OSBP-related protein-2 (ORP2) : a novel Akt effector that controls cellular energy metabolism

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.Peer reviewe

Role for formin-like 1-dependent acto-myosin assembly in lipid droplet dynamics and lipid storage

Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.Peer reviewe

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)—lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3β(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER–LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER–LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281–1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation

HSP70 induces liver X receptor pathway activation and cholesterol reduction in vitro and in vivo

Objective: Heat Shock Proteins (HSPs) maintain cellular homeostasis under stress. HSP70 represents a major stress-inducible family member and has been identified as a druggable target in inherited cholesterol-sphingolipid storage diseases. We investigated if HSP70 modulates cholesterol accumulation in more common conditions related to atherogenesis. Methods: We studied the effects of recombinant HSP70 in cholesterol-laden primary macrophages from human blood donors and pharmacological HSP70 upregulation in high-cholesterol diet fed zebrafish. Results: Recombinant HSP70 facilitated cholesterol removal from primary human macrophage foam cells. RNA sequencing revealed that HSP70 induced a robust transcriptional re-programming, including upregulation of key targets of liver X receptors (LXR), master regulators of whole-body cholesterol removal. Mechanistically, HSP70 interacted with the macrophage LXRalpha promoter, increased LXRalpha and its target mRNAs, and led to elevated levels of key proteins facilitating cholesterol efflux, including ATP-binding cassette transporters A1 and G1. Pharmacological augmentation of endogenous HSP70 in high-cholesterol diet fed zebrafish activated LXR and its target mRNAs and reduced cholesterol storage at the whole organism level. Conclusion: These data demonstrate that HSP70 exerts a cholesterol lowering effect in primary human cells and animals and uncover a nuclear action of HSP70 in mediating cross-talk between HSP and LXR transcriptional regulation. (C) 2019 The Authors. Published by Elsevier GmbH.Peer reviewe

Ensuring the ductility and rotational ability of a fin plate connection

Ripalevyliitoksia (fin plate connection) käsitellään rungon globaalissa tarkastelussa tavallisesti nivelinä. Todellisuudessa ruuviryhmällä on kuitenkin liitoksen mitoituksen kannalta merkittävä kiertymisjäykkyys ja momenttikapasiteetti, minkä vuoksi todellisten ruuvivoimien laskeminen on vaikea tehtävä. Kantavan rakenneosan ja ruuvikokoonpanon kiertymisjäykkyydet yhdessä määräävät todellisen momenttikuvion muodon. Jotta liitos ei menettäisi kantokykyään enneaikaisesti palkin pakottaman kiertymän takia, liitoksella on oltava riittävä sitkeys ja kiertymiskyky. Sitkeän liitoksen suunnittelussa avainasemassa on oikeiden ruuvikokojen ja levypaksuuksien valinta siten, että reunapuristusmyötääminen on mahdollista ennen kuin ruuvit leikkaantuvat. Eräs kirjallisuudesta löytyvä ripalevyliitoksen kiertymiskyvyn ja sitkeyden Eurokoodiin perustuva varmentamismenettely löytyy kirjasta ECCS No 126. Siinä ruuvikokoonpanoa kuormitetaan puhtaalla taivutusmomentilla ja määrätään murtotapa sitkeäksi. Reunapuristusmyötäämisen ollessa sitkeyden perustana on epäselvää, kuinka suuri muodonmuutos reiälle voidaan sallia, kun reunapuristuskestävyys Eurokoodin mukaan laskettuna on suurempi kuin ruuvin leikkauskestävyys. Ljubljanan yliopistossa tehtyjen tutkimusten mukaan Eurokoodin mukainen reunapuristuskestävyys on konservatiivisella puolella, mikä sitkeyden kannalta johtaa epävarmalle puolelle. Erityisesti kysymys koskee korkeita liitoksia, joissa pienikin kiertymä vaatii suuren muodonmuutoksen kauimpana ruuviryhmän painopisteestä olevissa rei’issä. Asiaa tutkittiin epälineaariseen elementtimenetelmään perustuvin vertailulaskelmin. Havaittiin, että kun reunapuristuskestävyys mitoitetaan yhtä suureksi ruuvin leikkauskestävyyden kanssa, reiän maksimimuodonmuutos ruuvin murtuessa on enimmillään noin 0,1d riippuen voiman suunnasta. Reunapuristuksesta myötäävien reikien osuus liitoksen kokonaiskiertymästä vaihtelee suuresti, mutta enimmäisarvio 80 – 90 % kattaa suurimman osan käytännön tapauksista. Näiden lähtötietojen pohjalta kehitettiin uusi yksinkertaisempi menettely kiertymiskyvyn varmentamiseen. Lisäksi havaittiin, että yksileikkeisissä liitoksissa ruuveille kehittyy huomattavia vetovoimia ja että korkeissa liitoksissa ruuvivoimien pystykomponenttien jakauma noudattaa lujuusopin mukaista poikkileikkauksen leikkausjännitysjakaumaa

Continuous Grading of Early Fibrosis in NAFLD Using Label-Free Imaging : A Proof-of- Concept Study

Maksan rasvoittumisen ajatellaan joskus olevan vain metabolisen oireyhtymän hyvälaatuinen maksamanifestaatio, vaikka se johtaa, erityisesti maksan fibrotisoituessa, kasvaneeseen kardiovaskulaarikuolleisuuteen ja pahimmillaan maksasyöpään. Tästä kansanterveydellisesti merkittävästä roolista huolimatta rasvamaksa on alidiagnosoitu ja sen patogeneettiset mekanismit ovat huonosti ymmärrettyjä, molemmat osin hyvien diagnostisten työkalujen puutteesta johtuen. Päätimme selvittää uusien non-lineaaristen kuvantamismodaliteettien soveltuvuutta rasvamaksan varhaisessa diagnostiikassa. Selvitimme 32 lihavuusleikkauspotilaan maksakudoskoepalojen kudosarkkitehtuurin perinteisin kudosvärjäysmenetelmin ja vertasimme tuloksia samojen potilaiden non-lineaarisesti kuvannettuihin näytteisiin. Kuvansimme minimaalisesti käsitellyistä jääleikkeistä coherent anti-Stokes Raman scattering (CARS) ja second harmonic generation (SHG) signaalit, joita hyödyntäen arvioimme näytteiden rasva- ja kollageenipitoisuuden. Kehitimme myös kuva-analytiikka-algoritmin, joka mahdollistaa kollageenisignaalin automatisoidun ja objektiivisen arvioimisen. Näytteistä mittaamamme SHG- ja CARS-signaalit korreloivat hyvin patologien arvioihin näytteiden fibrotisoitumis- ja rasvoittumisasteista, minkä lisäksi automatisoitu algoritmimme erotteli luotettavasti jopa varhaisen fibroosin normaalista kudoksesta. Myös terveiksi luokiteltujen näytteiden maksakudoksen keskellä esiintyi hienoa fibrillaarista SHG-signaalia. Tunnistamamme SHG-signaali oli spesifiä kolokalisoituen hyvin kollageeni-I- ja III-vasta-ainevärjäyksien kanssa. Tuloksemme osoittavat, että multimodaalisten kuvantamismenetelmien avulla voidaan tunnistaa maksan fibrillaarista kollageenia perinteisiä kudostutkimusmenetelmiä herkemmin. Kehittämämme kuva-analyysi-algoritmi puolestaan arvioi varhaisen fibroosin määrän nopeasti, objektiivisesti ja jatkuvalla skaalalla. Yhdessä nämä löydökset luovat edellytykset tutkia esimerkiksi maksan vähäisen fibroosin kliinistä merkitystä ja paranemistaipumusta isommissa potilasaineistoissa

A loss-of-function variant in OSBPL1A predisposes to low plasma HDL cholesterol levels and impaired cholesterol efflux capacity

Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. Methods: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. Results: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p <0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1-mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a loss-of-function. Conclusions: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias. (C) 2016 Elsevier Ireland Ltd. All rights reserved.Peer reviewe

Lipid Metabolic Reprogramming Extends beyond Histologic Tumor Demarcations in Operable Human Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignancies and potentially curable only with radical surgical resection at early stages. The tumor microenvironment has been shown to be central to the development and progression of PDAC.A better understanding of how early human PDAC metabolically communicates with its environment and differs from healthy pancreas could help improve PDAC diagnosis and treatment. Here we performed deep proteomic analyses from diagnostic specimens of operable, treatment-naive PDAC patients (n 14), isolating four tissue compartments by laser-capture microdissection: PDAC lesions, tumor-adjacent but morphologically benign exocrine glands, and connective tissues neighboring each of these compartments. Protein and pathway levels were compared between compartments and with control pancreatic proteomes. Selected targets were studied immunohistochemically in the 14 patients and in additional tumor microarrays, and lipid deposition was assessed by nonlinear label-free imaging (n = 16). Widespread downregulation of pancreatic secretory functions was observed, which was paralleled by high cholesterol biosynthetic activity without prominent lipid storage in the neoplastic cells. Stromal compartments harbored ample blood apolipoproteins, indicating abundant microvasculature at the time of tumor removal. The features best differentiating the tumor-adjacent exocrine tissue from healthy control pancreas were defined by upregulation of proteins related to lipid transport. Importantly, histologically benign exocrine regions harbored the most significant prognostic pathways, with proteins involved in lipid transport and metabolism, such as neutral cholesteryl ester hydrolase 1, associating with shorter survival. In conclusion, this study reveals prognostic molecular changes in the exocrine tissue neighboring pancreatic cancer and identifies enhanced lipid transport and metabolism as its defining features