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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 27, 2001 as doi:10.1096/fj.00-0830fje. |
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* Center for Research on Energy Metabolism, Department of Anatomy and Physiology, School of Medicine, Laval University, Québec, QC, Canada G1K 7P4; and
Lipid Research Unit, CHUQ, Laval University Hospital Research Center, Québec, QC, Canada, G1V 4G2
2Correspondence: Department of Anatomy and Physiology, School of Medicine, Laval University, Quèbec, QC, Canada G1K 7P4. E-mail: yves.deshaies{at}phs.ulaval.ca
SPECIFIC AIMS
Severe endotoxemia such as that induced by administration of bacterial lipopolysaccharide (LPS) causes hypertriglyceridemia through a generalized decrease in the activity of lipoprotein lipase (LPL; EC 3.1.1.34), the rate-limiting enzyme responsible for the intravascular hydrolysis of triglycerides (TG), and stimulates the expression and activity of inducible nitric oxide synthase (iNOS) in key TG-hydrolyzing tissues (adipose tissue and skeletal muscle), which leads to overproduction of nitric oxide (NO). The purpose of this study was to address, in two models of iNOS invalidation (pharmacological and genetic), the hypothesis that the endotoxin-induced reduction in tissue LPL activity and consequent hypertriglyceridemia are caused by iNOS-mediated NO overproduction.
PRINCIPAL FINDINGS
1. Hypertriglyceridemia induced by LPS is due to reduced
intravascular TG clearance
Lipopolysaccharide is known to bring about hypertriglyceridemia by
either increasing hepatic secretion of TG-rich lipoproteins or reducing
LPL-mediated intravascular TG hydrolysis. To ascertain that at the dose
used, LPS [from Escherichia coli, serotype 055:B5,
intraperitoneal (i.p.) 15 mg/kg] altered triglyceridemia solely
through the hydrolytic component of intravascular TG metabolism, the TG
secretion rate was measured after administration of Triton 1339, which
blocks intravascular TG clearance. LPS not only failed to increase TG
secretion, but diminished the latter by 50% (5.5±0.7 vs. 2.8±0.8
µmol/min in control and LPS-treated groups, respectively;
P<0.05). Therefore, LPS-induced hypertriglyceridemia was
not the consequence of an increase in TG output into the circulation,
but was due entirely to reduced TG clearance.
2. LPS-induced hypertriglyceridemia is abolished by pharmacological
inhibition of iNOS with aminoguanidine
In fed Sprague-Dawley rats without access to food and injected
6 h earlier with LPS, plasma TG concentration was close to
threefold higher (3.5 vs. 1.2 mmol/l) than in control animals
(Fig. 1A
). Whereas the iNOS inhibitor aminoguanidine (AGN, 100
mg/kg, i.p. 30 min before LPS administration, a dose that does not
interfere with other NOS isotypes) had no effect by itself, the drug
completely blunted LPS-induced hypertriglyceridemia (LPS–AGN
interaction: P<0.0002). Plasma
NO3/NO2 levels, an index of
NO production, were greatly elevated 6 h after LPS injection
(17±1 vs. 78±2 µmol/l in control and LPS-treated groups,
respectively; P<0.0001). Whereas AGN alone had no effect on
plasma NO3/NO2 levels
(17±1 vs. 20±1 µmol/l in control and AGN-treated groups,
respectively), the LPS-induced increase in plasma
NO3/NO2 was significantly
reduced by prior AGN treatment (78±2 vs. 50±3 µmol/l in LPS- and
LPS+AGN-treated groups, respectively; LPS–AGN interaction:
P<0.0001).
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3. The LPS-induced decrease in LPL activity is abrogated by
AGN in muscle, but not in adipose tissue
LPS treatment induced iNOS protein levels by 
four- to fivefold
in both skeletal muscle and WAT (data not shown). LPS decreased LPL
specific activity by at least 50% in the gastrocnemius muscle and
epididymal white adipose tissue (WAT) of rats (Fig. 1B
, both
P<0.0001). The effect of LPS on LPL activity was also
observed in other muscles and adipose depots including the soleus,
tibialis, and vastus lateralis muscles and perirenal WAT (not shown).
In the gastrocnemius muscle, AGN was without effect in saline-treated
rats but completely prevented the LPS-induced decrease in LPL activity
(LPS–AGN interaction: P<0.05), whereas in epididymal WAT,
AGN did not affect LPL activity in either saline- or LPS-injected rats.
LPL mRNA (adjusted for that of GAPDH mRNA) in skeletal muscle and WAT
was not significantly affected by either LPS or AGN (Fig. 1C
).
4. LPS fails to induce hypertriglyceridemia in iNOS knockout mice
As in normal rats, LPS injection to wild-type mice
(C57BL/6Jx129/SvJ background) resulted in a large increase in plasma
TG concentration (from 0.8 to 1.3 mmol/l, P<0.05,
Fig. 2A
). In contrast, LPS failed to induce hypertriglyceridemia in
iNOS knockout mice. As expected, a strong genotype–LPS interaction
(P<0.0001) was observed in the increase in plasma
NO3/NO2 levels after LPS
administration (wild-type control, 18±1; wild-type LPS, 53±8; iNOS
knockout control, 22±5; iNOS knockout LPS, 18±8 µmol/l). The
interaction reflected the inability of iNOS knockout mice to
overproduce NO during endotoxemia.
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5. LPS fails to decrease LPL activity in skeletal muscle of iNOS
knockout mice
LPS administration to wild-type mice reduced LPL activity in the
gastrocnemius muscle and epididymal WAT (Fig. 2B
) as well as
in the vastus lateralis muscle and perirenal WAT (not shown). Targeted
disruption of the iNOS gene prevented the LPS-induced decrease in LPL
activity in the gastrocnemius muscle, whereas LPS decreased LPL
activity in epididymal WAT (-78%) in both wild-type and iNOS knockout
mice.
CONCLUSIONS
Hypertriglyceridemia is among the major changes in lipid metabolism that occur during endotoxemia. This process occurs as part of the host defense, as it serves to neutralize endotoxins, which can bind to circulating lipoproteins, thereby lowering the endotoxin-induced production of cytokines and controlling the inflammatory response. The increase in circulating TG levels during endotoxemia can be the result of both higher hepatic secretion and lower clearance at the periphery. As the dose of endotoxin increases, the contribution of TG hepatic secretion to hypertriglyceridemia decreases and that of TG clearance becomes more important. The results obtained here demonstrate that LPS at a dose of 15 mg/kg decreased the rate of TG secretion in rats while giving rise to hypertriglyceridemia, which was therefore entirely due to impaired intravascular TG hydrolysis.
Two recent studies have reported that the reduction in LPL activity in
brown adipocytes exposed to the cytokine tumor necrosis factor 
(TNF-) for 24 h was mediated by overproduction of NO. The
present findings demonstrate for the first time that the decrease in
muscle LPL activity in LPS-treated rats is mediated by overproduction
of NO via iNOS stimulation. This was demonstrated pharmacologically and
by genetic invalidation of iNOS. Quantitation of LPL mRNA indicated
that the LPS-induced reduction in LPL activity was caused by
post-transcriptional mechanisms, as previously suggested. It is also
important to note that AGN abrogated the LPS-induced reduction in
muscle LPL activity while inhibiting LPS-induced NO overproduction by
only half, as reflected by plasma
NO3/NO2 levels. This is
consistent with previous studies, which have clearly shown that
iNOS-mediated NO production does not have to be completely abolished in
order to counteract NO-induced effects. Finally, that AGN exerted its
action through iNOS inhibition is strongly supported by confirmation of
the findings in the iNOS knockout mice.
In WAT, it is clear from the present results that the LPS-induced reduction in LPL activity was not related to iNOS-mediated NO production, although we have shown in other studies that NO is overproduced in response to LPS in this tissue as well as in skeletal muscle. Indeed, AGN failed to prevent the decrease in LPL in all adipose depots studied. In addition, LPL activity of LPS-treated iNOS knockout mice was half that of saline-injected animals, further demonstrating that iNOS-mediated NO production is not implicated in the LPS-induced reduction in adipose LPL. As with muscle, the lack of change in LPL mRNA in response to LPS points to post-transcriptional mechanisms of action.
The present findings show that skeletal muscle LPL is an important determinant of triglyceridemia during high-dose endotoxemia. Indeed, in both animal models of iNOS invalidation, only muscle LPL was correlated with TG levels. Adipose tissue LPL did not appear to play a role in the process, since hypertriglyceridemia was reversed by iNOS inhibition although adipose LPL remained unchanged. It can be speculated that the preponderant influence of muscle LPL over that of adipose LPL on triglyceridemia in LPS-treated rats may be related to alterations in local blood flows and their consequences on lipoprotein–LPL interactions. Although skeletal muscle and WAT are the major sites of LPL production and action on lipoprotein-bound TG, other tissues such as the heart and brown adipose tissue contain substantial amounts of LPL and may have contributed to LPS-induced hypertriglyceridemia. However, this was not the case, since iNOS inhibition prevented hypertriglyceridemia without affecting heart LPL and brown adipose LPL was modulated in an opposite manner in mice and rats by LPS (data not shown). The present studies therefore demonstrate that hypertriglyceridemia induced by high-dose LPS is mediated by NO through its action on skeletal muscle LPL.
It has been shown in vivo that the effects of endotoxins on LPL are mediated by cytokines, since no decrease in tissue LPL activity is seen in C3H/HeJ mice whose macrophages do not produce cytokines in response to LPS. The molecular mechanisms responsible for the acute, generalized reduction in LPL activity after LPS administration remain unclear. The lack of short-term changes in LPL mRNA observed here and in several previous studies using various cell and animal models indicate that LPL is altered in the short term at post-transcriptional levels. Furthermore, total immunoreactive mass of LPL appears to be conserved for an extended period of time after LPS exposure, suggesting post-translational modes of action. It can be argued that since LPL exists in both inactive monomeric and active homodimeric forms, LPS may decrease the ratio of the dimeric to monomeric forms of the enzyme by affecting the covalent binding between the monomers. Alternatively, increased NO production results in the nitrosation of various proteins, perhaps including LPL, which may interfere with their biological activity. Finally, the effect of LPS and/or cytokines on protein degradation may extend to that of LPL. Further studies are clearly needed to unravel the iNOS-independent mode of action of LPS on LPL activity in tissues such as WAT, as well as the means whereby iNOS-mediated NO production reduces LPL activity in skeletal muscle.
In conclusion, the present studies show that the LPS-induced reduction
in skeletal muscle LPL activity is post-transcriptionally mediated by
iNOS-induced NO production. They also demonstrate that NO is a key
factor in the LPS-mediated increase in plasma TG concentration through
its modulation of LPL activity in skeletal muscle. The studies have
therefore identified a new role for NO in the alterations in lipid
metabolism typically associated with an immune challenge, as summarized
in Fig. 3
. The findings emphasize the need for future research into the possible
involvement of NO in disturbances of lipid and lipoprotein metabolism
that characterize chronic conditions associated with increased cytokine
production such as obesity, insulin resistance, and
inflammation.
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FOOTNOTES
1 To read the full text of this
article, go to
http://www.fasebj.org/cgi/doi/10.1096/fj.00-0830fje ; to cite this
article, use FASEB J. (June 27, 2001)
10.1096/fj.00-0830fje 
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lack of effect on variable below.