ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload

Abstract

Palmitate overload induces hepatic cell dysfunction characterized by enhanced apoptosis and altered citric acid cycle (CAC) metabolism; however, the mechanism of how this occurs is incompletely understood. We hypothesize that elevated doses of palmitate disrupt intracellular calcium homeostasis resulting in a net flux of calcium from the ER to mitochondria, activating aberrant oxidative metabolism. We treated primary hepatocytes and H4IIEC3 cells with palmitate and calcium chelators to identify the roles of intracellular calcium flux in lipotoxicity. We then applied 13C metabolic flux analysis (MFA) to determine the impact of calcium in promoting palmitate-stimulated mitochondrial alterations. Co-treatment with the calcium-specific chelator BAPTA resulted in a suppression of markers for apoptosis and oxygen consumption. Additionally, 13C MFA revealed that BAPTA co-treated cells had reduced CAC fluxes compared to cells treated with palmitate alone. Our results demonstrate that palmitate-induced lipoapoptosis is dependent on calcium-stimulated mitochondrial activation, which induces oxidative stress.

Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver

“…Most importantly, we found that calculated oxygen consumption in humans, previously shown to have elevated anaplerosis (7), correlated with poorer NAS and necroinflammatory scores. These data are similar to results by Egnatchik et al (26,52), who found that exposure of liver cells to elevated NEFA caused increased TCA cycle flux and oxidative stress.…”

“…Since GNG mediated by TCA cycle cataplerosis is a significant consumer of hepatic energetics (16), we examined whether suppressing cataplerosis reduces oxidative stress in vitro. H4IIE cells incubated in the presence of high NEFA developed a 2-fold increase in ROS detected by 2′,7′-dichlorofluorescein, which is similar to results reported in primary hepatocytes (26). However, this effect was attenuated when anaplerotic/cataplerotic flux was blocked by the PEPCK inhibitor mercaptopicolinic acid ( Figure 3H).…”

The role of ER stress in lipid metabolism and lipotoxicity

“…Palmitate-induced lipotoxicity may occur through generation of reactive oxygen species (ROS) (97,98). Elevated levels of palmitate compromised the ER's capability to maintain Ca 2+ stores, resulting in the stimulation of mitochondrial oxidative metabolism, ROS production, and ER stress-mediated cellular dysfunction (98).…”

“…Elevated levels of palmitate compromised the ER's capability to maintain Ca 2+ stores, resulting in the stimulation of mitochondrial oxidative metabolism, ROS production, and ER stress-mediated cellular dysfunction (98). It is wellknown that protein misfolding in the ER and subsequent induction of ER stress contribute to ROS production and cellular dysfunction, which can be prevented by antioxidant treatment (29,31,32).…”

Elevated circulating stearic acid leads to a major lipotoxic effect on mouse pancreatic beta cells in hyperlipidaemia via a miR-34a-5p-mediated PERK/p53-dependent pathway

“…Although the molecular mechanisms of stearic-acidinduced lipotoxicity are not fully understood, it is evident that enhanced endoplasmic reticulum (ER) stress is a central contributor to lipotoxicity resulting from transcriptional reprogramming [12], activation of c-Jun N-terminal kinase [13], CCAAT/enhancer binding protein homologous protein (CHOP) [14], p53 and mitochondrial apoptosis pathways [15,16]. Importantly, microRNAs (miRNAs), which are involved in post-transcriptional regulation under ER stress, have emerged as key regulators of hepatocyte and cardiac muscle lipotoxicity [17,18].…”