Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.     

Dietary-induced increase in lactase activity and in immunoreactive lactase in adult rat jejunum.

Adult rats that had been fed on a low-starch high-fat diet for 7 days were force-fed with either the same diet or isoenergetic diets containing 40% of energy as either sucrose or lactose. Within 12h, the increase in jejunal lactase activity in sucrose- and lactose-fed rats was accompanied by a corresponding increase in immunoreactive lactase protein.     

Sucrase-alpha-dextrinase in the spontaneously diabetic BioBreed Wistar rat: altered intracellular carbohydrate processing

Sucrase-alpha-dextrinase (S-D), a glycoprotein hydrolase in the border surface of the enterocyte, is altered in congenitally diabetic BioBreed Wistar (BB(d)) rats. Its intracellular assembly was examined in the current studies. Following pulse-chase experiments, S--D was specifically immuno-isolated from ER-Golgi and brush border membranes, and examined by SDS-PAGE and autoradiography. While synthesis and co-translational glycosylation of an immature species, P(i), in the ER proceeded normally, post-translational maturation of the N-linked carbohydrate chains was altered in the BB(d) rat. The mature species, P(m), was 10 kDa larger in BB(d) than in normal rats, and approximately 25% of its N-linked chains remained immature. The difference in mass was attributed to an appreciably larger mass of the O-linked chains of P(m) in BB(d) rats. In vivo kinetics of intracellular assembly displayed a delay in the appearance of P(m) in Golgi (Wistar, 15 min; BB(d), 30--60 min). These experiments, revealing structural alterations in congenital diabetes suggest an important role for intracellular glycosylation in the orderly assembly and processing of brush border S-D in the enterocyte.     

Intestinal aminooligopeptidase in diabetic BioBreed rat: altered posttranslational processing and trafficking

The structure of aminooligopeptidase (AOP), an intestinal brush-border digestive hydrolase, is abnormal in human diabetes and in the congenitally diabetic BioBreed Wistar (BB(d)) rat. Its assembly in the BB(d) rat was examined. After normal initial synthesis and assembly of immature AOP precursor (AOP(i)) with high-mannose N-linked chains in the endoplasmic reticulum (ER), processing of N-linked glycans in Golgi yielded a smaller than normal mature AOP precursor (AOP(m)) with persistence of some high-mannose N-linked chains. Deglycosylation analyses suggested that the mass difference could be attributed to a lower mass of N-linked with unaltered O-linked glycans in AOP(m) of the diabetic rat. Intrajejunal pulse-chase experiments revealed that the conversion of AOP(i) to AOP(m) occurred at 30 min of chase in normal rats but at 60-90 min in diabetic rats, reflecting delay in ER-to-Golgi transport or a slower processing of high-mannose chains. Once maximal transport to Golgi was achieved, the residence time in Golgi was shortened in diabetes. This altered processing of the precursor accounted for the altered structure of AOP in diabetes.     

Intestinal uptake and transport of proteins in the adult rat.

The transport of immunoglobulin and ferritin across the intestinal mucosa of adult rats provides an excellent model for transcellular protein transport study. Intestinal uptake and transcellular transport have been extensively studied in the neonatal rat, but not to such an extent in the adult rat. The transport of 125I labelled bovine immunoglobulin G and ferritin was studied in 100 days old rats using intestinally administered proteins. Antigen was estimated in the tissues by reacting extracts against specific immune antiserum prepared in rats, and visualization studies were carried out by fluorescence microscopy and direct deposition autoradiography at electron microscopic level. From these studies, it can be seen that these proteins are taken up by the intestinal cells and transported, antigenically intact, across the barriers to the body organs.     

Stimulation of lactase synthesis induced by starvation in the jejunum of adult rat

The effects of actinomycin D and of cycloheximide administration have been investigated on the enzyme activities of the jejunal brush border membrane in adult rats after a 48-hour period of starvation. The modifications in the protein and enzyme patterns of the brush border membrane and the incorporation of radiolabelled amino acid in the protein band corresponding to lactase have been studied in the nourished and in the starved animal. The results show that actinomycin D administration did not modify the stimulation of lactase activity caused by starvation whereas cycloheximide completely inhibited this process. The stimulation of lactase activity, in the starved animal, is related to a quantitative increase of the corresponding protein band and with enhanced incorporation of L-[3H]valine in this protein band after separation of brush border proteins by gel electrophoresis. It is concluded that the stimulation of lactase activity observed during starvation is the consequence of de novo synthesis of lactase molecules and that this process is regulated at a translational level. A general hypothesis is proposed in order to clear up partly the mechanism involved in the stimulation of lactase activity by food deprivation in the adult rat.     

Biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase in rat hepatocytes.

The biosynthesis, processing, and intracellular transport of lysosomal acid phosphatase was studied using an in vitro cell-free translation system, pulse-chase experiments with primary cultured rat hepatocytes and subcellular fractionation techniques of rat liver after pulse-labeling with [35S]methionine in vivo. The single polypeptide of 45 kDa translated in the cell-free system from membrane-bound polysomal RNAs was converted to the 64 kDa form when the translation was carried out in the presence of microsomal vesicles. Pulse-chase experiments using cultured rat hepatocytes showed that acid phosphatase is initially synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form of 64 kDa, and processed via an Endo H-sensitive intermediate form of 62 kDa to an Endo H-resistant form with a 67 kDa mass. Phase separation with Triton X-114 showed that both the 64 and 67 kDa forms have hydrophobic properties. Treatment of the cells with chloroquine or tunicamycin, drugs which enhance the secretion of lysosomal hydrolases, had no effect on the normal transport of acid phosphatase to lysosomes. Acid phosphatase did not contain the phosphorylated high mannose type of oligosaccharide chains observed in cathepsin D. Subcellular fractionation experiments in conjunction with pulse-labeling in vivo showed that the acid phosphatase of the 67 kDa form was present in the Golgi heavy fraction (GF3) and the Golgi light fraction (GF1+2) enriched in cis and trans Golgi elements, respectively, at 30 min after the administration of [35S]methionine. Simultaneously, this polypeptide was also found in the lysosomal membrane fraction, thereby indicating that acid phosphatase is delivered to lysosomes in a membrane-bound form, immediately after reaching the trans-Golgi region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of adult lactase phenotypes in the Tuareg of Niger.

The adult lactase phenotype, lactose absorber or malabsorber, was determined using the lactose tolerance test with breath hydrogen assay in a group of Tuareg, a traditionally nomadic pastoralist population in the central Sahara. Out of a total of 118 subjects, 103 (87.3%) were lactose absorbers and 15 (12.7%) lactose malabsorbers. The frequency of the "lactase suppression gene" in this population sample was .357 (SD .043). The low frequency of lactase suppression in the Tuareg supports the hypothesis of natural selection in favor of the "lactase persistence gene" in milk-dependent nomadic pastoralist.     

Time- and dose-dependency of intestinal lactase activity in adult rat on starch intake

Although it is generally accepted that lactase (β-d-galactosidase, EC 3.2.1.23) activity is not influenced by intake of saccharides containing α-linkages, an effect of these carbohydrates on lactase activity was never thoroughly investigated. Activity of lactase and sucrase (sucrose α-d-glucohydrolase, EC 3.2.1.48) was determined in proximal, middle and distal thirds of the jejunoilem of female, 12-week-old rats, fed for 2 weeks a low-starch (5 cal%), high-fat (73%) diet, and in rats, that after this introduction period were fed for 1,2 and 3 days, an isocaloric middle-starch (40%), middle-fat (36%) diet or an isocaloric high-starch (70%), low-fat (7%) diet. During the entire experimental period, the body weight changes, food intake and the amount of protein per segment were practically the same in all three dietary groups. In all intestinal segments, increased intake of starch was followed by an increase of lactase and sucroase activity (both expressed as per tissue protein or per intestinal segment) within the first day. The increase continued during the second day and leveled off during the third day. A highly significant linear correlation was found between the search content of the diets and the lactase activity in all three segments. A highly significant correlation was also established in all three segments between sucrase and lactase activities. These studies thus demonstrated a dose- and time-dependency between the intake of starch (a carbohydrate containing only α-linkages) and the activity of lactase, a neutral β-galactosidase in adult rats.     

High intestinal lactase concentrations in adult Arbs in Saudi Arabia.

The maximum rise in blood glucose after 50 g lactose by mouth was determined in 40 adult Arabs. Out of 30 Bedouin, urban Saudi, and Yemeni and 9 of mixed ancestry (usually partly African), 25 (83%) and 2 (22%) respectively showed an increase of over 1-1 mmol/1 (20 mg/100 ml). In common with most northern Europeans and Hamitic people of northern Africa, Arabs in Saudi Arabia usually have high intestinal lactase concentrations in adult life. This persistence of high levels probably originated in the Arabian peninsula. Its selective advantage may have been associated with the fluid and calorie content of camels'' milk, which is important for survival in desert nomads.     

Recessive inheritance of the adult type of intestinal lactase deficiency.

In order to investigate the genetic control of the adult type of intestinal lactase deficiency, 61 families with 177 children over 6 years of age were investigated. The results strongly suggest that this deficiency is inherited as a simple Mendelian recessive trait.     

Intestinal glycosidase activities in one adult and two suckling echidnas: absence of a neutral lactase (beta-D-galactosidase)

The activities of various glycosidases in homogenates of the small-intestinal mucosa of one adult and two suckling echidnas, Tachyglossus aculeatus, were investigated. The activities of lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, neuraminidase and alpha-L-fucosidase were higher in the sucklings than in the adult animal. Maltase and isomaltase activities were lower. Sucrase and cellobiase activities were absent or present in trace amounts only. The lactase activity had a pH optimum of 4.0-4.5, was predominantly in the soluble fraction following ultracentrifugation and was inhibited by p-chloromercuribenzene sulfonate, suggesting that it was due to a lysosomal acid beta-galactosidase and not a brush-border neutral lactase. The maltase activity of the sucklings also had the characteristics predominantly of a lysosomal acid hydrolase. It is proposed that in suckling echidnas, the oligosaccharides (mainly neuraminyllactose and fucosyllactose) of the mother's milk are digested intracellularly by lysosomal enzymes, rather than at the brush border, of the epithelial cells of the small-intestinal mucosa.     

Intestinal lactase (beta-galactosidase) and other glycosidase activities in suckling and adult tammar wallabies (Macropus eugenii)

The activities of various glycosidases in homogenates of the small intestinal mucosa of two adult and 18 suckling tammar wallabies (M. eugenii) aged from 6 to 50 weeks were investigated. Lactase (beta-D-galactosidase), beta-N-acetylglucosaminidase, alpha-L-fucosidase and neuraminidase activities were high during the first 34 weeks post partum and then declined to very low levels. Maltase, isomaltase, sucrase and trehalase activities were very low or absent during the first 34 weeks, and then increased. The lactase activity was unusual in being greater in the distal than the middle or proximal thirds of the intestine, and in its low pH optimum (pH 4.6), inhibition by p-chloromercuribenzene sulfonate but not by Tris, and lack of cellobiase activity. These properties are those of a lysosomal acid beta-galactosidase rather than of a brush border neutral lactase. The maltase activity had the characteristics of a lysosomal acid alpha-glucosidase early in lactation and of a brush border neutral maltase in adult animals. The significance of these findings is discussed in relation to changes in dietary carbohydrates during weaning and to the mode of digestion of milk carbohydrates by the pouch young.