Identification and characterization of Drosophila lipid droplet-associated proteins

An essential component of organismic energy homeostasis is the incorporation of storage fat (triacylglycerol/TAG) in intracellular lipid droplets (LD) in fat storage tissues. LD share a stereotyped structure of a hydrophobic storage fat core surrounded by a phospholipid monolayer and associated proteins which form the lipid droplet-associated proteome (LDP). A regulatory function in fat storage has been demonstrated for certain vertebrate members of the LDP. This study characterizes the evolutionary conserved function of a member of the LDP, the Drosophila Perilipin-like PAT-domain protein LSD-2. As observed in Perilipin mutant mice, Lsd-2 mutant flies are lean, whereas fat storage tissue specific overexpression of LSD-2 causes a dosage-dependant increase of the organismic TAG-content. The studies provide evidence for the conserved functional potential of the lipid droplet surface for TAG-storage in Drosophila. In order to assess the LDP composition in a systematic manner, the proteins from isolated LDs from larval fat storage tissue of individuals with different TAG-content were investigated by mass spectroscopy. In total, 271 proteins were identified, reflecting the metabolically active character of these cellular organelles. Tissue culture transfection studies with GFP-tagged candidate proteins confirm their LD-association in vivo and imply heterogeneity of the lipid droplet population in individual cells indicating functional specialization of single LDs.Essentieller Bestandteil der organismischen Energie-Homöostase ist die Einlagerung von Speicherfett (Triazylglyzerol/TAG) im Fettspeichergewebe in Form von intrazellulären Lipidtröpfchen (LT). Diese besitzen einen stereotypen Aufbau aus einem hydrophoben Speicherfett-Kern der von einer Phospholipid-Einzelmembran mit assoziierten Proteinen umgeben ist. Diese Proteine werden ingesamt als Lipidtröpfchen-Proteom (LTP) bezeichnet von dem für einzelne Vertreter in Vertebraten eine regulatorische Funktion in der Fettspeicherung demonstriert wurde. Die vorliegende Arbeit charakterisiert die evolutionär konservierte Funktion eines Vertreters des LTP, des Perilipin-verwandten Drosophila PAT-Domänen Proteins LSD-2. Ebenso wie Perilipin-mutante Mäuse sind Lsd-2 mutante Fliegen dünn, wohingegen die Überexpression von LSD-2 im Fettspeichergewebe zu einer dosis-abhängigen Zunahme des organismischen TAG-Gehalts führt. Die Studien an LSD-2 belegen die konservierte funktionelle Bedeutung der LT-Oberfläche für die Fettspeicherung in Drosophila. Um die Zusammensetzung des LTP zu bestimmen, wurden die Proteine isolierter Tröpfchen aus larvalem Fettspeichergewebe von Fliegen unterschiedlichem Speicherfettgehalts mittels Massenspektroskopie systematisch untersucht. Insgesamt wurden 271 Proteine identifiziert, die den metabolisch aktiven Charakter dieser Organellen widerspiegeln. Transfektionsexperimente mit GFP-markierten Kandidatenproteinen in Zellkultur bestätigen ihre LT-Assoziation in vivo und legen eine Heterogenität der LT-Population in einzelnen Zellen nahe, die möglicherweise auf eine funktionelle Spezialisierung einzelner LT hindeutet

A stem cell based in vitro model of NAFLD enables the analysis of patient specific individual metabolic adaptations in response to a high fat diet and AdipoRon interference

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disease. Its development and progression depend on genetically predisposed susceptibility of the patient towards several ‘hits’ that induce fat storage first and later inflammation and fibrosis. Here, we differentiated induced pluripotent stem cells (iPSCs) derived from four distinct donors with varying disease stages into hepatocyte like cells (HLCs) and determined fat storage as well as metabolic adaptations after stimulations with oleic acid. We could recapitulate the complex networks that control lipid and glucose metabolism and we identified distinct gene expression profiles related to the steatosis phenotype of the donor. In an attempt to reverse the steatotic phenotype, cells were treated with the small molecule AdipoRon, a synthetic analogue of adiponectin. Although the responses varied between cells lines, they suggest a general influence of AdipoRon on metabolism, transport, immune system, cell stress and signalling

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate E NADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research

Fiscal Centralization, Limited Government, and Public Revenues in Europe, 1650-1913

Old Regime polities typically suffered from fiscal fragmentation and absolutist rule. By the start of World War I, however, many such countries had centralized institutions and limited government. This article uses a new panel data set to perform a statistical analysis of political regimes and public revenues in Europe from 1650 to 1913. Panel regressions indicate that centralized and limited regimes were associated with significantly higher revenues than fragmented and absolutist ones. Structural break tests also suggest close relationships between major turning points in revenue series and political transformations

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. $^{1}$$^{,}$$^{2}$), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. $^{3-8}$); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure

High-Content C. elegans Screen Identifies Natural Compounds Impacting Mitochondria-Lipid Homeostasis and Promoting Healthspan

The aging process is concurrently shaped by genetic and extrinsic factors. In this work, we screened a small library of natural compounds, many of marine origin, to identify novel possible anti-aging interventions in Caenorhabditis elegans, a powerful model organism for aging studies. To this aim, we exploited a high-content microscopy platform to search for interventions able to induce phenotypes associated with mild mitochondrial stress, which is known to promote animal’s health- and lifespan. Worms were initially exposed to three different concentrations of the drugs in liquid culture, in search of those affecting animal size and expression of mitochondrial stress response genes. This was followed by a validation step with nine compounds on solid media to refine compounds concentration, which led to the identification of four compounds (namely isobavachalcone, manzamine A, kahalalide F and lutein) consistently affecting development, fertility, size and lipid content of the nematodes. Treatment of Drosophila cells with the four hits confirmed their effects on mitochondria activity and lipid content. Out of these four, two were specifically chosen for analysis of age-related parameters, kahalalide F and lutein, which conferred increased resistance to heat and oxidative stress and extended animals’ healthspan. We also found that, out of different mitochondrial stress response genes, only the C. elegans ortholog of the synaptic regulatory proteins neuroligins, nlg-1, was consistently induced by the two compounds and mediated lutein healthspan effects

Characterization of the Drosophila lipid droplet subproteome.

Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states

COPI complex is a regulator of lipid homeostasis.

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets