Combination of vildagliptin and rosiglitazone ameliorates nonalcoholic fatty liver disease in C57BL/6 mice.

OBJECTIVES: To evaluate the effect of vildagliptin alone and in combination with metformin or rosiglitazone on murine hepatic steatosis in diet-induced nonalcoholic fatty liver disease (NAFLD).
MATERIALS AND METHODS: Male C57BL/6 mice were fed with high fat diet (60 Kcal %) and fructose (40%) in drinking water for 60 days to induce NAFLD. After the induction period, animals were divided into different groups and treated with vildagliptin (10 mg/kg), metformin (350 mg/kg), rosiglitazone (10 mg/kg), vildagliptin (10 mg/kg) + metformin (350 mg/kg), or vildagliptin (10 mg/kg) + rosiglitazone (10 mg/kg) orally for 28 days. Following parameters were measured: body weight, food intake, plasma glucose, triglyceride (TG), total cholesterol, liver function tests, and liver TG. Liver histopathology was also examined.
RESULTS: Oral administration of vildagliptin and rosiglitazone in combination showed a significant reduction in fasting plasma glucose, hepatic steatosis, and liver TGs. While other treatments showed less or no improvement in the measured parameters.
CONCLUSIONS: These preliminary results demonstrate that administration of vildagliptin in combination with rosiglitazone could be a promising therapeutic strategy for the treatment of NAFLD.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease that occurs in the absence of evident infection or significant consumption of alcohol. NAFLD includes different forms of chronic liver damage ranging from a simple fatty infiltration of hepatocytes (steatosis) to steatohepatitis and fibrosis, and may evolve to cirrhosis and hepatocellular carcinoma.[1] The prevalence of NAFLD is around 15%-40% in western and 9%-40% in Asian populations,[23] reaching levels as high as 75%-100% in obese, type-II diabetes, and other metabolic disease patients.[4]

The biological mechanism underlying steatosis occurrence and progression to NAFLD is not yet fully understood. Association of NAFLD with obesity, type 2 diabetes, and other metabolic syndrome suggests hyperglycemia, hyperlipidaemia, and insulin resistance as triggering factors in pathogenesis of NAFLD.[5] NAFLD can be considered as a hepatic manifestation of a metabolic syndrome. NAFLD patients have increase subclinical atherosclerosis and it present a higher risk of mortality from cardiovascular disease compared with those without steatosis.[6] These findings have created a surge toward development of strategies to: control obesity, improve glycemic control, enhance insulin sensitivity, and improve β-cell and hepatocyte function.[7] Nutritional counseling or diet prescription to reduce body weight, coupled with physical exercise remains the first line of treatment. Drug therapy has typically been focused on the management of associated risk factors such as diabetes, obesity, and hyperlipidemia, being predisposing factors for development of NAFLD.[8] So far, no pharmacological remedy has been approved for treatment of NAFLD. Therefore, for effective treatment of NAFLD, combination of antidiabetic agents which are acting through different mechanisms may be a logical approach.

Vildagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor which increases plasma levels of active glucogon-like peptide-1 (GLP-1), improves glucose-dependent insulin secretion, β-cell function, improves insulin sensitivity, reduces inappropriate glucagon secretion, and reduces fasting and postprandial glucose.[9] Rosiglitazone is a potent member of the thiazolidinedione class and acts as an insulin sensitizer improves glycemic control by increasing peripheral glucose disposal and reduces hepatic glucose output through activation of peroxisome proliferator activator receptor-gamma (PPAR-γ).[10] Activation of PPAR-g results in increased free fatty acid uptake by adipocytes, sparing the liver, skeletal muscle, and beta cells from the harmful metabolic effects of lipid toxicity.[5] Metformin is a biguanide, suppresses hepatic gluconeogenesis, and enhances insulin-mediated glucose disposal in muscle and fat. Its antilipolytic effect could be beneficial in reducing free fatty acid concentration via restricting efflux from adipose tissue and improving insulin sensitivity.[11] The present study was designed to assess the effect of vildagliptin alone and in combination with metformin or rosiglitazone on hepatic steatosis in diet-induced NAFLD in C57BL/6 mice.

Materials and Methods

Animals

Male C57BL/6 mice aged 4-6 weeks were obtained from the animal facility of Orchid Chemicals and Pharmaceutical Ltd. (Chennai, India). They were maintained in 12 h light/dark cycle with standard laboratory chow diet and water ad libitum in a controlled environment. All animals were handled according to the guidelines of experimental animal care issued by the committee for the purpose of control and supervision of experiments on animals, Government of India. The experimental protocol was approved by Institutional Animal Ethics Committee.

Chemical and Reagents

Normal chow diet was procured from Nutri Lab® Rodent (Tetragon Chemical Pvt. Ltd., Bangalore, India) and 60 Kcal % high fat diet (HFD) procured from Open Source diets, (New Brunswik, NJ, USA). Fructose was procured from Sisco Research Laboratories (Mumbai, India). Vildagliptin was synthesized at Orchid Chemicals and Pharmaceuticals Ltd. (Chennai, India). Metformin and rosiglitazone were received as a gift from Sri Sainath chemicals (India) and Dr Reddy's laboratory (Hyderabad, India). All other chemicals were of reagent grade obtained from standard sources.

HFD and High Fructose Liquid Induced NAFLD in Mice

Male C57BL/6 mice aged 4-6 weeks and body weight ranges 24.29 ± 0.29 g were used for the study. The animals were divided into two groups, first group (n = 12) fed normal chow diet and the second group (n = 65) fed with 60 Kcal % HFD and high fructose liquid 40% (HFL) for 60 days to induce NAFLD. On 50th day, two animals from normal chow diet and three from HFD + HFL were sacrificed to confirm development of NAFLD After the induction period, the animals were grouped based on body weight. Animals fed with normal chow diet served as Group I: Normal control (0.5% sodium carboxymethyl cellulose (CMC), n = 10) and the animals fed HFD + HFL were further divided homogenously according to body weight (35-37 g), as follows, Group II: NAFLD control (0.5% CMC, n = 10), Group III: vildagliptin (10 mg/kg, n = 7), Group IV: metformin (350 mg/kg, n = 9), Group V: rosiglitazone: (10 mg/kg, n = 7), Group VI: vildagliptin (10 mg/kg) in combination with metformin (350 mg/-kg, n = 8), Group VII: vildagliptin (10 mg/kg) in combination with rosiglitazone (10 mg/kg, n = 9). The animals were treated orally once daily for 28 days, during this period food intake and body weight were recorded on a daily basis.

Assessment of Biochemical Parameters in Plasma

Blood was collected from the retroorbital plexus of overnight fasted animals using microcapillary tubes on 29th day and plasma was separated by centrifugation at 3019 G (6000 rpm) for 10 min. The animals were then sacrificed by cervical dislocation; adipose tissues and liver tissue were excised and weighed. A liver sample from each mouse was kept in 10% formalin for histopathological examination, while the remaining tissue was frozen (-80°C) for further evaluation. The plasma glucose, triglyceride (TG), total cholesterol, total bilirubin, total protein, alanine aminotransaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase were measured from plasma by random access clinical chemistry analyzer Erba XL 300 using a commercially available ERBA diagnostics kit (ERBA diagnostics, Germany).

Assessment of Liver TGs

The liver lipids were extracted using a modified Folch extraction protocol.[12] Briefly, approximately 100 mg of liver tissue was homogenized with methanol (1 mL) then centrifuged at 1342 G (4000 rpm) for 5 min and the supernatant transferred into a separate tube (15 mL). The pellet was again homogenized with chloroform: methanol (2:1) for 2 min. The homogenate was then centrifuged and the supernatant separated and mixed with first supernatant. Potassium chloride (0.1 M) was added to supernatant and mixed well by vortexing. After centrifugation of the mixture, the bottom phase (organic phase) was transferred to a new tube (2 mL). The samples were evaporated in a Turbovap LV evaporator. Residue was reconstituted in 400 μL of mixture of N-butyl alcohol: triton X-100: methanol (3:1:1) and mixed properly by vortexing. The samples were used for TG estimation using a commercially available kit (ERBA diagnostics, Germany).

Assessment of Liver Tissue Histopathology

Liver tissue samples were fixed in 10 % formalin and embedded in paraffin. Sections measuring 5 μm thickness were cut and stained with hematoxylin and eosin (H & E). Liver histology was examined using the light microscope (NIKON, ECLIPSE- E200, Japan) and steatosis scoring was done according to the NAFLD histological scoring system.[13]

Statistical Analysis

All values are expressed as mean ± standard error mean and the graphs were generated using Graph-Pad Prism® (Version 4). Statistical analysis was performed by two-way analysis of variance (ANOVA), followed by Bonferroni test or one-way ANOVA followed by Dunnett's test for all parameters. Results were considered statistically significant at P < 0.05.

Results

Effect of Treatment on Body Weight and Fat Distribution

The animals fed with HFD + HFL diet for 60 days showed significant increase in body weight compared with chow fed animals (P < 0.05). Following 28 days treatment, there was significant reduction (P < 0.01) in body weight gain and cumulative feed intake (P < 0.05) in Group VI compared with Group II and no changes in body weight gain and feed intake was observed in other treatment groups [Table 1]. In NAFLD (Group II) control animals, both epididymal and inguinal fat weight was significantly more (P < 0.01) [Table 1] than normal control animals. None of the treatments had any effect on fat distribution.

Table 1

Effect of vildagliptin, metformin, and rosiglitazone (alone and in combination) on body weight, fat distribution, and liver weight in high fat diet-induced nonalcoholic fatty liver disease in mice

Effect of Treatment on Biochemical Parameters

The animals fed with HFD + HFL (Group II) developed significant hyperglycemia (P < 0.01) [Table 2] compared with chow fed animals (Group I). Rosiglitazone (Group V) treated animals did not showed change in AST levels; however, ALT levels increased which was not significant as compared with Group II. A significant improvement (P < 0.05) in plasma glucose level was observed in the group treated with vildagliptin and rosiglitazone combination (Group VII) as compared with Group II. Metformin treatment (Group IV) significantly reduced AST level (P < 0.05) [Table 2]. There were no significant changes observed in other biochemical parameters across the treated groups.

Table 2

Effect of vildagliptin, metformin, and rosiglitazone (alone and in combination) on plasma biochemical parameters in high fat diet-induced nonalcoholic fatty liver disease in mice

Effect of Different Treatments on Liver Weight

The animals fed with HFD+HFL (Group II) showed an increase in liver weight compared with Group I animals. The Group VII animals treated with vildagliptin in combination with rosiglitazone showed significant reduction in liver weight (P < 0.05) compared with Group II [Table 1]. No significant change in liver weight was observed in the other treatment groups.

Effect of Treatment on Liver TG Content

The animals fed with HFD + HFL (Group II) showed more TG accumulation as compared with normal chow fed animals (Group I). Vildagliptin and rosiglitazone combination treatment (Group VII) reduced TG accumulation as compared with group II [P < 0.01, Figure 1].

Figure 1

Effect of vildagliptin, metformin, and rosiglitazone (alone and in combination) on liver triglyceride in high fat diet-induced nonalcoholic fatty liver disease in mice. Group I: Normal control; Group II: Nonalcoholic fatty liver disease control; Group III: vildagliptin (10 mg/kg); Group IV: metformin (350 mg/kg); Group V: rosiglitazone (10 mg/kg); Group VI: vildagliptin (10 mg/kg) + metformin (350 mg/kg); Group VII: vildagliptin (10 mg/kg) + rosiglitazone (10 mg/kg). Values are expressed as mean ± standard error of the mean, n= 7−10. **P < 0.01 as compared with Group II

Effect of Treatment on Liver Histopathology

Development of hepatic steatosis was confirmed by histopathological scoring of H & E-stained liver sections. Liver sections from regular chow fed animals (Group I) had normal morphological appearance and scored 0 and HFD + HFL fed animals (Group II) developed moderate to severe macrovesicular steatosis and hepatocyte ballooning [Figure 2b, c] when examined histopathologically and had an average score (P < 0.01) compared with Group I [Figure 3]. The animals treated with vildagliptin and rosiglitazone combination (Group VII) showed prominent improvement in steatosis [Figure 2h] and significant reduction in steatosis score (P < 0.01) [Figure 3], as compared with Group II. No significant change in hepatic steatosis score was observed in other treatment groups.

Figure 2

Effect of vildagliptin, metformin, and rosiglitazone (alone and in combination) on liver histology in high fat diet-induced nonalcoholic fatty liver disease in mice (a) Normal control, (b and c) Nonalcoholic fatty liver disease control: profound marked hepatic steatosis and ballooning, (d) vildagliptin (10 mg/kg), (e) metformin (350 mg/kg), (f) rosiglitazone (10 mg/kg), (g) vildagliptin (10 mg/kg) + metformin (350 mg/kg), (h) vildagliptin (10 mg/kg) + rosiglitazone (10 mg/kg): reduced hepatic steatosis and ballooning. H & E staining of liver tissue (×10, 50 μm)

Figure 3

Effect of vildagliptin, metformin, and rosiglitazone (alone and in combination) on liver histopathological score in high fat diet-induced nonalcoholic fatty liver disease in mice

Discussion

The study focused on the effect of vildagliptin alone and in combination with metformin and rosiglitazone in high fat and fructose liquid diet-induced NAFLD in C57BL/6 mice. The NAFLD animals developed marked obesity, hyperglycemia, and fatty liver. Histopathological examination of the livers of NAFLD control mice revealed severe hepatic fat accumulation along with increased liver weight; however, this was accompanied with only mild elevation of liver specific enzymes. A lack of correlation between the degree of NAFLD and levels of liver enzymes is not surprising, since in a clinical situation the liver enzyme levels do not readily correlate with severity of hepatic steatosis.[14] These observed features are similar to the pathological features of human NAFLD. In the NAFLD control group, the animals showed significant reduction in plasma TG with corresponding increase in liver TG levels as compared with normal controls. Though such a reduction seems contrary with human metabolic syndrome condition, it has already been reported by other researchers.[15] In response to sudden excessive fat ingestion when the plasma lipid level exceeds oxidative capacity of energy requiring tissues like skeletal muscle, the liver acts as an effective buffer organ to avoid accumulation of circulating lipid and starts taking up lipid from plasma to store as TG.[14] Excessive stored TG in hepatocytes is the hallmark of NAFLD which is strongly associated with hyperinsulinemia and hyperglycemia.[16]

In the present study, the doses administered to the treatment groups were based on previous studies in rodents.[171819] Previous reports revealed metformin improved fatty liver disease, reversing hepatomegaly, steatosis, and aminotransferase abnormalities in ob/ob mice with fatty liver.[17] In this study, treatment with metformin showed significant reduction in body weight gain without much change in food intake and showed significant reduction in AST levels, but there was no improvement in other biochemical parameters tested, consistent with previous report,[20] and metformin in combination with vildagliptin also showed significant reduction in gain in body weight and food intake and only slight reduction in other biochemical parameters. The reduction in body weight and food intake may be because of the combined effect of metformin and vildagliptin on incretin hormone glucagon like peptide-1 (GLP-1).[21] Histopathological examination of liver sections revealed only mild improvement in hepatic vacuolation (steatosis) in metformin-treated animals and also in vildagliptin and metformin combination-treated animals.

Rosiglitazone has been reported to reverse hepatic steatosis and lower intramyocellular lipids in Zucker fatty rat,[22] attenuate liver inflammation and insulin resistance in methionine and choline-deficient diet induced steatosis in Wistar rats.[23] Ironically, it causes an increase in microvesicular steatosis and liver enzymes in ob/ob mice.[22] Perhaps in db/db mice treated with rosiglitazone in combination with vildagliptin showed improvement in plasma TG and glucose. In this study, treatment with rosiglitazone failed to show improvement in liver weight and histopathology or any other parameter measured. The mild elevation in ALT level observed may be because of the adverse effect of the rosiglitazone.[24] Interestingly, the animals treated with combination of vildagliptin and rosiglitazone showed significant reduction in fasting plasma glucose, liver weight, and hepatic TG levels. Also, the histological examination of liver sections showed significant improvement in hepatic steatosis. This improvement in histopathology may be attributed to decreased liver TG eventually leading to reduction in fasting glucose level.[7] Clinically, the combination of PPAR-γ agonist and DPP-4 inhibitor showed good efficacy in terms of controlling fasting glucose compared with monotherapy in diabetic patients.[25] Consistence with these results, vildagliptin and rosiglitazone combination showed improvement in the fatty liver disease as compared with monotherapy.

In conclusion, our study indicates that the animals treated with vildagliptin and rosiglitazone combination showed significant reduction in hepatic steatosis and TG s. However, further studies are needed to be done to evaluate the mechanism involved in reversal of hepatic steatosis. In this preliminary study, the results suggest that combination therapy of a DPP-4 inhibitor with a PPAR-γ agonist may be a new therapeutic strategy for the treatment of NAFLD.