1. Investigación

Fructose intake from added sugars correlates with the epidemic rise in obesity, metabolic syndrome and cardiovascular diseases. Fructose intake also causes features of metabolic syndrome in laboratory animals. Therefore, we have investigated whether fructose modifies lipidemia in pregnant rats and produces changes in their fetuses. Thus, fructose administration (10% wt/vol) in the drinking water of rats throughout gestation, leads to maternal hypertriglyceridemia. This change was not observed in glucose-fed rats, although both carbohydrates produced similar changes in liver triglycerides and in the expression of transcription factors and enzymes involved in lipogenesis. After fasting overnight, mothers fed carbohydrates were found to be hyperleptinemic. However, after a bolus of glucose, leptinemia in fructose-fed mothers showed no-response, whereas it increased in parallel in glucose-fed and control mothers. Fetuses from fructose-fed mothers showed hypotriglyceridemia and a higher hepatic triglyceride content than fetuses from control or glucose-fed mothers. A higher expression of genes related to lipogenesis and a lower expression of fatty acid catabolism genes were also found in fetuses from fructose-fed mothers. Moreover, although hyperleptinemic, these fetuses exhibited increased tyrosine phosphorylation of the signal transducer and activator of transcription-3 (STAT-3) protein, without a parallel increase in the serine phosphorylation of STAT-3 nor in the suppressor of cytokine signaling-3 (SOCS-3) protein levels whose expression is regulated by leptin through STAT-3 activation. Thus, fructose intake during gestation provoked a diminished maternal leptin-response to fasting and re-feeding, and an impairment in the transduction of the leptin signal in the fetuses which could be responsible for their hepatic steatosis.

Postnatal development of the glucose and insulin balance in offspring of ethanol-treated and control rats has been studied. Newborn rats were separated from their mothers and placed with normal lactating, nonethanol-treated dams. Prenatal exposure to ethanol led to hypoglycemia on the first day of extrauterine life and a general tendency to hyperinsulinemia during the entire postnatal period studied. The glucose-tolerance test in weaned rats (30 days old) gave a greater and faster increase than controls in levels of both glucose and plasma insulin. At adult age (90 days) the response of blood glucose to an oral glucose load in offspring from ethanol-treated mothers was not different from that in offspring from controls, but the insulin response was higher. This abnormal insulin response, such a long time after the end of ethanol exposure, suggests either a permanent alteration in the pancreatic response, or a peripheral insulin resistance and/or differences in the rate of insulin degradation in these animals.

Effects of ethanol intake on lipid metabolism in the lactating rat. ALCOHOL 13(5) 443-448. 1996.-Femalc rals receiving alcohol (20%) in drinking water during lactation (AL) were compared lo pair-fed ,inimals (l'F) and normal controls (C) fed ad lib. All animals were killed on the 12th day of lactation. When compared to C mis, food intake decreased in both AL and PF groups. and this effect was followed by a lower body weight nnd mammary gland (MG). liver. and parametrial adipose tissue weights. Mammary glands tri,1cylglyceride concentration (TG) was much lower in l'F lhan in AL. although in the latter. values <.lid not reach lhose of C'. and had higher liver TG conccnlration than ;1ny of the other group.~. Both l'F and AL rats had lower plasma TG. glycerol, and free folly acid concentrations ur1<.I higher (3;-hydroxybutyrnte concenlrntion than C rats. When compared to C rals. the rnte of lipogencsis in MG was higher in bolh PF and AL mis. whereas in liver it was higher in PF and lower in AL rats, and in adipose tissue it wns higher in PF and unchanged in AL nils. The appearance of uc lipids 4 h after ornl (1 4 jlriolcin in both MG and liver was lower in AL and PF rnts and only lower in adipose tissue of AL rats as compared lo the C rnts. Lipopmtein lipase and hormone-sensitive lipase activities were lower in MG in holh PF and AL rats lhan in C. whereas in adipose tissue the activity of lipoprolein lipase did nol differ between AL and C rats and the activity of I ISL was lower in the former. These findings therefore show that in spite of re<.luce<.1 upt,1ke of ornlly administered lriglyceri<.les due lo decreased LPL aclivily, maternal alcohol feeding during lactation in the rat preserves the mammary !!land triglyceride content thanks to enhanced lipogcnetic activity. On the olher hand. it causes liver triglycerides accumulation. probably as a result of the decreased rate of triglycerides released into circulation. and these changes are not caused hy the reduced food in lake of the animals.

From parturition, lactating Wistar rats were given 20% ethanol in drinking water and solid diet ad lihitum (ET group) or were pair-fed to the ET group (PF group) or were given water and solid diet ad lihitum (control group, C). Animals were studied on day 12 of lactation and/or treatment, when dams were separated from their litters and 2-4 hours latter they were i.p. injected with oxytocin for milk collection under anaesthesia. Maternal food intake, weight gain and pup's body weight were lower in ET than in C rats. When compared to C rats, milk of ET dams had a decreased proportion of C14:0 and C22:6 n-3 fatty acids while an increase in C18:0, C16:1 and C18:1 n-9 was detected, whereas when compared to PF it had higher CS:0, Cl0:0, C18:0, C18:1 n-9, C18:2 n-6 and lower C20:5 n-3 and C22:6 n-3. Body weight was lower in pups from ET than in those from C or PF, and whereas brain weight and brain lipid content was lower in ET and PF pups than in C, total phospholipid (PL) brain content was similar among the groups. The ratio of C20:3 n-9 to C20:4 n-6 in brain PL was higher in ET pups than in either C or PF, indicating an essential fatty acid deficiency in the formers. Ethanol treatment also decreased the proportional amount of C18:2 n-6, C18:3 n-3, C20:4 n-6, C20:5 n-3 and C22:6 n-3 in brain PL as compared to C, whereas from these fatty acids only C18:3 n-3, C20:5 n-3 and C22:6 n-3 were decreased in PF. On the other hand, the proportion of C22:6 n-3 was significantly lower in the pups of ET group than in PF animals. Present results show that maternal intake of ethanol during lactation in the rat modifies milk lipid composition and that these effects are not caused by the undemutrition condition of the animals. These effects alter fatty acid composition of brain PL in pups, and such effect may contribute to its abnormal development.

Diabetes during pregnancy results in congenital malformations and long-term postnatal diseases. Experimental models are still needed to investigate the mechanism responsible for these alterations. Thus, by the administration of different doses of streptozotocin (STZ) (0, 25, 30, or 35 mg/kg body weight, intravenous) at the onset of pregnancy in rats, the present study sought an appropriate animal model for this pathology. At day 6 of pregnancy, plasma glucose was progressively higher with an increasing STZ dose, and in rats receiving the 35-mg dose, 2 subgroups were detected: some animals had plasma glucose levels above controls but below 200 mg/dL (mildly diabetic, MD), whereas others had levels above 400 mg/dL (severely diabetic, SD). At day 20 of pregnancy, the MD rats had normal glycemia, but after an oral glucose load (2 g/kg body weight), plasma glucose increased more and insulin increased less than in controls. The SD rats maintained their hyperglycemia and had a greatly impaired oral glucose tolerance. At day 20, fetuses of SD dams were fewer, weighed less, and had enhanced plasma glucose and triglycerides and decreased insulin, whereas those from MD dams did not differ from controls. At birth, newborns from MD dams had higher body weight, plasma insulin, and liver triglycerides as well as total body lipid concentrations than controls, and on day 21, remained macrosomic and showed higher plasma glucose and liver triglyceride concentrations. At 70 days of age, offspring of MD dams had impaired oral glucose tolerance but normal plasma insulin change in the case of females, whereas plasma insulin increased less in males. These alterations were manifest more in those offspring from dams that had >50% macrosomic newborns than in those from dams that had <50% macrosomic newborns. In conclusion, whereas our MD rats mimic the changes taking place in gestational diabetic women and show the long-term risk of macrosomia, the SD rats are more similar to uncontrolled diabetics. Thus these two rat models, obtained with moderate amounts of STZ, could be used to study the pathophysiological consequences of these different diabetic conditions.

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.

Glutathione plays a central role in metabolism and antioxidant defence. Several factors can influence the analytical efficiency and rapidity of the quantitative determination of glutathione. Procedures in sample pre-treatment have been compared in order to minimize analytical errors. Capillary electrophoresis has been chosen as a more adequate technique for obtaining a rapid and simple method for glutathione and glutathione disulfide determination in the blood and liver of the rat. The methods, once optimised, have been validated and applied for monitoring the oxidative stress in an animal model, such as the rat made diabetic by streptozotocin injection, when the animals are treated with antioxidants and compared with the corresponding controls.

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.

To determine whether changes in uterine blood flow affect placental glucose transfer in the diabetic pregnant rat, on the 7th day of gestation rats were intravenously treated with either streptozotocin (45 mg/kg) (diabetics) or buffer (controls). On the 20th day of gestation, fetal body weight and uterine blood flow appeared reduced whereas fetal/maternal plasma glucose was enhanced and lactate ratios were unchanged in diabetics versus controls. After 20 min of (U- 14C)-D-glucose infusion through the maternal left uterine artery, plasma values of fetuses from left and right uterine horns were higher for 14C-glucose and lower for 14C-lactate in diabetics versus controls, and placental glucose transfer was greatly augmented in diabetics whether or not uterine blood flow was included in its calculation. Whereas a linear correlation existed between placental glucose transfer and maternal plasma glucose concentration, transferred glucose conversion into lactate remained stable even when the maternal glucose level was high. It was concluded that enhanced placental glucose transfer in the pregnant diabetic rat is not modified by reduced uterine blood flow. The limited capacity of the fetus to handle the great incoming flux of glucose through the placenta of a severely diabetic mother produces permanent hyperglycemia which may impair fetal growth.