ABSTRACT
In mammalian cells, mitochondrial respiration produces reactive oxygen species (ROS) such as superoxide (O2-), which is then converted by the SOD1 enzyme into hydrogen peroxide (H2O2), the predominant form of cytosolic ROS. ROS at high levels can be toxic, but below this threshold are important for physiological processes acting as a second messenger similar to Ca2+. Mitochondrial Ca2+ influx from the ER increases ATP and ROS production, while ATP and ROS can regulate Ca2+ homeostasis, leading to an intricate interplay between Ca2+, ROS, and ATP synthesis. The Unfolded Protein Response (UPR) proteins ATF6α and XBP1 contribute to protection from oxidative stress through upregulation of Sod1 and Catalase genes. Here, UPR-associated protein CREB3 is shown to play a role in balancing Ca2+, ROS, and ATP homeostasis. Creb3-deficient mouse embryonic fibroblast cells (MEF-/-) were susceptible to H2O2-induced oxidative stress while having a functioning antioxidant gene expression response compared to MEF+/+. MEF-/- cells also contained elevated basal cytosolic ROS levels, which was attributed to drastically increased basal mitochondrial respiration and spare respiratory capacity relative to MEF+/+. MEF-/- cells also showed an increase in endoplasmic reticulum Ca2+ release and mitochondrial Ca2+ levels hinting at a potential cause for MEF-/- cell mitochondrial dysfunction. These results suggest that CREB3 is essential for maintaining proper Ca2+, ATP, and ROS homeostasis in mammalian cells.
Subject(s)
Hydrogen Peroxide , Transcription Factors , Animals , Mice , Adenosine Triphosphate/metabolism , Calcium/metabolism , Fibroblasts/metabolism , Homeostasis , Hydrogen Peroxide/toxicity , Hydrogen Peroxide/metabolism , Mammals , Oxidative Stress , Reactive Oxygen Species/metabolism , Superoxide Dismutase-1/metabolism , Transcription Factors/metabolismABSTRACT
BACKGROUND: The endoplasmic reticulum senses alterations to cellular homeostasis that activates the unfolded protein response (UPR). UPR proteins are known to aid in regulating glucose and lipid metabolism. CREB3 is a UPR-associated transcription factor whose potential role in regulating energy metabolism remains unclear. METHODS: Eight-week-old wild-type (WT) and Creb3+/- mice were placed on control and high-fat diets (HFD) for 8 weeks, and metabolic phenotypes characterized by weekly weighing, indirect calorimetry, body composition scans, glucose tolerance tests, plasma analysis, tissue lipid quantifications and gene/protein expression analysis. RESULTS: HFD weight gain in Creb3+/- males was reduced by 34% (p < 0.0001) and females by 39.5% (p = 0.014) from their WT counterparts. No differences were found in HFD food intake or total fecal lipids between genotypes. Creb3+/- mice had increased energy expenditure and respiratory exchange ratios (p = 0.002) relative to WT. Creb3+/- mice had significant reductions in absolute fat and lean tissue, while Creb3+/- females had significant reductions in body fat% and increased lean% composition (p < 0.0001) compared to WT females. Creb3+/- mice were protected from HFD-induced basal hyperglycemia (males p < 0.0001; females p = 0.0181). Creb3+/- males resisted HFD-induced hepatic lipid accumulation (p = 0.025) and glucose intolerance compared to WT (p < 0.0001) while Creb3+/- females were protected from lipid accumulation in skeletal muscle (p = 0.001). Despite the metabolic differences of Creb3+/- mice on HFD, lipid plasma profiles did not significantly differ from WT. Fasted Creb3+/- mice additionally revealed upregulation of hepatic energy expenditure and gluconeogenic genes such as Pgc-1a and Gr (glucocorticoid receptor) (p < 0.05), respectively. CONCLUSIONS: Reduced expression of CREB3 increased energy expenditure and the respiratory exchange ratio, and protected mice from HFD-induced weight gain, basal hyperglycemia, and sex-specific tissue lipid accumulation. We postulate that CREB3 is a novel key regulator of diet-induced obesity and energy metabolism that warrants further investigation as a potential therapeutic target in metabolic disorders.
Subject(s)
Cyclic AMP Response Element-Binding Protein , Diet, High-Fat , Energy Metabolism , Obesity , Animals , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Diet, High-Fat/adverse effects , Energy Metabolism/genetics , Female , Glucose Intolerance/genetics , Lipid Metabolism , Lipids , Male , Mice , Mice, Inbred C57BL , Obesity/genetics , Obesity/metabolism , Transcription Factors/metabolism , Weight GainABSTRACT
Genetic factors contribute to the development of autism spectrum disorder (ASD), and although non-protein-coding regions of the genome are being increasingly implicated in ASD, the functional consequences of these variants remain largely uncharacterized. Induced pluripotent stem cells (iPSCs) enable the production of personalized neurons that are genetically matched to people with ASD and can therefore be used to directly test the effects of genomic variation on neuronal gene expression, synapse function, and connectivity. The combined use of human pluripotent stem cells with genome editing to introduce or correct specific variants has proved to be a powerful approach for exploring the functional consequences of ASD-associated variants in protein-coding genes and, more recently, long non-coding RNAs (lncRNAs). Here, we review recent studies that implicate lncRNAs, other non-coding mutations, and regulatory variants in ASD susceptibility. We also discuss experimental design considerations for using iPSCs and genome editing to study the role of the non-protein-coding genome in ASD.
