Immunocytochemical localization of a brush border hydrolase, lactase, in newborn suckling rat jejunum

Author(s):  
László G. Kömüves ◽  
Mary A. Dudley ◽  
Buford L. Nichols

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23), an integral membrane glycoprotein of the small intestinal brush border, converts lactose, the main carbohydrate in milk, to its monosaccharide components. Although the activity of LPH is high in suckling rats, little is known about its distribution within the intracellular compartments of the secretory pathway and brush border. We present the first description of the ultrastructural localization of LPH in the neonatal jejunum of suckling rat pups.Pieces of jejunum from 12- to 14-d-old suckling rat pups from three litters of Sprague- Dawley rats were fixed with 4% freshly prepared paraformaldehyde in 100 mM phosphate buffer, pH=7.40, for 4 h, and stored in 1% paraformaldehyde, at 4°C, until further processing. The samples were sectioned after cryoprotection in 2.3 M sucrose in an RMC MT-7 ultramicrotome equipped with CR21 cryoattachment. Ultrathin cryosections were collected on Formvar-coated, carbon-evaporated nickel grids. The nonspecific binding sites were blocked by 1% heat-inactivated newborn calf serum in 10 mM Trizma buffer, pH=7.60, containing 500 mM NaCl, 0.05% NaN3 and 20 mM glycine (buffer A).

Author(s):  
A.R. Beaudoin ◽  
G. Grondin ◽  
A. Lord ◽  
M. Pelletier

We have recently described the ultrastructural localization of NADPase activity in the exocrine pancreas of rat. The enzyme was found in the intermediate saccules of the Golgi apparatus, in dense bodies and lysosomes but was absent from zymogen granules. A very intense reaction was noticed in a peculiar structure which was termed “Snake-Like Tubule” (SLT). The purposes of the present study were firstly to delineate SLT distribution in the acinar cell and secondly to define any possible relationship or association with other cellular organelles.NADPase cytochemical reaction was performed on the pancreas of adult Sprague Dawley rats. Small lobules were excised and fixed for 50 min, at 4°C, in 2% glutaraldehyde buffered with 0.1M cacodylate at pH 7.2. Lobules were rinsed several times with the same buffer containing 570 sucrose and cut with a Mcllwayn tissue chopper. Sections were washed several times with buffer and incubated for 2 hr at 37°C in the following medium: 4mM NADPH; 40mM sodium acetate buffer, pH 5.0; 4mM lead acetate and 5% sucrose.


2002 ◽  
Vol 282 (4) ◽  
pp. F730-F740 ◽  
Author(s):  
Li Yang ◽  
Patrick K. K. Leong ◽  
Jennifer O. Chen ◽  
Nilem Patel ◽  
Sarah F. Hamm-Alvarez ◽  
...  

Acute hypertension rapidly decreases proximal tubule (PT) Na+ reabsorption, facilitated by a redistribution of PT Na+/H+exchangers (NHE3) out of the apical brush border, increasing NaCl at the macula densa, the signal for autoregulation of renal blood flow and GFR. This study aimed to determine whether NHE3 activity per transporter decreases during acute hypertension and the time dependence of the response. Blood pressure was elevated by 50–60 mmHg in male Sprague-Dawley rats for 5 or 30 min by constricting arteries. Renal cortical membranes were fractionated by density gradient centrifugation. NHE3 transport activity was assayed as the rate of appearance of acridine orange (AO) from AO-loaded vesicles in response to an inwardly directed Na+ gradient. After 5-min hypertension, 20% of total NHE3 protein, assayed by immunoblot, redistributed from low-density apical membranes to middensity membranes enriched in intermicrovillar cleft markers; by 30 min, a similar percentage shifted to heavier density membranes containing markers of endosomes. NHE3 activity shifted to higher density membranes along with NHE3 protein, that is, no change in activity/transporter during acute hypertension. Confocal analysis of NHE3 distribution also verified removal from apical microvilli and appearance in subapical vesicles. We conclude that the decrease in renal PT Na+ transport during acute hypertension is mediated by removal of transport-competent NHE3 from the apical brush border to subapical and internal reserves.


2006 ◽  
pp. 208
Author(s):  
Rustadi Sosrosumihardjo ◽  
Agus Firmansyah ◽  
Asri Rasad ◽  
Daldiyono Harjodisastro ◽  
Endi Ridwan ◽  
...  

2014 ◽  
Vol 306 (12) ◽  
pp. G1108-G1116 ◽  
Author(s):  
Joost Overduin ◽  
Tracy S. Tylee ◽  
R. Scott Frayo ◽  
David E. Cummings

Plasma levels of the orexigenic hormone ghrelin are suppressed by meals with an efficacy dependent on their macronutrient composition. We hypothesized that heterogeneity in osmolarity among macronutrient classes contributes to these differences. In three studies, the impact of small intestinal hyperosmolarity was examined in Sprague-Dawley rats. In study 1, isotonic, 2.5×, and 5× hypertonic solutions of several agents with diverse absorption and metabolism properties were infused duodenally at a physiological rate (3 ml/10 min). Jugular vein blood was sampled before and at 30, 60, 90, 120, 180, 240, and 300 min after infusion. Plasma ghrelin was suppressed dose dependently and most strongly by glucose. Hyperosmolar infusions of lactulose, which transits the small intestine unabsorbed, and 3- O-methylglucose (3- O-MG), which is absorbed like glucose but remains unmetabolized, also suppressed ghrelin. Glucose, but not lactulose or 3- O-MG, infusions increased plasma insulin. In study 2, intestinal infusions of hyperosmolar NaCl suppressed ghrelin, a response that was not attenuated by coinfusion with the neural blocker lidocaine. In study 3, we reconfirmed that the low-osmolar lipid emulsion Intralipid suppresses ghrelin more weakly than isocaloric (but hypertonic) glucose. Importantly, raising Intralipid's osmolarity to that of the glucose solution by nonabsorbable lactulose supplementation enhanced ghrelin suppression to that seen after glucose. Hyperosmolar ghrelin occurred particularly during the initial 3 postinfusion hours. We conclude that small intestinal hyperosmolarity 1) is sufficient to suppress ghrelin, 2) may combine with other postprandial mechanisms to suppress ghrelin, 3) might contribute to altered ghrelin regulation after gastric bypass surgery, and 4) may inform dietary modifications for metabolic health.


2000 ◽  
Vol 350 (3) ◽  
pp. 805-814 ◽  
Author(s):  
Niclas G. KARLSSON ◽  
Fredrik J. OLSON ◽  
Per-Åke JOVALL ◽  
Ylva ANDERSCH ◽  
Lennart ENERBÄCK ◽  
...  

The sialylation of the oligosaccharides from small-intestinal mucins during a 13-day infectious cycle was studied in Sprague–Dawley rats with the parasite Nippostrongylus brasiliensis. Sialic acid analysis and release, permethylation and analysis by GC-MS of the sialylated oligosaccharides isolated from the ‘insoluble’ mucin complex revealed a relative decrease (4–7-fold) of N-glycolylneuraminic acid compared with N-acetylneuraminic acid just before parasite expulsion. Northern blots showed that this effect was due to the decreased expression of a hydroxylase converting CMP-N-acetylneuraminic acid into CMP-N-glycolylneuraminic acid. Analysis of other rat strains showed that this parasite infection also caused the same effect in these animals. Detailed analysis of infected Sprague–Dawley rats revealed four sialylated oligosaccharides not found in the uninfected animals. These new oligosaccharides were characterized in detail and all shown to contain the trisaccharide epitope NeuAc/NeuGcα2-3(GalNAcβ1-4)Galβ1 (where NeuGc is N-glycolyl neuraminic acid). This epitope is similar to the Sda- and Cad-type blood-group antigens and suggests that the infection causes the induction of a GalNAcβ1-4 glycosyltransferase. This model for an intestinal infection suggests that the glycosylation of intestinal mucins is a dynamic process being modulated by the expression of specific enzymes during an infection process.


1999 ◽  
Vol 87 (2) ◽  
pp. 484-490 ◽  
Author(s):  
Jalal M. Abu-Shaweesh ◽  
Ismail A. Dreshaj ◽  
Agnes J. Thomas ◽  
Musa A. Haxhiu ◽  
Kingman P. Strohl ◽  
...  

Premature infants respond to hypercapnia by an attenuated ventilatory response that is characterized by a decrease in respiratory frequency. We hypothesized that this impaired hypercapnic ventilatory response is of central origin and is mediated via γ-aminobutyric acid-ergic (GABAergic) pathways. We therefore studied two groups of maturing Sprague-Dawley rats: unrestrained rats in a whole body plethysmograph at four postnatal ages (5, 16–17, 22–23, and 41–42 days); and ventilated, decerebrate, vagotomized, paralyzed rats in which phrenic nerve responses to hypercapnia were measured at 4–6 and 37–39 days of age. In the unrestrained group, the increase in minute ventilation induced by hypercapnia was significantly lower at 5 days vs. beyond 16 days. Although there was an increase in tidal volume at all ages, frequency decreased significantly from baseline at 5 days, whereas it increased significantly at 16–17, 22–23, and 41–42 days. The decrease in frequency at 5 days of age was mainly due to a significant prolongation in expiratory duration (Te). In the ventilated group, hypercapnia also caused prolongation in Te at 4–6 days but not at 37–39 days of age. Intravenous administration of bicuculline (GABAA-receptor blocker) abolished the prolongation of Te in response to hypercapnia in the newborn rats. We conclude that newborn rat pups exhibit a characteristic ventilatory response to CO2 expressed as a centrally mediated prolongation of Te that appears to be mediated by GABAergic mechanisms.


1997 ◽  
Vol 152 (3) ◽  
pp. 423-430 ◽  
Author(s):  
K Kondo ◽  
A Levy ◽  
S L Lightman

We have used in situ hybridization histochemistry to investigate the effects of maternal thyroidectomy and chronic maternal iodine deficiency on basal neuroendocrine function in rat pups. Specifically, we have measured hypothalamic thyrotrophin-releasing hormone (TRH) and pituitary thyroid-stimulating hormone (TSH) expression together with circulating levels of tri-iodothyronine (T3) in rat pups delivered from and suckled by thyroidectomized or iodine-deficient dams. Because of the close interaction between the thyroid, adrenal and growth hormone axes, we have also examined hypothalamic corticotrophin-releasing hormone (CRH) and growth hormone-releasing hormone (GRH) transcripts at the same time points: birth, 1 month and 2 months of age. Three weeks after surgical thyroidectomy, adult female Sprague–Dawley rats proved unable to carry pups to term and lactate successfully. Pups delivered from thyroidectomized dams given a small replacement dose of T3 during pregnancy were significantly lighter than controls (84 ± 3%) and had markedly depressed plasma T3 levels (36 ±6% of control). Hypothalamic CRH and GRH transcript levels were significantly decreased in pups at birth (to 8±2·5% and 24 ± 8% of control respectively) but had returned to normal by 1 month after delivery. Pituitary TSH transcript levels and hypothalamic levels of TRH transcripts, however, were similar to those of controls. Only one of seven dams fed a low-iodine diet for 6 months produced live pups, and these were too few in number to produce significant data. Dams fed a lowiodine diet from 4 months before mating, however, did produce live pups and although they were not significantly lighter than control pups at birth, by 1 month after birth, they were significantly lighter (72 ± 3% of controls). Circulating T3 levels were not significantly different from control at any time point examined. Hypothalamic TRH levels were significantly elevated at birth (451 ± 138% of control), but this difference was not maintained at 1 or 2 months after birth despite the lactating dams being maintained on the low-iodine diet. Pituitary TSH levels showed an upward trend at all time points that reached significance at 1 month after birth (204 ± 19%; P<0·05). Hypothalamic CRH and GRH transcript levels were not different from controls at any time point. In summary, chronic iodine deficiency or thyroidectomy with low-level T3 replacement in Sprague–Dawley rats markedly impaired fertility and the ability to carry pups to term, and produced an unexpectedly modest up-regulation of the hypothalamo–pituitary–thyroid axis and down-regulation of the hypothalamo–pituitary–adrenal axis. Journal of Endocrinology (1997) 152, 423–430


2021 ◽  
Author(s):  
Delei Cai ◽  
Qing Chen ◽  
Jianlong Han ◽  
Yanhua Song ◽  
Zhen Meng ◽  
...  

Abstract In this study, we used Sprague–Dawley rats to observe the intervention effects of curcumin on bioavailability of polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs). We reported the bioavailability of tetra- to hexa-chlorinated PCDD/Fs rose gradually, while that of hepta- and octa-chlorinated PCDD/Fs declined, and no obvious change was found in the bioavailability of DL-PCBs. Curcumin markedly reduced the toxic equivalent (TEQ) of PCDD/Fs in rats, illustrating it might competitively inhibit absorption of PCDD/Fs in small intestinal epithelial cells due to the similar chemical structure (diphenyl) between curcumin and PCDD/Fs. Moreover, curcumin was capable of lowering the TEQ of DL-PCBs in the liver of male rats, but caused no changes in female rats. In conclusion, the prominent decline in the bioavailability of PCDD/Fs and DL-PCBs induced by curcumin may serve as one of the detoxification mechanisms of curcumin for these pollutants.


2007 ◽  
pp. 183-192
Author(s):  
š Mozeš ◽  
z Šefčíková ◽  
Ľ Lenhardt

To investigate the relationship between early nutritional experience, ontogeny of the small intestinal functions and predisposition to obesity development, the following experimental models of male Sprague-Dawley rats were used: 1) rats in which the quantity of nutrition was manipulated from birth to weaning (day 30) by adjusting the number of pups in the nest to 4 (SL), 10 (NL) and 16 pups (LL) and 2) littermates of SL, NL and LL rats fed either a standard or a hypercaloric diet from days 80 to 135 of age. The overfed SL pups were overweight after day 15 and became permanently obese, whereas the underfed smaller LL pups, due to accelerated growth and enhanced food intake from day 30 to day 35, attained a body fat level that did not differ from normally fed NL rats. Moreover, a significantly increased duodenal and jejunal alkaline phosphatase (AP) activity was found in SL and LL rats and these acquired somatic and intestinal characteristics persisted from weaning throughout life. Eight weeks of high-energy diet feeding elicited a similar pattern of intestinal response in SL and LL rats that was clearly different from NL rats. Despite energy over-consumption in these three groups, both SL and LL rats still displayed enhanced AP activity and showed a significant increase in protein/DNA ratio accompanied with a significant body fat accretion. These results indicate that the postnatally acquired small intestinal changes induced by over- and undernutrition could be involved in the similar predisposition to obesity risk in later life when caloric density of the diet is raised.


2020 ◽  
Vol 4 (8) ◽  
Author(s):  
Libo Tan ◽  
Yanqi Zhang ◽  
Kristi M Crowe-White ◽  
Katelyn E Senkus ◽  
Maddy E Erwin ◽  
...  

ABSTRACT Background Vitamin A (VA) has been demonstrated to be a regulator of adipose tissue (AT) development in adult obese models. However, little is known about the effect of VA on obesity-associated developmental and metabolic conditions in early life. Objectives We aimed to assess the effects of dietary VA supplementation during suckling and postweaning periods on the adiposity and metabolic health of neonatal and weanling rats from mothers consuming a high-fat diet (HFD). Methods Pregnant Sprague-Dawley rats were fed a normal-fat diet (NFD; 25% fat; n = 2) or an HFD (50% fat; n = 2), both with 2.6 mg VA/kg. Upon delivery, half of the rat mothers were switched to diets with supplemented VA at 129 mg/kg, whereas the other half remained at 2.6 mg VA/kg. Four groups of rat pups were designated as NFD, NFD + VA, HFD, and HFD + VA, respectively. At postnatal day (P)14, P25, and P35, pups (n = 4 or 3/group) were killed. Body weight (BW), visceral white AT (WAT) mass, brown AT (BAT) mass, uncoupling protein 1 mRNA expression in BAT, serum glucose, lipids, adipokines, and inflammatory biomarkers, as well as serum and AT redox status were assessed. Results Rat pups in the HFD group exhibited significantly higher BW, WAT mass, and serum glucose and leptin but reduced BAT mass compared with the NFD group. Without affecting the dietary intake, supplementing the HFD with VA significantly reduced the BW and WAT mass of pups but increased the BAT mass, significantly lowered the systemic and WAT oxidative stress, and modulated serum adipokines and lipids to some extent. Conclusions VA supplementation during suckling and postweaning periods attenuated metabolic perturbations caused by excessive fat intake. Supplementing maternal or infant obesogenic diets with VA or establishing a higher RDA of VA for specific populations should be studied further for managing overweight/obesity in early life.


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