Lipocalin 2 regulates mitochondrial phospholipidome remodeling, dynamics, and function in brown adipose tissue in male mice.

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.

The Role of NAD+ in Metabolic Regulation of Adipose Tissue: Implications for Obesity-Induced Insulin Resistance.

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.

The αC-ß4 loop controls the allosteric cooperativity between nucleotide and substrate in the catalytic subunit of protein kinase A.

Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-ß4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-CF100A toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-ß4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-ß4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.

TP53/p53 Facilitates Stress-Induced Exosome and Protein Secretion by Adipocytes.

Besides the secretion of fatty acids, lipolytic stimulation of adipocytes results in the secretion of triglyceride-rich extracellular vesicles and some free proteins (e.g., fatty acid binding protein 4) that, in sum, affect adipose homeostasis as well as the development of metabolic disease. At the mechanistic level, lipolytic signals activate p53 in an adipose triglyceride lipase-dependent manner, and pharmacologic inhibition of p53 attenuates adipocyte-derived extracellular vesicle (AdEV) protein and FABP4 secretion. Mass spectrometry analyses of the lipolytic secretome identified proteins involved in glucose and fatty acid metabolism, translation, chaperone activities, and redox control. Consistent with a role for p53 in adipocyte protein secretion, activation of p53 by the MDM2 antagonist nutlin potentiated AdEV particles and non-AdEV protein secretion from cultured 3T3-L1 or OP9 adipocytes while the levels of FABP4 and AdEV proteins were significantly reduced in serum from p53-/- mice compared with wild-type controls. The genotoxin doxorubicin increased AdEV protein and FABP4 secretion in a p53-dependent manner and DNA repair-depleted ERCC1-/Δ-haploinsufficient mice expressed elevated p53 in adipose depots, along with significantly increased serum FABP4. In sum, these data suggest that lipolytic signals, and cellular stressors such as DNA damage, facilitate AdEV protein and FABP4 secretion by adipocytes in a p53-dependent manner.

Desmoglein 2 Functions as a Receptor for Fatty Acid Binding Protein 4 in Breast Cancer Epithelial Cells.

Fatty acid binding protein 4 (FABP4) is a secreted adipokine linked to obesity and progression of a variety of cancers. Obesity increases extracellular FABP4 (eFABP4) levels in animal models and in obese breast cancer patients compared with lean healthy controls. Using MCF-7 and T47D breast cancer epithelial cells, we show herein that eFABP4 stimulates cellular proliferation in a time and concentration dependent manner while the non-fatty acid-binding mutant, R126Q, failed to potentiate growth. When E0771 murine breast cancer cells were injected into mice, FABP4 null animals exhibited delayed tumor growth and enhanced survival compared with injections into control C57Bl/6J animals. eFABP4 treatment of MCF-7 cells resulted in a significant increase in phosphorylation of extracellular signal-regulated kinase 1/2 (pERK), transcriptional activation of nuclear factor E2-related factor 2 (NRF2) and corresponding gene targets ALDH1A1, CYP1A1, HMOX1, SOD1 and decreased oxidative stress, while R126Q treatment did not show any effects. Proximity-labeling employing an APEX2-FABP4 fusion protein revealed several proteins functioning in desmosomes as eFABP4 receptor candidates including desmoglein (DSG), desmocollin, junction plankoglobin, desomoplankin, and cytokeratins. AlphaFold modeling predicted an interaction between eFABP4, and the extracellular cadherin repeats of DSG2 and pull-down and immunoprecipitation assays confirmed complex formation that was potentiated by oleic acid. Silencing of DSG2 in MCF-7 cells attenuated eFABP4 effects on cellular proliferation, pERK levels, and ALDH1A1 expression compared with controls. IMPLICATIONS: These results suggest desmosomal proteins, and in particular desmoglein 2, may function as receptors of eFABP4 and provide new insight into the development and progression of obesity-associated cancers.

Inflammatory Macrophage Interleukin-1ß Mediates High-Fat Diet-Induced Heart Failure With Preserved Ejection Fraction.

Diabetes mellitus (DM) is a main risk factor for diastolic dysfunction (DD) and heart failure with preserved ejection fraction. High-fat diet (HFD) mice presented with diabetes mellitus, DD, higher cardiac interleukin (IL)-1ß levels, and proinflammatory cardiac macrophage accumulation. DD was significantly ameliorated by suppressing IL-1ß signaling or depleting macrophages. Mice with macrophages unable to adopt a proinflammatory phenotype were low in cardiac IL-1ß levels and were resistant to HFD-induced DD. IL-1ß enhanced mitochondrial reactive oxygen species (mitoROS) in cardiomyocytes, and scavenging mitoROS improved HFD-induced DD. In conclusion, macrophage-mediated inflammation contributed to HFD-associated DD through IL-1ß and mitoROS production.

An NMR portrait of functional and dysfunctional allosteric cooperativity in cAMP-dependent protein kinase A.

The cAMP-dependent protein kinase A (PKA) is the archetypical eukaryotic kinase. The catalytic subunit (PKA-C) structure is highly conserved among the AGC-kinase family. PKA-C is a bilobal enzyme with a dynamic N-lobe, harbouring the Adenosine-5'-triphosphate (ATP) binding site and a more rigid helical C-lobe. The substrate-binding groove resides at the interface of the two lobes. A distinct feature of PKA-C is the positive binding cooperativity between nucleotide and substrate. Several PKA-C mutations lead to the development of adenocarcinomas, myxomas, and other rare forms of liver tumours. Nuclear magnetic resonance (NMR) spectroscopy shows that these mutations disrupt the allosteric communication between the two lobes, causing a drastic decrease in binding cooperativity. The loss of cooperativity correlates with changes in substrate fidelity and reduced kinase affinity for the endogenous protein kinase inhibitor (PKI). The similarity between PKI and the inhibitory sequence of the kinase regulatory subunits suggests that the overall mechanism of regulation of the kinase may be disrupted. We surmise that a reduced or obliterated cooperativity may constitute a common trait for both orthosteric and allosteric mutations of PKA-C that may lead to dysregulation and disease.

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.

ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.

Obesity and Pancreatic Cancer: Recent Progress in Epidemiology, Mechanisms and Bariatric Surgery.

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.

Immune Modulation of Adipocyte Mitochondrial Metabolism.

Immune cells infiltrate adipose tissue as a function of age, sex, and diet, leading to a variety of regulatory processes linked to metabolic disease and dysfunction. Cytokines and chemokines produced by resident macrophages, B cells, T cells and eosinophils play major role(s) in fat cell mitochondrial functions modulating pyruvate oxidation, electron transport and oxidative stress, branched chain amino acid metabolism, fatty acid oxidation, and apoptosis. Indeed, cytokine-dependent downregulation of numerous genes affecting mitochondrial metabolism is strongly linked to the development of the metabolic syndrome, whereas the potentiation of mitochondrial metabolism represents a counterregulatory process improving metabolic outcomes. In contrast, inflammatory cytokines activate mitochondrially linked cell death pathways such as apoptosis, pyroptosis, necroptosis, and ferroptosis. As such, the adipocyte mitochondrion represents a major intersection point for immunometabolic regulation of central metabolism.

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.

Inflammasome Activation and Pyroptosis via a Lipid-regulated SIRT1-p53-ASC Axis in Macrophages From Male Mice and Humans.

Obesity-linked diabetes is associated with accumulation of proinflammatory macrophages into adipose tissue leading to inflammasome activation and pyroptotic secretion of interleukin (IL)-1ß and IL-18. Targeting fatty acid binding protein 4 (FABP4) uncouples obesity from inflammation, attenuates characteristics of type 2 diabetes and is mechanistically linked to the cellular accumulation of monounsaturated fatty acids in macrophages. Herein we show that pharmacologic inhibition or genetic deletion of FABP4 activates silent mating type information regulation 2 homolog 1 (SIRT1) and deacetylates its downstream targets p53 and signal transducer and activator of transcription 3 (STAT3). Pharmacologic inhibition of fatty acid synthase or stearoyl-coenzyme A desaturase inhibits, whereas exogenous addition of C16:1 or C18:1 but not their saturated acyl chain counterparts, activates SIRT1 and p53/STAT3 signaling and IL-1ß/IL-18 release. Expression of the p53 target gene ASC [apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (CARD)] required for assembly of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is downregulated in FABP4 null mice and macrophage cell lines leading to loss of procaspase 1 activation and pyroptosis. Concomitant with loss of ASC expression in FABP4-/- macrophages, inflammasome activation, gasdermin D processing, and functional activation of pyroptosis are all diminished in FABP4 null macrophages but can be rescued by silencing SIRT1 or exogenous expression of ASC. Taken together, these results reveal a novel lipid-regulated pathway linking to SIRT1-p53-ASC signaling and activation of inflammasome action and pyroptosis.

Fatty acid binding protein 4 regulates pancreatic cancer cell proliferation via activation of nuclear factor E2-related factor 2.

BACKGROUND: Obesity and diabetes are associated with an increased incidence of pancreatic cancer. Fatty acid binding protein 4 (FABP4), noted to be higher in patients with severe obesity, is linked to the development and progression of several cancers, and its level in the circulation decreases after bariatric surgery. OBJECTIVE: In this paper, we evaluate the role of FABP4 in pancreatic cancer progression. SETTING: University Hospital and Laboratories, United States. METHODS AND RESULTS: When Panc-1 (human) and Pan02 (mouse) pancreatic cancer cells were treated with FABP4 or the-single-point mutant FABP4 (R126Q, fatty acid binding site mutant), only FABP4 stimulated cellular proliferation. The transcriptional activity of nuclear factor E2-related factor 2 (NRF2) was increased in response to FABP4 but not the R126Q. FABP4 treatment also led to downregulation of reactive oxygen species (ROS) activity. Consistent with induced cell propagation by FABP4, the growth of Pan02 tumor was decreased in FABP4-null animals compared with C57BL/6J controls. CONCLUSION: These results suggest that FABP4 increases pancreatic cancer proliferation via activation of NRF2 and downregulation of ROS activity.

Is Disrupted Nucleotide-Substrate Cooperativity a Common Trait for Cushing's Syndrome Driving Mutations of Protein Kinase A?

Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing's syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure-activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

Stearoyl-CoA Desaturase-Mediated Monounsaturated Fatty Acid Availability Supports Humoral Immunity.

Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The roles of different FA species and their impacts on humoral immunity remain poorly understood. Here, we report that proliferating B cells require monounsaturated FAs (MUFAs) to maintain mitochondrial metabolism and mTOR activity and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Furthermore, B cell-extrinsic stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity.