The thyroid receptor ß modulator GC-1 reduces atherosclerosis in ApoE deficient mice.

Thyroid hormone reduces plasma cholesterol and increases expression of low-density lipoprotein receptor (LDL-R) in liver, an effect mediated by thyroid receptor ß (TRß). The selective TRß modulator GC-1 also enhances several steps in reverse cholesterol transport and can decrease serum cholesterol independently of LDL-R. To test whether GC-1 reduces atherosclerosis and to determine which mechanisms are active, we treated ApoE deficient mice with atherogenic diet ± GC-1. GC-1 reduced cholesteryl esters in aorta after 20 weeks. Serum free and esterified cholesterol were reduced after 1 and 10 weeks, but not 20 weeks. Hepatic bile acid synthesis and LDL-R expression was elevated after 1, 10 and 20 weeks, without changes in hepatic de novo cholesterol synthesis. GC-1 increased faecal neutral sterols and reduced serum campesterol after 1 week, indicating reduced intestinal cholesterol absorption. After 20 weeks, GC-1 increased faecal bile acids, but not faecal neutral sterols. Hepatic scavenger receptor B1 (SR-B1) expression was decreased by GC-1. We conclude that GC-1 delays the onset of atherosclerosis in ApoE deficient mice. Since ApoE is needed for hepatic cholesterol reabsorption by LDL-R, this supports the idea that GC-1 reduces serum cholesterol independently of LDL-R by increasing hepatic bile acid synthesis. GC-1 lipid-lowering effects in ApoE deficient mice may also be partly due to reduced intestinal cholesterol absorption. Since reductions in serum cholesterol are reversed at longer times, these GC-1 dependent effects may not be enough for sustained cholesterol reduction in long term treatments.

Clinical and biochemical implications of low thyroid hormone levels (total and free forms) in euthyroid patients with chronic kidney disease.

OBJECTIVES: In this study, we explore the associations of decreased thyroid hormone levels with inflammation, wasting and survival in biochemically euthyroid patients with end-stage renal disease (ESRD). DESIGN: After exclusion of 23 patients with thyroid-stimulating hormone (TSH) values outside the normal range (0.1-4.5 mIU L(-1)), 187 clinically and biochemically euthyroid incident ESRD stage 5 patients starting dialysis were followed for a median of 20 (range 1-60) months. Measurements of total and free forms of thyroid hormones, s-albumin, hs-CRP, interleukin (IL)-6, vascular adhesion molecule (VCAM)-1 and insulin-like growth factor 1 (IGF-1) were performed at baseline. RESULTS: In this population, 17 out of 210 patients (8%) were defined as subclinically hypothyroid. Multivariate analysis, according to receiver operating characteristic (ROC) curves, showed that mortality was best predicted by total triiodothyronine (T3). When using the cut-off levels derived from ROC, low T3 levels were associated with increased inflammation (higher hs-CRP, IL-6 and VCAM-1) and lower concentration of both s-albumin and IGF-1. Finally, low T3 but not low free triiodothyronine was associated with worse all-cause (Likelihood ratio = 45.4; P < 0.0001) and cardiovascular mortality (Likelihood ratio = 47.8; P < 0.0001) after adjustment for confounding factors. CONCLUSION: This study showed that low T3 levels are independent predictors of all-cause and also cardiovascular disease mortality in biochemically euthyroid patients, perhaps due to an intimate association with inflammation. Based on these results, the use of T3 levels in studies assessing the relationship between thyroid dysfunction and mortality risk is recommended.

Beta 3- and alpha1-adrenergic Erk1/2 activation is Src- but not Gi-mediated in Brown adipocytes.

A novel signaling pathway for mediation of beta(3)-adrenergic activation of the mitogen-activated protein kinases Erk1/2 (associated with proliferation, differentiation, and apoptosis) has recently been proposed, which implies mediation via constitutively coupled G(i)-proteins and Gbetagamma-subunits, distinct from the classical cAMP pathway of beta-adrenergic stimulation. To verify the significance of this pathway in cells in primary cultures that entopically express beta(3)-adrenoreceptors, we examined the functionality of this pathway in cultured brown adipocytes. Norepinephrine activated Erk1/2 via both beta(3) receptors and alpha(1) receptors but not via alpha(2) receptors. Forskolin induced Erk1/2 activation similarly to beta(3) activation, indicating cAMP-mediation; this induction could be inhibited with H89, implying protein kinase A mediation. The G(i)-pathway was functional in these cells, as pertussis toxin increased agonist-induced cAMP accumulation. However, pertussis toxin was unable to affect adrenergically induced Erk1/2 activation. Also, wortmannin was without effect, implying that Gbetagamma activation of the phosphatidylinositol 3-kinase pathway was not involved. PP1/2, which inhibits Src, abolished both beta(3)- and alpha(1)-induced Erk1/2 activation. Thus, the proposed novel G(i) pathway for beta(3) mediation is not universal, because it is not functional in the untransformed primary cell culture system with entopically expressed beta(3) receptors examined here. Here, the beta(3) signal is mediated classically via cAMP/protein kinase A. beta(3) and alpha(1) signals converge at Src, which thus mediates Erk1/2 activation in both pathways.

Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization.

Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.

The fatty liver dystrophy mutant mouse: microvesicular steatosis associated with altered expression levels of peroxisome proliferator-regulated proteins.

Fatty liver dystrophy ( fld) is an autosomal recessive mutation in mice characterized by hypertriglyceridemia and fatty liver during neonatal development. The fatty liver in fld/fld mice spontaneously resolves between the age of 14-18 days, at which point the animals develop a neuropathy associated with abnormal myelin formation in peripheral nerve. We have investigated the morphological and biochemical alterations that occur in the fatty liver of neonatal fld/fld mice. Studies at the light and electron microscopic level demonstrated the accumulation of lipid droplets and hypertrophic parenchymal cells in fld neonates, with no apparent liver pathology after resolution of the fatty liver. To better characterize the biochemical basis for the development of fatty liver in fld mice, we compared protein expression patterns in the fatty liver of fld mice and in the liver of phenotypically normal (wild-type) littermates using quantitative two-dimensional gel electrophoresis. We detected 24 proteins with significantly altered expression levels (P < 0.001) in the fld fatty liver, 15 of which are proteins that are altered in abundance by peroxisome proliferating chemicals. As these compounds characteristically elicit changes in the expression of mitochondrial and peroxisomal enzymes involved in fatty acid oxidation, we quantitated rates of fatty acid oxidation in hepatocytes isolated from fld and wild-type mice. These studies revealed that hepatic fatty acid oxidation in fld neonates is reduced by 60% compared to wild-type littermates. In hepatocytes from adult fld mice that no longer exhibit a fatty liver, oxidation rates were similar to those in hepatocytes from age-matched wild-type mice. These findings indicate that altered expression of proteins involved in fatty acid oxidation is associated with triglyceride accumulation in the fld fatty liver.

Ambient temperature regulation of apoptosis in brown adipose tissue. Erk1/2 promotes norepinephrine-dependent cell survival.

Brown adipose tissue hyperplasia is a fundamental response to low ambient temperature. We show here that cold exposure of an animal markedly increased the phosphorylation of mitogen-activated protein kinase (p42/p44) Erk1 and Erk2 in brown adipose tissue, and protected cells in the tissue from apoptosis. We also show that cessation of the sympathetic stimulus, by transferring cold-adapted animals to 28 degreesC, caused an increased rate of apoptosis in the tissue. In primary cultures of brown adipose tissue, norepinephrine (NE) stimulated both the phosphorylation and the activity of Erk1/2 via the Erk kinase MEK, and protected the cells form apoptosis. Similarly, agonist stimulation of alpha1- and beta-adrenergic receptors and increases in the intracellular level of Ca2+ and cAMP stimulated the phosphorylation of Erk1/2. Agonist stimulation of alpha1- and beta-adrenergic receptors, and increased intracellular cAMP level also promoted the cell survival. Furthermore, NE stimulated the expression and secretion of basic fibroblast growth factor (bFGF), which further promoted the cell survival via MEK-dependent activation of Erk1/2. In essence, we show that Erk1/2 has a critical role in promoting NE- and bFGF-dependent survival of brown adipocytes, and propose that NE- and bFGF-dependent regulation of the cell survival is involved in the cold-induced hyperplasia of brown adipose tissue.

Contrasting adrenergic effects on lipoprotein lipase gene expression in the brown adipose tissue of intact mice and in cultured brown adipocytes from mice.

To examine the regulation of lipoprotein lipase (LPL) gene expression, LPL mRNA levels in the brown adipose tissue of intact mice and in mouse brown adipocyte cultures were examined. In intact mice, exposure to cold resulted in a rapid, transient, 5-fold increase in LPL mRNA level. Norepinephrine (NE) injection could fully mimic the effect of acute exposure to cold, and LPL mRNA and enzymatic activity were increased in parallel after NE injection. These results indicated positive adrenergic control of LPL gene expression in the brown adipose tissue of intact mice. In cultured mouse brown adipocytes, the level of spontaneously expressed LPL mRNA decreased in parallel with the progression of brown adipocyte differentiation. NE treatment of undifferentiated cells led to a decrease in LPL mRNA levels. In brown adipocytes that had reached a mature state, NE had a small negative or no effect on LPL mRNA levels, irrespective of whether the experiment was performed in the presence or absence of insulin or of newborn-calf serum. It was concluded that LPL gene expression in brown adipose tissue in intact mice is under adrenergic control but that this gene is not under positive adrenergic control in cultured brown adipocytes from mice, although these cells are otherwise adrenergically sensitive. The presence of additional factors may be necessary to confer adrenergic sensitivity to the LPL gene in the cultured brown adipocytes; alternatively, cells other than the mature brown adipocytes may confer the positive adrenergic sensitivity to the brown adipose tissue depots in situ.

Adrenergic stimulation of lipoprotein lipase gene expression in rat brown adipocytes differentiated in culture: mediation via beta3- and alpha1-adrenergic receptors.

In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control.

Signal transduction in brown adipose tissue recruitment: noradrenaline and beyond.

The classical effect of noradrenaline on brown adipose tissue is stimulation of heat production. However, it is likely that noradrenaline is also the major regulator of proliferation and differentiation. The adrenergic receptors involved include at least beta 1, beta 3, alpha 2 and alpha 1. Heat production is mainly stimulated via beta 3 receptors and cAMP. Cell proliferation is mainly stimulated via beta 1 receptors and cAMP. Cell differentiation is also adrenergically promoted; at least the expression of the gene for the tissue-specific uncoupling protein thermogenin is controlled via beta 3 receptors and cAMP. There is a switch in beta-receptor endowment between young (beta 1) and mature (beta 3) cells. The expression of several transcription factors is also under adrenergic control: c-Fos gene expression depends synergistically on beta- and alpha 1-stimulation mediated via cAMP and [Ca2+]i increases. C/EBP beta gene expression is regulated only via beta-receptors, but the expression of the C/EBP alpha gene shows a switch during differentiation: in young cells, the expression is represented through both beta- and alpha 1-receptors; in mature cells, the expression is stimulated via b-receptors. It is likely that noradrenaline exerts its proliferation- and differentiation-promoting action through alterations in the expression of these or other transcription factors.

Norepinephrine as a morphogen?: its unique interaction with brown adipose tissue.

Norepinephrine is normally considered a neurotransmitter mediating acute metabolic effects in target cells. However, analysis of the regulation of the recruitment process in brown adipose tissue has indicated that norepinephrine may interact with this tissue in such a way that it could be considered a morphogen for this tissue. Besides stimulating the acute thermogenic processes, norepinephrine can induce the expression of tissue-specific proteins such as the uncoupling protein, induce expression of non-tissue specific proteins necessary of the thermogenic processes (e.g. lipoprotein lipase) and repress the expression of non-essential proteins (e.g. subunit c of the ATP-synthase). Upon chronic adrenergic stimulation, the general differentiation state of the tissue is advanced, indicating that the expression of factors with a more general effect on brown adipocyte differentiation is also under adrenergic control. It may even be discussed that norepinephrine may be involved early in the embryonal determination process directing cell clones into this line. The molecular basis for these effects of norepinephrine are only poorly known at present, but adrenergic effects on the expression level of many transcription factors, such as C/EBPalpha, C/EBPbeta, and PPARgamma 2, have been noted. These collective recruitment effects of norepinephrine are well suited to allow the tissue to grow or atrophy in response to the physiological needs of the organism.

Differential adrenergic regulation of C/EBP alpha and C/EBP beta in brown adipose tissue.

We investigated the regulation of the expression of two members of the C/EBP family of transcriptional activators, C/EBP alpha and C/EBP beta, in brown adipose tissue in mice. Less than one hour of cold exposure led to dramatic changes in the expression of both genes. C/EBP alpha steady-state mRNA and protein levels were drastically and rapidly reduced whereas C/EBP beta mRNA and protein levels were induced severalfold. Also norepinephrine injection affected the expression of the transcription factors. Preconfluent cells in brown fat primary cultures responded to norepinephrine with a decrease in C/EBP alpha and an increase in C/EBP beta mRNA; in confluent cells the expression of both factors was increased. Thus, C/EBP alpha and C/EBP beta gene expression is under adrenergic control both in vivo and in vitro but the type of response is directed by the degree of differentiation of the cells.

Transcriptional and posttranscriptional mechanisms in uncoupling protein mRNA response to cold.

Three mechanisms account for the rapid elevation and maintenance of uncoupling protein (UCP) mRNA levels in cold-exposed rats, namely, an increase in the rate of transcription initiation, an increase in the fraction of nascent UCP transcripts undergoing elongation, and stabilization of the mature UCP mRNA. The second mechanism precedes and outlasts the increase in the rate of UCP gene transcription, which is brisk but short lived. After 48 h of cold exposure, mature UCP mRNA levels are maintained elevated solely on the basis of stabilization, since the levels of both transcription initiation and fifth intron-containing transcripts (precursors) have returned to basal. Results in hypothyroid rats given 3,5,3'-triiodothyronine (T3) and in dispersed brown adipocytes show that T3 is involved both in the increase in UCP mRNA precursor level and stabilization of mature UCP mRNA. These mechanisms are rapidly reversed when the rats are returned to thermoneutrality. These coordinated transcriptional and post-transcriptional mechanisms modulating UCP gene expression ensure a rapid increase in the concentration of UCP and prevent further accumulation of the protein as physiologically adequate levels are attained.

Effect of iodine deficiency and cold exposure on thyroxine 5'-deiodinase activity in various rat tissues.

We measured thyroxine 5'-deiodinase I (T(4)5'D-I) activity in thyroid, liver, and kidney and thyroxine 5'-deiodinase II (T(4)5'D-II) activity in brown adipose tissue (BAT) in rats on a low-iodine diet (LID) to test the possibility that increased deiodinase activity in these tissues might contribute to the maintenance of ther serum 3,5,3'-triiodothyronine (T3) level. Control rats received LID plus KI. Experiments were also performed with LID and LID plus KI rats exposed to cold. T(4)5'D-I activity was greatly increased in the thyroids of LID rats but not in liver or kidney. We consider it likely that increased thyroxine (T4)-to-T3 conversion in the greatly enlarged thyroids of LID rats contributed to the maintenance of serum T3. T(4)5'D-II activity in BAT was markedly increased in LID rats and was further greatly increased on cold exposure. However, we were unable to demonstrate an increase in uncoupling protein mRNA levels in BAT in response to cold in LID rats. We attribute this to the very low serum T4 level, which limits substrate availability. This factor also makes it unlikely that BAT contributes to maintenance of serum T3 in LID rats.

Alpha- and beta-adrenergic induction of the expression of the uncoupling protein thermogenin in brown adipocytes differentiated in culture.

In order to examine the control of expression of the gene coding for the brown fat specific uncoupling protein thermogenin (UCP), brown fat cells isolated as undifferentiated precursors from the interscapular brown adipose tissue of young mice were grown in culture. In these cells, it was possible by norepinephrine (NE) addition to induce specifically the expression of the UCP gene. The effect of NE was due to activation of transcription. The ability to express the UCP gene was maximal in cells around confluence; cell cultures younger or older than this showed a lower response. The response to NE showed a sharp optimum around 0.1 microM and was linear with time over the 4-h period studied. The presence of insulin or thyroid hormones facilitated the NE response. Pharmacological analysis of the adrenergic response indicated that UCP gene expression could be induced both via beta-receptors (probably beta 3) and via alpha 1-receptors; these effects were synergistic. It was concluded that it is possible to promote these precursor cells to advance to such a state of differentiation that they can demonstrate the selective feature of the brown fat cell, i.e. the ability to express UCP. The expression of the UCP gene is regulated via interacting adrenergic mechanisms.

Norepinephrine-induced synthesis of the uncoupling protein thermogenin (UCP) and its mitochondrial targeting in brown adipocytes differentiated in culture.

Synthesis of the brown adipocyte-specific mitochondrial uncoupling protein thermogenin (UCP) is demonstrated here in brown adipocytes differentiated in culture from precursor cells. By immunoblotting, no UCP was detectable in untreated multilocular adipocytes. The synthesis of UCP was stimulated by norepinephrine at physiological concentrations and was observable already after 2 h. It was evident from immunoelectron microscopy that the newly synthesised protein was targeted to the mitochondrial inner membrane, demonstrating the functional competence of these cultured cells.

Effects of cholera toxin on gene expression in brown preadipocytes differentiating in culture.

To investigate the cellular control of the recruitment process in brown adipose tissue, the ability of cholera toxin to influence the differentiation of brown preadipocytes developing in culture was investigated. Stromalvascular cells obtained from the brown adipose tissue of 3-wk-old rats were grown in culture for 6-7 days in the presence or absence of cholera toxin. It was found that cholera toxin treatment decreased the expression of the actin gene (indicating an increased degree of differentiation), while at the same time promoting the expression of the genes coding for the mitochondriogenesis marker cytochrome-c oxidase and for the adipocyte conversion marker lipoprotein lipase (all followed at the mRNA level). Chronic cholera toxin treatment also increased the total amount of protein per cell in culture, and a specific cholera toxin-induced 35-kDa protein was identified. It was concluded that (in contrast to the case suggested for white preadipocytes) cholera toxin treatment of brown preadipocytes may not only affect the activity of catabolic enzymes but may also directly promote the differentiation process, indicating that this process is under beta-adrenergic control in the adapting animal.

Brown adipocytes differentiated in vitro can express the gene for the uncoupling protein thermogenin: effects of hypothyroidism and norepinephrine.

Expression of the gene for the brown-fat specific uncoupling protein thermogenin was investigated in cell cultures by hybridization of isolated RNA with a cDNA clone corresponding to mouse thermogenin. The RNA was isolated 3-4 days after confluence from cells differentiated in culture from precursors isolated from the interscapular brown adipose tissue of 5-week-old mice. Very low thermogenin mRNA levels were found in cells derived from untreated mice, and there was only little effect of added norepinephrine on thermogenin gene expression in these cells. However, in cells derived from hypothyroid (methimazole-treated) mice there was a higher expression of thermogenin, and norepinephrine had a marked augmenting effect on the thermogenin mRNA level in these cells. These effects of thermogenin mRNA levels were specific, in that they contrasted with the effects of hypothyroidism and norepinephrine on the level of other mRNA species in these cells (coding for beta-actin, lipoprotein lipase, cytochrome-c oxidase, and glycerol-3-phosphate dehydrogenase). It was concluded that brown-fat cells in culture can reach a differentiated state, sufficiently advanced that the unique properties of these cells can be expressed, and that thermogenin gene expression (i.e., the level of thermogenin mRNA) is under direct control of norepinephrine.

DNA synthesis in mouse brown adipose tissue is under beta-adrenergic control.

The rate of DNA synthesis in mouse brown adipose tissue was followed with injections of [3H]thymidine. Cold exposure led to a large increase in the rate of [3H]thymidine incorporation, reaching a maximum after 8 days, whereafter the activity abruptly ceased. A series of norepinephrine injections was in itself able to increase [3H]thymidine incorporation. When norepinephrine was injected in combination with the alpha-adrenergic antagonist phentolamine or with the beta-adrenergic antagonist propranolol, the stimulation was fully blocked by propranolol. It is suggested that stimulation of DNA synthesis in brown adipose tissue is a beta-adrenergically mediated process and that the tissue is an interesting model for studies of physiological control of DNA synthesis.

Optimal processing of Ektaspeed dental film.

Three rapid and two standard developing solutions have been tested. A jaw specimen and an aluminium step wedge were exposed on Kodak Ektaspeed film with a 60 kVp dental x-ray machine. The films were developed during varying times at 21 degrees C under controlled conditions. The image quality was tested with a densitometer as well as visually. The characteristic density curves were constructed and the relative speed, average density, fog and contrast were calculated for each test film. The time at which the contrast specified by the film manufacturer was reached, and beyond which the speed and contrast increased only marginally was regarded as the optimal developing time. With longer developing times no increase in image quality could be perceived visual by three trained observers. The optimal developing time at room temperature 21 degrees C, for the different solutions were found to be Kodak Dental 1:1 1.5 minutes Elfwing Rapid 1:3 1.0 minutes Scanfors Dental 1:2 1.5 minutes Kodak Dental 1:3 3.0 minutes Gevaert G 150 1:5 3.0 minutes The characteristic curves were identical for all developing solutions at the times recommended above and the total fog was below 0.20. However, the image quality was maintained and only an insignificant increase of fog was found even when those times were doubled.