Effect of impaired intestinal lipid transport on serum lipoproteins.

<p>Lipoprotein distribution measured by fast protein liquid chromatography in control (A) and <i>Mttp-IKO</i> (B) mice. Pooled samples of serum from n = 5–10 mice per genotype were analyzed in sham animals and both before and 24 hr after induction of pneumonia in the experimental groups. Cholesterol was assayed enzymatically and peaks corresponding to fractions 10–16 indicate particles in the low density lipoprotein (LDL) range while fractions 19–26 correspond to high density lipoprotein (HDL). (C) Expression of genes implicated in hepatic cholesterol efflux were analyzed by qRT-PCR on samples of RNA from the indicated groups (n = 4 mice per genotype and treatment) (D) and (E). Expression of hepatic Abca1, apoA1 and apoA4 protein by SDS-PAGE and western blot. Gapdh was used as loading control. Panel D shows representative Western blotting results. Panel E shows densitometric scanning from groups of 4 mice per genotype and treatment. (F) Expression of genes implicated in intestinal lipid metabolism were analyzed by qRT-PCR on samples of small intestinal RNA from the indicated groups (n = 4 mice per genotype and treatment). *p<0.05.</p

Effect of impaired intestinal lipid transport on lung cytokines.

<p>Cytokines were measured in bronchoalveolar lavage (BAL) fluid 24 hr after induction of pneumonia. Although several cytokines were elevated during pneumonia, there were no differences between septic <i>Mttp-IKO</i> and control mice. n = 8–10/group.</p

Effect of impaired intestinal lipid transport on intestinal morphology and proliferation.

<p>Intestinal morphology (A) was evaluated in H&E-stained sections. Magnification 20×. Villus length (B) and crypt depth (C) were quantified in jejunal sections. Control mice subjected to <i>P. aeruginosa</i> pneumonia had significantly shorter villi and smaller crypts than sham mice, while both villus length and crypt depth was restored or nearly restored to sham levels in septic <i>Mttp-IKO</i> mice. n = 9 shams/genotype, n = 17 septics/genotype. (D) S-phase cells were quantified in 100 crypts. Control mice subjected to <i>P. aeruginosa</i> pneumonia had significantly decreased intestinal proliferation compared to control sham mice, while septic <i>Mttp-IKO</i> mice exhibited increased proliferative capacity. n = 6–7 shams/genotype, n = 9–11 septics/genotype. (E) Intestinal tissue was stained with osmium to detect intracellular lipid droplets, which appear as dark black staining material. (F) Mucosal concentrations of triglycerides and (G) cholesterol were measured in jejunum, the data expressed as µg/mg protein. n = 5/group.</p

Effect of impaired intestinal lipid transport on serum lipopolysaccharide (LPS).

<p>Serum LPS was increased in septic control mice, but not in septic <i>Mttp-IKO</i> mice. Serum LPS concentration was measured using the LAL chromogenic endotoxin kit (Methods). N = 5–8 per group. *p<0.05.</p

Impaired intestinal lipid transport alters serum lipid concentrations in septic mice.

<p>TG = triglycerides, Chol = cholesterol, PL = phospholipid, FFA = free fatty acids.</p><p>All results are expressed as mean ± SEM.</p>a<p>p<0.001 vs. Pre Control Sham;</p>b<p>p<0.001 vs. Post Control Sham;</p>c<p>p<0.001 vs. Post Control Septic;</p>d<p>p<0.001 vs. Pre <i>Mttp-IKO</i> Septic;</p>e<p>p<0.001 vs. Pre Control Septic;</p>f<p>p<0.01 vs. Post <i>Mttp-IKO</i> Sham.</p

Effect of impaired intestinal lipid transport on systemic cytokines.

<p>Cytokines were measured in serum 24 hr after induction of pneumonia. The proinflammatory cytokines IL-6 and G-CSF were increased in septic control mice; however, these cytokines were reduced in septic <i>Mttp-IKO</i> mice. n = 4–5 shams/genotype, n = 13–15 septics/genotype.</p

Effect of impaired intestinal lipid transport on pulmonary bacterial clearance and neutrophil infiltration.

<p>Control mice with pneumonia had increased bacterial burden in the lungs compared to shams (A). There was less bacteria in the BAL fluid of <i>Mttp-IKO</i> mice with pneumonia, but the difference was not statistically significant compared to control septic mice. Myeloperoxidase (MPO) activity was evaluated as an index of neutrophil infiltration and degranulation in BAL fluid (B). Mice subjected to <i>P. aeruginosa</i> pneumonia exhibited elevated MPO activity compared to shams; however, the lack of intestinal lipid absorption did not significantly alter neutrophil activation. n = 3–5 shams/genotype, n = 8–10 septics/genotype.</p

Effect of impaired intestinal lipid transport on mortality in <i>P. aeruginosa</i> pneumonia.

<p>Mice with intestine-specific deletion of microsomal triglyceride transfer protein (<i>Mttp-IKO</i>) and control mice were subjected to <i>P. aeruginosa</i> pneumonia. Sham mice received intratracheal injections of saline. All mice were followed for survival for 7 days. Mice with impaired intestinal lipid transport exhibited significantly improved survival compared to control mice (p<0.05). All sham mice survived.</p

Effect of impaired intestinal lipid transport on intestinal epithelial apoptosis.

<p>Intestinal epithelial apoptosis was evaluated by active caspase-3 staining (A) and H&E staining (B) in 100 crypts. Control mice subjected to <i>P. aeruginosa</i> pneumonia exhibited increased intestinal apoptosis by both methods. In contrast, <i>Mttp-IKO</i> mice with pneumonia had similar levels of intestinal apoptosis as sham mice.n = 6–7 shams/genotype, n = 16–18 septics/genotype. The gene expression ratios of pro-apoptotic Bax to anti-apoptotic Bcl-2 (C) and Bcl-xL (D) were evaluated. Septic <i>Mttp-IKO</i> mice had significantly decreased ratios compared to septic control mice. n = 11/group.</p

Effect of impaired intestinal lipid transport on serum lipoproteins.

<p>Triglyceride (A) and cholesterol (B) in serum were measured before and 24 hours after induction of pneumonia in the same mice. Sepsis decreased both triglyceride and cholesterol after induction of pneumonia in WT mice (C, D, n = 16–18/group, p<0.0001 for both). Inhibiting chylomicron assembly also resulted in lower triglyceride and cholesterol at baseline (n = 17–18/group, p<0.0001 for both). In contrast, sepsis did not alter triglyceride levels in <i>Mttp-IKO</i> mice (n = 11–17/group, p>0.05). Additionally, sepsis increased cholesterol levels in <i>Mttp-IKO</i> mice after induction of pneumonia (n = 11–17/group, p<0.0001). Sepsis virtually eliminated triglyceride from large, VLDL and LDL size lipoproteins in WT mice (note fractions 10 and 25, respectively). By contrast, Mttp-IKO mice exhibited virtually no triglyceride in lipoprotein particles, as expected. Sepsis resulted in an increase in HDL cholesterol content in <i>Mttp-IKO</i> mice (fractions 38–40). Serum bile acids (E) were similar between WT and <i>Mttp-IKO</i> mice (n = 7–8/group, p>0.05).</p