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Increased gastrointestinal ethanol production in obese mice: Implications for fatty liver disease pathogenesis

      Abstract

      Background & Aims: Similarities in the hepatic responses to obesity and ethanol exposure suggest that these conditions evoke common pathogenic mechanisms. Thus, it is possible that ethanol exposure is increased in obesity. Given that intestinal bacteria can produce ethanol, the aim of this study was to determine if the intestinal production of ethanol is increased in obesity. Methods: Breath was collected from genetically obese, ob/ob male C57BL/6 mice and lean male littermates at different ages (14, 20, and 24 weeks) and times of the day (9 AM, 3 PM, and 9 PM). Obese mice (24 weeks old) were then treated with neomycin (1 mg/mL) for 5 days, and sampling was repeated. Results: Breath collected in the morning from 24-week-old obese mice had a higher ethanol content than breath from their lean littermates (271 vs. 78 pmol/mL CO2; P < 0.0001). Subsequent studies in 14- and 20-week-old mice showed that exhaled ethanol increased with age in obese (from 26 to 107 pmol/mL CO2; P < 0.002) but not lean (29 and 12 pmol/mL CO2) mice and was greater in older obese mice than in older lean mice (P < 0.0006). Obese mice showed a diurnal increase in breath ethanol in the morning that decreased through the afternoon and evening (107 to 33 to 13 pmol/mL CO2). Neomycin treatment decreased morning breath ethanol levels by 50% (from 220 to 110 pmol/mL CO2; P < 0.0003). Conclusions: Even in the absence of ethanol ingestion, ethanol can be detected in exhaled breath. In obesity, an age-related increase in breath ethanol content reflects increased production of ethanol by the intestinal microflora. Hence, intestinal production of ethanol may contribute to the genesis of obesity-related fatty liver.
      GASTROENTEROLOGY 2000;119:1340-1347

      Abbreviations:

      NASH (nonalcoholic steatohepatitis), RQ (respiratory quotient), TNF-α (tumor necrosis factor α)
      See editorial on page 1399.
      Obesity is a major risk factor for fatty liver diseases, including nonalcoholic steatohepatitis (NASH).
      • Matteoni C
      • Younossi ZM
      • McCullough A
      Nonalcoholic fatty liver disease: a spectrum of clinical pathological severity.
      • Caldwell SH
      • Oelsner DH
      • Iezzoni JC
      • Hespenheide EE
      • Battle EH
      • Driscoll CJ
      Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease.
      Obesity also increases the risk of both fatty liver and cirrhosis in patients who consume ethanol.
      • Giraud NS
      • Borotto E
      • Aubert A
      • Capron F
      • Chaput JC
      Excess weight risk factor for alcoholic liver disease.
      The mechanisms for obesity-related liver disease remain obscure.
      • Sheth SG
      • Gordon FD
      • Chopar S
      Nonalcoholic steatohepatitis.
      However, similarities between the histologic features of obesity-related liver disease and alcohol-induced liver damage
      • Ludwig J
      • Viggiano RT
      • McGill DB
      Nonalcoholic steatohepatitis. Mayo Clinic experiences with a hitherto unnamed disease.
      suggest that common pathogenic mechanisms may be involved. It is even conceivable that obesity increases hepatic exposure to ethanol. Theoretically, ingested ethanol might accumulate in the excessive adipose tissue of obese individuals because of its lipid solubility, prolonging ethanol exposure from “social” drinking. Alternatively, as discussed below, endogenous ethanol production might be increased in obese individuals.
      With reference to this possibility, the intestinal bacterial flora are capable of both ethanol production and metabolism.
      • Nosova T
      • Jokelainen K
      • Kaihovaara P
      • Jousimies-somer H
      • Siitonen A
      • Heine R
      • Salaspuro M
      Aldehyde dehydrogenase activity and acetate production by aerobic bacteria representing the normal flora of human large intestine.
      Intestinal ethanol production increases when generalized or segmental decreases in intestinal motility permit bacterial overgrowth in static intestinal segments.
      • Baraona E
      • Julkunen R
      • Tannenbaum L
      • Lieber CS
      Role of intestinal bacterial overgrowth in ethanol production and metabolism in rats.
      At least one group of investigators
      • Mezey E
      • Imbembo AL
      • Potter JJ
      • Rent KC
      • Lombardo R
      • Holt PR
      Endogenous ethanol production and hepatic disease following jejunoileal bypass for morbid obesity.
      detected ethanol in the blood of some morbidly obese patients who had undergone jejunal-ileal bypass surgery to induce weight loss. The same group showed that jejunal-ileal bypass surgery could increase blood ethanol concentrations in dogs. However, because patients with the highest blood ethanol concentrations did not have the most severe steatohepatitis, the authors concluded that intestinal ethanol production was not likely to be the cause of jejunal-ileal bypass–related liver disease.
      • Mezey E
      • Imbembo AL
      • Potter JJ
      • Rent KC
      • Lombardo R
      • Holt PR
      Endogenous ethanol production and hepatic disease following jejunoileal bypass for morbid obesity.
      The possibility that increases in intestinal ethanol production play a more general role in the genesis of obesity-related fatty liver disease merits further consideration. In particular, even small increases in intestine-derived ethanol might increase portal blood ethanol levels enough to induce hepatic steatosis, given evidence that ethanol metabolism produces obligatory redox changes that promote the accumulation of triglyceride in hepatocytes.
      • Lieber C
      Hepatic, metabolic and toxic effect of ethanol.
      Moreover, efficient hepatic metabolism of ethanol might eliminate most of the toxin before it enters the systemic circulation, causing blood ethanol determinations to underestimate the actual production of ethanol in the gastrointestinal tract. In addition, obesity has been associated with alterations in gut motility that might favor bacterial overgrowth. For example, an increased prevalence of irritable bowel syndrome
      • Crowell MD
      • Cheskin LJ
      • Musial F
      Prevalence of gastrointestinal symptoms in obese and normal weight binge eaters.
      and colonic pseudo-obstruction
      • Weiner P
      • Shlum H
      • Ganam R
      • Plavnick L
      Colonic pseudo-ob-struction: a late complication of jejunoileal bypass.
      • O'Malley KJ
      • Flechner SM
      • Kapoor A
      • Rhodes RA
      • Modlin CS
      • Goldfarb DA
      • Novick AC
      Acute colonic pseudo-obstruction (Ogilvie's syndrome) after renal transplantation.
      have been reported in obese individuals. Other evidence suggests that obesity decreases sensitivity to certain neuropeptides, such as cholecystokinin
      • Niederau C
      • Meereis-Schwanke K
      • Klonowski-Stumpe H
      • Herberg L
      CCK-resistance in Zucker obese versus lean rats.
      • Fink H
      • Rex A
      • Voits M
      • Voigt JP
      Major biological actions of CCK—a critical evaluation of research findings.
      and bombesin,
      • Lieverse RJ
      • Masclee AA
      • Jansen JB
      • Lam WF
      • Lamers CB
      Obese women are less sensitive for the satiety effects of bombesin than lean women.
      that regulate intestinal motility.
      Although noninvasive methods (e.g., breath analysis) exist to measure endogenous ethanol production, several factors confound efforts to evaluate the importance of gut-derived ethanol as a pathogenic factor for fatty liver in obese humans, including difficulties in selecting a study population with proven fatty liver and reliably documenting abstinence from ethanol ingestion. However, the question can be addressed easily by studying animal models under carefully controlled conditions. Genetically obese ob/ob mice are an inbred strain of C57BL/6 mice with spontaneous mutations in the leptin gene.
      • Campfield LA
      • Smith FJ
      • Burn P
      The OB protein (leptin) pathway— a link between adipose tissue mass and central neural networks.
      The resulting deficiency in this appetite-suppressing hormone causes the mice to overeat; they become obese in early adulthood and develop obesity-related fatty livers.
      • Yang SQ
      • Lin HZ
      • Lane MD
      • Clemens M
      • Diehl AM
      Obesity increases sensitivity to endotoxin liver injury: implications for pathogenesis of steatohepatitis.
      The aim of this study was to compare breath ethanol production in obese ob/ob mice and their lean littermates to determine if endogenous ethanol production is increased in obesity.

      Materials and methods

       Animals

      The animal protocol was approved by the Johns Hopkins Research Center Animal Care and Use Committee. Six genetically obese ob/ob male C57BL/6 mice and 6 lean male C57BL/6 control mice were obtained from Jackson Laboratories (Bar Harbor, ME). The mice were housed in the same facility, fed ad libitum on a standard laboratory rodent diet (PMI Nutrition International, Brentwood, MO) that consisted of 60% carbohydrates, 28% protein, and 12% fat and contained no ethanol. Mice were studied at 24 weeks of age.
      An additional 5 ob/ob male C57BL/6 mice and 5 lean male C57BL/6 mice were purchased from the same provider, fed the same diet, but housed in a different facility. These mice were studied at different ages (14 and 20 weeks). Both facilities were approved by the institution's animal care committee. Mice were housed in groups of 5 or 6 in plastic cages under conventional conditions. Food and water were provided ad libitum, and a regular 12:12-hour light-dark photo cycle was maintained throughout each study period.
      Breath was sampled from mice using a minor modification of the methods described previously for rats.
      • Risby T
      • Jiang L
      • Stoll S
      • Ingram D
      • Spangler E
      • Heim J
      • Cutler R
      • Roth GS
      • Rifkind JM
      Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats.
      To study mice, a smaller, 1.2-L flow-through Pyrex glass chamber was used to maximize breath concentration. Five to 6 mice can be accommodated comfortably in this chamber. The mice were maintained under high air flow (exceeding 2 L/min) for 15 minutes, allowing for 30 chamber volume changes. The mice were allowed to equilibrate in the chamber for 30 minutes under a constant air flow of 208 mL/min. After equilibration, the outflow gas of the chamber was sampled for 1 minute at 160 mL/min and concentrated onto 2 separate glass thermal desorption tubes (Supelco Corp., Bellefonte, PA).
      • Risby TH
      • Sehnert SS
      Clinical application of breath biomarkers of oxidative stress status.
      Chamber air was collected from each group on 5 different days and analyzed by thermal desorption capillary gas chromatography.

       Antibiotic treatment

      To inhibit the growth of gastrointestinal tract microflora, neomycin sulfate (1 mg/mL; Sigma Chemical Co., St. Louis, MO) was mixed in the deionized drinking water. This dose was selected based on previous reports indicating that it prevented fatty liver disease in lean rats that were fed ethanol-containing diets.
      • Adachi Y
      • Moore LE
      • Bradford BU
      • Gao W
      • Thurman RG
      Antibiotics prevent liver injury in rats following long-term exposure to ethanol.
      A group of 24-week-old ob/ob mice consumed neomycin-containing water for 5 days, while an identical group of ob/ob mice consumed deioinized water without antibiotic. Average water consumption was 5 mL · mouse−1 · day−1 in both groups. Morning breath ethanol production was measured 1 day before either group started antibiotics, and results were compared with breath ethanol content on each of the subsequent 5 days.

       Chromatographic analysis

      Sample analysis was performed by thermal desorption capillary gas chromatography (Perkin-Elmer, Norwalk, CT). The samples were thermally desorbed at 300°C for 15 minutes. Capillary gas chromatography was performed using a 60-m fused silica open tubular column (0.32-mm internal diameter) wall-coated with 7 μm of film dimethyl silicone. The separation was performed using the following temperature profile: isothermal at 35°C for 10 minutes, 35–200°C at 5°C/min, and isothermal at 200°C for 10 minutes.
      • Risby TH
      • Sehnert SS
      Clinical application of breath biomarkers of oxidative stress status.

       Evaluation of hepatic histopathology

      Liver sections were obtained from representative 24-week-old ob/ob and lean mice. After formalin fixation and embedding in paraffin, H&E-stained sections were evaluated under code by a single observer who was unaware of the sample source.

       Statistical analysis

      Data were analyzed by a statistical program (STATA) using 2-tailed unpaired t tests of the average differences between obese and lean or neomycin-treated and untreated animals.

      Results

       Liver histology

      Obese mice have fatty livers, with accumulations of small and large droplets of triglyceride in hepatocytes. Similar to the earliest stage of fatty liver disease in obese patients,
      • Samazasinghe D
      • Tasman-Jones C
      The clinical associations with hepatic steatosis: a retrospective study.
      • Lee RG
      Nonalcoholic steatohepatitis: tightening the morphological screws on a hepatic rambler.
      obesity-related steatosis in mice is not accompanied by obvious inflammatory cell infiltration or hepatocyte death (Figure 1).
      Figure thumbnail gr1
      Fig. 1Liver histology. (B) Obese ob/ob mice have microvesicular and macrovesicular steatosis, whereas (A) histology is normal in their lean littermates. Original magnification 100×.

       Body weight and metabolism

      Fat accumulation is believed to result when caloric intake chronically exceeds caloric expenditure. Genetically obese ob/ob mice are deficient in the appetite-suppressing hormone leptin, and overeat.
      • Campfield LA
      • Smith FJ
      • Burn P
      The OB protein (leptin) pathway— a link between adipose tissue mass and central neural networks.
      Hence, ob/ob mice have more fat and weigh more than age- and sex-matched lean control mice (Figure 2A).
      Figure thumbnail gr2
      Fig. 2Body weight and metabolism. Obese ob/ob mice and their lean littermates were maintained under identical conditions. (A) Body weights, (B) O2 consumption, and (C) CO2 production were evaluated as described in Materials and Methods. (D) The respiratory quotient (CO2 production/O2 consumption) was calculated. Results are shown as means ± SE for 6 ob/ob mice and 6 lean littermate controls (24 weeks old). *Significant differences between the experimental and control groups.
      Moreover, ob/ob mice continue to gain weight when their food consumption is restricted to that of lean mice, suggesting that hypometabolism also contributes to their obesity.
      • Pelleymounter MA
      • Cullen MF
      • Baker MB
      • Hecht R
      • Winters D
      • Boone T
      • Collins F
      Effects of the obese gene product on body weight regulation in ob/ob mice.
      Consistent with the latter concept, when corrected for total body weight, O2 consumption (Figure 2B) and CO2 production (Figure 2C) are lower in ob/ob mice than in lean controls. Others have reported that when O2 consumption is assessed in individual mice, it is also somewhat lower in ob/ob mice (72 mL · mouse−1 · h−1) than in lean control mice (85 mL · mouse−1 · h−1).
      • Breslow MJ
      • Min-Lee K
      • Brown DR
      • Chacko VP
      • Palmer D
      • Berkowitz DE
      Effect of leptin deficiency on metabolic rate in ob/ob mice.
      Further analysis of those published results suggests that the expected O2 consumption in mice varies between approximately 1.3 and 3.2 mL · g−1 · h−1, somewhat lower than the O2 consumption results from our mice. We suspect that these differences reflect differences in the oxygen detection systems in the 2 laboratories. If our system overestimated O2 consumption, this could explain the low respiratory quotient (Q) values in our study (Figure 2D). It is also conceivable that the low RQ values indicate that our mice were preferentially metabolizing lipid as an energy substrate.
      • Hwa JJ
      • Ghibaudi L
      • Compton D
      • Fawzi AB
      • Strader CD
      Intrace-rebroventricular injection of leptin increases thermogenesis and moilizes fat metabolism in ob/ob mice.
      However, unusually low RQ values are not typical of obesity. For example, RQ values of 0.77–0.92 have been reported in obese, leptin-resistant Zucker fa/fa rats. Interestingly, similar RQ values were noted in the lean control rats in that study.
      • Refinetti R
      Effect of ambient temperature on respiratory quotient of lean and obese Zucker rats.
      We also found no significant difference between the RQ values of obese ob/ob mice and lean control mice. Taken together, these findings suggest that the masses of metabolically active tissues are similar in obese and lean rodents. Consistent with this possibility, others have shown that the O2 consumption results in ob/ob mice and lean mice differ by only approximately 10% when O2 consumption is expressed as a function of lean body mass.
      • Breslow MJ
      • Min-Lee K
      • Brown DR
      • Chacko VP
      • Palmer D
      • Berkowitz DE
      Effect of leptin deficiency on metabolic rate in ob/ob mice.
      Hence, CO2 production is a more reliable measure of metabolism than body mass per se, particularly in ob/ob mice. Therefore, it is necessary to normalize ethanol production for steady-state CO2.
      • Risby T
      • Jiang L
      • Stoll S
      • Ingram D
      • Spangler E
      • Heim J
      • Cutler R
      • Roth GS
      • Rifkind JM
      Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats.

       CO2-normalized ethanol production in untreated mice

      In an initial effort to evaluate the possibility that obesity might increase endogenous ethanol production, breath was collected in the morning from a group (n = 6) of 24-week-old male ob/ob mice and a group (n = 6) of age- and gender-matched lean control mice. As shown in Figure 3 A, the ethanol content in expired air (expressed as pmol/mL CO2) is greater in obese mice than in lean mice (P < 0.0001).
      Figure thumbnail gr3
      Fig. 3Exhaled ethanol in breath. (A) Breath was collected in the morning from 6 24-week-old ob/ob mice and 6 lean littermate controls. Results are means ± SE of samples collected on 5 different days. *Significant differences between the experimental and control groups. (B) Gas chromatograms from representative experiments with ob/ob (top) and lean (bottom) mice; y-axis, response (millivolts); x-axis, time (minutes).
      Figure 3B illustrates a representative gas chromatogram from the chamber air collected from obese and lean mice. To determine the proportion of ethanol that was formed in vivo, as opposed to ethanol that might have been generated from urine or fecal material in the chamber, the measurements were repeated after mice were removed from the chamber. Relatively low concentrations of ethanol were noted in such “mouseless” chambers, and we determined that ex vivo ethanol production contributed no more than 15%–20% of the signal observed for the lean mice and therefore was negligible.
      To determine if unsuspected environmental factors might have contributed to the initial results, a second group of ob/ob and lean control mice (n = 5 mice/group) were obtained from the same provider. These mice were fed the same chow that had been given to the mice in the first study, but they were maintained in a different facility. The mice in this second group were also studied at earlier ages (14 and 20 weeks) to determine if aging, which is known to increase obesity,
      • Williamson DF
      • Kahn HS
      • Byers T
      The 10-y incidence of obesity and major weight gain in black and white US women aged 30-55 y.
      • Pi-Sunyer FX
      Medical hazards of obesity.
      • Rosenbaum M
      • Leibel RL
      • Hirsch J
      Obesity.
      also affects breath ethanol content. As shown in Figure 4A, breath ethanol levels increase with age in obese mice but not in lean controls and are greater in older obese mice than in older lean mice (P < 0.0006).
      Figure thumbnail gr4
      Fig. 4Exhaled ethanol in breath. (A) Effect of age. Ethanol was measured in exhaled breath in the morning from another group of 5 ob/ob mice and 5 lean littermate controls. Both groups were housed in the same facility, which was different from the facility where the mice described in had been kept. The mice were studied at 2 different ages (14 and 20 weeks). (B) Diurnal effects. Exhaled breath was collected from 20-week-old mice at different times of the day (9 AM, 3 PM, and 9 PM). Results are means ± SE of samples collected on 5 different days at each time point. *Significant differences between the experimental and control groups.
      The latter findings confirm the results of the initial experiments and demonstrate an environment-independent, obesity-related increase in breath ethanol content. To assure that these conclusions were not an artifact of the way the ethanol data were expressed, ethanol concentration was also expressed as parts per billion or as picomoles per gram of total body weight. Regardless of how the data were expressed, differences in ethanol production between lean and obese mice persisted. For example, early morning ethanol production in 20-week-old mice was 9.97 ± 4.9 parts per billion in lean mice and 101.4 ± 5.9 parts per billion in obese mice (24 ± 3.7 pmol · g−1 · h−1 in lean mice and 137.9 ± 23.2 pmol · g−1 · h−1 in obese mice).
      Mice feed nocturnally. Thus, if breath ethanol content is influenced by feeding, there may be diurnal variations in the ethanol content in expired breath. To evaluate this possibility, breath samples were obtained from 20-week-old ob/ob mice and lean control mice at 3 different times of the day. In ob/ob mice, breath ethanol content was consistently greater in the morning than at other times of the day (P < 0.0001). There was also a tendency for lean mice to have higher breath ethanol levels in the morning than at night. However, at each time of the day, ob/ob mice produced significantly more ethanol than lean control mice (Figure 4B).

       Effect of oral antibiotics on CO2-normalized ethanol production

      The relationship between feeding behavior and breath ethanol levels in obese mice suggested that intestinal microflora might be fermenting dietary carbohydrates to produce ethanol. To evaluate the importance of gut bacteria as a potential source of endogenous ethanol production, 22-week-old obese mice were given drinking water that contained neomycin. Morning breath samples were obtained 1 day before antibiotic treatment and daily during the 5-day antibiotic treatment period. One day after antibiotic administration was begun, breath ethanol content decreased by more than 30% (from 220 ± 18 to 139 ± 20 pmol ethanol/mL CO2). By the end of 5 days of antibiotic treatment, morning breath ethanol levels were half the initial values (Figure 5).
      Figure thumbnail gr5
      Fig. 5Effect of neomycin on breath ethanol content. Ten 24-week-old ob/ob mice consumed neomycin-containing water (1 mg/mL; n = 5 mice) or untreated water (n = 5 mice) for 5 days. Breath ethanol content in the 2 groups was compared at the end of the treatment period. Results are shown as means ± SE. *Significant differences between the experimental and control groups.
      Because oral treatment with a poorly absorbed antibiotic decreases the content of ethanol in expired breath, it is likely that intestinal microflora are a major endogenous source of obesity-related ethanol production.

      Discussion

      This study identifies a previously unsuspected factor, i.e., ethanol, that might contribute to the pathogenesis of obesity-related fatty liver disease. Although measurement of the ethanol concentration in exhaled breath is an accepted, albeit indirect, method for assessing the concentration of ethanol in blood,
      • Dubowski KM
      • Essary NA
      Measurement of low breath-alcohol concentrations: laboratory studies and field experience.
      it is important to acknowledge that blood ethanol concentrations were not measured in this study. Thus, we cannot predict whether the livers of obese mice actually experienced doses of ethanol sufficient to produce injury. However, given the recent experimental evidence that ethanol does not function as a direct hepatotoxin to cause alcoholic fatty liver disease,
      • Yin M
      • Wheeler MD
      • Kono H
      • Bradford BU
      • Gallucci RM
      • Luster MI
      • Thurman RG
      Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
      the possibility that slight increases in ethanol exposure promote a cascade of events that culminate in hepatic steatosis remains plausible. Moreover, because the mice were studied under carefully controlled experimental conditions, the possibility that surreptitious consumption of alcoholic beverages influenced the present findings can be excluded with confidence. Hence, these results provide a rationale for future studies to evaluate the possibility that endogenous production of ethanol also plays a role in obesity-related fatty liver disease in humans.
      Short-term treatment with a poorly absorbable oral antibiotic significantly reduced ethanol production in obese mice; therefore, intestinal bacteria are likely to be the predominant endogenous source for ethanol. This finding is consistent with evidence that bacterial strains that typically reside in the colons of rodents
      • Baraona E
      • Julkunen R
      • Tannenbaum L
      • Lieber CS
      Role of intestinal bacterial overgrowth in ethanol production and metabolism in rats.
      and humans
      • Nosova T
      • Jokelainen K
      • Kaihovaara P
      • Jousimies-somer H
      • Siitonen A
      • Heine R
      • Salaspuro M
      Aldehyde dehydrogenase activity and acetate production by aerobic bacteria representing the normal flora of human large intestine.
      can produce ethanol during the fermentation of dietary carbohydrates when intestinal stasis permits their overgrowth in the upper gastrointestinal tract.
      • Leung FW
      • Drenick EJ
      • Stanley TM
      Intestinal bypass complications involving the excluded small bowel segment.
      • Jorizzo JL
      • Apisarnthanarax P
      • Subrt P
      • Hebert AA
      • Henry JC
      • Raimer SS
      • Dinehart SM
      • Reinarz JA
      Bowel-bypass syndrome without bowel bypass. Bowel-associated dermatosis-arthritis syndrom.
      • Drenick EJ
      • Roslyn JJ
      Cure of arthritis-dermatitis syndrome due to intestinal bypass by resection of nonfunction segment of blind loop.
      Disordered intestinal motility has been associated with obesity in humans,
      • Crowell MD
      • Cheskin LJ
      • Musial F
      Prevalence of gastrointestinal symptoms in obese and normal weight binge eaters.
      • Weiner P
      • Shlum H
      • Ganam R
      • Plavnick L
      Colonic pseudo-ob-struction: a late complication of jejunoileal bypass.
      • O'Malley KJ
      • Flechner SM
      • Kapoor A
      • Rhodes RA
      • Modlin CS
      • Goldfarb DA
      • Novick AC
      Acute colonic pseudo-obstruction (Ogilvie's syndrome) after renal transplantation.
      and we have observed that colonic pseudo-obstruction increases as adult ob/ob mice age. The mechanisms for obesity-related abnormalities in intestinal motility are poorly understood and might be multifactorial. For example, in humans and ob/ob mice, obesity is associated with diabetes,
      • Pi-Sunyer FX
      Medical hazards of obesity.
      • Rosenbaum M
      • Leibel RL
      • Hirsch J
      Obesity.
      a common cause of visceral autonomic neuropathy. In addition, hyporesponsiveness to peptides that regulate gastrointestinal tract motility has been observed in some obese subjects.
      • Niederau C
      • Meereis-Schwanke K
      • Klonowski-Stumpe H
      • Herberg L
      CCK-resistance in Zucker obese versus lean rats.
      • Fink H
      • Rex A
      • Voits M
      • Voigt JP
      Major biological actions of CCK—a critical evaluation of research findings.
      • Lieverse RJ
      • Masclee AA
      • Jansen JB
      • Lam WF
      • Lamers CB
      Obese women are less sensitive for the satiety effects of bombesin than lean women.
      Thus, there is literature supporting an association between obesity, intestinal dysmotility, and small bowel bacterial overgrowth; the last would be expected to increase endogenous ethanol production. Moreover, small bowel bacterial overgrowth sometimes causes a clinical syndrome with features of dermatitis and arthritis that is cured by oral antibiotics or segmental surgical resection of the intestine,
      • Jorizzo JL
      • Apisarnthanarax P
      • Subrt P
      • Hebert AA
      • Henry JC
      • Raimer SS
      • Dinehart SM
      • Reinarz JA
      Bowel-bypass syndrome without bowel bypass. Bowel-associated dermatosis-arthritis syndrom.
      • Drenick EJ
      • Roslyn JJ
      Cure of arthritis-dermatitis syndrome due to intestinal bypass by resection of nonfunction segment of blind loop.
      showing that systemic effects accompany the absorption of gut bacterial products. Bacterial lipopolysaccharide (endotoxin) is the putative agent for the bowel-associated dermatosis-arthritis syndrome.
      • Mayeux PR
      Pathobiology of lipopolysaccharide.
      Thus there is evidence in humans that situations that promote intestinal bacterial overgrowth enhance both intestinal ethanol production
      • Mezey E
      • Imbembo AL
      • Potter JJ
      • Rent KC
      • Lombardo R
      • Holt PR
      Endogenous ethanol production and hepatic disease following jejunoileal bypass for morbid obesity.
      and endotoxemia.
      • Jorizzo JL
      • Apisarnthanarax P
      • Subrt P
      • Hebert AA
      • Henry JC
      • Raimer SS
      • Dinehart SM
      • Reinarz JA
      Bowel-bypass syndrome without bowel bypass. Bowel-associated dermatosis-arthritis syndrom.
      • Drenick EJ
      • Roslyn JJ
      Cure of arthritis-dermatitis syndrome due to intestinal bypass by resection of nonfunction segment of blind loop.
      • Mayeux PR
      Pathobiology of lipopolysaccharide.
      This association between endogenous production of ethanol in the intestine and endotoxemia may be relevant to the pathogenesis of obesity-related fatty liver disease because several lines of evidence indicate that gut-derived endotoxin promotes alcoholic fatty liver disease. For example, oral administration of neomycin
      • Adachi Y
      • Moore LE
      • Bradford BU
      • Gao W
      • Thurman RG
      Antibiotics prevent liver injury in rats following long-term exposure to ethanol.
      or lactobacillus,
      • Nanji AA
      • Khettry U
      • Sadrzadeh SM
      Lactobacillus feeding reduces endotoxemia and severity of experimental alcoholic liver disease.
      which decontaminates the gastrointestinal tract, decreases ethanol-related liver damage in experimental animals. Because normal gastrointestinal tract microflora can metabolize ethanol to acetaldehyde,
      • Baraona E
      • Julkunen R
      • Tannenbaum L
      • Lieber CS
      Role of intestinal bacterial overgrowth in ethanol production and metabolism in rats.
      the toxic by-product of ethanol oxidation,
      • Lieber C
      Hepatic, metabolic and toxic effect of ethanol.
      oral antibiotics may also reduce endogenously generated ethanol and/or acetaldehyde. Given that the intestinal mucosa is likely to have the highest concentrations of these toxins, it has been suggested that ethanol and its metabolites alter intestinal permeability and enhance the systemic absorption of endotoxin.
      • Bjarnason I
      • Waard K
      • Peters TJ
      The leaky gut of alcoholism: possible route of entry for toxic compounds.
      The latter is believed to activate macrophages in the splanchnic bed, including the liver, and stimulate local production of endotoxin-inducible cytokines, such as tumor necrosis factor α (TNF-α),
      • Mayeux PR
      Pathobiology of lipopolysaccharide.
      • Nolan JP
      Intestinal endotoxins as mediators of hepatic injury—an idea whose time has come again.
      • Fox ES
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      • Thomas P
      Bacterial endotoxins and the liver.
      which are increased in patients
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      Plasma tumor necrosis factor alpha predicts decreased long-term survival in severe alcoholic hepatitis.
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      Increased plasma tumor necrosis factor in severe alcoholic hepa-titis.
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      Plasma endotoxin concentrations in patients with alcoholic and non-alcoholic liver disease: reevaluation with an improved chromogenic assay.
      • Hill D
      • Marsano L
      • Cohen D
      • Allen J
      • Shedlosfsky S
      • McClain CJ
      Increased plasma interleukin-6 activity in alcoholic hepatitis.
      and experimental animals
      • Earnest DL
      • Evril ER
      • Jolley C
      • Martinez F
      Ethanol and diet-induced alterations in Kupffer cell function.
      • Kamimura S
      • Tsukamoto H
      Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease.
      • Nanji AA
      • Zakim D
      • Rahemtulla A
      • Daly T
      • Miao L
      • Zhao S
      • Khwaja S
      • Tahan SR
      • Dannenberg AJ
      Dietary saturated fatty acids down-regulate cyclooxygenase-2 and TNF-alpha and reverse fibrosis in experi-mental alcoholic liver disease.
      with alcohol-induced liver disease. Indeed, TNF-α is required for ingested ethanol to produce hepatic steatosis because long-term alcohol ingestion does not cause fatty liver disease in normal rats that are pretreated with neutralizing anti-TNF antibodies
      • Iimuro Y
      • Gallucci RM
      • Luster MI
      • Kono H
      • Thurman RG
      Antibodies to tumor necrosis factor alfa attenuates hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat.
      or in mice with inherited disruption of the gene for the type 1 TNF receptor.
      • Yin M
      • Wheeler MD
      • Kono H
      • Bradford BU
      • Gallucci RM
      • Luster MI
      • Thurman RG
      Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
      Obese humans and ob/ob mice with fatty livers also have increased serum TNF-α concentrations and express high levels of TNF-α messenger RNA (mRNA) in white adipose tissue.
      • Kern PA
      • Saghizaheh M
      • Ong JM
      • Bosch RJ
      • Deem R
      • Simsolo RB
      The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase.
      • Hotamisligil GS
      • Peraldi SP
      • Budavari A
      • Ellis R
      • White MF
      • Spiegelman BA
      IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance.
      Moreover, preliminary work in our laboratory shows that TNF-α and type 1 TNF receptor transcript levels are increased in the fatty livers of ob/ob mice. The role of TNF-α in obesity-related fatty liver disease has not been established nearly as definitively as it has in alcohol-related fatty liver disease. Nevertheless, evidence linking TNF-α to obesity-related insulin resistance
      • Uysal KT
      • Wiesbrock SM
      • Marino MW
      • Hotamisligil GS
      Protection from obesity-induced insulin-resistance in mice lacking TNF alpha function.
      and insulin resistance to obesity-related fatty liver disease
      • Marchesini G
      • Brizi M
      • Morselli-Labate AM
      • Bianchi G
      • Bugianesi E
      • McCullough AJ
      • Forlani G
      • Melchiondo N
      Association of non-alcoholic fatty liver disease with insulin resistance.
      • Shimomura I
      • Hammer RE
      • Ikemoto S
      • Brown MS
      • Goldstein JL
      Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy.
      indirectly supports the possibility that increased TNF-α contributes to obesity-related hepatic steatosis. Until now, it has been difficult to explain why TNF-α expression is increased in obesity. However, the present results suggest a unifying mechanism that might explain these observations, at least in obese mice with fatty livers: Obesity-related increases in the production (and presumably metabolism) of ethanol by intestinal bacteria might injure the intestinal barrier and promote the escape of endotoxin into the mesenteric venous circulation. Consequently, tissues, including mesenteric fat and the liver, that are exposed to this blood flow are stimulated to produce endotoxin-inducible cytokines, such as TNF-α. TNF-α, in turn, inhibits insulin-initiated signal transduction
      • Hotamisligil GS
      • Peraldi SP
      • Budavari A
      • Ellis R
      • White MF
      • Spiegelman BA
      IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF-alpha- and obesity-induced insulin resistance.
      and promotes insulin resistance. This interferes with the intermediary metabolism of carbohydrates and lipids, leading to multiple consequences, including hepatocyte lipid accumulation,
      • Reaven GM
      Role of insulin resistance in human disease.
      • Moller DE
      • Flier JS
      Insulin resistance. Mechanisms, syndromes, and implications.
      • De Fronzo RA
      • Ferranini E
      Insulin resistance. A multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and atherosclerotic cardiovascular disease.
      in the absence of exogenous ethanol consumption.
      Additional studies are necessary to test this concept more directly. However, if it is validated and a similar mechanism occurs in obese humans, then a number of novel strategies, including treatment with oral antibiotics or lactobacillus, might be considered to manage obesity-related insulin resistance and prevent fatty liver. Safe and inexpensive therapies for fatty liver are desirable because hepatic steatosis is no longer considered a trivial problem. Fatty livers have been demonstrated in more than a quarter of adults in industrialized countries,
      • Nomura H
      • Kashiwagi S
      • Hayashi J
      • Kajiyama W
      • Tani S
      • Goto M
      Prevalence of fatty liver in a general population of Okinawa, Japan.
      and some of these individuals develop more serious liver damage and have considerable liver-related morbidity and mortality.
      • Matteoni C
      • Younossi ZM
      • McCullough A
      Nonalcoholic fatty liver disease: a spectrum of clinical pathological severity.
      • Caldwell SH
      • Oelsner DH
      • Iezzoni JC
      • Hespenheide EE
      • Battle EH
      • Driscoll CJ
      Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease.

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