Low-dose UV radiation before running wheel access ...

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Journal of Endocrinology

244:3 473–486T S Allemann et al. UV and running activate brown adipose tissue

-19-0470

RESEARCH

Low-dose UV radiation before running wheel access activates brown adipose tissue

Tristan S Allemann1, Gursimran K Dhamrait1, Naomi J Fleury1, Tamara N Abel1, Prue H Hart1, Robyn M Lucas2,3, Vance B Matthews4 and Shelley Gorman1

1Telethon Kids Institute, University of Western Australia, Perth, Australia2National Centre for Epidemiology and Population Health, Research School of Population Health, Australian National University, Canberra, Australia3Centre for Ophthalmology and Visual Science, University of Western Australia, Perth, Australia4School of Biomedical Science – Royal Perth Hospital Unit, The University of Western Australia, Perth, Australia

Correspondence should be addressed to S Gorman: [email protected]

Abstract

In previous preclinical studies, low (non-burning) doses of UV radiation (UVR) limited weight gain and metabolic dysfunction in mice fed with a high-fat diet. Here, we explored the effects of low-dose UVR on physical activity and food intake and mechanistic pathways in interscapular brown adipose tissue (iBAT). Young adult C57Bl/6J male mice, housed as individuals, were fed a high-fat diet and exposed to low-dose UVR (sub-oedemal, 1 kJ/m2 UVB, twice-a-week) or ‘mock’ treatment, with or without running wheel access (2 h, for ‘moderate’ physical activity) immediately after phototherapy. There was no difference in distance run in mice exposed to UVR or mock-treated over 12 weeks of exposure to running wheels (P = 0.14). UVR (alone) did not significantly affect food intake, adiposity, or signs of glucose dysfunction. Access to running wheels increased food intake (after 10 weeks, P ≤ 0.02) and reduced gonadal white adipose tissue and iBAT mass (P ≤ 0.03). Body weight and hepatic steatosis were lowest in mice exposed to UVR with running wheel access. In the iBAT of mice exposed to UVR and running wheels, elevated Atgl, Cd36, Fasn, Igf1, Pparγ, and Ucp1 mRNAs and reduced CD11c on F4-80 + MHC class II+ macrophages were observed, while renal Sglt2 mRNA levels were increased, compared to high-fat diet alone (P ≤ 0.03). Blood levels of 25-hydroxyvitamin D were not increased by exposure to UVR and/or access to running wheels. In conclusion, when combined with physical activity, low-dose UVR may more effectively limit adiposity (specifically, body weight and hepatic steatosis) and modulate metabolic and immune pathways in iBAT.

Introduction

Current strategies to prevent obesity often aim to facilitate lifestyle change, promote energy balance to halt fat accumulation and enable weight management. These frequently involve pursuing a healthy lifestyle through decreased dietary intake of sugar and fat and increased physical activity. On its own, physical activity can be beneficial for metabolic health, improving glucose

tolerance and insulin sensitivity (Sigal et al. 2006, Lee et al. 2013). Similarly, diet can be an effective means of weight control in the short-term, but long-term results remain elusive due to difficulties with adherence (Mauro et al. 2008). These lifestyle strategies may be more effective when used together; however, new approaches are needed to improve their efficacy and lessen the impact of obesity.

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Key Words

f obesity

f adiposity

f metabolic dysfunction

f mice

f high-fat diet

f ultraviolet radiation

f physical activity

f running wheels

f brown adipose tissue

Journal of Endocrinology (2020) 244, 473–486

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474UV and running activate brown adipose tissue

T S Allemann et al. 244:3Journal of Endocrinology

Limited observational studies in humans suggest that higher levels of sun exposure are associated with better metabolic health (Gorman et al. 2017). We have previously shown that exposure to low doses (non-burning) of UVR suppressed weight gain and metabolic dysfunction in mice fed with a high-fat diet (Geldenhuys et al. 2014, Fleury et al. 2017, Teng et al. 2019). These findings were independent of circulating 25-hydroxyvitamin D (25(OH)D) and partially mediated by the release of nitric oxide from UV-irradiated skin. Interestingly, physical activity may increase the bioavailability of nitric oxide systemically, lowering blood pressure (Lin & Lee 2018) and improving endothelial function (Maiorana et al. 2003).

Physical activity may promote metabolic health in other ways by improving the functional capacity of brown adipose tissue (BAT). As part of an early response to exercise, BAT secretes the ‘lipokine’ 12,13-diHOME to increase fatty acid uptake by skeletal muscle (Stanford et al. 2018). The functional effects of physical activity on other aspects of BAT metabolism are less certain, with rodent and human studies generally demonstrating reduced BAT functionality (e.g. glucose and lipid uptake, fatty acid biosynthesis), with the effects of exercise on mitochondrial activity not well described (Peres Valgas da Silva et al. 2019). We have recently characterised the effects of low-dose UVR on metabolic functions in interscapular BAT (iBAT) (Dhamrait et al. 2020), which lies beneath irradiated dorsal skin of mice exposed to UVR in our previous studies (Geldenhuys et al. 2014, Fleury et al. 2017, Teng et al. 2019). Low-dose UVR curtailed whitening of iBAT through nitric oxide, while also modulating the expression of core gene regulators of BAT function through non-nitric oxide-dependent pathways (Dhamrait et al. 2020).

Here, we examined the effects of regular skin exposure to low-dose (non-burning) UVR on physical activity levels and food intake by C57Bl/6J male mice fed with a high-fat diet. Some mice were allowed access to running wheels for 2 h after each UVR exposure, with distance run recorded. Young adult mice were housed separately to record individual physical activity levels and avoid excessive fighting (unlike our previous studies (Geldenhuys et al. 2014, Fleury et al. 2017)). A second aim was to examine the separate and combined effects of ongoing treatment with low-dose UVR and access to running wheels (to induce moderate levels of physical activity) on weight gain and metabolic dysfunction in mice fed with a high-fat diet.

Materials and methods

Mice

All experiments were performed in accordance with the ethical guidelines of the National Health and Medical Research Council of Australia with approval from the Telethon Kids Institute Animal Ethics Committee (TKI AEC, AEC315) and reported according to ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Four-week-old C57Bl/6J male specific-pathogen-free (naive) mice were obtained from the Animal Resources Centre (Murdoch, Western Australia). Mice were housed individually (an ethical requirement stipulated by the TKI AEC) in the Bioresources Centre at the Telethon Kids Institute in temperature-controlled rooms (22 ± 1°C, mean ± range). Mice were housed as previously described (Dhamrait et al. 2020) with access to food and water ad libitum.

Diet

All diets were obtained from Specialty Feeds (Glenn Forrest, Western Australia). Mice were fed either a high-fat diet (23% lard with canola oil, SF12-031) or a low-fat diet (5% canola oil, SF12-029, control diet) from 4 weeks of age with detailed dietary content previously published (Dhamrait et al. 2020).

UVR

Mice with clean-shaven dorsal skin (8 cm2) were irradiated with sub-erythemal/-oedemal UVB radiation (1 kJ/m2; 3.3 ± 0.3 min (mean ± s.d.)); 40 W FS40 lamps (Philips TL UV-B, Eindhoven, The Netherlands), or mock irradiated as previously described (Dhamrait et al. 2020) usually on Tuesday and Friday mornings between 09:00 and 10:00 h. These lamps emit broadband UVR (250–360 nm), with 65% of the output in the UVB range (280–315 nm) (for spectral output (Goettsch et al. 1999)). PVC plastic was used to block wavelengths <280 nm (UVC). For mock treatments, mice were handled the same way, except that they were exposed to standard, non-UVR emitting fluorescent lighting. Mice were exposed to these treatments twice-a-week from 8 until 20 weeks of age.

Physical activity

Mice received access to a running wheel immediately following each UVR (n = 24) (or mock, n = 24) exposure






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T S Allemann et al. UV and running activate brown adipose tissue

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for 2 h (two sessions/week) from 8 until 20 weeks of age. This time was based upon recommendations for physical activity for adults in Australia (https ://ww w1.he alth. gov.a u/int ernet /main /publ ishin g.nsf /Cont ent/h ealth -pubh lth-s trate g-phy s-act -guid eline s), balanced with logistical constraints of exposing mice to UVR twice-a-week. Mice were placed individually for 2 h in the same open-top cage throughout the experiment, which contained a wireless running wheel (Med Associates, ENV-044, San Diego, VT, USA), and then returned to their home cage. Distance run (voluntary, weekly, and cumulative) was measured using Wheel Manager software (Med Associates, Version 1.7, 2015), calculating total distance run using the wheel radius and number of wheel revolutions.

Experiment design

Four-week-old C57Bl/6J male mice (n = 120) were fed the low-fat diet for 4 weeks (Fig. 1A). From 8 weeks of age, mice (n = 24/group) were randomly allocated into five treatment groups (Fig. 1A). Experiments were performed between February and August 2017. From 8 weeks of age, some mice were fed the low-fat diet (treatment 1, n = 24), while most were switched to a high-fat diet (treatments 2–5, n = 96) (Fig. 1A). From 8 weeks of age, the shaved

dorsal skin of mice was mock irradiated (treatments 1, 2 and 4); exposed to sub-erythemal UVR (treatments 3 and 5); and/or mice were allowed access to running wheels immediately following UVR or mock exposure (treatments 4 and 5). Mice were fed the low-fat diet or the high-fat diet with or without UVR and physical activity twice-a-week for 12 weeks until 20 weeks of age. The experiment was staggered (with results combined) with two lots of 12 mice per treatment and a 10-week difference between each experimental batch. This was done because of space limitations in the Bioresources Centre. For similar reasons, it was not possible to allow control mice access to cages with ‘locked’ running wheels. Three adverse events occurred in this study, which resulted in the killing of three mice, including a self-inflicted neck injury during ear-tagging; a broken paw from a limb being trapped in a cage-lid; and a dermatitis, which did not resolve. Data from mice experiencing these adverse events were excluded.

Measuring weight gain

Mice were weighed weekly on Monday mornings using a digital micro-weighing scale (CAS MWP-3000H, >0.05 g sensitivity). Percentage weight gain was calculated from body weight at 8 weeks of age.

Figure 1The effects of low-dose UVR on distance run in mice fed with a high-fat diet. In (A) the experimental design. Four week-old C57Bl/6J male mice were fed a low-fat diet (LFD) for 4 weeks. From 8 weeks-of-age, mice were fed the LFD (treatment 1) or switched to the high-fat diet (HFD, treatments 2–5). From 8 weeks-of-age, the shaved dorsal skin of mice were treated twice-a-week with: mock-irradiation (treatments 1 and 2); sub-erythemal UVR (1 kJ/m2 UVB, UVR) (treatment 3); mock-irradiation and then allowed access to running wheels for 2 h (physical activity, PA) (treatment 4); or sub-erythemal UVR and then mock-irradiation and then allowed access to running wheels for 2 h (UVR + PA) (treatment 5). Mice were fed the low- or high-fat diets and administered the treatments for 12 weeks until 20 weeks-of-age (n = 24 mice per treatment). In (B), weekly distance run (two-way repeated measures ANOVA with Bonferroni’s multiple comparison test), and (C) cumulative distance run are shown (*P < 0.05, Mann-Whitney test). Mice were weighed weekly with body weights shown in (D) and weight gain in (E) with data compared using one-way ANOVA with Tukey’s post hoc (*P < 0.05, comparing LFD with HFD; **P < 0.05, comparing LFD with UVR + PA; †P < 0.05, comparing HFD with PA; ††P < 0.05, comparing HFD with UVR + PA). Data are shown as mean ± s.d. (n = 23–24/treatment).




https://www1.health.gov.au/internet/main/publishing.nsf/Content/health-pubhlth-strateg-phys-act-guidelines







Food intake

For all mice, food and energy intake were measured 1 week prior to commencing treatments (7 weeks of age, n = 24/treatment) and for the first 4 days during weeks 2, 4, 6, and 10 of treatments as previously described (Dhamrait et al. 2020).

Glucose and insulin tolerance tests

Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were done after mice were fed the high-fat diet for 10 or 11 weeks as described previously (Dhamrait et al. 2020).

Specimen acquisition

At the conclusion of each experiment, mice were humanely killed and the specimens were obtained and weighed as previously (Dhamrait et al. 2020).

Serum lipids and metabolites

Fasting insulin levels were measured in plasma obtained after mice were fed the high-fat diet for 9 weeks as described previously (Dhamrait et al. 2020). At the end of the experiment, 25(OH)D levels were quantified in serum via liquid chromatography-tandem mass spectrometry (Clarke et al. 2013). Total cholesterol, HDL cholesterol, LDL cholesterol, triglyceride, calcium, phosphate, and creatinine levels were measured in serum by standard colorimetric reactions using the Architect c16000 Analyzer (Abbott Diagnostics). Interleukin (IL)-6 and TNF concentrations were measured as described previously using a modified ELISA (Dhamrait et al. 2020).

Histopathological assessment of liver

Non-alcoholic fatty liver disease (NAFLD) severity was assessed in formalin-fixed liver sections by blinded-scoring of formalin-fixed, stained liver sections for the degree of steatosis (H&E) and fibrosis (Masson’s) as previously described (Kleiner et al. 2005, Geldenhuys et al. 2014, Fleury et al. 2017, Teng et al. 2019, Dhamrait et al. 2020). Steatosis (≤6; hepatocellular ballooning, scored 0–3; and, steatosis, scored 0–3) was combined with fibrosis (≤4) for a total score of ≤10 (3–4 mice/treatment). For each sample, five fields-of-view were examined, with scores averaged for each mouse. Inflammatory foci/field were identified as tight bundles of eosinophilic staining

and multinucleated cells, with the mean number per field (at ×20 magnification, H&E) counted in a blinded fashion for five fields per mouse (Teng et al. 2019).

Confocal microscopy of mitochondria in iBAT

As previously described (Dhamrait et al. 2020), mitochondrial content (amount) and activity (membrane potential) were quantified using Mitospy Green FM (Biolegend, San Diego, CA, USA) and Mitospy Orange CMTMRos (Biolegend), respectively. A Nikon C2+ Confocal Microscope was used to image fluorochromes using a 40× objective (Nikon Plan Apo 40x NA 0.95 dry objective) and 405, 488, and 561 nm lasers. Three independent tissue sections were imaged per sample with ImageJ software (v1.45s, 2013) used to quantify fluorescent signals as previously described (Dhamrait et al. 2020).

Flow cytometry of immune cells from vascular stromal compartment of iBAT

This methodology has been previously described in detail, including source, type, dilutions, and catalogue numbers for all antibodies (Dhamrait et al. 2020). Briefly, surface and intracellular antigens were identified in a single cell suspension of vascular stromal cells from iBAT using fluorochrome-conjugated antibodies, after blocking Fc receptors. Cells were stained with antibodies to identify: (i) macrophages (CD86-BIO (with streptavidin-PE-Cy5), CD11b-APC-Cy7, CD11c-APC, MHC Class II-PE, CD301-PE-Cy7 and F4/80-FITC) or (ii) regulatory T cells (CD3e-PE-Cy7, CD25-APC, CD4-PE-Cy5, and CD8a-APC-Cy7 (surface), and, IL-10-PE and FOXP3-FITC (intracellular)). Cells stained with isotype control antibodies were used for IL-10 (rat IgG2b-PE) and FOXP3 (Rat IgG2a-FITC), as well as fluorescence-minus-one controls. An LSRII (BD Biosciences) flow cytometer was used to collect data whereby 100,000 events were collected per sample, analysed using FlowJo software (v10.2 TreeStar Inc, Ashland OR, USA).

mRNA expression in iBAT

At the end of the experiment, mRNA levels were quantified as previously described (Dhamrait et al. 2020). Quantitect Primer Assays (Qiagen) were used for detection of Atgl, Bmp7, Cd36, Dio2, Fasn, Fatp2, Fgf21, Glut4, Fatp2, Igf1, Igfbp2, Pgc1α, Pla2, Pparγ, Tnf, and Ucp1 with internal primers for detection of Eef1α, a house-keeping gene






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(Gorman et al. 2007). Pla2 mRNA was not detected in iBAT using this method.

mRNA expression in kidney

At the end of the experiment, mRNA was isolated from kidney specimens after homogenising in TRIzol Reagent (according to manufacturer’s instructions, Invitrogen). RNA samples were DNase-treated using the RQ1 RNase-Free DNase (Cat# M6101, Promega) and a TaqMan RT Reagents kit (Applied Biosystems) was used to generate cDNA on a Gene Amp PCR system 9700 Thermocycler (Applied Biosystems). To quantify the relative expression of genes of interest (Hprt (house-keeping, Mm01545399_m1), Sglt2 (Mm00453831_m1), Il6 (Mm00446190_m1) and Tnf (Mm00443260_g1, previously known as Tnfα)), TaqMan Primer Assays (Life Technologies) were performed using the Rotor Gene-2000 real-time PCR thermal cycler (Corbett Research) as per the manufacturer’s instructions. Real-time PCR data were analysed using the comparative critical threshold (also known as threshold cycle; CT) method.

Statistical analyses

Results were analysed using GraphPad Prism (v8.1.2 for MAC OS10, 2019). Descriptive statistics, mean and s.d. are reported with each mouse considered an experimental unit. Area under the curve (AUC) analyses used zero as baseline. All data were subjected to normality tests (D’Agostino & Pearson, Shapiro–Wilk) to determine if parametric data analyses were appropriate. Unless otherwise stated, data were compared using ANOVA with Tukey’s post hoc (or Students t test if only two treatments were compared, if normally-distributed) or Kruskal–Wallis test with Dunn’s post hoc (or Mann–Whitney test if only two treatments were compared, if not normally distributed) analyses to define differences between treatments. These post hoc tests correct for multiple comparisons (as previously (Fleury et al. 2017)). A two-way repeated-measures ANOVA (with Bonferroni’s multiple comparison test) was used to compare weekly distance run. Results were considered as statistically significant for P values <0.05. Power analyses (using G*Power v3.1.3, at 1-ß = 0.8) were done to determine the minimum number of mice required per treatment for each outcome, based on effect sizes and error using data previously published (Geldenhuys et al. 2014, Fleury et al. 2017, Teng et al. 2019). A one-way ANOVA (P < 0.05) was used to ensure that there was no significant difference in

starting body weights of mice, which could contribute to inter-group differences as the experiment proceeded.

Results

The effects of low-dose UVR on distance run in mice fed a high-fat diet

Mice allowed access to running wheels for 2 h, twice-a-week, increased their distance run for the first 4 weeks (P < 0.0001, week 0 vs week 4, Student’s t test) before these levels plateaued (Fig. 1B, P = 0.06, week 4 vs week 5, Student’s t test). There was no statistically significant difference in distance run between mice exposed to low-dose UVR immediately prior to running wheel access compared to mock-exposed mice (Fig. 1B, two-way repeated measures ANOVA: time P < 0.0001; PA v UVR + PA P = 0.14). There was some evidence that cumulative distance run (when combined across all 2-h access periods) was greater for mice exposed to low-dose UVR than mock-exposed mice (Fig. 1C, P = 0.07). A ROUT (Q = 10%) outlier analysis did not identify any statistically significant outliers in this dataset.

Moderate physical activity but not low-dose UVR modified food intake in mice fed a high-fat diet

Food intake was transiently reduced in mice fed a high-fat diet (compared to low-fat diet) during the 2- and 4-week timepoints (Table 1A, P ≤ 0.006), while energy intake was increased for mice fed a high-fat diet at nearly all timepoints (Table 1B, P < 0.001). There was no effect of ongoing exposure to UVR (alone) on food intake in mice fed a high-fat diet (compared to mock controls, P ≥ 0.49). After 10 weeks, mice allowed access to running wheels (without UVR) had significantly increased food and energy intake, compared to mice fed the high-fat diet (Table 1, P ≤ 0.02).

Body weight and weight gain were significantly reduced in mice exposed to UVR and allowed access to running wheels

Unlike previous studies in young adult C57Bl/6J mice (Geldenhuys et al. 2014, Fleury et al. 2017), all mice in this experiment were housed as individuals (as opposed to group-housing). After 1 week on the high-fat diet, mice had higher body weight (Fig. 1D, P ≤ 0.05) compared to mice fed with the low-fat diet, except for mice exposed to








UVR and allowed access to running wheels. There was no difference in body weight of mice of this treatment (UVR with running wheels) compared to mice fed the low-fat diet until week 7 (Fig. 1D, P ≤ 0.04). Moderate physical activity (running wheel access) following exposure to UVR significantly reduced body weight (Fig. 1D, P ≤ 0.02) but not percentage weight gain (Fig. 1E, p ≥ 0.10) from week 2 until week 12 in mice fed the high-fat diet (see also Table 2). Mice allowed access to running wheels (not exposed to UVR) had significantly lower body weights than mice fed the high-fat diet at week 8 only (Fig. 1D, P = 0.04).

Physical activity reduced adipose tissue weights in mice fed a high-fat diet

Mice fed the high-fat diet had increased gonadal white adipose tissue (WAT) and iBAT weights, compared to

those fed with the low-fat diet (Table 2). Mice allowed access to running wheels (both with and without UVR) had significantly reduced gonadal WAT (P ≤ 0.008) and iBAT (P ≤ 0.03) weights compared to those fed the high-fat diet (only), with no significant effect of low-dose UVR on its own (Table 2).

Effects of low-dose UVR and/or physical activity on measures of glucose metabolism and blood fats

There was no effect of eating a high-fat diet on fasting insulin levels in plasma (week 9) or fasting glucose in blood (week 10, before GTT) or blood glucose measured during the ITT (week 11) (Table 3, P > 0.05). Blood glucose measured during the GTT was greater from 15 to 60 min (P < 0.02) post-glucose injection in mice fed the high-fat diet and when comparing AUC of this assay with low-fat diet controls

Table 1 Food and energy intake.

Treatment groupa (Time) LFD (g/day) HFD (g/day) UVR (g/day) PA (g/day) UVR + PA (g/day)

A. Food intake Week 0 (baseline) 2.68 ± 0.23b 2.65 ± 0.18 2.53 ± 0.19 2.62 ± 0.17 2.55 ± 0.19 Week 2 2.65 ± 0.26 2.37 ± 0.23c 2.42 ± 0.25c 2.41 ± 0.22c 2.27 ± 0.17c

Week 4 2.63 ± 0.18 2.37 ± 0.20c 2.35 ± 0.21c 2.38 ± 0.13c 2.41 ± 0.23c

Week 6 2.65 ± 0.16 2.62 ± 0.19 2.62 ± 0.26 2.66 ± 0.21 2.55 ± 0.23 Week 8 2.57 ± 0.25 2.48 ± 0.33 2.40 ± 0.25c 2.51 ± 0.26 2.57 ± 0.31 Week 10 2.67 ± 0.20 2.48 ± 0.21 2.58 ± 0.28 2.72 ± 0.23d 2.62 ± 0.33

Treatment group (Time) LFD (kJ/day) HFD (kJ/day) UVR (kJ/day) PA (kJ/day) UVR + PA (kJ/day)

B. Energy intake Week 0 (baseline) 40.2 ± 3.5 39.8 ± 2.7 38.0 ± 2.9 39.3 ± 2.6 38.3 ± 2.9 Week 2 39.8 ± 3.9 45.0 ± 4.4c 46.0 ± 4.8c 45.8 ± 4.2c 43.1 ± 3.2 Week 4 39.5 ± 2.7 45.0 ± 3.8c 44.7 ± 4.0c 45.2 ± 2.5c 45.8 ± 4.4c

Week 6 39.8 ± 2.4 49.8 ± 3.6c 49.8 ± 4.9c 50.5 ± 4.0c 48.5 ± 4.4c

Week 8 38.6 ± 3.8 47.1 ± 6.3c 45.6 ± 4.8c 47.7 ± 4.9c 48.8 ± 5.9c

Week 10 40.1 ± 3.0 47.1 ± 4.0c 49.0 ± 5.3c 51.7 ± 4.4cd 49.8 ± 6.3c

aFour-week old male C57Bl/6J mice were fed a low-fat diet (LFD) for 4 weeks, until 8 weeks of age. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (1 kJ/m2 UVB radiation, twice a week; UVR); (4.) HFD with access to running wheels (2 h, twice a week; PA); or (5.) HFD with UVR followed by access to running wheels (UVR + PA). Data were acquired throughout 12 weeks of interventions with UVR and/or running wheels. bAll data are presented as mean ± s.d. (n = 23–24 per treatment) and were compared at each time-point using a one-way ANOVA with Tukey’s post-hoc analysis or Kruskal–Wallis test with Dunn’s post-hoc analysis (for non-normally distributed data), cp < 0.05 compared to LFD, dp < 0.05 compared to HFD.

Table 2 Final body and tissue weights.

Treatment groupa Body weight (g) Weight gain (%) WAT weight (g) iBAT weight (g) Liver weight (g)

LFD 25.4 ± 1.5b 126 ± 9 0.46 ± 0.08 0.18 ± 0.04 1.14 ± 0.14HFD 30.0 ± 2.1c 141 ± 11c 1.22 ± 0.42c 0.32 ± 0.05c 1.08 ± 0.13UVR 29.7 ± 2.3c 142 ± 12c 1.18 ± 0.35c 0.33 ± 0.09c 1.02 ± 0.14c

PA 28.3 ± 2.6c 135 ± 12 0.90 ± 0.36cd 0.26 ± 0.08cd 1.02 ± 0.14c

UVR + PA 27.8 ± 2.2cd 135 ± 12 0.87 ± 0.31cd 0.27 ± 0.08cd 0.97 ± 0.15cd

aFour-week old male C57Bl/6J mice were fed a low-fat diet (LFD) for 4 weeks, until 8 weeks of age. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (1 kJ/m2 UVB radiation, twice a week; UVR); (4.) HFD with access to running wheels (2 h, twice a week; PA); or (5.) HFD with UVR followed by access to running wheels (UVR + PA). All data reported here was obtained at the end of the experiment (after 12 weeks of treatments). bAll data are presented as mean ± s.d. (n = 21–24 per treatment) and were compared using a one-way ANOVA with Tukey’s post-hoc analysis, cP < 0.05 compared to LFD, dP < 0.05 compared to HFD.






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(P < 0.0001, Fig. 2A). There were no significant reductions in blood glucose during this assay in mice exposed to UVR with or without running wheel access compared to mice fed the high-fat diet (Fig. 2A). However, lower AUC (glucose levels measured during GTT) were observed for mice allowed access to running wheels compared to mice exposed to UVR (Fig. 2A, P ≤ 0.02). Cholesterol (total, LDL, HDL cholesterol) levels (but not triglyceride nor IL-6) were significantly increased in the blood of mice fed the high-fat diet (compared to low-fat diet, P < 0.05) acquired at 12 weeks, with no beneficial effects of UVR, physical activity, or both (Table 4, p > 0.05). Serum TNF levels were below the detection limit of the assay.

UVR combined with physical activity reduced hepatic steatosis

Compared to mice fed the high-fat diet, liver weights (Table 2, P = 0.04) and the degree of hepatic steatosis (Fig. 2B, C, D and E, P = 0.03) were reduced in mice exposed to UVR and allowed access to running wheels.

Serum 25-hydroxyvitamin D levels were not modified by physical activity

As previously mentioned, eating this high-fat diet increased serum levels of 25(OH)D (Table 5, P < 0.0001) (Geldenhuys et al. 2014, Fleury et al. 2017). Exposure to UVR did not further increase serum 25(OH)D levels, as previously observed (Geldenhuys et al. 2014, Fleury et al. 2017), which we have previously linked to reduced levels of the vitamin D precursor, 7-dehydrocholesterol in the skin of male mice (Gorman et al. 2012). There was no effect of physical activity on serum 25(OH)D levels in mice fed a high-fat diet (P ≥ 0.66). Serum levels of calcium, phosphate, creatinine, or albumin did not differ between treatments (Table 5, P ≥ 0.21).

Mitochondrial activity in iBAT increased by high-fat diet

To examine whether the beneficial effects of UVR and physical activity (combined) on body weight and hepatic steatosis may have been due to enhanced metabolic activity in iBAT, we measured the content (amount) and activity of mitochondria using confocal microscopy. Feeding mice with the high-fat diet for 12 weeks increased signs of mitochondrial activity (P < 0.03; but not content, P > 0.21), with no additional effects of low-dose UVR or physical activity or both in mice fed a high-fat diet (Fig. 3, p ≥ 0.62).

Low-dose UVR and physical activity suppressed markers of macrophage activity in iBAT

The relative numbers and phenotype of macrophages (Fig. 4A) in vascular stromal cells isolated from iBAT were determined by flow cytometry. Macrophages may regulate thermogenesis and inflammation in iBAT, while conversely, whitening induced by high energy diets may induce macrophage infiltration (Kotzbeck et al. 2018). Exposure to UVR, and/or physical activity significantly increased proportions of F4-80 + MHC classII+ cells (putative macrophages) in iBAT (Fig. 4B, P ≤ 0.04) compared to mice fed the low-fat (but not high-fat) diet. Consuming a high-fat diet reduced the surface expression of macrophage type-1 (M1) and macrophage type-2 markers (Braune et al. 2017) on these cells, including CD11c, CD86, CD301 and CD11b (P < 0.001; except CD301, P = 0.06). Physical activity further reduced the expression of CD86 (P = 0.005), while CD11c expression was lowest on F4-80 + MHC classII+ cells from mice exposed to UVR with running wheel access (P = 0.01, compared to high-fat diet only) (Fig. 4B). There were very few CD4+ T cells (CD3+CD4+CD8- cells) in iBAT (<0.1% of vascular stromal cells) and no significant differences in CD4+ T regulatory cell proportions (FOXP3+CD25+ or IL-10+ cells) between treatments (P ≥ 0.05, data not shown).

Table 3 Fasting levels of insulin in plasma and glucose in blood, and area under the curve (AUC) values for insulin tolerance tests (ITT).

Treatment groupa

Fasting insulin (ng/mL, week 9) (n = 10)

Fasting glucose (mmol/L, week 10) (n = 12)

ITT (AUC) (mmol/L × min, week 11) (n = 12)

LFD 1.55 ± 0.19b 10.2 ± 0.3 747 ± 33HFD 2.28 ± 0.23 10.4 ± 0.3 853 ± 68UVR 2.11 ± 0.30 9.7 ± 0.4 793 ± 50PA 1.92 ± 0.21 10.0 ± 0.3 869 ± 76UVR + PA 2.30 ± 0.35 9.4 ± 0.4 806 ± 61

aFour-week old male C57Bl/6J mice were fed a low-fat diet (LFD) for 4 weeks, until 8 weeks of age. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (1 kJ/m2 UVB radiation, twice a week; UVR); (4.) HFD with access to running wheels (2 h, twice a week; PA); or (5.) HFD with UVR followed by access to running wheels (UVR + PA). bAll data are presented as mean ± s.d. (n = 10–12 per treatment) and were compared using a one-way ANOVA with Tukey’s post hoc analysis.








UVR and physical activity increased Atgl, Cd36, Fasn, Igf1, Pparγ, and Ucp1 mRNA in iBAT, and, Sglt2 mRNA in kidney

In these individually housed mice, there were limited effects of high-fat diet on mRNA levels of Ucp1 (uncoupling protein-1), Pgc1α (peroxisome proliferator-activated

receptor gamma coactivator 1-α) and Dio2 (type 2 iodothyronine diodinase), core gene regulators of BAT function (Fig. 5). Exposure to UVR (alone) significantly upregulated expression of Pgc1α mRNA in iBAT compared to that observed in control mice fed the low or high-fat diet (consistent with our previous studies (Dhamrait et al. 2020)) or mice allowed access to running wheels. Combined treatment of mice fed the high-fat diet with UVR and running wheel access significantly increased Ucp1 expression in iBAT, as well as Fasn (fatty acid synthase, a lipogenesis gene), Atgl (adipose triglyceride lipase, a lipolysis gene), Igf1 (insulin-like growth factor-1, an insulin-signalling gene) (Valverde et al. 2005, Aldiss et al. 2019), Cd36 (linked with insulin-signalling (Samovski et al. 2018), mitochondrial function and fatty acid transport (Lehnig et al. 2019)) and Pparγ (peroxisome proliferator-activated receptor γ (Valverde et al. 2005)) (Fig. 5, P ≤ 0.03). There was some evidence for increased Bmp7 (bone morphogenetic protein-7, involved in insulin-signalling (Ma et al. 2019)) mRNA in iBAT of mice treated with UVR and allowed running wheel access compared to mice only fed the high-fat diet (Fig. 5, P = 0.06). In mice fed the high-fat diet, there was no significant difference in the expression of Dio2, Glut4 (glucose transporter-4), Tnf (pro-inflammatory gene), Fgf21 (fibroblast growth factor-21, a ‘batokine’), Fatp2 (fatty acid transport protein-2) or Igfbp2 (insulin-like growth factor binding protein-2, negative regulator of insulin-signalling (Baxter & Twigg 2009, Bredella et al. 2013)) mRNA with UVR and/or running wheel access. In further exploratory analyses, mRNA levels of Sglt2 (sodium glucose transporter-2; but not Tnf nor Il6, interleukin-6) were increased in the kidneys of mice exposed to UVR combined with physical activity (Supplementary Fig. 1, see section on supplementary materials given at the end of this article). Combined, these findings suggest that beneficial effects of low-dose UVR together with physical activity (provided through running wheel access) may be linked to increased signs of thermogenesis, lipid synthesis and turnover and insulin-signalling in iBAT and glucose metabolism in kidneys.

Discussion

Here we observed that low-dose UVR did not significantly increase distance run and did not affect food intake by mice fed a high-fat diet. In these individually housed mice, we did not observe significant effects of UVR on any measure of adiposity or marker of glucose metabolism. This contrasts with our previous observations in young

Figure 2Glucose tolerance test and liver histopathology. Four-week-old male C57Bl/6J male mice were fed a low-fat diet (LFD) for 4 weeks. At 8 weeks-of-age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (UVR); (4.) HFD with physical activity (PA); or (5.) HFD with UVR and physical activity (UVR + PA). A GTT was conducted after 10 weeks post-intervention, with blood glucose levels (*P < 0.05, comparing LFD with HFD) and area under the curve (AUC) shown in (A) (n = 12/treatment). After 12 weeks of treatment, livers were histopathologically assessed. In (B) representative sections (×20 oil immersion objective, with white scale bar = 30 µm) are shown for liver specimens stained with H&E (left) or Masson’s Trichrome (right) obtained from mice from each treatment. Livers were histopathologically assessed following staining with H&E or Masson’s Trichrome with a combined score for steatosis (yellow arrow in B), hepatocellular ballooning and fibrosis (blue arrow in B) shown in (C), and in (D) for steatosis only. Data in (E) are for liver sections scored for the presence of inflammatory foci (H&E, red arrow in B). Data in (C, D and E) are shown as mean + s.d. (n = 10/treatment); *P < 0.05 (one-way ANOVA with Tukey’s post hoc).






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C57Bl/6J mice housed communally (Geldenhuys et al. 2014, Fleury et al. 2017) and implies that beneficial effects of UVR may be linked to thermoregulation and/or animal interactions post-exposure. However, housing mice as individuals likely limited the capacity of the high-fat diet to induce metabolic dysfunction, and no significant increases in fasting glucose or insulin or signs of insulin resistance were observed. Furthermore, mice housed as individuals fed with the high-fat diet attained a reduced body weight (10% lower) compared to the co-housed mice of our previous studies (Geldenhuys et al. 2014). Even so, combining exposure to UVR with running wheel access prevented increases in adiposity (specifically, body weight and hepatic steatosis) in mice housed as individuals.

Although no significant effects of UVR were observed on distance run, it is possible that increased nitric oxide bioavailability following exposure to UVR could mediate improvements in physical activity performance. Physical activity increases the bioavailability of nitric oxide and is linked to its cardioprotective effects (Calvert 2011). Similarly, we previously observed that the same low doses of UVR (1 kJ/m2 UVB) ‘normalised’ blood nitrite levels in mice fed with a high-fat diet (Dhamrait et al. 2020). This dose of UVR also increased dermal concentrations

of nitric oxide 5 min after a single exposure (Geldenhuys et al. 2014). Nitric oxide release from skin is multi-phasic and involves a range of cells, including keratinocytes and endothelial cells (Holliman et al. 2017). This may allow for sustained release of nitric oxide potentially mediating improvements in physical activity performance. Increased sun exposure did not modify exercise performance in male military recruits (compared to placebo controls) (Carswell et al. 2018). However, exposure to UVA (315–400 nm) radiation in combination with nitrate supplementation (but not UVA alone) improved time trial performances achieved by cyclists immediately after exposure (Muggeridge et al. 2015). Additional dietary nitrate may provide optimal skin stores for release of nitric oxide bioactivity following exposure to UVR. Other potential mediators of the combined effects of UVR and physical activity could include those involved in regulating the hypothalamic-pituitary-adrenal axis and other neuroendocrinological pathways. Like physical activity (Chen et al. 2017), exposure to UVR can induce systemic (and dermal) synthesis of neuro-regulatory molecules (e.g. corticotropin-releasing hormone, neuropeptides, β-endorphin and corticosterone) to potentially affect the brain’s capacity to centrally control homeostasis (Gorman et al. 2017, Slominski et al. 2018).

Table 4 Lipid and cytokine levels in serum.

Treatment groupa

Total cholesterol (mmol/L) (n = 9–10)

HDL-cholesterol (mmol/L) (n = 9–10)

LDL-cholesterol (mmol/L) (n = 9–10)

Triglyceride (mmol/L) (n = 9–10) IL-6d (pg/mL) (n = 4)

LFD 2.3 ± 0.1b 1.6 ± 0.1 0.10 ± 0.01 1.1 ± 0.1 27.2 ± 1.5HFD 3.5 ± 0.1c 2.1 ± 0.1c 0.17 ± 0.01c 1.2 ± 0.1 36.5 ± 4.5UVR 3.4 ± 0.1c 2.1 ± 0.1c 0.18 ± 0.01c 0.9 ± 0.2 34.7 ± 4.5PA 3.2 ± 0.2c 2.0 ± 0.1c 0.16 ± 0.01c 0.9 ± 0.1 32.3 ± 3.9UVR + PA 3.4 ± 0.1c 2.1 ± 0.1c 0.18 ± 0.02c 1.2 ± 0.2 37.9 ± 5.6

aFour-week old male C57Bl/6J mice were fed a low-fat diet (LFD) for 4 weeks, until 8 weeks of age. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (1 kJ/m2 UVB radiation, twice a week; UVR); (4.) HFD with access to running wheels (2 h, twice a week; PA); or (5.) HFD with UVR followed by access to running wheels (UVR + PA). Serum was obtained at the end of the experiment (after 12 weeks of treatments). bAll data are presented as mean ± s.d. (n = 9–10 per treatment) and compared using a one-way ANOVA with Tukey’s post hoc analysis, cP < 0.05 compared to LFD. dThe lower limit of detection for the IL-6 and TNF assays were 15 and 8 pg/mL, respectively.

Table 5 Serum levels of 25-hydroxvitamin D (25(OH)D), calcium, phosphate, creatinine and albumin.

Treatment groupa

25 (OH)D (nmol/L) (n = 6)

Calcium (mmol/L) (n = 10)

Phosphate (mmol/L) (n = 10)

Creatinine (µmol/L) (n = 10)

Albumin (g/L) (n = 10)

LFD 0.0 ± 0.0b 2.00 ± 0.06 2.06 ± 0.11 29.2 ± 1.1 23.9 ± 0.4HFD 36.2 ± 3.1c 2.12 ± 0.03 2.16 ± 0.11 28.3 ± 0.8 24.8 ± 0.3UVR 34.0 ± 2.0c 2.01 ± 0.06 2.12 ± 0.12 29.2 ± 1.5 23.5 ± 0.6PA 38.4 ± 1.5c 2.08 ± 0.05 2.14 ± 0.11 29.2 ± 1.2 24.3 ± 0.2UVR + PA 36.5 ± 2.8c 2.10 ± 0.08 2.43 ± 0.12 30.8 ± 1.2 24.2 ± 0.5

aFour-week old male C57Bl/6J mice were fed a low-fat diet (LFD) for 4 weeks, until 8 weeks of age. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (1 kJ/m2 UVB radiation, twice a week; UVR); (4.) HFD with access to running wheels (2 h, twice a week; PA); or (5.) HFD with UVR followed by access to running wheels (UVR + PA). Serum was obtained at the end of the experiment (after 12 weeks of treatments). bAll data are presented as mean ± s.d. (n = 6–10 per treatment) and compared using a one-way ANOVA with Tukey’s post hoc analysis, cP < 0.05 compared to LFD.








Figure 3High-fat diet increased membrane potential of mitochondria in iBAT. Four-week old male C57Bl/6J male mice were fed a low-fat diet (LFD) for 4 weeks. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (UVR); (4.) HFD with physical activity (PA); or (5.) HFD with UVR and physical activity (UVR + PA). After 12 weeks of treatment, mitochondrial content (green staining) and membrane potential (orange staining) was determined in iBAT, with DAPI used to identify cell nuclei. Representative three colour merged images of iBAT from mouse of each treatment (×40 objective, with scale bar = 20 µm) are shown. ImageJ analysis was used to quantify fluorescence, with mitochondrial content and membrane potential shown for 8–10 mice/treatment. Data are shown as mean + s.d.; *P < 0.05 via one-way ANOVA with Tukey’s post hoc.

Figure 4Exposure to UVR or physical activity increased macrophages proportions in iBAT. Four-week old male C57Bl/6J male mice were fed a low-fat diet (LFD) for 4 weeks. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (UVR); (4.) HFD with physical activity (PA); or (5.) HFD with UVR and physical activity (UVR + PA). After 12 weeks of treatment, macrophages (F4-80+ MHC classII+ cells) were identified in vascular stromal cells isolated from iBAT using flow cytometry. In (A), forward and side scatter properties of the vascular stromal cells from a representative iBAT sample (a mouse fed the LFD), and the gating strategy used to identify macrophages, comparing cells stained for surface expression of F4-80, MHC class II, CD11c, CD86, CD11b and CD301, with isotype controls. In (B), the proportions of macrophages (F4-80+ MHC class II+ cells) and the geometric mean fluorescence intensity of CD11c, CD86, CD11b and CD301 detected on these cells. Data are shown as mean + s.d. (n = 10/treatment; *P < 0.05, one-way ANOVA with Tukey’s post-hoc).






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Mice allowed running wheel access had significantly reduced adipose tissue depot weights and increased food intake compared to controls fed a high-fat diet. Most studies point towards similar findings of reduced adiposity and increased or no change in food intake with similar ‘moderate’ (short) running wheel access protocols (reviewed by Cordeira & Monahan 2019). Physical activity may alter a deranged fat metabolism induced by eating a high-fat diet by improving lipolysis (Higa et al. 2014), fat mobilisation (Thompson et al. 2012) and fatty acid oxidation (Laye et al. 2009) in WAT to expend rather than store fatty acids. Combined together, UVR and physical activity reduced expression of the M1 marker, CD11c, on macrophages in iBAT. Reduced CD11c expression in s.c. WAT (but not iBAT) was also observed in ovariectomized rats with low aerobic fitness undertaking voluntary wheel running (Zidon et al. 2018). We also observed increases in Atgl, Cd36, Fasn, Igf1, Pparγ and Ucp1 mRNAs in iBAT of mice from the UVR and physical activity treatment, suggesting that related fatty acid transport, insulin-signalling, lipogenesis, lipolysis and thermogenesis pathways may be upregulated by combining these treatments. Increased Sglt2 mRNA in kidneys of mice exposed to UVR and running wheels may point towards modulation of glucose resorption pathways in the kidney and potentially an increased requirement for glucose by muscle, liver and other tissues (DeFronzo et al. 2012). We have shown that oral gavage with dapagliflozin, an SGLT-2 inhibitor, increased SGLT-2 protein expression in proximal tubule cells of the kidneys of mice fed with a high-fat diet and may be a compensatory response to

SGLT-2 inhibition (Matthews et al. 2017). However, it will be necessary to confirm all mRNA changes by measuring protein and perform additional experiments that measure markers of whole-body energy expenditure (e.g. oxygen utilization, CO2 production), tissue respiration, insulin-signalling and the activity of mitochondrial complexes in iBAT and muscle using freshly isolated tissue, cultured cells and/or post acute stimulation with insulin in vivo (Tsuchida et al. 2001). To better understand the mechanism by which UVR and physical activity combine to modulate metabolism, a time course experiment might be considered, in which tissue is sampled throughout the experiment period, at both early and later times, as changes in insulin signalling and mitochondrial activity may precede other effects (e.g. Ucp1 expression).

Clinical trials suggest that vitamin D supplementation is of limited benefit for reducing adiposity (Chandler et al. 2015, Golzarand et al. 2018). In our previous studies, vitamin D supplementation reduced the extent of hepatic steatosis but did not affect weight gain and other markers of adiposity in mice fed a high-fat diet (Geldenhuys et al. 2014). Many epidemiological studies report inverse correlations between blood 25(OH)D levels and BMI (Walsh et al. 2017). These observations may be explained by multiple hypotheses, including that: vitamin D accumulates in adipose tissue; blood 25(OH)D levels are volumetrically diluted with increasing BMI; and/or that sun exposure is reduced in individuals with a higher BMI (reviewed in Walsh et al. 2017). Other studies suggest that physical activity may mobilise vitamin D from adipose tissue (Hengist et al. 2019). Serum 25(OH)D levels were

Figure 5Low-dose UVR and access to running wheels increased Ucp-1, Fasn, Atgl, Igf1, CD36 and Pparγ mRNA in iBAT of mice fed with a high-fat diet (individually housed). Four-week old male C57Bl/6J male mice were fed with a low-fat diet (LFD) for 4 weeks. At 8 weeks of age, mice were separated into one of five treatment groups: (1.) LFD; (2.) High-fat diet (HFD); (3.) HFD with UVR (UVR); (4.) HFD with physical activity (PA); or (5.) HFD with UVR and physical activity (UVR + PA). After 12 weeks of treatment, mRNAs were quantified in iBAT via real-time PCR, specifically those for Ucp1, Pgc1α, Dio2, Fasn, Atgl, Igf1, Cd36, Pparγ, Bmp7, Glut4, Tnf, Fgf21, Fatp2 and Igfbp2. Data are shown as mean + s.d. (n = 9/treatment); *P < 0.05 (one-way ANOVA with Tukey’s post-hoc or Kruskal–Wallace with Dunn’s post hoc analysis).








increased in young adults up to 24 h after an acute endurance exercise (Sun et al. 2017). There is also evidence for uptake and retention of 25(OH)D in muscle (Abboud et al. 2013). We did not observe significant changes in blood 25(OH)D levels in mice frequently exposed to UVR (consistent with previous studies (Geldenhuys et al. 2014)) or with running wheel access. Male mice have reduced 7-dehydrocholesterol levels in skin and increased expression of enzymes involved in the catabolism of 25(OH)D (Gorman et al. 2012). Further studies are needed to determine whether similar sex-dependent processes may prevent changes in blood 25(OH)D that could otherwise be regulated by increasing physical activity in these male mice.

In addition to the ethical requirement to cage mice in the current study as individuals, other limitations include no measurement of incidental physical activity and that mice were housed at temperatures likely below thermoneutrality (at 22°C). Speakman & Keijer suggest that for thermoneutrality, mice caged as individuals should be housed at 23–25°C (Speakman & Keijer 2012). Mice are also nocturnal and furry animals and when free-living are unlikely to be exposed to (much) UVR.

In conclusion, low-dose UVR administered to mice allowed access to running wheels which did not significantly affect distance run or food intake. In mice fed with a high-fat diet, signs of adiposity were lowest in mice both exposed to UVR and allowed running wheel access and linked with reduced expression of an M1-type marker (CD11c) on F4-80 + MHC ClassII+ cells, and, increased Atgl, Cd36, Fasn, Igf1, Pparγ, and Ucp1 mRNA in iBAT. These findings suggest that metabolic benefits of physical activity may be enhanced by frequent and controlled exposure to low-dose (non-burning) UVR such as that found in sunlight, with future clinical studies needed to determine how these preclinical observations are relevant to people.

Supplementary materialsThis is linked to the online version of the paper at https://doi.org/10.1530/JOE-19-0470.

Declaration of interestThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

FundingThis research was supported by the Diabetes Research Foundation of Western Australia and the Telethon Kids Institute. RML is supported by a NHMRC Senior Research Fellowship. SG is funded by an Al and Val

Rosenstrauss Research Fellowship from the Rebecca L Cooper Medical Research Foundation.

Author contribution statementS G conceived and designed this study with input from T S A and all authors. T S A acquired and analysed the data for the study with help from S G. T N A performed the confocal microscopy data acquisition and analyses with help from S G. All authors have contributed towards the interpretation of findings from this study, have played a role in drafting the article or revising it critically for its intellectual content and have given their final approval for this version of the paper to be published.

AcknowledgementsThe authors thank the Bioresources staff at the Telethon Kids Institute for day-to-day care of experimental animals; Dr Emily Barrick (Telethon Kids Institute) for animal welfare; Mr Luke Berry (Telethon Kids Institute) for help preparing the histological liver sections; Ms Linda Gregory and Ms Andriene Cheen (PathWest Laboratory at Fiona Stanley Hospital, Murdoch) for performing serum cholesterol, triglyceride, phosphate, creatinine and calcium assays; A/Prof Michael Clarke (Centre for Metabolomics, University of Western Australia) for measuring serum 25(OH)D levels. The authors also gratefully acknowledge Bright Blue for donation of the Nikon C2 Confocal Microscope. The authors thank Professor Rebecca Mason (University of Sydney), Associate Professor Nathan Pavlos (University of Western Australia), and Dr Joshua Lewis (University of Western Australia) for their advice on measurement and interpretation of blood creatinine.

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Received in final form 9 December 2019Accepted 6 January 2020Accepted Manuscript published online 6 January 2020




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