Low-calcium diet in mice leads to reduced gut colonization by Enterococcus faecium.
Top Janetta,Hendrickx Antoni P A,van Ampting Marleen T J,van Limpt Kees,Knol Jan,van de Kamer Denise,Braat Johanna C,Viveen Marco,Rogers Malbert R,Kemperman Hans,Willems Rob J L,Paganelli Fernanda L
The aim of this study was to determine whether dietary intervention influenced luminal Ca levels and Enterococcus faecium gut colonization in mice. For this purpose, mice fed semi-synthetic food AIN93 were compared to mice fed AIN93-low calcium (LC). Administration of AIN93-LC resulted in lower luminal Ca levels independent of the presence of E. faecium. Furthermore, E. faecium gut colonization was reduced in mice fed AIN93-LC based on culture, and which was in concordance with a reduction of Enterococcaceae in microbiota analysis. In conclusion, diet intervention might be a strategy for controlling gut colonization of E. faecium, an important opportunistic nosocomial pathogen.
Dietary galacto-oligosaccharides and calcium: effects on energy intake, fat-pad weight and satiety-related, gastrointestinal hormones in rats.
Overduin Joost,Schoterman Margriet H C,Calame Wim,Schonewille Arjan J,Ten Bruggencate Sandra J M
The British journal of nutrition
Galacto-oligosaccharides (GOS) are carbohydrates that are fermented by colonic microbiota. The present study examined effects of a 3-week dietary enrichment with 6 % (w/w) GOS on parameters of energy balance in forty-three male Wistar rats. GOS was tested with two doses of calcium phosphate (30 and 100 mmol/kg), known to differently affect colonic fermentation. After 17 d, isoenergetic test meals were presented and plasma responses of ghrelin, glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) were measured. On day 21 (study termination) epididymal fat pads and caecum were weighed. Additionally, gastrointestinal mucosal samples and proximal colonic contents were analysed for gene expression (ghrelin, proglucagon and PYY) and fermentation metabolites (SCFA and lactate), respectively. GOS reduced energy intake most prominently during the first week, without provoking compensatory overeating later on (average intake reduction: 14 %). The GOS-fed rats showed increased caecal and reduced fat-pad weight and increased gene expression of the satiety-related peptides, PYY (1.7-fold) and proglucagon (3.5-fold). Pre-meal baseline and post-meal plasma levels of PYY, but not of ghrelin or GLP-1, were higher in GOS-fed rats than in control rats. Ca enrichment resulted in higher energy intake (average 4.5 %). GOS diets increased lactic acid levels and slightly reduced butyric acid in proximal colonic contents. Ca abolished the GOS-related elevation of lactic acid, while increasing propionic acid levels, but did not inhibit GOS-related effects on energy intake, fat-pad weight or gene expression. These results indicate that dietary GOS stimulate a number of physiological mechanisms that can reduce energy intake, regardless of the calcium phosphate content of the diet.
Damage to the intestinal epithelial barrier by antibiotic pretreatment of salmonella-infected rats is lessened by dietary calcium or tannic acid.
van Ampting Marleen T J,Schonewille Arjan J,Vink Carolien,Brummer Robert Jan M,van der Meer Roelof,Bovee-Oudenhoven Ingeborg M J
The Journal of nutrition
Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.
Could the beneficial effects of dietary calcium on obesity and diabetes control be mediated by changes in intestinal microbiota and integrity?
Gomes J M G,Costa J A,Alfenas R C
The British journal of nutrition
Evidence from animal and human studies has associated gut microbiota, increased translocation of lipopolysaccharide (LPS) and reduced intestinal integrity (II) with the inflammatory state that occurs in obesity and type 2 diabetes mellitus (T2DM). Consumption of Ca may favour body weight reduction and glycaemic control, but its influence on II and gut microbiota is not well understood. Considering the impact of metabolic diseases on public health and the role of Ca on the pathophysiology of these diseases, this review critically discusses possible mechanisms by which high-Ca diets could affect gut microbiota and II. Published studies from 1993 to 2015 about this topic were searched and selected from Medline/PubMed, Scielo and Lilacs databases. High-Ca diets seem to favour the growth of lactobacilli, maintain II (especially in the colon), reduce translocation of LPS and regulate tight-junction gene expression. We conclude that dietary Ca might interfere with gut microbiota and II modulations and it can partly explain the effect of Ca on obesity and T2DM control. However, further research is required to define the supplementation period, the dose and the type of Ca supplement (milk or salt) required for more effective results. As Ca interacts with other components of the diet, these interactions must also be considered in future studies. We believe that more complex mechanisms involving extraintestinal disorders (hormones, cytokines and other biomarkers) also need to be studied.
Calcium and colorectal cancer: some questions remain.
Chia Victoria,Newcomb Polly A
There is convincing laboratory evidence that calcium reduces the risk of colorectal cancer, but previous epidemiologic studies have reported somewhat inconsistent results. A recent large prospective study confirms that higher calcium intake is associated with a modestly reduced risk of distal colorectal cancer. There was little additional risk reduction associated with consumers of more than 700 mg calcium/day. This study also suggests that certain subgroups, such as males, smokers, and people who consume low levels of vitamin D, may be at differential risk. Because colon cancer is a common disease, even a modest decrease in risk has the potential for preventing a substantial number of cases.
Dietary calcium attenuation of body fat gain during high-fat feeding in mice.
Parra Pilar,Bruni Giovanni,Palou Andreu,Serra Francisca
The Journal of nutritional biochemistry
Human epidemiological studies have supported the hypothesis that a dairy food-rich diet is associated with lower fat accumulation, although prospective studies and intervention trials are not so conclusive and contradictory data exist in animal models. The purpose of this study was to assess the effects on body weight and fat depots of dairy calcium (12 g/kg diet) in wild-type mice under ad libitum high-fat (43%) and normal-fat (12%) diets and to gain comprehension on the underlying mechanism of dairy calcium effects. Our results show that calcium intake decreases body weight and body fat depot gain under high-fat diet and accelerates weight loss under normal-fat diet, without differences in food intake. No differences in gene or protein expression of UCP1 in brown adipose tissue or UCP2 in white adipose tissue were found that could be related with calcium feeding, suggesting that calcium intake contributed to modulate body weight in wild-type mice by a mechanism that is not associated with activation of brown adipose tissue thermogenesis. UCP3 protein but not gene expression increased in muscle due to calcium feeding. In white adipose tissue there were effects of calcium intake decreasing the expression of proteins related to calcium signalling, in particular of stanniocalcin 2. CaSR levels could play a role in decreasing cytosolic calcium in adipocytes and, therefore, contribute to the diminution of fat accretion. Results support the anti-obesity effect of dietary calcium in male mice and indicate that, at least at the time-point studied, activation of thermogenesis is not involved.
Methylation analysis in fatty-acid-related genes reveals their plasticity associated with conjugated linoleic acid and calcium supplementation in adult mice.
Chaplin Alice,Palou Andreu,Serra Francisca
European journal of nutrition
PURPOSE:DNA methylation is one of the most extensively studied mechanisms within epigenetics, and it is suggested that diet-induced changes in methylation status could be involved in energy metabolism regulation. Conjugated linoleic acid (CLA) and calcium supplementation counteract body weight gain, particularly under a high-fat (HF) diet, in adult mice. The aim was to determine whether the modulation of DNA methylation pattern in target genes and tissues could be an underlying mechanism of action. METHODS:Mice (C57BL/6J) were divided into five groups according to diet and treatment: normal fat as the control group (12 % kJ content as fat), HF group (43 % kJ content as fat), HF + CLA (6 mg CLA/day), HF + calcium (12 g/kg of calcium) and HF with both compounds. Gene expression and methylation degree of CpG sites in promoter sequences of genes involved in fatty acid metabolism, including adiponectin (Adipoq), stearoyl-CoA desaturase (Scd1) and fatty acid synthase (Fasn), were determined by bisulphite sequencing in liver and epididymal white adipose tissue. RESULTS:Results showed that the methylation profile of promoters was significantly altered by dietary supplementation in a gene- and tissue-specific manner, whereas only slight changes were observed in the HF group. Furthermore, changes in specific CpG sites were also associated with an overall healthier metabolic profile, in particular for calcium-receiving groups. CONCLUSIONS:Both CLA and calcium were able to modify the methylation pattern of genes involved in energy balance in adulthood, which opens a novel area for increasing efficiency in body weight management strategies.
Calcium ameliorates obesity induced by high-fat diet and its potential correlation with p38 MAPK pathway.
Sun Chao,Wang Li,Yan Jun,Liu Shumin
Molecular biology reports
To investigate whether and on which pathway dietary calcium influence the obesity induced by high-fat diet, thirty male Kunming mice were fed in six groups for 4 weeks and mouse preadipocytes were divided into eight groups for different treatment. Body weight gain was measured each week. Calcium in serum and tissues, intracellular free Ca(2+) concentration ([Ca(2+)]i), blood fat and intracellular lipid content were also measured. The expression of Lipid metabolism-related genes were measured by q RT-PCR. Compared with control group, body weight gain (P < 0.05) and fat pad weight (P < 0.01) in Low calcium group decreased. Triglycerides (TG) and total Cholesterol (TC) level decreased (P < 0.01), while HDL-Cholesterol (HDL) level increased (P < 0.01). And calcium supply increased calcium content in blood serum and tissues. In tissues, adipogenesis and vitamin D receptor (VDR) genes expression decreased but lipoclasis genes expression increased. These anti-obesity effects were more obvious when supplying with 2.8% calcium, but the effects were reduced while supplying Nifedipine at the same time. The results in preadipocytes indicated that calcium-treated can reduce intracellular lipid content, along with adipogenesis and lipoclasis genes expression decrease, promoted the expression levels of p38 MAPK pathway upstream gene MKK6 (P < 0.01) and downstream gene MAPKAPK2 (P < 0.01). Treated with SB203580 could increase adipogenesis genes expression, decrease lipoclasis genes expression and ([Ca(2+)]i) (P < 0.01). These results implied that dietary calcium had remarkable effect on anti-obesity effect and p38 MAPK pathway potentially participated in calcium-mediated lipid accumulation and lipolysis in mouse preadipocytes.
Effect of Early Overfeeding on Palatable Food Preference and Brain Dopaminergic Reward System at Adulthood: Role of Calcium Supplementation.
Conceição E P S,Carvalho J C,Manhães A C,Guarda D S,Figueiredo M S,Quitete F T,Oliveira E,Moura E G,Lisboa P C
Journal of neuroendocrinology
Rats raised in small litters (SL) are obese and hyperphagic. In the present study, we evaluated whether obesity is associated with changes in the mesocorticolimbic dopaminergic reward system in these animals at adulthood. We also assessed the anti-obesity effects of dietary calcium supplementation. To induce early overfeeding, litters were adjusted to three pups on postnatal day (PN)3 (SL group). Control litters were kept with 10 pups each until weaning (NL group). On PN120, SL animals were subdivided into two groups: SL (standard diet) and SL-Ca [SL with calcium supplementation (10 g calcium carbonate/kg rat chow) for 60 days]. On PN175, animals were subjected to a food challenge: animals could choose between a high-fat (HFD) or a high-sugar diet (HSD). Food intake was recorded after 30 min and 12 h. Euthanasia occurred on PN180. SL rats had higher food intake, body mass and central adiposity. Sixty days of dietary calcium supplementation (SL-Ca) prevented these changes. Only SL animals preferred the HFD at 12 h. Both SL groups had lower tyrosine hydroxylase content in the ventral tegmental area, lower dopaminergic transporter content in the nucleus accumbens, and higher type 2 dopamine receptor (D2R) content in the hypothalamic arcuate nucleus (ARC). They also had higher neuropeptide Y (NPY) and lower pro-opiomelanocortin contents in the ARC. Calcium treatment normalised only D2R and NPY contents. Precocious obesity induces long-term effects in the brain dopaminergic system, which can be associated with an increased preference for fat at adulthood. Calcium treatment prevents this last alteration, partially through its actions on ARC D2R and NPY proteins.
Anti-obesogenic effects of calcium prevent changes in the GLP-1 profile in adult rats primed by early weaning.
Quitete Fernanda Torres,Nobre Jessica Lopes,Peixoto-Silva Nayara,de Moura Egberto Gaspar,Lisboa Patricia Cristina,de Oliveira Elaine
Molecular nutrition & food research
SCOPE:Gut peptides regulate appetite and adipogenesis. Early weaning (EW) leads to later development of obesity that can be prevented by calcium supplementation. We evaluated gut peptides that may have a role in the establishment of this dysfunction. METHODS AND RESULTS:At birth, lactating Wistar rats were separated in: EW, lactating rats involved with a bandage interrupting the lactation during the last 4 days of standard lactation, and C (control) dams whose pups had free access to milk during throughout lactation. At 120 days old, half of EW group received calcium supplementation (EWCa); EW and C received standard diet. At 21 days old, EW presented higher glucagon-like peptide 1 (GLP-1) in plasma and glucagon-like peptide 1 receptor (GLP1-R) in adipose tissue and hypothalamus, but lower GLP-1 and GLP1-R in the gut. At 180 days old, GLP-1 response to food intake was blunted in EW and restored by calcium. GLP-1 in the gut was lower in EW and its receptor was lower in adipose tissue, and GLP1-R was higher in the gut of calcium EW group. CONCLUSION:Thus, EW had short- and long-term effects upon GLP-1 profile, which may have contributed to obesity development, hyperphagia, and insulin resistance due to its adipogenic and appetite control roles. Calcium supplementation was able to prevent most of the changes in GLP-1 caused by EW.
Food ingredients as anti-obesity agents: a review.
Trigueros L,Peña S,Ugidos A V,Sayas-Barberá E,Pérez-Álvarez J A,Sendra E
Critical reviews in food science and nutrition
Overweight and obesity have a major impact on global health; their prevalence has rapidly increased in all industrialized countries in the past few decades and diabetes and hypertension are their direct consequences. Pharmacotherapy provides reinforcement for obesity treatment, but should be an adjunctive support to diet, exercise, and lifestyle modification. At present, only orlistat and sibutramine have been approved by the US Food and Drug Administration for long-term use, but sibutramine was withdrawn for sale by the European Medicines Agency. The development of functional foods for the prevention and/or treatment of obesity suppose an opportunity for the food market and involve the knowledge of the mechanisms of appetite and energy expenditure as well as the metabolic sensation of satiety. Strategies for weight control management affect gut hormones as potential targets for the appetite metabolic regulation, stimulation of energy expenditure (thermogenesis), and modifications in the metabolic activity of the gut microbiota. Functional foods for obesity may also include bioactive fatty acids, phenolic compounds, soybean, plant sterols, dietary calcium, and dietary fiber. This review intends to offer an overview of the present situation of the anti-obesity agents currently used in dietary therapy as well as some functional food ingredients with potentially anti-obesity effects.
Regulation of adiposity and obesity risk by dietary calcium: mechanisms and implications.
Zemel Michael B
Journal of the American College of Nutrition
Dietary calcium plays a pivotal role in the regulation of energy metabolism; high calcium diets attenuate adipocyte lipid accretion and weight gain during periods of overconsumption of an energy-dense diet and increase lipolysis and preserve thermogenesis during caloric restriction, thereby markedly accelerating weight loss. Intracellular Ca2+ has a key role in regulating adipocyte lipid metabolism and triglyceride storage, with increased intracellular Ca2+ resulting in stimulation of lipogenic gene expression and lipogenesis, suppression of lipolysis, and increased lipid filling and adiposity. Moreover, we have recently demonstrated that the increased calcitriol released in response to low calcium diets stimulates Ca2+ influx in human adipocytes and thereby promotes adiposity. Accordingly, suppressing calcitriol levels by increasing dietary calcium is an attractive target for the prevention and management of obesity. In support of this concept, transgenic mice expressing the agouti gene specifically in adipocytes (a human-like pattern) respond to low calcium diets with accelerated weight gain and fat accretion, while high calcium diets markedly inhibit lipogenesis, accelerate lipolysis, increase thermogenesis and suppress fat accretion and weight gain in animals maintained at identical caloric intakes. Further, low calcium diets impede body fat loss, while high calcium diets markedly accelerate fat loss in transgenic mice subjected to caloric restriction. These findings are further supported by clinical and epidemiological data demonstrating a profound reduction in the odds of being obese associated with increasing dietary calcium intake. Notably, dairy sources of calcium exert a significantly greater anti-obesity effect than supplemental sources in each of these studies, possibly due to the effects of other bioactive compounds, such as the angiotensin converting enzyme inhibitor found in milk, on adipocyte metabolism, indicating an important role for dairy products in the control of obesity.
Small lipidated anti-obesity compounds derived from neuromedin U.
Micewicz Ewa D,Bahattab Omar S O,Willars Gary B,Waring Alan J,Navab Mohamad,Whitelegge Julian P,McBride William H,Ruchala Piotr
European journal of medicinal chemistry
A small library of truncated/lipid-conjugated neuromedin U (NmU) analogs was synthesized and tested in vitro using an intracellular calcium signaling assay. The selected, most active analogs were then tested in vivo, and showed potent anorexigenic effects in a diet-induced obese (DIO) mouse model. The most promising compound, NM4-C16 was effective in a once-weekly-dose regimen. Collectively, our findings suggest that short, lipidated analogs of NmU are suitable leads for the development of novel anti-obesity therapeutics.
Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression.
Sun Xiaocun,Zemel Michael B
Obesity (Silver Spring, Md.)
OBJECTIVE:Obesity is associated with elevated oxidative stress and low-grade systemic inflammation. We have demonstrated recently that 1alpha,25-(OH)(2)-D(3) promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1alpha,25-(OH)(2)-D(3) by increasing dietary calcium down-regulates diet-induced oxidative stress in aP2-agouti transgenic mice. However, whether the anti-obesity effect of dietary calcium plays a role in regulation of obesity-associated inflammation is not clear. RESEARCH METHODS AND PROCEDURES:We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2-agouti transgenic mice fed low- and high-calcium obesigenic diets and in the modulation of cytokine production by 1alpha,25-(OH)(2)-D(3) in cultured murine and human adipocytes. RESULTS:The high-calcium diet inhibited the expression of pro-inflammatory factors tumor necrosis factor alpha and interleukin (IL)-6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti-inflammatory factors IL-15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2-fold increase in IL-15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low-calcium diet. 1alpha,25-(OH)(2)-D(3) also markedly stimulated the expression of tumor necrosis factor alpha (p < 0.001) and IL-6 (p = 0.016) in differentiated 3T3-L1 adipocytes and increased IL-6 (p = 0.004) and IL-8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. DISCUSSION:These data demonstrate that 1alpha,25-(OH)(2)-D(3) favors inflammatory cytokine expression and inhibits anti-inflammatory cytokine expression; accordingly, suppression of 1alpha,25-(OH)(2)-D(3) by dietary calcium inhibits adipocyte-derived inflammation associated with obesity.
Calcium supplementation prevents obesity, hyperleptinaemia and hyperglycaemia in adult rats programmed by early weaning.
Nobre Jessica Lopes,Lisboa Patricia Cristina,Lima Natália da Silva,Franco Juliana Gastão,Nogueira Neto José Firmino,de Moura Egberto Gaspar,de Oliveira Elaine
The British journal of nutrition
It is known that Ca therapy may have anti-obesity effects. Since early weaning leads to obesity, hyperleptinaemia and insulin resistance, we studied the effect of dietary Ca supplementation in a rat model. Lactating rats were separated into two groups: early weaning (EW) - dams were wrapped with a bandage to interrupt lactation in the last 3 d of lactation and control (C) - dams whose pups had free access to milk during the entire lactation period (21 d). At 120 d, EW and C offspring were subdivided into four groups: (1) C, received standard diet; (2) CCa, received Ca supplementation (10 g of calcium carbonate/kg of rat chow); (3) EW, received standard diet; (4) EWCa, received Ca supplementation similar to CCa. The rats were killed at 180 d. The significance level was at P < 0·05. Adult EW offspring displayed hyperphagia (28 %), higher body weight (9 %) and adiposity (77 %), hyperleptinaemia (twofold increase), hypertriacylglycerolaemia (64 %), hyperglycaemia (16 %), higher insulin resistance index (38 %) and higher serum 25-hydroxyvitamin D₃ (fourfold increase), but lower adiponectinaemia:adipose tissue ratio (44 %). In addition, they showed Janus tyrosine kinase 2 and phosphorylated signal transducer and activator of transcription 3 underexpression in hypothalamus (36 and 34 %, respectively), suggesting leptin resistance. Supplementation of Ca for 2 months normalised these disorders. The EW group had no change in serum insulin, thyroxine or triiodothyronine, and Ca treatment did not alter these hormones. In conclusion, we reinforced that early weaning leads to late development of some components of the metabolic syndrome and leptin resistance. Dietary Ca supplementation seems to protect against the development of endocrine and metabolic disorders in EW offspring, maybe through vitamin D inhibition.
Calcium and vitamin D in obesity.
Song Qingming,Sergeev Igor N
Nutrition research reviews
New and more effective nutritional measures are urgently needed for the prevention of obesity. The role of Ca and vitamin D in obesity has been recently implicated. Low Ca intake and low vitamin D status have been linked with an increased risk of obesity in epidemiological studies; however, clinical intervention trials designed to test this association have produced controversial results. The suggested anti-obesity mechanisms of Ca and vitamin D include the regulation of adipocyte death (apoptosis), adipogenesis and lipid metabolism. Dietary Ca has been also shown to increase faecal fat excretion. The potential role of Ca and vitamin D in shifting energy balance towards a more negative state is an area of considerable interest. Ultimately, a review of recent research findings does not allow the reaching of a definitive conclusion that increasing Ca intake and rising vitamin D status will influence fat mass and body weight or decrease the risk of obesity and overweight.
Risk of keratinocyte carcinomas with vitamin D and calcium supplementation: a secondary analysis of a randomized clinical trial.
Passarelli Michael N,Karagas Margaret R,Mott Leila A,Rees Judy R,Barry Elizabeth L,Baron John A
The American journal of clinical nutrition
BACKGROUND:It is unknown whether dietary supplementation with vitamin D or calcium prevents keratinocyte carcinomas, also known as nonmelanoma skin cancers. OBJECTIVES:This study aimed to determine whether daily vitamin D or calcium supplementation alters the risk of basal cell carcinoma (BCC) or invasive cutaneous squamous cell carcinoma (SCC). METHODS:The Vitamin D/Calcium Polyp Prevention Study is a completed multicenter, double-blind, placebo-controlled, partial 2 × 2 factorial, randomized clinical trial of vitamin D, calcium, or both for the prevention of colorectal adenomas. During 2004-2008, a total of 2259 men and women, 45-75 y of age, recently diagnosed with a colorectal adenoma, were randomly assigned to 1000 IU/d of vitamin D3 or placebo and 1200 mg/d of calcium carbonate or placebo for 3 or 5 y, and followed after treatment ended. Reports of incident BCC or SCC were confirmed from pathology records. RESULTS:During a median follow-up of 8 y, 200 (9%) participants were diagnosed with BCC and 68 (3%) participants were diagnosed with SCC. BCC incidence was unrelated to treatment with vitamin D compared with no vitamin D (HR: 0.96; 95% CI: 0.73, 1.26), calcium compared with no calcium (HR: 1.01; 95% CI: 0.74, 1.39), and both agents compared with neither (HR: 0.99; 95% CI: 0.65, 1.51). SCC incidence was unrelated to treatment with vitamin D compared with no vitamin D (HR: 0.79; 95% CI: 0.49, 1.27), but there was suggestive evidence of beneficial treatment effects for calcium compared with no calcium (HR: 0.60; 95% CI: 0.36, 1.01) and both agents compared with neither (HR: 0.42; 95% CI: 0.19, 0.91). CONCLUSIONS:Calcium alone or in combination with vitamin D may reduce the risk of SCC, but not BCC. This trial was registered at clinicaltrials.gov as NCT00153816.