Insulin pathway components and insulin effects on rho kinase in rat colon.

The RhoA/Rho-associated protein kinase (ROCK) signaling pathway plays a central role in regulating smooth muscle contraction by inhibiting myosin light chain phosphatase. While insulin has been shown to modulate ROCK activity in vascular and airway smooth muscle, its effects on gastrointestinal (GI) smooth muscle remain largely unexplored. Insulin exerts its effects through a cascade involving the insulin receptor (IR), insulin receptor substrates (IRS), phosphoinositide 3-kinase (PI3K) subunits such as p110, and downstream effectors including ROCK. The aim of the current study was to characterize the expression profile of key insulin signaling components, including IR, IRS isoforms (IRS1/2), PI3K catalytic subunits (p110α/β), and ROCK isoforms (ROCK1/2), in normal rat colon, and to determine whether insulin affects ROCK activity. Adult Sprague-Dawley rats were used to obtain colonic tissue samples. Immunohistochemistry (IHC) was performed to localize IR protein expression. Quantitative PCR was used to evaluate the mRNA expression levels of IRA/B, IRS1/2, p110α/β, and ROCK1/2. The effect of insulin on acetylcholine-induced activation of ROCK was measured using a specifically designed activity assay kit. IHC confirmed the expression of IR protein in both mucosal and muscle layers of the colon wall tissue. Messenger RNA expression levels of IRA, IRS2, p110α, and ROCK2 isoforms were greater than IRB, IRS1, p110β, and ROCK1 isoforms respectively. Insulin significantly reduced acetylcholine-induced activation of ROCK. the results suggest that the colon is responsive to insulin and exhibits a distinct insulin signaling pattern. Insulin may modulate colonic contractile signaling through ROCK activity.