The serine/threonine-protein kinase Akt/protein kinase B (PKB) plays a critical role in insulin receptor-coupled phosphatidylinositol 3-kinase-mediated signaling (1
). There are three Akt mammalian isoforms (Akt1-3), of which Akt2 is the most important in glucose metabolism (1
). Mice deficient in Akt2 exhibit fed and fasting hyperglycemia, hyperinsulinemia, glucose intolerance, and impaired muscle glucose uptake (2
). We identified a missense mutation in the kinase domain of Akt2 (R274H) in a single family with autosomal dominantly inherited severe insulin resistance and diabetes (4
). The proband of this family had partial lipodystrophy (4
), suggesting that Akt2 may play a role in adipogenesis. To date, detailed genetic association studies of AKT2
have not been reported.
We undertook studies to determine 1
) whether other missense/nonsense mutations in AKT2 might result in human syndromes of severe insulin resistance with or without accompanying lipodystrophy and 2
) whether common genetic variants in AKT2
might be associated with metabolic phenotypes related to insulin resistance. The entire coding sequence and splice junctions of AKT2
were screened in 94 probands with severe insulin resistance, 35 of which had partial lipodystrophy. The results of this screen are shown in online appendix (available at http://dx.doi.org/10.2337/db06-0921
). We identified two novel missense mutations (). R467W was found in a white female patient with type 2 diabetes and partial lipodystrophy. This variant was present in neither 47 ethnically matched control subjects nor in 2 unaffected sons of the carrier. R208K was identified in a white female patient with severe insulin resistance and acanthosis nigricans. This variant was not present in her affected son but was present in 1 of 47 white control subjects. Unfortunately, parental DNA was not available to determine whether these mutations were inherited or spontaneous.
Relationship between genotype in AKT2 and type 2 diabetes status in a U.K. population
FIG. 1 Rare mutations in Akt2 in human individuals. A: Location of the identified mutations R208K and R467W in relation to functional domains and known phosphorylation sites. B: In vitro kinase assay of Akt2 mutants. HA-Akt2 and HA-Akt2 mutants were immunoprecipitated (more ...)
We investigated whether these mutations might cause functional impairment of the Akt2 kinase in vitro. CHO-T cells overexpressing the insulin receptor were transfected with either wild-type HA-Akt2 or mutant HA-Akt2. The ability of Akt2 to phosphorylate an artificial peptide substrate based on glycogen synthase kinase-3 was measured in an in vitro kinase assay using anti-HA immunoprecipitates from the transiently transfected cells, which were serum starved and then stimulated with insulin or left untreated. The known kinase-dead R274H mutant (4
) was used as a negative control. Neither R208K nor R467W significantly altered either the basal or the insulin-stimulated kinase activity of Akt2 on the peptide substrate (). The mutations did not affect expression levels (). Upon stimulation with insulin, both Akt2 mutants were phosphorylated on T309 and S474 to a similar extent as wild-type Akt2 ().
Although it is possible that these in vitro assays may fail to detect subtle changes in function of the Akt2 kinase, including, for example, selective impairments of activity at specific substrates, the normality of responses of the mutant kinases in two different assay systems suggests that the their function is likely to be unimpaired. Taken together, these data suggest these mutations are unlikely to be directly implicated in the severe insulin resistance of the probands.
To explore whether common single nucleotide polymorphisms (SNPs) in AKT2 are associated with type 2 diabetes, we studied two U.K. case-control studies: the Cambridgeshire Case-Control (CCC) Study and the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk Study. Based on the criteria described (see research design and methods), we obtained results for 11 polymorphic SNPs (). Average call rates were 93.3 and 97.7% for the CCC and EPIC-Norfolk studies, respectively. All SNPs were in Hardy-Weinberg equilibrium (HWE) (P > 0.01) in these populations. To increase power, we performed a joint analysis of the two case-control studies, with a term for study, and tested for heterogeneity between the studies (). When the genotype frequencies were compared between the combined case and control subjects, no statistically significant associations were detected (). Additional genotyping of other loci in these populations did not show evidence for heterogeneity; thus, the nominally significant associations in the separate case-control studies are likely to be due to chance. However, the heterogeneity could also be due to subtle differences in population substructure between study populations.
We next evaluated the degree of linkage disequilibrium among the 11 SNPs genotyped (). As shown, all but two SNPs (rs4273150 and rs3730256) fall into two linkage disequilibrium blocks (5
). Haplotype analysis in each of these blocks did not provide evidence of statistically significant associations with disease risk (data not shown). Furthermore, to estimate coverage of the genetic variation captured, we compared our selected SNPs with those in Centre d’Etude du Polymorphism Humain samples in HapMap II (release no. 20). There are 21 SNPs with minor allele frequency (MAF) >5%, spanning from 22 kb upstream of the start of the transcript to the 3′ untranslated region of the gene. We have tagged all SNPs present in HapMap (pairwise tagging with r2
= 0.8 and MAF >5%) except for SNPs rs10426842 and rs12460555 (each of these just tag themselves). Taken together, our data suggest that common variants in AKT2
do not significantly contribute to the type 2 diabetes status in the U.K. population.
FIG. 2 Location and linkage disequilibrium map of AKT2 SNPs genotyped. Thirteen exons of AKT2 are represented by solid bars (numbered 1-13); intronic regions and 5′ and 3′ regions are represented by solid lines. The positions of SNPs 1-11 are (more ...)
To examine whether the common variants in AKT2
are associated with metabolic parameters related to type 2 diabetes, we further genotyped 1,721 unrelated U.K. white participants in the Medical Research Council (MRC) Ely Study, a prospective population-based study of the etiology of type 2 diabetes (7
). Eleven SNPs were genotyped with an average call rate of 96.6%, although SNP8 and SNP11 were not in HWE (P
< 0.01) (). We tested for association of these SNPs with fasting and 2-h post-challenge plasma glucose levels, fasting plasma insulin levels, and 30-min insulin incremental response, a measure of insulin secretion (8
) (). None of the SNPs showed any association (P
< 0.01) with glucose or insulin levels. Therefore, these data suggest that common polymorphisms in AKT2
do not significantly contribute to plasma glucose or insulin levels.
Relationship between genotype in AKT2 and glucose and insulin levels in a U.K. population
In summary, we screened AKT2 as a candidate gene for human insulin resistance and partial lipodystrophy and detected two novel missense mutations, which did not affect function, at least in the assays used. We conducted the first association study of common variants in the AKT2 gene with human type 2 diabetes and related metabolic phenotypes. No statistically significant associations were found, suggesting that common variants in AKT2 are not associated with type 2 diabetes, in the populations studied. For a SNP with a MAF of 0.2, the power to detect an effect with an odds ratio >1.2 (or <0.82) is 80%, α = 0.05 (960 case and 1,386 control subjects). Therefore, small to moderate effects, if they exist, would be detected by our study. Further studies will be required to completely rule out small effects of AKT2 on type 2 diabetes risk.
Evidence from recent years has suggested that studying rare monogenic forms of diabetes not only contributes to our understanding of the mechanisms of glucose homeostasis but also might constitute an effective strategy to identify genes involved in more common and complex forms of diabetes. Indeed, while rare highly penetrant mutations in HNF4A, PPARG
, and KIR6.2
lead to monogenic forms of diabetes, common variants in each of these genes have been shown to increase disease risk in type 2 diabetes (9
). This study suggests that this is not the case for AKT2