October 1, 2022

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Health & Fitness

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

GLP-1-mediated delivery of tesaglitazar improves obesity and glucose metabolism in male mice

Compound synthesis

Peptidyl resin with modified GLP-1 backbone sequence of Boc-His(Trt)-aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Glu(OtBu)-Gln(Trt)-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Lys(Boc)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Lys(Mtt)-amide resin was assembled by automated Fmoc/tBu solid-phase chemistry starting with 0.1 mM of H-Rink Amide ChemMatrix (PCAS BioMatrix Inc.) on a Symphony Peptide Synthesizer (Peptide Technology). All Fmoc-amino acids were coupled with 6-Cl-HOBt/DIC activation in dimethylformamide (DMF). The Fmoc were removed by 20% piperidine in DMF. After the Mtt removal by treating the peptidyl resin with 1–2% trifluoroacetic acid (TFA)/5% Tis in dichloromethane, Fmoc-Glu-OtBu was coupled with 6-Cl-HOBt/DIC in DMF. Fmoc was deprotected and tesaglitazar/(S)-2-ethoxy-3-(4-(4-((methylsulfonyl)oxy) phenethoxy) phenyl) propanoic acid (Astatech Inc.) was coupled with 6-Cl-HOBt/DIC in DMF with threefold excess. GLP-1 Aib2 Glu16 CEX Lys40–tesaglitazar conjugate peptide was cleaved from the resin with 10 ml TFA cleavage cocktail containing 8.5 ml of TFA, 0.5 ml of water, 0.5 ml Tis (triisopropylsilane), 0.25 g phenol and 0.25 ml 2-mercaptoethanol for 2 h. Peptide was precipitated with cold ether, dissolved in 20% acetonitrile (ACN) containing 2% acetic acid and injected to a Luna 19 × 250 nm/10 μm C8 column (Phenomenex) to purify with 0.1%TFA/ACN eluent solvents in a Waters 2545 preparative high-performance liquid chromatography instrument. Peptide molecular weight characterization was measured by liquid chromatography–mass spectrometry on an Agilent 1260 Infinity/6120 Quadrupole instrument with a Kinetex C8 column with an eluent gradient of 10–80% 0.05% ACN. The purified GLP-1 Aib2 Glu16 CEX Lys40–tesaglitazar conjugate (high-performance liquid chromatography >95%) was characterized with a molecular mass of 1,178.5/[M+4H]4+ and 1,570.8/[M+3H]3+, which is consistent with the theoretically calculated molecular weight of 4,710.1 with formula C214H315N49O69S. For assessment of brain penetrance, the GLP-1RA and the GLP-1RA/tesaglitazar conjugate were covalently attached to a cyanine 5 (Cy5) fluorophore (Lumiprobe no. 13380). The Cy5 was attached to the peptide by engineering a single cysteine into the peptide followed by conjugation of the fluorophore using a maleimide-containing building block.

Animals

C57BL/6J mice and Leprdb (db/db) mice were purchased from the Jackson Laboratory (no. 000697). Glp1r−/− mice were kindly provided by D. Drucker (University of Toronto, Canada). All wildtype and knockout mice used in our studies were in-house bred on a C57BL/6J background. Mice were double-housed and kept in a constant environment with the ambient temperature set to 22 ± 2 °C with constant humidity (45–65%) and a 12 h/12 h light/dark cycle with lights off from 06:00 until 18:00. For studies in DIO mice, male C57BL/6J mice were fed with a high-fat diet comprising 58% kilocalories from fat (D12331, Research Diets). db/db mice were fed with a normal chow diet (T1314, Altromin GmbH) throughout the study. At the beginning of each experiment, mice were equally distributed into experimental groups according to their body weight and body composition. All animal studies were approved by the State of Bavaria, Germany, or the Insitutional Animal Care and Use Committee of the University of Cincinnati, OH, USA and conducted on the basis of the underlying animal welfare law of the respective countries. Compounds were dissolved in PBS and were s.c. administered with a volume of 5 μl g−1 body weight in the indicated doses between 15:00 and 16:00.

Body composition analysis

Fat and lean tissue mass were measured via nuclear magnetic resonance technology (EchoMRI).

Glucose- and insulin-tolerance tests (GTT/ITT)

Glucose tolerance was assessed in 6 h fasted mice after i.p injection of 1.75 g glucose per kg body weight (DIO mice). Insulin tolerance was assessed in 6 h fasted mice after i.p. injection of either 0.75 U Insulin per gram body weight (DIO mice) or 1 U Insulin per gram body weight (db/db mice) (Humalog, Eli Lilly). Tail vein blood glucose was subsequently measured using a handheld glucometer (TheraSense Freestyle) at baseline and after 15, 30, 60 and 120 min. Data were subsequently graphed as baseline-corrected AOC as previously suggested36.

Plasma creatinine

Plasma creatinine was measured by a fluorometric creatinine assay kit (catalogue no. ab65340, Abcam) based on the manufacturer’s instructions.

Urinary albumin and creatinine measurement

Spot urine samples were collected from male 49-week-old C57BL/6J mice treated for 14 days with either Vhcl (n = 8) or 50 nmol kg−1 of GLP-1RA/tesaglitazar (n = 5), the GLP-1RA (n = 6), Tezaglitazar (n = 7) or the fixed dose combination of the GLP-1RA and tesaglitazar (n = 8). Frozen urine samples from heterozygous GIPRdn mice in a CD1 background (n = 3) served as positive controls. Urinary albumin concentrations were measured using a mouse albumin enzyme-linked immunosorbent assay kit (catalogue no. 80630, Crystal Chem) based on the manufacturer’s instructions. Urinary creatinine levels were quantified using a mouse creatinine enzymatic assay kit (catalogue no. 80350, Crystal Chem) based on the manufacturer’s instructions. For each treatment group, uACR was computed as a ratio of urinary albumin (mg ml−1) to urinary creatinine (mg ml−1).

Pancreatic section immunostaining and confocal imaging

Adult pancreata were dissected and fixed in 4% PFA in PBS overnight at 4 °C. The samples were cryoprotected by sequential incubation with 7.5, 15 and 30% sucrose-PBS solutions at room temperature (2 h incubation for each solution). Pancreata were then incubated in 30% sucrose and tissue embedded medium (1:1) at 4 °C overnight. Tissues were then embedded in a cryoblock using a tissue-freezing medium, and stored at −80 °C. From each sample, 20-μm-thick sections were cut, mounted on a glass slide and dried for 10 min at room temperature before being used or stored at −20 °C.

Following three washes with 1× PBS, the cryosections were permeabilized with 0.2–0.15% Triton X-100 in H2O for 30 min. The sections were then blocked in blocking solution (PBS, 0.1% Tween-20, 1% donkey serum, 5% FCS) for 1 h, followed by incubation with the primary antibodies overnight at 4 °C. The following primary antibodies were used: insulin (Cell Signaling, no. 3014, 1:300), glucagon (TAKARA, no. M182, 1:3,500) and somatostatin (Invitrogen, no. MA5-16987, 1:300). After being rinsed three times and washed three times with 1× PBS, the sections were incubated with secondary antibodies in the blocking buffer. We used the following secondary antibodies: antirabbit-Alexa Flour 488 (Invitrogen, no. A11055, 1:800), antiguinea pig-Cy™3 (Dianova/Jackson, no. 706-165-148, 1:800) and anti-goat-Alexa Flour 633 (Invitrogen, no. A21082, 1:800). After 4–5 h of incubation with the secondary antibodies at room temperature, the pancreatic sections were subsequently stained with DAPI (1:500 in 1× PBS) for 30 min at room temperature, then rinsed and washed three times with 1× PBS and mounted. The images were obtained by using a Leica microscope of the type DMI 6000 and Leica’s LAS AF software. ImageJ and/or LAS AF were used to analyse the images. Mean signal intensity per islet area of confocal images were used to quantify the hormone contents. Nine to 12 islets were quantified for each animal.

Measurement of islet size area

Dissected pancreata were fixed in Formalin (Formalin 10% neutral buffered, HT501128, Sigma-Aldrich) for 24 h at room temperature and standardly processed for paraffin embedding (Tissue Tec VIP.6, Sakura Europe) Paraffinized pancreata were exhaustively cross-sectioned into four parallel, equidistant slices per case. Maintaining their orientation, the tissue slices were vertically embedded in paraffin. After a costaining for insulin (monoclonal rabbit anti-insulin, no. 3014, Cell Signaling 1:300; AlexaFluor750-conjugated goat antirabbit, A21039, Invitrogen 1:100) and for glucagon (polyclonal guinea pig anti-glucagon, M182, Takara 1:3,500; goat antiguinea pig AlexaFluor555, A21435, Invitrogen 1:200) nuclei were labelled with Hoechst33342 (H1399, Thermo Fischer, 7.5 μg ml−1). The stained tissue sections were scanned with an AxioScan.Z1 digital slide scanner (Zeiss, ZEN Blue v.3.5) equipped with a ×20 magnification objective. Insulin expression cells were performed on the entire tissue sections by using image analysis software Definiens Developer XD2 (Definiens AG, v.2.7.0). The insulin expressing cells were classified automatically using the fluorescence intensity. β cell volume (mg) was calculated by multiplying the detected relative insulin-positive cell area by total pancreatic weight. α cell volume (mg) was similarly calculated on the basis of the detected glucagon-positive cell area. The area of the pancreatic islet was calculated on the basis of the insulin and glucagon-positive area.

Dynamic glucose-stimulated insulin secretion in primary murine islets

After isolation and overnight incubation in RPMI (10% foetal bovine serum (FBS), 1% penicillin/streptomycin), 75 islets were handpicked and placed into chambers containing 2.7 mM glucose Krebs–Ringer phosphate-HEPES (KRPH) buffer (140 mM NaCl, 4.7 mM KCl, 1.5 mM CaCl2, 1 mM NaH2PO4, 1 mM MgSO4, 2 mM NaHCO3, 5 mM HEPES and 0.1% FA-free BSA (pH 7.4)) with 100 μl of Bio-Gel P-4 Media (Bio-Rad). Islets were equilibrated for 48 min and then perifused in intervals on the basis of the experimental conditions. All treatments were prepared in KRPH buffer +0.1% BSA. Islet proteins were extracted in acid ethanol to assess total insulin levels. Insulin secretion was assayed with Lumit Insulin Immunoassay Kit (Promega, CS3037A01) and measured using the EnVision plate reader (PerkinElmer).

Cell culture and transfection

HEK293T cells (ATCC, catalogue no. CRL-3216) or MIN6 cells (AddexBio, catalogue no. C0018008,) were cultured in DMEM (catalogue no. 11995073, Life Technologies) supplemented with 10% (HEK293T) or 15% (MIN6) heat-inactivated FBS (catalogue no. 10500064, Life Technologies), 100 IU ml−1 of penicillin and 100 μg ml−1 of streptomycin solution (Pen-Strep, catalogue no. P4333, Sigma-Aldrich). HEK293T cells (700,000 per well) were seeded in six-well plates and incubated to 70% confluency in DMEM (10% FBS, 1% Pen/Strep). Twenty-four hours following seeding, transient transfections were performed using Lipofectamine 2000 (catalogue no. 11668019, Invitrogen) according to the manufacturer’s protocol without including additional transformation carrier DNA.

Ligand-induced bioluminescence resonance energy transfer (BRET) assays

Twenty-four hours following transfection, HEK293T cells or MIN6 cells were washed with PBS and resuspended in FluoroBrite phenol red-free complete media (catalogue no. A1896701, Life Technologies) containing 5% FBS and 2 mM of l-glutamine (catalogue no. 25030081, Life Technologies). 100,000 cells per well were plated into poly-d-lysine-coated (catalogue no. P6403, Sigma-Aldrich) 96-well white polystyrene LumiNunc plates (catalogue no. 10072151, Thermo Fisher Scientific). After 24 h, the media was replaced with PBS (catalogue no. 10010056, Gibco) containing 10 μM of coelenterazine-h (catalogue no. S2011, Promega) or 1:500 dilution of NanoGlo (catalogue no. N1110, Promega). BRET measurements were taken every 30 s–2 min using a PHERAstar FS multi-mode microplate reader. Baseline measurements were taken after an initial 5 min of incubation with coelenterazine-h or NanoGlo-containing PBS after which cells were then treated with either vehicle (PBS) or respective ligands. The resulting ligand-specific ratiometric BRET signals were normalized to vehicle producing the ‘ligand-induced BRET ratio’37, followed by an additional normalization step to well-specific baseline readings. Ligand-induced measurements on the temporal scale is represented as the subsequent measurement after time point zero. Positive or negative incremental AUC (+iAUC/−iAUC) were calculated where noted. Each experiment was independently performed at least three times, each with at least three technical replicates for each group.

Receptor signalling and trafficking BRET assays

Untagged hGLP-1R was purchased from Sino Biological Inc. (catalogue no. HG13944-UT), hGLP-1R-GFP was a kind gift from D. Hodson (University of Birmingham), and hGLP-1R-Rluc8 was a kind gift from P. Sexton (Monash University, Melbourne, Australia). Gαs recruitment to the GLP-1R-GFP was quantified using mini-Gαs, a protein probe that translocates to ligand-bound Gαs-coupled G-protein coupled receptors38. NES-Nluc-MiniGαs was a gift from K. Pfleger (Harry Perkins Institute of Medical Research, Nedlands, Western Australia, Australia). Intracellular cAMP was measured using the YFP-Epac-Rluc CAMYEL sensor39. hGLP-1R-RLUC8 internalization was quantified using the intracellular plasma membrane marker Venus-KRAS40. Venus-KRAS was a kind gift from K. Pfleger. GLP-1R-Rluc8 lysosomal colocalization was measured using Lamp1-mNeonGreen. Lamp1-mNeonGreen was a gift from D. Gadella (Addgene plasmid no. 98882). Time-dependent RXR/PPARγ heterodimerization was measured using RXR-Rluc8 and PPARγ2-YFP, both of which were kind gifts from V. Ollendorf41.

Gene expression analysis

For assessment of acute drug effects, animals were treated with the respective compounds 4 h before tissue harvesting. Dissected tissues were frozen immediately on dry ice, and RNA was isolated using RNeasy Mini Kits (Qiagen). mRNA levels were determined using TaqMan probes for fatty acid binding protein 4 (Fabp4, Mm00445878_m1), perilipin-2 (Plin2, Mm00475794_m1), aryl hydrocarbon receptor nuclear translocator-like protein 1 (Arntl, Mm00500223_m), semaphorin 3 C (Sema3c, Mm00443121_m1) and hypoxanthine phosphoribosyltransferase 1 (Hprt, Mm03024075_m1) in custom-made low density array cards (Thermo Fisher) or in single assays on a QuantStudio 7 Real-Time PCR system. Target gene expression was normalized to HPRT and fold change was calculated relative to vehicle-treated controls.

Proteomics sample preparation

Frozen tissue samples were homogenized in 0.1 M Tris-HCl, pH 7.6 and 2% SDC, heated for 5 min at 95 °C and sonicated (Diagenode Bioruptor at high intensity, 15 × 30 s). Then 50 µg of protein was reduced and alkylated with 10 mM TCEP,40 mM CAA for 5 min at 40 °C in the dark. Samples were digested with trypsin and LysC 1:50 (enzyme:protein) overnight at 37 °C. Digested peptides were acidified to a final concentration of 1% TFA and loaded onto activated triple layer styrenedivinylbenzene–reversed phase sulfonated (SDB–RPS, 3M Empore) STAGE tips. STAGE tips were washed with 100 µl ethylacetate 1% TFA, 100 µl of 30% methanol 1% TFA and 150 µl of 0.2% TFA. Peptides were eluted with 60 µl of SDB–RPS elution buffer (80% ACN, 5% NH4OH), concentrated in a SpeedVac for 40 min at 45 °C and dissolved in 10 µl of MS loading buffer (2% ACN, 0.1% TFA).

LC–MS/MS analysis

Single-shot measurements were performed with 500 ng of purified peptides, determined by absorbance at 280 nm on a NanoDrop 2000. Peptides were loaded onto a 50-cm column, packed in-house with 1.9 µm C18 ReproSil particles (Dr. Maisch GmbH) with an EASY-nLC 1200 system (Thermo Fisher Scientific). Column temperature was kept at 60 °C using a column oven. Peptides were eluted over 60 min using a binary buffer system consisting of buffer A (0.1% formic acid) and buffer B (80% ACN, 0.1% formic acid). In brief, the gradient started with 5% buffer B and increased stepwise to 45% over 45 min, followed by a wash-out at 95% buffer B, all at a flowrate of 300 nl min−1. Peptides were then transferred to the gas phase using electrospray ionization, prefiltered by a FAIMS device (coefficient of variation −50 V) before entering the Orbitrap Exploris 480 (Thermo Fisher Scientific) mass spectrometer. A data-independent (DIA) acquisition method was used, in which one full scan (300–1,650 m/z, maximum ion fill time of 45 ms, normalized AGC target 300%, R = 120.000 at 200 m/z) was followed by 66 tMS2 fragment scans of unequally spaced windows with 1 Th overlap, covering the same m/z range (fill time 22 ms, normalized AGC target 1,000%, normalized high-collision density energy of 30%, R = 15.000).

Pharmacokinetic analysis

Plasma concentration-time profiles were analysed in C57BL6J mice and Sprague Dawley rats by a non-compartmental method using sparse sampling in Pharsight Phoenix WinNonLin v.6.4 software, and the resulting terminal half-life (T1/2), maximum plasma concentration (Cmax), time for maximum plasma concentration (Tmax), and AUC from zero to last valid measurable concentration-time point (AUC0-t) were determined. Criteria for estimation of T1/2 were at least three concentration-time points in the terminal phase not including Cmax, with an R2 ≥ 0.85.

Immunhistochemical analysis

For cFOS analysis, mice were treated s.c. with a single dose of 150 nmol kg−1. Tissues were harvested 90 min following drug exposure. Fixed brains were coronally cryosectioned and 35-μm-thick slices were immunolabelled with the monoclonal rabbit anti-cFOS antibody (1:400, Invitrogen, no. MA5-15055) and the antirabbit Alexa546 secondary antibody (1:4,000, Invitrogen, no. A10040). According to the Allen mouse brain atlas, the ARC and the dorsomedial and ventromedial hypothalamic nuclei were captured at ×20 magnification using the Leica SP8 confocal microscope. In each region, the number of cFOS positive cells was counted in a blinded manner using Fiji/ImageJ software. For assessment of central nervous system drug appearance, mice (n = 3–4 each group) were treated with a single s.c. dose of the Cy5-labelled peptides (150 nmol kg−1). After 90 min, tissues were harvested and processed similar to the cFOS analysis.

Bioinformatic workflow and proteomic data analysis

DIA raw files were processed using Spectronaut v.14.5.200813.47784 (ref. 42) using the directDIA function with default settings. Raw files from samples of the same tissue were processed together and searched against the mouse UniProt FASTA database (September 2014, 51,210 entries) and the provided MaxQuant contaminants list (245 entries). Spectronaut report files were then loaded into Perseus (v.1.6.2.3). In brief, quantified proteins were filtered for ≥3 valid values in at least one biological condition and annotations mapped from UniProtKB, Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. Significantly up- or downregulated proteins between the conditions were determined by ANOVA (FDR 0.025).

Statistics

Statistical analyses were performed using the statistical tools implemented in GraphPad Prism8 (v.8.3.0 or 9.00). All data are shown as mean ± s.e.m. Differences between groups were assessed by one- or two-way ANOVA with time and treatment as covariants followed by Bonferroni’s post hoc multiple comparison testing as indicated in the figure legends. A P value <0.05 was considered statistically significant with asterisks indicating *P < 0.05, **P < 0.01 and ***P < 0.001.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

https://www.nature.com/articles/s42255-022-00617-6