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

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 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.

Thyroid Hormone and Energy Expenditure 283

The dynamic equilibrium between cytosolic Ca+2 ([Ca+2]c) and endoplasmic reticulum (ER) Ca+2 ([Ca+2]ER) is maintained by an interplay between the inositol 1,4,5-trisphosphate receptor (IP3R1) and the sarcoplasmic Ca+2 ATPase (SERCA) that catalyzes ER Ca+2 efflux and influx, respectively (119). IP3R1 is activated by binding of the IP3 ligand and may further be modulated by its phosphorylation by PKA, PKC or CaMKII, its dephosphorylation by PP2B, or by its association with one or more of about 50 proteins, including FKBP12 or Bcl2 (120, 121). The putative role played by IP3R1 in TH-induced PTP gating was evaluated by verifying the effect of TH in cells lacking IP3R1 (113). Thus, PTP opening, dephosphorylation of mitochondrial Bcl2(S70) and depletion of mitochondrial Bcl2 are all abrogated in cells lacking IP3R1, indicating that IP3R1 is indeed required for THinduced mitochondrial uncoupling. Similarly, T3 is ineffective in increasing [Ca+2]c upon inhibition of IP3R1 by 2APB, indicating a specific requirement for IP3R1 activity in modulating [Ca+2]c by TH. Furthermore, T3-induced gating of IP3R1 is accounted for by both, increase in IP3R1 expression and protein levels, complemented by IP3R1 truncation into channel-only isoforms. Truncated IP3R1 isoforms have been reported to serve as channel-only peptides capable of carrying out [Ca+2]ER efflux in the absence of added IP3 (122-126). IP3R1 truncation by TH may reflect TH activation of IP3R1 proteases that remain to be further verified. The IP3R1 / PP2B crosstalk in mediating TH-induced PTP gating is supported by constitutive PP2B-induced PTP gating under conditions of suppressing IP3R1 expression by siRNA (127). Hence, PP2B is acting downstream to TH-induced IP3R1,

Over all, TH-induced expression of the IP3R1 channel accompanied by its truncation is proposed to result in [Ca+2]ER efflux, increase in [Ca+2]c and [Ca+2]c-activated PP2B, followed by dephosphorylation of mitochondrial Bcl2(S70) with concomitant decrease in mitochondrial Bcl2 protein levels and increase in mitochondrial free Bax (Scheme 1). The decrease in mitochondrial Bcl2 and/or the respective increase in mitochondrial free Bax may initiate and promote variable PTP gating, resulting in physiological LC-PTP– induced calorigenesis. LC-PTP gating may drift to HC-PTP–induced apoptosis as function of additional prevailing conditions that may affect mitochondrial permeability

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

and is obligatory as well as sufficient in mediating PTP by [Ca+2]c.

**9. Thyromimetic agents and energy expenditure** 

transition.

hyperthermia (129).
