Alzet micro osmotic pump model 1004

After 2 weeks of diet treatment (NFD or HFD), mice received subcutaneous osmotic minipump implants (Alzet Micro-Osmotic Pump Model 1004, DURECT, Cupertino, CA, USA) administering the PRO20 peptide (LPTRTATFERIPLKKMPSVREI) or scrambled peptide control (LRTETPITMIPSAERVFRKKPL) at 700 μg/kg/d for the remaining 4 weeks of treatment. For the MCD diet, mice received a similar osmotic minipump implant (Alzet Micro-Osmotic Pump Model 2006, DURECT, Cupertino, CA, USA) after 2 weeks of MCD treatment that delivered the same dose of PRO20 or scrambled peptide over the remaining 6 weeks of diet treatment. Mice were anesthetized by isoflurane inhalation and then subcutaneously implanted with osmotic minipumps infusing PRO20 or scrambled (control) peptide. A separate study using losartan, an angiotensin II type 1a receptor (AT1aR) blocker, was performed using the aforementioned 6-week HFD and NFD diet treatment regimen. Specifically, after 2 weeks of diet treatment, mice received subcutaneous osmotic minipump implants that delivered either losartan (10 mg/kg/d) or 0.9% saline (control) for the subsequent 4 weeks. All animals were housed singly in standard forced-air shoebox cages and were maintained on their respective diets until the end of the treatment period.