Hyper-variability in Insulin Secretion Across Two Cohorts.

<p>(<b>a</b>) Experimental design showing two cohorts (A and B) of <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ and <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male littermates fed chow diet (CD) or high fat diet (HFD). (<b>b</b>) Periodic measurements of 4-h fasted insulin levels is shown for both cohorts, with scatter points to indicate individual values (cohort A: closed points, n = 10–11; cohort B: open points, n = 6–10). In addition, periodic measurements of glucose-stimulated insulin secretion in (<b>c</b>) cohort A (n = 10–11) and (<b>d</b>) cohort B (n = 6–8) is shown, with area under the curve (y-axis units of ng/mL•min) in panel insets. Data are means ± SEM. Dark blue and red represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice, respectively; pale blue and orange represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice, respectively. <i>p</i> ≤ 0.05 denoted by * for CD vs HFD, *^+/+ for CD- vs HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ mice, # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-, § for cohort A vs cohort B.</p

Characterization of <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ and <i>Ins1</i>^-/-:<i>Ins2</i>^+/- Islets.

<p>Separate groups of male mice (discrete from cohorts A and B) were used for islet analyses. (<b>a</b>) <i>In vivo</i> 4-h fasted insulin is shown from 25 week-old mice prior to collection of islets to assess (<b>b</b>) <i>Ins2</i> mRNA, corrected against ß-actin and normalized to CD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ mice, (<b>c</b>) islet insulin content, and (<b>d</b>) insulin secretion by 150 islets perifused with 3 mM glucose (basal), 15 mM glucose (Glu), and 30 mM KCl, with area under the curve (y-axis units of ng/mL•min) depicted for phases I/II of glucose and KCl stimulation, minus basal secretion. (<b>e</b>) <i>in vivo</i> 4-h fasted insulin is shown from 52 week-old mice prior to collection of islets at 70 weeks to assess (<b>f</b>) <i>Ins2</i> mRNA, corrected against ß-actin and normalized to HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ mice, (<b>g</b>) islet insulin content, and (<b>h</b>) insulin secretion by 150 islets perifused with 3 mM glucose (basal), 15 mM glucose (Glu), and 30 mM KCl, with area under the curve (y-axis units of ng/mL•min) depicted for phases I/II of glucose and KCl stimulation, minus basal secretion. n = 3–7. Data are means ± SEM, with scatter points to indicate individual values. Dark blue and red represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice, respectively; pale blue and orange represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice, respectively. <i>p</i> ≤ 0.05 denoted by * for CD vs HFD, # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-. (#) indicates <i>p</i> = 0.056 for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-.</p

Attenuated Adiposity and Fat-free Mass in Cohort B <i>Ins1</i>^-/-:<i>Ins2</i>^+/- Males.

<p>Periodic DEXA-measured (<b>a</b>) fat mass and (<b>b</b>) fat-free mass is shown in cohort B male mice (n = 5–7). This corresponded to (<b>c</b>) wet masses of inguinal, epigonadal, and mesenteric WAT depots, the interscapular BAT depot, whole liver, and the triceps surae hindlimb mixed muscle group, with all tissues from a group of 25 week-old male mice (n = 3–7) that was separate yet similar in phenotype to cohort B mice. In addition, 4-h fasted (<b>d</b>) cholesterol, (<b>e</b>) triglycerides, (<b>f</b>) non-esterified fatty acids (NEFAs), (<b>g</b>) leptin, (<b>h</b>) resistin, (<b>i</b>) interleukin 6, (<b>j</b>) glucose-dependent insulinotropic polypeptide (GIP), and (<b>k</b>) peptide YY, is shown in 40 week-old mice from cohort B (n = 7–9). Data are means ± SEM, with scatter points to indicate individual values. Dark blue and red represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice, respectively; pale blue and orange represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice, respectively. <i>p</i> ≤ 0.05 denoted by * for CD vs HFD, # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-.</p

Cross-cohort Variation in Fasting Glucose, Body Mass, and Corticosterone.

<p>Periodic measurements of 4-h fasted blood glucose in (<b>a</b>) cohort A (n = 12–18, most time points) and (<b>b</b>) cohort B (n = 8–10, most time points) is shown, in addition to weekly body mass in (<b>c</b>) cohort A (n = 12–18, most time points) and (<b>d</b>) cohort B (n = 8–10, most time points). (<b>e</b>) Body mass was also tracked in a subset of pre-weaned male pups from cohort B (n = 12–22). (<b>f</b>) Litter sizes were compared between cohorts, with scatter points indicating the number of pups per individual litter (n = 12–16). (<b>g</b>) 4-h fasted corticosterone levels, measured in plasma collected during early afternoon from 27 week-old mice, were assessed between cohorts. Data are means ± SEM. (<b>h</b>) The relationship between corticosterone and insulin levels across cohorts in 27 week-old HFD-fed males (cohort A: closed points, n = 10; cohort B: open points, n = 9-10), with r² = 0.38 and <i>p</i> < 0.01 for <i>Ins1</i>^-/-:<i>Ins2</i>^+/- mice. (<b>i</b>) The relationship between body mass and 4-h fasted insulin levels at one year of age, r² = 0.55 and <i>p</i> < 0.0001 (cohort A: closed points, n = 9–12; cohort B: open points, n = 6–7). Dark blue and red represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice, respectively; pale blue and orange represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice, respectively. <i>p</i> ≤ 0.05 denoted by * for CD vs HFD, # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-.</p

<i>In Vivo</i> Energy Homeostasis of HFD-fed Cohort B Males.

<p>In HFD-fed 17 week-old mice (n = 6–7), (<b>a</b>) 24-h activity, with inset showing mean beam breaks across the first 4 h of the dark period (y-axis units of beam breaks), as well as (<b>b</b>) energy expenditure, (<b>c</b>) respiratory exchange ratio, and (<b>d</b>) food intake were averaged across 48–84 h, with grey indicating dark cycles. Energy expenditure is shown as estimated marginal means ± SEM, adjusted for covariates of lean and fat mass; other data are simple means ± SEM. Dark red represents HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice; orange represents HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice. <i>p</i> ≤ 0.05 denoted by # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-.</p

Variability in Glucose Homeostasis Across Two Cohorts.

<p>Periodic measurements of blood glucose response to intraperitoneal delivery of (<b>a</b>) an insulin analog (n = 10–19) in cohort A mice, and (<b>b</b>) glucose (n = 11–19) in cohort A mice, as well as (<b>c</b>) an insulin analog (n = 7–11) in cohort B mice, and (<b>d</b>) glucose (n = 7–11) in cohort B mice. Area under or over the curve (y-axis units of <b>a</b>,<b>c</b> percent•min, <b>b</b>,<b>d</b> mmol/L•min) is shown in panel insets. Data are means ± SEM. Dark blue and red represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ male mice, respectively; pale blue and orange represent CD- and HFD-fed <i>Ins1</i>^-/-:<i>Ins2</i>^+/- male mice, respectively. <i>p</i> ≤ 0.05 denoted by * for CD vs HFD, # for <i>Ins1</i>^-/-:<i>Ins2</i>^+/+ vs <i>Ins1</i>^-/-:<i>Ins2</i>^+/-.</p