Aryl hydrocarbon receptor nuclear translocator-like (BMAL1) is associated with susceptibility to hypertension and type 2 diabetes

Abstract

Many aspects of physiology and behavior follow a circadian rhythm. Brain and muscle Arnt-like protein-1 (BMAL1) is a key component of the mammalian molecular clock, which controls circadian oscillations. In the rat, the gene encoding Bmal1 is located within hypertension susceptibility loci. We analyzed the SNP distribution pattern in a congenic interval associated with hypertension in the spontaneously hypertensive rat (SHR), and we show that Bmal1 maps close to a region genetically divergent between SHR and its normotensive (Wistar-Kyoto) counterpart. Bmal1 sequencing in rat strains identified 19 polymorphisms, including an SHR promoter variant that significantly affects Gata-4 activation of transcription in transient transfection experiments. A genetic association study designed to test the relevance of these findings in 1,304 individuals from 424 families primarily selected for type 2 diabetes showed that two BMAL1 haplotypes are associated with type 2 diabetes and hypertension. This comparative genetics finding translated from mouse and rat models to human provides evidence of a causative role of Bmal1 variants in pathological components of the metabolic syndrome.

Fig. 1.

Genotype data in WKY and SHR strains for SNPs localized in the 3.06-Mb genomic region of chromosome 1 (169.25 Mb at marker D1Njs11; 172.31 Mb at marker D1Njs17) associated with changes in blood pressure regulation in the Sisa1a congenic strain (www.snp-star.eu). Arrows indicate the localization of markers flanking the congenic interval (black) and their genotypes in the congenic strain as reported in ref. and SNPs showing allele variations (blue) or no variations (pink) between SHR and WKY strains. Solid bar represents the chromosomal region carrying WKY alleles in the Sisa1a strain, and open bars at the end of the congenic segment are regions of crossover where genotype is unknown. SNP details are given in SI Table 4. Gene representation and location are from Ensembl (www.ensembl.org/Rattus_norvegicus/index.html). Chromosomal locations refer to the most recent rat genome assembly (RGSC 3.4, genebuild, December 2004).

Fig. 2.

Effects of Bmal1 promoter sequence variants on relative luciferase activity of human 293 embryonic kidney (HEK293) or hepatoma (Hep3B) cells. (A) Embryonic kidney (HEK293) cells. (B) Hepatoma (Hep3B) cells. Proliferating cells were transfected with the control vector pGL3-Basic or Bmal1 promoter-luciferase reporter designed to test the effect of promoter SNPs 18477-T/G and 18814-C/T (WKY/SHR allele) on gene transcription (see Materials and Methods). Luciferase reporter gene expression was determined 48 h after transfection. Transfection efficiencies were normalized to lacZ activity of cotransfected pCMX-lacZ plasmid. Results are expressed as the increase in luciferase activity relative to the pGL3-Basic. Results are the mean ± SEM and are representative of four separate experiments performed in duplicate.

Fig. 3.

Gata-4-mediated activation of Bmal1 promoter reporter constructs. Hep3B hepatoma cells were cotransfected with Bmal1 promoter constructs containing either the WKY alleles (18477-T and 18814-C) or the SHR alleles (18477-G and 18814-T) and increasing doses of plasmid expressing Gata-4 to determine the maximal activation of Bmal1 promoter. Results are expressed as the increase in luciferase activity relative to the pGL3-Basic. Results are the mean ± SEM and are representative of three separate experiments performed in duplicate.

Fig. 4.

Activation of Bmal1 promoter reporter constructs by Gata-5, Pax6, and Gata-4. Hep3B hepatoma cells were cotransfected with Bmal1 promoter constructs containing either the WKY alleles (18477-T and 18814-C) or the SHR alleles (18477-G and 18814-T) and the indicated expression plasmids. Two concentrations (20 and 40 ng) of Pax6 expressing vector were used. Results are expressed as the increase in luciferase activity relative to the pGL3-Basic. Results are the mean ± SEM and are representative of two separate experiments performed in duplicate.