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Abstract Background Aging is a complex biological process characterized by obesity and immunosenescence throughout the organism. Immunosenescence involves a decline in immune function and the increase in chronic-low grade inflammation, called inflammaging. Adipose tissue expansion, particularly that of visceral adipose tissue (VAT), is associated with an increase in pro-inflammatory macrophages that play an important role in modulating immune responses and producing inflammatory cytokines. The leukotriene B4 receptor 1 (BLT1) is a regulator of obesity-induced inflammation. Its ligand, LTB4, acts as a chemoattractant for immune cells and induces inflammation. Studies have shown that BLT1 is crucial for cytokine production during lipopolysaccharide (LPS) endotoxemia challenge in younger organisms. However, the expression patterns and function of BLT1 in older organisms remains unknown. Results In this study, we investigated BLT1 expression in immune cell subsets within the VAT of aged male and female mice. Moreover, we examined how antagonizing BLT1 signaling could alter the inflammatory response to LPS in aged mice. Our results demonstrate that aged mice exhibit increased adiposity and inflammation, characterized by elevated frequencies of B and T cells, along with pro-inflammatory macrophages in VAT. BLT1 expression is the highest in VAT macrophages. LPS and LTB4 treatment result in increased BLT1 in young and aged bone marrow-derived macrophages (BMDMs). However, LTB4 treatment resulted in amplified Il6 from aged, but not young BMDMs. Treatment of aged mice with the BLT1 antagonist, U75302, followed by LPS-induced endotoxemia resulted in an increase in anti-inflammatory macrophages, reduced phosphorylated NFκB and reduced Il6. Conclusions This study provides valuable insights into the age- and sex- specific changes in BLT1 expression on immune cell subsets within VAT. This study offers support for the potential of BLT1 in modulating inflammation in aging.

Abstract Mitochondrial function is vital for energy metabolism in thermogenic adipocytes. Impaired mitochondrial bioenergetics in brown adipocytes are linked to disrupted thermogenesis and energy balance in obesity and aging. Phospholipid cardiolipin (CL) and phosphatidic acid (PA) jointly regulate mitochondrial membrane architecture and dynamics, with mitochondria-associated endoplasmic reticulum membranes (MAMs) serving as the platform for phospholipid biosynthesis and metabolism. However, little is known about the regulators of MAM phospholipid metabolism and their connection to mitochondrial function. We discover that LCN2 is a PA binding protein recruited to the MAM during inflammation and metabolic stimulation. Lcn2 deficiency disrupts mitochondrial fusion-fission balance and alters the acyl-chain composition of mitochondrial phospholipids in brown adipose tissue (BAT) of male mice. Lcn2 KO male mice exhibit an increase in the levels of CLs containing long-chain polyunsaturated fatty acids (LC-PUFA), a decrease in CLs containing monounsaturated fatty acids, resulting in mitochondrial dysfunction. This dysfunction triggers compensatory activation of peroxisomal function and the biosynthesis of LC-PUFA-containing plasmalogens in BAT. Additionally, Lcn2 deficiency alters PA production, correlating with changes in PA-regulated phospholipid-metabolizing enzymes and the mTOR signaling pathway. In conclusion, LCN2 plays a critical role in the acyl-chain remodeling of phospholipids and mitochondrial bioenergetics by regulating PA production and its function in activating signaling pathways.

Obesity-induced insulin resistance is among the key factors in the development of type 2 diabetes, atherogenic dyslipidemia and cardiovascular disease. Adipose tissue plays a key role in the regulation of whole-body metabolism and insulin sensitivity. In obesity, adipose tissue becomes inflamed and dysfunctional, exhibiting a modified biochemical signature and adipokine secretion pattern that promotes insulin resistance in peripheral tissues. An important hallmark of dysfunctional obese adipose tissue is impaired NAD+/sirtuin signaling. In this chapter, we summarize the evidence for impairment of the NAD+/sirtuin pathway in obesity, not only in white adipose tissue but also in brown adipose tissue and during the process of beiging, together with correlative evidence from human studies. We also describe the role of PARPs and CD38 as important NAD+ consumers and discuss findings from experimental studies that investigated potential NAD+ boosting strategies and their efficacy in restoring impaired NAD+ metabolism in dysfunctional obese adipose tissue. In sum, these studies suggest a critical role of NAD+ metabolism in adipose biology and provide a basis for the potential development of strategies to restore metabolic health in obesity.

Olivieri, Walker, Karamafrooz et al. show that the fusion of the dynamic J-domain to PKA-C (PKA-CDNAJB1) disrupts the internal allosteric network, attenuating the nucleotide/PKI binding cooperativity. This study suggests that the reduced allosteric cooperativity may contribute to the pathology that PKA-CDNAJB1 drives.

Abstract Bariatric surgery is the most effective treatment for weight loss. Vertical sleeve gastrectomy (VSG) involves the resection of ~ 80% of the stomach and was conceived to purely restrict oral intake. However, evidence suggests more complex mechanisms, particularly postoperative changes in gut microbiota, in facilitating weight loss and resolving associated comorbidities. VSG in humans is a complex procedure and includes peri-operative antibiotics and caloric restriction in addition to the altered anatomy. The impact of each of these factors on the intestinal microbiota have not been evaluated. The aim of this study was to determine the relative contributions of each of these factors on intestinal microbiota composition following VSG prior to substantial weight loss. Thirty-two obese patients underwent one of three treatments: (1) VSG plus routine intravenous peri-operative antibiotics (n = 12), (2) VSG with intravenous vancomycin chosen for its low intestinal penetrance (n = 12), and (3) caloric restriction (n = 8). Fecal samples were evaluated for bacterial composition prior to and 7 days following each intervention. Only patients undergoing VSG with routine peri-operative antibiotics showed a significant shift in community composition. Our data support the single dose of routine peri-operative antibiotics as the most influential factor of intestinal microbial composition acutely following VSG.

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.

Objectives: Aging increases the risk for development of adipose tissue dysfunction, insulin resistance, dyslipidemia, and liver steatosis. Lipocalin 2 (Lcn2) deficient mice are more prone to diet-induced obesity and metabolic dysfunction, indicating a protective role for Lcn2 in younger mice. In this study, we determined whether overexpressing Lcn2 in adipose tissue can protect against age-related metabolic deterioration. Methods: We developed ap2-promoter-driven Lcn2 transgenic (Tg) mice and aged Lcn2 Tg mice for the metabolic assessments. Results: We found decreased adipocyte size in inguinal white adipose tissue (iWAT) from 10-month-old Lcn2 Tg mice relative to WT. This was accompanied by increased markers of adipogenesis in iWAT and attenuation of the age-related decline in AMP-activated protein kinase (AMPK) phosphorylation in adipose tissue depots. In addition to improvements in adipose tissue function, whole-body metabolic homeostasis was maintained in aged Lcn2 Tg mice. This included improved glucose tolerance and reduced serum triglycerides in older Lcn2 Tg mice relative to WT mice. Further, liver morphology and liver lipid levels were improved in older Lcn2 Tg mice, alongside a decrease in markers of liver inflammation and fibrosis. Conclusions: We demonstrate that overexpression of Lcn2 in adipose tissue not only preserves adipose tissue function during aging but also promotes maintenance of glucose tolerance, decreases dyslipidemia, and prevents liver lipid accumulation and steatosis. Keywords: Lipocalin 2, Adipose tissue, Adipose beiging, Healthspan

More than 30% of people in the United States (US) are classified as obese, and over 50% are considered significantly overweight. Importantly, obesity is a risk factor not only for the development of metabolic syndrome but also for many cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third leading cause of cancer-related death, and 5-year survival of PDAC remains around 9% in the U.S. Obesity is a known risk factor for PDAC. Metabolic control and bariatric surgery, which is an effective treatment for severe obesity and allows massive weight loss, have been shown to reduce the risk of PDAC. It is therefore clear that elucidating the connection between obesity and PDAC is important for the identification of a novel marker and/or intervention point for obesity-related PDAC risk. In this review, we discussed recent progress in obesity-related PDAC in epidemiology, mechanisms, and potential cancer prevention effects of interventions, including bariatric surgery with preclinical and clinical studies.

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.

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.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-02006-3

The α,β 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.

Oxidative stress is a hallmark of metabolic disease, though the mechanisms that define this link are not fully understood. Irreversible modification of proteins by reactive lipid aldehydes (protein carbonylation) is a major consequence of oxidative stress in adipose tissue and the substrates and specificity of this modification are largely unexplored. Here we show that histones are avidly modified by 4-hydroxynonenal (4-HNE) in vitro and in vivo. Carbonylation of histones by 4-HNE increased with age in male flies and visceral fat depots of mice and was potentiated in genetic (ob/ob) and high-fat feeding models of obesity. Proteomic evaluation of in vitro 4-HNE- modified histones led to the identification of both Michael and Schiff base adducts. In contrast, mapping of sites in vivo from obese mice exclusively revealed Michael adducts. In total, we identified 11 sites of 4-hydroxy hexenal (4-HHE) and 10 sites of 4-HNE histone modification in visceral adipose tissue. In summary, these results characterize adipose histone carbonylation as a redox-linked epigenomic mark associated with metabolic disease and aging.

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 transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD+/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function.

The role of fatty acid transport protein 1 (FATP1) and FATP4 in facilitating adipocyte fatty acid metabolism was investigated using stable FATP1 or FATP4 knockdown (kd) 3T3-L1 cell lines derived from retrovirus-delivered short hairpin RNA (shRNA). Decreased expression of FATP1 or FATP4 did not affect preadipocyte differentiation or the expression of FATP1 (in FATP4 kd), FATP4 (in FATP1 kd), fatty acid translocase, acyl-coenzyme A synthetase 1, and adipocyte fatty acid binding protein but did lead to increased levels of peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding protein α. Both FATP1 and FATP4 kd adipocytes exhibited reduced triacylglycerol deposition and corresponding reductions in diacylglycerol and monoacylglycerol levels compared with control cells. FATP1 kd adipocytes displayed an ∼25% reduction in basal 3H-labeled fatty acid uptake and a complete loss of insulin-stimulated 3H-labeled fatty acid uptake compared with control adipocytes. In contrast, FATP4 kd adipocytes as well as HEK-293 cells overexpressing FATP4 did not display any changes in fatty acid influx. FATP4 kd cells exhibited increased basal lipolysis, whereas FATP1 kd cells exhibited no change in lipolytic capacity. Consistent with reduced triacylglycerol accumulation, FATP1 and FATP4 kd adipocytes exhibited enhanced 2-deoxyglucose uptake compared with control adipocytes. These findings define unique and distinct roles for FATP1 and FATP4 in adipose fatty acid metabolism.

Leukotriene A4 (LTA4) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37°C and ∼40 s at 4°C. Regardless of this aqueous instability, LTA4 is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA4 to ∼20 min at 4°C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA4 at 4°C to ∼20 min with protein present. Stabilization of LTA4 was examined when arachidonic acid was present to compete with LTA4 for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA4 and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA4.Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B4, were found to have no effect on the stability of LTA4 induced by E-FABP even when present at concentrations 3-fold higher than LTA4.

By consensus, the acyl-CoA synthetase (ACS) community, with the advice of the human and mouse genome nomenclature committees, has revised the nomenclature for the mammalian long-chain acyl-CoA synthetases. ACS is the family root name, and the human and mouse genes for the long-chain ACSs are termed ACSL1,3–6 and Acsl1,3–6, respectively. Splice variants of ACSL3, -4, -5, and -6 are cataloged.Suggestions for naming other family members and for the nonmammalian acyl-CoA synthetases are made.

Fatty acid binding proteins (FAB2222251) are low-molecular-mass, soluble, intracellular lipid carriers. Previous studies on adipocytes from adipocyte fatty acid binding protein (A-FABP)-deficient mice have revealed that both basal and isoproterenol-stimulated lipolysis were markedly reduced (Coe et al. 1999. J. Lipid Res. 40: 967–972). Herein, we report the construction of transgenic mice overexpressing the FABP5 gene encoding the epithelial fatty acid binding protein (E-FABP) in adipocytes, thereby allowing evaluation of the effects on lipolysis of increased FABP levels and of type specificity. In adipocytes from FABP5 transgenic mice, the total FABP protein level in the adipocyte was increased to 150% as compared to the wild type due to a 10-fold increase in the level of E-FABP and an unanticipated 2-fold down-regulation of the A-FABP. There were no significant differences in body weight, serum FFA, or fat pad mass between wild-type and FABP5 transgenic mice. Importantly, both basal and hormone-stimulated lipolysis increased in adipocytes from the FABP5 transgenic animals. The molecular composition of the fatty acid pool from either the intracellular compartment or that effluxed from the adipocyte was unaltered.These results demonstrate that there is a positive relationship between lipolysis and the total level of FABP but not between lipolysis and a specific FABP type.

Biosynthesis of lipids was investigated in growing 293 cells stably expressing fatty acid (FA) transport protein 1 (FATP1), a bifunctional polypeptide with FA transport as well as fatty acyl-CoA synthetase activity. In short-term (30 s) incubations, FA uptake was increased in FATP1 expressing cells (C8 cells) compared with the vector (as determined by BODIPY 3823 staining and radioactive FA uptake). In long-term (4 h) incubations, incorporation of [14C]acetate, [3H]oleic acid, or [14C]lignoceric acid into 1,2,3-triacyl-sn-glycerol (TG) was elevated in C8 cells compared with vector, whereas incorporation of radiolabel into glycerophospholipids was unaltered. The increase in TG biosynthesis correlated with an increase in 1,2-diacyl-sn-glycerol acyltransferase activity in C8 cells compared with vector. In contrast, incorporation of [14C]acetate into sphingomyelin (SM) and cholesterol, and [3H]oleic acid or [14C]lignoceric acid into SM was reduced due to a reduction in de novo biosynthesis of these lipids in C8 cells compared with vector.The results indicate that exogenously supplied FAs, and their subsequently produced acyl-CoAs, are preferentially channeled by an FATP1 linked mechanism into the TG biosynthetic pathway and that such internalized lipids down-regulate de novo SM and cholesterol metabolism in actively growing 293 cells.

Peroxisome proliferator-activated receptors (PPARs) are activated by a variety of fatty acids, eicosanoids, and hypolipidemic and insulin-sensitizing drugs. Many of these compounds bind avidly to members of a family of small lipid-binding proteins, the fatty acid-binding proteins (FABPs). Fatty acids are activated to CoA esters, which bind with high affinity to the acyl-CoA-binding protein (ACBP). Thus, the availability of known and potential PPAR ligands may be regulated by lipid-binding proteins. In this report we show by transient transfection of CV-1 cells that coexpression of ACBP and adipocyte lipid-binding protein (ALBP) exerts a ligand- and PPAR subtype-specific attenuation of PPAR-mediated trans-activation, suggesting that lipid-binding proteins, when expressed at high levels, may function as negative regulators of PPAR activation by certain ligands. Expression of ACBP, ALBP, and keratinocyte lipid-binding protein (KLBP) is induced during adipocyte differentiation, a process during which PPARγ plays a prominent role. We present evidence that endogenous ACBP, ALBP, and KLBP not only localize to the cytoplasm but also exhibit a prominent nuclear localization in 3T3-L1 adipocytes. In addition, forced expression of ACBP, ALBP, and KLBP in CV-1 cells resulted in a substantial accumulation of all three proteins in the nucleus. These results suggest that lipid-binding proteins, contrary to the general assumption, may exert their action in the nucleus as well as in the cytoplasm.—Helledie, T., M. Antonius, R. V. Sørensen, A. V. Hertzel, D. A. Bernlohr, S. Kølvraa, K. Kristiansen, and S. Mandrup. Lipid-binding proteins modulate ligand-dependent trans-activation by peroxisome proliferator-activated receptors and localize to the nucleus as well as the cytoplasm. J. Lipid Res. 2000. 41: 1740–1751.

The transfer of genes into primary murine adipocytes using an adenovirus system has been developed. A recombinant adenovirus was constructed (expressing green fluorescent protein [GFP] under the control of the strong cytomegalovirus [CMV] promoter and a luciferase reporter gene under the control of the weak adipocyte promoter keratinocyte lipid-binding protein [KLBP/FABP5]) and incubated with primary adipocytes from C57BL/6J mice. Analysis of infected cells by confocal microscopy detected GFP expression in both the cytoplasm and nucleus of adipocytes with a 64% efficiency of infection. To demonstrate the applicability of this method in the study of gene regulation, adenovirus-infected adipocytes exhibited significant levels of luciferase activity even from a weak promoter. TPA treatment of infected adipocytes increased luciferase activity, consistent with previous studies indicating that the KLBP/FABP5 gene is up-regulated by phorbol esters.These results provide an efficient, convenient, and sensitive method to transiently infect primary murine adipocytes, facilitating protein expression or permitting analysis of reporter gene activity from both viral and endogenous promoters.

The availability of mice containing an adipocyte lipid-binding protein (ALBP/aP2) gene disruption allowed for a direct examination of the presumed role of lipid-binding proteins in the mobilization and trafficking of intracellular fatty acids. Total body and epididymal fat pad weights, as well as adipose cell morphology, were unaltered in male ALBP/aP2 disrupted mice when compared to their wild-type littermates. Analysis of adipocytes isolated from wild-type and ALBP/aP2 null mice revealed that a selective 40- and 13-fold increase in the level of the keratinocyte lipid-binding protein (KLBP) mRNA and protein, respectively, accompanied the ALBP/aP2 gene disruption. Although KLBP protein was significantly up-regulated, the total lipid-binding protein level decreased 8-fold as a consequence of the disruption. There was no appreciable difference in the rate of fatty acid influx or esterification in adipocytes of wild-type and ALBP/aP2 null animals. To the contrary, basal lipolysis decreased approximately 40% in ALBP/aP2 nulls as compared to wild-type littermates. The glycerol release from isproterenol-stimulated ALBP/aP2 null fat cells was similarly reduced by ∼35%. Consistent with a decrease in basal efflux, the non-esterified fatty acid (NEFA) level was nearly 3-fold greater in adipocytes from ALBP/aP2 nulls as compared to wild-type animals. The significant decrease in both basal and isoproterenol-stimulated lipolysis in adipose tissue of ALBP/aP2 null mice supports the model whereby intracellular lipid-binding proteins function as lipid chaperones, facilitating the movement of fatty acids out of the fat cell.—Ribarik Coe, N., M. A. Simpson, and D. A. Bernlohr. Targeted disruption of the adipocyte lipid-binding protein (aP2 protein) gene impairs fat cell lipolysis and increases cellular fatty acid levels. J. Lipid Res. 1999. 40: 967–972.

Lipocalin 2 (Lcn2) has been recently characterized as a new adipokine having a role in innate immunity and energy metabolism. Nonetheless, the metabolic regulation of Lcn2 production in adipocytes has not been comprehensively studied. To better understand the Lcn2 biology, we investigated the regulation of Lcn2 expression in adipose tissue in response to metabolic stress in mice as well as the control of Lcn2 expression and secretion by cytokines and nutrients in 3T3-L1 adipocytes. Our results showed that the mRNA expression of Lcn2 was upregulated in white and brown adipose tissues as well as liver during fasting and cold stress in mice. Among pro-inflammatory cytokines TNFα, IL-1β, and IL-6, IL-1β showed most profound effect on Lcn2 expression and secretion in 3T3-L1 adipocytes. Insulin stimulated Lcn2 expression and secretion in a dose-dependent manner; this insulin effect was significantly abolished in the presence of low concentration of glucose. Moreover, insulin-stimulated Lcn2 expression and secretion was also attenuated when glucose was replaced by 3-O-methyl-d-glucose or by blocking NFκB pathway activation. Additionally, we showed that palmitate and oleate induced Lcn2 expression and secretion more significantly than EPA, while phytanic acid reduced Lcn2 production. Our results demonstrated that Lcn2 production in adipocytes is highly responsive to metabolic stress, cytokines, and nutrient signals, suggesting an important role of Lcn2 in adipocyte metabolism and inflammation.