Ameliorative effect of riboflavin on hyperglycemia, oxidative stress and DNA damage in type-2 diabetic mice: Mechanistic and therapeutic strategies

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Highlights

  • Riboflavin (RF) ameliorates oxidative stress and DNA damage in diabetic mice.

  • Photo-illuminated RF act as pro-oxidant while non-illuminated act as an antioxidant.

  • RF showed a significant recovery in liver and kidney marker enzymes and tissue damage.

  • RF as a dietary compound might help in the reduction of damage caused by metabolic diseases.

Abstract

Increasing evidence in both experimental and clinical studies suggests that oxidative stress play a major role in the pathogenesis of type-2 diabetes mellitus (T2DM). Abnormally high levels of free radicals and the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular organelles and enzymes. Riboflavin constitutes an essential nutrient for humans and is also an important food additive for animals. It is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which serves as a coenzyme for several enzymes. The aim of this study was to observe the effects of illuminated and non-illuminated riboflavin in a diabetic mice model. The protocol included treatment of diabetic mice with illuminated RF and a control set without light. To our surprise, group receiving RF without light gave better results in a dose dependent manner. Significant amelioration of oxidative stress was observed with an increased glucose uptake in skeletal muscles and white adipose tissue. Histological studies showed recovery in the liver and kidney tissue injury. Cellular DNA damage was also recovered. Therefore, it is suggested that supplementation with dietary riboflavin might help in the reduction of diabetic complications. A possible mechanism of action is also proposed.

Introduction

Diabetes mellitus (DM) is a group of metabolic diseases characterized by hyperglycemia resulting from defects of insulin action, insulin secretion or both. DM is classified into two main types, type 1 and type 2 diabetes mellitus (T1DM and T2DM); both are characterized by defects in the body's ability to control glucose and insulin homeostasis. In particular, T2DM, which accounts for more than 90% of all diagnosed DM cases, is primarily the result of insulin resistance, and has been linked to risk factors including smoking, ageing, sedentary lifestyle, obesity, and unhealthy eating habits. Although the underlying pathogenic mechanisms have not yet been fully clarified, oxidative stress is unanimously considered to contribute significantly to the onset and progression of T2DM [1]. In general, the development of T2DM is associated with pancreatic β-cell dysfunction and insulin resistance. Normal β-cells can compensate for insulin resistance by increasing insulin secretion, but insufficient compensation leads to the onset of glucose intolerance. There is no doubt that T2DM and its related complications are associated with increased oxidative stress resulting from the imbalance in the production of free radicals and the inability of the body's antioxidant defence system [2]. Since, reactive oxygen species (ROS) has an important role in the aetiology of diabetes and its complications; antioxidant therapy has been proposed to preserve β-cell function by suppression of β-cell apoptosis [3].

Currently, many drugs are being used for the treatment of diabetes; however, their continuous use is associated with adverse effects. The high cost of these drugs is another issue in both developed and developing countries, thereby paving the way for alternative approaches in the management of diabetes [4], [5]. Numerous of studies conducted in the past have shown promising results with the treatment or use of herbal and natural products [6], [7], [8]. One such vitamin riboflavin (RF), also known as vitamin B2, is essential for normal cellular functions, growth and development. RF is widely distributed in a variety of food products, where it is found as exclusively bound to proteins, mostly in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). In its coenzyme forms of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), RF performs key metabolic functions as an intermediary in the transfer of electrons in biological oxidation–reduction reactions. RF is also essential for the intermediary metabolism of carbohydrates, amino acids and lipids in addition to its role in supporting cellular antioxidant potential. During periods of dietary deprivation or physiological and pathological stress, humans are vulnerable to developing RF deficiency. This may lead to a variety of clinical abnormalities, including growth retardation, anaemia, skin lesions, renal damage and degenerative changes in the nervous system. RF deficiency has been observed in the elderly, and in people having eating disorders, diabetes, chronic heart disease, inflammatory bowel disease and HIV [9], [10], [11]. Recent studies have shown that RF can protect tissues from oxidative damage. In a murine heart transplantation model, RF reduces myocardial lipid peroxidation, leukocyte infiltration, cytokine production, and cardiac allograft vasculopathy [12]. RF has also been reported to have a protective role on the rat brain after ischaemia, on rabbit myocardium during re-oxygenation [13], [14]. Moreover, riboflavin has been found to increase the survival rate of mice suffering endotoxin-induced sepsis and gram-negative and gram-positive bacterial infection. It was thus hypothesise that riboflavin may protect the diabetes from the progressive damage caused by ROS in the long run. To test this hypothesis, we investigated the effects of riboflavin on alterations of hyperglycemia, oxidative stress, tissue and DNA damage in diabetic mice.

Section snippets

Ethical statement for animal experimentation

Animal experimentations were permitted by Ministry of Environment and Forests, Government of India under registration no. 714/02/a/CPCSEA issued by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) dated 25th October, 2012 and approved 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).

Experimental animals

Forty two adult Swiss albino mice of six month's age

Effect of riboflavin on FBG level

Results in Fig. 1 show that fasting blood glucose (FBG) levels in treated and untreated groups vary significantly. The FBG levels were found to be increased in the diabetic group (244.63 ± 4.97 mg/dl), confirming the establishment of diabetes compared to the control group (105.30 ± 2.43 mg/dl) and normal group supplemented with riboflavin (108.50 ± 2.62 mg/dl). The diabetic group supplemented with riboflavin showed dose dependent decrease in the FBG levels. Riboflavin at 20 and 10 mg/kg body

Discussion

Riboflavin (Vitamin B2) deficiency is a common feature of modern diets, but the symptoms of riboflavin deficiency are prevented due to its production by the gut microflora. The consumption of variety of drugs including anti-diabetics and antibiotics are known to have an adverse effect on the intestinal microflora. This can have far reaching metabolic consequences as riboflavin is the precursor of coenzymes FAD and FMN, which play an important role in carbohydrate and fat metabolism. Riboflavin

Conclusions

It is concluded that riboflavin could act as an antioxidant against oxidative stress, especially lipid peroxidation, protein carbonylation and oxidative DNA damage. The mechanisms by which riboflavin protects the body against oxidative stress may be attributed to the glutathione redox cycle and also to other possible mechanisms such as conversion of reduced riboflavin to the oxidised form. However, most of the investigations in this area are limited to experimental studies and, therefore,

Conflict of interest

The authors declare there are no conflicts of interest.

Acknowledgements

The authors acknowledge the financial assistance provided by the University Grant Commission (UGC), New Delhi under SAP programme and the facilities provided by the Department of Biochemistry, Aligarh Muslim University. We are grateful to Professor S.M.A. Abidi and Ahammed Shareef (Department of Zoology, AMU, Aligarh) for histopathology. We are also thankful to Dr. Sandesh Chibbar, all the friends, lab colleagues and fellows who directly or indirectly helped us during different phases of

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