Deficit of tRNA(Lys) modification by Cdkal1 causes the development of type 2 diabetes in mice

Abstract

The worldwide prevalence of type 2 diabetes (T2D), which is caused by a combination of environmental and genetic factors, is increasing. With regard to genetic factors, variations in the gene encoding Cdk5 regulatory associated protein 1-like 1 (Cdkal1) have been associated with an impaired insulin response and increased risk of T2D across different ethnic populations, but the molecular function of this protein has not been characterized. Here, we show that Cdkal1 is a mammalian methylthiotransferase that biosynthesizes 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A) in tRNA(Lys)(UUU) and that it is required for the accurate translation of AAA and AAG codons. Mice with pancreatic β cell-specific KO of Cdkal1 (referred to herein as β cell KO mice) showed pancreatic islet hypertrophy, a decrease in insulin secretion, and impaired blood glucose control. In Cdkal1-deficient β cells, misreading of Lys codon in proinsulin occurred, resulting in a reduction of glucose-stimulated proinsulin synthesis. Moreover, expression of ER stress-related genes was upregulated in these cells, and abnormally structured ER was observed. Further, the β cell KO mice were hypersensitive to high fat diet-induced ER stress. These findings suggest that glucose-stimulated translation of proinsulin may require fully modified tRNA(Lys)(UUU), which could potentially explain the molecular pathogenesis of T2D in patients carrying cdkal1 risk alleles.

Figure 1. Methylthiolation of tRNA^Lys(UUU) by Cdkal1 controls the decoding accuracy of the lysine codon.

(A) The molecular structure of tRNA^Lys(UUU) and ms²t⁶A. (B) Results of a mass spectrometric analysis of the ms²t⁶A modification of tRNA in MIN6 and HeLa cells. The upper panels show the UV trace, and the middle and lower panels show the mass chromatograms for detecting t⁶A (m/z 413, arrow) and ms²t⁶A (m/z 459, arrow), respectively. (C) Results of a mass spectrometric analysis of the ms²t⁶A modification of tRNA isolated from the pancreas of Cdkal1^–/– and WT mice. The arrow indicates ms²t⁶A (m/z 459). (D) Modification of tRNA^Lys(UUU) isolated from the liver of Cdkal1^–/– and WT mice. The upper panels show mass chromatograms of GGDp fragments in tRNA^Lys(UUU). The middle and lower panels show mass chromatograms of ms2t6AAΨp fragments and t6AAΨp fragments, respectively. (E) WT and ΔyqeV cells were transformed with a reporter plasmid in which both Renilla renilla and firefly luciferases are cloned with the lac promoter (upper panel). Relative activity was determined by normalizing firefly luciferase intensity to renilla luciferase intensity (F/R, lower panel). Data are presented as the mean ± SEM, and asterisks indicate statistical significance determined by Student’s t test. ***P < 0.001; n = 4. (F) The expression level of the fusion protein of firefly and renilla luciferase after IPTG treatment induction was determined in WT and ΔyqeV cells (E) by Western blot.

Figure 2. Cdkal1 localizes on ER through its hydrophobic domain.

(A) Colocalization of overexpressed Cdkal1-EGFP (green) and ER-tracker (red) on ER in HEK293 cells and MIN6 cell. Scale bars: 10 μm. (B) Colocalization of overexpressed Cdkal1-EGFP (green) with endogenous Bip in HEK293 cells. Scale bar: 10 μm. (C) The domain structure of Cdkal1 protein. (D) EGFP-tagged full-length Cdkal1 or Cdkal1 with truncation of C terminus hydrophobic domain (Cdkal1ΔC) was transfected in HeLa cells together with ER-tracker. Localization of full-length Cdkal1 or Cdkal1ΔC was visualized using confocal microscope. Scale bars: 10 μm. (E) MIN6 cells were transfected with Cdkal1-EGFP, and the localization of Cdkal1 was determined by immunoelectronic microscopic examination. Arrows indicate EGFP-Cdkal1 signal on ER. Scale bar: 0.5 μm.

Figure 3. Conditional deletion of the Cdkal1 gene causes glucose intolerance.

(A) Conditional deletion of Cdkal1 in pancreatic islets in β cell KO (KO) mouse. (B) Comparison of the body weights of β cell KO and Flox mice. (C) Pancreatic sections obtained from β cell KO and Flox mice at 5 weeks of age were immunostained with anti-insulin (red) and anti-glucagon (green) antibodies. Nuclei were counterstained with DAPI. (D) Comparison of relative islet area in pancreas of β cell KO and Flox mice. Area of 529 islets from 3 Flox mice and 572 islets from 3 β cell KO mice were examined and classified into small, medium, and large islet area. The relative distribution of each islet area was compared between β cell KO and Flox. (E) Blood glucose during glucose tolerance test at 5 weeks (upper) and 10 weeks (lower). n = 4–7. (F) Plasma insulin levels during a glucose tolerance test at 15 weeks. n = 10–11. (G) Glucose-stimulated insulin secretion in islets (n = 8) isolated from β cell KO or Flox mice was determined. (H) Glucose-stimulated insulin secretion in perfused islets of Flox and β cell KO mice. n = 4–5. (I) Plasma insulin levels in Flox or β cell KO mice fasted for 14 hours and re-fed for 1.5 hours. n = 7. Significant difference was examined by repeated measure of 2-way ANOVA (E and F) or 2-way ANOVA (D, G, and I) followed by Bonferroni’s post-test or Mann-Whitney U test. Data are presented as mean ± sEM. *P < 0.05; **P < 0.01; ***P < 0.001 versus Flox.

Figure 4. Aberrant insulin synthesis in the pancreatic β cells of β cell KO mice.

(A) Relative incorporation of 14C-lysine to 3H-leucine in immunoprecipitated (pro)insulin in islets of β cell KO or Flox mice in KRB buffer containing 16.7 mM glucose for 1 hour. (B) Pancreatic C-peptide content of β cell KO or Flox mice was measured by ELISA, and value was normalized to total protein concentration. n = 5–8; *P < 0.05 by Student’s t test. (C) Plasma C-peptide concentrations in Flox and β cell KO mice fasted for 7 hours. n = 10. ***P < 0.001 by Student’s t test. (D) Relative total protein synthesis under basal condition (2.8 mM) or stimulated condition (16.7 mM) was determined by normalizing 35S incorporation to the total protein concentration. n = 4; *P < 0.05 by Student’s t test. (E) Proinsulin synthesis in KO or Flox islets under basal condition (2.8 mM) or stimulated condition (16.7 mM) is shown in top panel. n = 4; *P < 0.05 by Student’s t test. (F) Expression of actin, SUR1, Kir6.2, and Pdx1 protein in islets of Flox or β cell KO mice determined by Western blotting. Results representative of 3 independent experiments are shown. All data are presented as mean ± SEM.

Figure 5. ER stress response in the pancreatic β cell KO mice.

(A) Quantitative analysis of the mRNA expression of insulin, glucagon, and Cdkal1 in isolated islets of β cell KO and Flox mice. *P < 0.05; n = 4. (B) Comparison of the expression of β cell–related genes between β cell KO and Flox mice. *P < 0.05; n = 4. (C) Subcellular distribution of GLUT2 in islets of β cell KO and Flox mice. Scale bars: 50 μm. (D) Quantitative analysis of ER stress–related genes in β cell KO and Flox mice. *P < 0.05; n = 4. (E) Transmission electron microscopic examination of the ultrastructure of β cells in pancreatic sections of β cell KO mice and Flox mice. Arrows indicate the ER distention in the β cells of KO mice. Scale bar: 5 μm. Significant differences were examined by Student’s t test (A, B, and D). All data are presented as mean ± SEM.

Figure 6. β cell KO mice exhibit increased ER stress and glucose intolerance after consuming an HFD.

(A) Changes in body weight of β cell KO and Flox mice on an HFD and a LFD starting from 20 weeks old. (B and C) Results of the glucose tolerance test after 3 weeks (B) and 8 weeks (C) of consuming an HFD or a LFD. Mice were fasted for 7 hours from 8 am and injected with glucose (1 g/kg body weight). *P < 0.05; ***P < 0.001, KO-HFD versus Flox-HFD mice. n = 4–6. (D and E) Nonfasting blood glucose (D) and 7-hour fasting blood glucose (E) levels after 3 weeks on an HFD or an LFD. *P < 0.05; n = 6. (F) The insulin tolerance test was performed in mice fed an HFD or an LFD for 7 weeks. (G) β cell KO mice and Flox mice were fed an HFD for 8 weeks. Plasma insulin level during IPGTT (1 g/kg body weight) was examined in β cell KO mice and Flox mice fasted for 14 hours. **P < 0.01; n = 6. (H) Relative expression of ER stress–related genes in β cell KO mice and Flox mice fed an HFD for 8 weeks. **P < 0.01; n = 4–5. Significant differences between groups were examined by repeated measure of ANOVA (A–C, F, and G), 2-way ANOVA (D and E), or Student’s t test (H). All data are presented as mean ± SEM.