Hyperglycemic Medium Research Articles

Fasting and Glucose Metabolism Differentially Impact Peripheral Inflammation in Human Type 2 Diabetes.

Cytokines produced by peripheral T-helper 1/17 cells disproportionately contribute to the inflammation (i.e., metaflammation) that fuels type 2 diabetes (T2D) pathogenesis. Shifts in the nutrient milieu could influence inflammation through changes in T-cell metabolism. We aimed to determine whether changes in glucose utilization alter cytokine profiles in T2D. Peripheral blood mononuclear cells (PBMCs), CD4+ T-cells, and CD4+CD25- T-effector (Teff) cells were isolated from age-matched humans classified by glycemic control and BMI. Cytokines secreted by CD3/CD28-stimulated PBMCs and Teff were measured in supernatants with multiplex cytokine assays and a FLEXMAP-3D. Metabolic activity of stimulated CD4+ T-cells was measured by a Seahorse XFe96 analyzer. In this study, we demonstrated that T-cell stimulated PBMCs from non-fasted people with T2D produced higher amounts of cytokines compared to fasting. Although dysglycemia characterizes T2D, cytokine production by PBMCs or CD4+ T-cells in T2D was unaltered by hyperglycemic media. Moreover, pharmacological suppression of mitochondrial glucose oxidation did not change T-cell metabolism in T2D, yet enhanced cytokine competency. In conclusion, fasting and glucose metabolism differentially impact peripheral inflammation in human T2D, suggesting that glucose, along with fatty acid metabolites per our previous work, partner to regulate metaflammation. These data expose a major disconnect in the use of glycemic control drugs to target T2D-associated metaflammation.

Pharmacological activation of nuclear factor erythroid 2-related factor-2 prevents hyperglycemia-induced renal oxidative damage: Possible involvement of O-GlcNAcylation

Hyperglycemia is a major risk factor for kidney diseases. Oxidative stress, caused by reactive oxygen species, is a key factor in the development of kidney abnormalities related to hyperglycemia. The nuclear factor erythroid 2-related factor-2 (Nrf2) plays a crucial role in defending cells against oxidative stress by activating genes that produce antioxidants. L-sulforaphane (SFN), a drug that activates Nrf2, reduces damage caused by hyperglycemia. Hyperglycemic Wistar rats and HEK 293 cells maintained in hyperglycemic medium exhibited decreased Nrf2 nuclear translocation and reduced expression and activity of antioxidant enzymes. SFN treatment increased Nrf2 activity and reversed decreased renal function, oxidative stress and cell death associated with hyperglycemia. To investigate mechanisms involved in hyperglycemia-induced reduced Nrf2 activity, we addressed whether Nrf2 is modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification that is fueled in hyperglycemic conditions. In vivo, hyperglycemia increased O-GlcNAc-modified Nrf2 expression. Increased O-GlcNAc levels, induced by pharmacological inhibition of OGA, decreased Nrf2 activity in HEK 293 cells. In conclusion, hyperglycemia reduces Nrf2 activity, promoting oxidative stress, cell apoptosis and structural and functional renal damage. Pharmacological treatment with SFN attenuates renal injury. O-GlcNAcylation negatively modulates Nrf2 activity and represents a potential mechanism leading to oxidative stress and renal damage in hyperglycemic conditions.

Qufeng tongluo decoction decreased proteinuria in diabetic mice by protecting podocytes via promoting autophagy

BackgroundDiabetic kidney disease (DKD) is one of diabetic complications, which has become the leading cause of end-stage kidney disease. In addition to angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker(ACEI/ARB) and sodium-glucose cotransporter-2 inhibitor (SGLT2i), traditional Chinese medicine (TCM) is an effective alternative treatment for DKD. In this study, the effect of Qufeng Tongluo (QFTL) decoction in decreasing proteinuria has been observed and its mechanism has been explored based on autophagy regulation in podocyte. MethodsIn vivo study, db/db mice were used as diabetes model and db/m mice as blank control. Db/db mice were treated with QFTL decoction, rapamycin, QFTL + 3-Methyladenine (3-MA), trehalose, chloroquine (CQ) and QFTL + CQ. Mice urinary albumin/creatinine (UACR), nephrin and autophagy related proteins (LC3 and p62) in kidney tissue were detected after intervention of 9 weeks. Transcriptomics was operated with the kidney tissue from model group and QFTL group. In vitro study, mouse podocyte clone-5 (MPC-5) cells were stimulated with hyperglycemic media (30 mmol/L glucose) or cultured with normal media. High-glucose-stimulated MPC-5 cells were treated with QFTL freeze-drying powder, rapamycin, CQ, trehalose, QFTL+3-MA and QFTL + CQ. Cytoskeletal actin, nephrin, ATG-5, ATG-7, Beclin-1, cathepsin L and cathepsin B were assessed. mRFP-GFP-LC3 was established by stubRFP-sensGFP-LC3 lentivirus transfection. ResultsQFTL decoction decreased the UACR and increased the nephrin level in kidney tissue and high-glucose-stimulated podocytes. Autophagy inhibitors, including 3-MA and chloroquine blocked the effects of QFTL decoction. Further study showed that QFTL decoction increased the LC3 expression and relieved p62 accumulation in podocytes of db/db mice. In high-glucose-stimulated MPC-5 cells, QFTL decoction rescued the inhibited LC3 and promoted the expression of ATG-5, ATG-7, and Beclin-1, while had no effect on the activity of cathepsin L and cathepsin B. Results of transcriptomics also showed that 51 autophagy related genes were regulated by QFTL decoction, including the genes of ATG10, SCOC, ATG4C, AMPK catalytic subunit, PI3K catalytic subunit, ATG3 and DRAM2. ConclusionQFTL decoction decreased proteinuria and protected podocytes in db/db mice by regulating autophagy.

Normoglycemia partially recovers the disrupted osteoblast differentiation of mesenchymal stem cells induced by type 1 but not type 2 diabetes mellitus.

Type 1 (T1DM) and type 2 (T2DM) diabetes mellitus are characterized by changes in glucose metabolism and cause bone damage via a variety of mechanisms, including effects on osteoblasts. We aimed to evaluate the osteoblast differentiation of mesenchymal stem cells (MSCs) from rats with T1DM or T2DM and the effects of removing the hyperglycemic stimulus on the osteogenic potential of these cells. MSCs from healthy rats were cultured in normoglycemic medium, whereas MSCs from rats with T1DM or T2DM were cultured in hyperglycemic or normoglycemic medium. T1DM and T2DM reduced osteoblast differentiation of MSCs grown in hyperglycemic media, with T1DM having a more pronounced effect, as evidenced by alkaline phosphatase activity, RUNX2 protein expression, and extracellular matrix mineralization, and modulated the gene expression of several components of the bone morphogenetic protein signaling pathway. The restoration of the normoglycemic environment partially recovers the osteogenic potential of MSCs from rats with T1DM but not with T2DM. Our findings highlight the need for specific therapies to treat T1DM- or T2DM-induced bone loss, as both disrupt osteoblast differentiation at distinct levels and likely through different mechanisms.

The influence of high glucose conditions on macrophages and its effect on the autophagy pathway.

Macrophages are central cells in mediating the inflammatory response. We evaluated the effect of high glucose conditions on the inflammatory profile and the autophagy pathway in Bone-Marrow Derived Macrophages (BMDM) from diabetic (D-BMDM) (alloxan: 60mg/kg, i.v.) and non-diabetic (ND-BMDM) C57BL/6 mice. BMDM were cultured in medium with normal glucose (5.5 mM), or high glucose (25 mM) concentration and were primed with Nigericin (20µM) stimulated with LPS (100 ng/mL) at times of 30 minutes; 2; 4; 6 and 24 hours, with the measurement of IL-6, IL-1β and TNF-α cytokines. We have further identified changes in the secretion of pro-inflammatory cytokines IL-6, IL-1β and TNF-α, where BMDM showed increased secretion of these cytokines after LPS + Nigericin stimulation. In addition, changes were observed in the autophagy pathway, where the increase in the autophagic protein LC3b and Beclin-1 occurred by macrophages of non-diabetic animals in hyperglycemic medium, without LPS stimulation. D-BMDM showed a reduction on the expression of LC3b and Beclin-1, suggesting an impaired autophagic process in these cells. The results suggest that hyperglycemia alters the inflammatory pathways in macrophages stimulated by LPS, playing an important role in the inflammatory response of diabetic individuals.

Dual effect of the herbal matcha green tea (Camellia sinensis L. kuntze) supplement in EA.hy926 endothelial cells and Artemia salina

Ethnopharmacological relevanceMatcha green tea (Camellia sinensis) based-supplements have been widely used since they present a greater content of phenolic compounds than traditional green tea, which is popularly used in the treatment of diabetes. However, there are few studies on the effectiveness and safety of matcha supplements. Aim of the studyThis work aimed to evaluate the efficacy and safety of this supplement in endothelial cells (EA.hy926) in the hyperglycemic model and in vivo Artemia salina. Materials and methodsTo assess the effect of Matcha herbal supplement (MHS), EA. hy926 endothelial cells were treated with 20 μg/mL of MHS for 24 h, in a hyperglycemic medium with 35 mM glucose. After treatment, cells were trypsinized and centrifuged at 4 °C and 47×g for 5 min. The pellet was used to determine the reaction products to thiobarbituric acid and the levels of nitric oxide. Electron transport chain activity and ATP levels were also evaluated. Intracellular pH, apoptosis, and mitochondrial membrane depolarization were evaluated by flow cytometry. MHS chemical characterization was performed by HPLC-UV and total phenolic content analysis. The evaluation of the antioxidant capacity of MHS was performed by 2,2-diphenyl-1-picrylhydrazyl radical scavenger assay. To determine the in vivo acute toxicity of MHS, an A. salina assay was conducted, using 0,2 mL of different concentrations of MHS (10, 50, 100, 250, 500, 750 and 1000 μg/mL). The LD50 values were obtained by interpolation of 50% (y = 50) of the dead individuals in the trend curves. ResultsOur data showed that MHS was able to avoid oxidative and nitrosative stress induced by hyperglycemia, demonstrating important antioxidant activity. However, it was observed that MHS reduced up to 90% the activity of the four-electron transport complexes, reducing the ATP production of the endothelial cells. In the toxicity assay performed in Artemia salina, MHS showed mild toxicity (LD50 = 0,4 mg/mL). The major compounds found in MHS were epigallocatechin gallate, epicatechin, rutin, kaempferol, and quercetin. ConclusionsThis data draws attention to the fact that supplements with high content of phenolic compounds, capable of avoiding oxidative and nitrosative stress can have a dual effect and, simultaneously to antioxidant activity, can induce toxicity in different cell types.

An Insulin-like Growth Factor-1 Conjugated Bombyx mori Silk Fibroin Film for Diabetic Wound Healing: Fabrication, Physicochemical Property Characterization, and Dosage Optimization In Vitro and In Vivo.

This study aimed to develop a silk fibroin (SF)-film for the treatment of chronic diabetic wounds. Silk fibroin was purified through a newly developed heating degumming (HD) process and casted on a hydrophobic surface to form SF-films. The process allowed the fabricated film to achieve a 42% increase in transparency and a 32% higher proliferation rate for BALB/3T3 fibroblasts compared to that obtained by conventional alkaline degumming treatment. Fourier transform infrared analysis demonstrated that secondary structure was retained in both HD- and alkaline degumming-derived SF preparations, although the crystallinity of beta-sheet in SF-film after the HD processing was slightly increased. This study also investigated whether conjugating insulin-like growth factor-1 (IGF-1) would promote diabetic wound healing and what the optimal dosage is. Using BALB/3T3 cells grown in hyperglycemic medium as a model, it was demonstrated that the optimal IGF-1 dosage to promote the cell growth was approximately 0.65 pmol. Further analysis of wound healing in a diabetic mouse model indicated that SF-film loaded with 3.25 pmol of IGF-1 showed significantly superior wound closure, a 13% increase at the 13th day after treatment relative to treatment with 65 pmol of free IGF-1. Improvement in diabetic wound healing was exerted synergistically by SF-film and IGF-1, as reflected by parameters including levels of re-epithelialization, epithelial tissue area, and angiogenesis. Finally, IGF-1 increased the epithelial tissue area and micro-vessel formation in a dose-dependent manner in a low dosage range (3.25 pmol) when loaded to SF-films. Together, these results strongly suggest that SF-film produced using HD and loaded with a low dosage of IGF-1 is a promising dressing for diabetic wound therapy.

Exendin-4 differentially modulates essential functions of human dermal fibroblasts under normoglycemic and hyperglycemic conditions.

Evidence that exendin-4, a glucagon-like peptide-1 analog, might be used to treat poorly healing wounds under diabetic and nondiabetic conditions has gained increasing interest. Little is known, however, about the effects of the drug on the production by dermal fibroblasts of key extracellular matrix and regulatory compounds. Therefore, we used human skin fibroblasts cultured in normo- (1 g/l = 5.6 mmol/l glucose) or hyperglycemic (4.5 g/l = 25 mmol/l glucose) culture medium to test the effects of exendin-4 (0 - 100 nmol/l) on fibroblast functions crucial for the wound healing process. Exendin-4 increased the proliferative and metabolic activities, as measured by the BrdU (bromodeoxyuridine) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays, respectively, of fibroblasts cultured in normoglycemic medium. Under hyperglycemic conditions, the drug had no effect on proliferation and reduced metabolic fibroblast activity. Exendin-4 decreased metalloproteinase-9 (MMP-9) secretion in the normoglycemic milieu only and increased tissue inhibitor of metalloproteinase-1 (TIMP-1) concentration in fibroblast colonies under both normo- and hyperglycemic experimental conditions. Exendin-4 increased the fibroblast growth factor-1 (FGF-1) concentration in cell colonies maintained in the normoglycemic milieu but decreased FGF-1 release when fibroblasts were grown in hyperglycemic medium. High glucose caused lactic dehydrogenase (LDH) leakage when compared with normoglycemic conditions, and exendin-4 was not able to prevent this effect, although it reduced LDH release from fibroblasts cultured in normoglycemic medium. Finally, exendin-4 increased glycosaminoglycan (GAG) content under both experimental conditions. Our results indicate that exendin-4 effects on the production of the extracellular matrix and regulatory proteins differ in human skin fibroblasts exposed to either normal or high glucose. In general, the beneficial effects of the drug, which may be important for the improvement of wound healing, are more pronounced under normoglycemic conditions, thus indicating that hyperglycemia attenuates the positive effects of exendin-4 on fibroblasts.

Impaired soft and hard callus formation during fracture healing in diet-induced obese mice as revealed by 3D contrast-enhanced computed tomography imaging.

The impact of diabetes mellitus on bone fracture healing is clinically relevant as the patients experience delayed fracture healing. Even though efforts have been made to understand the detrimental effects of type 2 diabetes mellitus (T2DM) on the fracture healing process, the exact mechanisms causing the pathophysiological outcomes remain unclear. The aim of this study was to assess alterations in bone fracture healing (tibial fracture surgery, intramedullary pinning) of diet-induced obese (DIO) mice, and to investigate the in vitro properties of osteochondroprogenitors derived from the diabetic micro-environment. High-resolution contrast-enhanced microfocus X-ray computed tomography (CE-CT) enabled a simultaneous 3D assessment of the amount and spatial distribution of the regenerated soft and hard tissues during fracture healing and revealed that osteogenesis as well as chondrogenesis are altered in DIO mice. Compared to age-matched lean controls, DIO mice presented a decreased bone volume fraction and increased callus volume and adiposity at day 14 post-fracture. Of note, bone turnover was found altered in DIO mice relative to controls, evidenced by decreased blood serum osteocalcin and increased serum CTX levels. The in vitro data revealed that not only the osteogenic and adipogenic differentiation of periosteum-derived cells (PDCs) were altered by hyperglycemic (HG) conditions, but also the chondrogenic differentiation. Elevated PPARγ expression in HG conditions confirmed the observed increase in differentiated adipocytes in vitro. Finally, chondrogenesis-related genes COL2 and COL10 were downregulated for PDCs treated with HG medium, confirming that chondrogenic differentiation is compromised in vitro and suggesting that this may affect callus formation and maturation during the fracture healing process in vivo. Altogether, these results provide novel insights into the alterations of long bone fracture repair and suggest a link between HG-induced dysfunctionality of osteochondroprogenitor differentiation and fracture healing impairment under T2DM conditions.

An oxalate-bridged oxidovanadium(IV) binuclear complex that improves the in vitro cell uptake of a fluorescent glucose analog

The centrosymmetric oxidovanadium(IV) complex (Et3NH)2[{VO(OH2)(ox)}2(μ–ox)] (I), where ox2− = oxalate, was synthesized and characterized by X-ray diffraction (single-crystal and powder, PXRD), thermogravimetric (TGA), magnetic susceptibility (at room temperature) and spectroscopic analyses (infrared, Raman and electron paramagnetic resonance, EPR, spectroscopies). In the solid state, each vanadium center is coordinated by the oxygen atoms of a bis-bidentate oxalate bridging ligand, a terminal oxalate, an oxo group and one water molecule. The electronic structure of the binuclear complex was investigated by density functional theory (DFT) calculations, both in vacuum and in a simulated aqueous environment, employing the ωB97XD functional and the def2TZVP basis set. The cytotoxicity of I was evaluated in vitro in the human hepatocellular carcinoma cell line HepG2, giving an IC50 value of 15.67 µmol L−1 after incubation for 24 h. The EPR analysis of I in aqueous solution suggested the maintenance of the binuclear structure, while in the hyperglycemic medium DMEM the complex suffers dissociation to give a mononuclear oxidovanadium(IV) species. HepG2 cell treatment with 0.10 and 0.50 µmol L−1 of I in DMEM increased 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose) uptake significantly (up to 91% as compared to HepG2 in hyperglycemic condition, 59%). These results indicate a promising activity of I to be investigated further in additional antidiabetic studies.

Chymase inhibition: A key factor in the anti-inflammatory activity of ethanolic extracts and spilanthol isolated from Acmella oleracea

Ethnopharmacological relevanceAcmella oleracea (L.) R. K. Jansen (Asteraceae), known as jambú in Brazil, is used in traditional medicine as analgesic and for inflammatory conditions, characterized by the presence of N-alkylamides, mainly spilanthol. This bioactive compound is responsible for the above-described pharmacological properties, including sialagogue and anesthetic. Aim of the studyThis study aimed to characterize the anti-inflammatory effects of A. oleracea leaves (AOEE-L) and flowers (AOEE-F) extracts, including an isolated alkylamide (spilanthol), using in vitro and in vivo models. The mechanism underlying this effect was also investigated. Materials and methodsExtracts were analyzed by HPLC-ESI-MS/MS in order to characterize the N-alkylamides content. AOEE-L, AOEE-F (25–100 μg/mL) and spilanthol (50–200 μM) were tested in vitro on VSMC after stimulation with hyperglycemic medium (25 mM glucose). Their effects over nitric oxide (NO) generation, chymase inhibition and expression, catalase (CAT), superoxide anion (SOD) radical activity were evaluated. After an acute administration of extracts (10–100 mg/mL) and spilanthol (6.2 mg/mL), the anti-inflammatory effects were evaluated by applying the formalin test in rats. Blood was collected to measure serum aminotransferases activities, NO activity, creatinine and urea. ResultsA number of distinct N-alkylamides were detected and quantified in AOEE-L and AOEE-F. Spilanthol was identified in both extracts and selected for experimental tests. Hyperglycemic stimulation in VSMC promoted the expression of inflammatory parameters, including chymase, NO, CAT and SOD activity and chymase expression, all of them attenuated by the presence of the extracts and spilanthol. The administration of extracts or spilanthol significantly inhibited edema formation, NO production and cell tissue infiltration in the formalin test, without causing kidney and liver toxicity. ConclusionTaken together, these results provide evidence for the anti-inflammatory activity of leaves and flowers extracts of jambú associated distinctly with their chemical profile. The effects appear to be associated with the inhibition of chymase activity, suppression of the proinflammatory cytokine NO and antioxidant activities.

In Vitro Effect of Crude Extract from Traganum Nudatum on Glucose-Uptake in Liver Slices Isolated from Westar Rats

Background: Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both. There are many classes of drugs used for treatment, and these include insulin sensitizers, insulin secretagogues, and agents that delay the absorption of carbohydrates from the bowel. This study intends to investigate the effect of crude extract from a plant from South Algeria Traganum nudatum (Chenopodiaceae) on glucose uptake in liver slices isolated from Wistar rats. Methods: The liver slices were incubated for 90 min at 37° in normoglycaemic (1g/l of glucose) and hyperglycaemic (3g/l of glucose) KRBA Krebs Ringer Bicarbonate Albumin 4% media using 24 well-polyethylene plates. In each, well different concentrations of insulin (10, 50 and 100µU/ml) and hydromethanolic crude extract (100, 200 and 500µg/ml) were added. After every 30 minutes, aliquots of the culture media were assayed for the determination of glucose left. Results: Tests showed that the glucose left after 90 minutes in the media which contained insulin at 100µg/ml was the lowest (0.44 and 1.41 )g/l in the normo and hyperglycaemic media respectively, which reflect that insulin at this concentration was the most effective on the stimulation of glucose uptake. The extract had the highest effect at 500µg/ml, the concentrations of glucose left after 90 minutes of incubation were found to be (0.38 and 1.31)g/l in the normoglycaemic and hyperglycaemic media respectively. Conclusion: From the obtained results, it can be concluded that our extract seems to have an insulin-like effect on glucose uptake in liver slices isolated from Wistar rats.

P0982PTEN-INDUCED KINASE 1 (PINK1) DEPLETION ALTERS MITOCHONDRIAL EFFICIENCY AND GLYCOLYSIS IN HUMAN PODOCYTES

Abstract Background and Aims Podocytes are terminally differentiated cells, which constitute an inner layer of the renal filtration barrier. Podocytes are characterized by high metabolic activity, and their elevated energy requirements are met by maintaining the appropriate mitochondrial number and quality, which depend on mitochondrial biogenesis and mitophagy. Alteration of mitochondrial dynamics is linked to the development of insulin resistance and diabetes. The main goal of the research was to determine the role of mitophagy in podocytes bioenergetics and to elucidate the effects of hyperglycemia on mitochondrial dynamics. Method In order to inhibit mitophagy, we generated a human podocyte cell line stably expressing PINK1 shRNA through lentiviral transduction. Biochemical analyses were performed to assess the oxidative phosphorylation efficiency (by measurement of oxygen consumption rate; OCR) and glycolysis contribution to the total cell energy production (by measurement of extracellular acidification rate; ECAR) in the differentiated shPINK1 podocytes. The expression levels of mRNAs and proteins were evaluated in podocytes cultured in standard glucose (11 mM) and high glucose (30 mM) concentrations using real-time PCR and western blot. Intracellular mitochondrial network was visualized by MitoTrackertTM staining. The co-localization of proteins in podocytes was analysed by double immunofluorescence labelling and confocal microscopy. Results PINK1-deficiency resulted in the significant decrease of maximal respiration and spare respiratory capacity in podocytes (by 40% and 70%, respectively). Non-mitochondrial respiration was also decreased by 45% in shPINK1 cells. Interestingly, basal respiration and ATP production appeared similar in shPINK1 and control podocytes. PINK1 depletion increased glycolytic flux by 70%, in addition, the accompanying decline in glycolytic capacity and glycolytic reserve was observed (by 38% and 63%, respectively). Moreover, we observed accumulation of small and ring-shaped mitochondria in PINK1-deficient podocytes compared with the control cells. We showed a decreased expression of PINK1 and Parkin (mRNA and protein) in normal human podocytes cultured in hyperglycemic medium, which was associated with an elevated levels of mitochondrial fission markers (DRP1, FIS1) and with a decreased levels of PGC1α and TFAM, which play a role in mitochondrial biogenesis and mtDNA replication. Conclusion In this research, we demonstrated a novel role of mitophagy in podocyte bioenergetics. Moreover, we showed that high glucose inhibits mitophagy and promotes mitochondrial fission leading to the accumulation of damaged mitochondria and podocyte injury, which underlies the pathogenesis of diabetic nephropathy.

SUN-664 Effects of Acute Hyperglycemia on Monocytes in Type 2 Diabetes

Background: Type 2 diabetes is linked to accelerated atherosclerosis, however the mechanisms behind this association are unclear. Inadequate glycemic control is an important contributor for cardiovascular disease (CVD). Episodic hyperglycemia as a surrogate for glycemic variability (GV) promotes monocyte adhesion and increases proinflammatory monocytes within plaques of patients with diabetes. Monocytes are precursors to macrophages which play a vital role in atherosclerosis and are capable of delivering cholesterol content to plaques. Therefore, elucidating the mechanism by which acute hyperglycemia promotes proatherogenic monocytes in diabetes can lead to novel therapies slowing atherosclerosis progression in these patients. Methods: To assess the effects of acute hyperglycemia on monocyte cholesterol metabolism, we isolated monocytes from 28 patients with Type 2 diabetes cultured for 2 and 6 hours in normoglycemic (90mg/dL) or hyperglycemic media (300mg/dL). Cholesterol content was measured by lipid extraction and fluorometric cholesterol oxidase assay. Cholesterol uptake was assessed by incubating monocytes with Dil-oxidized LDL (Dil-OxLDL). Cholesterol synthesis was performed by incubating monocytes with 2-14C-Acetate in both glycemic conditions for 6 hours followed by lipid saponification, and separated via TLC on silica gel plates. Cholesterol efflux was assessed by labeling cells with 3H-Cholesterol-OxLDL in both glycemic conditions for 6 hours followed by APO-A1 or HDL stimulation for 4 hours. Results: Acute hyperglycemia for 2 and 6 hours induced foamy monocytes with increased total cholesterol content (22 and 32%, respectively), cholesterol ester (40% and 43%, respectively), and free cholesterol content (14% and 25%, respectively) due to enhanced DiI-OxLDL uptake. There was no significant increase in monocyte cholesterol synthesis or cholesterol efflux after stimulation with HDL or APO A-1. Conclusions: Increased monocyte cholesterol content during acute hyperglycemia is a potential mechanism promoting atherosclerosis in patients with Type 2 diabetes.

OR14-01 FSTL3 Neutralizing Antibodies Restore Function to Diabetic Mouse and Human Islets: A New Approach for Treating Diabetes

Activin, GDF11 and myostatin are structurally related members of the TGFbeta superfamily of growth factors with many biological roles in animal models and humans. Their actions are neutralized by extracellular proteins such as follistatin and follistatin like-3 (FSTL3). We have previously demonstrated that genetic inactivation of Fstl3 results in enlarged pancreatic islets containing increased numbers of beta cells that produce more insulin in response to glucose compared to wild type litter mates. We further discovered that at least some of these new beta cells arise via transdifferentiation from alpha cells. We also demonstrated that functional human islets from normal donors produce very high levels of activin. In contrast, activin biosynthesis is vastly reduced and FSTL3 synthesis is significantly increased in human islets from diabetic donors suggesting that activin is critical for normal insulin production. This was substantiated by direct treatment of human diabetic islets with activin which restored their response to glucose. These observations support the hypothesis that an FSTL3 neutralizing antibody would constitute a novel therapeutic approach to curing diabetes through restoring beta cell function as well as accelerating generation of new beta cells through transdifferentiation. To test this hypothesis, we produced a mouse monoclonal antibody that neutralized hFSTL3 (FP-101), thereby releasing bioactive activin, GDF11, and myostatin. We have now tested this antibody for biological activity in vitro on mouse and human islets. We used islets from high fat diet (HFD) treated mice to model diabetes-inducing effects of obesity as well as 24-hour incubation in hyperglycemic (33 mM glucose) medium to create human islets that lose responsiveness to high glucose as a model for human diabetes. In mouse islets we found that stimulation of normal (chow diet) islets by high glucose produced a stimulation index (SI) of 3.5 that was reduced to 2 in HFD islets. Treatment with activin, FP-101, or a commercial polyclonal antibody to mFSTL3 all increased response of HFD islets to elevated glucose and partially restored SI to normal levels. In human islets, hyperglycemia eliminated the normal (2.5 SI) response to high glucose while activin or FP-101 treatments dose-responsively restored this response. These results demonstrate that anti-FSTL3 therapy can restore function to compromised beta cells from mouse and human diabetes models. The observation that activin has the same action as anti-FSTL3 antibody indicates that FP-101 works through enhancing the activin signaling pathway. Finally, these results demonstrate that the FSTL3-activin pathway is an important regulator of beta cell function in humans as well as mice, supporting further development of this therapy as a diabetes treatment.

Identification Of A Novel Histone Derived Antimicrobial Peptide In Airway Surface Liquid.

The airway surface liquid (ASL), a protective layer secreted by the airway epithelium, represents the first line of defence against inhaled infectious material. It contains a complex array of secreted proteins and peptides that aid the neutralisation and removal of inhaled microbes and toxicants. The ASL also contains many proteases that can cleave proteins to generate further bioactive peptides. We therefore hypothesised that the airway ASL sustained an extensive peptidome containing novel bioactive peptides. We examined the ASL peptide profile of normal human bronchial epithelial cells transformed with BMI‐1 (NHBE‐BMI1) and cell‐lines Calu3 (submucosal adenocarcinoma) and H441 (Clara cell like adenocarcinoma). ASL was acquired by washing the apical surface of epithelial monolayers grown at air‐liquid interface with PBS (100μl) at 0, 24 and 120 hours after exposure to hyperglycaemic (25mM glucose) or normoglycaemic (5mM glucose and 20mM mannitol) basolateral medium. The ASL peptides < 10kDa, were subsequently isolated and analysed using the Q Exactive™ HF‐X Hybrid Quadrupole‐Orbitrap™ Mass Spectrometer. The resulting spectra of native peptides and their subsequent fragments were analysed using the pNovo De novo sequencing tool. Our preliminary results identified 4765 unique peptides (NHBE‐BMI1: 646, Calu3: 3150, H441: 1197). Of these, 37 peptides were common to all samples regardless of glycaemic state. A number of histone derived peptides were identified in all samples and cell lines. These peptides had a proline‐alanine rich N‐terminal region and a cationic C‐terminal, similar to antimicrobial peptides from histone H1 found in Atlantic salmon skin mucous. Synthetic peptides were produced by SPOT synthesis and the antimicrobial activity tested against luminescent Pseudomonas aeruginosa over 18 hours at 37°C in (100mM Tris (pH 7) and 20mM glucose). One of the crude peptides elicited a dose dependent inhibition of bacterial luminescence at 4.63±1.34 μM (n=3), indicating no bacteria survived. Truncated variants of this peptide containing the alanine‐proline rich N‐terminal sequence or the cationic C‐terminal sequence were produced to determine if a specific region of the peptide was required for antimicrobial activity. The N and the C terminal peptides had their antimicrobial activity significantly decreased, only reducing bacterial luminescence by 28.06±2.27% and 35.28±9.45% respectively compared to the full‐length peptide; 99.99±0.001% (all normalised to untreated bacterial luminescence, n=4, p<0.0001, ±SD). This indicates that both regions are required for antimicrobial activity. An immunoblot of concentrated native ASL peptides and cell lysates showed the presence of known histone protein epitopes in our peptide of interest. Furthermore, we identified this histone‐derived peptide in sputum samples from both Cystic Fibrosis and normal healthy patients. Our preliminary data indicates that this novel histone derived peptide may serve an antimicrobial function in the ASL.Support or Funding InformationMedical Research Council London Intercollegiate Doctoral Training Programme

A Comorbidity Model of Myocardial Ischemia/Reperfusion Injury and Hypercholesterolemia in Rat Cardiac Myocyte Cultures

IntroductionThe use of comorbidity models is crucial in cardioprotective drug development. Hypercholesterolemia causes endothelial and myocardial dysfunction, as well as aggravates ischemia/reperfusion (I/R)-induced myocardial injury. Endogenous cardioprotective mechanisms against I/R are impaired in hyperlipidemic and hyperglycemic in vivo animal models. Therefore, our aim was to develop a medium throughput comorbidity cell-based test system of myocardial I/R injury, hypercholesterolemia and hyperglycemia that mimics comorbidity conditions.MethodsCardiac myocytes isolated from neonatal or adult rat hearts were cultured in control or in three different hypercholesterolemic media with increasing cholesterol content (hiChol) or hiChol + hyperglycemic medium, respectively. Each group was then subjected to simulated ischemia/reperfusion (SI/R) or corresponding normoxic condition, respectively. Cholesterol uptake was tested by Filipin staining in neonatal cardiac myocytes. Cell viability, total cell count and oxidative stress, i.e., total reactive oxygen species (ROS) and superoxide level were measured by fluorescent assays.ResultsNeonatal cardiac myocytes took up cholesterol from the different hiChol media at a concentration-dependent manner. In normoxia, viability of hiChol neonatal cardiac myocytes was not significantly changed, however, superoxide levels were increased as compared to vehicle. After SI/R, the viability of hiChol neonatal cardiac myocytes was decreased and total ROS level was increased as compared to vehicle. HiChol combined with hyperglycemia further aggravated cell death and oxidative stress in normoxic as well as in SI/R conditions. Viability of hiChol adult cardiac myocytes was significantly decreased and superoxide level was increased in normoxia and these changes were further aggravated by SI/R. HiChol combined with hyperglycemia further aggravated cell death, however level of oxidative stress increased only in normoxic condition.ConclusionHiChol rat cardiac myocytes showed reduction of cell viability and increased oxidative stress, which were further aggravated by SI/R and with additional hyperglycemia. This is the first demonstration that the combination of the current hypercholesterolemic medium and SI/R in cardiac myocytes mimics the cardiac pathology of the comorbid heart with I/R and hypercholesterolemia.

IN VITRO STUDY OF THE EFFECTS OF BACTERIAL CELLULOSE, GLUCOSE, AND TESTOSTERONE ON THE OSTEO‐1 CELLS MULTIPLICATION

The objective of this study was to analyze the cell multiplication during 15 days of culture, in the absence or presence of bacterial cellulose (BC), in hyperglycemic and hyperandrogenic mediums. OSTEO‐1 cells were cultured with 1.5 mg / L and 4.5 mg / L glucose (mimicking normal glycemia and hyperglycemia), in the absence and presence of two concentrations of testosterone (T) (10−9 M and 10−3 M, mimicking normal androgenemia and hyperandrogenemia), in the absence and presence of BC. Cells were cultured at an initial density (day 0) of 2000 cells per 200 μL of osteogenic medium (DMEM + FBS + ascorbic acid + dexamethasone + b‐glycerophosphate), in 48‐well culture plates, for 5, 10, and 15 days, in triplicates, with exchanges of the medium every 72 h. Cell counting was performed with a Bio‐Rad TC20™ cell counter. The data were analyzed using ANOVA and Fisher's test for multiple comparisons. Culture time effects: The highest cell counts were observed at 15 days, except for the BC group in the presence of T at 10−9 M in hyperglycemic medium, which presented a higher cell count at 10 days of culture. The lowest cell counts were observed at 5 days of culture. Hyperglycemic medium effects: At 5 days of culture, the only treatment that did not present a higher cell count was BC control. At 10 days, all the treatments without BC, as well as the control with BC, showed lower cell counts, while both treatments with BC and T presented higher cell counts. At 15 days, lower cell counts were observed for the control group without BC and for the BC + T at 10−9 M and BC + T at 10−3 M groups. The other treatments presented higher cell counts in the presence of the hyperglycemic medium. BC and T effects. At 5 days of culture, the highest cell counts were observed in the presence of BC and T, while the lowest counts were in the absence of BC. At 10 days, the highest cell counts were observed for the BC + T groups, while the lowest counts were observed for the groups with BC, in the absence of T. At 15 days of culture, the highest cell counts were observed for the group with BC, in the absence of T, and for the group with BC + T at 10−3 M, while the lowest cell counts were observed for the groups without both CB and T. The results indicated that the cells presented higher multiplication activity at 15 days of culture in the presence of BC. In addition, the cell multiplication activity was stimulated by the hyperglycemic medium and by the T in a time‐dependent and dose‐dependent manner. Hence, BC can be used in OSTEO‐1 cultures for studies of hormonal and metabolic effects related to cell multiplication.Support or Funding InformationFAPESP (Grant 2018/14840‐4)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Impact of hemoglobin A1c levels on asymptomatic bacteriuria in type 2 diabetes mellitus

Background: Diabetes mellitus (DM) refers to a group of common metabolic disorders that share the phenotype of hyperglycemia. Infections occur more commonly in patients with DM due to the hyperglycemic medium that increases the virulence of pathogen. Aims and Objectives: The present study was aimed to determine the impact of poor glycemic control as a risk factor for symptomatic urinary tract infection (UTI) in diabetic patients with asymptomatic bacteriuria (ASB) during follow-up. Materials and Methods: This was a cross-sectional observational study conducted at Vinayaka Missions Medical College on diabetic patients. Hemoglobin Alc (HbA1c) was quantified spectrophotometrically using HbA1c test kits. All participants were instructed strictly to collect midstream urine sample. Urine samples collected from participants were inoculated on blood agar and MacConkey agar and incubated for overnight at 370°C. If growth occurs, organism was identified by standard microbiological procedures. Symptomatic UTI surveillance was done by instructing participants to contact research personnel if any new symptom/symptoms occurred (follow-up period 3 months) that suggested UTI. Results: A total of 225 participants were included in the study and categorized as diabetic (n = 175) and non-diabetic as control group (n = 50). A total of 107 diabetic patients had

Fern-9(11)-ene-2α,3β-diol Action on Insulin Secretion under Hyperglycemic Conditions.

The objective of this study was to investigate the effect and the mechanism of action of fernenediol as an insulin secretagogue. Wistar rats were treated with 0.1, 1, and 10 mg/kg fernenediol before inducing hyperglycemia by oral glucose. The glycaemia, insulin, LDH, calcium, and hepatic glycogen were analyzed. Considering the intestine and pancreas as targets for the triterpene action, the duodenum was used to verify the influence of fernenediol on intestinal glycosidases. Additionally, pancreatic islets were used for studies of 14C-deoxyglucose uptake and the influx of 45Ca2+ in hyperglycemic media with/without fernenediol in the presence/absence of an inhibitor/activator of KATP channels, glibenclamide, diazoxide, nifedipine, calcium chelator (BAPTA-AM), and H-89 and ST, the inhibitors of the PKA and PKC enzymes. Fernenediol significantly reduced glycaemia, potentiated glucose-induced insulin secretion, and stimulated liver glycogen deposition in hyperglycemic rats after an in vivo treatment without changing intestinal disaccharidases activities and showing no influence on intestinal glucose absorption. Also, it stimulated the glucose uptake and calcium influx in pancreatic islets. The involvement of voltage-dependent L-type calcium channels and ATP-dependent potassium channels and the release of calcium from intracellular stores are mandatory for the stimulatory effect of fernenediol on calcium influx. Fernenediol did not change PKA and PKC activities or modify calcium levels. This triterpene is a potent antihyperglycemic agent with a strong insulin secretagogue effect on glycogen accumulation as well. As a whole, this compound presents significant perspectives as a future new drug for the treatment of insulin resistance and/or diabetes.