Plasma metabolomics supports non-fasted sampling for metabolic profiling across a spectrum of glucose tolerance in the Nile rat model for type 2 diabetes

Articles https://doi.org/10.1038/s41684-023-01268-0 Plasma metabolomics supports non-fasted sampling for metabolic profiling across a spectrum of glucose tolerance in the Nile rat model for type 2 diabetes Benton J. Anderson 1, Anne M. Curtis2,3, Annie Jen 4, James A. Thomson2,3,5, Dennis O. Clegg2,3, Peng Jiang6,7,8, Joshua J. Coon1,4,5, Katherine A. Overmyer 4,5 ✉ and Huishi Toh 3 ✉ Type 2 diabetes is a challenge in modern healthcare, and animal models are necessary to identify underlying mechanisms. The Nile rat (Arvicanthis niloticus) develops diet-induced diabetes rapidly on a conventional rodent chow diet without genetic or chemical manipulation. Unlike common laboratory models, the outbred Nile rat model is diurnal and has a wide range of overt diabetes onset and diabetes progression patterns in both sexes, better mimicking the heterogeneous diabetic phenotype in humans. While fasted blood glucose has historically been used to monitor diabetic progression, postprandial blood glucose is more sensitive to the initial stages of diabetes. However, there is a long-held assumption that ad libitum feeding in rodent models leads to increased variance, thus masking diabetes-related metabolic changes in the plasma. Here we compared repeatability within triplicates of non-fasted or fasted plasma samples and assessed metabolic changes relevant to glucose tolerance in fasted and non-fasted plasma of 8–10-week-old male Nile rats. We used liquid chromatography–mass spectrometry lipidomics and polar metabolomics to measure relative metabolite abundances in the plasma samples. We found that, compared to fasted metabolites, non-fasted plasma metabolites are not only more strongly associated with glucose tolerance on the basis of unsupervised clustering and elastic net regression model, but also have a lower replicate variance. Between the two sampling groups, we detected 66 non-fasted metabolites and 32 fasted metabolites that were associated with glucose tolerance using a combined approach with multivariable elastic net and individual metabolite linear models. Further, to test if metabolite replicate variance is affected by age and sex, we measured non-fasted replicate variance in a cohort of mature 30-week-old male and female Nile rats. Our results support using non-fasted plasma metabolomics to study glucose tolerance in Nile rats across the progression of diabetes. D iabetes is an urgent global health challenge with an accelerating incidence rate in recent decades. Currently, 537 million adults are living with diabetes and 541 million adults have impaired glucose tolerance with a high risk of developing type 2 diabetes1. To better understand metabolic changes associated with impaired glucose tolerance, we need suitable animal models and experimental methods that can capture these changes. The Nile rat (Arvicanthis niloticus) is a model of type 2 diabetes with key benefits over other rodent models. First, diabetes is rapidly induced in both sexes by conventional laboratory rodent chow that is hypercaloric for the Nile rat compared to its native fiber-rich diet2,3. On conventional rodent chow, the onset of diabetes can range from a month to a year of age, and by 6 months of age, most of the Nile rats would have developed diabetes4. By contrast, common laboratory mice and rats are relatively resistant to diet-alone induced diabetes, and additional chemical or genetic manipulations are used to promote diabetes5. Second, diabetic Nile rats can develop long-term diabetic complications mimicking clinical features of patients with diabetes6–8, including diabetic retinopathy9,10. Third, the Nile rat model is outbred and displays a wide range of diabetic phenotypes11, reflecting its underlying genetic diversity. Fourth, the Nile rats, like humans, are active during the day12, unlike common nocturnal rodent models. Additionally, the Nile rat has a reference genome for mechanistic studies13. Overall, the Nile rat is highly suited to study the underlying mechanisms of glucose intolerance in diet-induced diabetes. When considering experimental methods for studying diabetes, a majority of studies looking for metabolic changes will use blood that has been sampled under fasted state to avoid excess variability from unrestricted eating behavior. However, for the early progression of diabetes, it is known that postprandial hyperglycemia precedes fasted hyperglycemia, and thus is a more sensitive measurement for early diabetes, as demonstrated in human studies14–16 and in the Nile rat model11,17–19. In addition, there is some evidence that the postprandial state might be associated with reduced variability in blood metabolites20. For rodent models, non-fasted state probably represents a postprandial state given the high frequency of food intake. Yet, so far, no study has validated the use of non-fasted sampling Department of Chemistry, University of Wisconsin-Madison, Madison, WI, USA. 2Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA, USA. 3Neuroscience Research Institute, University of California, Santa Barbara, CA, USA. 4Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, USA. 5Morgridge Institute for Research, Madison, WI, USA. 6Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH, USA. 7Center for Gene Regulation in Health and Disease, Cleveland State University, Cleveland, OH, USA. 8Center for RNA Science and Therapeutics, School of Medicine, Case Western Reserve University, Cleveland, OH, USA. ✉e-mail: ; 1 Lab Animal | VOL 52 | November 2023 | 269–277 | www.nature.com/laban 269 Articles for metabolomics studies in rodent models. Specifically, reproducibility and replicate variability between fasted and non-fasted states have not been sufficiently analyzed. Therefore, this study compares metabolite variance between non-fasted and fasted blood sampling for studying progressive glucose intolerance in Nile rats. To investigate the metabolic differences between the fasted state and non-fasted state, we performed metabolomics using liquid chromatography coupled to mass spectrometry (LC–MS) to measure a broad range of plasma biomolecules21. LC–MS has been used previously to analyze variance of plasma sampling across metabolites20,22–25 and to detect plasma biomarkers relevant to diabetes in mice and humans26,27. To assess replicate variance in fasted versus non-fasted samples and to capture markers of diabetes, we measured metabolites in non-fasted and fasted plasma samples in triplicate. The cohort consisted of male Nile rats aged 8–10 weeks displaying varied levels of glucose tolerance—from non-diabetic to overtly diabetic—at 12 weeks old. We found that metabolites in non-fasted plasma sampling had better predictive power of impaired glucose tolerance. Counter to the accepted wisdom that ad libitum feeding leads to increased variability, we f (...truncated)