**2. Hiperlipemia of sepsis**

Elevation of plasma lipid levels is an early hallmark of sepsis, clinically defined as lipemia of sepsis. The rise in circulating lipids is mainly caused by a rapid accumulation of triglycerides within very low density lipoproteins (Esteve et al., 2005; Khovidhunkit et al., 2004), although other lipids such as non-esterified fatty acids coming from peripheral tissue lipolysis (Khovidhunkit et al., 2004), or cholesterol, in the case of rodents, can also be elevated (Feingold et al., 1993). However, decreases in cholesterol associated to high density lipoproteins (HDL) have been reported as a characteristic associated to sepsis in primates and rodents (Khovidhunkit et al., 2004).

Disrupted VLDL Features and Lipoprotein Metabolism in Sepsis 201

Notwithstanding the beneficial role of VLDL, many of the sepsis-associated changes in lipoprotein characteristics and metabolism are similar to those promoting atherogenesis, and has been proposed that the APR associated changes in lipoproteins can be one possible link between infection/inflammation and atherosclerosis. During acute phase reaction, apart from changes in HDL-cholesterol, alterations in HDL associated proteins have been reported. These changes alter cholesterol reverse transport, leading to diminished HDLcholesterol, and reduce their antioxidant properties (Esteve et al., 2005; Khovidhunkit et al., 2004). In fact, following acute infection low density lipoproteins (LDL) become more

One factor that may influence lipoprotein metabolism and is known to be altered during sepsis is food intake (Grunfeld et al., 1996). In order to avoid this variability, animals are

We have shown that endotoxin administration to fasted rats induced hipertriglyceridemia in a time-dependent pattern, related to different systemic fuels (Bartolome et al., 2010). Metabolic background is an important factor contributing to the sepsis-promoted VLDL abundance. We have demonstrated a biphasic response to endotoxin in systemic fuels of fasted rats, with two 12 h differentiated phases. We found that during the first phase serum fatty acids were markedly increased and glucose levels decreased, whereas in the second period hyperglycemia was recorded and fatty acid levels were bellow controls. Impaired glucose metabolism has been reported (McGuinness, 2005). During the second phase of the response we detected increased levels of insulin, that together with the high glucose levels indicate the reported sepsis induced insulin resistance (Andersen et al., 2004). It is well known that overproduction of VLDL is a characteristic of insulin-resistant states (Adeli et

**VLDL** 31,3-64 8,21\*\* 3,16\*\* 3,29\*\*\* 2,24\* large 44,5-64 9,21\*\*\* 2,74\* 4,69\*\*\* 2,37\* medium 36,8 6,62\*\*\* 4,26\*\* 2,89\*\*\* 2,27\* small 31,3 5,83\*\* 4,18\*\* 1,76 2,00 **LDL** 16,7-28,6 3,66\*\* 2,88\*\* 0,97 1,36 large 28,6 4,37\*\* 3,49\* 1,37 1,78 medium 26,5 3,35\*\* 2,87\* 1,19 1,63 small 23 3,08\*\* 2,61\* 1,05 1,48 very small 16,7-20,7 3,82\*\* 2,42\*\* 0,83 1,21 **HDL** 7,6-15 4,64\*\* 1,53\* 0,98 0,72\* very large 13,5-15 7,28\*\* 1,93\*\* 1,11 0,72 large 12,1 5,78\*\* 1,80\*\* 1,03 0,69\*\* medium 10,9 4,15\*\* 1,50\*\* 0,79\* 0,75\*\* small 9,8 4,84\*\*\* 2,13\*\* 0,83 0,76\*\* very small 7,6-8,8 4,12\*\* 1,24 0,93 0,79\* Table 1. Fold-changes induced by LPS administration in triglyceride and cholesterol concentration in lipoprotein classes. Rats were injected with LPS and blood collected for

Triglycerides Cholesterol 8 h 18 h 8 h 18 h

susceptible to oxidation (Memon et al., 2000).

al., 2001).

food-deprived from the time of administration of endotoxin.

mean diamenter (nm)

lipoprotein triglyceride and total cholesterol measurements.

The accumulation of VLDL particles in plasma is attributable to complex disturbances in their metabolism, including increased hepatic production (Feingold et al., 1992; Khovidhunkit et al., 2004; Lanza-Jacoby et al., 1998) and depressed peripheral clearance in the bloodstream by lipoprotein lipase (LPL) depending upon the dose (Feingold et al., 1992; Khovidhunkit et al., 2004; Lanza-Jacoby et al., 1998). Initially, the sepsis-induced hypertriglyceridemia was thought to constitute a mechanism for supplying high-energy substrates to cells involved in host defence (Hardardottir et al., 1994). However, it is increasingly believed that TG-rich lipoproteins are also components of an innate, nonadaptive host immune reaction against infection in humans and animal models (Barcia & Harris, 2005; Harris et al., 2000; Harris et al., 2002).

Both in vitro (Levels et al., 2001; Van Lenten et al., 1986) and in vivo (Kitchens et al., 2003) studies have demonstrated that all lipoprotein classes are able to bind LPS, through their phospholipid (Kitchens et al., 2003) or apolipoprotein (Levels et al., 2001; Vreugdenhil et al., 2001) components, in such a way that lipoprotein-bound LPS is subsequently cleared from the circulation by hepatic parenchymal cells (Harris et al., 2002). Most of the LPS-binding ability corresponds to HDL particles (Levels et al., 2001); however, when levels of VLDL are increased and HDL diminished, as may occur in endotoxemia, the binding appears to shift towards VLDL (Kitchens & Thompson, 2003; Vreugdenhil et al., 2001) and partially depends on interacting with apolipoprotein B (apoB) (Vreugdenhil et al., 2001). Therefore, higher secretion levels of VLDL may be regarded as a protective mechanism against infection. We have shown in LPS-treated rats that plasma VLDL-apoB is rapidly elevated, and this can represent a defence mechanism to neutralize and remove LPS from the circulation (Fig. 1).

Fig. 1. Proposed role of VLDL in the host inflammatory response. Inflammatory mediators released from Kupffer cells act on hepatocytes inducing the production of VLDL and the synthesis of APR proteins, among them LBP. LBP mediates the binding of LPS to VLDL, the complex is taken up by the hepatocyte and LPS is eliminated through the bile.

The accumulation of VLDL particles in plasma is attributable to complex disturbances in their metabolism, including increased hepatic production (Feingold et al., 1992; Khovidhunkit et al., 2004; Lanza-Jacoby et al., 1998) and depressed peripheral clearance in the bloodstream by lipoprotein lipase (LPL) depending upon the dose (Feingold et al., 1992; Khovidhunkit et al., 2004; Lanza-Jacoby et al., 1998). Initially, the sepsis-induced hypertriglyceridemia was thought to constitute a mechanism for supplying high-energy substrates to cells involved in host defence (Hardardottir et al., 1994). However, it is increasingly believed that TG-rich lipoproteins are also components of an innate, nonadaptive host immune reaction against infection in humans and animal models (Barcia &

Both in vitro (Levels et al., 2001; Van Lenten et al., 1986) and in vivo (Kitchens et al., 2003) studies have demonstrated that all lipoprotein classes are able to bind LPS, through their phospholipid (Kitchens et al., 2003) or apolipoprotein (Levels et al., 2001; Vreugdenhil et al., 2001) components, in such a way that lipoprotein-bound LPS is subsequently cleared from the circulation by hepatic parenchymal cells (Harris et al., 2002). Most of the LPS-binding ability corresponds to HDL particles (Levels et al., 2001); however, when levels of VLDL are increased and HDL diminished, as may occur in endotoxemia, the binding appears to shift towards VLDL (Kitchens & Thompson, 2003; Vreugdenhil et al., 2001) and partially depends on interacting with apolipoprotein B (apoB) (Vreugdenhil et al., 2001). Therefore, higher secretion levels of VLDL may be regarded as a protective mechanism against infection. We have shown in LPS-treated rats that plasma VLDL-apoB is rapidly elevated, and this can represent a defence mechanism to neutralize and remove LPS from the circulation (Fig. 1).

Fig. 1. Proposed role of VLDL in the host inflammatory response. Inflammatory mediators released from Kupffer cells act on hepatocytes inducing the production of VLDL and the synthesis of APR proteins, among them LBP. LBP mediates the binding of LPS to VLDL, the

complex is taken up by the hepatocyte and LPS is eliminated through the bile.

Harris, 2005; Harris et al., 2000; Harris et al., 2002).

Notwithstanding the beneficial role of VLDL, many of the sepsis-associated changes in lipoprotein characteristics and metabolism are similar to those promoting atherogenesis, and has been proposed that the APR associated changes in lipoproteins can be one possible link between infection/inflammation and atherosclerosis. During acute phase reaction, apart from changes in HDL-cholesterol, alterations in HDL associated proteins have been reported. These changes alter cholesterol reverse transport, leading to diminished HDLcholesterol, and reduce their antioxidant properties (Esteve et al., 2005; Khovidhunkit et al., 2004). In fact, following acute infection low density lipoproteins (LDL) become more susceptible to oxidation (Memon et al., 2000).

One factor that may influence lipoprotein metabolism and is known to be altered during sepsis is food intake (Grunfeld et al., 1996). In order to avoid this variability, animals are food-deprived from the time of administration of endotoxin.

We have shown that endotoxin administration to fasted rats induced hipertriglyceridemia in a time-dependent pattern, related to different systemic fuels (Bartolome et al., 2010). Metabolic background is an important factor contributing to the sepsis-promoted VLDL abundance. We have demonstrated a biphasic response to endotoxin in systemic fuels of fasted rats, with two 12 h differentiated phases. We found that during the first phase serum fatty acids were markedly increased and glucose levels decreased, whereas in the second period hyperglycemia was recorded and fatty acid levels were bellow controls. Impaired glucose metabolism has been reported (McGuinness, 2005). During the second phase of the response we detected increased levels of insulin, that together with the high glucose levels indicate the reported sepsis induced insulin resistance (Andersen et al., 2004). It is well known that overproduction of VLDL is a characteristic of insulin-resistant states (Adeli et al., 2001).


Table 1. Fold-changes induced by LPS administration in triglyceride and cholesterol concentration in lipoprotein classes. Rats were injected with LPS and blood collected for lipoprotein triglyceride and total cholesterol measurements.

Disrupted VLDL Features and Lipoprotein Metabolism in Sepsis 203

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,

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, making them more proatherogenic.

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 HDLcholesterol levels fell in all HDL subclasses.
