulina versicolor* on (a) lipid peroxidation, (b) reduced glutathione, (c) superoxide dismutase and (d) glutathione peroxidase in liver, kidney and heart. Results are mean ± standard error of the mean (n = 6). \**P* \< 0.05, \*\**P* \< 0.01, and \*\*\**P* \< 0.001 versus control, and ^\#^*P* \< 0.05, ^\#\#^*P* \< 0.01, and ^\#\#\#^*P* \< 0.001 versus diabetic group](JIE-5-57-g005){#F3}

On the contrary, fructose supplementation significantly decreased liver (*P* \< 0.001), kidney (*P* \< 0.05) and heart (*P* \< 0.001) GSH content when compared with the control group, as represented in [Figure 3b](#F3){ref-type="fig"}. Similarly, SOD activity was significantly decreased in the liver (*P* \< 0.05), kidney (*P* \< 0.05) and heart (*P* \< 0.01) of fructose-induced diabetic rats when compared with the control group \[[Figure 3c](#F3){ref-type="fig"}\]. GPx activity showed a similar pattern where it was significantly decreased in the liver (*P* \< 0.01), kidney (*P* \< 0.001) and heart (*P* \< 0.05) of fructose-induced diabetic rats, as depicted in [Figure 3d](#F3){ref-type="fig"}. On the other hand, treatment of the fructose-induced diabetic rats with *S. versicolor* extract potentially ameliorated GSH content as well as activities of SOD and GPx in the liver, kidney and heart.

DISCUSSION {#sec1-4}
==========

Several studies have demonstrated the deleterious effects of fructose on insulin sensitivity and glucose metabolism \[[@ref38]\]. In the present study, fructose-fed rats showed significantly impaired glucose tolerance accompanied with hyperinsulinemia and increased HOMA-IR. Therefore, it is suggested that insulin resistance has been developed in these animals. This would closely reflect the natural history and metabolic characteristics of human diabetes, and it is further sensitive to pharmacological testing \[[@ref2]\]. Long term fructose feeding has been demonstrated to induce diabetes associated with insulin resistance in experimental animals \[[@ref38]-[@ref41]\]. The fructose-induced insulin resistance may be linked to alteration of insulin signaling. In this context, high fructose feeding has been reported to decrease insulin receptor substrate (IRS)-1 phosphorylation in rat skeletal muscles \[[@ref42]\]. In addition, fructose-induced hyperlipidemia \[[@ref43]\] and fat deposition \[[@ref44]\] may generate lipid-derived metabolites which reduce insulin signaling via increasing serine/threonine phosphorylation of IRS-1 \[[@ref45]\]. Oral supplementation of *S. versicolor* extract markedly reduced blood glucose and improved insulin sensitivity in fructose-fed rats. Although the anti-hyperglycemic effect of different *Spirulina* species has been previously reported, studies demonstrating the anti-diabetic efficacy of *S. versicolor* are scarce. In this context, Mani *et al*. \[[@ref46]\] showed a significant decrease in the fasting blood sugar level of patients received 2 g/day *Spirulina* for 21 days, and Layam *et al*. \[[@ref47]\] proved the same effect in diabetic rats treated with 15 mg/kg *Spirulina* for 45 days. The hypoglycemic effect of *Spirulina* could perhaps attributed to its high fiber content that diminish glucose absorption \[[@ref48]\], or to the possible action of peptides generated by the digestion of *Spirulina* proteins \[[@ref49]\].

Insulin resistance in Type 2 diabetes is also associated with hyperlipidemia and atherosclerosis \[[@ref50]\]. Fructose-fed rats in the present investigation exhibited hypercholesterolemia and hypertriglyceridemia. The fructose-induced hyperlipidemia may be attributed to the increased *de novo* hepatic lipogenesis through providing large amounts of hepatic triose-phosphate for fatty acid synthesis \[[@ref14]\]. In addition, fructose increases the expression of key lipogenic enzymes and induces the expression of sterol regulatory element binding protein-1c which is the principal inducer of hepatic lipogenesis \[[@ref51],[@ref52]\]. Moreover, fructose has been demonstrated to activate carbohydrate-responsive element binding protein (ChREBP), leading to up-regulated expression of hepatic fatty acid synthase and acetyl-CoA carboxylase \[[@ref53]\]. Activation of ChREBP may be attributed to the fructose-induced expression of glucose-6-phosphate dehydrogenase and intermediary substrates of the hexose-monophosphate shunt \[[@ref54]\].

The elevated triglycerides and cholesterol levels in the fructose-induced diabetic rats represent atherogenic lipid profile. The recorded values of atherogenic indices in the present study showed the bad impact of fructose-induced dyslipidemia on the cardiovascular system. These findings were confirmed by the elevated serum levels of AST, CK-MB, and LDH. Treatment of the diabetic rats with *S. versicolor* extract significantly ameliorated the altered lipid profile and atherogenic indices. Reduction of these indices in treated fructose-fed rats strongly supported the notion that dietary supplementation with *S. versicolor* may reduce the risk of developing heart diseases. These findings were further confirmed by the significantly decreased serum activities of the cardiac markers, CK-MB, LDH and AST, in *S. versicolor* treated fructose-fed rats. The anti-hyperlipidemic effects of *Spirulina sp*. have been demonstrated in animal \[[@ref55],[@ref56]\] and human studies \[[@ref57]-[@ref59]\].

The beneficial effects of *S. versicolor* extract in fructose-induced diabetic rats might be explained, at least in part, through its ability to increase serum adiponectin levels. Serum level of adiponectin is in agreement with insulin sensitivity and its reduced levels are associated with insulin resistance \[[@ref60]\]. Adiponectin regulates glucose metabolism \[[@ref61]\], increases muscle fat oxidation and glucose transport mediated \[[@ref62]\], inhibits hepatic gluconeogenesis \[[@ref63]\] and activates peroxisome proliferator activated receptor-a leading to decreased triglyceride content in skeletal muscles and liver \[[@ref64]\]. We also assume that suppression of the release of TNF-α following *S. versicolor* administration could be a direct result of increased serum adiponectin levels. Adiponectin is well known to inhibit the expression of the pro-inflammatory cytokine TNF-α in various tissues \[[@ref65]\]. TNF-α diminishes the ability of insulin to stimulate peripheral glucose uptake and to suppress hepatic glucose production \[[@ref66]\], and increases circulating free fatty acids; thus contributes to the pathogenesis of insulin resistance \[[@ref67]\]. In the present study, treatment of the fructose-induced diabetic rats with *S. versicolor* markedly decreased serum levels of TNF-α, confirming its anti-inflammatory efficacy.

Oxidative stress has been implicated in fructose-induced insulin resistance and Type 2 diabetes in rats \[[@ref13]\]. Oxidative stress can cause oxidation and damage to many cellular components such as DNA, lipids and proteins \[[@ref68]\]. Reactive oxygen species (ROS) in diabetes could react with polyunsaturated fatty acids leading to lipid peroxidation \[[@ref69]\]. In addition, high levels of free radicals and the simultaneous decline in endogenous antioxidants can lead to damage of cellular organelles, and development of insulin resistance \[[@ref70]\]. Hence, it was recommended by Mahmoud *et al*. \[[@ref2]\] that therapy with antioxidants represents a useful pharmacologic overture to the management of diabetes. The present findings showed significant elevation in lipid peroxidation levels in liver, kidney and heart of fructose-administered rats. Treatment of the fructose-fed rats with *S. versicolor* extract significantly decreased lipid peroxidation levels, reflecting its radical scavenging property.

In contrary, GSH and the antioxidant enzymatic defenses showed a simultaneous decrease in the liver, kidney and heart of fructose-induced diabetic rats. Antioxidant defenses are known to decrease under hyperglycemia \[[@ref71]\] and oxidative stress \[[@ref72]\]. Treatment of diabetic rats with *S. versicolor* significantly increased levels of GSH and activity of the antioxidant enzymes SOD and GPx. GSH is an endogenous antioxidant that protects against oxidative stress-induced cellular damage by reacting with oxidants or as a substrate for GPx. SOD and GPx provide a defense system against ROS-induced cellular damage \[[@ref73]\]. The antioxidant effect of *Spirulina* and their constituents has been previously demonstrated. Ahmed *et al*. \[[@ref74]\] reported that *S. versicolor* extract protected against diethylnitrosamine-induced hepatocarcinogenesis through potentiating the antioxidant defense system.

Several studies have reported the *in vitro* and *in vivo* antioxidant and/or anti-inflammatory efficacies of *Spirulina* and its extracts, suggesting the beneficial effects of *Spirulina* in managing insulin resistance and diabetes. The antioxidant and anti-inflammatory effects of *Spirulina Sp*. could be attributed to its active constituents. *Spirulina* contains a relative high concentration of β-carotene, provitamin A, vitamin B, vitamin C, vitamin D, vitamin E, ω-3 and ω-6 polyunsaturated fatty acids, and phycocyanin \[[@ref75]\]. Phycocyanin has the ability to scavenge free radicals, decrease nitrite production, suppress inducible nitric oxide synthase expression, and inhibit liver microsomal lipid peroxidation. In addition, phycocyanin has been reported to inhibit pro-inflammatory cytokine formation, suppress cyclooxygeanase-2 expression and decrease prostaglandin E2 production \[[@ref76]-[@ref78]\]. Another constituent, β-carotene, has been reported to have antioxidant and anti-inflammatory activities \[[@ref79],[@ref80]\].

CONCLUSION {#sec1-5}
==========

The current findings provide new information on the antidiabetic mechanism of *S. versicolor* in fructose-fed rats. High fructose feeding induces insulin resistance, inflammation and oxidative stress. Oral administration of *S. versicolor* ameliorates insulin sensitivity, increases serum adiponectin, and attenuates oxidative stress and inflammation in diabetic rats. Our findings suggest that *S. versicolor* extract could be used as a dietary supplement in diabetes management, pending further studies to trace out its exact mechanistic pathways.

**Source of Support: Nil**

**Conflict of Interest: None declared.**
