Uptake of vitamin D

VD from food is partially absorbed in the distal part of the small intestine, in emulsion with bile salts⁽ Reference Borel, Caillaud and Cano ^{16 )}. Its intestinal absorption occurs not only by passive diffusion but also via at least two cholesterol carriers⁽ Reference Reboul, Goncalves and Comera ^{17 ,} Reference Reboul ^{18 )}. VD and its metabolites are majorly transported in the plasma bound to vitamin D-binding protein (DBP), a globulin produced in the liver⁽ Reference Daiger, Schanfield and Cavalli-Sforza ^{19 )}, but also to albumin, LDL and chylomicrons for dietary VD⁽ Reference Haddad, Jennings and Aw ^{20 )}. Plasma DBP is in large excess compared with VD and metabolites, thus leaving only a very limited amount of circulating unbound VD and metabolites⁽ Reference Speeckaert, Huang and Delanghe ^{21 )}. Interestingly, only the unbound part of VD is considered biologically active and able to diffuse in any target cells⁽ Reference Speeckaert, Huang and Delanghe ^{21 )}, thus leading to the free-hormone hypothesis⁽ Reference Mendel ^{22 )}. Indeed, despite having low plasma levels of the different forms of VD compared with wild-type animals, Dbp-null mice do not show any signs of disrupted calcium homeostasis, suggesting that free VD levels can cover the needs of physiological functions as long as diet is VD-sufficient⁽ Reference Safadi, Thornton and Magiera ^{23 )}. Subsequent work showed that kidney content of 1,25(OH)₂D was not different from that of wild-type animals⁽ Reference Zella, Shevde and Hollis ^{24 )}. Taken together, these data suggest that tissues may uptake VD and metabolites from the free pool through a DBP-independent mechanism.

The molecular mechanisms involved in VD uptake/secretion by adipose tissue have not yet been investigated but may well involve the megalin/cubulin pathway (described later) as suggested by Abboud et al.⁽ Reference Abboud, Gordon-Thomson and Hoy ^{25 )} and/or cholesterol transporters as described in the intestine⁽ Reference Reboul, Goncalves and Comera ^{17 )} or for other lipophilic micronutrients in adipose tissue⁽ Reference Moussa, Gouranton and Gleize ^{26 )}.

25-hydroxylation

Whatever its origin (endogenous or exogenous), calciferol is taken to the liver via the circulation where the VD 25-hydroxylase enzyme catalyses the synthesis of 25(OH)D. 25(OH)D is the major circulating form of VD and its serum concentration is classically used as a marker of VD status. 25(OH)D has a relatively long half-life (15 d), and mean plasma 25(OH)D concentration varies between 20 and 50 ng/ml (50–125 nm/l)⁽ Reference Jones, Schoenmakers and Bluck ^{27 )}. Several enzymes can accomplish this first hydroxylation of 25(OH)D, but CYP2R1 seems to be the key one⁽ Reference Cheng, Motola and Mangelsdorf ^{28 ,} Reference Schuster ^{29 )}. Interestingly, Cyp2r1^−/− mice only display a 50 % reduction in serum 25(OH)D compared with wild-type or heterozygous animals, suggesting that other enzymes help maintain circulating 25(OH)D levels and/or compensate for CYP2R1 dysfunction⁽ Reference Zhu and DeLuca ^{30 )}.

In human subjects, other P450 cytochromes such as CYP3A4⁽ Reference Gupta, Hollis and Patel ^{31 )}, CYP2J2⁽ Reference Aiba, Yamasaki and Shinki ^{32 )} and CYP27A1⁽ Reference Guo, Strugnell and Back ^{33 )} display 25-hydroxylase activity towards VD molecules, but less efficiently (i.e. with a high K _M relative to physiological substrate concentration)⁽ Reference Zhu and DeLuca ^{30 )}. CYP2J3⁽ Reference Yamasaki, Izumi and Ide ^{34 )}, CYP2D25 and CYP2C11 also show VD 25-hydroxylase activity but are only expressed in pigs and male rats, respectively⁽ Reference Postlind, Axen and Bergman ^{35 ,} Reference Rahmaniyan, Patrick and Bell ^{36 )}.

25-Hydroxylation seems to be functional in adipose tissue, as Zoico et al.⁽ Reference Zoico, Franceschetti and Chirumbolo ^{37 )} recently reported that 25(OH)D release in the cell culture medium increased after 24 h incubation of 3T3-L1 adipocytes with VD. This production of 25(OH)D could be due to the presence of Cyp27A1, which is up-regulated by VD treatment. Interestingly, human adipose tissue biopsies have confirmed the expression of CYP27A1, CYP2R1 and CYP2J2⁽ Reference Wamberg, Christiansen and Paulsen ^{38 )}, suggesting that human adipose tissue and adipocytes are able to convert VD to 25(OH)D.

1α-hydroxylation

In renal proximal tubule cells, urinary loss of DBP–25(OH)D complexes is prevented by uptake via the membrane receptors megalin (also known as low-density lipoprotein receptor-related protein 2) and cubilin⁽ Reference Nykjaer, Dragun and Walther ^{39 ,} Reference Dusso, Brown and Slatopolsky ^{40 )}. After internalisation into vesicles, DBP is degraded into lysosomes and 25(OH)D is handled by intracellular DBP. Intracellular DBP have been identified in VD-resistant new-world primates (four isoforms have been reported so far), are related to the human heat-shock protein 70 family, and are thought to mediate 25(OH)D interactions with intracellular proteins⁽ Reference Gacad, Chen and Arbelle ^{41 )}. An additional binding protein termed cytosolic DBP has also been isolated from human intestinal cells⁽ Reference Teegarden, Nickel and Shi ^{42 )}. 25(OH)D is then either secreted into circulation or directed towards mitochondrial 1α-hydroxylase CYP27B1 to be metabolised into 1,25(OH)₂D, the active form of VD. CYP27B1 is the key enzyme of 1α-hydroxylation and its activity is regulated by parathyroid hormone, fibroblast growth factor 23, calcium and phosphorus and self-regulated by 1,25(OH)₂D via a negative-feedback mechanism⁽ Reference Holick ^{1 )}. 1,25(OH)₂D has a very short half-life (about 4 h) and is 1000 times less concentrated than 25(OH)D in the plasma.

The ability of adipocytes to convert 25(OH)D into 1,25(OH)₂D was initially demonstrated in 3T3-L1 cells via the activation of a gene reporter system and through the identification of radiolabelled 1,25(OH)₂D derived from radiolabelled 25(OH)D⁽ Reference Li, Byrne and Chang ^{43 )}. The production of 1,25(OH)₂D from 25(OH)D was then confirmed by Ching et al.⁽ Reference Ching, Kashinkunti and Niehaus ^{44 )} and Nimitphong et al.⁽ Reference Nimitphong, Holick and Fried ^{45 )}. CYP27B1 expression has also been detected in murine adipocytes⁽ Reference Li, Byrne and Chang ^{43 )} and in human adipose tissue biopsies⁽ Reference Wamberg, Christiansen and Paulsen ^{38 )}.

24-hydroxylation

Finally, vitamin D 24-hydroxylase (CYP24A1) is in charge of inactivating 1,25(OH)₂D. This inactivation is self-regulated, since 1,25(OH)₂D induces the expression of CYP24A1 that converts 25(OH)D and 1,25(OH)₂D into less-active metabolites (e.g. 24,25(OH)₂D and 1,24,25(OH)₃D), which are further catabolised into inactive calcitroic acid⁽ Reference Dusso, Brown and Slatopolsky ^{40 )}.

In adipose tissue, CYP24A1 expression has been detected in murine and human adipocytes⁽ Reference Li, Byrne and Chang ^{43 ,} Reference Nimitphong, Holick and Fried ^{45 )}. In addition, the mRNA levels of CYP24A1 are strongly induced by 1,25(OH)₂D incubation⁽ Reference Li, Byrne and Chang ^{43 ,} Reference Nimitphong, Holick and Fried ^{45 )}. CYP24A1 expression has also been confirmed in human adipose tissue biopsies⁽ Reference Wamberg, Christiansen and Paulsen ^{38 )}.

Vitamin D signalling

Even if few VD receptor (VDR)-independent effects of 1,25(OH)₂D have been documented⁽ Reference Lips ^{46 )}, most biological activities of VD are mediated by the VDR, a member of the nuclear receptor superfamily that is the only nuclear receptor that binds 1,25(OH)₂D with high affinity⁽ Reference Carlberg and Seuter ^{47 ,} Reference Bikle, Gee and Pillai ^{48 )}. VDR expression has been demonstrated in almost all human tissues⁽ Reference Bouillon, Carmeliet and Verlinden ^{49 )}, which means that all cells are potential targets of 1,25(OH)₂D action. The VDR–1,25(OH)₂D complex is associated with the retinoid X receptor⁽ Reference Issa, Leong and Eisman ^{50 )}, and the  retinoid X receptor–VDR–1,25(OH)₂D complex binds to the DNA of sites called VD response elements in the promoter region of genes whose expression is either activated or repressed⁽ Reference Carlberg and Seuter ^{47 )}. There are more than 1000 genes that are directly or indirectly regulated by 1,25(OH)₂D and involved in various physiological processes such as cell proliferation, differentiation, apoptosis and angiogenesis⁽ Reference Plum and DeLuca ^{51 )}.

The presence of VDR in adipose tissue was first reported in the early 1990s⁽ Reference Kamei, Kawada and Kazuki ^{52 )} and has since been widely confirmed. Interestingly, it was recently found that VDR expression is increased in obese compared with lean subjects⁽ Reference Wamberg, Christiansen and Paulsen ^{38 ,} Reference Clemente-Postigo, Munoz-Garach and Serrano ^{53 )}, but the physiological relevance of this up-regulation has not yet been elucidated.

Another VD-dependent signalling pathway has been described that involves ERp57 (also known as GRP58 or 1,25D3-MARRS), a protein disulfide isomerase involved in stress response⁽ Reference Turano, Gaucci and Grillo ^{54 )} that mediates rapid cellular responses (i.e. within seconds or minutes) to 1,25(OH)₂D stimulation⁽ Reference Nemere, Safford and Rohe ^{55 ,} Reference Nemere and Hintze ^{56 )}. ERp57–1,25(OH)₂D complexes are internalised, which opens the possibility that ERp57 might also participate in 1,25(OH)₂D intracellular trafficking, especially since ERp57 has been found to participate in nuclear complexes, including heat-shock protein 70, one of the human intracellular DBP. However, it is not yet known whether this signalling pathway is active in other tissues, particularly adipose tissue.

Taken together, these data demonstrate that on top of being a major storage site for VD, adipose tissue also expresses enzymes involved in VD metabolism and signalling, which points to the hypothesis that adipose tissue could be a target tissue that is also able to synthesise 25(OH)D and 1,25(OH)₂D that could be locally active via paracrine, autocrine or even intracrine processes⁽ Reference Lisse, Adams and Hewison ^{57 )}. The regulation of this local metabolism has never been studied but certainly warrants future investigation. Nevertheless, there is increasing evidence of the impact of VD and its active metabolites on adipose tissue, notably in terms of adipogenesis control, adipokine expression and a host of other metabolic regulations.

Regulation of adipogenesis

Many studies have examined the role of 1,25(OH)₂D in the proliferation and differentiation of murine 3T3-L1 pre-adipocytes⁽ Reference Kamei, Kawada and Kazuki ^{52 ,} Reference Ishida, Taniguchi and Baba ^{58 –} Reference Mutt, Hypponen and Saarnio ^{61 )}. Low 1,25(OH)₂D concentrations were associated with an inhibition of adipogenesis and a reduction of TAG accumulation in 3T3-L1 cells, even if the opposite effects, i.e. induction of adipogenesis, have also been depicted in this cellular model⁽ Reference Vu, Ong and Clemens ^{62 )}. The mechanism governing these effects implies 1,25(OH)₂D-mediated down-regulation⁽ Reference Kong and Li ^{59 ,} Reference Blumberg, Tzameli and Astapova ^{60 )} of C/EBPα and PPARγ, the two master regulators of adipogenesis⁽ Reference Farmer ^{63 )}. Other mechanisms such as antagonisation of PPARγ activity and stabilisation of the VDR protein are also components of this complex regulation⁽ Reference Kong and Li ^{59 )}. Similar results, i.e. inhibited differentiation under 1,25(OH)₂D, have also been reported in brown adipocytes⁽ Reference Ricciardi, Bae and Esposito ^{64 )}.

However, these data have been recently challenged in human adipocytes where 1,25(OH)₂D enhanced adipocyte differentiation and lipid accumulation⁽ Reference Nimitphong, Holick and Fried ^{45 )}. Interestingly, a similar activation of adipogenesis was found in primary mouse pre-adipocytes (albeit at a more advanced stage of differentiation compared with 3T3-L1 cells, suggesting that stage of differentiation is a key factor in the nature of the effect of 1,25(OH)₂D on adipogenesis). Similarly, 1,25(OH)₂D also increased adipogenesis in human adult stem cells derived from adipose tissue, as revealed by lipid accumulation and expression of adipogenic markers⁽ Reference Narvaez, Simmons and Brunton ^{65 )}.

To summarise, the effects of 1,25(OH)₂D and VDR on adipogenesis are not fully consistent: VDR appears to act as a promoter of adipogenesis, but 1,25(OH)₂D has less clear effects. It is currently difficult to firmly conclude in favour of an anti- or pro-adipogenic effect, and further in vivo studies are required to clarify this point.

Regulation of gene expression linked to energy metabolism

VD and particularly 1,25(OH)₂D may also influence adipose tissue and systemic biology. Indeed, 1,25(OH)₂D directly up-regulated leptin expression and secretion, independently of fat mass modifications⁽ Reference Kong, Chen and Zhu ^{66 )}. 1,25(OH)₂D was found to promote glucose uptake by adipocytes⁽ Reference Marcotorchino, Gouranton and Romier ^{67 )}, which could be related to the induction of GLUT4 protein expression and translocation observed in 3T3-L1 adipocytes⁽ Reference Manna and Jain ^{68 )}. We showed that VD supplementation in mice led to an increase of fatty oxidation (especially in brown adipose tissue) that could be responsible for a high-fat diet-induced limitation of body weight gain in VD-supplemented mice⁽ Reference Marcotorchino, Tourniaire and Astier ^{69 )}. Note that similar weight gain limitations in response to VD supplementation have been reported in mice⁽ Reference Sergeev and Song ^{70 )} while VD-deficient old rats showed fat mass gain⁽ Reference Domingues-Faria, Chanet and Salles ^{71 )}. Likewise, a global dietary vitamin restriction was associated with an increase of adiposity and a disruption of glucose homeostasis⁽ Reference Amara, Marcotorchino and Tourniaire ^{72 )} in mice.

Taken together, these data suggest that VD regulates energy expenditure, notably via its impact on adipose tissue biology.

Regulation of inflammation

Initial studies performed on human and mouse adipocytes (3T3-L1) found that 1,25(OH)₂D up-regulates several inflammatory cytokines and down-regulates anti-inflammatory cytokines in both cell types⁽ Reference Sun and Zemel ^{73 ,} Reference Sun and Zemel ^{74 )}. These results have since been challenged by several groups⁽ Reference Marcotorchino, Gouranton and Romier ^{67 ,} Reference Lorente-Cebrian, Eriksson and Dunlop ^{75 –} Reference Mutt, Karhu and Lehtonen ^{77 )} who consistently report anti-inflammatory effects of 1,25(OH)₂D whatever the model studied, which fits better with the well-described anti-inflammatory effect of VD in many other cell types⁽ Reference Guillot, Semerano and Saidenberg-Kermanac'h ^{78 )}. Indeed, it was shown that 1,25(OH)₂D significantly decreased the release of IL-8, monocyte chemoattractant protein (MCP)-1 and IL-6 by human preadipocytes⁽ Reference Gao, Trayhurn and Bing ^{76 )} and MCP-1 by human adipocytes⁽ Reference Lorente-Cebrian, Eriksson and Dunlop ^{75 )}. These anti-inflammatory effects were associated with inhibition of the NF-κB signalling pathway⁽ Reference Mutt, Karhu and Lehtonen ^{77 )}. We also demonstrated the anti-inflammatory properties of 1,25(OH)₂D in murine and human adipocytes⁽ Reference Marcotorchino, Gouranton and Romier ^{67 )}. In these various models, 1,25(OH)₂D was able to decrease the expression of inflammatory markers such as IL-6, IL-1β and MCP-1 in both basal and TNFα-stimulated conditions⁽ Reference Marcotorchino, Gouranton and Romier ^{67 )}. Similarly, 1,25(OH)₂D reduced the expression of IL-6, IL-8 and MCP-1 in human adipose tissue biopsies submitted to IL-1β stimulation in vitro ⁽ Reference Wamberg, Cullberg and Rejnmark ^{79 )}. The molecular mechanisms have been investigated, and VDR and NF-κB signalling pathways and p38 mitogen-activated protein kinases were shown to be involved in 3T3-L1 adipocytes⁽ Reference Marcotorchino, Gouranton and Romier ^{67 )}. Interestingly, Zoico et al. recently demonstrated that VD, similarly to 1,25(OH)₂D, was able to blunt the lipopolysaccharide-mediated pro-inflammatory effect in human adipocytes⁽ Reference Zoico, Franceschetti and Chirumbolo ^{37 )}. Using a microarray approach, we recently demonstrated that 1,25(OH)₂D was able to down-regulate a large set of chemokines⁽ Reference Karkeni, Marcotorchino and Tourniaire ^{80 )} induced by inflammatory stimulus⁽ Reference Tourniaire, Romier-Crouzet and Lee ^{81 )} in human and murine adipocytes. This effect was accompanied by a reduction of macrophage migration mediated by an adipocyte-conditioned medium⁽ Reference Karkeni, Marcotorchino and Tourniaire ^{80 )}.

These anti-inflammatory properties of 1,25(OH)₂D have also been established in several types of immune cells found in the adipose tissue, including lymphocytes and macrophages⁽ Reference Guillot, Semerano and Saidenberg-Kermanac'h ^{78 )}. Furthermore, 1,25(OH)₂D reduced macrophage-induced inflammatory response in human adipocytes via inhibition of NF-κB and mitogen-activated protein kinase activation together with monocyte migration mediated by the adipocyte-conditioned medium⁽ Reference Ding, Wilding and Bing ^{82 )}. These data suggest that VD not only blunts adipocyte response to inflammatory stimulus, but also interferes with macrophage/adipocyte cross-talk, a key element of the propagation of metabolic inflammation in obesity⁽ Reference Suganami, Nishida and Ogawa ^{83 )}.

The anti-inflammatory effect of VD in adipose tissue has also been observed in vivo. It was reported that dietary treatment with 1,25(OH)₂D-reduced IL6 protein content in the epididymal adipose tissue of obese mice⁽ Reference Lira, Rosa and Cunha ^{84 )}, whereas feeding with a VD-deficient high-fat diet was found to increase Il-6 expression in rat adipose tissue⁽ Reference Roth, Elfers and Figlewicz ^{85 )}. In addition, we recently demonstrated that VD supplementation of high-fat diet reduced the expression of proinflammatory cytokines and chemokines and inhibited macrophage infiltration in the adipose tissue of obese mice⁽ Reference Karkeni, Marcotorchino and Tourniaire ^{80 )}. Similar effects of VD supplementation were found in an acute inflammation model (intraperitoneal injection of LPS) where no modification of body weight was measured⁽ Reference Karkeni, Marcotorchino and Tourniaire ^{80 )}, which strongly suggests that the decreased inflammatory status of adipose tissue observed in obese mice is not only a consequence of reduced fat mass⁽ Reference Marcotorchino, Tourniaire and Astier ^{69 )} but is also driven by an anti-inflammatory effect of VD per se.

To summarise, it has been demonstrated in vitro and in vivo that VD has a limiting effect on adipose tissue inflammation, acting on both inflammatory status in preadipocytes and adipocytes and on leucocyte infiltration.