Cross-sectional association between blood cholesterol and calcium levels in genetically diverse strains of mice

Genetically diverse outbred mice allow for the study of genetic variation in the context of high dietary and environmental control. Using a machine learning approach we investigated clinical and morphometric factors that associate with serum cholesterol levels in 840 genetically unique mice of both sexes, and on both a control chow and high fat high sucrose diet. We find expected elevations of cholesterol in male mice, those with elevated serum triglycerides and/or fed a high fat high sucrose diet. The third strongest predictor was serum calcium which correlated with serum cholesterol across both diets and sexes (r=0.39–0.48). This is in-line with several human cohort studies which show associations between calcium and cholesterol, and calcium as an independent predictor of cardiovascular events.


Introduction
Elevated blood cholesterol, particularly in the forms of atherogenic LDL particles are causal of cardiovascular disease, the major cause of death in Western societies [1]. Cholesterol levels in humans vary widely depending on multiple factors including genetics, diet and other lifestyle factors, with genetics and lifestyle each contributing roughly equally to cardiovascular disease risk [2].
Human genetics has led to a sophisticated understanding of how cholesterol synthesis and excretion is regulated, and how this varies between individuals and diets [3]. Understanding complex genetic traits like cholesterol is a challenge in human studies where diet and lifestyle data is subject to substantial instrument error. Here genetically diverse panels of mice, where the diet and environment are tightly controlled, and genetics can be well defined can provide solutions. Among several resources, the Diversity Outbred [4] and UM-HET3 [5] mice represent genetically diverse outbred populations, while the Collaborative Cross [6] and BXD [7] resources provide data on recombinant inbred mice. Collectively these resources are valuable tools to understand complex traits such as cholesterol.

Diversity outbred mice exhibit diet and sex dependent variation in cholesterol levels
We first evaluated the cholesterol levels in the diversity outbred mice measured at 8 and 19 weeks. Cholesterol levels for each group were similar at both time points (p=0.474 by pairwise Wilcoxon test, see Supplementary Figure 1). This indicates that cholesterol levels are stable between both time points. We stratified cholesterol levels by sex and diet. Via multivariate regression, we found the expected cholesterol elevations in mice on a HFHS diet (33.7 +/-2.0 mg/dL, p=1.4 x 10 -56 ), and male sex (16.9 +/-2.0 mg/dL, p=3.0 x 10 -17 ; Figure 1A). There was no evidence of a significant interaction between diet and sex (p=0.636).
Diet, triglycerides and calcium associate with cholesterol levels.
To define other potential associations between cholesterol and measured phenotypes in this dataset we generated a regression tree using the 165 phenotypes in this dataset ( Figure 1B). The major classifier of cholesterol levels was the diet, and the second was triglycerides measured at 19 weeks. Serum calcium measured at 19 weeks was the third phenotype that associated with cholesterol levels, and body weight measured at 19 weeks was the fourth ( Figure 1B).
Dyslipidemia often includes elevations of both triglyceride and cholesterol levels in both mice and humans, so the association of triglycerides with cholesterol was not unexpected. Via multivariate modelling accounting for the effects of diet and sex, a 100 mg/dL increase in triglycerides was associated with a 17.7 +/-1.7 mg/dL elevation in cholesterol (p=3.8 x 10 -24 , Figure 2A).
The strong cross-sectional association of calcium with cholesterol was not predicted by our research team. As shown in Figure 2B and Table 1, after adjusting for diet and sex, a one mg/dL increase in calcium is associated with a 12.7 +/-0.8 mg/dL increase in cholesterol (p=3.0 x 10 -43 ). Linear models predicting serum cholesterol including sex and diet, and calcium as covariates had an adjusted R 2 of 0.45 with a partial effect size of 0.22 for serum calcium. We performed Above the box is the algorithmically generated cutoff for triglycerides (abbreviated TG in mg/dL), calcium (Ca in mg/dL), and body weight (BW in g). Each predictor was a phenotype measured at 19 weeks. Within each box, the value represents the average cholesterol level in that group (in mg/dL) and the number of mice in that group (n=822 in total). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 9, 2023. ; https://doi.org/10.1101/2023.02.08.527123 doi: bioRxiv preprint sub-group analyses and found that each diet-sex combination had broadly similar estimates for Spearman's rho (ranging from 0.39 for HFHS females to 0.48 for HFHS males), each of which had a p-value of less than 2.2 x 10 -7 ).
To externally test these findings, we evaluated a distinct dataset of genetically diverse mice, the BXD mouse collection. In a secondary data analysis using data in [10], we replicated this finding, showing a cross-sectional association between cholesterol and calcium levels after adjusting for sex differences. Similar to the data from the diversity outbred mice, we estimate a 14.8 +/-5.1 mg/dL increase in cholesterol was observed for every 1 mg/dL increase in calcium (p=0.005). In the smaller BXD dataset there appeared to be a stronger relationship in male mice (Spearman's rho=0.393 p=0.018, n=36 strains) than female mice (rho=0.269; p=0.297, n=17 strains), but in multivariate modelling there was no significant modifying effect of sex (p=0.178, likely due to the relatively small number of female strains).
In the diversity outbred mice, serum calcium levels are not significantly altered by sex (p=0.59), and only modestly increased by HFHS diets (0.30 +/-0.07 mg/dL; p=5.1 x 10 -5 ; Supplementary Figure 2A). Since calcium is normally tightly controlled by homeostatic mechanisms regulating calcium absorption and bone remodeling, we tested whether bone mineral content and density in these mice was associated with cholesterol levels. As shown in Supplementary Figure 2B and C there was no evidence of an association between bone mass or density measured at 21 weeks and cholesterol levels measured at 19 weeks (p=0.93 and 0.90 respectively) in the diversity outbred dataset.

Discussion
In this study we report an association between calcium and cholesterol in two distinct mouse datasets. This relationship was similar across both sexes and over both normal chow and . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 9, 2023. ; https://doi.org/10.1101/2023.02.08.527123 doi: bioRxiv preprint obesogenic high fat, high sucrose diets. The novel association between calcium and cholesterol study is strengthened by the expected findings that cholesterol is elevated in mice of male sex, with high triglycerides and fed HFHS diets. The finding that this association is largely independent of sex, diets or triglyceride levels suggests that serum calcium may represent a novel biomarker of dysfunctional cholesterol homeostasis and may point to novel mechanisms by which cholesterol could be controlled.
The present study does not speak to the directionality of this association, but there are some hints in the literature. A meta-analysis by demonstrates a 31% increased risk of myocardial infarction in patients with calcium supplementing with calcium compared to placebo [35]. Patients with primary hyperparathyroidism have elevated parathyroid hormone and calcium levels and are an interesting population to examine. The results of case-control studies evaluating cholesterol levels are mixed and there is limited evidence that PTH causes elevated cholesterol levels. While reports show that these patients also have significantly elevated total and/or LDL-cholesterol [36,37], though most others show either non-significant effect or even decreases [38][39][40][41][42][43][44].
In terms of whether cholesterol could be driving hypercalcemia, there is some positive evidence. Two interventional studies using statins have showed a reductions in calcium levels [45,46], whereas another single-arm study showed declines that did not reach significance [47] . A Mendelian Randomization approach using LDL-C as the instrument was also associated with elevated calcium levels [48], further supporting a potential causal relationship with cholesterol driving calcium levels. As this is a cross-sectional associative relationship we are not able at present to define directionality, much less the underlying biological mechanism(s). Whether calcium can modify serum cholesterol, or cholesterol can modify calcium are both important nutritional and pathophysiological questions, and future controlled mouse studies should shed light on the directions and mechanisms explaining this association.
There are several other strengths of this study. We present data on a large number of mice roughly equally divided between sexes and two diets and find consistent results across all groups. We have exceptional control of confounders such as diets, environment, activity levels, and other exposures that could affect the interpretation of the human studies. Our supervised machine learning approach used a large number of measured phenotypes to predict calcium levels, and set cutoffs in a data-driven manner. We consider the support of data from two independent genetically diverse mice populations another strength of this work. Calciumcholesterol associations appear to be robust over a wide variation in genetics and not restricted to findings in inbred mouse populations. As such, this relationship holds over multiple diets, sexes, investigators, sites, and genetic backgrounds.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 9, 2023. ; https://doi.org/10.1101/2023.02.08.527123 doi: bioRxiv preprint

Limitations of the present study
While there were multiple measurements of calcium and cholesterol in this dataset (at week 8 and week 19, after 5 and 16 weeks of HFHS/NCD respectively), cholesterol levels were stable through at these points. Therefore, it was possible to effectively evaluate the longitudinal association between cholesterol and calcium. In addition, this cross-sectional association does ascribe a directionality to this relationship, at this stage we think it plausible that calcium may increase cholesterol, that cholesterol might increase calcium, or that a third, unmeasured factor drives both factors. As this is a secondary data analysis, we are unable to evaluate differences in calcium-regulatory hormones, which we predict would vary more than the relatively homeostatic blood calcium levels. Another limitation is that cholesterol homeostasis is substantially different in mice and humans, especially in the fraction of cholesterol present in the HDL versus LDL fractions, due to the absence of CETP in mice. These data therefore largely reflect associations between calcium and the HDL pool.
In conclusion, in this work we use a machine learning approach to describe that diet, sex, triglyceride levels and calcium all contribute independently to the serum cholesterol levels in Diversity Outbred mice, and that the relationship between calcium and cholesterol holds true in BXD mice. These data support that the observed human relationships between serum and cholesterol levels are true in mice, and present an opportunity for further physiological and genetic dissection of this relationship.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 9, 2023. ; https://doi.org/10.1101/2023.02.08.527123 doi: bioRxiv preprint