B. Role of Deiodinases

The intracellular action of TH is regulated by the amount of local T₃ available for receptor binding (90, 149). The iodothyronine deiodinases include two activating enzymes, D1 and D2, and one inactivating enzyme, D3, which are differentially expressed developmentally and in adult tissues. Developmentally, D3 is generally expressed first, followed by D2, and D1 is expressed last. D1 is expressed at high levels in the liver, kidney, and thyroid; D2 in brain, pituitary, thyroid, and BAT; and D3 in the skin, vascular tissue, and placenta. The deiodinase enzymes also differ in subcellular localization, with D1 and D3 expressed on the cell membrane and D2 in the endoplasmic reticulum. D1 is not essential for TH action in the euthyroid mouse as was studied in a D1 gene knockout mouse (220). D1 functions predominantly as a scavenger enzyme that deiodinates sulfated TH while being cleared in the bile and urine (220). D1, therefore, may be important for the adaptation to iodine deficiency and to diminish the impact of elevated thyroid hormone levels in hyperthyroidism. D3 is expressed in the placenta, where it can protect a developing fetus from excessive maternal TH, as well as in the skin and vascular tissue. D3 expression is stimulated in hypoxia as mediated by hypoxia-inducible factor (HIF-1) (236). All of the deiodinases require selenium for catalytic activity, and defects in the synthesis of selenoproteins can lead to abnormal thyroid hormone metabolism and defects in the hypothalamic pituitary feedback mechanism (65). Defects in the selenocysteine insertion sequence binding protein 2 (SECISBP2) is associated with a range of defects, including azoospermia, myopathy, and reduced T-cell proliferation (221). Nutritional selenium deficiency is also associated with reduced deiodinase activity (90).

D2 is the primary enzyme responsible for the rapid increases in intracellular T₃ in specific tissues as well as the primary producer of serum T₃ in humans (166). The D2 enzyme has a short half-life due to ubiquitination and proteasome degradation (8, 90). Deubiquitination, which increases D2 activity, is stimulated by adrenergic activation or by low levels of serum T₄ (91, 212). D2 is expressed in key thyroid-responsive tissues, such as brain, skeletal muscle, and brown fat, which preserves T₃ in these tissues as serum T₄ levels fall. The T₃ generated intracellularly by D2 is transferred to the nucleus and then regulates gene transcription. D2 activity is critical for the synergism of TH and signaling in regulating thermogenesis in BAT (233).

D2 activity has been shown in one study to be stimulated by bile acids, through activation of the G protein-coupled receptor for bile acids (TGR5) receptor, which potentially links TH action with bile acid signaling (269). Administration of bile acids to mice resulted in increased energy expenditure in BAT, prevented obesity, and improved insulin sensitivity. This action was independent of the FXR but required D2 gene expression. D2 and TGR5 are coexpressed in key metabolic tissues, and this may be relevant for the regulation of energy expenditure. Bile acids may have an action in addition to bile acid homeostasis to function more broadly in metabolism (269). The TGR5 receptor is expressed in human adipose tissue, and its expression was correlated with basal metabolic rate (246).

A further link of D2 to a metabolic phenotype has come through studies of associations with D2 gene polymorphisms. Polymorphisms in the D2 gene have been associated with type 2 diabetes, insulin resistance, and obesity in some (70, 174), but not all (42, 175), studies. In those studies with a positive association between D2 polymorphisms and metabolic disease, it is strongest when combining the D2 gene polymorphism with a polymorphism in a gene from another interacting metabolic pathway, such as the β3 adrenergic receptor (174), or PPARγ (70). These findings also support the importance of the interaction of TH signaling with other metabolic pathways.

A recent study in hypothyroid patients found that those with a specific D2 gene polymorphism had improvement of symptoms on T₄/T₃ combined therapy compared with T₄ monotherapy (192). This suggests that individuals with reduced T₄ to T₃ conversion due to D2 gene polymorphisms may benefit from the addition of the active form of TH, T₃, to their replacement therapy. Recently, a direct crossover comparison of monotherapy with T₄ to desiccated thyroid extract, which contains both T₄ and T₃, was made in hypothyroid patients (115). The serum TSH was kept in the reference range for patients while on both forms of thyroid replacement therapy. Patients, while on desiccated thyroid, had a modest, but significant, weight loss compared with the time period when they were on T₄ monotherapy.

C. Intracellular Transport

It had generally been assumed that thyroid hormone, due to its hydrophobicity, enters cells via passive diffusion. In vitro studies have identified multiple transporters with the ability to transport thyroid hormone, including the monocarboxylate and organic ion transporter families; however, the physiological significance was not known (130). On the basis of association of a transporter defect with a clinical disorder, it was shown that thyroid hormone transport is required in specific tissues, especially the brain (135, 265). The genetic disorder Allan-Herndon-Dudley Syndrome, with serum thyroid hormone abnormalities, low serum T, and elevated serum T₃, and severe neurologic deficits, was shown to be due to a mutation in the monocarboxylate transporter 8 (MCT8) gene. This proved an important role for the MCT8 transporter in normal human brain development. Thyroid hormone transporters are expressed in a specific temporal and spatial pattern in the developing brain (224, 256, 265). The MCT8 gene is located on the X chromosome (66, 84), and males with MCT8 gene mutations have neurologic abnormalities including dystonia and developmental delay, with progression to quadriplegia (18).

Mouse models of MCT8 gene knockout show thyroid function study changes similar to those in patients with Allan-Herndon-Dudley Syndrome, but modest changes in brain function (67, 255, 265). Mice likely have redundant thyroid hormone transporters, such that the MCT8 gene inactivation does not have the same consequences as is seen in humans. MCT8 is highly expressed in the hypothalamus, resulting in impaired central regulation and blunted thyroid hormone feedback when mutated (5). Without a functioning MCT8 transporter in the brain, specific brain areas become hypothyroid. Conversely, the liver remains sensitive to TH action when MCT8 is inactivated, such that the excess hormone produced due to impaired negative feedback in the hypothalamus results in tissue-specific hyperthyroidism and hypermetabolism and profound weight loss (114). Treatment with diiodothyropropionic acid (DITPA), in animal models and humans with inactivation or mutation in the MCT8 gene, results in reduction in serum TSH and serum T₃, and an improvement in hypermetabolism with weight gain and reduced caloric needs (60, 262). There was, however, limited improvement in cognitive or developmental delay in the human studies. The robust metabolic response to treatment that lowers serum T₃ in these patients shows the important role T₃ plays in hypermetabolism, but also that the tissues that mediate T₃ action in metabolism remain sensitive to TH even in the absence of the MCT8 transporter.

B. Integrating Signals Regulating TRH/TSH

The sympathetic nervous system (SNS) and TH regulate a number of metabolic processes in a complementary fashion (233). The earliest observations of central sympathetic nervous system regulation of TH action came from clinical management of patients with hyper- and hypothyroidism. Thyrotoxic patients have normal plasma norepinephrine (NE) levels, while hypothyroid patients have elevated plasma NE levels, perhaps to compensate for reduced adrenergic sensitivity (48). Epinephrine levels were not different in hyperthyroid or hypothyroid patients compared with normal (48). Direct measurement of epinephrine secretion shows no difference in hyperthyroid or hypothyroid patients (47). Follicular cells of the thyroid gland are also innervated by sympathetic fibers containing NE, which can influence the mitotic response to TSH stimulation (245). Catecholamines increase T₄ to T₃ conversion, by stimulating activity of a specific deubiquitinase that acts on the D2 protein, upregulates D2 activity, and increases T₃ levels in the nucleus (90). The synergism between the SNS and TH is best characterized in studies of facultative thermogenesis in BAT (207). The role of TH within the central nervous system is evolving and now includes alterations in neuroendocrine peptides with relation to energy intake, adipokines, nongenomic actions of TH within the hypothalamus, and the action of decarboxylated and deiodinated analogs of TH (163).

In rats, fasting has been shown to decrease pituitary D2 levels and liver D1 levels, and correlates with reduced peripheral T₃ isolated from liver homogenates (22, 23). Despite this reduction in pituitary and liver T₃, hypothalamic D2 activity is actually increased with fasting, resulting in an increase in the orexigenic proteins neuropeptide Y (NPY) and agouti-related peptide (AgRP) from the arcuate nucleus. Thus, despite fasting associated reductions in peripheral TH levels, there is still a localized increase in T₃ within the hypothalamus during fasting with a marked increase in orexigenic signals, which in turn act upon the paraventricular nucleus to decrease TRH production. This is thought to be the mechanism for most such patients having a normal serum TSH despite a reduced serum T₄ concentration. Humans who are anorexic or undergo severe caloric restriction exhibit similar reductions in TH levels, which likely functions to protect energy stores (148, 204, 268). The administration of leptin (51), or α-MSH (73), can abolish the fasting-induced reductions in TRH.

Leptin is an adipokine that circulates in both the free and bound forms, and the serum concentration is proportional to body fat content. While both leptin and TH regulate signaling in the arcuate nucleus and reflect changes in energy stores, data regarding the correlation between leptin levels and hypo- and hyperthyroidism are inconsistent. In hypothyroid and hyperthyroid patients, followed before and after treatment, leptin levels were elevated in hypothyroidism and reduced in hyperthyroidism, correlating with BMI and with TSH levels (190). Adipocytes and preadipocytes express the TSH receptor, and acute administration of recombinant TSH in thyroid cancer patients has been shown to have an acute stimulatory effect on serum leptin, and the increase was proportional to the fat mass (215). In an obese animal model, and obese humans, there is an increase in free leptin with an increase in BMI (118). Within the hypothalamus, leptin is a known regulator of TRH and TSH secretion via direct action on the paraventricular nucleus and indirect action on the arcuate nucleus (223). In the direct pathway, leptin stimulates TRH neurons by inducing signal transducer and activator of transcription (STAT)3 phosphorylation, and regulating prepro-TRH transcription. In the indirect pathway, leptin inhibits NPY and AgRP and stimulates proopiomelanocortin (POMC). The POMC product α-MSH stimulates CREB in the TRH neuron. However, in the obese state, there is a significant amount of leptin resistance in the arcuate nucleus of the hypothalamus such that the indirect pathway of leptin stimulation of TRH is not active. This leptin resistance allows for maintenance of euthyroidism in the setting of diet-induced obesity (195).

The role of T₃ in adrenergic-mediated thermogenesis is thought to be due primarily to direct actions on BAT tissue. A recent study, however, has supported an important central role for T₃ in stimulating adrenergic-mediated thermogenesis. Rats given T₃ systemically or administered centrally in the cerebral ventricles showed reduced TRH/TSH, significant weight loss despite hyperphagia, and increased BAT thermogenesis (164). Within the hypothalamus, T₃ treatment selectively reduced AMPK phosphorylation, which was colocalized with TRα within the hypothalamus, and reduced activity (164). This resulted in increased lipogenesis and sympathetic output to BAT with a net effect of increased thermogenesis and energy expenditure (164). This action was blocked by selective expression of a mutant TR in the ventral medial hypothalamus, establishing a significant central role for T₃ in thermogenesis. The importance of TRα in mediating local adrenergic action in vivo in white fat and BAT was previously shown using an isoform-selective agonist (207), and a TRα mutant mouse model (158).

The influence of TH on central regulation of the autonomic nervous system has recently been localized to a previously unknown population of parvalbuminergic neurons (PBN) located in the anterior hypothalamus (179). These neurons are required for regulation of cardiovascular function and ablation results in hypertension and temperature-dependent tachycardia. Both TR isoforms are required for normal development of these neurons in the hypothalamus.

Thyroid hormone influences appetite and feeding through several pathways. T₃-treated rats have reduced POMC expression, accumulation of malonyl-CoA, and inactivation of CPT1 in the hypothalamus, which should produce anorexia, but the rats were resistant to this signal and remained hyperphagic (164). The increased energy demand may override the anorexic stimuli (164). Local TH metabolism also plays a role in appetite regulation. In the arcuate nucleus, D2 expressed in glial cells increase T₃ production during fasting, which stimulates UCP2 and mitochondrial proliferation in orexigenic NPY/AgRP neurons and stimulates rebound feeding after food deprivation (46).

A. Basal Metabolic Rate

Basal metabolic rate (BMR) is the primary source of energy expenditure in humans, and reductions in BMR can result in obesity and weight gain (201). TH is a key regulator of BMR, but the targets are not clearly established (137). BMR correlates with lean body mass (132) and thyroid hormone levels (52, 230). Cold and heat intolerance are hallmark clinical features of patients with hypothyroidism and hyperthyroidism, respectively. In addition, resting energy expenditure (REE) is remarkably sensitive to TH, especially in athyreotic individuals (4).

TH stimulates BMR by increasing ATP production for metabolic processes and by generating and maintaining ion gradients (82, 104, 231). TH stimulates metabolic cycles involving fat, glucose, and protein catabolism and anabolism, but these are minor contributions to BMR. The two ion gradients that TH stimulates, either directly or indirectly, are the Na⁺/K⁺ gradient across the cell membrane and the Ca²⁺ gradient between the cytoplasm and sarcoplasmic reticulum. TH can alter the levels of Na⁺ within the cell and K⁺ outside of the cell, thus requiring ATP consumption in the form of Na⁺-K⁺-ATPase to maintain the gradient. In addition, TH directly stimulates the Na⁺-K⁺-ATPase, but this effect has more impact on BMR in hyperthyroidism than in euthyroid or hypothyroid individuals (44, 68, 126). TH also regulates the expression of the sarcoplasmic/endoplasmic reticulum Ca²⁺-dependent ATPase (SERCA) in skeletal muscle (235, 237, 284). Stimulation of the Ca²⁺-ATPase produces heat during ATP hydrolysis (57). TH increases the amount and activity of ryanodine receptors in heart and skeletal muscle, which then stimulates Ca²⁺ efflux into the cytosol, requiring more ATP to return the Ca²⁺ to the sarcoplasmic reticulum (131).

TH maintains BMR by uncoupling oxidative phosphorylation in the mitochondria (107), or reducing the activity of shuttle molecules that transfer reducing equivalents into the mitochondria (72, 109). In skeletal muscle, TH increases the leak of protons through the mitochondrial inner membrane, stimulating more oxidation to maintain ATP synthesis, since the proton-motive force driving ATP production is compromised. The presence of uncoupling protein (UCP) 2 and 3 in skeletal and cardiac muscle initially suggested that these proteins were mediators of the TH-stimulated proton leak. Further investigation revealed that TH treatment produced upregulation of UCP2 and UCP3, but this was not associated with changes in the proton gradient in human muscle (13). Clinically, when transitioning from hypothyroidism to euthyroidism, TH induced energy expenditure results in heat production without a significant increase in ATP generation. In hyperthyroidism, there is an increase in both ATP synthesis and heat production (108). T₃ also regulates the efficiency of ATP synthesis induction of mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), a shuttle enzyme that contributes to the generation of ATP by transferring reducing equivalents generated in the cytoplasm into the mitochondrial membrane. Mice homozygous for a GPD gene knockout have higher levels of T₄ and T₃ and impaired ability to maintain core body temperature, consistent with a defect in thermogenesis (63). T₃ induction of UCP3 in skeletal muscle may play a role in thermogenesis. In mice lacking beta 1, 2, and 3 adrenergic receptor (beta-less), which are cold intolerant, T₃ treatment during cold exposure resulted in maintenance of body temperature (77). T₃ treatment of UCP3 knockout mice, compared with wild-type, had slightly less thermogenesis, indicating that T₃ induction of UCP3 may be important for thermogenesis in some settings (77).

B. Facultative Thermogenesis

Homeothermic species have developed a nonshivering or facultative thermogenesis to maintain core body temperature after cold exposure and increase energy expenditure after eating. The primary site of this adaptive thermogenesis in rodents is in BAT (33). Both the SNS and TH are required for maintenance of core body temperature (234). Hypothyroid rodents develop marked hypothermia with cold exposure, and T₄ treatment reverses this via induction of BAT activity (35). Expression of UCP1 is required for BAT thermogenesis, and UCP1 is synergistically regulated by both NE and T₃. While T₃ and NE each increase UCP1 expression by 2-fold separately, there is a 20-fold induction of UCP1 when both agents are combined (19). The UCP1 gene contains several cAMP response elements (CRE) that enhance the responsiveness of adjacent TREs to T₃ (234). It is important to note that while TRβ regulates UCP1 expression in BAT, TRα mediates sensitivity to adrenergic simulation (207). This demonstrates TR isoform specificity in metabolic regulation within a single tissue, and both TR isoforms are required for a normal thermogenic response. A recent study showed that TH induced UCP1 expression in WAT via TRβ and increased both mitochondrial biogenesis and the oxygen consumption rate (151).

UCP1 expression is critical to BAT thermogenesis, although it is now well established that D2 activity, required for the local conversion of T₄ to T₃, is also essential (56). D2KO mice develop hypothermia and must rely on shivering to maintain core body temperature (56). In addition, with cold exposure, D2KO mice preferentially oxidize fat. They are resistant to diet-induced obesity and have normal glucose tolerance due to increased sympathetic tone. The evaluation of D2 knockout mice provided an important insight into difference between rodents and humans with respect to the thermoneutral temperature. Mice, raised at room temperature 22°C, activate heat production pathways since this is colder than their thermoneutral temperature of 30°C (36). When thermal stress is eliminated by raising D2 knockout animals to be raised in an environment at 30°C, they develop obesity, glucose intolerance, and hepatic steatosis that is the result of impaired BAT T₃-induced thermogenesis (36).

There is both visceral and subcutaneous BAT in humans, which may have specific functions that relate to the anatomical location (211). Until recently, human BAT was considered to be important in neonates, but not likely to be important in the adult. Recent studies utilizing PET and CT imaging have shown a significant amount of BAT, especially in the subscapular and chest region (50, 257). In general, there is more BAT in younger and leaner individuals, and it is induced by cold. Treatment with β-adrenergic blockers reduces BAT activity, due to the importance of catecholamines for the development and regulation of BAT.

The relative importance of BAT for metabolic regulation in adults remains controversial, although significant effort has been focused on agents that stimulate BAT activity in humans, as well as the ability to convert white fat to more metabolically active “beige” or brown fat (211, 264). A recent study showed that direct biopsy of adipose tissue from the supraclavicular areas, thought to contain BAT tissue, had increased oxidative capacity and increased expression of UCP-1 (263). Activation of BAT in humans has been reported after overnight exposure to 19°C compared with those exposed to 24°C (39). Resting energy expenditure, as well as functional imaging by PET scan, was performed to demonstrate BAT activation.

C. Skeletal Muscle

Skeletal muscle has been recognized as a key TH target for contractile function, regeneration, and transport as well as for metabolism and glucose disposal (237, 238). TH stimulation favors transition to fast-twitch fibers and transition to a faster myosin heavy chain (MHC) form. The significant regulation of D2 is a key factor that modulates T₃ levels in skeletal muscle. In skeletal muscle development and regeneration after injury, FoxO3 stimulates D2 expression (170). Skeletal muscle injury is associated with a twofold increase in local T₃ levels, not seen in D2 knockout animals. There has also been interest in the common Myf 5 expressing precursor cell for both skeletal muscle and brown adipose tissue (150). The zinc finger protein, PRDM16, directly represses white fat genes and activates brown fat genes (133). D2 levels are higher in slow-twitch compared with fast-twitch muscle fibers and are stimulated by hypothyroidism, but not by cold exposure (169).

A. Regulation of Cholesterol Synthesis

TH regulates cholesterol synthesis through multiple mechanisms. A major pathway is TH stimulation of transcription of the LDL-R gene resulting in increased uptake of cholesterol and enhanced cholesterol synthesis (162). This has been a major pathway of T₄-mediated cholesterol lowering after T₄ treatment of patients with hypothyroidism (139). Another regulator of the LDL-R gene is the sterol response element binding protein (SREBP)-2 (97). SREBP-2 is a member of a family of transcription factors that regulate glucose metabolism, fatty acid synthesis, and cholesterol metabolism. Specifically, TH induces SREBP-2 gene expression that in turn modulates LDL-R expression. In hypothyroid rats, SREBP-2 mRNA is suppressed, but this is reversed when T₃ levels are restored (226). This nuclear coregulation is further highlighted by the fact that several genes have a tandem arrangement of the TRE and SREBP response element (SRE) (282). Other nuclear hormone receptors, such as PPARα, have opposing effects on LDL and cholesterol synthesis (144), which underscore the role of nuclear crosstalk in TH regulation of metabolism (157). The isoform-specific induction of LDL-R highlights the role of T₃ action in the liver.

TH also reduces cholesterol through non-LDL receptor-mediated pathways. Mice with hypercholesterolemia due to LDL-receptor gene knockouts were treated with high dose T₃ or 3,5-diiodo-l-thyronine (T₂) (95). Under these conditions, the reduction in LDL-cholesterol was linked to reductions in apolipoprotein (apo) B48 and apoB11. Hepatic triglyceride production was increased. The high doses of T₂ used were associated with cardiac toxicity and increased heart weight, but these findings suggest mechanisms for T₃, in addition to stimulation of the LDL-receptor, for cholesterol lowering.

B. Cholesterol Efflux

Reverse cholesterol transport is a complex process that results in transfer of cholesterol to the liver for elimination as bile acids or neutral steroids. ATP-binding cassette transporter A1 (ABCA1) is required for high-density lipoprotein (HDL) assembly and carriage of esterified cholesterol back to the liver for excretion. ABCA1 utilizes two separate promoters that are responsive to LXR and SREBP-2, both of which increase ABCA-1 transcription (249). The LXR-response element (LXRE) also permits TR binding. Cotransfection of the human ABCA1 promoter and an expression vector for TRβ resulted in suppression of the ABCA1 promoter in the presence of T₃ (122). In addition, TR competes with LXR for binding, resulting in T₃-induced inhibition of ABCA-1 and decreased HDL levels (122). ABCA1 mRNA is induced by overexpression of SREBP-2, but is completely absent in hepatic cells that are SREBP-2 null (272).

C. Bile Acid Synthesis

The conversion of cholesterol to bile acids is required to maintain cholesterol homeostasis. This cholesterol clearance pathway is regulated by a number of nuclear receptors that control the expression of cholesterol 7-hydroxylase (CYP7a1), the rate-limiting step in bile acid synthesis (41). Human and murine CYP7a1 are regulated by different nuclear receptors and their ligands (157). In murine models of impaired TRβ action, LXR is induced with a high-cholesterol diet that stimulates CYP7a1 gene expression and bile acid synthesis (103, 110). LXR has no effect on human CYP7A1 mRNA levels (2); however, T₃ treatment reduces CYP7A1 mRNA and cholic and chenodecoycholic acid synthesis in human hepatocytes (69). While there is no role for LXR in human CYP7A1 expression, both PPARα and hepatic nuclear factor (HNF) 4α have response elements that are located in close proximity to the TRE. In addition, HNF4α positively regulates CYP7A1 gene expression while PPARα inhibits HNF4α activity resulting in lower CYP7A1 levels (194).

Bile acids are now recognized as a regulatory pathway, stimulating both the TGR5 membrane receptor and the nuclear receptor FXR, as well as other related nuclear receptors including VDR, PXR, and CAR (279). Bile acids bind the TGR5 receptor on enteroendocrine L cells in the small intestine, which stimulates production of the incretin GLP-1 improving insulin sensitivity and increasing satiety. In BAT, as previously described, bile acids bind TGR5 and stimulate expression of D2 increasing energy expenditure and promoting resistance to diet-induced obesity (251, 269). Bile acids combine with the nuclear FXR receptor and stimulate target genes regulating cholesterol and bile acid metabolism (279). A recent clinical study in both healthy and cirrhotic subjects revealed that bile acid synthesis correlated positively with energy expenditure, and postprandially, serum TSH decreased in both groups (188), suggesting that the bile acid serum level influences the thyroid pituitary axis set point.

Bile acids are increasingly linked to glucose homeostasis mediated by both the TGR5 and FXR receptors. Animals studies have shown that a TGR5 agonist, EMCA, increases intracellular ATP/ADP, stimulates GLP-1, and attenuates diet-induced obesity (252). Activation of FXR by bile acids also improves diabetes in animal models. An FXR knockout mouse model resulted in glucose intolerance and insulin insensitivity (280). Treatment of diabetic mice with a synthetic FXR agonist repressed hepatic gluconeogenesis and enhanced liver sensitivity to insulin (280). Bile acids, like thyroid hormone, impact the metabolism of lipids and glucose and are linked by activation of D2 in specific tissues.

D. Fatty Acid Metabolism

TH stimulates both lipolysis and lipogenesis, although the direct action is lipolysis with lipogenesis thought to be stimulated to restore fat stores (191). A time course study in rats carefully measured whole body lipid content and thermogenesis after T₃ treatment and concluded that the TH-induced lipogenesis is primarily to maintain fat loss that occurs with TH-induced lipolysis (191). Fatty acids produced from TH-induced lipolysis are the substrate for the increase in thermogenesis (191). T₃ regulation of these divergent metabolic pathways is subject to nuclear receptor crosstalk, ligand-binding, nutritional status, and competition for RXR heterodimers (157). TH plays a significant role in the conversion of preadipocytes to adipocytes (187).

Malonyl CoA production in the liver promotes lipogenesis and directly inhibits carnitine palmitoyl transferase (CPT)-Iα, which converts long-chain fatty acyl-CoAs to acylcarnitines for translocation from the cytosol into inner mitochondrial matrix where β-oxidation occurs (172). T₃ also induces the transcription of acetyl CoA carboxylase (ACC)-1, which generates malonyl CoA from acetyl CoA. ACC-1 is regulated by TR, LXR, and SREBP-1 (121). While LXR can directly stimulate ACC-1 (248), TR and SREBP1 must form a complex that stabilizes SREBP-1 on the binding site (275). SREBP-1 action is also enhanced by a PPARα agonist, which can potentiate SREBP-1c nuclear activity (142).

CPT-Iα mRNA and enzyme activity is greatly increased in the livers of hyperthyroid animals, and a functional TRE and CCAAT enhancer binding protein (C/EBP) are both necessary for T₃ induction of CPT-Iα (129). The PPARα and TR response elements are in close proximity on the CPT-Iα gene. A PPARα agonist can induce CPT-Iα mRNA and reduced serum triglyceride levels after high fat feeding (177). PPARγ coactivator PGC-1α enhances both PPARα and TR induction of CPT-Iα (281). In vivo studies of a mutant TRα mouse model demonstrated crosstalk between PPARα and T₃ signals in CPT-Iα regulation. The TRα-P398H mutant mouse model has impaired fatty acid oxidation because the mutant TRα occupies the CPT-Iα PPRE and inhibits PPARα-induced CPT-Iα expression (158). Another in vivo study demonstrated nuclear crosstalk via treatment with polyunsaturated fatty acids. These fatty acids induce hepatic TRβ expression and decreases both serum cholesterol and serum triglycerides; however, in the hypothyroid state, polyunsaturated fatty acid failed to induce TRβ, but stimulate PPARα expression, resulting in decreased serum cholesterol, but persistent hypertriglyceridemia (244).

Finally, the actual mobilization of lipid droplets into the hepatocyte, termed “lipophagy,” has been shown to be T₃ regulated (239). Impairment of this process is associated with hepatic steatosis and insulin resistance (274). T₃-mediated autophagy is tightly coupled with β-oxidation to promote ketosis, is T₃ dependent, and in the unliganded state is repressed by NCoR (241).

E. Hepatic Steatosis

Nonalcoholic fatty liver disease (NAFLD) is associated with diminished thyroid action. The TRα mutation analogous to a resistance to thyroid hormone (RTH)-associated mutation in TRβ, TRα-P398H mutant, with impaired fatty acid metabolism, also had evidence of hepatic steatosis (158). There is significant evidence for nuclear hormone crosstalk in the development and treatment of hepatic steatosis. In fact, subclinical hypothyroidism, with TSH levels in the upper normal range, were found to be associated with NAFLD, with greater TSH elevations correlated with more extensive steatosis (43). In a gene expression array study of human hepatic steatosis samples, there was downregulation of T₃-responsive genes in the steatosis samples compared with normal liver (198). In a diabetic rat model, a TRβ selective analog was effective at reducing hepatic steatosis (32). These findings suggest that NAFLD is associated with impaired TH signaling. A recent study showed that a general, GC1, and liver-selective TRβ agonist, KB-2115, reduced hepatic steatosis, but both impaired insulin sensitivity by different pathways (260). GC1 treatment was associated with increased endogenous glucose production and KB-2115 with reduced insulin-stimulated glucose uptake in skeletal muscle due to reduced GLUT4 expression.

Fibroblast growth factor (FGF)-21 is expressed primarily in the liver, adipose tissue, and pancreas and is regulated by T₃ in a PPARα-dependent manner (1). FGF-21 stimulates glucose uptake in fat and enhances mitochondrial oxidation through AMPK activation. Transgenic mice overexpressing FGF21 in liver have reduced plasma triglyceride concentrations and are resistant to weight gain after high-fat feeding. In addition, treatment with FGF21 in diet-induced obesity mice led to increased β-oxidation, improved serum lipid concentrations, and decreased hepatic triglycerides (136). FGF21 expression is known to be downstream of the nuclear receptor PPARα, and fibrate treatment, the PPARα ligand, causes an increase in FGF21 expression in rodents (12). T₃ treatment in mice acutely induces hepatic expression of FGF21, but this induction is abolished in PPARα knockout mice (1).

In a study of healthy adults, 12 h of cold exposure at 24 or 19°C resulted in an increase in plasma FGF21 and enhanced lipolysis and energy expenditure (152). In a study of serum FGF-21 levels in obese youth with steatohepatitis, FGF-21 levels were elevated compared with control and correlated with hepatic fat content (94). In these patients with obesity and liver damage, the elevated FGF-21 levels may not be adequate to increase energy expenditure, although this was not directly studied.

One of the mechanisms that links the metabolic syndrome with hepatic steatosis is insulin stimulation of lipogenesis, which can lead to fatty liver and worsening insulin resistance, leading to greater stimulation of lipogenesis (182). The lipogenic transcription factor, SREBP-1c, is a mediator of this cycle and is itself influenced by a range of nuclear receptors, including CAR, TRβ, LRH-1, ERα, and FXR/SHP (182). Nuclear receptors have the potential to suppress SREBP-1c, which is a pathway that promotes insulin sensitivity.

A. Gluconeogenesis

It has been previously established that T₃ stimulates gluconeogenesis, especially in the hyperthyroid state, and that hypothyroidism is associated with reduced gluconeogenesis (45). Treatment with T₄ increases alanine transport into hepatocytes, increasing production of metabolic intermediate of the gluconeogenic pathway and ultimately conversion of alanine into glucose (240). Evaluation of T₃ treatment on target genes in the liver reveals that there is an increase in genes regulating glycogenolysis and gluconeogenesis (75). Specifically, regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting step in gluconeogenesis, is critical for glucose homeostasis and has been shown to be regulated by TRβ and CCAAT enhancer-binding protein in the liver (193). In thyrotoxic rats, hepatic PEPCK mRNA was stimulated 3.5-fold, and they were resistant to insulin suppression of hepatic glucose production compared with euthyroid rats (141). The hepatic insulin resistance was mediated by sympathetic stimulation. In a follow-up study, administration of T₃ to the hypothalamic paraventricular nucleus, through interaction with the SNS, increases glucose production via sympathetic input to the liver, showing that the T₃ effect was central (140).

Rodents with the RTH-associated Δ337T mutation in TRβ, conferring a dominant negative TR, have impaired gluconeogenesis with lower levels of PEPCK mRNA compared with wild-type (214). These animals are more sensitive to insulin and became hypoglycemic after an insulin injection that did not produce hypoglycemia in the wild-type animals.

B. Insulin Production and Action

Several studies have linked thyroid hormone action with pancreatic islet cell development and function. Pancreatic islets contain TRα1 and TRβ1 which are important for normal islet development (3). T₃ acts by stimulating the islet transcription factor Mafa. T₃ is required for the transition of islets to glucose-responsive insulin-secreting cells. In pancreatic islet cells studied in culture, T₃ and TRα promote proliferation (87). Inactivation of D2 gene is associated with insulin resistance and diet-induced obesity (168). Thyroid hormone acts to impair glucose-stimulated insulin release, despite increased islet glucose utilization and oxidation. Hyperthyroidism and high-fat feeding result in significant impairment of islet function (85). In contrast, physiological T₃ treatment prevents streptozocin-induced islet deterioration and maintains islet structure, size, and consistency (261). T₃ induces these anti-apoptotic effects via nongenomic activation of the AKT signaling pathway.

Hepatic glucose output is increased in hyperthyroidism due to increased gluconeogenesis. The rates of insulin-stimulated glucose disposal in peripheral tissues, therefore, must be altered to maintain euglycemia. In the hyperthyroid state, skeletal muscle glucose uptake is increased to overcome a depletion in glycogen stores (62). A TRE has been characterized in the promoter region of the GLUT-4 gene (271). T₃ treatment in rats increased GLUT-4 mRNA in skeletal muscle and, possibly through posttranscriptional splicing, and also augmented the levels of GLUT-4 protein in skeletal muscle. Similar results were reproduced in Zucker rats, but a notable finding was that T₃ treatment reversed hyperinsulinemia, but not hyperglycemia, in obese animals (254).

A study in rats supports a dissociation of thyroid hormone effects on BAT thermogenesis from glucose uptake and control (171). This group had previously shown in the streptozotocin (STZ)-induced uncontrolled diabetic rat that intracerebroventricular administration of leptin returned glucose to normal, restored BAT glucose uptake, and normalized serum T₄ and BAT UCP1 mRNA levels (92). Treatment of the STZ rat with T₃, or the selective TRβ agonist GC1, however, stimulated energy expenditure, but did not increase BAT glucose uptake (171). Although thyroid hormone can influence glucose levels, the primary action in the context of this rat diabetes model was on energy expenditure, which did not influence the hyperglycemia.

C. Thyroid Status and Diabetes

The interaction of thyroid status and diabetes is complex. Patients with type 1 diabetes have an increase in prevalence rates of autoimmune thyroid disorders compared with the nondiabetic population, especially among women (267). This is thought to be due to similar genetic susceptibility to both autoimmune conditions (253). Studies investigating the interaction of type 2 diabetes and thyroid dysfunction, however, have not shown a consistent association (61, 99, 124). Abnormal serum TSH concentrations were seen in ~30% of poorly controlled type 2 diabetic patients (37). Among those patients with an abnormal low or high TSH levels, who were negative for thyroid autoantibodies, serum TSH normalized in all but one patient when their glucose level was controlled for ~2 mo (37). Conversely, in severely thyrotoxic patients, the calculated metabolic clearance rate of insulin is markedly higher than control patients, contributing to hyperglycemia in the thyrotoxic state (200). In a recent case report, a patient with severe insulin resistance improved dramatically after suppressive dose levothyroxine for thyroid cancer (242). Imaging of the patient when hypothyroid and then after replacement was restored showed induction of BAT, highlighting the role of TH in insulin sensitivity and energy expenditure.

A. Integrating Mechanisms of Thyroid Hormone Regulation of Metabolism

TH directly regulates metabolic rate, body weight, and cholesterol metabolism (Table 2). TH regulates the expression of target genes directly through TR binding to specific TREs, as well as nongenomic modification of cell signaling. New evidence highlights the coordinate roles of central and peripheral regulation of TH in modulating metabolic pathways. TH interacts with the SNS in a synergistic and complementary fashion to maintain homeostasis. In addition, adipokine and neuropeptide regulation of the HPT axis and thermogenesis integrates information on energy availability, storage, and utilization to gauge the regulation of appetite, basal metabolic rate, and body weight. TR nuclear hormone crosstalk with other metabolic pathways, especially the nuclear receptors PPARα, LXR, and PGC-1α, is essential for the T₃ regulation of cholesterol metabolism and transcription of lipogenic and lipolytic genes. Bile acid stimulation of D2 and local thyroid hormone activation is another unexpected signaling link. Interference with thyroid hormone signaling is associated with obesity and hepatic steatosis. Finally, emerging evidence identifies a role for TH in glucose metabolism including actions in pancreatic islet development, gluconeogenesis, and insulin signaling.

Address for reprint requests and other correspondence: G. A. Brent, Dept. of Medicine, 111, VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd., Los Angeles, CA 90073 (e-mail: ude.alcu@tnerbg).