CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED ON AN ELEMENTAL DIET

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurrred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

Changes in growth kinetics of jejunal epithelium in mice maintained on an elemental diet.

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurred 16 hr later in diet-fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet-fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.     

A study of diurnal proliferative activity in tumour and small intestine of C3H mice bearing a transplanted mammary carcinoma

Diurnal changes in proliferative activity were investigated in tumour and small intestinal epithelium of mice bearing a transplanted mammary carcinoma. In addition to mitotic and labelling index studies, the metaphase-arrest technique with vincristine (VCR) was employed. In the tumour there was no clear evidence of a significant diurnal rhythm in proliferative activity but in the small intestinal epithelium such a rhythm was clearly demonstrated. A higher cell production rate (kB) measured by metaphase-arrest and higher labelling and mitotic indices were seen in the mid to late part of the dark period. The peak mitotic index was seen 3 to 6 h after the labelling peak in the small intestine. The basal third of the crypt which is believed to include the stem cell compartment of this tissue showed larger diurnal fluctuations in both labelling index and kB than the rest of the proliferative compartment.     

THE EFFECT OF CONTINUOUS IRRADIATION ON CELL PROLIFERATION AND MATURATION IN SMALL INTESTINAL EPITHELIUM

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with ³H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36–48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

Radioprotection by pretreatment with deuterated water: cytokinetic changes in the small intestine of the mouse

All mice partially deuterated by ingestion of 29% heavy water for 12 days survived whole body gamma irradiation (8.5 Gy) from a 60Co source, whereas 42% of nondeuterated control animals died from bone marrow failure. The incorporation of 3HTdR into enterocytic DNA, as measured by autoradiography and liquid scintillation spectrometry, was used to assess the proliferative activity of small intestinal epithelium. The sequence and the magnitude of changes in tritium activity were in good agreement. Deuteration alone resulted in a reduced proliferative activity of small intestinal crypt epithelium, particularly in the basal cell positions and the first positions of the proliferation compartment. The number of positions occupied by the proliferative compartment and the crypt length were, however, barely affected by deuteration. The radiation-induced depression of DNA synthesis in the proliferative compartment was of similar magnitude in both groups. Crypt epithelium in deuterated mice, however, displayed signs of an accelerated and/or enhanced regeneration. The cytokinetic changes in deuterated animals are consistent with a protective effect for clonogenic intestinal epithelium at the time of irradiation.     

The effect of continuous irradiation on cell proliferation and maturation in small intestinal epithelium.

Autoradiographic studies and scintillation counting of crypt material after pulse labelling with 3H-thymidine showed that during continuous irradiation with 290 rads/day a reduced proliferative activity is present in the crypts of rat small intestine after 1 day of irradiation and of normal activity during the remaining period (5 days) irradiation. After cessation of irradiation an increase in proliferative activity can be observed after 1 day of recovery. From the time (36-48 hr after starting of the irradiation) that the number of villus cells is reduced an expansion of the proliferation zone in the crypt was observed. Both effects last until 1 day of recovery after cessation of irradiation. The process of crypt cell maturation and of villus cell function has also been studied during and after continuous irradiation by micro-chemical enzyme analyses in isolated crypts and villi. It was found that the expansion of the proliferation zone in the crypt is accompanied by a decrease in activity of only those enzymes (i.e. non-specific esterases) which normally become active during crypt cell maturation. The activity of enzymes normally present mainly in the functional villus cells remained relatively unaffected by changes in crypt cell kinetics. A hypothesis of different regulation mechanisms of the proliferative activity in the intestinal crypt and a possible explanation of the different behaviour of various enzyme activities as a result of changes in crypt cell proliferation is discussed.     

EFFECT OF VILLUS LENGTH ON CELL PROLIFERATION AND MIGRATION IN SMALL INTESTINAL EPITHELIUM

For the interpretation of data supporting the hypothesis of a feedback regulation of proliferative activity in intestinal crypts by the functional villus cell compartment the life span and migration rate of epithelial cells on villi of experimentally reduced length should be known. Autoradiographic studies and scintillation counting of isolated villi at different time intervals after ³H-thymidine labelling were carried out 36, 48 and 60 hr intervals after X-irradiation. The results showed that the life span of epithelial cells in rat small intestine (36–48 hr) is independent of the villus length. In villi of reduced length the migration rate of the epithelial cells was found to be decreased compared with controls. Changes in the migration rate in turn seem to be dependent on the production of epithelial cells in the crypt. Comparative studies on the recovery of crypt and villus epithelium after various doses (300 and 700 R) of X-radiation support the hypothesis that increased proliferative activity in the crypt cell compartment is related to a reduction of the number of functional villus cells below a critical villus length. The importance of these findings in the interpretation of data on (micro) biochemical analyses of certain cell differentiation characteristics during increased proliferative activity is discussed.     

The influence of 400 r x-irradiation on the number and the localization of mature and immature goblet cells and Paneth cells in intestinal crypt and villus.

The influence of 400 R X-irradiation on the localization and the number of mature and immature goblet cells and Paneth cells in rat duodenal epithelium has been studied. At short times after irradiation, when the total proliferative activity in the crypts of Lieberkuhn is reduced, the proportion of mature and immature goblet cells of the total number of crypt cells was increased; also an absolute increase in the number of goblet cells in the crypts was found. The immature goblet cells were localized in the lower half of the crypt as in control animals, whereas the number of the mature cells increased over the whole crypt length. When the proliferative activity of the crypt cells increases again from 12 to 48 hr after irradiation the number of both types of goblet cells decreases. Between 48 and 72 hr, when the whole crypt is involved in proliferation, a second increase of both types of goblet cells was found. However, the localization of the immature goblet cells is no longer restricted to the lower half of the crypt but they also appear at the higher cell positions. On the villus no immature goblet cells were found and the changes in the numbers of mature goblet cells do reflect the changes induced by irradiation in the goblet cell population in the crypt. The absolute number and localization of Paneth cells did not change under the experimental conditions. The findings are discussed in relation to cell proliferation and differentiation processes in intestinal crypts.     

Interleukin-10-independent anti-inflammatory actions of glucagon-like peptide 2

Glucagon-like peptide 2 (GLP-2) is an important intestinal growth factor with anti-inflammatory activity. We hypothesized that GLP-2 decreases mucosal inflammation and the associated increased epithelial proliferation by downregulation of Th1 cytokines attributable to reprogramming of lamina propria immune regulatory cells via an interleukin-10 (IL-10)-independent pathway. The effects of GLP-2 treatment were studied using the IL-10-deficient (IL-10(-/-)) mouse model of colitis. Wild-type and IL-10(-/-) mice received saline or GLP-2 (50 microg/kg sc) treatment for 5 days. GLP-2 treatment resulted in significant amelioration of animal weight loss and reduced intestinal inflammation as assessed by histopathology and myeloperoxidase levels compared with saline-treated animals. In colitis animals, GLP-2 treatment also reduced crypt cell proliferation and crypt cell apoptosis. Proinflammatory (IL-1beta, TNF-alpha, IFN-gamma,) cytokine protein levels were significantly reduced after GLP-2 treatment, whereas IL-4 was significantly increased and IL-6 production was unchanged. Fluorescence-activated cell sorting analysis of lamina propria cells demonstrated a decrease in the CD4(+) T cell population following GLP-2 treatment in colitic mice and an increase in CD11b(+)/F4/80(+) macrophages but no change in CD25(+)FoxP3 T cells or CD11c(+) dendritic cells. In colitis animals, intracellular cytokine analysis demonstrated that GLP-2 decreased lamina propria macrophage TNF-alpha production but increased IGF-1 production, whereas transforming growth factor-beta was unchanged. GLP-2-mediated reduction of crypt cell proliferation was associated with an increase in intestinal epithelial cell suppressor of cytokine signaling (SOCS)-3 expression and reduced STAT-3 signaling. This study shows that the anti-inflammatory effects of GLP-2 are IL-10 independent and that GLP-2 alters the mucosal response of inflamed intestinal epithelial cells and macrophages. In addition, the suggested mechanism of the reduction in inflammation-induced proliferation is attributable to GLP-2 activation of the SOCS-3 pathway, which antagonizes the IL-6-mediated increase in STAT-3 signaling.     

THE INFLUENCE OF 400 R X-IRRADIATION ON THE NUMBER and THE OCALIZATION OF MATURE and IMMATURE GOBLET CELLS and PANETH CELLS IN INTESTINAL CRYPT and VILLUS

The influence of 400 R X-irradiation on the localization and the number of mature and immature goblet cells and Paneth cells in rat duodenal epithelium has been studied. At short times after irradiation, when the total proliferative activity in the crypts of Lieberkiihn is reduced, the proportion of mature and immature goblet cells of the total number of crypt cells was increased; also an absolute increase in the number of goblet cells in the crypts was found. The immature goblet cells were localized in the lower half of the crypt as in control animals, whereas the number of the mature cells increased over the whole crypt length. When the proliferative activity of the crypt cells increases again from 12 to 48 hr after irradiation the number of both types of goblet cells decreases. Between 48 and 72 hr, when the whole crypt is involved in proliferation, a second increase of both types of goblet cells was found. However, the localization of the immature goblet cells is no longer restricted to the lower half of the crypt but they also appear at the higher cell positions. On the villus no immature goblet cells were found and the changes in the numbers of mature goblet cells do reflect the changes induced by irradiation in the goblet cell population in the crypt. The absolute number and localization of Paneth cells did not change under the experimental conditions. The findings are discussed in relation to cell proliferation and differentiation processes in intestinal crypts.     

Leptin, cell proliferation and crypt fission in the gastrointestinal tract of intravenously fed rats

Many peptides, hormones and growth factors have been implicated in the control of cell renewal in the gastrointestinal epithelium. Leptin is present in the stomach and salivary glands and leptin receptors are seen throughout the gut. Leptin can stimulate mitogen-activated protein kinase activity in vitro and short-term infusion has been reported to have a proliferative action on the colon in vivo, suggesting a biological link between obesity, physical activity and colon cancer. Food intake is one of the most important determinants of intestinal mucosal cell renewal, thus any direct effects of leptin on the gut may be hidden. This problem has been avoided experimentally by maintaining animals on total parenteral nutrition (TPN). Male Wistar rats were anaesthetized and cannulae were inserted into the jugular vein to deliver the TPN diet to which had been added 0, 0.5, 2.5, or 10 mg/kg of recombinant murine leptin. Orally fed rats were also studied. After 6 days of treatment, all animals were injected with vincristine and killed 2 h later. Tissue weight was recorded and crypt cell proliferation (arrested metaphases) and crypt fission were scored in 'microdissected' crypts. Leptin infusion led to a small decrease in body weight and in the weight of the caecum. Intestinal cell proliferation was significantly reduced by TPN when compared to the orally fed rats, but the addition of leptin had no effect on the small intestine or colon. Crypt fission was also significantly lowered in the TPN group. Fission was slightly but significantly increased in the proximal and mid-colon of the leptin-treated rats, but was decreased in the distal colon. Although leptin did not significantly alter cell proliferation, it had significant effects on the process of crypt fission in the colon, which varied according to the exact locality.     

Effect of radiotherapy and chemoradiotherapy on circulating antioxidant system of human uterine cervical carcinoma

Rats bearing the Yoshida AH-130 ascites hepatoma showed important changes in lipid metabolism. The presence of this rapidly growing tumour induced a significant reduction in the intestinal absorption of an oral [^l4C]triolein load but without changes in whole body oxidation of the tracer to CO₃. Both white (WAT) and brown (BAT) adipose tissue lipoprotein lipase (LPL) activities were increased at day 4 of tumour growth, changes that seem to be related with those observed in [¹⁴C] lipid accumulation; however, heart LPL activity was increased at day 7 but there was no change at day 4. In addition, there was a marked hyperlipemia in the tumour-bearing animals, whereas the blood ketone body concentrations were lower in these animals in comparison with the corresponding pair-fed group. The in vivo lipogenic rate was increased in liver of the tumour-bearing animals (day 4); conversely, it was decreased in WAT and skeletal muscle (day 4) and IBAT (day 7) of the AH-130-bearing rats. It may be suggested that the increased liver lipogenic rate associated with tumour burden is the main factor contributing to the hyperlipidaemia present in the Yoshida AH-130 bearing rats.     

CONTROL OF EPITHELIAL CELL PROLIFERATION IN THE SMALL INTESTINAL CRYPT

A heat labile factor which has been shown to inhibit proliferative activity in crypt epithelium both in rat jejunum in vivo and in explants of rat jejunum maintained in organ culture has been prepared from the soluble fraction of homogenized epithelial cells isolated from rat small intestinal crypts. The factor appears to have tissue specificity, for it has no influence on epithelial cell proliferation in colonic crypts, oesophagus or skin. Extracts of rat intestinal villous cells prepared using identical techniques were without effect on proliferative activity of small intestinal crypt epithelium.
Isoprenalin, which was also found to suppress cell proliferation, did not potentiate the effect of the factor and its effects were evanescent.     

The effect of bile and of sodium taurocholate on the epithelial cell dynamics of the rat small intestine.

The absence of bile from the intestinal lumen of rats for a period of 48 hr led to: a decreased proliferative cell pool, reduced cell shedding, and a 50% decrease of the labeling index in the ileum. The constant duodenal perfusion of Na taurocholate for periods of 48, 60, 72, and 96 hr in animals with a biliary diversion was associated with: deepening of crypts and decreased crypt/villus ratios as well as with acceleration of the epithelial cell migration rate on the crypt-villus units. The data suggest that bile and bile acids constitute important regulatory factors influencing enterocyte proliferation, migration and loss.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

Hindlimb suspension suppresses muscle growth and satellite cell proliferation

The effects of long-term hindlimb unweighting by tail suspension on postnatal growth of 20-day rat extensor digitorum longus (EDL) and soleus muscles were studied. Morphological assay indicated that radial growth of soleus myofibers was completely inhibited between 3 and 10 days of suspension and reduced thereafter, leading to a severe attenuation (-76% from control) over the total experimental period. Longitudinal growth rate, however, was accelerated 40% over weight-bearing controls. In addition, myofibers were arranged parallel to the long axis of the muscle, an orientation associated with chronologically younger muscles, suggesting morphological maturation of the soleus muscle had been delayed by suspension. In contrast, radial and longitudinal growth of EDL myofibers were minimally affected under similar conditions and remained within approximately 5% of control at all times. Suspension also influenced the normal changes that occur in satellite cell and myonuclear populations during postnatal growth. Both the number and proliferative activity of satellite cells were severely reduced in individual myofibers after only 3 days in both soleus and EDL muscles. The reduced number of satellite cells within 3 days of initiating hindlimb suspension appeared to be the result of their incorporation into myofibers while the long-lasting reduction appeared to be the added effects of decreased proliferative activity. In the soleus, this reduction in number and proliferation of satellite cells persisted throughout the experimental period and resulted in an overall 43% fewer myonuclei and 45% fewer satellite cells than control at 50 days of age. In contrast, both the total number and mitotic activity of satellite cells in the EDL rapidly returned to weight-bearing control levels by day 10 of suspension, resulting in no overall reduction in myonuclear accretion.     

Cyclic nucleotide metabolism in rat colonic epithelial cells with different proliferative activities

Cyclic nucleotide metabolism was examined in rat distal colonic epithelial cells with different proliferative activities. Lower crypt cells had DNA synthetic rates 7-10-fold higher than surface cells. Without a phosphodiesterase inhibitor proliferative cells had reduced basal cyclic AMP-, cyclic GMP-, and cyclic AMP-dependent protein kinase activity ratios, as well as blunted cyclic AMP responses to prostaglandin E2 and vasoactive intestinal peptide compared to superficial cells. In the presence of 3-isobutyl-1-methylxanthine, basal cyclic AMP and responses to prostaglandin E2 and vasoactive intestinal peptide of proliferative cells exceeded values in superficial cells. This correlated with higher membrane adenylate cyclase activity in the proliferative cells. By contrast, particulate and soluble guanylate cyclase activities of superficial cells were higher than in proliferative cells. The apparent high Km soluble and particulate cyclic AMP and cyclic GMP phosphodiesterase activities of proliferative cells were 4-7-fold higher than those in superficial cells. Moreover, the apparent low Km soluble activity was absent in superficial cells. Thus, an altered rate of nucleotide degradation may mediate reduced cyclic AMP and cyclic GMP in proliferative versus superficial cells. Dibutyryl cyclic AMP, prostaglandin E2 or vasoactive intestinal peptide inhibited [3H]thymidine incorporation into DNA of colonic segments. Thus, reduced cyclic AMP in lower crypt cells may be a determinant of their greater proliferative activity.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (T_C) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G₁, S and G₂ phases of the crypt cell cycle are altered by sympathectomy, G₁ accounting for most of the increase in T_C. In addition, the percentage of [³H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.