Lipid sensor components

Comprehensive design and construction details for all expression vectors are provided in Supplementary Table S1. Key plasmids include the following: pKR135 encoding constitutive expression of the LSR (P hCMV -LSR-pA); pMG10 (ref. 30) harbouring a SEAP expression unit driven by the LSR-specific phloretin-responsive promoter (P TtgR1 ; P TtgR1 -SEAP-pA); and pKR146 containing a P TtgR1 -driven pramlintide expression unit (P TtgR1 -Pram-pA).

Cell culture and transfection

Human embryonic kidney cells (HEK-293T, ATCC: CRL-11268), Baby hamster kidney cells (BHK-21, ATCC: CCL-10), African green monkey kidney cells (COS-7, ATCC: CRL-1651), human cervical adenocarcinoma cells (HeLa, ATCC: CCL-2), human fibrosarcoma cells (HT-1080, ATCC: CCL-121) and immortalized human mesenchymal stem cells53) were cultivated in DMEM (Invitrogen, Basel, Switzerland) supplemented with 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich, Munich, Germany). Wild-type Chinese hamster ovary cells (CHO-K1, ATCC: CCL-61) were cultured in ChoMaster HTS (Cell Culture Technologies GmbH, Gravesano, Switzerland) containing 1% penicillin/streptomycin. All cell types were cultivated at 37 °C in a humidified atmosphere containing 5% CO 2 . For (co-)transfection of BHK-21, CHO-K1, COS-7, HeLa, HT-1080 and human mesenchymal stem cellss, 40,000 cells seeded per well of a 24-well plate 12 h beforetransfection were incubated for 6 h with a 4:1 PEI:DNA mixture (Polyethyleneimine; MW 40,000, Polysciences, Inc., Warrington, USA). After transfection, all cells were cultivated in their specific media containing different inducer concentrations and reporter protein levels were profiled after 48 h unless stated otherwise. For cotransfection of circuit components (pKR135 and pKR153) into HT-1080, a typical transfection efficiency of 61% was reached (Supplementary Fig. S5).

SEAP production

Production levels of the human placental SEAP were quantified in cell culture supernatants using a p-nitrophenylphosphate-based light-absorbance time course54. One hundred microlitres of cell-culture supernatant were heat inactivated for 30 min at 65 °C and centrifuged for 2 min at 14,000g to remove cell debris. Eighty microlitres of the sample were transferred into a well of a 96-well plate and adjusted to 37 °C. One hundred microlitres of 2 × SEAP buffer (20 mM homoarginine, 1 mM MgCl 2 , 21% (v/v) diethanolamine, pH 9.8) were mixed with 20 μl substrate solution (120 mM para-nitrophenyl phosphate), adjusted to 37 °C and added to the well containing the heat-inactivated sample. The absorbance time course was recorded at 405 nm using an Envision 2104 multilabel plate reader (Perkin Elmer, Waltham, USA). SEAP concentrations in blood samples were quantified using a chemiluminescence-based assay (Roche Diagnostics GmbH, Mannheim, Germany). In brief, 50 μl of heat-inactivated serum (30 min, 65 °C), centrifuged for 30 s at 14,000g, were transferred to a well of a 96-well plate containing 50 μl inactivation buffer and incubated for 10 min at 22 °C. Next, 50 μl of freshly prepared substrate reagent (50 μl CSPD [3-(4-methoxyspiro[1,2-dioxetane-3,2′(5′-chloro)-tricyclo(3.3.1.13,7 decane]-4-yl)phenyl phosphate] mixed with 950 μl substrate buffer) was added to each well and incubated for 10 min at 22 °C before light emission was recored at 477 nm using the Envision 2104 multilabel plate reader (Perkin Elmer).

Flow cytometry

pDSRed-N1- or pKR135/pKR153-(co)-transfected HT-1080 cell populations were diluted in 1% BSA (Sigma-Aldrich)-containing PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.4; 1 × 106 cells per ml) and analysed using a LSRII Fortessa flow cytometer (Becton Dickinson, Allschwil, Switzerland) equipped for DsRed (561 nm laser excitation and 586/15 emission filter) detection and set to exclude dead cells and cell doublets. At least 10,000 cells were recorded per data set and analysed using FACSDiva (BD Biosciences, Franklin Lakes, NJ, USA, version no. 6.1.3.).

Blood fat levels

Blood cholesterol levels were profiled using a total cholesterol assay kit (Fluoro Cholesterol; Cell Technology, Mountain View, USA). In brief, 50 μl of appropriately ddH 2 O-diluted serum was added per well of a clear-bottom black 96-well plate containing 50 μl of freshly prepared reaction cocktail (20 μl resuspended enzyme mix, 20 μl resuspended cholesterol probe and 960 μl reaction buffer). The plate was incubated in the dark for 1 h at 22 °C and fluorescence was quantified at excitation 530 nm and emission 585 nm using the Envision 2104 multilabel plate reader (Perkin Elmer). Blood phospholipid levels were quantified using a phospholipid assay kit (Abnova GmbH, Heidelberg, Germany). In brief, 20 μl of appropriately Triton X-100 (Sigma-Aldrich)-diluted serum was added per well of a clear-bottom black 96-well plate containing 80 μl of freshly prepared working reagent (85 μl assay buffer, 1 μl PLD enzyme, 1 μl Enzyme Mix and 1 μl Dye Reagent). The plate was incubated in the dark for 30 min at 22 °C and fluorescence was quantified at excitation 530 nm and emission 585 using the Envision 2104 multilabel reader (Perkin Elmer).

Pramlintide quantification and activity tests

In vitro activity of pramlintide was measured by (co-)transfecting HT-1080 with pKR135/pKR146 or pJWS17 (6 μg DNA, 800,000 cells, 10 cm Petri dish). Forty-eight hours after transfection, the culture medium was transferred to 60,000 HEK-293 cells that had been cotransfected with pCALCR, pRAMP3, pCK53 and pcDNA3.1 at a ratio of 1:1:0.5:7.5 (0.6 μg of total DNA) and SEAP levels were profiled after 48 h. Alternatively, pramlintide was quantified using the human amylin EIA ELISA kit (Phoenix Pharmaceuticals, Burlingame, USA) and pure pramlintide as standard (Feldan, Quebec, Canada). For quantification of pramlintide in mouse serum samples, 96-well plates were coated with 5 μg ml−1 (100 μl per well) rabbit polyclonal anti-human amylin (Amylin H-50, Santa Cruz Biotechnology, Santa Cruz, USA; catalogue number sc-20936, lot. no. I0303; dilution 1:300) in dilution buffer (PBS, 0.5% BSA, 0.01% Tween80, pH 7.4) at 4 °C overnight. Plates were blocked with 200 μl per well PBS/1% BSA, pH 7.4, for 1 h at 22 °C. Pramlintide standard (1–500 μg ml−1) and samples (100 μl per well) were diluted (PBS, 0.5% BSA, 0.01% Tween80, pH 7.4) and incubated for 1.5 h at 22 °C. Plates were washed three times with 250 μl per well PBS containing 0.15% Tween-20 and then incubated with 100 μl per well of a mouse monoclonal anti-Amylin (Abcam, Cambridge, UK; catalogue number ab115766, lot no. GR81110-2; dilution 1:450) for 1 h at 22 °C. After three washing steps (250 μl PBS/0.15% Tween-20), the plates were incubated with an anti-mouse horseradish peroxidase-conjugated IgG (GE Healthcare, Buckinghamshire, UK, catalogue number NA931V, lot no. 399402; dilution 1:1,000) for 1 h at 22 °C, washed again (250 μl PBS/0.15% Tween-20) and bound pramlintide was visualized by incubation of the samples with 100 μl per well of 3,3′,5,5′-tetramethylbenzidine substrate (Interchim, Montluçon, France) at 22 °C for 6 min. Reactions were stopped by addition of 2 M sulphuric acid (100 μl per well) and pramlintide was quantified at 450 nm using Envision 2104 multilabel reader (Perkin Elmer).

Co-immunoprecipitation

Six micrograms of Protein A Dynabeads (Invitrogen Dynal AS, Oslo, Norway) were washed twice with 300 μl co-immunoprecipitation (CoIP) buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) containing 6 μl protease inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland) and incubated with 6 μg of the rabbit polyclonal anti-HA antibody (Y-11; Santa Cruz Biotechnology Inc; catalogue number sc-805, lot no. G1210; dilution 1:3) for 4 h while mixing at room temperature on an end-over rotator. pKR151/pMG10-cotransfected HT-1080 cells (1.2 × 108) were washed twice with 30 ml cold PBS and the cell nuclei were extracted with 45 ml nuclear isolation buffer (300 mM sucrose, 10 mM Tris, pH 8, 5 mM MgCl 2 and 0.5% Triton X-100, 0.1% protease mix) for 10 min at 4 °C. Isolated cell nuclei were resuspended in 2 ml CoIP buffer containing 200 μl protease inhibitor cocktail (Roche Diagnostics GmbH) and 1 μl ml−1 Benzonase (Invitrogen) and sonicated for 15 min and 30 s at 4 °C (320 W, Biorupter Plus, Diagenode S.A, Liège, Belgium). Two millilitres of the resulting nuclear extract was incubated overnight at 4 °C with 30 μl of anti-HA antibody-linked Protein A Dynabeads while mixing on an end-over rotator. The immunocomplexes were washed three times with 500 μl CoIP buffer for 10 min and the immunoprecipitated protein complexes were then visualized by western blot analysis.

Western blotting

Twenty microlitres of the immunoprecipitated protein complexes were mixed with 5 μl of NuPAGE LSD sample buffer (4 × ; Invitrogen) and 1 μl of 1 M dithiothreitol, resolved on a 4–12% Bis-Tris gel (NuPAGE; Invitrogen) or a 3–8% Tris-Acetate Gel (NuPAGE Novex; Invitrogen) and electroblotted (Trans-Blot SD, Bio-Rad, Reinach, Switzerland) onto an Amersham Hybond-ECL membrane (GE Healthcare, Glattbrugg, Switzerland). The membrane was blocked with 5% (w/v) non-fat dried milk (AppliChem GmbH, Darmstadt, Germany) diluted in PBST (PBS, 0.1% Tween-20) for 30 min at 22 °C and incubated overnight at 4 °C with recommended dilutions of the following primary antibodies: mouse monoclonal anti-GPS2 antibody (Abcam; catalogue number ab53406, lot no. GR61606-3; dilution 1:500), goat polyclonal anti-NCoR antibody (C-20; Santa Cruz Biotechnology Inc.; catalogue number sc-1609, lot no. A2813; dilution 1:200), goat polyclonal anti-HDAC3 antibody (N-19; Santa Cruz Biotechnology Inc.; catalogue number sc-8138, lot no. C1913; dilution 1:200) and goat polyclonal anti-HA antibody (F-7; Santa Cruz Biotechnology Inc.; catalogue number sc-7392, lot no. C081; dilution 1:1,000). The membrane was then washed three times in PBST and incubated for 1 h with a horseradish peroxidase-coupled sheep anti-mouse IgG (GE Healthcare; catalogue number NA931V, lot no. 399402; dilution 1:1,000) or rabbit anti-goat IgG (Sigma-Aldrich; catalogue number A5420, lot no. 043M4763; dilution 1:2,000). The super signal West Femto Western blot detection reagent (Thermo Scientific, Rockford, USA) and the X-ray apparatus Konika Minolta SRX-101A (Konika Minolta GmbH, Langenhagen, Germany) were used for detection.

Cytokine measurements

TNF-α and IL-6 levels were profiled in the blood of treated animals using murine TNF-α- and IL-6-specific ELISA assays (PeproTech, Rocky Hill, NJ, USA, catalogue numbers 900-M54 (TNF-α) and 900-M50 (IL-6)) according to the manufacturer’s instructions. Intraperitoneal injections of 1 ml 1% (w/v) thioglycollate in ddH 2 O (Sigma-Aldrich) were used as an inflammation-inducing positive control set-up.

Animal experiments

Intraperitoneal implants were produced by encapsulating pKR135/pMG10- or pKR135/146-transgenic HT-1080 into coherent alginate-poly-(l-lysine)-alginate beads (400 μm; 200 cells per capsule) using an Inotech Encapsulator Research Unit IE-50R (Buchi Labortechnik AG, Flawil, Switzerland) set to the following parameters: 200 μm nozzle with a vibration frequency of 1,023 Hz and 900 V for bead dispersion, 20 ml syringe operated at a flow rate of 403 units. Four hundred microlitres of serum-free DMEM containing 2 × 106 microencapsulated transgenic cells (pKR135/pMG10- or pKR135/pKR146-transgenic HT-1080; 200 cells per capsule) were injected intraperitoneally into 14-week-old female CD1 (normal 5 kcal% fat diet; Janvier S.A.S., Le Genest-Saint-Isle, France) or 14-week-old diet-induced obese mice (C57BL/6J, The Jackson Laboratory, Maine, USA) that were on a 10-kcal% (D12450Bi, Research Diets, Inc., NJ) or a 60-kcal% fat diet (D12492i, Research Diets, New Brunswick, USA). CD1 mice implanted with pKR135/pMG10-transgenic HT-1080 received twice daily injections of linoleic or palmitic acid (200 μl, 0-200 mg kg−1; linoleic acid, Thermo Fisher Scientific, Geel, Belgium; palmitic acid: Sigma-Aldrich) or oral doses of rapeseed oil (100 and 200 μl; Naturaplan Rapsöl, Coop, Basel, Switzerland). Control mice were treated with capsules containing non-engineered parental HT-1080. Every day, the food intake and body weight of the animals was profiled. After a starvation period of 4 h, blood samples were collected and the serum was isolated using microtainer SST tubes according to the manufacturer’s protocol (Beckton Dickinson, Plymouth, UK) before serum SEAP, blood fat and pramlintide levels were scored as described above. All experiments involving animals were performed according to the directives of the European Community Council (2010/63/EU), approved by the French Republic (no. 69266309), and carried out by Ghislaine Charpin-El Hamri at the Institut Universitaire de Technology, IUTA, F-69622 Villeurbanne Cedex, France.