Experimental ethics for Care and Use of Experimental

Experimental Animals

Healthy male Wistar rats, weighting about 180–210 g,
were obtained from Experimental Animal Centre of Kashan University of Medical
Sciences, kept at standard conditions 22–24C, 40–60% relative humidity and 12h
light–dark cycle and fed with standard rat food and water ad libitum. The
animals were cared in agreement with the ethics for Care and Use of
Experimental Animals. The study protocol was permitted by the ethics committee
of Kashan University of Medical Sciences (IR.KAUMS.REC.1395.29).

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Induction of type 2 diabetes in rats

For the first time in literature, we used a typical diet
of high fat (includes 400 grams of sheep fat, 200 grams of sucrose, 18 egg
yolks and 5 egg whites and 400 grams of rat chow, well combined and homogenized)
and high fructose (including 25% fructose in drinking water) (HFFD: high
at and high fructose diet) for 12 weeks to generate
type 2 diabetic model.

Studied groups design

40 rats were randomly divided into 4 groups
as follows. The first group was normal rats that received standard rat chow and
water without any HFFD/vanadyl sulfate (VS), and considered as control group.
The 2nd group received HFFD without any treatment (non-treated diabetic
group), the 3rd group, received HFFD and so 25mg/kg vanadyl sulfate (diabetic
treated with VS25) and finally, the 4th group received HFFD and
50mg/kg vanadyl sulfate (diabetic treated with VS50). Each group had 10 rats.

 Sera and
liver tissue preparation

After 12 weeks, at the end of their treatment periods,
the animals were weighed and anesthetized using ether and killed by
decapitation. Blood samples were collected from the abdominal vein with a micro
syringe. For metabolic profiles assessment, sera were separated from blood cells
by centrifugation (Hettich D-78532, Tuttlingen, Germany) at 3000 rpm for 15 min,
and sored at -80 oC until analysis. The liver of each rat was
immediately excised. After washing with  cold (+4oC) saline solution, pieces
of the liver were dried up and weighed, collected and stored in -80 oC,
so that each liver sample for analysis was not thawed and re-frozen until


Metabolic profiles


Fasting blood glucose level and lipid profiles
including triglycerides (TG), Total Cholesterol (TC), high density lipoproteins
(HDL) and low density lipoproteins (LDL) were determined in serum using
commercially available kits (Pars Azmun, Tehran, Iran)  by BT-3000 auto analyzer. Insulin level was
quantified by ELISA method using kit of DiaMetra Company (Spello, Perugia,
Italy). To determine the HOMA-IR and the quantitative insulin sensitivity check
index (QUICKI), the suggested formulas were used(15).