Fetal deficiency of lin28 programs life-long aberrations in growth and glucose metabolism

Abstract

LIN28A/B are RNA binding proteins implicated by genetic association studies in human growth and glucose metabolism. Mice with ectopic over-expression of Lin28a have shown related phenotypes. Here, we describe the first comprehensive analysis of the physiologic consequences of Lin28a and Lin28b deficiency in knockout (KO) mice. Lin28a/b-deficiency led to dwarfism starting at different ages, and compound gene deletions showed a cumulative dosage effect on organismal growth. Conditional gene deletion at specific developmental stages revealed that fetal but neither neonatal nor adult deficiency resulted in growth defects and aberrations in glucose metabolism. Tissue-specific KO mice implicated skeletal muscle-deficiency in the abnormal programming of adult growth and metabolism. The effects of Lin28b KO could be rescued by Tsc1 haplo-insufficiency in skeletal muscles. Our data implicate fetal expression of Lin28a/b in the regulation of life-long effects on metabolism and growth, and demonstrate that fetal Lin28b acts at least in part via mTORC1 signaling.

Keywords: Diabetes; Dwarfism; Glucose metabolism; Growth; Lin28a; Lin28b; let-7; mTOR.

Figure 1. Constitutive deletion of Lin28a leads to persistent growth retardation from embryogenesis through adulthood

(A) Body weights of E13.5 Lin28a +/+, +/− and −/− embryos. (B) Body weights of E18.5 Lin28a +/+, +/− and −/− embryos. (C) Representative images of a heterozygous mouse and its Lin28a KO littermate. Bar 1cm. (D) Viability of P0.5 newborns and 21-day-old pups born from an inbreeding cross of Lin28a heterozygotes. P values were calculated by Chi-square test. (E–F) Growth curves for Lin28a heterozygous (solid) vs KO (dashed) mice, both male (blue) and female (red). N=4–15. (E) Body weight. (F) Body length. (G) Organ weights normalized to total body weight from 4 to 5-month-old males. Epididymal fat pads were used to measure the fat weight. N=3. (H) DEXA measurements of % lean and % fat weights, relative to total body weight. N=5–8. (I) DEXA measurement of bone mineral density. N=5–8. * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 2. Lin28a and Lin28b dosage regulates postnatal growth

(A–B) Postnatal growth curves of Lin28b heterozygous vs. KO mice. N=8–21. (A) Body weight. (B) Body length. (C–D) Postnatal growth curves of Lin28a +/+ vs. +/− mice on a Lin28b KO background. N=11–22. (C) Body weight. (D) Body length. * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 3. Double knockout of Lin28a/b leads to synthetic lethality in E10.5–E12.5 embryos

(A) Representative images, and (B) Number of somites, in Lin28a +/+ vs. Lin28a +/− vs. Lin28a −/− embryos on a Lin28b KO background at E9.5–10.5. N=4–15. (C) Representative images of Lin28a +/− vs. Lin28a −/− embryos on a Lin28b KO background at E11.5. Arrow indicates an open neural tube. (D) Hematoxylin-eosin (H&E) staining of Lin28a +/− vs. Lin28a −/− embryos on a Lin28b KO background at E11.5. (E) Frequency of viable Lin28a −/−; Lin28b −/− embryos at different ages. See the Supplementary Table. Scale bars 1 mm (A and C), 500 µm (D). * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 4. Fetal Lin28a/b, not neonatal or adult Lin28a/b, regulates postnatal growth

(A) Breeding strategy to test Lin28a fl/− mice at different ages. N=3–12. (B–D) Postnatal growth curves of Lin28a fl/−; UBC-CreER (+) vs. Lin28a fl/− mice. Body weights after tamoxifen (TAM) injections at (B) E15.5, (C) P7–9 and (D) 6 weeks. (E) Breeding strategy to test Lin28b fl/− mice at different ages. N=5–13. (F–H) Postnatal growth curves of Lin28b fl/−; UBC-CreER (+) vs. Lin28b fl/− mice. Body weights after TAM injections at (F) E15.5, (G) P7–9 and (H) 6 weeks. Arrows indicate ages at TAM injection. * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 5. Glucose metabolism dysfunction in Lin28a KO and Lin28b KO mice

(A) Glucose tolerance tests (GTT) (N=9–10) and (B) Insulin tolerance tests (ITT) (N=8–11) of Lin28b fl/fl; Myf5-Cre mice. (C–E) Metabolomic changes in Lin28a +/− vs. −/− embryos at E10.5. (C) Glycolysis pathway. (D) Bioenergetics and redox balance. (E) Nucleotide synthesis. + p<0.1, * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 6. Muscle-specific Lin28b/let-7 regulates postnatal growth

(A–C) Let-7 levels in (A) Lin28a +/− vs. −/− E8.5 embryos, (B) Lin28a +/− vs. −/− neonatal muscles, and (C) Lin28b +/− vs. −/− adult skeletal muscles. N=3–5. (D–E) Postnatal growth curves of LSL-iLet-7s; Myf5-Cre(+) males given DOX from conception. N=6–13. Statistical significance was shown for LSL-iLet-7s (controls) vs. LSL-iLet-7s; Myf5-Cre(+) mice. (D) Body weights. (E) Body lengths. (F–G) Postnatal growth curves of Lin28b fl/fl; Myf5-Cre males. N=13–17. (F) Body weights. (G) Body lengths. * p<0.05, ** p<0.01. Error bars represent SEM.

Figure 7. Tsc1/mTOR signaling in skeletal muscle can partially rescue aberrant programing of glucose metabolism and organismal growth in Lin28b KO mice

(A–B) Postnatal growth curves of Lin28b KO males, with or without Tsc1 haploinsufficiency. N=6–14. (A) Body weights. (B) Body lengths. Statistical significance was shown for Lin28b −/−; Tsc1 +/+ vs. Lin28b −/−; Tsc1 +/− mice. (C–D) Postnatal growth curves of Lin28b fl/fl; Myf5-Cre males, with or without Tsc1 haploinsufficiency. N=21–29. (C) Body weights. (D) Body lengths. Statistical significance was shown for Lin28b fl/fl; Tsc1 fl/+ vs. Lin28b fl/fl; Tsc1 fl/−; Myf5-Cre(+) mice on the top, and for Lin28b fl/fl; Tsc1 +/+; Myf5-Cre(+) vs. Lin28b fl/fl; Tsc1 fl/−; Myf5-Cre(+) mice on the bottom. (E) Glucose tolerance tests (GTT) of Lin28b fl/fl; Tsc1 fl/+; Myf5-Cre males. N=8–11. (F) Summary of the timing and tissue of Lin28a/b expression and the effect on growth and glucose metabolism. Note that pink color denotes the positive effect on normal growth and glucose metabolism. The intensity of color and the number of + signs reflect the magnitude of the effect. “+/−“ indicates that Lin28b has the effect on growth only if combined with Lin28a loss. * p<0.05, ** p<0.01. Error bars represent SEM.