oa Understanding Diabetes and Cardiovascular Risk Biomarkers in Clinical Samples - The FIELD Trial and the DAMAGE Study

People in developing countries of the world are facing a changing environment due to urbanization – changes that include lifestyle changes as well as changes in the dietary habits (and content). These lifestyle changes are now known to contribute to an epidemic of metabolic disease. The underlying mechanisms are unclear. The burden of type 2 diabetes mellitus (T2D) is increasing worldwide, particularly in developing countries, where it is predicted that over 70% of the global burden of T2D will exist by 2030 (Echouffo-Tcheugui and Dagogo-Jack, 2012). Although reasons for the increasing rates of T2D in developing countries are not fully elucidated, important factors include lifestyle changes involving rural-to-urban migration (“urbanization”), intra-uterine undernutrition and foetal programming (epigenetic changes progressively introduced over multiple generations and associated with maternal undernutrition). During the past two decades, increasing evidence arising from multiple clinical studies conducted by the research teams of Yajnik and Barker support an important role of early life undernutrition, and specifically disturbances of one-carbon metabolism, in the heightened susceptibility of Indians to diabetes at a younger age, and in the absence of generalized obesity (Kulkarni et al., 2007; Yajnik et al., 2014; Yajnik and Deshmukh, 2012; Yajnik et al., 2003; Yajnik et al., 1995). The Pune Maternal Nutrition Studies have highlighted the body composition and nutritional-metabolic peculiarities of multigenerationally undernourished Indians: a thin-fat (low lean mass, high fat mass) phenotype compared to Europeans, with predominant visceral deposition of fat. This body composition is strongly associated with insulin resistance and related metabolic-endocrine abnormalities. Importantly, this ‘thin-fat’ phenotype was present at birth and therefore, programmed during intrauterine life, possibly through epigenetic mechanisms over multiple generations. Maternal intergenerational undernutrition, evident in stunting, low BMI, and a disturbance of dietary methyl donors (low protein and vitamin B12 and high folate status related to vegetarian food habits) appear contributory to the increased risk of diabetes and CVD in Indians (Yajnik, 2004; Yajnik and Deshmukh, 2012; Yajnik et al., 2008; Yajnik et al., 2003). It is now well-appreciated that the intra-uterine environment can induce heritable alterations that may be retained over generations (Aiken and Ozanne, 2014; Good speed et al., 2014; Ng et al., 2010). A primate maternal high-fat diet supplemented with calorically dense treats leading to obesity has been shown to epigenetically alter chromatin structure in their progeny via SIRT1 mediated covalent modifications of histones (Aagaard-Tillery et al., 2008; Cox et al., 2009; Suter et al., 2012). Increased adiposity and insulin resistance have also been reported in high fat diet fed rodent models. Intra-uterine programming may involve epigenetic changes, which are passed over generations, and may promote the development of adiposity and T2D. Recently, we demonstrated (cover story; August 2015 issue of Cell Metabolism) that the metabolic effects introduced over multiple (50) generations of undernutrition cannot be reversed after two generations of unrestricted access to nutrients. Undernourished rats demonstrated low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age/gender-matched Control rats. Relative to Controls, Undernourished rats had higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an eight-fold increased susceptibility to diabetes, after Streptozotocin (STZ) exposure. These metabolic abnormalities were not reversed after two generations of recuperation. Adverse epigenetic (histone modification) profiles in insulin gene promoter region of Undernourished rats are not reversed following two generations of macro-nutrient availability and might explain the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations. One of the major strengths of this study is that the current animal model of diabetes was not generated by genetic manipulations but rather by feeding Control rats with an undernourished died for 50 generations (over 12 years) and then a normal diet for two generations. The Undernourished rats showed visceral adiposity and were less sensitivity to insulin. They also demonstrated several markers of metabolic disease. In order to correct this state, we allowed Undernourished rats to have unrestricted access to Control chow and water (now called as “Recuperation” rats). Recuperation rats, surprisingly, show increased visceral adiposity, continue to be less sensitive to insulin, and have the same level of diabetes susceptibility as Undernourished rats. Analysis of epigenetic signatures at insulin gene promoter region in the pancreatic islet cells confirms that although a slight improvement is seen in histone modifications as well as the recruitment of histone methyl transferases at the insulin gene promoter region, two generations of normal nutrient availability is in itself not sufficient to reverse the epigenetic changes to Control levels. These studies underscore the importance of epigenetic regulation of gene expression and indicate that micronutrient or other appropriate supplementation should be considered in dietary intervention studies on populations that have faced undernutrition over multiple generations. I will present our recent data from animal models as well as our data from two independent clinical cohorts; first, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) trial of around 10,000 Type 2 diabetic individuals followed over 12 years and secondly, the Pune Maternal Nutrition Study comprising of around 700 women and their babies over a period of 18 years of follow-up. Our studies (Discovery Analysis of MicroRNAs in Gene-Environment Interactions: DAMAGE-I study) involve analysis of DNA methylation changes (using the Illumina 450 K methylation arrays), GWAS analysis (using the Affymetrix platform), Telomere length assay (using quantitative real-time PCR) and microRNA analysis (using high throughput high sensitivity real-time TaqMan-PCR panels/chips, in addition to several (over 100) clinical biomarkers. Studies are also in progress to understand the role of gut microbiota – our inner environment that significantly influences human physiology. These studies demonstrate that the change in diet and lifestyle (urbanization) leads to significant changes in the short-chain fatty acid-producing gut bacteria and influence our lifestyle through epigenetic programming of gut cells. Overall, the studies discussed above, demonstrate the potential for integrative research technologies that we have used in understanding newer molecular biomarkers of diabetes and cardiovascular health and will allow future application of these molecular biomarkers to understanding the progression of diabetes and its complications in other (non-Australian) cohorts of at-risk of diabetes individuals worldwide. Hopefully, these technologies will help in better understanding the progression of Diabetes in individuals at risk of diabetes, provide newer tools to clinicians for predicting treatment efficacies and patient stratification and enable the development of newer and effective strategies to manage and cure diabetes.

Declarations

The current research discussed here is funded via three different grants to Professor Hardikar from the Australian Research Council (ARC), the National Health and Medical Research Council (NHMRC) and the Juvenile Diabetes Research Foundation (JDRF). Professor Hardikar is also on the advisory committee for Abbott Pharmaceuticals in relation to a probiotic formulation that has the potential for improving metabolic health in diabetes.