Journal: Nature Cell Biology

Article Title: mTORC2–NDRG1–CDC42 axis couples fasting to mitochondrial fission

doi: 10.1038/s41556-023-01163-3

Figure Lengend Snippet: (a) Experimental plan for generation of insulin-deficient diabetic mice using streptozotocin (STZ). (b) Serum insulin levels in 3 mo-old STZ-treated C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. N values for number of mice in each group are in parenthesis. Fed (n = 7); fed + STZ (n = 7); fed + corn oil (n = 8); fed + STZ + corn oil (n = 8); fasted (n = 8); fasted + STZ (n = 8); fasted + corn oil (n = 8); fasted + STZ + corn oil (n = 8). (c) Blood glucose levels 6 weeks after the first injection with STZ (n = 16 mice). (d) Representative IB, and (e-g) quantification for indicated proteins normalized to corresponding total protein in livers of 3 mo-old STZ-treated C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. For e , fed (n = 6); fed + STZ (n = 6); fed + corn oil (n = 6); fed + STZ + corn oil (n = 6); fasted (n = 5); fasted + STZ (n = 6); fasted + corn oil (n = 5); fasted + STZ + corn oil (n = 5). For f and g , all indicated groups consist of n = 5 mice each. (h, i) Serum (h) leptin and (i) IGF-1 levels in 3 mo-old C57BL/6 male mice that were fed or fasted for 14–16 h and then gavaged with corn oil for 30 min. For h , fed (n = 8); fed + corn oil (n = 8); fasted (n = 6); fasted + corn oil (n = 9). For i , all indicated groups consist of n = 9 mice each. (j) IB and (k) quantification for the indicated proteins in livers of 2–10 mo-old mice fed or fasted for the indicated durations. N values for number of mice analyzed at each time-point for individual phosphoproteins are indicated in parentheses. P-PKA Thr197 /PKA: 0 h (n = 3), 3 h-20 h (n = 5); P-PKCα/βII Thr638/641 /PKCα (all time-points are n = 5); P-PKCδ Thr505 /PKCδ (all time-points are n = 5); P-PKCδ/θ Ser643/676 /PKCδ (all time-points are n = 5); and P-PKCζ/λ Thr410/403 /PKCζ: 0 h (n = 4), 3 h-20 h (n = 5). Ponceau is the loading control. Individual replicates and mean values are shown. *P < 0.05, **P < 0.01 and ****P < 0.0001, 2-way ANOVA followed by Tukey’s multiple comparisons test (b, e, f, h and i) ; two-tailed unpaired Student’s t-test (c) . ns=not significant. Please refer to Supplementary Table _statistical summary. Source numerical data are available in SourceData_Table 1, and unprocessed blots are available in Source Data Extended Data Fig. .

Article Snippet: Serum insulin (ALPCO, 80-INSNS-E01), leptin (R&D Systems, DY49805), IGF-1 (R&D Systems, DY791), FFAs (FUJIFILM, NEFA-HR (2)), serum triglyceride (Sigma-Aldrich, T2449, F6428) and liver triglycerides (BioVision, K622) were evaluated as per the manufacturer’s instructions.

Techniques: Injection, Control, Two Tailed Test

Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 6. Molecular models of binding site II in mouse leptin. (A) Molecular surface map of leptin, coloured according to surface hydrophobicity (blue, hydrophilic; green, hydrophobic). A hydrophobic cleft is formed by residues L13, L86, L89 and F92. (B) Residues in binding site II that affect binding to CRH2 are coloured yellow. Residues in binding site II that affect both binding to CRH2 and LR activation are coloured orange. (C) Residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2).

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques: Binding Assay, Activation Assay

Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 9. Model of the mouse leptin/CRH2 complex. The molecular surface of leptin is coloured according to the surface hydrophobicity (blue, hydrophilic; green, hydrophobic). The CRH2 model is presented as ribbons, the Cα atom and heavy side chain atoms of residues I501, F502, L503, L504S and D615 are displayed as white sticks. L504 of CRH2 fits into the hydrophobic cleft of leptin and interacts with L13 and L86 of leptin.

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques:

Journal: Journal of cell science

Article Title: Mapping of the interface between leptin and the leptin receptor CRH2 domain.

doi: 10.1242/jcs.02386

Figure Lengend Snippet: Fig. 10. Molecular models of mouse LR CRH2. (A) CRH2 residues that become buried in the leptin/CRH2 interface, coloured according to the area that becomes buried (cyan, <25 Å2; blue, 25-50 Å2; green, >50 Å2). (B) CRH2 residues that lead to a drastic increase of leptin binding to CRH2 and LR activation upon mutation.

Article Snippet: Expression was checked by anti-HA western blot analysis and quantified using an anti-mouse leptin ELISA kit (R&D systems) (Peelman et al., 2004b).

Techniques: Binding Assay, Activation Assay, Mutagenesis

Journal: Molecular psychiatry

Article Title: Leptin enhances social motivation and reverses chronic unpredictable stress-induced social anhedonia during adolescence.

doi: 10.1038/s41380-022-01778-2

Figure Lengend Snippet: Fig. 2 Leptin enhances social reward-conditioned place preference under non-stressed conditions in adolescent male mice. a Schematic diagram of the experimental design in saline control and leptin-injected (1.0 mg/kg) mice. Illustrations were created with BioRender.com. b Time spent in social bedding shown in individual mice. c Average time spent in the compartment with social bedding. d Normalized social preference. e Subtracted social preference. Saline treatment, n = 8 male mice; leptin treatment, n = 7 male mice. *P < 0.05, **P < 0.01, ***P < 0.001 compared with pre-conditioning or saline-treated group.

Article Snippet: Recombinant mouse leptin (#498-OB, R&D Systems, Minneapolis, MN, USA) was dissolved in sterile saline at a concentration of 1.0 mg/ml and administered intraperitoneally (i.p.) at a dose of 1.0 mg/kg or 5.0 mg/kg body weight.

Techniques: Conditioned Place Preference, Saline, Control, Injection

Journal: Molecular psychiatry

Article Title: Leptin enhances social motivation and reverses chronic unpredictable stress-induced social anhedonia during adolescence.

doi: 10.1038/s41380-022-01778-2

Figure Lengend Snippet: Fig. 3 Leptin reverses chronic unpredictable stress (CUS)-induced social anhedonia in adolescent male mice. a Schematic diagram of the experimental design in saline control and leptin-injected (1.0 mg/kg) mice. Illustrations were created with BioRender.com. b Time spent in the compartment with social bedding. c Average time spent in the compartment with social bedding. d Normalized social preference. e Subtracted social preference. Saline treatment, n = 12 male mice; leptin treatment, n = 14 male mice. *P < 0.05, **P < 0.01 compared with pre-conditioning or saline-treated group; ns, no statistical significance.

Article Snippet: Recombinant mouse leptin (#498-OB, R&D Systems, Minneapolis, MN, USA) was dissolved in sterile saline at a concentration of 1.0 mg/ml and administered intraperitoneally (i.p.) at a dose of 1.0 mg/kg or 5.0 mg/kg body weight.

Techniques: Saline, Control, Injection

Journal: Molecular psychiatry

Article Title: Leptin enhances social motivation and reverses chronic unpredictable stress-induced social anhedonia during adolescence.

doi: 10.1038/s41380-022-01778-2

Figure Lengend Snippet: Fig. 4 Effects of leptin treatment on social reward and isolation aversion in male mice. a Schematic diagram of the social reward experimental design in saline control and leptin-injected (1.0 mg/kg) mice. Illustrations were created with BioRender.com. b Time spent in the compartment with social bedding. c Left: average time spent in the compartment with social bedding; right: time spent in social bedding in post-trial normalized by pre-trial. d Body weight. Saline, n = 10 male mice; leptin, n = 12 male mice. e Schematic diagram of the isolation aversion experimental design in saline control and leptin-injected (1.0 mg/kg) mice. f Time spent in the compartment with isolate bedding. g Left: average time spent in the compartment with isolate bedding; right: time spent in isolate bedding in post-trial normalized by pre-trial. h Body weight. Saline, n = 8 male mice; leptin, n = 9 male mice. *P < 0.05, **P < 0.01 compared with pre-conditioning or saline-treated group; ns, no statistical significance.

Article Snippet: Recombinant mouse leptin (#498-OB, R&D Systems, Minneapolis, MN, USA) was dissolved in sterile saline at a concentration of 1.0 mg/ml and administered intraperitoneally (i.p.) at a dose of 1.0 mg/kg or 5.0 mg/kg body weight.

Techniques: Isolation, Saline, Control, Injection

Journal: Molecular psychiatry

Article Title: Leptin enhances social motivation and reverses chronic unpredictable stress-induced social anhedonia during adolescence.

doi: 10.1038/s41380-022-01778-2

Figure Lengend Snippet: Fig. 5 Leptin induces oxytocin expression in the paraventricular nucleus (PVN) of the hypothalamus in male mice. a Distribution of leptin receptor (LepRb)-expressing cells (red) and oxytocin-immunoreactive neurons (green) in the different levels of PVN. Scale bars, 500 µm for low magnification and 100 µm for high magnification. b High-magnification image showing close apposition of puncta of LepRb-expressing neurons with oxytocin-positive neurons. Scale bar, 10 µm. c Oxytocin RNA expression levels 15 min after i.p. leptin injection. Left, hnRNA expression levels; right, mRNA expression levels. n = 8 male mice per group. **P < 0.01 compared with saline-treated group.

Article Snippet: Recombinant mouse leptin (#498-OB, R&D Systems, Minneapolis, MN, USA) was dissolved in sterile saline at a concentration of 1.0 mg/ml and administered intraperitoneally (i.p.) at a dose of 1.0 mg/kg or 5.0 mg/kg body weight.

Techniques: Expressing, RNA Expression, Injection, Saline

Journal: Molecular psychiatry

Article Title: Leptin enhances social motivation and reverses chronic unpredictable stress-induced social anhedonia during adolescence.

doi: 10.1038/s41380-022-01778-2

Figure Lengend Snippet: Fig. 6 Blockade of oxytocin receptors abolishes leptin-induced enhancement of social preference. a Schematic diagram of the experimental design. Illustrations were created with BioRender.com. b Representative images of heat maps depict the time spent by the mice from three treatment groups in the chamber containing social bedding versus the chamber containing isolate bedding during the pre- conditioning trial (upper panel) and post-conditioning trial (lower panel). c Time spent in the compartment with social bedding. d Average time spent in the compartment with social bedding. e Normalized social preference. f Subtracted social preference. Saline + Saline, n = 10 male mice; Saline + Leptin, n = 13 male mice; OTR-A + Leptin, n = 10 male mice. *P < 0.05, **P < 0.01 compared with relative control groups; ns, no statistical significance. OTR-A, oxytocin receptor antagonist L-368,899.

Article Snippet: Recombinant mouse leptin (#498-OB, R&D Systems, Minneapolis, MN, USA) was dissolved in sterile saline at a concentration of 1.0 mg/ml and administered intraperitoneally (i.p.) at a dose of 1.0 mg/kg or 5.0 mg/kg body weight.

Techniques: Saline, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 4. Inhibition of mTOR with rapamycin inhibits the expression of leptin and LepR in Teffs and at the systemic level. (A) Proliferation of hu- man Teffs, pretreated or not with rapamycin or anti- leptin neutralizing mAb or both, before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 20). *p , 0.005, **p , 0.0001. (B) Proliferation of Teffs, pretreated or not with rapamycin before treatment with leptin (100 ng/ml), before anti-CD3/ CD28 stimulation. Data are mean 6 SD (n = 10). *p , 0.005, **p , 0.0001. (C) Confocal micros- copy of human Teffs, pretreated or not with rapamycin in the presence of anti-CD3/CD28 stimulation, and stained for leptin (green) and LepR (red). Representative of three independent experi- ments. Immunofluorescence images were acquired in the green, red, and blue channels at a resolution of 1024 3 1024 pixels. (D) Real-time PCR for leptin in human Teffs, pretreated or not with rapa- mycin, in the presence of anti-CD3/CD28 stimu- lation. Data are mean 6 SD (n = 5). *p , 0.001. (E) Serum leptin levels in patients with acquired cystic kidney disease, chronically treated with rapamycin, analyzed after 6 and 12 mo of mTOR inhibition treatment (black line). BMI in all of the treated patients at the different time points is represented by the gray bars. Data are mean 6 SD (n = 15 patients/group). *p , 0.05. (F) Serum leptin levels in vehicle or rapamycin-treated mice analyzed at different time points. Data are mean 6 SD (n = 5 mice/group). *p , 0.05.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Inhibition, Expressing, Staining, Real-time Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 5. Leptin activates the mTOR pathway and controls Teff pro- liferation. (A) Immunoblot for p-STAT3, p-AKT, and p-S6 on Baf/3 cells stably transfected with the long form of human leptin receptor, treated or not with leptin for 30 or 60 min, in the presence or absence of LY294002 or rapamycin. One representative of three independent experiments. (B) Im- munoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on unstimulated Teffs treated or not with recombinant leptin for 1 h. One representative of five independent experiments. (C) Immunoblot for p-mTOR, p-p70S6K, p-S6, and p-STAT3 on Teffs in the presence or absence of anti-CD3/28 stimula- tion and treated or not with recombinant leptin or leptin-neutralizing mAb for 1 h. One representative of five independent experiments. (D) Ex vivo p-S6 expression in Teffs from db/+ mice or db/db mice (n = 3 mice/ group, representative of three independent experiments). (E) Ex vivo p-S6 expression in Teffs from ob/ob mice treated or not with recombinant leptin after 2 h from i.p. injection (n = 3 mice/group, representative of three independent experiments). (F) Flow cytometry for BrdU incorpo- ration in Teffs from the lymph nodes of ad libitum fed, 48-h fasted, and 48-h fasted + leptin eGFP-Foxp3 mice. Representative of two independent experiments (n = 3). The gray shaded graph represents the isotype-matched negative control. *p , 0.05 versus 48-h fasted mice, **p , 0.001 versus ad

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Western Blot, Stable Transfection, Transfection, Recombinant, Ex Vivo, Expressing, Injection, Flow Cytometry, Negative Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 6. In vivo leptin enhances Teff proliferation through mTOR activation. (A) Schematic model of the experimental design. Briefly, mice were treated daily with BrdU in basal conditions and upon Ag immunization with CFA; they were injected with a single dose of rapamycin or vehicle or leptin or rapamycin plus leptin 12 h before CFA priming, and the proliferation of Teffs was followed over time. Blood samples were obtained at day 5, and draining lymph nodes were harvested at days 8 and 12. Percentage (B) and absolute number (C) of Teffs gated on CD4+ cells in the lymph nodes from all of the groups of mice immunized with CFA. Data are mean 6 SD (n = 6). *p , 0.05, **p , 0.001. (D) Flow cytometry for BrdU incorporation in Teffs from the lymph nodes of mice pretreated in vivo with vehicle, rapamycin (RAPA), leptin, or RAPA and leptin 12 d after immunization with CFA. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle. No significant difference between vehicle versus leptin or vehicle versus RAPA + leptin was detected. The gray shaded graph represents the isotype-matched negative control. (E) p-S6 expression in Teffs from lymph nodes of all groups of mice. Representative of three independent experiments (n = 3 mice/group). *p , 0.005 versus RAPA pretreatment, **p , 0.001 versus vehicle.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, BrdU Incorporation Assay, Negative Control, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Leptin-induced mTOR activation defines a specific molecular and transcriptional signature controlling CD4+ effector T cell responses.

doi: 10.4049/jimmunol.1200935

Figure Lengend Snippet: FIGURE 8. Schematic model of leptin-induced mTOR activation in CD4+CD252FOXP32 Teffs. Under normal conditions (left panel), leptin, by binding its receptor (LepR), activates the mTOR pathway in CD4+CD252FOXP32 Teffs, resulting in an increase in T cell proliferation, Th1/Th17 cytokine se- cretion, and TCR signaling activation. The mTOR pathway itself is responsible for the autocrine leptin secretion by Teffs, which, in turn, sustains their own proliferation. Rapamycin pretreatment (middle) inhibits activation of the leptin-mediated mTOR pathway, resulting in the inhibition of Teff proliferation, a decrease in Th1/Th17 cytokine production, and TCR activation. Moreover, mTOR inhibition deceases leptin production and secretion by Teffs. Similar results can be detected in LepR-deficient Teffs, in which mTOR-pathway activity is impaired because of a lack of proper leptin signaling. These data sustain the hypothesis that rapamycin pretreatment and LepR deficiency share a common cellular, biochemical, and gene-expression profile, suggesting the presence of a convergence between leptin and mTOR at the signaling-pathway level to drive and control Teff responses.

Article Snippet: For fasting experiments, eGFP-Foxp3 mice, treated daily with BrdU (1 mg/mouse), were fasted for 48 h in the presence or absence of exogenous mouse recombinant leptin (R&D Systems) dissolved in 200 ml PBS at a dose of 1 mg/g initial body weight twice daily at 9 AM and at G eorge W ashington U niversity M edical C enter L ibrar on February 16, 2015 http://w w w .jim m unol.org/ D ow nloaded from 6 PM; they were compared with mice fed ad libitum (24).

Techniques: Activation Assay, Binding Assay, Inhibition, Activity Assay, Gene Expression, Control

Journal: PLoS Genetics

Article Title: Congenital lipodystrophy induces severe osteosclerosis

doi: 10.1371/journal.pgen.1008244

Figure Lengend Snippet: A) Transplanted fat depots 3 months after surgery. B) Fat depot weight 3 months after transplantation. C) Serum leptin and adiponectin of FF mice 3 months after fat depot transplantation. WT and non-transplanted FF mice serve as control. D) μCT analysis of trabecular bone volume and bone mineral density of distal femurs of FF mice 3 months after sham operation or transplantation of fat derived from WT or adipokine-deficient mice. Data are presented as mean ± SD. **p<0.01; *** p<0.001 as determined by ANOVA with Holm-Sidak's post hoc analysis for multiple comparisons test. D) comparison with FF Sham except where detailed.

Article Snippet: The primary antibody cocktail contained rat anti-mouse CD45-BUV395 (BD Horizon, clone 30-F11, final dilution factor 1:200), rat anti-mouse TER-119-APC (BioLegend, clone TER-119, 1:200), rat anti-mouse CD41-BV421 (BioLegend, clone MWReg30, 1:300), rat anti-mouse/human CD11b (BioLegend, clone M1/70, 1:400), and rat-anti mouse Leptin receptor (LepR)-biotin (R&D Systems, polyclonal, 1:50) in Brilliant Stain Buffer (BD Biosciences) containing 10 μg/mL FcBlock.

Techniques: Transplantation Assay, Control, Derivative Assay, Comparison

Journal: PloS one

Article Title: Reduced body weight and increased energy expenditure in transgenic mice over-expressing soluble leptin receptor.

doi: 10.1371/journal.pone.0011669

Figure Lengend Snippet: Figure 1. Structure of the OBRe transgene and its expression in hSAP-OBRe mice. (A) Full-length mouse OBRe cDNA and poly-adenylation signal, flanked by rabbit b-globin introns, were placed downstream of the hSAP component promoter. The size of each fragment is indicated in the diagram. Hind III and Sal I were used to linearize the transgene fragment before the microinjection procedure. (B) A representative Western blot (OBRe) of blood plasma from a 20- to 24-week old male hSAP-OBRe transgenic mouse and its littermate control. Below the Western blot is the relative densitometric quantification of OBRe from 4 independent pairs of hSAP-OBRe transgenic and control mouse plasma samples. (C) Quantification of soluble leptin receptors from plasma samples of male hSAP-OBRe transgenic (N = 13) and control (N = 11) mice using ELISA. Data are presented as means 6 SEM. **, P,0.01. a.u.: arbitrary units. doi:10.1371/journal.pone.0011669.g001

Article Snippet: Quantification of plasma OBRe OBRe concentration in plasma was measured by Mouse Leptin R DuoSet ELISA assay (R&D Systems) with a detection limit of 15.6 pg/ml.

Techniques: Expressing, Microinjection, Western Blot, Clinical Proteomics, Transgenic Assay, Control, Enzyme-linked Immunosorbent Assay