PET/CT imaging reveals unrivaled placental avidity for glucose compared to other tissues.

INTRODUCTION: The goal of this study was to define the kinetics of glucose transport from maternal blood to placenta to fetus using real time imaging. METHODS: Positron emission tomography (PET) imaging of the glucose-tracer [(18)F]fluorodeoxyglucose (FDG) was used to temporally and spatially define, in vivo, the kinetics of glucose transport from maternal blood into placentae and fetuses, in the late gestational gravid rat. Computed tomography (CT), with intravenous contrast, co-registered to the PET images allowed anatomic differentiation of placentae from fetal and maternal tissues. RESULTS: FDG was rapidly taken up by placentae and subsequently appeared in fetuses with minimal temporal lag. FDG standardized uptake values in placentae and fetuses approached that of maternal brain. In both anesthetized and awake dams, one quarter of the administered FDG ultimately was accrued in the collective fetuses and placentae. Accordingly, kinetic modeling demonstrated that the placentae had very high avidity for FDG, 2-fold greater than that of the fetus and maternal brain, when accounting for the fact that fetal FDG necessarily must first be taken up by placentae. Consistent with this, placental expression of glucose transporter 1 exceeded that of all other tissues. DISCUSSION: Fetal and placental tissues place a substantial glucose metabolic burden on the mother, owing to very high avidity of placentae for glucose coupled with the large relative mass of fetal and placental tissues. CONCLUSIONS: The placenta has a tremendous capacity to uptake and transport glucose. PET/CT imaging is an ideal means to study metabolite transport kinetics in the fetoplacental unit.

Modification of high saturated fat diet with n-3 polyunsaturated fat improves glucose intolerance and vascular dysfunction.

AIMS: The ability of dietary enrichment with monounsaturated fatty acid (MUFA), n-3 or n-6 polyunsaturated fatty acids (PUFAs) to reverse glucose intolerance and vascular dysfunction resulting from excessive dietary saturated fatty acids is not resolved. We hypothesized that partial replacement of dietary saturated fats with n-3 PUFA-enriched menhaden oil (MO) would provide greater improvement in glucose tolerance and vascular function compared to n-6 enriched safflower oil (SO) or MUFA-enriched olive oil (OO). METHODS: We fed mice a high saturated fat diet (HF) (60% kcal from lard) for 12 weeks before substituting half the lard with MO, SO or OO for an additional 4 weeks. At the end of 4 weeks, we assessed glucose tolerance, insulin signalling and reactivity of isolated pressurized gracilis arteries. RESULTS: After 12 weeks of saturated fat diet, body weights were elevated and glucose tolerance was abnormal compared to mice on control diet (13% kcal lard). Diet substituted with MO restored basal glucose levels, glucose tolerance and indices of insulin signalling (phosphorylated Akt) to normal, whereas restoration was limited for SO and OO substitutions. Although dilation to acetylcholine was reduced in arteries from mice on HF, OO and SO diets compared to normal diet, dilation to acetylcholine was fully restored and constriction to phenylephrine was reduced in MO-fed mice compared to normal. CONCLUSION: We conclude that short-term enrichment of an ongoing high fat diet with n-3 PUFA rich MO, but not MUFA rich OO or n-6 PUFA rich SO, reverses glucose tolerance, insulin signalling and vascular dysfunction.

Long-chain polyunsaturated fatty acid levels in US donor human milk: meeting the needs of premature infants?

OBJECTIVE: To determine fatty acid levels in the US donor milk supply. STUDY DESIGN: Donor human milk samples from Iowa (n=62), Texas (n=5), North Carolina (n=5) and California (n=5) were analyzed by gas chromatography. Levels in the Iowa donor milk were compared before and after pasteurization using Student's t-test. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels were compared among all milk banks using analysis of variance. RESULT: ARA (0.4 pre, 0.4 post, P=0.18) and DHA (0.073 pre, 0.073 post, P=0.84) were not affected by pasteurization. DHA varied between banks (P<0.0001), whereas ARA did not (P=0.3). DHA levels from all banks were lower than published values for maternal milk and infant formula (P<0.0001). CONCLUSION: Pasteurization of breastmilk does not affect DHA or ARA levels. However, DHA content in US donor milk varies with bank location and may not meet the recommended provision for preterm infants.

Binding of cytochrome P450 monooxygenase and lipoxygenase pathway products by heart fatty acid-binding protein.

Arachidonic acid metabolism by lipoxygenases and cytochrome P450 monooxygenases produces regioisomeric hydroperoxyeicosatetraenoic acids (HPETEs), hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs), which serve as components of cell signaling cascades. Intracellular fatty acid-binding proteins (FABPs) may differentially bind these nonprostanoid oxygenated fatty acids, thus modulating their metabolism and activities. Vascular cells, which express heart FABP (H-FABP), utilize oxygenated fatty acids for regulation of vascular tone. Therefore, the relative affinities of H-FABP for several isomeric series of these compounds were measured by fluorescent displacement of 1-anilinonaphthalene-8-sulfonic acid (ANS). In general, H-FABP rank order affinities (arachidonic acid > EETs > HETEs > DHETs) paralleled reversed-phase high-performance liquid chromatography retention times, indicating that the differences in H-FABP affinity were determined largely by polarity. H-FABP displayed a similar rank order of affinity for compounds derived from linoleic acid. H-FABP affinity for 20-HETE [apparent dissociation constant (K(d)') of 0.44 microM] was much greater than expected from its polarity, indicating unique binding interactions for this HETE. H-FABP affinity for 5,6-EET and 11,12-EET (K(d)' of approximately 0.4 microM) was approximately 20-fold greater than for DHETs (K(d)' of approximately 8 microM). The homologous proteins, liver FABP and intestinal FABP, also displayed selective affinity for EET versus DHET. Thus, FABP binding of EETs may facilitate their intracellular retention whereas the lack of FABP affinity for DHETs may partially explain their release from cells. The affinity of H-FABP for EETs suggests that this family of intracellular proteins may modulate the metabolism, activities, and targeting of these potent eicosanoid biomediators.

The isolation and characterization of purified heterocomplexes of recombinant retinoic acid receptor and retinoid X receptor ligand binding domains.

Retinoic acid exerts many of its biological effects by interaction with heterocomplexes of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). To further examine this interaction, a glutathione S-transferase (GST) fusion protein containing the ligand binding domain of human RXR alpha has been used to copurify the ligand binding domain of human RAR gamma by affinity chromatography over glutathione-agarose. Complexes of recombinant RAR-RXR ligand binding domains retaining full ligand binding capacity were purified, and their interactions with various retinoids were characterized by fluorometric titration and photoaffinity labeling. Analyses of the distribution of limiting amounts of [3H]-all-trans-retinoic acid between cytoplasmic retinoic acid binding proteins, CRABP-I and CRABP-II, and the purified heterocomplexes indicate that all-trans-retinoic acid binds with comparable affinity to CRABP-I and the heterocomplexes, but with approximately 10-fold less affinity to CRABP-II. The aromatic retinoid acitretin, which is used in the treatment of psoriasis, binds relatively poorly to the purified heterocomplexes, although it binds with high affinity to the CRABPs. Acitretin displaces [3H]-all-trans-retinoic acid from the CRABPs and increases retinoic acid occupancy of the heterocomplexes. These results suggest that certain retinoids could potentially perturb the distribution of endogenous retinoic acid between the CRABPs and the nuclear receptors and thus affect retinoid signaling. The purified recombinant complexes should provide a useful model system for further structural analysis of the dimerization interface between the RAR and RXR ligand binding domains.

Conformationally defined 6-s-trans-retinoic acid analogs. 3. Structure-activity relationships for nuclear receptor binding, transcriptional activity, and cancer chemopreventive activity.

We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.

Structure/function of cytoplasmic vitamin A-binding proteins.

Two cytoplasmic retinol-binding proteins, CRBP and CRBP II, and two cytoplasmic retinoic acid-binding proteins, CRABP-I and CRABP-II, have been well characterized. There has been significant progress in the structural analysis of these four proteins with X-ray crystallography, nuclear magnetic resonance, mutagenesis, and binding studies. In contrast, the cellular functions of these cytoplasmic vitamin A-binding proteins are less well understood. Since these proteins bind their respective ligands with high affinity, they are likely to influence retinoid signaling pathways. Analysis of retinoid metabolism in the presence or absence of these proteins provides support for the hypothesis that these proteins are involved in modulating intracellular retinoid metabolism. Molecular genetic approaches to alteration of the levels of these proteins in tissue culture cells and in whole animals have provided a powerful means toward defining the physiological roles of the cytoplasmic vitamin A-binding proteins in vivo.

Nuclear magnetic resonance studies demonstrate differences in the interaction of retinoic acid with two highly homologous cellular retinoic acid binding proteins.

Cellular retinoic acid binding protein-I (CRABP-I) and cellular retinoic acid binding protein-II (CRABP-II) are highly homologous, 15 kDa proteins which bind all-trans-retinoic acid. In the adult, CRABP-II is expressed predominately in the epidermis, while CRAPB-I is expressed in a variety of tissues. To obtain structural information which could aid the design of more selective ligands, isotope-directed NMR methods were employed to observe the CRABP-bound conformation of 13C-labeled retinoic acid and to identify its contact points with neighboring amino acids. Analysis of HMQC, HMQC-TOCSY, and 13C-TOCSY-REVINEPT on CRABP-bound (2,3,6,7,8,9,10,11,19-13C)- and (1,4,5,8,9,16, 17,18,19-13C)-all trans-retinoic acid allowed the unambiguous assignment of all labeled protons and their attached 13C resonances. The volumes of 16 olefinic proton-methyl NOE cross-peaks measured from 30-ms 13C-(omega 2)-filtered 1H NOESY experiments were used to determine the conformations about the 6-, 8-, and 10-single bonds of the retinoic acid polyene chain. These spectra show qualitatively distinct NOE patterns for the two CRABPs. Measured cross-peak volumes for CRABP-II bound retinoic acid were well predicted by a single, static conformational having a 6-s torsion angle of -60 degrees skewed from a cis conformation. In contrast, for CRABP-I no single, static conformation was able to match the pattern of cross-peaks, suggesting motion about the 6-s bond. The measured cross-peaks were best described by 8-s and 10-s torsion angles of 180 degrees +/- 30 degrees, a trans configuration, for both proteins. The pattern of intermolecular NOESY cross-peaks between 13C-labeled protons in the ring portion of retinoic acid and protein protons were different between CRABP-I and CRABP-II. These differences coincide well with nearby amino acid substitutions in the recently reported X-ray structures of crystalline CRABP-I and CRABP-II and may assist rational design of selective ligands.

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration.

Cellular retinoic acid binding protein I (CRABP-I) and cellular retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity. Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing. Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination. The apparent dissociation constants, K'd, of mouse CRABP-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than previously reported. The K'd of mCRABP-I and mCRABP-II complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- 1 nM and 15 +/- 11 nM. Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid.

Characterization of the ligand binding domain of human retinoid X receptor alpha expressed in Escherichia coli.

In order to study the structural details of ligand protein interactions of the human retinoid X receptor alpha (hRXR alpha), the DEF and EF domains of the receptor were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The fusion proteins were expressed at high levels and were affinity-purified by chromatography over glutathione-agarose. The DEF and EF domains were cleaved from the fusion proteins by digestion with thrombin. Retinoic acid binding was quantitated using two different methods. The apparent dissociation constant (Kd) and the stoichiometry of 9-cis-retinoic acid binding were performed by monitoring quenching of protein fluorescence. To directly compare the binding affinity of the E. coli-derived truncated hRXR alpha with full-length hRXR alpha expressed in transiently transfected COS cells, Scatchard analyses of [3H]9-cis-retinoic acid binding assays were performed. Both methods of analysis indicate that while the cleaved DEF peptide bound 9-cis-retinoic acid tightly, the cleaved EF peptide exhibited variable binding activity between preparations. By fluorimetric analysis, the Kd of the cleaved DEF peptide was estimated to be 3 +/- 0.5 nM with a stoichiometry of 1:1.1 +/- 0.1. By Scatchard analysis, the Kd values for [3H]9-cis-retinoic acid to the GST-hRXR alpha (DEF) peptide and the cleaved DEF peptide were estimated to be 1.8 nM and 5.6 nM, respectively. The estimated molecular mass from high speed sedimentation equilibrium experiments was 36 +/- 2 kDa for the apo-DEF peptide alone and 38 +/- 3 kDa for the holo-DEF peptide complexed with 9-cis-retinoic acid. This suggests that the recombinant ligand binding domain was predominantly in the monomer form. However, dimers of the cleaved DEF peptides were detected in chemical cross-linking experiments both in the presence and absence of 9-cis-retinoic acid. Since the purified E. coli-derived truncated hRXR alpha DEF peptide appears to fully retain its ligand binding activity, it should provide a useful model system for further structural analysis of ligand-protein interactions.