Experiment
Glucose-stimulated insulin secretion (GSIS) is a critical function in the regulation of glucose homeostasis, and its deregulation is associated with the development of type 2 diabetes. Here, the authors performed a genetic screen using islets isolated from 36 different lines of BXD advanced recombinant inbred (RI) mice to search for novel regulators of insulin production and secretion. This number of BXD lines is sufficient to identify QTL accounting for approximately 40% of the variance of the trait with a power of ~0.8.
Pancreatic islets were isolated from 36 RI BXD lines and insulin secretion was measured following exposure to 2.8 or 16.7 mM glucose with or without exendin-4. Islets from the same RI lines were used for RNA extraction and transcript profiling. QTL mapping was performed for each secretion condition using the R package R/qtl with an extended genotype map from the BXD panel composed of GeneNetwork (www.genenetwork.org) genotypes merged with variants detected from RNA-Seq variant calling analysis. QTL interval mapping was calculated using the expected-maximization algorithm, a 5% genotyping error rate, and pseudomarkers were generated every cM. QTL location was obtained by 6.915 likelihood ratio statistics (LRS) support intervals. Significant QTL were determined for each trait using a 5% false discovery rate threshold estimated from 1000 permutations.
For islet insulin content, a suggestive QTL was identified on the distal part of Chr 2 (between 167.77 and 172.56 Mb; LRS = 13.6).
For the 16.7 mM GSIS trait, two suggestive QTL were identified: one on Chr 2 (between 25.73 and 39.67 Mb; LRS = 13.20) and the other on Chr 7 (between 25.31 and 28.31 Mb; LRS = 12.51).
One genome-wide significant QTL for the 16.7 mM glucose plus exendin-4 insulin secretion data was identified:
Gluil3 (glucose insulin level 3, glucose plus exendin-4) maps to Chr 2: 26.78 - 35.77 (rs337220626 - c2.loc30) with a peak LRS score of 19.36. Gluil3 has an LRS of 19.36 and is located between SNP markers rs337220626 and c2.loc30. Gluil3 explains 41 percent of the variance of the trait and maps at the same position as the suggestive QTL for the 16.7 mM GSIS trait.
Within the Gluil3 QTL, RNA-Seq data prioritized Crat (carnitine O-acetyl transferase) as a strong candidate regulator of the insulin secretion trait. Silencing Crat expression in MIN6B1 cells reduced insulin content and insulin secretion by ~30%. Conversely, Crat overexpression enhanced insulin content and secretion by ~30%. When islets from mice with beta-cell-specific Crat inactivation were exposed to high glucose, they displayed a 30% reduction of insulin content as compared to control islets. The authors further showed that decreased Crat expression in both MIN6B1 cells and pancreatic islets reduced the oxygen consumption rate in a glucose concentration-dependent manner.
The authors identified Crat as a regulator of insulin secretion whose action is mediated by an effect on total cellular insulin content; this effect also depends on the genetic background of the RI mouse lines. These data show that in the presence of the stimulatory conditions used the insulin secretion rate is directly related to the insulin content.
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