Nancy L. R. Bucher

Regeneration of Mammalian Liver

Publisher Summary This chapter focuses on the regeneration of mammalian liver. Regeneration is commonly employed to describe the compensatory hypertrophy and hyperplasia that occur in liver in response to damage or loss of tissue. The chapter reviews experimental studies on the homeostatic control of hepatic growth, as exemplified primarily by regeneration in response to partial hepatectomy, with particular attention to the first cycle of cellular growth and division. Modifications of the regenerative response have been examined with particular attention to the control of variables. In addition to the usual environmental, dietary, genetic, and endocrine factors that may affect certain aspects of the process, attention is called to the fact that the times at which mitosis, and DNA synthesis reach their peaks vary significantly with the age of the animal. The degree of liver deficiency determines the extent of response above a certain threshold and the latter is much lower in young, rapidly growing rats. In a similar vein, techniques of evaluating the regenerative response are discussed with emphasis on the sources of error and drawbacks inherent in each. DNA synthesis, which is prerequisite to mitosis and of central importance in cellular proliferation, is discussed with emphasis on the interplay of various component reactions and points at which the process might be subject to regulation.

Induction of peroxisome proliferator-activated receptor gamma during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPbeta, C/EBPdelta, and glucocorticoids.

The differentiation of 3T3 preadipocytes into adipocytes is accompanied by a transient induction of C/EBPbeta and C/EBPdelta expression in response to treatment of the cells with methylisobutylxanthine (MIX) and dexamethasone (DEX), respectively. In this report, we demonstrate that peroxisome proliferator-activated receptor gamma (PPARgamma) expression in 3T3-L1 preadipocytes is induced by MIX and DEX, suggesting that C/EBPbeta and C/EBPdelta may be involved in this process. Using a tetracycline-responsive expression system, we have recently shown that the conditional ectopic expression of C/EBPbeta in NIH 3T3 fibroblasts (beta2 cells) in the presence of DEX activates the synthesis of peroxisome PPARgamma mRNA. Subsequent exposure of these cells to PPAR activators stimulates their conversion into adipocytes; however, neither the expression of C/EBPbeta nor exposure to DEX alone is capable of inducing PPARgamma expression in the beta2 cell line. We find that unlike the case for 3T3 preadipocytes, C/EBPdelta is not induced by DEX in these 3T3 fibroblasts and therefore is not relaying the effect of this glucocorticoid to the PPARgamma gene. To define the role of glucocorticoids in regulating PPARgamma expression and the possible involvement of C/EBPdelta, we have established an additional set of NIH 3T3 cell lines expressing either C/EBPdelta alone (delta23 cells) or C/EBPdelta and C/EBPbeta together (beta/delta39 cells), using the tetracycline-responsive system. Culture of these cells in tetracycline-deficient medium containing DEX, MIX, insulin, and fetal bovine serum shows that the beta/delta39 cells express PPARgamma and aP2 mRNAs at levels that are almost equivalent to those observed in fully differentiated 3T3-L1 adipocytes. These levels are approximately threefold higher than their levels of expression in the beta2 cells. Despite the fact that these beta/delta39 cells produce abundant amounts of C/EBPbeta and C/EBPdelta (in the absence of tetracycline), they still require glucocorticoids to attain maximum expression of PPARgamma mRNA. Furthermore, the induction of PPARgamma mRNA by exposure of these cells to DEX occurs in the absence of ongoing protein synthesis. The delta23 cells, on the other hand, are not capable of activating PPARgamma gene expression when exposed to the same adipogenic inducers. Finally, attenuation of ectopic C/EBPbeta production at various stages during the differentiation process results in a concomitant inhibition of PPARgamma and the adipogenic program. These data strongly suggest that the induction of PPARgamma gene expression in multipotential mesenchymal stem cells (NIH 3T3 fibroblasts) is dependent on elevated levels of C/EBPbeta throughout the differentiation process, as well as an initial exposure to glucocorticoids. C/EBPdelta may function by synergizing with C/EBPbeta to enhance the level of PPARgamma expression.

Regeneration of rat liver: transfer of humoral agent by cross circulation.

Carotid-to-jugular cross circulation between partially hepatectomized and normal rats, via polyethylene cannulas, stimulated incorporation of 14C-thymidine into hepatic DNA in the normal partners when it was maintained for 19 hours at a flow rate of about 2 milliliters per minute. Cross circulation for 7 hours or less was ineffective.

β-hydroxy-β-methylglutaryl coenzyme a reductase, cleavage and condensing enzymes in relation to cholesterol formation in rat liver

Abstract Investigation of the intracellular localization of the principal enzymes involved in the formation and breakdown of β-hydroxy-β-methyl-glutaryl CoA (HMG CoA) in rat liver has shown that HMG CoA condensing and cleavage enzymes are both preponderantly in the mitochondria. HMG CoA reductase, which leads to the pathway of cholesterol synthesis, is in the microsomes, and is only one twentieth as active as the cleavage enzyme which leads to acetoacetate production. In spite of this unfavorable ratio, cholesterol synthesis does occur—possibly because a small amount of condensing enzyme and most or all of the reductase are in the microsomes which are low in cleavage activity. Stimulation of cholesterol synthesis by injection of Triton, or inhibition by fasting does not importantly alter the amount or distribution of the condensing and cleavage enzymes. However, a severe depression of reductase activity in fasting suggests that this may be a significant factor in the rate limitation of cholesterol synthesis in this condition.

Cell-extracellular matrix interactions can regulate the switch between growth and differentiation in rat hepatocytes: reciprocal expression of C/EBP alpha and immediate-early growth response transcription factors.

Previous investigations have shown that culture of freshly isolated hepatocytes under conventional conditions, i.e., on dried rat tail collagen in the presence of growth factors, facilitates cell growth but also causes an extensive down-regulation of most liver-specific functions. This dedifferentiation process can be prevented if the cells are cultured on a reconstituted basement membrane gel matrix derived from the Englebreth-Holm-Swarm mouse sarcoma tumor (EHS gel). To gain insight into the mechanisms regulating this response to extracellular matrix, we are analyzing the activities of two families of transcription factors, C/EBP and AP-1, which control the transcription of hepatic and growth-responsive genes, respectively. We demonstrate that isolation of hepatocytes from the normal quiescent rat liver by collagenase perfusion activates the immediate-early growth response program, as indicated by increased expression of c-jun, junB, c-fos, and c-myc mRNAs. Adhesion of these activated cells to dried rat tail collagen augments the elevated levels of these mRNAs for the initial 1 to 2 h postplating; junB and c-myc mRNA levels then drop steeply, with junB returning to normal quiescence and the c-myc level remaining slightly elevated during the 3-day culture period. Levels of c-jun mRNA and AP-1 DNA binding activity, however, remain elevated from the outset, while C/EBP alpha mRNA expression is down-regulated, resulting in a decrease in the steady-state levels of the 42- and 30-kDa C/EBP alpha polypeptides and C/EBP alpha DNA binding activity. In contrast, C/EBP beta mRNA production remains at near-normal hepatic levels for 5 to 8 days of culture, although its DNA binding activity decreases severalfold during this time. Adhesion of hepatocytes to the EHS gel for the same period of time dramatically alters this program: it arrests growth and inhibits AP-1 DNA binding activity and the expression of c-jun, junB, and c-myc mRNAs, but, in addition, it restores C/EBP alpha mRNA and protein as well as C/EBP alpha and C/EBP beta DNA binding activities to the abundant levels present in freshly isolated hepatocytes. These changes are not due merely to growth inhibition, because suppression of hepatocyte proliferation on collagen by epidermal growth factor starvation or addition of transforming growth factor beta does not inhibit AP-1 activity or restore C/EBP alpha DNA binding activity to normal hepatic levels. These data suggest that expression of the normal hepatic phenotype requires that hepatocytes exist in a G0 state of growth arrest, facilitated here by adhesion of cells to the EHS gel, in order to express high levels of hepatic transcription factors such as C/EBP alpha.

Growth-dependent inhibition of CCAAT enhancer-binding protein (C/EBP alpha) gene expression during hepatocyte proliferation in the regenerating liver and in culture.

As an approach to understanding physiological mechanisms that control the proliferation of highly differentiated cells, we are addressing whether certain hepatic transcription factors participate in mechanisms that control the growth of hepatocytes. We have focused on CCAAT enhancer-binding protein (C/EBP alpha), a transcription factor which is highly abundant in normal liver and is considered to regulate expression of many genes, including some involved in energy metabolism (S. L. McKnight, M. D. Lane, and S. Gluecksohn-Walsh. Genes Dev. 3:2021-2024, 1989). Using Northern (RNA) blot analysis, we have examined the expression of C/EBP alpha mRNA during liver regeneration and in primary cultures of hepatocytes. C/EBP alpha mRNA levels decrease 60 to 80% within 1 to 3 h after partial hepatectomy as the cells move from G0 to G1 and decrease further when cells progress into S phase. Run-on transcription analysis is in agreement with the Northern blot data, thus suggesting that C/EBP alpha is transcriptionally regulated in regenerating liver. C/EBP alpha mRNA expression also decreases dramatically during the growth of freshly isolated normal hepatocytes cultured under conventional conditions (on dried rat tail collagen; stimulated to proliferate by epidermal growth factor [EGF] and insulin). Cultures of hepatocytes on rat tail collagen in the presence or absence of EGF clearly show that within 3 h, EGF depresses C/EBP alpha mRNA expression and that this effect is substantially greater by 4 h. Inhibition of protein synthesis in the liver by cycloheximide or in cultured hepatocytes by puromycin or cycloheximide effectively blocks the down-regulation of C/EBP alpha gene expression, apparently by stabilizing the normal rapid turnover of the C/EBP alpha mRNA (half-life of <2 h). This drop in C/EBP alpha gene expression in response to activation of hepatocyte growth is consistent with the proposal that C/EBP alpha has an antiproliferative role to play in highly differentiated cells (R. M. Umek, A. D. Friedman, and S. L. McKnight, Science 251: 288-292, 1991).

Stimulation of DNA synthesis in primary cultures of adult rat hepatocytes by rat platelet-associated substance(s)

SummaryExperiments in whole animals have shown that normally quiescent adult rat hepatocytes are induced to proliferate by blood borne substances, which we are now probing in primary monolayer cultures. Under our conditions, freshly isolated adult hepatocytes do not proliferate actively in a defined medium, but are stimulated to synthesize DNA — an essential first step — by either serum or an EGF-hormone combination.Stimulation of [3H]thymidine incorporation into hepatocyte DNA by addition of dialyzed mouse, human, horse, or bovine (fetal, newborn, or calf) serum, whose activities are all similar, is regularly surpassed by an EGF-insulin mixture without serum. This, in turn, is exceeded by dialyzed normal rat serum, which is several times more potent than the other sera tested.Removal of blood platelets reduces the activity of normal rat serum by over 50%. Heat inactivation (56° C) causes a similar loss, but heat treatment of platelet-poor serum fails to cause further reduction. The activity of mouse and human serum is not reduced by platelet removal.Serum from partially hepatectomized rats is not significantly more stimulatory than normal rat serum, and its activity is depressed in the same way by platelet deprivation and heat inactivation. Lack of enhancement by partial hepatectomy is not consonant with whole animal studies and requires further investigation.The heat-labile portion of the DNA synthesis-stimulating activity of rat serum appears to derive from platelets. This activity differs from the well-characterized heat-stable human PDGF. Its relation to other reported platelet-associated growth factors is still undetermined.

Ornithine decarboxylase activity in relation to growth of rat liver: Effects of partial hepatectomy, hypertonic infusions, celite injection or other stressful procedures

Abstract The specificity of association between ornithine decarboxylase activity and growth of rat liver as indicated by incorporation of labeled thymidine into DNA was studied in animals subjected to a variety of experimental conditions. 1. 2. Major elevations of the enzyme (25–70 times normal) were produced by partial hepatectomies ranging from 9 to 77%, cross circulation of a normal with a 77%-hepatectomized rat, large infusions of hypertonic glucose or mannitol, or intraperitoneal injection of Celite. 2. 3. Minor elevations (2–9 times normal) resulted from fasting and refeeding, fasting and sham hepatectomy, cross circulation of normal-to-normal or normal-to-Celite-injected partners, smaller hypertonic infusions, injection of albumin, epinephrine or norepinephrine, or confinement in restraining cages. 3. 4. While enhanced ornithine decarboxylase activity was an early feature of liver growth in these experiments, stimuli not inducing growth also caused the enzyme to increase; furthermore, the extent of enzyme augmentation was not necessarily proportional to the rate of growth when it did occur. 4. 5. Although putrescine formation seems not to be directly linked to DNA replication, it may be involved with some earlier step in the growth process, perhaps enhancement of RNA synthesis or function.

Liver regeneration: An overview

Nearly 60 years have elapsed since Higgens and Anderson published their classic procedure for partial hepatectomy in the rat,’ thereby opening the way for quantitative, reproducible studies of normal growth control. A veritable cascade of research has ensued, using this model system to address the problem of how an outburst of active proliferation is brought about and regulated in an organ that is normally quiescent. What have we learned during the interim? It is generally accepted that the liver grows in response to the workload imposed by the needs of the body. The problem is how and under what conditions a metabolic load is translated into proliferative activity, and why, as the needs are met, the growth subsides. What are the mechanisms that co-ordinate and control liver growth? Years of experimentation in whole animals have pinpointed the salient features of the growth process. The hepatocytes in the adult liver are essentially non-cycling cells, arrested in the so-called Go state. They are induced to re-engage in the cell cycle by liver loss due to excision or injury from infections, toxins or traumas. They also grow in response to hormonal or metabolic imbalances or to the poorly understood effects of certain drugs. The proliferative rate is proportional to the workload; if the need is small, the liver grows slowly despite its high growth potential. Following two-thirds hepatectomy, which is the most potent stimulus yet devised, growth associated events start almost instantly as the cells undergo the transition from Go to the pre-replicative (GI) phase, which lasts for 12-14 h, at which point DNA synthesis (S phase) begins, peaking at 22-24 h. Mitosis follows a similar course 6-8 h later. This initial growth wave involves only hepatocytes, non-parenchymal cells peaking nearly 1 day later. The growth occurs throughout the liver remnant, preserving the histological architecture. It diminishes and finally ceases in a week or so when the original cell complement is restored. The growth potential is retained and the liver remnant can be induced to undergo repeated episodes of regeneration. (This work has been repeatedly and extensively r e~ iewed .~ -~) Major attention has focused on the signals from the body that control the regenerative process. The signals consist of molecules that circulate in the blood, primarily hormones and growth factors. The responsiveness of the cells to these signals is strongly influenced by their prior metabolic state; indeed, the same signal may act positively or negatively, depending on It is important to note that all of the hepatic signalling molecules so far proposed are mitogenic for various types of cells, whereas in the case of a hepatic deficit the signals act only on the liver; other organs do not grow. The signals are multiple and a second important point is that they interact synergisti~ally,~~~ so that under appropriate conditions a seemingly weak signal, which may have little effect by itself, may substantially augment other effectors.” The multiple functions of the liver may well call for multiple signals and, moreover, a synergistic interaction among signals may constitute the key to specificity of the signalling mechanism. The signals may prime or initiate, inhibit, modulate or promote passage of liver cells through the cell cycle. They probably do all of these. Hepatic growth control may well rest on a balanced interplay of positive and negative All these features of the growth process and the signalling mechanism and their ramifications must be reckoned with to gain a full understanding of the physiology of liver In this review, the term ‘hepatocyte’ signifies hepatic parenchymal cells, and ‘hepatocyte cultures’ refers to primary cultures of adult rat hepatocytes.

Nucleotide pools and [6-14C]orotic acid incorporation in early regenerating rat liver

Abstract 1. 1. Investigation of nucleotide pools was undertaken as part of a continuing study of changes relevant to nucleic acid synthesis in regenerating rat liver that may occur during the early hours after partial hepatectomy. 2. 2. It has been reported that the ATP/ADP ratio is a useful index of alterations in labile metabolites resulting from unphysiological conditions during tissue sampling. When ether anesthesia was supplemented with 100 % oxygen and liver samples were rapidly frozen in situ , we obtained ATP/ADP ratios that compared favorably with reported values. 3. 3. Under the above conditions, the incorporation of [6- 14 C]orotic acid into uridine phosphates in vivo was determined in 45-min regenerating, normal, and sham hepatectomized control livers of 32–35-day-old female rats fasting 12 h. 4. 4. The 45-min regenerating livers did not differ appreciably from controls in size of ATP or UTP pools, and 70–80 % of both adenosine and uridine phosphates were present in the triphosphate form. 5. 5. The 45-min regenerating livers did differ from controls in the pattern of labeling of uridine phosphates and UDP-sugars. At 2–4 min after injection labeling was similar in all groups, but by 8–16 min activity levels were falling in the controls and still rising in the regenerating livers. The similarity in initial rates implied that these uridine compounds were synthesized at about the same rates in 45-min regenerating and control livers. The divergence at 8–16 min suggested more rapid depletion of a UMP precursor (possibly [ 14 C]orotic acid itself) in the control animals. 6. 6. The specific activity of UTP was higher than UDP, which was higher than UMP. Compartmentation of cellular pools of UMP, UDP (and possibly also UTP) seems the most likely explanation. 7. 7. The results underline the importance of knowledge of the precursor pools for evaluation of biosynthetic processes.