Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Sustains Hepatic G6Pase in Response to Systemic Polymicrobial Infection (A) Volcano plot of mean RNA expression from RNA microarray screen in the liver of Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice, 48 hr after CLP (n = 4 mice per group). (B) Validation of G6pc1 mRNA expression by qRT-PCR in the liver of Mx1 Cre Fth Δ/Δ mice not subjected to CLP (n = 13), 48 hr after CLP (n = 12) versus Fth lox/lox mice not subjected to CLP (n = 11), or 48 hr after CLP (n = 11). Data pooled from three independent experiments. (C and D) Representative western blot of G6pc1 in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice (C) and relative quantification by densitometry (D) before (Control; Ctr) or 48 hr after CLP (n = 6 per group). (E) Quantification of liver Gpt1 mRNA by qRT-PCR, same mice as (B). (F) Liver G6Pase enzymatic activity, same mice as (B). (G) Quantification of G6PC1 mRNA levels by qRT-PCR in HepG2 cells untreated (−) or treated (+) with heme and/or TNF. Mean ± SEM from eight independent experiments with similar trend. (H) G6PC1 protein levels in HepG2 cells treated as in (G). (I) Relative quantification G6PC1 protein levels by densitometry of western blot from HepG2 cells treated as in (G) and (H). (J) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and CMV Renilla luciferase reporters. Control cells were transfected with a promoterless firefly luciferase reporter. Cells were treated, 48 hr after transfection, with heme and TNF. Data are shown as mean RLU ± SD from four independent experiments with similar trend. (K) Liver mRNA quantification by qRT-PCR in C57BL/6 mice receiving heme (+; n = 8) (i.p. 30 mg/kg BW) or not (−; n = 9). Data pooled from three independent experiments. (L) Liver mRNA quantification by qRT-PCR in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice receiving heme (+; i.p. 15 mg/kg BW; n = 3 per genotype) or not (−; n = 6–8 per genotype). Data pooled from one to three independent experiments. (M) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and a CMV Renilla luciferase reporters plus a human FTH expression vector. Cells transfected with a promoterless firefly luciferase reporter were used as baseline RLU. Transfected cells were treated 48 hr thereafter with heme and TNF and analyzed 12 hr thereafter. Data shown as mean RLU ± SD from five independent experiments with similar trend. Red bars in (B), (D)–(F), (J), and (K) represent mean values and doted circles indicate individual mice. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.01. See also Figure S6 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Infection, RNA Expression, Microarray, Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Required to Establish Disease Tolerance to Sepsis, Related to and ( a ) Western Blot of G6PC1 and p21 in HepG2 cells untreated (-) or treated (+) with heme and TNF in the presence (+) or absence (-) of Lactacystin (Lact.) or MG132. Actin was used as loading control. Data are representative of three independent experiments with the same trend. ( b ) G6Pase enzymatic activity in HepG2 cells transduced (+) or not (-) with a G6PC1 Rec.Ad. and when indicated (+) co-transduced with a FTH Rec.Ad. Transduction with a LacZ Rec. Ad. was used as a control. NT: Not transduced. Cells were treated (+) or not (-) with heme and TNF 72 hr after Rec.Ad. transduction. Data are shown as mean ± SD, pooled from 2 independent experiments with four replicates each. ( c ) Temperature and ( d ) weight of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM, pooled from 3 independent experiments. ( e ) Blood glucose and ( f ) clinical severity score in Control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to CLP. Mean ± SD pooled from 2 independent experiments. ( g ) Serological markers of organ injury in C57BL/6 (n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice 6 hr mice after CLP. ( h ) H&E stained paraffin sections representative of at least four G6pc1 lox/lox and Alb Cre ER T2 G6pc1 Δ/Δ mice 9 hr after PCI. B: bronchus; CV: central vein; G: glomerulus. Magnification 400x. ( i ) Temperature and ( h ) weight and ( j ) blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme ( i.p ; 30 mg/kg BW). Mean ± SD from 2 independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Western Blot, Control, Activity Assay, Transduction, Staining

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Critical to Establish Disease Tolerance to Sepsis (A) G6pc1 protein expression in the liver of control ( G6pc1 lox/lox ; Ctr) and Alb Cre ER T2 G6pc1 Δ/Δ ( G6pc1 Δ/Δ ) mice. (B) Blood glucose levels of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM from three independent experiments. (C) Survival of control ( G6pc1 lox/lox ; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 10) mice subjected to CLP. Data were pooled from two independent experiments with similar trend. (D) Blood glucose of control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to PCI. Data are shown as mean ± SD from two independent experiments with the same trend. (E) Survival of control (C57BL/6; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 8) subjected to PCI. Data pooled from three independent experiments with similar trend. (F) CFU for aerobic (Ae) and anaerobic (An) bacteria, 6 hr after PCI. Red bars represent mean values and dotted circles represent individual mice. PC, peritoneal cavity. (G) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme (i.p. 30 mg/kg BW). Mean ± SD from two independent experiments. (H) Survival of same mice as (G). (I) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 9) mice receiving LPS (i.p. 5 mg/kg BW). Mean ± SD from two independent experiments. (J) Survival of same mice as (I). (K) Blood glucose levels in G6pc1 lox/lox (n = 9) and Alb Cre ER T2 Gpc1 Δ/Δ (n = 10) mice receiving poly(I:C) (intra-retro orbital [i.r.o.] 30 mg/kg BW). Mean ± SD from two independent experiments. (L) Survival of same mice as (K). Numbers in gray (B, D, G, I, and K) are live/total mice at each time point. ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S6 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Expressing, Control, Bacteria

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Overexpression or Ferritin Administration Induce Disease Tolerance to Sepsis (A) Survival of C57BL/6 mice transduced 48 hr before severe CLP with FTH Rec.Ad. (n = 11), ferroxidase-deficient FTH Rec.Ad. (FTH m ; n = 11), LacZ Rec.Ad. (n = 12), or receiving vehicle (PBS; n = 19). Data pooled from six independent experiments with similar trend. (B) CFU for aerobic (Ae) and anaerobic (An) bacteria, 12 hr after CLP in mice treated as in (A). (C) Representative western blot in HepG2 cells transduced with LacZ, FTH, or mutated FTH (FTH m ) Rec.Ad. (D and E) Densitometry of G6PC1 (D) and FTH protein (E) expression normalized to α-tubulin (n = 4 per group). (F) Expression of G6pc1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and deferoxamine (DFO). Mean ± SEM from three independent experiments done in duplicates. (G) Survival of C57BL/6 mice receiving apoferritin (n = 16), ferritin (n = 10), BSA (n = 12), or saline (n = 12), 24 hr before severe CLP. Data pooled from five independent experiments. (H) CFU for aerobic (Ae) and anaerobic (An) bacteria 12 hr after severe CLP in mice treated as in (G). Data from three independent experiments. (I) Survival of C57BL/6 mice receiving apoferritin (n = 22), BSA (n = 20), or saline (n = 16) 6 hr after severe CLP. Data pooled from seven independent experiments. Red bars represent mean values and dotted circles represent individual mice (B and G). PC, peritoneal cavity; NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. See also Figure S7 .

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Over Expression, Bacteria, Western Blot, Transduction, Expressing, Quantitative RT-PCR, Saline

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Antioxidants Bypass the Requirement of FTH in the Establishment of Normoglycemia and Disease Tolerance to Sepsis (A–C) Relative weight (A), blood glucose (B), and survival (C) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving NAC (n = 7; 15 mg/kg) or vehicle (n = 6). Data pooled from two independent experiments. (D) Colony forming units (CFU) for aerobic (Ae) and anaerobic (An) bacteria, 48 hr after CLP. PC, peritoneal cavity. Red bars represent mean values and doted circles represent individual mice. Pooled from two independent experiments. (E) Expression of G6PC1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and/or NAC. Data shown as mean ± SD from four independent experiments. (F and G) Blood glucose (F) and survival (G) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving BHA (n = 11; 50 mg/kg) or vehicle (n = 9). Data shown as mean ± SD. Pooled from three independent experiments. (H–J) Relative weight (H), blood glucose (I), and survival (J) of Alb Cre ER T2 G6pc1 Δ/Δ mice subjected to CLP and receiving NAC (n = 5) or vehicle (n = 5). Mean ± SD from one experiment. NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. Numbers in gray (A, B, F, I, and J) indicate live/total mice.

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Bacteria, Expressing, Quantitative RT-PCR

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet:

Article Snippet: At 80%–90% of confluence, cells were transduced at 50 or 100 pfu with LacZ, G6pc1 (Vector Biolabs; Ref: SKU#: ADV-259766), FTH and FTH m Rec.Ad. ( , ).

Techniques: Virus, Recombinant, cDNA Synthesis, Luciferase, SYBR Green Assay, Labeling, Enzyme-linked Immunosorbent Assay, Bioassay, Kinase Assay, Gene Expression, Software, Combined Bisulfite Restriction Analysis Assay, Stripping Membranes, Control

Journal: Nature Communications

Article Title: Base-editing corrects metabolic abnormalities in a humanized mouse model for glycogen storage disease type-Ia

doi: 10.1038/s41467-024-54108-1

Figure Lengend Snippet: The 10–12-week-old heterozygous (mR83/huR83C) mice, not fasted, were treated by systemic administration of BEAM-301 and the target site sequence was analyzed one week later via next-generation sequencing (NGS). a Target site sequence for p.R83C to p.R83 correction, the p.R83 synonymous variant and the pathogenic p.Y85H bystander variant. Nucleotides on the complementary strand edited by BEAM-301 are bracketed. b Correlation of dose levels of BEAM-301 to the editing efficiency for the conversion of the p.R83C ( G6PC1 - c.247C > T ) variant to wild-type p.R83 ( G6PC1 - c.247 C ) along with the p.Y85H mutation. The n numbers for each dosage of BEAM-301 in mg/kg are listed. c Allele frequencies resulting from dosing heterozygous mice at 1.5 mg/kg (301H): p.R83C to p.R83 (correction); p.Y85H bystander mutation; Uncorrected (synonymous) editing (R83C); and Indels ( n = 5). Statistics were performed using a two-tailed unpaired T test. Data are presented as Mean values ± SEM, and individual data points for each animal are displayed. * denotes p < 0.05, ** denotes p value < 0.005.

Article Snippet: A human cDNA encoding the open reading frame for G6PC1 - c.247C > T ( G6PC1 -R83C) were inserted into exon 1 of the mouse G6pc gene at the ATG start codon (Taconic Biosciences) in a way that created a premature STOP codon in the coding sequence of the mouse G6pc exon 1.

Techniques: Sequencing, Next-Generation Sequencing, Variant Assay, Mutagenesis, Two Tailed Test

Journal: Frontiers in Endocrinology

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

doi: 10.3389/fendo.2023.1265698

Figure Lengend Snippet: Increased gene expression of G6PC3 , SLC37A2 , and SLC37A4 in clinically-annotated glioblastoma tumor tissues. In silico analysis of transcript levels was performed for (A) three members of the G6PC family (G6PC1, G6PC2, G6PC3) and (B) four members of the SLC37A family (SLC37A1, SLC37A2, SLC37A3, SLC37A4) using RNA extracted from clinical samples from glioblastoma (GBM) and low-grade glioma (LGG) (red boxes) and compared to healthy tissue (grey boxes). (* p < 0.05).

Article Snippet: The following QuantiTect primer sets were provided by QIAGEN: G6PC1 (Hs_G6PC_1_SG QT00031913), G6PC2 (Hs_G6PC2_va.1_SG QT01664152), G6PC3 (Hs_G6PC3_1_SG QT00033453), SLC37A1 (SLC37A1_1_SG QT00073094), SLC37A2 (Hs_SLC37A2_1_SG QT00056203), SLC37A3 (Hs_SLC37A3_1_SG QT00057148), SLC37A4 (Hs_SLC37A4_1_SG QT00024325), GAPDH (Hs_GAPDH_2_SG QT01192646), β-actin (Hs_Actb_2_SG QT01680476) and PPIA (Hs_PPIA_4_SG QT01866137).

Techniques: Expressing, In Silico

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Comparison of human G6PC1 and G6PC2 protein expression driven by the pcDNA3.1D and pJPA5 vectors. 832/13 cells were transiently transfected with either pcDNA3.1D or pJPA5 expression vectors encoding human G6PC1 or G6PC2 with a C terminal V5 His Tag. Following transfection, cells were incubated for 18–20 h in serum-containing media. Cells were subsequently harvested and protein expression assayed by Western blotting as described in . A, G6PC expression was assessed using an anti-V5 antibody and equal protein loading was confirmed using both Ponceaus staining and measurement of actin expression. B, the conditions used to assess G6PC2 and actin expression are semiquantitative. Representative blots are shown. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Comparison, Expressing, Transfection, Incubation, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Conservation of amino acids between human G6PC2, mouse G6PC2, human G6PC1, and mouse G6PC1. Sequence alignment showing the conservation of AAs between human (h) and mouse (m) G6PC1 and G6PC2. Residues highlighted in yellow represent conserved AAs that are changed by a human G6PC2 SNP and where mutation in G6PC1 can cause GSD type 1a ( Table 1 ). Residues highlighted in pink represent conserved AAs that are changed by human G6PC2 SNPs but where mutation in G6PC1 has not been associated with GSD type 1a. Residues highlighted in green represent nonconserved AAs that are changed by human G6PC2 SNPs. Identities are indicated by filled circles and similarities by vertical bars. Gray boxes represent putative transmembrane domains determined using the TMHMM algorithm ( https://services.healthtech.dtu.dk/ ) . The predictions made by this program differ from earlier models ( , ). The figure shows the predicted domains for human G6PC1. G6PC1 and G6PC2 have an extended sequence motif (KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXXHXXXD) ( boxed ), similar to that found in bacterial vanadate-sensitive haloperoxidases and mammalian phosphatidic acid phosphatases, which constitutes the active site of these enzymes . G6PC1 and G6PC2 also share a C terminal ER retention sequence (KK; boxed ) . Adapted from Ref. . G6PC, glucose-6-phosphatase catalytic subunit; GSD1a, glycogen storage disease type 1a; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Sequencing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Mutagenesis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1, on G6PC2 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 where a mutation is known to cause GSD1a in the context of human G6PC1 ( Table 1 ). The effect of these G6PC2 SNPs on G6PC2 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1, on G6PC2 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2, which are not associated with GSD1a in the context of human G6PC1, as well as amino acids that are not conserved in G6PC1 ( Table 2 ). The effect of these G6PC2 SNPs on G6PC2 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4 to 10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the suppression of glucose-stimulated fusion gene expression by G6PC2 in 832/13 cells. 832/13 cells were transiently cotransfected with the −7248/+62 G6pc1 - luciferase fusion gene (2 μg), an expression vector encoding Renilla luciferase (0.5 μg) and expression vectors (1 μg) encoding either wild type (WT), catalytically dead (D; AA 174 Ala), or variant (AA8, AA114) G6PC2. Following transfection, cells were incubated for 18 to 20 h in serum-free medium in the presence of 2 or 11 mM glucose. Cells were then harvested and luciferase activity assayed as described in . Results show mean data −/+ SD (n = 3–6) and were calculated as the ratio of firefly: Renilla luciferase activity. Statistical comparisons to 11 mM WT were made using ANOVA with a Dunnett's Multiple Comparisons post-hoc test; ∗ p < 0.05. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Gene Expression, Luciferase, Expressing, Plasmid Preparation, Variant Assay, Transfection, Incubation, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs on G6PC1 protein expression. 832/13 cells were transiently transfected with pcDNA3.1D expression vectors encoding human G6PC1 with a C terminal V5 His Tag. Following transfection, cells were incubated for 18–20 h in serum-containing media. Cells were subsequently harvested and protein expression assayed by Western blotting as described in . G6PC1 expression was assessed using an anti-V5 antibody and equal protein loading was confirmed by measurement of actin expression. Representative blots are shown. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Transfection, Incubation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect conserved amino acids associated with GSD1a in the context of human G6PC1, on G6PC1 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 where a mutation is known to cause GSD1a in the context of human G6PC1 ( Table 1 ). The effect of these G6PC2 SNPs on G6PC1 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–8. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Analysis of the effect of human G6PC2 SNPs that affect amino acids that are not associated with GSD1a in the context of human G6PC1, on G6PC1 protein expression. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2, that are not associated with GSD1a in the context of human G6PC1 ( Table 2 ). The effect of these G6PC2 SNPs on G6PC1 protein expression was determined as described in Fig. 3 . Protein expression was quantified as described in . Results show mean data −/+ SD, n = 4–10. ∗ p < 0.05 vs. WT. G6PC, glucose-6-phosphatase catalytic subunit; SNP, single-nucleotide polymorphism.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Nonsynonymous single-nucleotide polymorphisms in the G6PC2 gene affect protein expression, enzyme activity, and fasting blood glucose

doi: 10.1016/j.jbc.2021.101534

Figure Lengend Snippet: Comparison of the effect of specific amino acid changes on the expression of mouse and human G6PC1. Human G6PC2 SNPs were identified that affect AAs conserved between mouse and human G6PC1 and mouse and human G6PC2 ( Tables 1 and ). The effect of these G6PC2 SNPs on mouse and human G6PC1 protein expression was determined as described in Fig. 3 . A representative blot is shown. G6PC, glucose-6-phosphatase catalytic subunit.

Article Snippet: A human G6PC1 cDNA (Accession number BC130478 ; IMAGE clone number 40146509) was purchased from Transomic technologies.

Techniques: Comparison, Expressing

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Sustains Hepatic G6Pase in Response to Systemic Polymicrobial Infection (A) Volcano plot of mean RNA expression from RNA microarray screen in the liver of Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice, 48 hr after CLP (n = 4 mice per group). (B) Validation of G6pc1 mRNA expression by qRT-PCR in the liver of Mx1 Cre Fth Δ/Δ mice not subjected to CLP (n = 13), 48 hr after CLP (n = 12) versus Fth lox/lox mice not subjected to CLP (n = 11), or 48 hr after CLP (n = 11). Data pooled from three independent experiments. (C and D) Representative western blot of G6pc1 in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice (C) and relative quantification by densitometry (D) before (Control; Ctr) or 48 hr after CLP (n = 6 per group). (E) Quantification of liver Gpt1 mRNA by qRT-PCR, same mice as (B). (F) Liver G6Pase enzymatic activity, same mice as (B). (G) Quantification of G6PC1 mRNA levels by qRT-PCR in HepG2 cells untreated (−) or treated (+) with heme and/or TNF. Mean ± SEM from eight independent experiments with similar trend. (H) G6PC1 protein levels in HepG2 cells treated as in (G). (I) Relative quantification G6PC1 protein levels by densitometry of western blot from HepG2 cells treated as in (G) and (H). (J) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and CMV Renilla luciferase reporters. Control cells were transfected with a promoterless firefly luciferase reporter. Cells were treated, 48 hr after transfection, with heme and TNF. Data are shown as mean RLU ± SD from four independent experiments with similar trend. (K) Liver mRNA quantification by qRT-PCR in C57BL/6 mice receiving heme (+; n = 8) (i.p. 30 mg/kg BW) or not (−; n = 9). Data pooled from three independent experiments. (L) Liver mRNA quantification by qRT-PCR in Mx1 Cre Fth Δ/Δ versus Fth lox/lox mice receiving heme (+; i.p. 15 mg/kg BW; n = 3 per genotype) or not (−; n = 6–8 per genotype). Data pooled from one to three independent experiments. (M) Relative luciferase units (RLU) in HepG2 cells transiently co-transfected with a rat G6pc1 firefly luciferase and a CMV Renilla luciferase reporters plus a human FTH expression vector. Cells transfected with a promoterless firefly luciferase reporter were used as baseline RLU. Transfected cells were treated 48 hr thereafter with heme and TNF and analyzed 12 hr thereafter. Data shown as mean RLU ± SD from five independent experiments with similar trend. Red bars in (B), (D)–(F), (J), and (K) represent mean values and doted circles indicate individual mice. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.01. See also Figure S6 .

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Infection, RNA Expression, Microarray, Biomarker Discovery, Expressing, Quantitative RT-PCR, Western Blot, Quantitative Proteomics, Control, Activity Assay, Luciferase, Transfection, Plasmid Preparation

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Required to Establish Disease Tolerance to Sepsis, Related to and ( a ) Western Blot of G6PC1 and p21 in HepG2 cells untreated (-) or treated (+) with heme and TNF in the presence (+) or absence (-) of Lactacystin (Lact.) or MG132. Actin was used as loading control. Data are representative of three independent experiments with the same trend. ( b ) G6Pase enzymatic activity in HepG2 cells transduced (+) or not (-) with a G6PC1 Rec.Ad. and when indicated (+) co-transduced with a FTH Rec.Ad. Transduction with a LacZ Rec. Ad. was used as a control. NT: Not transduced. Cells were treated (+) or not (-) with heme and TNF 72 hr after Rec.Ad. transduction. Data are shown as mean ± SD, pooled from 2 independent experiments with four replicates each. ( c ) Temperature and ( d ) weight of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM, pooled from 3 independent experiments. ( e ) Blood glucose and ( f ) clinical severity score in Control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to CLP. Mean ± SD pooled from 2 independent experiments. ( g ) Serological markers of organ injury in C57BL/6 (n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice 6 hr mice after CLP. ( h ) H&E stained paraffin sections representative of at least four G6pc1 lox/lox and Alb Cre ER T2 G6pc1 Δ/Δ mice 9 hr after PCI. B: bronchus; CV: central vein; G: glomerulus. Magnification 400x. ( i ) Temperature and ( h ) weight and ( j ) blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme ( i.p ; 30 mg/kg BW). Mean ± SD from 2 independent experiments. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Western Blot, Control, Activity Assay, Transduction, Staining

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Liver Gluconeogenesis Is Critical to Establish Disease Tolerance to Sepsis (A) G6pc1 protein expression in the liver of control ( G6pc1 lox/lox ; Ctr) and Alb Cre ER T2 G6pc1 Δ/Δ ( G6pc1 Δ/Δ ) mice. (B) Blood glucose levels of G6pc1 lox/lox (n = 12) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 12) mice subjected to CLP. Data are shown as mean ± SEM from three independent experiments. (C) Survival of control ( G6pc1 lox/lox ; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 10) mice subjected to CLP. Data were pooled from two independent experiments with similar trend. (D) Blood glucose of control (C57BL/6; n = 7) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 5) mice subjected to PCI. Data are shown as mean ± SD from two independent experiments with the same trend. (E) Survival of control (C57BL/6; n = 10) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 8) subjected to PCI. Data pooled from three independent experiments with similar trend. (F) CFU for aerobic (Ae) and anaerobic (An) bacteria, 6 hr after PCI. Red bars represent mean values and dotted circles represent individual mice. PC, peritoneal cavity. (G) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 7) mice receiving heme (i.p. 30 mg/kg BW). Mean ± SD from two independent experiments. (H) Survival of same mice as (G). (I) Blood glucose levels in G6pc1 lox/lox (n = 6) and Alb Cre ER T2 G6pc1 Δ/Δ (n = 9) mice receiving LPS (i.p. 5 mg/kg BW). Mean ± SD from two independent experiments. (J) Survival of same mice as (I). (K) Blood glucose levels in G6pc1 lox/lox (n = 9) and Alb Cre ER T2 Gpc1 Δ/Δ (n = 10) mice receiving poly(I:C) (intra-retro orbital [i.r.o.] 30 mg/kg BW). Mean ± SD from two independent experiments. (L) Survival of same mice as (K). Numbers in gray (B, D, G, I, and K) are live/total mice at each time point. ∗ p < 0.05; ∗∗ p < 0.01. See also Figure S6 .

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Expressing, Control, Bacteria

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: FTH Overexpression or Ferritin Administration Induce Disease Tolerance to Sepsis (A) Survival of C57BL/6 mice transduced 48 hr before severe CLP with FTH Rec.Ad. (n = 11), ferroxidase-deficient FTH Rec.Ad. (FTH m ; n = 11), LacZ Rec.Ad. (n = 12), or receiving vehicle (PBS; n = 19). Data pooled from six independent experiments with similar trend. (B) CFU for aerobic (Ae) and anaerobic (An) bacteria, 12 hr after CLP in mice treated as in (A). (C) Representative western blot in HepG2 cells transduced with LacZ, FTH, or mutated FTH (FTH m ) Rec.Ad. (D and E) Densitometry of G6PC1 (D) and FTH protein (E) expression normalized to α-tubulin (n = 4 per group). (F) Expression of G6pc1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and deferoxamine (DFO). Mean ± SEM from three independent experiments done in duplicates. (G) Survival of C57BL/6 mice receiving apoferritin (n = 16), ferritin (n = 10), BSA (n = 12), or saline (n = 12), 24 hr before severe CLP. Data pooled from five independent experiments. (H) CFU for aerobic (Ae) and anaerobic (An) bacteria 12 hr after severe CLP in mice treated as in (G). Data from three independent experiments. (I) Survival of C57BL/6 mice receiving apoferritin (n = 22), BSA (n = 20), or saline (n = 16) 6 hr after severe CLP. Data pooled from seven independent experiments. Red bars represent mean values and dotted circles represent individual mice (B and G). PC, peritoneal cavity; NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. See also Figure S7 .

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Over Expression, Bacteria, Western Blot, Transduction, Expressing, Quantitative RT-PCR, Saline

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet: Antioxidants Bypass the Requirement of FTH in the Establishment of Normoglycemia and Disease Tolerance to Sepsis (A–C) Relative weight (A), blood glucose (B), and survival (C) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving NAC (n = 7; 15 mg/kg) or vehicle (n = 6). Data pooled from two independent experiments. (D) Colony forming units (CFU) for aerobic (Ae) and anaerobic (An) bacteria, 48 hr after CLP. PC, peritoneal cavity. Red bars represent mean values and doted circles represent individual mice. Pooled from two independent experiments. (E) Expression of G6PC1 mRNA quantified by qRT-PCR in HepG2 cells exposed (+) or not (−) to heme, TNF, and/or NAC. Data shown as mean ± SD from four independent experiments. (F and G) Blood glucose (F) and survival (G) of Mx1 Cre Fth Δ/Δ mice subjected to CLP and receiving BHA (n = 11; 50 mg/kg) or vehicle (n = 9). Data shown as mean ± SD. Pooled from three independent experiments. (H–J) Relative weight (H), blood glucose (I), and survival (J) of Alb Cre ER T2 G6pc1 Δ/Δ mice subjected to CLP and receiving NAC (n = 5) or vehicle (n = 5). Mean ± SD from one experiment. NS, non-significant. ∗ p < 0.05, ∗∗ p < 0.01. Numbers in gray (A, B, F, I, and J) indicate live/total mice.

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Bacteria, Expressing, Quantitative RT-PCR

Journal: Cell

Article Title: Metabolic Adaptation Establishes Disease Tolerance to Sepsis

doi: 10.1016/j.cell.2017.05.031

Figure Lengend Snippet:

Article Snippet: Anti-mouse G6PC1 (1:5000), anti-FTH1 (clone D1D4) (1:1000) (Cell Signaling), anti-p21 (1:1000) (2947, Cell Signaling), anti- β-actin (1:5000) (A5441, Sigma) and anti-α-tubulin-HRP (clone DM1A) (Sigma) (1:10000) were detected using peroxidase conjugated secondary antibodies (1 hr; room temperature) and developed with ECL western blotting substrate (ThermoFisher Scientific) for anti-β-tubulin and SuperSignal West Pico Chemiluminiscent substrate (ThermoFisher Scientific) for anti-G6PC and anti-FTH1 antibodies.

Techniques: Virus, Recombinant, cDNA Synthesis, Luciferase, SYBR Green Assay, Labeling, Enzyme-linked Immunosorbent Assay, Bioassay, Kinase Assay, Gene Expression, Software, Combined Bisulfite Restriction Analysis Assay, Stripping Membranes, Control