The MODY-associated KCNK16 L114P mutation increases islet glucagon secretion and limits insulin secretion resulting in transient neonatal diabetes and glucose dyshomeostasis in adults
- Department of Molecular Physiology and Biophysics, Vanderbilt University, United States
- Department of Chemistry, Vanderbilt University, United States
- Center for Stem Cell Biology, Vanderbilt University, United States
- Department of Cell and Developmental Biology, Vanderbilt University, United States
Figures
Figure 1 with 2 supplements
Kcnk16L114P 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 Kcnk1…
Figure 1—figure supplement 1
Generation of Kcnk16 L114P model and assessment of neonatal glucose homeostasis and lethality.
(A) Targeted region of Kcnk16 exon 3 using CRISPR/Cas9 leading to the introduction of HinfI restriction enzyme site (red arrows) and CTG to CCA mutation in codon 337 corresponding to p.TALK-1 L114P …
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Figure 1—figure supplement 1—source data 1
Original file for the DNA gel analysis in Figure 1—figure supplement 1B (PCR confirmation of the male founder Kcnk16 L114P (L/P) mouse using HinfI restriction digestion).
- https://cdn.elifesciences.org/articles/89967/elife-89967-fig1-figsupp1-data1-v1.tif
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Figure 1—figure supplement 1—source data 2
Original file for the DNA gel analysis in Figure 1—figure supplement 1B (PCR confirmation of the male founder Kcnk16 L114P (L/P) mouse using HinfI restriction digestion) with bands and DNA ladder labeled.
- https://cdn.elifesciences.org/articles/89967/elife-89967-fig1-figsupp1-data2-v1.tif
Figure 1—figure supplement 2
Kcnk16 L114P male and female mice show transient neonatal hyperglycemia.
(A) Average blood glucose measurements of B6;CD-1 male mice at P1, P4, P10, P15, and P21 including: WT (black; P1 N=9, P4 N=27, P10 N=9, P15 N=9, P21 N=8), Kcnk16 L114P (L/P; green; P1 n=15, P4 …
Figure 2 with 3 supplements
Adult Kcnk16 L114P mice exhibit fasting hyperglycemia and glucose intolerance.
(A, B) Intraperitoneal glucose tolerance test (i.p. GTT) performed in 15-week-old and 18-week-old male mice following a 4 hr fast in response to 2 mg/g glucose injection (WT; black; N=8–10 and Kcnk16…
Figure 2—figure supplement 1
Oral glucose tolerance is impaired in Kcnk16 L114P (L/P) mice in the B6;CD-1 background.
Oral glucose tolerance test (OGTT) performed in 4- (A) and 14-week-old (B) male mice following a 4 hr fast in response to oral gavage of 2 g/kg glucose. (C) Average area under the curve (AUC) of the …
Figure 2—figure supplement 2
Glucose homeostasis is impaired in Kcnk16 L114P (L/P) mice in the B6 background.
(A) Body weight measurements of male WT (black; N=8) and Kcnk16 L114P (L/P; green; N=4) mice in the C57Bl/6J background. (B) Intraperitoneal glucose tolerance test (i.p. GTT) performed in …
Figure 2—figure supplement 3
Body composition measurements and assessment of plasma and liver triglycerides and total cholesterol.
(A–F) Body composition analysis of male and female B6;CD-1 WT and Kcnk16 L114P (L/P) mice assessing weight (g), body fat (g), and lean mass (g) (N=5–9 mice/genotype). (G–J) Average liver and plasma …
Figure 3
Adult Kcnk16 L114P mice show disrupted islet hormone secretion and islet composition.
(A, B) Plasma insulin levels in male (A) and female (B) WT and Kcnk16 L114P (L/P) mice following a 4 hr fast at the indicated time points before and after a 2 mg/g glucose injection. (C) Glucose (%) …
Figure 4 with 2 supplements
Kcnk16 L114P blunts β-cell glucose-stimulated electrical excitability and increases whole-cell two-pore domain K+ channel currents.
(A) Representative perforated patch-clamp Vm 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 …
Figure 4—figure supplement 1
Kcnk16 L114P attenuates β-cell glucose-stimulated electrical excitability in a heterogenous manner.
(A) Representative perforated patch-clamp Vm recording in response to 11 mM G and 11 mM G+30 mM K+ from a WT β-cell expressing GCaMP6s driven by an optimized RIP within an islet cluster …
Figure 4—figure supplement 2
Two-pore domain K+ channel currents in β-cells from homozygous Kcnk16 L114P (P/P) mice also exhibit a modest increase.
(A) Representative whole-cell two-pore domain K+ channel current density (pA/pF) recorded using a voltage ramp (–120 mV to +60 mV) in 11 mM G in islets from B6; CD-1 WT and Kcnk16 L114P (P/P) P4 …
Figure 5 with 2 supplements
Kcnk16 L114P reduces glucose- and tolbutamide-stimulated islet Ca2+ entry and augments IP3-induced islet [Ca2+]ER release.
(A, 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) …
Figure 5—figure supplement 1
Islets from Kcnk16 L114P (L/P) mice on the B6 background also exhibit blunted glucose-stimulated Ca2+ entry.
(A) Representative glucose-stimulated [Ca2+]c influx traces in islets from male WT and Kcnk16 L114P (L/P) mice in the C57BL/6J background in response to 2 mM G, 10 mM G, and 20 mM G (N=3 …
Figure 5—figure supplement 2
Kcnk16 L114P (L/P) islets exhibit prolonged glucose-stimulated phase 0 [Ca2+]ER uptake and show a complete absence of Ca2+ oscillations.
(A) Average relative [Ca2+]c at 2 mM G in islets from B6; CD-1 WT and Kcnk16 L114P (L/P) mice (N=4 mice/genotype). (B) Percent islets that exhibit the corresponding phase 0 response (drop in [Ca2+]c)…
Figure 6 with 2 supplements
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 …
Figure 6—figure supplement 1
Liraglutide increased Ca2+ oscillation frequency in WT islets but does not impact Kcnk16 L114P (L/P) mouse islet Ca2+ handling.
(A) Representative islet Ca2+ recording from a WT islet in 11 mM glucose treated with 200 nM liraglutide. (B) Average Ca2+ oscillation frequency of WT islets in 11 mM glucose and in 11 mM glucose …
Figure 6—figure supplement 2
RNA sequencing showed equivalent expression of a subset of genes encoding proteins known to control [Ca2+]ER in WT and Kcnk16 L114P (L/P) male islets.
(A) Average normalized RNA levels (baseMean) of Kcnk3, Itpr1, Itpr2, and Itpr3 from WT islets (gray bars, N=4) and Kcnk16 L114P (L/P) (green bars, N=4). Data are presented as mean ± SEM. Data were …
Additional files
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Supplementary file 1
All mouse primer sequences utilized for qRT-PCR.
- https://cdn.elifesciences.org/articles/89967/elife-89967-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89967/elife-89967-mdarchecklist1-v1.docx
