Diabetic Wild-type Mice Research Articles

Endothelial eNOS deficiency causes podocyte injury through NFAT2 and heparanase in diabetic mice

The pivotal role of endothelial nitric oxide synthase (eNOS) in diabetic nephropathy (DN) has been demonstrated using global eNOS knockout (eNOSGKO) mice. However, the precise role of endothelially expressed eNOS and how its deficiency advances DN are still unclear. Here, we targeted endothelial eNOS expression (E-eNOSKO) after the onset of diabetes using the floxed eNOS and endSCL-CreERT alleles. Diabetes was induced by low-dose streptozotocin injections. To evaluate the role of nuclear factor of activated T cells-2 (NFAT2) in podocyte injury in this condition, podocyte-specific NFAT2KO mice were also generated on eNOSGKO mice. The mechanisms of podocyte injury were investigated using cultured podocytes. Compared with diabetic wild-type mice, diabetic E-eNOSKO mice showed more advanced DN accompanied by NFAT2 expression in podocytes. NO donor suppressed NFAT2 expression and activation in high-glucose cultured podocytes as well as in diabetic E-eNOSKO mice. Furthermore, podocyte-specific deletion of NFAT2 attenuated DN in diabetic eNOSGKO mice accompanied by decreased heparanase (HPSE) expression in podocytes. Consistent with this finding, HPSE was upregulated by NFAT2 transfection and suppressed by NFAT2 siRNA or NO donor treatment in cultured podocytes. HPSE transfection reduced podocyte attachment to extracellular matrix concurrent with syndecan-4 (SDC4) shedding, and this effect was attenuated by co-transfection of SDC4. Finally, HPSE inhibitor treatment attenuated podocyte injury in diabetic E-eNOSKO mice with increased SDC4 expression in podocytes. Collectively, our data suggest that endothelial eNOS deficiency causes podocyte HPSE expression in diabetic mice through NFAT2, and HPSE promotes podocyte detachment in part through SDC4 shedding, advancing DN.

PPARβ/δ prevents inflammation and fibrosis during diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is a specific type of myocardial disease that often develops in patients suffering from diabetes, which has become the foremost cause of death among them. It is an insidious multifactorial disease caused by complex and partially unknown mechanisms that include metabolic dysregulation, local inflammation, fibrosis, and cardiomyocyte apoptosis. Despite its severity and poor prognosis, it often goes undiagnosed, and there are currently no approved specific drugs to prevent or even treat it. Peroxisome proliferator-activated receptor (PPAR)β/δ is a key metabolic regulator that has been proposed as a potential target for DCM due to its pleiotropic anti-inflammatory properties. Diabetes was induced by multiple low-dose streptozotocin (STZ) administration in wild-type and PPARβ/δ knockout male mice treated with the PPARβ/δ agonist GW0742 or vehicle. Human cardiomyocytes (AC16) and mouse atrial myocytes (HL-1) exposed to hyperglycemia and treated with PPARβ/δ agonists were also used. PPARβ/δ deletion in mice negatively impacted cardiac morphology and function, which was accompanied by interstitial fibrosis and structural remodeling of the heart. This phenotype was further exacerbated in knockout diabetic mice. At the molecular level, PPARβ/δ suppression resulted in increased expression of pro-inflammatory and pro-fibrotic markers. Some of these markers were also induced by diabetes in wild-type mice and were exacerbated in diabetic knockout mice. The activity of the transcription factors nuclear factor κB (NF-κB) and activator protein-1 (AP-1) correlated with most of these changes. Remarkably, PPARβ/δ activation partially prevented inflammation and fibrosis in the heart, as well as cardiac atrophy, induced during diabetes in mice, and also in cultured cardiomyocytes exposed to hyperglycemia. Finally, our results suggest that the beneficial effects of PPARβ/δ activation are mediated by the inhibition of mitogen-activated protein kinases (MAPK) activity and subsequent downregulation of the transcriptional activities of NF-κB and AP-1. Overall, the data suggest that PPARβ/δ agonists might be useful in preventing inflammation and fibrosis progression in DCM.

Early expression of osteopontin glycoprotein on the ocular surface and in tear fluid contributes to ocular surface diseases in type 2 diabetic mice.

Osteopontin (OPN) is a glycosylated, secreted phosphoprotein known to be elevated in both human and mouse retinas during various stages of diabetic retinopathy. However, its specific roles in modulating ocular surface dynamics and immune responses in diabetes remain unexplored. This study aims to investigate the role of OPN in the development of ocular surface disease (OSD) in type 2 diabetic (T2D) mice. Three- to four-week-old C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice were fed a high-fat diet (HFD) and were rendered diabetic by streptozotocin (STZ; 40 mg/kg body weight) in citrate buffer (vehicle); non-diabetic controls were injected with vehicle alone. Diabetes was confirmed if blood glucose levels were >200 mg/dL, measured 1-2 weeks post-STZ injection. Control, age- and sex-matched db/db diabetic mice fed a standard chow diet were also included in this study. Ocular surface inflammation was assessed using ELISA to quantify inflammatory cytokine proteins and wheat germ agglutinin (WGA) staining was utilized to highlight corneal surface irregularities. Clinical signs were evaluated by corneal fluorescein staining, tear production measurements, and tear sodium (Na+) concentration assessments. These evaluations were conducted 4, 6, 8 and 16-weeks post-diabetes onset in WT and OPN-/- mice and were compared to those obtained in non-diabetic controls. Statistical analysis was performed using a two-way ANOVA, with significance set at P < 0.05. Both WT and OPN-/- mice developed T2D within 4 and 8 weeks, respectively, following HFD + STZ treatment. Corneal OPN levels in WT diabetic mice increased ~2-fold at 2 weeks and ~4-fold at 16 weeks compared to non-diabetic controls, with similar elevations observed in their tear fluid. Diabetic db/db mice also exhibited elevated OPN levels in the blood and ocular surface, which persisted as diabetes progressed. Enhanced fluorescein staining, indicating corneal irregularities, appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. Additionally, WGA staining showed a significant reduction in fluorescence intensity in WT mice treated with HFD and STZ, confirming corneal surface irregularities that were delayed in OPN-/- mice. Elevated tear sodium concentration was observed in both WT and OPN-/- diabetic mice without affecting tear production rates. Notably, OPN levels increased early, at week 2, following HFD and STZ treatment, preceding changes in interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP-9). Upregulation of IL-6 became apparent at 6 weeks in WT mice and was delayed until 10 weeks in OPN-/- mice post-T2D induction. Elevated OPN levels were detected early post-T2D induction in diabetic WT and db/db mice corneas without initial subclinical changes. This early increase in OPN precedes other proinflammatory cytokines associated with eventual ocular surface inflammation as diabetes progresses. Persistence of OPN also correlated with clinical signs such as increased corneal surface irregularities and elevated tear Na+ concentration. Future research will explore OPN's role as a biomarker in ocular surface disease (OSD), including dry eye disease (DED), and investigate its impact on inflammatory processes and other mechanistic pathways in diabetic ocular complications.

Chronic hyperglycemia aggravates lung function in a Scnn1b-Tg murine model.

Cystic fibrosis-related diabetes (CFRD), the most common comorbidity in cystic fibrosis (CF), leads to increased mortality by accelerating the decline in lung function. Scnn1b-Tg transgenic mice overexpressing the epithelial sodium channel β subunit exhibit spontaneous CF-like lung disease, including airway mucus obstruction and chronic inflammation. Here, we established a chronic CFRD-like model using Scnn1b-Tg mice made diabetic by injection of streptozotocin (STZ). In Ussing chamber recordings of the trachea, Scnn1b-Tg mice exhibited larger amiloride-sensitive currents and forskolin-activated currents, without a difference in adenosine triphosphate (ATP)-activated currents compared with wild-type (WT) littermates. Both diabetic WT (WT-D) and diabetic Scnn1b-Tg (Scnn1b-Tg-D) mice on the same genetic background exhibited substantially elevated blood glucose at 8 wk; glucose levels also were elevated in bronchoalveolar lavage fluid (BALF). Bulk lung RNA-seq data showed significant differences between WT-D and Scnn1b-Tg-D mice. Neutrophil counts in BALF were substantially increased in Scnn1b-Tg-D lungs compared with controls (Scnn1b-Tg-con) and compared with WT-D lungs. Lung histology data showed enhanced parenchymal destruction, alveolar wall thickening, and neutrophilic infiltration in Scnn1b-Tg-D mice compared with WT-D mice, consistent with the development of a spontaneous lung infection. We intranasally administered Pseudomonas aeruginosa to induce lung infection in these mice for 24 h, which led to severe lung leukocytic infiltration and an increase in pro-inflammatory cytokine levels in the BALF. In summary, we established a chronic CFRD-like lung mouse model using the Scnn1b-Tg mice. The model can be used for future studies toward understanding the mechanisms underlying the lung pathophysiology associated with CFRD and developing novel therapeutics.NEW & NOTEWORTHY We established a chronic CFRD-like mouse model using the Scnn1b-Tg transgenic mice overexpressing the epithelial sodium channel β subunit made diabetic by injection of streptozotocin. The results underscore the urgent need to develop novel therapeutics for CF lung disease.

ENAMPT is a novel therapeutic target for mitigation of coronary microvascular disease in type 2 diabetes

Aims/hypothesisIndividuals with diabetes are at high risk of cardiovascular complications, which significantly increase morbidity/mortality. Coronary microvascular disease (CMD) is recognised as a critical contributor to the increased cardiac mortality observed in people with diabetes. Therefore, there is an urgent need for treatments that are specific to CMD. eNAMPT (extracellular nicotinamide phosphoribosyltransferase) is a damage-associated molecular pattern and TLR4 ligand, whose plasma levels are elevated in people with diabetes. This study was thus designed to investigate the pathogenic role of intracellular nicotinamide phosphoribosyltransferase (iNAMPT) and eNAMPT in promoting the development of CMD in a preclinical murine model of type 2 diabetes.MethodsAn inducible type 2 diabetic mouse model was generated by a single injection of low-dose streptozocin (75 mg/kg, i.p.) combined with a high-fat diet for 16 weeks. The in vivo effects of i/eNAMPT inhibition on cardiac endothelial cell (CEC) function were evaluated by using Nampt+/− heterozygous mice, chronic administration of eNAMPT-neutralising monoclonal antibody (mAb) or use of an NAMPT enzymatic inhibitor (FK866).ResultsAs expected, diabetic wild-type mice exhibited significantly lower coronary flow velocity reserve (CFVR), a determinant of coronary microvascular function, compared with control wild-type mice. eNAMPT plasma levels or expression in CECs were significantly greater in diabetic mice than in control mice. Furthermore, in comparison with diabetic wild-type mice, diabetic Nampt+/− heterozygous mice showed markedly improved CFVR, accompanied by increased left ventricular capillary density and augmented endothelium-dependent relaxation (EDR) in the coronary artery. NAMPT inhibition by FK866 or an eNAMPT-neutralising mAb significantly increased CFVR in diabetic mice. Furthermore, administration of the eNAMPT mAb upregulated expression of angiogenesis- and EDR-related genes in CECs from diabetic mice. Treatment with either eNAMPT or NAD+ significantly decreased CEC migration and reduced EDR in coronary arteries, partly linked to increased production of mitochondrial reactive oxygen species.Conclusions/interpretationThese data indicate that increased i/eNAMPT expression contributes to the development of diabetic coronary microvascular dysfunction, and provide compelling support for eNAMPT inhibition as a novel and effective therapeutic strategy for CMD in diabetes.Graphical

Melatonin attenuates diabetic cardiomyopathy by increasing autophagy of cardiomyocytes via regulation of VEGF-B/GRP78/PERK signaling pathway

AimsDiabetic cardiomyopathy (DCM) is a major cause of mortality in patients with diabetes, and the potential strategies for treating DCM are insufficient. Melatonin (Mel) has been shown to attenuate DCM, however, the underlying mechanism remains unclear. The role of vascular endothelial growth factor-B (VEGF-B) in DCM is little known. In present study, we aimed to investigate whether Mel alleviated DCM via regulation of VEGF-B and explored its underlying mechanisms.Methods and resultsWe found that Mel significantly alleviated cardiac dysfunction and improved autophagy of cardiomyocytes in type 1 diabetes mellitus (T1DM) induced cardiomyopathy mice. VEGF-B was highly expressed in DCM mice in comparison with normal mice, and its expression was markedly reduced after Mel treatment. Mel treatment diminished the interaction of VEGF-B and Glucose-regulated protein 78 (GRP78) and reduced the interaction of GRP78 and protein kinase RNA -like ER kinase (PERK). Furthermore, Mel increased phosphorylation of PERK and eIF2α, then up-regulated the expression of ATF4. VEGF-B−/− mice imitated the effect of Mel on wild type diabetic mice. Interestingly, injection with Recombinant adeno-associated virus serotype 9 (AAV9)-VEGF-B or administration of GSK2656157 (GSK), an inhibitor of phosphorylated PERK abolished the protective effect of Mel on DCM. Furthermore, rapamycin, an autophagy agonist displayed similar effect with Mel treatment; while 3-Methyladenine (3-MA), an autophagy inhibitor neutralized the effect of Mel on high glucose-treated neonatal rat ventricular myocytes.ConclusionsThese results demonstrated that Mel attenuated DCM via increasing autophagy of cardiomyocytes, and this cardio-protective effect of Mel was dependent on VEGF-B/GRP78/PERK signaling pathway.

MASP-2 deficiency does not prevent the progression of diabetic kidney disease in a mouse model of type 1 diabetes.

Mannan-binding lectin (MBL) initiates the lectin pathway of complement and has been linked to albuminuria and mortality in diabetes. We hypothesize that MBL-associated serine protease 2 (MASP-2) deficiency will protect against diabetes-induced kidney damage. Male C57BL/6J MASP-2 knockout (Masp2-/-) mice and wildtype (WT) mice were divided into a diabetic group and a non-diabetic group. Renal hypertrophy, albumin excretion, mesangial area and specific mRNA expressions in the renal cortex were measured after 8 and 12 weeks of diabetes. By two-way ANOVA it was tested if MASP-2 modulated the renal effects of diabetes, that is interaction. After 12 weeks of diabetes Masp2-/- diabetic mice had a smaller mesangium at 21.1% of the glomerular area (95% CI 19.7, 22.6) compared with WT diabetic mice, 25.2% (23.2, 27.2), p(interaction) = 0.001. After 8 weeks of diabetes, plasma cystatin C was 261.5 ng/mL (229.6, 297.8) in the WT diabetic group compared to 459.9 ng/mL (385.7, 548.3) in non-diabetic WT mice, p < 0.001. By contrast, no difference in plasma cystatin C levels was found between the Masp2-/- diabetic mice, 288.2 ng/mL (260.6, 318.6) and Masp2-/- non-diabetic mice, 293.5 ng/mL (221.0, 389.7), p = 0.86 and p(interaction) = 0.001. We demonstrated a protective effect of MASP-2 deficiency on mesangial hypertrophy after 12 weeks of diabetes and an effect on plasma cystatin C level. MASP-2 deficiency did, however, fail to protect against diabetic-induced alterations of kidney weight, albuminuria and renal mRNA expression of fibrotic- and oxidative stress markers.

Diabetic Müller-Glial-Cell-Specific Il6ra Knockout Mice Exhibit Accelerated Retinal Functional Decline and Thinning of the Inner Nuclear Layer.

Interleukin-6 (IL-6) is implicated in the pathology of diabetic retinopathy (DR). IL-6 trans-signaling via soluble IL-6 receptor (IL-6R) is primarily responsible for its pro-inflammatory functions, whereas cis-signaling via membrane-bound IL-6R is anti-inflammatory. Using a Müller-glial-cell-specific Il6ra-/- mouse, we examined how loss of IL-6 cis-signaling in Müller glial cells (MGCs) affected retinal thinning and electroretinography (ERG) response over 9months of diabetes. Diabetes was induced in wildtype and knockout mice with streptozotocin (40mg/kg, daily for 5days). Spectral domain optical coherence tomography (SD-OCT), ERG, and fundoscopy/fluorescein angiography (FA) were assessed at 2, 6, and 9months of diabetes. MGCs and bipolar neurons were examined in retinal tissue sections by immunofluorescence. Diabetic MGC Il6ra-/- mice had significantly thinner retinas than diabetic wildtype mice at 2 (-7.6µm), 6 (-12.0µm), and 9months (-5.0µm) of diabetes, as well as significant thinning of the inner nuclear layer (INL). Diabetic MGC Il6ra-/- mice also showed a reduction in scotopic B-wave amplitude and B-wave/A-wave ratio earlier than wildtype diabetic mice. In retinal sections, we found a decrease in bipolar neuronal marker PKCα only in diabetic MGC Il6ra-/- mice, which was significantly lower than both controls and diabetic wildtype mice. Glutamine synthetase, a Müller cell marker, was reduced in both wildtype and MGC Il6ra-/- diabetic mice compared to their respective controls. IL-6 cis-signaling in MGCs contributes to maintenance of the INL in diabetes, and loss of the IL-6 receptor reduces MGC-mediated neuroprotection of bipolar neurons in the diabetic retina.

Abstract 11795: CXCL5 Inhibition Recovers Neovascularization in Diabetes Mellitus

Introduction: Diabetes mellitus (DM) is a chronic inflammatory disease accompanied by dysfunction of vascular endothelial cells. Vascular complications are the main causes of death in patients with DM. CXCL5 is an inflammatory chemokine that upregulated in patients with type 2 DM. However, its role in diabetic vasculopathy was not clarified. This study aimed to explore the impacts and mechanistic insights of CXCL5 in neovasculogenesis in DM. Methods: Endothelial progenitor cells (EPCs) and human aortic endothelial cells (HAECs) were used in vitro. Streptozotocin-induced diabetic mice and dbdb mice were used as type 1 and type 2 DM models. CXCL5 knockout mice were also used to generate the diabetic animal model. The hindlimb ischemia surgery, matrigel plug assay, and aortic ring assay were conducted. Flow cytometry were used to evaluate the circulating number of EPCs. Results: CXCL5 concentrations were increased in plasma and EPCs culture medium from type 2 DM patients. CXCL5 inhibition by neutralizing antibody upregulated VEGF/SDF-1 expression and improved migration and tube formation abilities in EPCs from type 2 DM patients and high glucose-treated EPCs from non-diabetic subjects as well as HAECs. Intraperitoneal injection of CXCL5 neutralizing antibody increased circulating EPC number, enhanced capillary density, and recovered the blood flow after hindlimb ischemia surgery in both type 1 and type 2 diabetic animals. Protein levels of VEGF and SDF-1 in ischemic muscle were upregulated in the CXCL5 inhibition group. Additionally, diabetic mice injected with CXCL5 neutralizing antibody showed higher hemoglobin contents and neovascular branches in the matrigel plug assay and aortic ring assay, respectively. Importantly, CXCL5 knockout diabetic mice showed improved ischemia-induced angiogenesis and higher expression of angiogenic proteins in ischemic muscle compared with wild-type diabetic mice. Conclusion: CXCL5 inhibition can improve neovasculogenesis via protecting vascular endothelial function and upregulating angiogenic protein expressions in both type 1 and type 2 DM. Further clinical studies are needed to verify that CXCL5 may be a potential therapeutic target for DM vascular complications.

Abstract 11344: Targeted Deletion of Arrdc4 Mitigates Cardiac Injury in Insulin-Deficient Diabetes

Introduction: Diabetic cardiomyopathy is an important contributor to mortality in the diabetic population. However, the pathogenesis of diabetic cardiomyopathy is unclear. Hypothesis: We recently reported that the interaction between glucose transporter 1 (GLUT1) and its adaptor protein Arrdc4 mediates metabolic stress in the ischemic heart. Here, we tested whether this mechanism plays a role in diabetic cardiomyopathy. Methods: To determine glucose responsiveness, mouse neonatal cardiomyocytes were treated with high glucose (25 mM). To investigate cardiac phenotype in vivo, we engineered Arrdc4-knockout (KO) mice by CRISPR/Cas9 technology and created an insulin-deficient diabetic model by intraperitoneal injection of streptozotocin. Results: Arrdc4 expression was robustly stimulated by D-glucose (324±10%, p&lt;0.01) in cardiomyocytes. The glucose responsiveness was mediated by MondoA, a nutrient-sensing transcriptional factor, through a cytoplasmic-nuclear shuttling mechanism. Confocal live imaging showed that Arrdc4 triggered endocytic trafficking of GLUT1 into lysosomes. In the animal model, levels of insulin depletion were comparable between the genotypes. Nevertheless, Arrdc4-KO diabetic mice had enhanced glucose uptake in multiple organs including the heart (139±13%, p&lt;0.01) compared to wild-type (WT) diabetic mice. Arrdc4-KO diabetic hearts had lower induction of oxidative stress, ER stress, and autophagy by hyperglycemia, leading to better mitochondrial function and less cardiomyocyte apoptosis (70±5%, p&lt;0.01) than WT diabetic hearts. Although resting echocardiography could not detect early signs of left ventricular dysfunction, stress hemodynamic analysis uncovered the masked mechanical dysfunction by diabetes and found that Arrdc4-KO diabetic mice retained a greater cardiac response to beta-adrenergic stimulation than wild-type mice (149±5%, p&lt;0.01). Conclusion: The metabolic rearrangements by Arrdc4 deletion improve glucose transport due to the absence of its inhibition towards GLUT1 function, protecting the heart against diabetic damage. These results provide a novel link between GLUT1 and Arrdc4 as a fundamental mechanism of the regulation of glucose homeostasis in diabetic cardiomyopathy.

Interaction of Fabry Disease and Diabetes Mellitus: Suboptimal Recruitment of Kidney Protective Factors.

Fabry disease is a lysosomal disease characterized by globotriaosylceramide (Gb3) accumulation. It may coexist with diabetes mellitus and both cause potentially lethal kidney end-organ damage. However, there is little information on their interaction with kidney disease. We have addressed the interaction between Fabry disease and diabetes in data mining of human kidney transcriptomics databases and in Fabry (Gla-/-) and wild type mice with or without streptozotocin-induced diabetes. Data mining was consistent with differential expression of genes encoding enzymes from the Gb3 metabolic pathway in human diabetic kidney disease, including upregulation of UGCG, the gene encoding the upstream and rate-limiting enzyme glucosyl ceramide synthase. Diabetic Fabry mice displayed the most severe kidney infiltration by F4/80+ macrophages, and a lower kidney expression of kidney protective genes (Pgc1α and Tfeb) than diabetic wild type mice, without a further increase in kidney fibrosis. Moreover, only diabetic Fabry mice developed kidney insufficiency and these mice with kidney insufficiency had a high expression of Ugcg. In conclusion, we found evidence of interaction between diabetes and Fabry disease that may increase the severity of the kidney phenotype through modulation of the Gb3 synthesis pathway and downregulation of kidney protective genes.

Abstract P3197: Sarm1 Deficiency Protects Mice Against Diabetic Cardiomyopathy: Potential Mechanisms Via Ameliorating Lipotoxicity

Heart failure occurs at nearly twice the rate in diabetic patients as compared to normal patients. NAD levels are decreased in human and murine heart disease models including diabetic hearts. Strategies to raise NAD in these models have primarily focused on increasing NAD synthesis. Alternatively, inhibiting NAD consumption provides another viable path to increasing NAD levels in diabetic hearts. Sterile Alpha and Tir Motif Containing 1 (SARM1) is an NAD hydrolase that mediates axonal degeneration through NAD degradation and promotion of mitochondrial dysfunction. However, the role SARM1 plays in heart disease has not been investigated. We subjected male wild type (WT) and global SARM1 knockout mice (KO) to chronic diabetic stress induced by streptozotocin injections. We showed that 16-week diabetic stress promoted progressive decline in systolic and diastolic function as measured by longitudinal echocardiography. SARM1 deletion (diabetic KO) improved systolic and diastolic function of diabetic mice, despite similar glucose, and plasma aqueous and lipid metabolite levels as the diabetic wild-type mice. Diabetic KO hearts showed increased NAD levels, suggesting that SARM1 may be activated to promote NAD decline in diabetic hearts. Transcriptomic analysis identified 1948 differentially expressed genes in diabetic WT hearts, compared to non-diabetic WT hearts. Gene Module Network Analysis identified upregulation of fatty acid metabolic processes and suppression of genes involved in mitochondrial processes in diabetic hearts. We measured mitochondrial oxygen consumption rate and found suppressed mitochondrial respiration in diabetic WT hearts. SARM1 deficiency reversed the upregulated fatty acid metabolic genes, improved mitochondrial respiration, and elevated NAD levels in diabetic hearts. Genes such as CD36, HADHA, Acot2, and malonyl-CoA are all downregulated by SARM1 deficiency, indicating that SARM1 deficiency may relieve lipotoxicity by improving mitochondrial function and modifying metabolic transcriptional networks in diabetic hearts. Future analysis will shed light on how SARM1 contributes to lipotoxicity as a new pathogenic mechanism of diabetic cardiomyopathy.

P2X7R/AKT/mTOR signaling mediates high glucose-induced decrease in podocyte autophagy.

Diabetic nephropathy is one of the leading causes of end-stage renal disease worldwide. In our study we found that Adenosine triphosphate (ATP) content was significantly increased in the urine of diabetic mice. We examined the expression of all purinergic receptors in the renal cortex and found that only purinergic P2X7 receptor (P2X7R) expression was significantly increased in the renal cortex of wild-type diabetic mice and that the P2X7R protein partially co-localized with podocytes. Compared with P2X7R(-/-) non-diabetic mice, P2X7R(-/-) diabetic mice showed stable expression of the podocyte marker protein podocin in the renal cortex. The renal expression of microtubule associated protein light chain 3 (LC-3II) in wild-type diabetic mice was significantly lower than in wild-type controls, whereas the expression of LC-3II in the kidneys of P2X7R(-/-) diabetic mice was not significantly different from that of P2X7R(-/-) non-diabetic mice. In vitro, high glucose induced an increase in p-Akt/Akt, p-mTOR/mTOR and p62 protein expression along with a decrease in LC-3II levels in podocytes, whereas after transfection with P2X7R siRNA, Phosphorylated protein kinase B (p-Akt)/Akt, Phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, and p62 expression were restored and LC-3II expression was increased. In addition, LC-3II expression was also restored after inhibition of Akt and mTOR signaling with MK2206 and rapamycin, respectively. Our results suggest that P2X7R expression is increased in podocytes in diabetes, and that P2X7R is involved in the inhibition of podocyte autophagy by high glucose, at least in part through the Akt-mTOR pathway, thereby exacerbating podocyte damage and promoting the onset of diabetic nephropathy. Targeting P2X7R may be a potential treatment for diabetic nephropathy.

#3566 ACSS2 AGGRAVATES RENAL TUBULAR INFLAMMATION BY INDUCING THE DYSREGULATION OF FATTY ACID METABOLISM AND MITOCHONDRIAL OXIDATIVE STRESS IN DIABETIC MICE

Abstract Background and Aims Treatment options for diabetic nephropathy (DN) are currently limited. Chronic renal inflammation has been widely recognized as a major promoter of DN. The tubular epithelial cells are a major source of IL-1β in diabetic db/db mice. Inhibition of Acetyl CoA Synthase 2 (ACSS2) has been shown to promote high-fat diet-induced liver injuries, but the underlying mechanism of action of ACSS2 in diabetic renal injuries has not been elucidated. Method Streptozotocin (STZ) was intraperitoneally injected to establish the diabetic mouse model. We investigated the effects of ACSS2 on diabetes-induced renal tubular injury in transgenic mice with global acss2 knockout (acss2−/−). PAS staining was carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. HK-2 cell culture model was achieved by high glucose stimulation in vitro. Real-time PCR analysis was applied to evaluate gene expression of inflammatory factors IL-1β, TNF-α, and MCP-1. Western Blot and immunofluorescence staining were conducted to the activation of the NLRP3 pathway and mitochondrial reactive oxygen species (ROS). Results ACSS2 was upregulated in STZ-induced diabetic renal tubular cells, which is significantly co-expressed in IL-1β-positive renal tubular cells of STZ-induced diabetic mice. The mice with acss2−/- exhibited decreased renal tubular inflammation and inhibited NOD-like receptor protein 3 (NLRP3) inflammasome activation as evidenced by elevated protein levels of NLRP3 and cleaved caspase-1 when compared with diabetic wild-type mice. The selective inhibition of ACSS2 protected HK-2 cells in vitro when exposed to a high glucose environment by downregulating NLRP3 inflammasome and ameliorating mitochondrial oxidative stress. Mechanistically, we showed that mitochondrial dysfunction preceded inflammatory response in vitro. The elimination of mitochondrial ROS using mitochondria-targeted antioxidants (mito-TEMPO) reversed HG-mediated NLRP3 inflammation activation in HK-2 cells, which recapitalized the phenotype observed in acss2-/- mice. We also found that cytoplasm ACSS2, which was increased after HG stimulation, induced fatty acid synthase (FASN) mediated overproduction of free fatty acid. Downregulating FASN using FASN-small interfering RNA restored mitochondrial function, and suppressed NLRP3 inflammasome activation. Conclusion In conclusion, diabetes-induced ACSS2 upregulation led to subsequent FASN-mediated dysregulation of fatty acid metabolism and mitochondrial ROS overproduction, eventually causing NLRP3 inflammasome activation and renal inflammation. Therefore, our findings established the role of ACSS2 in diabetes-induced inflammation of renal tubular epithelium and unveiled the underlying mechanism of its action. ACSS2 appears as a promising target for treating diabetic renal tubular epithelial inflammation.

CD93 Ameliorates Diabetic Wounds by Promoting Angiogenesis via the p38MAPK/MK2/HSP27 Axis

Diabetic wounds are a complication of diabetes mellitus, which is characterised by microcirculation dysfunction caused by decreased local blood supply and insufficient metabolic exchange. Clinically, in addition to glycaemic control, the most important treatment for diabetic wounds is to promote local angiogenesis, which accelerates wound healing. The authors previous study demonstrated that CD93, which is specifically expressed on vascular endothelial cells (ECs), redundantly regulates angiogenesis in zebrafish, suggesting that CD93 is a potential angiogenic molecule. However, the role of CD93 in diabetic wounds has not yet been elucidated. The angiogenic effects of CD93 were studied from four aspects: exogenous, endogenous, invitro, and invivo. CD93 recombinant protein was used in microvascular ECs and in mice to observe angiogenesis invitro and invivo. The wound model was established in CD93-/- and wild type diabetic mice, and the degree of wound healing as well as the amount and maturity of neovascularisation were investigated. The possible mechanism of CD93 in angiogenesis was determined by CD93 overexpression in cultured ECs. CD93 recombinant protein was found to exogenously promote tube formation and sprouting of ECs. It also recruited cells to promote the formation of vascular like structures in subcutaneous tissue and accelerated wound healing by optimising angiogenesis and re-epithelisation. Furthermore, CD93 deficiency was observed to delay wound repair, characterised by reduced neovascularisation, vascular maturity, and re-epithelisation level. Mechanically, CD93 activated the p38MAPK/MK2/HSP27 signalling pathway, positively affecting the angiogenic functions of ECs. This study demonstrated that CD93 promotes angiogenesis both invitro and invivo and that its angiogenic role invitro is mediated by the p38MAPK/MK2/HSP27 signalling pathway. It was also found that CD93 exerts beneficial effects on wound healing in diabetic mice by promoting angiogenesis and re-epithelisation.

Deletion of the stress response protein REDD1 prevents impaired cardiac function in diabetic mice

The overwhelming impact of heart failure on morbidity and mortality in obesity and type 2 diabetes requires a deeper understanding of the molecular events that mediate cardiac dysfunction. We recently demonstrated using in vivo and in vitro models of diabetes that upregulation of endoplasmic reticulum stress in cardiomyocytes promoted expression of the stress response protein regulated in development and DNA damage 1 (REDD1). REDD1 has been implicated in diabetes-induced functional deficits in both the retina and kidney; however, a role for REDD1 in diabetes-induced cardiac dysfunction has not been investigated. Thus, we explored the hypothesis that REDD1 contributes to the development of diabetes-induced cardiac dysfunction. To induce diabetes, 6-week old male wild-type and REDD1 knock-out (KO) B6;129 mice were fed a high-fat high-sugar diet (42% kcal fat, 34% sucrose by weight) for 16 weeks; 2 weeks after initiation of the diet, mice were administered low-dose streptozotocin (STZ) for five consecutive days. Non-diabetic control mice were fed a standard rodent diet and received vehicle injections. Mice with a fasting blood glucose level &gt;250mg/dL were defined as diabetic. At 10 weeks of age, transthoracic echocardiogram was evaluated every 2 weeks using a GE Logique small animal echocardiography machine. After 12 weeks of diabetes, left ventricular ejection fraction (EF) was reduced in diabetic wild-type mice as compared to non-diabetic wild-type mice (51.0 +/-9.8% versus 39.5 +/- 5.5%, p = 0.015). Meanwhile, EF in diabetic REDD1 KO mice (47.4 +/- 6.6%) was similar to that observed in non-diabetic REDD1 KO mice (49.6 +/- 6.7%). To investigate a potential cause of the preserved EF in diabetic REDD1 KO mice, tissue homogenates from the left ventricle were examined by PCR analysis. REDD1 mRNA expression in diabetic wild-type mice was enhanced as compared to non-diabetic controls in coordination with increased expression of Tgfb1, as well as expression of the pro-fibrotic genes Fn1, Col1a1, and Col3a1. By contrast, there was no increase in pro-fibrotic mRNA expression in cardiac tissue homogenates from diabetic REDD1 KO mice, as compared to non-diabetic REDD1 KO mice. Overall, the data support a role for REDD1 expression in the development of impaired cardiac function and the pro-fibrotic response of the heart to diabetes. This is the full abstract presented at the American Physiology Summit 2023 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.

BMF-AS1/BMF Promotes Diabetic Vascular Calcification and Aging both In Vitro and In Vivo.

Vascular calcification and aging often increase morbidity and mortality in patients with diabetes mellitus (DM); however, the underlying mechanisms are still unknown. In the present study, we found that Bcl-2 modifying factor (BMF) and BMF antisense RNA 1 (BMF-AS1) were significantly increased in high glucose-induced calcified and senescent vascular smooth muscle cells (VSMCs) as well as artery tissues from diabetic mice. Inhibition of BMF-AS1 and BMF reduced the calcification and senescence of VSMCs, whereas overexpression of BMF-AS1 and BMF generates the opposite results. Mechanistic analysis showed that BMF-AS1 interacted with BMF directly and up-regulated BMF at both mRNA and protein levels, but BMF did not affect the expression of BMF-AS1. Moreover, knocking down BMF-AS1 and BMF suppressed the calcification and senescence of VSMCs, and BMF knockout (BMF-/-) diabetic mice presented less vascular calcification and aging compared with wild type diabetic mice. In addition, higher coronary artery calcification scores (CACs) and increased plasma BMF concentration were found in patients with DM, and there was a positive correlation between CACs and plasma BMF concentration. Thus, BMF-AS1/BMF plays a key role in promoting high glucose-induced vascular calcification and aging both in vitro and in vivo. BMF-AS1 and BMF represent potential therapeutic targets in diabetic vascular calcification and aging.

Tripartite motif containing 23 functions as a critical regulator in macrophages to control the pathological feature of diabetic nephropathy.

Diabetic nephropathy (DN) is a kidney disease resulting from diabetes. Macrophages and macrophage-mediated inflammation contributed to the development of DN. Tripartite motif containing 23 (TRIM23) is E3 ligase and has been involved in inflammation. Until now, the precise roles of TRIM23 in DN are not described yet. Therefore, we evaluated the functions of TRIM23 in DN. We generated mice with TRIM23 deficiency in macrophages. We also established diabetic mice model. The expression of TRIM23 was measured in diabetic animals. The DN symptoms were compared between diabetic wild-type (WT) mice and TRIM23 conditional knock out mice. The cytokine expression, ubiquitination of TAB2, and interactions between TAB2 and IKK were compared in oxidized low-density lipoprotein (oxLDL) or lipopolysaccharides (LPS)-treated WT and TRIM23-deficient macrophages. Upregulation of TRIM23 was observed in diabetic mice and LPS or oxLDL-treated macrophages. Diabetic mice with TRIM23 deficiency in macrophages had attenuated DN symptoms. TRIM23-deficient macrophages had decreased pro-inflammatory cytokines production after oxLDL or LPS stimulation. TRIM23 was predicted to interact with TAB2. The ubiquitination of TAB2 was abolished in oxLDL-treated TRIM23-deficient macrophages, which correlated with decreased activation of IKK. TRIM23 regulates inflammation in macrophages and plays important role in DN.

Transforming Growth Factorβ1 Overexpression Is Associated with Insulin Resistance and Rapidly Progressive Kidney Fibrosis under Diabetic Conditions.

Diabetes mellitus is a global health problem. Diabetic nephropathy is a common complication of diabetes mellitus and the leading cause of end-stage renal disease. The clinical course, response to therapy, and prognosis of nephropathy are worse in diabetic than in non-diabetic patients. The role of transforming growth factorβ1 in kidney fibrosis is undebatable. This study assessed whether the overexpression of transforming growth factorβ1 is associated with insulin resistance and the rapid progression of transforming growth factorβ1-mediated nephropathy under diabetic conditions. Diabetes mellitus was induced with streptozotocin in wild-type mice and transgenic mice with the kidney-specific overexpression of human transforming growth factorβ1. Mice treated with saline were the controls. Glucose tolerance and kidney fibrosis were evaluated. The blood glucose levels, the values of the homeostasis model assessment for insulin resistance, and the area of kidney fibrosis were significantly increased, and the renal function was significantly impaired in the diabetic transforming growth factorβ1 transgenic mice compared to the non-diabetic transgenic mice, diabetic wild-type mice, and non-diabetic mice. Transforming growth factorβ1 impaired the regulatory effect of insulin on glucose in the hepatocyte and skeletal muscle cell lines. This study shows that transforming growth factorβ1 overexpression is associated with insulin resistance and rapidly progressive kidney fibrosis under diabetic conditions in mice.

Hepatic cytokine-inducible SH2-containing protein (CISH) regulates gluconeogenesis via cAMP-responsive element binding protein (CREB).

Impairment of gluconeogenesis is a key factor responsible for hyperglycemia in patients with type 2 diabetes. As an important member of the suppressors of cytokine signaling (SOCS) protein family, many physiological functions of cytokine-inducible SH2-containing protein (CISH) have been described; however, the role of hepatic CISH in gluconeogenesis is poorly understood. In the present study, we observed that hepatic CISH expression was reduced in fasted wild-type (WT) mice. Overexpression of CISH decreased glucose production in mouse primary hepatocytes, while silencing of CISH had the opposite effects. In addition, adenovirus-mediated hepatic CISH overexpression resulted in improved glucose tolerance and decreased gluconeogenesis in WT and leptin receptor-deficient diabetic (db/db) mice. In contrast, adenovirus-mediated hepatic CISH knockdown impaired glucose tolerance and increased gluconeogenesis in WT mice. We also generated liver-specific CISH knockout (LV-CISH KO) mice and discovered that these mice had a similar phenotype in glucose tolerance and gluconeogenesis as mice injected with adenoviruses that knockdown CISH expression. Mechanistically, we found that CISH overexpression decreased and CISH knockdown increased the mRNA and protein levels of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase 1 (PEPCK), two key enzymes involved in gluconeogenesis, in vitro, and in vivo. Moreover, we discovered that the phosphorylation of cAMP-responsive element binding protein 1 (CREB), a transcription factor of G6pase and Pepck, was required for regulating gluconeogenesis by CISH. Taken together, this study identifies hepatic CISH as an important regulator of gluconeogenesis. Our results also provide important insights into the metabolic functions of the SOCS protein family and the potential targets for the treatment of diabetes.