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. 2009 May 22;284(21):14087–14095. doi: 10.1074/jbc.M807992200

FIGURE 8.

FIGURE 8.

Insulin suppresses hepatic mitochondrial production and function through cyclic nucleotide phosphodiesterase (PDE). Primary hepatocytes were pretreated with IBMX for 30 min, and then incubated with insulin for 24 h for measuring mRNAs or for 24 h for quantifying mDNA, proteins, and ATP content. A, levels of mtDNA were evaluated as described under “Materials and Methods.” B, intracellular ATP was quantified as described under “Materials and Methods.” C, ATP synthase protein was detected by immunoblotting. D, transcripts of mitochondrion-related genes were measured by TaqMan real-time PCR. Results of A, B, and D represent mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus vehicle treatment. #, p < 0.05; ##, p < 0.01; ###, p < 0.001 versus insulin alone. E, IBMX did not affect insulin signaling. Hepatoma cells (1c1c7) were pre-treated with 0.25 mm IBMX for 30 min followed by incubation with insulin for 5 min. Akt phosphorylation at Ser473 was measured by immunoblotting and normalized to levels of total Akt. Results shown represent two independent experiments. F and G, quantification of intracellular cAMP and cGMP. Hepatoma cells (1c1c7) were pre-treated with IBMX for 30 min and then incubated with insulin for 24 h in the presence or absence of IBMX. Intracellular levels of cAMP and cGMP were measured by enzyme immunoassays as described under “Materials and Methods.” Results represent mean ± S.D. of two independent experiments, each in 4–6 replicates. *, #, p < 0.05 versus vehicle or insulin alone, respectively. H, insulin neutralizes cAMP activation of CREB via PDE. Hepa1c1c7 cells were pre-treated with IBMX (0.1 and 0.25 mm) for 30 min followed by exposure to 10 nm cAMP in the presence and absence of 1 nm insulin for 30 min. CREB phosphorylation at Ser133 and Akt phosphorylation at Ser473 were determined by immunoblotting, and normalized to levels of total Akt. Results shown are a set of typical immunoblots of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.