ROS stimulates insulin secretion independent of calcium.

That would be pretty novel. Our current knowledge suggests that all signalling pathways converge at the level of intracytoplasmic ca increase to direct insulin containing vesicles to plasma membrane. Have you used ca channel blockers or anything that would inhibit ER release of ca from pancreatic beta cells ?

Dear Dr. Salih Sanlioglu, thank you for your answer. Yes, I tried nifedipine (L-type ca2+ channel blocker) and also thapsigargin (SERCA blocker) and that molecule-induced insulin secretion completely blocked. Consequently I speculated that it might increase intracellular Ca2+ influx. But I found no changes of Fura 2 AM (340/380) ratio in a perfusion system of MIN6 cells. May be I need to optimize my experimental conditions (for example: modification of buffer constituents or duration of cell culture). Do you have any further suggestion regarding optimum condition for Ca+ influx measurement. I also found fibroblast like structure (weak response to glucose) besides round shape structure (good response to glucose) even after overnight culture of MIN6 cells. How to keep insulin secretory property of pancreatic beta cells for long time?

You should have a look at the following reference "Mechanism of ethanol-induced insulin secretion from INS-1 and INS-1E tumor cell lines" by Roman Hafko et al. I came upon this kind of topic a while back. There are other papers out there and I think the mechanism is believed to be due to hypotonic swelling due to ethanol's cell membrane permeability and its sphere of hydration (urea can do it also). Whether intracellular Ca plays a role I'm not sure, but it does point to mechano-sensory receptors possibly be activated by the swelling. Maybe your molecule is acting in this manner.

ROS stimulates insulin secretion independent of calcium.

Hi.. to be sure your Fura2AM ratio-metric analysis working well with your model Min6; hope you have also induced with some positive control like glucose or KCl (~50mM) for Ca flux......then you can also try some culture condition like 1h starvation (just in KRBH buffer) or culture O/N in low glucose(2.8mm) medium...then induced with you molecule...

You may also try some advanced sensitive dye like Fura4 NW ..by FACS or live cell imaging

Next you can ask for the Ca independent mechanism for insulin secretion....good luck

It is important to define what you mean by "calcium-independent". Calcium is somewhat fundamental to the secretory process (among many cellular processes), so very little in a cell will function properly in the complete absence of calcium. That said, there are numerous examples of pathways that can potentiate insulin release without changes in calcium levels in the beta-cell provided there is sufficient calcium for exocytosis to occur. I’ve listed a few references as examples of this below. Also, note that the endoplasmic reticulum is such a huge source and sink for calcium that it has the potential to significantly alter calcium levels in the cytosol, mitochondria and nucleus. We touch upon this in a review article on the role of calcium in cytokine action in beta-cells (JW Ramadan et al. Cell Calcium, 50:481-90, 2011). Good luck with your studies!

Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Ammälä C, Ashcroft FM, Rorsman P. Nature. 1993 May 27;363(6427):356-8.

Relative contribution of Ca2+-dependent and Ca2+-independent mechanisms to the regulation of insulin secretion by glucose. Sato Y, Nenquin M, Henquin JC. FEBS Lett. 1998 Jan 9;421(2):115-9.

Glucose-dependent increase in mitochondrial membrane potential, but not cytoplasmic calcium, correlates with insulin secretion in single islet cells. Heart E, Corkey RF, Wikstrom JD, Shirihai OS, Corkey BE. Am J Physiol Endocrinol Metab. 2006 Jan;290(1):E143-E148. Epub 2005 Sep 6. PMID: 16144817

Dear Sir, I couldn't download the paper entitled "Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells."Cuold you please send me that paper

Thank you for your reply.

Dr. Niamh Cawley, thank you for your comment I have studied few papers that you suggested. Cell swelling induced hormone secretion is not associated with a rise in cAMP level [wang et al., Metabolism; 42: 435-439]. This resembles my data as I also found no significance change of cAMP in response to my compound. But this data remains paradoxical since Ca2+ independent effect of glucose (or other secratogogue) to augment insulin secretion involves the activation of PKA and PKC. Moreover hypotonicity induced swelling activates L-type Ca2+ channel and chloride channel. That means intracellular ca2+ should be increased in a magnitude that could be detectable by ratio metric analyses. So still it remains elusive how my desired molecule augment insulin secretion? In case of mechanoreceptors I found that gadolinium (GdCl3) is its most specific inhibitor [Cell Physiol Biochem 2000; 10: 393-402]. I will appreciate your suggestions regarding this issue.

To Dr. Vikash Chandra, thank you for your comment. I used FLURO dish to culture MIN6 cells and cells were perfused for 30 min with basal glucose (5.5 mM glucose).I used glucagon like peptide-1 (GLP-1, the incretin hormone) as a positive control in my experiment. After 20 min perfusion with my desired molecule I exposed the cells in tolbutamide. Cells that exhibited more than 200% of the 340-to-380 fluorescence ratio to tolbutamide were used for the analysis [Moin et al., Biochem Biophys Res Commun. 2012 Nov 30; 428(4): 512-7]

To Dr. Barbara Corkey, thank you for your comment. According to your paper [Marylana et al., PLoS One, 2012: 7(1), e30200] Mono-acyl-glycerol (MOG) lead to accumulation of intracellular mediators resulting in time-dependent impaired insulin release and this process is Ca2+ independent. My current research involves GSIS in response to my desired molecule in presence glucose (in fact in presence of high glucose only). In basal condition it showed no response. I am planning to check the effect of that molecule in presence of FFA or in HFD mice. Though we did not find any GPCR involvement for this molecule in pancreatic β cell, it’s worth checking cellular redox state like NAD(P)H: NAD(P) ratio, ROS or LC-CoA in response to my molecule.

To Dr. Craig Nunemaker, thank you for your comment. I have only checked the cytosolic Ca2+ changes in response to my desired compound. I appreciate your points that the kinetics of Ca2+ movement between ER, mitochondria and nucleus could play significant role for insulin secretion. I had already listed your paper [JW Ramadan et al., Cell Calcium, 50:481-90, 2011] for citation in our group’s coming manuscript. Simultaneous imaging of free [Ca2+] in mitochondria and in the cytosol could be performed using the mitochondrial pericam 2mt8RP and Fura-Red, respectively. [Ca2+] could also be measured simultaneously in the ER and cytosol using D4ER cameleon and Indo-1 respectively [Tarasov et al., Pflugers Arch - Eur J Physiol (2013) 465:543–554]. But our lab system may not support these experiments. I will appreciate your any further suggestions regarding this issue.

To Abu Saleh Md Moin, the methods you discuss for parsing out sources of calcium are among the best available. As you mention, these are also fairly difficult to perform without sophisticated equipment. Acutely blocking SERCA pumps with thapsigargin or cyclopiazonic acid while making your intracellular calcium recording is a simple approach that can provide an estimate of ER calcium storage/utilization. We have used this technique to show that reduced calcium release from the ER is indicative of ER dysfunction in islets from diabetes-prone mice (O’Neill CM et al., Endocrinology 154(9):3077-88, 2013). Since you are looking at cells in stimulatory glucose conditions, you should also make your calcium recordings in the continuous presence of diazoxide (to keep KATP channels open and keep the membrane hyperpolarized to prevent calcium influx). This approach should be able to tell you whether your drug of interest appears to impact ER calcium handling. You could then move on to more sophisticated techniques for more direct measurements of ER calcium. For mitochondrial calcium, the calcium changes would likely be very small and require a mitochondrial-specific probe. I hope this helps.

At a threshold level of glucose near 5mM, the glucose-induced depolarization is sufficient to trigger the opening of voltage dependent calcium channels in the β cell membrane. This admits extracellular Ca2+ into the intracellular pool of free Ca2+ where it interacts directly with the insulin exocytotic machinery to fuse the insulin secetory granules to the plasma membrane and release insulin.  The actual release of insulin occurs by exocytosis, a process in which the granule membrane fuses with the cell membrane, the membranes are disrupted at the point of fusion, and the insulin crystal is discharged to the extracellular space, leaving the granule membrane inserted into the cellular plasma membrane.  The granule membrane and the proteins it contains are thus inserted into and become part of the cellular plasma membrane.  The process of exocytosis is the rate limiting step for physiologic insulin secretion.