Peptide Hydrolysate of Telfairia occidentalis Hook f. Seed Protein Promotes Effective Glucose Homeostasis by Improving β -cell Dysfunction and Abating Carbohydrate Metabolic Disturbance in Diabetic Rats

Rising concerns with the use of synthetic antidiabetic drugs have promoted a shift towards the use of natural products. Tis study therefore investigated the antidiabetic activity of peptide hydrolysate of Telfairia occidentalis ( T . occidentalis ) seed protein (PHTOSP) in streptozotocin-induced diabetic rats. Tirty-six (36) experimental animals were randomly distributed into six groups (A–F) of six rats each ( n =6). Group A served as normal control while groups B, C, D, E, and F were treated with streptozotocin (STZ, 50mg/kg body weight, b.w, i.p) dissolved in cold citrate bufer (0.1M, pH 4.5) to induce type 2 diabetes. Groups C, D, and E were administered 50, 100, and 150mg/kg b.w PHTOSP, respectively, while groups B and F were diabetic untreated and 5mg/kg b.w glibenclamide-treated controls, respectively, in the experiment that lasted for 21days. Subsequently, the analysis of biochemical parameters demonstrated a signifcant ( p < 0 . 05) increase in serum insulin, hepatic glycogen, hexokinase, and glucose-6-phosphate dehydrogenase activities, accompanied by a signifcant ( p < 0 . 05) reduction in fasting serum glucose, glucose-6-phosphatase, and fructose-1,6-bisphosphatase, along with an enhancement in relative body weight. Similarly, PHTOSP demonstrated a signifcant ( p < 0 . 05) improvement on high-density (HDL) and low-density (LDL) lipoproteins, triglyceride (TG), total cholesterol (TC), and atherogenic index (AI) signifcantly ( p < 0 . 05). In addition, histoarchitectural analysis revealed a reversal of congestion and proliferation of infammatory cells in the pancreatic tissue following treatment with PHTOSP. Terefore, PHTOSP might possess potential antidiabetic properties such that it improves glycolytic pathway and promotes cell survival that are helpful in the management of diabetes mellitus (DM).


Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by persistent high blood sugar levels and disruptions in the metabolism of carbohydrates, lipids, and proteins due to abnormalities in insulin production, insulin action, or both [1,2]. It occurs when the pancreas does not produce enough insulin or when the body is unable to efectively use the insulin it produces [3]. In the twenty-frst century, diabetes has emerged as one of the most challenging health conditions [4]. It afects approximately 4% of the global population and is projected to increase by 27% by 2025 [5]. In many high-income nations, it ranks as the fourth leading cause of mortality, while in economically developing and industrialized countries, it poses a signifcant burden [6]. It is estimated that by 2030, the number of people with diabetes will exceed 552 million, with a 176% increase in developed nations [7,8]. Tis rise can be attributed to factors such as rapid urbanization, westernization, and associated lifestyle changes [9].
DM is classifed into two types: insulin-dependent diabetes mellitus (type 1) and non-insulin-dependent diabetes mellitus (type 2) [2]. Type 1 diabetes is an autoimmune disease characterized by infammation and selective destruction of insulin-secreting cells in and around the islets of Langerhans [10]. Type 2 diabetes is characterized by peripheral insulin resistance and dysfunction. Both types of diabetes pose signifcant risks to afected individuals and society due to their potential life-threatening complications. Complications associated with DM, including disturbances in body protein balance, can lead to conditions such as retinopathy, nephropathy, neuropathy, and atherosclerotic vascular disease [11][12][13]. Possible risks associated with diabetes include heart attack, stroke, kidney failure, lower limb amputation, vision loss, and nerve damage [11].
Cardiovascular diseases are a prevalent complication that often arises in association with DM [14]. Tese diseases are responsible for approximately 65% of diabetes-related deaths attributed to heart disease and stroke [15]. Managing DM involves various approaches, including dietary modifcations, medication, physical activity, regular screening, and care for complications that may arise [16]. While several oral hypoglycemic drugs and insulin are available to control diabetes, there is currently no defnitive treatment to cure the condition [17]. Furthermore, many of these drugs are costly and can lead to signifcant side efects such as hypoglycemia, weight gain, headaches, and gastrointestinal discomfort. Although medicinal plants have been traditionally used to manage DM, only a limited number of them have undergone scientifc and medical evaluation to assess their efectiveness [18][19][20].
T. occidentalis is a tropical vine cultivated for its edible seeds and leafy vegetable in West Africa [21]. Common names include Ugu, futed gourd, and futed pumpkin. It is a creeping vegetative shrub, with big lobed leaves and long twisting tendrils spreading low across the ground [22]. Te plant belongs to the Cucurbitaceae family and has a simple dark green veined leaf that is up to 35 cm long and as wide as 18 cm. Nutrients such as vitamins, proteins, carbohydrates, fber, and minerals are reportedly found in T. occidentalis [23]. More so, studies have highlighted antidiabetic properties of the plant [24][25][26], as well as hepatoprotective efect against oxidative stress caused by garlic (Allium sativum) in rats [25]. More so, with an increase in the use of this plant as one of the herbal remedies in the folkloric management of DM, this study therefore aimed at investigating the antidiabetic potential of peptide hydrolysate of T. occidentalis seed protein in STZ-induced diabetic rats.

Chemicals Used.
Pepsin, trichloroacetic acid (TCA), NaOH, bovine serum albumin (BSA) (standard protein), streptozotocin, and glibenclamide were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. All other chemicals were of analytical grade and prepared in distilled water using all-glass apparatus.

Sample Preparation.
Te fat-free four sample of T. occidentalis was prepared according to the method of Siddeeg et al. [27]. Te seeds obtained from T. occidentalis were decorticated and air-dried until a consistent weight was obtained. Te seeds were blended to a powdery form and sieved to obtain fne four. Te resulting T. occidentalis four was subsequently defatted with n-hexane at room temperature (25°C). Seed four was dispersed in n-hexane (1 : 10 w/v) and continuously agitated for 7 h (this was repeated twice). Te residue of n-hexane was removed by placing the defatted T. occidentalis four in a fume hood overnight. Te fat-free four was then dried and stored at −20°C for further treatments.

Preparation of T. occidentalis Seed Protein Isolate.
Protein was extracted from the defatted T. occidentalis seed four using acid precipitation method described by Adebowale et al. [28] with some modifcations by Arise et al. [29] and a modifed isoelectric precipitation procedure [30]. Defatted four was dispersed in ultrapure water, Milli-Q water (MQW) (1 : 10 w/v). Te pH was adjusted to 9.0 using 1.0 M NaOH in order to solubilize the protein. Te mixture was then stirred for 1 h at 1,000 rpm and centrifuged (5000 × g, 4°C) for 20 min. Te supernatant was stored at −4°C, and the procedure was repeated with a pellet to MQW (1 : 5 w/v). Supernatants from both extractions were combined and pH was adjusted to 4.6 with 1.0 M HCl to achieve protein precipitation. Te precipitate was collected after centrifugation (5000 × g, 4°C) for 20 min, washed with 25 mL MQW, frozen at −80°C, and freeze-dried to obtain a free-fowing powder.

Preparation of Peptide Hydrolysate of T. occidentalis Seed
Protein (PHTOSP). Te preparation of peptide hydrolysate of T. occidentalis seed protein was carried out using the modifed method of Radha et al. [31]. T. occidentalis seed protein isolate was enzymatically hydrolyzed using pepsin under optimal conditions at an enzyme/substrate ratio of 1 : 100 (w/w). Te resulting mixture was stirred and incubated for 8 h at 25°C. Te enzyme was deactivated after hydrolysis by heating the mixture at 100°C for 10 min in a water bath. Tis was centrifuged for 10 min at 7000 × g, and the supernatant was collected. Te hydrolysate was collected, lyophilized, and stored at −20°C for further bioassays.
2.6. Ethics Approval. Te use of experimental animals involved in this study was carried out with a strict compliance to the ethical guidelines for the best practice issued by Ethical Review Committee (UERC) of the University of Ilorin, Ilorin, Kwara State, Nigeria, on the use of laboratory animals with protocol approval number of UERC/ASN/ 2017/903.

Animal Treatment Protocol.
Tirty-six (36) healthy male Wistar albino rats weighing between 150 and 160 g used for this study were obtained from the Animal Breeding Unit at the Department of Biochemistry, University of Ilorin, Ilorin, Kwara State, Nigeria. Te animals were acclimatized for 2 weeks before the experiment and housed in clean wooden cages with a standard diet and distilled water ad libitum.

Induction of Diabetes.
Diabetes induction (type 2 DM) was carried out through single intraperitoneal injection of freshly prepared STZ (50 mg/kg b.w) dissolved in cold citrate bufer (0.1 M, pH 4.5) [32]. Te rats were fasted overnight prior the induction of diabetes. After 72 h of STZ injection, diabetic status was established following an overnight fasting using an Accu-Chek glucometer to measure blood glucose concentration (mg/dl). Rat tail was punctured to acquire blood sample for fasting serum glucose (FSG). Animals with blood glucose levels ≥ 200 mg/dl were classifed as diabetic. Te 21-day treatment commenced after diabetes has been established.

Animal
Grouping. Tirty-six (36) experimental animals were randomly distributed into six groups (A-F) of six rats each (n � 6) as follows. Treatment with PHTOSP was administered orally. Te choice of diferent doses (moderately lower doses of 50-150 mg/kg b.w) used was based on the pilot studies (in vitro and in vivo) conducted prior to the commencement of the experiment.

Collection and Preparation of Tissue and Homogenate.
Tissue and homogenate samples were obtained according to the method of Akanji et al. [33]. Te experimental animals were euthanized by mild exposure to chloroform in an airtight chamber, after which they were dissected. Blood samples were immediately collected through a jugular vein cut. Blood samples were then allowed to stand and clot for 30 min to obtain sera. Liver and pancreatic tissues were immediately isolated, washed (in normal saline), weighed, and placed on ice. Subsequently, the tissues were homogenized in 0.25 M sucrose solution (1 : 5 w/v) at 3000 × g for 10 min. Te supernatant was aspirated into sample bottles and appropriately diluted and later kept frozen at 4°C before being used for various biochemical parameters analyzed. Pancreatic tissue was also cut out and preserved (in 10% neutral bufered formalin solution) at 25°C for histopathological examination.

Biochemical
Assays. Biochemical parameters such as serum insulin concentration were determined using the method described in Dallak et al. [34], liver glycogen content was determined by the method of Murat and Serfaty [35], hexokinase activity was determined as described by Zhang et al. [36], glucose-6-phosphate dehydrogenase activity was determined by the modifed method of Maurya et al. [37], fructose-1,6-bisphosphatase activity was determined by the method of Riou et al. [38], and glucose-6-phosphatase activity was determined by the method of Maurya et al. [37]. Also, serum lipid profle such as TC was determined by the method of Siedel et al. [39], HDL was determined by the precipitation method of Hirano et al. [40], and TG level was determined by the method described by Fossati et al. [41], while LDL and atherogenic index (AI) were calculated as follows [42,43]: (1)

Histopathological Examination.
Te histological status of the pancreatic tissues of the rats was assessed using the method described by Oloyede et al. [44].

Statistical Analyses.
Data were analyzed using one-way ANOVA, followed by Tukey's test for post hoc analysis, and graphical representation of results was performed using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). All values were expressed as mean ± SEM (n � 6). Statistical diferences were considered at p < 0.05. Table 1 illustrate the efect of PHTOSP on fasting serum glucose (FSG) levels in STZ-induced diabetic rats. Te results revealed that the FSG levels of untreated STZ-induced diabetic rats were signifcantly (p < 0.05) higher than those of the normal control. However, rats treated with PHTOSP at doses of 50, 100, and 150 mg/kg for 7, 14, and 21 days exhibited a signifcant (p < 0.05) decrease in FSG levels compared to both the untreated STZ-induced diabetic control and the normal control. Additionally, the efect of PHTOSP treatment was found to be comparable to that of the 5 mg/kg glibenclamide-treated control group. Table 2 demonstrate the efect of PHTOSP on the body weight of in STZ-induced diabetic rats. Te data show a signifcant (p < 0.05) decrease in body weight (% change in body weight) in the untreated STZinduced diabetic control group compared to the normal control group. However, treatment with PHTOSP at doses of 50, 100, and 150 mg/kg for 21 days resulted in a signifcant (p < 0.05) increase in body weight in the treated groups compared to both the untreated STZ-induced diabetic control and the normal control. After 21-day treatment, it was discovered that this efect was comparable to that of the control group receiving 5 mg/kg glibenclamide. Table 3 illustrates the efect of PHTOSP on the lipid profle and atherogenic index (AI) of STZ-induced diabetic rats. Te results revealed a signifcant (p < 0.05) rise in the levels of serum TC, LDL, TG, and AI, as well as a signifcant (p < 0.05) reduction in HDL in the untreated STZ-induced diabetic control in contrast to the normal control. However, treatment with 50, 100, and 150 mg/kg PHTOSP for 21 days resulted in a signifcant (p < 0.05) decrease in the levels of serum TC, LDL, TG, as well as a signifcant (p < 0.05) reduction in AI compared to the untreated STZ-induced diabetic control, normal control with a benefcial comparison to the group treated with 5 mg/kg of glibenclamide. Figure 1 shows the efect of PHTOSP on serum insulin levels in STZ-induced diabetic rats. Te results demonstrate a signifcant (p < 0.05) reduction in serum insulin levels of the untreated STZ-induced diabetic rats when compared to the normal control. Conversely, groups that received treatment with 50, 100, and 150 mg/kg PHTOSP for 21 days showed a signifcant (p < 0.05) dosedependent increase in serum insulin levels when compared to the untreated STZ-induced diabetic control and normal control. Tis efect was also found comparably benefcial to diabetic groups treated with 5 mg/kg glibenclamide for 21 days. Figure 2 presents the efect of PHTOSP on hepatic glycogen levels of STZ-induced diabetic rats. As indicated in the result, there was an observed reduction signifcantly (p < 0.05) in the hepatic glycogen level of untreated STZ-induced diabetic control compared to the normal control. However, following the 21-day treatment with 50, 100, and 150 mg/kg PHTOSP, there was a dosedependent non-related signifcant (p < 0.05) increase in the hepatic glycogen level in the treated groups compared to untreated STZ-induced diabetic control and normal control. Tis observation demonstrated a favorable comparison with the efect seen in the diabetic group treated with 5 mg/kg glibenclamide after 21-day treatment. Figure 3 illustrates the efect of PHTOSP on the activity of hepatic hexokinase in STZinduced diabetic rats. According to the results, there was a noteworthy (p < 0.05) reduction in hepatic hexokinase activity in the untreated STZ-induced diabetic control compared to the normal control. However, groups treated with 50, 100, and 150 mg/kg PHTOSP for 21 days exhibited a signifcant (p < 0.05) reduction in hepatic hexokinase activity, compared to both the untreated STZ-induced diabetic control and normal control. Additionally, this efect contrasted favorably with the fndings in the group that received 5 mg/kg glibenclamide over the period of 21 days.

Efect of PHTOSP on the Hepatic Glucose-6-Phosphate
Dehydrogenase Activity of STZ-Induced Diabetic Rats. Figure 4 depicts the efect of PHTOSP on the hepatic glucose-6-phosphate dehydrogenase activity of STZ-induced diabetic rats. In the results, a signifcant (p < 0.05) decrease was evident in the activity of hepatic glucose-6phosphate dehydrogenase of untreated STZ-induced diabetic control compared to the normal control. Conversely, groups treated with diferent doses of 50, 100, and 150 mg/kg PHTOSP showed an increase signifcantly (p < 0.05) in the activity of glucose-6-phosphate dehydrogenase compared to that of untreated STZ-induced diabetic control as well as a favorable comparison with the normal control and 5 mg/kg glibenclamide-treated group after 21 days.

Efect of PHTOSP on Hepatic Fructose-1,6-Biphosphatase
Activity of STZ-Induced Diabetic Rats. Figure 5 represents the efect of PHTOSP on hepatic fructose-1,6-biphosphatase activity of STZ-induced diabetic rats. As indicated in the results, there was a signifcant (p < 0.05) increase in the hepatic activity of fructose-1,6-biphosphatase of the untreated STZ-induced diabetic control compared to that of normal control. However, treatment with 50, 100, and 150 mg/kg PHTOSP resulted in a signifcant (p < 0.05) dosedependent reversal compared to the untreated STZ-induced diabetic control group after 21 days, as well as a favorable comparison with the normal control group. Similarly, this efect was favorably compared to the observation in the group treated with 5 mg/kg glibenclamide for 21 days.
3.9. Efect of PHTOSP on the Glucose-6-Phosphatase Activity of STZ-Induced Diabetic Rats. Figure 6 reveals the efect of PHTOSP on the glucose-6-phosphatase activity of STZinduced diabetic rats. Te results demonstrate that the untreated STZ-induced diabetic control group displayed a signifcant (p < 0.05) increase in hepatic glucose-6-phosphatase activity compared to the normal control. However, treatment with PHTOSP at doses of 50, 100, and 150 mg/kg for 21 days caused a signifcant (p < 0.05) dose-dependent reduction in hepatic glucose-6-phosphatase activity compared to the untreated STZ-induced diabetic control and normal control groups. Furthermore, this observation   showed a good comparison with the group treated with 5 mg/kg glibenclamide. Figures 7(a)-7(f), the efect of PHTOSP on the histopathological condition of the pancreatic tissue of rats with STZinduced diabetes is presented. Te results indicate that the untreated STZ-induced diabetic control group (Figure 7(b)) exhibited pancreatic histology with pockets of congestion within the islet cells, which difers from the normal control (Figure 7(a)) that showed normal pancreatic histology characterized by normal islet cells, acinar cells (AC), and interstitium free from infammatory cells. However, treatment with PHTOSP at doses of 50, 100, and 150 mg/kg for 21 days (Figures 7(c)-7(e)) revealed a pancreatic histology free from congestions and infammatory cells with a favorable comparison with the normal and 5 mg/kg glibenclamide-treated groups.

Discussion
Several methods have been used to screen for antidiabetic activity of natural products and synthetic drugs in experimental animal models following diabetes induction [45,46]. STZ, a diabetogenic agent, well known for its selective pancreatic β-cell cytotoxicity is commonly used for the induction of diabetes [47]. It causes alkylation in the β-cell via glucose transporter 2 (GLUT 2) and thereby induces poly ADP-ribosylation, which causes cellular NAD + and ATP depletion [48]. As a result, pancreatic β-cell necrosis is induced by the generation of free radicals [49]. Te increased glucose levels observed in the STZ-induced diabetic rats in this study (Table 1) may be due to the detrimental efect of STZ on pancreatic β-cells, resulting in a signifcant reduction in insulin secretion [50,51]. However, administration of PHTOSP revealed a signifcant glucose-lowering efect, which could be credited to its ability to stimulate insulin secretion [52]. Uncontrolled diabetes has been linked to severe muscle breakdown and wasting, which can result in weight loss [53,54]. Te results in Table 2 indicate that untreated STZinduced diabetic animals experienced signifcant weight loss, possibly due to STZ-induced muscle wasting as reported in previous studies [55,56]. Tis weight loss could be attributed to insulin resistance or defciency, which is known to   decrease muscle protein turnover and contribute to muscle wasting [57]. However, the observed increase in body weight after treatment with PHTOSP may be due to its ability to reduce muscle wasting, which may be exacerbated by STZinduced destruction of pancreatic β-cells.
Lipids play a crucial role in the development of cardiovascular diseases (CVDs) that are associated with DM [58]. Disorder in lipid metabolism, often described as dyslipidemia, has been reported to contribute majorly to CVDs [59]. Also, according to recent studies, insulin resistance and islet dysfunction are signifcantly worsened by variations in the plasma and islet lipoprotein levels [60,61]. According to the result in Table 3, there was an alteration in the levels of serum lipoproteins in the untreated diabetic control, which perhaps indicates STZ-induced diabetic dyslipidemia according to the report of Afolabi et al. [58]. More so, it has been reported in a previous study that changes in total cholesterol (TC), total triglycerides (TG), HDL/LDL ratio, and atherogenic index (AI) are indicators of susceptibility to CVDs in DM [62]. Tese changes have been linked to the activation of hepatic lipase, which leads to lipolysis. However, the administration of PHTOSP signifcantly mitigated the STZ-induced variations in lipoproteins in treated diabetic rats. Tis suggests that the hydrolysate may have the ability to enhance pancreatic release of insulin and reduce lipolysis-induced insulin resistance by inhibiting hepatic lipase [63]. Likewise, this study found that the untreated STZ-induced diabetic control group exhibited an increase in the AI, a lipidrelated indicator of the potential for developing cardiovascular diseases such as arteriosclerosis [64]. However, the reduction in AI levels observed after PHTOSP administration may suggest the hydrolysate's efectiveness in preventing the risk of CVDs and related complications in STZ-induced diabetes, possibly by restoring normal lipid parameters. Tis efect aligns with the fndings of Nasri et al. [65].  Normal control Untreated diabetic control Diabetic + 50 mg/kg PHTOSP Diabetic + 100 mg/kg PHTOSP Diabetic + 150 mg/kg PHTOSP Diabetic + 5 mg/kg glibenclamide Figure 4: Efect of PHTOSP on the hepatic glucose-6-phosphate dehydrogenase activity of STZ-induced diabetic rats. Results were expressed as mean ± SD of six trials (n � 6). * A signifcant diference at p < 0.05 vs. normal control; # p < 0.05 vs. untreated diabetic control; a p < 0.05 vs. untreated diabetic control and p < 0.05 vs. PHTOSP treated groups.
It is also worthy of note that insulin regulates proper glucose homeostasis by enhancing its transport to peripheral tissues and muscles [66]. Chronic exposure of β-cells to high level of reactive oxygen species (ROS) has been shown to cause a progressive loss of β-cells and deterioration of its activities and failure [67]. In this condition, the capacity of the β-cell to produce sufcient insulin needed for proper glucose uptake in the physiological milieu is hampered [68]. As indicated in the result (Figure 1), there was a reduction signifcantly in insulin levels of the untreated STZ-induced diabetic control, an efect that could possibly refect STZinduced pancreatic β-cell dysfunction [69]. However, oral administration of PHTOSP evidently reversed this condition either by the ability of the hydrolysate to stimulate proper secretion of insulin or mitigate STZ-occasioned ROS implicated in the deleterious activities of STZ, thereby protecting the β-cell from STZ-induced oxidative damage [70].
Hepatocytes are the major site for the storage and metabolism of glycogen [71]. Glycogen is a branched polymer of glucose, which acts as a store for glucose units [72]. Imbalances in the hepatic glycogen accumulation and production of glucose through glycogenolysis have been reported in DM [73,74]. Te decrease observed in the hepatic glycogen levels among the STZ-induced diabetic control could possibly be a refection of STZ-stimulated insulin reduction in insulin, resulting in uncontrollable glycogenolysis probably by activating hepatic glycogen phosphorylase activity [75]. More so, an increase in glycogen phosphorylase and failure of glycogen synthase activating mechanism has been documented in DM [76]. However, treatment with PHTOSP signifcantly improved hepatic glycogen possibly by its ability to potentiate glycolysis and thereby enhancing secretion of insulin as well as inhibiting the frst rate-limiting enzyme of glycogenolysis. Te fnding of this report is consistent with the fndings of Chatterjee et al. [77].  Normal control Untreated diabetic control Diabetic + 50 mg/kg PHTOSP Diabetic + 100 mg/kg PHTOSP Diabetic + 150 mg/kg PHTOSP Diabetic + 5 mg/kg glibenclamide Figure 6: Efect of PHTOSP on hepatic hepatic glucose-6-phosphatase activity of STZ-induced diabetic rats. Results were expressed as mean ± SD of six trials (n � 6). * A signifcant diference at p < 0.05 vs. normal control; # p < 0.05 vs. untreated diabetic control; a p < 0.05 vs. PHTOSP treated group. 8 Journal of Food Biochemistry A defect in carbohydrate-metabolizing enzymes as a result of deteriorated endocrinal control has been implicated in DM [78]. Hexokinase is a rate-limiting enzyme of glycolysis and plays a central role in the maintenance of glucose homeostasis by catalyzing the phosphorylation of glucose to glucose-6-phosphate [79]. As observed in our study, a reduction was noticed in the activity of this protein among the untreated STZ-induced diabetic control, an efect that could be linked to the STZ-induced glucose metabolic derangement [2]. Besides, a decrease in the activity of hexokinase being an insulin-dependent enzyme has been reported in DM [80]. In the result, the administration of PHTOSP however signifcantly increased hexokinase activity. Tis observation might possibly suggest the ability of the extract to restore proper glucose utilization via glycolysis resulting in the reduction of blood glucose [81].
Glucose-6-phosphate dehydrogenase (G6PDH) is the rate-limiting enzyme in the pentose phosphate pathway (PPP), which leads to the production of ribose-5-phosphate and NADPH equivalents [82]. Te PPP is an alternate pathway for glucose-6-phosphate from glycolysis, and NADPH is the only source of antioxidant defense mechanisms, heavily relying on G6PDH activity [83]. Our fndings showed a reduction in hepatic G6PDH activity in untreated STZ-induced diabetic rats, which is consistent with the report of Karuna et al. [84]. Additionally, previous studies have reported a reduction in hepatic G6PDH activity in DM [85], leading to a decrease in the production of NADPH equivalents necessary for maintaining cellular reduced glutathione [86].
Te decrease in hepatic G6PDH activity in untreated STZ-induced diabetic rats leads to an accumulation of glucose-6-phosphate, which acts as a potential glycosylating agent that exacerbates GSH depletion and promotes the fnal step of gluconeogenesis. Tis ultimately results in oxidative auto-glycation [87,88]. However, administration of PHTOSP in diferent doses to diabetic groups led to a signifcant increase in hepatic G6PDH activity (Figure 4). Tis suggests that the hydrolysate has the ability to enhance the activation of hepatic G6PDH, possibly through a mechanism that promotes cell proliferation and survival and restores proper insulin-mediated hepatic glucose oxidation and PPP. Tis pathway may play a role in regulating glucose levels in DM [89].
Gluconeogenesis is the primary cause of an increased hepatic glucose release in diabetic condition [90]. As seen in this report, fructose-1,6-biphosphatase, a rate-controlling enzyme of gluconeogenesis [90], and glucose-6-phosphatase that catalyzes the fnal stage of hepatic glucose production were signifcantly increased among the untreated STZ-induced diabetic rats. Previous documented reports have shown that the absence of insulin in DM results in the activation of these gluconeogenic enzymes [91,92]. In a normal physiological condition, insulin acts as a suppressor of hepatic gluconeogenic enzymes [93]. However, following the administration of PHTOSP, there was a substantial reduction in the activities of these enzymes, an efect that perhaps signals the ability of the hydrolysate to control hepatic glucose production, thus suppressing gluconeogenesis possibly via a mechanism that stimulates insulin secretion and facilitates proper glucose utilization in the peripheral tissues [94].
Histological changes in pancreatic islet have previously been noted in STZ-treated experimental animals [95]. Tese alterations are reportedly mediated by the reduction in nicotinamide adenine dinucleotide (NAD + ) of the β-cells [96]. As shown in this study, a marked vacuolation of Langerhans islets with β-cells revealing an unstained vacuolated cytoplasm and dark-stained degenerated nuclei was noticeable in the pancreatic tissues of the untreated STZinduced diabetic rats, an efect that could possibly indicate STZ-occasioned infammation and cytotoxicity to the β-cells according to a previous report of Sangi et al. [97] and Gundala et al. [98]. However, STZ-induced diabetic groups treated with PHTOSP showed a reversal of the STZ-induced cytotoxic efect with a normal population of β-cells in the islets of Langerhans. Tis observation could also suggest cytoprotective ability of the hydrolysate against STZinduced pancreatic oxidative damage.

Conclusion
Tis study evaluated the possible antidiabetic potentials of peptide hydrolysate of Telfairia occidentalis seed protein (PHTOSP) in streptozotocin-induced diabetic rats through the assessment of some diabetes-related biochemical parameters and pancreatic histological examination. According to our fndings, PHTOSP lowered fasting serum glucose and improved insulin levels, lipid profle, and liver parameters such as glycogen contents and glycolytic enzymes as well as suppressed gluconeogenetic enzymes. Similarly, PHTOSP modulated body weight and pancreatic histoarchitecture. Terefore, PHTOSP might possess drug-able antidiabetic properties such that it restores glycolytic pathway and promotes cell survival that are helpful in the management of diabetes mellitus (DM).

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
Olasehinde Oluwaseun Ruth and Arise Rotimi Olusanya conceptualized the research. Olasehinde Oluwaseun Ruth and Afolabi Olakunle Bamikole were responsible for data curation. Olasehinde Oluwaseun Ruth, Arise Rotimi Olusaanya, and Afolabi Olakunle Bamikole were responsible for reagents and analytical tools. Arise Rotimi Olusanya supervised the study. Olasehinde Oluwaseun Ruth and Afolabi Olakunle Bamikole drafted the original manuscript. All authors were responsible for formal analysis and data validation and reviewed, edited, and confrmed the authorship of the fnal manuscript.

Supplementary Materials
Tis illustrates STZ-induced diabetic rats administered with peptide hydrolysate of Telfairia occidentalis (T. occidentalis) seed protein (PHTOSP). Evaluated biochemical parameter indicated an increase in serum insulin, hepatic glycogen, hexokinase, and glucose-6-phosphate dehydrogenase activities, accompanied by a reduction in fasting serum glucose, glucose-6-phosphatase, and fructose-1,6bisphosphatase. Also, rats treated with PHTOSP demonstrated an improved lipid profle and relative body weight. (Supplementary Materials)