|Year : 2021 | Volume
| Issue : 2 | Page : 95-102
Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus)
Evelyn Hassan-Danboyi1, Abdulazeez Jimoh1, Abdulwahab Alhassan1, Timothy Danboyi2, Kabir Ahmed Mohammed2, Augustine Banlibo Dubo1, Jamilu Haruna3, Bulus Billy Yakubu3
1 Department of Human Physiology, College of Medical Sciences, Ahmadu Bello University Zaria, Kaduna, Nigeria
2 Department of Human Physiology, College of Medicine, Kaduna State University, Kaduna, Nigeria
3 Department of Human Anatomy, College of Medicine, Kaduna State University, Kaduna, Nigeria
|Date of Submission||27-Feb-2021|
|Date of Decision||11-May-2021|
|Date of Acceptance||13-May-2021|
|Date of Web Publication||10-Aug-2021|
Dr. Timothy Danboyi
Department of Human Physiology, Kaduna State University, Kaduna
Source of Support: None, Conflict of Interest: None
Background: Oxidative stress is one of the major mechanisms underlying the onset and development of type-2 diabetes mellitus (T2DM). Although L-citrulline possesses antioxidant effect, little or no data exist linking such effects in diabetic setting. Objective: This study aimed to evaluate the effect of L-citrulline on biomarkers of oxidative stress in diabetic Wistar rats. Materials and Methods: Thirty male Wistar rats 10–12 weeks old and weighing 200–250 g were randomly assigned into six groups of five rats each. Group I rats were fed normal diet, while diabetes was induced in the other groups with high-fat diet (HFD) and dexamethasone intraperitoneally (1 mg/kg) for 21 days. Thereafter, Group III received metformin 100 mg/kg/day orally, and Groups IV, V, and VI received 200, 400, and 800 mg/kg/day L-citrulline, respectively, for another 21 days. Data were analyzed using SPSS and values at P < 0.05 were considered statistically significant. Results: The malondialdehyde concentrations were significantly reversed from 42.0 ± 0.42 μmol/mL in the diabetic group to 20.7 ± 0.81, 22.2 ± 0.75 and 22.1 ± 0.39 μmol/mL at 200, 400, and 800 mg/kg/day, respectively. The L-citrulline remarkably ameliorated the reduction in superoxide dismutase activity noted in the diabetic group (13.0 ± 0.44 μmol/mL) at all doses (17.8 ± 0.37, 16.0 ± 0.51, and 23.7 ± 0.78 μmol/mL at 200, 400, and 800 mg/kg, respectively). Similarly, there was a corresponding significant increase in the catalase activity, especially at 400 mg/kg (13.7 ± 0.43 ng/mL) and 800 mg/kg (14.6 ± 0.54 ng/mL) compared to the diabetic group (10.8 ± 0.41 ng/mL). The marked reduction in reduced glutathione level observed in the diabetic group (22.9 ± 0.69 mg/mL) was markedly ameliorated by L-citrulline supplementation at all doses (42.9 ± 1.08, 46.4 ± 0.53, and 45.2 ± 1.00 mg/mL at 200, 400, and 800 mg/kg, respectively). Conclusion: This study shows that L-citrulline supplementation has antioxidant effects in HFD- and dexamethasone-induced T2DM in male Wistar rats.
Keywords: Antioxidant, L-citrulline, oxidative stress, type-2 diabetes mellitus
|How to cite this article:|
Hassan-Danboyi E, Jimoh A, Alhassan A, Danboyi T, Mohammed KA, Dubo AB, Haruna J, Yakubu BB. Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus). Niger J Exp Clin Biosci 2021;9:95-102
|How to cite this URL:|
Hassan-Danboyi E, Jimoh A, Alhassan A, Danboyi T, Mohammed KA, Dubo AB, Haruna J, Yakubu BB. Antioxidant effects of L-citrulline supplementation in high-fat diet- and dexamethasone-induced Type-2 diabetes mellitus in wistar rats (Rattus norvegicus). Niger J Exp Clin Biosci [serial online] 2021 [cited 2022 Jan 23];9:95-102. Available from: https://www.njecbonline.org/text.asp?2021/9/2/95/323670
| Introduction|| |
Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia (high blood glucose level) resulting from defects in insulin secretion, insulin action, or both. About 422 million people had DM as of 2014. DM had an estimated global prevalence of about 8.8% in 2015 with a projected rise of about 10.4% by 2040 (affecting an estimated 642 million people). In sub-Saharan Africa, about 20 million people (projected to be 41.4 million by 2035) have DM, with Nigeria having the highest number (5 million) of people with DM. DM is divided into two major types: type-1 (insulin-dependent and associated with absolute insulin deficiency) and type-2 (90%–95% of DM, due to insulin resistance).
High-fat diets (HFDs) had been shown to increase serum glucose levels as well as insulin resistance in mice and rats, thereby contributing to the onset and progression of type-2 DM (T2DM). It is associated with increase in determine malondialdehyde (MDA) concentration and decrease in the activities of antioxidant enzymes and reduced glutathione (GSH) level in some tissues. HFD is a model of T2DM, and it is an established “side effect” of glucocorticoid therapy. Exogenous glucocorticoids such as dexamethasone cause insulin resistance and hyperglycemia, which may lead to the development of T2DM.,, Dexamethasone causes insulin resistance via direct impairment of beta-cell functions, decreasing key mediators of insulin action in peripheral tissues and anti-insulin effects on the liver, skeletal muscle, and adipose tissue.,, In our previous study, we demonstrated that combining both HFD and dexamethasone induces diabetes in albino Wistar rats.
The conventional antidiabetic drugs have been unable to halt the progression of T2DM, and coupled with their high cost as well as their associated adverse effects, the management has been a great challenge to both clinicians and patients or their relatives. In addition, most of these drugs may cause serious side effects such as severe hypoglycemia, and adverse drug–drug interactions. Hence, it is only logical to seek newer and (more) effective antidiabetic agents or supplements, with little or no side effects that can gain universal acceptance.
Oxidative stress is an imbalance between the production and clearance of free radicals (reactive oxygen and/or nitrogen species) with a potential damage to nucleic and cellular components. Oxidative stress occurs when there is an imbalance between the body's pro-oxidant system and antioxidant defense system in favor of the former, leading to the generation of excessive amount of free radicals that attack and damage biological membranes causing lipid peroxidation, protein glycation, and nucleic acid fragmentation. It leads to insulin resistance, beta-cell dysfunction, and impaired glucose tolerance and plays a significant role in the development and progression of T2DM together with its attendant complications.,,
L-citrulline derives its name from Citrullus lanatus as one of the major components of watermelon. L-citrulline is a nonessential amino acid mainly produced by the liver. It prevents the excessive and uncontrolled production of nitric oxide (NO)., It also augments the functions of NO, which include maintenance of normal endothelial function, and increase in peripheral and hepatic insulin sensitivity,, which are disrupted in the setting of T2DM., L-citrulline may be a potential supplement in the treatment of T2DM and prevention of complications associated with it.,,,
L-citrulline bypasses hepatic and intestinal metabolism,, and enhances plasma L-citrulline concentration, hence a better substrate than arginine in restoring NO production. It has no side effects even at high doses when taken orally.,, It is affordable and readily available, especially in its natural source (C. lanatus). It possesses an antioxidant activity as it significantly decreases reactive oxygen species (ROS) production. However, only very few studies exist on its effects on biomarkers of oxidative stress, especially in the setting of T2DM. This study aimed to investigate the effect of L-citrulline on biomarkers of oxidative stress in the setting of T2DM in male Wistar rats. We, therefore, hypothesized that L-citrulline has no significant effect on some biomarkers of oxidative stress in T2DM.
| Materials and Methods|| |
This study was carried out according to the guidelines for the care and use of laboratory animals provided and approved by the Ahmadu Bello University (ABU) Committee on Animal Use and Care (approval number: ABUCAUC/2020/72).
Materials used were 1 ml, 2 ml, and 5 ml disposable syringes; distilled water, Accu-Chek advantage glucometer and strips (Roche Diagnostics, Germany); electronic weighing balance, surgical blades, cotton wool, methylated spirit, ketamine, plain tubes, ice packs, masking tape, and marker. Others include commercial grower feeds (Chikun Feeds Company, Nigeria), distilled water, dissecting board, latex gloves, bench centrifuge, and feeding bowls. Dexamethasone injection and L-citrulline oral supplement (MedChemXpress, USA) were of analytical grade.
Experimental animals and housing conditions
A total of 30 male albino Wistar rats (Rattus norvegicus) 8–12 weeks old and weighing 200–250 g were used due to their genetic, biological, and behavioral similarities with humans and are the most commonly used strains in metabolic and other research. They were sourced from the animal house, Department of Human Physiology, ABU, Zaria. They were housed in plastic cages with commercial bedding materials which were changed every other day and allowed free access to commercial grower mash feed and water under standard laboratory conditions (temperature- and humidity controlled with 12-h light-dark cycle and well ventilated). There were 5–10 rats per cage depending on the grouping. They were all drug-and test-naïve and certified healthy.
Preparation of high-fat diet
This study was part of a multistage research using the same experimental design. HFD was prepared by mixing normal feed (crude fats: 18%, crude proteins: 47%, carbohydrates: 28%, crude fiber: 5%, methionine: 0.5%, lysine: 1.1%, and phosphorus: 0.4%) with margarine (99.9% fats) in the ratio of 10:1, i.e., 10 g of normal feed to 1 g margarine with some modifications.
Drug formulation, dosage, and route of administration
The L-citrulline was constituted with distilled water at 100 mg citrulline/ml distilled water. It was administered orally because of its water solubility, ready absorbability, and by-pass of the first-pass effect following oral ingestion. The dexamethasone was constituted with distilled water at 2 mg/ml. It was administered intraperitoneally (i.p.) at a dose of 1 mg/kg. The rats received the L-citrulline and dexamethasone in the mornings after their breakfast at a well-ventilated area within the laboratory, where they were kept for about 1–2 h after the administration before returning them to their cubicles. The remaining formulated drugs after administration were returned to the refrigerator maintained at 2°C–5°C.
Induction of type-2 diabetes mellitus
The rats were grouped into six groups (I–VI), but those in Groups II–IV were fed with HFD for 21 days in addition to dexamethasone 1 mg/kg body weight i.p. daily starting from day 15 to 21 of the experiment according to a previously described method with some modifications.
Determination of fasting blood glucose levels
Fasting blood glucose (FBG) levels were measured (after overnight fasting for 8 h) using the glucometer and strips and expressed in mg/dL prior to induction of diabetes. It is according to the glucose oxidase principle. The measurement of the FBG levels took place on day 0 (after induction of diabetes) and day 22. A drop of blood obtained from slightly piercing the tail veins was placed on the glucometer strip. Blood glucose levels >200 mg/dL were considered diabetic.,
Experimental animal grouping
The study involved 30 male Wistar rats randomly assigned into six groups of five rats each.
- Group I (n = 5): received normal diet for 21 days. This group serves as normal control
- Group II (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15). This group serves as diabetic group
- Group III (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral metformin (a biguanide) 100 mg/kg daily for 21 days
- Group IV (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 200 mg/kg body weight daily for 21 days
- Group V (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 400 mg/kg body weight daily for 21 days
- Group VI (n = 5): were fed HFD daily for 21 days plus i.p. dexamethasone 1 mg/kg body weight daily for 7 days (starting from day 15) to induce diabetes as in Group II above, then treated with oral L-citrulline 800 mg/kg body weight daily for 21 days.
Collection of blood samples
The rats were anesthetized using 75 mg/kg ketamine and 50 mg/kg diazepam i.p. Two milliliter of blood was drawn from each rat via cardiac puncture using a 5 mL syringe. Blood collected was allowed to stand and clot for 30 min and the resulting samples were centrifuged at 3000 g for 10 min. Centrifuged samples were stored in plain tubes kept in ice packs (temperature approximately between −2°C and −20°C) until ready for analysis.
Assay for biomarkers of oxidative stress
The centrifuged blood samples were used to determine MDA concentrations; activities of superoxide dismutase (SOD) and catalase (CAT); and GSH levels.
Determination of malondialdehyde concentrations
The level of thiobarbituric-acid reactive substance (TBARS), MDA concentrations, as an index of lipid peroxidation was determined. The principle is based on the reaction of MDA with thiobarbituric acid (TBA), forming an MDA-TBA or TBARS adduct that will absorb strongly at 532 nm. It was based on the colorimetric method described by Satoh. Exactly 150 μl of serum was treated with 2 ml of TBA-tricarboxylic acid-hydrochloric acid reagent (1:1:1 ratio) and placed in a water bath at 90°C for 60 min. The mixture was cooled and centrifuged at 3000 rpm for 5 min and the absorbance of the pink supernatant (TBA-MDA complex) was then measured at 535 nm. The MDA formed was then calculated using the molar extinction coefficient of 1.56 × 10−5 cm−1 M−1.
Determination of superoxide dismutase activity
Activities of SOD in the rat serum were determined according to the method described by Misra and Fridovich. This is based on the principle of superoxide inhibition of auto-oxidation of adrenaline at pH 10.2. Serum of 0.1 ml was diluted in 0.9 ml of distilled water to make 1:10 dilution of microsome. An aliquant mixture of 0.2 ml of the diluted microsome was added to 2.5 ml of 0.05 M carbonate buffer. The reaction was started with the addition of 0.3 ml of 0.3 mM adrenaline. The reference mixture contained 2.5 ml of 0.05 M carbonate buffer, 0.3 ml of 0.3 mM adrenaline, and 0.2 ml of distilled water. The absorbance was measured over 30 s up to 150 s at 480 nm.
Determination of catalase activity
CAT activity was measured according to the method described by Aebi. Exactly 100 μl of serum was added to a test tube containing 2.80 ml of 50 mM potassium phosphate buffer (pH 7.0). The reaction was initiated by adding 1 ml of freshly prepared 30 mM H2O2 and the decomposition rate of hydrogen peroxide (H2O2) was measured at 240 nm for 5 min on a spectrophotometer. A molar extinction coefficient (E) of 0.041 mM−1 mm−1 was used to calculate the CAT activity. One unit is the amount of CAT that decomposes 1 μmol of H2O2 per minute at pH 7.0.
Determination of reduced glutathione level
GSH concentration measurement was done according to the method described by Rukkumani et al. The procedure was based on the reaction of 5, 5-dithiobis nitro benzoic acid and GSH. To 150 μl of serum or tissue homogenate (in phosphate-saline buffer pH 7.4), 1.5 ml of 10% TCA was added and centrifuged at 1500 g for 5 min. One milliliter of the supernatant was treated with 0.5 ml of Ellman's reagent and 3 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. The quantity of GSH was obtained from the graph of the GSH standard curve.
Data obtained were analyzed using International Business Machine (IBM) Statistical Package for Social Sciences (SPSS) Statistics for Windows, Version 23.0 (Armonk, NY: IBM Corp., United States of America, 2015), using one-way analysis of variance, followed by Tukey's post hoc test. The format of result presentation was mean ± standard error of the mean. Any value at P < 0.05 is statistically significant.
| Results|| |
Serum malondialdehyde concentrations
There was a significant increase in the MDA concentrations observed in the diabetic group (42.0 ± 0.42 μmol/mL) compared to the normal control (30.7 ± 0.78 μmol/mL). Marked decreases were recorded in the citrulline 200 mg/kg (20.7 ± 0.81 μmol/mL), citrulline 400 mg/kg (22.2 ± 0.75 μmol/mL) and citrulline 800 mg/kg (22.1 ± 0.39 μmol/mL), compared to the diabetic group as well as metformin group (25.6 ± 0.56 μmol/mL). F =160.2; P < 0.0001 [Figure 1].
|Figure 1: Effect of L-citrulline on serum malondialdehyde concentrations of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, and c denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, and metformin group, respectively|
Click here to view
Level of serum superoxide dismutase activity
There was a marked decrease in SOD activity in the diabetic group (13.0 ± 0.44 μmol/mL) compared to the normal control (18.4 ± 0.54 μmol/mL). The activity in the citrulline 400 mg/kg group (16.0 ± 0.51 μmol/mL) was significantly increased compared to the diabetic group and decreased compared to metformin (19.5 ± 0.89 μmol/mL) and citrulline 800 mg/kg groups (23.7 ± 0.78 μmol/mL) (F = 33.7; P < 0.0001) [Figure 2].
|Figure 2: Effect of L-citrulline on level of serum superoxide dismutase activity of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, c, d, and e denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, metformin, CIT 200 mg/kg, and CIT 400 mg/kg groups, respectively|
Click here to view
Level of serum catalase activity
The CAT activity was significantly increased in the citrulline 400 mg/kg (13.7 ± 0.43 ng/mL) and citrulline 800 mg/kg (14.6 ± 0.54 ng/mL) groups, compared to the diabetic (10.8 ± 0.41 ng/mL) and metformin (11.0 ± 0.69 ng/mL) groups (F = 14.6; P < 0.0001) [Figure 3].
|Figure 3: Effect of L-citrulline on level of serum CAT activity of high-fat diet and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with L-citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, c, and d denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, metformin, and CIT 200 mg/kg groups, respectively|
Click here to view
Serum-reduced glutathione level
There was a significant reduction in the level of GSH seen in the diabetic group (22.9 ± 0.69 mg/mL) compared to the normal control (28.3 ± 1.17 mg/mL). There was a significant increase seen in the metformin (39.7 ± 0.69 mg/mL), citrulline 200 mg/kg (42.9 ± 1.08 mg/mL), citrulline 400 mg (46.4 ± 0.53 mg/mL), and citrulline 800 mg (45.2 ± 1.0 mg/mL) groups (F = 118.2; P < 0.0001) [Figure 4].
|Figure 4: Effect of L-citrulline on serum reduced glutathione level of high-fat diet- and dexamethasone-induced diabetic rats. MET 100 mg/kg: diabetic rats treated with metformin 100 mg/kg/day; CIT 200 mg/kg, 400 mg/kg, and 800 mg/kg: diabetic rats treated with citrulline at doses 200 mg/kg/day, 400 mg/kg/day, and 800 mg/kg/day, respectively. a, b, and c denote statistically significant difference (P < 0.0001) compared to normal control, diabetic control, and metformin group respectively|
Click here to view
| Discussion|| |
All doses of L-citrulline employed in this study were able to lower the FBG levels on day 22 compared to the diabetic control, with the greatest effect observed at 400 mg/kg. This hypoglycemic effect of L-citrulline is probably due to its ability to improve peripheral and hepatic insulin sensitivity., However, in a study among overweight and obese individuals, a daily intake of watermelon (the main natural source of L-citrulline) over a period of 4 weeks did not produce any significant change in the blood glucose level compared to the baseline. Metformin, one of the most commonly used hypoglycemic medications, known to ameliorate hepatic and peripheral insulin resistance, and improved endothelial dysfunction in type-2 diabetes, had shown a superior effect compared to L-citrulline in the present study.
There was a marked increase in the MDA concentration, a biomarker for lipid peroxidation in the diabetic control group compared to the normal control in our study. A study reported a marked increase in MDA level among diabetic patients compared to the nondiabetic control. Nevertheless, L-citrulline was able to ameliorate this in a dose-dependent pattern, suggesting a protective effect. In a study by Liu et al., L-citrulline was able to protect against glycerol-induced oxidative damage characterized by significant dose-dependent decreases in MDA concentrations in rats. Similarly, Lum et al. demonstrated a significant decrease in the MDA concentration after 4 weeks of watermelon consumption compared to the baseline in overweight and obese individuals.
In the present study, there were decreases in SOD activity and GSH level in the diabetic group compared to the normal control, which is probably due to excessive generation of free radicals leading to oxidative/nitrosative stress (though not measured in this study). As the SOD was consumed in the diabetic group, less amount of hydrogen peroxide (H2O2) is expected to be produced, which may explain the slight increase in CAT activity in the diabetic group. The significant decrease in GSH level is probably due to its depletion while scavenging free radicals that were likely generated. Yarube and Gwarzo had also demonstrated such decreased antioxidant capacity among diabetic patients compared to their nondiabetic counterparts.
The supplementation with L-citrulline was able to augment the endogenous antioxidant enzymes (SOD and CAT) activities and level of GSH at all doses from this study. This is in line with the findings of Oseni et al. that demonstrated a significant increase in GSH level and SOD activity with a mild increase in CAT activity after a week of administration of watermelon crude extract containing a large amount of L-citrulline. It may be due to the sparing of the endogenous antioxidant enzymes by the L-citrulline, especially at 800 mg/kg. As the activity of SOD increases (probably to scavenge the excessive superoxide ion generated), a large amount of H2O2 is produced which might have triggered the increase in the CAT activity (to scavenge the H2O2). This is necessary because, in the presence of reduced metals such as ferrous iron (Fe2+), H2O2 can be converted by the Fenton reaction into the highly reactive hydroxyl radical (OH−), one of the most harmful of all ROS. To further support the antioxidant efficacy of L-citrulline, Liu et al. also found a significant increase in SOD activity and GSH level in rats when supplemented with L-citrulline.
Endogenous GSH can augment the effect of oral L-citrulline on NO synthesis, protecting against the oxidative reactions of NO by increasing the activity of NO synthase in rodents and humans. GSH plays a significant role in intracellular defense against oxidative stress. In the present study, there was a marked reduction in the GSH level in the diabetic group. This is in line with some studies,, which reported reduced synthesis and level in patients with uncontrolled diabetes. However, with L-citrulline supplementation at all doses, there was a significant increase in the GSH level. This is probably due to sparing of the GSH by L-citrulline in the rats. In addition, L-citrulline might enhance the synthesis of cysteine and glycine, which are amino acid precursors for GSH synthesis.
L-citrulline supplementation, from our finding, had shown a superior effect compared to metformin in ameliorating the oxidative stress noted in the diabetic group. Although not measured in this study, L-citrulline could exert such effects via its ability to significantly decrease ROS formation and at the same time, regulate the production and metabolism of NO, preventing its excessive and uncontrolled production.
Some studies showed an improvement in antioxidant status and vascular function by watermelon consumption, preventing oxidative stress., It causes such effects via the production of NO that has a vasodilator effect and prevents oxidative stress by scavenging hydroxyl radicals. These results show that L-citrulline has a great potential in the treatment of cardiovascular diseases and diabetic complications with endothelial dysfunction as the underlying mechanism.,
| Conclusion|| |
The present study revealed that L-citrulline possesses antioxidant effects in diabetic rats in an almost dose-dependent manner. This shows a great potential in reversing the vascular complications as well as halting the progression of T2DM. Where the conventional antidiabetic drugs fail, L-citrulline may be a good alternative.
Limitations of the study
The levels of NO, endothelial NO synthase expression, ROS such as superoxide, and reactive nitrogen species such as peroxynitrite were not assessed in this study. NO may have oxidant effects when combined with superoxide (in the presence of oxidative stress), forming a reactive NO and other reactive nitrogen species. In type-2 diabetes, in which there is an enhanced production of superoxide, the superoxide-NO interaction may reduce flow-mediated dilation of the coronary arterioles which may contribute to the development of coronary artery disease. The question is could L-citrulline alter such interaction? Moreover, could diabetic angiopathy (the main cause of morbidity and mortality in DM), which is caused by endothelial dysfunction,, be due to such interaction? Tissues such as the liver, kidneys, brain, and heart and adipose tissues that are involved in glucose metabolism are also potential areas to explore in further studies, especially the effect of L-citrulline on the tissue markers for oxidative stress.
Furthermore, the mechanism(s) by which L-citrulline ameliorate the biomarkers of oxidative stress in the diabetic setting need is not explored. Other parameters such as serum insulin level or expression, HOMA-IR, and HbA1C not measured in this study are the next targets in future studies.
The authors would like to thank all staff of the Department of Physiology, College of Medical Sciences, Ahmadu Bello University, Zaria, who contributed to the success of this work.
Financial support and sponsorship
This research work was carried out through personal funding.
Conflicts of interest
There are no conflicts of interest.
| References|| |
American Diabetes Association. Classification and diagnosis of diabetes. Diabetes Care 2017;40 Suppl 1:S11-24.
NCD Risk Factor Collaboration (NCD-RisC). Worldwide trends in diabetes since 1980: A pooled analysis of 751 population-based studies with 4.4 million participants. Lancet 2016;387:1513-30.
Carracher AM, Marathe PH, Close KL. International diabetes federation 2017. J Diabetes 2018;10:353-6.
Dahiru T, Aliyu AA Shehu AU. A review of population-based studies on diabetes mellitus in Nigeria. Sub Saharan Afr J Med 2016;3:9-66.
Yamamoto K, Shuang E, Hatakeyama Y, Sakamoto Y, Tsuduki T. High-fat diet intake from senescence inhibits the attenuation of cell functions and the degeneration of villi with aging in the small intestine, and inhibits the attenuation of lipid absorption ability in SAMP8 mice. J Clin Biochem Nutr 2015;57:204-11.
Lozano I, Van der Werf R, Bietiger W, Seyfritz E, Peronet C, Pinget M, et al.
High-fructose and high-fat diet-induced disorders in rats: Impact on diabetes risk, hepatic and vascular complications. Nutr Metab (Lond) 2016;13:15.
Noeman SA, Hamooda HE, Baalash AA. Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats. Diabetol Metab Syndr 2011;3:17.
Schultz H, Engelholm SA, Harder E, Pedersen-Bjergaard U, Kristensen PL. Glucocorticoid-induced diabetes in patients with metastatic spinal cord compression. Endocr Connect 2018;7:719-26.
Shpilberg Y, Beaudry JL, D'Souza A, Campbell JE, Peckett A, Riddell MC. A rodent model of rapid-onset diabetes induced by glucocorticoids and high-fat feeding. Dis Model Mech 2012;5:671-80.
Perez A, Jansen-Chaparro S, Saigi I, Bernal-Lopez MR, Miñambres I, Gomez-Huelgas R. Glucocorticoid-induced hyperglycemia. J Diabetes 2014;6:9-20.
Suh S, Park MK. Glucocorticoid-induced diabetes mellitus: An important but overlooked problem. Endocrinol Metab (Seoul) 2017;32:180-9.
van Raalte DH, Diamant M. Steroid diabetes: From mechanism to treatment? Neth J Med 2014;72:62-72.
Danboyi T, Alhassan AW, Jimoh A, Hassan-Danboyi E. Effect of L-citrulline supplementation on blood glucose level and lipid profile in high-fat diet -and dexamethasone-induced type-2 diabetes in male Wistar rats. Nig J Exp Clin Biosci 2020;8:100-7.
Gurib-Fakim A. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol Aspects Med 2006;27:1-93.
Rosenblit PD. Common medications used by patients with type 2 diabetes mellitus: What are their effects on the lipid profile? Cardiovasc Diabetol 2016;15:95.
Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J Diabetes 2015;6:456-80.
Pitocco D, Tesauro M, Alessandro R, Ghirlanda G, Cardillo C. Oxidative stress in diabetes: Implications for vascular and other complications. Int J Mol Sci 2013;14:21525-50.
Shinde SN, Dhadke VN, Suryakar AN. Evaluation of oxidative stress in Type 2 diabetes mellitus and follow-up along with vitamin E supplementation. Indian J Clin Biochem 2011;26:74-7.
Vats P, Sagar N, Singh TP, Banerjee M. Association of superoxide dismutases (SOD 1 and SOD 2) and glutathione peroxidase 1 (GPx1) gene polymorphism with type-2 diabetes mellitus. Free Radical Res 2015;49:17-24.
Lum T, Connolly M, Marx A, Beidler J, Hooshmand S, Kern M, et al.
Effects of fresh watermelon consumption on the acute satiety response and cardiometabolic risk factors in overweight and obese adults. Nutrients 2019;11:595.
Azizi S, Mahdavi R, Vaghef-Mehrabany E, Maleki V, Karamzad N, Ebrahimi-Mameghani M. Potential roles of Citrulline and watermelon extract on metabolic and inflammatory variables in diabetes mellitus, current evidence and future directions: A systematic review. Clin Exp Pharmacol Physiol 2020;47:187-98.
Jabłecka A, Bogdański P, Balcer N, Cieślewicz A, Skołuda A, Musialik K. The effect of oral L-arginine supplementation on fasting glucose, HbA1c, nitric oxide and total antioxidant status in diabetic patients with atherosclerotic peripheral arterial disease of lower extremities. Eur Rev Med Pharmacol Sci 2012;16:342-50.
Papadia C, Osowska S, Cynober L, Forbes A. Citrulline in health and disease. Review on human studies. Clin Nutr 2018;37:1823-8.
Schwedhelm E, Maas R, Freese R, Jung D, Lukacs Z, Jambrecina A, et al.
Pharmacokinetic and pharmacodynamic properties of oral L-citrulline and L-arginine: Impact on nitric oxide metabolism. Br J Clin Pharmacol 2008;65:51-9.
Pop-Busui R, Sima A, Stevens M. Diabetic neuropathy and oxidative stress. Diabetes Metab Res Rev 2006;22:257-73.
Romero MJ, Platt DH, Caldwell RB, Caldwell RW. Therapeutic use of citrulline in cardiovascular disease. Cardiovasc Drug Rev 2006;24:275-90.
Piatti PM, Monti LD, Valsecchi G, Magni F, Setola E, Marchesi F, et al.
Long-term oral L-arginine administration improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients. Diabetes Care 2001;24:875-80.
Yoshitomi H, Momoo M, Ma X, Huang Y, Suguro S, Yamagishi Y, et al.
L-Citrulline increases hepatic sensitivity to insulin by reducing the phosphorylation of serine 1101 in insulin receptor substrate-1. BMC Complement Altern Med 2015;15:188.
Assmann TS, Brondani LA, Bouças AP, Rheinheimer J, de Souza BM, Canani LH, et al.
Nitric oxide levels in patients with diabetes mellitus: A systematic review and meta-analysis. Nitric Oxide 2016;61:1-9.
Shi Y, Vanhoutte PM. Macro- and microvascular endothelial dysfunction in diabetes. J Diabetes 2017;9:434-49.
Costa J, Borges M, David C, vaz Carneiro A. Efficacy of lipid lowering drug treatment for diabetic and non-diabetic patients: Meta-analysis of randomized controlled trials. Br Med J 2006;332:1115-24.
Low Wang CC, Hess CN, Hiatt WR, Goldfine AB. Clinical update: Cardiovascular disease in diabetes mellitus: Atherosclerotic cardiovascular disease and heart failure in Type 2 diabetes mellitus – Mechanisms, management, and clinical considerations. Circulation 2016;133:2459-502.
Allerton TD, Proctor DN, Stephens JM, Dugas TR, Spielmann G, Irving BA. l-Citrulline Supplementation: Impact on Cardiometabolic Health. Nutrients 2018;10:921-44.
Wijnands KA, Vink H, Briede JJ, van Faassen EE, Lamers WH, Buurman WA, et al.
Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia. PLoS One 2012;7:e37439.
Barr FE, Tirona RG, Taylor MB, Rice G, Arnold J, Cunningham G, et al.
Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: Potential therapy for postoperative pulmonary hypertension. J Thorac Cardiovasc Surg 2007;134:319-26.
Tsuboi T, Maeda M, Hayashi T. Administration of L-arginine plus L-citrulline or L-citrulline alone successfully retarded endothelial senescence. PLoS One 2018;13:e0192252.
Okoduwa SI, Umar IA, James DB, Inuwa HM. Appropriate insulin level in selecting fortified diet-fed, streptozotocin-treated rat model of type 2 diabetes for anti-diabetic studies. PLoS One 2017;12:e0170971.
Martínez BB, Pereira AC, Muzetti JH, Telles FP, Mundim FG, Teixeira MA. Experimental model of glucocorticoid-induced insulin resistance. Acta Cir Bras 2016;31:645-9.
Beach EF, Turner JJ. An enzymatic method for glucose determination uptake in body fluids. Clin Chem 1958;4:462-75.
Adeoye AT, Oyagbemi AA, Adedapo AD, Omobowale TO, Ayodele AE, Adedapo AA. Antidiabetic and antioxidant activities of the methanol leaf extract of Vernonia amygdalina in alloxan-induced diabetes in Wistar rats. J Med Plants Econ Devt 2017;1:a30.
Matteucci E, Giampietro O. Proposal open for discussion: Defining agreed diagnostic procedures in experimental diabetes research. J Ethnopharmacol 2008;115:163-72.
Wessels B, Ciapaite J, van den Broek NM, Nicolay K, Prompers JJ. Metformin impairs mitochondirial function in skeletal muscle of both lean and diabetic rats in a dose-dependent manner. PLoS One 2014;9:e100525.
McKinley-Barnard S, Andre T, Morita M, Willoughby DS. Combined L-citrulline and glutathione supplementation increases the concentration of markers indicative of nitric oxide synthesis. J Int Soc Sports Nutr 2015;12:27.
Satoh K. Serum lipid peroxide in cerebrovascular disorders determined by a new colorimetric method. Clin Chim Acta 1978;90:37-43.
Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170-5.
Aebi H. Catalase in vitro
. Methods Enzymol 1984;105:121-6.
Rukkumani R, Aruna K, Varma PS, Rajasekaran KN, Menon VP. Comparative effects of curcumin and an analog of curcumin on alcohol and PUFA induced oxidative stress. J Pharm Pharm Sci 2004;83:2747-52.
Staels B. Metformin and pioglitazone: Effectively treating insulin resistance. Curr Med Res Opin 2006;22 Suppl 2:S27-37.
Fidan E, Onder Ersoz H, Yilmaz M, Yilmaz H, Kocak M, Karahan C, et al.
The effects of rosiglitazone and metformin on inflammation and endothelial dysfunction in patients with type 2 diabetes mellitus. Acta Diabetol 2011;48:297-302.
Yarube IU, Gwarzo IM. Cognitive impairment and reduced antioxidant capacity in patients with type 2 diabetes. Sahel Med J 2019;22:171-8. [Full text]
Liu Y, Fu X, Gou L, Li S, Lan N, Zheng Y, et al.
L-citrulline protects against glycerol-induced acute renal failure in rats. Ren Fail 2013;35:367-73.
Oseni OA, Odesanmi OE, Oladele FC. Antioxidative and antidiabetic activities of watermelon (Citrullus lanatus
) juice on oxidative stress in alloxan-induced diabetic male Wistar albino rats. Niger Med J 2015;56:272-7.
] [Full text]
Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007;39:44-84.
Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother 2003;57:145-55.
Sekhar RV, McKay SV, Patel SG, Guthikonda AP, Reddy VT, Balasubramanyam A, et al.
Glutathione synthesis is diminished in patients with uncontrolled diabetes and restored by dietary supplementation with cysteine and glycine. Diabetes Care 2011;34:162-7.
Lutchmansingh FK, Hsu JW, Bennett FI, Badaloo AV, McFarlane-Anderson N, Gordon-Strachan GM, et al.
Glutathione metabolism in type 2 diabetes and its relationship with microvascular complications and glycemia. PLoS One 2018;13:e0198626.
Hong MY, Hartig N, Kaufman K, Hooshmand S, Figueroa A, Kern M. Watermelon consumption improves inflammation and antioxidant capacity in rats fed an atherogenic diet. Nutr Res 2015;35:251-8.
Figueroa A, Wong A, Jaime SJ, Gonzales JU. Influence of L-citrulline and watermelon supplementation on vascular function and exercise performance. Curr Opin Clin Nutr Metab Care 2017;20:92-8.
Wink DA, Miranda KM, Espey MG, Pluta RM, Hewett SJ, Colton C, et al
. Mechanisms of the antioxidant effects of nitric oxide. Antioxid Redox Signal 2001;3:203-13.
Chikezie PC, Ojiako O, Ogbuji AC. Oxidative stress in diabetes mellitus. Int J Biol Chem 2015;9:92-109.
Santili F, Cipollone F, Mezzetti A, Chiarelli F. The role of NO in the development of diabetic angiopathy. Horm Metab Res 2004;36:319-35.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]