CELL POPULATION CHANGES IN THE INTESTINAL MUCOSA OF PROTEIN-DEPLETED OR STARVED RATS : I. Changes in Mitotic Cycle Time

The effect of a protein-free diet and starvation on the duration of the rat ileal crypt cell cycle time was studied by Quastler's technique of labeled mitoses. Rats were fed a protein-free diet for 3, 7, or 11 wk or were starved for 7 or 10 days. Progressive protein depletion resulted in a progressive lengthening of the cycle time (GT), due primarily to a lengthening of the synthetic phase (S) of the cycle. The presynthetic gap (G₁) was the same as the control value after 3 wk and lower, but not significantly so, due to the large variability, after 11 wk. The duration of the postsynthetic gap (G₂) plus mitotic phase (M) was not affected by the diet. As the dietary stress became more severe, the cell cycle also became more variable. Although the GT of rats starved for as long as 10 days was only slightly different from the control, the relative duration of the components of the cycle changed significantly. S and G₂ were longer in the starved animals while G₁ was of shorter duration.     

PANETH AND GOBLET CELL RENEWAL IN MOUSE DUODENAL CRYPTS

Proliferation of Paneth and goblet cells of mouse duodenal crypts was studied by high resolution light microscope radioautography. In one group of mice, blood levels of thymidine-³H were sustained for up to 12 hr by repeated injections of isotope to facilitate identification of proliferating cells. In these animals, many goblet cell nuclei incorporated thymidine-³H whereas only 1 of 6261 tabulated Paneth cells was labeled. Cells intermediate in structure between undifferentiated and goblet cells and between undifferentiated and Paneth cells were identified and their light and electron microscopic features are described. A significant number of these "intermediate" cells incorporated thymidine-³H into their nuclei. Another group of mice received a single injection of thymidine-³H. These animals were killed 4 hr to 29 days after isotope administration. Goblet cells and intermediate cells with labeled nuclei were identified 4 hr after thymidine-³H but could not be seen after 15 days. In contrast, Paneth cells with labeled nuclei were not observed until 24 hr after thymidine-³H but were still present at 29 days, long after labeled undifferentiated, goblet, and intermediate cells had disappeared. We conclude that differentiated Paneth cells in mouse duodenum do not normally proliferate, but, instead, arise by differentiation from undifferentiated crypt cells or from intermediate cells. Moreover, once formed, Paneth cells persist in crypts for a prolonged period. In contrast, intermediate cells and crypt goblet cells proliferate actively and are less stable cell populations than differentiated Paneth cells. The precise function of the intermediate cells is not known, but they may represent transition forms between undifferentiated cells and the more matrure secretory cells. Damage of crypt epithelial cells, thought to be due to radiation effects, was evident in both groups of mice.     

Effects of a protein-free diet on worm recovery, growth, and distribution of Echinostoma caproni in ICR mice.

The effects of a protein-free diet on the host-parasite relationship of Echinostoma caproni in ICR mice were studied. The experimental diet was a customized protein-free diet (PFD) in pellet form containing 0% protein. The control diet consisted of a standard laboratory diet containing 23% casein as a source of protein. A total of 24 mice were each infected with 15 metacercarial cysts of E. caproni. Twelve mice were placed on the experimental diet (experimentals) and the remaining mice (controls) were placed on the control diet. Experimental and control mice were necropsied at 2, 3, and 4 weeks postinfection (p.i.). The weight of mice on the PFD was markedly lower than that of mice on the control diet. The length and circumference of the small intestine of infected mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. (Student's t-test; P < 0.05). Worm recoveries from mice on the PFD were significantly lower than those of the controls at 3 weeks p.i. There was a significant decline in worm body area in worms from the mice on the PFD compared with those on the control diet at 2, 3, and 4 weeks p.i. Worm dry weights from mice on the PFD were significantly lower than those on the control diet at 2 weeks p.i. Worms from hosts on the PFD were located more posteriad in the gut than those recovered from mice on the control diet. The findings suggest that the PFD contributes to growth retardation of E. caproni in ICR mice.     

On protein deficiency in mice with special reference to liver nucleolar size

Summary Mice were starved for 2 days and then fed on either a protein-free diet or a 25 per cent casein diet for 7 days and then sacrificed. The former group, in contrast to the latter, decreased in weight during these 7 days and showed significantly lower weight absolute and relative (g/100 g initial body weight) of kidneys, liver and spleen. In the animals deprived of protein, the ratio between total liver nucleolar volume per nucleus and the nuclear volume was larger, than in the casein fed animals. The results suggest that the liver nucleolar apparatus reacts to a protein-free diet in principally the same way in mice as in rats.The investigation was supported by a grant from the Swedish Medical Research Foundation.     

pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-³H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-³H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-³H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-³H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-³H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-³H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.     

Ultrasonography of the developing reproductive tract in ram lambs: effects of a GnRH agonist

In spring-born ram lambs, the testes (from 2 wk), prostate and vesicular glands (from 4 wk) were examined by ultrasonography every 2 wk up to 26 wk of age. Image analysis was done (numerical pixel values). Ram lambs were treated with a long acting formulation of a GnRH superagonist (Leuprolide acetate; 1.5 mg/kg) at 3 and 7 wk of age. In blood samples taken every 15 min for 8 h, mean serum LH, LH pulse amplitude, and basal and mean serum FSH concentrations were lower at 5 wk of age, and LH pulse frequency was lower at 15 wk of age in animals given Leuprolide acetate compared with those of the controls. There were no differences (P>0.05) in testis, prostate or vesicular gland development between treated and control animals. Testicular diameter of the left and right testes in transverse and longitudinal planes increased slowly to 8 wk of age, more rapidly to 18 wk of age, then more slowly to 26 wk of age (P<0.05). Numerical pixel values of testicular images decreased from 2 to 8 wk of age, increased to 22 wk of age and then plateaued. Width of the prostate increased from 4 to 26 wk of age, but length and width of the vesicular glands increased slowly to 8 wk of age, more rapidly to 18 wk of age and then plateaued (P<0.05). Numerical pixel values for the prostate declined from 4 to 8 wk and for the vesicular glands, declined from 4 to 10 wk of age; numerical pixel values increased to 12 wk and then decreased to a nadir at 18 wk, followed by a steady increase to 26 wk of age (P<0.05). We concluded that developmental patterns of numerical pixel values of the testes, prostate and vesicular glands in ram lambs reflect stages of development, but treatment with a GnRH superagonist at 3 and 7 weeks of age did not affect growth of testes, vesicular or prostate glands.     

Carbohydrate metabolism in starved fifth instar larvae of Manduca sexta

The influence of starvation on carbohydrate metabolism in fifth instar larvae of Manduca sexta was studied. The percentage of active fat body glycogen phosphorylase increased from 10% to approximately 50% within 3 h of starvation; afterward the enzyme was slowly inactivated. The increase of phosphorylase activity might have been caused by a peptide(s) from the CC. The amount of fat body glycogen in starved animals decreased over 24 h by approximately 20 mg. The released glucose molecules seem to be converted mainly to trehalose because the hemolymph trehalose concentration in starved animals was always slightly higher than in the fed controls, and the glucose concentration decreased even when phosphorylase was activated. The chitosan content in starved larvae increased during the first 9 h of treatment to the same extent as in fed controls. It is suggested that fat body glycogen phosphorylase was activated during starvation to provide substrates for chitin synthesis and energy metabolism.     

The effect of diet and 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on microsomal hydroxylating enzymes and on sensitivity of rats to carbon tetrachloride poisoning

1. Protein-depleted rats are resistant to the lethal effects of carbon tetrachloride. The LD₅₀ is 6·4ml./kg. in stock rats and 14·7ml./kg. in rats fed on protein-free diets. 2. Protein-depleted rats are resistant to carbon tetrachloride in its effect on the liver as judged by histology, accumulation of liver water, and plasma enzyme and bilirubin measurement. 3. The protection is present after feeding rats on a no-protein diet for 4 days. It is present after feeding rats on a 3%-casein diet, and partly found after feeding rats on a 6%-casein diet. 4. The activities of the microsomal enzymes that demethylate Pyramidon and hydroxylate benzopyrene in the liver fall by over 80% in rats fed on the no-protein diet for 4 days or more, or in rats fed on a 3%-casein diet. A 50% fall is found in rats fed on a 6%-casein diet. 5. A single dose of DDT or three doses of phenobarbitone cause increased microsomal enzyme activity in protein-depleted rats. 6. The animals are then sensitive to the lethal and liver-damaging effects of carbon tetrachloride. 7. DDT dosage also leads to increased sensitivity to carbon tetrachloride in rats fed on stock diets. 8. These findings support the hypothesis that carbon tetrachloride is metabolized by microsomal enzymes to form the true toxic compound.     

LYMPHOCYTE POPULATIONS IN MOUSE BONE MARROW: QUANTITATIVE KINETIC STUDIES IN YOUNG, PUBERTAL AND ADULT C3H MICE

Continuous ³H-thymidine infusion was used to characterize two kinetic subpopula-tions of small lymphocytes in mouse bone marrow during normal growth and development. Young (4 wk), pubertal (8 wk) and mature (16 wk) C3H mice were infused subcutaneously with ³H-thymidine for periods up to 10 days. Femoral marrow was then examined in radioautographic smears. During the first 3 days the proportion of marrow small lymphocytes labelled by ³H-thymidine showed a rapid exponential increase to 93%, 81% and 72% in 4 wk, 8 wk and 16 wk mice respectively. The rate of appearance of labelled small lymphocytes then declined markedly but remained higher in younger than in older animals. The labelling curves were found to represent the summation of two exponential curves from which the proportions and renewal rate of corresponding cell populations were calculated. Most marrow small lymphocytes comprised a rapidly renewing population but in mice of increasing age the relative incidence of these cells fell (93-3% at 4 wk; 88-0% at 8 wk; 78-5% at 16 wk) and their half-renewal time (T_½) lengthened (14 hr at 4 wk; 18 hr at 8 wk; 24 hr at 16 wk). The remaining small lymphocytes were slowly renewing with mean T_½ of 4, 7 and 14 days in 4, 8 and 16 wk mice, respectively. Some heavily labelled small lymphocytes persisted in the marrow up to 10 wk after fourteen daily ³H-thymidine injections in 10–12 wk mice. The numbers of rapidly renewing cells decreased from 604 times 10³ to 228 times 10³ per mm³ of marrow from 4 wk to 16 wk, respectively, while slowly renewing cells increased from 44 times 10³ to 61 times 10³ per mm³. The total number of nucleated marrow cells per femur increased from 4 wk to 16 wk but the rapidly renewing small lymphocytes per femur fell in numbers by 36% and in renewal rate by 63%. The results demonstrate a selective change in bone marrow small lymphocytes with age; rapidly renewing cells decline in number and renewal rate while the number of slowly renewing cells increases. The concept of bone marrow as a primary lymphoid organ is discussed.     

Effects of starvation on lung mechanics and biochemistry in young and old rats

Two groups of rats (young and old) were food-deprived for 3 wk and were compared with age-matched fed groups. Final body weight and dry and wet weights of lungs were significantly reduced in both young and old starved rats. As determined by saline volume-pressure (VP) curves, lungs of young starved rats accepted significantly less volume at all pressure levels compared with lungs of young fed rats. When expressed as a percent of maximum lung volume, the VP curve in young starved rats was significantly shifted upward at low lung volumes. In the old rats, the VP curves were similar in fed and starved rats. Total lung content of protein, DNA, crude connective tissue, hydroxyproline, and elastin were significantly reduced in young starved compared with young fed rats, whereas in old starved rats only protein and DNA contents were lower than those in old fed animals. It appears that in rapidly growing young rats starvation leads to growth retardation, loss of connective tissue components, and possibly reduction in tissue elastic forces at low lung volumes, whereas starvation has no significant effects on lung mechanics and connective tissue in old rats.     

Lacto-N-neotetraosylceramide ("paragloboside") as a possible tumor-associated surface antigen of hamster NILPY tumor.

Isolated rat hepatocytes from fed and starved rats synthesized net glucose from various precursors at similar rates. [3-¹⁴C]-lactate incorporation into glucose was also similar in hepatocytes from fed and starved rats, as was ketone body formation from oleate and octanoate. Rates of gluconeogenesis in hepatocytes from fed rats compare to rates seen in perfused livers from starved rats rather than perfused livers from fed rats. Thus metabolic rates and possibly controls may be different between perfused livers and isolated hepatocytes when using fed rats.     

TUMOR ANGIOGENESIS : Rapid Induction of Endothelial Mitoses Demonstrated by Autoradiography

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-³H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1µ thick Epon sections showed thymidine-³H labeling in endothelial cells of small vessels 1–3 mm from the site of implantation, as early as 6–8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-³H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.     

Circadian phase relationships of thymidine-3H uptake, labeled nuclei, grain counts, and cell division rate in rat corneal epithelium

Significant differences in the uptake of thymidine-³H, percentage of labeled cells, numbers of grains per labeled nucleus, and mitotic rate were noted in rat corneal epithelium along a 24-hr time scale. These were demonstrated by injecting subgroups of five animals every hour during a 24-hr period with thymidine-³H, sacrificing them 2 hr later, and analyzing the corneal epithelium by scintillation counter and radioautographic techniques. The increase in uptake during specific periods of the 24-hr time scale is attributed to an acceleration in the rate of DNA synthesis by individual cells and to an increase in the percentage of cells in the population actively synthesizing DNA.     

Effect of starvation and a protein diet on the amino acid metabolism enzyme activities of the organs of domestic fowl hatchlings

The activities of alanine and aspartate transaminases, adenylate deaminase, glutamine synthetase and glutamate and xanthine dehydrogenases have been measured in liver, yolk sac membrane, intestine and breast and leg muscle of domestic fowl hatchlings receiving for 3 or 5 days either a standard diet or hard boiled eggwhite as well as in 3 or 5 days starved animals. The patterns of activation of amino acid metabolism enzymes were fully comparable in protein-fed and starved groups with respect to fed controls; the differences with respect to the latter became more marked in 5- than in 3-days old chicks. In 5-days old chicks intestine alanine transaminase activity increased in parallel to that of liver in protein-fed animals but not in those starved, in agreement with an enhanced alanine transfer between both organs under this situation. Both, starvation and protein-feeding, induced a general decrease in the amino acid metabolizing ability of muscle. Glutamine (but not alanine) synthetizing capabilities were enhanced.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

A Cytochemical Study on the Pancreas of the Guinea Pig : II. Functional Variations in the Enzymatic Activity of Microsomes

Microsomes were isolated from the pancreas of starved and fed guinea pigs. In the first case, the gland was removed from animals starved for 48 hours; in the second, the pancreas was excised 1 hour after the beginning of a meal that ended a fast of 48 hours. These are referred to below as fed animals. In both cases the tissue was homogenized in 0.88 M sucrose and the microsomes obtained by centrifuging the mitochondrial supernatant at 105,000 g for 60 minutes. In starved animals the content of the endoplasmic reticulum of the exocrine cells and the content of the microsomes were found to be of low or moderate density. In fed guinea pigs the cavities of the reticulum frequently contained dense intracisternal granules and the microsomes were distinguished by a content of high density sometimes in the form of recognizable intracisternal granules. In starved animals, the microsomes were found to account for 5 to 20 per cent of the trypsin-activatable proteolytic activity and ribonuclease activity of the whole cell, whereas in fed animals they contained uniformly almost 30 per cent of these activities. In fed animals the dense, cohesive content of the microsomes (intracisternal granules) could be isolated by breaking up the microsomes with dilute (0.1 per cent) deoxycholate solutions and separating microsomal subfractions by differential centrifugation. The specific enzymatic activities of a heavy microsomal subfraction rich in intracisternal granules were almost equal to those of isolated purified zymogen granules. The ribonucleoprotein particles attached to the microsomal membranes could be isolated by the same technique and found also to exhibit some of the same enzymatic activities. Corresponding subfractions isolated from the microsomes of starved animals were considerably less active. The relevance of these findings for the synthesis and intracellular transport of protein in the exocrine cell of the pancreas is discussed.     

Metabolism of amino acids in protein-calorie-deficient rats

The overall oxidative degradation of leucine and phenylalanine, measured in vivo in rats fed on a 2%-casein diet for 8 weeks, is markedly decreased as compared with controls, whereas that of glutamate and alanine is apparently unaffected. The decrease in leucine degradation is due, at least in part, to a block before the formation of 3-methylbutyryl-CoA (isovaleryl-CoA) in the catabolic pathway. This phenomenon is accompanied by increased incorporation of [¹⁴C]leucine into liver proteins, decreased urinary excretion of leucine and increased urinary excretion of 4-methyl-2-oxopentanoate (α-oxoisocaproate) by protein-depleted animals. The results suggest the existence of adaptive mechanisms that function to conserve an indispensable carbon skeleton.     

A cytochemical study on the pancreas of the guinea pig. II. Functional variations in the enzymatic activity of microsomes

Microsomes were isolated from the pancreas of starved and fed guinea pigs. In the first case, the gland was removed from animals starved for 48 hours; in the second, the pancreas was excised 1 hour after the beginning of a meal that ended a fast of 48 hours. These are referred to below as fed animals. In both cases the tissue was homogenized in 0.88 M sucrose and the microsomes obtained by centrifuging the mitochondrial supernatant at 105,000 g for 60 minutes. In starved animals the content of the endoplasmic reticulum of the exocrine cells and the content of the microsomes were found to be of low or moderate density. In fed guinea pigs the cavities of the reticulum frequently contained dense intracisternal granules and the microsomes were distinguished by a content of high density sometimes in the form of recognizable intracisternal granules. In starved animals, the microsomes were found to account for 5 to 20 per cent of the trypsin-activatable proteolytic activity and ribonuclease activity of the whole cell, whereas in fed animals they contained uniformly almost 30 per cent of these activities. In fed animals the dense, cohesive content of the microsomes (intracisternal granules) could be isolated by breaking up the microsomes with dilute (0.1 per cent) deoxycholate solutions and separating microsomal subfractions by differential centrifugation. The specific enzymatic activities of a heavy microsomal subfraction rich in intracisternal granules were almost equal to those of isolated purified zymogen granules. The ribonucleoprotein particles attached to the microsomal membranes could be isolated by the same technique and found also to exhibit some of the same enzymatic activities. Corresponding subfractions isolated from the microsomes of starved animals were considerably less active. The relevance of these findings for the synthesis and intracellular transport of protein in the exocrine cell of the pancreas is discussed.     

Carbohydrate metabolism of hepatocytes from starved Japanese quail

Hepatocytes were isolated from livers of mature male and female starved Japanese quail (Coturnix coturnix japonica). The hepatocytes take up lactate and dihydroxyacetone extensively, and have a very high rate of glucose synthesis from these substrates. Fructose uptake and incorporation into glucose is much less. Pyruvate and alanine are taken up extensively, but form little glucose. There is negligible lipogenesis in cells of starved quail. Alanine increases up to 10-fold incorporation of ³HOH and ¹⁴C from several substrates into fatty acids, but it remains insignificant as compared to lipogenesis by cells of fed quail. There is little utilization of glucose, even in the presence of alanine, in marked contrast to hepatocytes from fed quail. However, glucose is phosphorylated at high rates, but most of the glucose 6-phosphate is recycled to glucose. There is a marked difference in the metabolism of polyols between the sexes. Glycerol, xylitol, and sorbitol are converted nearly quantitatively into glucose by hepatocytes of starved female quail. In cells of starved males, the uptake of polyols is higher, but conversion to glucose less efficient. In cells of starved male quail, alanine markedly stimulates the uptake of glycerol and xylitol and their conversion to glucose, but has no effect on sorbitol metabolism. In cells of female quail, alanine is without a significant effect on polyol metabolism.     

Effect of a protein-free diet on muscle protein turnover and nitrogen conservation in euthyroid and hyperthyroid rats.

Although protein turnover in skeletal muscle is increased in hyperthyroidism and decreased in hypothyroidism, a deficient protein intake tends to increase serum T3 (tri-iodothyronine) while decreasing muscle protein turnover. To determine whether this diet-induced decrease in protein turnover can occur independent of thyroid status, we have examined muscle protein turnover and nitrogen conservation in hyperthyroid rats fed on a protein-free diet. After inducing hyperthyroidism by giving 20 micrograms of T3/100g body wt. daily for 7 days, groups of euthyroid and hyperthyroid animals were divided into subgroups fed on basal and protein-free diets. Muscle protein turnover was measured by N tau-methylhistidine excretion and [14C]tyrosine infusion. Urinary nitrogen output of euthyroid and hyperthyroid animals fed on the protein-free diet was also measured. Although hyperthyroidism increased the baseline rates of muscle protein synthesis and degradation, it did not prevent a decrease in these values in response to protein depletion. Furthermore, hyperthyroid rats showed greatly decreased nitrogen excretion in response to the protein-free diet, although not to values for euthyroid rats. These findings suggest that protein depletion made the experimental animals less responsive to the protein-catabolic effects of T3.