Ames Dwarf (Prop1^df/Prop1^df ) mice derived from breeders were obtained from Southern Illinois University School of Medicine and described previously (7) and obtained from Dr. Michal Masternak at the University of Central Florida. All experiments were conducted at Joslin Diabetes Center with the approval of its Animal Care and Use Committee; mice were kept on a 12-h light/12-h dark cycle and had free access to water and food (LabDiet #5020; fat 21.6%, protein 23.2%, and carbohydrates 55.2%). The body weight, pancreas weight, blood glucose (BG), insulin, and IGF-1 were measured after 4 h of fasting at the age of 9–34 months in 18 Dwarf^+/+ and 11–14 months in nine Dwarf^+/− mice.

We used a mouse model in which Igf1r is specifically decreased in β-cells by crossing mice floxed for exon 3 of the Igf1r gene with mice expressing tamoxifen-inducible Cre recombinase under a rat insulin promoter (8, 9). Tamoxifen (Sigma-Aldrich, St. Louis, MO, USA; #10540-29-1) was dissolved by corn oil (Sigma; C8267) and administered via intraperitoneal injection (0.075 mg/g body weight) once every 24 h for five consecutive days to generate βIgf1rKD (Ins1-CreERT2^+/− Igf1r^flox/flox) mice. For comparison, βIgf1r (Ins1-CreERT2^−/− Igf1r^flox/flox) mice were treated with corn oil. Seven days after the final injection, islets were isolated and analyzed. To create an insulin resistance model, βIgf1rKD mice and littermate controls were fed a high-fat diet (Rodent Diet #D12492; fat 60 kcal%, protein 20%, and carbohydrate 20%) (HFD-βIgf1rKD) for a period of 5 weeks, which has been shown to induce insulin resistance in mice (10) and accelerate the appearance of senescent β-cells (5). Tamoxifen was administered during the first 5 days of the high-fat diet (HFD). Also, to test the effects of tamoxifen treatment in non-transgenic mice, 8–12-month-old male C57Bl/6 mice were used with and without treatment. All experiments included males and females, except where stated.

Quantitative reverse-transcription PCR (qRT-PCR) was carried out as previously described (5). Briefly, total RNA isolated with RNEasy Plus Mini Kit (QIAGEN, Valencia, CA, USA) was reverse transcribed (SuperScript reverse transcriptase; Invitrogen, Carlsbad, CA, USA). SYBR green (Thermo Scientific, Waltham, MA, USA; #A25741) and specific primers were used to detect the expression of specific genes. Expression levels were normalized using Actb as an internal control, and the comparative delta-delta threshold cycle (ΔΔCt) method was used to calculate relative gene expression levels. The sequences of each primer are listed in Supplementary Table 1.

Immunostaining of the pancreas in the Dwarf^+/+, Dwarf^+/−, βIgf1rKD, and βIgf1r mice was performed as described (5). Briefly, paraffin sections were deparaffinized with ethanol gradients, followed by washing with phosphate-buffered saline (PBS) and antigen retrieval with citric acid and permeabilization with a Triton X 0.3% solution. After washing with PBS and blocking with 1% normal donkey serum (NDS), the slides were incubated overnight with primary antibody IGF1R (1:100; sc-713 Rbt anti-Igf1rβ), INSULIN (1:400; Bio-Rad; 5330-0104G), and P21CIP1 (1:100; Cell Signaling, Danvers, MA, USA; 2947). Biotin-streptavidin amplification was used for IGF1R detection. Following subsequent washes, the slides were incubated overnight with a primary anti-insulin antibody. Incubations for 1 h with a secondary antibody for insulin were coupled with DAPI for nuclear staining. For each antibody, sections were stained and imaged in parallel such that the staining intensity reflected protein expression. For quantification, images were captured systematically covering the whole section in confocal mode on a Zeiss LSM 710 microscope.

Paraffin sections from 7-month-old male Dwarf^+/+ and Dwarf^+/− mice were stained in parallel and analyzed using confocal microscopy. Between 899 and 2,739 cells from 10 islets were counted from five animals per condition stained for IGF1R, INSULIN, and P21CIP1. The imaging settings were kept consistent between samples to allow for comparison of protein concentration based on intensity.

Glucose tolerance tests were conducted after 6 h of fasting. Blood samples were collected from the tails at 0, 15, 30, 60, 90, and 120 min after an intraperitoneal injection of glucose (2 g/kg body weight). For in vivo glucose-stimulated insulin secretion (GSIS), the insulin levels were measured from serum collected at the 0- and 15-min time points of the intraperitoneal glucose tolerance test (IPGTT). Four mice in each group were used for IPGTT in βIgf1rKD and βIgf1r. In the HFD study, seven mice were tested in the following groups: HFD-βIgf1rKD, HFD-βIgf1r, and Chow-βIgf1r. The serum levels of mouse insulin (Mercodia, Winston Salem, NC, USA; 10-1247-01) and Igf-1 (Invitrogen; EMIGF1) were measured using ELISA kits.

Islets were isolated by injecting collagenase Vitacyte (Indianapolis, IN, USA; CIzyme RI; 005-1030) into the pancreatic duct, followed by digestion in a 37°C water bath. After several washes with Medium199 (Thermo Scientific; 21180021) with 10% newborn calf serum (Sigma-Aldrich; N4762), islets were filtered through a 425-µm-diameter wire mesh (Cole-Parmer, Vernon Hills, IL, USA; SI59987-16) and collected by gradient separation with Lymphocyte Separation Medium (Corning, Glendale, AZ, USA; 25-072-CV). Islets from individual animals were kept separate and analyzed by qPCR or RNA-seq.

All Western blotting buffers and reagents were purchased from Bio-Rad (CA, USA). Anti-BETA ACTIN (Cat no. ab8226) antibody was purchased from Abcam (Cambridge, UK). Anti-IGF-I Receptor β (Cat no. 9750), anti-P21 (Cat no. 2947), and anti-PDX1 (Cat no. 5679) antibodies were obtained from Cell Signaling Technologies (MA, USA). The dilutions of all primary antibodies were 1:1,000.

Proteins were extracted from mouse islets using a 10X radioimmunoprecipitation assay buffer (Cell Signaling Technologies, MA, USA). All the mice used in Western blotting were male and female aged 4–9 months, with five mice per group. The concentration of the samples was quantified using bicinchoninic acid (BCA) assay. Equal amounts of protein for every sample (20 µg) were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto an Immuno-Blot polyvinylidene difluoride (PVDF) membrane using Bio-Rad wet transfer system. The blocking step was performed for 1 h at room temperature using 5% non-fat dry powder milk in TBST (Bio-Rad, CA, USA). After the blocking step, the membranes were incubated with the respective diluted primary antibodies overnight at 4°C. Following three washing steps, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1.5 h at room temperature. After three final washing steps, membranes were incubated with enhanced chemiluminescence (ECL) for 2 min, and the signals on the blots were visualized using ChemiDoc MP Imaging System (Bio-Rad, CA, USA).

Islets were isolated from four βIgf1rKD and four βIgf1r mice aged 17 months, including both genders in each group. The islets were resuspended in an RNA extraction buffer, and the RNeasy Micro Kit (No. 74004) was used to purify RNA and submitted for sequencing to DNA Link (Los Angeles, CA, USA). Data analysis was performed by the Bioinformatics and Biostatistics Core at Joslin Diabetes Center. The sequencing data have been deposited into Gene Expression Omnibus (GEO) from National Center for Biotechnology Information (NCBI) (accession GSE229709).

The statistical analysis was performed by using Graph Pad Prism. All the data were determined to be consistent with a normal distribution, and measurements were compared by t-test or one-way ANOVA followed by post-hoc Tukey’s test. The differences of p < 0.05 were considered to be statistically significant. In all the data, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To evaluate the differences between Ames Dwarf (Prop1^df /Prop1^df ) (Dwarf^+/+) mice and their heterozygous littermates (Dwarf^+/−), we measured body and pancreas weight, and blood glucose levels after 4 h of fasting. There was no significant difference in fed blood glucose levels between Dwarf^+/+ and Dwarf^+/− mice (Figure 1A). In Dwarf^+/+, body weight was decreased by 40%, while pancreatic weight was decreased by 50% (Figures 1B, C). Consistent with decreased pituitary growth hormone axis, Dwarf^+/+ mice had significantly lower circulating IGF-1 and insulin levels than Dwarf^+/− mice (Figures 1D, E). There were no gender differences in these parameters (Supplementary Figures 1A–H); therefore, the results are shown for both sexes combined.

To understand the effects of the Dwarf^+/+ genotype on β-cell IGF1R protein levels, pancreatic slides were stained and quantified for INSULIN and IGF1R. There was a 40% decrease in IGF1R protein intensity in the Dwarf^+/+ islets (Figures 1F–G′, J). Interestingly, INSULIN protein intensity levels in islet β-cells were also decreased by 40% (Figure 1K). These results are consistent with the downregulation of the IGF1R expression in β-cells. To examine the effects of this downregulation on senescence, the same sections were stained for P21CIP1, a marker and effector of senescence, which showed a significant decrease of 50% in β-cells (Figures 1H–I′, L). These results are consistent with decreased β-cell senescence in a genetic mouse model of decreased IGF1R in the same cell type. Dwarf^+/+ mice are a well-accepted and recognized model of longevity and delayed aging where the participation of the IGF1 pathway is well established (11, 12). However, these mice are global knockout that also lack other pituitary hormones (prolactin and thyroid stimulatory hormone). Therefore, to dissect the specific mechanistic role of IGF1R in β-cell senescence, we developed a tissue-specific conditional deletion model.

To understand whether senescent β-cells had increased levels of Igf1r, our previously published RNA-seq data (5) from senescent (βGal-positive) and non-senescent (βGal-negative) cell populations of mouse β-cells, were queried for Igf1r levels and pathway analysis. Pathway analysis revealed a significant decrease in the IGF1 pathway in the βGal-negative group compared to the βGal-positive group (Figure 2A). The levels of Igf1r were lower in the βGal-negative group (Figure 2B). These results support higher levels of IGF1R in senescent β-cells.

To specifically test the role of IGF1R in β-cell function, identity, and senescence, a conditional mouse model to knockdown IGF1R in β-cells was developed and induced by tamoxifen administration. To control for the acute and direct effects of tamoxifen treatment in islets, male C57Bl/6 mice aged 8–12 months were treated with the drug and with the vehicle. There were no significant differences in gene expression of key β-cell genes (Figure 2C), consistent with previous reports of a lack of effects of tamoxifen administration on glucose homeostasis (13, 14).

When βIgf1rKD (Ins1-CreERT2^+/− Igf1r^flox/flox) mice were treated with tamoxifen, there was a 30% reduction of Igf1r mRNA levels when compared to βIgf1r (Ins1-CreERT2^−/− Igf1r^flox/flox) (p < 0.05) (Figure 2D). IGF1R protein expression levels were decreased by 69% in islets of βIgf1rKD compared to βIgf1r (Figures 2E, F). These results were normalized to PDX1, which is only expressed in β-cells and therefore excludes potentially confounding results coming from IGF1R expression from other islet cell types, such as α, δ, or PP cells. In addition, P21CIP1 expression was significantly reduced in the islets of βIgf1rKD compared to that of the βIgf1r mice (Figures 2E, G). We also performed immunostaining of pancreas sections for P21CIP1 and insulin, which showed a significant decrease in their expression levels in βIgf1rKD compared to βIgf1r mice (Figures 2H–J). Consistent with decreased senescence when IGF1R is downregulated, P21CIP1 levels were decreased by 50% in β-cells in the islets from βIgf1rKD mice (Figure 2J).

To understand the effects of decreased IGF1R levels on β-cell function and glucose homeostasis, we performed IPGTT in βIgf1rKD and βIgf1r mice. βIgf1rKD showed significantly lower glucose levels at 15 and 60 min after glucose injection, also seen in the area under the curve (AUC), consistent with improved glucose clearance (Figures 2K, L). Additionally, in vivo evaluation of GSIS revealed a loss of glucose responsiveness in βIgf1r mice, which tended to be restored after decreasing levels of Igf1r in β-cells (Figure 2M). These results suggest that the downregulation of Igf1r in β-cells decreases senescence, potentially improving glucose homeostasis.

To understand the overall transcriptional changes in islets from βIgf1rKD, bulk RNA-seq analysis was performed and showed that 945 genes out of 14,806 were differentially expressed between the βIgf1rKD and βIgf1r groups. Since there are no specific universal senescent markers that are valid across all cell types and tissues, it is recommended that a combination of markers be used to identify senescent cells. Analysis of senescence and SASP factor gene expression included a subset of the recently published SenMayo panel (15) (Figures 3A, B), which showed overall downregulation in islets from βIgf1rKD mice. The expression level of the p16^Ink4a , a known senescence marker and effector, was significantly decreased in βIgf1rKD mice compared to that of the control (Figure 3E). βIgf1rKD mice showed increased expression of β-cell function and hallmark genes when compared to βIgf1r mice (Figures 3C, D), consistent with improved glucose clearance and in vivo GSIS. Additionally, pathway analysis revealed increased expression of three pathways that are known to go down with aging: sulfide oxidation to sulfate, autophagy, and mTOR signaling (Figure 3F). Specific gene analysis of the autophagy pathway (Supplementary Figures 2A, B) revealed the upregulation of many of these genes. Given that autophagy is among the most important protective catabolic processes in the cells activated when the cells experience different stressors that disturb homeostasis, it is suggested that IGF1R in β-cells might be interacting with other hallmarks of aging (autophagy) in addition to senescence. Further, Reactome pathway analysis for the RNA-seq of the two groups, βIgf1r and βIgf1rKD, revealed two additional and relevant upregulated pathways when Igf1r is knocked down: G2-M DNA damage checkpoint and DNA double-strand break response (Supplementary Figures 2C–F), both of which are directly connected to senescence. Collectively, these data suggest that conditional downregulation of Igf1r in β-cells in adulthood ameliorated cellular senescence through the enhancement of DNA damage mechanisms. Additionally, it decreased markers of senescence and increased functional and hallmark genes in the cells that are potentially primed to undergo senescence, as shown by a recovery of their transcriptional identity and decreased expression of senescence genes.

The induction of senescence in β-cells and adipose tissue by HFD has been previously shown by us and others (5, 16–18). Herein, mice were fed HFD for 5 weeks to evaluate the effect of βIgf1r knockdown in an adult model of insulin resistance (which is known to increase β-gal activity) and transcription of senescence and SASP genes. Tamoxifen or vehicle was administered during the first 5 days of HFD to induce knockdown of the Igf1r in β-cells. Since the effects of βIgf1r knockdown in the chow diet are shown in Figures 2, 3, this section only compared the knockdown group during HFD. During the 5 weeks, the AUC of fed glucose level in HFD fed βIgf1rKD (HFD-βIgf1rKD) was significantly lower than that in HFD fed βIgf1r (HFD-βIgf1r) (Figures 4A, B). HFD-βIgf1rKD mice had improved glucose clearance during IPGTT (Figures 4C, D). During the first 10 days, body weight was decreased in the HFD-βIgf1rKD when compared to HFD-βIgf1r (Supplementary Figures 3A, B); however, there were no differences in the food consumption measured between days 11 and 15 (Supplementary Figures 3C, D). This initial weight loss might have been caused by nausea, which is known to be induced by tamoxifen treatment, which preceded the switch in diet (19).

In the HFD group, βIgf1rKD led to a significant downregulation in the expression of senescence and SASP genes in pancreatic islets: p16lnk4a, p21Cip1, Il1a, and Il-6 (Figure 4E). The results of the βIgf1rKD experiment under a normal chow are shown in Figure 2.

Overall, these results show that the downregulation of Igf1r in both global and β-cell-specific mouse models is associated with decreased β-cell senescence, improved function, and cellular identity (Figure 5). This establishes a link between two hallmarks of aging, cellular senescence and deregulated nutrient-sensing, which might be relevant for the loss of cell identity and increased senescence during diabetes progression.