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Aims/hypothesis: The activation of NADPH oxidase has been implicated in NEFA-induced beta cell dysfunction. However, the causal role of this activation in vivo remains unclear. Here, using rodents, we investigated whether pharmacological or genetic inhibition of NADPH oxidase could prevent NEFA-induced beta cell dysfunction in vivo., Methods: Normal rats were infused for 48 h with saline or oleate with or without the NADPH oxidase inhibitor apocynin. In addition, NADPH oxidase subunit p47(phox)-null mice and wild-type littermate controls were infused with saline or oleate for 48 h. This was followed by measurement of NADPH oxidase activity, reactive oxygen species (ROS) and superoxide imaging and assessment of beta cell function in isolated islets and hyperglycaemic clamps., Results: Oleate infusion in rats increased NADPH oxidase activity, consistent with increased total but not mitochondrial superoxide in islets and impaired beta cell function in isolated islets and during hyperglycaemic clamps. Co-infusion of apocynin with oleate normalised NADPH oxidase activity and total superoxide levels and prevented beta cell dysfunction. Similarly, 48 h NEFA elevation in wild-type mice increased total but not mitochondrial superoxide and impaired beta cell function in isolated islets. p47(phox)-null mice were protected against these effects when subjected to 48 h oleate infusion. Finally, oleate increased the levels of total ROS, in both models, whereas inhibition of NADPH oxidase prevented this increase, suggesting that NADPH oxidase is the main source of ROS in this model., Conclusions/interpretation: These data show that NADPH-oxidase-derived cytosolic superoxide is increased in islets upon oleate infusion in vivo; and whole-body NADPH-oxidase inhibition decreases superoxide in concert with restoration of islet function.

Aims/hypothesis: Insulin effects reportedly involve reactive oxygen species (ROS) and oxidative stress in vitro, but skeletal muscle oxidative stress is an emerging negative regulator of insulin action following high-fat feeding. NEFA may enhance oxidative stress and insulin resistance. We investigated the acute impact of insulin with or without NEFA elevation on muscle ROS generation and insulin signalling, and the potential association with altered muscle mitochondrial function., Methods: We used hyperinsulinaemic-euglycaemic clamping, 150 min, without or with lipid infusion to modulate plasma NEFA concentration in lean rats., Results: Insulin and glucose (Ins) infusion selectively enhanced xanthine oxidase-dependent muscle ROS generation. Ins with lipid infusion (Ins+NEFA) lowered whole-body glucose disposal and muscle insulin signalling, and these effects were associated with high muscle mitochondrial ROS generation and activation of the proinflammatory nuclear factor-κB inhibitor (IκB)-nuclear factor-κB (NFκB) pathway. Antioxidant infusion prevented NEFA-induced systemic insulin resistance and changes in muscle mitochondrial ROS generation, IκB-NFκB pathway and insulin signalling. Changes in insulin sensitivity and signalling were independent of changes in mitochondrial enzyme activity and ATP production, which, in turn, were not impaired by changes in ROS generation under any condition., Conclusions/interpretation: Acute muscle insulin effects include enhanced ROS generation through xanthine oxidase. Additional NEFA elevation enhances mitochondrial ROS generation, activates IκB-NFκB and reduces insulin signalling. These alterations are not associated with acute reductions in mitochondrial enzyme activity and ATP production, and are reversed by antioxidant infusion. Thus, NEFA acutely cause systemic and muscle insulin resistance by enhancing muscle oxidative stress through mitochondrial ROS generation and IκB-NFκB activation.