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Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases (P.A.P., D.L., S.Y.), and Laboratory of Experimental Carcinogenesis, Center of Cancer Research, National Cancer Institute (S.T.), National Institutes of Health, Bethesda, Maryland 20892-1758; and Edison Biotechnology Institute, Ohio University College of Osteopathic Medicine, Ohio University (J.J.K.), Athens, Ohio 45701
Address all correspondence and requests for reprints to: Dr. Shoshana Yakar, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Room 8D12, Building 10, National Institutes of Health, Bethesda, Maryland 20892-1758. E-mail: shoshanay{at}intra.niddk.nih.gov.
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Abstract |
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Introduction |
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Liver regeneration may be initiated by the activation of one or more cytokine receptors, whereas growth factors usually act later on the already primed hepatocytes (4, 5). TNF
and IL-6 are thought to play major roles in initiating the process of liver regeneration. Mice lacking TNF receptor type 1 (TNFR-1) exhibited severely impaired liver regeneration after partial hepatectomy (6). This was found to be due to a defect in DNA synthesis and could be corrected by treatment with IL-6 (6). Similarly, IL-6 knockout mice showed impaired liver regeneration characterized by liver necrosis, liver failure, and a blunted DNA synthetic response in hepatocytes (7). Hepatocyte growth factor and TGF are potent hepatic mitogens in vitro that are highly expressed after hepatectomy (4, 8, 9). In addition, epidermal growth factor (EGF) is a primary mitogen for hepatocytes in culture, and its expression increases after liver resection (5, 10). After partial hepatectomy, the activation of cytokine and growth factor signaling pathways leads to the induction of transcription factor complexes such as nuclear factor-
B, c-Myc, signal transducer and activator of transcription-3 (STAT3), cAMP-responsive element modulator, and activating protein-1 (1, 4, 5, 6, 7, 8, 9). Mice with conditional knockouts of either STAT3 (11) or c-jun (12), specifically in the liver, exhibited impaired DNA synthesis, but liver regeneration still occurred.
GH is a member of the cytokine superfamily of polypeptide regulators (13). The growth-promoting effects of GH can be direct in selected target tissues, such as liver, or indirectly, via its endocrine mediator IGF-I. GH is the primary regulator of IGF-I synthesis and secretion in hepatocytes; in turn, IGF-I regulates GH secretion through a classical negative feedback loop (14, 15). In the circulation, IGF-I is bound to specific IGF-binding proteins (IGFBPs). Approximately 70–80% of the circulating IGF-I is found within a large ternary complex composed of the acid-labile subunit (ALS) and IGFBP-3 (16). A smaller proportion (15–20%) circulates as a binary complex with other IGFBPs, and less than 5% of IGF-I circulates in the free form (16, 17).
Despite the fact that the liver is the main source of circulating IGF-I and IGFBPs, hepatocytes have not been considered to be a primary target for IGF-I, because they do not express the IGF-I receptor (18). However, nonparenchymal cells within the liver, such as Kupffer cells and hepatic stellate cells, do express the IGF-I receptor and respond to IGF-I stimulation (19, 20), suggesting that IGF-I can act in a paracrine fashion in the liver. Interestingly, IGFBP-1 was shown to be one of the most rapidly induced and highly expressed proteins in regenerating liver (21). Mice that lacked IGFBP-1 exhibited normal development, but showed abnormal liver regeneration after partial hepatectomy, characterized by liver necrosis and reduced and delayed hepatocyte DNA synthesis (22, 23).
Recent evidence shows that GH can regulate EGF receptor (EGFR) expression in the liver as well as suppressors of cytokine signaling (SOCS) and glycoprotein 130 gene expression (24). It has also been shown that GH can stimulate tyrosine phosphorylation of the EGFR in cultured hepatocytes (25, 26, 27), raising the possibility that cross-talk between the GH receptor (GHR) and EGFR could also be important in liver regeneration.
The aim of our study was to investigate the role of GH (in the presence of reduced IGF-I levels) in liver regeneration. To that end we used three different mice models. The first group included liver IGF-I-deficient (LID) mice that also lack the ALS (LID+ALSKO mice). The LID+ALSKO mice have very low levels of circulating IGF-I and markedly elevated GH levels, which correlate with increased liver weight (28). The second group included GH antagonist transgenic mice (GHa mice), which have low levels of circulating IGF-I and no detectable GH activity (29). Due to their low levels of GH activity, these mice show growth retardation and reduced liver weight (30). The third group, control mice, had normal circulating GH and IGF-I levels.
Here we demonstrate that GHa mice, which lack GH activity, show a retarded response to hepatectomy and are not able to restore liver mass even 7 d after partial hepatectomy. Taken together, our results suggest that GH plays a major role in the process of liver regeneration.
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Materials and Methods |
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Partial hepatectomy
Male mice (6–8 wk old) were anesthetized with an ip injection of sodium phenobarbital (40 mg/kg). The abdominal area of the mice was shaved, and incisions of 2 cm were made. A partial hepatectomy was performed, in which approximately 70% or 30% of the liver was removed. The left lateral and median lobes of the liver were ligated before removal to prevent bleeding, as described previously (32). These lobes were removed intact, without damaging the remaining lobes. The remaining lobes were gently returned to the abdominal cavity. The incision was closed in two layers. The muscle layer was closed with catgut, and the skin was closed with stainless steel wound clips. The wound clips were removed 7 d later. The body weights of the mice were determined before and after surgery and during the follow-up period. Surgeries were performed in all mice at the same time of the day (during the morning hours). Mice were killed at the indicated time points, and livers were removed for histological analysis and isolation of RNA and protein preparations. Surgically removed liver lobes and the regenerating livers were weighed, and the relative percentage of body weight and the percentage of the corresponding original liver mass were calculated. To obtain dry weight data, three small pieces taken randomly from the removed liver lobes or regenerating livers were weighed and exhaustively lyophilized. Dried specimens were weighed again, and the dry/wet weight ratio was calculated.
Solution hybridization/ribonuclease (RNase) protection assay (RPA)
Livers were homogenized with a Polytron homogenizer (Brinkmann, Westbury, NY) in TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA), and total RNA was isolated according to the manufacturer’s instructions. Samples containing 50 µg total RNA were hybridized to 32P-labeled riboprobes corresponding to exon 4 of the IGF-I gene (pMI4 probe), exon 4 of the mouse GHR gene, exon 3 of the mouse IGF-I receptor (IGF-IR) gene, and ß-actin (Ambion, Inc., Austin, TX) overnight at 45 C. Protected fragments were separated on a 6% Tris-borate and EDTA/urea polyacrylamide gel (Invitrogen Life Technologies) and exposed to Kodak X-OMAT MS film (Eastman Kodak Co., Rochester, NY) overnight. The protected bands corresponding to the IGF-I mRNA, GHR mRNA, and ß-actin were analyzed with a Fuji phosphorimager (BAS-1800 II, Fujifilm, Tokyo, Japan). Levels of IGF-I and GHR mRNA were normalized to the amounts of ß-actin mRNA to correct for any differences in sample loading.
5-Bromo-2'-deoxyuridine (BrdU) incorporation and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL) assay
Mice were injected with a solution of BrdU (Amersham Biosciences, Piscataway, NJ; 100 mg/kg body weight) and were killed 2 h later. The remaining livers and portions of the gut were prepared and fixed in 4% paraformaldehyde for 14–16 h. For detection of incorporated BrdU, 5-µm sections from liver and gut (as an internal control), were processed according to the manufacturer’s instructions (Amersham Biosciences). On adjacent sections from the same liver samples, apoptotic cells were detected by the TUNEL assay with the ApopTag Plus peroxidase kit, according to the manufacturer’s instructions (Intergen, Purchase, NY). Mammary gland at an involutionary stage was used as an internal positive control. Hepatocytes were counted (between 1500 and 2000) from three different animals per group at each time point, and the average percentages of TUNEL-positive cells were calculated.
Statistical analysis
The experimental data were analyzed by t test, 
2 test, and one-way ANOVA with Tukey’s test for additional comparisons when appropriate. P < 0.05 was considered statistically significant. Results are expressed as the mean ± SEM, except when indicated.
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Results |
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70% of the liver) were performed in control, LID+ALSKO, and GHa male mice. GHa mice, which have low levels of circulating IGF-I and blunted GH activity, showed a dramatic increase in mortality after partial hepatectomy compared with control mice (P < 0.05, GHa vs. control; Table 1
). In contrast, the mortality of LID+ALSKO mice, which exhibit low levels of circulating IGF-I, but increased levels of GH, did not differ significantly from that of control mice [P = not significant (NS), LID+ALSKO vs. control; Table 1]. To rule out the possibility that the general stress of the surgery contributed to the increased mortality, we performed partial hepatectomies in which only approximately 30% of the liver mass was removed in control and GHa mice. All mice that underwent removal of only 30% of the liver mass were able to recover completely during the first 12 h after surgery, demonstrating that neither the surgical procedure nor the anesthetic is the cause of premature death. Accordingly, the number of mice that died during surgery was the same in the groups, suggesting that there were no differences in the response to anesthesia between the groups.
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), with only 60% of the mice surviving until 72 h after the surgery. A much smaller percentage of LID+ALSKO mice died during the first 48 h, and no control mice died after surgery. These results suggest that GHa mice were less able to overcome the loss of liver mass after partial hepatectomy.
Defective liver regeneration in the absence of GH activity
We next determined whether the reason for the premature death of GHa mice was the lack of their ability to regenerate liver mass. To do this, mice were killed at different time points after partial hepatectomy over a period of 7–10 d, and total body weight was measured before death. At death, the remaining liver lobes were harvested, and wet and dry weights were measured. The dry/wet weight ratios were calculated (see Materials and Methods) before and after surgery. There were no significant differences in the ratios between the groups before or after hepatectomy [0.263 ± 0.039 (control) vs. 0.263 ± 0.056 (LID+ALSKO), P = NS; 0.263 ± 0.039 (control) vs. 0.267 ± 0.025 (GHa), P = NS; 0.263 ± 0.039 (control) vs. 0.276 ± 0.035 (C after surgery), P = NS; 0.267 ± 0.025 (GHa) vs. 0.227 ± 0.040 (GHa after surgery), P = NS]. Thus, wet weights of lobes or regenerating livers were used for additional calculations. Because the three groups of animals started with different absolute and relative liver masses (28, 31, 33), the mass of the liver regenerated during the study was calculated as a percentage of the average total liver mass of the nonoperated mice. As shown in Fig. 1A, control mice completely regenerated their original liver mass (5.0% of total body weight) after 4 d. LID+ALSKO mice were able to restore their original liver mass after 7 d (6.1% of total body weight). In contrast, GHa mice recovered only 70% of their original liver mass (4.6% of total body weight) after 4 d, and the original liver mass was not reached until the end of the study (P < 0.05, control vs. GHa). Control and GHa mice did not exhibit any significant changes in total body weight over a course of 7 d (Fig. 1B). However, LID+ALSKO mice showed an initial decrease in body weight (24 h after hepatectomy) [P < 0.05, by one-way ANOVA; highest significant difference, 1.43, by Tukey’s test), with no further reductions during the study.
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, all three groups of mice exhibited a significant decrease in GHR and IGF-I mRNA levels during the first 24 h after partial hepatectomy compared with basal levels (P < 0.05, basal vs. d 1 control, GHa, and LID+ALSKO for GHR mRNA). In control and LID+ALSKO mice, GHR levels returned to basal expression levels 4 d after surgery, whereas GHa mice required 7 d to recover the original levels of GHR expression.
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) was observed 1 d after partial hepatectomy in control mice (P < 0.05, basal vs. d 1 in control and GHa), and their IGF-I expression levels returned to normal 4 d after surgery. In contrast, GHa mice, which express low levels of IGF-I due to blunted GH activity (40–50% of control), did not regain their original levels of IGF-I mRNA at any time point during the follow-up period after partial hepatectomy. LID+ALSKO mice lack IGF-I expression in the liver, because exon 4 of the IGF-I gene is specifically deleted in the livers of these mice.
IGF-IR mRNA was not detectable in the livers of any of the mice before surgery or after partial hepatectomy at any time during the study (Fig. 2A).
Necrosis without apoptosis in livers of GHa mice after partial hepatectomy
Normal liver architecture was observed in control, LID+ALSKO, and GHa mice in the basal state (data not shown). One day after surgery, little or no necrotic areas were observed in control (Fig. 3A, left panel) or LID+ALSKO (middle panel) mice. However, necrotic areas were apparent in the livers of most of the GHa mice (right panel, areas indicated by arrows) 24 h after surgery. On d 4, no necrosis was detected in the livers of control, LID+ALSKO, or GHa mice (Fig. 3B, left, middle, and right panels, respectively).
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), suggesting that the lack of regeneration in the liver of GHa mice was not caused by increased apoptosis.
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, left and middle panels, respectively). In contrast, there was dramatically less BrdU incorporation in GHa mice. Furthermore, maximal levels of BrdU incorporation in GHa mice (at 48 h) were 15% and significantly lower (P < 0.03 vs. control) than the maximal levels of BrdU incorporation observed in control mice (66%, at 36 h; P < 005 vs. basal; Fig. 4A, right panel). Portions of the gut from each animal were processed as internal controls for the entire procedure. The quantitation and kinetics of BrdU incorporation are shown in Fig. 4B. Although maximal BrdU incorporation was delayed in LID+ALSKO mice compared with control mice, this maximum was followed by a sharp decrease, and both groups exhibited similar low levels of BrdU incorporation by 72 h. Like LID+ALSKO mice, GHa mice exhibited a maximum incorporation at 48 h. However, in GHa mice this low level of incorporation did not decrease, and was sustained at least until 72 h post surgery. On d 4 after partial hepatectomy, there was no detectable BrdU incorporation in control or LID+ALSKO mice (data not shown). In contrast, a small, but significant, level of BrdU staining was observed in GHa mice even 7 d after surgery. Calculation of the area under the curve (see inset in Fig. 4B) was similar in control and LID+ALSKO mice [13.8 (control) and 13.6 (LID+ALSKO)], but was reduced approximately 25% in GHa mice [13.8 (control) vs. 10.5 (GHa)]. Thus, a low level of sustained cellular proliferation occurs in GHa mice at a time when neither control nor LID+ALSKO mice exhibit such activity, presumably because these mice have completed the process of liver regeneration. Taken together, the BrdU experiment indicates a prolonged G0/G1 or G1/S transition in the LID+ALSKO mice and essentially very low levels of DNA synthesis in GHa mice. |
Discussion |
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As the liver is a vital organ, the inability to regenerate the liver mass would be expected to prevent recovery after surgery. Therefore, it was important to evaluate the mortality levels of the mice that underwent partial hepatectomy. During the first 48 h after surgery, 100% of control mice and 88% of LID+ALSKO mice survived. In contrast, 57% of the GHa mice died during the first 48 h after surgery. A similar temporal pattern and a high percentage of mortality (50%) were reported in at least three other knockout animal models associated with impaired liver regeneration: IL-6–/– mice (7), TNFR-1–/– mice (6), and mice with a conditional liver-specific knockout of c-jun (c-jun 
Li mice) (12). Remarkably, IL-6, TNF-, and c-jun have all been identified as key regulatory factors in the proliferation and survival of hepatocytes during liver regeneration. TNFR-1–/– mice exhibited some distortion of the liver architecture and a decreased ratio of liver to body weight that persisted even 14 d after surgery (6). Both IL-6–/– mice and c-jun Li mice showed large areas of necrosis 36–72 h after surgery, but in both cases the mice that survived reversed those lesions (7, 12). In the current study necrotic foci were observed only in GHa mice 24 h after surgery. These lesions were not seen at later time points; thus, it is possible that although liver regeneration is impaired in GHa mice, they are still able to resolve necrotic lesions.
In the rat after partial hepatectomy of 70%, the original liver mass is completely restored within 7–10 d (1, 9). Even though the original shape of the liver is not recovered, the cellular architecture ultimately appears normal. In the present study the original liver mass was restored 4 d after surgery in control mice. As expected, normal cellular architecture was achieved by the end of the study, indicating that in control mice, 7 d were sufficient to complete the process. LID+ALSKO mice recovered both their original liver mass and cellular architecture 7 d after surgery. LID+ALSKO mice have an increased liver weight compared with controls, most likely due to the excess GH in the circulation. It has been well documented that liver size is proportional to body size, and that signals from the body can both positively and negatively regulate liver mass until the appropriate size is reached (38). In addition, studies demonstrated that GH affects liver mass (39, 40, 41). Therefore, it is possible that the delay observed in reaching the original liver mass in LID+ALSKO mice is the consequence of the higher original mass that needed to be restored in these mice.
The gain in liver weight after partial hepatectomy exhibited by the three groups of mice in this study was mainly the consequence of hepatocyte proliferation, as shown by BrdU incorporation and TUNEL assay. The regenerative process has been generally assumed to be similar in all rodents. However, rats and mice differ in their temporal responses after partial hepatectomy. In young rats, hepatocytes show a peak level of DNA synthesis about 24 h after resection, whereas in mice this peak is reached 36–40 h after partial hepatectomy (1). In our study the maximal levels of DNA synthesis in control mice were reached 36 h after surgery. Hepatocytes from LID+ALSKO animals proliferated to the same extent as in controls, but they reached this point approximately 12 h later. A similar delayed pattern was reported in IGFBP-1 knockout mice (IGFBP-1–/– mice) after partial hepatectomy (22). Hepatocytes of IGFBP-1–/– mice showed a delay of approximately 12 h and a mild reduction in the percentage of cells in S phase compared with wild-type mice. However, these mice could completely restore their original liver mass, and no significant level of mortality was observed. The pathway proposed to mediate these effects was that up-regulation of IGFBP-1 expression leads to the activation of ERK1/2 and stabilization of CCAAT/enhancer-binding protein-ß, which coordinates the progression of hepatocytes into S phase by regulating the expression of cyclins A, B1, and E (22).
Interestingly, LID+ALSKO mice, which have very low levels of circulating IGF-I and IGFBP-1 (35), exhibit a delay in the peak of proliferation, raising the possibility that this reduction in IGFBP-1 may contribute to the delayed entry of hepatocytes into S phase exhibited by these mice. However, this does not explain why hepatocytes from LID+ALSKO mice showed no reduction in DNA synthesis as IGFBP-1–/– mice do. It is well known that cyclins play a critical role in hepatocytic growth and proliferation (7, 12, 42) and that its expression can be modulated by nonessential amino acids (43), c-Jun N-terminal kinase (44), and GH (45) among several factors. Thus, it is possible that in our model the effect of reducing IGFBP-1 levels could be partially overcome by GH, thereby leading to a less severe phenotype, as shown by LID+ALSKO mice after surgery.
GHa mice showed not only delayed DNA synthesis, but a 75% reduction in the maximum levels of BrdU incorporation, compared with control and LID+ALSKO mice. Similarly impaired proliferation responses were reported in IL-6–/–, c-jun Li, and in TNFR-1–/– mice after partial hepatectomy (6, 7, 12). Krupczak-Hollys and colleagues (45) have shown that GH stimulates the proliferation of elderly regenerating liver and that injection of GH alone was sufficient to stimulate proliferation of hepatocytes without hepatectomy, providing evidence that GH can stimulate the proliferation of hepatocytes in vivo. Our finding that GHR expression in the liver after partial hepatectomy decreases to a similar extent in the three groups of mice 24 h after surgery suggests that this decrease in GHR expression might not be implicated in the subsequent different responses exhibited by the three groups. Therefore, if GH is having an effect on the ability of the liver to regenerate, it must be exerted either sometime before down-regulation of the GHR occurs or sometime after GHR expression has been restored. Because most of the genes that have been shown to be regulated by GH are involved in the earliest events of the regenerative process, the first alternative seems to be the most viable one. Basal levels of IGF-I mRNA in GHa mice were approximately 50% of those in control mice. Therefore, this level of IGF-I mRNA expression could be considered to be driven independently from GH. In this context, the lack of total recovery of IGF-I mRNA in GHa mice after partial hepatectomy could indicate that despite the increase in liver mass observed after surgery, the regenerating hepatocytes in GHa mice are not fully functional at the end of the study. This is also consistent with the fact that hepatocytes are still dividing at this point, and the regeneration of liver mass lags behind that in control or LID+ALSKO mice. IGF-I mRNA levels were decreased 24 h after surgery in both control and GHa mice, most likely due to the decrease in food intake during the first hours of recovery from surgery. Because LID+ALSKO mice lack IGF-I expression in the liver, no IGF-I mRNA was detected in the livers of these mice at any time.
The role of GH in liver regeneration is not yet fully defined. Our findings suggest that the effect of GH on liver regeneration is not mediated through IGF-I, because both LID+ALSKO and GHa mice exhibit very low levels of circulating IGF-I. GH may affect several signaling pathways involved in this regenerative process. It could exert its effect at the transcriptional level by regulating the expression of receptors or transcription factors or by modulating the activity of receptors other than the GHR. Additional work is needed to delineate the mechanisms by which GH regulates the process of liver regeneration.
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Footnotes |
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Received May 24, 2004.
Accepted for publication June 29, 2004.
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), LID+ALSKO (
), and GHa (
) animals at the indicated times. **, P < 0.05, GHa vs. control. B, Total body weight of the mice before and after surgery. Significant differences in body weight between the groups were observed at the beginning and end of the study. *, P < 0.05, LID+ALSKO vs. control; **, P < 0.05, GHa vs. control; #, P < 0.05, LID+ALSKO vs. GHa. No changes in body weight were observed in control and GHa mice during the days following partial hepatectomy (P = NS, by one-way ANOVA). Statistically significant changes were observed in LID+ALSKO mice on d 1, with no further reductions during the study (P < 0.05, by one-way ANOVA; 
, P < 0.05, d 0 vs. d1, vs. d 4, vs. d 7). Results are expressed as the mean ± SEM of at least three mice per time point.
), and GHa (
) mice were normalized to basal levels of GHR (B) and IGF-I (C) in control mice (arbitrarily designated a value of 1.0), and the results were expressed as the ratio over control levels. Data are expressed as the mean ± SEM of at least three mice per time point. *, P < 0.05, d 1 vs. basal control mice (B and C); **, P < 0.05, d 1 vs. basal GHa mice (B and C); #, P < 0.05, d 1 vs. basal LID+ALSKO mice (B and C); 
, P < 0.05, d 4 vs. basal GHa mice (B and C); 
, P < 0.05, d 7 vs. basal GHa mice (B).
