Molecular mediators of hepatic steatosis and liver injury

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap-/- livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic/knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.

AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (alpha) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase alpha or beta. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood.