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Sian Geldenhuys,
1
Prue H. Hart,
1
Raelene Endersby,
1
Peter Jacoby,
1
Martin Feelisch,
2
Richard B. Weller,
3
Vance Matthews,
4
and Shelley Gorman
1
Ultraviolet Radiation
Suppresses Obesity and
Symptoms of Metabolic
Syndrome Independently
of Vitamin D in Mice Fed a
High-Fat Diet
Diabetes 2014;63:3759–3769 | DOI: 10.2337/db13-1675
The role of vitamin D in curtailing the development of obe-
sity and comorbidities such as the metabolic syndrome
(MetS) and type 2 diabetes has received much attention
recently. However, clinical trials have failed to conclu-
sively demonstrate the benefits of vitamin D supple-
mentation. In most studies, serum 25-hydroxyvitamin D
[25(OH)D] decreases with increasing BMI above nor-
mal weight. These low 25(OH)D levels may also be
a proxy for reduced exposure to sunlight-derived ultra-
violet radiation (UVR). Here we investigate whether UVR
and/or vitamin D supplementation modifies the devel-
opment of obesity and type 2 diabetes in a murine model
of obesity. Long-term suberythemal and erythemal UVR
significantly suppressed weight gain, glucose intoler-
ance, insulin resistance, nonalcoholic fatty liver disease
measures; and serum levels of fasting insulin, glucose,
and cholesterol in C57BL/6 male mice fed a high-fat
diet. However, many of the benefits of UVR were not
reproduced by vitamin D supplementation. In further
mechanistic studies, skin induction of the UVR-induced
mediator nitric oxide (NO) reproduced many of the
effects of UVR. These studies suggest that UVR (sun-
light exposure) may be an effective means of suppress-
ing the development of obesity and MetS, through
mechanisms that are independent of vitamin D but
dependent on other UVR-induced mediators such as NO.
Obesity has significant effects on our health and well-
being: obese people have increased comorbidities resulting
from cardiovascular disease, type 2 diabetes, breast and
colon cancers, dementia, and depression. Vitamin D defi-
ciency is recognized as a health problem affecting many
individuals worldwide (1) and may contribute to the devel-
opment of obesity. Insufficient levels of vitamin D are as-
sociated with obesity, and obese people are more likely than
others to be vitamin D deficient (reviewed in Earthman
et al. [2] and Autier et al. [3]). Vitamin D is synthesized
from dermal 7-dehydrocholesterol after cutaneous ex-
posure to the ultraviolet radiation (UVR) of sunlight.
Vitamin D is transported to the liver bound to the vita-
min D–binding protein for conversion into the storage
form 25-hydroxyvitamin D [25(OH)D], before further
conversion into the active form 1,25-dihydroxyvitamin D
[1,25(OH)
2
D] in the kidneys. Many cells in other tissues
express the enzymatic machinery required to convert 25
(OH)D into active 1,25(OH)
2
D (2).
1
Telethon Kids Institute, The University of Western Australia, Perth, Western
Australia, Australia
2
Clinical and Experimental Sciences, Faculty of Medicine, University of South-
ampton, Southampton General Hospital, Southampton, U.K.
3
University of Edinburgh, MRC Centre for Inflammation Research, Edinburgh,
Scotland
4
Laboratory for Metabolic Dysfunction, Harry Perkins Institute of Medical Research,
Centre for Medical Research, The University of Western Australia, Perth, Western
Australia, Australia
Corresponding author: Shelley Gorman, shelley.gorman@telethonkids.org.au.
Received 30 October 2013 and accepted 27 May 2014.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1675/-/DC1.
© 2014 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
Diabetes Volume 63, November 2014 3759
OBESITY STUDIES
It is not known whether vitamin D deficiency is a causal
pathway for the development of obesity and the metabolic
syndrome (MetS). Serum 25(OH)D levels generally de-
crease with increasing BMI above normal weight (4), and
results from a genetic association study (5) suggest that
a higher BMI leads to reduced circulating 25(OH)D levels.
Furthermore, randomized controlled trials that test the
efficacy of vitamin D supplementation for weight loss (2)
or for curbing MetS-related diseases like type 2 diabetes
and cardiovascular disease (3,6,7) have had little success.
Even so, there is currently much interest in vitamin D
supplementation as a clinical means of controlling obesity
and MetS, with .100 clinical trials underway assessing
vitamin D supplementation (ClinicalTrials.gov).
Increased storage of fat-soluble vitamin D in obese
individuals may reduce circulating 25(OH)D levels (8). Also,
obese people exercise less and spend less time in the sun
(9). Our increasingly “indoor”lifestyles, coupled with con-
cerns about rising skin cancer rates for light-skinned pop-
ulations, have resulted in concomitant decreases in sun
exposure (10) and increased prevalence of vitamin D de-
ficiency (11) worldwide, including countries like Australia,
which experiences some of the highest obesity rates in the
world. Long-term sunlight exposure (particularly suberythe-
mal UVR) itself may be beneficial for obesity and MetS
outcomes like type 2 diabetes (12) and nonalcoholic fatty
liver disease (NAFLD) (13).
In this article, we present data further defining the role
of sunlight-induced vitamin D in modulating the de-
velopment of obesity and aberrant metabolic outputs,
including glucose intolerance, insulin resistance, and
NAFLD. We directly compared the abilities of long-term
UVR and/or dietary vitamin D to alter the development of
obesity using a physiologically relevant model induced by
feeding a high-fat diet to C57BL/6 male mice. Our
previous studies have shown that long-term UVR expo-
sure does not modify serum 25(OH)D levels in male mice
(14), allowing us to investigate the ability of UVR to modu-
late obesity and MetS independent of circulating 25(OH)D
levels. Here, long-term UVR exposure but not dietary vi-
tamin D suppressed weight gain and various measures of
MetS (circulating cholesterol levels, glucose intolerance,
and insulin resistance). Further, while vitamin D supple-
mentation did improve NAFLD, UVR suppressed its de-
velopment even more effectively. Vitamin D supplementation
suppressed circulating tumor necrosis factor-a(TNF-a)
levels, identifying a possible mechanism for the control
of NAFLD. In further mechanistic studies, UVR-induced
nitric oxide (NO) significantly suppressed some measures
of obesity and MetS development, including weight, white
adipose tissue (WAT) accumulation, fasting glucose level,
the development of insulin resistance, and NAFLD. These
studies suggest that while vitamin D supplementation
may be useful for preventing NAFLD development, sun-
light exposure may be more effective, and have the added
benefits of suppressing obesity and MetS through NO-
dependent pathways.
RESEARCH DESIGN AND METHODS
Mice
All experiments were performed according to the ethical
guidelines of the National Health and Medical Research
Council of Australia and with approval from the Telethon
Institute for Child Health Research Animal Ethics Com-
mittee. C57BL/6 male mice were purchased from the
Animal Resources Centre (Murdoch, Western Australia,
Australia). The temperature and lighting were controlled,
with a normal 12-h light/dark cycle to mimic day and
night. Mice were housed under Perspex-filtered fluorescent
lighting, which emitted no detectable UVR B as measured
using an ultraviolet (UV) radiometer (UVX Digital Radio-
meter; Ultraviolet Products Inc., Upland, CA). Mice were
allowedaccesstofoodandacidified water ad libitum.
Diet
All diets were obtained from Specialty Feeds (Glen Forrest,
Western Australia, Australia) and included two semipure
low-fat diets (5% fat; canola oil), which were supplemented
with vitamin D
3
(2,280 or 0 IU vitamin D
3
/kg) (LF-D
+
)or
not (LF-D
2
) and two high-fat diets (23%; lard [20.7%] and
canola oil [2.9%]) that were supplemented with vitamin D
3
(2,280 or 0 IU vitamin D
3
/kg) (HF-D
+
)orwerenot(HF-D
2
).
Mice that started on a vitamin D
3
–supplemented diet were
continued on diets supplemented with vitamin D
3
through-
out. The LF-D
2
and HF-D
2
were also supplemented with
2% calcium (vs. 1% for the LF-D
+
and HF-D
+
)toensure
normocalcemia.
UVR and Topical Skin Treatments
A bank of six 40-W lamps (TL UV-B; Philips, Eindhoven,
the Netherlands) emitting broadband UVR (250–360 nm),
with 65% of the output in the UVB range (280–315 nm),
was used to irradiate mice to deliver suberythemal (1 kJ/m
2
)
(15) or erythemal (4 or 8 kJ/m
2
) UVR onto a clean-shaven
8-cm
2
dorsal skin area, as previously described (16). Al-
ternatively, skin was treated with 0.1 mmoles S-nitroso-N-
acetylpenicillamine (SNAP; Sigma-Aldrich) (17), a NO
donor. In other treatments, a NO scavenger, carboxy-PTIO
potassium salt (cPTIO; 0.1 mmoles; Sigma-Aldrich) (18), or
1,25(OH)
2
D (11.4 pmol/cm
2
; Sigma-Aldrich) (19) were
applied immediately after delivery of suberythemal UVR
(1 kJ/m
2
). This dose of 1,25(OH)
2
D was previously reported
to not induce hypercalcemia (19). All topical reagents were
diluted with a vehicle consisting of ethanol, propylene glycol,
and water (2:1:1) (20). All topical treatments were per-
formed in the morning.
Measuring Weight Gain
Mice were weighed weekly on the same day in the
morning using a digital scale (.0.1 g sensitivity; Scout;
Ohaus). The percentage weight gain was calculated from
8 weeks of age.
Glucose and Insulin Tolerance Tests
Mice were fasted for 5 h and then intraperitoneally
challenged with either 1 g/kg glucose (Phebra, Lane Cove,
New South Wales, Australia), for glucose tolerance tests
3760 UV Inhibits Obesity Independently of Vitamin D Diabetes Volume 63, November 2014
(GTTs), or 0.5–0.75 IU/kg insulin (Lilly, Indianapolis, IN),
for insulin tolerance tests (ITTs). Glucose levels were
recorded at 0, 15, 30, 45, 60, and 90 min postinjection
using the Accu-Chek Performa glucometer (Roche).
Serum Metabolites
Serum 25(OH)D levels were measured using IDS EIA kits
(Immunodiagnostic Systems Ltd., Fountain Hills, AZ) as
described by the manufacturer (limit of detection 5–7
nmol/L; coefficient of variation 0.08 for internal controls).
For confirmation, 25(OH)D levels in selected samples were
measured using a liquid chromatography-tandem mass spec-
trometry method (21), which significantly correlated with
immunoassay 25(OH)D levels (n=8;r= 0.99, P#
0.0001). Serum calcium, cholesterol, HDL cholesterol, LDL
cholesterol, and triglyceride levels were measured by standard
colorimetric reactions using the Architect c16000 Analyzer
(AbbottDiagnostics,AbbottPark,IL).Glucose,insulin,adipo-
nectin, and leptin levels were measured in serum after fasting
mice for 5 h. Fasting glucose level was measured using the
Accu-Chek Performa glucometer (Roche, Castle Hill, New
South Wales, Australia). Fasting insulin, adiponectin, and lep-
tin levels were measured using rat/mouse insulin, mouse adi-
ponectin, and mouse leptin ELISA kits, respectively, as
described by the manufacturer (EMD Millipore Corporation,
Billerica, MA). Serum interleukin (IL)-6, TNF-a,andIL-10
concentrations were measured in serum using ELISA as pre-
viously described (15,22) with antibody pairs supplied by
Figure 1—The experimental approach. The 4-week-old C57BL/6
male mice were fed a low-fat diet (either LF-D
+
or LF-D
2
) for 4
weeks. At 8 weeks of age, mice were either continued on these
diets or switched to an HF-D
+
or an HF-D
2
. At the same time,
each dietary group was further divided into three treatment groups
of mice that received long-term irradiation with suberythemal UVR
(1 kJ/m
2
twice a week [biweekly]), erythemal UVR (4 kJ/m
2
once
a fortnight [fortnightly]), or no UVR. Mice were fed these diets and
irradiated with these UVR regimens for a further 12 weeks until mice
were 20 weeks of age. There were a total of 12 treatments, with 18
mice per treatment. The experiment was performed two times.
Figure 2—The effects of long-term skin exposure to UVR, dietary
vitamin D, and a high-fat diet on serum 25(OH)D levels. A: The 4-
week-old C57BL/6 male mice were fed a low-fat diet (either LF-D
+
or LF-D
2
) for 4 weeks. B–D: At 8 weeks of age (week 0), mice were
either continued on these diets or switched to an HF-D
+
or an
HF-D
2
. At the same time, each dietary group was further divided
into three treatment groups of mice that received long-term irradi-
ation with no UVR (B), suberythemal UVR (1 kJ/m
2
twice a week)
(C), or erythemal UVR (4 kJ/m
2
once a fortnight) (D) for a further
12 weeks. In B–D, serum 25(OH)D levels are depicted for mice that
underwent these UVR/dietary interventions for 12 weeks. Data are
shown as the mean 6SEM for n=4–9 mice at each time, pooled
from two independent experiments (*P<0.05).
diabetes.diabetesjournals.org Geldenhuys and Associates 3761
BD Biosciences (Franklin Lakes, NJ). The levels of detec-
tion for the IL-6, TNF-a, and IL-10 assays were 12, 3, and
14 pg/mL, respectively. Serum nitrite and nitrate levels
were measured as previously described (23).
Histopathological Assessment of Liver Pathology
The severity of NAFLD was assessed by grading formalin-
fixed and hematoxylin-eosin–stained liver sections. Steato-
sis and hepatocellular ballooning were scored using a scoring
system based on the nonalcoholic steatohepatitis (NASH)
scoring system (24). A separate score was given for stea-
tosis (0–3) and hepatocellular ballooning (0–3). These
scores were added together for an overall score (#6).
Measurement of Skin NO Levels
Formation of NO in the skin was measured by a non-
invasive in vivo assay using the substrate DAF-2 (applied
in the form of the membrane-permeable precursor 4,5-
diaminofluorescein diacetate [DAF-2DA]; Millipore [cleaved
by intracellular esterases to generate DAF-2, which then
chemically reacts with NO to form the highly fluorescent
compound DAF-2T) (25). DAF-2DA [1 mmole in an etha-
nol, propylene glycol, and water (2:1:1) vehicle (20)] was
applied to shaved dorsal skin for absorption for 1 h prior
to skin treatment with UVR and/or the topical reagent.
Serial images of skin fluorescence (excitation at 488 nm,
emission at 515 nm) were taken every 5 min over 20 min
using the IVIS Spectrum Bioimager (PerkinElmer).
Statistical Analyses
Area under the curve (AUC) was calculated for GTT and
ITT using GraphPad Prism (version 5) using 0 as the
baseline. Student ttests and ANOVA were used to com-
pare treatments with Tukey post hoc analyses. Because of
a significantly greater variance in weight gain among high-
fat diet–fed mice, the effects of vitamin D intake and UVR
treatment (and their interaction) on weight gain were
analyzed separately from the low-fat diet–fed mice using
SPSS (version 21.0.0). Results were considered to be sta-
tistically significant for Pvalues ,0.05.
RESULTS
Tracking the Effects of Long-term UVR Exposure and
Dietary Fat on Serum 25(OH)D
To confirm our previous findings that UVR does not
modify serum 25(OH)D levels in male mice (14), vitamin
D–deficient male or female C57BL/6 mice were exposed to
a single erythemal dose (4 or 8 kJ/m
2
) of UVR, and serum
25(OH)D levels were tracked over 17 days. Serum 25(OH)D
levels were raised in a dose-related fashion by skin ex-
posure to erythemal UVR in female but not male mice
Figure 3—Long-term UVR exposure suppressed weight gain in
mice fed high-fat or low-fat diets not supplemented with vitamin
D (VitD). The 4-week-old C57BL/6 male mice were fed a low-fat
diet (either LF-D
+
or LF-D
2
) for 4 weeks. Aand B: At 8 weeks of
age (week 0), mice were either continued on these diets or switched
to an HF-D
+
or an HF-D
2
. At the same time, each dietary group was
further divided into three treatment groups of mice that received
long-term irradiation with no UVR, suberythemal UVR (1 kJ/m
2
twice a week), or erythemal UVR (4 kJ/m
2
once a fortnight). The
percentage weight gain is shown for mice that underwent these
UVR/dietary interventions for 12 weeks (until 20 weeks of age) for
mice fed a high-fat diet (A) or a low-fat diet (B). Data are shown as
the mean 6SEM for n= 18 mice/treatment from a representative of
two independent experiments. C: Total weight gain after 12 weeks
of these UVR/dietary interventions (at 20 weeks of age) is shown for
all treatments (mean 6SEM). D: After 12 weeks of these UVR/dietary
interventions (at 20 weeks of age) gonadal fat-pad (n= 18/treatment)
weights were measured. Data are representative of two independent
experiments (mean + SEM). *P<0.05.
3762 UV Inhibits Obesity Independently of Vitamin D Diabetes Volume 63, November 2014
(Supplementary Fig. 1). To determine the relative roles of
dietary vitamin D and/or UVR-induced vitamin D in the
regulation of obesity and related cardiometabolic disease
outcomes, we performed the following experiment using
C57BL/6 mice (Fig. 1). Male mice were fed a vitamin D–
supplemented or nonsupplemented (low-fat) diet from 4
to 8 weeks of age to establish vitamin D sufficiency or
deficiency (Fig. 2A). From 8 weeks of age, mice were con-
tinued on the supplemented or nonsupplemented diets,
but some were switched to a diet that was high in fat.
Each of these four dietary treatments were further di-
vided into three treatments, with the shaved skin of
mice exposed to long-term irradiation with no UVR,
suberythemal UVR (1 kJ/m
2
twice a week) or erythemal
UVR (4 kJ/m
2
once a fortnight), as indicated in Fig. 1.
Mice were treated from 8 to 20 weeks of age with these
UVR and dietary interventions. A high-fat diet signifi-
cantly increased serum 25(OH)D levels in mice fed diets
not specifically supplemented with vitamin D (HF-D
2
,
LF-D
2
) (Fig. 2B). Mice fed either diet that was further
supplemented with vitamin D (HF-D
+
, LF-D
+
) had signif-
icantly higher serum 25(OH)D levels than those mice fed
a diet that was not supplemented with vitamin D (Fig. 2B).
There was no additive effect of a high-fat diet and vitamin D
supplementation on serum 25(OH)D level (Fig. 2B). Al-
though not observed in our preliminary (Supplementary
Table 1—AUC values for GTTs and ITTs, and fasting glucose, insulin, leptin, and adiponectin levels measured 9–11 weeks after
UVR/dietary intervention
Treatment Diet
UVR
(kJ/m
2
)
GTT (AUC,
% basal
glucose)
ITT (AUC,
% basal
glucose)
Fasting
glucose
(mmol/L)
Fasting
insulin
(ng/mL)
Fasting
leptin
(ng/mL)
Fasting
adiponectin
(ng/mL)
1 HF-D
+
0 2,190 683 1,200 663 9.8 60.5 8.2 63.5 36.7 63.0 10.4 60.3
2 HF-D
+
1 1,770 649* 1,060 646 8.8 60.4 7.1 60.4 29.8 65.7 11.9 61.8
3 HF-D
+
4 1,880 6180 1,370 634 10.2 60.4 3.6 61.1 19.7 67.3 15.8 63.9
4 LF-D
+
0 1,470 667 800 638 7.9 60.3 1.0 60.4 1.5 60.6 12.9 62.8
5 LF-D
+
1 1,510 665 760 637 8.0 60.4 4.9 62.8 2.6 61.1 8.8 62.5
6 LF-D
+
4 1,390 656 770 679 7.8 60.4 1.8 61.0 2.2 60.7 11.9 61.0
7 HF-D
2
0 2,120 6130 1,230 615 9.8 60.3 11.1 61.9 29.8 63.5 13.0 62.6
8 HF-D
2
1 1,760 665†1,050 643†8.7 60.3†3.8 61.1†32.6 65.6 11.3 60.9
9 HF-D
2
4 1,690 673†960 672†8.1 60.4†3.9 62.8†14.0 65.3†13.0 61.1
10 LF-D
2
0 1,260 651 680 648 6.3 60.2 3.4 61.6 5.9 62.5 16.6 66.2
11 LF-D
2
1 1,280 6102 600 627 6.0 60.2 1.6 61.1 1.0 60.5 10.8 60.6
12 LF-D
2
4 1,480 636 760 660 7.7 60.4 4.3 61.8 1.9 60.2 11.7 61.9
Data are the mean 6SEM; n=4–8 mice/treatment. *P,0.05 vs. no UVR and HF-D
+
with data representative of two experiments.
†P,0.05 relative to no UVR and HF-D
2
with data representative of two experiments.
Table 2—Circulating triglyceride and cholesterol levels at 12 weeks after dietary and UVR interventions
Treatment Diet
UVR
(kJ/m
2
)
Triglycerides
(mmol/L)
HDL cholesterol
(mmol/L)
LDL cholesterol
(mmol/L)
Total cholesterol
(mmol/L)
1 HF-D
+
0 0.7 60.1 2.1 60.2 0.3 60.0 4.2 60.4
2 HF-D
+
1 0.6 60.0 2.0 60.2 0.2 60.0 3.8 60.4
3 HF-D
+
4 0.8 60.1 2.1 60.1 0.2 60.0 4.3 60.2
4 LF-D
+
0 1.0 60.1 1.5 60.1 0.2 60.0 2.5 60.2
5 LF-D
+
1 1.2 60.1 1.8 60.1 0.2 60.0 2.9 60.1
6 LF-D
+
4 1.1 60.3 1.3 60.2 0.1 60.0 2.2 60.3
7 HF-D
2
0 0.9 60.1 2.1 60.1 0.4 60.0 4.3 60.1
8 HF-D
2
1 0.6 60.0 2.1 60.0 0.3 60.0 4.2 60.2
9 HF-D
2
4 0.9 60.1 1.5 60.2* 0.2 60.0* 2.6 60.3*
10 LF-D
2
0 1.2 60.1 1.6 60.3 0.1 60.0 2.4 60.4
11 LF-D
2
1 0.9 60.1 1.4 60.1 0.1 60.0 2.0 60.1
12 LF-D
2
4 1.1 60.1 1.5 60.1 0.1 60.0 2.3 60.1
n= 4 mice/treatment. *P,0.05 relative to no UVR and HF-D
2
with data representative of two experiments.
diabetes.diabetesjournals.org Geldenhuys and Associates 3763
Fig. 1) and past investigations (14), long-term suberythe-
mal (Fig. 2C) or erythemal (Fig. 2D) UVR exposure signif-
icantly (but transiently) enhanced serum 25(OH)D levels,
when administered to mice fed an LF-D
+
(but not HF-D
+
,
LF-D
2
, or HF-D
2
) (Supplementary Fig. 2). The effects
were more pronounced for mice administered the long-
term erythemal UVR, but returned to baseline levels after
6 weeks of UVR/dietary intervention (Fig. 2Dand Supple-
mentary Fig. 2B).
Long-term UVR Exposure Suppressed Weight Gain in
Mice Fed a Vitamin D–Nonsupplemented Diet
There was no effect of vitamin D supplementation on
weight gain (Fig. 3Aand B). Both long-term suberythemal
UVR (1 kJ/m
2
twice a week) and erythemal UVR (4 kJ/m
2
once a fortnight) treatment suppressed weight gain in mice
fed the HF-D
2
(Fig. 3A)by$40%. Long-term erythemal
UVR exposure also suppressed weight gain in mice fed the
LF-D
2
(Fig. 3B). The effects of long-term skin exposure
to UVR were less apparent for mice fed the vitamin
D–supplemented diet, where UVR exposure suppressed
weight gain in a transient fashion in mice fed the HF-D
+
(Supplementary Fig. 3A). At the end of the UVR/dietary
intervention period (12 weeks), gonadal fat-pad weights
were not affected by dietary vitamin D supplementation
but were significantly suppressed in mice irradiated with
UVR and fed the HF-D
2
(Fig. 3D).
Long-term UVR Exposure Suppressed Glucose
Intolerance and Insulin Resistance in Mice Fed
a Vitamin D–Nonsupplemented Diet
After 10 and 11 weeks of UVR/dietary intervention, GTTs
and ITTs were performed (Table 1). Mice fed the high-fat
diets developed glucose intolerance (Supplementary Fig.
3B) and insulin resistance (Supplementary Fig. 3C), with
no suppressive effect of vitamin D supplementation (Sup-
plementary Fig. 3Band C; Table 1 for AUC). Both mea-
sures were suppressed in mice receiving long-term irradiation
with UVR (either suberythemal or erythemal) and fed the
HF-D
2
(Table 1). Glucose intolerance was significantly
suppressed by long-term suberythemal UVR in mice fed
the HF-D
+
only (Table 1). In addition, fasting glucose and
insulin levels were also reduced by UVR treatment in
mice fed the HF-D
2
, with fasting leptin levels also sup-
pressed in mice that received long-term irradiation with
erythemal UVR (Table 1). There were no effects of long-
term UVR (or dietary vitamin D) on fasting adiponectin
levels (Table 1).
Long-term Erythemal UVR Exposure Suppressed
Circulating Cholesterol Levels in Mice Fed a High-Fat
Diet Not Supplemented With Vitamin D
After 12 weeks of UVR/dietary intervention, circulating
levels of triglycerides and cholesterol (HDL, LDL, and
total) were measured (Table 2). Triglyceride levels were
Figure 4—Long-term UVR significantly reduced the extent of liver steatosis and lobular ballooning in mice fed a high-fat diet. The 4-week-
old C57BL/6 male mice were fed a low-fat diet (either LF-D
+
or LF-D
2
) for 4 weeks. At 8 weeks of age, mice were either continued on these
diets or switched to an HF-D
+
or an HF-D
2
. At the same time, each dietary group was further divided into three treatment groups of mice
that received long-term irradiation with no UVR (A,D,G, and J), suberythemal UVR (1 kJ/m
2
twice a week; B,E,H,K), or erythemal UVR
(4 kJ/m
2
once a fortnight; C,F,I, and L). After 12 weeks of these UVR/dietary interventions (at 20 weeks of age), the extent of liver
histopathology was measured in liver specimens (n= 10/treatment for data pooled from two independent experiments). A–L: Represen-
tative hematoxylin-eosin–stained sections of liver for each treatment (Band C, original magnification 320 [equivalent to 150 mm]).
Examples of liver steatosis (blue arrow) and lobular ballooning (red arrow) are shown in G.
3764 UV Inhibits Obesity Independently of Vitamin D Diabetes Volume 63, November 2014
not modified by vitamin D supplementation or long-term
UVR (Table 2). HDL, LDL, and total cholesterol levels
were suppressed in mice fed the HF-D
2
and also receiving
long-term irradiation with erythemal UVR (Table 2).
Long-term UVR Exposure More Effectively Suppressed
the Development of NAFLD Than Vitamin D
Supplementation
The development of markers of NAFLD was measured by
analyzing the degree of liver steatosis and lobular
ballooning after 12 weeks of UVR/dietary intervention
(Figs. 4 and 5A). Long-term skin exposure to UVR sub-
stantially suppressed liver histopathology in mice fed the
high-fat diets (Fig. 4A–C, HF-D
+
; Fig. 4G–I, HF-D
2
; Fig.
5A) to a greater degree than that achieved by dietary
vitamin D supplementation alone (Fig. 4A, HF-D
+
; Fig.
4G,HF-D
2
; Fig. 5A). Vitamin D supplementation had
no effect on liver weight, whereas long-term erythemal
UVR suppressed liver weight in mice fed the HF-D
2
(Fig. 5B).
Vitamin D Supplementation Prevented the Suppressive
Effects of UVR Upon Weight Gain and Markers of MetS
The results presented above suggest that many of the
effects of UVR were more prominent in mice not further
supplemented with vitamin D. We used a general linear
model to assess whether there may be interactions within
the high-fat diet treatments, such that dietary vitamin D
may have inhibited the suppressive ability of UVR.
Significant interactions between dietary vitamin D and
long-term UVR exposure were detected for weight gain
(Fig. 3C)(P= 0.05), gonadal fat-pad weights (Fig. 3D)(P=
0.03), and fasting glucose levels (Table 1) (P= 0.01), but
not the other measures, including liver histopathology
(Figs. 4 and 5A)(P.0.05).
Serum Vitamin D or Calcium Levels Were Not Related
to Weight Loss or Suppression of MetS in UVR-
Irradiated Mice
Long-term UVR exposure suppressed aspects of weight
gain and measures of MetS, independently of changes to
circulating 25(OH)D levels (Fig. 2 and Supplementary Fig.
2). Therefore, it is unlikely that the mechanism through
which UVR acted was dependent on vitamin D. As calcium
levels can be modified by vitamin D and have been asso-
ciated with weight loss (26), we also assessed circulating
calcium levels after 12 weeks of UVR/dietary intervention,
but observed no significant effects of dietary vitamin D or
long-term skin exposure to UVR in mice fed the high-fat
diets (Fig. 5C). Long-term skin exposure to UVR reduced
calcium levels in mice fed a low-fat diet (Fig. 5C).
Figure 5—Long-term UVR exposure significantly reduced the ex-
tent of liver histopathology in mice fed a high-fat diet. The 4-week-
old C57BL/6 male mice were fed a low-fat diet (either LF-D
+
or
LF-D
2
) for 4 weeks. At 8 weeks of age, mice were either continued
on these diets or switched to an HF-D
+
or HF-D
2
. At the same
time, each dietary group was further divided into three treatment
groups of mice that received long-term irradiation with no UVR,
suberythemal UVR (1 kJ/m
2
twice a week), or erythemal UVR
(4 kJ/m
2
once a fortnight). After 12 weeks of these UVR/dietary
interventions (at 20 weeks of age), the extent of liver histopathology
(n= 10/treatment for data pooled from two independent experi-
ments) (A), liver weights (n= 18/treatment for data from a repre-
sentative experiment) (B), and serum levels of calcium (n=4–8/
treatment for data pooled from two independent experiments) (C)
and TNF-a(n=12–18/treatment for data pooled from two in-
dependent experiments) (D) are shown. Data are shown as the
mean 6SEM. *P<0.05. VitD, vitamin D.
diabetes.diabetesjournals.org Geldenhuys and Associates 3765
Figure 6—The UVR-induced mediator NO may regulate body weight, WAT accumulation, glucose metabolism, and the development of
NAFLD in mice fed a high-fat diet. Aand B: Using the DAF-2DA substrate, skin NO levels are shown for adult C57BL/6 male mice fed a low-
fat diet (LF-D
2
), 5 min after skin treatment with vehicle, 1 kJ/m
2
UVR, or the NO donor SNAP, with a quantitative measure (in photons per
second) (A) and representative skin fluorescence (B) shown. The 4-week-old C57BL/6 male mice were fed an LF-D
2
for 4 weeks. At 8
weeks of age, mice were either continued on these diets or switched to the HF-D
2
. Within the HF-D
2
treatments, mice were further divided
into five treatment groups. The shaved dorsal skin of these mice 1) was treated with vehicle only, 2) received long-term irradiation with
suberythemal UVR (1 kJ/m
2
twice a week) and then vehicle, 3) was topically treated with SNAP, 4) received long-term irradiation with
3766 UV Inhibits Obesity Independently of Vitamin D Diabetes Volume 63, November 2014
Circulating TNF-aLevel Was Linked With Improved
Markers of NAFLD in the Absence of Dietary Vitamin D
Supplementation But Not Skin Exposure to UVR
The ability of phototherapy to suppress the development of
NAFLD has been associated with reduced expression of TNF-a
(13). However, long-term UVR did not modify serum TNF-a
levels after 12 weeks of UVR/dietary intervention in mice fed
a high-fat diet (Fig. 5D). Vitamin D supplementation reduced
circulating TNF-alevels in mice fed an HF-D
+
when compared
with those fed an HF-D
2
(Fig. 5D). Serum levels of IL-6 and
IL-10 were below the level of detection of the ELISA.
UV-Induced NO Suppresses the Development of
Obesity and Symptoms of MetS
A role for NO, an alternate (non–vitamin D) mediator
induced by UVR, was examined. Skin levels of NO in-
creased from as early as 5 min after UVR/SNAP (Fig. 6A
and B) treatment as determined using DAF-2. To examine
a role for UVR-induced NO in modulating obesity and
MetS symptoms, 4-week-old C57BL/6 male mice were
fed an LF-D
2
for 4 weeks. From 8 weeks of age, mice
were either continued on this diet or switched to the
HF-D
2
, with mice fed an HF-D
2
further divided into
groups receiving the following five dorsal skin treatments:
1) vehicle only; 2) suberythemal UVR (1 kJ/m
2
) and then
vehicle; 3) SNAP; 4) suberythemal UVR and then cPTIO;
or 5) suberythemal UVR and then 1,25(OH)
2
D. This final
treatment was selected to test whether active 1,25(OH)
2
D
could prevent the suppressive effects of UVR on obesity
and MetS development (like dietary vitamin D in Supple-
mentary Fig. 3A) through inhibition of skin-induced NO.
Indeed, vitamin D may repair UV-induced DNA damage in
skin by suppressing NO (27).
After 12 weeks of feeding mice the HF-D
2
, skin NO
levels were assessed 10 min after a final treatment with
one of the five topical treatments detailed above. Skin NO
levels increased with UVR or SNAP (Fig. 6C). The NO
scavenger cPTIO reduced levels of NO in skin after UVR
treatment, but, unexpectedly, 1,25(OH)
2
D did not. Serum
nitrite/nitrate concentrations, measured 20 min after the
final skin treatment, were not altered by treatment with
long-term low-dose UVR or SNAP (data not shown). Long-
term UVR suppressed weight gain and the accumulation
of WAT after 12 weeks of the HF-D
2
(Fig. 6D). Long-term
SNAP treatment also effectively suppressed mouse weights
(although not weight gain) and WAT accumulation (Fig. 6D).
However, neither the NO scavenger cPTIO nor 1,25(OH)
2
D
reversed the suppressive effects of UVR on weight gain
or WAT accumulation. Indeed, the UVR and 1,25(OH)
2
D
treatment was more effective than UVR treatment alone,
but this observation may reflect the hypercalcemia observed
early on with topical 1,25(OH)
2
Dtreatment(4weekspost-
UVR [2.4 60.03 mmol/L] vs. post-UVR+1,25(OH)
2
D
[3.5 60.07]; *P,0.001 for serum calcium). In response
to these observations, we halved the dose of 1,25(OH)
2
Dad-
ministered, and mice were treated only once per week after 4
weeks of intervention. Despite this change, 1,25(OH)
2
D-
treated mice were still modestly hypercalcemic at the end
of the experiment (12 weeks post-UVR [2.4 60.03] vs.
post-UVR+1,25(OH)
2
D[2.760.07]; *P,0.001 for serum
calcium).
As observed previously, long-term UVR exposure sup-
pressed fasting glucose and insulin levels, and the de-
velopment of glucose intolerance and insulin resistance
(Fig. 6Eand F). Here, long-term SNAP treatment also
suppressed the development of insulin resistance (Fig.
6F). Furthermore, cPTIO treatment after UVR reversed
the suppressive effects of UVR alone upon fasting glucose
levels (Fig. 6E). Finally, both long-term UVR and SNAP
treatment suppressed the development of NAFLD, while
cPTIO reversed the effects of UVR upon liver histopathol-
ogy (Fig. 6G). Cumulatively, these data suggest that UVR-
induced NO may play an important role in modulating the
development of obesity and MetS through effects on
weight, WAT accumulation, fasting glucose level, and
the development of insulin resistance and NAFLD.
DISCUSSION
Here we present evidence that long-term skin exposure to
low-dose (suberythemal) and high-dose (erythemal) UVR
suppresses the development of obesity and measures of
MetS in mice fed a high-fat diet. Vitamin D supplemen-
tation alone did not reproduce these effects. In addition,
the suppressive effects of UVR on obesity and MetS
development were not observed to the same degree in
mice that were further supplemented with vitamin D (i.e.,
HF-D
+
). For mice fed a high-fat diet, serum 25(OH)D
levels were not enhanced by long-term UVR exposure,
suggesting that any effects induced by UVR in these
mice were independent of circulating 25(OH)D levels.
The HF-D
2
increased circulating 25(OH)D levels; it is
likely that this diet contains vitamin D, perhaps within
the lard-derived fat fraction. Supplementation of this diet
with vitamin D (i.e., the HF-D
+
) further increased serum
25(OH)D levels. Both UV irradiation and vitamin D sup-
plementation reduced the severity of NAFLD, suggesting
that vitamin D can recapitulate the effects of UVR for the
prevention of certain obesity-related pathologies. We also
showed that some of the effects of UVR may occur
through NO production. In particular, it is likely that
suberythemal UVR and then cPTIO, or 5) received long-term irradiation with suberythemal UVR and then 1,25(OH)
2
D. Mice were treated for
12 weeks with these skin/dietary interventions until 20 weeks of age. C: Skin NO levels, 10 min after skin treatment (n= 8 mice/treatment).
D: Mouse weights, weight gain, and WAT weights (n= 18 mice/treatment). E: Fasting glucose and GTT AUC (n= 8 mice/treatment). F:
Fasting insulin and ITT AUC (n= 8 mice/treatment). G: Liver histopathology scores (n= 8 mice/treatment). Data are shown as the mean 6
SEM from one experiment. *P<0.05. VitD, vitamin D.
diabetes.diabetesjournals.org Geldenhuys and Associates 3767
UVR-induced NO may have profound effects on the de-
velopment of NAFLD, as topical SNAP suppressed liver
pathology, and cPTIO antagonized the effects of UVR.
Various non–vitamin D immunomodulators induced by
UVR, like NO (28), may be important for the regulation
of immunity (29) and obesity/MetS development (30).
Skin exposure to UVR releases NO from skin (28) and
could control obesity through NO-dependent effects on
mitochondria biogenesis within brown adipose tissue (31).
We have recently shown that UVR-induced NO reduces
blood pressure in healthy human volunteers (28). NO may
also be a crucial modulator of insulin and glucose transport,
and inhibition of NO may cause insulin resistance (32).
Combined with our results, these studies point to topically
induced NO as a potentially important clinical means to
suppress obesity and type 2 diabetes development.
The capacity of long-term UVR to suppress the devel-
opment of obesity and metrics of MetS was less effective
in mice orally supplemented with vitamin D [but not with
topical 1,25(OH)
2
D]. This was an unexpected finding but
could be explained by potential interactions of UVR-induced
mediators and dietary vitamin D, including NO (27). The
different effects of dietary vitamin D and topical 1,25
(OH)
2
D could be accounted for by the hypercalcemia in-
duced by long-term topical 1,25(OH)
2
D. In addition, after
12 weeks of treatment, serum 25(OH)D levels were signif-
icantly reduced by topical 1,25(OH)
2
D but not by the other
treatments (data not shown). Others have also observed
(33) that vitamin D suppressed weight gain in vivo after
intraperitoneal injections of 1,25(OH)
2
D(5mg/kg every 2
days), although the effects on circulating levels of calcium
[and 25(OH)D] were not reported. Others have shown (34)
that UVR may increase cortisol production in skin, which
has the potential to impact the hypothalamic-pituitary-
adrenal axis. While this might be hypothesized to alter
physical activity, no obvious behavioral effects were ob-
served in this study. However, we cannot exclude the
possibility that UVR alters neuroendocrine signaling net-
works in the skin (35) that might have a systemic impact.
Nakano et al. (13) showed that phototherapy sup-
pressed NAFLD but failed to reduce obesity, steatosis,
and blood glucose levels in Zucker fa-fa rats. These results
may differ from our own through significant differences
in the phototherapies delivered and the mouse model of
obesity. Dietary vitamin D has also previously been shown
to suppress the development of NAFLD in Sprague-
Dawley rats fed a “westernized”(high-fat/fructose) diet
(36), and in Lewis rats fed a choline-deficient and iron-
supplemented L-amino acid–defined diet (13). We also
observed that dietary vitamin D suppressed circulating
TNF-alevels in mice fed a high-fat diet. UVR did not
suppress serum TNF-alevels, suggesting that dietary vi-
tamin D and UVR may suppress NAFLD through differing
mechanisms. For control of NAFLD, the role of other
players within the vitamin D pathway is worthy of further
consideration. For example, circulating levels of the vita-
min D binding protein GC inversely correlate with liver
steatosis, and may determine the ability of vitamin D to
modulate the development of NAFLD (37). In addition,
1,25(OH)
2
D may act through the vitamin D receptor to
improve insulin sensitivity (38).
Our observations suggest that not all of the effects of
UVR on disease prevention can be achieved through
dietary vitamin D and that the role of other UV-induced
mediators like NO deserve further consideration. Fur-
thermore, by using a mouse modeling approach we were
able to remove the confounding effects of activity out of
doors, which might explain the observed associations of
reduced obesity and increased serum 25(OH)D levels. A
caveat is that while mice have conserved the ability to
synthesize vitamin D and NO in the skin and systemically
post-UVR, as fur-covered nocturnal animals they are not
usually exposed to much sunlight. Further studies are
required to translate the findings of our murine studies to
humans. However, our results support recent calls for
clinical trials that test the efficacy of skin exposure to
sunlight or UVR for the control of chronic diseases like
multiple sclerosis (39) and depression (40), which, like
obesity and MetS, may take years to develop. In conclu-
sion, our studies show that long-term low-dose sunlight
exposure may be an effective means of suppressing obe-
sity and MetS in mice fed a high-fat diet, through path-
ways that are independent of vitamin D and at least
partially dependent on skin-derived NO.
Acknowledgments. The authors thank Drs. Bernadette Fernandez and
Magda Minnion for measuring serum nitrite/nitrate levels; Professor Michael
Clarke at the Centre for Metabolomics (University of Western Australia) for
performing the liquid chromatography-mass spectrometry detection of serum
25(OH)D; Linda Gregory at the PathWest Laboratory at Royal Perth Hospital
(Perth, Western Australia, Australia) for performing the serum calcium,
cholesterol, HDL cholesterol, LDL cholesterol, and triglyceride analyses; and
Maxine Crook at Princess Margaret Hospital Pathology (Subiaco, Western
Australia, Australia) for embedding, sectioning, and staining the liver specimens.
Funding. This research was supported by the BrightSpark Foundation and the
Telethon Institute for Child Health Research.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. S.Ge. performed the majority of the experiments
and statistical analyses, and reviewed and edited the manuscript. P.H.H.
contributed to the discussion, and reviewed and edited the manuscript. R.E.
helped to optimize the skin NO assay and reviewed and edited the manuscript.
P.J. provided the statistical expertise for the experimental design and data
analysis. M.F. helped to design the study, supervised the analysis of serum NO
metabolites, and reviewed and edited the manuscript. R.B.W. helped to design
the study and reviewed and edited the manuscript. V.M. helped to design the
study, contributed to the discussion, and reviewed and edited the manuscript.
S.Go. envisaged and designed the study and wrote the manuscript. S.Go. is the
guarantor of this work and, as such, had full access to all the data in the study
and takes responsibility for the integrity of the data and the accuracy of the data
analysis.
Prior Presentation. Parts of this study were presented in abstract form at
the Australian Society for Medical Research Western Australia Scientific Symposium,
Perth, Western Australia, Australia, 5 June 2013; the Murdoch Children’s Research
Institute Molecular Medicine Series, Melbourne, Victoria, Australia, 12 July 2013;
3768 UV Inhibits Obesity Independently of Vitamin D Diabetes Volume 63, November 2014
and the 6th Asia and Oceania Conference on Photobiology, Sydney, New South
Wales, Australia, 10–13 November 2013.
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