Identification and Characterization of a Non-retinoid Ligand for Retinol-binding Protein 4 Which Lowers Serum Retinol-binding Protein 4 Levels in Vivo

Abstract

Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (Ki = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4-/- mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes. RBP4 2The abbreviations used are: RBP4, retinol-binding protein 4; TTR, transthyretin; A1120, 2-(4-(2-(trifluoromethyl)phenyl)piperidine-1-carboxamido)benzoic acid; LC, liquid chromatography; MS, mass spectrometry; FRET, fluorescence resonance energy transfer; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance; CRBP1, cellular retinol-binding protein 1. is a serum protein that transports retinol (vitamin A) from the liver to extrahepatic tissues (1Goodman D.S. Sporn M.B. Roberts A.B. Goodman D.S. The Retinoids. vol. 2. Academic Press, New York1984: 41-88Crossref Google Scholar). The majority of RBP4 is expressed in the liver, with ∼15–20% expressed in adipose (2Makover A. Soprano D.R. Wyatt M.L. Goodman D.S. J. Lipid Res. 1989; 30: 171-180Abstract Full Text PDF PubMed Google Scholar). In the serum, RBP4 is present as a complex with transthyretin (TTR), which effectively increases the molecular weight of RBP4 and protects it from glomerular filtration. Thus disruption of the RBP4·TTR complex in vivo by administration of the synthetic retinoid fenretinide (N-(4-hydroxyphenyl)retinamide) results in a rapid reduction in serum RBP4 levels (3Peng Y.M. Dalton W.S. Alberts D.S. Xu M.J. Lim H. Meyskens Jr., F.L. Int. J. Cancer. 1989; 43: 22-26Crossref PubMed Scopus (55) Google Scholar, 4Berni R. Formelli F. FEBS Lett. 1992; 308: 43-45Crossref PubMed Scopus (91) Google Scholar). Although the major physiological ligand for RBP4 appears to be retinol, RBP4 can bind to other endogenous and synthetic retinoids. For example, using biochemical assays, RBP4 has been shown to bind to retinol, all-trans- and 13-cis-retinoic acid, retinyl acetate, N-(ethyl)retinamide, and fenretinide (4Berni R. Formelli F. FEBS Lett. 1992; 308: 43-45Crossref PubMed Scopus (91) Google Scholar, 5Berni R. Clerici M. Malpeli G. Cleris L. Formelli F. FASEB J. 1993; 7: 1179-1184Crossref PubMed Scopus (58) Google Scholar, 6Cogan U. Kopelman M. Mokady S. Shinitzky M. Eur. J. Biochem. 1976; 65: 71-78Crossref PubMed Scopus (348) Google Scholar). In addition, x-ray diffraction analysis of a variety of RBP4-retinoid co-crystal structures has demonstrated that these retinoids bind to the same site as retinol, with the cyclohexene ring buried within the internal cavity and the polar head group pointing toward the exterior of the protein (7Cowan S.W. Newcomer M.E. Jones T.A. Proteins. 1990; 8: 44-61Crossref PubMed Scopus (298) Google Scholar, 8Zanotti G. Malpeli G. Berni R. J. Biol. Chem. 1993; 268: 24873-24879Abstract Full Text PDF PubMed Google Scholar, 9Zanotti G. Marcello M. Malpeli G. Folli C. Sartori G. Berni R. J. Biol. Chem. 1994; 269: 29613-29620Abstract Full Text PDF PubMed Google Scholar). The loop regions of RBP4 surrounding the entrance of the binding cavity form the binding site for TTR, with the binding of retinol (in particular the presence of the hydroxyl group) increasing the affinity of RBP4 for TTR by a factor of ∼4 (10Monaco H.L. Rizzi M. Coda A. Science. 1995; 268: 1039-1041Crossref PubMed Scopus (353) Google Scholar). The binding of fenretinide, however, has the opposite effect. Through a combination of steric hindrance (from the bulky phenylamide head group) and changes in the position of the loop regions of RBP4 located at the TTR binding interface, fenretinide completely disrupts the binding of RBP4 to TTR (4Berni R. Formelli F. FEBS Lett. 1992; 308: 43-45Crossref PubMed Scopus (91) Google Scholar, 9Zanotti G. Marcello M. Malpeli G. Folli C. Sartori G. Berni R. J. Biol. Chem. 1994; 269: 29613-29620Abstract Full Text PDF PubMed Google Scholar). Recent reports have suggested that, in addition to its role in vitamin A transport, RBP4 may also be involved in the development of insulin resistance. For example, Yang et al. reported that mice overexpressing an RBP4 transgene and mice injected with recombinant RBP4 protein become insulin-resistant, and that reduction of RBP4 levels in mice either by gene ablation or treatment with fenretinide improves insulin sensitivity (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). In humans, Graham et al. showed that elevated serum RBP4 levels are correlated with the magnitude of insulin resistance in subjects with obesity, impaired glucose tolerance, and type 2 diabetes (12Graham T.E. Yang Q. Bluher M. Hammarstedt A. Ciaraldi T.P. Henry R.R. Wason C.J. Oberbach A. Jansson P.A. Smith U. Kahn B.B. N. Engl. J. Med. 2006; 354: 2552-2563Crossref PubMed Scopus (1120) Google Scholar). This has been confirmed by some studies (13Balagopal P. Graham T.E. Kahn B.B. Altomare A. Funanage V. George D. J. Clin. Endocrinol. Metab. 2007; 92: 1971-1974Crossref PubMed Scopus (205) Google Scholar, 14Cho Y.M. Youn B.S. Lee H. Lee N. Min S.S. Kwak S.H. Lee H.K. Park K.S. Diabetes Care. 2006; 29: 2457-2461Crossref PubMed Scopus (355) Google Scholar, 15Stefan N. Hennige A.M. Staiger H. Machann J. Schick F. Schleicher E. Fritsche A. Haring H.U. Diabetes Care. 2007; 30: 1173-1178Crossref PubMed Scopus (202) Google Scholar, 16Qi Q. Yu Z. Ye X. Zhao F. Huang P. Hu F.B. Franco O.H. Wang J. Li H. Liu Y. Lin X. J. Clin. Endocrinol. Metab. 2007; 92: 4827-4834Crossref PubMed Scopus (179) Google Scholar) but not by others (17Janke J. Engeli S. Boschmann M. Adams F. Bohnke J. Luft F.C. Sharma A.M. Jordan J. Diabetes. 2006; 55: 2805-2810Crossref PubMed Scopus (302) Google Scholar, 18Lewis J.G. Shand B.I. Frampton C.M. Elder P.A. Scott R.S. Clin. Biochem. 2008; 41: 1034-1038Crossref PubMed Scopus (29) Google Scholar, 19Petersen K.F. Dufour S. Savage D.B. Bilz S. Solomon G. Yonemitsu S. Cline G.W. Befroy D. Zemany L. Kahn B.B. Papademetris X. Rothman D.L. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12587-12594Crossref PubMed Scopus (528) Google Scholar, 20Promintzer M. Krebs M. Todoric J. Luger A. Bischof M.G. Nowotny P. Wagner O. Esterbauer H. Anderwald C. J. Clin. Endocrinol. Metab. 2007; 92: 4306-4312Crossref PubMed Scopus (80) Google Scholar), leaving in question the relevance of RBP4 as a marker or therapeutic target for insulin resistance in humans. To further understand the involvement of RBP4 in insulin resistance, we conducted a high throughput screen to identify non-retinoid ligands for RBP4. We report here the identification and characterization of A1120, a non-retinoid small molecule that binds with high affinity to RBP4 and displaces TTR from an RBP4·TTR complex. The co-crystal structure of A1120 with RBP4 shows that A1120 binds to the same site as retinol and induces changes in the orientation (compared with the retinol bound form) of loops at the RBP4·TTR interaction interface. When administered to mice, A1120 lowers RBP4 and retinol levels in a dose-dependent manner, to a similar extent as seen with fenretinide. However, unlike fenretinide, A1120 does not have beneficial effects on insulin resistance. In addition, our experiments show that Rpb4-/- mice are not resistant to high fat diet-induced insulin resistance and are indistinguishable from their wild-type littermates with respect to insulin sensitivity. These results suggest that the insulin-sensitizing effects of fenretinide are not mediated by RBP4 and indicate that lowering RBP4 levels does not significantly improve the diabetic state in mice. Synthesis of A1120-A solution of methyl 2-isocyanatobenzoate (10.00 g, 56.4 mmol) in tetrahydrofuran (30 ml) was slowly added to a solution of 4-(2-(trifluoromethyl)phenyl)piperidine hydrochloride (14.3 g, 53.8 mmol, Sigma) and triethylamine 99% (8.99 ml, 64.5 mmol) in tetrahydrofuran (120 ml) at 0 °C. The mixture was removed from the cooling bath and stirred at room temperature for 15 min, at which time LC/MS analysis indicated that the reaction was complete. EtOH (75 ml) and aqueous LiOH (2N, 95 ml) were then added, and the solution was stirred for 6 h at room temperature. Subsequently, aqueous HCl (2N, 150 ml) was added, and the resulting mixture was extracted with EtOAc (2 × 600 ml). The EtOAc extract was dried over MgSO4 and concentrated to an off-white solid. Recrystallization from EtOAc yielded 14.0 g (66%) of 2-(4-(2-(trifluoromethyl)phenyl)piperidine-1-carboxamido) benzoic acid as a white solid, which was homogeneous by analytical high-performance liquid chromatography (>99%): mp: 156.3–157.1 °C; MS electrospray ionization (pos.) m/e: 393.0 (M+H)+ (calcd. for C20H20F3N2O3, 393.1); 1H NMR (400 = = = = (2 NMR and RBP4 and were as P. M. J. F. A. Biochem. PubMed Scopus Google Scholar). was from and recombinant protein was in on acid and as for RBP4 P. M. J. F. A. Biochem. PubMed Scopus Google Scholar). The was as for RBP4. and protein was as K. Sci. PubMed Scopus Google Scholar). In the form of RBP4 was a by The recombinant protein with a was expressed in E. in at and by for 4 h recombinant RBP4 were by and with and then with The protein was in and and then to with HCl in of RBP4 was by in 4 and and by a over The protein was further by over by over The was then to a and in a of and The protein was then concentrated to 15 for The RBP4 with A1120 were at in a with of the protein solution and of the solution Bis-Tris, The were the with and in liquid and The x-ray diffraction for the co-crystal was on a at the in to a of The were using on 1992; Scholar) and in Biol. 1994; PubMed Scopus Google Scholar). The structure was by molecular with Adams J. 2007; PubMed Scopus Google Scholar) using a retinol binding protein structure as a of were using The Biol. PubMed Scopus Google Scholar) in was used for The have been in the for RBP4 and RBP4 was using the RBP4 from to the To serum retinol, were with and were extracted with of internal and the was and to liquid chromatography with mass with a with a mass The of and a The was a × at °C. acid in and acid in The with for min, and then to in by a to over 2 min, and at for The was were at in the MS was in ionization with reaction were as for and and °C. The reaction used for were and For retinol, was by to its of were by the by as The of were and were by of to internal the of the In mice of were on normal or high fat and with by were in a room to a at and room temperature and were in with the for the and of Press, D. and studies were in with and and and by the and For mice of were administered in in at the indicated in a of For was from a For mice were and was for of serum by For glucose and insulin mice were for 4 h from the of the were then with glucose (2 or insulin and was at the indicated time glucose levels were in by or in serum by was in serum by For treatment were as on a high fat for 6 were treatment on state and insulin mice on were used as weight and were Rpb4-/- and mice were from of mice from were by as L. W.S. S. R. P. S. V. M.E. J. PubMed Scopus Google Scholar), and these results were confirmed by For studies were conducted using wild-type were on or with high fat are expressed as for the indicated of treatment were by or ANOVA, by for or in of a RBP4 a P. M. J. F. A. Biochem. PubMed Scopus Google Scholar), we the for that retinol from RBP4. were and yielded A1120 The of A1120 was on RBP4 and on RBP4 We also the affinity of A1120 for cellular retinol-binding protein is an retinol but studies have shown that the retinol binding site is not between and RBP4 S.W. Newcomer M.E. Jones T.A. J. Biol. 1993; PubMed Scopus Google Scholar). shows that A1120 does not bind to In addition, A1120 was a of and not Thus A1120 appears to bind to RBP4. We have a fluorescence resonance energy that the interaction between RBP4 and TTR P. M. J. F. A. Biochem. PubMed Scopus Google Scholar). retinol increases the interaction between RBP4 and TTR, it increases the in a dose-dependent fenretinide disrupts the interaction between RBP4 and TTR, it the in a dose-dependent In addition, fenretinide and retinol bind to the same site on RBP4, fenretinide also a retinol shown in A1120 similar to fenretinide in the assay; it lowers the and the retinol that A1120 also disrupts the interaction between RBP4 and the we the affinity of A1120 for RBP4 to be 8.3 Thus A1120 binds to RBP4 with affinity either retinol or fenretinide (Ki = for P. M. J. F. A. Biochem. PubMed Scopus Google The of A1120 to co-crystal structure of RBP4 with A1120 was by molecular to using a reported RBP4 structure as a (7Cowan S.W. Newcomer M.E. Jones T.A. Proteins. 1990; 8: 44-61Crossref PubMed Scopus (298) Google Scholar). The x-ray are in and = = = = of of = and are and structure was from a of form the and was from the of in is the from in is the from = = and are and structure was from a of form the and was from the of is the from in a The of RBP4 in the A1120 co-crystal is the same as reported for RBP4 (7Cowan S.W. Newcomer M.E. Jones T.A. Proteins. 1990; 8: 44-61Crossref PubMed Scopus (298) Google Scholar, M.E. Jones T.A. J. J. U. L. P.A. J. PubMed Scopus Google Scholar, G. S. Berni R. H.L. J. Biol. 1993; PubMed Scopus Google Scholar). of a of an and a by a The co-crystal structure showed for A1120 in the cavity of the same site by with the ring binding in the and the benzoic acid at the The of the a from the of The of the benzoic acid a with as as a with the hydroxyl group of The of the molecule with the Although the structure of RBP4 shows A1120 conformational changes are in the loop regions surrounding the of the binding loop from to and loop from to These loop regions form of the TTR interaction (10Monaco H.L. Rizzi M. Coda A. Science. 1995; 268: 1039-1041Crossref PubMed Scopus (353) Google Scholar, Newcomer M.E. PubMed Scopus Google Scholar), and the conformational changes by A1120 the between RBP4 and TTR that is for high affinity shows loops and in the structure with TTR in the structure (10Monaco H.L. Rizzi M. Coda A. Science. 1995; 268: 1039-1041Crossref PubMed Scopus (353) Google Scholar, Newcomer M.E. PubMed Scopus Google Scholar). This a for the disruption of RBP4·TTR binding by A1120 in the is from that seen with fenretinide, the in particular the phenylamide head the of the RBP4·TTR interface, a steric hindrance that the RBP4·TTR complex from G. Marcello M. Malpeli G. Folli C. Sartori G. Berni R. J. Biol. Chem. 1994; 269: 29613-29620Abstract Full Text PDF PubMed Google Scholar). A1120 RBP4 and in vivo administration of fenretinide results in serum RBP4 and retinol levels (3Peng Y.M. Dalton W.S. Alberts D.S. Xu M.J. Lim H. Meyskens Jr., F.L. Int. J. Cancer. 1989; 43: 22-26Crossref PubMed Scopus (55) Google Scholar, F. R. A. F. G. A. M. Res. 1989; Google Scholar), we experiments to A1120 show the same effect. For our in vivo we used mice, a between and These are for insulin resistance on a high fat and shows that administration of an of A1120 to mice in a in serum RBP4 and retinol with the reduction in h Administration of fenretinide in in RBP4 and retinol that a similar time the magnitude of the was not as as for shows that the RBP4 lowering by A1120 is with the seen at and similar to that seen with fenretinide 4 h the in show that A1120 is at as and as fenretinide in serum levels of RBP4 and of the of et al. showed that fenretinide improves insulin sensitivity in diet-induced mice (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar), and we used a similar to the of mice a high fat for 6 were administered fenretinide, A1120, the or as a A group was as a of serum was for RBP4, retinol, and glucose and the were to glucose and insulin fenretinide and A1120 showed RBP4 and retinol lowering A and treatment in RBP4 a with in mice (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). However, the was and similar results were not seen in studies in diet-induced and of diabetes and not RBP4 levels the high fat a with in the (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google of Rpb4-/- mice. insulin in wild-type or Rpb4-/- mice a insulin in wild-type or Rpb4-/- mice a high fat for and serum glucose and insulin levels an glucose on the same mice in an on the high fat was in of the between the wild-type and Rpb4-/- mice by insulin in wild-type or Rpb4-/- mice with for 2 and for for wild-type or by by serum RBP4 levels the of mice in E. with by by shows that, 4 glucose levels with This is with (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). However, levels in the group glucose levels were not significantly from showed a lowering of glucose to a similar to that seen in insulin was also conducted 4 of glucose in to an of insulin with showed an in glucose levels at the time and not show an at time However, in the and significantly glucose levels at time glucose was conducted 6 of glucose and insulin levels in in to administration of glucose and of administration on glucose were in of the treatment with the with fenretinide or A1120 not in glucose levels the glucose showed in glucose levels at time the seen in levels the glucose were significantly at time in with However, not show lowering of insulin lowering fenretinide with insulin levels to The in 4 that, fenretinide and A1120 RBP4 fenretinide is to improve insulin sensitivity shown by insulin levels the glucose the magnitude and of RBP4 lowering is similar for it is that in these can the it that the insulin-sensitizing effects of fenretinide are not mediated RBP4. A may of retinoid which have been shown to to fenretinide D. D. Yu M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Rpb4-/- further the role of in insulin resistance, we Rpb4-/- which been reported to have glucose levels their wild-type littermates an insulin (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). shows that glucose levels in Rpb4-/- mice on a were the same as in wild-type an insulin This is with the in fenretinide A1120 treatment glucose levels in to However, the is not with the results (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). Although the Rpb4-/- used in studies is the same L. W.S. S. R. P. S. V. M.E. J. PubMed Scopus Google Scholar), the is not and may the To effects of we a of = in the wild-type group and = 15 in the Rpb4-/- We an to the Rpb4-/- mice were protected from high fat diet-induced insulin resistance. with the from the was in glucose an insulin between the wild-type and Rpb4-/- of high fat was in glucose or insulin an glucose and Therefore, Rpb4-/- mice are not protected from high fat diet-induced insulin resistance and not show insulin sensitivity on either a or high fat of Rpb4-/- with mice are and have elevated levels of serum RBP4 (11Yang Q. Graham T.E. Mody N. Preitner F. Peroni O.D. Zabolotny J.M. Kotani K. Quadro L. Kahn B.B. Nature. 2005; 436: 356-362Crossref PubMed Scopus (1685) Google Scholar). of these mice with the the insulin resistance and RBP4 To RBP4 is involved in the insulin-sensitizing effects of we an insulin on wild-type and Rpb4-/- mice that been with shows that, of by in significantly glucose levels at and time insulin In addition, was in the magnitude of the between the wild-type and Rpb4-/- mice. with the shown in RBP4 levels but showed in insulin sensitivity and A1120 RBP4 levels but showed in insulin that the effects of are to RBP4. In we have identified a non-retinoid RBP4 ligand that lowers RBP4 and retinol levels in but does not improve insulin resistance. In addition, we Rpb4-/- mice and demonstrated that are indistinguishable from their wild-type littermates with respect to insulin sensitivity a or high fat or with the and insulin These indicate that RBP4 lowering does not significantly the diabetic state in mice. The relevance of these to is not but is in the over the involvement of RBP4 in insulin resistance, with studies results (12Graham T.E. Yang Q. Bluher M. Hammarstedt A. Ciaraldi T.P. Henry R.R. Wason C.J. Oberbach A. Jansson P.A. Smith U. Kahn B.B. N. Engl. J. Med. 2006; 354: 2552-2563Crossref PubMed Scopus (1120) Google Scholar, P. Graham T.E. Kahn B.B. Altomare A. Funanage V. George D. J. Clin. Endocrinol. Metab. 2007; 92: 1971-1974Crossref PubMed Scopus (205) Google Scholar, 14Cho Y.M. Youn B.S. Lee H. Lee N. Min S.S. Kwak S.H. Lee H.K. Park K.S. Diabetes Care. 2006; 29: 2457-2461Crossref PubMed Scopus (355) Google Scholar, 15Stefan N. Hennige A.M. Staiger H. Machann J. Schick F. Schleicher E. Fritsche A. Haring H.U. Diabetes Care. 2007; 30: 1173-1178Crossref PubMed Scopus (202) Google Scholar, 16Qi Q. Yu Z. Ye X. Zhao F. Huang P. Hu F.B. Franco O.H. Wang J. Li H. Liu Y. Lin X. J. Clin. Endocrinol. Metab. 2007; 92: 4827-4834Crossref PubMed Scopus (179) Google Scholar, J. Engeli S. Boschmann M. Adams F. Bohnke J. Luft F.C. Sharma A.M. Jordan J. Diabetes. 2006; 55: 2805-2810Crossref PubMed Scopus (302) Google Scholar, 18Lewis J.G. Shand B.I. Frampton C.M. Elder P.A. Scott R.S. Clin. Biochem. 2008; 41: 1034-1038Crossref PubMed Scopus (29) Google Scholar, 19Petersen K.F. Dufour S. Savage D.B. Bilz S. Solomon G. Yonemitsu S. Cline G.W. Befroy D. Zemany L. Kahn B.B. Papademetris X. Rothman D.L. Shulman G.I. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 12587-12594Crossref PubMed Scopus (528) Google Scholar, 20Promintzer M. Krebs M. Todoric J. Luger A. Bischof M.G. Nowotny P. Wagner O. Esterbauer H. Anderwald C. J. Clin. Endocrinol. Metab. 2007; 92: 4306-4312Crossref PubMed Scopus (80) Google Scholar). in a of normal and type 2 diabetic it was that but not diabetes serum RBP4 levels A. J. M. A. J. Diabetes. 2008; PubMed Scopus (91) Google Scholar). studies be to between RBP4 and insulin resistance in humans. We and for critical of the