A central role for GRB10 in regulation of islet function in man

Abstract

Variants in the growth factor receptor-bound protein 10 (GRB10) gene were in a GWAS meta-analysis associated with reduced glucose-stimulated insulin secretion and increased risk of type 2 diabetes (T2D) if inherited from the father, but inexplicably reduced fasting glucose when inherited from the mother. GRB10 is a negative regulator of insulin signaling and imprinted in a parent-of-origin fashion in different tissues. GRB10 knock-down in human pancreatic islets showed reduced insulin and glucagon secretion, which together with changes in insulin sensitivity may explain the paradoxical reduction of glucose despite a decrease in insulin secretion. Together, these findings suggest that tissue-specific methylation and possibly imprinting of GRB10 can influence glucose metabolism and contribute to T2D pathogenesis. The data also emphasize the need in genetic studies to consider whether risk alleles are inherited from the mother or the father.

Figure 1. GWAS plots identifying GRB10 rs933360.

Genome-wide quantile-quantile (Q-Q) (A) and Manhattan (B) plots for CIR in the meta-analysis. Regional plots of identified GRB10 genome-wide significant association for CIR in men (C) and women (D). Directly genotyped and/or imputed SNPs of GRB10 are plotted with their meta-analysis p-values (as −log10 values) as a function of genomic position (NCBI Build 36). In each panel, the strongest associated SNP is represented by a purple diamond (with meta-analysis p-value). Estimated recombination rates (taken from HapMap) are plotted to reflect the local linkage disequilibrium structure around the associated SNPs and their correlated proxies (according to a blue to red scale from r² = 0 to 1, based on pairwise r² values from HapMap CEU). Gene annotations were taken from the University of California, Santa Cruz, genome browser, as implemented in LocusZoom .

Figure 2. Parent-of-origin effect of GRB10 rs933360 on insulin secretion and glucose levels.

(A) No significant effect for CIR was observed from the paternally transmitted A-allele. (B) Carriers of the maternally transmitted A-allele showed lower CIR compared to the G-allele. (C) Carriers of the paternally transmitted A-allele had elevated fasting plasma glucose levels, whereas (D) the maternally transmitted A-allele was associated with lower fasting plasma glucose levels. Fin-Swe = Trios from Finland and Sweden, Amish = Amish Family Diabetes Study, Kuopio = Kuopio Offspring Study.

Figure 3. GRB10 expression in human islets.

(A) Immunostainings demonstrating that GRB10 (green, panel A, E) is abundantly expressed in β- (panel B, D), α- (panel C, D) and δ-cells (panel F, G) in human pancreatic islets. Arrows indicate co-localization with insulin (panel D) and somatostatin (panel G). Arrowheads indicate co-localization with glucagon (panel D). Scale bar = 50 µm. (B) Schematic representation of the GRB10 gene and SNPs investigated in the present study. Grey boxes = untranslated exons. Black boxes = translated exons. (C) Examples of RT-PCR on islet cDNA (top six rows) and PCR on genomic DNA (gDNA, bottom row) from two individuals heterozygous for the reporter SNP rs1800504. The first column states the forward primer location of each PCR and a forward primer in exon 3 captures all transcripts. The peaks show the Sanger sequencing trace across rs1800504, which is underlined (A: green trace, G: black trace). Percentages indicate the contribution from the paternal allele (P.A.) (G-allele in the first case, A-allele in the second case). The paternal genotype is identified assuming complete maternal imprinting of the UN2 promoter, in line with previous findings . A sequence of heterozygous genomic DNA (gDNA) is shown on the bottom for comparison (50%-50%). (D) To study if DNA methylation of GRB10 is tissue-specific, the degree of methylation was analyzed at 3 CpG sites located ∼31.7 kb downstream of rs933360 in both human pancreatic islets of 98 donors and PBL from 6 trios using EpiTYPER. The exact position of each analyzed CpG site in relation to rs933360 is given in the figure. Data are presented as mean ± SEM. * p<0.05 for difference in methylation between human islets and PBL. (E) The GRB10 mRNA levels correlated negatively with the degree of methylation at the CpG site located 31,675 bp downstream of GRB10 rs933360.

Figure 4. Effects of disrupted GRB10 through knock-down on islet function.

(A) Disrupted GRB10 in INS-1 rat β-cells markedly reduced glucose-stimulated insulin secretion. (B) GRB10 knock-down showed reduced glucose-stimulated insulin secretion at 20 mM glucose and glucagon secretion at 1 mM glucose in human pancreatic islets (N_insulin = 7, N_glucagon = 6 donors of human pancreatic islets; 3–6 measurements in each experiment for each donor). (C) GRB10 knock-down resulted in a reduction of insulin and glucagon mRNA expression (N = 3 donors of human pancreatic islets; 3 measurements in each experiment for each donor). * p<0.05; ** p<0.01, *** p<0.001. Error bars denote SEM.