1. Investigación

To study the effect of prolonged diabetes on protein synthesis and on the activities of initiation factors elF-2 and elF-2B in the liver, female rats were treated with streptozotocin. Some animals were mated and studied on day 20 of pregnancy, whereas others were kept virgin and studied in parallel. The protein synthesis rate was measured with an 'in vitro' cellfree system, and was lower in diabetic pregnant and virgin animals than in pregnant and virgin controls (30-60%). The fetuses of diabetic rats had a lower protein synthesis rate than those from controls, although they always showed a higher protein synthesis rate than their mothers or virgin rats. Protein synthesis rate, RNA concentration, and initiation factor 2 activity were higher in pregnant than in virgin rats. Both activity and level of elF-2 factor changed in parallel to the protein synthesis rate, although no differences could be detected between control and diabetic animals. The eIF-2B activity in tissue extracts from diabetic virgin rats and fetuses was lower than in extracts from their controls, whereas no differences could be detected between pregnant and virgin control rats nor between pregnant control and pregnant diabetic animals. The percentage of the phosphorylated form of eIF-2 factor, eIF-2(uP), was slightly lower in virgin than in pregnant rats but was unaffected by the diabetic condition, while in diabetic fetuses this parameter was lower than in their corresponding controls. The cyclic adenosine monophosphate dependent protein kinase level was lower in diabetic rats than in controls, whereas no changes in the activity of casein kinase II were found. The isoelectric forms of the 13 subunit of eIF-2 factor, eIF-213, were different in the diabetic and the control animals, indicating that insulin deficiency modifies the phosphorylation of specific substrates. Since no differences were detected in RNA or eIF-2 content between control and diabetic rats, translation may, at least partly, be inhibited in the liver by an impairment of peptide chain initiation caused by the decreased eIF-2B activity which nevertheless is independent of eIF2u phosphorylation.

Fibrates are peroxisome proliferator-activated receptor alpha (PPARa) ligands used to normalize lipid and glucose parameters and exert anti-inflammatory effects. The acute-phase response (APR) is an important inflammatory process. One of the most important acute-phase proteins in rats is a2-macroglobulin (A2Mg). Whereas normal adult rats present low serum levels, pregnant rats display high amounts. Therefore, we used pregnant rats to detect the effect of fenofibrate on hepatic A2Mg expression by RT-PCR and Northern blot. Virgin rats were used as controls. The expression of other APR genes, a known fibrate-responder gene, gamma-chain fibrinogen (g-Fib), and one gene from the same family as A2Mg, complement component 3 (C3), were also measured in liver. In order to determine whether the fibrateeffects were mediated by PPARa, wild-type mice and PPARa-null mice were also used and treated with WY-14,643 (WY) or di-2-ethylhexyl phthalate (DEHP). Fenofibrate depressed A2Mg expression in virgin rats, but expression was decreased more sharply in pregnant rats. Expression of C3 and g-Fib was diminished after treatment only in pregnant rats. On the other hand, WY, but not DEHP, reduced A2Mg and g-Fib expression in the livers of wild-type mice, without any effect in PPARa-null mice. WY or DEHP did not affect C3 expression. Therefore, A2Mg expression is modified by PPARa agonists not only in pregnant rats under augmented APR protein synthesis, but also in virgin rats and mice under basal conditions. Interestingly, our results also identify A2Mg as a novel PPARa agonist-regulated gene.

To study insulin/glucose relationship during gestation, rats were studied on days 6, 12, 15, 18, 20 or 21 of presnancy and the results were compared to values in sex.matched virgin control rats. Blood glucose levels were decreased on days 20 and 21 of gestation whereas plasma insulin levels appeared decreased on days 6 and 12, unchanged on day 15 and enhanced on days 18. 20 and 2L of gestation. Total pancreas insulin content was already augmented on day 6 of gestation and continued to increase with gestational time. \Vith the exception of an increase in tbe 6-day.pregnant rats 22.5 min after an oral glucose toad, blood glucose levels did not differ between 6• or 12-day.pregnant rats and virgin controls although plasma insulin levels reached higher values on these days. However. in the 15-day-pregnam rats, glucose tolera!lce after the glucose load was enhanced while plasma insulin levels did not differ from those in virgin rats during the first 30 mjn. In the IS-day-pregnant rat blood glucose was more increased but plasma insulin did not differ after the glucose load when compared to virgin rats, whereas 20. or 21-day-pregnant rats showed a gJucose tolerance similar to that of virgin rats but their insulin levels shonly after the glucose load were higher. The hY'I.»" glycemic response lo a high intravenous dose of insulin was decreased in 12-, 18-. 20- and 21-day-pregnant rats. TI,erefore, whereas in both the 6- and 12-day-pregnant rats there is an enhanced 13-,celJ response to the glucose insulinotropic effect and insulin responsiveness is reduced in J 2. day-pregnant rats. the 15-day pregnam rat is in a transitory stage where both insulin sensitivity and the J},-ccll response return to non pregnant vaJ. ues. Ho\•.revcr. from 18 da)'S of gcslation on, there is an intense insulin• resistanl condition which is only partially compensated by an enormous accumulation of insuJin in the pancreas followed by a faster and larger insulin release after a glucose load.

Plasma-triglyceride levels were higher in pregnant than in virgin rats. The glucose infusion did not modify this parameter, probably because ef the changes in LPL activity in other tissues which are known to occur in the opposite direction to those observed in this study for adipose tissue and mammary gland. The present results support the notion that the insulin resistant condition which normally occurs during late gestation is responsible for the decreased LPL acti11ity in adipose tissue, but that the mammary gland remains sensitive to insulin and so maternal hyperinsulinemia would contribute to the induction of LPL activity in this organ prior to parturition.

We wanted to determine whether administration of vitamin E could reduce the production of free radicals which could play a role in the teratogenic effects of diabetes mellitus. Diabetes was induced in Wistar rats by the intravenous administration of streptozotocin. The animals were divided into six groups: one with no supplement (D) and two, supplemented during pregnancy either with oral vitamin E (150 mg/day) (D + E) or with a placebo (safflower oil) (D + 0). Three other groups were kept under the same conditions, but were treated with insulin: D + I, D + I + E and D + I + 0. There were three groups of matched controls: C, C + E and C + 0. All animals were killed on day 11.5 of pregnancy. In C animals the percentages of reabsorptions and malformations were 1.3 and 2 %, respectively, comapred with 23.6, 24.3, 6.2 and 13.2 %, respectively in D and D + I groups. The crown-rump length, number of somites, and protein and DNA content were: higher in C animals than in the diabetic rats, independent of insulin treatment. When vitamin E was administered no changes in these parameters were observed in C and D + I animals; however, in the D mothers it reduced the rate of embryo malformations to 4.6 % and increased the crown-rump length and the number of somites. However, vitamin E did not modify the protein and DNA content and the percentage of reabsorptions. In conclusion, administration of vitamin E to diabetic animals decreases the rate of embryo malformations and increases their size and maturation, supporting a role for free radicals in the teratogenic effects of diabetes.

To determine how a reduction in maternal hypertriglyceridemia during late pregnancy may affect glucose/insulin relationships, pregnant and virgin rats were orally treated with acipimox, a potent antilipolytic agent. In 20-day pregnant rats receiving 80 mg of acipimox, plasma triglycerides (TG), free fatty acids (FFA), and glycerol decreased more than in virgin rats shortly after the drug (up to 7 hours), when compared with animals treated with distilled water, whereas plasma glucose level was unaffected by the treatment in either group of rats. When acipimox was given every 12 hours from day 17 to day 20 of pregnancy, plasma TG, FFA, and glycerol levels progressively increased, whereas they either decreased or did not change in virgin rats receiving the same treatment, with no effect in plasma glucose levels in either group. Fetal body weight was lower than in controls in 20-day pregnant rats that received acipimox for 3 days. On day 20 of pregnancy, 3 hours after receiving acipimox or distilled water, rats received a 2 g glucose/kg oral load and it was found that the change in plasma glucose was similar in both groups, whereas the increase in plasma insulin was greater in pregnant rats treated with acipimox. However, no difference was found in either variable after the oral glucose load in virgin rats receiving acipimox or distilled water. No differences in plasma glucose levels were found after intravenous (IV) administration of insulin in pregnant rats treated or not treated with acipimox. In conclusion, present results show that administration of acipimox during the last days of gestation inhibited lipolysis and decreased fetal weight. Over a short period of time, in pregnant rats, reductions of plasma FFA and TG after acipimox treatment improved the glucose-induced insulin release, but did not seem to have any effect in peripheral insulin resistance.

The level of maternal circulating triglycerides during late pregnancy has been correlated to newborns’ weight in humans. To investigate the response to fenofibrate, a hypotriglyceridemic agent, in pregnant rats, 0, 100, or 200 mg of fenofibrate/kg body weight as oral doses were given twice a day from day 16 of gestation and studied at day 20. Virgin rats were studied in parallel. Liver weight was higher in pregnant than in virgin rats, and either dose of fenofibrate increased this variable in both groups. The highest dose of fenofibrate decreased fetal weight. Although plasma triglycerides decreased during the first 2 days of fenofibrate treatment in pregnant rats, the effect disappeared on day 3, and plasma triglycerides were even enhanced at day 4. In virgin rats, fenofibrate decreased plasma triglycerides throughout the experiment. Plasma cholesterol levels in pregnant rats decreased during the first 3 days of treatment, and the effect disappeared on day 4, whereas in virgin rats, values remained decreased. Changes in plasma triglycerides paralleled those of VLDL triglycerides. In pregnant rats, VLDL cholesterol levels increased while LDL cholesterol decreased with the treatment, whereas in virgin rats, cholesterol levels decreased in all lipoprotein fractions. Only in virgin rats did liver triglyceride concentration increase with fenofibrate treatment. Lumbar adipose tissue LPL was lower in pregnant than in virgin rats, and fenofibrate treatment decreased this variable in both groups. Maternal fenofibrate treatment increased fetal plasma and liver triglyceride and cholesterol concentrations.

During early pregnancy, long-chain polyunsaturated fatty acids (LC-PUFA) may accumulate in maternal fat depots and become available for placental transfer during late pregnancy, when the fetal growth rate is maximal and fetal requirements for LC-PUFAs are greatly enhanced. During this late part of gestation, enhanced lipolytic activity in adipose tissue contributes to the development of maternal hyperlipidaemia; there is an increase in plasma triacylglycerol concentrations, with smaller rises in phospholipid and cholesterol concentrations. Besides the increase in plasma very-low-density lipoprotein, there is a proportional enrichment of triacylglycerols in both low-density lipoproteins and high-density lipoproteins. These lipoproteins transport LC-PUFA in the maternal circulation. The presence of lipoprotein receptors in the placenta allows their placental uptake, where they are hydrolysed by lipoprotein lipase, phospholipase A2 and intracellular lipase. The fatty acids that are released can be metabolized and diffuse into the fetal plasma. Although present in smaller proportions, maternal plasma non-esterified fatty acids are also a source of LC-PU FA for the fetus, their placental transfer being facilitated by the presence of a membrane fatty acid-binding protein. There is very little placental transfer of glycerol, whereas the transfer of ketone bodies may become quantitatively important under conditions of maternal hyperketonaemia, such as during fasting, a high-fat diet or diabetes. The demands for cholesterol in the fetus are high, but whereas maternal cholesterol substantially contributes to fetal cholesterol during early pregnancy, fetal cholesterol biosynthesis rather than cholesterol transfer from maternal lipoproteins seems to be the main mechanism for satisfying fetal requirements during late pregnancy.

Diabetes mellitus is associated with a reduction of lipoprotein lipase (LPL) activity in adipose tissue and development of · hypertriglyceridemia. To determine how a condition of severe insulin deficiency affects mammary gland LPL activity and mRNA expression during late pregnancy, streptozotocin (STZ) treated (40 mg/kg) and non-treated (control) virgin and 20 day pregnant rats were studied. In control rats, both LPL activity and mRNA were higher in pregnant than in virgin rats. When compared to control rats, STZ-treated rats, either pregnant or virgin, showed decreased LPL activity and mRNA content. Furthermore, mammary gland LPL activity was linearly correlated with mRNA content, and either variable was linearly correlated with plasma insulin levels. Thus, insulin deficiency impairs the expression of LPL in mammary glands, revealing the role of insulin as a modulator of the enzyme at the mRNA expression level.