Role of hepatic glucose signaling in the development of liver disease

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University of Groningen

Role of hepatic glucose signaling in the development of liver disease

Lei, Yu

DOI:

10.33612/diss.126530476

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.

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Publication date: 2020

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Citation for published version (APA):

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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Appendices

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English Summary

Metabolic homeostasis is tightly regulated by various factors, including nutrients, hormones and the nervous system. Energy overload or unbalanced nutritional intake disturb metabolic homeostasis and can result in diseases such as obesity, diabetes, non-alcoholic fatty liver disease (NAFLD) and even cancer. The liver is a key organ in whole-body metabolism and the metabolic activities of hepatocytes are tightly controlled at different regulatory levels by hormones, neuronal input, metabolites, enzymes and transcription factors. One of the key metabolic transcriptional factors expressed in the liver is Carbohydrate Response Element Binding Protein (ChREBP), a glucose sensor that plays a pivotal role in the regulation of glucose and lipid metabolism. Glycogen storage disease type 1a (GSD Ia) is an Inborn Error of Metabolism caused by mutations in the glucose-6-phoshatase (G6PC) enzyme that is biochemically characterized by fasting hypoglycemia, hyperlipidemia, hepatomegaly and NAFLD. Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) formation are major long-term hepatic complications of GSD Ia. Although GSD Ia is a rare disease with an incidence of 1 in 100,000, it is considered a valuable model to investigate the pathophysiology of more complex metabolic diseases that show disturbed intrahepatic glucose balance, such as type 2 diabetes.

In this thesis, we employed a mouse model for hepatic GSD Ia (i.e., L-G6pc-/- mice) to study the physiological and molecular mechanisms that link intrahepatic glucose (-6-phosphate; G6P) imbalance to liver dysfunction. The work was focused on the following aspects: 1) The role of ChREBP in the development of NAFLD in liver-specific GSD Ia mice (Chapter 2). 2) The role of hepatic G6P-ChREBP signaling in bile acid and cholesterol metabolism (Chapter 3). 3) The role of ChREBP in hepatocyte proliferation in vitro versus in vivo (G6pc-/- hepatocytes) (Chapter 4). 4) The impact of hepatic G6pc deficiency on liver regeneration in response to partial liver resection (Chapter 5). 5) The expression pattern of glucose transporters 1 and 2 (GLUT1/2) and ChREBP in different stages of human HCC (Chapter 6).

It has been previously shown that the activity of hepatic ChREBP is markedly increased in L-G6pc-/- mice. Hepatic ChREBP is also activated in type 2 diabetic mice and ChREBP knockdown in the liver of these mice protects against NAFLD. In light of the association between hepatic ChREBP activity and NAFLD, we evaluated in

Chapter 2 the metabolic consequences of enhanced hepatic ChREBP activity in GSD

Ia, especially with respect to development of NAFLD. We found that hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L-G6pc-/- mice. Hepatic accumulation of G6P, glycogen and lipids were largely increased, while glycolysis and de novo lipogenesis were reduced after ChREBP knockdown in both wildtype and L-G6pc-/- mice. Importantly, hepatic ChREBP knockdown strongly suppressed hepatic VLDL lipidation and the excretion of VLDL-triglyceride (TG) from the liver. Moreover, we found that increased VLDL-TG secretion was associated with a ChREBP-dependent induction of the VLDL lipidation proteins MTTP and TM6SF2. The latter was thus identified as a novel ChREBP target in mouse liver. Taken together, attenuation of hepatic ChREBP induction in murine GSD Ia liver aggravates

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English Summary

Metabolic homeostasis is tightly regulated by various factors, including nutrients, hormones and the nervous system. Energy overload or unbalanced nutritional intake disturb metabolic homeostasis and can result in diseases such as obesity, diabetes, non-alcoholic fatty liver disease (NAFLD) and even cancer. The liver is a key organ in whole-body metabolism and the metabolic activities of hepatocytes are tightly controlled at different regulatory levels by hormones, neuronal input, metabolites, enzymes and transcription factors. One of the key metabolic transcriptional factors expressed in the liver is Carbohydrate Response Element Binding Protein (ChREBP), a glucose sensor that plays a pivotal role in the regulation of glucose and lipid metabolism. Glycogen storage disease type 1a (GSD Ia) is an Inborn Error of Metabolism caused by mutations in the glucose-6-phoshatase (G6PC) enzyme that is biochemically characterized by fasting hypoglycemia, hyperlipidemia, hepatomegaly and NAFLD. Hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) formation are major long-term hepatic complications of GSD Ia. Although GSD Ia is a rare disease with an incidence of 1 in 100,000, it is considered a valuable model to investigate the pathophysiology of more complex metabolic diseases that show disturbed intrahepatic glucose balance, such as type 2 diabetes.

In this thesis, we employed a mouse model for hepatic GSD Ia (i.e., L-G6pc-/- mice) to study the physiological and molecular mechanisms that link intrahepatic glucose (-6-phosphate; G6P) imbalance to liver dysfunction. The work was focused on the following aspects: 1) The role of ChREBP in the development of NAFLD in liver-specific GSD Ia mice (Chapter 2). 2) The role of hepatic G6P-ChREBP signaling in bile acid and cholesterol metabolism (Chapter 3). 3) The role of ChREBP in hepatocyte proliferation in vitro versus in vivo (G6pc-/- hepatocytes) (Chapter 4). 4) The impact of hepatic G6pc deficiency on liver regeneration in response to partial liver resection (Chapter 5). 5) The expression pattern of glucose transporters 1 and 2 (GLUT1/2) and ChREBP in different stages of human HCC (Chapter 6).

It has been previously shown that the activity of hepatic ChREBP is markedly increased in L-G6pc-/- mice. Hepatic ChREBP is also activated in type 2 diabetic mice and ChREBP knockdown in the liver of these mice protects against NAFLD. In light of the association between hepatic ChREBP activity and NAFLD, we evaluated in

Chapter 2 the metabolic consequences of enhanced hepatic ChREBP activity in GSD

Ia, especially with respect to development of NAFLD. We found that hepatic ChREBP knockdown markedly increased liver weight and hepatocyte size in L-G6pc-/- mice. Hepatic accumulation of G6P, glycogen and lipids were largely increased, while glycolysis and de novo lipogenesis were reduced after ChREBP knockdown in both wildtype and L-G6pc-/- mice. Importantly, hepatic ChREBP knockdown strongly suppressed hepatic VLDL lipidation and the excretion of VLDL-triglyceride (TG) from the liver. Moreover, we found that increased VLDL-TG secretion was associated with a ChREBP-dependent induction of the VLDL lipidation proteins MTTP and TM6SF2. The latter was thus identified as a novel ChREBP target in mouse liver. Taken together, attenuation of hepatic ChREBP induction in murine GSD Ia liver aggravates

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hepatomegaly due to further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL-TG secretion. These data indicate that enhanced ChREBP activity limits NAFLD development in GSD Ia by balancing hepatic TG production and -secretion.

It has been reported that altered intrahepatic glucose signaling in human type 2 diabetes associates with perturbed bile acid synthesis. However, whether glucose independently regulates bile acid metabolism was unknown. In Chapter 3, we aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, G6P, on bile acid metabolism. We found that hepatic G6P accumulation induced

Cyp8b1 expression and that this was mediated by ChREBP. Enhanced intrahepatic

G6P-ChREBP-CYP8B1 signaling increased the relative abundance of cholic acid-derived bile acids, leading to higher hydrophobicity of the circulating bile acid pool. The G6P-ChREBP-dependent change in bile acid hydrophobicity was associated with reduced fecal neutral sterol loss, suggesting enhanced intestinal cholesterol absorption. This work identifies hepatic G6P-ChREBP-CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

A major long-term complication of GSD Ia is the development of HCA and HCC, but the underlying molecular mechanisms have remained unresolved. ChREBP has been reported to support cellular proliferation in vitro, while loss of hepatic ChREBP expression protects against AKT or AKT/c-Met induced liver tumor development in mice. Although ChREBP is known to be constitutively active in GSD I, its contribution to liver tumor susceptibility in GSD Ia has as yet not been addressed. In Chapter 4, we assessed the oncogenic role of ChREBP in immortalized human hepatocytes (IHHs) and L-G6pc-/- mice. Our data show that lowering of ChREBP expression induced p53-activation in both IHHs and G6pc-/- hepatocytes. Interestingly, ChREBP knockdown in IHHs induced cell cycle inhibition and proliferation arrest, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. Our findings therefore indicate that inhibition of ChREBP activity differentially affects hepatocyte fate in vitro and in vivo, and point to a context-dependent oncogenic role of ChREBP in hepatocytes.

GSD Ia patients show a high prevalence of HCA development in young adulthood which predisposes to HCC formation. The mechanisms underlying GSD Ia-associated tumorigenesis are poorly understood and it is unclear at which stage of the disease the actual pathophysiology of tumor formation starts. In Chapter 5, we monitored hepatocyte proliferation in 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,

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. In conclusion, we report signs of severe genomic instability

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and replication stress in GSD Ia hepatocytes at an early stage of the disease. These findings are 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.

In Chapter 6, we investigated the expression patterns of ChREBP and glucose transporters (GLUTs) in HCC and their association with HCC progression. ChREBP, GLUT2 and GLUT1 immunohistochemistry was performed on a liver tissue array containing normal liver tissue, tumor tissue of different HCC stages and HCC adjacent tissues. We found that ChREBP protein expression tended to be positively correlated to hepatic malignancy. GLUT2 protein expression was significantly reduced in human HCC as compared to normal liver tissue and its expression was inversely associated to malignancy. In contrast, GLUT1 was significantly increased in HCC and its expression was positively correlated to malignancy. Furthermore, GLUT1 expression was positively associated to ChREBP expression but negatively correlated to GLUT2 expression. Notably, ChREBP-expressing hepatocytes did not express GLUT2 but did show GLUT1 positivity. This is the first report unveiling expressions of ChREBP and GLUT2/GLUT1 in relation to HCC malignancy. Such information may potentially improve the future evaluation HCC progression and treatment.

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hepatomegaly due to further accumulation of glycogen and lipids as a result of reduced glycolysis and suppressed VLDL-TG secretion. These data indicate that enhanced ChREBP activity limits NAFLD development in GSD Ia by balancing hepatic TG production and -secretion.

It has been reported that altered intrahepatic glucose signaling in human type 2 diabetes associates with perturbed bile acid synthesis. However, whether glucose independently regulates bile acid metabolism was unknown. In Chapter 3, we aimed to characterize the regulatory role of the primary intracellular metabolite of glucose, G6P, on bile acid metabolism. We found that hepatic G6P accumulation induced

Cyp8b1 expression and that this was mediated by ChREBP. Enhanced intrahepatic

G6P-ChREBP-CYP8B1 signaling increased the relative abundance of cholic acid-derived bile acids, leading to higher hydrophobicity of the circulating bile acid pool. The G6P-ChREBP-dependent change in bile acid hydrophobicity was associated with reduced fecal neutral sterol loss, suggesting enhanced intestinal cholesterol absorption. This work identifies hepatic G6P-ChREBP-CYP8B1 signaling as a regulatory axis in control of bile acid and cholesterol metabolism.

A major long-term complication of GSD Ia is the development of HCA and HCC, but the underlying molecular mechanisms have remained unresolved. ChREBP has been reported to support cellular proliferation in vitro, while loss of hepatic ChREBP expression protects against AKT or AKT/c-Met induced liver tumor development in mice. Although ChREBP is known to be constitutively active in GSD I, its contribution to liver tumor susceptibility in GSD Ia has as yet not been addressed. In Chapter 4, we assessed the oncogenic role of ChREBP in immortalized human hepatocytes (IHHs) and L-G6pc-/- mice. Our data show that lowering of ChREBP expression induced p53-activation in both IHHs and G6pc-/- hepatocytes. Interestingly, ChREBP knockdown in IHHs induced cell cycle inhibition and proliferation arrest, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. Our findings therefore indicate that inhibition of ChREBP activity differentially affects hepatocyte fate in vitro and in vivo, and point to a context-dependent oncogenic role of ChREBP in hepatocytes.

GSD Ia patients show a high prevalence of HCA development in young adulthood which predisposes to HCC formation. The mechanisms underlying GSD Ia-associated tumorigenesis are poorly understood and it is unclear at which stage of the disease the actual pathophysiology of tumor formation starts. In Chapter 5, we monitored hepatocyte proliferation in 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,

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. In conclusion, we report signs of severe genomic instability

183

and replication stress in GSD Ia hepatocytes at an early stage of the disease. These findings are 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.

In Chapter 6, we investigated the expression patterns of ChREBP and glucose transporters (GLUTs) in HCC and their association with HCC progression. ChREBP, GLUT2 and GLUT1 immunohistochemistry was performed on a liver tissue array containing normal liver tissue, tumor tissue of different HCC stages and HCC adjacent tissues. We found that ChREBP protein expression tended to be positively correlated to hepatic malignancy. GLUT2 protein expression was significantly reduced in human HCC as compared to normal liver tissue and its expression was inversely associated to malignancy. In contrast, GLUT1 was significantly increased in HCC and its expression was positively correlated to malignancy. Furthermore, GLUT1 expression was positively associated to ChREBP expression but negatively correlated to GLUT2 expression. Notably, ChREBP-expressing hepatocytes did not express GLUT2 but did show GLUT1 positivity. This is the first report unveiling expressions of ChREBP and GLUT2/GLUT1 in relation to HCC malignancy. Such information may potentially improve the future evaluation HCC progression and treatment.

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Nederlandse samenvatting

Het metabole evenwicht van het lichaam wordt gereguleerd door verschillende factoren, waaronder voedingsstoffen, hormonen en de activiteit van het centrale zenuwstelsel. Overmatige consumptie van energierijke voeding en/of een onevenwichtige inname van bepaalde voedingsstoffen verstoren dit evenwicht en kunnen leiden tot ziekten zoals obesitas, diabetes, niet-alcoholische leververvetting (NAFLD) en zelfs kanker. De lever speelt een sleutelrol in handhaving van het de metabole evenwicht, en de verschillende metabole activiteiten van de lever parenchym cellen, de hepatocyten, worden op verschillende niveaus gereguleerd door hormonen, neuronale input, metabolieten, enzymen en transcriptiefactoren. Een belangrijke metabole transcriptie factoren in de lever is “Carbohydrate Response Element Binding Protein (ChREBP)”, een glucose sensor die een cruciale rol speelt in de regulering van het glucose- en lipidenmetabolisme. Glycogeenstapelingsziekte type 1a (GSD Ia) is een aangeboren stofwisselingsziekte veroorzaakt door mutaties in het gen dat codeert voor het enzym glucose-6-fosfatase (G6PC). GSD Ia wordt biochemisch gekenmerkt door hypoglycemie tijdens vasten, hyperlipidemie, hepatomegalie en NAFLD.Belangrijke lange-termijn complicaties van GSD Ia zijn de vorming van hepatocellulaire adenomen (HCA) en hepatocellulair carcinoom (HCC). Hoewel GSD Ia een zeldzame ziekte is met een incidentie van 1 op 100.000, wordt het beschouwd als een waardevol model om de pathofysiologie van complexere metabole ziekten waarbij de glucosebalans in de lever verstoord is, zoals bijvoorbeeld type 2 diabetes, te onderzoeken.

In dit proefschrift is een muismodel voor hepatische GSD Ia (L-G6pc-/- muizen)

gebruikt om de fysiologische en moleculaire mechanismen te bestuderen die bijdragen aan verstoringen van essentiele leverfuncties welke ontstaan door een onbalans van intrahepatische glucose (-6-fosfaat; G6P). Het experimentele werk was gericht op de volgende aspecten: 1) De rol van ChREBP in de ontwikkeling van NAFLD in lever-specifieke GSD Ia muizen (hoofdstuk 2). 2) De rol van G6P-ChREBP signalering in de lever in regulering van het galzout- en cholesterolmetabolisme (hoofdstuk 3). 3) De rol van ChREBP in de inductie van hepatocyt proliferatie in vitro versus in vivo (G6pc-/- hepatocyten) (Hoofdstuk 4). 4) De impact van G6pc deletie in

hepatocyten op de leverregeneratie die optreedt in respons op partiële leverresectie (hoofdstuk 5). 5) Het expressiepatroon van glucosetransporters 1 en 2 (GLUT1/2) en ChREBP in verschillende stadia van humaan HCC (hoofdstuk 6).

Eerder onderzoek heeft aangetoond dat de activiteit van ChREBP in de lever aanzienlijk is toegenomen in L-G6pc-/- muizen. Hepatisch ChREBP is tevens

geactiveerd in muizen met type 2 diabetes, en “ChREBP knockdown” in de lever van deze muizen beschermt tegen het ontstaan van NAFLD. Gezien de associatie tussen ChREBP activiteit in de lever en ontwikkeling van NAFLD, zijn in hoofdstuk 2 de metabole gevolgen van langdurige ChREBP activering in de lever in van GSD Ia muizen onderzocht, specifiek met betrekking tot de ontwikkeling van NAFLD.Ontdekt werd dat een verlaging van de hepatische ChREBP expressie leidde tot een aanzienlijke toename in het levergewicht en de grootte van hepatocyten in L-G6pc

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Nederlandse samenvatting

Het metabole evenwicht van het lichaam wordt gereguleerd door verschillende factoren, waaronder voedingsstoffen, hormonen en de activiteit van het centrale zenuwstelsel. Overmatige consumptie van energierijke voeding en/of een onevenwichtige inname van bepaalde voedingsstoffen verstoren dit evenwicht en kunnen leiden tot ziekten zoals obesitas, diabetes, niet-alcoholische leververvetting (NAFLD) en zelfs kanker. De lever speelt een sleutelrol in handhaving van het de metabole evenwicht, en de verschillende metabole activiteiten van de lever parenchym cellen, de hepatocyten, worden op verschillende niveaus gereguleerd door hormonen, neuronale input, metabolieten, enzymen en transcriptiefactoren. Een belangrijke metabole transcriptie factoren in de lever is “Carbohydrate Response Element Binding Protein (ChREBP)”, een glucose sensor die een cruciale rol speelt in de regulering van het glucose- en lipidenmetabolisme. Glycogeenstapelingsziekte type 1a (GSD Ia) is een aangeboren stofwisselingsziekte veroorzaakt door mutaties in het gen dat codeert voor het enzym glucose-6-fosfatase (G6PC). GSD Ia wordt biochemisch gekenmerkt door hypoglycemie tijdens vasten, hyperlipidemie, hepatomegalie en NAFLD.Belangrijke lange-termijn complicaties van GSD Ia zijn de vorming van hepatocellulaire adenomen (HCA) en hepatocellulair carcinoom (HCC). Hoewel GSD Ia een zeldzame ziekte is met een incidentie van 1 op 100.000, wordt het beschouwd als een waardevol model om de pathofysiologie van complexere metabole ziekten waarbij de glucosebalans in de lever verstoord is, zoals bijvoorbeeld type 2 diabetes, te onderzoeken.

In dit proefschrift is een muismodel voor hepatische GSD Ia (L-G6pc-/- muizen)

gebruikt om de fysiologische en moleculaire mechanismen te bestuderen die bijdragen aan verstoringen van essentiele leverfuncties welke ontstaan door een onbalans van intrahepatische glucose (-6-fosfaat; G6P). Het experimentele werk was gericht op de volgende aspecten: 1) De rol van ChREBP in de ontwikkeling van NAFLD in lever-specifieke GSD Ia muizen (hoofdstuk 2). 2) De rol van G6P-ChREBP signalering in de lever in regulering van het galzout- en cholesterolmetabolisme (hoofdstuk 3). 3) De rol van ChREBP in de inductie van hepatocyt proliferatie in vitro versus in vivo (G6pc-/- hepatocyten) (Hoofdstuk 4). 4) De impact van G6pc deletie in

hepatocyten op de leverregeneratie die optreedt in respons op partiële leverresectie (hoofdstuk 5). 5) Het expressiepatroon van glucosetransporters 1 en 2 (GLUT1/2) en ChREBP in verschillende stadia van humaan HCC (hoofdstuk 6).

Eerder onderzoek heeft aangetoond dat de activiteit van ChREBP in de lever aanzienlijk is toegenomen in L-G6pc-/- muizen. Hepatisch ChREBP is tevens

geactiveerd in muizen met type 2 diabetes, en “ChREBP knockdown” in de lever van deze muizen beschermt tegen het ontstaan van NAFLD. Gezien de associatie tussen ChREBP activiteit in de lever en ontwikkeling van NAFLD, zijn in hoofdstuk 2 de metabole gevolgen van langdurige ChREBP activering in de lever in van GSD Ia muizen onderzocht, specifiek met betrekking tot de ontwikkeling van NAFLD.Ontdekt werd dat een verlaging van de hepatische ChREBP expressie leidde tot een aanzienlijke toename in het levergewicht en de grootte van hepatocyten in L-G6pc

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muizen. De stapeling van G6P, glycogeen en lipiden in de lever was sterk toegenomen, terwijl de glycolyse en de novo lipogenese werden geremd na ChREBP knockdown in zowel L-G6pc-/- als wildtype muizen. Een belangrijke bevinding was dat

lever-specifieke ChREBP knockdown de lipidatie van VLDL deeltjes in de lever en de uitscheiding van VLDL-triglyceriden (TG) door de lever sterk onderdrukte. Bovendien ontdekten we dat de verhoogde VLDL-TG secretie in GSD Ia muizen geassocieerd is met een ChREBP-afhankelijke toename in de expressie van de VLDL-lipidatie eiwitten MTTP en TM6SF2. Dit laatste eiwit werd door ons daarmee voor het eerst geïdentificeerd als een “ChREBP-doelwit gen”. De conclusie van dit werk is dat

verlaging van ChREBP activiteit in de lever van GSD la muizen leidt tot een verergering van hepatomegalie. Dit is het gevolg van een toegenomen stapeling van glycogeen en lipiden door een verminderde glycolyse en een onderdrukte VLDL-TG secretie. Deze gegevens suggereren dat een chronische activering van ChREBP de ontwikkeling van NAFLD in GSD Ia beperkt door handhaving van de balans tussen

TG-productie en -secretie.

Het is bekend dat een veranderde intrahepatische glucose signalering in mensen met type 2 diabetes geassocieerd is met een verstoorde galzoutsynthese.Of glucose de galzout huishouding ook onafhankelijk reguleert was echter onduidelijk.In hoofdstuk

3 is de regulerende rol van het primaire intracellulaire metaboliet van glucose, G6P,

op het galzoutmetabolisme gekarakteriseerd.Ontdekt werd dat G6P stapeling in de lever leidt tot een verhoogde expressie van het enzym CYP8B1 en dat dit effect afhankelijk was van ChREBP expressie. Een toegenomen intrahepatische G6P-ChREBP-CYP8B1-signalering verhoogde de relatieve bijdrage van cholaat en daarvan afgeleide galzouten aan de circulerende galzout pool, hetgeen leidde tot een toename in de hydrofobiciteit van deze pool.Deze G6P-ChREBP afhankelijke toename van hydrofobe galzouten in de gal ging gepaard met een lagere excretie van neutrale sterolen via de feces, wat duidt op een hogere opname van (voedings)cholesterol in de darm.Dit werk identificeert G6P-ChREBP-CYP8B1-signalering als een belangrijke regulerende as voor handhaving van de galzout- en cholesterol homeostase.

Een belangrijke lange-termijn complicatie van GSD Ia is de ontwikkeling van HCA en HCC. Echter, de onderliggende moleculaire mechanismen zijn tot op heden onopgelost gebleven. Van ChREBP is bekend dat het cel proliferatie in vitro stimuleert terwijl verlies van ChREBP expressie in de lever beschermt tegen AKT of AKT / c-Met geïnduceerde ontwikkeling van levertumoren bij muizen. Hoewel bekend is dat hepatisch ChREBP constitutief actief is in GSD Ia, is de bijdrage aan het ontstaan van levertumoren in GSD Ia tot nu toe niet onderzocht. In hoofdstuk 4 is de oncogene rol van ChREBP in geïmmortaliseerde menselijke hepatocyten (IHH cellen) en in L-G6pc

-/-muizen onderzocht. Uit de resultaten blijkt dat verlaging van ChREBP-expressie leidt tot p53 activatie in zowel IHH cellen als G6pc-/- hepatocyten. Interessant is dat

ChREBP-knockdown in IHH cellen remming van de celcyclus en celproliferatie veroorzaakte, terwijl het in de hepatocyten van L-G6pc-/- muizen leidde tot celdood,

activering van celdeling, DNA-schade en chromosomale instabiliteit. Deze bevindingen geven aan dat remming van ChREBP activiteit het lot van de hepatocyten in vitro en in vivo op verschillende manieren beïnvloedt, en duiden op een context-afhankelijke oncogene rol van ChREBP in hepatocyten.

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GSD Ia-patiënten vertonen een hoge prevalentie van HCA ontwikkeling op jong-volwassen leeftijd, hetgeen predisponeert tot het ontstaan van HCC. De mechanismen die ten grondslag liggen aan GSD Ia-geassocieerde tumor ontwikkeling zijn niet bekend, en het is onduidelijk in welk stadium van de ziekte de processen van tumorvorming beginnen. In hoofdstuk 5 is de proliferatie van hepatocyten in L-G6pc-/- muizen in reactie op twee-derde partiële hepatectomie (PHx) bestudeerd.

De experimenten werden 10 dagen na inductie van G6pc deficientie in de hepatocyten van deze dieren uitgevoerd. De resultaten tonen een snellere aanwezigheid van mitotische figuren en Ki67-positive cellen in L-G6pc- - in vergelijking

tot L-G6pc+/+ hepatocyten. Ook vertoonden L-G6pc-/- hepatocyten een verhoogde

BrdU incorporatie in de pre-mitotische fase van leverregeneratie, terwijl BrdU-positiviteit vergelijkbaar was in mitotische L-G6pc-/- en L-G6pc+/+ hepatocyten.

Opvallend was dat bijna alle mitotische hepatocyten in L-G6pc-/- muizen anafase

bruggen vormden, hetgeen kan duiden op genomische instabiliteit en replicatie stress. Het herstel van levermassa tijdens leverregeneratie was vergelijkbaar in L-G6pc-/- en L-G6pc+/+ muizen, echter L-G6pc-/- hepatocyten vertoonden een grotere

mate van cellulaire hypertrofie.Concluderend kan worden gesteld dat reeds in een vroeg stadium van GSD Ia tekenen van ernstige genomische instabiliteit en replicatiestress in hepatocyten zichtbaar zijn.Deze bevindingen zijn compatibel met een model waarin genomische instabiliteit in L-G6pc-/- hepatocyten leidt tot een

aanvankelijke versnelling van lever regeneratie na gedeeltelijke leverresectie, waarna de hyperplasie van hepatocyten vervolgens wordt geremd en een (compenserende) hypertrofie wordt geinduceerd.

In hoofdstuk 6 zijn de expressiepatronen van ChREBP en glucose transporters (GLUT ewitten) in HCC onderzocht in relatie tot HCC progressie. Immunohistochemie van ChREBP, GLUT2 en GLUT1 werd uitgevoerd op een leverweefsel “array” van gezond leverweefsel, tumorweefsel van verschillende HCC-stadia, alsmede weefsel aangrenzend aan HCC. In sommige gevallen was een hogere ChREBP eiwit expressie zichtbaar in HCC ten opzichte van gezond leverweefsel. De expressie van het GLUT2 eiwit was significant verlaagd in HCC in vergelijking met gezond leverweefsel, en GLUT2 expressie was omgekeerd geassocieerd met de mate van maligniteit. GLUT1 expressie was daarentegen significant verhoogd in HCC weefsel, en de expressie was positief gecorreleerd met maligniteit.Hepatocyten met een hoge ChREBP expressie vertoonden een lage of afwezige GLUT2 expressie, maar vertoonden wel positiviteit voor GLUT1. Dit is de eerste studie waarin de eiwit expressies van ChREBP en GLUT2 / GLUT1 bestudeerd worden in relatie tot progressie van humane HCC. Deze informatie kan mogelijk bijdragen aan verbeterde diagnose en behandeling van HCC in de toekomst.

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muizen. De stapeling van G6P, glycogeen en lipiden in de lever was sterk toegenomen, terwijl de glycolyse en de novo lipogenese werden geremd na ChREBP knockdown in zowel L-G6pc-/- als wildtype muizen. Een belangrijke bevinding was dat

lever-specifieke ChREBP knockdown de lipidatie van VLDL deeltjes in de lever en de uitscheiding van VLDL-triglyceriden (TG) door de lever sterk onderdrukte. Bovendien ontdekten we dat de verhoogde VLDL-TG secretie in GSD Ia muizen geassocieerd is met een ChREBP-afhankelijke toename in de expressie van de VLDL-lipidatie eiwitten MTTP en TM6SF2. Dit laatste eiwit werd door ons daarmee voor het eerst geïdentificeerd als een “ChREBP-doelwit gen”. De conclusie van dit werk is dat

verlaging van ChREBP activiteit in de lever van GSD la muizen leidt tot een verergering van hepatomegalie. Dit is het gevolg van een toegenomen stapeling van glycogeen en lipiden door een verminderde glycolyse en een onderdrukte VLDL-TG secretie. Deze gegevens suggereren dat een chronische activering van ChREBP de ontwikkeling van NAFLD in GSD Ia beperkt door handhaving van de balans tussen

TG-productie en -secretie.

Het is bekend dat een veranderde intrahepatische glucose signalering in mensen met type 2 diabetes geassocieerd is met een verstoorde galzoutsynthese. Of glucose de galzout huishouding ook onafhankelijk reguleert was echter onduidelijk.In hoofdstuk

3 is de regulerende rol van het primaire intracellulaire metaboliet van glucose, G6P,

op het galzoutmetabolisme gekarakteriseerd.Ontdekt werd dat G6P stapeling in de lever leidt tot een verhoogde expressie van het enzym CYP8B1 en dat dit effect afhankelijk was van ChREBP expressie. Een toegenomen intrahepatische G6P-ChREBP-CYP8B1-signalering verhoogde de relatieve bijdrage van cholaat en daarvan afgeleide galzouten aan de circulerende galzout pool, hetgeen leidde tot een toename in de hydrofobiciteit van deze pool.Deze G6P-ChREBP afhankelijke toename van hydrofobe galzouten in de gal ging gepaard met een lagere excretie van neutrale sterolen via de feces, wat duidt op een hogere opname van (voedings)cholesterol in de darm.Dit werk identificeert G6P-ChREBP-CYP8B1-signalering als een belangrijke regulerende as voor handhaving van de galzout- en cholesterol homeostase.

Een belangrijke lange-termijn complicatie van GSD Ia is de ontwikkeling van HCA en HCC. Echter, de onderliggende moleculaire mechanismen zijn tot op heden onopgelost gebleven. Van ChREBP is bekend dat het cel proliferatie in vitro stimuleert terwijl verlies van ChREBP expressie in de lever beschermt tegen AKT of AKT / c-Met geïnduceerde ontwikkeling van levertumoren bij muizen. Hoewel bekend is dat hepatisch ChREBP constitutief actief is in GSD Ia, is de bijdrage aan het ontstaan van levertumoren in GSD Ia tot nu toe niet onderzocht. In hoofdstuk 4 is de oncogene rol van ChREBP in geïmmortaliseerde menselijke hepatocyten (IHH cellen) en in L-G6pc

-/-muizen onderzocht. Uit de resultaten blijkt dat verlaging van ChREBP-expressie leidt tot p53 activatie in zowel IHH cellen als G6pc-/- hepatocyten. Interessant is dat

ChREBP-knockdown in IHH cellen remming van de celcyclus en celproliferatie veroorzaakte, terwijl het in de hepatocyten van L-G6pc-/- muizen leidde tot celdood,

activering van celdeling, DNA-schade en chromosomale instabiliteit. Deze bevindingen geven aan dat remming van ChREBP activiteit het lot van de hepatocyten in vitro en in vivo op verschillende manieren beïnvloedt, en duiden op een context-afhankelijke oncogene rol van ChREBP in hepatocyten.

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GSD Ia-patiënten vertonen een hoge prevalentie van HCA ontwikkeling op jong-volwassen leeftijd, hetgeen predisponeert tot het ontstaan van HCC. De mechanismen die ten grondslag liggen aan GSD Ia-geassocieerde tumor ontwikkeling zijn niet bekend, en het is onduidelijk in welk stadium van de ziekte de processen van tumorvorming beginnen. In hoofdstuk 5 is de proliferatie van hepatocyten in L-G6pc-/- muizen in reactie op twee-derde partiële hepatectomie (PHx) bestudeerd.

De experimenten werden 10 dagen na inductie van G6pc deficientie in de hepatocyten van deze dieren uitgevoerd. De resultaten tonen een snellere aanwezigheid van mitotische figuren en Ki67-positive cellen in L-G6pc- - in vergelijking

tot L-G6pc+/+ hepatocyten. Ook vertoonden L-G6pc-/- hepatocyten een verhoogde

BrdU incorporatie in de pre-mitotische fase van leverregeneratie, terwijl BrdU-positiviteit vergelijkbaar was in mitotische L-G6pc-/- en L-G6pc+/+ hepatocyten.

Opvallend was dat bijna alle mitotische hepatocyten in L-G6pc-/- muizen anafase

bruggen vormden, hetgeen kan duiden op genomische instabiliteit en replicatie stress. Het herstel van levermassa tijdens leverregeneratie was vergelijkbaar in L-G6pc-/- en L-G6pc+/+ muizen, echter L-G6pc-/- hepatocyten vertoonden een grotere

mate van cellulaire hypertrofie.Concluderend kan worden gesteld dat reeds in een vroeg stadium van GSD Ia tekenen van ernstige genomische instabiliteit en replicatiestress in hepatocyten zichtbaar zijn.Deze bevindingen zijn compatibel met een model waarin genomische instabiliteit in L-G6pc-/- hepatocyten leidt tot een

aanvankelijke versnelling van lever regeneratie na gedeeltelijke leverresectie, waarna de hyperplasie van hepatocyten vervolgens wordt geremd en een (compenserende) hypertrofie wordt geinduceerd.

In hoofdstuk 6 zijn de expressiepatronen van ChREBP en glucose transporters (GLUT ewitten) in HCC onderzocht in relatie tot HCC progressie. Immunohistochemie van ChREBP, GLUT2 en GLUT1 werd uitgevoerd op een leverweefsel “array” van gezond leverweefsel, tumorweefsel van verschillende HCC-stadia, alsmede weefsel aangrenzend aan HCC. In sommige gevallen was een hogere ChREBP eiwit expressie zichtbaar in HCC ten opzichte van gezond leverweefsel. De expressie van het GLUT2 eiwit was significant verlaagd in HCC in vergelijking met gezond leverweefsel, en GLUT2 expressie was omgekeerd geassocieerd met de mate van maligniteit. GLUT1 expressie was daarentegen significant verhoogd in HCC weefsel, en de expressie was positief gecorreleerd met maligniteit.Hepatocyten met een hoge ChREBP expressie vertoonden een lage of afwezige GLUT2 expressie, maar vertoonden wel positiviteit voor GLUT1. Dit is de eerste studie waarin de eiwit expressies van ChREBP en GLUT2 / GLUT1 bestudeerd worden in relatie tot progressie van humane HCC. Deze informatie kan mogelijk bijdragen aan verbeterde diagnose en behandeling van HCC in de toekomst.

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Acknowledgements

This thesis would not have been possible without the help of many people within the last almost 6 years. Here, I would like to show my sincere appreciation to all of you, who have contributed to this thesis, including my supervisors, colleagues, friends and family.

Firstly, I would like to thank my supervisors in Groningen, Maaike and Folkert. Maaike, thank you so much for all your guidance in my projects, you have taught me not only knowledge and techniques, but also showed me how to overcome difficulties and collaborate with others. You always impressed me with your work attitude. You are positive, thorough, persistent, well-organized and hardworking. No matter how many obstacles we met, you never gave up and always could find ways to work it out. I really appreciate your help and support in finishing my thesis. Besides, from you I know it is possible to balance work and family. Folkert, thank you very much for accepting me as your PhD student. Without you, I would not have the experience of working in the Netherlands, which I enjoyed a lot. I am very grateful for your constructive suggestions on my projects, your encouragement and guidance for my thesis.

Prof. Jiang Gu, my Chinese supervisor, I really appreciate all your help and support in Shantou, in Chengdu and even when I was in The Netherlands. You were always positive and supportive to me. Without your kind and continued help, I would not be where I am now in my life. No matter how hard a situation seems to be, you always backed me up. I am very grateful for your trust and guidance. It is a real pleasure and a big honor to work with you.

Further, I want to express my appreciation to the members of my reading committee. Prof. dr. C. J. de Vries, Prof. dr. P. Olinga and Prof. dr. A.J. Moshage, thank you for the judgement of my thesis.

I would also like to thank my colleagues who have contributed to this thesis, and basically everyone at the Department of Pediatrics. Aycha, you really helped me a lot with my experiments, I have learned so much from you. You are kind and humorous and I really like your personality. It was always nice to have you around. Trijnie, thank you for helping me with my experiments. You are so accurate and efficient. Joanne, you were always there when I needed help, no matter if in the CDP or in the lab. You always gave good suggestions for my experiments, thank you a lot. Niels, I know I have bothered you quite a bit with all kinds of questions, but you were so patient and helpful. I really appreciate all your help. Angelika, thank you for helping me with the animal experiments and the genotyping work. Your good organization was very impressive. I would also like to thank Renze, Manon and Tjasso, you all have supported me so much. Rick, thank you for the mouse work you did for me.

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Acknowledgements

This thesis would not have been possible without the help of many people within the last almost 6 years. Here, I would like to show my sincere appreciation to all of you, who have contributed to this thesis, including my supervisors, colleagues, friends and family.

Firstly, I would like to thank my supervisors in Groningen, Maaike and Folkert. Maaike, thank you so much for all your guidance in my projects, you have taught me not only knowledge and techniques, but also showed me how to overcome difficulties and collaborate with others. You always impressed me with your work attitude. You are positive, thorough, persistent, well-organized and hardworking. No matter how many obstacles we met, you never gave up and always could find ways to work it out. I really appreciate your help and support in finishing my thesis. Besides, from you I know it is possible to balance work and family. Folkert, thank you very much for accepting me as your PhD student. Without you, I would not have the experience of working in the Netherlands, which I enjoyed a lot. I am very grateful for your constructive suggestions on my projects, your encouragement and guidance for my thesis.

Prof. Jiang Gu, my Chinese supervisor, I really appreciate all your help and support in Shantou, in Chengdu and even when I was in The Netherlands. You were always positive and supportive to me. Without your kind and continued help, I would not be where I am now in my life. No matter how hard a situation seems to be, you always backed me up. I am very grateful for your trust and guidance. It is a real pleasure and a big honor to work with you.

Further, I want to express my appreciation to the members of my reading committee. Prof. dr. C. J. de Vries, Prof. dr. P. Olinga and Prof. dr. A.J. Moshage, thank you for the judgement of my thesis.

I would also like to thank my colleagues who have contributed to this thesis, and basically everyone at the Department of Pediatrics. Aycha, you really helped me a lot with my experiments, I have learned so much from you. You are kind and humorous and I really like your personality. It was always nice to have you around. Trijnie, thank you for helping me with my experiments. You are so accurate and efficient. Joanne, you were always there when I needed help, no matter if in the CDP or in the lab. You always gave good suggestions for my experiments, thank you a lot. Niels, I know I have bothered you quite a bit with all kinds of questions, but you were so patient and helpful. I really appreciate all your help. Angelika, thank you for helping me with the animal experiments and the genotyping work. Your good organization was very impressive. I would also like to thank Renze, Manon and Tjasso, you all have supported me so much. Rick, thank you for the mouse work you did for me.

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Vincent, thank you for helping with statistics and all your valuable suggestions for my manuscript. Mirjam, thank you for processing all the fresh tissue samples and performing stainings. Brenda, you were the first colleague I worked with, thank you for introducing me to new techniques. Martijn, although we did not directly work together when I was in the UMCG, you have helped me a lot with the GSEA, thank you.

Lots of help was also provided by other departments and universities. Special thanks to Susanne, for performing the mouse partial hepatectomy in the CDP. Rachel and Alain at Utrecht University, thank you for analyzing all the histology and giving valuable suggestions.

Of course, all the lovely people in our office deserve special appreciation. Rima, you are always so funny and thoughtful, thank you for your help with all kinds of things and your comfort when I was frustrated. You have so much curiosity in all kinds of food and, believe me, you are an excellent cook. You are also a big fan of learning foreign languages, do you still remember the Chinese I taught you? It was so nice to have you around, I will neither forget the funny African party you brought me to, nor the coffee corner we liked to go. Mirjam, you are so kind and social. Many thanks for a lot of things, you have really helped me to adapt to the new environment. It was you who read Dutch letters for me and introduced me to Salsa dance. It was you who organized our gatherings from time to time. Many thanks for your generous help. Lidiya, you are so sportive, a big fan of yoga and mountain climbing, you always reminded me I should do more exercise. There would not be so much laughter in our office without you. Onne, the big fan of cosplay, did I say that it was actually very cool? Also thank you for your help with all kinds of questions I had. Sanam, you were always so nice, it was very joyful and relaxed to talk with you and your husband. I still remember the tasty dinner we had in your house. Henk, I also bothered you with a lot of questions, thanks for your help with my experiments and for being my nearest neighbor.

Appreciations also go to my Chinese friends. Yuan, thank you for being my good friend. Thank you for your help and all the dinners when I was in Groningen. You could always give suggestions when I had problems. We could talk about a lot of things. It was an unforgettable vacation that we spent together in Spain. Yingying, you have accompanied me a lot in Groningen, we went shopping, to the movies and traveled together. I treasure all the beautiful memories we had. Weilin, you were the only Chinese PhD student in our department when I arrived. It was great to know you, thank you for your help and for hosting dinners at your home. Siqi and Tiantian, you both are so warm-hearted and hospitable. I had a lot of fun with you.

Samiksia, Arwin, you were the early friends I had when I was in Groningen. We have done so much together. I liked the food we made, the climbing of the Martini tower, the travel to Amsterdam. I wish you all the best. Sandra, you are so nice and

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hardworking, I hope everything is going well with you. Violeta, I liked all the talks we had, still remember the small picnic near the lake? Jun Li, Yana Geng, thank you for being a part of my life in Groningen. It was very nice to have you there. Not to forget, also many thanks to Hilde, Paula, Evelien. Without you, life would be much more difficult.

I would also like to express my appreciation to colleagues and friends in Shantou. Qiaoling, thank you for working with me and your help in our experiments. I hope you like your new job in Shenzhen. Teacher Huang, thank you for all your help with experiments and your encouragement to me when I felt frustrated. I hope you are doing well in the US. Zuoqing, I liked the time when we worked in the same office and went to the gym together, I wish you all the best. Mengpei, it was nice to go swimming with you in Shantou and thank you for distributing my thesis in Groningen. I wish you a lot of success with your PhD studies. Xueling and Huang Jin, I also thank you a lot for your help when I was in Shantou.

Appreciations also go to colleagues and friends in Chengdu. Dean Zhong, thank you for giving me the opportunity to work in the hospital and all your support. Prof. Huang, thank you for your help, encouragement, and all the good feedback you provided. I hope you enjoy your life and stay healthy. Shuling, thank you for the help with the experiments, it was nice to work with you. Quting, you are so warm-hearted, thank you for all your help. I wish you all the best. Also many thanks to you, Penghao, I miss the good restaurants you brought us to and the spicy food. Yuyin, congratulations on your new baby! Martin, thank you for all your help and keep up your drone flying skills! Jianying, you are so funny, there is always laughter in the air when you are around. Jihua, Tingting, Xinyue, Lingxiao, Liaoxue, Ali, it was nice to have you around, I wish all of you lots of success.

Last but not the least, I want to thank my dear family. Mom and Dad, thank you so much for your continuous support to me. No matter what decision I make you are always on my side. No matter how far I am away, I always know there is a home I can go back to. Yang, my dear brother, I wish you can successfully finish your university studies, find the job you like and be the person you want to be. Grandmother and grandfather, let me also express my deepest appreciation for everything you have done for me.

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Vincent, thank you for helping with statistics and all your valuable suggestions for my manuscript. Mirjam, thank you for processing all the fresh tissue samples and performing stainings. Brenda, you were the first colleague I worked with, thank you for introducing me to new techniques. Martijn, although we did not directly work together when I was in the UMCG, you have helped me a lot with the GSEA, thank you.

Lots of help was also provided by other departments and universities. Special thanks to Susanne, for performing the mouse partial hepatectomy in the CDP. Rachel and Alain at Utrecht University, thank you for analyzing all the histology and giving valuable suggestions.

Of course, all the lovely people in our office deserve special appreciation. Rima, you are always so funny and thoughtful, thank you for your help with all kinds of things and your comfort when I was frustrated. You have so much curiosity in all kinds of food and, believe me, you are an excellent cook. You are also a big fan of learning foreign languages, do you still remember the Chinese I taught you? It was so nice to have you around, I will neither forget the funny African party you brought me to, nor the coffee corner we liked to go. Mirjam, you are so kind and social. Many thanks for a lot of things, you have really helped me to adapt to the new environment. It was you who read Dutch letters for me and introduced me to Salsa dance. It was you who organized our gatherings from time to time. Many thanks for your generous help. Lidiya, you are so sportive, a big fan of yoga and mountain climbing, you always reminded me I should do more exercise. There would not be so much laughter in our office without you. Onne, the big fan of cosplay, did I say that it was actually very cool? Also thank you for your help with all kinds of questions I had. Sanam, you were always so nice, it was very joyful and relaxed to talk with you and your husband. I still remember the tasty dinner we had in your house. Henk, I also bothered you with a lot of questions, thanks for your help with my experiments and for being my nearest neighbor.

Appreciations also go to my Chinese friends. Yuan, thank you for being my good friend. Thank you for your help and all the dinners when I was in Groningen. You could always give suggestions when I had problems. We could talk about a lot of things. It was an unforgettable vacation that we spent together in Spain. Yingying, you have accompanied me a lot in Groningen, we went shopping, to the movies and traveled together. I treasure all the beautiful memories we had. Weilin, you were the only Chinese PhD student in our department when I arrived. It was great to know you, thank you for your help and for hosting dinners at your home. Siqi and Tiantian, you both are so warm-hearted and hospitable. I had a lot of fun with you.

Samiksia, Arwin, you were the early friends I had when I was in Groningen. We have done so much together. I liked the food we made, the climbing of the Martini tower, the travel to Amsterdam. I wish you all the best. Sandra, you are so nice and

191

hardworking, I hope everything is going well with you. Violeta, I liked all the talks we had, still remember the small picnic near the lake? Jun Li, Yana Geng, thank you for being a part of my life in Groningen. It was very nice to have you there. Not to forget, also many thanks to Hilde, Paula, Evelien. Without you, life would be much more difficult.

I would also like to express my appreciation to colleagues and friends in Shantou. Qiaoling, thank you for working with me and your help in our experiments. I hope you like your new job in Shenzhen. Teacher Huang, thank you for all your help with experiments and your encouragement to me when I felt frustrated. I hope you are doing well in the US. Zuoqing, I liked the time when we worked in the same office and went to the gym together, I wish you all the best. Mengpei, it was nice to go swimming with you in Shantou and thank you for distributing my thesis in Groningen. I wish you a lot of success with your PhD studies. Xueling and Huang Jin, I also thank you a lot for your help when I was in Shantou.

Appreciations also go to colleagues and friends in Chengdu. Dean Zhong, thank you for giving me the opportunity to work in the hospital and all your support. Prof. Huang, thank you for your help, encouragement, and all the good feedback you provided. I hope you enjoy your life and stay healthy. Shuling, thank you for the help with the experiments, it was nice to work with you. Quting, you are so warm-hearted, thank you for all your help. I wish you all the best. Also many thanks to you, Penghao, I miss the good restaurants you brought us to and the spicy food. Yuyin, congratulations on your new baby! Martin, thank you for all your help and keep up your drone flying skills! Jianying, you are so funny, there is always laughter in the air when you are around. Jihua, Tingting, Xinyue, Lingxiao, Liaoxue, Ali, it was nice to have you around, I wish all of you lots of success.

Last but not the least, I want to thank my dear family. Mom and Dad, thank you so much for your continuous support to me. No matter what decision I make you are always on my side. No matter how far I am away, I always know there is a home I can go back to. Yang, my dear brother, I wish you can successfully finish your university studies, find the job you like and be the person you want to be. Grandmother and grandfather, let me also express my deepest appreciation for everything you have done for me.

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Biography

Yu Lei was born on the 24th of Feb 1989 in Chongqing, China. In 2004, she finished high school and started to study Nursing at Zunyi Medical University, China. After 4 years, she graduated with the merit of an outstanding undergraduate student in the Guizhou Province. In 2011, she began to study Pathology under the supervision of Prof. Jiang Gu at Shantou University Medical College. In 2014, she obtained her Master degree and was admitted to a joint PhD program between Shantou University Medical College, China, and Groningen University, The Netherlands. She spent the first 2 ½ years working on glucose and lipid metabolism in the Department of Pediatrics, University Medical Center Groningen under the supervision of Dr. Maaike Oosterveer and Prof. Folkert Kuipers. In 2017, she continued her PhD study at Shantou University Medical College with Prof. Jiang Gu. In mid-2018, she started to work as a junior researcher in Chengdu Jinjiang Hospital for Maternal and Child Health Care, China. Currently, she is a post-doctoral researcher in the Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

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Biography

Yu Lei was born on the 24th of Feb 1989 in Chongqing, China. In 2004, she finished high school and started to study Nursing at Zunyi Medical University, China. After 4 years, she graduated with the merit of an outstanding undergraduate student in the Guizhou Province. In 2011, she began to study Pathology under the supervision of Prof. Jiang Gu at Shantou University Medical College. In 2014, she obtained her Master degree and was admitted to a joint PhD program between Shantou University Medical College, China, and Groningen University, The Netherlands. She spent the first 2 ½ years working on glucose and lipid metabolism in the Department of Pediatrics, University Medical Center Groningen under the supervision of Dr. Maaike Oosterveer and Prof. Folkert Kuipers. In 2017, she continued her PhD study at Shantou University Medical College with Prof. Jiang Gu. In mid-2018, she started to work as a junior researcher in Chengdu Jinjiang Hospital for Maternal and Child Health Care, China. Currently, she is a post-doctoral researcher in the Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.

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List of Publications

1. Lei Y, Huang T, Su M, Luo J, Korteweg C, Li J, Chen Z, et al. Expression and

distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver. Lab Invest 2014;94:1283-1295.

2. Wan X*, Lei Y*, Li Z, Wang J, Chen Z, McNutt M, Lin D, et al. Pancreatic Expression of Immunoglobulin G in Human Pancreatic Cancer and Associated Diabetes. Pancreas 2015;44:1304-1313.

3. Gu J*, Lei Y*, Huang YP, Zhao YY, Li J, Huang T, Zhang JJ, et al. Fab fragment glycosylated IgG may play a central role in placental immune evasion. Human Reproduction 2015;30:380-391.

4. Lei Y, Hu Q, Gu J. Expressions of Carbohydrate Response Element Binding

Protein and Glucose Transporters in Liver Cancer and Clinical Significance. Pathol Oncol Res 2019;doi: 10.1007/s12253-019-00708-y.

5. Hoogerland JA, Lei Y, Wolters JC, de Boer JF, Bos T, Bleeker A, Mulder NL, et al. Glucose-6-phosphate regulates hepatic bile acid synthesis in mice. Hepatology 2019;70:2171-2184.

6. Lei Y, Hoogerland JA, Bloks VW, Bos T, Bleeker A, Wolters H, Wolters JC, et al.

Hepatic ChREBP activation limits NAFLD development in a mouse model for Glycogen Storage Disease type Ia. Hepatology 2020;doi: 10.1002/hep.31198. 7. Lei Y*, Zhou S*, Hu Q, Chen X, Gu J. Carbohydrate response element binding

protein (ChREBP) correlates with colon cancer progression and contributes to cell proliferation. Sci Rep 2020;10:4233.

8. Lei Y, Rutten MGS, et al. ChREBP promotes hepatocyte proliferation in vitro

but protects against oncogenic hepatocyte transformation in a mouse model for Glycogen Storage Disease type 1a (in preparation)

9. Lei Y, Hoogerland JA, et al. Glucose-6-phosphatase deficiency shifts the balance between hepatocyte hyperplasia and hypertrophy during liver regeneration (in preparation)

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List of Publications

1. Lei Y, Huang T, Su M, Luo J, Korteweg C, Li J, Chen Z, et al. Expression and

distribution of immunoglobulin G in the normal liver, hepatocarcinoma and postpartial hepatectomy liver. Lab Invest 2014;94:1283-1295.

2. Wan X*, Lei Y*, Li Z, Wang J, Chen Z, McNutt M, Lin D, et al. Pancreatic Expression of Immunoglobulin G in Human Pancreatic Cancer and Associated Diabetes. Pancreas 2015;44:1304-1313.

3. Gu J*, Lei Y*, Huang YP, Zhao YY, Li J, Huang T, Zhang JJ, et al. Fab fragment glycosylated IgG may play a central role in placental immune evasion. Human Reproduction 2015;30:380-391.

4. Lei Y, Hu Q, Gu J. Expressions of Carbohydrate Response Element Binding

Protein and Glucose Transporters in Liver Cancer and Clinical Significance. Pathol Oncol Res 2019;doi: 10.1007/s12253-019-00708-y.

5. Hoogerland JA, Lei Y, Wolters JC, de Boer JF, Bos T, Bleeker A, Mulder NL, et al. Glucose-6-phosphate regulates hepatic bile acid synthesis in mice. Hepatology 2019;70:2171-2184.

6. Lei Y, Hoogerland JA, Bloks VW, Bos T, Bleeker A, Wolters H, Wolters JC, et al.

Hepatic ChREBP activation limits NAFLD development in a mouse model for Glycogen Storage Disease type Ia. Hepatology 2020;doi: 10.1002/hep.31198. 7. Lei Y*, Zhou S*, Hu Q, Chen X, Gu J. Carbohydrate response element binding

protein (ChREBP) correlates with colon cancer progression and contributes to cell proliferation. Sci Rep 2020;10:4233.

8. Lei Y, Rutten MGS, et al. ChREBP promotes hepatocyte proliferation in vitro

but protects against oncogenic hepatocyte transformation in a mouse model for Glycogen Storage Disease type 1a (in preparation)

9. Lei Y, Hoogerland JA, et al. Glucose-6-phosphatase deficiency shifts the balance between hepatocyte hyperplasia and hypertrophy during liver regeneration (in preparation)

* these authors contributed equally

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