The endoplasmic reticulum (ER) is a subcellular organelle where the vast majority of secreted and membrane proteins are folded. Many kinds of cellular perturbations, such as imbalances in calcium, loss of the luminal oxidizing environment and/or nutrient homeostasis, can lead to the accumulation of unfolded proteins and apoptosis[¹,²]. The apoptosis is involved in the pathogenesis of many diseases and conditions including ischemia-reperfusion injury, diabetes and nonalcoholic fatty liver disease (NAFLD) [³,⁴].

Hepatocyte death is a main feature of almost every liver disease, with apoptosis characterized by biochemical and morphological features being one of its modes. Over the past ten years, thapsigargin (TG), has become widely used as selective inhibitor of the ubiquitous sarco-endoplasmic reticulum Ca²⁺-ATPases (SERCAs), which pump Ca²⁺ from the cytosol into the lumen of the endoplasmic reticulum (ER) in mammalian cells [^5-7] The latter mechanism of TG action has been described in detail during the last twenty years [⁸]. It has long been known that elevation of intracellular free calcium ([Ca²⁺]i) has cytotoxic consequence in many cells including hepatocytes [⁹]. ER function is mediated, in part, by intraluminal Ca²⁺-binding proteins, which include the glucose-regulated proteins GRP78 and GRP94 [¹⁰,¹¹].

Several studies have linked ER dysfunction and activation of the unfolded protein response (UPR) to impairments in glucose homeostasis and diabetes [¹²,¹³]. ER stress and activation of the UPR in the liver have also been observed in genetic and dietary murine models of obesity, dietary models of NAFLD, and in humans with NAFLD [^13-15]. A large portion of the elevated hepatic triglyceride stores in NAFLD appear to arise from re-esterification of circulating free fatty acids [¹⁶]. Elevated circulating free fatty acids are positively correlated with liver disease severity in individuals with NAFLD [¹⁷]. Increased non-esterified fatty acids, in particular long chain saturated fatty acids, induce ER stress and activate the UPR in a number of cell types, including hepatocytes [¹⁵,¹⁸,¹⁹].

The metabolic signals that elicit ER stress are still poorly defined. Recent studies imply that long-chain saturated fatty acids, may induce ER stress in breast cancer cells and liver cells [¹⁵,³,²⁰]. Elevated lipids also induce apoptosis in many cell types, thus ER stress and an inability of the UPR to reestablish ER homeostasis may be upstream components of lipotoxicity [²⁰,²¹]. Therefore, impairments in ER homeostasis and the UPR have been implicated in a number of disease states including obesity, diabetes, hepatitis C, and NAFLD.

The present study had three aims: (1) To examine the characteristics of fatty acid-mediated ER stress and apoptosis in primary hepatocytes; (2) To determine whether α-linolenic acid, an unsaturated fatty acid, could provide protection against the cell death induced by stearic acid; (3) once the beneficial actions of α-linolenic acid were confirmed, this study inquiryed whether these effects were mediated via modification of the ER stress process with specific attention given to the role of GRP78, GRP94 expression and induction of CHOP.

The delivery and accumulation of lipids in non-adipose tissues leads to cellular dysfunction and death. This phenomenon, termed lipotoxicity, has been implicated in the pathogenesis of diabetes, cardiac failure and NAFLD [¹³,²⁶,²⁷]. Disruption of ER homeostasis and activation of the UPR has been observed in murine models of obesity, cardiac dysfunction and NAFLD [¹³,¹⁵,²⁸].

Increased free fatty acids, in particular long chain saturated fatty acids, induce ER stress, activate the UPR and promote cell death in a number of cell types, including hepatocytes [²⁹,^26-32]. Thus, impairments in ER function appear to contribute to the pathogenesis of several diseases and to cellular impairments associated with lipotoxicity. The present study was undertaken to begin to examine the mechanisms that link saturated fatty acids to ER stress, In this study, we report that (1) stearic acid causes significant cell death in primary rat hepatocytes. (2) stearic acid causes a significant degree of ER stress in primary rat hepatocytes. (3) α-linolenic acid protects primary rat hepatocytes against stearic acid lipotoxicity by reducing ER stress and apoptosis. The effects of stearic acid were similar to those observed using generated by saturated dietary free fatty acids. α-Linolenic acid was also able to reduce cellular dysfunction and apoptosis caused by thapsigargin -- one recognised inducer of ER stress. Thapsigargin causes apoptosis in many other cells such as neuronal cells, pancreatic beta-cells and cardiomyocytes [^33-35]. The mechanism underlying the necrotic cell death produced by thapsigargin is still not clear. It is possible that by protecting cells against thapsigargin-induced apoptosis, α-linolenic acid alleviated primary rat hepatocytes damaged.

Recent evidence has linked saturated fatty acid-induced apoptosis to the activation of JNK, pro-apoptotic Bcl-2 proteins, Bim and Bax, and the mitochondrial apoptotic pathway in liver cells [³⁶]. Long-chain saturated fatty acids also induce the ER-associated pro-apoptotic factor, CHOP, which suggests that disruption of ER homeostasis may also be directly linked to apoptosis. In several cell types, including liver, co-supplementation of oleate and palmitate reduces palmitate-mediated ER stress and apoptosis [³⁰,³⁷,³⁸]. Toward this end, the ability of long chain saturated fatty acids to activate the ER-associated caspase-3 was examined.

In this study, we have demonstrated for the first time that ER stress produced by stearic acid in primary rat hepatocytes can be significantly reduced by α-linolenic acid, an unsaturated fatty acid. We also report that the protection afforded by an unsaturated fatty acid involves a reduction of ER stress. The mechanism involves a reduction in the raised levels of caspase-3 and CHOP associated with stearic acid, however the results of our investigation appears to rule out a protective mechanism mediated by GRP78 as α-linolenic acid did not significantly affect levels of this chaperone molecule whereas levels of CHOP were significantly reduced.

To summarize, the results presented here suggest that unsaturated fatty acids such as α-linolenic acid may be able to offer protection of hepatocytes against the lipotoxicity of saturated fatty acids such as dietary stearic acid and nutrient overload associated 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 carried out analytical work, HS carried out analytical work and contributed in drafting the manuscript. LZ carried out analytical and statistical analysis. 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.