Genistein is a naturally occurring isoflavone found in soy. Genistein has been shown to increase the open probability of the most common cystic fibrosis (CF) disease-associated mutation, ∆F508-CFTR. Mice homozygous for the ∆F508 mutation are characterized with severe intestinal disease and require constant laxative treatment for survival. This pathology mimics the intestinal obstruction (meconium ileus) seen in some cystic fibrosis patients. This study tested whether dietary supplementation with genistein would reduce the dependence of the ∆F508 CF mouse model on laxatives for survival, thereby improving mortality rates. At weaning (21 days), homozygous ∆F508 mice were maintained on one of three diet regimens for a period of up to 65 days: normal diet, normal diet plus colyte, or genistein diet. Survival rates for males were as follows: standard diet (38%, n = 21), standard diet plus colyte (83%, n = 42) and genistein diet (60%, n = 15). Survival rates for females were as follows: standard diet (47%, n = 19), standard diet plus colyte (71%, n = 38), and genistein diet (87%, n = 15). Average weight of male mice fed genistein diet increased by ~2.5 g more (p = 0.006) compared to those with colyte treatment. Genistein diet did not change final body weight of females. Expression of intestinal SGLT-1 increased 2-fold (p = 0.0005) with genistein diet in females (no change in males, p = 0.722). Expression of GLUT2 and GLUT5 was comparable between all diet groups. Genistein diet reduced the number of goblet cells per micrometer of crypt depth in female (p = 0.0483), yet was without effect in males (p = 0.7267). The results from this study demonstrate that supplementation of diet with genistein for ~45 days increases the survival rate of female ∆F508-CF mice (precluding the requirement for laxatives), and genistein only improves weight gain in males.
Muscarinic acetylcholine receptor modulation contributes to changes in hypoglossal motoneuron excitability through pre- and post-synaptic effects, an important mechanism for regulation of airway tone during sleep. Previous data indicate a net excitatory effect of muscarinic modulation of inspiratory bursting in neonatal mouse brainstem slices, yet a net inhibitory effect on inspiratory bursting in adult rats. We tested the hypothesis that a developmental shift in muscarinic modulation impacts hypoglossal motoneurons, using neuroanatomical and electrophysiological methods. Immunofluorescence confirmed hypoglossal motoneurons express M1, M2, M3, and M5 muscarinic subtypes. We observed maturational decreases in M1 and M5 labeling intensity, M3 labeling intensity generally decreased with postnatal (P) age, although there was a transient increase in expression at P17. M2 labeling intensity did not change, although distribution of M2 labeling with postnatal maturation may change. Next, using the in-vitro rhythmic slice preparation from CD1 mice of either sex, local application of muscarine (100 μM) into the hypoglossal nucleus was tested in mice P0-14. The net effect of applying muscarine results in excitatory output after local application into the rhythmic slice because each data point is a positive percent change from control. The magnitude of this potentiation appears to lessen as the mice age toward P14. To evaluate the mechanisms through which the magnitude of muscarinic potentiation of inspiratory bursting changes with postnatal maturation, we next assessed the role of excitatory and inhibitory muscarinic receptor subtypes. Blocking M1 locally (pirenzepine, 100 μM) significantly decreased muscarine-mediated potentiation of inspiratory bursting (to 72±14% of control muscarine response, n=9, P0-5). Preliminary data indicate bath application of 4-DAMP (M3 antagonist) attenuated muscarinic potentiation of inspiratory bursting (range: to 7%-25% of control burst response, n=3, P0-5). Modulating M5 locally with a positive allosteric modulator VU 0238429 (1000 μM) had little effect on burst amplitude (119±4% of control burst response, n=3, P0-6). Blocking M2 locally (methoctramine, 2μM) showed no effect on inspiratory burst potentiation after applying muscarine (181±65% vs 187±69%, n=7, P0-5). Bath application of methoctramine did not have a significant effect on inspiratory burst potentiation by muscarine (179±65% vs control 182±90%, n=10) in P0-5 mice while preliminary analysis suggests an increasing effect of blocking M2 with postnatal maturation (147±56% vs control 134±30%, n=4, P9-14). These data support excitatory muscarinic receptor subtypes contributing to the excitatory response early in postnatal maturation. Based on our neuroanatomical data, we speculate the increasing inhibitory response to muscarinic modulation with postnatal maturation is due to a decreased contribution of excitatory muscarinic receptor subtypes. NIH R15HL148870 (Revill) and NIH/NHLBI R25 HL126140 (Moreno, Garcia, Parthasarathy) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Genistein is a naturally occurring isoflavone found in soy. Mice homozygous for the ∆F508 mutation are characterized with severe intestinal disease and require constant laxative treatment for survival. This pathology mimics the intestinal obstruction (meconium ileus) seen in some cystic fibrosis (CF) patients. We therefore tested whether dietary supplementation with genistein would reduce the dependence of the ∆F508 CF mouse model on laxatives for survival, thereby improving mortality rates. At weaning (21 days), we maintained homozygous ∆F508 mice on three diet regimens for a period of up to 65 days; normal diet, normal diet + Colyte or genistein diet. Survival rates for males were as follows: standard diet (38%), standard diet plus Colyte (83%) or genistein diet (60%). Survival rates for females were as follows: standard diet (47%), standard diet plus Colyte (71%), or genistein diet (87%). Average weight of male mice fed genistein diet increased by ~2.5 g more compared to those with Colyte treatment. Genistein diet did not change final body weight of females. Expression of SGLT-1 increased 2-fold with genistein diet in females (no change in males). Expression of GLUT2 and GLUT5 was comparable between all diet groups. Genistein diet reduced the number of goblet cells per micometer of crypt depth in female, yet was without effect in males. We conclude that supplementation of diet with genistein for ~45 days increases the survival rate of female ∆F508-CF mice precluding the requirement for laxatives, and only improves weight gain in males.
Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.
Chronic consumption of a western diet (high fat with high sugar, HFHS) is associated with type 2 diabetes, inflammation. Genistein is a naturally occurring isoflavone known to exert anti‐inflammatory properties and improve insulin sensitivity. Similar benefits have also been associated with moderate exercise. This study aimed to determine whether dietary genistein (600 mg genistein/kg diet, Gen) or moderate exercise (Ex), or both (Gen+Ex) would reduce the obese‐diabetic phenotype and thus mitigate the intestinal dysfunction induced by chronic consumption of a HFHS “western diet”, in C57BL/6J male and female mice. C57BL/6J mice (6 weeks old) were randomly assigned to one of the following groups (n=10/group): lean control, HFHS, HFHS+Gen, HFHS+Ex, and HFHS+Gen+Ex. The HF diet consisted of 60% saturated fat, 20% carbohydrate, 20% protein. The HS drinking water contained sucrose and fructose. Moderate exercise comprised daily treadmill running for 150 minutes/week for 12 weeks. We characterized jejunum function in this clinically relevant mouse model. We measured transepithelial short circuit current (Isc, a measure of chloride secretion), across freshly isolated segments of jejunum from the mice. Basal Isc was decreased 3‐fold in the HFHS males (19.3±1.7 μA/cm2, n=5, P<0.05) compared to leans (66.8±8.1 μA/cm2, n=5). Basal Isc was decreased 4‐fold in the HFHS females (16.9±5.4 μA/cm2, n=5, P<0.05) compared to leans (67.1±6.2 μA/cm2, n=5). Exercise alone significantly increased basal Isc for both sexes. We evaluated several key proteins involved in regulating Isc and/or intestinal motility: (1) expression of the adenosine receptor, A2BR was increased in HFHS females (5‐fold, P<0.05) and males (25‐fold, P<0.05) compared to lean controls. While there was no treatment effect in males, in females A2BR expression was reduced with Gen and Gen+Ex to ‘lean‐like’ levels. (2) expression of KCa (needed to create the driving force for chloride secretion) was decreased in HFHS males (3.7‐fold, P<0.05) compared to leans controls. Evaluation of tight junction proteins (essential for intestinal epithelial barrier integrity) indicated that desmoglein‐1a, claudin‐1 and desmocollin were all significantly decreased in HFHS females compared to leans, and treatments were without effect. Levels of claudin‐1 were also decreased in HFHS males with recovery by Gen+Ex: indicating that intestinal barrier integrity is likely improved. These benefits are associated with improvements in serum levels of TNFα and IL‐6 (additional cytokines are under evaluation). A comparison of jejunum morphology including assessment of Edu positive proliferative cells (i.e. villi length, number of goblet cells/villi, crypt depth, number of goblet cells/crypt, number of EdU cells/crypt) is in progress. These data suggest that jejunum Cl secretion and intestinal barrier function is markedly dysregulated by consumption of HFHS and may explain the gastrointestinal disturbances associated with diet‐induced diabetic obesity. We find that genistein supplementation and/or m...
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