Project Summary Obesity is a risk factor for the development of insulin resistance (IR), a hallmark of type 2 diabetes (T2D). Weight loss improves obesity-evoked IR; however, the majority of individuals who lose weight, regain the weight within 1-5 years. This ‘weight cycling’ further increases risk of metabolic disease compared to weight maintenance. Our group developed a mouse model of weight cycling to uncover mechanisms by which weight regain poses additional risk of metabolic disease. We show that weight cycled diet-induced obese (WC-DIO) animals have worsened glucose tolerance than equally obese mice that have not weight cycled. A unique finding is that glucose intolerance in WC-DIO mice is linked to impaired insulin secretion (in vivo during a hyperglycemic clamp and ex vivo in perifused islets). This key finding indicates that β-cell compensation fails to completely adapt to the physiological IR evoked by weight regain in the same way it does during the first bout of weight gain. At the cellular level, WC-DIO mice manifest with atypical β-cell mitochondrial morphology and decreases in gene signatures linked with mitophagy, redox metabolism, and TCA cycle regulation. Mitochondrial metabolism is fundamental for normal nutrient stimulated insulin secretion. Thus, the mitochondrial alterations evoked by weight cycling support a mechanism for impaired insulin secretion. Poor functioning mitochondria are also linked with disruptions to redox control, which can increase oxidative stress and impair β-cell function. We find that a major regulator of pro-oxidant status in β-cells, thioredoxin interacting protein (TXNIP), is increased in WC-DIO islets and inversely associates with insulin secretion. Together, these preliminary studies support that in response to repeated nutrient overload, β-cells are less efficient at coupling metabolic processes to insulin secretion. The central hypothesis is that repeated nutrient overload decreases mitochondrial function and evokes oxidative impairment in β-cells. This loss of β-cell adaptation to nutrient overload impairs insulin secretion and in vivo glucose regulation. This proposal will: i) determine whether impaired mitochondrial function evoked by repeated nutrient excess is central to loss of pancreatic function and ii) examine whether TXNIP is responsible for driving oxidative stress and loss of pancreatic function with weight cycling. Stable isotopes will be used to measure metabolic flux rates in isolated islets. 13C-labeled metabolites will be administered in vivo and incorporation in β-cells quantified using imaging mass spectrometry. Pharmacological inhibition and β-cell specific deletion of TXNIP will be used to determine whether attenuation of oxidative stress restores insulin production and secretion. In vivo insulin secretion and insulin sensitivity will be determined using the frequently- sampled intravenous glucose tolerance test coupled with glucose tracers to quantify glucose fluxes. Ex vivo...