Abstract
Obesity is classically associated with low serum total and free 25(OH)D. Hypotheses have been advanced to explain this observation but mechanisms remain poorly understood, and notably priming events that could explain such association. We investigated the impact of short-term high fat (HF) diet to investigate early events occurring in vitamin D metabolism. Male C57BL/6J mice were fed with a control diet (control group) and HF diet for 4 days. HF fed mice displayed similar body weight to control mice but significantly increased adiposity, together with a decrease of free 25(OH)D concentrations, which could be explained at least in part by a decrease of Cyp2r1 and Cyp3a11 expression in the liver. An increase of 1,25(OH)2D concentration was also observed and could be explained by a decrease of Cyp24a1 expression observed in the kidney. In white adipose tissue (WAT), no modification of vitamin D metabolites quantity detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nevertheless, an increase of Cyp2r1 and Cyp27a1 mRNA expression and a decrease of Cyp27b1 mRNA expression could suggest a possible storage of 25(OH)D in WAT at long-term. Our data are supportive of an active role of HF diet in mediating a priming effect leading the well-established perturbation of the vitamin D metabolism associated with obesity, including a decrease of free 25(OH)D and modulation of expression of genes involved in vitamin D metabolism.
Introduction
Low levels of total circulating 25-hydroxy-vitamin D (25(OH)D) are strongly associated with obesity and more specially with increased fat mass and BMI (Vilarrasa et al. 2007, Earthman et al. 2012, Landrier et al. 2016). Recently it has been shown that the free form of 25(OH)D was also low in obesity (Walsh et al. 2016), as well as 1,25 dihydroxy-vitamin D (1,25(OH)2D). Several hypotheses have been emitted to explain the observations. Bell et al. reported that hepatic 25-hydroxylation was inhibited by 1,25(OH)2D and parathyroid hormone (PTH) (Bell 1985). Wortsman et al. suggested a possible sequestration of vitamin D in white adipose tissue (WAT) caused by a passive phenomenon due to the hydrophobic nature of vitamin D (Wortsman et al. 2000). Drincic et al. proposed that 25(OH)D was simply diluted in a higher volume in obese and hypothesized a volumetric dilution (Drincic et al. 2012). Indeed, these authors showed an inverse correlation between plasma 25(OH)D concentration and total volume (lean and fat mass), which could be the best predictive marker than fat mass. Wamberg et al. suggested that vitamin D metabolism in WAT is modified by obesity and reported a modification of Cyp2j2 expression in biopsies of obese compared to lean patients (Wamberg et al. 2013).
In mice, Park et al. reported that high fat diet-induced obesity influenced vitamin D metabolizing enzymes expression in liver, kidney and WAT, which could provide a possible mechanism for altered vitamin D metabolism in obesity (Park et al. 2015). Roizen et al. and Aatsinki et al. recently reported an impact of a high fat diet and obesity/diabetes on hepatic 25 hydroxylase activity in mice liver, that could explain the low 25(OH)D level (Aatsinki et al. 2019, Roizen et al. 2019). In addition, we showed recently that adipose tissue could play an important active role in the modulation of vitamin D metabolism observed during obesity. Indeed, we found a decrease of free 25(OH)D, strongly associated with the induction of Cyp2r1 in adipose tissue, which could be responsible for the active production and storage of 25(OH)D highlighted in adipose tissue by direct quantification (Bonnet et al. 2019a).
It is noteworthy that all these observations have been made in obese mice models or in obese subjects and it is presently unclear if modulation of expression of genes involved in vitamin D metabolism have a priming effect in vitamin D metabolites modifications (total and free 25(OH)D and 1,25(OH)2D), or are just consequences of obesity.
The objective of this study was to identify early events that could explain the modulation of 25(OH)D (total and/or free) and 1,25(OH)2D levels observed in obesity. To this aim, a short term high fat diet (4 days) was implemented to initiate obesity and to study early modifications of vitamin D status. In parallel, a profile of expression of genes coding for enzymes involved in vitamin D metabolism was established in the liver, kidney and WAT in order to bring mechanistic keys.
Materials and methods
Reagents
TRIzol reagent, random primers, and Moloney murine leukemia virus reverse transcriptase were obtained from Life Technologies (Courtaboeuf, France). SYBR Green reaction buffer was purchased from Eurogentec (Liege, Belgium).
Animal experiments
The protocol received the agreement of the local ethics committee and by the French Ministry of Research (APAFIS#2595-2016091911217758). Six-week-old male C57BL/6J mice were obtained from Janvier (Le Genest Saint Isle, France), fed with control food (chow diet A04 from Safe-diets) ad libitum during the 1-week acclimation period and with full access to drinking water. The animals were maintained at 22°C under a 12 h light:12 h darkness cycle a 20% humidity level. Mice were divided into control diet group (control: chow diet A04 from Safe-diets, n = 10) or high fat diet group (HF group: 60% energy from lipids from Test Diet 58Y1, n = 10). The cholecalciferol concentration was 900 UI/kg for the control diet and 1300 UI/g for the high fat diet. The daily food intake of mice has been determined by giving them a known (i.e. pre-weighed) amount of food in their cage and weigh the remaining food once per day. We used adapted containers that limit the risk of spillage and hoarding. The intake of cholecalciferol is calculated by multiplying the food intake but the concentration of cholecalciferol in the food. After 4 days, mice were fasted overnight and blood was collected by cardiac puncture anesthesia, serum was isolated by centrifugation at 350 g for 15 min at 4°C and was stored at −80°C. Cervical dislocation was used to scarify animals and tissue (kidney, liver and WAT (epididymal (eWAT), subcutaneous and retroperitoneal pads)) were collected, weighted and stored at 80°C.
Free 25(OH)D, albumin and VDBP quantification
To determinate the free 25(OH)D and VDBP quantity in mice plasma, specific ELISA kits were used (mice free form of 25(OH)D, DIAsources, Louvain-La-Neuve, Belgique, Mouse VDBP DuoSet ELISA, R&S systems, Minneapolis, USA, respectively) according to manufacturer protocol. The direct assay for free 25(OH)D has been recently validated (Heureux et al. 2017). Albumin concentration in plasma was determined using BCG method (Biolabo, Maizy, France). The free 25(OH)D calculation was performed as previously reported (Bikle et al. 2017).
Cholecalciferol, 25(OH)D and 1,25(OH)2D quantification in plasma and eWAT
All quantifications were performed using LC-MS/MS according to the protocol as previously reported (Bonnet et al. 2018, 2019b) and detailed in Supplementary data (see section on supplementary materials given at the end of this article).
RNA extraction and real-time PCR
Total RNA was extracted from the liver, kidney and eWAT or from cells using TRIzol reagent (Life Technologies). One microgram of total RNA was used to synthetize cDNAs using random primers and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Courtaboeuf, France). Real-time quantitative PCR analyses were performed using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, USA) as previously described (Karkeni et al. 2017a). For each condition, expression was quantified in duplicate, and 18S rRNA was used as the endogenous control in the comparative cycle threshold (CT) method (Livak & Schmittgen 2001). The sequences of the primers used in this study are reported in Supplementary Table 1.
Statistical analysis
The data are communicated as the mean ± s.e.m. Significant differences between the control and treated group were determined using Student’s t-test with P < 0.05 was considered statistically significant.
Results
Morphological data of 4 days high fat diet fed mice
The effect of a high fat diet (60% energy from fat, HF group) compared to control diet (control group) was evaluated in WT C57BL/6J male mice for 4 days. Body weight and liver weight were not modified by the diet (Table 1). Adiposity index (sum of epididymal, inguinal and retroperitoneal adipose tissue mass relative to total body mass), as well as fat pads weight of subcutaneous, epididymal and peritoneal WAT and ratio of organ, weigh on body weight of these three organs, were increased in HF group (1.58-fold for adiposity index, Table 1). Energy intake was increased in HF group (1.2-fold, Table 1). No difference was observed in vitamin D intake between groups (Table 1).
Mice morphologic parameters.
| Control | HF | |
|---|---|---|
| Body weight (g) | 21.45 ± 0.28 | 22.06 ± 0.45 |
| Liver (mg) | 875 ± 28.7 | 850 ± 29.59 |
| Subcutaneous AT (mg) | 36.3 ± 5.06 | 71 ± 3.7a |
| Epididymal AT (mg) | 187.4 ± 17.7 | 282.2 ± 18.2a |
| Retroperitoneal AT (g) | 31.7 ± 3.9 | 63.7 ± 5.5a |
| Adiposity index | 1.185 ± 0.10 | 1.87 ± 0.09a |
| Energy intake (kcal/g) | 14.38 ± 0.44 | 17.28 ± 0.40a |
| Vitamin D intake (UI/g/day) | 4.46 ± 0.24 | 4.52 ± 0.08 |
Values are presented as means ± s.e.m. For unpaired Student’s t-test.
a P < 0.05 between control and high fat group (HF) for each condition.
Plasma concentrations of cholecalciferol and metabolites parameters of 4 days high fat diet fed mice
Plasma cholecalciferol concentration was decreased in HF group compared to control group (0.68-fold, Table 2) as well as measuredplasma free form of 25(OH)D concentration (0.31-fold, Table 2) and VDBP plasma level (0.3-fold, Table 2). Surprisingly the calculated free 25(OH)D was increased in HF group compared to control group (1.4-fold, Table 2). Conversely, no difference was observed in plasma total of 25(OH)D concentrations between control group (27.9 ± 0.9 ng/mL) and HF group (29.9 ± 0.9 ng/mL). Plasma 1,25(OH)2D concentration was increased in HF group (2.78-fold, Table 1). Interestingly, we observed a negative correlation between measured plasma free 25(OH)D concentrations and adiposity index (R² = 0.4002, P = 0.0085; Fig. 1E) and a positive correlation between adiposity index and plasma 1,25(OH)2D concentrations (R² = 0.335, P = 0.012; Fig. 1G).
Relationship between adiposity index, body weight and serum concentration of vitamin D metabolites in mice fed with high fat diet. Relationship between cholecalciferol and adiposity index (A) or body weight (B), total 25(OH)D and adiposity index (C) or body weight (D), free 25(OH)D and adiposity index (E) or body weight (F), 1,25(OH)2D and adiposity index (G) or body weight (H), in mice fed with control or high fat diet. For unpaired Student’s t-test, one-tailed probability.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Relationship between adiposity index, body weight and serum concentration of vitamin D metabolites in mice fed with high fat diet. Relationship between cholecalciferol and adiposity index (A) or body weight (B), total 25(OH)D and adiposity index (C) or body weight (D), free 25(OH)D and adiposity index (E) or body weight (F), 1,25(OH)2D and adiposity index (G) or body weight (H), in mice fed with control or high fat diet. For unpaired Student’s t-test, one-tailed probability.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Relationship between adiposity index, body weight and serum concentration of vitamin D metabolites in mice fed with high fat diet. Relationship between cholecalciferol and adiposity index (A) or body weight (B), total 25(OH)D and adiposity index (C) or body weight (D), free 25(OH)D and adiposity index (E) or body weight (F), 1,25(OH)2D and adiposity index (G) or body weight (H), in mice fed with control or high fat diet. For unpaired Student’s t-test, one-tailed probability.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Plasma and epididymal white adipose tissue vitamin D metabolites and VDBP quantification.
| Control | HF | |
|---|---|---|
| Plasma cholecalciferol concentration (ng/mL) | 1.196 ± 0.148 | 0.387 ± 0.098a |
| Plasma total 25(OH)D concentration (ng/mL) | 27.986 ± 0.998 | 29.987 ± 0.925 |
| Plasma free 25(OH)D concentration (pg/mL) | 10.879 ± 0.48 | 7.46 ± 0.60a |
| Plasma 1,25(OH)2D concentration (ng/mL) | 0.548 ± 0.051 | 1.524 ± 0.137a |
| Plasma VDBP quantification (µg/mL) | 114 ± 10.6 | 83 ± 8.5a |
| Plasma albumin concentration (g/dL) | 3.85 ± 0.09 | 5.46 ± 0.28a |
| Calculated free 25(OH)D (pg/mL) | 2.43 ± 0.14 | 3.48 ± 0.21a |
| eWAT cholecalciferol quantity (ng) | 174.1 ± 28.6 | 122.8 ± 12.5 |
| eWAT 25(OH)D quantity (ng) | 13.1 ± 1 | 15.3 ± 0.8 |
| eWAT 1,25(OH)2D quantity (ng) | 14.1 ± 1.4 | 17.7 ± 2.5 |
| eWAT cholecalciferol concentration (ng/g of tissue) | 949.2 ± 193.3 | 471.9 ± 73.9a |
| eWAT 25(OH)D concentration (ng/g of tissue) | 76 ± 6.1 | 57.1 ± 3.5a |
| eWAT 1,25(OH)2D concentration (ng/g of tissue) | 68.4 ± 6.8 | 63.1 ± 7.8 |
Values are presented as means ± s.e.m. For unpaired Student’s t-test.
aP < 0.05 between control and high fat group (HF) for each condition.
Effect of a 4 days high fat diet on expression of genes coding for enzymes of the vitamin D metabolism in mice
Expression of genes coding major actors of vitamin D metabolism were studied in the liver, kidney and eWAT of mice (all data presented in supplemental Table 2). We observed a significant decrease of mRNA expression of Cyp2r1 and Cyp3a11, involved in 25-hydroxylation (0.15 and 0.76-fold, respectively, Fig. 2A), in the liver. In kidney, a decrease of Cyp24a1 and Cyp27b1 expression decreased in HF group (0.42 and 0.34-fold, respectively, Fig. 2B). In eWAT, relative RNA expression of Cyp27a1 and Cyp2r1 were significantly increased in HF group compared to control group (1.48- and 1.98-fold, respectively, Fig. 2C). Conversely, expression of Cyp27b1 was decreased in HF group (0.78, Fig. 2C).
Effect of high fat diet for 4 days on gene expression of vitamin D metabolism in eWAT in mice. Expression of genes coding proteins of vitamin D metabolism relative to 18S ribosomal RNA in liver (A), kidney (B) and eWAT (C) of mice fed with control diet (control) or high fat diet 60% (HF) for 4 days (n = 10 per group). Values are presented as means ± s.e.m. For unpaired Student’s t-test, *P < 0.05.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Effect of high fat diet for 4 days on gene expression of vitamin D metabolism in eWAT in mice. Expression of genes coding proteins of vitamin D metabolism relative to 18S ribosomal RNA in liver (A), kidney (B) and eWAT (C) of mice fed with control diet (control) or high fat diet 60% (HF) for 4 days (n = 10 per group). Values are presented as means ± s.e.m. For unpaired Student’s t-test, *P < 0.05.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Effect of high fat diet for 4 days on gene expression of vitamin D metabolism in eWAT in mice. Expression of genes coding proteins of vitamin D metabolism relative to 18S ribosomal RNA in liver (A), kidney (B) and eWAT (C) of mice fed with control diet (control) or high fat diet 60% (HF) for 4 days (n = 10 per group). Values are presented as means ± s.e.m. For unpaired Student’s t-test, *P < 0.05.
Citation: Journal of Endocrinology 248, 1; 10.1530/JOE-20-0198
Quantification of vitamin D and metabolites in eWAT of 4 days high fat diet fed mice
Vitamin D and metabolites were quantified in eWAT by LC-MS/MS. No difference of 1,25(OH)2D concentration was observed whereas concentrations of cholecalciferol and 25(OH)D were decreased in HF group (0.5- and 0.25-fold, respectively, Table 2). Quantities of cholecalciferol, 25(OH)D and 1,25(OH)2D in eWAT were not modified by the short-term high fat diet (Table 2).
Discussion
In this present study, we report for the first time the effect of a short-term high fat diet on vitamin D and metabolites plasma and eWAT levels as well as expression of genes involved in vitamin D metabolism in the liver, kidney and WAT.
In order to mimic a food imbalance state associated with weight gain, we have used a short-time (4 days) high fat diet. This model allows to study the early effects of an unbalanced diet on weight gain and/or fat mass gain (Voigt et al. 2013). It is thus used to study the causal events that are concomitant with the initiation of weight gain, in contrast to a classical model of high fat diet-induced (generally 10 weeks or more) which mainly reflect consequences of weight gain. As expected, this diet did not affect body weight but increased adiposity index of HF fed mice.
It is also important to note that the two diets used in this protocol brought the same quantity of cholecalciferol, which is a crucial point for this experiment. To this aim, we increased cholecalciferol content of HF diet, to account for the lower food consumption that occurs during HF feeding. Despite similar vitamin D supplementation it is intriguing that cholecalciferol plasma levels were strongly reduced in HF group. The origin of such discrepancy is presently not well understood, but could be due to a reduced ability of intestine to absorb cholecalciferol, as a consequence of deregulation of proteins involved in vitamin D uptake (Reboul et al. 2011), such assumption will require further investigations.
In these experimental conditions, we did not observe any modification of the total 25(OH)D serum levels, which is consistent with previous reports that adjusted the cholecalciferol content in chow and HF diets (Park et al. 2015, Seldeen et al. 2017, Bonnet et al. 2019a). Such observation is not in the favour of the classical hypothesis of passive sequestration proposed by Wortsman et al. (2000) which suggests that the 25(OH)D plasma decrease observed during obesity is the direct result of adipose tissue expansion, since we observed an increase of adiposity index without modification of total 25(OH)D concentration.
In HF group, we observed an increase of serum 1,25(OH)2D concentration compared to control group, that was correlated to adiposity. Such increase has already been reported (Park et al. 2015), and could be linked to the downregulation of Cyp24a1 in kidney of HF group, leading to a lower catabolism of 1,25(OH)2D, which in turn lead to the decrease of Cyp27b1 mRNA levels in HF fed mice kidneys through a well-established negative feedback mechanism (Bikle et al. 1975, 1986). Interestingly 1,25(OH)2D has been demonstrated to repress hepatic 25-hydroxylation (Bell et al. 1984). Such observation is consistent with the observed decrease of Cyp2r1 and Cyp3a11 mRNA levels in the liver of HF fed mice. Note that similar hepatic repression of Cyp2r1 and Cyp3a11 have previously been reported in long-term HF diets (Park et al. 2015, Aatsinki et al. 2019, Roizen et al. 2019). Surprisingly, the downregulation of Cyp2r1 mRNA levels is not strictly associated with reduction of 25(OH)D plasma levels as reported by Park et al. (2015) and the present study, whereas Aatsinki et al. (2019) and Roizen et al. (2019) reported a decrease in 25(OH)D plasma level in parallel to a strong decrease of Cyp2r1. The origin of such discrepancy is presently not understood, but could be due to the different types of regimens used and will deserve further investigations. Concerning our experimentation, we have to keep in mind that only mRNA levels were reported and the decrease was relatively low notably for Cyp2r1, whereas Roizen et al. and Aatsinki et al. reported stronger downregulation of the Cyp2r1 mRNA levels (Aatsinki et al. 2019, Roizen et al. 2019). Nevertheless, our observation suggests that the downregulation of Cyp2r1 could precede the decrease of the 25(OH)D level and is thus in agreement with a causal role of HF diet on Cyp2r1 expression with consequences on 25(OH)D levels.
Even if we did not observe modification of the total 25(OH)D, we reported an impact of short-term HF diet on the VDBP plasma level and liver mRNA coding for VBDP (Gc mRNA). Such effect was not observed in a recently published article (Elkhwanky et al. 2020) but could be due not to the reduction of total 25(OH)D as suggested (Carpenter et al. 2013), but to the reduction of free 25(OH)D observed. This last assumption will require further investigations. In addition, we reported a reduced measured free 25(OH)D plasma level under short term HF diet, as previously reported in long-term HF diet (Bonnet et al. 2019a), and in agreement with published data in obese subjects (Walsh et al. 2016). Surprisingly, when calculating the free 25(OH)D, we did not found a correlation between measured and calculated free 25(OH)D as previously reported (Bikle et al. 2017). This observation could be due to the fact that such correlations were observed in normal subjects. We cannot exclude that in other pathophysiological situations (including high fat diet), affinity constants of VDBP and/or albumin regarding 25(OH)D are modified resulting in erroneous calculations. Such assumption will require further experimentations. Nevertheless, it is noteworthy that such reduction of measured free 25(OH)D could participate in the amplification of obesity and/or metabolic inflammation (Marcotorchino et al. 2012, 2014, Karkeni et al. 2015, 2017b, Marziou et al. 2020). Interestingly the reduction of free 25(OH)D was inversely correlated to adiposity but not to body weight, suggesting a specific role of adipose tissue accretion in the level of free 25(OH)D. In agreement, we reported in eWAT of HF fed mice, an increase of mRNA levels of two enzymes involved in 25-hydroxylation, Cyp2r1 and Cyp27a1 (Schuster 2011). This data related to the expression of Cyp2r1, which encodes a major enzyme of 25-hydroxylation (Zhu et al. 2013), under short-term effect of HF diet is innovative. Similarly to data generated in long-term HF diet, it suggests an increased ability of adipose tissue to store 25(OH)D due to the enhanced expression of 25-hydroxylation enzymes (Wamberg et al. 2013, Park et al. 2015, Bonnet et al. 2019a). In addition, mRNA expression of Cyp27b1 was decreased in HF group, suggesting that at term 1,25(OH)2D will decrease locally, even if not yet observed (no modification of quantity and concentration of 1,25(OH)2D in eWAT). Altogether, these data suggested that short-term HF diet induces the ability of adipose tissue to synthesis and to store 25(OH)D. Nevertheless, such assumption is not yet supported by the LC-MS/MS vitamin D metabolites quantifications in eWAT. Indeed, no cholecalciferol and metabolites quantity modifications were observed between control and HF groups, whereas cholecalciferol and 25(OH)D concentration decreased in HF group. Such decrease of concentration is simply due to the increase of adipose tissue quantity in absence of metabolite quantity modification. In fact, we hypothesized the modulation of genes expression reported in eWAT could precede the modulation of cholecalciferol and metabolites accumulations in eWAT. It could thus be considered as a priming event, induced by HF diet at an early stage, that could lead to the active storage of cholecalciferol and 25(OH)D in eWAT, via an active mechanism, as previously depicted (Bonnet et al. 2019a). Note that all quantification of gene expression and metabolites have been performed only in eWAT, which could be considered as a limitation to the present work, since we cannot exclude that other fat pads would have different responses.
To conclude, short-term HF is able to modulate vitamin D metabolism and vitamin D metabolites content in plasma and in eWAT. Our data are supportive of an active role of HF diet in mediating a priming effect leading the well-established perturbation of the vitamin D metabolism associated with obesity (a decrease of free 25(OH)D) and modulation of expression of genes involved in vitamin D metabolism (Cyp in the liver and in eWAT).
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/JOE-20-0198.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by INRAE, INSERM and AMU.
References
Aatsinki SM, Elkhwanky MS, Kummu O, Karpale M, Buler M, Viitala P, Rinne V, Mutikainen M, Tavi P & Franko A et al. 2019 Fasting-induced transcription factors repress vitamin D bioactivation, a mechanism for vitamin D deficiency in diabetes. Diabetes 68 918–931. (https://doi.org/10.2337/db18-1050)
Bell NH 1985 Vitamin D-endocrine system. Journal of Clinical Investigation 76 1–6. (https://doi.org/10.1172/JCI111930)
Bell NH, Shaw S & Turner RT 1984 Evidence that 1,25-dihydroxyvitamin D3 inhibits the hepatic production of 25-hydroxyvitamin D in man. Journal of Clinical Investigation 74 1540–154 4. (https://doi.org/10.1172/JCI111568)
Bikle DD, Murphy EW & Rasmussen H 1975 The ionic control of 1,25-dihydroxyvitamin D3 synthesis in isolated chick renal mitochondria. The role of calcium as influenced by inorganic phosphate and hydrogen-ion. Journal of Clinical Investigation 55 299–304. (https://doi.org/10.1172/JCI107933)
Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E & Haddad JG 1986 Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. Journal of Clinical Endocrinology and Metabolism 63 954–9 59. (https://doi.org/10.1210/jcem-63-4-954)
Bikle D, Bouillon R, Thadhani R & Schoenmakers I 2017 Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? Journal of Steroid Biochemistry and Molecular Biology 173 105–116. (https://doi.org/10.1016/j.jsbmb.2017.01.007)
Bonnet L, Karkeni E, Couturier C, Astier J, Dalifard J, Defoort C, Svilar L, Martin JC, Tourniaire F & Landrier JF 2018 Gene expression pattern in response to cholecalciferol supplementation highlights cubilin as a major protein of 25(OH)D uptake in adipocytes and male mice white adipose tissue. Endocrinology 159 957–966. (https://doi.org/10.1210/en.2017-00650)
Bonnet L, Hachemi MA, Karkeni E, Couturier C, Astier J, Defoort C, Svilar L, Martin JC, Tourniaire F & Landrier JF 2019a Diet induced obesity modifies vitamin D metabolism and adipose tissue storage in mice. Journal of Steroid Biochemistry and Molecular Biology 185 39–46. (https://doi.org/10.1016/j.jsbmb.2018.07.006)
Bonnet L, Margier M, Svilar L, Couturier C, Reboul E, Martin JC, Landrier JF & Defoort C 2019b Simple fast quantification of cholecalciferol, 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D in adipose tissue using LC-HRMS/MS. Nutrients 11. (https://doi.org/10.3390/nu11091977)
Carpenter TO, Zhang JH, Parra E, Ellis BK, Simpson C, Lee WM, Balko J, Fu L, Wong BYL & Cole DEC 2013 Vitamin D binding protein is a key determinant of 25-hydroxyvitamin D levels in infants and toddlers. Journal of Bone and Mineral Research 28 213–221. (https://doi.org/10.1002/jbmr.1735)
Drincic AT, Armas LA, Van Diest EE & Heaney RP 2012 Volumetric dilution, rather than sequestration best explains the low vitamin D status of obesity. Obesity 20 1444–144 8. (https://doi.org/10.1038/oby.2011.404)
Earthman CP, Beckman LM, Masodkar K & Sibley SD 2012 The link between obesity and low circulating 25-hydroxyvitamin D concentrations: considerations and implications. International Journal of Obesity 36 387–3 96. (https://doi.org/10.1038/ijo.2011.119)
Elkhwanky MS, Kummu O, Piltonen TT, Laru J, Morin‐Papunen L, Mutikainen M, Tavi P & Hakkola J 2020 Obesity represses CYP2R1, the vitamin D 25‐hydroxylase, in the liver and extrahepatic tissues. JBMR Plus 4 e10397. (https://doi.org/10.1002/jbm4.10397)
Heureux N, Lindhout E & Swinkels L 2017 A direct assay for measuring free 25-hydroxyvitamin D. Journal of AOAC International 100 1318–1322. (https://doi.org/10.5740/jaoacint.17-0084)
Karkeni E, Marcotorchino J, Tourniaire F, Astier J, Peiretti F, Darmon P & Landrier JF 2015 Vitamin D limits chemokine expression in adipocytes and macrophage migration in vitro and in male mice. Endocrinology 156 1782–17 93. (https://doi.org/10.1210/en.2014-1647)
Karkeni E, Bonnet L, Astier J, Couturier C, Dalifard J, Tourniaire F & Landrier JF 2017a All-trans-retinoic acid represses chemokine expression in adipocytes and adipose tissue by inhibiting NF-kappaB signaling. Journal of Nutritional Biochemistry 42 101–107. (https://doi.org/10.1016/j.jnutbio.2017.01.004)
Karkeni E, Bonnet L, Marcotorchino J, Tourniaire F, Astier J, Ye J & Landrier JF 2017b Vitamin D limits inflammation-linked microRNA expression in adipocytes in vitro and in vivo: a new mechanism for the regulation of inflammation by vitamin D. Epigenetics 13 156–162. (https://doi.org/10.1080/15592294.2016.1276681)
Landrier JF, Karkeni E, Marcotorchino J, Bonnet L & Tourniaire F 2016 Vitamin D modulates adipose tissue biology: possible consequences for obesity? Proceedings of the Nutrition Society 75 38–46. (https://doi.org/10.1017/S0029665115004164)
Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25 402–40 8. (https://doi.org/10.1006/meth.2001.1262)
Marcotorchino J, Gouranton E, Romier B, Tourniaire F, Astier J, Malezet C, Amiot MJ & Landrier JF 2012 Vitamin D reduces the inflammatory response and restores glucose uptake in adipocytes. Molecular Nutrition and Food Research 56 1771–17 82. (https://doi.org/10.1002/mnfr.201200383)
Marcotorchino J, Tourniaire F, Astier J, Karkeni E, Canault M, Amiot MJ, Bendahan D, Bernard M, Martin JC & Giannesini B et al. 2014 Vitamin D protects against diet-induced obesity by enhancing fatty acid oxidation. Journal of Nutritional Biochemistry 25 1077–10 83. (https://doi.org/10.1016/j.jnutbio.2014.05.010)
Marziou A, Philouze C, Couturier C, Astier J, Obert P, Landrier JF & Riva C 2020 Vitamin D supplementation improves adipose tissue inflammation and reduces hepatic steatosis in obese C57BL/6J mice. Nutrients 12 342. (https://doi.org/10.3390/nu12020342)
Park JM, Park CY & Han SN 2015 High fat diet-Induced obesity alters vitamin D metabolizing enzyme expression in mice. BioFactors 41 175–1 82. (https://doi.org/10.1002/biof.1211)
Reboul E, Goncalves A, Comera C, Bott R, Nowicki M, Landrier JF, Jourdheuil-Rahmani D, Dufour C, Collet X & Borel P 2011 Vitamin D intestinal absorption is not a simple passive diffusion: evidences for involvement of cholesterol transporters. Molecular Nutrition and Food Research 55 691–702. (https://doi.org/10.1002/mnfr.201000553)
Roizen JD, Long C, Casella A, O’Lear L, Caplan I, Lai M, Sasson I, Singh R, Makowski AJ & Simmons R et al. 2019 Obesity decreases hepatic 25-hydroxylase activity causing low serum 25-hydroxyvitamin D. Journal of Bone and Mineral Research 34 1068–1073. (https://doi.org/10.1002/jbmr.3686)
Schuster I 2011 Cytochromes P450 are essential players in the vitamin D signaling system. Biochimica et Biophysica Acta 1814 186–1 99. (https://doi.org/10.1016/j.bbapap.2010.06.022)
Seldeen KL, Pang M, Rodriguez-Gonzalez M, Hernandez M, Sheridan Z, Yu P & Troen BR 2017 A mouse model of vitamin D insufficiency: is there a relationship between 25(OH) vitamin D levels and obesity? Nutrition and Metabolism 14 26. (https://doi.org/10.1186/s12986-017-0174-6)
Vilarrasa N, Maravall J, Estepa A, Sanchez R, Masdevall C, Navarro MA, Alia P, Soler J & Gomez JM 2007 Low 25-hydroxyvitamin D concentrations in obese women: their clinical significance and relationship with anthropometric and body composition variables. Journal of Endocrinological Investigation 30 653–65 8. (https://doi.org/10.1007/BF03347445)
Voigt A, Agnew K, Van Schothorst EM, Keijer J & Klaus S 2013 Short-term, high fat feeding-induced changes in white adipose tissue gene expression are highly predictive for long-term changes. Molecular Nutrition and Food Research 57 1423–14 34. (https://doi.org/10.1002/mnfr.201200671)
Walsh JS, Evans AL, Bowles S, Naylor KE, Jones KS, Schoenmakers I, Jacques RM & Eastell R 2016 Free 25-hydroxyvitamin D is low in obesity, but there are no adverse associations with bone health. American Journal of Clinical Nutrition 103 1465–14 71. (https://doi.org/10.3945/ajcn.115.120139)
Wamberg L, Christiansen T, Paulsen SK, Fisker S, Rask P, Rejnmark L, Richelsen B & Pedersen SB 2013 Expression of vitamin D-metabolizing enzymes in human adipose tissue – the effect of obesity and diet-induced weight loss. International Journal of Obesity 37 651–65 7. (https://doi.org/10.1038/ijo.2012.112)
Wortsman J, Matsuoka LY, Chen TC, Lu Z & Holick MF 2000 Decreased bioavailability of vitamin D in obesity. American Journal of Clinical Nutrition 72 690–69 3. (https://doi.org/10.1093/ajcn/72.3.690)
Zhu JG, Ochalek JT, Kaufmann M, Jones G & Deluca HF 2013 CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo. PNAS 110 15650–1565 5. (https://doi.org/10.1073/pnas.1315006110)
