Fig 1: OC expression is related to Pygl expression in mouse Hepa1c1c7 cells. (A, B) mRNA (A) and protein (B) expression of Pygl and OC in Hepa1c1c7 cells treated with GluOC (0–50 ng/ml) for 24 h. Levels of Actb/ß-actin were determined as internal controls. (B) Left, immunoblot analysis. Right, quantification of the blot (ratio of Pygl or OC to ß-actin). (C) ELISA assays of GlaOC and GluOC in Hepa1c1c7 cell extracts (left) and supernatants (right) after 50 µM forskolin treatment for 24 h. Cells (7.5 × 106) were pre-cultured in 150 mm plates overnight, then incubated with 50 µM forskolin or DMSO for 24 h. The supernatants were lyophilized and dissolved in a small quantity of water, dialyzed against water, and then assayed for Gla- or GluOC by ELISA. (D) Top: knockdown of Gprc6A by siRNA; Middle: expression of OC and Pygl in Hepa1c1c7 cells transfected with Gprc6a siRNA or control siRNA for 24 h in the presence of GluOC (0–50 ng/ml); Bottom: Quantification of the immunoblots (ratio of Pygl or AHR to ß-actin). Data are expressed as mean ± SEM of three independent experiments. The blot shown is the typical one. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: Assessment of the metabolism of the P24 mice. Biological assays for (A) Pygl activity, (B) hepatic glycogen, (C) hepatic free glucose, (D) serum glucose, and (E) hepatic and (F) serum triglyceride. The numbers in the columns are the number of samples measured. Data are expressed as mean ± SEM. (G) PAS staining of P24 liver from female offspring in each group, with or without a-amylase treatment, using consecutive tissue sections. Typical staining is shown from more than 10 specimens. (H, I) Body (H) and liver (I) mass of P24 offspring. The numbers in the columns are the number of samples measured. The horizontal bar indicates the mean with the value. Statistical analysis was performed using one-way ANOVA and Dunnett's post-hoc test. *P < 0.05 and **P < 0.01.
Fig 3: A Summary of the Observed Metabolic States of Quiescent, LPS Stimulated, and COPD NeutrophilsA diagram showing the metabolic states of resting (A), stimulated (B), and COPD (C) neutrophils showing increased glycolytic activity and glycogen synthesis in response to LPS and defective glycogen cycling and glycolysis in COPD. Genes identified to actively regulate neutrophil glucose transport (Glut1), gluconeogenesis (GNG) (Fbp1 and Pck2), glycogenesis (Gys1, Gbe1, and Ugp2), and glycogenolysis (Pygl) are highlighted in red. Arrow thickness indicates the relative flux through metabolic pathways with glycogenolysis and glucose oxidation highlighted in blue and glycogenesis and gluconeogenesis in green.
Fig 4: Regulation of Glycogen Stores and Gluconeogenesis Pathway Activity during Human Neutrophil Activation(A) Glycogen level quantification in freshly isolated neutrophils (0 h) and following 6 h of culture in glucose-deplete and -replete media under normoxia and hypoxia. n = 4.(B) Glycogen content of neutrophils following 6 h of normoxic culture with the glycogen phosphorylase inhibitor CP-91149 preventing glycogen breakdown. n = 4.(C) Assessment of apoptosis rates using flow cytometry following culture in glucose-deprived media under normoxia and hypoxia with CP-91149 and LPS for 12 h. n = 5.(D and E) Liquid scintillation count measurement of radioactive U-14C glucose (D; n = 4) and U-14C lactate (E; n = 5) incorporation into neutrophil glycogen stores following 6 h of culture.(F) Transcript expression of glycogen metabolism and gluconeogenesis machinery: muscle glycogen synthase (GYS1, n = 4), glycogen branching enzyme (GBE1, n = 4), UDP-glucose pyrophosphorylase 2 (UGP2, n = 3), liver glycogen phosphorylase (PYGL, n = 4), fructose-1,6-bisphosphatase 1 (FBP1, n = 4), and phosphoenolpyruvate carboxykinase 2 (PEPCK2, n = 4).(G) Protein expression of glycogen metabolism and gluconeogenesis machinery in freshly isolated neutrophils (0 h) and neutrophils cultured for 6 or 20 h. Positive controls for FBP1 and PEPCK2- MCF7 lysate, PYGL- mouse liver lysate, phospho-GYS (p-GYS), and GYS- NIH/3T3 cell lysate. Representative western blots are shown. n = 3.(H) Diagrammatic representation of U-13C glucose (black circles) and U-13C glutamine (gray circles) labeling in human neutrophils. GNG, gluconeogenesis.(I) G6P/F6P isotopologue abundance following culture in U-13C glucose media for 4 h under conditions of normoxia, normoxia with LPS, and hypoxia. n = 4.(J–L) 13C percentage labeling of glycolytic intermediaries (J; n = 4) and isotopologue labeling of TCA cycle and glycolytic intermediaries (K and L; n = 4) following 4 h of culture in U-13C glutamine containing media.(M) Percentage heavy labeling of G6P/F6P following 4 h of culture in the presence of U-13C palmitic acid. n = 4.(N) Schematic diagram and relative abundance of glucose m+3 isotopologue following U-13C pyruvate tracing in neutrophils derived by LC-MS analysis of hydrolyzed glycogen. n = 4.Data represent mean ± SEM. Statistical significance was determined by paired t tests (A, B, and D) or two-way ANOVA with Tukey’s multiple comparisons test (C and J–L) and a one-way ANOVA with Tukey’s multiple comparisons test (N). *p < 0.05, **p < 0.01, ***p < 0.005. (J) Significance shown for unstimulated versus LPS stimulated cells across all isotopologues.
Fig 5: Experimental protocol and whole-genome bisulfite sequence (WGBS) analysis. (A) Experimental protocol. Female 8-week-old C57Bl/6N mice were mated with age-matched males and allocated randomly to three groups: a normal diet (ND) group or a high-fat, high-sucrose diet (HFS, F2HFHSD, Oriental Yeast, Tokyo, Japan; containing 20% sucrose and 30% fat) group, which was administered either saline (control) or GluOC (10 ng/g body mass) from the day of mating to that of delivery, and the dams were fed ND after the delivery. The offspring groups were designated as ND-, HFS-saline-, and HFS-GluOC-offspring, according to the diet and GluOC administration status of their dams determined during pregnancy. Tissues and blood were collected without fasting. (B) Quantitative RT-PCR analysis of 38 metabolic genes that had significantly differently methylated promoters in HFS-offspring, according to WGBS analysis. Of these, nine genes (red-colored) displayed lower mRNA expression and hypermethylation of their promoters: Apob (apolipoprotein B), Atg9a (autophagy-related 9A), Col13a1 (collagen type 13 alpha 1 chain), Hif3a (hypoxia-inducible factor 3 alpha subunit), Hist1h3c (histone cluster 1H3 family member C), Magi1 (membrane-associated guanylate kinase, WW and PDZ domain-containing 1), Plg (plasminogen), Ppp1cc (protein phosphatase 1 catalytic subunit gamma), and Pygl (glycogen phosphorylase L). In addition, there were three genes for which the promoter methylation in the HFS-fed samples was lower (blue-colored): Itgb6 (integrin beta 6), Plagl1 (pleomorphic adenoma gene-like 1), and Slc27a1 (solute carrier family 27, member 1). (C) Quantitative RT-PCR analysis of the above nine genes in the livers of the offspring from the three groups. RNA (n = 12 per group) was extracted from the P24 mouse liver and the expression of Actb was determined as the reference gene. Data are expressed as mean ± SEM of three independent experiments. Student's t-test (A) or one-way ANOVA and Dunnett's post-hoc test (C) were used. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant.
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