LIVER PHYSIOLOGY AND DISEASE

Transcription

1 GASTROENTEROLOGY 64: , by The Williams & Wilkins Co. Vol. 64, No.4 Printed in U.S.A. LIVER PHYSIOLOGY AND DISEASE ETHANOL-INDUCED FATTY LIVER: EFFECT OF INTESTINAL LYMPH FISTULA ROBERT K. OCKNER, M.D., STEVEN P. MISTILIS, M.D., RUTH B. POPPENHAUSEN, AND ADOLF F. STIEHL, M.D. Department of Medicine, University of California, School of Medicine, San Francisco, California Previous studies have shown that intraduodenal ethanol enhanced the production of nondietary very low density lipoprotein by rat intestine. In this study, this source of endogenous lipid was examined in regard to its possible contributing role in the pathogenesis of ethanol-induced fatty liver. Rats received constant intraduodenal infusions of ethanol (5 g per kg) or isocaloric glucose during the 0 - to 8-hr interval. After intraduodenal ethanol, rats without lymph fistula developed the expected fatty liver at 24 hr. In marked contrast, fistula rats had virtually normal liver triglycerides at this time. The effect of lymph diversion was not due to lipid losses prior to ethanol, nor to minor lymphatic losses of ethanol during the experiment. A similar effect was present at 16 hr in some rats, but not in others. The basis for the variable response at this earlier time was not elucidated, but correlated with plasma free fatty acid concentration. Nondietary intestinal lipoproteins do not seem quantitatively sufficient, by themselves, to account for the ethanol-induced fatty liver. However, they playa significant role in the over-all lipid balance of the rat, and may contribute, with other endogenous sources, to hepatic lipid accumulation in this disorder. It is well established that ethanolinduced fatty liver reflects, in part, a direct effect of ethanol on hepatic metabolism 1-4 and that ethanol enhances Received September 29, Accepted December 14, Address requests for reprints to: Dr. Robert K. Ockner, Department of Medicine, University of California, School of Medicine, San Francisco, California Supported by National Institutes of Health Grants AM and AM Dr. Ockner is the recipient of a National Institutes of Health Research Career Development Award AM The triglyceride fatty acid analyses were performed through the kindness of Dr. Richard Havel. Dr. Steven P. Mistilis' present address is: Royal 603 hepatic triglyceride synthesis. 5,6 However, it is not certain whether the triglyceride fatty acids which accumulate are primarily derived from (1) increased adipose tissue lipolysis, leading to increased uptake of free fatty acids by the liver, 7-10 (2) reduced fatty acid oxidation in the liver, (3) increased hepatic fatty acid synthesis,13 or (4) some combination of these processes. Furthermore, dietary fat, when Prince Alfred Hospital, Missenden Road, Camperdown 2050, NSW, Australia. Dr. Adolph Stiehl's present address is: Medizinische Universitatsklinik, Abteilung fur Gastroenterologie, Bergheimer Strasse 58, 6900 Heidelberg, Germany.

2 604 OCKNER ET AL. Vol. 64, No.4 available, has also been shown to contribute to alcoholic fatty liver Recently we examined the effects of ethanol on the metabolism of endogenous lipids and lipoproteins by rat intestine. 17 Those studies showed that intraduodenal ethanol administration led to a significant increase in the intestinal secretion of nondietary very low density lipoproteins 'S-2' into lymph, and suggested the possibility that this additional source of endogenous lipid might contribute to the development of ethanol-induced fatty liver. In this study, the relationship between the availability of these nondietary intestinal lipoproteins and the development of the ethanol-induced fatty liver is explored. Portions of this work have been reported in abstract form.22 Methods Experimental animals, diet, and operative procedures including cannulation of intestinal lymph duct and duodenum were as described previously. 17 Operated animals were placed in restraining cages and allowed to stabilize overnight, during which time they received 0.85% NaCI by constant intraduodenal infusion at a rate of 2.5 to 3.5 ml per hr. In some studies, animals with intestinal lymph fistulae received, during the postoperative period, intravenous infusions of whole defibrinated intestinal lymph (collected from donor rats with lymph fistulae), in addition to intraduodenal saline. During the experiments, rats received constant intraduodenal infusions of 10% (w/v) ethanol in 0.85% NaCI over 8 hr (0.63 g per kg; total dose 5 g per kg), or isocaloric glucose in 0.85% N acl. After the 8-hr test infusion period, all animals received intraduodenal 0.85% NaCI for the remainder of the experiment, up to 48 hr. Restrained animals were warmed by means of an infrared lamp. The liver was removed immediately after death, blotted, and weighed. Lipids were extracted from homogenates of liver and adipose tissue by the method of Folch et al. 23 Triglycerides were isolated from lipid extracts by thin layer chromatography on silica gel as described previously. 17 Analytical methods. The enzymatic method of Eggstein and Kreutz 24 was used for the determination of glyceride glycerol in extracts of liver. Phospholipids were determined by the method of Bartlett25 as modified by Marinetti,26 and total cholesterol by the method of Zlatkis et al. 27 Fatty acid composition of triglycerides in lymph, adipose tissue, and liver was determined by a modification of the procedure of Lipsky and Landowne. 28 Fatty acid methyl esters were analyzed using a Perkin-Elmer gas chromatograph model F 11, hydrogen flame detector, on a 12-foot column packed with 12% ethylene glycol succinate on acid-washed Chromosorb W (100 to 120 mesh), at 187 C with nitrogen at 60 psi. Fatty acid composition (percentage of total) of each sample was determined by computer analysis.29 Ethanol concentration in intestinal lymph was determined by a modification of the method of Bonnichsen and Theore1l 30 as described by Lundquist. 31 Serum ethanol averaged 323 mg per 100 ml at 8 hr, 113 mg per 100 ml at 16 hr, and was not detectable at 24 hr.'7 Output of ethanol in lymph was calculated from concentration and lymph volumes over 8-hr periods. In three experiments, the amount of ethanol lost over 24 hr via lymph fistula was 11.6% (10.4 to 13.4) of the administered dose. Statistical analysis. The standard error of the mean was calculated for groups of three or more observations, and significance of differences between groups was determined by means of Student's t-test. 32 Results Effect of lymph fistula on hepatic triglycerides after intraduodenal glucose or ethanol. In glucose-infused animals (fig. 1), hepatic triglyceride concentration at 24 hr was not significantly affected by diversion of intestinal lymph, and in both fistula and nonfistula rats was similar to that found in 10 nonfasted, unoperated, unrestrained rats (4.5 ± 0.65 mg per g of wet tissue). In ethanol-infused rats, however, the effect of lymph fistula was striking. Although ethanol-infused rats without lymph fistula showed the expected fatty infiltration of the liver when examined at 24 hr (fig. 1), those animals with intestinal lymph fistula had hepatic triglyceride concentrations at this time which were virtually normal. Hepatic triglyceride concentrations in these ethanol-infused fistula rats not only differed markedly from those

3 April 1973 LYMPH FISTULA AND ETHANOL-INDUCED FATTY LIVER 605 LIVER TRIGl YCERIDE mg/ g I ± SEM} wei weioht " FIG. 1. Effect of intestinal lymph fistula on hepatic triglycerides at 24 hr after ethanol or isocaloric glucose. Rats were given ethanol (5 g per kg) or isocaloric glucose by a constant intra duodenal infusion over B hr. Hepatic triglycerides were measured 24 hr after starting the infusion. in the nonfistula rats (P < ), but in addition did not differ significantly from hepatic triglycerides in glucose-infused rats, with or without lymph fistula. Moreover, loss via lymph of approximately 10 to 13% of the administered ethanol (see "Methods") was not sufficient to account for the effect of lymph diversion, since a corresponding reduction in the amount administered (i.e_, to a dose of 4.5 g per kg) still caused fatty livers in the rats without fistulae. In 7 such animals, liver triglycerides at 24 hr were 21.0 ± 4.6 mg per g (206 ± 47 mg per liver). Because lymph fistula rats were losing endogenous lipid not only during the experimental period itself but also during the preceeding overnight stabilization, the possibility was examined that the reduced levels of hepatic triglyceride at 24 hr were an artifact of this additional loss (prior to the actual experiment). In a group of rats prepared with lymph fistulae, defibrinated normal donor lymph was infused intravenously during the period between operation and the beginning of the ethanol infusion. In these rats, hepatic triglyceride concentration at 24 hr was 6.54 ± 1.33 mg per g of wet weight, and did not differ significantly from levels in ethanolinfused fistula rats (fig. 1) which had not received lymph replacement prior to the start of the experiment. Thus, protection of lymph fistula animals against fatty liver could not be attributed to the loss of intestinal lymph lipid during the stabilization period prior to the experiment itself. In table 1, total hepatic triglyceride content at 24 hr is compared in the various groups. Differences between ethanolinfused fistula and nonfistula rats are similar to those observed when hepatic triglyceride concentrations were compared (fig. 1). At 48 hr hepatic triglyceride concentration and content in all groups of animals were below that found in any group at 24 hr, and there were no significant differences among the various groups. These experiments show that ethanol infusion failed to produce a fatty liver at 24 hr in those animals with intestinal lymph fistula. Although this suggested that lymph fistula may have completely prevented the fatty liver, the possibility was also considered that lymph diversion resulted instead in a more rapid removal of hepatic lipid which had accumulated prior to 24 hr. To examine this possibility, an additional group of fistula and nonfistula rats was studied under identical conditions at 16 hr. In table 2 it is seen that in the nonfistula rats, the hepatic triglycerides at 16 hr (8 hr after completion of ethanol infusions) were TABLE 1. Effect of intestinal lymph fistula on hepatic triglyceride content at 24 hr after intraduodenal glucose or ethanol" No fistula Fistula Total hepatic triglyceride Glucose 33.0 ± 6.3 (n = 3) 40.1 ± 7.9 (n = 4) mglliver Ethanol ± 19.6 (n = 6) 63.3 ± 22.6 (n = 6) a Restrained rats with or without intestinal lymph fistula received constant intraduodenal infusions of ethanol (5 glkg) or isocaloric 'glucose over an B-hr period followed by intraduodenal isotonic saline in both groups. At 24 hr after the start of the infusion, rats were killed and livers were analyzed for triglyceride content. Mean ± SE.

4 606 OCKNER ET AL. VoI.64,No.4 TABLE 2. Effect of intestirwllymph fistula on hepatic triglycerides at 16 hr after intraduoderwl ethanol" No fistula (n ~ 6) Fistula (n ~ 6) Group A plus group B Group A (n ~ 3) Group B (n ~ 3) Hepatic triglyceride mg/g 23.0 ± ± ( ) 24.3 ( ) mg/liver 222 ± ± ( ) 226 ( ) a Restrained rats with or without intestinal lymph fistula received constant intraduodenal infusions of ethanol (5 g per kg) over an 8-hr period followed by intraduodenal isotonic saline in both groups. At 16 hr (8 hr after completion of ethanol infusion), rats were killed and livers analyzed for triglyceride. Groups A and B are entirely arbitrary designations, based on the observed hepatic triglyceride levels in the 6 lymph fistula rats. greater than at 24 hr (compare with fig. 1 and table 1). Moreover, in rats with lymph fistulae, considered as a single group, there was significant accumulation of triglyceride in the liver, although these levels were on the average less than those in the nonfistula rats. However, it was noteworthy that these animals appeared to be separable into two distinct groups, on the basis of the strikingly different response which they demonstrated. In the first, arbitrarily designated "group A," hepatic triglyceride concentration and content was virtually normal, as in the fistula rats studied at 24 hr (fig. 1, table 1). In "group B," however, lymph fistula appeared to have no effect on fatty liver development whatever; hepatic triglycerides in these rats were elevated, and did not differ from those in ethanol-infused rats without lymph fistula. These differences in response among lymph fistula rats at 16 hr could not be correlated with lymph triglyceride output, for in this respect the two groups were similar (unpublished observations). It was determined, however, that those lymph fistula rats which developed fatty livers at 16 hr (group B) had serum free fatty acid (FFA) levels (1.234 ± ~Eq per ml) which were greater than those in the group A rats which did not develop fatty liver (0.991 ± ~Eq per ml, P < 0.01). The reasons for these differences are not clear. These observations regarding the response of hepatic triglyceride levels to ethanol administration in rats with intestinal lymph fistula can be summarized as follows: at 24 hr after ethanol, liver: triglycerides in these rats were virtually normal, at which time rats without lymph fistula had persistence of marked fatty infiltration (fig. 1, table 1). At 16 hr, however, liver triglycerides in some rats were normal, while in others they were elevated. While these findings at 16 hr would be consistent with two different response patterns, it is more likely that at this earlier time there may be a spectrum of responses, unlike the more uniform effect seen later (24 hr). The effect of intestinal lymph diversion on fatty liver, therefore, was to result either in its "prevention" or its more rapid resolution. The ameliorating effect of lymph diversion was not attributable either to loss of lymph lipid prior to the experiment, or to lymphatic loss of ethanol during the experiment. The degree of protection afforded at 16 hr correlated with serum FFA levels, possibly reflecting the mobilization of adipose tissue. Fatty acid composition of triglycerides in intestinal lymph, ethanol-induced fatty liver, and adipose tissue. The possiblity that endogenous lipids in intestinal lymph might participate in the development of fatty liver was further examined by comparing triglyceride fatty acid (TGFA) composition of lymph (at 16 to 24 hr after ethanol) with that of liver and adipose tissue. The results are shown in table 3. After ethanol, liver TG FA were similar to lymph TGF A in relative amounts of 14:0,14:1,16:0,16:1,18:2, and 18:3. However, liver TGFA more closely resembled adipose TGFA in relative content of 18: 1 and 20: 4. Although these findings do not permit any definite conclusion as to which might be the more important source of hepatic triglyceride, they are consistent with the possibility that intestinal lymph

5 April 1973 LYMPH FISTULA AND ETHANOL-INDUCED FATTY LIVER 607 TABLE 3. Fatty acid composition of triglyceride in rat liver, intestinal lymph, and adipose tissue after ethanol" Tissue Fatty acid 14:0 14: 1 16:0 16: 1 18:0 18: 1 18: 2 18:3 20:4 % oftotal Liver Trace 2.2 ls.s S Trace 2.3 (n = 3) ( ) ( ) (0-3.2) ( ) (20.5-3S. 1) ) ( ) Lymph Trace Trace S Trace 10.9 (n = 3) ( ) (15.5-1S.3) ) ( ) ) ( ) Adipose 1.7 Trace 2S (n = 2) (1.4, 1.9) (27.3,28.6) (5.0,5.5 (4.2, 4.3) (27.7,30.2) (25.4, 2S.1) (1.5, 1.5) (0,7.4) " At 24 hr (16 hr after the infusion of ethanol) animals were killed and samples ofliver and adipose taken for analysis of t~iglyceride fatty acids as described in "Methods." Lymph samples represent those collected from ethanol-infused rats between 16 and 24 hr (S to 16 hr after completion of ethanol infusion). Mean and range or individual observations. contributes TGFA to the ethanol-induced fatty liver. Differences between lymph and liver in the composition of some of the fatty acids may reflect additional fatty acid sources such as adipose tissue or de novo hepatic synthesis, differences in the rates at which individual fatty acids are metabolized in the liver, or both. Furthermore, over-all adipose tissue TGFA composition may not necessarily reflect the composition of those fatty acids which are mobilized and transported to the liver as FFA.33 Discussion These experiments show that in contrast to the fatty liver characteristically observed at 24 hr after ethanol, animals with intestinal lymph fistula had virtually normal hepatic triglyceride levels. This effect was not attributable either to loss of lymph lipoproteins prior to the experiment or to lymphatic loss of ethanol during the experiments. At face value, these findings might be interpreted as suggesting that lymph triglyceride-rich lipoproteins were the immediate source of the lipid which accumulates in ethanol-induced fatty liver, and that fatty liver was prevented by their removal. Although the animals in these studies were fasting, this interpretation would be compatible with the earlier suggestion that in animals ingesting fat, the triglyceride carried in intestinal lipoproteins (chylomicrons) contributed to the ethanol-induced fatty liver. 16 However, for these data, such an interpretation would constitute a gross oversimplification for several reasons. First, our own data show that the increment in 24-hr lymph triglyceride output attributable to ethanol (24 mg per 24 hr), 17 was not sufficient to account for the increment in hepatic triglycerides after ethanol (97 mg per liver, table 1). Second, in some lymph fistula animals studied at 16 hr after ethanol, hepatic triglycerides were increased, clearly indicating that fatty liver was not being "prevented," at least in these particular animals. Third, it is generally accepted that most of the triglyceride in very low density lipoproteins or chylomicrons does not enter the liver directly, but rather is removed peripherally in adipose tissue after hydrolysis by lipoprotein lipase. 3 ' Therefore, it cannot be concluded from these data that increased intestinal lipoprotein production is the "cause'" of ethanol-induced fatty liver. However, our observations do indicate that diversion of intestinal lymph significantly modified the severity and duration of ethanolinduced fatty liver. This effect was consistent and predictable at 24 hr, and in some cases was present at 16 hr as well. The variable response at 16 hr correlated with the serum FFA levels, suggesting that

6 608 OCKNER ET AL. VoI.64, No. 4 the mobilization of adipose tissue fatty acids to the liver reduced the degree of "protection" afforded by lymph fistula. However, direct measurement of FFA flux is required to confirm or exclude this possibility. In either case, the ameliorating effect of lymph diversion on the ethanol-induced fatty liver indicated that endogenous intestinal lipids played a quantitatively significant role in the overall lipid balance of the animals, under the conditions studied. A possible effect due to the loss of some component of lymph other than lipids (e.g., lipoprotein peptides) is not excluded. The findings at 16 hr (8 hr after completion of ethanol) are of importance in one additional respect. Because the hepatic triglyceride content in ethanol-treated nonfistula animals at 16 hr is approximately 200 mg greater than in normal animals, it is evident that a lipid source other than intestinal lymph must have been responsible for the development of the fatty liver during this earlier time period (up to 16 hr). Most likely, this is a reflection of FF A mobilization from adipose tissue, decreased fatty acid oxidation, or both. Conversely, the contribution by intestinal lymph lipid appears to be most significant in the persistence of the fatty liver during the 16- to 24-hr interval. Comparisons of triglyceride fatty acid compositions in lymph and liver are consistent with this concept, but the differences between them in regard to certain fatty acids could reflect an additional lipid source, differences in the rates at which individual fatty acids are metabolized, or both. Conclusions These studies show that diversion of intestinal lymph results in the amelioration of the ethanol-induced fatty liver. They are consistent with the concept that endogenous lipids, carried by intestinal lipoproteins, play a contributing or permissive role in its persistence after 16 hr, but are not by themselves sufficient cause for the fatty liver. It is not clear from these studies whether intestinal lymph triglyceride enters the liver directly, or after first undergoing hydrolysis in the periphery, subsequently returning to the liver as FF A. In either case, these observations are of importance in view of earlier evidence suggesting that the intestinal contribution of nondietary triglyceride fatty acid to plasma lipid turnover in the rat was relatively insignificant, i.e., approximately 10% of plasma triglyceride turnover36 or 5% of plasma FF A turnover. 36 The effect of intestinal lymph diversion, observed in these experiments, supports the concept that endogenous intestinal lipoproteins may be of greater quantitative significance in over-all lipid balance than has been assumed. REFERENCES 1. Diluzio NR: Effect of acute ethanol intoxication on liver and plasma lipid fractions of the rat. Am J Physiol 194: , Lieber CS, Jones DP, DeCarli LM: Effects of prolonged ethanol intake: production of fatty liver despite adequate diets. J Clin Invest 44: , Mallov S: Effects of chronic ethanol intoxication on liver lipid content of rats. Proc Soc Exp BioI Med 88: , Rubin E, Lieber CS: Alcohol-induced hepatic injury to nonalcoholic volunteers. N Engl J Med 278: , Lundquist F, Tygstrup N, Winkler K, et al: Glycerol metabolism in the human liver: inhibition by ethanol. Science 150: , Nikkila EA, Ojala K: Role of hepatic L-aglycerophosphate and triglyceride synthesis in production of fatty liver by ethanol. Proc Soc Exp Bioi Med 113: , Brodie BB, Butler WM, Horning MG, et al: Alcohol-induced triglyceride deposition in liver through derangement of fat transport. Am J Clin N utr 9: , Maling HM, Horning MG, Butler WM, et al. Triglyceride deposition in rat liver through derangement of fat transport by various chemical agents. Fed Proc 19:229, Reboucas G, Isselbacher KJ: Studies on t he pathogenesis of the ethanol-induced fatty liver. 1. Synthesis and oxidation of fatty acids by the liver. J Clin Invest 40: , Schapiro RH, Scheig RL, Drummey GD, et al: Effect of prolonged ethanol on the transport and metabolism of lipids in man. N Engl J Med 272: , 1965

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