Regulation of the GPR40 locus: towards a molecular understanding.

GPR40 {FFAR1 [non-esterified ('free') fatty acid receptor 1]} is a G-protein-coupled receptor expressed preferentially in pancreatic beta-cells. GPR40 functions as a receptor for medium and long-chain fatty acids, and has been implicated in mediating both physiological and pathological effects of fatty acids on beta-cells. The GPR40 gene is encoded at an interesting chromosomal locus that contains several genes: at the 5'-end of the locus, located approximately 4 kb upstream of GPR40, is CD22, a gene encoding a receptor expressed selectively in lymphocytes and involved in B-lymphocyte maturation and function. At the 3'-end of the locus are the GPR41 (FFAR3) and GPR43 (FFAR2) genes encoding receptors activated by short-chain fatty acids. The intergenic region between CD22 and GPR40 contains several evolutionarily conserved sequence blocks, among them HR2 and HR3. beta-Cell-specific expression of GPR40 is controlled at the transcriptional level through HR2, a potent beta-cell-specific enhancer. The mechanisms controlling cell-specific expression of the remaining genes in the cluster are unknown. Given the divergent modes of expression of the genes within the locus and their demonstrated physiological significance, it is important to analyse further the locus with a view to fully understanding the basis for transcriptional regulation of the encoded genes.

Regulation of the gene encoding GPR40, a fatty acid receptor expressed selectively in pancreatic beta cells.

GPR40 is a G protein-coupled receptor expressed preferentially in pancreatic beta cells. It is activated by long-chain fatty acids and has been implicated in mediating physiological and pathological effects of long-chain fatty acids on beta cells. We mapped the GPR40 transcription start site to a location 1044 bp upstream of the translation start site. This permitted definition of the GPR40 core promoter and the organization of the gene, which comprises a 24-bp non-coding exon, a 698-bp intron and a 4402-bp second exon, containing the entire protein coding sequence. Sequence analysis of the GPR40 locus revealed three evolutionarily conserved regions upstream to the translation start site (HR1-HR3). DNase I-hypersensitive sites were present in the HR2 and HR3 regions in beta cells but not in non-beta cells. The 5'-flanking region of the GPR40 gene was capable of directing transcriptional activity selectively in beta cells. An important component of this is attributable to the HR2 region, which showed strong beta cell-specific enhancer activity. Systematic mutagenesis of HR2 revealed several important sub-regions. Mutagenesis of sub-regions 4-5, and 9 reduced transcriptional activity by approximately 60 and 40%, respectively. These sub-regions can bind the beta cell-specific transcription factors PDX1 and BETA2, respectively, both in vitro and in vivo. Thus, cell-specific expression of the GPR40 gene involves a characteristic chromatin organization of the locus and is controlled at the transcriptional level through HR2, a potent beta cell-specific enhancer.

The FFA receptor GPR40 links hyperinsulinemia, hepatic steatosis, and impaired glucose homeostasis in mouse.

Obesity is typically associated with elevated levels of free fatty acids (FFAs) and is linked to glucose intolerance and type 2 diabetes. FFAs exert divergent effects on insulin secretion from beta cells: acute exposure to FFAs stimulates insulin secretion, whereas chronic exposure impairs insulin secretion. The G protein-coupled receptor GPR40 is selectively expressed in beta cells and is activated by FFAs. We show here that GPR40 mediates both acute and chronic effects of FFAs on insulin secretion and that GPR40 signaling is linked to impaired glucose homeostasis. GPR40-deficient beta cells secrete less insulin in response to FFAs, and loss of GPR40 protects mice from obesity-induced hyperinsulinemia, hepatic steatosis, hypertriglyceridemia, increased hepatic glucose output, hyperglycemia, and glucose intolerance. Conversely, overexpression of GPR40 in beta cells of mice leads to impaired beta cell function, hypoinsulinemia, and diabetes. These results suggest that GPR40 plays an important role in the chain of events linking obesity and type 2 diabetes.

Activation of the insulin gene promoter through a direct effect of hepatocyte nuclear factor 4 alpha.

Maturity onset diabetes of the young, subtype 1 (MODY1), is associated with defective glucose-dependent insulin secretion from pancreatic beta cells. MODY1 is caused by mutation in the transcription factor hepatocyte nuclear factor 4 alpha (HNF4 alpha). To understand better the MODY1 phenotype, we tested whether HNF4 alpha was able to modulate directly the insulin gene promoter. Transfection of cultured 293T cells with an HNF4 alpha expression vector led to 10-fold activation of a cotransfected reporter plasmid containing the rat insulin I gene promoter. Computer analysis revealed a potential HNF4 alpha-binding site between nucleotides -57 and -69 of the promoter; mutation of this sequence led to reduced ability of HNF4 alpha to activate the promoter. The ability of HNF4 alpha to bind this sequence was confirmed using gel shift analysis. In transfected INS-1 beta cells, mutation of either the HNF1 alpha site or the HNF4 alpha site in the insulin gene promoter led to 50-75% reduction in reporter gene activity; expression of dominant negative HNF4 alpha led to significant reduction in the activity of wild type and both mutated promoters. Thus, in addition to the previously described indirect action of HNF4 alpha on insulin gene expression mediated through elevated HNF1 alpha levels, HNF4 alpha also activates the insulin gene directly, through a previously unrecognized cis element.