Nutrition xxx (2016) 1–7

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Nutrition
journal homepage: www.nutritionjrnl.com

Basic nutritional investigation

Vitamin D decreases adipocyte lipid storage and increases

NAD-SIRT1 pathway in 3T3-L1 adipocytes
Eugene Chang Ph.D., Yangha Kim Ph.D. *
Department of Nutritional Science and Food Management, Ewha Womans University, Seoul, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Previous studies suggest that low vitamin D status is associated with obesity charac-
Received 5 August 2015 terized by excess lipid storage in adipocytes. The aim of the present study was to determine the
Accepted 12 December 2015 effects of the most hormonally active form of vitamin D 1,25-dihydroxyvitamin D [1,25(OH)2D] on
adipocyte fat storage and lipid metabolism in mature 3T3-L1 cells.
Keywords: Methods: Differentiated 3T3-L1 cells were treated with various concentrations of 1,25(OH)2D.
1,25(OH)2D
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation, intracellular lipid
Adipocyte
content, and basal and isoproterenol-stimulated lipolysis were measured to investigate the regu-
Lipid storage
Lipolysis latory role of 1,25(OH)2D in adipocyte lipid metabolism. Reverse transcription polymerase chain
SIRT1 reaction was performed to determine the effects of 1,25(OH)2D on adipogenesis-related markers,
NAD fatty acid oxidation-associated genes, and lipolytic enzymes. Sirtulin 1 (SIRT1) activity, nicotin-
Obesity amide adenine dinucleotide (NAD) and NADH were measured.
Results: 1,25(OH)2D treatment (24 h, 100 nmol/L) induced a decrease in intracellular fat accumu-
lation and an increase of basal and isoproterenol-stimulated lipolysis without cell toxicity in adi-
pocytes. Adipogenic gene levels were decreased. In contrast, mRNA levels of b-oxidation–related
genes, lipolytic enzymes, and vitamin D responsive gene were elevated by 1,25(OH)2D. Addi-
tionally, significant incremental changes in NAD levels, the ratio of NAD to NADH, and SIRT1
expression and activity were noted in 1,25(OH)2D-treated 3T3-L1 adipocytes.
Conclusions: The observed potent inhibitory effect of 1,25(OH)2D on adipocyte fat storage in
mature 3T3-L1 cells suggests that vitamin D might improve adipocyte metabolic function and
protect against obesity. Increased NAD concentrations and SIRT1 activity may play a role in the
mechanism of 1,25(OH)2D action.
Ó 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-
ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction altered, which, in turn, leads to systemic insulin resistance and

type 2 diabetes [5]. Thus, an understanding of the molecular
Obesity is recognized as a major risk factor for type 2 diabetes, mechanisms of adipose tissue formation and alterations during
dyslipidemia, hypertension, and heart disease [1,2]. It is charac- the progression of obesity is required for the prevention and
terized by a significant increase of adiposity, depending on treatment of obesity.
hypertrophy of preexisting individual adipocytes and/or hyper- Accumulating evidence shows that vitamin D deficiency
plasia due to the formation of new adipocytes from precursor is highly prevalent in obese people, indicating obesity might
cells (adipogenesis) [3,4]. Additionally, the synthesis and release be an independent risk factor for vitamin D deficiency [6,7].
of adipocyte-derived factors regulating insulin sensitivity are Indeed, body fat content is negatively correlated with
25-hydroxyvitamin D [25(OH)D] concentrations, a commonly
This research was supported by the Basic Science Research Program through accepted marker for vitamin D status [8–10]. In contrast, weight
the National Research Foundation of Korea (NRF) funded by the Ministry of loss leads to a significant increase of 25(OH)D in obese children
Science, ICT & Future Planning (2014 R1 A1 A3050953) and Brain Korea 21 Plus
(22 A20130012143).
and adults [11,12]. Following human epidemiologic and clinical
* Corresponding author. Tel./fax: þ82 2 3277 4425. studies, numerous studies using in vitro adipocytes have
E-mail address: [email protected] (Y. Kim). demonstrated the regulation of 1,25-dihydroxyvitamin D [1,
http://dx.doi.org/10.1016/j.nut.2015.12.032
0899-9007/Ó 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-
nd/4.0/).
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2 E. Chang, Y. Kim / Nutrition xxx (2016) 1–7

25(OH)2D, calcitriol], the most active form of vitamin D, in carbon dioxide. After the preadipocytes reached confluence (day 0), fibroblasts
adipocyte differentiation. In cultured 3T3-L1 adipocytes, 1, were incubated with 10% fetal bovine serum (FBS)-containing HG-DMEM in the
presence of 0.5 mmol/L of 3-isobutyl-1-methylxanthine (Sigma, St. Louis, MO,
25(OH)2D treatment significantly inhibits adipogenesis [13–15] USA), 1 mmol/L of dexamethasone (Sigma), and 5 mg/mL of bovine insulin (Sigma)
and decreases fat content by increasing the expression and for 2 d to induce differentiation (day 2), followed by incubation with insulin for
activity of lipolysis by lipoprotein lipase (LPL), which is involved an additional 2 d (day 4). The medium was replaced with DMEM containing 10%
in adipocyte fat uptake and storage [16]. However, 1,25(OH) FBS every 2 d until >95% of the cells contained lipid droplets (day 6 or 7). Mature
adipocytes were treated with vehicle control (0.1% ethanol) or 1,25(OH)2D
2D–reduced adipogenesis is not observed in primary mouse or
(Sigma) dissolved in absolute ethanol at the given concentrations for the indi-
human preadipocytes [17]. Given the high prevalence of vitamin cated time period.
D deficiency in obese individuals and the strong association
between vitamin D and body fat mass, a better understanding of MTT assay
vitamin D action and regulation in adipocyte physiology and
pathophysiology is critical for the prevention of obesity and its Differentiated 3T3-L1 adipocytes were treated with different concentrations
of 1,25(OH)2D in 10% FBS-containing medium for 24 or 48 h. To determine the
associated health problems. However, the mechanisms by which relative viable levels, further incubation with fresh medium containing 3-(4,5-
1,25(OH)2D influences adipocyte differentiation, lipid meta- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/
bolism, and the energy balance are largely unknown. mL) was executed for 1 h. After dissolving in DMSO, the absorbance was
Sirtulin 1 (SIRT1), the mammalian homolog of yeast silent measured by a Varioskan plate reader (Thermo Electron, Waltham, MA, USA) at a
wavelength of 595 nm. The results are expressed as the fold-change compared
information regulator 2, is a nicotinamide adenine dinucleotide
with the vehicle-treated cells.
(NAD)-dependent protein deacetylase and an important metabolic
sensor in response to cellular energy status [18–20]. Fasting or Oil Red O staining
caloric restriction induces an increase of intracellular NAD, which
in turn stimulates SIRT1 activity, leading to hepatic glucose pro- The intracellular lipid contents in differentiated adipocytes were measured
duction and an increase of oxidative machinery activity [18,21,22]. by Oil Red O (ORO) staining. Briefly, the cells were washed with phosphate-
buffered saline (PBS), fixed with 10% formaldehyde in PBS for 1 h, washed with
SIRT1 promotes fat mobilization from adipose tissue to support
distilled water, and completely dried. The cells were subsequently stained with
lipid oxidation and inflammation [23]. Additionally, the genetic ORO for 1 h at room temperature, washed with distilled water, and air-dried.
deletion of SIRT1 in adipose tissue induces an increase in adipocyte ORO-stained adipocytes were observed under a microscope (Olympus, Tokyo,
mass, inflammation, and metabolic disorders [24,25]. Thus, tar- Japan) and digital images were captured at 40 magnification. For a quantitative
analysis of intracellular lipid accumulation, ORO-stained lipids were eluted by
geting SIRT1 activation has been proposed as a therapeutic tool for
adding 100% isopropanol and measured at a wavelength of 490 nm.
obesity and its associated metabolic dysfunction [26].
In the present study, we investigated the mechanisms by Lipolysis
which active vitamin D affects adipocyte fat storage, lipolysis,
and SIRT1 levels in 3T3-L1 cells. We hypothesized that 1,25(OH) The adipocytes were washed with PBS and incubated in the absence or
2D treatment inhibits intracellular fat accumulation and presence of 10 mmol/L b-adrenergic agonist isoproterenol in 2% fatty acid free
bovine serum albumin containing medium. Basal and isoproterenol-stimulated
enhances the NAD-to-NADH ratio and SIRT1 activity, accompa-
lipolysis was assessed by the release of glycerol in the culture medium using a
nied by changes in gene expression related to adipogenesis, fatty free glycerol reagent (Sigma) as previously described [27].
acid oxidation, and lipolytic enzymes in 3T3-L1 adipocytes.
RNA analysis
Materials and methods
Total RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA)
Cell culture according to manufacturer’s instruction. Reverse transcription (RT) of the total
RNA was performed using MMLV reverse transcriptase (Bioneer, Daejeon, Korea)
Murine 3T3-L1 cells obtained from the American Type Culture Collection and the reaction was performed at 37 C for 60 min followed by incubation at
(ATCC CL-173; Manassas, VA, USA) were maintained in high glucose (HG)-DMEM 95 C for 5 min. The primers used for RT-polymerase chain reaction (PRC) are
(Gibco, Grand Island, NY, USA) containing 10% bovine calf serum (Gibco), 100 U/ presented in Table 1. Real-time quantitative PCR was performed using the
mL of penicillin, and 100 mg/mL of streptomycin (Gibco) at 37 C in 95% air and 5% AccuPower 2 Greenstar qPCR Master Mix (Bioneer) and a fluorometric thermal

Table 1
Primers used for real-time polymerase chain reaction

Gene GeneBank no. Forward sequence Reverse sequence Product size (bp)
ACO NM_015729.3 ATC CAG ACT TCC AAC ATG AG AAC CAC ATG ATT TCT TCA GG 136
aP2 NM_024406.2 CGA CAG GAA GGT GAA GAG CA ATT CCA CCA CCA GCT TGT CA 128
b-actin NM_007393.3 GGA CCT GAC AGA CTA CCT CA GTT GCC AAT AGT GAT GAC CT 208
CEBPa NM_001287523.1 ATA GAC ATC AGC GCC TAC AT TCC CGG GTA GTC AAA GTC AC 147
CPT1a NM_013495.2 GTG TTG GAG GTG ACA GAC TT CAC TTT CTC TTT CCA CAA GG 100
CYP24 NM_009996 AGT GAA CCT GTG GAG ATG CTG AGT TGT CCC ATG CTC TGG TC 200
FAS NM_007988.3 AAC TCA CTG GCA GAA GAG AA CTT CAA GAA GAT AGC CAT GC 134
HSL NM_010719.5 TAT GGA GTG AGG GTG CCA GA ATG GTC CTC TGC CTC TGT CC 173
Leptin NM_008493.3 TTC CTG TGG CTT TGG TCC TA ACC GAC TGC GTG TGT GAA AT 127
LPL NM_008509.2 ACT TGT CAT CTC ATT CCT GG TCT CAT ACA TTC CCG TTA CC 112
PGC1a NM_008904.2 GGG CCA AAC AGA GAG AGA GG GTT TCG TTC GAC CTG CGT AA 266
PPARa NM_011144.6 ATC CCA TCA CTC TCT CTG TG AAC TAC CTG CTC AGG ACT CA 191
PPARg NM_001127330.1 TTG ATT TCT CCA GCA TTT CT TGT TGT AAG GCT GGG TCT TT 171
SCD-1 NM_009127.4 ATG GAT ATC GCC CCT ACG AC GAT GTG CCA GCG GTA CTC AC 147
SIRT1 NM_019812.2 TAC CCC ATG AAG TGC CTC AA AAC CAA TTC CTT TTG TGG GC 215
UCP1 NM_009463.3 CAG GCT TCC AGT ACC ATT AG CTT GGA CTG AGT CGT AGA GG 160

ACO, acetyl-CoA oxidase; aP2, fatty acid-binding protein 2; CEBPa, CCAAT/enhancer binding protein a; CPT1a, carnitine palmitoyltransferase 1a; CYP24, 1,25-
dihydroxyvitamin D3 24-hydroxylase; FAS, fatty acid synthase; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; PGC1a, peroxisome proliferative-activated re-
ceptor gamma coactivator 1a; PPAR, peroxisome proliferator-activated receptor; SCD-1, stearoyl-CoA desaturase 1; SIRT1, sirtuin 1; Tfam, mitochondrial transcription
factor A; UCP1, uncoupling protein 1

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 E. Chang, Y. Kim / Nutrition xxx (2016) 1–7 3

cycler (Rotor-GeneTM 2000; Corbett Research, Mortlake, NSW, Australia). The of Oil Red O staining illustrate that 1,25(OH)2D inhibits lipid
data were analyzed using the comparative Ct (DDCT) method for relative quan- accumulation (Fig. 2B). As Figure 2A shows, 24 h of 1,25(OH)2D
tification [28]. Each target gene was normalized to that of b-actin and expressed
as a fold-change compared with controls.
treatment inhibited adipogenesis in a dose-dependent manner
with a 15% significant reduction evident at 100 nmol/L 1,25(OH)
SIRT1 activity assay 2D as measured by Oil Red O staining.

SIRT1 activity was measured using a SIRT1 activity assay kit (Abcam, Cam-
bridge, MA, USA) according to the manufacturer’s instructions. In brief, nuclear 1,25(OH)2D promotes basal and isoproterenol-stimulated lipid
fraction from differentiated 3T3-L1 adipocytes was extracted and purified. Next, utilization in 3T3-L1 adipocytes
SIRT1 activity was measured in the presence of NAD and fluoro-substrate pep-
tides using a fluorometric microplate reader at 340 nm/460 nm wavelengths. To demonstrate whether the 1,25(OH)2D-decreased intra-
cellular fat content is due to lipolysis, we measured glycerol
Measurement of the NAD-to-NADH ratio
levels in the culture medium after 24 h treatment. Figure 3
A NAD/NADH assay kit was used to evaluate the levels of NAD, NADH, and demonstrates that there were significant reduction of the basal
their ratio (Abcam). Differentiated adipocytes were snapped and frozen in liquid lipolysis rates in 1,25(OH)2D-treated 3T3-L1 adipocytes. We next
nitrogen and extracted on ice with extraction buffer from the NAD/NADH assay sought to determine whether 1,25(OH)2D leads to lipolysis in the
kit. Protein concentration was determined using a BCA protein assay kit (Pierce,
isoproterenol-stimulated state. Incubation of 3T3-L1 adipocytes
Rockford, IL, USA). The NAD and NADH values were normalized to their respec-
tive protein concentrations. with a b-adrenergic agonist (isoproterenol, 10 mmol/L) on day 7
induced a significant increase of 51% compared with the control
Statistical analysis in the basal state. 1,25(OH)2D-treated adipocytes stimulated
with isoproterenol significantly promoted lipid mobilization.
The data are presented as the mean  SEM of at least two independent
Thus, 1,25(OH)2D might increase fatty acid availability due to
triplicate experiments. Statistical analysis was performed using PASW Statistics
21 (SPSS Inc., Chicago, IL, USA). Differences were determined using Student’s t enhanced basal and isoproterenol-stimulated lipolysis from
test. Statistical significance was defined as P < 0.05. adipocyte lipid accumulation.

Results Effects of 1,25(OH)2D on gene expression involved in

adipogenesis, fatty acid oxidation, lipolytic enzymes, vitamin D
1,25(OH)2D treatment (24 h) did not alter cell viability in mature metabolism, and obesity-associated adipocytokine
3T3-L1 adipocytes
We evaluated whether 1,25(OH)2D diminished lipid accu-
The affect of 1,25(OH)2D on cell toxicity was examined by the mulation by regulating the gene involved in adipogenesis, fatty
MTT cell proliferation assay following treatment of differentiated acid oxidation, and adipocyte-specific lipolytic enzymes.
adipocytes with vehicle or 1,25(OH)2D treatment. As shown in 1,25(OH)2D (24 h, 100 nmol/L) significantly decreased aP2,
Figure 1A, 24-h incubation with 1,25(OH)2D up to 100 nmol/L CEBPa, FAS, PPARg, and SCD-1 adipogenic mRNA expression by
had no effect on cell viability. In contrast, 48-h incubation with 43.9%, 63.4%, 60.2%, 49.2%, and 62%, respectively (Fig. 4A).
1,25(OH)2D induced a significant reduction, starting at a dose of Additionally, mRNA levels of CPT1a, PGC1a, PPARa, and UCP1
1 nmol/L. Thus, the dose of 100 nmol/L 1,25(OH)2D and 24 h involved in b-oxidation were significantly upregulated by 1.5-,
incubation was chosen for further experiments. 4.2-, 3.2-, and 1.9-fold in 1,25(OH)2D-treated cells compared
with the vehicle control (Fig. 4B). Furthermore, 1,25(OH)2D
1,25(OH)2D significantly decreases lipid accumulation in a dose- increased lipolytic enzymes hormone-sensitive lipase (HSL) and
dependent manner lipoprotein lipase (LPL) mRNA levels by 76.5% and 95.4%,
respectively, in mature adipocytes (P < 0.05). 1,25(OH)2D
Next, we investigated the effect of 1,25(OH)2D on adipo- responsive gene, CYP24 was significantly increased, but leptin,
genesis. Differentiated 3T3-L1 adipocytes were incubated with an obesity-associated adipocytokine secreted by adipose tissue,
1,25(OH)2D for 24 h in a dose-dependent manner on day 6 or 7 was not significantly changed by 24 h incubation of 1,25(OH)
after adipocyte differentiation induction. Representative images 2D.

Fig. 1. The effect of 1,25(OH)2D on cell viability in differentiated 3T3-L1 adipocytes. On day 6 or 7 after differention induction, the cells were incubated with 0 (vehicle), 1, 10, or
100 nmol/L of 1,25(OH)2D for 24 h (A) or 48 h (B). The value of each bar represents the mean  SEM (n ¼ 6w12/group). *P < 0.05; yP < 0.01 compared with the vehicle control.

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Fig. 2. The inhibitory effect of 1,25(OH)2D on lipid accumulation. 3T3-L1 adipocytes were treated with 1,25(OH)2D for 24 h in a dose-dependent manner (0, 1, 10, or
100 nmol/L) on day 6 or 7. Incubated mature 3T3-L1 adipocytes with 1,25(OH)2D was subjected to Oil Red O (ORO) staining and quantitative analysis was conducted (A). The
data are expressed as the mean  SEM (n ¼ 6w9/group). *P < 0.05; yP < 0.01 compared with the vehicle control. Intracellular lipid accumulation was observed by ORO
staining and photographed under light microscopy (magnification 40), Scale bars ¼ 50 mm (B).

1,25(OH)2D treatment significantly increases SIRT1 mRNA and shown in Figure 6, 100 nmol/L 1,25(OH)2D treatment signifi-
activity cantly increased the NAD-to-NADH ratio but decreased NADH
levels in differentiated 3T3-L1 cells.
The effect of 1,25(OH)2D on SIRT1 involved in lipid metabolism
through the regulation in fat mobilization has never been re-
ported. Therefore, we examined the influence of 1,25(OH)2D on Discussion
gene expression and activity of SIRT1 in 3T3-L1 adipocytes.
1,25(OH)2D-treated adipocytes had a 2-fold increase in SIRT1 Associations between vitamin D status and obesity have been
mRNA expression compared with vehicle (100% ethanol)-treated well documented in both epidemiologic and intervention
control cells (Fig. 5A). Moreover, 1,25(OH)2D significantly studies. There is a high prevalence of vitamin D inadequacy in
increased SIRT1 activity in a dose-dependent manner, started at a obese individuals. The present study demonstrates reduced fat
dose of 1 nmol/L. The maximum SIRT1 activity was at a concen- accumulation and increased lipid mobilization with 1,25(OH)2D
tration of 100 nmol/L by 3.3-fold compared with vehicle (Fig. 5B). treatment in murine mature adipocytes. To the best of our
knowledge, this study describes the first time that 1,25(OH)2D
treatment significantly increases adipocyte SIRT1 activity and
1,25(OH)2D treatment increases NAD and the NAD-to-NADH the NAD-to-NADH ratio. These results suggest that vitamin D
ratio might promote fat mobilization and hence decrease intracellular
fat accumulation and increase lipolysis, concurrently with an
Upon observing the results showing that 1,25(OH)2D- increase of activity in the NAD-SIRT1 pathway.
increased SIRT1 expression and activity, we investigated whether Adipose tissue is now recognized as one of the most important
1,25(OH)2D regulates adipocyte NAD biology by measuring NAD, tissues in obesity due to its dynamic changes during the pro-
NADH, and the NAD-to-NADH ratio in 3T3-L1 adipocytes. As gression of obesity: hyperplasia (an increase in cell number),
hypertrophy (enlarged cell size), and adipocyte-derived peptides
[3–5]. Targeting adipose development during the progression of
obesity has been considered for the prevention and/or treatment
of obesity and its associated metabolic disorders. First, we
examined whether 1,25(OH)2D modulates adipocyte lipid meta-
bolism. Adipocyte lipid accumulation was significantly decreased
in 1,25(OH)2D-treated adipocytes without intracellular toxicity in
the present study. A published paper showed that 1,25(OH)
2D-induced apoptosis which, in turn, might result in the pre-
vention and treatment of obesity [29]. Treatment of 1,25(OH)2D
(12.5–400 nmol/L) was started at day 0, when differentiation was
induced and 1,25(OH)2D was incubated for 1, 3, or 6 d. However, in
the current study, mature adipocytes on day 6 or 7 was incubated
with 1,25(OH)2D for 24 h (1–100 nmol/L). Despite this discrep-
ancy, a study to investigate whether 1,25(OH)2D treatment in-
duces apoptosis and its-associated decrease of lipid deposition in
Fig. 3. The influence of 1,25(OH)2D on adipocyte lipid utilization. Cell culture su-
pernatant was collected after 24 h of 1,25(OH)2D treatment (0, 1, 10, or 100 nmol/L) mature adipocyte is warranted. Additionally, our data illustrate
on day 7 and the glycerol content was determined. Lipolysis was stimulated by that 1,25(OH)2D treatment increases fat mobilization by reducing
adding 10 mmol/L isoproterenol in 2% fatty acid-free albumin to adipocytes pre- intracellular fat contents and increasing both basal and
treated for 24 h with 1,25(OH)2D. Lipolysis is presented as the mean glycerol isoproterenol-stimulated lipolysis. These results support human
release/h normalized to the total intracellular protein. The data are expressed as the
mean  SEM (n ¼ 6/group). yP < 0.01 compared with the vehicle control in the
epidemiological and clinical studies demonstrating the negative
absence of isoproterenol. zP < 0.05; xP < 0.01 compared with the vehicle control in association between adiposity/obesity and vitamin D status
the present of 10 mmol/L isoproterenol. [8–10].

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Fig. 4. The effects of 1,25(OH)2D on gene expression involved in adipogenesis, fatty acid oxidation, lipolytic enzymes, vitamin D metabolism and obesity-associated adi-
pocytokine. 3T3-L1 adipocytes were incubated with 100 nmol/L 1,25(OH)2D for 24 h on day 6 or 7 after differentiation induction. Gene expression related to adipogenesis (A),
fatty acid oxidation (B), and lipolytic enzymes (C) was analyzed by RT-PCR. CYP24 (D) and leptin (E) mRNA levels were also evaluated. mRNA gene expression was normalized
with respect to the b-actin level. The results are presented as the mean  SEM (n ¼ 9/group). *P < 0.05; yP < 0.01 compared with the vehicle control.

The inclusion of 1,25(OH)2D to a differentiation cocktail, monounsaturated fatty acids, which are the major components
significantly inhibited adipocyte differentiation by suppressing of triacylglycerol (TG) [33]. In the present study, 1,25(OH)2D
the expression of adipogenic genes such as CEBPa and PPARg in significantly decreased these three lipogenic mRNA levels, sug-
murine 3T3-L1 and primary porcine cells [13,14,30]. Consistent gesting the role of 1,25(OH)2D in the decrement of adipocyte fat
with these studies, results from the present study also demon- storage capacity. Additionally, our results show that the gene
strated that 1,25(OH)2D treatment significantly inhibited adi- expression involved in fatty acid oxidation was upregulated by 1,
pogenic transcription factors, despite the addition of 1,25(OH)2D 25(OH)2D treatment. Fatty acid oxidation is regulated by the
in the mature status after differentiation (day 6 or 7) and only for CPT1-mediated entry of LCFAs into mitochondria [34] and a
24 h. The lipogenic mRNA expression related to lipid accumu- transcription factor, PGC1a. PGC1a promotes lipid catabolism by
lation was measured. aP2 indicates the extent of fat accumula- regulating mitochondrial biogenesis and metabolism in associ-
tion in adipose tissue [31]. Fatty acid synthase (FAS) induces de ation with its nuclear receptors, PPARa and UCP1, and
novo lipogenesis [32]. Additionally, stearoyl-coenzyme desatur- gene-encoding respiratory chain proteins involved in thermo-
ase (SCD) converts saturated long-chain Fas (LCFAs) to genesis [35–37]. These data suggest that 1,25(OH)2D-increased

Fig. 5. The effect of 1,25(OH)2D on the mRNA expression and activity of SIRT1. On day 6, 3T3-L1 adipocytes were incubated with 1,25(OH)2D for 24 h at the indicated
concentrations. The mRNA levels of SIRT1 were determined by reverse transcription polymerase chain reaction (RT-PCR) (A). Quantitative RT-PCR was normalized for all
samples to b-actin. (B) SIRT1 activity was analyzed by a fluorometric SIRT1 activity assay kit. The results are expressed as the mean  SEM (n ¼ 6w9/group). *P < 0.05;
y
P < 0.01 compared with the vehicle control.

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Fig. 6. The influence of 1,25(OH)2D on the levels of NAD (A) and NADH (B) and the NAD-to-NADH ratio (C) in 3T3-L1 adipocytes. 1,25(OH)2D (10 or 100 nmol/L, 24 (h) was
used to treat 3T3-L1 adipocytes on day 6. Cell lysates containing NAD and NADH or NAD-decomposed samples including only NADH were used for the measurement. The
results are expressed as the mean  SEM (n ¼ 6/group). *P < 0.05; yP < 0.01 compared with the vehicle-treated 3T3-L1 adipocytes.

lipolysis might be associated with increased fatty acid oxidation metabolism and energy homeostasis, which have been linked to
capacity. In the present study, lipolytic enzymes HSL and LPL protection against obesity [44,45]. In this study, we revealed that
were upregulated by 1,25(OH)2D, which is consistent with the 1,25(OH)2D increased intracellular NAD levels and the NAD/-
findings of a cross-sectional study showing a positive correlation NADH ratio in 3T3-L1 adipocytes. We also found that 1,25(OH)2D
between vitamin D status and LPL [38], and in vitro studies treatment increased both SIRT1 expression and activity. A
demonstrating the increased expression of LPL in 1,25(OH) NAD-dependent deacetlyase, SIRT1, regulates adipose energy
2D-treated 3T3-L1 and human adipocytes [16,17,39]. The homeostasis and metabolism, which could be a potential thera-
reduction of adipogenic gene expression and the increase of peutic strategy to protect against obesity and obesity-associated
b-oxidation and lipolytic mRNA expression suggest that these metabolic disorders [46,47]. Additionally, SIRT1 inhibits pre-
may play a role in the changes of 1,25(OH)2D-induced adipocyte adipocyte differentiation and increases the mobilization of free
fat storage and lipolysis. Moreover, vitamin D target gene, CYP24, fatty acids from adipose tissues [23]. Furthermore, there is
is significantly induced in the current study, consistent with accumulating evidence showing an inverse association between
dose-responsive increments in 1,25(OH)2D-treated adipocytes SIRT1 levels and adipose tissue mass and inflammation [24,25,
[17,40]. However, further studies to investigate whether 1, 48]. Moreover, targeting SIRT1 activation by resveratrol supple-
25(OH)2D-induced changes in gene expression is mentation shows the favorable effect on body composition in
dose-responsive and how 1,25(OH)2D affects the expression of humans [26]. A recent research proposes SIRT1 activation is
genes and/or their enzyme activities are needed. Close associa- closely related to the activation of a steroid hormone receptor,
tion between obesity-induced production of leptin and vitamin vitamin D receptor (VDR), which mediates 1,25(OH)2D-induced
D metabolism have been observed in both humans and in vitro genomic changes [49]. SIRT1 activator, resveratrol, significantly
systems [41,42]. However, in the present study, 1,25(OH)2D stimulates 1,25(OH)2D binding to VDR, the recruitment of its
treatment did not change leptin gene expression. Therefore, co-receptor, retinoid X receptor (RXR), and SIRT1 activation in
further study to investigate whether not only 1,25(OH)2D but human colorectal adenocarcinoma cells (Caco-2, HCT116),
also different vitamin D metabolite, such as 25(OH)D affects human embryonic kidney cells (HEK293), and mouse myoblast
production and secretion of leptin in adipocytes needs to be cells (C2 C12) cells [50]. Thus, because NAD and SIRT1 protect
clarified. Additionally, 1,25(OH)2D-mediated adipocyte adipo- against adipose tissue mass and the derangement of metabolic
genesis and adipocyte marker gene regulation are discrepant functions that are known to related to obesity, we speculate that
according to cell type/origin. In 3T3-L1 cells, 1,25(OH)2D treat- 1,25(OH)2D-induced adipocyte fat storage and lipolysis might be
ment started at day 0 significantly inhibits adipogenesis and related to a NAD-sirtulin pathway. Further research is necessary
intracellular fat accumulation by down-regulating adipocyte to delineate whether 1,25(OH)2D-elevated NAD levels lead to
markers such as CEBPa, CEBPb, PPARg, or sterol regulatory changes in PGC-1a deacetylation, mitochondria biogenesis,
element-binding protein 1 (SREBP1) [13,14]. In contrast, 1,25(OH) oxidative respiration, fatty acid oxidation, and energy homeo-
2D-treated human subcutaneous preadipocytes demonstrated stasis in a SIRT1-dependent manner and/or in a VDR-mediated
increased adipogenesis determined by increased gene expres- manner in adipocytes and in vivo animal studies.
sion of PPARg and TG accumulation. There is no distinct change
in CEBPa and CEBPb levels [17]. Therefore, it is necessary to Conclusion
investigate the influence of 1,25(OH)2D on fat utilization in
human adipocytes. The present data demonstrated that the favorable effects of
Accumulating evidence demonstrates that SIRT1 activation vitamin D, 1,25(OH)2D on adipocyte metabolism and function are
plays an important role in metabolic functions by increasing the associated with decreased adipocyte fat storage and increased
b-oxidation of free fatty acids and mitochondrial biogenesis in a lipolysis. 1,25(OH)2D treatment modulates adipogenic, fatty acid
NAD-dependent manner [18,21,22,43]. The major metabolic oxidation, or lipolytic related gene expression and increases the
roles of NAD and NADH are associated with oxidative NAD-sirutlin pathway activity in 3T3-L1 adipocytes. Findings

Please cite this article in press as: Chang E, Kim Y, Vitamin D decreases adipocyte lipid storage and increases NAD-SIRT1..., Nutrition (2016),
http://dx.doi.org/10.1016/j.nut.2015.12.032
 E. Chang, Y. Kim / Nutrition xxx (2016) 1–7 7

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Please cite this article in press as: Chang E, Kim Y, Vitamin D decreases adipocyte lipid storage and increases NAD-SIRT1..., Nutrition (2016),
http://dx.doi.org/10.1016/j.nut.2015.12.032