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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161
Chapter
General Discussion
161 161Chapter
General Discussion
162
General discussion
Metabolic homeostasis is tightly regulated by various factors, including the availability of nutrients (e.g., glucose, fatty acids and amino acids), hormones (e.g., insulin and glucagon) and neuronal control (e.g., sympatic and parasympatic systems). The regulatory proteins that modulate metabolic changes respond to changes in nutrient availability are called nutrient sensors. Transcription factors represent an important group of nutrient sensors. So far, several transcription factors that regulate the transcription of metabolic genes have been identified, including CREB, FOXO1, ChREBP, SREBP, PGC-1α, CRTC2, LXR, FXR, PPARs. Chronic metabolic imbalances (such as energy overload or unbalanced nutrition) disturb systemic physiology leading to the development of metabolic diseases such as obesity, diabetes, NAFLD and even cancer (1-3).
In this thesis, a mouse model for GSD Ia was employed to study the physiological and molecular mechanisms that link hepatic glucose (G6P) imbalance to liver function and -health. Although GSD Ia itself is a rare disease it is associated with several ‘general’ metabolic perturbations such as hypoglycemia, hyperlipidaemia, NAFLD, lactic acidaemia and hyperuricaemia (4). Therefore, GSD I can be considered as a valuable model to investigate the pathophysiology of common metabolic diseases that show hepatic glucose imbalance, such as type 2 diabetes (4, 5). Metabolic diseases such as diabetes and NAFLD become more and more prevalent (4). In this thesis, we focused on the consequences of enhanced ChREBP activity for hepatocyte function and liver disease development in GSD Ia, the impact of metabolic imbalance on liver regeneration in GSD Ia, and the expression of glucose transporters and ChREBP during HCC progression in humans. Based on our findings, we will discuss these different aspects.
1. ChREBP inhibition aggravates metabolic liver disease in GSD Ia mice
The activity of ChREBP and its target genes, which are involved in glycolysis and lipogenesis, are markedly increased in liver-specific GSD Ia mice (L-G6pc-/-) (6-8).
ChREBP silencing in hepatocytes (9) and in ChREBP null mice (10) leads to a significant reduction in lipid synthesis and TG accumulation in liver. Since GSD I is characterized by massive hepatic fat accumulation, we hypothesized that downregulation of hepatic ChREBP expression in GSD Ia mice alleviates steatosis. However, in Chapter 2, upon specific knockdown of hepatic ChREBP in L-G6pc-/- mice,
we observed enlarged livers and a higher content of G6P, glycogen and lipids in livers compared to controls, although the expression of glycolytic and de novo lipogenic genes (L-pk, Acly, Acc, Fas, Elovl6, and Scd1) was significantly reduced upon ChREBP knockdown. Hepatic TG metabolism is balanced by fatty acid uptake (either through dietary sources or from lipolysis of fat tissue), DNL, fatty acid oxidation, and export of lipids from the liver via VLDL secretion (11). We found that DNL was suppressed, fatty acid oxidation appeared to be increased but adipose tissue lipolysis and plasma NEFA levels were unchanged upon ChREBP knockdown in L-G6pc-/- mice. Moreover, we
found VLDL-TG secretion and VLDL particle size to be markedly decreased after
163
ChREBP knockdown in both L-G6pc+/+ and L-G6pc-/- mice. Therefore, the increased TG
accumulation in GSD Ia livers after ChREBP knockdown was mainly caused by the strong inhibition of hepatic VLDL-TG secretion. The changes in hepatic TG metabolism upon ChREBP knockdown in L-G6pc-/- mice are depicted in Figure 1.
Taken together, although hepatic ChREBP downregulation has been reported to be beneficial in diabetic mice, it apparently aggravates liver disease in L-G6pc-/- mice by
decreasing hepatic VLDL-TG secretion. Therefore, ChREBP inhibition is not promising for the treatment of liver disease in GSD Ia.
Figure 1. Increased liver TG accumulation upon ChREBP knockdown was mainly caused by
decreased VLDL-TG export in L-G6pc-/- mice. FFA: free fatty acids; CM: chylomicron; TG:
triglycerides; FAO: fatty acid oxidation; DNL: de novo lipogenesis; VLDL: very low density lipoprotein.
Hepatic ChREBP: “friend” or “foe” in metabolic liver disease development?
Hepatic steatosis is a big health threat as it can progress to chronic liver inflammation. Severe inflammation can further develop to fibrosis and cirrhosis and, in a long term, increases the risk of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) formation (12, 13). ChREBP and SREBP-1c play essential roles in hepatic lipid synthesis (14). Adenoviral-mediated ChREBP overexpression has been shown to result in increased hepatic fat accumulation (15). Both whole-body deletion of ChREBP in high-carbohydrate diet fed and obese diabetic mice, or a liver-specific ChREBP knockdown in obese diabetic mice reduced hepatic steatosis (16, 17). Based on these findings, ChREBP has been considered a “foe” in NAFLD development, and inhibition of hepatic ChREBP could potentially be used to prevent hepatic steatosis. However, in our study (Chapter 2), ChREBP inhibition aggravated the phenotype of fatty liver in L-G6pc-/- mice. In other words, ChREBP protects against hepatic
metabolic disease in L-G6pc-/- mice. In this aspect, ChREBP can be considered as a
“friend” in GSD Ia mice. Moreover, ChREBP has also been reported to protect the
162
General discussion
Metabolic homeostasis is tightly regulated by various factors, including the availability of nutrients (e.g., glucose, fatty acids and amino acids), hormones (e.g., insulin and glucagon) and neuronal control (e.g., sympatic and parasympatic systems). The regulatory proteins that modulate metabolic changes respond to changes in nutrient availability are called nutrient sensors. Transcription factors represent an important group of nutrient sensors. So far, several transcription factors that regulate the transcription of metabolic genes have been identified, including CREB, FOXO1, ChREBP, SREBP, PGC-1α, CRTC2, LXR, FXR, PPARs. Chronic metabolic imbalances (such as energy overload or unbalanced nutrition) disturb systemic physiology leading to the development of metabolic diseases such as obesity, diabetes, NAFLD and even cancer (1-3).
In this thesis, a mouse model for GSD Ia was employed to study the physiological and molecular mechanisms that link hepatic glucose (G6P) imbalance to liver function and -health. Although GSD Ia itself is a rare disease it is associated with several ‘general’ metabolic perturbations such as hypoglycemia, hyperlipidaemia, NAFLD, lactic acidaemia and hyperuricaemia (4). Therefore, GSD I can be considered as a valuable model to investigate the pathophysiology of common metabolic diseases that show hepatic glucose imbalance, such as type 2 diabetes (4, 5). Metabolic diseases such as diabetes and NAFLD become more and more prevalent (4). In this thesis, we focused on the consequences of enhanced ChREBP activity for hepatocyte function and liver disease development in GSD Ia, the impact of metabolic imbalance on liver regeneration in GSD Ia, and the expression of glucose transporters and ChREBP during HCC progression in humans. Based on our findings, we will discuss these different aspects.
1. ChREBP inhibition aggravates metabolic liver disease in GSD Ia mice
The activity of ChREBP and its target genes, which are involved in glycolysis and lipogenesis, are markedly increased in liver-specific GSD Ia mice (L-G6pc-/-) (6-8).
ChREBP silencing in hepatocytes (9) and in ChREBP null mice (10) leads to a significant reduction in lipid synthesis and TG accumulation in liver. Since GSD I is characterized by massive hepatic fat accumulation, we hypothesized that downregulation of hepatic ChREBP expression in GSD Ia mice alleviates steatosis. However, in Chapter 2, upon specific knockdown of hepatic ChREBP in L-G6pc-/- mice,
we observed enlarged livers and a higher content of G6P, glycogen and lipids in livers compared to controls, although the expression of glycolytic and de novo lipogenic genes (L-pk, Acly, Acc, Fas, Elovl6, and Scd1) was significantly reduced upon ChREBP knockdown. Hepatic TG metabolism is balanced by fatty acid uptake (either through dietary sources or from lipolysis of fat tissue), DNL, fatty acid oxidation, and export of lipids from the liver via VLDL secretion (11). We found that DNL was suppressed, fatty acid oxidation appeared to be increased but adipose tissue lipolysis and plasma NEFA levels were unchanged upon ChREBP knockdown in L-G6pc-/- mice. Moreover, we
found VLDL-TG secretion and VLDL particle size to be markedly decreased after
163
ChREBP knockdown in both L-G6pc+/+ and L-G6pc-/- mice. Therefore, the increased TG
accumulation in GSD Ia livers after ChREBP knockdown was mainly caused by the strong inhibition of hepatic VLDL-TG secretion. The changes in hepatic TG metabolism upon ChREBP knockdown in L-G6pc-/- mice are depicted in Figure 1.
Taken together, although hepatic ChREBP downregulation has been reported to be beneficial in diabetic mice, it apparently aggravates liver disease in L-G6pc-/- mice by
decreasing hepatic VLDL-TG secretion. Therefore, ChREBP inhibition is not promising for the treatment of liver disease in GSD Ia.
Figure 1. Increased liver TG accumulation upon ChREBP knockdown was mainly caused by
decreased VLDL-TG export in L-G6pc-/- mice. FFA: free fatty acids; CM: chylomicron; TG:
triglycerides; FAO: fatty acid oxidation; DNL: de novo lipogenesis; VLDL: very low density lipoprotein.
Hepatic ChREBP: “friend” or “foe” in metabolic liver disease development?
Hepatic steatosis is a big health threat as it can progress to chronic liver inflammation. Severe inflammation can further develop to fibrosis and cirrhosis and, in a long term, increases the risk of hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) formation (12, 13). ChREBP and SREBP-1c play essential roles in hepatic lipid synthesis (14). Adenoviral-mediated ChREBP overexpression has been shown to result in increased hepatic fat accumulation (15). Both whole-body deletion of ChREBP in high-carbohydrate diet fed and obese diabetic mice, or a liver-specific ChREBP knockdown in obese diabetic mice reduced hepatic steatosis (16, 17). Based on these findings, ChREBP has been considered a “foe” in NAFLD development, and inhibition of hepatic ChREBP could potentially be used to prevent hepatic steatosis. However, in our study (Chapter 2), ChREBP inhibition aggravated the phenotype of fatty liver in L-G6pc-/- mice. In other words, ChREBP protects against hepatic
metabolic disease in L-G6pc-/- mice. In this aspect, ChREBP can be considered as a
“friend” in GSD Ia mice. Moreover, ChREBP has also been reported to protect the
163
164
liver from hepatotoxicity following highfructose diets (18, 19). Therefore, the impact of hepatic ChREBP on liver disease is context-dependent, which limits its therapeutic potential.
2. ChREBP-regulated pathways
Tm6sf2: a novel ChREBP target gene
In Chapter 2, we identified TM6SF2 as a target of ChREBP in mouse liver. Human TM6SF2 p.E167K variant carriers are at increased risk of developing NAFLD (20, 21). The Tm6sf2 gene encodes transmembrane 6 superfamily member 2 is involved in liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content (22). In human hepatoma Huh7 and HepG2 cells, TM6SF2 downregulation reduced the secretion of TG-rich lipoproteins and increased hepatic lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis (23). Knockdown of murine Tm6sf2 increased liver triglyceride content 3-fold and decreased VLDL secretion by 50% (24). These results indicate that hepatic TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function contributes to NAFLD. Therefore, the liver-specific ChREBP knockdown in our experiment very likely inhibited VLDL excretion via Tm6sf2 suppression and promoted the further accumulation of TG in the liver while lowering plasma TG (Figure 2). TM6SF2 is also highly expressed in the small intestine. Similar to what was observed upon hepatic TM6SF2 deletion (23, 24), it has been reported that depletion of TM6SF2 in human Caco-2 enterocyte cells, or of its homolog in zebrafish, lead to lipid accumulation in enterocytes and lowered postprandial serum TG upon a high fat challenge (25). As circulating TG originates from both liver-derived VLDL or intestinally-derived chylomicrons, TM6SF2 not only regulates lipid homeostasis in hepatoyctes and enterocytes, but also controls plasma TG concentrations by regulating the intracellular packaging TG-rich lipoproteins and their secretion into the circulation. These results furthermore indicate that ChREBP may play an essential role in control of TG release into the circulation by enterocytes via regulating TM6SF2.
Hepatic G6P-ChREBP-CYP8B1 signaling regulates bile acid synthesis
Besides promoting the absorption of dietary lipids and vitamins, bile acids act as signaling molecules that play important roles in control of glucose, lipid and energy metabolism (26, 27). In Chapter 4, we found that hepatic mRNA levels of Cyp8b1, a key enzyme controlling bile acid synthesis, were significantly increased in S4048-induced GSD Ib and L-G6pc-/- mice. This induction of Cyp8b1 expression was
abolished upon hepatic ChREBP knockdown, indicating a ChREBP dependent regulation of Cyp8b1. However, ChREBP did not directly control hepatic Cyp8b1 expression, because ChREBP did not transactivate the Cyp8b1 promoter in a cell reporter assay. CYP8B1 determines the production of cholic acid (CA) and its hydrophobic metabolite deoxycholic acid (DCA) (28). Therefore increased Cyp8b1 activity in L-G6pc-/- mice was predicted to increase bile hydrophobicity and this was
proved in our experiment. Moreover, hydrophobic bile acids effectively promote the
165
absorption of dietary lipids and sterols (29, 30) while a more hydrophilic bile acid pool is associated with enhanced intestinal cholesterol excretion (31). Accordingly, L-G6pc-/- mice exhibited less fecal cholesterol excretion as compared to the wildtype
mice, consistent with enhanced cholesterol absorption upon increased G6P-ChREBP-Cyp8b1 signaling. Taken together, we found that intrahepatic G6P regulates bile acid metabolism via a ChREBP-dependent induction of Cyp8b1, resulting in an increased hydrophobicity of the bile acid pool and then causes reduced fecal cholesterol loss (Figure 2).
Figure 2. Revised model for ChREBP-dependent regulation of liver metabolism with newly
identified targets indicated in red. ChREBP regulates glucose metabolism via promoting glycolysis, lipogenesis, and PPP. When G6P level is high in hepatocytes, ChREBP inhibits sweet taste preferences via FGF21 secretion to brain. FGF21 also promotes weight loss and improves insulin sensitivity through increased thermogenesis, glucose uptake, and lipid oxidation. ChREBP controls VLDL-TG secretion via targeting hepatic TM6SF2. Bile acids are also regulated by ChREBP by the controlling of CYP8B1, which contributes to increased hydrophobic bile acid pool in intestine, thus inhibiting intestine cholesterol excretion.
It was previously reported that insulin inhibits hepatic Cyp8b1 expression via transcription factor FOXO1 (28) while in our study, we found that intrahepatic glucose (G6P) induces hepatic Cyp8b1 expression to promote hydrophobic bile acid synthesis. Thus, intrahepatic glucose and insulin appear to have an opposite effect on Cyp8b1 expression. In contrast, glucose and insulin both promote the expression of Cyp7a1,
164
liver from hepatotoxicity following highfructose diets (18, 19). Therefore, the impact of hepatic ChREBP on liver disease is context-dependent, which limits its therapeutic potential.
2. ChREBP-regulated pathways
Tm6sf2: a novel ChREBP target gene
In Chapter 2, we identified TM6SF2 as a target of ChREBP in mouse liver. Human TM6SF2 p.E167K variant carriers are at increased risk of developing NAFLD (20, 21). The Tm6sf2 gene encodes transmembrane 6 superfamily member 2 is involved in liver fat metabolism influencing triglyceride secretion and hepatic lipid droplet content (22). In human hepatoma Huh7 and HepG2 cells, TM6SF2 downregulation reduced the secretion of TG-rich lipoproteins and increased hepatic lipid droplet content, whereas TM6SF2 overexpression reduced liver cell steatosis (23). Knockdown of murine Tm6sf2 increased liver triglyceride content 3-fold and decreased VLDL secretion by 50% (24). These results indicate that hepatic TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function contributes to NAFLD. Therefore, the liver-specific ChREBP knockdown in our experiment very likely inhibited VLDL excretion via Tm6sf2 suppression and promoted the further accumulation of TG in the liver while lowering plasma TG (Figure 2). TM6SF2 is also highly expressed in the small intestine. Similar to what was observed upon hepatic TM6SF2 deletion (23, 24), it has been reported that depletion of TM6SF2 in human Caco-2 enterocyte cells, or of its homolog in zebrafish, lead to lipid accumulation in enterocytes and lowered postprandial serum TG upon a high fat challenge (25). As circulating TG originates from both liver-derived VLDL or intestinally-derived chylomicrons, TM6SF2 not only regulates lipid homeostasis in hepatoyctes and enterocytes, but also controls plasma TG concentrations by regulating the intracellular packaging TG-rich lipoproteins and their secretion into the circulation. These results furthermore indicate that ChREBP may play an essential role in control of TG release into the circulation by enterocytes via regulating TM6SF2.
Hepatic G6P-ChREBP-CYP8B1 signaling regulates bile acid synthesis
Besides promoting the absorption of dietary lipids and vitamins, bile acids act as signaling molecules that play important roles in control of glucose, lipid and energy metabolism (26, 27). In Chapter 4, we found that hepatic mRNA levels of Cyp8b1, a key enzyme controlling bile acid synthesis, were significantly increased in S4048-induced GSD Ib and L-G6pc-/- mice. This induction of Cyp8b1 expression was
abolished upon hepatic ChREBP knockdown, indicating a ChREBP dependent regulation of Cyp8b1. However, ChREBP did not directly control hepatic Cyp8b1 expression, because ChREBP did not transactivate the Cyp8b1 promoter in a cell reporter assay. CYP8B1 determines the production of cholic acid (CA) and its hydrophobic metabolite deoxycholic acid (DCA) (28). Therefore increased Cyp8b1 activity in L-G6pc-/- mice was predicted to increase bile hydrophobicity and this was
proved in our experiment. Moreover, hydrophobic bile acids effectively promote the
165
absorption of dietary lipids and sterols (29, 30) while a more hydrophilic bile acid pool is associated with enhanced intestinal cholesterol excretion (31). Accordingly, L-G6pc-/- mice exhibited less fecal cholesterol excretion as compared to the wildtype
mice, consistent with enhanced cholesterol absorption upon increased G6P-ChREBP-Cyp8b1 signaling. Taken together, we found that intrahepatic G6P regulates bile acid metabolism via a ChREBP-dependent induction of Cyp8b1, resulting in an increased hydrophobicity of the bile acid pool and then causes reduced fecal cholesterol loss (Figure 2).
Figure 2. Revised model for ChREBP-dependent regulation of liver metabolism with newly
identified targets indicated in red. ChREBP regulates glucose metabolism via promoting glycolysis, lipogenesis, and PPP. When G6P level is high in hepatocytes, ChREBP inhibits sweet taste preferences via FGF21 secretion to brain. FGF21 also promotes weight loss and improves insulin sensitivity through increased thermogenesis, glucose uptake, and lipid oxidation. ChREBP controls VLDL-TG secretion via targeting hepatic TM6SF2. Bile acids are also regulated by ChREBP by the controlling of CYP8B1, which contributes to increased hydrophobic bile acid pool in intestine, thus inhibiting intestine cholesterol excretion.
It was previously reported that insulin inhibits hepatic Cyp8b1 expression via transcription factor FOXO1 (28) while in our study, we found that intrahepatic glucose (G6P) induces hepatic Cyp8b1 expression to promote hydrophobic bile acid synthesis. Thus, intrahepatic glucose and insulin appear to have an opposite effect on Cyp8b1 expression. In contrast, glucose and insulin both promote the expression of Cyp7a1,
165
166
which encodes the rate-limiting enzyme in the classical bile acid biosynthetic pathway. Type 1 and type 2 diabetic rodents exhibit increased hepatic expression levels of Cyp7a1 (32) and an enlarged bile acid pool (33), indicating that blood glucose levels regulate Cyp7a1 expression independently of hepatic G6P accumulation. Type 2 diabetic mice exhibit elevated hepatic Cyp8b1 expression and a corresponding increase in 12-hydroxylated bile acids (28, 33), which has been attributed to insulin resistance and consequent FOXO activation (28). As hepatic ChREBP is also activated in type 2 diabetic mice and humans (17, 34), increased G6P-ChREBP signaling potentially contributes to perturbed bile acid metabolism in type 2 diabetes. Follow-up research is therefore essential to establish the impact of intrahepatic G6P-ChREBP signaling on bile acid pool composition in humans and its contribution to perturbed bile acid metabolism in type 2 diabetes.
3. ChREBP inhibition sensitizes to advanced liver disease in L-G6pc-/- mice
ChREBP inhibition induces hepatocyte death, hepatocyte regeneration, and DNA damage in GSD Ia mice
It has been reported that ChREBP directs glucose metabolism to anabolic pathways and is required for cellular proliferation in vitro (35-38) and in vivo (39, 40). ChREBP knockdown in HCT116 colorectal cancer cells and in HepG2 hepatoblastoma cells inhibited cell proliferation and induced p53-dependent cell cycle arrest. The reduction of ChREBP expression concomitantly inhibited aerobic glycolysis as well as lipid and nucleotide synthesis (35). ChREBP suppression furthermore reduced glucose-induced pancreatic β-cell proliferation and caused cell cycle arrest, while overexpression of ChREBP promoted glucose-induced pancreatic β-cell proliferation (36, 41). These results indicate an important role of ChREBP in regulating cell cycle and -proliferation. However, how ChREBP influences these cell biological functions in non-tumor derived human liver cells and in vivo has not been reported. In Chapter 3, we confirmed that ChREBP knockdown inhibited cell proliferation and induced cell cycle arrest in immortalized human hepatocytes (IHH cells), and showed that this was associated with p53 pathway activation. On the other hand, short-term (10 day) knockdown of hepatic ChREBP expression in L-G6pc-/- mice increased the number of
ki67 and pH3 positive hepatocytes as well as mitotic hepatocytes, indicating enhanced hepatocyte proliferation and mitosis upon normalization of hepatic ChREBP activity in L-G6pc-/- mice while at the same time, there was more hepatocyte
death. The expression of γH2AX, a marker of DNA damage (42), tended to be increased upon ChREBP knockdown in L-G6pc-/- mice. Gene set enrichment analysis
(GSEA) of RNA-sequencing data indicated that hepatic ChREBP knockdown resulted in a significant enrichment of gene signatures marking p53 activation and cell cycle regulation in both L-G6pc+/+ and L-G6pc-/- mice. In addition, GSEA revealed a
significant enrichment of genes associated with chromosomal instability in L-G6pc
-/-mice upon ChREBP knockdown. The latter finding suggests that hepatic ChREBP downregulation increased genomic instability in L-G6pc-/- mice. Taken together, our
data indicate that short-term hepatic ChREBP knockdown in GSD Ia simultaneously induces proliferation and death, while aggravating genomic instability. These findings
167
may suggest that hepatic ChREBP activation in GSD Ia delays liver tumor development.
Increased susceptibility for NASH and liver tumor development upon hepatic ChREBP knockdown in L-G6pc-/- mice
In Chapter 2 we observed enhanced hepatic TG accumulation upon ChREBP inhibition in L-G6pc-/- mice. Increased TG content can induce chronic inflammation
which in turn promotes the development of non-alcoholic steatohepatitis (NASH) (12). γH2AX staining and GSEA analysis (Chapter 3) showed increased DNA damage upon hepatic ChREBP knockdown in L-G6pc-/- mice. It has been reported that 70% of
adult GSD I patients develop multiple hepatocellular adenomas (HCAs), with about 10% risk of transformation to hepatocellular carcinomas (HCC) (43-45). Moreover, it has been shown that G2/M DNA damage checkpoint regulation is the top-ranked pathways at the early stage of HCC by Ingenuity Pathway Analysis (46). Therefore, it is conceivable that HCA and HCC development is accelerated in L-G6pc-/- mice after
hepatic ChREBP knockdown. However, to prove this hypothesis, long-term studies are warranted.
Role of ChREBP on hepatocyte proliferation in vivo versus in vitro
In Chapter 3, we found that ChREBP knockdown promoted hepatocyte proliferation in GSD Ia mice but inhibited cell proliferation in IHH cells. Thus ChREBP appears to exert differential effects on cell proliferation in vivo and in vitro. This difference may be related to the induction of compensatory hepatocyte regeneration in response to cell death in vivo (47) but not in vitro. Thus, the role of ChREBP in hepatocyte proliferation might be context-dependent. In vivo, glucose is metabolized in hepatocytes to provide energy, stored as glycogen or TGs, or redistributed to extra-hepatic tissues in the form of VLDL-TG. In vitro, however, glucose is mainly metabolized to support cell proliferation. This discrepancy may contribute to the differential effects of ChREBP knockdown in vivo versus in vitro. Cellular apoptosis, assessed by caspase 3 activity in vivo and by AnnexinV/PI staining in vitro, was not altered upon ChREBP knockdown either in vivo or in vitro. These findings indicate that ChREBP has no significant effect on hepatocyte apoptosis, even though it has been reported that ChREBP down-regulation promoted cell apoptosis in HCT116 colorectal cancer cells (35) and murine macrophages (48). The induction of p21 upon ChREBP knockdown occured both in vivo and in vitro, and is suggestive of G2/M cell cycle arrest under these conditions.
Role of ChREBP in liver tumorigenesis
Many cancer cells are characterized by the “Warburg effect”, which refers to increased glucose uptake, glycolysis, PPP activity and lactate production despite sufficient oxygen supply (49, 50). Increased glycolysis and PPP activity promote the synthesis of amino acids, lipids, nucleotides and NADPH to support cell growth and proliferation and maintain cellular redox balance (49, 50). Growing evidence indicates that ChREBP plays an essential role in tumorigenesis. Tong et al. reported that lowering ChREBP expression 1) redirected glucose metabolism from aerobic
166
which encodes the rate-limiting enzyme in the classical bile acid biosynthetic pathway. Type 1 and type 2 diabetic rodents exhibit increased hepatic expression levels of Cyp7a1 (32) and an enlarged bile acid pool (33), indicating that blood glucose levels regulate Cyp7a1 expression independently of hepatic G6P accumulation. Type 2 diabetic mice exhibit elevated hepatic Cyp8b1 expression and a corresponding increase in 12-hydroxylated bile acids (28, 33), which has been attributed to insulin resistance and consequent FOXO activation (28). As hepatic ChREBP is also activated in type 2 diabetic mice and humans (17, 34), increased G6P-ChREBP signaling potentially contributes to perturbed bile acid metabolism in type 2 diabetes. Follow-up research is therefore essential to establish the impact of intrahepatic G6P-ChREBP signaling on bile acid pool composition in humans and its contribution to perturbed bile acid metabolism in type 2 diabetes.
3. ChREBP inhibition sensitizes to advanced liver disease in L-G6pc-/- mice
ChREBP inhibition induces hepatocyte death, hepatocyte regeneration, and DNA damage in GSD Ia mice
It has been reported that ChREBP directs glucose metabolism to anabolic pathways and is required for cellular proliferation in vitro (35-38) and in vivo (39, 40). ChREBP knockdown in HCT116 colorectal cancer cells and in HepG2 hepatoblastoma cells inhibited cell proliferation and induced p53-dependent cell cycle arrest. The reduction of ChREBP expression concomitantly inhibited aerobic glycolysis as well as lipid and nucleotide synthesis (35). ChREBP suppression furthermore reduced glucose-induced pancreatic β-cell proliferation and caused cell cycle arrest, while overexpression of ChREBP promoted glucose-induced pancreatic β-cell proliferation (36, 41). These results indicate an important role of ChREBP in regulating cell cycle and -proliferation. However, how ChREBP influences these cell biological functions in non-tumor derived human liver cells and in vivo has not been reported. In Chapter 3, we confirmed that ChREBP knockdown inhibited cell proliferation and induced cell cycle arrest in immortalized human hepatocytes (IHH cells), and showed that this was associated with p53 pathway activation. On the other hand, short-term (10 day) knockdown of hepatic ChREBP expression in L-G6pc-/- mice increased the number of
ki67 and pH3 positive hepatocytes as well as mitotic hepatocytes, indicating enhanced hepatocyte proliferation and mitosis upon normalization of hepatic ChREBP activity in L-G6pc-/- mice while at the same time, there was more hepatocyte
death. The expression of γH2AX, a marker of DNA damage (42), tended to be increased upon ChREBP knockdown in L-G6pc-/- mice. Gene set enrichment analysis
(GSEA) of RNA-sequencing data indicated that hepatic ChREBP knockdown resulted in a significant enrichment of gene signatures marking p53 activation and cell cycle regulation in both L-G6pc+/+ and L-G6pc-/- mice. In addition, GSEA revealed a
significant enrichment of genes associated with chromosomal instability in L-G6pc
-/-mice upon ChREBP knockdown. The latter finding suggests that hepatic ChREBP downregulation increased genomic instability in L-G6pc-/- mice. Taken together, our
data indicate that short-term hepatic ChREBP knockdown in GSD Ia simultaneously induces proliferation and death, while aggravating genomic instability. These findings
167
may suggest that hepatic ChREBP activation in GSD Ia delays liver tumor development.
Increased susceptibility for NASH and liver tumor development upon hepatic ChREBP knockdown in L-G6pc-/- mice
In Chapter 2 we observed enhanced hepatic TG accumulation upon ChREBP inhibition in L-G6pc-/- mice. Increased TG content can induce chronic inflammation
which in turn promotes the development of non-alcoholic steatohepatitis (NASH) (12). γH2AX staining and GSEA analysis (Chapter 3) showed increased DNA damage upon hepatic ChREBP knockdown in L-G6pc-/- mice. It has been reported that 70% of
adult GSD I patients develop multiple hepatocellular adenomas (HCAs), with about 10% risk of transformation to hepatocellular carcinomas (HCC) (43-45). Moreover, it has been shown that G2/M DNA damage checkpoint regulation is the top-ranked pathways at the early stage of HCC by Ingenuity Pathway Analysis (46). Therefore, it is conceivable that HCA and HCC development is accelerated in L-G6pc-/- mice after
hepatic ChREBP knockdown. However, to prove this hypothesis, long-term studies are warranted.
Role of ChREBP on hepatocyte proliferation in vivo versus in vitro
In Chapter 3, we found that ChREBP knockdown promoted hepatocyte proliferation in GSD Ia mice but inhibited cell proliferation in IHH cells. Thus ChREBP appears to exert differential effects on cell proliferation in vivo and in vitro. This difference may be related to the induction of compensatory hepatocyte regeneration in response to cell death in vivo (47) but not in vitro. Thus, the role of ChREBP in hepatocyte proliferation might be context-dependent. In vivo, glucose is metabolized in hepatocytes to provide energy, stored as glycogen or TGs, or redistributed to extra-hepatic tissues in the form of VLDL-TG. In vitro, however, glucose is mainly metabolized to support cell proliferation. This discrepancy may contribute to the differential effects of ChREBP knockdown in vivo versus in vitro. Cellular apoptosis, assessed by caspase 3 activity in vivo and by AnnexinV/PI staining in vitro, was not altered upon ChREBP knockdown either in vivo or in vitro. These findings indicate that ChREBP has no significant effect on hepatocyte apoptosis, even though it has been reported that ChREBP down-regulation promoted cell apoptosis in HCT116 colorectal cancer cells (35) and murine macrophages (48). The induction of p21 upon ChREBP knockdown occured both in vivo and in vitro, and is suggestive of G2/M cell cycle arrest under these conditions.
Role of ChREBP in liver tumorigenesis
Many cancer cells are characterized by the “Warburg effect”, which refers to increased glucose uptake, glycolysis, PPP activity and lactate production despite sufficient oxygen supply (49, 50). Increased glycolysis and PPP activity promote the synthesis of amino acids, lipids, nucleotides and NADPH to support cell growth and proliferation and maintain cellular redox balance (49, 50). Growing evidence indicates that ChREBP plays an essential role in tumorigenesis. Tong et al. reported that lowering ChREBP expression 1) redirected glucose metabolism from aerobic
167
168
glycolysis to mitochondrial respiration; 2) decreased the synthesis of lipids and nucleotides; 3) reduced the tumorigenic potential of xenografts in nude mice; 4) reduced cell proliferation and induced p53-dependent cell cycle arrest in colorectal cancer cells (35). It has also been reported that ChREBP regulates normal and neoplastic hepatocyte proliferation in mice (39, 40). Similarly, in our study (Chapter 3), we found that ChREBP suppression in IHHs inhibited glycolytic and lipogenic gene expression, activated p53, and reduced cell proliferation. These findings suggest that ChREBP plays a key role in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity in proliferating cells. Thus, enhanced ChREBP activity likely promotes cell proliferation and tumorigenesis. Such a tumor-supporting role is consistent with the reported positive correlation between ChREBP protein expression and tumor progression in breast cancer and prostate cancer (51). Moreover, ChREBP deletion in leukemic mice promoted the differentiation of leukemia-initiating cells and markedly reduced the survival, thus ChREBP could act as a tumor suppressor (52). With regard to the molecular mechanisms, ChREBP was reported to regulate cell cycle-related genes, but not by direct binding, in benign β cell proliferation and malignant prostate cancer (36, 53) and some studies proposed that ChREBP interacts with cancer-related factors (e.g. oncogens, c-myc and HIF) to mediate tumor-related microenvironment changes (38, 54). Recently, it was reported that ChREBP deletion strongly delayed or impaired hepatocarcinogenesis driven by AKT or AKT/c-Met or β-catenin/YAP overexpression in mice, further supporting the tumor-promoting effect of ChREBP (38-40, 55-57), which is in agreement with our finding that ChREBP expression tended to be increased in some cases of human HCC (Chapter 5). However, in the same report, it was also found that ChREBP deletion had no effect on HCC development driven by the co-expression of AKT and N-Ras proto-oncogenes (39). These findings may explain our findings in Chapter 5 that some, but not all, human HCC samples show increased ChREBP expression compared to normal liver, due to the heterogeneity in underlying gene mutations, i.e., some HCC are c-Met-, while others are N-Ras mutated. Moreover, ChREBP downregulation was found to play an essential role during the epithelial-to-mesenchymal transformation in non-small-cell lung carcinoma metastasis (58). Combined, these data suggest that ChREBP expression follows spatial and temporal dynamics in a tumor-specific manner.
ChREBP and GLUT1 expression increased while GLUT2 decreased upon human HCC progression
Increased glycolysis in cancer cells is accompanied by enhanced glucose uptake (59), and this is mediated mainly by increased expression of glucose transporters (GLUTs) (60, 61) and hexokinase (HK) enzymes (62, 63). For example, GLUT1 expression has been shown to be increased in a variety of malignancies and GLUT1 overexpression is associated with invasiveness and poor overall survival in various malignant tumors [44-48]. In our study (Chapter 5), we found that GLUT1 was significantly increased in human HCC compared to normal liver tissues and that its expression was positively correlated to liver malignancy. On the contrary, GLUT2 protein expression was significantly reduced in HCC as compared to normal liver tissue, and its expression in HCC was inversely associated with liver malignancy. Under normal conditions, GLUT2
169
is the most abundant glucose transporter in hepatocytes (64). Our findings are interesting given that GLUT1 has a high affinity for glucose (65, 66) but GLUT2 is a low-affinity glucose transporter (67, 68). Thus, these changes indicate that upon HCC progression the liver loses its sensitivity to circulating glucose levels. Moreover, the transition from GLUT2- to GLUT1-mediated glucose metabolism may mark hepatocellular differentiation upon HCC progression.
4. Consequences of glucose and lipid imbalance on hepatocyte proliferation in GSD Ia mice
Disturbed cell proliferation before and after partial hepatectomy in GSD Ia mice
A long-term complication of GSD1a is the frequent development of hepatocellular adenomas (HCAs) with the risk of progressing into hepatocellular carcinoma (44, 69). The mechanisms underlying GSD Ia-associated tumorigenesis are unknown and it is unclear which early events drive the transformation from G6pc-deficient hepatocytes into tumorous cells. Therefore, in Chapter 6, we monitored hepatocyte proliferation in L-G6pc-/- mice in response to 2/3 hepatectomy (PHx) performed at 10 days after
gene deletion. We found that the L-G6pc-/- mice exhibited more cell proliferation than
wildtype mice prior to the surgery. This may reflect a compensatory mechanism for the increased cell death in L-G6pc-/- mice as reported in Chapter 3. Besides, enhanced
proliferation may also mark tumor initiation in G6pc deficient hepatocytes. It has been reported that chronic hepatocyte loss caused by toxic agents, alcohol and steatohepatitis lead to continuous hepatocyte proliferation and that these constitutive proliferating hepatocytes undergo transition from polyploidy to diploidy. Consequently, the stochastically genomic alteration in growth regulatory genes (such as p53, EGFR, telomerase promoter) of the diploid hepatocyte results in the formation of HCC (70).
Our data (Chapter 6) furthermore showed an accelerated presence of mitotic figures in G6pc-deficient as compared to wildtypehepatocytes. This may be caused by the increased TG accumulation and recurrent hypoglycemic episodes in G6pcdeficient livers (71). TGs provide energy for DNA replication and TG-derived fatty acids provide substrates for phospholipid synthesis, which is the main component of cell membranes (72, 73). Intravenous or enteral glucose supplementation suppresses both PHx- (74, 75) and toxin- (76, 77) induced hepatocellular proliferation. Similarly, caloric restriction accelerates the onset of hepatocellular proliferation in response to surgical- or toxin-induced hepatic insufficiency (78, 79). Strikingly, we found that almost all mitotic hepatocytes in L-G6pc-/- mice formed anaphase bridges, indicating
genomic instability and replication stress. The presence of anaphase bridges is highly associated with cancer (80). Therefore, we speculate that severe genome instability in G6pc-deficient livers contributed to mitosis inhibition during liver regeneration. The genome instability may be caused by hepatic lipid accumulation in the livers of L-G6pc-/- mice, because it has been reported that lipid peroxidation products and
reactive oxygen species can result in DNA damage and genomic alterations (70).
168
glycolysis to mitochondrial respiration; 2) decreased the synthesis of lipids and nucleotides; 3) reduced the tumorigenic potential of xenografts in nude mice; 4) reduced cell proliferation and induced p53-dependent cell cycle arrest in colorectal cancer cells (35). It has also been reported that ChREBP regulates normal and neoplastic hepatocyte proliferation in mice (39, 40). Similarly, in our study (Chapter 3), we found that ChREBP suppression in IHHs inhibited glycolytic and lipogenic gene expression, activated p53, and reduced cell proliferation. These findings suggest that ChREBP plays a key role in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity in proliferating cells. Thus, enhanced ChREBP activity likely promotes cell proliferation and tumorigenesis. Such a tumor-supporting role is consistent with the reported positive correlation between ChREBP protein expression and tumor progression in breast cancer and prostate cancer (51). Moreover, ChREBP deletion in leukemic mice promoted the differentiation of leukemia-initiating cells and markedly reduced the survival, thus ChREBP could act as a tumor suppressor (52). With regard to the molecular mechanisms, ChREBP was reported to regulate cell cycle-related genes, but not by direct binding, in benign β cell proliferation and malignant prostate cancer (36, 53) and some studies proposed that ChREBP interacts with cancer-related factors (e.g. oncogens, c-myc and HIF) to mediate tumor-related microenvironment changes (38, 54). Recently, it was reported that ChREBP deletion strongly delayed or impaired hepatocarcinogenesis driven by AKT or AKT/c-Met or β-catenin/YAP overexpression in mice, further supporting the tumor-promoting effect of ChREBP (38-40, 55-57), which is in agreement with our finding that ChREBP expression tended to be increased in some cases of human HCC (Chapter 5). However, in the same report, it was also found that ChREBP deletion had no effect on HCC development driven by the co-expression of AKT and N-Ras proto-oncogenes (39). These findings may explain our findings in Chapter 5 that some, but not all, human HCC samples show increased ChREBP expression compared to normal liver, due to the heterogeneity in underlying gene mutations, i.e., some HCC are c-Met-, while others are N-Ras mutated. Moreover, ChREBP downregulation was found to play an essential role during the epithelial-to-mesenchymal transformation in non-small-cell lung carcinoma metastasis (58). Combined, these data suggest that ChREBP expression follows spatial and temporal dynamics in a tumor-specific manner.
ChREBP and GLUT1 expression increased while GLUT2 decreased upon human HCC progression
Increased glycolysis in cancer cells is accompanied by enhanced glucose uptake (59), and this is mediated mainly by increased expression of glucose transporters (GLUTs) (60, 61) and hexokinase (HK) enzymes (62, 63). For example, GLUT1 expression has been shown to be increased in a variety of malignancies and GLUT1 overexpression is associated with invasiveness and poor overall survival in various malignant tumors [44-48]. In our study (Chapter 5), we found that GLUT1 was significantly increased in human HCC compared to normal liver tissues and that its expression was positively correlated to liver malignancy. On the contrary, GLUT2 protein expression was significantly reduced in HCC as compared to normal liver tissue, and its expression in HCC was inversely associated with liver malignancy. Under normal conditions, GLUT2
169
is the most abundant glucose transporter in hepatocytes (64). Our findings are interesting given that GLUT1 has a high affinity for glucose (65, 66) but GLUT2 is a low-affinity glucose transporter (67, 68). Thus, these changes indicate that upon HCC progression the liver loses its sensitivity to circulating glucose levels. Moreover, the transition from GLUT2- to GLUT1-mediated glucose metabolism may mark hepatocellular differentiation upon HCC progression.
4. Consequences of glucose and lipid imbalance on hepatocyte proliferation in GSD Ia mice
Disturbed cell proliferation before and after partial hepatectomy in GSD Ia mice
A long-term complication of GSD1a is the frequent development of hepatocellular adenomas (HCAs) with the risk of progressing into hepatocellular carcinoma (44, 69). The mechanisms underlying GSD Ia-associated tumorigenesis are unknown and it is unclear which early events drive the transformation from G6pc-deficient hepatocytes into tumorous cells. Therefore, in Chapter 6, we monitored hepatocyte proliferation in L-G6pc-/- mice in response to 2/3 hepatectomy (PHx) performed at 10 days after
gene deletion. We found that the L-G6pc-/- mice exhibited more cell proliferation than
wildtype mice prior to the surgery. This may reflect a compensatory mechanism for the increased cell death in L-G6pc-/- mice as reported in Chapter 3. Besides, enhanced
proliferation may also mark tumor initiation in G6pc deficient hepatocytes. It has been reported that chronic hepatocyte loss caused by toxic agents, alcohol and steatohepatitis lead to continuous hepatocyte proliferation and that these constitutive proliferating hepatocytes undergo transition from polyploidy to diploidy. Consequently, the stochastically genomic alteration in growth regulatory genes (such as p53, EGFR, telomerase promoter) of the diploid hepatocyte results in the formation of HCC (70).
Our data (Chapter 6) furthermore showed an accelerated presence of mitotic figures in G6pc-deficient as compared to wildtypehepatocytes. This may be caused by the increased TG accumulation and recurrent hypoglycemic episodes in G6pcdeficient livers (71). TGs provide energy for DNA replication and TG-derived fatty acids provide substrates for phospholipid synthesis, which is the main component of cell membranes (72, 73). Intravenous or enteral glucose supplementation suppresses both PHx- (74, 75) and toxin- (76, 77) induced hepatocellular proliferation. Similarly, caloric restriction accelerates the onset of hepatocellular proliferation in response to surgical- or toxin-induced hepatic insufficiency (78, 79). Strikingly, we found that almost all mitotic hepatocytes in L-G6pc-/- mice formed anaphase bridges, indicating
genomic instability and replication stress. The presence of anaphase bridges is highly associated with cancer (80). Therefore, we speculate that severe genome instability in G6pc-deficient livers contributed to mitosis inhibition during liver regeneration. The genome instability may be caused by hepatic lipid accumulation in the livers of L-G6pc-/- mice, because it has been reported that lipid peroxidation products and
reactive oxygen species can result in DNA damage and genomic alterations (70).
169
170
Moreover, ER stress, which can directly cause genome instability (80), is more marked in fatty livers (81). ER stress-related genes, GRP78, sXBP-1, and CHOP were significantly increased upon PHx in wild type and mice fed with high fat diet (81). Interestingly, the IRE1/XBP1 and ATF6 ER stress pathways are induced in L-G6pc
-/-livers (82). Therefore, the occurance of anaphase bridges in G6pc-/- hepatocytes may
be related to increased ER stress.
Normal hepatocyte ploidy ranges from 2n (diploid), 4n, to 8n (polyploidy). Since in our experiment, we found more BrDU incorporation but impaired mitosis in L-G6pc-/-
livers, increased polyploidy of L-G6pc-/- hepatocytes may be expected. On the
contrary, it has been reported that chronic liver injury and liver steatosis are associated with reduced ploidy because of the “ploidy conveyor”, which is activated by continuous compensatory hepatocyte proliferation (83, 84). Altogether, it would be interesting to analyze hepatocyte ploidy in L-G6pc-/- miceafter PHx.
Compensatory induction of hepatocyte hypertrophy ensures adequate liver regeneration after PH in L-G6pc-/- mice
In our experiment (Chapter 6), we found that hepatocyte size increased (hypertrophy)
in response to PHx, both in wildtype and L-G6pc-/- livers, but the increase was more
pronounced in L-G6pc-/- mice, especially at 7 days post-PHx. Therefore, both
hepatocyte proliferation and hypertrophy contributed to liver regeneration in reponse to PHx, a concept that was introduced by the Miyajima laboratory (85). He and his colleagues reported that hepatocyte hypertrophy instead of cell division is responsible for liver regeneration after 30% PHx, while both hypertrophy and hyperplasia contribute after 70% PHx (85). Thus, besides proliferation of hepatocytes
(47, 86, 87) or stem-cell activation upon liver injury (88-90), hypertrophy is an
important mechanism to recover liver mass post-PHx. Hepatocyte hypertrophy can start as early as 3 hour after 70% PHx (85), indicating that the induction of hypertrophy is a rapid response. The enlargement of hepatocytes may be explained by mitochondrial swelling. It has been reported that mitochondria are significantly swollen in response to dexamethasone treatment, which inhibits hepatocyte proliferation and stem-cell activation (91). Mitochondrial swelling can be a sign of increased permeability of the inner mitochondrial membrane referred to as “mitochondrial permeability transition” (92). In general, this increased permeability is induced by oxidative stress (93). As oxidative stress is induced upon PHx (94), it would be interesting to investigate oxidative stress and hepatocyte ultrastructure in
post-PHx L-G6pc-/- liver. Hepatocyte hypertrophy could also be caused by
hepatocellular lipid accumulation.Previous research has shown that both micro- and
macrovesicular lipid accumulation are increased in response to PHx (81). This could also enhance oxidative stress (82) and, in turn, increase mitochondrial permeability. It has been proposed that epidermal growth factor, HGF and TGF-α, which drive hepatocyte proliferation, also regulate hepatocyte hypertrophy (95-97). Besides, c-Myc may also play a role in hepatocyte hypertrophy because it is overexpressed in the hypertrophic liver before the initiation of proliferation (98, 99). Taken together,
impaired mitosis and hepatocyte hyperplasia in L-G6pc-/- hepatocytes could be
compensated by hepatocyte hypertrophy to ensure adequate liver recovery in
171
reponse to PHx. However, elucidation of the exact molecular mechanisms underlying
differential liver regeneration in L-G6pc-/- mice warrants further investigation. Finally,
an interesting and remaining question is how hepatic ChREBP knockdown in L-G6pc
-/-mice subjected to PHx will affect liver tumor formation on the long term. This will be addressed in our future work.
170
Moreover, ER stress, which can directly cause genome instability (80), is more marked in fatty livers (81). ER stress-related genes, GRP78, sXBP-1, and CHOP were significantly increased upon PHx in wild type and mice fed with high fat diet (81). Interestingly, the IRE1/XBP1 and ATF6 ER stress pathways are induced in L-G6pc
-/-livers (82). Therefore, the occurance of anaphase bridges in G6pc-/- hepatocytes may
be related to increased ER stress.
Normal hepatocyte ploidy ranges from 2n (diploid), 4n, to 8n (polyploidy). Since in our experiment, we found more BrDU incorporation but impaired mitosis in L-G6pc-/-
livers, increased polyploidy of L-G6pc-/- hepatocytes may be expected. On the
contrary, it has been reported that chronic liver injury and liver steatosis are associated with reduced ploidy because of the “ploidy conveyor”, which is activated by continuous compensatory hepatocyte proliferation (83, 84). Altogether, it would be interesting to analyze hepatocyte ploidy in L-G6pc-/- miceafter PHx.
Compensatory induction of hepatocyte hypertrophy ensures adequate liver regeneration after PH in L-G6pc-/- mice
In our experiment (Chapter 6), we found that hepatocyte size increased (hypertrophy)
in response to PHx, both in wildtype and L-G6pc-/- livers, but the increase was more
pronounced in L-G6pc-/- mice, especially at 7 days post-PHx. Therefore, both
hepatocyte proliferation and hypertrophy contributed to liver regeneration in reponse to PHx, a concept that was introduced by the Miyajima laboratory (85). He and his colleagues reported that hepatocyte hypertrophy instead of cell division is responsible for liver regeneration after 30% PHx, while both hypertrophy and hyperplasia contribute after 70% PHx (85). Thus, besides proliferation of hepatocytes
(47, 86, 87) or stem-cell activation upon liver injury (88-90), hypertrophy is an
important mechanism to recover liver mass post-PHx. Hepatocyte hypertrophy can start as early as 3 hour after 70% PHx (85), indicating that the induction of hypertrophy is a rapid response. The enlargement of hepatocytes may be explained by mitochondrial swelling. It has been reported that mitochondria are significantly swollen in response to dexamethasone treatment, which inhibits hepatocyte proliferation and stem-cell activation (91). Mitochondrial swelling can be a sign of increased permeability of the inner mitochondrial membrane referred to as “mitochondrial permeability transition” (92). In general, this increased permeability is induced by oxidative stress (93). As oxidative stress is induced upon PHx (94), it would be interesting to investigate oxidative stress and hepatocyte ultrastructure in
post-PHx L-G6pc-/- liver. Hepatocyte hypertrophy could also be caused by
hepatocellular lipid accumulation.Previous research has shown that both micro- and
macrovesicular lipid accumulation are increased in response to PHx (81). This could also enhance oxidative stress (82) and, in turn, increase mitochondrial permeability. It has been proposed that epidermal growth factor, HGF and TGF-α, which drive hepatocyte proliferation, also regulate hepatocyte hypertrophy (95-97). Besides, c-Myc may also play a role in hepatocyte hypertrophy because it is overexpressed in the hypertrophic liver before the initiation of proliferation (98, 99). Taken together,
impaired mitosis and hepatocyte hyperplasia in L-G6pc-/- hepatocytes could be
compensated by hepatocyte hypertrophy to ensure adequate liver recovery in
171
reponse to PHx. However, elucidation of the exact molecular mechanisms underlying
differential liver regeneration in L-G6pc-/- mice warrants further investigation. Finally,
an interesting and remaining question is how hepatic ChREBP knockdown in L-G6pc
-/-mice subjected to PHx will affect liver tumor formation on the long term. This will be addressed in our future work.
171
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