The fluxes are several times as great as the net movement which is their difference. ON THE PATHOGENESIS OF FATTY LIVER

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1 January 1966 EDITORIALS 137 hexose and amino acid accumulates during absorption in vitro, with development of high concentration gradients between the cell and ECF, it is unlikely that this occurs in vivo. It is more likely that nutrient molecules pass rapidly through the serosal surface of the cell to be carried away in the circulation under in vivo conditions. Membrane pore size, which regulates permeability, is susceptible to metabolic effects such as hormone action. Antidiuretic hormone (ADH) regulates pore size of relatively impermeable membranes as in the distal renal tubule cells. The kidney is an organ of water excretion and receives large volumes of solutions. During dehydration, when water retention is necessary for homeostasis, the water must be recovered from the solutions. ADH increases pore size and permeability of renal tubule cells and promotes water retention by increasing its recovery from the lumen. In contradiction to earlier work, recent studies of the effect of ADH on the intestinal epithelium show a paradoxical decrease in permeability, particularly in the proximal portion of the small intestine. 9 Although these findings require confirmation, if the same agent produces opposite effects in different organs, this may be a valuable clue as to the chemical and structural nature of the opening and closing mechanism of the pores. Finally, it should be emphasized that transport has been discussed as net movement. The net is actually the difference between mucosal to serosal and serosal to mucosal unidirectional movements (fluxes). The fluxes are several times as great as the net movement which is their difference. Harold P. Schedl, M.D., Ph.D. Department of Internal Medicine University of Iowa College of Medicine Iowa City, Iowa REFERENCES 1. Woodbury, J. W The cell membrane: Ionic and potential gradients and active transport, p. 2. In T. C. Ruch and J. F. Fulton [ed.] Medical physiology and biophysics. W. B. Saunders Company, Philadelphia. 2. Curran, P. F Na, Cl, and water transport by the rat ileum in vitro. J. Gen. Physiol. 43: Durbin, R. P Osmotic flow of water across permeable cellulose membranes. J. Gen. Physiol. 44: Parsons, D. S., and D. L. Wingate Fluid movements across the wall of the rat intestine in vitro. Biochim. Biophys. Acta 30 : Curran, P. F., and J. R. Mac Intosh A model system for biological water transport. Nature (London) 193: Soter, N. A., J. P. Kinney, J. S. Fordtran, and F. Rector Permeability characteristics of the human small intestine. J. Lab. Clin. Med. 64: Schedl, H. P., and J. A. Clifton Cortisol absorption in man. Gastroenterology 44: Schedl, H. P., and J. A. Clifton Solute and water absorption by the human small intestine. Nature (London) 199: Soergel, K. H., G. E. Whalen, and J. A. Harris Effects of antidiuretic hormone (ADH) on the human small intestine. Clin. Res. 13: 261. ON THE PATHOGENESIS OF FATTY LIVER Recent advances in our knowledge of lipid metabolism have given new insight into the mechanisms of lipid absorption, transport, and regulation in higher animals as well as in man. New approaches to the Address requests for reprints to: Dr. Emmanuel Farber, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania study of disease, now made possible by this knowledge, give every indication of clarifying the pathogenesis of a common pathological manifestation which has intrigued students of the liver for a long time, namely, fatty liver. In the fasting organism, lipid is transported from the adipose tissue as free (F) (or nonesterified (NE)) fatty acid (FA)

2 138 EDITORIALS Vol. 50, No.1 bound to serum albumin. The various cells of the body utilize some of this FF A as a source of energy. In the liver also, some of it is oxidized but the bulk is re-esterified, predominantly to triglyceride (TG), and is then transferred to the blood mainly as,b-lipoprotein. The combination with protein, phospholipid, and other non-tg lipids appears to be essential for the transfer of the TG from the liver to the blood. The TG transported in the blood is utilized by the tissues of the body, presumably with the aid of lipoprotein lipase, which hydrolyzes the TG in the lipoprotein to free fatty acids. In the jed organism, the liver actively takes chylomicra TG coming from the intestine and again converts at least some of this lipid to plasma lipoprotein. Thus the liver plays an active role in the handling and preparation of lipid from both the diet and the storage depots for transport and utilization by the body. Important in this concept is the realization that the bulk of the free fatty acids taken up by the liver appears as blood TG and that the over-all rate of this conversion is very rapid. The first maj or breakthrough in our understanding of the pathogenesis of liver steatosis was the discovery in 1960 that the fatty liver induced by such agents as carbon tetrachloride was due to a block in the transfer of triglyceride from the liver to the plasma. 1,2 According to this formulation, the liver continues to take up FF A and dietary lipid from the blood and to esterify them to triglycerides but cannot release the newly synthesized triglyceride into the blood at a rate sufficiently rapid to balance the rate of uptake of its precursor. The second important step in our understanding was the suggestion that the underlying mechanism for such a block might be the interference with the synthesis by the liver of the protein moiety of the plasma lipoproteins. 3-5 Although some intimate relationship between fatty liver and disturbed protein synthesis was suspected some years earlier (cf. reference 6), it is now possible to formulate this in molecular terms which can be subjected to experimental test. In the case of at least four agents, CCl 4 ; phosphorus; the methionine antagonist, ethionine; and the antibiotic, puromycin, the accumulation of excess triglyceride is preceded by a significant inhibition of hepatic protein synthesis. With ethionine, this disturbance in protein metabolism in the liver is the result of a striking decrease in hepatic ATP concentration;7 while with puromycin, the amino acids, although activated normally, are prevented from being linked together to form the peptide chains of protein. 8 The underlying molecular mechanisms for the inhibitions of protein synthesis by CCl 4 9 and plo remain to be clarified. If further research substantiates the essential validity of this hypothesis, one may formulate the basic defect in one group of fatty livers as an inability of the liver either to package the lipoprotein for export or to transfer it through the various membranes from the interior of the liver cell to the plasma. Since the plasma lipoproteins, including the very low density,b-lipoproteins, are composed not only of triglycerides and protein but also of phospholipids, cholesterol, cholesterol esters, and some carbohydrate, and since the decrease in the level of such lipoproteins in the plasma following CCl 4 or ethionine administration involves all lipid components,ll, 12 it naturally follows that fatty liver may result from an interference with the ability of the liver either to synthesize anyone of these component parts or to conjugate them in the appropriate manner to make the complete lipoprotein molecule. There is a growing realization that, although the delineation of the chemical alterations in cells is fundamental to our eventual understanding of the pathogenesis of most disease, such information, without due regard to intracellular compartments, will probably be insufficient per se to explain disease. The beginning elucidation of the intricate subcellular structure of cells makes it evident that complex intracellular chemical compartmentation is a phenomenon that must be integrated with the overall biochemical activities of cells and tissues in order to explain many pathological as well as physiological phenomena. This concept is well illustrated by recent information

3 139 EDITORIALS January 1966 FIG. 1. Portions of three hepatocytes (H1, H 2, and H 3) 4\12 hr after ethionine. M embranebound osmiophilic bodies are apparent in each hepatocyte. Sites of continuity between the membrane enclosing the osmiophilic bodies and the agranular and rough endoplasmic reticulum are indicated by arrows. A peg-and-socket type of contact is seen between two hepatocytes (H2 and H.). ar agranular reticulum; ER rough endoplasmic reticulum ; r ribosomes. Tissue fixed in osmium tetroxide, buffered with Veronal acetate, embedded in Epon, and stained with lead citrate (X 14,250). about ultrastructural alterations in fatty liver as observed with the electron microscope. Work from several laboratories during the past year has shown that triglyceride accumulates first inside the endoplasmic reticulum early in the genesis of several forms of fatty liver. A representative illustration of this, as seen within a few hours after ethionine administration, is given in figure 1. As more and more TG appears in the liver, these small lipid bodies fuse to form larger lipid collections which are visible under light microscopy. It now appears that small lipid bodies are easily and rapidly removed from the liver when the basic chemical defect is reversed,

4 140 EDITORIALS Vol. 50, No. 1 whereas the larger accumulations are metabolically much less labile. This is observed, for example, in animals in which an early fatty liver has been induced by the administration of ethionine 3 to 4 hi' previously. By counteracting the A TP deficiency with the administration of an A TP precursor such as adenine, the small lipid bodies rapidly disappear from the endoplasmic reticulum and appear, transitorily, in the space of Disse. 13 According to the thesis outlined above, these lipid bodies accumulate in the ~ : m d o p lreticulum a s m i c as a result of the inability of the cell to transform them into plasma lipoprotein. Since triglyceride is synthesized from FF A and glycerophosphate in the endoplasmic reticulum, the TG naturally accumulates first in this site in the cell. This hypothesis is represented diagrammatically in figure 2. If it is basically valid, TG may accumulate in the liver as a result of an interference with the production of either protein, as with ethionine, puromycm, or CCI 4 ; phospholipid, as is probably the case in choline deficiency, cholesterol, or cholesterol esters. However, one would expect that the lipid accumulating in some fatty livers might consist not only of TG but also of phospholipid or cholesterol, or both. Yet, why do the majority of fatty livers contain only or predominantly excess triglyceride? No clear-cut answer to this important problem has been presented. However, it must be pointed out that the liver plays a unique role in plasma cholesterol and phospholipid metabolism in that this organ not only synthesizes these components but plays a very active part in their removal from the blood and in their further metabolism or breakdown. Also, triglyceride is utilized as a major energy source for many cells while phospholipid and cholesterol are mainly important as components of the structure of cell membranes or other cellular components or, as in the case of cholesterol, are precursors for metabolic regulators such as the steroid hormones. In view of this, it would not be at all surprising if a PLASMA F LIPID GLYCEROPHOSPHATE \... RETICULUM ETHIONINE, CCI4.., PUROMYCIN PHOSPHORUS CHOLINE ~ { DEFICIENCY? VERY LOW ~ Y P L A S M A ~ - - ~ ' LIPOPROTEIN FIG. 2. Diagrammatic representation of the synthesis of plasma ~ - l i p o p r o t e i n by the liver cell and the possible derangements during the pathogenesis of some forms of fatty liver. The fatty acids derived either from adipose tissue or from the diet are coupled with glycerophosphate to form triglyceride (TG) in the endoplasmic reticulum. This is somehow conjugated with protein, phospholipid, cholesterol, and cholesterol esters at some place along the route between the site of synthesis and the site of transfer into the blood. It is suggested that the interference with anyone of these conjugation steps may result in accumulation of triglyceride in the liver.

5 January 1966 EDITORIALS 14 I sensitive feedback inhibition of synthesis exists for phospholipid as well as for cholesterol, and that such a mechanism may be one factor in preventing any significant increase in either of these lipid components in most fatty livers. It should be emphasized that the pathogenetic mechanism here discussed is still a working hypothesis applicable to only some forms of fatty liver. A major exception among several is the case of the ethanol fatty liver,14 the genesis of which is still unclear. A major consideration for the experimental pathologist is the applicability to human disease of information obtained in experimental animals. In the case of fatty livers, it appears likely that the hypothesis discussed above may be useful in explaining human disease because of the essential similarity between higher animals and man in their over-all handling of lipids and in their lipid metabolism. Since several tests of the hypothesis could be relatively simply set up (e.g. serum lipid and lipoprotein determinations, conversion' of radioactive fatty acid to plasma triglyceride, etc.), it is anticipated that the appropriate studies will soon be performed to observe whether any forms of human fatty liver prove to have a pathogenetic mechanism similar to those discussed here. The results of such critical tests of the usefulness of this hypothesis in human disease will be awaited with great interest. Emmanuel Farber, M.D., Ph.D. Department of Pathology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania REFERENCES 1. Recknagel, R. 0., B. Lombardi, and M. C. Shotz A new insight into the pathogenesis of carbon tetrachloride fat infiltration. Proc. Soc. Exp. BioI. Med. 104: Recknagel, R. 0., and B. Lombardi Studies of biochemical changes in subcellular particles of rat liver and their relationship to a new hypothesis regarding the pathogenesis of carbon tetrachloride fat accumulation. J. BioI. Chern. 236: Lombardi, B., and R. O. Recknagel On the pathogenesis of experimental fatty infiltration of the liver. Fed. Proc. 20: Lombardi, B., and R. O. Reeknagel Interference with secretion of triglycerides by the liver as a common factor in toxic liver injury. Amer. J. Path. 40: Harris, P. M., and D. S. Robinson Ethionine administration in the rat. 1. Effects on the liver and plasma lipids and on the disposal of dietary fat. Biochem. J. 80: Farber, E Studies on the chemical pathology of lesions produced by ethionine. Arch. Path. (Chicago) 67: Farber, E., K. H. Shull, S. Villa-Trevino, B. Lombardi, and M. Thomas The biochemical pathology of acute hepatic adenosine triphosphate deficiency. Nature (London) 203: Darken, M. A Puromycin inhibition of protein synthesis. Pharmacol. Rev. 16: Smuckler, E. A., O. A. Iseri, and E. P. Benditt An intracellular defect in protein synthesis induced by carbon tetrachloride. J. E xp. Med. 116: Seakins, A., and D. S. Robinson Changes associated with the production of fatty livers by white phosphorus and ethanol in the rat. Biochem. J. 92: Lombardi, B., and G. Ugazio. Serum lipoproteins in rats with carbon tetrachloride induced fatty liver. J. Lipid R es. In press. 12. Ugazio, G., and B. Lombardi Serum lipoproteins in rats with ethionine-induced fatty liver. Lab. Invest. 14 : Baglio, C. M., and E. F arber Ultrastructural consequences of biochemical lesions in the liver induced by ethionine. Fed. Proc. 24: Isselbacher, K. J., and N. J. Greenberger Metabolic effects of alcohol on the liver. New Eng. J. Med. 270: 351.