M. High-sucrose diet increases ROS generation, FFA accumulation, UCP2 level, and proton leak in liver mitochondria. Am J Physiol Endocrinol Metab 301: E1198 -E1207, 2011. First published September 13, 2011 doi:10.1152/ajpendo.00631.2010.-Obesity, a risk factor for insulin resistance, contributes to the development of type 2 diabetes and cardiovascular diseases. The relationship between increased levels of free fatty acids in the liver mitochondria, mitochondrial function, and ROS generation in rat model of obesity induced by a high-sucrose diet was not sufficiently established. We determined how the bioenergetic functions and ROS generation of the mitochondria respond to a hyperlipidemic environment. Mitochondria from sucrose-fed rats generated H2O2 at a higher rate than the control mitochondria. Adding fatty acid-free bovine serum albumin to mitochondria from sucrose-fed rats significantly reduced the rate of H2O2 generation. In contrast, adding exogenous oleic or linoleic acid exacerbated the rate of H2O2 generation in both sucrose-fed and control mitochondria, and the mitochondria from sucrose-fed rats were more sensitive than the control mitochondria. The increased rate of H2O2 generation in sucrose-fed mitochondria corresponded to decreased levels of reduced GSH and vitamin E and increased levels of Cu/Zn-SOD in the intermembrane space. There was no difference between the levels of lipid peroxidation and protein carbonylation in the two types of mitochondria. In addition to the normal activity of Mn-SOD, GPX and catalase detected an increased activity of complex II, and upregulation of UCP2 was observed in mitochondria from sucrose-fed rats, all of which may accelerate respiration rates and reduce generation of ROS. In turn, these effects may protect the mitochondria of sucrose-fed rats from oxidative stress and preserve their function and integrity. However, in whole liver these adaptive mechanisms of the mitochondria were inefficient at counteracting redox imbalances and inhibiting oxidative stress outside of the mitochondria. reactive oxygen species; free fatty acid; metabolic syndrome; mitochondrial function; oxidative stress; uncoupling protein 2 ABDOMINAL OBESITY AND HIGH LEVELS of circulating free fatty acids (FFAs) have been indicated as primary contributors to acquired insulin resistance and hypertension because they induce oxidative stress that affects insulin signaling and nitric oxide availability (30, 11). Insulin resistance leads to continuous lipolysis within adipocytes, releasing FFAs into the local circulation, where they are transported into the liver. In the liver, FFAs can be incorporated into triglycerides that accumulate and lead to nonalcoholic fatty liver disease, the primary hepatic complication of obesity (13, 57). Oxidative stress and mitochondrial dysfunction have been shown to play a critical role in the initiation and progression of fatty liver to the more serious condition of nonalcoholic steatohepatitis in obesity models (15,21,38). Several studies using an animal model of fa...