Fig. 3.

Inhibition of insulin gene expression by PA is JNK-dependent. (A) Cultured mouse islets were incubated with PA for the indicated durations, and JNK activity and abundance were determined by a kinase assay and immunoblotting, respectively. (B) S1 nuclease-protection assay. Cytoplasmic RNA was isolated from wt or Jnk1^−/− islets that were cultured in low (5.5 mM) or high (16.7 mM) glucose and treated with PA or D-JNKi as indicated for 12 h. Relative insulin mRNA levels are indicated above the panel. (C) Palmitate inhibition of insulin-induced insulin transcription is JNK-dependent. wt or Jnk1^−/− islets were kept in low glucose (5.5 mM). PA with or without D-JNKi was added 12 h before the addition of insulin. RNA was prepared 2 h later. Relative insulin mRNA levels are indicated above the panel. (D) Immunoblot analysis of AKT Ser-473 phosphorylation. Primary mouse islets were treated with PA with or without D-JNKi, as above, and treated with insulin for 5 min. AKT phosphorylation and abundance were measured by immunoblotting.