Pair-fed Control Diet Research Articles

Effects of probiotics on methionine cholinedeficient diet-induced steatohepatitis in rats

Intestinal bacteria induce endogenous signals that play a pathogenic role in hepatic insulin resistance and non-alcoholic fatty liver disease. Probiotics could modulate the gut flora and could influence the gut-liver axis. We aimed to investigate the preventive effect of two probiotic mixtures on the methionine choline-deficient diet-induced non-alcoholic steatohepatitis model in rats. Two studies, short-term (2 weeks) and long-term (6 weeks), were carried out using 60 male Wistar rats. The 2-week study included six groups. Rats were fed with methionine choline-deficient diet or pair-fed control diet and were given a placebo or one of two probiotic mixtures (Pro-1 and Pro-2) by orogastric gavage. In the 6-week study, rats were allocated into four groups and were fed with methionine choline-deficient diet or pair-fed control diet and given a placebo or Pro-2. At the end of the 2- and 6-week periods, blood samples were obtained, the animals were sacrificed, and liver tissues were removed. Serum alanine aminotransferase activity was determined; histologic and immunohistochemical analysis was performed for steatosis, inflammation, protein expression of tumor necrosis factor-α, and apoptosis markers. In both studies, methionine choline-deficient diet caused an elevation of serum alanine aminotransferase activity, which was slightly reduced by Pro-1 and Pro-2. In the 2- and 6-week studies, feeding with methionine choline-deficient diet resulted in steatosis and inflammation, but not fibrosis, in all rats. In the 2-week study, in rats fed with methionine choline-deficient diet and given Pro-1, steatosis and inflammation were present in 2 of 6 rats. In rats fed with methionine choline-deficient diet and given Pro-2, steatosis was detected in 3 of 6 rats, while inflammation was present in 2 of 6 rats. In the 6-week study, in rats fed with methionine choline-deficient diet and given Pro-2, steatosis and inflammation were present in 3 of 6 rat livers. In both the 2- and 6-week studies, methionine choline-deficient diet resulted in tumor necrosis factor-α, proapoptotic Bax, caspase 3, caspase 8, and anti-apoptotic Bcl-2 expression in all rat livers. Pro-1 and Pro-2 treatment influenced protein expression involved in apoptosis and tumor necrosis factor-α in varying degrees. Pro-1 and Pro-2 decrease methionine choline-deficient diet-induced steatohepatitis in rats. The preventive effect of probiotics may be due, in part, to modulation of apoptosis and their anti-inflammatory activity.

Chronic Alcohol Exposure Stimulates Adipose Tissue Lipolysis in Mice: Role of Reverse Triglyceride Transport in the Pathogenesis of Alcoholic Steatosis

Alcohol consumption induces liver steatosis; therefore, this study investigated the possible role of adipose tissue dysfunction in the pathogenesis of alcoholic steatosis. Mice were pair-fed an alcohol or control liquid diet for 8 weeks to evaluate the alcohol effects on lipid metabolism at the adipose tissue-liver axis. Chronic alcohol exposure reduced adipose tissue mass and adipocyte size. Fatty acid release from adipose tissue explants was significantly increased in alcohol-fed mice in association with the activation of adipose triglyceride lipase and hormone-sensitive lipase. Alcohol exposure induced insulin intolerance and inactivated adipose protein phosphatase 1 in association with the up-regulation of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 3 (SOCS3). Alcohol exposure up-regulated fatty acid transport proteins and caused lipid accumulation in the liver. To define the mechanistic link between adipose triglyceride loss and hepatic triglyceride gain, mice were first administered heavy water for 5 weeks to label adipose triglycerides with deuterium, and then pair-fed alcohol or control diet for 2 weeks. Deposition of deuterium-labeled adipose triglycerides in the liver was analyzed using Fourier transform ion cyclotron mass spectrometry. Alcohol exposure increased more than a dozen deuterium-labeled triglyceride molecules in the liver by up to 6.3-fold. These data demonstrate for the first time that adipose triglycerides due to alcohol-induced hyperlipolysis are reverse transported and deposited in the liver.

Betaine treatment attenuates chronic ethanol-induced hepatic steatosis and alterations to the mitochondrial respiratory chain proteome.

Introduction. Mitochondrial damage and disruption in oxidative phosphorylation contributes to the pathogenesis of alcoholic liver injury. Herein, we tested the hypothesis that the hepatoprotective actions of betaine against alcoholic liver injury occur at the level of the mitochondrial proteome. Methods. Male Wister rats were pair-fed control or ethanol-containing liquid diets supplemented with or without betaine (10 mg/mL) for 4-5 wks. Liver was examined for triglyceride accumulation, levels of methionine cycle metabolites, and alterations in mitochondrial proteins. Results. Chronic ethanol ingestion resulted in triglyceride accumulation which was attenuated in the ethanol plus betaine group. Blue native gel electrophoresis (BN-PAGE) revealed significant decreases in the content of the intact oxidative phosphorylation complexes in mitochondria from ethanol-fed animals. The alcohol-dependent loss in many of the low molecular weight oxidative phosphorylation proteins was prevented by betaine supplementation. This protection by betaine was associated with normalization of SAM : S-adenosylhomocysteine (SAH) ratios and the attenuation of the ethanol-induced increase in inducible nitric oxide synthase and nitric oxide generation in the liver. Discussion/Conclusion. In summary, betaine attenuates alcoholic steatosis and alterations to the oxidative phosphorylation system. Therefore, preservation of mitochondrial function may be another key molecular mechanism responsible for betaine hepatoprotection.

Cholinergic Mediation of Alcohol‐Induced Experimental Pancreatitis

The mechanisms initiating pancreatitis in patients with chronic alcohol abuse are poorly understood. Although alcohol feeding has been previously suggested to alter cholinergic pathways, the effects of these cholinergic alterations in promoting pancreatitis have not been characterized. For this study, we determined the role of the cholinergic system in ethanol-induced sensitizing effects on cerulein pancreatitis. Rats were pair-fed control and ethanol-containing Lieber-DeCarli diets for 6 weeks followed by parenteral administration of 4 hourly intraperitoneal injections of the cholecystokinin analog, cerulein at 0.5 μg/kg. This dose of cerulein was selected because it caused pancreatic injury in ethanol-fed but not in control-fed rats. Pancreatitis was preceded by treatment with the muscarinic receptor antagonist atropine or by bilateral subdiaphragmatic vagotomy. Measurement of pancreatic pathology included serum lipase activity, pancreatic trypsin, and caspase-3 activities, and markers of pancreatic necrosis, apoptosis, and autophagy. In addition, we measured the effects of ethanol feeding on pancreatic acetylcholinesterase activity and pancreatic levels of the muscarinic acetylcholine receptors m1 and m3. Finally, we examined the synergistic effects of ethanol and carbachol on inducing acinar cell damage. We found that atropine blocked almost completely pancreatic pathology caused by cerulein administration in ethanol-fed rats, while vagotomy was less effective. Ethanol feeding did not alter expression levels of cholinergic muscarinic receptors in the pancreas but significantly decreased pancreatic acetylcholinesterase activity, suggesting that acetylcholine levels and cholinergic input within the pancreas can be higher in ethanol-fed rats. We further found that ethanol treatment of pancreatic acinar cells augmented pancreatic injury responses caused by the cholinergic agonist, carbachol. These results demonstrate key roles for the cholinergic system in the mechanisms of alcoholic pancreatitis.

Ultrastructural changes of cornea after ethanol ingestion in Otsuka Long-Evans Tokushima fatty (OLETF) and Long-Evans Tokushima Otsuka (LETO) rats

To compare ethanol-induced ultrastructural changes in corneas of Otsuka Long-Evans Tokushima fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) rats. Forty, five-week-old male LETO and OLETF rats were fed an ethanol-containing liquid diet (LETO alc and OLETF alc) for 11 weeks, and another forty OLETF and LETO rats were put on a pair-fed control diet (LETO con and OLETF con) for 11 weeks. The body weight and fasting blood glucose levels, and 2-hour blood glucose levels after glucose tolerance test (GTT) were checked every week. Central corneal thickness (CCT) was measured with an ultrasound pachymeter after 11 weeks. After corneal buttons were excised, transmission electron microscopy (TEM) examination was performed. The CCTs in groups LETO-alc (179.39 +/- 28.64 microm) and OLETF-alc (189.46 +/- 44.44 microm) were significantly thicker than those in groups LETO-con (168.07 +/- 15.57 microm) and OLETF-con (179.28 +/- 14.68 microm) respectively (P < 0.05). The percentage of basement membrane occupied by hemidesmosome (HD/BM) in the LETO-alc (43.2 +/- 3.7%) and OLETF-alc (35.3 +/- 4.1%) was lower than that in the LETO-con (51.5 +/- 2.6%) and OLETF-con (41.6 +/- 3.5%) group respectively (P < 0.05). Descemet's membrane thickness of the LETO-alc (4.45 +/- 0.3 microm) and OLETF-alc (4.98 +/- 0.5 microm) was greater than that of the LETO-con (4.11 +/- 0.2 microm) and OLETF-con (4.57 +/- 0.3 microm) group respectively (P < 0.05). In the OLETF group, the hemidesmosomes and endothelial tight junctions were less electron-dense; interstromal edema was more prominent than that in the LETO group. Corneal ultrastructural damage can develop in the pre-diabetic stage and when alcohol is ingested chronically in rats.

Complement and Alcoholic Liver Disease: Role of C1q in the Pathogenesis of Ethanol-Induced Liver Injury in Mice

Complement is involved in the development of alcoholic liver disease in mice; however, the mechanisms for complement activation during ethanol exposure have not been identified. C1q, the recognition subunit of the first complement component, binds to apoptotic cells, thereby activating the classical complement pathway. Because ethanol exposure increases hepatocellular apoptosis, we hypothesized that ethanol-induced apoptosis would lead to activation of complement via the classical pathway. Wild-type and C1qa-/- mice were allowed free access to ethanol-containing diets or pair-fed control diets for 4 or 25 days. Ethanol feeding for 4 days increased apoptosis of Kupffer cells in both wild-type and C1qa-/- mice. Ethanol-induced deposition of C1q and C3b/iC3b/C3c was colocalized with apoptotic Kupffer cells in wild-type, but not C1qa-/-, mice. Furthermore, ethanol-induced increases in tumor necrosis factor-alpha and interleukin-6 expression at this early time point were suppressed in C1q-deficient mice. Chronic ethanol feeding (25 days) increased steatosis, hepatocyte apoptosis, and activity of serum alanine and aspartate aminotransferases in wild-type mice. These markers of hepatocyte injury were attenuated in C1qa-/- mice. In contrast, chronic ethanol (25 days)-induced increases in cytochrome P450 2E1 expression and oxidative stress did not differ between wild-type and C1qa-/- mice. For the first time, these data indicate that ethanol activates the classical complement pathway via C1q binding to apoptotic cells in the liver and that C1q contributes to the pathogenesis of ethanol-induced liver injury.

Abstract 2872: Effects of ethanol and folate on peripubertal mouse mammary gland

Abstract Alcohol consumption has been clearly linked with increased breast cancer risk in women, with risks incurred even at low levels of consumption. The proposed mechanisms whereby ethanol exerts its effects include decreased folate levels and diminished DNA repair capacity, coupled with acetaldehyde-generated DNA damage. Based on these proposed mechanisms, we hypothesized that ethanol would have increased deleterious effects during periods of rapid mammary gland epithelial proliferation, such as peripuberty. We also hypothesized that folate deficiency alone might mimic the effects of ethanol, and that folate deficiency would exacerbate the effects of ethanol. CD2/F1 female mice (N=8 per group) were pair-fed control and ethanol-containing liquid diets +/− 0.2g/L folate supplementation from 5 weeks until 9 weeks of age. Mice were sacrificed after this 4 week feeding regimen, and mammary glands were harvested for preparation of whole mounts and paraffin sections. Folate levels of liver and kidney were assessed to verify folate status. Analysis of mammary gland whole mounts revealed that mammary gland structure was significantly altered in folate-replete mice fed ethanol, compared to control-fed mice (no ethanol/ folate-replete). These ethanol/folate replete mice showed an increased total number of ductal branches, due to an increased number of terminal short branches; no effect was seen on the number of terminal end buds or primitive alveolar buds. Systematic histological analysis of paraffin sections using NIH Image J software revealed that diets containing ethanol in the presence of folate significantly increased the total number of ducts, ductal epithelial area, and number of ducts with epithelial multilayering. These results demonstrate that folate deficiency alone does not mimic the effects of ethanol, and that folate deficiency in the presence of ethanol blocks proliferative effects of ethanol on the mammary ductal tree. Future studies are examining the effects of peripubertal ethanol and folate exposure on gene expression in the mouse mammary gland. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2872.

Involvement of adiponectin-SIRT1-AMPK signaling in the protective action of rosiglitazone against alcoholic fatty liver in mice

The development of alcoholic fatty liver is associated with reduced adipocyte-derived adiponectin levels, decreased hepatic adiponectin receptors, and deranged hepatic adiponectin signaling in animals. Peroxisomal proliferator-activated receptor-gamma (PPAR-gamma) plays a key role in the regulation of adiponectin in adipose tissue. The aim of the present study was to test the ability of rosiglitazone, a known PPAR-gamma agonist, to reverse the inhibitory effects of ethanol on adiponectin expression and its hepatic signaling, and to attenuate alcoholic liver steatosis in mice. Mice were fed modified Lieber-DeCarli ethanol-containing liquid diets for 4 wk or pair-fed control diets. Four groups of mice were given a dose of either 3 or 10 mg.kg body wt(-1).day(-1) of rosiglitazone with or without ethanol in their diets for the last 2 wk of the feeding study. Coadministration of rosiglitazone and ethanol increased the expression and circulating levels of adiponectin and enhanced the expression of hepatic adiponectin receptors (AdipoRs) in mice. These increases correlated closely with the activation of a hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling system. In concordance with stimulated SIRT1-AMPK signaling, rosiglitazone administration enhanced expression of fatty acid oxidation enzymes, normalized lipin 1 expression, and blocked elevated expression of genes encoding lipogenic enzymes which, in turn, led to increased fatty acid oxidation, reduced lipogenesis, and alleviation of steatosis in the livers of ethanol-fed mice. Enhanced hepatic adiponectin-SIRT1-AMPK signaling contributes, at least in part, to the protective action of rosiglitazone against alcoholic fatty liver in mice.

Low caloric value of ethanol itself increases alveolar bone loss in ligature-induced periodontitis in male rats

This study aimed at morphometrically evaluating the influence of variable caloric values of ethanol consumption on alveolar bone loss in periodontitis in male rats. Thirty-six male rats were randomized into four groups of nine rats each, as follows: Test group A (low) - rats were fed an ethanol-containing liquid diet (ethanol representing 22% of total caloric value); Control group A -rats were fed a pair-fed control diet (ethanol replaced by isocaloric amounts of carbohydrate); Test group B (high) -rats were fed an ethanol-containing liquid diet (ethanol representing 36% of total caloric value); Control group B -rats were fed a pair-fed control diet for Test B. Following anesthesia, cotton ligatures were placed around the cervix of the right upper second molar. At eight weeks, the maxillary bones were removed and alveolar bone loss was analyzed by measuring the distance between the cementoenamel junction and the alveolar bone crest at buccal and palatal sites of the upper second molar. The unligated groups showed no significant differences between the bone loss values observed for the low and high caloric values of ethanol (p > 0.05). In the ligated groups, the rats receiving low caloric values of ethanol showed significantly greater bone loss compared to the isocaloric rats (p < 0.05); however, the rats receiving high caloric values of ethanol showed no significant differences compared to the controls. Analysis of the results demonstrated that, in male rats, ethanol itself affected ligature-induced bone loss when representing a low value in the total caloric value.

Formation of γ-ketoaldehyde–protein adducts during ethanol-induced liver injury in mice

Ethanol metabolism promotes the formation of a variety of reactive aldehydes in the liver. These aldehydes can rapidly form covalent protein adducts. Accumulating evidence indicates that these protein adducts may contribute to ethanol-mediated liver injury. Overproduction of γ-ketoaldehydes, levuglandins (LGs) and isolevuglandins, is implicated in the pathogenesis of several chronic inflammatory diseases. γ-Ketoaldehydes can form protein adducts orders of magnitude more quickly than 4-hydroxynonenal (4-HNE) or malondialdehyde. We hypothesized that ethanol-induced oxidative stress in vivo results in overproduction of LGE 2– and iso[4]LGE 2–protein adducts in mouse liver. Female C57BL/6 mice were allowed free access to an ethanol-containing diet for up to 39 days or pair-fed control diets. Pathological markers of ethanol-induced hepatic injury including serum alanine aminotransferase, hepatic triglyceride, and CYP2E1 were elevated in response to ethanol feeding. Ethanol-induced formation of iso[4]LGE 2–, LGE 2–, and 4-HNE–protein adducts in mouse liver was dependent on both dose and duration of ethanol feeding. Deficiency of cyclooxygenase 1 or 2 did not prevent ethanol-induced iso[4]LGE 2 or LGE 2 adducts in the liver, but adduct formation was reduced in both TNFR1- and CYP2E1-deficient mice. In summary, ethanol feeding enhanced γ-ketoaldehyde–protein adduct production via a TNFR1/CYP2E1-dependent, but cyclooxygenase-independent, mechanism in mouse liver.

Alcohol alters whole body composition, inhibits bone formation, and increases bone marrow adiposity in rats

Chronic alcohol abuse is a risk factor for osteoporosis and sarcopenia, but the long-term effects of alcohol on the immature musculoskeletal system are less clear. The present investigation in growing rats was designed to determine the effects of alcohol consumption on body composition, muscle mass, and bone mass, architecture, and turnover. Few studies have focused on the long-term effects of drinking on bone and muscle during skeletal maturation. Alcohol was included in the diet of 4-week-old male Sprague-Dawley rats (35% caloric intake) for 3 months. The controls were fed an isocaloric alcohol-free liquid diet ad libitum. A second study was performed in which the controls were pair-fed to the alcohol-fed animals. Compared to ad libitum-fed age-matched controls, alcohol-fed rats weighed less and had lower lean mass, fat mass, and percent body fat. In addition, they had lower slow- and fast-twitch muscle mass, lower total body bone mineral content and bone mineral density, and lower cancellous bone volume in the lumbar vertebra and proximal tibia. The effects of alcohol consumption on body composition were reduced when compared to the pair-fed control diet, indicating that caloric restriction was a comorbidity factor. In contrast, the effects of alcohol to decrease bone formation and serum leptin and IGF-I levels and to increase bone marrow adiposity appeared independent of caloric restriction. The skeletal abnormalities in growing alcohol-fed rats were due to a combination of effects specific to alcohol consumption and alcohol-induced caloric restriction.

Exogenous thioredoxin prevents ethanol-induced oxidative damage and apoptosis in mouse liver #

Ethanol-induced liver injury is characterized by increased formation of reactive oxygen species (ROS) and inflammatory cytokines, resulting in the development of hepatic steatosis, injury, and cell death by necrosis and apoptosis. Thioredoxin (Trx), a potent antioxidant and antiinflammatory molecule with antiapoptotic properties, protects animals from a number of inflammatory diseases. However, the effects of ethanol on Trx or its role in ethanol-induced liver injury are not known. Female C57BL/6 mice were allowed ad libitum access to a Lieber-deCarli ethanol diet with 5.4% of calories as ethanol for 2 days to acclimate them to the diet, followed by 2 days with 32.4% of calories as ethanol or pair-fed control diet. Hepatic Trx-1 was decreased by ethanol feeding; daily supplementation with recombinant human Trx (rhTrx) prevented this ethanol-induced decrease. Therefore, we tested the hypothesis that administration of rhTrx during ethanol exposure would attenuate ethanol-induced oxidative stress, inflammatory cytokine production, and apoptosis. Mice were treated with a daily intraperitoneal injection of either 5 g/kg of rhTrx or phosphate-buffered saline (PBS). Ethanol feeding increased accumulation of hepatic 4-hydroxynonenal protein adducts, expression of hepatic tumor necrosis factor alpha, and resulted in hepatic steatosis and increased plasma aspartate aminotransferase and alanine aminotransferase. In ethanol-fed mice, treatment with rhTrx reduced 4-hydroxynonenal adduct accumulation, inflammatory cytokine expression, decreased hepatic triglyceride, and improved liver enzyme profiles. Ethanol feeding also increased transferase-mediated dUTP-biotin nick-end labeling-positive cells, caspase-3 activity, and cytokeratin-18 staining in the liver. rhTrx treatment prevented these increases. In summary, rhTrx attenuated ethanol-induced increases in markers of oxidative stress, inflammatory cytokine expression, and apoptosis.

Oxidative damage of rat liver induced by ligature-induced periodontitis and chronic ethanol consumption

Objective Oxidative stress plays a central role in the initiation and progression of liver disease. Chronic ethanol consumption induces oxidative damage of the liver. Using a rat model, we previously showed that chronic administration of lipopolysaccharide and proteases to gingival sulcus induced both periodontal inflammation and liver injury. Periodontitis and ethanol consumption may have an additional effect on hepatic oxidative damage. The present study investigated the effects of periodontitis on ethanol-induced oxidative damage of the liver using a rat model. Design Male Wistar rats were divided into four groups (six rats/group). During the experimental period of eight weeks, two groups were fed an ethanol-containing liquid diet, and two groups were on a pair-fed control diet. Four weeks prior to the end of the experimental period, one group from each dietary treatment was ligated to induce periodontitis, while the other group was left unligated. In order to evaluate hepatic oxidative damage, the level of hexanoyl-lysine and the ratio of reduced form glutathione/oxidized form glutathione (GSH/GSSG) was determined. The concentration of blood hexanoyl-lysine was also measured as an index of circulating lipid peroxide. Results Ligature-induced periodontitis increased plasma levels of hexanoyl-lysine. In the liver, periodontitis decreased the GSH/GSSG ratio, and the combination of periodontitis and ethanol consumption induced a significant increase in hexanoyl-lysine level compared to ethanol consumption alone. Conclusion In the rat model, ligature-induced periodontitis increased plasma lipid peroxide, decreased the hepatic GSH/GSSG ratio and augmented ethanol-induced lipid peroxidation in the liver.

Effects of Ethanol Consumption on Periodontal Inflammation in Rats

Studies suggest a correlation between ethanol consumption and periodontal disease. We hypothesized that elevated levels of blood reactive oxygen species following ethanol consumption may increase inflammation in periodontal tissue. Rats were divided into 4 groups (6-7 rats/group). Two groups were fed an ethanol-containing liquid diet, and 2 groups were fed a pair-fed control diet. In one of each dietary group, periodontitis was ligature-induced, while the other group was left unligated. Chronic ethanol feeding alone decreased the ratio of reduced/oxidized glutathione and increased 8-hydroxydeoxy-guanosine and tumor necrosis factor (TNF)-alpha levels in the gingiva. Blood hydroperoxides were also increased. In ligature-induced periodontitis lesions, ethanol feeding enhanced polymorpho-nuclear leukocyte infiltration and TNF-alpha expression. The results suggest that chronic alcohol consumption increased periodontal inflammation, oxidative damage, and TNF-alpha production and had an additive effect on polymorphonuclear leukocyte infiltration and gingival oxidative damage, increasing the severity of periodontal inflammation in the ligature model.

Niacin Supplementation Decreases the Incidence of Alkylation-Induced Nonlymphocytic Leukemia in Long-Evans Rats

Niacin deficiency impairs poly(ADP-ribose) formation and enhances ethylnitrosourea (ENU)-induced carcinogenesis. Previous experiments were compromised by rapid progression of cancer, and the current study was designed with half the number of ENU doses. Weanling male Long-Evans rats were fed niacin deficient (ND), pair-fed (PF) control (30 mg nicotinic acid/kg), or pharmacological niacin (NA; 4 g nicotinic acid/kg) diets. After 2 wk, rats were gavaged every other day with ENU [30 mg/kg body weight (bw)] or vehicle (6 doses). Four days after the last dose of ENU, all rats were switched to AIN-93M diet and mildly feed restricted to maintain a constant food intake per bw. Rats were monitored for termination criteria and assessed for cancer development. Total cancers developed more rapidly in rats on the ND diet compared to those receiving high dose supplements of NA (P = 0.02; Gehan's generalized Wilcoxon test). Importantly, all of these differences occurred in the leukemias, especially the nonlymphocytic leukemia fraction (P = 0.008; Gehan's generalized Wilcoxon test), with incidences of 36%, 17%, and 11% in ND, PF, and NA rats, respectively. Because nonlymphocytic leukemias represent the majority of secondary cancers, these data support the concept that niacin supplementation may help protect cancer patients from the deleterious side effects of chemotherapy.

Hepatocyte Retinoid X Receptor α-Dependent Regulation of Lipid Homeostasis and Inflammatory Cytokine Expression Contributes to Alcohol-Induced Liver Injury

Hepatocyte retinoid X receptor alpha (RXRalpha)-deficient mice are more sensitive to ethanol toxicity than wild-type mice. Because RXRalpha-mediated pathways are implicated in lipid homeostasis and the inflammatory response, we hypothesized that a compromise in lipid metabolism and associated production of proinflammatory mediators are responsible for the hepatotoxicity observed in ethanol-treated hepatocyte RXRalpha-deficient mice. Wild-type and hepatocyte RXRalpha-deficient mice were fed ethanol-containing diets or pair-fed control diets for 6 weeks. After ethanol treatment, serum ALT levels increased significantly (4-fold) in hepatocyte RXRalpha-deficient mice, but not in the wild-type mice. Hepatic liver fatty acid binding protein (L-FABP) mRNA and protein levels were reduced due to RXRalpha deficiency. Ethanol induced L-FABP mRNA and protein in wild-type mice and provided protection against nonesterified fatty acid toxicity; however, this effect was absent in the mutant mice. Accordingly, hepatic nonesterified fatty acid level was increased in ethanol-fed mutant mice. Ethanol increased nuclear factor (NF)-kappaB binding activity in hepatocyte RXRalpha-deficient mice, but not in wild-type mice. In agreement, hepatic mRNA levels of proinflammatory cytokines and chemokines were increased to a greater extent in the mutant than in wild-type mice. Furthermore, signal transducer and activator of transcription factor (STAT) 3 and associated Bcl-xL induction was observed in ethanol-fed wild-type mice but not in ethanol-fed hepatocyte RXRalpha-deficient mice. Taken together, after ethanol treatment, hepatocyte RXRalpha deficiency results in lack of L-FABP induction, increased hepatic free fatty acids, NF-kappaB activation, and proinflammatory cytokines production and a lack of STAT3 activation, which in part may contribute to alcohol-induced liver damage.

Acute Ethanol Exposure Inhibits Renal Folate Transport, but Repeated Exposure Upregulates Folate Transport Proteins in Rats and Human Cells

Deficiency of folate in heavy-drinking alcoholic populations can occur partly because of an increased urinary folate excretion. Ethanol directly reduces the reabsorption of folate in the renal proximal tubule (PT) by acting on either of 2 folate transport proteins, the reduced folate carrier (RFC) and the folate receptor (FR). This study was designed to determine the effects of ethanol on the transport of folate by PT cells and to examine the effects of ethanol on RFC and the FR protein expression. Normal human PT (HPT) cells were cultured on membrane inserts to study intracellular transport of 5-methyltetrahydrofolate from the apical or basolateral direction in the presence of ethanol [11–109 mmol/L (50–500 mg/dL)]. The long-term effect of ethanol on the renal folate transport protein content was determined by western blot in treated HPT cells and in vivo in rats pair-fed control diets or ethanol-containing liquid diets. A 1-h treatment of HPT cells with ethanol (≥65 mmol/L) reduced the apically directed transport of folate by 20–25% without affecting the basolateral transport. A 5-d exposure of HPT cells to ethanol dose-dependently increased the content of both the FR and RFC proteins, with a greater effect on the RFC. Similarly, a 14-d exposure of rats to ethanol increased the in vivo expression of both the RFC and FR. These studies demonstrate that ethanol decreases the reabsorptive transport of folate by renal PT cells, which would increase urinary folate excretion. In contrast, subchronic exposure of PT cells, both in vivo and in vitro, to folate-depleting concentrations of ethanol leads to an upregulation of the 2 folate transport proteins. The increase in folate transporters partly counteracts the inhibitory effects of ethanol on folate transport activity, which explains the lower magnitude of ethanol’s effect on transport with subchronic exposure compared with that with acute exposure.

Differential Contributions of C3, C5, and Decay-Accelerating Factor to Ethanol-Induced Fatty Liver in Mice

The complement pathway is an important component of the innate and adaptive immune response. Here we tested the hypothesis that activation of complement is required for development of ethanol-induced fatty liver. Wild-type mice and mice lacking the third (C3) or fifth (C5) components of the complement activation pathway, as well as mice lacking decay-accelerating factor (CD55/DAF), a complement regulatory protein, were fed Lieber-DeCarli ethanol-containing diets for 6 weeks or pair-fed control diets. Ethanol feeding to wild-type mice increased C3a in plasma. Wild-type and C5-/- mice fed the ethanol diet developed hepatic steatosis characterized by microvesicular and macrovesicular lipid accumulation and increased triglyceride content. C3-/- mice did not develop steatosis, while CD55/DAF-/- mice accumulated even more hepatic triglyceride after ethanol feeding than wild-type mice. Levels of serum alanine aminotransferase and hepatic tumor necrosis factor alpha, indicators of hepatocyte injury and inflammation, respectively, were increased in wild-type and CD55/DAF-/- mice but not in C5-/- mice after ethanol feeding. In contrast to the protective effect of C3-/- against ethanol-induced steatosis, levels of both alanine aminotransferase and tumor necrosis factor alpha were increased in C3-/- mice after ethanol feeding. Here we have identified several elements of the complement system as important contributors to ethanol-induced fatty liver. C3 contributed primarily to the accumulation of triglyceride in the liver, whereas C5 was involved in inflammation and injury to hepatocytes. Further, the absence of CD55/DAF exacerbated these responses, suggesting that CD55/DAF serves as a barrier to ethanol-induced fatty liver.

Chronic Ethanol Consumption Impairs Cellular Immune Responses Against HCV NS5 Protein Due to Dendritic Cell Dysfunction

Alcoholic patients with and without chronic liver disease have a high incidence of infection with hepatitis C virus (HCV). Long-term ethanol consumption in mice has been associated with a strikingly reduced CD8(+) cytotoxic T-lymphocyte (CTL) response to HCV nonstructural proteins following DNA-based immunization. This study evaluated the effect of ethanol on dendritic cells (DCs) as a mechanism(s) for reduced CTL activity. Mice were fed an ethanol-containing or isocaloric pair-fed control diet for 8 weeks, followed by DC isolation from the spleen. DCs were evaluated with respect to endocytosis properties, cell surface markers, allostimulatory activity, and cytokine production following stimulation. Immune responses to HCV NS5 protein were generated by genetic immunization. Syngeneic transfer was used to determine if DC dysfunction contributed to abnormal cellular immune responses. Long-term ethanol exposure resulted in a reduced number of splenic DCs but did not alter endocytosis capacity. There was an increase in the myeloid and a reduction in the lymphoid DC population. Ethanol reduced expression of CD40 and CD86 costimulatory molecules on resting DCs, which was corrected following stimulation with lipopolysaccharide or poly I:C. There was impaired allostimulatory activity. Cytokine profiles of DCs isolated from ethanol-fed mice were characterized by enhanced interleukin (IL)-1beta and IL-10 and decreased tumor necrosis factor alpha, IL-12, interferon gamma, and IL-6 secretion. Impaired CTL responses to NS5 were corrected by syngeneic transfer of control DCs. Altered DC function is one of the major changes induced by long-term ethanol consumption, which subsequently impairs the cellular immune response necessary for viral clearance.

Chronic ethanol feeding to rats decreases adiponectin secretion by subcutaneous adipocytes

Chronic ethanol feeding to mice and rats decreases serum adiponectin concentration and adiponectin treatment attenuates chronic ethanol-induced liver injury. Although it is clear that lowered adiponectin has pathophysiological importance, the mechanisms by which chronic ethanol decreases adiponectin are not known. Here, we have investigated the impact of chronic ethanol feeding on adiponectin expression and secretion by adipose tissue. Rats were fed a 36% Lieber-DeCarli ethanol-containing liquid diet or pair-fed control diet for 4 wk. Chronic ethanol feeding decreased adiponectin mRNA but had no effect on adiponectin protein in subcutaneous adipose tissue. Chronic ethanol feeding also reduced adiponectin secretion by isolated subcutaneous and retroperitoneal adipocytes despite the maintenance of equivalent intracellular concentrations of adiponectin between subcutaneous adipocytes from ethanol- and pair-fed rats. Treatment with brefeldin A suppressed adiponectin secretion by subcutaneous adipocytes from pair-fed rats but had little effect after ethanol feeding. In subcutaneous adipocytes from pair-fed rats, adiponectin was enriched in endoplasmic reticulum (ER)/Golgi relative to plasma membrane; however, after chronic ethanol feeding, adiponectin was equally distributed between plasma membrane and ER/Golgi fractions. In conclusion, chronic ethanol feeding impaired adiponectin secretion by subcutaneous and retroperitoneal adipocytes; impaired secretion likely contributes to decreased adiponectin concentrations after chronic ethanol feeding.