5 U/kg) and tissues were harvested under anesthesia 20 minutes postinjection. Hepatocytes from 12-month-old mice were isolated by collagenase perfusion and cultured for 5 days in a thin-layer collagen matrix as described with minor changes.16, 17 On the day of experiments, cells were serum starved for 5 hours. Cells for determination of insulin action were stimulated with 150 nM insulin for 15 minutes, lysed, and frozen at −80°C. All data were generated in 6 to 8 experiments; each experiment was performed using primary hepatocytes isolated from individual animals.

Mitochondrial suspensions were prepared according to modified methods of Koves et al.18 as described previously by our group.19 Palmitate oxidation (14CO2, representing complete fatty acid oxidation) was measured with radiolabeled [1-14C]palmitate (American Radiochemicals) in freshly isolated liver mitochondria and in serum starved primary hepatocytes as described.17, 19-21 Tanespimycin cost Intrahepatic lipids were extracted, quantified, and expressed as nmol/g tissue wet weight as described.20 Hepatic DAG content was determined after TLC isolation by methanolysis and measurement of fatty acid methyl esters HDAC inhibitor by gas chromatography with flame ionization detection, as previously described by our group.22 Hepatic glycogen content was assessed as previously described by our group.20 Hepatic ceramides were extracted by the method of Bligh and

Dyer.23 Ceramide (Cer) species were measured relative to a C8:0-Cer internal standard by negative-ion electrospray ionization tandem mass spectrometry (ESI/MS/MS) analysis (as [M-H]− ions) employing neutral loss of 256 with a Thermo TSQ Vantage triple quadrupole instrument (San Jose, CA) as described,24 and normalized to sample protein content. Hyperinsulinemic-euglycemic clamps were performed in conscious mice

following a 5-hour fast as described.25 After mice were anesthetized with sodium pentobarbital (50-75 mg/kg), the left common medchemexpress carotid artery and the right jugular vein were catheterized, free ends of catheters tunneled under the skin to the back of the neck where they were exteriorized and sealed with stainless steel plugs. Experiments were performed when mice were within 2 g of presurgery weight (∼5 days). Baseline blood samples were taken, followed by a priming bolus (1 μCi) and then a constant infusion (0.05 μCi/min) of 3H-3-glucose for a 2-hour period and a second blood sample was taken to assess basal hepatic glucose output. A priming bolus of insulin (16 mU/kg) was given and a constant infusion of insulin (4 mU/kg/min) and glucose (50g/100mL) infusion rate was adjusted to maintain euglycemia. In addition, a constant infusion of 3H-3-glucose (0.1 μCi/min) was maintained to measure insulin-suppression of hepatic glucose output. Mice received saline-washed erythrocytes from donors throughout (5-6 μL/min) to prevent a fall of >5% hematocrit. At the end of clamps the animals were anesthetized and liver was taken and frozen immediately.