Phosphatidylethanolamine positively regulates autophagy and longevity

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.Phosphatidylethanolamine (PE) is a phospholipid found in all living organisms. Together with phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI), PE represents the backbone of most biological membranes. PE is the second-most abundant phospholipid in mammalian membranes ranging from 20 to 50%.¹ In yeast, PE is essential for growth and is generated through four different enzymatic pathways:² PE can be produced by decarboxylation of PS, as a first option at the mitochondrial membrane via phosphatidylserine decarboxylase 1 (Psd1)^{3, 4} or, as a second, option at the Golgi and vacuolar membranes through phosphatidylserine decarboxylase 2 (Psd2).⁵ As a third possibility, PE can be produced from actively retrieved extracellular ethanolamine,^{6, 7} which is cytidine 5''-diphosphate-activated⁸ and then coupled to diacylglycerol to generate PE.⁹ The fourth, scarcely employed PE-generating pathway is based on the lysophospholipid acylation of lyso-PE. Importantly, PE does not spontaneously assemble in bilayers and rather incorporates into curved structures, such as the inverted hexagonal phase.¹⁰ The physiological function of non-bilayer lipids in membranes is considered to reside in their interaction with membrane proteins via the membrane lateral pressure¹⁰ and membrane tethering and fusion processes, which are relevant for autophagy.¹¹The term ‘autophagy'' describes a degradation process affecting intracellular components (for a review see, 12 13) which as an important cytoprotective mechanism, is closely linked to ageing. Autophagy mainly differs from the proteasomal pathway, the other major cellular degradation mechanism, in two aspects. First, autophagy can degrade large particles or whole organelles and second, the final degradation occurs in the lysosome/vacuole and not at the proteasome. Prior to the actual degradation, the cargo is gathered in autophagic particles, which are surrounded by a characteristic double-membrane. However, the origin of these autophagosomal membranes is still controversial and might actually depend on the mode of autophagy induction.^{14, 15} Among the discussed membrane sources are the Golgi apparatus, the endosplamic reticulum (ER) or the mitochondrion-associated membrane, which is formed at the interface between the ER.¹⁶ In higher eukaryotes autophagic membranes are enriched in PE with a high degree of unsaturation,¹⁷ similarly to the PE species found in mitochondria.^{14, 18} Moreover, the pre-autophagosomal structure or phagophore assembly site (PAS), which appears at the very beginning of autophagosome formation, already harbours Atg9, an autophagy-related transmembrane protein that shuttles between mitochondria and the PAS structure in yeast.¹⁹Importantly, PE also functions as an anchor to autophagosomal membranes for the autophagy-related protein Atg8 in yeast²⁰ and its mammalian orthologue LC3.^{21, 22} This PE anchor is provided to LC3/Atg8 post-translationally in a process called lipidation. First, LC3/Atg8 is carboxy-terminally cleaved by proteases from the Atg4 family.^{23, 24} Subsequently, the remaining C-terminal glycine is coupled to PE in a series of ubiquitination-like reactions involving diverse Atg-proteins.^{20, 25, 26, 27} In vitro, Atg8-PE causes hemifusion of vesicles, which argues for its potential role in autophagosomal phagophore expansion.^{11, 28} Consistently, semisynthetic LC3-PE has recently been described to stimulate membrane tethering and fusion.²⁹ We thus reasoned that the overall abundance of PE might be critical for PE-lipidation of LC3/Atg8 and could thus regulate autophagosomal membrane formation. Therefore, we tested whether increasing cellular PE levels might have an impact on autophagy and lifespan regulation.Here, we report that knock-out of PSD1 or PSD2 shortens the chronological lifespan of S. cerevisiae, whereas PSD1-overexpression enhances the autophagic capacity and increases longevity. Furthermore, external administration of ethanolamine increases endogenous PE levels, enhances autophagic flux and extends the lifespan of yeast, mammalian cells in culture and flies (Drosophila melanogaster).     

Cysteine 253 of UCP1 regulates energy expenditure and sex-dependent adipose tissue inflammation

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Enhanced insulin sensitivity, energy expenditure and thermogenesis in adipose-specific Pten suppression in mice

Pten is an important phosphatase, suppressing the phosphatidylinositol-3 kinase/Akt pathway. Here, we generated adipose-specific Pten-deficient (AdipoPten-KO) mice, using newly generated Acdc promoter-driven Cre transgenic mice. AdipoPten-KO mice showed lower body and adipose tissue weights despite hyperphagia and enhanced insulin sensitivity with induced phosphorylation of Akt in adipose tissue. AdipoPten-KO mice also showed marked hyperthermia and increased energy expenditure with induced mitochondriagenesis in adipose tissue, associated with marked reduction of p53, inactivation of Rb, phosphorylation of cyclic AMP response element binding protein (CREB) and increased expression of Ppargc1a, the gene that encodes peroxisome proliferative activated receptor gamma coactivator 1 alpha. Physiologically, adipose Pten mRNA decreased with exposure to cold and increased with obesity, which were linked to the mRNA alterations of mitochondriagenesis. Our results suggest that altered expression of adipose Pten could regulate insulin sensitivity and energy expenditure. Suppression of adipose Pten may become a beneficial strategy to treat type 2 diabetes and obesity.     

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.     

Orexin is required for brown adipose tissue development, differentiation, and function

Orexin (OX) neuropeptides stimulate feeding and arousal. Deficiency of orexin is implicated in narcolepsy, a disease associated with obesity, paradoxically in the face of reduced food intake. Here, we show that obesity in orexin-null mice is associated with impaired brown adipose tissue (BAT) thermogenesis. Failure of thermogenesis in OX-null mice is due to inability of brown preadipocytes to differentiate. The differentiation defect in OX-null neonates is circumvented by OX injections to OX-null dams. In?vitro, OX, triggers the full differentiation program in mesenchymal progenitor stem cells, embryonic fibroblasts and brown preadipocytes via p38 mitogen activated protein (MAP) kinase and bone morphogenetic protein receptor-1a (BMPR1A)-dependent Smad1/5 signaling. Our study suggests that obesity associated with OX depletion is linked to brown-fat hypoactivity, which leads to dampening of energy expenditure. Thus, orexin plays an integral role in adaptive thermogenesis and body weight regulation via effects on BAT differentiation and function.     

Nuclear receptor coactivator PNRC2 regulates energy expenditure and adiposity

PNRC2 was identified in our laboratory as a general cofactor for nuclear receptors. To better characterize the physiological function of PNRC2, we used gene-targeting technology to generate PNRC2-null mice (PNRC2(-/-) mice). These PNRC2(-/-) mice are viable and fertile. PNRC2-null mice, especially male mice, are lean and are resistant to high fat diet-induced obesity but without the induction of insulin resistance. Male mice devoid of PNRC2 protein have a higher metabolic rate than wild-type mice. They consume more oxygen and produce more heat. Consistent with reduced adipose mass, the levels of leptin are lower in PNRC2(-/-) mice. This study provides evidence that PNRC2 plays one or more important roles in controlling the energy balance between energy storage and energy expenditure. PNRC2 may be a new target in the treatment of obesity and related metabolic diseases.     

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.     

Hypothalamic melanocortin system regulates sympathetic nerve activity in brown adipose tissue

To clarify the neuronal mechanism of the hypothalamic melanocortin system in regulating energy metabolism, we investigated the effects of centrally administered alpha-melanocyte-stimulating hormone (alpha-MSH) and agouti-related protein (AGRP), an agonist and an antagonist for the melanocortin 4 receptor (MC4-R), respectively, on the activity of sympathetic nerves innervating brown adipose tissue (BAT) and on BAT temperature. A bolus infusion of alpha-MSH (1 nmol) into the third cerebral ventricle (i3vt) significantly increased sympathetic nerve activity and elevated BAT temperature (P<0.05). The i3vt infusion of AGRP (1 nmol) gradually suppressed BAT sympathetic nerve activity and was accompanied by a significant reduction in BAT temperature (P<0.05). In conclusion, the hypothalamic melanocortin system may regulate peripheral energy expenditure, as well as thermogenesis, through its influence on BAT sympathetic nerve activity.     

The arrestin domain-containing 3 protein regulates body mass and energy expenditure

A human genome-wide linkage scan for obesity identified a linkage peak on chromosome 5q13-15. Positional cloning revealed an association of a rare haplotype to high body-mass index (BMI) in males but not?females. The risk locus contains a single gene, "arrestin domain-containing 3" (ARRDC3), an uncharacterized α-arrestin. Inactivating Arrdc3 in mice led to a striking resistance to obesity, with greater impact on male mice. Mice with decreased ARRDC3 levels were protected from obesity due to increased energy expenditure through increased activity levels and increased thermogenesis of both brown and white adipose tissues. ARRDC3 interacted directly with β-adrenergic receptors, and loss of ARRDC3 increased the response to β-adrenergic stimulation in isolated adipose tissue. These results demonstrate that ARRDC3 is a gender-sensitive regulator of obesity and energy expenditure and reveal?a surprising diversity for arrestin family protein functions.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

A longevity protein,Lag2, interacts with SCF complex and regulates SCF function

SCF‐type E3‐ubiquitin ligases control numerous cellular processes through the ubiquitin‐proteasome pathway. However, the regulation of SCF function remains largely uncharacterized. Here, we report a novel SCF complex‐interacting protein, Lag2, in Saccharomyces cerevisiae. Lag2 interacts with the SCF complex under physiological conditions. Lag2 negatively controls the ubiquitylation activities of SCF E3 ligase by interrupting the association of Cdc34 to SCF complex. Overexpression of Lag2 increases unrubylated Cdc53, whereas deletion of lag2, together with the deletions of dcn1 and jab1, results in the accumulation of Rub1‐modified Cdc53. In vitro rubylation assays show that Lag2 inhibits the conjugation of Rub1 to Cdc53 in competition with Dcn1, which suggest that Lag2 down‐regulates the rubylation of Cdc53 rather than promoting derubylation. Furthermore, Dcn1 hinders the association of Lag2 to Cdc53 in vivo. Finally, the deletion of lag2 combined with the deletion of either dcn1 or rub1 suppresses the growth of yeast cells. These observations thus indicate that Lag2 has a significant function in regulating the SCF complex by controlling its ubiquitin ligase activities and its rubylation cycle.     

Ghrelin regulates adiposity in white adipose tissue and UCP1 mRNA expression in brown adipose tissue in mice

To examine the involvement of ghrelin in obesity, we investigated the effects of treatment with peripherally administered ghrelin on food intake, adiposity, and expression of uncoupling protein (UCP) mRNA in brown (BAT) and white (WAT) adipose tissue in mice. Acute bolus administration of ghrelin at a dose of 120 nmol/kg increased cumulative food intake over 4 and 24 h as compared to controls (p<0.05 for each), whereas 12 nmol/kg/day ghrelin showed no remarkable effect (p>0.1). Chronic repeated treatment with 12 nmol/kg/day ghrelin for 7 days increased body weight and adiposity assessed by the weight of adipose tissue, triglyceride content in WAT (p<0.05 for each versus control). In addition, the same treatment decreased and increased mRNA expression of BAT UCP1 and WAT UCP2, respectively (p<0.05 for each). In conclusion, ghrelin can regulate body weight, adiposity and UCPs mRNA expression in mice. The present results provide evidence for a new regulatory loop involving ghrelin and UCP, and add novel insights into the regulatory mechanisms of obesity.     

Lipidomics of brown and white adipose tissue: Implications for energy metabolism

Adipose tissue exerts multiple vital functions that critically maintain energy balance, including storing and expending energy, as well as secreting factors that systemically modulate nutrient metabolism. Since lipids are the major constituents of the adipocytes, it is unsurprising that the lipid composition of these cells plays a critical role in maintaining their functions and communicating with other organs and cells. In both positive and negative energy balance conditions, lipids and free fatty acids secreted from adipocytes exert either beneficial or detrimental effects in other tissues, such as the liver, pancreas and muscle. The way the adipocytes communicate with other organs tightly depends on the nature of their lipidome composition. Notwithstanding, the lipidome composition of the adipocytes is affected by physiological factors such as adipocyte type, gender and age, but also by environmental cues such as diet composition, thermal stress and physical activity. Here we provide an updated overview on how the adipose tissue lipidome profile is shaped by different physiological and environmental factors and how these changes impact the way the adipocytes regulate whole-body energy metabolism.     

Enhanced polyamine catabolism alters homeostatic control of white adipose tissue mass, energy expenditure, and glucose metabolism

Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha) is an attractive candidate gene for type 2 diabetes, as genes of the oxidative phosphorylation (OXPHOS) pathway are coordinatively downregulated by reduced expression of PGC-1 alpha in skeletal muscle and adipose tissue of patients with type 2 diabetes. Here we demonstrate that transgenic mice with activated polyamine catabolism due to overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) had reduced white adipose tissue (WAT) mass, high basal metabolic rate, improved glucose tolerance, high insulin sensitivity, and enhanced expression of the OXPHOS genes, coordinated by increased levels of PGC-1 alpha and 5'-AMP-activated protein kinase (AMPK) in WAT. As accelerated polyamine flux caused by SSAT overexpression depleted the ATP pool in adipocytes of SSAT mice and N(1),N(11)-diethylnorspermine-treated wild-type fetal fibroblasts, we propose that low ATP levels lead to the induction of AMPK, which in turn activates PGC-1 alpha in WAT of SSAT mice. Our hypothesis is supported by the finding that the phenotype of SSAT mice was reversed when the accelerated polyamine flux was reduced by the inhibition of polyamine biosynthesis in WAT. The involvement of polyamine catabolism in the regulation of energy and glucose metabolism may offer a novel target for drug development for obesity and type 2 diabetes.     

Modulation of adipose tissue thermogenesis as a method for increasing energy expenditure

There is a renewed interest in the role of adipose tissue in energy utilization and thermogenesis and its potential application in the treatment of metabolic disorders such as obesity and diabetes. The last few years have seen the identification of brown adipose tissue capable of metabolic activation in adult humans, the possibility of recruiting ‘beige’ adipocytes to increase energy expenditure, and the implication of molecules such as FGF21 and irisin in inducing increases in energy expenditure in adipose tissue. The translation of these findings into human trials to deliver safe, efficacious medicines remains a challenge.     

Mfn2 is critical for brown adipose tissue thermogenic function

Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine‐tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin‐resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose‐specific Mfn2 knockout (Mfn2‐adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2‐adKO mice were protected from high‐fat diet‐induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole‐body energy homeostasis.