Diabetes Care Volume 43, September 2020 2183 – Ele Ferrannini,1 Ashwin C. Murthy,2 Mechanisms of Sodium Glucose Yong-hoLee,3 ElzaMuscelli,1 SophieWeiss,4 Rachel M. Ostroff,4 Naveed Sattar,5 Cotransporter 2 Inhibition: Stephen A. Williams,4 and Peter Ganz6 Insights From Large-Scale Proteomics Diabetes Care 2020;43:2183–2189 | https://doi.org/10.2337/dc20-0456 OBJECTIVE To assess the effects of empagliflozin, a selective sodium–glucose cotransporter 2 (SGLT2) inhibitor, on broad biological systems through proteomics. RESEARCH DESIGN AND METHODS Aptamer-based proteomics was used to quantify 3,713 proteins in 144 paired plasma samples obtained from 72 participants across the spectrum of glucose tolerance before and after 4 weeks of empagliflozin 25 mg/day. The biology of the plasma proteins significantly changed by empagliflozin (at false discovery rate– corrected P < 0.05) was discerned through Ingenuity Pathway Analysis. RESULTS Empagliflozin significantly affected levels of 43 proteins, 6 related to cardiomyocyte function(fattyacid–bindingprotein3and4[FABPA],neurotrophic receptortyrosine kinase, renin, thrombospondin 4, and leptin receptor), 5 to iron handling (ferritin heavy chain 1, transferrin receptor protein 1, neogenin, growth differentiation CARDIOVASCULAR AND METABOLIC RISK factor 2 [GDF2], and b2-microglobulin), and 1 to sphingosine/ceramide metabolism 1CNR Institute of Clinical Physiology, Pisa, Italy (neutral ceramidase), a known pathway of cardiovascular disease. Among the 2 fi Cardiovascular Division, Department of Medi- protein changes achieving the strongest statistical signi cance, insulin-like binding cine, Hospital of the University of Pennsylvania, factor protein-1 (IGFBP-1), transgelin-2, FABPA, GDF15, and sulphydryl oxidase Philadelphia, PA 2 precursor were increased, while ferritin, thrombospondin 3, and Rearranged 3Department of Medicine, Severance Hospital, during Transfection (RET) were decreased by empagliflozin administration. Yonsei University College of Medicine, Seoul, South Korea 4 CONCLUSIONS SomaLogic, Inc., Boulder, CO 5Institute of Cardiovascular and Medical Scien- SGLT2 inhibition is associated, directly or indirectly, with multiple biological effects, ces, University of Glasgow, Glasgow, U.K. including changes in markers of cardiomyocyte contraction/relaxation, iron han- 6Zuckerberg San Francisco General Hospital, dling, and other metabolic and renal targets. The most significant differences were University of California, San Francisco, CA detected in protein species (GDF15, ferritin, IGFBP-1, and FABP) potentially related Corresponding author: Ele Ferrannini, ferranni@ ifc.cnr.it to the clinical and metabolic changes that were actually measured in the same Received 5 March 2020 and accepted 24 April patients. These novel results may inform further studies using targeted proteomics 2020 and a prospective design. This article contains supplementary material online at https://doi.org/10.2337/figshare.12195018. Sodium–glucose cotransporter 2 (SGLT2) is a transmembrane protein encoded by © 2020 by the American Diabetes Association. genes of the SLC5A family (1). SGLT2 is a high-capacity, low-affinity transporter almost Readers may use this article as long as the work is properly cited, the use is educational and not for exclusively expressed on the luminal side of the S1 segment of the proximal renal profit, and the work is not altered. More infor- tubule. Its function is to reabsorb glucose and sodium from the glomerular filtrate into mation is available at https://www.diabetesjournals the circulation. Convincing evidence shows that SGLT2 is functionally overactive in .org/content/license. 2184 Proteomics of SGLT2 Inhibitors Diabetes Care Volume 43, September 2020 patients with type 2 diabetes (T2D), in T2D or impaired glucose tolerance (IGT) modified aptamers to generate mod- whom it therefore contributes to the receiving SGLT2i treatment by applying ified aptamer-protein complexes un- hyperglycemia (2). SGLT2 inhibition re- a large-scale version of an aptamer- der equilibrium conditions. Unbound sults in urinary excretion of glucose, based multiplex proteomics platform modified aptamers and unbound or sodium, and water, leading to hemody- (SOMAscan) to quantify plasma proteins nonspecifically bound proteins were namic changes as well as glycemic im- (11). eliminated by two bead-based immo- provements in patients with T2D (1). bilization steps. After elution of the SGLT2 inhibitors (SGLT2i) have been de- RESEARCH DESIGN AND METHODS modified aptamers from the target veloped as glucose-lowering drugs for Study Population protein, the fluorescently labeled mod- the treatment of T2D. Somewhat un- A total of 72 subjects (25 women and ified aptamers were directly quantified expectedly, large cardiovascular (CV) 47 men) were recruited into the study on hybridization array (Agilent Tech- safety trials testing SGLT2i against pla- (Supplementary Table 1). Of these, nologies). Calibrators were included so cebo in patients with T2D at high CV risk 61 were patients with T2D (29 were that the degree of fluorescence was a have consistently demonstrated clini- drug naive or off any glucose-lowering consistentreflection of protein concen- cally relevant benefit for both CV and agent for at least 12 weeks, and 32 were tration. Protein concentration was ex- renal outcomes (3). This has led to on a stable dose of metformin of $1,500 pressed as relative fluorescence units. changes in clinical recommendations and mg/day for at least 12 weeks). Inclusion All samples were placed randomly on practice (4). Because the glucose-lowering criteria were either sex, age .18 years, 96-well plates, runin a singlebatch, and fi 2 ef cacy of SGLT2i is generally modest BMI 20–40 kg/m , HbA1c 6.5–10.5% [48– normalized against protein calibrator (i.e., HbA1c reductions in the range of 91 mmol/mol], and estimated glomeru- samplesincludedoneachplate.Tech- 0.6–0.8%), it is widely believed that lar filtration rate (eGFR) .60 mL/min/ nicianswereblindedtothebeforeand mechanisms other than (or in addition 1.73 m2 (by the Chronic Kidney Disease after status of samples. Details of scal- to) glycemic control must be at work to Epidemiology Collaboration equation). ing, normalization, and control of batch explain the CV and renal protection of Duration of diabetes was ,1 year in effects are given in Williams et al. (8). SGLT2i. The recently published Dapagli- 3%, ,5 years in 18%, ,10 years in Median intra- and interassay coeffi- flozin and Prevention of Adverse Out- 26%, and $10 years in 19% of the cients of variation are ;5% (16). A comes in Heart Failure (DAPA-HF) results patients. Exclusion criteria were history total of 4,005 proteins were measured; also suggest that SGLT2i lessen risk of of malignancy in the past 5 years, sig- 292 of them failed to pass SomaLogic’s heart failure by mechanisms indepen- nificant CV disorder within the past quality control metrics, leaving 3,713 dent of glycemia, since benefits were 6 months, pregnancy or women expect- proteins for analysis. Differences be- identical in those with and without di- ing to conceive within the study duration, tween the baseline and 4-week samples abetes (5). Multiple hypotheses have bariatric surgery within the past 2 years, were expressed as log2 ratios for each of been advanced, not necessarily exclusive treatment with antiobesity drugs in the the proteins measured. of one another. Thus, SGLT2i-induced past 3 months, neurogenic bladder dis- Plasma glucose and creatinine concen- natriuresis and osmotic diuresis with orders, ALT and AST .3.03 the upper trations were measured by standard blood volume contraction and increased limit of normal, changes in thyroid hor- laboratory methods. Creatinine clear- hematocrit may be responsible for the mone dosage within 6 weeks or any other ance was measured on carefully col- reduction in blood pressure and arterial endocrine disease except T2D, and alco- lected urine samples as the ratio of stiffness, which can improve cardiac hol or drug abuse. Detailed metabolic urinary creatinine excretion and plasma function by decreasing both pre- and analysis of these patients has been pub- creatinine levels. afterload (6). Also, SGLT2i may optimize lished previously (12). Eleven subjects tissue energy metabolism and mitochon- with IGT (by American Diabetes Associ- Statistical Analysis drial bioenergetics in impaired heart and ation criteria) were included; their an- Differences between on-treatment and kidney by supplying an excess of “thrifty” thropometric and metabolic characteristics baseline values were analyzed by the substrates (e.g., ketones) (7). Reduction have been previously reported (13). Wilcoxon signed rank test for paired of uricemia, modulation of the tubu- comparisons in 3,713 protein measure- loglomerular feedback, and lowering of Protocol ments. P values were corrected for inflammation and fibrosis biomarkers (8) One hundred forty-four fasting EDTA multiple comparisons using both the are additional putative contributors to plasma samples were obtained from Bonferroni familywise error rate cor- reported benefits (9). the subjects at baseline and after 4 weeks rection and the Benjamini-Hochberg From the in vivo mechanistic informa- of treatment with 25 mg/day empagli- false discovery rate (FDR) correction. tion so far gathered, it is likely that the flozin. The modified aptamer binding Proteins with an FDR-adjusted value of primary action of SGLT2i on the proximal reagents and SOMAscan assay and its P , 0.05 were considered statistically renal tubule induces a