Figure 2. Effects of HC and HC+4%gluten diets on insulin secretion.

(A, B) Intraperitoneal Glucose Tolerance Test (IPGTT) performed on 6–8-week-old NOD female mice either fed chow or HC diets shown as (A) the change of blood glucose levels after subtraction of the zero-time-point for every mouse (delta) and (B) calculation of Incremental Area Under the Curve (iAUC) for the same experiments. Combined data from 5 independent experiments, mean±sem. p values were calculated using Student’s t test. n=number of animals per group.

(C, D) Test for insulin resistance – (C) blood glucose clearance in 6–8-week-old mice fed indicated diets and injected with insulin after 6 hrs fasting and (D) AUC calculated for the same experiments. Combined data from two experiments, mean±sem. p values calculated using Student’s t test. n=number of animals per group.

(E, F) Distribution of relative Ins1 and Ins2 gene expression levels in β-cells from 8–10-week-old NOD.scid mice fed regular chow (blue line) or HC diet (red line). Data is based on single-cell RNA sequencing (SCS) of NOD.scid islets and is plotted as a kernel density estimation function. Dashed vertical line indicates the border between low and high expressors of insulin in islets of chow fed mice. The p values calculated by Mann-Whitney U test were 4.9x10⁻¹ and 1.4x10⁻¹⁴ for Ins 1 and Ins 2, respectively.

(G) Volcano plot showing differential gene expression between Ins1-High and Ins1-Low beta cells isolated from the islets of NOD.scid mice fed regular chow. Genes indicated have adjusted p-values less than 0.05 and log2 fold change magnitude greater than 0.5.

(H) Gene networks built using genes upregulated in β-cells with high insulin expression compared to β-cells with low insulin expression using STRING database ⁶⁵.

(I) Glutens from two different manufacturers were added to HC diet replacing 1/5^th of hydrolyzed casein weight (or 4% of the total diet weight, thus termed ‘HC−4%gluten diet’) and T1D incidence in female NOD mice was observed in the same experiment.

(J) Direct measurements of plasma insulin in NOD mice fed chow, HC, or HC+4%gluten at fasting baseline and 5 minutes after glucose challenge. Mean±sem. p values were calculated using Student’s t test.

(K) Proliferation of CFSE-labeled BDC2.5 T cells in the pancreatic lymph nodes of mice on chow and HC+4%gluten diets. Mean±sem. Combined data from 2 experiments, p value was calculated using Student’s t test.