Jean-Hervé Lignot

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.

Effects of fasting and refeeding on Jejunal morphology and cellular activity in rats in relation to depletion of body stores

Background: Intestinal mucosa atrophy following a period of starvation characterized by the mobilization of fat stores for energy expenditure (phase II) worsen after a long fast marked by an increase in protein catabolism (phase III). However, the morphology of the jejunum is completely restored after 3 days of refeeding. The aim of this study was to determine the mechanisms involved in the rapid jejunal restoration following the critical phase III. Methods: Jejunal structure was observed through conventional and environmental scanning electron microscopy, whilst cellular dynamics were studied using classical optic microscopy tools and immunohistochemistry. Results: Mucosal structural atrophy during fasting proved to worsen over the two phases. During phase II, apoptosis is still present at the tip of the villi, the number of mitosis in crypts showed a 30% decrease and a transient drop in cell migration is observed. During phase III, however, an 85% rise in mitosis was noticed along with an increase in cell migration and the disappearance of apoptotic cells at the villus tips. This increased cell renewal continues after food ingestion. Conclusions: Starved rats appeared to be in a phase of energy sparing in phase II, with depressed cellular events in the intestinal mucosa. In phase III, however, the preservation of functional cells and the early increase in crypt cell proliferation should prepare the mucosa to refeeding and could explain why jejunal repairs are complete after 3 days of refeeding following either phase II or phase III.

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.

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 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.

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-&bgr;1, TGF-&bgr;2 and TGF-&agr; and the proinflammatory cytokines tumor necrosis factor-&agr; (TNF-&agr;) and interleukin-1&bgr; (IL-1&bgr;) 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-&bgr;s 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-&bgr;1 and TGF-&bgr;2 messenger RNAs and proteins decreased in the entire colon from weaning to adult stages, whereas the amount of TGF-&agr; 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-&agr; and IL-1&bgr; gene expression but did not affect TGF-&bgr;s 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-&agr; and a decrease in TGF-&bgr;. Glucocorticoids may control the developmental profile of proinflammatory cytokines.

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.