On the basis of studies of signatures of selection, in vitro promoter assays, and glucose challenge tests in humans, we show that it is possible to identify causal variants related to energy-balance regulation by focusing on genic SNPs that were subject to environmental selection in a subset of candidate genes. Specifically, we demonstrated that GIP variants at the 5′ gene region represent metabolic modifiers that contribute to phenotypic variation in GIP response and glucose metabolism. Further characterization of these causal variants would open a new venue for understanding the molecular mechanisms that underlie phenotypic variations in energy-balance regulation and improve our ability to stratify and interpret clinical outcomes associated with the GIP signaling pathway.
For decades, adaptive selection was assumed to be rare; however, recent studies have demonstrated that adaptive substitution is pervasive in human genomes (1
). Despite this progress, it is obvious that population differentiation characteristics of human genomes have yet to be fully explored because physiologic consequences of almost all of these past gene–environmental interactions remain to be verified experimentally (1
). On the other hand, because many selection pressures could be heterogeneous or reversible in a short time, the signature of selection may have eroded in genes that were responsive to cultural selection pressures (12
) compared with those shielded from heterogeneous selection (e.g., the adaptation to environmental oxygen levels and altitude) (5
). We therefore reasoned that important metabolic modifiers could be hidden in the trove of SNPs that showed limited evidence of positive selection and that this limitation could be particularly pertinent to modifiers associated with adaptations in response to shifts of subsistence cultures.
Consistent with this hypothesis, a survey of earlier studies of genome-wide or chromosome-wide positive selection using the so-called outlier approaches—in which candidate loci are identified in the extreme tails of empiric distributions (40
)—showed that GIP
variants have not been reported as positively selected (1
). The selection of GIP
variants was inferred after we focused the analysis on local genomic regions and assessed the significance of integrated haplotype score using coalescent simulations (22
). Therefore, our investigation provided a proof-of-concept study for identifying causal mutations that underlie phenotypic variation of complex disease-related traits. This approach could open new venues for improving the translation of common variant association signals into biologic mechanisms that underlie physiologic variability or disease risk.
Neel hypothesized that mismatches between prior adaptations and new environments, or a “conflict of adaptations,” could lead to changes in fitness or health risks (11
). Because ancient variants could have been selected for the organism’s reproductive success but not for its health or longevity, the ancient alleles could confer disease risks as selection pressures change. Therefore, studies of positively selected variants that are associated with adaptations in energy-balance regulation could point not only to novel genotype–phenotype relationships but also to novel molecular mechanisms that mediate the potential phenotypic variation, thereby providing much-needed insight into how and which phenotypic variations in energy-balance regulation can be attributed to the selected variants. In support of the thrifty genotype hypothesis, human CAPN10
and house-mouse insulin genes have been shown to exhibit characteristics of adaptive evolution after the emergence of agricultural societies (41
). Conceptually, the high GIP response associated with the ancestral GIP−1920G
haplotype could have been a beneficial energy-conserving mechanism when the food supply was irregular. The ancestral haplotype could become deleterious in the last 10 millenniums as agricultural practice became widespread. One possible deleterious effect of the ancestral GIP−1920G
haplotype in an environment that supplies abundant high-starch food resources is the hypersecretion of insulin and insulin resistance (43
On the other hand, a reduced GIP response associated with the derived GIP−1920A
haplotype could be protective by decreasing the extent of insulin secretion in the face of oversupply of energy inputs (45
). In support of this speculation, it has been well documented that in the absence of modern medicine, diabetes-associated complications and, possibly, obesity posed detrimental effects on survival when human culture shifted (47
), even though type 2 diabetes is generally considered a chronic disease in modern society. Alternatively, an elevated glucose level associated with the derived haplotype may have improved the survival of fetuses if the population faced serious famine—an event frequently experienced by agricultural societies (48
)—despite the reality that an impaired glucose-tolerance response represents a risk to both the mother and fetus under normal circumstances. Moreover, the derived haplotype could have been beneficial by reducing the obesity-promoting effect of GIP (16
). In vitro and in vivo studies have shown that GIP promotes fatty liver and other obesity-associated metabolic disorders, whereas GIP antagonists suppress lipid accumulation induced by a high-fat diet (23
). Therefore, the derived GIP−1920A
haplotype could be selected for its effects on the enteroadipocyte or the enteroinsular axis, or both.
Although we speculated that the derived GIP−1920A
haplotype may have provided protective effects in famine-plagued agricultural societies, the observation that the derived haplotype has not been fixed in any population suggests that the selection of the derived GIP
haplotype(s) (e.g., cycles of famine) could be opposed (balance selection) as populations experienced temporal changes in selection pressure (e.g., resumption of population growth with stable food supply). Alternatively, the derived GIP
haplotype could simply be too young to become fixed, or the spread could be limited by the transgenerational effects associated with abnormal gestational glucose metabolism, which raise the risk of macrosomia and diabetes in the offspring (11
Although an association between GIP
variants and glucose-metabolism regulation has not been reported, GIPR
variants were associated with glucose and insulin levels after challenge tests as well as with BMI in GWA studies that evaluated >29,000 individuals (35
). The finding that patients with a homozygous GIP−1920A/A
genotype have significantly higher glucose levels compared with those carrying an ancestral GIP−1920G
haplotype within the pool of GIPR1159G/G
homozygotes suggested that there is a confounding effect stemming from interactions of GIP
variants, and that GIP
variants represent novel markers for the stratification of the capability to maintain glucose homeostasis during pregnancy.
We also speculate that the significant results observed in pregnant women could be related to the fact that the success of pregnancy has a significant impact on reproductive fitness and that a major fraction of gene–environmental selections probably occurred before birth (49
). Recent studies have corroborated this idea by showing that associations between many risk alleles and type 2 diabetes can be replicated with smaller sample sizes in patients with gestational diabetes mellitus (50
Furthermore, given the evolutionary signatures at the GIP
locus, the plausible molecular mechanism, and the significant results in East Asian women, we speculate that the GIP
variant–mediated phenotypic divergence could also exist in most human populations. It is also important to note that the selection of GIP
variants represents a unique example in which the selection process involves regulatory variants that alter the glucose-induced GIP response as well as a nonsynonymous variant that affects peptide bioactivity (22
). Thus, the GIP signaling pathway could represent a hotspot for selection in recent human history and play an important role in the manifestation of phenotypic variation in energy-balance regulation among individuals.
In addition to GIP
variants, our study identified several CDKAL1
, and PPARG
variants as potential metabolic modifiers. Recent studies have shown that variants in CDKAL1
, and more than two dozen genes are associated with glycemic traits in diabetic patients (10
). Surprisingly, none of the CDKAL1
, and PPARG
variants identified here have been implicated in earlier GWA studies, which suggests that these variants could be related to novel energy-balance regulatory mechanisms that operate at certain life stages or under specific physiologic conditions that have not been specifically investigated. Future investigations of these variants could reveal additional metabolic modifiers that have arisen recently and their contributions to phenotypic variation in normal human physiology and metabolic syndrome–related traits.
In conclusion, our data demonstrated a strong association between regulatory GIP variants, and GIP response and glucose metabolism, reinforcing the indication of an important role of GIP signaling in diabetes-related traits from earlier GWA studies of GIPR. Importantly, our study also provided a novel approach to reveal metabolic modifiers by studying consequences of previous mismatches of physiologic capabilities and environments.