POLSKI
Introduction
Type 2 diabetes mellitus (T2DM) is a classification of carbohydrate metabolism disorders characterized by common hyperglycemic symptoms, such as polyuria, polydipsia, polyphagia, and unexplained weight loss [1]. In 2021, the global population diagnosed with diabetes was estimated at 537 million, anticipated to increase to 643 million by 2030 and 783 million by 2045. Recent breakthroughs regarding T2DM suggest a significant increase in global prevalence, with the affected population expected to double within the next decade. These data are primarily due to an aging population and an increasing prevalence of obesity [3–5].
Type 2 diabetes mellitus develops when the body produces insufficient insulin or fails to use it effectively, resulting in hyperglycemia. Pancreatic β cells regulate insulin synthesis by controlling the concentration of GLUT-2. As a member of the GLUT family, GLUT-2 exhibits high glucose transporter activity and primarily operates in pancreatic β cells, as well as in the liver, intestines, kidneys, and nervous system.
A notable association has been reported between a high-fat diet and dysfunction of the GLUT-2 pathway in T2DM. A high-fat diet may impair glucose metabolism and insulin sensitivity, therefore promoting the development of T2DM.
GLUT-2 physiologically enables glucose transport into cells, interacts with glucokinase, and serves as a glucose sensor to swiftly adjust glucose concentrations across the cell membrane, attaining equilibrium with external glucose levels. GLUT-2 in pancreatic β cells operates through the conventional insulin secretion mechanism, primarily stimulated by increased blood glucose concentrations. Pancreatic β cells uptake glucose by GLUT-2, initiating a series of events that increase intracellular Ca2+ concentrations, leading to the fusion of insulin-containing granules with the cell membrane and subsequent exocytosis for insulin secretion.
GLUT-2 in the liver accounts for approximately 97% of all glucose transporters in hepatocytes. GLUT-2 enables bidirectional transport in hepatocytes. In fasting or famine conditions, the inactivation or reduced expression of GLUT-2 on the hepatic surface can affect lipid and energy metabolism in the organism. Researchers have associated alterations in GLUT-2 levels with many endocrine and metabolic disorders [6].
Unmanaged hyperglycemia in type 2 diabetes mellitus can lead to organ damage and may pose a life-threatening risk if sustained. However, proficient diabetes care can delay or even prevent these serious consequences. Medicinal herbs utilized in diabetic therapy often exhibit considerable efficacy and safety [7]. Prominent examples include figs (Ficus carica L.), sweet potatoes (Ipomoea batatas L.), garlic (Allium sativum), cinnamon (Cinnamomum cassia), and aloe (Aloe vera) [8].
Recent research has examined the phytochemical content of the ethanol extract of Ficus carica L. leaves, which display a multitude of notable metabolites, including tannins, phenols, glycosides, flavonoids, resins, alkaloids, steroids, and terpenoids. These compounds demonstrate biological activity, play roles in pharmacological and physiological processes, and show considerable antioxidant, antidiabetic, and hypolipidemic properties. Previous studies have elucidated the flavonoid content of F. carica L.
The leaves possess 73 ±4.01 mg RE/g. An ethanolic extract of F. carica L. leaves effectively sequesters hydrogen peroxide due to the phenolic group that converts it to water via electron donation [9]. Ramadan et al. assessed the effectiveness of methanol extracts of Lepidium sativum, F. carica, and Punica granatum in rats with diabetes induced by streptozotocin (STZ); the findings suggested that the extract of F. carica exhibited superior effectiveness compared to L. sativum. L. sativum and P. granatum showed efficacy in the prevention and management of T2DM, significantly reducing glucose levels, lipid profiles, and renal and hepatic enzyme levels [10].
This research employed fig leaf extract (F. carica L.) in a diabetic animal model study. We specifically induced male Wistar rats aged 8 to 12 weeks through the administration of streptozotocin and nicotinamide injections. This study aimed to investigate the effect of fig leaf extract on fasting blood glucose (FBG) levels and GLUT-2 concentrations in the pancreas and liver, administered at doses of 300 mg/kg body weight (BW), 500 mg/kg BW, and 700 mg/kg BW, over a period of 2 weeks. Glimepiride functioned as a positive control to validate the efficacy of fig leaf extract.
Material and methods
Subjects and experimental framework
This study employed 36 male Wistar rats, aged 8 to 12 weeks, with BW ranging from 150 to 250 grams. The rats employed were primarily healthy and had no abnormalities. The rats were kept in plastic cages measuring 1,500–1,800 cm2 and 22 cm in height, with each cage housing six rodents.
The researchers constructed the cage roof using 1 cm iron mesh and lined the base with rice husks, which they replenished every three days. They maintained the ambient temperature between 22 and 26°C and regulated the relative humidity between 40% and 70%. They provided the rats with regular meals and water ad libitum twice daily throughout the trial. Before starting the treatment, the researchers acclimatized the rats for three days. After acclimatization, the researchers divided the rats into six groups, each consisting of six animals. The first group served as the standard control group (KK), followed by the negative control group (KN) and the positive control group (KP). They administered fig leaf extract (F. carica L.) to the treatment groups at different doses: group 1 (P1) received 300 mg/kg BW, group 2 (P2) received 500 mg/kg BW, and group 3 (P3) received 700 mg/kg BW.
The researchers administered a 1:1 mixture of conventional and high-fat feed to groups KN, KP, P1, P2, and P3 for 3 days, then transitioned them to exclusive high-fat feed for four days to induce type 2 diabetes. They persisted in providing group KK with standard feed. After 10 days, the researchers induced diabetes in the groups KN, KP, P1, P2, and P3 using streptozotocin (STZ) and nicotinamide (NA). The researchers fasted the rats overnight before induction. They then administered an intraperitoneal injection of NA at a dosage of 110 mg/kg BW, followed by an injection of STZ at 45 mg/kg BW 15 minutes later. The recommended dosage of STZ for an adult weighing 70 kg is 45 mg. The dose conversion ratio between humans and rats is 0.018. Thus, for a rat weighing 200 g, the dosage of STZ is 0.81.11
The researchers evaluated FBG levels in the rats 72 hours after inducing diabetes with STZ-NA. Streptozotocin provides greater efficacy and causes fewer undesirable effects than other methods of inducing diabetes. By more accurately mimicking the complexity of type 2 diabetes mellitus in humans, STZ-NA improves the evaluation of both pharmaceutical and natural agents for their potential antidiabetic effects.
The researchers administered 1 ml of sodium carboxymethyl cellulose (Na-CMC, placebo) to the KK and KN groups while they gave glimepiride (0.18 mg/kg BW) to the KP group. They also administered 1 ml of Na-CMC to the P1 group. Treatment group 1 (P1) received fig leaf extract at a dose of 300 mg/kg BW, treatment group 2 (P2) received 500 mg/kg BW, and treatment group 3 (P3) received 700 mg/kg BW. The researchers administered the treatments to all groups via a nasogastric tube for 14 days, as illustrated in Figure 1, which presents a schematic representation of the research methodology.
This study has been approved by the Hasanuddin University Research Ethics Committee (registration number 709/UN4.6.4.5.31/PP36/2024, September 2024) in accordance with international guidelines for the care and use of animals in biomedical research.
Preparation of Ficus carica leaf extract
We gathered fig leaves from the Tin Al Figs garden situated in Lamongan Regency, East Java Province, Indonesia. We sanitized the fig leaves and air-dried them, subsequently placing them in an oven at roughly 40–50°C to maintain the active compound content. We then pulverized them into a fine powder prior to extraction. We conducted the extraction using maceration, immersing finely powdered fig leaves in a 70% ethanol solution at a predetermined ratio for three days. After soaking the mixture in a 70% ethanol solution, we treated it and filtered it to separate the extract solution from the leaf pulp. During the final concentration phase, we concentrated the extract by evaporating the solvent using a rotary evaporator to maintain the stability of the active components.
Evaluation of blood glucose levels
The researchers evaluated FBG levels using a GlucoDr Biosensor AGM 2100 glucometer manufactured by PT Tekno Medicalogy, Korea. They collected blood samples at three time points: before STZ-NA induction, during STZ-NA induction, and after 14 days of treatment via a nasogastric tube. They subjected all rats to overnight fasting before each measurement. To obtain the blood samples, they drew blood from the lateral vein at the distal end of the rat tail. They identified hyperglycemia in rats by FBG levels of 126 mg/dl or higher.
Evaluation of GLUT-2 levels in pancreatic β cells and hepatic cells
We prepared the samples by dissecting Wistar rats of the same strain, following animal ethics protocols. After dissection, we obtained pancreas and liver specimens and preserved them at a designated temperature to maintain protein stability. We homogenized and lysed the tissues by placing them in microcentrifuge tubes, adding lysis buffer, and sonicating the mixture until complete dissolution occurred. After dissolution, we incubated the samples and then centrifuged them to separate the supernatant (containing protein) from the pellet (tissue debris). We measured protein concentrations in pancreatic β cells and hepatic cells using an Enzyme-Linked Immunosorbent Assay (ELISA) kit (MyBioSource) according to the manufacturer’s instructions.
Data analysis
Data are presented as mean ± standard deviation (SD) and analyzed using SPSS 26 software (IBM Corporation, NY, USA). To compare rat body weight and FBG levels among groups, we subsequently analyzed the data’s significance using a dependent t-test for normally distributed data (parametric) or a Wilcoxon test for non-normally distributed data (non-parametric). We performed a one-way ANOVA for regularly distributed data (parametric) or a Kruskal-Wallis test for non-normally distributed data (non-parametric) to compare FBG levels and GLUT-2 levels across all groups. A p-value below 0.05 was considered statistically significant. Finally, we conducted a post hoc analysis of the prior test, which produced significant results.
Results
Table I illustrates the weight of rats before and during induction, with all groups except for group P2 exhibiting weight gain. However, there was no significant difference between the groups. Furthermore, Table I demonstrates that the starting weight characteristics of the rats exhibited no significant differences across groups (P = 0.656, p > 0.05), nor did the weight respond to high-fat feed induction (P = 0.382, p > 0.05). The weight variation of rats before and after induction among groups did not demonstrate a statistically significant difference (P = 0.395, p > 0.05). We evaluated the data utilizing the dependent t-test.
Table I
Characteristic of experimental animals
Initially, a significant disparity in FBG levels was observed between the groups (P = 0.005**, p < 0.05); however, after induction with STZ-NA, no statistically significant difference was seen (P = 0.479, p > 0.05).
Table II shows that the FBG levels in the KN, KP, P1, P2, and P3 groups exceeded 126 mg/dl following STZ-NA induction, indicating that the rats developed type 2 diabetes mellitus. The KN, KP, and P1 groups already had baseline FBG levels above 126 mg/dl, because the researchers had previously subjected these rats to a high-fat diet before administering STZ-NA. This syndrome underpins the pathophysiology of diabetes mellitus, wherein obesity facilitates the onset of type 2 diabetes. The Wilcoxon test indicated that FBG levels in each group did not exhibit significant differences before and after STZ-NA induction (p > 0.05). Following therapy administration, the FBG levels in the P1, P2, and P3 groups diminished; however, the reductions lacked statistical significance. Likewise, the KK, KN, and KP groups exhibited no significant differences (p > 0.05). The Kruskal-Wallis test indicated that FBG levels before and after STZ-NA induction, as well as after treatment, did not exhibit significant differences among the groups (p > 0.05).
Table II
Fasting blood glucose levels of rats
Table III presents the findings on GLUT-2 levels in pancreatic β cells and hepatic cells. Group P3 (fig leaf extract dose, 700 mg/kg) showed the highest GLUT-2 levels in pancreatic β cells. In contrast, group P1 (fig leaf extract dose, 300 mg/kg) showed increased GLUT-2 levels in liver cells. The Kruskal-Wallis test and subsequent post hoc analysis revealed no statistically significant differences in GLUT-2 levels among the treatment groups in either pancreatic β cells or liver cells.
Table III
GLUT-2 concentrations in pancreatic β cells and hepatic cells
Discussion
This study investigated the effect of fig leaf extract (F. carica L.) on modulating GLUT-2 levels in pancreatic β cells and hepatic cells of Wistar rats induced by streptozotocin and nicotinamide. GLUT-2 is a glucose transporter crucial for regulating blood glucose levels, particularly in pancreatic β cells, which are responsible for insulin secretion. Prior studies have shown that administering herbal extracts to rats can increase the number of pancreatic β cells and lower blood glucose levels in a diabetes model [10]. Despite our anticipation that fig leaf extract would elevate GLUT-2 levels, the findings indicated no significant difference between the treatment group and the control group (Table III).
Prior research has demonstrated that fig leaf extract may have a positive influence on glucose metabolism. Nonetheless, the results fluctuate based on the dosage and extraction technique employed. This study suggests that the dosage administered by the researchers may have been inadequate to substantially influence GLUT-2 levels, highlighting the necessity for additional research to ascertain the ideal dosage and a more efficacious extraction procedure. Ficus carica L. includes bioactive components, including flavonoids and polyphenols, which possess antioxidant and anti-inflammatory characteristics. These compounds may enhance cellular activity and reduce oxidative stress in pancreatic and hepatic β cells [11].
Prior research has shown that extracts from F. carica L. can significantly reduce blood glucose levels in diabetic animals, indicating an improvement in insulin sensitivity and glucose absorption. The fundamental mechanisms may entail the modulation of glucose transporters, especially GLUT-2, which is crucial for glucose sensing and transport in pancreatic β-cells and the liver. Researchers have demonstrated that polysaccharides from F. carica enhance the maturation and functionality of dendritic cells, potentially impacting metabolic pathways and immunological responses associated with diabetes. Furthermore, they have discovered that the bioactive chemicals present in F. carica L. inhibit enzymes implicated in carbohydrate metabolism, including α-glucosidases and α-amylases, therefore validating their function in controlling postprandial glucose levels.
Nonetheless, this investigation may involve additional factors that impede the efficacy of the chemical. Multiple variables have impacted the efficacy of fig leaf extracts, necessitating additional investigation. Table I displays characteristic data indicating that the groups of rats were homogeneous, with no significant differences observed before and after induction with STZ-NA, except for initial FBG levels (P = 0.005; p < 0.05). The data reveal that the normal control group (KK) exhibited significantly lower first FBG levels, at 74 mg/dl, in contrast to the KN, KP, P1, and P2 groups, which had initial FBG values above 126 mg/dl. The P3 group constituted an anomaly. The disparity arose because the KN, KP, P1, P2, and P3 groups had previously been administered a high-fat diet before STZ-NA induction. This food experiment illustrates the biology of T2DM, wherein obesity significantly contributes to disease onset. The findings corroborate previous research [12,13], indicating that a high-fat diet reduces expression of GLUT-2 – a vital glucose transporter in the liver and pancreatic β cells – resulting in reduced glucose-stimulated insulin production and β cell impairment [12, 13].
A decrease in GLUT-2 expression reduces glucose uptake and disrupts glucose metabolism, both of which play key roles in the development of insulin resistance and T2DM [13]. High-fat diets impair β-cell function by reducing the expression of GLUT-2 and glucokinase, two proteins essential for glucose sensing and insulin secretion. This dysfunction leads to hyperglycemia, a defining feature of T2DM. A high-fat diet also increases fat accumulation in the liver and downregulates glucose metabolic pathways, including GLUT-2, thereby promoting insulin resistance. Additionally, it elevates fatty acid metabolism, which further worsens glucose intolerance [13].
High-fat diets induce oxidative stress and apoptosis in β-cells, leading to reduced mass and function, which exacerbates deficiencies in glucose sensing and insulin secretion. High-fat diets promote systemic insulin resistance, affecting the liver and several tissues, including the brain, where they impair synapse formation and glucose transporter function. The Kruskal-Wallis test revealed that FBG levels before STZ-NA induction, after STZ-NA induction, and after treatment with several dosages of fig leaf extract were not statistically significant (p > 0.05). The Kruskal-Wallis test similarly indicated no statistically significant differences in average GLUT-2 levels between pancreatic β cells and liver cells across treatment groups. The data suggest that the STZ-NA dosage employed in this study may not have been sufficient to impair the GLUT-2 pathway, which plays a crucial role in insulin secretion. Researchers commonly use STZ-NA model to study T2DM in animals, as it effectively mimics the pathogenesis of T2DM. This model replicates key pathological features of human T2DM and enables researchers to evaluate various antidiabetic and renoprotective drugs. Compared to the low-dose STZ model, the STZ-NA model offers greater stability and reproducibility in reproducing the morphological and functional kidney changes associated with T2DM. However, it may fall short of reflecting the significant insulin resistance observed in the high-fat diet/STZ model [14].
We recognize that many factors may have contributed to the absence of statistically significant differences between the treatment groups. The dosage of fig leaf extract (F. carica L.) may have been insufficient to elicit a significant effect on GLUT-2 expression, as previous research has demonstrated efficacy with greater doses or prolonged treatment durations [9, 10]. The high-fat meal given prior to STZ-NA induction may have reduced GLUT-2 expression and exacerbated insulin resistance, thus attenuating the extract’s benefits [13].
A weakness of this study is that the dosage of fig leaf extract (F. carica L.) administered may have been inadequate to elicit a statistically significant enhancement in GLUT-2 expression in pancreatic β cells and hepatic cells. This concern aligns with data [15, 16] indicating a dose-dependent influence on glucose metabolism control.
Furthermore, while researchers frequently employ STZ-NA induction model, it may inadequately represent the complex pathophysiology of type 2 diabetes mellitus in humans, particularly in terms of insulin resistance mechanisms [17].
The brief treatment duration (14 days) may have limited the observation of long-term physiological alterations, as sustained effects on GLUT-2 expression and pancreatic β-cell functionality may require continuous exposure [12, 13].
The small sample size in our study (n = 6 per group) may result in low statistical power, limiting our ability to detect differences. Moreover, neglecting to assess downstream insulin signaling pathways limits the understanding of functional outcomes beyond GLUT-2 expression. This research highlights the importance of future studies incorporating molecular markers of insulin sensitivity and glucose uptake, thereby providing a more comprehensive understanding of the underlying mechanisms.
The heterogeneity in metabolic and immunological responses among rats may impact the potency of the extract. The high-fat meal administered to the rat cohort before induction may influence the study’s results by altering GLUT-2 expression and glucose metabolism; therefore, the researcher must consider external variables, such as nutrition, in the study design. The method for assessing GLUT-2 levels may have limitations in sensitivity and specificity, thereby undermining the validity of the data. The researcher requires additional precise and repeated measurements to obtain more consistent data.
This study does not include long-term evaluations to assess the sustained effects of fig leaf extract on GLUT-2 levels and pancreatic β-cell function, which are essential for understanding its long-term therapeutic potential. It also does not investigate potential interactions between fig leaf extract and other antidiabetic drugs, which may influence its efficacy in combination therapy.
Conclusions
The findings of this study demonstrate that fig leaf extract (F. carica L.) significantly reduces FBG levels. The data on glucose levels indicate a non-significant reduction in glucose levels. Conversely, elevated GLUT-2 levels were observed in the pancreatic β cells of Wistar rats subjected to STZ-NA induction; however, there was no statistically significant difference as compared to the control group. The inadequate extract dosage employed in this study may explain the absence of the expected impact.
