How many oxygens does thyroxine have

As a proof of concept, we performed an unbiased microarray approach using samples from 3 typical experiments. Thus, the transcriptional analysis was performed on the exact same cells demonstrating the energetic effect. Cells that were excluded in the baselining process were also excluded from microarray analysis. Differential expression analysis was first used to determine differences in the enrichment in individual genes between cells with and without T 3 treatment Figure 7A.

Of these, 52 coding genes were enriched in the control group at a fold change more than or equal to 1. A heatmap and dendrogram created by hierarchical clustering show that 1 the 3 samples of each treatment class cluster together based on their gene expression profiles and that 2 the gene expression patterns can be generally defined by 6 clusters Supplemental Figure 5.

LH86 cells treated to final concentrations of T 3 in the near-physiologic range display a unique transcriptome that underlies changes in OCR. Warm colors indicate a higher density of genes expressed, whereas cool colors represent more sparse density of expression.

C, Custom heat map of the 25 genes with the highest and lowest fold change where expression values are represented as colors red, increased expression; blue, decreased expression with the degree of color saturation indicating the degree of expression.

These gene expression data from all 6 samples were then used to create a classifier to predict the class T 3 vs vehicle of deidentified samples Supplemental Table 3.

In order to evaluate the physiologic context of the transcriptional changes that occur after T 3 treatment, the microarray data were then analyzed by GSEA. A heatmap of the 50 top genes for each treatment class was generated Supplemental Figure 6.

Gene sets enriched in T 3 -treated cells were related to angiogenesis, lipid metabolism, nucleotide metabolism, immune signaling and cellular proliferation. Gene sets down-regulated in T 3 -treated cells were related to transcription, cell membrane components and neuron differentiation.

The current data indicate that as hypothyroid hepatoma cells transition into hyperthyroidism, significant changes in energy expenditure occur, and this rapid metabolic effect of T 3 is associated with distinct changes in the transcriptome, including many genes known to be important for metabolism, as well as some whose role has not previously been established.

Importantly, the changes in O 2 consumption were seen as hypothyroid cells were treated with 0. Furthermore, the nondestructive experimental design allowed the transcriptional changes to be measured simultaneously as the changes in O 2 consumption, in the exact same cells. Thus, the set of genes identified as having changed acutely in this study is likely to be enriched in terms of directly responsive control elements for T 3 -dependent O 2 consumption.

That thyroid hormone modulates energy expenditure is not in question: a positive association between T 3 and O 2 consumption is readily observed in studies of animals with experimentally induced alterations of thyroid status 20 — When animals or humans are treated with T 3 , distinct transcriptional changes have been described for a number of tissues, including many genes with well-established roles in metabolism eg, Refs.

What then are the factors that prevent a consensus gene set from being identified? First of all, gene sets obtained from in vivo treatments cannot distinguish between direct and indirect effects of T 3 , the latter arising from downstream second messengers. For example, T 3 in the liver has direct effects on hepatic metabolism, but T 3 also indirectly alters liver metabolic pathways via direct effects on the brain; the latter arise because T 3 in the brain alters sympathetic input to the liver Thus, animal studies are not sufficient for isolating the effects of T 3 on peripheral tissues.

Finally, it is notable that concern about possible off-target effects of T 3 at suprapharmacologic levels are often raised when considering the literature on nongenomic effects of T 3 ; in theory, same concerns should apply to all studies of thyroid hormone. Although thyroid hormone can ultimately promote large changes in O 2 consumption in the chronic setting, it is perhaps not surprising that the acute effects may be much smaller in magnitude.

For comparison, norepinephrine has more potent acute effects, eg, increasing O 2 consumption by more than 5-fold within a few hours in brown adipocytes this effect of norepinephrine is T 3 dependent Extracellular flux technology has advantages over these methods due to miniaturization, multiwell format, high-sensitivity chemiluminescent sensors, and nondestructive measurement capabilities Thus, it is possible to quantify the effects of T 3 at much more physiologic concentrations as seen in the current study.

This variability is likely related to differences in cell seeding density, although other methodological sources of error are possible such as unequal incubation temperature across the plate surface. Because minute variations in seeding and culture conditions cannot be entirely eliminated, the experimental protocol must somehow account for this variability. In essence, the baselining protocol accomplishes this simply by creating 2 experimental groups matched for mean OCR and ECAR, with smaller SDs than would be achieved randomly.

Quantitatively, this improves accuracy as reflected in the smaller SD of the pooled experiments Figure 6. The process of creating the baselining groups could be done in a number of ways, and it should be noted that it does not really matter how the 2 groups are derived, as long as the subsequent treatments are randomized. However, using such a high number of replicates routinely would be cost inefficient. Thus, the baselining approach could be critical for demonstrating statistical significance for any Seahorse experiment featuring 2 experimental groups on a plate where the final differences in OCR or ECAR are close in magnitude to the basal variability.

For example, in Figure 6 , experiment F, the T 3 effect on OCR and ECAR was qualitatively different than for all other studies, suggesting some artifact compromised this particular experiment although of course the data cannot be dropped.

Contamination of the media is one possibility, accidental damage to the cells via pipetting is another, and cell detachment is a third. Regardless of mechanism, it important to recognize that such events do occur, and constitute a limitation of the extracellular flux methodology that is not corrected by the baselining protocol.

If the investigator hypothetically eliminated these wells from the data analysis post hoc and the qualitative result changed, this would indicate that the results were skewed by the chaotic wells, and would suggest that the results might not represent true biology.

However, because this type of analysis could be subject to bias, this was not done in reporting the baselined data in this study. Because identifying the metabolic genes that respond directly to T 3 acutely in each tissue is the ultimate goal of this line of research, we performed a limited microarray analysis as a proof of concept for the overall method.

In the current study, the genetic analysis was performed in exactly the same cells demonstrating the change in OCR, increasing the chance that the transcriptional changes identified are relevant to energy expenditure. The transcriptional fingerprint obtained after T 3 treatment was strikingly different compared with vehicle.

GSEA Supplemental Table 4 and pathway analysis Figure 7E were employed to further explore physiologic expression patterns of altered genes. Gene sets down-regulated in T 3 -treated cells were related to cell structure, chromosomal structure and maintenance, neuron differentiation and negative regulation of protein metabolism and cell differentiation. Thus the GSEA led to several hypotheses regarding mechanisms that may be contributing to the increased energy expenditure exhibited by these cells.

In all, these findings suggest a major role played by fatty acid synthesis and oxidation in T 3 -treated LH86 cells. These data are reminiscent of purely physiological findings that the molecular underpinnings of T 3 -induced O 2 consumption rely on increased turnover of fatty acid synthesis and oxidation in liver and brown adipose tissue 24 , Of course, there are commonalities comparing the transcriptome identified in the current study with those obtained using human and animal subjects in vivo thyrotoxicosis , as well as studies of cell lines, including the hepatoma line HepG2 Supplemental Tables 2 and 3 25 , For example, glucosephosphatase was induced in the current dataset Supplemental Table 2 , in HepG2 cells 26 and in hyperthyroid mouse liver 5.

Proving whether or not the genes uniquely identified in this study are truly more physiologically relevant will require additional studies, eg, using knockdown experiments; replication of these experiments as presented in additional hepatic models will also be important, eg, primary hepatocytes. In addition, there are several other noteworthy limitations to the current findings. The general caveat that immortalized cells are different from primary cells both genetically and metabolically certainly applies, although LH86 and other hepatoma cells are routinely used in the literature, because they maintain important features of hepatocytes Extracellular flux technology has been applied to primary hepatocytes, but to date most published experiments have focused on conditions with large OCR differences, such as hepatocytes from different genetically modified strains of mice It is tempting to speculate that the transcriptome identified here is enriched in genes that respond to T 3 during TR depression rather than transactivation.

However, to directly prove this point in future studies euthyroid vs increasingly hypothyroid cells will have to be compared. Finally, although our genome-wide analysis was performed as a proof of concept, in future studies the number of replicates used for microarray analysis could be increased to better account for multiple hypothesis testing.

Alterations in thyroid signaling likely modulate both central nervous system pathways for metabolic control as well as metabolically important pathways in peripheral tissues; any working model will have to incorporate both elements. It is thus necessary to take a deconstructionist approach to this problem; to understand how T 3 regulates the metabolic rate, one must first understand how T 3 affects metabolism in each tissue of the body.

The current work represents an important step in this process, revealing not only that treatment with near-physiologic final concentrations of T 3 triggers transcriptional changes that alter O 2 consumption within a few hours but also illustrating how the important genes in all other tissues may be identified thanks to the generalizability of the method.

We also thank Vicente Lau and Lorne Wasserman for general laboratory assistance. Magnus-Levy A. Energy metabolism in health and disease. J Hist Med Allied Sci. Google Scholar.

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Arch Toxicol. Chronic enrichment of hepatic endoplasmic reticulum-mitochondria contact leads to mitochondrial dysfunction in obesity. Nat Med. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Thyroid hormones enter cells through membrane transporter proteins.

A number of plasma membrane transporters have been identified, some of which require ATP hydrolysis; the relative importance of different carrier systems is not yet clear and may differ among tissues. Once inside the nucleus, the hormone binds its receptor, and the hormone-receptor complex interacts with specific sequences of DNA in the promoters of responsive genes. The effect of the hormone-receptor complex binding to DNA is to modulate gene expression , either by stimulating or inhibiting transcription of specific genes.

For the purpose of illustration, consider one mechanism by which thyroid hormones increase the strength of contraction of the heart. Cardiac contractility depends, in part, on the relative ratio of different types of myosin proteins in cardiac muscle.

Transcription of some myosin genes is stimulated by thyroid hormones, while transcription of others in inhibited. The net effect is to alter the ratio toward increased contractility. For additional details on mechanism of action and how these receptors interact with other transcription factors, examine the section Thyroid Hormone Receptors.

It is likely that all cells in the body are targets for thyroid hormones. While not strictly necessary for life, thyroid hormones have profound effects on many "big time" physiologic processes, such as development, growth and metabolism, and deficiency in thyroid hormones is not compatible with normal health.

Additionally, many of the effects of thyroid hormone have been delineated by study of deficiency and excess states, as discussed briefly below. Metabolism : Thyroid hormones stimulate diverse metabolic activities most tissues, leading to an increase in basal metabolic rate.

One consequence of this activity is to increase body heat production, which seems to result, at least in part, from increased oxygen consumption and rates of ATP hydrolysis. By way of analogy, the action of thyroid hormones is akin to blowing on a smouldering fire. A few examples of specific metabolic effects of thyroid hormones include:. Growth: Thyroid hormones are clearly necessary for normal growth in children and young animals, as evidenced by the growth-retardation observed in thyroid deficiency.

Not surprisingly, the growth-promoting effect of thyroid hormones is intimately intertwined with that of growth hormone , a clear indiction that complex physiologic processes like growth depend upon multiple endocrine controls.

Development: A classical experiment in endocrinology was the demonstration that tadpoles deprived of thyroid hormone failed to undergo metamorphosis into frogs. Of critical importance in mammals is the fact that normal levels of thyroid hormone are essential to the development of the fetal and neonatal brain.

Other Effects: As mentioned above, there do not seem to be organs and tissues that are not affected by thyroid hormones.

A few additional, well-documented effects of thyroid hormones include:.