Effect of dietary herbacetin on hyperglycaemia and hyperlipidemia in high percent dietary fat-induced C57BL/6J mice
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
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 < 0.05. Results Efficacy of herbacetin on the growth rate Table 1 represents that the mean body weight and calorie intake in normal and OIR mice. Herbacetin treated in OIR mice showed significant reduction in body weight when compared to that of OIR control mice. Further, there were observed distinct differences in calorie intake between the herbacetin treaded in OIR mice and OIR control mice. In addition, herbacetin significantly reduced the food efficiency ratio when compared to OIR control mice. Effectiveness of herbacetin on plasma glucose, insulin, HOMA and HbA1c Fig. 2 demonstrates that the plasma glucose, insulin, HOMA and HbA1c in normal and OIR mice. The plasma glucose and insulin levels were significantly increased in OIR mice and these parameters were reversed to near normal control mice while treated with herbacetin. At three different doses, 40 mg showed optimum lowering activity. A HOMA index mean value was calculated and it was increased significantly in OIR mice when compared to normal mice. Administration of herbacetin to OIR mice significantly decreased HOMA index value to near normal values. HbA1c significantly increased in OIR mice and treated with herbacetin significantly reversed to near normal mice values. Effectiveness of herbacetin on hepatic function markers Fig. 3 represents the effectiveness of herbacetin on hepatic function markers in normal and OIR mice. Hepatic function markers activities such as AST and ALT were increased significantly in OIR mice and these enzymes were reversed to near normal mice values by 10-15 weeks adminsration of herbacetin. Effectiveness of herbacetin on plasma and liver lipid pro?les Fig 4. reveals that the plasma and hepatic TG, TC and FFA in normal and OIR mice. The levels of hepatic and plasma TG, TC and FFA significantly increased in OIR mice when compared to normal mice and administration of herbacetin to OIR mice significantly reversed these TG, TC and FFA to near normal mice values. Effectiveness on hepatic lipid-regulating enzyme activities Fig. 5 and table 2 demonstrates that the effect of herbacetin on SREBP-1a, 1c, and 2, fatty acid synthase (FAS), fatty acid ?-oxidation activity (?-oxidation), malic enzyme and glucose6-phosphate dehydrogenase (G6PD) in normal and OIR experimental mice. The mRNA expression of SREBP-1a, 1c, and 2 and fatty acid synthase (FAS) significantly increased in OIR experimental mice and these mRNA expression significantly reduced by 10-15 weeks administration of herbacetin. The activity of hepatic ?-oxidation and malic enzyme significantly increased, whereas G6PD activity significantly reduced in OIR mice. Administration of herbacetin to OIR mice significantly reversed these hepatic lipid-regulating enzyme activities to near normal values. Histological scrutiny Histological analysis of liver section in OIR mice showed (Fig. 6) macro and macro-vesicular fatty lipid droplets. Herbacetin (40 mg/kg BW) treated in OIR mice showed reduced lipid droplets and regenerative hepatocytes. Discussion Obesity and insulin resistance and are the fastest-growing metabolic disease in worldwide; therefore, the adjuvant therapy without side effect is urgent needed. Dietary plants and their components have been used for alternative therapy to control obesity associated diabetics due to minimal side effect. Flax seed is consuming as a food around the several years and this major constituent is herbacetin 31. Flax seed has been used as a traditional medicine to control hyperglycemia and also it was proved that in experimental study 32. Therefore, we have designed to evaluate the herbacetin, a novel flavonoid found in flax seed, improves insulin resistance, hepatic lipids accumulation and biochemical parameters. High-fat and cholesterol-enriched diets induced obesity have been associated with the development of glucose intolerance, hyperglycemia, insulin resistance, hepatic steatosis and dyslipidemia in animal models 33,34. This animal model is shown similar metabolic features of T2D patients. In the recent years, this model has been used to development of obesity linked type 2 diabetics 35,36. In this study, high-fat diet fed mice had significantly increased in body weight, food intake, blood glucose level, insulin level, HOMA-IR and biochemical parameters. Administration of herbacetin by intragastric for 5 weeks results showed decreases in body weight and food intake when compared to OIR mice which could be due to the enhancing the insulin sensitivity. The liver is major site of glucose and fatty acids productions. Excessive chronic consumption of high fat diet has attributed in over production of glucose and fatty acids in liver which is responsible for the development of hyperglycemia, hyperinsulinemia, insulin resistance and T2DM. In this study, OIR mice had observed increased levels of plasma glucose, insulin and HOMA-IR which is ascertained the state of hyperglycemia, hyperinsulinemia and T2DM. Administration of herbacetin to OIR mice, the level of plasma glucose and insulin decreased and also index of HOMA was reduced when compared to normal control mice and thus, results has assured that the antihyperglycaemic activity of herbacetin due to the improvement in insulin sensitivity and lipid lowering activity. Pervious study documented that the flavonoid reducing the plasma glucose level due its antihyperglycaemic activity 37. Epidemiological and clinical studies have revealed that the protein glycation increases in diabetic conditions due to poorly controlled blood glucose concentration. Therefore, glycosylated haemoglobin has been used as an admirable marker to control hyperglycemia 38. Herbacetin have reduced the elevated glycosylated haemoglobin in OIR mice, which confirmed that the herbacetin prevented the hemoglobin glycation due to its antihyperglycaemic activity 39. Recently, non alcoholic fatty liver diseases in humans are one of most numerous health issues in worldwide, which are usually attributed to excessive consumption of high fat foods with less physical activities 40. Chronic consumption of high fat has attributed to the accumulation of lipids in liver, insulin resistance and T2DM, metabolic abnormalities and finally leads to cellular damages 41. Serum AST and ALT are the identified markers of liver damage because which are located in cytoplasmic membrane and released into the circulation while occurs in cellular damage 42. In the current study, the activities of serum AST and ALT were increased significantly in HF-induced group which are due to the leakage of these enzymes from the cytosol into the blood stream. Our results showed that administration of herbacetin along with high fat diet the above enzymes activities were restored to near normal mice that could be due to the improvement in insulin sensitivity and lipid lowering activity. Excessive chronic consumption of high fat diet is one of the numerous risk factors for the development of coronary heart disease. Experimental and clinical studies also have documented that the chronic obesity is associated with the increased risk of coronary heart disease due to in creased level of cholesterol and triglycerides 43,44. Oversupply and production of endogenous fatty acids and free fatty acids during chronic high fat diet consumption seems to accumulation in liver 45. In our study, lipids had accumulated in liver as evidenced by increased levels of TC, TG, and FFA and also clearly confirmed by histopathalogical examination of liver showed lipid droplets. Although, treatment of herbacetin to OIR mice significantly prevented the increased levels of TC, TG, and FFA and also in histopathalogical examination showed clear evidence that herbacetin decrease in lipid droplets in liver tissue. A hypolipidemic effective drug has seemed to be a primary and secondary prevention of coronary artery disease and minimize the risk of pancreatitis. Therefore, administration of herbacetin to EFD mice prevented the further increase in lipid accumulation and the related risk of coronary artery disease due to their hypolipidemic effects. Recently, studies have documented that the cellular lipid metabolism and homeostasis are primarily controlled by a number of proteins and transcription factors including sterol regulatory element-binding protein (SREBP) 46. Generally, SREBP is in two distinct forms such as SREBP-1 and -2. SREBP-1 gene is expressed in two isoforms 1a and 1c, which are playing a major role to regulating the fatty acid metabolism. Moreover, SREBP-2 is involved in the regulation of cholesterol metabolism. Recent studies have revealed that the SREBP-1a, 1c, and 2 are implicated as a numerous pathological conditions such as hyperlipidemia and liver steatosis during chronic administration of high fat diet 47, 48. In our study also observed that the high fat diet control mice has implemented in pathogenesis of hyperlipidemia and liver steatosis due to increased mRNA expression of SREBP-1a, 1c, and 2. Studies have suggested that the inhibition of SREBP gene expressions is a new molecular basis mechanical strategy as a remedy of obesity and its related metabolic diseases 49. Administration of herbacetin to OIR mice showed significantly decreased mRNA expressions of SREBP-1a, 1c, and 2 when compared to normal diet control mice, which could be due to the herbacetin might involved inhibition of cellular cholesterol and FA synthesis. Furthermore, fatty acid synthase (FAS), ?-oxidation and Malic enzyme are the rate limiting cellular metabolic enzymes that involved catalyze hepatic fatty acid synthesis. Studies have proved that the fatty acid synthase (FAS), ?-oxidation and Malic enzyme have been suppressed by altered glucose-6-phosphate dehydrogenase (G6PD) activity during obesity associated insulin resistance condition 50. Generally, the cellular NADPH produced by G6PD, which plays a vital role for cholesterol and fatty acids biosynthesis. Although G6PD is mainly required for energy homeostasis, it is proved that altered G6PD activity suppresses cellular metabolic enzymes thereby inducing dysregulated lipid metabolism and insulin resistance during chronic high fat induced obesity 51. In our study, fatty acid synthase (FAS), ?-oxidation and Malic enzyme activities were decreased and G6PD was increased in high fat diet mice and it was restored to near normal diet mice by administration of herbacetin. Therefore, we conclude that the herbacetin is having anti-hyperlipidemic activity which could be due to the controlled action of lipid metabolizing and lipid-regulating enzymes activities. In conclusion, based on our observed results, we suggest that herbacetin is having good anti-hyperglycemic and anti-hyperlipidemic action against HFD induced T2DM which might be due to the controlled effects of lipid metabolizing and lipid-regulating enzymes activities. The aim of our future studies should be conduct the role of herbacetin in human subjects and it should be a safe new novel drug against the obesity associated insulin resistance.