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A pancreatic islet-specific microRNA regulates insulin secretion

Abstract

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression1. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes2,3,4. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.

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Figure 1: miR-375 is expressed in pancreatic β-cells and regulates insulin secretion.
Figure 2: Expression of miR-375 using recombinant adenovirus (Ad-375) leads to impaired glucose-, KCl- and tolbutamide-induced insulin secretion in MIN6 cells.
Figure 3: No effect of miR-375 on intracellular Ca2+ signalling in β-cells.
Figure 4: Identification of target genes of miR-375.

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Acknowledgements

We thank K. Borglid, J. Chen, J. Galvanovskis, M. Lagos-Quintana, M. Landthaler, A. Lingqvist, G. Meister, B. M. Nilsson, A. Wendt and C. Wolfrum for advice and technical assistance. This work was supported by an unrestricted grant from Bristol Myers Squibb, the Juvenile Diabetes Research Foundation, the Deutsche Forschungsgemeinschaft and grants from the Swedish Research Council, the Swedish Diabetes Association, the Göran Gustafsson Stiftelse for Natural Sciences and Medicine and the Swedish Strategic Research Foundation (SSF).

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Correspondence to Markus Stoffel.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

Oligoribonucleotide 2’-O-me-375 anneals to endogenous miR-375 in MIN6 cells. Northern blot of MIN6 cells that were transfected with either 2’O-me-eGFP (control) or 2’-O-me-375. (PDF 84 kb)

Supplementary Figure 2

Increased [ATP]i levels in MIN6 cells infected with Ad-375 compared to Ad-eGFP. Total ATP measurements in MIN6 cells that were transfected with Ad-375 or Ad-eGFP. (PDF 42 kb)

Supplementary Figure 3

No apparent effects of miR-375 overexpression on submembrane actin network. Structural analysis using fluorescence microscopy of the submembrane actin network in MIN6 cells and primary β-cells that were infected with Ad-eGFP or Ad-375. (PDF 717 kb)

Supplementary Figure 4

Increased number of docked granules in Ad-375 infected β-cells. Electron micrographs of β-cells infected with Ad-eGFP or Ad-375 and quantitative analysis of granule density. (PDF 855 kb)

Supplementary Table 1

Mature and precursor sequences of mouse (mmu-miR) and human (hsa-miR) novel miRNAs, identified in mouse pancreatic α- and β-cell lines TC1 and MIN6, respectively. (DOC 182 kb)

Supplementary Table 2

Foldback precursor sequences of novel mouse miRNAs, identified in mouse pancreatic β-cell line MIN6. (DOC 24 kb)

Supplementary Table 3

Similar [Ca++]i in primary β-cells and MIN6 cells infected with Ad-GFP and Ad-375. Ca2+-measurements in primary β-cells and MIN6 cells infected with Ad-GFP and Ad-375 in response to 25 mM glucose and to 30 mM K+. (DOC 23 kb)

Supplementary Table 4

Similar [Ca++]i in response to glucose and K+ in primary b-cells and MIN6 cells infected with control siRNAs, si-375 and si-Mtpn. Ca2+-measurements in MIN6 cells co-transfected with CMV-eGFP and si-ApoM (control), si-375 or si-Mtpn in response to glucose K+. (DOC 20 kb)

Supplementary Methods

Description of methods used to identify miR-375 targets. (DOC 24 kb)

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Poy, M., Eliasson, L., Krutzfeldt, J. et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature 432, 226–230 (2004). https://doi.org/10.1038/nature03076

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