Infusion of a lipid emulsion modulates AMPK and related proteins in rat liver, muscle, and adipose tissues.

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate-buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCbeta, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARgamma-co-activator 1alpha (PGC1alpha) protein levels were also increased in LE-treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCalpha, PPARalpha, AdipoR1, AdipoR2, and sterol regulatory element-binding protein-1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.

Nutritional lipid-induced oxidative stress leads to mitochondrial dysfunction followed by necrotic death in FaO hepatocytes.

OBJECTIVE: Mitochondrial dysfunction and hepatocyte cell death have been reported in fatty liver and non-alcoholic steatohepatitis. Our aim in this study was to evaluate whether direct exposure of hepatocytes to extracellular fat could facilitate such deleterious effects. METHODS: FaO hepatic cells treated with fat was used as an in vitro model for steatosis. FaO hepatocytes were exposed to 0.1% triacylglycerols using commercially available lipid emulsion (LE) for various periods and studied for production of reactive oxygen species (ROS), mitochondrial function, and cell death parameters. To study the type of cell death, high-mobility group box chromosomal protein 1cellular levels, DNA fragmentation, and caspase activity were evaluated. RESULTS: Cells incubated with LE for 6 h exhibited a marked increase in the production of intracellular ROS. Using treatments with peroxisome proliferator-activated receptor activators, mitochondrial electron-transfer chain inhibitor, and different sources of LE that did or did not contain medium-chain triacylglycerols, the mitochondria were found to be the source of ROS. LE treatment resulted in phosphorylation of adenosine monophosphate-activated protein kinase, accompanied by a decrease in adenosine triphosphate levels. Changes in intracellular ROS and energy levels were followed by cell death. FaO hepatocytes showed a significant reduction in high-mobility group box chromosomal protein-1 and little DNA fragmentation. Incubation with LE for 24 h did not change caspase-3 activity, indicating that hepatocyte death was necrotic. The antioxidant N-acetylcysteine was able to attenuate the changes in intracellular energy levels and ROS levels and to prevent cell death after exposure to LE. CONCLUSION: These results suggest that exposure of FaO cells to LE leads to an increase in mitochondrial ROS production and a decrease in cellular energy levels followed by necrotic cell death.

Common inhibitory serine sites phosphorylated by IRS-1 kinases, triggered by insulin and inducers of insulin resistance.

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IkappaB kinase beta (IKKbeta) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKKbeta shares a repertoire of seven potential target sites on IRS-1 with protein kinase C zeta (PKCzeta), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKKbeta and PKCzeta when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKCzeta overlap with IRS kinases triggered by inducers of insulin resistance, such as IKKbeta, to phosphorylate IRS-1 on common Ser sites.

Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes.

This study was designed to evaluate the effects of triacylglycerol (TG) on nitric oxide (NO) production, expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) and variables related to oxidative stress in rat isolated hepatocytes. Hepatocytes were isolated and exposed to TG in the form of a lipid emulsion (0.01-0.1% LE). Exposure to LE dose dependently decreased nitrite levels. Nitrite levels were inhibited 67% and intracellular reactive oxygen species (ROS) levels were increased 250% at 0.1% LE. The decline in nitrite levels was accompanied by 37 and 67% reductions in iNOS and eNOS expressions, respectively. To evaluate whether the increased oxidative stress inhibited NOS synthesis, cells were treated for 48 h with rotenone (a mitochondrial complex 1 inhibitor) or buthionine sulfoximine (a glutathione synthesis inhibitor). Both compounds elevated ROS production, which was followed by inhibition of nitrite production. To determine whether there is an association between LE-mediated ROS production and the inhibition of NO synthesis by the LE, hepatocytes were treated with antioxidants. N-Acetyl-l-cysteine (NAC), ascorbate, and resveratrol attenuated the reduction of nitrite levels due to LE alone. NAC inhibited the reductions in eNOS and iNOS transcription and protein levels. Nuclear factor-kappaB (NF-kappaB), one of the transcription factors involved in eNOS and iNOS transcriptional regulation, was decreased 15% in the nucleus by LE treatment. These results suggest that TG reduces nitrite production by elevating intracellular ROS levels (prolonged oxidative stress), and the downregulation of NOS enzymes may occur at least in part via the NFkappaB pathway.