Jianrong Yao

IUGR decreases PPARγ and SETD8 Expression in neonatal rat lung and these effects are ameliorated by maternal DHA supplementation

Intrauterine growth restriction (IUGR) is associated with altered lung development in human and rat. The transcription factor PPARγ, is thought to contribute to lung development. PPARγ is activated by docosahexanoic acid (DHA). One contribution of PPARγ to lung development may be its direct regulation of chromatin modifying enzymes, such as Setd8. In this study, we hypothesized that IUGR would result in a gender-specific reduction in PPARγ, Setd8 and associated H4K20Me levels in the neonatal rat lung. Because DHA activates PPARγ, we also hypothesized that maternal DHA supplementation would normalize PPARγ, Setd8, and H4K20Me levels in the IUGR rat lung. We found that IUGR decreased PPARγ levels, with an associated decrease in Setd8 levels in both male and female rat lungs. Levels of the Setd8-dependent histone modification, H4K20Me, were reduced on the PPARγ gene in both males and females while whole lung H4K20Me was only reduced in male lung. Maternal DHA supplementation ameliorated these effects in offspring. We conclude that IUGR decreases lung PPARγ, Setd8 and PPARγ H4K20Me independent of gender, while decreasing whole lung H4K20Me in males only. These outcomes are offset by maternal DHA. We speculate that maintenance of the epigenetic milieu may be one role of PPARγ in the lung and suggests a novel benefit of maternal DHA supplementation in IUGR.

Glycaemic regulation and insulin secretion are abnormal in cystic fibrosis pigs despite sparing of islet cell mass

Diabetes is a common and significant co-morbidity in cystic fibrosis (CF). The pathogenesis of cystic fibrosis related diabetes (CFRD) is incompletely understood. Because exocrine pancreatic disease is similar between humans and pigs with CF, the CF pig model has the potential to contribute significantly to the understanding of CFRD pathogenesis. We determined the structure of the endocrine pancreas in fetal, newborn and older CF and non-CF pigs and assessed endocrine pancreas function by intravenous glucose tolerance test (IV-GTT). In fetal pigs, pancreatic insulin and glucagon density was similar between CF and non-CF. In newborn and older pigs, the insulin and glucagon density was unchanged between CF and non-CF per total pancreatic area, but increased per remnant lobular tissue in CF reflecting exocrine pancreatic loss. Although fasting glucose levels were not different between CF and non-CF newborns, CF newborns demonstrated impaired glucose tolerance and increased glucose area under the curve during IV-GTT. Second phase insulin secretion responsiveness was impaired in CF newborn pigs and significantly lower than that observed in non-CF newborns. Older CF pigs had elevated random blood glucose levels compared with non-CF. In summary, glycaemic abnormalities and insulin secretion defects were present in newborn CF pigs and spontaneous hyperglycaemia developed over time. Functional changes in CF pig pancreas were not associated with a decline in islet cell mass. Our results suggest that functional islet abnormalities, independent of structural islet loss, contribute to the early pathogenesis of CFRD.

Long-chain polyunsaturated fatty acid levels in US donor human milk: meeting the needs of premature infants?

Objective:To determine fatty acid levels in the US donor milk supply.Study Design:Donor human milk samples from Iowa (n=62), Texas (n=5), North Carolina (n=5) and California (n=5) were analyzed by gas chromatography. Levels in the Iowa donor milk were compared before and after pasteurization using Students t-test. Docosahexaenoic acid (DHA) and arachidonic acid (ARA) levels were compared among all milk banks using analysis of variance.Result:ARA (0.4 pre, 0.4 post, P=0.18) and DHA (0.073 pre, 0.073 post, P=0.84) were not affected by pasteurization. DHA varied between banks (P<0.0001), whereas ARA did not (P=0.3). DHA levels from all banks were lower than published values for maternal milk and infant formula (P<0.0001).Conclusion:Pasteurization of breastmilk does not affect DHA or ARA levels. However, DHA content in US donor milk varies with bank location and may not meet the recommended provision for preterm infants.

Effect of Insulin and Dexamethasone on Fetal Assimilation of Maternal Glucose

The growing fetus depends upon transfer of glucose from maternal blood to fetal tissues. Insulin and glucocorticoid impact maternal glucose metabolism, but the effects of these hormones on fetal glucose assimilation in vivo are understudied. We thus used positron emission tomography imaging to determine the disposition of [(18)F]fluorodeoxyglucose (FDG) in rats on gestational d 20, quantifying the kinetic competition of maternal tissues and fetus for glucose. Three fasting maternal states were studied: after 2-d dexamethasone (DEX), during euglycemic hyperinsulinemic clamp insulin receiving (INS), and control (CON). In CON and DEX mothers, FDG accumulation in fetuses and placentae was substantial, rivaling that of maternal brain. By contrast, FDG accumulation was reduced in INS fetuses, placentae, and maternal brain by approximately 2-fold, despite no diminution in FDG extraction kinetics from maternal blood into these structures. The reduced FDG accumulation was due to more rapid clearance of FDG from the circulation in INS mothers, related to increased FDG avidity in INS select maternal tissues, including skeletal muscle, brown adipose tissue, and heart. DEX treatment of mothers reduced fetal weight by nearly 10%. Nonetheless, the accumulation of FDG into placentae and fetuses was similar in DEX and CON mothers. In our rat model, fetal growth restriction induced by DEX does not involve diminished glucose transport to the fetus. Maternal insulin action has little effect on the inherent avidity of the fetal-placental unit for glucose but increases glucose utilization by maternal tissues, thus indirectly reducing the glucose available to the fetus.

Endogenous peroxisome proliferator-activated receptor-γ augments fatty acid uptake in oxidative muscle

In the setting of insulin resistance, agonists of peroxisome proliferator-activated receptor (PPAR)-gamma restore insulin action in muscle and promote lipid redistribution. Mice with muscle-specific knockout of PPARgamma (MuPPARgammaKO) develop excess adiposity, despite reduced food intake and normal glucose disposal in muscle. To understand the relation between muscle PPARgamma and lipid accumulation, we studied the fuel energetics of MuPPARgammaKO mice. Compared with controls, MuPPARgammaKO mice exhibited significantly increased ambulatory activity, muscle mitochondrial uncoupling, and respiratory quotient. Fitting with this latter finding, MuPPARgammaKO animals compared with control siblings exhibited a 25% reduction in the uptake of the fatty acid tracer 2-bromo-palmitate (P < 0.05) and a 13% increase in serum nonesterified fatty acids (P = 0.05). These abnormalities were associated with no change in AMP kinase (AMPK) phosphorylation, AMPK activity, or phosphorylation of acetyl-CoA carboxylase in muscle and occurred despite increased expression of fatty acid transport protein 1. Palmitate oxidation was not significantly altered in MuPPARgammaKO mice despite the increased expression of several genes promoting lipid oxidation. These data demonstrate that PPARgamma, even in the absence of exogenous activators, is required for normal rates of fatty acid uptake in oxidative skeletal muscle via mechanisms independent of AMPK and fatty acid transport protein 1. Thus, when PPARgamma activity in muscle is absent or reduced, there will be decreased fatty acid disposal leading to diminished energy utilization and ultimately adiposity.

Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography

Background Complex but common maternal diseases such as diabetes and obesity contribute to adverse fetal outcomes. Understanding of the mechanisms involved is hampered by difficulty in isolating individual elements of complex maternal states in vivo. We approached this problem in the context of maternal diabetes and sought an approach to expose the developing fetus in vivo to isolated hyperglycemia in the pregnant rat. Methodology and Principal Findings We hypothesized that glucose infused into the arterial supply of one uterine horn would more highly expose fetuses in the ipsilateral versus contralateral uterine horn. To test this, the glucose tracer [18F]fluorodeoxyglucose (FDG) was infused via the left uterine artery. Regional glucose uptake into maternal tissues and fetuses was quantified using positron emission tomography (PET). Upon infusion, FDG accumulation began in the left-sided placentae, subsequently spreading to the fetuses. Over two hours after completion of the infusion, FDG accumulation was significantly greater in left compared to right uterine horn fetuses, favoring the left by 1.9±0.1 and 2.8±0.3 fold under fasted and hyperinsulinemic conditions (p<10−11 n = 32-35 and p<10−12 n = 27–45) respectively. By contrast, centrally administered [3H]-2-deoxyglucose accumulated equally between the fetuses of the two uterine horns. Induction of significant hyperglycemia (103 mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained. Conclusions and Significance This approach exposes selected fetuses to localized hyperglycemia in vivo, minimizing exposure of the mother and thus secondary effects. Additionally, a set of less exposed internal control fetuses are maintained for comparison, allowing direct study of the in vivo fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health.

Maternal Hyperglycemia Directly and Rapidly Induces Cardiac Septal Overgrowth in Fetal Rats

Cardiac septal overgrowth complicates 10–40% of births from diabetic mothers, but perplexingly hyperglycemia markers during pregnancy are not reliably predictive. We thus tested whether fetal exposure to hyperglycemia is sufficient to induce fetal cardiac septal overgrowth even in the absence of systemic maternal diabetes. To isolate the effects of hyperglycemia, we infused glucose into the blood supply of the left but not right uterine horn in nondiabetic pregnant rats starting on gestational day 19. After 24 h infusion, right-sided fetuses and dams remained euglycemic while left-sided fetuses were moderately hyperglycemic. Echocardiograms in utero demonstrated a thickened cardiac septum among left-sided (glucose-exposed, 0.592 ± 0.016 mm) compared to right-sided (control, 0.482 ± 0.016 mm) fetuses. Myocardial proliferation was increased 1.5 ± 0.2-fold among left-sided compared to right-sided fetuses. Transcriptional markers of glucose-derived anabolism were not different between sides. However, left-sided fetuses exhibited higher serum insulin and greater JNK phosphorylation compared to controls. These results show that hyperglycemic exposure is sufficient to rapidly induce septal overgrowth even in the absence of the myriad other factors of maternal diabetes. This suggests that even transient spikes in glucose may incite cardiac overgrowth, perhaps explaining the poor clinical correlation of septal hypertrophy with chronic hyperglycemia.

Quantifying Insulin Sensitivity and Entero-Insular Responsiveness to Hyper- and Hypoglycemia in Ferrets

Ferrets are an important emerging model of cystic fibrosis related diabetes. However, there is little documented experience in the use of advanced techniques to quantify aspects of diabetes pathophysiology in the ferret. Glycemic clamps are the gold standard technique to assess both insulin sensitivity and insulin secretion in humans and animal models of diabetes. We therefore sought to develop techniques for glycemic clamps in ferrets. To assess insulin sensitivity, we performed euglycemic hyperinsulinemic clamps in 5–6 week old ferrets in the anesthetized and conscious states. To assess insulin secretion, we performed hyperglycemic clamps in conscious ferrets. To evaluate responsiveness of ferret islet and entero-insular hormones to low glucose, a portion of the hyperglycemic clamps were followed by a hypoglycemic clamp. The euglycemic hyperinsulinemic clamps demonstrated insulin responsiveness in ferrets similar to that previously observed in humans and rats. The anesthetic isoflurane induced marked insulin resistance, whereas lipid emulsion induced mild insulin resistance. In conscious ferrets, glucose appearance was largely suppressed at 4 mU/kg/min insulin infusion, whereas glucose disposal was progressively increased at 4 and 20 mU/kg/min insulin. Hyperglycemic clamp induced first phase insulin secretion. Hypoglycemia induced a rapid diminishment of insulin, as well as a rise in glucagon and pancreatic polypeptide levels. The incretins GLP-1 and GIP were affected minimally by hyperglycemic and hypoglycemic clamp. These techniques will prove useful in better defining the pathophysiology in ferrets with cystic fibrosis related diabetes.

PET/CT imaging reveals unrivaled placental avidity for glucose compared to other tissues.

INTRODUCTION The goal of this study was to define the kinetics of glucose transport from maternal blood to placenta to fetus using real time imaging. METHODS Positron emission tomography (PET) imaging of the glucose-tracer [(18)F]fluorodeoxyglucose (FDG) was used to temporally and spatially define, in vivo, the kinetics of glucose transport from maternal blood into placentae and fetuses, in the late gestational gravid rat. Computed tomography (CT), with intravenous contrast, co-registered to the PET images allowed anatomic differentiation of placentae from fetal and maternal tissues. RESULTS FDG was rapidly taken up by placentae and subsequently appeared in fetuses with minimal temporal lag. FDG standardized uptake values in placentae and fetuses approached that of maternal brain. In both anesthetized and awake dams, one quarter of the administered FDG ultimately was accrued in the collective fetuses and placentae. Accordingly, kinetic modeling demonstrated that the placentae had very high avidity for FDG, 2-fold greater than that of the fetus and maternal brain, when accounting for the fact that fetal FDG necessarily must first be taken up by placentae. Consistent with this, placental expression of glucose transporter 1 exceeded that of all other tissues. DISCUSSION Fetal and placental tissues place a substantial glucose metabolic burden on the mother, owing to very high avidity of placentae for glucose coupled with the large relative mass of fetal and placental tissues. CONCLUSIONS The placenta has a tremendous capacity to uptake and transport glucose. PET/CT imaging is an ideal means to study metabolite transport kinetics in the fetoplacental unit.

Development of a polarized pancreatic ductular cell epithelium for physiological studies

Pancreatic ductular epithelial cells comprise the majority of duct cells in pancreas, control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate ([Formula: see text]) secretion, but are difficult to grow as a polarized monolayer. Using NIH-3T3-J2 fibroblast feeder cells and a Rho-associated kinase inhibitor, we produced well-differentiated and polarized porcine pancreatic ductular epithelial cells. Cells grown on semipermeable filters at the air-liquid interface developed typical epithelial cell morphology and stable transepithelial resistance and expressed epithelial cell markers (zona occludens-1 and β-catenin), duct cell markers (SOX-9 and CFTR), but no acinar (amylase) or islet cell (chromogranin) markers. Polarized cells were studied in Ussing chambers bathed in Krebs-Ringer [Formula: see text] solution at 37°C gassed with 5% CO2 to measure short-circuit currents ( Isc). Ratiometric measurement of extracellular pH was performed with fluorescent SNARF-conjugated dextran at 5% CO2. Cells demonstrated a baseline Isc (12.2 ± 3.2 μA/cm2) that increased significantly in response to apical forskolin-IBMX (∆ Isc: 35.4 ± 3.8 μA/cm2, P < 0.001) or basolateral secretin (∆ Isc: 31.4 ± 2.5 μA/cm2, P < 0.001), both of which increase cellular levels of cAMP. Subsequent addition of apical GlyH-101, a CFTR inhibitor, decreased the current (∆ Isc: 20.4 ± 3.8 μA/cm2, P < 0.01). Extracellular pH and [Formula: see text] concentration increased significantly after forskolin-IBMX (pH: 7.18 ± 0.23 vs. 7.53 ± 0.19; [Formula: see text] concentration, 14.5 ± 5.9 vs. 31.8 ± 13.4 mM; P < 0.05 for both). We demonstrate the development of a polarized pancreatic ductular epithelial cell epithelium with CFTR-dependent [Formula: see text] secretion in response to secretin and cAMP. This model is highly relevant, as porcine pancreas physiology is very similar to humans and pancreatic damage in the cystic fibrosis pig model recapitulates that of humans. NEW & NOTEWORTHY Pancreas ductular epithelial cells control cystic fibrosis transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion. Their function is critical because when CFTR is deficient in cystic fibrosis bicarbonate secretion is lost and the pancreas is damaged. Mechanisms that control pancreatic bicarbonate secretion are incompletely understood. We generated well-differentiated and polarized porcine pancreatic ductular epithelial cells and demonstrated feasibility of bicarbonate secretion. This novel method will advance our understanding of pancreas physiology and mechanisms of bicarbonate secretion.