AIP Conference Proceedings 2092, 020004 (2019); https://doi.org/10.1063/1.5096672 2092, 020004
© 2019 Author(s).
Developing vitamin B12 deficient rat model based on duration of restriction diet: Assessment of plasma vitamin B12, homocysteine (Hcy), and blood glucose levels
Cite as: AIP Conference Proceedings 2092, 020004 (2019); https://doi.org/10.1063/1.5096672 Published Online: 09 April 2019
Imelda Rosalyn Sianipar, Irena Ujianti, Sophie Yolanda, Aditya K. Murthi, Patwa Amani, and Dewi Irawati Soeria Santoso
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Developing Vitamin B12 Deficient Rat Model Based on Duration of Restriction Diet: Assessment of Plasma Vitamin
B12, Homocysteine (Hcy), and Blood Glucose Levels
Imelda Rosalyn Sianipar
1,a), Irena Ujianti
2, Sophie Yolanda
1, Aditya K Murthi
2,3, Patwa Amani
3,4, Dewi Irawati Soeria Santoso
11Department of Physiology, Faculty of Medicine, Universitas Indonesia
2Master Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia
3Department of Physiology, Faculty of Medicine, Trisakti University
4Doctoral Program in Biomedical Science, Faculty of Medicine, Universitas Indonesia
a)Corresponding author: [email protected]
Abstract. Many studies indicate a crucial role of vitamin B12 in hyperhomocysteinemia and hyperglicemia. In this study, through dietary restriction, we developed rat models of vitamin B12 deficiencies to elucidate the impact on hyperhomocysteinemia and hyperglycemia. We investigated whether there is a correlation between plasma Vitamin B12 with homocysteine and the presence of hyperglycemia in male rats. Adult male mice were divided into 3 groups (a) Control diet for 16 weeks (b) vitamin B12-restricted diet with pectin as dietary fiber for 8 weeks (K1) (c) vitamin B12- restricted diet with pectin as dietary fiber for 16 weeks (K2). After 8 weeks of feeding, plasma concentrations of vitamin B12, homocysteine, and glucose were assessed. Blood samples for vitamin B12, homocysteine and glucose were obtained after overnight fasting. After 16 weeks of continued feeding on their respective diets, plasma concentrations of vitamin B12, homocysteine, and glucose were assessed. Plasma vitamin B12 levels were lower in K2 compared to both the control and K1 groups. On the other hand, the deficient B12 group had significantly higher levels of homocysteine and glucose than control on 8 and 16 weeks. In this study, we concluded that hyperglycemia was associated with lower serum vitamin B12 concentration and serum homocysteine in the development of vitamin B12 deficient rat model.
Keywords: blood glucose, homocysteine, hyperglycemia, rat model, vitamin B12.
INTRODUCTION
Vitamin B12 (B12), the largest and most complex vitamin molecule, is exclusively synthesized by certain bacteria, and is most abundant in higher order predators in the natural food chain. After B12 is taken up by living cells, it is converted into two coenzyme forms, 5-deoxyadenosylcobalamin (Ado-B12) and methylcobalamin (CH3- B12, which function as the coenzymes for methylmalonyl-CoA mutase and methionine synthase, respectively [1].
The major dietary source of B12 is food derived from animal product; therefore, strict vegetarians and/or elderly people are at a high risk of developing B12 deficiency [2]. The major symptoms of B12 deficiency are developmental disorders, megaloblastic anemia, metabolic abnormalities, and neuropathy, although the underlying disease mechanisms are poorly understood [3]. Developing animal models of B12-deficiency is essential for investigating the molecular mechanisms that are defective in this metabolic disorder. However, such animal models have proven difficult to generate because animals must be fed with a B12-deficient diet for long periods of time to achieve B12 deficiency.
Sprague Dawley rats offer several advantages for genetic and biochemical studies, because of their short lifespan, and complete sequenced genome that is similar to human. In addition, many molecular and cellular processes are conserved between humans and rats. Most human disease genes and pathways are present in the rat.
Thus, this animal has been widely used as a model for studying a variety of biological processes including apoptosis, cell signaling, cell cycle regulation, gene regulation, metabolism, and aging [4].
The purpose of this study is to develop a B12 deficient animal model and also to study its effect on hyperhomocysteinemia and hyperglycemia condition. Methionine synthase and methylmalonil coAmutase are enzymes that require vitamin B12 as a co-factor for its activity [5]. Methionine synthase activity is reduced in vitamin B12 deficiency conditions. This results in disruption of homocysteine (Hcy) metabolism, homocysteine accumulation and formation of intra-cell homocysteine thiolactone. The interaction of homocysteine thiolactone with Insulin Receptor Substrates or p85 of PIP3K results in the inhibition of insulin signaling [6,7]. For this purpose, Sprague Dawley rat were given special vitamin B12-deficient diet. Inhibition of insulin signaling was observed through examination of plasma B12 concentration, plasma homocysteine concentration and fasting blood glucose levels.
METHOD
Animal Maintenance and Feeding
This study was part of a larger research project at the Department of Medical Physiology FMUI. This in-vivo experimental study used male Sprague Dawley rats aged 36-40 weeks with body weight ranging from 300-350 grams (purchased from Badan Penelitian dan Kesehatan RI). Based on the Mead formula, total samples of 18 rats were used. Rats were treated in accordance with the Helsinki convention. This research has been approved by the Health Research Ethics Committee - Faculty of Medicine Universitas Indonesia/Cipto Mangunkusumo Hospital (FMUI/RSCM) No. 184/UN2.F1/ETIK/2017.
Rats were placed in individual cages in a room with proper ventilation. Room temperature was maintained between 18-26°C with humidity of 30-70% and a 12 hours light and dark cycle. Rat cages were cleaned every day to ensure the rat’s health. After one week of acclimatization, they were randomly divided into three groups. The first group was fed with AIN-93M (Research Diets Inc., USA) ad libitum as the control group (Group 1, n = 6, designated as C). The second group was fed with modified AIN-93M ad libitum (AIN-93M with rescricted-B12) with additional pectin/cellulose as a source of fiber for eight weeks (Group 2, n = 6). The third group was fed with the same modified AIN-93M diet ad-libitum (Group 3, n =6) for 16 weeks. The content of vitamin B12 modified diet was much lower than the control diet (0.016 vs. 28 mcg/kg diet); the modified diet contained 50 g pectin/kg to bind the intrinsic factor in the intestine and to makes vitamin B12 less bioavailable. Food intake was recorded daily, and body weight was measured every week.
After 8 weeks and 16 weeks of feeding, concentrations of plasma vitamin B12, homocysteine and glucose were measured.
Biochemical Parameters
At the end of 8 and 16 weeks of feeding the respective diets, blood was collected from the supraorbital sinus, after overnight fasting. Fasting blood glucose was measured using hexokinase assay method. Plasma vitamin B12 and homocysteine level examinations were performed using a standard ELISA kit (Rat insulin ELISA, MyBiosource, USA). Vitamin B12 and homocysteine assays were performed at the immuno-endocrinology laboratory, Faculty of Medicine, Universitas Indonesia.
Statistical analysis
Statistical analysis was performed using one-way ANOVA test followed by post-hoc analysis for significance of difference among the groups. T test was performed for significance of difference between the control and treatment group. Data were previously analyzed for normality with ShapiroWilk test. Data processing was performed using SPSS 12 (Statistical Social Sciences 12).
RESULTS
Establishing a Rat Model for Vitamin B12 Deficiency
After 8 weeks and 16 weeks of feeding the respective diets, we measured plasma vitamin B12 and homocysteine levels. The result showed that after only 8 weeks of feeding modified AIN-93M diet, we successfully established vitamin B12-deficient rat models.
There was a significant decrease in plasma vitamin B12 levels in the eight-week treatment group when compared to the eight-week control group. Plasma vitamin B12 in the eight-week treatment group was significantly lower compared to the eight-week control group (463.52 ± 28.03 pg/ml vs 670.2 ± 19.06pg/ml, p< 0.001). Plasma vitamin B12 levels in the sixteen-week treatment group were significantly lower compared to the sixteen-week control group (472.24±24.09 pg/ml vs 666.5 ± 52.63 pg/ml pg/ml, p < 0.05). No significant difference was observed between the 8-week and 16-week treatment groups.
FIGURE 1. Means of plasma Vitamin B12 levels
* p < 0.001 vs. control group at 8-week, ** p < 0.05 vs. control group at 16-week
Plasma homocysteine level in the eight-week treatment group was significantly higher compared to the eight- week control group (455.48 ± 39.056 nmol/ml vs 219.2 ± 28.072 nmol/ml, p <0.001). Plasma homocysteine level in the sixteen-week treatment group was significantly higher compared to the sixteen-week control group (1055.05 ± 172.75 nmol/ml vs 282.37 ± 43.38 nmol/ml, p <0.05).
FIGURE 2. Means of plasma homocysteine levels.
* p < 0.001 vs. control group at week 8, ** p < 0.05 vs. control group at week 16
Plasma Vitamin B12 (pg/ml)Plasma homocysteine (nmol/ml)
Fasting Blood Glucose
There was a significant difference in fasting blood glucose levels between the control groups compared to the treatment group. Fasting blood glucose level in the eight-week treatment group was significantly higher compared to the eight-week control group (189.66 ± 15.97 mg/dl vs 104.83 ± 3.19mg/dl, p<0.05). Fasting glucose in the sixteen- week treatment group was also significantly higher compared to the sixteen-week control group (144.6 ± 5.79 vs 100 ± 6.3 mg/dl, p<0.001).
FIGURE 3. Means of plasma glucose levels
* p < 0.05 vs. control group at week 8, ** p < 0.001 vs. control group at week 16
DISCUSSION
Vitamin B12 deficiency is silent and common in general population [8]. Causes of Vitamin B12 deficiencies are multifactorial and associated with many health problems [9]. Vitamins and minerals (micronutrients) play a central role in cellular metabolism, maintenance, and growth throughout life and are helpful in prevention and/or cure of various disorders during the course of life [10]. Vitamin B12 is an important micronutrient essential for numerous vital processes in our body including normal functioning of the brain and nervous system and for the formation of blood. A regular dietary supply of vitamin B12 across the life course is essential due to its role as methyl donor and in vital functions of normal growth, development, and maintenance of various physiological functions [5, 10, 11].
Deficiency of vitamin B12 is associated with pernicious anemia, neural defects, and atherosclerosis [12, 13].
Considering that vitamin B12 deficiency is widespread in developing countries, in this study, we assessed the effect of specific micronutrient vitamin B12 deficiency in 9-month-old Sprague Dawley male rats by feeding them with a modified AIN-93M (vitamin B12 restricted) diet. Pectin/cellulose were added in the modified diet to induce deficiency as it makes vitamin B12 less bioavailable and also promotes depletion of endogenous vitamin B12 due to its enterohepatic circulation [14]. Male Sprague Dawley rats were used in this study because the plasma levels of vitamin B12 observed in these rats were similar to those reported in vitamin-B12-deficient subjects [5, 15].
Our results showed that B12 deficient diet causes a gradual decrease in the B12 plasma concentration in rats.
Hcy is an indicator of B12 deficiency on both control and B12-deficient conditions. There was a significantly higher Hcy levels in the eight-week and sixteen-week treatment groups compared to the control group. Hcy levels were approximately four and five times greater, after eight weeks and sixteen weeks of B12-deficient treatment respectively, compared to the control group [14]. This study supports the use of vitamin B12 deficient rat as an animal model for hyperhomocysteinemia. In vitamin B12 deficient rats, methionine metabolism is impaired [16].
Methyl group of B12 functions as a coenzyme of Methionine Synthase which catalyzes the methyl transfer from methyltetrahydrofolate to homocysteine, resulting in the donation of a methyl group to homocysteine, forming methionine [1]. Methionine Synthase is important to re-synthesize methionine and to metabolize methyltetrahydrofolate. In patients with cobalamin deficiency leading to decreased liver transsulfuration pathways in homocysteine metabolism, plasma homocysteine will increase [17, 18]. Increased plasma homocysteine causes oxidative stress, resulting in insulin resistance through the ROS pathway [19, 20].
The oxidative stress produced by the conversion of Hcy to Hcy-thiolactone leads to the inhibitory activity of the tyrosine kinase insulin receptor [21-23]. An action of homocysteine on the insulin secretory pathway was
Plasma glucose (mg/dl)
demonstrated [22] which found that homocysteine thiolactone, the active form of homocysteine, inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor b-subunit and its substrates insulin receptor substrate- 1 and p60-70 in rat hepatoma cells. In addition, they showed that homocysteine thiolactone decreased the p85 regulatory subunit of phosphatidylinositol 3-kinase activity, inducing a reduction in insulin-stimulated glycogen synthesis. In that in vitro study, the effects of 100 nM insulin were completely blocked by 50mM homocysteine thiolactone, suggesting that 1mM homocysteine thiolactone inhibited 300 mU/ml insulin. This partly explains the result of our in vivo study in which a 3.4 mmol/l decrement of homocysteine levels was associated with a decrease in circulating insulin levels of 5 mU/ml; but this important issue deserves further investigation [22]. Insulin signaling disorder results in an increase in plasma blood glucose. The significantly higher fasting blood glucose levels in rats receiving fed restricted B12 diet compared to the control group indicated that the Vitamin B12 restricted diet ad libitum for 16 weeks had led to the development of insulin resistance. This is consistent with several studies that suggest a link between increased homocysteine due to vitamin B12 deficiency with insulin resistance [21, 22].
In summary, in this study, we have provided insights on the effects of vitamin B12 deficiencies in 9-month-old male Sprague Dawley rats on plasma vitamin B12, homocysteine and glucose levels. We also succeeded in developing Vitamin B12 deficient rat model after 8 weeks of restricted diet, which led to hyperhomocysteinemia condition and hyperglycemia.
ACKNOWLEDGMENTS
This work was supported by HIBAH PITTA 2018 funded by DRPM Universitas Indonesia No.2089/UN2.R3.1/HKP.05.00/2018.
REFERENCES
1. Chen Z, Crippen K, Gulati S, Banerjee R. Purification and kinetic mechanism of a mammalian methionine synthase from pig liver. J Biol Chem. 1994; 269(44):27193–7.
2. Moreira ES. Vascular Biochemistry of Vitamin B12: Exploring the Relationship between Intracellular Cobalamin and Redox Status in Human Aortic Endothelial Cells. Kent State University; 2010.
3. Wilson, Berenthal M. Dietary Reference Intake. Washington DC : National Academic Press; 2009:6-9
4. Shanks N, Greek R, Greek J. Are animal models predictive for humans? Philos Ethics, Humanit Med.
2009;4(1):1–20.
5. Guéant JL, Caillerez-Fofou M, Battaglia-Hsu S, Alberto JM, Freund JN, Dulluc I, et al. Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase.
Biochimie . 2013;95(5):1033–40.
6. Jakubowski H. Metabolism of homocysteine thiolactone in human cell cultures. J Biol Chem.
1997;272(3):1935–42.
7. Jakubowski H. Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci. 2004;61(4):470–87.
8. Baltaci D, Kutlucan A, Turker Y, Yilmaz A, Karacam S, Deler H, et al. Association of vitamin B12 with obesity, overweight, insulin resistance and metabolic syndrome, and body fat composition; primary care-based study. Med Glas (Zenica) . 2013;10(2):203–10.
9. Solomon LR. Disorders of cobalamin (Vitamin B12 ) metabolism : Emerging concepts in pathophysiology , diagnosis and treatment. 2007;113–30.
10. Pooya S, Blaise S, Garcia MM, Giudicelli J, Alberto J, Jeannesson E, et al. Methyl donor deficiency impairs fatty acid oxidation through PGC-1 a hypomethylation and decreased ER- a , ERR- a , and HNF-4 a in the rat liver. J Hepatol. 2012;57(2):344–51.
11. Bito T, Matsunaga Y, Yabuta Y, Kawano T, Watanabe F. Vitamin B12 deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and reduced lifespan. FEBS Open Bio [Internet]. 2013;3:112–7.
12. Gherasim C, Lofgren M, Banerjee R. Navigating the B12 road: Assimilation, delivery, and disorders of cobalamin. J Biol Chem. 2013;288(19):13186–93.
13. Yousaf F, Spinowitz B, Charytan C. Pernicious Anemia Associated Cobalamin Deficiency and Thrombotic Microangiopathy: Case Report and Review of the Literature. Case Reports. 2017;2017.
14. Cullen RW, Oace SM. Dietary pectin shortens the biologic half-life of vitamin B-12 in rats by increasing fecal and urinary losses. J Nutr. 1989;119(8):1121–7.
15. Bottle A, Millett C, Antonysunil A, Kumsaiyai W, Voyias P, Gates S, et al. Basic and clinical science posters : lipids and fatty liver Basic and clinical science posters : molecular basis and treatment of complications Basic and clinical science posters : new insulins , technology , therapies and treatment. 2017;34:67–8.
16. Kovierov H, Vidomanov E, Mahmood S, Sopkov J, Drgov A, Erve ov T, et al. The molecular and cellular effect of homocysteine metabolism imbalance on human health. Int J Mol Sci. 2016;17(10):1–18.
17. Gamble M V., Ahsan H, Liu X, Factor-Litvak P, Ilievski V, Slavkovich V, et al. Folate and cobalamin deficiencies and hyperhomocysteinemia in Bangladesh. Am J Clin Nutr. 2005;81(6):1372–7.
18. Mahalle N, Kulkarni M V, Garg MK, Naik SS. Vitamin B12 deficiency and hyperhomocysteinemia as correlates of cardiovascular risk factors in Indian subjects with coronary artery disease. 2013;61(4):289–94.
19. Tyagi N, Sedoris KC, Steed M, Ovechkin A V, Moshal KS, Tyagi SC, et al. Mechanisms of homocysteine- induced oxidative stress. Am J Physiol Heart Circ Physiol. 2005;289(6):H2649-56.
20. Hayden MR, Tyagi SC. Homocysteine and reactive oxygen species in metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy: The pleiotropic effects of folate supplementation. Nutr J. 2004;3-4
21. Najib S, Sánchez-Margalet V. Homocysteine thiolactone inhibits insulin-stimulated DNA and protein synthesis: Possible role of mitogen-activated protein kinase (MAPK), glycogen synthase kinase-3 (GSK-3) and p70 S6K phosphorylation. J Mol Endocrinol. 2005;34(1):119–26.
22. Najib S, Sánchez-Margalet V. Homocysteine thiolactone inhibits insulin signaling, and glutathione has a protective effect. J Mol Endocrinol. 2001;27(1):85–91.
23. Jakubowski H. The molecular basis of homocysteine thiolactone-mediated vascular disease. Clin Chem Lab Med. 2007;45(12):1704–16.
