- Email: [email protected]
Vitamin D increases IGF-I and insulin levels in experimental diabetic rats
Accepted Manuscript Vitamin D increases IGF-I and insulin levels in experimental diabetic rats
H. Derakhshanian, M.H. Javanbakht, M. Zarei, E. Djalali, M. Djalali PII: DOI: Reference:
S1096-6374(17)30077-1 doi: 10.1016/j.ghir.2017.09.002 YGHIR 1195
To appear in:
Growth Hormone & IGF Research
Received date: Revised date: Accepted date:
21 March 2017 27 July 2017 11 September 2017
Please cite this article as: H. Derakhshanian, M.H. Javanbakht, M. Zarei, E. Djalali, M. Djalali , Vitamin D increases IGF-I and insulin levels in experimental diabetic rats, Growth Hormone & IGF Research (2017), doi: 10.1016/j.ghir.2017.09.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Vitamin D increases IGF-I and insulin levels in experimental diabetic rats
H. Derakhshanian a, M.H. Javanbakht a, M. Zarei a, E. Djalali b, M. Djalalia,* a
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Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran b
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Department of Veterinary, Science and Research Branch of Islamic Azad University, Tehran, Iran
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Running title: Vitamin D ameliorated IGF-1 and insulin
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*Corresponding Author: Dr. Mahmoud Djalali Postal Address: Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Poorsina Street, Enghelab Avenue, Tehran, Iran. PO Box: 14155-6446 Tel: +982188954911 Fax: +982188974462
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Email: [email protected]
1
ACCEPTED MANUSCRIPT Vitamin D increases IGF-I and insulin levels in experimental diabetic rats Abstract Introduction and objective: Previous studies have found that IGF-I may play an important role in glucose metabolism. The aim of this study is to examine the effect of vitamin D intake on the serum levels of glucose, insulin, and IGF-I in experimental diabetic rats. Material and methods:
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A total of 24 male Sprague–Dawley rats aged six to seven months, with an average weight of
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300±30 g, were randomly divided into three groups (eight rats per group). The first group served
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as control and the other two groups received an intraperitoneal injection of 45 mg/kg streptozotocin (STZ) to develop diabetes. Then groups were treated for four weeks either with
US
placebo or vitamin D (two injections of 20,000 IU/kg). Results: At the end of the experiment, two injection of vitamin D were found to result in a significant increase in plasma
AN
cholecalciferol, which could improve hyperglycaemia and hypoinsulinemia in diabetic rats. HbA1c concentration had a slight and insignificant decrease following vitamin D intake. In
M
addition, a significant decline was observed in the serum IGF-I level of STZ-treated rats in comparison to the controls, which was compensated in the vitamin D group. The serum vitamin
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D concentration was positively correlated to the changes in IGF-I level by Pearson test. Conclusions: These data showed for the first time that vitamin D intake could significantly
PT
improve fasting plasma glucose, insulin, and IGF-I in an experimental type 1 diabetes model.
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Key words: Vitamin D, Cholecalciferol, Diabetes Mellitus, Insulin, IGF-І
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ACCEPTED MANUSCRIPT 1. Introduction Insulin-like growth factor-1 (IGF-I)—also known as somatomedin C—is produced by the liver and is classically considered as a pivotal growth factor that mediates the effects of growth hormone (GH) on the body. It can regulate growth, proliferation, and differentiation in endocrine, paracrine, and autocrine manners [1, 2]. IGF-I works as a part of a complex system—
T
often referred to as the IGF axis—which consists of GH, IGF-I, and IGF-II peptides, their
IP
receptors (IGF1R and IGF2R), a family of six high-affinity IGF-binding proteins (IGFBP-1 to -
CR
6), and IGFBP proteases [3]. IGF-I has a significant structural similarity to insulin. Although they have different receptors, IGF-I has an affinity to the insulin receptor as well [4]. Previous studies have found that IGF-I may play an important role in glucose metabolism by promoting
US
the glucose uptake in peripheral tissues, suppressing the hepatic glucose release, and increasing the insulin sensitivity [5-8]. Low levels of IGF-I have been reported in patients with type 1
AN
diabetes mellitus (T1DM), whereas the concentration was found to be elevated in T2DM [9-11]. This association may be either causal or pathological. Generally, a great body of evidence
M
suggests that the IGF-I axis plays a role in normal glucose homeostasis.
ED
Vitamin D (cholecalciferol) is a fat-soluble steroid hormone that is mainly synthesized through the skin by ultraviolet irradiation in sunlight and also derived from dietary intake [12]. Its
PT
relation to bone health has been clearly demonstrated. However, the discovery of the potential non-skeletal functions of vitamin D—mostly regulated via its nuclear receptor—has received
CE
tremendous amount of attention, with increasing number of reports of association between vitamin D deficiency and several health conditions such as hypertension, cancer, cardiovascular
AC
disease, and diabetes mellitus [13]. Vitamin D deficiency, recognized as a global health problem, has been linked to the onset and progression of both type 1 and type 2 diabetes mellitus. This might be related to the vitamin’s immunomodulatory properties. The relationship of vitamin D with insulin secretion, insulin resistance, and β-cell dysfunction in the pancreas has been highlighted [14]. In addition, vitamin D regulates insulin sensitivity by stimulating the expression of insulin receptors and promoting the expression of peroxisome proliferatoractivated receptor (PPAR), which is a nuclear receptor that is involved in fatty acid and glucose metabolism [15].
3
ACCEPTED MANUSCRIPT Considering the high prevalence of both vitamin D deficiency and diabetes mellitus and to clarify their relationship, this study is designed to examine the effect of vitamin D intake on the serum levels of insulin and IGF-I in experimental diabetic rats. 2. Material and methods 2.1. Animals and Chemicals
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A total of 24 male Sprague–Dawley rats aged six to seven months, with an average weight of
IP
300±30 g, were obtained and housed under standard laboratory conditions at a temperature of
CR
20–25 °C with a 12-hour light/dark cycle. All animals were acclimatized for 10 days before the experiment and had free access to tap water and pelleted rodent chow diet. The animals were
US
cared for in accordance with the Guide to the Care and Use of Experimental Animals [16]. Efforts were made to use least possible number of animals and to minimize their pain and
AN
suffering. Body weight was checked every week and food intake was estimated by weighting the remaining amount of food per cage on a daily basis (Seca scale, Hamburg, Germany).
M
Streptozotocin (STZ) was obtained from Sigma Aldrich Chemicals (St Louis, MO, USA) and
ED
was dissolved in sterile citrate buffer with a PH of 5–6 immediately before injection.
2.2. Study design
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Animals were randomly divided into three groups of eight (1: control; 2: diabetic; 3: diabetic + vitamin D) using a block randomization scheme, and treated for four weeks. All rats were
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injected intraperitoneally (i.p.) with a single dose of STZ (45 mg/kg, ~20 μl), except for the first group that served as control and received only citrate buffer. One week later, all rats were tested
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for fasting plasma glucose (FPG). Rats with a fasting blood glucose level above 250 mg/dl were considered as diabetic and included in the study. The vitamin D group was injected with 20,000 IU/kg of cholecalciferol on Days 1 and 14, while the other groups received sesame oil as placebo. After four weeks, all the animals were anesthetized and sacrificed with an intraperitoneal injection of ketamine (50 mg/kg) along with xylazine (30 mg/kg). 2.3. Biochemical testing Fasting samples were collected (between 0800 and 1000 hours) by cardiac puncture and were immediately centrifuged for serum isolation and stored at −80 °C until analysis for biochemical parameters. Commercially available ELISA kits were used to measure the levels of vitamin D, 4
ACCEPTED MANUSCRIPT insulin and IGF-I (IBL International, Hamburg, Germany). Serum levels of glucose, HbA1c and calcium were evaluated by auto-analyzer (BT-1500, Biotecnica Instruments, Italy).
2.4. Statistical analysis Data were presented as mean ± standard deviation (SD). The Statistical Package for Social
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Sciences (version 21.0; SPSS Inc., Chicago, Illinois, USA) was used for all analyses. Statistical
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differences between groups were assessed using analysis of covariance (ANCOVA), followed by
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a Bonferroni post-hoc test. Pearson correlation was used for exploring the association of serum vitamin D concentrations with IGF-I level. All assumptions, such as normality and equality of
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variances, were fulfilled. Values of p < 0.05 were considered statistically significant. 3. Results
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Following four weeks of treatment, the mean FPG level was magnified drastically in diabetic animals in comparison to the control group (p < 0.001). Two injection of vitamin D resulted in a
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significant increase in plasma cholecalciferol and could improve hyperglycaemia and hypoinsulinemia in comparison with diabetic rats (p = 0.005 and p = 0.01 respectively). Calcium
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level was checked and hypercalcaemia was not observed in any group. HbA1c concentration had a slight but insignificant decrease. In addition, at the end of the experiment, a significant decline
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was observed in the serum IGF-I level of STZ-treated rats in comparison to the controls, which was totally compensated in the vitamin D group (p = 0.01, Table 1). The serum vitamin D
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concentration was positively correlated to the changes in IGF-I level by the Pearson test (r = 0.39; p = 0.05). At the beginning and end of this study, groups were compared regarding body
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weight, food intake and serum calcium level. No significant difference was observed between groups at the beginning. Calcium level was also the same at the end of study. However, the two diabetic groups had a lower body weight and food intake at the endpoint. Therefore, ANCOVA test was used to control the possible confounding factors. 4. Discussion Recently, there has been increasing evidence from animal and human studies to suggest that vitamin D might be a potential risk modifier for both type 1 and type 2 diabetes mellitus, via direct and indirect mechanisms [17]. Some studies have shown that low vitamin D 5
ACCEPTED MANUSCRIPT concentrations are associated with an increased risk of T1DM incidence and T2DM development in the general population [18, 19]. However, the evidence from experimental studies and clinical trials is sparse and inconclusive; hence, it is not adequate to prove the causal relation between vitamin D intake and glycaemic control. In our experiment, vitamin D intake resulted in a significant decrease in FPG. Nevertheless, the
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decline in HbA1c was marginal and statistically insignificant, which might be due to the short
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period of experiment. In addition, a conspicuous enhancement was observed in the insulin level.
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Previous studies suggest several different mechanisms to explain the role of vitamin D in normalizing the glucose level. It has been shown that vitamin D deficiency results in impairment of glucose metabolism by increasing insulin resistance, which in turn takes place due to the
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decrease in adipose PPAR-γ expression and deterioration of β-cell function and mass [20]. Supplementation with cholecalciferol was found to restore the alteration in IP3 and AMPA
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receptor—a non-NMDA-type ionotropic transmembrane receptor for glutamate—expression in the pancreatic islets, which helped to restore the calcium-mediated insulin secretion, indicating
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the therapeutic role of vitamin through the regulation of glutamatergic function in diabetic subjects [21]. Moreover, it has been reported that 1, 25(OH)2 D3 treatment reversed the
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high glucose-induced pathological changes in mTOR signalling pathway, therefore effectively inhibiting β-cell apoptosis [22]. In addition, vitamin D appears to enhance the intracellular
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mechanisms of insulin action mediated by IRS-1 and to up-regulate GLUT4 total protein expression [23, 24]. Some investigations suggest that orally ingested dietary vitamin D in aged
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mice improves glucose metabolism as a GLP-1 enhancer [25]. Some reports also indicate that drug-induced reduction in Pik3r1 expression—a critical gene acting downstream
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of insulin receptor—is reversed by cholecalciferol supplementation in skeletal muscle, and that impaired insulin-stimulated glucose uptake into the myotube is improved by the concomitant calcitriol in a PI3K-dependent manner [26]. Another important finding of the present study was the remarkable improvement in IGF-I level following vitamin D injection in diabetic rats. Low levels of IGF-I have been reported in patients with T1DM, whereas the concentration was elevated in T2DM patients [9-11]. This association may be either causal or pathological. A great body of evidence suggests that IGF-I axis plays a role in normal glucose homeostasis by 6
ACCEPTED MANUSCRIPT promoting the glucose uptake in peripheral tissues, suppressing the hepatic glucose release, and increasing the insulin sensitivity. At the same time, insulin modulates hepatic IGF-I production through a direct regulation of the transcript levels of IGF-I [27]. Therefore, it seems that insulin and IGF-I are in a tight relationship in terms of their intracellular activities. However, there are very limited data about the effect of vitamin D on IGF-I in diabetic subjects. Kamycheva et al. found that one-year randomized controlled intervention with cholecalciferol had no
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corresponding effect on the insulin resistance, but significantly improved the elevated IGF-
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I/IGFBP-3 ratio in overweight subjects [28].
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According to the study by Soliman et al., treatment of children with nutritional vitamin D deficiency with one intramuscular injection of vitamin D (300000 IU) resulted in a significant
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increase in circulating concentrations of IGF-I [29]. In addition, a correlation between free IGF-I and 25(OH) D3 has been observed in randomly selected healthy population [30].
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Altogether, this study showed for the first time that vitamin D intake could significantly improve FPG, insulin, and IGF-I in an experimental diabetes model. However, there is still
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little scientific evidence about the possible benefits of vitamin D in diabetes through IGF-I axis;
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hence, more interventional studies and clinical trial are needed in this direction.
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ACCEPTED MANUSCRIPT References [1] Kim MS, Lee D-Y, Insulin-like growth factor (IGF)-I and IGF binding proteins axis in diabetes mellitus, Ann Pediatr Endocrinol Metab. 20 (2015) 69-73. [2] Butler AA, Roith DL, Control of growth by the somatropic axis: growth hormone and the insulin-like growth factors have related and independent roles, Annu Rev Physiol. 63 (2001) 141-64.
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[3] Le Roith D, Insulin-like growth factors, N Engl J Med. 336 (1997) 633-40.
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[4] Rinderknecht E, Humbel RE, The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin, J Biol Chem. 253(1978) 2769-76.
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[5] Holt R, Simpson H, Sönksen P, The role of the growth hormone–insulin‐like growth factor axis in glucose homeostasis, Diabet Med. 20 (2003) 3-15.
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[6] Saltiel AR, Kahn CR, Insulin signalling and the regulation of glucose and lipid metabolism, Nature. 414 (2001) 799-806.
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[7] Moses AC, Young SC, Morrow LA, O'Brien M, Clemmons DR, Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes, Diabetes. 45 (1996) 91-100.
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[8] Boulware S, Tamborlane W, Matthews L, Sherwin R, Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism, Am J Physiol Endocrinol Metab. 262 (1992) E130-E3.
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[9] Munoz M, Barrios V, Pozo J, Argente J, Insulin-Like Growth Factor I, Its Binding Proteins 1 and 3, and Growth Hormone-Binding Protein in Children and Adolescents with InsulinDependent Diabetes Mellitus: Clinical Implications1, Pediatr Res. 39 (1996) 992-8.
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[10] Wędrychowicz A, Dziatkowiak H, Nazim J, Sztefko K, Insulin-like growth factor-1 and its binding proteins, IGFBP-1 and IGFBP-3, in adolescents with type-1 diabetes mellitus and microalbuminuria, Horm Res Paediatr. 63 (2005) 245-51. [11] Rajpathak SN, Gunter MJ, Wylie‐Rosett J, Ho GY, Kaplan RC, Muzumdar R, et al., The role of insulin‐like growth factor‐I and its binding proteins in glucose homeostasis and type 2 diabetes, Diabetes Metab Res Rev. 25 (2009) 3-12. [12] Holick M, DeLuca H, Vitamin D metabolism, Annu Rev Med. 25 (1974) 349-67. [13] Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al., Vitamin D: beyond bone, Ann N Y Acad Sci. 1287 (2013) 45-58.
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ACCEPTED MANUSCRIPT [14] Nakashima A, Yokoyama K, Yokoo T, Urashima M, Role of vitamin D in diabetes mellitus and chronic kidney disease, World J Diabetes. 7 (2016) 89. [15] Pittas AG, Dawson-Hughes B, Vitamin D and diabetes, J Steroid Biochem Mol Biol. 121 (2010) 425-9. [16] Olfert ED, Cross BM, McWilliam AA, Guide to the care and use of experimental animals: Canadian Council on Animal Care Ottawa. 1993.
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[17] Pittas AG, Lau J, Hu FB, Dawson-Hughes B, The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis, J Clin Endocrinol Metab. 92 (2007) 2017-29.
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[18] Cooper JD, Smyth DJ, Walker NM, Stevens H, Burren OS, Wallace C, et al., Inherited variation in vitamin D genes is associated with predisposition to autoimmune disease type 1 diabetes, Diabetes 60 (2011) 1624-31.
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[19] Forouhi N, Ye Z, Rickard A, Khaw K, Luben R, Langenberg C, et al., Circulating 25hydroxyvitamin D concentration and the risk of type 2 diabetes: results from the European Prospective Investigation into Cancer (EPIC)-Norfolk cohort and updated meta-analysis of prospective studies, Diabetologia. 55 (2012) 2173-82.
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[20] Park S, Kim DS, Kang S, Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin resistance by reducing PPAR-γ expression in nonobese Type 2 diabetic rats, J Nutr Biochem. 27 (2016) 257-65.
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[21] Jayanarayanan S, Anju TR, Smijin S, Paulose CS, Vitamin D 3 supplementation increases insulin level by regulating altered IP3 and AMPA receptor expression in the pancreatic islets of streptozotocin-induced diabetic rat, J Nutr Biochem. 26 (2015) 1041-9.
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[22] Yang Z, Liu F, Qu H, Wang H, Xiao X, Deng H, 1, 25 (OH) 2 D 3 protects β cell against high glucose-induced apoptosis through mTOR suppressing, Mol Cell Endocrinol. 414 (2015) 111-9.
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[23] Alkharfy KM, Al-Daghri NM, Yakout SM, Hussain T, Mohammed AK, Krishnaswamy S, Influence of vitamin D treatment on transcriptional regulation of insulin-sensitive genes, Metab Syndr Relat Disord. 11 (2013) 283-8. [24] Manna P, Jain SK, Vitamin D up-regulates glucose transporter 4 (GLUT4) translocation and glucose utilization mediated by cystathionine-γ-lyase (CSE) activation and H2S formation in 3T3L1 adipocytes, J Biol Chem. 287 (2012) 42324-32. [25] Enciso PL, Wang L, Kawahara Y, Sakamoto S, Shimada S, Takeichi Y, et al., Dietary vitamin D 3 improves postprandial hyperglycemia in aged mice, Biochem Biophys Res Commun. 461 (2015) 165-71.
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ACCEPTED MANUSCRIPT [26] Nagashima T, Shirakawa H, Nakagawa T, Kaneko S, Prevention of antipsychotic-induced hyperglycaemia by vitamin D: a data mining prediction followed by experimental exploration of the molecular mechanism, Sci Rep. 6 (2016). [27] Boni-Schnetzler M, Schmid C, Meier P, Froesch E, Insulin regulates insulin-like growth factor I mRNA in rat hepatocytes, Am J Physiol Endocrinol Metab. 260 (1991) E846-E51.
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[28] Kamycheva E, Berg V, Jorde R, Insulin-like growth factor I, growth hormone, and insulin sensitivity: the effects of a one-year cholecalciferol supplementation in middle-aged overweight and obese subjects, Endocrine. 43 (2013) 412-8.
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[29] Soliman AT, Al Khalaf F, AlHemaidi N, Al Ali M, Al Zyoud M, Yakoot K, Linear growth in relation to the circulating concentrations of insulin-like growth factor I, parathyroid hormone, and 25-hydroxy vitamin D in children with nutritional rickets before and after treatment: endocrine adaptation to vitamin D deficiency, Metabolism. 57 (2008) 95-102.
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[30] Gomez J, Maravall F, Gomez N, Navarro M, Casamitjana R, Soler J, Relationship between 25-(OH) D3, the IGF-I system, leptin, anthropometric and body composition variables in a healthy, randomly selected population, Horm Metab Res. 36 (2004) 48-53.
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ACCEPTED MANUSCRIPT Table 1 Serum levels of vitamin D, FPG, HbA1c, Insulin and IGF-I in different experimental groups Groups
Vitamin D (ng/mL)
FPG (mg/dL)
HbA1c (%)
#
4.65±0.50
Insulin (mIU/L)
#
3.37±0.83
#
IGF-1 (ng/mL) 125.82±15.59#
Control
20.93±2.49
85.87±12.63
Diabetic
21.33±2.44
479.37±27.90*
8.75±0.48*
2.15±0.79*
96.71±7.12*
Diabetic+vitamin D
35.42±3.96*#
428.87±37.74*#
8.30±0.55*
3.31±0.65#
128.15±28.41#
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Data are presented as the mean ± SD (n = 8 for all groups). FPG, fasting plasma glucose; IGF-I, insulin like growth
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compared with the control group; #, p < 0.05compared with the diabetic group.
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factor-I. Statistical differences were determined using ANCOVA followed by a Bonferroni post-hoc test; *, p < 0.05
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ACCEPTED MANUSCRIPT Table 2 Body weight and food intake of different experimental groups Groups Initial weight (gram) Final weight (gram)
Food intake (gram/day)
#
29.0±1.2#
Control
294.3±15.4
304.0±22.1
Diabetic
295.5±14.5
222.8±30.1*
22.9±2.4*
Diabetic + vitamin D
296.5±11.1
198.0±40.0*
24.9±4.0*
Data are presented as the mean ± SD (n = 8 for all groups). Statistical differences were determined using ANOVA
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followed by a Bonferroni post-hoc test; *, p < 0.05 compared with the control group; #, p < 0.05 compared with the
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diabetic group
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ACCEPTED MANUSCRIPT
Highlights
Vitamin D significantly improved hyperglycemia and hypoinsulinemia in diabetic rats.
Serum IGF-1 decline in STZ-treated rats was compensated following vitamin D intake.
The serum vitamin D concentration was positively correlated to the changes in IGF-1
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T
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AN
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level.
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H. Derakhshanian, M.H. Javanbakht, M. Zarei, E. Djalali, M. Djalali PII: DOI: Reference:
S1096-6374(17)30077-1 doi: 10.1016/j.ghir.2017.09.002 YGHIR 1195
To appear in:
Growth Hormone & IGF Research
Received date: Revised date: Accepted date:
21 March 2017 27 July 2017 11 September 2017
Please cite this article as: H. Derakhshanian, M.H. Javanbakht, M. Zarei, E. Djalali, M. Djalali , Vitamin D increases IGF-I and insulin levels in experimental diabetic rats, Growth Hormone & IGF Research (2017), doi: 10.1016/j.ghir.2017.09.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Vitamin D increases IGF-I and insulin levels in experimental diabetic rats
H. Derakhshanian a, M.H. Javanbakht a, M. Zarei a, E. Djalali b, M. Djalalia,* a
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Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Tehran, Iran b
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Department of Veterinary, Science and Research Branch of Islamic Azad University, Tehran, Iran
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Running title: Vitamin D ameliorated IGF-1 and insulin
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ED
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*Corresponding Author: Dr. Mahmoud Djalali Postal Address: Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences, Poorsina Street, Enghelab Avenue, Tehran, Iran. PO Box: 14155-6446 Tel: +982188954911 Fax: +982188974462
AC
CE
Email: [email protected]
1
ACCEPTED MANUSCRIPT Vitamin D increases IGF-I and insulin levels in experimental diabetic rats Abstract Introduction and objective: Previous studies have found that IGF-I may play an important role in glucose metabolism. The aim of this study is to examine the effect of vitamin D intake on the serum levels of glucose, insulin, and IGF-I in experimental diabetic rats. Material and methods:
T
A total of 24 male Sprague–Dawley rats aged six to seven months, with an average weight of
IP
300±30 g, were randomly divided into three groups (eight rats per group). The first group served
CR
as control and the other two groups received an intraperitoneal injection of 45 mg/kg streptozotocin (STZ) to develop diabetes. Then groups were treated for four weeks either with
US
placebo or vitamin D (two injections of 20,000 IU/kg). Results: At the end of the experiment, two injection of vitamin D were found to result in a significant increase in plasma
AN
cholecalciferol, which could improve hyperglycaemia and hypoinsulinemia in diabetic rats. HbA1c concentration had a slight and insignificant decrease following vitamin D intake. In
M
addition, a significant decline was observed in the serum IGF-I level of STZ-treated rats in comparison to the controls, which was compensated in the vitamin D group. The serum vitamin
ED
D concentration was positively correlated to the changes in IGF-I level by Pearson test. Conclusions: These data showed for the first time that vitamin D intake could significantly
PT
improve fasting plasma glucose, insulin, and IGF-I in an experimental type 1 diabetes model.
AC
CE
Key words: Vitamin D, Cholecalciferol, Diabetes Mellitus, Insulin, IGF-І
2
ACCEPTED MANUSCRIPT 1. Introduction Insulin-like growth factor-1 (IGF-I)—also known as somatomedin C—is produced by the liver and is classically considered as a pivotal growth factor that mediates the effects of growth hormone (GH) on the body. It can regulate growth, proliferation, and differentiation in endocrine, paracrine, and autocrine manners [1, 2]. IGF-I works as a part of a complex system—
T
often referred to as the IGF axis—which consists of GH, IGF-I, and IGF-II peptides, their
IP
receptors (IGF1R and IGF2R), a family of six high-affinity IGF-binding proteins (IGFBP-1 to -
CR
6), and IGFBP proteases [3]. IGF-I has a significant structural similarity to insulin. Although they have different receptors, IGF-I has an affinity to the insulin receptor as well [4]. Previous studies have found that IGF-I may play an important role in glucose metabolism by promoting
US
the glucose uptake in peripheral tissues, suppressing the hepatic glucose release, and increasing the insulin sensitivity [5-8]. Low levels of IGF-I have been reported in patients with type 1
AN
diabetes mellitus (T1DM), whereas the concentration was found to be elevated in T2DM [9-11]. This association may be either causal or pathological. Generally, a great body of evidence
M
suggests that the IGF-I axis plays a role in normal glucose homeostasis.
ED
Vitamin D (cholecalciferol) is a fat-soluble steroid hormone that is mainly synthesized through the skin by ultraviolet irradiation in sunlight and also derived from dietary intake [12]. Its
PT
relation to bone health has been clearly demonstrated. However, the discovery of the potential non-skeletal functions of vitamin D—mostly regulated via its nuclear receptor—has received
CE
tremendous amount of attention, with increasing number of reports of association between vitamin D deficiency and several health conditions such as hypertension, cancer, cardiovascular
AC
disease, and diabetes mellitus [13]. Vitamin D deficiency, recognized as a global health problem, has been linked to the onset and progression of both type 1 and type 2 diabetes mellitus. This might be related to the vitamin’s immunomodulatory properties. The relationship of vitamin D with insulin secretion, insulin resistance, and β-cell dysfunction in the pancreas has been highlighted [14]. In addition, vitamin D regulates insulin sensitivity by stimulating the expression of insulin receptors and promoting the expression of peroxisome proliferatoractivated receptor (PPAR), which is a nuclear receptor that is involved in fatty acid and glucose metabolism [15].
3
ACCEPTED MANUSCRIPT Considering the high prevalence of both vitamin D deficiency and diabetes mellitus and to clarify their relationship, this study is designed to examine the effect of vitamin D intake on the serum levels of insulin and IGF-I in experimental diabetic rats. 2. Material and methods 2.1. Animals and Chemicals
T
A total of 24 male Sprague–Dawley rats aged six to seven months, with an average weight of
IP
300±30 g, were obtained and housed under standard laboratory conditions at a temperature of
CR
20–25 °C with a 12-hour light/dark cycle. All animals were acclimatized for 10 days before the experiment and had free access to tap water and pelleted rodent chow diet. The animals were
US
cared for in accordance with the Guide to the Care and Use of Experimental Animals [16]. Efforts were made to use least possible number of animals and to minimize their pain and
AN
suffering. Body weight was checked every week and food intake was estimated by weighting the remaining amount of food per cage on a daily basis (Seca scale, Hamburg, Germany).
M
Streptozotocin (STZ) was obtained from Sigma Aldrich Chemicals (St Louis, MO, USA) and
ED
was dissolved in sterile citrate buffer with a PH of 5–6 immediately before injection.
2.2. Study design
PT
Animals were randomly divided into three groups of eight (1: control; 2: diabetic; 3: diabetic + vitamin D) using a block randomization scheme, and treated for four weeks. All rats were
CE
injected intraperitoneally (i.p.) with a single dose of STZ (45 mg/kg, ~20 μl), except for the first group that served as control and received only citrate buffer. One week later, all rats were tested
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for fasting plasma glucose (FPG). Rats with a fasting blood glucose level above 250 mg/dl were considered as diabetic and included in the study. The vitamin D group was injected with 20,000 IU/kg of cholecalciferol on Days 1 and 14, while the other groups received sesame oil as placebo. After four weeks, all the animals were anesthetized and sacrificed with an intraperitoneal injection of ketamine (50 mg/kg) along with xylazine (30 mg/kg). 2.3. Biochemical testing Fasting samples were collected (between 0800 and 1000 hours) by cardiac puncture and were immediately centrifuged for serum isolation and stored at −80 °C until analysis for biochemical parameters. Commercially available ELISA kits were used to measure the levels of vitamin D, 4
ACCEPTED MANUSCRIPT insulin and IGF-I (IBL International, Hamburg, Germany). Serum levels of glucose, HbA1c and calcium were evaluated by auto-analyzer (BT-1500, Biotecnica Instruments, Italy).
2.4. Statistical analysis Data were presented as mean ± standard deviation (SD). The Statistical Package for Social
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Sciences (version 21.0; SPSS Inc., Chicago, Illinois, USA) was used for all analyses. Statistical
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differences between groups were assessed using analysis of covariance (ANCOVA), followed by
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a Bonferroni post-hoc test. Pearson correlation was used for exploring the association of serum vitamin D concentrations with IGF-I level. All assumptions, such as normality and equality of
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variances, were fulfilled. Values of p < 0.05 were considered statistically significant. 3. Results
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Following four weeks of treatment, the mean FPG level was magnified drastically in diabetic animals in comparison to the control group (p < 0.001). Two injection of vitamin D resulted in a
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significant increase in plasma cholecalciferol and could improve hyperglycaemia and hypoinsulinemia in comparison with diabetic rats (p = 0.005 and p = 0.01 respectively). Calcium
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level was checked and hypercalcaemia was not observed in any group. HbA1c concentration had a slight but insignificant decrease. In addition, at the end of the experiment, a significant decline
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was observed in the serum IGF-I level of STZ-treated rats in comparison to the controls, which was totally compensated in the vitamin D group (p = 0.01, Table 1). The serum vitamin D
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concentration was positively correlated to the changes in IGF-I level by the Pearson test (r = 0.39; p = 0.05). At the beginning and end of this study, groups were compared regarding body
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weight, food intake and serum calcium level. No significant difference was observed between groups at the beginning. Calcium level was also the same at the end of study. However, the two diabetic groups had a lower body weight and food intake at the endpoint. Therefore, ANCOVA test was used to control the possible confounding factors. 4. Discussion Recently, there has been increasing evidence from animal and human studies to suggest that vitamin D might be a potential risk modifier for both type 1 and type 2 diabetes mellitus, via direct and indirect mechanisms [17]. Some studies have shown that low vitamin D 5
ACCEPTED MANUSCRIPT concentrations are associated with an increased risk of T1DM incidence and T2DM development in the general population [18, 19]. However, the evidence from experimental studies and clinical trials is sparse and inconclusive; hence, it is not adequate to prove the causal relation between vitamin D intake and glycaemic control. In our experiment, vitamin D intake resulted in a significant decrease in FPG. Nevertheless, the
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decline in HbA1c was marginal and statistically insignificant, which might be due to the short
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period of experiment. In addition, a conspicuous enhancement was observed in the insulin level.
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Previous studies suggest several different mechanisms to explain the role of vitamin D in normalizing the glucose level. It has been shown that vitamin D deficiency results in impairment of glucose metabolism by increasing insulin resistance, which in turn takes place due to the
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decrease in adipose PPAR-γ expression and deterioration of β-cell function and mass [20]. Supplementation with cholecalciferol was found to restore the alteration in IP3 and AMPA
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receptor—a non-NMDA-type ionotropic transmembrane receptor for glutamate—expression in the pancreatic islets, which helped to restore the calcium-mediated insulin secretion, indicating
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the therapeutic role of vitamin through the regulation of glutamatergic function in diabetic subjects [21]. Moreover, it has been reported that 1, 25(OH)2 D3 treatment reversed the
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high glucose-induced pathological changes in mTOR signalling pathway, therefore effectively inhibiting β-cell apoptosis [22]. In addition, vitamin D appears to enhance the intracellular
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mechanisms of insulin action mediated by IRS-1 and to up-regulate GLUT4 total protein expression [23, 24]. Some investigations suggest that orally ingested dietary vitamin D in aged
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mice improves glucose metabolism as a GLP-1 enhancer [25]. Some reports also indicate that drug-induced reduction in Pik3r1 expression—a critical gene acting downstream
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of insulin receptor—is reversed by cholecalciferol supplementation in skeletal muscle, and that impaired insulin-stimulated glucose uptake into the myotube is improved by the concomitant calcitriol in a PI3K-dependent manner [26]. Another important finding of the present study was the remarkable improvement in IGF-I level following vitamin D injection in diabetic rats. Low levels of IGF-I have been reported in patients with T1DM, whereas the concentration was elevated in T2DM patients [9-11]. This association may be either causal or pathological. A great body of evidence suggests that IGF-I axis plays a role in normal glucose homeostasis by 6
ACCEPTED MANUSCRIPT promoting the glucose uptake in peripheral tissues, suppressing the hepatic glucose release, and increasing the insulin sensitivity. At the same time, insulin modulates hepatic IGF-I production through a direct regulation of the transcript levels of IGF-I [27]. Therefore, it seems that insulin and IGF-I are in a tight relationship in terms of their intracellular activities. However, there are very limited data about the effect of vitamin D on IGF-I in diabetic subjects. Kamycheva et al. found that one-year randomized controlled intervention with cholecalciferol had no
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corresponding effect on the insulin resistance, but significantly improved the elevated IGF-
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I/IGFBP-3 ratio in overweight subjects [28].
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According to the study by Soliman et al., treatment of children with nutritional vitamin D deficiency with one intramuscular injection of vitamin D (300000 IU) resulted in a significant
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increase in circulating concentrations of IGF-I [29]. In addition, a correlation between free IGF-I and 25(OH) D3 has been observed in randomly selected healthy population [30].
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Altogether, this study showed for the first time that vitamin D intake could significantly improve FPG, insulin, and IGF-I in an experimental diabetes model. However, there is still
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little scientific evidence about the possible benefits of vitamin D in diabetes through IGF-I axis;
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hence, more interventional studies and clinical trial are needed in this direction.
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ACCEPTED MANUSCRIPT References [1] Kim MS, Lee D-Y, Insulin-like growth factor (IGF)-I and IGF binding proteins axis in diabetes mellitus, Ann Pediatr Endocrinol Metab. 20 (2015) 69-73. [2] Butler AA, Roith DL, Control of growth by the somatropic axis: growth hormone and the insulin-like growth factors have related and independent roles, Annu Rev Physiol. 63 (2001) 141-64.
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[3] Le Roith D, Insulin-like growth factors, N Engl J Med. 336 (1997) 633-40.
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[4] Rinderknecht E, Humbel RE, The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin, J Biol Chem. 253(1978) 2769-76.
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[5] Holt R, Simpson H, Sönksen P, The role of the growth hormone–insulin‐like growth factor axis in glucose homeostasis, Diabet Med. 20 (2003) 3-15.
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[6] Saltiel AR, Kahn CR, Insulin signalling and the regulation of glucose and lipid metabolism, Nature. 414 (2001) 799-806.
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[7] Moses AC, Young SC, Morrow LA, O'Brien M, Clemmons DR, Recombinant human insulin-like growth factor I increases insulin sensitivity and improves glycemic control in type II diabetes, Diabetes. 45 (1996) 91-100.
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[8] Boulware S, Tamborlane W, Matthews L, Sherwin R, Diverse effects of insulin-like growth factor I on glucose, lipid, and amino acid metabolism, Am J Physiol Endocrinol Metab. 262 (1992) E130-E3.
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[9] Munoz M, Barrios V, Pozo J, Argente J, Insulin-Like Growth Factor I, Its Binding Proteins 1 and 3, and Growth Hormone-Binding Protein in Children and Adolescents with InsulinDependent Diabetes Mellitus: Clinical Implications1, Pediatr Res. 39 (1996) 992-8.
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[10] Wędrychowicz A, Dziatkowiak H, Nazim J, Sztefko K, Insulin-like growth factor-1 and its binding proteins, IGFBP-1 and IGFBP-3, in adolescents with type-1 diabetes mellitus and microalbuminuria, Horm Res Paediatr. 63 (2005) 245-51. [11] Rajpathak SN, Gunter MJ, Wylie‐Rosett J, Ho GY, Kaplan RC, Muzumdar R, et al., The role of insulin‐like growth factor‐I and its binding proteins in glucose homeostasis and type 2 diabetes, Diabetes Metab Res Rev. 25 (2009) 3-12. [12] Holick M, DeLuca H, Vitamin D metabolism, Annu Rev Med. 25 (1974) 349-67. [13] Christakos S, Hewison M, Gardner DG, Wagner CL, Sergeev IN, Rutten E, et al., Vitamin D: beyond bone, Ann N Y Acad Sci. 1287 (2013) 45-58.
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ACCEPTED MANUSCRIPT [14] Nakashima A, Yokoyama K, Yokoo T, Urashima M, Role of vitamin D in diabetes mellitus and chronic kidney disease, World J Diabetes. 7 (2016) 89. [15] Pittas AG, Dawson-Hughes B, Vitamin D and diabetes, J Steroid Biochem Mol Biol. 121 (2010) 425-9. [16] Olfert ED, Cross BM, McWilliam AA, Guide to the care and use of experimental animals: Canadian Council on Animal Care Ottawa. 1993.
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[17] Pittas AG, Lau J, Hu FB, Dawson-Hughes B, The role of vitamin D and calcium in type 2 diabetes. A systematic review and meta-analysis, J Clin Endocrinol Metab. 92 (2007) 2017-29.
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[18] Cooper JD, Smyth DJ, Walker NM, Stevens H, Burren OS, Wallace C, et al., Inherited variation in vitamin D genes is associated with predisposition to autoimmune disease type 1 diabetes, Diabetes 60 (2011) 1624-31.
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[19] Forouhi N, Ye Z, Rickard A, Khaw K, Luben R, Langenberg C, et al., Circulating 25hydroxyvitamin D concentration and the risk of type 2 diabetes: results from the European Prospective Investigation into Cancer (EPIC)-Norfolk cohort and updated meta-analysis of prospective studies, Diabetologia. 55 (2012) 2173-82.
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[20] Park S, Kim DS, Kang S, Vitamin D deficiency impairs glucose-stimulated insulin secretion and increases insulin resistance by reducing PPAR-γ expression in nonobese Type 2 diabetic rats, J Nutr Biochem. 27 (2016) 257-65.
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[21] Jayanarayanan S, Anju TR, Smijin S, Paulose CS, Vitamin D 3 supplementation increases insulin level by regulating altered IP3 and AMPA receptor expression in the pancreatic islets of streptozotocin-induced diabetic rat, J Nutr Biochem. 26 (2015) 1041-9.
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[22] Yang Z, Liu F, Qu H, Wang H, Xiao X, Deng H, 1, 25 (OH) 2 D 3 protects β cell against high glucose-induced apoptosis through mTOR suppressing, Mol Cell Endocrinol. 414 (2015) 111-9.
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[23] Alkharfy KM, Al-Daghri NM, Yakout SM, Hussain T, Mohammed AK, Krishnaswamy S, Influence of vitamin D treatment on transcriptional regulation of insulin-sensitive genes, Metab Syndr Relat Disord. 11 (2013) 283-8. [24] Manna P, Jain SK, Vitamin D up-regulates glucose transporter 4 (GLUT4) translocation and glucose utilization mediated by cystathionine-γ-lyase (CSE) activation and H2S formation in 3T3L1 adipocytes, J Biol Chem. 287 (2012) 42324-32. [25] Enciso PL, Wang L, Kawahara Y, Sakamoto S, Shimada S, Takeichi Y, et al., Dietary vitamin D 3 improves postprandial hyperglycemia in aged mice, Biochem Biophys Res Commun. 461 (2015) 165-71.
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ACCEPTED MANUSCRIPT [26] Nagashima T, Shirakawa H, Nakagawa T, Kaneko S, Prevention of antipsychotic-induced hyperglycaemia by vitamin D: a data mining prediction followed by experimental exploration of the molecular mechanism, Sci Rep. 6 (2016). [27] Boni-Schnetzler M, Schmid C, Meier P, Froesch E, Insulin regulates insulin-like growth factor I mRNA in rat hepatocytes, Am J Physiol Endocrinol Metab. 260 (1991) E846-E51.
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[28] Kamycheva E, Berg V, Jorde R, Insulin-like growth factor I, growth hormone, and insulin sensitivity: the effects of a one-year cholecalciferol supplementation in middle-aged overweight and obese subjects, Endocrine. 43 (2013) 412-8.
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[29] Soliman AT, Al Khalaf F, AlHemaidi N, Al Ali M, Al Zyoud M, Yakoot K, Linear growth in relation to the circulating concentrations of insulin-like growth factor I, parathyroid hormone, and 25-hydroxy vitamin D in children with nutritional rickets before and after treatment: endocrine adaptation to vitamin D deficiency, Metabolism. 57 (2008) 95-102.
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[30] Gomez J, Maravall F, Gomez N, Navarro M, Casamitjana R, Soler J, Relationship between 25-(OH) D3, the IGF-I system, leptin, anthropometric and body composition variables in a healthy, randomly selected population, Horm Metab Res. 36 (2004) 48-53.
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ACCEPTED MANUSCRIPT Table 1 Serum levels of vitamin D, FPG, HbA1c, Insulin and IGF-I in different experimental groups Groups
Vitamin D (ng/mL)
FPG (mg/dL)
HbA1c (%)
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4.65±0.50
Insulin (mIU/L)
#
3.37±0.83
#
IGF-1 (ng/mL) 125.82±15.59#
Control
20.93±2.49
85.87±12.63
Diabetic
21.33±2.44
479.37±27.90*
8.75±0.48*
2.15±0.79*
96.71±7.12*
Diabetic+vitamin D
35.42±3.96*#
428.87±37.74*#
8.30±0.55*
3.31±0.65#
128.15±28.41#
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Data are presented as the mean ± SD (n = 8 for all groups). FPG, fasting plasma glucose; IGF-I, insulin like growth
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compared with the control group; #, p < 0.05compared with the diabetic group.
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factor-I. Statistical differences were determined using ANCOVA followed by a Bonferroni post-hoc test; *, p < 0.05
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Food intake (gram/day)
#
29.0±1.2#
Control
294.3±15.4
304.0±22.1
Diabetic
295.5±14.5
222.8±30.1*
22.9±2.4*
Diabetic + vitamin D
296.5±11.1
198.0±40.0*
24.9±4.0*
Data are presented as the mean ± SD (n = 8 for all groups). Statistical differences were determined using ANOVA
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followed by a Bonferroni post-hoc test; *, p < 0.05 compared with the control group; #, p < 0.05 compared with the
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diabetic group
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Highlights
Vitamin D significantly improved hyperglycemia and hypoinsulinemia in diabetic rats.
Serum IGF-1 decline in STZ-treated rats was compensated following vitamin D intake.
The serum vitamin D concentration was positively correlated to the changes in IGF-1
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level.
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