Thyroid Hormone Metabolism and Action

Paige Meizlik, MD, MSTR

Teva Pharmceuticals, PA, United States

Abstract

Background: Subclinical hypothyroidism is common in older individuals, but the physiologic responses to treatment with levothyroxine (LT4) and liothyronine (LT3) are not well defined in this age group. Methods: We conducted a randomized, double-blind, cross-over study of LT4 and LT3 treatment in men and women aged 70 years and over without anti-thyroid peroxidase antibodies with persistent subclinical hypothyroidism, defined as having a TSH level between 4.5 and 19.9 µIU/mL with a normal free thyroxine (FT4) level at two consecutive time points. Physiologic outcome measures assessed after achieving a TSH level of 0.5-1.5 µIU/mL with each therapy included vital signs, weight and body composition, bone mineral content and bone density, lipids, resting energy expenditure (REE), cognitive function, quality of life, and thyroid symptoms. Results: Thirteen participants [mean (SD) age 77 (5) years], 4 women and 9 men, completed the study. Baseline mean TSH was 4.84 (1.29) µIU/mL. The mean LT4 dose was 105 (36) µg/day [1.4 (0.5) µg/kg/day] and LT4 dose was 34 (9) µg/day [0.4 (0.1) µg/kg/day]. Mean time on LT4 was 200 days and on LT3 was 231 days, with a 28 day washout period. Compared with baseline, participants had an average weight loss of 1.1 kg on LT4 (p<0.02) and 2.5 kg on LT3 (p<0.001), which was significantly different between the two treatments (p=0.01). Fat mass decreased by an average of 0.7 kg on LT4 (p=0.03 vs. baseline) and 1.5 kg on LT3 (p<0.01 vs. baseline) and differed between treatments (p=0.01). There was a significant difference in total cholesterol of 13.3 mg/dL (p<0.001) and in low-density lipoprotein cholesterol (LDL) of 10.8 mg/dL (p<0.001) between LT4 and LT3 treatment arms; for both, the levels were lower on LT3 than LT4. No differences were seen in the other assessed outcomes. Conclusions: In a cross-over study of treatment of LT4 or LT3 in persistent subclinical hypothyroidism, participants lost fat mass and weight after each treatment, with a greater decrease after treatment with LT3. These findings support different physiologic responses to LT4 compared with LT3.

Young-Wook Cho, Ph.D.

NIH NIDDK, MD, United States

Abstract

Background: The pituitary is a key target for thyroid hormone but underlying transcriptional mechanisms are poorly understood. Thyroid hormone modifies expression of hormones, including growth hormone (GH) and thyroid-stimulating hormone (TSH, thyrotropin). Wider transcriptome responses are undefined. Thyroid hormone receptor beta (TRb) encoded by THRB are expressed in the anterior pituitary and THRB mutations cause human resistance to thyroid hormone. Method: To investigate genomic genomic regulation by TRb, we derived Thrb-HAB knockin mice that express TRb protein with a tag that is biotinylated in vivo in presence of an R26-BirA allele. Specific, sensitive streptavidin pull-down facilitated Chromatin-Affinity-Purification-sequencing (ChAPseq) to identify genomic TRβ binding sites in pituitary of male mice. Hypo- and hyperthyroidism were produced using methimazole (MMI) in drinking water for 4 weeks with/without added thyroid hormone (T3) for the 4th week. Pituitaries from wild type and Thrb-KO mice were also isolated for RNA-sequencing (RNA-seq). Selected expression changes were confirmed by quantitative PCR. Epigenetic changes were determined by ChIPseq for histone acetylation and methylation and open chromatin analysis (ATAC-seq). Results: Transcriptome analysis revealed genes with statistically different expression induced by T3, including known response genes such as Tshb, Hr and Gh. Responses were impaired in Thrb-KO mice. T3 induced recruitment of TRb binding, chromatin opening and specific histone acetylation marks. Conclusion: Most T3 response genes in pituitary depend to some extent upon TRb. T3-dependent chromatin modifications indicate properties of TRb-dependent enhancer regions and a critical role for TRb in transcriptional regulation of pituitary function.

Gregory A Brent, MD

UCLA/VA Greater LA, CA, United States

Abstract

Joselyne Tessa Tonelu, BS

NIEHS, National Institutes of Health, NC, United States

Abstract

Background: Intermittent energy restriction (IER) is gaining popularity as a weight-loss strategy. However, the effect of short-term energy restriction on thyroid hormone dynamics is not well characterized. Methods: Nineteen healthy women age 23.36± 2.08 yr (mean ± SD) with normal baseline thyroid function and negative anti-thyroid antibodies underwent two 5-day interventions of a prescribed diet and identical standardized exercise in the early follicular phase of two menstrual cycles - neutral energy availability (NEA) 45 kCal/kg*LBM/d followed by deficient energy availability (DEA) 20 kCal/kg*LBM/d. Energy requirements were estimated as previously described (doi.org/10.1210/jendso/bvaa046.1468) and were used to generate a diet and exercise regimen for each participant. On day 5 of both interventions, body composition was assessed by BodPod®. Standardized NEA or DEA breakfast and lunch were provided as appropriate as well as a standardized NEA snack on both sampling visits. Blood sampling was performed for 8 hours starting at ~0800 h with measurement of TSH and growth hormone (GH) every 10 min, cortisol every 30 min, total T3 (TT3), reverse T3 (rT3) and total T4 (TT4) every 60 min, free T3 (FT3), free T4 (FT4) and TBG at the beginning and end of sampling. Liquid chromatography-tandem mass spectrometry (LC-MS) was used for measurements of all thyroid hormones, with the exception of TSH and TBG which were measured by ELISA as were GH and cortisol. Data were analyzed using ANOVA-RM and linear mixed models. Results are presented as mean or least squared mean ± sem. Results: Body mass index, bodyweight and % fat mass were not different between interventions. GH and cortisol were unaffected by DEA (p=0.46, p=0.63). TBG was not affected by time of day or dietary intervention (p=0.95, p=0.41). However, compared with NEA, TT3 (89.15 ± 2.89 vs 95.55 ± 2.89 ng/dL for DEA and NEA, respectively; p<0.0001) and TSH (0.92 ± 0.08 vs 1.03 ± 0.09 μIU/mL; p=0.0011) were lower after DEA, while TT4 (6.26 ± 0.25 vs 6.06 ± 0.25 μg/dL; p=0.04), FT4 (3.37 ± 0.26 vs 2.94 ± 0.25 ng/d;, p=0.0052) and rT3 (11.77 ± 0.58 vs 8.85 ± 0.51 ng/dL; p<0.0001) were higher. Regardless of dietary intervention, FT3 (p=0.0005), TT3 (p<0.0001), TT4 (p<0.0001) and TSH (p<0.0001) decreased across the day. Conclusion: Using LC-MS for as a more robust measure of thyroid hormones, we have now shown that changes in thyroid hormone dynamics occur after only 5 days of 55% energy restriction in the absence of alterations in body composition, cortisol, GH, TBG or the circadian pattern of thyroid hormone secretion. The decrease in TSH combined with the decrease in TT3 and increase in rT3 support the contribution of both central and peripheral mechanisms to these changes. Taken together these results provide support for a multi-level adaptation in thyroid hormone dynamics to conserve energy expenditure in response to short-term energy restriction.

Noelle Gillis, BS

University of Vermont, VT, United States

Abstract

Transcriptional regulation in response to thyroid hormone (T3) is a dynamic and cell-type specific process that maintains cellular homeostasis and identity. A bimodal switch model, where T3 binding alters the co-regulator profile of a constitutively DNA-bound thyroid hormone receptor (TR) to affect downstream gene expression, is widely used to describe the interaction between TRβ and chromatin. To test this model on a genome-wide scale, we used an integrated genomics approach to profile and characterize the cistrome of TRβ by CUT&RUN, map changes in chromatin accessibility by ATAC-seq, and capture the transcriptomic changes in response to T3 by RNA-seq in the normal thyroid cell line, Nthy-ORI. Our CUT&RUN data demonstrated that T3 binding causes significant shifts in TRβ genomic occupancy; these shifts are associated with differential chromatin accessibility. Most of the T3-induced differentially expressed genes have a TRβ binding site associated with the proximal protomer region within one kilobase of the transcriptional start site, suggesting that these are direct TRβ regulatory target genes. Remarkably, the majority of TRβ binding sites were found in transgenic regions and distal regulatory elements. In order to identify the co-regulatory proteins that are required for execution of a T3-dependent transcriptional program in our thyroid cells, we used a TRβ-miniTurboID fusion construct to perform a proximity ligation assay followed by mass spectrometry. We identified 1,138 nuclear proteins that interact with TRβ. Of these proteins, 75 interact preferentially in the presence of T3 and 68 in the absence of T3. All of the core SWI/SNF complex subunits from both of the major subtypes (BAF and PBAF) were identified as TRβ. Interestingly, we found that the PBAF-specific subunit, PBRM1, was significantly enriched in the presence of T3. BAF complex-specific subunits, such as ARID1A, were also present in our dataset. To test whether TRβ could differentially recruit BAF and PBAF complexes to its binding sites, we performed CUT&RUN targeting BRG1, PBRM, and ARID1A to determine the degree of co-occupancy with TRβ. Based on our comprehensive genomic and proteomic analyses, we propose a new model for selective recruitment of BAF and PBAF SWI/SNF complexes to TRβ binding sites for differential functions in regulating chromatin accessibility.