Acute nitric oxide synthase inhibition enhances trans‐endothelial insulin efflux and muscle insulin sensitivity in vivo
Authors: Williams Ian M. , McClatchey Penn Mason , Mignemi Nicholas A. , McGuinness Owen P. , Wasserman David H. 
Published: June 2021

Before insulin can stimulate muscle glucose uptake, it must first traverse the continuous endothelium of skeletal muscle (SkM) capillaries. Despite the importance of trans‐endothelial insulin transport to SkM insulin sensitivity, little is known about the regulation of this process in vivo. We previously developed a technique by which we can measure the rate of trans‐endothelial insulin efflux using intravital microscopy of a fully bioactive, fluorescent insulin probe (ins‐647). Using this technique, we showed that the movement of insulin across the endothelium in SkM occurs by a fluid‐phase transport mechanism which is not saturable and does not involve the insulin receptor. Furthermore, we have demonstrated that trans‐endothelial insulin efflux is impaired in obese, insulin resistant mice. Insulin resistance in humans and rodents is associated with endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability. Studies in cultured endothelial cells suggest that insulin transport by endothelial cells is positively regulated by NO. These findings led to the hypothesis that NO is a positive regulator of trans‐endothelial insulin efflux.To test this hypothesis, we treated male C57Bl/6 mice with an intravenous bolus of either 30 mg/kg L‐NG‐Nitroarginine methyl ester (L‐NAME; NO synthase inhibitor), or saline. L‐NAME treatment increased mean arterial blood pressure by ~30 mmHg, indicating that NO synthase was successfully inhibited. Intravital ins‐647 imaging demonstrated that L‐NAME enhanced the rate at which plasma insulin equilibrated with the interstitial space by ~25%, indicating enhanced trans‐endothelial insulin efflux. The more rapid trans‐endothelial movement of insulin was associated with a 25% increase in SkM insulin sensitivity as determined by the accumulation of 2[14C]2‐deoxyglucose (2DG) in the gastrocnemius. To test the effects of increasing NO availability on insulin efflux, mice were treated with 100 μg/kg/min sodium nitroprusside (SNP; NO donor) or saline. SNP had a small, but non‐significant blood pressure‐lowering effect. SNP had no effect on either trans‐endothelial insulin efflux kinetics or gastrocnemius 2DG accumulation. To test whether the effects of NO modulation were due to changes in blood pressure, we infused a primed, continuous infusion of either phenylephrine (300 μg/kg bolus; 300 μg/kg/min infusion) or hydralazine (1 mg/kg bolus; 1 mg/kg/min infusion) into mice. Phenylephrine and hydralazine are direct smooth‐muscle acting drugs which raise and lower blood pressure independent of NO, respectively. Intravital ins‐647 imaging demonstrated that neither phenylephrine nor hydralazine had any effect on trans‐endothelial insulin efflux. Taken together, these results suggest that, surprisingly, reduction of NO by L‐NAME treatment increases trans‐endothelial insulin efflux. The mechanism by which lowering NO enhances the movement of insulin may involve effects of L‐NAME on SkM hemodynamics or direct effects on the capillary endothelium.Support or Funding InformationThis work was supported by NIH grants R01DK054902 and U24 DK059637 to D.H.W.; grant F31DK109594 to I.M.W.; and grant T32DK007563 to I.M.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.