www.nature.com/scientificreports OPEN Bats: Body mass index, forearm mass index, blood glucose levels and SLC2A2 genes for diabetes Received: 26 February 2016 Fanxing Meng1, Lei Zhu1,2, Wenjie Huang1, David M. Irwin3 & Shuyi Zhang1,4 Accepted: 28 June 2016 Bats have an unusually large volume of endocrine tissue, with a large population of beta cells, and Published: 21 July 2016 an elevated sensitivity to glucose and insulin. This makes them excellent animal models for studying diabetes mellitus. We evaluated bats as models for diabetes in terms of lifestyle and genetic factors. For lifestyle factors, we generated data sets of 149 body mass index (BMI) and 860 forearm mass index (FMI) measurements for different species of bats. Both showed negative inter-species correlations with blood glucose levels in sixteen bats examined. The negative inter-species correlations may reflect adaptation of a small insectivorous ancestor to a larger frugivore. We identified an 11 bp deletion in the proximal promoter of SLC2A2 that we predicted would disrupt binding sites for the transcription repressor ZNF354C. In frugivorous bats this could explain the relatively high expression of this gene, resulting in a better capacity to absorb glucose and decrease blood glucose levels. The World Health Organization (WHO) defines diabetes mellitus as a group of metabolic diseases in which blood glucose levels are elevated over a prolonged period of time. Diabetes mellitus is due either to the pancreas not producing enough insulin or the cells of the body not responding properly to insulin1. According to WHO, type 2 diabetes, the most common type, represents ~ 90% of diabetic cases worldwide. Insulin resistance is fundamental to the etiology of type 2 diabetes2 because of a combination of lifestyle3, including being overweight (a body mass index (BMI) > 25) or obese, lack of physical activity, poor diet, stress, and urbanization. Genetic factors, includ- ing more than three dozen genes4 also may be involved. For type 2 diabetes in humans, obesity is an important lifestyle factor that provides a paradigm for estimating risk factors for health problems5. Bats, order Chiroptera, are the second largest group of mammals. The diets of bats range from fruits, flowers and pollen to insects and other animals as well as blood6. This dietary diversity is reflected by different approaches to obtain, store and use energy7. The diversity of bats also is reflected by different responses to fasting. Mammals fed protein-rich diets typically show steady plasma glucose levels during fasting. Those fed a carbohydrate-rich diet experience a severe drop in fasting glucose levels8,9. Bats show different patterns. Pallas’s mastiff bats (Molossus molossus) eat insects and maintain constant blood glucose levels through a 48 h fast showing no increase in the concentration of free fatty acids in their blood10. Similarly, two new species of New World fruit-eating bats with carbohydrate rich diets maintain relatively high blood glucose levels after a short-term fasts of 2–6 days11. These data suggest that frugivorous and insectivorous bats share an ability to maintain glucose homeostasis. Old World fruit-eating bats (e.g., Rousettus aegyptiacus) have an unusually large volume (9.1%) of endocrine tissue12. New world fruit-eating bats (e.g., Artibeus lituratus) have large population of beta cells and elevated sensitivity to glucose and insulin13. The Old World great roundleaf bats Hipposideros( armiger) generate higher amounts of cold-induced beige fat from white adipose tissue than mice14. These data suggest that bats have a good capacity for maintaining glucose homeostasis, lowing blood glucose levels through insulin, and browning the white adipose tissue. Together these features suggest that bats could be good models for understanding lifestyle and genetic factors regulating glucose metabolism, especially for type 2 diabetes. To aid in diabetes research we have investigated the good properties of sugar metabolism in bats as well as the molecular mechanism underlying them. In Old World Fruit bats, we have previously shown adaptive changes in 1State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062, China. 2Kunming Institute of Zoology, Chinese Academy of Sciences & University of Chinese Academy of Sciences, Kunming 650223, China. 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 2E8, Canada. 4Key Laboratory of Zoonosis of Liaoning Province, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang 110866, China. Correspondence and requests for materials should be addressed to S. Z. (email: [email protected]) SCIENTIFIC REPORTS | 6:29960 | DOI: 10.1038/srep29960 1 www.nature.com/scientificreports/ Figure 1. Scatter diagrams of the associations of BMI (left) and FMI (right) to body weight in bats with differing diets. Bats were grouped as frugivorous, omnivorous, sanguivorous, insectivorous, and carnivorous for BMI and indicated by green upward triangles, blue pentagons, red leftward triangles, purple downward triangles, and yellow rightward triangles, respectively. SLC2A4, which encodes glucose transporter 4 and is expressed in adipose and muscles15. These changes appear to have improved the ability of bats to respond to high sugar diets15. In humans, single nucleotide polymorphisms (SNPs) within SLC2A2 are associated with the conversion from impaired glucose tolerance (IGT) to type 2 dia- betes16. Glut2 (encoded by SLC2A2), another member of the glucose transport family, functions in the pancreas to mediate glucose uptake for the regulation of insulin secretion and in hepatic cells to allow the generation of hepatic glycogen stores17. This could mean that changes in the transcription or translation of SLC2A2 might affect the abundance of its protein product (Glut2) as well as their efficiency of transporting glucose from blood to the liver during hyperglycemia. Here, to explore the value of Chiroptera as a model for diabetes in terms of lifestyle and genetic factors, we calculated the body mass index (BMI) and the forearm mass index (FMI) for 149 and 860 species of bats, respec- tively. We then used both BMI as well as FMI to establish baselines for estimating risk factors for sixteen species of bats with different diets and associated levels of blood glucose. To determine whether fruit-eating bats have better adapted to periodic acute glucose stress than insect-eating bats, we conducted acute glucose tolerance tests on representative species. We then investigated the sequences of the proximal promoters of SLC2A2 genes as well as the relative abundance of its mRNA product in frugivorous and insectivorous bats to examine the molecular basis for the better capability of fruit-eating bats, as shown by the acute glucose tolerance tests, to lowing blood glucose levels. Results Body mass index and forearm mass index in bats. Body fat is a risk factor for diabetes in humans and can be estimated using BMI. Standards for normal and overweight bats are lacking. To establish baselines and determine whether BMI, can be used to assess the body mass quantity in bats, we collected a data set of mean weights and sizes of bats from a total of 149 species (see Appendix S1). BMI values were calculated for each of the 149 species of bats, which could be categorized into frugivore, insectivore, and others (sanguivore, carnivore, and omnivore) dietary groups, with sample sizes of 88, 49, and 12 species, respectively. Among these categories, the frugivore group had the highest average BMI, of 7.0 kg/m2, with the insectivore group being lowest, with a mean of 3.9 kg/m2, and the other bats having an intermediate mean BMI of 5.7 kg/m2. For bats, the number of available forearm length measurements is much greater than the number of full body lengths. Forearm lengths and full body lengths show a positive correlation with each other (R2 = 0.933, see Appendix S2), thus we assessed the use of body weight and forearm length to quantify bat body mass. Similar to BMI, FMI is defined as body mass (kg) divided by the square of the forearm length (m2). FMI might be a more appropriate measure for body mass quality in bats, as BMI is calculated using total body lengths, which for some, but not all, species include the tail. Thus BMI for species with tails would be lower than that for similar sized bats that lack tails. FMI values were calculated for 860 species of bats, which could be classified as having frugivore, insectivore, carnivore, sanguivore, and omnivore diets and being represented by 244, 581, 8, 3, and 24 species, respectively (see Appendix S1). Similar to BMI, the frugivore group had the highest average FMI value (13.9 kg/m2) and the insectivores were lowest (6.1 kg/m2), while omnivores (11.6 kg/m2) and others (9.6 kg/m2) had intermedi- ate FMI values. Both the mean BMI and mean FMI of frugivorous bats are significantly higher than those of the insectivorous bats (p-values of 1.021E-013 and 1.263E-098, respectively). BMI, FMI and body sizes in bats. A scatter diagram of BMI and body weights is used to illustrate the differences in the body sizes of bats with differing diets (Fig. 1A). Among insectivorous bats, 48 of the 49 species group near the origin as they have small body sizes (weight < 40 g and BMI < 8 kg/m2). In contrast, a majority (79.5%, 70 of 88 species) of the frugivorous bats are large, being heavier than 40 g and have BMI values larger than 4 kg/m2 (89.8%, 79 of 88). The distribution of FMI and body sizes shows a similar pattern (Fig. 1B). The vast majority of insectivorous bats (85.4%, 496 of 581 species) have a small body size (weight < 40 g and FMI SCIENTIFIC REPORTS | 6:29960 | DOI: 10.1038/srep29960 2 www.nature.com/scientificreports/ Figure 2. Scatter diagrams of the associations of blood glucose to BMI (above) and FMI (below), and linear regression curves for sixteen bats as a single group or divided into two dietary groups.