1. Investigación

The amount of peroxisome proliferator-activated receptor-ex (PPARcx) protein was markedly augmented in the liver of suckling rats compared to adult rats. This different PPARcx abundance was used to study the sensitivity to nutritional changes in the expression and activity of this receptor. Thus, 10-day-old and adult rats were orally given either glucose, Intralipid or a combination of both diets, and liver mRNA levels of PPARcx and the PPAR related genes, acyl-CoA oxidase (ACO) and phosphoenolpyruvate carboxykinase (PEPCK), and plasma metabolites were measured. In neonates, the expression of PPARcx and ACO was seen to increase when the level of FF A in plasma was also high, unless an elevated level of insulin was also present. However, this fatty acid-induced effect was not detected in adult rats. On the contrary, the hepatic expression of PEPCK was modulated by the nutritional changes similarly in both neonates and adult rats. Thus, it may be concluded that the expression of the PPARcx gene in adult rats seems to be less sensitive to nutritional changes than in neonates.

It was previously found that the expression of peroxisome proliferator-activated receptor-a (PP ARa) was markedly augmented in the liver of suckling rats, in comparison to the fetuses and most notably to adult rats and it paralleled similar changes in hepatic lipid concentration. To determine whether these changes could be related to the high lipid content of the maternal milk and/or to hormonal status, the role of changes in nutrient availability and in plasma insulin concentration on liver expression during the perinatal stage in vivo in the rat was studied. When suckling rats were weaned on day 17, instead of on day 20, the level of hepatic PPARa mRNA decreased earlier than in rats weaned later. When 10-day-old rats were force-fed with either glucose or Intralipid or a combination of both diets, it was found that, at similar low levels of plasma insulin, a high level of FFA stimulated PPARa expression, whereas, at similar high plasma FF A concentrations, an elevated insulin level attenuated the increase in PPARa expression. It is proposed that both the high lipid intake and decreased plasma insulin level are responsible for the high PP ARa expression detected in rat neonates.

By using different experimental designs in the rat we have been able to answer several unanswered questions on the short- and long-term effects of alterations of lipid metabolism during the perinatal stage. The first was to demonstrate the importance of maternal body fat accumulation during the first half of pregnancy, since undernutrition in this critical period when fetal growth is slow, impedes fat depot accumulation and not only restrains intrauterine development but has long-term consequences, as shown by an impaired glucose tolerance when adults. Secondly, undernutrition during suckling has major long-term effect of decreasing body weight, even though food intake is kept normal from the weaning period. Our findings also show that a diet rich in n-3 fatty acids during pregnancy and lactation has adverse effects on offspring development, but cross fostered experiments showed that this effect was a consequence of the intake of these fatty acids during the lactation period rather than during pregnancy. Pups from dams that were fed a fish oil-rich diet during pregnancy and lactation were found to have altered glucose/insulin relationship at the age of 10 weeks. Since a n-3 fatty acid-rich diet decreases milk yield during lactation, additional experiments were carried out to determine whether decreased food intake or altered dietary fatty acid composition, or both, were responsible for the long-term effects on the glucose/insulin axis. Results show that the decreased food intake caused by a n-3 fatty acidrich diet rather than the change in milk composition during suckling was responsible for the reduced pancreatic glucose responsiveness to insulin release at 16 weeks of age. In conclusion, present findings indicate that impaired maternal fat accumulation during early pregnancy and food intake during lactation, rather than a difference in dietary fatty acid composition, have major effects on postnatal development and affect glucose/insulin relationships in adult rats.

The present study was addressed to determine whether the high expression of peroxisome proliferator-activated receptor-a (PPAR-a) in rat liver during the perinatal stage plays a role in the induction of liver lipoprotein lipase (LPL) expression and activity. Parallel increases in liver mRNA PPAR-a and LPL activity were found in newborn rats, and after a slight decline, values remained elevated until weaning. Anticipated weaning for 3 days caused a decline in those two variables as well as in the mRNA LPL level, and a similar change was also found in liver triacylglycerol concentration. Force-feeding with Intralipid in 10-day-old rats or animals kept fasted for 5 h showed high mRNA-PPARa and -LPL levels as well as LPL activity with low plasma insulin and high FFA levels, whereas glucose and a combination of glucose and Intralipid produced low mRNA-PPARa and -LPL levels as well as LPL activity. Under these latter conditions, plasma insulin and FFA levels were high in those rats receiving the combination of glucose and Intralipid, whereas plasma FF A levels were low in those force-fed with glucose. It is proposed that the hormonal and nutritional induction of liver PPAR-a expression around birth and its maintained elevated level throughout suckling is responsible for the induction of liver LPLexpression and activity during suckling.

During pregnancy, lipid metabolism plays a major role to warrant the availability of substrates to the foetus. By using different experimental designs in the rat we have been able to answer several questions that were open about the short- and long-term effects of alterations of lipid metabolism during the perinatal stage. The first one was to demonstrate the importance of maternal body fat depot accumulation during the first half of pregnancy. We found that conditions like undernutrition circumscribed to this specific period when foetal growth is still small, that impede such fat accumulation not only restrain intrauterine development but also have long-term consequences, as shown by an impaired glucose tolerance when adults. Secondly, undernutrition during suckling has major long-term effect decreasing body weight, even though food intake was kept normal from the weaning period. Present findings also show that a diet rich in co-3 fatty acids during pregnancy and lactation has negative effects on offspring development, but cross fostered experiments showed that the effect was a consequence of the intake of these fatty acids during the lactation period rather than during pregnancy. Pups from dams that were fed a fish oil-rich diet during pregnancy and lactation were found to have altered glucose/insulin relationship at the age of 10 weeks. Since a ro-3 fatty acid-rich diet decreases milk yield during lactation, additional experiments were carried out to determine whether decreased food intake, altered dietary fatty acid composition, or both were responsible for the long-term effects on the glucose/insulin axis. Results show that the decreased food intake caused by a ro-3 fatty acid-rich diet rather than the change in milk composition during suckling was responsible for the reduced pancreatic glucose responsiveness to insulin release at 16 weeks of age. In conclusion, present findings indicate that impaired maternal fat accumulation during early pregnancy and food intake during lactation, rather than a difference in dietary fatty acid composition have a greater influence on postnatal development and affect glucose/insulin relationships in adult rats.

Prolonged hyperinsulinemia in the pregnant rat, caused either by daily treatment with exogenous insulin of the STZ-diabetic rat or a continous infusion of glucose, produces an increase of adipose tissue LPL activity. These findings support the hypothesis that under normal conditions, insulin resistance during late pregnancy is responsible for the decreased LPL activity normally seen in maternal adipose tissue.

To establish the temporal stages at which changes in insulin/glucose interactions may appear during gestation in the rat, unanesthetized animals were subjected to oral glucose tolerance tests (2 g glucose/kg) at days 15 and 21 of gestation and were compared to virgin female controls. On day 15 glucose tolerance is enhanced in the pregnant rat whereas plasma insulin levels are like those in control animals. On day 21 glucose tolerance does not differ between the two groups although insulin is higher in the pregnant animals. Results show 2 differenciated stages of insulin/glucose relationships throughout gestation in the rat with enhanced insulin sensitivity on day 15 and enhanced insulin resistance during late gestation. It is suggested that these changes contribute to the anabolic tendencies of the mother during mid gestation and her catabolic condition during late gestation.

I. During the first two thirds of gestation, coinciding with a minimal accretion by the conceptus, the mother is in an anabolic state which is supported by her hyperphagia and the more efficient conservation of exogenous nutrients when she eats. During this phase maternal fat deposits are accumulated thanks to the enhancement in adipose tissue lipogenic and glycerolgenic activity. In contrast, in the latter part of gestation, the rapid fetal growth is sustained by the intense transfer of nutrients from maternal circulation. 2. Glucose is quantitatively the most abundant of the several substrates that cross the placenta and despite increased maternal gluconeogenesis this transfer is responsible for the maternal tendency to hypoglycemia. This causes a switch to a net catabolic state which is especially evident in the net breakdown of fat depots. 3. Enhanced release of adipose tissue Iipolytic products, free fatty acids (FFA) and glycerol, facilitates the liver synthesis of triglycerides and their later release into circulation associated to very low-density lipoprotein (VLDL). Glycerol is also used as an important gluconeogenic substrate and FF As are broken down through 13-oxidation for ketone body synthesis. Flow through these pathways becomes increased when food is withheld and this actively contributes to the availability of fuels to the fetus which becomes partially preserved from maternal metabolic insult. Increased liver production of VLDL-triglycerides and decreased extrahepatic lipoprotein lipase contribute to exaggerated maternal hypertriglyceridemia which, besides being a floating metabolic reserve for emergency conditions such as starvation, constitutes an essential substrate for milk synthesis around parturition in preparation for lactation. 4. While the maternal anabolic tendencies found during the first two-thirds of gestation seem to be facilitated by hyperinsulinemia in the presence of a normal responsiveness to the hormone, it is proposed that most of the metabolic changes taking place during the last third of gestation seem to be caused by the insulin-resistant state which is consistently present at this stage, since its reversion caused by sustained exaggerated hyperinsulinemia also reverts several of these metabolic adaptations.

In sham-operated rats, intravenous administration of 14C-very low density lipoprotein triglycerides (with labelled esterified fatty acids) caused an initial decrease and subsequent increase in plasma 14C-lipids of both very low density lipoproteins (VLDL) (density < 1.006) and lipoproteins of density > 1.019. There was a similar change in 14C-lipids in adipose tissue and heart whereas in kidney, spleen and liver, 14C-lipids increased initially and then decreased. Insulin treatment in sham-operated animals decreased circulating 14C-lipids in VLDL and in lipoproteins of density > 1.019, while intermediate density (1.006-1.019) lipoproteins increased. Insulin also enhanced the radioactivity retained in spleen. In functionally hepatectomized rats, 14C-lipids progressively increased in heart. Insulin treatment in these rats enhanced the disappearance from circulation of 14C-VLDL and of lipoproteins of density > 1.019, as well as the appearance of 14C-intermediate density lipoproteins. Toe appearance of 14C-lipids in white adipose tissue also was augmented, while it decreased in heart and lung. Thus, in sham-operated animals, insulin apparently stimulates the uptake of products of VLDL metabolism by cells in the reticuloendothelial system, while in functionally hepatectomized rats there is increased heart utilization of VLDL triglycerides, and insulin enhances the net extrahepatic catabolism of these lipoproteins.

To understand the mechanism of exaggerated hypertriglyceridemia in diabetic pregnancy, streptozotocin-treated rats receiving a daily insulin supplement were mated with nom1al males and divided into four groups: i) kept under this regime until the 20th day of gestation (Dl+ll), ii) the same regime until the 12th day of gestation (DI), iii) the insulin treatment was suspended during the first half of gestation (days 0-12) and then restored on a daily basis until the 20th day (DU), and iv) no insulin treatment was given after mating (D). All animals were studied on day 20. Despite increased food intake, maternal conceptus-free body weight was greatly reduced in the D animals as compared with the other groups whose values did not differ. Both the plasma glucose and ~-hydroxybutyrate levels were increased more in D than in DI rats and values in both groups were greater than in the others. Insulin levels showed an opposite trend to that of glucose, but the values in DI+ll rats were higher than in untreated intact control rats (C). The plasma triglyceride concentration was highest in the DI rats, followed by the D group whose values were still significantly higher than in either C or DI+ll rats. Plasma free fatty acid levels were lower in D than in any of the other groups, although they were also lower in Dl+II and DI than in C animals. Adipose tissue lipoprotein lipase activity wa~ highest in DI+II animals and their values were very similar lo those found in DII, whereas the values in the C, D and DI animals were all similar and much lower. Results indicate that reductions in fat accumulation during the first half of gestation impair the activation of lipolytic activity in the severe diabetic mother during late gestation. During this period lipolysis helps sustain maximal hypertriglyceridemia, which develops in animals whose diabetes was circumscribed to the second half of gestation. In general, our findings show that anabolic changes during the first half of gestation affect metabolic events during late gestation.