Protective Role of the Farnesoid X Receptor Ligand Ivermectin in Rats with Diabetic Dyslipidemia

Diabetic dyslipidemia has been used to describe the pathophysiology surrounding the effects of insulin resistance on abnormal lipid levels which shows increased plasma concentration of triglycerides (TG), reduced concentration of high density lipoprotein cholesterol (HDL-C) and a qualitative alteration of low density lipoprotein (LDL) fraction due to the preponderance of the small and dense LDL (sdLDL).1 According to National Cholesterol Education Panel Guidelines, Diabetes is considered a CHD equivalent.2 Although the first priority of treatment is to decrease LDL-C level it may require an advanced treatment that normalize the lipid profile which minimize the risk of cardiovascular event.3 Ligand-activated nuclear receptors provide promising new targets for both glucose and lipid homeostatis.4 The Farnesoid X receptor (FXR) is one of the most important member of the nuclear receptor superfamily and expressed in liver, intestine and adipose tissue. FXR is activated by bile acids. After ligand binding, FXR binds to DNA elements which is called as FXR response elements (FXREs).5 On activation of FXR, it regulates bile acid synthesis, conjugation, transportation and other aspects of lipid and glucose metabolism.6 Serum triglyceride level reflect the balance between production and clearance of triglyceride-rich lipoproteins such as very-low-density lipoprotein and chylomicrons. FXR induces the expression of the very-low density lipoprotein receptor.7 FXR regulates the expression of peroxisome proliferator-activated receptor (PPAR)- α.8 FXR down regulates sterol response element-binding proteins (SREBP)-1c expression, which is responsible for decrease in hepatic triglyceride secretion.9 FXR decreases triglyceride levels by: increasing their clearance by modulating LPL activity, inducing PPARα and inhibiting SREBP1c. Activation of FXR cause increase in LDL receptor (LDLR) and scavenger receptor-BI (SR-BI) involved in cholesterol uptake.10, 11 FXR also regulate carbohydrate metabolism. FXR participates in the regulation of the fasting induced gluconeogenic response via induction of the small heterodimer partner (SHP).12 In this way, FXR indirectly affects Phosphoenolpyruvate carboxykinase (PEPCK) and modulating gluconeogenesis.13 FXR activation may improves peripheral insulin sensitivity.14 and promotes glycogen synthesis.15 In view of its central roles in coordinating regulation of both glucose and lipid metabolism, we proposed, FXR agonists are promising therapeutic agents for treatment of diabetic dyslipidemia. On the basis of above, objective of the present study is to find Protective Role of the Farnesoid X Receptor Ligand Ivermectin in Wistar Rats with Diabetic Dyslipidemia.

Several diabetic dyslipidemia parameters namely body weight, food intake, oral glucose tolerance test, serum glucose level, lipid profile, creatinine, albumin, urea and antioxidant parameter were measured. Throughtout the study, there was no ststistical significant (p<0.05) different in body weight (Table 1) and food intake (Table 2) among the groups.

Table 1 - Effect of Ivermectin on body weight in experimentally induced diabetic dyslipidemia in rats

 Values are expressed as mean ± SEM. (N=6).Values are statistically evaluated using ANOVA analysis followed by Dunnett’s Post hoc test.

Table 2 - Effect of Ivermectin on food intake in experimentally induced diabetic dyslipidemia in rats

Values are expressed as mean ± SEM. (N=6).Values are statistically evaluated using ANOVA analysis followed by Dunnett’s Post hoc test.

Analysis of oral glucose tolerance pattern during 120mins test period in normal control and model control rats showed statistical significant (P<0.05) difference in glucose levels. The glucose level was not found to be decreased in model control rats at 60, 90 and 120 min. However, it was significantly greater than normal control rats. Ivermectin supplemented rats showed lower glucose elevation and faster disposal rates thereby displaying significant improvement (P<0.05) in glucose tolerance pattern as compared to model control (Table 3).

Table 3 - Effect of Ivermectin on glucose levels in experimentally induced diabetic dyslipidemia in rats

Results were presented as mean±SEM, with n=6 animals in each group. One way Analysis of Variance (ANOVA) was used to determine the significant statistical difference between the groups followed by Dunnett’s Post-hoc test. P<0.05 value was set as the level of statistical significance.

At 28^th day serum level of glucose, TC, TG, LDL, VLDL, creatinine, and urea are significantly (p<0.05) higher and HDL level is significantly (p<0.05) lower in model control group as compare to normal control group. Standard (atorvastatin+ metformin) and Ivermectin (0.2, 0.6, 1.8 mg/kg) treated group shows significantly (p<0.05) decreased serum level of glucose, TC, TG, LDL, VLDL, creatinine, and urea and significantly (p<0.05) increased level of HDL as compare to model control group. No significant different were noted in serum albumin levels of all groups of animal. Model control animal shows significant (p<0.05) increased serum MDA levels and decreased free radical scavenging enzyme (SOD and catalyse) activity in serum compared to normal control group. Standard (atorvastatin+metformin) and Ivermectin (0.2, 0.6, 1.8 mg/kg) treated group shows significant(P<0.05) reduction in MDA level and enhance activity of SOD and Catalyse level as compare to model control group (Table 4)

Table 4 - Effect of Ivermectin on biochemical and antioxidant parameter in experimentally induced diabetic dyslipidemia in rats

Results were presented as mean±SEM, with n=6 animals in each group. One way Analysis of Variance (ANOVA) was used to determine the significant statistical difference between the groups followed by Dunnett’s Post-hoc test. P<0.05 value was set as the level of statistical significance.

Diabetes and dyslipidemia are associated with a high risk of cardiovascular disease. The most common lipid pattern in people with type 2 diabetes consists of hypertriglyceridemia (hyper-TG), low high density lipoprotein cholesterol (HDL-C), and relatively normal plasma concentrations of low-density lipoprotein cholesterol (LDL-C). The Farnesoid X receptor (FXR) is one of the most important members of the nuclear receptor superfamily.8 On its activation by bile acids, FXR regulates bile acid synthesis, conjugation, and transport, as well as various aspects of lipid and glucose metabolism.9 Thus upon modulation, FXR can be attractive target for diabetic dyslipidemia. Thus this research attempts to evaluate the FXR ligand Ivermectin in diabetic dyslipidemia. Recent evidences tend to identify consumption of carbohydrates more so of refined sugars with high fructose content, as an important culprit in development of hyperglycemia, insulin resistance, hyperinsulinemia, and dyslipidemia, hence a suitable model for evaluating the efficacy of preventive/ameliorating agents. Therefore In current study, fructose diet model was used for induction of diabetes dyslipidemia.28 High dietary fructose intake does not result in a significant weight gain. Supporting evidence, in present study Body weight and food intake was not significantly different between groups.

Diabetes consists of array of dysfunctions characterised by hyperglycaemia hence measurement of blood glucose levels can serve as important tool for diagnosis of diabetes.  Ivermectin decrease the blood glucose level in treatment group as compare to model control group. The present study portray prevalence of insulin resistance in fructose fed rats which is clearly indicated by poor the glucose tolerance curve as recorded in oral glucose tolerance test. Ivermectin improve glucose tolerance pattern as compare to model control group. The dyslipidemia accompanying insulin resistance is characterized by distinct changes from a normal plasma lipid and lipoprotein profile. The most commonly observed lipid abnormalities in diabetes are hypercholesterolemia. A deficiency of insulin is associated with increase in cholesterol levels due to the enhanced mobilization of lipids from the adipose tissue to the plasma.29 The inability of the fat cell to adequately store excess triacylglycerol is a likely first step in the underlying hypertriglyceridemia of insulin resistance.30 Hypertriglyceridemia is a common finding in patients with diabetes mellitus and in insulin resistant states results from accumulation of VLDL particles, either by overproduction, decreased catabolism or both. Increased LDL concentration in the plasma of diabetic rats might be due to the defect in insulin secretion. In particular, many studies have found LDL to be the most dangerous among the plasma lipids, and the oxidation of LDL leads to its increased penetration of arterial walls. 31

In contrast to higher levels of other cholesterol subtypes being a risk factor of dyslipidemia, HDL lower levels are harmful. The decreased plasma HDL level in diabetic rats may be due to the decreased production of HDL by intestine and liver and or due to the glycation of HDL or its apoproteins by high glucose rendering it more pro-atherogenic.30 The present study showed that treatment with Ivermectin significantly decrease the level of TC,TG,LDL,VLDL and significantly increase HDL level. Serum albumin has been suggested to be associated with insulin resistance. Both the antioxidant and anti-inflammatory properties of albumin have been suggested as possible mechanisms for this association.32 In this present study there was no significant change in serum albumin level. In diabetes the delicate filtering system in the kidney becomes gradually destroyed. Urea & creatinine are the parameters to diagnose functioning of the kidney. Changes in serum creatinine concentration more reliably reflect changes in GFR than do changes in serum urea concentrations. Serum concentrations of creatinine depend almost solely upon GFR. Urea formation is influenced by a number of factors such as liver function, protein intake and rate of protein catabolism.33 Ivermectin treatment significantly reduced the serum creatinine and urea levels compared to the model control animals. Hyperglycemia and characteristic dyslipidemia of diabetes mellitus (DM) along with increased oxidative stress leading to endothelial dysfunction have been implicated as early events in the pathogenesis of atherothrombotic macrovascular disease. Oxidative stress results from imbalance between the production of free radicals and antioxidant defense mechanisms and is an important causative factor in several chronic diseases viz diabetes and associated complications. Lipid peroxidation is a marker of cellular oxidative damage initiated by reactive oxygen species. The increased level of lipid peroxidation induces oxidative damage by increasing peroxy radicals and hydroxyl radicals. Malondialdehyde, an end product of lipid peroxidation, is extensively used as a biomarker of lipid peroxidation. 34 Administration of a fructose-rich diet resulted in higher lipid peroxidation by the induction of free radical production in model group as compared to normal animals. A decrease in the rise of MDA level after administration of Ivermectin suggest that Ivermectin decrease the lipid peroxidation. Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide (H2O2) which is then transformed into water. Hydrogen peroxide can also give rise to hydroxyl radicals in the cells. Thus the removal of H2O2 is very important for antioxidant defense in cell or food systems. H2O2 can cross membranes and may oxidize a number of compounds. Glucose oxidation is believed to be a major factor adding to the level of oxidative stress, as glucose is oxidized in a transition-metal dependent reaction to an enediol radical anion that is converted into reactive ketoaldehydes and to superoxide anion radicals. The superoxide anion radicals undergo dismutation to hydrogen peroxide by SOD, which if not degraded by catalase or glutathione peroxidase, and in the presence of transition metals, can lead to production of extremely reactive hydroxyl radicals. 24 Hydrogen peroxide is enzymatically processed by catalase. It was reported that endogenous catalase, antioxidant plays an important role in protecting the kidney from diabetic stress through maintaining peroxisomal and mitochondrial fitness.34 Ivermectin significantly increase the level of SOD and catalyse.

From the above finding, it can be concluded that Ivermectin can be used for management of diabetic dyslipidemia.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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