Assessment of factors associated with pre-diabetes in HCV infection including direct and dynamic measurements of insulin action.

Although hepatitis C (HCV) is associated with diabetes, few studies have examined pre-diabetes in this population. We aimed to evaluate factors associated with pre-diabetes in HCV-infected patients, including direct measurement of insulin action. Ninety-seven non-cirrhotic, non-diabetic and HCV-infected patients underwent clinical evaluation and oral glucose tolerance testing (OGTT). Insulin sensitivity was measured directly by steady-state plasma glucose (SSPG) concentration during insulin suppression test. Early phase and total insulin secretion were determined using OGTT. Rates of pre-diabetes were as follows: 21% impaired fasting glucose (IFG), 7% impaired glucose tolerance (IGT) and 9% combined IFG/IGT. Twelve percent of Caucasians, 50% of African Americans and 70% of Latinos had pre-diabetes (P = 0.002). Patient characteristics among the glucose metabolism categories were similar except those with combined IFG/IGT had a higher body mass index (BMI) vs normal glucose tolerance (NGT) (30 vs 26 kg/m(2), P = 0.007) and lower LDL vs NGT and IGT (74, 104 and 112 mg/dL, respectively, P ≤ 0.01). On multivariable analysis, non-Caucasian race (OR 23.1, P = 0.003), BMI (OR 3.4, P = 0.02) and greater liver inflammation (OR 7.9, P = 0.03) predicted IFG, whereas non-Caucasian race (OR 14.8, P = 0.01) and SSPG (OR 1.1 per 10 units, P = 0.01) predicted IGT. Early and total insulin secretion adjusted for the degree of insulin resistance was decreased in pre-diabetes compared with NGT (P = 0.01 and P = 0.02, respectively). Pre-diabetes is highly prevalent among HCV-infected patients, and in some instances, coincides with host responses to the virus. In most cases, however, factors that are associated with pre-diabetes in HCV-infected patients are similar to those observed in the non-HCV population.

Interactions between hepatic stellate cells and the immune system.

Stellate cells and immune cells are both active participants in the pathogenesis of liver disease. Interactions between these two populations are important determinants of disease outcome. Kupffer cells, neutrophils, and lymphocytes all have the potential to influence stellate cells. They produce a host of humoral mediators, including oxidants, nitric oxide, cytokines, eicosanoids, and proteinases, which can affect stellate cell proliferation, gene expression, and contractility. One important feature of stellate cell-immune cell interactions is that they are bidirectional. Not only do stellate cells receive signals from leukocytes, but they also elaborate signals that target leukocytes. Specifically, stellate cells can promote leukocyte chemotaxis and adherence, and they may also influence leukocyte activation by producing regulatory cytokines. Studies in culture provide an important background for understanding the effects of specific mediators on stellate cells and immune cells. Experiments in vivo offer an important adjunct, but often lead to confounding effects that limit interpretation. Both types of studies are required to develop a better understanding of the complex interplay between stellate cells and leukocytes.

Anti-Fas induces hepatic chemokines and promotes inflammation by an NF-kappa B-independent, caspase-3-dependent pathway.

Agonistic antibodies against the Fas receptor, when administered to mice in vivo, cause significant apoptosis in the liver. In this study we show that anti-Fas antibody not only causes apoptosis of liver cells but also provokes hepatic inflammation. Two hours after injection of anti-Fas, when mice displayed evidence of caspase-3 activation and apoptosis, we found significant hepatic induction of the CXC chemokines macrophage inflammatory protein-2 and KC. Coincident with the chemokine induction was infiltration of the hepatic parenchyma by neutrophils. Neutralization experiments identified that chemokines were the cause of Fas-induced hepatic inflammation, with KC having the predominant effect. Chemokine induction in the livers of anti-Fas-treated mice was not associated with activation of NF-kappa B. Instead, it coincided with nuclear translocation of activator protein-1 (AP-1). AP-1 activation in liver was detected 1-2 h after anti-Fas treatment, suggesting a connection to the onset of apoptosis. When apoptosis was prevented by pretreating mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced. Hepatic inflammation was also reduced by 70%. Taken together, these findings indicate that Fas ligation can induce inflammation in the liver in vivo. Inflammation does not arise from Fas-mediated signaling through NF-kappa B; rather, it represents an indirect effect, requiring activation of caspase-3 and nuclear translocation of AP-1.

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii.

Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.

Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice.

Overexpression is the most common abnormality of receptor tyrosine kinases (RTKs) in human tumors. It is presumed that overexpression leads to constitutive activation of RTKs, but the mechanism of that activation has been uncertain. Here we show that overexpression of the Met RTK allows activation of the receptor by cell attachment and that this form of activation can be tumorigenic. Transgenic mice that overexpressed Met in hepatocytes developed hepatocellular carcinoma (HCC), one of the human tumors in which Met has been implicated previously. The tumorigenic Met was activated by cell attachment rather than by ligand. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. These results reveal a previously unappreciated mechanism by which the tumorigenic action of RTKs can be mediated, provide evidence that Met may play a role in both the genesis and maintenance of HCC, and suggest that Met may be a beneficial therapeutic target in tumors that overexpress the receptor.

Bile duct ligation in rats induces biliary expression of cytokine-induced neutrophil chemoattractant.

BACKGROUND & AIMS: Bile duct obstruction causes neutrophilic inflammation of the liver and leads to hepatic fibrosis. In obstructive liver disease, the localization of neutrophils in portal tracts suggests that cells within this region produce neutrophil chemoattractants. In this study, we investigated whether bile duct obstruction in rats induces portal expression of cytokine-induced neutrophil chemoattractant (CINC). METHODS: Rats underwent bile duct ligation for 3 hours to 8 days. CINC regulation was examined in vivo at various intervals by immunohistochemistry, ribonuclease protection, and in situ hybridization. CINC production was also investigated in cell culture, in response to putative stimuli from obstructed liver. RESULTS: Bile duct ligation caused neutrophilic infiltration of the liver within 3 hours. CINC was also rapidly induced, with specific expression identified in biliary cells. Rat intrahepatic biliary cells produced CINC constitutively in culture; when exposed to cholestatic bile, they showed a 12-fold increase in CINC secretion. The effect of bile was not attributable to toxicity or to dissolved cytokines or endotoxin. Mechanical strain, designed to mimic the stretching of biliary cells during obstruction, did not induce CINC. CONCLUSIONS: Biliary cells contribute to hepatic inflammation during cholestasis by producing neutrophil chemoattractants. A major stimulus to biliary chemoattractant production in vivo may be bile itself.

Cytokines: overview.

Cytokines are involved in a number of physiologic and pathophysiologic processes in the liver. These small molecules impact hepatic growth and repair, regulate inflammation, and influence fibrogenesis. Cytokines acting within the liver derive from both resident and recruited cells; the pattern of cytokines produced in a given clinical situation depends upon the inciting stimulus and the background cellular milieu. Experimental animals display inherited differences in cytokine responses to environmental insults. If these differences are operative in human beings, they may account in part for variations in susceptibility among individuals or groups to certain types of liver disease.

Leukocytes as modulators of stellate cell activation.

Activation of stellate cells is central to the process of hepatic fibrogenesis. Stellate cell activation can be influenced by many factors, including cytokines, oxidants, and alterations in the perisinusoidal extracellular matrix. These factors can be produced by resident liver cells (hepatocytes, Kupffer cells, or stellate cells themselves); however, infiltrating leukocytes may also play an important role. Because liver fibrosis often follows a prolonged period of hepatic inflammation, investigators have begun to study leukocytes as modulators of stellate cell activation. The following data summarize recent investigations in this area that focus on neutrophils as well as mononuclear cells.

Rat hepatic stellate cells produce cytokine-induced neutrophil chemoattractant in culture and in vivo.

Hepatic stellate cells are widely recognized for their contribution to liver fibrosis. This study investigated whether these cells also promote hepatic inflammation by producing neutrophil chemoattractants. Specifically, stellate cells were examined as potential sources of cytokine-induced neutrophil chemoattractant (CINC), a rat chemokine resembling human interleukin-8. Stellate cells from normal rat liver expressed little or no CINC. In culture, CINC mRNA was induced rapidly, coinciding with the phenomenon of culture activation. CINC mRNA rose 4.6-fold within 3 days and was accompanied by secretion of immunoreactive and biologically active CINC protein (4.1 ng . microgram DNA-1 . day-1). Studies in vivo demonstrated that CINC could be induced in stellate cells during liver injury. CINC mRNA rose significantly (4- to 6-fold) in two models of liver disease, both of which cause stellate cell activation. In summary, the data indicate that CINC is induced during stellate cell activation in culture and in vivo. They suggest that stellate cell-derived CINC can promote hepatic inflammation in vivo.

Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli.

Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors. Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes. While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking. In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA. An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose. Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E.coli by disrupting transcriptional interference. Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo , despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro . Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.

Manipulation of glutathione stores in rat hepatic stellate cells does not alter collagen synthesis.

Hepatic stellate cells are resistant to the fibrogenic effects of lipid hydroperoxides in primary culture. Recent studies from our laboratory suggest that antioxidants, particularly glutathione, play a role in this resistance. We have observed that glutathione accumulates rapidly in stellate cells during primary culture; in the current study, we investigated whether glutathione modulates stellate cell collagen synthesis. Stellate cells from normal rat liver were plated in primary culture and maintained for 7 days. From day 4 through day 7, the cells were treated with L-buthionine sulfoximine (BSO) to deplete glutathione stores. BSO profoundly diminished stellate cell glutathione but had no effect on morphology, viability, or basal levels of collagen synthesis and gene expression. When cultured stellate cells were incubated with the putative fibrogenic mediator 4-hydroxynonenal or iron/ascorbate, little or no increase in collagen synthesis occurred regardless of glutathione content. In contrast, iron/ascorbate induced collagen synthesis by cultured fibroblasts. The data indicate that stellate cells strongly resist oxidant- and lipid peroxide-induced collagen synthesis in primary culture. They demonstrate that the mechanism of this resistance does not involve glutathione.

Adenovirus-mediated expression of cytokine-induced neutrophil chemoattractant in rat liver induces a neutrophilic hepatitis.

C-X-C chemokines are potent chemoattractants that are believed to mediate neutrophilic inflammation in several organs. Recent studies suggest a role for C-X-C chemokines in the pathogenesis of neutrophilic hepatitis but do not prove causation. We investigated the biological consequences of hepatic chemokine production in vivo by transiently overexpressing cytokine-induced neutrophil chemoattractant (CINC), a member of the C-X-C chemokine family, in intact rats. Rats were injected intraportally with a replication-defective recombinant adenovirus containing the CINC complementary DNA (cDNA). Within 4 days, treated animals had high levels of CINC in both liver tissue and plasma Rats overexpressing CINC exhibited an eightfold increase in circulating neutrophils; they also developed severe hepatic injury, characterized by a 6- to 25-fold increase in plasma transaminases and marked hepatic inflammation on biopsy. Liver disease in CINC-producing rats correlated positively with the number of neutrophils sequestered in the hepatic parenchyma. Tissue injury was attributed directly to chemokine overproduction, because control rats infected with adenoviruses lacking the CINC cDNA did not produce CINC and developed only minor hepatic abnormalities. These experiments provide direct evidence that C-X-C chemokines, when expressed in sufficient quantity in the liver in vivo, induce neutrophil recruitment and tissue invasion and provoke severe liver injury. The data suggest that C-X-C chemokines have important pathogenic potential in both clinical and experimental liver disease.

Glutathione regulation in rat hepatic stellate cells. Comparative studies in primary culture and in liver injury in vivo.

Lipid peroxidation accompanies many types of liver injury and is believed to promote liver fibrosis. Cellular antioxidants are likely to play an important role in modulating this process; however, little is known about antioxidants in hepatic stellate cells, the major collagen-producing cells of liver. In this study, we measured glutathione homeostasis in stellate cells isolated from rat liver. Glutathione, measured by HPLC in stellate cell homogenates, increased significantly when the cells were plated in primary culture. The rise in glutathione coincided with pretranslational up-regulation of the synthetic enzyme gamma-glutamylcysteine synthetase (GCS). Additional experiments were performed to determine whether stellate cell glutathione and GCS are similarly altered during liver injury in vivo. Two types of hepatic insults, namely, bile duct ligation (8 days) and carbon tetrachloride treatment (4 weeks), failed to provoke an increase in either stellate cell glutathione or GCS. This disparate behavior of stellate cells in culture and in vivo is unusual; the data suggest that stellate cells might respond variably to oxidants depending on their glutathione status.

Exploring alcohol's effects on liver function.

A large proportion of heavy drinkers develop serious alcoholic liver disease. Susceptibility to alcoholic hepatitis and cirrhosis appears to be influenced by heredity, gender, diet, and co-occurring liver illness. Most alcoholic liver damage is attributed to alcohol metabolism. Liver injury may be caused by direct toxicity of metabolic by-products of alcohol as well as by inflammation induced by these by-products. Exposure of liver cells to bacterial toxins may contribute to liver disease. Escalating liver injury can lead to fibrosis and, ultimately, to cirrhosis. Increased understanding of the mechanisms of liver injury has led to innovative treatments for alcoholic liver disease, including the use of corticosteroids, antioxidants, antibiotics, and certain polyunsaturated fats.

Rat hepatocytes and Kupffer cells interact to produce interleukin-8 (CINC) in the setting of ethanol.

Interleukin-8 is a neutrophil chemoattractant that has been implicated in the pathogenesis of alcoholic hepatitis. The mechanism of ethanol-induced interleukin-8 production in liver is uncertain, although hepatocytes and Kupffer cells have both been proposed as sources of the chemokine. In this study we investigated whether short-term ethanol exposure stimulates production of rat interleukin-8 [cytokine-induced neutrophil chemoattractant (CINC)] by normal rat hepatocytes and Kupffer cells in primary culture. Initial experiments verified that hepatocytes and Kupffer cells produce CINC in response to cytokines or lipopolysaccharide. Ethanol, by contrast, failed to stimulate CINC secretion by either cell type even at concentrations as high as 100 mM. Although ethanol had no direct effect on liver cell CINC production, conditioned medium from ethanol-treated hepatocytes induced a threefold rise in CINC production by Kupffer cells. The increase was abrogated when hepatocytes were treated with ethanol and the metabolic inhibitor 4-methylpyrazole. The results suggest that the mechanism of ethanol-induced CINC production is indirect, involving ethanol oxidation and communication between hepatocytes and Kupffer cells.

Malondialdehyde stimulates collagen production by hepatic lipocytes only upon activation in primary culture.

Lipid aldehydes have been proposed as mediators of hepatic fibrosis in alcoholics. In this study we examined whether hepatic lipocytes, the principal matrix-producing cells in liver, exhibit enhanced collagen synthesis in response to the lipid aldehyde malondialdehyde. Lipocytes isolated from normal rat liver and plated in primary culture for 3 days were not affected by malondialdehyde in concentrations ranging from 2 to 200 microM. Cells cultured for 7 days displayed a modest increase in collagen synthesis (137% of control levels) in response to malondialdehyde, but only at a concentration of 200 microM. The malondialdehyde-induced increase in collagen synthesis was paralleled by a rise in type I procollagen mRNA. Subcultured rat fibroblasts at confluent density responded better to malondialdehyde than did 7-day lipocytes. The results indicate that lipocytes respond to the fibrogenic effects of malondialdehyde only after activation in primary culture. This delayed response suggests that lipid aldehydes may enhance, but do not initiate, alcoholic liver fibrosis in vivo.