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Current biochemical studies of Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) suggest a new therapeutic approach
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2003 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 98, No. 2, 2003 ISSN 0002-9270/03/$30.00
WORLD LITERATURE REVIEW Editor: David Johnson, M.D., F.A.C.G. REVIEW PANEL Luis Balart Jamie Barkin David Bjorkman Cedric Bremner Randall Burt Harold Conn Jack DiPalma Hashem El-Serag M. Brian Fennerty
Mark Flemmer Christopher Gostout Robert Hawes Jorge Herrera Brenda Hoffman Kelvin Hornbuckle Doug Howerton Sunanda Kane Philip Katz
Current Biochemical Studies of NonAlcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) Suggest a New Therapeutic Approach Lin Hz, Yang SQ, Chuckaree C, et al. Metformin’s Reverses Fatty Liver Disease in Obese LeptinDeficient Mice Nature Medicine 2000;6(8):998 –1003
ABSTRACT The authors’ study population was ob/ob mice that have an inherited genetic deficiency of the appetite suppressing hormone leptin. These mice develop hyperinsulinemia, insulin resistance and fatty livers. Compared with their lean littermates and wild-type C57BL-6 mice, ob/ob mice have hepatomegaly. In this study, the authors compared three different groups of adult mice (age 8 –10 wk), including male ob/ob C57BL-6 mice, their lean littermates, and wild type C57BL-6 mice of the same age and sex. The primary purpose of this study was to test the efficacy of Metformin for treatment of fatty liver disease in obese, ob/ob mice that develop hyperinsulinemia or insulin resistance and fatty livers. Metformin therapy was found to eliminate fatty liver disease in this model. The potential mechanisms of the action of metformin was the inhibition of hepatic tumor necrosis factor (TNF)-␣ and inhibition of several TNF-inducible responses, which are likely to promote hepatic steatosis and necrosis. In these experiments, ob/ob mice were divided into three treatment groups. Group 1 consisted of eight mice that were treated with Metformin and permitted to consume a nutritiously replete liquid mouse diet ad libitum. Mice in Group 2 (n ⫽ 8) did not receive Metformin, but were pair-fed the same volume of liquid diet that the mice in the Metformin treated group had consumed on the previous day. Obese ob/ob mice in group 3 (n ⫽ 4) and lean mice received no metformin as the mice in group 2, but were permitted to
Timothy Koch Mark Lawson Theodore Levin Edward C. Oldfield III David Ott C. S. Pitchumoni K. Rajender Reddy Douglas Rex Albert Roach
Arvey Rogers Richard Sampliner Prateek Sharma Paul Souney Christina Surawicz Nimish Vakil Harlan Vingan Maurits Wiersema
consume the liquid diet ad libitum. Liquid diet was given to facilitate accurate daily comparison of food intake among the various treatment groups. All mice were weighed at the beginning of the study and weekly thereafter until killed and then sera, fat and liver tissues were collected. Tissues were either fixed in buffered formalin and processed from the deceased mice for histology or snap frozen in liquid nitrogen and stored until RNA and proteins were isolated. The feeding protocol was repeated with a second group of 18 ob/ob mice. After 4 wk hepatocytes were obtained by in situ liver perfusion with collagenase and assayed for cellular ATP content. In each experiment hepatocytes isolated from 2–3 mice feeding group were suspended in a medium and pooled for subsequent analysis to evaluate cell viability, determine the number of obtained cells, and to assay cellular ATP content. These experiments were repeated using another 2–3 mice feeding group so that analysis of hepatocytes took place from six ob/ob mice in each feeding group. Thus the final ATP results reflected analysis of hepatocytes from six ob/ob mice in each feeding group. Hepatic steatosis was decreased significantly only in the Metformin treated group. The authors found that metformin’s beneficial effect on the fatty liver disease of mice was not due to its ability to constrain hyperphagia, nor due to decreased caloric ingestion, as the daily caloric intakes of the metformin treated mice and the pair-fed control mice were virtually identical. These caloric intakes were consistently about 20% less than that of another obese control group that was permitted to consume diet ad lib. The authors also observed no significant effect of metformin on serum glucose concentration from fed, ob/ob mice. Metformin is known to reduce hyperinsulinemia by about 40% in both of these obese hyperinsulinemic and insulin-resistant rodent strains. In conclusion, they documented that Metformin improves fatty liver disease, and reverses hepatomegaly, steatosis and aminotransferase abnormalities in mice. In addition, the authors suggest that Metformin may inhibit dieting-induced redistribution of lipid from the liver to adipose tissue depots. In summary, this study identifies a potential treatment for fatty liver disease in humans. (Am J
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Gastroenterol 2003;98:495– 499. © 2003 by Am. Coll. of Gastroenterology)
COMMENT Fatty liver is a ubiquitous cause of chronic liver disease. It is potentially progressive and may lead to NASH (Nonalcoholic steatohepatitis). Ludwig et al. first described NASH (1), as a “hitherto unnamed disease that mimics alcoholic hepatitis histologically,” and not related to the consumption of alcohol. NASH is part of the spectrum of steatosis, better known as non-alcoholic fatty liver disease (NAFLD). NAFLD may be the most prevalent form of hepatic disease in the United States (2, 3) with a spectrum ranging from relatively benign hepatic steatosis to the more severe NASH and ultimately to cirrhosis. The prevalence of NAFLD ranges between 15–39% (4, 5), whereas the prevalence of NASH in the general population is about 2% (6). In patients undergoing liver biopsies, presumably for evaluation of elevated LFTs, NASH was found in 7–9% in Western countries (1, 4) and in 1.2% of Japanese patients (7). The majority of cases are found between the ages of 40 and 60 yr, although there have been reports in children as young as 10 yr of age (8, 9). NASH is the most common hepatic disease among adolescents in North America (10). Some maintain that NASH progresses to cirrhosis less frequently when compared with alcoholic hepatitis (11–16) but firm evidence is lacking. Interestingly, in a recent study, 73% of 70 consecutive patients with cryptogenic cirrhosis were found to be obese and 53% were diabetic. Both obesity and diabetes are predisposing factors for NASH (17). Thus, the pathological changes terminating in cryptogenic cirrhosis may have had their start with NASH. NASH is a significant cause of morbidity and mortality in patients with obesity-related type-2 diabetes (18). There is a strong association between hepatic steatosis and insulin resistance in humans (18, 19) as well as experimental animals (20 –22). This suggests that insulin resistance is critically involved in the pathogenesis of fatty liver disease and NASH. The model of genetically obese, ob/ob C57BL-6 mice used in this study provides a well-characterized model of hyperinsulinemia and insulin-resistance (21, 23–26) in which the steatosis mimics the evolution of fatty liver in obese human beings with hyperinsulinemia and increased insulin resistance. Insulin resistance is uniformly present in patients with fatty liver or NASH and it is present even in those patients with NASH without diabetes. NASH seems to develop as a progression of hepatic disease from NAFLD and may progress to end-stage cirrhosis. It is believed that this progression involves several pathological processes. The first (10) is the accumulation of fatty acids in the liver and the development of steatosis associated with resistance to insulin, which is strongly linked to obesity and the metabolic syndrome. The metabolic syndrome consists of increased insulin resistance, hy-
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pertriglyceridemia, hypertension, and central obesity with and without the presence of diabetes mellitus. The mechanism of this first process involves oxidation of mitochondrial fatty acid which is blocked by insulin. This ultimately results in an increased concentration of intracellular fatty acids within hepatocytes (10). Insulin resistance also leads to increased mobilization of fatty acids in the hepatocytes (10) thus leading to steatosis. The second pathological process leads to the occurrence of necrosis, inflammation, and fibrosis. This involves oxidative stress in which cytochrome P450 2E1 (CYP2E1) is induced by fatty acids. CYP2E1 generates free radicals from endogenous ketones and aldehydes as well as dietary Nnitrosamines that damage the hepatocytes and result in the secondary recruitment of inflammatory cells (27, 28). The latter is associated with abnormal production of inflammatory cytokines, such as enhanced production of tumor necrosis factor-␣ (TNF-␣) by adipose and other peripheral tissues. In these ob/ob mice this occurs in the presence of leptin deficiency. Conversely, the presence of increased leptin in human NAFLD is associated with obesity and has been reported to lead to increased fibrosis and cirrhosis. Ikejima, et al. have recently shown that leptin presumably produced by hepatic satellite cells may play an important role in liver fibrosis (29, 30). Thus, leptin is profibrogenic and may modulate the liver’s inflammatory response (31, 32). Since hyperlipidemia is a common finding in obese patients (29, 33), Marra believes that leptin is one of the factors linking patients with increased body fat to a higher risk of developing cirrhosis (34). He also speculates that leptin is directly involved in the pathogenesis of fibrosis in patients with NASH and may even be the second process that contributes to the progression from the benign forms of simple steatosis to steatohepatitis, and subsequently cirrhosis. The role of leptin in NAFLD is becoming a more complicated story— because chronic leptin treatment has been shown by Petersen et al. (35) to improve insulin resistance via insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients. Similarly, NASH is also a disease associated with dyslipidemia, hepatic steatosis, with the common denominator of insulin resistance and which often results in diabetes. Savage and O’Rahilly (36) have studied patients with congenital leptin deficiency disorders and generalized lipodystrophy and speculate that, “the major therapeutic impact of leptin is to reduce fat mass with a marked amelioration of resulting insulin resistance.” We need further studies that detail effects of leptin administration on patients with low leptin levels resulting in increased insulin resistance. The Diehl study is important as it demonstrated that treatment with Metformin reverses severe fatty liver disease in an animal model. This study also provides important pathophysiological information including:
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1. Though overeating might promote the genesis of fatty liver, modest caloric restriction did little to improve the problem in this model of obesity. This feeding is particularly important because it is standard medical practice to encourage obese patients with hepatic steatosis to diet as a therapy for fatty liver disease. 2. Ob/ob mice exhibited increased hepatic expression of TNF–␣—a cytokine that is known to inhibit the propagation of insulin receptor initiated signals in hepatocytes (37). TNF-␣ promotes insulin resistance in ob/ob mice (21). Hepatic expression of TNF-␣ is also increased in alcohol induced fatty liver disease, which closely resembles obesity related hepatic steatosis (38). The reason we believe that TNF-␣ is required for alcohol to produce fatty liver disease is because transgenetic mice that are deficient in type-1 TNF receptors are completely protected from this disorder (39). 3. Additional mechanisms that could also help to explain the association between increased hepatic TNF-␣ and obesity related liver disease is (a) the hepatic expression of fatty acid synthase (FAS) and (b) fatty acid synthesis, which were both increased in the fatty livers of ob/ob mice (22). 4. It is important to note that Metformin also has been shown to reverse TNF-␣ induced insulin resistance in cultured rat hepatocytes (37). 5. Finally, this study demonstrates that Metformin reduces both hepatic steatosis and hepatic TNF-␣ expression in insulin resistant ob/ob mice. This suggests that the use of metformin in humans with NAFLD and NASH may be useful in decreasing inflammation and steatofibrosis. One possible defect of this study is that the authors did not directly evaluate serum insulin concentrations and hepatic glucose output. However the authors point out that it is, “reasonable to assume that the Metformin treated ob/ob mice experienced typical Metformin induced improvements in hyperinsulinemia and insulin sensitivity that have been reported in other studies of hyperinsulinemic animals and humans.” This study was the basis of an evaluation of metformin as a treatment for NASH in human patients with obesityrelated insulin resistance performed by Marchesini et al. (39). They treated 20 patients with biopsy proven NASH with Metformin at a dose of 500 mg t.i.d. for 4 months. They compared the six individuals who did not comply with treatment, to the group of 14 patients who completed the Metformin protocol. They found the latter group to have significantly reduced mean transaminase concentrations. In seven of 14 who completed Metformin therapy, transaminase levels returned to normal, insulin sensitivity improved and liver volume decreased by 20% compared with baseline studies. This report on the efficacy of Metformin in human patients with NASH adds to Metformin’s clinical credibility to treat future patients with NASH; a disease for which there has not yet been a well-defined treatment. There is need for
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more studies on liver safety of Metformin, compared to thiazolidinediones, another family of insulin sensitizing agents. Thiazolidinediones includes troglitazone (Rezulin), which has proven to be a safe agent. In addition, there needs to be a double-blind, cross-over controlled study of Metformin in NASH focusing on its potential role in delaying, preventing or reversing liver fibrosis. However, such a study would be very difficult to perform as to achieve statistical significance, in the absence of any currently known noninvasive predictor of liver fibrosis, it would require approximately 2000 –3000 patients with NAFLD to screen with a baseline liver biopsy to find enough patients with biopsyproven NASH (K. Lindor, personal communication, June 27, 2002). Therefore, such a study is still far off in the future. NASH, which up to now has been essentially untreated for the lack of a definitive therapy, remains a significant cause of morbidity and mortality (18). Bugianesi et al. have shown that features suggestive of NASH are more frequently observed in hepatocellular carcinoma (HCC) arising in patients with cryptogenic cirrhosis than in age- and sexmatched HCC patients of well-defined viral or alcoholic etiology (40). Therefore, HCC may represent a late complication of NASH-related cirrhosis according to these authors. Therefore, we believe that NASH should be treated with whatever treatment has been shown to have beneficial effects as soon as the diagnosis is made. Zhou et al. have shown that Metformin’s glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization (41). They also found that Metformin has beneficial effects on circulating lipids that have been linked to the occurrence of fatty liver. Metformin activates AMPactivated protein kinase (AMPK) in hepatocytes, which is a major cellular regulator of lipid and glucose metabolism. As a result, they reported, that acetyl-CoA carboxylase (ACC) activity is reduced, so that fatty acid oxidation is induced, and thus expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin also suppresses expression of SREBP-1, a key lipogenic transcription factor. The authors also report that in metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced and activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, they found that AMPK activation is required for metformin’s inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. In the authors’ opinion activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug. Knowler et al. showed that Metformin reduced the incidence of Diabetes in non-diabetics or “pre-diabetics” (42). They randomly assigned 3234 non-diabetic persons with elevated fasting and post-load plasma glucose concentrations (postprandial glucose levels 140 to 199 mg/dL or fasting levels 110 to 125 mg/dL) to one of these groups: placebo, metformin (850 mg b.i.d.), or a lifestyle-modifica-
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tion program. The goals were at least a 7% weight loss and at least 150 min of physical activity per week. The mean age of the participants was 51 yr, and the mean body-mass index (the weight in kilograms divided by the square of the height in meters) was 34.0; 68% were women, and 45% were members of minority groups. They reported that the incidence of diabetes was 11.0, 7.8, and 4.8 cases per 100 person-years in the placebo, metformin, and lifestyle groups respectively. Lifestyle intervention reduced the incidence of diabetes by 58% (95% confidence interval, 48 – 66%) and metformin reduced the incidence of diabetes by 31% (95% confidence interval, 17– 43%), compared with placebo. Thus we believe that there is enough evidence to begin using this relatively safe drug in those NASH patients with documented insulin resistance and pre-diabetes of the metabolic syndrome. Metformin should not be utilized in patients with potential lactic acidosis, renal insufficiency and congestive heart failure. Perry Hookman, M.D., F.A.C.P., F.A.C.G. Jamie S. Barkin, M.D., F.A.C.P., M.A.C.G. University of Miami School of Medicine Mount Sinai Medical Center Division of Gastroenterology Miami, Florida
REFERENCES 1. Ludwig J, Viggiano TR, et al. Nonalcoholic steatohepatitis. Mayo Clinic Proc 1980;55:434 –38. 2. Falck-Ytter Y, Younossi ZM, et al. Clinical features and natural history of nonalcoholic steatosis syndromes. Semin Liver Dis 2002;21:17–26. 3. McCollough AJ, Falck-Ytter Y. Body composition and hepatic steatosis as precursors for fibrotic liver disease. Hepatology 1999;29:1328 –30. 4. Propst A, Propst T, et al. Prognosis in nonalcoholic steatohepatitis. Gastroenterology 1995;108:1607 [letter]. 5. Hultcrantz R, Glaumann H, et al. Liver investigation in 149 asymptomatic patients with moderately elevated activities of serum aminotransferases. Scand J Gastroenterol 1986;21:109 – 13. 6. Hilden M, Christofferson P, et al. Liver histology in a normal population — examinations of 503 consecutive fatal traffic casualties. Scand J Gastroenterol 1977;12:593–97. 7. Nonomura A, Mizukami Y, Unoura M, et al. Clinicopathologic study of alcohol-like liver disease in non-alcoholics; non-alcoholic steatohepatitis and fibrosis. Gastroenterol Jpn 1992;27:521. 8. Baldridge AD, Perez-Atayde AR, Graeme-Cook F, et al. Idiopathic steatohepatitis: An expanded clinical entity. Gastroenterology 1994;107:1103. 9. Moran JR, Ghishan FK, Halter SA, Graeme-Cook HL. Steatohepatitis in obese children: A cause of chronic liver dysfunction. Am J Gastroenterol 1983;78:374. 10. James O, Day C. Non-alcoholic steatohepatitis. Another disease of affluence. Lancet 1999;353:1634 –6. 11. Galambos JT. Natural history of alcoholic hepatitis III. Histologic changes. Gastroenterology 1972;63:1026. 12. Marbet UA, Bianchi L, Meury U, Stadler GA. Long-term histological evaluation of the natural history and prognostic factors of alcoholic liver disease. J Hepatol 1987;4:364.
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13. Powell EE, Cooksley WG, Hanson R, et al. The natural history of nonalcoholic steatohepatitis: A follow-up study of forty-two patients for up to 21 years. Hepatology 1990;11:74. 14. Lee RG. Nonalcoholic steatohepatitis: A study of 49 patients. Hum Pathol 1989;20:594. 15. Pinto HC, Baptista A, Camilo ME, et al. Nonalcoholic steatohepatitis. Clinicopathological comparison with alcoholic hepatitis in ambulatory and hospitalized patients. Dig Dis Sci 1996;41:172. 16. Matteoni CA, Younossi ZM, Gramlich T, et al. Nonalcoholic fatty liver disease: A spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413. 17. Galdwell SH, Oelsner DH, et al. Cryptogenic cirrhosis: Clinical characterization and risk factors for underlying disease. Hepatology 1998;29:664 –9. 18. Marchesini G, et al. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med 1999;107:450 –55. 19. Marceau P, et al. Liver pathology and metabolic syndrome x in severe obesity. Clin Endocrinol Metab 1999;84:1513–7. 20. Yang SQ, Lin HZ, Lane MD, et al. Obesity increases sensitivity of endotoxin liver injury: Implications for pathogenesis of steatohepatitis. Proc Natl Acad Sci USA 1997;2557– 62. 21. Uysai KT, Weisbrok SM, Marino MW, Motamislgil GS. Protection from obesity-induced insulin-resistance in mice lacking TNF-␣ function. Nature 1997;389:610 –4. 22. Shimoura I, Basmokow Y, Horton JD. Increased nuclear levels of SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 1999;274:30028 –8832. 23. Megalsson MD, et al. Antihyperglycemic actions of guanidenoakanoic acids; 3-guanidlnoproplonic acid and ameliorates hyperglycemia in diabetic KKAy and CS7BL-61/ob/ob mice and increases glucose disappearance in rhesus monkeys. J Pharmaol Exp Ther 1993;266:1454 –62. 24. Campfield IA, Smith FI, Bum P. The OB protein (leptin) pathway—a link between adipose tissue mass and central neural networks. Horm Metab Res 1996;28:619 –32. 25. Rouru I, Pesonen V, Kaulu M. Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats. Life Sci 1992;50: 1813–20. 26. Paolisso G, et al. Effect of metformin on food intake in obese subjects. Eur J Clin Invest 1998;28:441–6. 27. Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis 2001;21:27–41. 28. Day CP, Oliver FWJ. Steatohepatitis: A tale of two ‘hits’? Gastroenterology 1998;114:842–5. 29. Ikejima K, Honda H, Yoshikawa M, et al. Leptin augments inflammatory and profibrogenic responses in the murine liverinduced by hepatotoxic chemicals. Hepatology 2001;34:288 – 97. 30. Ikejima K, Takei Y, Honda H, et al. Leptin receptor-mediated signaling regulates hepatic profibrogenic and remodeling of extracellular matrix in the rat. Gastroenterology 2002;122: 1399 –410. 31. Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J 2001;15:2565–71. 32. Diehl AM. Nonalcoholic steatosis and steatohepatitis IV. Nonalcoholic fatty liver disease abnormalities in macrophage function and cytokines. Am J Physiol Gastrointest Liver Physiol 2002;282:G1–G5. 33. Potter JJ, Womack L, Mezey E, Anania FA. Transdifferentiation of rat hepatic stellate cells results in leptin expression. Biochem Biophys Res Commun 1998;244:178 –82. 34. Marra F. Leptin and fatty liver fibrosis: A matter of fat. Gastroenterology 2002;122:1529 –30. 35. Petersen KF, Oral EA, Dufour S, et al. Leptin reversed insulin
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36. 37.
38. 39. 40.
41. 42.
resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest 2002;109:1345–50. Savage DB, O’Rahilly SO. Leptin: A novel therapeutic role in lipodystrophy. J Clin Invest 2002;109:1285–6. Soloman SS, Mishra SK, Cwik C, et al. Pioglitazone and metformin reverse insulin resistance induced by tumor necrosis factor-alpha in liver cells. Horm Metab Res 1997;29:370 – 82. Mc Clain Cl, Hill DB, Schmidt J, Diehl AM. Cytokines and alcoholic liver disease. Semin Liv Dis 1993;13:170 –82. Yin M, Wheeler MD, Kono H, et al. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 1999;117:942–52. Bugianesi E, Leone N, Vanni E, et al. Expanding the natural history of nonalcoholic steatohepatitis: From cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 2002; 123:134 –40. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108:1167–74. Knowler WC, Barrett-Connor E, Fowler SE, et al. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393–403.
The Road to Resistance: Antibiotics as Growth Promoters for Animals White DG, Zhao S, Sudler R, et al. The Isolation of Antibiotic Resistant Salmonella From Retail Ground Meats N Engl J Med 2001;345:1147–54
ABSTRACT White et al. report their research on Salmonella strains isolated from ground meats purchased at grocery stores in the Washington, DC area. The authors isolated Salmonella from 20% of the 200 sampled products including 35% of chicken, 24% of turkey, 16% of pork and 6% of all beef samples. Resistance to at least one antibiotic was found in 84% of the isolates and 53% of the isolates were resistant to at least three antibiotics (some isolates were resistant to nine different antibiotics). Ceftriaxone-resistant Salmonella and multidrug resistant S. enterica serotype typhimurium DT 104 were also isolated. (Am J Gastroenterol 2003;98:499. © 2003 by Am. Coll. of Gastroenterology)
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resistance which was also documented. A major concern is that the high levels of antibiotic resistance are a result of the use of antibiotics in food animals. Previously it had been suggested that half of all antibiotics were used in animals; however, a more recent estimate suggests that 24.6 million pounds of antibiotics are given to animals each year as growth promoters at subtherapeutic amounts in their feed compared to 3 million pounds consumed by humans (1). Currently, chickens spend 40 of their 42 days of life on antibiotics. Especially problematic is the broad spectrum of antibiotics allowed in animals, including expanded spectrum cephalosporins and quinolones. Even more disconcerting is the use of antibiotics from the same classes as our newest antibiotics in the fight against vancomycin-resistant Enterococci (VRE), including virginiamycin (a streptogramin related to Synercid) which has been used as a growth promoter in chickens since 1974. In the same issue of the Journal, McDonald et al. reported sampling chickens from 26 stores in four states. They found 82% of chicken carcasses had quinupristin– dalfopristin (Synercid, Aventis Pharm) resistant Enterococcus faecium (2). These resistant isolates were found in 81% of supermarket chains and 59% of different brands. These results raise significant concerns that Synercid, which was only approved by the Food and Drug Administration (FDA) as an option to treat VRE in 1999 may be compromised by the use of antibiotics in animal feed. This concern was supported by Sorensen et al. who found that healthy volunteers who ingested streptogramin resistant E. faecium strains isolated from chickens had the organism isolated from stool for up to 14 days (3). The European Union banned all antibiotics that are related to those used in humans from use in animals for growth promotion in 1998. How long will it take the United States to realize we run a significant risk for developing resistance in foodborne pathogens by the continued use of important antibiotics as growth promoters in animals? Edward C. Oldfield, III, M.D. Division of Infectious Disease Eastern Virginia School of Medicine Norfolk, Virginia
COMMENT In the United States, there are an estimated 2 to 4 million annual cases of Salmonellosis. Salmonella are widespread in animal sources, in particular poultry and pigs. A wide range of food sources have been implicated in outbreaks including raw shell eggs, poultry, ground beef, ice cream, powdered milk, cheese, cantaloupes, tomatoes, sprouts, and numerous other products of animal origin. White et al. document the widespread contamination of ground meat products in grocery stores. Even more disconcerting was the extremely high level of antibiotic
REFERENCES 1. Mellon M, Benbrook C, Benbrook KL. Hogging it: Estimates of antimicrobial abuse in livestock. Cambridge: Mass: Union of Concerned Scientists, 2001. 2. McDonald LC, Rossiter S, Mackinson C. Quinupristin-dalfopristin-resistant Enterococcus faecium on chicken and in human stool specimens. N Engl J Med 2001;354:16. 3. Sorensen TL, Blom M, Monnet DL, et al. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. N Engl J Med 2001;345: 1161– 6.
Vol. 98, No. 2, 2003 ISSN 0002-9270/03/$30.00
WORLD LITERATURE REVIEW Editor: David Johnson, M.D., F.A.C.G. REVIEW PANEL Luis Balart Jamie Barkin David Bjorkman Cedric Bremner Randall Burt Harold Conn Jack DiPalma Hashem El-Serag M. Brian Fennerty
Mark Flemmer Christopher Gostout Robert Hawes Jorge Herrera Brenda Hoffman Kelvin Hornbuckle Doug Howerton Sunanda Kane Philip Katz
Current Biochemical Studies of NonAlcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) Suggest a New Therapeutic Approach Lin Hz, Yang SQ, Chuckaree C, et al. Metformin’s Reverses Fatty Liver Disease in Obese LeptinDeficient Mice Nature Medicine 2000;6(8):998 –1003
ABSTRACT The authors’ study population was ob/ob mice that have an inherited genetic deficiency of the appetite suppressing hormone leptin. These mice develop hyperinsulinemia, insulin resistance and fatty livers. Compared with their lean littermates and wild-type C57BL-6 mice, ob/ob mice have hepatomegaly. In this study, the authors compared three different groups of adult mice (age 8 –10 wk), including male ob/ob C57BL-6 mice, their lean littermates, and wild type C57BL-6 mice of the same age and sex. The primary purpose of this study was to test the efficacy of Metformin for treatment of fatty liver disease in obese, ob/ob mice that develop hyperinsulinemia or insulin resistance and fatty livers. Metformin therapy was found to eliminate fatty liver disease in this model. The potential mechanisms of the action of metformin was the inhibition of hepatic tumor necrosis factor (TNF)-␣ and inhibition of several TNF-inducible responses, which are likely to promote hepatic steatosis and necrosis. In these experiments, ob/ob mice were divided into three treatment groups. Group 1 consisted of eight mice that were treated with Metformin and permitted to consume a nutritiously replete liquid mouse diet ad libitum. Mice in Group 2 (n ⫽ 8) did not receive Metformin, but were pair-fed the same volume of liquid diet that the mice in the Metformin treated group had consumed on the previous day. Obese ob/ob mice in group 3 (n ⫽ 4) and lean mice received no metformin as the mice in group 2, but were permitted to
Timothy Koch Mark Lawson Theodore Levin Edward C. Oldfield III David Ott C. S. Pitchumoni K. Rajender Reddy Douglas Rex Albert Roach
Arvey Rogers Richard Sampliner Prateek Sharma Paul Souney Christina Surawicz Nimish Vakil Harlan Vingan Maurits Wiersema
consume the liquid diet ad libitum. Liquid diet was given to facilitate accurate daily comparison of food intake among the various treatment groups. All mice were weighed at the beginning of the study and weekly thereafter until killed and then sera, fat and liver tissues were collected. Tissues were either fixed in buffered formalin and processed from the deceased mice for histology or snap frozen in liquid nitrogen and stored until RNA and proteins were isolated. The feeding protocol was repeated with a second group of 18 ob/ob mice. After 4 wk hepatocytes were obtained by in situ liver perfusion with collagenase and assayed for cellular ATP content. In each experiment hepatocytes isolated from 2–3 mice feeding group were suspended in a medium and pooled for subsequent analysis to evaluate cell viability, determine the number of obtained cells, and to assay cellular ATP content. These experiments were repeated using another 2–3 mice feeding group so that analysis of hepatocytes took place from six ob/ob mice in each feeding group. Thus the final ATP results reflected analysis of hepatocytes from six ob/ob mice in each feeding group. Hepatic steatosis was decreased significantly only in the Metformin treated group. The authors found that metformin’s beneficial effect on the fatty liver disease of mice was not due to its ability to constrain hyperphagia, nor due to decreased caloric ingestion, as the daily caloric intakes of the metformin treated mice and the pair-fed control mice were virtually identical. These caloric intakes were consistently about 20% less than that of another obese control group that was permitted to consume diet ad lib. The authors also observed no significant effect of metformin on serum glucose concentration from fed, ob/ob mice. Metformin is known to reduce hyperinsulinemia by about 40% in both of these obese hyperinsulinemic and insulin-resistant rodent strains. In conclusion, they documented that Metformin improves fatty liver disease, and reverses hepatomegaly, steatosis and aminotransferase abnormalities in mice. In addition, the authors suggest that Metformin may inhibit dieting-induced redistribution of lipid from the liver to adipose tissue depots. In summary, this study identifies a potential treatment for fatty liver disease in humans. (Am J
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Gastroenterol 2003;98:495– 499. © 2003 by Am. Coll. of Gastroenterology)
COMMENT Fatty liver is a ubiquitous cause of chronic liver disease. It is potentially progressive and may lead to NASH (Nonalcoholic steatohepatitis). Ludwig et al. first described NASH (1), as a “hitherto unnamed disease that mimics alcoholic hepatitis histologically,” and not related to the consumption of alcohol. NASH is part of the spectrum of steatosis, better known as non-alcoholic fatty liver disease (NAFLD). NAFLD may be the most prevalent form of hepatic disease in the United States (2, 3) with a spectrum ranging from relatively benign hepatic steatosis to the more severe NASH and ultimately to cirrhosis. The prevalence of NAFLD ranges between 15–39% (4, 5), whereas the prevalence of NASH in the general population is about 2% (6). In patients undergoing liver biopsies, presumably for evaluation of elevated LFTs, NASH was found in 7–9% in Western countries (1, 4) and in 1.2% of Japanese patients (7). The majority of cases are found between the ages of 40 and 60 yr, although there have been reports in children as young as 10 yr of age (8, 9). NASH is the most common hepatic disease among adolescents in North America (10). Some maintain that NASH progresses to cirrhosis less frequently when compared with alcoholic hepatitis (11–16) but firm evidence is lacking. Interestingly, in a recent study, 73% of 70 consecutive patients with cryptogenic cirrhosis were found to be obese and 53% were diabetic. Both obesity and diabetes are predisposing factors for NASH (17). Thus, the pathological changes terminating in cryptogenic cirrhosis may have had their start with NASH. NASH is a significant cause of morbidity and mortality in patients with obesity-related type-2 diabetes (18). There is a strong association between hepatic steatosis and insulin resistance in humans (18, 19) as well as experimental animals (20 –22). This suggests that insulin resistance is critically involved in the pathogenesis of fatty liver disease and NASH. The model of genetically obese, ob/ob C57BL-6 mice used in this study provides a well-characterized model of hyperinsulinemia and insulin-resistance (21, 23–26) in which the steatosis mimics the evolution of fatty liver in obese human beings with hyperinsulinemia and increased insulin resistance. Insulin resistance is uniformly present in patients with fatty liver or NASH and it is present even in those patients with NASH without diabetes. NASH seems to develop as a progression of hepatic disease from NAFLD and may progress to end-stage cirrhosis. It is believed that this progression involves several pathological processes. The first (10) is the accumulation of fatty acids in the liver and the development of steatosis associated with resistance to insulin, which is strongly linked to obesity and the metabolic syndrome. The metabolic syndrome consists of increased insulin resistance, hy-
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pertriglyceridemia, hypertension, and central obesity with and without the presence of diabetes mellitus. The mechanism of this first process involves oxidation of mitochondrial fatty acid which is blocked by insulin. This ultimately results in an increased concentration of intracellular fatty acids within hepatocytes (10). Insulin resistance also leads to increased mobilization of fatty acids in the hepatocytes (10) thus leading to steatosis. The second pathological process leads to the occurrence of necrosis, inflammation, and fibrosis. This involves oxidative stress in which cytochrome P450 2E1 (CYP2E1) is induced by fatty acids. CYP2E1 generates free radicals from endogenous ketones and aldehydes as well as dietary Nnitrosamines that damage the hepatocytes and result in the secondary recruitment of inflammatory cells (27, 28). The latter is associated with abnormal production of inflammatory cytokines, such as enhanced production of tumor necrosis factor-␣ (TNF-␣) by adipose and other peripheral tissues. In these ob/ob mice this occurs in the presence of leptin deficiency. Conversely, the presence of increased leptin in human NAFLD is associated with obesity and has been reported to lead to increased fibrosis and cirrhosis. Ikejima, et al. have recently shown that leptin presumably produced by hepatic satellite cells may play an important role in liver fibrosis (29, 30). Thus, leptin is profibrogenic and may modulate the liver’s inflammatory response (31, 32). Since hyperlipidemia is a common finding in obese patients (29, 33), Marra believes that leptin is one of the factors linking patients with increased body fat to a higher risk of developing cirrhosis (34). He also speculates that leptin is directly involved in the pathogenesis of fibrosis in patients with NASH and may even be the second process that contributes to the progression from the benign forms of simple steatosis to steatohepatitis, and subsequently cirrhosis. The role of leptin in NAFLD is becoming a more complicated story— because chronic leptin treatment has been shown by Petersen et al. (35) to improve insulin resistance via insulin-stimulated hepatic and peripheral glucose metabolism in severely insulin-resistant lipodystrophic patients. Similarly, NASH is also a disease associated with dyslipidemia, hepatic steatosis, with the common denominator of insulin resistance and which often results in diabetes. Savage and O’Rahilly (36) have studied patients with congenital leptin deficiency disorders and generalized lipodystrophy and speculate that, “the major therapeutic impact of leptin is to reduce fat mass with a marked amelioration of resulting insulin resistance.” We need further studies that detail effects of leptin administration on patients with low leptin levels resulting in increased insulin resistance. The Diehl study is important as it demonstrated that treatment with Metformin reverses severe fatty liver disease in an animal model. This study also provides important pathophysiological information including:
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1. Though overeating might promote the genesis of fatty liver, modest caloric restriction did little to improve the problem in this model of obesity. This feeding is particularly important because it is standard medical practice to encourage obese patients with hepatic steatosis to diet as a therapy for fatty liver disease. 2. Ob/ob mice exhibited increased hepatic expression of TNF–␣—a cytokine that is known to inhibit the propagation of insulin receptor initiated signals in hepatocytes (37). TNF-␣ promotes insulin resistance in ob/ob mice (21). Hepatic expression of TNF-␣ is also increased in alcohol induced fatty liver disease, which closely resembles obesity related hepatic steatosis (38). The reason we believe that TNF-␣ is required for alcohol to produce fatty liver disease is because transgenetic mice that are deficient in type-1 TNF receptors are completely protected from this disorder (39). 3. Additional mechanisms that could also help to explain the association between increased hepatic TNF-␣ and obesity related liver disease is (a) the hepatic expression of fatty acid synthase (FAS) and (b) fatty acid synthesis, which were both increased in the fatty livers of ob/ob mice (22). 4. It is important to note that Metformin also has been shown to reverse TNF-␣ induced insulin resistance in cultured rat hepatocytes (37). 5. Finally, this study demonstrates that Metformin reduces both hepatic steatosis and hepatic TNF-␣ expression in insulin resistant ob/ob mice. This suggests that the use of metformin in humans with NAFLD and NASH may be useful in decreasing inflammation and steatofibrosis. One possible defect of this study is that the authors did not directly evaluate serum insulin concentrations and hepatic glucose output. However the authors point out that it is, “reasonable to assume that the Metformin treated ob/ob mice experienced typical Metformin induced improvements in hyperinsulinemia and insulin sensitivity that have been reported in other studies of hyperinsulinemic animals and humans.” This study was the basis of an evaluation of metformin as a treatment for NASH in human patients with obesityrelated insulin resistance performed by Marchesini et al. (39). They treated 20 patients with biopsy proven NASH with Metformin at a dose of 500 mg t.i.d. for 4 months. They compared the six individuals who did not comply with treatment, to the group of 14 patients who completed the Metformin protocol. They found the latter group to have significantly reduced mean transaminase concentrations. In seven of 14 who completed Metformin therapy, transaminase levels returned to normal, insulin sensitivity improved and liver volume decreased by 20% compared with baseline studies. This report on the efficacy of Metformin in human patients with NASH adds to Metformin’s clinical credibility to treat future patients with NASH; a disease for which there has not yet been a well-defined treatment. There is need for
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more studies on liver safety of Metformin, compared to thiazolidinediones, another family of insulin sensitizing agents. Thiazolidinediones includes troglitazone (Rezulin), which has proven to be a safe agent. In addition, there needs to be a double-blind, cross-over controlled study of Metformin in NASH focusing on its potential role in delaying, preventing or reversing liver fibrosis. However, such a study would be very difficult to perform as to achieve statistical significance, in the absence of any currently known noninvasive predictor of liver fibrosis, it would require approximately 2000 –3000 patients with NAFLD to screen with a baseline liver biopsy to find enough patients with biopsyproven NASH (K. Lindor, personal communication, June 27, 2002). Therefore, such a study is still far off in the future. NASH, which up to now has been essentially untreated for the lack of a definitive therapy, remains a significant cause of morbidity and mortality (18). Bugianesi et al. have shown that features suggestive of NASH are more frequently observed in hepatocellular carcinoma (HCC) arising in patients with cryptogenic cirrhosis than in age- and sexmatched HCC patients of well-defined viral or alcoholic etiology (40). Therefore, HCC may represent a late complication of NASH-related cirrhosis according to these authors. Therefore, we believe that NASH should be treated with whatever treatment has been shown to have beneficial effects as soon as the diagnosis is made. Zhou et al. have shown that Metformin’s glucose-lowering effect results from decreased hepatic glucose production and increased glucose utilization (41). They also found that Metformin has beneficial effects on circulating lipids that have been linked to the occurrence of fatty liver. Metformin activates AMPactivated protein kinase (AMPK) in hepatocytes, which is a major cellular regulator of lipid and glucose metabolism. As a result, they reported, that acetyl-CoA carboxylase (ACC) activity is reduced, so that fatty acid oxidation is induced, and thus expression of lipogenic enzymes is suppressed. Activation of AMPK by metformin also suppresses expression of SREBP-1, a key lipogenic transcription factor. The authors also report that in metformin-treated rats, hepatic expression of SREBP-1 (and other lipogenic) mRNAs and protein is reduced and activity of the AMPK target, ACC, is also reduced. Using a novel AMPK inhibitor, they found that AMPK activation is required for metformin’s inhibitory effect on glucose production by hepatocytes. In isolated rat skeletal muscles, metformin stimulates glucose uptake coincident with AMPK activation. In the authors’ opinion activation of AMPK provides a unified explanation for the pleiotropic beneficial effects of this drug. Knowler et al. showed that Metformin reduced the incidence of Diabetes in non-diabetics or “pre-diabetics” (42). They randomly assigned 3234 non-diabetic persons with elevated fasting and post-load plasma glucose concentrations (postprandial glucose levels 140 to 199 mg/dL or fasting levels 110 to 125 mg/dL) to one of these groups: placebo, metformin (850 mg b.i.d.), or a lifestyle-modifica-
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tion program. The goals were at least a 7% weight loss and at least 150 min of physical activity per week. The mean age of the participants was 51 yr, and the mean body-mass index (the weight in kilograms divided by the square of the height in meters) was 34.0; 68% were women, and 45% were members of minority groups. They reported that the incidence of diabetes was 11.0, 7.8, and 4.8 cases per 100 person-years in the placebo, metformin, and lifestyle groups respectively. Lifestyle intervention reduced the incidence of diabetes by 58% (95% confidence interval, 48 – 66%) and metformin reduced the incidence of diabetes by 31% (95% confidence interval, 17– 43%), compared with placebo. Thus we believe that there is enough evidence to begin using this relatively safe drug in those NASH patients with documented insulin resistance and pre-diabetes of the metabolic syndrome. Metformin should not be utilized in patients with potential lactic acidosis, renal insufficiency and congestive heart failure. Perry Hookman, M.D., F.A.C.P., F.A.C.G. Jamie S. Barkin, M.D., F.A.C.P., M.A.C.G. University of Miami School of Medicine Mount Sinai Medical Center Division of Gastroenterology Miami, Florida
REFERENCES 1. Ludwig J, Viggiano TR, et al. Nonalcoholic steatohepatitis. Mayo Clinic Proc 1980;55:434 –38. 2. Falck-Ytter Y, Younossi ZM, et al. Clinical features and natural history of nonalcoholic steatosis syndromes. Semin Liver Dis 2002;21:17–26. 3. McCollough AJ, Falck-Ytter Y. Body composition and hepatic steatosis as precursors for fibrotic liver disease. Hepatology 1999;29:1328 –30. 4. Propst A, Propst T, et al. Prognosis in nonalcoholic steatohepatitis. Gastroenterology 1995;108:1607 [letter]. 5. Hultcrantz R, Glaumann H, et al. Liver investigation in 149 asymptomatic patients with moderately elevated activities of serum aminotransferases. Scand J Gastroenterol 1986;21:109 – 13. 6. Hilden M, Christofferson P, et al. Liver histology in a normal population — examinations of 503 consecutive fatal traffic casualties. Scand J Gastroenterol 1977;12:593–97. 7. Nonomura A, Mizukami Y, Unoura M, et al. Clinicopathologic study of alcohol-like liver disease in non-alcoholics; non-alcoholic steatohepatitis and fibrosis. Gastroenterol Jpn 1992;27:521. 8. Baldridge AD, Perez-Atayde AR, Graeme-Cook F, et al. Idiopathic steatohepatitis: An expanded clinical entity. Gastroenterology 1994;107:1103. 9. Moran JR, Ghishan FK, Halter SA, Graeme-Cook HL. Steatohepatitis in obese children: A cause of chronic liver dysfunction. Am J Gastroenterol 1983;78:374. 10. James O, Day C. Non-alcoholic steatohepatitis. Another disease of affluence. Lancet 1999;353:1634 –6. 11. Galambos JT. Natural history of alcoholic hepatitis III. Histologic changes. Gastroenterology 1972;63:1026. 12. Marbet UA, Bianchi L, Meury U, Stadler GA. Long-term histological evaluation of the natural history and prognostic factors of alcoholic liver disease. J Hepatol 1987;4:364.
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13. Powell EE, Cooksley WG, Hanson R, et al. The natural history of nonalcoholic steatohepatitis: A follow-up study of forty-two patients for up to 21 years. Hepatology 1990;11:74. 14. Lee RG. Nonalcoholic steatohepatitis: A study of 49 patients. Hum Pathol 1989;20:594. 15. Pinto HC, Baptista A, Camilo ME, et al. Nonalcoholic steatohepatitis. Clinicopathological comparison with alcoholic hepatitis in ambulatory and hospitalized patients. Dig Dis Sci 1996;41:172. 16. Matteoni CA, Younossi ZM, Gramlich T, et al. Nonalcoholic fatty liver disease: A spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413. 17. Galdwell SH, Oelsner DH, et al. Cryptogenic cirrhosis: Clinical characterization and risk factors for underlying disease. Hepatology 1998;29:664 –9. 18. Marchesini G, et al. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med 1999;107:450 –55. 19. Marceau P, et al. Liver pathology and metabolic syndrome x in severe obesity. Clin Endocrinol Metab 1999;84:1513–7. 20. Yang SQ, Lin HZ, Lane MD, et al. Obesity increases sensitivity of endotoxin liver injury: Implications for pathogenesis of steatohepatitis. Proc Natl Acad Sci USA 1997;2557– 62. 21. Uysai KT, Weisbrok SM, Marino MW, Motamislgil GS. Protection from obesity-induced insulin-resistance in mice lacking TNF-␣ function. Nature 1997;389:610 –4. 22. Shimoura I, Basmokow Y, Horton JD. Increased nuclear levels of SREBP-1c associated with fatty livers in two mouse models of diabetes mellitus. J Biol Chem 1999;274:30028 –8832. 23. Megalsson MD, et al. Antihyperglycemic actions of guanidenoakanoic acids; 3-guanidlnoproplonic acid and ameliorates hyperglycemia in diabetic KKAy and CS7BL-61/ob/ob mice and increases glucose disappearance in rhesus monkeys. J Pharmaol Exp Ther 1993;266:1454 –62. 24. Campfield IA, Smith FI, Bum P. The OB protein (leptin) pathway—a link between adipose tissue mass and central neural networks. Horm Metab Res 1996;28:619 –32. 25. Rouru I, Pesonen V, Kaulu M. Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats. Life Sci 1992;50: 1813–20. 26. Paolisso G, et al. Effect of metformin on food intake in obese subjects. Eur J Clin Invest 1998;28:441–6. 27. Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis 2001;21:27–41. 28. Day CP, Oliver FWJ. Steatohepatitis: A tale of two ‘hits’? Gastroenterology 1998;114:842–5. 29. Ikejima K, Honda H, Yoshikawa M, et al. Leptin augments inflammatory and profibrogenic responses in the murine liverinduced by hepatotoxic chemicals. Hepatology 2001;34:288 – 97. 30. Ikejima K, Takei Y, Honda H, et al. Leptin receptor-mediated signaling regulates hepatic profibrogenic and remodeling of extracellular matrix in the rat. Gastroenterology 2002;122: 1399 –410. 31. Faggioni R, Feingold KR, Grunfeld C. Leptin regulation of the immune response and the immunodeficiency of malnutrition. FASEB J 2001;15:2565–71. 32. Diehl AM. Nonalcoholic steatosis and steatohepatitis IV. Nonalcoholic fatty liver disease abnormalities in macrophage function and cytokines. Am J Physiol Gastrointest Liver Physiol 2002;282:G1–G5. 33. Potter JJ, Womack L, Mezey E, Anania FA. Transdifferentiation of rat hepatic stellate cells results in leptin expression. Biochem Biophys Res Commun 1998;244:178 –82. 34. Marra F. Leptin and fatty liver fibrosis: A matter of fat. Gastroenterology 2002;122:1529 –30. 35. Petersen KF, Oral EA, Dufour S, et al. Leptin reversed insulin
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36. 37.
38. 39. 40.
41. 42.
resistance and hepatic steatosis in patients with severe lipodystrophy. J Clin Invest 2002;109:1345–50. Savage DB, O’Rahilly SO. Leptin: A novel therapeutic role in lipodystrophy. J Clin Invest 2002;109:1285–6. Soloman SS, Mishra SK, Cwik C, et al. Pioglitazone and metformin reverse insulin resistance induced by tumor necrosis factor-alpha in liver cells. Horm Metab Res 1997;29:370 – 82. Mc Clain Cl, Hill DB, Schmidt J, Diehl AM. Cytokines and alcoholic liver disease. Semin Liv Dis 1993;13:170 –82. Yin M, Wheeler MD, Kono H, et al. Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice. Gastroenterology 1999;117:942–52. Bugianesi E, Leone N, Vanni E, et al. Expanding the natural history of nonalcoholic steatohepatitis: From cryptogenic cirrhosis to hepatocellular carcinoma. Gastroenterology 2002; 123:134 –40. Zhou G, Myers R, Li Y, et al. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001; 108:1167–74. Knowler WC, Barrett-Connor E, Fowler SE, et al. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002;346:393–403.
The Road to Resistance: Antibiotics as Growth Promoters for Animals White DG, Zhao S, Sudler R, et al. The Isolation of Antibiotic Resistant Salmonella From Retail Ground Meats N Engl J Med 2001;345:1147–54
ABSTRACT White et al. report their research on Salmonella strains isolated from ground meats purchased at grocery stores in the Washington, DC area. The authors isolated Salmonella from 20% of the 200 sampled products including 35% of chicken, 24% of turkey, 16% of pork and 6% of all beef samples. Resistance to at least one antibiotic was found in 84% of the isolates and 53% of the isolates were resistant to at least three antibiotics (some isolates were resistant to nine different antibiotics). Ceftriaxone-resistant Salmonella and multidrug resistant S. enterica serotype typhimurium DT 104 were also isolated. (Am J Gastroenterol 2003;98:499. © 2003 by Am. Coll. of Gastroenterology)
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resistance which was also documented. A major concern is that the high levels of antibiotic resistance are a result of the use of antibiotics in food animals. Previously it had been suggested that half of all antibiotics were used in animals; however, a more recent estimate suggests that 24.6 million pounds of antibiotics are given to animals each year as growth promoters at subtherapeutic amounts in their feed compared to 3 million pounds consumed by humans (1). Currently, chickens spend 40 of their 42 days of life on antibiotics. Especially problematic is the broad spectrum of antibiotics allowed in animals, including expanded spectrum cephalosporins and quinolones. Even more disconcerting is the use of antibiotics from the same classes as our newest antibiotics in the fight against vancomycin-resistant Enterococci (VRE), including virginiamycin (a streptogramin related to Synercid) which has been used as a growth promoter in chickens since 1974. In the same issue of the Journal, McDonald et al. reported sampling chickens from 26 stores in four states. They found 82% of chicken carcasses had quinupristin– dalfopristin (Synercid, Aventis Pharm) resistant Enterococcus faecium (2). These resistant isolates were found in 81% of supermarket chains and 59% of different brands. These results raise significant concerns that Synercid, which was only approved by the Food and Drug Administration (FDA) as an option to treat VRE in 1999 may be compromised by the use of antibiotics in animal feed. This concern was supported by Sorensen et al. who found that healthy volunteers who ingested streptogramin resistant E. faecium strains isolated from chickens had the organism isolated from stool for up to 14 days (3). The European Union banned all antibiotics that are related to those used in humans from use in animals for growth promotion in 1998. How long will it take the United States to realize we run a significant risk for developing resistance in foodborne pathogens by the continued use of important antibiotics as growth promoters in animals? Edward C. Oldfield, III, M.D. Division of Infectious Disease Eastern Virginia School of Medicine Norfolk, Virginia
COMMENT In the United States, there are an estimated 2 to 4 million annual cases of Salmonellosis. Salmonella are widespread in animal sources, in particular poultry and pigs. A wide range of food sources have been implicated in outbreaks including raw shell eggs, poultry, ground beef, ice cream, powdered milk, cheese, cantaloupes, tomatoes, sprouts, and numerous other products of animal origin. White et al. document the widespread contamination of ground meat products in grocery stores. Even more disconcerting was the extremely high level of antibiotic
REFERENCES 1. Mellon M, Benbrook C, Benbrook KL. Hogging it: Estimates of antimicrobial abuse in livestock. Cambridge: Mass: Union of Concerned Scientists, 2001. 2. McDonald LC, Rossiter S, Mackinson C. Quinupristin-dalfopristin-resistant Enterococcus faecium on chicken and in human stool specimens. N Engl J Med 2001;354:16. 3. Sorensen TL, Blom M, Monnet DL, et al. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. N Engl J Med 2001;345: 1161– 6.