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Inhibition of mTORC1 by ER stress impairs neonatal β-cell expansion and predisposes to diabetes in the Akita mouse

Research Article
Cite this article as: eLife 2018;7:e38472 doi: 10.7554/eLife.38472
11 figures, 1 table, 4 data sets and 1 additional file

Figures

Figure 1 with 2 supplements
β-Cell mass, turnover and differentiation in adult Akita mice.

Analyses were performed on 2- to 3-month-old Akita mice and age-matched controls. (a) β-cell mass (n = 6 in each group); (b) β-cell proliferation and apoptosis assessed by staining for insulin and Ki67 (n = 6–7 mice in each group; a total of 4909 wild type (WT) and 2523 Akita β-cells were quantified) or TUNEL (n = 4–5 mice in each group; 2592 WT and 1754 Akita β-cells). The percentage of Ki67+ and TUNEL+β-cells is shown in the table above; (c–d) β-cell differentiation was assessed by lineage tracing. Wild-type and Akita mice were crossed with RIP-Cre:Rosa26-Yfp reporter mice; (c) pancreatic sections of Akita mice were immunostained for insulin and somatostatin or glucagon. Lineage-traced β-cells (YFP+) expressing somatostatin or glucagon is shown in squares and zoomed in; (d) quantification of insulin-expressing β-cells (percentage of insulin+/YFP+ cells), insulin-degranulated β-cells (percentage of insulin-/YFP+ cells) and of cells with misexpression of somatostatin or glucagon (percentage of somatostatin+ or glucagon+/YFP+ cells) in WT and Akita mice is shown; *p<0.05.

https://doi.org/10.7554/eLife.38472.003
Figure 1—figure supplement 1
Glycemia and β-cell function in adult Akita and control mice.

(a) Fed blood glucose (n = 11–15 in each group); (b) IPGTT- glucose (1.5 g/kg) was injected intraperitoneally after an overnight or 4 hr fast (n = 4–5 in each group); (c) pancreatic insulin content analyzed by ELISA on whole pancreas extracts (n = 4–5 in each group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.004
Figure 1—figure supplement 2
NKX6.1 and PDX-1 expression in adult Akitaβ-cells.

Pancreatic sections of 2- to 3-month-old Akita and control mice were stained for NKX6.1 (n = 5 mice in each group; 3348 WT and 2370 Akita β-cells) or PDX-1 and insulin (n = 5–7 mice in each group; 4580 WT and 2320 Akita β-cells). Values are mean ± SE. **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.005
Dynamic changes of body and pancreas growth and glycemia, β-cell mass, proliferation and differentiation in Akita and control mice at P1-2.

(a) body weight, (b) pancreas weight of wild-type and Akita mice at P1-2, P19-21 and at the age of 2–3 months. (a) P1-2: WT (n = 8); Akita mice (n = 4), P19-21: WT (n = 21); Akita mice (n = 23), 2–3 months: WT (n = 33); Akita mice (n = 39); (b) P1-2: WT (n = 8); Akita mice (n = 4), P19-21: n = 14 in each group, 2–3 months: n = 17 mice in each group). (c) fed blood glucose (n = 7–8 mice in each group); (d) β-cell mass (n = 5 mice in each group); (e) β-cell proliferation assessed by immunostaining for insulin and Ki67 (n = 4 mice in each group; 1886 WT and 1483 Akita β-cells). The percentage of Ki67β-cells is shown in the table above; (f–g) quantification of β-cells (insulin+) expressing NKX6.1 (n = 3–4 mice in each group; 1148 WT and 1808 Akita β-cells) and PDX-1 (n = 3–5 mice in each group; 1364 WT and 1507 Akita β-cells). *p<0.05, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.006
Metabolic state, β-cell function and mass in pre-weaning (P19-21) Akita mice and age-matched controls.

(a) fed blood glucose (n = 7 in each group); (b) IPGTT- glucose (1.5 g/kg) was injected intraperitoneally after an overnight fast (n = 5 in each group); (c) glucose-stimulated insulin secretion in vivo. Insulin was measured before and 15 min following IP glucose injection (1.5 g/kg); (d) pancreatic insulin content (n = 4–5 in each group); (e) basal (3.3 mmol/l glucose) and stimulated (16.7 mmol/l glucose) insulin secretion and insulin content of Akita and control islets analyzed by static incubations. Islets were divided into 4 batches of 25 islets per group (n = 3); (f) β-cell mass (n = 6 mice in each group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.007
Figure 4 with 1 supplement
Dynamic changes in β-cell expansion in Akita and control mice.

(a) β-cell proliferation assessed by immunostaining for insulin and Ki67 (n = 6 mice in each group; 2541 WT and 3391 Akita β−cells); (b) β-cell size at P1-2 (newborn, n = 4–5 mice in each group; 334 WT and 435 Akita β-cells), P19-21 (pre-weaning, n = 3 mice in each group; 330 WT and 364 Akita β−cells) and in adult mice (2–3 month-old, n = 3 mice in each group; 266 WT and 417 Akita β−cells) assessed by immunostaining for E-cadherin and insulin. Quantifications of β-cell size (c), proliferation (d), and mass (e) are shown. *p<0.05, **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.008
Figure 4—figure supplement 1
Proliferation of β-cells and exocrine cells in pre-weaning Akita and control mice.

Analyses were performed on pancreatic sections of Akita mice and age-matched controls at 19–21 days stained for proliferation markers. (a) β-Cell proliferation was assessed by staining for insulin and PCNA (n = 3 mice in each group; 1241 WT and 1944 Akita β cells), or phospho-Histone H3 (H3P n = 3 mice in each group; 1176 WT and 1982 Akita β−cells). (b) Proliferation of pancreatic exocrine cells in pre-weaning Akita and control mice. Pancreatic sections of pre-weaning Akita and control mice were stained for insulin and Ki67 and exocrine cells surrounding the islets were used for quantification. The percentage of proliferating exocrine cells (Ki67+/INS-) is shown (n = 6 mice in each group; 2486 WT and 2670 Akita cells). *p<0.05, **p<0.01.

https://doi.org/10.7554/eLife.38472.009
Figure 5 with 1 supplement
Transcriptomic analysis of ER stress markers and β-cell gene signature in neonate Akita islets.

(a) RNA-seq comparing the transcriptome of islets from P19-21 Akita and age-matched control mice (n = 3 samples in each group, each sample is a pool of islets from three mice). Columns represent pathways that are differentially regulated in Akita mice; (b) expression of UPR and apoptosis genes and of Nkx6.1 and Pdx1 in islets of Akita compared to control mice at P19-21. Spliced and total Xbp1 were also quantified by qPCR. The spliced/total Xbp1 ratio is shown beside (n = 3); (c–d) heat map of genes regulated by NKX6.1 (c) and PDX-1 (d) in Akita islets and controls. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.010
Figure 5—figure supplement 1
Islet composition of wild-type and Akita mice.

β/α cell ratio and number of β and α cells per islet area in P19-21 wild-type and Akita mice (n = 44 and 35 islets isolated from three to four mice in each group).

https://doi.org/10.7554/eLife.38472.011
Effects of ER stress on the expression of β-cell transcription factors in neonate Akita islets (P19-21) and islets treated with thapsigargin.

(a) PDX-1 and BiP protein level analyzed by Western blotting (n = 3, each sample is a pool of islets from four to six mice); (b) quantification of NKX6.1 (n = 3 mice in each group; 1646 WT and 728 Akita β−cells), and PDX-1 (n = 3 mice in each group; 1534 WT and 844 Akita β−cells) expressing β-cells. Pancreatic sections were immunostained for NKX6.1 or PDX-1 and insulin. The percentage of NKX6.1- and PDX-1-positive β-cells is shown. (c) Islets from young (P19-21) and adult wild-type mice were treated with low-dose thapsigargin (50 nmol/l) and TUDCA (250 µmol/l) or PBA (2.5 mmol/l) for 48 hr with daily media changes and further analyzed by western blotting for PDX-1 and BiP (n = 3, each sample is a pool of islets from six to nine mice); (d) INS-1E cells were treated with 20 nmol/l thapsigargin for 24 and 48 hr followed by western blotting for PDX-1. **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.012
Effects of ER stress on IRS2/Akt signaling in Akita islets and in INS-1E treated with low-dose thapsigargin.

(a) IRS2/Akt signaling in islets from neonate (P19-21) and adult wild-type and Akita mice. Each sample is a pool of islets from 4 to 15 mice (n = 4 for neonate islets and n = 2 for adult islets). (b–c) INS-1E cells were treated with 20 nmol/l thapsigargin for 24 and 48 hr followed by western blotting for IRS2, total and phosphorylated Akt (Ser473 and Thr308) and S6 (Ser240/244). A representative experiment (b) and quantification (c) are shown (n = 4–6). *p<0.05.

https://doi.org/10.7554/eLife.38472.014
Figure 8 with 1 supplement
mTORC1 signaling in neonate and adult Akita islets.

(a) Western blot analysis of S6 and 4EBP1 phosphorylation in islets of neonate (P19-21) and adult wild-type and Akita mice. Quantification of phosphorylated S6 in neonate Akita compared to control islets is shown (n = 3, each sample is a pool of islets from 4 to 7 mice); (b) immunostaining for phospho-S6 on pancreatic sections of P1-2, P19-21 and adult Akita mice and age-matched controls and quantifications of the percentage of S6β-cells (P1-2: n = 4 mice in each group; 1159 WT and 1655 Akita β−cells; P19-21: n = 6 mice in each group; 2259 WT and 1567 Akita β−cells; adult: n = 4–5 mice in each group; 2391 WT and 1383 Akita β-cells). Islet boundaries are marked by dotted line; (c) adult Akita mice were treated with 25 mg/kg dapagliflozin in drinking water for 72 hr. Blood glucose in dapagliflozin-treated Akita mice was ~ 200 mg/dl compared to ~ 500 mg/dl in control Akita mice. Pancreatic sections were immunostained for insulin and phospho-S6 (n = 3 mice in each group). *p<0.05, **p<0.01, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.015
Figure 8—figure supplement 1
Effects of chemical chaperones on mTORC1 activity in neonate Akita islets and controls.

(a) islets of P16-19 Akita and wild-type (WT) mice were treated with 250 μmol/l TUDCA or 2.5 mmol/l PBA for 48 hr followed by western blotting for BiP and phosphorylated S6 (n = 3, each sample is a pool of islets from 4 to 9 mice); (b) effects of TUDCA on β-cell proliferation in neonate Akita mice (P18-20). TUDCA (1 mg/kg) was injected IP twice daily for 48 hr followed by immunostaining of pancreatic sections for Ki67 and insulin (n = 4–6 mice in each group; 1403 control Akita and 2183 TUDCA-treated Akitaβ-cells). **p<0.01.

https://doi.org/10.7554/eLife.38472.016
Figure 9 with 1 supplement
Effects of mTORC1 activation in neonate Akita β-cells on β-cell size and proliferation.

Studies were performed on heterozygous and homozygous βTsc1 knockout Akita mice (RIP-Cre:Tsc1flox/+:Akita (Akita, βTsc1+/-) and RIP-Cre:Tsc1flox/flox:Akita (Akita, βTsc1-/-). Tsc1flox/+:Akita and Tsc1flox/flox:Akita were used as Akita controls. RIP-Cre:Tsc1flox/+ mice (βTsc1+/-) and RIP-Cre:Tsc1flox/flox mice (βTsc1-/-) were used as WT controls (a, b). (a) Western blotting for phospho-S6 on islets from homozygous and heterozygous knockout mice and matched controls (n = 4, each sample is a pool of islets from two to four mice); (b) Western blotting and quantification of BiP expression in wild-type, Akita and Akita, βTsc1 +/- mice (n = 4, each sample is a pool of islets from two to four mice); (c) β-cell size was assessed by immunostaining for insulin and E-cadherin (n = 400–500 β-cells per group), (d) β-cell proliferation was assessed by immunostaining for insulin and Ki67 (n = 1200–1400 β−cells per group). Quantifications and representative images are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

https://doi.org/10.7554/eLife.38472.017
Figure 9—figure supplement 1
Metabolic characterization of RIP-Cre mouse.

(a) IPGTT- glucose (1.5 gr/kg) was injected after an overnight fast to adult RIP-Cre and non-transgenic control mice (n = 3); (b) fed blood glucose of adult RIP-Cre mice and RIP-Cre:Akita mice compared to non-transgenic controls (n = 6–8); (c) insulin tolerance test on Akita and RIP-Cre:Akita mice (n = 3). *p<0.05.

https://doi.org/10.7554/eLife.38472.018
Figure 10 with 2 supplements
Effects of mTORC1 activation in neonate Akita β-cells on diabetes.

(a–b) IPGTT at P30-35: glucose (1 g/kg) was injected IP after an overnight fast; (a) heterozygous Tsc1 knockout Akita mice (RIP-Cre:Tsc1flox/+:Akita (Akita, βTsc1+/-) and matched controls: Tsc1flox/+ mice (Tsc1+/+), RIP-Cre:Tsc1flox/+ mice (βTsc1+/-), and Tsc1flox/+:Akita (Akita) (n = 3–5 mice in each group); (b) homozygous Tsc1 knockout Akita mice (RIP-Cre:Tsc1flox/flox:Akita (Akita, βTsc1-/-) and matched controls: Tsc1flox/flox mice (Tsc+/+), RIP-Cre:Tsc1flox/flox mice (βTsc1-/-), and Tsc1flox/flox:Akita (Akita) (n = 3–5 in each group); (c–d) pancreatic insulin content of heterozygous and homozygous Tsc1 knockout Akita mice and matched controls at P30-35 (WT (n = 7), Akita (n = 11), Akita, βTsc1+/- (n = 3) and Akita, βTsc1-/- (n = 4); (e) islet insulin content. (f–g) Effects of mTORC1 activation in neonate Akita β-cells on insulin secretion in vivo and ex vivo. (f) insulin secretion in response to IP glucose injection (n = 6 mice in each group); (g) islets were isolated from Tsc1flox/+ WT mice (WT), Tsc1flox/+:Akita (Akita) and RIP-Cre:Tsc1flox/+:Akita (Akita, βTsc1+/-) mice and insulin secretion assessed following static incubations at basal (3.3 mmol/l) and stimulated (16.7) mmol/l glucose. (h) a model of the pathophysiology of permanent neonatal diabetes. *p<0.05 **, p<0.01, ***, p<0.001****, p<0.0001.

https://doi.org/10.7554/eLife.38472.019
Figure 10—figure supplement 1
Effects of mTORC1 activation in Akitaβ-cells on PDX-1 (a, b) and NKX6.1 expression (c, d).

Heterozygous (a, c) and homozygous (b, d) βTsc1 knockout Akita and age-matched controls were sacrificed at P18. Pancreatic sections were stained for insulin and PDX-1 (Akita (n = 1760 β-cells); Akita, βTsc1+/- (n = 814 β-cells); Akita, βTsc1-/- (n = 1458 β−cells) or NKX6.1 (Akita (n = 1500 β-cells); Akita, βTsc1+/- (n = 1438 β-cells); Akita, βTsc1-/- (n = 1584 β-cells).

https://doi.org/10.7554/eLife.38472.020
Figure 10—figure supplement 2
Fed blood glucose of Tsc1flox/+ mice (WT), Tsc1flox/+:Akita (Akita) and heterozygous Tsc1 knockout RIP-Cre:Tsc1flox/+:Akita (Akita,βTsc1+/-) mice at the age of 2–3 months.

Blood glucose levels are the mean of the last three consecutive glucose measurements.

**p<0.01, ***p<0.001.

https://doi.org/10.7554/eLife.38472.021
Author response image 1
Effects of 4 and 16h fast on IPGTT.

Wildtype (WT) and Akita mice were fasted for 4 or 16h followed by IPGTT (1.5 gr/kg glucose) (n=4 in each group).

Tables

Table 1
Transcriptome changes in P19-21 Akita islets compared to age-matched controls (n = 3 per each group).
https://doi.org/10.7554/eLife.38472.013
Gene symbollog2 Fold changep valueGene symbollog2 Fold changep value
β cell signatureGrowth factors and mTOR signaling
Pcsk1−2.11011.1341E-13Dapp1−2.05547.7092E-09
Mafa−1.98012.1124E-12Egfr−2.01852.0824E-08
Igsf11−1.76322.8185E-05Cth−1.94454.9302E-05
Insulin II−1.70705.9006E-14Igf2−1.58464.3954E-03
Ucn3−1.59792.8193E-06Tubg1−1.51343.6446E-06
Nkx6-1−1.55425.0005E-11Sqle−1.47585.5074E-07
Vdr−1.42993.0451E-08IGF1r−1.47351.5160E-05
Slc2a2−1.43525.3528E-04Tpi1−1.45161.4280E-15
Insulin I−1.39881.7436E-11Btg2−1.43501.5855E-03
Nkx2-2−1.24088.1578E-04Elovl6−1.34843.1052E-03
Elovl5−1.34846.7970E-08
Insulin secretion, Insulin granulesMllt11−1.32839.1320E-04
Sytl4−2.19036.6331E-18Ppa1−1.31624.9816E-04
Pcsk1−2.11011.1341E-13Uchl5−1.30858.1939E-03
Vgf−2.10032.1515E-18IGF2r−1.24401.9131E-05
Gng12−1.62543.0041E-07
Syt5−1.51431.3141E-04Cell cycle, replication
Iqgap1−1.49972.8284E-08Pak6−2.16021.0026E-12
Chrm3−1.45711.2136E-05Tmem71−2.06973.8133E-12
Gng4−1.43652.7040E-06S100a10−2.05199.5186E-07
Chgb−1.37391.4523E-03Spc25−1.87391.1632E-05
Gpr119−1.37122.6136E-03Mpp6−1.84573.7249E-06
Ptprn−1.33421.7018E-04Plagl1−1.82531.5119E-12
Nup93−1.78501.1110E-06
Calcium signalingOrc6−1.62601.5634E-04
Npy−3.20263.9755E-13Tmem144−1.60912.9040E-03
Crem−1.92042.4035E-09Vrk1−1.55781.0175E-08
Gem−1.81872.6277E-05Shmt1−1.54101.6394E-03
Dusp1−1.51829.3142E-10Mcm3−1.53304.7341E-04
Plat−1.51378.2853E-03Plch1−1.53056.9677E-11
Tpcn2−1.49063.5030E-03Hells−1.52872.7324E-03
Mif−1.45979.0441E-04Mns1−1.52705.1788E-06
Vcl−1.38703.6093E-03Plat−1.51378.2853E-03
Serca2−1.25295.7379E-03Tubg1−1.51343.6446E-06
Dnmt1−1.48203.7757E-06
ER sressJunb−1.46515.6951E-04
Herpud11.84614.6638E-24Pcna−1.45577.8393E-04
Nucb11.40453.0361E-08Cast−1.45629.4477E-04
Hspa51.35174.1005E-04Net1−1.45071.6503E-03
Dnajc31.34683.2649E-06Myo5a−1.42521.2734E-03
Ddit31.31537.5196E-03Alms1−1.42291.1049E-03
Manf1.20093.2893E-02Chaf1a−1.41376.1889E-03
Lig1−1.41019.1110E-04
Oxidative stressRamp2−1.38604.0237E-03
Gstp11.61776.5804E-06Nphp4−1.38548.2551E-03
Txnip1.50751.0152E-02Mcm6−1.35875.1079E-03
Gstz11.43884.9681E-04Ywhah−1.35617.3805E-05
Tubb4b−1.35431.8881E-04
Cell deathRgs3−1.34243.2137E-04
Card144.34508.5040E-32Bex2−1.33895.1152E-04
Gdf153.14101.6785E-27Clic1−1.32913.9543E-04
Bmp32.50515.7201E-18Polh−1.31699.9122E-03
Proc2.06281.5909E-07Tpm4−1.31253.3364E-04
Rorc1.96127.4298E-07Uchl5−1.30858.1939E-03
Bdnf1.84878.2780E-05Kpnb1−1.30813.7374E-05
Herpud11.84614.6638E-24Phf6−1.30485.1520E-05
Creb3l11.77755.6240E-11Pitpnm1−1.30325.6223E-04
Eph71.74611.7641E-04Aim1−1.30239.1456E-03
Pde3a1.72362.3400E-04Cdk5rap2−1.30221.7257E-03
Ascl11.72196.1969E-04
Mpz1.70491.2768E-04Mitochondria and electron transport chain
Relt1.69923.9164E-07Ndufs2−1.62496.6049E-16
Cnr11.69061.3432E-03Sdhc−1.20089.8858E-04
Osgin11.67321.0137E-06Cox6a2−5.10881.1287E-24
Vip1.67211.5422E-03Cox6c−1.21502.0212E-02
Gstp11.61776.5804E-06Pdk12.00524.3578E-11
Klf111.61386.0024E-05Pdk21.58971.6104E-04
Rgn1.57844.3567E-03Pdk41.30106.7512E-02
Dlc11.56829.9181E-05Pcx−1.39784.8333E-09
Rass2f1.56515.6005E-03Fh1−1.85891.7194E-07
Wnt41.55734.4795E-08
Tle11.54881.0352E-13Non-beta cell hormones
Fgb1.53567.8501E-03Glucagon1.37277.0468E-03
Bmpr1b1.53353.1844E-03Somatostatin1.15106.5084E-02
Pycr11.53084.0752E-04Pancreatic polypeptide1.35461.9982E-03
Cd441.52659.8606E-03Ghrelin1.21951.3036E-01
Nod11.52595.4041E-05
Rasgrf21.50682.4891E-04
Dapk11.50367.5147E-06

Data availability

The RNA-seq data is available through NCBI. The accession number is: GSE114927

The following data sets were generated
  1. 1
    Gene Expression Omnibus
    1. Y Riahi
    2. T Israeli
    3. R Yeroslaviz
    4. S Chimenez
    5. D Avrahami
    6. M Stolovich-Rain
    7. I Alter
    8. M Sebag
    9. N Polin
    10. E Bernal-Mizrachi
    11. Y Dor
    12. E Cerasi
    13. G Leibowitz
    (2018)
    ID GSE114927. RNAseq analysis of whole islets from pre-weaning wild type and Akita mice.
The following previously published data sets were used
  1. 1
  2. 2
    Gene Expression Omnibus
    1. BL Taylor
    2. FF Liu
    3. M Sander
    (2013)
    ID GSE40470. Identification of Nkx6.1 regulated genes in mature pancreatic islets.
  3. 3
    ArrayExpress Archive of Functional Genomics Data
    1. MM Sachdeva
    2. KC Claiborn
    3. C Khoo
    4. J Yang
    5. DN Groff
    6. RG Mirmira
    7. DA Stoffers
    (2009)
    ID E-MTAB-134. Chromatin immunoprecipitation of mouse MIN6 pancreatic beta cells to identify Pdx1 targets.

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