Roles of nitric oxide and angiotensin II in the impaired baroreflex gain of pregnancy

The present study tested the hypothesis that nitric oxide (NO) contributes to impaired baroreflex gain of pregnancy and that this action is enhanced by angiotensin II. To test these hypotheses, we quantified baroreflex control of heart rate in nonpregnant and pregnant conscious rabbits before and after: 1) blockade of NO synthase (NOS) with Nomega-nitro-L-arginine (20 mg/kg iv); 2) blockade of the angiotensin II AT1 receptor with L-158,809 (5 microg x kg(-1) x min(-1) iv); 3) infusion of angiotensin II (1 ng x kg(-1) x min(-1) nonpregnant, 1.6-4 ng x kg(-1) x min(-1) pregnant iv); 4) combined blockade of angiotensin II AT(1) receptors and NOS; and 5) combined infusion of angiotensin II and blockade of NOS. To determine the potential role of brain neuronal NOS (nNOS), mRNA and protein levels were measured in the paraventricular nucleus, nucleus of the solitary tract, caudal ventrolateral medulla, and rostral ventrolateral medulla in pregnant and nonpregnant rabbits. The decrease in baroreflex gain observed in pregnant rabbits (from 23.3 +/- 3.6 to 7.1 +/- 0.9 beats x min(-1) x mmHg(-1), P < 0.05) was not reversed by NOS blockade (to 8.3 +/- 2.5 beats x min(-1) x mmHg(-1)), angiotensin II blockade (to 5.0 +/- 1.1 beats x min(-1) x mmHg(-1)), or combined blockade (to 12.3 +/- 4.8 beats x min(-1) x mmHg(-1)). Angiotensin II infusion with (to 5.7 +/- 1.0 beats x min(-1) x mmHg(-1)) or without (to 8.4 +/- 2.4 beats x min(-1) x mmHg(-1)) NOS blockade also failed to improve baroreflex gain in pregnant or nonpregnant rabbits. In addition, nNOS mRNA and protein levels in cardiovascular brain regions were not different between nonpregnant and pregnant rabbits. Therefore, we conclude that NO, either alone or via an interaction with angiotensin II, is not responsible for decrease in baroreflex gain during pregnancy.     

Pregnancy and acute baroreflex resetting in conscious rabbits

To test the hypothesis that acute resetting of baroreflex control of heart rate (HR) is enhanced during pregnancy, we determined whether the rightward shift in the baroreflex relationship between arterial pressure and HR after arterial pressure is raised [~25 mmHg for 30 min, due to infusion of phenylephrine (PE) or methoxamine (Meth)] is greater in late pregnant compared with nonpregnant conscious rabbits. Baroreflex function was assessed by monitoring HR responses to both stepwise steady-state changes (n = 14) and rapid ramp changes (n = 10) in arterial pressure. Pregnancy decreased reflex gain, increased reflex minimum HR, and shifted the curves to a lower pressure level, when either the steady-state or ramp method was used (all changes, P < 0.05). When PE was used to increase pressure, resetting of steady-state curves was observed both before and during pregnancy, but the magnitude of the resetting was less in the pregnant rabbits. Further inspection of the data revealed that the size of the shift in pregnant rabbits was inversely related to the dose of PE. Because the pressure rise was the same in all experiments, PE appears to nonspecifically counteract acute resetting. When Meth was used instead to increase pressure, resetting of steady-state curves was similar in pregnant and nonpregnant rabbits and was unrelated to dose. Similarly, when reflex curves were generated using the ramp method, and either Meth or low doses of PE were used to increase pressure, no differences in the degree of resetting were observed between pregnant and nonpregnant rabbits. In summary, high doses of PE counteract acute resetting of baroreflex control of HR. More importantly, while baroreflex function is depressed, the ability of the baroreflex to reset appears to be preserved during pregnancy.     

Downregulated IRS-1 and PPARgamma in obese women with gestational diabetes: relationship to FFA during pregnancy

Gestational diabetes mellitus (GDM) is associated with elevated postprandial free fatty acids (FFA) and insulin resistance; however, little is known about the cellular mechanisms underlying insulin resistance to suppress lipolysis during gestation. We evaluated the longitudinal changes in insulin suppression of FFA before pregnancy and in early (12-14 wk) and late (34-36 wk) gestation in obese subjects with normal glucose tolerance and in obese GDM subjects. Abdominal subcutaneous adipose tissue biopsies were also obtained during cesarean delivery from normal obese pregnant (Preg-Con), GDM, and nonpregnant obese control (Non-Preg-Con) subjects during gynecological surgery. GDM subjects had higher basal plasma FFA before pregnancy (P = 0.055). Insulin's ability to suppress FFA levels declined from early to late gestation in both GDM and Preg-Con subjects and was significantly less in GDM subjects compared with Preg-Con subjects over time (P = 0.025). Adipose tissue insulin receptor substrate (IRS)-1 protein levels were 43% lower (P = 0.02) and p85alpha subunit of phosphatidylinositol 3-kinase was twofold higher (P = 0.03) in GDM compared with Preg-Con subjects. The levels of peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA and protein were lower by 38% in Preg-Con (P = 0.006) and by 48% in GDM subjects (P = 0.005) compared with Non-Preg controls. Lipoprotein lipase and fatty acid-binding protein-2 mRNA levels were 73 and 52% lower in GDM compared with Preg-Con subjects (P < 0.002). Thus GDM women have decreased IRS-1, which may contribute to reduced insulin suppression of lipolysis with advancing gestation. Decreased PPARgamma and its target genes may be part of the molecular mechanism to accelerate fat catabolism to meet fetal nutrient demand in late gestation.     

Cerebral Blood Flow Links Insulin Resistance and Baroreflex Sensitivity

Insulin resistance confers risk for diabetes mellitus and associates with a reduced capacity of the arterial baroreflex to regulate blood pressure. Importantly, several brain regions that comprise the central autonomic network, which controls the baroreflex, are also sensitive to the neuromodulatory effects of insulin. However, it is unknown whether peripheral insulin resistance relates to activity within central autonomic network regions, which may in turn relate to reduced baroreflex regulation. Accordingly, we tested whether resting cerebral blood flow within central autonomic regions statistically mediated the relationship between insulin resistance and an indirect indicator of baroreflex regulation; namely, baroreflex sensitivity. Subjects were 92 community-dwelling adults free of confounding medical illnesses (48 men, 30-50 years old) who completed protocols to assess fasting insulin and glucose levels, resting baroreflex sensitivity, and resting cerebral blood flow. Baroreflex sensitivity was quantified by measuring the magnitude of spontaneous and sequential associations between beat-by-beat systolic blood pressure and heart rate changes. Individuals with greater insulin resistance, as measured by the homeostatic model assessment, exhibited reduced baroreflex sensitivity (b = -0.16, p < .05). Moreover, the relationship between insulin resistance and baroreflex sensitivity was statistically mediated by cerebral blood flow in central autonomic regions, including the insula and cingulate cortex (mediation coefficients < -0.06, p-values < .01). Activity within the central autonomic network may link insulin resistance to reduced baroreflex sensitivity. Our observations may help to characterize the neural pathways by which insulin resistance, and possibly diabetes mellitus, relates to adverse cardiovascular outcomes.     

Accretion of visceral fat and hepatic insulin resistance in pregnant rats

Insulin resistance (IR) is a hallmark of pregnancy. Because increased visceral fat (VF) is associated with IR in nonpregnant states, we reasoned that fat accretion might be important in the development of IR during pregnancy. To determine whether VF depots increase in pregnancy and whether VF contributes to IR, we studied three groups of 6-mo-old female Sprague-Dawley rats: 1) nonpregnant sham-operated rats (Nonpreg; n = 6), 2) pregnant sham-operated rats (Preg; n = 6), and 3) pregnant rats in which VF was surgically removed 1 mo before mating (PVF-; n = 6). VF doubled by day 19 of pregnancy (Nonpreg 5.1 +/- 0.3, Preg 10.0 +/- 1.0 g, P < 0.01), and PVF- had similar amounts of VF compared with Nonpreg (PVF- 4.6 +/- 0.8 g). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp in late gestation in chronically catheterized unstressed rats. Glucose IR (mg.kg(-1).min(-1)) was highest in Nonpreg (19.4 +/- 2.0), lowest in Preg (11.1 +/- 1.4), and intermediate in PVF- (14.7 +/- 0.6; P < 0.001 between all groups). During the clamp, Nonpreg had greater hepatic insulin sensitivity than Preg [hepatic glucose production (HGP): Nonpreg 4.5 +/- 1.3, Preg 9.3 +/- 0.5 mg.kg(-1).min(-1); P < 0.001]. With decreased VF, hepatic insulin sensitivity was similar to nonpregnant levels in PVF- (HGP 4.9 +/- 0.8 mg.kg(-1).min(-1)). Both pregnant groups had lower peripheral glucose uptake compared with Nonpreg. In parallel with hepatic insulin sensitivity, hepatic triglyceride content was increased in pregnancy (Nonpreg 1.9 +/- 0.4 vs. Preg 3.2 +/- 0.3 mg/g) and decreased with removal of VF (PVF- 1.3 +/- 0.4 mg/g; P < 0.05). Accretion of visceral fat is an important component in the development of hepatic IR in pregnancy, and accumulation of hepatic triglycerides is a mechanism by which visceral fat may modulate insulin action in pregnancy.     

Impairment of T cell-mediated immunity to Listeria monocytogenes in pregnant mice

In order to study pregnancy-induced changes in cell-mediated immunity to Listeria monocytogenes, acquired resistance and T cell functions in pregnant mice were compared with those in nonpregnant mice after immunization with viable listerial cells. Impaired generation of acquired resistance was evident in pregnant mice from the impaired elimination of bacteria and poor survival after secondary challenge. Delayed footpad reactivity to listerial antigen was also lower in the pregnant mice. When immune spleen cells were examined for their ability to produce macrophage activating factor in vitro, culture supernatants from pregnant-mouse spleen cells with listerial antigen showed far less ability to render macrophages cytostatic for P815 mastocytoma cells. To elucidate further the impairment of listeria-immune T cell generation in pregnant mice, a local transfer experiment was carried out. When a given number of immune spleen cells was transferred locally into the footpads of naive mice, both delayed footpad reaction and local protection were much lower in the pregnant mice. This local transferability of the reactions was abrogated after treatment of cells with anti-Thy 1 antibody plus complement. These findings indicate that pregnancy impairs the generation of specific T cells capable of contributing to acquired resistance to L. monocytogenes. Possible mechanisms for this impairment and the relationship to macrophage functions are discussed.     

Pregnancy alters hemodynamic responses to hemorrhage in conscious rabbits

Pregnant animals are less able to maintain mean arterial pressure (MAP) during hemorrhage compared with nonpregnant animals, but the hemodynamic basis of this difference is unknown. The hypothesis that pregnancy attenuates responses of cardiac output, as well as total peripheral resistance (TPR) and femoral conductance, to hemorrhage was tested in conscious rabbits in both the pregnant and nonpregnant state (n = 10). During continuous slow blood loss (2% of the initial blood volume per minute), MAP was maintained initially in both groups. However, MAP then abruptly decreased to <45 mmHg in all animals after a smaller percentage of the initial blood volume was removed in pregnant compared with nonpregnant rabbits (43.6 +/- 1.7%, nonpregnant; 29.6 +/- 2.2%, pregnant; P < 0.005). The more rapid transition to hypotension exhibited by pregnant rabbits was associated with greater initial falls in cardiac output (-56 +/- 10 ml/min, nonpregnant; -216 +/- 33 ml/min, pregnant; P < 0.005) and stroke volume (0.8 +/- 0.1 ml/beat, nonpregnant; -1.3 +/- 0.1 ml/beat, pregnant; P < 0.05). In addition, the increase in TPR as a function of the decrease in cardiac output was markedly attenuated (P < 0.0001) during pregnancy. Whereas femoral conductance decreased in nonpregnant rabbits, it did not change significantly in pregnant animals. In conclusion, the lesser ability of conscious pregnant rabbits to maintain MAP during hemorrhage is due largely to a greater decrease in cardiac output but also to inadequate reflex increases in TPR, possibly in part in the femoral vascular bed.     

Effect of pregnancy on joint contracture in the rat knee.

As there is evidence that ligamentous laxity is affected by the female hormones, we hypothesized that hormonal changes occurring during pregnancy could have a therapeutic role in preventing the development of a joint contracture. Knee joint contractures were created in pregnant and nonpregnant rats. After 2 wk of immobilization, the degree of contracture was measured with structural properties of the medial collateral and anterior cruciate ligaments and the pubic symphysis. Although not statistically significant, there was a general trend toward reduced contracture in pregnant compared with nonpregnant rats. Cutting the posterior capsule significantly decreased contracture for both the pregnant and nonpregnant groups, confirming the contribution of capsular structures to contracture. Ultimate loads of the medial collateral and anterior cruciate ligaments significantly decreased after immobilization compared with control, but there was no significant effect due to pregnancy. Stiffness and ultimate load of the pubic symphysis were not significantly different between pregnant and nonpregnant groups. The trend toward reduced contracture with pregnancy points toward a possible therapeutic role for female hormones in the prevention of postoperative and/or posttraumatic joint contracture.     

Thermoregulatory and acute-phase responses to endotoxin of full- term-pregnant rabbits

Fever, a fall in the plasma level of iron (Fe), and rises in the levels of certain plasma glycoproteins (indexed by protein-bound-N-acetylneuraminic acid [NANA]) normally occur during infection; they are thought to be mutually enhancing in host defense. It has been reported that fever is suppressed at full term of pregnancy; however, it is not known whether the blood chemical changes are similarly affected. Also, the mechanism for the suppression of fever is controversial. Since uteroplacental blood flow is at its maximum near term, competition between the demands of the fetoplacental unit and of thermoregulatory effectors might result in underperfusion of thermogenic tissues and therefore provide a basis for the lack of fever. To examine these questions, the changes in colonic temperature (Tco) and regional blood flow induced by Salmonella enteritidis endotoxin (LPS, 2 micrograms/kg iv) were compared in conscious nonpregnant and 30-day-pregnant rabbits 35 min after injection, using 15-microgram radiolabeled microspheres. In different rabbits, the effects of LPS on Tco and plasma Fe and NANA levels were measured before mating and at term. LPS induced fevers similar in heights and courses in both nonpregnant and full-term pregnant rabbits It caused decreases in the blood flows to brain, tongue, mammary gland, small intestine, and ear and increases in the blood flows to masseter muscle, bone, liver (hepatic artery), and left ventricle; blood flows to the kidneys, spleen, right ventricle, ovaries, and myometrium did not change. There were no significant differences in these vascular responses between nonpregnant and 30-day-pregnant rabbits, except a 28% reduction in the blood flow to the placentas.     

Differential sensitivity of pancreatic beta-cells to phorbol ester TPA and C-kinase inhibitor H-7 in nonpregnant and pregnant rats

In order to elucidate the possible role of C-kinase in exaggerated insulin release in pregnancy, the effects of phorbol ester TPA and a C-kinase inhibitor H-7 were investigated using the isolated perfused pancreas from nonpregnant and pregnant rats. At the termination of perfusion, the insulin content of the perfused pancreas was determined to estimate insulin biosynthesis. Insulin release from the perfused pancreas was markedly augmented by 20 nM TPA in the presence of 4.4 mM glucose in pregnant rats, but not in nonpregnant rats. When glucose concentrations in the perfusate were raised to 16.7 mM, insulin release from the perfused pancreas was profoundly enhanced in pregnant rats. TPA further augmented insulin release, but the insulin content was not affected by TPA. In contrast to the considerable effect of TPA in the presence of 4.4 mM glucose, the potentiating effect of TPA on insulin release was rather weaker in pregnant than in non-pregnant rats in the presence of 16.7 mM glucose. The release of insulin induced by 16.7 mM glucose was inhibited by the addition of 100 microM H-7 in nonpregnant rats, whereas insulin release from pregnant rat pancreases was not altered. Thus, the effect of TPA and H-7 on insulin release can be more clearly observed in the beta-cells of nonpregnant rats than those of pregnant ones when maximal concentrations of glucose are used as a stimulant. Exaggerated insulin release caused by glucose in pregnancy may be due to already fully activated C-kinase in the beta-cells.     

Functional and histochemical characterization of a uterine adrenergic denervation process in pregnant rats

The time course of pregnancy-induced changes in the contractile responses of isolated uterine rings and sympathetic innervation pattern were studied using electric field stimulation and histofluorescence techniques, respectively, in intact and 6-hydroxydopamine-treated rats. Neurally mediated contractions elicited by field stimulation (0.6 msec, 1-70 Hz, 40 V) were measured in uterine preparations obtained from nonpregnant, 6-hydroxydopamine-treated and 5-, 10-, 15-, 18-, and 22-day (term) pregnant rats. At all frequencies, the amplitudes of contractions were highest in nonpregnant uteri. Stimulation at 1-2.5 Hz evoked contractions in 10-day pregnant uteri but failed to cause contractions on Day 5 and from Day 15 onward. In uterine preparations obtained from term and from 6-hydroxydopamine-treated rats, contractions could not be evoked by stimulation at 1-20 Hz. Fluorescence histochemistry of uterine adrenergic nerves revealed rich perivascular and myometrial innervation in nonpregnant and in pregnant rats through Day 10. Degeneration and loss of adrenergic nerve fibers was apparent by Day 15, and fluorescent myometrial and perivascular nerves were practically absent by Day 22. These findings demonstrate a progressive, frequency-related reduction of nerve-mediated uterine contractions beginning in midterm pregnancy, in parallel with a gradual loss of adrenergic nerve fibers. Pregnancy-induced nerve degeneration may promote the development of nonsynaptic alpha-adrenergic uterine contractile activity towards term. The reduced responsiveness of uterine smooth muscle to electric field stimulation in early pregnancy appears to be unrelated to alterations in uterine innervation but may be related to changes associated with implantation.     

Effect of high levels of insulin on glucose utilization and glucose production in pregnant and nonpregnant sheep

The present study was conducted to test the hypothesis that pregnancy in sheep alters the effects of insulin on glucose utilization and glucose production. Euglycemic, hyperinsulinemic glucose clamp experiments were performed in chronically catheterized, unstressed, fed or 24-hr fasted, nonpregnant sheep and fed, pregnant sheep. Endogenous glucose production rate for the whole sheep and glucose utilization rate of the uterine and nonuterine maternal tissues were measured in control and high-insulin periods by tracer technique using [6-3H]glucose. Control glucose utilization rate in the fed, nonpregnant sheep was significantly (P less than 0.05) greater than that in the fasted, nonpregnant sheep, 2.29 +/- 0.17 and 1.86 +/- 0.11 mg/min/kg, respectively, and also in the nonuterine maternal tissues of the pregnant sheep (1.71 +/- 0.18 mg/min/kg). Insulin stimulated glucose utilization 116.4 +/- 14.8% in the fed, nonpregnant sheep but only 82.8 +/- 11.0% in the fasted, nonpregnant sheep and 94.2 +/- 14.3% in the nonuterine tissues of the fed, pregnant sheep. Also, insulin suppressed endogenous glucose production to 53.2 +/- 5.6% in the fed, nonpregnant sheep, to 3.9 +/- 3.1% in the fasted, nonpregnant sheep, and to 9.0 +/- 3.7% in the fed, pregnant sheep. In the pregnant animals, uterine glucose uptake and uterine glucose utilization were not different and were not altered by changes in maternal insulin concentration. The results indicate that during late pregnancy glucose utilization is reduced and resistance to the effect of insulin to enhance glucose utilization is present in the nonuterine maternal tissues compared with nonpregnant, fed sheep. In contrast, the effectiveness of insulin to suppress glucose production in the pregnant sheep is greater than that in nonpregnant, fed sheep. These results also demonstrate that differential changes in the effect of insulin can exist simultaneously between peripheral (glucose consuming) and central (glucose producing) tissues. The changes in glucose utilization and in insulin effect in the pregnant sheep are both qualitatively and quantitatively similar to those of the nonpregnant sheep when fasted, suggesting that similar substrate and/or hormonal factors may be involved.     

AT1 receptor block does not affect arterial baroreflex during pregnancy in rabbits

The role of ANG II in the arterial baroreflex control of renal sympathetic nerve activity (RSNA) in eight term-pregnant (P) and eight nonpregnant (NP) conscious rabbits was assessed using sequential intracerebroventricular and intravenous infusions of losartan, an AT1 receptor antagonist. The blood pressure (BP)-RSNA relationship was generated by sequential inflations of aortic and vena caval perivascular occluders. Pregnant rabbits exhibited a lower maximal RSNA reflex gain (-44%) that was primarily due to a reduction in the maximal sympathetic response to hypotension (P, 248 +/- 20% vs. NP, 357 +/- 41% of rest RSNA, P < 0.05). Intracerebroventricular losartan decreased resting BP in P (by 9 +/- 3 mmHg, P < 0.05) but not NP rabbits, and had no effect on the RSNA baroreflex in either group. Subsequent intravenous losartan decreased resting BP in NP and further decreased BP in P rabbits, but had no significant effect on the maximal RSNA reflex gain. ANG II may have an enhanced role in the tonic support of BP in pregnancy, but does not mediate the gestational depression in the arterial baroreflex control of RSNA in rabbits.     

Enhanced vascular effects of the Ca(2+) channel agonist Bay K 8644 in pregnant rabbits

Hemodynamic studies were performed to determine if blunting of vascular pressor responsiveness to vasoconstrictors during pregnancy may be due to impaired L-type voltage-dependent calcium channels (L-VDCC). Bay K 8644 (BAY), an L-VDCC agonist, was infused in pregnant and nonpregnant anesthetized rabbits (10, 20, 40, and 60 microg/kg) and pregnant and nonpregnant conscious, chronically instrumented (conscious) rabbits (10, 25, and 50 microg/kg). BAY infusions resulted in greater elevation of mean arterial pressure in both anesthetized pregnant (n = 6) vs. nonpregnant (n = 6) (P < 0.05) and conscious pregnant (n = 10) vs. nonpregnant (n = 10) rabbits (P < 0.05). Fractional increase over baseline of total peripheral resistance index was greater in pregnant (36 +/- 5 to 78 +/- 14%) vs. nonpregnant rabbits (14 +/- 4 to 52 +/- 6%) (P < 0.02). Cardiac output index did not differ. There was a single high-affinity L-VDCC antagonist aortic binding site with similar number and affinity in pregnant (n = 7) and nonpregnant (n = 7) rabbits. In conclusion, stimulation of L-VDCC induces greater pressor responses in pregnant rabbits with heightened peripheral vasoconstriction. This does not appear to be due to a change in L-VDCC receptor parameters.     

Systemic vascular reactivity during high-altitude pregnancy

There is an increased incidence of preeclampsia at high compared with low altitude. Increased vasoreactivity, possibly due to a deficiency of vasodilator prostaglandins, is thought to contribute to the etiology of preeclampsia. We sought to determine whether high-altitude exposure increased systemic vascular reactivity during pregnancy. We measured systemic vascular reactivity and contractile sensitivity of isolated aortic rings from pregnant and nonpregnant guinea pigs kept for 6 wk at either simulated high altitude (3,900 m) or low altitude (1,600 m). We found that pregnancy at high compared with low altitude increased baseline systemic vascular resistance (SVR) but not the SVR response to angiotensin II in awake unstressed guinea pigs. Contractile sensitivity to norepinephrine was also increased in aortic rings isolated from high-altitude compared with low-altitude pregnant animals. Meclofenamate, a prostaglandin synthesis inhibitor, did not equalize vasoreactivity in the high- and low-altitude pregnant guinea pigs or in their isolated aortic rings. We concluded that pregnancy at high compared with low altitude increased base-line SVR and aortic contractile sensitivity but that mechanisms other than decreased vasodilator prostaglandin production were responsible.     

Normal pregnancy: mechanisms underlying the paradox of a ouabain-resistant state with elevated endogenous ouabain, suppressed arterial sodium calcium exchange, and low blood pressure

Endogenous cardiotonic steroids (CTS) raise blood pressure (BP) via vascular sodium calcium exchange (NCX1.3) and transient receptor-operated channels (TRPCs). Circulating CTS are superelevated in pregnancy-induced hypertension and preeclampsia. However, their significance in normal pregnancy, where BP is low, is paradoxical. Here we test the hypothesis that vascular resistance to endogenous ouabain (EO) develops in normal pregnancy and is mediated by reduced expression of NCX1.3 and TRPCs. We determined plasma and adrenal levels of EO and the impact of exogenous ouabain in pregnancy on arterial expression of Na(+) pumps, NCX1.3, TRPC3, and TRPC6 and BP. Pregnant (embryonic day 4) and nonpregnant rats received infusions of ouabain or vehicle. At 14-16 days, tissues and plasma were collected for blotting and EO assay by radioimmunoassay (RIA), liquid chromatography (LC)-RIA, and LC-multidimensional mass spectrometry (MS3). BP (-8 mmHg; P < 0.05) and NCX1.3 expression fell (aorta -60% and mesenteric artery -30%; P < 0.001) in pregnancy while TRPC expression was unchanged. Circulating EO increased (1.14 ± 0.13 nM) vs. nonpregnant (0.6 ± 0.08 nM; P < 0.05) and was confirmed by LC-MS3 and LC-RIA. LC-MS3 revealed two previously unknown isomers of EO; one increased ～90-fold in pregnancy. Adrenal EO but not isomers were increased in pregnancy. In nonpregnant rats, similar infusions of ouabain raised BP (+24 ± 3 mmHg; P < 0.001). In ouabain-infused rats, impaired fetal and placental growth occurred with no BP increase. In summary, normal pregnancy is an ouabain-resistant state associated with low BP, elevated circulating levels of EO, two novel steroidal EO isomers, and increased adrenal mass and EO content. Ouabain raises BP only in nonpregnant animals. Vascular resistance to the chronic pressor activity of endogenous and exogenous ouabain is mediated by suppressed NCX1.3 and reduced sensitivity of events downstream of Ca(2+) entry. The mechanisms of EO resistance and the impaired fetal and placental growth due to elevated ouabain may be important in pregnancy-induced hypertension (PIH) and preeclampsia (PE).     

Pregnancy diagnosis in sows: direct ELISA for estrone in feces and its prospects for an on-farm test, in comparison to ultrasonography

The usefulness of fecal estrone (E1) measurement as a tool for pregnancy diagnosis was investigated. Concentrations of E1 were measured in feces from pregnant and nonpregnant sows by a direct ELISA without extraction. Highly significant differences in E1 concentrations were found in feces from nonpregnant and pregnant sows (P = 0.016 to < 0.001). Pregnancy diagnosis on Days 26 to 32 after insemination, based both on fecal E1 concentrations as measured by ELISA and ultrasonography using a 5.0 MHz linear-array transducer, was performed in a group of 496 gilts and sows. The fecal E1 test had a sensitivity (correct diagnosis of pregnancy) of 96.5% and a specificity (correct diagnosis of nonpregnancy) of 93.6%, using 3.65 ng E1/g feces as a cut-off value. For ultrasonographic pregnancy diagnosis the test sensitivity and specificity were 99.3 and 92.5%, respectively. Although an increase of fecal E1 concentrations was noticed for increasing litter sizes, the results indicated that these concentrations could not be used to predict litter size. It is concluded that the distribution of fecal E1 concentrations in both nonpregnant and pregnant sows offers a suitable basis for the development of a simple, sow-side pregnancy test.     

17β-Estradiol treatment is unable to reproduce p85α redistribution associated with gestational insulin resistance in rats

Maternal metabolic adaptations are essential to ensure proper fetal development. According to changes in insulin sensitivity, pregnancy can be divided into two periods: early pregnancy, characterized by an increase in maternal insulin sensitivity, and late pregnancy, in which there is a significant increase in insulin resistance. The aims of the present work were two-fold: firstly, the molecular mechanisms associated with the development of pregnancy-related insulin resistance in peripheral tissues, mainly retroperitoneal adipose tissue and skeletal muscle, were studied in pregnant rats at 6, 11, and 16 days gestation. Secondly, the role of 17β-estradiol in this process was elucidated in an animal model consisting of ovariectomized rats treated with 17β-estradiol to mimic plasma gestational levels. The results support the conclusion that retroperitoneal adipose tissue plays a pivotal role in the decrease in insulin sensitivity during pregnancy, through a mechanism that involves p85α redistribution to the insulin receptor and impairment of Glut4 translocation to the plasma membrane. Treatment with 17β-estradiol did not reproduce the molecular adaptations that occur during pregnancy, suggesting that other hormonal factors presents in gestation but absent in our experimental model are responsible for p85α redistribution to the insulin receptor.     

Validation of simple indexes to assess insulin sensitivity during pregnancy in Wistar and Sprague-Dawley rats

Insulin resistance plays a role in the pathogenesis of diabetes, including gestational diabetes. The glucose clamp is considered the gold standard for determining in vivo insulin sensitivity, both in human and in animal models. However, the clamp is laborious, time consuming and, in animals, requires anesthesia and collection of multiple blood samples. In human studies, a number of simple indexes, derived from fasting glucose and insulin levels, have been obtained and validated against the glucose clamp. However, these indexes have not been validated in rats and their accuracy in predicting altered insulin sensitivity remains to be established. In the present study, we have evaluated whether indirect estimates based on fasting glucose and insulin levels are valid predictors of insulin sensitivity in nonpregnant and 20-day-pregnant Wistar and Sprague-Dawley rats. We have analyzed the homeostasis model assessment of insulin resistance (HOMA-IR), the quantitative insulin sensitivity check index (QUICKI), and the fasting glucose-to-insulin ratio (FGIR) by comparing them with the insulin sensitivity (SI(Clamp)) values obtained during the hyperinsulinemic-isoglycemic clamp. We have performed a calibration analysis to evaluate the ability of these indexes to accurately predict insulin sensitivity as determined by the reference glucose clamp. Finally, to assess the reliability of these indexes for the identification of animals with impaired insulin sensitivity, performance of the indexes was analyzed by receiver operating characteristic (ROC) curves in Wistar and Sprague-Dawley rats. We found that HOMA-IR, QUICKI, and FGIR correlated significantly with SI(Clamp), exhibited good sensitivity and specificity, accurately predicted SI(Clamp), and yielded lower insulin sensitivity in pregnant than in nonpregnant rats. Together, our data demonstrate that these indexes provide an easy and accurate measure of insulin sensitivity during pregnancy in the rat.