**5. Kupffer cell mediators and VLDL secretion**

During the acute phase of the septic response, Kupffer cells, the resident macrophages in the liver, release a plethora of soluble bioactive molecules, among them soluble proinflammatory mediators as cytokines, particularly TNF-, IL-6 and IL-1. These cytokines would act locally on nearby hepatocytes as paracrine factors promoting VLDL secretion and many other metabolic changes that accompany the inflammatory reaction.

We confirmed a rapid release of TNF- and IL-6 into the bloodstream in rats after 2 h of LPS injection (1 mg/Kg bw), whereas the IL-1 maximum level was observed at 6 h and the rise was less accentuated. Basal levels were recovered in all cases at 12 h from the LPS treatment (Bartolome et al., 2008).

Administration of cytokines to animals has been shown to mimic the sepsis induced alterations in lipid metabolism. TNF-, IL-6 and IL-1 administered to rodents induce triglyceride synthesis and promote rises in VLDL-TG (Khovidhunkit et al., 2004). In the case of TNF- and IL-6 administration, the hypertriglyceridemia has been reported to be secondary to higher lipolysis in peripheral tissues; consequently more fatty acids in blood are available and can be recruited by liver. In some studies, the three cytokines have been shown to stimulate hepatic de novo fatty acid synthesis (Feingold et al., 1989) and TG secretion (Feingold et al., 1991; Nonogaki et al., 1995), and decrease adipose tissue LPL activity (Feingold et al., 1994; Popa et al., 2007). Nevertheless, the reduction in LPL was delayed several hours with respect to hypertriglyceridemia (Esteve et al., 2005; Popa et al., 2007) and blockade of TNF- or IL-6 function in septic mice inhibited hypertriglyceridemia without affecting LPL activity (Feingold et al., 1994).

In order to address the role of Kupffer cells in VLDL oversecretion during endotoxemia, we analyzed the response of rat primary hepatocytes to the direct effect of LPS-stimulated KC or unstimulated products. Hepatocytes were cultured for 8 h with the conditioned medium containing the mediators generated in 16 h by Kupffer cells after a previous 4 h culture with or without LPS. The exposure of hepatocytes to unstimulated KC conditioned medium resulted in doubled the secretion VLDL particles of normal composition. Cells cultured in LPS stimulated KC medium secreted further more VLDL particles that were enriched in PL (Bartolome et al., 2008). Regarding to apoB expression, KC products multiplied by two the abundance of apoB mRNA and no further increment was caused by specific LPS-triggered products. In any case was MTP mRNA modified. The high PL available would protect apoB from degradation, explaining the increase in apoB secretion in cells challenged by LPS-KC medium.

There are few studies investigating the direct effect of cytokines on VLDL secretion, and contradictory results have been reported using different cell types. In HepG2 cells IL-6 and IL-1 were found to reduce apoB secretion although apoB mRNA levels were increased (Yokoyama et al., 1998). However, in IL-6 treated murine hepatocytes, enhanced apoB synthesis, which corresponded with high apoB mRNA levels, was found to be the primary mechanism for increased lipoprotein secretion (Sparks et al., 2010). They also found that IL-6 did not alter the decay rate of apoB mRNA transcripts, concluding that it favours secretion of apoB-containing lipoproteins by increasing availability of apoB through changes in *apob* gene transcription (Sparks et al., 2010).

Our studies in rat hepatocyte cultures, have demonstrated that the inflammatory cytokines TNF-α, IL-6 and IL-1β, over a wide range of concentrations, enhanced VLDL-apoB secretion linked to upregulation of apoB mRNA expression (Bartolome et al., 2007; Bartolome et al.,

During the acute phase of the septic response, Kupffer cells, the resident macrophages in the liver, release a plethora of soluble bioactive molecules, among them soluble proinflammatory mediators as cytokines, particularly TNF-, IL-6 and IL-1. These cytokines would act locally on nearby hepatocytes as paracrine factors promoting VLDL secretion and

We confirmed a rapid release of TNF- and IL-6 into the bloodstream in rats after 2 h of LPS injection (1 mg/Kg bw), whereas the IL-1 maximum level was observed at 6 h and the rise was less accentuated. Basal levels were recovered in all cases at 12 h from the LPS treatment

Administration of cytokines to animals has been shown to mimic the sepsis induced alterations in lipid metabolism. TNF-, IL-6 and IL-1 administered to rodents induce triglyceride synthesis and promote rises in VLDL-TG (Khovidhunkit et al., 2004). In the case of TNF- and IL-6 administration, the hypertriglyceridemia has been reported to be secondary to higher lipolysis in peripheral tissues; consequently more fatty acids in blood are available and can be recruited by liver. In some studies, the three cytokines have been shown to stimulate hepatic de novo fatty acid synthesis (Feingold et al., 1989) and TG secretion (Feingold et al., 1991; Nonogaki et al., 1995), and decrease adipose tissue LPL activity (Feingold et al., 1994; Popa et al., 2007). Nevertheless, the reduction in LPL was delayed several hours with respect to hypertriglyceridemia (Esteve et al., 2005; Popa et al., 2007) and blockade of TNF- or IL-6 function in septic mice inhibited hypertriglyceridemia

In order to address the role of Kupffer cells in VLDL oversecretion during endotoxemia, we analyzed the response of rat primary hepatocytes to the direct effect of LPS-stimulated KC or unstimulated products. Hepatocytes were cultured for 8 h with the conditioned medium containing the mediators generated in 16 h by Kupffer cells after a previous 4 h culture with or without LPS. The exposure of hepatocytes to unstimulated KC conditioned medium resulted in doubled the secretion VLDL particles of normal composition. Cells cultured in LPS stimulated KC medium secreted further more VLDL particles that were enriched in PL (Bartolome et al., 2008). Regarding to apoB expression, KC products multiplied by two the abundance of apoB mRNA and no further increment was caused by specific LPS-triggered products. In any case was MTP mRNA modified. The high PL available would protect apoB from degradation, explaining the increase in apoB secretion in cells challenged by LPS-KC

There are few studies investigating the direct effect of cytokines on VLDL secretion, and contradictory results have been reported using different cell types. In HepG2 cells IL-6 and IL-1 were found to reduce apoB secretion although apoB mRNA levels were increased (Yokoyama et al., 1998). However, in IL-6 treated murine hepatocytes, enhanced apoB synthesis, which corresponded with high apoB mRNA levels, was found to be the primary mechanism for increased lipoprotein secretion (Sparks et al., 2010). They also found that IL-6 did not alter the decay rate of apoB mRNA transcripts, concluding that it favours secretion of apoB-containing lipoproteins by increasing availability of apoB through changes in *apob*

Our studies in rat hepatocyte cultures, have demonstrated that the inflammatory cytokines TNF-α, IL-6 and IL-1β, over a wide range of concentrations, enhanced VLDL-apoB secretion linked to upregulation of apoB mRNA expression (Bartolome et al., 2007; Bartolome et al.,

many other metabolic changes that accompany the inflammatory reaction.

**5. Kupffer cell mediators and VLDL secretion** 

without affecting LPL activity (Feingold et al., 1994).

gene transcription (Sparks et al., 2010).

(Bartolome et al., 2008).

medium.

2008; Perez et al., 2006). IL-1β was the most potent and was the only one presenting a doseresponse effect. The effect of the three cytokines was redundant, as the increase was not additive when they were combined. However, none of the treatments with cytokines modified the amount of TG and total lipids secreted as components of VLDL, suggesting that these particles are lipid poor.

We conclude that Kupffer cells play a role in the rise of VLDL secretion detected during the inflammatory processes and that the three cytokines TNF-α, IL-6 and IL-1β may be involved, nevertheless other Kupffer cells mediators are necessary to accomplish increased lipid association.
