Diet as an environmental factor influences age of onset in models of spontaneous insulin-dependent diabetes mellitus. We reported recently that a protein-rich diet accelerated diabetes incidence in nonobese diabetic (NOD) mice. In the present study, we investigated the effect of diet on beta-cells and glucose metabolism in NOD mice before diabetes onset. Three different diets were maintained from 4 weeks on: low fat (LF; 12% fat, 21% protein, and 68% carbohydrates), high fat (HF; 39% fat, 17% protein, and 43% carbohydrate), and high fat-high protein (HFHP; 43% fat, 38% protein, and 19% carbohydrates) diet. The cumulative incidence of diabetes was 92% for HFHP (P < 0.01 vs. LF), 80% for HF (P = NS), and 65% for the LF cohort. At 20 weeks of age insulin secretion in the isolated pancreas was doubled for the HF diet and 4.4 times higher for the HFHP-fed mice compared with the LF group. Feeding HF and HFHP reduced total glucose utilization during continuous insulin infusion (1 mU/kg) by 34% (P < 0.05). HFHP, but not HF, diet elevated endogenous glucose production by 48% (P < 0.05) compared with that in the LF group. Beta-cell mass, estimated by imaging analysis, was initially high in young HFHP-fed mice, aged 10 weeks, but declined rapidly thereafter [HFHP, 1.6 +/- 0.2 (P < 0.05 vs. LF); HF, 2.4 +/- 0.4 (P = NS vs. LF); LF, 2.1 +/- 0.5 mg at 30 weeks]. A reduction of beta-cell mass was associated with HF 14% (P < 0.05 vs. LF) and HFHP 82% (P < 0.01 vs. LF) more apoptotic beta-cells at 30 weeks. Depending on age, 1.2-3.1 of 1000 beta-cells were in a stage of proliferation without significant differences among the dietary groups. In conclusion, HFHP diet was associated with impaired glucose metabolism and high insulin release followed by enhanced diabetes incidence. Diabetes was promoted by increased rate of cell death over beta-cell neogenesis.