Abstract
The pathogenesis of diabetes-associated motility disorders are multifactorial and attributed to abnormalities in extrinsic and intrinsic innervation, and a decrease in the number of interstitial cells of Cajal, and nNOS expression and activity. Here we studied the effect of hyperglycemia on smooth muscle function. Using smooth muscles from the fundus of ob/ob mice and of wild type (WT) mice treated with 30 mM glucose (HG), we identified the molecular mechanism by which hyperglycemia upregulates RhoA/Rho kinase pathway and muscle contraction. RhoA expression, Rho kinase activity and muscle contraction were increased, while miR-133a expression was decreased in smooth muscle of ob/ob mice and in smooth muscle treated with HG. Intraperitoneal injections of pre-miR-133a decreased RhoA expression in WT mice and reversed the increase in RhoA expression in ob/ob mice. Intraperitoneal injections of antagomiR-133a increased RhoA expression in WT mice and augmented the increase in RhoA expression in ob/ob mice. The effect of pre-miR-133a or antagomiR-133a in vitro in smooth muscle treated with HG was similar to that obtained in vivo, suggesting that the expression of RhoA is negatively regulated by miR-133a and a decrease in miR-133a expression in diabetes causes an increase in RhoA expression. Oxidative stress (levels of reactive oxygen species and hydrogen peroxide, and expression of superoxide dismutase 1 and NADPH oxidase 4) was increased in smooth muscle of ob/ob mice and in HG-treated smooth muscle. Treatment of ob/ob mice with N-acetylcysteine (NAC) in vivo or addition of NAC in vitro to HG-treated smooth muscle reversed the effect of glucose on the expression of miR-133a and RhoA, Rho kinase activity and muscle contraction. NAC treatment also reversed the decrease in gastric emptying in ob/ob mice. We conclude that oxidative stress in diabetes causes a decrease in miR-133a expression leading to an increase in RhoA/Rho kinase pathway and muscle contraction.
Highlights
Diabetes Mellitus is associated various gastrointestinal complications including delayed gastric emptying, constipation, diarrhea and fecal incontinence [1,2,3]
ACh (1 μM)induced Rho kinase activity and Rho kinase-mediated phosphorylation of MYPT1 at Thr696 were augmented (69±8% and 96±10% increase in Rho kinase activity and MYPT1 phosphorylation, respectively) in smooth muscle of ob/ob mice compared to wild type (WT) mice (Fig 1C and 1D)
The pathogenesis of the diabetes-induced motility disorders has been linked to the decrease in interstitial cells of Cajal (ICC), extrinsic and intrinsic neuropathy, and decrease in nNOS expression and activity [18,19,20,21,22,23,24,25,26,27]
Summary
Diabetes Mellitus is associated various gastrointestinal complications including delayed gastric emptying, constipation, diarrhea and fecal incontinence [1,2,3]. The role of altered intrinsic signaling pathways that lead to smooth muscle contraction in motility disorders is largely unknown. Smooth muscle contraction is regulated by phosphorylation of Ser on the 20 kDa regulatory chain of myosin II (MLC20) by two enzymes known as myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) [28,29,30,31,32,33,34,35,36]. An increase in intracellular cytosolic Ca2+, either through Ca2+ influx and/or release from intracellular stores, by contractile agonists leads to Ca2+/CaM-dependent activation of MLCK which is essential for activation of actin-activated myosin ATPase, interaction of actin and myosin, and smooth muscle contraction. One pathway involves phosphorylation of the regulatory subunit of MLCP by Rho kinase and the other pathway involves phosphorylation of CPI-17 by protein kinase C (PKC), resulting in inhibition of MLCP activity and increase in MLC20 phosphorylation [28,29,31,32]
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