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Thyroid hormone regulation of hepatic lipid and carbohydrate metabolism Rohit A. Sinha1*, Brijesh K. Singh1*, and Paul M. Yen1,2 1

Cardiovascular and Metabolic Disorders Program, Duke-NUS Graduate Medical School, 8 College Road, Singapore 169547, Singapore 2 Sarah W. Stedman Nutrition and Metabolism Center, Departments of Medicine and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27705, USA

Thyroid hormone (TH) has important roles in regulating hepatic lipid, cholesterol, and glucose metabolism. Recent findings suggest that clinical conditions such as non-alcoholic fatty liver disease and type 2 diabetes mellitus, which are associated with dysregulated hepatic metabolism, may involve altered intracellular TH action. In addition, TH has key roles in lipophagy in lipid metabolism, mitochondrial quality control, and the regulation of metabolic genes. In this review, we discuss recent findings regarding the functions of TH in hepatic metabolism, the relationship between TH and metabolic disorders, and the potential therapeutic use of thyromimetics to treat metabolic dysfunction in the liver. Thyroid hormone and metabolic homeostasis Thyroid hormone (TH, see Glossary) mediates important physiological processes such as development, growth, and metabolism [1,2]. Intracellular triiodothyronine (T3) is the active form of TH and binds to the thyroid hormone receptor (TR), which is a transcription factor that belongs to the nuclear receptor superfamily (Figure 1). The TR exists in two isoforms: TRa and TRb. The TRa isoform is highly expressed in the heart, muscle, and adipose tissue, whereas TRb is the predominant isoform in the liver [3]. The TR acts as a ligand-dependent transcription factor to regulate genes involved in the biological functions of TH (Figure 1) [4]. TH has profound effects on body weight, thermogenesis, and lipolysis; these effects are mediated primarily through the action of TH on skeletal muscle and adipose tissue [1]. TH can also regulate fatty acid (FA), cholesterol, and carbohydrate homeostasis through its actions on the liver (Figure 2) [1]. Thus, thyroid dysfunctions can result in intrahepatic as well as systemic dysregulation of the metabolism of nutrients that serve as important energy sources for cells and adversely affect hepatic lipid and carbohydrate metabolism [1]. In clinical hypothyroidism, patients gain weight owing to a decreased basal metabolic rate, particularly in muscle and brown fat [1]. Additionally, hypothyroidism is associated Corresponding author: Yen, P.M. ([email protected]). Keywords: thyroid hormone; lipid metabolism; glucose metabolism; liver. * Sinha, R.A. and Singh, B.K. contributed equally to this review 1043-2760/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tem.2014.07.001

with non-alcoholic fatty liver disease (NAFLD) [5], a condition that is estimated to occur in more than 30% of American adults, many of whom also have obesity and/or type 2 diabetes mellitus (T2DM) [6]. Hypothyroidism is also considered a risk factor for developing NAFLD because hypothyroid patients have an increased prevalence of NAFLD [5,7], and both hypothyroidism and NAFLD are associated with hyperlipidemia, obesity, and insulin resistance. In support of their mutual effects, studies have shown that

Glossary Carbohydrate metabolism: refers to the production, storage, and use of carbohydrates in cells to maintain energy homeostasis. It is highly conserved and is essentially glucose metabolism to control blood glucose levels. De novo lipogenesis: the process in which acetyl-CoA is converted to FAs, which are then esterified with glycerol to form TGs that are packaged in VLDLs and secreted from the liver. Gluconeogenesis: during starvation, glucose is generated from non-carbohydrate carbon substrates (such as pyruvate, lactate, and FAs) in the liver to maintain blood glucose levels. Hyperlipidemia: a medical condition characterized by abnormally high levels of lipid or lipoproteins in the blood. Hyperthyroidism: a condition in which the thyroid gland produces and secretes excessive amounts of the free thyroid hormones T3 and/or T4. Graves’ disease is the most common cause of hyperthyroidism. Hypothyroidism: a common endocrine disorder of underactive thyroid or low thyroid, in which the thyroid gland does not produce enough thyroid hormone. It causes several symptoms including tiredness, cold intolerance, and weight gain. Lipid metabolism: refers to the regulation of lipids in cells such as their degradation (lipolysis) or formation (lipogenesis). TGs and cholesterol are the major components of lipid metabolism. TGs are important for energy storage in adipocytes and muscle cells, whereas cholesterol is a ubiquitous constituent of cell membranes, steroids, bile acids, and as signaling molecules. Lipolysis: a highly regulated process of lipid breakdown that involves the hydrolysis of TGs into glycerol and FAs. Lipophagy: the autophagic degradation of cellular lipids, which involves the sequestration of lipid droplets and their degradation via lysosomal lipase. Non-alcoholic fatty liver disease (NAFLD): a condition in which fat accumulates in the liver independent of alcohol intake. It is generally related to metabolic disorders and is one of the major causes for fatty liver. Non-alcoholic steatohepatitis is the most extreme form of NAFLD and a major cause of liver cirrhosis. Nuclear receptor superfamily: a family of transcription factors, most of which directly bind to DNA in a hormone-dependent manner to regulate gene expression. b-Oxidation: a multi-step process of FA breakdown to form acetyl-CoA in the mitochondrial matrix, which subsequently enters into the tricarboxylic acid cycle. Reverse cholesterol transport (RCT): a process resulting in the net movement of cholesterol from peripheral tissues back to the liver via the plasma compartment. Thyroid hormone (TH): includes thyroxin (T4) and triiodothyronine (T3). T3 is a ligand for TRs, which modulate cell proliferation, development, and metabolism. TH is produced by the thyroid gland.

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Figure 1. Thyroid hormone signaling. Thyroxine (T4), the precursor of 3,5,30 triiodothyronine (T3), or T3 enters the cytoplasm via various thyroid hormone (TH) transporters (e.g., MCT8, MCT10, and OATP1C1). The activation and inactivation of TH occur through iodothyronine deiodinases (Dio1, Dio2, and Dio3), which constitute a subfamily of deiodinase enzymes. Dio1 and Dio2 convert prohormone thyroxine (T4) to the active hormone triiodothyronine (T3). Inactivation of TH occurs through Dio3, which converts T4 to the inactive reverse triiodothyronine (rT3) or converts active T3 to the inactive diiodothyronine (T2). Active T3 enters the nucleus and binds to TH receptors (TRs), resulting in the recruitment of co-activator complexes (Co-Act) with histone acetyl transferase (HAT) activity. The HAT activity of Co-Act increases acetylation of histone protein tails, thus creating a permissive chromatin environment, near TREs on the target gene, and promoting gene transcription. T3 trans-activates the expression of genes in nearly every vertebrate cell. TRs, in the absence of T3, recruit nuclear co-repressor complexes (NCo-R) with histone deacetylase activity (HDAC), which ultimately repress target gene transcription. Abbreviations: RXR, retinoid X receptor; TRE, thyroid hormone response element; MCT8, monocarboxylate transporter 8; MCT10, monocarboxylate transporter 10; OATP1C1, organic anion-transporting polypeptide 1c1.

key target genes of TH are downregulated in fatty livers of morbidly obese patients undergoing bariatric surgery [8]. Hypothyroid patients also commonly have hypercholesterolemia due to decreased hepatic low-density lipoprotein receptor (LDL-R) expression, which leads to an impairment in cholesterol uptake [9]. This, in turn, leads to a decrease in cholesterol turnover and excretion, and a marked increase in serum apolipoprotein B (ApoB) [10], total cholesterol, and LDL cholesterol levels. A decrease in the clearance of triglycerides (TGs) from plasma and an accumulation of intermediate LDL particles has also been observed in hypothyroid patients [10]. In hyperthyroidism, the basal metabolic rate of patients is increased and often accompanied by weight loss when caloric intake cannot keep pace with increased energy consumption [11]. However, serum total cholesterol and TG levels are decreased owing to increased clearance via the reverse cholesterol transport (RCT) pathway and hepatic b-oxidation is increased due to lipolysis from adipose 2

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Figure 2. Thyroid hormone regulation of fatty acid, cholesterol, and glucose metabolism. (i) Thyroid hormone (T3) may increase fatty acid (FA) uptake in the liver via the regulation of FA transporter proteins such as FA translocase (FAT; also known as CD36). T3 is known to increase hepatic lipogenesis by upregulating proteins including Spot14, FA synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), liver X receptor (LXR), carbohydrate-responsive element-binding protein (CHREBP), and malic enzyme (ME). Activation of hepatic lipases and lipophagy has been implicated in the intrahepatic lipolysis induced by T3. FAs released via lipolysis are shuttled into hepatic mitochondria for oxidation; this process is positively regulated via the T3 induction of carnitine palmitoyltransferase-1 (CPT1), sirtuin 1 (SirT1), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1a) and fibroblast growth factor-21 (FGF-21). (ii) Cholesterol uptake via low-density lipoprotein receptor (LDL-R) endocytosis is promoted by T3. Other proteins involved in cholesterol uptake, such as sterol regulatory element-binding protein-1c (SREBP-2) and scavenger receptor class B1 (SR-B1), are also induced by T3. Key genes involved in cholesterol turnover and bile production, such as cytochrome P450 7A1 (Cyp7A1; also known as cholesterol 7-alphamonooxygenase) and ATP-binding cassette sub-family G member 8 (ABCG5/8), are also induced by T3 to increase net reverse cholesterol transport. (iii) T3 bound thyroid receptor (TR) directly upregulate TR target genes and key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase 1 (PCK1) and glucose-6phosphatase (G6PC) in the liver. Increased deacetylation and activation of the master gluconeogenic transcription factor forkhead box O1 (FoxO1) by SirT1 is also modulated by T3 to increase gluconeogenesis. Apart from regulating gluconeogenic gene transcription, increased alanine transport and inhibition of insulin signaling may also contribute to TH-induced hepatic glucose production. + indicates positively regulated.

tissue [11]. In the hyperthyroid state, excessive hepatic lipid oxidation and oxidative phosphorylation can generate reactive oxygen species that cause hepatic tissue damage [12]. Indeed, hepatitis and liver failure have been reported to occur in uncontrolled and/or prolonged hyperthyroidism [13]. Additionally, hyperthyroidism can induce gluconeogenesis and glycogenolysis. It is a well-known clinical phenomenon that hyperthyroidism can induce hyperglycemia as well as worsen glycemic control in diabetic patients [14]. With the advent of new molecular biological methods, knockout and knock-in mouse models, and metabolic characterization of different physiological and genetic states, we are beginning to gain a much deeper understanding of TH actions on liver metabolism. In this review, we describe the molecular and intracellular mechanisms utilized by TH that impact the hepatic regulation of lipids and carbohydrates in normal and pathological conditions. These findings not only enhance our understanding of liver

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Review dysfunction associated with metabolic syndrome, but may also provide new tools for the diagnoses and treatment of NAFLD and related disorders. TH regulation of hepatic FA uptake Circulating free FAs (FFAs) generated through the lipolysis of adipose TG stores are an important source of lipid for the liver. FFAs are taken up by hepatocytes via protein transporters such as fatty acid transporter proteins (FATPs), liver FA binding proteins (L-FABPs), and FA translocase (FAT; also known as CD36). Studies have shown that knockdown of FATP2 and FATP5 protected against high fat diet-induced hepatosteatosis. Knock down of FATP5 also partitioned lipids towards skeletal muscle and heart, and away from the liver [15]. Similarly, upregulation of FAT in the livers of lean mice increased hepatic FA uptake, TG storage, and secretion. [16]. Additionally, L-FABP knockout mice were resistant to diet-induced hepatic TG accumulation [17,18]. Although FA transporters are transcriptionally induced by peroxisome proliferator-activated receptors (PPARs) [19], recent data suggest that FA transporters may also be regulated by TRs. Using radiolabeled FA infusion techniques, it was shown that FA uptake from TG-rich lipoproteins was increased by TH in a tissue-specific manner [19]. Hyperthyroidism also increased TG-derived FA uptake in all oxidative tissues except brown adipose tissue, whereas hypothyroidism increased TG-derived FA uptake in lipid-storing white adipose tissue and decreased its uptake in liver [20]. Moreover, hepatic FAT expression was suppressed in animal models of postnatal hypothyroidism [21]. Although these studies indicate that TH may be important in regulating FA uptake in different tissues such as the liver, the precise mechanism by which TH alters FATP activities needs further investigation. TH regulation of hepatic lipogenesis The synthesis of FFA from acetyl-CoA (de novo lipogenesis) is regulated through various nuclear hormone receptors, including liver X receptor (LXR), PPAR, and TR. TH regulates hepatic lipogenesis by stimulating the transcription of three key lipogenic transcription factors that are known to regulate the expression of genes involved in lipogenesis: sterol regulatory element-binding protein-1c (SREBP-1c) [22], LXR [23], and carbohydrate-responsive element-binding protein (CHREBP) [24]. TH can also directly increase the transcription of lipogenic enzymes such as acetyl-CoA carboxylase (ACC), malic enzyme (ME), fatty acid synthase (FAS), and Spot14 (S14) [25]. Liganded TR associates with co-activator complex on TH response elements (TREs) in the promoter region of these genes to enhance transcription. However, contrary to its positive regulation of most of the lipogenic genes, TH negatively regulates stearoyl-CoA desaturase-1 (SCD-1) via a TREindependent mechanism in mice [26]. Distinct abnormalities in hepatic lipid metabolism in the PV mutant mouse model, which mimics a TR mutation in patients with resistance to TH, have also been reported [27]. Interestingly, whereas livers from TRbPV mice had markedly increased lipid accumulation, liver mass and lipogenic gene expression were decreased in TRaPV mice. This

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result suggests that, at least in these mouse models, dominant negative TRb and TRa1 may act on different repertoires of genes with contrasting effects on hepatic lipid metabolism [28]. TH regulation of hepatic lipolysis and b-oxidation Although TH can stimulate lipogenesis, prolonged treatment leads to increased FA oxidation [29]. However, the mechanism for this switch is currently unknown. Most of the FAs used for oxidation are derived from FFAs from adipose tissue via TH-mediated lipolysis. FAs are also released from TG stores during hepatic lipolysis. Lipases hydrolyze lipids to form FAs and glycerol. TH does not seem to significantly alter the gene or protein expression of classic lipases in the liver, with the exception of hepatic lipase, which is transcriptionally upregulated by TH [30]. The effect of TH on adipose TG lipase in liver is presently unclear. TH can also increase the gene expression of zinca2-glycoprotein, a protein that contributes to lipolysis in hepatocytes [31]. Although it is not known whether hepatic lipases and zinc-a2-glycoprotein are direct targets of TR, these findings shed light on a relatively less-studied aspect of hepatic lipid hydrolysis, which might have a crucial role in both normal and diseased conditions [32]. Recent studies have shown that autophagy is a key process in hepatic lipolysis and lipid droplet degradation [33]. The resultant FFAs released after hydrolysis are catabolized by b-oxidation within nearby mitochondria. Moreover, TH was shown to increase lipophagy, a specific autophagic process used to traffic lipids to the lysosome, in hepatocytes [34]. In particular, TH significantly increased the number of lipid-laden autophagosomes and lysosomes in both human hepatic cells and mouse liver. Although the precise mechanism used by TH to induce hepatic lipophagy is not well understood, it is TR-dependent [33]. Additionally, blockade of autophagic flux in cell culture [34] and in vivo significantly reduced TH-induced ketogenesis, which is the final step in b-oxidation. Collectively, these findings suggest that lipophagy is a major mechanism for supplying FFAs for b-oxidation by TH [34]. Moreover, loss of lipophagy in TRbPV mice correlated with a diminished boxidation capacity and hepatosteatosis [27,34]. Thus, a decrease in lipophagy due to decreased intracellular TH levels or TH action may contribute to hepatosteatosis. Oxidation of FAs to acetyl-CoA occurs within mitochondria, peroxisomes, and the endoplasmic reticulum. FAs are activated by acyl-CoA-synthetase to acyl-CoA in the cytosol. This process is essential for enabling FAs to cross membranes and enter organelles. Short (aliphatic tails of fewer than six carbons in length) and medium-chain FAs (aliphatic tails of 6–12 carbons in length) pass the mitochondrial membrane without activation, whereas activated long-chain FAs are shuttled across the membrane by carnitine palmitoyltransferase-1 (CPT-1) [35]. The gene expression of CPT-1 and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), which leads to decreased glycolysis by phosphorylating pyruvate dehydrogenase, are positively regulated by TH. Recently, TR was shown to assemble with the histone deacetylase SirT1 and peroxisome proliferator-activated receptor gamma coactivator 1a (PGC-1a) on the TREs of their promoters [36]. SirT1 is 3

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Review crucial for maintaining normal hepatic lipid and glucose metabolism [37]. TH-bound TR can also increase the expression of fibroblast growth factor-21 (FGF21) in a PPARa-dependent manner [38]; FGF21 is a pivotal regulator of mitochondrial activity and b-oxidation in the liver [39]. However, more recent findings suggest that FGF21 levels may be regulated independent of TH action in vivo [40]. Finally, in addition to increasing substrate availability through increased lipolysis and CPT-1 expression, TH also increases the biogenesis of hepatic mitochondria by stimulating PGC-1a expression [41]. Collectively, these findings suggest that TH strongly induces coordinated lipid catabolism by stimulating lipophagy-mediated lipolysis and mitochondrial b-oxidation of FA. TH and regulation of cholesterol homeostasis Normal serum levels of TH are essential for maintaining a sufficient pool of cholesterol to meet the body’s requirements as well as for regulating the crucial steps of cholesterol synthesis, uptake, and metabolism [1]. TH regulates serum cholesterol levels by stimulating its hepatic synthesis, serum uptake, and intrahepatic conversion to bile acids. TH modestly induces 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG CoA) reductase and farnesyl pyrophosphate gene expression to promote cholesterol synthesis [42]. However, TH strongly induces the gene and protein expressions of LDL-R and ApoA1, which increase cholesterol uptake into the liver [43]. By contrast, patients with hypothyroidism typically have hypercholesterolemia and LDL accumulation in the liver, which can be normalized after TH replacement [9]. Apart from direct stimulation by TH, the LDL-R gene is subject to regulation by SREBP-2, which, in turn, is directly regulated by TH [44]. Additionally, the transcription of LDL-R-related protein 1 (LRP-1), a lipoprotein involved in the removal of chylomicron remnants and very LDL (VLDL), is increased by TH [45]. Recently, other LDL-R-independent mechanisms, such as increased expression of cholesterol 7a-hydroxylase (Cyp7A1), the rate-limiting enzyme in the synthesis of bile acid from cholesterol, or decreased ApoB protein levels, have been proposed to explain the TH-mediated reduction of LDL [46,47]. Furthermore, TH-induced secretion of cholesterol into bile acids is dependent on TH stimulation of ATP-binding cassette transporter G5/G8 (ABCG5/G8) complex gene transcription [48]. Finally, in addition to the transcriptional regulation of genes involved in cholesterol synthesis and bile secretion, TH may also utilize miRNAs to regulate these processes. Recently, miRNA-181d was shown to decrease the expression of caudal type homeobox 2 (cdx2), a regulator of sterol O-acyltransferase 2 (Soat2), a gene that is crucial for the hepatic secretion of cholesterol esters; this may represent a novel mechanism for TH to lower serum cholesterol [49]. Crosstalk of TR-mediated hepatic lipid metabolism with other nuclear receptor signaling pathways TR often forms heterodimers with retinoid X receptors (RXRs) to regulate target gene expression. However, because RXRs also heterodimerize with several other nuclear receptors, such as PPAR and LXR, TR heterodimerization with RXR can influence gene regulation by other nuclear 4

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receptors through their mutual competition for a limited pool of nuclear RXRs [25]. Hashimoto and Mori [50] demonstrated that the D337T TRb mutation, which diminishes TRRXR heterodimerization, promoted RXR interaction with LXR and induced the hypocholesterolemic effects of LXR. Similarly, the dominant-negative TRa P398H mutation, which still enables the mutated TR to heterodimerize with RXR, interfered with PPARa signaling and activation of its target genes to impair b-oxidation [51]. Interestingly, LXRRXR dimers negatively regulated TH signaling by downregulating the expression of hepatic deiodinase 2 [52]. Apart from competition among nuclear receptors to form heterodimers with RXR, there can also be competition for binding to common DNA response elements. In this regard, transcription of the ATP-binding cassette transporter A1 (A1), which is involved in cholesterol efflux from cells, is reciprocally regulated by the competitive binding of TR and LXR to a common direct repeat 4 (DR4) element located on its promoter [50]. Besides other nuclear receptors, direct interaction with nuclear co-repressors, such as silencing mediator for retinoid and thyroid hormone receptors (SMRT) [53] and nuclear receptor co-repressor (NCoR) [34,54,55], can also regulate hepatic lipid and cholesterol metabolism and potentially modify TH signaling. Moreover, distinct isoform-specific effects of TR were also attributed to have selective affinity to NCoR [28]. Thus, competition among nuclear receptors for RXR, common DNA sites, and co-repressors and/or co-activators, enables intricate crosstalk between nuclear receptor signaling pathways. Regulation of hepatic lipid metabolism via non-classical TH signaling Although most actions of TH are mediated by transcriptional regulation through nuclear TRs, there is evidence for a non-classical TH signaling pathway that does not require nuclear TRs [56]. The lipid-lowering effect of TH on FaO rat hepatoma cells, which are devoid of TRs, engages a nonreceptor-mediated mechanism involving both short-term stimulation of mitochondrial O2 consumption and longterm transcriptional effects on PPARs [57]. Additionally, in contrast to its suppressive effect on mouse SREBP-1c, TH appears to upregulate the human homolog via a nongenomic ERK-mediated pathway in HepG2 cells [58]. TH also increases b-oxidation in HeLa cells through a mechanism that involves an increase in cytosolic calcium and activation of 50 AMP-activated protein kinase (AMPK) [59]. A deeper mechanistic understanding of non-genomic TH signaling may help explain some of the acute effects of TH on hepatic lipid and mitochondrial metabolism, which have remained elusive so far. TH regulation of hepatic carbohydrate metabolism Although the role of TH in carbohydrate metabolism has been studied for nearly a century, the molecular and intracellular mechanisms of its regulation have only recently begun to be understood. Clinically, hyperthyroidism is associated with increased glucose production, absorption, and utilization, whereas hypothyroidism is characterized by decreased glucose utilization by peripheral tissues [1]. TH influences glucose metabolism peripherally

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Review through its actions on many organs, particularly the pancreas, muscle, adipose tissue, and the liver [60]. TH can also act centrally to modulate hepatic glucose production and insulin sensitivity via a sympathetic pathway connecting the paraventricular hypothalamus to the liver, without any changes in circulating glucoregulatory hormone levels [61,62]. Hyperthyroidism is known to stimulate hepatic gluconeogenesis by increasing alanine transport and its conversion to glucose. TH also directly increases the expression of both the rate-limiting enzyme in gluconeogenesis, phosphoenolpyruvate carboxykinase (PCK1) [63], and glucose6-phosphatase (G6PC) [64]. The TH analog GC-1 can activate transcription of these gluconeogenic genes [25]. Recently, several groups studied the effect of TH on hepatic glucose production in more detail. It was shown that SirT1 contributes to TH regulation of CPT-1, the acyl-CoA carrier protein that is important for b-oxidation of FAs [36,64]. SirT1 interacts with liganded TRb1 in vitro to promote its deacetylation and activation while enhancing ubiquitindependent TRb1 turnover. which is a common response of nuclear receptors (NRs) to ligand binding [36,64]. Similarly, it was recently reported that FoxO1 is crucial for the TH-mediated transcription of PCK1 and G6PC [65]. In vivo, FoxO1 siRNA knockdown markedly decreased the TH-mediated transcription of gluconeogenic genes in mice [65]. TH also was unable to induce FoxO1 deacetylation or hepatic PCK1 gene expression in TRb-null (TRb/) mice [65], suggesting that SirT1 deacetylation and activation of FoxO1 were TR-dependent. Collectively, these results raise the possibility that drugs targeting the SirT1 pathway in the liver may also modulate TH regulation of hepatic target genes involved in lipid and carbohydrate metabolism. TH and hepatic insulin resistance Glucose homeostasis in humans is regulated by insulin secretion from pancreatic b-cells and glucose metabolism by insulin-sensitive tissues such as the liver and muscle [66]. Insulin facilitates glucose utilization in peripheral tissues and suppresses hepatic glucose production (HGP). Insulin resistance is often associated with obesity and predisposes affected individuals to glucose intolerance and T2DM [67]. Clinically, hyperthyroidism is associated with impaired glucose tolerance, hepatic insulin resistance, and increased HGP [14,25]. One major connection between glucose metabolism and TR signaling is the stimulation of ChREBP by TH, a basic helix–loop–helix leucine zipper transcription factor that stimulates the expression of enzymes promoting lipogenesis in response to glucose and insulin [68]. Thus, TH can potentiate inappropriate HGP during insulin resistance as well as stimulate lipogenesis during hyperinsulinemia. The overall effects of TH on glucose tolerance and insulin sensitivity are complex and mixed [14,37,69,70], and some of its actions on other tissues may counteract, in part, the insulin resistance and glucose intolerance in the liver. For example, TH reduces body fat and increases mitochondrial oxidative metabolism in skeletal muscle, which can increase insulin sensitivity and glucose utilization while it promotes HGP [25].

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Isoform-and liver-specific TH analogs As mentioned earlier, the tissue-specific distribution of TR isoforms, TRa and TRb, provides a therapeutic opportunity for selective TH action in specific tissues such as the liver [71]. In particular, liver-selective or TRb-specific agonists can uncouple the beneficial effect of TH on hepatic and plasma lipids from its deleterious effects on heart and bone. In this regard, selective thyroid receptor modulators could potentially be used to treat major metabolic disorders such as obesity, NAFLD, hypercholesterolemia, and T2DM [72–74]. For example, treatment of leptin-deficient ob/ob mice with a TRb-selective agonist reduced plasma glucose and improved insulin resistance [25,73,74]. A liverselective pro-drug that is metabolized into a TH mimetic within the liver was also effective in lowering serum cholesterol and TG levels [75], suggesting that activating TR specifically in the liver may be a therapeutic approach towards improving lipid profiles. TH analogs, hypercholesterolemia, and RCT TH can decrease serum cholesterol levels in animals and humans, particularly in combination with statin therapy [76,77]. The primary mechanism for this beneficial effect is the enhancement of reverse cholesterol transport (RCT). Studies in rodents showed that TH and the thyromimetic compound CGS-23425 increased the plasma levels of ApoA1 [78], suggesting that TH may promote the synthesis of high-density lipoprotein (HDL) and influence the initial step of RCT. Additionally, TH and thyromimetics increased RCT by increasing the expression and activities of several key proteins involved in cholesterol metabolism, including the following: scavenger receptor class B member 1 (SRB1), which is responsible for the uptake of cholesterolenriched HDL; CYP7A1, which converts cholesterol into bile acids in the liver; and ABCG5/G8, which promotes biliary cholesterol excretion [75,79–82]. One of the first chemically synthesized TH analogs was 3,5-diiodothyropropionic acid (DITPA). Although beneficial effects were noted on cholesterol and weight, DITPA resulted in increased serum markers of bone turnover, suggesting that DITPA has a negative effect on bone [83]. Moreover, cholesterol-independent positive effects of thyromimetics have been observed in rodent models of atherosclerosis [84]. TH analogs and hepatic carbohydrate metabolism TRb-specific analogs have favorable effects on lipid metabolism; however, it appears that TRa may have some opposite actions to TRb with respect to metabolism. In contrast to TRbKO and TRbPV knock-in mice, which have increased hepatosteatosis, TRa knockout (Tra-null) mice were protected from hepatosteatosis and high fat dietinduced hepatic insulin resistance [85]. Thra-0/0 mice were also leaner, less sensitive to high fat diet-induced obesity, and had significantly decreased liver TG and cytosolic diacylglycerol levels. Along with these changes, insulinstimulated p-Akt/Akt ratios were also increased in these mice compared with wild-type mice. Interestingly, a similar phenotype was observed in TRaPV knock-in mice when compared with TRbPV knock-in mice [86]. Collectively, these findings suggest that development of a TRa antagonist may have therapeutic potential. However, it should be 5

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Review noted that knock-in mice expressing another mutant TRa with a decreased binding affinity for TH exhibited decreased metabolic rates and increased visceral fat [87]. These finding raise the interesting possibility that specific TRa mutations may have variable effects on the metabolic phenotype. Notably, patients with TRa mutations have recently been identified, and these patients show increased body mass index values and decreased metabolic rates [88,89]. In addition to TR isoform-specific or liver-specific analogs, TH metabolites may also have beneficial effects on hepatic metabolism. Recently, the diiodothyronine (T2) mimetic TRC150094 was developed for the treatment of heart failure, dyslipidemia, and diabetes. As TRC150094 has very low affinity for both TRa and TRb isoforms, it is thought to enhance the mitochondrial oxidative capacity in liver via the increased activity of complex V [74]. The development and use of TR-independent analogs opens new avenues for the treatment of metabolic disorders through their non-genomic actions. TH and analogs in treating NAFLD NAFLD is a major global health problem that is associated with obesity and insulin resistance [90]. Its pathogenesis remains poorly understood, and, currently, there are no approved drug therapies [91]. As mentioned earlier, there exists a strong association between hypothyroidism and NAFLD [7,92]. This association seems plausible because clinical thyroid dysfunction can lead to hyperlipidemia, obesity, and insulin resistance, all of which are major components of metabolic syndrome [93] and are implicated in the pathogenesis of NAFLD. In human NAFLD microarray studies, hepatic lipid accumulation caused the downregulation of a set of TH-responsive genes, including some that were involved in energy metabolism [8]. In rodent models of NAFLD, TH analogs were effective in reducing hepatosteatosis [71,94,95]. In this regard, TH induction of lipolysis via lipophagy and its concomitant increase in boxidation may help reduce TG loads in the liver [34]. However, because increased peripheral lipolysis occurring in adipose tissue and adverse cardiac side effects are matters of concern, receptor isoform-specific and hepatictargeted TH analogs have been developed [96]. Interestingly, TH analogs that are unable to bind TRs were also effective in reducing hepatosteatosis and in increasing hepatic lipid metabolism [97–99]. Thus, utilization of TH analogs, inhibition of unliganded TR action [85], or modulation of co-repressor activity [53,100] may be viable therapeutic strategies for treating NAFLD. Concluding remarks and future perspectives The mammalian liver has been a classic organ for studying TH action. TH regulates both hepatic lipid and carbohydrate metabolism in a multi-layered and complex manner (Figure 2). Our current understanding of the actions of TH on the liver has progressed from the early characterization of its physiological effects to the elucidation of the regulation of specific target genes and the complex molecular interactions involved in metabolic programming. In particular, we have begun to understand the network of hormonal stimuli, intracellular energy status, cell 6

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Box 1. Outstanding questions  What is the mechanism(s) by which TH activates SIRT1 to regulate lipid and glucose homeostasis?  What is the role of miRNAs in the TH regulation of hepatic lipid and carbohydrate metabolism?  How does TH signaling crosstalk with hepatic insulin signaling and how does it contribute to insulin resistance?  Is there intrahepatic TH resistance that triggers fatty liver development?

signaling pathways, and transcription factor crosstalk that are affected by TH regulation of carbohydrate and lipid metabolism in normal and pathological conditions. Given the global rise of metabolic disease, a better understanding of the physiological, metabolic, and molecular relationships between TH action and metabolism may lead to improved therapies for these diseases. Indeed, future studies in these areas (Box 1) may help determine whether NAFLD, insulin resistance, and dyslipidemia in subclinical hypothyroidism, or hyperglycemia in subclinical hyperthyroidism, warrant correction of serum or intrahepatic TH levels. Intrahepatic TH levels could potentially be assessed by measuring serum protein and metabolomic markers. Finally, a better understanding of TH action and hepatic metabolism may also lead to the development of new and safe thyromimetics that could be beneficial for the treatment of metabolic conditions such as hyperlipidemia, NAFLD, T2DM, and obesity. Acknowledgments This manuscript was supported by grant NMRC/CIRG/1340/2012 to P.M.Y. from the National Medical Research Council, Singapore.

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