Novel regulators of islet function identified from genetic variation in mouse islet Ca²⁺ oscillations

Christopher H. Emfinger
Lauren E. Clark
Brian Yandell
Kathryn L. Schueler
Shane P. Simonett
Donnie S. Stapleton
Kelly A. Mitok
Matthew J. Merrins
Mark P. Keller
Alan D. Attie author has email address

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA
Department of Statistics, University of Wisconsin-Madison, Madison, WI, 53706, USA
Department of Medicine, Division of Endocrinology, University of Wisconsin-Madison, Madison, WI, 53705, USA
William S. Middleton Memorial Veterans Hospital, Madison, WI 53705 USA

https://doi.org/10.7554/eLife.88189.1

Open access
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Figures and data

Imaging medium formula.
Components are indicated by chemical abbreviation on the left and final concentration in mM is indicated in the right column.

Categories included in SNP queries.
These terms were considered as glycemia-related and are categorized as such on the Common Metabolic Diseases Knowledge portal, which was queried for the relevant SNPs. Also included but not listed here were variations of these terms that were adjusted for BMI.

High diversity in Ca²⁺ oscillation across eight genetically distinct mouse strains.
(A) Male and female mice from eight strains (A/J; C57BL/6J (B6); 129S1/SvImJ (129); NOD/ShiLtJ (NOD); NZO/HILtJ (NZO); CAST/EiJ (CAST); PWK/PhJ (PWK); and WSB/EiJ (WSB)) were placed on a Western Diet (WD) for 16 weeks, before their islets were isolated. The islets were then imaged on a confocal microscope using Fura Red dye under conditions of 8 mM glucose; 8 mM glucose + 2 mM L-glutamine, 0.5 mM L-leucine, and 1.25 mM L-alanine (QLA); 8 mM glucose + QLA + 10 nM GIP; and 2 mM glucose. (B) Representative Ca²⁺ traces for male mice (n = 3-8 mice per strain, and 15-83 islets per mouse), with the transitions between solution conditions indicated by dashed lines. Abbreviations: ‘[Glu]’ = ‘concentration of glucose in mM’; ‘Sol.’ = ‘solution’; ‘SNPs’= ‘single-nucleotide polymorphisms’

Ca²⁺ wave breakdown reveals mechanisms underlying Ca²⁺ responses.
(A) In the example B6 female Ca²⁺ wave, the islet oscillations can change in their average peak and average baseline in response to different nutrients. Additionally, shifts in wave shape (green box) can be broken down into changes in time between peaks (period), the time in the active phase (active duration, A_D), and the length of the oscillation (pulse duration, P_D). From these, the relative time in active phase, or plateau-fraction (P_F), and the time the islet is inactive between oscillations (silent duration, S_D) can be calculated. Each parameter can be changed by different underlying mechanisms. (B) For islets that plateaued, as in the example islet in 8/QLA, they were assigned a plateau-fraction of one and a period of zero. For islets that ceased to oscillate, such as the example islet in 2 mM glucose, they were assigned a plateau-fraction of zero and a period of the time of measurement (40 minutes). (C) For trace 1 (left), which has a longer period (red bars) than trace 2 (right), but the same active duration (blue bars), the silent duration is greater and consequently the P_F is shorter, in contrast to the trace in (A) where the P_F increases between 8mM and 8/QLA are largely due to increases in A_D. (D) Changes in specific Ca²⁺ wave parameters can reflect different mechanisms in β-cells. For example, changing K_ATP activity pharmacologically (upper panels) predominantly increases P_F by altering S_D, whereas increasing glucose concentrations by elevating glucose or activating GK cause significant alterations in both A_D and S_D to increase P_F. Abbreviations: ‘[Glc]’ = ‘concentration of glucose in mM’; ‘GK’ = ‘glucokinase’

WSB mice secrete significantly more insulin than 129 mice.
(A) Insulin secretion was measured for perifused islets from WSB (n=6, magenta circles) and 129 (n=5, yellow squares) male mice in 2 mM glucose, 8 mM glucose, 8 mM glucose + QLA, and 8 mM glucose + QLA + GIP. Transitions between solutions are indicated by dotted lines and the conditions for each are indicated above the graph. “[Glc]” denotes the concentration of glucose in mM. Data are shown as a percentage of total islet insulin (mean ± SEM). (B) Average total insulin per islet for the WSB and 129 males used in (A) with one exception: islets from one of the 129 mice were excluded from perifusion analysis due to technical issues with perifusion system on the day those animals’ islets were perifused. Dots represent individual values, and the mean is denoted by the black line. For (A), asterisks denote strain effect for the area-und-the-curve of the section determined by 2-way ANOVA, mixed effects model; ** p < 0.01, *** p < 0.001. For (B), asterisk denotes p < 0.05 from Student’s T-test with Welch’s correction.

Comparing sex and strain patterns for Ca²⁺ metrics, insulin secretion, and clinical traits nominates Ca²⁺ metrics of interest. (A) The z-score correlation coefficient was calculated for Ca²⁺ parameters and raw insulin secreted and % total insulin secreted. Insulin measurements were previously collected for seven different secretagogues (16.7 mM glucose + 0.5 mM palmitic acid (16.7G/PA); 3.3 mM glucose + 50 mM KCl (3.3G/KCl); 16.7 mM glucose (16.7G); 8.3 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8.3G/QLA); 8.3 mM glucose + 100 nM GLP-1 (8.3G/GLP-1); 8.3 mM glucose (8.3G); and 3.3 mM glucose (3.3G)) (32). (B) Correlation of the Ca²⁺ parameters to the clinical measurements in the founder mice which include 1) plasma insulin, triglycerides, and glucose at 6, 10, and 14 weeks as well as at time of sacrifice; 2) number of islets; 3) whole-pancreas insulin content (WPIC); and 5) islet content for insulin and glucagon. For (A) and (B), the Ca²⁺ parameters shown here include average Ca²⁺ in 2 mM glucose (basal Ca²⁺); average Ca²⁺ in 8 mM glucose (8G avg.); average Ca²⁺ in 8 mM glucose + 1.25 mM L-alanine, 2 mM L-glutamine, and 0.5 mM L-leucine (8G/QLA avg); average Ca²⁺ in 8 mM glucose + QLA + 10 nM GIP (8G/QLA/GIP avg.); pulse duration in 8 mM glucose (8G P_D); active duration in 8G (8G A_D); silent duration in 8G (8G S_D), 8G/QLA (8G/QLA/S_D), and 8G/QLA/GIP (8G/QLA/GIP S_D); and 1^st component frequency in 8 mM glucose (8G 1^st freq.). Other parameters analyzed are indicated in Supplemental Figure 4. (B-E) Sex and strain variability for (C) average Ca²⁺ determined by the fura-ratio (FR) in 2 mM glucose, (D) pulse duration of oscillations in 8G, (E) 1^st component frequency in 8G and (F) silent duration of oscillations in 8G, 8G/QLA, and 8G/QLA/GIP.

Islet proteins show correlation architecture to specific Ca²⁺ parameters.
(A) Unsupervised clustering of correlation coefficients between protein abundance z-scores and z-scores for the Ca²⁺ parameters indicated. Islet proteins show differential correlation values to basal Ca²⁺, excitatory Ca²⁺ (detrended average values for 8mM, 8/QLA, and 8/QLA/GIP), active duration and pulse duration in 8mM glucose (8G P_D & A_D), and silent durations (S_D) in 8G, 8G/QLA, and 8G/QLA/GIP. Correlation coefficients for other parameters are indicated in Supplemental Figure 5. (B) Histograms representing the number of proteins that are correlated (red) and anti-correlated (blue) to 8G A_D. TRRUST transcription factor motif database and ARCHS4 Tissue signature database (C) as well as pathway enrichments for the Elsevier Pathway database and KEGG 2021 Human pathway database (D) (-log₁₀(p-values)), for the highly correlated (red) and anticorrelated (blue) proteins to 8 A_D metric. Databases were queried using Enrichr (39, 40).

Identifying candidate protein targets by integrating human GWAS.
(A) An example gene, COBLL1, orthologous to a gene coding for a protein identified as highly correlated to Ca²⁺ wave parameters in the founder mice. The recombination rate is indicated by the solid blue line. Significant SNPs ( 8 < -log₁₀(p), red) decorate the gene body for multiple glycemia-related parameters (in bold). Human islet chromatin data (34) for histone methylation (H3K4me3), histone acetylation (H3K27ac), ATAC-sequencing (ATAC-seq), and regulome score suggest active transcription of the gene within a topologically-associated domain (TAD). Human islet promoter-capture HiC data (pc-HiC) (34) show contacts between the SNP-containing regions decorating the gene and its promoter. The highest SNP for 2hr glucose () and the other parameters () are indicated. (B) Some orthologues did not show SNPs decorating the gene itself but did show looping to regions with SNPs for glycemic traits. The promoter of ACP1, for example, loops to a region within its topologically associated domain (black bar) with strong SNPs for type 2 diabetes risk and near-threshold SNPs for fasting insulin adjusted for BMI. Some SNPs (, ) lie directly on the contact regions identified by HiC, whereas others lie immediately proximal to these contacts. For both panels, the significance of association (-log₁₀ of the p-value) for the individual SNPs is on the left y axis and the recombination rate per megabasepair (Mbp) is on the right y axis. Chromosomal position in Mbp is aligned to Hg19. SNP data were provided by the Common Metabolic Diseases Knowledge Portal (cmdkp.org).

Identifying candidate protein targets by integrating human GWAS.
(A) An example gene, COBLL1, orthologous to a gene coding for a protein identified as highly correlated to Ca²⁺ wave parameters in the founder mice. The recombination rate is indicated by the solid blue line. Significant SNPs ( 8 < -log₁₀(p), red) decorate the gene body for multiple glycemia-related parameters (in bold). Human islet chromatin data (34) for histone methylation (H3K4me3), histone acetylation (H3K27ac), ATAC-sequencing (ATAC-seq), and regulome score suggest active transcription of the gene within a topologically-associated domain (TAD). Human islet promoter-capture HiC data (pc-HiC) (34) show contacts between the SNP-containing regions decorating the gene and its promoter. The highest SNP for 2hr glucose () and the other parameters () are indicated. (B) Some orthologues did not show SNPs decorating the gene itself but did show looping to regions with SNPs for glycemic traits. The promoter of ACP1, for example, loops to a region within its topologically associated domain (black bar) with strong SNPs for type 2 diabetes risk and near-threshold SNPs for fasting insulin adjusted for BMI. Some SNPs (, ) lie directly on the contact regions identified by HiC, whereas others lie immediately proximal to these contacts. For both panels, the significance of association (-log₁₀ of the p-value) for the individual SNPs is on the left y axis and the recombination rate per megabasepair (Mbp) is on the right y axis. Chromosomal position in Mbp is aligned to Hg19. SNP data were provided by the Common Metabolic Diseases Knowledge Portal (cmdkp.org).

Mining Ca²⁺ data using a novel online resource (preceding page):
(A) 3073 islet proteins significantly correlated to islet Ca²⁺ parameters of interest. Among the proteins, 647 had orthologues containing SNPs for glycemic traits. Of these, 478 showed no results in our starting triage (see Methods) under any alias, suggesting they may be understudied in islet biology. (B) Of these 478 proteins, 198 were found to be secreted either as soluble proteins or in exosomes (66, 67, 69-73), 52 have existing knockout mice with annotated glycemia or pancreatic phenotypes (74, 91), and 45 have existing compounds that target them (60-65). To make these data more accessible, we have developed an online resource that enables individuals to query the Ca²⁺ and proteomic data simultaneously. The user can select proteins and calcium traits (C) and display strain and sex distribution of these traits to determine the ideal backgrounds on which to test their traits or proteins of interest. In this example, GALNS is highly correlated to 8G/QLA/GIP A_D, with the highest and lowest abundance strains for GALNS being AJ (yellow arrow) and CAST (green arrow), respectively. (D) The user can also query for the correlations between Ca²⁺ traits and proteins against one another or other traits of the same category. (E) The user can also see which of the traits or proteins has the largest change and most significant effects by sex, strain, or sex and strain.

(on following pages): Proteins correlated with Ca²⁺ parameters that have glycemic-related SNPs.
This includes protein IDs, gene names, gene IDs, and human orthologues for each of the proteins that correlate to one of the following metrics and have a glycemic-related SNP (see Table 2): basal Ca²⁺, 8G S_D, 8G/QLA S_D, 8G/QLA/GIP S_D, 8G A_D, 8G P_D, and 8G 1^st freq.

(on following pages): Proteins correlated with Ca²⁺ parameters that have glycemic-related SNPs.
This includes protein IDs, gene names, gene IDs, and human orthologues for each of the proteins that correlate to one of the following metrics and have a glycemic-related SNP (see Table 2): basal Ca²⁺, 8G S_D, 8G/QLA S_D, 8G/QLA/GIP S_D, 8G A_D, 8G P_D, and 8G 1^st freq.

(on following pages): Proteins understudied in islet biology.
This table of proteins indicates the subset of proteins meeting our selection criteria (Table 3) that did not have any results in Pubmed, Google Scholar, or Google for any alias and the term “insulin secretion,” suggesting that they may be understudied in islet biology. The gene symbols for the mouse gene and human orthologue are indicated. The “Mouse?” column indicates whether a knockout mouse with metabolic phenotypes exists (identified from sources in the subsequent “Source” column). The “Drug?” column similarly indicates whether any source (indicated in the following “Source” column) shows existing compound(s) targeting the protein. Finally, the “Secreted?” column indicates whether any source (indicated in the subsequent “Source” column) shows an isoform of the protein to be secreted.

(on following pages): Proteins understudied in islet biology.
This table of proteins indicates the subset of proteins meeting our selection criteria (Table 3) that did not have any results in Pubmed, Google Scholar, or Google for any alias and the term “insulin secretion,” suggesting that they may be understudied in islet biology. The gene symbols for the mouse gene and human orthologue are indicated. The “Mouse?” column indicates whether a knockout mouse with metabolic phenotypes exists (identified from sources in the subsequent “Source” column). The “Drug?” column similarly indicates whether any source (indicated in the following “Source” column) shows existing compound(s) targeting the protein. Finally, the “Secreted?” column indicates whether any source (indicated in the subsequent “Source” column) shows an isoform of the protein to be secreted.

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