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. 2018 Feb;67(2):256-264.
doi: 10.2337/db17-0506. Epub 2017 Nov 3.

miR-204 Controls Glucagon-Like Peptide 1 Receptor Expression and Agonist Function

SeongHo Jo  1 , Junqin Chen  1 , Guanlan Xu  1 , Truman B Grayson  1 , Lance A Thielen  1 , Anath Shalev  2
Affiliations

miR-204 Controls Glucagon-Like Peptide 1 Receptor Expression and Agonist Function

SeongHo Jo et al. Diabetes. 2018 Feb.

Abstract

Glucagon-like peptide 1 receptor (GLP1R) agonists are widely used to treat diabetes. However, their function is dependent on adequate GLP1R expression, which is downregulated in diabetes. GLP1R is highly expressed on pancreatic β-cells, and activation by endogenous incretin or GLP1R agonists increases cAMP generation, which stimulates glucose-induced β-cell insulin secretion and helps maintain glucose homeostasis. We now have discovered that the highly β-cell-enriched microRNA, miR-204, directly targets the 3' UTR of GLP1R and thereby downregulates its expression in the β-cell-derived rat INS-1 cell line and primary mouse and human islets. Furthermore, in vivo deletion of miR-204 promoted islet GLP1R expression and enhanced responsiveness to GLP1R agonists, resulting in improved glucose tolerance, cAMP production, and insulin secretion as well as protection against diabetes. Since we recently identified thioredoxin-interacting protein (TXNIP) as an upstream regulator of miR-204, we also assessed whether in vivo deletion of TXNIP could mimic that of miR-204. Indeed, it also enhanced islet GLP1R expression and GLP1R agonist-induced insulin secretion and glucose tolerance. Thus, the present studies show for the first time that GLP1R is under the control of a microRNA, miR-204, and uncover a previously unappreciated link between TXNIP and incretin action.

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Figures

Figure 1
Figure 1
miR-204 targets the GLP1R 3′ UTR. Alignment of the miR-204 seed sequence (arrow) with target sequences (bold) identified in the WT GLP1R 3′ UTR of human (hGLP1R-WT, site 1 and site 2) (A) or rat (rGlp1r WT) (B); mutated (M) target sequences are shown in red or blue, respectively. HEK293 cells were cotransfected with WT or mutant 3′ UTR luciferase reporter plasmids and with miR-204 mimic or negative control, and miR-204–directed repression of the luciferase reporter gene bearing human (C) or rat (D) WT or mutant 3′ UTR segments was assessed 48 h after transfection. Bars represent means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01.
Figure 2
Figure 2
miR-204 regulates GLP1R expression in primary human and mouse islets and in rat INS-1 cells. Primary human islets (A), mouse islets (B), and rat INS-1 cells (C) were transfected with miR-204 mimic or negative control, and 72 h later, GLP1R mRNA expression was assessed by qRT-PCR. D: GLP1R protein was measured by immunoblotting and quantified by ImageQuant in INS-1 cells 72 h after transfection with miR-204 mimic or negative control. E: INS-1 cells were transfected with miR-204 inhibitor or inhibitor negative control, and GLP1R mRNA expression was assessed 48 h later. All data are shown as the mean ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01.
Figure 3
Figure 3
Deletion of miR-204 enhances islet GLP1R expression and promotes Ex-4–induced insulin secretion and glucose control in vivo in mice. miR-204 (A) and GLP1R (B) expression in islets of 204KO mice and control littermates as assessed by qRT-PCR and corrected for U6 and 18S, respectively. C: Glucose tolerance tests with or without injection of Ex-4 (10 nmol/kg i.p. at −30 min) using age- and sex-matched 204KO or control mice. Glucose (1.5 g/kg i.p.) was injected at 0 min, and blood glucose was measured at the designated time points; glucose AUC (D) and Ex-4–induced changes in serum insulin (E). F: Insulin secretion from isolated islets of 204KO or control mice in response to Ex-4. G: Intracellular cAMP production in response to Ex-4 was assessed in isolated islets of 204KO or control mice. All data are shown as the mean ± SEM; n = 3–6 mice per group. *P < 0.05; ***P < 0.001.
Figure 4
Figure 4
204KO mice exhibit improved glucose control and increased serum insulin levels in the context of diabetes. To induce diabetes, male 204KO and control mice received multiple low-dose STZ injections with or without the GLP1R antagonist Ex-9. A: The blood glucose on day 1 prior to STZ and on day 15 after the start of the STZ injections is shown. B: Comparison of serum insulin levels on day 15. Bars represent means ± SEM; n = 3–6 mice per group. *P < 0.05; ***P ≤ 0.001.
Figure 5
Figure 5
miR-204 expression is low or absent in stomach or exocrine pancreas, and GLP1R expression remains unaltered in response to miR-204 deletion in these tissues. A: Low or absent expression of miR-204 in stomach and exocrine pancreas of control and 204KO mice as compared with miR-204 expression in control islets. GLP1R expression in stomach (B) and exocrine pancreas (C) of 204KO mice and control mice. All data are shown as the mean ± SEM of at least three mice.
Figure 6
Figure 6
β-Cell–specific TXNIP knockout mice have lower miR-204 and higher GLP1R expression and exhibit enhanced insulin secretion and glucose control in response to Ex-4. miR-204 (A) and GLP1R (B) expression was measured in islets of bTKO mice and control mice using qRT-PCR. C: Glucose tolerance tests were performed in age- and sex-matched bTKO or control mice by glucose administration (1.5 g/kg i.p. at 0 min) and with or without Ex-4 (10 nmol/kg i.p. at −30 min); blood glucose was measured at the designated time points; glucose AUC (D) and Ex-4–induced changes in serum insulin (E). F: Intracellular cAMP production in response to Ex-4 was assessed in isolated islets of bTKO or control mice; n = 3–9 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.
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