Figure 4. Comparing sex and strain patterns for Ca²⁺ metrics, insulin secretion, and clinical traits nominates Ca²⁺ metrics of interest.

(A) The Z-score correlation coefficient was calculated for Ca²⁺ parameters and raw insulin secreted and % total insulin secreted. Insulin measurements were previously collected for seven different secretagogues (16.7 mM glucose + 0.5 mM palmitic acid (16.7G/PA); 3.3 mM glucose + 50 mM KCl (3.3G/KCl); 16.7 mM glucose (16.7G); 8.3 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8.3G/QLA); 8.3 mM glucose + 100 nM GLP-1 (8.3G/GLP-1); 8.3 mM glucose (8.3G); and 3.3 mM glucose (3.3G)) (Mitok et al., 2018). (B) Correlation of the Ca²⁺ parameters to the clinical measurements in the founder mice which include (1) plasma insulin, triglycerides (TG), and glucose at 6, 10, and 14 weeks as well as at time of sacrifice; (2) number of islets; (3) whole-pancreas insulin content (WPIC); and (5) islet content for insulin and glucagon. For (A) and (B), the Ca²⁺ parameters shown here include average Ca²⁺ in 2 mM glucose (basal Ca²⁺); average Ca²⁺ in 8 mM glucose (8G avg.); average Ca²⁺ in 8 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8G/QLA avg); average Ca²⁺ in 8 mM glucose + QLA + 10 nM GIP (8G/QLA/GIP avg.); pulse duration in 8 mM glucose (8G P_D); active duration in 8G (8G A_D); silent duration in 8G (8G S_D), 8G/QLA (8G/QLA/S_D), and 8G/QLA/GIP (8G/QLA/GIP S_D); and 1st component frequency in 8 mM glucose (8G 1st freq.). Other parameters analyzed are indicated in Figure 4—figure supplement 2 and Figure 4—figure supplement 3. (B–E) Sex and strain variability for (C) average Ca²⁺ determined by the Fura-ratio (FR) in 2 mM glucose, (D) pulse duration of oscillations in 8G, (E) 1st component frequency in 8G, and (F) silent duration of oscillations in 8G, 8G/QLA, and 8G/QLA/GIP.

Figure 4—figure supplement 1. Differential strain and sex effects in correlations between traits.

The correlations between Z-scores for the calcium trait (x axis) and the other indicated trait (y axis) are shown for the eight strains (key, upper right). Males are indicated as boxes (□), females as circles (○) in each plot, where the color indicates the strain. (A) The correlation between the Z-scores for the 1st frequency component in 8 mM glucose (8G) and the percent insulin secreted in 8.3 mM glucose with 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8.3G/QLA) reveal clustering of strains into the disease susceptible strains (blue ellipse), classic inbred strains (gray ellipse), and wild-derived strains (green ellipse). (B) The correlation between the Z-scores for silent duration (S_D) in 8G/QLA and the ng of insulin secreted per islet in 8.3G/QLA show less of a separation between the three groups. (C) Some correlations, such as that between Z-scores for the S_D in 8 mM glucose with 1.25 mM L-alanine, 2 mM L-glutamine, 0.5 mM L-leucine, and 10 nM GIP (8G/QLA/GIP) and the plasma insulin level at sacrifice show strong effects of single strains (e.g. NZO, indicated by blue arrow). In each plot, the Pearson’s R value and slope are indicated. Related to Figure 4.

Figure 4—figure supplement 2. Correlation reveals specific Ca²⁺ parameters highly associated with insulin secretion.

Heatmap displaying the correlation coefficients between the Z-scores for Ca²⁺ wave metrics and the Z-scores for raw insulin secreted, fold-change (FC) over basal insulin secreted, and % of total islet insulin secreted. Insulin measurements were previously collected (Mitok et al., 2018) for seven different secretagogues (16.7 mM glucose + 0.5 mM palmitic acid (16.7G/PA); 3.3 mM glucose + 50 mM KCl (3.3G/KCl); 16.7 mM glucose (16.7G); 8.3 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8.3G/QLA); 8.3 mM glucose + 100 nM GLP-1 (8.3G/GLP-1); 8.3 mM glucose (8.3G); and 3.3 mM glucose (3.3G)). Perifusion conditions included 8 mM glucose (8G); 8 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8G/QLA); 8 mM glucose + QLA + 10 nM GIP (8G/QLA/GIP). Unsupervised clustering of the Ca²⁺ wave parameters revealed several parameters highly correlated to multiple insulin secretion conditions. Parameters included: average Ca²⁺ in 2 mM glucose (basal Ca²⁺), in 8 mM glucose (8G avg.), in 8G/QLA (8G/QLA avg), and in 8G/QLA/GIP (8G/QLA/GIP avg.); average detrended Ca²⁺ in 8 mM glucose (8G detr. avg.), in 8G/QLA (8G/QLA detr. avg), and in 8G/QLA/GIP (8G/QLA/GIP detr. avg.); average change in Ca²⁺ vs. basal in 8 mM glucose (8G avg. Δ vs. 2G), in 8G/QLA (8G/QLA avg. Δ vs. 2G), and in 8G/QLA/GIP (8G/QLA/GIP avg. Δ vs. 2G); change in detrended average Ca²⁺ vs. basal in 8 mM glucose (8G detr. Δ vs. 2G), in 8/QLA (8G/QLA detr. Δ vs. 2G), and in 8G/QLA/GIP (8G/QLA/GIP detr. Δ vs. 2G); average oscillation peak Ca²⁺ in 8G (8G peak), in 8G/QLA (8G/QLA peak), and in 8G/QLA/GIP (8G/QLA/GIP peak); average oscillation baseline Ca²⁺ in 8G (8G baseline), in 8G/QLA (8G/QLA baseline), and in 8G/QLA/GIP (8G/QLA/GIP baseline); pulse duration in 8G (8G P_D), in 8G/QLA (8G/QLA P_D), and in 8G/QLA/GIP (8G/QLA/GIP P_D); active duration in 8 mM glucose (8G A_D), in 8G/QLA (8G/QLA A_D), and in 8G/QLA/GIP (8G/QLA/GIP A_D); silent duration in 8 mM glucose (8G S_D), in 8G/QLA (8G/QLA S_D), and in 8G/QLA/GIP (8G/QLA/GIP S_D); plateau fraction in 8 mM glucose (8G P_F), in 8G/QLA (8G/QLA P_F), and in 8G/QLA/GIP (8G/QLA/GIP P_F); spectral density 1st component frequency in 8 mM glucose (8G 1st freq.), in 8G/QLA (8G/QLA 1st freq.), and in 8G/QLA/GIP (8G/QLA/GIP 1st freq.); spectral density 2nd component frequency in 8 mM glucose (8G 2nd freq.), in 8G/QLA (8G/QLA 2nd freq.), and in 8G/QLA/GIP (8G/QLA/GIP 2nd freq.); contribution of the 1st component to the Ca²⁺ waveform for 8 mM glucose (8G 1st freq. amp.), for 8G/QLA (8G/QLA 1st freq. amp.), and for 8G/QLA/GIP (8G/QLA/GIP 1st freq. amp.); and contribution of the 2nd component to the Ca²⁺ waveform for 8 mM glucose (8G 2nd freq. amp.), for 8G/QLA (8G/QLA 2nd freq. amp.), and for 8G/QLA/GIP (8G/QLA/GIP 2nd freq. amp.). Related to Figure 4.

Figure 4—figure supplement 3. Correlation reveals specific Ca²⁺ parameters highly associated with in vivo traits.

Heatmap displaying the correlation coefficients between the Z-scores for Ca²⁺ wave metrics and the Z-scores for mouse in vivo metrics previously collected (Mitok et al., 2018) for the same strains and sexes. These traits included plasma insulin, glucose, and triglycerides (TGs) (measured at 6, 10, and 14 weeks of age as well as at sacrifice) as well as whole body weights at those time points. Whole-pancreas insulin content (WPIC), islet glucagon, islet number, and islet insulin content were also determined. Ca²⁺ perifusion conditions included 8 mM glucose (8G); 8 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8G/QLA); 8 mM glucose + QLA + 10 nM GIP (8G/QLA/GIP). Unsupervised clustering of the Ca²⁺ wave parameters revealed several parameters highly correlated to multiple in vivo traits. Parameters included: average Ca²⁺ in 2 mM glucose (basal Ca²⁺), in 8 mM glucose (8G), in 8G/QLA (8G/QLA), and in 8G/QLA/GIP (8G/QLA/GIP); average detrended Ca²⁺ in 8 mM glucose (8G detr.), in 8G/QLA (8G/QLA detr.), and in 8G/QLA/GIP (8G/QLA/GIP detr.); average change in Ca²⁺ vs. basal in 8 mM glucose (8G Δ vs. basal), in 8G/QLA (8G/QLA Δ vs. basal), and in 8G/QLA/GIP (8G/QLA/GIP Δ vs. basal); change in detrended average Ca²⁺ vs. basal in 8 mM glucose (8G detr. Δ vs. basal), in 8/QLA (8G/QLA detr. Δ vs. basal), and in 8G/QLA/GIP (8G/QLA/GIP detr. Δ vs. basal); average oscillation peak Ca²⁺ in 8G (8G peak), in 8G/QLA (8G/QLA peak), and in 8G/QLA/GIP (8G/QLA/GIP peak); average oscillation baseline Ca²⁺ in 8G (8G baseline), in 8G/QLA (8G/QLA baseline), and in 8G/QLA/GIP (8G/QLA/GIP baseline); pulse duration in 8G (8G P_D), in 8G/QLA (8G/QLA P_D), and in 8G/QLA/GIP (8G/QLA/GIP P_D); active duration in 8 mM glucose (8G A_D), in 8G/QLA (8G/QLA A_D), and in 8G/QLA/GIP (8G/QLA/GIP A_D); silent duration in 8 mM glucose (8G S_D), in 8G/QLA (8G/QLA S_D), and in 8G/QLA/GIP (8G/QLA/GIP S_D); plateau fraction in 8 mM glucose (8G P_F), in 8G/QLA (8G/QLA P_F), and in 8G/QLA/GIP (8G/QLA/GIP P_F); spectral density 1st component frequency in 8 mM glucose (8G 1st freq.), in 8G/QLA (8G/QLA 1st freq.), and in 8G/QLA/GIP (8G/QLA/GIP 1st freq.); spectral density 2nd component frequency in 8 mM glucose (8G 2nd freq.), in 8G/QLA (8G/QLA 2nd freq.), and in 8G/QLA/GIP (8G/QLA/GIP 2nd freq.); contribution of the 1st component to the Ca²⁺ waveform for 8 mM glucose (8G 1st freq. amp.), for 8G/QLA (8G/QLA 1st freq. amp.), and for 8G/QLA/GIP (8G/QLA/GIP 1st freq. amp.); and contribution of the 2nd component to the Ca²⁺ waveform for 8 mM glucose (8G 2nd freq. amp.), for 8G/QLA (8G/QLA 2nd freq. amp.), and for 8G/QLA/GIP (8G/QLA/GIP 2nd freq. amp.). Related to Figure 4.