Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) HepG2 cells were treated with DMSO, NOG (1 mM), or DMOG (0.1 mM) for 12 hr. (B) shRNA-mediated screen. Each bar represents a single shRNA construct. Data were presented as fold relative to the scramble control. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Left, gene expression data were presented as fold relative to the scramble control from three experiments. Full names of individual genes are listed in . Right, levels of PEPCK and G6Pase protein were determined. (D) Jhdm1a knockdown or scramble HepG2 cells were treated with NOG (1 mM) for 12 hr. (E) Jhdm1a knockdown or scramble HepG2 cells were treated with a combination of dibutyryl cyclic-AMP (cAMP, 0.5 mM) and dexamethasone (Dex, 1 µM) in DMEM medium for 6 hr. Data were from two experiments. (F) Lentiviral knockdown of Jhdm1a in mouse hepatoma HepA1-6 cells. Data were from two experiments. (G) Adenoviral knockdown of Jhdm1a in mouse primary hepatocytes. Experiments were repeated three times with similar results.

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: shRNA, Construct, Control, Gene Expression, Knockdown

Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) Jhdm1a knockdown or scramble adenoviruses were transduced into the liver of wild-type male C57BL/6J mice (n = 5 per group). Mice fed ad libitum were sacrificed at Day 5 after viral infusion. (Left) mRNA levels of PEPCK, G6Pase and Jhdm1a in the liver were measured and normalized to U36b4. **, P<0.005. (Right) PEPCK and G6Pase protein. (B) Blood insulin levels at fed state were measured at Day 5. (C) Jhdm1a knockdown or scramble adenoviruses were transduced into the liver of wild-type male C57BL/6J mice (n = 10 per group). At Day 5, mice were i.p. injected with pyruvate (2 g/kg body weight) after a starvation for 16 hr and blood glucose levels were measured. *, P<0.05. (D and E) Adenoviruses expressing wild-type Jhdm1a, H212A point mutant, or GFP were transduced into the liver of male ob/ob mice (n = 5 per group). Gene expression was measured on Day 5 and blood glucose levels were measured on Day 3 after a 5-hr fasting. Changes of blood glucose level relative to Day 0 are presented. *, P<0.03; **, P<0.01. ( F ) Hepatic Jhdm1a mRNA levels in male C57BL/6J mice (n = 5 per group) fed ad libitum , or fasted for 5 hr or 20 hr. (G) Male C57BL/6J mice (n = 4) were i.p. injected with glucagon (300 µg/kg), insulin (0.75 U/Kg), or PBS. Hepatic Jhdm1a mRNA levels were examined 6 hr after injection. (H) Hepatic Jhdm1a mRNA levels in lean mice and diabetic ob/ob mice (n = 3 per group).

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: Knockdown, Injection, Expressing, Mutagenesis, Gene Expression

Journal: PLoS Genetics

Article Title: The Histone Demethylase Jhdm1a Regulates Hepatic Gluconeogenesis

doi: 10.1371/journal.pgen.1002761

Figure Lengend Snippet: (A) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Expression levels of known transcriptional regulators for gluconeogenesis were examined. Data are presented as fold relative to the scramble control from three experiments. ***, P<0.00005. (B) C/EBPα expression in Jhdm1a knockdown mouse primary hepatocytes. (C) Jhdm1a was knocked down in HepG2 cells with shRNA lentiviruses. Endogenous C/EBPα association with known binding sites on the PEPCK and G6Pase promoters was examined by ChIP assay. (D) Gene expression in HepG2 cells infected with lentiviruses expressing C/EBPα or vector. (E) Increased C/EBPα expression in the liver of wild-type C57BL/6J mice (n = 5 per group) with Jhdm1a knockdown. C/EBPα mRNA level and protein level were shown from independent groups of mice. **, P<0.02. (F) Decreased C/EBPα expression in the liver of ob/ob mice (n = 5 per group) ectopically expressing wild-type Jhdm1a, but not in the liver expressing H212A point mutant. ***, P<0.001. (G) HepG2 cells were infected with lentiviruses expressing C/EBPα shRNA and selected with puromycine. Cells were then infected with lentiviruses expressing Jhdm1a shRNA without selection. Data were shown from one representative of four experiments. Note, the low induction of PEPCK and G6Pase expression by Jhdm1a knockdown is due to the lack of selection pressure.

Article Snippet: 26 μg protein extracts were separated by SDS-PAGE and probed with antibody against C/EBPα (Santa Cruz, sc-61), PEPCK (ABcam, ab28455) or G6Pase (Santa Cruz, sc-25840).

Techniques: shRNA, Expressing, Control, Knockdown, Binding Assay, Gene Expression, Infection, Plasmid Preparation, Mutagenesis, Selection

Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S4. (A) Protein expression levels of missense G6PC variants were determined in Huh7 cells (n=4-5) and (B) HEK293 cells (n=5) by western blot densitometric analysis of FLAG-tagged G6PC constructs relative to tubulin control, with representative blots shown. (C) Protein expression levels of PTV Q347X were determined in Huh7 cells (n=3) and (D) HEK293 cells (n=4) by western blot densitometric analysis of V5-tagged G6PC constructs relative to tubulin control, with representative blots shown. Bars in red indicate variants that are statistical drivers of the gene-based signal. (E) Cellular localisation of V5-tagged G6PC-Q347X was assessed in Huh7 cells and overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Glucose-6-phosphatase activity of unglycosylated WT G6PC protein obtained from tunicamycin-treated (Tuni) HEK293 microsomes (n=2), with representative western blot of microsomal protein shown. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Western Blot, Construct, Activity Assay

Journal: bioRxiv

Article Title: Tissue-Specific Alteration of Metabolic Pathways Influences Glycemic Regulation

doi: 10.1101/790618

Figure Lengend Snippet: Related to Figure S5. (A) Expression levels of the glycosylated forms (upper bands only) of G6PC2 variant proteins were determined in INS-1 832/13 cells by western blot densitometric analysis of Myc-tagged G6PC2 constructs relative to tubulin control (n=5). Representative blots are shown for untreated cells together with cells treated with proteasomal inhibitor MG-132 or lysosomal inhibitor chloroquine. (B) Glucose-6-phosphatase activity of L173T and L173V variants in G6PC (proxy for I171T and I171V in G6PC2 respectively) in HEK293 against increasing glucose-6-phosphate concentrations (n=4), with mean Vmax ± SEM and Km ± SEM values shown for WT and each variant. (C) Glucose-6-phosphatase activity of F258L variant in G6PC (proxy for F256L in G6PC2) in HEK293 against increasing glucose-6-phosphate concentrations (n=3), with mean Vmax ± SEM and Km ± SEM values shown. Vmax and Km results were computed based on the Michaelis-Menten kinetic model. (D) Effect of G6PC2 WT and variant protein expression on luciferase activity driven by ER stress response elements in HEK293 cells. Relative luciferase units corrected for background activity were normalised to WT for each reporter, from n=6 across two independent experiments (except for F256L, n=3 in one experiment) using two-way ANOVA with Fisher’s LSD test comparing each variant to WT. (E) Cellular localisation of R283X in EndoC-βH1 overlaid with markers for the ER (calreticulin) and the trans-golgi network (TGN46). White arrows point to positions of the Golgi apparatus. Scale bar indicates 10μm. (F) Insulin secretion normalised to total content at basal and high glucose conditions (with and without drug treatments) following 96-120h G6PC2 knockdown in EndoC-βH1. Unpaired two-tailed Students’ t tests were used to compare G6PC2 knockdown to control for each condition, from n=16 across 4 independent experiments. Tol: tolbutamide; Diaz: diazoxide. All data presented as mean ± SEM. * p=0.01-0.05; ** p=0.001-0.01; *** p<0.001.

Article Snippet: Human G6PC (NM_000151.3) and G6PC2 cDNA (NM_021176.2) within a pCMV6-Entry vector (with a C-terminal Myc-FLAG-tag) was purchased from OriGene (RC215623 and RC211146 respectively).

Techniques: Expressing, Variant Assay, Western Blot, Construct, Activity Assay, Luciferase, Two Tailed Test

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Primer sequences of target genes

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Primary antibodies used for immunofluorescence

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques:

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Immunofluorescence staining of proliferation and differentiation biomarkers in typical (A), ISC (B), enterocyte (C), and goblet (D) organoids. Nuclei are stained blue. Organoids were stained with stem cell marker OLFM4 (red), Paneth cell marker lysozyme (LYZ; green), enterocyte marker sucrase isomaltase (SI; red), goblet cell marker mucin 2 (MUC2; green), or enteroendocrine marker chromogranin-A (CHGA; green). Representative typical, ISC, enterocyte, and goblet organoids are shown at ×60 magnification. Bars = 25 μm. Occasional basolateral staining has been shown (personal observations) to be caused largely by autofluorescence of the Matrigel. Frequency of marked cells from at least 5 organoids from 3 mice (vi of each organoid type, a,b,c,dP ≤ 0.05). Immunocytochemistry of the fructose-responsive enzyme glucose-6-phosphatase (G6Pase) in enterocyte, ISC, and secretory goblet organoids is shown (E). All organoid types were analyzed on the 7th day of experiment, which for goblet, Paneth, and enterocyte organoids, is the 4th day after differentiation (see Fig. 1). The control panel is an image of an enterocyte organoid incubated without the primary antibody.

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Immunofluorescence, Staining, Marker, Immunocytochemistry, Control, Incubation

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Effect of cell type on the mRNA expression of classical fructose-responsive genes Glut5, G6Pase, and Khk. Typical and ISC organoids were incubated in their respective culture media for 6 days after generation from crypt precursors. Enterocyte and goblet organoids were incubated in their respective culture media for 3 days after directed differentiation from ISC precursors. Stem cells with very few Paneth cells constituted ISC organoids; enterocytes made up almost entirely enterocyte organoids, with few goblet and occasional enteroendocrine cells, whereas goblet organoids consisted primarily of goblet with some enteroendocrine cells. Media in each organoid type was supplemented with an additional 5 mM glucose, 5 mM fructose, or 10 mM fructose for 24 h. Levels of mRNA in all organoids were normalized to that in typical organoids exposed to 5 mM glucose (1.0). Only typical, enterocyte, and goblet organoids responded to supplemental fructose. Expression of the control gene Sglt1 did not vary with fructose. These results were confirmed 3 other times, using ISC, enterocyte, and goblet organoids from different mice. Analysis was by 2-way ANOVA, a,b,cP ≤ 0.05 within cell type, n = 3.

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Expressing, Incubation, Control

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Effect of fructose incubation of organoids enriched in Paneth cells on fructose-responsive genes Glut5, G6Pase, and Khk. Because Paneth cells also arise from secretory progenitors, we made (i to iv) then challenged Paneth cell-enriched organoids with 10 mM fructose (10 mM glucose as control, v). *P < 0.05, n = 3; for i-iii, a,b,c,dP < 0.05). Fructose induced marked increases in expression of Glut5, G6Pase, Khk, and Si genes. vi: Challenging ISC-enriched organoids with 20 mM fructose to increase the concentration gradient still had no effect on glucose-responsive genes.

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Incubation, Control, Expressing, Concentration Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Effect of glucose concentration in the incubation medium on fructose-responsive genes. The media in typical and enterocyte organoids was changed from 17.5 to 11.5 mM glucose (control: 17.5 to 17.5 mM) at the same time that fructose was added 24 h before the termination of the experiment. We added 5 mM glucose or 5 mM or 10 mM fructose to organoids in normal 17.5 mM or low 11.5 mM glucose media (results separated by a dashed line). Although there was an expected effect of fructose on expression of Glut5 (i) and G6Pase (ii), there was no effect of glucose concentration in the media. Alpi (iii) expression was independent of media and sugar in both typical and enterocyte organoids. There was also an effect of cell type on the expression of these genes that are highly expressed in enterocytes in vivo. Expression was normalized to that in 5 mM glucose of the control 17.5 to 17.5 mM experiment (1.0). Analysis was by 3-way ANOVA (see text for details), n = 3.

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Concentration Assay, Incubation, Control, Expressing, In Vivo

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Effect of Glut5 and Khk deletion on fructose sensing by typical organoids. Typical organoids from wild-type (WT), Glut5−/−, and Khk−/− mice were cultured for 6 days then incubated in media supplemented with 5 mM glucose, 5 mM fructose, or 10 mM fructose for 24 h on the final day of culture. There was no effect of genotype on expression of undeleted genes. Levels of mRNA in 5 and 10 mM fructose were normalized to that in 5 mM glucose (1.0) for each genotype. Except for G6Pase expression, which did not require glucose transporter 5 (GLUT5), fructose transport and metabolism were clearly necessary for fructose sensing. a,b,cP ≤ 0.05 within genotype, n = 3.

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Cell Culture, Incubation, Expressing

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Nutrient sensing by absorptive and secretory progenies of small intestinal stem cells

doi: 10.1152/ajpgi.00416.2016

Figure Lengend Snippet: Effect of CHIR-induced dedifferentiation of enterocyte organoids on expression of genes involved in the fructose response and in marking cell types. From ISC precursors, enterocyte organoids were cultured for 3 days with C59 +VPA to full differentiation as indicated by peak expression of fructose-responsive genes. To force dedifferentiation, the Wnt inhibitor C59 was removed from the medium, and enterocyte organoids were then exposed for 36 h to 3 (dENT1) or 6 μM CHIR (dENT2) or for 6 days to 6 μM CHIR (dENT3). ENT, dENT1, dENT2, dENT3, and ISC organoids from the same mouse were then challenged with 5 mM fructose in the last 12 h before harvest. Gene expression was normalized to glucose-incubated, enterocyte organoids. Although expression of fructose-responsive genes decreased markedly with CHIR treatment, Glut5 and G6Pase expression still responded to fructose incubation in all dedifferentiated enterocyte organoids. Lgr5+ levels increased almost 100-fold with CHIR-induced dedifferentiation but never reached levels observed in ISC organoids. Lyz and Muc2 expression were also affected by dedifferentiation (n = 3 per treatment).

Article Snippet: Glucose-6-phosphatase (G6PASE) , Santa Cruz Biotechnology , sc-25840 , 1:200.

Techniques: Expressing, Cell Culture, Gene Expression, Incubation

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Expression of gluconeogenic enzymes and 11β-hydroxysteroid dehydrogenase type 1 in liver of diabetic mice after acute exercise

doi: 10.2147/DMSO.S70767

Figure Lengend Snippet: Hepatic protein expression of glucose-6-phosphatase α (G6Pase α) measured 8 hours after acute exercise in db/db mice. Notes: Values are reported as a mean ± standard error of the mean for 6–9 mice in each group, and normalized to internal control.

Article Snippet: Blots were incubated with primary antibody to GR (1:1000; Cell Signaling Technology, Danvers, MA, USA), PEPCK (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 11β-HSD1 (1:1000; Abcam Cambridge, MA, USA), G6Pase α (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and G6Pase β (1:200; Santa Cruz Biotechnology Inc.) overnight at 4°C.

Techniques: Expressing, Control

Journal: Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy

Article Title: Expression of gluconeogenic enzymes and 11β-hydroxysteroid dehydrogenase type 1 in liver of diabetic mice after acute exercise

doi: 10.2147/DMSO.S70767

Figure Lengend Snippet: Hepatic protein expression of glucose-6-phosphatase β (G6Pase β) measured 8 hours after acute exercise in db/db mice. Notes: Values are reported as a mean ± standard error of the mean for 6–9 mice in each group, and normalized to internal control.

Article Snippet: Blots were incubated with primary antibody to GR (1:1000; Cell Signaling Technology, Danvers, MA, USA), PEPCK (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), 11β-HSD1 (1:1000; Abcam Cambridge, MA, USA), G6Pase α (1:200; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and G6Pase β (1:200; Santa Cruz Biotechnology Inc.) overnight at 4°C.

Techniques: Expressing, Control