Kaolinite ingestion facilitates restoration of body energy reserves during refeeding after prolonged fasting.

Clay consumption is a spontaneous behavior currently observed in animals and humans, particularly during undernutrition. Often regarded as intestinal care products, ingested clays also enhance food efficiency, notably by increasing intestinal lipid uptake. Clay complementation could then optimize the reconstitution of energy reserves in animals with low lipid stocks consecutive to intensive fasting. The aim of this study was therefore to observe the effects of voluntarily kaolinite complementation during the refeeding of fasted rats to determine whether body mass, food uptake, lipid and mineral contents as intestinal morphology and protein profile were modified. This study examined two types of refeeding experiments after prolonged fasting. Firstly, rats with ad libitum access to food were compared to rats with ad libitum access to food and kaolinite pellets. Animals were randomly put into the different groups when the third phase of fasting (phase III) reached by each individual was detected. In a second set of experiments, rats starting phase III were refed with free access to food and kaolinite pellets. When animals had regained their body mass prior to fasting, they were euthanized for chemical, morphological, and proteomic analyses. Although kaolinite ingestion did not change the time needed for regaining prefasting body mass, daily food ingestion was seen to decrease by 6.8% compared with normally refed rats, without affecting lipid composition. Along the intestinal lining, enterocytes of complemented animals contained abundant lipid droplets and a structural modification of the brushborder was observed. Moreover, the expression of two apolipoproteins involved in lipid transport and satiety (ApoA-I and ApoA-IV) increased in complemented rats. These results suggest that kaolinite complementation favors intestinal nutrient absorption during refeeding despite reduced food uptake. Within the intestinal lumen, clay particles could increase the passive absorption capacity and/or nutrient availability that induce mucosal morphological changes. Therefore, clay ingestion appears to be beneficial for individuals undergoing extreme nutritional conditions such as refeeding and limited food supplies.

Effects of controlled ingestion of kaolinite (5%) on food intake, gut morphology and in vitro motility in rats.

Geophagia is found in various animal species and in humans. We have previously shown that spontaneously ingested kaolinite interacts with the intestinal mucosa modifies nutrient absorption and slows down gastric emptying and intestinal transit in rats in vivo. However, the precise mechanisms involved are not elucidated. The aim of this work was to investigate the effects of controlled kaolinite ingestion on food intake, gut morphology and in vitro motility in rats. Male Wistar rats were fed with 5% kaolinite in standard food pellets during 7, 14 and 28 days. Body mass and food consumption were measured daily. Intestinal morphological and proteomic analyses were conducted. The length of mucosal lacteals was evaluated. Plasmatic levels of leptin and adiponectin were determined. Finally, organ bath studies were conducted to evaluate smooth muscle contractility. Food consumption was significantly increased during the first two weeks of kaolinite ingestion without any mass gain compared to controls. Kaolinite induced weak variations in proteins that are involved in various biological processes. Compared to control animals, the length of intestinal lacteals was significantly reduced in kaolinite group whatever the duration of the experiment. Leptin and adiponectin plasmatic levels were significantly increased after 14 days of kaolinite consumption. Changes in spontaneous motility and responses to electrical nerve stimulation of the jejunum and proximal colon were observed at day 14. Altogether, the present data give evidence for a modulation by kaolinite-controlled ingestion on satiety and anorexigenic signals as well as on intestinal and colonic motility.

Morphological plasticity of vertebrate aestivation.

Aestivation or daily torpor is an adaptive tactic to survive hot and dry periods of low food availability, and has been documented for species of lungfishes, teleost fishes, amphibians, reptiles, birds, and mammals. Among these species, aestivation is characterized by inactivity and fasting, and for lungfishes and amphibians the formation of a cocoon around the body to retard water loss. While metabolic and physiological changes to aestivation have been well examined, few studies have explored the morphological responses of organs and tissues to aestivation. Predictably, inactive tissues such as skeletal muscles and those of the gastrointestinal tract would regress during aestivation, and thus aid in the reduction of metabolic rate. African lungfishes experience changes in the structure of their skin, gills, lungs, and heart during aestivation. For anurans, the group most thoroughly examined for morphological responses, aestivation generates significant decreases in gut mass and modification of the intestinal epithelium. Intestinal mucosal thickness, enterocyte size, and microvillus length of anurans are characteristically reduced during aestivation. We can surmise from laboratory studies on fasting reptiles, birds, and mammals that they likewise experience atrophy of their digestive tissues during torpor or aestivation. Aestivation-induced loss of tissue structure may be matched with a loss of cellular function generating an integrative decrease in tissue performance and metabolism. Ample opportunity exists to remedy the paucity of studies on the morphological plasticity of organs and tissues to aestivation and examine how such responses dictate tissue function during and immediately following aestivation.

Functional changes with feeding in the gastro-intestinal epithelia of the Burmese python (Python molurus).

The morphology of the digestive system in fasting and refed Burmese pythons was determined, as well as the localization of the proton (H(+), K(+)-ATPase) and sodium (Na(+), K(+)-ATPase) pumps. In fasting pythons, oxyntopeptic cells located within the fundic glands are typically non-active, with a thick apical tubulovesicular system and numerous zymogen granules. They become active Immediately after feeding but return to a non-active state 3 days after the Ingestion of the prey. The proton pump, expressed throughout the different fasting/feeding states, is either sequestered in the tubulovesicular system in non-active cells or located along the apical digitations extending within the crypt lumen in active cells. The sodium pump is rapidly upregulated in fed animals and is classically located along the baso-lateral membranes of the gastric oxyntopeptic cells. In the Intestine, it is only expressed along the lateral membranes of the enterocytes, i.e., above the lateral spaces and not along the basal side of the cells. Thus, solute transport within the Intestinal lining is mainly achieved through the apical part of the cells and across the lateral spaces while absorbed fat massively crosses the entire height of the cells and flows into the Intercellular spaces. Therefore, in the Burmese python, the gastrointestinal cellular system quickly upregulates after feeding, due to Inexpensive cellular changes, passive mechanisms, and the progressive activation and synthesis of key enzymes such as the sodium pump. This cell plasticity also allows anticipation of the next fasting and feeding periods.

Interactions between ingested kaolinite and the intestinal mucosa in rat: proteomic and cellular evidences.

Although some of the effects of clay ingestion by humans and animals, such as gastrointestinal wellness and the increase in food efficiency are well known, the underlying mechanisms are not yet fully understood. Therefore, the interactions between the intestinal mucosa and kaolinite particles and their effects on mucosal morphology were observed using light microscopy (LM), transmission electron microscopy (TEM), conventional (CSEM) and environmental (ESEM) scanning electron microscopy combined with an EDX micro-analysis system. Kaolinite consumption, given with free access to rats, varied considerably from one animal to the other but was regular through time for each individual. Some kaolinite particles appeared chemically dissociated in the lumen and within the mucus barrier. Aluminium (Al) originating from ingested clay and present in the mucus layer could directly cross the intestinal mucosa. A significant increase in the thickness of the villi with large vacuoles at the base of the mucosal cells and a decrease in the length of enterocyte microvilli characterized complemented animals. The proteomic analyses of the intestinal mucosa of complemented rats also revealed several modifications in the expression level of cytoskeleton proteins. In summary, kaolinite particles ingested as food complement interact with the intestinal mucosa and modify nutrient absorption. However, these data, together with the potential neurotoxicity of Al, need further investigation.

Clay ingestion enhances intestinal triacylglycerol hydrolysis and non-esterified fatty acid absorption.

Consumption by animals and humans of earthy materials such as clay is often related to gut pathologies. Our aim was to determine the impact of kaolinite ingestion on glucose and NEFA transport through the intestinal mucosa. The expression of hexose transporters (Na/glucose co-transporter 1 (SGLT1), GLUT2, GLUT5) and of proteins involved in NEFA absorption (fatty acid transporter/cluster of differentiation 36 (FAT/CD36), fatty acid transport protein 4 (FATP4) and liver fatty acid binding protein (L-FABP)) was measured (1) in rats whose jejunum was perfused with a solution of kaolinite, and (2) in rats who ate spontaneously kaolinite pellets during 7 and 28 d. Also, we determined TAG and glucose absorption in the kaolinite-perfused group, and pancreatic lipase activity, gastric emptying and intestinal transit in rats orally administered with kaolinite. Glucose absorption was not affected by kaolinite perfusion or ingestion. However, kaolinite induced a significant increase in intestinal TAG hydrolysis and NEFA absorption. The cytoplasmic expression of L-FABP and FATP4 also increased due to kaolinite ingestion. NEFA may enter the enterocytes via endocytosis mainly since expression of NEFA transporters in the brush-border membrane was not affected by kaolinite. After uptake, rapid binding of NEFA by L-FABP and FATP4 could act as an intracellular NEFA buffer to prevent NEFA efflux. Increased TAG hydrolysis and NEFA absorption may be due to the adsorption properties of clay and also because kaolinite ingestion caused a slowing down of gastric emptying and intestinal transit.

Morphological changes of the rat intestinal lining in relation to body stores depletion during fasting and after refeeding.

Intestinal villus atrophy through prolonged fasting was studied according to two different metabolic phases reached by fasting animals and characterized by (a) the mobilization of fat stores as body fuel and (b) an increase in protein catabolism for energy expenditure. The mechanisms involved in the rapid jejunal restoration after refeeding were also determined. Mucosal structural atrophy during fasting proved to worsen over the two phases due mainly to the retraction of the lacteals in the lamina propria, as observed through the immunolocalization of aquaporin 1 in the endothelial cells of the lymphatic vessels and the detachment of the basal membrane of the epithelial lining at the tip of the villi. Microvilli surface area is preserved through fasting, and apical PepT1 expression increases during both metabolic fasting phases. Refeeding after both fasting phases induces an increase in FATP4 accompanied by a rapid lipid uptake by the enterocytes at the tip of the villi and a rapid extension of the lamina propria due to inflated lymphatic vessels. These mechanisms were more prevalent in animals refed after the phase III fast and could be considered as the major processes allowing complete morphological restoration of the jejunum within only 3 days after refeeding.

Cytokine expression in rat colon during postnatal development: regulation by glucocorticoids.

OBJECTIVES: Cytokine expression and regulation by glucocorticoids and retinoic acid were investigated in the colon during postnatal development. MATERIALS AND METHODS: Gene expression of the transforming growth factors (TGFs) TGF-beta1, TGF-beta2 and TGF-alpha and the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) in rat colon mucosa during weaning and in adult rats. Protein expression and distribution of TGF-betas was analysed in the colon from 14- and 60-day-old animals. The effect of hydrocortisone administration on mucosal cytokine transcripts (RT-PCR) and of dexamethasone on the expression of cytokines by the epithelial cell line IEC-18 and 2 subepithelial myofibroblasts (MIC 307-1 and 316) was examined. RESULTS: TGF-beta1 and TGF-beta2 messenger RNAs and proteins decreased in the entire colon from weaning to adult stages, whereas the amount of TGF-alpha messenger RNA increased in the proximal colon and decreased in the distal part of the colon in adult rats in comparison with weanlings. However, proinflammatory cytokines showed no postnatal changes in the proximal colon but decreased in the distal part in comparison with weaning rats. Hydrocortisone treatment did not affect growth factor expression but decreased proinflammatory cytokines. Likewise, dexamethasone decreased TNF-alpha and IL-1beta gene expression but did not affect TGF-betas in either epithelial or myofibroblast cells. CONCLUSIONS: During postnatal maturation, the expression of growth factors and proinflammatory cytokines decreased in the distal colon, whereas in the proximal colon, a differential maturation occurs with no changes in proinflammatory cytokines, an increase in TGF-alpha and a decrease in TGF-beta. Glucocorticoids may control the developmental profile of proinflammatory cytokines.

Intestinal apoptotic changes linked to metabolic status in fasted and refed rats.

Intestinal apoptosis and expression of apoptosis inducers--the cytokines TNFalpha, TGFbeta1--and the intestinal transcription factor Cdx2, were studied according to two different metabolic and hormonal phases which characterize long-term fasting: the long period of protein sparing during which energy expenditure is derived from lipid oxidation (phase II), and the later phase characterized by a rise in body protein utilization and plasma corticosterone (phase III). Apoptosis was further studied in 2, 6, and 24 h refed rats. Morphological apoptotic events were observed by environmental and conventional scanning electron microscopy and a TUNEL test was used to characterize the final stages of apoptotic death. The gene and protein expressions of TNFalpha, TGFbeta1, and Cdx2 were measured. Apoptotic events and TNFalpha, TGFbeta1, and Cdx2 gene and protein expressions did not vary significantly during phase II as compared to the normally fed animals. However, a phase III fasting induced a delay in intestinal epithelial apoptosis, along with a 92, 58, and 25% decrease in TNFalpha, TGFbeta1, and Cdx2 mRNAs, respectively. The amounts of TNFalpha, TGFbeta1, and Cdx2 proteins decreased by 70, 36, and 25%, respectively. Apoptosis was restored rapidly after a 2 h refeeding following the phase III, accompanied by a significant increase in TNFalpha, TGFbeta1, and Cdx2 mRNA and the protein levels, compared to the phase III fasting values. The concomitant decreases in cytokines and Cdx2 and in apoptotic cells during phase III suggest the preservation of enterocytes during this critical fasting period in order to optimize nutrient absorption as soon as food is available and thus, to rapidly restore body mass.

Postprandial morphological response of the intestinal epithelium of the Burmese python (Python molurus).

The postprandial morphological changes of the intestinal epithelium of Burmese pythons were examined using fasting pythons and at eight time points after feeding. In fasting pythons, tightly packed enterocytes possess very short microvilli and are arranged in a pseudostratified fashion. Enterocyte width increases by 23% within 24 h postfeeding, inducing significant increases in villus length and intestinal mass. By 6 days postfeeding, enterocyte volume had peaked, following as much as an 80% increase. Contributing to enterocyte hypertrophy is the cellular accumulation of lipid droplets at the tips and edges of the villi of the proximal and middle small intestine, but which were absent in the distal small intestine. At 3 days postfeeding, conventional and environmental scanning electron microscopy revealed cracks and lipid extrusion along the narrow edges of the villi and at the villus tips. Transmission electron microscopy demonstrated the rapid postprandial lengthening of enterocyte microvilli, increasing 4.8-fold in length within 24 h, and the maintaining of that length through digestion. Beginning at 24 h postfeeding, spherical particles were found embedded apically within enterocytes of the proximal and middle small intestine. These particles possessed an annular-like construction and were stained with the calcium-stain Alizarine red S suggesting that they were bone in origin. Following the completion of digestion, many of the postprandial responses were reversed, as observed by the atrophy of enterocytes, the shortening of villi, and the retraction of the microvilli. Further exploration of the python intestine will reveal the underlying mechanisms of these trophic responses and the origin and fate of the engulfed particles.

Intestinal gluconeogenesis and glucose transport according to body fuel availability in rats.

Intestinal hexose absorption and gluconeogenesis have been studied in relation to refeeding after two different fasting phases: a long period of protein sparing during which energy expenditure is derived from lipid oxidation (phase II), and a later phase characterized by a rise in plasma corticosterone triggering protein catabolism (phase III). Such a switch in body fuel uses, leading to changes in body reserves and gluconeogenic precursors, could modulate intestinal gluconeogenesis and glucose transport. The gene and protein levels, and the cellular localization of the sodium-glucose cotransporter SGLT1, and of GLUT5 and GLUT2, as well as that of the key gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (Glc6Pase) were measured. PEPCK and Glc6Pase activities were also determined. In phase III fasted rats, SGLT1 was up-regulated and intestinal glucose uptake rates were higher than in phase II fasted and fed rats. PEPCK and Glc6Pase mRNA, protein levels and activities also increased in phase III. GLUT5 and GLUT2 were down-regulated throughout the fast, but increased after refeeding, with GLUT2 recruited to the apical membrane. The increase in SGLT1 expression during phase III may allow glucose absorption at low concentrations as soon as food is available. Furthermore, an increased epithelial permeability due to fasting may induce a paracellular movement of glucose. In the absence of intestinal GLUT2 during fasting, Glc6Pase could be involved in glucose release to the bloodstream via membrane trafficking. Finally, refeeding triggered GLUT2 and GLUT5 synthesis and apical recruitment of GLUT2, to absorb larger amounts of hexoses.

Immunolocalization of Na+,K+-ATPase in the branchial cavity during the early development of the crayfish Astacus leptodactylus (Crustacea, Decapoda).

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium-potassium adenosine triphosphatase (Na(+),K(+)-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430-450 microm at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgGalpha5 raised against the avian alpha-subunit of the Na(+),K(+)-ATPase. Immunoreactivity staining, indicating the presence of Na(+), K(+)-ATPase appeared in the gills of late embryos (EI>/=400 microm), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410-420 microm and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na(+),K(+)-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na(+)active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.

Observations of the intestinal mucosa using environmental scanning electron microscopy (ESEM); comparison with conventional scanning electron microscopy (CSEM).

In order to evaluate the potential use of environmental scanning electron microscopy (ESEM) in biology, structural changes of the jejunal villi of rats were studied after periods of fasting and refeeding, using a conventional scanning electron microscope (CSEM) and ESEM. While observation using the CSEM, involves chemical fixation, drying and coating, observation of fresh, unprepared materials can be directly realized with the ESEM. Environmental microscopy provides a relatively new technology for imaging hydrated materials without specimen preparation and conductive coating. Direct observation of biological samples in their native state is therefore possible with an ESEM. After fasting, the jejunal mucosa is dramatically reduced in size, splits and holes appearing at the tip of the villi. These changes were observed whatever the type of technique used. Artifacts due to the sample preparation for CSEM observation (drying, coating) can therefore be excluded. However, CSEM and ESEM must be used jointly. While, CSEM must be preferred for surface analysis involving high magnifications, ESEM observation, on the other hand, can prove valuable for determining the living aspect of the samples.