Abstract
This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02vs 0.15 ± 0.02), medial (0.30 ± 0.06vs 0.14 ± 0.01) and distal (0.43 ± 0.07vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.
mRNA enzyme abundance; Maternal protein restriction; Disaccharidase activity; Fetal programming
Introduction
Recent studies have shown that nutritional changes during critical periods of prenatal development can lead to permanent alterations in the metabolism of the offspring, which can become more susceptible to chronic diseases 11. Waterland RA, Michels KB. Epigenetic epidemiology of the developmental origins hypothesis. Annu Rev Nutr 2007; 27: 363-388, doi: 10.1146/annurev.nutr.27.061406.093705.
https://doi.org/10.1146/annurev.nutr.27....
. Disorders such as arterial hypertension, diabetes mellitus and obesity can be programmed during fetal life as a consequence of the maternal dietary regime during pregnancy 22. Pinney SE, Simmons RA. Epigenetic mechanisms in the development of type 2 diabetes. Trends Endocrinol Metab 2010; 21: 223-229, doi: 10.1016/j.tem.2009.10.002.
https://doi.org/10.1016/j.tem.2009.10.00...
3. Rinaudo P, Wang E. Fetal programming and metabolic syndrome. Annu Rev Physiol 2012; 74: 107-130, doi: 10.1146/annurev-physiol-020911-153245.
https://doi.org/10.1146/annurev-physiol-...
-44. Remmers F, Delemarre-van de Waal HA. Developmental programming of energy balance and its hypothalamic regulation. Endocr Rev 2011; 32: 272-311, doi: 10.1210/er.2009-0028.
https://doi.org/10.1210/er.2009-0028...
. According to Hales and Barker 55. Hales CN, Barker DJ. The thrifty phenotype hypothesis. Br Med Bull 2001; 60: 5-20, doi: 10.1093/bmb/60.1.5.
https://doi.org/10.1093/bmb/60.1.5...
, fetuses receiving insufficient nutrient levels in the intrauterine environment develop adaptive mechanisms to ensure survival, optimizing nutrient use and exhibiting a “thrifty” phenotype. After birth, however, individuals may develop physiological modifications if their nutrient offer is proportionally higher than that received during intrauterine life66. Gicquel C, El-Osta A, Le Bouc Y. Epigenetic regulation and fetal programming. Best Pract Res Clin Endocrinol Metab 2008; 22: 1-16, doi: 10.1016/j.beem.2007.07.009.
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7. McArdle HJ, Andersen HS, Jones H, Gambling L. Fetal programming: causes and consequences as revealed by studies of dietary manipulation in rats - a review. Placenta 2006; 27 (Suppl A): S56-S60, doi: 10.1016/j.placenta.2006.01.014.
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.
The effects of fetal programming on offspring metabolism and obesity are well known99. Ross MG, Beall MH. Adult sequelae of intrauterine growth restriction. Semin Perinatol 2008; 32: 213-218, doi: 10.1053/j.semperi.2007.11.005.
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10. Chernausek SD. Update: consequences of abnormal fetal growth. J Clin Endocrinol Metab 2012; 97: 689-695, doi: 10.1210/jc.2011-2741.
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-1111. Lumey LH, Stein AD, Susser E. Prenatal famine and adult health. Annu Rev Public Health 2011; 32: 237-262, doi: 10.1146/annurev-publhealth-031210-101230.
https://doi.org/10.1146/annurev-publheal...
. However, the consequences regarding nutrient digestion, particularly at the molecular level, have yet to be studied. This information is required to understand food processing, the first step in nutrient metabolism 77. McArdle HJ, Andersen HS, Jones H, Gambling L. Fetal programming: causes and consequences as revealed by studies of dietary manipulation in rats - a review. Placenta 2006; 27 (Suppl A): S56-S60, doi: 10.1016/j.placenta.2006.01.014.
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.
Intestinal enzymes are partially responsible for food processing, and their activity can drastically change because of the high intestinal phenotypic flexibility 1212. Drozdowski LA, Clandinin T, Thomson AB. Ontogeny, growth and development of the small intestine: Understanding pediatric gastroenterology. World J Gastroenterol 2010; 16: 787-799.. Disaccharidases are intestinal brush border enzymes that vary as a function of stimulus such as diet and animal age 1313. Jang I, Jung K, Cho J. Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats. Exp Anim 2000; 49: 281-287, doi: 10.1538/expanim.49.281.
https://doi.org/10.1538/expanim.49.281...
and particularly protein malnutrition 1414. Dudley MA, Schoknecht PA, Dudley AW Jr, Jiang L, Ferraris RP, Rosenberger JN, et al. Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet. Am J Physiol Gastrointest Liver Physiol 2001; 280: G621-G628.. These enzymes are directly involved in supplying energy to the organism and some of them, like sucrase, can be increased in obese individuals 1515. Kiely JM, Noh JH, Svatek CL, Pitt HA, Swartz-Basile DA. Altered small intestinal absorptive enzyme activities in leptin-deficient obese mice: influence of bowel resection. J Pediatr Surg 2006; 41: 1243-1249, doi: 10.1016/j.jpedsurg.2006.03.047.
https://doi.org/10.1016/j.jpedsurg.2006....
,1616. Scoaris CR, Rizo GV, Roldi LP, de Moraes SM, de Proenca AR, Peralta RM, et al. Effects of cafeteria diet on the jejunum in sedentary and physically trained rats. Nutrition 2010; 26: 312-320, doi: 10.1016/j.nut.2009.04.012.
https://doi.org/10.1016/j.nut.2009.04.01...
.
The short-term consequences of maternal protein restriction for the disaccharidases of weaned offspring include increased lactase activity 1717. Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998; 27: 553-559, doi: 10.1097/00005176-199811000-00012.
https://doi.org/10.1097/00005176-1998110...
,1818. Timofeeva NM, Gordova LA, Egorova VV, Nikitina AA. Protein-deficient diet in pregnant females impairs functional activity of enzyme systems in digestive and non-digestive organs in the offspring. Bull Exp Biol Med 2003; 135: 117-119, doi: 10.1023/A:1023847226313.
https://doi.org/10.1023/A:1023847226313...
. However, studies about the long-term effects of maternal dietary restriction during pregnancy on the intestinal enzymes of offspring are scarce, and results are contradictory. For instance, in these cases sucrase and maltase activity is decreased in offspring aged 2 and 4 months and markedly increased in 6-month-old rats 1818. Timofeeva NM, Gordova LA, Egorova VV, Nikitina AA. Protein-deficient diet in pregnant females impairs functional activity of enzyme systems in digestive and non-digestive organs in the offspring. Bull Exp Biol Med 2003; 135: 117-119, doi: 10.1023/A:1023847226313.
https://doi.org/10.1023/A:1023847226313...
.
The mechanisms underlying the control of enzyme activity remain to be elucidated. Molecular studies that assess the responses to maternal dietary restriction are not available, although the determination of mRNA levels of enzymes can provide information on enzyme synthesis. Enzyme synthesis and activity are crucial parameters from a developmental standpoint, but one does not necessarily reflect the other 1919. Marion J, Petersen YM, Rome V, Thomas F, Sangild PT, Le Dividich J, et al. Early weaning stimulates intestinal brush border enzyme activities in piglets, mainly at the posttranscriptional level. J Pediatr Gastroenterol Nutr 2005; 41: 401-410, doi: 10.1097/01.mpg.0000177704.99786.07.
https://doi.org/10.1097/01.mpg.000017770...
.
The present study investigated the effects of fetal programming, determined by gestational protein restriction, on gene expression and activity of the intestinal enzymes sucrose and maltase in the duodenum, jejunum and ileum of 3- and 16-week-old offspring rats and lactase in 3-week-old rats. Gastrointestinal organ weight was also determined.
Material and Methods
Animals
The male and female rats used to produce the offspring were provided by the central animal facility of Universidade Estadual Paulista, Botucatu, SP, Brazil. The procedures were previously approved by the Research Ethics Committee of Instituto de Biociências of the same institution.
Sexually mature Wistar rats were housed in maternity cages (33 × 45 × 17 cm) with 2 females to 1 male to allow mating. On the morning of the following day, females were inspected for the presence of sperm in their vaginal smear, which indicated the first gestational day (day 0). This protocol was repeated until the number of pregnant females in each group was adequate.
From day 0 to the day they gave birth, female rats were fed an isocaloric diet [400 kcal/100 g, AIN-93G 2020. Reeves PG, Nielsen FH, Fahey GC Jr. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutritionad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993; 123: 1939-1951., manufactured by Pragsoluções Biociências, Brazil; Table 1] with normal protein content (17% protein, control group, n = 8) or low-protein content (6% protein, protein-restricted group, n = 8). After giving birth, females were fed a standard rodent chow (Purina, Brazil).
Offspring groups
After birth, litters were reduced to 4 male and 4 female rats to ensure the same litter size per dam. Rats born to dams fed the low-protein (6% protein) and control diet (17% protein) were studied at 3 weeks (PR3 and C3 groups, respectively) or 16 weeks of age (PR16 and C16 groups, respectively). Each group comprised 8 male rats born to different dams. Female offpring rats were not included in this study, because we did not intend to investigate the differences between genders. After weaning (3 weeks), the animals were transferred to individual cages and fed the standard diet ad libitum. All rats were weighed at birth and at 3 and 16 weeks of age. Epididymal and visceral white adipose tissues were dissected and weighed at the end of the study and the adiposity index was calculated (the sum of fat depots divided by body weight x 100). All offspring were culled at the same time of day to prevent the influence of circadian rhythm on enzyme activity. During the experiments, dams and offspring rats were housed in a temperature-controlled environment (22°C) on a 12-h light/dark cycle with free access to food and water.
Gastrointestinal organs
At the age of 3 and 16 weeks, rats from each group were sacrified by decapitation. The abdominal cavity was then opened and the gastrointestinal organs removed, washed with saline solution and weighed.
Intestinal enzymes
After median laparotomy, the entire small intestine, from the pyloric sphincter to the ileocecal junction, was removed and washed in ice-cold saline solution. Segments roughly corresponding to the duodenum (0-12 cm distal to the pyloric valve), jejunum (0-12 cm in the middle intestine), and ileum (0-12 cm proximal to the cecum) were excised. The mucosa of each segment was scraped and frozen in liquid nitrogen. The digestive enzymes lactase (EC 3.2.1.23), sucrase (EC 3.2.1.48) and maltase (EC 3.2.1.20) were determined by the method of Dahlquist2121. Dahlquist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964; 7: 447-454.. Briefly, this procedure consists of measuring glucose levels of homogenized intestinal mucosa aliquots and water incubated with the specific substrates (lactose, sucrose or maltose). The glucose content released during the reaction is determined by the glucose oxidase method, which allows the calculation of specific enzyme activity, expressed as units per gram of mucosa.
Gene expression analysis: real-time PCR
Gene expression in the duodenum, jejunum and ileum was determined by RNA extraction with Trizol reagent (Invitrogen Life Technologies, USA) according to manufacturer recommendations. Complementary DNA (cDNA) synthesis was performed from total RNA using the SuperScript First kit - Synthesis Strand System for RT-PCR (Invitrogen Life Technologies).
The amplification protocol of the Power SYBR® Green Master Mix kit (Applied Biosystems, USA) was performed to evaluate the gene expression of target genes, lactase (sense: S5′-TACATCAACGAGGTGCTCAAGGCT-3′, antisense: A5′-TAGCCGTCAATGAGGGAACG<1?show=[fo]?>AACA-3′); sucrase (sense: S5′-AACAATCAAAGTCCTTGGGCTGCG-3′, antisense: A5′-AAGCTTCCGGCAAGGTTGAAGTTG-3′) and maltase (sense: S5′-TGACAACCAAATGGCACAAGGGAC-3′, antisense: A5′- ATGCTGCCAAGTCTCATCTCCTGT-3′) [GenBank accession Nos.: lactase NM_053841.1; sucrase X15546.1; maltase XM_002726326.3]. The mRNA expression of the constitutive gene α-actin was used to normalize the gene expression of lactase, sucrase and maltase.
Amplification reactions of the target genes were performed on 96-well plates containing Power SYBR® Green Master Mix (Applied Biosystems), cDNA and the specific oligonucleotide primers. Differences in gene expression rates were normalized by the expression frequency of α-actin, which proved to be the best endogenous control. The relative expression values for each gene were calculated using the ΔΔCt method with efficiency correction and using one control sample as calibrator 2222. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29: e45, doi: 10.1093/nar/29.9.e45.
https://doi.org/10.1093/nar/29.9.e45...
. Mean efficiency values for each gene were calculated from the amplification profile of individual samples with the LinRegPCR software 2323. Ramakers C, Ruijter JM, Deprez RH, Moorman AF. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 2003; 339: 62-66, doi: 10.1016/S0304-3940(02)01423-4.
https://doi.org/10.1016/S0304-3940(02)01...
.
Statistical analysis
Data are reported as means ± SE. Differences between control and experimental animals at each age were analyzed by the Student t-test. Statistical significance was set at P < 0.05.
Results
Body measures
Animals born to protein-restricted dams were lighter (P < 0.05) than those born to control dams at birth (5.7 ± 0.1 vs 6.3 ± 0.1 g, respectively) and weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g, respectively). At 16 weeks of age, however, offspring from protein-restricted and control groups had similar weight (421.7 ± 8.9 and 428.5 ± 8.5 g, respectively). The adiposity index at 16 weeks of age was higher (P < 0.05, Student t-test) in animals born to protein-restricted dams (2.2 ± 0.2 vs 1.7 ± 0.3%). The organs of rats born to protein-restricted dams were also lighter at weaning, but at 16 weeks of age there was no difference between groups (Table 2). The relationship between organ and body weight was similar for the protein-deprived and control groups.
Intestinal enzyme activity. Lactase activity in the duodenum, jejunum and ileum was higher in PR3 than in C3 rats (Figure 1). There was no statistical difference between PR3 and C3 rats in maltase activity in proximal, middle, and distal segments. Similar results were obtained for sucrase activity in proximal, middle and distal intestine. PR16 rats had higher sucrase activity in the duodenum compared to C16 (Figure 2). However, maltase activity did not differ between PR16 and C16 in the proximal, middle or distal intestinal segments.
Lactase activity (A) and lactase mRNA abundance (B) in the intestine of 3-week-old rats (n = 5-8 animals) born to dams fed a protein-restricted (6% protein) or a control diet (17% protein). Data are reported as means ± SE. *P < 0.05 compared to control (Student t-test).
Sucrase activity (A) and sucrase mRNA abundance (B) in the intestine of 16-week-old rats (n = 5-8 animals) born to dams fed a protein-restricted (6% protein) or a control diet (17% protein). Data are reported as means ± SE. *P < 0.05 compared to control (Student t-test).
Gene expression of intestinal enzymes
Lactase mRNA expression in the duodenum was higher (P < 0.05) in PR3 than in C3 animals (Figure 1). Dam protein restriction during pregnancy did not affect the abundance of maltase and sucrase mRNA in the proximal, middle or distal intestinal segments of 3-week-old rats.
In PR16, the expression of sucrase mRNA in the duodenum was higher (P < 0.05) than in C16 (Figure 2). However, there was no significant increase in maltase mRNA expression in proximal, middle or distal segments.
Discussion
The present study evaluated gene expression and intestinal disaccharidase activity as a consequence of intrauterine protein restriction. The responses varied as a function of the intestinal segment, enzyme type and animal age.
Rats born to protein-restricted dams showed higher lactase mRNA abundance at 3 weeks of age and higher sucrase mRNA abundance at 16 weeks. Furthermore, 3-week-old animals born to protein-restricted dams showed higher lactase activity in the three intestinal segments. Sixteen-week-old rats showed higher sucrase activity in the duodenum.
The up-regulation of lactase and sucrase mRNA can be considered to be a specific adaptive response to intrauterine protein restriction. The positive association between lactase and sucrase activity and gene expression suggests that transcriptional controls are involved in the fetal programming of these genes. Transcriptional regulation is considered to play a pivotal role in the carbohydrate-induced enhancement of sucrase and lactase expression 2424. Honma K, Mochizuki K, Goda T. Carbohydrate/fat ratio in the diet alters histone acetylation on the sucrase-isomaltase gene and its expression in mouse small intestine. Biochem Biophys Res Commun 2007; 357: 1124-1129, doi: 10.1016/j.bbrc.2007.04.070.
https://doi.org/10.1016/j.bbrc.2007.04.0...
. Moreover, transcriptional control is considered to be the major mechanism in the regulation of lactase-phlorizin hydrolase 2525. Naim HY. Molecular and cellular aspects and regulation of intestinal lactase-phlorizin hydrolase. Histol Histopathol 2001; 16: 553-561..
The increase in disaccharidase activity is consistent with the prediction that animals suffering from intrauterine restriction optimize nutrient use to ensure survival 2626. Armitage JA, Khan IY, Taylor PD, Nathanielsz PW, Poston L. Developmental programming of the metabolic syndrome by maternal nutritional imbalance: how strong is the evidence from experimental models in mammals? J Physiol 2004; 561: 355-377, doi: 10.1113/jphysiol.2004.072009.
https://doi.org/10.1113/jphysiol.2004.07...
. Similar results were observed for lactase in offspring rats born to dams fed only 50% of the adequate food supply1717. Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998; 27: 553-559, doi: 10.1097/00005176-199811000-00012.
https://doi.org/10.1097/00005176-1998110...
and for sucrase and maltase in offspring rats born to dams receiving only 60% of the adequate dietary protein content 1818. Timofeeva NM, Gordova LA, Egorova VV, Nikitina AA. Protein-deficient diet in pregnant females impairs functional activity of enzyme systems in digestive and non-digestive organs in the offspring. Bull Exp Biol Med 2003; 135: 117-119, doi: 10.1023/A:1023847226313.
https://doi.org/10.1023/A:1023847226313...
. However, they contradict the findings of Weaver et al. 1717. Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998; 27: 553-559, doi: 10.1097/00005176-199811000-00012.
https://doi.org/10.1097/00005176-1998110...
for 42-day-old and 1-year-old rats subjected to similar intrauterine protein restriction. These different results can be attributed to the age of the animals used by Weaver et al.1717. Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998; 27: 553-559, doi: 10.1097/00005176-199811000-00012.
https://doi.org/10.1097/00005176-1998110...
. Enzyme activity is changing at both ages. In fact, at 42 days of age the activity is increasing toward its highest level and at 1 year it is decreasing toward its lowest level 1313. Jang I, Jung K, Cho J. Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats. Exp Anim 2000; 49: 281-287, doi: 10.1538/expanim.49.281.
https://doi.org/10.1538/expanim.49.281...
. Moreover, an early study showed that the adaptive response of the intestine is reduced in old age 2727. Detel D, Baticic L, Varljen J. The influence of age on intestinal dipeptidyl peptidase IV (DPP IV/CD26), disaccharidases, and alkaline phosphatase enzyme activity in C57BL/6 mice. Exp Aging Res 2008; 34: 49-62, doi: 10.1080/03610730701761957.
https://doi.org/10.1080/0361073070176195...
.
The high enzyme activity in rats born to protein-restricted dams can increase individual short- and mid-term viability. However, increased disaccharidase activity may also contribute to the development of obesity by fetal programming since it can lead to increased carbohydrate absorption in the small intestine 1515. Kiely JM, Noh JH, Svatek CL, Pitt HA, Swartz-Basile DA. Altered small intestinal absorptive enzyme activities in leptin-deficient obese mice: influence of bowel resection. J Pediatr Surg 2006; 41: 1243-1249, doi: 10.1016/j.jpedsurg.2006.03.047.
https://doi.org/10.1016/j.jpedsurg.2006....
. In fact, leptin-deficient obese mice show increased sucrase activity 2828. Flores CA, Bezerra J, Bustamante SA, Goda T, MacDonald MP, Kaplan ML, et al. Age-related changes in sucrase and lactase activity in the small intestine of 3- and 10-week-old obese mice (C57BL/6Jobob). J Am Coll Nutr 1990; 9: 255-260. and obese rats fed a high-fat diet show an increase in maltase activity 2929. Woods SC, Seeley RJ, Rushing PA, D'Alessio D, Tso P. A controlled high-fat diet induces an obese syndrome in rats. J Nutr 2003; 133: 1081-1087..
In the present study, rats born to protein-restricted dams showed a higher adiposity index at 16 weeks of age although their body weight was similar to that of the control group. Body fat has been considered to be a more sensitive criterion than body weight for assessing obesity 3030. Liu R, Wei N, Guo W, Qiang O, Li X, Ou Y, et al. Octreotide alleviates obesity by reducing intestinal glucose absorption and inhibiting low-grade inflammation. Eur J Nutr 2012 [Epub ahead of print].. These investigators verified a 35-40% increase in body fat but a 10% increase in total body weight in rats fed a high-fat diet. Dissociation between body weight and adiposity index was also observed by Lopes et al. 3131. Lopes KL, Furukawa LN, de Oliveira I, Dolnikoff MS, Heimann JC. Perinatal salt restriction: a new pathway to programming adiposity indices in adult female Wistar rats. Life Sci 2008; 82: 728-732, doi: 10.1016/j.lfs.2008.01.003.
https://doi.org/10.1016/j.lfs.2008.01.00...
in a study about the obesity caused by salt restriction during the perinatal phase. It was not an objective of the present study to determine the mechanisms of such dissociation, but further studies on this subject would be worth developing.
The effects investigated in the present study were primarily identified in the duodenum, corroborating earlier findings from our research group on the fetal programming of intestinal transporters (Pinheiro DF, Pinheiro PFF, Buratini Jr J, Castilho ACS, Lima PF, Trinca LA, et al., unpublished results) and other studies on the consequences of dietary changes. For instance, fructose absorption by animals subjected to food restriction for 24 and 270 days was higher in the proximal intestine portion 3232. Ferraris RP, Cao QX, Prabhakaram S. Chronic but not acute energy restriction increases intestinal nutrient transport in mice. J Nutr 2001; 131: 779-786., and the effects of a carbohydrate-rich diet on sucrase activity in mice was also higher in the duodenum3333. Ferraris RP, Villenas SA, Diamond J. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am J Physiol 1992; 262: G1047-G1059.. The activity of disaccharidases occurs along the longitudinal axes of the small intestine, with the highest activity occurring in the jejunum 1313. Jang I, Jung K, Cho J. Influence of age on duodenal brush border membrane and specific activities of brush border membrane enzymes in Wistar rats. Exp Anim 2000; 49: 281-287, doi: 10.1538/expanim.49.281.
https://doi.org/10.1538/expanim.49.281...
. An increase in enzyme activity could be expected in rats born to protein-restricted dams since it could improve the digestion and assimilation of nutrients. However, the adaptive response affecting only the duodenum observed here deserves further investigation. Enzymatic activity and mRNA abundance increased in the duodenum of 3-week-old rats from the protein-deprived groups, although they had a lighter small intestine. This specific increase in enzyme activity may therefore compensate for weight reduction, favoring better nutrient use. The weight of other body organs in 3-week-old animals was also affected by intrauterine protein restriction. The 16-week-old rats showed a heavier small and large intestine.
Alterations in enzymatic activity have been associated with morphofunctional changes in the mucosa. Montoya et al. 3434. Montoya CA, Leterme P, Lalles JP. A protein-free diet alters small intestinal architecture and digestive enzyme activities in rats. Reprod Nutr Dev 2006; 46: 49-56, doi: 10.1051/rnd:2005063.
https://doi.org/10.1051/rnd:2005063...
detected a decrease in enzyme activity in the intestine of rats with a smaller villus height due to the chronic consumption of a protein-free diet. In addition, Ferraris et al.3333. Ferraris RP, Villenas SA, Diamond J. Regulation of brush-border enzyme activities and enterocyte migration rates in mouse small intestine. Am J Physiol 1992; 262: G1047-G1059. suggested that the proportion of immature and mature differentiated intestinal cells (the latter in higher amounts) in the atrophic mucosa of adult animals subjected to feed restriction accounts for the increase in enzymatic activity. In contrast, studies carried out in our laboratory have shown that intrauterine restriction increases enterocyte proliferation in 3- and 16-week-old rats (Pinheiro DF, Pinheiro PFF, Buratini Jr J, Castilho ACS, Lima PF, Trinca LA, et al., unpublished results). Therefore, we believe that the increased lactase activity in the jejunum and ileum, which was not associated with an increase in mRNA abundance, may be attributed at least in part to increased cell proliferation.
The present study shows for the first time that intrauterine protein restriction affects the gene expression of intestinal enzymes in the offspring. Moreover, it reinforces the idea that specific enzymatic activity can be programmed during early development.
Further studies are necessary to determine if other digestive enzymes, including pancreatic enzymes, can also be affected by a low-protein diet during gestation.
Research supported by FAPESP (fellowship #2009/5008-2 and research grant #2010/50616-0).
References
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1Waterland RA, Michels KB. Epigenetic epidemiology of the developmental origins hypothesis. Annu Rev Nutr 2007; 27: 363-388, doi: 10.1146/annurev.nutr.27.061406.093705.
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2Pinney SE, Simmons RA. Epigenetic mechanisms in the development of type 2 diabetes. Trends Endocrinol Metab 2010; 21: 223-229, doi: 10.1016/j.tem.2009.10.002.
-
3Rinaudo P, Wang E. Fetal programming and metabolic syndrome. Annu Rev Physiol 2012; 74: 107-130, doi: 10.1146/annurev-physiol-020911-153245.
-
4Remmers F, Delemarre-van de Waal HA. Developmental programming of energy balance and its hypothalamic regulation. Endocr Rev 2011; 32: 272-311, doi: 10.1210/er.2009-0028.
-
5Hales CN, Barker DJ. The thrifty phenotype hypothesis. Br Med Bull 2001; 60: 5-20, doi: 10.1093/bmb/60.1.5.
-
6Gicquel C, El-Osta A, Le Bouc Y. Epigenetic regulation and fetal programming. Best Pract Res Clin Endocrinol Metab 2008; 22: 1-16, doi: 10.1016/j.beem.2007.07.009.
-
7McArdle HJ, Andersen HS, Jones H, Gambling L. Fetal programming: causes and consequences as revealed by studies of dietary manipulation in rats - a review. Placenta 2006; 27 (Suppl A): S56-S60, doi: 10.1016/j.placenta.2006.01.014.
-
8Barker DJ. The origins of the developmental origins theory. J Intern Med 2007; 261: 412-417, doi: 10.1111/j.1365-2796.2007.01809.x.
-
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Publication Dates
-
Publication in this collection
15 Mar 2013 -
Date of issue
Mar 2013
History
-
Received
14 Oct 2012 -
Accepted
3 Dec 2012