John Fuller

Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters.

The expression of leptin, the ob gene product, is increased in adipose tissue in response to feeding and energy repletion, while leptin decreases food intake. Because adipose tissue gene expression is regulated by cytokines induced during infection and because infection is associated with anorexia, we tested whether induction of leptin might occur during the host response to infection. Administration of endotoxin (LPS), a model for gram negative infections, induces profound anorexia and weight loss in hamsters. In fasted adipose tissue to levels similar to fed control animals. There is a strong inverse correlation between mRNA levels of leptin and subsequent food intake. TNF and IL-1, mediators of the host response to LPS, also induced anorexia and increased levels of leptin in mRNA in adipose tissue. As assessed by immuknoprecipitation and Western blotting, circulating leptin protein is regulated by LPS and cytokines in parallel to regulation of adipose tissue leptin mRNA. Induction of leptin during the host response to infection may contribute to the anorexia of infection.

IL-1β mediates leptin induction during inflammation

Interleukins (IL) are key mediators of the host response to infection and inflammation. Leptin is secreted by adipose tissue and plays an important role in the control of food intake. Administration of lipopolysaccharide (LPS), tumor necrosis factor (TNF), or IL-1 acutely increases leptin mRNA and protein levels. To investigate the role of IL-1β and IL-6 in leptin expression during inflammation, we used IL-1β-deficient (-/-) and IL-6 -/- mice. Mice were injected intraperitoneally with LPS or subcutaneously with turpentine, as models of systemic or local inflammation, respectively. In IL-1β +/+ mice, both LPS and turpentine increased leptin mRNA and circulating leptin. In contrast, neither LPS nor turpentine increased leptin levels in IL-1β -/- mice. In IL-6 +/+ or IL-6 -/- mice, turpentine increased leptin protein to comparable levels. We conclude that IL-1β is essential for leptin induction by both LPS and turpentine in mice, but IL-6 is not.

Endotoxin and Cytokines Increase Hepatic Sphingolipid Biosynthesis and Produce Lipoproteins Enriched in Ceramides and Sphingomyelin

Alterations in triglyceride and cholesterol metabolism often accompany inflammatory diseases and infections. We studied the effects of endotoxin (lipopolysaccharide [LPS]) and cytokines on hepatic sphingolipid synthesis, activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid synthesis, and lipoprotein sphingolipid content in Syrian hamsters. Administration of LPS induced a 2-fold increase in hepatic SPT activity. The increase in activity first occurred at 16 hours, peaked at 24 hours, and was sustained for at least 48 hours. Low doses of LPS produced maximal increases in SPT activity, with half-maximal effect seen at approximately 0.3 microg LPS/100 g body weight. LPS increased hepatic SPT mRNA levels 2-fold, suggesting that the increase in SPT activity was due to an increase in SPT mRNA. LPS treatment also produced 75% and 2.5-fold increases in hepatic sphingomyelin and ceramide synthesis, respectively. Many of the metabolic effects of LPS are mediated by cytokines. Interleukin 1 (IL-1), but not tumor necrosis factor, increased both SPT activity and mRNA levels in the liver of intact animals, whereas both IL-1 and tumor necrosis factor increased SPT mRNA levels in HepG2 cells. IL- produced a 3-fold increase in SPT mRNA in HepG2 cells, and the half-maximal dose was 2 ng/mL. IL-1 also increased the secretion of sphingolipids into the medium. Analysis of serum lipoprotein fractions demonstrated that very low density lipoprotein, intermediate density lipoprotein, and low density lipoprotein isolated from animals treated with LPS contained significantly higher amounts of ceramide, glucosylceramide, and sphingomyelin. Taken together, these results indicate that LPS and cytokines stimulate hepatic sphingolipid synthesis, which results in an altered structure of circulating lipoproteins and may promote atherogenesis.

Regulation of fatty acid transport protein and fatty acid translocase mRNA levels by endotoxin and cytokines.

The cloning of two novel fatty acid (FA) transport proteins, FA transport protein (FATP) and FA translocase (FAT), has recently been reported; however, little is known about their in vivo regulation. Endotoxin [lipopolysaccharide (LPS)], tumor necrosis factor (TNF), and interleukin-1 (IL-1) stimulate adipose tissue lipolysis and enhance hepatic lipogenesis and reesterification while suppressing FA oxidation in multiple tissues. Hence, in this study we examined their effects on FATP and FAT mRNA levels in Syrian hamsters. Our results demonstrate that LPS decreased FATP and FAT mRNA expression in adipose tissue, heart, skeletal muscle, brain, spleen, and kidney, tissues in which FA uptake and/or oxidation is decreased during sepsis. In the liver, where FA oxidation is decreased during sepsis but the uptake of peripherally derived FA is increased to support reesterification, LPS decreased FATP mRNA expression by 70-80% but increased FAT mRNA levels by four- to fivefold. The effects of LPS on FATP and FAT mRNA levels in liver were observed as early as 4 h after administration and were maximal by 16 h. TNF and IL-1 mimicked the effect of LPS on FATP and FAT mRNA levels in both liver and adipose tissue. These results indicate that the mRNAs for both transport proteins are downregulated by LPS in tissues in which FA uptake and/or oxidation are decreased during sepsis. On the other hand, differential regulation of FATP and FAT mRNA in liver raises the possibility that these proteins may be involved in transporting FA to different locations inside the cell. FATP may transport FA toward mitochondria for oxidation, which is decreased in sepsis, whereas FAT may transport FA to cytosol for reesterification, which is enhanced in sepsis.The cloning of two novel fatty acid (FA) transport proteins, FA transport protein (FATP) and FA translocase (FAT), has recently been reported; however, little is known about their in vivo regulation. Endotoxin [lipopolysaccharide (LPS)], tumor necrosis factor (TNF), and interleukin-1 (IL-1) stimulate adipose tissue lipolysis and enhance hepatic lipogenesis and reesterification while suppressing FA oxidation in multiple tissues. Hence, in this study we examined their effects on FATP and FAT mRNA levels in Syrian hamsters. Our results demonstrate that LPS decreased FATP and FAT mRNA expression in adipose tissue, heart, skeletal muscle, brain, spleen, and kidney, tissues in which FA uptake and/or oxidation is decreased during sepsis. In the liver, where FA oxidation is decreased during sepsis but the uptake of peripherally derived FA is increased to support reesterifiation, LPS decreased FATP mRNA expression by 70-80% but increased FAT mRNA levels by four- to fivefold. The effects of LPS on FATP and FAT mRNA levels in liver were observed as early as 4 h after administration and were maximal by 16 h. TNF and IL-1 mimicked the effect of LPS on FATP and FAT mRNA levels in both liver and adipose tissue. These results indicate that the mRNAs for both transport proteins are downregulated by LPS in tissues in which FA uptake and/or oxidation are decreased during sepsis. On the other hand, differential regulation of FATP and FAT mRNA in liver raises the possibility that these proteins may be involved in transporting FA to different locations inside the cell. FATP may transport FA toward mitochondria for oxidation, which is decreased in sepsis, whereas FAT may transport FA to cytosol for reesterification, which is enhanced in sepsis.

Endotoxin and Cytokines Decrease Serum Levels and Extra Hepatic Protein and mRNA Levels of Cholesteryl Ester Transfer Protein in Syrian Hamsters

Endotoxin alters the metabolism of lipoproteins, including that of high density lipoprotein (HDL). Cholesteryl ester transfer protein (CETP) facilitates exchange of HDL cholesterol for very low density lipoprotein (VLDL) triglyceride, leading to catabolism of HDL. We investigated the effects of endotoxin and cytokines on CETP in Syrian hamsters. Endotoxin induced a rapid and progressive decrease in serum CETP levels, by 48 h CETP had decreased to < 20% of control levels. Endotoxin also decreased CETP mRNA and protein levels in adipose tissue, heart, and muscle, the tissues with highest levels of CETP mRNA, providing a plausible mechanism for the endotoxin-induced decrease in circulating CETP. Dexamethasone did not mimic the effects of endotoxin on CETP, but the combination of tumor necrosis factor and interleukin-1 did, indicating that these cytokines may in part mediate the effects of endotoxin on CETP. The endotoxin-induced decrease in CETP may help maintain HDL cholesterol levels during infection and inflammation when increased triglyceride levels could drive the exchange of HDL cholesteryl ester for VLDL triglyceride. Maintaining circulating HDL may be important because HDL protects against the toxic effects of endotoxin and provides cholesterol for peripheral cells involved in the immune response and tissue repair.

In vivo regulation of acyl-CoA synthetase mRNA and activity by endotoxin and cytokines

Acyl-CoA synthetase (ACS) catalyzes the activation of fatty acids (FA) to acyl-CoA esters, which are further metabolized in either anabolic or catabolic pathways. Endotoxin [lipopolysaccharide (LPS)], tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhance hepatic FA synthesis and reesterification and inhibit FA oxidation. LPS also decreases triglyceride storage in adipose tissue and inhibits the uptake of FA by heart and muscle. Therefore, in this study we examined the effects of LPS and cytokines on ACS (now also known as ACS1) mRNA expression and activity in multiple tissues in Syrian hamsters. LPS markedly decreased ACS1 mRNA levels in liver, adipose tissue, heart, and skeletal muscle. The inhibitory effects of LPS on ACS1 mRNA levels in liver and adipose tissue were observed as early as 2-4 h after administration, became maximal by 4-8 h, and were sustained for ≥24 h. Very low doses of LPS (0.1-1 μg/100 g body wt) were needed to reduce ACS1 mRNA levels in liver and adipose tissue. TNF and IL-1 mimicked the effect of LPS on ACS1 mRNA levels in liver and adipose tissue. LPS decreased ACS activity in adipose tissue, heart, and muscle. In liver, where ACS is localized in several subcellular organelles, both LPS and cytokines decreased mitochondrial ACS activity, whereas they increased microsomal ACS activity. Taken together, these results indicate that LPS and cytokines decrease ACS1 mRNA expression and ACS activity in tissues where FA uptake and/or oxidation is decreased during sepsis. In liver, where FA oxidation is decreased during sepsis but the reesterification of FA is increased, LPS and cytokines decrease ACS1 mRNA and mitochondrial ACS activity, which may inhibit FA oxidation, but increase microsomal ACS activity, which may support the reesterification of peripherally derived FA for triglyceride synthesis.Acyl-CoA synthetase (ACS) catalyzes the activation of fatty acids (FA) to acyl-CoA esters, which are further metabolized in either anabolic or catabolic pathways. Endotoxin [lipopolysaccharide (LPS)], tumor necrosis factor (TNF), and interleukin-1 (IL-1) enhance hepatic FA synthesis and reesterification and inhibit FA oxidation. LPS also decreases triglyceride storage in adipose tissue and inhibits the uptake of FA by heart and muscle. Therefore, in this study we examined the effects of LPS and cytokines on ACS (now also known as ACS1) mRNA expression and activity in multiple tissues in Syrian hamsters. LPS markedly decreased ACS1 mRNA levels in liver, adipose tissue, heart, and skeletal muscle. The inhibitory effects of LPS on ACS1 mRNA levels in liver and adipose tissue were observed as early as 2-4 h after administration, became maximal by 4-8 h, and were sustained for >/=24 h. Very low doses of LPS (0.1-1 microg/100 g body wt) were needed to reduce ACS1 mRNA levels in liver and adipose tissue. TNF and IL-1 mimicked the effect of LPS on ACS1 mRNA levels in liver and adipose tissue. LPS decreased ACS activity in adipose tissue, heart, and muscle. In liver, where ACS is localized in several subcellular organelles, both LPS and cytokines decreased mitochondrial ACS activity, whereas they increased microsomal ACS activity. Taken together, these results indicate that LPS and cytokines decrease ACS1 mRNA expression and ACS activity in tissues where FA uptake and/or oxidation is decreased during sepsis. In liver, where FA oxidation is decreased during sepsis but the reesterification of FA is increased, LPS and cytokines decrease ACS1 mRNA and mitochondrial ACS activity, which may inhibit FA oxidation, but increase microsomal ACS activity, which may support the reesterification of peripherally derived FA for triglyceride synthesis.

In vivo regulation of plasma platelet-activating factor acetylhydrolase during the acute phase response

Plasma platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes PAF and oxidized phospholipids and is associated with lipoproteins in the circulation. Endotoxin [lipopolysaccharide (LPS)], a potent inducer of the acute phase response (APR), produces marked changes in several proteins that play important roles in lipoprotein metabolism. We now demonstrate that LPS produces a 2.5- to 3-fold increase in plasma PAF-AH activity in Syrian hamsters. The plasma PAF-AH activity is found in the high-density lipoprotein (HDL) fraction and is increased threefold with LPS treatment despite a decrease in plasma HDL levels, indicating that plasma PAF-AH activity is increased per HDL particle. LPS markedly increased PAF-AH mRNA levels in liver, spleen, lung, and small intestine. The maximal increase in plasma PAF-AH activity and mRNA expression in liver and spleen is seen 24 h after LPS treatment. Both tumor necrosis factor and interleukin-1 modestly increased plasma PAF-AH activity and mRNA levels in liver and spleen, suggesting that they may partly mediate the effect of LPS on PAF-AH. Surgical removal of spleen had no effect on basal or LPS-induced plasma PAF-AH activity, suggesting that spleen per se may not contribute to plasma PAF-AH activity. Finally, LPS, turpentine and zymosan increased plasma PAF-AH activity in mice and/or rats, indicating that multiple APR inducers upregulate plasma PAF-AH and this effect is consistent across different rodent species. Taken together, our results indicate that plasma PAF-AH activity and mRNA expression is markedly upregulated during the host response to infection and inflammation. An increase in plasma PAF-AH may enhance the degradation of PAF as well as alter the structure and function of HDL during infection and inflammation.Plasma platelet-activating factor acetylhydrolase (PAF-AH) hydrolyzes PAF and oxidized phospholipids and is associated with lipoproteins in the circulation. Endotoxin [lipopolysaccharide (LPS)], a potent inducer of the acute phase response (APR), produces marked changes in several proteins that play important roles in lipoprotein metabolism. We now demonstrate that LPS produces a 2.5- to 3-fold increase in plasma PAF-AH activity in Syrian hamsters. The plasma PAF-AH activity is found in the high-density lipoprotein (HDL) fraction and is increased threefold with LPS treatment despite a decrease in plasma HDL levels, indicating that plasma PAF-AH activity is increased per HDL particle. LPS markedly increased PAF-AH mRNA levels in liver, spleen, lung, and small intestine. The maximal increase in plasma PAF-AH activity and mRNA expression in liver and spleen is seen 24 h after LPS treatment. Both tumor necrosis factor and interleukin-1 modestly increased plasma PAF-AH activity and mRNA levels in liver and spleen, suggesting that they may partly mediate the effect of LPS on PAF-AH. Surgical removal of spleen had no effect on basal or LPS-induced plasma PAF-AH activity, suggesting that spleen per se may not contribute to plasma PAF-AH activity. Finally, LPS, turpentine and zymosan increased plasma PAF-AH activity in mice and/or rats, indicating that multiple APR inducers upregulate plasma PAF-AH and this effect is consistent across different rodent species. Taken together, our results indicate that plasma PAF-AH activity and mRNA expression is markedly upregulated during the host response to infection and inflammation. An increase in plasma PAF-AH may enhance the degradation of PAF as well as alter the structure and function of HDL during infection and inflammation.

Down-regulation of liver and heart specific fatty acid binding proteins by endotoxin and cytokines in vivo.

Fatty acid binding proteins (FABPs) are abundantly present in tissues that actively metabolize fatty acids (FA). While their precise physiological function is not known, FABPs have been shown to play a role in the uptake and/or utilization of FA within the cell. FA metabolism is markedly altered during the host response to infection and inflammation. Previous studies have demonstrated that endotoxin or bacterial lipopolysaccharide (LPS) enhances hepatic FA synthesis and re-esterification while inhibiting FA oxidation in liver, heart and muscle. Now, we have examined the in vivo effects of LPS and cytokines on FABPs in liver (L-FABP), heart and muscle (H-FABP). Syrian hamsters were injected with LPS, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and the mRNA and protein content for L-FABP and H-FABP were analyzed. 16 h after administration, LPS (100 microg/100 g body weight) produced a 72% decrease in L-FABP mRNA levels in liver and this effect was sustained for 24 h. LPS also produced a 41% decrease in the protein content of L-FABP in liver after 24 h of treatment. TNF-alpha and IL-1beta decreased L-FABP mRNA levels in liver by 30 and 45%, respectively. LPS decreased H-FABP mRNA levels in skeletal muscle by 60% and in heart by 65%. LPS also produced a 49% decrease in H-FABP protein content in muscle. Neither TNF-alpha nor IL-1beta had any significant effect on H-FABP mRNA expression in heart and muscle. Taken together, these results indicate that LPS decreases FABP mRNA and protein levels in liver, heart and muscle, tissues that normally utilize FA as their primary fuel, whereas the inhibitory effect of cytokines is limited to the liver. The LPS-induced decrease in L-FABP and H-FABP may be an additional mechanism contributing to the decrease in FA oxidation that is associated with the host response to infection and inflammation.