The causal relationships between insulin levels, insulin resistance, and longevity are not fully elucidated. Genetic downregulation of insulin/insulin-like growth factor 1 (Igf1) signaling components can extend invertebrate and mammalian lifespan, but insulin resistance, a natural form of decreased insulin signaling, is associated with greater risk of age-related disease in mammals. We compared Ins2 +/− mice to Ins2 +/+ littermate controls, on a genetically stable Ins1 null background. Proteomic and transcriptomic analyses of livers from 25-week-old mice suggested potential for healthier aging and altered insulin sensitivity in Ins2 +/− mice. Halving Ins2 lowered circulating insulin by 25%–34% in aged female mice, without altering Igf1 or circulating Igf1. Remarkably, decreased insulin led to lower fasting glucose and improved insulin sensitivity in aged mice. Moreover, lowered insulin caused significant lifespan extension, observed across two diverse diets. Our study indicates that elevated insulin contributes to age-dependent insulin resistance and that limiting basal insulin levels can extend lifespan.

Remarkably, we found that across both diets, mice with reduced circulating insulin levels had improved insulin sensitivity with advanced age and exhibited lifespan extension without changing Igf1 levels. These results suggest a causal contribution for hyperinsulinemia in age-dependent insulin resistance and point to the modest suppression of insulin as a safe and attainable strategy for extending lifespan.

To determine how moderately lowering the insulin ligand would affect late-life glucose homeostasis and longevity in mammals, we compared mice with either full or partial expression of the ancestral insulin gene Ins2. Since altering insulin gene dosage does not affect circulating insulin levels in all contexts (), we used a mouse model in which the rodent-specific insulin gene (Ins1) was fully inactivated to prevent compensatory Ins1 expression. We also provided the mice with diet formulations that support relatively robust insulin production and secretion, because reducing insulin gene dosage cannot always diminish circulating insulin levels under the minimally stimulatory conditions of a low-energy, low-fat diet () or in male Ins1 null mice (). We designed our experiment to evaluate these animals in the context of two distinct diets (diet A: moderate-energy diet of 4.68 kcal/g, with 20% of calories from protein, 25% from fat, and 55% from carbohydrate; diet B: high-energy diet of 5.56 kcal/g, with 16% of calories from protein, 58% from fat, and 26% from carbohydrate) to allow us to assess the effects of insulin levels across diverse dietary parameters that were not limited to a specific macronutrient and micronutrient composition ().

The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice.

Hyper-variability in circulating insulin, high fat feeding outcomes, and effects of reducing Ins2 dosage in male Ins1-null mice in a specific pathogen-free facility.

Hyper-variability in circulating insulin, high fat feeding outcomes, and effects of reducing Ins2 dosage in male Ins1-null mice in a specific pathogen-free facility.

Insulin resistance is a common feature of mammalian aging and a risk factor for numerous age-related diseases (). Although this suggests that reducing insulin signaling could be detrimental for mammalian healthspan, it is important to consider that conventionally defined insulin resistance (i.e., impaired insulin-stimulated glucose disposal) is not a generalized reduction of all insulin signaling. Instead, some insulin-regulated processes are maintained at normal capacity in the “insulin resistant” state, while others are downregulated (). Moreover, circulating levels of the insulin ligand are elevated with insulin resistance. The commonly accepted paradigm posits that insulin levels rise as a compensatory response to prevent hyperglycemia when there is insufficient insulin-stimulated glucose uptake (). However, causality between the closely associated conditions of systemic insulin resistance and insulin hypersecretion has remained controversial, and it has been suggested that hyperinsulinemia could be an early, primary cause of insulin resistance, obesity, and eventually type 2 diabetes (). Genetic loss-of-function experiments targeting insulin itself present an ideal opportunity to disentangle hyperinsulinemia from insulin resistance and to evaluate the lifelong effects of limiting endogenous insulin production and secretion.

Insulin resistance and hyperinsulinemia: is hyperinsulinemia the cart or the horse?.

Basal state hyperinsulinemia in healthy normoglycemic adults heralds dysglycemia after more than two decades of follow up.

Metabolism and lifespan are influenced by evolutionarily conserved gene networks (). In both invertebrates and mammals, genetic downregulation of the insulin/insulin-like growth factor 1 (Igf1) signaling pathway can extend lifespan (). As insulin and Igf1 share nearly identical downstream signaling pathways in mammals, and act in part via hybrid insulin/Igf1 heterodimer receptors that can bind to either ligand, the relative functions of these two ligands have not been completely delineated (). Many studies have focused on Igf1 as the primary ligand which mediates the lifespan-altering effects through this signaling cascade in mammals (), but the impact of directly altering insulin levels on longevity had not been evaluated.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production.

Histopathological evaluation of post-mortem tissues was conducted for >90% of all mice to determine if lowering insulin protected mice from specific conditions or whether it had a more general effect on health. Interestingly, diet B-fed Ins2female mice tended to show delayed and reduced occurrence of malignant cancer-associated mortality compared to Ins2littermates ( Figures 5 D–5F). There was also a statistically significant delay in mortality associated with renal degeneration and kidney diseases (e.g., glomerulonephritis, glomerular sclerosis, tubule atrophy or dilation, protein deposits or mineralization, cysts, and renal infarcts) in Ins2female mice compared to their Ins2littermates ( Figure 5 H). Diet A appeared to induce disease in middle-aged female mice ( Figure 5 A), but since detected lesions were not considered severe enough to have caused mortality or morbidity for many diet A-fed mice ( Figure 5 I), we were unable to link any one disease category to diet A consumption. It is important to note that diets A and B were not matched for the type of fat content, protein levels, or micronutrient composition, so there are numerous potential factors that could have impacted diet-dependent outcomes. It is well known that diet composition has profound impacts on lifespan (e.g.,) and on metabolic parameters, such as circulating insulin levels. Therefore, it was not a goal of our study to evaluate the effects of different diet formulations on lifespan, but rather to ascertain whether genetically limiting production and secretion of insulin, a key metabolic enzyme, could have consistent effects on lifespan under diverse dietary circumstances. As the relative proportion of mice experiencing any of the identified major pathologies was largely comparable between Ins2and Ins2mice within each tested diet, our results indicate that lowering insulin levels appears to provide a general extension of healthspan and lifespan, rather than alleviating one specific type of disease.

The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice.

One evolutionarily conserved feature of aging is increased reactive oxygen species and oxidative damage, and many long-lived organisms exhibit resistance to oxidative stress (). Although there were no differences observed in 30-week-old mice, 90-week-old Ins2female mice showed diminished effects of oxidative damage compared to their Ins2littermates across both diets, as indicated by reduced plasma levels of 8-isoprostane, a circulating marker of oxidative stress ( Figure 5 C).

Our metabolic data revealed that reducing Ins2 gene dosage had only modest effects on circulating insulin levels, unlike the extensive endocrine disruption evident in many long-lived mouse models (). Remarkably, we found that this slight change in circulating insulin was sufficient for Ins2mice to have a significant increase in lifespan, compared to Ins2littermates (p ≤ 0.05; Figure 5 A). The maximum lifespan of Ins2mice also tended to be higher than Ins2controls across both diets, whether evaluated as the average lifespan of the longest-lived decile (p = 0.059; Figure 5 B) or as an assessment of distribution differences in the 90percentile of the population (p = 0.058; Figure 5 A). The degree of median lifespan extension afforded by reduced Ins2 dosage was similar for female mice on both diets, if slightly blunted on diet B (diet A: 11% and diet B: 3%), suggesting that attenuation of diet B-associated obesity () was mechanistically distinct from lifespan extension in this study. Other than a tendency for delayed and reduced occurrence of mortality associated with malignant cancers in Ins2mice (p = 0.058; Figure S4 H), there was no clear effect of genotype on lifespan of male mice ( Figure S4 K); since our experimental manipulation did not consistently lower plasma insulin levels in the Ins2males, we were unable to ascertain whether reduced circulating insulin could affect longevity in males.

(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.

(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.

Ins2mice with modestly lowered circulating insulin had reduced body mass ( Figure 4 A) and fat mass ( Figure 4 B) at 85 weeks of age compared to their Ins2littermates, consistent with measurements of these same animals at younger ages () and confirming the causal requirement for elevated insulin in obesity (). Other parameters of body composition were unchanged between genotypes, including skeletal muscle strength and bone mineral density ( Figures 4 C–4E and S5 B–S5D). At 90 weeks of age, we also did not observe differences between genotypes in metabolism-regulating hormones such as leptin, resistin, interleukin-6, glucose-dependent insulinotropic polypeptide, peptide YY, or glucagon ( Figures S5 E–S5J). Similarly, circulating cholesterol, triglycerides, and non-esterified fatty acids in 90-week-old Ins2mice were not different from Ins2controls ( Figures S5 K–S5M). Nonetheless, post-mortem histopathological analyses revealed that while 12.5% (four of 32 analyzed mice) of diet B-fed Ins2mice had moderate or severe hepatic lipidosis, none of the 29 analyzed diet B-fed Ins2mice showed evidence of lipid accumulation in liver (p ≤ 0.05; Figure 4 F). Thus, diet B-fed Ins2mice with lowered circulating insulin experienced a lifelong attenuation of high-fat diet-induced obesity and protection from some obesity-associated sequelae, compared to Ins2controls ().

(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.

(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 +/− .

We previously reported that reduced circulating insulin in female Ins2mice does not have a prolonged negative impact on glucose homeostasis across the first year of life (). However, aging has been reported to be associated with insulin secretory defects and increased demand on pancreatic β cells (), so lowering Ins2 dosage could potentially have had adverse effects on glucose homeostasis late in life. Counter to this possibility, aged Ins2female mice exhibited a significant reduction in fasting glucose compared to Ins2controls ( Figure 3 D) and at 80 weeks had lower value for the homeostatic model assessment-insulin resistance (HOMA-IR), a measure of insulin resistance ( Figure 3 E). Insulin tolerance tests confirmed that aged Ins2female mice on both diets had superior peripheral insulin sensitivity compared to their Ins2littermates ( Figure 3 F). In a preliminary experiment, we were unable to observe marked differences in Akt or Erk phosphorylation in liver, skeletal muscle, or white adipose tissue in a subset of 70-week-old Ins2mice ( Figure S5 A), although this result does not preclude alterations that could have been more subtle, state-dependent, or involved other insulin signaling components or phosphorylation sites. There were no statistically significant effects of genotype detected for the insulin secretory response ( Figure 3 G) or blood glucose response ( Figure 3 H) to intraperitoneal glucose in 1.5-year-old female mice. Collectively, these data demonstrate that slightly lowering basal insulin levels led to lower fasting glucose levels via improved insulin sensitivity, suggesting a healthier metabolic state with age.

Our previous studies of Ins2mice and their Ins2littermate controls were limited to mice that were less than 1 year of age () and therefore provided no information about age-dependent insulin resistance and longevity. Here, we assessed the effects of reduced Ins2 gene dosage on circulating insulin levels in aged mice. At 1.5 years of age, Ins2female mice showed only a modest reduction in fasting insulin compared to Ins2littermates under both diets. Specifically, Ins2mice had on average 25% lower insulin compared to their Ins2littermates when fed diet A ( Figure 3 A), which was in fact a greater divergence in circulating insulin than any preceding measurements in these cohorts of diet A-fed mice (). On diet B, Ins2mice had 34% lower insulin than Ins2littermate controls ( Figure 3 A), consistent with the magnitude of reduction evident at young ages (). Importantly, we did not observe changes in Igf1 expression or total circulating Igf1 with reduced Ins2 dosage in young or aged female mice on either diet ( Figures 3 B and 3C), nor were there changes in the expression of Igf1, Igfbp3, or Igf1r in muscle or liver ( Tables S2 S3 , and S4 ). Together, these observations suggest that our model allowed us to evaluate for the first time the effects of altering levels of the insulin ligand independent from detectable changes in circulating Igf1 levels. It should be noted that inactivating one Ins2 allele in the male littermates did not consistently or robustly reduce circulating insulin in old age ( Figure S4 A), or at younger ages (). Sex-dependent metabolic differences () could potentially contribute to the often-observed differences in longevity between sexes (), but our model did not allow us to evaluate whether changing circulating insulin would affect insulin sensitivity or lifespan in male mice. Therefore, we used female mice as the means to test our hypotheses that lowering circulating insulin levels might modulate age-dependent insulin resistance and extend lifespan.

(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 +/− .

(B) Igf1 expression was unaltered by diet or genotype at 25 weeks of age, based on RNA sequencing data ( Tables S2 S3 , and S4 ).

Hyper-variability in circulating insulin, high fat feeding outcomes, and effects of reducing Ins2 dosage in male Ins1-null mice in a specific pathogen-free facility.

When examining liver alone, we found that many other networks of genes related to cellular metabolism, oxidative stress, circadian rhythm, proteostasis, and cell cycle progression were altered by reducing Ins2 dosage under one or both diets ( Figures 2 D and 2E; Table S5 ). Many of the altered genes are known to be associated with insulin signaling or glucocorticoid signaling ( Figure 2 E) and are established regulators of longevity, cancer, and metabolic homeostasis ( Table S5 ). Interestingly, functional clustering identified cholesterol metabolic processes as significantly enriched (FDR < 1.35 × 10) with changed Ins2 gene dosage in the context of diet B ( Figure S2 ). Notably, several of the significantly altered genes have been previously associated with aging or longevity in humans or other animals, or in cellular models ( Table S5 ). Therefore, these unbiased transcriptomic data suggested the possibility that Ins2mice would eventually exhibit improved metabolic homeostasis and perhaps extended lifespan.

We were only able to examine a limited part (<10%) of the proteome from our liver samples. To provide a more comprehensive survey, we turned to RNA sequencing analyses of muscle and liver tissue from 25-week-old mice ( Figures 2 S1 , and S2 S3 , and S4 ). A multifactorial analysis of all samples found that a single gene, Grpel2, was consistently and significantly downregulated in mice with reduced Ins2 gene dosage across both diets and both tissues ( Figure 2 A). Grpel2 is an essential component of the presequence translocase-associated motor (PAM) complex that regulates the import of proteins into the mitochondrial matrix () and is predicted by String or Biological General Repository for Interaction Datasets (BioGRID) () to bind to several essential regulators of mitochondrial function ( Figures 2 B and S3 ). By querying published transcriptomic datasets, we found that liver Grpel2 mRNA is consistently decreased with caloric restriction and increased in a number of high-fat diet studies, as well as in obese ob/ob mice and Clock or Cyr knockout mice ( Figure 2 C).

(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.

(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.

(C) Analysis of Grpel2 mRNA values in public microarray data reveal consistent reciprocal effects of caloric restriction and high-fat diets.

(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.

We have previously shown that genetically reducing circulating insulin leads to sustained protection from high-fat diet-induced obesity in female mice (). However, we did not thoroughly characterize the proteome and transcriptome profiles of metabolically relevant tissues in these mice. To gain insight into the molecular mechanisms associated with the phenotype, we focused initially on the liver as a key organ controlling glucose homeostasis. We conducted quantitative proteomics on a pilot cohort of female Ins2mice and their Ins2littermate controls, randomized to either the moderate-fat diet A or the high-fat diet B ( Figure 1 A). We obtained reliable identification of 1,074 unique proteins across all groups from livers isolated from 25-week-old mice ( Table S1 ). The abundances of a total of 52 proteins were significantly different between either diet groups or genotype groups, with false discovery rate (FDR) <0.05 (four proteins differed between genotypes, Figure 1 B; and 48 proteins differed between diets, Figure 1 C). Regardless of diet, livers from Ins2mice showed elevated levels of Slco1b2, Dld, Cyb5b, and Rpl23a relative to their Ins2littermate controls ( Figure 1 B). Interestingly, Slco1b2 (Oatp1b2) knockout mice have impaired glucose tolerance, reduced liver Glut2 expression, and reduced liver glucose content (), which suggests that this protein normally plays a positive role in glucose homeostasis. Dld was previously identified in a proteomic screen for factors involved in insulin signaling (), and reduced Dld activity has been shown to lead to accelerated oxidative damage (). Cyb5b is enriched in liver mitochondria and may participate in lipid synthesis (). Collectively, this unbiased proteomic analysis suggested that mice with reduced circulating insulin levels might exhibit improved insulin sensitivity and metabolic health.

(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 log 2 transformed values between genotype (B) or diet (C).

(A) Schematic of cohorts used for proteomic and transcriptomic analyses (top) and physiology and lifespan tracking (bottom).

Amidoxime reductase system containing cytochrome b5 type B (CYB5B) and MOSC2 is of importance for lipid synthesis in adipocyte mitochondria.

Mutations in the dimer interface of dihydrolipoamide dehydrogenase promote site-specific oxidative damages in yeast and human cells.

Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC).

Discussion

The aims of this study were to examine molecular sequelae of moderately reduced circulating insulin and to test the hypotheses that directly reducing insulin levels could improve metabolic homeostasis, healthspan, and longevity in old mice. We compared Ins2+/− mice to Ins2+/+ littermate controls on a genetic background that was stable between experimental groups and assessed mice that were exposed to two diverse diets. Protein and mRNA changes consistent with improvements to insulin sensitivity and healthspan were already evident at a young age in mice with reduced insulin gene dosage. Furthermore, we found that when reducing insulin gene dosage successfully suppressed circulating levels of the hormone, mice showed enhanced insulin sensitivity with age and lifespan extension.

+/− mice from this study did exhibit a slight, temporary elevation in fasting glucose relative to their Ins2+/+ littermate controls, indicating that their insulin levels may have been not quite sufficient for full glycemic control when they were young ( Templeman et al., 2015 Templeman N.M.

Clee S.M.

Johnson J.D. Suppression of hyperinsulinaemia in growing female mice provides long-term protection against obesity. +/− mice. Since aging has been reported to be associated with the emergence of insulin secretory defects and worsening insulin responsiveness ( Chang and Halter, 2003 Chang A.M.

Halter J.B. Aging and insulin secretion. +/− mice. Although many investigators in the type 2 diabetes research field believe that insulin resistance is an early and causal factor in the progression to hyperinsulinemia and type 2 diabetes, there is evidence that insulin resistance can instead be the result of elevated or continuous exposure to the insulin ligand, causing tissue “desensitization” at the receptor or post-receptor level ( Shanik et al., 2008 Shanik M.H.

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et al. A major role of insulin in promoting obesity-associated adipose tissue inflammation. The concept that limiting circulating insulin levels could improve glucose homeostasis may seem counterintuitive. Indeed, at 6 weeks of age, the female Ins2mice from this study did exhibit a slight, temporary elevation in fasting glucose relative to their Ins2littermate controls, indicating that their insulin levels may have been not quite sufficient for full glycemic control when they were young (). However, these differences were only evident for a very brief period in young mice, and rather than having lasting adverse effects on glucose homeostasis, we found that a moderate reduction in basal insulin levels led to improved insulin responsiveness and lowered fasting glucose in aged Ins2mice. Since aging has been reported to be associated with the emergence of insulin secretory defects and worsening insulin responsiveness (), a lifetime of lessening the demands of insulin production in the β cell and also limiting insulin stimulation in the periphery could have contributed to improvements in metabolic health of aged Ins2mice. Although many investigators in the type 2 diabetes research field believe that insulin resistance is an early and causal factor in the progression to hyperinsulinemia and type 2 diabetes, there is evidence that insulin resistance can instead be the result of elevated or continuous exposure to the insulin ligand, causing tissue “desensitization” at the receptor or post-receptor level (). Our observation that moderately lowering insulin improved insulin responsiveness in aged mice suggests that insulin hypersecretion is a key upstream contributor to the insulin resistance that commonly develops with aging in mammals. Similarly, alleviating insulin hypersecretion in obese mammals has been shown to lead to improvements in insulin signaling and sensitivity (). Together, these findings indicate that hyperinsulinemia and insulin resistance have a more complex relationship with each other than widely appreciated.

+/− mice across both diets. Grpel2 has been reported to interact with the longevity-regulating mitochondrial Hsp70 ( Naylor et al., 1998 Naylor D.J.

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Finkel T. The mitochondrial basis of aging. Our data suggest that mild insulin reduction has general effects on healthspan, rather than affecting the risk of a specific set of ailments. Thus, we expected the underlying molecular mechanisms to be mediated through multiple pathways. Indeed, proteomic and transcriptomic analyses identified a broad range of pathways that were modulated in the direction expected for longevity and improved glucose homeostasis before these phenotypes became evident. Specifically, we noted differences in genes related to cellular metabolism and redox state, circadian rhythm, cell cycle, cholesterol/lipid homeostasis, and proteostasis. We identified Grpel2 as a transcript that was consistently and significantly decreased in both skeletal muscle and liver in Ins2mice across both diets. Grpel2 has been reported to interact with the longevity-regulating mitochondrial Hsp70 () and is linked to mitochondrial function and protein import into the matrix, which is particularly interesting in light of the putative roles for the mitochondrial unfolded protein response and mild energy stress in mediating longevity ().