Journal: Frontiers in Pharmacology

Article Title: Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet

doi: 10.3389/fphar.2024.1409313

Figure Lengend Snippet: Average body weight (A) , food intake (B) , and water intake (C) in metabolic cages, and the urine volume (D) of male obese Zucker rats before the initiation of HSD feeding and treatment with the AT 2 R agonist C21 and/or mas receptor antagonist A779 and after the study period. The body weight normalized weight of the right (E) and left kidneys (F) of the rats are reported. The water intake was determined by housing the rats individually in a metabolic cage before (days −3, −2, and −1) and after the study period (days 11, 12, and 13). The daily food intake, water intake, and urine volume were averaged separately and represented for the study period, i.e., “before” and “after.” Food intake (B) : two rats were housed in a cage; hence, each value applies to two rats. The values are represented as the mean ± SEM and analyzed via two-way ANOVA, followed by Fisher’s LSD test, whereas the values “before” and “after” were compared via a non-parametric, two-tailed, Wilcoxon matched paired t -test; * p < 0.05, n = 6–7.

Article Snippet: Male obese Zucker rats (HsdHlr:ZUCKER- Lepr fa , 6–7 weeks of age) weighing ∼270 g were purchased from Envigo.

Techniques: Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet

doi: 10.3389/fphar.2024.1409313

Figure Lengend Snippet: Urinary excretion of hydrogen peroxide (H 2 O 2 ) (A) , NGAL (B) , and KIM-1 (C) and the renal expression of NGAL (D) and KIM-1 (E) and proteinuria (F) in male obese Zucker rats. The A–C, F indices were measured before the initiation of HSD feeding and treatment with the AT 2 R agonist C21 and/or mas receptor antagonist A779 and after the study period. D, E: The indices were measured terminally in the kidney homogenate after the treatment period. Proteinuria was determined in the urine collected before (days −3, −2, and −1) and after the study period (days 11, 12, and 13), averaged, and represented for the study period, i.e., “before” and “after.” The values are represented as the mean ± SEM and analyzed via two-way ANOVA (A–C,F) or one-way ANOVA (D,E) , followed by Fisher’s LSD test, whereas the values “before” and “after” were compared via a non-parametric, two-tailed, Wilcoxon matched paired t -test; * p < 0.05, n = 6–7.

Article Snippet: Male obese Zucker rats (HsdHlr:ZUCKER- Lepr fa , 6–7 weeks of age) weighing ∼270 g were purchased from Envigo.

Techniques: Expressing, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet

doi: 10.3389/fphar.2024.1409313

Figure Lengend Snippet: The urinary excretion of creatinine (A) , the plasma concentrations of creatinine (B) , urinary urea nitrogen (C) , plasma urea nitrogen (D) , the estimated glomerular filtration rate (eGFR) (E) , urinary osmolality (F) , urinary sodium (G) , and urinary potassium levels (H) were measured in male obese Zucker rats before the initiation of HSD feeding and treatment with the AT 2 R agonist C21 and/or mas receptor antagonist A779 and after the study period. The plasma values are used to calculate the eGFR, as described previously (Besseling et al., 2021). The urinary osmolality and excretion of sodium and potassium were determined in urine collected before (days −3, −2, and −1) and after the study period (days 11, 12, and 13), averaged, and represented for the study period, i.e., “before” and “after.” The values are represented as the mean ± SEM and analyzed via two-way ANOVA, followed by Fisher’s LSD test, whereas the values “before” and “after” were compared via a non-parametric, two-tailed, Wilcoxon matched paired t -test; * p < 0.05, n = 5–7.

Article Snippet: Male obese Zucker rats (HsdHlr:ZUCKER- Lepr fa , 6–7 weeks of age) weighing ∼270 g were purchased from Envigo.

Techniques: Clinical Proteomics, Filtration, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: Angiotensin-II type 2 receptor-mediated renoprotection is independent of receptor Mas in obese Zucker rats fed high-sodium diet

doi: 10.3389/fphar.2024.1409313

Figure Lengend Snippet: Sandwich-based semi-quantitative inflammation array in the kidney homogenate of male obese Zucker rats after the subacute feeding of HSD and treatment with the AT 2 R agonist C21 and/or MasR antagonist A779. The electrochemiluminescent integrated density values are normalized to two positive controls (POS1 and POS2). The values are represented as the mean ± SEM and analyzed via one-way ANOVA, followed by Fisher’s LSD test; * p < 0.1, n = 6.

Article Snippet: Male obese Zucker rats (HsdHlr:ZUCKER- Lepr fa , 6–7 weeks of age) weighing ∼270 g were purchased from Envigo.

Techniques:

Journal: bioRxiv

Article Title: Atg8-LC3 controls systemic nutrient surplus signaling from flies to humans

doi: 10.1101/2023.05.31.543119

Figure Lengend Snippet: A) A GFP sandwich ELISA assay was performed on conditioned media of HEK293T cells that were transiently transfected with hLep-WT::EGFP, hLep-AIM1::EGFP (WGTL◊AGTA), hLep-AIM2::EGFP (WPYL◊ APYA) to quantitatively assess Leptin secretion. Normalized percent fold change of the secreted GFP signal is plotted, with hLep-WT::EGFP (grey) as the control. Error bars represent %SD. Statistical significance was measured by an unpaired two-tailed t-test on 6 biological replicates per condition. B) Confocal micrography of single optical slices was performed for HEK293T cells, that were transfected with a Myc-tagged Leptin-WT (top panel) or Leptin-AIM (both AIMs mutated; bottom panel). Cells were fixed and immunostained with anti-Myc (green), anti-GM130 (red) and DAPI (blue). Leptin’s intracellular accumulation (quantified in B’), and colocalization with GM130 (quantified in B’’). Scale bar is 10um. Each dot in the corresponding quantitation represents a cell. 28-44 cells were counted per condition. Data is shown as mean ± SEM. Statistical significance was measured by a non-parametric two-tailed Mann Whitney U test. C) A polarization histogram for Leptin::Myc was calculated to describe and quantify its distribution and is pictorially represented in C’. Each dot represents a cell. 32-44 cells were counted per condition. Data is shown as mean ± SEM. Statistical significance was measured by a non-parametric two-tailed Mann Whitney U test. Also see for additional plots. D) A Venn diagram depicting the number of interacting partners identified in an IP-MS screen of Leptin-WT::GFP vs Leptin-AIM::GFP. HEK293T cells were transfected with either Leptin-WT::GFP or Leptin-AIM::GFP, and a GFP pulldown followed by a mass spectrometric analysis was done to investigate differences in Leptin’s interactome. D’ shows STRING analysis of the 31 proteins that were unique interactors of Lepin-WT. D’’ lists the GO terms of proteins associated with Leptin-WT. Enrichment for the GO term is displayed on the X-axis and the color of the dot represents the significance, with blue indicating an FDR=0.05 and red indicating an FDR<0.0001. E) Endogenous Leptin ELISA from primary human adipocytes that incubated with either nontargeted siRNA (positive control; grey), Leptin siRNA (negative control, green), LC3 siRNA (mauve), GABARAP siRNA (lilac), and LC3 + GABARAP siRNA (purple). siRNA treatment did not affect adiposity of the cells (See ). Error bars represent %SD. Statistical significance was measured by an unpaired two-tailed t-test on 6 biological replicates per condition. F) Summary model. Leptin/Upd2 secretion requires LC3/GABARAP: A conserved model for adipokine secretion from human adipocytes to fly fat, showing that Leptin/Upd2 (adipokine) requires interaction with LC3/GABARAP/Atg8 (transporter) to be efficiently secreted from adipocytes to signal nutrient status to the brain. Disrupting the interaction of the adipokine with its transporter results in intracellular retention, reduced secretion, and impaired signaling to the brain resulting in large scale transcriptomic/metabolic alterations.

Article Snippet: For human leptin ORF clone (Origene; pCMV-leptin-DDK-Myc; CAT#: RC209259) was used for mutagenizing the AIM sequences.

Techniques: Sandwich ELISA, Transfection, Control, Two Tailed Test, Quantitation Assay, MANN-WHITNEY, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Atg8-LC3 controls systemic nutrient surplus signaling from flies to humans

doi: 10.1101/2023.05.31.543119

Figure Lengend Snippet: A, A’. Additional representative polarization plots for Leptin::Myc was calculated to describe and quantify its distribution. Leptin-WT::Myc (A’) shows a significantly higher polarization factor relative to Lepin-AIM::Myc. B. Endogenous Leptin ELISA from primary human adipocytes that were incubated with either non-targeted siRNA (positive control; grey), Leptin siRNA (negative control, green), LC3 siRNA (mauve), GABARAP siRNA (lilac), and LC3 + GABARAP siRNA (purple). Raw values of leptin are shown here, and see for % fold change. B’ AdipoRed staining of two independent siRNA treatments of adipocytes. Error bars represent % SD. Statistical significance was measured by a One-way ANOVA with Tukey corrections for multiple comparisons on 6 biological replicates per condition.

Article Snippet: For human leptin ORF clone (Origene; pCMV-leptin-DDK-Myc; CAT#: RC209259) was used for mutagenizing the AIM sequences.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Negative Control, Staining

Journal: iScience

Article Title: Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

doi: 10.1016/j.isci.2024.109779

Figure Lengend Snippet:

Article Snippet: CD295/LEPR human antibody (clone REA361) , Miltenyi , Cat#130-125-203; RRID: AB_2889537.

Techniques: Recombinant, Adhesive, Sequencing, Modification, DC Protein Assay, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Gene Expression, Software, Saline