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Retinol and retinyl esters: biochemistry and physiology

Thematic Review Series: Fat-Soluble Vitamins: Vitamin A
Open AccessPublished:April 26, 2013DOI:https://doi.org/10.1194/jlr.R037648
      By definition, a vitamin is a substance that must be obtained regularly from the diet. Vitamin A must be acquired from the diet, but unlike most vitamins, it can also be stored within the body in relatively high levels. For humans living in developed nations or animals living in present-day vivariums, stored vitamin A concentrations can become relatively high, reaching levels that can protect against the adverse effects of insufficient vitamin A dietary intake for six months, or even much longer. The ability to accumulate vitamin A stores lessens the need for routinely consuming vitamin A in the diet, and this provides a selective advantage to the organism. The molecular processes that underlie this selective advantage include efficient mechanisms to acquire vitamin A from the diet, efficient and overlapping mechanisms for the transport of vitamin A in the circulation, a specific mechanism allowing for vitamin A storage, and a mechanism for mobilizing vitamin A from these stores in response to tissue needs. These processes are considered in this review.
      Since the identification of fat-soluble A a century ago (
      • McCollum E.V.
      • Davis M.
      The necessity of certain lipins in the diet during growth.
      ), retinoids (vitamin A and its natural and synthetic analogs) have been the most extensively studied of the fat-soluble vitamins. This research has identified essential roles for retinoids in many different aspects of mammalian physiology, including embryonic development, adult growth and development, maintenance of immunity, maintenance of epithelial barriers, and vision (
      • McCollum E.V.
      • Davis M.
      The necessity of certain lipins in the diet during growth.
      ,
      • Gudas L.
      • Sporn M.B.
      • Roberts A.B.
      Cellular biology and biochemistry of the retinoids.
      ,
      • Wald G.
      Molecular basis of visual excitation.
      ,
      • Saari J.C.
      Retinoids in mammalian vision.
      ,
      • Ross A.C.
      • Hammerling U.
      Retinoids and the immune system.
      ). This review focuses on retinoid biochemistry in mammals, primarily on retinol and retinyl ester metabolism. However, we also consider other retinol metabolites, including retro- and anhydro-retinoids and retinoid-β-glucuronides.

      RETINOID CHEMICAL FORMS

      The different retinoid forms present within the body (see Fig. 1) are generated by and large through modifications to the terminal polar end group of the molecule. Retinol and retinyl esters are the most abundant retinoid forms present in the body. All-trans-retinol is by definition vitamin A. When a fatty acyl group is esterified to the hydroxyl terminus of retinol, a storage form of retinol, the retinyl ester, is formed. The most abundant retinyl esters present in the body are those of palmitic acid, oleic acid, stearic acid, and linoleic acid (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). Although retinyl acetate can be found in supplements to foods and vitamin formulations, only long-chain acyl groups are esterified to retinol by animals. Retinyl esters have no known biological activity aside from retinol storage and for serving as the substrate for the formation of the visual chromophore 11-cis-retinal, which must be formed from all-trans-retinyl ester through linked hydrolysis and isomerization reactions catalyzed by the enzyme RPE65 (
      • Wald G.
      Molecular basis of visual excitation.
      ,
      • Saari J.C.
      Retinoids in mammalian vision.
      ,
      • Travis G.H.
      • Golczak M.
      • Moise A.R.
      • Palczewski K.
      Diseases caused by defects in the visual cycle: retinoids as potential therapeutic agents.
      ,
      • Palczewski K.
      Chemistry and biology of vision.
      ). Retinol is a transport form and a precursor form, which is enzymatically activated to retinoic acid via a two-step oxidation process. The primary role of retinal is in the eye where 11-cis-retinal is needed for visual pigment formation. In tissues, retinal serves as an intermediate in the synthesis of retinoic acid from retinol (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). The literature also suggests a direct role for retinal in adipose tissue, where it was shown to inhibit adipogenesis and suppress peroxisome proliferator-activated receptor-γ (PPARγ) and retinoid X receptor (RXR) responses in cell culture models and in mouse models fed high-fat diets (
      • Ziouzenkova O.
      • Orasanu G.
      • Sharlach M.
      • Akiyama T.E.
      • Berger J.P.
      • Viereck J.
      • Hamilton J.A.
      • Tang G.
      • Dolnikowski G.G.
      • Vogel S.
      • et al.
      Retinaldehyde represses adipogenesis and diet-induced obesity.
      ).
      Figure thumbnail gr1
      Fig. 1Chemical structures of different retinoid chemical species. The chemical structure of the major proretinoid carotenoid, β-carotene, is shown at the top. The chemical structures for all-trans-retinol (which by definition is vitamin A), an all-trans-retinyl ester, 11-cis-retinal (the active retinoid in vision), and the all-trans-, 9-cis-, and 13-cis-isomers of retinoic acid are shown.
      The all-trans- (tretinoin, Retin-A) and 9-cis- (alitretinoin) isomers of retinoic acid are transcriptionally active retinoids and are thought to account for the gene regulatory properties of retinoids within cells and tissues (
      • Chambon P.
      The retinoid signaling pathway: molecular and genetic analyses.
      ,
      • Mangelsdorf D.J. UK
      • Evans R.M.
      The retinoid receptors.
      ). The concentration of retinoic acid within tissues is generally very low and usually 100 to 1,000 times less than that of retinol (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). All-trans-retinoic acid can be isomerized through a nonenzymatic process to form the 9-cis- or 13-cis-retinoic acid (isotretinoin, Accutane) isomers (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). Although 13-cis-retinoic acid is a naturally occurring retinoid, it is less transcriptionally active than either the all-trans- or 9-cis-isomer (
      • Chambon P.
      The retinoid signaling pathway: molecular and genetic analyses.
      ,
      • Mangelsdorf D.J. UK
      • Evans R.M.
      The retinoid receptors.
      ). The actions of retinoic acid in regulating transcription are considered in detail in this thematic series by Rochette-Egly and colleagues (
      • Al Tanoury Z.
      • Piskunov A.
      • Rochette-Egly C.
      Vitamin A and retinoid signaling: genomic and non-genomic effects.
      ).
      Various oxo- and hydroxy- forms of retinol and retinoic acid as well as glucuronides of both retinol and retinoic acid are present in the body, albeit at very low concentrations relative to retinol and retinyl esters (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). Although some of these oxidized and conjugated retinoid forms may have biologic/transcriptional activity, it appears likely that most of these forms are catabolic in nature and destined for elimination from the body. Since there are no known enzymes that can reduce retinoic acid to retinal, excessive or unneeded retinoic acid is not recycled back to retinol/retinyl ester and must be catabolized and eliminated from the body. This catabolism is catalyzed by one of several cytochrome P450 (CYP) enzymes (
      • White J.A.
      • Beckett-Jones B.
      • Guo Y.D.
      • Dilworth F.J.
      • Bonasoro J.
      • Jones G.
      • Petkovich M.
      cDNA cloning of human retinoic acid-metabolizing enzyme (hP450RAI) identifies a novel family of cytochromes P450.
      ,
      • White J.A.
      • Guo Y.D.
      • Baetz K.
      • Beckett-Jones B.
      • Bonasoro J.
      • Hsu K.E.
      • Dilworth F.J.
      • Jones G.
      • Petkovich M.
      Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.
      ,
      • Fujii H.
      • Sato T.
      • Kaneko S.
      • Gotoh O.
      • Fujii-Kuriyama Y.
      • Osawa K.
      • Kato S.
      • Hamada H.
      Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos.
      ,
      • Ray W.J.
      • Bain G.
      • Yao M.
      • Gottlieb D.I.
      CYP26, a novel mammalian cytochrome P450, is induced by retinoic acid and defines a new family.
      ,
      • Ross A.C.
      Retinoid production and catabolism: role of diet in regulating retinol esterification and retinoic acid oxidation.
      ), giving rise to more water-soluble oxidized and conjugated retinoid forms that can be more easily excreted. These CYPs are discussed in depth by Kedishvili in a review in this thematic review series (
      • Kedishvili N.Y.
      Enzymology of retinoic acid biosynthesis and degradation.
      ). The metabolism of retinoids described above and the enzymes responsible for catalyzing this metabolism are summarized in Fig. 2.
      Figure thumbnail gr2
      Fig. 2The metabolism of β-carotene and retinoids. All retinoid is originally derived from proretinoid carotenoids such as β-carotene (1). Retinal (2) can be formed by the central cleavage of β-carotene by the enzyme BCMO1. Retinol (3) is formed by the reversible reduction of retinal (2) by one of the retinal reductase family members. The enzyme LRAT synthesizes retinyl esters (4) by transferring a fatty acyl moiety from the sn-1 position of membrane phosphatidyl choline to retinol. Unesterified retinol is liberated from retinyl ester stores through the action of a REH. Retinol is oxidized to retinal by one of several RDHs, which is then irreversibly oxidized (by one of three RALDHs) to form transcriptionally active retinoic acid (5). Retinoic acid is oxidized/catabolized to more water-soluble hydroxy- and oxo- forms by one of several cytochrome P450 enzyme family members.
      Retro- and anhydro-retinoids are also naturally occurring retinoid forms that can be synthesized by cells and tissues that are present within the body (
      • Vakiani E.
      • Buck J.
      Retro-retinoids: metabolism and action.
      ,
      • Moise A.R.
      • Kuksa V.
      • Imanishi Y.
      • Palczewski K.
      Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase.
      ). Enzymes able to catalyze the formation of retro- and anhydro-retinoids have been identified (
      • Vakiani E.
      • Buck J.
      Retro-retinoids: metabolism and action.
      ,
      • Moise A.R.
      • Kuksa V.
      • Imanishi Y.
      • Palczewski K.
      Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase.
      ). It has been proposed that the retro- and anhydro-retinoids may have actions in regulating immune function, but the biochemical mechanisms responsible for these actions have not been elucidated (
      • Ross A.C.
      • Hammerling U.
      Retinoids and the immune system.
      ,
      • Vakiani E.
      • Buck J.
      Retro-retinoids: metabolism and action.
      ,
      • Moise A.R.
      • Kuksa V.
      • Imanishi Y.
      • Palczewski K.
      Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase.
      ). The enzyme responsible for the saturation of the 13-14 double bond of all-trans-retinol to produce all-trans-13,14-dihydroretinol, termed retinoid saturase (RetSat), was described and cloned several years ago (
      • Moise A.R.
      • Kuksa V.
      • Imanishi Y.
      • Palczewski K.
      Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase.
      ,
      • Moise A.R.
      • Kuksa V.
      • Blaner W.S.
      • Baehr W.
      • Palczewski K.
      Metabolism and transactivation activity of 13,14-dihydroretinoic acid.
      ,
      • Moise A.R.
      • Isken A.
      • Dominguez M.
      • de Lera A.R.
      • von Lintig J.
      • Palczewski K.
      Specificity of zebrafish retinol saturase: formation of all-trans-13,14-dihydroretinol and all-trans-7,8-dihydroretinol.
      ). Expression of RetSat is regulated by PPARγ, a key transcriptional regulator of adipogenesis. RetSat activity has been implicated in the regulation of adipocyte development and differentiation. Ablation of RetSat expression in a cell culture model of adipocyte differentiation inhibited adipogenesis, whereas ectopic expression of RetSat enhanced differentiation (
      • Schupp M.
      • Lefterova M.I.
      • Janke J.
      • Leitner K.
      • Cristancho A.G.
      • Mullican S.E.
      • Qatanani M.
      • Szwergold N.
      • Steger D.J.
      • Curtin J.C.
      • et al.
      Retinol saturase promotes adipogenesis and is downregulated in obesity.
      ). This block in adipocyte differentiation could not be rescued by addition of 13,14-dihydroretinol to the cells, implying that this enzyme may have other, unknown substrates (
      • Schupp M.
      • Lefterova M.I.
      • Janke J.
      • Leitner K.
      • Cristancho A.G.
      • Mullican S.E.
      • Qatanani M.
      • Szwergold N.
      • Steger D.J.
      • Curtin J.C.
      • et al.
      Retinol saturase promotes adipogenesis and is downregulated in obesity.
      ). Mice totally lacking RetSat exhibit increased adiposity, which is associated with an upregulation of PPARγ (
      • Moise A.R.
      • Lobo G.P.
      • Erokwu B.
      • Wilson D.L.
      • Peck D.
      • Alvarez S.
      • Dominguez M.
      • Alvarez R.
      • Flask C.A.
      • de Lera A.R.
      • et al.
      Increased adiposity in the retinol saturase-knockout mouse.
      ). Future studies may identify other novel metabolites of retinol, possibly generated by RetSat, which may explain these phenotypes.
      Over the last four decades, many retinoid metabolites have been identified (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Vakiani E.
      • Buck J.
      Retro-retinoids: metabolism and action.
      ,
      • Moise A.R.
      • Kuksa V.
      • Imanishi Y.
      • Palczewski K.
      Identification of all-trans-retinol:all-trans-13,14-dihydroretinol saturase.
      ). Some of these are proposed but not proved to have important physiological roles, whereas others are simply catabolic products destined for elimination from the body. It is possible that some of these metabolites may yet prove to be very important physiologically, but we have chosen to consider in our review only those associated with a relatively substantial literature. The reader should turn to earlier review Refs.
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      , and
      • Vakiani E.
      • Buck J.
      Retro-retinoids: metabolism and action.
      for more details regarding these other retinoid metabolites.

      RETINOL ESTERIFICATION AND RETINYL ESTER HYDROLYSIS

      Although the liver and intestine are the major tissue sites of retinol esterification in the body, many tissues are able to esterify retinol and accumulate some retinyl ester stores, including the eye, lung, adipose tissue, testes, skin, and spleen (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). It is now understood that the enzyme responsible for the preponderance of retinyl ester formation in the body is lecithin:retinol acyltransferase (LRAT). This understanding was obtained from study of mutant mice in which the gene encoding LRAT had been totally ablated (
      • Batten M.L.
      • Imanishi Y.
      • Maeda T.
      • Tu D.C.
      • Moise A.R.
      • Bronson D.
      • Possin D.
      • Van Gelder R.N.
      • Baehr W.
      • Palczewski K.
      Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.
      ,
      • Liu L.
      • Gudas L.J.
      Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.
      ,
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). Lrat-deficient mice possess significant retinyl ester stores in adipose tissue but not in liver, eyes, lungs, testes, skin, or spleen (
      • Batten M.L.
      • Imanishi Y.
      • Maeda T.
      • Tu D.C.
      • Moise A.R.
      • Bronson D.
      • Possin D.
      • Van Gelder R.N.
      • Baehr W.
      • Palczewski K.
      Lecithin-retinol acyltransferase is essential for accumulation of all-trans-retinyl esters in the eye and in the liver.
      ,
      • Liu L.
      • Gudas L.J.
      Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.
      ,
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ,
      • Liu L.
      • Tang X.H.
      • Gudas L.J.
      Homeostasis of retinol in lecithin:retinol acyltransferase gene knockout mice fed a high retinol diet.
      ). LRAT catalyzes a transesterification, transferring long-chain fatty acyl moieties (primarily palmitic, stearic, oleic, and linoleic acids) present at the sn-1 position of membrane bilayer phosphatidylcholine to retinol, forming retinyl esters (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Yost R.W.
      • Harrison E.H.
      • Ross A.C.
      Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein.
      ,
      • Ong D.E.
      • MacDonald P.N.
      • Gumbitosi A.M.
      Esterificatrion of retinol in rat liver. Possible participation by cellular retinol-binding protein and cellular retinol-binding protein II.
      ,
      • Saari J.C.
      • Bredberg D.L.
      Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes.
      ,
      • Ross A.C.
      Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A:retinol acyltransferase.
      ,
      • Randolph R.K.
      • Winkler K.E.
      • Ross A.C.
      Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes.
      ). LRAT is one of six distinct proteins/genes that compose the vertebrate members of the NIpC/P60 protein family (
      • Anantharaman V.
      • Aravind L.
      Evolutionary history, structural features and biochemical diversity of the NIpC/P60 superfamily of enzymes.
      ). Members of this protein family share a common property of having conserved cysteine, histidine, and polar amino acid residues that are required for catalytic activity (
      • Anantharaman V.
      • Aravind L.
      Evolutionary history, structural features and biochemical diversity of the NIpC/P60 superfamily of enzymes.
      ). The other vertebrate member of this family that is relatively well studied is adipose-specific phospholipase A2 (AdPLA) (
      • Duncan R.E.
      • Sarkadi-Nagy E.
      • Jaworski K.
      • Ahmadian M.
      • Sul H.S.
      Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA).
      ,
      • Jaworski K.
      • Ahmadian M.
      • Duncan R.E.
      • Sarkadi-Nagy E.
      • Varady K.A.
      • Hellerstein M.K.
      • Lee H.Y.
      • Samuel V.T.
      • Shulman G.I.
      • Kim K.H.
      • et al.
      AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency.
      ). AdPLA was described by Sul and colleagues as a phospholipase associated with the development of obesity induced by high-fat feeding (
      • Jaworski K.
      • Ahmadian M.
      • Duncan R.E.
      • Sarkadi-Nagy E.
      • Varady K.A.
      • Hellerstein M.K.
      • Lee H.Y.
      • Samuel V.T.
      • Shulman G.I.
      • Kim K.H.
      • et al.
      AdPLA ablation increases lipolysis and prevents obesity induced by high-fat feeding or leptin deficiency.
      ). The other vertebrate members of this protein family are less well studied and are reported to possess either phospholipase and/or phosphatidylcholine-dependent acyltransferase activities (
      • Uyama T.
      • Jin X.H.
      • Tsuboi K.
      • Tonai T.
      • Ueda N.
      Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.
      ,
      • Jin X.H.
      • Okamoto Y.
      • Morishita J.
      • Tsuboi K.
      • Tonai T.
      • Ueda N.
      Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.
      ,
      • Uyama T.
      • Morishita J.
      • Jin X.H.
      • Okamoto Y.
      • Tsuboi K.
      • Ueda N.
      The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type.
      ,
      • Ueda N.
      • Tsuboi K.
      • Uyama T.
      Enzymological studies on the biosynthesis of N-acylethanolamines.
      ,
      • Golczak M.
      • Kiser P.D.
      • Sears A.E.
      • Lodowski D.T.
      • Blaner W.S.
      • Palczewski K.
      Structural basis for the acyltransferase activity of lecithin:retinol acyltransferase-like proteins.
      ). Some show a sn-1 preference and others a preference for the sn-2 acyl moiety (
      • Uyama T.
      • Jin X.H.
      • Tsuboi K.
      • Tonai T.
      • Ueda N.
      Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipid-metabolizing enzymes.
      ,
      • Jin X.H.
      • Okamoto Y.
      • Morishita J.
      • Tsuboi K.
      • Tonai T.
      • Ueda N.
      Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners.
      ,
      • Uyama T.
      • Morishita J.
      • Jin X.H.
      • Okamoto Y.
      • Tsuboi K.
      • Ueda N.
      The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type.
      ,
      • Ueda N.
      • Tsuboi K.
      • Uyama T.
      Enzymological studies on the biosynthesis of N-acylethanolamines.
      ,
      • Golczak M.
      • Kiser P.D.
      • Sears A.E.
      • Lodowski D.T.
      • Blaner W.S.
      • Palczewski K.
      Structural basis for the acyltransferase activity of lecithin:retinol acyltransferase-like proteins.
      ). It should be noted that LRAT is completely distinct from and shares no relationship with lecithin:cholesterol acyltransferase (LCAT).
      The older literature suggests that another enzymatic activity, acyl-CoA:retinol acyltransferase (ARAT), may be physiologically important for catalyzing retinyl ester formation. ARAT is proposed to esterify retinol using fatty acyl groups present in the acyl-CoA pool (
      • Ross A.C.
      Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A:retinol acyltransferase.
      ). ARAT also is reported to differ from LRAT with regard to its ability to acquire retinol within the cell. LRAT is capable of esterifying retinol when it is bound to one of the cellular retinol-binding proteins (CRBPI, CRBPII, or CRBPIII), whereas ARAT is not (
      • Randolph R.K.
      • Winkler K.E.
      • Ross A.C.
      Fatty acyl CoA-dependent and -independent retinol esterification by rat liver and lactating mammary gland microsomes.
      ). Several published studies have established that one of the two enzymes responsible for the final step in triglyceride synthesis, diacylglycerol acyltransferase 1 (DGAT1), possesses ARAT activity in vitro (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ,
      • Orland M.D.
      • Anwar K.
      • Cromley D.
      • Chu C.H.
      • Chen L.
      • Billheimer J.T.
      • Hussain M.M.
      • Cheng D.
      Acyl coenzyme A dependent retinol esterification by acyl coenzyme A: diacylglycerol acyltransferase 1.
      ,
      • Shih M.Y.
      • Kane M.A.
      • Zhou P.
      • Yen C.L.
      • Streeper R.S.
      • Napoli J.L.
      • Farese Jr, R.V.
      Retinol esterification by DGAT1 is essential for retinoid homeostasis in murine skin.
      ,
      • Yen C.L.
      • Brown C.H.t.
      • Monetti M.
      • Farese Jr, R.V.
      A human skin multifunctional O-acyltransferase that catalyzes the synthesis of acylglycerols, waxes, and retinyl esters.
      ,
      • Yen C.L.
      • Monetti M.
      • Burri B.J.
      • Farese Jr, R.V.
      The triacylglycerol synthesis enzyme DGAT1 also catalyzes the synthesis of diacylglycerols, waxes, and retinyl esters.
      ). DGAT1, when expressed in vitro, will catalyze retinyl ester formation using nonprotein-bound retinol as a substrate, but it is unable to catalyze retinyl ester formation when the retinol is supplied bound to CRBPI or CRBPII (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). When Lrat-deficient mice were challenged with an oral physiological dose (6 µg) of retinol provided in oil, some retinyl esters were still present in nascent chylomicrons (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). When this same oral challenge was given to mice totally lacking expression of both Lrat and Dgat1, no retinyl esters could be detected in newly synthesized chylomicrons (
      • Wongsiriroj N.
      • Piantedosi R.
      • Palczewski K.
      • Goldberg I.J.
      • Johnston T.P.
      • Li E.
      • Blaner W.S.
      The molecular basis of retinoid absorption: a genetic dissection.
      ). This observation was taken to indicate that DGAT1 acts in vivo as an intestinal ARAT (
      • Wongsiriroj N.
      • Piantedosi R.
      • Palczewski K.
      • Goldberg I.J.
      • Johnston T.P.
      • Li E.
      • Blaner W.S.
      The molecular basis of retinoid absorption: a genetic dissection.
      ). Interestingly, when an oral pharmacological dose (1.0 mg) of retinol in oil was given to the mice lacking both Lrat and Dgat1, some retinyl esters were observed in the postprandial circulation (
      • Wongsiriroj N.
      • Piantedosi R.
      • Palczewski K.
      • Goldberg I.J.
      • Johnston T.P.
      • Li E.
      • Blaner W.S.
      The molecular basis of retinoid absorption: a genetic dissection.
      ). This observation suggests that a third enzyme is able to esterify retinol in the intestine when very high levels of retinol have been consumed. Studying Dgat1-deficient mice, Farese and colleagues independently established that DGAT1 can influence retinoid homeostasis in the skin of mice (
      • Shih M.Y.
      • Kane M.A.
      • Zhou P.
      • Yen C.L.
      • Streeper R.S.
      • Napoli J.L.
      • Farese Jr, R.V.
      Retinol esterification by DGAT1 is essential for retinoid homeostasis in murine skin.
      ). Thus, in at least two mouse tissues, intestine and skin, DGAT1 acts physiologically in catalyzing retinyl ester formation. As noted above, Lrat-deficient mice possess significant retinyl ester stores in adipose tissue (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ,
      • Liu L.
      • Tang X.H.
      • Gudas L.J.
      Homeostasis of retinol in lecithin:retinol acyltransferase gene knockout mice fed a high retinol diet.
      ). This is also true for mice totally lacking both Lrat and Dgat1 (
      • Wongsiriroj N.
      • Piantedosi R.
      • Palczewski K.
      • Goldberg I.J.
      • Johnston T.P.
      • Li E.
      • Blaner W.S.
      The molecular basis of retinoid absorption: a genetic dissection.
      ). This observation implies that another, unidentified enzyme acts in adipose tissue to catalyze retinyl ester formation.
      LRAT in the liver is thought to be structurally identical to intestinal LRAT, which synthesizes retinyl esters from dietary retinol for incorporation into nascent chylomicrons. Interestingly though, hepatic but not intestinal LRAT expression is regulated by retinoid nutritional status (
      • Matsuura T.
      • Ross A.C.
      Regulation of hepatic lecithin:retinol acyltransferase activity by retinoic acid.
      ). The regulation of expression of the Lrat gene in the liver involves the presence of a retinoic acid response element present in the Lrat gene, and probably the actions of retinoic acid receptors (RAR) and/or RXRs (
      • Matsuura T.
      • Ross A.C.
      Regulation of hepatic lecithin:retinol acyltransferase activity by retinoic acid.
      ,
      • Cai K.
      • Gudas L.J.
      Retinoic acid receptors and GATA transcription factors activate the transcription of the human lecithin:retinol acyltransferase gene.
      ). This retinoic acid-responsive regulation is proposed to give rise to a positive feedback loop when cellular retinoic acid levels are high, turning on Lrat expression and increasing the synthesis of retinyl esters and preventing the synthesis of additional retinoic acid. Supporting this proposal is the observation that hepatic expression levels of the retinoic acid catabolic enzyme Cyp26A (which is also a retinoic acid-responsive gene) are markedly upregulated in Lrat-deficient mice (
      • Liu L.
      • Gudas L.J.
      Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.
      ,
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). In addition to a role for retinoic acid in regulating Lrat gene expression, studies carried out in the PC-3 prostate cancer cell line indicate that GATA transcription factors act importantly in regulating Lrat transcription (
      • Cai K.
      • Gudas L.J.
      Retinoic acid receptors and GATA transcription factors activate the transcription of the human lecithin:retinol acyltransferase gene.
      ).
      Since retinyl esters represent a retinoid storage form, they must be hydrolyzed to retinol before conversion to retinoic acid. Unlike LRAT, which is accepted to be the major enzyme responsible for retinyl ester formation, there are many retinyl ester hydrolases (REH) that are proposed to be responsible physiologically for the generation of free retinol from retinyl ester stores (
      • Harrison E.H.
      Lipases and carboxylesterases: possible roles in the hepatic utilization of vitamin A.
      ,
      • Matsuura T.
      • Gad M.Z.
      • Harrison E.H.
      • Ross A.C.
      Lecithin:retinol acyltransferase and retinyl ester hydrolase activities are differentially regulated by retinoids and have distinct distributions between hepatocyte and nonparenchymal cell fractions of rat liver.
      ,
      • Sanghani S.P.
      • Davis W.I.
      • Dumaual N.G.
      • Mahrenholz A.
      • Bosron W.F.
      Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.
      ). One is a bile salt-dependent retinyl ester hydrolase. Most or all of this enzymatic activity in the liver probably arises from the actions of bile salt-activated carboxylester lipase (CEL) (
      • van Bennekum A.M.
      • Li L.
      • Piantedosi R.
      • Shamir R.
      • Vogel S.
      • Fisher E.A.
      • Blaner W.S.
      • Harrison E.H.
      Carboxyl ester lipase overexpression in rat hepatoma cells and CEL deficiency in mice have no impact on heaptic uptake or metabolism of chylomicron-retinyl ester.
      ). However, since mice lacking CEL display no alteration in retinoid storage, metabolism, or action, this enzyme cannot be the sole physiologically relevant REH (
      • van Bennekum A.M.
      • Li L.
      • Piantedosi R.
      • Shamir R.
      • Vogel S.
      • Fisher E.A.
      • Blaner W.S.
      • Harrison E.H.
      Carboxyl ester lipase overexpression in rat hepatoma cells and CEL deficiency in mice have no impact on heaptic uptake or metabolism of chylomicron-retinyl ester.
      ). Another group of enzymes, collectively known as bile salt-independent REHs, has been described. Based on their pH optima, there are two groups of bile salt-independent REHs: neutral REHs and acidic REHs. It has been reported that the activities of the neutral and acidic REHs are unaffected by retinoid nutritional status. There is evidence demonstrating that three known hepatic carboxylesterases (also known in the literature as ES-2, ES-4, and ES-10) act as REHs in vitro (
      • Harrison E.H.
      Lipases and carboxylesterases: possible roles in the hepatic utilization of vitamin A.
      ,
      • Matsuura T.
      • Gad M.Z.
      • Harrison E.H.
      • Ross A.C.
      Lecithin:retinol acyltransferase and retinyl ester hydrolase activities are differentially regulated by retinoids and have distinct distributions between hepatocyte and nonparenchymal cell fractions of rat liver.
      ,
      • Sanghani S.P.
      • Davis W.I.
      • Dumaual N.G.
      • Mahrenholz A.
      • Bosron W.F.
      Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.
      ). However, it is not yet established whether any or all of these REHs are physiologically important in retinyl ester/retinol metabolism. The reader is referred to the review of Eroglu and Harrison (
      • Eroglu A.
      • Harrison E.H.
      Carotenoid metabolism in mammals including man: formation, occurrence and function of apocarotenoids.
      ) in this thematic review series for further consideration of this point.

      RETINOID-BINDING PROTEINS

      To solubilize, protect, and detoxify retinoids in the aqueous intracellular and extracellular environment, retinol, retinal, and retinoic acid are bound to specific retinoid-binding proteins (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ). These binding proteins can be classified using several different criteria. Some of these proteins, specifically retinol-binding protein 4 (RBP4), interphotoreceptor matrix retinoid-binding protein (IRBP), epididymal retinoid-binding protein (ERBP), and β-trace, are found in extracellular fluids, whereas the remaining are found only intracellularly (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Ong D.E.
      • Newcomer M.E.
      • Lareyre J.J.
      • Orgebin-Crist M.C.
      Epididymal retinoic acid-binding protein.
      ,
      • Fujimori K.
      • Aritake K.
      • Urade Y.
      A novel pathway to enhance adipocyte differentiation of 3T3–L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c.
      ,
      • Tanaka T.
      • Urade Y.
      • Kimura H.
      • Eguchi N.
      • Nishikawa A.
      • Hayaishi O.
      Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter.
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ,
      • Levin M.S.
      • Locke B.
      • Yang N.C.
      • Li E.
      • Gordon J.I.
      Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.
      ,
      • Saari J.C.
      • Bredberg L.
      • Garwin G.G.
      Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.
      ,
      • Vogel S.
      • Mendelsohn C.L.
      • Mertz J.R.
      • Piantedosi R.
      • Waldburger C.
      • Gottesman M.E.
      • Blaner W.S.
      Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
      ,
      • Folli C.
      • Calderone V.
      • Ramazzina I.
      • Zanotti G.
      • Berni R.
      Ligand binding and structural analysis of a human putative cellular retinol-binding protein.
      ,
      • Panagabko C.
      • Morley S.
      • Hernandez M.
      • Cassolato P.
      • Gordon H.
      • Parsons R.
      • Manor D.
      • Atkinson J.
      Ligand specificity in the CRAL-TRIO protein family.
      ). Of the intracellular-binding proteins, some bind only retinoic acid [cellular retinoic acid-binding protein, type I (CRABPI) and cellular retinoic acid-binding protein, type II (CRABPII)] (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ); some preferentially bind both retinol and retinal [cellular retinol-binding protein, type I (CRBPI) (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Levin M.S.
      • Locke B.
      • Yang N.C.
      • Li E.
      • Gordon J.I.
      Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.
      ,
      • Saari J.C.
      • Bredberg L.
      • Garwin G.G.
      Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.
      ,
      • Vogel S.
      • Mendelsohn C.L.
      • Mertz J.R.
      • Piantedosi R.
      • Waldburger C.
      • Gottesman M.E.
      • Blaner W.S.
      Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
      ) and cellular retinol-binding protein, type II (CRBPII) (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ,
      • Levin M.S.
      • Locke B.
      • Yang N.C.
      • Li E.
      • Gordon J.I.
      Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.
      )]; some preferentially bind retinol [cellular retinol-binding protein, type III (CRBPIII) (
      • Vogel S.
      • Mendelsohn C.L.
      • Mertz J.R.
      • Piantedosi R.
      • Waldburger C.
      • Gottesman M.E.
      • Blaner W.S.
      Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
      ) and cellular retinol-binding protein, type IV (CRBPIV) (
      • Folli C.
      • Calderone V.
      • Ramazzina I.
      • Zanotti G.
      • Berni R.
      Ligand binding and structural analysis of a human putative cellular retinol-binding protein.
      )]; and one preferentially binds retinal [cellular retinal-binding protein (CRALBP) (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ,
      • Saari J.C.
      • Bredberg L.
      • Garwin G.G.
      Identification of the endogenous retinoids associated with three cellular retinoid-binding proteins from bovine retina and retinal pigment epithelium.
      ,
      • Panagabko C.
      • Morley S.
      • Hernandez M.
      • Cassolato P.
      • Gordon H.
      • Parsons R.
      • Manor D.
      • Atkinson J.
      Ligand specificity in the CRAL-TRIO protein family.
      )]. These proteins can also be grouped by the protein families to which they belong. RBP4, ERBP, and β-trace are all members of the lipocalin protein family (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Ong D.E.
      • Newcomer M.E.
      • Lareyre J.J.
      • Orgebin-Crist M.C.
      Epididymal retinoic acid-binding protein.
      ,
      • Fujimori K.
      • Aritake K.
      • Urade Y.
      A novel pathway to enhance adipocyte differentiation of 3T3–L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c.
      ,
      • Tanaka T.
      • Urade Y.
      • Kimura H.
      • Eguchi N.
      • Nishikawa A.
      • Hayaishi O.
      Lipocalin-type prostaglandin D synthase (beta-trace) is a newly recognized type of retinoid transporter.
      ). CRBPI–IV as well as CRABPI and CRABPII are members of the fatty acid-binding protein family of proteins (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Ong D.E.
      • Newcomer M.E.
      • Lareyre J.J.
      • Orgebin-Crist M.C.
      Epididymal retinoic acid-binding protein.
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ,
      • Levin M.S.
      • Locke B.
      • Yang N.C.
      • Li E.
      • Gordon J.I.
      Comparison of the ligand binding properties of two homologous rat apocellular retinol-binding proteins expressed in Escherichia coli.
      ,
      • Vogel S.
      • Mendelsohn C.L.
      • Mertz J.R.
      • Piantedosi R.
      • Waldburger C.
      • Gottesman M.E.
      • Blaner W.S.
      Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
      ,
      • Folli C.
      • Calderone V.
      • Ramazzina I.
      • Zanotti G.
      • Berni R.
      Ligand binding and structural analysis of a human putative cellular retinol-binding protein.
      ). CRALBP is a member of the CRAL-TRIO protein family that also contains the vitamin E-binding protein, α-tocopherol-transport protein (TTP) (
      • Panagabko C.
      • Morley S.
      • Hernandez M.
      • Cassolato P.
      • Gordon H.
      • Parsons R.
      • Manor D.
      • Atkinson J.
      Ligand specificity in the CRAL-TRIO protein family.
      ). For more details on TTP and vitamin E, the reader is referred to the review from Traber in this thematic review series (
      • Traber M.D.
      Mechanisms for the prevention of vitamin E excess.
      ).
      Each of the known retinoid-binding proteins has been proposed to have a role in facilitating retinoid transport and/or metabolism. However, most if not all of these proteins do not have essential roles in facilitating these processes, as the genes for nearly all of these retinoid-binding proteins have been ablated in mouse models and none of the gene disruptions is lethal or associated with a severe phenotype (
      • Ghyselinck N.B.
      • Bavik C.
      • Sapin V.
      • Mark M.
      • Bonnier D.
      • Hindelang C.
      • Dierich A.
      • Nilsson C.B.
      • Hakansson H.
      • Sauvant P.
      • et al.
      Cellular retinol-binding protein I is essential for vitamin A homeostasis.
      ,
      • Gorry P.
      • Lufkin T.
      • Dierich A.
      • Rochette-Egly C.
      • Decimo D.
      • Dolle P.
      • Mark M.
      • Durand B.
      • Chambon P.
      The cellular retinoic acid binding protein I is dispensable.
      ,
      • Lampron C.
      • Rochette-Egly C.
      • Gorry P.
      • Dolle P.
      • Mark M.
      • Lufkin T.
      • LeMeur M.
      • Chambon P.
      Mice deficient in cellular retinoic acid binding protein II (CRABPII) or in both CRABPI and CRABPII are essentially normal.
      ,
      • Liou G.I.
      • Fei Y.
      • Peachey N.S.
      • Matragoon S.
      • Wei S.
      • Blaner W.S.
      • Wang Y.
      • Liu C.
      • Gottesman M.E.
      • Ripps H.
      Early onset photoreceptor abnormalities induced by targeted disruption of the interphotoreceptor retinoid-binding protein gene.
      ,
      • Quadro L.
      • Blaner W.S.
      • Salchow D.J.
      • Vogel S.
      • Piantedosi R.
      • Gouras P.
      • Freeman S.
      • Cosma M.P.
      • Colantuoni V.
      • Gottesman M.E.
      Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein.
      ,
      • Saari J.C.
      • Nawrot M.
      • Kennedy B.N.
      • Garwin G.G.
      • Hurley J.B.
      • Huang J.
      • Possin D.E.
      • Crabb J.W.
      Visual cycle impairment in cellular retinaldehyde binding protein (CRALBP) knockout mice results in delayed dark adaptation.
      ,
      • Lu E.X.J.
      • Tso P.
      • Blaner W.S.
      • Levine M.S.
      • Li E.
      Increased neonatal mortality in mice lacking cellular retinol-binding protein II.
      ). It seems likely that these proteins are needed to facilitate optimal retinoid retention, transport, and metabolism. When dietary retinoid is plentiful, the actions of the binding proteins are not essential for maintaining retinoid status of the body or the health of the organism. However, in times of dietary retinoid insufficiency, the binding proteins and the enhanced metabolic efficiency and retention that they afford convey an advantage to the organism.

      INTESTINAL ABSORPTION

      The two most abundant retinoid forms that are present in the diet are retinol and retinyl esters. Dietary retinol is taken up directly by mucosal cells. However, dietary retinyl esters are unable to enter the intestinal mucosa and must first be acted upon by a luminal REH to yield free retinol. Retinyl esters can be hydrolyzed within the intestinal lumen by nonspecific pancreatic enzymes, such as pancreatic triglyceride lipase and cholesteryl ester hydrolase, or at the mucosal cell surface where a retinyl ester hydrolase is associated with the intestinal brush boarder (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). The free retinol formed upon hydrolysis of the retinyl ester or unesterified retinol arriving as such from the diet is taken up into the intestinal cells (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). In contrast to dietary preformed retinoid, dietary proretinoid carotenoids, such as β-carotene, can be either converted to retinal within the enterocyte or absorbed unmodified by these cells. The intestinal enzyme responsible for the cleavage of proretinoid carotenoids to retinal is β-carotene-15,15′-monooxygenase (BCMO1) (see Fig. 2) (
      • von Lintig J.
      Colors with functions: elucidating the biochemical and molecular basis of carotenoid metabolism.
      ). CRBPII is present at high concentrations in the enterocytes and binds both retinal and retinol (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Herr F.M.
      • Ong D.E.
      Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.
      ). Retinal, formed upon proretinoid carotenoid cleavage by BCMO1, binds to CRBPII, and this is proposed to be the preferred substrate for reduction to retinol by an intestinal retinal reductase. Retinol bound to CRBPII is then reesterified to long-chain fatty acids predominantly through the action of LRAT, which utilizes retinol bound to CRBPII as a substrate for esterification (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      Retinoid-binding proteins: mediators of retinoid action.
      ,
      • Herr F.M.
      • Ong D.E.
      Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins.
      ). The resulting retinyl esters are then packaged along with the rest of the dietary lipids into nascent chylomicrons and secreted into the lymphatic system for uptake into the general circulation (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). The molecules and molecular events responsible for the intestinal absorption of retinoids and carotenoids are considered in more detail elsewhere in this thematic review series (
      • Eroglu A.
      • Harrison E.H.
      Carotenoid metabolism in mammals including man: formation, occurrence and function of apocarotenoids.
      ).

      TRANSPORT IN THE POSTPRANDIAL AND FASTING CIRCULATIONS

      A number of different retinoids are found in the circulation, and these differ in the fasting and postprandial states. These potential retinoid delivery pathways are summarized in Fig. 3. They include retinyl esters in chylomicrons, chylomicron remnants, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high density lipoprotein (HDL); retinol bound to retinol-binding protein (RBP4); retinoic acid bound to albumin; and the water-soluble β-glucuronides of retinol and retinoic acid. In the postprandial circulation following consumption of a retinoid-rich meal, retinyl ester concentrations in the 5–10 µM range can be reached (
      • Arnhold T.
      • Tzimas G.
      • Wittfoht W.
      • Plonait S.
      • Nau H.
      Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption.
      ), although this will depend directly on the quantity of retinoid consumed in the meal. The liver secretes some retinyl ester bound to nascent VLDL, and upon metabolism of the VLDL, some of this retinyl ester can be found in LDL or transferred to HDL in species that express cholesteryl ester transfer protein. Concentrations of retinyl esters in the fasting circulation can vary but are generally in the range of 100–200 nM (
      • Relas H.
      • Gylling H.
      • Miettinen T.A.
      Effect of stanol ester on postabsorptive squalene and retinyl palmitate.
      ). In the fasting circulation, retinol bound to RBP4 is the predominant retinoid species, with normal concentrations ranging from 2–4 µM in humans and around 1 µM in mice (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ,
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Chuwers P.
      • Barnhart S.
      • Blanc P.
      • Brodkin C.A.
      • Cullen M.
      • Kelly T.
      • Keogh J.
      • Omenn G.
      • Williams J.
      • Balmers J.R.
      The protective effect of beta-carotene and retinol on ventilatory function in an asbestos-exposed cohort.
      ). In the circulation, the retinol-RBP4 complex binds another plasma protein, transthyretin (TTR) and this stabilizes the complex, reducing renal filtration of the retinoid (
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • van Bennekum A.M.
      • Wei S.
      • Gamble M.V.
      • Vogel S.
      • Piantedosi R.
      • Gottesman M.
      • Episkopou V.
      • Blaner W.S.
      Biochemical basis for depressed serum retinol levels in transthyretin-deficient mice.
      ).
      Figure thumbnail gr3
      Fig. 3The delivery of retinoids and carotenoids through the circulation to cells. Retinoids and proretinoid carotenoids are delivered to cells and tissues through a number of alternative delivery pathways. In the fasting circulation, retinol delivered via RBP4 (1) accounts for most delivery to cells/tissues. However, in the postprandial state, retinyl esters present in chylomicrons and their remnants (2) can contribute substantially to the retinoid taken up by cells. Similarly, proretinoid carotenoids, such as β-carotene, are present in the postprandial circulation (4) and this can be taken up by cells/tissues and converted to retinoid. Retinyl esters and carotenoids (3) are also present in VLDL, LDL, and HDL in the fasting circulation. Retinoic acid is present in both the fasting and postprandial circulation (7), albeit at relatively low levels compared with retinol and retinyl esters. The water-soluble retinyl- (5) and retinoyl-β-glucuronides (6) are also present at relatively low levels in the circulation.
      Retinoic acid is present in both the fasting and postprandial circulations where it is found bound to albumin. Nau and colleagues have reported that immediately following consumption of a retinoid-rich meal consisting of 100 g of turkey liver, human blood levels of retinoic acid can reach 80–90 nM (
      • Arnhold T.
      • Tzimas G.
      • Wittfoht W.
      • Plonait S.
      • Nau H.
      Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption.
      ). However, this level is quickly restored by the body to fasting levels that range 1–3 nM (
      • Arnhold T.
      • Tzimas G.
      • Wittfoht W.
      • Plonait S.
      • Nau H.
      Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption.
      ,
      • Kurlandsky S.B.
      • Gamble M.V.
      • Ramakrishnan R.
      • Blaner W.S.
      Plasma delivery of retinoic acid to tissues in the rat.
      ). The half-life of plasma all-trans-retinoic acid in a fasted, chow-fed rat is very short, with the plasma pool completely turning over every 2 min (
      • Kurlandsky S.B.
      • Gamble M.V.
      • Ramakrishnan R.
      • Blaner W.S.
      Plasma delivery of retinoic acid to tissues in the rat.
      ). The studies of Nau and colleagues (
      • Arnhold T.
      • Tzimas G.
      • Wittfoht W.
      • Plonait S.
      • Nau H.
      Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption.
      ) suggest that the intestine contributes significantly to the retinoic acid that is present in the postprandial circulation. The tissues responsible for contributing retinoic acid to the fasting circulation remain to be established. At present, it is unclear whether only one or a few tissues contribute to the circulating retinoic acid pool or whether retinoic acid is simply “leaking” into the fasting circulation from most or all tissues.
      Plasma/serum concentrations of retinyl- and retinoyl-β-glucuronides were reported by Olson and colleagues to be in the range of 5–15 nM (
      • Barua A.B.
      • Batres R.O.
      • Olson J.A.
      Characterization of retinyl beta-glucuronide in human blood.
      ,
      • Barua A.B.
      • Olson J.A.
      Retinoyl beta-glucuronide: an endogenous compound of human blood.
      ). Although it has been proposed that retinyl- and retinoyl-β-glucuronides, which are known to be readily hydrolyzed by a number of β-glucuronidases (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ), may serve as sources of retinoids for tissues, it is generally believed that these fully water-soluble metabolites are filtered in the kidney and eliminated quickly from the body.
      In addition to preformed retinoid delivery through the circulation, proretinoid carotenoids, such as β-carotene, are absorbed intact by the intestine, and these too can be found in the blood bound to chylomicrons and their remnants, VLDL, LDL, and HDL (
      • Redlich C.A.
      • Grauer J.N.
      • Van Bennekum A.M.
      • Clever S.L.
      • Ponn R.B.
      • Blaner W.S.
      Characterization of carotenoid, vitamin A, and alpha-tocopheral levels in human lung tissue and pulmonary macrophages.
      ,
      • Redlich C.A.
      • Chung J.S.
      • Cullen M.R.
      • Blaner W.S.
      • Van Bennekum A.M.
      • Berglund L.
      Effect of long-term beta-carotene and vitamin A on serum cholesterol and triglyceride levels among participants in the Carotene and Retinol Efficacy Trial (CARET).
      ). Since many tissues, including liver, lungs, and testes, express BCMO1, intact carotenoid delivered to these tissues can be converted in situ to retinoids that may be needed for supporting retinoid-dependent functions. Fasting blood levels of the canonical proretinoid carotenoid β-carotene in humans, a species that absorbs carotenoids well, can be as great as 5–8 µM (
      • Redlich C.A.
      • Chung J.S.
      • Cullen M.R.
      • Blaner W.S.
      • Van Bennekum A.M.
      • Berglund L.
      Effect of long-term beta-carotene and vitamin A on serum cholesterol and triglyceride levels among participants in the Carotene and Retinol Efficacy Trial (CARET).
      ). Although the mouse, owing to its ability to be genetically manipulated, is presently being used to study β-carotene uptake from the diet as well as β-carotene metabolism and physiologic actions, it should be noted that rodents are very poor absorbers of β-carotene. Consequently, studies involving the feeding of β-carotene to mice must employ very high concentrations of β-carotene in the diet, concentrations that are many times greater than those that would be found in any human diet.
      As can be surmised from the text above, the delivery of retinoids to tissues is complex, involving many different retinoid forms and carriers. Quantitatively the two most important pathways are those involving retinol bound to RBP4 and the postprandial delivery pathway. However, the importance of retinoic acid delivery to tissues from the blood should not be discounted, since the accumulation of retinoic acid from the blood is tissue dependent (
      • Kurlandsky S.B.
      • Gamble M.V.
      • Ramakrishnan R.
      • Blaner W.S.
      Plasma delivery of retinoic acid to tissues in the rat.
      ). In this regard, the delivery of retinoid to tissues, as either retinol or retinoic acid, is not different from the delivery of vitamin D or thyroid hormone, involving the presence of relatively large concentrations of the transcriptionally inactive precursor (retinol, 25-hydroxy-vitamin D, or T4) and relatively low concentrations of the transcriptionally active metabolite (retinoic acid, 1,25-dihydroxy-vitamin D, or T3).

      CELLULAR UPTAKE OF RETINOIDS

      Retinyl esters in chylomicrons enter the circulation and are taken up by tissues as the chylomicron undergoes lipolysis and remodeling. Approximately 66–75% of chylomicron retinyl ester is cleared by the liver, and the remainder is cleared by peripheral tissues (
      • Goodman D.W.
      • Huang H.S.
      • Shiratori T.
      Tissue distribution and metabolism of newly absorbed vitamin A in the rat.
      ). Prior to uptake by peripheral tissues, chylomicron retinyl ester must undergo hydrolysis. It has been proposed that the enzyme lipoprotein lipase (LpL) performs this function in peripheral tissues, facilitating retinol uptake (
      • Blaner W.S.
      • Obunike J.C.
      • Kurlandsky S.B.
      • al-Haideri M.
      • Piantedosi R.
      • Deckelbaum R.J.
      • Goldberg I.J.
      Lipoprotein lipase hydrolysis of retinyl ester. Possible implications for retinoid uptake by cells.
      ). Postprandial unesterified retinol taken up by cells is thought to bind immediately to CRBPs that are present in tissues (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      • Blaner W.S.
      Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein.
      ). As noted above, it has been suggested that CRBPs facilitate/optimize retinol uptake process and facilitate its subsequent metabolism (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Noy N.
      • Blaner W.S.
      Interactions of retinol with binding proteins: studies with rat cellular retinol-binding protein and with rat retinol-binding protein.
      ). In the liver, retinyl ester hydrolysis occurs as the chylomicron remnant particle is internalized by hepatocytes during the early stages of endosome formation (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ). However, it is not presently established what enzyme(s) is responsible for hydrolysis, although one of several carboxyesterases may play a role in this process. Once retinol is formed upon retinyl ester hydrolysis within the hepatocyte, it is quickly bound by apo-CRBPI, which is in molar excess of retinol in these cells (
      • Blaner W.S.
      Retinoid (Vitamin A) metabolism and the liver.
      ,
      • Harrison E.H.
      • Blaner W.S.
      • Goodman D.S.
      • Ross A.C.
      Subcellular localization of retinoids, retinoid-binding proteins, and acyl-CoA:retinol acyltransferase in rat liver.
      ).
      How retinol is taken up by cells from the circulating retinol-RBP4 complex has been the subject of much research interest for many years. Studies of intestinal cells imply that retinol enters by diffusion, and this is likely true for other cell types (
      • During A.
      • Hussain M.M.
      • Morel D.W.
      • Harrison E.H.
      Carotenoid uptake and secretion by CaCo-2 cells: beta-carotene isomer selectivity and carotenoid interactions.
      ,
      • During A.
      • Harrison E.H.
      Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells.
      ,
      • During A.
      • Harrison E.H.
      Intestinal absorption and metabolism of carotenoids: insights from cell culture.
      ,
      • During A.
      • Dawson H.D.
      • Harrison E.H.
      Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe.
      ). However, in 2007, a cell surface receptor for RBP4 termed STRA6 (stimulated by retinoic acid 6) was identified (
      • Blaner W.S.
      STRA6, a cell-surface receptor for retinol-binding protein: the plot thickens.
      ,
      • Kawaguchi R.
      • Yu J.
      • Honda J.
      • Hu J.
      • Whitelegge J.
      • Ping P.
      • Wiita P.
      • Bok D.
      • Sun H.
      A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.
      ). STRA6 is expressed in a number of tissues/cells that have a high demand for retinoid, especially the retinal pigmented epithelium (RPE) cells of the eye, but it is not expressed in many others, including liver cell types (
      • Blaner W.S.
      STRA6, a cell-surface receptor for retinol-binding protein: the plot thickens.
      ,
      • Kawaguchi R.
      • Yu J.
      • Honda J.
      • Hu J.
      • Whitelegge J.
      • Ping P.
      • Wiita P.
      • Bok D.
      • Sun H.
      A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.
      ,
      • Pasutto F.
      • Sticht H.
      • Hammersen G.
      • Gillessen-Kaesbach G.
      • Fitzpatrick D.R.
      • Nurnberg G.
      • Brasch F.
      • Schirmer-Zimmermann H.
      • Tolmie J.L.
      • Chitayat D.
      • et al.
      Mutations in STRA6 cause a broad spectrum of malformations including anophthalmia, congenital heart defects, diaphragmatic hernia, alveolar capillary dysplasia, lung hypoplasia, and mental retardation.
      ,
      • Bouillet P.
      • Sapin V.
      • Chazaud C.
      • Messaddeq N.
      • Decimo D.
      • Dolle P.
      • Chambon P.
      Developmental expression pattern of Stra6, a retinoic acid-responsive gene encoding a new type of membrane protein.
      ). This receptor interacts with RBP4 and increases cellular uptake of retinol (
      • Kawaguchi R.
      • Yu J.
      • Honda J.
      • Hu J.
      • Whitelegge J.
      • Ping P.
      • Wiita P.
      • Bok D.
      • Sun H.
      A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.
      ). In addition, STRA6 is reported to facilitate retinol efflux from cells (
      • Isken A.
      • Golczak M.
      • Oberhauser V.
      • Hunzelmann S.
      • Driever W.
      • Imanishi Y.
      • Palczewski K.
      • von Lintig J.
      RBP4 disrupts vitamin A uptake homeostasis in a STRA6-deficient animal model for Matthew-Wood syndrome.
      ,
      • Kim Y.K.
      • Wassef L.
      • Hamberger L.
      • Piantedosi R.
      • Palczewski K.
      • Blaner W.S.
      • Quadro L.
      Retinyl ester formation by lecithin:retinol acyltransferase is a key regulator of retinoid homeostasis in mouse embryogenesis.
      ,
      • Kawaguchi R.
      • Zhong M.
      • Kassai M.
      • Ter-Stepanian M.
      • Sun H.
      STRA6-catalyzed vitamin A influx, efflux, and exchange.
      ). Both LRAT and CRBPI are proposed to couple with STRA6 within the cell (
      • Kawaguchi R.
      • Yu J.
      • Honda J.
      • Hu J.
      • Whitelegge J.
      • Ping P.
      • Wiita P.
      • Bok D.
      • Sun H.
      A membrane receptor for retinol binding protein mediates cellular uptake of vitamin A.
      ,
      • Kawaguchi R.
      • Zhong M.
      • Kassai M.
      • Ter-Stepanian M.
      • Sun H.
      STRA6-catalyzed vitamin A influx, efflux, and exchange.
      ,
      • Amengual J.
      • Golczak M.
      • Palczewski K.
      • von Lintig J.
      Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein.
      ). von Lintig and colleagues have convincingly established that the functional coupling of LRAT with STRA6 increases cellular retinol uptake into tissues and have proposed that LRAT is a critical component of this process (
      • Amengual J.
      • Golczak M.
      • Palczewski K.
      • von Lintig J.
      Lecithin:retinol acyltransferase is critical for cellular uptake of vitamin A from serum retinol-binding protein.
      ). In vitro experiments have established that STRA6 facilitates efficient retinol exchange between intercellular CRBPI and extracellular RBP4 (
      • Kawaguchi R.
      • Zhong M.
      • Kassai M.
      • Ter-Stepanian M.
      • Sun H.
      STRA6-catalyzed vitamin A influx, efflux, and exchange.
      ). Recently, mice lacking STRA6 were generated and studied (
      • Ruiz A.
      • Mark M.
      • Jacobs H.
      • Klopfenstein M.
      • Hu J.
      • Lloyd M.
      • Habib S.
      • Tosha C.
      • Radu R.A.
      • Ghyselinck N.B.
      • et al.
      Retinoid content, visual responses, and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6.
      ). These mice are viable and generally normal. The primary phenotype of the Stra6-deficient mice is a visual one, similar to the mice lacking Rbp4 (
      • Quadro L.
      • Blaner W.S.
      • Salchow D.J.
      • Vogel S.
      • Piantedosi R.
      • Gouras P.
      • Freeman S.
      • Cosma M.P.
      • Colantuoni V.
      • Gottesman M.E.
      Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein.
      ,
      • Ruiz A.
      • Mark M.
      • Jacobs H.
      • Klopfenstein M.
      • Hu J.
      • Lloyd M.
      • Habib S.
      • Tosha C.
      • Radu R.A.
      • Ghyselinck N.B.
      • et al.
      Retinoid content, visual responses, and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6.
      ). Interestingly, the results of studies of Stra6-deficient mice suggest that there are other pathways facilitating retinol uptake into the RPE, but that the one involving STRA6 is the most important one (
      • Ruiz A.
      • Mark M.
      • Jacobs H.
      • Klopfenstein M.
      • Hu J.
      • Lloyd M.
      • Habib S.
      • Tosha C.
      • Radu R.A.
      • Ghyselinck N.B.
      • et al.
      Retinoid content, visual responses, and ocular morphology are compromised in the retinas of mice lacking the retinol-binding protein receptor, STRA6.
      ).
      Over the last several years, a large number of published reports have suggested a link between RBP4 and obesity, diabetes, and insulin signaling. The first of these studies looked at the impact of RBP4 levels in various mouse and human models (
      • Yang Q.
      • Graham T.E.
      • Mody N.
      • Preitner F.
      • Peroni O.D.
      • Zabolotny J.M.
      • Kotani K.
      • Quadro L.
      • Kahn B.B.
      Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.
      ,
      • Graham T.E.
      • Yang Q.
      • Bluher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • et al.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Polonsky K.S.
      Retinol-binding protein 4, insulin resistance, and type 2 diabetes.
      ). RBP4 levels appear to be increased in human obesity and this increase impairs insulin signaling, possibly contributing to the development of type 2 diabetes, although the published studies are sometimes not in agreement (
      • Yang Q.
      • Graham T.E.
      • Mody N.
      • Preitner F.
      • Peroni O.D.
      • Zabolotny J.M.
      • Kotani K.
      • Quadro L.
      • Kahn B.B.
      Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.
      ,
      • Graham T.E.
      • Yang Q.
      • Bluher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • et al.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Polonsky K.S.
      Retinol-binding protein 4, insulin resistance, and type 2 diabetes.
      ,
      • Munkhtulga L.
      • Nakayama K.
      • Utsumi N.
      • Yanagisawa Y.
      • Gotoh T.
      • Omi T.
      • Kumada M.
      • Erdenebulgan B.
      • Zolzaya K.
      • Lkhagvasuren T.
      • et al.
      Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia.
      ,
      • Janke J.
      • Engeli S.
      • Boschmann M.
      • Adams F.
      • Bohnke J.
      • Luft F.C.
      • Sharma A.M.
      • Jordan J.
      Retinol-binding protein 4 in human obesity.
      ,
      • Gomez-Ambrosi J.
      • Rodriguez A.
      • Catalan V.
      • Ramirez B.
      • Silva C.
      • Rotellar F.
      • Gil M.J.
      • Salvador J.
      • Fruhbeck G.
      Serum retinol-binding protein 4 is not increased in obesity or obesity-associated type 2 diabetes mellitus, but is reduced after relevant reductions in body fat following gastric bypass.
      ,
      • Kotnik P.
      • Fischer-Posovszky P.
      • Wabitsch M.
      RBP4: a controversial adipokine.
      ). There is also evidence that STRA6 may be involved in mediating this effect of RBP4 on insulin signaling (
      • Berry D.C.
      • Jin H.
      • Majumdar A.
      • Noy N.
      Signaling by vitamin A and retinol-binding protein regulates gene expression to inhibit insulin responses.
      ,
      • Berry D.C.
      • Noy N.
      Signaling by vitamin A and retinol-binding protein in regulation of insulin responses and lipid homeostasis.
      ). We will further consider this topic below in the section Retinoids in Adipose Tissue.
      Unlike retinol delivered bound to RBP4, which is thought to be taken up by cells through a process involving a cell surface receptor, RA uptake into tissues is not presently thought to involve a cell surface receptor. It is well established experimentally that RA can “flip-flop” across a phospholipid bilayer, and it is generally assumed that it is taken up into cells from the circulation through this process (
      • Noy N.
      The ionization behavior of retinoic acid in lipid bilayers and in membranes.
      ,
      • Noy N.
      The ionization behavior of retinoic acid in aqueous environments and bound to serum albumin.
      ). This same “flip-flop” mechanism was earlier proposed to account for most unesterified fatty acid uptake by cells across the plasma membrane from the circulation (
      • Kamp F.
      • Hamilton J.A.
      • Kamp F.
      • Westerhoff H.V.
      • Hamilton J.A.
      Movement of fatty acids, fatty acid analogues, and bile acids across phospholipid bilayers.
      ). However, it is now widely accepted that several plasma membrane proteins are responsible for most unesterified fatty acid uptake by tissues (
      • Schwenk R.W.
      • Holloway G.P.
      • Luiken J.J.
      • Bonen A.
      • Glatz J.F.
      Fatty acid transport across the cell membrane: regulation by fatty acid transporters.
      ). The possibility that RA also may be taken up into cells by one or more cell surface receptors has not been systematically explored and remains to be established.
      Retinoids and proretinoid carotenoids present in VLDL and LDL are presumably taken up along with the lipoprotein particles by their cell surface receptors, but this too has not been systematically investigated. Retinyl- and retinoyl-β-glucuronides are fully water soluble and have been proposed to serve as a source of retinoids for use by tissues, but it is unclear whether and how these may be taken up by cells (
      • Barua A.B.
      • Batres R.O.
      • Olson J.A.
      Characterization of retinyl beta-glucuronide in human blood.
      ,
      • Barua A.B.
      • Olson J.A.
      Retinoyl beta-glucuronide: an endogenous compound of human blood.
      ).

      HEPATIC STORAGE

      It is well established that hepatocytes are responsible for the uptake of chylomicron remnant retinoid into the liver (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ,
      • Geerts A.
      • Bleser P.D.
      • Hautekeete M.L.
      • Niki T.
      • Wisse E.
      Fat storing (Ito) cell biology.
      ). Hepatocytes not only take up postprandial retinoid into the liver but also account for about 10–20% of all of the retinoid stored within the liver, and they are the sole cellular site of RBP4 synthesis in the liver (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Moreover, hepatocytes possess enzymatic activities needed for the hydrolysis of retinyl esters and the synthesis and catabolism of retinoic acid. After postprandial retinoid is taken up by the hepatocyte, this retinoid is either secreted back into the circulation bound to RBP4 (see above for more details) or is transferred to the hepatic stellate cells (HSC) for storage (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). It has been estimated that for healthy, well-nourished individuals, approximately 70% of the retinoid present in the body will be stored in the liver and approximately 70–90% of this is found in HSCs (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Within HSCs, retinoid is stored as retinyl esters in the large lipid droplets that are characteristic of these cells (see Fig. 4) (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ,
      • Geerts A.
      • Bleser P.D.
      • Hautekeete M.L.
      • Niki T.
      • Wisse E.
      Fat storing (Ito) cell biology.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Nearly all of the retinoid present in HSCs is retinyl ester (primarily retinyl palmitate, with smaller amounts of retinyl stearate, retinyl oleate, and retinyl linoleate) (
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ,
      • Geerts A.
      • Bleser P.D.
      • Hautekeete M.L.
      • Niki T.
      • Wisse E.
      Fat storing (Ito) cell biology.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Normally, unesterified retinol accounts for less than 1% of the total retinoid present within these cells.
      Figure thumbnail gr4
      Fig. 4The genetic ablation of Lrat results in the total absence of lipid droplets in HSCs. Electron micrographs of liver sections obtained from age-, gender-, and genetic background matched wild-type (left panel) and Lrat-deficient (right panel) mice maintained throughout life on a retinoid-sufficient chow diet. The large lipid droplets, indicated by the arrow in the left panel, are a characteristic morphological feature of HSCs, which are clearly present in the wild-type liver, whereas but totally absent in liver from Lrat-deficient mice. H, hepatocyte.
      When the body senses a need for retinoid, hepatic retinyl esters are hydrolyzed by REHs to free retinol, which through some poorly characterized process, is mobilized from the liver bound to its plasma transport protein, RBP4. Mice lacking RBP4 are unable to mobilize their retinoid stores, which are effectively trapped within the liver (
      • Quadro L.
      • Blaner W.S.
      • Salchow D.J.
      • Vogel S.
      • Piantedosi R.
      • Gouras P.
      • Freeman S.
      • Cosma M.P.
      • Colantuoni V.
      • Gottesman M.E.
      Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein.
      ,
      • Quadro L.
      • Blaner W.S.
      • Hamberger L.
      • Novikoff P.M.
      • Vogel S.
      • Piantedosi R.
      • Gottesman M.E.
      • Colantuoni V.
      The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.
      ,
      • Quadro L.
      • Hamberger L.
      • Colantuoni V.
      • Gottesman M.E.
      • Blaner W.S.
      Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models.
      ). It remains to be established how a signal is conveyed from peripheral tissues to the liver regarding their need for retinoid, thus stimulating retinoid mobilization from hepatic stores. Retinol-RBP4 is secreted from the liver into the circulation as a means of delivering retinol to peripheral tissues (
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Quadro L.
      • Blaner W.S.
      • Salchow D.J.
      • Vogel S.
      • Piantedosi R.
      • Gouras P.
      • Freeman S.
      • Cosma M.P.
      • Colantuoni V.
      • Gottesman M.E.
      Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein.
      ). The liver is the major site of synthesis of RBP4 in the body, and within the liver, the hepatocyte is the sole cellular site of RBP4 synthesis (
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Other tissues, including adipose tissue, kidney, lung, heart, skeletal muscle, spleen, eyes, and testes, express RBP4 (
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ), which may be important for recycling retinoid from peripheral tissues back to the liver (
      • Soprano D.R.
      • Blaner W.S.
      Plasma retinol-binding protein.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ).
      One of the key unanswered questions regarding hepatic retinoid storage and metabolism is how retinoid(s) is transported between hepatocytes and HSCs. It is well established that postprandial retinoid is taken up by hepatocytes and equally well established that HSCs are the major cellular storage site for retinoid in the liver (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • Hendriks H.F.
      • Brouwer A.
      • de Leeuw A.M.
      • Knook D.L.
      • Goodman D.S.
      Retinoids, retinoid-binding proteins, and retinyl palmitate hydrolase distributions in different types of rat liver cells.
      ,
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). This immediately raises the question as to how dietary retinoid is transferred from hepatocytes to HSCs for storage. The early literature proposed that RBP4 was responsible for this intercellular movement, and it was generally accepted that RBP4 was responsible for intercellular retinoid movement within the liver (
      • Blomhoff R.
      • Green M.H.
      • Green J.B.
      • Berg T.
      • Norum K.R.
      Vitamin A metabolism: new perspectives on absorption, transport, and storage.
      ). However, studies of Rbp4-deficient mice indicate that this cannot be correct, since these mutant mice possess normal HSC lipid droplet retinyl ester stores (
      • Quadro L.
      • Blaner W.S.
      • Salchow D.J.
      • Vogel S.
      • Piantedosi R.
      • Gouras P.
      • Freeman S.
      • Cosma M.P.
      • Colantuoni V.
      • Gottesman M.E.
      Impaired retinal function and vitamin A availability in mice lacking retinol-binding protein.
      ,
      • Quadro L.
      • Blaner W.S.
      • Hamberger L.
      • Novikoff P.M.
      • Vogel S.
      • Piantedosi R.
      • Gottesman M.E.
      • Colantuoni V.
      The role of extrahepatic retinol binding protein in the mobilization of retinoid stores.
      ,
      • Quadro L.
      • Hamberger L.
      • Colantuoni V.
      • Gottesman M.E.
      • Blaner W.S.
      Understanding the physiological role of retinol-binding protein in vitamin A metabolism using transgenic and knockout mouse models.
      ). This implies that other proteins/factors must be responsible for retinoid transport between these two hepatic cell types. Moreover, this transport needs to be bidirectional. Since the hepatocyte is the site of RBP4 synthesis in the liver, when dietary retinoid is insufficient, retinol newly released from HSC retinyl ester stores must be transported back to hepatocytes where it binds nascent apo-RBP4 for secretion into the circulation and delivery to peripheral tissues. A few early investigators proposed that CRBPI, which is found in high abundance in both hepatocytes and HSCs, mediates retinol transfer possibly by moving through/across cell-to-cell contacts that exist between hepatocytes and HSCs (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      Retinoid (Vitamin A) metabolism and the liver.
      ). Other early authors suggested that some unidentified protein was involved in facilitating this process (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      Retinoid (Vitamin A) metabolism and the liver.
      ). In late 2012, Alapatt et al. reported the identification of a novel retinol transporter that is expressed primarily in mouse liver and intestine and that is able to bind RBP4 (this receptor is named RBP4 receptor-2 or RBPR2) (
      • Alapatt P.
      • Guo F.
      • Komanetsky S.M.
      • Wang S.
      • Cai J.
      • Sargsyan A.
      • Rodriguez Diaz E.
      • Bacon B.T.
      • Aryal P.
      • Graham T.E.
      Liver retinol transporter and receptor for serum retinol-binding protein (RBP4).
      ). It is possible that this protein has a role in intercellular retinol transfer within the liver. Interestingly, Alapatt et al. report that RBPR2 shares considerable structural similarity with STRA6 (
      • Alapatt P.
      • Guo F.
      • Komanetsky S.M.
      • Wang S.
      • Cai J.
      • Sargsyan A.
      • Rodriguez Diaz E.
      • Bacon B.T.
      • Aryal P.
      • Graham T.E.
      Liver retinol transporter and receptor for serum retinol-binding protein (RBP4).
      ). However, at present, these and other possibilities for explaining how retinol is transported between hepatocytes and HSCs remain to be established.
      The lipid droplets present within HSCs are specialized for retinoid storage (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ,
      • Moriwaki H.
      • Blaner W.S.
      • Piantedosi R.
      • Goodman D.S.
      Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets.
      ). The lipid composition of HSC lipid droplets isolated from control fed rats is unique and consists of approximately 40% retinyl ester, 32% triglyceride, 20% cholesteryl ester and cholesterol, 6% phospholipid, and 2% unesterified fatty acids (
      • Moriwaki H.
      • Blaner W.S.
      • Piantedosi R.
      • Goodman D.S.
      Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets.
      ). The relative lipid composition of these lipid droplets is dependent on dietary retinoid intake, with greater retinyl ester accumulation seen in rats fed an excess-retinol diet, and diminished accumulation seen for rats fed a retinol-restricted diet (
      • Moriwaki H.
      • Blaner W.S.
      • Piantedosi R.
      • Goodman D.S.
      Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets.
      ). Changes in the triglyceride composition of the diet do not affect either the relative or absolute lipid composition of rat liver HSC lipid droplets (
      • Moriwaki H.
      • Blaner W.S.
      • Piantedosi R.
      • Goodman D.S.
      Effects of dietary retinoid and triglyceride on the lipid composition of rat liver stellate cells and stellate cell lipid droplets.
      ). A number of lipid droplet-associated proteins, members of the perilipin family, are reported to be expressed by HSCs, including perilipin 2 and perilipin 3 (
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ). Given the central role of HSC lipid droplets in retinoid storage and metabolism, both LRAT and REHs must be able to associate with these lipid droplets, but this possibility remains to be elucidated. Interestingly, as illustrated in Fig. 4, the HSCs of Lrat-deficient mice lack lipid droplets. This observation was unexpected since the majority of lipid present in the HSC lipid droplets is not retinyl ester. This suggests that the synthesis of HSC lipid droplets is regulated in some manner that takes into account either LRAT presence and/or hepatic retinoid status.
      One of the most intriguing questions regarding hepatic retinoid storage concerns why more than 50% of all retinoid present in the body is stored in HSCs (for a healthy individual, 70% of what is present in the body is in the liver, and 80–90% of this is in HSCs). Thus, although HSCs account for only approximately 8% of the total cells in the liver and only 1% of hepatic protein, they account for more than half of all retinoid present in the body (
      • Blaner W.S.
      Retinoid (Vitamin A) metabolism and the liver.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ,
      • Geerts A.
      History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.
      ,
      • Friedman S.L.
      Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.
      ). What evolutionary factors or processes are responsible for this? HSCs, upon activation, are known to play a major causal role in the development of hepatic disease (
      • Blaner W.S.
      Retinoid (Vitamin A) metabolism and the liver.
      ,
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ,
      • Geerts A.
      History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.
      ,
      • Friedman S.L.
      Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.
      ). HSCs quickly lose their lipid droplets and retinoid stores during the process of activation (
      • Blaner W.S.
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Kluwe J.
      • D'Ambrosio D.M.
      • Jiang H.
      • Schwabe R.F.
      • Hillman E.M.
      • Piantedosi R.
      • Libien J.
      Hepatic stellate cell lipid droplets: a specialized lipid droplet for retinoid storage.
      ,
      • Geerts A.
      History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells.
      ,
      • Friedman S.L.
      Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.
      ). Possibly HSC lipid-droplet retinoid stores are needed to buffer against disease development, but this hypothesis remains to be established. In this regard, recently published data indicate that HSC retinoid stores do not protect against CCl4-induced fibrosis in mice (
      • Kluwe J.
      • Wongsiriroj N.
      • Troeger J.S.
      • Gwak G.Y.
      • Dapito D.H.
      • Pradere J.P.
      • Jiang H.
      • Siddiqi M.
      • Piantedosi R.
      • O'Byrne S.M.
      • et al.
      Absence of hepatic stellate cell retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis.
      ) but that they are required to ensure optimal liver regeneration in response to a 70% partial hepatectomy (
      • Shmarakov I.O.
      • Jiang H.
      • Yang K.J.
      • Goldberg I.J.
      • Blaner W.S.
      Hepatic retinoid stores are required for normal liver regeneration.
      ).

      RETINOIDS IN ADIPOSE TISSUE

      The major tissue storage site for retinoid in the body is the liver. Other tissues, including the eyes, lungs, adipose tissue, skin, testes, and spleen, have the capacity to store retinoid, albeit at a lesser concentration than liver. Adipose tissue is able to accumulate significant retinyl ester stores (
      • Blaner W.S.
      • Olson J.A.
      Retinol and retinoic acid metabolism.
      ,
      • Vogel S.
      • Gamble M.
      • Blaner W.S.
      Retinoid uptake, metabolism and transport.
      ,
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ). The concentrations of retinol/retinyl esters in adipose tissue of rats and mice maintained on a control retinoid-sufficient diet are reported to be in the range of 6–7 µg total retinol (retinol + retinyl ester)/g tissue wet weight (
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ). On a per gram of tissue basis, this is much less than in the liver. For this same study (
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ), hepatic total retinol levels were reported to average approximately 150 µg/g tissue wet weight. But considering that the liver contributes only 3–4% of the total mass of the body and adipose tissue can contribute a much greater percentage of total body mass, it was estimated that adipose tissue may account for as much as 15–20% of the total retinoid present in the body of a healthy, well-nourished rat maintained on a control chow diet (
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ). Moreover, adipose tissue expresses RBP4, CRBPI, and CRBPIII (
      • Vogel S.
      • Mendelsohn C.L.
      • Mertz J.R.
      • Piantedosi R.
      • Waldburger C.
      • Gottesman M.E.
      • Blaner W.S.
      Characterization of a new member of the fatty acid-binding protein family that binds all-trans-retinol.
      ,
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ,
      • Zovich D.C.
      • Orologa A.
      • Okuno M.
      • Wong Yen Kong L.
      • Talmage D.A.
      • Piantedosi R.
      • Goodman D.S.
      • Blaner W.S.
      Differentiation-dependent expression of retinoid-binding proteins in BFC-1β adipoycytes.
      ). The adipocyte is the cellular site of retinyl ester accumulation within adipose tissue, and adipocytes are able to synthesize and secrete RBP4 (
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ,
      • Zovich D.C.
      • Orologa A.
      • Okuno M.
      • Wong Yen Kong L.
      • Talmage D.A.
      • Piantedosi R.
      • Goodman D.S.
      • Blaner W.S.
      Differentiation-dependent expression of retinoid-binding proteins in BFC-1β adipoycytes.
      ). Thus, the adipocyte must be considered to be an important cell type in the body for retinoid storage and mobilization.
      As discussed above, mice totally lacking Lrat have no detectable retinyl esters in nearly all tissues, with the notable exception of adipose tissue (
      • Liu L.
      • Gudas L.J.
      Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency.
      ,
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). The adipose tissue of Lrat-deficient mice has, in fact, an elevation in retinyl esters compared with chow-fed wild-type mice. This is probably due to the inability of other tissues to store retinol. Moreover, this finding implies the presence of another enzyme capable of synthesizing retinyl esters within adipose tissue (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ). Although DGAT1 has been shown to be capable of synthesizing retinyl esters in intestine, mice lacking both Lrat and Dgat1 expressions display the same elevation of retinyl esters in adipose tissue, implying that there is another enzyme active in adipocytes (
      • O'Byrne S.M.
      • Wongsiriroj N.
      • Libien J.
      • Vogel S.
      • Goldberg I.J.
      • Baehr W.
      • Palczewski K.
      • Blaner W.S.
      Retinoid absorption and storage is impaired in mice lacking lecithin:retinol acyltransferase (LRAT).
      ,
      • Wongsiriroj N.
      • Piantedosi R.
      • Palczewski K.
      • Goldberg I.J.
      • Johnston T.P.
      • Li E.
      • Blaner W.S.
      The molecular basis of retinoid absorption: a genetic dissection.
      ). As in the liver, retinyl esters stored in adipose tissue can be mobilized and secreted back into the circulation bound to RBP4 synthesized in adipocytes (
      • Ong D.E.
      • Newcomer M.E.
      • Chytil F.
      Cellular retinoid-binding proteins..
      ,
      • Tsutsumi C.
      • Okuno M.
      • Tannous L.
      • Piantedosi R.
      • Allan M.
      • Goodman D.S.
      • Blaner W.S.
      Retinoids and retinoid-binding protein expression in rat adipocytes.
      ,
      • Zovich D.C.
      • Orologa A.
      • Okuno M.
      • Wong Yen Kong L.
      • Talmage D.A.
      • Piantedosi R.
      • Goodman D.S.
      • Blaner W.S.
      Differentiation-dependent expression of retinoid-binding proteins in BFC-1β adipoycytes.
      ,
      • Wei S.
      • Lai K.
      • Patel S.
      • Piantedosi R.
      • Shen H.
      • Colantuoni V.
      • Kraemer F.B.
      • Blaner W.S.
      Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes.
      ). These retinyl esters are first hydrolyzed by hormone-sensitive lipase (HSL), which acts as a physiologically relevant REH in adipocytes (
      • Wei S.
      • Lai K.
      • Patel S.
      • Piantedosi R.
      • Shen H.
      • Colantuoni V.
      • Kraemer F.B.
      • Blaner W.S.
      Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes.
      ,
      • Strom K.
      • Gundersen T.E.
      • Hansson O.
      • Lucas S.
      • Fernandez C.
      • Blomhoff R.
      • Holm C.
      Hormone-sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL.
      ). Mice lacking HSL expression are unable to hydrolyze adipocyte retinyl esters; this results in abnormal retinoid signaling, leading to aberrant adipocyte differentiation (
      • Strom K.
      • Gundersen T.E.
      • Hansson O.
      • Lucas S.
      • Fernandez C.
      • Blomhoff R.
      • Holm C.
      Hormone-sensitive lipase (HSL) is also a retinyl ester hydrolase: evidence from mice lacking HSL.
      ).
      One of the most exciting recent findings from research focused on adipose retinoid physiology has been the observation by Kahn and colleagues that RBP4 synthesized by adipocytes and secreted into the circulation may have a role in modulating tissue responsiveness to insulin (
      • Yang Q.
      • Graham T.E.
      • Mody N.
      • Preitner F.
      • Peroni O.D.
      • Zabolotny J.M.
      • Kotani K.
      • Quadro L.
      • Kahn B.B.
      Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.
      ,
      • Graham T.E.
      • Yang Q.
      • Bluher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • et al.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Polonsky K.S.
      Retinol-binding protein 4, insulin resistance, and type 2 diabetes.
      ). Since the first report of this relationship by Kahn and colleagues in 2005, there have been reports involving studies in humans, animals, and cell culture models from many independent research laboratories confirming and extending this work, which links adipocyte RBP4, obesity, and impaired peripheral tissue insulin responsiveness (
      • Yang Q.
      • Graham T.E.
      • Mody N.
      • Preitner F.
      • Peroni O.D.
      • Zabolotny J.M.
      • Kotani K.
      • Quadro L.
      • Kahn B.B.
      Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.
      ,
      • Graham T.E.
      • Yang Q.
      • Bluher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • et al.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Polonsky K.S.
      Retinol-binding protein 4, insulin resistance, and type 2 diabetes.
      ,
      • Munkhtulga L.
      • Nakayama K.
      • Utsumi N.
      • Yanagisawa Y.
      • Gotoh T.
      • Omi T.
      • Kumada M.
      • Erdenebulgan B.
      • Zolzaya K.
      • Lkhagvasuren T.
      • et al.
      Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia.
      ,
      • Janke J.
      • Engeli S.
      • Boschmann M.
      • Adams F.
      • Bohnke J.
      • Luft F.C.
      • Sharma A.M.
      • Jordan J.
      Retinol-binding protein 4 in human obesity.
      ,
      • Kotnik P.
      • Fischer-Posovszky P.
      • Wabitsch M.
      RBP4: a controversial adipokine.
      ,
      • Tan Y.
      • Sun L.Q.
      • Kamal M.A.
      • Wang X.
      • Seale J.P.
      • Qu X.
      Suppression of retinol-binding protein 4 with RNA oligonucleotide prevents high-fat diet-induced metabolic syndrome and non-alcoholic fatty liver disease in mice.
      ). However, not all investigators have been able to confirm this linkage, and it remains an active and somewhat controversial research area (
      • Gomez-Ambrosi J.
      • Rodriguez A.
      • Catalan V.
      • Ramirez B.
      • Silva C.
      • Rotellar F.
      • Gil M.J.
      • Salvador J.
      • Fruhbeck G.
      Serum retinol-binding protein 4 is not increased in obesity or obesity-associated type 2 diabetes mellitus, but is reduced after relevant reductions in body fat following gastric bypass.
      ,
      • Kotnik P.
      • Fischer-Posovszky P.
      • Wabitsch M.
      RBP4: a controversial adipokine.
      ,
      • von Eynatten M.
      • Lepper P.M.
      • Liu D.
      • Lang K.
      • Baumann M.
      • Nawroth P.P.
      • Bierhaus A.
      • Dugi K.A.
      • Heemann U.
      • Allolio B.
      • et al.
      Retinol-binding protein 4 is associated with components of the metabolic syndrome, but not with insulin resistance, in men with type 2 diabetes or coronary artery disease.
      ).
      Our goal is not to summarize in a detailed manner the extensive literature concerning adipose-derived RBP4 and insulin responsiveness but rather simply to familiarize the reader with some of this literature, which in our view ultimately is biochemically linked to adipose retinoid storage. Since 2005, the literature has suggested linkages between serum RBP4 concentrations, obesity, impaired insulin responsiveness (
      • Yang Q.
      • Graham T.E.
      • Mody N.
      • Preitner F.
      • Peroni O.D.
      • Zabolotny J.M.
      • Kotani K.
      • Quadro L.
      • Kahn B.B.
      Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes.
      ,
      • Graham T.E.
      • Yang Q.
      • Bluher M.
      • Hammarstedt A.
      • Ciaraldi T.P.
      • Henry R.R.
      • Wason C.J.
      • Oberbach A.
      • Jansson P.A.
      • Smith U.
      • et al.
      Retinol-binding protein 4 and insulin resistance in lean, obese, and diabetic subjects.
      ,
      • Polonsky K.S.
      Retinol-binding protein 4, insulin resistance, and type 2 diabetes.
      ,
      • Munkhtulga L.
      • Nakayama K.
      • Utsumi N.
      • Yanagisawa Y.
      • Gotoh T.
      • Omi T.
      • Kumada M.
      • Erdenebulgan B.
      • Zolzaya K.
      • Lkhagvasuren T.
      • et al.
      Identification of a regulatory SNP in the retinol binding protein 4 gene associated with type 2 diabetes in Mongolia.
      ,
      • Janke J.
      • Engeli S.
      • Boschmann M.
      • Adams F.
      • Bohnke J.
      • Luft F.C.
      • Sharma A.M.
      • Jordan J.
      Retinol-binding protein 4 in human obesity.
      ,
      • Tan Y.
      • Sun L.Q.
      • Kamal M.A.
      • Wang X.
      • Seale J.P.
      • Qu X.
      Suppression of retinol-binding protein 4 with RNA oligonucleotide prevents high-fat diet-induced metabolic syndrome and non-alcoholic fatty liver disease in mice.
      ), fatty liver development (

      Xia, M., Liu, Y., Guo, H., Wang, D., Wang, Y., Ling, W., . Retinol binding protein 4 stimulates hepatic SREBP-1 and increases lipogenesis through PGC-1beta-dependent pathway. Hepatology. Epub ahead of print. January 8, 2013; doi:10.1002/hep.26227.

      ,
      • Petta S.
      • Camma C.
      • Di Marco V.
      • Alessi N.
      • Barbaria F.
      • Cabibi D.
      • Caldarella R.
      • Ciminnisi S.
      • Licata A.
      • Massenti M.F.
      • et al.
      Retinol-binding protein 4: a new marker of virus-induced steatosis in patients infected with hepatitis C virus genotype 1.