Glucose Conditions Research Articles

Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. 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.

Histone Deacetylase Inhibition Increases Glycerol Uptake in Normal and Hyperglycemic Conditions in Human Corneal Epithelial Cells

Delayed corneal wound healing, such as is seen in diabetes, can lead to visual impairment. Diabetes afflicts over 30 million Americans and leads to multiple comorbidities. Histone deacetylase (HDAC) expression and activity are increased in diabetes in correlation with an upregulation of inflammatory molecules and reactive oxygen species (ROS). HDACs have been previously found to suppress the expression of the glycerol channel aquaporin-3 (AQP3) in epithelial cells of the skin, the epidermal keratinocytes. Global deletion of AQP3 in mice has been found to delay corneal wound healing. We hypothesized that hyperglycemic conditions would decrease AQP3 levels and function while increasing the expression of HDAC(s), ROS scavenger proteins, and inflammatory mediators in human corneal epithelial cells (HCECs). HCECs were incubated for 48-hours in high glucose (25.5 mM) or normal glucose (~5.0 mM osmotically matched by mannitol) conditions. Under hyperglycemic conditions, the mRNA expression of interleukin-1α and -1β was significantly elevated, providing evidence for a pro-inflammatory effect of acute hyperglycemia in HCECs. Under these conditions, there was a significant increase in the mRNA expression of the ROS scavenger genes, superoxide dismutase-1 and -2 and peroxiredoxin-3 and -6, suggesting a compensatory response to an increase in ROS generation. Under acute hyperglycemic conditions, HDAC3 and HDAC8 mRNA expression also tended to increase while HDAC1 and HDAC2 mRNA levels remained unchanged. Although an upregulation of HDAC expression would be predicted to decrease AQP3 expression, AQP3 mRNA and protein expression levels were also unchanged. In addition, acute hyperglycemia (48 hours) did not alter glycerol transport into the HCECs, as measured by their uptake of radiolabeled [14C]-glycerol. However, HDAC inhibition using the broad-spectrum inhibitor SAHA for 24 hours increased [14C]-glycerol uptake under both normoglycemic and hyperglycemic conditions, suggesting that HDAC activity plays a role in AQP3 function. Overall, acute hyperglycemic conditions were pro-inflammatory and led to increased expression of ROS-scavenging genes; hyperglycemia also tended to upregulate HDAC expression, although without affecting AQP3 expression. On the other hand, HDAC inhibition increased glycerol uptake, an indicator of AQP3 function, suggesting an ability of HDAC activity to regulate AQP3 glycerol transport activity. National Institutes of Health/National Eye Institute award #R01EY030576 (to MW and WBB) VA Merit #BX005055 (to WBB). 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.

Abstract 3020: The Effects Of High Glucose On The Release Of Exosomes From Adipocytes

Type 2 Diabetes Mellitus is primarily characterized by hyperglycemia accompanied by adipose tissue dysfunction. More importantly, diabetes is a risk factor for stroke and other cerebrovascular diseases, which might be related to poorly explored adipose tissue dysfunction. Even though adipose tissue is comprised of various cells, it is found that predominantly adipocyte dysfunction contributes to diabetes and related complications. Exosomes are extracellular vesicles released from almost all cells with varying functions in different conditions. Here, we aim to determine the function of adipocyte-derived exosomes (Adp-EXs) in high glucose (HG) conditions. We hypothesized that Adp-EXs from the HG group would induce cytotoxicity and oxidative stress in primary human brain microvascular endothelial cells (HBMECs). First, the level of Adp-EXs was measured after HG treatment. Secondly, their effects were studied on HBMECs after co-incubation. Adipocytes were cultured in normal glucose (NG) or HG (25mM) media to isolate Adp-EXs by serial ultracentrifugation method. The size and concentration of Adp-EXs were determined via the Nanoparticle Tracking Analysis system. Adp-EXs at three concentrations (1x10 8 , 5x10 8 and 1x10 9 particles/mL) were co-incubated with HBMECs for 24 to 48 hours to evaluate optimal cellular communication. PKH26-labelling (2uL/mL) of Adp-EX was performed to track its incorporation into HBMECs. Dihydroethidium staining and flow cytometry quantified reactive oxygen species production and cytotoxicity was determined by lactate dehydrogenase assay. For all experiments, n=5-10 and mean SEM=8.600±6.194. Results showed: 1) HG increased Adp-EX levels without size differences between NG and HG groups. 2) Adp-EXs integrated with HBMECs in a time and dose-dependent manner, more prominently in the HG group. 3) Adp-EXs from the HG group increased Dihydroethidium-positive HBMECs. 4) Adp-EXs from the HG group increased fluorescence for lactate dehydrogenase. In conclusion, the data suggest that HG could promote Adp-EX release, and trigger oxidative stress and cytotoxicity in HBMECs.

Protective Effect of Curcumin on the Tight Junction Integrity and Cellular Senescence in Human Retinal Pigment Epithelium of Early Diabetic Retinopathy.

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

Abstract 2058: Endothelial Cell Regeneration By Secretome Obtained From P53 Silenced Bone Marrow Derived MSCs (BMSCs).

Introduction: Diabetes is associated with impaired endothelial function. Studies have demonstrated that endothelial progenitor cells (EPCs) and mature endothelial cells (EC) are susceptible to apoptosis in hyperglycemic environment. Hyperglycemia promotes p53 gene over-expression and p53 gene silencing prior to hyperglycemia exposure prevents cellular senescence and helps vascular regeneration. Previously we have shown that p53 silenced human CD34+ve progenitor cell therapy and even associated cell derived secretome, helped tissue regeneration. Our goal in this project was to test whether conditioned media (CM) obtained from p53 silenced human stem cells (other than CD34+ve cells) can also improve endothelial regeneration exposed to hyperglycemia. We compared CM obtained from adipose and bone marrow derived mesenchymal stem cells on endothelial regeneration Methods: Ad-human-P53 (TP53)-shRNA was used to silence P53 and Ad-scrambled-null-shRNA was used as control in bone marrow derived mesenchymal stromal cells. To collect conditioned media BM-MSCs were cultured in exosome free FBS media for 5 days, following transduction with Ad-p53sh or Ad-null, as the case may be. After transduction, cells were grown in media containing 2% exosome free FBS for only 48h and the collected supernatant was concentrated 10-fold to obtain the CM. Endothelial regeneration was tested using endothelial wound healing assay kit ( from Cell Biolabs # CBA-120-T). We compared CM obtained from null and p53sh in adipose and bone marrow derived mesenchymal stem cells on human endothelial cells in normal and high glucose conditions Results: Proliferation rate of endothelial cells was enhanced when we used CM from bone marrow derived cells as compared to CM from adipose derived mesenchymal stem cells, by nearly two fold. We are currently analyzing the secretome by mass spectroscopy. Conclusion: P53 silencing appears to improve endothelial regeneration on using CM obtained from bone marrow derived stem cells. This maybe secondary to improved mitochondrial function post p53 gene silencing. The CM thus obtained are in sufficient quantities and may have therapeutic role in treating diabetes vascular complications such as diabetic wound healing.

Berberine alleviates diabetic retinopathy by regulating the Th17/Treg ratio

BackgroundDiabetic retinopathy (DR) stands as a prominent complication of diabetes. Berberine (BBR) has reported to be effective to ameliorate the retinal damage of DR. Studying the potential immunological mechanisms of BBR on the streptozotocin (STZ) induced DR mouse model will explain the therapeutic mechanisms of BBR and provide theoretical basis for the clinical application of this drug. MethodsC57BL/6 J mice were induced into a diabetic state using a 50 mg/(kg·d) dose of STZ over a 5-day period. Subsequently, they were subjected to a high-fat diet (HFD) for one month. Following a 5-week treatment with 100 mg/(kg·d) BBR, the concentrations of inflammatory factors in the mice's peripheral blood were determined using an enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin staining was employed to scrutinize pathological changes in the mice's retinas, while flow cytometry assessed the proportions of T-lymphocyte subsets and the activation status of dendritic cells (DCs) in the spleen and lymph nodes. CD4+T cells and DC2.4 cell lines were utilized to investigate the direct and indirect effects of BBR on T cells under high glucose conditions in vitro. ResultsFollowing 5 weeks of BBR treatment in the streptozotocin (STZ) mouse model of DR, we observed alleviation of retinal lesions and a down-regulation in the secretion of inflammatory cytokines, namely TNF-α, IL-1β, and IL-6, in the serum of these mice. And in the spleen and lymph nodes of these mice, BBR inhibited the proportion of Th17 cells and promoted the proportion of Treg cells, thereby down-regulating the Th17/Treg ratio. Additionally, in vitro experiments, BBR directly inhibited the expression of the transcription factor RORγt and promoted the expression of the transcription factor Foxp3 in T cells, resulting in a down-regulation of the Th17/Treg ratio. Furthermore, BBR indirectly modulated the Th17/Treg ratio by suppressing the secretion of TNF-α, IL-1β, and IL-6 by DCs and enhancing the secretion of indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta (TGF-β) by DCs. This dual action inhibited Th17 cell differentiation while promoting Treg cells. ConclusionOur findings indicate that BBR regulate T cell subpopulation differentiation, reducing the Th17/Treg ratio by directly or indirectly pathway. This represents a potential therapeutic avenue of BBR for improving diabetic retinopathy.

Resveratrol protects against diabetic retinal ganglion cell damage by activating the Nrf2 signaling pathway

ObjectiveOxidative stress-induced retinal neurodegenerative changes are among the pathological alterations observed in diabetic retinopathy. Resveratrol (RSV), a polyphenolic compound with diverse pharmacological effects, has shown preventive qualities in several neurodegenerative illnesses, including anti-inflammatory, anti-aging, and antioxidant benefits. However, its therapeutic efficacy in diabetic retinal neurodegeneration has not yet been thoroughly elucidated. Our study aimed to explore the protective mechanisms and therapeutic benefits of RSV on diabetic retinal neurodegeneration alterations. Materials and methodsUsing streptozotocin, we created a diabetic mouse model and conducted visual electrophysiological examinations on mice from the normal group, diabetic group, and diabetic group treated with RSV. Retinas were harvested for histological staining. Additionally, primary retinal ganglion cells cultured in high glucose conditions were used to assess malondialdehyde (MDA) levels and superoxide dismutase (SOD) levels upon siRNA-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) interference. Protein levels of Nrf-2, heme oxygenase-1 (HO-1), and transcriptional levels of them were also measured. ResultsWe demonstrated that RSV significantly improved the retinal morphology and function in the diabetic retinopathy model mice. The treated mice exhibited notable improvements in visual electrophysiology, with a significant reduction in retinal ganglion cell apoptosis. Following RSV treatment, the high glucose-cultured ganglion cells demonstrated a considerable rise in SOD levels and a substantial drop in MOD. Moreover, the protein expression of solute carrier family 7 member 11 (SLC7A11) and Nrf2 significantly increased. RT-PCR and Western blot results indicated a significant attenuation of RSV's therapeutic effects upon Nrf2 inhibition. ConclusionOur findings suggest that RSV may reduce oxidative stress levels in the retina and inhibit retinal ganglion cell apoptosis via reducing the Nrf2/HO-1 pathway, which lessens the harm that excessive glucose causes to the retina.

Abstract 2005: Interrogating The Cross-talk: Endothelial-Macrophage Interactions In Diabetic Vascular Dysfunction

Cardiovascular disease (CVD) occurs 15 years earlier in diabetics compared to those without, but the underlying mechanisms driving diabetes-accelerated CVDs (DACVDs) pathogenesis remains incompletely understood. Endothelial cells (ECs) and macrophages (MΦ) are key players in vascular wall and their crosstalk is crucial in DACVD. In diabetes, ECs activation enables monocytes recruitment, which transmigrate across the intima and differentiate into macrophages (MΦ). Beyond this established diapedesis model, EC-MΦ interplay is highly intricate and heterogenous. Here, we leveraged human mesenteric arteries of varied diabetic state and used single cell (sc)- and spatial transcriptome mapping to interrogate the functional crosstalk between MΦ subtypes and ECs in diabetes. We identified diabetes-induced EC-MΦ interactions and explored their consequences in vitro and in vivo, in peripheral artery disease (PAD), a prominent DACVD. Integrative scRNA-seq and spatial transcriptome (Visium) profiling identified a remarkable increase of MΦ subtype expressing triggering receptor expressed on myeloid cells (TREM2) with concomitant increased EC-derived TREM2 ligands in human diabetic vs non-diabetic vessels. Such induction of TREM2 MΦ was consistently observed in arteries from type I and II diabetes mouse models. Compared to the condition medium from MΦ cultured under normal glucose, that from MΦ cultured under high glucose conditions reduced EC migration and increased inflammatory markers, which were reversed by the medium from MΦ with TREM2-knockdown. We further evaluated the functional importance of EC-TREM2 interaction in vivo in a mouse hindlimb ischemia (HLI) model. In diabetic mice under HLI, a TREM2 neutralizing antibody promoted tissue recovery and perfusion. Finally, we showed that TREM2 was increased, especially in proximity to ECs, in ischemic sites, as compared to non-ischemic regions in limb muscles from human PAD patients.Taken together, our study presents a diabetic artery atlas and identifies EC-TREM2 interaction as a crucial event in DACVDs, such as PAD. Our findings provide novel insights into the EC-MΦ interplay in diabetes and vascular inflammation and how these cellular interactions may be targeted to ameliorate DACVD.

Upregulation of the Urotensin II System in Diabetic Kidney Disease Includes Urotensin II-Related Peptide

The urotensin II (UII) system consists of endogenous ligands UII and its paralog urotensin II-related peptide (URP) and their receptor, UT. Originally isolated from the rat brain, URP is conserved across all vertebrates and shares the same cyclic hexapeptide core sequence with UII. However, UII and URP exhibit different N-terminal amino acid sequences. Beyond the central nervous system, UII and URP are functionally expressed in peripheral tissues. Alterations in tissue expression levels of UII and UT and blood or urinary UII concentrations have been associated with cardiovascular, pulmonary, and kidney dysfunctions, including hypertension, chronic heart failure, pulmonary hypertension, glomerulonephritis, and diabetes, making the UII system a potential therapeutic target for cardiovascular and kidney disease. The skeletal muscle and plasma levels of UII are increased in diabetic mice. Increased kidney tissue expression levels of UII and UT have also been demonstrated in humans and animals with diabetic kidney disease (DKD). Our recent work showed that UT is functionally expressed in cultured mouse glomerular mesangial cells (GMCs). Activation of UT by UII contributes to Ca2+-dependent GMC proliferation and extracellular matrix protein accumulation under high glucose conditions. We have also shown that DKD is attenuated in UT knockout mice. Despite the structural homology between UII and URP, studies have shown that the peptides may exhibit differential central and peripheral expression and pathophysiological function. However, whether URP is dysregulated in DKD remains unclear. Here, we examined whether URP production and kidney expression levels are altered in a hyperglycemic milieu and associated with DKD. Using a streptozotocin (STZ)-induced diabetes model, we examined the kidney tissue, plasma, and urinary levels of UII peptides. We also tested the hypothesis that, like UII, URP stimulates GMC proliferation and matrix protein synthesis. Our data show that protein and mRNA expression levels of both UII and URP were increased in the kidneys of hyperglycemic mice that developed albuminuria. Urinary and plasma UII and URP concentrations were also elevated in the mice. Although both UII and URP stimulated proliferation and type IV collagen production in cultured mouse GMCs by activating UT, the effect of UII was greater. Our data suggests that changes in the UII system in DKD are not limited to UII and UT but include the less-studied URP. GMC proliferation and extracellular matrix accumulation driven by UII and URP may contribute to DKD. NHLBI: R01 HL151735. 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.

Hyperglycemia Induces Remodeling of Nitric Oxide Synthase Activity in Glomerular Podocytes

Background: The regulation of glomerular filtration is influenced by the renin-angiotensin system (RAS) and the release of nitric oxide (NO) by glomerular cells. Several studies suggest that NO levels in glomeruli decrease as renal pathology develops, and restoring NO levels can be beneficial for glomerular function. Despite these findings, the expression of nitric oxide synthase (NOS) subunits in podocytes, particularly under pathological conditions, still requires further investigation. We hypothesized that decreased NO bioavailability in diabetes and hyperglycemia is linked to pathological NOS remodeling, thus influencing redox balance in podocytes and promoting renal damage. Methods: Live-cell confocal imaging was used to investigate NO production and the distribution of NOS in both control and hyperglycemic conditions. First, we used immortalized human podocytes (obtained from M. Saleem lab) loaded with the DAF-FM fluorescent marker in control and hyperglycemic (exposed to high glucose for 12 hours) conditions. Next, freshly isolated glomeruli from Type 2 Diabetic Nephropathy (T2DN) and Wistar male rats (age 26-30 weeks) were used to confirm our data ex vivo. NO production was monitored following Angiotensin II (Ang II) stimulation, and the distribution of NOS was determined using specific NOS1 and NOS2 commercially available inhibitors (Nω-Propyl-L-arginine hydrochloride and L-NIL, respectively). One-way ANOVA (OriginPro) was used for statistical analysis. Results: Upon application of Ang II, rapid NO production was observed in cultured and Wistar rats isolated glomeruli podocytes. The release of NO was blocked by preincubation with corresponding inhibitors targeting NOS. Our findings reveal that NOS1 primarily contributes to signaling in healthy podocytes, accounting for 69±5% of the total NO response, while the remaining NO production mediated by the RAS is attributed to NOS2 (39±5%). In diabetic conditions, the maximal amplitude of NO production mediated by Ang II was decreased by 22±10% compared to the control (one-way ANOVA, n≥24, p<0.001). Furthermore, high glucose conditions significantly altered NOS distribution in podocytes, where NOS2 signaling became predominant (89±7% of the total NO response), and the contribution of NOS1 became less significant. Conclusions: The changes in the distribution of NO sources could be associated with heightened oxidative and nitrosative stress, promoting renal damage. Our research demonstrates that in diabetes, NOS2 activity becomes predominant, potentially playing a direct role in advancing pathological processes in podocytes. Revealing the mechanisms of this fundamental pathway in the glomerulus impacts our progress toward new treatment and preventive measures for diabetic kidney disease development. This research was supported by National Institutes of Health Grants R01 DK126720 (to OP), R01 DK129227 (to AS and OP), and R01 DK115749 (to KS), US Department of Veteran Affairs grants I01CX002391 (to JHL), I01CX001248 (JCO) and I01 BX004024 (to AS). 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.

FGF21 overexpression alleviates VSMC senescence in diabetic mice by modulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway.

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with progressive senescence in vascular smooth muscle cells (VSMCs). The vascular protective effect of FGF21 has gradually gained increasing attention, but its role in diabetes-induced vascular senescence needs further investigation. In this study, diabetic mice and primary VSMCs are transfected with an FGF21 activation plasmid and treated with a peroxisome proliferator-activated receptor γ (PPARγ) agonist (rosiglitazone), an NLRP3 inhibitor (MCC950), and a spleen tyrosine kinase (SYK)-specific inhibitor, R406, to detect senescence-associated markers. We find that FGF21 overexpression significantly restores the level of catalase (CAT), vascular relaxation, inhibits the intensity of ROSgreen fluorescence and p21 immunofluorescence, and reduces the area of SA-β-gal staining and collagen deposition in the aortas of diabetic mice. FGF21 overexpression restores CAT, inhibits the expression of p21, and limits the area of SA-β-gal staining in VSMCs under high glucose conditions. Mechanistically, FGF21 inhibits SYK phosphorylation, the production of the NLRP3 dimer, the expression of NLRP3, and the colocalization of NLRP3 with PYCARD (ASC), as well as NLRP3 with caspase-1, to reverse the cleavage of PPARγ, preserve CAT levels, suppress ROSgreen density, and reduce the expression of p21 in VSMCs under high glucose conditions. Our results suggest that FGF21 alleviates vascular senescence by regulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway in diabetic mice.

Abstract 1032: Development Of A Bio-hybrid Endovascular Stent-graft That Enables Immunosuppression Free Islet Cell Transplantation

Introduction: Islet cell transplantation has had limited clinical success freeing type 1 diabetics from exogenous insulin. Currently, islet cell transplants are performed via infusion of islet cells into the recipient's portal vein. Approximately 60% of transplanted cells die within 3 days of transplantation due to ischemic injury. Significant immunosuppression is needed to avoid rejection but is also toxic to islet cells. We have developed a novel endovascular biologic stent-graft that provides cellular immunoisolation while preventing ischemic injury through endovascular deployment (Fig 1). Hypothesis: We hypothesize islet cells seeded within the semipermeable biohybrid endovascular graft will survive and secrete insulin in an in-vitro model of perfusion. Methods: The semipermeable cell chamber was made from ePTFE with a pore size of 0.22 um, and was seeded with islet cells harvested from BALB/c mice. A normothermic machine perfusion circuit was used to model intravascular deployment. Devices were exposed to varying glucose concentrations and insulin was detected by ELISA. Results: There were significantly higher levels of insulin secretion after exposure to high vs low glucose media (p = 0.005). At hour 3 of low glucose (2.8 mM), the average insulin concentration was 76 ± 14 mIU/ml, and at hour 3 of high glucose (28 mM), the average insulin concentration was 134 ± 30 mIU/ml (Fig 2). The rate of insulin secretion was significantly higher in high vs low glucose conditions (6759 ± 503 mIU/h vs. 5344 ± 144 mIU/hr, p = 0.008). Conclusions: The endovascular biohybrid device enables islet cell function and survival. In-vivo studies are needed to study the immune interactions.

Quantification of total phenols in Coleus forskohlii and inhibition of aldose reductase by rosmarinic acid

Many complications of diabetes have been associated with the accumulation of intracellular sorbitol caused by excessive activity of aldose reductase (ALR2). ALR2 is a member of the aldo-keto reductases (AKR) superfamily and has gained significant attention due to its role in the development of diabetic complications. Inhibiting ALR2 could help prevent or delay some of these problems. Coleus forskohlii is a natural medicinal plant that is known for its medicinal effects and is also used as a beverage in several European countries. High-performance liquid chromatography (HPLC) was used to measure the polyphenol content of aqueous leaf extracts of C. forskohlii, and it was found that rosmarinic acid (RA) had the highest concentration (1.25 mg/g) compared to other polyphenols. In vitro bioassays were conducted to evaluate the inhibitory effect of RA on ALR2. RA significantly and dose-dependently reduced the build-up of sorbitol in human erythrocytes under high glucose conditions. Molecular docking studies suggest that RA, which is an essential polyphenol, could be a potential ALR2 inhibitor. This study is the first to show that RA has an IC50 value of 56 % to exhibit an inhibitory effect against ALR2.

Effects of high glucose and severe hypoxia on the biological behavior of mesenchymal stem cells at various passages.

Mesenchymal stem cells (MSCs) have been extensively studied for therapeutic potential, due to their regenerative and immunomodulatory properties. Serial passage and stress factors may affect the biological characteristics of MSCs, but the details of these effects have not been recognized yet. To investigate the effects of stress factors (high glucose and severe hypoxia) on the biological characteristics of MSCs at different passages, in order to optimize the therapeutic applications of MSCs. In this study, we investigated the impact of two stress conditions; severe hypoxia and high glucose on human adipose-tissue derived MSCs (hAD-MSCs) at passages 6 (P6), P8, and P10. Proliferation, senescence and apoptosis were evaluated measuring WST-1, senescence-associated beta-galactosidase, and annexin V, respectively. Cells at P6 showed decreased proliferation and increased apoptosis under conditions of high glucose and hypoxia compared to control, while the extent of senescence did not change significantly under stress conditions. At P8 hAD-MSCs cultured in stress conditions had a significant decrease in proliferation and apoptosis and a significant increase in senescence compared to counterpart cells at P6. Cells cultured in high glucose at P10 had lower proliferation and higher senescence than their counterparts in the previous passage, while no change in apoptosis was observed. On the other hand, MSCs cultured under hypoxia showed decreased senescence, increased apoptosis and no significant change in proliferation when compared to the same conditions at P8. These results indicate that stress factors had distinct effects on the biological processes of MSCs at different passages, and suggest that senescence may be a protective mechanism for MSCs to survive under stress conditions at higher passage numbers.

Discovery of a SUCNR1 antagonist for potential treatment of diabetic nephropathy: In silico and in vitro studies

Diabetic nephropathy (DN) is one of the most severe complications of diabetes mellitus. Succinate Receptor 1 (SUCNR1), a member of the G-protein-coupled receptor (GPCR) family, represents a potential target for treatment of DN. Here, utilizing multi-strategy in silico virtual screening methods containing AlphaFold2 modelling, molecular dynamics (MD) simulation, ligand-based pharmacophore screening, molecular docking and machine learning-based similarity clustering, we successfully identified a novel antagonist of SUCNR1, AK-968/12117473 (Cpd3). Through extensive in vitro experiments, including dual-luciferase reporter assay, cellular thermal shift assay, immunofluorescence, and western blotting, we substantiated that Cpd3 could specifically target SUCNR1, inhibit the activation of NF-κB pathway, and ameliorate epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition in renal tubular epithelial cells (NRK-52E) under high glucose conditions. Further in silico simulations revealed the molecular basis of the SUCNR1-Cpd3 interaction, and the in vitro metabolic stability assay indicated favorable drug-like pharmacokinetic properties of Cpd3. This work not only successfully pinpointed Cpd3 as a specific antagonist of SUCNR1 to serve as a promising candidate in the realm of therapeutic interventions for DN, but also provides a paradigm of dry-wet combined discovery strategies for GPCR-based therapeutics.

Mitigation of ROS-triggered endoplasmic reticulum stress by upregulating Nrf2 retards diabetic nephropathy

Endoplasmic reticulum stress (ERS) plays a crucial role in the pathogenesis of diabetic nephropathy (DN), and it is often accompanied by an increase in reactive oxygen species (ROS) production. However, the precise relationship between NFE2-related factor-2 (Nrf2), a key regulator of ROS balance, and ERS in DN remains elusive. This study aimed to investigate the impact of Nrf2 on ERS and its therapeutic potential in DN. Herein, ERS-related changes, including increased activating transcription factor-6 (ATF6), glucose-regulated protein 78 (GRP78), and transcription factor C/EBP homologous protein (CHOP) expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose cultured human renal proximal tubular (HK-2) cells. Nrf2 knockdown increased the sensitivity of HK-2 cells to ERS under high glucose conditions, underscoring the regulatory role of Nrf2 in ERS modulation. Notably, upregulating Nrf2 in ezetimibe-treated diabetic mice restored ERS markers and ameliorated albuminuria, glomerular hypertrophy, mesangial expansion, and tubulointerstitial fibrosis. Furthermore, the inhibition of ERS in HK-2 cells by the ROS scavenger, N-acetylcysteine, highlights the interplay between ROS and ERS. This study, for the first time, elucidates that the upregulation of Nrf2 may alleviate the negative influence of ROS-mediated ERS, presenting a promising therapeutic avenue for delaying the progression of DN. These findings suggest a potential strategy for targeting Nrf2 and ERS in developing novel therapeutic interventions for DN.

Beta hydroxybutyrate induces lung cancer cell death, mitochondrial impairment and oxidative stress in a long term glucose-restricted condition.

Metabolic plasticity gives cancer cells the ability to shift between signaling pathways to facilitate their growth and survival. This study investigates the role of glucose deprivation in the presence and absence of beta-hydroxybutyrate (BHB) in growth, death, oxidative stress and the stemness features of lung cancer cells. A549 cells were exposed to various glucose conditions, both with and without beta-hydroxybutyrate (BHB), to evaluate their effects on apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) levels using flow cytometry, and the expression of CD133, CD44, SOX-9, and β-Catenin through Quantitative PCR. The activity of superoxide dismutase, glutathione peroxidase, and malondialdehyde was assessed using colorimetric assays. Treatment with therapeutic doses of BHB triggered apoptosis in A549 cells, particularly in cells adapted to glucose deprivation. The elevated ROS levels, combined with reduced levels of SOD and GPx, indicate that oxidative stress contributes to the cell arrest induced by BHB. Notably, BHB treatment under glucose-restricted conditions notably decreased CD133 expression, suggesting a potential inhibition of cell survival through the downregulation of CD133 levels. Additionally, the simultaneous decrease in mitochondrial membrane potential and increase in ROS levels indicate the potential for creating oxidative stress conditions to impede tumor cell growth in such environmental settings. The induced cell death, oxidative stress and mitochondria impairment beside attenuated levels of cancer stem cell markers following BHB administration emphasize on the distinctive role of metabolic plasticity of cancer cells and propose possible therapeutic approaches to control cancer cell growth through metabolic fuels.

Exosomes from MicroRNA-125b-Modified Adipose-Derived Stem Cells Promote Wound Healing of Diabetic Foot Ulcers.

Exosomes derived from Adipose-Derived Stem Cells (ADSCs-Exo) have been implicated in the enhancement of wound repair in Diabetic Foot Ulcers (DFU). The current research was designed to explore the therapeutic potential and underlying mechanisms of ADSCs-Exo modified with microRNA-125b (miR-125b) in the context of DFU. Rat models with DFU and human umbilical vein endothelial cells (HUVECs) subjected to high glucose (HG) conditions served as experimental systems and were administered miR-125b-engineered ADSCs-Exo. Then, the expressions of CD34, Ki-67, angiogenesis-related factors (VEGF and TGFβ-1), angiogenesis inhibitor DLL-4, and inflammation-related proteins (TLR-4 and IL-6) were detected. MiR-125b was upregulated in ADSCs-Exo. MiR-125b-mimics transfection in ADSCs- Exo reduced inflammatory infiltration and promoted granulation formation and wound healing in wound tissues. MiR-125b-mimics-modified ADSCs-Exo injection increased the expression of CD34, Ki-67, VEGF, and TGFβ-1, whereas decreased the expression of DLL-4, TLR-4, and IL-6 in wound tissues of DFU rats. In addition, miR-125b-mimics-ADSCs-Exo injection reversed the negative effects of HG on the proliferation, migration, and angiogenesis of HUVECs, as well as the positive effects of cell apoptosis. Moreover, miR-125b-inhibitor-ADSCs-Exo injection had the opposite effects to miR-125b-mimics-ADSCs-Exo. ADSCs-Exo promoted wound healing of DFU rats, especially when overexpressing miR-125b.

Zinc oxide nanoparticles prevent the onset of diabetic nephropathy by inhibiting multiple pathways associated with oxidative stress

BackgroundZinc deficiency is strongly correlated with prolonged diabetes mellitus and diabetic nephropathy (DN). Previously, glucose-lowering, insulinomimetic, and β-cell proliferative activities of zinc oxide nanoparticles (ZON) have been reported. Considering these pleiotropic effects, we hypothesized that ZON modulates multiple cellular pathways associated with necroptosis, inflammation, and renal fibrosis, which are involved in progressive loss of renal function. AimThis study evaluated the effect of ZON on renal function, leading to the alleviation of DN in streptozotocin (STZ)-induced type 1 diabetic Wistar rats and proposed a probable mechanism for its activity. MethodsWistar rats (n = 6/group) were used as healthy controls, diabetic controls, diabetic rats treated with ZON (1, 3, and 10 mg/kg), and insulin controls. Urine and serum biochemical parameters, glomerular filtration rate (GFR), and renal histology were also evaluated. Cultured E11 podocytes were evaluated in vitro for markers of oxidative stress, proteins associated with the loss of renal function, and genes associated with renal damage. Key findingsSTZ-treated rats receiving oral doses of ZON showed enhanced renal function, with no histological alterations in the kidney tissue. ZON inhibited the TGF-β/Samd3 pathway in renal fibrosis; blocked Ripk1/Ripk3/Mlkl mediated necroptosis and protected against hyperglycemia-induced pyroptosis. In E11 podocytes, ZON reduced oxidative stress under high glucose conditions and retained podocyte-specific proteins. SignificanceA probable mechanism by which ZON prevents DN has been proposed, suggesting its use as a complementary therapeutic agent for the treatment of diabetic complications. To the best of our knowledge, this is the first study to demonstrate the in vitro effects of ZON in cultured podocytes.

Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1β, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-β1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.