Antidiabetic activity of avocado seeds (Persea americana Mill.) in diabetic rats via activation of PI3K/AKT signaling pathway

The treatment of diabetes involves the use of herbal plants, attracting interest in their cost-effectiveness and efficacy. An aqueous extract of Persea americana seeds (AEPAS) was explored in this study as a possible therapeutic agent in rats with diabetes mellitus. The induction of diabetes in the rats was achieved by injecting 65 mg/kg body weight (BWt) of alloxan along with 5% glucose. This study was conducted using thirty-six (36) male Wistar rats. The animals were divided into 6 equal groups, (n = 6) and treated for 14 days. In vitro assays for total flavonoid, phenols, FRAP, DPPH, NO, α-amylase, and α-glucosidase, were performed. Biochemical indices fasting blood sugar (FBS), BWt, serum insulin, liver hexokinase, G6P, FBP, liver glycogen, IL-6, TNF-α, and NF-ĸB in the serum, were investigated as well as the mRNA expressions of PCNA, Bcl2, PI3K/Akt in the liver and pancreas. The in vitro analyses showed the potency of AEPAS against free radicals and its enzyme inhibitory potential as compared with the positive controls. AEPAS showed a marked decrease in alloxan-induced increases in FBG, TG, LDL-c, G6P, F-1, 6-BP, MDA, IL-6, TNF-α, and NF-ĸB and increased alloxan-induced decreases in liver glycogen, hexokinase, and HDL-c. The diabetic control group exhibited pancreatic dysfunction as evidenced by a reduction in serum insulin, HOMA-β, expressions of PI3K/AKT, Bcl-2, and PCNA combined with an elevation in HOMA-IR. The HPLC revealed luteolin and myricetin to be the phytochemicals that were present in the highest concentration in AEPAS. The outcome of this research showed that the administration of AEPAS can promote the activation of the PI3K/AkT pathway and the inhibition of β-cell death, which may be the primary mechanism by which AEPAS promotes insulin sensitivity and regulates glycolipid metabolism.

In vitro antioxidant activity of AEPAS. As shown in Fig. 1, we compared the phenol and flavonoid contents of the AEPAS with standard gallic acid and quercetin. The graph shows the total phenolic content in the sample and the standard, which increases with concentration. The total flavonoid content was concentration dependent as the results showed an increase in flavonoid content with increased concentration. Figure 2 shows    www.nature.com/scientificreports/ Fasting blood glucose (FBG) level of diabetic rats administered AEPAS. The FBG level of the male rats was elevated 72 h after induction of alloxan to values above 250 mg/dl, which were maintained in the untreated group for the duration of the experiment. A decline in FBG levels was observed after the administration of AEPAS at 26.7 mg/kg, 53.3 mg/kg, and 106.6 mg/kg BWt ( Table 2). The reduction in the FBG level in the animal groups treated with 53.3 mg/kg and 106.6 mg/kg BWt yielded values in alignment with the nondiabetic group (Table 2). We found the most profound effect at 106.6 mg/kg BWt.
Body weight of diabetic rats administered AEPAS. We witnessed a notable decrease in the body weight in the animals, apparently resulting from the administration of alloxan (Table 3), as evidenced by the weight gain in the normal control group. Treatment with AEPAS at 26.7 mg/kg, 53.3 mg/kg, and 106.6 mg/kg BWt resulted in slight increases in the BWt of the rats (Table 3) with the increases being dose-dependent.

Serum insulin levels, HOMA-IR and HOMA-β levels of diabetic rats administered AEPAS.
From the results presented in Table 4, the serum insulin and HOMA-β levels of the diabetic untreated group showed a remarkable decrease whereas the HOMA-IR levels increased. Administration of AEPAS at 26.7 mg/kg, 53.3 mg/ kg, and 106.6 mg/kg resulted in elevated serum insulin and HOMA-β levels with a noteworthy decrease in the HOMA-IR levels.
Antioxidant markers in experimental DM. The activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) in the liver were in contrast to the untreated diabetic rats when either AEPAS or metformin was administered to diabetic rats. Lipid peroxidation (MDA) levels decreased after administration of either metformin or AEPAS, in contrast to the elevated levels in the untreated diabetic rats, and the results were dose-dependent ( Table 5). The activities of GPx, GST, GSH, CAT, and SOD in the pancreas were elevated and the MDA levels decreased after administration of AEPAS and metformin compared with the diabetic untreated rats (Table 6).

Serum lipid parameters in experimental DM administered AEPAS. Administration of alloxan led
to an increased concentrations of VLDL-c, LDL-c, TG, CRI, AI, and total cholesterol with a reduction in HDL-c levels ( Table 7). Administration of the various dosages of AEPAS at 26.7 mg/kg, 53.3 mg/kg, and 106.6 mg/kg resulted in a decrease in triglycerides, LDL-c, total cholesterol, CRI, VLDL-c, and AI levels in contrast to the diabetes-induced rats. The doses of the extract elevated the serum HDL-c levels when compared to the diabetic rats. Metformin treatment resulted in a significant decrease in triglycerides, LDL-c, total cholesterol, CRI, VLDL-c, and AI levels with a corresponding improvement in HDL-c levels. Table 2. Fasting blood glucose levels of alloxan-induced diabetic rats before and after oral administration of aqueous extract of P. americana seeds. Data are expressed as mean ± SEM (n = 6). *Statistically significant (p < 0.05) to DC; # Statistically significant (p < 0.05) to NC; **P < 0.01 is considered as very significant. AEPAS: Aqueous extract of P. americana seeds.  www.nature.com/scientificreports/ Hepatic glycogen and carbohydrate metabolizing enzymes in diabetic rats administered AEPAS. The levels of hepatic glycogen and the glycolytic enzyme, hexokinase, were diminished in the diabetic rats, and we observed a remarkable increase of these in the treatment groups after administration of either metformin or AEPAS (Table 8). We found that the activities of the gluconeogenesis enzymes G6Pase and F-1,6-BPase increased in the diabetic control group, but administration of either AEPAS or metformin yielded a notable decrease in the treatment groups (Table 8).

Pro-and anti-inflammatory markers of diabetic rats administered AEPAS. Administration of
alloxan increased the levels of IL-6, TNF-α, and NF-κB ( Fig. 4) in the plasma of the rats (p < 0.05) compared with those of the control rats. AEPAS reduced the levels of IL-6, TNF-α, and NF-κB (p < 0.05) compared to the diabetic rats, as did treatment with metformin. AEPAS at 26.7, 53.3, and 106.6 mg/kg BWt showed the reversal of the alloxan treatment-related rises in the concentrations of IL6, TNF-α, and NF-κB (Fig. 4).
Gene expressions of PI3K, AKT, and apoptotic markers of diabetic rats administered AEPAS. The PI3K, AKT, Bcl-2, and PCNA mRNA expression levels in the liver and pancreas are shown in Histological study of pancreatic tissues. Alloxan induction resulted in the partial destruction of the β-cells of the pancreatic islets when compared to control rats (Fig. 6). Treatment with AEPAS, on the other hand, improves and restores the damaged pancreatic islets cells at all doses. Table 7. Lipid profile of alloxan-induced diabetic rats after oral administration of aqueous extract of P. americana seeds. Data are expressed as mean ± SEM (n = 6). *Statistically significant (p < 0.05) to DC;   Table 8. Hepatic glycogen and carbohydrate metabolizing enzyme levels after oral administration of aqueous extract of P. americana seeds. Data are expressed as mean ± SEM (n = 6). *Statistically significant (p < 0.05) to DC; # Statistically significant (p < 0.05) to NC. AEPAS: Aqueous extract of P. americana seeds; α : Unit for glycogen (mg of glucose/g of wet tissue); β : Unit for hexokinase (µmole glucose-6-phosphate formed/min/mg protein); γ : Unit for fructose-1,6-bisphosphatase and glucose-6-phosphatase (µmole phosphate liberated/min/ mg protein).

Discussion
Researchers have related the antidiabetic properties of several medicinal plants to bioactive compounds such as phenolics, flavonoids, and tannins 17 . Over the years, scientists have explored these chemical compounds in managing several diseases by folklore medicine [18][19][20] . The HPLC of AEPAS confirmed the presence of ascorbic acid, myricetin, luteolin, and gallic acid. Ascorbic acid is available as a natural antioxidant in some systems. Advanced glycation end products, glycosylation of proteins, the polyol pathway, and auto-oxidation of glucose are involved in the pathogenesis of T1DM and T2DM. Thus, one important defense against such impairment is antioxidant compounds such as ascorbic acid 21 .
Myricetin, a flavonoid compound, was documented to have antioxidant and antidiabetic activities. Myricetin facilitated the metabolic action of insulin by stimulating phosphatidylinositol 3-kinase (PI3K) and its effectors 22 .
Luteolin is an active flavonoid with an extensive array of pharmacological properties. The well-known pharmacological properties of luteolin, for example, its antioxidant, anti-inflammatory, anti-apoptotic, and antidiabetic properties, have been demonstrated 23 .
The antioxidant property and inhibitory activities of gallic acid on marker enzymes (α-glucosidase and α-amylase) linked to diabetes mellitus have been shown by Mackensie et al. 24 , and improvement in IR sensitivity by gallic acid may account for its anti-diabetic property 25 . All the activities of ascorbic acid, myricetin, luteolin, and gallic acid summarized above could have accounted for the antioxidant and antidiabetic properties we observed for AEPAS.
Recently, phenolic compounds have attracted considerable attention for their promising use as novel nutraceuticals or biological products because of their notable antiradical and anti-inflammatory properties, which may be concentration dependent 26 . Flavonoids are a major group of secondary metabolites, and many experiments have reported their biological properties 27 . This study showed that the total phenolic and flavonoid content of AEPAS operated in a concentration-dependent manner.
Free radicals have been linked to diseases such as T2DM 28 . Researchers documented that hyperglycemia produces free radicals that turn on the free radical/antioxidant defense system, producing oxidative stress. We can evaluate the antioxidant activities of therapeutic plants via their total antioxidant capacity and their reducing and radical scavenging properties 29 . Recent studies reported that strong antioxidants present in therapeutic foods and plants can be useful in neutralizing the effects of stress in diseases such as type-2 diabetes 30 . The efficiency of AEPAS as an antioxidant could be due to phenolics and flavonoids, a conclusion that is further supported by the ability of AEPAS to reduce ferric ions and to neutralize NO and DPPH radicals. From this study, AEPAS revealed a high total antioxidant capacity, reducing capacity, and scavenging power. An overabundance of nitric oxide can lead to tissue injury and Type-2 diabetes-linked cardiovascular problems 31 . Hence, AEPAS could inhibit nitric oxide radicals in a concentration-dependent manner. This could be because of the compounds, including ascorbic acid, myricetin, luteolin, and gallic acid, present in AEPAS, which have been reported to have these properties.
The treatment of hyperglycemia is the essential goal for managing diabetes mellitus. One of the most significant beneficial methods is reducing postprandial hyperglycemia by hindering carbohydrate absorption and digestion 32 . Anti-diabetic agents function by inhibiting α-amylase and α-glucosidase enzymes relevant to T2DM 33 . These enzymes perform a key role in the breakdown of nutritional carbohydrates to glucose 33 . The inhibitory activity of the AEPAS on α-amylase and α-glucosidase indicates its potential as an antidiabetic treatment. The inhibition of these enzymes by AEPAS clarifies its capability to delay the breakdown of carbohydrates, hence controlling the FBG level. We can credit these activities to the metabolites, phenolics, and flavonoids that have been described as components of herbal extracts with anti-diabetic properties 34 . It is important to note that the α-amylase activity of AEPAS compared with that of acarbose, whose mechanism of action involves inhibiting these enzymes.
Alloxan-induced diabetes is a valuable experimental model for examining the antidiabetic properties of many agents 35 . The mechanism by which alloxan induces diabetes is that it selectively inhibits glucose-induced insulin production via a specific hexokinase inhibition that triggers a condition similar to T2DM via its capacity to induce ROS, resulting in pancreatic β-cell toxicity 36 . The reference antidiabetic agent, metformin, acts in many ways, including decreasing glucose generation, improving lipid oxidation in liver cells, and/or increasing the uptake of glucose 36 . The increased FGB level observed after 72 h of alloxan administration supports the idea  www.nature.com/scientificreports/ that glucose-induced insulin production is inhibited through specific hexokinase inhibition and selective toxicity on the β-cells 36 . The decreased level of serum FBG in both the AEPAS and metformin-treated groups compared with the diabetic group supports the view that the therapeutic properties displayed by AEPAS could result from phenolics components such as gallic acid and myricetin. The reduction in serum glucose level by the AEPAS could be due to the increased uptake of glucose evidenced by the improved glycolytic pathway in this study. A severe loss in bodyweight typifies alloxan-induced diabetes 37 . The difference in energy consumption and usage leads to an alteration in body weight 37 . The decrease in body weight in diabetic rats might be the result of a decrease in glucose metabolism, elevated metabolism of fats, or the structural breakdown of proteins that provide an alternate source of energy 37 . The increased body weight of the diabetic rats treated with AEPAS at the highest dose could have resulted from greater glycemic control through improved insulin secretion. This antidiabetic property indicates that AEPAS may promote insulin production from the residual β-cells or restored β-cells which could activate the hormones involved in fat storage.
Insulin resistance and pancreatic β-cell dysfunction typifies diabetes mellitus 38 . The quantitative assessment of insulin resistance (HOMA-IR) and insulin production/β-cell function (HOMA-β) are vital keys for measuring IR and assessing β-cell function 38 . Improved insulin production and insulin sensitivity in response to AEPAS showed its ability to improve HOMA-IR and HOMA-β. It would be reasonable to credit this effect to the flavonoids in AEPAS since they have been shown to promote the restoration of pancreatic β-cells and increase insulin release in diabetes-induced rats 38 . This corresponds to the findings of a previous study from our laboratory.
Enzymatic antioxidants (CAT, SOD, GPx, GSH, and GST) and non-enzymatic antioxidants (MDA) perform a crucial function in maintaining the biological levels of oxygen and hydrogen peroxide via improving the dismutation of O 2 radicals and destroying organic peroxides produced from alloxan exposure 39 . This current study showed that alloxan-induced diabetes unbalanced the activity of liver marker enzymes. The observed reduction in the activities of CAT, SOD, GPx, and GST and the levels of GSH in the liver and pancreas of diabetic rats may be due to the chemical reduction of alloxan to dialuric acid to produce redox intermediates and of oxidized glutathione (GSSG) to produce radicals, such as the OH radical that is the main toxic ROS species 36 . This may account for the inadequacy of the defense system in alleviating ROS facilitated injury 40 . The improved activities of these enzymes by AEPAS appears to have reduced the difference between the production of ROS and enzymatic antioxidant activities in the diabetic rats. Also, the formation of ROS is prevented by the destruction of radicals by these antioxidant enzymes. Thus, the ability of AEPAS to attenuate the changed antioxidant enzymes in alloxan-induced diabetic rats indicates its radical scavenging property. This may be due to the phenolics and flavonoids in AEPAS, which have been identified as having radical scavenging effects 27 .
Dyslipidemia, increments in the concentrations of TC, TG, and alterations in lipoprotein components, is a recognized challenge in diabetic patients 41,42 . Also, the high concentrations of LDL-c and AI induced by alloxan imply a propensity for cardiovascular issues 42 . The increased levels of TC in diabetic animals are due to the Glycogen concentrations in tissues such as the liver are a direct indication of insulin action in that insulin improves internal glycogen deposition by inhibiting glycogen phosphorylase 43 . Depletion of glycogen by alloxan administration could result from a decrease in the activity of hexokinase. The restoration of hepatic glycogen after the administration of AEPAS may have resulted from its ability to improve insulin release from the pancreas 43 . However, this could also have been because of polyphenols, which have been reported to have insulin mimetic activity thus, giving rise to direct peripheral glucose uptake 43 .
The liver is a significant organ that performs a vital function in the glycolytic and gluconeogenic pathways. Hexokinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase are key enzymes in glucose metabolism 44 . The decrease in hexokinase activity in diabetic animals could be because of reduced glycolysis and diminished use of glucose for energy creation. Increased activity resulting from AEPAS appears to have enhanced the use of glucose for energy creation, which suggests a higher glucose uptake from the blood by hepatocytes and improved glycolysis.
The activity of these enzymes (F-1,6-BPase and G-6-Pase may be elevated in diabetes 45 . The increase in F-1,6-BPase and G-6-Pase in the hepatic tissue of diabetic rats may be related to insulin inadequacy and overproduction of glucose 46 . In addition, the decrease in F-1,6-BPase and G-6-Pase after AEPAS treatment could have resulted from inhibiting gluconeogenesis through improved insulin production, modulating the activity of F-1,6-BPase and G-6-Pase via the control of cyclic adenosine monophosphate (cAMP) or inhibition of glycolysis 47 . The ability of AEPAS to modulate F-1,6-BPase and G-6-Pase could be the result of the action of flavonoids and phenolics, which play a vital role in reversing F-1,6-BPase and G-6-Pase to near normal through enhanced insulin secretion by their antioxidant potential. This further supported the antihyperglycemic activity of the AEPAS.
The PI3K/AKT signaling pathway is linked to the glucose metabolism essential for insulin stimulated glucose intake in the liver, as earlier documented by Nandipati et al. 48 , and Yu et al. 49 Akt activation stimulated the cell's existence by phosphorylation 50 . It facilitated the metabolic action of insulin through the activation of PI3K and its effectors, the protein kinase B (PKB/Akt) kinases. In addition, the AMPK signaling pathway may facilitate the impact of the insulin-independent response to glucose uptake. AEPAS could protect against alloxan-induced damage through an anti-apoptotic effect by increasing the mRNA expression of the phosphorylation of Akt 50 . AEPAS reversed the reduced mRNA expression of PI3K and AKT levels in diabetic rats (Fig. 7). www.nature.com/scientificreports/ The antiapoptotic Bcl-2 is a major molecule involved in apoptosis. Alloxan-induced diabetic rats had reduced Bcl-2 expression in the liver and pancreas tissues while treatment with AEPAS altered the balance of the antiapoptotic (Bcl-2) molecules and prevented cell death of the hepatic and pancreatic cells at all doses, as did metformin. The findings from this study (elevated MDA level and reduced SOD activity in the liver and pancreatic tissues of diabetic rats) agree with a study describing the elevated generation of radicals resulting in cell death of hepatic and pancreatic cells 51 . In this case, the apoptotic pathway requires additional examination to determine whether caspase(s) and cytochrome c are involved, as documented in other research experiments 52 .
As a valuable proliferation marker, proliferating cell nuclear antigen (PCNA) expression performs exclusive functions at the start of cell propagation by facilitating DNA polymerase. PCNA also performs key functions in the eukaryotic cell cycle in addition to stimulating the development of antibodies towards foreign compounds 53 . In this study, the mRNA expression of PCNA was up-regulated in the hepatocytes and pancreatic tissues of the normal rats and downregulated in the diabetic rats. AEPAS improved the PCNA expression in the liver and pancreatic tissues of diabetic rats.
In conclusion, AEPAS attenuates insulin resistance in alloxan-induced diabetic rats. The potential molecular mechanisms of AEPAS are an increase in glucose uptake, increased hexokinase activity, increased insulin levels, and a decrease in pancreatic β-cells apoptosis via activation of the PI3K/Akt signaling pathway in diabetic rats (Fig. 7). AEPAS could be an excellent source of antidiabetic agents as it controls the hyperglycemic index and other associated biochemical indices.

Preparation of aqueous extract of P. americana seeds (AEPAS). Persea americana seeds were sliced
into smaller parts and dried for 4 weeks at 25 °C, after which they were ground into powder using an electric blender (Kenwood, Model BL490, China). 35 g of dried seed was soaked in distilled water for 48 h to obtain the aqueous extracts 17 . We concentrated the aqueous extract obtained using a freeze-dryer (Modulyo Freeze Dryer, Edward, England) to obtain an 18.5 g yield.

High-performance liquid chromatography (HPLC-UV) analysis.
We evaluated the HPLC analysis of AEPAS by a chromatographic system (N 2000, Korea) utilizing an Autosampler (YL 9150) with 100 μl fixed loop and a YL9120 UV-visible detector. We did the separation on an SGE Protocol PC18GP120 (250 mm × 4.6 mm, 5 μm) column at 25 °C. The mobile phase contailed methanol to water (70:30 v/v), and we utilized the isocratic mode; elution was achieved at a flow rate of 1 ml/min. The samples were run for 15 min and absorbance was read at 254 nm. The chromatographic data were assessed using the Autochro-2000 software.
Total phenolic content determination. The technique described by Singleton et al. 54 was used to determine the phenolic content. 200 µl of AEPAS at varying concentrations of 15-240 µg/ml was added to test tubes containing 2 ml of NaHCO 3 . 200 µl of Folin-Ciocalteu (Folin-C) reagent was added two minutes later, after which the mixtures were well mixed and incubated in the water bath for 30 min at 50 °C. We read the absorbance at 760 nm. The standard gallic acid was prepared in the same way as the AEPAS stock solution. The phenolic content was determined using the same protocol as used for AEPAS with the standard being used in place of AEPAS.
Total flavonoid content determination. The aluminum chloride (AlCl 3 ) procedure was used to determine the total flavonoid content of AEPAS 55 . The stock solutions of AEPAS were prepared at concentrations of 15-240 µg/ml and 1 ml of AEPAS was measured into clean tubes, after which 3 ml of distilled water and 0.3 ml of 5% NaNO 2 were added. 0.3 ml of 10% AlCl 3 and 2 ml of 1 M NaOH were added 5 min later and the volume of the solution in the test tube was diluted to 10 ml by adding distilled water. We read the absorbance at 510 nm. Quercetin was used as a reference and prepared by dissolving 4 mg in 1 ml of methanol. Various concentrations were prepared, and we assayed the flavonoid content using the same method outlined above for AEPAS.

Assessment of in vitro antioxidant capacity. Total antioxidant activity (TAC).
The protocol described by Prieto et al. 56 was used to determine the TAC. 1 ml of the reagent (28 mM sodium phosphate, 4 mM ammonium molybdate, and 0.6 M sulfuric acid) and 100 µl (0.1 ml) of AEPAS at various concentrations were placed in test tubes. The test tubes were then capped using foil paper and incubated in a water bath at 95 °C for about 90 min. The samples were then cooled, and we read the absorbance at 695 nm.
Assay of DPPH radical scavenging ability. We adopted the methodology described by Oboh and Rocha 57 to determine the 2, 2-diphenyl-1-picrylhydrazyl scavenging effect of AEPAS. In brief, the extract (1 ml) at con- www.nature.com/scientificreports/ centrations ranging from 15 to 240 µg/ml was added to 0.4 mM of DPPH solution prepared with methanol. Before reading, we kept the solution in the dark for 30 min, after which we read the absorbance of the mixture at 517 nm. This was done in triplicate. The reference used was ascorbic acid. The percentage of DPPH discoloration was then further calculated.
Assay of NO radical scavenging ability. The method described by Jagetia and Baliga 58 estimated the capacity of nitric oxide to scavenge radicals in AEPAS. Varying concentrations (15-240 µg/ml) of AEPAS and the reference were prepared. 2.5 ml of 10 mM sodium nitroprusside (SNP) prepared in PBS (phosphate buffered saline) was added to 0.5 ml of varying concentrations of AEPAS and standard. The mixture was then further incubated for 150 min at 25 °C. A 0.5 ml aliquot was removed after the incubation period, and 0.5 ml of Griess reagent, which contains 1% (w/v) of sulphanilamide, 2% (v/v) of H 3 PO 4 , and 0.1% (w/v) of naphthylethylenediamine dihydrochloride, was added. We used 2 ml of sodium nitroprusside in PBS as the reference sample. The NO scavenging radical activities of AEPAS and gallic acid were both calculated and expressed as percentages.
Reducing ability. The reducing ability of AEPAS was assessed using Pulido et al. 's procedure 59 . We dissolved 2.5 ml of the sample (powdered) in about 2.5 ml of 200 mM sodium phosphate buffer of pH 6.6 and about 2.5 ml of 1% potassium ferricyanide. The mixtures were further incubated for 20 min at 50 °C, and then about 2.5 ml of 10% TCA was introduced to terminate the reaction. This was centrifuged for 10 min at 3000×g after which we added about 5 ml of the supernatant, an equivalent volume of water, and about 1 ml of 0.1% ferric chloride. The procedure followed above was also repeated, but in this case, it was for ascorbic acid, which was used as the reference sample and the reaction absorbance was read at 700 nm, followed by the calculation of the reducing ability which was expressed as an ascorbic acid equivalent.
α-Amylase inhibitory activity. The inhibitory activity of α-amylase in AEPAS was determined following Experimental animals. 36  Organ harvesting and analysis of samples. This research study lasted 14 days, after which we euthanized the rats using halothane. Then, the liver and pancreas were harvested and homogenized in a cold phosphate buffer, before being kept at a temperature of − 4°. The homogenized liver and pancreas were both centrifuged at 3000×g for 10 min to obtain a solution clear enough to evaluate some selected oxidative stress biomarkers. The blood was also collected and left for 1 h before centrifuging for 10 min at 3000×g to obtain a clear solution. We utilized this blood to determine the selected biochemical parameters.
Biochemical indices. We determined serum insulin concentration based on the method described by Ojo et al. 17 , which uses an ELISA kit from Sweden in a multiple plate ELISA reader (Winooski, Vermont, in the USA). We evaluated serum total cholesterol using the protocol from Fredrickson et al. 62 Triglyceride was determined using the procedure described by Tietz 63  Determination of biomarkers of oxidative stress. The supernatants of both the liver and pancreas were used to assay for reduced glutathione (GSH) level 68 , GPx 69 , catalase (CAT) 37 , and superoxide dismutase (SOD) activities 70 , and MDA level 71 .
Determination of the activities of glycolytic enzymes and glycogen level. The liver supernatant was used to analyze the activities of the glycolytic enzymes, including hexokinase 72 , glucose-6-phosphatase (G6Pase) 73 , and fructose 1,6-bisphosphatase 74 . We estimated the liver glycogen following the procedure described by Morales et al. 75 .
Determination of inflammatory biomarkers. TNF-α, IL-6, and NF-κB, were determined in the serum utilizing the procedure delineated in ELISA (Sigma Chemical Company Inc. (St. Louis, MO, USA)) kits.
Total RNA isolation. We removed total RNA from entire organs following a technique described by Omotuyi et al. 76 The organs were homogenized in cool ( PCR amplification and agarose gel electrophoresis. PCR intensification for the assessment of genes whose primers (Primer3 software) are recorded below were performed using the accompanying procedure: PCR enhancement was achieved in a 25 µl volume mixture containing 2 µl cDNA (10 ng), 2 µl primer (100 pmol) 12.5 µl Ready Mix Taq PCR master mix (One Taq Quick-Load 2x, master mix, NEB, Cat: M0486S) and 8.5 µl nuclease-free water. Early denaturation at 95 °C for 5 min was followed by 20 cycles of amplification (denaturation at 95 °C for 30 s, annealing for 30 s and amplified at 72 °C for 60 s), concluding with a final amplification at 72 °C for 10 min. In all the tests, we incorporated negative controls, in which the mixture had no cDNA. The amplicons were separated on 1.5% agarose gel (Cleaver Scientific Limited: www.nature.com/scientificreports/