ABSTRACT Preclinical studies indicate that the neurobiology of obesity may be related to diet-induced cellular inflammatory responses in hypothalamic areas that regulate body weight. In rodent models of diet-induced obesity, overfeeding by high-fat diet rapidly induces inflammation and gliosis in the arcuate nucleus of the hypothalamus. Moreover, these glial cell inflammatory responses are both necessary and sufficient to induce hyperphagia and weight gain, suggesting that gliosis plays a mechanistic role in the ability of diet to induce obesity in rodent models. Using magnetic resonance imaging (MRI), the investigators discovered the first evidence of hypothalamic gliosis in humans with obesity. These findings have since been replicated in adults and children with obesity as well as in type 2 diabetes and insulin resistance. However, the current evidence in humans is observational, and the lack of controlled clinical experiments limits our understanding of whether findings of hypothalamic gliosis are diet-induced in humans. The overall goal of the proposed research is to determine the feasibility, promise, and safety of an experimental model in humans to test the effects of hypercaloric, obesogenic diets on hypothalamic gliosis as measured by MRI. The proposed mechanistic clinical trial is in response to PA-20-160 (Small R01s for Clinical Trials Targeting Diseases within the Mission of NIDDK). Using a randomized clinical trial design and a eucaloric control group, the proposal aims to compare the effect of two overfeeding regimens that vary in caloric load (but not macronutrient composition) on hypothalamic gliosis as measured by MRI. A second goal is to determine whether a standardized hypocaloric diet following the overfeeding regimen is a feasible strategy to mitigate any observed weight gain and/or hypothalamic gliosis due to overfeeding. Finally, the feasibility, acceptability, and long-term weight effects of study participation will be assessed. Forty-two participants with overweight will undergo serial MRI scans before, during, and at completion of 7 days of overfeeding or eucaloric diet as well as before, during, and at completion of a 7-day hypocaloric or eucaloric diet period. Diet composition during overfeeding will be based on preclinical studies of gliosis and utilize a robust stimulus that is high in dietary fat, saturated fat, and refined carbohydrates and most would consider obesogenic. Effect sizes, feasibility, and safety data will contribute to the design of future mechanistic clinical trials. Additional future directions include parsing which diet elements (e.g., caloric excess, saturated fatty acids) elicit radiologic evidence of inflammation and gliosis in the mediobasal hypothalamus. The proposed study represents a necessary first step in testing the hypothesis that a hypercaloric, obesogenic diet induces inflammation and structural changes in body-weight-regulating areas of the brain in humans—changes that promote the de...