Sitagliptin

For GTTs, mice received a glucose bolus i.p. (2 g/kg body weight, Braun) after 6 h of fasting. Blood glucose was measured after 0, 15, 30, 60, 90 and 120 min using a freestyle lite glucometer (Cat#7091870, Abbott). Blood was collected at time points 0, 15 and 30 min for insulin measurements.
For ITTs, mice were fasted 3 h and injected with 1U/kg body weight insulin (Actrapid Penfill Insulin 100 IU/mL, Novo Nordisk). Glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection.
For GLP-1 measurements, mice were i.p. injected with 25 mg/kg body weight Sitagliptin (Cat# sc-364620, Santa Cruz) 30 min prior to oral glucose administration and blood collected in diprotein A (Cat# I9759, Bachem). To block GLP-1 signaling, synthetic exendin (9–39) 25 nM/kg body weight (Cat# H-8740, Bachem) was injected i.p. 1 min prior to GTT.

Metabolic phenotype was assessed by glucose and insulin tolerance tests (GTT/ITT) performed at 1, 4, and 12 weeks of HFD feeding. For a GTT, mice fasted 6 h. After intraperitoneal (IPGTT) or oral (OGTT) injection of glucose (2 g/kg body weight) blood glucose was monitored from the tail vein after 15, 30, 60, 90, and 120 min by using a glucometer (Freestyle, Abbot). For active GLP-1 measurements, 25 mg/kg sitagliptin (Cat#sc-364620, Santa Cruz) was injected i.p. 30 min before oral glucose application. To block GLP-1 or parasympaticus action, 236 µg/kg exendin (9-39) (#H-8740, Bachem) or 5 mg/kg atropine (#A0257-5G, Sigma), respectively, were i.p. injected at timepoint −30 min prior glucose application. ITT was performed after 3 h of fasting by injecting 1–2 U/kg body weight insulin i.p. (Actrapid HM Penfill, Novo Nordisk). Glucose levels were measured at 0, 15, 30, 60, 90, and 120 min after injection.
Plasma insulin, GLP-1, TNF, and IL-6 were quantified by electrochemiluminescence (MESO SECTOR S 600) by using kits from Meso Scale Diagnostics (MSD, Rockville, MD, USA), according to the manufacturer’s instructions: Mouse/Rat Insulin Kit (#K152BZC), V-PLEX Plus Proinflammatory Panel 1 Mouse Kit (#K15048G), V-PLEX GLP-1 Active Kit vers. 2 (#K15030D).

Sitagliptin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ERK1/2 inhibitor (PD98059) and FITC-conjugated anti-α-smooth muscle actin (α-SMA) monoclonal antibody were obtained from Sigma-Aldrich (St Louis, MO, USA). Mouse monoclonal core-binding factor α 1 (cbfa1) (ab54868) and mouse monoclonal α-SMA antibody (ab7817) were obtained from Abcam (Cambridge, MA, USA). Trypsin, fetal bovine serum (FBS) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Gibco (of Thermo Fisher Scientific, Waltham, MA, USA). Rabbit polyclonal antibody against ERK1/2 and a mouse monoclonal antibody against p-ERK1/2 were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Secondary antibodies were purchased from Vector Labs (Burlingame, CA, USA). Western blot bands were detected using the ECL Advance Western blotting detection kit from GE Healthcare (Chalfont St Giles, UK). Cell counting kit-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Transwell plates were purchased from Millipore (Bedford, MA, USA). Annexin V-FITC Kit was purchased from BD Biosciences (San Jose, CA, USA). The alkaline phosphatase (ALP) activity kit was from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Calcium Colorimetric Assay Kit was obtained from Biovision (Mountain View, CA, USA).

Proinsulin-related peptides were assessed by a proinsulin ELISA (10-1232-01, Mercodia, Uppsala, Sweden). According to the manufacturer’s instructions, this assay does not cross-react with mouse mature insulin or C-peptide but our data suggest that it also detects split proinsulins. IAPP was measured by ELISA (human mature IAPP, EZHA-52K, Millipore, St. Charles, MO, USA). This assay employs a monoclonal capture antibody (F002; binds all molecular forms of proIAPP) and a monoclonal detection antibody (F025; binds amidated IAPP at its C terminus)^{77 (link)} and is 100% cross-reactive to rodent IAPP. To determine active GLP-1, mice were injected with 25 mg kg⁻¹ sitagliptin (sc-364620, Santa Cruz Biotechnology, Dallas, USA) 30 min prior to oral glucose loading (2 g kg⁻¹ glucose) and blood was collected in tubes containing Diprotin A (H-38250050, Bachem, Bubendorf, Switzerland). Active GLP-1 was assessed by the V-PLEX assay (K1503OD-1, Meso Scale Diagnostics) and serum levels of ghrelin (active), leptin, and PYY (total) were analyzed using the U-PLEX metabolic kit (K152ACL-1, Meso Scale Diagnostics). To determine the ileal content of active GLP-1, 3 cm of the terminal ileum was excised, rinsed with PBS, extracted in 0.18 N HCl in 70% EtOH, and assessed with the kit described above.