Effects of naringenin on metformin disposition in a diabetic rat model.

Abstract

Diabetes mellitus (DM) is one of the largest global health emergencies of the 21st century. It is a major cause of blindness, kidney failure, cardiovascular diseases, lower limb amputation and accounted for 10,7 % of global all-cause mortality among people aged between 20 and 79 years old. Metformin is currently the most widely prescribed anti-diabetic drug. It exists as a hydrophilic cation at physiological pH. As such, membrane transporters play a substantial role in its oral absorption, hepatic uptake, and renal elimination. Among these transporters, organic cation transporters OCT 1 (SLC22A1) and OCT 2 (SLC22A2) are known to be important determinants of the pharmacokinetics of metformin. Naringenin, which is a plant-derived compound found in citrus fruits and vegetables, has been presumed to interact with conventional drugs and influence their disposition by modification of drug-metabolizing enzymes and transporters. The aim of this study was to investigate the effects of naringenin on organic cations transporters OCT1 and OCT2 protein expression and subsequently on metformin disposition in streptozotocin- induced diabetic rats. Methods Forty-nine male Sprague Dawley rats 250–300 g body weight (BW) were randomly divided into 7 experimental groups (n = 7). They were orally treated daily with 3.0 ml/kg body weight (BW) of distilled water (group 1) or 250 mg/kg BW of metformin (groups 3, 6 and 7) or 60 mg/kg BW of naringenin (groups 2, 5 and 7) dissolved in distilled water. Groups 4, 5, 6 and 7 were given a single intraperitoneal injection of 60 mg/kg BW of streptozotocin to induce diabetes. Animal body weights and water intake were recorded daily. Fasting blood glucose (FBG) and glucose tolerance tests (GTT) were subsequently done. Urine samples were collected from rats kept in individual metabolic cages for 24 hours, to determine output, electrolytes, albumin, creatinine and metformin levels. Thereafter, the animals were sacrificed by halothane overdose and blood was collected via cardiac puncture. Liver and kidneys were excised, rinsed in normal saline, blotted dry, weighed, snap frozen in liquid nitrogen and stored at -80°c for analysis of OCT 1 and OCT 2 protein expression by Western blot. OCT 1 and OCT 2 proteins were extracted and separated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Then, the gel was blotted electrophoretically onto a nitrocellulose membrane which was then probed with a primary antibody and ultimately an enzyme conjugated secondary antibody and substrate to visualize the bands representing the target proteins. Results Diabetic rats treated with naringenin and metformin either alone or in combination exhibited weight gain, improved creatinine clearance and reduced polydipsia, albuminuria, serum creatinine and blood urea nitrogen compared to untreated diabetic rats. By contrast, metformin with/without naringenin did not significantly ameliorate hyperglycemia in diabetic rats. Treatment with naringenin increased hepatic uptake and renal clearance of metformin in diabetic rats compared to untreated groups. In addition, naringenin significantly increased lactate concentrations and metabolic acidosis in rats treated with metformin compared to those that were not treated with metformin. Furthermore, diabetic rats exhibited lower OCT1 and OCT2 protein expressions but naringenin treatment significantly increased hepatic OCT1 and renal OCT2 protein expressions in the presence of metformin. Conclusion Collectively, our data suggest that metformin disposition could be affected by naringenin through the upregulation of OCT1 and OCT2 protein expressions. Upregulation of OCT1 expression may be associated with metformin-induced lactic acidosis while increased renal OCT2 expression might facilitate metformin excretion and reduce the risk of lactic acid. However, increased renal excretion of metformin by naringenin may not be sufficient to avert metformin-induced lactic acidosis.