β Cell Aging Markers Have Heterogeneous Distribution and Are Induced by Insulin Resistance

Abstract

We hypothesized that the known heterogeneity of pancreatic β cells was due to subpopulations of β cells at different stages of their life cycle with different functional capacities and that further changes occur with metabolic stress and aging. We identified new markers of aging in β cells, including IGF1R. In β cells IGF1R expression correlated with age, dysfunction, and expression of known age markers p16^ink4a, p53BP1, and senescence-associated β-galactosidase. The new markers showed striking heterogeneity both within and between islets in both mouse and human pancreas. Acute induction of insulin resistance with an insulin receptor antagonist or chronic ER stress resulted in increased expression of aging markers, providing insight into how metabolic stress might accelerate dysfunction and decline of β cells. These novel findings about β cell and islet heterogeneity, and how they change with age, open up an entirely new set of questions about the pathogenesis of type 2 diabetes.

Keywords: aging markers; beta-cell heterogeneity; islets.

Figure 1. Development of insulin resistance, loss of glucose tolerance and changes of insulin secretion as C57Bl/6J mice age

Elevation of fasting blood glucose (A), glucose intolerance (B), hyperinsulinemia (C), peripheral insulin resistance as determined by an insulin tolerance test (D) and weight gain (E) were observed in 2y mice compared to 1.5m mice. n=6 for 1.5m; n=11 for 2y; *p<0.05 to 1.5m F. By static incubation, glucose stimulated insulin secretion (GSIS) increased with age. n=3 independent experiments in triplicate, *p<0.01 respect to 12w. G. By RHPA, aged mice had decreased proportion of cells secreting at high glucose, particularly those secreting medium and large plaques (arrows). n= 495–1008 cells counted/condition, 600–700 fields/condition from two independent experiments. Mean±SEM. Table 1 shows the aggregate data and the applicable statistics.

Figure 2. Heterogeneity patterns of β cells change with age as seen with FACS analysis of GFP+ cells and measurement of aging markers

Subpopulations of β-cells based on GFP intensity change with age (A) and have increasing expression of p16^Ink4a with age (B), supporting the notion of aging heterogeneity. 5–9w:n=3; 4–10m: 7; 1–2y:8; *p<0.04 to previous stage; +p<0.04 to 5–9w. For p16^Ink4a: e15.5-5w:n=7;2.5–7.5m:3;1.5–2y:3. ANOVA < 0.0001; *p<0.0001 to youngest group. ND, not detected. Mean±SEM C. Heatmaps of expression data from microarrays of β cells of MIP:GFP mice showing changes in key β cell and senescence signature genes (see Suppl Fig 3). D. Volcano plot of differentially expressed cell surface genes (see Suppl Table 1). E. Expression of Igf1r mRNA increased in GFP ^high/medium cells at 1.5- 2y. Same samples as for p16^ink4a (F) Potential aging surface markers verified by qPCR on isolated islets: p16^Ink4a, Igf1r, Bambi, Kcnq5, and Fgfr1. Samples were from individual mice; n=10–18 per age; *p<0.05 to previous age. Mean±SEM

Figure 3. Islet heterogeneity for markers of aging in mouse and human pancreas

A. By immunostaining increased IGF1R and decreased KCNQ5 was found in islets from older mice compared to young. Magnification bar= 25 μm Intensity of individual islet staining quantified for IGF-1R (B), KCNQ5 (C) and Insulin (D). n=3 animals per age. *p<0.05 respect to previous age. E. Age dependent IGF-1R expression in human islets supports the validity of this marker. Pancreas from 2 donors per decade (aged fetal to 68 y); 15–92 islets/cell clusters per donor; total 400 human islets. Quantification given in Suppl Fig 6A. Magnification bar= 25 μm F. FACS plot showing sorting by IGF1R for β cells. G. Percentage of IGF1R+ β cells per age. n=7 independent cell preparations. H. IGF1R+ β cells express higher p16^Ink4a, Ldha and Flattop mRNA as compared to unsorted. n=5 samples, each pooled from 3–7 1.5–2y mice. #p<0.05. I. GSIS profiles comparing IGF1R- and IGF1R+ subpopulations from 4 independent cell preparations of 4 m mice. J. Knockdown experiments in MIN6 cells transfected with p16^Ink4a, Igf1r or scrambled siRNA (siSc) show p16^ink4a affects Igf1r expression. n=3 independent experiments in triplicate. Mean±SEM

Figure 4. Senescent β cells increase in proportion through adulthood

A. Representative images for the senescence marker nuclear P53BP1 in β cells across ages. Magnification bar= 25 μm B. Cellular co-localization of IGF1R and p53BP1 increased with age. C. Representative images of adjacent sections of islet from 1y MIP:GFP mouse costained for GFP, Insulin, and glucagon/somatostatin/pancreatic polypeptide or for GFP, p53BP1 and DAPI showing p53BP1+ cells are mostly GFP^lowinsulin+. Magnification bar= 30 μm D. FACS plot showing sorting criteria for β cells stained for acidic β-Galactosidase (B-Gal) activity. E. Proportion of B-Gal ^positive β cells increased with age. n=5 independent preparations, each pooled of 3–5 mice. F. B-Gal ^positive and ^negative subpopulations from mature (3–6 m, n=3) and aged (1.8–2y,n=4) mice show higher Igf1r and p16^Ink4a mRNA levels in B-Gal+ cells at both ages. Data normalized to negative cells. #p<0.05. Mean±SEM

Figure 5. Islet heterogeneity at both protein and mRNA levels

Adjacent islets showed heterogeneous staining of aging markers CD99 and IGF1R (A–C, merged and split channels) and KCNQ5 (D) indicating islet heterogeneity in adult pancreas. Magnification bar=50 μm. Heterogeneity of islets was further supported by similar insulin content (E) and marked differences in single islet mRNA content for Igf1r (F) and Kcnq5 (G). Handpicked 150 μM diameter islets: for RNA, 8 islets from same 1.5y mouse; for insulin content, 12 islets, 4 from each of 3 1.5y mice. Individual islet values shown.

Figure 6. Acceleration of the appearance of aging markers in β cells by insulin resistance and metabolic stress

In 7m C57Bl/6J mice, 7 d of S961 administration induced hyperglycemia (A) and hyperinsulinemia (B), which were reversed by 2 w recovery period. C. Islets of these mice had increased expression of p16^Ink4a, Igf1r and Bambi mRNA that reversed in 2w recovery period. Data related to control values. n= 4–12mice / group;# *p<0.01 D. In islets from Ins2^Akita(Akita) mice, a model of ER stress, Igf1r mRNA increased and both p16^Ink4a and Bambi mRNA tended to increase, compared to wildtype (WT). n=4 mice/group *p< .005. Islets from INK-ATTAC mice show heterogeneity of both GFP and FLAG protein at 10 m (E) as well as age-related changes in Igf1r and Kcnq5 mRNA (F). n=4 mice/age. *p<0.02 Magnification bar=40 μm. Mean±SEM