Purinergic and cholinergic neuro-neuronal transmission underlying reflexes activated by mucosal stimulation in the isolated guinea-pig ileum

We present evidence that adenosine triphosphate (ATP) plays a major role in excitatory neuro-neuronal transmission in ascending and descending reflex pathways to the longitudinal (LM) and circular muscle (CM).A partitioned bath was used for the pharmacological isolation of a segment of guinea-pig ileum (∼6 cm in length), allowing drugs to be selectively applied to an intermediate region between the region where mucosal stimulation was applied and that where mechanical recordings were made.Brush stroking the mucosa (3 strokes) elicited a synchronous contraction of the LM and CM both above (ascending excitation) and below (descending excitation) the site of stimulation. All reflexes were abolished when tetrodotoxin (1 μm) was applied to the intermediate chamber.Hexamethonium (300 μm) added to the intermediate chamber abolished the ascending contraction in 15 % of oral preparations (from 26 preparations, 18 animals) and the descending contraction in 13 % of anal preparations studied (from 53 preparations, 48 animals). In the remaining 85 % of oral preparations, hexamethonium usually attenuated the oral contraction of the LM and CM. However, in the remaining 87 % of anal preparations, hexamethonium had no effect on the anal contraction of the LM and CM.Oral and anal reflexes that were hexamethonium resistant were either abolished or attenuated by the further addition of the P2 purinergic receptor antagonist pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 10 μm) or α,β-methylene ATP (50–100 μm) to the intermediate chamber.1,1-Dimethyl-4-phenyl-piperazinium iodide (DMPP, 20 μm) or α,β-methylene ATP (50–100 μm) stimulated both ascending and descending excitatory pathways, when applied to the intermediate chamber.In conclusion, ascending and descending neuro-neuronal transmission in excitatory nervous pathways to the LM and CM is complex and clearly involves neurotransmitter(s) other than acetylcholine (ACh). We suggest mucosal stimulation releases ACh and ATP in both ascending and descending excitatory reflex pathways that synapse with excitatory motoneurons to the LM and CM

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 (p2 = 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.</p

Exercise Prevents Weight Gain and Alters the Gut Microbiota in a Mouse Model of High Fat Diet-Induced Obesity

<div><p>Background</p><p>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.</p><p>Objective</p><p>Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.</p><p>Methods</p><p>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.</p><p>Results</p><p>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).</p><p>Conclusion</p><p>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.</p></div

Exercise, Body Weight and Glucose Tolerance.

<p>A. Weekly exercise was recorded for low fat/exercise (LF/Ex) and high fat/exercise (HF/Ex) mice. B. Weekly body weight starting at week 0 of the diet and activity protocol (age  = 5 weeks) through week 12 (age  = 17 weeks) was recorded. C. Oral glucose tolerance was tested at week 12 of the diet and activity protocol. D. The area under the curve (AUC) was calculated for oral glucose tolerance. Data are presented as mean ± SEM. Groups are abbreviated as follows: low-fat/sedentary (LF/Sed), low-fat/exercise (LF/Ex), high-fat/sedentary (HF/Sed) and high-fat/exercise (HF/Ex). Data were analyzed by 2-way ANOVA with a Sidak post hoc test. Significant differences indicated as follows: “*” p<0.05 for diet effect, “†” p<0.05 activity effect and “‡” p<0.05 diet and activity interaction. n = 6 mice/group.</p

Diet and Activity Altered the Relative Level of Bacteroidetes and Firmicutes.

<p>A. The fold change in Bacteroidetes and Firmicutes was determined from the 2^−ΔΔCt values calculated from the ΔCt values generated by quantitative polymerase chain reaction (qPCR) using primers specific to each phyla (one-way ANOVA). B. Criterion validity of qPCR was examined by correlating the ΔCt-Bacteroidetes: ΔCt-Firmicutes ratio with the %-Bacteroidetes: %- Firmicutes ratios from sequencing. Data was analyzed by Pearson product-moment correlation coefficient and alpha level of p<0.05.</p

Phylum Level Changes with Diet and Activity.

<p>At week 12, diet and activity changed the levels of two major and two minor phyla of bacteria. Data were analyzed by 2-way ANOVA with a Sidak post hoc test. Significant differences indicated as follows: “*” p<0.05 for diet effect, “†” p<0.05 activity effect and “‡” p<0.05 diet and activity interaction. n = 6 mice/group.</p