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Role of the SIK2–p35–PJA2 complex in pancreatic β-cell functional compensation

An Erratum to this article was published on 01 April 2014

A Corrigendum to this article was published on 01 April 2014

This article has been updated

Abstract

Energy sensing by the AMP-activated protein kinase (AMPK) is of fundamental importance in cell biology. In the pancreatic β-cell, AMPK is a central regulator of insulin secretion. The capacity of the β-cell to increase insulin output is a critical compensatory mechanism in prediabetes, yet its molecular underpinnings are unclear. Here we delineate a complex consisting of the AMPK-related kinase SIK2, the CDK5 activator CDK5R1 (also known as p35) and the E3 ligase PJA2 essential for β-cell functional compensation. Following glucose stimulation, SIK2 phosphorylates p35 at Ser 91, to trigger its ubiquitylation by PJA2 and promote insulin secretion. Furthermore, SIK2 accumulates in β-cells in models of metabolic syndrome to permit compensatory secretion; in contrast, β-cell knockout of SIK2 leads to accumulation of p35 and impaired secretion. This work demonstrates that the SIK2–p35–PJA2 complex is essential for glucose homeostasis and provides a link between p35–CDK5 and the AMPK family in excitable cells.

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Figure 1: Deletion of Sik2 in adult β-cells leads to glucose intolerance due to impaired stimulus-dependent insulin secretion.
Figure 2: SIK2 is required for glucose-stimulated insulin secretion at a step downstream of depolarization.
Figure 3: SIK2 phosphorylates p35.
Figure 4: PJA2 ubiquitylates CDK5R1.
Figure 5: The SIK2–p35–PJA2 complex is required for calcium mobilization in the β-cell.
Figure 6: The SIK2–p35–PJA2 complex is required for insulin secretion.
Figure 7: SIK2 is essential for β-cells to meet insulin demand in models of metabolic syndrome.
Figure 8: Regulation of SIK2 and AMPK by glucose in the β-cell.

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Change history

  • 26 February 2014

    In the version of this Article originally published, the two labels 'SIK2' in Fig. 5g should have read 'PJA2'. This error has now been corrected in the online versions of the Article.

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Acknowledgements

We would like to thank members of the R.A.S. laboratory and M. Wheeler for helpful comments and Z-Y. Lin for proteomic analysis. We acknowledge the Department of Laboratory Medicine and Pathology Core Facility at the University of Ottawa and K. Yates at the University of Ottawa Animal Care for technical assistance. This work was supported by grants from the Canadian Diabetes Association (number OG-3-11-3328-RS), the Canadian Institutes of Health Research (CIHR) MOP-97772 and the Canadian Foundation for Innovation (CFI) to R.A.S., operating grant from CIHR (MOP-84314) to A-C.G., by a Japan Society for the Promotion of Science Fellows to J.S. (10J00366), and a Canadian Diabetes Studentship to A.F. A-C.G. holds the Canada Research Chair in Functional Proteomics and the Lea Reichmann Chair in Cancer Proteomics. R.A.S. holds the Canada Research Chair in Apoptotic Signalling.

Author information

Authors and Affiliations

Authors

Contributions

J.S. performed most of the experiments. A.F. helped with physiological analyses and performed metabolic analyses in Supplementary Fig. 4f, g. C.R. assisted with animal husbandry and physiological analyses. S.B. performed imaging based analysis of MIN6 cell proliferation in Supplementary Figs 3h–j and β-cell mass in Fig. 1h and Supplementary Fig. 3g. C.D., M.A. and N.B assisted with the design and generation of the Sik2 knockout construct. A-C.G. performed proteomic analyses of the p35 complex. S-P.Y. generated Sik2fl/fl and Sik2ko/+ animals. R.A.S. conceived and supervised the study. J.S. and R.A.S. wrote the manuscript.

Corresponding author

Correspondence to Robert A. Screaton.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Normal AMPK-mTOR signalling and CRTC2-CREB activity in SABKO mice.

(a). QPCR analysis showing reduction of SIK1, SIK2 and SIK3 mRNA levels in total RNA from islets isolated from S/S and SABKO mice. RNA was pooled from 3 mice per genotype. Sik mRNA levels are normalized to levels of 36B4 mRNA as internal control. (b). Western blot analysis showing the phosphorylation status and total protein levels of AMPK in islets from control S/S and SABKO mice. (c). Western blot analysis of mTOR signalling pathway in SABKO islets. Western blot analysis of the phosphorylation status of mTORC1 substrate S6 kinase (S6K) at Thr389 in control S/S and SABKO islets cultured in 2.8 mM or 16.7 mM glucose for 1 h. (d). Western blots showing levels of S6K pThr389, S6 pSer240/pSer244, and eEF2 pThr56 and their respective total protein controls in islets cultured in RPMI-1640 medium. (e). Western blot analysis of CRTC2 phosphorylation at regulatory sites Ser171 and Ser275 in islets from control S/S and SABKO mice. (f). Western blot anlysis of CRTC2 phosphorylation at regulatory sites Ser171 and Ser275 in islets from control S/S and SABKO mice cultured in low glucose or stimulated with 16.7 mM glucose + 10 nM exendin-4 for 1 h. (g). QPCR analysis of mRNA levels of CREB target genes NR4A2 (left) and IRS2 (right) in islets isolated from control S/S and SABKO animals. RNA was pooled from 3 mice per genotype. (a and g): Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results. All western blot data are representative of three independent experiments with consistent results.

Supplementary Figure 2 Absence of sex difference and normal insulin tolerance in SABKO mice.

(a) Blood glucose levels of fasted and random fed control (S/S, n=35 mice) and SABKO (n=33 mice) mice were measured before tamoxifen treatment. (b, c) Fasted and refed (2 h) blood glucose levels in (b) male S/S (n=17 mice) and SABKO (n=16 mice) and (c) female S/S (n=5 mice) and SABKO (n=5 mice) mice. (d, e). Fasted and refed (2 h) plasma insulin levels in (d) male S/S (n=14 mice) and SABKO (n=18 mice) and (e) female S/S (n=5 mice) and SABKO (n=5 mice) animals. (f) Body weights of control (S/S, n=14 mice) and SABKO (n=9 mice) animals at age 9 weeks. (g) Insulin tolerance test in S/S (n=7 mice) and SABKO (n=11 mice) mice. (h, i) Glucose tolerance test in (h) male S/S (n=8 mice) and SABKO (n=7 mice) and (i) female S/S (n=8 mice) and SABKO (n=4 mice) mice. (j, k) Plasma insulin levels during glucose tolerance test in (j) male S/S (n=9 mice) and SABKO (n=9 mice) and (k) female S/S (n=4 mice) and SABKO (n=4 mice) animals. All error bars represent s.e.m. Statistical significance for all data was determined using two-tailed unpaired Students t-test (p < 0.01,p < 0.05).

Supplementary Figure 3 Sik2 does not affect beta cell mass, proliferation, survival.

(a) Body weights of control (S/S, n=14 mice) and SABKO (n=8 mice) animals on high fat diet for 16 weeks. (b) Food intake in control (S/S, n=11 mice) and SABKO (n=8 mice) animals after 18 weeks on HFD. (c) Blood glucose levels in control (S/S, n=14 mice) and SABKO (n=8 mice) animals on high fat diet for 16 weeks following an overnight fast or 2 h refeeding. (d) Plasma insulin levels for animals shown in n. (e) Plasma insulin levels at indicated times postinjection of glucose during GTT assay shown in Fig. 1g for control (S/S, n=8 mice) and SABKO (n=8 mice) HFD-fed mice. (f) Ratio of AUC from HFD-fed SABKO mice at indicated times to AUC for normal diet (pre-HFD) SABKO mice. (g) (Left) Immunostaining of pancreatic sections of control S/S and SABKO mice on normal chow diet. Sections were stained with insulin antibody (red) and CellMask Blue cytoplasmic/nuclear stain (blue). Bar =100 um. (Right) Quantification of beta cell area in S/S (n=3 mice) and SABKO (n=3 mice) mice. (h) Quantification of Ki-67 positive MIN6 cells infected with non-targeting control (CON1, n=10,509 cells; CON2, n=11,044 cells) and SIK2 (shRNA1, n=13,153 cells; shRNA2, n=11,224 cells) shRNA assessed from 60 fields from a single experiment, representative of two independent experiments. (i) Quantification of EdU positive MIN6 cells infected with non-targeting control (j) Quantification of the cell number of cells infected with non-targeting control (CON) and SIK2 shRNA. For i and j, CON1; n=11,860 cells; CON2; n=11,905 cells) and SIK2 (shRNA1, n=11,990 cells; shRNA2, n=11,147 cells) shRNA, assessed from 60 fields from a single experiment, representative of two independent experiments. (k) Western blot anaylsis of cleaved caspase-3 levels in MIN6 cells infected with non-targeting control (CON) and SIK2 shRNA. Data is representative of two independent experiments. (ag): Error bars represent s.e.m. (hj): Error bars represent s.d. Statistical significance for all data was determined using two-tailed unpaired Students t-test (p < 0.01,p < 0.05). The statistics source data for (g) is provided in Supplementary Table 1.

Supplementary Figure 4 SIK2 status does not affect β cell insulin content or mitochondrial function.

(a) Insulin content from islets shown in Fig. 2b (n=3 mice for each condition). (b) Insulin content from islets treated with HG-9-91-1 shown in Fig. 2c (n=3 mice for each condition). (c) (left) GSIS assay showing percent insulin secretion (normalized to total insulin content) in control or SIK2 knockdown MIN6 cells cultured in 1 mM (black bars) or 20 mM (white bars) glucose. (middle) Insulin content. (right) Western blot anaylsis of SIK2 levels in MIN6 cells stably infected with lentivirus expressing non-targeting control (CON) or SIK2 shRNA. (d) Western blot showing levels of SIK2 protein in MIN6 cell extracts following infection with lentivirus expressing SIK2 WT, kinase-dead K49M or constitutively active S587A mutants compared to empty vector control. (e) Insulin content in MIN6 cells infected with lentivirus encoding an empty vector (CON) or SIK2 WT, K49M, or S587A cDNA. (f) Oxygen consumption in control and SIK2 knockdown MIN6 cells during mitochondrial stress test. Oxygen consumption rates are normalized to ug protein. (g) Extracellular acidification rate in cells from f, (ECAR, a proxy for glycolytic activity). Effects of addition of glucose (G) and the ATP synthase inhibitor oligomycin (O) are shown. All error bars represent s.d. (c, e, f and g): Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results. All western blot data are representative of three independent experiments with consistent results. The statistics source data for (a) and (b) are provided in Supplementary Table 1.

Supplementary Figure 5 Effect on p35 protein accumulation is specific to SIK2.

(a) Western blot analysis of p35 levels in MIN6 cells infected with lentivirus encoding shRNA targeting SIK2, AMPK alpha 1, LKB1, or non-silencing control (CON). (b) Western blot analysis of p35 and SIK2 levels in MIN6 cells infected with lentivirus encoding non silencing control shRNA (CON) or shRNAs targeting SIK1, SIK2 or SIK3. (c) QPCR data showing knockdown of SIK1 and SIK3 mRNAs from experiment shown in b. Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results. All western blot data are representative of three independent experiments with consistent results.

Supplementary Figure 6 p35 status does not affect β cell insulin content and SABKO islets show increased phospho-VDCC.

(a) Insulin content from cells shown in Fig. 5a. (b) Insulin content from cells shown in Fig. 5b. (c) Western blot analysis showing pSer783 levels on VDCC immunoprecipitated from S/S and SABKO islets. Western blot data is representative of two independent experiments with consistent results. (a and b): Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results.

Supplementary Figure 7 PJA2 is required for insulin secretion.

(a) Western blot analysis of endogenous p35 levels in PJA2 knockdown MIN6 cells. Data is representative of three independent experiments with consistent results. (b) Insulin secretion in PJA2 knockdown MIN6 cells after treatment with 1 mM and 20 mM glucose. Data is from a single experiment performed in triplicate, representative of three experiments. (c) Insulin content in MIN6 cells from Figure S6b that were infected with lentivirus encoding control non-silencing (CON) or two independent shRNAs targeting PJA2. (b and c) Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results.

Supplementary Figure 8 Sik2 gene expression is unaffected by nutrient status and SIK2-PJA2-p35 pathway is conserved in the brain.

(a) QPCR analysis of SIK2 mRNA levels normalized to 36B4 internal control in islets isolated from control C57BL6/J (B6) (n=4 mice) and ob/ob (n=3 mice) mice. Data are mean ± s.d. from n=3 or 4 mice from a singleexperiment, and are representative of three independent experiments with consistent results. (b) Insulin content in islets isolated from C57BL6/J (B6) (n=3 mice for each condition) or ob/ob (n=3 mice for each condition) mice infected with control non silencing (CON) or SIK2 shRNA. Error bars represent s.d. (c) QPCR analysis of SIK2 mRNA levels normalized to 36B4 internal control in MIN6 cells cultured for 15 h in 0, 5 or 25 mM glucose in the presence or absence of 2DG (5, 10, or 25 mM). Data are mean ± s.d. from n=3 technical replicates from a singleexperiment, and are representative of three independent experiments with consistent results. (d) Insulin secretion from mouse islets infected with lentivirus encoding control non-silencing (CON), SIK1, SIK2, or SIK3 (n=3 mice for each condition). (e) Insulin content from islets shown in a (n=3 mice for each condition). (c) Western blot showing p35 and SIK2 levels in indicated mouse tissues. (f) Western blot analysis showing levels of p35 and SIK2 in brain extracts from WT and SIK2 whole animal knockout mice. All error bars represent s.d. Statistical significance for all data was determined using two-tailed unpaired Students t-test (p < 0.05). All western blot data are representative of three independent experiments with consistent results. The statistics source data for (b, d and e) are provided in Supplementary Table 1.

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Sakamaki, JI., Fu, A., Reeks, C. et al. Role of the SIK2–p35–PJA2 complex in pancreatic β-cell functional compensation. Nat Cell Biol 16, 234–244 (2014). https://doi.org/10.1038/ncb2919

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