S6K1 controls pancreatic β cell size independently of intrauterine growth restriction

Abstract

Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of β cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased β cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic β cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore β cell size or insulin levels in S6K1-/- embryos, suggesting that loss of S6K1 leads to an intrinsic β cell lesion. Consistent with this hypothesis, reexpression of S6K1 in β cells of S6K1-/- mice restored embryonic β cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic β cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced β cell growth and eventual development of T2DM later in life.

Figure 7. Pancreatic β cell–specific expression of S6K1 restores diminished β cell size and S6 phosphorylation in adult S6K1^–/– mice.

(A and B) Pancreas tissue sections from adult mice at 3–6 months of age: (A) histology, by H&E staining; (B) β cell size in islets, by Hoechst staining for DNA (blue) and immunofluorescence (IF) staining for insulin (red). (C) β cell density in islets was assessed by counting the number of nuclei normalized for islet area (n = 32–42 islets in 3–5 mice per genotype). (D) Endocrine mass calculated from 13–24 determinations (n = 3–5 mice per genotype). (E) Phosphorylated S6K1 T389 (pS6K1 T389), phosphorylated S6 S240/S244 (pS6 S240/S244), phosphorylated Akt (pAkt S473), and phosphorylated ERK 1 and 2 T202/Y204 (pERK T202/Y204) by Western blots analyses. Scale bars: 50 μm. Values in C and D are given as mean ± SEM. *P < 0.05 vs. other genotypes, ANOVA.

Figure 6. Pancreatic β cell–specific expression of S6K1 improves glucose tolerance.

(A) RIP-S6K1 transgene expression in islet extracts by Western blot analysis detecting Myc-tag and S6K1 protein. (B) Body weight was measured in 8-month-old male mice (n = 6–12 per genotype). (C–H) Male mice were analyzed at 3–6 months of age for (C) percent of fat-mass and (D) lean mass. They were normalized by body weight and determined by NMR analysis (n = 5–9 mice of per genotype); (E) increased oxygen consumption in S6K1 transgenic mice measured either during the day or night as indicated (n = 4–9 mice per genotype). (F) Plasma insulin concentrations in mice fasted for 6 hours (n = 6–10 mice of per genotype; experiments were performed twice). (G) Blood glucose concentrations before and after i.p. injection of 2 g d-glucose per kg body weight in mice fasted overnight (n = 5–9 mice per genotype). (H) Insulin tolerance test after 3 hours fasting: plasma glucose concentration before and after intraperitoneal injection of insulin (0.75 U per kg body weight; n = 5–9 mice per genotype). Values in B–H are given as mean ± SEM. *P < 0.05 vs. other genotypes, **P < 0.01 vs. other genotypes, ANOVA.

Figure 5. Pancreatic β cell–specific expression of S6K1 restores diminished β cell size and insulin content in S6K1^–/– embryos.

(A–D) Pancreas sections from WT Tg(RIP-S6K1), S6K1^–/–, and S6K1^–/– Tg(RIP-S6K1) embryos at E16.5: (A) histology by H&E staining; (B) GLUT2 by immunofluorescence (IF) staining; (C) cleaved caspase-3 (red), insulin (green), and DAPI (blue), by IF; and (D) insulin (red) and glucagon (green), by IF staining. (E) β cell number in pancreas. A total of 18 sections from the middle of the pancreas was analyzed (n=3 for per genotype from 3 independent crosses). (F) β cell size, obtained from 29–51 determinations (n = 3 for per genotype). (G) Insulin content from embryonic pancreata (n = 3–10 for per genotype). Scale bars: 25 μm. Values in E–G are given as mean ± SEM. *P < 0.05, **P < 0.01 vs. other genotypes, ANOVA.

Figure 4. Tetraploid WT placenta rescues growth retardation but not β cell size in S6K1^–/– embryos.

(A–D) WT, S6K1^–/–, and S6K1^–/– TR placentas and embryos at E13.5: (A) pictures representative of sizes; (B) histology by H&E staining: S6K1^–/– TR placenta reveals correct intermingling of maternal (open arrows) and embryonic blood vessels (filled arrows) in labyrinthine trophoblast (Lb); (C) Body weights (n = 3–9 per genotype). (D) Placenta weights (n = 3–9 per genotype). (E) Genotype of the tetraploid placenta and the ES cell–derived embryo from an S6K1^–/– TR mouse by Southern blot analysis. The lanes were run on the same gel but were noncontiguous. (F and G) Pancreas sections from E16.5 WT and S6K1^–/– TR embryos: (F) histology by H&E staining; (G) glucose transporter GLUT2 by immunofluorescence (IF) staining; and (H) insulin (red) and glucagon (green) by IF staining. (I) Diminished β cell size in S6K1^–/– TR pancreata, calculated on 56 determinations from WT embryos and 43 from S6K1^–/– TR embryos (n = 3 per genotype). (J) β cell number in embryonic pancreata (n = 3 for per genotype, 18 sections per genotype). (K) Reduced insulin content in E16.5 S6K1^–/– TR embryos (n = 3, from independent experiments) compared with WT embryos (n = 6) developed in surrogate mother mice. Scale bars: 25 μm. In C–K, values are given as mean ± SEM. *P < 0.05; **P < 0.01 vs. WT, unpaired t test.

Figure 3. Abnormal placental development in S6K1^–/– mice.

(A) Reduced placental weight in S6K1-deficient mice from E11.5–E13.5 (n = 5–14 per genotype). (B–E) Placenta sections from WT and S6K1^–/– embryos at E13.5. (B) Labyrinthine trophoblast (Lb) and spongiotrophoblast (Sp) layers by H&E staining; (C) Tpbp Sp-specific marker by ISH labelling. Note that the Sp layer is thinner in S6K1^–/– placenta (right). (D) Reduced intermingling of maternal (containing enucleated erythrocytes, open arrows) and embryonic blood vessels (identified by nucleated erythrocytes, filled arrows) in the Lb layer of S6K1^–/– placenta (right). (E) Anti-PECAM staining (brown) shows dilated fetal blood vessels, irregular in diameter in S6K1^–/– placenta (right). Filled arrows indicate nucleated erythrocytes. Scale bars: 25 μm. In A, values in are given as mean ± SEM. *P < 0.05 vs. WT, unpaired t test.

Figure 2. S6K1^–/– embryos are developmentally retarded and display IUGR.

(A) Weight of WT and S6K1^–/– embryos at the indicated gestational ages. A total of 234 WT and 223 S6K1^–/– embryos was analyzed. n = 25–33 at each time point, for both genotypes. (B) Bone development in WT and S6K1^–/– embryos. Top panel: Lateral view of skeleton at E16.5 analyzed for ossification, using alcian blue (cartilage) and alizarine red (bone). Arrows indicate ossification in the frontal bone of the cranium; in the fibula and tibia bone on the hind limb of WT embryo, the corresponding areas on the S6K1^–/– embryo are still cartilaginous. Bottom panels: Hypertrophied chondrocytes in cervical vertebrae from WT (left) but not in S6K1^–/– (right) at E14.5. (C) Lateral view of E9.5 embryos (asterisk indicates somite) by scanning electron microscopy (left) and number of somites (right) (n = 12 per genotype). Note the open otic pit (open arrow) and the absence of a limb bud (filled arrow) in the S6K1^–/– embryo. (D) Preimplantation development–stage analysis using 106 eggs from 7 WT mice and 45 eggs from 4 S6K1^–/– mice. Scale bar: 25 μm. In A, C, and D, values are given as mean ± SEM. *P < 0.05 vs. WT, unpaired t test.

Figure 1. Diminished pancreatic β cell size and number in S6K1^–/– embryos.

(A–D) Pancreas sections from WT or S6K1^–/– embryos at E16.5: (A) histology, by H&E staining; (B) GLUT2 glucose transporter, by immunofluorescence (IF) staining; (C) cleaved caspase-3 (red), insulin (green) by IF staining and DAPI (blue) staining; and (D) insulin (red) and glucagon (green), by IF staining. (E) β cell number in embryonic pancreata (n = 3 per genotype). (F) β cell size in embryonic pancreata from 45–52 determinations in WT embryos and 29–44 determinations in S6K1^–/– embryos (n = 3 per genotype; β cell surface: WT, 95.60 ± 2.69 μm²; S6K1^–/–, 69.54 ± 1.5 μm²). (G) Reduced insulin content, normalized for body weight, in embryonic pancreata (n = 6–9 per genotype). Scale bars: 25 μm. In E–G, values are given as mean ± SEM. *P < 0.05, **P < 0.01 vs. WT, unpaired t test.