Cells In Diabetic Mice Research Articles

IL-10 Enhances the Inhibitory Effect of Adipose-Derived Stromal Cells on Insulin Resistance/Liver Gluconeogenesis by Treg Cell Induction.

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus.

LRG1 loss effectively restrains glomerular TGF-β signaling to attenuate diabetic kidney disease

Transforming growth factor (TGF)-β signaling is a well-established pathogenic mediator of diabetic kidney disease (DKD). However, owing to its pleiotropic actions, its systemic blockade is not therapeutically optimal. The expression of TGF-β signaling regulators can substantially influence TGF-β's effects in a cell- or context-specific manner. Among these, leucine-rich α2-glycoprotein 1 (LRG1) is significantly increased in glomerular endothelial cells (GECs) in DKD. As LRG1 is a secreted molecule that can exert autocrine and paracrine effects, we examined the effects of LRG1 loss in kidney cells in diabetic OVE26 mice by single-cell transcriptomic analysis. Gene expression analysis confirmed a predominant expression of Lrg1 in GECs, which further increased in diabetic kidneys. Loss of Lrg1 led to the reversal of angiogenic and TGF-β-induced gene expression in GECs, which were associated with DKD attenuation. Notably, Lrg1 loss also mitigated the increased TGF-β-mediated gene expression in both podocytes and mesangial cells in diabetic mice, indicating that GEC-derived LRG1 potentiates TGF-β signaling in glomerular cells in an autocrine and paracrine manner. Indeed, a significant reduction in phospho-Smad proteins was observed in the glomerular cells of OVE26 mice with LRG1 loss. These results indicate that specific antagonisms of LRG1 may be an effective approach to curb the hyperactive glomerular TGF-β signaling to attenuate DKD.

Juxtaglomerular (JG) cell-specific deletion of the NADPH oxidase accessory protein, NOXA1, decreases pro-renin and proteinuria in vivo in a mouse model of diabetes

A large fraction of patients with diabetes develops diabetic nephropathy. Plasma pro-renin levels are elevated in diabetic patients and thought to contribute to hypertension, renal damage and inflammation. Studying the mechanisms by which diabetes reprograms JG cells to increase pro-renin release could provide new targets for kidney disease. We previously found that JG cells from diabetic mice had higher reactive oxygen species production (superoxide and H2O2), pro-renin expression and pro-renin and renin release. We hypothesized that increased ROS production by specific NADPH oxidases (NOXs) in JG cells is responsible for higher pro-renin release and contributes to glomerular damage in diabetes. Western Blots showed that the NADPH oxidase isoform NOX1 and NOXA1 are upregulated in JG cells from diabetic mice (C57=100%; Akita=147±10%; n=4; p<0.05), whereas NOX2 or NOX4 were not different. Confocal microscopy showed that NOX1 and NOXA1 are present in JG cells and co-localize with renin-containing granules (n=3). Silencing NOX1 in primary cultures of JG cells from diabetic mice decreased renin release to C57 control levels; (n=6; p<0.01) whereas silencing NOX4 had no effect (n=6). To study the role of NOXA1 and NOX1 in JG cells in vivo, we used Cas9 mediated silencing in kidneys of control mice. Adenoviruses expressing Cas9 under control of the Ren1 promoter (AV-Ren1-Cas9) and AAV-gRNA-NOXA1 were loaded into 28-day minipumps and delivered into the renal subcapsular space in C57 mice. After 5 weeks, we obtained control (non-transduced) and Ren-Cas9-NOXA1-edited kidneys and isolated JG cells. NOXA1 expression was reduced by 60±22% in isolated JG cells and pro-renin expression was decreased by 46±19% compared to non-transduced kidneys (n=3). We then specifically deleted NOXA1 from JG cells in diabetic mice by subcapsular delivery of AV-Ren1-Cre in the kidney cortex of transgenic NOXA1 floxed diabetic Akita mice. JG cell-specific deletion of NOXA1 in diabetic mice decreased urinary ROS (8-Isoprostane pg/24hrs: Akita=4091±307; NOXA1 JG/KO=3086 ±137; n=4; p<0.05), and glomerular damage as indicated by decrease proteinuria (24h Ualbumin Akita= 98.6± 9.4; NOXA1 JG/KO= 57.8± 16.4 μg/24h; n=4; p<0.05). We conclude that enhanced NOX1/NOXA1 induced ROS in JG cells drive enhanced pro-renin release from JG cells which contribute to glomerular damage in diabetes. Thus, NOXA1/NOX1 in JG cells are a potential new target for diabetic nephropathy. DK105300-RO3 Henry Ford Hospital Institutional Funds. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hyperbaric oxygen therapy promotes the browning of white fat and contributes to the healing of diabetic wounds.

Non-healing wounds are one of the chronic complications of diabetes and have remained a worldwide challenge as one of the major health problems. Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic wound treatment, for which the molecular basis is not understood. Adipocytes regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes. Endothelial cell-derived extracellular vesicles could promote wound healing in diabetes. To study the mechanism by which HBO promotes wound healing in diabetes, we investigated the effect of HBO on fat cells in diabetic mice. A diabetic wound mouse model was established and treated with HBO. Haematoxylin and eosin (H&E) staining and immunofluorescence were used for the analysis of wound healing. To further explore the mechanism, we performed whole-genome sequencing on extracellular vesicles (EVs). Furthermore, we conducted in vitro experiments. Specifically, exosomes were collected from human umbilical vein endothelial cell (HUVEC) cells after HBO treatment, and then these exosomes were co-incubated with adipose tissue. The wound healing rate in diabetic mice treated with HBO was significantly higher. HBO therapy promotes the proliferation of adipose precursor cells. HUVEC-derived exosomes treated with HBO significantly promoted fat cell browning. These data clarify that HBO therapy may promote vascular endothelial cell proliferation and migration, and promote browning of fat cells through vascular endothelial cells derived exosomes, thereby promoting diabetic wound healing. This provides new ideas for the application of HBO therapy in the treatment of diabetic trauma.

Inhibition of Ferroptosis Ameliorates Photoreceptor Degeneration in Experimental Diabetic Mice

Diabetic retinopathy (DR) is a leading cause of vision impairment in the working-age population worldwide. Various modes of photoreceptor cell death contribute to the development of DR, including apoptosis and autophagy. However, whether ferroptosis is involved in the pathogenesis of photoreceptor degeneration in DR is still unclear. High-glucose (HG)-stimulated 661W cells and diabetic mice models were used for in vitro and in vivo experiments, respectively. The levels of intracellular iron, glutathione (GSH), reactive oxygen species (ROS), lipid peroxidation (MDA), and ferroptosis-related proteins (GPX4, SLC7A11, ACSL4, FTH1, and NCOA4) were quantified to indicate ferroptosis. The effect of ferroptosis inhibition was also assessed. Our data showed the levels of iron, ROS, and MDA were enhanced and GSH concentration was reduced in HG-induced 661W cells and diabetic retinas. The expression of GPX4 and SLC7A11 was downregulated, while the expression of ACSL4, FTH1, and NCOA4 was upregulated in the 661W cells cultured under HG conditions and in the photoreceptor cells in diabetic mice. Furthermore, the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1) obviously alleviated ferroptosis-related changes in HG-cultured 661W cells and in retinal photoreceptor cells in diabetic mice. Taken together, our findings suggest that ferroptosis is involved in photoreceptor degeneration in the development of the early stages of DR.

Sodium tanshinone IIA sulfonate ameliorates neointima by protecting endothelial progenitor cells in diabetic mice

BackgroundEndothelial progenitor cells (EPCs) transplantation is one of the effective therapies for neointima associated with endothelial injury. Diabetes impairs the function of EPCs and cumbers neointima prevention of EPC transplantation with an ambiguous mechanism. Sodium Tanshinone IIA Sulfonate (STS) is an endothelium-protective drug but whether STS protects EPCs in diabetes is still unknown.MethodsEPCs were treated with High Glucose (HG), STS, and Nucleotide-binding Domain-(NOD) like Receptor 3 (NLRP3), caspase-1, the Receptor of Advanced Glycation End products (AGEs) (RAGE) inhibitors, Thioredoxin-Interacting Protein (TXNIP) siRNA, and EPC proliferation, differentiation functions, and senescence were detected. The treated EPCs were transplanted into db/db mice with the wire-injured Common Carotid Artery (CCA), and the CD31 expression and neointima were detected in the CCA inner wall.ResultsWe found that STS inhibited HG-induced expression of NLRP3, the production of active caspase-1 (p20) and mature IL-1β, the expression of catalase (CAT) cleavage, γ-H2AX, and p21 in EPCs. STS restored the expression of Ki67, CD31 and von Willebrand Factor (vWF) in EPCs; AGEs were found in the HG-treated EPCs supernatant, and RAGE blocking inhibited the expression of TXNIP and the production of p20, which was mimicked by STS. STS recovered the expression of CD31 in the wire-injured CCA inner wall and the prevention of neointima in diabetic mice with EPCs transplantation.ConclusionSTS inhibits the aggravated neointima hyperplasia by protecting the proliferation and differentiation functions of EPC and inhibiting EPC senescence in diabetic mice. The mechanism is related to the preservation of CAT activity by inhibiting the RAGE-TXNIP-NLRP3 inflammasome pathway.

Greb1 Transiently Accelerates Pancreatic β-Cell Proliferation in Diabetic Mice Exposed to Estradiol

Decrease of pancreatic β cells leads to diabetes. In an inducible cAMP early suppressor (ICER-Iγ) transgenic mouse model of severe type 2 diabetes with reduced insulin production and depleted β cells, supplementation with high concentrations of 17β-estradiol (E2) markedly enhances β-cell proliferation and normalizes glucose levels. The current study explored the underlying mechanisms leading to a dynamic increase of β cells and pathologic changes in diabetic mice exposed to E2. Gene expression profiling of pancreatic islets of 6-month-old ICER-transgenic mice recovering from diabetes due to elevated E2 levels identified growth regulation by estrogen in breast cancer 1 (Greb1) as a gene significantly up-regulated during the recovery phase. To substantiate this, β-cell-specific Greb1-deficient mice were generated, and Greb1 was shown to be essential for recovery of depleted β cells in diabetic mice. Graft growth and glucose lowering were observed in 50 islets with increased Greb1 expression transplanted adjacent to E2 pellets beneath the kidney capsule of streptozotocin-induced diabetic mice. Greb1 expression due to a drastic increase in exogenous or endogenous E2 was transient and closely correlated with changes in E2-related and some cell cycle-related genes. These findings provide new insights into invivo proliferation of deficient β cells and suggest the possibility of new therapeutic approaches targeting pancreatic β cells that could revolutionize the concept of diabetes treatment, which has been considered difficult to cure completely.

Salidroside Inhibits Ganglion Cell Apoptosis by Suppressing the Müller Cell Inflammatory Response in Diabetic Retinopathy

Purpose This study aimed to investigate the role of salidroside (SAL) in the cellular communication between Müller cells and retinal ganglion cells in diabetic mice. Methods The diabetes mellitus (DM) animal models were established by the intraperitoneal injection of streptozotocin and treatment with SAL via gavage or by the injection of IL-22BP into the vitreous cavity. Immunohistochemistry was used to measure the expression of the glial fibrillary acidic protein in Müller cells. The expression of IL-22 and IL-22Rα1 in retinal tissues was assessed by immunofluorescence. Western blotting was used to measure the expression of inflammatory and apoptosis-related proteins. Hematoxylin–eosin staining, TUNEL staining, and flow cytometry were used to analyze the apoptosis of retinal ganglion cells. The effect of cellular interactions was explored by Transwell assays. Results Western blotting showed that glial fibrillary acidic protein, IL-22 protein expression was significantly upregulated in the DM animal models compared with the control mice. Immunofluorescence showed that IL-22 was highly expressed in Müller cells and IL-22Rα1 was expressed in ganglion cells in the retina of DM mice. Hematoxylin–eosin and TUNEL staining results showed an increase in the number of ganglion cells apoptotic in DM. However, SAL reversed these phenomena. Meanwhile, after coculture with Müller cells, Western blotting suggested that ganglion cells secreted p-STAT3, and c-caspase3 protein expression was increased. More interestingly, the treatment of IL-22BP and SAL inhibited the expression of the p-STAT3 and c-caspase3 proteins. Flow cytometry indicates that compared with the control group, the apoptosis rate of ganglion cells was increased in the high glucose group, while the apoptosis rate of cells in the recombinant IL-22 protein group was significantly increased, while the SAL inhibited ganglion cells apoptosis. Conclusion SAL inhibits the apoptosis of retinal ganglion cells via the IL-22/STAT3 pathway in Müller cells.

Hyperglycemia Induces Tear Reduction and Dry Eye in Diabetic Mice through the Norepinephrine–α1 Adrenergic Receptor–Mitochondrial Impairment Axis of Lacrimal Gland

Dry eye syndrome is a common complication in diabetic patients with a prevalence of up to 54.3%. However, the pathogenic mechanisms underlying hyperglycemia-induced tear reduction and dry eye remain less understood. In the present study, we found that both norepinephrine (NE) and tyrosine hydroxylase levels were elevated in the lacrimal gland of diabetic mice, accompanied by increased c-FOS+ cells in the superior cervical ganglion. However, the elimination of NE accumulation by surgical and chemical sympathectomy significantly ameliorated the reduction in tear production, suppressed abnormal inflammation of the lacrimal gland, and improved the severity of dry eye symptoms in diabetic mice. Among various adrenoceptors (ARs), the α1 subtype played the predominant role in the regulation of tear production, as treatments of α1AR antagonists improved tear secretion in diabetic mice compared with βAR antagonist propranolol. Moreover, the α1AR antagonist alfuzosin treatment also alleviated functional impairments of the meibomian gland and goblet cells in diabetic mice. Mechanically, the α1AR antagonist rescued the mitochondrial bioenergetic deficit, increased the mitochondrial DNA copy numbers, and elevated the glutathione levels of the diabetic lacrimal gland. Overall, these results deciphered a previously unrecognized involvement of the NE-α1AR-mitochondrial bioenergetics axis in the regulation of tear production in the lacrimal gland, which may provide a potential strategy to counteract diabetic dry eye by interfering with the α1AR activity.

In vivo drug discovery for increasing incretin-expressing cells identifies DYRK inhibitors that reinforce the enteroendocrine system

Analogs of the incretin hormones Gip and Glp-1 are used to treat type 2 diabetes and obesity. Findings in experimental models suggest that manipulating several hormones simultaneously may be more effective. To identify small molecules that increase the number of incretin-expressing cells, we established a high-throughput invivo chemical screen by using the gip promoter to drive the expression of luciferase in zebrafish. All hits increased the numbers of neurogenin 3-expressing enteroendocrine progenitors, Gip-expressing K-cells, and Glp-1-expressing L-cells. One of the hits, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor, additionally decreased glucose levels in both larval and juvenile fish. Knock-down experiments indicated that nfatc4, a downstream mediator of DYRKs, regulates incretin+ cell number in zebrafish, and that Dyrk1b regulates Glp-1 expression in an enteroendocrine cell line. DYRK inhibition also increased the number of incretin-expressing cells in diabetic mice, suggesting a conserved reinforcement of the enteroendocrine system, with possible implications for diabetes.

Nicotinamide mononucleotide improves spermatogenic function in streptozotocin-induced diabetic mice via modulating the glycolysis pathway.

Spermatogenic dysfunction is one of the major secondary complications of diabetes; however, the underlying mechanisms remain ill-defined, and there is no available drug or strategy for the radical treatment of diabetic spermatogenic dysfunction. Therefore, the objective of this study is to investigate the protective effects of nicotinamide mononucleotide (NMN) on testicular spermatogenic function in streptozotocin (STZ)-induced diabetic mice. The results show that oral administration of NMN significantly increases the body and testis weight and the number of sperms. Moreover, the abnormal sperm count and the rate of sperm malformation are significantly decreased compared with the saline-treated diabetic mice. Histological analysis reveals that NMN treatment significantly increases the area and diameter of seminiferous tubules, accompanied by an increased number of spermatogenic cells and sperms. Immunohistochemistry and qRT-PCR results show that NMN increases Bcl-2 expression and decreases Bax expression in the testis. NMN also increases the protein expression of Vimentin and the mRNA expressions of WT1 and GATA4. In addition, qRT-PCR, western blot analysis and immunohistochemistry results also show that NMN increases the expressions of glycolysis-related rate-limiting enzymes including HK2, PKM2, and LDHA. In summary, this study demonstrates the protective effects of NMN on the testis in an STZ-induced diabetic mice model. NMN exerts its protective effects via reducing spermatogenic cell apoptosis by regulating glycolysis of Sertoli cells in diabetic mice. This study provides an experimental basis for the future clinical application of NMN in diabetes-induced spermatogenic dysfunction.

Interleukin-35 Prevents the Elevation of the M1/M2 Ratio of Macrophages in Experimental Type 1 Diabetes.

Macrophages play an important role in the early development of type 1 diabetes (T1D). Based on the phenotype, macrophages can be classified into pro-inflammatory (M1) and anti-inflammatory (M2) macrophages. Despite intensive research in the field of macrophages and T1D, the kinetic response of M1/M2 ratio has not been studied in T1D. Thus, herein, we studied the M1 and M2 macrophages in the early development of T1D using the multiple low dose streptozotocin (MLDSTZ) mouse model. We determined the proportions of M1 and M2 macrophages in thymic glands, pancreatic lymph nodes and spleens on days 3, 7 and 10 after the first injection of STZ. In addition, we investigated the effect of IL-35 in vivo on the M1/M2 ratio and IL-35+ plasmacytoid dendritic cells in diabetic mice and in vitro on the sorted macrophages. Our results revealed that the M1/M2 ratio is higher in STZ-treated mice but this was lowered upon the treatment with IL-35. Furthermore, IL-35 treated mice had lower blood glucose levels and a higher proportion of IL-35+ cells among pDCs. Macrophages treated with IL-35 in vitro also had a higher proportion of M2 macrophages. Together, our data indicate that, under diabetic conditions, pro-inflammatory macrophages increased, but IL-35 treatment decreased the pro-inflammatory macrophages and increased anti-inflammatory macrophages, further suggesting that IL-35 prevents hyperglycemia by maintaining the anti-inflammatory phenotype of macrophages and other immune cells. Thus, IL-35 should be further investigated for the treatment of T1D and other autoimmune disorders.

Celastrol inhibits TXNIP expression to protect pancreatic β cells in diabetic mice

BackgroundCelastrol (CEL) has a great potential in the treatment of a wide variety of metabolic diseases. However, whether CEL protects pancreatic β cells and its underlying mechanism are not yet clear. PurposeThis study investigates to determine the effects of CEL on the pathogenesis of pancreatic β cells damage. MethodsC57BLKS/Leprdb (db/db) mice and rat insulinoma INS-1 cell line or mouse J774A.1 cell line were used as in vivo and in vitro models for investigating the protective effect of CEL on pancreatic β cells under high glucose environment and the related mechanism. The phenotypic changes were evaluated by immunofluorescence, immunohistochemical staining, flow cytometry and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-qPCR, co-immunoprecipitation and lentivirus infection. ResultsOur results showed that CEL at the high dose (CEL-H, 0.2 mg/kg) protects db/db mice against increased body weight and blood glucose. CEL-H inhibits pancreatic β cell apoptosis in db/db mice and high glucose-induced INS-1 cells. CEL-H also reduced IL-1β production in islet macrophages. The further study found that CEL suppressed TXNIP expression and NLRP3 inflammasome activation in pancreatic β cells and islet macrophages. Importantly, the inhibitory effect of CEL on pancreatic β cell apoptosis and IL-1β production was also dependent on TXNIP. Mechanically, CEL inhibits Txnip transcription by promoting the degradation of ChREBP. ConclusionCelastrol inhibits TXNIP expression to protect pancreatic β cells in vivo and in vitro. Our research pointed out another mechanism by which celastrol functions under the condition leptin signaling is ineffective.

Role of diabetes in lung injury from acute exposure to electronic cigarette, heated tobacco product, and combustible cigarette aerosols in an animal model.

BackgroundPatients with diabetes are more vulnerable to the detrimental respiratory effects of combustible cigarette smoke (CS) when compared to the general population. Electronic cigarettes (ECIG) and heated tobacco products (HTP) are marketed as less harmful alternatives to CS. In this study, we compared the effects of acute ECIG, HTP and CS exposure on the lungs of type II diabetes versus non-diabetic mice in an animal model.MethodsType II Diabetic (Diab) and Non-Diabetic (Non-Diab) mice were divided into Control, ECIG, HTP and CS groups. Animals were exposed for 6 hrs./day to either air, ECIG, HTP or CS for seven days. Lung injury was determined by a) histopathology, b) wet to dry ratio, c) albumin concentration in bronchoalveolar lavage fluid, d) expression of TNF-α, IL-6, and IL-1 β, e) reactive oxygen species production (ROS), and f) assessment of cellular apoptosis.ResultsLung histology revealed increased edema and inflammatory cells in diabetic mice exposed to ECIG, HTP and CS. The expression of Inflammatory mediators was, in general, more significant in the Diabetic groups as well. TNF-α expression, for example, was upregulated in Diab + ECIG but not in Non-Diab + ECIG. ROS was significantly increased in Diab + CS, less in Non-Diab + CS and weakly noted in ECIG + Diab. Significant albumin leak was observed in Diab and Non-Diab HTP-exposed animals. CS exposure worsened lung injury in Diab when compared to Non-Diab mice.ConclusionComorbid medical conditions like diabetes may amplify ill effects of CS, ECIG or HTP exposure.

Development of Advanced Cell-Based Therapy by Regulating Cell-Cell Interactions.

Cell-based therapy for disease treatment involves the transplantation of cells obtained either from self or others into relevant patients. While cells constituting the body tissues maintain homeostasis by performing remarkable functions through complicated cell-cell interactions, transplanted cells, which are generally cultured as a monolayer, are unable to recapitulate similar interactions in vivo. The regulation of cell-cell interactions can immensely increase the function and therapeutic effect of transplanted cells. This review aims to summarize the methods of regulating cell-cell interactions that could significantly increase the therapeutic effects of transplanted cells. The first method involves the generation of multicellular spheroids by three-dimensional cell culture. Spheroid formation greatly improved the survival and therapeutic effects of insulin-secreting cells in diabetic mice after transplantation. Moreover, mixed multicellular spheroids, composed of insulin-secreting cells and aorta endothelial cells or fibroblasts, were found to significantly improve insulin secretion. Secondly, adhesamine derivatives, which are low-molecular-weight compounds that accelerate cell adhesion and avoid anoikis and anchorage-dependent apoptosis, have been used to improve the survival of bone marrow-derived cells and significantly enhanced the therapeutic effects in a diabetic mouse model of delayed wound healing. Finally, the avidin-biotin complex method, a cell surface modification method, has been applied to endow tumor-homing mesenchymal stem cells with anti-tumor ability by modifying them with doxorubicin-encapsulated liposomes. The modified cells showed excellent effectiveness in cell-based cancer-targeting therapy. The discussed methods can be useful tools for advanced cell-based therapy, promising future clinical applications.

Selection for CD26- and CD49A+ Cells From Pluripotent Stem Cells-Derived Islet-Like Clusters Improves Therapeutic Activity in Diabetic Mice.

BackgroundCell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the β-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells.MethodsFunctional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice.ResultsCD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A+ cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26+ cells followed by enrichment for CD49A+ cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26-/CD49A+ enriched ILC exhibited improved function over non-sorted ILC or CD49A+ cells in diabetic mice and maintain prolonged blood C-peptide levels.ConclusionsRefining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

Secretome effect of adipose tissue-derived stem cells cultured two-dimensionally and three-dimensionally in mice with streptozocin induced type 1 diabetes

AimsTo analyze therapeutic potential of the conditioned medium from adipose tissue-derived stem cells (ASC) cultivated in 2D (CM-2D) and 3D (CM-3D) models, in mice with Type 1 diabetes (T1D) induced by streptozotocin. Main methodsViability andCD105 expression of 2D and 3D ASC were analyzed by flow cytometry. T1D was induced in mice by multiple injections of streptozocin. On the 28th and 29th days after the first injection of streptozocin, diabetic animals received CM-2D or CM-3D. Pancreatic, CM-2D, and CM-3D cytokines were analyzed by cytometric bead array (CBA) and insulin and PDX-1 were observed and quantified by immunohistochemistry. Apoptosis-related proteins were quantified by Western Blotting. Key findingsASC in three-dimensional culture released increased levels of IL-6 and IL-2, while IL-4 was decreased. CM-2D induced pancreatic PDX-1 expression and was able to reduce glycemia in diabetic mice one week after injections but not CM-3D. On the other hand, CM-2D and CM-3D were not able to reverse apoptosis of pancreatic cells in diabetic mice nor to increase insulin expression. SignificanceTogether, these results demonstrate that the 3D cell culture secretome was not able to improve diabetes type 1 symptoms at the times observed, while 2D cell secretome improved glycemic levels in T1D mice.

Epoxyeicosatrienoic acids improve glucose homeostasis by preventing NF-κB-mediated transcription of SGLT2 in renal tubular epithelial cells

Studies have shown that epoxyeicosatrienoic acids (EETs) can regulate glucose homeostasis, but the specific mechanisms need further exploration. The sodium-glucose co-transporter 2 (SGLT2) is highly expressed in diabetic kidneys, which further promotes renal reabsorption of glucose to respond to the hyperglycemic state of diabetes. Herein, whether EETs can be a latent inhibitor of SGLT2 to regulate glucose homeostasis in diabetic state needs to be elucidated. Our study demonstrated that EETs attenuated the glucose reabsorption via renal tubular epithelial cells in diabetic mice, which partly accounted for the beneficial effects of EETs on glucose homeostasis. Moreover, 14,15-EET suppressed SGLT2 expression in both diabetic kidney and renal tubular epithelial cells. Further, inhibition of NF-κB with BAY 11-7082 decreased insulin-induced SGLT2 expression while NF-κB overexpression reversed the above effects. In addition, 14,15-EET attenuated SGLT2 expression via inactivating NF-κB. Mechanistically, 14,15-EET attenuated NF-κB mediated SGLT2 transcription at the −1821/-1812 P65-binding site. These results showed that EETs ameliorated glucose homeostasis via preventing NF-κB-mediated transcription of SGLT2 in renal tubular epithelial cells, providing a unique therapeutic strategy for insulin resistance and diabetes.

Inhibition of histone acetyltransferase by naringenin and hesperetin suppresses Txnip expression and protects pancreatic β cells in diabetic mice

BackgroundThe damage of pancreatic β cells is a major pathogenesis of the development and progression of type 2 diabetes and there is still no effective therapy to protect pancreatic β cells clinically. In our previous study, we found that Quzhou Fructus Aurantii (QFA), which is rich in flavanones, had the protective effect of pancreatic β cells in diabetic mice. However, the underlying mechanism is still unclear. PurposeIn the current study, we administered naringenin and hesperetin, two major active components of QFA, to protect pancreatic β cells and to investigate the underlying molecular mechanism focusing on the epigenetic modifications. MethodsWe used diabetic db/db mouse and INS-1 pancreatic β cell line as in vivo and in vitro models to investigate the protective effect of naringenin and hesperetin on pancreatic β cells under high glucose environment and the related mechanism. The phenotypic changes were evaluatedby immunostaining and the measurement of biochemical indexes. The molecular mechanism was explored by biological techniques such as western blotting, qPCR, ChIP-seq and ChIP-qPCR, flow cytometry and lentivirus infection. ResultsWe found that naringenin and hesperetin had an inhibitory effect on histone acetylation. We showed that naringenin and hesperetin protected pancreatic β cells in vivo and in vitro, and this effect was independent of their direct antioxidant capacity. The further study found that the inhibition of thioredoxin-interacting protein (Txnip) expression regulated by histone acetylation was critical for the protective role of naringenin and hesperetin. Mechanistically, the histone acetylation inhibition by naringenin and hesperetin was achieved through regulating AMPK-mediated p300 inactivation. ConclusionThese findings highlight flavanones and the phytomedicine rich in flavanones as important dietary supplements in protecting pancreatic β cells in advanced diabetes. In addition, targeting histone acetylation by phytomedicine is a potential strategy to delay the development and progression of diabetes.

Assessment of Anti-Diabetic Activity of Vinca Rosea Extract on Induced Diabetic Mice

Background and Objective: Vinca rosea is a medicinal plant that has been investigated and applied as analternative treatment for disorders. This study aimed to investigate the anti-diabetic activity of ethanolicextract of Vinca rosea on induced diabetes in mice.Methods: Forty male albino mice were randomly divided into four groups, including control that wasadministered a standard physiologic saline and served as control, diabetic group, Streptozotocin & Vincarosea, and only Vinca rosea; each group contained ten mice.Results: No significant lesions were observed in pathological sections of the control; however, the lesions indiabetic group showed massive lesions in the islets of Langerhans and reduced dimensions of islets due to thedamage of beta-cells. Furthermore, the Pancreas in Streptozotocin & Vinca rosea demonstrated a moderatedvacuolation and Langerhans of islet cells. In contrast, the histopathological sections of the pancreas, whichtreated by ethanolic extracts of Vinca rosea, illustrated increasing number of beta-cells hypercellularity. Thevalues of blood glucose were increased in the diabetic group; however, the significant decreases (P<0.05)in blood glucose detected in treated by ethanolic extract as represented by increased Beta-cells. While, theblood glucose levels were tolerated in treated Streptozotocin & Vinca rosea; thus, these values were closelyfor the control results.Conclusion: The Vinca rosea extracts induced a significant impact on diabetic mice, and they can improveand protect pancreatic cells in diabetic mice. Therefore, ethanolic extracts of Vinca rosea could be beneficialfor the diabetes.