Blood samples for assessment of blood glucose and HbA1c were collected from the tail vein in heparin-coated capillary tubes (Vitrex Medical, Herlev, Denmark) once weekly throughout the treatment period. Blood glucose was assessed with the glucose oxidase method at a Biosen Apparatus (EKF Diagnostics, Barleben, Germany) according to manufacturer’s instructions. HbA1c was determined using a Cobas 6000 c501 instrument (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer’s instructions. In addition, larger blood samples were collected into K3-EDTA microvette tubes (Sarstedt AG & Co, Nümbrecht, Germany) from the sublingual vein in conscious, non-fasted animals on the morning of the day of euthanization. After centrifugation, plasma was isolated and kept at − 20 °C until further analysis. Quantification of very-low-density lipoprotein triglyceride (VLDL-TG), low-density lipoprotein cholesterol (LDL-C), high-density-lipoprotein cholesterol (HDL-C) and total cholesterol in plasma was done using gel-filtration high performance liquid chromatography at LipoSEARCH (Skylight Biotech Inc, Akita, Japan). Plasma levels of free fatty acids (FFA), haptoglobin, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using a Cobas 6000 c501 instrument (Roche Diagnostics GmbH, 68206 Mannheim, Germany) according to the manufacturer’s instructions. Plasma concentration of endogenous hamster insulin was measured as described previously with an assay designed for detection of rat insulin [34]. The results are therefore presented as “rat insulin immunoactivity equivalents” (RIIE) as the cross reactivity to hamster insulin was not determined. In samples where equivalents were below the lower limit of quantification (LLOQ, 20 pM), these were set to be LLOQ/2 (10 pM) for subsequent statistical analysis, as described previously [35]. Plasma concentration of human insulin was measured in the NASH-STZ-HI group as described previously [36]. The LLOQ for the human insulin assay was 15 pM.

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