**4. Mitochondrial uncoupling proteins (UCPs)**

278 Thyroid Hormone

metabolic efficiency *in vivo*.

mitochondrial targets still remained enigmatic.

**3. Nuclear / mitochondrial targets of TH** 

evidence in support of 'mitochondrial uncoupling' has however indicated increase in oxygen consumption and mitochondrial proton permeability of isolated liver mitochondria derived from hyperthyroid rats, with concomitant decrease in mitochondrial phosphate potential and inner mitochondrial membrane (IMM) potential (9, 10). These observations were further corroborated by increase in liver oxidizing capacity of hyperthyroid rats accompanied by decrease in phosphate and cytosolic redox potential (11), while opposite effects were reported in livers of hypothyroid rats (12). Similarly, hepatocytes isolated from T3-treated rats show higher oxygen consumption and lower IMM potential as compared with non-treated control (13-16). Also, a decrease in IMM potential has been reported in TH-treated human lymphocytes or those derived from hyperthyroid patients (17). Overall, these findings suggested that TH indeed induces mitochondrial uncoupling, and that mitochondrial uncoupling may account for the cellular mode-of-action of TH in modulating

Concomitantly with the proposed mitochondrial paradigm of TH, others have proposed non-mitochondrial activity of TH in modulating metabolic efficiency. Thus, TH was claimed to induce "futile substrate cycles", namely, opposing energy-requiring metabolic pathways that proceed simultaneously without generating net products, e.g. glycolysis accompanied by gluconeogenesis, lipolysis with lipogenesis (18, 19), Na+/K+ ATPase with concomitant Na+/K+ leakage (13), or glycerol-3- phosphate/NADH mitochondrial shuttling (20). However, these proposed mechanisms could account for only a small fraction (about 15%) of the total increase in oxygen consumption induced by TH (13, 21), resulting in a mitochondrial paradigm consensus for TH-induced calorigenesis. Yet, the concerned

TH effects in the mitochondrial context may consist of long-term effects that are dependent upon gene expression, and short-term effects that are refractory to inhibitors of protein synthesis. An important finding reported by Tata et al (2), indicated that the calorigenic activity of TH was abrogated by actinomycin D, implying that TH-induced mitochondrial uncoupling is mediated by modulating nuclear gene expression of respective mitochondrial and/or extra-mitochondrial proteins. These results were followed by extensive studies ((22, 23) and others) that resulted in discovering the nuclear TH receptors (THR) of the superfamily of nuclear receptors, acting as target for TH in modulating nuclear gene expression. THR are encoded by two distinct tissue-dependent genes coding for splice variants of the THRbrain, bone, heart) and THRliver, brain, heart) isoforms. THR homodimers or heterodimers with other members of the superfamily of nuclear receptors (e.g., retinoic X receptor (RXR)) may interact with TH-response elements (TRE) in the promoters of TH-responsive genes, resulting in transcriptional activation or suppression as function of transcriptional co-activators or co-suppressors recruited by TH/THR to the transcriptional complex of respective promoters. Indeed, TH binding to nuclear THR results in direct transcriptional activation of the expression of genes coding for components of mitochondrial oxidative phosphorylation (i.e. βF1-adenosine triphosphatase, adenine With the discovery of UCPs, extensive efforts were invested in verifying their putative role in mediating the calorigenic effect of TH. In fact, the UCP-coding genes have TREs in their promoters and their expression level is increased by TH treatment, implying their putative role in mediating TH-induced calorigenesis (40). UCP1 (29) mediates proton leak in brown adipose tissue IMM (30), resulting in uncoupling fuel oxidation from ATP synthesis and in dissipating IMM potential as heat. The adaptive thermogenic response of UCP1 is driven by the sympathetic nervous system in response to cold temperature or high-energy cafeteria diet, and could apparently serve as target for TH in modulating total body energy expenditure. Indeed, recent findings by Lopez et al (31) have indicated that TH treatment results in suppressing hypothalamic AMP-activated protein kinase (AMPK) activity, resulting in SNS-induced thermogenic response of brown adipose fat. However, UCP1 is specifically expressed in brown adipose tissue, which is sparse in adult humans. While recent findings point to some brown adipose islets in adult humans (32-37), their putative impact on total body energy expenditure still remains to be resolved. Hence, other proteins that share sequence homology with UCP1(38), including the ubiquitously expressed UCP2, and in particular the UCP3 that is expressed in skeletal muscle, were pursued for their role in mediating TH-induced calorigenesis (39). However, the following observations may indicate that UCP2 and UCP3 may not account for TH-induced mitochondrial uncoupling (41). Thus, findings suggest that UCP2/3 do not contribute to adaptive thermogenesis (42), but may have a role in ROS signaling (43) and/or in exporting fatty acid anions from the mitochondrial matrix (44). Also, the expression of liver UCP2/3 proteins is restricted to Kupffer cells, implying that the uncoupling effect of TH in liver parenchymal cells is not due to UCPs. Most importantly, UCP3 knock-out mice are lean and show normal response to TH (45), leaving unresolved the specific proteins that may mediate TH metabolic effects in the mitochondrial context.

### **5. Mitochondrial permeability transition pore (PTP)**

In analogy to UCPs, mitochondria consist of Permeability Transition Pores (PTP) (46-50) located at the contact sites of the inner (IMM) and outer (OMM) mitochondrial membranes. The molecular composition and structure of mitochondrial PTP still remains to be resolved. The current model of PTP consists of the integral proteins ANT (in the IMM), the voltagedependent anion channel (VDAC, in the OMM), cyclophylin D (CypD, in the mitochondrial matrix) and the Bcl2 family of proteins (in the OMM). PTP gating may present itself in definitive or transient modes, differing in reversibility and synchronization (51, 52). Definitive synchronized PTP gating is induced by intramitochondrial Ca+2 load (53), and is enhanced by oxidative stress, depletion of adenine nucleotides, increased inorganic phosphate, increased matrix pH, and depolarization of the IMM (54-56). This opening/gating mode results in high-conductance PTP (HC-PTP), extensive depolarization of the IMM (~70% decrease in IMM potential), rapid passage of ions and solutes of less than 1500 Da across the IMM, and mitochondrial swelling. These may lead to rupture of the OMM, release of mitochondrial proapoptotic proteins (such as cytochrome c, apoptotic intrinsic factor), followed by programmed cell death/apoptosis (57). Alternatively, spontaneous, non-synchronized, transient/flickering PTP gating due to cyclic opening and closure of individual PTP channels may result in reversible and limited depolarization of the IMM (~30% decrease in IMM potential), moderate decrease in proton motive force, and passage of solutes of less than 300 Da, accompanied by mitochondrial contraction rather than swelling (58-63). Most importantly, in contrast to the irreversible proapoptotic depolarization inflicted by definitive PTP gating, transient low conductance PTP (LC-PTP) gating is innocuous and reversible, leading to mild mitochondrial uncoupling. These findings may indicate that LC-PTP may serve as mitochondrial target of TH in inducing physiological mitochondrial uncoupling and calorigenesis.

Thyroid Hormone and Energy Expenditure 281

mitochondrial IMM potential, with high ANT levels resulting in IMM depolarization and mitochondrial proton leak (71, 77, 79). Hence, in light of ANT structural and regulatory functions in the PTP context, and since the expression level of ANT2, the only ANT isoform expressed in liver, is increased in hyperthyroidism and decreased by hypothyroidism (80), TH-induced ANT expression could apparently account for liver TH-induced LC-PTP gating. However, over-expression of ANT2 in HeLa cell line or in rat primary hepatocytes resulted in extensive mitochondrial depolarization that was not inhibited by CSA (71), implying the formation of PTP-nonrelated ANT channels (81), or of CSA-insensitive PTP (82). Lack of an obligatory linkage between PTP and ANT conforms to other findings pointing to PTP gating

**Cyclophilin D (CypD):** CypD is a member of the family of peptidyl-prolyl cis-trans isomerases (PPIase) (83). The CypD protein contains a mitochondrial-targeting sequence that directs it specifically to the mitochondrial matrix. The link between CypD and PTP has been verified by CypD direct association with ANT (72) as well as by CSA inhibition of PTP gating due to its interaction and inhibition of CypD activity (64, 65, 84). Moreover, PTP opening by Ca+2 and oxidative stress was enhanced in isolated mitochondria of neurons over-expressing CypD (85), while CSA-sensitive PTP opening was abrogated in isolated mitochondria of CypD knock-out mice (86). These CypD characteristics may indicate that CypD could apparently serve as protein target of TH in inducing PTP opening and mitochondrial uncoupling. Indeed, liver mitochondria of hyperthyroid rats show increased expression of CypD as well as its PPIase enzymatic activity (71), while opposite effects prevailed in liver mitochondria isolated from hypothyroid rats. However, over-expression of CypD in HeLa cell line, or in rat primary hepatocytes, resulted in mitochondrial hyperpolarization rather than PTP opening (71, 87). Moreover, over-expressed CypD was found to desensitize cells to apoptotic stimuli or to protect cells from mitochondrial depolarization and apoptosis induced by over-expression of ANT1 (77, 78). Hence, THinduced CypD expression may not account for TH-induced PTP gating and calorigenesis. CypD induction by TH may reflect TH activity in inducing peptidyl-prolyl cis-trans

**Voltage Dependent Anion Channel (VDAC):** VDAC is a highly abundant protein of the OMM. Its primary function consists of exchanging anions between the cytosol and the intermembrane mitochondrial space (89). Previous findings have indicated its putative role in gating mitochondrial PTP (90-94). However, its expression level is not changed by *in vivo* TH treatment (71), excluding VDAC from being a molecular target of TH in inducing

The family of Bcl2 proteins consists of more than 20 proteins that were extensively studied in terms of their role in cell death (95). The Bcl2 family is grouped into two main subfamilies: proapoptotic proteins (e.g. Bax, Bak, and others) and anti-apoptotic proteins (e.g. Bcl2), which promote or inhibit PTP gating, respectively (95-97). Bcl2-family proteins

by proapoptotic ligands in liver cells or isolated mitochondria that lack ANT (76).

isomerase activity and protein folding rather than PTP opening (88).

**7. Modulation of Bcl2-family proteins by TH** 

mitochondrial uncoupling.
