Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures

Diabetes Ther DOI 10.1007/s13300-016-0210-y ORIGINAL RESEARCH Simvastatin Rapidly and Reversibly Inhibits Insulin Secretion in Intact Single-Islet Cultures Valentina Scattolini . Camilla Luni . Alessandro Zambon . Silvia Galvanin . Onelia Gagliano . Catalin Dacian Ciubotaru . Angelo Avogaro . Fabio Mammano . Nicola Elvassore . Gian Paolo Fadini Received: September 23, 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com ABSTRACT are non-physiologic and have limited clinical transferability. Introduction: Epidemiological studies suggest that statins may promote the development or Methods: Here, we study the effect of simvastatin on insulin secretion using exacerbation of diabetes, but whether this single-islet cultures, an optimal compromise occurs through inhibition of insulin secretion is unclear. This lack of understanding is partly between biological observability and physiologic fidelity. We develop and validate a due to the cellular models used to explore this phenomenon (cell lines or pooled islets), which microfluidic device to study single-islet function ex vivo, which allows for switching between media of different compositions with a Enhanced content The online video files are available to view at http://www.medengine.com/Redeem/ 6317F060735F7A3B. resolution of seconds. In parallel, fluorescence imaging provides real-time analysis of the membrane voltage potential, cytosolic Ca2? dynamics, and insulin release during perfusion Electronic supplementary material The online version of this article (doi:10.1007/s13300-016-0210-y) contains supplementary material, which is available to authorized users. under 3 or 11 mM glucose. V. Scattolini
A. Avogaro
G. P. Fadini (&) Department of Medicine, University of Padova, Via Giustiniani 2, 35129 Padua, Italy e-mail: C. Luni
A. Zambon
S. Galvanin
O. Gagliano
N. Elvassore Department of Industrial Engineering, University of Padova, Via Marzolo 9, 35131 Padua, Italy V. Scattolini
C. Luni
A. Zambon
S. Galvanin
O. Gagliano
N. Elvassore (&)
G. P. Fadini Venetian Institute of Molecular Medicine, Via Orus 2, 35128 Padua, Italy e-mail: C. D. Ciubotaru
F. Mammano CNR Institute of Cell Biology and Neurobiology, 00015 Monterotondo, Italy C. Luni Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 99 Haike Road, Shanghai 201210, China F. Mammano Department of Physics, University of Padova, Via Marzolo 8, 35131 Padua, Italy Diabetes Ther Results: We found that simvastatin reversibly accelerate the onset of diabetes remain unclear, inhibits insulin secretion, even in high-glucose. This phenomenon is very rapid (\60 s), occurs and whether statins truly exert any action on 2? without affecting Ca concentrations, and is likely unrelated to cholesterol biosynthesis and protein isoprenylation, which occur on a time span of hours. Conclusions: Our insulin secretion is debated [16, 17]. This is in part because cellular models used to explore this phenomenon, either cell lines [13, 14] or pooled islets [14], are non-physiologic and lack clinical transferability. Cell lines may behave differently data provide the first real-time live demonstration that a statin inhibits insulin secretion in intact islets and that single islets respond differently from cell lines on a short time scale. Funding: University of from mature beta cells or have incomplete glucose sensor or insulin secretion machinery, whereas pooled islet cultures are poorly suitable to provide information on dynamic insulin secretion because islet Padova, EASD response may be heterogeneous and Foundation. asynchronous. A single islet is the minimum fully functional Keywords: Insulin; unit of the endocrine pancreas. A precise understanding of islet response to exogenous Islet; Microfluidic; Simvastatin; Statin stimuli is better captured in vitro at the single-islet level, devoid of complex and not fully understood islet–islet interactions. INTRODUCTION Microfluidics is a technology suitable for cell culture at micrometer scale. Because a single islet Statins, hydroxy-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, are widely used has an approximate size of 50–500 lm, it is particularly suitable for microfluidic culture. to lower cholesterol and prevent cardiovascular disease [1, 2]. As diabetes is characterized by high Accordingly, a number of microfluidic devices guidelines have been developed for understanding single islet behavior, as recently reviewed [18]. However recommend that most diabetic patients should receive statin therapy [3–5]. Statins effectively the advantages of microfluidic technology go well beyond the size of these devices [19]. For example, reduce cardiovascular morbidity and mortality in patients with diabetes [6], but epidemiological Benninger et al. used a microfluidic device to cardiovascular risk, international studies suggest they increase the risk of developing generate precise glucose concentration gradients in a single islet microenvironment to study diabetes in healthy individuals [7–10] and may worsen glycemia in diabetic patients [11]. cell–cell gap junctions in the regulation of coordinated insulin release [20]. Microfluidic Pathophysiologic studies in humans indicate that statins may affect both insulin sensitivity and devices favor the accumulation of endogenously insulin secretion [12]. In vitro studies show that secreted factors and were previously used to detect islet amyloid polypeptide secretion from as few as statins impair insulin secretion by affecting multiple pathways [13], including cellular ten islets [21] and glucose flux in live myoblasts [22], besides insulin [23]. cholesterol synthesis, membrane fluidity, and isoprenylation of proteins [14]. Though their Here, we study the effects of simvastatin on diabetes insulin secretion from single murine islets. A microfluidic system able to temporally control hazard [15], the mechanisms whereby statins the single-islet soluble microenvironment is cardiovascular benefit exceeds the Diabetes Ther presented. The setup has been technically and Annexin-V (BD Biosciences) was added in the biologically perfusion medium. Prior to microfluidic device validated, before analyzing simvastatin action on intact isolated islets. insertion, islets were loaded with 3-lM Fura Red acetoxymethyl (AM) (ThermoFisher) or 10 lM METHODS Fluo-4 AM (ThermoFisher) for 50 min in RPMI medium at 37 °C for intracellular Ca2? Microfluidic Setup detection; 2-lM FluoZin-3 (ThermoFisher) was The setup was produced by standard photo- and added at the same concentration to the different media used for islet stimulation. soft-lithographic techniques. Details on the fabrication of the islet culture and the multi-inlet chips are given in the Electronic Extracellular Zn2? concentration detection was used as an indirect measure for in (...truncated)