SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Lipid Droplet-Derived Monounsaturated Fatty Acids Traffic via PLIN5 to Allosterically Activate SIRT1.

Lipid droplets (LDs) provide a reservoir for triacylglycerol storage and are a central hub for fatty acid trafficking and signaling in cells. Lipolysis promotes mitochondrial biogenesis and oxidative metabolism via a SIRT1/PGC-1α/PPARα-dependent pathway through an unknown mechanism. Herein, we identify that monounsaturated fatty acids (MUFAs) allosterically activate SIRT1 toward select peptide-substrates such as PGC-1α. MUFAs enhance PGC-1α/PPARα signaling and promote oxidative metabolism in cells and animal models in a SIRT1-dependent manner. Moreover, we characterize the LD protein perilipin 5 (PLIN5), which is known to enhance mitochondrial biogenesis and function, to be a fatty-acid-binding protein that preferentially binds LD-derived monounsaturated fatty acids and traffics them to the nucleus following cAMP/PKA-mediated lipolytic stimulation. Thus, these studies identify the first-known endogenous allosteric modulators of SIRT1 and characterize a LD-nuclear signaling axis that underlies the known metabolic benefits of MUFAs and PLIN5.

RNAseq Analysis of FABP4 Knockout Mouse Hippocampal Transcriptome Suggests a Role for WNT/ß-Catenin in Preventing Obesity-Induced Cognitive Impairment.

Microglial fatty-acid binding protein 4 (FABP4) is a regulator of neuroinflammation. We hypothesized that the link between lipid metabolism and inflammation indicates a role for FABP4 in regulating high fat diet (HFD)-induced cognitive decline. We have previously shown that obese FABP4 knockout mice exhibit decreased neuroinflammation and cognitive decline. FABP4 knockout and wild type mice were fed 60% HFD for 12 weeks starting at 15 weeks old. Hippocampal tissue was dissected and RNA-seq was performed to measure differentially expressed transcripts. Reactome molecular pathway analysis was utilized to examine differentially expressed pathways. Results showed that HFD-fed FABP4 knockout mice have a hippocampal transcriptome consistent with neuroprotection, including associations with decreased proinflammatory signaling, ER stress, apoptosis, and cognitive decline. This is accompanied by an increase in transcripts upregulating neurogenesis, synaptic plasticity, long-term potentiation, and spatial working memory. Pathway analysis revealed that mice lacking FABP4 had changes in metabolic function that support reduction in oxidative stress and inflammation, and improved energy homeostasis and cognitive function. Analysis suggested a role for WNT/ß-Catenin signaling in the protection against insulin resistance, alleviating neuroinflammation and cognitive decline. Collectively, our work shows that FABP4 represents a potential target in alleviating HFD-induced neuroinflammation and cognitive decline and suggests a role for WNT/ß-Catenin in this protection.

Cellular Senescence in Obesity and Associated Complications: a New Therapeutic Target.

PURPOSE OF REVIEW: Obesity has increased worldwide recently and represents a major global health challenge. This review focuses on the obesity-associated cellular senescence in various organs and the role of these senescent cells (SnCs) in driving complications associated with obesity. Also, the ability to target SnCs pharmacologically with drugs termed senotherapeutics as a therapy for these complications is discussed. RECENT FINDINGS: Several studies have shown a positive correlation between obesity and SnC burden in organs such as adipose tissue, liver, and pancreatic-ß-cells. These SnCs produce several secretory factors which affect other cells and tissues in a paracrine manner resulting in organ dysfunction. The accumulation of SnCs in adipocytes affects their lipid storage and impairs adipogenesis. The inflammatory senescence-associated secretory phenotype (SASP) of SnCs downregulates the antioxidant capacity and mitochondrial function in tissues. Senescent hepatocytes cannot oxidize fatty acids, which leads to lipid deposition and senescence in ß-cells decrease function. These and other adverse effects of SnCs contribute to insulin resistance and type-2 diabetes. The reduction in the SnC burden genetically or pharmacologically improves the complications associated with obesity. The accumulation of SnCs with age and disease accelerates aging. Obesity is a key driver of SnC accumulation, and the complications associated with obesity can be controlled by reducing the SnC burden. Thus, senotherapeutic drugs have the potential to be an effective therapeutic option.

Microglial FABP4-UCP2 Axis Modulates Neuroinflammation and Cognitive Decline in Obese Mice.

The microglial fatty-acid-binding protein 4-uncoupling protein 2 (FABP4-UCP2) axis is a key regulator of neuroinflammation in high-fat-diet (HFD)-fed animals, indicating a role for FABP4 in brain immune response. We hypothesized that the FABP4-UCP2 axis is involved in regulating diet-induced cognitive decline. We tested cognitive function in mice lacking microglial FABP4 (AKO mice). Fifteen-week-old male AKO and wild-type (WT) mice were maintained on 60% HFD or normal chow (NC) for 12 weeks. Body composition was measured using EchoMRI. Locomotor activity, working memory, and spatial memory were assessed using behavioral tests (open field, T-maze, and Barnes maze, respectively). Hippocampal microgliosis was assessed via immunohistochemical staining. An inflammatory cytokine panel was assayed using hippocampal tissue. Real-time RT-PCR was performed to measure microglial UCP2 mRNA expression. Our data support that loss of FABP4 prevents cognitive decline in vivo. HFD-fed WT mice exhibited impaired long- and short-term memory, in contrast with HFD-fed AKO mice. HFD-fed WT mice had an increase in hippocampal inflammatory cytokine expression (IFNγ, IL-1ß, IL-5, IL-6, KC/GRO(CXCL1), IL-10, and TNFα) and microgliosis, and decreased microglial UCP2 expression. HFD-fed AKO mice had decreased hippocampal inflammatory cytokine expression and microgliosis and increased microglial UCP2 expression compared to HFD-fed WT mice. Collectively, our work supports the idea that the FABP4-UCP2 axis represents a potential therapeutic target in preventing diet-induced cognitive decline.

Identification of gut microbiota and microbial metabolites regulated by an antimicrobial peptide lipocalin 2 in high fat diet-induced obesity.

Lipocalin 2 (Lcn2), as an antimicrobial peptide is expressed in intestine, and the upregulation of intestinal Lcn2 has been linked to inflammatory bowel disease. However, the role of Lcn2 in shaping gut microbiota during diet-induced obesity (DIO) remains unknown. We found that short-term high fat diet (HFD) feeding strongly stimulates intestinal Lcn2 expression and secretion into the gut lumen. As the HFD feeding prolongs, fecal Lcn2 levels turn to decrease. Lcn2 deficiency accelerates the development of HFD-induced intestinal inflammation and microbiota dysbiosis. Moreover, Lcn2 deficiency leads to the remodeling of microbiota-derived metabolome, including decreased production of short-chain fatty acids (SCFAs) and SCFA-producing microbes. Most importantly, we have identified Lcn2-targeted bacteria and microbiota-derived metabolites that potentially play roles in DIO and metabolic dysregulation. Correlation analyses suggest that Lcn2-targeted Dubosiella and Angelakisella have a novel role in regulating SCFAs production and obesity. Our results provide a novel mechanism involving Lcn2 as an antimicrobial host factor in the control of gut microbiota symbiosis during DIO.

Slc43a3 is a regulator of free fatty acid flux.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

Adipose oxidative stress and protein carbonylation.

Increased oxidative stress and abundance of reactive oxygen species (ROS) are positively correlated with a variety of pathophysiologies, including cardiovascular disease, type 2 diabetes, Alzheimer's disease, and neuroinflammation. In adipose biology, diabetic obesity is correlated with increased ROS in an age- and depot-specific manner and is mechanistically linked to mitochondrial dysfunction, endoplasmic reticulum (ER) stress, potentiated lipolysis, and insulin resistance. The cellular quality control systems that homeostatically regulate oxidative stress in the lean state are down-regulated in obesity as a consequence of inflammatory cytokine pressure leading to the accumulation of oxidized biomolecules. New findings have linked protein, DNA, and lipid oxidation at the biochemical level, and the structures and potential functions of protein adducts such as carbonylation that accumulate in stressed cells have been characterized. The sum total of such regulation and biochemical changes results in alteration of cellular metabolism and function in the obese state relative to the lean state and underlies metabolic disease progression. In this review, we discuss the molecular mechanisms and events underlying these processes and their implications for human health and disease.

Stearic Acid Induces CD11c Expression in Proinflammatory Macrophages via Epidermal Fatty Acid Binding Protein.

Obesity is associated with elevated levels of free fatty acids (FAs) and proinflammatory CD11c+ macrophages. However, whether and how free FAs contribute to CD11c+ macrophage differentiation and proinflammatory functions remain unclear. Here we report that dietary saturated FAs, but not unsaturated FAs, promoted the differentiation and function of CD11c+ macrophages. Specifically, we demonstrated that stearic acid (SA) significantly induced CD11c expression in monocytes through activation of the nuclear retinoid acid receptor. More importantly, cytosolic expression of epidermal FA binding protein (E-FABP) in monocytes/macrophages was shown to be critical to the mediation of the SA-induced effect. Depletion of E-FABP not only inhibited SA-induced CD11c upregulation in macrophages in vitro but also abrogated high-saturated-fat diet-induced skin lesions in obese mouse models in vivo. Altogether, our data demonstrate a novel mechanism by which saturated FAs promote obesity-associated inflammation through inducing E-FABP/retinoid acid receptor-mediated differentiation of CD11c+ macrophages.

Obesity-induced protein carbonylation in murine adipose tissue regulates the DNA-binding domain of nuclear zinc finger proteins.

In obesity-linked insulin resistance, oxidative stress in adipocytes leads to lipid peroxidation and subsequent carbonylation of proteins by diffusible lipid electrophiles. Reduction in oxidative stress attenuates protein carbonylation and insulin resistance, suggesting that lipid modification of proteins may play a role in metabolic disease, but the mechanisms remain incompletely understood. Herein, we show that in vivo, diet-induced obesity in mice surprisingly results in preferential carbonylation of nuclear proteins by 4-hydroxy-trans-2,3-nonenal (4-HNE) or 4-hydroxy-trans-2,3-hexenal (4-HHE). Proteomic and structural analyses revealed that residues in or around the sites of zinc coordination of zinc finger proteins, such as those containing the C2H2 or MATRIN, RING, C3H1, or N4-type DNA-binding domains, are particularly susceptible to carbonylation by lipid aldehydes. These observations strongly suggest that carbonylation functionally disrupts protein secondary structure supported by metal coordination. Analysis of one such target, the nuclear protein estrogen-related receptor γ (ERR-γ), showed that ERR-γ is modified by 4-HHE in the obese state. In vitro carbonylation decreased the DNA-binding capacity of ERR-γ and correlated with the obesity-linked down-regulation of many key genes promoting mitochondrial bioenergetics. Taken together, these findings reveal a novel mechanistic connection between oxidative stress and metabolic dysfunction arising from carbonylation of nuclear zinc finger proteins, such as the transcriptional regulator ERR-γ.

Antibiotic-induced Disruption of Intestinal Microbiota Contributes to Failure of Vertical Sleeve Gastrectomy.

OBJECTIVE: The aim of this study was to test whether the perioperative composition of intestinal microbiota can contribute to variable outcomes following vertical sleeve gastrectomy (VSG). SUMMARY OF BACKGROUND DATA: Although bariatric surgery is the most effective treatment for obesity, metabolic outcomes are variable. METHODS: Diet-induced obese mice were randomized to VSG or sham surgery, with or without exposure to antibiotics that selectively suppress mainly gram-positive (fidaxomicin, streptomycin) or gram-negative (ceftriaxone) bacteria on postoperative days (POD) 1-4. Fecal microbiota was characterized before surgery and on POD 7 and 28. Mice were metabolically characterized on POD 30-32 and euthanized on POD 35. RESULTS: VSG resulted in weight loss and shifts in the intestinal microbiota composition relative to sham-operated mice. Antibiotic exposure resulted in sustained reductions in alpha (within-sample) diversity of microbiota and shifts in its composition. All antibiotic treatments proved to be detrimental to metabolic VSG outcomes, regardless of antimicrobial specificity of antibiotics. These effects involved functionally distinct pathways. Specifically, fidaxomicin and streptomycin markedly altered hepatic bile acid signaling and lipid metabolism, while ceftriaxone resulted in greater reduction of key antimicrobial peptides. However, VSG mice exposed to antibiotics, regardless of their specificity, had significantly increased subcutaneous adiposity and impaired glucose homeostasis without changes in food intake relative to control VSG mice. CONCLUSION: Dysbiosis induced by brief perioperative antibiotic exposure attenuates weight loss and metabolic improvement following VSG. Potential mechanisms include disruption of bile acid homeostasis and reduction in the production of gut antimicrobial peptides. Results of this study implicate the intestinal microbiota as an important contributor to metabolic homeostasis and a potentially modifiable target influencing clinical outcomes following VSG.

Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-Induced Macrophage Cell Death through Enhancing Ceramide Production.

Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. In this article, we report a new mechanism by which dietary FAs, in particular, saturated FAs (sFAs), are able to directly trigger macrophage cell death. We demonstrated that excess sFAs, but not unsaturated FAs, induced the production of cytotoxic ceramides (Cers) in macrophage cell lines. Most importantly, expression of adipose FA binding protein (A-FABP) in macrophages facilitated metabolism of excess sFAs for Cer synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased sFA-induced Cer production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting sFA-induced macrophage cell death with primary bone marrow-derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary sFAs may serve as direct triggers in induction of Cer production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity.

Perilipin 5 and liver fatty acid binding protein function to restore quiescence in mouse hepatic stellate cells.

Hepatic stellate cell (HSC) activation occurs along with decreased Perilipin5 (Plin5) and liver fatty acid-binding protein (L-Fabp) expression and coincident lipid droplet (LD) depletion. Conversely, the activated phenotype is reversible in WT HSCs upon forced expression of Plin5. Here, we asked if L-Fabp expression is required for Plin5-mediated rescue of the quiescent phenotype. Lentiviral Plin5 transduction of passaged L-Fabp-/- HSCs failed to reverse activation markers or restore lipogenic gene expression and LD formation. However, adenoviral L-Fabp infection of lentiviral Plin5 transduced L-Fabp-/- HSCs restored both the quiescent phenotype and LD formation, an effect also mediated by adenoviral intestine-Fabp or adipocyte-Fabp. Expression of exogenous Plin5 in activated WT HSCs induced a transcriptional program of lipogenic gene expression including endogenous L-Fabp, but none of the other FABPs. We further demonstrated that selective, small molecule inhibition of endogenous L-Fabp also eliminated the ability of exogenous Plin5 to rescue LD formation and reverse activation of WT HSCs. This functional coordination of L-Fabp with Plin5 was 5'-AMP-activated protein kinase (AMPK)-dependent and was eliminated by AMPK inhibition. Taken together, our results indicate that L-Fabp is required for Plin5 to activate a transcriptional program that restores LD formation and reverses HSC activation.

FABP4 regulates eosinophil recruitment and activation in allergic airway inflammation.

Fatty acid binding protein 4 (FABP4), a member of a family of lipid-binding proteins, is known to play a role in inflammation by virtue of its ability to regulate intracellular events such as lipid fluxes and signaling. Studies have indicated a proinflammatory role for FABP4 in allergic asthma although its expression and function in eosinophils, the predominant inflammatory cells recruited to allergic airways, were not investigated. We examined expression of FABP4 in murine eosinophils and its role in regulating cell recruitment in vitro as well as in cockroach antigen (CRA)-induced allergic airway inflammation. CRA exposure led to airway recruitment of FABP4-expressing inflammatory cells, specifically eosinophils, in wild-type (WT) mice. FABP4 expression in eosinophils was induced by TNF-α as well as IL-4 and IL-13. FABP4-deficient eosinophils exhibited markedly decreased cell spreading/formation of leading edges on vascular cell adhesion molecule-1 and significantly decreased adhesion to intercellular adhesion molecule-1 associated with reduced ß2-integrin expression relative to WT cells. Furthermore, FABP4-deficient eosinophils exhibited decreased migration, F-actin polymerization, calcium flux, and ERK(1/2) phosphorylation in response to eotaxin-1. In vivo, CRA-challenged FABP4-deficient mice exhibited attenuated eosinophilia and significantly reduced airway inflammation (improved airway reactivity, lower IL-5, IL-13, TNF-α, and cysteinyl leukotriene C4 levels, decreased airway structural changes) compared with WT mice. In conclusion, expression of FABP4 in eosinophils is induced during conditions of inflammation and plays a proinflammatory role in the development of allergic asthma by promoting eosinophil adhesion and migration and contributing to the development of various aspects of airway inflammation.

Identification of a fatty acid binding protein4-UCP2 axis regulating microglial mediated neuroinflammation.

Hypothalamic inflammation contributes to metabolic dysregulation and the onset of obesity. Dietary saturated fats activate microglia via a nuclear factor-kappa B (NFκB) mediated pathway to release pro-inflammatory cytokines resulting in dysfunction or death of surrounding neurons. Fatty acid binding proteins (FABPs) are lipid chaperones regulating metabolic and inflammatory pathways in response to fatty acids. Loss of FABP4 in peripheral macrophages via either molecular or pharmacologic mechanisms results in reduced obesity-induced inflammation via a UCP2-redox based mechanism. Despite the widespread appreciation for the role of FABP4 in mediating peripheral inflammation, the expression of FABP4 and a potential FABP4-UCP2 axis regulating microglial inflammatory capacity is largely uncharacterized. To that end, we hypothesized that microglial cells express FABP4 and that inhibition would upregulate UCP2 and attenuate palmitic acid (PA)-induced pro-inflammatory response. Gene expression confirmed expression of FABP4 in brain tissue lysate from C57Bl/6J mice and BV2 microglia. Treatment of microglial cells with an FABP inhibitor (HTS01037) increased expression of Ucp2 and arginase in the presence or absence of PA. Moreover, cells exposed to HTS01037 exhibited attenuated expression of inducible nitric oxide synthase (iNOS) compared to PA alone indicating reduced NFκB signaling. Hypothalamic tissue from mice lacking FABP4 exhibit increased UCP2 expression and reduced iNOS, tumor necrosis factor-alpha (TNF-α), and ionized calcium-binding adapter molecule 1 (Iba1; microglial activation marker) expression compared to wild type mice. Further, this effect is negated in microglia lacking UCP2, indicating the FABP4-UCP2 axis is pivotal in obesity induced neuroinflammation. To our knowledge, this is the first report demonstrating a FABP4-UCP2 axis with the potential to modulate the microglial inflammatory response.

Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy.

Protein-protein interactions (PPIs) regulate a plethora of cellular processes and NMR spectroscopy has been a leading technique for characterizing them at the atomic resolution. Technically, however, PPIs characterization has been challenging due to multiple samples required to characterize the hot spots at the protein interface. In this paper, we review our recently developed methods that greatly simplify PPI studies, which minimize the number of samples required to fully characterize residues involved in the protein-protein binding interface. This original strategy combines asymmetric labeling of two binding partners and the carbonyl-carbon label selective (CCLS) pulse sequence element implemented into the heteronuclear single quantum correlation (¹H-15N HSQC) spectra. The CCLS scheme removes signals of the J-coupled 15N⁻13C resonances and records simultaneously two individual amide fingerprints for each binding partner. We show the application to the measurements of chemical shift correlations, residual dipolar couplings (RDCs), and paramagnetic relaxation enhancements (PRE). These experiments open an avenue for further modifications of existing experiments facilitating the NMR analysis of PPIs.

Fatty acid binding protein 4/aP2-dependent BLT1R expression and signaling.

Previous studies have shown that reduced levels of the adipocyte fatty acid binding protein (FABP)4 (AFABP/aP2), result in metabolic improvement including potentiated insulin sensitivity and attenuated atherosclerosis. Mechanistically, pharmacologic or genetic inhibition of FABP4 in macrophages upregulates UCP2, attenuates reactive oxygen species (ROS) production, polarizes cells toward the anti-inflammatory M2 state, and reduces leukotriene (LT) secretion. At the protein level, FABP4 stabilizes LTA4 toward chemical hydrolysis, thereby potentiating inflammatory LTC4 synthesis. Herein, we extend the FABP4-LT axis and demonstrate that genetic knockout of FABP4 reduces expression of the major macrophage LT receptor, LTB4 receptor 1 (BLT1R), via a ROS-dependent mechanism. Consistent with inflammation driving BLT1R expression, M1 polarized macrophages express increased levels of BLT1R relative to M2 polarized macrophages and treatment with proinflammatory lipopolysaccharide increased BLT1R mRNA and protein expression. In FABP4 knockout macrophages, silencing of UCP2, increased ROS levels and led to increased expression of BLT1R mRNA. Similarly, addition of exogenous H2O2 upregulated BLT1R expression, whereas the addition of a ROS scavenger, N-acetyl cysteine, decreased BLT1R levels. As compared with WT macrophages, LTB4-BLT1R-dependent JAK2-phosphorylation was reduced in FABP4 knockout macrophages. In summary, these results indicate that FABP4 regulates the expression of BLT1R and its downstream signaling via control of oxidative stress in macrophages.

Lipocalin 2, a Regulator of Retinoid Homeostasis and Retinoid-mediated Thermogenic Activation in Adipose Tissue.

We have recently characterized the role of lipocalin 2 (Lcn2) as a new adipose-derived cytokine in the regulation of adaptive thermogenesis via a non-adrenergic pathway. Herein, we explored a potential non-adrenergic mechanism by which Lcn2 regulates thermogenesis and lipid metabolism. We found that Lcn2 is a retinoic acid target gene, and retinoic acid concurrently stimulated UCP1 and Lcn2 expression in adipocytes. Lcn2 KO mice exhibited a blunted effect of all-trans-retinoic acid (ATRA) on body weight and fat mass, lipid metabolism, and retinoic acid signaling pathway activation in adipose tissue under the high fat diet-induced obese condition. We further demonstrated that Lcn2 is required for the full action of ATRA on the induction of UCP1 and PGC-1α expression in brown adipocytes and the restoration of cold intolerance in Lcn2 KO mice. Interestingly, we discovered that Lcn2 KO mice have decreased levels of retinoic acid and retinol in adipose tissue. The protein levels of STRA6 responsible for retinol uptake were significantly decreased in adipose tissue. The retinol transporter RBP4 was increased in adipose tissue but decreased in the circulation, suggesting the impairment of RBP4 secretion in Lcn2 KO adipose tissue. Moreover, Lcn2 deficiency abolished the ATRA effect on RBP4 expression in adipocytes. All the data suggest that the decreased retinoid level and action are associated with impaired retinol transport and storage in adipose tissue in Lcn2 KO mice. We conclude that Lcn2 plays a critical role in regulating metabolic homeostasis of retinoids and retinoid-mediated thermogenesis in adipose tissue.

Down regulation of Peroxiredoxin-3 in 3T3-L1 adipocytes leads to oxidation of Rictor in the mammalian-target of rapamycin complex 2 (mTORC2).

Mitochondrially-derived oxidative stress has been implicated in the development of obesity-induced insulin resistance and is correlated with down regulation of Peroxiredoxin-3 (Prdx3). Prdx3 knockout mice exhibit whole-body insulin resistance, while Prdx3 transgenic animals remain insulin sensitive when placed on a high fat diet. To define the molecular events linking mitochondrial oxidative stress to insulin action, Prdx3 was silenced in 3T3-L1 adipocytes (Prdx3 KD) and the resultant cells evaluated for mitochondrial function, endoplasmic reticulum stress (ER stress), mitochondrial unfolded protein response (mtUPR) and insulin signaling. Prdx3 KD cells exhibit a two-fold increase in H2O2, reduced insulin-stimulated glucose transport and attenuated S473 phosphorylation of the mTORC2 substrate, Akt. Importantly, the decrease in glucose uptake can be rescued by pre-treatment with the antioxidant N-acetyl-cysteine (NAC). The changes in insulin sensitivity occur independently from activation of the ER stress or mtUPR pathways. Analysis of mTORC2, the complex responsible for phosphorylating Akt at S473, reveals increased cysteine oxidation of Rictor in Prdx3 KD cells that can be rescued with NAC. Taken together, these data suggest mitochondrial dysfunction in adipocytes may attenuate insulin signaling via oxidation of the mammalian-target of rapamycin complex 2 (mTORC2).

Oxidative stress and lipotoxicity.

The α,ß polyunsaturated lipid aldehydes are potent lipid electrophiles that covalently modify lipids, proteins, and nucleic acids. Recent work highlights the critical role these lipids play under both physiological and pathological conditions. Protein carbonylation resulting from nucleophilic attack of lysine, histidine, and cysteine residues is a major outcome of oxidative stress and functions as a redox-sensitive signaling mechanism with roles in autophagy, cell proliferation, transcriptional control, and apoptosis. In addition, protein carbonylation is implicated as an initiating factor in mitochondrial dysfunction and endoplasmic reticulum stress, providing a mechanistic connection between oxidative stress and metabolic disease. In this review, we discuss the generation and metabolism of reactive lipid aldehydes, as well as their signaling roles.