Metabolic Messengers: Thyroid Hormones

Abstract

Thyroid hormones (THs) are key hormones that regulate development and metabolism in mammals. In man, the major target tissues for TH action are the brain, liver, muscle, heart, and adipose tissue. Defects in TH synthesis, transport, metabolism, and nuclear action have been associated with genetic and endocrine diseases in man. Over the past few years, there has been renewed interest in TH action and the therapeutic potential of THs and thyromimetics to treat several metabolic disorders such as hypercholesterolemia, dyslipidaemia, non-alcoholic fatty liver disease (NAFLD), and TH transporter defects. Recent advances in the development of tissue and TH receptor isoform-targeted thyromimetics have kindled new hope for translating our fundamental understanding of TH action into an effective therapy. This review provides a concise overview of the historical development of our understanding of TH action, its physiological and pathophysiological effects on metabolism, and future therapeutic applications to treat metabolic dysfunction.

History & Clinical Manifestations

The thyroid hormones (THs), 3,5,3’,5’ tetraiodo-L-thyronine (T₄) and 3,5,3’ triiodo-L-thyronine (T₃), are iodinated tyrosine-based hormones produced by the thyroid gland. THs play active and permissive roles in the metabolism, growth, and development of almost all tissues in the body. In 1820 the essential role of iodine for normal thyroid function was first reported¹ (Fig. 1). In 1883, the renowned Swiss surgeon, Emil Kocher, noted the development of myxoedema in patients after he performed thyroidectomies². Around the same time, the Clinical Society of London reported that cretinism and myxoedema were associated with thyroid gland destruction³. Later, sheep thyroid extracts were used to treat myxoedema successfully and this soon was adopted as standard therapy⁴. Kendall isolated and identified an iodine-containing substance from human thyroid extracts that improved symptoms in myxoedematous patients and thyroidectomized animals in 1915⁵. Harington later determined that the active compound was a p-hydroxyphenyl ester of tyrosine that contained iodine in the 3,5,3′ and 5′ positions, and named it ‘thyroxine’ (T₄)⁶. It was not until 30 years later that Pitt-Rivers and Gross identified and characterized T₃ as the most active form of TH in the serum and thyroid gland in the early 1950s⁷. The discovery of the radioimmunoassay by Berson and Yalow⁸ soon led to the development of sensitive assays to measure serum T₃ and T₄, and pituitary-generated thyroid stimulating hormone (TSH, thyrotropin) concentrations^9,10.

Fig. 1. Timeline of TH discovery and clinical development.

Soon after the cloning of T₃ receptors in 1986, significant progress has been made to understand the mechanism of TH metabolic action and to develop TH mimetics for the treatment of metabolic and neurological diseases.

In the early 1960s, Tata showed that T₃ induced RNA and protein synthesis¹¹, and in the 1970s, several laboratories demonstrated that thyroid hormone receptors (THRs) were localized within the nucleus^12,13. These findings suggested that THRs could be transcriptional regulators. Furthermore, 5’ deiodination of T₄ into T₃ was shown to be essential to exert its cellular actions¹⁴. In 1986, the Evans and Vennstrom laboratories first cloned the THRs and identified them as the cellular homologs of a viral oncogene, v-erbA^15,16. Surprisingly at that time, THRs also shared structural and sequence similarities with the previously cloned glucocorticoid and oestrogen receptors to comprise a superfamily of nuclear receptors.

Previous reviews have examined the role of TH on development and gene transcription of specific enzymes involved in metabolism; however, recent advances in metabolomics and genomics have enabled deeper and broader understanding of TH’s global impact on mammalian physiology in peripheral tissues. When combined with in vivo mouse models under metabolic or nutritional stress, they also showed that TH or thyromimetics may have therapeutic benefits when targeted to tissues that have inappropriately low intracellular TH concentration/action. These findings suggest that understanding TH’s metabolic actions in specific tissues are important to determine overall TH status clinically, and take us beyond the current practice of using the hypothalamic-pituitary-thyroid axis as the sole assessor of TH status. In this review, we will focus on this more extensive and integrated view of metabolism of major tissues and metabolic cross-talk between tissues by TH.

TH synthesis, secretion, cell transport, and metabolism

The hypothalamus secretes thyrotropin releasing hormone (TRH), a tripeptide that stimulates pituitary secretion of thyroid stimulating hormone (TSH) that, in turn, induces the production and release of TH into the circulation by thyrocytes¹⁷. Significantly, circulating TH concentrations can inhibit both TRH and TSH secretion to enable the co-ordinated negative feedback regulation of the hypothalamic-pituitary-thyroid axis¹⁸. The thyroid gland itself actively concentrates circulating iodide into thyrocytes via sodium/iodide symporters (NIS) located on their basolateral plasma membranes. The thyrocytes themselves are organized as follicles that surround intraluminal depots of colloid. Intracellular iodide then is shuttled into the colloid lumen of thyroid follicles by the pendrin protein encoded by the SLC26A4 gene (solute carrier family 26, member 4) on the apical plasma membrane where it is conjugated to tyrosine residues of the thyroglobulin (Tg) protein by two enzymes, dual oxidase (DUOX) and thyroid peroxidase (TPO) to form monoiodotyrosine (MIT) and diiodotyrosine (DIT). MIT and DIT are coupled by TPO to generate T₄ and T₃ and stored in the colloid. Upon TSH stimulation, Tg is internalized at the apical pole of thyrocytes and digested by lysosomal proteases to release T₄ and T₃ into the circulation¹⁹ via the bidirectional MCT8 and MCT10 transporters. MIT and DIT also are released from Tg and deiodinated by iodotyrosine dehalogenase 1(Dehal1) on the apical pole to recycle iodide required for new TH synthesis²⁰. The majority of circulating T₄ and T₃ is transported by carrier proteins such as thyroxine-binding globulin [TBG or thyropexin], transthyretin [TTR], and albumin (HAS, human serum albumin)²¹ that serve as TH reservoirs. The TH bound to these proteins are inactive and are in dynamic equilibrium with the circulating levels of free T₄ and T₃ (FT₃, FT₄). The circulating free T₃ and T₄ then enter target tissues to exert their biological effects.

Several plasma membrane transporter proteins such as monocarboxylate transporter MCT8, MCT10, the organic anion transporting polypeptide (OATP)1C1, SLC17A4 and L-type amino acid transporters (LAT1 and LAT2)²² enable free TH to enter target cells (Fig. 2A). The expression of these transporters varies among different tissues. Many of the TH transporters also act as both influx and efflux transporters to dynamically regulate intracellular TH levels^23,24. Of note, several human mutations in MCT8 and in OATP1C1 have been reported and are associated with severe neurological defects^25,26.

Fig. 2. Cellular and metabolic actions of TH.

(a) Both T₃ and T₄ after their secretion from the thyroid gland enter target cells via specific transporters expressed on plasma membrane. Once inside the cells the deiodinase enzymes DIO1 and DIO2 convert T₄ into T₃ thereby promoting TH’s biological action. However, intracellular action of TH is negatively regulated by Dio3 which converts T₄ and T₃ into rT₃ and T₂ respectively. Intracellular T₃ primarily acts by binding to nuclear TH receptors (THRα/β). T₃ bound THRs associate with cognate DNA sequence or TREs as either homodimers or heterodimers with RXRs. THRs, upon T₃ binding, recruit nuclear Coactivators (CoA) which are associated with Histone acetylase (HAT) thereby promoting gene transcription. Transcriptional targets of THRs can be primary responsive genes which harbor TREs on their promoter/enhancer genes. However, THRs can also modulate secondary gene target expression by regulating the expression of other transcription factors or regulatory non-coding RNAs which, once transcribed by THRs, regulate secondary TH responsive gene targets. Apart from the classical nuclear genomic pathway, THs including T₄, T₃ and T₂ also act on cytosolic proteins, mitochondria and integrin at cell surface to induce non-genomic action on cells. Of note, mitochondrial action of T₃ and T₂ has been associated with increased respiration, ROS-mediated autophagy induction and SirT1 activation (b). Transcriptional regulation of metabolic pathways by TH. T₃-mediated nuclear action in the hepatocytes induces both anabolic (lipogenesis) and catabolic (lipolysis) action on hepatic lipid metabolism (Table 1b). TH increases the expression of several pro-lipogenic genes including FAS, ME etc. to increase de novo lipogenesis in hepatocytes; however, it also down-regulates genes which are required for lipid droplet assembly such as SCD1 and GPAT3. Compared to its effect on hepatic lipogenesis, TH effects on lipid catabolism are more profound as evidenced by the potent stimulation of autophagy-mediated lipolysis and mitochondrial β-oxidation observed by T₃ in rodent and human hepatocytes. TH also regulate cholesterol synthesis, uptake, and reverse cholesterol transport (RCT) via its effects on different genes involved in hepatic cholesterol metabolism. TH also is potent inducer of hepatic glucose output through direct and SIRT1-FOXO1-mediated stimulation of gluconeogenic gene transcription. Additionally, TH increases hepatic gluconeogenesis by inhibiting insulin-stimulated AKT activation in rodents. Genes regulated by TH are italicized and pathways are in bold font. Green arrows denote upregulation and red arrows denote downregulated genes and pathways.

Deiodinases (DIOs) are selenoproteins that play crucial roles in maintaining circulating T₃ concentration as well as intracellular levels of TH and their metabolites²⁷. Thus, the DIOs involved in TH metabolism fine-tune the intracellular concentration and biological activity of TH. DIO1 and DIO2 convert T₄ to T₃ whereas DIO3 inactivates T₃ to 3,3′-diiodo-L-thyronine (T2) and modifies T₄ to reverse triiodothyronine (rT₃)²⁷. DIO1 and DIO2 have additional enzymatic activity and metabolize rT₃ to T₂ (Fig. 2A). The expression of DIOs varies among different tissues with DIO1 predominating in liver and DIO2 in brown adipose tissue (BAT), skeletal muscle, and brain. Interestingly, DIO1 activity also can by inhibited by β-blockers, propylthiouracil, and iodine whereas DIO2 is insensitive to these compounds. Additionally, hepatic DIO1 plays a significant role in modulating circulating T₃ levels²⁷. Human DIO1 mutations have been linked to abnormally elevated serum rT₃ levels and increased rT₃/T₃ ratios²⁸ and epigenetic overexpression of DIO3 in certain tumours causes ‘consumptive’ hypothyroidism requiring high amounts of TH supplementation²⁹.

A crucial component of selenoprotein production required for deiodinase enzyme activity is selenocysteine insertion sequence binding protein 2 (SBP2). In addition to the typical hypothyroid phenotype, patients with SBP2 mutations also exhibit developmental delay, poor motor coordination, male infertility, and growth retardation^30,31. In summary, the production of circulating T₃ and T₄ by the thyroid gland is regulated by iodide intake and the negative control of TSH. The expression of specific TH transporters and deiodinases in peripheral tissues help determine the intra-tissue concentration of T₃, the more potent form of TH.

Genomic Actions of T₃

Identification and characterization of THR

In 1986 the laboratories of Bjorn Vennstrom and Ronald Evans first reported the gene sequences of THRs^15,16. Sequence comparison revealed there are two THR genes: thyroid hormone receptor α/β (Thra and Thrb in mice; THRA and THRB in man) that encode two different THRs (designated as THRα and THRβ in mice and humans) and their splice variants³². THRs belong to a large superfamily of nuclear receptors, some of which bind to other hormones (for example, vitamin D and retinoic acid) or endogenous intracellular ligands (for example, peroxisome proliferator-activated receptors) whereas others do not appear to bind to any detectable ligands (‘orphan’ receptors). The hormone-binding specificities of THRs and other nuclear receptors are determined by key differences in the amino acid sequences of their carboxy-terminal ligand-binding domains. THRs also bind to DNA enhancer elements located in the promoters of target genes via a central DNA-binding domain containing two ‘zinc fingers’ that interact with the major grooves of DNA formed by thyroid hormone response elements (TREs) typically located within the promoters of target genes (Fig. 2A). Thus, THRs regulate the transcription of target genes in a hormone-dependent manner³³. THRα is the predominant isoform in heart, bone, skeletal muscle, gastrointestinal tract, and brain. THRβ1 is the major isoform encoded by THRB and is widely expressed; however, it is the predominant isoform in the liver and kidney. Another THRβ isoform, THRβ2 has a different amino-terminal sequence than THRβ1 and is expressed restrictively in the hypothalamus, pituitary, cochlea, and retina.

THR as ligand-inducible transcription factors

T₃ binds to nuclear THRs with a 10-fold higher affinity than T₄ and is the major TH involved in its genomic action³⁴. Upon binding T₃, THRs heterodimerize with another nuclear hormone receptor, retinoic acid X receptors (RXRs) and interact with TREs containing relatively well-preserved canonical half-site sequences frequently arranged as direct repeats separated by four nucleotides^35,36. Interestingly, THRs can positively- or negatively-regulate different target genes. In positively regulated genes, T₃-bound THR/RXR heterodimers bind to TREs whereupon they recruit and assemble THR-coactivator complexes with intrinsic histone acetyltransferase (HAT) activity that induce nucleosome uncoiling and RNA POL II-mediated transcription (Fig. 2A). In the absence of TH, THRs can repress the transcription of positively regulated target genes by binding to TREs and recruiting nuclear co-repressor complexes containing histone deacetylases³⁷. Negative regulation by T₃ remains enigmatic; however, different models have been proposed that include direct THR binding to a negative TRE³⁸ and co-recruitment of inhibitory transcription factors and corepressors³⁹. In summary, THRs are nuclear receptors (NRs) expressed in almost all cell types of the body. The differential expression of THRα and THRβ isoforms, relative amounts of various co-activators/corepressors, and the quantity of specific TH transporters and deiodinases can contribute to the tissue-specific regulation of transcription by T₃⁴⁰.

Amplification of gene repertoire by TH

Interestingly, the gene repertoire regulated by TH is much larger than the number of genes predicted to harbour a TRE based upon ChIP seq analyses⁴¹. This discrepancy is likely explained by the transcriptional regulation of secondary transcription factors⁴¹ or other mechanisms such as generation of gene-regulatory non-coding RNA species⁴² by THRs to amplify the biological effect(s) of TH. Indeed, many of the effects of TH on mitochondrial function are mediated by its down-stream induction of other nuclear hormone receptors such as PPARG coactivator 1 alpha (PPARGC1A; PGC1α), and oestrogen receptor-related receptor-α (ESRRA)⁴³. Additionally, TH induction of sirtuin 1 (SIRT1) activity deacetylates FOXO1 and PGC1a to promote their nuclear localization and transcriptional activity^44,45. In summary, secondary induction of transcription factors or miRNAs, and/or post-translational modifications of transcription factors can amplify the number of genes regulated by TH.

Human mutations of THRs

Human mutations in THRA and THRB genes confer resistance to thyroid hormone (RTH) with different phenotypes, depending upon the relative expression of the two isoforms in different tissues as the mutant THR typically has dominant negative effects on the THRs from normal alleles. These findings also highlight the importance of TH actions in specific tissues, particularly where a specific THR isoform is predominantly expressed. Patients with THRB mutations in exons encoding the ligand binding domain exhibit increased TH levels, and inappropriate TSH secretion⁴⁶ reflecting the critical role of THRβ2 isoform in the suppression of TSH in the pituitary. They also have hypercholesterolemia, hypertriglyceridemia, insulin resistance, and non-alcoholic fatty liver disease, increased prevalence of attention-deficit disorder, and reduced colour vision and hearing^46,47 reflecting the important roles of THRβ1 in the adult liver and THRβ2 in the development of brain, retina, and cochlea. Additionally, they have increased BMR, heart rate, and osteoporosis owing to the elevated circulating TH levels activating the THRα isoform which is highly expressed in brown adipose tissue (BAT), skeletal muscle, heart, and bone. In contrast, patients with THRA mutations usually have normal or slightly increased FT₃ levels, decreased or normal FT₄ levels, increased T₃/rT₃ ratio, and normal or slightly elevated TSH levels^48–50. They typically present with dysmorphic features such macroglossia and flat nasal bridge, as well as short stature, constipation, bradycardia, and neurodevelopmental impairment, and normocytic anaemia^48–50. Patients usually also have lower BMR and elevated serum muscle creatine kinase, LDL, and cholesterol levels⁴⁸.Many of these characteristics resemble features of hypothyroidism and are due to the relatively higher expression of THRα in bone, gastrointestinal tract, heart, and brain⁴⁹. Interestingly, some of the phenotypic and metabolic changes in RTH also may be due to epigenetic changes that occur from exposure to abnormal TH levels in utero. For example, Thra mutant mice had aberrant DNA methylation of ion channel genes that could be corrected by maternal administration of T₃ during pregnancy⁵⁰. In general, mouse models of Thra and Thrb knockouts and dominant negative mutants have shown similar phenotypes as patients with RTH and TRHA and THRB mutations⁵¹.

Non-genomic actions by THs

Extranuclear biological actions of TH that do not require nuclear THR-mediated gene transcription and protein synthesis are defined as ‘non-genomic’ actions⁵². Non-genomic actions of THs can involve binding to intracellular proteins other than THRs or cell surface proteins such as integrins. Additionally, unlike the genomic actions of THRs which are regulated primarily by T₃, other ligands such as T₄ and TH metabolites, for example, TRIAC, Reverse T₃ (3,3’,5’-triiodothyronine or rT3) and 3,5-diiodothyronine (T₂) can be potent inducers of non-genomic signaling in cells⁵². Furthermore, T₃ exerts non-genomic actions on a cytosolic THRβ variant by activating phosphatidylinositol 3-kinase (PI3K)⁵³. Some of the other non-genomic actions of THs that have been reported include changes in the neuro-glial cytoskeleton⁵⁴, proliferation of airway smooth muscle cells⁵⁵, proliferation of human bone cells in vitro⁵⁶, regulation of angiogenesis⁵⁷, modulation of ion pumps⁵⁸, and induction of rapid mitochondrial respiration⁵⁹.

Metabolic effects of TH

Liver

TH regulation of lipogenesis

THRβ is the predominant THR isoform expressed in the liver⁶⁰, and it plays a major role in the regulation of metabolic processes such as de novo lipogenesis (DNL), fatty acid β-oxidation (FAO), cholesterol biosynthesis and reverse transport, and carbohydrate metabolism of the liver⁶¹ by TH. TH increases lipogenesis in liver via THR-regulated transcription of key lipogenic genes: fatty acid synthase (Fasn), acetyl-CoA carboxylase alpha (Acc1; also known as Acaca), malic enzyme (Me) and thyroid hormone-responsive Spot14 homologue (Thrsp; also known as Spot14)^61,62 (Fig. 2B). Additionally, TH also induces the expression of other pro-lipogenic transcription factors such as sterol regulatory element-binding protein 1 C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (CHREBP) which further increase the expression of lipogenic genes^61,62 (Fig. 2B). Interestingly, despite its induction of pro-lipogenic genes, TH negatively regulates the expression of genes involved in triglyceride synthesis and packaging such as stearoyl-CoA desaturase 1 (SCD1) in human hepatic cells, and glycerol-3-phosphate acyltransferase 3 (GPAT3) and apolipoprotein B100 (APOB100) (Fig. 2B) in rat hepatocytes^61,62 so excess fatty acid synthesis by TH typically does not cause increased triglyceride accumulation in the liver. These latter genes also are positively regulated by the nuclear receptor, LXR, as both THRs and LXRs counter-regulate each other via competitive binding to common hormone response elements in the promoters of these genes involved in fatty acid and triglyceride synthesis⁶³.

TH regulation of fatty acid β-oxidation

TH mobilizes FFAs from triacylglycerols (TAGs) stored in fat droplets and stimulates their fatty acid β-oxidation (FAO) in the mitochondria (Fig. 2B). Although TH induction of hepatic lipases has been documented, the major pathway for lipolysis of stored hepatic lipids is the autophagy-lysosomal pathway⁶⁴ (Fig. 2B). TH-induced autophagy of lipid or ‘lipophagy’ begins with the engulfment of TAG stored in fat droplets by double membrane autophagosomes followed by their fusion with lysosomes to form autolysosomes⁶⁴. The lipids inside the autolysosomes are hydrolyzed by lysosomal acid lipase (LIPA) to release the resultant glycerol and free fatty acids into the cytosol⁶⁴. This lipophagy-mediated lipolysis provides most of the free fatty acids for mitochondrial β-oxidation since abrogation of autophagy by siRNA significantly reduces TH-induced FAO and ketogenesis in the mouse liver⁶⁴.

In addition to lipophagy, TH can induce the expression of two major cytosolic lipases in the liver, hepatic lipase and adipose triglyceride lipase (ATGL; also known as PNPLA2), to increase intrahepatic lipolysis⁶¹ Furthermore, TH increases FAO and suppresses glycolysis by inducing the expression of hepatic carnitine O-palmitoyl transferase 1, (CPT1-Lα)⁶⁵, medium-chain acyl-CoA dehydrogenase (MCAD)⁶⁶, and pyruvate dehydrogenase kinase isoform 4 (PDK4) (Fig. 2B). TH increases mitochondrial activity and biosynthesis by stimulating mitochondrial uncoupling protein 2 (UCP2), PGC1α, and ESRRA^61,67. Concurrently, TH increases autophagy of mitochondria (mitophagy) to remove mitochondria damaged by reactive oxygen species (ROS)⁶⁸ (Fig. 2B). Thus, TH maintains efficient and long-term FAO by regulating mitochondria turnover and quality⁶⁸.

Interestingly, although TH induces lipogenesis, it also paradoxically increases β-oxidation of fatty acids in the liver. However, in general, hepatic catabolism of lipids predominates over lipid synthesis, most likely due to TH upregulation of AMP-activated protein kinase (AMPK)⁶¹ signaling which leads to phosphorylation of acetyl-coA carboxylase 1 (ACC1) and activation of carnitine palmitoyl transferase-1α (CPT-1α). In summary, TH controls multiple steps by regulating fatty acid synthesis and FAO via the phosphorylation of ACC by AMPK, and inducing lipophagy, mitophagy, hepatic lipases, mitochondrial biogenesis, and transcription of genes involved in these two major lipid pathways.

TH regulation of cholesterol synthesis and reverse cholesterol transport

TH has significant effects on serum cholesterol levels⁶⁹ through its effects on hepatic cholesterol metabolism, particularly cholesterol biosynthesis, endocytosis via the LDL-receptor (LDLR), peripheral uptake and hepatic excretion via reverse cholesterol transport (RCT)^61,62. Cholesterol biosynthesis is regulated by a rate-limiting enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMGCR). TH increases both HMGCR mRNA and protein levels in rats^61,62; however, TH regulation of HMGR expression may be indirect and depend upon induction of secondary transcription factors such as upstream stimulatory factor-2 (USF-2), sterol regulatory element binding protein 2 (SREBP2), and nuclear factor-y (NF-Y)^61,62 (Fig. 2B). Despite its stimulation of HMGCR expression, TH lowers circulating cholesterol levels by inducing LDL-R expression in concert with SREBP2 and enhancing clearance of circulating LDL by endocytosis^70,71 It also reduces pro-protein convertase subtilisin/kexin type 9 (PCSK9) levels in rodent and human livers⁷² (Fig. 2B). Interestingly, mice lacking Mct8 exhibit increased hepatic T₃ signalling and lower serum cholesterol levels as observed in patients with MCT8 mutations⁷³.

TH further increases cholesterol clearance by stimulating reverse cholesterol transport (RCT) at several steps^61,62 First, it strongly induces the gene and protein expression of Apo A1, scavenger receptor class B member 1 (SRB1), and cholesteryl ester transfer protein (CETP) to increase cholesterol efflux from peripheral tissues to HDL and enhance hepatic uptake in the RCT pathway^61,62 (Fig. 2B). Second, it increases hepatic expression of the rate-limiting enzyme, cholesterol 7α-hydroxylase (CYP7A1), that converts cholesterol into bile acids in both rats and humans⁷⁴. Finally, TH upregulates the expression of ATP-binding cassette transporters, subfamily G, member 5 (ABCG5) and 8 (ABCG8) to increase biliary cholesterol excretion in mice^61,62 (Fig. 2B). In summary, TH lowers serum cholesterol by inducing the expression of LDL-R and decreasing PCSK9, and by promoting cholesterol efflux from peripheral tissues and reverse cholesterol transport.

TH effects on glucose metabolism

Besides its effects on fatty acid and cholesterol metabolism, T₃ stimulates glycogenolysis and gluconeogenesis in both rodents and humans^75–77. In this connection, impaired glucose tolerance and hepatic insulin action and increased hepatic glucose output are observed in patients with hyperthyroidism^78,79. These effects are due to T₃-mediated induction of hepatic phosphorylase kinase and lysosomal alpha-glucosidase to increase glycogenolysis⁷⁶ and rate-limiting enzymes such as phosphoenolpyruvate carboxykinase (PCK1)⁸⁰ and glucose-6-phosphatase (G6PC)⁸¹ to increase gluconeogenesis (Fig. 2B). TH also regulates PDK4 to inhibit pyruvate dehydrogenase (PDH), the key enzyme controlling glycolysis⁸². Additionally, TH can impede insulin’s inhibitory effects on gluconeogenesis by reducing phosphatidylinositol 3-kinase (PI3K) signalling and promoting FOXO1 nuclear localization by activating SIRT1⁸³ (Fig. 2B). Thus, TH has hyperglycaemic activity in the liver by inhibiting insulin action and increasing hepatic glucose output in both rodents and humans.

Skeletal muscle

THRα is the predominant THR isoform in skeletal muscle⁸⁴ and regulates the contractile function, metabolism, myogenesis, regeneration, and thermogenic activity of skeletal muscle⁸⁵ (Fig. 3). Intramuscular T₃ is primarily generated by the local conversion of T₄ to T₃ by Dio2. T₃ mediates a fibre type switch from oxidative slow type I-expressing MYH7 fibres that primarily utilize fatty acids to oxidative rapid type II-expressing MYH-2, -1, and -4 expressing fibres that favour glycolysis mostly due to its transcriptional induction of myosin heavy chain 2 A (MHC2A) gene⁸⁶. This fibre type switching also is accompanied by increased expression of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA1a) and SERCA2a to promote muscle relaxation⁸⁶. Thus, the relative proportion of fast/slow switch skeletal muscle fiber types is regulated by TH and can change the fuel utilization and metabolic phenotype of skeletal muscle. TH also induces Na⁺/K⁺-ATPase and UCP3 protein expression by genomic and non-genomic signalling to increase skeletal muscle thermogenesis^87,88. In conjunction with the fibre type switch, TH also increases FAO in muscle cells by stimulating mitochondrial biogenesis and turnover via induction of PGC1α and mitophagy^89,90. Additionally, T₃ enhances insulin signalling in muscles by upregulating the expression of Slc2a4 (GLUT4) in both basal and hyperinsulinemic conditions⁹¹. In summary, TH increases glucose uptake and mitochondrial biogenesis, and induces the switch from slow to fast twitch fibres in skeletal muscle.

Fig. 3. Target tissues and metabolic effects of THs in rodents and humans.

THs exert multiple metabolic effects in rodent and human tissues such as the central nervous system (CNS), heart, liver, skeletal muscle, and brown adipose tissue.

Heart

THRα is the predominant THR isoform in the heart and mediates TH’s effects on gene expression in this organ. Significantly, mice lacking Thra gene have decreased basal heart rate confirming its key role in heart function⁹². Previous studies showed that circulating TH levels have profound effects on heart contractility, rate, and metabolism⁹³ (Fig. 3). TH positively regulates the contractile apparatus of cardiac myocytes’ by stimulating alpha-myosin heavy chain (α-MHC) and sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2) gene expression. In contrast, TH negatively regulates β-MHC and phospholamban (PLB) expression. The expression of voltage-gated potassium ion (Kþ) channels (Kv1.5 and Kv4.2), and the sodium/calcium ion (Na⁺/Ca²⁺) exchanger (NCX1) also are transcriptionally regulated by TH to maintain heart contractility⁹³. Recently, a study demonstrated that T₃ influences cardiovascular physiology by a noncanonical TRα-mediated mechanism by involving activation of PI3K and eNOS to cause endothelium-dependent vasodilation⁹⁴. Moreover, a non-DNA-binding THRα mutant also regulated basal heart rate to suggest that THRs may have additional actions besides direct transcriptional regulation⁹⁵.

The foetal and perinatal heart primarily undergo anaerobic glycolysis and then shift to FAO during the neonatal surge in serum T₃ concentration that occurs shortly after birth. This switch occurs concomitantly with TH-mediated inductions of cardiomyocyte proliferation and mitochondrial biogenesis, and a shift from alpha-myosin to b-myosin heavy chain gene expression⁹⁶. TH also stimulates gene expression of muscle-carnitine palmitoyl-transferase I (M-CPTI), pyruvate dehydrogenase kinase-2 (PDK2), and AMPK to promote the preferential usage of fatty acids over glucose⁹⁷. In summary, THRα plays a key role in TH’s effects on heart contractility, rate, and metabolism by its transcriptional regulation of target genes and its non-canonical actions involved in these functions. The neonatal surge in T₃ causes major changes in cardiomyocyte proliferation, metabolism, and contractile protein isoform expression.

Brown adipose tissue (BAT)

TH effects on adaptive thermogenesis

TH is required for proper BAT function and adaptive thermogenesis⁹⁸ (Fig. 3). BAT is primarily located in the interscapular region in mice and the supraclavicular and paravertebral areas in man⁹⁹. Intracellular T₃ concentrations in BAT are tightly regulated by DIO2¹⁰⁰. Norepinephrine and cold exposure increase the expression of DIO2 to elevate intracellular T₃ concentration and induce BAT activity and thermogenesis by stimulating UCP1 gene expression¹⁰¹. Both THR isoforms are expressed in BAT; however, THRα is the major THR isoform since dominant negative Thra mutant mice have increased body fat and deficient adaptive thermogenesis¹⁰². Further evidence that Thra is primarily involved in norepinephrine-induced BAT activity is the observation that Thra knockout mice have diminished BAT response to norepinephrine stimulation¹⁰³.

Central and peripheral regulation of BAT by TH

TH increases BAT activity in rodents and humans^100,104,105. Unlike WAT cells, BAT cells are derived from myogenic precursors rather than preadipocytes. Recent studies demonstrated TH has both central¹⁰⁶ and local regulation of BAT activity although their relative contributions to thermogenesis are not fully understood. Both sympathetic induction of Dio2 and increased circulating T₃ levels induce thermogenesis¹⁰⁷. Despite this controversy, it is clear that TH induction of mitochondrial biogenesis and mitophagy are necessary for sustained stimulation of BAT activity by TH¹⁰⁸. Regardless of whether TH’s central or peripheral actions on BAT are dominant, TH causes thermogenesis by directly regulating the gene expression of thermogenin/uncoupling protein 1(Ucp1), the protein responsible for disassociating ATP synthesis from chemiosmotic coupling of proton flow across the inner mitochondrial membrane¹⁰⁹.

In addition to stimulating BAT activity, TH increases both the differentiation and proliferation of progenitor cells¹¹⁰. A recent study in mice showed that loss of Dio3 leads to precocious exposure of T₃ in developing BAT and induction of selective epigenetic changes of genes involved in thermogenesis¹¹¹. Given TH’s ability to induce classical BAT activity and regulate adipose tissue plasticity, new TH-based interventions that target BAT are being investigated for the treatment of metabolic diseases and obesity¹¹². In summary, TH appears to regulate BAT activity at both central and peripheral tissue levels. Thus, TH and adrenergic regulation of Dio2 expression controls intracellular T₃ concentration and helps regulate the expression of Ucp1, thermogenesis, mitochondrial biogenesis, mitophagy, and BAT differentiation.

White adipose tissue (WAT)

T₃ regulation of glycolysis, lipogenesis, and lipolysis

TH regulates both adipogenesis and lipolysis in white adipose tissue WAT¹¹³ (Fig. 3). WAT and BAT arise from different cell lineages and TH appears to play an important role in the differentiation of both cell types. Although both THRα and THRβ isoforms are present in mature adipocytes, THRβ appears to be the predominant isoform since it is required for induction of genes involved in lipogenesis (for example, malic enzyme, fatty acid synthase), lipolysis (ATGL, DGAT2, CPT1b), glucose uptake (GLUT4) and glycolysis (PDK4) in control mice vs. adipose-specific Thrb KO mice¹¹⁴. TH induction of adipose triglyceride lipase (ATGL) and sympathetic activation of hormone sensitive lipase (HSL) gene expression regulate lipolysis in WAT; however, the co-ordinated regulation of these genes by TH to effect lipolysis is not well understood. Additionally, it is not known what causes preferential lipogenesis vs, lipolysis or vice versa under hypo- and hyperthyroid conditions in WAT although it is possible that T₃ regulation by leptin and/or dietary conditions could play an important modulatory role¹¹⁵. TH also has effects on glucose metabolism in WAT as TH stimulates both GLUT4 and glycolysis gene expression to increase fatty acid synthesis during conditions of carbohydrate excess¹¹⁴.

The role of TH status on plasma leptin levels is not clear as different findings have been reported^116,117. However, significantly more is known about the effects of leptin on TH action. In humans, circadian TSH levels track closely with leptin levels in normal subjects and are perturbed in patients with leptin deficiency¹¹⁸. In rodent studies, leptin acts on the arcuate nucleus to activate the melanocortin pathway and stimulate TRH release from TRH neurons located in the paraventricular nucleus¹¹⁹. Thus, serum T₄ and T₃ fall during starvation or leptin deficiency due to the development of central hypothyroidism via this pathway¹¹⁹. The regulation of TH action by WAT and leptin is an excellent example of nutritional status and adiposity (leptin) playing co-ordinated and integrated roles in directing the hypothalamus-pituitary-thyroid axis to modulate the circulating levels of TH to regulate BMR and cellular metabolism throughout the entire body.

T₃ regulation of WAT browning

Certain stimuli such as exercise, cold exposure, and TH stimulate the differentiation of white adipose tissue to beige/brite adipose cells that are thermogenic and express UCP-1^120,121. These cells are located in specific depots of subcutaneous WAT¹²². Currently, little is known about the mechanism of browning by TH. Deletion of p38 MAPK activator (MKK6) leads to increased T₃-mediated thermogenesis in WAT¹²³. THs also induce browning of WAT via the central effects on the hypothalamus¹²⁴. Interestingly, one report claimed that TH-induction of browning did not increase thermogenesis or glucose utilization¹²⁵ so the precise contribution of browning to T₃-induced thermogenesis still is not known.

Brain

The brain normally relies upon glucose as its primary source of fuel although it can metabolize ketones during starvation. TH effects on glucose utilization in peripheral tissues, and on gluconeogenesis and β-oxidation in the liver during fasting conditions modulate the availability of these fuel substrates to the brain.

Although TH has direct actions on the metabolism of many tissues⁶¹, several studies have suggested that it may regulate energy metabolism via central actions on the hypothalamus¹²⁶ (Fig. 3). Indeed, TH appears to have central effects on food intake¹²⁷, BAT thermogenesis¹²⁸, glucose homeostasis¹²⁶ and insulin sensitivity¹²⁶. More recently, Martínez-Sánchez et al., showed that central T₃ action regulates de novo lipogenesis in liver and FAO in BAT through the parasympathetic (PSNS) and sympathetic nervous (SNS) systems¹⁰⁶. Additionally, TH induction of AMPK activity stimulates BAT thermogenesis via two signalling pathways in the ventromedial nucleus of the hypothalamus (VMH): ceramide-induced endoplasmic reticulum (ER) stress and lipogenesis by c-Jun N-terminal kinase (JNK) activation, respectively¹⁰⁶. In this connection, T₃ stimulation of the hypothalamus potentiates SNS output to limit weight gain from an obesogenic diet¹²⁹. Metabolism in other parts of the developing and adult brain are regulated indirectly by TH by virtue of its effect on mitochondrial respiration. In particular, TH is essential for mitochondrial biogenesis in the developing neocortex and cerebellum of rats, and may help explain some of the neurodevelopmental abnormalities associated with congenital hypothyroidism¹³⁰. Similarly, TH directs neural stem cells (NSCs) towards a neuronal phenotype in the mouse subventricular zone (SVZ) in the adult hippocampus through its effects on mitochondrial metabolism¹³¹. Therefore, TH not only regulates peripheral metabolism via its effect on CNS but also mediates several aspects of neuronal metabolism required for their differentiation and survival in the brain.

Pharmacological applications of TH, TH-metabolites and TH-analogs

Levothyroxine replacement is frequently used to treat patients with hypothyroidism. Currently, the replacement levothyroxine dose for patients is primarily dictated by measurement of serum TSH levels. However, the identification of tissue-specific biomarkers suggests there can be variable TH function in different tissues, particularly during early transitions between thyroid states and in metabolic diseases^132,133. Thus, it is possible that TH or thyromimetics that target specific tissues or THR isoforms need to be titrated to both TSH and tissue-specific biomarker levels¹³⁴. These issues notwithstanding, there are several major conditions that are candidates for targeted TH or thyromimetic therapy such as:

Hypercholesterolemia

Hypercholesterolemia is a lipid disorder with elevated circulating low-density lipoprotein-cholesterol (LDL-C). It is a known risk factor for coronary artery disease and stroke. Initial studies showed there was an inverse relationship between TH status and serum LDL-C levels, and high LDL-C levels in hypothyroid patients were corrected successfully by TH supplementation¹³⁵. Liver-selective TH analogs for example, L-94901, CGH-509A, CGS-23425, T-0681 and GC-1 also effectively lowered serum levels of LDL-C in animal studies¹³⁶. The THRβ-specific analog, KB2115 (eprotirome) decreases serum levels of LDL-C, TAGs, and lipoprotein(s) in patients on maximal statin treatment¹³⁷. Unfortunately, further development of this thyromimetic was terminated due to its adverse effects on cartilage in preclinical studies¹³⁷. More recently, the newest generation of THRβ-specific TH analogues, MB0781 and MGL-3196¹³⁶ are being evaluated in clinical trials for the treatment of hypercholesterolemia.

Obesity

3-Iodothyronamine (T1AM) is produced by deiodination and decarboxylation of THs and exhibits anti-obesity effects in animal models¹³⁸. T1AM do not bind THRs but to a specific G-protein coupled receptor known as trace amine-associated receptor 1 (TAAR1) to exert most of its biological effects. In a recent study T1AM administration in animals was found to increase glucose and lipid utilization along with suppression in lipogenesis resulting in significant weight loss¹³⁸. Further, trials in humans with endogenous thyronamines, their metabolites and synthetic thyronamine-like derivatives¹³⁹ will be needed to establish them as an anti-obesity drug.

Non-alcoholic fatty liver disease (NAFLD)/Metabolic dysfunction-associated steatotic liver disease (MASLD)

NAFLD is now considered a hepatic manifestation of metabolic syndrome¹⁴⁰. NAFLD initially starts with steatosis but can progress to a more serious condition, non-alcoholic steatohepatitis (NASH), that can further lead to liver fibrosis and cirrhosis. NAFLD frequently co-exists with insulin resistance, coronary artery disease (CAD), and hypercholesterolemia. Its prevalence among adults currently is approximately 30% worldwide and has risen in parallel with the rapid global increases in type II diabetes and obesity during the past four decades¹⁴⁰. Unfortunately, there is no approved pharmacological treatment for NAFLD¹⁴¹. Several clinical studies have demonstrated associations between NAFLD and hypothyroidism and vice versa in humans¹⁴². A clinical study showed that low dose levothyroxine decreased hepatosteatosis in diabetic patients with NAFLD suggesting that TH might be useful to treat hepatosteatosis and reduce lipotoxicity in man (Fig. 1)¹⁴³. Furthermore, combined T₄/T₃ treatment significantly reduced hepatic steatosis, inflammation, and fibrosis in pre-clinical models of NASH^144,145. Interestingly, 3,5-diiodo-l-thyronine (T₂), a TH metabolite with weak affinity for THR, significantly reduced steatosis in animal models of NAFLD but the mechanism(s) is not known^146,147. Since the major THR isoform in the liver is THRβ, THRβ- and liver-specific thyromimetics have been developed and examined as potential therapies in rodent models of NAFLD. These compounds improve hepatic specificity and often reduce the untoward effects of T₃ on heart and bone, GC-1 and KB2115 reduced hepatic steatosis in rodent models of NAFLD such as ob/ob mice and rats fed high-fat diet (HFD)¹⁴⁸. Of note, newer thyromimetics such as VK2809/MB07811 and Resmetirom (MGL-3196)¹⁴⁹ which exhibit cell-selective transport into hepatocytes¹⁵⁰, showed very promising results for improving NAFLD/NASH in man. In particular, the Phase 3 MAESTRO-NASH Clinical Trial with Resmetirom showed robust improvement in both steatohepatitis and fibrosis to attenuate NASH progression and with relatively few downstream complications¹⁵¹. Resmetirom recently underwent fast-track evaluation and approval by the FDA for the treatment of NASH. Additionally, nanoparticle delivery of a THRβ-selective thyromimetic to the liver showed promising results in a mouse model of NASH and obesity, and suggest this may be an additional method to obtain liver-selective delivery of thyromimetics while reducing side effects¹⁵².

Neurological Disorders

Mutations in the TH transporter gene, MCT8 (SLC16A2 on chromosome Xq13.2) cause Allan-Herndon-Dudley syndrome, a condition characterized by profound intellectual and motor disability in man¹⁵³. Diiodothyropropionic acid (DITPA) and triiodothyroacetic acid (Triac) are two different TH analogues that improved the neurological phenotype in a mouse model of MCT8 deficiency¹⁵⁴. Results from a phase II clinical trial demonstrated that in both paediatric and adult patients with MCT8 deficiency, Triac appears to have decreased incidence of adverse effects such as thyrotoxicosis¹⁵⁵. X-linked adrenoleukodystrophy is a rare genetic disorder marked by adrenal insufficiency and central nervous system demyelination due to mutations in ABCD1 ATP binding cassette subfamily D member 1 (ABCD1), which encodes a transporter of very long chain fatty acids (VLCFAs) into peroxisomes to undergo oxidation. GC-1 (Sobiterome) induces the cerebral expression of ABCD2 transporter to promote VLCFAs uptake into cerebral peroxisomes and lower VCLFA levels in serum and peripheral tissues, and recently has been approved as an orphan drug for this condition¹⁵⁶.

Concluding remarks

THs play major roles in the regulation of metabolic and physiological processes such as basal metabolic rate, caloric intake, fat deposition, lipolysis and lipid utilization, reverse cholesterol transport, gluconeogenesis, and glycolysis depending upon the metabolic program of specific tissues and their response to the nutritional environment and hormonal state. Each of these physiological processes may require the co-ordination of multiple tissues that are directly or indirectly affected by THs or vice versa. For example, down-regulation of the HPT axis during starvation has downstream effects on TH action in peripheral tissues and BMR. Indeed, the entire HPT axis appears to be regulated by nutrient conditions and fat storage by virtue of leptin’s stimulation of TRH release in conjunction with its effects on appetite suppression. In addition to the global and interconnecting effects at the systemic level, TH has direct effects on the metabolism of key tissues such as liver, muscle, skeletal muscle, and both BAT and WAT. These effects on individual tissues also may be modulated by nutrition and TH status to promote their crosstalk with other tissues. For example, TH is the key driver for promoting fatty acid β-oxidation by stimulating autophagy, mitochondrial turnover, and transcription of genes involved in promoting β-oxidation and inhibiting glycolysis in BAT, heart, and liver. It also promotes gluconeogenesis and ketogenesis in the liver. The resultant glucose can be utilized by skeletal muscle and brain. On the other hand, during carbohydrate excess, the liver favors glycolysis and fatty acid synthesis with the excess triglycerides stored in fat droplets in white adipose tissue and liver. In skeletal muscle, TH stimulates glucose uptake and glycolysis in skeletal muscle and induces a switch from slow twitch fibers that preferentially use primarily fatty acids to fast twitch fibers that primarily use glucose as fuel.

Previously, lack or excess of TH was thought to correlate well with clinical signs and symptoms in patients. The treatment of hypo- and hyperthyroid disorders was considered relatively straight forward by titrating to circulating FT₄ and TSH levels. However, disparities in TH levels and actions in different tissues under certain clinical conditions may require measurements of metabolic profiles and tissue-specific markers of TH action to fully understand thyroid hormone status in individual patients¹³⁴, and will likely become a feature of optimal clinical management of TH status in the future, particularly if thyromimetics are used to treat metabolic diseases. However, understanding this complexity also presents new opportunities to harness and maximize the potential beneficial effects of TH and/or thyromimetics that selectively target particular tissues and/or THR isoforms, especially when TH and metabolic dysfunction occur in specific tissues. Thus, it may be possible in the future to employ tissue-specific delivery and isoform-specific targeting of thyromimetics and TH metabolites that have relatively minor side effects to treat metabolic conditions such as hypercholesterolemia, NAFLD, obesity, and CVDs. These new applications of THs and their related compounds will enlarge the therapeutical repertoire available to treat metabolic disorders. Moreover, it is likely that the current practice of using THs to maintain euthyroid status based upon serum TH and TSH levels may need to be expanded with a more refined and nuanced approach that enables targeted TH therapy or thyromimetics to treat TH hypofunction in specific tissues and/or correct dysfunctional metabolic conditions.