AimsHypoglycemia is a severe side effect of intensive insulin therapy. Recurrent hypoglycemia (RH) impairs the counter-regulatory response (CRR) which restores euglycemia. During hypoglycemia, ventromedial hypothalamus (VMH) production of nitric oxide (NO) and activation of its receptor soluble guanylyl cyclase (sGC) are critical for the CRR. Hypoglycemia also increases brain reactive oxygen species (ROS) production. NO production in the presence of ROS causes protein S-nitrosylation. S-nitrosylation of sGC impairs its function and induces desensitization to NO. We hypothesized that during hypoglycemia, the interaction between NO and ROS increases VMH sGC S-nitrosylation levels and impairs the CRR to subsequent episodes of hypoglycemia. VMH ROS production and S-nitrosylation were quantified following three consecutive daily episodes of insulin-hypoglycemia (RH model). The CRR was evaluated in rats in response to acute insulin-induced hypoglycemia or via hypoglycemic-hyperinsulinemic clamps. Pretreatment with the anti-oxidant N-acetyl-cysteine (NAC) was used to prevent increased VMH S-nitrosylation.ResultsAcute insulin-hypoglycemia increased VMH ROS levels by 49±6.3%. RH increased VMH sGC S-nitrosylation. Increasing VMH S-nitrosylation with intracerebroventricular injection of the nitrosylating agent S-nitroso-L-cysteine (CSNO) was associated with decreased glucagon secretion during hypoglycemic clamp. Finally, in RH rats pre-treated with NAC (0.5% in drinking water for 9 days) hypoglycemia-induced VMH ROS production was prevented and glucagon and epinephrine production was not blunted in response to subsequent insulin-hypoglycemia.ConclusionThese data suggest that NAC may be clinically useful in preventing impaired CRR in patients undergoing intensive-insulin therapy.

In Type 1 and advanced Type 2 diabetes mellitus, elevation of plasma epinephrine plays a key role in normalizing plasma glucose during hypoglycaemia. However, recurrent hypoglycaemia blunts this elevation of plasma epinephrine. To determine whether recurrent hypoglycaemia affects peripheral components of the sympatho-adrenal system responsible for epinephrine release, male rats were administered subcutaneous insulin daily for 3 days. These recurrent hypoglycaemic animals showed a smaller elevation of plasma epinephrine than saline-injected controls when subjected to insulin-induced hypoglycaemia. Electrical stimulation of an adrenal branch of the splanchnic nerve in recurrent hypoglycaemic animals elicited less release of epinephrine and norepinephrine than in controls, without a change in adrenal catecholamine content. Responsiveness of isolated, perfused adrenal glands to acetylcholine and other acetylcholine receptor agonists was also unchanged. These results indicate that recurrent hypoglycaemia compromised the efficacy with which peripheral neuronal activity stimulates adrenal catecholamine release and demonstrate that peripheral components of the sympathoadrenal system were directly affected by recurrent hypoglycaemia.

This study examined how the quaternary organic ammonium ion, benzyltriethylamine (BTEA), binds to the Na,K-ATPase to produce membrane potential (V M )-dependent inhibition and tested the prediction that such a V M -dependent inhibitor would display electrogenic binding kinetics. BTEA competitively inhibited K + activation of Na,K-ATPase activity and steady-state 86 Rb + occlusion. The initial rate of 86 Rb + occlusion was decreased by BTEA to a similar degree whether it was added to the enzyme prior to or simultaneously with Rb + , a demonstration that BTEA inhibits the Na,KATPase without being occluded. Several BTEA structural analogues reversibly inhibited Na,K-pump current, but none blocked current in a V M -dependent manner except BTEA and its para-nitro derivative, pNBTEA. Under conditions that promoted electroneutral K + -K + exchange by the Na,KATPase, step changes in V M elicited pNBTEA-activated ouabain-sensitive transient currents that had similarities to those produced with the K + congener, Tl + . pNBTEA-and Tl + -dependent transient currents both displayed saturation of charge moved at extreme negative and positive V M , equivalence of charge moved during and after step changes in V M , and similar apparent valence. The rate constant (k tot ) for Tl + -dependent transient current asymptotically approached a minimum value at positive V M . In contrast, k tot for pNBTEA-dependent transient current was a "U"-shaped function of V M with a minimum value near 0 mV. Homology models of the Na,K-ATPase alpha subunit suggested that quaternary amines can bind to two extracellularly-accessible sites, one of them located at K + binding sites positioned between transmembrane helices 4, 5, and 6. Altogether, these data revealed important † This research was supported by the National Institutes of Health (R01 HL-076392 to RDP; R01 GM-057253 to JRB), the American Heart Association (RDP, JRB), and by Universidad de Buenos Aires, Agencia Nacional de Promoción Científica y Tecnológica and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. RMGL and RCR are established investigators of CONICET.Contact author: Joshua R. Berlin, Ph.D., Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, 195 S. Orange Ave., Newark, NJ, 07101-1709; Tel: 973-972-1618; Fax: 973-972-7950; berlinjr@umdnj.edu. SUPPORTING INFORMATION AVAILABLE Supplemental material contains the following: (1) derivation of a general time-dependent solution for a 3 state model of the Na,K-ATPase in the presence of an activating ligand (K + ) and a competitive inhibitor; (2) a brief review of the V M dependence of Na,K-pump current inhibition by TEA and BTEA and an explanation of how eq. 4 can be used to show V M -dependent inhibition of the Na,K-ATPase, (3) a figure comparing Na,K-pump current in TMA and NMG-containing superfusion solutions; (4) a figure showing the synthetic scheme for para-nitrobenzyltriethylammonium bromide; (5) a figure showing the K + o and V M dependence of Na,K-pump current in the presence...