- Email: [email protected]
Oral administration of 1,25-dihydroxyvitamin D3 completely protects NOD mice from insulin-dependent diabetes mellitus
ABB Archives of Biochemistry and Biophysics 417 (2003) 77–80 www.elsevier.com/locate/yabbi
Oral administration of 1,25-dihydroxyvitamin D3 completely protects NOD mice from insulin-dependent diabetes mellitusq Julia B. Zella, Laura C. McCary,1 and Hector F. DeLuca* Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Received 15 April 2003, and in revised form 25 June 2003
Abstract 1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ), the biologically active form of vitamin D, is widely recognized as a modulator of the immune system as well as a regulator of mineral metabolism. The objective of this study was to determine the effects of vitamin D status and treatment with 1,25(OH)2 D3 on diabetes onset in non-obese diabetic (NOD) mice, a murine model of human type I diabetes. We have found that vitamin D-deficiency increases the incidence of diabetes in female mice from 46% (n ¼ 13) to 88% (n ¼ 8) and from 0% (n ¼ 10) to 44% (n ¼ 9) in male mice as of 200 days of age when compared to vitamin D-sufficient animals. Addition of 50 ng of 1,25(OH)2 D3 /day to the diet prevented disease onset as of 200 days and caused a significant rise in serum calcium levels, regardless of gender or vitamin D status. Our results indicate that vitamin D status is a determining factor of disease susceptibility and oral administration of 1,25(OH)2 D3 prevents diabetes onset in NOD mice through 200 days of age. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Type I diabetes; IDDM; Vitamin D; Calcium
Vitamin D is traditionally recognized as a potent regulator of calcium and phosphorus metabolism. More appropriately considered a prohormone, vitamin D3 is either manufactured in the epidermis from 7-dehydrocholesterol by the absorption of ultraviolet light, or obtained from the diet in a limited number of foods such as eggs, fish oils, and fortified milk [1]. Inactive in this form, vitamin D3 requires enzymatic hydroxylation of the 25- and 1-carbons in the liver and kidney, respectively, to generate the active form of the hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2 D3 )2 [2]. The biological actions of 1,25(OH)2 D3 are mediated through binding to the vitamin D receptor (VDR), a member of the nuclear steroid hormone receptor family [3]. In conq This work was supported by funds from the Wisconsin Alumni Research Foundation. * Corresponding author. Fax: 1-608-262-7122. E-mail address: [email protected] (H.F. DeLuca). 1 Present address: Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA. 2 Abbreviations used: 1,25(OH)2 D3 , 1,25-dihydroxyvitamin D3 ; VDR, vitamin D receptor; RXR, retinoid X receptor; VDRE, vitamin D response elements; IDDM, insulin-dependent (type I) diabetes mellitus; NOD, non-obese diabetic.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-9861(03)00338-2
junction with its heterodimeric partner, the retinoid X receptor (RXR), the ligand-bound VDR alters transcription through binding to specific vitamin D-response elements (VDRE) in the promoter regions of vitamin D target genes. Once thought to exert its effects solely on bone, kidney, and intestine, vitamin D is now appreciated in a wider variety of roles including cellular differentiation, reproduction, and insulin secretion. In 1983, the VDR was discovered in human peripheral mononuclear leukocytes and activated T lymphocytes, suggesting that vitamin D may also act upon the immune system [4,5]. Since this initial finding, further investigations have provided evidence that vitamin D is a modulator of immune function, particularly in the prevention of autoimmune disorders in both humans and animal models of autoimmunity [6–9]. In the current study, we focused on the role of vitamin D in insulin-dependent (type I) diabetes mellitus (IDDM), using the non-obese diabetic (NOD) mouse. In man, IDDM is classified as an autoimmune disorder with both genetic and environmental determinants. A state of hyperglycemia results from the T-cell mediated destruction of insulin-secreting b-cells in the pancreatic Islets of
78
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
Langerhans. The disease pathogenesis is not fully understood, although the elucidation of some mechanisms is due to studies with animal models such as the NOD mouse. These mice are genetically susceptible to diabetes with preponderance in females. Similar to the human condition, the NOD mouse exhibits hyperglycemia, polyuria, polydipsia, glucosuria, insulitis, and a dependence on exogenous insulin to sustain life [10]. Such characteristics have made the NOD mouse an excellent and widely accepted murine model of human IDDM. From our initial findings, we report that a vitamin Ddeficient state alone potentiates diabetic onset in the NOD mouse. The incidence of diabetes in vitamin Ddeficient mice was higher in both males and females, and appeared to onset earlier than the vitamin D-sufficient counterparts. In addition, we found that daily dietary supplementation with 50 ng of 1,25(OH)2 D3 from weaning completely prevents diabetes in both genders through 200 days of age, regardless of vitamin D status.
Materials and methods Animal production and maintenance A NOD/LtJ mouse colony was produced from breeding pairs purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained in our facility in temperature- (70–72 °F) and humidity (40– 50%)-controlled rooms under a 12-h light, 12-h dark cycle. The mice were housed in plastic cages lined with wooden shavings and consumed distilled water ad libitum. All experimental protocols were approved by the University of Wisconsin-Madison Research Animal Resources Center Committee Review Board. Studies in vitamin D-sufficiency Breeding pairs were maintained on LabDiet Mouse Diet 5015 (PMI Nutrition International, St. Louis, MO) containing 0.8% calcium and 3.3 IU vitamin D3 /g diet. The breeding colony and vitamin D-sufficient (+D) experimental animals were housed under fluorescent lighting. Both male and female pups were weaned at 21 days of age and immediately fed one of the following: (1) a vitamin D-free purified diet containing 0.47% calcium as previously described [11], or (2) the identical purified diet with the addition of 50 ng of 1,25(OH)2 D3 /3.5 g diet. Both purified diets were produced in our facility. The diets were solidified with molten agar (Harlan Teklad, Madison, WI) and supplemented with fat-soluble vitamins A (as b-carotene), E, K, and 175 IU vitamin D3 / mouse/week in vegetable oil. The 1,25(OH)2 D3 -containing diet was administered at 3.5 g/mouse/day, 3 times/week, and monitored for total food consumption. All other diets were fed ad libitum.
Studies in vitamin D-deficiency When visibly pregnant, the female breeder alone was transferred to a vitamin D-free room with UV-filtered lighting that does not allow for the synthesis of vitamin D from the skin. The dam was fed a vitamin D-free purified diet containing 1.2% calcium [11]. The offspring were weaned at 21 days of age and randomized into the study. These vitamin D-deficient ()D) experimental animals were also housed under UV-filtered lighting and placed on the same purified dietary regimens as described for studies in vitamin D-sufficiency with the omission of vitamin D3 from the fat-soluble vitamin oil supplement. Evaluation of diabetic status All experimental mice were bled from the orbital sinus following a 4-h fast approximately every 10 days from 50 to 200 days of age. Whole blood was immediately centrifuged to obtain serum. The concentration of glucose in the serum was measured using an enzymaticcolorimetric assay (Glucose Liquicolor, Stanbio Laboratory, Boerne, TX) followed by spectrophotometric analysis. Animals with fasting glucose levels greater than 300 mg/dl serum were diagnosed as diabetic. Diabetic animals were sacrificed immediately following diagnosis. The study was terminated when the remaining mice reached 200 days of age. Serum calcium analysis At 100 days of age, blood was collected from the orbital sinus of each mouse. The serum was separated by centrifugation and diluted 1:40 in a 0.1% LaCl2 solution. The concentration of calcium was determined by atomic absorption spectrometry (Spectrometer 3110, Perkin Elmer, Norwalk, CT). Statistics The two-tailed, unpaired t test was performed on mean age of onset and serum calcium data using a computer-based statistics program (GraphPad Software). P values less than 0.05 (P < 0:05) were considered to be significant.
Results Vitamin D-deficiency increases diabetes incidence and age of onset in NOD mice To examine the effect of vitamin D status on diabetes in the NOD mouse, we examined the incidence of disease and age of onset in both vitamin D-sufficient
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
and -deficient mice. As shown in Fig. 1, vitamin Ddeficient animals ()D) exhibit a higher incidence of diabetes at 200 days of age than mice maintained in a vitamin D-sufficient (+D) state. Specifically, 88% (7/8) of vitamin D-deficient female mice were diabetic at 200 days of age, while only 46% (6/13) of vitamin D-sufficient female mice had developed diabetes by that time. As expected, the overall incidence in male NOD mice was lower than in females, regardless of vitamin D status. By 200 days of age, 44% (4/9) of vitamin D-deficient male mice were diabetic, whereas no mouse (0/10) in the vitamin D-sufficient group had developed the disease. In addition to a higher incidence, vitamin D-deficient NOD mice display an earlier onset of disease. Female and male vitamin D-deficient NOD mice began to develop diabetes at approximately the same age, 110 and 111 days, respectively. In contrast, male vitamin D-sufficient mice did not exhibit any symp-
100
80 70 60 50 40 30 20
Diabetes Incidence (%)
90
10 0
-D Female -D Male +D Female +D Male
100
110
130
120
140
150
160
170
190
180
200
Age at Diabetes Onset (days)
Fig. 1. Vitamin D-deficiency increases diabetic incidence in NOD mice. Vitamin D-deficiency ()D) increases the incidence of diabetes in female mice from 46% (n ¼ 13) to 88% (n ¼ 8) and from 0% (n ¼ 10) to 44% (n ¼ 9) in male mice as of 200 days of age when compared to vitamin D-sufficient (+D) animals. Whole blood was collected from fasted animals approximately every 10 days beginning at 50 days of age. Animals with serum levels of 300 mg glucose/dl serum or greater were diagnosed as diabetic. Non-diabetic animals that did not survive through 200 days of age were omitted from the total number of animals.
79
toms of diabetes by 200 days of age, whereas diabetes was diagnosed in vitamin D-sufficient female mice as early as 145 days. The mean age of diabetic onset also occurs earlier in a vitamin D-deficient state (Table 1). Male and female vitamin D-deficient NOD mice showed mean onsets of 138 and 130 days, respectively, whereas the mean day of onset in vitamin D-sufficient females was significantly delayed until 174 days of age (P < 0:05). Oral administration of 50 ng of 1,25(OH)2 D3 is necessary to protect NOD mice completely from developing diabetes through 200 days of age To study the protective effect of pharmacological levels of the biologically active metabolite of vitamin D, we administered a daily dose of 50 ng of 1,25(OH)2 D3 by incorporating the hormone into the diet. Using this method, we were able to protect completely all mice in these groups from developing diabetes through 200 days of age (Table 1). We also tested lower dietary levels of 1,25(OH)2 D3 in vitamin D-deficient mice and found that 10 ng of 1,25(OH)2 D3 was insufficient to protect either males or females from diabetes onset. In addition, 20 ng of 1,25(OH)2 D3 offered complete protection to the males, although not to the females (data not shown). In a separate study, we examined the ability of two different delivery methods of 1,25(OH)2 D3 to protect vitamin D-sufficient mice from developing diabetes. From weaning, both male and female mice were fed a purified diet with or without 50 ng of 1,25(OH)2 D3 by incorporation of the hormone into the diet, or injected with 5 lg 1,25(OH)2 D3 /kg body weight or vehicle (arachis oil) i.p. every 48 h. We found that oral administration of 50 ng of 1,25(OH)2 D3 was necessary and sufficient to protect both male and female mice from developing diabetes through 200 days of age. Although a reduction in diabetes onset was observed in mice injected with 1,25(OH)2 D3 as opposed to vehicle as previously reported [12], the level of protection did not match that
Table 1 Diabetic incidence, mean age of onset, and serum calcium values for vitamin D-sufficient and -deficient NOD mice fed a diet with or without 50 ng of 1,25(OH)2 D3 Group
1,25(OH)2 D3 /day (ng)
Diabetic incidence (%)a
Mean age onset (days)b
Serum calcium (mg/dl)c
)D Male )D Male )D Female )D Female +D Male +D Male +D Female +D Female
0 50 0 50 0 50 0 50
44 0 88 0 0 0 46 0
139 29 NA 130 26 NA NA NA 174 27 NA
7.1 1.1 10.9 0.3 7.3 1.0 11.9 0.5 9.3 0.7 11.9 0.3 9.1 0.9 12.4 0.6
a
(4/9) (0/7) (7/8) (0/5) (0/10) (0/10) (6/13) (0/10)
(8) (7) (13) (5) (11) (10) (12) (9)
Reported as the total number of diabetic mice out of the total number of mice in a given group that survived through 200 days of age. Mean age of onset SD. c Average serum calcium value SD (n). b
80
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
seen with the daily dietary administration of 50 ng 1,25(OH)2 D3 (data not shown). Protection from diabetes by 1,25(OH)2 D3 is accompanied by hypercalcemia As an indirect measure of vitamin D status, we monitored serum calcium levels in all mice approximately every 10 days from 50 to 200 days of age. The results reported in Table 1 represent means SD serum calcium values measured from blood collected when the mice reached 100 days of age. The level of calcium in the serum significantly increased in all mice fed a daily level of 50 ng 1,25(OH)2 D3 , regardless of vitamin D status (P < 0:0001). The average serum calcium values of vitamin D-deficient males and females increased from 7.1 and 7.3 mg% to 10.9 and 11.9 mg%, respectively, when the diet was supplemented with 1,25(OH)2 D3 . In the vitamin D-sufficient state, the addition of 1,25(OH)2 D3 to the diet elevated the average serum calcium values of male and female mice from 9.3 and 9.1 mg% to 11.9 and 12.4 mg%, respectively. Our method of producing vitamin D-deficient mice resulted in a significant decrease in serum calcium levels by approximately 2 mg% in both genders (P < 0:0001). It is also important to note that at no time during the study did the serum calcium values significantly differ from those reported at day 100.
Discussion In the present study, we have demonstrated that a vitamin D-sufficient status alone confers partial protection against the development of diabetes in the NOD mouse. Vitamin D-deficiency resulted in an earlier mean day of onset and greater overall incidence of IDDM in both male and female mice when compared to vitamin D-sufficient animals. These results support the observations in humans that vitamin D status may be an environmental factor in determining IDDM risk. Similar to other autoimmune diseases including multiple sclerosis and rheumatoid arthritis, the incidence of IDDM has been associated with geographic patterns [13,14]. Specifically, there is a significant correlation between increased IDDM risk and lower average yearly temperatures, which are strongly associated with latitude, levels of sunlight, and production of 25(OH)D3 [14,15]. In addition, several recent reports have shown a negative correlation between dietary intake of vitamin D3 during pregnancy or infancy with and incidence of IDDM [16–18]. We have also shown that incorporation of 50 ng 1,25(OH)2 D3 /day into the diet completely protects both
male and female NOD mice from IDDM through 200 days of age, regardless of vitamin D status. This result agrees with earlier reports of the ability of 1,25(OH)2 D3 to partially prevent IDDM in female NOD mice (18). The level of protection we have observed may be due to the method by which the 1,25(OH)2 D3 was administered. Frequent consumption of 1,25(OH)2 D3 should result in a consistent circulating level of the hormone, which would argue that oral administration of 1,25(OH)2 D3 or an analog would be more clinically relevant in the prevention of IDDM. We also observed a non-lethal, although significant increase in serum calcium concentrations in mice fed 50 ng 1,25(OH)2 D3 . A lower, less calcemic dose of 10 ng of 1,25(OH)2 D3 offered only partial protection against IDDM in male and female NOD mice, arguing that hypercalcemia may be a contributing factor to complete prevention of IDDM in NOD mice. The present study cannot provide the information required to assess the relative roles of hypercalcemia and 1,25(OH)2 D3 itself in preventing the onset of diabetes in the NOD mouse and remains to be investigated.
References [1] H.F. DeLuca, Nutr. Today 28 (1993) 6–11. [2] G. Jones, S.A. Strugnell, H.F. DeLuca, Physiol. Rev. 74 (1998) 1193–1231. [3] S.A. Strugnell, H.F. DeLuca, Proc. Soc. Exp. Biol. Med. 215 (1997) 223–228. [4] A.K. Bhalla, E.P. Amento, T.L. Clemens, M.F. Holick, S.M. Krane, J. Clin. Endocrinol. Metab. 57 (1983) 1308–1310. [5] D.M. Provvedini, C.D. Tsoukas, L.J. Deftos, S.C. Manolagas, Science 221 (1983) 1181–1183. [6] M.T. Cantorna, C.E. Hayes, H.F. DeLuca, J. Nutr. 128 (1998) 68–72. [7] M.T. Cantorna, C.E. Hayes, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 93 (1996) 7861–7864. [8] M.T. Cantorna, C. Munsick, C. Bemiss, B.D. Mahon, J. Nutr. 120 (2000) 2648–2652. [9] J.M. Lemire, A. Ince, M. Takashima, Autoimmunity 12 (1992) 143–148. [10] Y. Tochino, Crit. Rev. Immunol. 8 (1987) 49–81. [11] T. Suda, H.F. DeLuca, Y. Tanaka, J. Nutr. 100 (1970) 1049–1052. [12] C. Mathieu, M. Waer, J. Laureys, O. Rutgeerts, R. Bouillon, Diabetologia 37 (1994) 552–558. [13] M.T. Cantorna, Proc. Soc. Exp. Biol. Med. 223 (2000) 230–233. [14] Diabetes Epidemiology Research International Group, Diabetes 37 (1988) 1113–1119. [15] R.P. Stryd, T.J. Gilbertson, M.N. Brunden, J. Clin. Endocrinol. Metab. 48 (1979) 771–775. [16] The EURODIAB Substudy 2 Study Group, Diabetologia 42 (1999) 51–54. [17] E. Hypponen, E. Laara, A. Reunanen, M.R. Jarvelin, S.M. Virtanen, Lancet 358 (2001) 1500–1503. [18] L.C. Stene, J. Ulriksen, P. Magnus, G. Joner, Diabetologia 43 (2000) 1093–1098.
Oral administration of 1,25-dihydroxyvitamin D3 completely protects NOD mice from insulin-dependent diabetes mellitusq Julia B. Zella, Laura C. McCary,1 and Hector F. DeLuca* Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA Received 15 April 2003, and in revised form 25 June 2003
Abstract 1,25-Dihydroxyvitamin D3 (1,25(OH)2 D3 ), the biologically active form of vitamin D, is widely recognized as a modulator of the immune system as well as a regulator of mineral metabolism. The objective of this study was to determine the effects of vitamin D status and treatment with 1,25(OH)2 D3 on diabetes onset in non-obese diabetic (NOD) mice, a murine model of human type I diabetes. We have found that vitamin D-deficiency increases the incidence of diabetes in female mice from 46% (n ¼ 13) to 88% (n ¼ 8) and from 0% (n ¼ 10) to 44% (n ¼ 9) in male mice as of 200 days of age when compared to vitamin D-sufficient animals. Addition of 50 ng of 1,25(OH)2 D3 /day to the diet prevented disease onset as of 200 days and caused a significant rise in serum calcium levels, regardless of gender or vitamin D status. Our results indicate that vitamin D status is a determining factor of disease susceptibility and oral administration of 1,25(OH)2 D3 prevents diabetes onset in NOD mice through 200 days of age. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Type I diabetes; IDDM; Vitamin D; Calcium
Vitamin D is traditionally recognized as a potent regulator of calcium and phosphorus metabolism. More appropriately considered a prohormone, vitamin D3 is either manufactured in the epidermis from 7-dehydrocholesterol by the absorption of ultraviolet light, or obtained from the diet in a limited number of foods such as eggs, fish oils, and fortified milk [1]. Inactive in this form, vitamin D3 requires enzymatic hydroxylation of the 25- and 1-carbons in the liver and kidney, respectively, to generate the active form of the hormone, 1,25dihydroxyvitamin D3 (1,25(OH)2 D3 )2 [2]. The biological actions of 1,25(OH)2 D3 are mediated through binding to the vitamin D receptor (VDR), a member of the nuclear steroid hormone receptor family [3]. In conq This work was supported by funds from the Wisconsin Alumni Research Foundation. * Corresponding author. Fax: 1-608-262-7122. E-mail address: [email protected] (H.F. DeLuca). 1 Present address: Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA. 2 Abbreviations used: 1,25(OH)2 D3 , 1,25-dihydroxyvitamin D3 ; VDR, vitamin D receptor; RXR, retinoid X receptor; VDRE, vitamin D response elements; IDDM, insulin-dependent (type I) diabetes mellitus; NOD, non-obese diabetic.
0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0003-9861(03)00338-2
junction with its heterodimeric partner, the retinoid X receptor (RXR), the ligand-bound VDR alters transcription through binding to specific vitamin D-response elements (VDRE) in the promoter regions of vitamin D target genes. Once thought to exert its effects solely on bone, kidney, and intestine, vitamin D is now appreciated in a wider variety of roles including cellular differentiation, reproduction, and insulin secretion. In 1983, the VDR was discovered in human peripheral mononuclear leukocytes and activated T lymphocytes, suggesting that vitamin D may also act upon the immune system [4,5]. Since this initial finding, further investigations have provided evidence that vitamin D is a modulator of immune function, particularly in the prevention of autoimmune disorders in both humans and animal models of autoimmunity [6–9]. In the current study, we focused on the role of vitamin D in insulin-dependent (type I) diabetes mellitus (IDDM), using the non-obese diabetic (NOD) mouse. In man, IDDM is classified as an autoimmune disorder with both genetic and environmental determinants. A state of hyperglycemia results from the T-cell mediated destruction of insulin-secreting b-cells in the pancreatic Islets of
78
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
Langerhans. The disease pathogenesis is not fully understood, although the elucidation of some mechanisms is due to studies with animal models such as the NOD mouse. These mice are genetically susceptible to diabetes with preponderance in females. Similar to the human condition, the NOD mouse exhibits hyperglycemia, polyuria, polydipsia, glucosuria, insulitis, and a dependence on exogenous insulin to sustain life [10]. Such characteristics have made the NOD mouse an excellent and widely accepted murine model of human IDDM. From our initial findings, we report that a vitamin Ddeficient state alone potentiates diabetic onset in the NOD mouse. The incidence of diabetes in vitamin Ddeficient mice was higher in both males and females, and appeared to onset earlier than the vitamin D-sufficient counterparts. In addition, we found that daily dietary supplementation with 50 ng of 1,25(OH)2 D3 from weaning completely prevents diabetes in both genders through 200 days of age, regardless of vitamin D status.
Materials and methods Animal production and maintenance A NOD/LtJ mouse colony was produced from breeding pairs purchased from The Jackson Laboratory (Bar Harbor, ME). The animals were maintained in our facility in temperature- (70–72 °F) and humidity (40– 50%)-controlled rooms under a 12-h light, 12-h dark cycle. The mice were housed in plastic cages lined with wooden shavings and consumed distilled water ad libitum. All experimental protocols were approved by the University of Wisconsin-Madison Research Animal Resources Center Committee Review Board. Studies in vitamin D-sufficiency Breeding pairs were maintained on LabDiet Mouse Diet 5015 (PMI Nutrition International, St. Louis, MO) containing 0.8% calcium and 3.3 IU vitamin D3 /g diet. The breeding colony and vitamin D-sufficient (+D) experimental animals were housed under fluorescent lighting. Both male and female pups were weaned at 21 days of age and immediately fed one of the following: (1) a vitamin D-free purified diet containing 0.47% calcium as previously described [11], or (2) the identical purified diet with the addition of 50 ng of 1,25(OH)2 D3 /3.5 g diet. Both purified diets were produced in our facility. The diets were solidified with molten agar (Harlan Teklad, Madison, WI) and supplemented with fat-soluble vitamins A (as b-carotene), E, K, and 175 IU vitamin D3 / mouse/week in vegetable oil. The 1,25(OH)2 D3 -containing diet was administered at 3.5 g/mouse/day, 3 times/week, and monitored for total food consumption. All other diets were fed ad libitum.
Studies in vitamin D-deficiency When visibly pregnant, the female breeder alone was transferred to a vitamin D-free room with UV-filtered lighting that does not allow for the synthesis of vitamin D from the skin. The dam was fed a vitamin D-free purified diet containing 1.2% calcium [11]. The offspring were weaned at 21 days of age and randomized into the study. These vitamin D-deficient ()D) experimental animals were also housed under UV-filtered lighting and placed on the same purified dietary regimens as described for studies in vitamin D-sufficiency with the omission of vitamin D3 from the fat-soluble vitamin oil supplement. Evaluation of diabetic status All experimental mice were bled from the orbital sinus following a 4-h fast approximately every 10 days from 50 to 200 days of age. Whole blood was immediately centrifuged to obtain serum. The concentration of glucose in the serum was measured using an enzymaticcolorimetric assay (Glucose Liquicolor, Stanbio Laboratory, Boerne, TX) followed by spectrophotometric analysis. Animals with fasting glucose levels greater than 300 mg/dl serum were diagnosed as diabetic. Diabetic animals were sacrificed immediately following diagnosis. The study was terminated when the remaining mice reached 200 days of age. Serum calcium analysis At 100 days of age, blood was collected from the orbital sinus of each mouse. The serum was separated by centrifugation and diluted 1:40 in a 0.1% LaCl2 solution. The concentration of calcium was determined by atomic absorption spectrometry (Spectrometer 3110, Perkin Elmer, Norwalk, CT). Statistics The two-tailed, unpaired t test was performed on mean age of onset and serum calcium data using a computer-based statistics program (GraphPad Software). P values less than 0.05 (P < 0:05) were considered to be significant.
Results Vitamin D-deficiency increases diabetes incidence and age of onset in NOD mice To examine the effect of vitamin D status on diabetes in the NOD mouse, we examined the incidence of disease and age of onset in both vitamin D-sufficient
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
and -deficient mice. As shown in Fig. 1, vitamin Ddeficient animals ()D) exhibit a higher incidence of diabetes at 200 days of age than mice maintained in a vitamin D-sufficient (+D) state. Specifically, 88% (7/8) of vitamin D-deficient female mice were diabetic at 200 days of age, while only 46% (6/13) of vitamin D-sufficient female mice had developed diabetes by that time. As expected, the overall incidence in male NOD mice was lower than in females, regardless of vitamin D status. By 200 days of age, 44% (4/9) of vitamin D-deficient male mice were diabetic, whereas no mouse (0/10) in the vitamin D-sufficient group had developed the disease. In addition to a higher incidence, vitamin D-deficient NOD mice display an earlier onset of disease. Female and male vitamin D-deficient NOD mice began to develop diabetes at approximately the same age, 110 and 111 days, respectively. In contrast, male vitamin D-sufficient mice did not exhibit any symp-
100
80 70 60 50 40 30 20
Diabetes Incidence (%)
90
10 0
-D Female -D Male +D Female +D Male
100
110
130
120
140
150
160
170
190
180
200
Age at Diabetes Onset (days)
Fig. 1. Vitamin D-deficiency increases diabetic incidence in NOD mice. Vitamin D-deficiency ()D) increases the incidence of diabetes in female mice from 46% (n ¼ 13) to 88% (n ¼ 8) and from 0% (n ¼ 10) to 44% (n ¼ 9) in male mice as of 200 days of age when compared to vitamin D-sufficient (+D) animals. Whole blood was collected from fasted animals approximately every 10 days beginning at 50 days of age. Animals with serum levels of 300 mg glucose/dl serum or greater were diagnosed as diabetic. Non-diabetic animals that did not survive through 200 days of age were omitted from the total number of animals.
79
toms of diabetes by 200 days of age, whereas diabetes was diagnosed in vitamin D-sufficient female mice as early as 145 days. The mean age of diabetic onset also occurs earlier in a vitamin D-deficient state (Table 1). Male and female vitamin D-deficient NOD mice showed mean onsets of 138 and 130 days, respectively, whereas the mean day of onset in vitamin D-sufficient females was significantly delayed until 174 days of age (P < 0:05). Oral administration of 50 ng of 1,25(OH)2 D3 is necessary to protect NOD mice completely from developing diabetes through 200 days of age To study the protective effect of pharmacological levels of the biologically active metabolite of vitamin D, we administered a daily dose of 50 ng of 1,25(OH)2 D3 by incorporating the hormone into the diet. Using this method, we were able to protect completely all mice in these groups from developing diabetes through 200 days of age (Table 1). We also tested lower dietary levels of 1,25(OH)2 D3 in vitamin D-deficient mice and found that 10 ng of 1,25(OH)2 D3 was insufficient to protect either males or females from diabetes onset. In addition, 20 ng of 1,25(OH)2 D3 offered complete protection to the males, although not to the females (data not shown). In a separate study, we examined the ability of two different delivery methods of 1,25(OH)2 D3 to protect vitamin D-sufficient mice from developing diabetes. From weaning, both male and female mice were fed a purified diet with or without 50 ng of 1,25(OH)2 D3 by incorporation of the hormone into the diet, or injected with 5 lg 1,25(OH)2 D3 /kg body weight or vehicle (arachis oil) i.p. every 48 h. We found that oral administration of 50 ng of 1,25(OH)2 D3 was necessary and sufficient to protect both male and female mice from developing diabetes through 200 days of age. Although a reduction in diabetes onset was observed in mice injected with 1,25(OH)2 D3 as opposed to vehicle as previously reported [12], the level of protection did not match that
Table 1 Diabetic incidence, mean age of onset, and serum calcium values for vitamin D-sufficient and -deficient NOD mice fed a diet with or without 50 ng of 1,25(OH)2 D3 Group
1,25(OH)2 D3 /day (ng)
Diabetic incidence (%)a
Mean age onset (days)b
Serum calcium (mg/dl)c
)D Male )D Male )D Female )D Female +D Male +D Male +D Female +D Female
0 50 0 50 0 50 0 50
44 0 88 0 0 0 46 0
139 29 NA 130 26 NA NA NA 174 27 NA
7.1 1.1 10.9 0.3 7.3 1.0 11.9 0.5 9.3 0.7 11.9 0.3 9.1 0.9 12.4 0.6
a
(4/9) (0/7) (7/8) (0/5) (0/10) (0/10) (6/13) (0/10)
(8) (7) (13) (5) (11) (10) (12) (9)
Reported as the total number of diabetic mice out of the total number of mice in a given group that survived through 200 days of age. Mean age of onset SD. c Average serum calcium value SD (n). b
80
J.B. Zella et al. / Archives of Biochemistry and Biophysics 417 (2003) 77–80
seen with the daily dietary administration of 50 ng 1,25(OH)2 D3 (data not shown). Protection from diabetes by 1,25(OH)2 D3 is accompanied by hypercalcemia As an indirect measure of vitamin D status, we monitored serum calcium levels in all mice approximately every 10 days from 50 to 200 days of age. The results reported in Table 1 represent means SD serum calcium values measured from blood collected when the mice reached 100 days of age. The level of calcium in the serum significantly increased in all mice fed a daily level of 50 ng 1,25(OH)2 D3 , regardless of vitamin D status (P < 0:0001). The average serum calcium values of vitamin D-deficient males and females increased from 7.1 and 7.3 mg% to 10.9 and 11.9 mg%, respectively, when the diet was supplemented with 1,25(OH)2 D3 . In the vitamin D-sufficient state, the addition of 1,25(OH)2 D3 to the diet elevated the average serum calcium values of male and female mice from 9.3 and 9.1 mg% to 11.9 and 12.4 mg%, respectively. Our method of producing vitamin D-deficient mice resulted in a significant decrease in serum calcium levels by approximately 2 mg% in both genders (P < 0:0001). It is also important to note that at no time during the study did the serum calcium values significantly differ from those reported at day 100.
Discussion In the present study, we have demonstrated that a vitamin D-sufficient status alone confers partial protection against the development of diabetes in the NOD mouse. Vitamin D-deficiency resulted in an earlier mean day of onset and greater overall incidence of IDDM in both male and female mice when compared to vitamin D-sufficient animals. These results support the observations in humans that vitamin D status may be an environmental factor in determining IDDM risk. Similar to other autoimmune diseases including multiple sclerosis and rheumatoid arthritis, the incidence of IDDM has been associated with geographic patterns [13,14]. Specifically, there is a significant correlation between increased IDDM risk and lower average yearly temperatures, which are strongly associated with latitude, levels of sunlight, and production of 25(OH)D3 [14,15]. In addition, several recent reports have shown a negative correlation between dietary intake of vitamin D3 during pregnancy or infancy with and incidence of IDDM [16–18]. We have also shown that incorporation of 50 ng 1,25(OH)2 D3 /day into the diet completely protects both
male and female NOD mice from IDDM through 200 days of age, regardless of vitamin D status. This result agrees with earlier reports of the ability of 1,25(OH)2 D3 to partially prevent IDDM in female NOD mice (18). The level of protection we have observed may be due to the method by which the 1,25(OH)2 D3 was administered. Frequent consumption of 1,25(OH)2 D3 should result in a consistent circulating level of the hormone, which would argue that oral administration of 1,25(OH)2 D3 or an analog would be more clinically relevant in the prevention of IDDM. We also observed a non-lethal, although significant increase in serum calcium concentrations in mice fed 50 ng 1,25(OH)2 D3 . A lower, less calcemic dose of 10 ng of 1,25(OH)2 D3 offered only partial protection against IDDM in male and female NOD mice, arguing that hypercalcemia may be a contributing factor to complete prevention of IDDM in NOD mice. The present study cannot provide the information required to assess the relative roles of hypercalcemia and 1,25(OH)2 D3 itself in preventing the onset of diabetes in the NOD mouse and remains to be investigated.
References [1] H.F. DeLuca, Nutr. Today 28 (1993) 6–11. [2] G. Jones, S.A. Strugnell, H.F. DeLuca, Physiol. Rev. 74 (1998) 1193–1231. [3] S.A. Strugnell, H.F. DeLuca, Proc. Soc. Exp. Biol. Med. 215 (1997) 223–228. [4] A.K. Bhalla, E.P. Amento, T.L. Clemens, M.F. Holick, S.M. Krane, J. Clin. Endocrinol. Metab. 57 (1983) 1308–1310. [5] D.M. Provvedini, C.D. Tsoukas, L.J. Deftos, S.C. Manolagas, Science 221 (1983) 1181–1183. [6] M.T. Cantorna, C.E. Hayes, H.F. DeLuca, J. Nutr. 128 (1998) 68–72. [7] M.T. Cantorna, C.E. Hayes, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 93 (1996) 7861–7864. [8] M.T. Cantorna, C. Munsick, C. Bemiss, B.D. Mahon, J. Nutr. 120 (2000) 2648–2652. [9] J.M. Lemire, A. Ince, M. Takashima, Autoimmunity 12 (1992) 143–148. [10] Y. Tochino, Crit. Rev. Immunol. 8 (1987) 49–81. [11] T. Suda, H.F. DeLuca, Y. Tanaka, J. Nutr. 100 (1970) 1049–1052. [12] C. Mathieu, M. Waer, J. Laureys, O. Rutgeerts, R. Bouillon, Diabetologia 37 (1994) 552–558. [13] M.T. Cantorna, Proc. Soc. Exp. Biol. Med. 223 (2000) 230–233. [14] Diabetes Epidemiology Research International Group, Diabetes 37 (1988) 1113–1119. [15] R.P. Stryd, T.J. Gilbertson, M.N. Brunden, J. Clin. Endocrinol. Metab. 48 (1979) 771–775. [16] The EURODIAB Substudy 2 Study Group, Diabetologia 42 (1999) 51–54. [17] E. Hypponen, E. Laara, A. Reunanen, M.R. Jarvelin, S.M. Virtanen, Lancet 358 (2001) 1500–1503. [18] L.C. Stene, J. Ulriksen, P. Magnus, G. Joner, Diabetologia 43 (2000) 1093–1098.