**6. Higher apoB availability within the hepatocytes**

As stated before, the assembly of VLDL is a complex process that depends on the availability of lipids and apoB (Davidson & Shelness, 2000).

Since we have found that under LPS treatment VLDL-apoB secretion was always increased, and given that not always enhanced apoB secretion is linked to high levels of apoB transcript, we hypothesized that during the acute phase response, transcriptional or posttranscriptional regulation affecting apoB mRNA levels might occur supplying more apoB for VLDL assembly.

During the first phase of the septic response we detected elevated circulating VLDL-apoB and -TG after 8 h of LPS treatment without altered apoB transcript levels. Taking into account that at this time point of the septic response, circulating fatty acid levels were elevated, we propose that fatty acid uptake by the liver is increased and large amounts of TG are synthesized. Since the N-terminus of apoB acquires neutral lipids in the endoplasmic reticulum membrane (Hussain et al., 2008), more nucleation sites are expected to be generated in apoB leading to increased apoB secretion. This could result in an increased hepatic secretion of triglycerides in VLDL particles, which would accumulate in the circulation, even in the absence of augmented levels of hepatic apoB mRNA (Bartolome et al., 2010).

In the second phase of the septic response, after 18 h of LPS challenge, enhanced VLDLapoB secretion is accompanied by increased apoB mRNA levels (Bartolome et al., 2010). In addition, hepatocytes isolated 18 h after LPS administration presented higher levels of apoB transcript and secreted more VLDL particles, been this effect more marked in PP cells. At this time point of the septic response, lipid poor VLDL particles are secreted (Aspichueta et al., 2005) and lipid synthesis is not modified (Aspichueta et al., 2006). Therefore, the increase in *apob* gene transcript would provide the additional apoB necessary to enhance VLDL-apoB secretion. Similarly, the inflammatory cytokines TNF-α (Bartolome et al., 2007), IL1-β (Bartolome et al., 2010) and IL-6 (Perez et al., 2006) augmented the levels of apoB mRNA and secretion of VLDL particles without changing the amounts of lipid secreted in the VLDL.

Our hypothesis was that endotoxin-enhanced VLDL-apoB secretion was driven by higher transcription rates. However, we did not find a rise in transcription rate of *apob* gene when we measured the incorporation of 5'-[α-32P]-UTP into newly synthesized RNA in liver nuclei from 16 h LPS-treated rats (Bartolome et al., 2010). We reported that global transcription rate in endotoxic liver was nearly two times higher than in control rats as expected in the acute phase response for up-regulating the positive proteins. However, the transcription rate of

Disrupted VLDL Features and Lipoprotein Metabolism in Sepsis 209

poor VLDL accumulation. In the later stage, the endotoxin induced VLDL secretion is more accentuated in periportal cells. Kupffer cells released products directly promote VLDL assembly and secretion and increase apoB mRNA levels, among these products the cytokines TNF–α, IL-6 and IL-1β and other mediator/s could play a role in the enhancement

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This work was supported by the Basque Government (IT336-10).

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*Endotoxin. Res*, Vol.12, No.3, pp. 181-192, ISSN 0968-0519

*Innate. Immun.*, ISSN 1753-4259 Epub ahead of print

Vol.1096, pp. 55-69, ISSN 0077-8923

168, ISSN 0013-9432

0199-9885

of VLD secretion.

**9. References** 

**8. Acknowledgments** 

ISSN 0741-5400

1753-4259

pp. 1017-1026, ISSN 0022-2275

apoB gene was unaffected after 16 h of LPS challenge in the treated animals. It cannot be discarded that transcriptional activation may occur transiently during other stages of the APR.

Another aspect involved in regulating mRNA level is the modulation of mRNA stability through regulatory elements residing in the 3'- and 5'-untranslated region (UTR) and adequate RNA binding proteins. HuR is an important protein in stabilizing inflammatory AU-rich elements (ARE)-bearing RNAs. Human apoB mRNA has been reported to contain ARE sequences at 3'-UTRs and bioinformatic analysis of rat apoB transcript revealed the presence of AU-rich regions. Our results demonstrated the specific binding of stabilizing HuR protein to the rat apoB mRNA, although there were no superior binding in livers from LPS treated rats. Consequently, in our conditions it is not likely that apoB mRNA half-life was extended by HuR binding, but we can not discard a role for other stabilizing proteins or changes in the mRNA degradation pathway, but further analysis is need (Fig 5).

Fig. 5. Endotoxin induce increase in apoB mRNA without altering transcription rate or HuR protein binding.
