Transcript Slide - Texas Tech University Departments

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
2
Quisenberry ,
Leah
James
Reid
2
1,2
Daniel Hardy , Joaquin Lado-Abeal
2
Hutson ,
3
Norman ,
John
4
McGlone ,
Medicine 2Cell Biology and Biochemistry 3Pharmacology and Neurosciences 4Animal Sciences, Texas Tech University Health Sciences
Center, Lubbock, Texas, United States, 79430
Abstract
A
2
40
*
30
20
10
0
B E
control
B E
B E
control
LPS
B E
F Lobe Cortex
Lung
Heart LV
12
6
0
0
Kidney Cortex
Liver
LPS
Kidney
Medulla
control
LPS
-4
50
*
*
25
-6
*
-8
0
Liver
Kidney
Cortex
Kidney
Medulla
**
*
LPS
Thrb
Mct8
*
*
-10
*
*
**
*
-15
0
-5
*
-10
*
-5
-10
-15
-15
0
0
-5
-10
***
*
-5
-10
*
**
-15
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]
NF-kB p65 Ab
- - - - + - - - - +
NF-kB p50 Ab
- - - + - - - - + -
NF-kB mutated probe
- - + - - - - + - -
32P-NF-kB
mutated probe
32P-NF-kB
32P-NF-kB
mutated probe
mutated probe
control
consensus probe + - + + + + - + + +
control
LPS
Liver
Adrenal Gland
Adrenal Gland
B
LPS
NF-kB
NF-kB
Spleen
FL HV Lu Lv Sp Ad KC
control
LPS
8
*
*
*
*
*
-5
*
*
-10
*
4
control
LPS
2 FL HV Lu Lv Sp Ad KC
0
*
-15
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.
Methods
- + - - - - + - - -
NF-kB
Log NF-kB band intensity
-2
*
0
32P-NF-kB
*
75
*
*
control
-10
-15
0
Dio1 mRNA expression
100
Kidney
Cortex
-5
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 Figure 5 A) NF-kB nuclear binding activity in pig tissues. Left:
kidney, and in Dio1activity in kidney cortex. Values are NF-kB p50-p65 nuclear binding was identified in pig tissues
by EMSA. Center: Adrenal gland from LPS treated pigs
represented as mean ± SD. [*, p<0.05].
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
*
control
LPS. B) Relationship between NF-kB nuclear binding (left)
LPS
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
FL Cb Hp Pit Th Lu Lv KC KM Ad SM
expression was observed between groups. Finally, in heart
Figure 3 Dio3 activity in tissues from control and LPS treated left ventricle and liver, LPS induced a decrease in Thrb
pigs. Dio3 activity was detected in all studied tissues. LPS mRNA expression that did not correspond with a significant
caused a statistically significant increase in Dio3 activity in liver, increase in NF-kB nuclear binding activity. Thrb mRNA
hypothalamus and thyroid gland. Values are represented as relative expression is represented as mean ΔCt values ± SD.
mean ± SD. [*, p<0.05].
[*, p<0.05].
Dio3 activity (fmol/min/mg)
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) activity8, 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 tissues8,11-13 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.
Dio1 activity (pmol/min/mg)
Liver
-5
-15
Sk muscle
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]
0
Thrb mRNA expression
0
0
ThrA2
0
0
FL Cb Hp Pit Th Lu HV Lv KC KM Sp Ad SM Du Co
Rxrb
2
ThrA1
1
6
Rxra
4
control
LPS
thyroid hormone ng/g tissue
FT3 (pg/ml)
*
1
FL Cb Hp Pit Th Lu HV Lv KC KM Sp Ad SM Du Co
12
cortisol (µg/dl)
FT4 (ng/dl)
6
*
A
Introduction
Conclusions
B
2
rT3 (ng/ml)
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 multitissue 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.
Results
mRNA relative expression
1Internal
1
CastroPiedras ,
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
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
RT2 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 [125I] generated when
[125I]-rT3 (for Dio1) and [125I]-T4 (for Dio2) was used as a substrate. Dio3 activity was measured by
HPLC, analyzing the amount of [125I]-T2 generated when [125I]-T3 was used as a substrate.
NF-kB nuclear binding activity For EMSA, nuclear extracts were incubated with γ32P-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 32P-labeled probe.
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Acknowledgements
This project was funded by the CH Foundation and Spanish Ministry of Education