Effects of oleate and insulin on glucose uptake, oxidation, and glucose transporter proteins in rat adipocytes.

We examined effects of Na oleate on glucose uptake, glucose transporter protein concentrations, and glucose oxidation in isolated adipocytes from fed rats. Na oleate increased basel 14C-glucose uptake in a dose-dependent manner (+42% with 1.0 mM, +79% with 2.8 mM Na oleate), but had no statistically significant effect on insulin-stimulated glucose uptake. Insulin (100 nM) resulted in a redistribution of GLUT4 protein concentration from the LDM fraction (-42%) to the PM fraction (+266%) but did not affect the distribution of GLUT1. Na oleate had no effect on basal or insulin-stimulated concentrations of GLUT1 or GLUT4 proteins in the PM or LDM fractions. Na oleate (2.8 mM) had no statistically significant effect on basal glucose oxidation, but inhibited insulin-stimulated glucose oxidation by 48% (P less than 0.01). In summary, Na oleate inhibited insulin-stimulated glucose oxidation and stimulated basal glucose uptake in isolated adipocytes without affecting PM or LDM distribution of GLUT1 or GLUT4 proteins. We conclude that the stimulatory effect of Na oleate on basal glucose uptake in adipocytes may be mediated by changes in the intrinsic activity of the glucose transporters.

A sustained elevation in retinoic acid receptor-beta 2 mRNA and protein occurs during retinoic acid-induced fetal dysmorphogenesis.

We have previously shown that oral treatment of pregnant mice with all-trans retinoic acid (RA) at doses which cause 100% fetal dysmorphogenesis results in a rapid elevation in the mRNA of one specific isoform of the RA receptor-beta, RAR-beta 2, in susceptible embryonic regions. To further investigate the involvement of RAR-beta 2 mRNA in teratogenesis, we have examined its expression in mouse embryos exposed to marginal/nonteratogenic and teratogenic dosing regimens of both 13-cis RA and all-trans RA. We have found that the mere elevation in embryonic RAR-beta 2 mRNA levels and free retinoid levels is not sufficient to result in dysmorphogenesis. Rather, retinoid-induced dysmorphogenesis of embryos appears to occur only when RAR-beta 2 mRNA and unbound retinoid levels remain elevated for at least 6-9 h following retinoid treatment resulting in a significant and prolonged elevation in RAR-beta protein levels.

Modulation of limb bud chondrogenesis by retinoic acid and retinoic acid receptors.

An excess of retinoic acid (RA) in the mouse embryo in utero produces hypochondrogenesis and severe limb bone deformities. Since one of the RA receptors--RAR-beta 2, is specifically induced in the limb bud cells upon treatment of embryos with teratogenic doses of RA, we investigated if this receptor played a role in teratogenesis by regulating the process of chondrogenesis. In micromass cultures of mouse limb bud mesenchymal cells, we found that a downregulation of RAR-beta 2 as well as several other RAR isoforms by supplementation of the culture medium with specific oligodeoxynucleotides stimulated chondrogenesis: cartilage nodule number, sulfated proteoglycans, and synthesis of collagen type IIB were all enhanced in a dose-dependent manner. However, only the antisense RAR-beta 2 probe efficiently prevented the strong inhibitory effects of exogenous RA on chondrogenesis in these cells. The data suggest that the RAR-RA complexes play a role in position-dependent patterning of the limb skeleton in normal development and that, in particular, RAR-beta 2 serves to prevent the mesenchymal cells from expressing their chondrogenic bias. Our results further strengthen the argument that RA-dependent elevation in RAR-beta 2 levels plays a unique role in RA-induced teratogenesis.

Macrophage colony-stimulating factor suppresses osteoblast formation.

We provide the first evidence that the bone marrow-derived cytokine, macrophage colony-stimulating factor (M-CSF), inhibits the formation of bone-forming osteoblasts. We examined both osteoclast and osteoblast formation in primary rat bone marrow cultures. As expected, M-CSF together with osteoprotegerin ligand (OPGL) markedly accelerated osteoclastogenesis. In contrast, treatment with M-CSF alone yielded no osteoclasts at any time. The most striking and novel observation was that M-CSF with or without OPGL dramatically suppressed osteoblast formation. In separate experiments, estradiol markedly suppressed osteoclast formation in the M-CSF/OPGL-treated cultures independently of osteoblasts. Consistent with this was the expression of estrogen receptor-alpha (ERalpha) and ERbeta mRNA in osteoclast precursors. We therefore conclude that in addition to the well-known action of M-CSF to modulate osteoclastogenesis, this cytokine may also regulate osteoblast formation.

Arg269 and Lys220 of retinoic acid receptor-beta are important for the binding of retinoic acid.

Retinoic acid receptors, RARs, are retinoic acid (RA)-inducible transcriptional regulatory proteins which transduce the RA signal at the level of gene expression via a retinoic acid response element. Three subtypes of RARs have been described termed RAR-alpha, RAR-beta, and RAR-gamma. RARs, like other members of the steroid/thyroid hormone receptor family, are composed of six structurally distinct domains, one of which is responsible for binding RA. No structural information is available concerning the nature of the amino acids which are responsible for binding of RA within the ligand binding domain of any RAR. In this report, the role of 2 positively charged amino acids of RAR-beta for binding of RA, Arg269 and Lys220, was examined using site-directed mutagenesis. When compared with wild type RAR-beta, mutation of either Arg269 or Lys220 singly to the small neutral amino acid Ala had only a small effect on both the EC50 value in all-trans-RA and 9-cis-RA transactivation assays and the apparent Kd for all-trans-RA. However, mutation of both of these positively charged amino acids simultaneously to Ala caused a 500- and 100-fold elevation in the EC50 for all-trans-RA and 9-cis-RA, respectively, compared with that of wild type RAR-beta. Similarly, the apparent Kd for all-trans-RA was increased 580-fold when that of the double mutant was compared with that of the wild type RAR-beta. Furthermore, this double mutant RAR-beta acted as a dominant negative mutant when transfected with wild type RAR-alpha, -beta, or -gamma in a RA concentration-dependent fashion. Taken together these data demonstrate the importance of both Arg269 and Lys220 of RAR-beta for the binding of RA, possibly by interacting with the negatively charged carboxyl group of RA.

New insights into the regulation of cathepsin K gene expression by osteoprotegerin ligand.

Cathepsin K plays a key role in bone resorption. We provide the first evidence that osteoprotegerin ligand (OPGL), a critical pro-resorptive cytokine, acutely stimulates the expression of cathepsin K in osteoclasts. We used in situ RT-PCR and real time quantitative RT-PCR to analyze cathepsin K gene expression. OPGL enhanced cathepsin K mRNA levels in mature osteoclasts isolated from rat neonatal long bones. OPGL together with macrophage colony-stimulating factor (M-CSF) also stimulated cathepsin K gene expression in monocytic cells and multinucleate osteoclasts in bone marrow cultures. Real time quantitative RT-PCR demonstrated high levels of cathepsin K mRNA in bone marrow cultures, paralleling the degree of osteoclastogenesis. We therefore suggest that OPGL enhances bone resorption, at least in part, by inducing cathepsin K gene expression.

Induction of RAR-beta 2 gene expression in embryos and RAR-beta 2 transactivation by the synthetic retinoid Ro 13-6307 correlates with its high teratogenic potency.

Vitamin A (retinol), its metabolite all-trans retinoic acid (RA), and many synthetic analogs (retinoids) express variable potencies as teratogens. Although biological activities of retinoids are mediated by nuclear RA receptors (RARs) and retinoid X receptors (RXRs), it is not known if any of these receptors mediate teratogenicity, and if the potency also depends on the nature of the ligand-receptor interactions. Previous evidence has implicated that one specific isoform, RAR-beta 2, does play a role in mediating retinoid teratogenicity. Here, we employed an aromatic retinoid with a triene side chain, Ro 13-6307, to study its interactions with RAR-beta 2 since its teratogenicity is much higher and its accessibility to the embryo is much lower than RA. A fully teratogenic dose of Ro 13-6307 (10 mg-kg) given to pregnant mice preferentially elevated the level of RAR-beta 2 mRNA in susceptible embryonic regions (maximal induction, 10- to 12-fold above control in limb buds) in a manner comparable to a fully teratogenic dose of all-trans RA (100 mg-kg). Using the RAR-beta 2 promoter linked to a reporter gene in cotransfection experiments, the efficacy of Ro 13-6307 and RAR-beta 2 in transcription transactivation was found to be 30-40 times greater than all-trans RA. Since the teratogenic potency of Ro 13-6307 is estimated from a previous study to be 44-fold greater than all-trans RA, we suggest that the teratogenicity of this synthetic retinoid is generally proportional to its ability to enhance receptor function.

Teratogenesis by retinoic acid analogs positively correlates with elevation of retinoic acid receptor-beta 2 mRNA levels in treated embryos.

Retinoic acid (RA) plays an important role during normal embryogenesis, however high doses of RA are teratogenic. Retinoic acid receptor-beta 2 (RAR-beta 2) mRNA and protein levels were previously demonstrated to undergo rapid elevation in susceptible tissues after treatment with teratogenic doses of RA. In this report we compared the effects of a number of retinoids, which represent a wide variety of chemical structures and which differ in their teratogenic potencies, on RAR-beta 2 mRNA levels in mouse embryos 6 hr after treatment. Retinoid treatments which result in a high incidence of limb defects elevated RAR-beta 2 mRNA levels similarly (10-14 fold in the limb buds, 4-8 fold in the head, and 2-4 fold in the remainder of the body). On the other hand, retinoid treatments which cause a low or no incidence of limb defects resulted in minor changes in RAR-beta 2 mRNA levels in each embryonic region. Therefore, a strong positive correlation was found between the elevation of RAR-beta 2 mRNA levels and the retinoids which produce limb defects. This provides further evidence that an elevation of RAR-beta 2 mRNA levels, and subsequently protein levels, is an important event involved in mediating the effects of RA during dysmorphogenesis.