Mol. Nutr. Food Res. 2014, 00, 1–7
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DOI 10.1002/mnfr.201300503
RESEARCH ARTICLE
High vitamin D and calcium intakes reduce diet-induced obesity in mice by increasing adipose tissue apoptosis Igor N. Sergeev and Qingming Song Department of Health and Nutritional Sciences, South Dakota State University, Brookings, SD, USA Scope: Modulation of apoptosis is emerging as a promising antiobesity strategy because removal of adipocytes through this process will result in reducing body fat. Effects of vitamin D on apoptosis are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca2+ . We have previously shown that 1,25-dihydroxyvitamin D3 induces the Ca2+ signal associated with activation of Ca2+ -dependent apoptotic proteases in mature adipocytes. In this study, a diet-induced obesity (DIO) mouse model was used to evaluate the role of vitamin D and calcium in adiposity. Methods and results: DIO mice fed high vitamin D3 , high Ca, and high D3 plus high Ca diets demonstrated a decreased body and fat weight gain, improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25-hydroxyvitamin D, 1,25dihydroxyvitamin D, parathyroid hormone (PTH)), but an increased plasma Ca2+ . High D3 and Ca intakes were associated with induction of apoptosis and activation of Ca2+ -dependent apoptotic proteases, calpain and caspase-12, in adipose tissue of DIO mice. The combination of D3 plus Ca was more effective than D3 or Ca alone in decreasing adiposity. Conclusion: The results imply that high vitamin D and Ca intakes activate the Ca2+ -mediated apoptotic pathway in adipose tissue. Targeting this pathway with vitamin D and Ca supplementation could contribute to the prevention and treatment of obesity. However, this potentially effective and affordable approach needs to be evaluated from a safety point of view.
Received: July 11, 2013 Revised: November 4, 2013 Accepted: December 13, 2013
Keywords: Apoptosis / Calcium / Calpain / Caspase-12 / Diet-induced obesity / Vitamin D
1
Introduction
The induction of the death of adipocytes through apoptosis is emerging as a promising strategy for the prevention and treatment of obesity [1–5]. The increase in adipose tissue mass is the result of both hypertrophy, an increase in adipocyte size, and hyperplasia, an increase in adipocyte number, and once adipocytes achieve a maximum size, further increase in the adipose tissue mass involves an increase in adipocyte number [6–8]. Thus, weight loss can be caused not only by a decrease in adipocyte size (i.e., increasing lipolysis with a potential for lipotoxic effects), but also in adipocyte number (e.g., by stimulating apoptosis). Even a Correspondence: Dr. Igor N. Sergeev, Department of Health and Nutritional Sciences, Box 2203, South Dakota State University, Brookings, SD 57007, USA E-mail: [email protected] Fax: +1-605-688-5603 Abbreviations: 1,25(OH)2 D3 , 1,25-dihydroxyvitamin D3 ; 25(OH)D, 25-hydroxyvitamin D; DIO, diet-induced obesity; HF, high fat; PTH, parathyroid hormone; WAT, white adipose tissue C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
small increase in the rate of adipocyte apoptosis will prevent excessive accumulation of adipose tissue and can result in a significant loss of adipose tissue mass over time. Therefore, removal of adipocytes through apoptosis will reduce body fat and can help with long-term maintenance of weight loss. The effects of vitamin D on apoptosis are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca2+ [9–13]. We have shown that the critical characteristic of the apoptotic Ca2+ signal is a sustained increase in concentration of intracellular Ca2+ , reaching elevated but not cytotoxic levels [12, 14–18]. Recently, we demonstrated that the Ca2+ regulatory hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) induces apoptosis in mature mouse 3T3-L1 adipocytes and the apoptosis-inducing effect of this hormone is mediated via Ca2+ signaling and Ca2+ -dependent apoptotic proteases, -calpain and caspase-12 [1, 3, 4, 19–21]. However, the critical question—can adipocyte apoptosis be induced and the apoptotic molecular targets activated in adipose tissue by increasing dietary intake levels of vitamin D and Ca?—remains unanswered. This is an important problem because it does www.mnf-journal.com
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not allow for the development of nutritional strategies for regulation of apoptosis in obesity. This study was undertaken to determine whether increased dietary vitamin D and Ca intakes can decrease the body and fat weight gain in mice fed high-fat (HF) diet. We hypothesized that high vitamin D and Ca intakes will lead to activation of Ca2+ -dependent apoptotic molecular targets through increasing circulating concentrations of 1,25(OH)2 D3 and Ca2+ , resulting in induction of Ca2+ -mediated apoptosis in adipocytes. The main findings obtained demonstrate that high vitamin D and Ca intakes decrease the body and fat weight gain in diet-induced obesity (DIO) and the mechanism of these effects involves activation of the Ca2+ -mediated apoptotic pathway in adipose tissue (specifically, induction of activities of apoptotic proteases, Ca2+ -dependent calpain and Ca2+ /calpain-dependent caspase-12).
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Materials and methods
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Dihydroxyvitamin D ELISA, 25-Hydroxyvitamin D ELISA (ImmunoDiagnosticSystems, Scottsdale, AZ), PTH (mouse) ELISA (Immutopics Int., San Clemente, CA).
2.3 Apoptosis and apoptotic proteases Apoptotic DNA/chromatin changes were detected by measuring the histone-associated DNA fragments (mono- and oligonucleosomes; Cell Death Detection ELISA, Roche Diagnostics, Indianapolis, IN) and ssDNA breaks (ssDNA Apoptosis ELISA, Chemicon/EMD Millipore) in white adipose tissue (WAT) extracts. The activities of apoptotic proteases, calpain and caspase-12, were measured in WAT extracts with the fluorogenic peptide substrates, Ac-Leu-Leu-Tyr-AFC and Ala-Thr-Ala-Asp-AFC, respectively (Calpain and Caspase-12 Fluorometric Assay Kits, BioVision) [3, 15, 19]. Fluorescence and absorbance were measured in FL ×800 and EL ×800 microplate readers (BioTek, Highland Park, VT), respectively.
2.1 Animals and diets Male 4-wk-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in ventilated plastic transparent cages with bedding in a 12:12-h light– dark cycle at a temperature of 22 ± 1⬚C. Weight-matched mice were randomly assigned to the experimental HF diets containing 60% energy as fat (the normal control diet contained 10% energy as fat). The treatment groups on HF diet were high calcium (Ca diet, 1.2% Ca), high vitamin D (D diet; vitamin D3 intake ten times higher than the recommended level of 1000 IU/kg) [22] and high Ca plus high D3 (Ca + D diet). The control (normal fat and HF) diets contained the recommended 0.6% Ca and 1000 IU D3 /kg [22]; Ca diet contained 1000 IU D3 /kg. The average intake of vitamin D3 by mice fed the normal and HF diets (1000 IU D3 /kg) was 2.5 IU per mouse per day. The mice fed D diet received per os 0.625 g (25 IU) vitamin D3 (Sigma-Aldrich, St. Louis, MO) in soybean oil per animal every day plus 1000 IU D3 /kg in HF diet. The duration of the experiment was 10 wk. The diets were purchased from Research Diets, Inc. (New Brunswick, NJ). Body weight was measured every 3 days and food intake was measured every day. Mice were food-deprived overnight prior to blood and tissue collection. The South Dakota State University Institutional Care and Use Committee approved the protocols and maintenance of the animals (IACUC Protocol number 12-021A).
2.4 Statistical analysis A one-way ANOVA with t-test was used for group comparisons (SAS 9.2). Data are expressed as mean ± SEM and differences considered to be statistically significant at p < 0.05.
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Results
3.1 Body and fat weight gain Body weight gain and terminal body weights were decreased in mice fed a Ca + D diet, as compared with the DIO control, while mice fed Ca or D diets demonstrated a trend to a reduced terminal body weights (Fig. 1, upper panel). Cumulative energy intakes in mice fed HF diets were not different (not shown) and, thus, it may not account for the differences in body weight between mice fed HF diets. The weights of epididymal and retroperitoneal fat pads were significantly lower in mice fed Ca, D, and especially Ca + D diets, as compared with the DIO control (Fig. 1, lower panel). Liver weights were significantly higher in the DIO control compared with all treatment groups (not shown). These data strongly indicate a reduced percentage of total body fat in the treatment groups compared with the control DIO group.
2.2 Plasma markers Biomarkers of adiposity, vitamin D status, and Ca metabolism were measured in blood plasma (collected in heparinized tubes) using commercially available kits. The following kits were used: Adiponectin (mouse) ELISA, Glucose Assay Kit, Calcium Colorimetric Assay Kit (BioVision, Milpitas, CA), Insulin (mouse) ELISA (EMD Millipore, Billerica, MA), 1,25 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Biomarkers of adiposity, vitamin D status, and Ca metabolism The fasting plasma glucose concentration was significantly lower in mice fed Ca and D diets, and it was at the level of the normal fat diet control in mice fed Ca + D diet (Fig. 2, upper www.mnf-journal.com
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panel). These changes in glucose concentration were accompanied by a corresponding decrease (Ca diet) or a complete normalization (D and Ca + D diets) of insulin concentration in plasma (Fig. 2, middle panel). The concentration of adiponectin, a polypeptide insulin-sensitizing hormone secreted by adipose tissue, was decreased in plasma of DIO mice, but the trend to an increased adiponectin concentration was observed in mice fed Ca, D, and Ca + D diets (Fig. 2, lower panel). DIO in mice was accompanied by a significant decrease in plasma concentration of the transport, 25-hydroxyvitamin D (25(OH)D), and the hormonal, 1,25(OH)2 D, forms of vitamin D (Fig. 3, upper panel). In mice fed a D diet, concentrations of these vitamin D metabolites were raised dramatically, exceeding those concentrations not only in the DIO group, but
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Figure 1. Body and fat weight gain in mice fed high-fat diets with increased levels of Ca and vitamin D. Upper panel: Data are means for each group and time point; **p < 0.1, as compared with the DIO group at week 10; p < 0.05 for all DIO groups as compared with the normal control group at week 10; n = 7–8 per group from week 1 to week 10. The inset shows (from left to right) mice from the DIO, control, and Ca + D groups at week 10. Lower panel: Absolute and relative weights of the epididymal and retroperitoneal fat pads. Means ± SEM, n = 7–8 per group, p < 0.05 for all DIO groups as compared with the normal control group, **p < 0.05 as compared with the DIO group.
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Figure 2. Glucose, insulin, and adiponectin concentrations in blood of DIO mice. Results are presented as means ± SEM, n = 7–8 per group. The indicated parameters were measured in plasma, as described in Section 2. *p < 0.05, as compared with the control group; **p < 0.05, as compared with the DIO group.
also in the normal control. In the Ca diet group, concentrations of 25(OH)D and 1,25(OH)2 D did not differ from those in the DIO group, although the trend to a further decrease in the 25(OH)D concentration was observed. Mice fed Ca + D diet demonstrated increases in 25(OH)D and 1,25(OH)2 D concentrations reaching the normal control (for 25(OH)D) or slightly below than normal controls (for 1,25(OH)2 D) levels. www.mnf-journal.com
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Figure 3. Biomarkers of vitamin D status and calcium metabolism in DIO mice. The concentrations of 25(OH)D, 1,25(OH)2 D, total Ca2+ , and PTH were measured in heparinized plasma, as described in Section 2. Results are means ± SEM, n = 7–8 per group. *p < 0.05, as compared with the control group; **p < 0.05, as compared with the DIO group.
The increases in 1,25(OH)2 D concentration were accompanied by a significant elevation of the plasma total Ca2+ concentration (D and Ca + D groups; Fig. 3, lower left panel). It appears that there was a modest trend of increasing plasma Ca2+ in the Ca diet group, as compared with DIO control, but the Ca2+ concentration was within normal values in this group. Interestingly, the plasma concentration of PTH was increased in the DIO group and it was markedly decreased in mice fed Ca (1.2%), but not D diet (Fig. 3, lower right panel).
However, the formation of ssDNA breaks was not decreased in WAT of DIO mice (see Fig. 4, left panel), probably, reflecting a low level of apoptosis and no significant DNA damage. High Ca alone, high vitamin D3 alone, and high Ca plus high D3 intakes induced apoptosis in adipose tissue of DIO mice, as demonstrated by an enrichment of WAT extracts with nucleosomal and ssDNA fragments.
3.4 Calpain and caspase-12 activities 3.3 Apoptosis Apoptosis in WAT was measured by oligonucleosomal fragmentation of the chromosome using antihistone plus antiDNA antibodies and by ssDNA breaks using monoclonal anti-ssDNA antibodies. These antibodies detect the apoptotic internucleosomal degradation of genomic DNA (mono- and oligonucleosomes) and the apoptosis-induced DNA breaks. DIO mice demonstrated a small, but significant decrease in apoptotic-like degradation of nuclei, as measured by the nucleosomal fragments in WAT extracts (Fig. 4, left panel). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DIO in mice was accompanied by a significant decrease in activities of Ca2+ -dependent apoptotic proteases, calpain and caspase-12, in the WAT extracts (Fig. 4, right panel). High Ca intake markedly raised calpain activity, as compared with the DIO and normal controls. High vitamin D3 intake increased calpain activity up to the levels of the normal control. Vitamin D3 was effective in increasing caspase-12 activity in adipose tissue of DIO mice, while increased Ca intake appeared to have no effect. The pattern of activities of these apoptotic proteases was generally consistent with the level of apoptosis in adipose tissue of DIO mice. It is also necessary to note that a www.mnf-journal.com
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Figure 4. Apoptosis and activities of calpain and caspase-12 in adipose tissue of DIO mice. Apoptosis was measured by the increase in nucleosomal and ssDNA fragments in WAT extracts, as described in Section 2. Results are expressed in the relative absorbance units (RAUs) per mg of protein, where RAU = (absorbance 405 nm − absorbance 490 nm) × (102 ). The calpain and caspase-12 activities were measured in the WAT extracts, as described in Section 2, and expressed in the relative fluorescence units (RFUs) per microgram of protein. Results are means ± SEM, n = 7–8 per group. *, **, and *** indicate statistically significant differences (p < 0.05), as compared with the control, DIO, and D3 group, respectively.
decreased (below the minimal, basal level of normal control) apoptosis in DIO was associated with decreased activity of calpain and caspase-12.
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Discussion
Obesity is associated with an increased adipocyte number leading to excessive accumulation of adipose tissue [1, 6]. Apoptosis is the main mechanism for regulating cell number in many tissues [4, 18]. Induction of the death of adipocytes through apoptosis is emerging as a promising strategy for the prevention and treatment of obesity because removal of adipocytes via this mechanism will result in reducing body fat [2–5]. Importantly, decreasing adiposity through induction of adipocyte apoptosis can result in long-lasting maintenance of weight loss, in contrast to that which occurs after energy restriction. Vitamin D insufficiency is prevalent in obesity, and low vitamin D status has been linked with the increased risk of adiposity in epidemiological studies (see [2] for a review; [23,24]). However, a recent study [25] based on a bidirectional genetic approach that limits confounding suggests that a higher BMI leads to lower circulating 25(OH)D, while any effects of lower 25(OH)D increasing BMI appear to be small. On the other hand, it is well established that 25(OH)D is accumulated in adipose tissue [26,27] making it less accessible for bioconversion to 1,25(OH)2 D3 , and this may causally contribute to the vitamin D–obesity link. Several experimental studies suggested the critical roles of 1,25(OH)2 D3 in regulation of the mitochondrial uncoupling proteins linked to modulation of thermogenesis [28,29] as well as C/EPB␣/PPAR␥ signaling linked to adipogenesis [30, 31]. Somewhat paradoxically, vitamin D receptor knockout mice are resistant to DIO [32], but this is, probably, due to the global effects causing growth disturbances and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
generalized tissue atrophy and impacting overall energy balance (it is worth mentioning that vitamin D receptors occupy over 2700 genomic positions and dramatically change expression of at least 229 human genes [33]). It is also important to emphasize that 1,25(OH)2 D3 induces nongenomic, cellular Ca2+ -mediated signaling in mature adipocytes [3]. The observations described implicate a possible role for the hormone 1,25(OH)2 D3 in regulation of adiposity but underscore the lack of mechanistic understanding of these effects. The potential role of calcium in regulation of adiposity has been investigated using animal models of obesity and in epidemiological studies (see [2] for a review). These observations suggest that a low calcium intake is associated with a greater fat mass, while a high calcium intake appears to promote weight loss [34–36], although the lack of relationship between calcium intake and body weight was also reported [37]. The proposed mechanisms include modulation of the fecal fat excretion, adipogenesis and lipid metabolism, and the mitochondrial uncoupling proteins [2, 34–36]. However, the role of calcium/vitamin D signaling in regulation of adiposity, particularly via apoptotic mechanism, has not been investigated. In this study, we employed a mouse model of DIO to investigate whether high vitamin D and calcium intakes reduce adiposity. The results obtained show that mice fed an HF diet with an increased level of Ca (1.2%) and high per os intake of vitamin D3 (25 g/kg of body weight) demonstrate a decreased body and fat weight gain (epididymal and peritoneal WAT depots) and significantly improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25(OH)D, 1,25(OH)2 D). However, high vitamin D3 intake led to an increased circulating Ca2+ concentration, which has reached hypercalcemic levels in mice fed a high Ca plus high vitamin D3 diet. It is important to note that the Ca2+ –PTH–vitamin D physiological interactions appear to be affected in DIO: mice fed an HF diet demonstrated lower Ca2+ and increased PTH concentrations; however, the www.mnf-journal.com
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chronic increased intakes of Ca and vitamin D did not reduce plasma Ca2+ and PTH to normal levels. The apoptosis (as indicated by the oligonucleosomal fragmentation of chromatin) and activities of calpain and caspase12 in WAT were significantly decreased in DIO, and high Ca and vitamin D intakes were effective in inducing apoptosis and increasing activities of these proteases. An increased Ca intake was particularly effective in inducing calpain activity, while a high vitamin D intake was associated with increased caspase-12 activity. These observations are important because they indicate that apoptosis is decreased in WAT of obese mice and that the activities of Ca2+ -dependent apoptotic proteases can be induced by increasing Ca and vitamin D intakes. It is tempting to hypothesize that an increased Ca intake can directly lead to an increased pool of intracellular and intercellular Ca2+ in and around adipocytes, while high levels of 1,25(OH)2 D3 due to high intake of vitamin D3 allow an increased Ca2+ influx from the extracellular space and Ca2+ mobilization from the intracellular stores and, thus, its subsequent utilization in the Ca2+ -mediated apoptotic signaling (which requires a sustained, prolonged increase in concentration of cytosolic Ca2+ ) [10,12]. Although the involvement of 1,25(OH)2 D3 in increasing cellular Ca2+ and inducing apoptosis has been previously demonstrated in adipocytes in vitro [3, 21], the findings presented here demonstrate, for the first time, that the vitamin D induced, Ca2+ -mediated signaling can be employed to trigger apoptosis in adipose tissue in vivo. Furthermore, the data obtained provide the identification of apoptotic molecular targets in WAT, Ca2+ -dependent calpain and caspase-12, which appear to be capable of maintaining apoptosis at the level associated with the reduced fat weight gain in DIO. Collectively, results reported here support the hypothesis that high vitamin D and calcium intakes reduce DIO by increasing Ca2+ -mediated apoptosis in adipose tissue via activation of the Ca2+ /calpain/caspase-dependent pathway and indicate that targeting this pathway with vitamin D and Ca supplementation can represent an effective and affordable approach to the prevention and treatment of obesity. Further studies are warranted to evaluate this approach from a safety point of view in preclinical animal studies as well as to identify the optimal levels of Ca and vitamin D intakes for the prevention and treatment of overweight and obesity in humans. This research was supported by the US Department of Agriculture (USDA) grants SD00H325 and 2009-35200-05008 to I.N.S. The authors have declared no conflict of interest.
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DOI 10.1002/mnfr.201300503
RESEARCH ARTICLE
High vitamin D and calcium intakes reduce diet-induced obesity in mice by increasing adipose tissue apoptosis Igor N. Sergeev and Qingming Song Department of Health and Nutritional Sciences, South Dakota State University, Brookings, SD, USA Scope: Modulation of apoptosis is emerging as a promising antiobesity strategy because removal of adipocytes through this process will result in reducing body fat. Effects of vitamin D on apoptosis are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca2+ . We have previously shown that 1,25-dihydroxyvitamin D3 induces the Ca2+ signal associated with activation of Ca2+ -dependent apoptotic proteases in mature adipocytes. In this study, a diet-induced obesity (DIO) mouse model was used to evaluate the role of vitamin D and calcium in adiposity. Methods and results: DIO mice fed high vitamin D3 , high Ca, and high D3 plus high Ca diets demonstrated a decreased body and fat weight gain, improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25-hydroxyvitamin D, 1,25dihydroxyvitamin D, parathyroid hormone (PTH)), but an increased plasma Ca2+ . High D3 and Ca intakes were associated with induction of apoptosis and activation of Ca2+ -dependent apoptotic proteases, calpain and caspase-12, in adipose tissue of DIO mice. The combination of D3 plus Ca was more effective than D3 or Ca alone in decreasing adiposity. Conclusion: The results imply that high vitamin D and Ca intakes activate the Ca2+ -mediated apoptotic pathway in adipose tissue. Targeting this pathway with vitamin D and Ca supplementation could contribute to the prevention and treatment of obesity. However, this potentially effective and affordable approach needs to be evaluated from a safety point of view.
Received: July 11, 2013 Revised: November 4, 2013 Accepted: December 13, 2013
Keywords: Apoptosis / Calcium / Calpain / Caspase-12 / Diet-induced obesity / Vitamin D
1
Introduction
The induction of the death of adipocytes through apoptosis is emerging as a promising strategy for the prevention and treatment of obesity [1–5]. The increase in adipose tissue mass is the result of both hypertrophy, an increase in adipocyte size, and hyperplasia, an increase in adipocyte number, and once adipocytes achieve a maximum size, further increase in the adipose tissue mass involves an increase in adipocyte number [6–8]. Thus, weight loss can be caused not only by a decrease in adipocyte size (i.e., increasing lipolysis with a potential for lipotoxic effects), but also in adipocyte number (e.g., by stimulating apoptosis). Even a Correspondence: Dr. Igor N. Sergeev, Department of Health and Nutritional Sciences, Box 2203, South Dakota State University, Brookings, SD 57007, USA E-mail: [email protected] Fax: +1-605-688-5603 Abbreviations: 1,25(OH)2 D3 , 1,25-dihydroxyvitamin D3 ; 25(OH)D, 25-hydroxyvitamin D; DIO, diet-induced obesity; HF, high fat; PTH, parathyroid hormone; WAT, white adipose tissue C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
small increase in the rate of adipocyte apoptosis will prevent excessive accumulation of adipose tissue and can result in a significant loss of adipose tissue mass over time. Therefore, removal of adipocytes through apoptosis will reduce body fat and can help with long-term maintenance of weight loss. The effects of vitamin D on apoptosis are mediated via multiple signaling pathways that involve common regulators and effectors converging on cellular Ca2+ [9–13]. We have shown that the critical characteristic of the apoptotic Ca2+ signal is a sustained increase in concentration of intracellular Ca2+ , reaching elevated but not cytotoxic levels [12, 14–18]. Recently, we demonstrated that the Ca2+ regulatory hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) induces apoptosis in mature mouse 3T3-L1 adipocytes and the apoptosis-inducing effect of this hormone is mediated via Ca2+ signaling and Ca2+ -dependent apoptotic proteases, -calpain and caspase-12 [1, 3, 4, 19–21]. However, the critical question—can adipocyte apoptosis be induced and the apoptotic molecular targets activated in adipose tissue by increasing dietary intake levels of vitamin D and Ca?—remains unanswered. This is an important problem because it does www.mnf-journal.com
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not allow for the development of nutritional strategies for regulation of apoptosis in obesity. This study was undertaken to determine whether increased dietary vitamin D and Ca intakes can decrease the body and fat weight gain in mice fed high-fat (HF) diet. We hypothesized that high vitamin D and Ca intakes will lead to activation of Ca2+ -dependent apoptotic molecular targets through increasing circulating concentrations of 1,25(OH)2 D3 and Ca2+ , resulting in induction of Ca2+ -mediated apoptosis in adipocytes. The main findings obtained demonstrate that high vitamin D and Ca intakes decrease the body and fat weight gain in diet-induced obesity (DIO) and the mechanism of these effects involves activation of the Ca2+ -mediated apoptotic pathway in adipose tissue (specifically, induction of activities of apoptotic proteases, Ca2+ -dependent calpain and Ca2+ /calpain-dependent caspase-12).
2
Materials and methods
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Dihydroxyvitamin D ELISA, 25-Hydroxyvitamin D ELISA (ImmunoDiagnosticSystems, Scottsdale, AZ), PTH (mouse) ELISA (Immutopics Int., San Clemente, CA).
2.3 Apoptosis and apoptotic proteases Apoptotic DNA/chromatin changes were detected by measuring the histone-associated DNA fragments (mono- and oligonucleosomes; Cell Death Detection ELISA, Roche Diagnostics, Indianapolis, IN) and ssDNA breaks (ssDNA Apoptosis ELISA, Chemicon/EMD Millipore) in white adipose tissue (WAT) extracts. The activities of apoptotic proteases, calpain and caspase-12, were measured in WAT extracts with the fluorogenic peptide substrates, Ac-Leu-Leu-Tyr-AFC and Ala-Thr-Ala-Asp-AFC, respectively (Calpain and Caspase-12 Fluorometric Assay Kits, BioVision) [3, 15, 19]. Fluorescence and absorbance were measured in FL ×800 and EL ×800 microplate readers (BioTek, Highland Park, VT), respectively.
2.1 Animals and diets Male 4-wk-old C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in ventilated plastic transparent cages with bedding in a 12:12-h light– dark cycle at a temperature of 22 ± 1⬚C. Weight-matched mice were randomly assigned to the experimental HF diets containing 60% energy as fat (the normal control diet contained 10% energy as fat). The treatment groups on HF diet were high calcium (Ca diet, 1.2% Ca), high vitamin D (D diet; vitamin D3 intake ten times higher than the recommended level of 1000 IU/kg) [22] and high Ca plus high D3 (Ca + D diet). The control (normal fat and HF) diets contained the recommended 0.6% Ca and 1000 IU D3 /kg [22]; Ca diet contained 1000 IU D3 /kg. The average intake of vitamin D3 by mice fed the normal and HF diets (1000 IU D3 /kg) was 2.5 IU per mouse per day. The mice fed D diet received per os 0.625 g (25 IU) vitamin D3 (Sigma-Aldrich, St. Louis, MO) in soybean oil per animal every day plus 1000 IU D3 /kg in HF diet. The duration of the experiment was 10 wk. The diets were purchased from Research Diets, Inc. (New Brunswick, NJ). Body weight was measured every 3 days and food intake was measured every day. Mice were food-deprived overnight prior to blood and tissue collection. The South Dakota State University Institutional Care and Use Committee approved the protocols and maintenance of the animals (IACUC Protocol number 12-021A).
2.4 Statistical analysis A one-way ANOVA with t-test was used for group comparisons (SAS 9.2). Data are expressed as mean ± SEM and differences considered to be statistically significant at p < 0.05.
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Results
3.1 Body and fat weight gain Body weight gain and terminal body weights were decreased in mice fed a Ca + D diet, as compared with the DIO control, while mice fed Ca or D diets demonstrated a trend to a reduced terminal body weights (Fig. 1, upper panel). Cumulative energy intakes in mice fed HF diets were not different (not shown) and, thus, it may not account for the differences in body weight between mice fed HF diets. The weights of epididymal and retroperitoneal fat pads were significantly lower in mice fed Ca, D, and especially Ca + D diets, as compared with the DIO control (Fig. 1, lower panel). Liver weights were significantly higher in the DIO control compared with all treatment groups (not shown). These data strongly indicate a reduced percentage of total body fat in the treatment groups compared with the control DIO group.
2.2 Plasma markers Biomarkers of adiposity, vitamin D status, and Ca metabolism were measured in blood plasma (collected in heparinized tubes) using commercially available kits. The following kits were used: Adiponectin (mouse) ELISA, Glucose Assay Kit, Calcium Colorimetric Assay Kit (BioVision, Milpitas, CA), Insulin (mouse) ELISA (EMD Millipore, Billerica, MA), 1,25 C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.2 Biomarkers of adiposity, vitamin D status, and Ca metabolism The fasting plasma glucose concentration was significantly lower in mice fed Ca and D diets, and it was at the level of the normal fat diet control in mice fed Ca + D diet (Fig. 2, upper www.mnf-journal.com
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panel). These changes in glucose concentration were accompanied by a corresponding decrease (Ca diet) or a complete normalization (D and Ca + D diets) of insulin concentration in plasma (Fig. 2, middle panel). The concentration of adiponectin, a polypeptide insulin-sensitizing hormone secreted by adipose tissue, was decreased in plasma of DIO mice, but the trend to an increased adiponectin concentration was observed in mice fed Ca, D, and Ca + D diets (Fig. 2, lower panel). DIO in mice was accompanied by a significant decrease in plasma concentration of the transport, 25-hydroxyvitamin D (25(OH)D), and the hormonal, 1,25(OH)2 D, forms of vitamin D (Fig. 3, upper panel). In mice fed a D diet, concentrations of these vitamin D metabolites were raised dramatically, exceeding those concentrations not only in the DIO group, but
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Figure 1. Body and fat weight gain in mice fed high-fat diets with increased levels of Ca and vitamin D. Upper panel: Data are means for each group and time point; **p < 0.1, as compared with the DIO group at week 10; p < 0.05 for all DIO groups as compared with the normal control group at week 10; n = 7–8 per group from week 1 to week 10. The inset shows (from left to right) mice from the DIO, control, and Ca + D groups at week 10. Lower panel: Absolute and relative weights of the epididymal and retroperitoneal fat pads. Means ± SEM, n = 7–8 per group, p < 0.05 for all DIO groups as compared with the normal control group, **p < 0.05 as compared with the DIO group.
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Figure 2. Glucose, insulin, and adiponectin concentrations in blood of DIO mice. Results are presented as means ± SEM, n = 7–8 per group. The indicated parameters were measured in plasma, as described in Section 2. *p < 0.05, as compared with the control group; **p < 0.05, as compared with the DIO group.
also in the normal control. In the Ca diet group, concentrations of 25(OH)D and 1,25(OH)2 D did not differ from those in the DIO group, although the trend to a further decrease in the 25(OH)D concentration was observed. Mice fed Ca + D diet demonstrated increases in 25(OH)D and 1,25(OH)2 D concentrations reaching the normal control (for 25(OH)D) or slightly below than normal controls (for 1,25(OH)2 D) levels. www.mnf-journal.com
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Figure 3. Biomarkers of vitamin D status and calcium metabolism in DIO mice. The concentrations of 25(OH)D, 1,25(OH)2 D, total Ca2+ , and PTH were measured in heparinized plasma, as described in Section 2. Results are means ± SEM, n = 7–8 per group. *p < 0.05, as compared with the control group; **p < 0.05, as compared with the DIO group.
The increases in 1,25(OH)2 D concentration were accompanied by a significant elevation of the plasma total Ca2+ concentration (D and Ca + D groups; Fig. 3, lower left panel). It appears that there was a modest trend of increasing plasma Ca2+ in the Ca diet group, as compared with DIO control, but the Ca2+ concentration was within normal values in this group. Interestingly, the plasma concentration of PTH was increased in the DIO group and it was markedly decreased in mice fed Ca (1.2%), but not D diet (Fig. 3, lower right panel).
However, the formation of ssDNA breaks was not decreased in WAT of DIO mice (see Fig. 4, left panel), probably, reflecting a low level of apoptosis and no significant DNA damage. High Ca alone, high vitamin D3 alone, and high Ca plus high D3 intakes induced apoptosis in adipose tissue of DIO mice, as demonstrated by an enrichment of WAT extracts with nucleosomal and ssDNA fragments.
3.4 Calpain and caspase-12 activities 3.3 Apoptosis Apoptosis in WAT was measured by oligonucleosomal fragmentation of the chromosome using antihistone plus antiDNA antibodies and by ssDNA breaks using monoclonal anti-ssDNA antibodies. These antibodies detect the apoptotic internucleosomal degradation of genomic DNA (mono- and oligonucleosomes) and the apoptosis-induced DNA breaks. DIO mice demonstrated a small, but significant decrease in apoptotic-like degradation of nuclei, as measured by the nucleosomal fragments in WAT extracts (Fig. 4, left panel). C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DIO in mice was accompanied by a significant decrease in activities of Ca2+ -dependent apoptotic proteases, calpain and caspase-12, in the WAT extracts (Fig. 4, right panel). High Ca intake markedly raised calpain activity, as compared with the DIO and normal controls. High vitamin D3 intake increased calpain activity up to the levels of the normal control. Vitamin D3 was effective in increasing caspase-12 activity in adipose tissue of DIO mice, while increased Ca intake appeared to have no effect. The pattern of activities of these apoptotic proteases was generally consistent with the level of apoptosis in adipose tissue of DIO mice. It is also necessary to note that a www.mnf-journal.com
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Figure 4. Apoptosis and activities of calpain and caspase-12 in adipose tissue of DIO mice. Apoptosis was measured by the increase in nucleosomal and ssDNA fragments in WAT extracts, as described in Section 2. Results are expressed in the relative absorbance units (RAUs) per mg of protein, where RAU = (absorbance 405 nm − absorbance 490 nm) × (102 ). The calpain and caspase-12 activities were measured in the WAT extracts, as described in Section 2, and expressed in the relative fluorescence units (RFUs) per microgram of protein. Results are means ± SEM, n = 7–8 per group. *, **, and *** indicate statistically significant differences (p < 0.05), as compared with the control, DIO, and D3 group, respectively.
decreased (below the minimal, basal level of normal control) apoptosis in DIO was associated with decreased activity of calpain and caspase-12.
4
Discussion
Obesity is associated with an increased adipocyte number leading to excessive accumulation of adipose tissue [1, 6]. Apoptosis is the main mechanism for regulating cell number in many tissues [4, 18]. Induction of the death of adipocytes through apoptosis is emerging as a promising strategy for the prevention and treatment of obesity because removal of adipocytes via this mechanism will result in reducing body fat [2–5]. Importantly, decreasing adiposity through induction of adipocyte apoptosis can result in long-lasting maintenance of weight loss, in contrast to that which occurs after energy restriction. Vitamin D insufficiency is prevalent in obesity, and low vitamin D status has been linked with the increased risk of adiposity in epidemiological studies (see [2] for a review; [23,24]). However, a recent study [25] based on a bidirectional genetic approach that limits confounding suggests that a higher BMI leads to lower circulating 25(OH)D, while any effects of lower 25(OH)D increasing BMI appear to be small. On the other hand, it is well established that 25(OH)D is accumulated in adipose tissue [26,27] making it less accessible for bioconversion to 1,25(OH)2 D3 , and this may causally contribute to the vitamin D–obesity link. Several experimental studies suggested the critical roles of 1,25(OH)2 D3 in regulation of the mitochondrial uncoupling proteins linked to modulation of thermogenesis [28,29] as well as C/EPB␣/PPAR␥ signaling linked to adipogenesis [30, 31]. Somewhat paradoxically, vitamin D receptor knockout mice are resistant to DIO [32], but this is, probably, due to the global effects causing growth disturbances and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
generalized tissue atrophy and impacting overall energy balance (it is worth mentioning that vitamin D receptors occupy over 2700 genomic positions and dramatically change expression of at least 229 human genes [33]). It is also important to emphasize that 1,25(OH)2 D3 induces nongenomic, cellular Ca2+ -mediated signaling in mature adipocytes [3]. The observations described implicate a possible role for the hormone 1,25(OH)2 D3 in regulation of adiposity but underscore the lack of mechanistic understanding of these effects. The potential role of calcium in regulation of adiposity has been investigated using animal models of obesity and in epidemiological studies (see [2] for a review). These observations suggest that a low calcium intake is associated with a greater fat mass, while a high calcium intake appears to promote weight loss [34–36], although the lack of relationship between calcium intake and body weight was also reported [37]. The proposed mechanisms include modulation of the fecal fat excretion, adipogenesis and lipid metabolism, and the mitochondrial uncoupling proteins [2, 34–36]. However, the role of calcium/vitamin D signaling in regulation of adiposity, particularly via apoptotic mechanism, has not been investigated. In this study, we employed a mouse model of DIO to investigate whether high vitamin D and calcium intakes reduce adiposity. The results obtained show that mice fed an HF diet with an increased level of Ca (1.2%) and high per os intake of vitamin D3 (25 g/kg of body weight) demonstrate a decreased body and fat weight gain (epididymal and peritoneal WAT depots) and significantly improved markers of adiposity and vitamin D status (plasma concentrations of glucose, insulin, adiponectin, 25(OH)D, 1,25(OH)2 D). However, high vitamin D3 intake led to an increased circulating Ca2+ concentration, which has reached hypercalcemic levels in mice fed a high Ca plus high vitamin D3 diet. It is important to note that the Ca2+ –PTH–vitamin D physiological interactions appear to be affected in DIO: mice fed an HF diet demonstrated lower Ca2+ and increased PTH concentrations; however, the www.mnf-journal.com
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chronic increased intakes of Ca and vitamin D did not reduce plasma Ca2+ and PTH to normal levels. The apoptosis (as indicated by the oligonucleosomal fragmentation of chromatin) and activities of calpain and caspase12 in WAT were significantly decreased in DIO, and high Ca and vitamin D intakes were effective in inducing apoptosis and increasing activities of these proteases. An increased Ca intake was particularly effective in inducing calpain activity, while a high vitamin D intake was associated with increased caspase-12 activity. These observations are important because they indicate that apoptosis is decreased in WAT of obese mice and that the activities of Ca2+ -dependent apoptotic proteases can be induced by increasing Ca and vitamin D intakes. It is tempting to hypothesize that an increased Ca intake can directly lead to an increased pool of intracellular and intercellular Ca2+ in and around adipocytes, while high levels of 1,25(OH)2 D3 due to high intake of vitamin D3 allow an increased Ca2+ influx from the extracellular space and Ca2+ mobilization from the intracellular stores and, thus, its subsequent utilization in the Ca2+ -mediated apoptotic signaling (which requires a sustained, prolonged increase in concentration of cytosolic Ca2+ ) [10,12]. Although the involvement of 1,25(OH)2 D3 in increasing cellular Ca2+ and inducing apoptosis has been previously demonstrated in adipocytes in vitro [3, 21], the findings presented here demonstrate, for the first time, that the vitamin D induced, Ca2+ -mediated signaling can be employed to trigger apoptosis in adipose tissue in vivo. Furthermore, the data obtained provide the identification of apoptotic molecular targets in WAT, Ca2+ -dependent calpain and caspase-12, which appear to be capable of maintaining apoptosis at the level associated with the reduced fat weight gain in DIO. Collectively, results reported here support the hypothesis that high vitamin D and calcium intakes reduce DIO by increasing Ca2+ -mediated apoptosis in adipose tissue via activation of the Ca2+ /calpain/caspase-dependent pathway and indicate that targeting this pathway with vitamin D and Ca supplementation can represent an effective and affordable approach to the prevention and treatment of obesity. Further studies are warranted to evaluate this approach from a safety point of view in preclinical animal studies as well as to identify the optimal levels of Ca and vitamin D intakes for the prevention and treatment of overweight and obesity in humans. This research was supported by the US Department of Agriculture (USDA) grants SD00H325 and 2009-35200-05008 to I.N.S. The authors have declared no conflict of interest.
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