Elsevier

Physiology & Behavior

Volume 94, Issue 5, 6 August 2008, Pages 649-659
Physiology & Behavior

Gene discovery and the genetic basis of calcium consumption

https://doi.org/10.1016/j.physbeh.2008.04.004Get rights and content

Abstract

This review makes the case that gene discovery is a worthwhile approach to the study of ingestive behavior in general and to calcium appetite in particular. A description of the methods used to discover genes is provided for non-geneticists. Areas covered include the characterization of an appropriate phenotype, the choice of suitable mouse strains, the generation of a hybrid cross, interval mapping, congenic strain production, and candidate gene analysis. The approach is illustrated with an example involving mice of the C57BL/6J and PWK/PhJ strains, which differ in avidity for calcium solutions. The variation between the strains can be attributed to at least seven quantitative trait loci (QTLs). One of these QTLs is most likely accounted for by Tas1r3, which is a gene involved in the detection of sweet and umami tastes. The discovery of a novel function for a gene with no previously known role in calcium consumption illustrates the power of gene discovery methods to uncover novel mechanisms.

Introduction

This review is based on a talk given at the annual meeting of the Society for the Study of Ingestive Behavior (SSIB), in Steamboat Springs, Colorado, in July 2007. Two aspects of the talk were unusual for members of the Society: First, was the topic; second, was the use of gene discovery methods. My goal here is to lay out for the non-geneticist the steps involved in gene discovery, together with some of the advantages and disadvantages of the approach. I will use my work on calcium intake as an example but the same methods can be applied to just about any ingestive behavior. For those looking for general overviews of the physiological and behavioral controls of calcium consumption, a comprehensive review [84] and a book [75] describes the animal literature, and there is a book chapter outlining some examples of calcium appetite in humans [85].

Before describing the methods used to discover genes, it is worth considering why calcium appetite has been largely ignored in the past and why the gene discovery method is a particularly appropriate way to approach it.

According to a quick count of abstracts of presentations at the 2007 SSIB meeting [53], there were 227 presentations on food intake or obesity, 12 on sodium or water intake, and only one on calcium intake. I doubt this is a bias of the Society or its membership; it is fairly typical of the field of ingestive behavior in general. According to PubMed, in 2007, there were published 9515 papers on ”obesity”, 1515 papers on “food intake”, 22 papers on “sodium appetite”, and none on “calcium appetite” [54].

Why is the large preponderance of research on ingestive behavior focused on food rather than salt or calcium? The first thing that comes to mind is that food intake and obesity may be bigger clinical problems than either sodium or calcium intake. There are, after all, an estimated 59 million obese Americans and another 65 million overweight. However, this argument by incidence is a misperception because 58 million Americans have hypertension [34], at least some of which is linked to high sodium intakes, and 44 million Americans have low bone mass or osteoporosis [55], at least some of which is linked to low calcium intakes. Moreover, low calcium intakes have been implicated in several other diseases, including hypertension, obesity, premenstrual syndrome, and various cancers. Thus, if research effort were to be dictated solely by disease prevalence some emphasis on studying energy intake would be warranted but not anywhere near the current level.

Instead, I believe the bias is part historical but largely irrational. It is easy to see that eating too much food will lead to obesity but not so easy to see the connection between mineral intake and hypertension or osteoporosis. Of course, simply because the connection is not obvious does not imply it is not important. More tellingly, obesity is historically considered a disease of energy balance (energy in–energy out) but hypertension has historically focused on the kidney, and osteoporosis on bone. I make it a habit to inspect the schematic diagrams of calcium homeostasis provided in textbooks. Many include sources of calcium loss (i.e., fecal and urinary loss, and sweat) but I have yet to find one that shows where this calcium enters the body. It seems strange that few would consider obesity as entirely a disease of energy loss whereas this is the norm for diseases of mineral homeostasis.

Another complication, at least in my opinion, is that research on the controls of sodium intake has lost contact with its clinical implications. Early work in this field emphasized the uniqueness of sodium appetite, particularly in contrast to the appetites for other minerals and vitamins. An unfortunate consequence of this was to dissuade investigation of calcium and other appetites. I have written about this elsewhere [84] so will not repeat it here. More recently, the main focus of sodium consumption research has been on controls exerted by the renin–angiotensin–aldosterone system. This too is unfortunate because there is little evidence to suggest that circulating levels of angiotensin or aldosterone are relevant for human sodium consumption [15]. The evidence suggests that the renin–angiotensin–aldosterone system is crucially involved in the motivation to ingest sodium during sodium deficiency but the human problem is one of avoiding, not finding, sodium [see [82] for discussion].

There are three factors that have led my group to approach the study of calcium appetite using a genetic approach. The first is that we have tried other approaches and, although they have been useful, they reveal how things can get complicated very easily. For example, a seemingly simple question is whether 1,25-dihydroxyvitamin D, the active metabolite of vitamin D, effects calcium intake of rats. The “obvious” approach is to infuse 1,25-dihydroxyvitamin D and observe the behavior. However, 1,25-dihydroxyvitamin D levels influence parathyroid hormone and calcitonin production, and these hormones may influence behavior, either by direct action on the brain or indirectly, by altering circulating calcium concentrations. Thus, parathyroid hormone and calcitonin must be controlled and the only effective way to do this is to remove the parathyroid and thyroid glands and infuse replacement doses. An unintended consequence of destroying the thyroid glands is that this removes the source of thyroxin, which also influences calcium metabolism, so this hormone must be replaced as well. There is another problem because 1,25-dihydroxyvitamin D influences calcium absorption from the gut, which in turn will influence circulating calcium concentrations, so the infused rats need special diets formulated so that final circulating calcium concentrations are normal. The original question is simple but the experiment needed to test it requires thyroparathyroidectomized rats receiving four hormone infusions simultaneously and a special diet. It would be necessary but technically formidable to replicate the circadian dynamics of each hormone, and even if this could be done it would be difficult to ensure the result would tell us much about normal behavior.

The second reason to use a genetic approach is that there is very little already known about the physiological controls of calcium appetite. There are hypotheses concerning circulating calcium levels, calcium-regulatory hormones, aching bones, and changes in taste sensitivity, but none that prove completely compelling [reviewed in [84]]. With virtually a blank slate, it is difficult to know where to look first. One of the strongest advantages of using a gene discovery approach is that it is, at least in the early stages, hypothesis-free. The results suggest the hypotheses, not the other way around.

The third factor that motivated us to take a gene discovery approach was the fortunate coincidence of time and place. The main field of ingestive behavior appears to have overlooked the gene discovery approach but there are three sub-fields that are exceptions: the study of taste receptors, alcohol consumption, and obesity genes. All three of these areas have been explored by my colleagues at Monell and their successes are clear [e.g., [3], [4], [6], [64], [65]]. Moreover, starting a gene discovery project even 10 years ago would have been difficult because of the limited number of polymorphic markers available and the high costs of genotyping. Now, there are large databases of markers and the costs of genotyping have dropped precipitously.

Section snippets

The gene discovery approach to study calcium appetite

The distinguishing feature of the gene discovery approach is the use of a genome screen to discover quantitative trait loci (QTLs) but to conduct a successful genome screen requires preparation, and to interpret the results requires follow up. Consequently, there are several steps involved, which are summarized below.

What next?

Characterizing the contribution of Tas1r3 and Casr to the control of calcium consumption will take several years, but even if things turn out as expected, there will still be a lot left to learn. Tas1r3 accounts for only 7–13% of the phenotypic variance in calcium and magnesium preference in the B6 × PWK cross and there are at least 6 other QTLs involved. Crosses of other mouse strains will undoubtedly reveal even more QTLs, and there are likely to be epistatic and gene × environment interactions

Acknowledgements

This is a review based on a keynote address given at the Society for the Study of Ingestive Behavior annual meeting in Steamboat Springs, Colorado in July 2007. I thank Masterfoods (now Mars) for sponsoring the talk and the members of SSIB for their attention and questions. The following have supported the project: most funding was provided by NIH RO1 grant DK-46791, and I particularly thank the program officer, Dr. Robert Karp, for taking an active interest in this project. Funding for

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