Leptin levels were supplemented by twice daily intraperitoneal injections of mouse recombinant leptin (Peprotech, UK; 12.5 μg/g dissolved in saline delivered at 09:00 and 18:00; adapted from (Halaas et al., 1995 (link))) for 10 days; controls received saline.
Mouse recombinant leptin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Mouse recombinant leptin is a protein produced in a laboratory setting that is structurally and functionally similar to the natural leptin hormone found in mice. Leptin is a hormone primarily secreted by fat cells and plays a role in regulating energy balance, appetite, and metabolism.
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6 protocols using mouse recombinant leptin
1
Leptin Supplementation Protocol
2
BV2 Microglial Cells in Normoxia/Hypoxia with Leptin
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The BV2 microglial cell line was purchased from ScienCell (CA, USA) and cultured in MEM medium with 10% FBS and 1% penicillin/streptomycin. For normoxia culture, cells were maintained in a humidified incubator containing 95% air and 5% CO2 at 37°C; for hypoxia culture, cells were maintained in a tri-gas incubator (Thermo Fisher Scientific, USA) with oxygen concentration at 1%. BV2 cells were cultured in the normoxia/hypoxia condition with/without the treatment of mouse recombinant leptin (Peprotech, UK) at the concentration of 1 μM for 24 h.
3
Leptin-Induced Changes in Mouse Brain
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Mice were fasted overnight (16 h) and injected intraperitoneally with mouse recombinant leptin (5 μg/g, PeproTech, 450-31, NJ, USA).38 (link) Forty-five minutes after injection, the animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal) and perfused via transcardial perfusion with 1× PBS followed by ice-cold 4% PFA. Brains were postfixed for 4 h in 4% PFA and cryoprotected in 20% and 30% sucrose in 1× PBS at 4 °C. For immunofluorescence analysis, sections were postfixed for 10 min in 4% PFA and then washed with 1× PBS prior to blocking with 5% normal horse serum/0.25% Triton X-100 in PBS (1 h). Sections were then incubated with PTP1B (ABclonal, A1590, Cambridge, MA, USA) primary antibodies diluted 1:100 in blocking solution overnight. The following day, sections were washed with 1× PBS and subsequently incubated in blocking solution containing secondary antibody for 1 h. Then, the sections were washed with 1× PBS and placed in DAPI staining solution for 10 min. After washing with 1× PBS, the sections were ready for examination.
4
ICV Injection and Hormonal Analysis in Mice
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2–3 days before the experiment, the mice were briefly anesthetized with isoflurane and a small hole was bored in the skull 1 mm lateral and 0.5 mm posterior to bregma with a Hamilton syringe attached to a 27-gauge needle fitted with polyethylene tubing, leaving 3.5 mm of the needle tip exposed. Once the initial hole was made, all subsequent injections were made at the same site. For ICV injections, mice were anesthetized with isoflurane for a total of 2–3 min, during which time 5 μl of solution were slowly and continuously injected into the lateral ventricle. The needle remained inserted for approximately 60 s after the injection to minimize backflow up the needle track. Mice typically recovered from the anesthesia within 3 min after the injection. For hormonal analyses, blood samples (4 µl) were obtained from the tail before and 30 min after injection. Recombinant mouse Leptin (2 ug/5 ul) was purchased from Preprotech. Mouse kisspeptin-10 (Kp-10) (1 nmol/5 ul) was obtained from Phoenix Pharmaceuticals. The drugs were dissolved in saline (0.9% NaCl). The dose and time of collection were selected based on our previous studies (Navarro et al., 2015 (link)).
5
Leptin Treatment in Diabetic Mice
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STZ-diabetic mice at 10 weeks of age received 20 μg/day of recombinant mouse leptin (Peprotech, Rocky Hill, NJ, USA) prepared in sterile water and administered via Alzet 1004 mini-osmotic pumps (DURECT Corporation, Cupertino, CA, USA). HFD/STZ-diabetic mice at 12 weeks of age received 20 μg/day PEG-ylated leptin (Protein Laboratories Rehovot Ltd., Rehovot, Israel) prepared in sterile water by daily i.p. injection while control HFD/STZ mice received water (vehicle).
6
Inhibition of p38 MAPK and PI3K in Leptin Signaling
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The p38 MAPK inhibitor SB 203,580 and PI3K inhibitor LY294002 were obtained from Cell Signaling Technology (Danvers, MA, USA). The leptin antagonist triple mutant (leptA), a mutant leptin analog that functions as a competitive inhibitor, was purchased from PROSPEC (Rehovot, Israel). Recombinant mouse leptin was purchased from PeproTech (Rocky Hill, NJ, USA).
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