Alzet model 1002

The 12-LO inhibitor, CDC and 12(S)-HETE were obtained from Biomol (Plymouth Meeting, PA, USA); p21, p27, AT1R, AMPK, p-AMPK, Ang I/II and ATRAP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); IRS-1, p-IRS-1 (Tyr612), Akt and p-Akt (Tyr308) antibodies and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling (Beverly, MA, USA); β-actin antibody, STZ and insulin were obtained from Sigma (St. Louis, MO, USA); the plasma membrane protein extraction kit and SuperSignal chemiluminescent reagents were obtained from Pierce (Rockford, IL, USA); RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA); SB202190 was obtained from Calbiochem (La Jolla, CA, USA); RNA STAT-60 reagent was obtained from Tel-Test (Friendswood, TX, USA); the RT-PCR kit was obtained from Ambion Inc. (Austin, TX, USA); the Urinary Albumin ELISA Kit was obtained from Exocell Inc. (Philadelphia, PA, USA); the Ang II ELISA kit and the 12(S)-HETE ELISA kit were obtained from Assay Designs (Ann Arbor, MI, USA); the insulin RIA Kit was obtained from Linco Research Inc. (St. Charles, MO, USA); the osmotic mini-pump (Alzet Model 1002) was obtained from Durect Corp. (Cupertino, CA, USA).

After an acclimatisation period of 7 days, mice were subjected to subcutaneous implantation of osmotic mini-pumps (ALZET model 1002, Durect Corporation, Cupertino, California, USA; supplied by Charles River UK Ltd., Kent, UK). After an incision was made on the back, slightly posterior to the scapulae, a distal subcutaneous pocket was created and the pump was located posterior to the right flank. ISO (DL-Isoproterenol hydrochloride, Sigma, St. Louis, MO, USA) was dissolved in 0.9% NaCl at a concentration calculated to deliver 30 μg/g/day for up to 15 days at an infusion rate of 0.25 μl/h. Pumps were filled with either the ISO solution or 0.9% NaCl (vehicle) and activated according to the manufacturer's instructions. During the surgical intervention, mice were anesthetised with 3% isoflurane mixed with 97% O₂ (flow rate 1 L/min) and the mini-pumps were implanted via a 0.5 cm interscapular incision under sterile surgical conditions. The wound was closed with an interrupted suture using a 6–0 silk thread.

Osmotic pumps (Alzet Model 1002, Durect Corporation, Cupertino, CA) were implanted subcutaneously in the particles + cLeptin mice immediately following particle implantation. The dorsal thoracic area was shaved, cleaned, and a one cm incision was made longitudinally across the cleaned region. The osmotic pump was inserted under the skin and the incision was closed with 7 mm surgical staples (Reflex 7 Wound Closure System).