Oleic Acid and Succinic Acid: A Potent Nutritional Supplement in Improving Hepatic Glycaemic Control in Type 2 Diabetic Sprague–Dawley Rats

Nutritional supplements are gaining traction for their effects in mitigating the impacts of various health conditions. In particular, many supplements are being proposed to reduce the impacts of type 2 diabetes (T2D), a metabolic condition that has reached global epidemic proportions. Recently, a supplement of oleic acid (OA) and succinic acid (SA; 1 : 1, w/w) was reported to improve glycaemic control in type 2 diabetic (T2D) Sprague–Dawley (S-D) rats through ameliorating insulin release and sensitivity. Here, we investigate the effects of the supplement (OA and SA) on hepatic and pancreatic function in T2D S-D rats. Eighteen (18) S-D rats were rendered diabetic and were divided into three equal groups: diabetic control, diabetic treatment, and diabetic glibenclamide. Another 12 S-D rats were obtained and served as the normal groups. The animals were treated daily with the vehicle, OA and SA (800 mg/kg body weight (bw); 1 : 1), or glibenclamide (10 mg/kg bw) which served as the positive control. The findings indicated that treatment with the supplement resulted in a 35.69 ± 4.22% reduction (p=0.006) in blood glucose levels (BGL). Analysis of hepatic enzymes depicted that the nutritional supplement reduced the activity of the gluconeogenesis enzyme, glucose-6-phosphatase (G6P) while improved the activity of catabolic enzymes such as glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK). Furthermore, the supplement attenuated oxidative stress through restoration of catalase (CAT) and superoxide dismutase (SOD), while reducing malondialdehyde (MDA) levels. Finally, the supplement showed no liver or kidney toxicity and improved the size and number of pancreatic islets of Langerhans, indicating its potential application in treating T2D. The study highlighted that a supplement of the two organic acids may be beneficial in reducing the rate of pathogenesis of type 2 diabetes. Therefore, it may offer therapeutic value as a dietary or nutritional supplement in the approach against diabetes and its complications.


Introduction
Tere has been a trend in the increase of type 2 diabetes (T2D) in both developing and developed societies [1,2].With the rate of increase in prevalence and incidence, the metabolic syndrome has reached epidemic proportions with over 422 million persons sufering from the disease [3].Tis number is predicted to double over the next decade, accounting for more than 4.4% of world's population.T2D, in particular, accounts for approximately 90% of all cases of diabetes and is generally characterized by initial insulin insensitivity followed by destruction of beta-cells of the pancreas [4][5][6][7].Despite the availability of oral synthetic agents that ameliorate several of these conditions, there is still a high demand for novel therapeutics with higher effcacy and fewer side efects.
In our previous study, it was highlighted that oleic acid (OA) and succinic acid (SA) found in the plant Desmodium canum synergistically improve glycaemic control in diabetic S-D rats [8].In this study, an improvement in insulin sensitivity was documented and played a pivotal role in alleviating the exacerbated state of hyperglycaemia.Te individual benefts of the organic acids in the treatment of T2D are well documented with a wealth of supporting evidence [9][10][11][12].In earlier studies, Saravanan and Pari [13] reported on the insulinotropic nature of SA and its esters and proposed the monomethyl ester to be a novel therapeutic agent in the fght against diabetes.Other research confrmed the mechanism of action of the dicarboxylic acid which includes a stimulation of the mevalonate pathway [14][15][16].Similarly, the literature purports the benefcial effects of OA in the treatment of T2D.Palomer et al. [17] documented that OA mitigates against insulin resistance and decreases the deposition of diacylglycerides in peripheral tissues.
In the current study, the efects of a supplement of the two organic acids on hepatic glucose metabolism and morphology of pancreatic tissue in diabetic rats are highlighted.Te role of the liver in glycaemic modulation is well documented, especially through regulation of gluconeogenesis, glycolysis, and glycogenolysis [18,19].Diabetes mellitus disrupts the tight regulation of these processes at both the transcriptional and translational levels.One of the leading factors that disrupts this homeostasis is insulin resistance, which negates the suppressive efects of insulin on gluconeogenesis and glycogenolysis.Te expression and activity of gluconeogenetic regulatory enzymes such as glucose-6-phosphatase (G6P) and fructose-1, 6bisphosphatase (FBP) are signifcantly elevated in T2D, consequently resulting in an elevated level of glucose hepatic output [20][21][22][23][24]. Tis partially explains the aberrant glucose levels reported in type 2 diabetics.Other studies have underscored the importance of maintaining a normal gluconeogenic rate in diabetics [25,26].Any agent that demonstrates potential to regulate this, whether at the transcriptional level or post-translation levels, may ofer therapeutic benefts in glycaemic control.
Moreover, oxidative stress is well documented to be a contributing factor in the progression of diabetes mellitus [27,28].An increase in the production of reactive oxygen species (ROS) is linked to the glycation of endogenous antioxidants as well as an increased production of ROS from the mitochondria.Studies have highlighted the role that reactive species play in the degradation of the pancreas and its exacerbation of insulin resistance [27].Tese are contributing factors towards the progressive nature of diabetes and ultimately results in the need for therapeutic agents to achieve glycaemic control.Furthermore, the ROS efect on hepatic function is well described in the literature [29][30][31].Studies have reported a marked elevation of biomarkers for oxidative stress such as malondialdehyde (MDA) and 8hydroxy-2′-deoxyguanosine, while decreasing the body's endogenous antioxidants such as catalase (CAT), glutathione, and superoxide dismutase (SOD) [27,32,33].Oxidative stress disrupts the metabolic processes within the liver including those important for glycaemic control.Consequently, increased oxidative stress acts as a progressive factor in exacerbating diabetes and complications associated with the metabolic syndrome.Novel antidiabetic therapeutics should, therefore, be able to mitigate this contributing factor.
Given the antidiabetic nature of a supplement containing both OA and SA, it warrants studying the efects on the hepatic carbohydrate metabolizing enzymes, along with any other efects that the supplement may have on liver function in type 2 diabetic S-D rats.Furthermore, given the importance of the pancreas, examining the potential amelioration of degeneration of the pancreas is noteworthy.It is being hypothesized that a synergy of the two organic acids will improve glycaemic control through an improvement if hepatic and pancreatic functioning.

Ethical Consideration. Te Faculty of Medical Sciences
Mona Campus Research Ethics Committee, UWI Mona Ethic Committee granted ethical approval for the use of S-D rats in the study (Ethical approval number: AN07, 2018).Te ethical guideline for their usage was strictly followed.

Diabetic Induction.
Tirty S-D rats (15 males and 15 females, mean weight of 155 ± 12 g) were retrieved from the Department of Basic Medical Sciences Animal House, UWI, Mona campus.Te animals were subsequently randomly divided into fve groups, with a sample size of 6 each.
Diabetes induction was done using a method described by [5,34].Te normal groups (1 and 2) were fed water ad libitum for two weeks, after which, they were subjected to an intraperitoneal (ip) injection of citrate bufer (0.3 mL, pH 4.5).Tis bufer served as the vehicle for STZ in the diabetic groups (3 to 5).In the diabetic induction, these animals were fed a 10% fructose solution for two weeks ad libitum.Subsequently, the animals were rendered diabetic through an intraperitoneal (ip) injection of 40 mg/kg bw STZ dissolved in 0.3 mL of cold citrate bufer.Teir glycaemic state was assessed by measuring their nonfasted BGL 1 week postadministration.Blood glucose values ≥ 15.6 mM were considered diabetic [35].
Nonfasting blood glucose levels were monitored 1 week post STZ or citrate bufer administration and at the end of the study.Te percentage change in BGL was calculated as reported by [36,37] shown below: 2.4.Animal Sacrifce.At the end of 28 d of treatment, the animals were anaesthetized through 65 mg/kg bw (ip) of sodium pentobarbital [8,38].Blood was collected from the renal arteries and analysed for liver and kidney biomarkers such as bilirubin, alanine aminotransferase, aspartate aminotransferase, creatinine, urea, and blood urea nitrogen.Te liver, pancreas, and kidneys were harvested, rinsed in distilled water, and then dried before being preserved either by storing at −80 °C (liver) or in 10% neutral bufered formalin (pancreas).

Analysis of Hepatic Carbohydrate Metabolic Enzymes.
Te activity of G6P and FBP was determined as described by [40] with minor modifcations.A mixture of citrate bufer (0.1 M, pH 6.6, 3 mL) and 1 mL of glucose 6 phosphate disodium salt (200 mM) were preheated at 37 °C for 5 min.Te liver homogenate was then added (0.1 mL) and further incubated at 37 °C for 5 min.Te resulting inorganic phosphate was developed using Taussky-Shor colour reagent and read at 600 nm using a Termo Scientifc GENESYS 30 (model no: 9A1X358128) UV/Vis spectrophotometer.In the analysis of FBP, a reaction mixture was prepared which contained 2.74 mL of glycine bufer (100 mM, pH 9.5), a solution of fructose 1,6-bisiphosphate solution (0.10 mL, 41 mM), manganese chloride solution (0.02 mL, 51 mM), phosphoglucose isomerase enzyme solution (catalogue number: P9455; 0.02 mL), G6PD enzyme solution (catalogue number: G7787-1KU, 0.01 mL), NADP + (0.10 mL, 12 mM), and liver homogenate solution (0.2 mL).Te mixture was incubated for 5 min and then absorbance readings were read at 340 nm.Te analysis of PK was done as described by Storey and Bailey [41] with modifcations.A reaction mixture of 3.0 mL containing 5 mM phosphoenol pyruvate, 5 mM ADP, 0.2 mM NADH, 10 mM MgCl 2 , 100 mM KCl, and 14 U of lactate dehydrogenase (catalogue number: 427217-M).Te reaction was initiated following the addition of 50 μL of a 5-fold diluted liver homogenate.Absorbance readings were measured at 340 nm using a UV/Vis spectrophotometer at 10 s intervals for 5 min.
G6PD was measured as described by Noltmann, Gubler, and Kuby [42] with modifcations.A stock solution containing a fnal concentration of 20 mM Tris-HCl (pH 7.5), 2 mM glucose-6-phosphate, 0.67 mM NADP + , and 10 mM MgCl 2 was prepared.To this reaction mixture, a 5-fold diluted liver homogenate was added (0.1 mL), and OD readings at 340 nm were monitored at 10 s intervals for 5 min to measure the formation of NADPH.

Lipid Peroxidation (Malondialdehdye (MDA)).
Te method was adapted from [43] with minor modifcations.MDA was extracted from liver homogenate using a solution of ferric chloride (100 mM) and ascorbic acid (100 mM).Te MDA was then reacted with 0.67% thiobarbituric acid (500 μL) and subsequently centrifuged.Te supernatant was analysed using a spectrophotometer at 535 nm against a suitable reagent blank.

Catalase (CAT).
Te CAT activity was determined based on the protocol described by [43].A reaction mixture contained 1.25 mL of 50 mM phosphate bufer (pH 5), 200 μL of 5.8 mM H 2 O 2 , and 70 μL of the homogenate was prepared.Te change in the absorbance at 240 nm after 1 min was noted.An absorbance change of 0.001 units/min represents 1 U of catalase activity.

Superoxide Dismutase (SOD).
Te activity of superoxide dismutase (SOD) was determined using pyrogallol autooxidation protocol.Te reaction mixture (2 mL) consisted of 1 mL of 100 mM sodium phosphate bufer (pH 8.2), 3.3 mM EDTA (62 μL), 60 μL of tissue homogenate diluted 20-fold and distilled water.Te reaction was initiated by the addition of 60 μL of freshly prepared pyrogallol (8.1 mM).Te change in the absorbance at 420 nm was measured at 30 s intervals for 3 min.Te percentage inhibition of autoxidation was calculated, and 1 U of SOD activity is amount of enzyme that is needed to inhibit the autoxidation of pyrogallol by a half [44].

Analysis of Serum Samples. Assay kits purchased from
Cayman Chemical Company or Crystal Chem Inc. were used to determine the levels of total bilirubin, creatine, urea, Advances in Pharmacological and Pharmaceutical Sciences aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein (HDL-C) within rats' sera.Blood samples were retrieved from the renal arteries, stored in vacutainers, and allowed to clot at 25 °C for 30 min.Te samples were then centrifuged (Labconco Refrigerated Centrifuge) at 2000 × g at 4 °C and the serum was collected.All serum analyses were done in triplicate.
2.8.1.Total Bilirubin Levels.Total bilirubin levels were assayed using a total and direct Bilirubin kit purchased from Cayman Chemical Company (catalogue no.701720) with a lower limit of detection (LLOD) and lower limit of quantifcation (LLOQ) of 0.045 mg/dL and 0.25 mg/dL, respectively.Briefy, bilirubin assay catalyst (100 μL) was added to wells of a 96 well microplate, to which samples/standard were added (50 μL) and left to incubate at room temperature for 10 min in the dark.Signal and background reagents were prepared and added to respective wells followed by gentle pipetting.Te samples were incubated for a further 15 min, followed by the addition of a total bilirubin probe (75 μL) and then read using a microplate reader (Agilent BioTek) at 600 nm.

Serum Creatinine Levels.
Serum creatinine levels were determined using a Rat Creatinine Assay Kit purchased from Crystal Chem Inc. (catalogue number: 80340, sensitivity: 0.15 mg/dL) based on the manufacturer's instructions.Samples/standards (8 μL) were added to microplate wells to which 270 μL of sarcosine oxidase solution were added.Te mixture was incubated at 37 °C for 5 min and read at 550 nm.Tis was followed by the addition of 90 μL of peroxidase solution and further incubation at 37 °C for 5 min, and subsequently rereading at 550 nm.
2.8.3.Serum Urea.Rats' serum urea was determined using the assay kit purchased from Cayman Chemical Company (catalogue number: 700620) which has a precision between 1.7% and 2.1% and a LLOD of 0.05 mM.A series of urea standards was prepared and 20 μL of each was added to individual wells in a microplate, to which 150 μL of diluted bufer were added.Te procedure was repeated with serum samples.Te reaction was initiated by the addition of 20 μL of urease and then the mixtures were incubated for 10 min at room temperature.Subsequently, ammonia detector reagent (10 μL) was added to each well and the microplate incubated for 15 min at room temperature.Finally, the fuorescence was read at excitation wavelength of 405 nm and an emission wavelength of 480 nm.Blood urea nitrogen (BUN) was calculated by dividing the serum urea by a factor of 2.14.

Serum Aspartate Aminotransferase (AST) and Alanine
Aminotransferase Kits (ALT).Sample AST levels were measured using an assay kit purchased from Cayman Chemicals Company (catalogue number: 701640; LLOQ: 0.01 U/mL and precision: 4.1%).Briefy, sample wells were prepared by the addition of 150 μL of AST substrate, 20 μL of AST cofactor, and 20 μL of samples.Standard was prepared in a similar fashion where the sample was substituted with 20 μL of standard.Te mixtures were incubated at 37 °C for 15 min, followed by the addition of the AST initiator.Samples and standards were immediately read at 340 nm once per min for 10 min.
ALT levels were determined in a similar manner using ALT substrate, ALT cofactor, and ALT initiator as described by the assay kit purchased from Cayman Chemical Company (catalogue number: 700260; LLOD: 0.006 U/mL and precision: 5.8%).

Serum HDL-C, LDL-C, Total Cholesterol (TC), and
Triacylglycerol (TAG).HDL-C (catalogue number: 79970; sensitivity: 1.1 mg/dL) and LDL-C (catalogue number: 79960; sensitivity: 4.5 mg/dL) assay kits were purchased from Crystal Chem Inc. and used to determine the levels of HDL-C and LDL-C, respectively.In the assays, serum samples (3 μL) were mixed with 225 μL of polyvinyl sulfonic acid (PVS)/polyethylene-glycol methyl ether (PGME) and left to incubate at 37 °C for 5 min.Subsequently, 75 μL of enzymatic solution was added and the mixtures incubated at 37 °C for 5 min then read at 600 nm against a series of HDL-C standard.In the LDL-C assay, serum samples (3 μL) were mixed with PVS/PGME and incubated at 37 °C for 5 min.After which, 75 μL of decomplexing reagent was added and the mixtures incubated at 37 °C for 5 min then read at 600 nm against a series of LDL-C standard.
2.8.6.Total Cholesterol.Te total cholesterol in rats' sera was estimated using Zak's method [45].Serum (0.1 mL) was added to 4 mL of FeCl 3 precipitating reagent.Te mixture was shaken on an automated shaker for approximately 3 min, after which, it was fltered using Whatman number 41 flter paper.To 3 mL of the fltrate, 2 mL of concentrated sulfuric acid was added.Te solution was mixed and allowed to stand at room temperature for 10 min.Te resultant red-violent solution was read at 560 nm using a UV/Vis spectrophotometer against a suitable reagent blank.A series of cholesterol standards was also prepared and read at 560 nm.Te data was used to obtain a standard curve which was used to estimate the TC in the unknowns.TAG was calculated by rearranging Friedewald's formula: LDL-C � (TC) − (HDL-C) − (TG/5) [46].
2.9.Histopathological Analysis of Pancreas.Pancreatic tissues were harvested, washed in cold saline, and then weighed.Tese were then fxed in 10% neutral bufered formalin and were subsequently dehydrated using a series of ethanol treatment.Te tissues were subsequently embedded in parafn wax and sectioned into 10 μm thick segments using a microtone.Te tissues were stained using haematoxylin and eosin and analysed using a Nikon Eclipse E 200 light microscope at ×40 objective lens.Te number of islets of Langerhans in ten random microscope felds were counted and documented while the diameters of the islets were estimated using ImageJ software bundled with 64 bit Java 1.8.0_172.

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Advances in Pharmacological and Pharmaceutical Sciences 2.10.Statistical Analysis.Data were expressed as mean-± standard error of the mean and analysed using IBM SPSS Statistics for Windows, version 27 (IBM Corp., Armonk, NY, USA).Comparisons among the groups were carried out using one-way analysis of variance (ANOVA), followed by the Tukey post hoc test, and p ≤ 0.05 was considered to be statistically signifcant.Additionally, the magnitude of the signifcant diferences obtained between the D-OA + SA and the DC group was further calculated using Cohen's d, with d ≥ 0.2 ≤ 0.499 considered a "small efect," d ≥ 0.5 ≤ 0.799 considered a "medium efect," and d ≥ 0.8 considered a "large efect."Tough there was no signifcant diference between the initial and the fnal data readings for these animals, there is a noticeable trend for increase in glycaemic state, indicating the progressive nature of T2D.Treatment with the supplement, however, had no efect on the normal rats, which may indicate that there is a threshold of BGL for the supplement to provoke a hypoglycaemic response.

Hepatic Enzymes in Carbohydrate Metabolism and Serum
Lipid Profle in Diabetic and Nondiabetic S-D Rats.As refected in Figure 1, untreated diabetic animals reported a marked increase in the activity of G6P, refecting an increased rate of gluconeogenesis.Tere was also a marked decrease in the activity of PK (0.45 ± 0.15 U/mg protein; p < 0.001) and G6PD (0.42 ± 0.02 U/mg protein; p � 0.043) when compared with the normal control S-D rats (2.11 ± 0.25 U/mg protein and 0.57 ± 0.02 U/mg protein, respectively).On the other hand, the mixture of the two carboxylic acids signifcantly retarded the activity of the gluconeogenetic enzyme, G6P to levels comparable to those seen in the normoglycaemic rats (1.31 vs 1.51 U/mg protein, p � 0.341).Tis was also signifcantly lower than the DC group with a large size efect as reported by Cohen's d of 1.89.Furthermore, the efcacy of the supplement resulted in a large signifcant increase in the activity of enzymes involved in the catabolic reaction of glucose (G6PD: p < 0.05, d � 1.73 and PK: p < 0.05, d � 1.02).However, there was no statistically signifcant efect reported in the normal animals that were treated with the cocktail of organic acids.Te 28 d treatment with glibenclamide demonstrated similar results to that of the animals treated with the naturally occurring organic acids.
Over the period of the study, there were no diferences among the groups for total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C, Figure 2).However, there was a signifcant increase in the serum triacylglycerol (TAG) for the diabetic control group when compared with the NC group (262.84 ± 9.41 mg/dL vs 211.97 ± 5.67 mg/dL, p < 0.05).Notable, the supplement of OA and SA reduced the serum TAG (206.97 ± 8.35 mg/dL, p < 0.05 and d � 0.68) when compared with the DC group and no signifcant diference when compared with the NC group.

Toxicological Efects on Renal and Hepatic Function.
Te analysis of serum biomarkers for liver and kidney function serves to highlight pathological changes in these key regulatory organs due to diabetes and due to the administration of the nutritional cocktail.Statistical analysis established that there were no signifcant alterations in the biomarkers assayed.

Oxidative Stress in Hepatic Tissues.
Oxidative stress augments many of the complications associated with diabetes mellitus.An increased level of hepatic oxidative stress is linked to a myriad of complications observed in diabetics.Te study highlighted this through an increased levels of reactive oxygen species within the hepatic tissues with a decrease in endogenous antioxidant enzymes such as SOD shown in Figure 3(a) (7.91 ± 1.40 vs 2.68 ± 0.80 U/mg protein; p < 0.001) and CAT in Figure 3(b) (93.33 ± 6.21 vs. 39.82 ± 8.41 U/mg protein; p < 0.001) when compared with the NC group.Similarly, the presence of high levels of MDA in the hepatic tissues of the DC group further supported the increased level of ROS present within the tissues.Treatment with the supplement reversed this with a large signifcant improvement of CAT (104.54 ± 12.84 U/mg protein, p � 0.028, d � 1.39) and SOD (9.14 ± 1.01, p � 0.001, d � 1.01) activities, ultimately preventing the oxidative degradation of lipids, and the formation of MDA (0.14 ± 0.012, p < 0.001, d � 1.08).A similar result was observed with the glibenclamide-treated group, exhibiting no signifcant diferences when compared with the NC group (Figures 3(a)-3(c)). 2 depicts a signifcant reduction in the number of islets of Langerhans in all diabetic groups.Tis was achieved through the destruction of the beta cells of the pancreas by the STZ administered.Te DC group showed the lowest number of islets with 1.00 ± 0.26 vs 3.88 ± 0.48 for the NC group.Tough the supplement failed to increase the number of islets, it promoted a signifcant restoration of the size of the islets (312.53 ± 23.60 vs. 383.61± 35.53 μm, respectively, p � 0.142) when compared with the NC group.Te supplement also showed a large signifcant increase in the size of the islets of Langerhans when compared with the DC group (p � 0.048, d = 1.07).On the other hand, glibenclamide failed to restore the size of the islets for its respective diabetic group.

Alterations in the Structure, Number, and Size of the Islets of Langerhans. Table
Histopathological analysis of the pancreatic tissues highlighted normal architecture of the pancreatic acinar cells and islets of Langerhans in the NC (A) and NT (B) groups.However, signifcant atrophy of the islets of Langerhans of the DC (C) and DGLIB (D) groups was observed (Table 2).Additionally, necrosis was evident in all the diabetic groups (C, D, and E) with more extensive necrosis being observed in the DC group with more eosinophilic cells, alluding to destruction of the nuclei of these cells.Treatment with the nutritional supplement resulted in less necrotic damage being observed in the DT group (E).
Te normal architecture of the acinar cells (denoted by the letter A) and the islets (denoted by the letter I) is shown in the NC and NT groups.However, the diabetic untreated group (DC) showed changes in the morphology of the islets with a reduction of stained nuclei along with necrosis (represented by arrows), and with atrophy of the endocrine cells.Te DGLIB animals also had distorted and atrophic endocrine cells which is consistent with diabetes, while in the DT group, there was only mild necrosis observed in the endocrine portion of the pancreatic tissues.

Discussion
End stage diabetes afects the liver adversely and reduces its role in maintaining homeostasis in carbohydrate metabolism.Tis paper sought to investigate the efect that a nutritional supplement of OA and SA has on liver function in type 2 diabetic rats.As previously documented, supplement possessed antidiabetic efect and showed  Te pharmacological efect being reported stemmed from the improvement in insulin sensitivity as described in our previous paper [8].In addition to improve absorption of glucose into peripheral tissues, improved responses to insulin have been reported to suppress the secretion of glucagon from the α-cells of the pancreas and ultimately reduced hepatic gluconeogenesis.An examination of some of the hepatic enzymes involved in carbohydrate metabolism suggested an overall elevation in the activity of glucose-6-phosphatase (G6P) in the diabetic control group.G6P plays a pivotal role in the synthesis of glucose in the gluconeogenesis and any increase in this enzyme, typically translates to an increase in the rate of gluconeogenesis [21,22].Several studies have documented that an increased hepatic glucose output contributes to the increased hyperglycaemic state and the current fndings supported this.An elevation of the rate of gluconeogenesis was observed through an increase in the activity of G6P (2.08 ± 0.01 vs. 1.51 ± 0.03 U/mg protein, p < 0.05 when compared with the NC group).Te increase in gluconeogenesis partially explains the 20.10 ± 13.57% increase in BGL as documented in Table 1.In comparison to this, the nutritional supplement signifcantly normalized the levels of the gluconeogenetic enzyme.Tis may have been achieved through the inhibition of expression of the enzyme at the transcription level.Free fatty acids have been documented to inhibit the expression of peroxisome-proliferator-activated receptor-c coactivator 1α (PPARc-1 α), a transcription cofactor necessary for the expression of gluconeogenetic enzymes [47].Te nutritional supplement consists of OA, resulting in an increase in the circulating free fatty acids, thus reducing the expression of PPARc-1 α.Moreover, the supplement increased the expression of G6PD, a regulatory enzyme in the pentose phosphate pathway.Te literature reports that hyperglycaemia reduces the activity of the enzyme through phosphorylation and thus inactivation of the enzyme [48,49].Te exacerbated state of glycaemia reported in the DC animals, therefore, accounted for the low activity of G6PD (0.42 ± 0.02 U/mg protein) observed.Successful reduction in the rate of gluconeogenesis is a hallmark for ameliorating the hyperglycaemic state of T2D, indicating the antidiabetic nature of the supplement.However, there were no diferences in the enzymes assayed in the NT group when compared with the NC animals, further suggesting the specifc nature of the supplement.Tat is, the supplement induces an antihyperglycaemic nature when BGLs are high.
Te nutritional supplement also improved oxidative stress within the hepatic tissues.Te correlation between diabetes and oxidative stress is well documented, where the elevation of reactive oxygen species (ROS) is responsible for several of the complications associated with the metabolic disease.Tese include insulin resistance, a condition that is well documented to be consistent with T2D, as well as liver Advances in Pharmacological and Pharmaceutical Sciences diseases and associated coronary heart diseases [50,51].As previously reported, the supplement resulted in an increase in the activity of G6PDH, hence an increase in the production of NADPH.Te coenzyme played a central role in the promotion and regeneration of reduced glutathione, a notorious antioxidant molecule.Te reducing power from NADPH may indirectly infuence the restoration of the activity of CAT and SOD documented within the diabetic groups treated with the supplement and the positive control, glibenclamide.Moreover, the levels of malondialdehyde, a marker for lipid peroxidation were normalized in both the aforementioned groups when compared with the diabetic  Other possible mechanisms include a reduction in the production of ROS due to the hypoglycaemic property of the supplement.Studies have confrmed a positive correlation between the BGL and the levels of ROS; thus, any reduction in BGL may followed by a reduction in ROS levels [27,52].Overall, a reduction in oxidative stress within the liver is advantageous in maintaining the integrity of the hepatocytes and the role they play in maintaining glycaemic homeostasis and detoxifcation.Moreover, there was no reported hepatic or renal toxicity due to the supplement.Te evidence of this lies within the biomarkers that were monitored, where there were no diferences among the groups.Generally, xenobiotics enter the liver through the hepatic portal vein and eventually to hepatocytes, where they may exert toxic efects [53,54].Tis often leads to an elevation of serum total bilirubin, alanine amino transferase, aspartate amino transferase, among others.Te study underscored that the supplement had no efect on these hepatic biomarkers (Table 3), indicating that there was no extensive damage done to the liver tissues.Similarly, many xenobiotics cause damage to renal cells and therefore afect the glomerular fltration rate.Consequently, there may be an elevation in metabolites that are fltered by the kidneys.In this study, we examined the serum levels of creatinine, urea, and blood urea nitrogen, none of which displayed an elevation due to the cocktail of organic acids.Tese tests confrm that the supplement had low toxicity and may therefore form a novel therapeutic approach to T2D.
Further to the efect of the nutritional supplement on liver function, the impact on serum lipid profle and the integrity of pancreatic tissues were highlighted.Tere was a signifcant increase in serum triglycerides in the diabetic untreated group, which was normalized in the supplementtreated group.Tis was possible through regulating the improved liver functions including the metabolism of lipids.
A mitigation of glycaemia aids in restoring homeostasis, thus accounting for the reduction of triglyceride.Additionally, OA has been documented to increase metabolism of fats and, therefore, reduce the levels of TAGs [55].
Te nutritional supplement also showed efcacy in protecting the endocrine portion of the pancreatic tissues from degeneration (Figures 4(a)-4(e)).Te DC animals had a marked increase in the destruction of the islets, especially cells towards the middle of the islets, where the beta-cells of the pancreas are found (Figure 4(c)).Tere was a noticeable increase in cytoplasm vacuolation, eosinophilia, and pyknosis of the nuclei, all of which indicate necrosis of the pancreatic tissue (Figure 4(c)).Tis is possibly as a result of the pharmacodynamics of STZ or oxidative stress.With the level of damages done within the DC groups when compared with the other diabetic groups, it is very likely that much of the damages done were due to oxidative stress.Te pancreatic β-cells are highly susceptible to ROS due to the limited antioxidants present [56].Consequently, an elevation in ROS, as was seen in the DC group, may have dire consequences on the pancreatic cells.Te supplement, however, prevented this through a reduction in the damage done to the β-cells (Figure 4(d)).Tis resulted in a general increase in size of the islets of Langerhans when compared with all the other diabetic groups, with no signifcant diference when compared with the normal groups (Table 2).Te pancreatic protective role coupled with the reduction of hepatic production of glucose nature due to the supplement was able to increase the secretion of insulin, thus assisting with the reduction of the blood glucose concentrations.
Te study, therefore, added further evidence of the previously reported antidiabetic nature of the nutritional supplement.OA and SA synergistically reduced the rate of depletion of the function of the liver and pancreas, both of which are essential for maintaining efcient glucose metabolism.In the DC animals, the dysfunction of these organs contributed to the aberrant glucose levels in the blood, which provoked diabetic symptoms.However, the nutritional supplement ameliorated the condition through a protection or reducing the rate of damage done to these vital organs.Consequently, resulting in an improved metabolism of glucose, and thus resulted in an alleviation of diabetic conditions of the animals.All data are expressed as mean ± standard error of the mean and were analysed by Tukey post hoc test (n � 6).NC: normal control, NT: normal treatment, DC: diabetic control, DGLIB: diabetic glibenclamide, DT: diabetic treatment group.Te superscript 'a' indicates a signifcant diference when compared with the superscript 'b' within the same column, while the superscript 'b' indicates a signifcant diference when compared with the superscript 'a' in the same column.
Advances in Pharmacological and Pharmaceutical Sciences

Conclusion
Te nutritional supplement of OA and SA served as an antidiabetic agent which aided in maintaining the glycaemic regulatory role of the liver.Te study underscored that the supplement mediated glycaemic control through a restoration of carbohydrate metabolic enzymes and protected against ROS destruction.Te role of the liver in homeostatic control has been well documented and the study established that the supplement ofered a protective role against damage in diabetic rats.Furthermore, the supplement also preserved the architecture of the pancreatic tissues and reduced STZ-related necrosis in the islets.Tis allowed for a successful maintenance in the role of the pancreas and therefore, resulted in an antihyperglycaemic efect of the supplement.Values were expressed as mean ± standard error of the mean (n � 6).AST: aspartate aminotransferase, ALT: alanine aminotransferase, NC: normal control, NT: normal treatment, DC: diabetic control, DGLIB: diabetic glibenclamide, DT: diabetic treatment group, where the superscript, " a " indicates there were no signifcant diferences among the groups studied.

Figure 1 :
Figure 1: Te efect of treatment on the activity of hepatic carbohydrate metabolizing enzymes.Data are expressed as mean ± standard error of the mean and were analysed by Tukey post hoc test (n � 6).G6P: glucose 6 phosphatase, G6PD: glucose-6-phosphate dehydrogenase, FBP: fructose-1,6-bisphosphatase, PK: pyruvate kinase, NC: normal control, NT: normal treatment, DC: diabetic control, DGLIB: diabetic glibenclamide, DT: diabetic treatment group, where the letters above the columns represent the following: a: statistically signifcant diferences when compared with the values of the same enzyme superscripts b and c. b: statistically signifcant diferences when compared with the values of the same enzyme superscripts a and b. c: statistically signifcant diferences when compared with the values of the same enzyme superscripts b and c. ab: No statistically signifcant diference when compared with the values indicated by superscripts a or b for the same enzyme.

Figure 2 :
Figure 2: Serum lipids in diabetic and nondiabetic rats fed the nutritional supplements.Data are expressed as mean ± standard error of the mean and were analysed by Tukey post hoc test (n � 6).TC: total cholesterol, HDL-C: high-density lipoprotein cholesterol, LDL-C: lowdensity lipoprotein cholesterol, TAG: triacylglycerol, NC: normal control, NT: normal treatment, DC: diabetic control, DGLIB: diabetic glibenclamide, DT: diabetic treatment group, where the letters above the columns represent the following: a: statistically signifcant diferences when compared with the values of the same parameter with superscript b. b: statistically signifcant diferences when compared with the values of the same parameter with superscript a.

Figure 3 :
Figure 3: (a) Te efect of the various treatments on the activity of superoxide dismutase.(b) Te efect of the treatment options on catalase activity in diabetic and normal S-D rats.(c) Te efect of the various treatments on the concentration of hepatic MDA in normal and diabetic Sprague-Dawley rats.All data are expressed as mean ± standard error of the mean and were analysed by Tukey post hoc test (n � 6).NC: normal control, NT: normal treatment, DC: diabetic control, DGLIB: diabetic glibenclamide, DT: diabetic treatment group, where the letters above the columns represent the following: A: statistically signifcant diferences when compared with the bars with letter B. B: statistically signifcant diferences when compared with the bars with letter A.
Nutritional Supplement on BGL in Diabetic and Nondiabetic S-D Rats.Te nutritional supplement of OA and SA synergistically improves glycaemic control in diabetic rats as noted in Table1.Treatment with the supplement resulted in a decrease in BGL from 22.92 to 14.48 mM, accounting for a 35.69 ± 4.22% reduction (p � 0.006) in blood glucose concentrations.Similar results were observed with the positive control, where glibenclamide resulted in a 39.88 ± 7.55% reduction in BGL of the diabetic animals (p � 0.004).Tere was a noticeable 20.10 ± 13.57% increase in the diabetic untreated animal.

Table 1 :
Change in BGL of diabetic and nondiabetic rats over the period of the study.Data are expressed as mean ± standard error of the mean.BGL: blood glucose levels, NC: normal control, NT: normal treatment, DC: diabetic control, DT: diabetic treatment, DGLIB: diabetic glibenclamide group.* p ≤ 0.05 when compared with the DC group.
6Advances in Pharmacological and Pharmaceutical Sciences a 35.69 ± 4.22% reduction in BGL.

Table 2 :
Te number of islets/10 microscope feld and diameter of pancreatic islets of diabetic and nondiabetic animals after 28 d of treatment with their respective regime.