SLEEP 2011 Abstract Supplement

A. Basic Science II. Cell and Molecular Biology and Genetics
Results: Viral nucleoprotein (NP) negative and positive sense RNA
was found in the olfactory bulb (OB) of both fMx1 and dMx1 mice
that received live virus at both time points although more was present
at 96 h post-inoculation. In contrast, in lungs by 96 h post-inoculation
significantly more viral NP - and + sense RNA was found in the dMx1
mice. Further, in lungs TNFα and IL1β mRNAs were higher in the fMx1
whereas the anti-inflammatory type I interferons were higher in the
dMx1strain at 15 h post infection. By 96 h, only dMx1 had elevated
TNFα mRNA in the OB. In the lungs, dMx1 mice had elevated IFNβ,
IL1β and TNFα mRNAs. The fMx1 strain also showed elevation of the
same genes but at significantly lower levels.
Conclusion: Influenza virus invaded the OB of both dMx1 and fMx1
mouse strains and there was little difference in viral levels between the
strains. In contrast, the lungs of the fMx1 mice had a much lower response
of innate immune mediators than the dMx1 mice. Data suggest
that the recovery of the fMx1 mice is likely due to what is happening in
the lung and not the OB.
Support (If Any): NIH HD036520
0043
THE EFFECT OF PRO-INFLAMMATORY MEDIATORS ON
HYPOCRETIN/OREXIN RECEPTOR EXPRESSION
Zhan S 1,2 , Cai G 1 , Fang F 1 , Hu M 1 , Ding Q 1
1
Medicine, University of Alabama at Birmingham, Birmingham, AL,
USA, 2 Neurology, Xuanwu Hospital, Beijing, China
Introduction: The important role of the Hypocretin/Orexin system in
regulation of the pattern of sleep and wakefulness, as well as the related
disorders, is recognized. Also, autoimmune diseases or/and enhanced inflammatory
mediators are found in patients with sleep and wakefulness
disorders. Thus, the crosstalk between neuroinflammation and regulators
of sleep and wakefulness needs to be further investigated. Here, we
present our research studies on the effect of pro-inflammation mediators
on expression of the Hypocretin/Orexin receptors.
Methods: Hypocretin/Orexin receptor 2 (HcrtR2) was inserted into
the MSCV vectors between the polylinker located within the multiple
cloning sites of the MSCV vector. Then, the MSCV- HcrtR2 vector was
transfected into the packaging cells by lipid-mediated transfection, to
produce retroviral particles, and the collected rtroviral vectors are used
to transfect the primary neuron cells. Cells then were treated with vehicle
only or pro-inflammation mediators, tumor necrosis factor alpha
(TNF-α) and IL-1. The expression of HcrtR2 was examined after cells
treated with or without TNF-α and IL-1.
Results: HcrtR2 expression in cells treated with TNF-α was decreased,
and the downregulation of HcrtR2 is in one persistent pattern. In contrast,
HcrtR2 expression in cells treated with IL-1 was in a slow-wave
pattern, and the effect of IL-1 on HcrtR2 expression is not persistent.
Conclusion: These results demonstrate that TNF-α and IL-1 can be
involved in regulation of the Hypocretin/Orexin system through manipulation
of the HcrtR2 expression. These results also suggest that
pro-inflammatory mediators may be involved in sleep and wakefulness
disorders through regulation of the Hypocretin/Orexin system.
0044
TEMPORAL CHANGES IN THE BRAIN CORTICAL
UNFOLDED PROTEIN RESPONSE (UPR) FOLLOWING
SLEEP FRAGMENTATION (SF) IN THE MOUSE
Kayali F, Gozal D
Section of Pediatric Sleep Medicine, Department of Pediatrics,
University of Chicago, Chicago, IL, USA
Introduction: The activation of the UPR is a critically important component
of the molecular response to stressful conditions such as ischemia/reperfusion,
hypoxia, trauma, and sleep deprivation. The UPR
occurs as the result of accumulation of misfolded proteins in the ER
lumen, which in turn activates the molecular events leading to either cell
survival or apoptosis. However, the effect on the UPR of conditions such
as sleep fragmentation, a frequent occurrence in many sleep disorders,
is unknown.
Methods: C57BL/6 mice (n=40; 6 week-old) were purchased from
Jackson Laboratories and were exposed to SF for different time periods
using a custom designed and validated device that does not require
social isolation, restricted access to food, increased physical activity or
increases corticosterone levels. . SF or control mice were sacrificed after
3 hrs, 6 hrs, 9 hrs, 12 hrs, 36 hrs, 60 hrs, and 1 week of exposure. The
SF paradigm was implemented during daylight hours from 7 am to 7
pm. The cortex was rapidly harvested, snap frozen, and subsequently
processed for protein extraction. Equal amounts of cortical lysate proteins
were run on 15% SDS gels and immunoblotted for detection of
phosphorylated eIF2(alpha) and CHOP using previously validated antibodies.
Results: An increase in eIF2(alpha) phosphorylation occurred at 6 hrs of
SF, persisted for 48 hours, and subsequently gradually returned to basal
levels at 1 week of SF. CHOP was not expressed in control conditions,
and became detectable at 2 days of SF, progressively decreasing thereafter
to basal levels after 1 week of SF.
Conclusion: Sleep fragmentation elicits transient and distinct activation
of the UPR in the brain cortex. We postulate that the UPR triggered by
SF may reflect induction of protective mechanisms aiming to minimize
neuronal cell dysfunction and initiation of apoptotic processes.
Support (If Any): DG is supported by National Institutes of Health
grant HL-086662; FK is supported by T32 Training Grant HL-094282.
0045
SLEEP FRAGMENTATION REDUCES VISCERAL FAT
INSULIN SENSITIVITY IN MICE
Khalyfa A 1 , Abdelkarim A 1 , Neel B 2 , Brady M 2 , Gozal D 1
1
Pediatrics, The University of Chicago, Chicago, IL, USA, 2 Medicine,
University of Chicago, Chicago, IL, USA
Introduction: Sleep fragmentation (SF) is one of the hallmarks of sleep
apnea (SA), which is associated with metabolic dysregulation independently
of obesity. SF has been proposed as contributing to the putative
adverse metabolic conse¬quences of SA via disruption of visceral adipose
tissue (VAT) homeostasis, and altered insulin sensitivity. We hypothesized
that chronic experimental SF in mice will lead to changes in
insulin signaling in visceral fat
Methods: Adult young male C57BL/6J mice were exposed to SF or
control sleep conditions (CO) for 7 days, after which visceral fat tissues
were harvested and treated with a series of incremental insulin concentrations
for 10 min. SF was performed for 12 hrs during daylight and
consisted of gentle, mechanically-induced arousals at 2 min intervals using
a custom developed automated device. Protein lysates were separated
by electrophoresis and probed with anti-phosphorylated-Akt (pAkt)
and total Akt antibodies. Antibodies to phosphorylated tyrosine residues,
insulin receptor substrate 1 (IRS1), and insulin receptor beta (IRb) were
also used. Serum lipid profiles were measured.
Results: A dose-response of insulin sensitivity emerged in CO intact
visceral fat in vitro. However, pAkt was decreased in mice exposed to
SF at lower doses of insulin compared to CO, indicating the presence of
insulin resistance. In addition, serum triglycerides were increased and
HDL levels were reduced in SF-exposed mice. Phosphorylated tyrosine
in the lysates, IRS1, and IRb proteins showed no changes in SF compared
to CO.
Conclusion: SF induces development of insulin resistance in mouse visceral
fat, and is also accompanied by dyslipidemia. Further delineation
of the pivotal molecular components that coordinate insulin action in
visceral fat, and the perturbations in these pathways that are associated
with SF, will be essential for further understanding of the mechanisms
underlying insulin resistance and metabolic dysfunction in sleep apnea.
Support (If Any): This work was supported by Comer Kids Run Classic
Fund Grant to (AK).
SLEEP, Volume 34, Abstract Supplement, 2011
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