1326 Diabetes Volume 63, April 2014 Richard A. Zuellig,1 Thorsten Hornemann,2,3,4 Alaa Othman,2,4 Adrian B. Hehl,5 Heiko Bode,2,3 Tanja Güntert,3,6 Omolara O. Ogunshola,6 Enrica Saponara,7 Kamile Grabliauskaite,7 Jae-Hwi Jang,7 Udo Ungethuem,7 Yu Wei,2,3,4 Arnold von Eckardstein,2,3,4 Rolf Graf,7 and Sabrina Sonda7 Deoxysphingolipids, Novel Biomarkers for Type 2 Diabetes, Are Cytotoxic for Insulin- Producing Cells Irreversible failure of pancreatic b-cells is the main 1-deoxysphinganine contribute to its cytotoxicity. culprit in the pathophysiology of diabetes, a disease Analyses of signaling pathways identified Jun that is now a global epidemic. Recently, elevated N-terminal kinase and p38 mitogen-activated plasma levels of deoxysphingolipids, including protein kinase as antagonistic effectors of cellular 1-deoxysphinganine, have been identified as a novel senescence. The results revealed that biomarker for the disease. In this study, we analyzed 1-deoxysphinganine is a cytotoxic lipid for insulin- whether deoxysphingolipids directly compromise producing cells, suggesting that the increased levels the functionality of insulin-producing Ins-1 cells and of this sphingolipid observed in diabetic patients primary islets. Treatment with 1-deoxysphinganine may contribute to the reduced functionality of PATHOPHYSIOLOGY induced dose-dependent cytotoxicity with pancreatic b-cells. Thus, targeting senescent, necrotic, and apoptotic characteristics deoxysphingolipid synthesis may complement the and compromised glucose-stimulated insulin currently available therapies for diabetes. secretion. In addition, 1-deoxysphinganine altered Diabetes 2014;63:1326–1339 | DOI: 10.2337/db13-1042 cytoskeleton dynamics, resulting in intracellular accumulation of filamentous actin and activation of the Rho family GTPase Rac1. Moreover, In the past 3 decades, the prevalence of diabetes has 1-deoxysphinganine selectively upregulated risen worldwide at a dramatic rate, with incidence pro- ceramide synthase 5 expression and was converted jections approaching 8% of the population by 2030 (1,2). to 1-deoxy-dihydroceramides without altering This remarkable increase is largely due to the epidemic normal ceramide levels. Inhibition of intracellular spread of type 2 diabetes (T2DM), which accounts for 1-deoxysphinganine trafficking and ceramide 90% of all cases of diabetes worldwide (3,4). Given the synthesis improved the viability of the cells, level of complexity associated with the pathophysiology indicating that the intracellular metabolites of of T2DM, understanding the mechanisms underlying this 1Division of Endocrinology, Diabetes and Clinical Nutrition, University Hospital Corresponding author: Sabrina Sonda, [email protected]. Zurich, Zurich, Switzerland Received 3 July 2013 and accepted 14 December 2013. 2Institute for Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland This article contains Supplementary Data online at http://diabetes 3Centre for Integrative Human Physiology, University of Zurich, Zurich, Switzerland .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1042/-/DC1. 4Competence Centre for Systems Physiology and Metabolic Diseases, Zurich, Switzerland R.A.Z. and T.H. contributed equally to this work. 5Institute of Parasitology, University of Zurich, Zurich, Switzerland © 2014 by the American Diabetes Association. See http://creativecommons 6Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland .org/licenses/by-nc-nd/3.0/ for details. 7Swiss Hepato-Pancreatico-Biliary (HPB)-Center, Division of Surgical Research, See accompanying article, p. 1191. Department of Visceral and Transplantation Surgery, University Hospital Zurich, Zurich, Switzerland diabetes.diabetesjournals.org Zuellig and Associates 1327 disease is necessary to design alternative strategies to Animal Experiments limit its progression. Recently, substantial improve- Wistar rats, leptin-deficient ob/ob mice on C57BL/6J ments occurred in the detection of early stage or un- background (B6.V-Lep/OlaHsd), and wild-type (WT) diagnosed T2DM, thus allowing appropriate treatments C57BL/6J (Harlan Laboratories) were kept under a light- in high-risk populations. One of the latest biomarkers dark regimen (16:8 h) at constant temperature and given identified in patients with diabetes and metabolic free access to food and water. All animal experiments syndromes are increased plasma levels of deoxy- were performed in accordance with Swiss federal animal sphingolipids (1-deoxySLs) (5,6), a type of sphingolipid regulations and approved by the cantonal veterinary of- characterized by an initial condensation of alanine or fice of Zurich. Islets were harvested from pancreata of glycine instead of serine with palmitic acid and the male Wistar rats (250–300 g) by collagenase (NB8 col- resultant absence of the hydroxyl group in position C1. lagenase; Serva, Heidelberg, Germany) followed by tryp- Consequently, although these deoxysphingoid bases can sin digestion to dissociate them into single cells as be acylated to deoxy-dihydroceramides, they cannot be previously described (13). further metabolized to complex sphingolipids or effi- ciently degraded by the canonical degradation pathway; Insulin Secretion thus, they tend to accumulate once produced. Impor- Dissociated islets cells were seeded in 12-well extracel- – tantly,1-deoxySLsdisplaytoxicpropertiesinvitro lular matrix (ECM) coated plates (Novamed, Jerusalem, m toward several cell lines (7–9), and in vivo, 1-deoxySLs Israel); treated for 24 h with 5 mol/L sphinganine, are believed to impair neuronal functionality in patients 1-deoxysphinganine, or BSA; and incubated in RPMI with the hereditary sensory and autonomic neuropathy medium containing 3.3 mmol/L glucose for 1 h. Follow- type I (10). In light of the increased plasma levels of ing sequential 1-h incubations with low (3.3 mmol/L), 1-deoxySLs found in diabetic patients and of the high (16.7 mmol/L), low (3.3 mmol/L) glucose concen- reported cytotoxic effects associated with the exposure trations, insulin secretion was measured by radioimmu- to increased 1-deoxySL concentrations, we investigated noassay (Insulin-CT; CIS Bio International, Schering AG, ’ whether these atypical sphingolipids directly compro- Baar, Switzerland) according to the manufacturer s mise pancreatic b-cells, the dysfunction of which plays instructions. an important role in the pathogenesis of both type 1 Quantitative RT-PCR diabetes and T2DM. Total RNA was extracted from Ins-1 cells cultured in m RESEARCH DESIGN AND METHODS 1 mol/L sphinganine or 1-deoxysphinganine for 24 h. Quality of RNA was assessed by a 2100 bioanalyzer Biochemical Reagents (Agilent Technologies, Basel, Switzerland). cDNA was Unless otherwise stated, all chemicals were purchased obtained with the RT2 First Strand Kit and profiled by from Sigma and cell culture reagents from Gibco-BRL. the Rat Cell Death Pathway Finder PCR Array (both from Inhibitor stock solutions were freshly diluted to the SABiosciences, Hombrechtikon, Switzerland) according concentrations required for the individual experiment to the manufacturer’s instructions. Ceramide synthase indicated in the figure legends. Lipid stock solutions were (CerS) primers for SYBR green quantitative PCR are prepared as previously described (10) as a bovine serum listed in the Supplementary Data. albumin (BSA) complex and added to the cells at the concentrations indicated in the figure legends. BSA was Immunohistochemistry and Flow Cytometry Analyses fi used as control. Pancreas specimens were xed in 4% formalin and par- affin embedded according to standard procedures (14). In Vitro Cell Culture Ins-1 cells were fixed in 3.6% formaldehyde and per- The Ins-1 rat insulinoma cell clone 832/13, provided by meabilized with 0.2% Triton X-100 in PBS. Primary C. Wollheim, was maintained in RPMI 1640 medium as antibodies used in this study are listed in the Supple- previously described (11,12). Cell metabolic activity was mentary Data. Apoptosis detection was performed with tested with the 0.5% tetrazolium salt solution 3-(4,5- an ApopTag peroxidase kit (MP Biomedicals, Illkirch, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide France). Immunofluorescence analysis and image data (MTT) or WST-1 (Roche) according to the manufacturer’s collection were performed on a Zeiss Axioplan 2 imaging instructions. Cell death was quantified by trypan blue fluorescence microscope (Carl Zeiss Microimaging, exclusion or lactate dehydrogenase (LDH) release in the Göttingen, Germany) or on a Leica SP2 AOBS confocal medium (Roche). Cellular senescence was quantified with laser-scanning microscope (Leica Microsystems, Wetzlar, the b-galactosidase assay kit (Cell Biolabs). Adenovirus- Germany) using a glycerol immersion objective lens expressing p21 (Adp21) (rat) and adenovirus-containing (Leica, HCX PL APO CS 633 1.3 Corr). Image z-stacks green fluorescent protein (AdGFP) were purchased from were collected with a pinhole setting of Airy 1 and two- Vector Biolabs (Philadelphia, PA). Rac1 activity was fold oversampling. Image stacks of optical sections were measured with G-LISA Rac1 activation assay (Cytoskeleton, processed with the Huygens deconvolution software Denver, CO). package version 2.7 (Scientific Volume Imaging, 1328 Deoxysphingolipids and Diabetes Diabetes Volume 63, April 2014 Hilversum, the Netherlands). Three-dimensional (3D) (5,6), we analyzed whether 1-deoxySLs can directly reconstruction, volume rendering, and quantification of affect the viability of insulin-secreting cells.