Chronic Effects of a High Sucrose Diet on Murine Gastrointestinal Nutrient Sensor Gene and Protein Expression Levels and Lipid Metabolism.

The gastrointestinal tract (GIT) plays a key role in regulating nutrient metabolism and appetite responses. This study aimed to identify changes in the GIT that are important in the development of diet related obesity and diabetes. GIT samples were obtained from C57BL/6J male mice chronically fed a control diet or a high sucrose diet (HSD) and analysed for changes in gene, protein and metabolite levels. In HSD mice, GIT expression levels of fat oxidation genes were reduced, and increased de novo lipogenesis was evident in ileum. Gene expression levels of the putative sugar sensor, slc5a4a and slc5a4b, and fat sensor, cd36, were downregulated in the small intestines of HSD mice. In HSD mice, there was also evidence of bacterial overgrowth and a lipopolysaccharide activated inflammatory pathway involving inducible nitric oxide synthase (iNOS). In Caco-2 cells, sucrose significantly increased the expression levels of the nos2, iNOS and nitric oxide (NO) gas levels. In conclusion, sucrose fed induced obesity/diabetes is associated with changes in GI macronutrient sensing, appetite regulation and nutrient metabolism and intestinal microflora. These may be important drivers, and thus therapeutic targets, of diet-related metabolic disease.

Demonstration of ethanol-induced protein adducts in oral leukoplakia (pre-cancer) and cancer.

BACKGROUND: Excessive alcohol consumption is a common cause for upper gastrointestinal tract cancers. However, the primary mechanisms of alcohol-induced carcinogenesis have remained poorly defined. METHOD: We examined the generation and subcellular distribution of protein adducts with acetaldehyde (AA), the first metabolite of ethanol, and end products of lipid peroxidation, malondialdehyde (MDA) and 4-hydroxynonenal (HNE), from oral biopsy specimens obtained from 36 subjects (11 British, 25 Japanese) reporting alcohol misuse. All patients had been diagnosed with oral pre-cancer (leukoplakia, n = 7) or squamous cell carcinoma (SCC; n = 29). Automated immunostaining for AA, MDA and HNE adducts was performed using monospecific antibodies. RESULTS: Positive staining for AA, MDA and HNE adducts was observed in the dysplastic or malignant epithelial cells, HNE being relatively the most abundant adduct species. The subgroup of Japanese patients had higher levels of AA and MDA, although not HNE, than the British sample. When the material was divided to those with SCC or leukoplakia, MDA adducts but not the other antigens were more prominent in the former group. Significant correlations were found between the different adducts (AA vs. MDA, r = 0.68, P < 0.001; AA vs. HNE, r = 0.47, P < 0.01 and MDA vs. HNE, r = 0.59, P < 0.001). In addition, cytochrome P450 2E1 staining was found in these samples, correlating with both AA and MDA adducts. CONCLUSION: The data indicates that AA- and lipid peroxidation-derived adducts are formed in oral tissues of alcohol misusers with oral leukoplakia and cancer. The findings also support a pathogenic role of AA and excessive oxidative stress in carcinogenesis.

Covert assessment of concurrent and construct validity of a chart to characterize fecal output and diarrhea in patients receiving enteral nutrition.

BACKGROUND: An accurate and convenient method for characterizing fecal output and a consistent threshold for classifying diarrhea in patients receiving enteral nutrition are required. The aim of this study is to covertly assess the construct and concurrent validity of a chart for characterizing fecal output and classifying diarrhea in patients receiving enteral nutrition. METHODS: The chart was used to monitor fecal output in patients receiving enteral nutrition for a total of 280 patient days. Nurses characterized 291 fecal samples, of which 84 underwent measurement of fecal water using lyophilization and 60 underwent Clostridium difficile enterotoxin analysis using enzyme-linked immunosorbent assay. Construct and concurrent validity was assessed covertly to measure the true performance of the chart in a real-life clinical and research context. RESULTS: Use of the chart demonstrated higher fecal frequency (P

Diet Quality : An Evidence-Based Approach, Volume 2

Diet quality is a broad term that encapsulatesboth perceived and actual practices, personal preferences andcultural diversity. Measuring dietary quality can be problematicand includes investigating food types, the number or size ofportions or their frequency. Diet quality may also be related tothe type of food being ingested, snacking and other eating habits.Manufactured beverages and fast food may also be included as wellas microbiological quality and attempts to improve single fooditems such as meats or vegetables. In this book, Diet Quality: AnEvidence-Based Approach, Volume 2 all of the major facets of dietquality in relation to health outcomes are covered. This importantnew text includes methods for determining diet quality whileadopting a holistic approach to impart information on the majorareas of concern or knowledge. Chapters link in measurable indicesof health such as obesity, pregnancy outcomes, cancer and canceroutcomes, and mortality. This book represents a diverse set ofsubject matters and seeks to fill a gap in the literature at a timewhen there is an increasing awareness that well being is associatedwith the qualitative nature of diets.ÿ Contributors areauthors of international and national standing and emerging fieldsof science are incorporated. Diet Quality: An Evidence-BasedApproach, Volume 2 is a useful new text designed for nutritionists,dietitians, clinicians, epidemiologist, policy makers and healthcare professionals of various disciplines.

Handbook of Food Fortification and Health : From Concepts to Public Health Applications Volume 2

Handbook of Food Fortification and Health: FromConcepts to Public Health Applications Volume 2 represents amultidisciplinary approach to food fortification. This book aims todisseminate important material pertaining to the fortification offoods from strategic initiatives to public health applications.Optimal nutritional intake is an essential component of health andwellbeing. Unfortunately situations arise on a local or nationalscale when nutrient supply or intake is deemed to be suboptimal. Asa consequence, ill health occurs affecting individual organs orcausing premature death. In terms of public health, malnutritiondue to micronutrient deficiency can be quite profound imposingeconomic and social burdens on individuals and whole communities.This comprehensive text examines the broad spectrum of foodfortification in all its manifestations. Coverage includes sectionson definitions of fortifications, fortified foods, beverages andnutrients, fortifications with micronutrients, biofortification,impact on individuals, public health concepts and issues, andselective methods and food chemistry. Handbook of FoodFortification and Health: From Concepts to Public HealthApplications Volume 2 is an indispensable text designed fornutritionists, dietitians, clinicians and health relatedprofessionals.

Alcoholic myopathy and acetaldehyde.

Alcoholic myopathy is characterized by biochemical and morphological lesions within muscle, ranging from impairment of muscle strength and loss of lean tissue to cellular disturbances and altered gene expression. The chronic form of the disease is five times more common than cirrhosis and is characterized by selective atrophy of type 11 (anaerobic) fibres: type I (aerobic) fibres are relatively protected. Although the causative agent is known (i.e. ethanol), the intervening steps between alcohol ingestion and the development of symptoms and lesions are poorly understood. However, acetaldehyde appears to have an important role in the aetiology of the disease. For example, alcohol is a potent perturbant of muscle protein synthesis in vivo, and this effect is exacerbated by cyanamide pre-dosage, which raises acetaldehyde concentrations. Acetaldehyde alone also reduces muscle protein synthesis in vivo and proteolytic activity in vitro. The formation of acetaldehyde protein adducts is another mechanism of putative importance in alcoholic myopathy. These adducts are formed within muscle in response to either acute or chronic alcohol exposure and the adducts are located preferentially within the sarcolemmal and sub-sarcolemmal regions. However, the significance of protein adduct formation is unclear since we do not currently know the identity of the adducted muscle proteins nor whether adduction alters the biochemical or functional properties of skeletal muscle proteins.

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver.

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Molecular and cellular events in alcohol-induced muscle disease.

Alcohol consumption induces a dose-dependent noxious effect on skeletal muscle, leading to progressive functional and structural damage of myocytes, with concomitant reductions in lean body mass. Nearly half of high-dose chronic alcohol consumers develop alcoholic skeletal myopathy. The pathogenic mechanisms that lie between alcohol intake and loss of muscle tissue involve multiple pathways, making the elucidation of the disease somewhat difficult. This review discusses the recent advances in basic and clinical research on the molecular and cellular events involved in the development of alcohol-induced muscle disease. The main areas of recent research interest on this field are as follows: (i) molecular mechanisms in alcohol exposed muscle in the rat model; (ii) gene expression changes in alcohol exposed muscle; (iii) the role of trace elements and oxidative stress in alcoholic myopathy; and (iv) the role of apoptosis and preapoptotic pathways in alcoholic myopathy. These aforementioned areas are crucial in understanding the pathogenesis of this disease. For example, there is overwhelming evidence that both chronic alcohol ingestion and acute alcohol intoxication impair the rate of protein synthesis of myofibrillar proteins, in particular, under both postabsorptive and postprandial conditions. Perturbations in gene expression are contributory factors to the development of alcoholic myopathy, as ethanol-induced alterations are detected in over 400 genes and the protein profile (i.e., the proteome) of muscle is also affected. There is supportive evidence that oxidative damage is involved in the pathogenesis of alcoholic myopathy. Increased lipid peroxidation is related to muscle fibre atrophy, and reduced serum levels of some antioxidants may be related to loss of muscle mass and muscle strength. Finally, ethanol induces skeletal muscle apoptosis and increases both pro- and antiapoptotic regulatory mechanisms.

Skeletal muscle and liver oxysterols during fasting and alcohol exposure.

Oxysterols are cytotoxic agents that have a range of cellular actions, including impairment of albumin synthesis, cell differentiation, and induction of apoptosis. Their regulations by nutritional factors are poorly described. Our objective was to test the hypothesis that the imposition of food withdrawal and alcohol exposure increases tissue oxysterol concentrations. We measured the concentrations of the oxysterols 7alpha-hydroxycholest-5-en-3beta-ol (7alpha-OH), 7beta-hydroxycholest-5-en-3beta-ol (7beta-OH), and 3beta-hydroxycholest-5-en-7-one (7-keto) in liver and skeletal muscle of fed and fasted (food withdrawal for 1 and 2 days) male Wistar rats. Both oxidative (type I; soleus) and glycolytic (type II; plantaris) muscles were analyzed. We also investigated the effects of a nutritional perturbant induced by a short-term bolus of ethanol (75 mmol/kg weight IP administered 2.5 hours before sacrifice). The results showed that in response to fasting there were significant increases in 7alpha-OH, 7beta-OH, and 7-keto in liver and both type I and II skeletal muscle (P < .001 in all instances). For skeletal muscle, the increases were blunted or ameliorated after 2 days when compared with data from rats starved for 1 day. In contrast, the increases in liver after 1 day's fasting were relatively sustained at 2 days. Short-term ethanol increased 7alpha-OH, 7beta-OH, and 7-keto in type I muscle of fed animals only (P < .001 in all instances) with a significant interaction between fasting and alcohol (P < .001 in all instances). For the first time, we have shown that oxysterols can increase in muscle and liver in response to food withdrawal and in response to an immediately imposed nutritional perturbant (ie, alcohol). Increased oxysterols represent elevated oxidative stress and/or disturbances in their formation or clearance. Because of the reported cytotoxic properties of oxysterols, these data are important in understanding cellular pathology because episodic anorexia and/or oxidative stress occur in a variety of disease conditions including sepsis, cancer cachexia, ischemia, and hormonal imbalance.

Alcohol alters skeletal muscle heat shock protein gene expression in rats: these effects are moderated by sex, raised endogenous acetaldehyde, and starvation.

Alcoholic myopathy is a common pathology characterized by wasting due to reduced protein synthesis, although the mechanisms involved remain unclear. Women are particularly sensitive and malnutrition exacerbates the myopathy. This study aimed to address (i) whether long-term alcohol feeding alters expression of heat shock proteins (HSPs) in male and female rats; (ii) the effect of immediate alcohol dosing with or without raised levels of endogenous acetaldehyde; and (iii) the effect of starvation. To address this, (i) male and female rats were fed alcohol in the long-term (6-7 weeks as 35% of energy in a liquid diet) and compared to controls fed the same diet with isoenergetic glucose; (ii) male rats given an immediate bolus (75 mmol ethanol per kilogram body weight intraperitoneally) 2.5 hours before sacrifice and compared to controls given a dose of saline (with or without pretreatment with cyanamide-an acetaldehyde dehydrogenase inhibitor which raises endogenous acetaldehyde); (iii) male rats starved for 1 or 2 days then immediately dosed with alcohol. Protein levels of HSP 27, HSP 60, and HSP 70 were measured in muscles of male rats fed alcohol and pair-fed control rats by SDS-PAGE and Western blotting in study I. Levels of HSP 27, HSP 60, HSP 70, and HSP 90 mRNA were analyzed in hind limb skeletal muscle by reverse transcription-polymerase chain reaction with an endogenous internal standard, glyceraldehyde-3-phosphate-dehydrogenase. (i) Long-term alcohol dosage reduced HSP 27 in male rats but not in females, whereas HSP 90 mRNA increased in long-term alcohol-fed female rats but not in male rats. These changes were reflected by a similar trend in HSP protein content, although statistical significance was not achieved. (ii) There was no effect on any of the HSP mRNAs in rats dosed immediately with alcohol or in combination with cyanamide. (iii) Starvation per se for 2 days was associated with an increase in HSP 27 mRNA. Alcohol administration after 2 days starvation caused a blunting of the increased HSP 27 mRNA in starvation alone. This suggests that long-term alcohol exposure affects HSP gene expression and that this effect is moderated by sex and starvation. This may contribute to, or reflect, the biochemical lesion in alcoholic myopathy.

Formula delivery in patients receiving enteral tube feeding on general hospital wards: the impact of nasogastric extubation and diarrhea.

OBJECTIVE: In contrast to the intensive care unit, little is known of the percentage of formula delivered to patients receiving enteral tube feeding (ETF) on general wards or of the complications that affect its delivery. This study prospectively investigated the incidence of nasogastric extubation and diarrhea in patients starting ETF on general wards and examined their effect on formula delivery. METHODS: In a prospective observational study, the volume of formula delivered to patients receiving ETF on general wards was compared with the volume prescribed. The incidence of nasogastric extubation and diarrhea was measured and its effect on formula delivery calculated. RESULTS: Twenty-eight patients were monitored for a total of 319 patient days. The mean +/- SD volume of formula prescribed was 1460 +/- 213 mL/d, whereas the mean volume delivered was only 1280 +/- 418 mL/d (P < 0.001), representing a mean percentage delivery of 88 +/- 25% of prescribed formula. Nasogastric extubation occurred in 17 of 28 patients (60%), affecting 53 of the 319 patient days (17%). The percentage of formula delivered on days when the nasogastric tube remained in situ was 96 +/- 12% and on days when nasogastric extubation occurred it was only 45 +/- 31% (P < 0.001). Diarrhea affected 39 of 319 patient days (12%) but there was no difference in formula delivery on days when diarrhea did or did not occur (78% versus 89%, P = 0.295). There was a significant, albeit small, negative correlation between the daily stool score and formula delivery (correlation coefficient -0.216, P < 0.001). CONCLUSIONS: Formula delivery is marginally suboptimal in patients receiving ETF on general wards. Nasogastric extubation is common and results in an inherent cessation of ETF until the nasogastric tube is replaced and is therefore a major factor impeding formula delivery. Diarrhea is also common but does not result in significant reductions in formula delivery.

Free radicals in alcoholic myopathy: indices of damage and preventive studies.

Chronic alcoholic myopathy affects up to two-thirds of all alcohol misusers and is characterized by selective atrophy of Type II (glycolytic, fast-twitch, anaerobic) fibers. In contrast, the Type I fibers (oxidative, slow-twitch, aerobic) are relatively protected. Alcohol increases the concentration of cholesterol hydroperoxides and malondialdehyde-protein adducts, though protein-carbonyl concentration levels do not appear to be overtly increased and may actually decrease in some studies. In alcoholics, plasma concentrations of alpha-tocopherol may be reduced in myopathic patients. However, alpha-tocopherol supplementation has failed to prevent either the loss of skeletal muscle protein or the reductions in protein synthesis in alcohol-dosed animals. The evidence for increased oxidative stress in alcohol-exposed skeletal muscle is thus inconsistent. Further work into the role of ROS in alcoholic myopathy is clearly warranted.

Generation of protein adducts with malondialdehyde and acetaldehyde in muscles with predominantly type I or type II fibers in rats exposed to ethanol and the acetaldehyde dehydrogenase inhibitor cyanamide.

BACKGROUND: Alcoholic myopathy is known to primarily affect type II muscle fibers (glycolytic, fast-twitch, anaerobic), whereas type I fibers (oxidative, slow-twitch, aerobic) are relatively protected. OBJECTIVE: We investigated whether aldehyde-derived adducts of proteins with malondialdehyde and acetaldehyde are formed in muscle of rats as a result of acute exposure to ethanol and acetaldehyde. The differences between type I muscle, type II muscle, and liver tissue were also assessed. DESIGN: The formation and distribution of malondialdehyde- and acetaldehyde-protein adducts were studied with immunohistochemistry in soleus (type I) muscle, plantaris (type II) muscle, and liver in 4 groups of rats. The different groups were administered saline (control), cyanamide (an acetaldehyde dehydrogenase inhibitor), ethanol, and cyanamide + ethanol. RESULTS: Treatment of rats with ethanol and cyanamide + ethanol increased the amount of aldehyde-derived protein adducts in both soleus and plantaris muscle. The greatest responses in malondialdehyde-protein and acetaldehyde-protein adducts were observed in plantaris muscle, in which the effect of alcohol was further potentiated by cyanamide pretreatment. Malondialdehyde- and acetaldehyde-protein adducts were also found in liver specimens from rats treated with ethanol and ethanol + cyanamide; the most abundant amounts were found in rats given cyanamide pretreatment. CONCLUSIONS: Acute ethanol administration increases protein adducts with malondialdehyde and acetaldehyde, primarily in type II muscle. This may be associated with the increased susceptibility of anaerobic muscle to alcohol toxicity. Higher acetaldehyde concentrations exacerbate adduct formation, especially in type II-predominant muscles. The present findings are relevant to studies on the pathogenesis of alcohol-induced myopathy.

Influence of starvation, surgery, and sepsis on cardiac protein synthesis in rats: effects of parenteral nutrition, glutamine, and growth hormone.

The effect of sepsis on the rate of protein synthesis in the heart is poorly described. We have investigated changes in protein synthesis in the ventricles of the heart over time after cecal ligation and puncture (CLP) in rats in comparison with sham-operated and unoperated animals (ad libitum). All operated animals were starved from the time of surgery to the time of sacrifice. When operated animals were compared with ad libitum animals, ventricular weight and ventricular protein, and DNA and RNA contents were unchanged at 24 h, but were invariably reduced at 72 and 96 h. Fractional rate of protein synthesis (FSR), RNA activity, and cellular efficiency were reduced at 24 h and further reduced at 72 and 96 h. There were no differences, however, between septic and sham-operated animals. Eighteen hours after CLP, additional groups of rats were infused intravenously with 0.9% sodium chloride, parenteral nutrition (PN), or PN with glutamine, and were given a single dose of 400 microg recombinant human growth hormone (rhGH) or an equal volume of 0.9% sodium chloride. FSR was higher in animals given PN when compared with those given 0.9% sodium chloride only, and did not differ from FSR measured in unoperated animals. There was no additional benefit from the acute administration of either glutamine-enriched PN or rhGH. These results indicate that ventricular protein synthesis is markedly reduced by surgery and starvation, but that superimposed sepsis does not further influence these changes. PN can prevent the fall in cardiac protein synthesis associated with starvation, surgery, and sepsis, but neither glutamine nor rhGH produced any additional benefit.

Ca2+-regulatory muscle proteins in the alcohol-fed rat.

Alcoholic myopathy is characterized by muscle weakness and difficulties in gait and locomotion. It is one of the most prevalent skeletal muscle disorders in the Western hemisphere, affecting between 40% and 60% of all chronic alcohol misusers. However, the pathogenic mechanisms are unknown, although recent studies have suggested that membrane defects occur as a consequence of chronic alcohol exposure. It was our hypothesis that alcohol ingestion perturbs membrane-located proteins associated with intracellular signalling and contractility, in particular those relating to calcium homeostasis. To test this, we fed male Wistar rats nutritionally complete liquid diets containing ethanol as 35% of total dietary energy. Controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At the end of 6 weeks, rats were killed and skeletal muscles dissected. These were used to determine important ion-regulatory skeletal muscle proteins including sarcalumenin (SAR), sarcoplasmic-endoplasmic reticulum Ca(2+)-adenosine triphosphatase (ATPase) (SERCA1), the junctional face protein of 90 kd (90-JFP), alpha(1)- and alpha(2)-dihydropyridine receptor (alpha(1)-DHPR and alpha(2)-DHPR), and calsequestrin (CSQ) by immunoblotting. The relative abundance of microsomal proteins was determined by immunoblotting using the enhanced chemiluminescence (ECL) technique. The data showed that alcohol-feeding significantly reduced gastrocnemius and hind limb muscle weights (P <.05 in both instances). Concomitant changes included increases in the relative amounts of SERCA1 (P <.05) and Ca(2+)-ATPase activity (P <.025). However, there were no statistically significant changes in either SAR, 90-JFP, alpha(1)-DHPR or alpha(2)-DHPR (P >.2 in all instances). Reductions in CSQ were of marginal significance (P =.0950). We conclude that upregulation of SERCA1 protein and Ca(2+)-ATPase activity may be an adaptive mechanism and/or a contributory process in the pathology of alcohol-induced muscle disease.

Chronic ethanol feeding increases 7-hydroperoxycholesterol and oxysterols in rat skeletal muscle.

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in skeletal muscles chronically exposed to ethanol. To test this we fed male Wistar rats (0.1 kg initial body weight) a nutritionally complete liquid diet containing ethanol as 35% of total calories: controls were pair-fed identical amounts of the same diet in which ethanol was replaced by isocaloric glucose. At the end of 1(1/2) months, soleus (type I fiber-predominant) and plantaris (type II fiber-predominant) skeletal muscles were dissected out. We measured 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH), as well as 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH), and 3 beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). We identified and confirmed by LC-MS the presence of 7 alpha-OOH, 7 beta-OOH and 7 alpha-OH, 7 beta-OH and 7-keto in skeletal muscle of rats. We also showed that in response to chronic alcohol feeding, there were significant increases in soleus 7 alpha-OH (P =.0005), 7 beta-OH (P =.0005), and 7-keto (P =.0007). In the plantaris, mean 7 alpha-OH and 7-keto were not significantly altered (P >.05), but 7 beta-OH increased (P =.0418). This is the first report of 7 alpha-OH, 7 beta-OH, and 7-keto oxysterols being identified in skeletal muscle of rats. Their elevation in chronic experimental alcoholism, together with increases in cholesterol hydroperoxides, may possibly represent evidence of increased oxidative stress.

Skeletal muscle and jejunal protein synthesis in normal and ethanol-treated rats: the effect of the nitric oxide synthase inhibitors, L-omega-nitro-L-arginine methyl ester and N(G)-nitro-L-arginine in vivo.

The nitric oxide synthase (NOS) inhibitors, L-omega-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg and 100 mg/kg) and N(G)-nitro-L-arginine (L-NNA; 100 mg/kg) were used to investigate the role of NO on in vivo skeletal muscle and jejunal (mucosa and seromuscular layer) protein synthesis rates in normal (ie, untreated) and ethanol-dosed (75 mmol/kg body weight) rats. Fractional rates of protein synthesis, ie, percentage of protein pool renewed each day, k(s), %/d) were measured with a flooding dose of L-[(3)H-4]phenylalanine. In response to both doses of L-NAME and L-NNA, k(s) in skeletal muscle of normal rats decreased by 9% to 31% (P between <.05 and <.001). In the mucosa, k(s) was significantly reduced only by the higher dose of L-NAME (-49%, P <.001). In the seromuscular layer, k(s) was reduced by 15% to 57% (P between <.05 and <.001) in response to both doses of L-NAME and L-NNA. Ethanol dosage reduced k(s) in skeletal muscle (-35%, P <.001), and small reductions also occurred in the jejunal mucosal and seromuscular layers (-14% P <.05 and -12% P <.05, respectively). However, in the presence of L-NAME, ethanol reduced k(s) in jejunal mucosal and seromuscular layers by -35% (P <.01) and -30% (P <.01), respectively, compared with controls. This exacerbating effect of L-NAME predosage in ethanol-treated rats was not demonstrable in skeletal muscle. The data thus suggest that (1) endogenous NO facilitates constitutive skeletal muscle and jejunal protein synthesis in control animals in vivo; (2) the sensitivity of jejunal (but not skeletal muscle) protein synthesis to acute ethanol is increased when inhibitors of NOS are used. This tentatively implies that, in response to ethanol, overproduction of NO is not involved in the ethanol-induced reduction of protein synthesis in skeletal muscle or the jejunum.

Liver dysfunction induced by bile duct ligation and galactosamine injection alters cardiac protein synthesis.

Liver disease has been shown to affect the cardiovascular system and may influence cardiac protein metabolism. This hypothesis was tested by measuring rates of cardiac protein synthesis in 2 models of liver disease in rats. The study consisted of 5 groups--group 1: control, injected with saline and fed ad libitum; group 2: acute liver injury, by dosage with 400 mg/kg galactosamine; group 3: injected with saline and pair-fed to group 2; group 4: chronic liver disease, using bile duct ligation; and group 5: sham-operated and pair-fed to group 4. Rates of cardiac protein synthesis were measured using the flooding dose technique. After 1 week, galactosamine injection caused the following cardiac changes, i.e. group (2) versus (3): an increased RNA content, RNA/DNA ratio, and RNA/protein ratio. However, there was no change in DNA or protein content, or protein/DNA ratio. There was an increase in the fractional rate of protein synthesis, and the absolute synthesis rate. Cellular efficiency was increased, but RNA activity remained unchanged. Comparison of groups 4 and 5 showed that bile duct ligation caused no change in any parameters measured. Although comparison of the ad libitum-fed group 1 with the bile duct ligation group 4 showed reduced cardiac weight, protein, and RNA content, with decreased right ventricular absolute synthesis rates; this was also seen in the pair-fed group 5, suggesting that these effects were due solely to reduced oral intake. Thus, although galactosamine-induced acute liver injury caused marked changes in cardiac biochemistry, bile duct ligation per se did not. This study also illustrates the importance of including a pair-fed group.

Chronic alcohol feeding and its influence on c-Fos and heat shock protein-70 gene expression in different brain regions of male and female rats.

Excessive ingestion of the macronutrient, alcohol, causes devastating complications in the brain leading to atrophy and impaired cognitive function with corresponding increases in morbidity and mortality and, consequently, reduced quality of life measures. The pathogenic mechanisms are unknown, but various studies have shown that the immediate early genes and heat shock (ie, stress or chaperone) proteins are increased in alcohol-exposed tissue. However, many of these studies have been performed in vitro or have failed to consider either the nutritional elements in the experimental design by appropriate use of pair-feeding or whether there are regional and/or gender differences. We hypothesized that (1) increased expression of heat shock proteins and/or oncogenes occur as a consequence of alcohol-feeding in vivo, and sensitivities are related to different (2) gender and (3) brain regions. To test this, we fed male and female rats nutritionally complete diets containing ethanol as 35% of total calories (treated) or isocaloric amounts of the same diet in which ethanol was replaced by isocaloric glucose (controls). At the end of 6 weeks, rats were killed and c-Fos and heat shock protein-70 (HSP70) mRNA analyzed in midbrain, cortex, brainstem, and cerebellum by reverse transcription-polymerase chain reaction (RT-PCR) with an endogenous internal standard, beta-actin. The results showed that there were distinct regional differences (P at least <.05) in both c-Fos (cerebellum > cortex > midbrain and brainstem) and HSP70 (brainstem and cerebellum > cortex and midbrain). However, the only significant effect of alcohol feeding occurred in the HSP70 mRNA in midbrain of male rats, which was reduced by approximately 50% (P <.01). In contrast, no corresponding effect of alcohol feeding was observed in c-Fos mRNA levels in either midbrain or other regions of female rats. These data show that chronic ethanol feeding has no demonstrable effect on c-Fos mRNA expression in the brain when using nutritionally complete liquid diet regimens with concomitant pair-feeding. HSP70 mRNA, in contrast, is reduced by alcohol feeding and appears to be regional and gender dependent.

Effect of chronic ethanol feeding on oxysterols in rat liver.

It was our hypothesis that, as a consequence of increased oxidative stress, cholesterol-derived hydroperoxides and oxysterols are increased in livers of rats exposed to ethanol. To test this we dosed Wistar rats (approximately 0.1 kg initial body weight) with ethanol chronically (rats fed a nutritionally complete liquid diet containing ethanol as 35% of total calories; sampled liver at approximately 6-7 weeks). We measured concentrations of 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH) as well as 7 alpha- and 7 beta-hydroxycholesterol (7 alpha-OH and 7 beta-OH), and 3 beta-hydroxycholest-5-en-7-one (also termed 7-ketocholesterol; 7-keto). In response to chronic alcohol feeding, there were significant elevations in the concentrations of 7 alpha-OOH (+169%, P = 0.005) and 7 beta-OOH (+199%, P = 0.011). Increases in the concentrations of hepatic 7-keto (+74%, P = 0.01) and decreases in cholesterol (-43%; P = 0.03) also occurred. In contrast, the concentrations of both 7 alpha-OH and 7 beta-OH were not significant (NS). However, when oxysterols in chronic ethanol-fed rats were expressed relative to cholesterol there were significant increases in 7-keto/cholesterol (P = 0.0006), 7 alpha-OH/cholesterol (P = 0.0018) and 7 beta-OH/cholesterol (P = 0.0047). In conclusion, this is the first report of increased 7 alpha-OOH, 7 beta-OOH, and 7-keto in liver of rats and their elevation in chronic experimental alcoholism represent evidence of increased oxidative stress.