Leptin Accelerates Autoimmune Diabetes in Female NOD Mice

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

Disclosures

Diabetes. 2002;51(5) 

In This Article

Results

NOD females show increased type 1 diabetes susceptibility compared with males [1,3]. We first compared basal serum leptin levels (at 6 weeks of age) of NOD female mice with normal age- and sex-matched females from other strains of mice that are not susceptible to type 1 diabetes (Fig. 1A). It is interesting that NOD females showed a statistically significant increase of serum leptin compared with all other strains of mice tested (NOD females serum leptin: 9.3 ± 3.3 vs. 1.96 ± 0.58 ng/ml in SJL females, P < 0.001; 2.6 ± 0.71 ng/ml in BALB/c females, P < 0.01; and 3.84 ± 1.2 ng/ml in C57BL/6J females, P < 0.01; Fig. 1A). Subsequently, we examined the serum leptin levels in (type 1 diabetes susceptible) female NOD mice at different time points corresponding to preclinical (where no animals exhibited disease signs), subclinical (where asymptomatic insulitis was present), and clinical diabetes (as assessed by hyperglycemia >300 mg/dl). Parallel measurement was also performed in diabetes-resistant NOD male mice. The results shown in Figs. 1B and C indicate that a surge of serum leptin occurred only in preclinical female mice before the onset of hyperglycemia, increasing 5- to 10-fold to a peak level of 35.8 ± 11.7 ng/ml in 21-week-old mice and 34.0 ± 9.9 in 23-week-old mice. After this surge, a drop of serum leptin accompanied the establishment of clinical diabetes (Fig. 1B). Conversely, leptin levels were significantly lower in male NOD mice (Fig. 1B) in which there were no significant changes of serum leptin and blood glucose (Figs. 1B and C).

NOD female mice have higher serum leptin levels than NOD males and other nonsusceptible strains of mice. A: Serum leptin at 6 weeks of age in female NOD mice is significantly higher than age- and sex-matched SJL (*P < 0.001), BALB/c (**P < 0.01), and C57BL/6J (**P < 0.01) mice. B: Serum leptin surge precedes appearance of clinical type 1 diabetes in female NOD mice but not in males. ELISA for serum leptin (B) and blood glucose measurement (C) in female and male NOD mice at different ages. Single mice are represented at each time point, and the horizontal line represents the mean value. For females, data are accumulated and averaged from four independent experiments that gave similar results. For males, data are accumulated and averaged from two independent experiments with similar results.

To understand whether the changes observed in the expression of serum leptin were related to diabetogenesis, we treated early in life young female and male NOD mice from the first week of age with i.p. injections of recombinant leptin (details of the protocol are indicated in RESEARCH DESIGN AND METHODS). Leptin administration led to rapid acceleration of diabetes, and by the 6th week of age ~90% of the leptin-treated female mice had developed diabetes, whereas all age- and sex-matched PBS-treated controls were free from diabetes (Fig. 2). More specific, we observed higher severity of disease in leptin-treated female mice (median time of onset, 6.5 ± 0.5 weeks of age in leptin-treated vs. 25.5 ± 0.5 in PBS-treated females; P < 0.0001) and a rapid evolution to ketoacidosis ( Table 1 ), this complication being responsible for a mortality rate of approximately one third in leptin-treated animals (n = 7 of 21 [33.3%]) within ~2–3 days of the onset of hyperglycemia ( Table 1 ). This phenomenon did not occur in any of the PBS-treated controls and in leptin-treated males ( Table 1 ). We also tested NOD male mice in parallel with females using the same protocol of leptin injection (see RESEARCH DESIGN AND METHODS). Administration of leptin did not result in an increase in incidence of either diabetes or mortality ( Table 1 , Fig. 2).

Early administration of leptin accelerates onset and progression of type 1 diabetes in female but not in male NOD mice. Cumulative incidence of diabetes in NOD mice treated with leptin or PBS. For females, the median time of onset was 6.5 ± 0.5 weeks of age in leptin-treated vs. 25.5 ± 0.5 in PBS-treated females (P < 0.0001). In males, nonsignificant differences were observed between the two groups (median time to onset was 45.6 ± 1.0 weeks of age for both groups). Injections were performed at times indicated by arrows (see RESEARCH DESIGN AND METHODS). BG was determined weekly, and mice were considered diabetic after two consecutive measurements of BG above 300 mg/dl. Data are representative of two experiments with similar results.

Finally, accelerated diabetes occurred in leptin-treated female mice only when leptin administration was started early in life. Indeed, the treatment regimen that caused diabetes in young female mice (Fig. 2) did not significantly influence development of type 1 diabetes in 10-week-old adult female NOD mice (n = 10) in comparison with age-matched PBS-treated controls (n = 8; not shown).

We also examined the pancreata of 5-week-old mice from both the leptin-treated and control groups. Although perivascular lymphomonocytic infiltration was sporadically observed in the islets of PBS-treated controls, morphological integrity of the islets was generally maintained in these mice (Fig. 3A). Conversely, leukocytic infiltrates invaded the islets of leptin-treated mice, with the result that widespread insulitis occurred, leading to erosion of the beta-cell mass as leukocytes penetrated into the islet core (Fig. 3B). Scoring for insulitis confirmed severe pancreatic damage in leptin-treated mice (2.25 ± 0.45 vs. 0.65 ± 0.15 in matched controls; P < 0.001; Fig. 3C). Finally, immunohistochemical analysis for insulin revealed insulin depletion in pancreata of leptin-treated mice but normal insulin content in the age-matched control group (not shown).

Leptin administration leads to early islet inflammatory infiltration in NOD mice. Hematoxylin and eosin staining of pancreata from control NOD mice (A) and leptin-treated mice (B) at 5 weeks of age. Magnification: 200x. C: Insulitis score of leptin- and PBS-treated control mice (*P < 0.001, Mann-Whitney U test) is expressed as mean ± SD. Pancreatic islets of leptin-treated mice are heavily infiltrated (B and C), whereas matched controls mostly have normal islets (A and C).

We next compared the cytokine expression profile of splenic T-cells from leptin-treated female NOD mice (n = 4) and PBS-treated age- and sex-matched controls (n = 4) stimulated in vitro with anti-CD3 Ab (2C11 hybridoma, ATCC). Nonsignificant differences were observed between these two groups of mice for the secretion of IFN-gamma (200 ± 49 vs. 190 ± 77 pg/ml, NS, respectively) or IL-4 (below the detection limit of the assay). However, in situ hybridization for the IFN-gamma mRNA expression in the spleen revealed that the periarteriolar sheaths of leptin-treated mice were rich in IFN-gamma mRNA-expressing cells (Figs. 4C and D), a phenomenon lacking in PBS-treated controls (Figs. 4A and B).

Early administration of leptin increases T-cell-mediated IFN-gamma mRNA expression in NOD mice. In situ hybridization for the presence of IFN-gamma in spleens of PBS-treated controls (A, B) and leptin-treated mice (C, D). More IFN-gamma mRNA expression (in brown) is found within the periarteriolar sheaths (T-cell areas) of leptin-treated mice (C, D) than controls (A, B). Magnification: 200x (A, C) and 800x (B, D). The white squared areas in A and C represent the zone of higher magnification shown in B and D, respectively. Representative experiment of three.

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