OB/GYN DAY 2017 poster
TNFα exposure induces neuroinflammation and insulin resistance in a rat-derived hypothalamic cell model, rHypoE-7 Matthew N. Clemenzi1, Makram E. Aljghami1, Leigh Wellhauser1, and Denise D. Belsham1,2,3. Departments of Physiology1, Obstetrics and Gynaecology2 and Medicine3; University of Toronto, Toronto, Ontario, Canada.
Results
Pre-treatment with 50 ng/mL TNFα significantly decreased Akt phosphorylation, implicating attenuated insulin signaling. Quantified by Western blot analysis. n = 4-5, * p < 0.05, *** p < 0.001, **** p < 0.0001
Figure designed by Neruja Loganathan
IL6/ Histone mRNA
e at
H 50
uM
O
G
le
nR
2O
nM 10
uM
O
le
H
e at
H nR
10
50
nM
G
le
uM
O
G
0.0
e at
H nR
2O
10
nM
O
G nM 10
H
H
2O
H
TN /m ng
0.5
50
H2O
24 hour
4 hour
24 hour
IL6 / Histone mRNA
1.5
qPCR Array Verification • Treatment with 50 ng/mL TNFα significantly increased gene expression of TNFR (cytokine receptor), IκBα, and IL6. n = 4, * p < 0.05, **** p < 0.0001
In conclusion, treatment with the inflammatory cytokine TNFα causes cellular insulin resistance in the rHypoE-7 neurons, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin- and palmitate-induced insulin resistance in hypothalamic neurons. Further, these results demonstrate that components of the IKK-β/NF-κB pathway appear to be activated upon exposure to TNFα, providing a putative target to reverse neuroinflammation and insulin resistance with anti-inflammatory agents. Co-treatment with GnRH or oleate did not strongly attenuate TNFα-induced neuroinflammation. Future experiments plan to pre-treat with these compounds, as well as to investigate whether oleate or GnRH treatment may restore insulin signaling in TNFα-treated neurons.
Acknowledgements
50 ng/mL TNFα
** *
1.0
We would like to thank Jennifer Chalmers for her technical assistance. This research was supported by grants from the CIHR, CRC, CFI, NSERC, APS and BBDC.
0.5
24
4
ho
ho
ur
ur
0.0
IκBα
TNFR
ur
0.0
ho
ur ho 24
ur
0.0
0.5
24
0.5
1.0
ur
1.0
**
1.5
ho
IκBα / Histone mRNA
**
**
2.0
****
4
1.5
ho
Subcloning
rHypoE-7 neurons were pre-treated with TNFα prior to insulin rechallenge. pAkt levels quantified by Western blot.
0
1.0
Conclusions
• Several genes of interest were identified by Qiagen qPCR cytokine array, outlining the genes whose mRNA expression is regulated by TNFα treatment
4
Population Selection: Geneticin
Total Akt
TNFR / Histone mRNA
SV-40 T-Antigen
1
1.5
pAkt
IL-6
Quantitative Real-Time PCR
**** **** ****
4 hour
- qPCR array data
rHypoE-7 neurons were treated with TNFα and mRNA expression was quantified by qPCR.
Dispersion
L
in nM 10
0
ng 50
Pre-treatment
TNFα exposure induces mRNA expression of inflammatory gene markers
Methods
Hypothalamic Extraction
Fα
lin su
2O
H
/m
L
H
TN
2O
Fα
0
2.0
2
nR
1
1
**** ****
uM
2
IκBα / Histone mRNA
***
2
50
pAkt / total Akt
3
50 ng/mL TNFα
50
****
Total Akt
We hypothesized that TNFα-treated rat hypothalamic neurons would exhibit cellular neuroinflammation and insulin resistance as evidenced by increased expression of proinflammatory genes and attenuated phosphorylation of Akt (pAkt) in the insulin signaling pathway. It is also hypothesized that co-treatment with GnRH or oleate will abrogate TNFαinduced inflammatory gene expression.
2.5
e
****
pAkt
Hypothesis
H2O
3
2O
*
3
Pre-treatment
Western Blot
10 nM insulin
at
4
H2O
16 hours
***
Co-treatment with GnRH or oleate with TNFα exposure did not strongly decrease IκBα expression. However, co-treatment with these compounds may decrease TNFα-induced IL6 expression, suggesting a potential anti-inflammatory effect. n = 2-4, **** p < 0.0001.
H
6 hours
0
Generation of rHypoE-7 Immortalized Cell Line
GnRH and Oleate do not strongly reduce inflammatory gene expression
TNFα Induces Insulin Resistance
pAkt / total Akt
Obesity is approaching epidemic levels. One of the main characteristics of obesity is the development of insulin resistance, leading to other comorbidities, such as type 2 diabetes mellitus and cardiovascular disease. Insulin resistance has been shown to be induced with obesity in peripheral tissues, such as liver, muscle, and adipocytes, by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In the hypothalamus, the main brain region for energy regulation, insulin resistance has been demonstrated to occur upon prolonged treatment with high levels of insulin and palmitate, and we are exploring whether TNFα, a downstream surrogate for palmitate, has similar effects. Gonadotropin releasing hormone (GnRH) and the monounsaturated fatty acid oleate have been hypothesized to act as potential anti-inflammatory agents in the brain, however it is not yet clear how these compounds may do so.
Results
le
Rationale
• Kolb H & Mandrup-Poulsen T (2010). The global diabetes epidemic as a consequence of lifestyle-induced low-grade inflammation. Diabetologia 53, 10–20. • Kwon et al (2014). Oleate prevents palmitate-induced mitochondrial dysfunction, insulin resistance and inflammatory signaling in neuronal cells. Biochim. Biophys. Acta 1843 (7), 1402-1413. • Mayer CM & Belsham, DD (2010). Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation. Endocrinology 151, 576-585. • Mayer CM & Belsham, DD (2010). Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation. Endocrinology 151, 75-84. • Schwartz MW (2001). Progress in the search for neuronal mechanisms coupling type 2 diabetes to obesity. J Clin Invest 108, 963-964.
Results
Pre-treatment with 50 ng/mL TNFα significantly decreased Akt phosphorylation, implicating attenuated insulin signaling. Quantified by Western blot analysis. n = 4-5, * p < 0.05, *** p < 0.001, **** p < 0.0001
Figure designed by Neruja Loganathan
IL6/ Histone mRNA
e at
H 50
uM
O
G
le
nR
2O
nM 10
uM
O
le
H
e at
H nR
10
50
nM
G
le
uM
O
G
0.0
e at
H nR
2O
10
nM
O
G nM 10
H
H
2O
H
TN /m ng
0.5
50
H2O
24 hour
4 hour
24 hour
IL6 / Histone mRNA
1.5
qPCR Array Verification • Treatment with 50 ng/mL TNFα significantly increased gene expression of TNFR (cytokine receptor), IκBα, and IL6. n = 4, * p < 0.05, **** p < 0.0001
In conclusion, treatment with the inflammatory cytokine TNFα causes cellular insulin resistance in the rHypoE-7 neurons, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin- and palmitate-induced insulin resistance in hypothalamic neurons. Further, these results demonstrate that components of the IKK-β/NF-κB pathway appear to be activated upon exposure to TNFα, providing a putative target to reverse neuroinflammation and insulin resistance with anti-inflammatory agents. Co-treatment with GnRH or oleate did not strongly attenuate TNFα-induced neuroinflammation. Future experiments plan to pre-treat with these compounds, as well as to investigate whether oleate or GnRH treatment may restore insulin signaling in TNFα-treated neurons.
Acknowledgements
50 ng/mL TNFα
** *
1.0
We would like to thank Jennifer Chalmers for her technical assistance. This research was supported by grants from the CIHR, CRC, CFI, NSERC, APS and BBDC.
0.5
24
4
ho
ho
ur
ur
0.0
IκBα
TNFR
ur
0.0
ho
ur ho 24
ur
0.0
0.5
24
0.5
1.0
ur
1.0
**
1.5
ho
IκBα / Histone mRNA
**
**
2.0
****
4
1.5
ho
Subcloning
rHypoE-7 neurons were pre-treated with TNFα prior to insulin rechallenge. pAkt levels quantified by Western blot.
0
1.0
Conclusions
• Several genes of interest were identified by Qiagen qPCR cytokine array, outlining the genes whose mRNA expression is regulated by TNFα treatment
4
Population Selection: Geneticin
Total Akt
TNFR / Histone mRNA
SV-40 T-Antigen
1
1.5
pAkt
IL-6
Quantitative Real-Time PCR
**** **** ****
4 hour
- qPCR array data
rHypoE-7 neurons were treated with TNFα and mRNA expression was quantified by qPCR.
Dispersion
L
in nM 10
0
ng 50
Pre-treatment
TNFα exposure induces mRNA expression of inflammatory gene markers
Methods
Hypothalamic Extraction
Fα
lin su
2O
H
/m
L
H
TN
2O
Fα
0
2.0
2
nR
1
1
**** ****
uM
2
IκBα / Histone mRNA
***
2
50
pAkt / total Akt
3
50 ng/mL TNFα
50
****
Total Akt
We hypothesized that TNFα-treated rat hypothalamic neurons would exhibit cellular neuroinflammation and insulin resistance as evidenced by increased expression of proinflammatory genes and attenuated phosphorylation of Akt (pAkt) in the insulin signaling pathway. It is also hypothesized that co-treatment with GnRH or oleate will abrogate TNFαinduced inflammatory gene expression.
2.5
e
****
pAkt
Hypothesis
H2O
3
2O
*
3
Pre-treatment
Western Blot
10 nM insulin
at
4
H2O
16 hours
***
Co-treatment with GnRH or oleate with TNFα exposure did not strongly decrease IκBα expression. However, co-treatment with these compounds may decrease TNFα-induced IL6 expression, suggesting a potential anti-inflammatory effect. n = 2-4, **** p < 0.0001.
H
6 hours
0
Generation of rHypoE-7 Immortalized Cell Line
GnRH and Oleate do not strongly reduce inflammatory gene expression
TNFα Induces Insulin Resistance
pAkt / total Akt
Obesity is approaching epidemic levels. One of the main characteristics of obesity is the development of insulin resistance, leading to other comorbidities, such as type 2 diabetes mellitus and cardiovascular disease. Insulin resistance has been shown to be induced with obesity in peripheral tissues, such as liver, muscle, and adipocytes, by the pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). In the hypothalamus, the main brain region for energy regulation, insulin resistance has been demonstrated to occur upon prolonged treatment with high levels of insulin and palmitate, and we are exploring whether TNFα, a downstream surrogate for palmitate, has similar effects. Gonadotropin releasing hormone (GnRH) and the monounsaturated fatty acid oleate have been hypothesized to act as potential anti-inflammatory agents in the brain, however it is not yet clear how these compounds may do so.
Results
le
Rationale
• Kolb H & Mandrup-Poulsen T (2010). The global diabetes epidemic as a consequence of lifestyle-induced low-grade inflammation. Diabetologia 53, 10–20. • Kwon et al (2014). Oleate prevents palmitate-induced mitochondrial dysfunction, insulin resistance and inflammatory signaling in neuronal cells. Biochim. Biophys. Acta 1843 (7), 1402-1413. • Mayer CM & Belsham, DD (2010). Palmitate attenuates insulin signaling and induces endoplasmic reticulum stress and apoptosis in hypothalamic neurons: rescue of resistance and apoptosis through adenosine 5' monophosphate-activated protein kinase activation. Endocrinology 151, 576-585. • Mayer CM & Belsham, DD (2010). Central insulin signaling is attenuated by long-term insulin exposure via insulin receptor substrate-1 serine phosphorylation, proteasomal degradation, and lysosomal insulin receptor degradation. Endocrinology 151, 75-84. • Schwartz MW (2001). Progress in the search for neuronal mechanisms coupling type 2 diabetes to obesity. J Clin Invest 108, 963-964.
