Modification of a GLITEROS Diabetes-Specific Enteral Formula Incorporating Tempeh, Jicama, and Sunflower Seed Flours Improves Insulin Levels and Reduces Fasting Blood Glucose in Rats with Streptozotocin and Nicotinamide-Induced Hyperglycemia

Introduction

Diabetes mellitus represents a long-standing metabolic condition manifested by a persistent increase in blood glucose concentration resulting from defective insulin secretion and/or resistance.¹ Suboptimal glycemic management may lead to progressive pancreatic β-cell dysfunction and increase the potential for microcirculatory and macrovascular complications.²  Patients with diabetes who experience decreased consciousness, dysphagia, or other conditions limiting oral food intake often require diabetes-specific enteral nutrition to maintain adequate nutritional status while improving glycemic control.^2,3

GLITEROS (glycemia, tempeh and Pachyrhizus erosus (L.) Urb.) modification is one of the innovative diabetes-specific enteral formulas developed for diabetic patients with decreased consciousness or swallowing disorders.^4–6 GLITEROS comes from glycemia, tempeh, and Pachyrhizus erosus (L.) Urb.⁴ The modified GLITEROS formula was prepared using tempeh, jicama, and sunflower flours. Tempeh is a traditional Indonesian fermented soybean food produced through controlled fermentation of cooked soybeans using Rhizopus species, resulting in a nutrient-dense product containing high-quality plant protein, bioactive peptides, essential amino acids, and beneficial fermentation-derived compounds.⁷  Although originally developed as a staple food in Indonesia, tempeh has gained international recognition as a functional food due to its glucose-lowering effects by protecting and improving pancreatic β-cell function.^8,9  Tempe flour is rich in the amino acid leucine, which increases pancreatic β-cell insulin secretion by promoting gene transcription and biosynthesis of proteins in pancreatic β cells.¹⁰

Jicama, also known as yam bean or Mexican turnip, is an edible root vegetable widely consumed in tropical regions, including Indonesia, Mexico, and other parts of Southeast Asia.¹¹  Jicama flour contains several bioactive compounds, including inulin, resistant starch, dietary fiber, and phenolic bioactive components, previously reported to exhibit exert antidiabetic effects by improving glycemic control through delayed glucose absorption, enhanced insulin sensitivity, modulation of gut microbiota, and antioxidative properties, which has been proven through both experimental and biological models.^12-15  Sunflower seed flour, known for its high oleic acid content (a monounsaturated fatty acid), is considered appropriate in patients with elevated blood glucose levels because it may decrease oxidative stress, which increases under hyperglycemic conditions.⁴  In addition, sunflower seeds are rich in vitamin E, which exhibits natural antioxidant properties and is thought to help protect the pancreas.¹⁶

The combination of these three ingredients is expected to provide complementary mechanisms that improve glycemic control by enhancing insulin release, increasing insulin responsiveness, protecting pancreatic β-cells, and reducing oxidative stress. However, although the antidiabetic properties of each individual ingredient have been widely reported, evidence regarding the combined effects of the GLITEROS enteral formula on insulin and fasting blood glucose levels remains limited.

Animal-based research models are crucial for investigating the metabolic mechanisms underlying diabetes and evaluating potential nutritional interventions. Wistar rats are widely utilized in diabetes research because they have been extensively studied for their physiological and metabolic responses, stable growth characteristics, and reproducible responses to diabetogenic agents.⁶ In particular, the streptozotocin–nicotinamide (STZ+NA) induction model in Wistar rats is widely used to establish a type 2 diabetes mellitus-like condition by inducing incomplete pancreatic β-cell dysfunction while sustaining remaining β-cell secretory function. Streptozotocin causes pancreatic β-cell impairment through oxidative stress and DNA integrity disruption; on the other hand, nicotinamide helps preserve β-cells from excessive destruction, resulting in moderate hyperglycemia that resembles several profiles of clinical type 2 diabetes mellitus in humans.⁷

Therefore, this research was designed to examine the role of a GLITEROS diabetes-specific enteral formula modification containing tempeh, jicama, and sunflower seed flours on insulin and blood glucose concentrations during fasting in streptozotocin–nicotinamide (STZ+NA)-induced hyperglycemic rats.

Materials and Methods

Research site

The research was performed at the Center for Food and Nutrition Studies, Universitas Gadjah Mada, Yogyakarta, Special Region of Yogyakarta, Indonesia, for a duration of a month.

Enteral formula composition

The enteral formula was prepared using tempeh flour (46 g) and jicama flour (23 g) sourced from Hasil Bumiku, an Indonesian commercial manufacturer of plant-based food ingredients; raw peeled sunflower seeds (23 g) obtained from Granology, an Indonesian commercial food manufacturer; skimmed milk powder (60 g) supplied by Indoprima, a commercial dairy ingredient manufacturer; maltodextrin (50 g); granulated sugar (12 g) from Gulaku, a commercial sugar brand; and soybean oil (7 g from Happy, a commercial cooking oil brand).⁶

Preparation of formula

Sunflower seed flour was prepared from undried, peeled sunflower seeds (Granology). The seeds were dried maintained at 100 °C for 30 minutes in an oven, ground using a grinder, then sieved through a sieve with a 100-mesh size to obtain a uniform flour. Tempeh and jicama flours were purchased as commercially prepared flours from Hasil Bumiku (Indonesia) and were passed through a sieve with a 100-mesh size before use. Granulated sugar was milled using a food processor to obtain powdered sugar and then passed through a 100-mesh sieve. The enteral formula was obtained through precise measurement of the ingredients based on their respective ratios (Table 1). Dry components consisting of three types of flour (jicama, tempeh, and sunflower seed flours), maltodextrin, skim milk powder, and ground sugar were homogenized for 3 minutes by hand. Subsequently, soybean oil was incorporated into the dry blend and mixed for 2 minutes. The mixture was subsequently blended using a mixer for 8 minutes to produce a uniform powdered enteral formula.

Prior to administration, the powdered formula was reconstituted with water at a concentration of 22.1 g/100 mL (221 g/L). Specifically, 221 g of powdered formula was dissolved in water and diluted to the desired final volume of 1,000 mL. The reconstituted formula was freshly prepared before administration and delivered orally by gastric gavage. ⁶

Table 1: Ingredient composition and nutritional characteristics of modified GLITEROS  diabetes-specific enteral formula per 1000 mL⁶

*(for 1000cc formula)

Study design

This study utilized a randomized controlled experimental design with a pre–posttest approach to evaluate the impacts of the modified GLITEROS enteral formula on insulin and FBG concentration in STZ+NA-induced diabetic rats. 

Intervention groups

The experimental treatment was provided to Wistar rats (male, 8 weeks of age, 150–200 g). All animal handling and experimental procedures were conducted following approval from the Health Research Ethics Committee, Faculty of Medicine, Universitas Diponegoro, Indonesia (Ethical Clearance No. 10/EC/H/FK-UNDIP/I/2022). All procedures involving experimental animals were carried out in compliance with the approved ethical guidelines. A hyperglycemic rat model was developed through the intraperitoneal route of streptozotocin (STZ) at a dose of 45 mg per kg of body weight and nicotinamide (NA) at a dose of 110 mg/kg body weight. Thirty rats were allocated to five experimental groups through random assignment as follows:

The commercial formula used in the positive control group was Diabetasol® (Nutricia Indonesia), a diabetes-specific enteral formula. The nutritional composition of the formula, according to the manufacturer’s label, included carbohydrates (37 g), fat (7 g), protein (11 g), dietary fiber (4 g), vitamins, and minerals formulated for glycemic control. The enteral formula dose was calculated using the effective doses of tempeh, jicama, and sunflower seed flours reported in prior studies. Because the initial dose contained less tempeh flour than the amount previously shown to improve pancreatic histopathology in hyperglycemic rats, a second dose equivalent to 2.2 times the initial dose was included to achieve the effective tempeh flour level. ⁷

Insulin Levels Measurement

The ELISA method used blood plasma samples to measure insulin levels before and after the intervention ended. Blood samples for measuring insulin levels were taken from the eye veins of rats using a Pasteur pipette after 1.5 hours of feeding the rats. The collected blood specimens were subjected to centrifugation at 3000×g for 10 minutes under refrigerated conditions (4 °C), and plasma samples containing insulin (the antigen) were collected. Then, the plasma samples were reacted with microplate wells pre-coated using antibodies against mouse insulin and the reagents provided in the mouse insulin ELISA kit. After processing these reactions, the spectrophotometric optical density was determined at 450 nm using a microplate reader.¹⁷

Measurement of Blood Glucose Levels

Blood glucose concentrations were quantified at three different time points during the study. The first measurement was performed before diabetes induction. The second measurement was conducted three days after induction with streptozotocin (STZ) and nicotinamide (NA), serving as a pre-test. The third measurement was performed on day 28 after administration of the enteral formula as a post-test measurement. Before fasting blood glucose measurement, rats were maintained under fasting conditions for 7–12 hours. Blood specimens were obtained from the retro-orbital vascular plexus, and a 10-µL aliquot from each blood specimen was used for blood glucose analysis.

The FBG concentrations of rats were quantified using the glucose oxidase method (GOD-PAP). Experimental animals were first given general anesthesia before taking blood samples, followed by incubation for 30 minutes at ambient temperature to collect serum. A 10 µL blood serum specimen was mixed with 1,000 µL of kit reagent. The specimen was then transferred into a test tube and homogenized using a vortex mixer. The mixture underwent incubation under two temperature conditions: 37 °C for 10 minutes or 30 °C for 20 minutes. Then, spectrophotometric analysis was performed by measuring absorbance at 500 nm. The principle underlying this method is based on glucose oxidation by oxygen with the enzyme glucose oxidase (GOD) as an enzymatic catalyst. Blood glucose levels (mg/dL) were determined during the adaptation period, before and after STZ+NA injection, calculated by the following formula: Blood Glucose Level = sample absorbance/standard absorbance ×100.¹⁸

Statistical Analysis

All data analyses were conducted by IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA). Data are reported as mean ± standard deviation (SD). The assumption of normality was assessed using the Shapiro–Wilk test before parametric analysis. Comparisons of fasting blood glucose and insulin levels before and after streptozotocin–nicotinamide (STZ–NA) induction within the same group were conducted using a paired t-test. The variations between groups were analyzed through an independent t-test based on delta changes (Δ), calculated as the difference between post-induction and pre-induction values.

For the intervention analysis, paired t-tests were applied to evaluate variations in fasting blood glucose and insulin levels before and after enteral formula administration within each group. Differences in delta values among multiple treatment groups were compared using one-way analysis of variance (ANOVA) and pairwise comparisons using Bonferroni post hoc analysis for multiple comparisons. Statistical significance was defined as a p-value < 0.05.

Results

Changes in Rat Insulin Levels Before and Following Streptozotocin and Nicotinamide Induction

The insulin levels of rats were measured before STZ+NA induction and 3 days post-STZ+NA  induction. The differences in mean insulin levels and statistical analysis between the non-induced and induced groups are reported in Table 2.

Table 2: Differences in mean insulin levels of rats between the non-STZ+NA group and the STZ+NA group

a = p-value prior to and following STZ/NA induction among groups (Paired T-Test)

b = p-value prior to and following STZ/NA induction between groups by Independent T-Test

* p-value < 0.05 = significant difference

Table 2 demonstrates a significant variation among the induction and non-induction groups pre- and post- STZ+NA induction (p<0.000). The induction group experienced a significant decline in insulin levels following STZ+NA induction, whereas the non-induction group tended to exhibit an elevated in insulin levels.

Fasting Insulin Levels of Rats Before and After Enteral Formula Intervention

Insulin levels were measured before the intervention on the third day after STZ+NA induction, while insulin concentration after the intervention were determined on the 28th day after administration of the modified GLITEROS formula and the commercial enteral formula. Differences in mean insulin levels and statistical analysis between the five groups of treated rats are presented in Table 3.

Table 3: Changes in Rat Insulin Levels Before and After Enteral Formula Intervention

Description: Values with diverse superscript annotations (a, b, c, d, and e) in the same column reveal statistically significant differences (post hoc Bonferroni test)

* p-value < 0.05 = significant difference with paired-t-test

** p-value < 0.05 = significant differences among groups were classified using one-way ANOVA

Table 3 shows changes in insulin levels before and after intervention in all experimental groups. Insulin levels decreased in the C and C(-) groups after the intervention. However, the decrease was not statistically significant in the C group (p = 0.076), whereas a significant reduction was identified in the C(-) group (p = 0.001). In contrast, insulin levels significantly increased in the C(+), T1, and T2 groups after the intervention (all p = 0.000).

The one-way ANOVA analyzing changes in insulin levels among groups revealed a significant disparity (p < 0.001). The Bonferroni multiple comparison test indicated significant differences in insulin changes between several treatment groups, as indicated by different superscript letters in Table 3. The C(-) group showed a reduction in insulin levels after STZ+NA induction, confirming the impaired insulin regulation associated with the diabetic model. Meanwhile, administration of the modified GLITEROS formula and commercial formula improved insulin levels, as demonstrated by the increased post-intervention insulin concentrations in the treatment groups.

Fasting Blood Glucose Levels Changes in Rats Following STZ+NA Induction

Fasting blood glucose levels of rats were measured before the induction of STZ+NA, three days after acclimatization. Blood glucose testing was performed 3 days after STZ+NA induction. The difference in average FBG concentration between the non-induction and induction groups is evident in Table 4.

Table 4: Differences in mean fasting blood glucose between the non-STZ+NA group and the STZ+NA group

a = p-value prior to and following STZ+NA induction among groups (paired-t-test)

b = p-value prior to and following STZ+NA induction between groups by independent-t-test)

* p value < 0.05 = significant difference

Table 4 presents the FBG levels of non-induction and induction rats before STZ+NA induction, which had normal FBG values within 30-135 mg/dL. Statistical findings revealed differences in FBG between non-induced and induced rats prior to STZ+NA induction. Following STZ+NA induction, the diabetes-induced group demonstrated a significant increase of 187.44 mg/dL. Significant differences were identified between the non-induction and induction groups post-STZ+NA induction (p < 0.000).

Fasting Blood Glucose Levels Changes in Rats Following Enteral Formula Intervention

Mean differences in FBG levels and statistical analysis between the five treatment groups of rats are presented in Table 5.

Table 5: Fasting Blood Glucose Changes in Rats Before and After Enteral Formula Intervention

* p-value < 0.05 = significant difference with paired-t-test

**p-value <0.05 = significantly different from the one-way ANOVA test

Values assigned diverse superscript letters (a, b, c, and d) within the same column are significantly different from each other (Bonferroni post hoc test, p < 0.05), Values with the same superscript letters indicate no statistically significant differences.

After the intervention, the C and C(-) groups showed a slight increase in fasting blood glucose (FBG) levels. However, the FBG level in the C group remained within the normal range, whereas the C(-) group remained in the hyperglycemic range. In contrast, the C(+), T1, and T2 groups showed marked reductions in FBG levels following the intervention, with the greatest decrease observed in the T2 group. Statistical analysis using one-way ANOVA identified significant differences in the changes in FBG levels within the experimental groups (p < 0.001). Bonferroni post hoc test further revealed differed significantly between the intervention groups and the untreated hyperglycemic group.

Discussion

Rat Insulin Levels Before and After STZ+NA Induction

Table 2 demonstrated that STZ+NA induction significantly altered insulin levels in Wistar rats. Before induction, insulin levels were comparable between the non-STZ+NA induction group and the STZ+NA induction group, with values of 547.20 ± 3.8 ng/mL and 556.39 ± 1.8 ng/mL, respectively. Following STZ+NA administration, insulin levels significantly decreased in the STZ+NA induction group from 556.39 ± 1.8 ng/mL to 451.71 ± 2.6 ng/mL (p < 0.001), representing a reduction of 104.68 ng/mL. On the contrary, the non-induced group indicated only a slight reduction in insulin levels (−8.59 ng/mL). These findings indicated that STZ+NA induction successfully impaired pancreatic β-cell function and produced a diabetic metabolic condition in the experimental animals.

The reduction in insulin levels following STZ+NA induction suggested that pancreatic β-cells were damaged or functionally impaired, resulting in decreased insulin secretion.¹⁹ Streptozotocin is a glucosamine-nitrosourea compound that is selectively delivered into pancreatic β-cells mediated by glucose transporter 2 (GLUT2). Inside the β-cells, STZ induces DNA alkylation, oxidative stress, and mitochondrial dysfunction, which subsequently impair insulin synthesis and secretion.²⁰ However, nicotinamide administration partially protects β-cells from excessive STZ-mediated destruction by acting as a precursor of nicotinamide adenine dinucleotide (NAD+) and reducing oxidative damage. Therefore, the STZ+NA model produces moderate β-cell dysfunction that resembles type 2 diabetes mellitus rather than complete insulin deficiency. ²¹

The significant decline in insulin levels observed after STZ+NA induction confirmed the suitability of this experimental model for evaluating nutritional interventions targeting glucose metabolism.²² The remaining insulin production in STZ+NA-induced rats indicated that β-cell function was partially preserved, allowing assessment of interventions that may promote insulin sensitivity, optimize insulin secretion, or preserve pancreatic β-cells from further dysfunction.²³ 

Fasting Insulin Levels of Rats Before and After Enteral Formula Intervention

Following the successful development of the STZ+NA-induced diabetes model, the effect of the modified enteral formula intervention on insulin levels was evaluated (Table 3). The results showed varied responses across experimental groups after the intervention period. The diabetic control group (C−) showed a further decrease in insulin levels from 451.21 ± 5.1 ng/mL to 438.14 ± 4.3 ng/mL, with a reduction of 13.07 ng/mL (p = 0.001). This finding indicated that pancreatic β-cell dysfunction induced by STZ+NA continued to progress in the absence of nutritional intervention. In contrast, the intervention groups receiving the modified GLITEROS formula showed significant increases in insulin levels. The T1 group increased from 451.21 ± 10.1 ng/mL to 490.61 ± 8.0 ng/mL, corresponding to an increase of 39.40 ng/mL (p < 0.001), while the T2 group demonstrated the greatest improvement, increasing from 450.32 ± 6.7 ng/mL to 520.52 ± 6.7 ng/mL, with an increase of 70.20 ng/mL (p < 0.001). These results suggested that administration of the modified GLITEROS formula improved insulin release capacity in STZ+NA-induced diabetic rats, with a greater effect observed at the higher intervention dose.

The increase in insulin levels observed in the T1 and T2 groups indicated that the modified GLITEROS formula may have contributed to the enhancement of pancreatic β-cell function and glucose metabolism. The dose-dependent response observed in the T2 group suggests that a higher intake of bioactive nutritional components may provide stronger metabolic benefits. Meanwhile, the absence of spontaneous recovery in the C− group confirmed that the improvement in insulin levels was associated with the nutritional intervention rather than natural restoration after STZ+NA induction.

The beneficial effects of the modified GLITEROS formula may be related to the synergistic action of its functional ingredients, including tempeh, jicama, and sunflower seed flours.⁷  Tempeh flour is rich in insulinotropic amino acids, particularly leucine and arginine, which can stimulate insulin secretion through nutrient-sensitive pathways in pancreatic β-cells.²⁴  Leucine activates mammalian target of rapamycin (mTOR)-related signaling, while arginine promotes insulin release through membrane depolarization and calcium-dependent exocytosis. These mechanisms may contribute to the restoration of insulin secretion capacity observed in the intervention groups.¹⁰

In addition, tempeh-derived isoflavones, including genistein and daidzein, may provide protective properties in preventing oxidative stress-induced β-cell damage.²⁵ Oxidative stress contributes a crucial role in diabetes progression by promoting β-cell apoptosis and reducing insulin secretory function. Therefore, the antioxidant activity of isoflavones may help preserve residual β-cell function following STZ+NA induction.²⁶ Also, arginine promotes insulin release by enhancing nitric oxide-related glucose metabolism, whereas leucine stimulates insulin secretion through nutrient-sensitive pathways involved in β-cell function.²⁵  

The jicama flour component may further support insulin regulation through its high inulin and dietary fiber content.^27-29 Inulin may also contribute to improved insulin regulation by enhancing glucagon-like peptide-1 (GLP-1) secretion, which promotes glucose-dependent insulin secretion and supports pancreatic β-cell function.^27,28 Furthermore, sunflower seed flour may further support insulin regulation through its bioactive compounds, including oleic acid, vitamin E, and phenolic compounds.^30,31 These components have antioxidant potential that may help preserve pancreatic β-cells from oxidative damage and preserve insulin secretory function under diabetic conditions.³²

The reference control group(C+) also showed a significant elevation in insulin levels, from 454.08 ± 5.2 ng/mL to 511.74 ± 4.6 ng/mL (increase of 57.66 ng/mL; p < 0.001). This improvement may be explained by the presence of functional nutrients in commercial diabetes-specific enteral formulas, particularly whey protein and unsaturated fatty acids.^33,34 Whey protein is rich in leucine and branched-chain amino acids, which stimulate insulin and incretin secretion and improve postprandial glucose regulation.³³ Therefore, the response observed in the C+ group supports the validity of the nutritional intervention model used in this study.

Overall, the findings in Table 3 demonstrated that the modified GLITEROS formula significantly improved insulin levels in STZ+NA-induced diabetic rats. The greater elevation observed in the T2 group suggests a potential dose-dependent effect, indicating that the formulation may have potential as a diabetes-specific enteral nutrition intervention through mechanisms involving enhanced insulin secretion, improved tissue responsiveness to insulin, and protection of pancreatic β-cell function. 

Fasting Blood Glucose Levels of Rats Before and After STZ+NA Induction

Table 4 demonstrated that STZ+NA induction significantly increased FBG levels in Wistar rats. Prior to induction, FBG levels were comparable between the non-STZ+NA induction group and the STZ+NA induction group (70.98 ± 1.47 mg/dL and 73.00 ± 0.67 mg/dL, respectively). Following induction, the STZ+NA group showed a marked elevation in FBG levels from 73.00 ± 0.67 mg/dL to 260.45 ± 1.23 mg/dL (p < 0.001), on the other hand the non-induced group exhibited no substantial alteration in glucose concentration. These results confirmed the development of a hyperglycemic condition following STZ+NA administration.

The increased FBG levels observed in the STZ+NA group were associated with impaired insulin regulation, as demonstrated by the reduction in insulin concentration described previously (Table 2). The reduction in insulin availability resulted in reduced glucose utilization uptake by peripheral target tissues and subsequently increased circulating glucose levels.³⁵ The underlying mechanism of STZ+NA-induced pancreatic β-cell dysfunction has been described in the previous section, in which STZ-mediated oxidative stress and cellular damage impaired insulin secretion, while nicotinamide partially preserved residual β-cell function. ²²

The simultaneous reduction in insulin levels and elevation of FBG concentrations indicated that the STZ+NA protocol successfully generated a moderate diabetic metabolic condition. This metabolic profile resembles type 2 diabetes mellitus, manifested by impaired insulin secretion accompanied by insufficient glucose regulation rather than complete insulin deficiency.¹⁹ Therefore, the STZ+NA-induced model was considered appropriate for evaluating the effectiveness of nutritional interventions targeting glucose homeostasis.²² 

Rats’ Fasting Blood Glucose Levels Before and After Enteral Formula Intervention

Following the improvement in insulin levels observed after intervention (Table 3), fasting blood glucose (FBG) levels showed significant reductions in the treated groups (Table 5).  The results demonstrated significant differences in FBG changes among the experimental groups after intervention. The diabetic control group (C−) showed a slight increase in FBG levels from 259.80 ± 2.85 mg/dL to 261.30 ± 3.38 mg/dL, with an increase of 1.50 mg/dL (p = 0.002). This finding indicated that hyperglycemia induced by STZ+NA was not spontaneously improved without nutritional intervention.

In contrast, all intervention groups receiving nutritional formulas demonstrated significant reductions in FBG levels. The positive control group (C+) showed a reduction from 258.72 ± 2.18 mg/dL to 99.41 ± 1.62 mg/dL, with a decrease of 159.31 mg/dL (p < 0.001). Similarly, administration of the modified GLITEROS formula significantly reduced FBG levels in both treatment groups. The T1 group decreased from 261.55 ± 1.51 mg/dL to 128.30 ± 2.69 mg/dL, corresponding to a reduction of 133.25 mg/dL (p < 0.001), whereas the T2 group demonstrated the greatest reduction, from 261.70 ± 2.55 mg/dL to 92.41 ± 1.99 mg/dL, with a decrease of 169.29 mg/dL (p < 0.001). These results indicated that the modified GLITEROS formula significantly improved glycemic control in STZ+NA-induced diabetic rats, with the higher-dose intervention (T2) showing a stronger glucose-lowering effect.

The decline in FBG levels documented in the T1 and T2 groups was consistent with the improvement in insulin levels reported in Table 3. Increased insulin availability may enhance glucose uptake by insulin-target tissues, especially adipose tissue and skeletal muscle, through activation of insulin signaling pathways, thereby promoting glucose clearance from circulation.³⁵ Furthermore, the greater reduction observed in the T2 group suggested a potential dose-dependent effect of the modified GLITEROS formula in improving glucose metabolism.

The antihyperglycemic effect of the modified GLITEROS formula may be related to the synergistic effects of its functional ingredients in improving glucose regulation. The inulin and dietary fiber content of jicama flour may contribute to reduced postprandial glucose elevation by delaying gastric emptying and limiting glucose uptake in the intestine.¹⁵ In addition, inulin has been reported to restore insulin responsiveness and enhance incretin responses, including glucagon-like peptide-1 (GLP-1), which has an essential role in glucose-dependent insulin secretion and glucose homeostasis.²⁷

Furthermore, sunflower seed flour may contribute to improved glycemic control through its bioactive components, particularly oleic acid, vitamin E, and phenolic compounds. Sunflower seeds provide substantial amounts of oleic acid, a monounsaturated fatty acid (MUFA) that has been reported to improve glucose metabolism and attenuate oxidative stress associated with hyperglycemia.^30,31 The insulin-sensitizing and antioxidant properties of oleic acid may enhance glucose utilization and improve metabolic regulation, thereby contributing to the attenuation of fasting blood glucose observed in the intervention groups.³³

In addition, sunflower seeds contain high levels of vitamin E, particularly α-tocopherol, which acts as a potent antioxidant. Other bioactive compounds, including quinic acid, chlorogenic acid, caffeic acid derivatives, glycosides, and phytosterols, have also been associated with glucose-lowering effects through modulation of oxidative stress and glucose metabolism.^22,24–27 These compounds may improve glycemic control by preserving insulin signaling pathways and reducing excessive hepatic glucose production. Therefore, together with the bioactive components from tempeh and jicama flour, the antioxidant and insulin-sensitizing properties of sunflower seed flour may have synergistically contributed to the improved glucose regulation observed in the GLITEROS-treated groups.

The improvement observed in the T2 group may be associated with the higher amount of bioactive components provided by the modified GLITEROS formula, resulting in stronger effects on insulin secretion, glucose utilization, and metabolic regulation. These findings suggest that the modified GLITEROS formula may have potential as a diabetes-specific enteral nutrition intervention for improving glycemic control in experimental diabetes. 

Conclusion

The modification of GLITEROS diabetes-specific enteral formula, formulated from tempeh, jicama, and sunflower seed flours, significantly enhanced insulin levels and suppressed FBG levels in STZ+NA-induced hyperglycemic rats. The higher-dose formulation (T2) demonstrated the greatest improvement in glycemic parameters, suggesting a dose-dependent effect. These findings indicate that GLITEROS modification shows promise as a functional, diabetes-specific enteral formula to improve glycemic control, possibly through the combined actions of its bioactive components, including isoflavones, inulin, dietary fiber, and monounsaturated fatty acids.

Although this study provides experimental evidence regarding the potential effects of the modified GLITEROS diabetes-specific enteral formula on glycemic regulation, several limitations need to be recognized. First, the present study utilized an STZ–NA-induced hyperglycemic rat model, which can mimic several metabolic characteristics of type 2 diabetes mellitus but cannot fully reproduce the complex pathophysiological mechanisms observed in humans, including variations in insulin resistance, dietary patterns, genetic factors, and disease progression. Therefore, the beneficial changes in fasting blood glucose and insulin levels should be interpreted cautiously when extrapolating these findings to clinical populations.

Second, the current study evaluated the short-term effects of GLITEROS modification and did not assess long-term safety, metabolic adaptation, or potential adverse effects associated with continuous administration. Parameters related to organ toxicity, inflammatory responses, lipid metabolism, and gut microbiota alterations were not evaluated, although these factors may contribute to the long-term effectiveness and safety of diabetes-specific enteral nutrition. Third, this study focused primarily on fasting blood glucose and insulin levels as indicators of glycemic improvement; therefore, additional metabolic parameters, such as insulin sensitivity indices, oxidative stress markers, inflammatory mediators, and pancreatic β-cell histopathological changes, are needed to provide deeper insights into the underlying mechanisms.

Furthermore, although the formulation utilized locally sourced functional food ingredients, including tempeh, jicama, and sunflower seed flours, variations in nutrient composition and bioactive compound concentrations may occur depending on raw material sources and processing methods. Future studies should therefore include standardized characterization of the bioactive components and conduct well-designed clinical trials to confirm the efficacy, safety, and applicability of the GLITEROS modification in patients with diabetes requiring enteral nutrition.

Acknowledgement  

The authors are grateful to the Department of Nutrition Science, Faculty of Medicine, Universitas Diponegoro, Indonesia, for providing research data support. The authors also thank the Center for Food and Nutrition Studies, Universitas Gadjah Mada, Yogyakarta, Special Region of Yogyakarta, Indonesia, as the research site for data collection. 

Funding Sources   

This research was fully funded by the Non-Tax Revenue of the Faculty of Medicine, Universitas Diponegoro in 2021. The publication of this research was supported by the Ministry of Higher Education, Science, and Technology of the Republic of Indonesia through the Decree of the Director General of Research and Development Number 279/C/C2/KPT/2025. 

Conflict of Interest   

The author(s) do not have any conflict of interest. 

Data Availability Statement  

The manuscript incorporates all datasets produced or examined throughout this research study.  

Ethics Statement 

This research has been approved by the Health Research Ethics Committee, Faculty of Medicine, Diponegoro University (No.10/EC/H/FK-UNDIP/1/2022). 

Informed Consent Statement 

This study did not involve human participants, and therefore, informed consent was not required. 

Clinical Trial Registration 

This research does not involve any clinical trials 

Permission to reproduce material from other sources 

Not Applicable

Author Contributions 

• Lilis Wijayanti: conducted the experiments, collected the data, and drafted the manuscript
• Muflihatul Muniroh: supervision and reviewed the manuscript
• Ahmad Ni’matullah Al-Baarri: reviewed and critically revised the manuscript
• Deny Yudi Fitranti: methodology development and validation
• Endang Mahati: performed data analysis and interpretation
• Irwandi Jaswir: reviewed and critically revised the manuscript
• Diana Nur Afifah: conceptualized and designed the study 

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