Dmd054254 2148..2157

Previous studies have revealed that the glucoincretin hormone glucagon-like peptide-1 (GLP-1)(7-36)amide is metabolized by dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) to yield GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, as the principal metabolites. Contrary to the previous notion that GLP-1(7-36)amide metabolites are pharmacologically inactive, recent studies have demonstrated cardioprotective and insulinomimetic effects with both GLP-1(9-36)amide and GLP-1(28-36)amide in animals and humans. In the present work, we examined the metabolic stability of the two GLP-1(7-36)amide metabolites in cryopreserved hepatocytes, which have been used to demonstrate the in vitro insulin-like effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis. To examine the metabolic stability of the GLP-1(7-36)amide metabolites, a liquid chromatography–tandem mass spectrometry assay was developed for the quantitation of the intact peptides in hepatocyte incubations. GLP-1(9-36)amide and GLP-1(28-36)amide were rapidly metabolized in mouse [GLP-1(9-36)amide: t1/2 = 52 minutes; GLP-1(28-36)amide: t1/2 = 13 minutes] and human hepatocytes [GLP-1(9-36)amide: t1/2 = 180 minutes; GLP-1(2836)amide: t1/2 = 24 minutes), yielding a variety of N-terminal cleavage products that were characterized using mass spectrometry. Metabolism at the C terminus was not observed for either peptides. The DPP-IV and NEP inhibitors diprotin A and phosphoramidon, respectively, did not induce resistance in the two peptides toward proteolytic cleavage. Overall, our in vitro findings raise the intriguing possibility that the insulinomimetic effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds. Introduction The glucoincretin hormone glucagon-like peptide-1 (GLP-1) is derived from a proglucagon precursor and secreted by intestinal enteroendocrine L cells in response to oral nutrient ingestion (Kieffer and Habener, 1999; Holst, 2007; Lovshin and Drucker, 2009; Mundil et al., 2012). The majority of circulating GLP-1 levels comprise the 30amino acid peptide GLP-1(7-36)amide, which acts through a seventransmembrane-spanning, heterotrimeric class B G protein–coupled receptor on pancreatic b cells to exert glucoregulatory and insulinotropic actions (Thorens, 1992). However, after its secretion from the intestine, native GLP-1(7-36)amide is rapidly degraded [half-life (t1/2) = 1–2 minutes) on its N and C termini by the ubiquitously expressed enzymes dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase 24.11 (NEP) (Hansen et al., 1999; Holst, 2007), respectively, to yield GLP-1(9-36)amide and nonapeptide GLP-1(28-36)amide as metabolites (Deacon et al., 1995a,b; Hupe-Sodmann et al., 1995; Mentlein, 1999). In patients with type 2 diabetes mellitus, secretion of GLP-1 is diminished, and administration of either DPP-IV inhibitors (e.g., sitagliptin) or exogenous GLP-1 analogs (e.g., exenatide and liraglutide) represents a potential therapeutic option in the treatment of type 2 diabetes mellitus (Moore and Saudek, 2008; Edwards, 2013). Until recently, GLP-1(9-36)amide, the major circulating human metabolite of GLP-1(7-36)amide (Deacon et al., 1995a,b), was thought to be an inactive metabolite of GLP-1 due to its weak, if any, insulinotropic activity. However, growing evidence has suggested that GLP-1(9-36)amide possesses unique extrapancreatic insulin-like actions in the heart, vasculature, and liver, which appear to be mediated independently of the GLP-1 receptor (Abu-Hamdah et al., 2009; Tomas and Habener, 2010). For instance, in vivo administration of GLP-1(936)amide has demonstrated cardioprotective effects in dogs, rats, and mice, which arise from elevation in myocardial glucose uptake and from protection against ischemia-reperfusion injury (Nikolaidis et al., 2005; dx.doi.org/10.1124/dmd.113.054254. s This article has supplemental material available at dmd.aspetjournals.org. ABBREVIATIONS: amu, atomic mass unit; BSA, bovine serum albumin; CID, collision-induced dissociation; CLint,app, apparent intrinsic clearance; DPP-IV, dipeptidyl peptidase-IV; GLP-1, glucagon-like peptide-1; LC-MS/MS, liquid chromatography–tandem mass spectrometry; (M+H), protonated molecular ion; NEP, neutral endopeptidase 24.11; t1/2, half-life; tR, retention time; UPLC, ultra-performance liquid chromatography; WEM, Williams’ E medium. 2148 http://dmd.aspetjournals.org/content/suppl/2013/09/20/dmd.113.054254.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from Ban et al., 2008; Sonne et al., 2008). Likewise, infusions of GLP-1(936)amide into obese, insulin-resistant human subjects significantly lowered hepatic glucose production in an insulin-independent fashion, suggesting that GLP-1(9-36)amide lowers plasma glucose in insulinresistant subjects via direct inhibition of hepatic glucose production (Elahi et al., 2008). The NEP cleavage product GLP-1(28-36)amide has also been shown to inhibit weight gain and accumulation of liver triglycerides and improve insulin sensitivity in diet-induced obese mice (Tomas et al., 2011a). Consistent with the in vivo observations, Tomas et al. (2010) recently demonstrated that GLP-1(9-36)amide suppressed glucose production in isolated mouse hepatocytes by 30% over a dose range of 0.1–100 mM independently of the GLP-1 receptor. Furthermore, in a follow-up study by the same authors (Tomas et al., 2011b), dosedependent (0.01–10 mM) suppression of mitochondrial glucose production and oxidative stress by ;50% was also demonstrated in mouse hepatocytes with GLP-1(28-36)amide. In the case of GLP-1(28-36)amide, cytoprotective action was enhanced (0.1–100 nM) when a “solubilizing” formulation was used (Liu et al., 2012). Toward this end, we became interested in examining the in vitro hepatocyte stability of GLP-1(9-36)amide and GLP-1(28-36)amide, especially since hepatic endopeptidases, including DPP-IV and/or NEP (Elovson, 1980; Roques et al., 1993; Yasojima et al., 2001; Itou et al., 2013), bear the potential to cleave the two GLP-1(736)amide metabolites into additional Nand C-truncated products with unique pharmacological activity. In the present work, we examined the metabolic stability of GLP-1(9-36)amide and GLP-1(28-36)amide in cryopreserved mouse and human hepatocytes using a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) assay for the quantitation of intact GLP-1(9-36)amide and GLP-1(28-36)amide, respectively, in the incubations. A facile decline of the GLP-1(736)amide metabolites in the hepatocyte incubations (e.g., t1/2 , 1 hour in mouse hepatocytes) led to a detailed characterization of their metabolic fate. Our in vitro findings raise the intriguing possibility that the inhibitory effects of GLP-1(9-36)amide and GLP-1(28-36)amide on gluconeogenesis and oxidative stress might be due, at least in part, to the actions of additional downstream metabolites, which are obtained from the enzymatic cleavage of the peptide backbone in the parent compounds. Materials and Methods GLP-1(9-36)amide (EGTFTSDVSSYLEGQAAKEFIAWLVKGRamide) and GLP-1(28-36)amide (FIAWLVKGRamide) were synthesized by solid-phase synthesis and purified by sequential high-performance liquid chromatography to .95% single component homogeneity. A description of the synthetic methodology is provided in the Supplemental Methods. Gibco Williams’ E medium (WEM) supplemented with L-glutamine and without phenol red or sodium bicarbonate, pooled hepatocytes from humans (pool of 10 livers from males/females), and pooled hepatocytes from male CD-1 mice were purchased from Celsis In Vitro Technologies (Baltimore, MD). Bovine serum albumin (BSA), sodium bicarbonate, diprotin A, and HEPES were purchased from Sigma-Aldrich (St. Louis, MO). Phosphoramidon was purchased from R&D Systems (Minneapolis, MN). Solvents used for analysis were of analytical or high-performance liquid chromatography grade (Fisher Scientific, Pittsburgh, PA). Incubations in Hepatocytes.WEM was prepared by adding 26 mM sodium carbonate and 50 mM HEPES, followed by 0.2 mm filtration then 30 minutes of CO2 bubbling at 37°C. This medium was used for thawing and suspension of hepatocytes. Stock solutions of GLP-1(9-36)amide and GLP-1(28-36)amide were prepared in water at 1 mM and diluted to 5 mM in WEM. Incubations were conducted in a 96-well flat-bottom polystyrene plate. Stability assessments were performed in duplicate. Mouse and human hepatocytes were suspended at 0.5 million viable cells per milliliter of WEM and prewarmed at 37°C for 30 minutes. Incubations were initiated with the addition of peptide stocks (final concentration in incubation: 1 mM) and were conducted at 37°C,75% relative humidity, and 5% CO2. The total incubation volume was 0.1 ml per well. To assess the role of NEP and DPP-IV in GLP-1(9-36)amide and GLP-1(2836)amide cleavage, human hepatocyte stability of the two peptides was also examined in the presence of the NEP inhibitor phosphoramidon (1000 mM) and the DPP-IV inhibitor diprotin A (500 mM) (Ura et al., 1987; Gandhi et al., 1993; Roden et al., 1994; Turner et al., 2001; Malm-Erjefält et al., 2010), respectively, in triplicate. Inhibitor concentrations were based on experiments described in aforementioned references, and were not optimized in the present work. Phosphoramidon was purchased as a solution in methanol, which was dried down and reconstituted in WEM prior to use. Aliquots (50 ml) of the reaction mixture at 0, 5, 10, 15, 30, 60, 90, and 120 min (time period associated with reaction linearity) were added to acetonitrile or ethanol (200 ml) containing terfenadine (mol. wt. = 472, 2.0 ng/ml) as an internal standard. The samples were centrifuged at 2000 g for 10 min before LC-MS/MS analysis for the disappearance of the peptides. For the purposes of qualitative metabolite identification studies, the concentration of peptides in the hepatocyte incubations was increased to 20 mM. Reactions were carried out in 24-well polystyrene plates for approximately two half-lives. A nonenzymatic matrix control consisted of 10 mg/ml BSA in WEM. After quenching the incubation mixtures (1 ml) with Fig. 1. Depletion of GLP-1(9-36)amide (A) and GLP-1(28-36)amide (B) in mouse and human hepatocyte incubations. TABLE 1 In vitro stability of GLP-1(9-36)amide and GLP-1(28-36)amide in mouse and human hepatocytes t1/2 Values are a mean of two independent incubations with individual replicate values in parentheses. Compound Hepatocyte Species t1/2 CLint,app min ml min million cells GLP-1(9-36)amide Human 180 (200,150) 8.2 Mouse 52 (48,56) 27 GLP-1(28-36)amide Human 24 (24,24) 57 Mouse 13 (12,13) 110 Metabolism of GLP-1(9-36) and GLP-1(28-36) Amides 2149 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from