Metabolic abnormalities in the bone marrow cells of young offspring born to mothers with obesity.

BACKGROUND/OBJECTIVES: Intrauterine metabolic reprogramming occurs in mothers with obesity during gestation, putting the offspring at high risk of developing obesity and associated metabolic disorders even before birth. We have generated a mouse model of maternal high-fat diet-induced obesity that recapitulates the metabolic changes seen in humans born to women with obesity. METHODS: Here, we profiled and compared the metabolic characteristics of bone marrow cells of newly weaned 3-week-old offspring of dams fed either a high-fat (Off-HFD) or a regular diet (Off-RD). We utilized a state-of-the-art flow cytometry, and targeted metabolomics approach coupled with a Seahorse metabolic analyzer. RESULTS: We revealed significant metabolic perturbation in the offspring of HFD-fed vs. RD-fed dams, including utilization of glucose primarily via oxidative phosphorylation. We also show a reduction in levels of amino acids, a phenomenon previously linked to bone marrow aging. Using flow cytometry, we found changes in the immune complexity of bone marrow cells and identified a unique B cell population expressing CD19 and CD11b in the bone marrow of three-week-old offspring of high-fat diet-fed mothers. Our data also revealed increased expression of Cyclooxygenase-2 (COX-2) on myeloid CD11b, and on CD11bhi B cells. CONCLUSIONS: Altogether, we demonstrate that the offspring of mothers with obesity show metabolic and immune changes in the bone marrow at a very young age and prior to any symptomatic metabolic disease.

Hypericum alpestre extract exhibits in vitro and in vivo anticancer properties by regulating the cellular antioxidant system and metabolic pathway of L-arginine.

Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the  l-arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG-hydroxy-nor- l-arginine and NO inhibitor NG-nitro-Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12-dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro-oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF-7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC-Q-Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer-like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with Nω -OH-nor- l-arginine and Nω -nitro- l-arginine methyl ester, H. alpestre extract exhibits pro- and antioxidant, antiangiogenic, and cytotoxic effects.

Maternal high-fat diet increases airway sensory innervation and reflex bronchoconstriction in adult offspring.

Children born to obese mothers are prone to develop asthma and airway hyperresponsiveness, but the mechanisms behind this are unclear. Here we developed a mouse model of maternal diet-induced obesity that recapitulates metabolic abnormalities seen in humans born to obese mothers. Offspring of dams fed a high-fat diet (HFD) showed increased adiposity, hyperinsulinemia, and insulin resistance at 16 wk of age despite being fed only a regular diet (RD). Bronchoconstriction induced by inhaled 5-hydroxytriptamine was also significantly increased in offspring of HFD-fed versus RD-fed dams. Increased bronchoconstriction was blocked by vagotomy, indicating this reflex was mediated by airway nerves. Three-dimensional (3-D) confocal imaging of tracheas collected from 16-wk-old offspring showed that both epithelial sensory innervation and substance P expression were increased in the offspring of HFD-fed dams compared with offspring of RD-fed dams. For the first time, we show that maternal high-fat diet increases airway sensory innervation in offspring, leading to reflex airway hyperresponsiveness.NEW & NOTEWORTHY Our study reveals a novel potential mechanism, by which maternal high-fat diet increases the risk and severity of asthma in offspring. We found that exposure to maternal high-fat diet in mice leads to hyperinnervation of airway sensory nerves and increased reflex bronchoconstriction in offspring fed a regular diet only. These findings have important clinical implications and provide new insights into the pathophysiology of asthma, highlighting the need for preventive strategies in this patient population.

Antihyperglycemic activity of L-norvaline and L-arginine in high-fat diet and streptozotocin-treated male rats.

BACKGROUND: A decrease in nitric oxide (NO) bioavailability has been shown to cause hyperglycemia, type II diabetes mellitus (DM), and chronic cardio-metabolic complications. In turn, hyperglycemia and hypercholesterolemia are associated with increased oxidative stress that leads to reduced nitric oxide bioavailability through disruption of L-arginine transport into cells, inactivation of nitric oxide synthase, and activation of arginase. Upregulation of arginase has been demonstrated in both diabetic patients and animal models of hyperglycemia and type 2 diabetes. L-norvaline is a nonselective inhibitor of arginase that increases NO production and promotes the normal functioning of the vascular endothelium. Another means of increasing NO bioavailability in the cardiovascular system is L-arginine supplementation. Whether L-norvaline and L-arginine have antihyperglycemic effects has not been studied. HYPOTHESIS: We hypothesized that inhibition of arginase will provide an antihyperglycemic effect and, as a result of the recovery of NO bioavailability, will protect against oxidative stress and hypercholesterolemia. METHODS: Rats were fed a high-fat diet (HFD) for three weeks concomitant with the two-time injection of 30 mg/kg of streptozotocin (STZ) to induce stable hyperglycemia. We studied the antihyperglycemic properties of arginase inhibition (via L-norvaline) and its combination with NOS substrate supplementation (via L-arginine). RESULTS: Treatment of HFD/STZ mice with L-norvaline and L-arginine reduced fasting blood glucose levels by 27.1% vs. untreated HFD/STZ rats (p < 0.001). Blood levels of total cholesterol, low-density lipoprotein (LDL), and malondialdehyde (MDA), a marker for oxidative stress, were significantly decreased in both L-norvaline- and L-norvaline+L-arginine-treated HFD/STZ rats when compared with untreated rats. In addition, administration of L-norvaline and L-arginine reversed the progression of pancreatic and kidney pathology in HFD/STZ rats as assessed by histology (p < 0.001). CONCLUSIONS: Both L-norvaline and L-arginine act as potent antihyperglycemic agents and can represent alternative therapeutic tools in individuals with hyperglycemia and pre-diabetes.

Sex-dependent vulnerability of fetal nonhuman primate cardiac mitochondria to moderate maternal nutrient reduction.

Poor maternal nutrition in pregnancy affects fetal development, predisposing offspring to cardiometabolic diseases. The role of mitochondria during fetal development on later-life cardiac dysfunction caused by maternal nutrient reduction (MNR) remains unexplored. We hypothesized that MNR during gestation causes fetal cardiac bioenergetic deficits, compromising cardiac mitochondrial metabolism and reserve capacity. To enable human translation, we developed a primate baboon model (Papio spp.) of moderate MNR in which mothers receive 70% of control nutrition during pregnancy, resulting in intrauterine growth restriction (IUGR) offspring and later exhibiting myocardial remodeling and heart failure at human equivalent ∼25 years. Term control and MNR baboon offspring were necropsied following cesarean-section, and left ventricle (LV) samples were collected. MNR adversely impacted fetal cardiac LV mitochondria in a sex-dependent fashion. Increased maternal plasma aspartate aminotransferase, creatine phosphokinase (CPK), and elevated cortisol levels in MNR concomitant with decreased blood insulin in male fetal MNR were measured. MNR resulted in a two-fold increase in fetal LV mitochondrial DNA (mtDNA). MNR resulted in increased transcripts for several respiratory chain (NDUFB8, UQCRC1, and cytochrome c) and adenosine triphosphate (ATP) synthase proteins. However, MNR fetal LV mitochondrial complex I and complex II/III activities were significantly decreased, possibly contributing to the 73% decreased ATP content and increased lipid peroxidation. MNR fetal LV showed mitochondria with sparse and disarranged cristae dysmorphology. Conclusion: MNR disruption of fetal cardiac mitochondrial fitness likely contributes to the documented developmental programming of adult cardiac dysfunction, indicating a programmed mitochondrial inability to deliver sufficient energy to cardiac tissues as a chronic mechanism for later-life heart failure.

Assessment of neonatal, cord, and adult platelet granule trafficking and secretion.

Despite the transient hyporeactivity of neonatal platelets, full-term neonates do not display a bleeding tendency, suggesting potential compensatory mechanisms which allow for balanced and efficient neonatal hemostasis. This study aimed to utilize small-volume, whole blood platelet functional assays to assess the neonatal platelet response downstream of the hemostatic platelet agonists thrombin and adenosine diphosphate (ADP). Thrombin activates platelets via the protease-activated receptors (PARs) 1 and 4, whereas ADP signals via the receptors P2Y1 and P2Y12 as a positive feedback mediator of platelet activation. We observed that neonatal and cord blood-derived platelets exhibited diminished PAR1-mediated granule secretion and integrin activation relative to adult platelets, correlating to reduced PAR1 expression by neonatal platelets. PAR4-mediated granule secretion was blunted in neonatal platelets, correlating to lower PAR4 expression as compared to adult platelets, while PAR4 mediated GPIIb/IIIa activation was similar between neonatal and adult platelets. Under high shear stress, cord blood-derived platelets yielded similar thrombin generation rates but reduced phosphatidylserine expression as compared to adult platelets. Interestingly, we observed enhanced P2Y1/P2Y12-mediated dense granule trafficking in neonatal platelets relative to adults, although P2Y1/P2Y12 expression in neonatal, cord, and adult platelets were similar, suggesting that neonatal platelets may employ an ADP-mediated positive feedback loop as a potential compensatory mechanism for neonatal platelet hyporeactivity.

Sexual dimorphism in the fetal cardiac response to maternal nutrient restriction.

Poor maternal nutrition causes intrauterine growth restriction (IUGR); however, its effects on fetal cardiac development are unclear. We have developed a baboon model of moderate maternal undernutrition, leading to IUGR. We hypothesized that the IUGR affects fetal cardiac structure and metabolism. Six control pregnant baboons ate ad-libitum (CTRL)) or 70% CTRL from 0.16 of gestation (G). Fetuses were euthanized at C-section at 0.9G under general anesthesia. Male but not female IUGR fetuses showed left ventricular fibrosis inversely correlated with birth weight. Expression of extracellular matrix protein TSP-1 was increased (p<0.05) in male IUGR. Expression of cardiac fibrotic markers TGFß, SMAD3 and ALK-1 were downregulated in male IUGRs with no difference in females. Autophagy was present in male IUGR evidenced by upregulation of ATG7 expression and lipidation LC3B. Global miRNA expression profiling revealed 56 annotated and novel cardiac miRNAs exclusively dysregulated in female IUGR, and 38 cardiac miRNAs were exclusively dysregulated in males (p<0.05). Fifteen (CTRL) and 23 (IUGR) miRNAs, were differentially expressed between males and females (p<0.05) suggesting sexual dimorphism, which can be at least partially explained by differential expression of upstream transcription factors (e.g. HNF4α, and NFκB p50). Lipidomics analysis of fetal cardiac tissue exhibited a net increase in diacylglycerol and plasmalogens and a decrease in triglycerides and phosphatidylcholines. In summary, IUGR resulting from decreased maternal nutrition is associated with sex-dependent dysregulations in cardiac structure, miRNA expression, and lipid metabolism. If these changes persist postnatally, they may program offspring for higher later life cardiac risk.

Mitochondrial function and glucose metabolism in the placenta with gestational diabetes mellitus: role of miR-143.

A predisposing factor for development of the hyperglycaemic state of gestational diabetes mellitus (GDM) is obesity. We previously showed that increasing maternal obesity is associated with significant reductions in placental mitochondrial respiration. MicroRNA (miR)-143 has been previously shown to regulate the metabolic switch from oxidative phosphorylation to aerobic glycolysis in cancer tissues. We hypothesized that mitochondrial respiration is reduced and aerobic glycolysis is up-regulated via changes in miR-143 expression in the placenta of women with GDM. Placental tissue was collected at term from women with A1GDM (controlled by diet), A2GDM (controlled by medication) and body mass index (BMI)-matched controls (CTRL). miR-143 expression was measured by RT-PCR. Expression of mitochondrial complexes, transcription factors peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α) and peroxisome proliferator-activated receptor γ (PPARγ), components of mammalian target of rapamycin (mTOR) signalling, glucose transporter GLUT1 and glycolytic enzymes [hexokinase-2 (HK-2), phosphofructokinase (PFK) and lactate dehydrogenase (LDH)] were measured by Western blot. Trophoblast respiration was measured by XF24 Analyser. Expression of miR-143, mitochondrial complexes, and PPARγ and PGC1α, which act downstream of miR-143, were significantly decreased in A2GDM placentae compared with A1GDM and CTRL (P<0.01). Placental hPL (human placental lactogen) levels, expression of glycolytic enzymes, GLUT1 and mTOR signalling were also significantly increased by more than 2-fold in A2GDM compared with A1GDM and CTRL (P<0.05). There was a 50% reduction in mitochondrial respiration in trophoblast cells isolated from A2GDM placentae. Overexpression of miR-143 was able to increase mitochondrial respiration, increase protein expression of mitochondrial complexes and decrease expression of glycolytic enzymes by 40% compared with A2GDM. Down-regulation of miR-143 mediates the metabolic switch from oxidative phosphorylation to aerobic glycolysis in placenta of women with A2GDM.

Impaired mitochondrial function in human placenta with increased maternal adiposity.

The placenta plays a key role in regulation of fetal growth and development and in mediating in utero developmental programming. Obesity, which is associated with chronic inflammation and mitochondrial dysfunction in many tissues, exerts a programming effect in pregnancy. We determined the effect of increasing maternal adiposity and of fetal sex on placental ATP generation, mitochondrial biogenesis, expression of electron transport chain subunits, and mitochondrial function in isolated trophoblasts. Placental tissue was collected from women with prepregnancy BMI ranging from 18.5 to 45 following C-section at term with no labor. Increasing maternal adiposity was associated with excessive production of reactive oxygen species and a significant reduction in placental ATP levels in placentae with male and female fetuses. To explore the potential mechanism of placental mitochondrial dysfunction, levels of transcription factors regulating the expression of genes involved in electron transport and mitochondrial biogenesis were measured. Our in vitro studies showed significant reduction in mitochondrial respiration in cultured primary trophoblasts with increasing maternal obesity along with an abnormal metabolic flexibility of these cells. This reduction in placental mitochondrial respiration in pregnancies complicated by maternal obesity could compromise placental function and potentially underlie the increased susceptibility of these pregnancies to fetal demise in late gestation and to developmental programming.

Effect of preeclampsia on placental function: influence of sexual dimorphism, microRNA's and mitochondria.

In pregnancy fetal growth and development occur in a sexually dimorphic manner. Male and female fetuses respond differently to the intrauterine environment with males disproportionately suffering from perinatal morbidity and mortality. We have demonstrated placental dysfunction and sexually dimorphic responses in pregnancies complicated by severe preeclampsia. Production of cytokines and apoptosis in the male placenta is heightened relative to that of the female placenta. We also find increased expression and stabilization and a sexual dimorphism in expression of the transcription factor HIF-1α, but a defect in binding to the hypoxia response element with corresponding reduced expression of HIF-1α target genes including VEGF and Glut-1. HIF-1α is involved in crosstalk with the redox sensitive transcription factor NFκB in regulation by cytokines, reactive oxygen species and expression of inflammatory genes. We find increased placental expression and DNA binding of NFκB and a sexually dimorphic response suggesting a role for NFκB in placental dysfunction with preeclampsia. Placental mitochondrial complex III activity and complex I and IV expression are reduced and alterations in mitochondrial morphology are found in preeclampsia and are linked to the hypoxamir miR-210. We propose that with severe PE placental HIF-1α is stabilized by excessive ROS, inflammation and relative hypoxia. This increases the expression of miR-210 in the placenta causing repression of mitochondria-associated target genes, potentially leading to mitochondrial and placental dysfunction. This placental dysfunction may lead to a fetal programming effect that results in disease in later life.

5-fluorouracil and Rumex obtusifolius extract combination trigger A549 cancer cell apoptosis: uncovering PI3K/Akt inhibition by in vitro and in silico approaches.

The continuous increase in cancer rates, failure of conventional chemotherapies to control the disease, and excessive toxicity of chemotherapies clearly demand alternative approaches. Natural products contain many constituents that can act on various bodily targets to induce pharmacodynamic responses. This study aimed to explore the combined anticancer effects of Rumex obtusifolius (RO) extract and the chemotherapeutic agent 5-fluorouracil (5-FU) on specific molecular targets involved in cancer progression. By focusing on the PI3K/Akt signaling pathway and its related components, such as cytokines, growth factors (TNFa, VEGFa), and enzymes (Arginase, NOS, COX-2, MMP-2), this research sought to elucidate the molecular mechanisms underlying the anticancer effects of RO extract, both independently and in combination with 5-FU, in non-small lung adenocarcinoma A549 cells. The study also investigated the potential interactions of compounds identified by HPLC/MS/MS of RO on PI3K/Akt in the active site pocket through an in silico analysis. The ultimate goal was to identify potent therapeutic combinations that effectively inhibit, prevent or delay cancer development with minimal side effects. The results revealed that the combined treatment of 5-FU and RO demonstrated a significant reduction in TNFa levels, comparable to the effect observed with RO alone. RO modulated the PI3K/Akt pathway, influencing the phosphorylated and total amounts of these proteins during the combined treatment. Notably, COX-2, a key player in inflammatory processes, substantially decreased with the combination treatment. Caspase-3 activity, indicative of apoptosis, increased by 1.8 times in the combined treatment compared to separate treatments. In addition, the in silico analyses explored the binding affinities and interactions of RO's major phytochemicals with intracellular targets, revealing a high affinity for PI3K and Akt. These findings suggest that the combined treatment induces apoptosis in A549 cells by regulating the PI3K/Akt pathway.

Identification and comparative analyses of myocardial miRNAs involved in the fetal response to maternal obesity.

Human and animal studies show that suboptimal intrauterine environments lead to fetal programming, predisposing offspring to disease in later life. Maternal obesity has been shown to program offspring for cardiovascular disease (CVD), diabetes, and obesity. MicroRNAs (miRNAs) are small, noncoding RNA molecules that act as key regulators of numerous cellular processes. Compelling evidence links miRNAs to the control of cardiac development and etiology of cardiac pathology; however, little is known about their role in the fetal cardiac response to maternal obesity. Our aim was to sequence and profile the cardiac miRNAs that are dysregulated in the hearts of baboon fetuses born to high fat/high fructose-diet (HFD) fed mothers for comparison with fetal hearts from mothers eating a regular diet. Eighty miRNAs were differentially expressed. Of those, 55 miRNAs were upregulated and 25 downregulated with HFD. Twenty-two miRNAs were mapped to human; 14 of these miRNAs were previously reported to be dysregulated in experimental or human CVD. We used an Ingenuity Pathway Analysis to integrate miRNA profiling and bioinformatics predictions to determine miRNA-regulated processes and genes potentially involved in fetal programming. We found a correlation between miRNA expression and putative gene targets involved in developmental disorders and CVD. Cellular death, growth, and proliferation were the most affected cellular functions in response to maternal obesity. Thus, the current study reveals significant alterations in cardiac miRNA expression in the fetus of obese baboons. The epigenetic modifications caused by adverse prenatal environment may represent one of the mechanisms underlying fetal programming of CVD.

Metabolic abnormalities in the bone marrow cells of young offspring born to obese mothers.

Intrauterine metabolic reprogramming occurs in obese mothers during gestation, putting the offspring at high risk of developing obesity and associated metabolic disorders even before birth. We have generated a mouse model of maternal high-fat diet-induced obesity that recapitulates the metabolic changes seen in humans. Here, we profiled and compared the metabolic characteristics of bone marrow cells of newly weaned 3-week-old offspring of dams fed either a high-fat (Off-HFD) or a regular diet (Off-RD). We utilized a state-of-the-art targeted metabolomics approach coupled with a Seahorse metabolic analyzer. We revealed significant metabolic perturbation in the offspring of HFD-fed vs. RD-fed dams, including utilization of glucose primarily via oxidative phosphorylation, and reduction in levels of amino acids, a phenomenon previously linked to aging. Furthermore, in the bone marrow of three-week-old offspring of high-fat diet-fed mothers, we identified a unique B cell population expressing CD19 and CD11b, and found increased expression of Cyclooxygenase-2 (COX-2) on myeloid CD11b, and on CD11b hi B cells, with all the populations being significantly more abundant in offspring of dams fed HFD but not a regular diet. Altogether, we demonstrate that the offspring of obese mothers show metabolic and immune changes in the bone marrow at a very young age and prior to any symptomatic metabolic disease.

Anti-cancer effect of Rumex obtusifolius in combination with arginase/nitric oxide synthase inhibitors via downregulation of oxidative stress, inflammation, and polyamine synthesis.

Cancer continues to be a leading cause of death worldwide, making the development of new treatment methods crucial in the fight against it. With cancer incidence rates increasing worldwide, ongoing research must focus on identifying new and effective ways to prevent and treat the disease. The combination of herbal extracts with chemotherapeutic agents has gained much interest as a novel strategy to combat cancer. Rumex obtusifolius L. is a wild plant known for its medicinal properties and is widely distributed worldwide. Our preclinical evaluations suggested that R. obtusifolius seed extracts possessed cancer-inhibiting properties and we also evaluated the beneficial effects of the arginase inhibitor NG-hydroxy-nor-L-arginine and nitric oxide inhibitor NG-nitro-L-arginine methyl ester in the treatment of breast cancer. The current study aimed to combine these observations and evaluate the antioxidant and antitumor properties of R. obtusifolius extracts alone and in combination with the arginase and nitric oxide synthase inhibitors. Metabolic characterization of the plant extract using a liquid chromatography/high-resolution mass spectrometry advanced system revealed the presence of 240 phenolic compounds many of which possess anticancer properties, according to the literature. In vitro studies revealed a significant cytotoxic effect of the R. obtusifolius extracts on the human colon (HT29) and breast cancer (MCF-7) cell lines. Thus, a new treatment approach of combining R. obtusifolius bioactive phytochemicals with the arginase and nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester and/or NG-hydroxy-nor-L-arginine, respectively, was proposed and could potentially be an effective way to treat breast cancer. Indeed, these combinations showed immunostimulatory, antiproliferative, antioxidant, anti-inflammatory, and antiangiogenic properties in a rat breast cancer model.

Manipulation of death pathways in desmin-related cardiomyopathy.

RATIONALE: Transgenic mice with cardiac specific overexpression of mutated alphaB-crystallin (CryAB(R120G)) display Desmin-related myopathy (DRM) with dilated cardiomyopathy and heart failure. Our previous studies showed the presence of progressive mitochondrial abnormalities and activation of apoptotic cell death in CryAB(R120G) transgenic hearts. However, the role of mitochondrial dysfunction and apoptosis in the overall course of the disease was unclear. OBJECTIVE: We tested the hypothesis that prevention of apoptosis would ameliorate CryAB(R120G) pathology and decrease morbidity. METHODS AND RESULTS: We crossed CryAB(R120G) mice to transgenic mice with cardiac specific overexpression of Bcl-2. Sustained Bcl-2 overexpression in CryAB(R120G) hearts prolonged CryAB(R120G) transgenic mice survival by 20%. This was associated with decreased mitochondrial abnormalities, restoration of cardiac function, prevention of cardiac hypertrophy, and attenuation of apoptosis. CryAB(R120G) misfolded protein aggregation was significantly reduced in the double transgenic. However, inhibition of apoptotic signaling resulted in the upregulation of autophagy and alternative death pathways, the net result being increased necrosis. CONCLUSION: Although Bcl-2 overexpression prolonged life in this DRM model, in the absence of apoptosis, another death pathway was activated.

Vitamin D Supplementation Improves Mitochondrial Function and Reduces Inflammation in Placentae of Obese Women.

Background: About 30% of women entering pregnancy in the US are obese. We have previously reported mitochondrial dysregulation and increased inflammation in the placentae of obese women. Vitamin D (VitD) is a major player in calcium uptake and was shown to modulate mitochondrial respiration and the immune/inflammation system. Studies show decreased VitD levels in obese individuals; however, the effect of maternal obesity on VitD metabolism and its association with placental function remains understudied. Methods: Maternal and cord blood plasma and placental samples were collected upon C-section from normal-weight (NW, body mass index [BMI]<25) and obese (OB, BMI>30) women with uncomplicated pregnancies at term. We measured 25(OH)D3 (calcidiol) levels in maternal and cord blood plasma using ELISA. We assessed the expression of CYP27B1, an activator of calcidiol, and Vitamin D receptor (VDR) in placentae from NW and OB, and women with gestational diabetes and preeclampsia. In addition, we examined the effects of VitD supplementation on mitochondrial function and inflammation in trophoblasts from NW and OB, using the Seahorse Bioanalyzer and Western blot, respectively. Results: Vitamin D levels in blood from OB but not NW women and in cord blood from babies born to NW and OB women showed a significant inverse correlation with maternal pre-pregnancy BMI (r=-0.50, p<0.1 and r=-0.55, p=0.004 respectively). Cord plasma VitD levels showed a positive correlation with placental efficiency, i.e., the ratio between fetal and placental weight, as well as with maternal blood VitD levels (r=0.69 and 0.83 respectively, p<0.00). While we found no changes in CYP27B1 in OB vs. NW women, VDR expression were decreased by 50% (p<0.03) independent of fetal sex. No changes in VDR expression relative to BMI-matched controls were observed in the placentae of women with gestational diabetes or preeclampsia. Cytotrophoblasts isolated from placentae of OB women showed a dose-dependent increase in VDR expression after 24-hour treatment with calcitriol (10 nM and 100 nM), an active form of VitD. Trophoblasts isolated from OB women and treated with calcitriol improved mitochondrial respiration (p<0.05). We also found a two-fold increase in expression of the NLRP3 inflammasome and the pro-inflammatory cytokine IL-18 in trophoblasts isolated from placentae of OB women (p<0.05), with IL-18 expression being reversed by calcitriol treatment (100 nM). Conclusions: We show that VitD deficiency is at least partially responsible for mitochondrial dysfunction and increased inflammation in the placentae of obese women. Vitamin D supplementation could be beneficial in improving placental dysfunction seen in obese women.

Dipeptidyl peptidase IV inhibition delays developmental programming of obesity and metabolic disease in male offspring of obese mothers.

Maternal obesity programs the offspring to metabolic diseases later in life; however, the mechanisms of programming are yet unclear, and no strategies exist for addressing its detrimental transgenerational effects. Obesity has been linked to dipeptidyl peptidase IV (DPPIV), an adipokine, and treatment of obese individuals with DPPIV inhibitors has been reported to prevent weight gain and improve metabolism. We hypothesized that DPPIV plays a role in maternal obesity-mediated programming. We measured plasma DPPIV activity in human maternal and cord blood samples from normal-weight and obese mothers at term. We found that maternal obesity increases maternal and cord blood plasma DPPIV activity but only in male offspring. Using two non-human primate models of maternal obesity, we confirmed the activation of DPPIV in the offspring of obese mothers. We then created a mouse model of maternal high-fat diet (HFD)-induced obesity, and found an early-life increase in plasma DPPIV activity in male offspring. Activation of DPPIV preceded the progression of obesity, glucose intolerance and insulin resistance in male offspring of HFD-fed mothers. We then administered sitagliptin, DPPIV inhibitor, to regular diet (RD)- and HFD-fed mothers, starting a week prior to breeding and continuing throughout pregnancy and lactation. We found that sitagliptin treatment of HFD-fed mothers delayed the progression of obesity and metabolic diseases in male offspring and had no effects on females. Our findings reveal that maternal obesity dysregulates plasma DPPIV activity in males and provide evidence that maternal inhibition of DPPIV has potential for addressing the transgenerational effects of maternal obesity.

Biochemical and mechanical dysfunction in a mouse model of desmin-related myopathy.

An R120G mutation in alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM). In mice with cardiomyocyte-specific expression of the mutation, CryAB(R120G)-mediated DRM is characterized by CryAB and desmin accumulations within cardiac muscle, mitochondrial deficiencies, activation of apoptosis, and heart failure (HF). Excessive production of reactive oxygen species (ROS) is often a hallmark of HF and treatment with antioxidants can sometimes prevent the progression of HF in terms of contractile dysfunction and cardiomyocyte survival. It is unknown whether blockade of ROS is beneficial for protein misfolding diseases such as DRM. We addressed this question by blocking the activity of xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in our model of DRM. The XO inhibitor oxypurinol was administered to CryAB(R120G) mice for a period of 1 or 3 months. Mitochondrial function was dramatically improved in treated animals in terms of complex I activity and conservation of mitochondrial membrane potential. Oxypurinol also largely restored normal mitochondrial morphology. Surprisingly, however, cardiac contractile function and cardiac compliance were unimproved, indicating that the contractile deficit might be independent of mitochondrial dysfunction and the initiation of apoptosis. Using magnetic bead microrheology at the single cardiomyocyte level, we demonstrated that sarcomeric disarray and accumulation of the physical aggregates resulted in significant changes in the cytoskeletal mechanical properties in the CryAB(R120G) cardiomyocytes. Our findings indicate that oxypurinol treatment largely prevented mitochondrial deficiency in DRM but that contractility was not improved because of mechanical deficits in passive cytoskeletal stiffness.

Dyslipidemia, insulin resistance, and impairment of placental metabolism in the offspring of obese mothers.

Obesity is a chronic condition associated with dyslipidemia and insulin resistance. Here, we show that the offspring of obese mothers are dyslipidemic and insulin resistant from the outset.Maternal and cord blood and placental tissues were collected following C-section at term. Patients were grouped as being normal weight (NW, BMI = 18-24.9) or obese (OB, BMI ≥ 30), and separated by fetal sex. We measured plasma lipids, insulin, and glucose in maternal and cord blood. Insulin resistance was quantified using the HOMA-IR. Placental markers of lipid and energy metabolism and relevant metabolites were measured by western blot and metabolomics, respectively.For OB women, total cholesterol was decreased in both maternal and cord blood, while HDL was decreased only in cord blood, independent of sex. In babies born to OB women, cord blood insulin and insulin resistance were increased. Placental protein expression of the energy and lipid metabolism regulators PGC1α, and SIRT3, ERRα, CPT1α, and CPT2 decreased with maternal obesity in a sex-dependent manner (P < 0.05). Metabolomics showed lower levels of acylcarnitines C16:0, C18:2, and C20:4 in OB women's placentas, suggesting a decrease in ß-oxidation. Glutamine, glutamate, alpha-ketoglutarate (αKG), and 2-hydroxyglutarate (2-HG) were increased, and the glutamine-to-glutamate ratio decreased (P < 0.05), in OB placentas, suggesting induction of glutamate into αKG conversion to maintain a normal metabolic flux.Newly-born offspring of obese mothers begin their lives dyslipidemic and insulin resistant. If not inherited genetically, such major metabolic perturbations might be explained by abnormal placental metabolism with potential long-term adverse consequences for the offspring's health and wellbeing.

Multiscale cardiac imaging spanning the whole heart and its internal cellular architecture in a small animal model.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

The heart is our hardest-working organ and beats around 100,000 times a day, pumping blood through a vast system of vessels to all areas of the body. Specialized heart cells make the heart contract rhythmically, enabling it to work efficiently. Contractile molecules inside these cells, called myofibrils, align within the heart cells, and heart cells align to each other, so that the heart tissue contracts effectively. However, when the heart has defects or is diseased this organization can be lost, and the heart may no longer pump blood efficiently, leading to sometimes life-threatening complications. For example, around one in a hundred newborn babies suffer from congenital heart defects, and despite medical advances, these conditions remain the main cause of non-infectious mortality in children. Many cases of congenital heart disease are diagnosed before a baby is born during an ultrasound scan. However, these scans, as well as subsequent diagnostic tools, lack the precision to detect problems within the heart cells. Now, Rykiel et al. used two complementary imaging techniques known as micro-computed tomography and scanning electron microscopy to acquire pictures of the whole heart as well as of the organization inside the heart cells. This made it possible to capture the structure of the heart tissue at both micrometer (the whole heart) and nanometer resolution (the inside of the cells), and to study what happens within the heart and its cells when the heart has a defect. Rykiel et al. tested the imaging technology on the hearts of chicken embryos, at stages equivalent to a five to six-month-old human fetus, and compared a healthy heart with a heart with a defect called tetralogy of Fallot. They found that the tissues in the heart with a defect had a sponge-like appearance, with increased space in between cells. Moreover, the myofibrils of the heart with a defect were aligned differently compared to those in the normal heart. More research is needed to fully understand what happens when the heart has a defect. However, the imaging technology used in this study offers the possibility of examining the heart at an unprecedented level of detail. This will deepen our understanding of how structural heart defects arise and how they affect the pumping of the heart, and will give us clues to design better treatments for patients with heart defects and other heart anomalies.