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The liver, under normal conditions, presents only one mitosis per 1,000 hepatocytes, and the cells contain a fair amount of glycogen, lipids, and proteins.
Table 1 indicates the two types of factors controlling normal liver physiology. It is very difficult to define the exact role of each of these factors because they are very closely interrelated. The typical alterations induced by a portacaval shunt constitute the best demonstration of the existence of these two types of factors.1,2 In fact, in addition to the typical signs of atrophy in this model, the presence of regeneration with increases in the number of mitosis from 1.14 to 6 can be observed. The appearance of atrophy and ultrastructural deterioration demonstrates a lowering of factors controlling the size and morphology of the liver, and the latter indicates the appearance of regulating factors of regeneration.
In a series of investigations during 1971 to 1977,3-5 we have established that insulin is the main factor that regulates the size, morphology, and structure of the liver. In fact, a nonhypoglycemic insulin infusion into the ligated left portal vein for four days after an Eck fistula greatly reduced the left lobular hepatocyte atrophy, preserved the hepatocyte ultrastructure, and increased the cell renewal (mitosis rose from six to 15 per 1,000 cells). This effect was not changed by the addition of glucagon, regardless of the dose. The finding that insulin increased the number of mitoses raised the question of the importance of hormones in the regeneration process.
After this study, several authors6-10 have described hormonal changes in both rats and dogs, ie, decreases in insulin, triiodothyronine, thyroxine, and calcitonin levels, whereas glucagon and corticosterone levels increase. Furthermore, we have recently reported that sexual hormones are involved in liver regeneration after 70% hepatectomy in rats.11-13 Estrogen levels increase in the serum with a corresponding increase in estrogen receptors, which correlates with the peak of the mitotic index. On the other hand, serum testosterone and androgen receptors display a very significant decrease.
On the basis of these hormonal changes, many speculations have been proposed to attempt to define the role of hormones in liver regeneration. Nevertheless, none of these hypotheses were conclusive for the following reasons: (1) it is possible to prevent the typical insulin-glucagon change after hepatectomy by glucose infusion without affecting liver regeneration in a major way; (2) insulin and glucagon in vitro do not affect hepatocyte proliferation; (3) triiodothyronine, thyroxine, calcitonin, and sex hormones in vitro do not show particular activity on hepatocyte proliferation; (4) the administration of all these hormones in animals after hepatectomy has never provided consistent data on liver regeneration; and finally (5) and even of further importance are the results we have obtained by transplanting small livers into much larger recipients. A rapid growth of small livers occurs during the first day after transplantation, which is similar to that observed after partial hepatectomy. None of the typical hormonal alterations observed after hepatectomy occurred during this process.
Because hormonal changes did not provide any conclusive answers as related to liver regeneration, in 1975 several investigations on growth factors in serum and tissue were initiated.
Table 2 illustrates the growth factors that have been partially isolated from serum14-19 and liver.19-32 As reported in Table 2, we have been working on the extraction and purification of hepatic stimulator substance (HSS) since 1980. HSS was first described by LaBrecque and Pesch20 in the cytosol of partially hepatectomized rats and subsequently identified in canine liver by Starzl et al.22
Table 3 reports the steps in the purification and the physicochemical characteristics of the active chromatographic fraction, referred to as F150, that is obtained from weanling rat liver cytosol using fast protein liquid chromatography (FPLC) apparatus. This fraction stimulated DNA synthesis in hepatocytes in a dose-dependent regimen when injected in 40% hepatectomized rats (Fig 1). The activity of this fraction is organ specific but not species specific.33
Similar to the findings with hormones, the considerable knowledge obtained on growth factors has not yet resulted in any conclusive theory on liver regeneration. A better understanding of this process is clinically very important, not only for cases of liver damage but also for hepatic transplantation.
It has been observed that in clinical liver transplantation a liver from a small donor, occasionally transplanted into a much larger recipient, rapidly increases in size and achieves a size comparable to that of a normal liver for that particular recipient in a period of 2 weeks. These phenomena have been extensively studied in dogs in which small-for-size livers were transplanted into larger recipients.34
In Fig 2, the increase in the volume of a small liver transplanted in a larger animal is compared with that of a normal-for-size liver transplanted into a dog of suitable size.
A great increase in thymidine kinase, ornithine, and decarboxylase levels and in the percentage of mitoses was detected only in the small liver transplanted in a host of a larger size. This indicates an active hepatocyte proliferation. Figure 3 compares the insulin glucagon changes observed in both models. Despite a different rate of hepatocyte proliferation, the hormonal changes observed were similar but at the same time different from the levels observed in a regenerating liver induced by partial hepatectomy.3
In this experiment, the lack of specific hormonal changes suggests that the role of hormones in liver regeneration has to be reevaluated, and it raises the question of the existence of inhibitors as additional factors of liver growth regulation. A small liver, when transplanted into a large animal, might be unable to produce enough inhibitor substance to keep a normal plasma concentration. Therefore, regeneration could be seen as a complex mechanism in which the initiating key is the plasma concentration of inhibitors. This possibility even further complicates the already complex view of the regeneration process. In fact, we know only a few pieces of the puzzle that are responsible for the complex system of regeneration, and even more discouraging, we do not fully understand how all these pieces fit together.
Supported by research grants from the Veterans Administration; Project Grant No. AM-29961 from the National Institutes of Health, Bethesda, MD; and by Grant No. 885/02 16544 from the Consiglio Nazionale delle Ricerche, Italy.