Deiodinases and the Metabolic Code for Thyroid Hormone Action

Abstract

Deiodinases modify the biological activity of thyroid hormone (TH) molecules, ie, they may activate thyroxine (T4) to 3,5,3′-triiodothyronine (T3), or they may inactivate T3 to 3,3′-diiodo-L-thyronine (T2) or T4 to reverse triiodothyronine (rT3). Although evidence of deiodination of T4 to T3 has been available since the 1950s, objective evidence of TH metabolism was not established until the 1970s. The modern paradigm considers that the deiodinases not only play a role in the homeostasis of circulating T3, but they also provide dynamic control of TH signaling: cells that express the activating type 2 deiodinase (D2) have enhanced TH signaling due to intracellular build-up of T3; the opposite is seen in cells that express type 3 deiodinase (D3), the inactivating deiodinase. D2 and D3 are expressed in metabolically relevant tissues such as brown adipose tissue, skeletal muscle and liver, and their roles have been investigated using cell, animal, and human models. During development, D2 and D3 expression customize for each tissue/organ the timing and intensity of TH signaling. In adult cells, D2 is induced by cyclic adenosine monophosphate (cAMP), and its expression is invariably associated with enhanced T3 signaling, expression of PGC1 and accelerated energy expenditure. In contrast, D3 expression is induced by hypoxia-inducible factor 1α (HIF-1a), dampening T3 signaling and the metabolic rate. The coordinated expression of these enzymes adjusts TH signaling in a time- and tissue-specific fashion, affecting metabolic pathways in health and disease states.

An unusual group of patients with “low basal metabolism without myxedema” or “euthyroid hypometabolism” was identified in 1917 as the application of indirect calorimetry to thyroid diseases was being developed (1, 2). After excluding other endocrinopathies associated with slower metabolism, several individuals with lower basal metabolic rates remained. These patients did not appear to have myxedema and did not improve with the administration of desiccated thyroid extract or levothyroxine (LT4). The syndrome was tentatively attributed to insensitivities to thyroid-stimulating hormone (TSH) or thyroid hormone (TH), accelerated TH catabolism, or poor intestinal absorption of TH. Many years later, in 1955, physicians in Boston reported a few patients with this syndrome (3, 4). Basal metabolic rate ranged from −20 to −25% (normal range, −5 to +5%), and the patients complained of lethargy, fatigue, irritability, and sensitivity to cold. Remarkably, the basal metabolic rate in these patients did not respond to oral administration of LT4, but treatment with L-triiodothyronine (LT3) and combinations of LT4+LT3 resulted in striking clinical improvement. Inspired by findings from Pitt-Rivers et al (5), who observed that athyreotic patients could activate thyroxine (T4) to the more potent triiodothyronine (T3), the authors postulated a peripheral defect in T4 metabolism. Unfortunately, double-blinded placebo-controlled studies were not performed, which added to the retraction of the in vivo deiodination findings (6) and dampened interest in the hypothesis of impaired activation of thyroxine. It took another 15 years before this issue was revisited when Braverman et al (7) demonstrated unequivocally that T4 is activated to T3 in athyreotic patients.

It is fascinating that the role played by TH deiodination in metabolism had such an interesting but often overlooked debut. Outer ring deiodination (ORD) of T4 to T3 and inner ring deiodination (IRD) of T4 and T3 adjust TH signaling ordinarily through modulating intracellular T3 levels. This model has been studied mostly in cellular and animal settings, yet it is anticipated that deiodinases play similar roles in humans and participate in disease mechanisms. Two dramatic examples crystalize the clinical role played by the ORD and IRD pathways in TH signaling. The first is the activation of ORD via type 2 deiodinase (D2) in the lungs of patients with acute lung injury (8) or idiopathic pulmonary fibrosis (9), which led to treatment of 2 patients with acute respiratory distress syndrome caused by COVID-19 infection with aerosolized T3, with encouraging results (10). The second is the syndrome of consumptive hypothyroidism caused by the ectopic IRD reactivation via type 3 deiodinase (D3) in newborns with large hemangiomas (11).

Two important signaling pathways place the genes encoding D2 and D3, respectively, Dio2 and Dio3, centerstage in the control of cellular metabolism. Dio2 is downstream of the cyclic adenosine monophosphate (cAMP) cascade (12, 13), the key pathway that accelerates oxidative phosphorylation. In turn, Dio3 expression is accelerated by hypoxia-inducible factor 1α (HIF-1a) (14), the key molecule that orchestrates a metabolic switch to anaerobic processes such as hepatic glucose production and glycolysis. Only recently loss-of-function mutations were identified in the gene that encodes the type 1 deiodinase (D1), DIO1, without any detectable metabolic phenotype (15). Nonetheless, a prevalent polymorphism in DIO2, Thr92Ala-DIO2, reduces catalytic activity (16, 17) and leaves a genetic fingerprint in the brain (18). Notably, Neanderthals and Denisovans carried only the Ala-DIO2 allele. Whether this could have played an adaptive dietary role—their diet was mainly based on the consumption of meat, with a low intake of carbohydrates—remains to be elucidated (19). In this regard, it is striking that carriers of this polymorphism exhibit clear metabolic consequences, such as an association with insulin resistance (20) and type 2 diabetes mellitus (21). In addition, individuals with compounded Thr92Ala-DIO2 and the Trp64Arg β3-adrenergic receptor polymorphism, a receptor variant that generates less cAMP (22), exhibited higher body mass index (23). Not surprisingly, these associations have not been reproduced in all populations studied (24-29), and more and larger trials are needed to identify relevant interfering linkage factors (28, 30, 31).

The purpose of this review article is to briefly summarize the metabolic roles played by the deiodinases with an emphasis on findings reported in the last 5 years (2016-2020). These were identified through PubMed searches using “deiodinase,” “deiodination,” “energy metabolism,” “adipose tissue,” “liver,” “skeletal muscle,” and “hypothalamus” as keywords.

Thyroidal Secretion and Deiodinases Are Coordinated

While circulating TH levels are largely stable (32), “local” deiodinase-mediated mechanisms within target cells modulate TH signaling (extensively reviewed elsewhere (33-38)). Deiodinases are dimeric integral membrane selenoproteins that contain a single N-terminal transmembrane segment connected to a larger globular area that contains a thioredoxin-fold catalytic domain (39-42). This group of enzymes can activate (ORD via D1 or D2) or inactivate (IRD via D1 and D3) TH molecules once they have entered target cells (33, 34, 43, 44). Mechanistic details of the deiodination reaction are available elsewhere (39, 40, 45, 46).

DIO2 is expressed in the brain, pituitary gland, brown adipose tissue (BAT), skin, and skeletal and smooth muscles. Alternatively, DIO3 expression is found in most fetal tissues, subsiding after birth (47, 48). In adults, DIO3 expression is mostly in the brain, placenta, pregnant uterus, skin, and pancreatic beta cells (49, 50). Nonetheless, DIO3 is also generally expressed during clinical illness (51, 52). D1 is unique in that it exhibits an affinity for T4 that is 3 orders of magnitude lower than that exhibited by D2. In fact, D1’s preferred substrate is reverse triiodothyronine (rT3), which is reflected in the elevated serum rT3 levels observed in the D1-deficient (D1KO) mouse (53) and in patients with loss-of-function mutations in DIO1 (15).

The integrated actions of the thyroid gland and the deiodinases stabilize plasma T3 levels, preserving TH signaling and clinical euthyroidism in most tissues. On one hand, the hypothalamus-pituitary-thyroid (HPT) axis is particularly driven to defend serum T3 levels (54, 55), and TSH-induced thyroidal T3 secretion is the gateway through which the HPT axis controls systemic TH signaling (56). On the other hand, deiodinases regulate T3 production and clearance outside the thyroid parenchyma (33, 57, 58). This is possible because T3 flows across cellular membranes via specific transmembrane transporters. Deiodinases have their catalytic active site located in the cytosol (39, 40, 59), and T3 molecules produced via D1 or D2 eventually exit cells and mix with the circulating pool of T3. In contrast, D3-expressing cells function as sinks for T4 and T3, dampening local TH signaling and consuming circulating TH (60).

In hypothyroidism, there is an increase in the D2-mediated fractional conversion of T4 to T3, and a decrease in the D3-mediated clearance of T3 (61-64). Regulation of D2 is mostly posttranslational via interaction with T4 (65, 66), which causes conjugation to ubiquitin via K48-linked ubiquitin chains (67) and inactivation of D2 (68, 69). Once D2 is ubiquitinated, it is retrotranslocated to the cytoplasm via interaction with the p97-ATPase complex and taken up by 26S proteasomes (67). This is the mechanism through which T3 production fluctuates according to the availability of T4 (69-78).

Deiodinases Customize TH Signaling

Distinct subcellular localization likely explains why D2-generated T3 remains in the cells where it was produced much longer than D1-generated T3. Thus, D2-expressing cells are subject to tissue-specific dynamic changes in TH signaling without antecedent changes in circulating levels of THs. For example, the sympathetic nervous system stimulates Dio2 expression and T3 production in BAT that adds to the intracellular T3 entering from the circulation. As a result, cellular T3 content and thyroid hormone receptor (TR) occupancy increases from its baseline level of about 75% (79, 80) to >95%, along with induction of T3-responsive genes (81).

The role played by Dio2 activation in TH signaling can be visualized through bioluminescence in the TH action indicator (THAI) mouse model (82), a transgenic mouse ubiquitously expressing a luciferase reporter gene regulated by a strong T3 response element that operates in the context of endogenously expressed levels of TH transporters, TRs, and transcriptional co-regulators. Exposing these mice to cold (4 °C) caused tissue-specific bioluminescence in the interscapular region (iBAT), along with a ~3.0- to 9.0-fold increase in luciferase activity and mRNA in iBAT, which was eliminated after surgical denervation of the iBAT (82). Such a role for D2 in defining local TH signaling is not unique to BAT (33), as it is also seen in the developing cochlea (83) and liver (84), where there is a surge in D2-generated T3. In the adult mouse, D2-generated T3 also plays a role in brain, lung, skeletal muscle, and skeleton (85-88).

D3 is located in the plasma membrane, but during hypoxic conditions it can relocate to the cell nucleus and more efficiently reduce TH signaling (60, 89, 90). D3 limits TH signaling because of T4 and T3 inactivation. For example, induction of DIO3 in skin cells by members of the Hedgehog family of proteins reduces local TH signaling. This is because the mRNA levels for cyclin-D1, a gene that is negatively regulated by T3, increase upon induction of DIO3, and this increase is followed by proliferation of keratinocytes (91). Most regions of the D3-deficient (D3KO) brain experience reduced TH signaling transiently during late neonatal life, while the opposite is true during early development and later life (92). However, the fact that D3 is relatively abundant in the brain by virtue of being expressed in neurons does not prevent TRs from being almost fully saturated with T3 (93). Thus, future studies should expand and clarify the role played by D3 in modulating local TH signaling.

Food Availability Adjusts Thyroidal and Deiodinase-Mediated T3 Production

Caloric intake stimulates the thyroid system, coupling availability of energy substrates and TH signaling (94, 95). During fasting, the default thyroid activity is low, along with a slow rate of energy expenditure. Once caloric intake is resumed, activity is accelerated and circulating TH levels increase; for example, in patients recovering from anorexia nervosa (96), weight gain and serum T3 are closely associated with a faster energy expenditure (96).

Fasting in rodents is associated with decreased thyroidal (97) and extrathyroidal D2-mediated T3 production (98). The activity of the D1 pathway is reduced as well (99, 100) but that may be a result of reduced T3 levels, not the cause (97, 101). Nonetheless, the inactivation of both Dio1 and Dio2 pathways in mice does not prevent the drop in circulating levels of T4 and T3 during fasting, pointing toward a major role played by other mechanisms (102). For example, upregulation of D3 in skeletal muscle, liver, and kidney play a role as well, along with enzymes involved in glucuronidation and sulfation of iodothyronines (103-105). Accordingly, in rats, semistarvation and refeeding in catch-up fat (a phase of weight regain after significant weight loss characterized by a disproportionately high rate of body fat recovery relative to lean tissue recovery) (106) slow down T3 formation in skeleton muscles due primarily to upregulation in the IRD enzymes, DIO1, and/or DIO3. Fasting has also been found to increase D3 activity in the liver and white adipose tissue (107). Fasting-induced constitutive androstane receptor activation increased Dio3 expression and activity in mice and rat hepatocytes. In addition, inhibition of the mammalian target of rapamycin (mTOR) by fasting increased Dio3 expression in mice liver and white adipose tissue, but only slightly increased D3 activity in rat hepatocytes.

Notwithstanding the different nuances associated with each individual animal model, all models of food restriction develop low levels of circulating T3 and diminished TH signaling in most tissues, explaining the reduction in the rate of metabolism.

Carbohydrates and carbohydrate-related hormones are potent modulators of circulating T3 levels through deiodination (94, 95). Insulin stimulates D2 activity in rat brown adipocytes (108), and insulin sensitizers stimulate Dio2 expression in cultures of skeletal myocytes (109). In addition, BAT D2 is upregulated by insulin-like growth factor 1 (IGF-1) and insulin (108, 110), which activate nutrient sensing pathways such as the phosphatidylinositol 3-kinase (PI3K)/mTOR (111, 112) pathways. Studies in cells and mice revealed that Dio2 is inhibited by forkhead box, subgroup O1 (Foxo1), a transcriptional regulator that binds the Dio2 promoter. In turn, insulin signals through the PI3K–mammalian target of rapamycin complex 2 (mTORC2)–serine/threonine kinase 1 (AKT) pathway and relieves Foxo1 repression. Dio2 expression in the brain is not modified by fasting, indicating that Dio2 regulation by nutrient availability is not universal, likely occurring in tissues such as BAT, skeletal muscle and neonatal liver where the metabolic pathways are responsive to T3 and insulin (43, 84). In addition, in neonatal rat ventricular myocytes, Foxo1 directly upregulates Dio2 and downregulates Dio3, indicating that these mechanisms are tissue-specific (113). Thus, the balance between PI3K-mTORC2-AKT and Foxo1 signaling in metabolically relevant tissues should provide nutritional input and fine-tuning to the regulation of circulating levels of T3 and T3-dependent processes (Fig. 1).

Figure 1.

Metabolic roles played by Dio2 in iBAT and skeletal muscle. D2 catalyzes T4 to T3 conversion inside cells, next to the nuclear compartment. D2-generated T3 enters the nucleus and binds to TRs, enhancing local thyroid hormone signaling. D2-T3 accelerates expression of PGC1 in both iBAT and skeletal muscle, increasing thermogenic capacity. In the iBAT, D2-T3 also activates expression of UCP1, the key uncoupling protein that increases heat production. Dio2 is a cAMP-inducible gene. Dio2 is also responsive to insulin, which in the skeletal muscle may contribute to circulating levels of T3. Dio2 expression is typically repressed by FOXO1 in skeletal muscle, which can be minimized by insulin-induced FOXO1 phosphorylation.

Tissue-Specific Metabolic Control

The expressions of Dio2 and Dio3 in the medial basal hypothalamus strategically place both enzymes at the crossroads of neural regulation of metabolism. For example, studies in mice show that food deprivation increases hypothalamic Dio2 mRNA levels and D2 activity (114). Hence, this localized increase in TH signaling could explain the reduction in thyrotropin-releasing hormone mRNA observed in fasted rats (115, 116). This mechanism might also regulate the torpor state in which there is reduction in metabolism and body temperature (117). In hamsters, hypothalamic Dio2 expression is decreased during spontaneous daily torpor as well as fasting-induced torpor (117). Notably, the global-D3KO mouse exhibits increased TH signaling in the hypothalamus with abnormal expression of genes in the melanocortin system, suggesting leptin resistance (118). They also have decreased adiposity, reduced BAT size, and accelerated fat loss in response to treatment with LT3. Global-D3KO mice display increased locomotor activity and an increased rate of energy expenditure along with expanded nighttime activity periods, suggesting a disrupted circadian rhythm (118).

Angiogenesis allows vessel density to reach the metabolic demands of the tissues via active sprouting of new vessels from endothelial cells, a process that in humans is regulated by TH (119). D2 is required for rapid stimulation of PI3K by T4 in human umbilical vein endothelial cells (120), which promotes endothelial cell migration. In addition, the production of pro-angiogenic factors such as vascular endothelial growth factor alpha is reduced in D2KO myoblasts. TH are also involved in neovascularization that occurs in tumor masses. Notably, D2 is highly expressed in intestinal polyps of a mouse model of familial adenomatous polyposis, and treatment with the deiodinase inhibitor iopanoic acid or chemical thyroidectomy suppresses tumor formation accompanied by reduced tumor cell proliferation and angiogenesis (121). Altogether, these data suggest that conversion of T4 to T3 by D2 plays a role in TH-induced angiogenesis in physiological and pathological conditions.

Brown Adipose Tissue

The ability to accelerate metabolic rate, a process known as adaptive thermogenesis (122), is accomplished through the release of norepinephrine in tissues such as BAT. There, norepinephrine induces the expression of cAMP-responsive genes that are part of the thermogenic program, such as Dio2, PGC1α, and UCP1 (123, 124) (Fig. 1). All 3 β-adrenergic receptor subtypes are involved, with each playing slightly different roles in adaptive thermogenesis and metabolic control (125-127).

Mice defend body temperature and survive in the cold thanks to the activation of a thermoregulatory response that critically depends on TH. Acutely cold-exposed thyroidectomized rats rapidly become hypothermic; LT4 is effective in restoring thermogenesis, given its activation to T3 in BAT via D2 (128). Circulating T3 plays a role, but norepinephrine-induced D2 acceleration and BAT T3 production are critical because they amplify cAMP production, therefore directly inducing UCP1 expression (129-131). Lipogenic enzymes are also stimulated (132-134) through a mechanism that involves T3 induction of the carbohydrate response element binding protein (135). BAT expresses both TRα and TRβ (136, 137) and activation of BAT thermogenesis depends on the coordinated effort between both TR isoforms (138, 139). Activation of TRβ induces the BAT thermogenic program eg, expression of Dio2, UCP1, and PGC1α, but amplification of cAMP production depends on activation of both TR isoforms (138). The activation of the thermogenic program in white adipose cells, also known as browning, can also be prompted by stimulation of TRβ and involves induction of Dio2 (140).

Survival of hypothyroid mice in the cold is only possible if cold exposure is gradually increased over time. In this case, it involves massive increase in sympathetic activity (141) and shivering (142). Animals in which Dio2 is globally inactivated (global-D2KO) are more sensitive to cold; they stimulate cold-induced thermogenesis even at room temperature (21°C), paradoxically rendering them resistant to diet-induced obesity. This phenotype is reversed by acclimatization at thermoneutrality (30 °C), which “turns off” sympathetic activity to the BAT. Only then, as a result of the unopposed reduction in TH signaling, global-D2KO mice become sensitized to diet-induced obesity, gaining excessive weight and also developing severe hepatic steatosis (143).

Having a fast-responding, cAMP-inducible, T3 generating system inside brown adipocytes (Dio2) is a key element of the extraordinary metabolic capacity exhibited by this tissue.

Liver

The global-D2KO mouse exhibits liver steatosis when placed at thermoneutrality and fed on a high fat diet (HFD) (143-145). Dio2 is not expressed in the normal adult liver, but it can be ectopically present in the liver of mice with targeted deletion of both liver X receptors (LXR) α and β (145). That LXR and RXR signaling inhibit Dio2 expression was confirmed by the observation that 22(R)-OH-cholesterol negatively regulates the human DIO2 promoter confirms that LXR and RXR signaling inhibit Dio2 expression (144). Remarkably, adipocyte fatty acid–binding protein (AFABP), an adipokine, induces expression of Dio2 in BAT via inhibition of the nuclear receptor LXRα, thereby increasing local TH signaling. AFABP accelerates thermogenesis by activating D2-mediated T3 production in brown adipocytes. The thermogenic responses to T4 are abrogated in Afabp-KO mice, but enhanced by AFABP (146).

Although not expressed in the adult mouse liver, a transient surge of Dio2 was detected at around the first day of life, activating local T4 to T3 conversion and TH signaling. This T3 surge occurs at a time in which serum T3 levels are half of what is observed in adult mice (147, 148); hence, it doubles local T3 concentration and modifies the expression of genes involved in broad aspects of hepatocyte function, including lipid metabolism (84, 149). Liver-specific Dio2 inactivation (Alb-D2KO) delays the expression of lipid-related genes and modifies baseline and long-term hepatic transcriptional response to a HFD. Feeding on a HFD normally affects the expression of approximately 400 hepatic genes involved in synthesis of fatty acids and triglycerides. In contrast, the response to HFD in the Alb-D2KO animals is restricted to a different set of only approximately 200 genes, which are associated with reverse cholesterol transport and lipase activity (84).

Subsequent studies revealed that the Alb-D2KO liver exhibits signs of epigenetic modifications, ie, about 1500 sites of DNA hypermethylation that explain the dramatic phenotype of the adult Alb-D2KO mouse with greatly reduced susceptibility to diet-induced steatosis, hypertriglyceridemia, and obesity (84). The sites of DNA hypermethylation seem to be caused by a perinatal increase in H3K9me3 levels in discrete chromatin areas (150). The change in DNA methylation pattern reduces chromatin accessibility, with reduction in the expression of several hundred genes. Thus, the postnatal surge in hepatic D2 activity and TH signaling prevents discrete DNA methylation and modifies the transcriptome of the adult mouse liver (150).

One of the repressed genes that underlies the Alb-D2KO phenotype is the gene encoding the zinc finger protein-125 (Zfp125) (84, 149). Zfp125 is a Foxo1-inducible transcriptional repressor that promotes lipid accumulation and liver steatosis in mice by reducing hepatic secretion of triglycerides and cholesterol (149). Zfp125 acts by repressing genes involved in lipoprotein structure and lipid binding and transport. While liver-specific knockdown of Zfp125 causes an “Alb-D2KO-like” metabolic phenotype, liver-specific normalization of Zfp125 expression in Alb-D2KO mice rescues the phenotype, restoring normal susceptibility to diet-induced obesity, liver steatosis, and hypercholesterolemia (149). In addition, mice with liver-specific Zfp125 knockdown exhibited a drop in respiratory quotient, reduced glycemia and pyruvate-stimulated liver glucose output, and higher levels of B-hydroxybutyrate (151). Liver-specific Zfp125 knockdown also amplified B-hydroxybutyrate production in response to 24 to 36 hours of fasting or feeding on a ketogenic diet. In isolated mouse hepatocytes, Zfp125 knockdown amplified the induction of ketogenesis by glucagon or insulin resistance, whereas Zfp125 overexpression amplified the expression of key gluconeogenic genes Pck1 and G6pc, placing Zfp125 at the center of fuel dysregulation of type 2 diabetes (151).

Overall, these studies indicate that the final differentiation of hepatoblasts to hepatocytes that occurs in the neonatal liver is influenced by TH signaling. The transient peak of D2-generated T3 is aligned with the overall positive effect of TH on liver lipogenesis and carries significant implications for future development of obesity and liver steatosis.

Zebrafish is another animal model in which this has been investigated, where Dio2 inactivation caused hyperglycemia due to reduced insulin sensitivity and expression of glycolytic enzymes in the skeletal muscles (152). As the fish aged, pancreatic islet size and β and α cell numbers increased along with insulin secretion. Glucagon receptors and downstream targets were also downregulated in the liver, indicating hepatic glucagon resistance and decreased gluconeogenesis (152).

Other Metabolically Relevant Tissues

The development of the flox-Dio2 and flox-Dio3 mice have allowed for a better understanding of the tissue-specific roles played by deiodinases (153, 154). Astro-D2KO mice (mice in which Dio2 has been inactivated in astrocytes) eat normally but exhibit a lower diurnal respiratory quotient and greater contributions of fatty acid oxidation to energy expenditure (155). In contrast, the Fat-D2KO mice (mice in which Dio2 has been inactivated in fat tissue) exhibit greater contribution of carbohydrate oxidation to energy expenditure. Furthermore, Fat-D2KO animals placed on HFDs gained more body weight and fat, suggesting impaired thermogenesis (possibly at the BAT level) and/or an inability to oxidize the fat excess (155).

DIO2 is expressed in human and murine skeletal muscle (33, 109, 156-158), but at levels at least 2 orders of magnitude lower than in the brain (159-161). While the role played by deiodinases in the context of skeletal muscle development seems clear (109, 158, 162-164), a metabolic role has not been well established. For example, Skm-D2KO mice (mice in which Dio2 has been inactivated in skeletal muscle) exhibited no apparent metabolic phenotype (155). The fact that there is BAT mixed with some muscle fibers (165), suggests that some of the local effects attributed to D2-generated T3 could be mediated by BAT tissue. With that in mind, Dio2 expression in skeletal muscle has been found to be induced by feeding on a methionine-restricted diet, which was associated with increased energy expenditure and protection against HFD-induced metabolic dysfunction (166).

Two models of induction of Dio2 in skeletal muscle have provided further understanding of the role played by this enzyme. The first is a cell model in which D2 expression is controlled by the “Tet-ON” system (167). D2 expression caused a shift from oxidative phosphorylation to glycolysis, with a consequent increase in the extracellular acidification rate; changes in metabolism coincided with a D2-induced shift from slow type-I to fast type-II myofibers, suggesting that D2 is essential in coordinating metabolic reprogramming of myocytes during myogenic differentiation (167). Indeed, in mice, Dio2 expression in the skeletal muscle can be induced by physical activity (86). A 20-minute treadmill exercise increased Dio2 expression/activity as well as PGC1α mRNA levels in rat and mouse skeletal muscle (Fig. 1). In contrast, induction of PGC1α was only partial (about 40% less) in the Skm-D2KO mice by acute and chronic treadmill exercise as well as in primary Skm-D2KO myocytes stimulated with cAMP (86). These findings that Dio2 expression mediates part of PGC1α induction by treadmill exercise are reminiscent of similar observation in BAT and lungs. Induction of PGC1α in BAT (168) and in the lung (9) is only partial in mice with Dio2 inactivation, suggesting a common pathway through which Dio2 affects mitochondrial biogenesis and metabolism.

Dio3 is expressed in embryonic and adult human and murine pancreatic β-cells (49, 50). It is conceivable that its presence minimizes the inhibitory effects of T3 on insulin secretion observed in isolated mature murine pancreatic islets (49, 50) and in MIN6 insulinoma cells (169). Dio3 expression in MIN6 cells is stimulated by glucagon-like peptide-1 and by Exendin-4, a glucagon-like peptide-1 receptor agonist (169). Mice with Dio3 inactivation in pancreatic islets are glucose intolerant and have impaired glucose-stimulated insulin secretion (49, 50).

Other Metabolic Signals Affect Dio2 Expression and Local TH Signaling

Endogenous and exogenous molecules that activate the Dio2 pathway include bile acids (170), flavonols (171), chemical chaperones (172), and the adipokine adipocyte-specific fatty acid–binding protein (AFABP) (146). These molecules have been linked to activation of D2-generated T3 and acceleration of energy expenditure (170), and protection against obesity (173). In vitro treatment of primary human brown adipocytes with chenodeoxycholic acid (CDCA) or specific TGR5 agonists increased mitochondrial uncoupling and DIO2 expression, an effect that was absent in human primary white adipocytes (174). Studies in humans support the idea that bile acids play a role in controlling energy expenditure (174, 175). Along the same lines but through a different pathway, kaempferol and other flavonols stimulate DIO2 expression via a cAMP-mediated mechanism in primary cultures of human skeletal myocytes, leading to D2-mediated T3 production, expression of thermogenic relevant genes, and acceleration of O₂ consumption (171).

Endoplasmic reticulum (ER) stress may be a link between metabolic homeostasis and D2-mediated TH signaling (17, 176). From a metabolic perspective, ER stress can be triggered by HFD and obesity, contributing with downregulation of insulin sensitivity (177). It is notable that ER stress leads to a rapid loss of D2 activity without affecting Dio2 mRNA levels, resulting in a drop in D2-mediated T3 production (176). The drop in D2 activity requires eukaryotic initiation factor 2, which blocks Dio2 mRNA translation, hence inhibiting synthesis of D2.

Conclusions

Deiodinases can augment and reduce TH signaling in a tissue-specific fashion while plasma T3 levels remain tranquil. Although a hypothetical metabolic role was linked to the pathway that converts T4 to T3 in the 1950s, objective evidence that this was the case was only developed decades later. Several settings using cell, animal and human models illuminate how activation of T4 to T3 and inactivation of T4 and T3 play physiological and pathophysiological roles at various life moments. Further understanding of these mechanisms will allow a more refined therapeutic control of TH signaling with enormous implications for health and disease states.

Glossary

Abbreviations

AFABP

adipocyte fatty acid-binding protein

BAT

brown adipose tissue

cAMP

cyclic adenosine monophosphate

D1

type 1 deiodinase

D2

type 2 deiodinase (activating)

D3

type 3 deiodinase (inactivating)

ER

endoplasmic reticulum

FOXO1

forkhead box O1

HFD

high-fat diet

iBAT

interscapular brown adipose tissue

IRD

inner ring deiodination

LT3

L-triiodothyronine

LT4

levothyroxine

LXR

liver X receptor

mTOR

mammalian target of rapamycin

ORD

outer ring deiodination

PI3K

phosphatidylinositol 3-kinase

rT3

reverse triiodothyronine

T3

triiodothyronine

T4

thyroxine

TH

thyroid hormone

TR

thyroid hormone receptor

TSH

thyrotropin (thyroid-stimulating hormone)

Zfp125

zinc finger protein-125

Additional Information

Disclosures: Dr. Bianco is a consultant for Synthonics Inc, Allergan Inc, and BLA Technology LLC; the other authors have no disclosures.

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.