Hint2−/− mice were generated by homologous recombination in embryonic stem cells (ESCs). To delete the five exons of Hint2, a distal LoxP site was inserted upstream of Hint2 exon 1 and an FRT-neomycin-FRT-LoxP selection cassette was inserted downstream of Hint2 exon 5. The targeting vector in a 129Sv/Pas genetic background was electroporated into 129Sv/Pas ESCs (GenOway, Lyon, France). G418-resistant ESC clones were screened via polymerase chain reaction (PCR) and Southern blotting. Recombined ESC clones were injected

into C57BL/6J-derived blastocysts. find more Germline transmission and deletion of the floxed region (exons 1-5) were assessed after breeding the chimeras with the Cre-expressing C57BL/6J deleter strain. The resulting Hint2 heterozygotes of mixed 129Sv/C57Bl6J genetic background were intercrossed, and constitutive Hint2−/− and control Hint2+/+ mice were selected by PCR and Southern blotting. Mice were subjected to 12-hour light/dark cycles and were fed ad libitum. The 2018

Teklad Global 18% protein diet (Harlan Laboratories Inc, Madison, WI) contained 17% calories derived from fat. Initially, male mice aged 10, 20, and 30 weeks were assessed for phenotypic changes. Additional experimentation was performed on male mice aged 20-28 weeks, except

for electron microscopy studies at 50 weeks. Experiments were approved by the selleck chemicals University of Bern Animal Care Committee. Activity of L-3-hydroxyacyl-coenzyme A (CoA) dehydrogenase short chain (Hadhsc) was measured in isolated liver mitochondria (250 medchemexpress μg/mL). The reaction mixture (37°C, pH 7.0) contained 0.1 M triethanolamine-HCl, 5 mM ethylene diamine tetraacetic acid, 0.45 mM reduced nicotinamide adenine dinucleotide (NADH), and 0.1 mM acetoacetyl-CoA. Enzyme activity was calculated as [(ΔA340nm/minute test − ΔA340nm/minute blank) · (mL)]/6.22, where 6.22 is the extinction coefficient of β-NADH.14 Glutamate dehydrogenase activity (GDH) was measured in tissue lysate using an assay kit that monitored the generation of NADH at 450 nm (Biovision, Mountain View, CA). Carnitine palmitoyltransferase (CPT) activity was measured in liver mitochondria according to Shimoda et al.15 with palmitoyl-CoA as the substrate. Sirtuin 3 activity was measured in liver mitochondria using the Cyclex SIRT3 Deacetylase Fluorometric kit (MBL International, Woburn, MA). Mice were fasted for 16 hours. Glucose (2 g/kg body weight) or insulin (1 U/kg body weight) was injected intraperitoneally.