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Histology and glycogen storage in the liver and kidneys of KO mice does not improve markedly with age. PAS-stained liver (a–d), and kidney (e–h), from KO pup, WT pup and KO adult. Relative glycogen deposition is indicated by accumulation of magenta staining within the cell cytoplasm. All shown at 400 total magnification.
Source publication
Glycogen storage disease type Ia (GSDIa) is caused by a genetic defect in the hepatic enzyme glucose-6-phosphatase (G6Pase-alpha), which manifests as life-threatening hypoglycemia with related metabolic complications. A G6Pase-alpha knockout (KO) mouse model was generated to study potential therapies for correcting this disorder. Since then, gene t...
Contexts in source publication
Context 1
... analysis of the liver tissue revealed distortion of the hepatic microarchitecture in both young and mature KO mice (Figure 3a and c) compared with that in WT animals (Figure 3b and d). PAS-stained sections revealed excess glycogen accumulation in GSDIa mice compared with that in WT mice, independent of age ( Figure 3a,c vs b,d). ...
Context 2
... analysis of the liver tissue revealed distortion of the hepatic microarchitecture in both young and mature KO mice (Figure 3a and c) compared with that in WT animals (Figure 3b and d). PAS-stained sections revealed excess glycogen accumulation in GSDIa mice compared with that in WT mice, independent of age ( Figure 3a,c vs b,d). ...
Context 3
... analysis of the liver tissue revealed distortion of the hepatic microarchitecture in both young and mature KO mice (Figure 3a and c) compared with that in WT animals (Figure 3b and d). PAS-stained sections revealed excess glycogen accumulation in GSDIa mice compared with that in WT mice, independent of age ( Figure 3a,c vs b,d). Analysis of histology and glycogen content revealed the same phenotypic manifestation in the kidney of KO animals compared with that in the kidney of WT animals (Figure 3eg vs f,h). ...
Context 4
... sections revealed excess glycogen accumulation in GSDIa mice compared with that in WT mice, independent of age ( Figure 3a,c vs b,d). Analysis of histology and glycogen content revealed the same phenotypic manifestation in the kidney of KO animals compared with that in the kidney of WT animals (Figure 3eg vs f,h). Aperio slide scanning and image analysis software were used to quantify relative staining intensity among mice of both genotypes and age brackets. ...
Citations
... Globally, 65 of the 69 amino acids at positions of the naturally occurring GSD type 1a missense/nonsense mutations, including all of those at the active site, are strictly conserved between hG6PC1 and mG6PC1. Moreover, a murine model of GSD type 1a (G6PC1 -/-) recapitulates a similar phenotype as human patients (34,35). Importantly, detergent-solubilized mouse G6PC1 demonstrates elevated structural and catalytic stability relative to human G6PC1 in biochemical assays (33), supporting an unequivocal interpretation of our in vitro studies. ...
Mediating the terminal reaction of gluconeogenesis and glycogenolysis, the integral membrane protein G6PC1 regulates hepatic glucose production by catalyzing hydrolysis of glucose-6-phosphate within the lumen of the endoplasmic reticulum. Because G6PC1 function is essential for blood glucose homeostasis, inactivating mutations cause glycogen storage disease (GSD) type 1a, which is characterized by severe hypoglycemia. Despite its physiological importance, the structural basis of G6P binding to G6PC1 and the molecular disruptions induced by missense mutations within the active site that give rise to GSD type 1a are unknown. Exploiting a computational model of G6PC1 derived from the groundbreaking structure prediction algorithm AlphaFold2 (AF2), we combine molecular dynamics (MD) simulations and computational predictions of thermodynamic stability with a robust in vitro screening platform to define the atomic interactions governing G6P binding within the active site as well as explore the energetic perturbations imposed by disease-linked variants. From over 15 μs of MD simulations, we identify a collection of side chains, including conserved residues from the signature phosphatidic acid phosphatase motif, that contribute to a hydrogen bonding and van der Waals network that stabilize G6P in the active site. Introduction of GSD type 1a mutations into the G6PC1 sequence causes changes in G6P binding energy, thermodynamic stability and structural properties, suggesting multiple mechanisms of catalytic impairment. Our results, which corroborate the high quality of the AF2 model as a guide for experimental design and to interpret outcomes, not only confirm active site structural organization but also suggest novel mechanistic contributions of catalytic side chains.
... A large number of studies have con rmed that the expression of G6Pase is sharply increased in diabetic mice [10]. However, G6Pase knockout mice exhibit increased glycogen accumulation [31]. Besides that, inhibiting G6Pase expression involved in the improvement of glucose metabolism disorder in T2DM. ...
Background: Abnormalities in lipid and glucose metabolism are are constantly occured in type 2 diabetes (T2DM). However, it can be ameliorated by gentiopicroside (GPS). Considering the key role of fibroblast growth factor receptor 1/phosphatidylinositol 3-kinase/protein kinase B (FGFR1/PI3K/AKT) pathway in T2DM, we explore the possible mechanism of GPS on lipid and glucose metabolism through its effects on FGFR1/PI3K/AKT pathway.
Methods: Palmitic acid (PA)-induced HepG2 cells and a db/db mice were used to clarify the role and mechanism of polydatin on lipid and glucose metabolism.
Results: GPS ameliorated glucose and lipid metabolism disorders in db/db mice and PA-induced HepG2 cells. Furthermore, GPS activated FGFR1/PI3K/AKT pathway including increased the protein expression of FGFR1 and promoted the phosphorylation of PI3K, AKT and FoxO1. Additionally, knockdown of FGFR1 reversed the activation of PI3K/AKT pathway by GPS.
Conclusions: The present study demontrates that GPS ameliorates glucose and lipid metabolism disorders via activation of FGFR1/PI3K/AKT pathway.
... No significant changes in expression levels of other gluconeogenic proteins CREB, PGC-1a, CRTC2, FOXO1, or FOXO3 were observed [29]. Hepatic knockdown of HDAC4, HDAC5 or HDAC4/5/7 in mice increased glycogen storage and lowered blood glucose [29], phenotyping hepatic deficiency of Foxo1 in mice [112], as well as the G6pc deficiency in mice and patients of Glycogen Storage Disease Type I [113,114]. Simultaneous triple knockdown of HDAC4/5/7 showed the most significant effect in ameliorating hyperglycemia in several diabetic mouse models, with improved glucose tolerance in a glucose tolerance test [29]. Mechanistic studies indicated that knockdown of class IIa HDACs led to FOXO hyperacetylation and decreased expression of FOXO targets, such as gluconeogenic genes (G6pc, Pck1 and Fbp1) [29]. ...
This review summarizes key literature defining the phenotypes of individual class IIa HDAC proteins and compounds that selectively target their enzymatic catalytic domain (CD). The focus is on the effects of class IIa HDACs in physiological and pathological conditions, both in vitro and in vivo, and their mode of action in regulating genes, upstream proteins and signaling pathways. Phenotype studies further demonstrate either beneficial or detrimental effects of silencing selected class IIa HDACs or their enzymatic properties. We also summarize the knowledge gained from structure-activity relationships of CD inhibitors as well as molecular mechanisms underpinning isozyme selectivity where crystal structures or modelling studies were available. Given that the numbers of genes affected by silencing class IIa HDACs are much smaller than class I, the role of gene regulation of class IIa HDACs could be much more selective. Since class IIa HDACs have restricted tissue distributions and multiple functions independent of their CD, targeting the CD of class IIa HDACs could lead to more selective therapeutic agents with significantly fewer side-effects than for other HDAC ligands.
... A limitation of our study is the use of adult, hepatocyte-specific G6pc1 deficient mice. Whole body G6pc1 À/À mice require daily glucose injections to survive, they rarely live longer than 3 months, and cannot be used to investigate the effects of G6pc1 deficiency during fasting [11,86,87]. Although kidney-specific and intestinal-specific G6pc1 deficient mice do not become hypoglycemic upon fasting [88e90], we cannot exclude that in GSD Ia patients, loss of G6pc activity in kidney and intestine contributes to the bleeding phenotype under conditions of poor glycemic control. ...
Objective
Glycogen storage disease type 1a (GSD Ia) is a rare inherited metabolic disorder caused by mutations in the glucose-6-phosphatase (G6PC1) gene. When untreated, GSD Ia leads to severe fasting-induced hypoglycemia. Although current intensive dietary management aims to prevent hypoglycemia, patients still experience hypoglycemic events. Poor glycemic control in GSD Ia is associated with hypertriglyceridemia, hepatocellular adenoma and carcinoma, and also with an increased bleeding tendency of unknown origin.
Methods
To evaluate the effect of glycemic control on leukocyte levels and coagulation in GSD Ia, we employed hepatocyte-specific G6pc1 deficient (L-G6pc-/-) mice under fed or fasted conditions, to match good or poor glycemic control in GSD Ia, respectively.
Results
We found that fasting-induced hypoglycemia in L-G6pc-/- mice decreased blood leukocytes, specifically pro-inflammatory Ly6Chi monocytes, compared to controls. Refeeding reversed this decrease. The decrease in Ly6Chi monocytes was accompanied by an increase in plasma corticosterone levels and was prevented by the glucocorticoid receptor antagonist mifepristone. Further, fasting-induced hypoglycemia in L-G6pc-/- mice prolonged bleeding time in the tail vein bleeding assay, with reversal by refeeding. This could not be explained by changes in coagulation factors V, VII, or VIII, or von Willebrand factor. While the prothrombin and activated partial thromboplastin time, as well as total platelet counts were not affected by fasting-induced hypoglycemia in L-G6pc-/- mice, ADP-induced platelet aggregation was disturbed.
Conclusions
These studies reveal a relationship between fasting-induced hypoglycemia, decreased blood monocytes, and disturbed platelet aggregation in L-G6pc-/- mice. While disturbed platelet aggregation likely accounts for the bleeding phenotype in GSD Ia, elevated plasma corticosterone decreases levels of pro-inflammatory monocytes. These studies highlight the necessity of maintaining good glycemic control in GSD Ia.
... Glycogen accumulation in the liver can be diet-dependent [7][8][9] and age-dependent [9], e.g., younger mice store slightly more glycogen than older ones [10]. Moreover, an in vitro study showed that the primary culture of hepatocytes derived from old rats (around 24 months) exhibited a higher potential for glucose production when compared with the hepatocytes derived from younger rats (around 4-months old) [11]. ...
Background:
A growing body of data indicates that the physiology of the liver is sex-hormone dependent, with some types of liver failure occurring more frequently in males, and some in females. In males, in physiological conditions, testosterone acts via androgen receptors (AR) to increase insulin receptor (IR) expression and glycogen synthesis, and to decrease glucose uptake controlled by liver-specific glucose transporter 2 (GLUT-2). Our previous study indicated that this mechanism may be impaired by finasteride, a popular drug used in urology and dermatology, inhibiting 5α-reductase 2, which converts testosterone (T) into dihydrotestosterone (DHT). Our research has also shown that the offspring of rats exposed to finasteride have an altered T-DHT ratio and show changes in their testes and epididymides. Therefore, the goal of this study was to assess whether the administration of finasteride had an trans-generational effect on (i) GLUT-2 dependent accumulation of glycogen in the liver, (ii) IR and AR expression in the hepatocytes of male rat offspring, (iii) a relation between serum T and DHT levels and the expression of GLUT2, IR, and AR mRNAs, (iv) a serum glucose level and it correlation with GLUT-2 mRNA.
Methods:
The study was conducted on the liver (an androgen-dependent organ) from 7, 14, 21, 28, and 90-day old Wistar male rats (F1:Fin) born by females fertilized by finasteride-treated rats. The control group was the offspring (F1:Control) of untreated Wistar parents. In the histological sections of liver the Periodic Acid Schiff (PAS) staining (to visualize glycogen) and IHC (to detect GLUT-2, IR, and AR) were performed. The liver homogenates were used in qRT-PCR to assess GLUT2, IR, and AR mRNA expression. The percentage of PAS-positive glycogen areas were correlated with the immunoexpression of GLUT-2, serum levels of T and DHT were correlated with GLUT-2, IR, and AR transcript levels, and serum glucose concentration was correlated with the age of animals and with the GLUT-2 mRNA by Spearman's rank correlation coefficients.
Results:
In each age group of F1:Fin rats, the accumulation of glycogen was elevated but did not correlate with changes in GLUT-2 expression. The levels of GLUT-2, IR, and AR transcripts and their immunoreactivity statistically significantly decreased in F1:Fin animals. In F1:Fin rats the serum levels of T and DHT negatively correlated with androgen receptor mRNA. The animals from F1:Fin group have statistically elevated level of glucose. Additionally, in adult F1:Fin rats, steatosis was observed in the liver (see Appendix A).
Conclusions:
It seems that treating male adult rats with finasteride causes changes in the carbohydrate metabolism in the liver of their offspring. This can lead to improper hepatic energy homeostasis or even hyperglycaemia, insulin resistance, as well as some symptoms of metabolic syndrome and liver steatosis.
... Although very few untreated mice lived beyond 6 months of age, surprisingly they did not show lethal hepatic or renal lesions. The significance of this apparently paradoxical finding has yet to be determined [118]. ...
GSD are a group of disorders characterized by a defect in gene expression of specific enzymes involved in glycogen breakdown or synthesis, commonly resulting in the accumulation of glycogen in various tissues (primarily the liver and skeletal muscle). Several different GSD animal models have been found to naturally present spontaneous mutations and others have been developed and characterized in order to further understand the physiopathology of these diseases and as a useful tool to evaluate potential therapeutic strategies. In the present work we have reviewed a total of 42 different animal models of GSD, including 26 genetically modified mouse models, 15 naturally occurring models (encompassing quails, cats, dogs, sheep, cattle and horses), and one genetically modified zebrafish model. To our knowledge, this is the most complete list of GSD animal models ever reviewed. Importantly, when all these animal models are analyzed together, we can observe some common traits, as well as model specific differences, that would be overlooked if each model was only studied in the context of a given GSD.
... However, HCC have not been observed in untreated G6pc À/À mice as old as 6 months, which limits the usefulness of this model for studying the prevention of HCC. 23 Based on these results, we have investigated whether a genome-editing-based correction approach with these two vectors can suppress hepatic tumors in the adult liver-specific G6pc À/À mouse model (L-G6pc À/À ), a model that consistently formed HCC by 1 year of age. 24 ...
Glycogen storage disease type Ia (GSD Ia) is caused by mutations in the glucose-6-phosphatase (G6Pase) catalytic subunit gene (G6PC). GSD Ia complications include hepatocellular adenomas (HCA) with a risk for hepatocellular carcinoma (HCC) formation. Genome editing with adeno-associated virus (AAV) vectors containing a zinc-finger nuclease (ZFN) and a G6PC donor transgene was evaluated in adult mice with GSD Ia. Although mouse livers expressed G6Pase, HCA and HCC occurred following AAV vector administration. Interestingly, vector genomes were almost undetectable in the tumors but remained relatively high in adjacent liver (p < 0.01). G6Pase activity was decreased in tumors, in comparison with adjacent liver (p < 0.01). Furthermore, AAV-G6Pase vector-treated dogs with GSD Ia developed HCC with lower G6Pase activity (p < 0.01) in comparison with adjacent liver. AAV integration and tumor marker analysis in mice revealed that tumors arose from the underlying disorder, not from vector administration. Similarly to human GSD Ia-related HCA and HCC, mouse and dog tumors did not express elevated α-fetoprotein. Taken together, these results suggest that AAV-mediated gene therapy not only corrects hepatic G6Pase deficiency, but also has potential to suppress HCA and HCC in the GSD Ia liver. Keywords: glycogen storage disorder type Ia, von Gierke disease, glucose-6-phosphatase, hepatocellular carcinoma, genome editing
... Elles développent tous les symptômes de la GSDI, mais leur espérance de vie est très faible puisque la plupart ne survivent pas au sevrage. La mise en place de soins palliatifs quotidiens tels que des injections fréquentes de glucose, l'ajout de glucose dans l'eau de boisson et une supplémentation alimentaire permet d'obtenir un taux de survie au sevrage uniquement de 60%, avec une espérance de vie courte(Salganik et al., 2009). Les souris G6pc -/ne survivent pas assez longtemps pour développer les pathologies hépatiques et rénales à long terme associées à la GSDI et ne permettent donc pas d'étudier les mécanismes moléculaires sous-jacents de ces complications. ...
La glycogénose de type Ia (GSDIa) est une maladie métabolique rare causée par une déficience en glucose-6-phosphatase (G6Pase), due à des mutations de la sous-unité catalytique (G6PC). Cette enzyme confère au foie, aux reins et à l’intestin la capacité de produire du glucose. Les patients atteints de GSDIa sont donc incapables de produire du glucose et souffrent d’hypoglycémies sévères lors de jeûnes courts. De plus, la déficience en G6Pase provoque une accumulation de glucose-6 phosphate dans le foie et les reins, conduisant à l’accumulation de glycogène et de lipides. A long terme, la plupart des patients souffre d’une maladie chronique rénale (MCR), qui peut évoluer en insuffisance rénale, nécessitant une mise sous dialyse ou une transplantation rénale. Cette MCR se caractérise par une fibrose, ainsi que par le développement de kystes dans les stades tardifs. Au niveau du foie, les patients développent une hépatomégalie et une stéatose hépatique qui peut évoluer vers le développement d’adénomes ou carcinomes hépatocellulaires. Le but de mes travaux de thèse a été d’identifier les mécanismes moléculaires impliqués dans l’établissement de la pathologie rénale et la formation des kystes, à l’aide de modèles murins invalidés pour le gène G6pc spécifiquement dans les reins (souris K.G6pc-/-). Alors que la GSDIa est une maladie caractérisée par l’accumulation hépatique et rénale de glycogène, nous avons d’abord montré que le développement de la fibrose, à l’origine de la perte de la fonction rénale, était induit par l’accumulation de lipides, indépendamment du contenu en glycogène. De plus, l’utilisation d’un agoniste de PPARα, le fénofibrate, en diminuant le contenu lipidique rénal, a ralenti l’installation de la fibrose et l’évolution de la MCR. Le mécanisme moléculaire impliqué est l’activation du système rénine angiotensine par les dérivés lipidiques, qui induit l’expression du facteur profibrotique TGFβ1. De même, le fénofibrate en limitant l’accumulation de lipides hépatiques a prévenu le développement d’atteintes hépatiques caractéristiques de la GSDI. Ainsi, l’activation du catabolisme des lipides par des agonistes de PPARα semble une stratégie thérapeutique intéressante pour réduire la progression des maladies rénales et hépatique de la GSDI. La deuxième partie de mes résultats suggèrent que le développement de kystes rénaux chez les patients atteints de la GSDI pourrait être causé par une altération du cil primaire, organelle jouant un rôle clé dans le maintien d’une structure et fonction normale des reins. En effet, une augmentation de la longueur du cil primaire a pu être observée dans les reins des souris K.G6pc-/- associée à une dérégulation de différentes protéines impliquées dans sa structure et sa fonction, par rapport aux souris contrôles. Nous avons également mis en évidence une reprogrammation métabolique de type Warburg, caractérisée par une activation accrue de la glycolyse aérobie, une inhibition de l’oxydation mitochondriale du pyruvate et une production de lipides. Ainsi, l’ensemble de ces perturbations va favoriser la prolifération cellulaire et le développement de kystes, et pourrait mener au développement de tumeur rénale comme observée chez une souris K.G6pc-/-. En conclusion nous avons démontré que, dans le cadre de la GSDI, l’accumulation de lipides dans les reins et le foie, secondaire à la déficience en G6Pase, joue un rôle clé dans le développement des complications hépatiques et rénales à long terme. Également, la reprogrammation métabolique rénale de type Warburg, prenant place dans le cadre de la GSDI, associée à un défaut du cil primaire pourrait être à l’origine de la formation des kystes et de tumeurs rénales. Ces études, en permettant une meilleure compréhension de la physiopathologie des complications à long terme de la GSDIa, offrent de nouvelles perspectives concernant les stratégies thérapeutiques à développer pour une meilleure prise en charge des patients atteints de GSDIa
... Since free radicals are also released in large amounts from inflammatory cells activated [Decker, 1990], their increased number reinforces the evidence of generation of ROS/RNS in liver of rats. Staining for the analysis of glycoproteins/glycogen revealed that changes in the hepatic microarchitecture are similar (although less severe) to histological phenotype to that observed in liver of patients with glycogen storage disease [Salganik et al., 2009], presenting a marked increase in the glycogen content in liver of proline-treated rats. Biochemical determination reaffirmed this finding, which demonstrate that proline increases glycogen concentration and the synthesis of glycogen from both direct (from D[U-14 C]glucose) and indirect (from L[U-14 C]alanine) pathways in the liver of rats. ...
The present study investigated the effects of chronic hyperprolinemia on oxidative and metabolic status in liver and serum of rats. Wistar rats received daily subcutaneous injections of proline from their 6th to 28th day of life. Twelve hours after the last injection the rats were sacrificed and liver and serum were collected. Results showed that hyperprolinemia induced a significant reduction in total antioxidant potential and thiobarbituric acid-reactive substances. The activities of the antioxidant enzymes catalase and superoxide dismutase were significantly increased after chronic proline administration, while glutathione (GSH) peroxidase activity, dichlorofluorescin oxidation, GSH, sulfhydryl, and carbonyl content remained unaltered. Histological analyses of the liver revealed that proline treatment induced changes of the hepatic microarchitecture and increased the number of inflammatory cells and the glycogen content. Biochemical determination also demonstrated an increase in glycogen concentration, as well as a higher synthesis of glycogen in liver of hyperprolinemic rats. Regarding to hepatic metabolism, it was observed an increase on glucose oxidation and a decrease on lipid synthesis from glucose. However, hepatic lipid content and serum glucose levels were not changed. Proline administration did not alter the aminotransferases activities and serum markers of hepatic injury. Our findings suggest that hyperprolinemia alters the liver homeostasis possibly by induction of a mild degree of oxidative stress and metabolic changes. The hepatic alterations caused by proline probably do not implicate in substantial hepatic tissue damage, but rather demonstrate a process of adaptation of this tissue to oxidative stress. However, the biological significance of these findings requires additional investigation.
... G6Pase is a rate-limiting enzyme of both gluconeogenesis and glycogenolysis (Hutton and O'Brien, 2009) and mutations in glucose-6-phophatase (G6pc) result in Glycogen Storage Disease Type I in humans (GSD Type I or Von Gierke's disease) characterized by aberrant glycogen storage and hypoglycemia, a phenotype also mimicked in genetic mouse models of G6Pase deletion (Salganik et al., 2009;Peng et al., 2009). Given the dramatic effect of HDAC4/5/7 depletion on G6Pase in hepatocytes, we sought to examine the effect of their loss in the intact mouse liver. ...
... These findings illuminate a mechanism by which glucagon can acutely stimulate FOXO activity, providing a molecular basis for how FOXO mediates effects of both fasting hormones and insulin on hepatic glucose production (Matsumoto et al., 2007). Consistent with this, hepatic knockdown of Class IIa HDACs in vivo results in lowered blood glucose and altered glycogen storage, phenocopying hepatic deficiency of Foxo1 in mice (Matsumoto et al., 2007), as well as the G6pc deficiency in mice and human Glycogen Storage Disease Type I (GSDI) patients (Salganik et al., 2009;Peng et al., 2009). ...
Class IIa histone deacetylases (HDACs) are signal-dependent modulators of transcription with established roles in muscle differentiation and neuronal survival. We show here that in liver, class IIa HDACs (HDAC4, 5, and 7) are phosphorylated and excluded from the nucleus by AMPK family kinases. In response to the fasting hormone glucagon, class IIa HDACs are rapidly dephosphorylated and translocated to the nucleus where they associate with the promoters of gluconeogenic enzymes such as G6Pase. In turn, HDAC4/5 recruit HDAC3, which results in the acute transcriptional induction of these genes via deacetylation and activation of FOXO family transcription factors. Loss of class IIa HDACs in murine liver results in inhibition of FOXO target genes and lowers blood glucose, resulting in increased glycogen storage. Finally, suppression of class IIa HDACs in mouse models of type 2 diabetes ameliorates hyperglycemia, suggesting that inhibitors of class I/II HDACs may be potential therapeutics for metabolic syndrome.
