Differential Lipotoxic Effects of Palmitate and Oleate in Activated Human Hepatic Stellate Cells and Epithelial Hepatoma Cells.

BACKGROUND/AIMS: Nonalcoholic fatty liver disease (NAFLD) progression to fibrosis, cirrhosis and hepatocellular carcinoma, alters the cellular composition of this organ. During late-stage NAFLD, fibrotic and possibly cancerous cells can proliferate and, like normal hepatocytes, are exposed to high concentrations of fatty acids from both surrounding tissue and circulating lipid sources. We hypothesized that primary human activated hepatic stellate cells and epithelial hepatoma (HepG2) cells respond differently to lipotoxic conditions, and investigated the mechanisms involved. METHODS: Primary activated hepatic stellate cells and HepG2 cells were exposed to pathophysiological concentrations of fatty acids and comparative studies of lipid metabolic and stress response pathways were performed. RESULTS: Both cell types remained proliferative during exposure to a combination of palmitate plus oleate reflective of the general saturated versus unsaturated fatty acid composition of western diets. However, exposure to either high palmitate or high oleate alone induced cytotoxicity in activated stellate cells, while only palmitate caused cytotoxicity in HepG2 cells. mRNA microarray and biochemical comparisons revealed that stellate cells stored markedly less fatty acids as neutral lipids, and had reduced capacity for beta-oxidation. Similar to previous observations in HepG2 cells, palmitate, but not oleate, induced ER stress and actin stress fiber formation in activated stellate cells. In contrast, oleate, but not palmitate, induced the inflammatory signal TXNIP, decreased cytoskeleton proteins, and decreased cell polarity preceding cell death in activated stellate cells. CONCLUSIONS: Palmitate-induced lipotoxicity was associated with ER stress pathways in both primary activated hepatic stellate cells and epithelial hepatoma cells, whereas high oleate caused lipotoxicity only in activated stellate cells, possibly through a distinct mechanism involving disruption of cytoskeleton components. This may have implications for optimal dietary fatty acid compositions during various stages of NAFLD.

Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

Niacin promotes revascularization and recovery of limb function in diet-induced obese mice with peripheral ischemia.

Niacin can reduce vascular disease risk in individuals with metabolic syndrome, but in light of recent large randomized controlled trials outcomes, its biological actions and clinical utility remain controversial. Niacin can improve endothelial function, vascular inflammation, and vascular regeneration, independent of correcting dyslipidemia, in various lean rodent models of vascular injury. Here, we tested whether niacin could directly improve endothelial cell angiogenic function during combined exposure to excess fatty acids and hypoxia, and whether intervention with niacin during continued feeding of western diet could improve revascularization and functional recovery in obese, hyperlipidemic mice with peripheral ischemia. Treatment with niacin (10 µmol/L) increased human microvascular endothelial cell angiogenic function during exposure to high fatty acids and hypoxia (2% oxygen), as determined by tube formation on Matrigel. To assess revascularization in vivo, we used western diet-induced obese mice with unilateral hind limb femoral artery ligation and excision. Treatment for 14 days postinjury with once daily i.p. injections of a low dose of niacin (50 mg/kg) improved recovery of hind limb use, in association with enhanced revascularization and decreased inflammation of the tibialis anterior muscle. These effects were concomitant with decreased plasma triglycerides, but not increased plasma apoAI. Thus, niacin improves endothelial tube formation under lipotoxic and hypoxic conditions, and moreover, promotes revascularization and functional hind limb recovery following ischemic injury in diet-induced obese mice with hyperlipidemia. These data may have implications for niacin therapy in the treatment of peripheral ischemic vascular disease associated with metabolic syndrome.

Elongation Factor 1A-1 Is a Mediator of Hepatocyte Lipotoxicity Partly through Its Canonical Function in Protein Synthesis.

Elongation factor 1A-1 (eEF1A-1) has non-canonical functions in regulation of the actin cytoskeleton and apoptosis. It was previously identified through a promoter-trap screen as a mediator of fatty acid-induced cell death (lipotoxicity), and was found to participate in this process downstream of ER stress. Since ER stress is implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD), we investigated the mechanism of action of eEF1A-1 in hepatocyte lipotoxicity. HepG2 cells were exposed to excess fatty acids, followed by assessments of ER stress, subcellular localization of eEF1A-1, and cell death. A specific inhibitor of eEF1A-1 elongation activity, didemnin B, was used to determine whether its function in protein synthesis is involved in lipotoxicity. Within 6 h, eEF1A-1 protein was modestly induced by high palmitate, and partially re-localized from its predominant location at the ER to polymerized actin at the cell periphery. This early induction and subcellular redistribution of eEF1A-1 coincided with the onset of ER stress, and was later followed by cell death. Didemnin B did not prevent the initiation of ER stress by high palmitate, as indicated by eIF2α phosphorylation. However, consistent with sustained inhibition of eEF1A-1-dependent elongation activity, didemnin B prevented the recovery of protein synthesis and increase in GRP78 protein that are normally associated with later phases of the response to ongoing ER stress. This resulted in decreased palmitate-induced cell death. Our data implicate eEF1A-1, and its function in protein synthesis, in hepatocyte lipotoxicity.

Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity.

OBJECTIVE: Niacin (nicotinic acid) as a monotherapy can reduce vascular disease risk, but its mechanism of action remains controversial, and may not be dependent on systemic lipid modifying effects. Niacin has recently been shown to improve endothelial function and vascular regeneration, independent of correcting dyslipidemia, in rodent models of vascular injury and metabolic disease. As a potential biosynthetic precursor for NAD(+), niacin could elicit these vascular benefits through NAD(+)-dependent, sirtuin (SIRT) mediated responses. Alternatively, niacin may act through its receptor, GPR109A, to promote endothelial function, though endothelial cells are not known to express this receptor. We hypothesized that niacin directly improves endothelial cell function during exposure to lipotoxic conditions and sought to determine the potential mechanism(s) involved. METHODS AND RESULTS: Angiogenic function in excess palmitate was assessed by tube formation following treatment of human microvascular endothelial cells (HMVEC) with either a relatively low concentration of niacin (10 µM), or nicotinamide mononucleotide (NMN) (1 µM), a direct NAD(+) precursor. Although both niacin and NMN improved HMVEC tube formation during palmitate overload, only NMN increased cellular NAD(+) and SIRT1 activity. We further observed that HMVEC express GRP109A. Activation of this receptor with either acifran or MK-1903 recapitulated niacin-induced improvements in HMVEC tube formation, while GPR109A siRNA diminished the effect of niacin. CONCLUSION: Niacin, at a low concentration, improves HMVEC angiogenic function under lipotoxic conditions, likely independent of NAD(+) biosynthesis and SIRT1 activation, but rather through niacin receptor activation.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity.

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.