**9. Thyromimetic agents and energy expenditure**

282 Thyroid Hormone

may directly interact with PTP components such as ANT (98-100) or VDAC (101), and when over-expressed or added to isolated mitochondria may specifically induce (e.g. Bax and Bak) (102-104) or antagonize (e.g. Bcl2) (105) PTP gating. Similarly, depletion of proapoptotic Bax or Bak results in failure of PTP gating (98, 105, 106), whereas Bcl2 inactivation results in definitive PTP gating triggered by oxidative stress (107). Thus, mitochondrial Bcl2-family proteins and their respective heterodimers (e.g., Bax/Bcl2, Bad/Bcl2) may apparently serve as candidate targets of TH in inducing mitochondrial uncoupling (108-110). Indeed, TH-induced PTP gating is accompanied by increase in mitochondrial Bax and Bak, together with decrease in mitochondrial Bcl2 content, whereas hypothyroidism results in opposite effects that are reversed by TH (71). Modulation of the mitochondrial content of Bcl2 proteins by TH is due to their specific translocation in/out of mitochondria, rather than reflecting modulation of their expression and total cellular content. Amplifying the ratio of mitochondrial pro- vs. anti-apoptotic proteins, results in robust decrease in mitochondrial Bax/Bcl2 heterodimer with concomitant increase in free Bax, leading to PTP gating by free mitochondrial Bax (111). Indeed, over-expression of Bcl2 protects against TH-induced mitochondrial PTP gating (71), implying that depletion of

mitochondrial Bcl2 by TH may account for TH-induced mitochondrial uncoupling.

**8. Extra-mitochondrial upstream signals that induce TH-induced** 

Since Bcl2-Bax hetrodimerization may depend on Bcl2(S70) phosphorylation state (112), mitochondrial Bcl2 depletion by T3 was further verified in terms of Bcl2(S70) phosphorylation profile. Indeed, concomitantly with decrease in mitochondrial Bcl2, T3 treatment results in decreased phosphorylation of monomeric mitochondrial Bcl2(S70) as well as of Bcl2(S70)-Bax heterodimer (113), indicating that mitochondrial Bcl2 depletion may reflect Bcl2(S70) dephosphorylation by TH. In pursuing kinases (e.g. PKA, PKC) or phosphatases (e.g. PP2A, PP2B/Calcineurin) reported to be involved in Bcl2(S70) phosphorylation (112, 114), neither PKA, PKC nor PP2A were found to mediate phosphorylation/dephosphorylation of Bcl2(S70) by TH (113). In contrast, dephosphorylation of Bcl2(S70) and the depletion of mitochondrial Bcl2 protein by T3 are both reversed by the FK506 inhibitor of PP2B, indicating that the TH effect may be mediated by activation of PP2B (113). Furthermore, added FK506 blocksT3-induced opening of PTP, indicating that dephosphorylation of Bcl2(S70) and its mitochondrial depletion by T3-activated PP2B may account for mitochondrial PTP opening by TH. Since TH-induced PP2B activation was not accompanied by increase in PP2B expression, PP2B activation was further pursued by searching for TH-induced increase in cytosolic Ca+2 (113). Indeed, Ca+2 activated PP2B has previously been reported to bind and dephosphorylate Bcl2(S70) (115, 116). Most importantly, T3 treatment resulted in pronounced increase in Ca+2, while Ca+2 chelation by BAPTA resulted in abrogating LC-PTP gating by TH, indicating that THinduced PP2B activity involved mobilization of intracellular Ca+2 (113). Indeed, T3-induced mobilization of intracellular Ca+2 has recently been reported to mediate a variety of non-

**PTP gating mediated by Bcl2-family proteins** 

genomic effects of TH (117, 118).

Increase in energy expenditure by TH has long been considered for treating obesity. Indeed, treating obesity by thyroid extracts was quite popular throughout the 20th century and well into the 1970s, being later abandoned due to severe side effects consisting of cardiac dysrhythmias, bone resorption / osteoporosis, electrolyte disturbances, and loss of lean body mass (128). Thus, a final ruling warning against the use of thyroid preparations for the treatment of obesity of euthyroid subjects has been issued by the FDA on 1978. Similarly to TH, treating obesity by uncoupling of mitochondrial oxidative phosphorylation by 2,4 dinitrophenol (DNP) has been introduced on 1933, but abandoned on 1938 due to fatal hyperthermia (129).

Thyroid Hormone and Energy Expenditure 285

of hypothalamic TSH, thyromimetics may suppress the production of endogenous TH, resulting in combined hypo- and hyperthyroidsm. These limitations, combined with our present view of the mode-of-action of TH in the mitochondrial context, may however point to an alternative strategy, namely synthesizing thyromimetics that may directly target

Long chain fatty acids (LCFA) have long been shown to induce mitochondrial uncoupling due to their protonophoric activity (81, 132) and/or PTP gating ((51, 133), and ref therein), implying a potential mitochondrial thyromimetic activity. However, the uncoupling activity of LCFA is confounded by their dual role as putative uncouplers of oxidative phosphorylation and as substrates for oxidation or esterification. MEDICA analogs consist of long chain dioic acids (HOOC-C(α′)-C(β′)-(CH2)n-C(β)-C(α)-COOH (n=10-14)) that are substituted in the αα′ or ββ′ carbons (134). MEDICA analogs may be thioesterified endogenously into their respective mono acyl-CoA thioesters (135), however, they are not esterified into lipids nor β-oxidized, thus dissociating between the substrate role and the

Similarly to TH, MEDICA analogs induce calorigenesis in animal models *in vivo*. Thus, treatment of rats with MEDICA analogs results in an increase in oxygen consumption accompanied by a decrease in liver mitochondrial phosphate potential and cytosolic redox potential, reflecting mitochondrial uncoupling *in vivo* (136). Furthermore, treatment of obese leptin receptor-deficient rats (*e.g.* Zucker, cp/cp) with MEDICA analogs results in increased oxygen consumption and food consumption together with weight loss, implying increased total body energy expenditure (137, 138). Also, the non-protonophoric mitochondrial activity of MEDICA analogs is similar to that of TH (71), in terms of promoting CSAsensitive decrease in phosphate and redox potentials with concomitant increase in oxygen consumption in cultured cells as well as *in vivo* (11, 16, 67, 139, 140), indicating that both MEDICA analogs and TH do converge onto LC/HC-PTP gating (11, 71). Indeed, similarly to TH, PTP gating by MEDICA analogs is mediated by modulating the profile of mitochondrial Bcl2-family proteins, resulting in decrease in mitochondrial Bcl2-Bax heterodimer with concomitant increase in mitochondrial free Bax (71, 113). However, different transduction pathways are involved in modulating the mitochondrial content of free Bax by TH or MEDICA analogs. Thus, dissociation of the Bcl2-Bax heterodimer by TH is driven by dephosphorylation of Bcl2(Ser-70) by T3-activated PP2B (113), whereas dissociation of the Bcl2/Bax heterodimer by MEDICA analogs is driven by dephosphorylation of Bad(Ser-112, Ser-155) (141). The decrease in phosphorylated Bad(Ser-112, Ser-155) results in its decreased binding to14-3-3 followed by its increased binding to mitochondrial Bcl2, resulting in Bax displacement and PTP gating (142, 143). Decrease in phosphorylated Bad by MEDICA analogs is due to suppression of the Raf1/MAPK/RSK1 and the adenylate cyclase/PKA transduction pathways, and their respective downstream targets Bad(Ser-112) and Bad(Ser-155) (141). Hence, the TH and MEDICA transduction pathways converge at their downstream Bax target but diverge upstream of the Bcl2/Bax heterodimer (Scheme 1). LC-PTP gating by MEDICA analogs may account for their

mitochondrial PTP while avoiding the TH/THR transduction pathway altogether.

putative uncoupling activity of natural LCFA.

thyromimetic calorigenic activity in *vivo*.

**Scheme 1.** PTP-induced calorigenesis by Thyroid Hormone and MEDICA Analogs

These early attempts were followed by rational drug design of synthetic structural analogs of TH that may avoid the lethal chronotropic cardiac effects of TH, while maintaining the beneficial effects of TH in the context of diseases of the Metabolic Syndrome (130, 131). Most efforts in that direction were invested in designing thyromimetics that selectively target the liver TH-receptor isoforms (THR) while avoiding the heart isoforms (THR). Tissue selectivity has been further pursued by designing thyromimetics that undergo selective hepatic uptake. These efforts have mainly resulted in thyromimetics effective in treating dyslipidemia, due to increased expression of hepatic LDL-receptors together with CYP7A1 / 7-alpha-cholesterol hydroxylase, resulting in enhancing hepatic uptake of LDL-cholesterol and its conversion into bile. Liver-specific thyromimetics were further found to induce the expression of the hepatic scavenger receptor SR-B1 that mediates reverse cholesterol transport. However, in contrast to the advances made in designing hypolipidemic thyromimetics, the efficacy of thyromimetics in treating obesity and obesity-induced diabetes type 2 still remains to be verified. Moreover, the use of thyromimetics for treating diseases of the Metabolic Syndrome involves potential harmful risks due to: a. The partial selectivity of thyromimetics for hepatic THR, resulting in positive chronotropic effects as well as enhanced bone and muscle catabolism induced by high-dose. Hence, the safety of hypolipidemic thyromimetics still remains to be verified in subjects suffering from congestive heart failure or coronary heart disease. b. Since THR regulates the feedback loop of hypothalamic TSH, thyromimetics may suppress the production of endogenous TH, resulting in combined hypo- and hyperthyroidsm. These limitations, combined with our present view of the mode-of-action of TH in the mitochondrial context, may however point to an alternative strategy, namely synthesizing thyromimetics that may directly target mitochondrial PTP while avoiding the TH/THR transduction pathway altogether.

284 Thyroid Hormone

**Scheme 1.** PTP-induced calorigenesis by Thyroid Hormone and MEDICA Analogs

These early attempts were followed by rational drug design of synthetic structural analogs of TH that may avoid the lethal chronotropic cardiac effects of TH, while maintaining the beneficial effects of TH in the context of diseases of the Metabolic Syndrome (130, 131). Most efforts in that direction were invested in designing thyromimetics that selectively target the liver TH-receptor isoforms (THR) while avoiding the heart isoforms (THR). Tissue selectivity has been further pursued by designing thyromimetics that undergo selective hepatic uptake. These efforts have mainly resulted in thyromimetics effective in treating dyslipidemia, due to increased expression of hepatic LDL-receptors together with CYP7A1 / 7-alpha-cholesterol hydroxylase, resulting in enhancing hepatic uptake of LDL-cholesterol and its conversion into bile. Liver-specific thyromimetics were further found to induce the expression of the hepatic scavenger receptor SR-B1 that mediates reverse cholesterol transport. However, in contrast to the advances made in designing hypolipidemic thyromimetics, the efficacy of thyromimetics in treating obesity and obesity-induced diabetes type 2 still remains to be verified. Moreover, the use of thyromimetics for treating diseases of the Metabolic Syndrome involves potential harmful risks due to: a. The partial selectivity of thyromimetics for hepatic THR, resulting in positive chronotropic effects as well as enhanced bone and muscle catabolism induced by high-dose. Hence, the safety of hypolipidemic thyromimetics still remains to be verified in subjects suffering from congestive heart failure or coronary heart disease. b. Since THR regulates the feedback loop

**Increased mitochondrial Bax**

**PTP opening**

**Mitochondrial uncoupling**

**Whole body energy expenditure, weight loss**

Long chain fatty acids (LCFA) have long been shown to induce mitochondrial uncoupling due to their protonophoric activity (81, 132) and/or PTP gating ((51, 133), and ref therein), implying a potential mitochondrial thyromimetic activity. However, the uncoupling activity of LCFA is confounded by their dual role as putative uncouplers of oxidative phosphorylation and as substrates for oxidation or esterification. MEDICA analogs consist of long chain dioic acids (HOOC-C(α′)-C(β′)-(CH2)n-C(β)-C(α)-COOH (n=10-14)) that are substituted in the αα′ or ββ′ carbons (134). MEDICA analogs may be thioesterified endogenously into their respective mono acyl-CoA thioesters (135), however, they are not esterified into lipids nor β-oxidized, thus dissociating between the substrate role and the putative uncoupling activity of natural LCFA.

Similarly to TH, MEDICA analogs induce calorigenesis in animal models *in vivo*. Thus, treatment of rats with MEDICA analogs results in an increase in oxygen consumption accompanied by a decrease in liver mitochondrial phosphate potential and cytosolic redox potential, reflecting mitochondrial uncoupling *in vivo* (136). Furthermore, treatment of obese leptin receptor-deficient rats (*e.g.* Zucker, cp/cp) with MEDICA analogs results in increased oxygen consumption and food consumption together with weight loss, implying increased total body energy expenditure (137, 138). Also, the non-protonophoric mitochondrial activity of MEDICA analogs is similar to that of TH (71), in terms of promoting CSAsensitive decrease in phosphate and redox potentials with concomitant increase in oxygen consumption in cultured cells as well as *in vivo* (11, 16, 67, 139, 140), indicating that both MEDICA analogs and TH do converge onto LC/HC-PTP gating (11, 71). Indeed, similarly to TH, PTP gating by MEDICA analogs is mediated by modulating the profile of mitochondrial Bcl2-family proteins, resulting in decrease in mitochondrial Bcl2-Bax heterodimer with concomitant increase in mitochondrial free Bax (71, 113). However, different transduction pathways are involved in modulating the mitochondrial content of free Bax by TH or MEDICA analogs. Thus, dissociation of the Bcl2-Bax heterodimer by TH is driven by dephosphorylation of Bcl2(Ser-70) by T3-activated PP2B (113), whereas dissociation of the Bcl2/Bax heterodimer by MEDICA analogs is driven by dephosphorylation of Bad(Ser-112, Ser-155) (141). The decrease in phosphorylated Bad(Ser-112, Ser-155) results in its decreased binding to14-3-3 followed by its increased binding to mitochondrial Bcl2, resulting in Bax displacement and PTP gating (142, 143). Decrease in phosphorylated Bad by MEDICA analogs is due to suppression of the Raf1/MAPK/RSK1 and the adenylate cyclase/PKA transduction pathways, and their respective downstream targets Bad(Ser-112) and Bad(Ser-155) (141). Hence, the TH and MEDICA transduction pathways converge at their downstream Bax target but diverge upstream of the Bcl2/Bax heterodimer (Scheme 1). LC-PTP gating by MEDICA analogs may account for their thyromimetic calorigenic activity in *vivo*.
