Nonalcoholic fatty liver disease (NAFLD) is a multifactorial disease [¹,²] that illustrates a variety of symptoms, ranging from mild steatosis, nonalcoholic steatohepatitis to cirrhosis in the liver. Although it affects millions of people all over the world, the etiology of NAFLD is still unknown. However, hepatic inflammation and degeneration induced by the deposition of lipid droplets in the organ is considered as one of the major reasons of the disease [^1-3]. In particular, certain saturated fatty acids (FA) such as palmitic acid can induce endoplasmic reticulum (ER) stress and apoptosis in rat and human liver cell lines [^4-8], leading to inflammation and/or degeneration in the liver. This hypothesis is further demonstrated by the fact that ER stress and apoptosis could be induced by palmitic acid in both primary cells and cell lines derived from mice and rats [⁹,¹⁰].

Different fatty acids have different effects on ER stress. Palmitic acid is an effective inducer of ER stress [¹¹][⁴]. Palmitic acid stimulates the synthesis of ceramides and increases reactive oxygen species [⁴][¹²], either of which may induce ER stress [^12-14]. Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and leads to apoptosis in mouse 3T3-L1 and rat primary preadipocytes [⁹].

We hypothesized that the cytoprotection provided by α-linolenic acid was a common function of rat primary hepatocytes and would be effective with clinically-relevant palmitic acid lipotoxicity. We have previously proved that α-linolenic acid protects against endoplasmic reticulum stress-mediated apoptosis of stearic acid lipotoxicity [¹⁵]. In this paper, we report that: (1) The characteristics of palmitic acid-mediated ER stress and apoptosis in primary hepatocytes; (2) α-linolenic acid could provide protection against the cell death induced by palmitic acid; (3) Take the role of GRP78, GRP94 expression and induction of CHOP into consideration, the beneficial effects were mediated via modification of the ER stress process with specific attention.

The present study was undertaken to elucidate the hypotheses that palmitic acid would induce ER stress and then cell death in liver cells, and that α-linolenic acid would inhibit these outcomes. Our study has three main findings. First, we have shown that the effects of a saturated and an unsaturated fatty acid, singly or in combination, upon induction of cell death, cell apoptosis, caspase-3 activity, ER stress in primary rat hepatocytes. Secondly, we have also illustrated the protein expression of three ER stress-associated genes in response to fatty acids treatment. The delivery and accumulation of lipids in non-adipose tissues leads to cellular dysfunction and death. This phenomenon, termed lipotoxicity, has been demonstrated in the pathogenesis of diabetes, cardiac failure and NAFLD [^20-22]. Studies has found that damage of ER homeostasis and activation of the UPR in murine models of cardiac dysfunction, obesity and NAFLD [²⁰,²³,²⁴].

Increased long chain saturated fatty acids induce ER stress, activate the UPR and promote cell death in a great amount of cell types, such as hepatocytes [²⁵][²¹][⁴][²⁶,²⁷]. Thus, disruption in ER function seems lead to the pathogenesis of some diseases and to cellular impairments coupled with lipotoxicity. This was supported by our observations. Previously, we found that palmitic acid causes obvious cell death in primary rat hepatocytes. To elucidate the underlying mechanism of these effects, we found that the palmitic acid causes an obvious degree of ER stress in primary rat hepatocytes. Our results demonstrated that the ER stress response contributes to palmitic acid lipotoxicity. In addition, the studies showed that α-linolenic acid protects primary rat hepatocytes against palmitic acid lipotoxicity via reducing ER stress and apoptosis. Comparing with our previous study about stearic acid, we prove that 150 μmol/l α-linolenic acid provides very little benefits, which was presumed as the result of palmitic acid's stronger ability to elicited ER stress [¹⁵]. Furthermore, α-linolenic acid can reduce cellular dysfunction and apoptosis caused by tunicamycin. Tunicamycin, a well-known ER stress inducer, leads to apoptosis in a number of cells, including intestinal epithelial cells, renal cells and liver cells [^28-30]. The mechanism on the basis of the cell necrosis induced by tunicamycin has not been clarified. These observations, together with our findings, suggest that α-linolenic acid protect primary rat hepatocytes through alleviation of tunicamycin-induced apoptosis.

The previous study sought to determine whether the ER stress and cell death in hepatocytes contributed to cell death in hepatocytes and to establish a link between these two [³¹]. Chop is among the best characterized of the UPR-regulated proapoptotic proteins [³²]. Chop plays a role in disruption of ER homeostasis and apoptosis induced by long-chain saturated fatty acids. In several cell types, including liver, co-supplementation of oleate and palmitate reduces palmitate-mediated ER stress and apoptosis [⁴][³³,³⁴]. For this purpose, the ability of long chain saturated fatty acids to activate the ER-associated caspase-3 was examined.

Our work sheds new light on the mechanisms behind that ER stress produced by palmitic acid in primary rat hepatocytes can be considerably reduced by α-linolenic acid, an unsaturated fatty acid. We also manifest that an unsaturated fatty acid involves a reduction of ER stress which result in protection. Moreover, we examined the effects of a reduction in the raised levels of caspase-3 and CHOP coupled with palmitic acid. However, the results of our investigation seems to exclude a protective mechanism regulated by GRP78 as α-linolenic acid did not significantly affect levels of this chaperone molecule whereas levels of CHOP were substantialy reduced. In short, we concluded that α-linolenic acid may provide a useful strategy to avoid the lipotoxicity of dietary palmitic acid and nutrient overload accompanied with obesity and NAFLD.

The authors declare that they have no competing interests.

YZ conceived, designed and coordinated the work, as well as prepared the manuscript. LD was involved in the co-design of the work as well as the draft of the manuscript. XY, HS and JB carried out analytical work and contributed in drafting the manuscript. All authors read and approved the final manuscript.

We thank laboratory of Tissue Engineering, the Fourth Military Medical University, for guidance of primary cell culture.