Diabetic Mice Research Articles

Using GeoMx DSP Spatial Proteomics to Investigate Immune Infiltration of NOD Mouse Islet and Exocrine Compartments

Abstract Purpose Type 1 Diabetes (T1D) pathogenesis involves immune cells infiltrating pancreatic Islets of Langerhans, leading to T cell activation, beta cell destruction, and impaired insulin production. However, infiltration has a heterogenic nature that isn’t described in detail, as not all islets are infiltrated. The aim of this study was to investigate if the observed heterogeneity is coupled to differences in immune and/or dysfunctional status of islets or exocrine cells, and if specific markers could elucidate mechanistic details of T1D pathogenesis. Procedures The GeoMx platform was used to spatially quantify protein levels in pancreatic islets and exocrine tissue in Non-Obese Diabetic (NOD) mice. The protein panel included 17 immune activity markers and nine dysfunction markers. Immunohistochemical (IHC) staining and digital image analysis was used to analyze select marker proteins. Results Use of the GeoMx platform to investigate T1D was shown to be possible, as Granzyme B protein levels were found to be lower in distal islet areas when compared to proximal areas. Smooth Muscle Actin protein levels were higher in exocrine areas proximal to immune-infiltrated islets, when compared to distally located exocrine areas. Findings from GeoMx were however not observed in IHC-stained sections. Conclusions This study demonstrates that investigating T1D is possible with spatial proteomics, as the assays revealed presence of heterogenic islet areas in NOD mice, which may play a role in T1D progression and escape from immune recognition. This study highlights the potential of spatial technologies for elucidating T1D pathogenesis and future treatment strategies.

Possible Role of Endothelial-Derived Cellular and Exosomal-miRNAs in Lipid-Mediated Diabetic Retinopathy: Microarray Studies

Diabetic retinopathy (DR) is a salient cause of blindness worldwide. There is still an immense need to understand the pathophysiology of DR to discover better diagnostic and therapeutic modalities. Human retinal endothelial cells (HRECs) were treated with 15-HETE or D-glucose, then miRNAs were isolated, and a microarray was performed. MirWALK 2 and Ingenuity Pathway Analysis (IPA) were used to analyze the microarray results. Exosomal miRNAs from 15-HETE-treated HRECs were isolated, microarrayed, and then imported into IPA for further analysis. The microarray results showed that 15-HETE downregulated 343 miRNAs and upregulated 297 miRNAs in HRECs. High glucose treatment induced a differential expression of HREC-miRNAs where 185 miRNAs were downregulated and 244 were upregulated. Comparing the impact of 15-HETE versus DG or diabetic mouse retina elaborated commonly changing miRNAs. Pathway and target analysis for miRNAs changed in 15-HETE-treated HRECs revealed multiple targets and pathways that may be involved in 15-HETE-induced retinal endothelial dysfunction. The HREC-exosomal miRNAs were differentially expressed after 15-HETE treatment, with 34 miRNAs downregulated and 45 miRNAs upregulated, impacting different cellular pathways. Here, we show that 15-HETE induces various changes in the cellular and exosomal miRNA profile of HRECs, highlighting the importance of targeting the 12/15 lipoxygenase pathway in DR.

Prolactin deficiency drives diabetes-associated cognitive dysfunction by inducing microglia-mediated synaptic loss.

Diabetes-associated cognitive dysfunction, characterized by hippocampal synaptic loss as an early pathological feature, seriously threatens patients' quality of life. Synapses are dynamic structures, and hormones play important roles in modulating the formation and elimination of synapses. The pituitary, the master gland of the body, releases several hormones with multiple roles in hippocampal synaptic regulation. In this study, we aimed to explore the relationship between pituitary hormones and cognitive decline in diabetes. A total of 744 patients with type 2 diabetes (T2DM) (445 men and 299 postmenopausal women) who underwent serum pituitary hormone level assessments, comprehensive cognitive evaluations and MRI scans were enrolled. Dynamic diet interventions were applied in both chow diet-fed mice and high-fat diet (HFD)-fed diabetic mice. The cognitive performance and hippocampal pathology of prolactin (PRL)-knockout mice, neuronal prolactin receptor(PRLR)-specific knockout mice and microglial PRLR-specific knockout mice were assessed. Microglial PRLR-specific knockout mice were fed an HFD to model diabetes. Diabetic mice received an intracerebroventricular infusion of recombinant PRL protein or vehicle. This clinical study revealed that decreased PRL levels were associated with cognitive impairment and hippocampal damage in T2DM patients. In diabetic mice, PRL levels diminished before hippocampal synaptic loss and cognitive decline occurred. PRL loss could directly cause cognitive dysfunction and decreased hippocampal synaptic density. Knockout of PRLR in microglia, rather than neurons, induced hippocampal synaptic loss and cognitive impairment. Furthermore, blockade of PRL/PRLR signaling in microglia exacerbated abnormal microglial phagocytosis of synapses, further aggravating hippocampal synaptic loss and cognitive impairment in diabetic mice. Moreover, PRL infusion reduced microglia-mediated synaptic loss, thereby alleviating cognitive impairment in diabetic mice. PRL is associated with cognitive dysfunction and hippocampal damage in T2DM patients. In diabetes, a decrease in PRL level drives hippocampal synaptic loss and cognitive impairment by increasing microglia-mediated synapse engulfment. Restoration of PRL levels ameliorates cognitive dysfunction and hippocampal synaptic loss in diabetic mice.

Enhancing Skin Wound Healing in Diabetic Mice Using SIKVAV-Modified Chitosan Hydrogels

Diabetic foot ulcers (DFUs), a prevalent chronic disease caused by various factors, significantly impact patients’ quality of life due to prolonged healing times and increased infection risks. Current treatment modalities, including pharmacological, physical, and surgical interventions, often yield limited efficacy and adverse effects, highlighting the urgent need for novel therapeutic strategies. The objective of this research is to create SIKVAV-modified chitosan hydrogels with the intention of improving the process of skin wound healing in diabetic mice. We synthesized the hydrogels and established a diabetic mice model with skin wounding to evaluate its healing effects and underlying mechanisms. The results of our study indicate that the SIKVAV-modified chitosan hydrogels markedly enhance the wound healing process in diabetic mice. This effect may be attributed to several mechanisms, including differentiation of fibroblasts, proliferation of keratinocytes, the promotion of angiogenesis, stimulation of collagen synthesis, upregulation of growth factor expression, and possible involvement of the TGF-β1/Smad3 signaling pathway. This research not only provides a new biomaterial for the treatment of diabetic wounds but also elucidates the related molecular mechanisms involved in wound healing of DFUs, offering valuable insights for future clinical applications.

The anti-inflammatory and anti-glycative effects of rosmarinic acid in the livers of type 1 diabetic mice

The anti-inflammatory and anti-glycative effects of rosmarinic acid in the livers of type 1 diabetic mice

Renal protective effects of Porphyra dentate aqueous extract in diabetic mice

Renal protective effects of Porphyra dentate aqueous extract in diabetic mice

Platelet-rich plasma promotes wound repair in diabetic foot ulcer mice via the VEGFA/VEGFR2/ERK pathway

Diabetic foot ulcers (DFUs) are a severe microvascular complication. Platelet-rich plasma (PRP) pitches in DFU treatment. This study explored the mechanism of PRP facilitating wound repair in DFU mice via vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2)/extracellular signal-regulated kinase (ERK) pathway. The DFU mouse model was established, with wound skin injected with PRP, followed by the detections of wound area, histopathological changes, and CD31-positive cells. IL-6/TNF-α/VEGFA/VEGFR2/p-VEGFR2/(ERK1/2)/(p-ERK1/2) levels in wound tissue homogenates were assessed. VEGFA-VEGFR2 interaction was evaluated. PRP-treated DFU mice were simultaneously treated with fruquintinib/PD98059. PRP reduced wound area, IL-6 and TNF-α levels, elevated epidermal dermal thickness, CD31-positive cell number, and aligned tissue structure, which were mitigated by fruquintinib/PD98059. PRP promoted VEGFR2 phosphorylation. PRP and fruquintinib/PD98059 abated p-VEGFR2/VEGFR2 or p-ERK1/2/ERK1/2 levels in DFU mice. PRP activated the ERK pathway through VEGFA/VEGFR2. Collectively, PRP promoted VEGFR2 phosphorylation and activated the ERK pathway, thereby facilitating wound repair in DFU mice.

Protective Effect of Mesenchymal Stromal Cells in Diabetic Nephropathy: The In vitro and In vivo Role of the M-Sec-Tunneling Nanotubes.

 Mitochondrial dysfunction plays an important role in the development of podocyte injury in diabetic nephropathy (DN). Tunnelling nanotubes (TNTs) are long channels that connect cells and allow organelle exchange. Mesenchymal stromal cells (MSCs) can transfer mitochondria to other cells through the M-Sec-TNTs system. However, it remains unexplored whether MSCs can form heterotypic TNTs with podocytes, thereby enabling the replacement of diabetes-damaged mitochondria. In this study, we analysed TNT formation, mitochondrial transfer, and markers of cell injury in podocytes that were pre-exposed to diabetes-related insults and then co-cultured with diabetic or non-diabetic MSCs. Furthermore, to assess the in vivo relevance, we treated DN mice with exogenous MSCs, either expressing or lacking M-Sec, carrying fluorescent-tagged mitochondria. MSCs formed heterotypic TNTs with podocytes, allowing mitochondrial transfer, via a M-Sec-dependent mechanism. This ameliorated mitochondrial function, nephrin expression, and reduced apoptosis in recipient podocytes. However, MSCs isolated from diabetic mice failed to confer cytoprotection due to Miro-1 downregulation. In experimental DN, treatment with exogenous MSCs significantly improved DN, but no benefit was observed in mice treated with MSCs lacking M-Sec. Mitochondrial transfer from exogenous MSCs to podocytes occurred in vivo in a M-Sec-dependent manner. These findings demonstrate that the M-Sec-TNT-mediated transfer of mitochondria from healthy MSCs to diabetes-injured podocytes can ameliorate podocyte damage. Moreover, M-Sec expression in exogenous MSCs is essential for providing renoprotection in vivo in experimental DN.

Abstract Sa1108: The Aberrant Autophagy-Apoptosis regulation Mediates the Severe Neurological Dysfunction after Cardiac Arrest in Diabetic Mice

Background: Patients with diabetes have lower survival and worse neurological outcome after cardiopulmonary resuscitation (CPR) compared with non-diabetes. Previous studies have mentioned the aberrant autophagy in diabetic mice. However, whether this abnormal autophagy exacerbates neuronal apoptosis and neurological outcome in diabetic mice remains unclear. Methods: Patients were divided into non-diabetic cardiac arrest (CA) patients and diabetic CA patients, and the survival and neurological outcomes at discharge and day 28 after ROSC were compared between these two groups. Diabetic mice were fed with high fat diet (HFD), and CA model was established by inducing ventricular fibrillation electrically. The neurological prognosis was assessed by open field test (OPT), novel object recognition (NOR) and Y-maze. Neuronal apoptosis and autophagy were evaluated by immunofluorescence and western blotting, including TUNEL, cleaved caspase 3, LC3B, p62 and beclin1. Results: Compared with non-diabetic patients, the survival at discharge and at day 28 after ROSC are significantly lower in IHCA patients with diabetes. Meanwhile, the neurological deficits at discharge and at day 28 after ROSC are much severer in diabetic-CA patients. In addition, the lower survival and worse neurological function have been discovered, and the cognition impairment is more significant after ROSC in HFD mice. Neuronal apoptosis is severer in HFD mice. In ND mice, the activated autophagy has been revealed after CA-CPR-ROSC, however, autophagy can not be activated after ROSC in HFD mice since the baseline autophagy is at a higher level when compared with ND mice. Neuronal apoptosis is exacerbated after autophagy inhibition by 3-MA in ND mice, while after autophagy activation by rapamycin in HFD mice, apoptosis is attenuated after ROSC. Conclusion: Diabetes is the risk factor for neurological outcome in IHCA patients. The aberrant autophagy regulation in cortex neurons contributes to the severe apoptosis and neurological deficits after ROSC in diabetic mice.

Abstract 4119062: A KLF2-BMPER-Smad1/5 checkpoint regulates high fluid shear stress-mediated artery remodeling

Background: Vascular remodeling to match arterial diameter to tissue metabolic requirements commonly fails in ischemic disease. Endothelial cell (EC) sensing of elevated fluid shear stress (FSS) from blood flow induces vessel outward remodeling to restore physiological FSS, but mechanisms are poorly understood. The Smad1/5 pathway, which is maximally activated at physiological FSS and suppressed at higher flow, opposes activation of Akt, suggesting that inhibiting Smad1/5 may be required for outward remodeling. Methods: In vitro flow studies used ECs in a parallel plate flow chamber. In vivo mouse studies used a carotid-jugular fistula model to induce high flow outward remodeling in the carotid artery, and femoral artery ligation to examine recovery from ischemia and arteriogenesis in the hindlimb. Results: Suppression of Smad1/5 at high FSS is mediated KLF2-dependent induction of the BMP pathway inhibitor BMPER, which suppresses Smad1/5 and de-inhibits Akt. In a mouse arteriovenous fistula (AVF) model, high FSS induces arterial outward remodeling coincident with elevated BMPER expression and Smad1/5 inactivation. Endothelial BMPER deletion impaired blood flow recovery and vascular remodeling in the AVF and a hindlimb ischemia (HLI) model, with the latter reversed by BMP9/10 blocking antibodies (bAbs). In both STZ-induced type 1 and HFD-induced type 2 diabetic mice that show poor recovery from HLI, BMP9/10 bAbs improved outcomes. Conclusions: Suppression of Smad1/5 through a KLF2-BMPER pathway is required for high FSS-mediated outward remodeling. Mimicking this pathway with BMP9/10 antibodies improves vascular remodeling in diabetic mice, suggesting a potential new therapeutic approach for ischemic disease.

Abstract 4129523: MicroRNA-1282 Rescues Diabetic Limb Ischemia Via a SERF2-Protein Aggregation Pathway

Introduction: Patients with diabetes are at higher risk of chronic limb-threatening ischemia (CLTI), a severe form of peripheral artery disease (PAD) causing restricted blood flow to the lower limbs due, in part, to impaired angiogenesis. However, the role of microRNAs (miRNAs) in diabetic CLTI remains poorly understood. By integrating plasma miRNA sequencing data from PAD patients with diabetes with a diabetic CLTI mouse model, we have recently identified the conserved miRNA miR-1282 that is in cis-antisense orientation to SERF2, a gene associated with amyloid aggregation. Therefore, we hypothesize that miR-1282 orchestrates endothelial angiogenesis and proteostasis during diabetic CLTI. Methods: Using miR-1282 overexpression or SERF2 knockdown studies, we characterized mouse orthologs of human miR-1282 and SERF2 for angiogenesis, apoptosis, protein aggregation, and oxidative stress in diabetic mouse skeletal muscle endothelial cells (ECs). In vivo, miR-1282 mimics were delivered intramuscularly to assess their impact on blood flow recovery, angiogenesis, and protein aggregation. Mechanistic insights in ECs were gained via RNA-seq, predictive algorithms, and proteomic analyses. Results: miR-1282 inhibited the expression of its cis-antisense target SERF2 by 98%. miR-1282 is a hypoxia-induced endothelial-enriched miRNA, and its expression was inversely correlated with SERF2 expression. miR-1282 levels were markedly reduced after femoral artery ligation (FAL) in diabetic db/db mice. Overexpression of miR-1282 or SERF2 knockdown enhanced angiogenesis and reduced protein aggregation, apoptosis, and oxidative stress in both normal and hypoxic conditions in vitro. Delivery of miR-1282 mimics in db/db mice improved blood flow recovery by 108% and angiogenesis by 98%, and reduced protein aggregation by 48% and tissue necrosis. Coupling RNA-seq profiling and prediction algorithms of ECs upon miR-1282 overexpression or SERF2 knockdown revealed EREG, BAG5, CASP3, ARG1, and HSP90AA1 as potential downstream regulators. Pathway enrichment analysis implicated inhibition of endothelial apoptosis, ER stress, and protein stability among the most dysregulated processes. Conclusion: A novel mouse ortholog of human miR-1282 augments endothelial functions and diminishes protein aggregation in diabetic CLTI via suppression of its cis-antisense target SERF2. These findings uncover new potential therapeutic targets in treating diabetic CLTI.

A microenvironment-modulating dressing with proliferative degradants for the healing of diabetic wounds.

Diabetic wounds are usually entangled in a disorganized and self-perpetuating microenvironment and accompanied by a prolonged delay in tissue repair. Sustained and coordinated microenvironment regulation and tissue regeneration are key to the healing process of diabetic wounds, yet they continue to pose a formidable challenge. Here we report a rational double-layered dressing design based on chitosan and a degradable conjugated polymer polydiacetylene, poly(deca-4,6-diynedioic acid) (PDDA), that can meet this intricate requirement. With an alternating ene-yne backbone, PDDA degrades when reacting with various types of reactive oxygen species (ROS), and more importantly, generates proliferative succinic acid as a major degradant. Inheriting from PDDA, the developed PDDA-chitosan double layer dressing (PCD) can eliminate ROS in the microenvironment of diabetic wounds, alleviate inflammation, and downregulate gene expression of innate immune receptors. PCD degradation also triggers simultaneous release of succinic acid in a sustainable manner, enabling long-term promotion on tissue regeneration. We have validated the biocompatibility and excellent performance of PCD in expediting the wound healing on both diabetic mouse and porcine models, which underscores the significant translational potential of this microenvironment-modulating, growth-promoting wound dressing in diabetic wounds care.

Loureirin B Accelerates Diabetic Wound Healing by Promoting TGFβ/Smad-Dependent Macrophage M2 Polarization: A Concerted Analytical Approach Through Single-Cell RNA Sequencing and Experimental Verification.

Diabetic wound (DW) represent a significant clinical challenge and often fail to heal effectively. Loureirin B (LB), a flavonoid extracted from dragon's blood, has shown potential by influencing macrophage polarization and promoting wound healing. However, its mechanisms and efficacy in DW remain to be explored. This study employed single-cell RNA sequencing to analyze the classification of cells in diabetic foot ulcers and to identify the related mechanisms influenced by macrophages. Molecular docking was used to predict the interactions of LB with key proteins in the TGFβ/Smad signaling pathway. The effects of LB on macrophage polarization and wound healing were further validated through invitro and invivo experiments using a DW model. Single-cell analysis identified specific macrophage subtypes involved in the DW healing process and highlighted the role of the TGFβ/Smad pathway. Molecular docking suggested the potential action within the TGFβ/Smad pathway. Invitro studies showed that under high glucose conditions, LB promoted macrophage polarization from pro-inflammatory M1 to healing-promoting M2 and ECM production in fibroblasts by activating TGF-β/Smad signaling. Invivo, LB treatment enhanced wound healing rates in diabetic mice and promoted macrophage M2 polarization and fibroblast synthesis of ECM by activating TGF-β/Smad signaling. LB regulates macrophage M2 polarization and fibroblast synthesis of ECM by activating TGF-β/Smad signaling to promote DW healing. These findings suggest that LB could be a potential therapeutic agent for improving DW healing, emphasizing the need for further clinical studies to explore its efficacy and mechanisms in human subjects.

Abstract 4122717: An Epigenetic Drug, GSK126 Mitigates Endothelial to Mesenchymal Transition Attenuating Atherosclerosis in Diabetes

Background: Endothelial to mesenchymal transition (EndMT) involves transformation of an endothelial to mesenchymal like cellular state. Recent studies implicate EndMT in atherosclerosis development. Diabetes is associated with both EndMT as well as accelerated atherosclerosis. Experimental evidence supports the use of epigenetic drugs that act on relevant epigenetic mechanisms to attenuate atherosclerosis. Recently, inhibition of a histone methyltransferase Enhancer of zest homolog 2 (EZH2) with GSK-126 attenuated atherosclerosis development. However, the role of EZH2 in induction of EndMT in diabetes induced atherosclerosis is not known. Methods: An in-vitro model of EndMT was generated using high glucose (HG) and TNF-α in human aortic endothelial cells (HAECs). EZH2 methyltransferase activity was inhibited using EZH2 specific inhibitor GSK-126. RNA sequencing was performed in this setting. EZH2 genetic knockdown (shRNA) in HAECs was also performed to validate role of EZH2 in EndMT. In addition, EZH2 mediated H3K27me3 and EndMT was assessed in atheroprone diabetic mouse model. Results: In HAECs, morphological changes as well as gene and protein expression confirmed TNF-α+HG induced EndMT, which was blunted by GSK-126. Elevated EZH2 mediated H3K27me3 activity by the TNF-α + HG stimulation was blunted with GSK-126 treatment. Furthermore, RNA sequencing identified several enriched pathways including AGE-RAGE signaling pathway in diabetic complications, focal adhesion, and leukocyte trans-endothelial migration directly relevant to EndMT and commonly deregulated in diabetic complications, which were rescued by EZH2 inhibition. Transcriptomic analysis showed that 242 genes including MMP2, NOS3 linked to EndMT were dysregulated. Interestingly, expression of 76 dysregulated genes in EndMT were rescued by GSK-126. EZH2 knockdown studies in HAECs also validated the role of EZH2 in EndMT. Furthermore, immunofluorescence staining identified elevated levels of H3K27me3 in the aortic endothelial layer of diabetic Apoe -/- mice. Co-localization of EndMT markers was observed in the endothelial layer of aortic sinus of diabetic Apoe -/- mice which was blunted with GSK-126 treatment. Conclusion: The study identified EZH2 inhibition as a novel therapeutic target in diabetes induced EndMT in atherosclerosis. These findings highlighted ongoing efforts to develop targeted therapies for diabetic patients who are at risk to develop cardiovascular disease.

Insulin-degrading enzyme regulates insulin-directed cellular autoimmunity in murine type 1 diabetes

Type 1 diabetes results from the destruction of pancreatic beta cells by autoreactive T cells. As an autoantigen with extremely high expression in beta cells, insulin triggers and sustains the autoimmune CD4+ and CD8+ T cell responses and islet inflammation. We have previously shown that deficiency for insulin-degrading enzyme (IDE), a ubiquitous cytosolic protease with very high affinity for insulin, induces endoplasmic reticulum (ER) stress and proliferation in islet cells and protects non-obese diabetic mice (NOD) from diabetes. Here we wondered whether IDE deficiency affects autoreactive CD8+ T cell responses to insulin and thereby immune pathogenesis in NOD mice. We find that Ide-/- NOD harbor fewer diabetogenic T cells and reduced numbers of CD8+ T cells recognizing the dominant autoantigen insulin and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Using in vitro digestions and cellular antigen presentation assays, we show that generation of the dominant insulin epitope B15-23 involves both the proteasome and IDE. IDE deficiency attenuates MHC-I presentation of the immunodominant insulin epitope by beta cells to cognate CD8+ T cells. Consequently, Ide-/- islets display reduced susceptibility to autoimmune destruction upon grafting, and to killing by insulin-specific CD8+ T cells. Moreover, Ide-/- mice are partly resistant to disease transfer by CD8+ T cells specific for insulin but not for IGRP. Thus, IDE has a dual role in beta cells, regulating ER stress and proliferation while at the same time promoting insulin-directed autoreactive CD8+ T cell responses.

Prolonged Photobiomodulation with Deep Red Light Mitigates Incipient Retinal Deterioration in a Mouse Model of Type 2 Diabetes

Diabetic retinopathy is a prevalent complication of type 2 diabetes mellitus, characterized by progressive damage to the retinal structure and function. Photobiomodulation therapy, using red or near-infrared light, has been proposed as a non-invasive intervention to mitigate retinal damage, but has been tested generally with short-term stimuli. This study aimed to evaluate the effects of prolonged photobiomodulation with deep red light on retinal structure and function in a type 2 diabetes mouse model. Transgenic LepRdb/J (db/db) mice were exposed to photobiomodulation therapy via LED devices emitting 654 nm light for 12 h daily over ten weeks and compared to untreated mice. Retinal function was evaluated by flash electroretinography, while structural changes were assessed through histology and immunohistochemistry to detect astro- and microgliosis. At 33 weeks of age, db/db mice were obese and severely diabetic, but exhibited only incipient indicators of retinal deterioration. Electroretinogram b-wave peak latencies were prolonged at intermediate flash intensities, while the outer plexiform layer displayed significantly elevated IBA1 expression. Photobiomodulation therapy prevented these two markers of early retinal deterioration but had no effect on other morphological and functional parameters. Photobiomodulation is well-tolerated and maintains potential as a complementary treatment option for diabetic retinopathy but requires further optimization of therapeutic settings and combinatory treatment approaches.

12-Lipoxygenase inhibition delays onset of autoimmune diabetes in human gene replacement mice.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing beta cells and involves an interplay between beta cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in beta cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our finding demonstrated that VLX-1005, a potent 12-LOX inhibitor, effectively delayed the onset of autoimmune diabetes in human gene replacement non-obese diabetic mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecule PD-L1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets and polarized proinflammatory macrophages revealed significant alteration of cytokine-responsive pathways and a reduction in interferon response after VLX-1005 treatment. Our studies demonstrated that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

Abstract 4138745: Blockade of Mineralocorticoid Receptor by Esaxerenone Improves Insulin Sensitivity in Obese Mice

Background: Aldosterone regulates blood pressure and electrolyte balance. However, overactivation of the mineralocorticoid receptor (MR) and elevated aldosterone levels are implicated in the pathogenesis of cardiometabolic disorders, though the underlying mechanisms are not fully understood. In this study, we investigated whether a novel, non-steroidal selective MR blocker, esaxerenone, ameliorates the development of metabolic disorders in obese mice models and examined the underlying mechanism. Methods: Esaxerenone (3 mg/kg/day) was orally administered to high-fat diet (HFD)-fed C57BL/6J or normal chow-fed db/db mice. Metabolic parameters, tissue morphology, and effects of esaxerenone on insulin sensitivity were assessed. Gene expression and insulin signaling, determined by Akt phosphorylation, were examined using quantitative RT-PCR and western blotting, respectively. In in-vitro experiments, insulin targets cell lines such as 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes were used. Insulin-responsive cells were treated with 10 nM or 100 nM esaxerenone or eplerenone for 30 minutes before 4-hour 1000 nM aldosterone stimulation. Insulin signaling was assessed 15 minutes after stimulation with 17 nM insulin. Results: Esaxerenone improved insulin sensitivity (P<0.05) without altering metabolic parameters in HFD-induced obese and db/db mice. Esaxerenone suppressed fat accumulation, adipocyte size, and liver lipid droplets compared with the vehicle-treated group (P<0.01). In adipose tissue, esaxerenone reduced macrophage infiltration (P<0.01) and the expression of inflammatory molecules (P<0.05). In in-vitro experiments, aldosterone significantly decreased the expression levels of PPARγ and adiponectin in 3T3-L1 adipocytes. Moreover, aldosterone attenuated insulin-induced Akt phosphorylation in 3T3-L1 adipocytes, HepG2 cells, and C2C12 myotubes in a dose-dependent manner (P<0.01). Pretreatment with esaxerenone reversed the effect of aldosterone, whereas eplerenone, an MR antagonist, did not show a similar effect at the same dose. Conclusion: Inhibition of MR by esaxerenone improved insulin sensitivity in HFD-induced obese and genetically diabetic mice. The reduction of inflammation and fat accumulation and enhancement of insulin signaling were suggested to be underlying mechanisms. Esaxerenone might be a beneficial therapeutic approach for managing metabolic disorders.

Hypoglycemic and intestinal microbiota-regulating effects of melanoidins in diabetic mice.

The aqueous extraction of sesame oil is a traditional process that generates a large amount of melanoidins. However, little is known about the characteristics and bioactive functions of these melanoidins. Electronic tongue, fluorescence emission spectroscopy, and Fourier transform infrared spectroscopy analyses indicated that melanoidins from sesame residues (MELs) are brown macromolecular compounds with protein skeletons and heteroaromatic ring structures, a bitter taste, and instability in strong oxidative and reductive environments. The MELs demonstrated inhibitory effects on α-glucosidase, α-amylase and pancreatic lipase in vitro. These MELs mitigated weight loss in mice with type 2 diabetes (T2DM), reduced their fasting blood glucose to 54.73% (500 mg kg-1 day-1) of the initial value, increased the glycogen levels in the liver and skeletal muscles, lowered blood lipid levels, and protected the liver. Western blot analysis revealed that MELs inhibited the activities of enzymes such as PEPCK, FBPase, and G6Pase through the IRS-1/PI3K/Akt and AMPK pathways, increased the activity of the enzymes hexokinase (HK) and pyruvate kinase (PK), enhanced liver glycolytic ability, and promoted liver glycogen synthesis, thereby reducing blood glucose levels in T2DM mice. Moreover, MELs reduced the ratio of Firmicutes to Bacteroides (F/B) in the intestines of T2DM mice, increased the relative abundance of beneficial bacteria such as Lactobacillus, Coprococcus, and Ruminococcus, and reduced the propionic acid content. Melanoidins can regulate T2DM by activating the IRS-1/PI3K/Akt and AMPK-signaling pathways and ameliorating gut microbiota imbalances in T2DM mice. © 2024 Society of Chemical Industry.

Antidiabetic activity of Inonotus obliquus water extract in alloxan-induced diabetic mice.

The Inonotus obliquus is a medicinal mushroom that grows on mature birch trees and is commonly used in traditional medicine for various ailments. The current study aimed to explore the hypoglycemic, hypolipidemic, and pancreatic protective effects of the water extract of Mongolian natural Inonotus obliquus on alloxan-induced diabetic mice. Alloxan monohydrate (200 mg/kg) was intraperitoneally single injected into C57BL/6 mice to induce diabetes. Alloxan-induced diabetic mice were orally given the water extract of inonotus obliquus (WEIO) at doses of 500 mg/kg and subcutaneously administered with insulatard 0.6 IU for 4weeks. Subsequently, the levels of factors related to blood glucose, insulin and lipids were assessed. WEIO significantly restored body weight, reduced fasting blood glucose levels, improved glucose tolerance ability, and increased insulin levels compared to the control diabetic mice. WEIO also enhanced cholesterol transportation in the liver, correlated with increased HDL-C levels and decreased TC, TG and LDL-C levels. Histologic examinations revealed that WEIO alleviated alloxan-induced pancreatic tissue damage. Natural Mongolian I. Obliquus water extracts have an antihyperglycemic effect and enhance islet cell function in alloxan-induced diabetic mice. It is a good candidate for a hypoglycemic functional food or medicine.