Anti-diabetic study of vitamin B6 on hyperglycaemia induced protein carbonylation, DNA damage and ROS production in alloxan induced diabetic rats

Study of nutraceutical and food supplements especially vitamins against diabetes.

Abstract

Oxidative stress performs an imperative role in the onset and progression of diabetes. Metabolic enzymes and cellular organelles are detrimental to increased levels of free radicals and the subsequent reduction in anti-oxidant defence. Pyridoxamine (vitamin B6) is an indispensible nutrient for humans and is considered to be an important food additive too. The aim of this research was to examine the effect of vitamin B6 in a diabetic environment. This study reports the effects of pyridoxamine supplementation in alloxan induced diabetic rats. Diabetes was induced by the single intra peritoneal dose of alloxan (120 mg per kg body weight). Diabetic rats were treated with pyridoxamine (10 and 15 mg per kg body weight) and compared with a control set of diabetic rats without supplementation. Pyridoxamine treatment showed dose dependent recovery in all parameters. A notable decline in oxidative stress parameters and ROS production with reductions in fasting blood glucose levels along with normal patterns of the glucose tolerance test has been reported here. Histological studies reveal damage recovery in the liver as well as kidney tissues. A notable amount of recovery was observed in cellular DNA distortion and damage. It is thus advocated that pyridoxamine might help in reducing problems associated with diabetes. A probable mechanism pertaining to the action of pyridoxamine is proposed as well.

1. Introduction

Diabetes mellitus (DM) is an global health problem, touching about 422 million lives around the globe and its prevalence has been soaring in low and middle income countries (WHO global report, 2014). DM is a collection of metabolic disorders characterized by persistent hyperglycaemia arising due to flaws in the process involving secretion of insulin or its mode of action or both.1 Prolonged diabetic status of an individual often leads to additional complications, such as atherosclerosis, neuropathy, retinopathy, cardiovascular disease etc.2,3 Numerous pathophysiological complications that lead to flawed glucose equilibrium in diabetic cases are consequences of environmental and genetic factors. In diabetes, chronic hyperglycaemia is associated with long term damage, dysfunction and eventually the failure of organs, especially the eyes, kidneys, cardiovascular system, and nerves.4 Diabetic nephropathy is viewed as the most well-known reason for end stage renal disease. However, insulin resistance and impaired insulin secretion remain the hallmark defects in diabetes. Several innovative medications are unfolding, but the greatest urgency is for agents that halt the progressive pancreatic β-cell failure and forbid or counter the microvascular complications.

Emerging evidence has shown that oxidative stress induced by a persistent hyperglycaemic condition can lead to various degrees of testicular dysfunctions.5 Moreover, ROS impair the biochemical structures of the cell membrane as they possess a high affinity to polyunsaturated fatty acids.6 Nowadays, the focus of exploration has shifted to green synthesis or natural products to cure various diseases due to their safety, efficacy and lesser side effects. Nutritional input of plant derived food and their ingredients could be a more effective strategy to manage DM. Thus, new chemical compounds from natural products have been explored as possibly safer antidiabetic agents that will have fewer side effects.7

A broad range of plant products have been used as conventional remedies for the treatment of diabetes throughout the world.8 Our laboratory has already analyzed and published the effect of some natural compounds and vitamins on the prognosis of diabetes through both in vitro as well as in vivo studies.9,10 Pyridoxamine (PM) is a metabolite of vitamin B6, which was known to reduce the diabetes related complications and the incidence of neurodegenerative diseases.11 The decrement in the levels of vitamin B6 has been coordinated with the establishment of both type 1 and type 2 diabetes.12,13 Several research lines have shown that the external dosing of vitamin B6 reduces the clinical signs of retinopathy and neuropathy in diabetic individuals.14,15

Pyridoxamine has been found to be an effective agent in targeting tissue and/or circulating AGE levels.16 It inhibits the production of AGEs from glycated proteins by trapping physiologically reactive carbonyl compounds and causes delay in the development of diabetic kidney disease along with reduction in albuminuria as evident from studies in animal models of both type 1 and type 2 diabetic nephropathy.17 William et al. (2007) conducted two phase II clinical studies in mild and moderate type 1 and type 2 diabetic nephropathy patients, and after analysing the merged results, it was found that pyridoxamine successfully provides better medication in reducing the slope of creatinine change from the baseline compared with a placebo (percent serum creatinine changed is 48%, P = 0.03), interestingly, the effect was even more pronounced and impressive in the type 2 diabetic group and baseline serum creatinine group,1.3 mg dl^–1, along with the significant reduction in the AGE concentration of plasma compared with the placebo group.18

When the screening of vitamin B6 was done on Streptozotocin (STZ)-mice, a reduction in the blood glucose level was observed,19 along with the lowering of HbA1c levels in type-2 diabetic patients.20In vitro evaluations of pyridoxamine had also been done which indicates an inhibition in the formation of AGE products.21 The present study reveals that the administration of pyridoxamine helps in improving the antioxidant capacity of diabetic rats by reducing free radical formation, increasing activities of antioxidative parameters such as superoxide dismutase and catalase, along with the elevation in glutathione levels. Furthermore, the results of histopathological studies, DAPI-staining, DCFH-DA ROS microscopy and the comet assay corroborate that tissue and DNA damage had also been controlled by the external supplementation of vitamin B6.

2. Materials and methods

2.1. Materials

Pyridoxamine, RPMI 1640, Histopaque-1077, alloxan, low melting point agarose and normal agarose were purchased from Sigma-Aldrich Chemical Company, USA. DTNB, DMSO, glucose, pyrogallol, H₂O₂, CDNB, NADPH, propylene glycol, NaCl, NaOH, and EDTA were purchased from SRL Chemicals, India. Creatinine, urea, and alkaline phosphatase kits were obtained from Span Diagnostics Limited, India. All other reagents used were of analytical grade.

2.2. Ethical statements

The authorization of experimental trials involving animals was done by the Ministry of Environment and Forests, Government of India under registration no. 714/GO/Re/S/02/CPCSEA and issued by the committee assigned for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The validation was given by the Institutional Animal Ethic Committee (IAEC) of Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, India (Order no: D. No. 4165).

2.3. Diabetes induction and pyridoxamine treatment

Twenty five male Wistar rats weighing around 100–120 grams and aged six months were bought from the central animal house of Jamia Hamdard University, New Delhi, India. According to the Departmental Ethical Committee for Animal Experimentation, the animals were sheltered in sufficiently spacious cases and handled under agreeable and wholesome conditions. Besides, the temperature and day : night cycles were managed as 25 ± 2 °C room temperature and 12 h respectively. For one week, the animals were adjusted on uniform dietary pellets for rats (Ashirwad Industries, Chandigarh, India). Fresh drinking water ad libitum was made available to them prior to the start of treatment. Diabetes was induced by alloxan (120 mg per kg body weight) via intra-peritoneal injection dissolved in normal saline. Diabetes was confirmed when glucose levels were high (>250 mg dL^–1), in the peripheral blood obtained from the tail vein. Treatment with pyridoxamine was started in diabetic rats and this was counted as day one. Pyridoxamine at 10 and 15 mg per kg body weight was supplemented daily for 30 consecutive days using an oral gavage tube.

Group I is the control which received normal diet and water.

Group II is a control set of rats which received pyridoxamine (15 mg per kg body weight) with normal diet and water.

Group III consists of diabetic rats induced by alloxan which received normal diet and water.

Group IV & V are diabetic rats supplemented with pyridoxamine at 10 & 15 mg per kg body weight respectively.

2.4. Sample preparation

Rats were allowed to fast overnight and thereafter sacrifice by cervical dislocation once the experimental protocol was over. Heparinised vials were used to store the blood drawn via cardiac puncture. The blood was centrifuged at 1000g for 15 minutes immediately after collection. After sacrifice, the following day 1 and day 2 were utilized for analysis of blood and serum parameters. However, the tissues or tissue homogenates from the liver, pancreas and kidney were stored at –20 °C for further analysis.

2.5. Isolation of rat peripheral blood lymphocytes

For all experiments, around 5 ml of blood was withdrawn from rats of all groups by cardiac puncture. With the help of Histopaque 1077, peripheral blood lymphocytes were isolated after diluting blood in Ca²⁺ and Mg²⁺ free phosphate buffered saline (PBS) and were used immediately after isolation. The trypan blue exclusion test was performed to check the viability of lymphocytes15 before the start of reaction.

2.6. Estimation of fasting blood glucose

The glucose oxidase–peroxidase method16 was employed to check fasting blood glucose levels using a Ranbaxy diagnostic kit (containing 6.7 U ml^–1 of glucose oxidase, 6.2 U ml^–1 of horseradish peroxidase, 0.2 mM of 4-aminoantipyrene, 8 M of phosphate buffer and 86 mM of phenol). Thereafter, with the help of a standard curve the concentration of glucose in the samples was estimated. Insulin levels were approximated using a commercially available kit (Span Diagnostics Limited, India).

2.6.1. Oral glucose tolerance test (OGTT)

After 30 days of treatment, oral glucose tolerance tests (OGTT) were performed on both diabetic as well as normal (control) rats. The animals were fasted overnight (12 hours), following fasting they were administered an oral dose of 30% glucose solution. Blood samples were collected from the tail vein at 0, 30, 60, and 120 min after feed and measured with an Accu-chek Active blood glucose meter (Model: GU Accu-chek is a trademark of Roche, Mannheim, Germany).

2.7. Glucose metabolic enzymes

2.7.1. Hexokinase activity

For checking the activity of enzyme hexokinase, the method of Crane and Sols17 was performed. 1 ml reaction mixture was prepared containing tris HCl (50 μM), MgCl₂ (10 μM), adenosine triphosphate (5 μM), and glucose (2 μM). The prepared reaction mixture was added to 1–1.5 mg of sample protein and after a time lapse of 60 minutes, the reaction was terminated by adding 0.5 ml each of 10% Ba(OH)₂ and ZnSO₄ solutions. Thereafter, the samples were centrifuged at 2000g for 10 minutes and glucose was measured in the supernatant (free from phosphorylated derivatives) by the earlier described method.

2.7.2. Glucose-6-phosphatase (G6Pase) activity

The protocol given by Shull19 was followed for measuring the activity of G6Pase. 1.5 ml reaction mixture comprising 50 mM of tris–HCl buffer, 10 mM of MgCl₂, 10 mM glucose-6-phosphate, and the sample was prepared. The reaction temperature was maintained at 37 °C for 1 h and stopped by the adding up of 1 ml 10% TCA. This step was followed by centrifugation of the samples at 2000g for 10 min and after that the phosphorus content in the protein free supernatant was evaluated.

2.7.3. Fructose-bisphosphatase activity (FBPase)

For estimating the activity of FBPase, the protocol of Freedland and Harper21 was followed with minor modifications. A 1.5 ml reaction mixture containing tris HCl (50 μM), MgCl₂ (10 μM), cysteine HCl (12 μM) and fructose 1,6-bisphosphate (10 μM) was prepared and added to the sample. The reaction was stopped by the addition of 1 ml of 10% TCA after 60 minutes, and the samples were then centrifuged at 2000g for 10 minutes. The temperature was set at 37 °C during the course of the experiment and on completion, TCA solution (10%) was added to stop the reaction after 1 hour. The phosphorus content was estimated in the protein free supernatant by the earlier described method.

2.8. Measurement of antioxidant and oxidative markers

2.8.1. Superoxide dismutase (CuZn SOD)

The method of Marklund and Marklund which is based on the autoxidation of pyrogallol22 was performed for the measurement of SOD activity. Here, 2.85 ml of tris-succinate buffer (0.05 M, pH 8.2) was added to 50 μl of sample followed by 100 μl of pyrogallol (8 mM) in the dark. The absorbance was recorded at 412 nm on a Centra-5 spectrophotometer for 3 minutes. One enzyme unit is defined as the amount of enzyme required to cause 50% inhibition of the auto-oxidation of pyrogallol per 3 mL of the assay mixture.

2.8.2. Catalase (CAT)

For estimating catalase activity, the protocol of Aebi was followed with minor modifications.23 The reaction was carried out under dark conditions containing 1.95 ml of potassium phosphate buffer (50 mM, pH 7.0), 50 μl of the sample and 1 ml of H₂O₂ (30 mM). Immediately upon completion of incubation, the absorbance was recorded at 240 nm for 3 minutes. The catalase activity was calculated in nmoles of H₂O₂ consumed per mg of protein per minute.

2.8.3. Reduced glutathione (GSH)

The method proposed by Jollow et al.38 with slight modifications as per requirements was used for measuring GSH levels. A sample homogenate (0.5 ml) was mixed with an equal volume of 4% (w/v) sulphosalicylic acid and incubated at 4 °C for 1 hour. This step was followed by centrifugation at 1200g for 15 minutes. The reaction was initiated by addition of 0.2 ml of DTNB which was read at 412 nm within 30 seconds. The GSH levels were reported as nmoles per gram of the tissue.

2.8.4. Lipid peroxidation and protein carbonylation

The method proposed by Buege et al. was used for the estimation of lipid peroxidation. In this method the major product of lipid peroxidation is measured i.e. total malondialdehyde (MDA).39 The volume of the reaction mixture was taken as 1.5 ml which comprises 0.5 ml of 10% TCA (trichloroacetic acid) and 0.5 ml of 0.6 M TBA (2-thiobarbituric acid). All constituents were then incubated in a boiling water bath for 20 minutes and the absorbance was read at 532 nm. The amount of thiobarbituric-reactive substances (TBARS) present was determined using the molar extinction co-efficient of 1.56 × 10^–5 M^–1 cm^–1 for the MDA–thiobarbituric acid colored complex. The amount of protein oxidation was estimated by measurement of the total carbonyl content which is the final product of the oxidation.26 One ml reaction mixture after treatment was blended with 0.5 ml of 10 mM 2,4-dinitrophenylhydrazine in 2.5 M HCl. The mixture was left for 1 h at room temperature and 0.5 ml of 20% trichloroacetic acid was added to a tube. The tube was left in an ice bucket for 10 min followed by centrifugation at 12 000g for 15 min. The supernatant was discarded and the protein pellet was washed with ethanol/ethyl acetate (1 : 1 v/v) and dissolved in 2 ml of 6 M guanidine (pH 2.3) and vortexed. The carbonyl content was calculated using the molar absorption coefficient of 22 000 M^–1 cm^–1.

2.9. Liver and kidney function markers

Aspartate transaminase (AST) and alanine transaminase (ALT) levels were estimated using a commercially available kit (Span Diagnostics Limited, India) based on the Reitman and Frankel method.27 The pyruvate formed as a result of AST activity reacts with DNPH to produce brown colored hydrazones which are read at 505 nm taking distilled water as the blank. Alkaline phosphatase (ALP) was also estimated using a commercially available kit based on Kind and King's method.28 The principle of the kit is that alkaline phosphatase converts phenyl phosphate to inorganic phosphate and phenol at pH 10. Phenol formed reacts at alkaline pH with 4-aminoantipyrene in the presence of the oxidising agent potassium ferricyanide and results in the formation of an orange-red coloured complex, which can be measured colorimetrically. The levels of urea were reported in mg per dl of the sample using a commercially available kit.

2.10. Lipid profile

Separated serum was tested on the day of sample collection; else the samples were preserved at –20 °C for future use. The serum lipid profile was evaluated using the Lab Life Chem Master semi-automated chemistry analyser (model no. BTR-830) and included total cholesterol, TG and HDL-C.

2.11. Histopathological study of the liver and kidneys

For histopathological analysis, liver and kidney samples of the treated and untreated animals were kept in 10% formalin for immersion fixation. For embedding in paraffin, 10 × 5 × 3 mm sized tissue blocks of the organs were processed. Around 5 μm thick sections of the embedded tissues were cut with a rotary microtome and further stained with haematoxylin and eosin stain. The observation of these sections was performed under a trinocular light microscope (Olympus BX40, Japan) and their photomicrographs depicting important features were snapped at the final magnification of 400×.

2.12. Detection of intracellular ROS

A DCFH-DA probe assay was performed for detection of intracellular ROS generation.29 The principle of this assay is based on the oxidation of non-fluorescent DCFH-DA to fluorescent 2,7-dichlorofluorescein (DCF) by the action of intracellular oxidants. All the samples of lymphocytes isolated from rats were incubated for 1 h with 10 μM of DCFH-DA at 37°. To visualize the intracellular fluorescence of DCF, a parallel set of treated lymphocytes were analyzed using a fluorescence microscope and the images were captured.

2.13. Apoptosis analysis by DAPI staining

Lymphocytes isolated from the rats of all groups were analyzed by staining with the fluorescent dye DAPI. All the samples were incubated with DAPI (20 mg L^–1) in a 1 : 1 ratio and were kept in the dark for 5 min at room temperature. After staining images were captured using a fluorescence microscope.

2.14. Comet assay (single cell alkaline gel electrophoresis)

Under alkaline conditions the comet assay was performed in accordance with the protocol of Singh et al.30 A detailed protocol for the assay has been described in our previous publication by K. M. Abdullah et al.40 Scoring of the comet slides was performed using an image analysis system (Komet 5.5, Kinetic Imaging, Liverpool, UK) attached to an Olympus (CX41) fluorescence microscope (Olympus Optical Co, Tokyo, Japan).

2.15. Statistical analysis

For all continuous variables the data have been expressed as mean ± SD. Student's t-test was performed using Microsoft Excel 2016, for making comparison among various groups. p ≤ 0.05 was chosen to be statistically significant for the treatment. All experiments have been repeated thrice for checking reproducibility.

3. Results

3.1. Blood glucose test and glucose metabolic enzymes

The level of fasting blood glucose (FBG) in different groups is shown in Fig. 1a, and it was found elevated in the diabetic group (229 ± 13.86 mg dl^–1) in comparison with the control group (122 ± 13.07 mg dl^–1) with a P value <0.001. There is a dose dependent decrease in FBG levels in the diabetic group supplemented with vitamin B6. Pyridoxamine when supplemented at 10 and 15 mg per kg body weight appreciably decreases the FBG level to 171.33 ± 9.71 mg dl^–1 (P < 0.03 w.r.t to the diabetic group) and 152.33 ± 13.86 (P < 0.04 w.r.t to the diabetic group) respectively. Likewise, an oral glucose tolerance test was performed and the level of blood glucose (30 min) was significantly increased in the diabetic group as can be seen in Fig. 1b. When treated with pyridoxamine (10 & 15 mg per kg body weight) preventive effects against hyperglycaemia induced by glucose were observed. The levels of blood glucose were considerably lower than the diabetic group and decreased mildly during the 2-hour experiment. Table 1 depicts the alterations in the activities of all the three glucose metabolic enzymes. Hexokinase activity was found to be decreased in the diabetic group in target organs (liver, kidney and pancreas), which recovered upon pyridoxamine supplementation. However, the activity of FBPase and G6Pase decreased significantly with the supplementation. The enzymes FBPase and G6Pase are involved in the gluconeogenic pathway of glucose metabolism and play an important role in regulating blood glucose levels.

Fig. 1. (A) Fasting blood glucose levels and (B) glucose tolerance test in the normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups of rats. P1 is pyridoxamine treatment at 10 mg per kg body weight and P2 is pyridoxamine treatment at 15 mg per kg body weight. Results presented are mean ± SD of three independent treatments. # is a p value ≤0.05 compared to the diabetic group.

Table 1. Dose dependent effect of pyridoxamine on glucose metabolic enzymes in alloxan induced diabetic rats. Hexokinase, FBPase and G6Pase activities were determined in normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups.

Parameters	Samples	Normal C	Normal + P2	Diabetic	Diabetic + P1	Diabetic + P2
Hexokinase (μmol per mg protein)	Liver	3.66 ± 0.11#	3.53 ± 0.18#	1.92 ± 0.17	2.57 ± 0.15#	3.17 ± 0.23#
	Kidney	3.43 ± 0.20#	3.29 ± 0.15#	1.67 ± 0.12	2.13 ± 0.16	2.72 ± 0.16#
	Pancreas	1.94 ± 0.11#	1.96 ± 0.09#	0.93 ± 0.07	1.42 ± 0.11#	1.71 ± 0.09#
FBPase (μmol per mg protein)	Liver	1.63 ± 0.09#	1.62 ± 0.10#	3.09 ± 0.15	2.32 ± 0.20#	1.80 ± 0.12#
	Kidney	0.74 ± 0.05#	0.74 ± 0.03#	2.15 ± 0.09	1.60 ± 0.07#	1.12 ± 0.11#
	Pancreas	0.59 ± 0.04#	0.58 ± 0.03#	1.43 ± 0.06	1.04 ± 0.07#	0.80 ± 0.06#
G6Pase (μmol per mg protein)	Liver	2.10 ± 0.10#	2.02 ± 0.10#	3.23 ± 0.15	2.74 ± 0.05#	2.32 ± 0.06#
	Kidney	1.69 ± 0.07#	1.70 ± 0.13#	2.71 ± 0.10	2.45 ± 0.15#	2.10 ± 0.12#
	Pancreas	1.32 ± 0.08#	1.38 ± 0.10#	3.01 ± 0.21	2.44 ± 0.20#	1.89 ± 0.21#

3.2. Antioxidant parameters

The antioxidant enzymes like CuZn-SOD, catalase and GSH, were assayed in liver, pancreas and kidney samples. The maximum activity of all the antioxidant enzymes was seen in the control group as well as the control group with pyridoxamine supplementation as shown in Table 2. The specific activity of the antioxidant enzymes decreased significantly in the diabetic group, whereas the diabetic group supplemented with pyridoxamine showed recovery of all the enzymatic activities in a dose dependent manner. Amongst all the organs, a maximum reduction of SOD levels was observed in the livers of the diabetic group as compared to the control group while the levels recovered by 43.33% and 77.6% upon pyridoxamine supplementation at a dose of 10 and 15 mg per kg body weight respectively. Almost a 2-fold decrease, which is the maximum decrease, was seen in catalase levels in the pancreas and nearly 44% & 38% in the kidneys and liver respectively. Maximum recovery was seen in the pancreas (76%), followed by the liver (74%) and kidneys (66%) in the diabetic group supplemented with higher doses i.e. 15 mg per kg body weight of pyridoxamine. In the assay for determining GSH levels, a decrease of about 49% was found in the pancreas followed by 43% in the kidneys and 42% in the liver in the diabetic group, while recovery in the pancreas was 72.6%, kidneys 63.4% and liver 73.74%.

Table 2. Dose dependent effect of pyridoxamine on antioxidant enzymes in alloxan induced diabetic rats. SOD, catalase and GSH activities were determined in normal, diabetic, pyridoxamine supplemented control and pyridoxamine supplemented diabetic groups.

Parameters	Samples	Control	Normal + P2	Diabetic	Diabetic + P1	Diabetic + P2
SOD (units per mg protein)	Liver	144.33 ± 9.70#	142.87 ± 8.49#	106.77 ± 5.52	123.04 ± 7.65	135.93 ± 7.07#
	Kidney	47.33 ± 2.72#	46.49 ± 3.79#	28.96 ± 3.22	34.82 ± 2.56	42.12 ± 2.02#
	Pancreas	62.55 ± 4.57#	60.55 ± 3.84#	29.56 ± 1.48	41.67 ± 2.50#	50.47 ± 3.23#
Catalase (units per mg protein)	Liver	60.62 ± 2.58#	59.23 ± 1.87#	37.25 ± 2.21	44.89 ± 2.32	54.52 ± 1.43#
	Kidney	51.17 ± 1.84#	50.31 ± 2.13#	28.19 ± 3.27	34.87 ± 2.24	43.44 ± 2.36#
	Pancreas	55.53 ± 3.15#	54.66 ± 3.22#	26.75 ± 1.67	39.34 ± 2.12#	48.63 ± 2.80#
Glutathione (units per mg protein)	Liver	34.05 ± 1.43#	33.58 ± 2.26#	19.49 ± 2.23	24.69 ± 1.90	30.23 ± 2.81#
	Kidney	30.52 ± 2.39#	29.34 ± 1.32#	17.15 ± 1.63	20.71 ± 1.85	25.64 ± 1.48#
	Pancreas	52.18 ± 3.50#	51.22 ± 3.86#	26.53 ± 0.70	37.13 ± 2.01#	45.18 ± 3.32#

3.3. Lipid peroxidation and protein carbonylation

The final products of lipid peroxidation i.e. MDA and protein oxidation, and their carbonyl contents were measured. A more than 2-fold increase in lipid peroxidation was seen in the liver and pancreas samples of the diabetic group, whereas in the kidneys a less than 2-fold increase was observed as compared to the control group. However, when pyridoxamine was given as supplementation, a significant decrease in the MDA levels in all the target organs was observed. Maximum recovery was shown in the kidneys (78%) followed by the pancreas (76%) and liver (54%) with respect to the diabetic group (Fig. 2a). A slightly less than two-fold increase in carbonyl content in the kidney & liver samples of the diabetic group and a more than two-fold increase in the pancreas compared to the respective control group were observed. There was approximately 70% recovery in the pyridoxamine supplemented (15 mg per kg bw) group in all the target organs i.e. liver, kidneys and pancreas (Fig. 2b).

Fig. 2. (A) MDA levels and (B) carbonyl contents in the liver, kidney and pancreas of the normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups of rats. P1 is pyridoxamine treatment at 10 mg per kg body weight and P2 is pyridoxamine treatment at 15 mg per kg body weight. Results presented are mean ± SD of three independent treatments. # is a p value ≤0.05 compared to the diabetic group.

3.4. Hepatic and renal markers in serum

The markers of liver function such as alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (ALT) were approximated in the serum (Fig. 3a). An increase of more than two fold in the AST and ALT levels of the diabetic group was observed, whereas a prominent recovery was seen in the pyridoxamine supplemented group at a higher dose. The maximum increase in the ALP level was seen in the diabetic group (more than 5-fold) while the B6 supplemented diabetic group showed a decrease of 62%. The marker of kidney function i.e. urea was assayed in the serum samples. As compared to the control group urea levels were found to be elevated by more than 2-fold in the diabetic group. Upon supplementation with pyridoxamine, momentous recovery (73%) was shown in urea levels at a higher dose (Fig. 3b).

Fig. 3. (A) Liver function markers and (B) urea levels in serum. Urea was measured as a kidney function marker and ALT, AST and ALP were measured as liver function markers in the serum of normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups of rats. P1 is pyridoxamine treatment at 10 mg per kg body weight and P2 is pyridoxamine treatment at 15 mg per kg body weight. Results presented are mean ± SD of three independent treatments. # is a p value ≤0.05 compared to the diabetic group.

3.5. Levels of serum biomarkers

HDL, cholesterol, and triglyceride are lipid biomarkers which play a vital role in insulin resistance, and their increased levels are imperative causes of secondary problems including cardiovascular disease, stroke, etc., in diabetes patients. The levels of HDL-C were compromised in diabetic serum (49%) when compared with the control group, which upon supplementation with vitamin B6 were restored to around 68% with respect to the control. On the other hand, serum cholesterol and triglyceride levels were significantly increased by less than 2-fold in the diabetic group when compared to the control group. When B6 was administered, the levels of cholesterol and triglycerides in diabetic rats were found to be inclined towards the normal control (Fig. 4).

Fig. 4. Lipid profiles of normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups of rats. P1 is pyridoxamine treatment at 10 mg per kg body weight and P2 is pyridoxamine treatment at 15 mg per kg body weight. Results presented are mean ± SD of three independent treatments. # is a p value ≤0.05 compared to the diabetic group.

3.6. Histopathological study of the liver and kidney

Different groups of animals were used to carry out histopathological studies. An increase in the build-up of lipid droplets was seen in the diabetic treated group. This increased accumulation is a characteristic of non-alcoholic fatty liver disease. Also, the markers of cirrhosis i.e. bile duct destruction, increased fibrous content, and centrilobular hepatocytic degeneration (indicative of parenchymal tissue cell loss) were observed in the diabetic group. Upon treatment with pyridoxamine, the markers of NASH, fibrosis and cirrhosis showed reduction and improvement was seen when compared with the control (Fig. 5a). For the kidney samples of the diabetic treated group, we observed that there is thickening of the glomerular basement membrane besides edema of distal convoluted tubules and also inflammation of interstitial tubules. All these observations are characteristic of kidney damage. These features were less obvious in the pyridoxamine supplemented tissues (Fig. 5b).

Fig. 5. Histopathology of the liver and kidney. Histopathology of the haematoxylin and eosin-stained sections of rat liver and kidney. Treated is niacin treatment at 15 mg per kg body weight. (A) Normal; (B) Diabetic; and (C) Treated.

3.7. Inhibition of intracellular ROS by pyridoxamine treatment

The levels of intracellular ROS produced in lymphocytes were assessed using a sensitive fluorescent probe i.e. DCFH-DA. DCFHDA is non-fluorescent in nature which diffuses inside the cell membrane and is converted to 2,7-dichlorofluorescein (DCF) in the presence of ROS that makes the cell fluorescent. Lymphocytes isolated from normal rats were not visible due to the lack of fluorescence (Fig. 6a) while lymphocytes from the diabetic group can be clearly seen with bright fluorescence. Lymphocytes from pyridoxamine treated rats exhibited weaker fluorescence indicating a reduced level of intracellular ROS production (Fig. 6c).

Fig. 6. Intracellular production of ROS in lymphocytes. Representative fluorescence microscopy images of lymphocytes isolated from (A) normal rats, (B) diabetic rats, (C) diabetic rats treated with a lower dose of pyridoxamine and (D) diabetic rats treated with a higher dose of pyridoxamine.

3.8. Apoptosis analysis by DAPI-treated lymphocytes using fluorescence microscopy

A deformed and fragmented nucleus is the hallmark of apoptosis which can be easily visualized by the DAPI staining technique. In Fig. 7a, it is clearly visible that the nuclear appearance of cells from normal rats is round and the nucleus which belongs to those cells comprising the diabetic group is fragmented and distorted in appearance (Fig. 7b). However, the photomicrographs of lymphocytes from the pyridoxamine treated group (10 & 15 mg per kg body weight) display concentration dependent revival in the fragmentation and distortion of the nucleus (Fig. 7c and d respectively). The recovery is evident upon comparison of cells to the control, thus this indicates that upon pyridoxamine treatment a significant decrease in apoptosis is perceptible.

Fig. 7. DAPI stained fluorescence images of lymphocytes isolated from (A) normal rats, (B) diabetic rats, (C) diabetic rats treated with a lower dose of pyridoxamine and (D) diabetic rats treated with a higher dose of pyridoxamine.

3.9. Role of pyridoxamine in DNA damage

Due to hyperglycaemia in diabetes, free radicals are produced which are damaging to DNA. This damage was observed by single cell alkaline gel electrophoresis (comet assay). The average tail length of comet is taken as the measure of cellular DNA damage. The tail length was found to be higher in all the target organs (Fig. 8) of the diabetic group when compared to the control group. However, pyridoxamine supplementation protects DNA against free radical mediated damage in all the samples (liver, kidney and pancreas) as evidenced by the decreased average tail length.

Fig. 8. Comet assay. Results of the comet assay are expressed based on the tail length of the normal, diabetic, pyridoxamine supplemented normal and pyridoxamine supplemented diabetic groups of rats. P1 is pyridoxamine treatment at 10 mg per kg body weight and P2 is pyridoxamine treatment at 15 mg per kg body weight. Results presented are mean ± SD of three independent treatments. # is a p value ≤0.05 compared to the diabetic group.

4. Discussion

The gut flora is capable of producing pyridoxamine (vitamin B6) in an appropriate amount, therefore, even if the modern diet is deficient in vitamin B6, an average person does not suffer from this vitamin deficiency. However, the population that is on prolonged medication of statin drugs, anti-diabetic drugs and antibiotics are likely to suffer from metabolic disorders which are resulting from vitamin B6 deficiency due to the altered intestinal flora. In our previous studies, we successfully revealed the antiglycating properties of pyridoxamine. It binds to the sub domain IIA of human serum albumin (HSA) which is one of the two major binding sites of HAS.21 Pyridoxamine was also shown as a protecting agent against DNA damage in lymphocytes and also an efficient quencher of ROS production. Therefore, it was hypothesized that pyridoxamine may prove helpful in controlling diabetes and complications arising from diabetes. For proving the hypothesis, we have examined the anti-diabetic effects of pyridoxamine through rats as animal models.

Diabetes results in significant alterations in the levels of various metabolic enzymes. A persistent hyperglycaemic condition may contribute to various metabolic alterations that pave the way for a number of diabetic complications including macro and micro-vascular complications.22 Diabetes was induced here by alloxan in experimental rats. Some limitations are there for inducing diabetes by alloxan/streptozotocin as sometimes these drugs entirely destroy the beta cells of the pancreas and sometimes at a low dose damaged beta cells revert to normal. Here in this study we have developed a model for diabetes to examine the effect of vitamin B6 in a dose dependent manner. Upon administration of vitamin B6 a significant decrease in blood glucose levels was observed and also the oral glucose intolerance was tested indicating that vitamin B6 is hypoglycaemic in nature. The physical activity of all the animals during the test was observed. The rats in the diabetic group were less active compared to the rats in the normal group. However, upon treatment of rats in the diabetic group with vitamin B6, almost similar activity like normal control rats was seen.

Hexokinase is an insulin dependent glucose metabolic enzyme which plays a key role in the maintenance of glucose homeostasis in cells by phosphorylating glucose to glucose-6-phosphate.23 In our study we have demonstrated that in tissues obtained from the kidney, liver and pancreas of diabetic animals the activity of HK was significantly decreased when compared to the control group, which may be due to insulin deficiency. Also, in our previous studies we have documented that there is a decrease in the activity of HK in the liver of diabetic animals.24,25 Upon treatment with vitamin B6 of diabetic rats an elevation in the HK activity in the liver, kidney and pancreas was seen which could be due to augmented insulin secretion. The increase in the activity of HK when vitamin B6 was supplemented might have caused increased glycolysis and ultimately leading to increase in the utilization of glucose for energy production leading to decreased blood glucose levels. The enzymes namely glucose-6-phosphatase and fructose-1-6-phosphatase are key regulatory enzymes of the gluconeogenic pathway.26 The activities of these key regulatory enzymes were found to be elevated in the liver and kidneys of diabetic rats compared to the control which results in decreased glycolytic flux. Under normal conditions, insulin acts as a suppressor of gluconeogenic enzymes.27 The activities of these gluconeogenic enzymes were recovered to near-normal levels after the supplementation of vitamin B6. The possible mechanism underlying the normalization of enzyme activity by B6 may be due to the increased flux of insulin from β-cells of the islets of Langerhans, which might in turn enhance glucose utilization.

An important role in insulin resistance is played by the serum lipids. The increased level of lipids in serum is an important factor contributing to secondary complications in diabetes including cardiovascular disease, stroke, etc. Phytochemicals are long known for appreciably reducing the lipid concentration in the 3T3-L1 adipocytes. Therefore, they could also be useful in reducing secondary complications caused by increased levels of lipids. Diabetes often goes hand in hand with dyslipidemia and hence, also with increased cardiovascular complications.28 Altered metabolism of triglyceride rich lipoproteins plays an essential role in the development of dyslipidemia. Levels of HDL-C decreased significantly in the diabetic group while in the B6 treated group, the levels tend towards the normal range. The levels of cholesterol and triglyceride were also found elevated in the diabetic group with respect to the control group. A dose dependent decrease was observed with vitamin B6 supplementation in both the levels.

According to Younes et al. excessive free radical generation may lead to lipid peroxidation that is oxidation of cell membrane lipids in the target organs deteriorating the membrane function.29 Proteins are also directly or indirectly inactivated or degraded due to oxidation by ROS affecting their biological functions. In the diabetic state, the glucose auto-oxidation leads to the formation of free radicals.30 This can be considered as an important factor responsible for an apparent decline in the activity of the antioxidant enzymes (SOD, catalase, GSH). The ROS are formed from pre-existing molecules via delocalization of π-electrons in a chemical bond keeping one electron with each fragment or by cleavage of a radical to generate another radical. Formation of these reactive radicals is considered to be a necessary evil because they are the part of various basic metabolic and defence pathways of cells.31 Vitamin B6 protects the antioxidant enzymes from ROS mediated damage due to its antioxidant properties. Reduced glutathione (GSH) is a sensitive oxidative stress marker because it helps to maintain the integrity of the mitochondria and cell membrane. Its compromised level in the cells may deteriorate the membrane permeability making the cell more prone to ROS induced damage.32 The redox state of the cell is maintained by many glutathione based antioxidant enzymes and proteins (GST, GR, GSH and –SH). All the aforementioned enzymes utilize glutathione (GSH) in the reactions. The levels of GSH and GR may decline in the living system under the condition of oxidative stress.

Supplementation with B6 resulted in very significant recovery in all the parameters in a dose dependent manner. The levels of antioxidant enzymes and proteins became normalized with an improvement in lipid peroxidation and protein oxidation in the diabetic group. Diabetes impairs the functions of the kidneys, so the increased blood levels of urea and creatinine are indicative of kidney health in glomerular filtration,33 which is due to oxidative stress. Also, Ravi et al. showed that the ROS generated in diabetic rats can damage the liver and kidney tissues along with the disruption in their functions.

With pyridoxamine supplementation the levels of liver function markers (ALT, AST, and ALP) and the kidney function marker (urea) in serum samples were restored to the normal range. Previously, Marzouk et al. (2013) showed that fenugreek and termis which are known antioxidants reverted the levels of AST, ALT, GSH and muscle glycogen.34 The findings are in agreement with previous results in which antioxidants reduced the level of urea.35 On histopathological grounds the diabetic group showed an increase in the accumulation of lipid droplets which are a characteristic of non-alcoholic fatty liver disease in hepatocytes. Along with this the destruction of bile duct, a marker of cirrhosis, a significant increase in fibrous content (fibrosis) and centrilobular hepatocytic degeneration (suggestive of parenchymal tissue cell death) were observed whereas in the group treated with pyridoxamine all the markers of NASH (nonalcoholic steatohepatitis), cirrhosis and fibrosis were improved showing similarity with the control group. We observed thickening of the glomerular basement membrane of the kidney in the diabetic group and also edema of distal convoluted tubules. However, all these changes were improved and tended towards normal in the kidney tissues of the group treated with B6. The hydroxyl radical is the main culprit in causing cellular DNA damage via the Fenton pathway. This was also appreciably ameliorated in the B6 supplemented group. Among all the concentrations studied for pyridoxamine, the concentration of 15 mg per kg bw of pyridoxamine was found most effective in inhibiting DNA strand breakage and nuclear deformation in all the target organs and lymphocytes as evident from the comet assay and DAPI-staining technique. Our results are in agreement with a number of earlier studies that reveal that vitamin B6 is capable of quenching ROS production and hydroxyl radicals to safeguard cellular DNA from damage resulting from diabetes.21,36,37

5. Conclusion

Hyperglycemia is the hallmark of diabetes and major cause of various diabetic complications viz. retinopathy, nephropathy, neuropathy and cardiovascular disease. The results of fasting blood glucose and glucose metabolic enzymes showed the positive effects of vitamin B6 in improving the status of the system in diabetes. Other parameters like antioxidant enzyme and oxidative stress markers like SOD, catalase, GSH, lipid peroxidation and protein oxidation and DCFH-DA microscopy showed significant recovery in the treated group in comparison with the diabetic group. Furthermore, the histopathological studies of liver and kidney tissues and cellular DNA damage and nuclear distortion analysis also support the protective role of vitamin B6 in diabetes. This study is likely to provide important insight into the remedial potential of pyridoxamine in the treatment of diabetes and may broaden the understanding of the role and distribution of B6 in nature and its therapeutic efficacy.

Author contributions

Prof. Imrana Naseem designed the experiment and all the experiments were conducted by Mr K.M. Abdullah along with data analysis. Hamza Hasan helped out in the experimental work as a lab intern and Faizan Abul Qais performed statistical analysis along with the paper writing of some experiments.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed and all methods were performed in accordance with the relevant guidelines and regulations.

Data availability statement

Data can be made available on request.

Abbreviations

DM

Diabetes mellitus

ROS

Reactive oxygen species

SOD

Superoxide dismutase

GSH

Reduced glutathione

FBPase

Fructose bis phosphatase

G6Pase

Glucose-6-phosphatase

MDA

Malondialdehyde

AST

Aspartate transaminase

ALT

Alanine transaminase

TBARS

Thiobarbituric-reactive substances

FBG

Fasting blood glucose

DCF

2,7-Dichlorofluorescein

DAPI

4′,6-Diamidino-2-phenylindole

Conflicts of interest

The authors declare that there is no conflict of interest in this work.