Effect to mice for 10 weeks, after that

Effect of dietary herbacetin on hyperglycaemia and hyperlipidemia in high percent dietary fat-induced C57BL/6J mice

 

Abstract

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Healthy plants and their constituents have been considered as a safe remedy for treatment of obesity and obesity associated diseases. Herbacetin is a dietary flavonoid that has explored many pharmacological activities, but anti-hyperglycaemic and anti-hyperlipidemic properties of herbacetin is not yet been explored. However, this study was performed to evaluate the ameliorative effect of herbacetin on high-fat diet-induced hyperglycemia and hyperlipidemia to 57BL/6J mice. Obesity associated insulin resistance was induced by continuously fed on high-fat diet to mice for 10 weeks, after that subjected to intragastric administration of herbacetin (different doses) daily along with high-fat diet for the next 5 weeks. At end of 106th day, the changes of body weight, blood glucose, insulin, HOMA-HR and lipids profiles and lipid-regulating enzymes were evaluated. Herbacetin significantly reduced the body weight, plasma glucose, plasma insulin and HOMA-HR activity in obesity associated insulin resistance mice (OIR). In addition, the herbacetin administration significantly reduced the plasma and hepatic total cholesterol, triglycerides and free fatty acids in OIR mice. Although, herbacetin significantly improved the altered hepatic lipid-regulating enzymes such as SREBP-1a, 1c, and 2, fatty acid synthase (FAS), fatty acid ?-oxidation activity (?-oxidation), malic enzyme and glucose6-phosphate dehydrogenase (G6PD) when compared to OIR control mice. In addition, the histopathalogical examination showed clear evidence that herbacetin decrease in lipid droplets in liver tissue. Thus observed results strongly indicate that herbacetin afforded remarkable protection against the chronic high-fat diet consumption due to its anti-hyperglycaemic and anti-hyperlipidemic properties.

Keywords:  57BL/6J mice. obesity. insulin resistance.  herbacetin. anti-hyperglycaemia. anti-hyperlipidemia

Introduction

Currently, obesity and diabetes mellitus are the foremost prevalent metabolic disorders in developed and developing countries. According to  World Health Organization report diabetic patients number have reached 422 million in 2014 and around 3.7 million patients have died from diabetics complications in 2012 1. Obesity prevalence has increased distressingly throughout the world over the past several decades. Uncontrolled obesity can cause insulin resistant, hyperglycemia, dyslipidemia and oxidative cellular damage finally leads to micro and macro vascular complications 2,3. Majority of obese patients have suffered from type 2 diabetics due to chronic consumption of high calorie foods with less physical activities. Several experimental studies also have proved that high-fat fed can cause lipid accumulation, insulin resistance, hyperglycemia and metabolic abnormalities 4,5.

The liver is a major organ for hormonal anabolic and catabolic counterpart of insulin and glucagons. Impaired insulin action or sensitivity in the liver is significantly contributed to the development of T2DM 6. Chronic consumption of high fat diet has attributed in numerous changes and deposition of fat in liver which is characterised by the insulin resistance and T2DM 7,8. Liver damage is associated with the excess deposition of fat in liver, systemic insulin resistance, metabolic syndromes and oxidative cellular damages and finally leads to cell death 9,10. Lipid metabolism improvement has become an important remedy for prevention and treatment of obesity associated insulin resistance 11.

            Dietary plants and their components are a potential resource of improved insulin sensitivity, tissues inflammation and deregulated lipids metabolism in animals rat models 12,13. Currently, many synthetic drugs are available for controls type 2 diabetics and obesity. Although, these drugs are having side effects including weight gain, dropsy, drug-resistance and hypoglycemia. Thus, there are needed antidiabetic drugs with minimal side effect and low cost. However, currently, studies have been focused to develop new alternative drugs from plants and their components because of its proved that have no side effects. Herbacetin a natural flavonoid compound found in flaxseed hulls. The chemical structure is shown in Fig. 1. Flax seed hulls have been reported to having several medicinal properties including antidiabetic, hypertension, cancer and antibacterial effect 14-17. Earlier studies have revealed that the herbacetin medicinal properties such as antioxidant activity, antitumor activity and also act as an inhibitor for phosphorylation of c-Met and AKT 18. To the best of our knowledge very few studies only have been explored the herbacetin biologic activities. Therefore, for the first study was desired to evaluate the whether herbacetin, a novel flavonol found in flax seed, improves insulin sensitivity by improving lipid metabolism. 

Materials and methods

Chemicals and solvents purchasing

Herbacetin and all other used chemicals, solvents and primers were procured from Sigma-Aldrich and other qualified chemicals Laboratories.

Animals purchasing and maintenance

C57BL/6J mice were acquired from Central Animal House at King Saud University and the throughout experiment were followed our University Research Centre animal care policy. All animals were kept 12-h cycling process of daylight/dark at persistent temperature (22 ± 2° C) throughout experiment.

Obesity associated insulin resistance induction (OIR)

Obesity associated insulin resistance was induced by administration of high percent beef tallow-containing fat diet to male C57BL/6J mice for 10 weeks. The diet compositions of high and normal fat are given in table 1. After 10 weeks of high fat or normal fat administration, all the animals were used for the further experiment to check effectiveness of herbacetin. Especially, we have chosen the blood glucose ranges more than180 mg/dl to be considered diabetic from the 10 weeks high fat administered animals. Herbacetin (dissolved in 0.5% dimethyl sulfoxide (DMSO)) was given daily along with high fat diet for the next 5 weeks.

At end of 106th day, mice were sacri?ced by cervical dislocation after kept overnight fasting. Blood was collected for the estimation of biochemical parameters. Liver tissue was expunged immediately from each animal and it was washed with ice-cold isotonic saline and blemished with a ?lter paper. A tissue was used for the assessment of histology and lipid parameters.

Biochemical and hematology measurements

Plasma glucose, insulin and glycosylated haemoglobin (HbA1c) were evaluated by Trinder 19, Burgi et al. 20 and Nayak and Pattabiraman 21 respectively. The insulin resistance activity was calculated by using the blood glucose and insulin levels according to the method of homeostasis model assessment (HOMA) (Matthews et al., 1985).

HOMA-IR = Insulin (mUI/l) × Blood glucose (mmol/l)/22.4.

 

 

Lipids parameters and hepatic markers and measurement

Tissue lipids were extracted by the followed method of Folch, et al. 22. FFA, TC and TG were evaluated by Falholt et al. 23, Allain et al. 24 and McGowan et al. 25 methods. AST and ALT activity were evaluated by Reitmanand Frankel method 26.

Lipid-regulating enzymes activity assessment

Fatty acid synthase (FAS) and fatty acid ?-oxidation activity (?-oxidation) were evaluated by Nepokroeff et al. 27 and Lazarow 28. The malic enzyme was evaluated by an established method of Ochoa 29. The glucose6-phosphate dehydrogenase (G6PD) activity was evaluated by Pitkanen et al. 30 (spectrophotometric methods).

Hepatic histopathological assessment

Estimated liver tissue was fixed with formaldehyde (10%) and dehydrated by using ethanol and then embedded with paraf?n. 5 ?m thick liver section was taken and then dewaxed followed by rehydrated. Finally, the liver section was stained with hematoxylin-eosin (H&E) and captured the liver histology by using the light microscope.

Evaluation of statistical significant

The obtained results are expressed as a means average of 6 reading from 10 mice (Mean ± S.E.). A statistical significant was evaluated by ANOVA followed by Tukey’s multiple comparison tests P value