Corticosterone

Corticosterone, picrotoxin, and ketoprofen were purchased from Cayman Chemical. FITC-AffiniPure Donkey Anti-Mouse IgG (H + L) and AlexaFluor-594-IgG Fraction Monoclonal Mouse Anti-Biotin were purchased from Jackson ImmunoResearch Laboratories. TTX was purchased from the Research Institute of the Aquatic Products of Hebei. All other regents and drugs were purchased from Sigma Millipore, Tocris Bioscience, or BBI Life Sciences.

We measured glucose, body weight, and food intake every 5 days as previously described (1 (link), 6 (link), 8 (link)). We plotted survival to determine if LEPRs in RIP-Cre^25Mgn neurons are required or sufficient for leptin's capacity to reduce lethality in insulin-deficient mice. Free fatty acids (FFAs), ketone bodies, TG, and glycerol in blood were measured by commercially available kits (Wako diagnose, US; and Cayman US for glycerol) as described previously (1 (link)). Corticosterone (Cayman, US) and glucagon (#10-1271-01, Mercodia, US) in the plasma were measured by commercially available ELISA kits.

Following anesthesia using Zoletil (25 mg/kg, Zoletil 50; Virbac, Cedex, France), the mice blood and brains were collected. Plasma was separated by centrifugation at at 4°C for 10 min (1,000 ×g). Plasma was stored at -80°C before use. Whole brain of each mouse was weighed and immediately homogenized on ice with 3-mL phosphate-buffered saline (PBS). The resulting homogenate was centrifuged at 4°C for 5 min (5,000 ×g). The supernatant was then used to determine serotonin levels. The plasma levels of corticosterone (Cayman Chemical Company, Ann Arbor, MI, USA) and the brain levels of serotonin were determined using enzyme-linked immunosorbent assay (ELISA) kits (Abcam, Cambridge, UK).

Mice were randomly divided into 8 groups with 8 animals each and the grouping was the same as the acute restraint stress test. All treatments were given p.o. for 21 days. After that, all mice except for group 1 (nonstress control) were exposed to cold restraint stress (placed under 4°C for 1 hour) continuously for another 7 days. Treatments were continued during this period. On the last day of the experiment, mice were treated, exposed to cold restraint stress, and sacrificed and blood was collected to obtain serum for determination of glucose, total cholesterol, triglyceride (TG) (Biovision, USA), and corticosterone (Cayman, USA) level according to the manufacturers' protocol. Moreover, adrenal gland was removed and weighted. Brains were frozen in liquid nitrogen and homogenized in mixture of HCl (0.01 N) and butanol. The aqueous phase acid extract was recovered by centrifugation and concentrations of dopamine (DA) (IBL, Hamburg, Germany) and 5-hydroxytryptamine (5-HT) (IBL, Hamburg, Germany) were quantified using ELISA [15 (link)] while brain superoxide dismutase (SOD) and malondialdehyde (MDA) levels were quantified according to the literature [16 ].

A total of 32 mice were randomly divided into four groups (n=8 per group), namely the no-stress control and the untreated control, positive control and VCO stress groups, which were administered 250 μl saline, 2 mg/kg bw diazepam and 10 ml/kg bw VCO, respectively, p.o. for a total of 28 days. From day 21 onward, the mice in the untreated control, positive control and VCO stress groups were daily subjected to 1 h of cold restraint stress at 4°C for a total of 7 days. At the end of the experiment, the mice were sacrificed via isoflurane and their sera were collected and subjected to total cholesterol, TG, total protein, glucose (all kits from BioVision, Mountain View, CA, USA) and corticosterone (Cayman Chemical Company, Ann Arbor, MI, USA) determination, according to the manufacturer’s instructions. The adrenal glands were also removed and weighed. The mouse brains were frozen in liquid nitrogen and homogenized in a mixture of HCl (0.01N) and butanol. Dopamine (DA) and serotonin (IBL International GmbH, Hamburg, Germany) from the aqueous phase of the acid extract were quantified using ELISA (2 ), while brain SOD and MDA were determined according to method described by Ho et al (8 ).