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

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Representative Western blot detection using the XPA antibody of different exogenously expressed LepR domains in HEK293 cells. Experiments were repeated at least twice. ( B ) Schematic representation of leptin receptor domains and the XPA binding site. ( C ) Top: schematic representation of the BRET assay to study the ligand-induced conformational change/interaction between LepR-Rluc and LepR-YFP. Bottom: BRET donor saturation curves in HEK293T cells with a constant expression level of LepR-Rluc and increasing levels of LepR-YFP, upon stimulation with vehicle, leptin (50nM) or XPA (100nM) for 30 min at 37?°C. ( D ) STAT3 and ERK1/2 phosphorylation in HEK293 cells stably expressing LepR after stimulation with 50 nM leptin or 100 nM XPA for 5, 15, 30 or 60 minutes. Representative Western blot out of at least 2 independent experiments. ( E ) STAT3 phosphorylation in tanycytes upon 50 nM leptin or 100 nM XPA stimulation for 30 minutes. Representative Western blot out of 2 independent primary cultures of tanycytes. ( F ) Leptin colocalizes with LepR in primary tanycytes. Representative confocal images of tanycytes treated for 5 min with 125 nM fluorescent leptin (red) together with 33 nM XPA antibodies against LepR labeled with fluorescent secondary antibodies (green). The extent of colocalization is represented by the mask on the right. Arrows point to examples of colocalized pixels. Scale bar: 10 μm. Experiments were repeated in 2 independent primary cultures of tanycytes. ( G ) Representative photomicrograph revealing sites of XPA fixation in tanycytes of the median eminence (vimentin-positive cells) 2 minutes after intravenous XPA injection (2 nmol/animal) in vivo (n=3 mice per group). White arrows show XPA (red) and vimentin (white) colocalization. 3V: third ventricle; ARH: arcuate nucleus of the hypothalamus; ME: median eminence. Scale bar: 200μm. ( H ) Representative image of a living brain slice containing the median eminence from a GCamp3 Trpm5 mouse under bright-field and fluorescence microscopy, showing the reversible increase in intracellular calcium levels in tanycytic cell bodies lining the third ventricle (3V) upon the local application of a puff of leptin (6μM) via a glass pipette. The experiment was repeated in 5 mice. ME: median eminence. Scale bar: 100 μm ( I ) Representative curves of GCamp3 fluorescence (calcium current) over time (Delta T) compared to the baseline in tanycytes in living hypothalamic slices during a puff of leptin (yellow rectangle, 6uM), alone (left curve) or after pre-treatment with leptin antagonist (LAN, 6μM, top black line; right curve), in a GCamp3 Trpm5 mouse. ( J ) Same measurement as in ( I ) in a GCamp3 Trpm5 ; LepR Trpm5 mouse lacking LepR in tanycytes after a puff of leptin (6μM, yellow rectangle). ( K ) Graph representing maximum difference in calcium concentration from baseline during the treatment of living brain slices in GCamp3 Trpm5 and GCamp3 Trpm5 ; LepR Trpm5 mice, described in ( I ) and ( J ). Krustal Wallis with Dunn’s; N=5 ( GCamp3 Trpm5 + leptin), 3 ( GCamp3 Trpm5 + LAN and leptin) and 4 ( GCamp3 Trpm5 ; LepR Trpm5 ) mice; each dot represents one cell (n=146,28,58). Values indicate means ± SEM. ( L ) Graph representing maximum c difference in calcium concentration from baseline during a puff of ATP (10 mM) in living brain slices from GCamp3 Trpm5 and GCamp3 Trpm5 ; LepR Trpm5 mice. Mann-Whitney test; N=3 mice per condition; each dot represents one cell (n=40,53). Values indicate means ± SEM. See also Supplementary Figure 1.

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Western Blot, Binding Assay, Bioluminescence Resonance Energy Transfer, Expressing, Phospho-proteomics, Stable Transfection, Labeling, Injection, In Vivo, Slice Preparation, Fluorescence, Microscopy, Transferring, Concentration Assay, MANN-WHITNEY

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Endocytosed leptin colocalizing with early endosomes. Representative confocal images showing primary tanycytes treated for 10 min with 125 nM fluorescent leptin (red) and antibodies to the early endosome marker EEA1 (green). The extent of colocalization is represented by the mask on the right. Arrows in inset point to examples of colocalized pixels. Scale bar: 10μm. ( B ) Percentage of leptin colocalizing with EEA1 over time following object-based detection of fluorescent leptin and EEA1 vesicles. Mann-Whitney test. n=10,10,15,15,12 cells as shown by individual dots from at least 2 independent primary cultures. Values represent means ± SEM. ( C ) Percentage of endocytosed leptin found in the EEA1-positive compartment over time. Mann-Whitney test. n=11,11,15,13,9 cells at 2,5,10,15 and 30 min, respectively, from at least 2 independent primary cultures. Values represent means ± SEM. ( D ) Leptin secreted into the medium by primary cultures of tanycytes as a percentage of total leptin concentration (intracellular and medium) 15, 30 and 45 minutes after fluorescent leptin addition. One-way ANOVA and Tukey’s test. n=4 and 6 wells cells as shown by individual dots from at least 2 independent primary cultures. Values represent means ± SEM. ( E ) Percentage (as % of 0 min time point) of endocytosed fluorescent leptin or fluorescent LAN found in EEA1 compartments over time in cells treated or not with U0126 (leptin) or EGF (LAN). Mann-Whitney test. n=18,19,17,14,14 (leptin), 30,28,31,22,12 (LAN), 15,8,7,8,9 (Leptin+U0126) 15,13,14,13,10 (LAN+EGF) at 0,5,15,30,60 min, respectively, from at least 2 independent primary cultures. Values represent means ± SEM. ( F ) Volcano plot showing differences in peptide phosphorylation between primary cultures of tanycytes treated for 2 min with vehicle (PBS pH 8.0) or leptin (1 μg/ml in PBS pH 8.0) (n=4 wells per condition). See also corresponding Source Data Files (Extended data Table 1 and 2). ( G ) Proximity Ligation Assay (PLA) between LepR and EGFR using XPA and a rabbit anti-EGFR antibody. PLA signal is seen in tanycytic cell bodies (empty arrows), processes (white arrows) and end-feet in the external zone of the median eminence, where they contact the fenestrated endothelium of the pituitary portal circulation (arrowheads). Scale bar: 100 μm. ( H ) Co‐immunoprecipitation of EGFR along with LepR in HEK293T cells; no co-immunoprecipitation of EGFR is observed when LepR is not expressed. IP, immunoprecipitation; Lys., cell lysate. ( I ) Schematic representation of the TR-FRET technique (left). Right: specific saturation curves of leptin-d2 binding to its cognate receptor LepR within the LepR:SNAP-EGFR complex at the cell surface are obtained after 3h at 37°C. Data are presented as means ± SD of 3 replicates of 1 representative experiment out of 3 independent experiments. ( J ) Phosphorylation of EGFR and ERK upon addition of leptin 50nM, EGF 10nM or both for 30min at 37°C in primary tanycytes. ( K ) Phosphorylation of STAT3 and ERK upon addition of leptin 10nM, EGF 1nM or both for 30min at 37°C in HEK293T cells expressing endogenous EGFR and transfected with LepRb in the presence or absence of the EGFR inhibitor AG1478 (1μM). Two-way ANOVA and Sidak’s multiple comparison. n=5,5,3,5,3,5,3 wells as shown by individual dots from 2 independent experiments.

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Marker, MANN-WHITNEY, Concentration Assay, Phospho-proteomics, Proximity Ligation Assay, Immunoprecipitation, Binding Assay, Expressing, Transfection, Comparison

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Schematic diagram and gating strategy for sorting Tomato positive cells following vehicle (top panel) and TAT-Cre infusion (bottom panel) into the third ventricle (3V) of LepR +/+ ; tdTomato l oxP-STOP-loxP or LepR loxP/loxP ; tdTomato loxP-STOP-loxP littermates. ( B-D ) mRNA expression levels of short forms ( B ) and the long form, LepRb ( C ), of the leptin receptor, and of Socs3 ( D ) in tdTomato-positive cells (left panels) and tdTomato-negative cells (right panels). A two-sided unpaired Student t-test or Mann-Whitney U test was applied, depending on Shapiro-Wilk normality test results. Values indicate means ± SEM. Each dot represents a mouse (n=6,8,7,7 in B, 6,6,6,6 in C , 5,6,5,6 in D ). ( E ) Food intake pattern (daily average of automatic measurements in metabolic cages over 24h), showing an increase at lights-on in LepR TanKO mice when compared to LepR l oxP/loxP littermates. The night was divided in two 6h time slots (Night 1 and Night 2). Two-sided unpaired t-tests. Values indicate means ± SEM. Each dot represents a mouse (n=10,8). ( F-H ) Curves representing the kinetics of the % change in body weight ( F ), % change in fat mass ( G ) and % change in lean mass ( H ) between LepR loxP/loxP , LepR TanHet and LepR TanKO through the 12 weeks following the TAT-Cre infusion into the 3V. Two-way ANOVA with Tukey’s correction; Values represent means ± SEM; n indicates the number of mice. ( I,J ) Visceral fat mass ( I ) and subcutaneous ( J ) 12 weeks after TAT-Cre infusion. Mann-Whitney U test. Values indicate means ± SEM. Each dot represents a mouse (n=7,9). ( K ) Cumulative food intake in LepR TanKO pair-fed mice 12 weeks after TAT-Cre infusion compared to their control littermates. Two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( L ) Cumulative body weight change. Two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( M ) Energy ratio (RER) over time. Two-way ANOVA with uncorrelated Fisher’s LSD test. Values indicate means ± SEM; n indicates the number of mice. ( N ) Mean energy ratio (RER) during light phase, dark phase and total.Two-way ANOVA with Tukey’s correction. Values indicate means ± SEM. Each dot represents a mouse (n=6,5,5). ( O ) Circulating leptin levels in LepR loxP/loxP and LepR TanKO animals at 4 weeks and 12 weeks after TAT-Cre infusion into the 3V. Mann-Whitney test (4 weeks) and two-sided unpaired t-test (12 weeks). Values represent means ± SEM. Each dot represents a mouse (n=6,7,8,8). ( P ) Basal serum EGF concentrations in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Mann-Whitney U test. Values represent means ± SEM. Each dot represents a mouse (n=6,5). ( Q ) Basal serum EGF concentrations in C57Bl/6J mice fed normal chow or those fed a high-fat diet for 8 weeks. Mann-Whitney U test. Values represent means ± SEM. Each dot represents a mouse (n=6,5).

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Expressing, MANN-WHITNEY, Control

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A-C ) Representative photomicrograph ( A ) and quantification of leptin-induced P-STAT3 immunofluorescence in the ventromedial (vm) ( B) and dorsomedial (dm) arcuate nucleus (ARH) ( C ). Scale bar: 200μm. Two-sided unpaired Student’s t-test. Values indicate means ± SEM. Each dot represents a mouse (n=4,4). ( D ) Relative mRNA expression levels of several genes known to be involved in the hypothalamic regulation of energy homeostasis and leptin activity in the microdissected mediobasal hypothalamus (MBH) of LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Student’s t-test or Mann-Whitney U test, depending on Shapiro-Wilk normality test results. Values indicate means ± SEM. Each dot represents a mouse (n=5,4). ( E ) Schematic diagram showing the design of the experiment investigating the anorectic response to either intraperitoneal (i.p) or intracerebroventricular (i.c.v) leptin administration. Bottom left graph represents food intake in LepR loxP/loxP (black and grey bars) and LepR TanKO mice (red and pink bars) 24h after i.p. leptin (3mg/kg, grey and pink bars) or vehicle (PBS pH 8.0, black and red bars) administration. Bottom right graph represents food intake in LepR loxP/loxP and LepR TanKO mice 24h after i.c.v. leptin (2μg in 2μL) or vehicle (2μL PBS pH 8.0) injection. Mann-Whitney U test; Values indicate means ± SEM. Each dot represents a mouse (n=7 per group). ( F ) Leptin concentrations in the ARH interstitial liquid collected by microdialysis every 20 minutes following i.p. vehicle (t -40 min ) or leptin (t -1 min ) injection in LepR loxP/loxP (n= 7) and LepR TanKO mice (n=6). Two-way ANOVA followed by Fisher’s LSD post hoc test analysis was applied; Values indicate means ± SEM; n indicates the number of mice. ( G ) Representative photomicrograph of in situ hybridation of EGFR in the median eminence using RNAscpe technology from mice injected with AAV1/2 Dio2::gfp or AAV(1+2)-GFP-U6-m-EGFR-shRNA . The experiment was performed in 3 animals per condition. The left panel shows vimentin-immunoreactivity in red. Arrowheads show the cells seen at higher magnification in insets. Scale bar: 100μm (25 μm in inset). ( H-J ) Curves representing the evolution of body weight ( H ), % change in fat mass ( I ) and % change in lean mass ( J ) between mice injected with AAV1/2 Dio2::gfp (control in black) or AAV(1+2)-GFP-U6-m-EGFR-shRNA (in orange) over 4 weeks following the beginning of the viral activity. Two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; each dot represents a mouse (n=9,8). ( K ) Graph representing food intake in mice injected with AAV1/2 Dio2::gfp or AAV(1+2)-GFP-U6-m-EGFR-shRNA 24h after i.p. leptin (1mg/kg) or vehicle (PBS pH 8.0) injection. An two-sided unpaired t-test was applied. Values indicate means ± SEM; each dot represents a mouse (n=6 per group).

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Immunofluorescence, Expressing, Activity Assay, MANN-WHITNEY, Injection, In Situ, shRNA, Control

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Fatty-acid (FA) oxidation over time. 2-way ANOVA with uncorrelated Fisher’s LSD test. Values indicate means ± SEM; n indicates the number of mice. ( B ) Graphs representing serum cholesterol and triglyceride concentrations in LepR loxP/loxP and LepR TanKO mice fed ad libitum on chow and LepR TanKO mice pair-fed with LepR loxP/loxP mice, 12 weeks after TAT-Cre infusion. One-way ANOVA with Tukey multiple comparison test or Kruskal-Wallis test with Dunn multiple comparison test were applied depending Shapiro-Wilk normality test results. Values indicate means ± SEM; each dot represents a mouse (n=15,14,6,14,14,6). ( C ) Graph representing serum non-esterified fatty acid (NEFAS) concentrations in LepR loxP/loxP and LepR TanKO mice fed ad libitum on chow and LepR TanKO mice pair-fed with LepR loxP/loxP mice, 12 weeks after TAT-Cre infusion. One-way ANOVA with Tukey multiple comparison test. Values indicate means ± SEM; each dot represents a mouse (n=15,14,6). ( D ) Representative images of histological hematoxylin-eosin staining of the adipose tissue in LepR loxP/loxP and LepR TanKO mice fed ad libitum on chow and LepR TanKO mice pair-fed with LepR loxP/loxP mice illustrating quantifications in the graph. Graph shows quantification of adipocyte size. One-way ANOVA with Tukey multiple comparison test. Values indicate means ± SEM; each dot represents a mouse (n=6,5,6). ( E ) Representative western blots of the different proteins mentioned in ( F ). ( F ) Graph representing protein expression levels of several proteins implicated in fatty acid synthesis or fatty acid lipolysis in white adipose tissue from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Lipoprotein lipase (LPL) is implicated in the lipids uptake from the circulation to the adipose tissue. One-way ANOVA with Tukey multiple comparison test or Kruskal-Wallis test with Dunn multiple comparison test. Values indicate means ± SEM; each dot represents a mouse (n=11,12,5). ( G ) Representative Oil-Red-stained images from the liver of LepR loxP/loxP and LepR TanKO mice fed ad libitum and LepR TanKO mice pair-fed with LepR loxP/loxP mice illustrating quantifications in H . ( H ) Quantification of triglycerides in the liver of LepR loxP/loxP and LepR TanKO mice fed ad libitum and LepR TanKO mice paired-fed with LepR loxP/loxP mice. One-way ANOVA with Tukey multiple comparison test. Values indicate means ± SEM; each dot represents a mouse (n=6,5,5). ( I ) Representative western blots of the different proteins mentioned in ( J ). ( J ) Graph representing protein expression levels of several proteins implicated in fatty acid synthesis and lipid uptake from the circulation into the liver in LepR loxP/loxP and LepR TanKO mice fed ad libitum and LepR TanKO mice paired-fed with LepR loxP/loxP mice, 12 weeks after TAT-Cre infusion. One-way ANOVA with Tukey multiple comparison test. Values indicate means ± SEM; each dot represents a mouse (n=9,10,4).

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Comparison, Staining, Western Blot, Expressing

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Curve representing glycemia during a glucose tolerance test in LepR loxP/loxP , LepR TanHet and LepR TanKO mice, 4 weeks after TAT-Cre infusion. Graph represents the area under the curve; two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( B ) Serum insulin concentrations during the first 30 mins of a glucose tolerance test in LepR loxP/loxP , LepR TanHet and LepR TanKO mice, 4 weeks after TAT-Cre infusion; two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( C ) Graph representing serum insulin concentrations at T0 of the glucose tolerance test; two-sided t-test. Values indicate means ± SEM; n indicates the number of mice. ( D ) Curve representing glycemia during a glucose tolerance test in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion; two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( E ) Serum insulin concentrations during the first 30 mins of a glucose tolerance test in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion; two-way ANOVA with Tukey’s correction. Values indicate means ± SEM; n indicates the number of mice. ( F ) Percentage change in basal glycemia during an insulin tolerance test in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Two-way ANOVA with Sidak’s multiple comparaison (two-sided unpaired t-test for AUC, inset). Values indicate means ± SEM; n indicates the number of mice. ( G ) HOMA-IR. Two-sided unpaired t-test. Values indicate means ± SEM; each dot represents a mouse (n=7,5,5,5). ( H ) Graph representing insulin secretion from total isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion, following treatment with low or high glucose concentrations. Two-sided unpaired t-test. Values indicate means ± SEM; each dot represents a mouse (n=3,4). ( I ) Graph representing insulin concentrations in isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Two-way ANOVA with Tukey’s multiple comparaison. Values indicate means ± SEM; each dot represents a mouse (n=3,4). ( J ) Relative mRNA expression levels of markers of β-cell function and identity in isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. A two-sided unpaired Student t-test or Mann-Whitney U test was applied, depending on Shapiro-Wilk normality test results. Values indicate means ± SEM; each dot represents a mouse (n=4,4). ( K ) Relative mRNA expression levels of ER stress markers in isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. A two-sided unpaired Student t-test or Mann-Whitney U test was applied, depending on Shapiro-Wilk normality test results. Values indicate means ± SEM; each dot represents a mouse (n=4,4). ( L ) Representative confocal images representing nuclei (blue), glucagon (green) and insulin (red) in isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion illustrating quantifications in M . Scale bar: 50 μm. ( M ) Graphs representing the ratio between insulin-positive (left) or glucagon-positive area (right) to the total islet surface area in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Two-way ANOVA with Tukey’s multiple comparison. Values indicate means ± SEM; each dot represents a mouse (n=4,4). ( N ) Graph representing the average surface area of pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Mann-Whitney test. Values indicate means ± SEM; each dot represents a mouse (n=4,4). ( O ) Curve representing glycemia during a glucose tolerance test in LepR loxP/loxP and LepR TanKO mice, before (black and red curves) and after (light and dark blue curves) i.c.v. leptin injection (2μg/animal). Graph represents the area under the curve; one-way ANOVA with Tukey’s correction. Values indicate means ± SEM. The n number of mice is identical in the main graph and bottom right graph, where each mouse is represented by a dot (n=4,4,6,6). ( P ) Serum insulin concentrations at 15 minutes during the glucose tolerance test presented in (O). A paired two-sided t-test was applied for comparisons between the same group before and after leptin injection and a two-sided unpaired t-test. Values indicate means ± SEM; each dot represents a mouse (n=4,4,6,6).

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Isolation, Expressing, Cell Function Assay, MANN-WHITNEY, Comparison, Injection

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: ( A ) Serum noradrenaline concentrations in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Two-sided unpaired Student’s t-test. Values indicate means ± SEM; each dot represents a mouse (n=7,8). ( B ) Relative mRNA expression levels of adrenergic receptors in isolated pancreatic islets from LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion. Two-sided unpaired Student’s t-test. Values indicate means ± SEM; each dot represents a mouse (n=4 in each group). ( C ) Rectal temperature mesured in LepR loxP/loxP and LepR TanKO mice, 12 weeks after TAT-Cre infusion, before and after 2h cold exposure. Two-sided paired Student’s t-test. Values indicate means ± SEM; each dot represents a mouse (n=8,8,6,6). ( D ) Ratio between the delta temperature after-before cold exposure to the serum noradrenaline concentration after cold exposure from LepR loxP/loxP and LepR TanKO mice. Two-sided unpaired Student’s t-test. Values indicate means ± SEM; each dot represents a mouse (n=8,6).

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Expressing, Isolation, Concentration Assay

Journal: Nature metabolism

Article Title: Leptin brain entry via a tanycytic LepR:EGFR shuttle controls lipid metabolism and pancreas function

doi: 10.1038/s42255-021-00432-5

Figure Lengend Snippet: Knocking out LepR in tanycytes blunts the leptin-mediated neuroendocrine response to fasting

Article Snippet: Twelve-week-old C57Bll6J mice were anesthetized with ketamine+xylazine and injected with the human anti mouse LepR monoclonal XPA antibody (2nmol/animal, #XPA.80.037, XOMA Corporation, Berkeley, California, USA) into the jugular vein.

Techniques: Saline

Journal: Cell Chemical Biology

Article Title: SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET

doi: 10.1016/j.chembiol.2021.06.008

Figure Lengend Snippet: Development of SARS-CoV-2 spike protein/ACE2 TR-FRET binding assay (A) Principle of TR-FRET assay. (B) Scheme illustrating the TR-FRET-based RBD-d2 binding assay to SNAP-tagged ACE2 labeled with Lumi4-Tb. (C) Saturation binding curve of RBD-d2 to Lumi4-Tb-labeled SNAP-ACE2 expressed in HEK293 cells (representative curve, expressed as mean ± SD of triplicates; n = 10). Non-specific binding was defined in the presence of an excess of non-labeled RBD (1 μM). (D and E) Association (D) and dissociation (E) kinetics of RBD-d2 binding (5 nM) to Lumi4-Tb-SNAP-ACE2 expressed in HEK293 cells (representative curve, expressed as mean ± SD of triplicates; n = 4). Dissociation was initiated by adding unlabeled RBD (1 μM). (F) Binding of RBD-d2 (20 nM) to Lumi4-Tb-SNAP-ACE2 but not Lumi4-Tb-SNAP-VEGFR2. Data are expressed as mean ± SEM of three independent experiments, each performed in triplicate. Insert: representative data of the expression level of Lumi4-Tb-labeled SNAP-ACE2 and SNAP-VEGFR2 assessed by Tb fluorescence measurement (620 nm). (G) Competition of RBD-d2 (5 nM) binding to Lumi4-Tb-SNAP-ACE2 by non-labeled RBD (1 μM), leptin, IL-6, MCP1, Gal3, insulin, EGF, VEGF (each at 100 nM). Data are expressed as mean ± SEM of three independent experiments, each performed in triplicate; ∗∗∗∗p = 0.0001 by one-way ANOVA. “RBD-d2 Binding” in (F and G) corresponds to the TR-FRET ratio and is expressed as percent of basal (absence of RBD-d2). (H) RBD-d2 (20 nM) interaction with HEK293 cells expressing either SNAP-ACE2 or SNAP-LepR monitored by fluorescence microscopy. Scale bar, 10 μm. See also Figure S1 .

Article Snippet: Plasmids encoding for SNAP-tagged proteins (human VEGFR2, human LepR) were obtained from Cisbio Bioassays (Codolet, France).

Techniques: Binding Assay, Labeling, Expressing, Fluorescence, Microscopy

Journal: Cell Chemical Biology

Article Title: SARS-COV-2 spike binding to ACE2 in living cells monitored by TR-FRET

doi: 10.1016/j.chembiol.2021.06.008

Figure Lengend Snippet:

Article Snippet: Plasmids encoding for SNAP-tagged proteins (human VEGFR2, human LepR) were obtained from Cisbio Bioassays (Codolet, France).

Techniques: Recombinant, Labeling, Transfection, Plasmid Preparation, Expressing, Software

Journal: Nature medicine

Article Title: Fasting selectively blocks development of acute lymphoblastic leukemia via leptin-receptor upregulation

doi: 10.1038/nm.4252

Figure Lengend Snippet: Fasting and LEPR signaling inhibit human ALL development. (a) LEPR mRNA levels in the indicated types of human lymphoid leukemia and myeloid leukemia samples relative to healthy bone marrow samples (GEO data set GSE13159, n = 2096). (b) Flow cytometry analysis showing surface LEPR on human patient T-ALL (CD3+, n = 5 patients), B-ALL (CD19+, n = 8 patients) and AML (CD33+, n = 52 patients) cells; on human cord blood-derived normal T cell subpopulations including DN (CD3+CD4−CD8−), CD3+CD4−, CD3+CD8−, DP (CD3+CD4+CD8+) and total CD3+ T cells; normal B cell subpopulations including Pro-B (CD19+CD34+CD38+), Pre-B (CD19+CD34+CD40+); immature-B (CD19+CD40+IgM+), mature-B (CD19+IgM+IgD+) and total CD19+ B cells; and myeloid subpopulations including CD33+CD34+ myeloid progenitors and CD33+ myeloid cells (for all normal cell populations, n = 5 healthy donors per group). (c) Overall survival of pediatric patients with pre-B-ALL patients (COG P9906, n = 206) and patients with AML (TCGA, n = 186) relative to LEPR mRNA expression levels (above (high) or below (low) the 50th percentile). (d) Correlation analysis of the expression of LEPR-signaling-related genes (118) and nonrelated genes (20933) with overall survival (OS) and event-free survival (EFS) of patients with B-ALL, or with the overall survival of patients with AML. (e) Correlation analysis of the expression of 29 leptin/LEPR-signaling related genes with OS of patients with AML, or OS and EFS of patients with B-ALL. Positive and negative correlations with patient survival are indicated by red and blue, respectively; genes that are negatively or positively associated with LEPR signaling are indicated in the bar on the far right as green or pink, respectively. (f) Expression of surface LEPR on human BM CD19+ NALM-6 cells, by flow cytometry, in scid mice xenografted with 5 × 106 human B-ALL NALM-6 cells, which were fed or subjected to a 48-h fasting regimen initiated at day 10 after cell injection (n = 5 per group). (g–i) Xenografted mice as in f were fed or subjected to three cycles of 2-d fasting/2-d feeding initiated on day 2 after cell injection, and analyses were performed on day 25. (g) Paraplegia occurrence (n = 5 per group). (h) Percentage of human CD19+ NALM-6 cells in PB, BM and SP, by flow cytometry (n = 5 per group). (i) Survival analysis (n = 5 per group). Data are presented as means ± s.e.m. in a, or as dot plot (means ± s.e.m.) in g, or as box-and-whisker plots (median values (line), 25th–75th percentiles (box outline) and minimum and maximum values (whiskers)) in b, f and h. Statistical significance was calculated by Student’s t-test, *P < 0.05. Statistical significance in d was calculated by the χ-squared test. Statistical significance for survival analysis in c, e and i was calculated by the log–rank test.

Article Snippet: Human patient samples were stained with anti-human CD19-PE (mouse, HIB19, 1:50 dilution, eBioscience), anti-human CD3-PE (mouse, HIT3a, 1:50 dilution eBioscience), anti-human CD33 (mouse, HIM3–4, 1:20 dilution, eBioscience), anti-human CD45 (mouse, HI30, 1:50 dilution, BD Pharmingen), anti-human CD34 (mouse, 4H11, 1:50 dilution, eBioscience), anti-human CD4 (mouse, OKT4, 1:50 dilution, eBioscience), anti-human CD8a (mouse, 1:20 dilution, R&D system), anti-human CD40 (mouse, 5C3, 1:20 dilution, BioLegend), anti-human IgM (mouse, SA-DA4, 1:20 dilution, eBioscience), anti-human IgD (mouse, IA6–2, 1:20 dilution, eBioscience), or anti-human LEPR (mouse, 52263, 1:50 dilution) monoclonal antibodies (BD Pharmingen).

Techniques: Flow Cytometry, Derivative Assay, Expressing, Injection, Whisker Assay

Journal: Nature medicine

Article Title: Fasting selectively blocks development of acute lymphoblastic leukemia via leptin-receptor upregulation

doi: 10.1038/nm.4252

Figure Lengend Snippet: Fasting upregulates LEPR expression and its downstream signaling. (a–h) N-Myc-infected Lin− cells were transplanted into lethally irradiated recipient mice, and a 48-h fast was initiated when the percentage of GFP+ cells in PB reached ~60% (at around 3–4 weeks). Analyses were conducted before initiation of fasting and at days 1, 2 and 5 after initiation of fasting. (a) Representative flow cytometry plots showing GFP and B220 staining in PB of fasted and fed mice at the indicated time points. Numerals in the outlined area indicate the percentage of cells in each quantile. (b) The percentages of GFP+ cells in PB, BM, SP and LV from fasted and fed mice at the indicated time points (n = 5 per group). (c) The percentages of B220+, Annexin V+ and Ki67+ cells in the GFP+ and GFP− compartments from BM of fasted and fed mice (n = 5 per group). (d) The percentages of CD43+, IgM+, Igκ+, Igλ+ and Tdt+ cells in the GFP+B220+ B-ALL cell compartment from BM of fasted and fed mice (n = 5 per group). A representative flow cytometry plot of Tdt staining is also shown (lower right). (e,f) GFP+B220+ B-ALL cells from 1-d (D1) or 2-d (D2) fasted mice, and control mice were sorted for RNA-seq analysis. (e) Fasting induced alterations of signature transcription factors of B cell terminal differentiation, shown for mRNA levels (fasted versus fed, left) and inferred activities (fasted versus fed, right). (f) Fasting-induced alterations in pathways (top) and transcription factor activities (fasted versus fed, bottom). (g) Relative mRNA expression (log2-fold change, FC) of all 42 cytokine receptors in the KEGG pathway database in the sorted cells by qPCR (fasted versus fed, left), the log2 values of their original hazard ratios (HRs) (middle) and their mRNA fold change normalized hazard ratios (log2 HR multiplied by fold change) for survival of human pediatric patients with pre-B-ALL. Up- and downregulation of mRNA levels are indicated by red and green, respectively; HRs below and above 1 are indicated by blue and red, respectively. (h) Representative flow cytometry plots (left and middle) and quantitation (right) of LEPR staining (mean fluorescence intensity, MFI) in GFP+ and GFP− BM cell populations from fasted and fed mice at the indicated time points. (n = 5 per group). (i) Ratio of Lepr mRNA isoforms Ob-Rb to Ob-Ra in GFP+ and GFP− PB cells of fed B-ALL mice, by qPCR (n = 4 per group). (j) Fold change of Ob-Rb mRNA in BM GFP+ cells of fasted mice at the indicat ed time points, by qPCR (n = 5 per group). (k) Quantitation of phospho-STAT3 staining in BM GFP+ cells from fasted mice at the indicated time points (n = 5 per group). (l) Fold change of surface LEPR in the indicated populations of BM cells relative to pre-fast levels (n = 5 per group). (m) Comparison of surface LEPR expression on N-Myc B-ALL cells, Notch1 T-ALL cells and MLL-AF9 AML cells with normal BM B, T and myeloid cell subpopulations: pro-B (B220+IgM−CD43+), Pre-B (B220+IgM−CD43−), immature-B (B220+IgM+IgD−), mature-B (B220+IgM+IgD+) and total B220+ cells (B cell subpopulations); DN (CD3+CD4−CD8−), CD3+CD4−, CD3+CD8−, DP (CD3+CD4+CD8+) and total CD3+ cells (T cell subpopulations); and Mac1+cKit+ myeloid progenitors and total Mac1+ cells (myeloid cell subpopulations) (n = 5 per group). Representative data from three independent experiments are presented as means ± s.e.m. in b–d, h and l, or as box-and-whisker plots (median values (line), 25th–75th percentiles (box outline) and minimum and maximum values (whiskers)) in i–k and m. In statistical significance was calculated by Student’s t-test, *P < 0.05.

Article Snippet: Human patient samples were stained with anti-human CD19-PE (mouse, HIB19, 1:50 dilution, eBioscience), anti-human CD3-PE (mouse, HIT3a, 1:50 dilution eBioscience), anti-human CD33 (mouse, HIM3–4, 1:20 dilution, eBioscience), anti-human CD45 (mouse, HI30, 1:50 dilution, BD Pharmingen), anti-human CD34 (mouse, 4H11, 1:50 dilution, eBioscience), anti-human CD4 (mouse, OKT4, 1:50 dilution, eBioscience), anti-human CD8a (mouse, 1:20 dilution, R&D system), anti-human CD40 (mouse, 5C3, 1:20 dilution, BioLegend), anti-human IgM (mouse, SA-DA4, 1:20 dilution, eBioscience), anti-human IgD (mouse, IA6–2, 1:20 dilution, eBioscience), or anti-human LEPR (mouse, 52263, 1:50 dilution) monoclonal antibodies (BD Pharmingen).

Techniques: Expressing, Infection, Irradiation, Flow Cytometry, Staining, RNA Sequencing Assay, Quantitation Assay, Fluorescence, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Leptin-mediated differential regulation of microsomal triglyceride transfer protein in the intestine and liver affects plasma lipids

doi: 10.1074/jbc.RA119.011881

Figure Lengend Snippet: Tissue-specific deletion of leptin receptors has differential effect on lipids and MTP expression in chow-fed mice. Body weight (A) and average daily food intake (B) of 10-week-old male C57BL/6J Leprf/f (n = 6), I-Lepr−/− (n = 3), and L-Lepr−/− (n = 3) mice on a chow diet were recorded. Mice were sacrificed after 4 h of fasting. Plasma was used to measure the levels of triglycerides (C), total cholesterol (D), HDL cholesterol (E), and non-HDL cholesterol (F). Triglycerides (G and I) and cholesterol (H and J) were also measured in the intestines (G and H) and livers (I and J) of these mice. Intestinal and hepatic tissues were also used to measure the activity (K and M) and the mRNA (L and N) levels of MTP. Data are plotted as mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 were determined by Student's t test.

Article Snippet: Human long-form LEPR (hObRb) plasmid (pRP.ExBi-CMV-LeptinReceptorB-T2A-Luc) was designed through VectorBuilder Inc. (Chicago, IL) using the pRP.Des2d as vector backbone under the CMV promoter.

Techniques: Expressing, Clinical Proteomics, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Leptin-mediated differential regulation of microsomal triglyceride transfer protein in the intestine and liver affects plasma lipids

doi: 10.1074/jbc.RA119.011881

Figure Lengend Snippet: Differential regulation of MTP expression by leptin in enterocytes from Lepr+/+, Leprdb/+, Leprdb/db, and Leprdb3j/db3j mice. Enterocytes were isolated from either WT Lepr+/+ (A), Leprdb/+ (B), Leprdb/db (C), or Leprdb3j/db3j mice (D) (n = 3) and incubated without (PBS) or with leptin (60 nm) for 3 h and used for MTP mRNA quantification. All the expression values were normalized to the related values of Lepr+/+ PBS-treated enterocytes. Data are plotted as mean ± S.D. *, p < 0.05 were determined by Student's t test. The data are representative of 2 independent experiments.

Article Snippet: Human long-form LEPR (hObRb) plasmid (pRP.ExBi-CMV-LeptinReceptorB-T2A-Luc) was designed through VectorBuilder Inc. (Chicago, IL) using the pRP.Des2d as vector backbone under the CMV promoter.

Techniques: Expressing, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Leptin-mediated differential regulation of microsomal triglyceride transfer protein in the intestine and liver affects plasma lipids

doi: 10.1074/jbc.RA119.011881

Figure Lengend Snippet: Leptin decreases MTP expression and apoB secretion in Huh7 cells overexpressing the long-form of leptin receptor. Human long-form of leptin receptor (hObRb) was overexpressed in Huh7 cells for 48 h (A) and then incubated in triplicate with 60 nm leptin for 24 h in serum-free media. Cells were used to measure the mRNA levels of MTP (B). Conditioned media from these cells were used to measure the secreted levels of apoB (C) and apoA1 (D) by ELISA. Data are plotted as mean ± S.D. *, p < 0.05 and ***, p < 0.001 were determined by Student's t test. The data are representative of 2 independent experiments.

Article Snippet: Human long-form LEPR (hObRb) plasmid (pRP.ExBi-CMV-LeptinReceptorB-T2A-Luc) was designed through VectorBuilder Inc. (Chicago, IL) using the pRP.Des2d as vector backbone under the CMV promoter.

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay