Leptin Accelerates Autoimmune Diabetes in Female NOD Mice

Giuseppe Matarese, Veronica Sanna, Robert I. Lechler, Nora Sarvetnick, Silvia Fontana, Serafino Zappacosta, Antonio La Cava


Diabetes. 2002;51(5) 

In This Article

Research Design and Methods

NOD/LtJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and isolator-reared at the Charles River Laboratories (Calco, Italy) before transfer to our animal facility, where they were kept under specific pathogen-free conditions. Mice had free access to autoclaved food and water. The cumulative incidence of type 1 diabetes in untreated female mice was ~85% by 6 months of age, whereas in males it was ~40% by 12 months of age. All of the experiments were performed under an approved protocol in accordance with the animal use guidelines of the Istituto Superiore di Sanità, Rome, Italy.

Young NOD/LtJ mice were treated with intraperitoneal (i.p.) injections of mouse recombinant leptin, purchased from R&D Systems Europe (Oxon, U.K.); purity was >97%, as determined by SDS-PAGE and visualized by silver staining analysis. The endotoxin level was <0.1 ng/µg leptin, as determined by the Limulus amebocyte lysate method. NOD mice received an injection if 1 µg of leptin/g body wt or PBS. Treatment was started at the 1st week of age, and injections were administered thereafter at weekly intervals until the 4th week of age. From the 4th week of age, i.p. injections were repeated every 36 h for 2 consecutive weeks.

Mice were monitored for diabetes by weekly measurement of blood glucose (BG) levels with a one-step glucometer (Bayer, Newbury, U.K.). Animals were considered diabetic when BG levels were >300 mg/dl after two sequential measurements. Ketoacidosis was assessed by measuring the concentration of urinary acetoacetate using the Ketostix kit (Bayer) according to the manufacturer’s instructions. The detection limit for the assay was <5 mg/dl.

Lymphocytic infiltration of pancreatic islets was evaluated on hematoxylin and eosin-stained 6-µm paraffin-embedded sections. Histological scoring for insulitis was performed on hematoxylin and eosin-stained pancreatic sections of 5-week-old leptin-treated or PBS-treated control mice. Scoring was assessed blind by two investigators according to the following scale of 0–3: 0, no insulitis; 1, peri-insulitis; 2, insulitis in <50% of the islet; 3, insulitis in >50% of the islet [13]. A mean score was derived from a number of 10 mice per group and 30–40 islets per individual pancreas from sections taken throughout the organ. Islets were also analyzed for insulin content by immunohistochemistry performed with anti-insulin antibodies (Dako, Carpinteria, CA).

In situ hybridization for IFN-gamma and IL-4 mRNAs was performed according to the technique of Kikuchi et al. [14] with the following modification. Briefly, 6-µm paraffin-embedded sections of spleens from leptin- and PBS-treated mice were incubated with 50 µg/ml proteinase K (Sigma-Aldrich, Milan, Italy), washed, and hybridized overnight at 42°C with biotinylated antisense or control sense probes for IFN-gamma and IL-4 (Primm s.r.l., Milan, Italy). Probe sequences corresponded to amino acids 885–920 for IFN-gamma (accession no. M28621) and to amino acids 140–185 for IL-4 (accession no. M25892), respectively. After hybridization, samples were treated with RNase A (Sigma-Aldrich), incubated with 3,3'-diaminobenzidine (Sigma-Aldrich) in the presence of H2O2, and then counterstained with hematoxylin.

Serum leptin levels and cytokine concentrations in cell culture supernatants were determined by colorimetric sandwich enzyme-linked immunosorbent assay (ELISA) with R&D Quantikine kits (Oxon, U.K.) following the manufacturer’s instructions. Briefly, samples were titrated in test solution and incubated overnight at 4°C in plates coated with capture antibody (Ab). Plates were then incubated with biotinylated detection Ab diluted in PBS/0.1% Tween 20/1% BSA. After washing and incubation for 30 min with peroxidase-conjugated avidin Ab, secondary Ab was detected by addition of substrate. Absorbance was read at 450 nm, and quantification was obtained from two to three titration points using standard curves of purified recombinant molecules. Detection limits were 25 pg/ml for leptin, 10 pg/ml for IL-4, and 20 pg/ml for IFN-gamma.

Analyses were performed using the Mann-Whitney U test (for unpaired two-group analyses). Results are expressed as means ± SD; P < 0.05 was considered to be statistically significant.