Riaz A. Memon

Infection and Inflammation-Induced Proatherogenic Changes of Lipoproteins

Epidemiologic studies suggest a link between infection/inflammation and atherosclerosis. During the acute-phase response to infection and inflammation, cytokines induce tissue and plasma events that lead to changes in lipoprotein. Many of these changes are similar to those proposed to promote atherogenesis. The changes of lipoproteins during infection and inflammation are reviewed with a focus on those that are potentially proatherogenic. Hypertriglyceridemia, elevated triglyceride-rich lipoproteins, the appearance of small dense low-density lipoproteins, increased platelet-activating factor acetylhydrolase activity, and secretory phospholipase A(2), sphingolipid-enriched lipoproteins, and decreased high-density lipoprotein (HDL) cholesterol are changes that could promote atherogenesis. Moreover, alterations of proteins associated with HDL metabolism (e.g., paraoxonase, apolipoprotein A-I, lecithin:cholesterol acyltransferase, cholesterol ester transfer protein, hepatic lipase, phospholipid transfer protein, and serum amyloid A) could decrease the ability of HDL to protect against atherogenesis through antioxidation and reverse cholesterol transport mechanisms. These proatherogenic changes of lipoproteins may contribute to the link between infection/inflammation and atherosclerosis.

Up-Regulation of Peroxisome Proliferator-Activated Receptors (PPAR-α) and PPAR-γ Messenger Ribonucleic Acid Expression in the Liver in Murine Obesity: Troglitazone Induces Expression of PPAR-γ-Responsive Adipose Tissue-Specific Genes in the Liver of Obese Diabetic Mice1

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that play an important role in the regulation of genes involved in lipid utilization and storage, lipoprotein metabolism, adipocyte differentiation, and insulin action. The three isoforms of the PPAR family, i.e. alpha, delta, and gamma, have distinct tissue distribution patterns. PPAR-alpha is predominantly present in the liver, and PPAR-gamma in adipose tissue, whereas PPAR-delta is ubiquitously expressed. A recent study reported increased PPAR-gamma messenger RNA (mRNA) expression in the liver in ob/ob mice; however, it is not known whether increased PPAR-gamma expression in the liver has any functional consequences. The expression of PPAR-alpha and -delta in the liver in obesity has not been determined. We have now examined the mRNA levels of PPAR-alpha, -delta, and -gamma in three murine models of obesity, namely, ob/ob (leptin-deficient), db/db (leptin-receptor deficient), and serotonin 5-HT2c receptor (5-HT2cR) mutant mice. 5-HT2cR mutant mice develop a late-onset obesity that is associated with higher plasma leptin levels. Our results show that PPAR-alpha mRNA levels in the liver are increased by 2- to 3-fold in all three obese models, whereas hepatic PPAR-gamma mRNA levels are increased by 7- to 9-fold in ob/ob and db/db mice and by 2-fold in obese 5-HT2cR mutant mice. PPAR-delta mRNA expression is not altered in ob/ob or db/db mice. To determine whether increased PPAR-gamma expression in the liver has any functional consequences, we examined the effect of troglitazone treatment on the hepatic mRNA levels of several PPAR-gamma-responsive adipose tissue-specific genes that have either no detectable or very low basal expression in the liver. The treatment of lean control mice with troglitazone significantly increased the expression of adipocyte fatty acid-binding protein (aP2) and fatty acid translocase (FAT/CD36) in the liver. This troglitazone-induced increase in the expression of aP2 and FAT/CD36 was markedly enhanced in the liver in ob/ob mice. Troglitazone also induced a pronounced increase in the expression of uncoupling protein-2 in the liver in ob/ob mice. In contrast to the liver, troglitazone did not increase the expression of aP2, FAT/CD36, and uncoupling protein-2 in adipose tissue in lean or ob/ob mice. Taken together, our results suggest that the effects of PPAR-gamma activators on lipid metabolism and energy homeostasis in obesity and type 2 diabetes may be partly mediated through their effects on PPAR-gamma in the liver.

Paraoxonase activity in the serum and hepatic mRNA levels decrease during the acute phase response

Numerous epidemiological studies have suggested an association between the acute phase response and atherosclerosis. Paraoxonase (PON) is an HDL associated enzyme that protects LDL from oxidative stress. Here we demonstrate that serum PON activity decreases following endotoxin (LPS) administration in Syrian hamsters. This decrease is seen within 24 h following LPS treatment and doses as low as 100 ng/100 g body weight of LPS elicit a reduction in serum PON activity. LPS also induces a marked decrease in PON1 mRNA in the liver (80% decrease). The decrease in mRNA levels is observed as early as 4 h and is sustained for at least 48 h after a single LPS treatment. Moreover, TNF and IL-1, cytokines which mediate the acute phase response, also decrease serum PON activity and PON mRNA levels in the liver. Additionally, TNF and IL-1 treatment of HepG2 cells results in a decrease in PON mRNA levels indicating that these cytokines are capable of directly affecting liver cells. Along with other changes in lipid metabolism that occur during the acute phase response, the decrease in PON could be another factor linking the acute phase response with increased atherogenesis.

Infection and Inflammation Induce LDL Oxidation In Vivo

Epidemiological studies have shown an increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders. Because oxidative modification of lipoproteins plays a major role in atherosclerosis, the present study was designed to test the hypothesis that the host response to infection and inflammation induces lipoprotein oxidation in vivo. Lipoprotein oxidation was measured in 3 distinct models of infection and inflammation. Syrian hamsters were injected with bacterial lipopolysaccharide (LPS), zymosan, or turpentine to mimic acute infection, acute systemic inflammation, and acute localized inflammation, respectively. Levels of oxidized fatty acids in serum and lipoprotein fractions were measured by determining levels of conjugated dienes, thiobarbituric acid-reactive substances, and lipid hydroperoxides. Our results demonstrate a significant increase in conjugated dienes and thiobarbituric acid-reactive substances in serum in all 3 models. Moreover, LPS and zymosan produced a 4-fold to 6-fold increase in conjugated diene and lipid hydroperoxide levels in LDL fraction. LPS also produced a 17-fold increase in LDL content of lysophosphatidylcholine that is formed during the oxidative modification of LDL. Finally, LDL isolated from animals treated with LPS was significantly more susceptible to ex vivo oxidation with copper than LDL isolated from saline-treated animals, and a 3-fold decrease occurred in the lag phase of oxidation. These results demonstrate that the host response to infection and inflammation increases oxidized lipids in serum and induces LDL oxidation in vivo. Increased LDL oxidation during infection and inflammation may promote atherogenesis and could be a mechanism for increased incidence of coronary artery disease in patients with chronic infections and inflammatory disorders.

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 interleukin-1 decrease hepatic lipase mRNA levels

The acute phase response induces a multitude of changes in lipoprotein metabolism including hypertriglyceridemia, triglyceride enriched LDL, and decreased HDL levels accompanied by changes in HDL composition including increased free cholesterol and triglycerides and a decrease in esterified cholesterol. Here we demonstrate that endotoxin (LPS) induces a 56% decrease in hepatic lipase activity in liver and a 45% decrease in hepatic lipase activity in post heparin plasma in Syrian hamsters. LPS treatment also produces a marked decrease in hepatic lipase mRNA levels in the liver. Half maximal reduction in hepatic lipase mRNA levels occurred at approximately 0.2 microg LPS/100 g BW with a maximal decrease at 1.0 microg/100 g BW ( > 90% decrease), indicating that inhibition of hepatic lipase is a sensitive host response to LPS. Additionally, IL-1 produced a marked decrease in hepatic lipase mRNA levels while TNF had no effect. Moreover, IL-1 treatment of HepG2 cells in vitro also decreased hepatic lipase mRNA levels suggesting that IL-1 can directly regulate hepatic lipase expression in liver cells. LPS decreased hepatic lipase mRNA levels in control as well as IL-1 type 1 receptor deficient mice indicating that IL-1 action is not absolutely essential and that several cytokines and/or small molecular mediators can regulate hepatic lipase during the acute phase response. The LPS and IL-1 induced decrease in hepatic lipase could have several consequences including decreasing the clearance of triglyceride rich lipoprotein particles and producing an increase in triglyceride rich HDL. The decrease in hepatic lipase activity and mRNA levels may be part of a series of coordinated changes in lipoprotein metabolism that occur during the acute phase response. These changes may be initially beneficial to the host but if present for an extended period may be proatherogenic.

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.