Hormonal control of rat liver regeneration

GASTROENTEROLOGY

PROGRESS

76:1470-1482,

1979

ARTICLE

Hormonal Control of Rat Liver Regeneration H. L. LEFFERT,

K. S. KOCH,

T. MORAN,

and B. RUBALCAVA

Cell Biology and Molecular Biology Laboratories, The Salk Institute, Department of Biochemistry, Centro de Investigation y de Estudios Politecnico National, Mexico City, Mexico

One problem of animal cell growth regulation which has generated tremendous controversy and speculation has been the search for “hepatotrophic” factors.’ Eagerly sought because of their postulated role in regulating liver regeneration-a rapid but transient proliferative process extensively studied in the rat following partial hepatectomy-it seemed reasonable to expect that by characterizing these substances, and their physiological actions upon the liver, a precise explanation of fundamental regulatory mechanisms would emerge. This review describes how recent developments have sustained this expectation. At the least, working solutions to some basic questions concerning regeneration now exist. What Blood-Borne Regeneration?

Factors Control Liver

Six, possibly seven, hormones, and a family of essential and non-essential amino acids, regulate rat liver regeneration (Table 1). These conclusions are supported by several kinds of experimental evidence. First, cell culture studies indicate directly that stationary phase adult rat hepatocytes (Figure l), obtained from normal liver and permitted to undergo one growth cycle in primary monolayer culture,’ can be reinitiated to synthesize DNA and to divide. This is accomplished by adding fresh growth medium supplemented only with insulin, glucagon and epi-

Received September 14, 1978. Accepted January 8, 1979. Address requests for reprints to: H. L. Leffert, M. D., The Salk Institute, Post Office Box 1809, San Diego, California 92112. This work was supported by the National Cancer Institute (CA21230) and the National Institute on Alcohol Abuse and Alcoholism (P50AA03504). We thank Drs. M. Brown and B. Roos for helpful discussions and Drs. S. Potter, J. Rivier, W. Bromer, and R. Chance for gifts of EGF, neural-gastric and pancreatic polypeptides. 0 1979 by the American Gastroenterological Association

0016-5085/79/061470-13$02.00

San Diego, California, and Avanzados de1 Instituto

dermal growth factor (EGF is one of the non-suppressible insulin-like peptides” chemically identical to urogastrone).4 These observations extend earlier findings” and exclude criticisms not considered by Richman et al. that in vitro conditions merely permit expression of antecedent in vivo “stimulatory” events, or that DNA synthesis initiating events occur during tissue disruption as hepatocytes become exposed to digesting proteases present in the perfusion buffers. Many peptides fail to stimulate proliferation using our assay. This group includes pancreatic polypeptide,” fibroblast,’ and nerve growth factor,* the neural-gastric peptides listed in Table 2 (except for enteroglucagon, which has not been tested), a putative hepatotrophic serum tripeptide,” all of the known hypothalamic pituitary-releasing hormones,‘” and all of the known pituitary glycoprotein hormones in addition to adrenocorticotropin and somatotropin. The possible significance of earlier ablation/repletion studies suggesting a role for in adult liver regeneration remains somatotropin” unclear because of uncertainty concerning the purity of the injected hormone preparation. Second, peripheral infusions of triiodothyronine (T3), glucagon and amino acids”; or of insulin, glucagon and EGF’” into intact adult rats induce small but significant amounts of hepatocyte DNA synthesis, Third, prior pancreatic ablation and evisceration severely suppress rat liver DNA synthesis 24 hr after 67% hepatectomy but not if such animals receive peripheral insulin and glucagon infusions.14 Fourth, peripheral insulin-antiserum infusions virtually inhibit rat hepatic DNA synthesis 24 hr after 67% hepatectomy.13 Similar experiments are warranted using neutralizing antibodies to EGF or, if obtainable, to glucagon. Fifth, split portacaval transposition studies in both intact and pancreatectomized animals indicate that hepatocyte proliferation critically depends upon insulin.’ Sixth, hepatic regeneration is significantly inhib-

June 1979

Table

PROGRESS

1.

Probable

Physiologic

Regulators

of Liver

ARTICLE

1471

Regeneration Source(s)

Factor(s)

Extrahepatic

lodothyronines

Pancreatic islets Pancreatic islets GI tract Brunner’s glands Submandibular glands Parathyroid glands Thyroid gland Pituitary glands1 Thyroid gland

Glucocorticoids

Adrenal

Amino

Blood Muscle

Insulin Glucagon EGF (urogastrone) Parathyroid Calcitonin

24-HOUR

hormone

acids

[3~]-~~~~~~~~~

PULSE:

cortex

DAY 12-13 in vitro

NO MEDIUM CHANGE

e

MEDIUM CHANGE PLUS i

INSULIN GLUCAGON EGF

None None

Direct Direct

Unknown

Direct

None

Indirect

None ? Binding proteins ? Binding proteins ? Protein turnover

Indirect Direct Direct Direct

ited after prior parathyroidectomy’” and thyroidectomy.‘” Repletion with parathyroid hormone, calcitonin and T,, or thyroxine (TJ corrects the growth defect. Glucocorticoids and T, or T, also affect in vivo hepatic proliferation”~‘” and cultured fetal’Y-23 and adult hepatocyte proliferationz~z2~z3under special conditions. The glucocorticoid effects are quite intriguing because of their paradoxical nature.22 When added at the time of plating, hydrocortisone reduces the saturation density attained by cultured fetal rat hepatocytes2z~z3 but at lower levels it promotes reinitiation of DNA synthesis under quiescent conditions.“.‘” If adult hepatocytes are cultured under our conditions, growth cannot ensue unless hydrocortisone is added at the time of plating.2~‘“~z3However, using the growth initiation assay shown in Figure 1, none of these hormones measurably affect DNA synthesis and cell division either alone, combined, or when added together with insulin, glucagon, and EGF. Table

i

Primary site of action

Hepatic

2.

Gastrointestinal and Glucagon

Peptides Secretion

Regulating

Insulin

Insulin Figure

1. Initiation

of cultured adult hepatocyte proliferation by insulin, glucagon, and epidermal growth factor. Stationary phase cultures 255m12 days after plating were labeled with [3H]thymidine51 (top panel) or given fresh serum-free growth medium containing 50 ng/ml each of insulin, glucagon, and epidermal growth factor, plus [“Hlthymidine (bottom panel). Twenty-four hours the cultures were prepared for radiolater, autography.“’ After 7 days, the film was developed; cultures were stained with crystal violet, and photographed (inset bars given magnification in micrometers). Approximate labeling indices”’ were 7% (top panel) and 35% (bottom panel). Arrows indicate cells which have synthesized DNA and undergone either metaphase formation, nuclear division or cytokinesis. Such cells were not observed under conditions of “no medium change.” (H. Leffert and K. S. Koch, manuscript in preparation.)

Factor ____ Bombesin Enteroglucagon Gastrin Gastric inhibitory peptide Neurotensin Pancreozymin Secretin Somatostatin Vasoactive intestinal peptide

(Relative

Glucagon changes

in portal

1” t t

(I? (D) (D)

t OC

0

: 1

(3 (?I) (D) (D) (D)

t

(?)

0

blood) (I)

: t

: :

(D)

(D) (D) (D) (D)

(11 = decrease; t = increase: 0 = no change; ? = unknown. bI = indirect action; D = direct action. c Central venous, but not portal, insulin levels decrease, suggesting increased hepatic insulinuptake (M. Brown, personal communication).

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Seventh, the 70% hepatectomized rat develops a specific pattern of blood hormone levels not observed in laparotomized controls (Figure Z).ZZ-Z6 This pattern displays low insulin and T, or T, levels; high glucagon and corticosterone” levels; and constant plasma calcitonin levels. Rat plasma parathyroid hormone radioimmunoassays have encountered technical problems (B. Roos, personal communication) but indirect evidenceZ8 suggests that this hormone rises rapidly after 67% hepatectomy. Blood insulin-like peptide levels apparently fall to 50-80% of control values after partial hepatectomy. This is suggested by indirect measurements of somatomedinCZ9 which promotes cultured fetal hepatocyte DNA synthesis initiation,‘” and by direct radioimmunoassay of EGF (S. Potter et al., unpublished observations; normal rat plasma levels are 800-1000 picograms EGF/ml). Most of these hormonal changes occur rapidly after surgery, many hours before DNA replication begins. They are proportional to the amount of excised live? and they persist for at least 24 hr, gradually returning to their initial steady-state.z5 More interestingly, decreased insulin and elevated glucagon levels characterize hepatoproliferative states in genera1.22.25 Nonetheless it has been difficult to prove rigorously that these alterations per se are necessary to stimulate rat liver regeneration. For example, attempts to suppress glucagon secretion and subsequent rat hepatic DNA synthesis after 70% hepatectomy, using somatostatin, have been inconclusive (H. Leffert, M. Brown, T. Moran, unpublished results). Somatostatin analogs which specifically inhibit glucagon secretion may obviate this problem, if such analogs do not directly perturb hepatocyte proliferation. Another assumption inherent to in vivo studies which require exogenous compounds to counteract altered hormone blood levels is that these compounds do not exert effects upon nonspecific tissue sites. Thus, although it could be argued that declining insulin levels after partial hepatectomy are necessary to stimulate liver growth, because peripherally elevated insulin retards regeneration3” this interpretation is complicated by insulin’s many extrahepatic effects. In summary, the combined evidence suggests that normal liver regeneration is controlled by five peptide and two nonpeptide hormones. Three-insulin, glucagon, and EGF-seem to be principle regulators because they act directly upon liver cells. Furthermore, these three peptides are sufficient, together with nutrient and ionic growth medium constituents, to stimulate adult hepatocyte proliferation in stationary cell culture. Two additional peptides, parathyroid hormone and calcitonin, are obligatory

GASTROENTEROLOGY

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Figure

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24

2. Hormonal changes in the bloodstream of 70% hepatectomized and laparotomized rats. The data arc taken from references 24 and 26 (panels A-D) and 27 (panel E) where the “100%control” values may be found. Calcitonin (in collaboration with Dr. B. Roos, Department of Medicine, Case Western Reserve School of Medicine; panel F) was measured by radioimmunoassay; 100% control values were 30-40 pg immunoreactive material/ml plasma. Two-way analysis of variance of these data indicate that in all cases, the curves for 70% hepatectomized rats (solid lines) significantly differ from laparotomized controls.

for growth but probably act extrahepatically because they are proliferatively inactive in primary hepatocyte cell culture. And two nonpeptide hormones (T, or T,, and glucocorticoid) modulate hepatic proliferation under special conditions and, in certain instances, with paradoxical effects. Although specific blood hormonal changes occur after 70% hepatectomy, and although they are observed during hepatoproliferative states in general, at least with respect to insulin and glucagon, it is unclear whether these changes cause or merely are associated with regeneration. What Are the Sources of RegenerationRegulating Hormones? Table 1 lists extrahepatic and hepatic sites which produce or store factors regulating regeneration. Many of the peptide factors are present in more than one tissue”’ and in more than one chemical form. Hepatocytes also produce intracellular proteins which bind large quantities of free hormone, for example, the glucocorticoids.“’ EGF has been localized by immunofluorescent

June 1979

PROGRESS

methods predominantly to the submandibular”,“3 and Brunner’s duodenal glands,“’ both in rodents and humans. Biological activity resides with a 6100 dalton peptide.“,“” This peptide is associated with a 29,300dalton macromolecule which contains arginyl-esterase activity needed to generate the active low moassay has detected lecular peptide.“” Radioreceptor small (amounts of EGF in various tissue extracts including liver.“’ But rigorous proof that EGF or other insulin-like peptides are synthesized or stored by parenchymal or nonparenchymal liver cells is lacking. Glucagon, a 3600-dalton peptide produced and secreted by pancreatic islet A-cells, also has multiple gastrointestinal sources. Both high and low molecular weight forms are detected in rat plasma. Biological activity resides with the low molecular weight species (see Reference 37 for review). Insulin is a 6000-dalton peptide produced and secreted by pancreatic islet B-cells. No other source for this hormone is known. While there is suggestive evidence that immunoreactive insulin circulates in macromolecular form,3R the exact chemical nature and biologic role of this “complex” remain uncertain. What Controls Hepatic Regeneration-Regulating

Levels of Hormones?

Because insulin, glucagon, and EGF directly stimulate adult rat hepatocyte proliferation, it is of interest to ask how their intrahepatic humoral levels are controlled after partial hepatectomy? These levels presumably depend upon rates of hepatic blood flow, and the ambient sinusoidal concentration of free peptide. The latter is mainly a function of secretion rates at the endocrine source as well as intrinsic rates of hepatic and extrahepatic uptake. Although hepatic blood flow rates are transiently elevated twofold shortly after partial hepatectomy, regenerative hyperplasia is not solely dependent upon these changes.“’ Extrahepatic uptake rates, notably renal clearance of specific hormones, are poorly characterized. Pancreatic islet secretion of insulin and glucagon appears decreased and increased, respectively, after 67% hepatectomy, as determined from measurements of portal hormone leve1s.22~“” No information is available concerning EGF secretion from either of its known sources in the partially hepatectomized rat. At least forty substances regulate insulin and glucagon secretion (for review, see reference 41). Many originate from the gastrointestinal tract (Table 2), brain, and adrenal medulla. How and which of these factors regulate the endocrine pancreas during regeneration is unknown. Glu-

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1473

coregulatory effects seem unlikely.“‘,” However, catecholamines may be involved because persistent adrenergic discharges occur after partial hepatectomy, as implicated from a number of physiologic sfudies.42m44Adrenergic transmitters also are potent secretogogues for mouse EGF derived from the submandibular gland.“” Such findings raise the intriguing possibility that hepatic EGF levels depend upon rapid and transitory elevations of arterial and portal EGF originating from the submandibular gland and the duodenum, respectively. This would not be inconsistent with pilot experiments indicating diminished plasma EGF levels (at 1 and 24 hr after 67% hepatectomy) if, in addition, the liver remnant increased its turnover of EGF as it does with thyroid Mechanisms controlling circadian hormones.2fi rhythms of blood hormone levels also may be invo1ved4’.4’ but are poorly understood at the present time. Hepatic uptake mechanisms also depend upon cellular membrane binding, internalization, and degradation processes. Preliminary evidence suggests that the liver remnant, after 67% hepatectomy, retains insulin and glucagon more efficiently than control tissue because the apparent half-lives (tH) for the disappearance of both ““I-labeled peptides increase by a factor of two (Table 3).” Transient renal failure seems not to account for this observation because a concomitant twofold decrease in the apparent t, for T, is observed.26 Whether these changes are caused by altered hormone-receptor interactions (discussed below); decreased hormone degradation; and/or enhanced hepatic pinocytotic activity49 occurring soon after 67% hepatectomy, is not yet clear. In summary, defined secretory changes at the major endocrine sources of insulin and glucagon (and possibly EGF) occur after partial hepatectomy. Concomitantly, altered hepatic uptake of these peptides Table

3.

Hepatic

Disappearance and Thyroxine

Glucagon,

of ‘2”I-Labeled Insulin, After 67%

Hepatectomy in Rats Hepatic ‘““l-Labeled Insulin Glucagon Thyroxine

‘IApparent

hormoneh

disappearance

Laparotomy

(t,+ min)”

67% Hepatectomy

2.0 + 0.6 (SEMI

3.6 f

0.4

3.7 * 0.7

6.4 f

0.9

674.0 f 42.0

348.0 f 70.0

half-lives were calculated from linear semilogarithmic curves, fit (r > 0.95) to at least three time-points, according to the relation t, = In Z/k,+ where kd is assumed to be a first-order rate constant determined graphically. ’Posthepatectomy time intervals for measurements of insulin, glucagon, and thyroxine disappearance were O-20,20-40, and O1440 min, respectively. Data points were obtained from measurements using 3-6 rats. See References 26 and 48 for further details.

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occurs. Delineating the mechanisms responsible for these changes and determining the effective extracellular peptide levels are challenges of future research.

How Might Insulin, Glucagon, and EGF Interact to Stimulate Liver Regeneration? While insulin and glucagon probably exert extrahepatic effects which modify liver regeneration, it is clear that these peptides and EGF may act directly to promote liver cell growth. How might these interactions occur? Do the peptides move a “switch” or do they interact with additional substances to facilitate ongoing processes? Do they act simultaneously or intermittently? Are their effects additive or synergistic? Do nonparenchymal cells collaborate in these interactions? Animal studies have provided some information. Cross-circulation experiments suggest that humoral changes occur during a lo-1.2hr interval after 67% hepatectomy in order to stimulate liver DNA synthesis and entrance of cells into S-phase (for review, see Reference 50). Specifically, hormone infusion studies using eviscerated rats show that insulin and glucagon become necessary within a few hours, but not immediately after the operation.14 These observations suggest, but do not prove, that at least two “programs” operate to initiate hepatic DNA synthesis, one which begins “earlier” than another. This type of hypothesis was discussed critically elsewhere.2”-23 Growth initiation assays with hepatocyte cultures”’ permit a direct analysis of the problem.20.21 Using the stationary-phase adult system (Figure l), we have obtained further evidence strongly supporting a two-program hypothesis (H Leffert and KS Koch, manuscript in preparation). The principle findings are: 1 The

combined addition of insulin, glucagon and EGF without a medium change is nonstimulatory, whereas a medium change (which includes provision of excess nutrients and ionic constituents) stimulates some DNA synthesis (ca. 15% of the maximal response measured at 24 hr). Therefore, fresh medium alone “sets” zero-time (Figure 3). These observations agree with earlier findings in the fetal system’“~Z1 and suggest that similar conditions participate to “start” regeneration in the liver remnant after partial hepatectomy. 2. Pulse-labeling studies using [3H]thymidine show that DNA synthesis rates increase lo-12 hr after the medium change (the “onset-time”), reach maximal levels between 20 and 24 hr and decline gradually after 48 hr. Similar kinetics are observed in the adult rat after 67% hepatectomy.5”

GASTROENTEROLOGY

NUTRIENT

0 t ALTERED

EXCESS ,,.

:

I

..

I

6

12

MONOVALENT

t ALTERED

Figure.

..

Vol. 76, No. 6

CATION

RNA AND PROTEIN

I8

24 I

FLUXES METABOLISM

3. Interactions among hepatotrophic factors required to control cultured adult hepatocyte proliferation (H. Leffert and K. S. Koch, manuscript in preparation). The time scale (O-24 hr) represents experimental conditions similar to those described in Figure 1. Rectongular bars illustrate the estimated duration and time intervals of action of the indicated factors: class I signals here represent EGF (-7-T ); class II signals represent insulin and glucagon (J-1; see reference 21 for further discussion); nutrient excess represents fresh serum-free growth medium (-1): and (bottom of diagram) DNA synthesis initiation inhibitors represent VLDL and amiloride (F-1). The vertical arrows indicate the approximate onset times of the various processes. See text for further details.

EGF enhances the action of the medium change if it alone is added O-12 hr. But its full effects are retained when washed from the cultures 3-6 hr after the initial change if insulin and glucagon are subsequently added (times earlier than 3 hr have not yet been studied). Insulin and glucagon effects are synergistic with EGF. Added singly, insulin and glucagon stimulate about 20-30s of the maximal [“Hlthymidine incorporation response. Exposure to the pancreatic peptides is unnecessary for at least 3 and can be delayed until 12 hr after the medium change. These results provide direct evidence to support observations from infusion studies in eviscerated rats.14 In vitro studies further indicate that insulin and glucagon exposure is necessary for at least 810 hr. Appropriate infusion intervals using the eviscerate preparation should be of further interest to validate these findings. Increasing amounts of peptide hormones increase the proportion of responding cells without shortening the onset-time. The in vivo counterpart of this finding is that increased percent hepatectomy also fails to shorten the prereplicative phase but instead increases the proportion of proliferating cells.5o

June 1979

Thus it appears that EGF promotes the effects of “nutrient excess” (see Figure 3) via a fairly shortlived interaction with the cells (53 hr). These events might constitute the first program. This seems to control one or more ongoing cellular reactions (or structures) associated with forming regulatory substances which normally are unstable and decay rapidly. These interactions then are stabilized or potentiated by insulin and glucagon. Such events might constitute the second program. The lo-12 hr delay, the minimal time before the fraction of cells entering S-phase increases, presumably is required to accumulate (or deplete) a critical pool of regulatory substance(s). Therefore, when the “clocks” run smoothly, hormone interactions operate intermittently during the 12-hr interval after the medium change. The cell population during this time is sensitive to DNA synthesis initiation inhibitors such as plasma very low density lipoprotein (VLDL; active in the fetal hepatoor the potassium-sparing diuretic cyte system)52 amiloride.“” Both are discussed below. If they are present after this interval, they are ineffective (Figure 3). Which of the two programs are disrupted by these substances is unknown. Preliminary experiments not shown suggest that amiloride delays the onset time but not the rates of entrance into S-phase. Interestingly, these observations mimic earlier in vivo findings with L-asparaginase,54 which delayed hepatic DNA synthesis initiation in 67% hepatectomized rats if given during (but not after) the prereplicative phase. Common mechanisms may be involved as discussed in the next section. It is worth noting that proliferating adult hepatocytes under our culture conditions are “growth-insensitive” to certain stimulatory hormones. For example, EGF and glucagon fail to stimulate DNA synthesis during the lag and logarithmic phases of the growth-cycle.’ This phenomenon manifests itself in still another way in that in vitro insulin and glucocorticoid growth-cycle requirements of regenerating liver cells are considerably reduced in comparison to normal hepatocytes.” Both findings suggest that, as a consequence of antecedent hormone action, growing cells become hormonally “desensitized.” How normal hepatocytes regain proliferative-responsiveness to hormones as they enter resting states is of crucial importance to our understanding of how hepatoma cells seem to “lose” it. Finally, it is unclear if nonparenchymal cell/hormone interactions are involved in liver regeneration.” For example, insulin and EGF might stimulate such cells to produce diffusible substances or low molecular weight factors which enter hepatocytes via membrane junctional connections. In the adult that a micultures shown in Figure 1, it is possible

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nority fraction of the population (JO-20%) consists of nonparenchymal cells.“’ Close inspection of [3H]thymidine labeled cultures by radioautography reveals what appears to be flattened cells occasionally situated underneath hepatocyte aggregates. The possibility that these underlapping cells are of mesodermal origin, survive the arginine-free plating conditions by contact cross-feeding, and contribute to the response can not be excluded. For the purposes of the remaining discussion, however, it will be assumed that stimulatory responses result from principle regulatory peptide action upon hepatocytes.

What Mechanisms Are Regulated by Insulin, Glucagon and EGF to Control Liver Regeneration? From the foregoing discussion, the problem of how hepatotrophic factors regulate liver regeneration may be formulated in terms of concerted hormone action.‘” Therefore, it is necessary to determine first, how insulin, glucagon, and EGF produce their signals by interacting with cellular receptors: and, second, how these signal-interactions subsequently lead to enhanced frequencies of DNA synthesis and cell division. Previously we discussed how Levitzki’s “signal strength” parameter (Y)“” might be applied to insulin and glucagon action after partial hepatectomy.” Briefly, the initial effects of a signal (hormone) are a complex function of its effective concentration, the numbers of available oligomeric membrane receptors, and the molecular subunit properties of these receptor systems. The calculations suggest that after 70% hepatectomy, insulin’s hepatic signal remains high (y = 1.0) despite hypoinsulinemia, because of compensatory increases in hepatic insulin “receptors” (see Figures 4 and 5; and Table 4, which gives quantitative binding constants) and because of decreased hormone degradation or pinocytosis (Table 3). Negative cooperative properties of the insulin recepto?’ may further maintain insulin signal strength after partial hepatectomy because the hormone “spends more time”“” on the receptor. Preliminary evidence supports this idea (Figure 6) because the ability of unlabeled excess insulin to accelerate dissociation of tracer amounts of ““I-labeled peptide is diminished in liver membranes from 70% hepatectomized rats. Glucagon signals are predicted to rise to Y- 1.0 in these persistently hyperglucagonemic animals (Figure 2) despite reduced numbers of glucagon membrane binding sites (Figures 4 and 5; Table 4). Reduced glucagon binding, first reported in 1975,“’ may represent a form of receptor “internalization,” a property exhibited by EGF during its interaction with fibroblast receptors.S” EGF binding studies us-

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ing liver membranes similar to those shown for insulin and glucagon in Figures 4 and 5, have yet to be reported. The significance of these observations could be that immediately after 70% hepatectomy, strong insulin and glucagon signals are received by the liver remnant while concomitant peripheral lipopolytic effects make stored fuels rapidly available. There is continuing controversy as to whether the entire spectrum of a peptide hormone’s effects result only from its binding to cell surface receptors, followed by production of one or more “second messengers” (e.g. cyclic AMP, in response to glucagon) and/or a membrane perturbation which itself exerts subsequent effects. Alternatively, the hormone-receptor complex may enter the cell (e.g., insulinao,61 and EGF”9.81.62 ), whereupon the hormone and/or receptor with or without subsequent modification exert effects which lead to the interesting biologic change. These are difficult problems to approach experimentally. The use of antireceptor antibodies, now available for insulin63 and, perhaps, soon for EGF, might exclude ligand internalization if such antisera are found to promote DNA synthesis. In

g::-:g

=

‘25l-

I I

Figure

’

0

4

8 HOURS

12 AFTER

BOUND

GLUCAGON

Figure

( PICOMOLES

BOUND

(

/ MG

PROTEIN

I

PlCOhmES / MG PROTEIN 1

5. Scatchard plots of ‘2”1-insulin and ““I-glucagon binding. Experimental conditions were as described in legend to Figure 4. Membranes were isolated 24 hr postoperatively; protein concentrations were 40 ag/tube. Measurement errors were + 5-10%. See also reference 22.

GLUCAGON

6 4_

INSULIN

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4. ‘251-insulin and ‘251-glucagon binding to liver plasma membranes after 70% hepatectomy. Membrane fractions (20-40 ag protein) were incubated in a reaction volume of 100 ~1 with either ligand (see reference 57 for methodologic details). Nonspecific binding (radioactivity retained by filters from parallel assay tubes incubated together with 10 PM unlabeled hormone) was subtracted to obtain “specific binding”; zero-hour values equal to “100% of control” were 6 and 46 fmol/mg protein for insulin and glucagon, respectively. Each point represents the average of 2-3 filters from two membrane pools, each derived from 4 rats (i.e. n = 4-6 filters f SEM). Data were subjected to two-way analysis of variance: experimental points significantly differcnt (P < 0.05) from controls were observed at 6, 8. 12, and 24 hr (insulin) and at 4, 6, 8, 10, 12, 24, and 144 hr (glucagon) postoperatively.

this regard, a provocative observation of increased hepatic proliferation has been made in diabetic patients whose blood contains high levels of antiinsulin receptor antibody (R. Kahn, personal communication). In summary, the primary mechanisms of hormonal induction of regeneration probably involve direct actions of at least two, possibly three, peptides to generate persistently increased hepatic signals. The molecular mechanisms involved, with respect to insulin and EGF, are poorly understood. More is known about glucagon’s initial signalling effects at the cell surface,W but no direct evidence excludes its possible internalization from also playing a functional role. Secondary mechanisms for hormone action after partial hepatectomy include two broad possible catagories. First, hormones may cause hepatocytes to produce less of a proliferation inhibitor. Second, hormones may alter one or more cellular processes or structures necessary to form critical substances which initiate DNA synthesis and mitosis. Obviously, both mechanisms could operate simultaneous1y.2” Although circulating”” and tissue-extractable”” regeneration inhibitors have been reported, much of this evidence remains indirect, conflicting, and unconfirmed.67 However, in studies with fetal hepatocyte cultures, VLDL and its lipid components show properties expected for tissue-specific “chalones.““’

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June 1979

0

20

40

I=

40 60 0 0 20 60 MINUTES OF DISSOCIATION

20

Thus, VLDL blocks the G,, + S transition by inhibiting prereplicative protein synthesis.52 Moreover, VLDL production is reduced by glucagonea (this could be secondary to the diversion of lipid precursors into pools for new membrane synthesis). Very low density lipoprotein blood levels are reduced in a dose-dependent manner rapidly after partial hepatectomy48 and by a variety of different experimental conditions which stimulate hepatic proliferation in the intact rat.Z3.*5.48 Mutant “fatty” rats with circulating hyper-very low density lipoproteinemia show diminished hepatic DNA synthesis after 67% hepatectomy.52 And, cultured adult hepatocytes produce less VLDL-lipid during logarithmic and more during stationary phase.” Inter-

Table

4.

Equilibrium

Constants

for Insulin

and Glucagon

40

ARTICLE

Figure 6. Negative cooperative binding of ‘?-insulin to regenerating liver membranes. Binding conditions and membranes used were as described in legend to Figure 5. Bound complexes were centrifuged (10 min at 50,000 g) and resuspended without dilution into buffer at 4’C. One-hundred 1.11 aliquots were added to a series of tubes containing 10 ml buffer without (panel at extreme left) or with varying concentrations of unlabeled insulin. Dissocation of bound complexes was monitored at 23’C for 65 min using the standard assay. Each point was corrected for nonspecific binding by running a parallel dissociation experiment in the presence of 10e5M insulin. Measurement errors were f5-10%. Results with laparotomized and 70% hepatectomized liver membranes are given in the middle and extreme right panels, respectively.

60

estingly, primary adult hepatocyte cultures from lipotrope-deficient rats, whose growing liversz3 produce less VLDL,‘” show reduced proliferative requirements of hormones.” Further studies are required to determine whether endogenous VLDL components directly block one or more prereplicative protein-synthetic processes. Cellular processes necessary to initiate proliferation are poorly defined, despite considerable effort to identify them using in vivo models (reviewed in detail elsewhere).21.5” While prominent hepatic changes are associated with the induction of regeneration, for example, the stimulation of ornithine decarboxylase activity” or cyclic AMP formation,72 it is unclear whether these changes are necessary to induce

Interactions

with Liver Plasma Membrane

Fractions

at 30°C”

Site-Class Treatment

Hormone Insulin

Anesthesia

Laparotomy

70% Hepatectomy Glucagon

Anesthesia

Laparotomy

70% Hepatectomy

0 Data are calculated

from Scatchard

Constant K, (apparent) K, (apparent) Binding capacity K, Kd Binding capacity K, Binding capacity K, (apparent) Kd (apparent) Binding capacity K, Kd Binding capacity K, Kd Binding capacity

1477

High affinity-low

capacity

1.5 x log M-’ 6.5 x 10“” M 14.0 X lo-l4 moles/mg 2.1 x log M-’ 4.9 x lo-lo M 17.9 X lo-l4 moles/mg 2.1 x lo9 M-’ 27.4 X lo-l4 moIes/mg 4.8 x 108 M-’ 2.1 x 1O-9M 1.1 X lo-” moles/mg 3.9 x 108 M-’ 2.5 x 1O-9M 1.4 X lo-l2 moles/mg 4.3 x 10’ M-’ 2.3 X10-’ M 0.7 X lo-” moles/mg

plots shown in Figure 5. See also Reference

22.

Low affinity-high

capacity

4.0 x 10’ M-’ 2.5 X 1O-8M 44.0 X lObI4 moles/mg 4.9 x lo7 M-’ 2.0 x 10” M 56.0 X lo-l4 moIes/mg 3.8 x10’ M-’ 97.0 X lo-l4 moles/mg 3.8 X lo7 M-’ 2.6 x lo-” M 2.0 X lo-l2 moles/mg 2.7 X lo7 M-’ 3.7 x lo-’ M 2.7 X 10.” moIes/mg 3.1 x 10’ M-’ 3.2 x lo-’ M 1.8 X 10.” moles/mg

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proliferation.‘“,” Figure 3 illustrates three changes brought about by exposure to nutrient excess and insulin, glucagon and EGF, which are necessary to induce cultured adult hepatocyte proliferation. These include (a) altered monovalent cation fluxes; (b) altered RNA metabolism; and (c) altered protein metabolism (H. Leffert and K.S. Koch, manuscript in preparation). Monovalent cation fluxes, particularly sodium ion, may play critical roles in controlling hepatic proliferation and retrodifferentiation.“” The evidence from both in vivo and in vitro studies that initiation of liver regeneration depends upon increased Na’ ion influx, stems from findings that amiloride (which specifically blocks Na’ ion influx in a variety of tissues) also blocks regeneration.6” In vitro studies with both fetal and adult hepatocyte systems show that amiloride exerts fully reversible effects during but not after the prereplicative interval. The consequences of inhibition of Na’ ion influx are rapid inhibition of protein and later inhibition of RNA synthesis, both of which are necessary for DNA synthesis initiation. These results are strikingly similar to those reported for VLDL and VLDL-lipids. Further work is necessary to see if these and othe? similarities are more than fortuitous. The “earliest” Na’ ion-dependent events have not been identified. They may include phosphate uptake and amino acid transport7”,7” via the hormonally controlled A-system (this includes methionine, glutamine, alanine, and glycine).” How the synthesis of deoxynucleotide synthesizing proteins, DNA synthesizing proteins,77 and proteins necessary for producing a functional mitotic apparatus, are linked to these or to other Na’ ion-dependent changes is unknown. Many cellular processes are, however, linked to phosphate ion and to A-system transports. These may include altered phosphorylation’“~“; new rRNA,” mRNA,“’ and highly phosphorylated nucleotideZ”,22,A2,“3 synthesis; altered transcriptional processingH4; and decreased protein degradation.“” Direct evidence is needed to prove that one or more of Table

5.

Changes Ligation

are from

Because most extrahepatic tissues in a partially hepatectomized rat do not show altered proliferative rates, liver regeneration usually is considered “specific.” But to a certain extent this is not the case. For biliary epithelial, endothelial, and Kuppfer cells

Time after manipulation

for 2 min for 10 min reference

(p atoms/g

0 min 5 hr 0 min 5 min 5 hr 5 hr 5 hr

67% Hepatectomy

crease.

How Is the “Specificity” of Liver Regeneration Controlled?

in Liver Na’ and DNA Formation as a Function of Time After 6% Branch to the Left Lateral and Median Lobes”

Laparotomy

(1Data

these “secondary” processes are causally related to initiating proliferation. A finding made more than 14 yr ago,“’ summarized in Table 5, is strikingly provocative with regard to the proposed model.69 It was observed that caudate lobe Na’ levels increase about 40% above controls 5 min after 67% hepatectomy. Similar changes followed a transient lo-min ligation of the portal vessels supplying the left lateral and median liver lobes. Both procedures induced hepatic DNA synthesis measured in the caudate lobe 24 hr later. However, Na’ levels were not elevated by laparotomy nor by 2 min portal ligation. And neither manipulation induced DNA synthesis in the caudate liver lobes. Such findings were supported by later studies of metal ion levels in regenerating liver,” and are consistent with observations that amiloride inhibits liver regeneration. Perhaps, a 16min ligation is sufficient time to permit an insulin-like peptide to exert its initial effects. During or after this time, caudate liver cells would transiently receive elevated insulin and glucagon signals. Thus one would expect elevated DNA synthesis, albeit unequal to the amount observed after 67% hepatectomy. This was observed. Reports with fibroblast cultures indicate that EGF activates the ouabain-sensitive membrane Na+-K’ ATPase”” and that insulin inhibits Na’ efflux.” Preliminary studies in this laboratory using adult hepatocyte cultures show that “Na+ uptake is enhanced by insulin, glucagon, and EGF. The possible net hormonal effects, an early elevation of intracellular Na’ or modulation of the Na’ ion gradient, warrant further investigation in hepatocyte systems.

Hepatectomy

or Temporary

of the Portal

Manipulation

Ligation Ligation

Vol. 76. No. 6

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86. Values

are averages

+ SD from

Na+ wet liver))

DNA formation at 24 hr (caudate lobes)b

34 f 2 (6) 35 f 2 (3)

E

34 f 3 (11) 45 f 6 (8)

Gz

43 f 4 (8)

the numbers

33 + 1 (6)

:

44 f 1 (6)

0

of rats shown

in parentheses.

“0

= no increase:

@ = in-

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all proliferate after 67% hepatectomy but with delayed kinetics”” (hepatocytes begin to synthesize DNA some 48-96 hr earlier). Moreover, brief proliferative responses of cornea1 and testicular (see reference 50) as well as hemopoietic”’ and adrenal cortical”’ tissues have been reported in partially hepatectomized rats. How are these responses selectively regulated with respect to cell type and to time after the operation? Three possibilities may account for the initial hepatocyte response. First, a specific stimulatory factor may enter or accumulate in the bloodstream. There is yet no evidence that this occurs for known insulin-like peptides: on the contrary, as discussed above, both somatomedin-like activity and EGF seem to fall when measured one hour post-operatively. Even if earlier measurements reveal transient increases, which could be sufficient from in vitro evidence to promote DNA synthesis initiation, neither somatomedin-C nor EGF are hepatocyte-specific. Alternatively, their plasma levels may not actually change, but instead become “saturating” with respect to receptor load because of reduced hepatic mass. Consequently synergistic and “specific” stimulation might occur during the potentiating phase (Figure 3) secondary to hyperglucagonemia (Figure 2). This is reasonable because glucagon possesses some degree of hepatocyte specificity. This latter possibility would satisfy the classic observations of Leong et al. that heterotopic liver autografts proliferate after 67% hepatectomy with similar kinetics to the remnant lobe.“” Second, an hepatic “chalone” may disappear from the bloodstream. This could be brought about by liver tissue loss; or, hormone/nutrient interactions could inhibit hepatocytes from producing a specific inhibitor.“” We think that with the possible exception of VLDL, no convincing evidence has yet been obtained to support the “chalone” concept. Much of the inhibitor work, including our own, will acquire more validity when adult hepatocyte cultures are used to test directly the putative actions of the reported substancesB5~BB;their putative growthstate dependent production; and their molecular mechanisms of action.25.R2 Third, hepatocytes may respond, like a “combination” lock, to a pattern of blood hormone alterations. As mentioned above, it is unclear if such an endocrine pattern could cause a proliferative response. Nevertheless a striking correlation exists between increased hepatic proliferation, resulting from a variety of different perturbations and increased plasma glucagon, decreased insulin, appropriate com“receptor changes,” and decreased pensatory VLDL.”

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All three possibilities or their combinations may be operating. Finally, regenerating hepatocytes or other liver cells may produce substances which control proliferation of an extrahepatic or intrahepatic cell type. The former possibility is supported by observations that regenerating liver releases erythropoietin which accounts for a transient hemopoiesis seen during regeneration.” The latter possibility is supported by preliminary studies in this laboratory which suggest that cultured adult hepatocytes produce one or more substances in response to glucagon which inhibit nonhepatocyte DNA synthesis. If substantiated, this might account for the delayed in vivo proliferative response during liver regeneration of the nonparenchymal hepatocytes (which would be expected to be proliferatively blocked by small molecules like E-series prostaglandins or CAMP).” Conclusions

and Future Prospects

Experimental results from different laboratories have converged upon an endocrine theory of how rat liver regeneration is controlled. The strength of a theory, however intriguing it may be, is its ability to unify apparently unrelated findings and to permit testable predictions. We have attempted in this review to provide examples along these lines. Only further work can show whether numerous hepatoproliferogenic factors not discussed here, including chemical carcinogens,y4 viruses,‘” proteases,% and xenobiotic compounds? stimulate regeneration via the endocrine and ionic mechanisms revealed thus far. The endocrine theory clearly is far from perfection and it may be in for additional surprises. These may involve nonmicroenvironmental” neuronal mechanisms.z0,42.4” The most outstanding current problems appear to be the exact manner by which hormones regulate “specificity” and the formation of critical intracellular growth-regulating molecules. These molecules have not yet been identified in animal cells. Progress here may depend upon advances with simpler systems.99~‘00With the advent of physiologic proliferation-competent hepatocyte culture systems,2.55 the next few years should reveal if a truly unique hepatocyte-specific growth-factor exists. For there are many claims, but less clear-cut evidence, that such substances circulate in the bloodstream.“‘~““~“~““-‘“” Whether or not the results from animal studies can be extrapolated to humans also is unknown. The clinical implications of these problems are obvious. But, however desirable it would seem, it is by no means clear that management of human hepatocellular carcinoma or of other liver disease can be

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accomplished by manipulating hormones whose effects also are required to sustain vital, extrahepatic tissue function. Generalized claims to the contrary should be interpreted with caution. Becker once stated that only the liver “knew” the secret to regeneration.lM In due respect to the tissue whose regenerative properties have attracted so many investigators’ efforts for so long, it would now seem that the “secret” is out.

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17. Castellano TJ, Schiffman RL, Jacob MC, Loeb JN: Suppression of liver cell proliferation by glucocorticoid hormone: a comparison of normally growing and regenerating tissue in the immature rat. Endocrinology 102:1107-1112, 1978 18. Short J, Armstrong NB, Zemel R, et al: Amino acids and the control of nuclear DNA replication in liver. In: Control of Proliferation in Animal Cells, Vol. I. Edited by B Clarkson, R Baserga. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory, 1974, p 37-48 fetal rat hepato19. Leffert, HL: Growth control of differentiated

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93. Leong GF, Grisham JW, Hole BV, Albright ML: Effect of partial hepatectomy on DNA synthesis and mitosis in heterotopic partial autografts of rat liver. Cancer Res 24:1495-1501, 1964 94. Epstein S, Nobuyuki I, Merkow L, Farber E: Cellular analysis of liver carcinogenesis: the induction of large hyperplastic nodules in the liver with 2-fluorenylacetamide or ethionine and some aspects of their morphology and glycogen metabolism. Cancer Res 27:1702-1711, 1967 95. Farivar M, Wands JR, Isseibacher KJ, Bucher NLR: Effect of insulin and glucagon in fulmainant murine hepatitis. N Engl J Med 295:1517-1519, 1976 96. Yamamoto K, Omata S, Ohnishi T, Terayama H: In vivo effects of proteases on cell surface architecture and cell proliferation in the liver. Cancer Res 33:587-572, 1973 97. Schulte-Hermann R: Induction of liver growth by xenobiotic compounds and other stimuli. CRC Crit Rev Tox 3:97-158, 1974 98. Rabes HM, Tuczek HV, Wirsching R: Kinetics of hepatocellular proliferation after partial hepatectomy as a function of structural and biochemical heterogeneity of the rat liver. In: Liver Regeneration After Experimental Injury. Edited by R Lesch, W Reutter. New York, Grune & Stratton, 1975, p 3355 99. Hartwell LH: Saccharomyces cerevisiae cell cycle. Bacterial Rev 38:164-198, 1974 106. Wickner SH: DNA replication proteins of Escherichio cob. Ann Rev Biochem 47:1163-1191, 1978 101. LaBrecque DR, Pesch LA: Preparation and partial characterization of hepatic regenerative stimulator substances (SS) from rat liver. J Physiol 248:273-284, 1975 102. Morley CGD, Boyer JL: Stimulation of hepatocellular proliferation by a serum factor from thioacetamide-treated rats. Biochem Biophys Acta 477:165-176, 1977 103. Preuss HG, Parris R, Goldin H: Effects of sera and liver extracts from partially hepatectomized rats on liver slice DNA synthesis. Experientia 33/5:630-631, 1977 104. Becker FF: Humoral aspects of liver regeneration. In: Humoral Control of Growth and Differentiation. Vol. I. Edited by J LoBue, AS Gordon. New York, Academic Press, Inc., 1973, p 249-256