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. 2017 Nov;5(21):e13493.
doi: 10.14814/phy2.13493.

MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles

Vishal A Salunkhe  1 , Jones K Ofori  1 , Nikhil R Gandasi  2 , Sofia A Salö  1 , Sofia Hansson  1 , Markus E Andersson  1 , Anna Wendt  1 , Sebastian Barg  2 , Jonathan L S Esguerra  3 , Lena Eliasson  3
Affiliations

MiR-335 overexpression impairs insulin secretion through defective priming of insulin vesicles

Vishal A Salunkhe et al. Physiol Rep. 2017 Nov.

Abstract

MicroRNAs contribute to the maintenance of optimal cellular functions by fine-tuning protein expression levels. In the pancreatic β-cells, imbalances in the exocytotic machinery components lead to impaired insulin secretion and type 2 diabetes (T2D). We hypothesize that dysregulated miRNA expression exacerbates β-cell dysfunction, and have earlier shown that islets from the diabetic GK-rat model have increased expression of miRNAs, including miR-335-5p (miR-335). Here, we aim to determine the specific role of miR-335 during development of T2D, and the influence of this miRNA on glucose-stimulated insulin secretion and Ca2+-dependent exocytosis. We found that the expression of miR-335 negatively correlated with secretion index in human islets of individuals with prediabetes. Overexpression of miR-335 in human EndoC-βH1 and in rat INS-1 832/13 cells (OE335) resulted in decreased glucose-stimulated insulin secretion, and OE335 cells showed concomitant reduction in three exocytotic proteins: SNAP25, Syntaxin-binding protein 1 (STXBP1), and synaptotagmin 11 (SYT11). Single-cell capacitance measurements, complemented with TIRF microscopy of the granule marker NPY-mEGFP demonstrated a significant reduction in exocytosis in OE335 cells. The reduction was not associated with defective docking or decreased Ca2+ current. More likely, it is a direct consequence of impaired priming of already docked granules. Earlier reports have proposed reduced granular priming as the cause of reduced first-phase insulin secretion during prediabetes. Here, we show a specific role of miR-335 in regulating insulin secretion during this transition period. Moreover, we can conclude that miR-335 has the capacity to modulate insulin secretion and Ca2+-dependent exocytosis through effects on granular priming.

Keywords: TIRF; Beta cell; SNAP25; STXBP1; Type 2 Diabetes; exocytosis; insulin secretion; microRNA; patch‐clamp.

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Figures

Figure 1
Figure 1
miR‐335 and insulin secretion in human islets and EndoC‐βH1 cells. (A) miR‐335 expression in human islets NGT donors against stimulation index, (B) As in A, but data are measured in human islets from IGT donors. (C) Insulin secretion in EndoC‐βH1 cells overexpressing miR‐335 (OE335(h); black bars) and in control cells (SCR(h); white bars) cells after stimulation for 1 h in 2.8 mmol/L or 16.7 mmol/L glucose (2.8G and 16.7 G) as indicated. n = 3; *P < 0.05. (D) As in C, but insulin secretion was measured after 15 min stimulation in 2.8 mmol/L glucose (2.8G) in the absence and presence of 50 mmol/L KCl (K+).
Figure 2
Figure 2
Overexpression of miR‐335 and its effect on the expression of three of its putative protein targets. (A) Expression of miR‐335 using mature miR‐335 mimic (OE335; black bar) relative to its expression using scramble control (SCR; white bar). Mir‐335 expression was normalized to endogenous controls U6 and U87. (n = 4; *P < 0.05). (B, C, D). Average protein levels of SNAP25, STXBP1 and SYT11 in OE335 (black bar) compared to SCR (white bar) cells (n = 4 each group; *P < 0.05). Representative western blots are shown for each protein. Expression levels were normalized to beta‐actin or cyclophilin B (PPIB).
Figure 3
Figure 3
Consequence on insulin secretion and exocytosis in INS‐1 832/13 cells by miR‐335 overexpression. (A) Insulin secretion in OE335 (black bars) and SCR (white bars) cells after stimulation for 1 h in 2.8 mmol/L or 16.7 mmol/L glucose as indicated. n = 3; **P < 0.01. Secreted insulin is measured using human insulin‐RIA.(B) Insulin content in OE335 (black bar) and SCR (white bars) cells. n = 3. (C) Insulin secretion in OE335 (black bars) and SCR (white bars) cells after stimulation for 15 min. 2.8 mmol/L glucose with or without 50 mmol/L K+ as indicated. n = 5; *P < 0.05. (D) Representative traces of depolarization‐induced increases of membrane capacitance in OE335 (black trace) and SCR (gray trace) cells. (E) Summary of capacitance changes presented as the summed increased in membrane capacitance during all ten depolarizations (ΣDepolall), increase evoked by the first two depolarizations (ΣDepol1‐2) or the latter eight depolarizations (ΣDepol3‐10). n = 13 for OE335 (black bar) and n = 16 for SCR (white bar) cells; *P < 0.05. (F) Representative traces of a voltage‐dependent Ca2+ currents evoked by a depolarization from −70 mV to 0 mV in a control cell (SCR) and a cell overexpressing miR‐335 (OE335), respectively. (G) Summary of the charge (Q)‐voltage (V) relationship. n = 15 for SCR and n = 17 for OE335 cells.
Figure 4
Figure 4
Influence of miR‐335 knockdown on insulin secretion and exocytosis in INS‐1 832/13 cells. (A) Expression of miR‐335 in cells after knockdown of miR‐335 by LNA against miR‐335 (LNA335; black bar) relative to expression in scramble control cells (SCR; white bar). n = 4; ***P < 0.001. (B) Insulin secretion in LNA335 (black bars) and SCR (white bars) cells after stimulation for 1 h in 2.8 mmol/L or 16.7 mmol/L glucose as indicated (n = 4). The amount of released insulin is measured using human/rat insulin‐ELISA. (C) Insulin content in LNA335 (black bars) and SCR (white bars) cells. n = 8; *P < 0.05. (D) Insulin secretion in OE335 (black bars) and SCR (white bars) cells after stimulation for 15 min. in 2.8 mmol/L glucose with 50 mmol/L K+. n = 4. (E) Representative traces of depolarization‐induced increases of membrane capacitance in LNA335 (black trace) and SCR (gray trace) cells. (F) Summary of capacitance changes presented as the summed increased in membrane capacitance during all ten depolarizations (ΣDepolall), increase evoked by the first two depolarizations (ΣDepol1‐2) or the latter eight depolarizations (ΣDepol3‐10). n = 10 for LNA335 (black bar) and n = 9 for SCR (white bar) cells; *P < 0.05
Figure 5
Figure 5
Function of miR‐335 on density of granules and exocytosis measured by TIRF microscopy in INS‐1 832/13 cells. (A) Representative TIRF images of OE335 and SCR cells co‐transfected with NPY‐mEGFP. (B) Density of granules in OE335 (black bar; n = 31) and SCR (white bar; n = 53) cells. (C) Examples of single exocytosis event measured in OE335 and SCR cells, frames are 100 msec apart. Exocytosis was stimulated through depolarization using elevated K+. (D, E) Cumulative number of exocytosis events per area in OE335 and SCR cells (n = 8 each group; ***P < 0.001). (F) Representative image of fluorescence time course during a single exocytosis event (black) with fit overlaid (red) illustrating the numerical analysis. The interval between the moments of exocytosis/fusion and release is taken as fusion pore lifetime (green). (G, H) Summary of fitted exocytosis and decay constants of individual events as described in (F) The lifetime of the events (*P=n.S.) and decay constant from individual granules during content release (*P < 0.05) was measured in OE335 and SCR cells (n = 8 each group). (I,J) Average fluorescence signal of granule fluorescence for exocytosis events in OE335 and SCR cells as in C.
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References

    1. Ammala, C. , Eliasson L., Bokvist K., Larsson O., Ashcroft F. M., and Rorsman P.. 1993. Exocytosis elicited by action potentials and voltage‐clamp calcium currents in individual mouse pancreatic B‐cells. J. Physiol. 472:665–688. - PMC - PubMed
    1. Andersson, S. A. , Olsson A. H., Esguerra J. L., Heimann E., Ladenvall C., Edlund A., et al. 2012. Reduced insulin secretion correlates with decreased expression of exocytotic genes in pancreatic islets from patients with type 2 diabetes. Mol. Cell. Endocrinol. 364:36–45. - PubMed
    1. Andersson, L. E. , Valtat B., Bagge A., Sharoyko V. V., Nicholls D. G., Ravassard P., et al. 2015. Characterization of stimulus‐secretion coupling in the human pancreatic EndoC‐betaH1 beta cell line. PLoS ONE 10:e0120879. - PMC - PubMed
    1. Arango Duque, G. , Fukuda M., and Descoteaux A.. 2013. Synaptotagmin XI regulates phagocytosis and cytokine secretion in macrophages. J. Immunol. 190:1737–1745. - PubMed
    1. Barg, S. , Olofsson C. S., Schriever‐Abeln J., Wendt A., Gebre‐Medhin S., Renstrom E., et al. 2002. Delay between fusion pore opening and peptide release from large dense‐core vesicles in neuroendocrine cells. Neuron 33:287–299. - PubMed