Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin–sensitizing

Enzymatic activity assays acc activity in liver or

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Enzymatic activity assays Acc activity in liver or gastrocnemius was measured by 14 CO 2 fixation into acid–stable products. Acc1 and Acc2 protein was precipitated from 2 mg of tissue homogenates using antibodies specific for either Acc1 or Acc2. Antibodies against Acc1 and Acc2 were generated by immunizing sheep with synthetic peptides coupled to keyhole limpet hemocyanin (CDEPSPLAKTLELNQ (rat Acc1 (1–15 Cys 1 ) and CEDKKQAPIKRQLMT (rat Acc2 (145–159 Cys 145 )) and purifying antibodies from the resulting sera by affinity chromatography on immobilized peptides. Immunoprecipitates were incubated for 1.5 h at room temperature with reaction buffer containing 125 μM acetyl–CoA, 12.5 mM NaHCO 3 and 16.7 μCi/ml [ 14 C]–NaHCO 3 with the indicated concentrations of citrate. The reactions were terminated by addition of HCl and dried overnight at 37 °C. Water was added to the Fullerton et al. Page 6 Nat Med . Author manuscript; available in PMC 2016 July 28. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscript
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dried sample and radioactivity measured by liquid scintillation counting. Purified Acc1 protein from WT and Acc1 KI livers were subjected to λ phosphatase (400 U in a 60 μl reaction) treatment for 25 min at 30 °C. After a wash removal of the phosphatase, a small aliquot was used for determination of Acc1 Ser79 phosphorylation via Western blot. The majority of the protein that remained was used to assess Acc activity, as described above. Mass spectrometry analyses Acc isoforms from WT and AccDKI livers were affinity purified by Straptavidin Sepharose. Proteins were eluted from beads, reduced and alkylated with iodacetamide, then precipitated with methanol:chloroform (1:1, v/v). The denatured proteins were digested overnight with trypsin in solution at 37 °C. Peptides were separated on a PepMap RSLC C18 column using a Dionex 3000 Series NCS–3500RS nano liquid chromatography system. Mass spectrometry was performed on a 5600 Triple TOF MS (Ab–Sciex). Malonyl–CoA Assay Quantification of short chain CoA species was performed as reported previously with modification 41 . Briefly, frozen muscle and liver samples (~15–20 mg) were homogenized for 20 sec in 300 μl of 6% (v/v) Perchloric acid. After homogenization, the samples were left on ice for 10 min and then centrifuged at 12,000 g for 5 min. 100 μl of the resulting supernatant was used for UPLC analysis by a Water’s Acquity system. Each sample was run at a flow rate of 0.4 ml/min through an Ascentis express column C18, 10 cm × 2.1 mm and 2.7 μm particle size from Supelco maintained at a temperature of 40 °C. The analyte detection occurred at an absorbance of 260 nm. The mobile phase consisted of a mixture of buffer A (0.25 M NaH 2 PO 4 and water) and buffer B (0.25 M NaH 2 PO 4 and acetonitrile). The gradient elution profile consisted of the following initial conditions (2% B; 2–4 min 25% B; 4–6 min 40% B; 6–8 min 100% B; 10–12 min) maintained for 15 min. All gradients were linear and peaks were acquired, integrated and analyzed using the Waters Empower Software.
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  • Winter '19
  • Robert S Kiss
  • Insulin resistance, Fatty acid metabolism

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