Programming of intermediary metabolism

Molecular and Cellular Endocrinology 185 (2001) 81 – 91 www.elsevier.com/locate/mce

Programming of intermediary metabolism C.J. Petry, S.E. Ozanne, C.N. Hales * Department of Clinical Biochemistry, Addenbrooke’s Hospital, Uni6ersity of Cambridge, Hills Road, Cambridge CB2 2QR, UK

Abstract Studies of animal models were carried out to explore mechanisms that might underlie epidemiological findings linking indices of poor early (fetal and early postnatal) growth to an increased risk of developing poor glucose tolerance, including the metabolic syndrome, in adult life. Adult obesity was also seen to play an important role in adding to these risks. We proposed the ‘thrifty phenotype’ hypothesis to provide a conceptual and mechanistic framework that could be tested by experimentation in animal models. Our main approach has been to feed a reduced protein diet to pregnant and/or lactating rat dams as a means of reducing growth in the fetal and/or preweaning stages of pup growth. Animals were weaned onto either a normal diet or an obesity-inducing highly palatable, cafeteria-style diet. Alterations in intermediary metabolism were noted in the rats with early growth restriction, which provide support for our hypothesis and clues to the mechanism. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetes mellitus; Insulin resistance; Hypertension; Obesity

1. Introduction As early as 1934, Kermack et al. noted that in certain populations, after people survived the neonatal period, the year in which they died seemed to have some relationship with the year in which they were born (Kermack et al., 1934). They speculated that the reason for this association was due, in some way, to the environment that the individuals were exposed to in early life having a long-term influence on their well being. In particular, maternal factors were foreseen to have a major influence on the environment experienced in early life. Over 40 years later, Forsdahl (1977) observed that geographical variations in current death rates from arteriosclerotic heart disease in Norway showed a significant positive relationship with geographical variations in past infant mortality rates (but not with current infant mortality rates). Williams et al. (1979), in a similar study, then found that in England and Wales, geographical variations in death rates from ischaemic heart disease showed positive correlations with geographical variations in both current and past * Corresponding author. Tel.: + 44-1223-336787; fax: + 44-1223330598. E-mail address: [email protected] (C.N. Hales).

infant mortality rates. Subsequently, Barker and Osmond (1986) found that the geographical pattern of mortality from cardiovascular disease in England and Wales resembled the pattern of maternal and neonatal mortality earlier in the century. Further to this, they found that the distribution was more closely related to neonatal and maternal death rates in the past than to more recent postneonatal death rates (Barker and Osmond, 1987). An association was, therefore, established between poor maternal and neonatal health and subsequent disease many years later. Forsdahl (1977) thought that the link was the result of poverty during adolescence, but Barker and Osmond (1986) suggested that the association was the result of poor nutrition in fetal and early life. To clarify this, Barker et al. (1989b) studied a population for which birth weight data were available and found that there were increased death rates from ischaemic heart disease in men with low birth weights and weights at one year of age, favouring their initial hypothesis. Poor fetal nutrition is accompanied by organ-selective changes in nutrient distribution such that the growth of some organs (e.g. the brain) is relatively protected whereas that of others (e.g. the viscera) suffers more (Winick and Noble, 1966; Desai et al., 1996). A consequence of the latter could be impaired develop-

0303-7207/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 0 1 ) 0 0 6 2 7 - X

82

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

ment of the b-cells of the islets of Langerhans, which multiply rapidly during fetal life (Rahier et al., 1981). Poor b-cell development could predispose to diabetes in later life. It was decided, therefore, to investigate whether indices of fetal growth restriction were related to the subsequent risk of developing type 2 diabetes. In 64 year-old men in Hertfordshire, England, the proportion of men with either impaired glucose tolerance or type 2 diabetes fell with increasing birth weight such that those with the lowest birth weights were over six times more likely to have these conditions than those with the highest birth weights (Hales et al., 1991). When this population was restudied with reference to the metabolic syndrome (defined in terms of impaired glucose tolerance, hypertension and hypertriglyceridaemia), it was found that those with the lowest birth weights were 18 times as likely to have the metabolic syndrome as those with the highest birth weights (Barker et al., 1993). A slightly younger population of men and women from Preston, England, were almost 14 times as likely to have the metabolic syndrome if they were born in the lightest category in comparison to those born in the heaviest category (Barker et al., 1993).

2. The thrifty phenotype hypothesis Over 20 studies have now found an association between some index of restricted fetal growth and the future development of glucose intolerance or some marker of insulin resistance (reviewed by Petry and Hales, 1999). In 1992, Hales and Barker proposed the thrifty phenotype hypothesis to provide a conceptual and mechanistic framework for these associations, which could be tested by experimentation in animal models. Central to this hypothesis, are the ‘thrifty’ adaptations that a developing fetus makes to malnutrition. These include a redistribution of fetal blood flow towards the brain (to preserve growth) and away from other organs such as the liver, muscles and pancreas. Such adaptations were foreseen as being able to aid survival in the short term (slowing visceral growth and therefore reducing nutrient requirements, while relatively preserving essential brain growth). They would also aid survival in the long term (the postnatal animal becoming metabolically ‘thrifty’ to store metabolic fuel when nutrition was good and for non-neural tissue to preferentially utilise substrates other than glucose when nutrition was poor, thus preserving glucose for use by the brain). The fetus would therefore be metabolically ‘programmed’, a concept which has been defined as the process whereby a stimulus or insult when applied at a critical or sensitive period of development results in a long-term or permanent alteration in the structure or function of an organ or metabolic action (Lucas, 1991).

While the fetal adaptations to malnutrition were foreseen as aiding survival with poor or cyclic availability of food, the thrifty phenotype hypothesis proposed that when nutrition was adequate or even over-abundant, these adaptations become detrimental to health (Hales and Barker, 1992). Effects associated with fetal adaptations to malnutrition were anticipated to interact with effects associated with the ageing process, the development of obesity and sedentary lifestyles to cause type 2 diabetes and other factors related to the development of the metabolic syndrome. It is further complicated by the possibility that obesity itself may result from fetal malnutrition (Hales et al., 1991; Law et al., 1992), possibly reflecting metabolic ‘thriftiness’. Fetal malnutrition can have a number of different causes, but world-wide maternal malnutrition is probably the most common. The actual nutritional deficiencies that cause a developing fetus to become nutritionally ‘thrifty’ are not fully known. However, attention has been drawn to the potential role of protein deficiency, due to the importance of amino acids in fetal growth, pancreatic b-cell development and insulin secretion (Hales and Barker, 1992). In addition, protein is generally a relatively scarce and expensive foodstuff in communities with a high prevalence of diabetes and the metabolic syndrome (Hales et al., 1997). Most of the research to test the thrifty phenotype hypothesis has been performed in rats that were subjected to maternal protein restriction, during pregnancy alone, pregnancy and lactation or a combination of pregnancy, lactation and after weaning up to a period of about 70 days of age. This review will, therefore, focus on the progress that has been made using variations of the maternal low protein rat model to test the thrifty phenotype hypothesis and examine the programming of intermediary metabolism. Progress made using other models of fetal malnutrition is then briefly addressed, to reflect different nutritional deficiencies and other causes of fetal malnutrition.

3. Low protein rat model

3.1. Fetal, neonatal and ju6enile life Feeding a pregnant rat, a diet containing a little under half (8% (w/w)) the protein content but all the energy of a control diet (20% (w/w) protein) (see Snoeck et al., 1990 for details of the diets) led to a couple of days of adaptation followed by an increased food intake for the following ten days of pregnancy (all PB 0.01) (Fig. 1a). The body weights of the pregnant rats being fed the two diets were indistinguishable at this stage (Fig. 1b). From the 11th to 18th day of pregnancy, there were no differences in food intakes or body weights. By the 19th day of pregnancy, while food

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

intakes of the two groups were indistinguishable, the rats fed on the low protein diet were significantly lighter than those that fed on the control diet (P B 0.05). From the following day onwards until parturition, the low protein rats ate less (PB0.05) and weighed less than those fed on the control diet (P B 0.01). The offspring of the dams fed on the low protein diet were then born smaller such that two days after birth they were around 15–20% lighter (with similar litter sizes) than the control offspring (Desai et al., 1996). Differences in weights between the two groups of pups then increased if the nursing dam continued to be fed the low protein diet (Desai et al., 1996; Holness, 1996a). Cellular differences are detectable very early in gestation since preimplanted

83

embryos (i.e. up to 4.5 days of age) from dams fed on a low protein diet since conception were shown to have significantly reduced cell numbers, probably resulting from a slower rate of cellular proliferation (Kwong et al., 2000). Even after such a short span of time, differences in maternal hormone concentrations were apparent, with the low protein-fed dams having reduced plasma insulin and amino acid concentrations, and slightly elevated circulating glucose concentrations. The endocrine pancreas showed a number of differences from controls in fetuses from dams fed the low protein diet, including reduced b-cell proliferation, islet size and islet vascularisation (Snoeck et al., 1990). Their islets demonstrated a depressed insulin secretion in vitro in response to different secretagogues (Dahri et al., 1991) and a total failure to secrete insulin in vitro in response to taurine (an amino acid with a lower circulating concentration than those of controls in these fetuses (Cherif et al., 1996)). Their insulin secretion was restored by adding taurine to the mother’s drinking water (Cherif et al., 1998). In neonatal life, these offspring showed increased rates of pancreatic b-cell apoptosis with decreased proliferation and an increased G1 phase of the cell cycle (Petrik et al., 1999). Morphological studies showed that offspring of dams fed on the low protein diet throughout pregnancy and lactation had reduced numbers of pancreatic b-cells but increased a-cells (Berney et al., 1997). At three days of age, the low protein offspring showed a reduced cerebral cortex blood vessel density, analogous to previous observations in the islets of Langerhans (Bennis-Taleb et al., 1999). They also had lower DNA concentrations in the forebrain and higher DNA concentrations in the cerebellum. These results suggest that despite the relative preservation of brain weight, the brains of these animals are still in some way altered. At six weeks of age, i.e. three weeks after weaning, these animals had better glucose tolerance than controls and increased expression of insulin receptors in epididymal adipocytes (Shepherd et al., 1997). Such findings were evident if the exposure to maternal protein restriction was during gestation alone or during gestation and suckling periods.

3.2. Young adult life

Fig. 1. The (a) food intakes, and (b) body weights of pregnant rats fed on isocaloric diets containing 8 or 20% (w/w) protein (as in Snoeck et al., 1990). n= 20 for control animals (symbolised by ‘X’) and n= 23 for low protein animals (symbolised by ‘ ’). Data are the mean values (standard error of the mean).

Most functional studies in low protein offspring have been performed in young adult life. At this age, their glucose tolerance has been reported to be either better than (Hales et al., 1996; Holness, 1996b; Petry et al., 2000b) or equivalent to (Holness, 1996a; Wilson and Hughes, 1997) that of controls. Circulating insulin concentrations were lower at this stage (Hales et al., 1996; Ozanne et al., 1998b; Petry et al., 2000b). Normal or lower plasma insulin concentrations plus the increased glucose infusion rates required to maintain euglycaemia

84

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

in hyperinsulinaemic clamps suggest an increased whole body insulin sensitivity (Holness, 1996a) making the circulating insulin concentrations entirely appropriate. Consistent with an increased insulin sensitivity, these animals also had lower circulating triglyceride concentrations (Lucas et al., 1996; Ozanne et al., 1998b). In addition, they had reduced circulating b-hydroxybutyrate concentrations, despite higher non-esterified fatty acid concentrations, which could reflect either increased utilisation (perhaps to preserve blood glucose concentrations in a ‘thrifty’ metabolism) or decreased production (Ozanne et al., 1998b). Both plasma adrenaline and noradrenaline concentrations were found to be raised in low protein offspring in the fed-state (Petry et al., 2000a). These concentrations did not rise after one day of starvation, however, unlike with controls in which the concentrations rose to the levels observed in the low protein offspring. These findings suggest that even in the fed-state, the metabolism of the low protein offspring operates in a quasi-starved fashion as would be predicted in the thrifty phenotype hypothesis (Hales and Barker, 1992). Variations in the low protein diet (such as differences in protein content and source and differences in other constituents and length of exposure) fed to the pregnant dams have important influences on the blood pressure of the offspring. Thus, while one diet (containing 9% (w/w) protein but supplemented with methionine) has consistently produced hypertension in the offspring (Langley and Jackson, 1994; Langley-Evans, 2000), another diet (containing 8% (w/w) protein without methionine supplementation) caused either no change or a slight lowering of blood pressure in the offspring (Lucas et al., 1996; Langley-Evans, 2000; Petry et al., 2000a). Where changes in blood pressure have been detected, their validity has been questioned due to the indirect nature of the tail-cuff procedure, but radiotelemetry has also been used to show mild hypertension in the offspring of rats fed on a low protein diet supplemented with methionine (Tonkiss et al., 1998). The smaller increase in blood pressure found using radiotelemetry was attributed to an augmented stressrelated increase in blood pressure in low protein offspring when restrained for the tail-cuff procedure. Where hypertension has been observed, its mechanism has been suggested to include impaired nephrogenesis and a hyperactive renin– angiotensin system (LangleyEvans et al., 1999; Woods, 2000) associated with an early increased exposure to glucocorticoids (LangleyEvans et al., 1996). The livers of low protein offspring have been observed to have larger hepatic lobules than those of controls (Burns et al., 1997). The consequences of this anatomical difference are at present unknown, but associated functional differences in the livers from these animals are numerous. Ex vivo perfusion studies have

shown a resistance of the liver to the effects of glucagon to stimulate glucose output, associated with a five-fold reduction in hepatic glucagon receptor expression (Ozanne et al., 1996a). Conversely, there was an increased expression of hepatic insulin receptors. This was associated with an increased internalisation of insulin into the hepatocyte endosomes and release of insulin degradation products. There was a biphasic response of hepatic glucose output to insulin with respect to time, with an initial stimulation followed by the expected inhibition. Over all, perfusion in the presence of physiological glucose concentrations led to a reduced hepatic glucose output (Ozanne et al., 1996a) and a reduced endogenous glucose production during euglycaemic clamps (Holness, 1996a) in the low protein offspring. These findings are consistent with the lower fasting blood glucose concentrations found in young adult life (Hales et al., 1996). Other observed changes in hepatic function in low protein offspring include a reduction in the expression of alpha-, beta- and gamma-fibrinogen genes (Zhang et al., 1997), fibrinogen protein, glucocorticoid receptor mRNA and glucocorticoid receptor binding affinity that is restricted to the left lobe of the liver (Zhang and Byrne, 2000). As in the liver, in tibialis anterior muscles low protein offspring had increased insulin receptor expression (Ozanne et al., 1996b). This was associated with increased insulin-stimulated glucose uptake and a greater fractional presence of GLUT4 protein in the plasma membranes (with no detectable difference in total GLUT4 protein). Such alterations are likely to have made a significant contribution to the improvement in glucose tolerance observed in young adult life (Hales et al., 1996). In vivo glucose utilisation (defined in terms of glucose uptake and phosphorylation) was enhanced when stimulated with euglycaemic hyperinsulinaemia in oxidative skeletal muscles of low protein offspring, despite being lower in the basal state (Holness, 1996a). In tibialis anterior muscles from low protein offspring there was a reduced ratio of docosahexanoic acid to docosapentaenic acid (Ozanne et al., 1998a), a factor that could potentially lead to a reduction in insulin sensitivity (Pan et al., 1995). In mesenteric adipocytes from low protein offspring, euglycaemic hyperinsulinaemia has been shown to cause an enhanced glucose utilisation (Holness, 1996a). Consistent with this, insulin receptor expression was increased in epididymal (Ozanne et al., 1997) (and intra-abdominal but not sub-cutaneous (Ozanne et al., 2000)) adipocytes from low protein offspring, as had been observed at a younger age (Shepherd et al., 1997). Basal glucose uptakes were higher in adipocytes from low protein offspring (Ozanne et al., 1997, 1999), from epididymal, intra-abdominal and sub-cutaneous fat depots (Ozanne et al., 2000). While insulin stimulated glucose uptakes in adipocytes from both low protein

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

offspring and controls, the magnitude of the stimulation in the presence of an elevated basal glucose uptake was smaller in low protein adipocytes. GLUT4 protein expression was unaltered in low protein offspring in adipocytes from the three different fat depots (Ozanne et al., 2000). In vivo studies showed that adipocytes isolated from pregnant low protein offspring exhibited enhanced glucose utilisation in response to hyperinsulinaemia, despite a lower basal utilisation (Holness et al., 1999). In vitro studies using adipocytes isolated from these animals showed increased glucose uptakes at higher insulin concentrations, but a reduced response to insulin at low physiological concentrations. It has been suggested that if at any time such animals became insulin deficient, hyperglycaemia may worsen by insulin resistance as well as by the initiating insulin deficiency (Holness, 1996a). Despite having increased insulin receptor expression, lipolysis studies have revealed a degree of selective insulin resistance in adipocytes from low protein offspring (Ozanne et al., 1999, 2000). This occurs at a time when these animals appear to exhibit increased whole body insulin sensitivity relative to controls. Basal lipolytic rates were the same in adipocytes from low protein offspring and controls using epididymal (Ozanne et al., 1999), intra-abdominal and sub-cutaneous adipocytes (Ozanne et al., 2000). The magnitude of stimulation by isoproterenol (and noradrenaline (Holness and Sugden, 1999)), however, was larger in adipocytes from low protein offspring (Ozanne et al., 1999), irrespective of the fat depot from which the adipocytes were isolated (Ozanne et al., 2000). Consistent with this fact, isolated adipocytes from these animals were shown to have increased beta-adrenergic receptor expression (Petry et al., 2000a). The magnitude of the inhibition of the isoproterenol-stimulated lipolysis by insulin was smaller in epididymal and intra-abdominal adipocytes from low protein offspring, suggesting a selective resistance to the metabolic actions of insulin (Ozanne et al., 1999, 2000). The exact mechanism of this resistance to the antilipolytic action of insulin in adipocytes from low protein offspring is unknown, but alterations in expression and activities of various components of the insulin signalling-pathway may provide clues. Adipocytes isolated from low protein offspring had significantly higher insulin receptor substrate-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activities, both in the basal state and after insulin-stimulation (Ozanne et al., 1997). PI 3-kinase activation has been shown to be necessary for the mediation of numerous metabolic actions of insulin, including the inhibition of lipolysis (Shepherd et al., 1998). Adipocytes from low protein offspring also exhibited higher p85-associated PI 3-kinase activities. While there were no differences in expressions of either p110a or p85 PI 3-kinase sub-units,

85

there was a six-fold reduction in the expression of the p110b sub-unit (Ozanne et al., 1997). This may suggest that the p110b isoform of the catalytic sub-unit of PI 3-kinase is required to mediate the anti-lipolytic action of insulin. Downstream of PI 3-kinase in the insulin signalling-cascade, protein kinase B activities were found to be higher in adipocytes from low protein offspring in both the basal state and when stimulated with insulin (Ozanne et al., 1999). These findings may, therefore, help us to understand how insulin signallingpathways diverge to facilitate the different metabolic actions that insulin can trigger (Ozanne et al., 1999). Other organs of low protein offspring in young adult life have been less well characterised. Female rats exposed to maternal and chronic protein restriction were shown to have a decreased pancreatic and specifically islet blood flow, with the flow increasing during a glucose challenge (Iglesias-Barreira et al., 1996). Consistent with changes in pancreatic islet cells noted at weaning (Berney et al., 1997), pancreatic insulin and amylin contents were reduced in such animals, whereas glucagon contents were raised (Petry et al., 2000b). They also exhibited reduced circulation in the glycerol phosphate shunt in islets (with low activity of mitochondrial glycerophosphate dehydrogenase (Rasschaert et al., 1995)), combined with a reduced transamination of L-leucine to 2-ketoisocaproate (Sener et al., 1996). The reduced islet vascularisation observed in fetal and neonatal life in low protein offspring was normalised by the consumption of a control diet after weaning (Bennis-Taleb et al., 1999). In the cerebral cortex of the brain, however, reduced vascularisation was still evident in adult life even after consumption of the control diet (Bennis-Taleb et al., 1999). In the heart, low protein offspring exhibited reduced glucose utilisation, suggesting that the heart was preferentially using alternative fuels to glucose (Holness et al., 1998). However, there was no apparent alteration in cardiac palmatoyltransferase activity or sensitivity to malonyl CoA inhibition (Holness et al., 1998).

3.3. Middle age and beyond Despite the low protein rat offspring being used to model and test a hypothesis that deals with the pathogenesis of diseases that become more prevalent in middle age and beyond, relatively few studies have been performed to investigate intermediary metabolism in such animals beyond young adult life. Longevity studies in male rats have revealed that exposure to maternal protein restriction in utero followed by suckling from a dam fed on a control diet leads to initial growth restriction, rapid catch-up growth and ultimately a reduced lifespan (Hales et al., 1996) associated with a rapid shortening of renal telomeres (Jennings et al., 1999). These premature deaths, however, appear not to

86

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

be diabetes-related (Hales et al., 1996) and may be more linked to the tendency that male rats have to die from renal disease (Iwasaki et al., 1988). Most of the studies that have been performed in older low protein offspring relate to their glucose tolerance. After a period in young adult animals, where any alteration in glucose tolerance from that of controls is an improvement (Hales et al., 1996; Holness, 1996b; Petry et al., 2000b), in middle age there appears to be no difference in the glucose tolerance of low protein offspring and controls (Langley et al., 1994; Hales et al., 1996; Petry et al., 1997b). By 15 to 16 months of age, however, there was a more rapid deterioration in the glucose tolerance of low protein offspring (Hales et al., 1996; Petry and Hales, 1999), such that they had significantly higher blood glucose concentrations at certain time points in an intra-peritoneal glucose tolerance test. These higher glucose concentrations were associated with relative hyperinsulinaemia in male low protein offspring (suggesting a degree of whole body insulin resistance) and, if anything, relative hypoinsulinaemia in female low protein offspring (suggesting a degree of insulin deficiency). Until recently, frank diabetes had never been observed in low protein offspring. Very recent experiments in male low protein offspring which were around 18 months of age (i.e. older than the average longevity in such animals (Hales et al., 1996)), however, found that their fasting plasma glucose concentrations were higher than that defined by the World Health Organisation (Alberti and Zimmet, 1998) as being diagnostic for diabetes in humans (Petry et al., manuscript in preparation). In intra-venous glucose tolerance tests, the differences in both glucose and insulin concentrations were larger than had previously been observed using intraperitoneal glucose tolerance tests in 15 month-old animals.

3.4. Additi6e effects of obesity and dietary-induced insulin resistance The thrifty phenotype hypothesis suggests that the increased risk of disease associated with adaptations to fetal malnutrition are added to by effects in adult life associated with the development of obesity, ageing and sedentary lifestyles (Hales and Barker, 1992). This aspect of the hypothesis has been tested in the low protein rat model using a variety of different dietary-induced interventions. Sucrose-feeding low protein offspring in adult life led to an impaired glucose-stimulated insulin secretion (as well as an impaired response to keto-isocaproate and tolbutamide (Wilson and Hughes, 1998)) from isolated islets, which was associated with a worsening of glucose tolerance that was similar in magnitude to that observed in sucrose-fed controls (Wilson and Hughes, 1997). Feeding a high fat diet to low

protein offspring caused a similar impairment in insulin secretion, but in contrast to sucrose-feeding, caused a worsening of glucose tolerance that was not observed in controls. In another study, four weeks of feeding a high fat diet to low protein offspring failed to produce the enhanced glucose-stimulated insulin secretion in intravenous glucose tolerance tests that was observed in controls (Holness, 1996b). However, the high fat-fed low protein offspring failed to show any difference in glucose disposal in the glucose tolerance tests, or in glucose production or whole body glucose disposal during euglycaemic hyperinsulinaemic clamps. In contrast, after eight weeks of high fat-feeding, low protein offspring showed impaired glucose tolerance associated with reduced insulin-stimulated glucose uptakes and an impaired effect of insulin to inhibit lipolysis in isolated adipocytes (Holness and Sugden, 1999). Obesity has been induced in female low protein offspring by feeding them a highly palatable, cafeteriastyle diet (Fig. 2; Petry et al., 1997a), as other dietary-induced changes in insulin sensitivity were not associated with large increases in body weight (Holness, 1996b; Wilson and Hughes, 1997; Holness and Sugden, 1999). After maternal and postweaning protein restriction until 70 days of age, ten weeks of cafeteria-feeding resulted in obesity and a significant worsening of glucose tolerance (with associated increases in fasting plasma insulin and pancreatic insulin contents). This meant that by 20 weeks of age the glucose tolerances of the low protein-cafeteria rats were not detectably different from those of cafeteria-fed control animals (Petry et al., 2000b). When cafeteria-feeding was continued until one year of age, both the early protein restriction and the cafeteria-feeding were associated with rises in systolic blood pressures (Petry et al., 1997b). These rises were independent of each other and additive, so that the highest systolic blood pressures were found in cafeteria-fed animals who were protein-restricted in early life and the lowest pressures were found in chow-fed controls. Such changes are reminiscent of effects associated with low birth weight and current obesity observed in humans (Barker et al., 1989a). At one year of age, the pattern of glucose tolerances was similar to that observed earlier in life (Petry et al., 2000b). This meant that in comparison to chow-fed controls, the cafeteriafed low protein offspring had hypertension, impaired glucose tolerance and hypertriglyceridaemia (Petry et al., 1997b). Such changes are obviously similar to those observed in humans with the metabolic syndrome, but with the exception of blood pressure, tended to be more associated with the cafeteria-feeding than with the early protein restriction. At 16 months of age, current cafeteria-feeding still had a greater associated effect on glucose tolerance than early protein restriction. However, there was a relative worsening of glucose tolerance in the low protein offspring compared with controls be-

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

87

Fig. 2. The obesity-inducing effect of eating the highly palatable, cafeteria-style diet. These two litter-mate female rats were born to a mother fed on the control (20% (w/w) protein) diet, and were weaned onto this diet until 70 days of age. From this time onwards, the sister on the right was fed the highly palatable diet, while her lighter sister was fed standard chow. The rats were nine months old when the photograph was taken (see Petry et al., 1997a,b).

tween 12 and 16 months of age (Petry and Hales, 1999). There have been no reported studies investigating these animals at ages similar to those where diabetes was observed with maternal protein restriction alone.

4. Other rat models While maternal protein restriction has produced many of the changes pertaining to the thrifty phenotype hypothesis in rat offspring, it is not the only factor, nutritional or otherwise, that causes fetal malnutrition and subsequent disease. Other potential factors have generally not been as widely investigated and it is not known yet whether adaptations to other factors act through similar mechanisms to those observed with adaptations to maternal protein restriction. The one exception to this is increased fetal exposure to glucocorticoids, which has been reported to be evident in fetuses of protein-restricted mothers (Langley-Evans et al., 1996) and has been shown through pharmacological interventions to lead to reduced birth weight, hypertension (Lindsay et al., 1996a) and impaired glucose tolerance (Lindsay et al., 1996b) in the offspring (reviewed by Seckl et al., 1999). Other nutritional factors that have been investigated to assess effects on the offspring include feeding the pregnant rat an iron-deficient diet. This led to reduced birth weights, altered cardiovascular development and increased blood pressure in the offspring by 40 days of age (Crowe et al., 1995). More widely studied, have

been the effects of a generalised malnutrition of a pregnant rat on its offspring. Eight week-old offspring of rats who were fed 50% of the food intakes of matched controls during the last trimester of pregnancy showed no deficit in glucose tolerance, glucose utilisation or glucose production (Bertin et al., 1999). For comparison, offspring of rats fed on an isocaloric low protein diet during the same period showed similar results, but in contrast also exhibited reduced pancreatic insulin contents and pancreatic b-cell masses. Previous to this, it had been demonstrated that underfeeding the pregnant rats during the first two weeks of pregnancy did not alter either insulin secretion or action in the young offspring (Portha et al., 1995). Increasing the period of exposure to maternal malnutrition from day 15 of pregnancy to the end of weaning was shown to be associated with impaired pancreatic b-cell development but not proliferation (Garofano et al., 1998) and at one year of age impaired glucose tolerance and insulinopenia (Garofano et al., 1999). In female offspring subjected to this regime, at eight months of age there was a failure to mount the usual changes in the endocrine pancreas to pregnancy (Blondeau et al., 1999). A more severe maternal food restriction, to 30% of that of controls, has been shown to lead to prolonged hypertension (Woodall et al., 1996) and hyperphagia, raised fasting insulin and leptin concentrations and obesity in adult offspring (Vickers et al., 2000). Experimental fetal malnutrition has also been induced in rats by ligating one or both maternal uterine arteries during late pregnancy. Such manipulations

88

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

caused a marked reduction in fetal body weights and a long-term increase in brain GLUT1 expression (Sadiq et al., 1999). This may reflect the shift of metabolism proposed in the thrifty phenotype hypothesis to preserve brain growth and metabolism at the potential expense of that of other organs (Hales and Barker, 1992). Uterine artery ligations also caused neonatal changes in the hepatic redox potential (Lane et al., 1996) as well as alterations in fetal and juvenile skeletal muscle mitochondrial gene expression that altered the mitochondrial redox potential (Lane et al., 1998). Three–four month-old female offspring from such manipulations have been shown to have an increased sympathetic nervous system activity, increased fasting blood glucose concentrations and higher glucose concentrations (with lower insulin) in response to a glucose load (Jansson and Lambert, 1999).

5. Concluding remarks The thrifty phenotype hypothesis (Hales and Barker, 1992) has proved a useful conceptual and mechanistic framework to test potential mechanisms that link indices of restricted fetal (and possibly early-postnatal) growth with subsequent disease. Variations of the maternal protein restriction model in rats have now successfully modelled the major components of the thrifty phenotype hypothesis including restricted fetal growth and the subsequent development of diabetes and hypertension (along with hypertriglyceridaemia when combined with dietary-induced obesity). In addition, factors in adult life, such as the development of obesity, have been shown to be able to interact with effects associated with the early growth restriction to increase risk. These findings, therefore, add to studies of human twins, which have shown that the twin who was most growthrestricted at birth has a higher risk of diabetes (Poulsen et al., 1997; Bo et al., 2000) and hypertension (Levine et al., 1994; Dwyer et al., 1999; Poulter et al., 1999). All these observations would suggest that the link between restricted fetal growth and subsequent disease could occur independently of a major genetic influence. This is important since such non-genetic mechanisms may be more amenable to treatment prior to the onset of disease in the future. The fact that many of the findings in humans in this area have now been modelled in rats with maternal protein restriction suggests that attention should be paid to observations in this model that have not been tested so far in humans. Of particular importance in this area, may be the observed alterations in the insulin signalling-pathways and the increased insulin receptor expression found in organs important for the maintenance of glucose tolerance in young adult life. In addition to providing details of molecular mechanisms, such

findings may in future provide markers that may be able to be used to predict those individuals at high risk of disease and may even provide targets for pharmacological intervention. Rat models of intra-uterine growth restriction, other than that using maternal protein restriction, have provided similar and sometimes additional information to that provided by the low protein rat model. It is unlikely that in humans you would find pregnant women with a protein deficiency in isolation; and in the other rat models, it is not currently known if these effects operate through similar or additional mechanisms to those found with maternal protein restriction. Certainly, nutritional deficiency models are important since potentially they are the simplest to rectify in humans. Models of maternal intra-uterine artery ligation, however, remind us that in the Western World placental insufficiency may play a more important role than maternal malnutrition in causing fetal malnutrition. As such pregnancies are not readily amenable to treatment, further mechanistic studies in this whole area are needed so that treatment can be targeted to individuals at an early stage to reduce the heavy burden of morbidity and mortality of type 2 diabetes and associated conditions (Turner, 1998).

Acknowledgements The cited studies from the research group of C.N.H. were funded by the Medical Research Council, the Parthenon Trust, the Wellcome Trust and Diabetes UK. The authors would like to thank M. Dorling, A. Flack, D. Hutt, L. James, N. Martensz, L. Smith, C.L. Wang and A. Wayman for their invaluable technical assistance in these studies.

References Alberti, K.G.M.M., Zimmet, P.Z., for the WHO Consultation, 1998. Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO Consultation. Diabet. Med. 15, 539 – 553. Barker, D.J.P., Hales, C.N., Fall, C.H., Osmond, C., Phipps, K., Clark, P.M., 1993. Type 2 (non-insulin-dependent) diabetes mellitus, hypertension and hyperlipidaemia (syndrome x): relation to reduced fetal growth. Diabetologia 36, 62 – 67. Barker, D.J., Osmond, C., 1986. Infant mortality, childhood nutrition, and ischaemic heart disease in England and Wales. Lancet i, 1077 – 1081. Barker, D.J., Osmond, C., 1987. Death rates from stroke in England and Wales predicted from past maternal mortality. Br. Med. J. 295, 83 – 86. Barker, D.J.P., Osmond, C., Golding, J., Kuh, D., Wadsworth, M.E., 1989a. Growth in utero, blood pressure in childhood and adult life, and mortality from cardiovascular disease. Br. Med. J. 298, 564 – 567.

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91 Barker, D.J., Winter, P.D., Osmond, C., Margetts, B., Simmonds, S.J., 1989b. Weight in infancy and death from ischaemic heart disease. Lancet ii, 577 –580. Bennis-Taleb, N., Remacle, C., Hoet, J.J., Reusens, B., 1999. A low-protein isocaloric diet during gestation affects brain development and alters permanently cerebral cortex blood vessels in rat offspring. J. Nutr. 129, 1613 –1619. Berney, D.M., Desai, M., Palmer, D.J., Greenwald, S., Brown, A., Hales, C.N., Berry, C.L., 1997. The effects of maternal protein deprivation on the fetal rat pancreas: major structural changes and their recuperation. J. Pathol. 183, 109 –115. Bertin, E., Gangnerau, M.N., Bailbe, D., Portha, B., 1999. Glucose metabolism and beta-cell mass in adult offspring of rats protein and/or energy restricted during the last week of pregnancy. Am. J. Physiol. 277, E11 – E17. Blondeau, B., Garofano, A., Czernichow, P., Breant, B., 1999. Age-dependent inability of the endocrine pancreas to adapt to pregnancy: a long-term consequence of perinatal malnutrition in the rat. Endocrinology 140, 4208 –4213. Bo, S., Cavallo-Perin, P., Scaglione, L., Ciccone, G., Pagano, G., 2000. Low birthweight and metabolic abnormalities in twins with increased susceptibility to Type 2 diabetes mellitus. Diabet. Med. 17, 365 – 370. Burns, S.P., Desai, M., Cohen, R.D., Hales, C.N., Iles, R.A., Germain, J.P., Going, T.C., Bailey, R.A., 1997. Gluconeogenesis, glucose handling, and structural changes in livers of the adult offspring of rats partially deprived of protein during pregnancy and lactation. J. Clin. Invest. 100, 1768 – 1774. Cherif, H., Reusens, B., Ahn, M.T., Hoet, J.J., Remacle, C., 1998. Effects of taurine on the insulin secretion of rat fetal islets from dams fed a low-protein diet. J. Endocrinol. 159, 341 –348. Cherif, H., Reusens, B., Dahri, S., Hoet, J.J., Remacle, C., 1996. Stimulatory effects of taurine on insulin secretion by fetal rat islets cultured in vitro. J. Endocrinol. 151, 501 –506. Crowe, C., Dandekar, P., Fox, M., Dhingra, K., Bennet, L., Hanson, M.A., 1995. The effects of anaemia on heart, placenta and body weight, and blood pressure in fetal and neonatal rats. J. Physiol. 488, 515 – 519. Dahri, S., Snoeck, A., Reusens-Billen, B., Remacle, C., Hoet, J.J., 1991. Islet function in offspring of mothers on low-protein diet during gestation. Diabetes 40 (Suppl. 2), 115 –120. Desai, M., Crowther, N.J., Lucas, A., Hales, C.N., 1996. Organ-selective growth in the offspring of protein-restricted mothers. Br. J. Nutr. 76, 591 – 603. Dwyer, T., Blizzard, L., Morley, R., Ponsonby, A.L., 1999. Within pair association between birth weight and blood pressure at age 8 in twins from a cohort study. Br. Med. J. 319, 1325 –1329. Forsdahl, A., 1977. Are poor living conditions in childhood and adolescence an important risk factor for arteriosclerotic heart disease? Br. J. Prev. Soc. Med. 31, 91 –95. Garofano, A., Czernichow, P., Breant, B., 1998. Beta-cell mass and proliferation following late fetal and early postnatal malnutrition in the rat. Diabetologia 41, 1114 –1120. Garofano, A., Czernichow, P., Breant, B., 1999. Effect of ageing on beta-cell mass and function in rats malnourished during the perinatal period. Diabetologia 42, 711 –718. Hales, C.N., Barker, D.J.P., 1992. Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty phenotype hypothesis. Diabetologia 35, 595 – 601. Hales, C.N., Barker, D.J.P., Clark, P.M.S., Cox, L.J., Fall, C., Osmond, C., Winter, P.D., 1991. Fetal and infant growth and impaired glucose tolerance at age 64. Br. Med. J. 303, 1019 – 1022. Hales, C.N., Desai, M., Ozanne, S.E., 1997. The thrifty phenotype hypothesis: how does it look after 5 years? Diabet. Med. 14, 189 – 195. Hales, C.N., Desai, M., Ozanne, S.E., Crowther, N.J., 1996. Fish-

89

ing in the stream of diabetes: from measuring insulin to the control of fetal organogenesis. Biochem. Soc. Trans. 24, 341 – 350. Holness, M.J., 1996a. Impact of early growth retardation on glucoregulatory control and insulin action in mature rats. Am. J. Physiol. 270, E946 – E954. Holness, M.J., 1996b. The influence of sub-optimal protein nutrition on insulin hypersecretion evoked by high-energy/high-fat feeding in rats. FEBS Lett. 396, 53 – 56. Holness, M.J., Priestman, D.A., Sugden, M.C., 1998. Impact of protein restriction on the regulation of cardiac carnitine palmitoyltransferase by malonyl-CoA. J. Mol. Cell. Cardiol. 30, 1381 – 1390. Holness, M.J., Fryer, L.G., Sugden, M.C., 1999. Protein restriction during early development enhances insulin responsiveness but selectively impairs sensitivity to insulin at low concentrations in white adipose tissue during a later pregnancy. Br. J. Nutr. 81, 481 – 489. Holness, M.J., Sugden, M.C., 1999. Antecedent protein restriction exacerbates development of impaired insulin action after highfat feeding. Am. J. Physiol. 276, E85 – E93. Iglesias-Barreira, V., Ahn, M.T., Reusens, B., Dahri, S., Hoet, J.J., Remacle, C., 1996. Pre- and post-natal low protein diet affect pancreatic islet blood flow and insulin release in adult rats. Endocrinology 137, 3797 – 3801. Iwasaki, K., Gleiser, C.A., Masoro, E.J., McMahan, C.A., Seo, E.J., Yu, B.P., 1988. The influence of dietary protein source on longevity and age-related disease processes of Fischer rats. J. Gerontol. 43, B5 – B12. Jansson, T., Lambert, G.W., 1999. Effect of intrauterine growth restriction on blood pressure, glucose tolerance and sympathetic nervous system activity in the rat at 3 – 4 months of age. J. Hypertens. 17, 1239 – 1248. Jennings, B.J., Ozanne, S.E., Dorling, M.W., Hales, C.N., 1999. Early growth determines longevity in male rats and may be related to telomere shortening in the kidney. FEBS Lett. 448, 4 – 8. Kermack, W.O., McKendrick, A.G., McKinlay, P.L., 1934. Deathrates in Great Britain and Sweden. Lancet i, 698 – 703. Kwong, W.Y., Wild, A.E., Roberts, P., Willis, A.C., Fleming, T.P., 2000. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 127, 4195 – 4202. Lane, R.H., Chandorkar, A.K., Flozak, A.S., Simmons, R.A., 1998. Intrauterine growth retardation alters mitochondrial gene expression and function in fetal and juvenile rat skeletal muscle. Pediatr. Res. 43, 563 – 570. Lane, R.H., Flozak, A.S., Ogata, E.S., Bell, G.I., Simmons, R.A., 1996. Altered hepatic gene expression of enzymes involved in energy metabolism in the growth-retarded fetal rat. Pediatr. Res. 39, 390 – 394. Langley, S.C., Browne, R.F., Jackson, A.A., 1994. Altered glucose tolerance in rats exposed to maternal low protein diets in utero. Comp. Biochem. Physiol. 109A, 223 – 229. Langley, S.C., Jackson, A.A., 1994. Increased systolic blood pressure in adult rats induced by fetal exposure to low protein diets. Clin. Sci. 86, 217 – 222. Langley-Evans, S.C., 2000. Critical differences between two low protein diet protocols in the programming of hypertension in the rat. Int. J. Food Sci. Nutr. 51, 11 –17. Langley-Evans, S.C., Philips, G., Benediktsson, R., Gardner, D.S., Edwards, C.R., Jackson, A.A., Seckl, J.R., 1996. Maternal dietary protein restriction, placental glucocorticoid metabolism and the programming of hypertension. Placenta 17, 169 –172.

90

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91

Langley-Evans, S.C., Sherman, R.C., Welham, S.J., Nwagwu, M.O., Gardner, D.S., Jackson, A.A., 1999. Intrauterine programming of hypertension: the role of the rennin –angiotensin system. Biochem. Soc. Trans. 27, 88 –93. Law, C.M., Barker, D.J., Osmond, C., Fall, C.H., Simmonds, S.J., 1992. Early growth and abdominal fatness in adult life. J. Epidemiol. Community Health 46, 184 –186. Levine, R.S., Hennekens, C.H., Jesse, M.J., 1994. Blood pressure in prospective population based cohort of newborn and infant twins. Br. Med. J. 308, 298 –302. Lindsay, R.S., Lindsay, R.M., Edwards, C.R., Seckl, J.R., 1996a. Inhibition of 11-beta-hydroxysteroid dehydrogenase in pregnant rats and the programming of blood pressure in the offspring. Hypertension 27, 1200 –1204. Lindsay, R.S., Lindsay, R.M., Waddell, B.J., Seckl, J.R., 1996b. Prenatal glucocorticoid exposure leads to offspring hyperglycaemia in the rat: studies with the 11 beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone. Diabetologia 39, 1299 – 1305. Lucas, A., 1991. Programming by early nutrition in man. In: Bock, G.R., Whelan, J. (Eds.), The childhood environment and adult disease (CIBA Foundation Symposium 156). Wiley, Chichester, UK, pp. 38– 55. Lucas, A., Baker, B.A., Desai, M., Hales, C.N., 1996. Nutrition in pregnant rats programs lipid metabolism in the offspring. Br. J. Nutr. 76, 605 – 612. Ozanne, S.E., Dorling, M.W., Wang, C.L., Petry, C.J., 2000. Depot-specific effects of early growth retardation on adipocyte insulin action. Horm. Metab. Res. 32, 71 – 75. Ozanne, S.E., Martensz, N.D., Petry, C.J., Loizou, C.L., Hales, C.N., 1998a. Maternal low protein diet in rats programmes fatty acid desaturase activities in the offspring. Diabetologia 41, 1337 – 1342. Ozanne, S.E., Nave, B.T., Wang, C.L., Shepherd, P.R., Prins, J., Smith, G.D., 1997. Poor fetal nutrition causes long-term changes in expression of insulin signaling components in adipocytes. Am. J. Physiol. 273, E46 –E51. Ozanne, S.E., Smith, G.D., Tikerpae, J., Hales, C.N., 1996a. Altered regulation of hepatic glucose output in the male offspring of protein-malnourished rat dams. Am. J. Physiol. 270, E559 – E564. Ozanne, S.E., Wang, C.L., Coleman, N., Smith, G.D., 1996b. Altered muscle insulin sensitivity in the male offspring of proteinmalnourished rats. Am. J. Physiol. 271, E1128 –E1134. Ozanne, S.E., Wang, C.L., Dorling, M.W., Petry, C.J., 1999. Dissection of the metabolic actions of insulin in adipocytes from early growth-retarded male rats. J. Endocrinol. 162, 313 – 319. Ozanne, S.E., Wang, C.L., Petry, C.J., Smith, J.M., Hales, C.N., 1998b. Ketosis resistance in the male offspring of protein-malnourished rat dams. Metabolism 47, 1450 –1454. Pan, D.A., Lillioja, S., Milner, M.R., Kriketos, A.D., Baur, L.A., Bogardus, C., Storlien, L.H., 1995. Skeletal muscle membrane lipid composition is related to adiposity and insulin action. J. Clin. Invest. 96, 2802 –2808. Petrik, J., Reusens, B., Arany, E., Remacle, C., Coelho, C., Hoet, J.J., Hill, D.J., 1999. A low protein diet alters the balance of islet cell replication and apoptosis in the fetal and neonatal rat and is associated with a reduced pancreatic expression of insulin-like growth factor-II. Endocrinology 140, 4861 – 4873. Petry, C.J., Desai, M., Ozanne, S.E., Hales, C.N., 1997a. Early and late nutritional windows for diabetes susceptibility. Proc. Nutr. Soc. 56, 233 – 242. Petry, C.J., Dorling, M.W., Wang, C.L., Pawlak, D.B., Ozanne, S.E., 2000a. Catecholamine levels and receptor expression in low protein rat offspring. Diabet. Med., 17, 848 – 853. Petry, C.J., Hales, C.N., 1999. Intrauterine development and its relationship with type 2 diabetes mellitus. In: Hitman, G.A. (Ed.), Type 2 Diabetes: Prediction and Prevention. Wiley,

Chichester, UK, pp. 153 – 168. Petry, C.J., Ozanne, S.E., Wang, C.L., Hales, C.N., 1997b. Early protein restriction and obesity independently induce hypertension in 1-year-old rats. Clin. Sci. 93, 147 – 152. Petry, C.J., Ozanne, S.E., Wang, C.L., Hales, C.N., 2000b. Effects of early protein restriction and adult obesity on rat pancreatic hormone content and glucose tolerance. Horm. Metab. Res. 32, 233 – 239. Portha, B., Kergoat, M., Blondel, O., Bailbe, D., 1995. Underfeeding of rat mothers during the first two trimesters of gestation does not alter insulin action and insulin secretion in the progeny. Eur. J. Endocrinol. 133, 475 – 482. Poulsen, P., Vaag, A.A., Kyvik, K.O., Møller-Jensen, D., BeckNielsen, H., 1997. Low birth weight is associated with NIDDM in discordant monozygotic and dizygotic twin pairs. Diabetologia 40, 439 – 446. Poulter, N.R., Chang, C.L., MacGregor, A.J., Snieder, H., Spector, T.D., 1999. Association between birth weight and adult blood pressure in twins: historical cohort study. Br. Med. J. 319, 1330 – 1333. Rahier, J., Wallon, J., Henquin, J.C., 1981. Cell populations in the endocrine pancreas of human neonates and infants. Diabetologia 20, 540 – 546. Rasschaert, J., Reusens, B., Dahri, S., Sener, A., Remacle, C., Hoet, J.J., Malaisse, W.J., 1995. Impaired activity of rat pancreatic islet mitochondrial glycerophosphate dehydrogenase in protein malnutrition. Endocrinology 136, 2631 – 2634. Sadiq, H.F., Das, U.G., Tracy, T.F., Devaskar, S.U., 1999. Intrauterine growth restriction differentially regulates perinatal brain and skeletal muscle glucose transporters. Brain Res. 823, 96 – 103. Seckl, J.R., Nyirenda, M.J., Walker, B.R., Chapman, K.E., 1999. Glucocorticoids and fetal programming. Biochem. Soc. Trans. 27, 74 – 78. Sener, A., Reusens, B., Remacle, C., Hoet, J.J., Malaisse, W.J., 1996. Nutrient metabolism in pancreatic islets from protein malnourished rats. Biochem. Mol. Med. 59, 62 – 67. Shepherd, P.R., Crowther, N.J., Desai, M., Hales, C.N., Ozanne, S.E., 1997. Altered adipocyte properties in the offspring of protein malnourished rats. Br. J. Nutr. 78, 121 – 129. Shepherd, P.R., Withers, D.J., Siddle, K., 1998. Phosphoinositide 3-kinase: the key switch in insulin signalling. Biochem. J. 333, 471 – 490. Snoeck, A., Remacle, C., Reusens, B., Hoet, J.J., 1990. Effect of a low protein diet during pregnancy on the fetal rat endocrine pancreas. Biol. Neonate 57, 107 – 118. Tonkiss, J., Trzcin˜ ska, M., Galler, J.R., Ruiz-Opazo, N., Herrera, V.L.M., 1998. Prenatal malnutrition-induced changes in blood pressure. Dissociation of stress and nonstress responses using radiotelemetry. Hypertension 32, 108 – 114. Turner, R.C., 1998. The U.K. prospective diabetes study. A review. Diabetes Care 21 (Suppl. 3), C35 – C38. Vickers, M.H., Breier, H., Cutfield, W.S., Hofman, P.L., Gluckman, P.D., 2000. Fetal origins of hyperphagia, obesity, and hypertension and postnatal amplification by hypercaloric nutrition. Am. J. Physiol. 279, E83 – E87. Williams, D.R.R., Roberts, S.J., Davies, T.W., 1979. Deaths from ischaemic heart disease and infant mortality in England and Wales. J. Epidemiol. Community Health 33, 199 – 202. Wilson, M.R., Hughes, S.J., 1997. The effect of maternal protein deficiency during pregnancy and lactation on glucose tolerance and pancreatic islet function in adult rat offspring. J. Endocrinol. 154, 177 – 185. Wilson, M.R., Hughes, S.J., 1998. Impaired glucose-stimulated insulin release in islets from adult rats malnourished during foetal-neonatal life. Mol. Cell. Endocrinol. 142, 41 – 48.

C.J. Petry et al. / Molecular and Cellular Endocrinology 185 (2001) 81–91 Winick, M., Noble, A., 1996. Cell responses in rats during malnutrition at various ages. J. Nutr. 89, 300 – 306. Woodall, S.M., Johnston, B.M., Breier, B.H., Gluckman, P.D., 1996. Chronic maternal undernutrition in the rat leads to delayed postnatal growth and elevated blood pressure of offspring. Pediatr. Res. 40, 438 – 443. Woods, L.L., 2000. Fetal origins of adult hypertension: a renal mechanism? Curr. Opin. Nephrol. Hypertens. 9, 419 –425.

91

Zhang, J., Byrne, C.D., 2000. Differential hepatic lobar gene expression in offspring exposed to altered maternal dietary protein intake. Am. J. Physiol. 278, G128 – G136. Zhang, J., Desai, M., Ozanne, S.E., Doherty, C., Hales, C.N., Byrne, C.D., 1997. Two variants of quantitative reverse transcriptase PCR used to show differential expression of alpha-, beta- and gamma-fibrinogen genes in rat liver lobes. Biochem. J. 321, 769 – 775.