Abstract
Abnormal dietary intake of macronutrients is implicated in the development of obesity and fatty liver disease. Steatosis develops in cultured hepatocytes exposed to medium containing either a high concentration of long chain free fatty acids (HFFA) or medium deficient in methionine and choline (MCD). This study examined the mitochondrial reactive oxygen species (ROS)-dependent regulation of the phosphoinositol (PI) 3-kinase pathway in steatosis induced by exposure of AML-12 mouse hepatocytes to MCD or HFFA medium. Exposure to either MCD or HFFA medium resulted in increased production of superoxide anions and H(2)O(2), transduction of the PI 3-kinase pathway and steatosis. Inhibition of PI 3-kinase with LY294002 prevented steatosis. Pharmacologically inhibiting electron transport chain complex III production of ROS prevented activation of PI 3-kinase during macronutrient perturbation, whereas pharmacologically promoting electron transport chain complex III ROS production activated PI 3-kinase independent of nutrient input. The data suggest that H(2)O(2) is the ROS species involved in signal transduction; promoting the rapid conversion of superoxide to H(2)O(2) does not inhibit PI 3-kinase pathway activation during nutrient perturbation, and exogenous H(2)O(2) activates it independent of nutrient input. In addition to transducing PI 3-kinase, the ROS-dependent signal cascade amplifies the PI 3-kinase signal by maintaining phosphatase and tensin homolog in its inactive phosphorylated state. Knockdown of phosphatase and tensin homolog by small interfering RNA independently activated the PI 3-kinase pathway. Our findings suggest a common path for response to altered nutrition involving mitochondrial ROS-dependent PI 3-kinase pathway regulation, leading to steatosis.
Highlights
Experimental NASH can be produced in laboratory rodents by dietary manipulation, including both restricted intake (i.e. methionine-choline deficient (MCD) diet) [5] and excess intake [6, 7]
The overall aim of the present study was to determine the signaling mechanisms involved in the development of hepatocyte steatosis resulting from altered macronutrient input, the role of mitochondrial reactive oxygen species (ROS) in the downstream activation and modulation of the PI 3-kinase pathway
The present study examined the signaling mechanisms involved in the development of hepatocyte steatosis resulting from altered macronutrient input to hepatocytes in culture and resulted in several important findings
Summary
Experimental NASH can be produced in laboratory rodents by dietary manipulation, including both restricted intake (i.e. methionine-choline deficient (MCD) diet) [5] and excess intake (i.e. various high fat diets) [6, 7]. To further explore the possibility that the MCD diet represented a signal leading to steatosis and other expressions of experimental NASH, including the overexpression of OPN, we examined the effects of substituting medium deficient in methionine and choline (MCD medium) on AML-12 mouse hepatocytes in vitro [12]. We hypothesized that nutrient perturbation in the form of macronutrient deprivation (MCD medium) or excess (HFFA medium) activates and modulates the PI 3-kinase cell signaling pathway to produce steatosis. Showing that perturbation of macronutrient input to hepatocytes, either deprivation or excess input, produces steatosis by mechanisms that are dependent upon mitochondrial ROS regulating the PI 3-kinase pathway would provide evidence for a novel link between abnormal dietary intake and NASH
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