Low-protein Offspring Research Articles

The Fetal Origins of Disease and Associations With Low Birthweight

After completing this article, readers should be able to: 1. Explain the fetal insulin hypothesis. 2. Explain the thrifty phenotype hypothesis. The importance of the early environment for long-term health has been recognized for many years. Seventy years ago, observations in England, Scotland, and Sweden showed that death rates decreased between 1751 and 1930 due to improved childhood living conditions. Death rates in specific age groups at any time depended more on the date of birth of the individuals than the year under consideration. A relationship between infant mortality and ischemic heart disease in the same geographic cohort of Norwegians was reported in the 1970s. There was a positive correlation between death rates from heart disease and geographic variations in past infant mortality rates. A study in the United Kingdom by Williams and colleagues showed similar findings. These relationships were linked to events in childhood and adolescence. A study by Wadsworth and associates in 1985 revealed that adult blood pressure was inversely related to birthweight in British men and women, which was linked to “social and familial factors, smoking, and body mass.” In 1986, Barker and Osmond examined mortality rates from stroke and cardiovascular diseases in different areas of England and Wales. They noticed a parallel relationship between neonatal mortality in the 1920s and 1930s and the mortality rates from strokes and cardiovascular diseases in the 1960s and 1970s from the same geographic areas. In the early 20th century, a high neonatal mortality rate was an indication of a high occurrence of low-birthweight babies and of poor nutrition and health of the mothers. Barker and Osmond concluded that the health of the mothers was important in determining the risk of stroke in their offspring and proposed that cardiovascular diseases might originate during fetal life or early childhood. To follow up these findings, …

Maternal protein restriction leads to hyperinsulinemia and reduced insulin-signaling protein expression in 21-mo-old female rat offspring

Human adult diseases such as cardiovascular disease, hypertension, and type 2 diabetes have been epidemiologically linked to poor fetal growth and development. Male offspring of rat dams fed a low-protein (LP) diet during pregnancy and lactation develop diabetes with concomitant alterations in their insulin-signaling mechanisms. Such associations have not been studied in female offspring. The aim of this study was to determine whether female LP offspring develop diabetes in later life. Control and LP female offspring groups were obtained from rat dams fed a control (20% protein) or an isocaloric (8% protein) diet, respectively, throughout pregnancy and lactation. Both groups were weaned and maintained on 20% normal laboratory chow until 21 mo of age when they underwent intravenous glucose tolerance testing (IVGTT). Fasting glucose was comparable between the two groups; however, LP fasting insulin was approximately twofold that of controls (P < 0.02). Glucose tolerance during IVGTT was comparable between the two groups; however, LP peak plasma insulin at 4 min was approximately threefold higher than in controls (P < 0.001). LP plasma insulin area under the curve was 1.9-fold higher than controls (P < 0.02). In Western blots, both muscle protein kinase C-zeta expression and p110beta-associated p85alpha in abdominal fat were reduced (P < 0.05) in LPs. Hyperinsulinemia in response to glucose challenge coupled with attenuation of certain insulin-signaling molecules imply the development of insulin resistance in LP muscle and fat. These observations suggest that intrauterine protein restriction leads to insulin resistance in females in old age and, hence, an increased risk of type 2 diabetes.

Early programming of weight gain in mice prevents the induction of obesity by a highly palatable diet.

Poor early growth is associated with Type II diabetes, hypertension and other features of the metabolic syndrome in adulthood. It has been suggested that this results from the development of a thrifty phenotype by a malnourished fetus. Such a phenotype would predispose the offspring to the development of obesity if born into conditions of over-nutrition. The present study aimed to determine if early nutrition affected subsequent development of obesity. Mice were established as follows: (a) controls (offspring of control dams), (b) recuperated (offspring of dams fed a low-protein diet during pregnancy, but nursed by control dams) and (c) postnatal low-protein (offspring of control dams nursed by low-protein-fed dams). Mice were weaned on to standard laboratory chow or a cafeteria diet. Recuperated offspring, although smaller at birth ( P <0.01), caught up and exceeded the weight of control offspring by 7 days of age ( P <0.001). Postnatal low-protein offspring were smaller than controls by 7 days of age ( P <0.001). Recuperated animals gained more weight than controls when given free access to a highly palatable diet ( P <0.01). Postnatal low-protein animals showed no additional weight gain when given a highly palatable diet compared with chow-fed litter-mates. These results suggest that the early environment has long-term consequences for weight gain. These programmed responses are powerful enough to block excess weight gain from a highly palatable diet and, thus, have major implications for the drug-free regulation of food intake and obesity.

The effects of maternal mild protein restriction on stroke incidence and blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP).

The effect of maternal protein restriction during pregnancy on the offspring's blood pressure was assessed in stroke-prone spontaneously hypertensive rats (SHRSP) which are genetically predisposed to hypertension and stroke. After the confirmation of pregnancy, the control group was given a 20% casein diet, and the low-protein group was fed a 9% casein diet. After the confirmation of delivery, commercial feed was given to both of the groups. No differences were seen between the control and low-protein offspring in regard to body weight, blood pressure elevation, or life span. One percent saline solution was put in the control and low-protein groups after the age of 11 weeks. Blood pressure increased markedly in the low-protein group, on the blood pressure level in the low-protein group on week 2 after salt loading (242+/-6 mmHg) was significantly higher than that in the control group (223+/-9 mmHg; p<0.05). The survival duration was significantly shorter in the low-protein group (113+/-4 days) than in the control group (135+/-22 days; p<0.05). These results suggest that maternal protein malnutrition in SHRSP exerted a high salt sensitivity and a malignant influence on stroke incidence on offspring.

The maternal diet during pregnancy programs altered expression of the glucocorticoid receptor and type 2 11beta-hydroxysteroid dehydrogenase: potential molecular mechanisms underlying the programming of hypertension in utero.

Potential mechanisms underlying prenatal programming of hypertension in adult life were investigated using a rat model in which maternal protein intake was restricted to 9% vs. 18% casein (control) during pregnancy. Maternal low protein (MLP) offspring exhibit glucocorticoid-dependent raised systolic blood pressure throughout life (20-30 mm Hg above the control). To determine the molecular mechanisms underlying the role of alterations in glucocorticoid hormone action in the prenatal programming of hypertension in MLP offspring, tissues were analyzed for expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11betaHSD1, 11betaHSD2, and corticosteroid-responsive Na/K-adenosine triphosphatase alpha1 and beta1. GR protein (95 kDa) and messenger RNA (mRNA) expression in kidney, liver, lung, and brain was more than 2-fold greater in MLP vs. control offspring during fetal and neonatal life and was more than 3-fold higher during subsequent juvenile and adult life (P < 0.01). This was associated with increased levels of Na/K-adenosine triphosphatase alpha1- and beta1-subunit mRNA expression. Levels of MR gene expression remained unchanged. Exposure to the MLP diet also resulted in markedly reduced levels of 11betaHSD2 expression in the MLP placenta on days 14 and 20 of gestation (P < 0.001), underpinning similar effects on 11betaHSD2 enzyme activity that we reported previously. Levels were also markedly reduced in the kidney and adrenal of MLP offspring during fetal and postnatal life (P < 0.001). This programmed decline in 11betaHSD2 probably contributes to marked increases in glucocorticoid hormone action in these tissues and potentiates both GR- and MR-mediated induction of raised blood pressure. In contrast, levels of 11betaHSD1 mRNA expression in offspring central and peripheral tissues remained unchanged. In conclusion, we have demonstrated that mild protein restriction during pregnancy programs tissue-specific increases in glucocorticoid hormone action that are mediated by persistently elevated expression of GR and decreased expression of 11betaHSD2 during adult life. As glucocorticoids are potent regulators not only of fetal growth but also of blood pressure, our data suggest important potential molecular mechanisms contributing to the prenatal programming of hypertension by maternal undernutrition in the rat.

The Maternal Diet during Pregnancy Programs Altered Expression of the Glucocorticoid Receptor and Type 2 11 -Hydroxysteroid Dehydrogenase: Potential Molecular Mechanisms Underlying the Programming of Hypertension in Utero

Potential mechanisms underlying prenatal programming of hypertension in adult life were investigated using a rat model in which maternal protein intake was restricted to 9% vs. 18% casein (control) during pregnancy. Maternal low protein (MLP) offspring exhibit glucocorticoid-dependent raised systolic blood pressure throughout life (20–30 mm Hg above the control). To determine the molecular mechanisms underlying the role of alterations in glucocorticoid hormone action in the prenatal programming of hypertension in MLP offspring, tissues were analyzed for expression of the glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11βHSD1, 11βHSD2, and corticosteroid-responsive Na/K-adenosine triphosphatase α1 and β1. GR protein (95 kDa) and messenger RNA (mRNA) expression in kidney, liver, lung, and brain was more than 2-fold greater in MLP vs. control offspring during fetal and neonatal life and was more than 3-fold higher during subsequent juvenile and adult life (P < 0.01). This was associated with increased levels of Na/K-adenosine triphosphatase α1- and β1-subunit mRNA expression. Levels of MR gene expression remained unchanged. Exposure to the MLP diet also resulted in markedly reduced levels of 11βHSD2 expression in the MLP placenta on days 14 and 20 of gestation (P < 0.001), underpinning similar effects on 11βHSD2 enzyme activity that we reported previously. Levels were also markedly reduced in the kidney and adrenal of MLP offspring during fetal and postnatal life (P < 0.001). This programmed decline in 11βHSD2 probably contributes to marked increases in glucocorticoid hormone action in these tissues and potentiates both GR- and MR-mediated induction of raised blood pressure. In contrast, levels of 11βHSD1 mRNA expression in offspring central and peripheral tissues remained unchanged. In conclusion, we have demonstrated that mild protein restriction during pregnancy programs tissue-specific increases in glucocorticoid hormone action that are mediated by persistently elevated expression of GR and decreased expression of 11βHSD2 during adult life. As glucocorticoids are potent regulators not only of fetal growth but also of blood pressure, our data suggest important potential molecular mechanisms contributing to the prenatal programming of hypertension by maternal undernutrition in the rat.

Impaired PI 3-kinase activation in adipocytes from early growth-restricted male rats.

Epidemiological studies have established a relationship between early growth restriction and subsequent development of type 2 diabetes. Animal studies have shown that offspring of protein-restricted rats undergo a greater age-related loss of glucose tolerance than controls. The aim of this study was to investigate the possibility that this deterioration of glucose tolerance is associated with changes in adipocyte insulin action. Adipocytes from low-protein offspring had higher basal levels of glucose uptake than controls. Insulin stimulated glucose uptake into control adipocytes but had little effect on low-protein adipocytes. Both groups had similar levels of basal and isoproterenol-stimulated lipolysis. Insulin inhibited lipolysis in control adipocytes but had a reduced effect on low-protein adipocytes. These changes in insulin action were not related to altered expression of insulin receptors or insulin receptor tyrosine phosphorylation; however, they were associated with reduced phosphatidylinositol 3-kinase and protein kinase B activation. These results demonstrate that reduced glucose tolerance observed in late adult life after early growth restriction is associated with adipocyte insulin resistance.

Diabetes in old male offspring of rat dams fed a reduced protein diet.

Restricted fetal growth is associated with increased risk for the future development of Type 2 diabetes in humans. The study aim was to assess the glucose tolerance of old (seventeen months) male rats, which were growth restricted in early life due to maternal protein restriction during gestation and lactation. Rat mothers were fed diets containing either 20% or 8% protein and all offspring weaned onto a standard rat diet. In old-age fasting plasma glucose concentrations were significantly higher in the low protein offspring: 8.4 (1.3)mmol/l v. 5.3 (1.3)mmol/l (p = 0.005), Areas under the curves were increased by 67% for glucose (p = 0.01) and 81% for insulin (p = 0.01) in these rats in intravenous glucose tolerance tests, suggesting (a degree of) insulin resistance. These results show that early growth retardation due to maternal protein restriction leads to the development of diabetes in old male rat offspring. The diabetes is predominantly associated with insulin resistance.

Catecholamine levels and receptor expression in low protein rat offspring.

Low birthweight in humans has been shown to lead to increased resting pulse rate in adult life, suggesting possible increased sympathoadrenal activity. The hypothesis that early growth restriction is associated with permanent alterations in catecholamine metabolism was tested. Circulating catecholamine concentrations (by radioimmunoassay) and adipocyte adrenoceptor expression from different fat depots (by Western blot) were estimated in 12-week-old male offspring of rat dams fed a reduced protein diet during pregnancy and lactation. In the fed state, median (interquartile range) plasma adrenaline concentrations for male control and low protein offspring rats were: 0.65 (0.48-0.86) vs. 1.42 (0.89-1.87) nmol/l (P < 0.005), respectively. Equivalent noradrenaline concentrations were: 2.71 (2.16-3.46) vs. 3.45 (3.00-4.28) nmol/l (P < 0.05). After 24 h starvation, plasma adrenaline concentrations of controls rose to become similar to those of low protein offspring: 1.03 (0.95-1.31) vs. 1.41 (0.69-1.62) nmol/l (P = 0.3), respectively. Noradrenaline concentrations rose in both groups to become similar: 3.84 (3.33-4.54) vs. 4.32 (3.70-6.54) nmol/l (P = 0.3). In epididymal adipocytes adrenoceptor expression (relative to that of controls) was: alpha2A 0.79 (0.66-0.94) (P = 0.08), beta1 2.60 (2.27-3.07) (P = 0.04), beta3 1.37 (1.27-1.46) (P = 0.02). Similar-pattern differences in adrenoceptor expression were observed in subcutaneous and intra-abdominal adipocytes. These results are consistent with the suggestion that long-term alterations in catecholamine metabolism are present in adult offspring of rats fed a reduced protein diet during pregnancy and lactation.

Depot-specific effects of early growth retardation on adipocyte insulin action.

Male offspring of rats protein restricted during pregnancy and lactation are growth restricted and have changes in insulin action on epididymal adipocytes. Adipocytes from different anatomical depots are thought to have distinct metabolic functions. The aim of the present study was to determine whether the alterations in metabolism of adipocytes from early growth restricted rats is depot-specific or more generalised. Epididymal, intra-abdominal and subcutaneous adipocytes were studied from three-month-old male offspring of control and protein restricted dams. Basal glucose uptakes were higher in low protein adipocytes (p<0.01) compared to controls. However, insulin stimulation was less in the low protein group in all depots compared to controls (p<0.05). Isoproterenol-stimulated lipolysis was greater in low protein adipocytes (p<0.0001), but the magnitude was greater in epididymal (p<0.0001) and intra-abdominal (p<0.0001) adipocytes than in subcutaneous adipocytes. Epididymal and intra-abdominal adipocytes from low protein offspring were also resistant to the anti-lipolytic action of insulin. These results suggest that certain changes associated with early growth retardation are depot-specific, being enhanced in the more metabolically active intra-abdominal and epididymal tissues.

Dissection of the metabolic actions of insulin in adipocytes from early growth-retarded male rats.

Numerous studies have shown a relationship between early growth restriction and Type 2 diabetes. Studies have shown that offspring of rats fed a low protein (LP) diet during pregnancy and lactation have a worse glucose tolerance in late adult life compared with controls. In contrast, in young adult life LP offspring have a better glucose tolerance which is associated with increased insulin-stimulated glucose uptake into skeletal muscle. The aim of the present study was to compare the regulation of glucose uptake and lipolysis in adipocytes by insulin in control and LP offspring. LP adipocytes had increased basal and insulin-stimulated glucose uptake compared with controls. There was no difference in basal rates of lipolysis. Isoproterenol stimulated lipolysis in both groups, but it was more effective on LP adipocytes. Insulin reduced lipolytic rates in controls to basal levels but had a reduced effect in LP adipocytes. Protein kinase B activity matched glucose uptake, with LP adipocytes having elevated activities. These results suggest that early growth retardation has long-term effects on adipocyte metabolism. In addition, they show selective resistance to different metabolic actions of insulin and provide insight into the mechanisms by which insulin regulates glucose uptake and lipolysis.

Ketosis resistance in the male offspring of protein-malnourished rat dams

Plasma β-hydroxybutyrate concentrations were measured in the offspring of rats that were fed either a control (20% protein) diet or low-protein (8% protein) diet during pregnancy and lactation. Low-protein offspring had significantly lower plasma β-hydroxybutyrate compared with controls in the fed state ( P < .04) and after fasting for 24 hours ( P < .001) and 48 hours ( P < .04). There were no differences in blood glucose, acetoacetate, plasma glucagon, cholesterol, or glycerol between control and low-protein offspring. However, plasma nonesterified fatty acids (NEFAs) were significantly higher in low-protein offspring in the fed state ( P < .05). In contrast, plasma triglycerides and insulin were significantly lower in low-protein offspring compared with controls when fed ( P < .001) and after a 24-hour fast ( P < .001). These results suggest that poor maternal and early postnatal nutrition can have long-term effects on ketone body metabolism in the offspring during adulthood. This apparent ketosis resistance is similar to that observed in some forms of human diabetes.

Maternal low protein diet in rats programmes fatty acid desaturase activities in the offspring.

Numerous studies show an association between poor fetal growth and adult insulin resistance. Recent studies have shown relation between the long chain polyunsaturated fatty acid composition of skeletal muscle membranes and insulin sensitivity. More detailed analysis has indicated that the activity of delta5 desaturase is inversely correlated to insulin resistance. The amount of docosahexaenoic acid (C22:6n3) is also thought to play a part in determining insulin sensitivity. The purpose of this study was to test the hypothesis that early growth retardation in the rat, as a result of maternal protein restriction, would lead to alterations in desaturase activities similar to those observed in human insulin resistance. There were no differences in phospholipid fatty acid composition in liver or muscle from control and low protein rats. In both muscle and liver the ratio of docosahexaenoic acid to docosapentaenoic acid was, however, reduced in low protein offspring. Direct measurement of delta5 desaturase activity in hepatic microsomes showed a reduction (p < 0.03) in the low protein offspring which was negatively correlated (r = -0.855) with fasting plasma insulin. No correlation was observed in controls. These results show that it is possible to programme the activity of key enzymes involved in the desaturation of long chain polyunsaturated fatty acids. This is possibly a mechanism linking fetal growth retardation to insulin resistance.

Poor fetal nutrition causes long-term changes in expression of insulin signaling components in adipocytes.

Insulin action on adipocytes was studied in the offspring of mothers who had been fed either a control (20% protein) or a low (8%)-protein diet during pregnancy and lactation. Adipocytes isolated from low-protein offspring had significantly higher basal and insulin-stimulated glucose uptakes than controls. This may be related to a threefold increase in insulin receptors in low-protein adipocytes. Consistent with these observed changes in glucose transport, adipocytes from low-protein animals had significantly higher basal and insulin-stimulated insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activities. There was also more p85-associated PI 3-kinase activity in these adipocytes. There was no difference in expression in the p85 regulatory subunit or the p110-alpha catalytic subunit of PI 3-kinase. In contrast, there was a sixfold reduction in the p110-beta catalytic subunit of PI 3-kinase in adipocytes from low-protein animals. These results suggest that poor fetal nutrition during pregnancy and lactation can have long-term effects on glucose transport and on the expression of key components of the insulin signaling pathway in adipocytes.

Programming of hepatic insulin-sensitive enzymes in offspring of rat dams fed a protein-restricted diet.

Hepatic enzymes associated with glucose hemostasis were studied in offspring of dams fed either a 20% protein (control) or an isocaloric 8% protein (low-protein) diet during pregnancy and lactation. Additionally, offspring were exposed to maternal 8% protein diet only during gestation (recuperated) or lactation (postnatal low-protein). Glucokinase activity decreased (approximately 50%), whereas phosphoenolpyruvate carboxykinase (PEPCK) activity increased (approximately 100%), in the low-protein and recuperated offspring compared with controls (P < 0.001) at 21 days of age. However, the postnatal low-protein offspring had enzyme activities comparable with those of controls. These changes were still evident in 11-mo-old offspring weaned onto a normal laboratory chow. Parallel changes were apparent in mRNA levels of glucokinase and PEPCK in the low-protein male offspring. Thus the effect of programming metabolism extends not only to protein biochemistry but possibly also to the regulation of gene expression. Furthermore, these changes could not be attributed to glucagon or insulin, because ratios of these hormones were comparable between the control and low-protein groups.