Medicine

The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults

Arya Y. Nakhe
Prasanna K. Dadi
Jinsun Kim
Matthew T. Dickerson
Soma Behera
Jordyn R. Dobson
Shristi Shrestha
Jean-Philippe Cartailler
Leesa Sampson
Mark A. Magnuson
David A. Jacobson author has email address

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232, USA
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
Center for Stem Cell Biology, Vanderbilt University, Nashville, TN 37232, USA
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 37232, USA

https://doi.org/10.7554/eLife.89967.2

Open access
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Figures and data

Kcnk16 L114P neonates exhibit loss of glucose stimulated Ca²⁺ entry and insulin secretion leading to transient neonatal hyperglycemia and death.
A. Schematic of the Kcnk16 L114P mouse line generation in the C57BL/6J background and the C57BL/6J:CD-1(ICR) mixed background. B. χ² analysis of the F1 progeny from C57BL/6J and heterozygous Kcnk16 L114P (L/P) crossings genotyped at weaning on post-natal day 21 (P21). C. Blood glucose measurements of male wildtype (WT; black; N=8), heterozygous Kcnk16 L114P (L/P; green; N=16), and homozygous Kcnk16 L114P (P/P; blue; N=9) mice on P4. D. Plasma insulin measurements performed on P4 in WT (N=4), Kcnk16 L114P (L/P; N=4), and Kcnk16 L114P (P/P; N=5) neonates. E. and F. In-vitro glucose-stimulated insulin secretion from P4 mouse islets stimulated with 2.8 mM glucose (G) or 20 mM G, respectively (WT; N=4, Kcnk16 L114P (L/P); N=8, and Kcnk16 L114P (P/P); N=6). G. Pancreas weight/ body weight measurements of P4 male mice (WT; N=6, Kcnk16 L114P (L/P); N=14, and Kcnk16 L114P (P/P); N=9). H. Representative immunostaining images of pancreas sections from P0 WT and Kcnk16 L114P (L/P) mice (N=3 mice/genotype), stained against insulin (INS; green), glucagon (GCG; red), somatostatin (SST; magenta), and Hoechst (blue); scale bar=100 μm. I. and J. Average islet area and area fraction of hormone staining per islet quantified using Fiji ImageJ software in P0 mouse pancreas sections. K. Representative glucose stimulated Ca²⁺ influx traces from P4 mouse islets sequentially stimulated with 2 mM glucose (G), 7 mM G, 20 mM G, and 20 mM G with 30 mM KCl (WT; N=6, Kcnk16 L114P (L/P); N=9, and Kcnk16 L114P (P/P); N=7). L-N. Average area under the curve (AUC) analysis of normalized Ca²⁺ during 7 mM G, 20 mM G, and 20 mM G+30 mM KCl stimulations. O. Survival curve for Kcnk16 L114P (P/P) mice treated with (N=6) or without (N=6) insulin (Lantus insulin glargine; 0.2 U/kg/day) starting at P0 up until death or weaning. Data are presented as mean±SEM. Data were analyzed using student’s t-test, one-way ANOVA, and two-way ANOVA as appropriate.

Adult Kcnk16 L114P mice exhibit fasting hyperglycemia and glucose intolerance.
A. and B. Intraperitoneal glucose tolerance test (i.p. GTT) performed in 15-week-old and 18-week-old male mice following a 4-hour fast in response to 2mg/g glucose injection (WT; black; N=8-10 and Kcnk16 L114P (L/P); green; N=9-10). C. Average AUC of the 2-hr GTT excursion profiles from ages 7 weeks up to 18 weeks in male mice. D. Weekly body weight measurements of male WT; N=5 mice and Kcnk16 L114P (L/P); N=5 mice E. and F. I.P. GTT performed in 15-week-old and 18-week-old female mice following a 4-hour fast in response to 2mg/g glucose injection WT; N=9-11 and Kcnk16 L114P (L/P); N=10-11. G. Average AUC of the 2-hr GTT excursion profiles from ages 7 weeks up to 18 weeks in female mice. H. Body weight measurements of female WT (black; N=4) and Kcnk16 L114P (L/P; blue; N=5) mice. Data are presented as mean±SEM. Data were analyzed using student’s t-test.

Adult Kcnk16 L114P mice show disrupted islet hormone secretion and islet composition.
A. and B. Plasma insulin levels in male (A) and female (B) WT and Kcnk16 L114P(L/P) mice following a 4-hour fast at the indicated time points before and after a 2mg/g glucose injection. c. Glucose (%) change in response to an i.p. human insulin injection (0.75 UI/kg body weight) was measured as an indicator of insulin sensitivity in WT and Kcnk16 L114P male mice after a 4-hour fast. D. and E. In-vitro insulin secretion (N=5 mice/genotype) and glucagon secretion (N=3 mice/genotype) from male mice at the specified glucose concentrations F. Representative immunostaining images of pancreas sections from WT and Kcnk16 L114P(L/P) male mice (N=3/genotype) stained against insulin (INS; green), glucagon (GCG; red), somatostatin (SST; magenta), and Hoechst (blue); scale bar = 100 μm. G-J. Average islet area and area of hormone staining/ islet for β-cells (INS), α-cells (GCG), and δ-cells (SST). Data are presented as mean±SEM. Data were analyzed using student’s t-test or two-way ANOVA as appropriate.

Kcnk16 L114P blunts β-cell glucose-stimulated electrical excitability and increases whole-cell two-pore domain K⁺ channel currents.
A. Representative perforated patch-clamp V_m recordings in response to 2 mM G and 11 mM G in islets from WT and Kcnk16 L114P(L/P) mice (N=6-10 islets from 5 mice/genotype). B. Average resting islet V_m under 2 mM G and plateau islet V_m at 11 mM G. C. Representative whole-cell two-pore domain K⁺ channel current density (pA/pF) recorded using a voltage ramp (–120 mV to +60 mV) at 11 mM G in β-cells from WT and Kcnk16 L114P(L/P) mice. D. Average current density (pA/pF) at the specified membrane voltages during the voltage ramp recordings shown in panel C. (N=9-13 cells/genotype). E. Representative whole-cell two-pore domain K⁺ channel current (pA) recorded using a voltage ramp (–120 mV to +60 mV) in 11 mM G in HEK293T cells expressing either Kcnk16 WT or Kcnk16 L114P(L/P). F. Average current (pA) at the specified membrane voltages during the voltage ramp recordings shown in panel E. (N=9-11 cells/ group). Data are presented as mean±SEM. Data were analyzed using two-way ANOVA.

Kcnk16 L114P reduces glucose– and tolbutamide-stimulated islet Ca²⁺ entry and augments IP₃-induced islet [Ca²⁺]_ER release.
A. and B. Representative [Ca²⁺]_c traces in islets from male (A) and female (B) WT and Kcnk16 L114P(L/P) mice in response to 2 mM G, 10 mM G, and 20 mM G. C-F. Average normalized Ca²⁺ peak (C. and E.) and total AUC (D. and F.) in response to the indicated glucose concentrations in islets from male and female WT and Kcnk16 L114P(L/P) mice (N=3-4/genotype). G. Representative [Ca²⁺]_c traces in islets from WT and Kcnk16 L114P(L/P) male mice in response to 100 μM tolbutamide followed by 30 mM KCl stimulation. H. Average AUC of normalized [Ca²⁺]_c during 100 μM tolbutamide or 100 μM tolbutamide with 30 mM KCl stimulation (N=3 mice/genotype). I. Representative [Ca²⁺]_c traces in response to 100 μM acetylcholine in the absence of extracellular Ca²⁺ in islets from male WT and Kcnk16 L114P(L/P) mice. J. Average AUC of normalized [Ca²⁺]_c following 100 μM acetylcholine stimulated [Ca²⁺]_ER release (N=876 cells; WT, N=513 cells; Kcnk16 L114P (L/P)). Data are presented as mean±SEM. Data were analyzed using student’s t-test.

Kcnk16 L114P islets exhibit altered expression of genes involved in β-cell identity and function, ion channel activity, hormone activity, inflammatory signaling, and extracellular matrix interaction pathways.
A. Heatmap of a selected gene subsets showing differential gene expression in WT and Kcnk16 L114P islets. Normalized expression levels were scaled and centered by rows. B. Volcano plot displays genes differentially expressed between WT and Kcnk16 L114P samples. Differentially expressed genes are defined by FDR <0.05 and log2FC (≥1). C. Dotplot represents the top 10 most significantly (FDR <0.05) altered Gene Ontology (Molecular Function). GeneRatio represent (count of enriched genes) / (count of genes in the GO term). The color represents FDR adjusted p-values and the size of the dot represents the number of genes that are significant from the experimental dataset. D. qRT-PCR validation of the gene expression differences in WT and Kcnk16 L114P samples for the selected genes observed through bulk-sequencing.

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