FIG. 8.

Chronic coculture exposure dampens myotube insulin response. Primary human skeletal myotubes from lean women were maintained 96 h in the absence or presence of human adipocytes under stimulated (100 μmol/L IBMX) conditions. Adipocytes were removed, and myotubes were washed and incubated with α-MEM ± 100 nmol/L insulin for 10 min. A: Protein extracts were used for immunoblot analysis of Akt Ser⁴⁷³ phosphorylation, corrected for total protein loading using Memcode staining. Measurement of total Akt protein showed no treatment effects. B: GSK-3β Ser⁹ phosphorylation, corrected for total protein using tubulin staining. C–F: Myotube extracts were used for measurement of total triacylglycerol (C), total diacylglycerol (D), total ceramide (E), and acylcarnitine profiling (F). C3DC and C5OH are isobaric species. The ratio of total medium-chain relative to total long-chain acylcarnitines was calculated to assess potential flux limitations at the medium-chain acyl-CoA dehydrogenase step of β-oxidation. Results are expressed as mean ± SEM and representative of at least three experiments performed in triplicate, except for the TAG, DAG, and ceramides, which represent two independent experiments performed in quadruplicate. *Significant (P < 0.05) effect of adipocyte exposure analyzed by Student t test. Abbreviations reflect carbon chain length (e.g., C2, acetylcarnitine). DC, dicarboxylic acid; OH, hydroxylated species.