Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Histogram of relative abundance of 3160 proteins detected (out of 5399 total, see ) by LC–MS/MS in 8.7 × 10 4 purified human ²-cells from n = 3 donors. Green lines indicate gene products related to the urea cycle, argininosuccinate and aspartate metabolism detected at the protein level. b, Expression levels of genes related to the urea cycle, pyruvate metabolism and arigninosuccinate shunt based on RNAseq analysis of FACS-sorted human β-cells from n=3 human donors, data are in Log2CPM. c, Co-immunostaining of insulin and individual metabolic enzymes related to the urea cycle in dispersed human islet cells from one donor representing similar results obtained from two additional donors. Representative images are shown (left), scale bar is 10 microns. Mean fluorescence intensity (FI) within the insulin positive region of interest (ROI) was calculated from 5 images per antibody (right). Neg denotes negative control for background Alexa Fluor 488 signal with insulin co-stain. Statistical analyses are student’s t-tests of each enzyme compared to Neg. Enzyme abbreviations in a–c are ARG2, arginase 2; ASL, argininosuccinate lyase; ASS1, argininosuccinate synthase 1; CPS1, carbamoyl-phosphate synthase 1; DDAH 1, dimethylarginine dimethylaminohydrolase 1; DDAH 2, dimethylarginine dimethylaminohydrolase 2; GOT1, aspartate aminotransferase 1; GOT2, aspartate aminotransferase 2; MDH1, malate dehydrogenase 1; MDH2, malate dehydrogenase 2; NAGS, N-acetyl-glutamate synthase; OAT, ornithine aminotransferase; PC, pyruvate carboxylase; SLC25A12, solute carrier family 25 member 12/calcium-binding mitochondrial carrier protein Aralar 1; SLC25A13, solute carrier family 25 member 13/calcium-binding mitochondrial carrier protein Aralar 2; SLC25A15, solute carrier family 25 member 15/mitochondrial ornithine transporter 1. d–e, Cytokine-induced changes in the partitioning of 15 N 2 -L-arginine to urea ( d ) and citrulline/NO ( e ) synthesis in human islets comparing protective vs non-protective glucose metabolism as modeled by BAD SAHB A SD vs RO0281675 treatment, respectively, n=4 donors. f, Chemical inhibition of arginase via ABH interferes with the protective effect of BAD SAHB A SD in human islets undergoing inflammation, n=5 donors. Data are means ± s.e.m. with one-way (d,e) and two-way (f) ANOVA statistical tests with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Purification, Expressing, Immunostaining, Fluorescence, Negative Control, Staining, Binding Assay, Inhibition

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, Schematic of the TCA and urea cycles and their connection via the aspartate-argininosuccinate shunt. Enzymes of interest are marked in red and their corresponding inhibitors in blue. b, Total aspartate levels in human islets treated with the indicated compounds and cultured in the absence or presence of inflammatory cytokines. Data are from the untargeted metabolomics analysis in , n=5 human donors pooled and split into 8 replicates. c, Contribution of glucose to total aspartate pools in mouse islets labeled with 13 C 6 glucose and treated with inflammatory cytokines in the context of protective vs non-protective glucose metabolism. Data are from n=5 (Veh, RO0281675), n=6 (BAD SAHB A SD) and n=4 (BAD SAHB A AAA) independent mouse islet isolations and experiments. See for isotopologue distribution of aspartate in an analogous labelling experiment. d–e, Quantification of urea and NO , and viability ( e ) in human islets subjected to shRNA-mediated GOT1 ( G1 ) or GOT2 ( G2 ) depletion and treated with cytokines in the context of protective vs non-protective glucose metabolism, n=4 donors for urea, n=3 donors for NO, and n=5 donors for viability measurements. Data for one hairpin per gene are displayed (sh#1 for GOT1 and sh#2 for GOT2 ) from the full data set of multiple hairpins, see – . f–g, Quantification of urea levels ( f ) and viability ( g ) in human islets supplemented with argininosuccinate (AS) in the presence of inflammatory cytokines, H 2 O serves as vehicle control for AS. Data are from 6 independent experiments from n=3 donors. Data are means ± s.e.m. with statistical analyses on means from independent experiments using one-way ANOVA with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Cell Culture, Labeling, shRNA

Journal: Nature metabolism

Article Title: Glucose-dependent partitioning of arginine to the urea cycle protects β-cells from inflammation

doi: 10.1038/s42255-020-0199-4

Figure Lengend Snippet: a, 13 C fractional labelling of aspartate from 13 C 6 glucose. Data are shown as non-normalized to vehicle PBS and display the fraction of each M+ n mass isotopomer out of the total pool of aspartate for each condition. For clarity, statistical comparisons are only shown for each M+ n of a given condition (RO0281675, BAD SAHB A SD and BAD SAHB A AAA) compared to the corresponding M+ n of vehicle control. Data are pooled means from n=6 (Veh), n=5 (RO0281675), and n=6 (BAD SAHB A SD, BAD SAHB A AAA) independent mouse islet isolations and experiments. b, Western blot analysis of GOT1/2 knockdown efficiency using multiple independent hairpins for data shown in – and – . Blots are representative of n=2 independent experiments with similar results. c–d, Aspartate ( c ), urea and NO ( d ) levels in human islets from the same experiments shown in – , displaying the complete set of data on all hairpins tested. Aspartate data are from n = 4 human donors for shCtrl samples and n = 3 donors for knockdown samples. Urea and NO data are from n = 4 and n = 3 donors, respectively. Statistical analyses in (a) are two-way ANOVA showing p-value comparisons for each condition to Veh, and one-way ANOVA in (c–d), both with Tukey adjustment for multiple comparisons.

Article Snippet: For all genetic manipulations, corresponding changes in protein expression were verified by western blot analysis using the following antibodies and dilutions; GOT1 (Sino Biological 14196-T52-50, 1:1000), GOT2 (My Bio Source MBS769801, 1:2000), glucokinase (Santa Cruz, 1:1000), pyruvate carboxylase (PCB, Santa Cruz, 1:1000), ARG2 (Life technologies, 1:1000), V5 (Cell signaling, 1:1000), MYC-tag (Cell Signaling, 1:1000), BAD (Abcam, 1:3000), and actin (Sigma, 1:20000).

Techniques: Western Blot

Journal: Cell reports

Article Title: Respiratory Supercomplexes Promote Mitochondrial Efficiency and Growth in Severely Hypoxic Pancreatic Cancer

doi: 10.1016/j.celrep.2020.108231

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-GOT1 , Novus Biologicals , Cat#NBPI-54778; Lot#QC19370–43530; RRID:AB_11006805.

Techniques: Plasmid Preparation, Virus, Recombinant, Clinical Proteomics, Membrane, Protease Inhibitor, Avidin-Biotin Assay, Fractionation, Software

Journal: Oncotarget

Article Title: Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles

doi:

Figure Lengend Snippet: (A) Functional analysis using FunRich software indicates the biological pathways overrepresented either in large (10,000 g) or nano-sized EVs (100,000 g). (B) Protein lysates from cells, large and nano-sized EVs were blotted with the indicated antibodies. CD63 was expressed specifically in nano-sized EVs, and GOT1 in large EVs. (C) Protein lysates from DU145 cells untreated or treated with large EVs for the indicated times, were blotted with GOT1 antibody. GAPDH was used as a loading control (top panel). GOT1 mRNA expression levels in DU145 cells untreated or treated with large EVs for the indicated times do not exhibit significant changes. The result is displayed as levels of GOT1 transcript after normalization to the housekeeping gene GAPDH in treated versus untreated cells at 2-48h (bottom panel). (D) GOT1 activity was measured in DU145 cells in 5% glutamine with or without treatment with large oncosomes (1.15 g/ml OptiPrep TM density fractions) or exosomes (1.10 g/ml) (20μg/ml of protein lysate). The results from 3 experiments are displayed as relative AST activity in treated cells in comparison with the baseline activity of the enzyme (p=0.024). (E) Cell-cycle was analyzed by flow cytometry in DU145 cells treated with large oncosomes or vehicle in the presence of 1% or 5% glutamine for 24 hours.

Article Snippet: Cells, purified large EVs and nano-sized EVs were lysed and analyzed by western blotting using the following antibodies: rabbit monoclonal GOT1 (Sigma, 1:1000 dilution), rabbit polyclonal CD63, clone H-193 (Santa Cruz, 1:1000 dilution), rabbit polyclonal CK18 (Abcam, 1:1000 dilution); rabbit polyclonal CD81, clone M38 (Abcam, 1:1000 dilution), mouse monoclonal TSG101, clone C-2 (Santa Cruz, 1:500 dilution), rabbit monoclonal HSPA5 (Cell Signaling, 1:1000), and GAPDH (Cell Signaling 1:2000).

Techniques: Functional Assay, Software, Expressing, Activity Assay, Flow Cytometry