Age-dependent slowing of enteric axonal transport in insulin-resistant mice.

AIM: To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin (Hb). METHODS: Under anesthesia, the retrograde tracer fluorogold was superficially injected into the fundus or antrum using a microsyringe in KK Cg-Ay/J mice prior to onset of type 2 diabetes mellitus (T2DM; 4 wk of age), at onset of T2DM (8 wk of age), and after 8, 16, or 24 wk of untreated T2DM and in age-matched KK/HIJ mice. Six days later, mice were sacrificed by CO2 narcosis followed by pneumothorax. Stomachs were removed and fixed. Sections from fundus, corpus and antrum were excised and mounted on a glass slide. Tracer-labeled neurons were viewed using a microscope and manually counted. Data were expressed as the number of neurons in short and long descending and ascending pathways and in local fundus and antrum pathways, and the number of neurons in all regions labeled after injection of tracer into either the fundus or the antrum. RESULTS: By 8 wk of age, body weights of KKAy mice (n = 12, 34 ± 1 g) were heavier than KK mice (n = 17, 29 ± 1 g; F (4, 120) = 4.414, P = 0.002] and glycosylated Hb was higher [KK: (n = 7), 4.97% ± 0.04%; KKAy: (n = 6), 6.57% ± 0.47%; F (1, 26) = 24.748, P < 0.001]. The number of tracer labeled enteric neurons was similar in KK and KKAy mice of all ages in the short descending pathway [F (1, 57) = 2.374, P = 0.129], long descending pathway [F (1, 57) = 0.922, P = 0.341], local fundus pathway [F (1, 53) = 2.464, P = 0.122], local antrum pathway [F (1, 57) = 0.728, P = 0.397], and short ascending pathway [F (1, 53) = 2.940, P = 0.092]. In the long ascending pathway, fewer tracer-labeled neurons were present in KKAy as compared to KK mice [KK: (n = 34), 302 ± 17; KKAy: (n = 29), 230 ± 15; F (1, 53) = 8.136, P = 0.006]. The number of tracer-labeled neurons was decreased in all mice by 16 wk as compared to 8 wk of age in the short descending pathway [8 wk: (n = 15), 305 ± 26; 16 wk: (n = 13), 210 ± 30; F (4, 57) = 9.336, P < 0.001], local antrum pathway [8 wk: (n = 15), 349 ± 20; 16 wk: (n = 13), 220 ± 33; F (4, 57) = 8.920, P < 0.001], short ascending pathway [8 wk: (n = 14), 392 ± 15; 16 wk: (n = 14), 257 ± 33; F (4, 53) = 17.188, P < 0.001], and long ascending pathway [8 wk: (n = 14), 379 ± 39; 16 wk: (n = 14), 235 ± 26; F (4, 53) = 24.936, P < 0.001. The number of tracer-labeled neurons decreased at 24 wk of age in the local fundus pathway [8 wk: (n = 14), 33 ± 11; 24 wk: (n = 12), 3 ± 2; F (4, 53) = 5.195, P = 0.001] and 32 wk of age in the long descending pathway [8 wk: (n = 15), 16 ± 3; 32 wk: (n = 12), 3 ± 2; F (4, 57) = 2.944, P = 0.028]. The number of tracer-labeled enteric neurons was correlated to final body weight for local fundus and ascending pathways [KK: (n = 34), r = -0.746, P < 0.001; KKAy: (n = 29), r = -0.842, P < 0.001] as well as local antrum and descending pathways [KK (n = 36), r = -0.660, P < 0.001; KKAy (n = 31), r = -0.622, P < 0.001). In contrast, glycosylated Hb was not significantly correlated to number of tracer-labeled neurons [KK (n = 17), r = -0.164, P = 0.528; KKAy (n = 16), r = -0.078, P = 0.774]. CONCLUSION: Since uncontrolled T2DM did not uniformly impair tracer transport in gastric neurons, long ascending neurons may be more susceptible to persistent hyperglycemia and low effective insulin.

Neostigmine-induced contraction and nitric oxide-induced relaxation of isolated ileum from STZ diabetic guinea pigs.

Both delayed gastrointestinal transit and autonomic neuropathy have been documented in patients with diabetes mellitus. The mechanism of neostigmine, an agent that mimics release of acetylcholine from autonomic neurons by prokinetic agents, to contract smooth muscle, despite dysfunctional enteric neural pathways, was determined using isolated ilea from STZ-treated and control guinea pigs. Both bethanechol- and neostigmine-induced contractions were stronger in diabetic ileum. Bethanechol-induced contractions of control but not diabetic ileum were increased by low dose scopolamine suggesting reduced activation of presynaptic muscarinic autoreceptors in diabetic ileum. The muscarinic receptor antagonist 4-DAMP strongly, but the nicotinic receptor antagonist hexamethonium only weakly, reduced neostigmine-induced contractions of control and diabetic ilea. The amount of acetylcholine, inferred from tissue choline content, was increased in diabetic ileum. Nicotinic neural and noncholinergic postjunctional smooth muscle receptors contributed more strongly to neostigmine-induced contractions in diabetic than control ileum. Relaxation of diabetic ileum by exogenous nitric oxide generated from sodium nitroprusside was comparable to control ileum, but smooth muscle relaxation by l-arginine using neuronal nitric oxide synthase to generate nitric oxide was weaker in diabetic ileum with evidence for a role for inducible nitric oxide synthase. Despite autonomic neuropathy, neostigmine strongly contracted ileum from diabetic animals but by a different mechanism including stronger activation of postjunctional muscarinic receptors, greater synaptic acetylcholine, stronger activation of noncholinergic excitatory pathways, and weaker activation of inhibitory pathways. A selective medication targeting a specific neural pathway may more effectively treat disordered gastrointestinal transit in patients with diabetes mellitus.

Regional differences in neostigmine-induced contraction and relaxation of stomach from diabetic guinea pig.

Delayed gastric emptying and autonomic neuropathy have been documented in patients with diabetes mellitus. Some medications used to treat delayed gastric emptying enhance release of acetylcholine from autonomic neurons to strengthen gastric contractions. Autonomic coordination among gastric regions may be altered in diabetes resulting in poor outcomes in response to prokinetic drugs. Fundus, antrum, and pylorus from STZ or control guinea pigs were treated with neostigmine to mimic release of acetylcholine from autonomic neurons by prokinetic agents. In diabetic animals, neostigmine-induced contractions were weaker in fundus and pylorus but similar in antrum. The muscarinic receptor antagonist 4-DAMP or the nicotinic receptor antagonist hexamethonium reduced neostigmine-induced contractions. Activation of presynaptic muscarinic receptors on nitrergic neurons was impaired in fundus and antrum from diabetic animals. Nerve-stimulated contractions and relaxations, number of nNOS myenteric neurons, and tissue choline content were reduced in fundus from diabetic animals. Despite reduced number of myenteric neurons, tissue choline content was increased in antrum from diabetic animals. Since cholinergic motility of each gastric region was affected differently by diabetes, prokinetic drugs that nondiscriminately enhance acetylcholine release from autonomic neurons may not effectively normalize delayed gastric emptying in patients with diabetes and more selective medications may be warranted.