The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

IntroductionHuman mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into fat, muscle, bone and cartilage cells. Exposure of subcutaneous abdominal adipose tissue derived AD-MSCs to high glucose (HG) leads to superoxide accumulation and up-regulation of inflammatory molecules. Our aim was to inquire how HG exposure affects MSCs differentiation and whether the mechanism is reversible.MethodsWe exposed human adipose tissue derived MSCs to HG (25 mM) and compared it to normal glucose (NG, 5.5 mM) exposed cells at 7, 10 and 14 days. We examined mitochondrial superoxide accumulation (Mitosox-Red), cellular oxygen consumption rate (OCR, Seahorse) and gene expression.ResultsHG increased reactive superoxide (ROS) accumulation noted by day 7 both in cytosol and mitochondria. The OCR between the NG and HG exposed groups however did not change until 10 days at which point OCR of HG exposed cells were reduced significantly. We noted that HG exposure upregulated mRNA expression of adipogenic (PPARG, FABP-4, CREBP alpha and beta), inflammatory (IL-6 and TNF alpha) and antioxidant (SOD2 and Catalase) genes. Next, we used AdSOD2 to upregulate SOD2 prior to HG exposure and thereby noted reduction in superoxide generation. SOD2 upregulation helped reduce mRNA over-expression of PPARG, FABP-4, IL-6 and TNFα. In a series of separate experiments, we delivered the eGFP and SOD2 upregulated MSCs (5 days post ex-vivo transduction) and saline intra-peritoneally (IP) to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSCs, delivered IP, to different inflamed fat pockets, particularly omental fat. Mice receiving SOD2-MSCs showed progressive reduction in body weight and improved glucose tolerance (GTT) at 4 weeks, post MSCs transplantation compared to the GFP-MSC group (control).ConclusionsHigh glucose evokes superoxide generation, OCR reduction and adipogenic differentiation. Mitochondrial superoxide dismutase upregulation quenches excess superoxide and reduces adipocyte inflammation. Delivery of superoxide dismutase (SOD2) using MSCs as a gene delivery vehicle reduces inflammation and improves glucose tolerance in vivo. Suppression of superoxide production and adipocyte inflammation using mitochondrial superoxide dismutase may be a novel and safe therapeutic tool to combat hyperglycemia mediated effects.

AimsType 2 diabetes is associated with endothelial dysfunction leading to cardiovascular disease. CD34+ endothelial Progenitor Cells (EPCs) are responsible for endothelial repair and neo-angiogenesis and can be used as a cardiovascular disease risk biomarker. This study investigated whether the addition of saxagliptin, a DPP-IV inhibitor, to metformin, may reduce cardiovascular disease risk in addition to improving glycemic control in Type 2 diabetes patients.MethodsIn 12 week, double-blind, randomized placebo-controlled trial, 42 subjects already taking metformin 1–2 grams/day were randomized to placebo or saxagliptin 5 mg. Subjects aged 40–70 years with diabetes for < 10 years, with no known cardiovascular disease, BMI 25–39.9, HbA1C 6–9% were included. We evaluated EPCs number, function, surface markers and gene expression, in addition to arterial stiffness, blood biochemistries, resting energy expenditure, and body composition parameters. A mixed model regression to examine saxagliptin vs placebo, accounting for within-subject autocorrelation, was done with SAS (p < 0.05).ResultsAlthough there was no significant increase in CD34+ cell number, CD31+ cells percentage increased. Saxagliptin increased migration (in response to SDF1α) with a trend of higher colony formation count. MNCs cytometry showed higher percentage of CXCR4 double positivity for both CD34 and CD31 positive cells, indicating a functional improvement. Gene expression analysis showed an upregulation in CD34+ cells for antioxidant SOD1 (p < 0.05) and a downregulation in CD34− cells for IL-6 (p < 0.01). For arterial stiffness, both augmentation index and systolic blood pressure measures went down in saxagliptin subjects (p < 0.05).ConclusionSaxagliptin, in combination with metformin, can help improve endothelial dysfunction in early diabetes before macrovascular complications appear.Trial registration Trial is registered under clinicaltrials.gov, NCT02024477Electronic supplementary materialThe online version of this article (10.1186/s12933-018-0709-9) contains supplementary material, which is available to authorized users.

Background The incidence of obesity and diabetes is increasing rapidly. Optimal management is still elusive. Obesity associated with type 2 diabetes is known to cause adipose tissue inflammation, increase oxidative stress, and cause white fat hyperplasia and mitochondrial dysfunction. In this study, we investigated whether mitochondrial and cytosolic antioxidant-upregulated mesenchymal stem cell (MSC) delivery reduces oxidative stress and subsequently improves glucose tolerance, reduce systemic inflammation, and improves fatty liver disease in diet-induced obese (DIO) mouse models. Methods Antioxidant genes Sod2 (mitochondrial) and catalase (cytosolic) or null (control) were upregulated in human adipose tissue-derived MSCs using adenoviral constructs. Modified MSCs were then delivered intraperitoneally into mice that were fed a 45% or 60% high-fat diet (HFD), and animals were followed for 4 weeks. Results Over 4 weeks, body weight remained stable; however, we noted a significant reduction in liver fat content by histological analysis and liver triglyceride assay. Triglyceride assay ( p < 0.01) confirmed reduced liver fat accumulation in animals that received either Sod2- or Cat-MSCs. There was a lower plasma level of inflammatory marker TNFα, measured in mice that were fed either 45% or 60% HFD and received Sod2- or Cat-MSCs, indicating reduced systemic inflammation. Ucp1 mRNA was upregulated approximately 100–1000-fold for omental fat and 10–100-fold for pericardial fat compared to the Null-MSC-receiving group. Pcgc1a and Prdm16 mRNA upregulation was also noted particularly for pericardial fat. Glucose tolerance showed a positive improvement trend with a lower area under the curve (AUC) values for both Sod2- and Cat-MSCs groups in comparison to control. For mice fed with 60% HFD and that received Sod2-MSCs, glucose levels were significantly lower than control (* p < 0.05) at a time point of 60 min in the glycemic curve during glucose tolerance test. Conclusion Reduction of oxidative stress post-antioxidant-upregulated MSC delivery, intraperitoneally, reduces systemic inflammation and fat accumulation in the liver. There is evidence of an increase in browning of white adipose tissue depots with concomitant improvement of glucose tolerance in a weight-independent fashion. Antioxidant-upregulated MSC delivery may be a safe yet effective therapy for obesity and prediabetes and improves related complication such as non-alcoholic fatty liver disease. Electronic supplementary material The online version of this article (10.1186/s13287-019-1393-8) contains supplementary material, which is available to authorized users.