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. 2020 Dec:42:101056.
doi: 10.1016/j.molmet.2020.101056. Epub 2020 Jul 28.

Lactate activation of α-cell KATP channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca2+ entry

Karolina E Zaborska  1 , Prasanna K Dadi  1 , Matthew T Dickerson  1 , Arya Y Nakhe  1 , Ariel S Thorson  1 , Charles M Schaub  1 , Sarah M Graff  1 , Jade E Stanley  1 , Roy S Kondapavuluru  1 , Jerod S Denton  2 , David A Jacobson  3
Affiliations

Lactate activation of α-cell KATP channels inhibits glucagon secretion by hyperpolarizing the membrane potential and reducing Ca2+ entry

Karolina E Zaborska et al. Mol Metab. 2020 Dec.

Abstract

Objective: Elevations in pancreatic α-cell intracellular Ca2+ ([Ca2+]i) lead to glucagon (GCG) secretion. Although glucose inhibits GCG secretion, how lactate and pyruvate control α-cell Ca2+ handling is unknown. Lactate enters cells through monocarboxylate transporters (MCTs) and is also produced during glycolysis by lactate dehydrogenase A (LDHA), an enzyme expressed in α-cells. As lactate activates ATP-sensitive K+ (KATP) channels in cardiomyocytes, lactate may also modulate α-cell KATP. Therefore, this study investigated how lactate signaling controls α-cell Ca2+ handling and GCG secretion.

Methods: Mouse and human islets were used in combination with confocal microscopy, electrophysiology, GCG immunoassays, and fluorescent thallium flux assays to assess α-cell Ca2+ handling, Vm, KATP currents, and GCG secretion.

Results: Lactate-inhibited mouse (75 ± 25%) and human (47 ± 9%) α-cell [Ca2+]i fluctuations only under low-glucose conditions (1 mM) but had no effect on β- or δ-cells [Ca2+]i. Glyburide inhibition of KATP channels restored α-cell [Ca2+]i fluctuations in the presence of lactate. Lactate transport into α-cells via MCTs hyperpolarized mouse (14 ± 1 mV) and human (12 ± 1 mV) α-cell Vm and activated KATP channels. Interestingly, pyruvate showed a similar KATP activation profile and α-cell [Ca2+]i inhibition as lactate. Lactate-induced inhibition of α-cell [Ca2+]i influx resulted in reduced GCG secretion in mouse (62 ± 6%) and human (43 ± 13%) islets.

Conclusions: These data demonstrate for the first time that lactate entry into α-cells through MCTs results in KATP activation, Vm hyperpolarization, reduced [Ca2+]i, and inhibition of GCG secretion. Thus, taken together, these data indicate that lactate either within α-cells and/or elevated in serum could serve as important modulators of α-cell function.

Keywords: Ca(2+) handling; Glucagon secretion; K(ATP) channels; Lactate; Pyruvate; α-cells.

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Figures

Image 1
Graphical abstract
Figure 1
Figure 1
Lactate reduces ⍺-cell [Ca2+]i. Representative α-cell GCaMP3 recording (A and B) and relative fluorescence AUC (C) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM lactate. Representative δ-cell GCaMP6 recording (D and E) and relative fluorescence AUC (F) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM lactate. Representative Fura-2 AM recording from β-cells (G and H) and relative fluorescence AUC (I) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM lactate. Representative α-cell Fura-2AM recording (J and K) and relative fluorescence AUC (L) at 1 mM and 11 mM glucose in dispersed human α-cells in the presence or absence of 10 mM lactate. N = 3–8. The statistical analysis was conducted using the two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗P < 0.01).
Figure 2
Figure 2
Pyruvate decreases ⍺-cell [Ca2+]i. Representative α-cell GCaMP3 recording (A and B) and relative fluorescence AUC (C) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM pyruvate. Representative δ-cell GCaMP6 recording (D and E) and relative fluorescence AUC (F) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM pyruvate. Representative Fura-2 AM recording from β-cells (G and H) and relative fluorescence AUC (I) at 1 mM and 11 mM glucose in whole mouse islets in the presence or absence of 10 mM pyruvate. N = 3. The statistical analysis was conducted using the two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗∗P < 0.001).
Figure 3
Figure 3
Lactate entry into ⍺-cell via monocarboxylate transporters reduces [Ca2+]i. Representative lactate entry recording (A) and relative fluorescence AUC (B) from dispersed red fluorescent protein-expressing (αRFP) α-cells in 1 mM glucose before and after the addition of 10 mM lactate. Representative α-cell GCaMP3 recording (C) and relative fluorescence AUC (D) at 1 mM glucose in whole islets in the presence or absence of 10 mM lactate continuously treated with a MCT1/2/4 inhibitor (2 μM BAY8002) or vehicle control (E,F). Representative α-cell GCaMP3 recording (G) and relative fluorescence AUC (H) at 1 mM glucose in whole islets in the presence or absence of 10 mM lactate continuously treated with a MCT1 inhibitor (100 nM 7ACC2). N = 3. The statistical analysis was conducted using the two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗∗∗P < 0.0001).
Figure 4
Figure 4
Lactate hyperpolarizes ⍺-cells. Representative membrane potential (Vm) recording (A) and average Vm (B) from dispersed fluorescent protein-expressing (αRFP) α-cells at 1 mM glucose in whole mouse islets (N = 3 cells/3 mice) before and after the addition of 20 mM lactate. Representative membrane potential (Vm) recordings (D and G) and average Vm (E) from α-cells at 1 mM glucose in human non-β-cells pseudoislets (N = 6 cells/3 islet donors) before and after the addition of 20 mM lactate. Average action potential frequency from αRFP α-cells at 1 mM glucose in whole mouse islets (N = 3 cells/3 mice; C) and human non-β-cell pseudoislets (N = 6 cells/3 islet donors; F) before and after the addition of 20 mM lactate. Representative α-cell GCaMP3 recording (H) and relative fluorescence AUC (I) at 1 mM glucose in whole islets in the presence or absence of 10 mM lactate continuously treated with 30 mM KCl and 200 μM diazoxide (N = 3 mice). The statistical analysis was conducted using the two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗P < 0.01).
Figure 5
Figure 5
Lactate reduces ⍺-cell [Ca2+]ithrough activation of KATPchannels. Representative α-cell GCaMP3 recording (A) and relative fluorescence AUC (B) at 1 mM glucose (without Cl−) in whole islets in the presence or absence of 10 mM lactate. Representative α-cell GCaMP3 recording and relative fluorescence AUC at 1 mM glucose in whole islets in the presence or absence of 10 mM lactate continuously treated with a BK channel inhibitor (100 nM Iberiotoxin; C and D) or GIRK channel inhibitor (100 nM Tertiapin-Q; E and F). Representative α-cell GCaMP3 recording (G) and relative fluorescence AUC (H) at 1 mM glucose in whole islets in the presence or absence of 10 mM lactate or KATP channel blocker (100 μM glyburide). N = 3 mice. The statistical analysis was conducted using two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗P < 0.01).
Figure 6
Figure 6
Lactate and pyruvate activate KATPchannels. Representative thallium (Tl+) flux recordings from T-REx-HEK-293 cells expressing KATP channels treated with lactate (A; N = 3) or pyruvate (C; N = 3) (0.5, 2.5, and 10 mM) and uninduced KATP cells. Fluorescence values were normalized (F/F0) to baseline values recorded before Tl+ addition. Dose-dependent activation of KATP by lactate (B; N = 3) or pyruvate (D; N = 3). Area under the curve data are expressed as % activation calculated as % change in KATP activation by lactate or pyruvate compared to KATP activation by vehicle control (assay buffer with 7 μM KATP activator). Estimated EC50 values were calculated using 3-parameter (lactate) or 4-parameter (pyruvate) logistic regression fit. Representative KATP current recorded from dispersed red fluorescent protein-expressing (αRFP) α-cells in the presence or absence of 20 mM lactate (E) or 20 mM pyruvate (G). Average KATP currents (N ≥ 28 cells/4 mice) from α-cells in the presence or absence of 20 mM lactate (F) or 20 mM pyruvate (H). The statistical analysis was conducted using the unpaired two-tailed Student's t-test, and uncertainty is expressed as SE (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001).
Figure 7
Figure 7
Lactate and pyruvate reduce islet glucagon secretion. Average GCG secretion from mouse (A) and human (B) islets treated with or without 10 mM lactate at 1 mM (mouse N = 3; human N = 5) and 11 mM glucose (mouse N = 6; human N = 5) normalized to the total islet number and 11 mM glucose. (B) Average GCG secretion from mouse (C) and human (D) islets treated with or without 10 mM pyruvate at 1 mM and 11 mM glucose normalized to the total islet number and 11 mM glucose (N = 3). The statistical analysis was conducted using the two-tailed unpaired Student's t-test, and uncertainty is expressed as SE (∗P < 0.05 and ∗∗P < 0.01).
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