Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of GDF15 knockout mice on a high fat diet (HFD) . (A and B) Body weight and percent body weight gain of wild type (WT) and GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (C) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT), harvested at the end of the study, 25 weeks of HFD (n = 13,9). (D) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 14,9). (E) Lipid droplet area (Percent liver area) determined from histological analyses of haematoxylin/eosin (H&E) stained liver sections (n = 9,8). (F) Plasma triglycerides (TG), cholesterol, leptin, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice after 16 weeks of HFD-feeding (n = 12,11). (G) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 16–29). (H and I) Blood glucose levels during intraperitoneal (ip) glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (J) Plasma FGF21 levels from random fed mice at 16 weeks of HFD feeding (n = 7,9). (K) FGF21 mRNA expression in tissues from WT and GDF15 KO mice after 25 weeks HFD feeding (n = 8–11). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Staining, Clinical Proteomics, Expressing

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: GDF15 mRNA expression within high fat diet fed mouse and human adipose tissue . (A) In situ hybridization analysis of GDF15 mRNA (red) from 18-week-old high fat diet (HFD) fed wild type (WT) mouse epididymal adipose tissue. Black arrows indicate GDF15 staining in foamy macrophages contributing to the formation of ‘crown-like structures’. Blue arrow indicates GDF15 staining within adipocytes. (B) Representative haematoxylin/eosin image of epididymal tissue from 18-week old HFD fed WT mouse. Black arrows indicate infiltrating cells. (C) Representative image (corresponding to the image in B) of epididymal tissue from 18-week-old HFD fed WT mouse stained with the macrophage marker F4/80 confirming that the cells contributing to the ‘crown like structures’ are macrophages. (D and E) GDF15 and EMR1 mRNA expression from 14 week old Chow or HFD fed WT mouse epididymal tissue fractionated into adipocytes, CD11b negative (−) and CD11b positive (+) stromal vascular fractions (SVF) (n = 3–4). (F) Correlation of GDF15 expression with EMR1 expression in epididymal tissue from 14 week Chow or HFD fed wild type mice (n = 8). (G–I) Human subcutaneous adipose tissue GDF15 gene expression levels in the TwinsUK adipose study associated with estimated macrophage proportion in adipose tissue (G), and macrophage markers CD68 and EMR1 (H and I). Each point represents data from a single individual. Plotted gene expression residuals of GDF15, CD68 and EMR1 were adjusted for age, BMI and RNA-Seq technical covariates. (J and K) GDF15 gene expression levels in the ‘Obese study’ associated with macrophage marker CD68 in human subcutaneous and visceral adipose tissue. (L and M) In situ hybridization analysis of Gdf15 mRNA (red) from 18-week old HFD fed WT mouse liver and brown adipose tissue. (N) FGF21 mRNA expression from 14 week Chow or HFD fed WT mouse epididymal tissue fractionated in to adipocytes, CD11b negative (−) and CD11b positive (+) stromal vascular fraction (SVF) (n = 3–4).

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Expressing, In Situ Hybridization, Staining, Marker, Gene Expression, RNA Sequencing

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of LysM-Cre mediated GDF15 macrophage knockout mouse on a high fat diet (HFD) . (A) GDF15 mRNA expression from 24-week-old high fat diet (HFD) fed wild type (WT) and LysM-GDF15 KO mouse epididymal adipose tissue fractionated into adipocytes and CD11b positive (+) stromal vascular fraction (SVF) (n = 4). (B) GDF15 mRNA expression in tissues from 24 week HFD fed WT and LysM-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), brown adipose tissue (BAT), liver and kidney (n = 4–7). (C) Plasma GDF15 levels from random fed mice at indicated weeks on HFD (n = 15–18). (D) FGF21 mRNA expression in tissues from 24-week HFD fed WT and LysM-GDF15 KO mice (n = 4–7). (E) Plasma FGF21 levels from random fed mice at indicated weeks on HFD (n = 11–18). (F and G) Body weight and percent body weight gain of WT and LysM-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (H) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 16–18). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 16,17). (J) Plasma leptin levels in mice from random fed mice after 16 weeks HFD-feeding (n = 16–18). (K) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 16,18). (L and M) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (N) Plasma triglycerides (TG), cholesterol, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 9–11). All data are means ± S.D ∗/∗∗ - p < 0.05/0.01.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of bone marrow deleted GDF15 knockout mouse on a high fat diet . (A) GDF15 mRNA expression in tissues from 24-week old high fat diet (HFD) fed WT and BMT-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT) and brown adipose tissue (BAT) (n = 8–12). (B) Plasma GDF15 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 12). (C) FGF21 mRNA expression from indicated tissues of 24 week HFD fed WT and BMT-GDF15 KO mice (n = 8–12). (D) Plasma FGF21 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 12). (E and F) Body weight and percent body weight gain of WT and BMT-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 3–12). (H) Plasma triglycerides (TG), cholesterol, leptin, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 12). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 12). (J) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice after 16 weeks of HFD feeding (n = 7,6). (K and L) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of Alb-Cre mediated GDF15 hepatocyte knockout mouse on a high fat diet . (A) GDF15 mRNA expression in tissues from 24-week old high fat diet (HFD) fed wild type (WT) and Alb-GDF15 KO mice. Epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver, brown adipose tissue (BAT) and kidney (n = 10–16). (B) Plasma GDF15 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 15,13). (C) FGF21 mRNA expression in indicated tissues from 24-week HFD fed WT and Alb-GDF15 KO mice (n = 10–13). (D) Plasma FGF21 levels from random fed mice at the onset (week 0) and after 12 weeks of HFD feeding (n = 15,13). (E and F) Body weight and percent body weight gain of WT and Alb-GDF15 KO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 15,11). (H) Plasma leptin, triglycerides (TG), cholesterol, alanine transaminase (ALT) and aspartate transaminase (AST) from random fed mice, after 16 weeks of HFD feeding (n = 15,13). (I) Weight of total hepatic lipids in g; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 15,16). (J and K) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (L) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice, after 16 weeks of HFD feeding (n = 14,13). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001.

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Clinical Proteomics

Journal: Molecular Metabolism

Article Title: Combined genetic deletion of GDF15 and FGF21 has modest effects on body weight, hepatic steatosis and insulin resistance in high fat fed mice

doi: 10.1016/j.molmet.2022.101589

Figure Lengend Snippet: Phenotypic characterization of dKO knockout mice on a high fat diet . (A) Liver GDF15 and (B) FGF21 mRNA expression from 24-week-old high fat diet (HFD) fed WT, FGF21 KO, GDF15 KO and FGF21/GDF15 double knockout (dKO) mice (n = 5). (C and D) Plasma GDF15 and FGF21 from random fed mice at indicated time after high fat feeding (n = 3–11). (E and F) Body weight and percent body weight gain of WT, FGF21 KO, GDF15 KO and dKO mice fed a 60% HFD; Inset, final body weight and percent body weight gain. (G and H) Blood glucose levels during ip glucose tolerance test (GTT) and percent change from initial blood glucose levels during insulin tolerance test (ITT) after 16 weeks of HFD feeding. Inset, area under the curve analysis of glucose over time. (I) Blood glucose, plasma insulin and HOMA-IR levels from 6 h fasted mice after 16 weeks of HFD feeding (n = 19–29). (J) Weight of epididymal white adipose tissue (eWAT), subcutaneous white adipose tissue (scWAT), liver and brown adipose tissue (BAT) harvested at the end of the study, 24 weeks of HFD (n = 11–17). (K) Weight of total hepatic lipids; total lipid extracted from 25 mg tissue was normalized to total liver weight (n = 9–17). (L) Lipid droplet area (Percent liver area) determined from histological analyses of H&E stained liver sections (n = 8–11). (M) Plasma triglycerides (TG) from random fed mice, after 24 weeks of HFD feeding (n = 8). (N) Hepatic mRNA expression of genes involved in lipid metabolism (n = 9–17). All data are means ± S.D ∗/∗∗/∗∗∗/∗∗∗∗ - p < 0.05/0.01/0.001/0.0001. Please note that the data for WT and GDF15KO overlaps with the data presented in Figure 1 .

Article Snippet: Briefly, one-cell stage embryos (obtained from super-ovulated wild type C57Bl/6N females fertilised in vitro with sperm from homozygous GDF15 Tm1a male) were injected into the pronucleus with 100ng/ul StemMACS Flp Recombinase mRNA (Miltenyi Biotec) then transferred into the uteri of pseudo pregnant recipient females (F1 hybrids from C57Bl/6J female × CBA/Ca male crosses).

Techniques: Knock-Out, Expressing, Double Knockout, Clinical Proteomics, Staining

Journal: The EMBO Journal

Article Title: Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

doi: 10.15252/embj.201796831

Figure Lengend Snippet: HeLa cells transfected with control (siLuc), APC11 or Fbw7 siRNA for 48 h were synchronised in mitotic arrest with 830 nM nocodazole for 2 and 6 h and samples analysed with antibodies as indicated. An untreated sample (−) was used as a control. DLD1 FBW7 +/+ (WT) and FBW7 −/− (KO) cells were released from a double thymidine block prior to addition of paclitaxel (500 nM). Samples were collected at times indicated after thymidine washout. DLD1 cells WT for FBW7 transfected with control (siLuc) or APC11 siRNA were treated as in (B). HeLa‐YFP‐Mcl‐1 cells were transfected with siRNA as indicated and treated with nocodazole 48 h later. YFP‐Mcl‐1 fluorescence was monitored from the time of mitotic entry. Error bars represent ±SD, n = 3. Scale bar, 10 μm. Source data are available online for this figure.

Article Snippet: To generate stable inducible cell lines, vectors were co‐transfected with pOG44 (encodes a FLP recombinase) into HeLa Flp‐In cells stably expressing a TetR using FuGene HD according to the manufacturer's instructions (Promega).

Techniques: Transfection, Control, Blocking Assay, Fluorescence

Journal: The EMBO Journal

Article Title: Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

doi: 10.15252/embj.201796831

Figure Lengend Snippet: Characterisation by Western blotting of DLD1 FBW7 +/+ (WT) and FBW7 −/− (KO) cells. *denotes a non‐specific band. Elevation of Myc, a substrate of Fbw7, demonstrates the effect of Fbw7 loss. Western blotting analysis of Mcl‐1 degradation in DLD1 WT and FBW7 KO in cells arrested with 100 nM paclitaxel (upper panels). The efficacy of 100 nM paclitaxel to induction of mitotic arrest is indicated by flow cytometric analysis of histone H3 phosphorylated on Ser10 (lower panel). Analysis of mitotic arrest induced by treatment of DLD1 WT and FBW7 KO cells with 500 nM paclitaxel as in (B). YFP‐Mcl‐1 is degraded as endogenous Mcl‐1 during a synchronised mitotic arrest. Live‐cell imaging of the degradation of cyclin A‐Venus and YFP‐Nek2A during mitotic arrest induced by treatment with 830 nM nocodazole. Error bars represent ±SD, n = 3. Confirmation of siRNA knockdown of Fbw7 or APC2 and APC11 and the stabilisation of cyclin E and Myc following Fbw7 depletion in HeLa cells stably expressing YFP‐Mcl‐1. *denotes a non‐specific band. Source data are available online for this figure.

Article Snippet: To generate stable inducible cell lines, vectors were co‐transfected with pOG44 (encodes a FLP recombinase) into HeLa Flp‐In cells stably expressing a TetR using FuGene HD according to the manufacturer's instructions (Promega).

Techniques: Western Blot, Live Cell Imaging, Knockdown, Stable Transfection, Expressing

Journal: The EMBO Journal

Article Title: Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

doi: 10.15252/embj.201796831

Figure Lengend Snippet: A, B Confirmation of siRNA knockdown of APC3, APC8 and APC11 in HeLa cells stably expressing (A) YFP‐Mcl‐1 or YFP‐Mcl‐1ΔIR or (B) cyclin A‐Venus or cyclin B1‐Venus. C, D APC3, APC8 or APC11 were knocked down by siRNA prior to treatment with nocodazole to induce mitotic arrest. The effect on the degradation of cyclin A‐Venus protein in 830 nM nocodazole (C) and cyclin B1‐Venus protein in 33 nM nocodazole (D) was analysed by time‐lapse microscopy. The nocodazole concentration was reduced to analyse cyclin B1 degradation since cyclin B1 was stable for the duration of the experiment at a higher nocodazole concentration (830 nM, data not shown). Representative images (left) and quantification (right) are shown. Error bars represent ±SD, n = 3. Scale bar, 10 μm. E Confirmation of siRNA knockdown of APC10 in HeLa cells stably expressing YFP‐Mcl‐1 WT or ΔIR, or cyclin B1‐Venus. F APC10 was knocked down by siRNA prior to treatment with nocodazole (33 nM) to induce mitotic arrest. The effect on the degradation of cyclin B1‐Venus was analysed by time‐lapse microscopy. Error bars represent ±SD, n = 3. Source data are available online for this figure.

Article Snippet: To generate stable inducible cell lines, vectors were co‐transfected with pOG44 (encodes a FLP recombinase) into HeLa Flp‐In cells stably expressing a TetR using FuGene HD according to the manufacturer's instructions (Promega).

Techniques: Knockdown, Stable Transfection, Expressing, Time-lapse Microscopy, Concentration Assay

Journal: The EMBO Journal

Article Title: Atypical APC/C‐dependent degradation of Mcl‐1 provides an apoptotic timer during mitotic arrest

doi: 10.15252/embj.201796831

Figure Lengend Snippet: Confirmation of siRNA knockdown of Cdc20 in HeLa cells stably expressing YFP‐Mcl‐1 WT. Western blotting of cells synchronised during mitotic arrest with nocodazole (830 nM) confirmed that the degradation of YFP‐Mcl‐1 protein reflected temporally that of endogenous Mcl‐1 and that both were unaffected by Cdc20 depletion. Mcl‐1 ubiquitination is inhibited by depletion of the catalytic APC/C subunits but not Cdc20. HeLa‐YFP‐Mcl‐1 cells transfected with His‐ubiquitin were depleted of APC2 and APC11 or Cdc20. Dox was added where indicated to induce expression of YFP‐Mcl‐1. Ubiquitinated proteins were purified and samples blotted for Mcl‐1. Cell lysates (input) were also blotted with indicated antibodies to demonstrate knockdown of protein expression. Luc1 denotes luciferase siRNA transfected for 48 h as a control for siAPC2 and 11; Luc2 indicates luciferase transfection for 24 h as a control for siCdc20. Cdc20 and Cdh1 were knocked down singly or in combination by siRNA prior to induction of mitotic arrest with nocodazole (830 nM). The effect on the degradation of YFP‐Mcl‐1 was analysed by time‐lapse microscopy. Error bars represent ±SD, n = 3. Efficiency of Cdc20 and Cdh1 knockdown is shown in the accompanying Western blot. Confirmation of siRNA knockdown of Cdc20 in HeLa cells stably expressing Mcl‐1ΔIR or cyclin A‐Venus. Cdc20 was knocked down by siRNA prior to treatment with nocodazole (830 nM) to induce mitotic arrest. The effect on the degradation of cyclin A‐Venus was analysed by time‐lapse microscopy. Error bars represent ±SD, n = 3. Scale bar, 10 μm. APC3 was knocked down by siRNA prior to treatment with nocodazole (830 nM) and reversine (500 nM) to generate a weak checkpoint. The effect on the degradation of YFP‐Mcl‐1ΔIR was analysed by time‐lapse microscopy. Quantification of 10 individual cells from one experiment is shown. The traces are overlaid with mean values (open characters, n = 3). Error bars represent ±SD. Representative images (left panels) and quantification (right panels) of the degradation of Mcl‐1ΔIR in nocodazole‐treated cells depleted of Cdc20, forced to exit mitosis by inhibiting Cdk1. Cdc20 was knocked down prior to treatment of cells with nocodazole (830 nM). After 6 h, cells already arrested in mitosis were imaged by time‐lapse microscopy for 20–28 min before adding Cdk1 inhibitor (R03306, 10 μM) to force mitotic exit. Cells were then imaged for another 1.5 h. Results are representative of three experiments. Scale bar, 10 μm. Western analysis demonstrating that Mcl‐1 UTR siRNA specifically knocked down endogenous Mcl‐1 and had no effect on the expression of YFP‐Mcl‐1 WT or ΔIR. Source data are available online for this figure.

Article Snippet: To generate stable inducible cell lines, vectors were co‐transfected with pOG44 (encodes a FLP recombinase) into HeLa Flp‐In cells stably expressing a TetR using FuGene HD according to the manufacturer's instructions (Promega).

Techniques: Knockdown, Stable Transfection, Expressing, Western Blot, Ubiquitin Proteomics, Transfection, Purification, Luciferase, Control, Time-lapse Microscopy