Yuren Wei

Linking Endoplasmic Reticulum Stress to Cell Death in Hepatocytes: Roles of C/EBP Homologous Protein and Chemical Chaperones in Palmitate-Mediated Cell Death

Prolonged endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) have been linked to apoptosis via several mechanisms, including increased expression of C/EBP homologous protein (Chop). Increased long-chain fatty acids, in particular saturated fatty acids, induce ER stress, Chop expression, and apoptosis in liver cells. The first aim of the present study was to determine the role of Chop in lipid-induced hepatocyte cell death and liver injury induced by a methionine-choline-deficient diet. Albumin-bound palmitate increased Chop gene and protein expression in a dose-dependent fashion in H4IIE liver cells. siRNA-mediated silencing of Chop in H4IIE liver cells reduced thapsigargin-mediated cell death by approximately 40% and delayed palmitate-mediated cell death, but only at high concentrations of palmitate (400-500 microM). Similar results were observed in primary hepatocytes isolated from Chop-knockout mice. Indices of liver injury were also not reduced in Chop-knockout mice provided a methionine-choline-deficient diet. To ascertain whether ER stress was linked to palmitate-induced cell death, primary hepatocytes were incubated in the absence or presence of the chemical chaperones taurine-conjugated ursodeoxycholic acid or 4-phenylbutyric acid. The presence of either of these chemical chaperones protected liver cells from palmitate-mediated ER stress and cell death, in part, via inhibition of JNK activation. These data suggest that ER stress is linked to palmitate-mediated cell death via mechanisms that include JNK activation.

Saturated fatty acid-mediated endoplasmic reticulum stress and apoptosis are augmented by trans-10, cis-12-conjugated linoleic acid in liver cells

Lipid accumulation in non-adipose tissues leads to cell dysfunction and apoptosis, a phenomenon known as lipotoxicity. Recent evidence suggests that lipotoxicity in hepatocytes involves endoplasmic reticulum (ER) stress and c-Jun NH2-terminal kinase-mediated apoptosis. The present study examined (1) the dose–response and time course characteristics of fatty acid-mediated ER stress and apoptosis in H4IIE liver cells; (2) whether saturated fatty acid-induced apoptosis involved the ER-associated caspase-12; and (3) whether trans-10, cis-12-conjugated linoleic acid, an inhibitor of stearoyl-CoA desaturase, influenced fatty acid-mediated ER stress and apoptosis. Saturated fatty acids induced ER stress in a dose-dependent manner with a time course that was delayed relative to chemical-induction of ER stress. Saturated fatty acids increased caspase-9 and caspase-3 activity, however increased caspase-12 activity was not observed. Inhibition of stearoyl-CoA desaturase, using conjugated linoleic acid (trans-10, cis-12), augmented saturated fatty acid-induced ER stress and apoptosis. These data suggest that saturated fatty acids induce ER stress and apoptosis at physiologic concentrations and with a relatively rapid time course. It would appear that saturated fatty acid-mediated apoptosis occurs independently of caspase-12 activation. Since conjugated linoleic acid inhibited stearoyl-CoA desaturase activity, it is hypothesized that saturation, per se, plays a role in lipotoxicity in liver cells.

Experimental evidence for therapeutic potential of taurine in the treatment of nonalcoholic fatty liver disease

The incidence of obesity is now at epidemic proportions and has resulted in the emergence of nonalcoholic fatty liver disease (NAFLD) as a common metabolic disorder that can lead to liver injury and cirrhosis. Excess sucrose and long-chain saturated fatty acids in the diet may play a role in the development and progression of NAFLD. One factor linking sucrose and saturated fatty acids to liver damage is dysfunction of the endoplasmic reticulum (ER). Although there is currently no proven, effective therapy for NAFLD, the amino sulfonic acid taurine is protective against various metabolic disturbances, including alcohol-induced liver damage. The present study was undertaken to evaluate the therapeutic potential of taurine to serve as a preventative treatment for diet-induced NAFLD. We report that taurine significantly mitigated palmitate-mediated caspase-3 activity, cell death, ER stress, and oxidative stress in H4IIE liver cells and primary hepatocytes. In rats fed a high-sucrose diet, dietary taurine supplementation significantly reduced hepatic lipid accumulation, liver injury, inflammation, plasma triglycerides, and insulin levels. The high-sucrose diet resulted in an induction of multiple components of the unfolded protein response in the liver consistent with ER stress, which was ameliorated by taurine supplementation. Treatment of mice with the ER stress-inducing agent tunicamycin resulted in liver injury, unfolded protein response induction, and hepatic lipid accumulation that was significantly ameliorated by dietary supplementation with taurine. Our results indicate that dietary supplementation with taurine offers significant potential as a preventative treatment for NAFLD.

Menhaden oil prevents but does not reverse sucrose-induced insulin resistance in rats

Although fish oil supplementation may prevent the onset of diet-induced insulin resistance in rats, it appears to worsen glycemic control in humans with existing insulin resistance. In the present study, the euglycemic, hyperinsulinemic (4× basal) clamp technique with [3-3H]glucose and 2-deoxy-[1-14C]glucose was used to directly compare the ability of fish oil to prevent and reverse sucrose-induced insulin resistance. In study 1 (prevention study), male Wistar rats were fed a purified high-starch diet (68% of total energy), high-sucrose diet (68% of total energy), or high-sucrose diet in which 6% of the fat content was replaced by menhaden oil for 5 wk. In study 2 (reversal study), animals were fed the high-starch or high-sucrose diets for 5 wk and then the sucrose animals were assigned to one of the following groups for an additional 5 wk: high starch, high sucrose, or high sucrose with 6% menhaden oil. Rats fed the high-starch diet for 10 wk served as controls. In study 3 (2nd reversal study), animals followed a similar diet protocol as in study 2; however, the reversal period was extended to 15 wk. In study 1, the presence of the fish oil in the high-sucrose diet prevented the development of insulin resistance. Glucose infusion rates (GIR, mg ⋅ kg-1 ⋅ min-1) were 17.0 ± 0.9 in starch, 10.6 ± 1.7 in sucrose, and 15.1 ± 1.5 in sucrose with fish oil animals. However, in study 2, this same diet was unable to reverse sucrose-induced insulin resistance (GIR, 16.7 ± 1.4 in starch, 7.1 ± 1.5 in sucrose, and 4.8 ± 0.9 in sucrose with fish oil animals). Sucrose-induced insulin resistance was reversed in rats that were switched back to the starch diet (GIR, 18.6 ± 3.0). Results from study 3 were similar to those observed in study 2. In summary, fish oil was effective in preventing diet-induced insulin resistance but not able to reverse it. A preexisting insulin-resistant environment interferes with the positive effects of menhaden oil on insulin action.

Rapamycin Inhibits Postprandial-Mediated X-Box-Binding Protein-1 Splicing in Rat Liver

Recent studies have linked the unfolded protein response (UPR), in particular the inositol-requiring, endoplasmic reticulum-to-nucleus signaling protein 1alpha (IRE1alpha)-X-box-binding protein-1 (XBP1) branch of the UPR, to the regulation of lipogenesis and hepatic steatosis. In this study, we examined the hypothesis that the postprandial environment can activate the IRE1alpha-XBP1 branch of the UPR in the liver via a mammalian target of rapamycin complex 1 (mTORC1)-dependent mechanism. Toward this end, rats were fed a high-carbohydrate diet (68% of energy from corn starch) for 3 h in the absence or presence of rapamycin (intraperitoneal injection of 1 mg/kg) and liver tissue was taken 1 or 7 h following the feeding period. Feeding activated the mTORC1 pathway and IRE1alpha, induced XBP1 splicing, and increased the expression of XBP1 target genes and lipogenic genes in the liver. The presence of rapamycin prevented the activation of mTORC1 and IRE1alpha, XBP1 splicing, and the increased expression of XBP1 target genes and lipogenic genes. Rapamycin also prevented the feeding-induced increase in nuclear sterol regulatory element binding protein 1c. These data suggest that the postprandial environment promotes activation of the IRE1-XBP1 branch of the UPR in the liver. This activation appears to be mediated in part by mTORC1.

Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance

The initial response of liver cells to insulin is mediated through exocytosis of Cl− channel‐containing vesicles and a subsequent opening of plasma membrane Cl− channels. Intracellular accumulation of fatty acids leads to profound defects in metabolism, and is closely associated with insulin resistance. It is not known whether the activity of Cl− channels is altered in insulin resistance and by which mechanisms. We studied the effects of fatty acid accumulation on Cl− channel opening in a model liver cell line. Overnight treatment with amiodarone increased the fat content by ∼2‐fold, and the rates of gluconeogenesis by ∼5‐fold. The ability of insulin to suppress gluconeogenesis was markedly reduced indicating that amiodarone treatment induces insulin resistance. Western blot analysis showed that these cells express the same number of insulin receptors as control cells. However, insulin failed to activate exocytosis and Cl− channel opening. These inhibitory effects were mimicked in control cells by exposures to arachidonic acid (15 μm). Further studies demonstrated that fatty acids stimulate the PKC activity, and inhibition of PKC partially restored exocytosis and Cl− channel opening in insulin‐resistant cells. Accordingly, activation of PKC with PMA in control cells potently inhibited the insulin responses. These results suggest that stimulation of PKC activity in insulin resistance contributes to the inhibition of cellular responses to insulin in liver cells.

Fuzhuan tea consumption imparts hepatoprotective effects and alters intestinal microbiota in high saturated fat diet-fed rats.

SCOPE Nonalcoholic fatty liver disease is an obesity-related disorder characterized by lipid infiltration of the liver. Management is limited to lifestyle modifications, highlighting the need for alternative therapeutic options. The objective of this study was to examine if fermented Fuzhuan tea prevents metabolic impairments associated with development of hepatic steatosis. METHODS AND RESULTS Rats consumed control (CON) or high saturated fat (SAT) diets with or without Fuzhuan tea for 8 weeks. Outcomes included enzymatic and gene expression measures of metabolic dysregulation in liver and adipose tissue. Pyrosequencing was used to assess intestinal microbiota adaptations. Fuzhuan tea prevented diet-induced inflammation in the liver. Liver triglycerides of ∼18 mg/g were observed in SAT-fed animals, but remained similar to CON diet levels (∼12 mg/g) when supplemented with Fuzhuan tea. In adipose tissue, tea treatment prevented SAT-induced inflammation and reduced plasma leptin approximately twofold. Fuzhuan tea also altered intestinal function and was associated with a threefold increase in two Lactobacillus spp. CONCLUSIONS These data suggest that Fuzhuan tea protects against liver and adipose tissue stress induced by a high SAT diet and positively influences intestinal function. Further investigation of the molecular targets of Fuzhuan tea is warranted.

Trehalose supplementation reduces hepatic endoplasmic reticulum stress and inflammatory signaling in old mice

The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging and cellular damage. Quality control systems, such as the unfolded protein response (UPR), inflammatory signaling and protein degradation, mitigate the residence time of damaged proteins. In the present study, we have examined the UPR and inflammatory signaling in the liver of young (~6 months) and old (~28 months) mice (n=8/group), and the ability of trehalose, a compound linked to increased protein stability and autophagy, to counteract age-induced effects on these systems. When used, trehalose was provided for 4 weeks in the drinking water immediately prior to sacrifice (n=7/group). Livers from old mice were characterized by activation of the UPR, increased inflammatory signaling and indices of liver injury. Trehalose treatment reduced the activation of the UPR and inflammatory signaling, and reduced liver injury. Reductions in proteins involved in autophagy and proteasome activity observed in old mice were restored following trehalose treatment. The autophagy marker, LC3B-II, was increased in old mice treated with trehalose. Metabolomics analyses demonstrated that reductions in hexosamine biosynthetic pathway metabolites and nicotinamide in old mice were restored following trehalose treatment. Trehalose appears to be an effective intervention to reduce age-associated liver injury and mitigate the need for activation of quality control systems that respond to disruption of proteostasis.

Saturated Fatty Acid-induced cytotoxicity in liver cells does not involve phosphatase and tensin homologue deleted on chromosome 10.

Liver specific deletion of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) induces steatosis and hypersensitivity to insulin. Saturated fatty acids, which induce endoplasmic reticulum stress and cell death, appear to increase PTEN, whereas unsaturated fatty acids which do not induce endoplasmic reticulum stress or cell death reduce this protein. In the present study, the role of PTEN in saturated fatty acid-induced cytotoxicity was examined in H4IIE and HepG2 liver cells. Palmitate and stearate increased the expression of PTEN, whereas the unsaturated fatty acids, oleate and linoleate, reduced PTEN expression in both cell types. SiRNA-mediated knockdown of PTEN did not increase liver cell triglyceride stores or reduce palmitate- or stearate-mediated ER stress or apoptosis. These results suggest that PTEN does not play a significant role in saturated fatty acid-induced cytotoxicity in these liver cell models and in the absence of insulin.

Short-term changes in diet composition do not affect in vivo hepatic protein synthesis in rats

Protein synthesis is critical to protein homeostasis (proteostasis), and modifications in protein synthesis influence lifespan and the development of comorbidities associated with obesity. In the present study, we examined the acute response of liver protein synthesis to either high-fat or high-sucrose diets in order to elucidate nutrient-mediated regulation of hepatic protein synthesis in the absence of body fat accumulation. Total and endoplasmic reticulum-associated protein syntheses were assessed by use of the stable isotope, deuterium oxide (2H2O), in rats provided a control diet or diets enriched in polyunsaturated fat, saturated fat, or sucrose for 2, 4, or 7 days. The three experimental diets increased hepatic triglycerides 46-91% on day 7 and fasting insulin levels 83-117% on day 7, but did not result in differences in body weight when compared with control ( n = 6/diet/time). The fraction of newly synthesized proteins in total liver lysates and microsomes was not significantly different among dietary groups ( n = 3/diet/time). To determine whether the experimental diets provoked a transcriptional response to enhance the capacity for protein synthesis, we also measured a panel of genes linked to amino acid transport, synthesis, and processing. There were no significant differences in any of the genes measured among groups. Therefore, dietary treatments that have been linked to impaired proteostasis and that promote hepatic steatosis and insulin resistance, did not result in significant changes in total or ER-associated protein synthesis in the liver over a 7-day period.