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Excessive exposure to glucocorticoids (GCs), either from endogenous overproduction of cortisol, or exogenous pharmacological GC treatment, potentiates the development of diabetes and obesity in a fat depot-specific manner. Undesirable metabolic side effects resulting from the activation of the glucocorticoid receptor (GR) remain a key limitation to the long-term therapeutic use of GCs as immunosuppressants. GC treatment disrupts the thermogenic function of brown adipose tissue (BAT) and enhances futile cycling within white adipose tissue (WAT). Mice lacking the liver X receptors (LXRs) were previously shown to have smaller AT depots with enhanced BAT activity compared to wildtype (WT) mice. We previously demonstrated that LXRβ is required to mediate the side effects of GCs in the liver but not the beneficial anti-inflammatory effects of GCs. The discovery of this GC/LXRβ cross-talk led to the hypothesis that LXRβ antagonism may be therapeutically beneficial to prevent GC-induced dysfunction in AT. To test this idea, LXRα-/- mice were treated for 5 days with vehicle, 5 mg/kg dexamethasone (Dex, a synthetic GC agonist), and/or 40 mg/kg GSK2033 (GSK, non-selective LXR antagonist). As expected, Dex-treated mice showed significant accumulation of lipids in BAT and were unable to maintain their body temperature when exposed to cold, yet GSK was able to protect against these effects. Dex increased body fat mass and caused adipocyte enlargement in WAT which was not observed in mice co-treated with GSK. At the transcriptional level, GSK attenuated Dex-mediated downregulation of thermogenic genes in BAT, and upregulation of lipogenic genes in WAT. Similar beneficial changes were confirmed in WT mice. The protection afforded by GSK against Dex-induced fat accumulation was confirmed to be cell-autonomous from studies in adipose-specific LXRβ-/- mice (AdβKO). Dex-dependent increases in lipolysis in gonadal WAT and plasma free fatty acids (FFA) were also attenuated by GSK co-treatment. With GSK co-treatment, Dex-induced liver steatosis was diminished suggesting that LXRβ antagonism attenuated FFA shuttling to the liver. The lipolytic and lipotoxic effects of Dex in AT and liver were largely abrogated in AdβKO along with improved systemic insulin sensitivity. Overall, our data suggest that LXRβ antagonism prevents disruption of BAT and WAT (and indirectly liver) function caused by GC treatment in an in vivo model, highlighting the potential role of LXRβ antagonists in combating the negative effects of excessive GC exposure on the development of diabetes and obesity. The identification of this novel mechanism of interrupting GC adipose tissue action suggests therapeutic targeting of LXRβ with an antagonist could improve the health of patients currently taking GCs to control inflammation but suffer the detrimental side effects of drug treatment.?? 

ENDO 2021

Disruption of Adipose Tissue Metabolism by Glucocorticoids Is Attenuated With LXRβ Antagonism

technical paper

The androgen receptor (AR) is a nuclear receptor with a main role in the development and maintenance of the male phenotype. To execute its role as transcription factor, the AR forms homodimers. Three dimerization modes have been described for the AR: one via the DNA binding domain, a second via the ligand binding domain (LBD) and a third via interactions between the LBD and the aminoterminus of the AR (N/C). Based exclusively on in vitro data, all three dimerization modes seem to contribute to full AR activity, albeit to a different extent. The in vivo role of the dimerization modes, however, remains unknown. To study the physiological relevance, we generated two mouse models using a CRISPR/Cas9 approach, in which either the N/C interaction (ARNoC) or LBD dimerization (ARLmon) was disrupted. Surprisingly, the male ARNoC mice have a normal phenotype, indicating that the N/C interaction is not crucial for male development. In contrast, ARLmon males have an external female phenotype with cryptorchid testes and high levels of circulating testosterone (T), androstenedione and luteinizing hormone (LH) (6-, 13- and 45-fold higher, respectively). They have no prostate, seminal vesicles or epididymis, illustrating the importance of LBD dimerization during male development. Phenotyping the ARLmon model furthermore provided evidence of a crucial role for the AR in bone homeostasis as well as steroidogenesis. The ARLmon males display a severe bone phenotype, similar to that of complete AR knockout (ARKO) mice. The bone phenotype of ARKO was postulated to be mainly due to lower estrogen levels. However, in contrast to ARKO mice, ARLmon mice have high circulating levels of T, which can still function as prohormone for estradiol and support bone function via the ERα. Immunohistological analysis of ARLmon testes showed hyperplasia of the Leydig cells and residual spermatogenesis. Analysis of the steroidogenic pathway revealed that while the expression of most genes is increased, the expression of Hsd17b3, encoding the enzyme responsible for conversion of androstenedione into T, is low in ARLmon testis. Reporter assays confirmed that the promotor of this gene is indeed upregulated by the AR itself. In conclusion, our work uncovers the physiological role of the N/C interaction and LBD dimerization of the AR. It furthermore demonstrates a direct role for AR in male bone development independent of T aromatization into estrogens. Finally, we show that the AR controls the final step in the synthesis of its own ligand. In contrast to the in vitro data, N/C interaction is not crucial for male development in vivo. The ARLmon model illustrates that LBD dimerization could be an excellent new therapeutic target for inhibiting AR activity for example in advanced prostate cancer that has developed resistance to the current AR-targeting therapies.?? 

LBD Dimerization of the Androgen Receptor but Not N/C Interaction Is Crucial for Normal Male Development in Mice

Progesterone is critical for normal breast development and function, and has been shown to stimulate proliferation of normal breast epithelial cells by increasing stem and progenitor cell numbers. Breast cancer incidence is increased in women exposed to progesterone analogues in combined estrogen plus progestin hormone replacement therapy, but not in women taking estrogen alone. Classical progesterone signaling is mediated through the nuclear progesterone receptor (PR), which occurs as two related but functionally different isoforms, PRA and PRB. PRA and PRB are co-expressed equally in normal breast tissue but become dysregulated in breast cancer where PRA often becomes predominant. PRA predominance in breast cancer is associated with poorer outcome and higher risk of distant metastasis in tamoxifen treated patients. We show using integrated analysis of ChIP-seq, ATAC-seq and transcriptomic profiling in a breast cancer cell line model of acquired PRA predominance that: 1) PRA and PRB have different requirements with regard to chromatin accessibility; 2) PRA predominance reshapes the PR cistrome and the associated transcriptome to affect genes not normally regulated by PR when PRA and PRB are equivalently expressed, possibly through assisted loading with multiple other transcription factors; 3) Genes regulated by PR only when PRA is predominant are associated with poorer breast cancer outcome and involved in multiple cancer-associated pathways including those that regulate cell proliferation and adhesion. Our data suggest a mechanism for the poorer disease outcome seen in breast cancers with a predominance of PRA.?? 

Differential Genomic Interactions Drive Progesterone Receptor Isoform Specific Functions in Breast Cancer

Transcriptional and Genomic Regulation of Pituitary Function by Thyroid Hormone Receptor ß (TRß)

Dependent Transcriptional Programming by TRβ in Thyroid Cells Requires SWI/SNF Chromatin Remodelers

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.?? 

The Effects of Energy Restriction on Thyroid Hormone Dynamics

Physiologic Effects of Levothyroxine and Liothyronine in Older Individuals With Persistent Subclinical Hypothyroidism: A Randomized, Double-Blind, Cross-Over Study

Introduction: There is a strong genetic association between autoimmune thyroiditis (AITD) and Type 1 diabetes (T1D), and the co-occurrence of AITD and T1D in the same individual is referred to as Autoimmune Polyglandular Syndrome type 3 variant (APS3v). We previously discovered a unique amino acid signature of the HLA-DR pocket (designated APS3v HLA-DR pocket) that predisposes to APS3v, and we found that both thyroid (Tg.1571, TPO.758) and islet (GAD.492) peptides can bind to this flexible APS3v HLA-DR pocket. The goal of the present study is to identify small molecules that can block the presentation of these thyroid and islet peptides by the APS3v HLA-DR pocket as a potential therapeutic approach for APS3v.Methods: We screened a panel of small molecules using an in vitro screening assay we developed using Baculovirus-generated recombinant APS3v-DR. To validate “hit” compounds discovered by the in vitro assay in a mouse model we established a novel mouse model of APS3v. The APS3v mouse model was established by immunizing humanized NOD-DR3 mice with Tg.1571, TPO.758 and GAD.492 combined, together with adjuvant. We then validated hit small molecules for their effectiveness in blocking stimulation of T-cell responses in the APS3v mouse model. Results: Our screen identified 4 small molecules (S5, S9, S15, S53) that showed more than 50% inhibition in our in vitro assay. We were also able to establish a novel APS3v mouse model. Immunized mice showed an increase in cytokine production in response to all 3 thyroidal and islet peptides. The immunized mice also developed antibody responses against each peptide. When we tested the 4 “hit” compounds, only Cepharanthine (S53) which we previously identified as a small molecule blocking AITD immune responses, could block all 3 thyroidal/islet peptides in the APS3v mouse model. Treating with Cepharanthine prior to inducing APS3v in the mice also reduced T-cell proliferation in the immunized mice.Conclusions: We established a novel humanized APS3v mouse model by co-immunizing 3 thyroidal and islet peptides. We also identified Cepharanthine as a small molecule that can block thyroid and islet specific T-cell responses in this model. Cepharanthine can be further tested in APS3v patients carrying the specific APS3v-DR pocket as a potential therapeutic treatment.?? 

Cepharanthine Blocks the Presentation of Thyroid & Islet Peptides in a Novel Humanized Autoimmune Polyglandular Syndrome Type 3 Variant (APS3v) Mouse Model

Background: Two-thirds of patients with metastatic differentiated thyroid cancer (DTC) become refractory to radioactive iodine (RAIR). The inhibition of the MAP-kinase pathway that is activated in case of BRAFV600E mutation might increase RAI incorporation into metastatic foci and reverse the RAI refractoriness.MERAIODE is a prospective multicentric open-label phase II trial, using a one-stage Fleming design, evaluating the efficacy and tolerance of trametinib (a MEK inhibitor) and dabrafenib (a BRAF inhibitor) treatment followed by the administration of RAI in metastatic RAIR DTC patients.?Methods: Patients with BRAFV600E mutated RAIR metastatic DTC with RECIST progression within 18 months prior to enrollment and no lesion > 3 cm were included. A baseline rhTSH-stimulated diagnostic whole body scan (dc WBS) was performed prior to treatment initiation. Patients were treated with dabrafenib (150 mg bid) and trametinib (2 mg per day) for 42 days. At day 28, a second rhTSH-stimulated dc WBS was performed. After 35 days, a therapeutic activity of RAI (5.5 GBq) was administered. Primary endpoint was objective response rate (ORR) at 6 months according to RECIST v1.1 (central review).?Patients: Among the 24 patients (mean age 67 years, 15 females) with a BRAFV600E mutated RAI refractory papillary DTC included between March 2018 and January 2020 in 8 French centers from the TUTHYREF netwok, 24 patients were treated and 21 patients were evaluable for the principal outcome at 6 months.?Results: Abnormal RAI uptake was present in only 1 of the 21 patients (5%; 95%CI 0-24%) on a RAI diagnostic whole body scan (dc-WBS) performed prior to treatment initiation, in 11 patients, 11/17 (65%; 95%CI 38-86) on a dc-WBS performed 4 weeks after dabrafenib-trametinib initiation and in 20/21 (95%; 95%CI 76-100) on the post-therapeutic WBS performed after 5.5 GBq of RAI. The RECIST 6-months tumor response (central review) was partial response (PR) in 38% (95%CI 18-61), stable disease (SD) in 52% (95% CI 30-74) and progressive disease (PD) in 10% (95% CI 1-30). The median change in the sum of target lesions was -22% (range: -79 to +46) at 6 months after baseline. The 6-month fluorodesoxyglucose metabolic PET response was PR in 11/17 (65% 95%CI 38-86), SD in 4/17 (23%) (95% CI 7-50) and PD in 2/17 (12%; 95% CI 1-36). Among the 15 patients without Tg antibodies, 7 (47%) patients had a decrease of serum thyroglobulin level on T4 treatment by more than 50%All patients experienced at least one grade 1-2 adverse event, mainly asthenia, nausea, fever, diarrhea and cutaneous eruption. Nine grade 3 toxicities occurred in 6 treated patients. No grade 4-5 adverse event occurred?Conclusion:The association of dabrafenib and trametinib in BRAFV600E mutated patients is effective for restoring RAI uptake and is followed by a tumor control in 90% of patients and by tumor response in 38% with limited adverse events. (PHRC 2015, NCT 03244956)?? 

MERAIODE: A Redifferentiation Phase II Trial With Trametinib and Dabrafenib Followed by Radioactive Iodine Administration for Metastatic Radioactive Iodine Refractory Differentiated Thyroid Cancer Patients With a BRAFV600E Mutation (NCT 03244956)

Background: The benefits of post-operative radioactive iodine (RAI) administration have not been demonstrated in patients with low risk differentiated thyroid cancer (DTC). The objective of this randomized phase III trial is to assess in low risk DTC patients the non-inferiority of a follow-up strategy as compared to a systematic adjuvant post-operative RAI administration.?Methods: ESTIMABL2 is a French multicentric randomized phase III trial in patients with low-risk DTC treated with total thyroidectomy with or without prophylactic neck lymph node dissection (pT1am N0 or Nx with a sum of the diameters of tumor lesions ≥ 10mm, pT1b N0 or Nx). Two to five months after surgery, in the absence of suspicious lateral neck lymph node on ultrasonography (US), patients were randomized either to the follow-up group (FU, no RAI administration) or to the ablation group and received post-operative RAI (1.1 GBq following rhTSH stimulation). Yearly controls under levothyroxine treatment consisted in thyroglobulin (Tg) and Tg antibodies (TgAb) determinations and neck-US. The primary objective was to assess at 3 years after randomization the non-inferiority of the proportion of patients without tumor-related event in the FU group as compared to the ablation group. Non-inferiority is demonstrated if the rate of patients without event at 3 years does not differ by more than ΔL=-5%. A tumor-related event was defined by the occurrence of subsequent treatment (RAI administration or surgery) for abnormal RAI uptake on the post-therapeutic WBS or by elevated Tg or TgAb levels and/or abnormal neck US during controls. Tg levels on levothyroxine treatment were considered elevated if > 2ng/mL in the FU group and > 1ng/mL in the ablation group. TgAb were considered elevated if > the upper limit range with an increase above 50% on 2 consecutive determinations performed 6 months apart.?Results: 776 low-risk DTC patients were included between 2013 and 2017 in 35 French centers within the TUTHYREF network; 83% females, mean age: 52 years, papillary TC: 96%, pT1bNx: 43.6%, pT1bN0: 37.5%, pT1amNx: 12.6%, pT1amN0: 6.3%. Among the 729 patients evaluable at 3 years after randomization, tumor-related events occurred in 18/367 patients (4.9% IC95%=[2.9 ; 7.6]) in the FU group and in 15/362 patients (4.1% IC95%=[2.3 ; 6.7]) in the ablation group. Thus, 95.1% of patients in the FU group had no event at 3 years and this percentage is not inferior from the 95.9% of patients observed in the ablation group (difference = -0.8% [95% CI:-3.3%; 1.8%]. The number of subsequent surgery and/or RAI administration was 6 (1.6% IC95%=[0.6 ; 3.5]) in the FU group and 9 (2.5% IC95%=[1.1 ; 4.7]) in the ablation group.?Conclusion: this phase III trial demonstrates the non-inferiority of a follow-up strategy compared to a systematic adjuvant post-operative administration of RAI (1.1GBq following rhTSH) in low risk DTC patients (PHRC 2012; NCT01837745).?? 

Estimabl2: Is There a Need for Radioiodine Ablation in Low Risk Differentiated Thyroid Cancer (DTC) Patients ? : Results From the French Randomized Phase III Prospective Trial on 776 Patients (NCT 01837745)

Disruption of Adipose Tissue Metabolism by Glucocorticoids Is Attenuated With LXRβ Antagonism

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