Possible association of G6PC2 and MUC6 induced by low­dose­rate irradiation in mouse intestine with inflammatory bowel disease.

Although there are several types of radiation exposure, it is debated whether low­dose­rate (LDR) irradiation (IR) affects the body. Since the small intestine is a radiation­sensitive organ, the present study aimed to evaluate how it changes when exposed to LDR IR and identify the genes sensitive to these doses. After undergoing LDR (6.0 mGy/h) γ radiation exposure, intestinal RNA from BALB/c mice was extracted 1 and 24 h later. Mouse whole genome microarrays were used to explore radiation­induced transcriptional alterations. Reverse transcription­quantitative (RT­q) PCR was used to examine time­ and dose­dependent radiation responses. The histopathological status of the jejunum in the radiated mouse was not changed by 10 mGy of LDR IR; however, 23 genes were upregulated in response to LDR IR of the jejunum in mice after 1 and 24 h of exposure. Upregulated genes were selected to validate the results of the RNA sequencing analysis for RT­qPCR detection and results showed that only Na+/K+ transporting subunit α4, glucose­6­phosphatase catalytic subunit 2 (G6PC2), mucin 6 (MUC6) and transient receptor potential cation channel subfamily V member 6 levels significantly increased after 24 h of LDR IR. Furthermore, G6PC2 and MUC6 were notable genes induced by LDR IR exposure according to protein expression via western blot analysis. The mRNA levels of G6PC2 and MUC6 were significantly elevated within 24 h under three conditions: i) Exposure to LDR IR, ii) repeated exposure to LDR IR and iii) exposure to LDR IR in the presence of inflammatory bowel disease. These results could contribute to an improved understanding of immediate radiation reactions and biomarker development to identify radiation­susceptible individuals before histopathological changes become noticeable. However, further investigation into the specific mechanisms involving G6PC2 and MUC6 is required to accomplish this.

Tanycytes release glucose using the glucose-6-phosphatase system during hypoglycemia to control hypothalamic energy balance.

OBJECTIVE: The liver releases glucose into the blood using the glucose-6-phosphatase (G6Pase) system, a multiprotein complex located in the endoplasmic reticulum (ER). Here, we show for the first time that the G6Pase system is also expressed in hypothalamic tanycytes, and it is required to regulate energy balance. METHODS: Using automatized qRT-PCR and immunohistochemical analyses, we evaluated the expression of the G6Pase system. Fluorescent glucose analogue (2-NBDG) uptake was evaluated by 4D live-cell microscopy. Glucose release was tested using a glucose detection kit and high-content live-cell analysis instrument, Incucyte s3. In vivo G6pt knockdown in tanycytes was performed by AAV1-shG6PT-mCherry intracerebroventricular injection. Body weight gain, adipose tissue weight, food intake, glucose metabolism, c-Fos, and neuropeptide expression were evaluated at 4 weeks post-transduction. RESULTS: Tanycytes sequester glucose-6-phosphate (G6P) into the ER through the G6Pase system and release glucose in hypoglycaemia via facilitative glucose transporters (GLUTs). Strikingly, in vivo tanycytic G6pt knockdown has a powerful peripheral anabolic effect observed through decreased body weight, white adipose tissue (WAT) tissue mass, and strong downregulation of lipogenesis genes. Selective deletion of G6pt in tanycytes also decreases food intake, c-Fos expression in the arcuate nucleus (ARC), and Npy mRNA expression in fasted mice. CONCLUSIONS: The tanycyte-associated G6Pase system is a central mechanism involved in controlling metabolism and energy balance.

Identifying G6PC3 as a Potential Key Molecule in Hypoxic Glucose Metabolism of Glioblastoma Derived from the Depiction of 18F-Fluoromisonidazole and 18F-Fluorodeoxyglucose Positron Emission Tomography.

Purpose: Glioblastoma is the most aggressive primary brain tumor, characterized by its distinctive intratumoral hypoxia. Sequential preoperative examinations using fluorine-18-fluoromisonidazole (18F-FMISO) and fluorine-18-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) could depict the degree of glucose metabolism with hypoxic condition. However, molecular mechanism of glucose metabolism under hypoxia in glioblastoma has been unclear. The aim of this study was to identify the key molecules of hypoxic glucose metabolism. Methods: Using surgically obtained specimens, gene expressions associated with glucose metabolism were analyzed in patients with glioblastoma (n = 33) who underwent preoperative 18F-FMISO and 18F-FDG PET to identify affected molecules according to hypoxic condition. Tumor in vivo metabolic activities were semiquantitatively evaluated by lesion-normal tissue ratio (LNR). Protein expression was confirmed by immunofluorescence staining. To evaluate prognostic value, relationship between gene expression and overall survival was explored in another independent nonoverlapping clinical cohort (n = 17) and validated by The Cancer Genome Atlas (TCGA) database (n = 167). Results: Among the genes involving glucose metabolic pathway, mRNA expression of glucose-6-phosphatase 3 (G6PC3) correlated with 18F-FDG LNR (P = 0.03). In addition, G6PC3 mRNA expression in 18F-FMISO high-accumulated glioblastomas was significantly higher than that in 18F-FMISO low-accumulated glioblastomas (P < 0.01). Protein expression of G6PC3 was consistent with mRNA expression, which was confirmed by immunofluorescence analysis. These findings indicated that the G6PC3 expression might be facilitated by hypoxic condition in glioblastomas. Next, we investigated the clinical relevance of G6PC3 in terms of prognosis. Among the glioblastoma patients who received gross total resection, mRNA expressions of G6PC3 in the patients with poor prognosis (less than 1-year survival) were significantly higher than that in the patients who survive more than 3 years. Moreover, high mRNA expression of G6PC3 was associated with poor overall survival in glioblastoma, as validated by TCGA database. Conclusion: G6PC3 was affluently expressed in glioblastoma tissues with coincidentally high 18F-FDG and 18F-FMISO accumulation. Further, it might work as a prognostic biomarker of glioblastoma. Therefore, G6PC3 is a potential key molecule of glucose metabolism under hypoxia in glioblastoma.

Extraislet expression of islet antigen boosts T cell exhaustion to partially prevent autoimmune diabetes.

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

Identification of structural motifs critical for human G6PC2 function informed by sequence analysis and an AlphaFold2-predicted model.

G6PC2 encodes a glucose-6-phosphatase (G6Pase) catalytic subunit, primarily expressed in pancreatic islet ß cells, which modulates the sensitivity of insulin secretion to glucose and thereby regulates fasting blood glucose (FBG). Mutational analyses were conducted to validate an AlphaFold2 (AF2)-predicted structure of human G6PC2 in conjunction with a novel method to solubilize and purify human G6PC2 from a heterologous expression system. These analyses show that residues forming a predicted intramolecular disulfide bond are essential for G6PC2 expression and that residues forming part of a type 2 phosphatidic acid phosphatase (PAP2) motif are critical for enzyme activity. Additional mutagenesis shows that residues forming a predicted substrate cavity modulate enzyme activity and substrate specificity and residues forming a putative cholesterol recognition amino acid consensus (CRAC) motif influence protein expression or enzyme activity. This CRAC motif begins at residue 219, the site of a common G6PC2 non-synonymous single-nucleotide polymorphism (SNP), rs492594 (Val219Leu), though the functional impact of this SNP is disputed. In microsomal membrane preparations, the L219 variant has greater activity than the V219 variant, but this difference disappears when G6PC2 is purified in detergent micelles. We hypothesize that this was due to a differential association of the two variants with cholesterol. This concept was supported by the observation that the addition of cholesteryl hemi-succinate to the purified enzymes decreased the Vmax of the V219 and L219 variants ∼8-fold and ∼3 fold, respectively. We anticipate that these observations should support the rational development of G6PC2 inhibitors designed to lower FBG.

Endogenous Glucose Production in Patients With Glycogen Storage Disease Type Ia Estimated by Oral D-[6,6-2H2]-glucose.

CONTEXT: Glycogen storage disease type Ia (GSDIa) is an inborn metabolic disorder characterized by impaired endogenous glucose production (EGP). Monitoring of patients with GSDIa is prioritized because of ongoing treatment developments. Stable isotope tracers may enable reliable EGP monitoring. OBJECTIVE: The aim of this study was to prospectively assess the rate of appearance of endogenous glucose into the bloodstream (Ra) in patients with GSDIa after a single oral D-[6,6-2H2]-glucose dose. METHODS: Ten adult patients with GSDIa and 10 age-, sex-, and body mass index-matched healthy volunteers (HVs) were enrolled. For each participant, 3 oral glucose tracer tests were performed: (1) preprandial/fasted, (2) postprandial, and (3) randomly fed states. Dried blood spots were collected before D-[6,6-2H2]-glucose administration and 10, 20, 30, 40, 50, 60, 75, 90, and 120 minutes thereafter. RESULTS: Glucose Ra in fasted HVs was consistent with previously reported data. The time-averaged glucose Ra was significantly higher in (1) preprandial/fasted patients with GSDIa than HV and (2) postprandial HV compared with fasted HV(P < .05). A progressive decrease in glucose Ra was observed in preprandial/fasted patients with GSDIa; the change in glucose Ra time-course was directly correlated with the change in capillary glucose (P < .05). CONCLUSION: This is the first study to quantify glucose Ra in patients with GSDIa using oral D-[6,6-2H2] glucose. The test can reliably estimate EGP under conditions in which fasting tolerance is unaffected but does not discriminate between relative contributions of EGP (eg, liver, kidney) and exogenous sources (eg, dietary cornstarch). Future application is warranted for longitudinal monitoring after novel genome based treatments in patients with GSDIa in whom nocturnal dietary management can be discontinued.

Mutational analysis and clinical investigations of medically diagnosed GSD 1a patients from Pakistan.

Glycogen storage disease type I (GSD I) is a rare autosomal recessive inborn error of carbohydrate metabolism caused by the defects of glucose-6-phosphatase complex (G6PC). Disease causing variants in the G6PC gene, located on chromosome 17q21 result in glycogen storage disease type Ia (GSD Ia). Age of onset of GSD Ia ranges from 0.5 to 25 years with presenting features including hemorrhage, hepatic, physical and blood related abnormalities. The overall goal of proposed study was clinical and genetic characterization of GSD Ia cases from Pakistani population. This study included forty GSD Ia cases presenting with heterogeneous clinical profile including hypoglycemia, hepatomegaly, lactic acidosis i.e., pH less than 7.2, hyperuricemia, seizures, epistaxis, hypertriglyceridemia (more than180 mg/dl) and sometimes short stature. All coding exons and intron-exon boundaries of G6PC gene were screened to identify pathogenic variant in 20 patients based on availability of DNA samples and willingness to participate in molecular analysis. Pathogenic variant analysis was done using PCR-Sanger sequencing method and pathogenic effect predictions for identified variants were carried out using PROVEAN, MutationTaster, Polyphen 2, HOPE, Varsome, CADD, DANN, SIFT and HSF software. Overall, 21 variants were detected including 8 novel disease causing variants i.e., G6PC (NM_000151.4):c.71A>C (p.Gln24Pro), c.109G>C(p.Ala37Pro), c.133G>C(p.Val45Leu), c.49_50insT c.205G>A(p.Asp69Asn), c.244C>A(p.Gln82Lys) c.322A>C(p.Thr108Pro) and c.322A>C(p.Cys284Tyr) in the screened regions of G6PC gene. Out of 13 identified polymorphisms, 3 were identified in heterozygous condition while 10 were found in homozygous condition. This study revealed clinical presentation of GSD Ia cases from Pakistan and identification of novel disease-causing sequence variants in coding region and intron-exon boundaries of G6PC gene.

A molecular signature for the G6PC3/SLC37A2/SLC37A4 interactors in glioblastoma disease progression and in the acquisition of a brain cancer stem cell phenotype.

Background: Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive. Objective: We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes. Methods: In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype. Results: Higher expression in G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-ß-induced EMT biomarker SNAIL and cell chemotaxis. Conclusion: Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.

Aberrant glucose metabolism underlies impaired macrophage differentiation in glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT) that is responsible for transporting G6P into the endoplasmic reticulum. GSD-Ib is characterized by disturbances in glucose homeostasis, neutropenia, and neutrophil dysfunction. Although some studies have explored neutrophils abnormalities in GSD-Ib, investigations regarding monocytes/macrophages remain limited so far. In this study, we examined the impact of G6PT deficiency on monocyte-to-macrophage differentiation using bone marrow-derived monocytes from G6pt-/- mice as well as G6PT-deficient human THP-1 monocytes. Our findings revealed that G6PT-deficient monocytes exhibited immature differentiation into macrophages. Notably, the impaired differentiation observed in G6PT-deficient monocytes seemed to be associated with abnormal glucose metabolism, characterized by enhanced glucose consumption through glycolysis, even under quiescent conditions with oxidative phosphorylation. Furthermore, we observed a reduced secretion of inflammatory cytokines in G6PT-deficient THP-1 monocytes during the inflammatory response, despite their elevated glucose consumption. In conclusion, this study sheds light on the significance of G6PT in monocyte-to-macrophage differentiation and underscores its importance in maintaining glucose homeostasis and supporting immune response in GSD-Ib. These findings may contribute to a better understanding of the pathogenesis of GSD-Ib and potentially pave the way for the development of targeted therapeutic interventions.

A splice-switching oligonucleotide treatment ameliorates glycogen storage disease type 1a in mice with G6PC c.648G>T.

Glycogen storage disease type 1a (GSD1a) is caused by a congenital deficiency of glucose-6-phosphatase-α (G6Pase-α, encoded by G6PC), which is primarily associated with life-threatening hypoglycemia. Although strict dietary management substantially improves life expectancy, patients still experience intermittent hypoglycemia and develop hepatic complications. Emerging therapies utilizing new modalities such as adeno-associated virus and mRNA with lipid nanoparticles are under development for GSD1a but potentially require complicated glycemic management throughout life. Here, we present an oligonucleotide-based therapy to produce intact G6Pase-α from a pathogenic human variant, G6PC c.648G>T, the most prevalent variant in East Asia causing aberrant splicing of G6PC. DS-4108b, a splice-switching oligonucleotide, was designed to correct this aberrant splicing, especially in liver. We generated a mouse strain with homozygous knockin of this variant that well reflected the pathophysiology of patients with GSD1a. DS-4108b recovered hepatic G6Pase activity through splicing correction and prevented hypoglycemia and various hepatic abnormalities in the mice. Moreover, DS-4108b had long-lasting efficacy of more than 12 weeks in mice that received a single dose and had favorable pharmacokinetics and tolerability in mice and monkeys. These findings together indicate that this oligonucleotide-based therapy could provide a sustainable and curative therapeutic option under easy disease management for GSD1a patients with G6PC c.648G>T.

Metabolomics Analysis and Biochemical Profiling of Arsenic-Induced Metabolic Impairment and Disease Susceptibility.

Our study aimed to conduct a comprehensive biochemical profiling and metabolomics analysis to investigate the effects of arsenic-induced metabolic disorders, with a specific focus on disruptions in lipid metabolism, amino acid metabolism, and carbohydrate metabolism. Additionally, we sought to assess the therapeutic potential of resveratrol (RSV) as a remedy for arsenic-induced diabetes, using metformin (MF) as a standard drug for comparison. We measured the total arsenic content in mouse serum by employing inductively coupled plasma mass spectrometry (ICP-MS) after administering a 50-ppm solution of sodium arsenate (50 mg/L) in purified water. Our findings revealed a substantial increase in total arsenic content in the exposed group, with a mean value of 166.80 ± 8.52 ppb (p < 0.05). Furthermore, we investigated the impact of arsenic exposure on various biomarkers using enzyme-linked immunosorbent assay (ELISA) methods. Arsenic exposed mice exhibited significant hyperglycemia (p < 0.001) and elevated levels of homeostatic model assessment of insulin resistance (HOMA-IR), hemoglobin A1c (Hb1Ac), Inflammatory biomarkers as well as liver and kidney function biomarkers (p < 0.05). Additionally, the levels of crucial enzymes linked to carbohydrate metabolism, including α-glucosidase, hexokinase, and glucose-6-phosphatase (G6PS), and oxidative stress biomarkers, such as levels of glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), catalase, and superoxide dismutase (SOD), were significantly reduced in the arsenic-exposed group compared to the control group (p < 0.05). However, the level of MDA was significantly increased. Molecular analysis of gene expression indicated significant upregulation of key enzymes involved in lipid metabolism, such as carnitine palmitoyl-transferase-I (CPT-I), carnitine palmitoyl-transferase-II (CPT-II), lecithin-cholesterol acyltransferase (LCAT), and others. Additionally, alterations in gene expression related to glucose transporter-2 (GLUT-2), glucose-6-phosphatase (G6PC), and glucokinase (GK), associated with carbohydrate metabolism, were observed. Amino acid analysis revealed significant decreases in nine amino acids in arsenic-exposed mice. Metabolomics analysis identified disruptions in lipid metabolomes, amino acids, and arsenic metabolites, highlighting their involvement in essential metabolic pathways. Histopathological observations revealed significant changes in liver architecture, hepatocyte degeneration, and increased Kupffer cells in the livers of arsenic-exposed mice. In conclusion, these findings enhance our comprehension of the impact of environmental toxins on metabolic health and offer potential avenues for remedies against such disruptions.

Systematic literature review of the epidemiology of glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is an inherited autosomal recessive metabolic disease caused by a deficiency in glucose-6-phosphatase activity. The objectives of this research were to systematically review the published literature on the epidemiology of GSD 1a and to assess the performance of reported epidemiology measures in a simulation model. In this systematic literature review 2,539 record titles and abstracts were screened. Of these, only 11 studies contained relevant data on GSD 1a disease epidemiology. Reported disease frequency ranged from 0.085/100,000 to 10.3/100,000 newborns when considering all the GSD literature. When this was narrowed to GSD 1 and GSD 1a, the range was tightened to 0.25-3.02/100,000 and 0.085-4.9/100,000 newborns, respectively. Most of the identified studies counted the number of diagnoses in a defined period and related to the number of births in the same (Dx method) or different time period (DoB method). The simulation model results indicate that in most of the situations, the Dx method provides a closer estimate to the true disease incidence than the DoB method. Despite the scarcity of epidemiology data, the results of this systematic review strongly support that GSD 1a and its parent disease groups (GSD and GSD 1) are rare diseases.

An Enhancer Within Abcb11 Regulates G6pc2 in C57BL/6 Mouse Pancreatic Islets.

G6PC2 is predominantly expressed in pancreatic islet ß-cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus. In isolated islets, G6pc2 and Spc25 expression were reduced ∼50%, and Gm13613 expression was abolished, whereas Cers6 and nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.

Rare functional variants in the CRP and G6PC genes modify the relationship between obesity and serum C-reactive protein in white British population.

BACKGROUND: C-reactive protein (CRP) is a sensitive biomarker of inflammation with moderate heritability. The role of rare functional genetic variants in relation to serum CRP is understudied. We aimed to examine gene mutation burden of protein-altering (PA) and loss-of-function (LOF) variants in association with serum CRP, and to further explore the clinical relevance. METHODS: We included 161,430 unrelated participants of European ancestry from the UK Biobank. Of the rare (minor allele frequency <0.1%) and functional variants, 1,776,249 PA and 266,226 LOF variants were identified. Gene-based burden tests, linear regressions, and logistic regressions were performed to identify the candidate mutations at the gene and variant levels, to estimate the potential interaction effect between the identified PA mutation and obesity, and to evaluate the relative risk of 16 CRP-associated diseases. RESULTS: At the gene level, PA mutation burdens of the CRP (ß = -0.685, p = 2.87e-28) and G6PC genes (ß = 0.203, p = 1.50e-06) were associated with reduced and increased serum CRP concentration, respectively. At the variant level, seven PA alleles in the CRP gene decreased serum CRP, of which the per-allele effects were approximately three to seven times greater than that of a common variant in the same locus. The effects of obesity and central obesity on serum CRP concentration were smaller among the PA mutation carriers in the CRP (pinteraction = 0.008) and G6PC gene (pinteraction = 0.034) compared to the corresponding non-carriers. CONCLUSION: PA mutation burdens in the CRP and G6PC genes are strongly associated with decreased serum CRP concentrations. As serum CRP and obesity are important predictors of cardiovascular risks in clinics, our observations suggest taking rare genetic factors into consideration might improve the delivery of precision medicine.

CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia.

Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.

[Correction of the pathogenic mutation in the G6PC3 gene by adenine base editing in mutant embryos].

Objective: To determine whether the adenine base editor (ABE7.10) can be used to fix harmful mutations in the human G6PC3 gene. Methods: To investigate the safety of base-edited embryos, off-target analysis by deep sequencing was used to examine the feasibility and editing efficiency of various sgRNA expression vectors. The human HEK293T mutation models and human embryos were also used to test the feasibility and editing efficiency of correction. Results: ①The G6PC3(C295T) mutant cell model was successfully created. ②In the G6PC3(C295T) mutant cell model, three distinct Re-sgRNAs were created and corrected, with base correction efficiency ranging from 8.79% to 19.56% . ③ ABE7.10 could successfully fix mutant bases in the human pathogenic embryo test; however, base editing events had also happened in other locations. ④ With the exception of one noncoding site, which had a high safety rate, deep sequencing analysis revealed that the detection of 32 probable off-target sites was <0.5% . Conclusion: This study proposes a new base correction strategy based on human pathogenic embryos; however, it also produces a certain nontarget site editing, which needs to be further analyzed on the PAM site or editor window.

Polyphenolic compounds of Phyllanthus amarus Schum & Thonn. (1827) and diabetes-related activity of an aqueous extract as affected by in vitro gastrointestinal digestion.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the aerial part of Phyllanthus amarus have been extensively used in several countries to cure diabetes. No data is available on the impact of gastrointestinal digestion of such crude extracts on their antidiabetic activity. AIM OF THE STUDY: The aim of this study was to identify active fractions and compounds of fresh aerial parts of P. amarus extracted by an infusion method that are responsible for antidiabetic effects occurring at the level of glucose homeostasis. MATERIALS AND METHODS: An aqueous extract was obtained by an infusion method and its polyphenolic composition was analysed by reverse phase UPLC-DAD-MS. The influence of in vitro gastrointestinal digestion was evaluated both on the chemical composition and on the antidiabetic effect of P. amarus infusion extract using glucose-6-phosphatase enzyme inhibition and stimulation of glucose uptake. RESULTS: Analysis of the chemical composition of the crude extract revealed the presence of polysaccharides and various families of polyphenols such as phenolic acids, tannins, flavonoids and lignans. After simulated digestion, the total content of polyphenols decreased by about 95%. Caffeoylglucaric acid derivates and lignans exhibited strong stimulation of glucose uptake similar to metformin with an increase of 35.62 ± 6.14% and 34.74 ± 5.33% respectively. Moreover, corilagin, geraniin, the enriched polysaccharides fraction and the bioaccessible fraction showed strong anti-hyperglycemic activity with about 39-62% of glucose-6-phosphatase inhibition. CONCLUSION: Caffeoylglucaric acid isomers, tannin acalyphidin M1 and lignan demethyleneniranthin were reported for the first time in the species. After in vitro gastroinstestinal digestion, the composition of the extract changed. The dialyzed fraction showed strong glucose-6-phosphatase inhibition.

Blood glucose trends in glycogen storage disease type Ia: A cross-sectional study.

BACKGROUND: Glycogen storage disease type Ia (GSDIa) is caused by biallelic pathogenic variants in the glucose-6-phosphatase gene (G6PC) and mainly characterized by hypoglycemia, hepatomegaly, and renal insufficiency. Although its symptoms are reportedly mild in patients carrying the G6PC c.648G>T variant, the predominant variant in Japanese patients, details remain unclear. Therefore, we examined continuous glucose monitoring (CGM) data and daily nutritional intake to clarify their associations in Japanese patients with GSDIa with G6PC c.648G>T. METHODS: This cross-sectional study enrolled 32 patients across 10 hospitals. CGM was performed for 14 days, and nutritional intake was recorded using electronic diaries. Patients were divided according to genotype (homozygous/compound heterozygous) and age. The durations of biochemical hypoglycemia and corresponding nutritional intake were analyzed. Multiple regression analysis was performed to identify factors associated with the duration of biochemical hypoglycemia. RESULTS: Data were analyzed for 30 patients. The mean daily duration of hypoglycemia (<4.0 mmol/L) in the homozygous group increased with age (2-11 years [N = 8]: 79.8 min; 12-18 years [5]: 84.8 min; ≥19 years [10]: 131.5 min). No severe hypoglycemic symptoms were recorded in the patients' diaries. The mean frequency of snack intake was approximately three times greater in patients aged 2-11 years (7.1 times/day) than in those aged 12-18 years (1.9 times/day) or ≥19 years (2.2 times/day). Total cholesterol and lactate were independently associated with the duration of biochemical hypoglycemia. CONCLUSION: Although nutritional therapy prevents severe hypoglycemia in patients with GSDIa with G6PC c.648G>T, patients often experience asymptomatic hypoglycemia.

A caveolin-1 dependent glucose-6-phosphatase trafficking contributes to hepatic glucose production.

OBJECTIVE: Deregulation of hepatic glucose production is a key driver in the pathogenesis of diabetes, but its short-term regulation is incompletely deciphered. According to textbooks, glucose is produced in the endoplasmic reticulum by glucose-6-phosphatase (G6Pase) and then exported in the blood by the glucose transporter GLUT2. However, in the absence of GLUT2, glucose can be produced by a cholesterol-dependent vesicular pathway, which remains to be deciphered. Interestingly, a similar mechanism relying on vesicle trafficking controls short-term G6Pase activity. We thus investigated whether Caveolin-1 (Cav1), a master regulator of cholesterol trafficking, might be the mechanistic link between glucose production by G6Pase in the ER and glucose export through a vesicular pathway. METHODS: Glucose production from fasted mice lacking Cav1, GLUT2 or both proteins was measured in vitro in primary culture of hepatocytes and in vivo by pyruvate tolerance tests. The cellular localization of Cav1 and the catalytic unit of glucose-6-phosphatase (G6PC1) were studied by western blotting from purified membranes, immunofluorescence on primary hepatocytes and fixed liver sections and by in vivo imaging of chimeric constructs overexpressed in cell lines. G6PC1 trafficking to the plasma membrane was inhibited by a broad inhibitor of vesicular pathways or by an anchoring system retaining G6PC1 specifically to the ER membrane. RESULTS: Hepatocyte glucose production is reduced at the step catalyzed by G6Pase in the absence of Cav1. In the absence of both GLUT2 and Cav1, gluconeogenesis is nearly abolished, indicating that these pathways can be considered as the two major pathways of de novo glucose production. Mechanistically, Cav1 colocalizes but does not interact with G6PC1 and controls its localization in the Golgi complex and at the plasma membrane. The localization of G6PC1 at the plasma membrane is correlated to glucose production. Accordingly, retaining G6PC1 in the ER reduces glucose production by hepatic cells. CONCLUSIONS: Our data evidence a pathway of glucose production that relies on Cav1-dependent trafficking of G6PC1 to the plasma membrane. This reveals a new cellular regulation of G6Pase activity that contributes to hepatic glucose production and glucose homeostasis.

Amnio acid substitution at position 298 of human glucose-6 phosphatase-α significantly impacts its stability in mammalian cells.

Glucose-6-phosphatase-α (G6Pase-α) catalyzes the hydrolysis of glucose-6-phosphate to glucose and functions as a key regulator in maintaining blood glucose homeostasis. Deficiency in G6Pase-α causes glycogen storage disease 1a (GSD1a), an inherited disorder characterized by life-threatening hypoglycemia and other long-term complications. We have developed a potential mRNA-based therapy for GSD1a and demonstrated that a human G6Pase-α (hG6Pase-α) variant harboring a single serine (S) to cysteine (C) substitution at the amino acid site 298 (S298C) had > twofold increase in protein expression, resulting in improved in vivo efficacy. Here, we sought to investigate the mechanisms contributing to the increased expression of the S298C variant. Mutagenesis of hG6Pase-α identified distinct protein variants at the 298 amino acid position with substantial reduction in protein expression in cultured cells. Kinetic analysis of expression and subcellular localization in mammalian cells, combined with cell-free in vitro translation assays, revealed that altered protein expression stemmed from differences in cellular protein stability rather than biosynthetic rates. Site-specific mutagenesis studies targeting other cysteines of the hG6Pase-α S298C variant suggest the observed improvements in stability are not due to additional disulfide bond formation. The glycosylation at Asparagine (N)-96 is critical in maintaining enzymatic activity and mutations at position 298 mainly affected glycosylated forms of hG6Pase-α. Finally, proteasome inhibition by lactacystin improved expression levels of unstable hG6Pase-α variants. Taken together, these data uncover a critical role for a single amino acid substitution impacting the stability of G6Pase-α and provide insights into the molecular genetics of GSD1a and protein engineering for therapeutic development.