Ins2 Akita Mice Research Articles

7581 Periodic Administration of Quercetin and Dasatinib Alleviates Skeletal Fragility in the INS2AKITA Mouse Model of Type 1 Diabetes

Abstract Disclosure: R. Dresner-Pollak: Consulting Fee; Self; Opko Biologics LTD.. Speaker; Self; Medison Pharma. N. Lishinsky: None. I. Gurt: None. J. Stokar: None. V. Temkin: None. P. Shivam: None. G. Zatarah: None. J. Milgram: None. R. Shahar: None. Skeletal fragility is a recognized complication of type 1 diabetes (T1D). The risk of hip and non-vertebral fractures is significantly higher in people living with T1D compared to age- and sex-matched non-diabetic controls and those living with type 2 diabetes (T2D). Hip fracture risk is up to six-fold higher in T1D patients; hip fractures occur 10-15 years earlier and the outcome is poorer compared to non-diabetics. Alterations in bone quality are considered major contributors to T1D-related skeletal fragility. As people with T1D live longer, there is a growing need to discover new therapeutics to reduce fracture risk. TID is associated with accelerated biological aging. Cellular senescence is a fundamental mechanism of aging. Senescent cells stop dividing, are resistant to apoptosis and acquire a distinct phenotype that includes secretome changes termed “senescence-associated secretory phenotype” (SASP). Clearing senescent cells with senolytics is recognized as a promising therapeutic approach for multiple conditions in mice and humans We explored the ability of senolytics dasatinib (D), a tyrosine kinase inhibitor, and quercetin (Q), a flavanol, present in fruit and vegetables, drug combination, to alleviate femur fragility in mice with T1D. Ins2Akita mice show a sexual dimorphic effect of the mutant Ins2 allele causing severe hyperglycemia in males. We treated Ins2Akita male and female mice and C57BL/6 wild type (WT) male mice with D + Q administered once monthly for four months beginning at age 3 months. 3-month-old Ins2Akita male mice had higher blood levels of the SASPs components HMGB1, PAI-1, MMP12, and increased gene expression of p53 and senescence effectors p16, p15 and SASPs Il1b, Mmp3, Mmp9, Mmp13, Serpineb2 at the femur diaphysis and in bone marrow mesenchymal stem cells, respectively compared to age-matched WT male mice. In vitro, exposure to glucose levels equivalent to those observed in blood in Ins2Akita male mice induced cellular senescence in IDG-SW3 osteocyte-like cells including a 4-5-fold increase in gene expression of p16INK4a, p21Cip[1], p53, double percentage of SA-β-gal+ cells and P21 protein level. Ingenuity pathway analysis of differentially expressed proteins revealed enrichment for the senescence pathway (Z=1.213; P=1.11E-05). D+Q administration did not affect bone microarchitecture at the spine (L2), distal femur metaphysis and diaphysis assessed by µCT. D+Q administration significantly increased femur resistance to fracture assessed by the 3-point-bending test by increasing whole femur work-to-fracture, the ultimate parameter of bone resistance to failure, and post-yield displacement, an index of matrix composition, organization, and bone quality in Ins2Akita males. No effect was observed in Ins2Akita females or WT male mice, indicating that the intervention is specific for T1D. Senolyitcs D+Q show promise in improving bone health in T1D. Presentation: 6/1/2024

Autoimmune uveitis attenuated in diabetic mice through imbalance of Th1/Th17 differentiation via suppression of AP-1 signaling pathway in Th cells.

Inflammation is involved in the pathogenesis of diabetes, however the impact of diabetes on organ-specific autoimmune diseases remains unexplored. Experimental autoimmune uveoretinitis (EAU) is a widely accepted animal model of human endogenous uveitis. In this study, we investigated the effects of diabetic conditions on the development of EAU using a mouse diabetes model. EAU was induced in wild-type C57BL/6 (WT) mice and Ins2Akita (Akita) mice with spontaneous diabetes by immunization with IRBP peptide. Clinical and histopathological examinations, and analysis of T cell activation state were conducted. In addition, alternations in the composition of immune cell types and gene expression profiles of relevant immune functions were identified using single-cell RNA sequencing. The development of EAU was significantly attenuated in immunized Akita (Akita-EAU) mice compared with immunized WT (WT-EAU) mice, although T cells were fully activated in Akita-EAU mice, and the differentiation into Th17 cells and regulatory T (Treg) cells was promoted. However, Th1 cell differentiation was inhibited in Akita-EAU mice, and single-cell analysis indicated that gene expression associated AP-1 signaling pathway (JUN, FOS, and FOSB) was downregulated not only in Th1 cells but also in Th17, and Treg cells in Akita-EAU mice at the onset of EAU. In diabetic mice, EAU was significantly attenuated. This was related to selective inhibition of Th1 cell differentiation and downregulated AP-1 signaling pathway in both Th1 and Th17 cells.

Complement Cascade Proteins Correlate with Fibrosis and Inflammation in Early-Stage Type 1 Diabetic Kidney Disease in the Ins2Akita Mouse Model.

Diabetic kidney disease (DKD) is characterized by histological changes including fibrosis and inflammation. Evidence supports that DKD is mediated by the innate immune system and more specifically by the complement system. Using Ins2Akita T1D diabetic mice, we studied the connection between the complement cascade, inflammation, and fibrosis in early DKD. Data were extracted from a previously published quantitative-mass-spectrometry-based proteomics analysis of kidney glomeruli of 2 (early DKD) and 4 months (moderately advanced DKD)-old Ins2Akita mice and their controls A Spearman rho correlation analysis of complement- versus inflammation- and fibrosis-related protein expression was performed. A cross-omics validation of the correlation analyses' results was performed using public-domain transcriptomics datasets (Nephroseq). Tissue sections from 43 patients with DKD were analyzed using immunofluorescence. Among the differentially expressed proteins, the complement cascade proteins C3, C4B, and IGHM were significantly increased in both early and later stages of DKD. Inflammation-related proteins were mainly upregulated in early DKD, and fibrotic proteins were induced in moderately advanced stages of DKD. The abundance of complement proteins with fibrosis- and inflammation-related proteins was mostly positively correlated in early stages of DKD. This was confirmed in seven additional human and mouse transcriptomics DKD datasets. Moreover, C3 and IGHM mRNA levels were found to be negatively correlated with the estimated glomerular filtration rate (range for C3 rs = -0.58 to -0.842 and range for IGHM rs = -0.6 to -0.74) in these datasets. Immunohistology of human kidney biopsies revealed that C3, C1q, and IGM proteins were induced in patients with DKD and were correlated with fibrosis and inflammation. Our study shows for the first time the potential activation of the complement cascade associated with inflammation-mediated kidney fibrosis in the Ins2Akita T1D mouse model. Our findings could provide new perspectives for the treatment of early DKD as well as support the use of Ins2Akita T1D in pre-clinical studies.

One therapy to protect them all: Controlling diabetic eye disease in anterior and posterior segment via mitophagy‐activating drugs

Aims/Purpose: The accumulation of dysfunctional mitochondria has been implicated in the pathogenesis of diabetic eye disease through the impairment of mitochondrial quality control (MQC). This study aimed to investigate whether mitophagy‐activating drugs can alleviate pathology in the anterior and posterior ocular tissues of type‐1 diabetic mice.Methods: Mitophagy was evaluated in corneal and retinal tissues from diabetic mitophagy reporter mice (mitoQC‐Ins2Akita). The therapeutic potential of targeting mitophagy was evaluated by treating diabetic Ins2Akita mice with PIA‐01 (a PINK1‐independent mitophagy activator). Treatment was delivered orally between 4 and 8 months of diabetes. Ocular degenerative changes were evaluated by scotopic electroretinography (ERG), along with immunohistochemical analysis of retinal neurons and corneal cytoarchitecture.Results: Indicative of deteriorated MQC, mitophagy and mitochondrial transcription factor A (TFAM) positive mitochondrial nucleoids (which contain mtDNA) significantly decreased in the cornea and retina by 8 months of diabetes. Oral delivery of PIA‐01 restored TFAM+ mitochondrial nucleoids, leading to improved visual outcomes in Ins2Akita mice. In the retina, this was reflected by improved ERG responses (e.g. increased scotopic a‐wave and b‐wave amplitudes), while also protecting retinal neurons, including cone‐photoreceptors, synaptic structures, amacrine cells and retinal ganglion cells. This protection extended to the anterior segment, where PIA‐01 rescued corneal cytoarchitecture (basal epithelium and stroma) in diabetic mice.Conclusions: Diabetes progresses with deterioration of MQC in anterior and posterior ocular tissues. Rescuing MQC via mitophagy‐activating drugs may allow for multisystem therapies to control diabetic eye disease.

Topical Nerve Growth Factor (NGF) restores electrophysiological alterations in the Ins2Akita mouse model of diabetic retinopathy

People suffering from diabetes mellitus commonly have to face diabetic retinopathy (DR), an eye disease characterized by early retinal neurodegeneration and microvascular damage, progressively leading to sight loss. The Ins2Akita (Akita) diabetic mouse presents the characteristics of DR and experimental drugs can be tested on this model to check their efficacy before going to the clinic. Topical administration of Nerve Growth Factor (NGF) has been recently demonstrated to prevent DR in the Akita mouse, reverting the thinning of retinal layers and protecting the retinal ganglion cells (RGCs) from death. In this study, we characterize the effects of topical NGF on neuroretina function, quantified with the electroretinogram (ERG). In particular, we show that NGF can ameliorate RGC conduction in the retina of Akita mice, which correlates with a recovery of retinal nerve fiber plus ganglion cell layer (RNFL-GCL) structure. Overall, our preclinical results highlight that topical administration of NGF could be a promising therapeutic approach for DR, being capable of exerting a beneficial impact on retinal functionality.

Blockade of Annexin A2 Prevents Early Microvasculopathy in Murine Models of Diabetic Retinopathy.

We examined the role of annexin A2 (A2) in the development of diabetic retinal vasculopathy by testing the effect of Anxa2 gene deletion as well as administration of anti-A2 antibodies on pericyte dropout and retinal neovascularization in diabetic Akita mice, and in mice subjected to oxygen-induced retinopathy. We analyzed diabetic Ins2AKITA mice with or without global deletion of Anxa2, as well as Ins2AKITA mice that received intravitreal anti-A2 IgG or control antibody at 2, 4, and 6 months, for retinal pericyte dropout at 7 months of age. In addition, we assessed the effect of intravitreal anti-A2 on oxygen-induced retinopathy (OIR) in neonatal mice by quantifying retinal neovascular and vaso-obliterative area, and by enumeration of neovascular tufts. Both deletion of the Anxa2 gene and immunologic blockade of A2 prevented pericyte depletion in retinas of diabetic Ins2AKITA mice. Blockade of A2 also reduced vaso-obliteration and neovascularization in the OIR model of vascular proliferation. This effect was amplified when a combination of antivascular endothelial growth factor (VEGF) and anti-A2 antibodies was used. Therapeutic approaches that target A2, alone or in combination with anti-VEGF therapy, are effective in mice, and may also curtail the progression of retinal vascular disease in humans with diabetes.

The association between altered intestinal microbiome, impaired systemic and ocular surface immunity, and impaired wound healing response after corneal alkaline-chemical injury in diabetic mice.

We aim to investigate the effect of sustained hyperglycemia on corneal epithelial wound healing, ocular surface and systemic immune response, and microbiome indices in diabetic mice compared to controls after alkaline chemical injury of the eye. Corneal alkaline injury was induced in the right eye of Ins2Akita (Akita) mice and wild-type mice. The groups were observed at baseline and subsequently days 0, 3, and 7 after injury. Corneal re-epithelialization was observed under slit lamp with fluorescein staining using a cobalt blue light filter. Enucleated cornea specimens were compared at baseline and after injury for changes in cornea thickness under hematoxylin and eosin staining. Tear cytokine and growth factor levels were measured using protein microarray assay and compared between groups and time points. Flow cytometry was conducted on peripheral blood and ocular surface samples to determine CD3+CD4+ cell count. Fecal samples were collected, and gut microbiota composition and diversity pattern were measured using shotgun sequencing. Akita mice had significantly delayed corneal wound healing compared to controls. This was associated with a reduction in tear levels of vascular endothelial growth factor A, angiopoietin 2, and insulin growth factor 1 on days 0, 3, and 7 after injury. Furthermore, there was a distinct lack of upregulation of peripheral blood and ocular surface CD3+CD4+ cell counts in response to injury in Akita mice compared to controls. This was associated with a reduction in intestinal microbiome diversity indices in Akita mice compared to controls after injury. Specifically, there was a lower abundance of Firmicutes bacterium M10-2 in Akita mice compared to controls after injury. In diabetic mice, impaired cornea wound healing was associated with an inability to mount systemic and local immune response to ocular chemical injury. Baseline and post-injury differences in intestinal microbial diversity and abundance patterns between diabetic mice and controls may potentially play a role in this altered response.

Diabetes has no additional impact on retinal ganglion cell loss in a mouse model of spontaneous glaucoma.

There is no valid medical treatment for diabetic retinopathy mostly because its pathogenesis remains largely unknown. Early stages of diabetic retinopathy, just like glaucoma, are characterized by the loss of retinal ganglion cells. Whether the two diseases may share a similar pathogenic background is unknown. To clarify this issue the thickness of retinal nerve fiber layer was studied in vivo by optical coherence tomography in 10 Ins2Akita (diabetic) and 10 C57BL/6J (control) mice. The number of retinal ganglion cells and retina's surface covered by neurofilaments were quantified ex vivo in 12 normoglycemic DBA/2J (glaucoma) and 11 diabetic (alloxan-induced) DBA/2J mice (glaucoma + diabetes). At 16 weeks of age retinal nerve fiber layer was significantly thinner in Ins2Akita mice confirming the neurodegenerative impact of diabetes. Number of retinal ganglion cells and retina's surface covered by neurofilaments were similar in normoglycemic and diabetic DBA/2J mice with the exception of the superior quadrant where the number of retinal ganglion cells was increased in animals with glaucoma + diabetes. In presence of glaucoma, diabetes is unable to induce further retinal ganglion cells loss. The hypothesis that the mechanism leading to retinal ganglion cells loss may be shared by the two diseases cannot be ruled out. Whether early diabetes-driven retinal neurodegeneration could be prevented by neuroprotective treatment proven to be effective in case of glaucoma, remains to be clarified.

Investigation of Retinal Metabolic Function in Type 1 Diabetic Akita Mice.

Diabetic retinopathy (DR) is the leading cause of vision loss in working age adults. Understanding the retinal metabolic response to circulating high glucose levels in diabetic patients is critical for development of new therapeutics to treat DR. Measuring retinal metabolic function using the Seahorse analyzer is a promising technique to investigate the effect of hyperglycemia on retinal glycolysis and mitochondrial respiration. Here, we analyzed the retinal metabolic function in young and old diabetic and control mice. We also compared the expression of key glycolytic enzymes between the two groups. The Seahorse XF analyzer was used to measure the metabolic function of retina explants from young and old type 1 diabetic Akita (Ins2Akita) mice and their control littermates. Rate-limiting glycolytic enzymes were analyzed in retina lysates from the two age groups by Western blotting. Retinas from young adult Akita mice showed a decreased glycolytic response as compared to control littermates. However, this was not observed in the older mice. Western blotting analysis showed decreased expression of the glycolytic enzyme PFKFB3 in the young Akita mice retinas. Measurement of the oxygen consumption rate showed no difference in retinal mitochondrial respiration between Akita and WT littermates under normal glucose conditions ex vivo despite mitochondrial fragmentation in the Akita retinas as examined by electron microscopy. However, Akita mice retinas showed decreased mitochondrial respiration under glucose-free conditions. In conclusion, diabetic retinas display a decreased glycolytic response during the early course of diabetes which is accompanied by a reduction in PFKFB3. Diabetic retinas exhibit decreased mitochondrial respiration under glucose deprivation.

In Vivo Capillary Structure and Blood Cell Flux in the Normal and Diabetic Mouse Eye.

PurposeTo characterize the early structural and functional changes in the retinal microvasculature in response to hyperglycemia in the Ins2Akita mouse.MethodsA custom phase-contrast adaptive optics scanning light ophthalmoscope was used to image retinal capillaries of 9 Ins2Akita positive (hyperglycemic) and 9 Ins2Akita negative (euglycemic) mice from postnatal weeks 5 to 18. A 15 kHz point scan was used to image capillaries and measure red blood cell flux at biweekly intervals; measurements were performed manually. Retinal thickness and fundus photos were captured monthly using a commercial scanning laser ophthalmoscope/optical coherence tomography. Retinal thickness was calculated using a custom algorithm. Blood glucose and weight were tracked throughout the duration of the study.ResultsElevated blood glucose (>250 mg/dL) was observed at 4 to 5 weeks of age in Ins2Akita mice and remained elevated throughout the study, whereas euglycemic littermates maintained normal glucose levels. There was no significant difference in red blood cell flux, capillary anatomy, lumen diameter, or occurrence of stalled capillaries between hyperglycemic and euglycemic mice between postnatal weeks 5 and 18. Hyperglycemic mice had a thinner retina than euglycemic littermates (p < 0.001), but retinal thickness did not change with duration of hyperglycemia despite glucose levels that were more than twice times normal.ConclusionsIn early stages of hyperglycemia, retinal microvasculature structure (lumen diameter, capillary anatomy) and function (red blood cell flux, capillary perfusion) were not impaired despite 3 months of chronically elevated blood glucose. These findings suggest that hyperglycemia alone for 3 months does not alter capillary structure or function in profoundly hyperglycemic mice.

Interleukin 17A deficiency alleviates neuroinflammation and cognitive impairment in an experimental model of diabetic encephalopathy.

Interleukin 17A (IL-17A) was previously shown to be a key pro-inflammatory factor in diabetes mellitus and associated complications. However, the role of IL-17A in diabetic encephalopathy remains poorly understood. In this study, we established a mouse model of diabetic encephalopathy that was deficient in IL-17A by crossing Il17a-/- mice with spontaneously diabetic Ins2Akita (Akita) mice. Blood glucose levels and body weights were monitored from 2-32 weeks of age. When mice were 32 weeks of age, behavioral tests were performed, including a novel object recognition test for assessing short-term memory and learning and a Morris water maze test for evaluating hippocampus-dependent spatial learning and memory. IL-17A levels in the serum, cerebrospinal fluid, and hippocampus were detected with enzyme-linked immunosorbent assays and real-time quantitative polymerase chain reaction. Moreover, proteins related to cognitive dysfunction (amyloid precursor protein, β-amyloid cleavage enzyme 1, p-tau, and tau), apoptosis (caspase-3 and -9), inflammation (inducible nitric oxide synthase and cyclooxygenase 2), and occludin were detected by western blot assays. Pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, and interferon-γ in serum and hippocampal tissues were measured by enzyme-linked immunosorbent assays. Microglial activation and hippocampal neuronal apoptosis were detected by immunofluorescent staining. Compared with that in wild-type mice, mice with diabetic encephalopathy had higher IL-17A levels in the serum, cerebrospinal fluid, and hippocampus; downregulation of occludin expression; lower cognitive ability; greater loss of hippocampal neurons; increased microglial activation; and higher expression of inflammatory factors in the serum and hippocampus. IL-17A knockout attenuated the abovementioned changes in mice with diabetic encephalopathy. These findings suggest that IL-17A participates in the pathological process of diabetic encephalopathy. Furthermore, IL-17A deficiency reduces diabetic encephalopathy-mediated neuroinflammation and cognitive defects. These results highlight a role for IL-17A as a mediator of diabetic encephalopathy and potential target for the treatment of cognitive impairment induced by diabetic encephalopathy.

Polyphenol Metabolite Pyrogallol-O-Sulfate Decreases Microglial Activation and VEGF in Retinal Pigment Epithelium Cells and Diabetic Mouse Retina.

(Poly)phenol-derived metabolites are small molecules resulting from (poly)phenol metabolization after ingestion that can be found in circulation. In the last decade, studies on the impact of (poly)phenol properties in health and cellular metabolism accumulated evidence that (poly)phenols are beneficial against human diseases. Diabetic retinopathy (DR) is characterized by inflammation and neovascularization and targeting these is of therapeutic interest. We aimed to study the effect of pyrogallol-O-sulfate (Pyr-s) metabolite in the expression of proteins involved in retinal glial activation, neovascularization, and glucose transport. The expression of PEDF, VEGF, and GLUT-1 were analyzed upon pyrogallol-O-sulfate treatment in RPE cells under high glucose and hypoxia. To test its effect on a diabetic mouse model, Ins2Akita mice were subjected to a single intraocular injection of the metabolite and the expression of PEDF, VEGF, GLUT-1, Iba1, or GFAP measured in the neural retina and/or retinal pigment epithelium (RPE), two weeks after treatment. We observed a significant decrease in the expression of pro-angiogenic VEGF in RPE cells. Moreover, pyrogallol-O-sulfate significantly decreased the expression of microglial marker Iba1 in the diabetic retina at different stages of disease progression. These results highlight the potential pyrogallol-O-sulfate metabolite as a preventive approach towards DR progression, targeting molecules involved in both inflammation and neovascularization.

Short-and Long-Term Expression of Vegf: A Temporal Regulation of a Key Factor in Diabetic Retinopathy.

To investigate the role of vascular endothelial growth factor (VEGF) at different phases of diabetic retinopathy (DR), we assessed the retinal protein expression of VEGF-A164 (corresponding to the VEGF165 isoform present in humans, which is the predominant member implicated in vascular hyperpermeability and proliferation), HIF-1α and PKCβ/HuR pathway in Ins2 Akita (diabetic) mice at different ages. We used C57BL6J mice (WT) at different ages as control. Retina status, in terms of tissue morphology and neovascularization, was monitored in vivo at different time points by optical coherence tomography (OCT) and fluorescein angiography (FA), respectively. The results showed that VEGF-A164 protein expression increased along time to become significantly elevated (p < 0.05) at 9 and 46 weeks of age compared to WT mice. The HIF-1α protein level was significantly (p < 0.05) increased at 9 weeks of age, while PKCβII and HuR protein levels were increased at 46 weeks of age compared to WT mice. The thickness of retinal nerve fiber layer as measured by OCT was decreased in Ins2 Akita mice at 9 and 46 weeks of age, while no difference in the retinal vasculature were observed by FA. The present findings show that the retina of the diabetic Ins2 Akita mice, as expected for mice, does not develop proliferative retinopathy even after 46 weeks. However, diabetic Ins2 Akita mice recapitulate the same evolution of patients with DR in terms of both retinal neurodegeneration and pro-angiogenic shift, this latter indicated by the progressive protein expression of the pro-angiogenic isoform VEGF-A164, which can be sustained by the PKCβII/HuR pathway acting at post-transcriptional level. In agreement with this last concept, this rise in VEGF-A164 protein is not paralleled by an increment of the corresponding transcript. Nevertheless, the observed increase in HIF-1α at 9 weeks indicates that this transcription factor may favor, in the early phase of the disease, the transcription of other isoforms, possibly neuroprotective, in the attempt to counteract the neurodegenerative effects of VEGF-A164. The time-dependent VEGF-A164 expression in the retina of diabetic Ins2 Akita mice suggests that pharmacological intervention in DR might be chosen, among other reasons, on the basis of the specific stages of the pathology in order to pursue the best clinical outcome.

Connectivity mapping of glomerular proteins identifies dimethylaminoparthenolide as a new inhibitor of diabetic kidney disease

While blocking the renin angiotensin aldosterone system (RAAS) has been the main therapeutic strategy to control diabetic kidney disease (DKD) for many years, 25–30% of diabetic patients still develop the disease. In the present work we adopted a systems biology strategy to analyze glomerular protein signatures to identify drugs with potential therapeutic properties in DKD acting through a RAAS-independent mechanism. Glomeruli were isolated from wild type and type 1 diabetic (Ins2Akita) mice treated or not with the angiotensin-converting enzyme inhibitor (ACEi) ramipril. Ramipril efficiently reduced the urinary albumin/creatine ratio (ACR) of Ins2Akita mice without modifying DKD-associated renal-injuries. Large scale quantitative proteomics was used to identify the DKD-associated glomerular proteins (DKD-GPs) that were ramipril-insensitive (RI-DKD-GPs). The raw data are publicly available via ProteomeXchange with identifier PXD018728. We then applied an in silico drug repurposing approach using a pattern-matching algorithm (Connectivity Mapping) to compare the RI-DKD-GPs’s signature with a collection of thousands of transcriptional signatures of bioactive compounds. The sesquiterpene lactone parthenolide was identified as one of the top compounds predicted to reverse the RI-DKD-GPs’s signature. Oral treatment of 2 months old Ins2Akita mice with dimethylaminoparthenolide (DMAPT, a water-soluble analogue of parthenolide) for two months at 10 mg/kg/d by gavage significantly reduced urinary ACR. However, in contrast to ramipril, DMAPT also significantly reduced glomerulosclerosis and tubulointerstitial fibrosis. Using a system biology approach, we identified DMAPT, as a compound with a potential add-on value to standard-of-care ACEi-treatment in DKD.

IL-17A is involved in diabetic inflammatory pathogenesis by its receptor IL-17RA.

The participation of interleukin (IL)-17A in diabetic pathogenesis is suggested in animal models of autoimmune diabetes and in patients with type 1 diabetes (T1D), but with some contradictory results. Particularly, evidence for a direct injury of IL-17A to pancreatic β cells is lacking. We showed that IL-17A deficiency alleviated diabetic signs including hyperglycemia, hypoinsulinemia, and inflammatory response in Ins2Akita (Akita) mice, a T1D model with spontaneous mutation in insulin 2 gene leading to β-cell apoptosis. IL-17A enhanced inflammatory reaction, oxidative stress, and cell apoptosis but attenuated insulin level in mouse insulin-producing MIN6 cells. IL-17A had also a synergistic destruction to MIN6 cells with streptozotocin (STZ), a pancreatic β-cell-specific cytotoxin. Blocking IL-17 receptor A (IL-17RA) reduced all these deleterious effects of IL-17A on MIN6 cells. The results demonstrate the role and the importance of IL-17A in T1D pathogenesis and suggest a potential therapeutic strategy for T1D targeting IL-17A and/or IL-17RA.

Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina

Intravitreal injections of anti-vascular endothelial growth factor drugs have become the gold standard treatment for diabetic retinopathy (DR). However, several patients are classified as non-responders or poor responders to treatment. Therefore, it is essential to study alternative target molecules. We have previously shown that the progression of DR in the Ins2Akita mouse reflects the imbalance between pro- and anti-angiogenic molecules found in the human retina. We report, for the first time, the therapeutic potential of a dual-acting antiangiogenic non-viral gene therapy. We have used an expressing vector encoding both the pigment epithelium-derived factor gene and a short hairpin RNA (shRNA) targeted to the placental growth factor to restore the balance between these factors in the retina. Twenty-one days after a single subretinal injection, we observed a marked decrease in the inflammatory response in the neural retina and in the retinal pigment epithelium, together with reduced vascular retinal permeability in the treated diabetic mouse. These results were accompanied by the restoration of the retinal capillary network and regression of neovascularization, with significant improvement of DR hallmarks. Concomitant with the favorable therapeutic effects, this approach did not affect retinal ganglion cells. Hence our results provide evidence toward the use of this approach in DR treatment.

Retinal changes in mice spontaneously developing diabetes by Th17-cell deviation

Elevated level of interleukin (IL)-17, predominantly produced by T helper (Th) 17 cells, has been implicated in diabetic retinopathy (DR), but it remains unclear whether IL-17 is involved in the pathogenesis of DR. Ins2Akita (Akita) mice spontaneously develop diabetes, and show early pathophysiological changes in diabetic complications. On the other hand, interferon-γ knock out (GKO) mice exhibit high differentiation and activation of Th2 and Th17 cells as a result of Th1 cell inhibition. In this study, Ins2Akita IFN-γ–deficient (Akita-GKO) mice were established by crossbreeding Akita mice with GKO mice, and Th17-mediated immune responses on DR were investigated. Blood glucose levels (BGL) of Akita mice and Akita-GKO mice were significantly higher than those of age-matched wild type (WT) or GKO mice, and there was no significant difference in BGL between Akita and Akita-GKO mice. Relative mRNA expression of ROR-γt that is a transcriptional factor of Th17 cells but not GATA-3 that is for Th2 cells was significantly upregulated only in Akita-GKO mice compared with WT mice, and the proportions of IL-17 and IL-22–producing splenic CD4+ cells were significantly higher in Akita-GKO mice than in wild type (WT), Akita, or GKO mice. In the retina, mRNA expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) were increased in Akita-GKO mice more than in Akita or GKO mice, and statistically significant differences were observed between Akita-GKO mice and WT mice. Leukostasis in retinal vessels and ocular level of VEGF protein increased significantly in Akita-GKO mice compared with the other groups. Edematous change in the retinal surface layer, retinal exudative lesions depicted as areas of hyperfluorescence in fluorescein angiography (FA), and vascular basement membrane thickening in all layers of the retina were also observed in Akita-GKO mice at 9-week-old but not in age-matched Akita or GKO mice. These results suggested that Th17 cell-mediated immune responses might be involved in promotion of functional and morphological changes in the retina of mice spontaneously developing diabetes.

Aspartic acid supplementation ameliorates symptoms of diabetic kidney disease in mice.

Diabetic kidney disease (DKD) is among the most common and serious complications of both type 1 and type 2 diabetes. In this study, we used KK/Ta‐Ins2Akita (KK‐Akita) mice as a model of DKD and KK/Ta (KK) mice as controls to identify novel factors related to the development/progression of DKD. Capillary electrophoresis coupled with mass spectrometry analysis revealed that circulating Asp (l‐aspartic acid) levels in diabetic KK‐Akita mice tend to be lower than those in control KK mice. Therefore, we evaluated the effect of Asp supplementation to prevent the progression of DKD in KK‐Akita mice. Mice were divided into three groups: (a) untreated KK mice (Control group), (b) untreated KK‐Akita mice (DKD group), and (c) treated (double‐volume Asp diet) KK‐Akita mice (Tx group). Kidney sections were stained with fluorescein isothiocyanate‐labeled lectins, wheat germ agglutinin (WGA), and anti‐endothelial nitric oxide synthase (eNOS) antibody for evaluation of endothelial surface layer (ESL) and NO synthesis. The mesangial area and glomerular size in the DKD group were significantly larger than those in the Control group; however, there was no significant difference in those between the DKD and Tx groups. Albuminuria, the ratio of foot process effacement, and thickness of glomerular basement membrane in the Tx group were significantly lower than those in the DKD group. Furthermore, the expression levels of glomerular WGA and microvascular eNOS in the Tx group improved significantly and approached the level in the Control group. In conclusion, the improvement of albuminuria in the Tx group may be caused by the reduction of oxidative stress in the kidneys, which may lead to the subsequent improvement of glomerular ESL.

Dysregulation of trophic factors contributes to diabetic retinopathy in the Ins2Akita mouse

Diabetic retinopathy (DR) is considered as a diabetes-related complication that can lead to severe visual impairments. By 2030, it is expected that 1 in 5 adults will suffer from the disease. Suitable animal models for chronic DR are essential for a better understanding of the pathophysiology and to further develop new treatments.The Ins2Akita mouse is a type 1 diabetes model that shows signs of both early and late stages of DR, including pericyte loss, increased vascular permeability, increased acellular capillaries and neovascularization. To further characterize DR in the Ins2Akita mouse model, we have evaluated the protein levels of the angiogenesis inducers vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) and the angiogenesis inhibitor pigment epithelium-derived factor (PEDF). Additionally, we have analyzed the protein expression profile of the glial markers ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) as well as of the chemokine monocyte chemoattractant protein 1 (MCP-1). In this study we demonstrate that, with disease progression, there is the development of an inflammatory response and an unbalanced expression of pro- and antiangiogenic factors in the neural retina and in the retinal pigment epithelium (RPE) of Ins2Akita mice. Therefore, our data provide support for the diabetic retinopathy features detected in the Ins2Akita retina, reflecting what is observed in the human pathology.

Supplementation of endogenous Ahr ligands reverses insulin resistance and associated inflammation in an insulin-dependent diabetic mouse model

Aryl-hydrocarbon receptor (Ahr) plays an important role in the regulation of intestinal homeostasis.Diabetes is characterized by vascular complications and intestinal dysfunction. We aimed at understanding the relationship between intestinal defense impairment and inflammation in diabetes and effects of Ahr ligands on diabetes-induced insulin resistance, endovascular inflammation, and intercellular adhesion molecule (ICAM) and flavin mono-oxygenase (FMO3) expression. Effects of Ahr ligands, such as tryptophan (Trp) and indole-3-carbinol (I3C) on intestinal barrier and inflammation of Ins2Akita mice were examined. Myeloid differentiation primary response 88 (MYD88) is the adaptor for inflammatory signaling pathways. Ins2Akita-MyD88-/- mice were used to study the role of MyD88. Ins2Akita mice demonstrated decreased Ahr and regenerating islet-derived 3-β (Reg3β) expression, and increased Klebsiella pneumoniae translocation. Ins2Akita mice demonstrated increased inducible nitric oxide synthase (iNOS) expression of intestine; ICAM, iNOS, interleukin 1 beta (IL-1β), and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta. Trp and I3C decreased diabetes-induced translocation and increased Ahr and Reg3β expression of intestine. Ahr ligands reduced diabetes-induced ICAM and FMO3 expression in liver and aorta; IL-6, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP4) activity; and insulin insensitivity. Ins2Akita-MyD88-/- mice demonstrated decreased expression of p-NF-κB of liver and ICAM of aorta compared with Ins2Akita mice. Altogether, our data suggest that diabetes induces ICAM and FMO3 expression through the decrease in intestinal defense and MyD88. Ahr ligands reverse diabetes-induced intestinal defense impairment, insulin insensitivity, FMO3/ICAM expression, and systemic inflammation.