Career Development Award: Center for Appalachian Research in Environmental Sciences: Arsenic Exposure, Inflammation and Type 2 Diabetes

Type 2 diabetes mellitus is an increasing worldwide problem. It is the leading cause of adult blindness, kidney failure and cardiovascular disease, which contributes to significant morbidity and mortality. In addition to the well-recognized risk factors (e.g. caloric excess, sedentary lifestyles, or genetics) for the development of type 2 diabetes, recently the nontraditional risk factors such as environmental pollutants (e.g. arsenic (As)) exposure) have been paid much attention. Epidemiological studies in many countries including U.S. demonstrate a strong diabetogenic effect of arsenic in human population mainly through As-containing drinking water. Animal as well as in vitro cell culture studies also support the pathological role of As in type 2 diabetes. However, the molecular mechanisms that link arsenic and diabetes remain to be determined. Multiple evidence indicates that arsenic can induce oxidative stress (ROS) in multiple cell types and relates to various diseases such as cancer, aging, and cardiovascular disease. Arsenics may induce oxidative stress by cycling between oxidative states of metals or by interacting with antioxidants, resulting in the accumulation of free radicals in cells. Mitochondria are suggested to be one of the important sources of ROS production. Arsenic exposure stimulated ROS production and resulted in insulin resistance in adipocytes and myotubes. It has been shown that ROS signaling leads to the activation of NFkb or NLRP3 inflammasome, resulting in production of proinflammatory cytokines (TNF-á, IL-1 â et al). The role of chronic inflammation in promoting the development of insulin resistance and type 2 diabetes is well established. Moreover, inflammation can also stimulate the ROS production. However, the role of inflammation in arsenic exposure induced impaired glucose homeostasis (e.g. reduced insulin production in pancreatic beta cells and decreased glucose uptake/utilization in adipocytes) is unknown. Based on these reports, we hypothesize that arsenic exposure-induced ROS production stimulates tissue inflammation and leads to impaired glucose homeostasis and the development of diabetes. To test this hypothesis, two specific aims are proposed as below: Aim 1: Determine whether arsenic-induced oxidative stress stimulates pro-inflammatory cytokine production and leads to reduced insulin production in pancreatic beta cells and decreased glucose uptake/utilization in adipocytes in vitro. In this aim, preadipocyte cell line 3T3-L1 or primary mouse preadipocyte will be cultured, differentiated into mature adipocytes, and used for the following studies. First, we will determine the temporal and dose dependent effect of arsenic exposure on ROS production, the proinflammatory cytokine production and insulin mediated glucose uptake in mature adipoctye. These cells will be treated with arsenic (0, 0.5, 1, and 2 ìM) for both short time (3h, 6h and 24 h) and long time periods (1 week, 2 weeks and 4 weeks) to mimic in vivo conditions. Second, we will determine whether arsenic induced ROS production is a causal factor for proinflammatory cytokine production in adipocytes. Adipocytes will be pretreated with the mitochondria ROS inhibitor-MitoQ and then exposed to arsenic for different time periods as described above. ROS production, inflammation and insulin mediated glucose uptake in these cells will be determined. Third, an effect of anti-inflammation on arsenic induced ROS production and insulin mediated glucose uptake will be determined. Fourth, we will test whether the strategy to antagonize both ROS and inflammation at the same time can achieve maximal improvement of arsenic induced insulin resistance in adipocytes. In addition, pancreatic beta cell line (INS-1 from ATCC) will be also used to determine whether arsenic-induced oxidative stress stimulates proinflammatory cytokine production and leads to reduced insulin production in these cells. These studies will provide important mechanistic mechanisms of arsenic induced impaired glucose homeostasis and will be used to direct in vivo intervention studies. Aim 2: Determine the temporal effect of arsenic exposure on ROS production and its association with tissue inflammation and the development of impaired glucose homeostasis in mice in vivo. In this aim, C57BL6 mice will be fed with arsenic (two doses: 0.1 ppm and 1 ppm) in the drinking water for different time period (2, 4, 8, 16, and 24 weeks). The time course of changes in ROS production and inflammation status from various tissues including pancreatic tissue, adipose, liver and muscle will be investigated. The association of ROS and tissue inflammation with arsenic-induced glucose intolerance and /or impaired insulin sensitivity will be also determined. These studies will allow us to identify key arsenic targeted tissues in the development of diabetes in the different stage of arsenic exposure and will also allow us to determine whether arsenic induced ROS is the early and primary contributor to tissue inflammation and the development of diabetes. These results will direct our future studies and will also sever as critic preliminary data for a R01 application. These integrated in vitro and in vivo studies will provide new mechanistic information on arsenic-induced insulin resistance and diabetes, and may lead to the development of novel therapies for this disease