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, 97 (8), 4023-8

Role of AMP-activated Protein Kinase in the Regulation by Glucose of Islet Beta Cell Gene Expression

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Role of AMP-activated Protein Kinase in the Regulation by Glucose of Islet Beta Cell Gene Expression

G da Silva Xavier et al. Proc Natl Acad Sci U S A.

Abstract

Elevated glucose concentrations stimulate the transcription of the pre-proinsulin (PPI), L-type pyruvate kinase (L-PK), and other genes in islet beta cells. In liver cells, pharmacological activation by 5-amino-4-imidazolecarboxamide riboside (AICAR) of AMP-activated protein kinase (AMPK), the mammalian homologue of the yeast SNF1 kinase complex, inhibits the effects of glucose, suggesting a key signaling role for this kinase. Here, we demonstrate that AMPK activity is inhibited by elevated glucose concentrations in MIN6 beta cells and that activation of the enzyme with AICAR prevents the activation of the L-PK gene by elevated glucose. Furthermore, microinjection of antibodies to the alpha2- (catalytic) or beta2-subunits of AMPK complex, but not to the alpha1-subunit or extracellular stimulus-regulated kinase, mimics the effects of elevated glucose on the L-PK and PPI promoter activities as assessed by single-cell imaging of promoter luciferase constructs. In each case, injection of antibodies into the nucleus and cytosol, but not the nucleus alone, was necessary, indicating the importance of either a cytosolic phosphorylation event or the subcellular localization of the alpha2-subunits. Incubation with AICAR diminished, but did not abolish, the effect of glucose on PPI transcription. These data suggest that glucose-induced changes in AMPK activity are necessary and sufficient for the regulation of the L-PK gene by the sugar and also play an important role in the regulation of the PPI promoter.

Figures

Figure 1
Figure 1
Immunolocalization of AMPK isoforms in MIN6 beta cells (a) and regulation of AMPK activity by glucose and AICAR (b). (a) Cells were incubated for 24 h at the indicated glucose concentrations before probing for AMPK α1 and α2 isoforms as detailed in Experimental Procedures. Shown are clusters of 30–40 cells; the dark areas in the center of each cell, most evident after probing with the anti-AMPK α1 antibody, correspond to the position of the nuclei, as identified in bright-field images (not shown). (Bar = 20 μm.) (b) After incubation for 6 h at the indicated glucose concentration, each isoform of AMPK activity was assayed after immunoprecipitation as given in Experimental Procedures. Data are normalized to activity vs. AMPK α1 in extracts cultured at 3 mM and represent the means ± SEM of four separate experiments. P < 0.05 for the effect of 30 mM glucose (*) and 200 μM AICAR (§).
Figure 2
Figure 2
Regulation of L-PK gene expression by glucose and AICAR. L-PK mRNA was determined by semiquantitative reverse transcription–PCR, as detailed in Experimental Procedures, with 25 elongation cycles. (Lower) Autoradiograph of PCR products from a single experiment, run in duplicate. (Upper) Data from four separate experiments, *, P < 0.05 for the effect of 30 vs. 3 mM glucose; **, P < 0.01 for the effect of AICAR. (b) L-PK promoter activity was assessed after microinjection of cells with plasmids pLPK.LucFF and pRL.CMV and incubation for 6 h at either 3 or 30 mM glucose, plus 200 μM AICAR, as indicated. Firefly and R. reniformis luciferase activities were measured as detailed in the Experimental Procedures. Data were from five separate experiments, involving 42–70 productively injected cells in each condition. P < 0.05 for the effect of 30 vs. 3 mM glucose (*) and AICAR (§).
Figure 3
Figure 3
Effect of anti-AMPK antibody injection on regulation of L-PK promoter by glucose. (a) MIN6 cells were microinjected with plasmids LPK.LucFF and pCMV.RL as detailed in Fig. 2, plus either preimmune IgG or anti-AMPK-α2 IgG (1.0 mg⋅ml−1 each) as shown. In all cases, cells were doubly injected into the cytosol and nucleus and cultured for 6 h at the indicated glucose concentrations before successive imaging of firefly and R. reniformis luciferase activities (see Experimental Procedures and ref. 38). The intensity of cell luminescence is given in pseudocolor (photon⋅pixel−1⋅s−1). The relative activity of the L-PK vs. cytomegalovirus promoters in individual cells is indicated in the ratiometric images (firefly/R. reniformis luciferase activities) which were calculated by using photek software. (Bar = 50 μm.) (b) Effect of nucleus-only injection of anti-α2-antibodies. (c) Same as b, but with nucleus plus cytosolic injection. (d) Same as c, but with anti-AMPK β2 antibodies. (e) Same as c, using anti-AMPK α1-IgG. (f) Same as c, using anti-Erk2 (p42 MAPK) antibodies. Data from three to five separate experiments, with 20–90 productively injected cells per condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001 for the effect of 30 vs. 3 mM glucose; §§, P < 0.01 for the effect of anti-AMPK antibodies.
Figure 4
Figure 4
Effect of anti-AMPK antibody injection on regulation of the insulin promoter by glucose. Transcriptional analysis was performed as described in Fig. 3, after microinjection with the antibodies indicated. Effects of AICAR (no IgG injected) (a); microinjection of control IgG or anti-AMPK-α2 IgG into the cytosol plus nucleus (b); nucleus-only microinjection of anti-AMPK-α2 IgG (c); microinjection of control IgG, anti-AMPK-α1, or β2 IgG into cytosol plus nucleus, as shown (d and e). Data from three to five separate experiments, 32–105 productively injected cells per condition. *, P < 0.05; *, P < 0.01 for the effect of 30 vs. 3 mM glucose; §, P < 0.05 for the effect of AICAR.

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