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Researchers gain new insights into hypothyroidism
Results suggest why standard treatments fail some patients and how to help them
An international research team led by physician-scientists at Rush University Medical Center has gained new insights into hypothyroidism - a condition affecting about 10 million people in the U.S. - that may lead to new treatment protocols for the disease, particularly among the approximately 15 percent of patients for whom standard treatments are less effective.
The researchers published their findings at the beginning of the new year in a pair of articles in the Journal of Clinical Investigation (JCI) and the Journal of Clinical Endocrinology & Metabolism (JCEM).
Hypothyroidism occurs when the thyroid gland fails to produce sufficient quantities of two hormones, thyroxine (known as T4) and its more active form, called T3. The condition can cause a number of health problems, including weight gain, fatigue and so-called "foggy brain."
For decades, the standard treatment has been a daily T4 supplement named levothyroxine. Once absorbed into the body, T4 is transformed to T3, in theory fully normalizing blood levels of T3. However, physicians have long been puzzled by why this type of treatment fails to relieve all symptoms in up to 15 percent of patients.
The puzzle persists in large part because the efficacy of treatments for hypothyroidism relies also on patients' subjective reports of how they feel - patients with a normal thyroid may experience symptoms similar to those of hypothyroidism but due to other conditions, such as post-menopause syndrome or clinical depression.
The study published in the JCI was performed on rats whose thyroid glands had been removed and explains the cellular basis for why circulating levels of T3 are not fully normalized by levothyroxine alone. In addition, the study reveals that circulating T3 levels and hypothyroidism can be corrected fully when the levothyroxine regimen is supplemented with T3.
The study found that some of the rats that only received levothyroxine had higher cholesterol levels in their blood than rats that received the combination T4 and T3 therapy. They also had signs of hypothyroidism in their brains, which could potentially explain the "foggy brain" that is a common symptom of hypothyroidism. Therefore, the combined therapy established normal thyroid hormone action in the areas of the body commonly affected by hypothyroidism -- the brain, the liver and also the skeletal muscles.
"Of course it's important to confirm these studies clinically," says Antonio Bianco, MD, PhD, head of Rush's Division of Endocrinology and Metabolism and senior author of both journal articles. Bianco also co-chaired an American Thyroid Association task force that updated the association's guidelines for the treatment of hypothyroidism published this past December in the journal Thyroid.
"Hypothyroid patients are not all the same. Some will do better on the combination therapy others not. The challenge is to identify these individuals and understand why these differences exist," Bianco says.
This point was explored in the study published in the JCEM, in which the researchers examined a common polymorphism (a frequent genetic mutation) in the enzyme known as D2 that transforms T4 to T3. In a previous study, hypothyroid patients with this polymorphism preferred the combination therapy, which led Bianco and his team to explore the relationship between the polymorphism and the failure of standard therapy for hypothyroidism.
Working with the brains of about 100 cadaver donors, the researchers found that the polymorphic D2 has a tendency to accumulate in a cell compartment that normally does not contain D2. This abnormal accumulation of D2 disrupts cell function in a way also observed in the brain of patients with neurodegenerative diseases such as Huntington disease.
"It is conceivable that the D2 polymorphism is a risk factor for neurodegenerative disease that could be aggravated when these patients develop hypothyroidism," Bianco says.
Fortunately, treatment may be possible for this condition. "Some of the genes affected by the polymorphic D2 were indicative of oxidative stress," Bianco says. "When we treated cells containing the polymorphic D2 with a substance that neutralizes oxidative stress, that [treatment] normalized the expression of those genes."
"If confirmed by additional studies, the findings with the D2 polymorphism explain why not all hypothyroid patients are the same, with some exhibiting one or additional risk factors for decreased cognition," Bianco says. "It would seem that personalized medicine has caught up with hypothyroidism and might be able to ensure that treatment is effective in 100 percent of patients."
In addition to Bianco, the leading Rush investigators involved in the studies included Elizabeth McAninch, MD, an endocrinologist that also sees patients with thyroid diseases, and staff scientists Sungro Jo, PhD, Joao Pedro Werneck de Castro, PhD, and Tatiana Fonseca PhD.
Researchers from Harvard Medical School and Tufts Medical Center, both in Boston;
the University of Miami Miller School of Medicine;
the Hungarian Academy of Sciences, Péter Pázmány Catholic University and Semmelweis University, all in Budapest, Hungary;
and Erasmus Medical Center, Rotterdam, the Netherlands, also participated in one or both of the studies.
Bron Eureka
EEN KLEIN GEDEELTE UIT HET ONDERZOEK IN JCI
J Clin Invest. doi:10.1172/JCI77588.
Copyright © 2015, The American Society for Clinical Investigation.
Differences in hypothalamic type 2 deiodinase ubiquitination explain localized sensitivity to thyroxine
Authorship note: Joao Pedro Werneck de Castro and Tatiana L. Fonseca contributed equally to this work.
Published January 2, 2015
Submitted: June 18, 2014; Accepted: November 20, 2014.
The current treatment for patients with hypothyroidism is levothyroxine (L-T4) along with normalization of serum thyroid-stimulating hormone (TSH). However, normalization of serum TSH with L-T4 monotherapy results in relatively low serum 3,5,3′-triiodothyronine (T3) and high serum thyroxine/T3 (T4/T3) ratio. In the hypothalamus-pituitary dyad as well as the rest of the brain, the majority of T3 present is generated locally by T4 deiodination via the type 2 deiodinase (D2); this pathway is self-limited by ubiquitination of D2 by the ubiquitin ligase WSB-1. Here, we determined that tissue-specific differences in D2 ubiquitination account for the high T4/T3 serum ratio in adult thyroidectomized (Tx) rats chronically implanted with subcutaneous L-T4 pellets. While L-T4 administration decreased whole-body D2-dependent T4 conversion to T3, D2 activity in the hypothalamus was only minimally affected by L-T4. In vivo studies in mice harboring an astrocyte-specific Wsb1 deletion as well as in vitro analysis of D2 ubiquitination driven by different tissue extracts indicated that D2 ubiquitination in the hypothalamus is relatively less. As a result, in contrast to other D2-expressing tissues, the hypothalamus is wired to have increased sensitivity to T4. These studies reveal that tissue-specific differences in D2 ubiquitination are an inherent property of the TRH/TSH feedback mechanism and indicate that only constant delivery of L-T4 and L-T3 fully normalizes T3-dependent metabolic markers and gene expression profiles in Tx rats.
Introduction
Hypothyroidism is a prevalent condition, affecting more than 10 million Americans (1, 2). Historically, the standard of care for treating hypothyroid patients was administration of thyroid extracts to resolve symptoms. The discovery that the main thyroid product thyroxine (T4) is largely activated to 3,5,3′-triiodothyronine (T3) outside of the thyroid parenchyma made treatment with levothyroxine (L-T4) and normalization of serum thyroid-stimulating hormone (TSH) levels the new, and current, standard of care (1, 2). However, patients on L-T4 monotherapy exhibit a relatively higher serum T4/T3 ratio (3), with unknown long-term consequences to thyroid hormone signaling and general health. This is particularly relevant given that clinical studies indicate that 5%–10% of L-T4–treated hypothyroid patients with normal serum TSH have persistent symptoms that can be related to the disease (4).
While the molecular basis for the residual hypothyroid symptoms is lacking, it is generally hypothesized that these patients suffer from tissue-specific states of hypothyroidism and that serum TSH might not adequately reflect thyroid status at the level of different tissues (5). This scenario is partially supported by 2 factors:
First, serum T3 is below the lower limit of the reference range in approximately 15% of the hypothyroid patients treated with L-T4 monotherapy, despite normal serum levels of TSH and a relatively higher serum free T4 (6, 7). In many cases, normalization of serum T3 with L-T4 monotherapy can only be achieved at the expense of having an elevated serum T4 and low/suppressed serum TSH (7). However, it is not clear that the relatively lower serum T3 is clinically relevant or plays a causal role in the residual hypothyroid-like symptoms, which can be nonspecific in nature, such that some investigators have implicated other prevalent conditions, such as perimenopause or depression, as possible contributing factors in these symptomatic patients (8).
Second, in many organs and tissues the type 2 deiodinase (D2, encoded by DIO2) pathway makes a substantial contribution to the local T3 content, as much as approximately 50% in some tissues (9). For instance, in both the brain and the brown adipose tissue (BAT), thyroid hormone signaling depends on both plasma T3 and T3 locally generated via the D2 pathway (10–12). In the brain, D2 is expressed in astrocytes (13, 14), while thyroid hormone receptors and type 3 deiodinase (D3), which inactivates T3, are found in neurons (15). D2-generated T3 exits the glial cells and acts in a paracrine fashion to modulate the expression of T3-responsive genes in the neighboring neurons (16). In a mouse with astrocyte-specific D2 inactivation (Dio2fl/fl crossed with GFAP-Cre) there is loss of more than 95% in brain D2 activity (17). D2 is a type I endoplasmic reticulum–resident thioredoxin fold–containing selenoprotein with a variable half-life that depends on the level of its natural substrate, T4. In the presence of T4, D2 is inactivated with an approximately 20-minute half-life, whereas in the absence of T4, its half-life is prolonged to hours (9). This provides a mechanism through which the production of T3, the biologically active thyroid hormone, can be regulated according to the availability of T4. For example, an accumulation of D2 in cells increases the fractional conversion of T4 to T3 when serum T4 levels are low, such as in the case of iodine deficiency or hypothyroidism. In contrast, because T4 inactivates D2 so efficiently, it is conceivable that thyroid hormone signaling is dampened if T4 levels are high (18). In other words, a high serum T4/T3 ratio in L-T4–treated patients could actually reduce thyroid hormone signaling in D2-expressing tissues such as brain and BAT.
D2 ubiquitination is the molecular mechanism that modifies D2 half-life where binding to ubiquitin inactivates the enzyme and targets it for degradation in the proteasomes (19, 20). Ubiquitination is thought to inactivate D2 by disrupting the conformation of the D2:D2 dimer, critical for enzyme activity. A unique 18–amino acid loop confers intrinsic metabolic instability to D2, facilitating binding to proteins involved in the ubiquitination process (21, 22). This loop is also the site where a prevalent genetic polymorphism in DIO2 causes a single amino acid substitution, Thr to Ala, in position 92 in humans (23). While the kinetic properties of the polymorphic D2 remain unaffected, the fact that it is associated with a large number of diseases and conditions, including obesity and intolerance to glucose, suggests that the Thr92AlaD2 compromises the ability of this pathway to mediate thyroid hormone signaling (24).
The ubiquitin-activating enzymes UBC6 (UBE2J) and UBC7 (UBE2G1) participate in the process of D2 ubiquitination (25, 26), as well as 2 ubiquitin ligases, the hedgehog-inducible WSB-1 (22) and TEB4, a ligase involved in the degradation of proteins in the endoplasmic reticulum (27). Ubiquitinated D2 (UbD2) is not immediately taken up by the proteasomes. Instead, UbD2 can be reactivated by deubiquitination, a process catalyzed by two USP-class D2-interacting deubiquitinases (DUBs), USP-20 and USP-33 (28). D2 ubiquitination occurs via K48-linked ubiquitin chains, and exposure to its natural substrate, T4, accelerates UbD2 formation (29). UbD2 is retrotranslocated to the cytoplasm via interaction with the p97-ATPase complex and deubiquitination by the p97-associated DUB ataxin-3. Once in the cytosol, D2 is delivered to the proteasomes and irreversibly degraded (29).
These observations that a lower serum T3 and higher serum T4 could independently dampen thyroid hormone signaling support a modified approach to treatment of hypothyroidism that includes combination therapy with L-T4 and liothyronine (L-T3), through administration of either L-T4 plus L-T3 or desiccated thyroid extracts, which inherently contain both compounds. Replacing some of the L-T4 given to patients with L-T3 would raise/normalize serum T3 while preserving serum TSH within the normal range. This rationale is supported by studies in rodents in which normalization of serum T4, T3, and TSH as well as tissue content of T4 and T3 can only be achieved if combination therapy with L-T4 and L-T3 was used (30, 31). Nevertheless, multiple randomized controlled clinical trials in hypothyroid patients have addressed objective and subjective clinical outcomes comparing L-T4 monotherapy and L-T4 plus L-T3 combination therapy. The large majority of these trials found that both forms of treatment are equivalent, despite elevation in serum T3 in the patients on combination therapy (32). A large study also considered the DIO2 polymorphism but only found a weak statistical association with preference for the combination therapy versus monotherapy among hypothyroid patients (33, 34). These trials have led major professional societies in the US and Europe to label the situation as “controversial” and to continue the recommendation that L-T4 monotherapy remain the standard of care for hypothyroid patients (1, 2, 4).
Insight into 3 basic questions is needed for a better understanding of the molecular basis for the treatment of hypothyroidism: (a) What is the mechanistic explanation for the lack of normalization of serum T3 in L-T4–treated hypothyroid individuals exhibiting a normal serum TSH? (b) Do these relatively low serum T3 levels and/or relatively elevated serum T4 levels affect thyroid hormone signaling? (c) Can thyroid hormone homeostasis and T3-dependent markers be normalized by L-T4 and L-T3 combination therapy?
To address these questions, the present studies modeled the situation in an animal system using a large number of thyroidectomized (Tx) rats that were treated with L-T4 alone or a combination of L-T4 and L-T3 for 7 weeks. Here we report that due to hypothalamus-specific differences in D2 ubiquitination there is a sensitivity gradient in the loss of D2 activity in response to T4 between the hypothalamus and the rest of the brain and body. This explains why treatment with L-T4 alone fails to normalize serum TSH and T3 simultaneously. The lack of normalization of serum T3 and the higher serum T4 levels in L-T4–treated Tx rats have clear metabolic implications, including persistent hypercholesterolemia and relatively lower mitochondria content in liver and skeletal muscle. Furthermore, the relatively high serum T4 levels reduce D2 activity in different areas of the CNS, which, combined with lower serum T3 levels, results in local hypothyroidism. Only combined therapy with constant delivery of both L-T4 and L-T3 fully normalized T3-dependent metabolic markers and gene expression in Tx rats. These findings have important implications that may support the role of combination therapy in the treatment strategy for humans with hypothyroidism and thus may drive the need for development of improved pharmacologic modes for L-T3 administration and for high-quality randomized controlled trials in humans.
Results
Serum thyroid function tests in Tx rats treated with different thyroid hormone replacement regimens
Placebo-Tx animals exhibited the expected elevation in serum TSH and decrease in serum T4, T3, and reverse T3 (rT3) levels, with a reduction in the serum T4/T3 ratio when compared with placebo-control animals (Table 1). At the same time, T4-mono animals had normal serum TSH (Table 1 and Figure 1A), but serum T4 was found to be higher, and serum T3 lower, in comparison with placebo-control animals (Table 1); thus the elevated serum T4/T3 ratio (Table 1 and Figure 1B). In contrast, T4/T3-comb animals exhibited serum levels of TSH, T4, T3, and T4/T3 ratio that were indistinguishable from those in placebo-control animals (Table 1). Notably, T4/T3-inj-comb animals exhibited normal serum T4 but serum TSH was approximately doubled, serum T3 slightly decreased, and serum T4/T3 ratio increased in comparison with placebo-control animals (Table 1). Serum rT3 levels were normalized by treatment with thyroid hormone independently of the replacement strategy used (Table 1).