Type 2 diabetes susceptibility gene expression in normal or diabetic sorted human alpha and beta cells: correlations with age or BMI of islet donors

Abstract

Background: Genome-wide association studies have identified susceptibility genes for development of type 2 diabetes. We aimed to examine whether a subset of these (comprising FTO, IDE, KCNJ11, PPARG and TCF7L2) were transcriptionally restricted to or enriched in human beta cells by sorting islet cells into alpha and beta - specific fractions. We also aimed to correlate expression of these transcripts in both alpha and beta cell types with phenotypic traits of the islet donors and to compare diabetic and non-diabetic cells.

Methodology/principal findings: Islet cells were sorted using a previously published method and RNA was extracted, reverse transcribed and used as the template for quantitative PCR. Sorted cells were also analysed for insulin and glucagon immunostaining and insulin secretion from the beta cells as well as insulin, glucagon and GLP-1 content. All five genes were expressed in both alpha and beta cells, with significant enrichment of KCNJ11 in the beta cells and of TCF7L2 in the alpha cells. The ratio of KCNJ11 in beta to alpha cells was negatively correlated with BMI, while KCNJ11 expression in alpha cells was negatively correlated with age but not associated with BMI. Beta cell expression of glucagon, TCF7L2 and IDE was increased in cells from islets that had spent more time in culture prior to cell sorting. In beta cells, KCNJ11, FTO and insulin were positively correlated with each other. Diabetic alpha and beta cells had decreased expression of insulin, glucagon and FTO.

Conclusions/significance: This study has identified novel patterns of expression of type 2 diabetes susceptibility genes within sorted islet cells and suggested interactions of gene expression with age or BMI of the islet donors. However, expression of these genes in islets is less associated with BMI than has been found for other tissues.

Figure 1. Characterization of alpha and beta cell fractions.

A: Representative image of the beta cell fraction stained for insulin (red) and glucagon (green). B: Representative image of the alpha cell fraction stained likewise. C: Insulin secretion from sorted beta cells. Data are displayed as mean + SEM (islets from separate donors, n = 6 for all glucose concentrations and 5 for KCl). Statistical significance was assessed by one-tailed paired Student's T test comparing stimulated to basal secretion each time.

Figure 2. Control gene expression in sorted islet cells.

A: RNA yield (picograms per cell) from the sorted islet cell fractions, expressed as mean + SEM (n = 4). B: Housekeeping gene RPS29 transcript quantity per cell, mean + SEM of quantitative RT-PCR measurements normalised to cell number (n = 6). C: Insulin and glucagon mRNA expression in both cell fractions, normalised to RPS29, mean + SEM from all non-diabetic islet donors (n = 16). D: Insulin, glucagon and GLP-1 content of beta cell fractions expressed in pg/ml (note logarithmic axis), mean + SEM of 6 non-diabetic islet isolations which were also used for insulin secretion measurements in Figure 1C. Statistical significance of differences in hormone concentrations was by Student's T test assuming equal variance.

Figure 3. Type 2 diabetes susceptibility gene expression in sorted islet cells.

A: mRNA expression of type 2 diabetes susceptibility genes in alpha cells (white bars) and beta cells (black bars), expressed as mean + SEM of all non-diabetic islet donors (n = 16). Statistical significance of pairwise comparisons between alpha and beta cell expression was by Student's T test assuming equal variance. B: Expression of the same data in alpha cells only with male (white bars) and female (black bars) islet donors considered separately (n = 8 each). C: Expression of the same data in beta cells only with male (white bars) and female (black bars) islet donors considered separately (n = 8 each).

Figure 4. Correlations of type 2 diabetes susceptibility gene expression with age or BMI.

Filled square data points represent non-diabetic donors, empty squares represent diabetic donors. Trendlines, Spearman correlation coefficients (rho) and their corresponding p values are calculated on the basis of the non-diabetic data points only. A: Correlation of the ratio of enrichment of KCNJ11 mRNA in beta cells relative to alpha cells with BMI. B: Correlation of alpha cell KCNJ11 mRNA with age.

Figure 5. Correlations of mRNA levels with each other.

Filled square data points represent non-diabetic donors, empty squares represent diabetic donors. Trendlines, Spearman correlation coefficients (rho) and their corresponding p values are calculated on the basis of the non-diabetic data points only. A: Correlation of beta cell insulin and FTO. B: Correlation of beta cell KCNJ11 and FTO. C: Correlation of beta cell insulin and KCNJ11. D: Correlation of alpha cell glucagon and KCNJ11. E: Correlation of alpha cell TCF7L2 and IDE. F: Correlation of beta cell TCF7L2 and IDE.

Figure 6. Effect of islet culture time on TCF7L2 and IDE mRNA expression in non-diabetic islets.

A: Scatter plot of beta cell TCF7L2 mRNA against islet culture time prior to cell sorting. B: Scatter plot of beta cell IDE mRNA against islet culture time prior to cell sorting.

Figure 7. Effect of islet culture time on beta cell glucagon transcript levels.

A: Scatter plot of beta cell glucagon mRNA against islet culture time prior to cell sorting (non-diabetic islets only). B: Average insulin and glucagon levels in non-diabetic islets excluding those which were cultured for 10 or 11 days (n = 14).

Figure 8. Insulin, glucagon and type 2 diabetes susceptibility gene expression in diabetic sorted islet cells.

A: Differential expression of beta cell insulin and alpha cell glucagon in non-diabetic (white bars) compared to diabetic (black bars) islet cells. B: Expression levels of type 2 diabetes susceptibility gene in diabetic alpha cells (white bars) and beta cells (black bars). C: Differential expression of FTO in both cell fractions from non-diabetic (white bars) compared to diabetic (black bars) islet cells. Statistical comparison of diabetic to non-diabetic samples was by unpaired T test assuming unequal variance. n = 16 non-diabetic and 3 diabetic samples.