Placental triglyceride accumulation in maternal type 1 diabetes is associated with increased lipase gene expression

Maternal diabetes can cause fetal macrosomia and increased risk of obesity, diabetes, and cardiovascular disease in adulthood of the offspring. Although increased transplacental lipid transport could be involved, the impact of maternal type 1 diabetes on molecular mechanisms for lipid transport in placenta is largely unknown. To examine whether maternal type 1 diabetes affects placental lipid metabolism, we measured lipids and mRNA expression of lipase-encoding genes in placentas from women with type 1 diabetes (n = 27) and a control group (n = 21). The placental triglyceride (TG) concentration and mRNA expression of endothelial lipase (EL) and hormone-sensitive lipase (HSL) were increased in placentas from women with diabetes. The differences were more pronounced in women with diabetes and suboptimal metabolic control than in women with diabetes and good metabolic control. Placental mRNA expression of lipoprotein lipase and lysosomal lipase were similar in women with diabetes and the control group. Immunohistochemistry showed EL protein in syncytiotrophoblasts facing the maternal blood and endothelial cells facing the fetal blood in placentas from both normal women and women with diabetes. These results suggest that maternal type 1 diabetes is associated with TG accumulation and increased EL and HSL gene expression in placenta and that optimal metabolic control reduces these effects.     

Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance

16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.     

Constitutively increased micronuclei are predominantly caused by acentric fragments

The frequency of kinetochore (centromere)-positive micronuclei (MN) was determined in 32 fibroblast cell lines. We tested 16 probands with spontaneously high MN levels (greater than or equal to 20 MN/500 cells (4%] and 8 probands (controls) with low MN levels (less than or equal to 13 MN/500 cells (2.6%]. To study whether the elevation of MN levels is due to increased chromosomal breakage we used the antikinetochore antibody fluorescent staining method. Probands with spontaneously high MN had kinetochore-positive MN increased by a factor 2.1 compared to the controls whereas the kinetochore-negative MN were increased by a factor 6.14. This shows that spontaneous elevation of MN is mainly caused by increased chromosomal breakage and only in a minor proportion by chromosome segregation errors as a consequence of spindle defects.     

Serum prolactin concentrations and the initiation of maternal behavior in the rat

Maternal behavior and serum prolactin were measured in pregnant and virgin female rats. Pregnant rats were either ovariectomized or shamovariectomized on Day 17 of pregnancy, while virgin females were ovariectomized at the same age. Two days after surgery nests were rated and the three treatment groups were tested for responsiveness to rat pups. Both pregnant treatment groups built superior nests compared to the virgin group and also responded more frequently to rat pups within a 1 hr test period than the virgin controls. In addition, significantly more ovariectomized pregnant subjects responded to pups than did intact pregnant females. Serum prolactin levels did not differ among the three treatments nor did exposure to pups affect serum prolactin levels. In each treatment group serum prolactin was less than 15 ng/ml, well below the 139.7 ng/ml mean found on Day 23 of pregnancy. These data suggest that high levels of serum prolactin during late pregnancy are not essential for the initiation of maternal behavior in the rat.     

Antidiuretic action of prolactin in the rat with diabetes insipidus.

Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.     

Placental structure in gestational diabetes mellitus

The placenta is a transitory organ, located between the mother and the foetus, which supports intrauterine life. This organ has nutritional, endocrine and immunologic functions to support foetal development. Several factors are related to the correct functioning of the placenta including foetal and maternal blood flow, appropriate nutrients, expression and function of receptors and transporters, and the morphology of the placenta itself. Placental morphology is crucial for understanding the pathophysiology of the organ as represents the physical structure where nutrient exchange occurs. In pathologies of pregnancy such as diabetes mellitus in humans and animal models, several changes in the placental morphology occur, related mainly with placental size, hypervascularization, higher branching capillaries of the villi and increased glycogen deposits among others. Gestational diabetes mellitus is associated with modifications in the structure of the human placenta including changes in the surface area and volume, as well as histological changes including an increased volume of intervillous space and terminal villi, syncytiotrophoblast number, fibrinoid areas, and glycogen deposits. These modifications may result in functional changes in this organ thus limiting the wellbeing of the developing foetus. This review gives an overview of recurrent morphological changes at macroscopic and histological levels seen in the placenta from gestational diabetes in humans and animal models. This article is part of a Special Issue entitled: Membrane Transporters and Receptors in Pregnancy Metabolic Complications edited by Luis Sobrevia.     

Effects of insulin and maternal diabetes on fetal lipogenesis in the rat

Offspring of diabetic mothers have been investigated with regard to fetal hepatic and brown adipose tissue lipogenesis in the rat. Results, which cannot be explained by existing theory, are obtained from offspring of subdiabetic mothers and manifest diabetic mothers. In re-evaluating the effect of exogenous insulin on perinatal lipogenesis, we find important differences in hormone sensitivity between liver and brown adipose tissue.     

Placental isoprostane is significantly increased in preeclampsia.

We determined placental tissue levels, production rates, and secretion rates of isoprostanes for placentas obtained from women with normal pregnancies and women with preeclampsia, a hypertensive disorder of pregnancy. Isoprostanes are markers of oxidative stress that exert biological actions such as vasoconstriction. Placental tissue was rinsed and immediately frozen in liquid nitrogen to determine tissue levels of total and free isoprostane. Placental tissue pieces were also incubated in serum-free DMEM for 48 h at 37 degrees C in 95% air/5% CO(2) to determine production rates. Isolated placental cotyledons were perfused for the determination of secretion rates. All samples were analyzed by EIA for isoprostane using an antibody specific for 8-Iso-PGF(2) (15-F(2t)-IsoP). In addition, medium samples were analyzed for malondialdehyde (MDA), a breakdown product of lipid peroxidation. We found that tissue levels of free isoprostane and total isoprostane (free plus esterified forms) were significantly higher for preeclamptic placentas than for normal placentas. Concentrations of isoprostane and MDA in the medium increased progressively during 48 h of incubation of placental explants. At 48 h of incubation, the mean concentrations of both isoprostane and MDA were significantly higher for the placentas from preeclamptic women than for the placentas from normal pregnant women. Concentrations of MDA were highly correlated with those of isoprostane. Induction of oxidative stress with xanthine plus xanthine oxidase increased placental production of isoprostane by normal tissue to a level similar to that of preeclamptic tissue. Placental secretion of isoprostane was eightfold greater toward the maternal side of the placenta than toward the fetal side, and was increased sixfold on the maternal side and twofold on the fetal side by inducing oxidative stress with t-butyl hydroperoxide. This study presents new information that isoprostanes are formed and secreted by the human placenta and provides convincing evidence that oxidative stress and lipid peroxidation are abnormally increased in placentas of preeclamptic women.     

Seasonal variations of gonadotropins and prolactin in the laboratory rat. Role of maternal pineal gland

The laboratory rat, a non-photoperiodic rodent, exhibits seasonal fluctuations of melatonin. Melatonin has been found to be readily transferred from the maternal to the fetal circulation. No data exist on the possible influence of maternal pineal gland upon seasonal variations of the offspring. The aim of the present study was to asses the influence of the maternal melatonin rhythm on the offspring postnatal development of the reproductive hormones LH, FSH and prolactin. Male offspring from control, pinealectomized (PIN-X) and PIN-X + melatonin (PIN-X+MEL) mother Wistar rats were studied at 21, 31, and 60 days of age. Seasonal age-dependent variations were found for all hormones studied in control offspring but PIN-X offspring showed a tendency to have reduced duration or altered seasonal variations. Maternal melatonin treatment to PIN-X mothers partially restored the effect of pinealectomy. The chronological study of LH, FSH, and prolactin in PIN-X offspring also showed an altered pattern as compared to control-offspring. Melatonin treatment to the mothers partially restored the developmental pattern of reproductive hormones. Results of this study indicate that maternal pineal gland of the laboratory rat is involved in the seasonal postnatal development variations of reproductive hormones of the offspring.     

Glucose transporters and maximal transport are increased in endurance-trained rat soleus.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of maternal obesity on diabetes development in adult rat offspring

This study aimed to evaluate whether maternal obesity leads to the onset of diabetes in adult Wistar rats offspring. MSG solution neonatally administration induced obesity in rats (F(1)MSG group, n=30); and saline solution was also administrated to control rats (F(1)CON group, n=13). In 3rd month of age, both control and MSG groups were mated for offspring (generation F(2)), named as F(2)CON, n=28 and F(2)MSG groups, n=15; and so both generations were studied until 7th month of life. Lee Index was measured for experimental obesity validation from 5th to 7th month. Glycemia was weekly determined during pregnancy and monthly from 3rd to 7th month. In the end of experimental period all rats were submitted to oral glucose tolerance test (OGTT), with estimation of total area under the curve (AUC); and insulin tolerance test (ITT). Rats were then anesthetized and killed. Data were statistically analyzed with significance level of p<0.05. Lee Index has confirmed obesity in all MSG rats. Glycemic levels comparisons between generations showed significant maternal interference in control and MSG groups. OGTT analysis showed higher glycemia in obese rats (F(1)MSG) and their offspring (F(2)MSG) as compared to their respective controls; and MSG groups increased AUC from OGTT. As regards ITT, F(2)MSG showed higher glycemia at 30 and 120 min, suggesting a delay of insulin action decreasing. Although glucose intolerance and insulin resistance clinical conditions represent as a factors for type 2 Diabetes mellitus development, this experimental model proposal was not efficient to induce type 2 Diabetes mellitus, but for obesity developing, glucose intolerance and insulin resistance in successive generations of rats.     

Influence of maternal diabetes on lipid metabolism in neonatal rat lung

The effect of maternal diabetes on tissue constituents, lipid metabolism and glucose utilization was examined in 1-day old rat lungs. Maternal diabetes was induced by intravenous injection of streptozotocin (50 mg/kg body weight) into pregnant Long-Evans hooded rats on the 10th day of gestation resulting in a maternal serum glucose concentration which was 3-fold higher than that of controls. Neonates from diabetic mothers showed a significant decrease in body weight (14%), lung weight (32%) and lung protein concentration (30%). Glycogen, DNA and lipid content of the lungs were significantly elevated in neonates from diabetic mothers. The percent of total phospholipid made up of phosphatidylcholine was not altered, but the percentage of disaturated phosphatidylcholine was decreased (25%). The activity of the CDPcholine pathway enzymes (choline kinase, cholinephosphate cytidylyltransferase and choline phosphotransferase) also showed a marked increase in lungs of neonates from diabetic mothers. Lung slices of neonates from diabetic mothers showed depressed in vitro incorporation of [U-¹⁴C]glucose into neutral lipids and decreased oxidation to CO₂. The results of these investigations show that maternal diabetes interferes with the structural and metabolic processes by which the undifferentiated lung becomes functional at birth.     

Suckling ability and maternal prolactin levels in hypothyroid rats

Long-Evans rats and their offspring were made hypothyroid by addition of the antithyroid goitrogen 6-N-propylthiouracil (PTU) to the drinking water (0.1%) from the day of parturition. Serum concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH) and thyroxine (T4) were determined by double radioimmunoassay (RIA). From the fifth postnatal day, body weight of PTU-treated pups was significantly lower than that of control rats, and a strikingly elevated serum TSH level and nondetectable amount of T4 were measured both in PTU-exposed mothers and their offspring at Day 10 postpartum. To test the youngs' suckling capability and the amount of maternal milk production, 10- and 15-day-old normal and PTU-treated pups were separated from their mothers for 4 hr in the morning and then reunited and allowed to suckle. Normal pups gained body weight at the end of both the first and second hour postreunion, while PTU pups gained only during the first hour and lost weight in the second hour of testing. When the pups were exchanged between normal and PTU mothers, opposite results were obtained, indicating that the reduced gain in hypothyroid rats was not due to impaired suckling capability, or insufficient sensory stimulation for milk secretion but to a decreased milk production of PTU mothers. In accordance with this, in lactating hypothyroid rats both the basal (presuckling) level and the suckling-induced rise of serum PRL were found significantly depressed.