e-spen, the European e-journal of Clinical Nutrition and Metabolism

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1 e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 Contents lists available at ScienceDirect e-spen, the European e-journal of Clinical Nutrition and Metabolism journal homepage: Review The role of vitamin D deficiency in the pathogenesis of type 2 diabetes mellitus Tracy S. Moreira a, Mazen J. Hamadeh b, * a Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada b School of Kinesiology and Health Science, Muscle Health Research Centre, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada article info summary Article history: Received 12 August 2009 Accepted 1 May 2010 Keywords: Type 2 diabetes mellitus 1,25 Dihydroxyvitamin-D 3 Insulin resistance b-cell apoptosis Vitamin D deficiency Physical inactivity, poor nutrition practices and obesity contribute significantly to the development of T2DM, however increasing evidence suggests that vitamin D deficiency may play a role in the pathogenesis of T2DM. T2DM manifests as a result of insulin resistance, increased hepatic glucose production and b-cell failure. Vitamin D deficiency increases insulin resistance and decreases insulin secretion in humans and animal models. Conversely, vitamin D supplementation restores insulin secretion and decreases insulin resistance and plasma glucose in humans and animal models. Vitamin D metabolites are thought to play a role in increasing insulin sensitivity and facilitating insulin exocytosis. Vitamin D has also been shown to decrease inflammatory cytokines which play a role in insulin resistance and b-cell apoptosis. Human studies have shown an inverse association between dairy intake and the risk of T2DM. The inverse association between vitamin D and dairy intake and the risk of T2DM may be due to the fact that dairy products are high in more readily absorbable calcium and are often fortified with vitamin D. Vitamin D deficiency is positively associated with insulin resistance and T2DM; however more research is needed to understand this relationship. Ó 2010 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. 1. Introduction Type 2 diabetes mellitus (T2DM) is a serious metabolic disorder that has become increasingly prevalent, not only in North America but throughout the developing world. The number of people with diabetes is expected to more than double by 2030 from 171 million to an astounding 366 million people worldwide, ninety percent of which will have T2DM. 1,2 The exponential increase of T2DM is a great cause for concern as it is no longer a disease found solely in adults, but has become increasingly evident in children as well. 3 If left untreated, T2DM can lead to a multitude of chronic microvascular and macrovascular conditions such as retinopathy, Abbreviations: BMI, body mass index; CVD, cardiovascular disease; FFA, free fatty acids; GLUT-4, glucose transporter-4; HOMA-IR, homeostasis model assessment of insulin resistance; HSL, hormone sensitive lipase; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; IL-6, interleukin-6; IR, insulin resistance; IRS, insulin receptor substrate; OGIS, oral glucose insulin sensitivity; RCT, randomized controlled trial; ROS, reactive oxygen species; RXR, retinoid X receptor; TNF-a, tumour necrosis factor-a; T2DM, type 2 diabetes mellitus; UVB, ultraviolet B; VDR, vitamin D receptor; VDRE, vitamin D response element; 1a(OH)ase, 25(OH) D 3-1a-hydroxylase; 1,25(OH) 2 D 3, 1,25-dihydroxyvitamin-D 3 ; 7-DHC, 7-dehydrocholesterol; 25(OH)ase, 25-hydroxylase; 25 (OH)D 3, 25-hydroxyvitamin-D 3. * Corresponding author. Tel.: þ x33552; fax: þ address: hamadeh@yorku.ca (M.J. Hamadeh). nephropathy, neuropathy and cardiovascular disease (CVD). 4 CVD is the leading cause of death in individuals with T2DM. 5 The pathogenesis of T2DM remains unknown as there are many malfunctioning mechanisms that occur simultaneously which can contribute to the development of the disease. 6,7 The rapid rise in obesity that has occurred in recent years is thought to have contributed significantly to the rise in T2DM. The enormity of the T2DM epidemic and its sequelae emphasize the importance of finding ways to prevent and/or ameliorate the deleterious effects of this disease. In addition to genetics which predisposes individuals to developing T2DM, there are also many environmental factors which contribute greatly to its development; these include physical inactivity, poor nutrition practices and obesity; however increasing evidence suggests that vitamin D deficiency (as measured by serum 25-hydroxyvitamin-D 3 concentration) may also contribute to the pathogenesis of T2DM. 8e14 Therefore, the focus of this review will be on the role of vitamin D deficiency in the pathogenesis of T2DM. 2. Vitamin D-synthesis, structure and function Vitamin D can be obtained either through dietary intake or produced endogenously. It is found in foods such as oily fish (salmon, sardines, mackerel), egg yolks and fortified milk and juice; 18 however dietary intake only accounts for about 30% of the vitamin D obtained. 19 The primary route via which people obtain /$36.00 Ó 2010 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved. doi: /j.eclnm

2 e156 T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 vitamin D is through exposure to ultraviolet B (UVB) sunlight at wavelengths between 290e315 nm, occurring predominantly in the summer months (JuneeJuly) in the Northern hemisphere (latitude 42 N). 20,21 UVB sunlight activates 7-dehydrocholesterol [7-DHC), a pre-cursor synthesized from cholesterol and found within the skin. 22 The activation of 7-DHC and synthesis to previtamin D 3 within the skin and its subsequent isomerization to the inactive form, vitamin D 3 (cholecalciferol), 15 produce the endogenous stores of vitamin D that, once hydroxylated twice, perform many integral physiological functions. 23 (see Fig. 1). The vitamin D 3 which is obtained in the diet or through endogenous production is not biologically active. 15 In order for vitamin D 3 to become biologically active, it must receive two successive hydroxylations from the liver by 25-hydroxylase (25 (OH)ase) to form 25(OH)D 3 (also known as calcidiol) and the kidneys by 25(OH)D 3-1a-hydroxylase (1a(OH)ase) to form 1,25 dihydroxyvitamin-d 3 (1,25(OH) 2 D 3 ) (also known as calcitriol). 15,24 Once formed, 1,25(OH) 2 D 3 must bind to its vitamin D receptor (VDR) which forms a complex with the retinoid X receptor (RXR). 15,23 The VDR has been identified in many cell types, including intestinal mucosal cells, immune cells (T and B cells), kidney cells and pancreatic b-cells. 15,25,26 The binding of 1,25 (OH) 2 D 3 to the VDR/RXR complex and subsequent binding to its specific DNA sequence known as the vitamin D response element (VDRE) leads to an increased expression of proteins, such as calbindin-d9k found in the intestine and calbindin-d28k found in pancreatic b-cells, thus facilitating calcium influx into these tissues. 27,28 Vitamin D, although traditionally classified as a vitamin, actually performs functions that are analogous to those of a hormone. 23 1,25(OH) 2 D 3 helps maintain calcium and Fig. 1. Vitamin D synthesis pathway e Exposure to UVB sunlight activates 7-dehydrocholesterol (7-DHC), a pre-cursor structure made from cholesterol and found predominantly within the epidermis, to form pre-vitamin D 3. Pre-vitamin D 3 then isomerizes to vitamin D 3 which is then carried to the liver where it is hydroxylated by 25-hydroxylase and then the kidney by 25(OH)D 3-1a-hydroxylase to form the biologically active 1,25-dihydroxyvitamin-D 3 (1,25(OH) 2 D 3 ). 1,25(OH) 2 D 3 is now free to exert its effects by binding to its vitamin D receptor (VDR) in target tissues. phosphorous homeostasis by increasing calcium absorption by the intestinal mucosa, decreasing calcium excretion by the kidney, and promoting bone resorption when serum calcium falls to suboptimal levels (<2.25e2.5 mmol/l). 1 1,25(OH) 2 D 3 also plays an integral role in preventing rickets and osteomalacia. 15 There has been much debate with regards to the levels of 25(OH)D 3 considered to be optimal; however maintaining serum 25(OH)D 3 levels of 80 nmol/l is thought to be ideal for healthy individuals; when levels drop below this value, calcium absorption is thought to be impaired and bone mineral density compromised. 22 Although serum 25(OH)D 3 concentrations of 80 nmol/l have shown to be efficacious in preventing many diseases associated with vitamin D deficiency, it is important to note that this is an arbitrary cut-off point, i.e., it may be sufficient for some individuals while grossly inadequate for others. These recommendations are based on data from individuals that do not spend a significant amount of time outdoors, and although they may be asymptomatic this does not mean that these recommendations are adequate for the general population; more research is needed to establish appropriate cutoff points for various populations. 22 Although 1,25(OH) 2 D 3 plays an important role in maintaining calcium homeostasis, there is evidence that it plays a role in immune function and more recently is thought to play a role in T2DM. 12,14e17,25,27,29e31 The most effective way of measuring vitamin D status is to measure serum concentration of 25(OH)D 3, not 1,25(OH) 2 D 3 ; this is due to the rapid clearance rate of the latter T2DM pathogenesis-insulin resistance In a normoglycemic individual, in response to a rise in blood glucose, the b-cells of the islets of Langerhans, found within the pancreas, will synthesize and secrete insulin into the blood in a biphasic pattern. 32,33 Insulin, an anabolic hormone, works to build tissues by promoting glucose uptake and synthesis of glycogen, protein and triglycerides. 1 Glucose transporters-4 (GLUT-4) are responsible for glucose uptake into tissues which work to decrease blood glucose to an optimal level between 3.9 and 5.8 mmol/l; however this mechanism is impaired in individuals with T2DM. (see Fig. 2). 1,4 The pathogenesis of T2DM is quite complex; it involves many different pathways, organs, tissues and hormones. 6,7 T2DM is a progressive chronic disease; it begins with insulin resistance, which leads to increases in hepatic glucose production and ends with b-cell failure. 4,7,34,35 Insulin resistance is defined as the inability of target tissues, such as skeletal muscle and adipose tissue, to respond adequately to the body s endogenous insulin secretions. As stated previously, the precise mechanisms responsible for the decrease in insulin sensitivity have not been elucidated; however some have implicated hyperglycemia or glucotoxicity for the decrease in insulin sensitivity observed in T2DM. 36 Hyperglycemia is thought to exacerbate insulin resistance by further decreasing tyrosine phosphorylation while increasing serine and threonine phosphorylation of the insulin receptor, which has an inhibitory effect. 33 It has been estimated that there is a 50% decrease in insulin receptor autophosphorylation in individuals with T2DM, this may be due, in part, to hyperglycemia. 33 The decrease in insulin signalling observed in individuals with T2DM has also been shown in a study conducted by Cline et al. who found that insulin-stimulated glycogen synthesis was 80% lower in individuals with T2DM when compared to healthy controls. 37 Moreover, hyperglycemia has been shown to increase the production of reactive oxygen species (ROS), which are beneficial at low levels but detrimental at supra-physiological levels. 38 Hyperglycemia decreases the activity of pertinent antioxidant enzymes, such

3 T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 e157 Fig. 2. Insulin signalling pathway e Insulin binding to its receptor results in autophosphorylation of the insulin receptor b-subunits on tyrosine amino acids. Insulin receptor autophosphorylation activates the insulin receptor substrates (IRS-1/2) and phosphatidylinositol (PI-3) kinases leading to a cascade of events that ultimately leads to GLUT-4 translocation and glucose uptake into tissues. as superoxide dismutase (SOD) and glutathione reductase, which are important in neutralizing ROS and may play a protective role by decreasing the low-grade inflammation that is characteristic of T2DM. 38 In addition to glucotoxicity, lipotoxicity is also thought to contribute to insulin resistance. Lipotoxicity refers to the high concentration of circulating free fatty acids (FFA) that occurs as a result of a suppressed inhibition of hormone sensitive lipase (HSL). 4,34 Normally, insulin inhibits lipolysis by inhibiting HSL; however in insulin resistant individuals this does not occur as efficiently. The result is an increase in lipolysis and an increase in circulating FFA; this is one of the reasons that obesity and increased adiposity, particularly in the visceral area, are of great concern. 39 HSL is more sensitive to visceral adiposity than subcutaneous adiposity, making visceral adipose tissue more detrimental to an individual s health and perhaps more likely to contribute to insulin resistance. 35,40 Also, FFA are thought to induce insulin resistance by promoting serine phosphorylation of the insulin receptor which again decreases the activity of the insulin signalling pathway. 6 Phosphorylation of the insulin receptor on tyrosine amino acids is essential in activating the insulin signalling pathway; if this does not occur, then GLUT-4 will fail to translocate, and glucose uptake into tissues will be attenuated, leading to persistent hyperglycemia or T2DM. 41 Furthermore, the production of tumour necrosis factor-a (TNFa), an inflammatory cytokine produced and secreted by adipocytes, is also thought to contribute to insulin resistance by inhibiting insulin action. 42,43 Elevated levels of TNF-a have been found in insulin resistant obese individuals without T2DM, suggesting that the increased cytokine production may contribute to the pervasiveness of insulin resistance and T2DM observed in this population. 42 Although not all insulin resistant individuals progress to T2DM, it is important to acknowledge the significant role insulin resistance plays in the pathogenesis of T2DM. 4. T2DM pathogenesis-b-cell dysfunction The role of b-cell dysfunction in the pathogenesis of T2DM has been established. 7,40,44 Normally in non-diabetic individuals, the b-cells are able to counteract insulin resistance by increasing insulin production and secretion. 45 Glucose sensors located on b-cells sense increases in blood glucose levels despite increases in insulin secretion; the persistent hyperglycemia triggers a series of events which ultimately leads to an increase in b-cell expression, b- cell mass and enhanced secretory capacity of the pancreas. 45,46 This compensatory increase in insulin secretion explains why some highly insulin resistant individuals never develop T2DM. In a study which examined pancreatic tissue from obese, non-diabetic individuals, relative b-cell volume of the pancreas was 50% greater in obese individuals than in their lean, non-diabetic counterparts ( % vs %, P ¼ 0.05), suggesting that these obese individuals did not progress to T2DM because they were able to increase their insulin production capacity by increasing b-cell mass. 47 Individuals with T2DM do not experience this increase in b-cell mass, in fact there is a significant decrease in b-cell mass. 47 In addition to a decrease in b-cell mass, there is a 41% decrease (P < 0.05) in relative b-cell volume in individuals with T2DM when compared to their lean non-diabetic counterparts, which can be partially explained by the increase in b-cell apoptosis that is also observed in individuals with T2DM. 44,47 The increase in b-cell apoptosis may be a result of an increase in any one of the following: excessive ROS production, cytokines (TNF-a, IL-6), glucotoxicity or lipotoxicity, which are often present in individuals with T2DM. 38,44,48 In vitro, islets from individuals with T2DM have an increased activity of the pro-apoptotic proteins caspase-3 and caspase-8 when compared to those from individuals without T2DM. 49 Cleaved caspase-3, the activated caspase, is responsible for translocating into the nucleus and dis-inhibiting the DNA fragmentation factor, an endonuclease responsible for fragmenting DNA resulting in apoptosis. This finding suggests that b-cell apoptosis may be more pronounced in individuals with T2DM partly due to an increase in caspase-3 activity, and that it may contribute to the decrease in b-cell mass observed in this study. 49 Moreover, the increased secretory demand placed on b-cells to produce insulin in the face of insulin resistance may also induce b-cell apoptosis via increased ROS, resulting in a decrease in b-cell mass in individuals with T2DM. 47 In addition to environmental factors, genetic predisposition may also play a role in precipitating b-cell dysfunction. Indeed, seventeen genes identified in genome wide

4 e158 T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 association scan (GWAS) studies have been associated with b-cell dysfunction and a greater incidence of T2DM Vitamin D and T2DM Although evidence for the relationship between vitamin D status and T2DM is sparse, its role seems physiologically plausible. One of the hallmarks of T2DM is low-grade inflammation which can be a result of an increase in circulating cytokines. High amounts of circulating inflammatory cytokines, such as TNF a and IL-6, contribute significantly to insulin resistance in muscle and adipose tissue. 42 To help establish a protective role for vitamin D and its respective metabolites against T2DM, Riachy et al. examined the effects of 1,25(OH) 2 D 3 on human islets in the presence of cytokines and found that islets incubated with cytokines and 1,25(OH) 2 D 3 were protected against apoptosis compared to those incubated with cytokines alone. 51 Moreover, the identification of the VDRE in the insulin receptor gene promoter has also helped establish a role for 1,25(OH) 2 D 3 in increasing insulin sensitivity by increasing insulin receptor gene expression. 52 Furthermore, vitamin D 3 supplementation has been shown to reduce inflammatory cytokines such as IL-6 and TNF-a, which play a significant role in inducing insulin resistance. 53 In keeping with the notion that T2DM cannot manifest without b-cell failure, it is important to examine the role of vitamin D metabolites (i.e., 1,25(OH) 2 D 3 ) in pancreatic b-cell function. The 1a (OH)ase enzyme, although originally thought to be present only in renal tissue, has been identified in many extra-renal tissues including pancreatic b-cells. 54 The identification of the 1a(OH)ase in b-cells suggests that 1,25(OH) 2 D 3 may play a role in overall b-cell function. In vitro and in vivo studies have ascertained that 1,25 (OH) 2 D 3 is essential for insulin secretion and glucose homeostasis. 54 VDR mutant mice show a significant decrease in insulin mrna levels when compared to controls, suggesting that 1,25 (OH) 2 D 3 may be required for insulin synthesis. 55 Moreover, 1,25 (OH) 2 D 3 is thought to be essential for insulin exocytosis by increasing the expression of calbindin-d28k in b-cells. 56 Calbindin- D28K plays an integral role in regulating intracellular calcium levels in b-cells, thus facilitating insulin exocytosis, a calcium-dependent process. 56 In addition, calbindin-d28k plays a protective role by decreasing inflammatory cytokine-induced b-cell apoptosis. 57 Calbindin-D28K is also thought to play a protective role by decreasing calcium-induced mitochondrial damage. 28 Mitochondrial damage leads to increased ROS production and subsequent apoptosis. 28 Caspase-3 activity is greater in islets from individuals with T2DM than in individuals without T2DM. 49 Calbindin-D28K is also thought to protect against apoptosis, therefore playing a protective role against b-cell apoptosis. 28 Vitamin D and its metabolites may play a role in preventing T2DM by increasing insulin production and secretion, and overall b-cell function. Lastly, obese individuals are often vitamin D deficient due to a decrease in the bio-availability of vitamin D metabolites which may explain why obesity is a risk factor for developing T2DM, although this association is only speculative. 6. Establishing a role for vitamin D deficiency in the pathogenesis of T2DM 6.1. Animal studies Animal studies have contributed significantly to the current knowledge on vitamin D deficiency and its potential role in T2DM. One of the first studies to establish a prospective role for vitamin D metabolites in b-cell function was conducted by Norman et al. in In this study, Norman and colleagues observed a 51% decrease in 1st phase insulin secretion and a 53% decrease in 2nd phase insulin secretion in the perfused pancreas of vitamin D deficient rats when compared to vitamin D replete rats in response to a glucose-arginine perfusion (P < 0.05). 58 These results suggest that the decline in insulin secretion observed in T2DM may be due in part to vitamin D deficiency; individuals with T2DM are often hypo-vitaminotic D. 9,59 In this study, serum calcium concentration was also assessed and found to be 55% lower in vitamin D deficient rats when compared to the vitamin D replete rats (P < 0.005). 58 In contrast to some later studies, this study observed a significantly lower serum calcium concentration which is important to note considering the integral role calcium plays in insulin exocytosis. In another study which examined the effects of vitamin D 3 supplementation on blood glucose concentration in rats with streptozotocin induced diabetes, high doses of vitamin D 3, equivalent to 12.5 mg/kg body weight, significantly reduced blood glucose levels by 40e60%; this study suggests that vitamin D repletion may help decrease blood glucose levels. 60 (a summary of animal studies can be found in Table 1) Although animal studies have helped to establish an association between vitamin D status and T2DM, they still do not provide enough concrete evidence for clinicians. Human studies are necessary to better understand this relationship, however the scarcity of randomized controlled trials (RCT) makes this arduous. To examine the role of vitamin D deficiency in the pathogenesis of T2DM, a review of recent cross-sectional, prospective and human intervention studies follows Cross-sectional studies Vitamin D deficiency has been linked to T2DM, with circulating levels of 25(OH)D 3 significantly lower in type 2 diabetics than in controls (Ref. 9 : 9 11 ng/ml vs ng/ml respectively, P < 0.008; Ref. 59 : vs ng/ml, respectively, P < 0.001) although both groups had mean values well below the level considered ideal (80 nmol/l). 9,59 Chiu et al. studied the Table 1 Animal studies. Reference n Outcome measures Supplementation Main results % Change Conclusion Norman et al., 1980 Weanling rats, n ¼ 12; 6 vitamin D deficient, 6 replete Vitamin D deficiency and repletion on insulin secretion from isolated pancreatic tissue 200 IU of vitamin D 3 72, 48 and 24 h prior to perfusion Pancreases from vitamin D deficient rats had a 51% and 53% decrease in FPIS and SPIS, respectively, compared to D replete animals (P < 0.05) 51% increase in FPIS and 53% increase in SPIS Vitamin D deficiency decreases insulin secretion. Insulin secretion can be partially restored after administering vitamin D De Souza Santos and Vianna, 2005 Wistar rats, n ¼ 6; SHR, n ¼ 6 Effect of vitamin D 3 supplementation on BG 500 IU/kg/d of vitamin D 3 for 14 days 40% of SHR rats had a 60% decrease in BG (P < 0.05); all wistar rats had a 40% decrease in BG after supplementation 40e60% reduction in BG Supplementation decreased BG levels Abbreviations: BG, blood glucose; FPIS, first phase insulin secretion; SHR, spontaneously hypertensive rats; SPIS, second phase insulin secretion.

5 T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 e159 relationship between 25(OH)D 3, insulin sensitivity and b-cell function in 126 normoglycemic individuals (age, 25 6 y; BMI, 25 2 kg/m 2 ). 61 Results revealed an inverse association between 25(OH)D 3 and plasma glucose concentration at 60 (r ¼ 0.29, P ¼ ), 90 (r ¼ 0.29, P ¼ ) and 120 min (r ¼ 0.30, P ¼ ) post-prandial, as well as a positive association between 25(OH)D 3 and insulin sensitivity (r ¼ 0.25, P ¼ ). 61 Another study comparing levels of 25(OH)D 3 in a group of type 1 and type 2 diabetics found that individuals with T2DM (46 3 nmol/l) had lower circulating levels of 25(OH)D 3 than type 1 diabetics (65 3 nmol/l, P < 0.05). 8 The mean concentrations of 25(OH)D 3 were below the ideal level of 80 nmol/l in both groups; however 64% of type 2 diabetics were considered to have serum 25(OH)D 3 levels of <54 nmol/l (which is indicative of vitamin D deficiency) compared to 36% of the type 1 diabetic patients. 8 In the Third National Health and Nutrition Examination Survey (NHANES III) conducted in 6228 men and women, serum 25(OH)D 3 was inversely related to T2DM in non-hispanic whites (OR ¼ 0.25; 95% CI 0.11e0.60, P < 0.01) and Mexican Americans (OR ¼ 0.17; 95% CI 0.08e0.37, P < 0.01), but not in non-hispanic blacks. 17 However, it is important to recognize that non-hispanic blacks had significantly lower levels of serum 25(OH)D 3 (49.1 nmol/l) compared to Mexican Americans (66.0 nmol/l) and non-hispanic whites (79.6 nmol/l). 17 Also, as serum 25(OH)D 3 increased, the incidence of T2DM decreased concomitantly. 17 After separating non-hispanic whites into groups based on their serum concentration of 25(OH)D 3, there was a four-fold decrease in the likelihood of having T2DM when comparing those in the highest quartile to those in the lowest (80 nmol/l vs nmol/l; the latter values indicative of vitamin D deficiency). 17 In addition, a significant inverse relationship between serum 25(OH)D 3 and fasting insulin was observed in Mexican Americans (P ¼ 0.015); although not significant, a trend towards an inverse relationship was observed in non-hispanic whites (P ¼ 0.061). However, no significant relationship was observed in non-hispanic blacks (P ¼ 0.69). 17 Moreover, an inverse relationship between serum 25 (OH)D 3 and insulin resistance (HOMA-IR) in non-hispanic whites (P ¼ 0.058) and Mexican Americans (P ¼ ), but not in non- Hispanic blacks (P ¼ 0.93) was observed. 17 The inverse relationship between serum 25(OH)D 3 concentration and insulin observed in this study suggests that 25(OH)D 3 may be involved in decreasing insulin resistance, which is a contributing factor to the development of T2DM. The low serum 25(OH)D 3 concentration observed in non-hispanic blacks was also observed in a study conducted by Harris and Dawson-Hughes where black women were found to have lower circulating levels of 25(OH)D 3 compared to Caucasian women irrespective of the time of year (winter, summer, etc.). 20 The data from Scragg et al. and Harris and Dawson-Hughes indicate that a large majority of black individuals are vitamin D deficient and thus may be at a greater risk of developing T2DM, although there is little evidence to support this notion. A more recent cross-sectional study conducted in non-diabetic individuals (n ¼ 808; M/F; age ¼ 60 9 y) observed an inverse association between plasma 25(OH)D 3 and fasting plasma glucose, insulin concentration and HOMA-IR and a positive association with the insulin sensitivity index (ISI) after adjusting for multiple factors (age, sex, BMI, waist circumference, smoking status). 62 After separating subjects into tertiles based on their serum 25 (OH)D 3 concentration, those in the highest tertile (64 nmol/l) had significantly lower fasting plasma glucose (P-trend ¼ 0.007), fasting plasma insulin (P-trend ¼ 0.001) and insulin resistance as measured by HOMA-IR (P-trend < 0.001) than those in the lowest tertile (30 nmol/l). 62 Again, it s important to note that those in the highest tertile still had serum levels below optimal (80 nmol/l). Vitamin D intake in this population of women was also examined and no effect on markers of insulin resistance was observed. 62 The above findings suggest that vitamin D deficiency may play a role in insulin resistance and b-cell dysfunction which may ultimately lead to the development of T2DM (a summary of cross-sectional studies can be found in Table 2) Prospective studies Prospective studies examining the relationship between vitamin D status and T2DM have ascertained a negative relationship between serum 25(OH)D 3 concentration and the risk of developing T2DM. In a recent study, the risk of developing T2DM was found to be significantly lower in men and women (40e74 y) in the highest serum 25(OH)D 3 quartile (w76 nmol/l [men]; w62 nmol/l [women]) when compared to the lowest quartile (w24 nmol/l [men]; 20 nmol/l [women]), the decreased risk of developing T2DM was particularly prominent in men (82% decrease in the risk of T2DM when comparing highest to lowest quartile). 12 These results coincide with the results from another larger study which examined serum 25(OH)D 3 concentrations in 4097 men and women aged 40e69 y. 14 In this study, a significant inverse association was observed between 25(OH)D 3 and T2DM after adjusting for age, sex and month in which blood sample was taken (RR 0.60; 95% CI 0.36e0.98, P ¼ 0.01), further adjustments for BMI, leisure time exercise, smoking and education attenuated this association (RR 0.70; 95% CI 0.42e1.16, P-trend ¼ 0.07). 14 In a study conducted in post-menopausal women, Isaia et al. observed that those with T2DM had significantly lower levels of 25(OH)D 3 compared to those without T2DM (24 30 nmol/l vs nmol/l, respectively, P < 0.008). 9 There were also more diabetic women with serum concentrations of 25(OH)D 3 of <14 nmol/l, which is indicative of severe vitamin D deficiency, than those in the control group (39% in T2DM vs. 25% in controls). 9 Because insulin resistance often precedes T2DM, it is important to identify potential modifiers of this condition. Forouhi et al. prospectively examined the association between serum 25(OH)D 3 and glycemic status in a cohort of nondiabetic individuals (n ¼ 524; M/F; age, 53 8 y). 63 Serum 25(OH) D 3 concentration was inversely associated with fasting glucose (P ¼ 0.019), 2 h glucose tolerance (P ¼ 0.006), fasting insulin (P ¼ 0.010) and HOMA-IR (P ¼ 0.005). 63 After adjusting for multiple factors (age, sex, smoking, BMI, season), 2 h glucose, fasting insulin, and HOMA-IR remained significant 63 (a summary of prospective studies can be found in Table 2) Prospective studies-vitamin D, calcium and dairy intake In 2006, Pittas et al. analyzed vitamin D and calcium intake data from 83,779 women (age 46 y 1 y; BMI 25 1 kg/m 2 ) who took part in the Nurse s Health Study. 64 The results showed that women with a vitamin D 3 intake >800 IU/d and a calcium intake >1200 mg/d had a 33% lower risk of developing T2DM compared to those who had a vitamin D 3 intake <400 IU/d and a calcium intake <600 mg/d (RR 0.67; 95% CI 0.49e0.90). 64 However, after adjusting for multiple factors (dietary magnesium, retinol and calcium) no significant relationship was observed between vitamin D 3 intake and T2DM (RR 0.87; 95% CI 0.75e1.00). 64 The preliminary results from this study are promising; however the study only accounted for dietary intake of vitamin D 3 and did not account for the endogenous production of vitamin D that occurs with UVB sunlight exposure. Perhaps the results from this study may have been more profound had serum 25(OH)D 3 been measured as well to identify vitamin D status. Pittas et al. also prospectively examined data from adult Caucasian men and women with normal fasting glucose (NFG) and impaired fasting glucose (IFG) (71 1 y; BMI 26 2 kg/ m 2 ) who were given either a placebo or a supplement containing

6 Table 2 Summary of cross-sectional, prospective and human intervention studies. e160 Reference Subjects (sex, age, n) Cross-sectional studies Scragg et al., 2004 M/F, 20 y, n ¼ 6228, white, black and Mexican Americans Liu et al., 2009 M/F, 60 9y, n ¼ 808 Chiu et al., 2004 M/F, 26 6y, n ¼ 126, normoglycemic Prospective studies Knekt et al., 2008 M/F, 40e74 y, n ¼ 27, 518 Mattila et al., 2007 M/F, 40e69 y, n ¼ 4097 Forouhi et al., 2008 M/F, 40e69 y, n ¼ 524 Pittas et al., 2006 F, 46 1y, n ¼ 83,779 Pittas et al., 2007 M/F, 71 1y, n ¼ 314 Outcome measure Main results Adjustments Conclusion serum 25(OH)D 3, IR and T2DM risk serum 25(OH)D 3, plasma insulin and glucose, HOMA-IR and ISI serum 25(OH)D 3 concentration, ISI, fasting and postprandial plasma glucose serum 25(OH)D 3 and T2DM serum 25(OH)D 3 and T2DM risk serum 25(OH)D 3 and glycemic status Vitamin D 3, calcium intake and T2DM risk Vitamin D 3 and calcium supplementation on blood glucose T2DM risk decreased as serum 25(OH)D 3 concentration increased in whites (OR 0.25; 95% CI 0.11e0.60) and Mexican Americans (OR 0.17; 95% CI 0.08e0.37); IR was inversely related to serum 25(OH)D 3 in white (P ¼ 0.058) and Mexican Americans (P ¼ ) 25(OH)D 3 was inversely associated with fasting plasma glucose (P-trend ¼ 0.007), insulin (P-trend ¼ 0.001) and HOMA-IR (P-trend < 0.001) 25(OH)D 3 was positively correlated with ISI (P ¼ , r ¼ 0.25) and negatively correlated with glucose concentration at 60 min (P ¼ , r ¼ ), 90 min (P ¼ , r ¼ ) and 120 min (P ¼ , r ¼ 0.30) post OGTT Odds of T2DM when comparing highest vs. lowest quartile (OR 0.28; 95% CI 0.10e0.81) for men and (OR 1.14; 95% CI 0.60e2.17) for women 25(OH)D 3 was inversely related to T2DM risk when comparing highest vs. lowest quartile (RR 0.70; 95% CI 0.42e1.16, P-trend < 0.07) Baseline serum 25(OH)D 3 was inversely associated with fasting glucose (b ¼ , P ¼ 0.019), 2 h glucose (b ¼ , P ¼ 0.006), fasting insulin (b ¼ , P ¼ 0.010), HOMA-IR (b ¼ , P ¼ 0.005) at 10 y follow-up No association between vitamin D intake and T2DM; RR for developing T2DM was 0.87 (95% CI 0.75e1.00) when comparing highest and lowest vitamin D intake; 33% lower risk of T2DM (RR 0.67; 95% CI 0.49e0.90) with intake of vitamin D 3 > 800 IU/d and 1200 mg/d calcium compared to < 400 IU/d vitamin D 3 and <600 mg/ d calcium In individuals with IFG (n ¼ 92) for >3 y, fasting glucose had an attenuated rise (0.02 mmol/l vs mmol/l) with supplementation compared to placebo (P ¼ 0.042); lower rise in IR (0.05 vs. 0.91, P ¼ 0.031) Age, sex, ethnicity, BMI, leisure activity, time of year Age, sex, BMI, waist circumference, smoking Age, sex, ethnicity, BMI, WHR, blood pressure, season Smoking, BMI, physical activity, education Age, sex, month blood sample collected, BMI, leisure-time, exercise, smoking, education Age, BMI, sex, smoking, season Age, BMI, non-dietary factors, magnesium, retinol and calcium intake Age, sex, BMI, physical activity, smoking An inverse association between serum 25(OH)D 3 and IR and T2DM risk was observed in white and Mexican Americans but not in black Americans Vitamin D status may be a determinant of T2DM risk Serum 25(OH)D 3 is positively correlated with ISI and negatively correlated with post-prandial glucose concentration High serum 25(OH)D 3 was inversely associated with the risk of T2DM, particularly in men, risk of T2DM decreased by 82% between highest and lowest quartiles of serum 25(OH)D 3 Serum 25(OH)D 3 concentration was inversely related to T2DM Serum 25(OH)D 3 is inversely associated with T2DM bio-markers Vitamin D combined with calcium supplementation may decrease the risk of developing T2DM Vitamin D 3 and calcium supplementation may attenuate the natural rise in glycaemia and IR that accompanies aging T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 Choi et al., 2005 M, 40e75 y, n ¼ 41,254 Pereira et al., 2002 M/F, 18e30 y, n ¼ 3157 Dairy intake and T2DM risk Dairy intake and incidence of IR syndrome High dairy intake (4 servings/d) decreased risk of developing T2DM compared to those with the lowest intake (mean ¼ 0.5 servings/d; RR 0.77; 95% CI 0.62e0.95, P ¼ 0.003); each additional dairy serving decreased risk of T2DM by 9% Odds of developing IR syndrome for BMI 25 kg/m 2 (OR 0.28; 95% CI 0.14e0.58, P < 0.001) when comparing the highest and lowest dairy intake; each additional serving of dairy/d decreased odds of IR syndrome by 21% (OR 0.79; 95% CI 0.70e0.88) BMI, physical activity, dietary factors Age, sex, race, caloric intake, study centre, and baseline BMI Dairy intake, particularly low-fat dairy may decrease the risk of T2DM Dairy consumption is associated with a decreased risk of developing T2DM in individuals with BMI 25 kg/m 2

7 Kirii et al., 2009 M/F, 57 8y, n ¼ 59,796 Calcium, vitamin D 3 intake and risk of T2DM No association between calcium intake and T2DM; inverse association with T2DM in high vitamin D intake þ calcium group in men (OR ¼ 0.62; 95% CI 0.41e0.94, P-trend ¼ 0.050) and women (OR ¼ 0.59; 95% CI 0.38e0.91, P-trend ¼ 0.043); dairy intake associated with a lower risk of T2DM in women (OR ¼ 0.65; 95% CI 0.49e0.88, P-trend ¼ 0.007) but not men BMI, family history of T2DM, smoking status, alcohol intake, history of hypertension, exercise frequency, coffee consumption, magnesium intake, total energy intake Calcium, vitamin D 3 intake independently not associated with a decrease in T2DM risk; dairy intake is associated with a decreased risk of T2DM Liu et al., 2006 F, 55 7y, n ¼ 37,183 Reference Experimental group (sex, age, n) Intervention studies Borissova et al., 2003 Gedik et al., 1986 Nyomba et al., 1986 De Boer et al., 2008 Von Hurst et al., 2010 Nagpal et al., 2009 F, 43e63 y, n ¼ 10, with T2DM F, 17e54 y, n ¼ 4 M/F, 56e78 y, n ¼ 25 institutionalized F, 50e79 y, n ¼ 16,999 (CaD group) F, y, n ¼ 42 M, 35 y, n ¼ 35 Tai et al., 2008 M/F, 55 3y, n ¼ 33, 12 with IGT Dairy intake and T2DM risk RR ¼ 0.79 (95% CI 0.67e0.93, P ¼ 0.007) when comparing highest and lowest dairy intake; each additional serving of dairy decreased risk of diabetes, by 4% (RR 0.96; 95% CI 0.93e1.01) BMI, smoking status, physical activity, family history of diabetes, alcohol consumption, hypertension, hormone therapy, high cholesterol Control group Study design Outcome measure Supplementation Main results % Change after supplementation F, n ¼ 17, age and BMI matched M/F, 24e46 y, n ¼ 10 n ¼ 95, 50e77 y; non-institutionalized Placebo groupn ¼ 16,952 Placebo group- 42 9y,n ¼ 39 Placebo group- M, 35 y, n ¼ 36 Clinical Effect of vitamin D 3 supplementation on insulin secretion and IR Clinical Clinical Double-blind randomized placebo controlled Randomized placebo controlled Double-blind randomized placebo control Effect of vitamin D deficiency and repletion on pancreatic alpha and beta-cell function Effect of Vitamin D deficiency and repletion with 25(OH) D 3 on insulin and glucagon secretion Effect of vitamin D 3 and calcium supplementation on incidence of T2DM Effect of vitamin D 3 supplementation on IR Effect of 3 doses of 120,000 IU of vitamin D 3 on HOMA-IR, insulin sensitivity, 3 h OGIS and insulin secretion n/a Clinical Effect of 2 doses of 100,000 IU of vitamin D 3 on plasma glucose, serum insulin, ISI, HOMA-IR 1332 IU D 3 /d for 1 month 2000 IU D 3 /d for 6 months 200 mg followed by 10 mg/d of 25(OH)D 3 for 2 weeks 400 IU/d vitamin D 3 ; 1000 mg/d calcium 4000 IU/d of vitamin D 3 for 6 months Three doses of 120,000 IU of vitamin D 3 over 6 weeks Two doses of 100,000 IU vitamin D 3 over 2 weeks Positive correlation between FPIS and changes in 25(OH)D 3 levels (P < 0.018, r ¼ 0.72); FPIS increased by 34% (P < 0.05); SPIS increased by 20% (P > 0.8); IR decreased by 21% (P < 0.001) Insulin AUC in response to oral glucose increased after supplementation (9 to 14 mu min, P < 0.05) No improvement in oral glucose tolerance after 25(OH)D 3 supplementation Vitamin D 3 and calcium supplementation did not reduce the risk of developing T2DM; hazard ratio 1.01 (95% CI 0.94e1.10) Increase in insulin sensitivity (P ¼ 0.003), and a decrease in fasting insulin (P ¼ 0.02) and IR (P ¼ 0.02) in supplemented vs. placebo group A significant increase in serum 25 (OH)D 3 and 3 h OGIS but no significant differences were observed for secondary outcome measures (insulin secretion, insulin sensitivity) No effect on plasma glucose, serum insulin, ISI and Homa-IR 34% increase in FPIS and 21% decrease in IR compared to controls 55% increase in insulin AUC in response to oral glucose 35% decrease in insulin secretion in epileptic patients No change 257% increase in serum 25(OH)D 3 Dairy intake may lower risk of T2DM, the decreased risk of T2DM is mainly attributed to low-fat dairy intake Conclusion Supplementation with vitamin D 3 increases FPIS and insulin sensitivity Vitamin D 3 supplementation increases insulin secretion in response to oral glucose 25(OH)D 3 supplementation has no effect on pancreatic function Vitamin D 3 and calcium supplementation does not reduce the risk of developing T2DM Vitamin D 3 decreases IR and increases insulin sensitivity 5% increase in OGIS Vitamin D 3 supplementation increases insulin sensitivity in response to an oral glucose load No change Vitamin D 3 supplementation has no effect on plasma glucose, serum insulin, ISI and HOMA-IR Abbreviations: AUC, area under the curve; BMI, body mass index; CI, confidence interval; d, day; FPIS, first phase insulin secretion; HOMA-IR, Homeostasis Model Assessment of Insulin Resistance; IFG, impaired fasting glucose; IGT, impaired glucose tolerance; IR, insulin resistance; ISI, insulin sensitivity index; n, number of subjects; n/a, not available; OGIS, oral glucose insulin sensitivity; OGTT, oral glucose tolerance test; OR, odds ratio; RR, relative risk; SPIS, second phase insulin secretion; WHR, waist-to-hip ratio. T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 e161

8 e162 T.S. Moreira, M.J. Hamadeh / e-spen, the European e-journal of Clinical Nutrition and Metabolism 5 (2010) e155ee165 7 mg/kg body weight (both NFG and IFG) of calcium and 10 IU/kg body weight (NFG) and 9 IU/kg body weight (IFG) of vitamin D 3 for a 3 year period. 30 The results showed that supplementation attenuated the rise in fasting glucose in subjects with IFG (5.6e6.9 mmol/l) compared to those given placebo (0.02 mmol/l vs mmol/l, respectively, P ¼ 0.042). 30 The group supplemented with vitamin D 3 and calcium also had a smaller increase in insulin resistance compared to the placebo group (0.05 vs. 0.91, respectively, P ¼ 0.031) as measured with the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). 30 In another large scale study conducted in 41,254 men (40e75 y; BMI 25 3 kg/m 2 ), those who had the greatest dairy intake (mean ¼ 4.1 servings/d) had the lowest risk of developing T2DM compared to those with the lowest intake (mean ¼ 0.5 servings/d; RR 0.77; 95% CI 0.62e0.95, P ¼ 0.003). The results also showed a 9% decrease in the risk of developing T2DM with each additional serving of dairy products consumed daily, with the greatest benefit attributed to an increase in low-fat dairy product consumption (RR 0.91; 95% CI 0.85e0.97). The CARDIA study conducted by Pereira et al. examined the relationship between dairy intake and IR syndrome in young adult men and women (18e30 y). 65 Dairy consumption was inversely related to IR syndrome only in those who were classified as overweight (BMI 25 kg/m 2 ) (OR 0.28; 95% CI 0.14e0.58, P < 0.001). 65 In addition, with each additional serving of dairy products there was a concomitant 21% decrease in the odds of having IR syndrome (OR 0.79; 95% CI 0.70e0.88). 65 One possible explanation for this inverse association between dairy intake and the risk of T2DM is that an increased consumption of dairy products particularly those that are low-fat could displace high-fat, highcalorie foods in the diet which contribute to obesity, a risk factor for T2DM. 66 A more recent study by Kirii et al. also examined the relationship between calcium and vitamin D intake and the risk of T2DM in a Japanese cohort of men and women (n ¼ 59, 796; M/F; age ¼ 57 8 y) and found no association between calcium and T2DM. 67 However, an inverse association between calcium intake and T2DM was observed in individuals in the higher quartile of vitamin D intake versus the lower quartile in men (OR ¼ 0.62; 95% CI 0.41e0.94, P-trend ¼ 0.050) and women (OR ¼ 0.59; 95% CI 0.38e0.91, P-trend ¼ 0.043). Dairy product intake was associated with a reduced risk of T2DM in women (OR ¼ 0.65; 95% CI 0.49e0.88, P-trend ¼ 0.007) but not men. 67 Vitamin D status was not assessed in these studies; however one could assume that the increased consumption of dairy products which are often fortified with vitamin D could play a protective role against the development of T2DM. The above studies help support the notion that there is an association between vitamin D, calcium, dairy intake and T2DM, whether this relationship plays a role in the pathogenesis of T2DM still remains unknown (a summary of prospective studies can be found in Table 2) Intervention studies To examine the effects of cholecalciferol supplementation on insulin resistance and insulin secretion, Borrissova et al. supplemented diabetic women (T2DM) with 1332 IU/d for one month. 10 First phase insulin secretion (FPIS) increased significantly (34%, P < 0.05) after supplementation while no significant change was seen in second phase insulin secretion (SPIS) (20% increase, P > 0.8). This is important considering the integral role that FPIS plays in reducing post-prandial glucose. In another study, Gedik et al. examined the effects of 2000 IU of vitamin D 3 /d for six months on pancreatic a and b-cell function in vitamin D deficient subjects (n ¼ 4; 17e54 y; BMI 23 1 kg/m 2 ). 68 Supplementation did not significantly alter serum calcium or glucagon; however serum 1,25 (OH) 2 D 3 rose exponentially, from 30 pg/ml to 70 pg/ml (P < 0.05), suggesting that a super-compensatory response had occurred in these vitamin D deficient subjects. 68 Insulin secretion rose significantly in subjects who received supplementation when compared to healthy controls as evident by an increase in the area under the insulin curve (from 9 1mUmin to 14 1mUmin in subjects supplemented with vitamin D 3 vs. 12 1mUmin in controls, P < 0.05). 68 These findings suggest that vitamin D deficiency may decrease insulin secretion and that supplementation may help restore insulin secretion. Moreover, a study conducted in twentyfive institutionalized patients (10 epileptic and 15 geriatric; age 56e78 y) examined the effect of 25(OH)D 3 supplementation on insulin secretion by administering patients a single dose of 200 mg followed by 10 mg/d of 25(OH)D 3 for two weeks. In this particular study, there was no effect on insulin secretion in geriatric patients; however, a 35% decrease in insulin secretion was observed in epileptic patients (P < 0.01). 69 This does not necessarily suggest that 25(OH)D 3 is not beneficial in this population because the supplementation period was too short (2 weeks) to elicit any significant physiological effects. It is also possible that the geriatric subjects were more insulin resistant and hence may have required a much higher dose to elicit any effects. Also, these patients were severely vitamin D deficient, with 50% of epileptic and 40% of geriatric patients having levels of 25(OH)D 3 thought to be well below the threshold for developing rickets and osteomalacia (<14 nmol/l). Hence, we hypothesize that they probably required either a greater dosage and/or a longer period of supplementation in order to replenish their vitamin D stores. 69 A randomized placebo-controlled study conducted by De Boer et al. examined the effect of vitamin D 3 (400 IU) and calcium (1000 mg) supplementation (CaD) on the risk of T2DM in 33,951 non-diabetic, postmenopausal women. 70 At the 7 year follow-up, there were 2291 newly diagnosed cases of T2DM; however no difference between the CaD and placebo groups was observed. Although this study did not show a significant negative relationship between vitamin D 3 and calcium supplementation and the risk of developing T2DM, it is important to note that the amount of vitamin D 3 given to these subjects was low compared to that used in previous studies (2000 IU/d). 30,68 A more recent randomized placebo-controlled trial conducted by von Hurst et al. examined the effect of vitamin D 3 supplementation (4000 IU) on insulin sensitivity and secretion in a cohort of insulin resistant, vitamin D deficient women (n ¼ 81). After six months of vitamin D 3 supplementation, serum 25(OH)D 3 concentration increased from 21 to 75 nmol/l. 71 Significant improvements in insulin sensitivity (P ¼ 0.003), and a decrease in fasting insulin (P ¼ 0.02) and IR (P ¼ 0.02) were also observed in the supplemented group when compared to placebo. 71 Perhaps, this study could have been strengthened by also measuring postprandial glucose response as this is thought to be a better indicator of glucose tolerance. In contrast to previous studies which investigated short-term supplementation, von Hurst et al. examined the effect of vitamin D 3 supplementation over a six month period in insulin resistant individuals who because of this may have been at a greater risk of developing T2DM. To date, studies have examined the effects of vitamin D 3 supplementation of various dosages (50e4000 IU) on markers of T2DM; however few have examined the effects of administering supra-physiological doses of vitamin D 3. A study conducted in healthy, centrally-obese, non-diabetic men examined the effect of three oral doses of 120,000 IU of vitamin D 3 or placebo over a period of six weeks on HOMA-IR, insulin sensitivity, OGIS and insulin secretion and found that supplementation improved OGIS compared to placebo (P ¼ 0.038; intention-to-treat analysis P ¼ 0.055). 72 However, no changes were observed in the other outcome measures despite a significant increase in serum 25(OH)D 3. In addition to the above study, Tai et al. found that 2 oral doses of 100,000 IU of vitamin D 3 given to