Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet

Mary A. Dudley1, Patricia A. Schoknecht2, Alden W. Dudley Jr.3, Lan Jiang1, Ronaldo P. Ferraris1, Judy N. Rosenberger4, Joseph F. Henry4, and Peter J. Reeds4

1 Department of Pharmacology and Physiology, New Jersey School of Medicine and Dentistry, Newark 07103; 2 Department of Animal Science, Cook College, Rutgers University, New Brunswick 08903; 3 Pathology and Laboratory Medicine Service, Veterans Affairs Medical Center and New Jersey School of Dentistry and Medicine, Newark, New Jersey 07103; and 4 United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [13C1]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha , but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

small intestine; messenger ribonucleic acid; fractional and total synthesis rates; gas chromatography-mass spectroscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

BRUSH BORDER (BB) lactase phlorizin hydrolase (LPH) is a membrane-bound glycoprotein (7). Because of the complexities of its synthesis, the level of BB LPH could be regulated by either LPH mRNA abundance, the rate of mRNA translation, or the relative rates and efficiencies of the multiple steps associated with posttranslational processing (7, 9-11).

We (13) showed previously that, in pigs, the small intestine responds to severe protein malnutrition (3% dietary protein) with a significant (P < 0.05) reduction of LPH mRNA abundance and a parallel reduction in BB LPH synthesis rates. However, this study (13) involved a prolonged (8 wk) period of very severe protein deficiency during which little growth occurred. For the present study, we wished to determine earlier responses to a more moderate level of chronic protein deficiency [10% vs. 3% dietary protein in the previous study (13)]. We also wished to determine the predominant intestinal mechanisms by which nutritional rehabilitation of protein-deficient animals restored gut growth and LPH synthesis rates to normal levels by comparing rehabilitated animals with an age-matched group that had never been malnourished. Because we (12) have demonstrated that jejunal lactase enzyme activity is highest in pigs fed diets containing large quantities of saturated fat, we designed this study using this dietary regime, theorizing that a high-saturated fat diet would accentuate the changes associated with protein malnutrition and rehabilitation.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials

Casein, dextrose, corn oil, and lard for the animal diets were obtained from ICN (Cleveland, OH). The dietary minerals and amino acids as well as leupeptin, aprotinin, and antipain were obtained from Sigma Chemical (St. Louis, MO). L-[13C1]leucine was purchased from Cambridge Isotope Laboratory (Andover, MA). Ultrapure hydrochloric acid was purchased from J. T. Baker Chemical (Phillipsburg, NJ). All other chemicals were of the highest analytical grade available. All aqueous solutions were prepared with deionized water (Millipore, Bedford, MA).

Experimental Design

The protocol for these studies was carried out in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892] and was approved by the Animal Care and Use Committee of the New Jersey Medical School. Twenty-four 5-day-old pigs (from 6 litters) were obtained from a local commercial swine herd as previously described (8, 12, 13). They were housed individually in rooms with an ambient temperature of 25°C. During a 1-day adaptation period, pigs were fed Soweena Litter Lite (Merrick, Madison, WI). The pigs were then divided into two dietary treatment groups and weaned to isocaloric diets containing 10% (protein-deficient diet) or 24% protein (protein-sufficient diet) (Table 1). To ensure that the effect of protein deficiency was not the result of different dietary amino acid patterns, the proportion of essential amino acids in each diet was adjusted to parallel the proportion found in body protein (Table 1; Refs. 24 and 34). Thus the amino acid supplements were calculated so that the contribution of each essential amino acid to dietary protein was equal to or greater than its contribution to porcine whole body protein (24, 34).

                              
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Table 1.   Diets for protein-deficient and -sufficient pigs

For 4 wk, the pigs were allowed free access to their respective diets and water. At the end of 4 wk, one-half of the animals in each dietary treatment group were anesthetized with 5% isoflurane (Aerrane, Anaquest, Liberty Corner, NJ), and catheters were inserted into the jugular vein and the carotid artery as previously described (8, 10). The remaining animals in each dietary treatment group were fed the 24% protein diet (Table 1) for an additional week, at which time they too were anesthetized and catheters were inserted into the jugular vein and carotid artery.

Two days after surgery, conscious pigs were given a continuous infusion of 20 µmol · kg-1 · h-1 of [13C1]leucine in 0.45 g/l saline via the jugular vein catheter for 6 h. Arterial blood samples (1 ml) were taken immediately before the infusion was started and at the end of the infusion. Blood was drawn into prechilled tubes containing Na2EDTA and centrifuged immediately at 4°C and 1,200 g for 15 min. Plasma was removed and stored at -70°C for later analysis.

At the end of the infusion, the pigs were killed by intravenous injection of 0.33 ml/kg body wt of Beuthenasia-D (Schering-Plough Animal Health, Kenilworth, NJ). The entire small intestine from the stomach to the ileal-cecal junction was excised immediately and placed in iced saline. The duodenum was defined as the segment of small intestine from the stomach to the peritoneal reflection (analogous to the ligament of Treitz) and was separated from the remainder of the intestine. The remainder of the small intestine was cut in half with the proximal portion defined as the jejunum and the distal portion as the ileum. Samples for mRNA analysis were taken from the middle portion of the midjejunum and immediately frozen in liquid nitrogen. Samples for histology were taken from the same location and placed in phosphate-buffered formalin. The remaining tissue was then flushed with cold saline (9 g/l) and weighed. The mucosa was scraped, homogenized in phosphate buffer containing protease inhibitors, and frozen at -70°C until analyses were performed (9).

Analyses

Histological analysis. The jejunal samples were processed and stained with hematoxylin and eosin as previously described (13, 35). Crypt depth and villus height were measured using a Microimage video analysis system using Image Pro Plus software in a system assembled by Micron Optics (Cedar Knolls, NJ). For each animal, 50 villi were measured, and the crypt depth was determined at 50 locations.

Measurement of plasma albumin levels and lactase enzyme activity. Plasma albumin levels were determined as previously described (37). Lactase enzyme activity (µmol glucose · min-1 · g protein-1) of mucosal homogenates was measured and converted to micromoles of glucose per minute per gram of mucosa, as previously described (2, 4, 13, 17). The latter value was then used to calculate lactase activity per jejunum (µmol glucose · min-1 · jejunum-1) and the absolute synthesis rate (synthesis rate in 1 g of mucosa) of BB LPH (see below).

Measurement of steady-state mRNA abundance. The 450-kb LPH probe for porcine LPH mRNA was kindly supplied by J. T. Troelsen, Department of Biochemistry C, University of Copenhagen, The Panum Institute, Copenhagen, Denmark (33). The probe for elongation factor-1alpha (EF-1alpha ; a ribosomal binding protein shown to be unaffected by diet or the animal's stage of development) was as previously described (13).

RNA was isolated by the guanidine isothiocyanate-cesium chloride method and fractionated and blotted as previously described (6, 13). In brief, total cellular RNA (20 µg/lane) was fractionated on 8 g/l agarose-2.2 mol/l formaldehyde gels in 0.02 mol/l MOPS buffer. Ethidium bromide staining was used to assess RNA integrity. All data in this study are derived from lanes in which the staining of the rRNA bands indicated that RNA was intact and the lanes were evenly loaded. Overnight blotting was used to transfer RNA to a nylon transfer membrane. RNA was ultraviolet cross-linked to the membrane.

Radioactive probes for LPH and EF-1alpha mRNA were prepared by random-primed oligolabeling of linearized plasmids (6, 13, 32). Blots were washed, and hybridization was detected as previously described (6, 13, 32). After autoradiography to visualize the positions of the bands, the relative abundance of LPH mRNA was quantified densitometrically as previously described (27) and reported in integrated density units (IDU) × 10-3. The abundance of LPH mRNA was then calculated relative to the abundance of EF-1alpha mRNA.

Measurement of mucosal protein and LPH polypeptide labeling. The immunoisolation of LPH polypeptides using the hybridoma PBB3/7/3/2 and their purification by SDS-PAGE were performed as previously described (7, 8, 13). The preparation of plasma and mucosal free amino acid pools, mucosal protein, and LPH polypeptides for gas chromatography-mass spectroscopy (GCMS) analysis was also performed as described previously (8, 10, 13). Briefly, amino acids from the tissue free amino acid pools and the protein hydrolysates were converted to the n-propyl ester, heptafluorobutyramide derivatives. GCMS was performed using methane negative chemical ionization with helium as the carrier gas on a Hewlett Packard 5988A instrument linked to a Hewlett Packard 5890 H gas chromatograph. Samples (1 µl) were injected onto a silica-based DB5 capillary column (30 m × 0.2 mm, 1-mm film thickness; J & W Scientific, Folsom, CA). Chromatography was effected with a linear temperature gradient (80-250°C at 10°/min). Isotopic abundance of ions at a mass-to-charge ratio of 349 and 350 was converted to a tracer-to-tracee ratio using the matrix method (19).

Calculations

Fractional synthesis rate of total protein and BB LPH. The fractional synthesis rate (FSR) of total mucosal protein and BB LPH synthesis was calculated using the isotopic enrichment of the first detectable LPH precursor polypeptide synthesized (proLPHh) as the denominator in the simplified equation FSR (%/day) = Sb/St × 24/t × 100, in which Sb is the tracer-to-tracee ratio of [13C1]leucine in total mucosal protein or BB LPH after 6 h of infusion, St is the tracer-to-tracee ratio of [13C1]leucine in proLPHh after 6 h of infusion, and t is labeling time (in h). We (10, 13) used this same method previously to estimate FSR in conscious, unrestrained older pigs.

The use of the equation involves two assumptions. First, as we (7-10, 13) have shown in rats and pigs, isotopic equilibrium is achieved rapidly in both the mucosal free amino acid pool and proLPHh. Second, once isotopic equilibrium is achieved in proLPHh, label incorporation into the BB protein is linear; this assumption has also been shown (7-10, 13) to be valid. However, we have also shown a delay of ~1 h as label moves from proLPHh to the second detectable LPH precursor (proLPHc) and finally to the mature BB protein. Thus, for all dietary treatment groups, estimating the FSR of BB LPH from 0 h results in a slight underestimate of the true rate of label incorporation into the BB protein and, hence, the true FSR.

Abundance of mucosal protein. The abundance of mucosal protein per gram of mucosa (mg protein/g mucosa) was determined as previously described (14). The abundance of mucosal protein per jejunum (g protein/jejunum) was calculated by multiplying the abundance per gram of mucosa by the mucosal mass as previously described (14).

Abundance of mature BB LPH. The abundance of BB LPH was estimated as previously described (7, 14). Briefly, for each animal, lactase enzyme activity (µmol glucose · min-1 · g mucosa-1) was divided by the relative abundance of BB LPH protein [i.e., the proportional contribution of the Coomassie blue-stained 160-kDa band (mature BB LPH) relative to the total amount of all Coomassie blue-stained LPH polypeptides observed by scanning a SDS-polyacrylamide gel (see Table 3)] to yield, in arbitrary units (AU), the total quantity of BB LPH protein in 1 g of mucosa. These values were then used to calculate absolute and total LPH synthesis rates (see below).

The calculation of the abundance of BB LPH is based on the assumption that lactase enzyme activity is attributable only to the mature BB form of the enzyme. Naim et al. (28) reported that the precursor forms of the enzyme appear to be enzymatically active in COS-1 cells transfected with the human cDNA for LPH. However, in COS-1 cells, the mature BB form of the enzyme seen in vivo is not synthesized; rather, a precursor form of LPH appears to be expressed at the cell surface. Because it has not been proven conclusively that the precursor polypeptides in the pig are enzymatically active and because the quantity of precursor relative to mature LPH is small (<15%), it seems reasonable to omit these proteins in calculations of total abundance.

Absolute synthesis rates. The absolute synthesis rates of mucosal protein and mature BB LPH (synthesis per g mucosa in AU) were calculated as the product of the abundance of the protein per gram of mucosa (see above) and FSR [expressed in day-1 as previously described (7, 14), not %day-1].

Total synthesis rates. The total synthesis rates (synthesis per segment of intestine in AU) of mucosal protein and BB LPH in the jejunum were calculated as the product of their absolute synthesis rate and the mass of the jejunal mucosa (7, 14).

Statistics

Data are means ± SE. Differences between means were determined using an unpaired Student's t-test. P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The body weights of the animals in the two treatment groups at 5 days of age were not significantly different (on average, 2,647 ± 50 g). Likewise, plasma albumin levels were not different (on average, 2.57 ± 0.16 g/dl). After 4 wk on their respective diets, the protein-sufficient pigs weighed significantly (P < 0.05) more than the protein-deficient animals (Table 2). Plasma albumin levels were also significantly (P < 0.05) different (Table 2). The protein-deficient pigs developed dry, scaly skin and sparse, dull hair and, toward the end of the study, showed signs of inactivity or ataxia compared with the protein-sufficient animals.

                              
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Table 2.   Body, jejunal, and mucosal weight, protein abundance, plasma albumin level, villus height, and crypt depth in PS, PD, and PD + PS pigs

After 5 wk of feeding, the body weights of protein-sufficient animals were still significantly (P < 0.05) higher than the body weights of protein-deficient animals fed a protein-sufficient diet for 1 wk (rehabilitated animals) (Table 2). However, ingestion of a diet containing adequate protein resulted in a weight gain of 139 ± 36 g/day in the protein-malnourished animals, a value that was not significantly different from that of the age-matched well-nourished animals (138 ± 3 g/day). The plasma albumin levels in these rehabilitated animals, although still significantly (P < 0.05) lower than in protein-sufficient pigs, increased markedly and were within the normal range (Table 2; Ref. 37).

The weight of the jejunum was significantly (P < 0.05) less in protein-deficient pigs compared with protein-sufficient animals (Table 2). Rehabilitation led to a rapid increase in jejunal mass, and after 7 days the mass was not significantly different from the age-matched controls (Table 2). The mucosal protein concentration per gram of mucosa was unaffected by the dietary treatments at 4 and 5 wk (average of all animals, 100 ± 4.6 mg protein/g mucosa; Table 2). However, mucosal protein abundance per jejunum (g protein/jejunum) was significantly (P < 0.05) higher in protein-sufficient pigs than in protein-deficient pigs fed for 4 wk (Table 2). Mucosal protein abundance per jejunum was not significantly different between rehabilitated pigs and their respective protein-sufficient controls (Table 2). Thus the effect of protein malnutrition on mucosal protein content paralleled its effect on jejunal mass (Table 2).

The jejunal enterocytes appeared to be morphologically normal in all dietary treatment groups. The cells were columnar with nuclei located close to the basal membrane. As shown in Table 2, villus height and crypt depth were significantly (P < 0.05) greater in the protein-sufficient pigs than in the protein-deficient pigs after 4 wk, although the crypt-to-villus ratio was unaltered. After 1 wk of ingestion of a protein-sufficient diet, jejunal mucosal morphometry of the rehabilitated animals was not significantly different from age-matched well-nourished controls.

Although lactase enzyme activity (µmol glucose · min-1 · g mucosa-1) was slightly different between protein-sufficient groups after 4 or 5 wk (Table 3), these differences were not significant. As previously reported (31), lactase enzyme activity in protein-sufficient animals was lower at 5 wk than at 4 wk but was not significantly different from that in rehabilitated animals. However, lactase activity per jejunum (µmol glucose · min-1 · jejunum-1) was significantly (P < 0.05) lower in protein-deficient animals compared with protein-sufficient controls (Table 3). In rehabilitated pigs, lactase activity per jejunum was not significantly different from that in protein-sufficient controls (Table 3).

                              
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Table 3.   BB lactase activity, mRNA ratio, and polypeptide relative amount in jejunum of PS, PD, and PD + PS pigs

After 4 wk of feeding, the abundance of EF-1alpha mRNA was not significantly different between protein-deficient and -sufficient pigs (15 ± 1.9 and 17.7 ± 1 IDU, respectively). Furthermore, the ratio of LPH mRNA abundance to EF-1alpha mRNA abundance was not significantly different between these dietary treatment groups (Fig. 1; Table 3). A similar comparison of rehabilitated pigs with pigs fed a protein-sufficient diet for 5 wk demonstrated that, although EF-1alpha mRNA abundance was not different between dietary treatment groups (15.1 ± 1.8 and 16.1 ± 1.8 IDU, respectively), the abundance of LPH mRNA relative to EF-1alpha mRNA was significantly (P < 0.05) higher in the rehabilitated pigs than in their protein-sufficient controls (Fig. 1; Table 3).


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Fig. 1.   Northern blots of a representative animal from each dietary treatment group. Lane 1, protein-sufficient pig fed for 4 wk; lane 2, protein-deficient pig fed for 4 wk; lane 3, protein-sufficient pig fed for 5 wk; lane 4, protein-deficient pig fed a protein-sufficient diet for 1 wk (rehabilitated). LPH, lactase phlorizin hydrolase; EF-1alpha , elongation factor-1alpha .

After SDS-PAGE, LPH polypeptides, immunoisolated from the solubilized, scraped mucosa, separated into four bands that we (7-10, 13) previously identified as two precursor forms of BB LPH (proLPHh, 200 kDa; proLPHc, 220 kDa) and two forms of BB LPH (the 160-kDa polypeptide and a dimer of BB LPH with an apparent molecular mass of ~240 kDa). In order of synthesis, the LPH polypeptides are proLPHh (the first detectable translation product), proLPHc (the complex glycosylated precursor), and, finally, the mature BB enzyme (7-10, 13).

The relative amounts (i.e., the amount of an individual LPH polypeptide relative to the total amount of all 4 LPH polypeptides on a gel) differed between dietary treatment groups (Table 3). The relative amount of proLPHh was significantly (P < 0.05) higher in protein-sufficient pigs than in protein-deficient pigs and was not altered by 1 wk of rehabilitation (Table 3). However, the relative amount of proLPHc was significantly (P < 0.05) increased in rehabilitated animals compared with protein-sufficient controls (Table 3).

The tracer-to-tracee ratios of the plasma leucine and mucosal free leucine (on average, 46 ± 0.8% of the plasma value) were not significantly different between dietary treatment groups (Table 4). Although the tracer-to-tracee ratio of mucosal protein-bound leucine in the protein-malnourished animals was lower than in their age-matched well-nourished controls, the difference was not significant. When assessed against the tracer-to-tracee ratio of pro-LPHh, the FSR and absolute synthesis rate of mucosal protein were also not significantly different between treatment groups at 4 or 5 wk (Table 5). Total mucosal protein synthesis paralleled mucosal protein mass and was significantly (P < 0.05) reduced in the protein-malnourished animals compared with well-nourished controls (Table 5).

                              
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Table 4.   Tracer-to-tracee ratio of [13C1]leucine in plasma and mucosal free amino acid pools, mucosal protein, and LPH polypeptides after 6-h infusion


                              
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Table 5.   Jejunal mucosa kinetics of mucosal protein and LPH synthesis

The tracer-to-tracee ratios of the earliest detectable BB LPH precursor polypeptide (proLPHh) are shown in Table 4. ProLPHh-bound leucine tracer-to-tracee ratios were not significantly different among groups and were 64 ± 8.7% (average of all pigs) of the mucosal free leucine free pool. Likewise, the tracer-to-tracee ratios of proLPHc-bound leucine were not significantly different among dietary treatment groups (Table 4). Both the tracer-to-tracee ratio and the FSR of BB LPH synthesis were significantly (P < 0.05) higher in protein-sufficient pigs than in protein-deficient pigs at 4 wk (Table 5). The tracer-to-tracee ratios of the dimer of BB LPH (240-kDa polypeptide) paralleled that of the 160-kDa BB protein (data not shown). In well-nourished animals, the FSR of BB LPH fell between 4 and 5 wk, whereas the values for the rehabilitated animals remained constant (Table 5). Thus the FSR of BB LPH in the rehabilitated and 5-wk-old well-nourished animals was similar.

The abundance of BB LPH in a gram of tissue (AU) was not significantly different between the dietary treatment groups at 4 or 5 wk (Table 5), although there was a small fall between 4 and 5 wk in the well-nourished groups. No differences were observed between rehabilitated pigs and their protein-sufficient controls (Table 5).

The absolute synthesis rate of BB LPH was not significantly different between protein-sufficient and -deficient pigs after 4 wk of feeding (Table 5). However, the absolute synthesis rate of BB LPH was significantly (P < 0.05) higher in rehabilitated pigs compared with their protein-sufficient controls (Table 5). The total synthesis rate of BB LPH was significantly (P < 0.05) lower in protein-deficient pigs after 4 wk of feeding compared with protein-sufficient animals but was not significantly different between rehabilitated pigs and their protein-sufficient controls (Table 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Severe (generally defined as an 80-85% reduction in dietary protein), chronic protein malnutrition, which causes a pronounced decrease in the rate of whole body protein turnover, is widely recognized as a major global nutritional problem (18, 22, 36). However, whether decreased whole body protein turnover reflects decreased turnover in specific proteins within an organ is not clear (5, 13-16, 20, 25, 26, 36, 37). The effect of protein malnutrition on the synthesis of individual proteins has not been widely studied; however, available data suggest that the effect may depend on which protein is examined (22). For example, in pigs, severe chronic protein undernutrition results in the reduction of the plasma albumin FSR and absolute synthesis rate but does not affect the synthesis rate of plasma fibrinogen (22). Although not strictly parallel because the animals were protein and calorie restricted, it has also been shown (21) in rats that intestinal alkaline phosphatase mRNA abundance, but not LPH mRNA abundance, is sharply lower after a 4-day fast.

LPH expression during development and the decline of expression that takes place in pigs, rats, and many humans during the postweaning period is widely believed to be controlled by transcriptional events (3, 15, 23). However, little is known about whether pretranslational or posttranslational steps of LPH synthesis are affected by diet during the same period. We (14) have shown that the rate constants of LPH synthesis are higher in enterally fed pigs compared with parenterally fed animals, suggesting that at least in some cases posttranslational regulation predominates. We have also shown that when pigs are severely protein malnourished, both LPH mRNA abundance and posttranslation synthesis rates are significantly lower than in well-nourished animals (13). However, for this study we could not determine the primary event during protein synthesis affected by changes in the protein content of the diet.

The present study further defines the steps of LPH synthesis regulated by the amount of dietary protein. The results demonstrated that moderate protein deficiency, induced with a diet containing 10% [as opposed to 3% in our (13) earlier study] protein (a 57% reduction in dietary protein relative to suggested requirements in young pigs) lowered body weight, serum albumin levels, and intestinal mass but had smaller effects on mucosal structure than more severe undernutrition. Villus length was significantly reduced in protein-malnourished animals compared with controls, presumably caused by a reduction in the number of enterocytes as shown in our (13) previous study. Crypt depth was likewise significantly less in the protein-deficient pigs compared with protein-sufficient controls, but the crypt-to-villus ratio was unaltered by nutritional state. Nutritional rehabilitation for a relatively short period of time was sufficient to rapidly restore villus morphometry to normal. It should be noted, however, that the present study is not strictly comparable to our (13) previous study of severe protein malnutrition because the diets for the two studies differed. The protein-sufficient diets in the present study contained a greater quantity of protein than the diets in our (13) study of older pigs. This change was necessary to meet the dietary requirements of young pigs. All diets in the present study were high in saturated fat, because we (12) have shown that lactase specific activity is higher if saturated fats are used. Finally, the diets contained dextrose instead of cornmeal and cornstarch.

In the present study, the FSR of mucosal protein in general was not different among the dietary treatment groups at 4 or 5 wk, although because of their greater mucosal protein mass, total mucosal protein synthesis was higher in the protein-sufficient pigs compared with protein-deficient animals. Despite a substantial reduction in the quantities of amino acids presented to the mucosa via the diet, the degree of isotopic dilution in the mucosal leucine free pool was unaffected either by protein deficiency or by rehabilitation, an observation that we (13) made in our earlier study. This observation is compatible with a rise in mucosal proteolysis associated with marginal protein deficiency. It was of particular interest that after the reintroduction of a protein-sufficient diet, mucosal protein synthesis did not increase whether expressed as a rate constant (%/day) or total synthesis rate (synthesis rate/jejunum). Adegoke et al. (1) reported similar findings in pigs using in situ intestinal loops (1). This implies that the rapid accretion of mucosal protein was achieved largely by a decrease in mucosal proteolysis rather than an increase in protein synthesis.

Lactase enzyme activity (µmol glucose · min-1 · g mucosa-1) was not significantly different among dietary treatment groups. Thus lactase activity per jejunum (µmol glucose · min-1 · jejunum-1) varied in parallel to jejunal mucosal mass. These findings are consistent with the data from our (13) earlier study that showed no significant differences in lactase enzyme activity after severe chronic protein malnutrition but significant differences in lactase activity per jejunum. Because lactase activity is found only in the villus enterocyte (11) and villus length in the protein-deficient pigs fed for 4 wk was reduced by 30% compared with that in protein-sufficient controls, it is interesting to speculate as to how enzyme activity could be maintained in the small intestine of protein-deficient animals. Although not proven by the present study, it is possible that lactase enzyme activity is maintained by a reduction in the rate of enzyme degradation or migration rate of the villus enterocyte. If the former explanation is correct, it implies that regulation of the degradation of LPH differs from that of total mucosal protein. These are issues that need further attention.

LPH mRNA abundance was not significantly different between the protein-deficient and -sufficient pigs after 4 wk of feeding. This observation contrasts markedly with the data from our (13) study of severe chronic protein malnutrition that demonstrated significantly lower LPH mRNA abundance in protein-deficient pigs compared with the well-nourished controls and raises the question as to how LPH mRNA abundance could be maintained in protein-malnourished animals when villus length is significantly reduced. Although the data from this study do not provide an answer, there are several possible explanations. Rings et al. (30) showed that in the rat LPH mRNA is restricted to the lower portion of the villus, whereas LPH protein shows uniform expression along the villus. Thus the reduction in villus length may result from shedding of cells at the tip of the villus, leaving the cells at the base of the villus unaffected. Alternatively, more LPH mRNA may be synthesized per enterocyte.

LPH mRNA relative abundance in rehabilitated animals increased significantly compared with protein-sufficient controls. Whether this is a reflection of increased LPH gene transcription or whether the LPH mRNA stability was increased during the rehabilitation period is unknown. It is interesting to note that, as in our (13) study of severe protein malnutrition, the absolute synthesis rate (synthesis rate/g mucosa) per unit of LPH mRNA (IDU LPH mRNA/IDU EF-1alpha mRNA), although not significantly different between treatment groups, was 14% lower in protein-malnourished than in control animals at 4 wk (0.048 ± 0.005 vs. 0.056 ± 0.006 absolute synthesis rate/unit LPH mRNA in protein-malnourished and -sufficient pigs, respectively). After rehabilitation, LPH mRNA abundance was 27% lower in the rehabilitated pigs than in controls (0.022 ± 0.004 vs. 0.030 ± 0.003 absolute synthesis rate/unit LPH mRNA in rehabilitated and protein-sufficient pigs, respectively). These calculations are undoubtedly imprecise because they were not based on the direct measurement of mRNA abundance, i.e., RNase protection assay. This observation suggests that 1 wk of rehabilitation does not permit full recovery of BB LPH synthesis from the effects of protein malnutrition.

The FSR and the total synthesis rate (synthesis rate per jejunum) of BB LPH were significantly higher in protein-sufficient pigs compared with protein-deficient pigs. Again, these results parallel those we (13) previously reported in severely protein-malnourished pigs compared with normally nourished controls. In contrast to the rapid increase in LPH mRNA, when protein-deficient pigs were rehabilitated for 1 wk with a protein-sufficient diet, neither the FSR nor the total synthesis rate of BB LPH were increased significantly. Because no differences in LPH mRNA abundance were found between protein-deficient and -sufficient pigs fed for 4 wk, it seems reasonable to conclude that the predominant intestinal response to moderate chronic protein malnutrition was posttranslational.

Together, the data from the present study suggest that under conditions of moderate chronic protein malnutrition, the initial jejunal response with respect to BB LPH, but not mucosal protein in general, is controlled by posttranslational events. There is no reduction in BB LPH mRNA but a marked drop in both the FSR and total synthesis rates of BB LPH. In contrast, when protein-deficient animals are rehabilitated with a protein-sufficient diet, the initial jejunal response appears to be pretranslational with an increase in the steady-state abundance of BB LPH mRNA.


    ACKNOWLEDGEMENTS

We are grateful to Michael Naumoff without whose technical assistance this project would not have been possible.


    FOOTNOTES

This study was funded by the United States Department of Agriculture, Agricultural Research Service under Cooperative Agreement no. 96-35206-3903 and by the National Science Foundation (IBN-9985808).

The contents of this publication do not necessarily reflect the views or policies of the United States Dept. of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States government.

Address for reprint requests and other correspondence: M. A. Dudley, Dept. of Pharmacology and Physiology, New Jersey School of Medicine and Dentistry, 185 South Orange Ave., Newark, NJ 07103.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 14 July 2000; accepted in final form 18 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 280(4):G621-G628




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