Kathy J. LePard

Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity.

Background Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown. Objective Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO. Methods Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation. Results HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043). Conclusion Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.

Choline acetyltransferase and inducible nitric oxide synthase are increased in myenteric plexus of diabetic guinea pig

Alterations in enzymes in myenteric neurons from ileum were investigated in guinea pigs treated with either the pancreatic beta cell toxin streptozotocin or vehicle. After 5-6 weeks, expressions of choline acetyltransferase, neuronal nitric oxide synthase and inducible nitric oxide synthase were determined in longitudinal and myenteric plexus preparations using indirect immunohistochemistry. In ileum from streptozotocin-treated animals, the density of choline acetyltransferase-immunoreactive nerve fibers within the tertiary plexus and the percent total myenteric neurons expressing inducible nitric oxide synthase were increased, but the percent total myenteric neurons expressing neuronal nitric oxide synthase was not changed. Diabetes resulted in selective alterations in myenteric neurons including an increased density of cholinergic tertiary fibers and percentage of neurons expressing the inducible isoform of nitric oxide synthase. These adaptive changes by myenteric neurons to diabetes may contribute to gastrointestinal dysfunctions associated with diabetes.

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.

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.

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.

Enhanced nerve-stimulated muscarinic and neurokinin contractions of ileum from streptozotocin guinea-pigs

Diabetes mellitus can lead to neuropathy of enteric neurons, resulting in abnormal gut motility. These studies investigated voltage-dependent contributions of muscarinic M₃ receptor activation by acetylcholine and neurokinin NK₁ receptor activation by neurokinins to nerve-stimulated contractions of longitudinal ileal strips from STZ guinea-pigs, a type 1 diabetic model with insulin deficiency, but mild hyperglycaemia. Contractions to bethanechol, substance P methyl ester, and nerve stimulation were greater in diabetic as compared to control ileum. The muscarinic M₃ receptor antagonist 4-DAMP at lower voltages and the neurokinin NK₁ receptor antagonist SR140333 at higher voltages, but not the neurokinin NK₁ receptor antagonist CP-96,345, were more effective at inhibiting nerve-stimulated immediate peak contractions and total areas of contraction of ileum from diabetic as compared to control animals. For diabetic ileum, voltage-dependent increases in the areas of nerve-stimulated contraction were observed in the presence of 4-DAMP and CP-96,345 but not SR140333. At low voltages only, nerve-stimulated release of acetylcholine was greater from diabetic as compared to control ileum. Fluorescence intensity of tachykinin-like immunoreactivity was increased in ileal myenteric ganglia from diabetic as compared to control animals. In diabetic guinea-pigs, stronger ileal nerve-stimulated contractions reflected increased release of acetylcholine at lower voltages and tachykinins at higher voltages, as well as increased sensitivity of smooth muscle M₃ and NK₁ receptors to acetylcholine and tachykinins. Hypoinsulinaemia may be a primary contributor to intestinal motility dysfunction in type 1 diabetes mellitus.

Tu1451 Retrograde Axonal Transport of Fluorogold Was Reduced in Gastric Ascending, but Not Descending, Enteric Neurons in Type 2 Diabetic Kkay as Compared to Control Kk Mice

Retrograde axonal transport was impaired in the gastric branch of the vagus nerve in animals with type 1 diabetes mellitus (DM). These studies evaluated active, retrograde axonal transport of the neural tracer fluorogold (FG) in gastric enteric neurons in KKAy mice 4 weeks prior to and at the onset of type 2 DM, and after 8, 16, and 24 weeks of uncontrolled DM. We hypothesized that the number of FG-labeled enteric neurons would progressively decrease with increasing durations of uncontrolled DM as compared to control KK mice. Under anesthesia, FG was injected into the ventral fundus or antrum. After 5-6 days, the stomach was removed and the numbers of FG-labeled enteric neurons with cell bodies in 2.8 mm2 of fundus, 1.7 mm2 of corpus, and 1.7 mm2 of antrum were counted. The total number of FG-labeled neurons in ascending pathways (cell bodies in corpus or antrum with nerve terminals in fundus), descending pathways (cell bodies in the fundus or corpus with nerve terminals in antrum), the local fundic pathway (cell body and nerve terminals in fundus), and the local antral pathway (cell body and nerve terminals in antrum) were compared in control and diabetic mice of different ages by 2-way ANOVA. Only main effects were significant; no interactions were significant. Data are mean±SEM with n representing the number of mice. In the ascending pathway, fewer FG-labeled neurons were observed in diabetic as compared to control mice [F(1,116)=10.668; control (n=68), 179±11; diabetic (n= 58), 145±11*; *p<0.05]. In contrast, similar numbers of FG-labeled neurons were observed in descending pathways [F(1,124)=0.489; p=0.486], local fundic pathways [F(1,53)=2.464; p=0.122], and local antral pathways [F(1,57)=0.728; p=0.397] of control and diabetic mice. Overall, the number of FG-labeled neurons decreased significantly with age in all pathways [ascending: F(4,116)=42.862; descending: F(4,124)=2.456; local fundic: F(4,53)=5.195; local antral: F(4,57)=8.920]. In the ascending pathway, the number of FG-labeled neurons was significantly less at 16 weeks of age [n=28, 146±12] as compared to 4 weeks [n=24, 246±8] or 8 weeks [n=28, 229±12] of age, reaching its lowest value at 24 weeks of age [n= 24, 80±10]. In conclusion, DM did not uniformly impair active tracer transport in all gastric enteric neurons suggesting specific axonal targets in ascending enteric neurons rendering them more susceptible to hyperglycemia and low effective insulin. Supported by ORSP at MWU.

T1272 Muscarinic Antagonist Reduced Tegaserod-Induced Contractions of Fundus But Not Antrum in Obese Type 2 Diabetic But Not Control Mice

Prokinetic drugs accelerate gastric emptying in diabetic patients with gastroparesis. Tegaserod, a 5-HT4 partial agonist, increases GI motility by enhancing Ach release from autonomic neurons innervating smooth muscle. Autonomic neuropathy, evident in animal models of diabetes, may alter the efficacy of prokinetic drugs which rely on effective neuromuscular transmission to increase motility. These studies examined the effect of tegaserod on motility of whole thickness rings of fundus and antrum from control (KK) and obese type 2 diabetic Agouti mice (KK.Cg-Aγ) using tissue bath set-up. Tissues were pretreated with vehicle or 4-DAMP (M3 muscarinic receptor antagonist), then treated with bethanechol or tegaserod (TG). Drug responses were quantified as area under the curve (g*s) or peak contraction (g). At 7 weeks after diabetes onset, diabetic mice were heavier (42±1g) than control mice (32±1g). Bethanechol (10 μM)-induced peak contractions of antrum, but not fundus, were reduced in diabetic as compared to control mice [antrum: control (n=11), 1.6±0.2; diabetic (n=10), 1.1±0.1*; *p<0.05][fundus: control (n=9), 2.9±0.3; diabetic (n=11), 2.8±0.3]. In both control and diabetic mice, TG treatment resulted in relaxation of antrum [0.1 μM: control (n=13), 1±1; diabetic (n=12), 3±2] [1.0 μM: control (n=14), -1±1; diabetic (n=12), -1±3] [10 μM: control (n=14), -6±2; diabetic (n=15), -5±3], but contraction of fundus [0.1 μM: control (n=13), 10±3; diabetic (n=11), 6±4] [1.0 μM: control (n=13), 18±6; diabetic (n=11), 31±11] [10 μM: control (n=13), 28±10; diabetic (n=13), 43±13]. 4-DAMP at 0.5 μM was equally effective, but at 1 μM was more effective, in reducing bethanechol (10 μM)induced contractions of fundus from diabetic mice [vehicle: (n=11), 380±58; 0.5 μM: (n= 8), 77±23; 1 μM: (n=8), 12±2*; *p<0.05] as compared to control mice [vehicle: (n=9), 374±53; 0.5 μM: (n=8), 55±14; 1 μM: (n=9), 20±3]. Using fundus, 4-DAMP at 0.5 μM, but not 1 μM, reduced TG (10 μM)-induced contractions in diabetic mice [vehicle: (n=13), 43±13; 0.5 μM: (n=14), 7±8*; 1 μM: (n=14), 13±7; *p<0.05], but not in control mice [vehicle: (n=13), 28±10; 0.5 μM: (n=11), 10±10; 1 μM: (n=11), 24±9]. 4-DAMP (0.5, 1, 10 μM) did not alter TG (10 μM)-induced relaxation of antrum from control or diabetic mice. In diabetic, but not control mice, TG-induced contractions of fundus were reduced by 4-DAMP, suggesting an additional non-cholinergicmechanism for TG-induced contraction in control mice. Both gastric region and type 2 diabetes may alter the effectiveness of prokinetic agents in accelerating gastric emptying. Supported by College of Health Sciences and ORSP at MWU.