Retraction Note: Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives.

This paper has been retracted at the request of the authors.

Super-Obese Patient-Derived iPSC Hypothalamic Neurons Exhibit Obesogenic Signatures and Hormone Responses.

The hypothalamus contains neurons that integrate hunger and satiety endocrine signals from the periphery and are implicated in the pathophysiology of obesity. The limited availability of human hypothalamic neurons hampers our understanding of obesity disease mechanisms. To address this, we generated human induced pluripotent stem cells (hiPSCs) from multiple normal body mass index (BMI; BMI ≤ 25) subjects and super-obese (OBS) donors (BMI ≥ 50) with polygenic coding variants in obesity-associated genes. We developed a method to reliably differentiate hiPSCs into hypothalamic-like neurons (iHTNs) capable of secreting orexigenic and anorexigenic neuropeptides. Transcriptomic profiling revealed that, although iHTNs maintain a fetal identity, they respond appropriately to metabolic hormones ghrelin and leptin. Notably, OBS iHTNs retained disease signatures and phenotypes of high BMI, exhibiting dysregulated respiratory function, ghrelin-leptin signaling, axonal guidance, glutamate receptors, and endoplasmic reticulum (ER) stress pathways. Thus, human iHTNs provide a powerful platform to study obesity and gene-environment interactions.

Endocrine disruptors induce perturbations in endoplasmic reticulum and mitochondria of human pluripotent stem cell derivatives.

Persistent exposure to man-made endocrine disrupting chemicals during fetal endocrine development may lead to disruption of metabolic homeostasis contributing to childhood obesity. Limited cellular platforms exist to test endocrine disrupting chemical-induced developmental abnormalities in human endocrine tissues. Here we use an human-induced pluripotent stem cell-based platform to demonstrate adverse impacts of obesogenic endocrine disrupting chemicals in the developing endocrine system. We delineate the effects upon physiological low-dose exposure to ubiquitous endocrine disrupting chemicals including, perfluoro-octanoic acid, tributyltin, and butylhydroxytoluene, in endocrine-active human-induced pluripotent stem cell-derived foregut epithelial cells and hypothalamic neurons. Endocrine disrupting chemicals induce endoplasmic reticulum stress, perturb NF-κB, and p53 signaling, and diminish mitochondrial respiratory gene expression, spare respiratory capacity, and ATP levels. As a result, normal production and secretion of appetite control hormones, PYY, α-MSH, and CART, are hampered. Blocking NF-κB rescues endocrine disrupting chemical-induced aberrant mitochondrial phenotypes and endocrine dysregulation, but not ER-stress and p53-phosphorylation changes.Harmful chemicals that disrupt the endocrine system and hormone regulation have been associated with obesity. Here the authors apply a human pluripotent stem cell-based platform to study the effects of such compounds on developing gut endocrine and neuroendocrine systems.

Erratum to "Hyperglycemia Induces Toll-Like Receptor-2 and -4 Expression and Activity in Human Microvascular Retinal Endothelial Cells: Implications for Diabetic Retinopathy".

[This corrects the article DOI: 10.1155/2014/790902.].

Involvement of NADPH oxidase in A2A adenosine receptor-mediated increase in coronary flow in isolated mouse hearts.

Adenosine increases coronary flow mainly through the activation of A2A and A2B adenosine receptors (ARs). However, the mechanisms for the regulation of coronary flow are not fully understood. We previously demonstrated that adenosine-induced increase in coronary flow is in part through NADPH oxidase (Nox) activation, which is independent of activation of either A1 or A3ARs. In this study, we hypothesize that adenosine-mediated increase in coronary flow through Nox activation depends on A2A but not A2BARs. Functional studies were conducted using isolated Langendorff-perfused mouse hearts. Hydrogen peroxide (H2O2) production was measured in isolated coronary arteries from WT, A2AAR knockout (KO), and A2BAR KO mice using dichlorofluorescein immunofluorescence. Adenosine-induced concentration-dependent increase in coronary flow was attenuated by the specific Nox2 inhibitor gp91 ds-tat or reactive oxygen species (ROS) scavenger EUK134 in both WT and A2B but not A2AAR KO isolated hearts. Similarly, the A2AAR selective agonist CGS-21680-induced increase in coronary flow was significantly blunted by Nox2 inhibition in both WT and A2BAR KO, while the A2BAR selective agonist BAY 60-6583-induced increase in coronary flow was not affected by Nox2 inhibition in WT. In intact isolated coronary arteries, adenosine-induced (10 µM) increase in H2O2 formation in both WT and A2BAR KO mice was attenuated by Nox2 inhibition, whereas adenosine failed to increase H2O2 production in A2AAR KO mice. In conclusion, adenosine-induced increase in coronary flow is partially mediated by Nox2-derived H2O2, which critically depends upon the presence of A2AAR.

Pseudoinsulinoma in a white man with autoimmune hypoglycemia due to anti-insulin antibodies: value of the free C-Peptide assay.

OBJECTIVES: Insulin autoimmune syndrome (IAS) is an extremely rare cause of hypoglycemia, particularly in non-Asian populations. METHODS: In this report, we describe a white male patient with elevated total insulin (>100.0 µIU/mL), C-peptide, and proinsulin levels who was diagnosed with IAS due to anti-insulin antibodies. He also had a small IgG κ M-protein. RESULTS: We show that anti-insulin antibodies and/or the monoclonal protein can significantly interfere with insulin and C-peptide immunoassays and propose polyethylene glycol precipitation to quantitate free C-peptide levels as a useful assay in differentiating IAS due to anti-insulin antibodies from insulinoma. CONCLUSIONS: In patients presenting with hypoglycemia with excessively high insulin levels, consideration needs to be given to autoimmune hypoglycemia due to anti-insulin antibodies as a cause. Additionally, if total C-peptide levels are increased, free C-peptide needs to be quantitated following polyethylene glycol precipitation.

Endotoxemia of metabolic syndrome: a pivotal mediator of meta-inflammation.

Endotoxemia, which is now emerging as a feature of metabolic syndrome, is believed to contribute to the chronic low-grade inflammatory status and insulin resistance of the syndrome. Lipopolysaccharides (LPS), or endotoxins, bind to LPS-binding protein and activate pattern recognition receptors, classically Toll-like receptor-4, mediating inflammation. Increased gut permeability and changes in composition and diversity of gut microbiome have been proposed as possible mechanisms to explain increased circulating endotoxins in metabolic syndrome. Endotoxins are also believed to be delivered into the circulation by chylomicrons. Weight loss and probiotic and prebiotic therapeutic strategies can reduce endotoxemia, inflammation, and insulin resistance in metabolic syndrome.

Hyperglycemia induces Toll-like receptor-2 and -4 expression and activity in human microvascular retinal endothelial cells: implications for diabetic retinopathy.

Diabetic retinopathy (DR) causes visual impairment in working age adults and hyperglycemia-mediated inflammation is central in DR. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, scanty data is available on their role in DR. Hence, in this study, we examined TLR2 and TLR4 mRNA and protein expression and activity in hyperglycemic human retinal endothelial cells (HMVRECs). HMVRECs were treated with hyperglycemia (HG) or euglycemia and mRNA and protein levels of TLR-2, TLR-4, MyD88, IRF3, and TRIF as well as NF-κB p65 activation were measured. IL-8, IL-1ß, TNF-α and MCP-1, ICAM-1, and VCAM-1 as well as monocyte adhesion to HMVRECs were also assayed. HG (25 mM) significantly induced TLR2 and TLR4 mRNA and protein in HMVRECs. It also increased both MyD88 and non-MyD88 pathways, nuclear factor-κB (NF-κB), biomediators, and monocyte adhesion. This inflammation was attenuated by TLR-4 or TLR-2 inhibition, and dual inhibition by a TLR inhibitory peptide as well as TLR2 and 4 siRNA. Additionally, antioxidant treatment reduced TLR-2 and TLR4 expression and downstream inflammatory markers. Collectively, our novel data suggest that hyperglycemia induces TLR-2 and TLR-4 activation and downstream signaling mediating increased inflammation possibly via reactive oxygen species (ROS) and could contribute to DR.

Dysregulation of monocyte biology in metabolic syndrome.

Metabolic syndrome (MetS), which constitutes a cardio-metabolic risk cluster, is becoming a global epidemic. It is a pro-inflammatory and pro-oxidant state that confers an increased risk of cardiovascular disease and diabetes. MetS is not only characterized by increased circulating biomarkers of inflammation and oxidative stress but also by dysregulation of a pivotal phagocyte, the circulating monocyte. Pertubations manifesting in monocytes of patients with MetS include increased Toll-like receptors, CD40-CD40L dyad, increased ER stress, increased CCR5 and Fc-γ receptors (CD32 and CD64). Additionally, the monocytes demonstrate increase in NADPH oxidase activity and decreased Nrf2, resulting in oxidative damage to biomolecules. Thus the dysregulated monocyte in MetS appears to be a critical cell in the predisposition of MetS patients to diabetes and CVD. Therapeutic strategies targeting monocytes can attenuate this risk and the most compelling data derives from studies with statin therapy.

Evidence for the involvement of NADPH oxidase in adenosine receptors-mediated control of coronary flow using A1 and A3 knockout mice.

The NADPH oxidase (Nox) subunits 1, 2 (gp91 phox) and 4 are the major sources for reactive oxygen species (ROS) in cardiovascular system. In conditions such as ischemia-reperfusion injury and hypoxia, both ROS and adenosine are released suggesting a possible interaction. We hypothesized that ROS generated through Nox is involved in adenosine-induced coronary flow (CF) responses. Adenosine (10-8-10-5.5 M) increased CF in isolated hearts from wild type (WT; C57/BL6), A1 adenosine receptor (AR) knockout (A1KO), A3AR KO (A3KO) and A1 and A3AR double KO (A1/A3DKO) mice. The Nox inhibitors apocynin (10-5 M) and gp91 ds-tat (10-6 M) or the SOD and catalase-mimicking agent EUK134 (50 µM) decreased the adenosine-enhanced CF in the WT and all the KOs. Additionally, adenosine increased phosphorylation of p47-phox subunit and ERK 1/2 without changing protein expression of Nox isoforms in WT. Moreover, intracellular superoxide production was increased by adenosine and CGS-21680 (a selective A2A agonist), but not BAY 60-6583 (a selective A2B agonist), in mouse coronary artery smooth muscle cells (CASMCs) and endothelial cells (CAECs). This superoxide increase was inhibited by the gp91 ds-tat and ERK 1/2 inhibitor (PD98059). In conclusion, adenosine-induced increase in CF in isolated heart involves Nox2-generated superoxide, possibly through ERK 1/2 phosphorylation with subsequent p47-phox subunit phosphorylation. This adenosine/Nox/ROS interaction occurs in both CASMCs and CAECs, and involves neither A1 nor A3 ARs, but possibly A2A ARs in mouse.

Oleanolic acid: a novel cardioprotective agent that blunts hyperglycemia-induced contractile dysfunction.

Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher in-hospital mortality. Previous studies implicate oxidative stress, excessive flux through the hexosamine biosynthetic pathway (HBP) and a dysfunctional ubiquitin-proteasome system (UPS) as potential mediators of this process. Since oleanolic acid (OA; a clove extract) possesses antioxidant properties, we hypothesized that it attenuates acute and chronic hyperglycemia-mediated pathophysiologic molecular events (oxidative stress, apoptosis, HBP, UPS) and thereby improves contractile function in response to ischemia-reperfusion. We employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 µM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) In vivo coronary ligations were performed on streptozotocin treated rats ± OA administration during reperfusion; and 4) Effects of long-term OA treatment (2 weeks) on heart function was assessed in streptozotocin-treated rats. Our data demonstrate that OA treatment blunted high glucose-induced oxidative stress and apoptosis in heart cells. OA therapy also resulted in cardioprotection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia-reperfusion under hyperglycemic conditions. In parallel, we found decreased oxidative stress, apoptosis, HBP flux and proteasomal activity following ischemia-reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. These findings are promising since it may eventually result in novel therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications.

Hyperglycemia-mediated activation of the hexosamine biosynthetic pathway results in myocardial apoptosis.

The mechanisms mediating hyperglycemia-mediated myocardial cell death are poorly defined. Since elevated flux through the hexosamine biosynthetic pathway (HBP) is closely linked with the diabetic phenotype, we hypothesized that hyperglycemia-mediated oxidative stress results in greater O-GlcNAcylation (HBP end product) of the proapoptotic peptide BAD, thereby increasing myocardial apoptosis. H9c2 cardiomyoblasts were exposed to high glucose (33 mM) +/- HBP modulators +/- antioxidant treatment for 5 days vs. matched controls (5.5 mM), and we subsequently evaluated apoptosis by immunoblotting, immunofluorescence staining, and caspase activity measurements. In vitro reactive oxygen species (ROS) levels were quantified by 2',7'-dichlorodihydrofluorescein diacetate staining (fluorescence microscopy and flow cytometry). We determined total and BAD O-GlcNAcylation, respectively, by immunoblotting and immunofluorescence microscopy. The current study shows that high glucose treatment of cells significantly increased the degree of apoptosis. In parallel, overall O-GlcNAcylation, BAD O-GlcNAcylation, and ROS levels were increased. HBP inhibition and antioxidant treatment attenuated these effects, while increased end product levels exacerbated it. As BAD-Bcl-2 dimer formation enhances apoptosis, we performed immunoprecipitation analysis and colocalization and found increased dimerization in cells exposed to hyperglycemia. Our study identified a novel pathway whereby hyperglycemia results in greater oxidative stress and increased HBP activation and BAD O-GlcNAcylation in H9c2 cardiomyoblasts. Since greater BAD-Bcl-2 dimerization increases myocardial apoptosis, this pathway may play a crucial role in diabetes-related onset of heart diseases.