p16(Ink4a)-induced senescence of pancreatic beta cells enhances insulin secretion

Abstract

Cellular senescence is thought to contribute to age-associated deterioration of tissue physiology. The senescence effector p16(Ink4a) is expressed in pancreatic beta cells during aging and limits their proliferative potential; however, its effects on beta cell function are poorly characterized. We found that beta cell-specific activation of p16(Ink4a) in transgenic mice enhances glucose-stimulated insulin secretion (GSIS). In mice with diabetes, this leads to improved glucose homeostasis, providing an unexpected functional benefit. Expression of p16(Ink4a) in beta cells induces hallmarks of senescence--including cell enlargement, and greater glucose uptake and mitochondrial activity--which promote increased insulin secretion. GSIS increases during the normal aging of mice and is driven by elevated p16(Ink4a) activity. We found that islets from human adults contain p16(Ink4a)-expressing senescent beta cells and that senescence induced by p16(Ink4a) in a human beta cell line increases insulin secretion in a manner dependent, in part, on the activity of the mechanistic target of rapamycin (mTOR) and the peroxisome proliferator-activated receptor (PPAR)-γ proteins. Our findings reveal a novel role for p16(Ink4a) and cellular senescence in promoting insulin secretion by beta cells and in regulating normal functional tissue maturation with age.

Figure 1

p16 induces senescence of beta cells, (a) FACS analysis of p16 and Insulin expression in islet cells from control Ins2-rtTA (left) and Ins2-rtTA;tet-p16 (right) mice after tet treatment. The experiment was done ten times, (b) Representative images showing Immunostaining of human p16 (red), the proliferation marker Ki67 (green) and Insulin (to label beta cells; blue) in pancreatic Islets (dotted line) of control Ins2-rtTA mice (left; of 12 images) and Ins2-rtTA;tet-p16 mice (right; of 21 images) (n = 3 mice per group). Arrows Indicate Ki67⁺p16⁻ cells, (c) FACS analysis of p16 and Ki67 expression in insulin⁺ cells from dissociated iindicated. The experiment wasslets of control Ins2-rtTA (left) and Ins2-rtTA;tet-p16 (right) mice. The experiment was repeated three times, (d) FACS analysis of SA–β-Gal activity (as measured by the fluorescent β-Gal substrate C₁₂FDG) In Islet cells from the Indicated mice. The ratio of SA–β-Gal⁺ cells in p16-expressing versus control mice (p16/Ctl) is Indicated. The experiment was done twice, (e) FACS analysis of the expression of the lysosomal protein Lamp2a In insulin⁺ cells from the Indicated mice. Red line shows p16⁺ cells, (f) Representative images of islets from the indicated mice that were stained, for the purpose of measurement of beta cell size, for human p16 (red), insulin (green) and E-cadherin (Cdh 1, white), (g) Quantification of the cross-sectional areas of beta cells from control Ins2-rtTA mice (left) and of p16⁺ beta cells from Ins2-rtTA;tet-p16 mice (right), done by image analysis of sections stained as in f (n = 6 mice per group; >100 cells were measured in each mouse). Data are mean ± s.d. **P < 0.005; by Student’s t-test. (h) Forward scatter (FSC-A) histograms of insulin⁺ cells from the Indicated mice. Red line shows p16⁺ cells. The experiment was repeated five times, (i) Representative images showing Pdx1 (blue) and phospho-S6 (pS6, green) expression in islets from control Ins2-rtTA (left; of 12 images) and Ins2-rtTA;tet-p16 (right; of 24 images) mice (n = 3 mice per group), (j) Enrichment significance of gene sets associated with senescence (sen) and proliferation (top graph), or with beta cell maturation and differentiation (bottom graph), among genes upregulated (red) or downregulated (blue) in p16-expressing beta cells. Values indicate −log₁₀ (P value); by hypergeometric test. MEFs, mouse embryo fibroblasts; IMR90, normal human fibroblasts; fibrosis-dn, genes downregulated in senescent stellate cells. Throughout, scale bars, 20 μm.

Figure 2

p16 expression enhances insulin secretion, (a) Insulin levels secreted by islets isolated from indicated tet-treated mice and incubated in medium with low (2.8 mM) or high (16.7 mM) glucose concentrations for 1h. Secreted insulin levels were normalized to the insulin content of each sample and are shown relative to the value observed for control islets in medium with high glucose (defined as 1). Dots indicate individual mice (n = 8 per group, combined from two independent experiments), and each shows the mean of three replicates per mouse. The experiment was done four times, (b) Mean insulin levels secreted by islets from the indicated mice and assayed as in a. Secreted insulin levels were normalized to beta cell number in each sample and are shown relative to the value observed for control islets in medium with high glucose (defined as 1). Islets were pooled from three mice and assayed in five replicates per group, (c) Insulin content in equal numbers of beta cells, sorted on the basis of GFP expression, from control (Ins2-rtTA;tet-GFP) or p16-expressing (Ins2-rtTA;tet-GFP;tet-p16) mice (n = 2 per group). Values are presented relative to those of the control, (d) Glucose tolerance test of wild-type (WT, n = 2), Pdx1-tTA (n = 2) and Pdx1-tTA; tet-p16 (n = 4) mice following p16 activation for 2 weeks. The experiment was repeated in three independent mouse cohorts, (e) Serum insulin concentrations in the indicated mice after overnight fasting (fast) or 10 min after glucose injection (glucose) (n = 3 mice per group), (f) Insulin tolerance test in the indicated mice following p16 activation for 10 d (n = 3 mice per group), (g,h) Glucose tolerance test of WT (n = 3), Pdx1-tTA (n = 4) and Pdx1-tTA;tet-p16 (n = 6) mice following p16 activation for 2 (g) or 5 (h) months, (i) Percentage of insulln⁺) area in pancreatic sections of the indicated mice at 2 weeks, 2 months or 5 months after p16 activation (n = 3 mice per group). Error bars indicate s.e.m. in all panels, except in b (in which they indicate s.d.). Throughout, *P< 0.05, ** P< 0.005, ***P< 0.0005; n.s., not significant; by Student’s t-test.

Figure 3

p16 expression increases glucose uptake and mitochondrial activity, (a) Relative mRNA levels of Gck and Aldob in GFP⁺ beta cells of control Ins2-rtTA;tet-GFP(n = 2) and p16-express¡ng Ins2-rtTA;tet-GFP;tet-p16 (n = 3) mice. Error bars indicate s.e.m. (b) Relative glucose uptake rates in dissociated islet cells from the indicated mice, as measured by FACS analysis after incubation with the fluorescent glucose analog 2-NBDG for 30 mm. Values are mean ± s.e.m. of five samples, each comprised of cells from three mice, (c) Relative mRNA levels of Ppargc1a in mice (indicated as in panel a) (left) and western blot of Pgc-1α in control and p16-expressing islets (right; done twice). Hsp90 is shown as a loading control. Error bars indicate s.e.m. (d) FACS analysis of islet cells from the indicated mice that were stained with MitoTracker to label mitochondria. p16/Ctl indicates the ratio of the mean fluorescence value from p16-expressing cells to that from control cells, (e) Representative electron microscopy images of beta cells from control Ins2-rtTA (left) and Ins2-rtTA;tet-p16 (middle) mice. Right, quantification of the mitochondrial area as a percentage of the analyzed images. Values indicate mean (± s.e.m.) of control cells (n = 20) and cells from p16-expressing islets (n = 23), obtained from a total of five mice per group. Yellow arrows indicate mitochondria. Scale bars, 2 μm. (f) FACS analysis of expression of the mitochondrial protein Atp5a in insulin⁺ islet cells from the indicated mice. Red line shows p16⁺ cells. The experiment was done once, (g) Mitochondrial DNA content, as measured by qPCR of the mitochondrial cytochrome b gene (mt-Cytb), in DNA extracted from equal numbers of GFP⁺ cells pooled from five mice per indicated group. Values were normalized to the levels of the L1 genomic repeat sequence and are presented as a mean of triplicate reactions ± s.e.m. (h) Left, FACS analyses of GFP⁺ cells isolated and pooled from five mice per group after incubation in medium with low (3 mM) or high (20 mM) concentrations of glucose (as indicated) and stained with the mitochondrial membrane potential indicator dye TMRE. Right, representative images of cells stained with TMRE after incubation in high-glucose medium as measured by FACS. The FACS experiment was repeated three times. Scale bars, 10 μm. (i) Oxygen-consumption rates of islets isolated from the indicated mice. Glucose (20 mM), the membrane uncoupler FCCP, and the electron transport chain inhibitors rotenone + antimycin A were added at the indicated times. Values were normalized to islet insulin content in each sample and are presented relative to the basal oxygen-consumption levels of control islets (defined as 1). Data are mean ± s.e.m. of five replicates, each containing 40 islets pooled from five mice. The experiment was repeated three times. Throughout, *P< 0.05, **P< 0.005, ***P< 0.0005; by Student’s t-test.

Figure 4

Increased insulin secretion in mature mice is driven by p16. (a) GSIS of islets isolated from WT mice of the indicated ages. Dots indicate individual mice, three replicates per mouse; values were normalized to insulin content and are presented relative to mean secretion levels in islets from 1-month-old mice incubated in medium with the high glucose concentration (defined as 1) (1-month-old mice, n = 10; 6-month-old mice, n = 5; 11-month-old mice, n = 5; 27-month-old mice, n = 3). (b) FACS analysis of endogenous p16 expression in insulin⁺ cells from WT mice of the indicated ages. The experiment was done twice, (c) FSC-A histogram of insulin⁺ cells from WT mice of the indicated ages. The experiment was done three times, (d) FACS analysis of SA–β-Gal activity (as measured by C₁₂FDG fluorescence) in islet cells from WT mice of the indicated ages. The experiment was done twice, (e) GSIS of islets from 6-month-old WT and p16-deficient mice (n = 6 per group). Values were normalized to insulin content and are presented relative to the secretion level of control islets incubated in medium with high glucose (defined as 1). (f) FSC-A histogram of insulin⁺ cells from 6-month-old control and p16-deficient mice. The experiment was done twice, p16-null/Ctl indicates the ratio of the mean fluorescence value from p16-deficient cells to that from the control cells, (g) GSIS of islets from 2-month-old MIP-CreER;CDK4^+/lsl-R24C and control MIP-CreER; CDK4^+/+ mice 2 weeks after Cre activation (n = 5 per group). Values were normalized to insulin content and are presented relative to the secretion level of control islets incubated in medium with high glucose (defined as 1). Throughout, error bars indicate mean ± s.e.m. *P< 0.05, ***P< 0.0005; by Student’s t-test.

Figure 5

Senescent beta cells in human islets, (a) Representative islet sections of juvenile (left; 5-month-old) and adult (middle; 45-year-old) human subjects stained for insulin (blue) and p16 (green), and quantification of the mean intensity of p16 staining in juvenile subjects (5-month-old to 10-year-old; n = 6; 46 sections stained) and adult subjects (30- to 60-year-old; n = 5; 40 sections stained) (right), a.u., arbitrary units, (b) Relative p16 mRNA levels in islets from human subjects aged 6 months to 60 years (n = 18). (c) FACS analysis of dissociated islets from a 42-year-old subject stained for insulin and p16. Red dots indicate insulin⁺p16⁺ cells. The experiment was done three times, (d) FSC-A histograms of insulin⁺p16⁺ and insulin+p16− cells within islets of a 56-year-old subject. The experiment was done three times, (e) Representative islet sections of juvenile (left; 5-month-old) and adult (right; 51-year-old) human subjects stained for insulin (blue) and pS6 (green). Subjects as in a; 61 islet sections from juvenile and 51 sections from adult human subjects were stained, (f) SA–β-Gal activity (measured by C₁₂FDG fluorescence) in live islet cells from a 53-year-old (left) and a 14-year-old (right) human subject. Five adults and one young subject were analyzed (see Supplementary Table 30). (g) FACS analysis of TMRE-stained SA–β-Gal⁺ and SA–β-Gal⁻ islet cells isolated from a 53-year-old human subject. The experiment was repeated three times, (h) Representative islet sections from a 5-year-old (left) and a 45-year-old (middle) human subject stained for insulin (blue) and the mitochondrial protein COX17 (green), and quantification of mean COX17 intensity In sections from subjects as in a (juvenile, n = 46 sections; adult, n = 46 sections) (right). Throughout, error bars indicate s.e.m. *P < 0.05, ***P < 0.0005; by Student’s t-test. Scale bars, 20 μm.

Figure 6

p16-induced senescence of human cells leads to enhanced GSIS. (a) FACS analysis of Ki67-stained EndoC-βH2 cells 3 weeks after infection with a lentivirus expressing GFP (blue line) or Cre + GFP(red line); GFP⁺ cells are shown. The experiment was done three times, (b) Representative images (out of ten taken) of EndoC-βH2 cells expressing GFP (left) or Cre + GFP (right) stained for SA–β-Gal activity (blue) in 3 weeks after infection, (c) Representative images (out of ten taken) of EndoC-βH2 cells expressing either GFP (left) or Cre + GFP (right) that were stained for insulin (red), (d) Insulin secretion levels by EndoC-βH2 cells expressing either GFP (white) or Cre+ GFP(gray) after incubation in medium containing low (2.8 mM) or high (16.7 mM) glucose concentrations for 1 h. Values are the mean of four replicates per group ± s.d., normalized to cell number and shown relative to insulin levels in control cells incubated in medium with high glucose (defined as 1). (e) FACS analyses of FSC-A, TMRE staining and 2-NBDG fluorescence in control and Cre-expressing cells. The ratios indicate the mean fluorescence value from Cre-expressing to that from control cells, (f) Representative images (out of ten taken) of SA–β-Gal activity (blue) in EndoC-βH2 cells infected with an empty vector alone (left), an empty vector followed by the Cre-expressing lentivirus (middle) or an shp16-expressing construct followed by the Cre-expressing lentivirus (right), (g) Insulin secretion levels by EndoC-βH2 cells infected with an empty vector alone, an empty vector followed by the Cre-expressing lentivirus or an shp16-expressing construct followed by the Cre-expressing lentivirus (black), analyzed as in d. Values are the mean of five replicates ± s.d. (h) FACS analysis of TMRE staining in the cells indicated in g. (i) Left, insulin secretion levels by control (GFPonly) and Cre-expressing cells treated with vehicle or the mTOR inhibitor Torin1 for 3 weeks. Right, similar analysis of control (vector only) and Cre-expressing cells treated with either vehicle or the PPAR-γ inhibitor GW9662 for 3 weeks. Values are the mean of five replicates ± s.d. (j) Schematic diagram summarizing the effects of p16-induced senescence on beta cell function. Components of the senescence program that contribute to increased insulin secretion are highlighted in red. Question mark represents potential additional effectors. Throughout, **P< 0.005, ***P< 0.0005; by Student’s t-test. Scale bars, 20 μm.