RARgamma and TRbeta expressions are decreased in PBMC and SWAT of obese subjects in weight gain.

In order to evaluate the expression of nuclear receptors at the peripheral level in obese subjects, messenger RNA (mRNA) levels of different isoforms of retinoic acid receptor (RAR), triiodothyronine (TR), and peroxisome proliferator-activated receptor (PPAR) were determined and compared in peripheral mononuclear blood cells (PBMC) and subcutaneous white adipose tissue (SWAT). Twelve lean subjects and 68 obese subjects divided into weight gain (WG), weight-stable (WS), and weight loss (WL) groups were studied. Nuclear receptor mRNA levels were assessed in PBMC and SWAT using a quantitative real-time reverse transcription polymerase chain reaction method. mRNA levels of RARgamma were significantly lower in PBMC of obese subjects (WG -19%, WS -30%, and WL -24.7%) as in SWAT of WG (-50%). Lower mRNA levels of TRbeta were observed in PBMC and SWAT of WG (-50.7% and -28%, respectively) just as for TRalpha in PBMC of WG (-19%). In contrast, retinoid X receptors alpha (RXRalpha) and RARalpha mRNA levels were higher in PBMC of obese subjects (+53% and +54.5% in WG, +56% and +67% in WS, and +68% and +49.7% in WL, respectively), while expression of RXRalpha was lower in SWAT of WG (-24.5%). As for PPARgamma, its mRNA level was significantly higher in PBMC of WG subjects (+34%) while its expression was not modified in SWAT, contrary to the PPARgamma2 isoform which was significantly higher. These data show that in both adipose tissue and blood compartment of obese subjects, expressions of RARgamma and TRbeta were downregulated. Thus, we suggest that the expression in PBMC of obese subjects may constitute new cellular indicators of nuclear receptor retinoid and thyroid status.

Role of adipogenic and thermogenic genes in susceptibility or resistance to develop diet-induced obesity in rats.

The aim of the present work was to assess whether changes in adipose tissue gene expression related with adipogenesis and/or thermogenesis could be involved in the mechanism conferring susceptibility or resistance to develop obesity in high-fat fed outbreed rats. For this purpose, male Wistar rats were fed with standard laboratory diet (control group) or high fat diet. After 15 days, two groups of rats with significant differences on body weight gain in response to the high fat diet were characterized and identified as diet-induced obesity (DIO) and diet resistant (DR) rats. A significant increase in visceral white adipose tissue (WAT) PPARgamma and aP2 (p < 0.05) mRNA levels associated to a decrease in RARgamma expression (p < 0.05) was observed in DIO rats, suggesting an increase of adipogenesis. Furthermore, our data showed a marked increase in brown adipose tissue (BAT) of UCP1 mRNA in DIO animals (p < 0.01) (without affecting PGC-1alpha gene expression), whereas no changes were found in WAT UCP2 gene expression. All these data suggest that the variations found in the expression pattern of PPARgamma, aP2 and RARgamma by high-fat diet could be involved, at least in part, in the differences in body weight gain and adiposity observed between DR and DIO animals. The compensatory adaptations through the increase in energy expenditure by changes on the expression levels of UCP1 seem not to be enough to avoid the obesity onset in the DIO group.

Alleviation of a selective age-related relational memory deficit in mice by pharmacologically induced normalization of brain retinoid signaling.

Vitamin A and its derivatives, the retinoids, have been implicated recently in the synaptic plasticity of the hippocampus and might therefore play a role in associated cognitive functions. Acting via transcription factors, retinoids can regulate gene expression via their nuclear receptors [retinoic acid receptors (RARs) and retinoid X receptors]. In a series of experiments, the present study investigated the possible role of age-related downregulation of retinoid-mediated transcription events in the cognitive decline seen in aged mice. We observed that the brain (and hippocampal) levels of retinoid receptors and the expression of specific associated target genes were restored to presenescent (adult) levels in aged mice after acute administration (150 microg/kg, s.c.) of retinoic acid (RA). These effects of RA, however, could be abolished by the coadministration of an RAR antagonist. RA was also demonstrated to alleviate the age-related deficit in the CA1 long-term potentiation efficacy of aged mice in vivo. Moreover, RA was found to alleviate completely the performance deficit of aged mice to the control level in a two-stage spatial discrimination paradigm designed to assess relational memory. This promnesic effect of RA was again susceptible to abolition by RAR antagonist treatment. The parallel molecular, cellular, and behavioral correlates associated with the decrease of retinoid receptor expression and its normalization demonstrated here suggest that the fine regulation of retinoid-mediated gene expression is fundamentally important to optimal brain functioning and higher cognition. Specifically, a naturally occurring dysregulation of retinoid-mediated molecular events might be a potential etiological factor for cognitive deterioration during senescence.

Differential effect of retinoic acid and triiodothyronine on the age-related hypo-expression of neurogranin in rat.

Given the important role of retinoids and thyroid hormone for optimal brain functioning and the tenuous relationship between retinoic acid (RA) and triiodothyronine (T3) signalings, we compared the effects of RA or T3 administrations on RA and T3 nuclear receptors (RAR, RXR and TR) and on their target genes, neuromodulin (GAP43) and neurogranin (RC3) in 24-month-old rats. Quantitative real time PCR and western blot analysis allowed us to verify that retinoid and thyroid signalings and GAP43 and RC3 expression are affected by age. By in situ hybridization we observed a decreased expression of RC3 in hippocampus, striatum and cerebral cortex. RARbeta, RXRbeta/gamma and GAP43 were up-regulated by RA as well as T3 treatment. The abundance of TRalpha/beta mRNA and RC3 expression were only increased by T3 administration in the whole brain. This up-regulator effect of T3 on RC3 was only observed in the striatum. During aging, T3 become a limiting factor alone able to correct the age-related concomitant hypo-activation of retinoid and thyroid signalings and alterations of synaptic plasticity.

Decreased expression of retinoid nuclear receptor (RAR alpha and RAR gamma) mRNA determined by real-time quantitative RT-PCR in peripheral blood mononuclear cells of hypothyroid patients.

In vivo assessment of the cellular impact of thyroid hormones on target tissues might be of help for physiological studies and to evaluate the consequences of various diseases of the thyroid gland in humans. Given the tenuous relationship between retinoid and tri-iodothyronine (T3) status and that retinoids have also intracellular roles, the aim of this study was to determine the effect of hypothyroidism on the expression of T3 nuclear receptors (TR) and retinoic acid nuclear receptors (RAR, RXR) in human peripheral blood mononuclear cells (PBMC). Using real time RT-PCR, we quantified the relative amount of mRNA of the thyroid (TR alpha and TR beta) and retinoid (RAR alpha, RAR gamma, and RXR alpha) nuclear receptors in PBMC of euthyroid (n = 22) compared with hypothyroid (n = 22) subjects. Classical plasma parameters (free T3 (FT3), free thyroxine (T4) (FT4), thyroid-stimulating hormone (TSH), retinol (ROH), retinol-binding protein (RBP) and transthyretin (TTR)) were also measured. In hypothyroid subjects, the concentration of TSH was elevated, and dramatically low T3 and T4 concentrations were associated with a decrease in the expression of TR beta. Expression of RAR alpha and RAR gamma significantly decreased in hypothyroid versus control subjects, while an increased concentration of ROH was emphasised by hypothyroidism. These results first indicated that primary hypothyroidism induces hypoactivation of the retinoid nuclear pathway in PBMC, which was not predicted by the plasma ROH level. Further investigations will be necessary to evaluate these parameters in very small changes in thyroid hormone production such as mild (subclinical) hypothyroidism.

Aging affects the retinoic acid and the triiodothyronine nuclear receptor mRNA expression in human peripheral blood mononuclear cells.

BACKGROUND: Inadequate retinoid status has often been described as occurring with aging. Moreover, subclinical hypothyroid status has also been evoked in the elderly. Several studies performed in animals have described the crucial incidence of age-related hypo-functioning of retinoid and thyroid signalling pathways, particularly in the brain. OBJECTIVE: The aim of the present study was to clarify whether aging modifies retinoid and thyroid signalling in humans. METHODS: Using real-time RT-PCR the relative amount of mRNA of the retinoid (RARalpha, RARgamma and RXRalpha) and thyroid (TRalpha and TRbeta) nuclear receptors in peripheral blood mononuclear cells (PBMC) of young (24-57 years old, n = 22) compared with elderly (69-90 years old, n = 24) healthy subjects was quantitated. Classical plasma parameters used to characterize the retinoid and thyroid status - retinol (ROH), retinol-binding protein (RBP), free triiodothyronine (FT3) and thyroxine (FT4), thyroid-stimulating hormone (TSH) and transthyretin (TTR) - were also assessed. RESULTS: RARgamma expression was significantly decreased in elderly versus young subjects while no modification of the retinoid-related plasma parameters ROH and RBP were emphasized by aging. Concerning thyroid criteria, the elderly exhibited an increase in TSH concentration (+39%) without significant modifications of FT3 and FT4, which indicated an age-related sub-clinical hypothyroidism. Concurrently, the amount of TR mRNA (alpha as well as beta subtypes) was significantly decreased in the elderly. CONCLUSION: These data constitute the first evidence of an age-related hypo-activation of the retinoid and thyroid nuclear pathways in PBMC. Further study of the possible association between the expression of the retinoid and thyroid nuclear receptors and age-related cognitive alterations in humans would be interesting.

Effect of vitamin A content in cafeteria diet on the expression of nuclear receptors in rat subcutaneous adipose tissue.

The aim of this study was to determine the effects of cafeteria diet containing control or elevated level of vitamin A on the expression of nuclear receptors in adipose tissue. Male Wistar rats were submitted to 3 experimental diets during 8 weeks, a standard diet and two hyper-energetic, hyperlipidic "cafeteria" diets containing normal (Caf) or higher (Caf+) vitamin A level. During the experiment, body weights and energy intakes were measured. At the end of the experimental period, subcutaneous adipose tissue (Swat) and all the fat mass were removed and weighted. Nuclear receptors mRNA levels of RARalpha, RARgamma, RXRalpha, PPARgamma were measured in the Swat by a real-time semi-quantitative RT-PCR method. We observed that energy intake of Caf+ and Caf groups was significantly higher than that of the control group. Despite a higher increase of the energy intake in the Caf group compared to the Caf+ group, no significant difference was observed in the body weight gain of the Caf+ compared to the Caf group. The Caf+ and Caf diets led to a significant increase of adipose tissue in cafeteria groups as observed in the Swat depot. The mRNA levels of PPARgamma and RXRalpha were significantly increased in the Caf+ group as compared to control group, with a significant positive correlation between these two parameters. Expressions of RARalpha and RARgamma were not modified in experimental groups compared to controls. In conclusion, 8-week exposure to cafeteria diets with normal and higher levels of vitamin A led to an increase of adiposity in rats, associated, only in the group fed with the higher vitamin A level cafeteria diet, with an increase of PPARgamma and RXRalpha expressions in subcutaneous adipose tissue.

c-erb-A mRNA content and triiodothyronine nuclear receptor binding capacity in rat liver according to vitamin A status.

Vitamin A is required for normal growth and development and synergistically acts with thyroid hormones in these processes. Effects of retinol dietary intake (0, 5, 50 U retinol/g food) on the triiodothyronine (T3) nuclear receptor were examined in rat liver. Properties of this receptor have been investigated by an in vitro binding method. The amount of c-erb-A mRNA has been deduced by reverse transcription and amplification methods (PCR). Results show that both retinol deficiency and excess leads to a reduced amount of c-erb-A mRNA and to a decreased T3 binding capacity, suggesting that retinol, or retinoic acid which is its natural metabolite, contributes to the regulation of the T3 nuclear receptor properties.

Aging decreases the abundance of retinoic acid (RAR) and triiodothyronine (TR) nuclear receptor mRNA in rat brain: effect of the administration of retinoids.

Aging is accompanied by troubles resulting from changes in hormonal and nutritional status. Therefore, the abundance of mRNA coding for triiodothyronine (TR) and retinoic acid (RA) nuclear receptors was studied in the brain of young, adult and aged (2.5, 6 and 24 months, respectively) rats. In the brain of aged rats, there was a lower abundance of TR and RAR mRNA and a lower activity of tissue transglutaminase (tTG), an enzyme the gene of which is a target for retinoids. Administration of RA in these rats restored TR and RAR mRNA and the activity of tTG in the brain. The importance of these observations to the function of the aged brain is discussed.

Chronic ethanol administration enhances retinoic acid and triiodothyronine receptor expression in mouse liver.

Chronic alcoholism induces perturbations of storage and metabolization of retinol and related compounds. After 6 months of ethanol consumption we have observed in mouse liver an increased expression of Tri-iodothyronine receptors (TR) while the expression of retinoic acid (RA) receptors (RAR) was unaffected. After 10 months of alcoholization the TR expression was strongly increased and the RAR expression was also increased. At this time the activity of aldehyde dehydrogenase and that of alcohol dehydrogenase, two enzymes involved in biosynthesis of RA from retinol, were similar in the liver of alcoholized and pair-fed mice. Thus it can be hypothesized that (i) the change of RAR expression was, at least in part, the result of a change of TR expression (result in agreement with previous data), (ii) the increased expression of RAR could induce apoptosis and subsequently liver necrosis.

Retinoic acid decreases retinoic acid and triiodothyronine nuclear receptor expression in the liver of hyperthyroidic rats.

Retinoic acid (RA) and triiodothyronine (T3) exert many of their actions by binding to specific nuclear receptors (respectively, RA receptor (RAR) and T3) receptor (TR) belonging to a 'superfamily' of receptors. Some heterologous regulation of these receptors has been shown, and in particular regulation of the maximum binding capacity of TR by either retinol or RA. Now, using hyperthyroidic rats as a model, the effect of RA on binding capacity and on the mRNA levels of TR and RAR was investigated. The results show that the benefit of vitamin A treatment for the hyperthyroidic state, which has been described for a long time, could be the result of a down-heteroregulation of TR by RA, the active metabolite of retinol.

Aging decreases retinoic acid and triiodothyronine nuclear expression in rat liver: exogenous retinol and retinoic acid differentially modulate this decreased expression.

The expression of nuclear receptors of retinoic acid (RAR) and triiodothyronine (TR) was analyzed in the liver of rats aged 2.5 (young), 6 (adult) and 24 (aged) months. In aged rats, decreased binding properties, binding capacity (Cmax) and affinity (Ka), of nuclear receptors were observed. This resulted, at least in part, from decreased transcription of receptor genes in that the amount of their mRNA also decreased. Moreover, the activity of malic enzyme (ME) and tissue transglutaminase (tTG), whose genes are TR and RAR responsive, respectively, was reduced in aged rats. These results are in agreement with the decreased binding capacity of these receptors. An inducer-related increase of RAR and TR expression was observed 24 h after a single dose of retinoic acid administration (5 mg/kg), while retinol administration (retinyl palmitate, 13 mg/kg) was without incidence on nuclear receptor expression in aged rats.

Free and protein-bound tri-iodothyronine in the serum of vitamin A-deficient rats.

Tri-iodothyronine (T3) transport in the serum of rats on a vitamin A-deficient diet was studied by electrophoresis after the incubation of serum samples with radioactive T3. The level of serum free tri-iodothyronine (fT3) was measured by radioimmunoassay after serum chromatography on Sephadex LH-20 gel. In the serum of control rats a major part of the radioactivity was bound to the albumin zone (61%) and a minor part to the prealbumin zone (32%). In the serum of vitamin A-deficient rats a larger amount of radioactivity was bound to the postalbumin zone; the lower the level of vitamin A in serum, the greater was the radioactivity in this zone of the electrophoretogram. In these animals there was also a negative correlation between the serum vitamin A and the fT3. There was also a positive correlation between the radioactivity bound to the postalbumin zone and the fT3. The possible causes and effects of these modifications in vitamin A-deficient rats are discussed.

Transport of thyroxine in the serum of vitamin A-deficient rats.

Thyroxine (T4) transport in the serum of rats deficient in vitamin A was studied by electrophoresis after incubation of serum samples with radioactive T4. In serum from vitamin A-deficient rats, the amount of radioactivity bound to the prealbumin zone of the electrophoretograms decreased, whereas there was an increase in the amount of radioactivity bound to the albumin zone. The serum from vitamin A-deficient rats subsequently fed a normal diet (repletion experiment) had an electrophoretic pattern similar to that of normal rats. The serum fraction which bound radioactive T4 appeared in the prealbumin rather than the albumin zone. These results suggest that vitamin A deficiency causes an early change in the transport of thyroid hormones in the plasma and a new level of serum thyroxine which may be responsible for the biochemical and physiological changes which occur in vitamin A-deficient rats.

Vitamin A deficiency and tri-iodothyronine action at the cellular level in the rat.

Young rats were given a diet with or without vitamin A for a period of 7 weeks. In rats on the vitamin A-deficient diet, the formation of tri-iodothyronine (T3) complexes with nuclear proteins was reduced, and Scatchard analysis showed a decreased capacity of nuclear T3 receptors. In these rats, a decrease in growth and a decrease in the activity of lipogenic enzymes (glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase and L-malate dehydrogenase) occurred, which could be related to the decreased formation of T3-nuclear protein complexes.

Effect of retinoids on protein kinase C activity and on the binding characteristics of the tri-iodothyronine nuclear receptor.

Retinoids and thyroid hormones exert profound effects on the development, growth and homeostasis of vertebrates. The receptor proteins which bind retinoic acid, tri-iodothyronine (T3) or steroid hormones and, as a result of this binding, interact with DNA to stimulate expression of specific genes, belong to the same recently discovered superfamily. The functionality of thyroid and steroid hormone receptors is thought to be related to a phosphorylation-dephosphorylation cycle. In the present work, the action of two retinoids (retinol and retinoic acid) was studied on the properties of T3-nuclear receptors and on protein kinase C (PKC) activity in the rat liver (PKC is known to be a phosphorylating enzyme for various proteins). The influence of 12-O-tetradecanoyl phorbol-13-acetate (TPA; known to enhance PKC activity) on the properties of T3-nuclear receptors was also investigated. Measurements of binding characteristics and enzyme activity were performed 4 or 12 h after a single i.p. injection of retinol or retinoic acid (6 mg/kg body weight) or 1 h after a single i.p. injection of TPA (0.7 mg/kg). The activity of PKC was increased 4 h after administration of the retinoids, and the affinity of the T3-nuclear receptor protein was increased markedly after 12 h. The activity of PKC and the affinity of nuclear T3 receptor were both increased 1 h after administration of TPA. These observations provide indirect evidence that retinoids, particularly retinoic acid, induce an increase in PKC activity and a subsequent increase in the affinity of the T3-nuclear receptor protein.

Vitamin A deficiency and glucocorticoid receptor activity in rat liver.

The influence of vitamin A on the binding properties of hepatic glucocorticoid receptors (GR) was studied in young rats 7 weeks after they had been given a diet with or without vitamin A. Scatchard analysis showed an increased capacity of cytosolic GRs to bind dexamethasone in vitamin A deficiency. In these rats, an increase in tyrosine aminotransferase also occurred, and this could be related to the increased formation of hormone-GR complexes. Measurement of protein kinase C activity showed an increase which might be related to the functional activity of GRs.

Retinoic acid differentially modulates triiodothyronine and retinoic acid receptors in rat liver according to thyroid status.

Triiodothyronine (T3) receptors (TRs) and retinoic acid (RA) receptors (RARs) exert their effects on growth, differentiation and cellular homeostasis by acting as transcription factors. The binding characteristics of these receptors have been studied in liver of hypothyroid and hyperthyroid rats, with or without treatment with T3, RA or T3 + RA together. The changes in binding induced by RA treatment depended on the hormonal status of the rat. In hypothyroid rats the T3 binding capacity was unaltered by administration of T3 or RA alone but increased by 48% after treatment with T3 and RA together. In these rats administration of RA, T3 or T3 + RA increased the RAR binding capacity by 45, 79 and 112%, respectively. In hyperthyroid rats the administration of RA reduced the TR and RAR binding capacities by 22 and 37%, respectively. We found also that the affinity constants of TRs and RARs were reduced in hypothyroid rats after treatment with T3 or T3 + RA. It is suggested that this change of the properties of receptors is related to a ligand-dependent conformational change in these receptors.

Expression of neurogranin and neuromodulin is affected in the striatum of vitamin A-deprived rats.

Our previous data showed that vitamin A deficiency (VAD) induces, in whole brain, a reduced amount of mRNA for brain retinoic acid (RA) and triiodothyronine (T3) nuclear receptors (i.e., RAR, RXR, and TR, respectively), which is accompanied by reduced amounts of mRNA and protein of neurogranin (RC3, a neuronal protein involved in synaptic plasticity) as well as selective behavioral impairment. Given the important role of retinoids for optimal brain functioning, the effects of vitamin A depletion and subsequent administration of RA or T3 on the mRNA levels of RA and T3 nuclear receptors and on two target genes' (RC3 and neuromodulin or GAP43) mRNA and protein levels were examined in the hippocampus, striatum, and cerebral cortex. A quantitative real-time polymerase chain reaction (PCR), in situ hybridization, and Western blot analysis demonstrated that the striatal region is the brain site where both RA and T3 signaling pathways are most affected by VAD. Indeed, rats fed a vitamin A-free diet for 10 weeks exhibited decreased expression of RAR, RXR, TR, RC3, and GAP43 in the striatum. The administration of T3 to these vitamin A-deprived rats reversed the reduction in mRNA levels of RA and T3 nuclear receptors and in mRNA and protein levels of target genes in this region. These data suggest that modifications that appear preferentially in the striatum, a region highly sensitive to vitamin A bioavailability, may contribute to neurobiological alterations and the spatial learning impairment that occurs in vitamin A-deprived animals.

Dexamethasone decreases the expression of retinoic acid receptors (RARs) in rat liver.

Although adrenalectomy was without effect on the expression of retinoic acid (RA) receptors (RARs), administration of the glucocorticoid analog dexamethasone (Dex) to both control and adrenalectomized rats reduced the expression of these receptors in rat liver. This effect can be mainly attributed to the action of Dex on 4-hydroxylation of RA. Dex, by enhancing 4-hydroxylation of RA, reduces its intracellular concentration thereby leading to a decreased expression of RARs, since RARbeta, the main type of RARs in liver, are known to be up-regulated by RA.