Synergistic Effects of Naringenin and Metformin on Dyslipidemia and Glucose Regulation in High Fructose-Fed Rats

Abstract

Background:

Excessive intake of fructose can lead to insulin resistance (IR). Metformin is commonly used as a first-line treatment for type 2 diabetes, and naringenin, a flavonoid known for its anti-inflammatory properties, can potentially improve insulin resistance. However, the effects of combining these two treatments have not been evaluated. Therefore, the present study aims to investigate the combined effects of naringenin and metformin on fructose-induced insulin resistance in male rats.

Materials and Methods:

Male Wistar rats were divided into five groups: control (without receiving fructose), fructose-induced insulin resistance; F group (with receiving fructose for 8 weeks), fructose+metformin; FM (receiving metformin for last 4 weeks), fructose+naringenin; FN (receiving naringenin for last 4 weeks) and fructose+metformine+naringenin; FMN (receiving metformin and naringenin for last 4 weeks). At the end of the experiment, after an oral glucose tolerance test (OGTT), following deep anesthesia, blood drawing was performed to measure glucose, insulin, total cholesterol (TC), high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), triglyceride (TG), tumor necrosis factor-alpha (TNF-a), interleukin 6 (IL-6), free fatty acid (FFA), homeostatic model assessment for insulin resistance (HOMA-IR), quantitative insulin sensitivity check index (QUICKI), and liver enzymes.

Results:

Although metformin and naringenin each alone showed anti-hyperglycemic effects, combined treatment of metformin with naringenin could more remarkably improve glucose intolerance, decrease glucose, TNF-alpha, IL-6, FFA, TG, and TG/HDL-C ratio, and alanine aminotransferase in FMN group compared to F group (P < .05 to P < .0001).

Conclusion:

Our findings demonstrated that the combination therapy of metformin and naringenin, as a novel therapeutic approach, reduced insulin resistance by improving glucose and lipid metabolism, as well as inhibiting inflammation.

INTRODUCTION

Insulin resistance is the main pathogenic cause of many metabolic disorders, such as type 2 diabetes mellitus. Type 2 diabetes mellitus (T2DM) is a common and complex metabolic disorder that is rapidly increasing worldwide. It is linked to unhealthy dietary habits, including high-carbohydrate diets, lifestyle, and obesity.[1,2,3] Insulin resistance is a key factor in the development of type 2 diabetes. It seems that insulin resistance precede the onset of T2DM by 10-15 years. Research has shown that significant consumption of fructose causes the accumulation of triglycerides and cholesterol (dyslipidemia), which subsequently leads to a decrease in insulin sensitivity, insulin resistance, and glucose intolerance.[4,5] Diabetes causes significant changes in cellular metabolism in various tissues, including the liver, muscle, and pancreatic tissue.[1] Geddawy et al. (2017) demonstrated that a high fructose diet (10% HF) over 6 weeks reduces insulin levels by 50% and impairs glucose tolerance, while increasing visceral fat in the liver tissue of male Wistar rats.[6] High-carbohydrate diet exposure can increase inflammatory cytokines directly and indirectly, by inducing oxidative stress which it can lead to reduced insulin sensitivity and insulin resistance.[7,8]

Metformin is an anti-hyperglycemic drug that plays a central role in the management of metabolism-related diseases, such as diabetes.[9] Metformin acts directly or indirectly on the liver to lower glucose production, enhances insulin sensitivity, reduces body weight, increases glucose uptake, and decreases fasting insulin levels.[10] Recently, several studies have reported the effectiveness of plant-derived bioflavonoids in managing metabolic diseases such as diabetes.[11] Naringenin is a flavonoid with anti-glycemic and anti-inflammatory properties that was observed in vitro and in vivo models of diabetes, reducing blood lipids, improving insulin resistance by improving insulin sensitivity and β-cell function, and also reducing the damage of pancreatic β-cells against apoptosis caused by glucotoxicity in diabetics.[12,13,14]

Given that metformin and naringenin with anti-inflammatory properties can affect insulin sensitivity and improve dyslipidemia and glucose dysregulation and because insulin resistance tends to worsen over time and also due to the low effectiveness of initial monotherapy, combined treatments may be necessary. Therefore, the present study aims to investigate the effect of metformin alone or in combination with naringenin, through the modulation of inflammation and lipid profile in a fructose-induced insulin resistance rat model.

MATERIALS AND METHODS

Animals

In the current study, 40 male Wistar rats with the weight of 180-200 g were randomly selected and housed in cages (2-3 rats/cage) with condition standard including a cycle of 12h-light/12h dark and temperature 22 ± 2°C with free access to tap water and standard food. Following acclimatization for 1 week, rats were separated into five groups (n = 8/group): control; rats received without fructose water with vehicle solution by gavage, fructose (F); rats received fructose 10% in drinking water with vehicle solution by gavage for 8 weeks, fructose + metformin (FM); rats received fructose 10% in drinking water for 8 weeks and metformin (as a standard drug) 200 mg/kg and vehicle solution by gavage daily for last 4 weeks, fructose + naringenin (FN); received fructose 10% in drinking water for 8 weeks and naringenin (50 mg/kg in 0.5% carboxymethyl cellulose) by gavage daily for last 4 weeks, fructose + metformin + naringenin (FMN); rats received fructose 10% in drinking water for 8 weeks and both metformin and naringenin daily for 4 last weeks. At the end of each week, the body weight of all animal groups was measured.

All procedures performed in the present study followed the local Ethics Committee of Mazandaran University of Medical Sciences, Sari, Iran (IR.MAZUMS.ACE.1402.076).

OGTT

After the last treatment, rats of all groups were fasted overnight for 12 h. Then, following gavage of a bolus of glucose solution 45% (2 g/kg), blood glucose levels were detected by glucometer through the cut tail method at 0, 15, 30, 60, 90, and 120 min after glucose injection.[15]

Blood sampling

After doing the OGTT, animals were sacrificed and decapitated following deep anesthesia. Then, blood samples were collected. After centrifugation, serum samples were separated and stored at − 80°C for measuring insulin, FFA, TG, LDL, HDL, cholesterol, IL-6, TNF-alpha, aspartate aminotransferase (AST), and alanine aminotransferase (ALT).

Serum biochemical assay

The serum parameters were detected by using a rat insulin, FFA, IL-6, TNF-alpha ELISA kit (ZellBio, Germany), ALT, AST, LDL, HDL, TG, and cholesterol using the IFFC method (ELISA kits; Pars Azmoon, Iran).

HOMA-IR

Homeostatic model assessment of insulin resistance is one of the insulin resistance indexes that are determined with a formula: HOMA-IR (ci × cg)/22.5,

where ci is fasting insulin level (μU/ml) and cg is fasting glucose level (mmol/L).[16]

QUICKI

QUICKI = 1/[log (insulin μU/mL) + log (glucose mg/dL)].[16]

TG/HDL-C ratio is one of the important insulin resistance markers and a cardiovascular disorder.[17]

Statistical analysis

All data were presented as standard error of the mean (mean ± SEM). The data were analyzed by GraphPad Prism 8 statistical software using two-way repeated measures and one-way ANOVA analysis, followed by the post hoc Tukey test. The significance level was set at P < .05. The Kolmogorov-Smirnov normality test was used for variables in this study.

RESULTS

Body weight

Body weight in all groups was measured weekly during the experiment period. Analytic results did not show any significant differences between the groups [Figure 1].

Figure 1.

The effects of metformin or naringenin on the body weight of rats during experiment. Groups: Control (C), Fructose (F), Fructose + Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin + Naringenin (FMN). Data are expressed as the mean ± SEM (n = 8)

Glucose and insulin levels, HOMA-IR, QUICKI

Fasting blood glucose level after 8 weeks of feeding with fructose 10% in drinking water showed a significant increase in the F group (P < .05, CI: 17.33 to -4.08), whereas treatment with metformin significantly reduced glucose compared to F and control groups (P < .001, CI: 8.16 to -29.58 and P < .05, CI:1.54 to -22.96, respectively). Naringenin alone lowered glucose in the FN group as compared to the F group (P < .01, CI: 2.41 to -22.83). However, naringenin in combination could more significantly decrease blood glucose level in the FMN group when compared to F and control groups (P < .0001, CI: 12.54 to -33.96 and P < .001, CI: 5.92 to -27.33) [Figure 2a]. No significant differences were observed in serum insulin levels between groups [Figure 2b].

Figure 2.

The effects of metformin or naringenin on the serum level of (a) glucose, (b) insulin, (c) HOMA-IR, (d) QUICKI. Groups: Control (C), Fructose (F), Fructose + Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin + Naringenin (FMN). Data are expressed as the mean ± SEM (n = 8). *** P < .001, * P < .05 vs the C group. ^$$$$ P < 0.0001, ^$$$ P < 0.001, ^$$P <.01 vs the F group

Fructose diet for 8 weeks significantly increased HOMA-IR compared to C rats (P < .001, CI: −0.651 to 0.188) and after treatment with metformin in the FM group (P < .0001, CI: 0.379 to -0.843). On the other hand, naringenin, alone and in combination with metformin in FN and FMN groups, lowered HOMA-IR in comparison with fructose-fed rats of the F group (P < .0001, CI: 0.379 to -0.843) [Figure 2c].

QUICKI in the F group had a marked reduction in comparison to the C group (P < .05, CI: 0.003 to -0.036), which increased by metformin (P < .0001, CI: 0.049 to -0.016). Naringenin alone and also in combination with metformin remarkably increased QUICKI when compared to the F group (P < .01, CI: 0.037 to -0.004 and P < .0001, CI: 0.049 to -0.016). Therefore, the increase in QUICKI in the FMN group was larger than in the FN group [Figure 2d].

OGTT

One day after the last treatment, an OGTT was performed, and the results were analyzed. Fructose-fed rats in the F group significantly had a higher glucose level than control rats at 30 and 60 min after ingestion of a glucose load (30 min: P <.0001, 60 min: P <.05). Treatment of fructose-fed rats with metformin, naringenin, and a combination of both for 4 weeks could significantly reduce the blood level of glucose increased by fructose at the time point of 30 min when compared to F group (P < .0001). At 60 min, glucose was significantly higher in the F group compared to the C group (P < .05), and just the combined treatment of metformin with naringenin in the FMN group was able to suppress this increased glucose level (P < .05) [Figure 3a]. AUC_glucose in the F group was significantly enhanced as compared to the control group (P < .01). 4 weeks after treatment in the F, M, and FMN groups, AUC_glucose significantly lowered in comparison to the F group (P < .01). All three treatments showed a similar effect [Figure 3b].

Figure 3.

The effects of metformin or naringenin on OGTT (oral glucose tolerance test). (a) Linear curve of blood glucose level during OGTT, (b) area under curve of blood glucose level during OGTT (AUC). Groups: Control (C), Fructose (F), Fructose + Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin + Naringenin (FMN). Data are expressed as the mean ± SEM (n = 8). **** P < .0001, ** P < .01, * P < .05 vs the C group. ^$$$$ P < 0.0001, ^$$ P < 0.01, ^$P <.05 vs the F group

TNF-alpha and IL-6, and FFA

One-way ANOVA analysis of results showed that serum concentration of TNF-alpha in F group significantly increased following high-fructose intake for 8 weeks (P < .05, CI: 34.21 to 3.58). After administration of metformin and naringenin alone in FM and FN groups, the increased TNF-alpha resulting from fructose significantly decreased compared to F group (P < .05, CI: 1.86 to -32.5). It was also observed that combined treatment of metformin and naringenin in the FMN group considerably reduced TNF-alpha levels as compared to the F group (P < .001, CI: 13.66 to -44.28) [Figure 4a]. Although a non-significant increase in IL-6 serum levels was observed in the F group compared to the C group, only in the FMN group, IL-6 concentration was significantly decreased when compared to the F group (P < .05, CI: 0.74 to -28.36) [Figure 4b].

Figure 4.

The effects of metformin or naringenin on serum level of (a) IL-6, (b) TNF-alpha, (c) free fatty acid (FFA). Groups: Control (C), Fructose (F), Fructose + Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin + Naringenin (FMN). Data are expressed as the mean ± SEM (n = 8). * P < .05 vs the C group. ^$$$ P < 0.001, ^$P <.05 vs the F group

Although FFA in serum level had a non-significant increase after a high-fructose diet in the F group, metformin and naringenin alone were not able to reduce FFA levels in comparison to the F group, but in the FMN group following administration of both of them, FFA concentration significantly decreased as compared to the F group (P < .05, CI: 0.094 to -3.90). These results confirm the synergistic effect of this combination therapy [Figure 4c].

Serum total cholesterol, TG, HDL-C, LDL-C, TG/HDL-C ratio

After 8 weeks of fructose supplementation, rats of the F group had a marked increase in total cholesterol level of serum as compared to the C group (P < .001, CI: −47.96 to 14.37). In the FM group, metformin was able to lower this high level of Chol in comparison to the F group (P < .01, CI: 5.20 to -38.79). Naringenin, both alone in the FN group and combination with metformin in the FMN group, significantly reduced cholesterol levels equally (P < .05, CI: 0.538 to -34.13, CI: 2.20 to -35.79). Therefore, a synergistic effect from this combination was not observed.

Fructose also remarkably enhanced the TG of serum in the F group compared to the C group (P < .001, CI: 41.60 to -11.06), which was decreased by treatment with metformin and naringenin alone (P < .05, CI: 1.23 to -31.77 and P < .01, CI: 7.73 to -38.27). However, the remarkable effect of this combination therapy was indicated in the FMN group when compared to the F group (P < .0001, CI: 16.73 to -41.27) [Table 1].

Table 1.

The effects of Metformin or naringenin on serum lipid levels

Groups		Total cholesterol (mg/dl)		TG (mg/dl)		LDL-C (mg/dl)		HDL-C (mg/dl)		TG/HDL-C ratio
C		84.3+4.5		46.1+2.2		37.2+1.2		32.3+1.9		1.45+0.11
F		115.5+6***		72.5+3.8***		48+1.8***		39.9+2.1*		1.85+0.12
FM		93.5+2.2$$		56.+5.2$		39.3+1.8$$		31+1.3$$		1.82+0.19
FN		98.2+4$		49.5+4.01$$		39.3+0.8$$$		34.5+1		1.45+0.15
FMN		96.5+2.3$		40.5+3.06$$$$		37.8+1.53$$$		37.5+1.8		1.08+0.08**&&

***P<0.001, **P<0.01, *P<.05 vs the C group. ^$$$$P<0.0001, ^$$$P<0.001, ^$$P<.01, ^$P<.05 vs the F group. ^&&P<0.01 vs F group. TG triglyceride (TG), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C) and TG/HDL-C ratio. Groups: Control (C), Fructose (F), Fructose+Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin+Naringenin (FMN). Data are expressed as the mean±SEM (n=5)

HDL concentration in the F group significantly increased (P < .05, CI: −13.69 to 1.31), and only treatment with metformin decreased that (P < .01, CI: 2.64 to -15.02). Naringenin in the FN and FMN groups did not change significantly. A fructose diet for 8 weeks had a significant effect on LDL levels in the F group (P < .001, CI: −17.25 to 4.41). While treatment for 4 weeks with metformin in the FM group, naringenin alone in the FN group, and combination with metformin in the FMN group, remarkably reduced LDL compared to the F group (P < .01, CI: 2.24 to -15.09, P < .001, CI: 3.74 to -16.59) [Table 1].

One of the lipid markers of insulin resistance is the TG/HDL ratio. In current research, this ratio in the F group was non-significantly higher than the control group. Neither naringenin nor metformin treatment alone was able to reduce this marker, but when they were administered in combination, this marker decreased in the FMN group as compared to the F and FM groups (P < .01, CI: 0.190 to -1.33) [Table 1].

Liver enzymes: ALT and AST

The function of the liver following a high-fructose diet was assayed by measuring the activity of ALT and AST in blood. Results analysis showed that AST did not have significant changes between groups [Figure 5a]. However, ALT concentration only in the FMN group after combination therapy for 4 weeks showed a significant reduction in comparison to the F group (P < .05, CI: 1.83 to -27.5) [Figure 5b].

Figure 5.

The effects of metformin or naringenin on serum level of (a) AST, (b) ALT. Groups: Control (C), Fructose (F), Fructose + Metformin (FM), Fructose + Naringenin (FN), Fructose + Metformin + Naringenin (FMN). Data are expressed as the mean ± SEM (n = 8). ^$P <.05 vs the F group

DISCUSSION

Results of current research showed that chronic fructose feeding in rats for 8 weeks as a solution in drinking water led to several indicators of insulin resistance, including dyslipidemia with overproduction of TG, LDL, cholesterol, impaired OGTT, HOMA-IR, and impaired liver function. For the last 4 weeks of 8 weeks of fructose feeding, rats were administered metformin, naringenin, and a combination of both treatments orally. Results of these measured metabolic parameters indicated improving effects of all three treatments, with these therapeutic effects being more evident in the combined treatment.

Administration of naringenin as a natural flavonoid and metformin as the first-line drug for the treatment of type 2 diabetes, either alone or in combination, was able to reduce blood glucose levels in all rats treated, but this reduction in the group that used the combined treatment was greater than that of them alone. It confirmed the additional impact of this combination on glucose reduction. There was no significant difference in insulin levels by fructose feeding and treatments. Several previous studies have shown the hypoglycemic effect of naringenin on different animal models of diabetes, such as high-fat or fructose-induced diabetes.[18,19,20,21,22,23] A human study conducted by Dallas et al.[23] (2014) demonstrated the hypoglycemic effect of naringenin on healthy overweight individuals. It seems that naringenin reduces fructose-induced hyperglycemia by decreasing gluconeogenesis, increasing glucose oxidation and FFA beta-oxidation through up-regulating AMP-activated protein kinase AMPK.[24,25] Moreover, OGTT is impaired by a high fructose diet. This result indicated that insulin resistance was induced by fructose. Following taking naringenin and metformin, OGTT was improved in treated rats, and the hypoglycemic effect of naringenin was more obvious in the combined treatment group. HOMA-IR as an index of insulin resistance enhanced by fructose feeding was reduced by metformin, naringenin, and a combination of them. Besides, QUICKI marker, as an insulin sensitivity index, was raised by both mono and combined therapy to the same extent. Furthermore, the results of the present study showed that two pro-inflammatory factors, TNF-alpha, IL-6, and FFA, which are involved in insulin resistance by impairing the insulin signaling pathway, declined more by naringenin in combination with metformin than by monotherapy for each of them. We found an additive effect of naringenin with metformin on high levels of these factors induced by a high-fructose diet. Naringenin diminishes glucose production from the liver. In confirmation of our study, the results of other studies indicate that naringenin can improve important indicators involved in insulin resistance, such as glucose intolerance, HOMA-IR, and QUICKI.[19,25,26] It seems that reduced glucose output from the liver is caused by inhibiting the activity of enzymes involved in glycogenolysis and gluconeogenesis. On the other hand, low blood glucose levels may result from increased glucose uptake due to elevated GLUT4 levels and insulin receptor activity.[26] In the current study, the insulin-sensitivity improvement effect of naringenin (as shown by QUICK) may be attributed to an increase in glucose uptake via lowering FFA,[27] resulting from augmenting FFA oxidation.[28] In another study, it was proved that naringenin directly increases glucose uptake in muscle cells in an AMPK-dependent manner.[8] In another in vitro study, naringenin reduced the secretion of FFA and TNF-α from adipocytes.[18,29,30]

In this study, high total cholesterol levels in rats fed a fructose diet were decreased by metformin and naringenin alone and combined therapy of both, However, no additional effects were observed from the combined administration. On the other hand, a marked combined therapeutic effect was created by naringenin and metformin in the reduction of TG levels in the FMN group. Naringenin alone and in combination did not produce any elevation in HDL-c, but was able to reduce the level of LDL-c both alone and in combination, even better than metformin. Furthermore, the TG/HDL ratio is an important index of insulin resistance, more remarkably decreased by the combination therapy of naringenin with metformin in fructose-fed rats. Regarding these results, it can be concluded that a larger improvement effect on dyslipidemia was seen by co-administration of naringenin and metformin in these lipid markers of insulin resistance. As previous studies reported, naringenin exhibited anti-hyperlipidemia effects in metabolic disorders such as insulin resistance or diabetic conditions.[24,25,26,31,32,33,34] Previous studies reported that there are several mechanisms for lowering hyperlipidemia by naringenin. In hepatocytes, naringenin was shown to diminish LDL production by increasing LDL-receptor expression and LDL degradation.[35] Moreover, administration of naringenin exerts its hypo-lipidemic impact by upregulating AMP-activated protein kinase AMPK in the liver, and also decreases TG production.[28,35] Therefore, it can be said that it has a metformin-like effect. Previous reports confirm that naringenin has an anti-lipidemic effect in both animal and human studies, particularly in conditions of insulin resistance or diabetes.[22,36,37,38]

In the current study, the other additive impact of naringenin with metformin on the function of the liver was confirmed. ALT concentration was also significantly decreased by combined therapy in FMN group. While naringenin and metformin alone were not able to lower ALT levels in the serum of rats fed fructose. Serum levels of AST did not display any change between groups. ALT and AST are usually indicators of liver function, which facilitate metabolism. ALT is generated mainly in the liver, but AST is produced not only by the liver but also by other tissues such as the kidney, heart, lung, muscle, and brain. A high ALT level is a clear indication of liver dysfunction. Thus, it may be suggested that a combined treatment with naringenin and metformin could improve the dysfunction of the liver in fructose-fed rats. Following our results, 21 days of treatment of diabetic rats with naringenin 50 mg/kg b.w daily, declined activity of ALT and AST in serum.[39]

Based on the findings of this research, it can be concluded that the combination therapy of metformin and naringenin is more effective in suppressing the secretion of pro-inflammatory cytokines, inhibiting lipogenesis, increasing insulin sensitivity, improving liver function, insulin resistance, and glucose metabolism, compared to using metformin or naringenin alone. Overall, the important findings of this study suggest that combining antidiabetic medications with natural bioactive compounds is an effective and novel therapeutic strategy for improving insulin resistance and addressing metabolic abnormalities caused by a high fructose diet.

Data availability

All data generated or analyzed during this study can be obtained by contacting me via email (sadeghi.f.ph@gmail.com).

Ethics approval and consent to participate

All procedures performed in the present study were in accordance with the local Ethics Committee of Mazandaran University of Medical Sciences, Sari, Iran (IR.MAZUMS.ACE.1402.076).

Conflicts of interest

There are no conflicts of interest.

Funding Statement

This research was supported by the Immunogenetics Research Center, Mazandaran University of Medical Sciences, Sari, Iran (grant number: 17698).