Research ArticlePhysiology

Hypusine biosynthesis in β cells links polyamine metabolism to facultative cellular proliferation to maintain glucose homeostasis

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Science Signaling  03 Dec 2019:
Vol. 12, Issue 610, eaax0715
DOI: 10.1126/scisignal.aax0715
  • Fig. 1 β Cell–specific knockout of Dhps impairs glucose tolerance after HFD feeding.

    Control [wild-type (WT), Cre+, or DhpsloxP/loxP] and Dhps∆β (βKO) mice were fed for 4 weeks with a normal chow diet (NCD) or high-fat diet (HFD), and metabolic parameters were assessed. (A) Schematic timeline showing the period of tamoxifen (TAM) injections and feeding. (B) Body weight at the conclusion of the feeding regimen. Data from n = 5 mice per group. (C) Percent body fat at the conclusion of the feeding regimen. Data from n = 4 mice per group. (D) Glucose tolerance test (GTT) in NCD-fed mice. (E) Area under the curve (AUC) analysis of GTTs in (D). (F) GTT in HFD-fed mice. (G) AUC analysis of GTTs in (F). (H) Insulin tolerance test in NCD- and HFD-fed mice. (I) Area over the curve (AOC) of insulin tolerance test in (H). (J) Serum insulin levels during a GTT. (K) Images of whole pancreatic sections from representative HFD-fed control and Dhps∆β mice immunostained for insulin (brown) and counterstained with hematoxylin. Scale bars, 1000 μm. (L) Quantitation of pancreatic β cell mass. Data from n = 3 to 6 mice per group. (M) Representative images of pancreata stained for TUNEL (red), insulin (green), and nuclei (DAPI, blue). Scale bars, 50 μm. (N) Quantification of TUNEL immunostaining (n = 3 mice per group). Data presented as means ± SEM; *P < 0.05 for the comparisons shown by one-way ANOVA.

  • Fig. 2 RNA sequencing and proteomics of control and β cell–specific Dhps knockout islets.

    Control (Cre+ and DhpsloxP/loxP) and Dhps∆β (βKO) mice were fed an NCD or HFD for 4 weeks. Data from n = 3 mice per group. (A) Principal components analysis displaying the variance of RNA sequencing data obtained from control and βKO islets. (B) Volcano plot of differentially expressed mRNAs in comparison between control and Dhps∆β islets. FC, fold change. (C) Gene Ontology Enrichment Analysis for mRNA sequencing data. (D) Heatmap of differentially expressed genes involved in cellular proliferation. (E) Volcano plot of differentially expressed proteins in comparison between control and Dhps∆β mice. Data from n = 3 to 6 mice per group. (F) Gene Ontology Enrichment Analysis from proteomics data.

  • Fig. 3 Hypusination of eIF5A is increased after 1 week of HFD feeding.

    Control Cre+ and Dhps∆β (βKO) mice were fed for 1 week with NCD or HFD, and eIF5AHyp and eIF5ATotal levels were assessed by immunostaining of pancreas sections. (A) Representative images of pancreata stained for eIF5AHyp (magenta), insulin (green), and nuclei (DAPI, blue). Scale bars, 50 μm. (B) Quantification of eIF5AHyp immunostaining in (A) from n = 3 animals per group. (C) Representative images of pancreata stained for eIF5ATotal (magenta), insulin (green), and nuclei (DAPI, blue). Scale bars, 50 μm. (D) Quantification of eIF5ATotal immunostaining in (C). Data from n = 3 animals per group. Data presented as means ± SEM; *P < 0.05 for the comparisons shown by one-way ANOVA.

  • Fig. 4 β Cell–specific Dhps knockout mice exhibit normal glucose tolerance after 1 week of HFD feeding.

    Control (Cre+, WT, and DhpsloxP/loxP) and Dhps∆β (βKO) mice were fed for 1 week with NCD or HFD, and metabolic parameters were assessed. (A) GTT in NCD-fed mice. (B) AUC analysis for GTT from NCD-fed mice in (A). (C) Serum insulin levels in NCD-fed mice in (A). Data from n = 3 Cre+ and DhpsloxP/loxP mice and n = 5 Dhps∆β mice. (D) β Cell mass in NCD-fed mice in (A). n = 3 mice per group. (E) GTT in HFD-fed mice. (F) AUC analysis for GTTs in (E). (G) Serum insulin levels in HFD-fed mice in (E). Data from n = 3 Cre+ and DhpsloxP/loxP mice and n = 4 Dhps∆β mice. (H) β Cell mass in HFD-fed mice in (E). Data from n =3 Cre+ and DhpsloxP/loxP mice and n = 5 Dhps∆β mice. (I) Representative images of pancreata from the indicated mice stained for Ki67 (magenta, arrow), insulin (green), and nuclei (DAPI, blue). Scale bar, 50 μm. (J) Quantification of Ki67 immunostaining in (I). Data from n = 3 to 5 animals per group. Data presented as means ± SEM; *P < 0.05 by one-way ANOVA.

  • Fig. 5 Decreased mRNA translation of cyclin D2 in Dhps∆β mice.

    (A) Schematic showing experimental design. Control (Cre+) and Dhps∆β (βKO) mice were fed an HFD for 1 week, and islets were isolated and subjected to RT-PCR and polyribosome profiling (PRP) analysis. (B) Quantitative RT-PCR from islets for the genes indicated. Data from n = 5 to 6 animals per group. (C) Representative immunoblot analysis from islets for cyclin D2, cyclin D1, and ERK1/2 (left) and quantification of immunoblots (right). Data from n = 3 to 5 animals per group. (D) PRP of isolated islets (left) and quantification of P/M ratio (right). Data from n = 3 mice per group. (E) Quantitative RT-PCR of Cycd2 in polyribosomal fractions. Data from n = 3 to 4 mice per group. (F) Quantitative RT-PCR of Cycd1 in polyribosomal fractions. Data from n = 3 to 4 mice per group. Data presented as means ± SEM; *P < 0.05 by t test.

  • Fig. 6 DHPS inhibition attenuates harmine-induced β cell proliferation in mouse and human islets.

    (A) Islets were isolated from male 8- to 9-week old CD1 mice and then treated in vitro with vehicle (V), harmine (H), and Gc7 (G). Representative immunoblot (top) and quantification of protein levels (bottom) for cyclin D2. Data are from n = 6 mice per group. (B) Islets were isolated from male 8- to 9-week old CD1 mice and then treated in vitro with vehicle (V), harmine (H), and Gc7, followed by flow cytometry analysis of dispersed islet cells immunostained for phospho–histone H3. Data are from n = 4 to 6 mice per group. (C). Islets were isolated from control (Cre+) and Dhps∆β (βKO) mice and then treated in vitro with vehicle (V) or harmine (H), and percent of β cells that immunostained for Ki67 was calculated. Data are from n = 3 animals per group. (D) Human islets from four donors were treated in vitro with vehicle, harmine, and/or Gc7 (G), dispersed, immunostained for phospho–histone H3, and then subjected to flow cytometry analysis. BMI, body mass index. Graphs show a single technical replicate for each donor. Data in (A) to (C) are presented as means ± SEM; *P < 0.05 by one-way ANOVA.

  • Fig. 7 Hypusine generation requires PKC-ζ.

    WT and kinase-dead PKC-ζ (KD) mice were fed an NCD or HFD for 1 week. (A) Representative images of pancreata stained for eIF5AHyp (magenta), insulin (green), and nuclei (DAPI, blue). (B) Quantification of eIF5AHyp levels from (A). Data are from n = 3 mice per group. (C) Representative images of pancreata stained for eIF5ATotal (magenta), insulin (green), and nuclei (DAPI, blue). (D) Quantification of eIF5ATotal levels from (B). Data are from n = 3 mice per group. (E) Representative images of NCD- and HFD-fed control and Dhps∆β (βKO) mouse pancreata immunostained for phospho-PKC-ζ (red), insulin (green), and nuclei (DAPI, blue). Scale bars, 50 μm. (F) Quantification of phospho-PKC-ζ immunostaining in insulin-positive cells shown in (E). Data are from n = 3 mice per group. Data presented as means ± SEM; *P < 0.05 for the comparisons shown by one-way ANOVA.

  • Fig. 8 Proposed pathway linking PKC-ζ, c-Myc, and polyamines to adaptive proliferation in β cells.

    The schematic diagram shows the proposed positioning of the PI3K-PKC-ζ-mTOR pathway (left arm of the diagram) and the polyamine-eIF5A pathway (right arm of the diagram) relative to the adaptive translational and proliferative responses. The hierarchy of the factors depicted in red (PKC-ζ, c-Myc, ODC, and DHPS) have been shown in this study or others to serve as key nodes through which proliferative responses are mediated—deletion or inhibition of these factors severely restricts the ability of the β cell to produce cyclin D2 and undergo replication. This study demonstrated that DHPS is downstream of PKC-ζ, c-Myc, and ODC functions. This study does not explicitly rule out the possibility that PKC-ζ might directly or indirectly regulate the function of DHPS (shown as a dashed line with a question mark).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/610/eaax0715/DC1

    Fig. S1. β Cell–specific deletion of Dhps and its effect on glucose homeostasis.

    Fig. S2. ODC inhibition attenuates HFD-induced β cell proliferation.

    Fig. S3. DHPS inhibition attenuates harmine-induced β cell proliferation in mouse islets.

    Fig. S4. DHPS inhibition decreases harmine-induced β cell proliferation in human islets.

  • This PDF file includes:

    • Fig. S1. β Cell–specific deletion of Dhps and its effect on glucose homeostasis.
    • Fig. S2. ODC inhibition attenuates HFD-induced β cell proliferation.
    • Fig. S3. DHPS inhibition attenuates harmine-induced β cell proliferation in mouse islets.
    • Fig. S4. DHPS inhibition decreases harmine-induced β cell proliferation in human islets.

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