University of Groningen
Role of hepatic glucose signaling in the development of liver disease
Lei, Yu
DOI:
10.33612/diss.126530476
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Publication date: 2020
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Lei, Y. (2020). Role of hepatic glucose signaling in the development of liver disease. University of Groningen. https://doi.org/10.33612/diss.126530476
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125
Chapter
Glucose-6-phosphatase deficiency shifts
the balance between hepatocyte
hyperplasia and hypertrophy during
liver regeneration
Yu Lei1,2, Joanne A. Hoogerland1, Susanne Veldhuis4, Rachel E. Thomas3,
Rick Havinga1, Mirjam H. Koster1,3, Fabienne Rajas5, Gilles Mithieux5, Ton
Lisman4, Terry G.J. Derks7, Alain de Bruin1,3, Folkert Kuipers1,6 and
Maaike H. Oosterveer1
1Departments of Pediatrics, 4Surgery, 6Laboratory Medicine and 7Section
of Metabolic Diseases, University of Groningen, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands. 2Department of
Pathology and Provincial Key Laboratory of Infectious Diseases and Immunopathology, Shantou University Medical College, Shantou 515041, Guangdong, China. 3Department of Pathobiology, Dutch Molecular
Pathology Center, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3509, the Netherlands. 5Institut National de la Santé et de la
Recherche Médicale U1213 and Université de Lyon, Lyon, France.
In preparation
125125
Chapter
Glucose-6-phosphatase deficiency shifts
the balance between hepatocyte
hyperplasia and hypertrophy during
liver regeneration
Yu Lei1,2, Joanne A. Hoogerland1, Susanne Veldhuis4, Rachel E. Thomas3,
Rick Havinga1, Mirjam H. Koster1,3, Fabienne Rajas5, Gilles Mithieux5, Ton
Lisman4, Terry G.J. Derks7, Alain de Bruin1,3, Folkert Kuipers1,6 and
Maaike H. Oosterveer1
1Departments of Pediatrics, 4Surgery, 6Laboratory Medicine and 7Section
of Metabolic Diseases, University of Groningen, University Medical Center Groningen, 9713 GZ Groningen, The Netherlands. 2Department of
Pathology and Provincial Key Laboratory of Infectious Diseases and Immunopathology, Shantou University Medical College, Shantou 515041, Guangdong, China. 3Department of Pathobiology, Dutch Molecular
Pathology Center, Faculty of Veterinary Medicine, Utrecht University, Utrecht 3509, the Netherlands. 5Institut National de la Santé et de la
Recherche Médicale U1213 and Université de Lyon, Lyon, France.
126
Abstract
Glycogen storage disease type 1a (GSD Ia) is an inherited metabolic disorder caused by defective activity of glucose-6-phosphatase (G6PC), the enzyme that catalyzes the conversion of glucose-6-phosphate (G6P) to glucose. GSD Ia patients show a high prevalence of hepatocellular adenoma (HCA) development in young adulthood which predisposes to hepatocellular carcinoma (HCC) formation. The mechanisms underlying GSD Ia-associated tumorigenesis are poorly understood, and it is unclear at what stage of the disease pathophysiology tumor formation starts. Here we monitored hepatocyte proliferation in liver-specific GSD Ia (L-G6pc-/-) mice in
response to two-thirds partial hepatectomy (PHx) performed at 10 days after hepatocyte G6pc deletion. Our data show an accelerated presence of mitotic figures and Ki67 positivity in L-G6pc-/- as compared to L-G6pc+/+ hepatocytes. We also
observed that L-G6pc-/- hepatocytes exhibited enhanced BrdU incorporation in the
pre-mitotic phase of liver regeneration, while BrdU positivity was comparable in mitotic L-G6pc-/- andL-G6pc+/+ hepatocytes. Strikingly, almost all mitotic hepatocytes
in L-G6pc-/- mice formed anaphase bridges (94% and 80% at 48 and 72 hours
post-PHx, respectively), indicative of genomic instability and replication stress. Liver mass restoration was comparable in L-G6pc-/- and L-G6pc+/+ mice, however L-G6pc
-/-hepatocytes displayed increased hypertrophy (215% vs 170% in L-G6pc-/- and
L-G6pc+/+ mice at 7 days post-PHx, respectively). In conclusion, we are the first to
report signs of severe genomic instability and replication stress in GSD Ia hepatocytes at an early stage of the disease. Our findings are, furthermore, compatible with a model in which genomic instability in L-G6pc-/-hepatocytes alters liver regeneration
after partial liver resection by initially accelerating, then arresting hepatocyte hyperplasia and inducing (compensatory) hypertrophy.
127
Introduction
Glycogen storage disease type 1a is an inborn error of metabolism caused by defective activity of glucose-6-phosphatase (G6PC), the enzyme that mediates the production of glucose in the last step of glycogenolysis and gluconeogenesis (1, 2). The primary consequence of G6PC deficiency is fasting hypoglycemia. Loss of hepatic G6PC furthermore promotes the accumulation of glucose-6-phosphate (G6P), glycogen and triglycerides (TG) in the liver, resulting in hepatomegaly and non-alcoholic fatty liver disease (NAFLD) (3). The introduction of dietary management has resulted in improved glycemic control and hence reduced mortality from hypoglycemia at young age (4). As a consequence, the patient population is ageing and the clinical focus has shifted to the long-term complications of the disease (5). These include focal nodular hyperplasia and hepatocellular adenomas (HCAs), with a risk of transformation into hepatocellular carcinomas (HCC) (3, 6, 7). HCA and HCC predominantly develop after puberty, affect 70% of adult GSD Ia patients, and have become a major reason for hospitalization of adult patients. In the European Study on Glycogen Storage Disease Type I, HCAs were detected in 44 out of 288 patients, hence the overall prevalence was 16% (3). HCAs were first detected at a median age of 15 years (range 2-30 years) and 64% of the patients exhibit multiple tumors.
The mechanisms underlying GSD Ia-associated tumorigenesis are incompletely understood. In particular, it is unclear at what stage of the disease liver tumor formation starts. Liver tumors are macroscopically detectable in hepatocyte-specific
G6pc deficient (L-G6pc-/-) mice starting from 9 months after gene deletion (8, 9).
However, several cellular processes that may promote tumor development, such as mitochondrial dysfunction, perturbed autophagy, ER stress, apoptosis, metabolic rewiring, epithelial-mesenchymal transition, have been reported from 3 months of gene deletion onwards (10-14). Mutel et al., reported that metabolic liver disease presents as early as 10 days after gene deletion in L-G6pc-/- mice, illustrated by
substantial hepatic G6P, glycogen and TG accumulation (8). Whether there are also signs of tumorigenesis at this early disease stage is as yet unknown.
It is well-established that mutation-driven cell proliferation is a key event in tumor initiation (15, 16). Under normal conditions, the adult liver shows a low degree of hepatocyte proliferation (17). To evaluate hepatocyte proliferation in GSD Ia at an early stage of the disease, we therefore performed partial hepatectomy (PHx) in L-G6pc-/- mice 10 days post gene deletion and their wildtype (L-G6pc+/+) controls. We
evaluated hepatocyte proliferation by means of morphological analysis and immunohistochemistry at serial time points after surgery, and assessed liver regeneration by quantifying liver mass recovery.
126
Abstract
Glycogen storage disease type 1a (GSD Ia) is an inherited metabolic disorder caused by defective activity of glucose-6-phosphatase (G6PC), the enzyme that catalyzes the conversion of glucose-6-phosphate (G6P) to glucose. GSD Ia patients show a high prevalence of hepatocellular adenoma (HCA) development in young adulthood which predisposes to hepatocellular carcinoma (HCC) formation. The mechanisms underlying GSD Ia-associated tumorigenesis are poorly understood, and it is unclear at what stage of the disease pathophysiology tumor formation starts. Here we monitored hepatocyte proliferation in liver-specific GSD Ia (L-G6pc-/-) mice in
response to two-thirds partial hepatectomy (PHx) performed at 10 days after hepatocyte G6pc deletion. Our data show an accelerated presence of mitotic figures and Ki67 positivity in L-G6pc-/- as compared to L-G6pc+/+ hepatocytes. We also
observed that L-G6pc-/- hepatocytes exhibited enhanced BrdU incorporation in the
pre-mitotic phase of liver regeneration, while BrdU positivity was comparable in mitotic L-G6pc-/- andL-G6pc+/+ hepatocytes. Strikingly, almost all mitotic hepatocytes
in L-G6pc-/- mice formed anaphase bridges (94% and 80% at 48 and 72 hours
post-PHx, respectively), indicative of genomic instability and replication stress. Liver mass restoration was comparable in L-G6pc-/- and L-G6pc+/+ mice, however L-G6pc
-/-hepatocytes displayed increased hypertrophy (215% vs 170% in L-G6pc-/- and
L-G6pc+/+ mice at 7 days post-PHx, respectively). In conclusion, we are the first to
report signs of severe genomic instability and replication stress in GSD Ia hepatocytes at an early stage of the disease. Our findings are, furthermore, compatible with a model in which genomic instability in L-G6pc-/-hepatocytes alters liver regeneration
after partial liver resection by initially accelerating, then arresting hepatocyte hyperplasia and inducing (compensatory) hypertrophy.
127
Introduction
Glycogen storage disease type 1a is an inborn error of metabolism caused by defective activity of glucose-6-phosphatase (G6PC), the enzyme that mediates the production of glucose in the last step of glycogenolysis and gluconeogenesis (1, 2). The primary consequence of G6PC deficiency is fasting hypoglycemia. Loss of hepatic G6PC furthermore promotes the accumulation of glucose-6-phosphate (G6P), glycogen and triglycerides (TG) in the liver, resulting in hepatomegaly and non-alcoholic fatty liver disease (NAFLD) (3). The introduction of dietary management has resulted in improved glycemic control and hence reduced mortality from hypoglycemia at young age (4). As a consequence, the patient population is ageing and the clinical focus has shifted to the long-term complications of the disease (5). These include focal nodular hyperplasia and hepatocellular adenomas (HCAs), with a risk of transformation into hepatocellular carcinomas (HCC) (3, 6, 7). HCA and HCC predominantly develop after puberty, affect 70% of adult GSD Ia patients, and have become a major reason for hospitalization of adult patients. In the European Study on Glycogen Storage Disease Type I, HCAs were detected in 44 out of 288 patients, hence the overall prevalence was 16% (3). HCAs were first detected at a median age of 15 years (range 2-30 years) and 64% of the patients exhibit multiple tumors.
The mechanisms underlying GSD Ia-associated tumorigenesis are incompletely understood. In particular, it is unclear at what stage of the disease liver tumor formation starts. Liver tumors are macroscopically detectable in hepatocyte-specific
G6pc deficient (L-G6pc-/-) mice starting from 9 months after gene deletion (8, 9).
However, several cellular processes that may promote tumor development, such as mitochondrial dysfunction, perturbed autophagy, ER stress, apoptosis, metabolic rewiring, epithelial-mesenchymal transition, have been reported from 3 months of gene deletion onwards (10-14). Mutel et al., reported that metabolic liver disease presents as early as 10 days after gene deletion in L-G6pc-/- mice, illustrated by
substantial hepatic G6P, glycogen and TG accumulation (8). Whether there are also signs of tumorigenesis at this early disease stage is as yet unknown.
It is well-established that mutation-driven cell proliferation is a key event in tumor initiation (15, 16). Under normal conditions, the adult liver shows a low degree of hepatocyte proliferation (17). To evaluate hepatocyte proliferation in GSD Ia at an early stage of the disease, we therefore performed partial hepatectomy (PHx) in L-G6pc-/- mice 10 days post gene deletion and their wildtype (L-G6pc+/+) controls. We
evaluated hepatocyte proliferation by means of morphological analysis and immunohistochemistry at serial time points after surgery, and assessed liver regeneration by quantifying liver mass recovery.
127
128
Materials and Methods
Generation of hepatocyte-specific G6pc null mice
To induce hepatocyte-specific excision of G6pc exon 3 (L-G6pc-/-), male adult (6-12
weeks old) C57BL/6 G6pclox/lox and G6pclox/lox.SAcreERT2/w mice (8) were injected
intraperitoneally with 100 μl of tamoxifen (10 mg/ml, Sigma–Aldrich) on five consecutive days (8). All mice were housed in a standard light- and temperature-controlled facility, and fed a standard laboratory chow diet (RMH-B, Abdiets, Woerden) ad libitum with free access to water. Surgeries were performed 10 days after the last tamoxifen injection. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen.
Experimental procedures
8-13-week old hepatocyte-specific G6pc knockout (L-G6pc-/-) and wildtype (L-G6pc+/+)
mice were subjected to sham surgery or to 2/3 PHx under isoflurane anesthesia as described previously (18). Briefly, the abdomen was opened with a midline incision, after which about 2/3 liver mass was removed (PHx), or the liver was gently touched using a cotton tip (sham). The wound was subsequently ligated with suture, the abdomen was closed, and animals received a subcutaneous injection of Carprofen (5mg/kg). After 22, 34, 46, 70 and 166 hours groups of L-G6pc+/+ and L-G6pc-/- mice
that underwent either PHx or sham surgery received an intraperitoneal injection of bromodeoxyuridine (BrdU) (100 μg/g body weight, Sigma Aldrich).Two hours later, blood glucose levels were measured using an One Touch Ultra glucose meter (LifeScan Inc.), after which animals were sacrificed by cardiac puncture. For each timepoint, 2-3 sham-operated control animals of both genotypes were included. Following cardiac puncture, liver remnants were isolated, and wet liver weights were recorded to calculate liver/body weight ratios. Because the absolute liver weight of L-G6pc-/- mice is increased compared to L-G6pc+/+ mice (8, 19), the ratio of relative
PHx liver weight to sham relative liver weight was used to compare liver mass recovery between L-G6pc+/+ and L-G6pc-/- mice. After excision and weighing, a piece
of liver was trimmed and fixed in 4% formalin solution for histological analysis while the remaining tissue was snap-frozen in liquid nitrogen and stored at -80 °C until further analysis.
Real-time PCR
Total RNA was isolated by TRI-Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. cDNA was obtained by reverse transcription (Life Technologies) using 1 μg of total RNA. qPCR reactions were performed using the TaqMan Universal PCR Master Mix Kitand an ABI 7500 PCR system (Applied Biosystems). mRNA levels were calculated based on a pooled calibration curve, expressed relative to the expression of Ppig (cyclophilin). The sequences of the primers and probes are listed in Table S1.
Liver immunohistochemistry
Formalin-fixed liver tissues were embedded in paraffin and cut to 4 μm thick sections.
Hematoxylin and eosin (H&E) staining was conducted according to routine
129
procedures (20). Briefly, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Then sections were stained with hematoxylin for 4 minutes after which they were rinsed using tap water during 10 minutes. Sections were subsequently stained with eosin for 1 minute and followed by dehydration with graded alcohol and clearing in xylene. Immunohistochemistry was carried out following an established protocol (21). Briefly, after deparaffinization and rehydration, antigen retrieval was performed using 10mM citrate buffer at pH 6.0. The BrdU sections were treated with 2N HCl for 30 minutes at 37°C. Endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 minutes. For Ki67,
the unspecific antigens on tissues were blocked in 10% normal goat serum, while BrdU sections were blocked in 10% normal horse serum, both for 30 minutes. Sections were incubated with primary antibodies, i.e., rabbit anti Ki67 (RM-9106, Thermo, at 1:50), and mouse anti BrdU (M0744, DAKO, at 1:50 dilution) at 4°C overnight and subsequently incubated with biotinylated goat-anti-rabbit secondary antibody for Ki67 (Vector Labs BA-1000 at 1:250) and horse-anti-mouse (Vector Labs BA-9200 at 1:250) for BrdU during 30 minutes at room temperature. After a subsequent 30 minute incubation with Avidin-biotin ABC complex (PK-4000, Vector) the sections were washed and colorized with a 3,3ʹ-diaminobenzidine (DAB) (Sigma, D5637) for 10 minutes and then counterstained with hematoxylin. Slides were scanned using a Nikon E800 microscope. Hepatocyte area was quantified on H&E stained sections using Image J software and the hepatocyte number per microscopic field was calculated based on the average cell area of each individual mouse. Ki67- and BrdU positive hepatocytes were counted by examination of 5 random microscopic fields (magnification: 200x for Ki67 and 400x for BrdU, respectively) in each tissue section by a veterinary pathologist. Positivity for Ki67 and BrdU were corrected for the average hepatocyte number per microscopic field.
Quantification of mitotic hepatocytes and anaphase bridges
The total number of mitotic figures in either 5 200x fields or 10 400x fields was counted and scores latterly adjusted to take into account variations in hepatocyte size. In addition, the number of cells in anaphase was counted and the percentage of those cells that showed lagging chromosomes or anaphase bridges calculated.
Statistical Analysis
Statistical analysis was performed using 2-way ANOVA with Bonferroni's multiple comparisons test. Differences were considered statistically significant when p < 0.05.
Results
Liver mass recovery is slightly impaired in L-G6pc-/- mice
We first confirmed that G6pc mRNA levels remained below detection levels in remnant livers from L-G6pc-/- mice at all time points after PHx, while hepatic G6pc
expression was induced at 24 hours after PHx in wildtype mice only (Fig. 1A). At 36, 48 and 72 hours post-PHx the liver-to-body weight ratios of L-G6pc-/- mice were
slightly reduced compared to controls, while they were similar after 24 hours, and after 168 hours even tended to be increased in L-G6pc-/- mice (Fig. 1B and Table 1).
128
Materials and Methods
Generation of hepatocyte-specific G6pc null mice
To induce hepatocyte-specific excision of G6pc exon 3 (L-G6pc-/-), male adult (6-12
weeks old) C57BL/6 G6pclox/lox and G6pclox/lox.SAcreERT2/w mice (8) were injected
intraperitoneally with 100 μl of tamoxifen (10 mg/ml, Sigma–Aldrich) on five consecutive days (8). All mice were housed in a standard light- and temperature-controlled facility, and fed a standard laboratory chow diet (RMH-B, Abdiets, Woerden) ad libitum with free access to water. Surgeries were performed 10 days after the last tamoxifen injection. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen.
Experimental procedures
8-13-week old hepatocyte-specific G6pc knockout (L-G6pc-/-) and wildtype (L-G6pc+/+)
mice were subjected to sham surgery or to 2/3 PHx under isoflurane anesthesia as described previously (18). Briefly, the abdomen was opened with a midline incision, after which about 2/3 liver mass was removed (PHx), or the liver was gently touched using a cotton tip (sham). The wound was subsequently ligated with suture, the abdomen was closed, and animals received a subcutaneous injection of Carprofen (5mg/kg). After 22, 34, 46, 70 and 166 hours groups of L-G6pc+/+ and L-G6pc-/- mice
that underwent either PHx or sham surgery received an intraperitoneal injection of bromodeoxyuridine (BrdU) (100 μg/g body weight, Sigma Aldrich).Two hours later, blood glucose levels were measured using an One Touch Ultra glucose meter (LifeScan Inc.), after which animals were sacrificed by cardiac puncture. For each timepoint, 2-3 sham-operated control animals of both genotypes were included. Following cardiac puncture, liver remnants were isolated, and wet liver weights were recorded to calculate liver/body weight ratios. Because the absolute liver weight of L-G6pc-/- mice is increased compared to L-G6pc+/+ mice (8, 19), the ratio of relative
PHx liver weight to sham relative liver weight was used to compare liver mass recovery between L-G6pc+/+ and L-G6pc-/- mice. After excision and weighing, a piece
of liver was trimmed and fixed in 4% formalin solution for histological analysis while the remaining tissue was snap-frozen in liquid nitrogen and stored at -80 °C until further analysis.
Real-time PCR
Total RNA was isolated by TRI-Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. cDNA was obtained by reverse transcription (Life Technologies) using 1 μg of total RNA. qPCR reactions were performed using the TaqMan Universal PCR Master Mix Kitand an ABI 7500 PCR system (Applied Biosystems). mRNA levels were calculated based on a pooled calibration curve, expressed relative to the expression of Ppig (cyclophilin). The sequences of the primers and probes are listed in Table S1.
Liver immunohistochemistry
Formalin-fixed liver tissues were embedded in paraffin and cut to 4 μm thick sections.
Hematoxylin and eosin (H&E) staining was conducted according to routine
129
procedures (20). Briefly, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Then sections were stained with hematoxylin for 4 minutes after which they were rinsed using tap water during 10 minutes. Sections were subsequently stained with eosin for 1 minute and followed by dehydration with graded alcohol and clearing in xylene. Immunohistochemistry was carried out following an established protocol (21). Briefly, after deparaffinization and rehydration, antigen retrieval was performed using 10mM citrate buffer at pH 6.0. The BrdU sections were treated with 2N HCl for 30 minutes at 37°C. Endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 minutes. For Ki67,
the unspecific antigens on tissues were blocked in 10% normal goat serum, while BrdU sections were blocked in 10% normal horse serum, both for 30 minutes. Sections were incubated with primary antibodies, i.e., rabbit anti Ki67 (RM-9106, Thermo, at 1:50), and mouse anti BrdU (M0744, DAKO, at 1:50 dilution) at 4°C overnight and subsequently incubated with biotinylated goat-anti-rabbit secondary antibody for Ki67 (Vector Labs BA-1000 at 1:250) and horse-anti-mouse (Vector Labs BA-9200 at 1:250) for BrdU during 30 minutes at room temperature. After a subsequent 30 minute incubation with Avidin-biotin ABC complex (PK-4000, Vector) the sections were washed and colorized with a 3,3ʹ-diaminobenzidine (DAB) (Sigma, D5637) for 10 minutes and then counterstained with hematoxylin. Slides were scanned using a Nikon E800 microscope. Hepatocyte area was quantified on H&E stained sections using Image J software and the hepatocyte number per microscopic field was calculated based on the average cell area of each individual mouse. Ki67- and BrdU positive hepatocytes were counted by examination of 5 random microscopic fields (magnification: 200x for Ki67 and 400x for BrdU, respectively) in each tissue section by a veterinary pathologist. Positivity for Ki67 and BrdU were corrected for the average hepatocyte number per microscopic field.
Quantification of mitotic hepatocytes and anaphase bridges
The total number of mitotic figures in either 5 200x fields or 10 400x fields was counted and scores latterly adjusted to take into account variations in hepatocyte size. In addition, the number of cells in anaphase was counted and the percentage of those cells that showed lagging chromosomes or anaphase bridges calculated.
Statistical Analysis
Statistical analysis was performed using 2-way ANOVA with Bonferroni's multiple comparisons test. Differences were considered statistically significant when p < 0.05.
Results
Liver mass recovery is slightly impaired in L-G6pc-/- mice
We first confirmed that G6pc mRNA levels remained below detection levels in remnant livers from L-G6pc-/- mice at all time points after PHx, while hepatic G6pc
expression was induced at 24 hours after PHx in wildtype mice only (Fig. 1A). At 36, 48 and 72 hours post-PHx the liver-to-body weight ratios of L-G6pc-/- mice were
slightly reduced compared to controls, while they were similar after 24 hours, and after 168 hours even tended to be increased in L-G6pc-/- mice (Fig. 1B and Table 1).
129
130
Blood glucose levels tended to be lower in sham-operated L-G6pc-/- mice as compared to L-G6pc+/+ controls (Table 1) while no differences in blood glucose
concentrations were observed between L-G6pc-/- and L-G6pc+/+ mice until 7 days post-PHx, when L-G6pc-/- mice again showed a tendency towards lower glucose levels (Table 1).
Figure 1. Relative liver weight and histological analysis of hepatocyte proliferation and genomic instability in liver sections of PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, relative
G6pc mRNA expression normalized to 18S expression at different time points after PHx. B, ratio of liver mass regeneration compared to the sham-operated livers at different time points after PHx (n=4-9/group). C, ratio of mitotic figures at different time points after PHx (n=11-12/group for the sham group; n=5-9/group for the PHx groups). D, Ki67 positivity at different time points after PHx (n=8-9/group for the sham groups; n=4-6/group for the PHx groups). E, BrdU positivity at different time points after PHx (n=8-9/group for the sham groups; n=4-6/group for the PHx groups). F, ratio of anaphase bridge to anaphase figures at different time points after PHx (n=8-9/group for the sham groups; n=3-9/group for the PHx groups). Mitotic figures and Ki67 positive-hepatocytes were counted in 5 microscopic fields at 200x magnification. BrdU-positive hepatocytes were counted in 5 microscopic fields at 400x magnification. For C-E, scoring was corrected for the average hepatocyte size of each individual mouse. Data is presented as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum. Sham-operated control groups are composed of 2-3 mice of both genotypes sacrificed at each indicated timepoint. *p<0.05, ***p<0.001.
Aberrant patterns of mitosis, proliferation and DNA synthesis in response to PHx in L-G6pc-/- hepatocytes
Livers of sham-operated L-G6pc-/- mice showed a higher percentage of mitotic
hepatocytes as compared to wildtype livers (Fig. 1C) and a slight non-significant increase in the number of Ki67 and BrdU positive cells (Fig. 1D and 1E). At 24 hours post-PHx, no mitotic or Ki67 positive cells could be detected in L-G6pc+/+ and
131
L-G6pc-/- livers (Fig. 1C and 1D), yet BrdU positivity was present in both groups (Fig.
1E). At this timepoint L-G6pc-/- mice showed a higher percentage of BrdU positive
hepatocytes as compared to wildtype controls (Fig. 1E). At 36 hours post-PHx, both groups of mice showed an increased frequency of mitotic figures, Ki67 and BrdU positive cells (Fig. 1C-1E). At this timepoint, L-G6pc-/- livers presented a higher frequency of mitotic figures and Ki67 positive cells as compared to L-G6pc+/+ livers while the percentage of BrdU positive cells was similar in both groups (Fig. 1C-1E and Fig. 2A). In contrast, 12 hours later, at 48 hours post-PHx, L-G6pc+/+ livers showed highest mitosis rates, as well as Ki67 and BrDU positivity (Fig. 1C-1E). At 72 hours mitosis as well as Ki67and BrdU positivity decreased in both groups, reaching baseline values after 1 week post-PHx (Fig. 1C-1E). Strikingly, we observed that a very high percentage of mitotic hepatocytes in L-G6pc-/- mice formed anaphase bridges
(Fig. 1F and Fig. 2B), which represent lagging chromosomes in anaphase. The incidence of these bridges was low in L-G6pc+/+ hepatocytes at the same timepoints. This indicates that L-G6pc-/- hepatocytes exhibited severe genomic instability and
replication stress.
Figure 2. Immunohistochemical staining of proliferation markers in liver sections of PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, representative immunohistochemical staining
of Ki67 and BrdU at 36 hours after PHx in L-G6pc+/+ and L-G6pc-/- mice. B, anaphase bridges at
48 hours after PHx in L-G6pc+/+ and L-G6pc-/- mice. The white circles indicate mitotic cells.
Hepatocytes in anaphase are pointed out with arrow heads in L-G6pc+/+ (a) and L-G6pc-/- (b)
mice. Anaphase bridges were detected in L-G6pc-/- hepatocytes (b). B (c) presents a higher
magnification of area in dashed-line in (b). All images were taken at 400x magnification. Increased hepatocyte hypertrophy in response to PHx in L-G6pc-/- mice
H&E staining and subsequent quantification of hepatocyte size showed that sham-operated L-G6pc-/- livers exhibited increased hepatocellular vacuolization and –
size as compared to wildtype livers (Fig. 3A, 3B, Table 2).At 24 hours post-PHx, the hepatocytes in both groups had a similar size as compared to their respective sham-operated groups, but after 36, 48 and 72 hours post-PHx, hepatocytes were enlarged in both L-G6pc+/+ and L-G6pc-/- mice by 25-46% (Fig. 3A, 3B, Table 2). At 7
days post-PHx, all groups showed the largest increase in hepatocyte size, with L-G6pc-/- hepatocytes being significantly larger as compared to wildtype controls (Fig.
3A, 3B, Table 2). Thus, livers from L-G6pc-/- mice show a significant increase in
hepatocyte hypertrophy in response to 2/3 PHx. 130
130
Blood glucose levels tended to be lower in sham-operated L-G6pc-/- mice as
compared to L-G6pc+/+ controls (Table 1) while no differences in blood glucose
concentrations were observed between L-G6pc-/- and L-G6pc+/+ mice until 7 days
post-PHx, when L-G6pc-/- mice again showed a tendency towards lower glucose levels
(Table 1).
Figure 1. Relative liver weight and histological analysis of hepatocyte proliferation and
genomic instability in liver sections of PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, relative
G6pc mRNA expression normalized to 18S expression at different time points after PHx. B, ratio of liver mass regeneration compared to the sham-operated livers at different time points after PHx (n=4-9/group). C, ratio of mitotic figures at different time points after PHx (n=11-12/group for the sham group; n=5-9/group for the PHx groups). D, Ki67 positivity at different time points after PHx (n=8-9/group for the sham groups; n=4-6/group for the PHx groups). E, BrdU positivity at different time points after PHx (n=8-9/group for the sham groups; n=4-6/group for the PHx groups). F, ratio of anaphase bridge to anaphase figures at different time points after PHx (n=8-9/group for the sham groups; n=3-9/group for the PHx groups). Mitotic figures and Ki67 positive-hepatocytes were counted in 5 microscopic fields at 200x magnification. BrdU-positive hepatocytes were counted in 5 microscopic fields at 400x magnification. For C-E, scoring was corrected for the average hepatocyte size of each individual mouse. Data is presented as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum. Sham-operated control groups are composed of 2-3 mice of both genotypes sacrificed at each indicated timepoint. *p<0.05, ***p<0.001.
Aberrant patterns of mitosis, proliferation and DNA synthesis in response to PHx in L-G6pc-/- hepatocytes
Livers of sham-operated L-G6pc-/- mice showed a higher percentage of mitotic
hepatocytes as compared to wildtype livers (Fig. 1C) and a slight non-significant increase in the number of Ki67 and BrdU positive cells (Fig. 1D and 1E). At 24 hours post-PHx, no mitotic or Ki67 positive cells could be detected in L-G6pc+/+ and
131
L-G6pc-/- livers (Fig. 1C and 1D), yet BrdU positivity was present in both groups (Fig.
1E). At this timepoint L-G6pc-/- mice showed a higher percentage of BrdU positive
hepatocytes as compared to wildtype controls (Fig. 1E). At 36 hours post-PHx, both groups of mice showed an increased frequency of mitotic figures, Ki67 and BrdU positive cells (Fig. 1C-1E). At this timepoint, L-G6pc-/- livers presented a higher
frequency of mitotic figures and Ki67 positive cells as compared to L-G6pc+/+ livers
while the percentage of BrdU positive cells was similar in both groups (Fig. 1C-1E and Fig. 2A). In contrast, 12 hours later, at 48 hours post-PHx, L-G6pc+/+ livers showed
highest mitosis rates, as well as Ki67 and BrDU positivity (Fig. 1C-1E). At 72 hours mitosis as well as Ki67and BrdU positivity decreased in both groups, reaching baseline values after 1 week post-PHx (Fig. 1C-1E). Strikingly, we observed that a very high percentage of mitotic hepatocytes in L-G6pc-/- mice formed anaphase bridges
(Fig. 1F and Fig. 2B), which represent lagging chromosomes in anaphase. The incidence of these bridges was low in L-G6pc+/+ hepatocytes at the same timepoints.
This indicates that L-G6pc-/- hepatocytes exhibited severe genomic instability and
replication stress.
Figure 2. Immunohistochemical staining of proliferation markers in liver sections of
PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, representative immunohistochemical staining
of Ki67 and BrdU at 36 hours after PHx in L-G6pc+/+ and L-G6pc-/- mice. B, anaphase bridges at
48 hours after PHx in L-G6pc+/+ and L-G6pc-/- mice. The white circles indicate mitotic cells.
Hepatocytes in anaphase are pointed out with arrow heads in L-G6pc+/+ (a) and L-G6pc-/- (b)
mice. Anaphase bridges were detected in L-G6pc-/- hepatocytes (b). B (c) presents a higher
magnification of area in dashed-line in (b). All images were taken at 400x magnification.
Increased hepatocyte hypertrophy in response to PHx in L-G6pc-/- mice
H&E staining and subsequent quantification of hepatocyte size showed that sham-operated L-G6pc-/- livers exhibited increased hepatocellular vacuolization and –
size as compared to wildtype livers (Fig. 3A, 3B, Table 2).At 24 hours post-PHx, the hepatocytes in both groups had a similar size as compared to their respective sham-operated groups, but after 36, 48 and 72 hours post-PHx, hepatocytes were enlarged in both L-G6pc+/+ and L-G6pc-/- mice by 25-46% (Fig. 3A, 3B, Table 2). At 7
days post-PHx, all groups showed the largest increase in hepatocyte size, with L-G6pc-/- hepatocytes being significantly larger as compared to wildtype controls (Fig.
3A, 3B, Table 2). Thus, livers from L-G6pc-/- mice show a significant increase in
hepatocyte hypertrophy in response to 2/3 PHx. 131
132 Ta bl e 1. G ene ral char ac te ris tic s o f s ham - an d PH x-ope rat ed L-G6 pc +/ + a nd L-G6 pc -/- mi ce . n = 1 0 a nd 1 2 i n sh am -o pe ra te d L-G6p c +/ + a nd L-G6p c -/ - m ice , re spe ct iv el y; n= 4-9/ gr oup in PH x-ope ra te d gr oup s. Da ta is p re se nt ed as m edi an (ra ng e) . S ha m -o pe ra te d co nt ro l g ro ups a re c om po se d of 2 -3 m ice o f bo th ge no ty pe s s ac rif ice d a t e ac h in di ca te d ti m ep oi nt. * p< 0. 05 , * *p <0 .0 1, a nd * ** p< 0. 00 1 i nd ica te s s ig ni fic an ce co m pa re d to L-G6p c+ /+ m ice . Bod y w ei gh t Liv er w eig ht Bl ood g lu cos e Ho ur s af te r P Hx L-G6p c +/ + L-G6p c -/ - L-G6p c +/ + L-G6p c -/ - L-G6p c +/ + L-G6p c -/ -sh am 26. 2 (23. 5-33. 0) 26. 1 (2 3. 2. 2) 1. 4 (0 .9 -1. 6) 2. 1 (1 .2 -2. 4) * ** 8. 8 (6. 1-11. 3) 6. 3 (3. 9-11. 2) 24 25. 1 (21. 5-25. 8) 24. 5 (2 2. 8-26. 4) 0. 6 (0 .5 -0. 6) 0. 8 (0 .7 -1. 0) 9. 6 (8. 4-11. 5) 10. 3 (6 .1 -10. 6) 36 25. 3 (23. 5-26. 6) 23. 3 (2 3. 1-26. 6) 0. 6 (0 .5 -0. 7) 0. 8 (0 .7 -0. 9) 7. 1 (6. 0-8. 9) 7. 0 (6. 5-9. 4) 48 23. 7 (23. 3-24. 4) 23. 2 (2 2. 0-25. 8) 0. 9 (0 .7 -1. 1) 1. 3 (1 .0 -1. 4) 6. 9 (6. 6-12. 2) 6. 5 (5. 0-10. 4) 72 25. 5 (21. 2-27. 2) 22. 7 (2 1. 7-25. 0) 1. 0 (0 .8 -1. 3) 1. 2 (1 .1 -1. 7) 7. 6 (6. 0-9. 4) 6. 3 (2. 8-10. 4) 168 24. 9 (22. 7-25. 8) 23. 7 (2 2. 5-26. 3) 1. 2 (1 .0 -1. 3) 1. 8 (1 .6 -2. 1) ** 10. 9 (10. 5-11. 7) 7. 5 (3. 5-10. 1) 133
Figure 3. Representative H&E stainings and hepatocyte size distributions in liver sections of sham- and PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, representative H&E stainings of
L-G6pc+/+ and L-G6pc-/- livers at different time points after PHx. Images were taken at 400x
magnification. B, hepatocyte size distribution of L-G6pc+/+ and L-G6pc-/- livers at different
time points after PHx. Hepatocyte areas were quantified in at least 150 cells per mouse liver using Image J software (National Institutes of Health, Bethesda, Maryland). Data were analyzed using the percent relative cumulative frequency (PRCF) approach (54) and EC50 values (50th percentile value) of the PCRF curves were derived following nonlinear regression with customized equation using GraphPad Prism version 7.00 (GraphPad Software, San Diego, California). Inset: EC50 values of the PCRF curves and their 95%-confidence intervals in parentheses. n = 10 and 12 in sham-operated L-G6pc+/+ and
L-G6pc-/- mice, respectively; n=4-9/group in PHx-treated groups. Sham-operated control
groups are composed of 2-3 mice of both genotypes sacrificed at each indicated timepoint.
132 Ta bl e 1. G ene ral char ac te ris tic s o f s ham - an d PH x-ope rat ed L-G6 pc +/ + a nd L-G6 pc -/- mi ce . n = 1 0 a nd 1 2 i n sh am -o pe ra te d L-G6p c +/ + a nd L-G6p c -/ - m ice , re spe ct iv el y; n= 4-9/ gr oup in PH x-ope ra te d gr oup s. Da ta is p re se nt ed as m edi an (ra ng e) . S ha m -o pe ra te d co nt ro l g ro ups a re c om po se d of 2 -3 m ice o f bo th ge no ty pe s s ac rif ice d a t e ac h in di ca te d ti m ep oi nt. * p< 0. 05 , * *p <0 .0 1, a nd * ** p< 0. 00 1 i nd ica te s s ig ni fic an ce co m pa re d to L-G6p c+ /+ m ice . Bod y w ei gh t Liv er w eig ht Bl ood g lu cos e Ho ur s af te r P Hx L-G6p c +/ + L-G6p c -/ - L-G6p c +/ + L-G6p c -/ - L-G6p c +/ + L-G6p c -/ -sh am 26. 2 (23. 5-33. 0) 26. 1 (2 3. 2. 2) 1. 4 (0 .9 -1. 6) 2. 1 (1 .2 -2. 4) * ** 8. 8 (6. 1-11. 3) 6. 3 (3. 9-11. 2) 24 25. 1 (21. 5-25. 8) 24. 5 (2 2. 8-26. 4) 0. 6 (0 .5 -0. 6) 0. 8 (0 .7 -1. 0) 9. 6 (8. 4-11. 5) 10. 3 (6 .1 -10. 6) 36 25. 3 (23. 5-26. 6) 23. 3 (2 3. 1-26. 6) 0. 6 (0 .5 -0. 7) 0. 8 (0 .7 -0. 9) 7. 1 (6. 0-8. 9) 7. 0 (6. 5-9. 4) 48 23. 7 (23. 3-24. 4) 23. 2 (2 2. 0-25. 8) 0. 9 (0 .7 -1. 1) 1. 3 (1 .0 -1. 4) 6. 9 (6. 6-12. 2) 6. 5 (5. 0-10. 4) 72 25. 5 (21. 2-27. 2) 22. 7 (2 1. 7-25. 0) 1. 0 (0 .8 -1. 3) 1. 2 (1 .1 -1. 7) 7. 6 (6. 0-9. 4) 6. 3 (2. 8-10. 4) 168 24. 9 (22. 7-25. 8) 23. 7 (2 2. 5-26. 3) 1. 2 (1 .0 -1. 3) 1. 8 (1 .6 -2. 1) ** 10. 9 (10. 5-11. 7) 7. 5 (3. 5-10. 1) 133
Figure 3. Representative H&E stainings and hepatocyte size distributions in liver sections of
sham- and PHx-operated L-G6pc+/+ and L-G6pc-/- mice. A, representative H&E stainings of
L-G6pc+/+ and L-G6pc-/- livers at different time points after PHx. Images were taken at 400x
magnification. B, hepatocyte size distribution of L-G6pc+/+ and L-G6pc-/- livers at different
time points after PHx. Hepatocyte areas were quantified in at least 150 cells per mouse liver using Image J software (National Institutes of Health, Bethesda, Maryland). Data were analyzed using the percent relative cumulative frequency (PRCF) approach (54) and EC50 values (50th percentile value) of the PCRF curves were derived following nonlinear regression with customized equation using GraphPad Prism version 7.00 (GraphPad Software, San Diego, California). Inset: EC50 values of the PCRF curves and their 95%-confidence intervals in parentheses. n = 10 and 12 in sham-operated L-G6pc+/+ and
L-G6pc-/- mice, respectively; n=4-9/group in PHx-treated groups. Sham-operated control
groups are composed of 2-3 mice of both genotypes sacrificed at each indicated timepoint.
133
134
Table 2. Changes in average hepatocyte size after PHx in L-G6pc+/+ and L-G6pc-/- mice.
Absolute EC50 values of the hepatocyte size PCRF curves presented in Fig. 3, and relative EC50 values compared to sham-operated controls of either genotype (in parentheses)
Discussion
Liver tumor development is serious long-term complication of GSD Ia and a major reason for hospitalization of adult patients. Partial liver resection or liver transplantation are currently the preferred options to reduce the risks associated with liver tumor formation (22-24). Although liver tumor prevention would be far more desirable than surgical intervention, development of preventive therapies requires understanding of the molecular basis of tumor development in GSD Ia, as well as its time course. The mechanisms underlying GSD Ia-associated tumorigenesis are, however, incompletely understood and it is currently unclear at what stage of the disease pathophysiology liver tumor formation is initiated. In the current study we evaluated hepatocyte proliferation in L-G6pc-/- mice at an early stage of the
disease and found that G6pc deficient hepatocytes show an accelerated induction of hepatocyte proliferation and mitosis in response to partial liver resection (PHx). In addition, we observed a very high incidence of anaphase bridges. The presence of these anaphase bridges indicates severe genomic instability and replication stress in
G6pc deficient hepatocytes as compared to wildtype controls. This may in turn have
triggered cell cycle arrest and a reduction in hepatocyte hyperplasia in response to PHx. On the other hand, hepatocytes from L-G6pc-/- mice showed increased
hypertrophy. The apparently comparable overall rates of proliferation in L-G6pc-/- and
L-G6p+/+ livers do not directly explain the similar recovery in liver weights post-PHx as
average hepatocyte size was significantly increased in L-G6pc-/- mice. It is conceivable
that increased hepatocyte hypertrophy in L-G6pc-/- mice reflects a compensatory
response to a post-proliferative loss of hepatocytes in which genomic stability was observed during mitosis.
Liver regeneration in response to PHx is a well-controlled process that involves different stages and is coordinated by intracellular signaling cascades, cytokines and growth factors, as well as metabolic signals (25-27). During PHx-induced liver regeneration in rodents, proliferation of mature hepatocytes involves both hypertrophy and hyperplasia, thereby enabling restoration of the original liver mass (28). When two-thirds of the liver is removed, there is a rapid hypertrophy of the
Hours after PHx L-G6pc+/+ L-G6pc -/-sham 213 (100%) 338 (100%) 24 227 (107%) 352 (104%) 36 267 (125%) 494 (146%) 48 262 (123%) 438 (129%) 72 272 (128%) 459 (136%) 168 363 (170%) 729 (215%) 135
remnant hepatocytes, followed by hyperplasia with almost all hepatocytes entering the cell cycle. In contrast, in the case of one-third partial hepatectomy, the liver almost exclusively restores its original mass through hepatocyte hypertrophy (29, 30).
In the current study, we found that hepatocyte size increased during liver regeneration, starting from 36 hours after two-thirds PHx, and that the hypertrophy was most pronounced at day 7 post-PHx. These time-dependent changes in hepatocyte hypertrophy are different compared to what has been reported previously after two-thirds hepatectomy in rodents, which is most likely explained by species-, genetic background, and context-specific regenerative responses (30, 31).
Notably, the increase in hepatocyte size in L-G6pc-/- mice was more pronounced as compared to wildtype controls (+ 25%) while liver mass recovery was comparable in both genotypes, indicating a larger contribution of hypertrophy to regeneration in G6pc deficient livers. Interestingly, research has shown that in animal models with aberrant cell cycle induction, liver mass recovery after PHx is maintained due an increase in hepatocyte hypertrophy that compensates for impaired hyperplasia (32-34). Our current findings that G6pc deficient hepatocytes showed signs of severe
genomic instability and replication stress, and displayed more hypertrophy in response to PHx while achieving a similar degree of liver generation, are consistent with the model in which impaired hepatocyte hyperplasia due to aberrant mitosis is compensated for by impaired hypertrophy (30).
In addition, we observed that the the timing of the regenerative response after PHx was altered in L-G6pc-/- mice as compared to wildtype controls. The presence of mitotic figures was increased at 36 hours post-PHx, a timepoint reflecting the first peak of DNA synthesis (35), and consistently, the number of Ki67 positive hepatocytes was more than doubled in L-G6pc-/- mice at this timepoint. In contrast, after 48 hours both the number of mitoses and Ki67 positive cells were lower in
L-G6pc-/- mice as compared to controls. Collectively, these data highly suggest that L-G6pc-/-hepatocytes entered mitosis faster. Interestingly, almost all mitotic L-G6pc
-/-hepatocytes showed severe signs of genomic instability and replication stress, as evidenced by a high prevalence of anaphase bridges in these cells. Moreover, the BrdU incorporation rate in L-G6pc-/-livers 24 hours after PHx was markedly increased.
Because no mitotic hepatocytes and/or Ki67 positive cells were detected at this timepoint, this may reflect enhanced DNA repair in quiescent L-G6pc-/-hepatocytes.
Alternatively, it may indicate that L-G6pc-/- hepatocytes were in S-phase at this
timepoint, i.e. synthesizing DNA but at a stage prior to significant Ki67 induction.
Taken together, we hypothesize that, despite the apparent accelerated mitotic entry, L-G6pc-/-hepatocytes progress slower through G2-M and undergo cell cycle arrest,
which may be due to the high degree of genomic instability observed in these cells. The molecular mechanism that drives the accelerated cell cycle entrance of L-G6pc
-/-hepatocytes is as yet unclear. Interestingly, research has shown that hypoglycemia is an important driver of hepatocyte proliferation after partial hepatectomy (36-38) and evidence is accumulating that improved metabolic control results in regression of HCAs in GSD Ia patients (4, 39, 40). We did not frequently monitor blood glucose levels following PHx, but observed lower blood glucose levels in L-G6pc-/- mice that were sham-operated, and in L-G6pc-/- mice at 7 days post-PHx. However, it is
134
Table 2. Changes in average hepatocyte size after PHx in L-G6pc+/+ and L-G6pc-/- mice.
Absolute EC50 values of the hepatocyte size PCRF curves presented in Fig. 3, and relative EC50 values compared to sham-operated controls of either genotype (in parentheses)
Discussion
Liver tumor development is serious long-term complication of GSD Ia and a major reason for hospitalization of adult patients. Partial liver resection or liver transplantation are currently the preferred options to reduce the risks associated with liver tumor formation (22-24). Although liver tumor prevention would be far more desirable than surgical intervention, development of preventive therapies requires understanding of the molecular basis of tumor development in GSD Ia, as well as its time course. The mechanisms underlying GSD Ia-associated tumorigenesis are, however, incompletely understood and it is currently unclear at what stage of the disease pathophysiology liver tumor formation is initiated. In the current study we evaluated hepatocyte proliferation in L-G6pc-/- mice at an early stage of the
disease and found that G6pc deficient hepatocytes show an accelerated induction of hepatocyte proliferation and mitosis in response to partial liver resection (PHx). In addition, we observed a very high incidence of anaphase bridges. The presence of these anaphase bridges indicates severe genomic instability and replication stress in
G6pc deficient hepatocytes as compared to wildtype controls. This may in turn have
triggered cell cycle arrest and a reduction in hepatocyte hyperplasia in response to PHx. On the other hand, hepatocytes from L-G6pc-/- mice showed increased
hypertrophy. The apparently comparable overall rates of proliferation in L-G6pc-/- and
L-G6p+/+ livers do not directly explain the similar recovery in liver weights post-PHx as
average hepatocyte size was significantly increased in L-G6pc-/- mice. It is conceivable
that increased hepatocyte hypertrophy in L-G6pc-/- mice reflects a compensatory
response to a post-proliferative loss of hepatocytes in which genomic stability was observed during mitosis.
Liver regeneration in response to PHx is a well-controlled process that involves different stages and is coordinated by intracellular signaling cascades, cytokines and growth factors, as well as metabolic signals (25-27). During PHx-induced liver regeneration in rodents, proliferation of mature hepatocytes involves both hypertrophy and hyperplasia, thereby enabling restoration of the original liver mass (28). When two-thirds of the liver is removed, there is a rapid hypertrophy of the
Hours after PHx L-G6pc+/+ L-G6pc -/-sham 213 (100%) 338 (100%) 24 227 (107%) 352 (104%) 36 267 (125%) 494 (146%) 48 262 (123%) 438 (129%) 72 272 (128%) 459 (136%) 168 363 (170%) 729 (215%) 135
remnant hepatocytes, followed by hyperplasia with almost all hepatocytes entering the cell cycle. In contrast, in the case of one-third partial hepatectomy, the liver almost exclusively restores its original mass through hepatocyte hypertrophy (29, 30).
In the current study, we found that hepatocyte size increased during liver regeneration, starting from 36 hours after two-thirds PHx, and that the hypertrophy was most pronounced at day 7 post-PHx. These time-dependent changes in hepatocyte hypertrophy are different compared to what has been reported
previously after two-thirds hepatectomy in rodents, which is most likely explained by
species-, genetic background, and context-specific regenerative responses (30, 31).
Notably, the increase in hepatocyte size in L-G6pc-/- mice was more pronounced as
compared to wildtype controls (+ 25%) while liver mass recovery was comparable in both genotypes, indicating a larger contribution of hypertrophy to regeneration in G6pc deficient livers. Interestingly, research has shown that in animal models with aberrant cell cycle induction, liver mass recovery after PHx is maintained due an
increase in hepatocyte hypertrophy that compensates for impaired hyperplasia
(32-34). Our current findings that G6pc deficient hepatocytes showed signs of severe
genomic instability and replication stress, and displayed more hypertrophy in response to PHx while achieving a similar degree of liver generation, are consistent with the model in which impaired hepatocyte hyperplasia due to aberrant mitosis is compensated for by impaired hypertrophy (30).
In addition, we observed that the the timing of the regenerative response after PHx was altered in L-G6pc-/- mice as compared to wildtype controls. The presence of mitotic figures was increased at 36 hours post-PHx, a timepoint reflecting the first peak of DNA synthesis (35), and consistently, the number of Ki67 positive
hepatocytes was more than doubled in L-G6pc-/- mice at this timepoint. In contrast,
after 48 hours both the number of mitoses and Ki67 positive cells were lower in
L-G6pc-/- mice as compared to controls. Collectively, these data highly suggest that
L-G6pc-/- hepatocytes entered mitosis faster. Interestingly, almost all mitotic L-G6pc
-/-hepatocytes showed severe signs of genomic instability and replication stress, as evidenced by a high prevalence of anaphase bridges in these cells. Moreover, the BrdU incorporation rate in L-G6pc-/-livers 24 hours after PHx was markedly increased.
Because no mitotic hepatocytes and/or Ki67 positive cells were detected at this timepoint, this may reflect enhanced DNA repair in quiescent L-G6pc-/-hepatocytes.
Alternatively, it may indicate that L-G6pc-/- hepatocytes were in S-phase at this
timepoint, i.e. synthesizing DNA but at a stage prior to significant Ki67 induction.
Taken together, we hypothesize that, despite the apparent accelerated mitotic entry, L-G6pc-/-hepatocytes progress slower through G2-M and undergo cell cycle arrest,
which may be due to the high degree of genomic instability observed in these cells. The molecular mechanism that drives the accelerated cell cycle entrance of L-G6pc
-/-hepatocytes is as yet unclear. Interestingly, research has shown that hypoglycemia is an important driver of hepatocyte proliferation after partial hepatectomy (36-38) and evidence is accumulating that improved metabolic control results in regression of HCAs in GSD Ia patients (4, 39, 40). We did not frequently monitor blood glucose levels following PHx, but observed lower blood glucose levels in L-G6pc-/- mice that
were sham-operated, and in L-G6pc-/- mice at 7 days post-PHx. However, it is
135
136
conceivable that compared to their wildtype littermates, L-G6pc-/- mice became more
hypoglycemic within the first day after surgery due to the loss of hepatic G6PC activity and the blunted induction of G6pc in response to PHx (37), triggering the accelerated mitotic entry in these animals.
A major and remarkable finding in our study was the high prevalence of anaphase bridges in L-G6pc-/-hepatocytes undergoing mitosis. Anaphase bridges are a sign of
genomic instability and replication stress, and commonly observed in cancer cells (41). However, evidence is accumulating that genomic instability and replication stress may also be a cause of cancer (42-44). To the best of our knowledge, our study is the first to point towards signs of genomic instability at a very early stage of GSD Ia. In addition, within 10 days after G6pc deletion, baseline hepatocyte proliferation, evidenced by a higher positivity for BrdU and Ki67, as well as the number of mitotic hepatocytes, were slightly increased while hepatocytes of sham-operated wildtype mice were in a quiescent state. Although the magnitude of these alterations was relatively low, the proposed accelerated entry into the proliferative state further supports our hypothesis of pro-tumorigenic events occurring at this early disease stage.
A key and unresolved question is via which mechanisms the loss of hepatocyte G6PC activity induces genomic instability and replication stress. The toxic accumulation of metabolites such as triglycerides, but possibly also glycogen in L-G6pc-/-liver, causes
cellular stress (12-14, 45) hence impacting DNA integrity- and replication, as well as hepatocyte proliferation (46-48). Follow-up studies are required to unravel the underlying mechanisms of genomic instability and replication stress at this early stage of hepatic GSD Ia.
In current clinical practice partial liver resection is the preferred strategy in case where HCAs reach a certain size and persist or when HCAs undergo malignant transformation into HCC (24). After liver resection, the human liver generally regenerates to its original size within a timeframe of 6 months to 1 year (49, 50) but it is as yet unclear to what extent hyperplasia and hypertrophy contribute to this process. It is well-documented that partial hepatectomy accelerates carcinogen-induced liver tumor development in animal models (51, 52). Moreover, it has been reported that GSD Ia patients have shorter time to adenoma progression after partial hepatectomy as compared to patients without GSD Ia (53). Taken together, although liver resection is currently one of the few options to prevent liver tumor progression in GSD Ia patients, it may promote tumor recurrence, as the remaining, genomically unstable hepatocytes re-enter the cell cycle, hence triggering tumorigenesis (52).
In conclusion, this study is the first to report signs of severe genomic instability and replication stress in L-G6pc-/-hepatocytes at an early stage of GSD Ia. Hepatocytes
from L-G6pc-/- mice also showed increased hypertrophy but comparable liver mass
restoration in response to partial hepatectomy as compared to wildtype controls. Combined, these findings may suggest that genomic instability in L-G6pc
-/-hepatocytes shifts the balance between hepatocyte hyperplasia and hypertrophy
137
during liver regeneration. By causally linking perturbed intrahepatic glucose metabolism to genomic instability our findings may help to uncover novel mechanisms that contribute to liver tumor formation in GSD Ia.
Acknowledgements
We thank Angelika Jurdzinski, Aycha Bleeker and Yang Yang for excellent technical assistanceand data acquisition. This work was supported by The Abel Tasman Talent Program (ATTP)of the University of Groningen to Y. Lei. M.H.O. is the recipient of a VIDI grant from the Dutch Scientific Organization, and holds a Rosalind Franklin Fellowship from the University of Groningen. F.K. is supported by CardioVasculair Onderzoek Nederland (CVON2012-03).
136
conceivable that compared to their wildtype littermates, L-G6pc-/- mice became more
hypoglycemic within the first day after surgery due to the loss of hepatic G6PC activity and the blunted induction of G6pc in response to PHx (37), triggering the accelerated mitotic entry in these animals.
A major and remarkable finding in our study was the high prevalence of anaphase bridges in L-G6pc-/-hepatocytes undergoing mitosis. Anaphase bridges are a sign of
genomic instability and replication stress, and commonly observed in cancer cells (41). However, evidence is accumulating that genomic instability and replication stress may also be a cause of cancer (42-44). To the best of our knowledge, our study is the first to point towards signs of genomic instability at a very early stage of GSD Ia. In addition, within 10 days after G6pc deletion, baseline hepatocyte proliferation, evidenced by a higher positivity for BrdU and Ki67, as well as the number of mitotic hepatocytes, were slightly increased while hepatocytes of sham-operated wildtype mice were in a quiescent state. Although the magnitude of these alterations was relatively low, the proposed accelerated entry into the proliferative state further supports our hypothesis of pro-tumorigenic events occurring at this early disease stage.
A key and unresolved question is via which mechanisms the loss of hepatocyte G6PC activity induces genomic instability and replication stress. The toxic accumulation of metabolites such as triglycerides, but possibly also glycogen in L-G6pc-/-liver, causes
cellular stress (12-14, 45) hence impacting DNA integrity- and replication, as well as hepatocyte proliferation (46-48). Follow-up studies are required to unravel the underlying mechanisms of genomic instability and replication stress at this early stage of hepatic GSD Ia.
In current clinical practice partial liver resection is the preferred strategy in case where HCAs reach a certain size and persist or when HCAs undergo malignant transformation into HCC (24). After liver resection, the human liver generally regenerates to its original size within a timeframe of 6 months to 1 year (49, 50) but it is as yet unclear to what extent hyperplasia and hypertrophy contribute to this process. It is well-documented that partial hepatectomy accelerates carcinogen-induced liver tumor development in animal models (51, 52). Moreover, it has been reported that GSD Ia patients have shorter time to adenoma progression after partial hepatectomy as compared to patients without GSD Ia (53). Taken together, although liver resection is currently one of the few options to prevent liver tumor progression in GSD Ia patients, it may promote tumor recurrence, as the remaining, genomically unstable hepatocytes re-enter the cell cycle, hence triggering tumorigenesis (52).
In conclusion, this study is the first to report signs of severe genomic instability and replication stress in L-G6pc-/-hepatocytes at an early stage of GSD Ia. Hepatocytes
from L-G6pc-/- mice also showed increased hypertrophy but comparable liver mass
restoration in response to partial hepatectomy as compared to wildtype controls. Combined, these findings may suggest that genomic instability in L-G6pc
-/-hepatocytes shifts the balance between hepatocyte hyperplasia and hypertrophy
137
during liver regeneration. By causally linking perturbed intrahepatic glucose metabolism to genomic instability our findings may help to uncover novel mechanisms that contribute to liver tumor formation in GSD Ia.
Acknowledgements
We thank Angelika Jurdzinski, Aycha Bleeker and Yang Yang for excellent technical assistanceand data acquisition. This work was supported by The Abel Tasman Talent Program (ATTP)of the University of Groningen to Y. Lei. M.H.O. is the recipient of a VIDI grant from the Dutch Scientific Organization, and holds a Rosalind Franklin Fellowship from the University of Groningen. F.K. is supported by CardioVasculair Onderzoek Nederland (CVON2012-03).
137
