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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 1, 2004 as doi:10.1096/fj.04-1971fje. |
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, 25-dihydroxyvitamin D3 in modulating adipocyte apoptosis
Department of Nutrition, University of Tennessee, Knoxville, Tennessee, USA
1Correspondence: Department of Nutrition, University of Tennessee, 1215 W. Cumberland Ave., #229, Knoxville, TN 37996-1900, USA. E-mail mzemel{at}utk.edu
SPECIFIC AIM
The present study was designed to determine effect of mitochondrial uncoupling and 1
, 25-(OH)2-D3 on apoptosis in adipocytes.
PRINCIPAL FINDINGS
1. 1, 25-(OH)2-D3 has a dual effect on adipocyte apoptosis, with physiological doses of 1, 25-(OH)2-D3 inhibiting apoptosis in differentiated 3T3 L1 cells; a pharmacological dose stimulates apoptosis
Bcl-2 and Bax are apoptotic proteins belonging to the Bcl-2 family. Because of their different roles in apoptotic signaling pathway, the ratio of protective Bcl-2 to apoptotic Bax is widely used to determine the susceptibility to apoptosis by regulating mitochondrial function after an apoptotic stimuli. Figure 1
shows the effect of 1, 25-(OH)2-D3 on the Bcl-2/ Bax expression ratio. We found that lower levels induced a dose-dependent increase in the Bcl-2/Bax ratio (P<0.01), indicating a protective role for a lower level of 1, 25-(OH)2-D3 against apoptosis. In contrast, a high dose of 1, 25-(OH)2-D3 resulted in a significant decrease in the Bcl-2/Bax ratio in control, DNP-treated, and UCP2-transfected 3T3-L1 cells (P<0.01).
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2. Mitochondrial uncoupling induces apoptosis in adipocyte
DNP-treated and UCP2-transfected cells showed a significantly lower the ratio of Bcl-2/Bax than nontransfected 3T3-L1 cells (P<0.02), indicating mitochondrial uncoupling induced a decrease in protection from apoptotic death (Fig, 1)
.
3. High calcium diets, which suppress 1, 25-(OH)2-D3 levels in vivo, stimulate adipose tissue apoptosis in aP2-agouti transgenic mice
Since physiological doses of 1, 25-(OH)2-D3 appear to inhibit adipocyte apoptosis, suppression of 1, 25-(OH)2-D3 with a high calcium diet may stimulate apoptosis. Accordingly, we compared the Bcl-2/Bax expression ratio in aP2-agouti transgenic mice fed diets with a different calcium content after energy restriction. As shown in Fig. 2
, we found significant decreases in the Bcl-2/Bax ratio in mice fed high (1.3%) vs. low calcium diets (0.4%) (P<0.01), suggesting that suppression 1, 25-(OH)2-D3 physiologically in vivo may stimulate adipocyte apoptosis.
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CONCLUSIONS
Obesity is characterized by an increase in adipocyte size and number, but data on the mechanisms and regulation of adipocyte apoptosis remain limited.
Previous data from our laboratory demonstrated that 1, 25-(OH)2-D3 modulates adipocyte lipid and energy metabolism via genomic and nongenomic mechanisms. We have reported that 1, 25-(OH)2-D3 plays a direct role in modulating adipocyte Ca2+ signaling, resulting in increased lipogenesis and decreased lipolysis. In addition, 1, 25-(OH)2-D3 plays a role in regulating human adipocyte UCP2 mRNA and protein levels, indicating that suppression of 1, 25-(OH)2-D3 and the resulting up-regulation of UCP2 may contribute to increased rates of lipid oxidation. Consequently, suppression of 1, 25-(OH)2-D3 by increasing dietary calcium attenuates adipocyte triglyceride accumulation and causes a net reduction in fat mass in mice and humans in the absence of caloric restriction, marked augmentation of body weight and fat loss during energy restriction in mice and humans, and a reduction in the rate of weight and fat regain after food restriction in mice. Although these anti-obesity effects of dietary calcium appear to be due in part to effects on lipolysis and lipogenesis, it is possible a loss of adipocytes could result in a cellular deficit for lipid esterification as the body recovers from energy restriction. Thus, a reduced number of available adipocytes may not be able to store excess energy from a rebound in food intake. Moreover, generating new cells would require extra energy, which would contribute to further metabolic enhancement. The present study demonstrates that lower (physiological) doses of 1, 25-(OH)2-D3 inhibit apoptosis in differentiated 3T3-L1 adipocytes and suppression of 1, 25-(OH)2-D3 in vivo by increasing dietary calcium stimulated adipocyte apoptosis during re-feeding after energy restriction in aP2 transgenic mice, suggesting that stimulation of adipocyte apoptosis contributes to reduced adipose tissue mass after administration of high calcium diets.
A key feature of apoptosis involves the proteolytic caspases as well as apoptotic proteins Bcl-2 and Bax. Activation of Bax induces apoptosis by disturbing mitochondrial electron transport chain, counters the death repressor activity of Bcl-2, and promotes release of cytochrome c into the cytoplasm. This in turn activates caspases, which initiate execution of apoptotic death. Localized to mitochondria, Bcl-2 inhibits Bax-induced apoptosis; thus, the Bcl-2/Bax ratio can be used to determine susceptibility to apoptosis. In the present study, we found that lower doses of 1, 25-(OH)2-D3 (0.1, 1, 5, and 10 nm) dose-dependently increase the Bcl-2/Bax expression ratio in control and UCP2-transfected 3T3-L1 adipocytes, indicating an inhibition of apoptosis. We found that UCP2-transfected 3T3-L1 adipocytes have a lower Bcl-2/Bax ratio than nontransfected 3T3-L1 cells. These results are consistent with our observation that UCP2 stimulates apoptosis by inducing mitochondrial potential collapse and inhibiting ATP production. We previously demonstrated that 1, 25-(OH)2-D3 inhibits UCP2 expression in human adipocytes. Consistent with this, data from this study demonstrate that 1, 25-(OH)2-D3 functionally inhibits UCP2 action by increasing mitochondrial potential and ATP production in nontransfected and UCP2 transfected 3T3-L1 cells. Thus, the suppression of apoptosis induced by 1, 25-(OH)2-D3 is mediated in part by inhibiting UCP2 expression and activity. Accordingly, we proposed that the suppression of 1, 25-(OH)2-D3 secondary to consumption of high calcium diets may stimulate adipocytes apoptosis in vivo. We evaluated this concept in aP2-agouti transgenic mice undergoing re-feeding after energy restriction and found a significant increase in white adipose tissue apoptosis in mice re-fed high (1.3%) vs. low calcium diets (0.4% Ca). These results further confirm that suppression of 1, 25-(OH)2-D3 stimulates apoptosis in white adipose tissue and suggest that this effect contributes to the anti-obesity effect of dietary calcium.
Pharmacological doses of 1, 25-(OH)2-D3 3- to 4-fold higher than physiological levels exert the opposite effect, inducing a decrease in the Bcl-2/Bax ratio. Although lower doses of 1, 25-(OH)2-D3 caused moderate increases in cytosolic [Ca2+] levels in adipocytes, the high dose of 1, 25-(OH)2-D3 induced extreme elevation, and excessive intracellular calcium has been reported to be a proapoptotic factor. In the present study, we monitored the mitochondrial Ca2+([Ca2+]m) concentration and found that a low dose of 1, 25-(OH)2-D3 decreased [Ca2+]m in a dose-dependent manner whereas high-dose 1, 25-(OH)2-D3 markedly elevated [Ca2+]m. We reported before that mitochondrial uncoupling stimulated the cytoplasmic calcium ([Ca2+]c) level, which may induce an increase in [Ca2+]m to maintain [Ca2+]c homeostasis. Low doses of 1, 25-(OH)2-D3 induce dose-dependent depletion [Ca2+]m by inhibiting UCP2, thereby protecting adipocytes from apoptotic death. High doses of 1, 25-(OH)2-D3, on the other hand, markedly increase [Ca2+]c, which may increase [Ca2+]m and [Ca2+]er. Since ER can open their Ca2+ release channels in response to elevations in [Ca2+]c and contribute to Ca2+-induced Ca2+ release, this may further increase [Ca2+]m and calcium overload in mitochondria, in turn triggering apoptosis.
In summary, the present study demonstrated dual effects of 1, 25-(OH)2-D3 on apoptotic death (summarized in Fig. 3
). By inhibiting UCP2 and decreasing mitochondrial calcium, lower (physiological) doses of 1, 25-(OH)2-D3 inhibit apoptosis in adipocytes whereas suppression of 1, 25-(OH)2-D3 using high calcium diets stimulates adipose apoptosis in mice. In contrast, a high dose of 1, 25-(OH)2-D3 stimulates mitochondrial calcium overload and apoptosis in adipocytes. These results indicate that dietary calcium not only regulates adipocyte size by increasing lipid accumulation, but modulates adipocyte number by stimulating apoptotic death. Thus, these results provide a new mechanism in support of the "anti-obesity" of dietary calcium.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1971fje;
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