**3. Altered VLDL metabolism in sepsis**

The assembly of VLDL particles is a complex and highly regulated process that occurs in the secretory pathway of hepatocytes. It represents an active export process of fuel carbons, mainly in the form of TG, and is an important route for cholesteryl ester and phospholipid secretion to the circulation. The biogenesis of VLDL has been mostly described as a two step process depending on the cellular availability of lipids, such as triglycerides, phospholipids, cholesterol, and cholesteryl esters, and it is absolutely dependent on the provision of functional apoB, which, in rodents, may be either the full length apoB-100 or the truncated form of apoB-48 (Davidson & Shelness, 2000). Firstly, during translocation to the lumen across the endoplasmic reticulum (ER) membrane, nascent apoB is lipidated by the essential chaperone microsomal triglyceride transfer protein (MTP) (Gordon & Jamil, 2000; Hussain et al., 2003; Liang & Ginsberg, 2001), originating a relatively small, dense, TG-poor lipoprotein particle. In the second stage, bulk of lipidation and final maturation of lipoprotein precursor occur in the ER and post-ER compartments to form mature VLDL (Gusarova et al., 2003; Kulinski et al., 2002). It is known that when MTP activity is low, or when lipid availability or synthesis is reduced, apoB is cotranslationally targeted for ER-associated degradation by both proteasomedependent and non-dependent pathways (Fisher et al., 2001; Fisher & Ginsberg, 2002; Ginsberg & Fisher, 2009).

The apoB gene has been considered to be constitutively expressed (Pullinger et al., 1989) and VLDL assembly regulation as a post-transcriptional event. However, increasing evidence from in vivo and in vitro studies over the last years has shown changes in hepatic steadystate mRNA levels for apoB in several pathophysiological conditions, particularly under a variety of inflammatory conditions (Jura et al., 2004; Yokoyama et al., 1998).

VLDL secretion rate and composition can be modulated by a variety of factors, such as nutritional state (Gibbons & Burnham, 1991), endotoxin and proinflammatory cytokines (Aspichueta et al., 2006; Bartolome et al., 2007; Perez et al., 2006). Different mechanisms may be involved in the sepsis enhanced VLDL secretion (Fig. 2).

We analyzed the lipoprotein lipid profile in serum of rats after 8 or 18 h of LPS treatment (Table 1). We found that hypertriglyceridemia was associated with different VLDL, LDL and HDL subclasses depending on the metabolic background of the APR. Although TG increased in all lipoprotein classes, VLDL particles were the major contributors. We did find transient proatherogenic changes in VLDL particles. During the first phase of the APR hypertriglyceridemia was predominantly associated to large VLDL, which were increased 8 fold after 8 h (Bartolome et al., 2010). These large TG-rich VLDL particles, more than normal VLDL, are able to cross the endothelial barrier and interact with lipoprotein receptors in macrophages, initiating a sequence of events that result in the atherosclerotic lesion and, in addition they give rise to small-dense atherogenic LDL (Gianturco et al., 1998; Ginsberg, 2002; Taskinen, 2003). In addition, large TG-rich VLDL were also enriched in cholesterol,

In the second phase, the rise in serum VLDL-TG corresponded mainly to medium and small VLDL particles. Endotoxin did not affected serum total cholesterol, however changes occurs in lipoprotein subclasses. Total cholesterol increased in large and medium VLDL and HDL-

The assembly of VLDL particles is a complex and highly regulated process that occurs in the secretory pathway of hepatocytes. It represents an active export process of fuel carbons, mainly in the form of TG, and is an important route for cholesteryl ester and phospholipid secretion to the circulation. The biogenesis of VLDL has been mostly described as a two step process depending on the cellular availability of lipids, such as triglycerides, phospholipids, cholesterol, and cholesteryl esters, and it is absolutely dependent on the provision of functional apoB, which, in rodents, may be either the full length apoB-100 or the truncated form of apoB-48 (Davidson & Shelness, 2000). Firstly, during translocation to the lumen across the endoplasmic reticulum (ER) membrane, nascent apoB is lipidated by the essential chaperone microsomal triglyceride transfer protein (MTP) (Gordon & Jamil, 2000; Hussain et al., 2003; Liang & Ginsberg, 2001), originating a relatively small, dense, TG-poor lipoprotein particle. In the second stage, bulk of lipidation and final maturation of lipoprotein precursor occur in the ER and post-ER compartments to form mature VLDL (Gusarova et al., 2003; Kulinski et al., 2002). It is known that when MTP activity is low, or when lipid availability or synthesis is reduced, apoB is cotranslationally targeted for ER-associated degradation by both proteasomedependent and non-dependent pathways (Fisher et al., 2001; Fisher & Ginsberg, 2002;

The apoB gene has been considered to be constitutively expressed (Pullinger et al., 1989) and VLDL assembly regulation as a post-transcriptional event. However, increasing evidence from in vivo and in vitro studies over the last years has shown changes in hepatic steadystate mRNA levels for apoB in several pathophysiological conditions, particularly under a

VLDL secretion rate and composition can be modulated by a variety of factors, such as nutritional state (Gibbons & Burnham, 1991), endotoxin and proinflammatory cytokines (Aspichueta et al., 2006; Bartolome et al., 2007; Perez et al., 2006). Different mechanisms may

variety of inflammatory conditions (Jura et al., 2004; Yokoyama et al., 1998).

be involved in the sepsis enhanced VLDL secretion (Fig. 2).

making them more proatherogenic.

Ginsberg & Fisher, 2009).

cholesterol levels fell in all HDL subclasses.

**3. Altered VLDL metabolism in sepsis** 

Fig. 2. Model of VLDL assembly and secretion during sepsis. FAT, fatty acid translocase; FAS, fatty acid synthase; ACC, Acetyl-CoA carboxylase; CPT, carnitine acyltransferase. Solid and blue arrows indicate increases, discontinuous and red arrows indicate decreases.

Our results suggest that specific mechanisms are involved in the temporal response to sepsis. In LPS treated rats we found that both fatty acids and hypertriglyceridemia, associated with VLDL-TG, peaked after 8 hours of endotoxin contact. During inflammation adipose tissue lipolysis is activated by pro-inflammatory mediators (Khovidhunkit et al., 2004; Zu et al., 2009) providing fatty acids for hepatic triglyceride synthesis, thus promoting VLDL secretion (Lanza-Jacoby et al., 1998). It has been reported that LPS enhance the expression of fatty acid translocase FAT/CD36, involved in fatty acid uptake (Memon et al., 1998a) and that endotoxin and cytokines suppressed mitochondrial acyl-CoA synthetase expression and activity (Memon et al., 1998b) but enhanced microsomal acyl-CoA synthetase. In addition, LPS administration to rats led to reduced carnitine acyltransferase I, lower ketogenic capacity (Takeyama et al., 1990) and decreased levels of serum ketone bodies (Bartolome et al., 2010). Evidences also established a relationship between increased de novo fatty acid synthesis and enhanced secretion of VLDL-TG in rodents treated with LPS or cytokines (Feingold et al., 1992; Lanza-Jacoby & Tabares, 1990). Taken all together, high amounts of fatty acids are directed away from mitochondrial oxidation and are available for their esterification into TG and secreted within VLDL. However, previous works done in our laboratory did not support the proposed hypothesis since levels of fatty acid synthase mRNA or rate of TG synthesis measured as the incorporation of [3H]acetate or [3H]oleate did not change after 18 h of LPS treatment (Aspichueta et al., 2006).

The high availability of lipids in the septic hepatocyte would protect apoB from degradation leading to an increased number of secreted VLDL particles (Phetteplace et al., 2000). In fact,

Disrupted VLDL Features and Lipoprotein Metabolism in Sepsis 205

genetic expression was of a lesser extend in PV cells. Regarding to de novo synthesis of lipids for VLDL assembly, the incorporation of [3H]acetate into TG and cholesterol did not

We concluded that periportal and perivenous hepatocytes exhibited similar capabilities for VLDL assembly and secretion in normal conditions; and, only the endotoxic condition led

Fig. 3. Proposed model for the biphasic response to endotoxin in VLDL metabolism. In the first phase the stimulation of lipolysis provides fatty acids that are taken by the liver and esterified to be secreted into TG-rich VLDL. In the second phase apoB mRNA levels are

Fig. 4. Model of VLDL secretion by periportal and perivenous hepatocytes in fed state and 18 h after fasting or endotoxin treatment. Endotoxin effect when compared with the fasted

increased providing the apolipoprotein for secretion of TG-poor VLDL.

PP hepatocytes to a marked increase in TG-poor VLDL secretion (Fig 4).

change by endotoxin challenge.

state is marked with

we detected an elevation of 5 fold in the number of circulating VLDL particles, measured as apoB quantities, at 8 h from LPS administration, without any modification in apoB transcript level. The increment in VLDL-TG is of greater magnitude (8 fold), indicating that during the first phase of the septic response TG-rich VLDL particles accumulate in the circulation (Bartolome et al., 2010).

Different mechanisms seem to be involved in the second phase of septic response. The serum fatty levels drop below controls, which would suggest a lower availability of fatty acids of extrahepatic origin for hepatic VLDL-TG secretion.

Endotoxic rats showed a higher number (10 fold) of circulating VLDL particles in rats at 18 h, but the content of the lipid in each VLDL is reduced (Aspichueta et al., 2006; Bartolome et al., 2010). This was accompanied by high levels of *apob* gene transcript, which could provide for high apoB availability increasing the secretion of lipid poor VLDL particles. Using intact rats in the fasted state, injected with the LPL inhibitor Triton WR-1339, we have shown that the TG and cholesterol secreted into VLDL released by the liver to the blood in 2 h was not enhanced by LPS administration to the same extent as the VLDL-apoB production was (Aspichueta et al., 2005). In this way, hepatocytes isolated from 18 hour LPS-treated rats secreted TG-poor VLDL, and although secretion was highly stimulated, global triglyceride secretion in VLDL remained unchanged. This was related to unchanged rates of fatty acid esterification, measured as [3H]oleate incorporation into TG (Aspichueta et al., 2005; Aspichueta et al., 2006).

In septic hypertriglyceridemic rats, 24 h after sepsis induction, the increase in plasma TG was associated to a decrease in VLDL-TG clearance rate, due to suppressed mRNA levels, protein mass and activity of LPL in peripheral tissues (Lanza-Jacoby et al., 1997; Lanza-Jacoby & Tabares, 1990). Thus, in the early phase of the septic reaction hypertriglyceridemia is mostly due to high VLDL secretion driven by availability of lipids in the hepatocyte; and during the second phase, hypertriglyceridemia would be the result of LPL inhibition, and the increase in apoB transcription would be responsible for the increased secretion of VLDL particles (Fig.3).
