Volume 20, Issue 2, Pages 451-463 (July 2017) Reduced Circulating Insulin Enhances Insulin Sensitivity in Old Mice and Extends Lifespan  Nicole M. Templeman, Stephane Flibotte, Jenny H.L. Chik, Sunita Sinha, Gareth E. Lim, Leonard J. Foster, Corey Nislow, James D. Johnson  Cell Reports  Volume 20, Issue 2, Pages 451-463 (July 2017) DOI: 10.1016/j.celrep.2017.06.048 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Experimental Design and Proteomic Analysis of Livers from Mice with Reduced Insulin Gene Dosage (A) Schematic of cohorts used for proteomic and transcriptomic analyses (top) and physiology and lifespan tracking (bottom). (B and C) Proteomic analyses of livers from 25-week-old mice revealed proteins with statistically significant differences in levels between Ins2+/+ mice and Ins2+/− mice (B) and between mice on diet A versus diet B (C). FDR < 0.05 (n = 5). Proteins were clustered on the heatmap by the magnitude of the average difference in log2 transformed values between genotype (B) or diet (C). Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Transcriptomic Analysis of Liver and Skeletal Muscle from Mice with Reduced Insulin Gene Dosage (A) Expression levels of Grpel2 in liver and muscle, with # denoting a significant effect of genotype (adjusted p value ≤ 0.05) across both diets and both tissues. The data are means ± SEM, with scatter points indicating individual values. (B) Analysis using String shows that GRPEL2 is linked to multiple mitochondrial proteins. (C) Analysis of Grpel2 mRNA values in public microarray data reveal consistent reciprocal effects of caloric restriction and high-fat diets. (D) Effects of reduced Ins2 dosage on gene expression across both diets in livers of 25-week-old female mice. ∗∗∗ denotes adjusted p value (pAdj) ≤ 0.05, ∗∗0.05 < pAdj ≤ 0.10, and ∗0.10 < pAdj ≤ 0.15 (n = 4–5). The data are means ± SEM. (E) Examples of genes with expression levels in liver affected by reduced Ins2 dosage under either diet A or diet B, arranged according to function and functional interactions. See Figures S1 and S2 for the full list of liver genes that varied by genotype significantly within each diet and Tables S2, S3, and S4 for adjusted p values. Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Effects of Reduced Insulin Gene Dosage on Circulating Insulin and Glucose Homeostasis in Aged Female Mice (A) Insulin levels in circulation after a 4 hr fast at 80 weeks of age (n = 16–26). (B) Igf1 expression was unaltered by diet or genotype at 25 weeks of age, based on RNA sequencing data (Tables S2, S3, and S4). (C) Total circulating Igf1 after a 4 hr fast at 30 and 80 weeks (n = 5–9). (D) 4 hr fasting blood glucose (n = 13–33). (E) HOMA-IR scores at 80 weeks (n = 16–26). (F–H) Blood glucose response to intraperitoneal insulin analog (F), glucose-stimulated insulin secretion (G), and blood glucose response to intraperitoneal glucose (H) for 1.5-year-old mice (n = 15–33), with area under the curve or over the curve (y axis units of percent × min F, ng/mL × min G, mmol/L × min H) in panel insets. The data are means ± SEM, with scatter points indicating individual values. p ≤ 0.05 denoted by ∗ for diets A versus B, # for Ins2+/+ versus Ins2+/−, and #A for diet A-fed Ins2+/+ versus Ins2+/−. Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Effects of Reduced Insulin Gene Dosage on Body Composition and Liver Steatosis in Aged Mice (A–E) 85-week-old (A) body mass (n = 17–30), as well as DEXA-measured fat mass (B), fat-free mass (C), and bone mineral density (D) (n = 7–8), in addition to forelimb grip strength (n = 7–14) (E). The data are means ± SEM, with scatter points indicating individual values. p ≤ 0.05 denoted by ∗ for diets A versus B and # for Ins2+/+ versus Ins2+/−. (F) Representative images of liver sections stained with H&E, from 2-year-old diet B-fed female mice (n = 3) in longevity study that did not exhibit signs of gross liver lesions at the time of euthanization. The scale bar represents 48 μm. Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Reduced Insulin Gene Dosage Extends Lifespan and Reduces Oxidative Stress in Female Mice (A) Survival curves with significant (p ≤ 0.05) lifespan extension in Ins2+/− females compared to Ins2+/+ littermates, assessed using a Kaplan-Meier log rank test stratified by diet and verified with a Cox proportional hazards regression analysis incorporating diet and cohort covariates. n = 40–43, with ticks indicating censored individuals. The red line indicates the age at which 90% of the population had died. (B) Average lifespan of the longest-lived decile. (C) 4 hr fasted 8-isoprostane at 30 and 90 weeks of age (n = 7–9). (D–I) In post-mortem assessments, we evaluated causes of death (D and G–I), as well as percentage of mice detected with malignant tumors (hatched), benign tumors (solid), or both (gray) (E), and the number of distinct tumor-types identified per mouse (F). Survival curves for malignant cancers (D), cardiac-associated pathologies (G), and kidney degeneration (H) indicate timelines of causative contributions to mortality by severe lesions in these categories, with unidentified pathologies (I) indicating deaths with unidentified cause. The data in bar graphs represent means ± SEM, with scatter points indicating individual values. p ≤ 0.05 denoted by #, p = 0.059 denoted by (#) for Ins2+/+ versus Ins2+/−, and p ≤ 0.05 denoted by ∗ for diets A versus B. Cell Reports 2017 20, 451-463DOI: (10.1016/j.celrep.2017.06.048) Copyright © 2017 The Author(s) Terms and Conditions