Elevated hepatic DPP4 activity promotes insulin resistance and non-alcoholic fatty liver disease

Baumeier, Christian; Schlüter, Luisa; Saussenthaler, Sophie; Laeger, Thomas; Rödiger, Maria; Alaze, Stella Amelie; Fritsche, Louise; Häring, Hans-Ulrich; Stefan, Norbert; Fritsche, Andreas; Schwenk, Robert Wolfgang; Schürmann, Annette

Plasma insulin concentrations were quantified from vena cava blood using a Mouse Ultrasensitive Insulin ELISA (Alpco). Plasma adiponectin and leptin levels were measured by Mouse Adiponectin/Acrp30 (DY1119, R&D Systems) and Mouse/Rat Leptin (MOB00, R&D Systems) ELISA kits. Active GLP-1 levels were detected after oral glucose administration using GLP-1 (Active) ELISA Kit (AKMGP-011, Shibayagi). Mice were fasted for 16 h, orally administered with glucose (2 mg/g body weight), and euthanized after 15 min by isoflurane. Following this, blood was taken with 0.5 M EDTA-coated syringe which was supplemented with 20 ml/ml DPP4 inhibitor (Cat. DPP4, Millipore) from either vena cava or vena portae. Plasma triglyceride (T2449, F6428, G7793, Sigma), free fatty acid (91096, 91898, 91696, Wako), cholesterol (10017, Human), ALT (12212, Human), AST (12211, Human), and GGT (12213, Human) levels were measured according to manufacturer's protocol. Soluble DPP4 concentration was determined using a Mouse DPP4 ELISA Kit (DY954, R&D Systems). Plasma DPP4 activity was measured by the conversion of glycin-prolin-p-nitroanilide (Sigma) to pnitroanilide. Twenty ml plasma, 90 ml cell supernatants, and 45 ml cell homogenates were filled to 90 ml with assay buffer (50 mmol/l glycine, 1 mmol/l EDTA, pH 8.7) and supplemented with 10 ml glycine-proline-pnitroanilide (5 mmol/l). Production of p-nitroanilide was measured by the absorbance at 405 nm in a kinetic measurement at 37 C. The DPP4 activity in the samples was calculated using p-nitroaniline (Sigma) standard curve over the concentration range of 20e100 mmol/l. The results are expressed as nmol/min/ml. 2.6. Liver glycogen, cholesterol, and triglyceride content Liver glycogen content was analyzed using a glucose colorimetric assay (10260, Human) subsequent to an amyloglucosidase (Fluka) digestion of liver homogenates. Hepatic triglyceride content was measured as described before [27] using the TR-210 kit (Randox). Liver cholesterol level was determined using a commercial kit (10017, Human).