Chronic exposure to elevated free fatty acids, in particular long chain saturated fatty acids, provokes endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR) and cell death in a number of cell types. The perturbations to the ER that instigate ER stress and activation of the unfolded protein response to fatty acids in hepatocytes have not been identified. The present study employed H4IIE liver cells and primary rat hepatocytes to examine the hypothesis that saturated fatty acids induce ER stress via effects on ER luminal calcium stores and reduced chaperone function. Exposure of H4IIE liver cells and primary hepatocytes to palmitate and stearate reduced thapsigargin‐sensitive calcium stores and induced multiple markers of ER stress and UPR activation over similar time courses (6h). Increased cell death was observed following 16h of exposure. Co‐incubation with oleate prevented the reduction in calcium stores, induction of ER stress markers and cell death. Since several ER molecular chaperones function in a calcium‐dependent manner, the effects of chemical chaperones on palmitate‐mediated ER stress and cell death was examined. The chemical chaperones, 4‐phenyl butyric acid and taurine‐conjugated ursodeoxycholic acid, reduced palmitate‐mediated ER stress and cell death. These data suggest that reduced ER luminal calcium and protein folding capacity contribute to long chain saturated fatty acid‐mediated perturbations in the ER.
Elevated fatty acids play a role in non‐alcoholic fatty liver disease (NAFLD). Disruption of endoplasmic reticulum (ER) homeostasis, or ER stress, is a characteristic feature of NAFLD. Elevated fatty acids, in particular saturated fatty acids, induce ER stress in numerous cell types including hepatocytes. However, the specific mechanisms that link fatty acids to ER stress in the liver are poorly understood. ER stress is the result of an imbalance between the protein load delivered to the ER from cellular protein synthesis, and the capacity of the ER to process, transport and/or degrade the client protein load. In this study, we examined the hypothesis that saturated fatty acid‐mediated ER stress resulted from increased cellular protein synthesis. H4IIE liver cells (n=3) were treated with palmitate (250µM), a saturated fatty acid that induces ER stress, oleate (250µM), an unsaturated fatty acid that does not induce ER stress, or a combination of both palmitate and oleate that does not induce ER stress. Protein synthesis was measured utilizing mass spectrometry based analysis of deuterated water incorporation into total cellular proteins and polysome profiling. Protein synthesis as determined by both techniques was not significantly different among groups. These data suggest that increased cellular protein synthesis does not mediate palmitate‐induced ER stress in H4IIE liver cells. Grant Funding Source : Supported by National Institutes of Health, DK‐072017, AG031829‐01
Fructose produces hepatic insulin resistance in humans and animals. We have proposed that the selective metabolism of fructose by the liver and the unique ability of fructose‐1‐phosphate to stimulate hepatic glucose uptake can, under conditions of elevated fructose delivery, inflict a metabolic insult that is localized to the hepatocyte. The present study was designed to identify potential cellular effectors of this insult. Primary hepatocytes were incubated with 8 mM glucose and 0.12% inulin (G, n=6) or 8 mM glucose, 0.12% inulin, and 28 mU of inulinase (GF, n=6) in the presence or absence of insulin for 0, 2, or 4h. GF produced fructose concentrations of ~0.7 mM over the 4h experiment. GF induced phosphorylation of MKK7 and JNK, phosphorylation of serine 307 on IRS‐1, and reduced tyrosine phosphorylation of IRS‐1 and ‐2. GF increased ceramide levels and reactive oxygen species (ROS); however inhibitors of ceramide synthesis or ROS accumulation did not prevent GF‐mediated changes in MKK7, JNK or IRS proteins. Low concentrations of methylglyoxal recapitulated the GF‐induced changes in MKK7, JNK and IRS proteins. We hypothesize that GF‐mediated changes in stress signaling involves methylglyoxal in primary hepatocytes.
The underlying causes of nonalcoholic fatty liver disease (NAFLD) are unclear, although recent evidence has implicated the endoplasmic reticulum (ER) in both the development of steatosis and progression to nonalcoholic steatohepatitis. Disruption of ER homeostasis, often termed "ER stress," has been observed in liver and adipose tissue of humans with NAFLD and/or obesity. Importantly, the signaling pathway activated by disruption of ER homeostasis, the unfolded protein response, has been linked to lipid biosynthesis, insulin action, inflammation, and apoptosis. Therefore, understanding the mechanisms that disrupt ER homeostasis in NAFLD and the role of ER-mediated signaling have become topics of intense investigation. The present review will examine the ER and the unfolded protein response in the context of NAFLD. Antioxid. Redox Signal. 15, 505–521.
Free fatty acid‐induced toxicity is a characteristic feature of obesity, type 2 diabetes and non‐alcoholic fatty liver disease. Long chain saturated fatty acids, including palmitate and stearate, are particularly cytotoxic. Diversion of saturated fatty acids into the triglyceride pool reduces saturated fatty acid‐induced cytotoxicity in CHO cells and β‐cells. LXR agonists and downregulation of Phosphatase and Tensin Homologue Deleted on Chromosome 10 (PTEN) promote triglyceride storage in the liver. In the present study we examined the role of LXR agonists and PTEN in saturated fatty acid‐induced cytotoxicity in HepG2 and H4IIE liver cells. The presence of the LXR agonists, TO901317 or GW3965, increased liver cell triglyceride stores and reduced palmitate‐ (250 μM) and stearate‐ (250 μM) mediated ER stress and apoptosis in both cell types. Palmitate and stearate increased the expression of PTEN, whereas the unsaturated fatty acids, oleate and linoleate (250 μM), reduced PTEN expression in both cell types. SiRNA‐mediated knockdown of PTEN did not increase liver cell triglyceride stores nor did it reduce palmitate‐ or stearate‐induced ER stress or apoptosis. These results suggest that LXR‐mediated triglyceride storage protects against saturated fatty acid‐induced cytotoxicity in these liver cell models.
High-fat meal (HFM) consumption may induce transient postprandial atherogenic responses, including impairment of vascular endothelial function, in individuals with overweight/obesity. Red beetroot juice (RBJ) may modulate endothelial function and other measures of cardiometabolic health.
