Acetyl-CoA Derived from Hepatic Peroxisomal ß-Oxidation Inhibits Autophagy and Promotes Steatosis via mTORC1 Activation.

Autophagy is activated by prolonged fasting but cannot overcome the ensuing hepatic lipid overload, resulting in fatty liver. Here, we describe a peroxisome-lysosome metabolic link that restricts autophagic degradation of lipids. Acyl-CoA oxidase 1 (Acox1), the enzyme that catalyzes the first step in peroxisomal ß-oxidation, is enriched in liver and further increases with fasting or high-fat diet (HFD). Liver-specific Acox1 knockout (Acox1-LKO) protected mice against hepatic steatosis caused by starvation or HFD due to induction of autophagic degradation of lipid droplets. Hepatic Acox1 deficiency markedly lowered total cytosolic acetyl-CoA levels, which led to decreased Raptor acetylation and reduced lysosomal localization of mTOR, resulting in impaired activation of mTORC1, a central regulator of autophagy. Dichloroacetic acid treatment elevated acetyl-CoA levels, restored mTORC1 activation, inhibited autophagy, and increased hepatic triglycerides in Acox1-LKO mice. These results identify peroxisome-derived acetyl-CoA as a key metabolic regulator of autophagy that controls hepatic lipid homeostasis.

Fatty liver disease protective MTARC1 p.A165T variant reduces the protein stability of MTARC1.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver disease worldwide. MTARC1, encoded by the MTARC1 gene, is a mitochondrial outer membrane-anchored enzyme. Interestingly, the MTARC1 p.A165T (rs2642438) variant is associated with a decreased risk of NAFLD, indicating that MTARC1 might be an effective target. It has been reported that the rs2642438 variant does not have altered enzymatic activity so we reasoned that this variation may affect MTARC1 stability. In this study, MTARC1 mutants were generated and stability was assessed using a protein stability reporter system both in vitro and in vivo. We found that the MTARC1 p.A165T variant has dramatically reduced the stability of MTARC1, as assessed in several cell lines. In mice, the MTARC1 A168T mutant, the equivalent of human MTARC1 A165T, had diminished stability in mouse liver. Additionally, several MTARC1 A165 mutants, including A165S, A165 N, A165V, A165G, and A165D, had dramatically decreased stability as well, suggesting that the alanine residue of MTARC1 165 site is essential for MTARC1 protein stability. Collectively, our data indicates that the MTARC1 p.A165T variant (rs2642438) leads to reduced stability of MTARC1. Given that carriers of rs2642438 show a decreased risk of NAFLD, the findings herein support the notion that MTARC1 inhibition may be a therapeutic target to combat NAFLD.

Autophagy modulates the stability of Wee1 and cell cycle G2/M transition.

The mammalian cell cycle is divided into four sequential phases, namely G1 (Gap 1), S (synthesis), G2 (Gap 2), and M (mitosis). Wee1, whose turnover is tightly and finely regulated, is a well-known kinase serving as a gatekeeper for the G2/M transition. However, the mechanism underlying the turnover of Wee1 is not fully understood. Autophagy, a highly conserved cellular process, maintains cellular homeostasis by eliminating intracellular aggregations, damaged organelles, and individual proteins. In the present study, we found autophagy deficiency in mouse liver caused G2/M arrest in two mouse models, namely Fip200 and Atg7 liver-specific knockout mice. To uncover the link between autophagy deficiency and G2/M transition, we combined transcriptomic and proteomic analysis for liver samples from control and Atg7 liver-specific knockout mice. The data suggest that the inhibition of autophagy increases the protein level of Wee1 without any alteration of its mRNA abundance. Serum starvation, an autophagy stimulus, downregulates the protein level of Wee1 in vitro. In addition, the half-life of Wee1 is extended by the addition of chloroquine, an autophagy inhibitor. LC3, a central autophagic protein functioning in autophagy substrate selection and autophagosome biogenesis, interacts with Wee1 as assessed by co-immunoprecipitation assay. Furthermore, overexpression of Wee1 leads to G2/M arrest both in vitro and in vivo. Collectively, our data indicate that autophagy could degrade Wee1-a gatekeeper of the G2/M transition, whereas the inhibition of autophagy leads to the accumulation of Wee1 and causes G2/M arrest in mouse liver.

Mechanisms and metabolic regulation of PPARα activation in Nile tilapia (Oreochromis niloticus).

Although the key metabolic regulatory functions of mammalian peroxisome proliferator-activated receptor α (PPARα) have been thoroughly studied, the molecular mechanisms and metabolic regulation of PPARα activation in fish are less known. In the first part of the present study, Nile tilapia (Nt)PPARα was cloned and identified, and high mRNA expression levels were detected in the brain, liver, and heart. NtPPARα was activated by an agonist (fenofibrate) and by fasting and was verified in primary hepatocytes and living fish by decreased phosphorylation of NtPPARα and/or increased NtPPARα mRNA and protein expression. In the second part of the present work, fenofibrate was fed to fish or fish were fasted for 4weeks to investigate the metabolic regulatory effects of NtPPARα. A transcriptomic study was also performed. The results indicated that fenofibrate decreased hepatic triglyceride and 18C-series fatty acid contents but increased the catabolic rate of intraperitoneally injected [1-(14)C] palmitate in vivo, hepatic mitochondrial ß-oxidation efficiency, the quantity of cytochrome b DNA, and carnitine palmitoyltransferase-1a mRNA expression. Fenofibrate also increased serum glucose, insulin, and lactate concentrations. Fasting had stronger hypolipidemic and gene regulatory effects than those of fenofibrate. Taken together, we conclude that: 1) liver is one of the main target tissues of the metabolic regulation of NtPPARα activation; 2) dephosphorylation is the basal NtPPARα activation mechanism rather than enhanced mRNA and protein expression; 3) activated NtPPARα has a hypolipidemic effect by increasing activity and the number of hepatic mitochondria; and 4) PPARα activation affects carbohydrate metabolism by altering energy homeostasis among nutrients.

Molecular characterization and immune response to lipopolysaccharide (LPS) of the suppressor of cytokine signaling (SOCS)-1, 2 and 3 genes in Nile tilapia (Oreochromis niloticus).

Suppressor of cytokine signaling (SOCS) proteins are inverse feedback regulators of cytokine and hormone signaling mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway that are involved in immunity, growth and development of organisms. In the present study, three SOCS genes, SOCS-1, SOCS-2 and SOCS-3, were identified in an economically important fish, Nile tilapia (Oreochromis niloticus) referred to as NtSOCS-1, NtSOCS-2 and NtSOCS-3. Multiple alignments showed that, the three SOCS molecules share highly conserved functional domains, including the SRC homology 2 (SH2) domain, the extended SH2 subdomain (ESS) and the SOCS box with others vertebrate counterparts. Phylogenetic analysis indicated that NtSOCS-1, 2 and 3 belong to the SOCS type II subfamily. Whereas NtSOCS-1 and 3 showed close evolutionary relationship with Perciformes, NtSOCS-2 was more related to Salmoniformes. Tissue specific expression results showed that, NtSOCS-1, 2 and 3 were constitutively expressed in all nine tissues examined. NtSOCS-1 and 3 were highly expressed in immune-related tissues, such as gills, foregut and head kidney. However, NtSOCS-2 was superlatively expressed in liver, brain and heart. In vivo, NtSOCS-1 and 3 mRNA levels were up-regulated after lipopolysaccharide (LPS) challenge while NtSOCS-2 was down-regulated. In vitro, LPS stimulation increased NtSOCS-3 mRNA expression, however it inhibited the transcription of NtSOCS-1 and 2. Collectively, our findings suggest that, the NtSOCS-1 and 3 might play significant role(s) in innate immune response, while NtSOCS-2 may be more involved in metabolic regulation.

Molecular characterization, transcriptional activity and nutritional regulation of peroxisome proliferator activated receptor gamma in Nile tilapia (Oreochromis niloticus).

Peroxisome proliferator activated receptor gamma (PPARγ) is a master regulator in lipid metabolism and widely exists in vertebrates. However, the molecular structure and transcriptional activity of PPARγ in fish are still unclear. This study cloned PPARγ from Nile tilapia (Oreochromis niloticus) referred as NtPPARγ and transfected the NtPPARγ plasmids into HEK-293 cells to explore its mechanism of transcriptional regulation in fish. The expression of NtPPARγ was compared in fed and fasted fish. Two transcripts of NtPPARγ varied at the 5'-untranslated region and the DNA binding domain was highly conserved. Thirty-nine amino acid residues in the ligand binding domain in Nile tilapia were different from those in human. Two transcripts showed different expression profiles in 11 tissues, but both were highly expressed in liver, intestine and kidney. The transcriptional activity assay showed that NtPPARγ collaborates with retinoid X-receptor α (NtRXRα) to regulate the expression of Nile tilapia fatty acid binding protein 4 (FABP4), the compartment of which have been identified as the target gene of PPARγ in human. In the fish fasting trial, the mRNA expression of NtPPARγ1 and NtPPARγ2 in intestine and liver at 3h post-feeding (HPF) was lower than those at 8 HPF, 24 HPF and in fish fasted for 36h, but was relatively stable in kidney among different feeding treatments. In conclusion, the DNA binding domain in PPARγ was highly conserved, while the ligand binding domain was moderately conserved. In Nile tilapia, the PPARγ collaborates with RXRα to perform transcriptional regulation of FABP4 at least in vitro. The plasmid system established in this study along with a cell line from Nile tilapia will be useful tools for the further functional study of PPARγ in fish.

Blockage of TMEM189 induces G2/M arrest and inhibits the growth of breast tumors.

Cancer is the major cause of premature death in humans worldwide, demanding more efficient therapeutics. Aberrant cell proliferation resulting from the loss of cell cycle regulation is the major hallmark of cancer, so targeting cell cycle is a promising strategy to combat cancer. However, the molecular mechanism underlying the dysregulation of cell cycle of cancer cells remains poorly understood. TMEM189, a newly identified protein, plays roles in the biosynthesis of ethanolamine plasmalogen and the regulation of autophagy. Here, we demonstrated that the expression level of TMEM189 was negatively correlated with the survival rate of the cancer patients. TMEM189 deficiency significantly suppresses the cancer cell proliferation and migration, and causes cell cycle G2/M arrest both in vitro and in vivo. Furthermore, TMEM189 depletion suppressed the growth of breast tumors in vivo. Taken together, our work indicated that TMEM189 promotes cancer progression by regulating cell cycle G2/M transition, suggesting that it is a promising target in cancer therapy.

Adipose tissue peroxisomal lipid synthesis orchestrates obesity and insulin resistance through LXR-dependent lipogenesis.

OBJECTIVE: Adipose tissue mass is maintained by a balance between lipolysis and lipid storage. The contribution of adipose tissue lipogenesis to fat mass, especially in the setting of high-fat feeding, is considered minor. Here we investigated the effect of adipose-specific inactivation of the peroxisomal lipid synthetic protein PexRAP on fatty acid synthase (FASN)-mediated lipogenesis and its impact on adiposity and metabolic homeostasis. METHODS: To explore the role of PexRAP in adipose tissue, we metabolically phenotyped mice with adipose-specific knockout of PexRAP. Bulk RNA sequencing was used to determine transcriptomic responses to PexRAP deletion and 14C-malonyl CoA allowed us to measure de novo lipogenic activity in adipose tissue of these mice. In vitro cell culture models were used to elucidate the mechanism of cellular responses to PexRAP deletion. RESULTS: Adipose-specific PexRAP deletion promoted diet-induced obesity and insulin resistance through activation of de novo lipogenesis. Mechanistically, PexRAP inactivation inhibited the flux of carbons to ethanolamine plasmalogens. This increased the nuclear PC/PE ratio and promoted cholesterol mislocalization, resulting in activation of liver X receptor (LXR), a nuclear receptor known to be activated by increased intracellular cholesterol. LXR activation led to increased expression of the phospholipid remodeling enzyme LPCAT3 and induced FASN-mediated lipogenesis, which promoted diet-induced obesity and insulin resistance. CONCLUSIONS: These studies reveal an unexpected role for peroxisome-derived lipids in regulating LXR-dependent lipogenesis and suggest that activation of lipogenesis, combined with dietary lipid overload, exacerbates obesity and metabolic dysregulation.

Liver ACOX1 regulates levels of circulating lipids that promote metabolic health through adipose remodeling.

The liver gene expression of the peroxisomal ß-oxidation enzyme acyl-coenzyme A oxidase 1 (ACOX1), which catabolizes very long chain fatty acids (VLCFA), increases in the context of obesity, but how this pathway impacts systemic energy metabolism remains unknown. Here, we show that hepatic ACOX1-mediated ß-oxidation regulates inter-organ communication involved in metabolic homeostasis. Liver-specific knockout of Acox1 (Acox1-LKO) protects mice from diet-induced obesity, adipose tissue inflammation, and systemic insulin resistance. Serum from Acox1-LKO mice promotes browning in cultured white adipocytes. Global serum lipidomics show increased circulating levels of several species of ω-3 VLCFAs (C24-C28) with previously uncharacterized physiological role that promote browning, mitochondrial biogenesis and Glut4 translocation through activation of the lipid sensor GPR120 in adipocytes. This work identifies hepatic peroxisomal ß-oxidation as an important regulator of metabolic homeostasis and suggests that manipulation of ACOX1 or its substrates may treat obesity-associated metabolic disorders.

Lipopolysaccharide binding protein resists hepatic oxidative stress by regulating lipid droplet homeostasis.

Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.

Cloning and molecular characterization of complement component 1 inhibitor (C1INH) and complement component 8ß (C8ß) in Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus), one of the most important groups of food fishes in the world, has frequently suffered from serious challenge from pathogens in recent years. Immune responses of Nile tilapia should be understood to protect the aquaculture industry of this fish. The complement system has an important function in recognizing bacteria, opsonizing these pathogens by phagocytes, or killing them by direct lysis. In this study, two Nile tilapia complement component genes, complement component 1 inhibitor (C1INH) and complement component 8ß subunit (C8ß), were cloned and their expression characteristics were analyzed. C1INH cDNA was found containing a 1791 bp open reading frame (ORF) encoding a putative protein with 597 amino acids, a 101 bp 5'-untranslated region (UTR) and a 236 bp 3'-UTR. The predicted protein structure for this gene consisted of two Ig-like domains and glycosyl hydrolase family-9 active site signature 2. The C8ß cDNA consisted of a 1761 bp ORF encoding 587 amino acids, a 15 bp 5'-UTR and a 170 bp 3'-UTR. The predicted protein of C8ß contained three motifs, thrombospondin type-1 repeat, membrane attack complex/perforin domain, and LDL-receptor class A. Expression analysis revealed that these two complement genes were highly expressed in the liver, however, were weakly expressed in the gill, heart, brain, kidney, intestine, spleen and dorsal muscle tissues. The present study provided insights into the complement system and immune functions of Nile tilapia.

Isolation and Differentiation of Adipocyte Precursors Derived from Neonatal Murine Brown Adipose Tissue.

Brown adipose tissue (BAT) is an important regulator of energy homeostasis. Primary brown adipocyte culture provides a powerful and physiologically relevant tool for in vitro studies related to BAT. Here, we describe a detailed procedure for isolation and differentiation of adipocyte precursors from neonatal murine interscapular BAT (iBAT).

Complete mitochondrial DNA sequences of the Nile tilapia (Oreochromis niloticus) and Blue tilapia (Oreochromis aureus): genome characterization and phylogeny applications.

Cichlid fishes have played an important role in evolutionary biology and aquaculture industry. Nile tilapia (Oreochromis niloticus), blue tilapia (Oreochromis aureus) and Mozambique tilapia (Oreochromis mossambicus), the useful models in studying evolutionary biology within Cichlid fishes, are also mainly cultured species in aquaculture with great economic importance. In this paper, the complete nucleotide sequence of the mitochondrial genome for O. niloticus and O. aureus were determined and phylogenetic analyses from mitochondrial protein-coding genes were conducted to explore their phylogenetic relationship within Cichlids. The mitogenome is 16,625 bp for O. niloticus and 16,628 bp for O. aureus, containing the same gene order and an identical number of genes or regions with the other Cichlid fishes, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one putative control region. Phylogenetic analyses using three different computational algorithms (maximum parsimony, maximum likelihood and Bayesian method) show O. niloticus and O. mossambicus are closely related, and O. aureus has remotely phylogenetic relationship from above two fishes.

Peroxisomes as cellular adaptors to metabolic and environmental stress.

Peroxisomes are involved in multiple metabolic processes, including fatty acid oxidation, ether lipid synthesis, and reactive oxygen species (ROS) metabolism. Recent studies suggest that peroxisomes are critical mediators of cellular responses to various forms of stress, including oxidative stress, hypoxia, starvation, cold exposure, and noise. As dynamic organelles, peroxisomes can modulate their proliferation, morphology, and movement within cells, and engage in crosstalk with other organelles in response to external cues. Although peroxisome-derived hydrogen peroxide has a key role in cellular signaling related to stress, emerging studies suggest that other products of peroxisomal metabolism, such as acetyl-CoA and ether lipids, are also important for metabolic adaptation to stress. Here, we review molecular mechanisms through which peroxisomes regulate metabolic and environmental stress.

Peroxisomal ß-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis.

To liberate fatty acids (FAs) from intracellular stores, lipolysis is regulated by the activity of the lipases adipose triglyceride lipase (ATGL), hormone-sensitive lipase and monoacylglycerol lipase. Excessive FA release as a result of uncontrolled lipolysis results in lipotoxicity, which can in turn promote the progression of metabolic disorders. However, whether cells can directly sense FAs to maintain cellular lipid homeostasis is unknown. Here we report a sensing mechanism for cellular FAs based on peroxisomal degradation of FAs and coupled with reactive oxygen species (ROS) production, which in turn regulates FA release by modulating lipolysis. Changes in ROS levels are sensed by PEX2, which modulates ATGL levels through post-translational ubiquitination. We demonstrate the importance of this pathway for non-alcoholic fatty liver disease progression using genetic and pharmacological approaches to alter ROS levels in vivo, which can be utilized to increase hepatic ATGL levels and ameliorate hepatic steatosis. The discovery of this peroxisomal ß-oxidation-mediated feedback mechanism, which is conserved in multiple organs, couples the functions of peroxisomes and lipid droplets and might serve as a new way to manipulate lipolysis to treat metabolic disorders.

Hepatic peroxisomal ß-oxidation suppresses lipophagy via RPTOR acetylation and MTOR activation.

Hepatic lipid homeostasis is controlled by a coordinated regulation of various metabolic pathways involved in de novo synthesis, uptake, storage, and catabolism of lipids. Disruption of this balance could lead to hepatic steatosis. Peroxisomes play an essential role in lipid metabolism, yet their importance is often overlooked. In a recent study, we demonstrated a role for hepatic peroxisomal ß-oxidation in autophagic degradation of lipid droplets. ACOX1 (acyl-Coenzyme A oxidase 1, palmitoyl), the rate-limiting enzyme of peroxisomal ß-oxidation, increases with fasting or high-fat diet (HFD). Liver-specific acox1 knockout (acox1-LKO) protects mice from hepatic steatosis induced by starvation or HFD via induction of lipophagy. Mechanistically, we showed that hepatic ACOX1 deficiency decreases the total cytosolic acetyl-CoA levels, which leads to reduced acetylation of RPTOR/RAPTOR, a component of MTORC1, which is a key regulator of macroautophagy/autophagy. These results identify peroxisome-derived acetyl-CoA as a critical metabolic regulator of autophagy that controls hepatic lipid homeostasis.

MED19 Regulates Adipogenesis and Maintenance of White Adipose Tissue Mass by Mediating PPARγ-Dependent Gene Expression.

The Mediator complex relays regulatory signals from gene-specific transcription factors to the basal transcriptional machinery. However, the role of individual Mediator subunits in different tissues remains unclear. Here, we demonstrate that MED19 is essential for adipogenesis and maintenance of white adipose tissue (WAT) by mediating peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity. MED19 knockdown blocks white adipogenesis, but not brown adipogenesis or C2C12 myoblast differentiation. Adipose-specific MED19 knockout (KO) in mice results in a striking loss of WAT, whitening of brown fat, hepatic steatosis, and insulin resistance. Inducible adipose-specific MED19 KO in adult animals also results in lipodystrophy, demonstrating its requirement for WAT maintenance. Global gene expression analysis reveals induction of genes involved in apoptosis and inflammation and impaired expression of adipose-specific genes, resulting from decreased PPARγ residency on adipocyte gene promoters and reduced association of PPARγ with RNA polymerase II. These results identify MED19 as a crucial facilitator of PPARγ-mediated gene expression in adipose tissue.

Lipid Regulators of Thermogenic Fat Activation.

The global prevalence of obesity continues to increase, suggesting a need for alternative treatment approaches. Targeting brown fat function to promote energy expenditure represents one such approach. Brown adipocytes and the related beige adipocytes oxidize fatty acids and glucose to generate heat and are activated by cold exposure or consumption of high-calorie diets. Alternative, more practical means to activate thermogenic fat are needed. Here, we review emerging data suggesting new roles for lipids in activating thermogenesis that extend beyond their serving as a fuel source for heat generation. Lipids have also been implicated in mediating interorgan communication, crosstalk between organelles, and cellular signaling regulating thermogenesis. Understanding how lipids regulate thermogenesis could identify innovative therapeutic interventions for obesity.

TFEB drives PGC-1α expression in adipocytes to protect against diet-induced metabolic dysfunction.

TFEB is a basic helix-loop-helix transcription factor that confers protection against metabolic diseases such as atherosclerosis by targeting a network of genes involved in autophagy-lysosomal biogenesis and lipid catabolism. In this study, we sought to characterize the role of TFEB in adipocyte and adipose tissue physiology and evaluate the therapeutic potential of adipocyte-specific TFEB overexpression in obesity. We demonstrated that mice with adipocyte-specific TFEB overexpression (Adipo-TFEB) were protected from diet-induced obesity, insulin resistance, and metabolic sequelae. Adipo-TFEB mice were lean primarily through increased metabolic rate, suggesting a role for adipose tissue browning and enhanced nonshivering thermogenesis in fat. Transcriptional characterization revealed that TFEB targeted genes involved in adipose tissue browning rather than those involved in autophagy. One such gene encoded PGC-1α, an established target of TFEB that promotes adipocyte browning. To dissect the role of PGC-1α in mediating the downstream effects of TFEB overexpression, we generated mice with adipocyte-specific PGC-1α deficiency and TFEB overexpression. Without PGC-1α, the ability of TFEB overexpression to brown adipose tissue and to elicit beneficial metabolic effects was blunted. Overall, these data implicate TFEB as a PGC-1α-dependent regulator of adipocyte browning and suggest its therapeutic potential in treating metabolic disease.