The Third Annual Symposium of the Midwest Aging Consortium.

The geroscience hypothesis suggests that addressing the fundamental mechanisms driving aging biology will prevent or mitigate the onset of multiple chronic diseases, for which the largest risk factor is advanced age. Research that investigates the root causes of aging is therefore of critical importance given the rising healthcare burden attributable to age-related diseases. The third annual Midwest Aging Consortium symposium was convened as a showcase of such research performed by investigators from institutions across the Midwestern United States. This report summarizes the work presented during a virtual conference across topics in aging biology, including immune function in the lung-particularly timely given the Corona Virus Immune Disease-2019 pandemic-along with the role of metabolism and nutrient-regulated pathways in cellular function with age, the influence of senescence on stem cell function and inflammation, and our evolving understanding of the mechanisms underlying observation of sex dimorphism in aging-related outcomes. The symposium focused on early-stage and emerging investigators, while including keynote presentations from leaders in the biology of aging field, highlighting the diversity and strength of aging research in the Midwest.

Organelle interactions compartmentalize hepatic fatty acid trafficking and metabolism.

Organelle interactions play a significant role in compartmentalizing metabolism and signaling. Lipid droplets (LDs) interact with numerous organelles, including mitochondria, which is largely assumed to facilitate lipid transfer and catabolism. However, quantitative proteomics of hepatic peridroplet mitochondria (PDM) and cytosolic mitochondria (CM) reveals that CM are enriched in proteins comprising various oxidative metabolism pathways, whereas PDM are enriched in proteins involved in lipid anabolism. Isotope tracing and super-resolution imaging confirms that fatty acids (FAs) are selectively trafficked to and oxidized in CM during fasting. In contrast, PDM facilitate FA esterification and LD expansion in nutrient-replete medium. Additionally, mitochondrion-associated membranes (MAM) around PDM and CM differ in their proteomes and ability to support distinct lipid metabolic pathways. We conclude that CM and CM-MAM support lipid catabolic pathways, whereas PDM and PDM-MAM allow hepatocytes to efficiently store excess lipids in LDs to prevent lipotoxicity.

Systemic lipolysis promotes physiological fitness in Drosophila melanogaster.

Since interventions such as caloric restriction or fasting robustly promote lipid catabolism and improve aging-related phenotypical markers, we investigated the direct effect of increased lipid catabolism via overexpression of bmm (brummer, FBgn0036449), the major triglyceride hydrolase in Drosophila, on lifespan and physiological fitness. Comprehensive characterization was carried out using RNA-seq, lipidomics and metabolomics analysis. Global overexpression of bmm strongly promoted numerous markers of physiological fitness, including increased female fecundity, fertility maintenance, preserved locomotion activity, increased mitochondrial biogenesis and oxidative metabolism. Increased bmm robustly upregulated the heat shock protein 70 (Hsp70) family of proteins, which equipped the flies with higher resistance to heat, cold, and ER stress via improved proteostasis. Despite improved physiological fitness, bmm overexpression did not extend lifespan. Taken together, these data show that bmm overexpression has broad beneficial effects on physiological fitness, but these effects did not impact lifespan.

Isolated and combined impact of dietary olive oil and exercise on markers of health and energy metabolism in female mice.

An olive oil (OO) rich diet or high-intensity interval training (HIIT) independently improve markers of health and energy metabolism, but it is unknown if combining OO and HIIT synergize to improve these markers. This study characterized the isolated and combined impact of OO and HIIT on markers of health and energy metabolism in various tissues in C57BL/6J female mice. Nine-week-old mice were divided into four groups for a 12-week diet and/or exercise intervention including: (1) Control Diet without HIIT (CD), (2) Control Diet with HIIT (CD+HIIT), (3) OO diet (10% kcal from olive oil) without HIIT, and (4) OO diet with HIIT (OO+HIIT). Neither dietary OO or HIIT altered body weight, glucose tolerance, or serum lipids. HIIT, regardless of diet, increased aerobic capacity and HDL cholesterol levels. In liver and heart tissue, OO resulted in similar adaptations as HIIT including increased mitochondrial content and fatty acid oxidation but combining OO with HIIT did not augment these effects. In skeletal muscle, HIIT increased mitochondrial content in type II fibers similarly between diets. An RNA sequencing analysis on type I fibers revealed OO reduced muscle regeneration and lipid metabolism gene abundance, whereas HIIT increased the abundance of these genes, independent of diet. HIIT training, independent of diet, induced subcutaneous white adipose tissue (sWAT) hypertrophy, whereas OO induced gonadal white adipose tissue (gWAT) hypertrophy, an effect that was augmented with HIIT. These data highlight the pleiotropic effects of OO and HIIT, although their combination does not synergize to further improve most markers of health and energy metabolism.

Perilipins at a glance.

Lipid droplets (LDs) are ubiquitous organelles that store and supply lipids for energy metabolism, membrane synthesis and production of lipid-derived signaling molecules. While compositional differences in the phospholipid monolayer or neutral lipid core of LDs impact their metabolism and function, the proteome of LDs has emerged as a major influencer in all aspects of LD biology. The perilipins (PLINs) are the most studied and abundant proteins residing on the LD surface. This Cell Science at a Glance and the accompanying poster summarize our current knowledge of the common and unique features of the mammalian PLIN family of proteins, the mechanisms through which they affect cell metabolism and signaling, and their links to disease.

Muscle Lipid Droplets: Cellular Signaling to Exercise Physiology and Beyond.

Conventionally viewed as energy storage depots, lipid droplets (LDs) play a central role in muscle lipid metabolism and intracellular signaling, as recognized by recent advances in our biological understanding. Specific subpopulations of muscle LDs, defined by location and associated proteins, are responsible for distinct biological functions. In this review, the traditional view of muscle LDs is examined, and the emerging role of LDs in intracellular signaling is highlighted. The effects of chronic and acute exercise on muscle LD metabolism and signaling is discussed. In conclusion, future directions for muscle LD research are identified. The primary focus will be on human studies, with inclusion of select animal/cellular/non-muscle studies as appropriate, to provide the underlying mechanisms driving the observed findings.

Application of Proteomics in Sarcoidosis.

Sarcoidosis is a multisystem disease with heterogeneity in manifestations and outcomes. System-level studies leveraging "omics" technologies are expected to define mechanisms contributing to sarcoidosis heterogeneous manifestations and course. With improvements in mass spectrometry (MS) and bioinformatics, it is possible to study protein abundance for a large number of proteins simultaneously. Contemporary fast-scanning MS enables the acquisition of spectral data for deep coverage of the proteins with data-dependent or data-independent acquisition MS modes. Studies leveraging MS-based proteomics in sarcoidosis have characterized BAL fluid (BALF), alveolar macrophages, plasma, and exosomes. These studies identified several differentially expressed proteins, including protocadherin-2 precursor, annexin A2, pulmonary surfactant A2, complement factors C3, vitamin-D-binding protein, cystatin B, and amyloid P, comparing subjects with sarcoidosis with control subjects. Other studies identified ceruloplasmin, complement factors B, C3, and 1, and others with differential abundance in sarcoidosis compared with other interstitial lung diseases. Using quantitative proteomics, most recent studies found differences in PI3K/Akt/mTOR, MAP kinase, pluripotency-associated transcriptional factor, and hypoxia response pathways. Other studies identified increased clathrin-mediated endocytosis and Fcγ receptor-mediated phagocytosis pathways in sarcoidosis alveolar macrophages. Although studies in mixed BAL and blood cells or plasma are limited, some of the changes in lung compartment are detected in the blood cells and plasma. We review proteomics for sarcoidosis with a focus on the existing MS data acquisition strategies, bioinformatics for spectral data analysis to infer protein identity and quantity, unique aspects about biospecimen collection and processing for lung-related proteomics, and proteomics studies conducted to date in sarcoidosis.

Hepatic PLIN5 signals via SIRT1 to promote autophagy and prevent inflammation during fasting.

Lipid droplets (LDs) are energy-storage organelles that are coated with hundreds of proteins, including members of the perilipin (PLIN) family. PLIN5 is highly expressed in oxidative tissues, including the liver, and is thought to play a key role in uncoupling LD accumulation from lipotoxicity; however, the mechanisms behind this action are incompletely defined. We investigated the role of hepatic PLIN5 in inflammation and lipotoxicity in a murine model under both fasting and refeeding conditions and in hepatocyte cultures. PLIN5 ablation with antisense oligonucleotides triggered a pro-inflammatory response in livers from mice only under fasting conditions. Similarly, PLIN5 mitigated lipopolysaccharide- or palmitic acid-induced inflammatory responses in hepatocytes. During fasting, PLIN5 was also required for the induction of autophagy, which contributed to its anti-inflammatory effects. The ability of PLIN5 to promote autophagy and prevent inflammation were dependent upon signaling through sirtuin 1 (SIRT1), which is known to be activated in response to nuclear PLIN5 under fasting conditions. Taken together, these data show that PLIN5 signals via SIRT1 to promote autophagy and prevent FA-induced inflammation as a means to maintain hepatocyte homeostasis during periods of fasting and FA mobilization.

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.

Sizing lipid droplets from adult and geriatric mouse liver tissue via nanoparticle tracking analysis.

The significance of lipid droplets in lipid metabolism, cell signaling, and regulating longevity is increasingly recognized, yet the lipid droplet's unique properties and architecture make it difficult to size and study using conventional methods. To begin to address this issue, we demonstrate the capabilities of nanoparticle tracking analysis (NTA) for sizing of lipid droplets. NTA was found to be adequate to assess lipid droplet stability over time, indicating that lipid droplet preparations are stable for up to 24 h. NTA had the ability to compare the size distributions of lipid droplets from adult and geriatric mouse liver tissue, suggesting an age-related decrease in lipid droplet size. This is the first report on the use of NTA to size intracellular organelles. Graphical Abstract Light scattering reveals the temporal positions of individual lipid droplets, which are recorded with a camera. The two-dimensional diffusion constant of each lipid droplet is extracted from the data set, which is then used to calculate a hydrodynamic radius using the Stokes-Einstein equation.

Liver-specific loss of Perilipin 2 alleviates diet-induced hepatic steatosis, inflammation, and fibrosis.

Hepatic inflammation and fibrosis are key elements in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive liver disease initiated by excess hepatic lipid accumulation. Lipid droplet protein Perilipin 2 (Plin2) alleviates dietary-induced hepatic steatosis when globally ablated; however, its role in the progression of NASH remains unknown. To investigate this further, we challenged Plin2 liver-specific knockout mice (designated L-KO) and their respective wild-type (WT) controls with a methionine-choline-deficient (MCD) diet for 15 days to induce a NASH phenotype of increased hepatic triglyceride levels through impaired phosphatidylcholine (PC) synthesis and very-low-density lipoprotein (VLDL) secretion. Results on liver weights, body weights, fat tissue mass, and histology in WT and L-KO mice fed the MCD diet revealed signs of hepatic steatosis, fibrosis, and inflammation; however, these effects were blunted in L-KO mice. In addition, levels of PC and VLDL were unchanged, and hepatic steatosis was reduced in L-KO mice fed the MCD diet, due in part to an increase in remodeling of PE to PC via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). These mice also exhibited decreased hepatic expression of proinflammatory markers cyclooxygenase 2, IL-6, TNF-α, IL-1ß, and reduced expression of endoplasmic reticulum (ER) stress proteins C/EBP homologous protein and cleaved caspase-1. Taken together, these results suggest that Plin2 liver-specific ablation alleviates diet-induced hepatic steatosis and inflammation via a PEMT-mediated mechanism that involves compensatory changes in proteins involved in phospholipid remodeling, inflammation, and ER stress that work to alleviate diet-induced NASH. Overall, these findings support a role for Plin2 as a target for NASH therapy.

Structural and functional assessment of perilipin 2 lipid binding domain(s).

Although perilipin 2 (Plin2) has been shown to bind lipids with high affinity, the Plin2 lipid binding site has yet to be defined. This is of interest since Plin2's affinity for lipids has been suggested to be important for lipid droplet biogenesis and intracellular triacylglycerol accumulation. To define these regions, mouse Plin2 and several deletion mutants expressed as recombinant proteins and in mammalian cells were assessed by molecular modeling, fluorescence binding, circular dichroic, and fluorescence resonance energy transfer techniques to identify the structural and functional requirements for lipid binding. Major findings of this study indicate (1) the N-terminal PAT domain does not bind cholesterol or stearic acid; (2) Plin2 residues 119-251, containing helix α4, the α-ß domain, and part of helix α6 form a Plin3-like cleft found to be important for highest affinity lipid binding; (3) both stearic acid and cholesterol interact favorably with the Plin2 cleft formed by conserved residues in helix α6 and adjacent strands, which is common to all the active lipid-binding constructs; and (4) discrete targeting of the Plin2 mutants to lipid droplets supports Plin2 containing two independent, nonoverlapping lipid droplet targeting domains in its central and C-terminal sequences. Thus, the current work reveals specific domains responsible for Plin2-lipid interactions that involves the protein's lipid binding and targeting functions.