Yixin Su

Early Pregnancy Maternal Lipid Profiles and the Risk of Gestational Diabetes Mellitus Stratified for Body Mass Index

Objective: To determine associations between lipid profiles in early pregnancy stratified by body mass index (BMI) and risk of developing gestational diabetes mellitus (GDM). Study Design: A total of 2488 healthy pregnant women were enrolled prospectively. Fasting plasma lipid profiles were measured at mean 11 weeks of gestation including triglycerides (TGs), total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and cholesterol (CHO). We assessed early pregnancy maternal lipid concentrations in different tertiles in association with the risk of GDM stratified for BMI. Multivariable logistic regression analyses were used to estimate the relative risk of GDM by calculating odds ratios and 95% confidence intervals (CIs). Results: In univariate analyses, pregnant women with GDM had significantly increased serum TG, CHO, LDL concentrations, LDL/HDL ratio, and decreased LDL concentrations, compared to control groups, each P < .01, respectively. After adjustment for confounders, there was a 1.8-fold increase in risk for GDM in the lean group (95% CI: 1.2-2.7) and 2.7-fold increase in the obese group (95% CI: 1.1-6.6), respectively, if TG ≥ 1.58 mmol/L. About a 50% decrease in the risk of GDM was observed in lean women with HDL ≥ 2.22 mmol/L (95% CI: 0.3-0.9). No significant correlations of other lipid profiles with the risk of developing GDM were observed. Conclusion: Early pregnancy dyslipidemia is associated with the risk of developing GDM. Lean or obese women with higher TG concentrations are at an increased risk for developing GDM while lean women with high HDL are protected.

Sex-specific effect of antenatal betamethasone exposure on renal oxidative stress induced by angiotensins in adult sheep

Prenatal glucocorticoid administration in clinically relevant doses reduces nephron number and renal function in adulthood and is associated with hypertension. Nephron loss in early life may predispose the kidney to other insults later but whether sex influences increases in renal susceptibility is unclear. Therefore, we determined, in male and female adult sheep, whether antenatal glucocorticoid (betamethasone) exposure increased 8-isoprostane (marker of oxidative stress) and protein excretion after acute nephron reduction and intrarenal infusions of angiotensin peptides. We also examined whether renal proximal tubule cells (PTCs) could contribute to alterations in 8-isoprostane excretion in a sex-specific fashion. In vivo, ANG II significantly increased 8-isoprostane excretion by 49% and protein excretion by 44% in male betamethasone- but not in female betamethasone- or vehicle-treated sheep. ANG-(1-7) decreased 8-isoprostane excretion but did not affect protein excretion in either group. In vitro, ANG II stimulated 8-isoprostane release from PTCs of male but not female betamethasone-treated sheep. Male betamethasone-exposed sheep had increased p47 phox abundance in the renal cortex while superoxide dismutase (SOD) activity was increased only in females. We conclude that antenatal glucocorticoid exposure enhances the susceptibility of the kidney to oxidative stress induced by ANG II in a sex-specific fashion and the renal proximal tubule is one target of the sex-specific effects of antenatal steroids. ANG-(1-7) may mitigate the impact of prenatal glucocorticoids on the kidney. P47 phox activation may be responsible for the increased oxidative stress and proteinuria in males. The protection from renal oxidative stress in females is associated with increased SOD activity.

Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

We have shown a sex-specific effect of fetal programming on Na(+) excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na(+) uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and female sheep, we measured Na(+) uptake under basal conditions and after stimulation with ANG II. To gain insight into the mechanisms involved, we also measured nitric oxide (NO) levels, ANG II receptor mRNA levels, and expression of Na(+)/H(+) exchanger 3. Basal Na(+) uptake increased more in cells from Beta-exposed male sheep than in cells from vehicle-exposed male sheep (400% vs. 300%, P < 0.00001). ANG II-stimulated Na(+) uptake was also greater in cells from Beta-exposed males. Beta exposure did not increase Na(+) uptake by RPTCs from female sheep. NO production was suppressed more by ANG II in RPTCs from Beta-exposed males than in RPTCs from either vehicle-exposed male or female sheep. Our data suggest that one site of the sex-specific effect of Beta-induced fetal programming in the kidney is the RPTC and that the enhanced Na(+) uptake induced by antenatal Beta in male RPTCs may be related to the suppression of NO in these cells.

An angiotensin-(1–7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme

Angiotensin 1-7 [ANG-(1-7)] is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic, and pro-oxidant effects of ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

Leptin Alters Adrenal Responsiveness by Decreasing Expression of ACTH-R, StAR, and P450c21 in Hypoxemic Fetal Sheep

The late gestation increase in adrenal responsiveness to adrenocorticotropin (ACTH) is dependent upon the upregulation of the ACTH receptor (ACTH-R), steroidogenic acute regulatory protein (StAR), and steroidogenic enzymes in the fetal adrenal. Long-term hypoxia decreases the expression of these and adrenal responsiveness to ACTH in vivo. Leptin, an adipocyte-derived hormone which attenuates the peripartum increase in fetal plasma cortisol is elevated in hypoxic fetuses. Therefore, we hypothesized that increases in plasma leptin will inhibit the expression of the ACTH-R, StAR, and steroidogenic enzymes and attenuate adrenal responsiveness in hypoxic fetuses. Spontaneously hypoxemic fetal sheep (132 days of gestation, PO2 ∼15 mm Hg) were infused with recombinant human leptin (n = 8) or saline (n = 7) for 96 hours. An ACTH challenge was performed at 72 hours of infusion to assess adrenal responsiveness. Plasma cortisol and ACTH were measured daily and adrenals were collected after 96 hours infusion for messenger RNA (mRNA) and protein expression measurement. Plasma cortisol concentrations were lower in leptin- compared with saline-infused fetuses (14.8 ± 3.2 vs 42.3 ± 9.6 ng/mL, P < .05), as was the cortisol:ACTH ratio (0.9 ± 0.074 vs 46 ± 1.49, P < .05). Increases in cortisol concentrations were blunted in the leptin-treated group after ACTH1-24 challenge (F = 12.2, P < .0001). Adrenal ACTH-R, StAR, and P450c21 expression levels were reduced in leptin-treated fetuses (P < .05), whereas the expression of Ob-Ra and Ob-Rb leptin receptor isoforms remained unchanged. Our results indicate that leptin blunts adrenal responsiveness in the late gestation hypoxemic fetus, and this effect appears mediated by decreased adrenal ACTH-R, StAR, and P450c21 expression.

Developmental changes in adrenocorticotrophin (ACTH)-induced expression of ACTH receptor and steroid acute regulatory protein mRNA in ovine fetal adrenal cells

Objectives: Adrenocorticotrophin (ACTH) plays an important role in mediating the increase in cortisol output in the late gestation sheep fetus. At the adrenal itself, heightened expression of ACTH receptor (ACTH-R) and steroid acute regulatory protein (StAR) appear to be important parallel changes. This study examined how ACTH affects ACTH-R and StAR mRNA expression, and cortisol production in adrenocortical cells isolated from fetuses of varying gestational age (dGA). We hypothesized that the ability of ACTH to stimulate its receptor and StAR mRNA expression would be greater close to term than earlier in development. Methods: Adrenals were obtained from fetuses (100-105, 120, or 135-139 dGA), and the cortical cells were dispersed. After 3 days of culture, cells were stimulated with ACTH1-24, and the cells and medium were collected at different time points (0, 3, 6, 9, 12, and 24 hours) for measurement of cortisol and ACTH-R and StAR mRNA. Results: Cortisol secretion was increased after ACTH treatment in all three age cohorts. Cells from the 135-139 dGA group secreted the most cortisol, followed by the 100-105 and then the 120 dGA groups (P < .05). ACTH-R mRNA levels before and after ACTH were higher in the late compared to both earlier groups. StAR mRNA levels before and after ACTH were higher in the 100-105 and 135 than in the 120 dGA group. The time to peak ACTH-R mRNA response was age-dependent, with the 100-105 dGA cells taking longer to attain maximum levels. Maximal StAR mRNA levels were not age-related. Conclusion: The data suggest that ACTH-R and StAR are indeed key mediators of fetal adrenocortical responsiveness, and that ACTH is able to up-regulate responsiveness, and hence cortisol production, by increasing their expression.

Developmental Aspects of Ovine Adrenal Adrenocorticotropic Hormone Receptor Expression

Objective: Maturation of adrenocortical function is important because a prepartum increase in fetal plasma glucocorticoids is required for survival after birth. Adrenal maturation may include alterations in the regulation of adrenocorticotropic hormone (ACTH) receptor expression. Therefore, we quantitated ACTH receptor expression in the ovine adrenal cortex during development and after manipulations to better understand the regulation of the adrenal receptor in vivo. Methods: For the ontogeny study, adrenals were obtained from fetuses at different stages of development, and the cortical tissue was stored at -80C until total RNA was extracted. The ACTH binding studies were done on adrenal membranes obtained from fetuses at two different ages using I125 (Phe-2, Nle-4) ACTH as the ligand. Plasma ACTH was measured by two-site immunoradiometric assay, and cortisol was measured by radioimmunoassay. ACTH receptor mRNA was quantitated by ribonuclease protection assay. The data were analyzed by analyses of variance. Results: ACTH receptor mRNa level progressively increased in fetal life; relative changes in receptor mRNA and binding were similar (3.0-fold and 2.4-fold, respectively). Physiologic increases in fetal plasma cortisol decreased adrenal ACTH receptor mRNA concentration, and there was a strong (r2 = 0.76, P < .002) linear relationship between fetal plasma ACTH concentration and receptor mRNA levels. Receptor mRNA stability increased in development, and message half-life was greater in adulthood than in fetal life. Conclusion: The data suggest that developmental increases in receptor expression are part of the maturation process in the fetal adrenal and that plasma ACTH concentration plays a major role in regulating ACTH receptor mRNA levels in vivo.

Antenatal glucocorticoid exposure enhances the inhibition of adrenal steroidogenesis by leptin in a sex-specific fashion.

Antenatal treatment with glucocorticoids (GC) poses long-lasting effects on endocrine and cardiovascular function. Given that leptin attenuates adrenal function and the reported sex differences in plasma leptin concentration, we hypothesized that antenatal GC will affect leptin levels and leptin modulation of adrenal function in a sex-specific manner. Pregnant sheep were randomly given betamethasone or vehicle at 80 days of gestational age, and offspring were allowed to deliver at term. Adrenocortical cells (ADC) were studied from male and female animals at 1.5 yr of age. Plasma leptin was increased 66% in male and 41% in female GC-treated animals (P < 0.05), but adrenal leptin mRNA was increased only in GC-treated males (P < 0.05). Whereas mRNA expression of adrenal leptin receptor isoforms showed sex (Ob-Ra and Ob-Rb) and treatment-dependent (Ob-Rb) differences, protein expression remained unchanged. GC-treated females showed greater plasma cortisol and greater ACTH-stimulated cortisol production (P < 0.05) in ADC. Leptin exerted a greater inhibitory effect on basal and stimulated cortisol by ADC from GC-treated males (P < 0.05), with no differences in females. Similarly, greater inhibitory effects on basal and ACTH-stimulated StAR and ACTH-R mRNA expression by leptin were observed in cells from GC males (P < 0.05), with no changes in females. Persistent effects of antenatal GC on leptin levels and leptin modulation of adrenal function are expressed in a sex-specific manner; males are more sensitive than females to the inhibitory influences of leptin on adrenal function, and this effect appears to be mediated by a greater inhibition of StAR and ACTH-R expression in adrenals of adult GC-treated males.

Angiotensinogen Import in Isolated Proximal Tubules: Evidence for Mitochondrial Trafficking and Uptake

The renal proximal tubules are a key functional component of the kidney and express the angiotensin precursor angiotensinogen; however, it is unclear the extent that tubular angiotensinogen reflects local synthesis or internalization. Therefore, the current study established the extent to which angiotensinogen is internalized by proximal tubules and the intracellular distribution. Proximal tubules were isolated from the kidney cortex of male sheep by enzymatic digestion and a discontinuous Percoll gradient. Tubules were incubated with radiolabeled 125I-angiotensinogen for 2 h at 37°C in serum/phenol-free DMEM/F12 media. Approximately 10% of exogenous 125I-angiotensinogen was internalized by sheep tubules. Subcellular fractionation revealed that 21 ± 4% of the internalized 125I-angiotensinogen associated with the mitochondrial fraction with additional labeling evident in the nucleus (60 ± 7%), endoplasmic reticulum (4 ± 0.5%), and cytosol (15 ± 4%; n = 4). Subsequent studies determined whether mitochondria directly internalized 125I-angiotensinogen using isolated mitochondria from renal cortex and human HK-2 proximal tubule cells. Sheep cortical and HK-2 mitochondria internalized 125I-angiotensinogen at a comparable rate of (33 ± 9 vs. 21 ± 10 pmol·min-1·mg protein-1; n = 3). Lastly, unlabeled angiotensinogen (100 nM) competed for 125I-angiotensinogen uptake to a greater extent than human albumin in HK-2 mitochondria (60 ± 2 vs. 16 ± 13%; P < 0.05, n = 3). Collectively, our data demonstrate angiotensinogen import and subsequent trafficking to the mitochondria in proximal tubules. We conclude that this pathway may constitute a source of the angiotensinogen precursor for the mitochondrial expression of angiotensin peptides.

The Impact of ACTH Receptor Knockdown on Fetal and Adult Ovine Adrenocortical Cell Function

Preparing the mammalian fetus for birth requires an increase in fetal plasma glucocorticoid levels. The mechanisms facilitating this increase are not fully known. It has been shown in sheep that the prepartum elevation in fetal plasma cortisol is accompanied by increases in adrenocorticotropin receptor (ACTH-R) expression in the fetal adrenal and in the adrenal responsiveness to stimulation. To determine the significance of the upregulation in ACTH-R expression on fetal adrenal function, the authors used small interfering RNA targeted to the ovine ACTH-R to reduce receptor expression and studied responses to stimulation in ovine adrenal cells. They studied fetal cells from late gestation after responsiveness had increased. They also studied adult cells to determine if maturation would influence the impact of receptor expression suppression on responsiveness. Fetal and adult cells were obtained, dispersed, transfected with receptor-targeted small interfering RNA or scrambled small interfering RNA, and subsequently stimulated with ACTH. Cells and media were harvested for measurements of gene and protein expression and cyclic adenosine monophosphate (cAMP) and cortisol levels. The ability of ACTH to upregulate its receptor or steroid acute regulatory protein was attenuated in fetal (P < .01) and adult cells (P < .01) by small interfering RNA treatment; the blockade was more pronounced in the adult cells (P < .01). The small interfering RNA treatment also blocked the cAMP response to ACTH in fetal (P < .001) and adult (P < .05) cells. This was accompanied by marked reductions in cortisol responses in both (P < .001 and P < .01, respectively). These data suggest that upregulation of the ACTH-R expression in late gestation is essential for the increase in adrenal steroidogenic capacity occurring then. The data also indicate that a reduction in the ACTH-R expression blocks the ability of the peptide to stimulate early steps in the steroidogenic pathway event after maturation is complete.