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Figure 5 | BMC Endocrine Disorders

Figure 5

From: Nutrient-stimulated insulin secretion in mouse islets is critically dependent on intracellular pH

Figure 5

Insulin secretion and pH i -changes produced by MP: A. MP does not stimulate insulin release, even with intracellular acidification: Insulin secretion in response to 20 mM MP with and without intracellular acidification, from freshly isolated islets. Controls were exposed to 2.8 mM and 16.7 mM glucose. All groups were first pre-incubated in basal glucose for one hour, and subsequently exposed to each condition denoted in the X axis for one hour, followed by sample collection for insulin assay. Insulin secretion is expressed as fractional release %, i.e. the percentage of the total insulin content released over the one-hour period of stimulation with different agents. Intracellular acidification was produced by addition of 40 μM DMA. G = mM glucose. n = 6. B. MP produces a drastic decrease in intracellular pH: Islets loaded with SNARF5-AM were placed in basal KRBH, excited at 514 nm and emission fluorescence recorded at 580 and 630 nm. After a steady baseline was established, 20 mM MP was added where indicated. One representative recording is shown. n = 16; average decrease in pHi = 1.0 ± 0.08. C and D. MP-induced acidification is not affected by traditional methods of alkalinization: Same procedure as in B. Islets were switched to Cl--free medium (C) or high pH (>8.3) medium (D), as indicated, before addition of MP. One representative recording for each condition is shown. C: n = 3; D: n = 4. E. MP-induced acidification is reversed by a Cl--free high pH medium: Islets loaded with SNARF5-AM were placed in Cl--free KRBH of pH above 8.3, excited at 514 nm and emission fluorescence recorded at 580 and 630 nm. 20 mM MP was added where indicated. One representative recording is shown. n = 8. Average pHi in Cl--free high pH medium = 7.5 ± 0.04; Average lowest pHi after addition of MP = 6.1 ± 0.12; Average pHi after recovery = 6.9 ± 0.1; pHi range after recovery = 6.4–7.0 F. MP stimulates insulin secretion when pHi is brought back to 6.4–7.0 by strong alkalinization: Same procedure as in A. Intracellular alkalinization was produced by exposure to a Cl--free high pH (>8.3) medium. G = mM glucose; n = 8; * = p < 0.001, compared to MP without alkalinization or basal glucose control.

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