Fatty Acid Profile of Mature Red Blood Cell Membranes and Dietary Intake as a New Approach to Characterize Children with Overweight and Obesity

Obesity is a chronic metabolic disease of high complexity and of multifactorial origin. Understanding the effects of nutrition on childhood obesity metabolism remains a challenge. The aim of this study was to determine the fatty acid (FA) profile of red blood cell (RBC) membranes as a comprehensive biomarker of children's obesity metabolism, together with the evaluation of their dietary intake. An observational study was carried out on 209 children (107 healthy controls, 41 who were overweight and 61 with obesity) between 6 and 16 years of age. Mature RBC membrane phospholipids were analyzed for FA composition by gas chromatography-mass spectrometry (GC-MS). Dietary habits were evaluated using validated food frequency questionnaires (FFQ) and the Mediterranean Diet Quality Index for children (KIDMED) test. Compared to children with normal weight, children with obesity showed an inflammatory profile in mature RBC FAs, evidenced by higher levels of omega-6 polyunsaturated FAs (mainly arachidonic acid, p < 0.001). Children who were overweight or obese presented lower levels of monounsaturated FA (MUFA) compared to children with normal weight (p = 0.001 and p = 0.03, respectively), resulting in an increased saturated fatty acid (SFA)/MUFA ratio. A lower intake of nuts was observed for children with obesity. A comprehensive membrane lipidomic profile approach in children with obesity will contribute to a better understanding of the metabolic differences present in these individuals.This work was supported by the Department of Environment: Territorial Planning: Agriculture and Fisheries of the Basque Country Government (ELKARTEK 2017: and Innovation Fund 2017); the Department of Health of the Basque Government (2017222033: OBESIA 2016-2019); the Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement: TECNOMIFOOD project (CER-20191010); the INC (INTERNATIONAL NUT AND DRIED FRUIT COUNCIL) under the grant agreement OBINUT project (2016(II)-R01)

Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients

This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m2, respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2± 11.0 and 40.7 ± 8.7 kg/m2, respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω-3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω-6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω-3 to ω-6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes.This work was supported in part by the Basque Government Department of Economic Development, grant No. KK-2019/00028 (OBINTER); the Basque Government Department of Education, grants No. IT1264-19, IT1281-19, IT1270-19, and IT1625-22; the Basque Government Department of Health, grants No. 2019-222030, 2020-333023; Fundación Ramón Areces; and by Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement: TECNOMIFOOD project (CER-20191010) and Basque Government: IT1625-22

Reversal of New Onset Type 1 Diabetes by Oral Salmonella-Based Combination Therapy and Mediated by Regulatory T-Cells in NOD Mice

Autoimmune diseases such as type 1 diabetes (T1D) involve the loss of regulatory mechanisms resulting in increased tissue-specific cytotoxicity. The result is destruction of pancreatic insulin-producing β-cells and loss of glucose homeostasis. We are developing a novel oral vaccine using live attenuated Salmonella to deliver TGFβ, IL10, and the diabetic autoantigen preproinsulin combined with low-doses of anti-CD3 mAb. Here we show that oral administration of Salmonella-based anti-CD3 mAb combined therapy reverses new-onset T1D in non-obese diabetic (NOD) mice. The therapeutic effect of the combined therapy was associated with induction of immune suppressive CD4+CD25+Foxp3+ Treg and CD4+CD49b+LAG3+ Tr1 cells. In adoptive transfer experiments, adding or depleting Treg or Tr1 cells indicated that both are important for preventing diabetes in combined therapy-treated mice, but that Tr1 cells may have a more central role. Furthermore, induced Tr1 cells were found to be antigen-specific responding to peptide stimulation by secreting tolerance inducing IL10. These preclinical data demonstrate a role for Treg and Tr1 cells in combined therapy-mediated induction of tolerance in NOD mice. These results also demonstrate the potential of oral Salmonella-based combined therapy in the treatment of early T1D

Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO_2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO_2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes

Fundamental properties of Ca²⁺ signals

Background Ca²⁺ is a ubiquitous and versatile second messenger that transmits information through changes of the cytosolic Ca²⁺ concentration. Recent investigations changed basic ideas on the dynamic character of Ca²⁺ signals and challenge traditional ideas on information transmission. Scope of review We present recent findings on key characteristics of the cytosolic Ca²⁺ dynamics and theoretical concepts that explain the wide range of experimentally observed Ca²⁺ signals. Further, we relate properties of the dynamical regulation of the cytosolic Ca²⁺ concentration to ideas about information transmission by stochastic signals. Major conclusions We demonstrate the importance of the hierarchal arrangement of Ca²⁺ release sites on the emergence of cellular Ca²⁺ spikes. Stochastic Ca²⁺ signals are functionally robust and adaptive to changing environmental conditions. Fluctuations of interspike intervals (ISIs) and the moment relation derived from ISI distributions contain information on the channel cluster open probability and on pathway properties. General significance Robust and reliable signal transduction pathways that entail Ca²⁺ dynamics are essential for eukaryotic organisms. Moreover, we expect that the design of a stochastic mechanism which provides robustness and adaptivity will be found also in other biological systems. Ca2 + dynamics demonstrate that the fluctuations of cellular signals contain information on molecular behavior. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling. Highlights ► We review recent findings on key characteristics of cytosolic Ca²⁺ dynamics. ► We demonstrate the importance of the hierarchal arrangement of Ca²⁺ release sites. ► New theoretical concepts exploit emergent behavior of cellular Ca²⁺ spikes. ► We relate the dynamical regulation of [Ca²⁺] to information transmission. ► Stochastic Ca²⁺ signals are functionally robust and adaptive to changing conditions

Insulin Gene Expression Is Regulated by DNA Methylation

BACKGROUND:Insulin is a critical component of metabolic control, and as such, insulin gene expression has been the focus of extensive study. DNA sequences that regulate transcription of the insulin gene and the majority of regulatory factors have already been identified. However, only recently have other components of insulin gene expression been investigated, and in this study we examine the role of DNA methylation in the regulation of mouse and human insulin gene expression. METHODOLOGY/PRINCIPAL FINDINGS:Genomic DNA samples from several tissues were bisulfite-treated and sequenced which revealed that cytosine-guanosine dinucleotide (CpG) sites in both the mouse Ins2 and human INS promoters are uniquely demethylated in insulin-producing pancreatic beta cells. Methylation of these CpG sites suppressed insulin promoter-driven reporter gene activity by almost 90% and specific methylation of the CpG site in the cAMP responsive element (CRE) in the promoter alone suppressed insulin promoter activity by 50%. Methylation did not directly inhibit factor binding to the CRE in vitro, but inhibited ATF2 and CREB binding in vivo and conversely increased the binding of methyl CpG binding protein 2 (MeCP2). Examination of the Ins2 gene in mouse embryonic stem cell cultures revealed that it is fully methylated and becomes demethylated as the cells differentiate into insulin-expressing cells in vitro. CONCLUSIONS/SIGNIFICANCE:Our findings suggest that insulin promoter CpG demethylation may play a crucial role in beta cell maturation and tissue-specific insulin gene expression

Studies on the regulation and structure of the ovarian high-density lipoprotein receptor.

The ovary utilizes the cholesterol from high density lipoproteins (HDL) for steroidogenesis. To further characterize the role of HDL, the regulation and structure of the ovarian HDL receptor was studied. To examine cholesterol regulation of HDL binding, luteal cells were cultured with increasing concentrations of low density lipoproteins (LDL). Treatment with LDL caused a dose-dependent increase in total cellular sterol content, progesterone secretion, and HDL binding (2.1-, 2.3-, and 2.2-fold, respectively). Treatment with 25-hydroxycholesterol (HOC) in the presence of 100 $\mu$g/ml aminoglutethimide (a cholesterol metabolism inhibitor) also elicited a dose-dependent increase in HDL binding to luteal cells (5.8-fold at 40 $\mu$g HOC/ml). Scatchard analysis of the HOC (10 $\mu$g/ml) up-regulation of HDL binding demonstrated a 2.1-fold increase in B\sb{\rm max} with no effect on the K\sb{\rm d}. These results suggest that HDL binding to luteal cells is regulated by cellular cholesterol levels. Rat ovarian membrane preparations exhibited binding of HDL to a single class of high affinity sites (K\sb{\rm d} of 15.0 $\mu$g HDL/ml). Ligand blotting analyses revealed the presence of a single 58 kDa HDL binding protein. The HDL binding characteristics of the protein were similar to those obtained using intact cells. The protein was up-regulated by gonadotropin and was present in liver and adrenal membranes. These results suggest that the rat HDL receptor is a 58 kDa protein. Purification of a HDL receptor was attempted using bovine luteal plasma membranes. Membrane preparations exhibited binding of HDL to a single class of high affinity sites (K\sb{\rm d} of 6.7 $\mu$g HDL/ml). HDL binding was unaffected by EDTA or changes in pH. HDL binding increased with increasing salt concentrations due to an increase in the number of HDL binding sites. Ligand blotting analyses revealed the presence of a single 108 kDa HDL binding protein. The protein was isolated by sequential solubilization, polyethylene glycol precipitation, DEAE-Sephadex chromatography, and preparative SDS-PAGE. The purified product appeared electrophoretically homogeneous and retained HDL binding activity. These studies will enable structural characterization of the HDL receptor and examination of its role in cholesterol transport.Ph.D.Biological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/105889/1/9226893.pdfDescription of 9226893.pdf : Restricted to UM users only

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

<div><p>The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.</p></div