Induction of mitochondrial biogenesis and respiration is associated with mTOR regulation in hepatocytes of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA)

Abstract

The hypolipidemic effect of peroxisome proliferator-activated receptor (PPAR) activators has been explained by increasing mitochondrial fatty acid oxidation, as observed in livers of rats treated with the pan-PPAR activator tetradecylthioacetic acid (TTA). PPAR-activation does, however, not fully explain the metabolic adaptations observed in hepatocytes after treatment with TTA. We therefore characterized the mitochondrial effects, and linked this to signalling by the metabolic sensor, the mammalian target of rapamycin (mTOR). In hepatocytes isolated from TTA-treated rats, the changes in cellular content and morphology were consistent with hypertrophy. This was associated with induction of multiple mitochondrial biomarkers, including mitochondrial DNA, citrate synthase and mRNAs of mitochondrial proteins. Transcription analysis further confirmed activation of PPARα-associated genes, in addition to genes related to mitochondrial biogenesis and function. Analysis of mitochondrial respiration revealed that the capacity of both electron transport and oxidative phosphorylation were increased. These effects coincided with activation of the stress related factor, ERK1/2, and mTOR. The protein level and phosphorylation of the downstream mTOR actors eIF4G and 4E-BP1 were induced. In summary, TTA increases mitochondrial respiration by inducing hypertrophy and mitochondrial biogenesis in rat hepatocytes, via adaptive regulation of PPARs as well as mTOR.

Introduction

Metabolic adaptation is linked to life span regulation [1] and important diseases, such as cancer [2], neurodegeneration [3] and obesity-related disorders [4]. Accordingly, mechanisms of metabolic adaptation represent potential targets for prevention and treatment of disease.

The peroxisome proliferator-activated receptors (PPARs) [5], [6] and the mammalian target of rapamycin (mTOR) [7], [8] are major regulators of metabolism. They converge on the cell survival-associated protein kinase AKT [9], which is involved in insulin signalling and metabolic balancing. The PPARs crosstalk with central metabolic sensors, such as AMP-dependent protein kinase (AMPK) activated upon low energy stress, and PPARγ co-activator 1α (PGC1α) involved in mitochondrial biogenesis, to control adaptive responses [5], [6]. The mTOR protein exists in two complexes, named mTOR complex 1/2 (mTORC1/2), where mTORC1 is the primary nutrient responder [8]. Upon activation, mTORC1 promotes protein translation and cell growth via regulation of the downstream eukaryotic translation initiation factor 4E (eIF4E) and the eIF4E-binding protein 1 (4E-BP1) [10]. Upstream regulators of mTORC1 include AKT [11], AMPK [12] and MAP-kinase-extracellular regulated kinase (ERK) [13].

Metabolic signalling is closely associated with regulation of mitochondrial function and biogenesis. Mitochondria are essential organelles in cellular energy metabolism, and house several catabolic pathways, including fatty acid oxidation, TCA-cycle and mitochondrial respiration. In mitochondrial respiration, electron transport by the respiratory protein complexes is coupled to oxidative phosphorylation yielding ATP from ADP. Regulation of mitochondrial metabolism and respiration is a crucial mechanism of cellular adaptation, e.g. in response to nutritional alterations or energy depletion. For instance, induction of mitochondrial biogenesis can be observed in muscle as a reaction to physical exercise [14] or mitochondrial mutations [15], and is linked to the actions of AMPK, PPARs and PGC1α. [16]. Multiple important functions of mitochondrial regulators have previously been characterized in rat liver after nutritional and pharmacological interventions [17].

Tetradecylthioacetic acid (TTA) is a modified fatty acid that has hypolipidemic effects in rats [18], [19]. This agent activates all the PPAR members, in the ranking order PPARα > PPARδ > PPARγ [19]. Treatment with TTA dramatically increases the mitochondrial oxidative capacity in rat hepatocytes, which seems only partly to be explained by PPAR-activation [17], [20]. Here, we used TTA-treatment to investigate the impact and mechanisms of mitochondrial adaptation in relation to key signalling pathways involved in metabolic regulation in rat hepatocytes.