1. SAT−391 Lipopolysaccharide (LPS)−Induced Non−Thyroidal Illness Syndrome (NTIS) in Pigs Causes Profound Hypothyroidism and Tissue−Specific Changes in Thyroid Hormone Metabolism and Molecular Targets Isabel CastroPiedras 1, Leah Quisenberry 2, James Hutson 2, Reid Norman 3, John McGlone 4, Daniel Hardy 2, Joaquin Lado-Abeal 1,2 1 Internal Medicine 2 Cell Biology and Biochemistry 3 Pharmacology and Neurosciences 4 Animal Sciences, Texas Tech University Health Sciences Center, Lubbock, Texas, United States, 79430 Methods Conclusions References Abstract Introduction Non-thyroidal illness syndrome (NTIS) is a component of the neuroendocrine and metabolic response to severe stress and starvation, characterized by low serum T3 and inappropriately normal or low TSH; in severe cases serum rT3 increases and T4 decreases. 1,2 During prolonged illness it is unclear if NTIS is beneficial and, in fact, patients with low serum T4 have an increased probability of death. 3-5 NTIS results from a central hypothyroidism together with tissue specific changes resembling consumptive hypothyroidism and reduced sensitivity to TH action. Central hypothyroidism in NTIS is a consequence of decreased expression of TRH mRNA in the hypothalamus. 6,7 A tissue specific increase in type 3 deiodinase (Dio3) activity 8, as observed in consumptive hypothyroidism, combined with decreased activity of type 1 deiodinase (Dio1) 9,10 results in inactivation of T4 into rT3 and reduced synthesis and increased degradation of T3. NTIS is associated with decreased expression of thyroid hormone receptors (Thrs) and their nuclear partners retinoid X receptors (Rxr) in some tissues 8,11-13 which may create a situation of reduced sensitivity to the action of TH, the relevance of which has not yet been explored. The molecular mechanisms at the root of NTIS are not completely understood, but NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation has been suggested to be responsible for the decrease in thyroid hormone receptor β1 (Thrb1) expression during inflammation. 14 Our aim was to establish a pig model for lipopolysaccharide (LPS) induced septic shock NTIS to investigate the effects on: 1) TH levels in serum and tissues; 2) changes in expression of TH molecular targets and activity of iodothyronine deiodinases at a multi-tissue level; 3) NF-kB nuclear binding activity and its correlation with the tissue specific decrease in Thrb gene expression observed during endotoxemia. The effect of lipopolysaccharide (LPS) on thyroid hormone (TH) levels and TH molecular targets, and the relationship between LPS-induced NF-kB activation and thyroid hormone receptor beta (Thrb) gene expression were investigated at a multi- tissue level in a pig model of septic shock non-thyroidal illness syndrome (NTIS). Pre-pubertal domestic pigs were given iv saline (control, n=8) or LPS (n=9) for 48 h. Serum free T4 (FT4), FT3, rT3 and cortisol, and tissues T4 and T3 were measured by chemiluminescence and RIA; expression of TH receptors and co-factors by real time PCR, deiodinases (Dio) activity by specific enzyme assays, and NF-kB nuclear binding activity by EMSA. LPS-treated pigs had decreased TH levels in serum and most tissues. Dio1 expression in liver and kidney and Dio1 activity in kidney decreased after LPS. No changes in Dio2 activity were observed between groups. LPS induced an increase in hypothalamus, thyroid and liver Dio3 activity. Tissue specific changes were also observed in expression of other studied genes. Among these, monocarboxylate transporter 8 (Mct8) and Thrb were the most commonly repressed in endotoxemic pigs. LPS-induced NF-kB activation was associated with a decrease in Thrb gene expression only in some tissues. Overall, LPS-induced NTIS in pigs is characterized by a profound hypothyroidism and tissue specific changes that create conditions for reduced TH sensitivity. The role of NF-kB in regulating Thrb expression during endotoxemia, if any, is restricted to a limited number of tissues. Animal Model Fifteen pre-pubertal female domestic pigs, weighting 9-11 kg, were randomly assigned to either a control group (n:8) or an endotoxemic group (Lipopolysaccharide –LPS, n:9). Animals received an initial bolus of 3.5 µg/kg/min for five minutes of LPS, or saline for controls, followed by 3.5 µg/kg/hour of LPS/saline for up to 48 hours. Tissue samples were collected immediately after euthanasia, frozen in liquid nitrogen, and stored at -80°C until use. Hormonal Studies Serum hormonal levels were measured in blood samples obtained immediately before infusion began and at the end of the infusion. Chemiluminescence was used to measure serum free T4 (FT4), free T3 (FT3) and cortisol. Serum rT3 was measured by RIA. Gene Expression mRNA expression was measured by real-time RT-PCR using M-MLV RT and RT 2 SYBRgreen ROX FAST Master mix. Ribosomal protein L4(RPL4)was used as internal control. Deiodinase activity Dio1 and Dio2 activity was measured by the amount of [ 125 I] generated when [ 125 I]-rT3 (for Dio1) and [ 125 I]-T4 (for Dio2) was used as a substrate. Dio3 activity was measured by HPLC, analyzing the amount of [ 125 I]-T2 generated when [ 125 I]-T3 was used as a substrate. NF-kB nuclear binding activity For EMSA, nuclear extracts were incubated with γ 32 P-labeled probes containing the NFκB consensus binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3') or mutant binding site (5’- AGTTGAATTCACTTTCCCAGGC-3’). In competition studies, 100-fold molar excess of mutant oligo was added in addition to wt probe. The protein-DNA complexes were resolved on a non-denaturing 6% polyacrylamide gel. For supershifts, 2 ug of antibody against p50 or p65 was added to the reaction buffer for 20 minutes before addition of 32 P-labeled probe. LPS administration induced a profound decrease in TH levels in serum and most tissues, as well as a tissue-specific decrease in Mct8, Dio, Rxr and Thr gene expression, resulting in a severe hypothyroidism with conditions for reduced sensitivity to thyroid hormone action in some tissues. Characterization of deiodinases revealed high Dio1 activity in liver and kidney, Dio2 activity in hypothalamus, pituitary and thyroid, and high Dio3 activity in hypothalamus, frontal lobe, pituitary and adrenal gland. LPS induced a decrease in Dio1 expression and activity in liver and kidney, and stimulated Dio3 activity in several tissues, contributing to NTIS hypothyroidism by decreasing T3 production, promoting conversion of T4 into rT3, and accelerating T3 clearance. LPS-induced NF-kB nuclear binding activity was present in most but not all analyzed tissues and, overall, a relationship between NF-kB activation and decreased Thrb gene expression was not observed, indicating activation of the NF-kB pathway is not solely responsible for decreased expression of Thrb. Results 1 Wartofsky L, Burman KD 1982. Endocr Rev 3:164-203 2 DeGroot LJ 1999. J Clin Endocrin Metab 84:151-164 3 Stathatos N, Wartofsky L 2003. J Endocrinol Invest 26:1174-1179 4 DeGroot LJ 2003. J Endocrinol Invest 26:1163-1170 5 Slag MF, Morley JE, Elson MK, Crowson TW, Nuttall FQ, Shafer RB 1981. JAMA 245:43-45 6 Fliers E, Guldenaar SE, Wiersinga WM, Swaab DF 1997. J Clin Endocrin Metab 82:4032- 4036 7 Van den Berghe G, de Zegher F, Baxter RC, Veldhuis JD, Wouters P, Schetz M, Verwaest C, Van der Vorst E, Lauwers P, Bouillon R, Bowers CY 1998. J Clin Endocrin Metab 83:309-319 8 Rodriguez-Perez A, Palos-Paz F, Kaptein E, Visser TJ, Dominguez-Gerpe L, Alvarez-Escudero J, Lado-Abeal J 2008. Clin Endocrinol 68:821- 827 9 Peeters RP, Wouters PJ, Kaptein E, Van Toor H, Visser TJ, Van den Berghe G 2003. J Clin Endocrin Metab 88:3202-3211 10 Debaveye Y, Ellger B, Mebis L, Van Herck E, Coopmans W, Darras V, Van den Berghe G 2005. Endocrinology 146:5604- 5611 11 Boelen A, Kwakkel J, Fliers E 2011. Endocr Rev 32:670-693 12 Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, Feingold KR 2003. Am J Physiol-Endoc M 284:E228–E236 13 Feingold K, Kim MS, Shigenaga J, Moser A, Grunfeld C 2004. Am J Physiol-Endoc M 286: E201-E207 14 Kwakkel J, Wiersinga WM, Boelen A 2006. J Endocrinol 189: 37-44 Acknowledgements This project was funded by the CH Foundation and Spanish Ministry of Education Figure 1 A) Serum levels of thyroid hormones and cortisol in pigs at beginning (B) and end (E) of infusion. In LPS-treated pigs, serum FT4 and FT3 decreased and cortisol increased. B) T4 and T3 levels in tissues from control and LPS treated pigs. Both T4 and T3 levels decreased in LPS treated pigs in most studied tissues. Values are represented as mean ± SD. [*, p<0.05; outlier] Figure 2 Dio1 activity (left) and relative gene expression (right) in liver and kidney cortex and medulla. LPS induced a significant decrease in Dio1 gene expression in liver and kidney, and in Dio1activity in kidney cortex. Values are represented as mean ± SD. [*, p<0.05]. Figure 3 Dio3 activity in tissues from control and LPS treated pigs. Dio3 activity was detected in all studied tissues. LPS caused a statistically significant increase in Dio3 activity in liver, hypothalamus and thyroid gland. Values are represented as mean ± SD. [*, p<0.05]. Figure 4 Monocarboxylate transporter 8 (Mct8), thyroid hormone receptors beta (Thrb), alpha1 (Thra1), and alpha2 (Thra2) and retinoid X receptors alpha (Rxra) and beta (Rxrb) gene expression changes between control and LPS treated pigs. Values are represented as mean ΔCt ± SD. [*, p<0.05] FL, frontal lobe; Cb, cerebellum; Hp, hypothalamus; Pit, pituitary; Th, thyroid gland; Lu, lung; HV, heart left ventricle; Lv, liver; KC, kidney cortex; KM, kidney medulla; Sp, spleen; Ad, adrenal gland; SM, skeletal muscle; Du, duodenum; Co, colon Figure 5 A) NF-kB nuclear binding activity in pig tissues. Left: NF-kB p50-p65 nuclear binding was identified in pig tissues by EMSA. Center: Adrenal gland from LPS treated pigs showed higher NF-kB nuclear binding activity than controls. Right: Spleen from control pigs had a high basal NF-kB nuclear binding activity that did not increase significantly after LPS. B) Relationship between NF-kB nuclear binding (left) and Thrb mRNA expression (right) in pig tissues. In frontal lobe, adrenal gland and kidney cortex, LPS induced a significant increase in NF-kB nuclear binding activity and a decrease in Thrb mRNA expression. In spleen no differences in NF-kB nuclear binding or Trhb gene expression were observed between groups. In lungs, LPS caused an increase in NF-kB nuclear binding activity but no change in Thrb gene expression was observed between groups. Finally, in heart left ventricle and liver, LPS induced a decrease in Thrb mRNA expression that did not correspond with a significant increase in NF-kB nuclear binding activity. Thrb mRNA relative expression is represented as mean ΔCt values ± SD. [*, p<0.05].