Backhed et al. 10.1073/pnas.0605374104. |
Table 4. Biochemical assays of various metabolites in gastrocnemius muscle and liver harvested from GF wild-type and Fiaf-deficient littermates
| Muscle | Liver | ||||
Metabolites, μmol/g of protein | Fiaf +/+ | Fiaf -/- | P value | Fiaf +/+ | Fiaf -/- | P value |
AMP | 2.70±0.18 | 2.00±0.0.33 | 0.11 | 5.72±0.41 | 5.24±0.28 | 0.30 |
ADP | 4.33±0.45 | 4.36±0.62 | 0.97 | 3.48±0.32 | 3.44±0.27 | 0.92 |
ATP | 24.02±1.42 | 27.13±0.87 | 0.08 | 9.29±0.43 | 13.23±1.25 | 0.07 |
NAD+ | 1.48±0.05 | 1.50±0.06 | 0.78 | 0.68±0.17 | 0.61±0.26 | 0.81 |
NADH | 0.26±0.02 | 0.24±0.02 | 0.5 | 0.36±0.02 | 0.37±0.03 | 0.71 |
Mean values ± SE are shown (n = six animals per group).
Table 5. Gene-specific primers used for qRT-PCR assays
Gene | GenBank accession no. | Primer | Primer sequences | Amplicon size, bp |
Cpt1 | NM_009948 | Forward | 5'- AGCACACCAGGCAGTAGCTT | 144 |
Reverse | 5'- AGGATGCCATTCTTGATTCG | |||
Fiaf | AF278699 | Forward | 5'- CAATGCCAAATTGCTCCAATT | 82 |
Reverse | 5'- TGGCCGTGGGCTCAGT | |||
L32 | NM_172086 | Forward | 5'-CCTCTGGTGAAGCCCAAGATC | 102 |
Reverse | 5'-TCTGGGTTTCCGCCAGTTT | |||
Mcad | NM_007382 | Forward | 5'-GCTGGAGACATTGCCAATCA | 106 |
Reverse | 5'- TCTTGGCGTCCCTCATCAG | |||
Pgc-1a | NM_008904 | Forward | 5'- AACCACACCCACAGGATCAGA | 73 |
Reverse | 5'- TCTTCGCTTTATTGCTCCATGA |
SI Text
Biochemical Assays of Enzyme Activities and Metabolite Levels
Pyridine nucleotide cycling methods were used to measure NADH, NAD, AMP, ADP, and ATP in freeze-clamped gastrocnemius muscle and liver (1). Glycogen was determined according to Lin et al. (2). Cpt activity was measured as follows. Muscle samples (2.5 mg) were homogenized on ice in 0.5 ml of extraction buffer (20 mM sodium phosphate, pH 7.4/0.02% BSA/0.5 mM EDTA/5 mM 2-mercaptoethanol/25% glycerol/0.6 M KCl/0.5% Triton X-100). A 1-ml aliquot of the resulting homogenate was added to 0.5 ml of assay reagent A [100 mM Tris[chemp]HCl, pH 8.1/1 mM MgCl2/1 mM carnitine (Sigma)/0.25 mM palmitoylCoA (Sigma)], and the samples were incubated for 60 min at 20°C before adding 0.5-ml assay reagent B [100 mM Tris succinate, pH 7.7/1 mM MgCl2/0.4 mM guanosine 5[prime] triphosphate/0.4 mM phosphoenolpyruvate/0.3 M KCl/30 mM NADH/5 mg/ml beef-heart lactate dehydrogenase (Sigma; specific activity, 500 units/mg protein)/40 mg/ml rabbit muscle pyruvate kinase (Sigma; 400 units/mg protein)/40 mg/ml pig-heart succinyl CoA synthetase (Sigma; 130 units/mg protein)]. Samples were incubated for 5 min at 25°C before reduction of NADH was measured by using a Farrand filter fluorometer (excitation 365 nm; emission 355 nm). CoA standards (2-5 nmol) were used to quantify enzyme activity, which was normalized to protein content (Bradford assay; BioRad, Hercules, CA).
To measure glycogen synthase activity, liver samples were homogenized on ice in 0.5 ml of lysis buffer consisting of 20 mM sodium phosphate, pH 7.4/5 mM EDTA/5 mM 2-mercaptoethanol/25% glycerol/0.5% Triton X-100/50 mM potassium fluoride. Enzyme activity was determined in the resulting homogenate according to ref. 2.