Journal: Cell Research

Article Title: In vivo self-assembled small RNAs as a new generation of RNAi therapeutics

doi: 10.1038/s41422-021-00491-z

Figure Lengend Snippet: a Flow chart of the experimental design. Male C57BL/6J mice at 3 weeks of age were placed on a HFD for 12 weeks. Mice rapidly gained weight and became obese. Mice were then maintained on a HFD and treated with PBS or 5 mg/kg CMV-scrR, CMV-siR P or CMV-RVG-siR P circuit through tail vein injection for a total of 12 times over 24 days. Body weights were monitored during treatment. After treatment, mice were divided into several groups and subjected to the evaluation of fat mass, energy expenditure, leptin sensitivity and glucose homoeostasis. b Body weight curves ( n = 14 in each group). c Weights of epididymal fat pads ( n = 14 in each group). d – k Energy expenditure parameters, including oxygen consumption (VO 2 ), respiratory exchange ratio (RER), total activity and heat production, were monitored ( n = 3 in each group). l , m Weight loss and food intake inhibition in response to leptin. Male mice were injected with leptin (0.5 μg/g body weight every 12 h) for the indicated periods. Food intake and body weight were monitored 2 days prior to the start of injection and normalized to 100% for day 0 values ( n = 6 in each group). n Basal serum leptin levels ( n = 6 in each group). o Western blot analysis of PTP1B in the hypothalamus. Mice were injected with or without leptin (to stimulate leptin signalling), and hypothalamus was collected for immunoblot analysis of PTP1B ( n = 3 in each group). SSG (200 mg/kg i.p. dose) serves as a control. Shown are representative western blots. p Mouse GTT results ( n = 8 in each group). q Mouse ITT results. Blood glucose values are expressed as the percentage of the initial concentration ( n = 8 in each group). r Western blot analysis of PTP1B and tyrosine phosphorylation of insulin receptors in the liver. Mice were injected with or without insulin (to stimulate insulin signalling), and liver was collected for immunoblot analysis with antibodies against PTP1B, p-IR (Tyr1162/Tyr1163) or total IR ( n = 3 in each group). SSG (200 mg/kg i.p. dose) serves as a control. Shown are representative western blots. Values are presented as the means ± SEM. Significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison in c , e , g , i , k , n , and using two-way ANOVA followed by Dunnett’s multiple comparison in b , l , m , p , q . * P < 0.05; ** P < 0.01; *** P < 0.005; ns, not significant.

Article Snippet: Mice were fasted overnight (16 h) and injected intraperitoneally with mouse recombinant leptin (5 μg/g, PeproTech, 450-31, NJ, USA).

Techniques: Injection, Activity Assay, Inhibition, Western Blot, Control, Concentration Assay, Phospho-proteomics, Comparison

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: Leptin regulates gallbladder genes related to absorption and secretion.

doi: 10.1152/ajpgi.00389.2006

Figure Lengend Snippet: Fig. 1. Leptin administration decreased gallbladder volume, bile sodium concentration, and pH.

Article Snippet: For leptin replacement, mice received daily intraperitoneal injections of either recombinant murine leptin (R&D Systems, Minneapolis, MN) at a dose of 5 g/g body wt or saline as a control for 4 wk.

Techniques: Concentration Assay

Journal: Diabetes

Article Title: Liver-Specific Loss of Lipolysis-Stimulated Lipoprotein Receptor Triggers Systemic Hyperlipidemia in Mice

doi: 10.2337/db08-1184

Figure Lengend Snippet: Hepatic LSR expression is repressed in leptin-resistant type 2 diabetes. A–C : Quantitative PCR analysis of cholesterol/bile acid transporter ( A ), fatty acid transporter ( B ), and triglyceride/cholesterol transporter ( C ) mRNA levels in livers of C57BKS (wild-type [wt]) or db/db diabetic (db) mice under fasted (24 h) or refed (24 h fasted, 6 h refed) conditions as indicated ( n = 4) (means ± SE). * P ≤ 0.05; ** P ≤ 0.01. D : Western blot of liver extracts from four representative wild-type or db/db ( upper panel ), ob/ob ( middle panel ), and NZB or NZO ( lower panel ) mice under refed conditions using LSR or VCP antibodies. E : Relative changes in body weight in wt and ob/ob mice treated daily with leptin (5 μg/g body weight) for a period of 21 days (means ± SE). F : Western blot of liver ( upper panel ) and intestinal ( lower panel ) extracts from the same mice as in E using LSR or VCP antibodies. Two representative animals per group are shown. G : Pearson correlation coefficient shown for relative hepatic LSR protein levels versus body weight in the same mice as in E . L, leptin; S, saline.

Article Snippet: For leptin replacement studies, wild-type C57BL/6J and ob/ob mice were daily injected intraperitoneally with recombinant mouse leptin (5 μg/g body weight) (R&D Systems, Wiesbaden, Germany) for 21 days.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Saline

Journal: iScience

Article Title: Allograft inflammatory factor-1-like is a situational regulator of leptin levels, hyperphagia, and obesity

doi: 10.1016/j.isci.2022.105058

Figure Lengend Snippet: The E2a-Cre transgene is integrated in a recognized obesity quantitative trait locus (QTL) on chromosome 6 that includes the leptin gene (A) Targeted locus amplification (TLA) sequence coverage across the mouse genome of the transgenic mice. (B) Precise location and expanded chromosomal region of the transgene integration site.

Article Snippet: Following this PBS administration period, mice were intraperitoneally injected with recombinant leptin (Peprotech, 450–31) with the following regime: 3 μg/g of BW for 2 days followed by 4 μg/g of BW for 2 days followed by 5 μg/g of BW for 2 days.

Techniques: Amplification, Sequencing, Transgenic Assay

Journal: iScience

Article Title: Allograft inflammatory factor-1-like is a situational regulator of leptin levels, hyperphagia, and obesity

doi: 10.1016/j.isci.2022.105058

Figure Lengend Snippet: Cre+ mice on HFD show higher responsiveness to exogenous leptin (A) Circulating leptin levels in serum from 16 weeks HFD-fed WT (n = 11) and Cre+ (n = 9) male mice. (B) Fat mass of WT (n = 11) and Cre+ (n = 9) male mice after 16 weeks of HFD feeding (a subset of this cohort is also a part of Figure 1 C). (C) Cumulative food intake of single housed 16 weeks HFD-fed male mice measured every 24 h in response to daily PBS or leptin administration (WT; n = 6) (Cre+; n = 4). (D) Cumulative food intake of single housed 2 weeks HFD-fed male mice measured every 24 h in response to daily PBS or leptin administration (WT; n = 7) (Cre+; n = 4). (E) Difference in food intake; food intake after leptin administration minus food intake after PBS administration (leptin-PBS) (WT; n = 7) (Cre+; n = 4). (F) Circulating leptin levels in serum from adult WT (n = 15) and Cre+ (n = 24) male mice at baseline on normal diet. Data are represented as mean ± SEM. See also Figures S1 and .

Article Snippet: Following this PBS administration period, mice were intraperitoneally injected with recombinant leptin (Peprotech, 450–31) with the following regime: 3 μg/g of BW for 2 days followed by 4 μg/g of BW for 2 days followed by 5 μg/g of BW for 2 days.

Techniques:

Journal: iScience

Article Title: Allograft inflammatory factor-1-like is a situational regulator of leptin levels, hyperphagia, and obesity

doi: 10.1016/j.isci.2022.105058

Figure Lengend Snippet: Concurrent loss of allograft inflammatory factor-1-like (AIF1L) in Cre+ mice reduces circulating leptin levels after short-term HFD feeding (A) AIF1L protein expression in eWAT using immunoblotting in 8–9.5 weeks old WT and Cre+ mice, at baseline on normal diet (n = 7). (B) Circulating leptin levels in serum from adult Cre+ (n = 24) and Aif1l flox ;Cre+ (n = 17) male mice at baseline (Repetition of Cre+ mice data from Figure 3 F, for a new comparison) and 2 weeks HFD-fed Cre+ (n = 12) and Aif1l flox ;Cre+ (n = 11) male mice. (C) Difference in food intake; food intake after leptin administration minus food intake after PBS administration (leptin-PBS) ( Aif1l flox ;Cre+; n = 5) (Repetition of WT and Cre+ mice data from Figure 3 E, for a new comparison). Data are represented as mean ± SEM. See also Figures S1 and .

Article Snippet: Following this PBS administration period, mice were intraperitoneally injected with recombinant leptin (Peprotech, 450–31) with the following regime: 3 μg/g of BW for 2 days followed by 4 μg/g of BW for 2 days followed by 5 μg/g of BW for 2 days.

Techniques: Expressing, Western Blot, Comparison

Journal: iScience

Article Title: Allograft inflammatory factor-1-like is a situational regulator of leptin levels, hyperphagia, and obesity

doi: 10.1016/j.isci.2022.105058

Figure Lengend Snippet:

Article Snippet: Following this PBS administration period, mice were intraperitoneally injected with recombinant leptin (Peprotech, 450–31) with the following regime: 3 μg/g of BW for 2 days followed by 4 μg/g of BW for 2 days followed by 5 μg/g of BW for 2 days.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software

Journal: Nature Communications

Article Title: Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus

doi: 10.1038/s41467-020-19638-4

Figure Lengend Snippet: a Cilia (AC3) staining in the hypothalamic ARH and hippocampal dentate gyrus of C57 neonates at P14 that received either saline or leptin antagonist SHLA injections from P4 to P13. The average lengths of 100 cilia and the ciliated cell percentage in each area per mouse are presented ( n = 5 for hypothalamus, n = 4 for hippocampus). Scale bars: 20 μm. b Cilia staining in the hypothalamus (Hypo) and hippocampus (Hippo) of ob/+ mice, ob/ob mice, and ob/ob mice with leptin treatment (P10–P14) ( n = 5). The average lengths of 100 cilia in each area per mouse are presented. Scale bars: 20 μm. c Upper panel: cilia (AC3) and POMC (β-END) costaining in the hypothalamic ARH in 7-week-old ob/+ mice, ob/ob mice, and ob/ob mice with adulthood leptin replacement (10 mg/kg/day for 7 days; n = 5). Lower panel: axonal projections of POMC neurons in the three groups ( n = 4). Scale bars: 50 μm. d Ciliogenesis in the ARH and POMC axonal projection to the PVH in ob/ob neonates injected with saline, leptin alone, or leptin + shIFT88-GFP-AAV ( n = 4 for AAV on-target group, n = 5 for the other 3 groups). The mice with off-target AAV injection were used as a control. Scale bars: 50 μm. e AC3 (cilia), functional leptin receptor (LepRb), GFP (AAV transfection), and DAPI costaining in the ARH of ob/ob neonates injected with saline, leptin, or leptin + shIFT88-GFP-AAV. The percentages of ARH cells and AAV-infected cells with periciliary leptin receptor accumulation were counted ( n = 5 for the ob/ob-saline group and n = 4 for the other 2 groups). Scale bars: 10 μm. Data values are presented as mean ± SEM. Statistics were performed using two-sided Student’s t test ( a ) and one-sided one-way ANOVA ( b – e ) followed by post hoc LSD test. ** p < 0.01, *** p < 0.001 between the indicated groups. ns not significant.

Article Snippet: Conversely, to inhibit postnatal leptin action, C57 neonates received intraperitoneal injections of the leptin antagonist SHLA (3 mg/kg/day, MyBioSource).

Techniques: Staining, Saline, Injection, Control, Functional Assay, Transfection, Infection

Journal: Nature Communications

Article Title: Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus

doi: 10.1038/s41467-020-19638-4

Figure Lengend Snippet: a Representative images of DQ-BSA/β-END staining and LAMP1/DAPI staining in the hypothalamic ARH of ob/+ and ob/ob mice at P14 ( n = 5). The graphs depict the average values of DQ-BSA fluorescence intensity (lysosomal protein degradation), LAMP1 intensity (lysosomal mass) and LAMP1 + puncta size (lysosomal size) in 100 ARH cells per mouse. Scale bars: 50 μm. 3V third ventricle. b DQ-BSA/β-END and LAMP1/DAPI staining in the ARH of ob/ob mice injected with saline, leptin alone, or leptin + shIFT88-GFP-AAV ( n = 5). The average values of DQ-BSA and LAMP1 intensity and puncta size in 100 ARH cells per mice are presented. Scale bars: 50 μm. c DQ-BSA fluorescence intensity in N1 cells transfected with GFP-AAV or shIFT88-GFP-AAV and treated with leptin (100 nM for 1 h) ( n = 4 wells for GFP-AAV, n = 5 wells for shIFT88-GFP-AAV). The DQ-BSA fluorescence intensity of 50 GFP + cells per well was analysed. Scale bars: 50 μm. d TFEB/DAPI double staining, as a measure of lysosomal biogenesis, in the ARH of ob/+ mice with saline and ob/ob mice with saline or leptin treatment ( n = 4 for ob/+ saline, n = 5 for ob/ob saline, and n = 6 for ob/ob leptin). Asterisks indicate cells with nuclear TFEB expression. Scale bars: 10 μm. e Lysosomal staining using LysoTracker and pH-sensitive LysoSensor dyes in N1 cells with or without leptin treatment ( n = 4 wells). The average fluorescent intensity of 100 cells per well was analysed. Scale bars: 50 μm. f The vacuolar H + -ATPase (v-ATPase) activity in N1 cells treated with leptin alone or leptin with lysosomal v-ATPase inhibitor bafilomycin ( n = 4 wells). g The v-ATPase activity in N1 cells transfected with either control small inhibitory RNA (siRNA) or IFT88 siRNA and then treated with leptin ( n = 4 wells). Data values are presented as mean ± SEM. Statistics were performed using two-sided Student’s t test ( a , e , g ) and one-sided one-way ANOVA ( b – d , f ) followed by post hoc LSD test. * p < 0.05 and *** p < 0.001 between the indicated groups. ns not significant.

Article Snippet: Conversely, to inhibit postnatal leptin action, C57 neonates received intraperitoneal injections of the leptin antagonist SHLA (3 mg/kg/day, MyBioSource).

Techniques: Staining, Fluorescence, Injection, Saline, Transfection, Double Staining, Expressing, Activity Assay, Control

Journal: Nature Communications

Article Title: Primary cilia mediate early life programming of adiposity through lysosomal regulation in the developing mouse hypothalamus

doi: 10.1038/s41467-020-19638-4

Figure Lengend Snippet: a Hypothalamic cilia images in the neonatal offspring (at P14) of dams fed a high-fat diet (HFD) or low protein diet (LPD) during gestation and lactation or during lactation ( n = 5 for M-CD, n = 4 for M-HFD and M-LPD). The average lengths of 100 ARH cilia per mouse are presented. Scale bars: 50 μm. b Changes in body weights, fat and lean mass, cumulated food intake, and energy expenditure (at 4 weeks) during the post-weaning period in the offspring of CD- or LPD-fed dams ( n = 4 for food intake monitoring, n = 8 for other measurement). c DQ-BSA/β-END and LAMP1/DAPI double staining in the hypothalamic ARH of offspring of dams on a HFD or LPD ( n = 5). The average values of 100 ARH cells per mouse are presented. Scale bars: 50 μm. d Plasma leptin concentrations in the offspring of CD-, HFD-, or LPD-fed dams during lactation at P10 ( n = 7). e Hypothalamic cilia staining (AC3), lysosomal protein degradation (DQ-BSA/β-END staining) in the ARH, and POMC axonal projection in the PVH in the pups nourished by dams on a HFD or LPD and received either saline or leptin injections during P4–P13 ( n = 5 for DQ-BSA study and n = 6 for all other analyses). Scale bars: 20 and 100 μm (axonal projections). f Hypothalamic leptin receptor ( Leprb ) mRNA expression in the offspring of CD-, LPD-, or HFD-fed dams at P14 ( n = 6 for M-CD, n = 5 for the other 2 groups). Data values are presented as mean ± SEM. Statistics were performed using one-sided one-way ANOVA ( a , c , d , f ) and one-sided two-way ANOVA ( b ) followed by post hoc LSD test and two-sided Student’s t test ( b —energy expenditure, e ). * p < 0.05, ** p < 0.01, and *** p < 0.001 between the indicated groups. ns not significant.

Article Snippet: Conversely, to inhibit postnatal leptin action, C57 neonates received intraperitoneal injections of the leptin antagonist SHLA (3 mg/kg/day, MyBioSource).

Techniques: Double Staining, Clinical Proteomics, Staining, Saline, Expressing