The protective effect of 1alpha, 25-dihydroxyvitamin d3 and metformin on liver in type 2 diabetic rats

https://doi.org/10.1016/j.jsbmb.2016.11.012Get rights and content

Highlights

  • 1,25(OH)2D3 has an insulin sensitizing effect and can attenuate insulin resistance.

  • 1,25(OH)2D3 decreases lipotoxicity and modulates the inflammatory response.

  • 1,25(OH)2D3 inhibits apoptosis and stimulates proliferation of liver cells.

  • Metformin had similar effects to that produced by 1,25(OH)2D3.

  • Using combination of both agents gives a better protection.

Abstract

There is an accumulating evidence suggesting an immunomodulatory role of 1α,25(OH)2D3. Altered 1α,25(OH)2D3 level may play a role in the development of T2DM and contribute to the pathogenesis of liver diseases. Our study was designed to study and compare the effect of metformin and 1α,25(OH)2D3 supplementation on liver injury in type 2 diabetic rat.

Sixty male Albino rats were divided into 5 groups; group 1: control rats. the remaining rats were fed high fat diet for 2 weeks and injected with streptozotocin (35 mg/kg BW, i.p.) to induce T2DM and were divided into: group 2: untreated diabetic rats, group 3: diabetic rats treated by metformin (100 mg/kgBW/d, orally), group 4: diabetic rats supplemented by 1α,25(OH)2D3 (0.5 μg/kg BW, i.p.) 3 times weekly and group 5: supplemented by both 1α,25(OH)2D3 and metformin. Eight weeks later, serum glucose and insulin levels were measured, HOMA IR was calculated, lipid profile, Ca2+, ALT and AST were estimated. Liver specimens were taken to investigate PPAR-α (regulator of lipid metabolism), NF-κB p65, caspase 3 and PCNA (proliferating cell nuclear antigen) and for histological examination.

The liver enzymes were elevated in the diabetic rats and the histological results revealed an injurious effect of diabetes on the liver. 1α,25(OH)2D3, metformin and both drugs treatment significantly improved liver enzymes as compared to the untreated rats. The improvement was associated with a significant improvement in the glycemic control, lipid profile and serum Ca2+ with a significant reduction in NF-κB p65 and caspase 3 and increased PPAR-α, and PCNA expression as compared to the untreated group. 1α,25(OH)2D3 induced a slightly better effect as compared to metformin. Both agents together had a synergistic action and almost completely protected the liver. Histological results confirmed the biochemical findings.

Our results showed a protective effect of 1α,25(OH)2D3 and metformin on liver in diabetic rats as indicated by an improvement of the level of the liver enzymes, decreased apoptosis and increased proliferation and this was confirmed histologically, with modulating NFkB and PPAR-α. Both agents together had a synergistic effect.

Introduction

Type 2 diabetes mellitus (T2DM) is one of the most common chronic diseases. Over the past three decades, its prevalence has more than doubled globally, therefore, it represents an eminent challenge to all nations [1]. Chronic complications of diabetes are the main causes of disability and mortality in diabetic patients [2].

A large spectrum of liver disease is documented in type 2 diabetic patients, including elevated liver enzymes, fatty liver disease, cirrhosis, hepatocellular carcinoma, and acute liver failure. The standardized mortality rate from uncompensated cirrhosis is higher than that from cardiovascular disease among diabetic patients [3].

Chronic hyperglycemia and insulin resistance induce an inflammatory response in the liver through accumulation of reactive oxygen species, which triggers the nuclear factor kappa beta (NF-κB) pathway [4]. Inflammation, together with disturbed systemic and hepatic fat metabolism, is the main causative factor underlying liver injury in diabetes [5]. Triglycerides accumulation within the hepatocytes is the main characteristic feature of fatty liver in T2DM [6].

Peroxisome proliferator-activated receptor α (PPAR-α), a ligand-activated transcription factor belonging to the nuclear receptor family, is highly expressed in the liver. It plays a crucial role in regulating hepatic lipid metabolism [7]. It has been reported that PPAR-α expression is decreased in diabetic liver [8].

Furthermore, caspase-3 is essential for the apoptotic process, being an effector caspase downstream of apoptotic pathways [9]. Haligur et al. found an increased apoptotic activity within the hepatocytes in streptozotocin induced diabetic rats and suggested that a possible mechanism of liver complications in diabetes may be linked to the increased apoptotic activity [10].

On the other hand, proliferating cell nuclear antigen (PCNA), a 36 kDa non-histone protein found in the nucleus, mediates DNA polymerase. Its expression has an extensive correlation with mitotic activity and can serve as a marker for cell proliferation [11], [12].

1,25(OH)2D3 receptors are disseminated in more than 38 tissues [13]. Animal studies demonstrated that vitamin D receptors (VDR) knockout mice showed spontaneous liver injury and fibrosis [14]. Also, VDR expression was found to be negatively correlated with the severity of non alcoholic fatty liver disease (NAFLD) [15].

Vitamin D insufficiency is frequent in patients with T2DM [16], [17]. In fact vitamin D deficiency was related to the development of insulin resistance and many pathological complications of T2DM [18] and has been associated with an impaired glycemic control [19]. Mathieu et al. suggested that vitamin D deficiency had a detrimental effect to beta cell function and this caused glucose intolerance and predisposed to type 2 diabetes [20].

Many evidences suggested the key role of 1,25(OH)2D3 in decreasing the risk of chronic diseases, including T2DM [21]. Administration of 1,25(OH)2D3, led to diabetes prevention through immunomodulatory effects [20]. Other studies showed that vitamin D, as a lipid metabolism modulator, can correct dyslipidemia in overweight subjects [22], [23]. Based on these information, it can be suggested that 1,25(OH)2D3 supplementation may help in modulating the hepatic inflammation and lipid metabolism in T2DM rat models and protect against the liver injury associated with prolonged diabetes.

Metformin is the most widely used oral hypoglycemic agent as first-line therapy for T2DM and proved its efficiency in attenuating many complications of T2DM [24].

The aim of this study is to examine the possible effect of 1α,25(OH)2D3 supplementation on the diabetic state and liver functions, to compare the role of 1α,25(OH)2D3 and metformin in providing protection for the liver in type 2 diabetic rat, and to explore the underlying mechanism for their actions.

Section snippets

Animals

The study was in agreement with the approved guidelines and its protocol was approved by the Committee on the Ethics of Animal Experiments of the Faculty of Medicine Cairo University.

Sixty adult male Wistar Albino rats weighting 100–120 gm, were included in this study and were housed in wire mesh cages at comfortable temperature about 22 °C, normal light and dark periods in Physiology Department, Faculty of Medicine, Cairo University. The rats were provided with veterinary care by the Laboratory

ALT

The 1,25(OH)2D3 supplementation, either alone or in combination with metformin, improved the ALT level as compared to the untreated diabetic group. Moreover, the combined treatment had no significant difference as compared to the control group, while the group treated with metformin only showed a non significant decrease in the level of ALT as compared to the untreated diabetic group (Table 1).

AST

All treated groups showed a significant decrease in the level of AST as compared to the untreated

Discussion

Some previous studies showed that vitamin D plays a key role in modulating metabolic homeostasis, insulin secretion and insulin sensitivity [35]. Insulin resistance, the main problem in type 2 diabetes mellitus, is significantly involved in the development of the associated liver diseases [36]. People with T2DM often have lower circulating vitamin D levels than those without diabetes [37], and this was believed to contribute to an increased risk of diabetic complications and mortality.

Thus this

Conclusion

In conclusion, we can suggest from the results of the present study that 1,25(OH)2D3 as well as metformin can have beneficial effects in protection against liver injury in T2DM. These effects may be through the modulation of glucose metabolism, correcting the disturbed lipid profile and hepatic PPARα. Each drug was able to ameliorate insulin secretion and sensitivity, attenuating the inflammatory response and promotes hepatocyte survival via inhibiting apoptosis and stimulation of cellular

Acknowledgement

This study was financially partially supported by Cairo University.

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    S. Elattar and S. Estaphan are joint first authors contributing equally to this manuscript.

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